Romosozumab (Romo) has a dual effect of increasing bone formation and decreasing bone resorption and is associated with vertebral and clinical fracture risk reduction within 12 months (1). We present the results of 12-months Romo retreatment in subjects initially treated with Romo followed by placebo (Pbo) or denosumab (DMAb).

Methods

Postmenopausal women with a lumbar spine (LS), total hip (TH), or femoral neck (FN) T score ≤−2.0 and ≥−3.5 were enrolled in a phase 2 study (2) (NCT00896532). A total of 139 subjects entered the 12-month Romo 210 mg QM retreatment phase at month 36 after being randomized to various Romo doses from baseline (BL) to month 24, followed by Pbo or DMAb (month 24‒36). Here we report the results of retreatment for the group that was initially randomized to Romo 210 mg QM (n=35).

Results

Romo retreatment after Pbo resulted in bone mineral density (BMD) increases at the LS, TH, and FN comparable to the initial Romo treatment. BMD increased by 12.7% from month 36‒48 (12.0% BL‒month 12; 17.6% BL‒month 48) at the LS, 5.8 % (5.5% BL‒month 12; 7.1% BL‒ month 48) at the TH, and 6.3% (5.4% BL‒month 12; 8.6% BL‒month 48) at the FN. In subjects initially treated with Romo followed by DMAb for 12 month, then retreated with Romo, BMD increased by 2.8% from month 36‒48 (12.6% BL‒month 12; 22.1% BL‒month 48) at the LS, while no further BMD increase was noted at TH (7.3% BL‒month 36 and BL‒month 48) or FN (6.3% BL‒month 36; 6.7% BL‒month 48) in the retreatment phase. Bone formation (P1NP) and bone resorption (CTX) patterns were similar in Romo retreatment subjects after Pbo to subjects who received Romo in the first 12 months. In subjects who had received DMAb, reduced levels of both P1NP and CTX increased gradually after Romo retreatment to 52.1% and 16.5%, respectively, at month 48 from BL. The safety profile of Romo retreatment was generally consistent with Romo treatment from BL‒month 24. Of the 140 subjects initially exposed to Romo, 2 subjects previously antibody negative developed binding antibodies during retreatment, none had neutralizing activity; 6 subjects had neutralizing antibodies at the start of retreatment, 1 subject remained positive at the end of retreatment.

Conclusion

After a 12-month treatment-free period, Romo retreatment increased BMD at the spine and hip to an extent similar to initial Romo treatment. Romo retreatment after DMAb resulted in further increases in BMD at the spine and maintenance of BMD at the hip. Safety findings were similar to the initial Romo treatment.

Romosozumab (Romo) increased bone mineral density (BMD) and bone formation, and decreased bone resorption, resulting in vertebral and clinical fracture risk reduction within 12 months in postmenopausal women with osteoporosis (1). Here we report the efficacy and safety of transitioning to zoledronic acid (Zol) following Romo.

Methods

A phase 2 trial (2) (NCT00896532) enrolled postmenopausal women with a lumbar spine (LS), total hip (TH), or femoral neck (FN) T-score ≤−2.0 and ≥−3.5. In this analysis, women received various Romo doses or placebo (Pbo) from baseline (BL) to month 24, were re-randomized to denosumab (DMAb) or Pbo (month 24‒36), and then all received Romo (210 mg QM) for 12 months (month 36‒48). At month 48, subjects on active treatment (Romo/DMAb/Romo) for 48 months were assigned to no intervention, unless they had a fracture from month 24‒48 or a T-score ≤–2.5 at LS, TH, or FN at month 48 and were subsequently assigned to Zol (5 mg IV). All other subjects received Zol and both groups were followed for 24 months to month 72.

Results

After Romo treatment (month 36‒48), 141 women (90 Zol, 51 no intervention) continued in the study. In the Zol group, LS BMD was maintained through month 48‒72 (percentage change from month 48: ‒0.8%; percentage change from BL at month 72: 12.8%). Similar patterns were observed at the TH (0.1% and 4.2%, respectively) and FN (0.5% and 4.4%, respectively). After no intervention, LS BMD decreased from month 48‒72 (‒10.8%), although remained above BL at month 72 (percentage change from BL at month 72: 4.2%). At the TH and FN, BMD decreased by 6.4% and 5.9% respectively from month 48‒72, reaching values close to BL at month 72. In the Zol group, both median P1NP and CTX levels decreased initially, then moved towards BL by month 72. In the no intervention group, PINP decreased while CTX increased initially and gradually returned towards BL. During month 48‒72, adverse events (AEs) were reported in 83.9% of subjects receiving Zol and 72.5% assigned to no intervention; serious AEs were reported in 13.8% and 15.7% of subjects, respectively. Fragility fractures were reported in 2 Zol (1 radius, 1 rib) and 2 no intervention (1 radius; 1 fibula) subjects, and 1 fatal event occurred in a no intervention subject.

Conclusion

BMD gains at the LS, TH, and FN with prior Romo treatment were generally maintained over 24 months with a single dose of Zol. In women who received no further intervention, BMD decreased, but remained above (LS) or near BL (TH, FN) from month 48‒72.

Methods: Subjects who discontinued investigational product (IP; PBO or DMAb 60 mg SC Q6M) from the 3-year FREEDOM trial and its 7-year Extension (Ext) were included in this analysis. Subjects receiving DMAb from FREEDOM or Ext were analyzed together. Off-Tx period was defined as 7 mo after last dose of IP through the end of study. Analyses assessed off-Tx new or worsening VFx; MVF were defined as ≥ 2 off-Tx new and/or worsening VFx.

Results: This analysis included 470 PBO and 1001 DMAb subjects. VFx were sustained by 6.6% subjects off PBO and 5.6% off DMAb, with 2.6% PBO and 3.4% DMAb sustaining MVF (including 0.9 and 1.6% with clinical MVF). Median follow-up time was 1.4/1.4 years (PBO/DMAb) and 0.5/0.3 years (PBO/DMAb) for those who did and did not sustain off-Tx VFx, respectively. More subjects who discontinued PBO (43%) than DMAb (15%) initiated alternative OP therapy. Subjects who initiated OP therapy before single VFx or MVF began OP therapy a median 4.4 or 6.3 months after last dose of PBO and 6.3 or 10.2 months after DMAb, respectively. Among those who initiated OP therapy, 5.0 and 4.0% from PBO and 4.1 and 9.0% from DMAb sustained a single VFx and MVF, respectively. Among subjects with available off-Tx BMD data (N = 297 PBO, N = 466 DMAb), mean annualized percent change in total hip BMD was +0.6% and –1.9% after stopping PBO and DMAb, respectively, for those with no off-Tx VFx; –1.3% and –2.2% for single VFx; and –1.2% and –3.5% for MVF.

Conclusion: This analysis confirms and expands previous results showing discontinuation of DMAb is associated with an increase in VFx rate, particularly clinical and morphometric MVF, whose incidence remained low and comparable to that of PBO. Fewer subjects who discontinued DMAb than PBO transitioned to alternative OP treatment, and transitioned later, suggesting that delaying a substitute therapy after DMAb may be detrimental. This is further corroborated by off-Tx BMD loss, which was greatest among subjects who sustained MVF and greater among those who sustained single VFx than those with no off-Tx VFx. Those who discontinue DMAb should transition to another OP therapy after the 6-mo dosing interval.

During FREEDOM, postmenopausal women with osteoporosis were randomized to Pbo or DMAb for 3 years. During the 7-year Extension, all participants were allocated to receive DMAb. In this analysis, we report subsequent osteoporotic fx (new vertebral or nonvertebral) in subjects who received ≥ 2 doses of DMAb during FREEDOM or the Extension, had an osteoporotic fx while on treatment, and continued treatment post-fx, compared with subsequent fx in FREEDOM Pbo subjects. These subsequent fx were analyzed as recurrent events using the stratified Cox model with the robust variance estimation adjusting for prior fx. The analysis was repeated in subgroups of subjects with or without prevalent vertebral fx, defined at treatment baseline, without adjusting for prior fx.

During FREEDOM, 438 Pbo and 272 DMAb subjects had an osteoporotic fx (mean age at first on-study fx: 74.1 and 74.5 years, respectively). Of these, there were 54 (12.3%) and 24 (8.8%) subjects who had ≥ 1 subsequent fx in the Pbo and DMAb groups, respectively. Adjusted subject incidence per 100 patient-years was lower for DMAb (6.7) vs Pbo (10.1). Combining all subjects on DMAb from FREEDOM and the Extension for up to 10 years (Combined-DMAb), 794 (13.7%) subjects had an osteoporotic fx while on DMAb (mean age at first on-study fx: 76.5 years); of these, one or more subsequent fx occurred in 144 (18.1%) subjects, with an adjusted subject incidence of 5.8 per 100 patient-years, similar to FREEDOM DMAb (6.7 per 100 patient-years). Among subjects with ≥ 1 subsequent fx, 90% had only 1 fx; vertebral fx was the most frequent. The risk of having subsequent on-study osteoporotic fx was lower in the Combined-DMAb group vs Pbo (HR 0.59 [95% CI: 0.43–0.81]; p = 0.0012), adjusting for prior fx. In this analysis, 33% of subjects had prevalent vertebral fx at treatment baseline. The effect of DMAb treatment on reduction of subsequent on-study fx was statistically greater in subjects with prevalent vertebral fx (Pbo = 17.4 vs Combined-DMAb = 7.8 per 100 patient-years; HR 0.41 [95% CI: 0.26–0.65]; p < 0.0001) compared with subjects without prevalent vertebral fx (Pbo = 6.8 vs Combined-DMAb = 4.9 per 100 patient-years; HR 0.81 [95% CI: 0.50, 1.30]; p = 0.3728), with an interaction p = 0.0347. In both subgroups, spine fx was the most frequent subsequent fx. These data demonstrate that DMAb decreases the risk of subsequent fx and a fx sustained while on DMAb is not necessarily indicative of treatment failure.

Clinical Case: A 48-year-old Mexican man with HIV was evaluated for severely low BMD and multiple atraumatic fractures starting in adulthood, including bilateral femur fractures. His viral load was suppressed on HAART. Transient hypercalcemia had followed hip surgery. He lost teeth at age 28. He had difficultly hearing the TV but never had formal audiometry. He did not report familial bone disease, but his father had short stature and multiple fractures. Physical exam noted a height of 5’2”, edentulous mouth, subjective right > left decreased hearing, white sclerae, thoracic kyphosis, enlarged fingers, and anteriorly bowed tibias. Serum alkaline phosphatase (ALP) was 330 U/L (n: 39-117 U/L) and bone-specific ALP was 87.6 ug/L (n: 6.5-20.1 ug/L), whereas PTH, Ca, Vit D and phosphorus were normal. Bone turnover markers were strikingly elevated: serum CTX 2477 pg/mL (n: 60-700 pg/mL) and osteocalcin 281 ng/mL (n: 11-50 ng/mL). DXA spine BMD was 0.488 g/cm2 with Z-score -5.5. Skeletal survey showed remarkably lucent bones with vertebral compression fractures, calvarial lucencies, and thinned long bone cortices. We identified a unique, heterozygous 27-base pair duplication (77dup27) in exon 1 of TNFRSF11A. Alendronate 70 mg/week modestly decreased bone turnover markers in 6 weeks. Family investigation is planned.

Conclusion: Our patient’s high-turnover bone disease involves a novel 27-base pair duplication in TNFRSF11A, the gene encoding the signal peptide of RANK. Similar duplications have been identified in Japanese (75dup27) and Chinese (78dup27) PBD2 cohorts, although they seem to predict the same 9-amino acid duplication (4). Our patient shares features of their PDB2 with presentation in his late 20s, painless enlarged fingers, transient hypercalcemia, and absence of childhood tooth or hearing loss. Bisphosphonate therapy improves bone turnover markers in PBD2 patients. Our experience supports mutation analysis in the diagnosis and management of high-turnover bone diseases, including those involving constitutively active RANK.

Four bone biomarkers (CTX = bone resorption; PINP = bone formation; sclerostin/FGF-23 = osteocyte function) were measured on q2h plasma samples over a 24-h interval at baseline and after a 3-wk intervention of sleep restriction (5.6h sleep/24-h) with concurrent circadian disruption (recurring 28-h ‘day’). Post-intervention samples were obtained when participants were at a similar circadian phase compared to the baseline samples.

Maximum likelihood estimates for repeated measures were obtained to assess the effects of sleep/circadian disruption and of age, on bone biomarker levels across a 24-h interval.

Except FGF-23, bone biomarker levels varied significantly by age at baseline (CTX/PINP higher in younger men (p = 0.01 and 0.02, respectively); sclerostin higher in older men (p = 0.005). This is consistent with higher bone (re)modeling rates in younger men during their consolidation phase and a nadir in BTMs in 50-60yo men. PINP levels were significantly lower post intervention compared to baseline; the decrease in PINP occurred at every time point and this decline was greater for the younger men with higher bone turnover at baseline (-27% or -20.73 ± 1.38 mcg/L, p<0.001), compared to older men (-18% or -10.01 ± 1.70 mcg/L, p<0.001). These decreases were of similar magnitude to the early increase seen with teriparatide treatment and occurred independently of changes in CTX (Δ = 2-4%, p = 0.56). Sclerostin levels were significantly higher post-intervention in the younger men only (Δ +25% or 5.92 ± 1.84 pmol/L, p=0.002) compared to older men (Δ +2.3% or 0.90 ± 2.26 pmol/L, p=0.69). Post intervention, FGF-23 levels were 6.5% lower (-2.46 ± 0.96 pg/mL; p = 0.01).

These results suggest that 3 weeks of sleep loss with circadian disruption can lead to an uncoupling of bone turnover and a potential “catabolic window,” wherein bone formation is decreased but bone resorption is unchanged. These data further suggest that sleep disruption may be most detrimental to bone during high bone turnover states (i.e. bone modeling, menopause). These changes could be due to direct effects on diurnal bone remodeling (either related to sleep restriction, or prior circadian disruption), indirect effects via changes in sex-hormone status, inflammation and/or sympathetic tone induced by sleep disruption, or less likely, the decreased physical activity inherent in study conditions. Further studies are needed to confirm these data independent of study conditions and to explore sex differences and mechanisms.

Country-specific FRAX models were used to calculate 10-year absolute fracture risks at baseline. Relative risk reductions for new vertebral fractures (VF) and hazard ratios for nonvertebral (NVF), clinical (CF), and major osteoporotic (MOF) fractures were calculated for each geographic and ethnic subgroup, and Forest plots were constructed to assess treatment-by-subgroup interactions.

Prevalent VF were observed at baseline in 20.0%, 18.0%, 25.3%, and 18.5% of total (placebo and ABL-SC) patients from North America, South America, Europe, and Asia, and at least one prior NVF was reported in 80.0%, 40.1%, 58.5%, and 32.2%, respectively. Mean FRAX 10-year probabilities were 16.5%, 8.8%, 13.9%, and 17.7% for MOF. The effects of ABL-SC versus placebo on the risks of VF, NVF, CF, and MOF did not differ significantly across geographic regions, as well as for Hispanic/Latino versus other ethnicities, with no significant treatment-by-region interactions (p=0.57, 0.89, 0.83, 0.76, respectively, for geographic subgroups; p=0.35, 0.57, 0.33, and 0.65, respectively, for Hispanic/Latino versus other ethnicities).

In conclusion, despite limitations of subgroup analyses and geographic variability in fracture incidence and risk at baseline, these analyses suggest that the effects of ABL-SC versus placebo on reducing the risk of vertebral, nonvertebral, clinical, and major osteoporotic fractures were consistent across prespecified geographic subgroups. Additionally, a post hoc analysis suggests that the effects of ABL-SC versus placebo on fracture risk were also similar for women of Hispanic/Latino ethnicity compared to other women enrolled in ACTIVE.

Methods: 160 patients with type 2 diabetes and microalbuminuria were assigned to conventional or intensified, multi-factorial therapy targeting multiple risk factors. Mean treatment duration was 7.8 years. After 7.8 years the study continued as an observational follow-up with all patients treated as the original intensive-therapy group. Time to event was modelled using Cox-regressions adjusted for age and sex.

The primary endpoint was survival time after randomization and survival time before first CVD event.

The secondary endpoint was the relative risk reduction in a 3 point MACE defined as death of CVD, coronary artery disease (CAD: non-fatal or fatal acute myocardial infarction or cardiac revascularization) and cerebrovascular disease (defined as non-fatal and fatal ischemic or hemorrhagic stroke) analyzed both as a composite- and separate endpoints.

Results: Overall median survival time was 7.9 [95% CI 2.2 – 9.6; p = 0.005] years longer in the original intensive-therapy group compared to standard treatment. Time before first CVD-event was 8.1 [4.0 – 12.6; p = 0.001] years longer in the intensive therapy group.

Hazard rates of both the composite and separate secondary endpoints were all decreased with intensified therapy. HR of the composite endpoint was 0.36 [95 % CI 0.23 – 0.57; p < 0.001], of CAD 0.43 [0.23 – 0.77; p = 0.005] and cerebrovascular disease 0.26 [0.12 – 0.55; p < 0.001]. In addition, the risk of recurrent events was significantly decreased in the intensive-therapy group (p = 0.049 for CAD and p = 0.003 for stroke).

Conclusions: At 21.2 years of follow up of 7.8 years of intensified, multifactorial, target driven treatment of type 2 diabetes mellitus with microalbuminuria, we demonstrate significantly increased life span and reduced risk of macrovascular complications.

(ClinicalTrials.gov number, NCT00320008.)

Results: There was no relationship between severe hypoglycemia and incident cognitive impairment after adjusting for baseline CVD, diabetes status, treatment allocation, a propensity score for severe hypoglycemia and the interaction of treatment allocation and severe hypoglycemia (HR 1.28, 95% 0.8, 2.05). Those individuals who experienced at least one episode of non-severe hypoglycemia were less likely to develop cognitive dysfunction than were those that did not, after adjustment for baseline CVD, diabetes status, treatment allocation, a propensity score for non-severe hypoglycemia and the interaction of treatment allocation and hypoglycemia (HR 0.71, 95% 0.56, 0.9).

To conclude: Among 11,495 individuals ~63 years of age with dysglycemia, after accounting for the risk factors of hypoglycemia, hypoglycemia itself was not associated with an increased incidence of cognitive dysfunction over 6.2 years.

Cross sectional studies in T2D patients were conducted separately in Chicago (latitude 42N) (n=194) and Thailand (latitude 13N) (n=282). Demographics, diabetes history and complications were collected. Depressive symptoms were assessed by the Center for Epidemiologic Studies Depression scale (CES-D). Chronotype was assessed using the Morningness-Eveningness Questionnaire (MEQ) in the Chicago cohort, and the Composite Score of Morningness (CSM) in the Thailand cohort. Sleep quality was assessed using the Pittsburg Sleep Quality Index (PSQI). HbA1c values were retrieved from medical records.

In the Chicago cohort, mean age was 58.3±13.0 years, 69.6% were female and 28.4% were whites. Higher CES-D scores (more depressive symptoms) were associated with later chronotype (lower MEQ score, r= -0.242, p=0.001), as well as younger age (p=0.005), female sex (p=0.047), non-whites (p=0.003), insulin use (p<0.001), higher HbA1c levels (p=0.005), poorer sleep quality (p <0.001). After adjusting for age, sex, ethnicity, HbA1c, insulin use and PSQI score, later chronotype was significantly associated with higher CES-D scores (B=-0.112, p=0.045). In Thailand cohort, mean age was 55.7±11.6 years and 57.4% were female. Higher CES-D scores were associated with later chronotype (lower CSM score, r= -0.227, p<0.001), as well as younger age (p=0.019), female sex (p=0.004), and poorer sleep quality (p<0.001). After adjusting for age, sex and PSQI score, later chronoytpe was significantly associated with higher CES-D score (B= -0.114, p=0.045).

In this study of two different ethnic cohorts, later chronotype was found to be independently associated with depressive symptoms in T2D. These data support an association between circadian regulation and psychological functioning in individuals with T2D. Intervention studies targeting circadian timing should be explored to determine whether altering circadian functioning may improve depressive symptoms in T2D patients.

Methods: Obese pregnant women (pre-pregnancy BMI >=25 kg/m²) with diet-controlled GDM were asked to participate. OSA was screened using an overnight home monitor. Those diagnosed with OSA were randomized to receive CPAP treatment for 2 weeks or be wait-list controls. After randomization period ended, all women were offered CPAP treatment until delivery. Pregnancy outcomes were collected in all women from medical records.

Results: Of the 82 women screened at a median gestational age (GA) of 29 weeks, 43 had OSA (apnea hypopnea index >=5). Of these, 28 had CPAP treatment and 15 did not. Pregnancy outcomes were available in 78 women (38 in No-OSA group, 28 in OSA/CPAP group and 11 OSA/no-CPAP group).

When comparing women among the three groups; No-OSA vs. OSA/CPAP vs. OSA/No-CPAP, there were no significant differences their age, pre-pregnancy BMI, gravida and para. There were also no significant differences in the pregnancy outcomes including subsequent rates of insulin use, preterm delivery (<37 weeks), preeclampsia, unplanned caesarean section, birth weight (3164±489 gm vs. 3099±509 gm vs. 3196±608, p=0.821), Apgar score at 1 minute and 5 minutes, or the rate of small for gestational age and large for gestational age.

Of the 28 women exposed to CPAP, 23 had used CPAP for >=2 weeks. There was no significant difference in GA at CPAP start between those using CPAP >=2 vs. <2 weeks (30.7±3.0 vs. 30.0 ± 3.7 weeks, p=0.619). Those using CPAP >=2 weeks used the device on average for 37±16 days and for 74±18% of the days.

When comparing women in No-OSA group (n=38) vs. OSA with CPAP use >=2 weeks (n=23) and OSA with CPAP use <2 weeks (n=16), the rate of preterm delivery was significantly less in those who used CPAP >=2 weeks (13.1% vs. 0% vs. 31.2%, p=0.017). In addition, unplanned caesarean section was also significantly less (34.2% vs. 13.0% vs 52.0%, p=0.026). Other pregnancy outcomes were similar among groups.

Conclusion: CPAP use in pregnant women with diet-controlled GDM and OSA was safe. CPAP use for >=2 weeks in the third trimester was associated with a significantly less risk for premature delivery and unplanned caesarean section. This information supported the benefits of CPAP treatment in this patient group and should be confirmed in a larger study.

Aim: To investigate the risk of T2D and prediabetes in lean women with PCOS, and whether to use age, ethnicity and androgen concentration as additional screening criteria.

Design:Cross-sectional study in 1068 premenopausal Scandinavian women with PCOS. All included women underwent a 2h OGTT measuring baseline fasting glucose and 2h glucose. T2D and prediabetes were diagnosed according to American Diabetes Association’s recommendations. 839 women were white-European (WE), 69 were Indian-Pakistani and 160 were of other ethnicities. None had previously been diagnosed with T2D, were pregnant, or had other serious endocrine diseases. Testosterone was measured using mass spectrometry.

Results: 0/356 (0%) lean women (BMI<25 kg/m²) with PCOS in our study had T2D, and 47/356 (13.2%) had prediabetes. In women with BMI 25-30 kg/m², 4/271 (1.5 %) had T2D and 76/271 (27.8%) had prediabetes. In women with BMI >30 kg/m², 28/438 (6.4%) had T2D and 148/438 (54.6%) had prediabetes.

BMI and waist to hip ratio (WHR) were significantly associated with the risk of T2D and prediabetes. Indian-Pakistani women had a 3.2 higher relative risk of T2D (p=0.006) and 1.7 higher relative risk of prediabetes (p=0.002) compared to WE women (mean BMI 28.8 kg/m² for both ethnicities). Age and androgen concentration (measured by both total testosterone and free androgen index) were not associated with the prevalence of T2D although older age and lower testosterone were significantly associated with the prevalence of prediabetes.

Conclusion: No women with PCOS and BMI<25 kg/m² had T2D. Our data suggest that OGTT is not necessary in normal-weight women with PCOS. The present data underline the need for prospective studies in well described study cohorts with PCOS.

To test this hypothesis, we combined RNA-seq and ChIP-seq analyses in liver tissue from vehicle- and BPA-exposed animals. RNA-seq identified a unique cassette of genes that were induced in response to HFD only in the livers of BPA-reprogrammed rats. Of these reprogrammed genes, the top pathways identified by gene set enrichment analysis (GSEA) were involved in fatty acid and/or lipid metabolism and are known to play a role in NAFLD. We have previously shown that acute, neonatal exposure to EDCs can reprogram the epigenome of developing tissues by increasing the activity of mixed lineage leukemia (MLL), the histone methyltransferase in the COMPASS complex responsible for methylation of histone H3 at lysine 4 (H3K4), an active chromatin mark. To determine the underlying mechanism responsible for the observed change in response to HFD, we performed ChIP-seq for histone modifications associated with active (H3K4me3, H3K4me1, H3K27ac) and repressive (H3K27me3) histone marks. The epigenome of 178 reprogrammed genes exhibited new H3K4me1, H3K4me3 and/or H3K27ac marks at their promoters. Importantly, these novel, active chromatin marks 1) appeared in the neonatal liver in response to BPA; 2) persisted into adulthood; and 3) were not associated with any change in gene expression in the absence to HFD (i.e. their persistence was not a consequenceof altered gene expression). Therefore, the neonatal BPA exposure resulted in an altered epigenetic landscape that persisted into adulthood and preceded the increase in gene expression seen in the reprogrammed rats in response to HFD.

H3K4me1 and H3K27ac marks are generally associated with active enhancer regions, and their presence at promoters is indicative of so-called “super promoters”. Together, these data suggest that early life EDC exposure modulates the activity of the COMPASS complex to reprogram the developing epigenome and generate new “super promoters” that are responsive to later life stimuli, such as HFD, resulting in a liver phenotype that is prone to NAFLD and metabolic disease.

Current in vitro approaches to studying EDCs mechanistically during adipogenesis are limited in that they combine chemical exposures with an adipogenic induction cocktail, creating difficulties in distinguishing obesogens that act early during MSC lineage commitment from those that might promote terminal differentiation. To overcome this limitation, we developed an in vitro assay to screen for obesogens that act during the commitment step whereby MSCs are pretreated with chemical ligands prior to being differentiated with a standard cocktail. Using this new approach, we found that TBT commits MSCs to the adipose lineage in a PPARγ-independent, RXR-dependent manner. Transcriptomic profiling of MSCs treated with TBT revealed genome-wide changes in gene expression that were replicated by an RXR agonist, but not a PPARγ agonist. Pathway analysis of altered transcripts indicated an enrichment of gene targets of the repressive histone modifier Enhancer of Zeste 2 (EZH2), which led us to explore how TBT acts through RXR to reshape the epigenome of unspecified MSCs. Taken together, our data show a critical and unreported role for RXR in early MSC specification. Furthermore, we have shown for the first time that TBT induces adipogenesis in two distinct phases: first during lineage commitment through RXR, and subsequently during differentiation through PPARγ and RXR.

Here, we recognize pyrethroids as a new environmental contributor to the observed secular trend toward earlier male sexual maturity. For the first time to our knowledge, this work reveal a highly significant and positive association between pyrethroids exposure and gonadotropins levels in 463 Chinese boys (p<0.001), in which a 10% increase in 3-PBA (a common urinary metabolite of pyrethroids) is associated with an approximate 4% increase in LH and FSH. Boys with increased urinary levels of 3-PBA have a significantly increased risk of earlier pubertal onset, in which the odds of being in an advanced pubertal stage are increase by 73% to 110%.

Because it is difficult to test the direct causality of environmental risk factors in humans, this investigation further sought to identify the mechanism(s) by which the pyrethroids act to alter the onset of puberty using in vitro and in vivo experimental rodent models. Consistent with the human data, our animal study shows that postnatal exposure to a widely used pyrethroid pesticide, cypermethrin (CP), can accelerate pubertal timing and induce circulating levels of gonadotropins and testosterone in male mice. We demonstrate that the acceleration of puberty onset by CP is independent of hypothalamic responsiveness and that the induction of gonadotropins and testosterone results from the direct effects of CP on pituitary gonadotropes and testis. CP regulates gonadotropins synthesis and the expression of gonadotropin subunit genes through the Ca²⁺/PKC/ERK/IEGs transduction pathways in pituitary gonadotropes. CP induces testosterone synthesis and steroidogenesis-related gene expression in testicular Leydig cells via interference with voltage-gated calcium channels (VGCCs) pathway. Our findings reveal the activation of VGCCs in pituitary gonadotropes and testicular Leydig cells as a newly discovered mechanism of pyrethroid-induced early puberty onset in the male.

In conclusion, this is the first study to provide evidence that environmental exposure to pyrethroids at levels actually present in human is associated with measurable effects on male pubertal development. Given the growing use of pyrethroid insecticides, our findings have important implications for the assessment of children’s health risk from these insecticides. More broadly, this study significantly expands the understanding of the possible mechanisms involved in humans.

Methods: Ninety-two patients (diagnosed with AGHD, male/female, 18–79 years, previously treated with GH for ≥6 months) were randomized to once-weekly somapacitan (n=61) or once-daily Norditropin^® FlexPro^® (n=31). Somapacitan and Norditropin^® FlexPro^® doses were titrated for the first 8 weeks based on serum insulin-like growth factor-I (IGF-I) to achieve serum IGF-I standard deviation scores (SDS; within the normal range [preferably 0–2 SDS]). Doses were fixed for the remaining 18 weeks. Convenience, effectiveness and global treatment satisfaction were assessed using the Treatment Satisfaction Questionnaire for Medication-9 (TSQM-9), with an increase in scores signifying an increase in treatment satisfaction. A mixed model for repeated measurements was used to estimate treatment differences in TSQM-9 scores.

Results: Serum IGF-I levels were maintained in both treatment arms after dose titration. Mean (SD) serum IGF-I SDS scores at week 25 were 0.22 (0.89) and 0.35 (0.82) for somapacitan and Norditropin^® FlexPro^®, respectively. No safety issues were identified with somapacitan; the pattern and rate of adverse events (AEs) and serious AEs were similar with the two treatments. More than 1500 somapacitan injections were administered; two mild, transient injection-site reactions were observed. No anti-somapacitan or anti-GH antibodies were detected. Mean (SD) convenience score increased from 68.3 (18.3) to 83.8 (12.9) with somapacitan, and from 71.7 (17.5) at baseline to 75.8 (19.1) at end of treatment with Norditropin^® FlexPro^®; estimated between-treatment difference in change from baseline to end of treatment (somapacitan–Norditropin^® FlexPro^®) was 8.22 (95% CI: 1.51; 14.93, P=0.0171), with somapacitan being more convenient than Norditropin^® FlexPro^®. Effectiveness and global satisfaction scores were not statistically significantly different between treatment arms.

Conclusions: As a once-weekly GH treatment for AGHD, somapacitan was well tolerated with no detected safety issues and may be more convenient for patients than once-daily treatment.

Design: Multicenter retrospective study by members of the French Society of Endocrinology.

Methods: Forty-three patients (14 women) treated with TMZ between 2006 and 2016 were included. Most tumors were corticotroph (n=23) or lactotroph (n=13), and 14 were carcinomas. Clinical/pathological characteristics of PT as well as data from treatment evaluation and at the last follow-up were recorded. A partial response was considered if the maximal tumor diameter had decreased by more than 30% and/or the hormonal rate by more than 50% at the end of treatment.

Results: The median treatment duration was 6.5 cycles (range 2-24), using a standard regimen for most and combined radiotherapy for six. Twenty-three patients (53.5%) were considered as responders. Silent tumor at diagnosis, though not MGMT expression, was associated with a poor response at multivariate analysis. The median follow-up after the end of treatment was 16 months [0-72] at which time 18 patients had deceased, 13 of whom were non-responders. Overall survival was significantly higher among responders (p=0.002) however ten patients relapsed 5 months (ranges 0-57) after the end of TMZ treatment, five in whom TMZ was reinitiated but without success.

Discussion: Patients in our series showed a 53.5% response rate to TMZ, with an improved survival among responders despite frequent relapses. Our study highlights the high variability and lack of standardization of treatment protocols.

Methods: Pts receiving clinical benefit at wk 22 could enter the extension and continue on the same dose of osilodrostat. Dose adjustments were permitted during the extension. Response rate (RR; defined as the proportion of pts with UFC≤ULN [controlled; C] or UFC>ULN but ≥50% decrease from baseline [BL] [partially controlled; PC]) was assessed with last observation carried forward (LOCF; imputation of missing values using the last available measurements) and observed measurements at 31 mo. Safety was assessed from core BL until the last pt reached mo 31 (maximum follow-up = 40.3 mo).

Results: Of the 16 pts (11 females) who entered the extension, 12 remained on treatment at mo 31. The overall RR (LOCF) for pts who entered the extension was 93.8% (C, 93.8% [15/16]) at wk 22 and 100% (C, 87.5% [14/16]; PC, 12.5% [2/16]) at mo 31. RR was lower at mo 31 when missing values were not imputed: 56.3% (C, 50.0% [8/16]; PC, 6.3% [1/16]). No pts had escape from response (UFC>ULN at ≥2 consecutive visits on maximum tolerated dose after initial UFC normalization) during the extension. Mean (SD) changes in clinical signs of CD from BL to mo 31 (n=11) were: SBP, –3.4 (18.5) mmHg; DBP, –5.4 (9.9) mmHg; weight, –4.5 (5.7) kg; and BMI, –1.8 (2.3) kg/m². The most common clinical AEs were diarrhea (n=6), headache, asthenia, and nausea (n=5 each). Six pts reported hypocortisolism-related AEs. Three pts discontinued from the extension; 1 other patient did not wish to participate. Mean (SD) plasma ACTH increased from 20.0 (10.4) pmol/L (normal 1.8–9.2; n=15) at BL to 80.5 (145.5) pmol/L (n=15) at wk 22 and 54.0 (35.1) pmol/L (n=10) at mo 31. Mean (SD) 11-deoxycortisol and 11-deoxycorticosterone increased from 4.5 (5.3) nmol/L (normal 0–3.92; n=15) and 0.3 (0.3) nmol/L (normal 0.1–0.4; n=13), respectively, at BL to 44.7 (39.5) nmol/L (n=15) and 4.8 (6.6) nmol/L (n=13) at wk 22, and 24.5 (18.8) nmol/L (n=9) and 2.0 (1.5) nmol/L (n=6) at mo 31. Mean (SD) aldosterone decreased from 168.1 (255.4) pmol/L (normal 55–250; n=15) at BL to 44.5 (72.0) pmol/L (n=15) at wk 22 and 19.0 (35.4) pmol/L (n=8) at mo 31.

Conclusion: Osilodrostat maintained normal UFC levels for >2.5 years in a majority of pts with CD, with a long-term safety profile similar to that after 22 wks; no new safety signals emerged. Osilodrostat is being evaluated in two ongoing Phase III studies in pts with CD.

The aim of our study is to compare the detection rate of GLP-1R PET/CT, GLP-1R SPECT/CT and standardized contrast enhanced 3T MRI in patients with a biochemically proven endogenous hyperinsulinemic hypoglycemia highly suspicious for an insulinoma. Preliminary results of an ongoing study are reported. 

Methods:

40 patients (31 females, 9 males, age range 18-80 years, mean 48 years) with neuroglycopenic symptoms due to endogenous hyperinsulinemic hypoglycemia were enrolled (ClinicalTrials.gov, NCT02127541). A standardized contrast enhanced 3T MRI was performed. Afterwards the patients received a SPECT/CT at 4 and 72 hours after injection of ¹¹¹In-DOTA-exendin-4 and a PET/CT 2,5 hours after injection of ⁶⁸Ga-DOTA-exendin-4 in a randomized order. Three independent blinded nuclear medicine physicians and three independent blinded radiologists reviewed the scans. Standard of comparison was the histological diagnosis after surgery. 

Results:

Previously performed cross-sectional imaging (CT/MRI) was negative or not conclusive in 27/40 (68%) of patients.

29 patients have been operated. In this collective, the histological diagnosis of a benign insulinoma was confirmed in 25 patients, 1 patient had adult islet cell hyperplasia. In 1 patient both intraoperative palpation as well as the histological diagnosis did not confirm an insulinoma. In 2 patient symptoms of endogenous hypoglycemia ceased postoperative but histological diagnosis did not confirm the diagnosis of a benign insulinoma or nesidioblastosis. Only these two patients were excluded from evaluation as the final diagnosis remained unclear.
Two patients refused surgery. Three patients are awaiting surgery. In five patients PET/CT, SPECT/CT as well as the previous performed conventional imaging did not find any suspicious lesion and were thus not operated up to date. One patient showed signs of malignancy in contrast enhanced MRI, thus did not meet the inclusion criteria and did not receive surgery up to date.
In this interim analysis of 27 operated patients PET/CT patients showed an overall pooled sensitivity of 92%, SPECT/CT at 72 hours an overall pooled sensitivity of 71% and contrast enhanced standardized 3T MRI an overall pooled sensitivity of 76%.  

Conclusion:

1) These preliminary data suggest that PET/CT performs better as standardized MRI imaging and SPECT/CT at lower irradiation dose and much shorter investigation time than the latter.

2) GLP-1R PET/CT will be a useful diagnostic tool in patients in which an insulinoma is suspected.

Results: This report details a novel series of highly potent SARDs with unique pharmacology that selectively bind to the AR-LBD and inhibit transactivation at nanomolar concentrations. The SARDs antagonize the AR with an IC₅₀ of ~50-250 nM in comparison to enzalutamide with an IC₅₀ of ~500 nM. The SARDs are selective for the AR with the only significant observed cross-reactivity being with the PR. In addition to their antagonistic activity, the SARDs degrade full length AR (AR-FL) in the high (100-500 nM) nanomolar range and variant AR around 1-5 μM. The SARDs inhibit the proliferation of AR-FL and AR-SV- dependent PCa cells with potencies better than that of the comparators. The SARDs robustly inhibit the growth of the LNCaP androgen-dependent prostate cancer (PCa) xenograft, the 22RV1 CRPC xenograft, and AR- and AR-SV- positive CRPC patient-derived xenografts (PDX). NMR analysis as well fluorescence quenching support an interaction between the SARDs and the AR activation function domain (AF-1), making these molecules first-in-class dual-interacting AR antagonists and degraders. SARDs prevent AR-FL nuclear translocation and export the constitutively nuclear AR-SV. Mechanistic studies indicate that these SARDs re-program the interaction of the AR with DNA elements (ChIP-Seq) and proteins.

Conclusions: These novel highly potent SARDs that interact with both the AF-1 and LBD of the AR represent potential next-generation treatment options for advanced prostate cancer. Clinical development of these compounds is ongoing.

Results: We report that the activated LXR significantly reduced AR mRNA and protein levels in C4-2 human CRPC cells upon treatment with a synthetic LXR ligand (T0901317 or GW3965) at low micro molar concentrations. Similar doses of the ligand strongly abated proliferation of C4-2 cells and their foci formation in culture. Reduced AR expression paralleled decreased AR activity, since androgen-induced expression of the AR target genes PSA and NKX3.1 diminished markedly. Activation of LXR in ligand-treated cells was confirmed from marked induction of ABCG1, a transporter-encoding gene and LXR target. We engineered C4-2 cells in which the endogenous AR gene was fused in-frame at its 3’UTR with the NanoLuc reporter cDNA by genome editing with CRISPR/Cas9. NanoLuc luciferase activity in the tagged C4-2 cells was markedly reduced upon LXR activation, indicating that LXR-mediated antagonism of AR expression was at least partly due to transcriptional suppression of the genomic AR. Cells were examined by atomic force microscopy (AFM) to analyze nanomechanical properties, which can reveal cell’s invasive potential (3). Ligand-activated LXR reduced the invasive phenotype of C4-2 cells in a dose dependent manner, since the AFM parameters demonstrated enhanced adhesion and higher young modulus –the latter indicating greater rigidity or lessened elasticity. Using DNAse1 footprinting and chromatin immunoprecipitation of the human AR promoter, we identified an LXR-responsive region, which was occupied by both LXR isoforms. Upon activation, LXR in C4-2 cells was associated with an HDAC1- and NCoR-associated corepressor complex. Molecular insights into the interplay of LXR with corepressors and various epigenetic modulators at the AR promoter can potentially unveil new anti-AR strategies, which, in a therapy setting, can stem prostate cancer progression.

For most nuclear receptors (including the estrogen receptor) a dimerization at the level of the ligand binding domain (LBD) has been demonstrated. However, for the oxysteroid receptors (AR, GR, MR and PR) the dimerization via the LBD has been controversial.

Here, we show the crystal structure of the AR-LBD homodimerization interface (1,000-Å2) of the agonist-coactivator peptide-bound LBD. This interface is different from the one described for other nuclear receptors (including the estrogen receptor) and would not prevent coactivator binding or N/C interactions. In fluorescence resonance energy transfer (FRET) experiments this AR-LBD dimerization is disrupted by a mutation described in an androgen insensitivity syndrome (AIS) patient although it does not disrupt ligand binding. In fact up to 40 mutations described in AIS or prostate cancer that were unexplained before reside in this new LBD dimer surface. Clinical data thus strongly corroborate our AR-LBD dimer model. FRET assays further demonstrated that the dimerization of the AR-LBD is inducible by androgen agonists but not by antagonists. Importantly, mutations that disrupt the FRET signal (dimerization) did not disrupt ligand binding so dimerization is dependent on hormone, but hormone binding does not dependent on dimerization. Mutations that are predicted to disrupt the AR-LBD dimerization were introduced in the full size AR. They disrupted its transactivation properties on reporter genes clearly demonstrating the crucial role of dimerization in transactivation.

In conclusion, we described a new dimerization surface in the AR-LBD with a crucial role in transactivation.

* EV and RØ have shared last authorship

Objective: We performed a systematic review, meta-analysis and meta-regression to evaluate 2009 IOM guidelines in contemporary maternal populations across all weight categories and broad ethnic groups to validate the guidelines.

Data Sources: EMBASE, All EBM Reviews, Medline and Medline in-process were searched from 1st January 1999-28th January 2016.

Study selection: Inclusion required observational studies stratify by prepregnancy body mass index (BMI) category and total pregnancy GWG. Authors were contacted for re-analysis given data heterogeneity. Odds ratios (OR) used recommended GWG within each BMI category as the reference. PROSPERO registration CRD42015023325.

Data extraction and synthesis: Data were extracted by two independent reviewers. OR were calculated using a random-effects model. Heterogeneity was assessed using I². Methodological quality was assessed using Monash Centre for Health Research and Implementation Evidence Synthesis appraisal.

Main outcomes and measures: Outcomes were small for gestational age (SGA), preterm birth, large for gestational age (LGA), macrosomia, caesarean section and gestational diabetes.

Results: 5354 studies were identified, 19 studies (n=1,137,464 women) met inclusion criteria. GWG was below, at or above guidelines in 20, 29 and 51% of pregnancies respectively. GWG below recommended had higher SGA [(OR) 1.49; 95% CI 1.39,1.61] and preterm birth (1.37;1.21,1.55), and lower LGA (0.62;0.57,0.67) and macrosomia (0.65; 0.57 0.73) compared to recommended GWG. GWG above recommended had lower SGA (0.65;0.62,0.68) and preterm birth (0.77;0.68,0.88) and higher LGA (1.89;1.79,2.01), macrosomia (1.83;1.69,1.99) and caesarean (1.29;1.23,1.35). Subgroup analyses stratified by obesity class I-III found similar risks for SGA, LGA, macrosomia and caesarean.

Conclusions: In this review of over 1,000,000 pregnancies in a contemporary population with high mean BMI and diverse ethnicity, GWG outside 2009 IOM recommendations is associated with greater maternal and infant adverse effects. This work attests the broad applicability of 2009 IOM guidelines and highlights the need to implement these GWG recommendations broadly across maternity care.

IVF is an effective therapy for infertility, but can result in the potentially life-threatening complication ovarian hyperstimulation syndrome (OHSS). We have previously reported that a single injection of kisspeptin results in an LH-surge of ~12-14hrs duration, sufficient to safely trigger oocyte maturation in women at high risk of OHSS. We investigated whether increasing the duration of LH-exposure by administering a second dose of kisspeptin could further optimise oocyte maturation.

Methods:

We conducted a phase2 single-blinded randomised placebo-controlled trial of 62 women at high risk of OHSS. Following a standard recFSH/GnRH-antagonist IVF protocol, all patients received a subcutaneous injection of kisspeptin-54(9.6nmol/kg) 36hrs prior to oocyte retrieval. Patients were then randomized 1:1 to receive either a second dose of kisspeptin 10hrs later (D;Double), or saline placebo (S;Single). IVF physicians, embryologists and participants were blinded to the randomization. Retrieved oocytes were assessed for maturation and fertilized by ICSI. Elective single embryo transfer (eSET) was carried out in all patients with at least one high quality blastocyst.

Outcomes:

Primary Outcome: Proportion of patients achieving a satisfactory oocyte yield (% of mature oocytes retrieved from follicles ≥14mm in diameter) of ≥60%.

Secondary Outcomes: Implantation Rate and occurrence of OHSS.

Discussion:

A second injection of kisspeptin at 10hrs following the first induced a significant further mean fold-rise in LH-secretion at 4hrs (S:3.3, D:14.7;P<0.0001) and 10hrs (S:1.5, D:3.1;P=0.0002) thereafter when compared to pre-trigger levels. The proportion of patients achieving a satisfactory oocyte yield was improved following two doses of kisspeptin (S:45%, D:71%; RD 25.8%, CI 2.1-49.5%). There was a trend towards a higher implantation rate following 2 doses of kisspeptin (S:23.3%; D:37.1%; P=0.20), but no difference in the frequency of OHSS.

Conclusion:

Prolonging the duration of LH-exposure by administering a second dose of kisspeptin safely improves oocyte yield in women at high risk of developing OHSS undergoing IVF treatment.

Ovarian hyperstimulation syndrome (OHSS) is a serious iatrogenic condition predominantly related to the mode of triggering oocyte maturation during IVF treatment. Kisspeptin is a novel trigger which stimulates the physiological release of GnRH from the hypothalamus. Kisspeptin has recently been shown to safely trigger oocyte maturation in a population at high risk of OHSS, but has yet to be directly compared with other triggers. Ovarian volume and ascitic fluid are commonly used to categorise the severity of OHSS in diagnostic guidelines. We therefore investigated these parameters in women undergoing IVF treatment with 3 different triggers of oocyte maturation in women at high risk of OHSS.

Method and Outcomes:

Women at high risk of OHSS (antral follicle count ≥23), aged <35yrs, BMI <30 kg/m² with both ovaries intact, were screened sonographically and for symptoms of OHSS at 2-5 days following oocyte retrieval. Patient outcomes were determined when patients were triggered with human chorionic gonodotrophin (hCG) (n=8), GnRH agonist (GnRHa) (n=54) or kisspeptin (n=122). Statistical analysis was performed using Kruskal-Wallis test with post-hoc Bonferroni correction.

Discussion:

Median ovarian volume (MOV) following GnRHa trigger (74.8mls) was significantly lower than in patients triggered with hCG (143.3mls; p <0.05). MOV following kisspeptin trigger (44.0mls) was significantly lower still when compared with GnRHa trigger (p <0.001).

Median ascitic volume was lower after GnRHa (2mls; p<0.01) and kisspeptin (0mls; p<0.001) when compared with hCG (42mls). Symptoms of OHSS were more frequently reported following GnRHa use than kisspeptin and more frequently still following hCG.

 Relevance / Impact:

Increased ovarian volume and ascitic fluid volume consistent with OHSS were less frequent following kisspeptin than GnRHa or hCG in a population undergoing IVF treatment at high risk of OHSS. Kisspeptin was also associated with less frequent reporting of symptoms of OHSS. Kisspeptin may thus present a safer alternative than GnRHa or hCG triggering in patients undergoing IVF treatment at high risk of OHSS.

Methods: Genotyping was performed in 200 unrelated healthy subjects of Ashkenazi Jewish descent (defined as having 4 Ashkenazi Jewish grandparents) and 200 healthy Caucasians who did not self-identify as a specific ethnicity. DNA was collected from The Foundation for Jewish Genetic Medicine (Ashkenazi Jews), and the Coriell Institute for Medical Research Biorepository (Caucasians). CYP21A2 was analyzed using multiplex minisequencing, real time PCR and junction site analysis. Proportions and comparisons between groups were analyzed using the binomial and Fisher’s exact tests, respectively.

Results: Of the 200 Ashkenazi Jews screened, 15% (95% CI: 10.4 - 20.7) were NCCAH carriers, and 2.5% (95% CI: 0.8 - 5.7) were classic CAH carriers. Of the Caucasians screened, 9.5% (95% CI: 5.8 - 14.4) were NCCAH carriers, and 1.5% (95% CI: 0.3 - 4.3) were classic CAH carriers. One subject in each cohort (0.5%, 95% CI: 0.01-2.8) had a genotype consistent with being affected with NCCAH. p.Q318X in the setting of gene duplication was not considered pathogenic. The proportion of Ashkenazi Jewish NCCAH carriers (0.15 vs. 0.309, P < .0001) and disease affected (0.005 vs. 0.037, P = .009) were not as high as previously reported and did not differ from our random sample of Caucasians (0.15 vs. 0.095, P = .13). Our study also suggests that nonclassic CAH is commonly found in general Caucasian populations, with an estimated prevalence of 1 in 200 (0.5%, 95% CI: 0.01-2.8).

Conclusion: Our study is the first to demonstrate, through state-of-the-art CYP21A2 analysis, that NCCAH is less common in Ashkenazi Jews than previously suggested. Our estimates of disease rates in the general U.S. Caucasian population argue for universal screening of NCCAH in the setting of female infertility or hyperandrogenism, regardless of ethnicity. These results have important implications with preconception and infertility counseling.

Objective: To analyze the metabolic impact of DEX therapy in adult CAH patients followed in a single tertiary center.

Patients and Methods: 60 well-controlled CAH patients (41M/30SW) received DEX therapy after the achievement of final height. All patients had adequate androgen levels and normal serum electrolytes. Mean daily DEX doses/m²was evaluated. Clinical and laboratorial data were compared before DEX introduction and at the last evaluation. Frequency of MS components were assessed as proposed by the NCEP-ATPIII criteria. Statistical analysis included paired t-tests, Wilcoxon signed-rank tests, Chi-square and regression analyses when appropriated.

Results: mean age at the last evaluation and mean duration of DEX therapy were 31.9 ± 9.6 yrs and 11.5 ± 4.9 yrs, respectively; mean daily DEX dose was 0.17±0.07mg/m². No significant differences were found regarding BMI-SDS, LDL-cholesterol, glucose and triglycerides levels before and after DEX therapy. A decrease in HDL-cholesterol levels (57 ± 12.8 vs 53.5 ± 13.6; p=0.008) and an increase in HOMA-IR (2.5 ± 1.3 vs 2.8 ± 1.7; p=0.03) were observed. Obesity prevalence remained unchanged before and after DEX and was lower than a previous study comprising a large cohort receiving different GCs (1) (26.7% vs 41%, respectively; p=0.042). However, waist to height ratio (WHtR) increased from 0.54 ± 0.08 to 0.56 ± 0.1 after DEX (p=0.001), although final waist circunference (WC) values were similar to the ones described in patients on different GCs (2). Regression analysis showed no correlation between duration/dose of DEX therapy and WC gain. Frequency of MS and hypertension remained similar before and after DEX (6.7% vs. 10%, p=0.7; and 15% vs 13.3%, p=0.8, respectively).

Conclusions: In our cohort, the use of dexamethasone didn’t lead to increased frequency of obesity and MS, although undesirable body fat distribution can occur. DEX appears to be a good option for the CAH treatment in adulthood.

Objectives: To assess the prevalence of obesity, diabetes, hypertension and dyslipidemia in postmenopausal hyperandrogenic women before and after testosterone levels normalization by bilateral oophorectomy.

Patients and Methods: 30 hyperandrogenic postmenopausal women, mean age at diagnosis of 57.5 + 7.8 yrs and submitted to bilateral oophorectomy were selected. All patients had elevated total testosterone (T) levels, mean 305 + 321 ng/dL, (IFMA, reference value < 98 ng/dl) before oophorectomy, and normalized T levels after surgery. Ovarian histopathology confirmed OH in 19/30 and OAST in 11/30 cases. Physical and biochemical parameters including BMI, blood pressure, glucose, A1c, cholesterol and triglycerides levels were assessed before and 2 years after oophorectomy. T-test, Mann-Whitney, Qui-square tests were performed for statistical analysis.

Results: There was an elevated prevalence of obesity (53.3%), diabetes (70%), hypertension (70%) and dyslipidemia (56.6%) in these patients. Despite the difference in T levels between the OH (median 173 ng/dl) and the OAST groups (median 448 ng/dl), p = 0.002, there were no differences between OH and OAST patients in the prevalence of obesity (58% x 66%, p = 0.24), diabetes (74% x 64%, p = 0.13) and hypertension (69% x 73%, p = 0.53). However, the prevalence of dyslipidemia was higher in the OAST (82%) than in the OH group (42%), p < 0.001. The mean glycated hemoglobin (A1c) levels in the diabetic patients was 7.0%, with no difference between OH (mean 7.4%) and OAST group (mean 7.0%), (p = 0.61). The A1c levels after oophorectomy did not change (mean 6.9%), (p = 0.81), and so did not the triglycerides (p = 0.51), LDL (p = 0.77) and HDL (p = 0.71) levels before and after treatment. There was no difference in the BMI before and 2 years after bilateral oophorectomy (p = 0.34).

Conclusion: Women with postmenopausal ovarian hyperandrogenism have a very high prevalence of insulin resistance and metabolic syndrome risk factors. Hyperinsulinemia might contribute to the ovarian hyperandrogenism by directly stimulating ovarian testosterone synthesis. The normalization of androgen levels did not improve the metabolic profile suggesting no impact of androgen excess in insulin resistance in postmenopausal hyperandrogenism.

The evaluation of clinical hyperandrogenism in women often includes the measurement of circulating concentrations of total testosterone (TT). Additional evaluation for androgen producing tumors (APTs) in women is generally recommended for TT greater than 200ng/dL. However, this is largely based on data using immunoassays to measure TT. Liquid chromatography/mass spectroscopy (LC/MS) assays are considered the gold standard for measuring TT due to its higher specificity. We hypothesized that a lower TT threshold for further APT evaluation might increase the detection of APTs without increasing evaluation of false negatives. To date, there have been no large series published regarding TT concentrations in women with APTs as measured by LC-MS.

Aim

To determine the best discriminatory threshold for TT concentrations measured by LC/MS to screen for APTs in women.

Methods

A retrospective analysis was conducted of all women with a TT concentration ≥ 100 ng/dL by LC-MS at the Mayo Clinic in Rochester, MN from 2004-2012(since assay availability). Medical records were reviewed to determine clinical presentation, laboratory and radiographic findings, diagnosis and treatment.

Results

A total of 366 adult female patients had TT concentrations ≥100ng/dL. Two hundred and eighty women were excluded from analysis (184 on exogenous testosterone, 22 liver failure, 12 gender dysphoria, 13 congenital adrenal hyperplasia/androgen insensitivity, 12 pregnant, 8 pediatric, 7 adrenocortical carcinoma and 22 had no further evaluation). Out of 86 who underwent final analysis, 55 women (64%) had polycystic ovarian syndrome (PCOS), 12 (14%) had ovarian hyperthecosis (OH) and 19 (22%) had an APT as the cause for hyperandrogenism. The mean age at presentation was 28±7 years in the PCOS group, 61±8 years in the OH group and 51±18 years in the APT group (p<0.001). APT patients had a significantly rapid onset of their symptoms (PCOS 60±7 months vs OH 35±15 months vs APT 19 ± 11 months; p<0.01). There was no difference among the three groups regarding symptoms of hyperandrogenism or examination findings. TT concentrations were significantly higher in the APT group (PCOS 125±35 ng/dL vs OH 183±103 ng/dL vs APT 287±135 ng/dL; p<0.001). Free testosterone concentrations were also higher in the APT group. DHEA-S was measured in 64 (74.4%) women. Significantly higher DHEA-S concentrations were found in the PCOS group (PCOS 186±15 µg/dL vs OH 77±34 µg/dL vs APT 93±31 µg/dL; p<0.01; not age adjusted). Eleven (73%) APTs localized to the ovary. A TT concentration ≥ 148ng/dL had a sensitivity of 84% and specificity of 85% for the diagnosis of APT as determined by the ROC curve. In the APT group, there was no difference between pre- and post-menopausal women in TT concentrations or tumor source.

Conclusion

A TT concentration threshold of ≥148 ng/dL identified 84% of APTs among women with TT concentrations ≥ 100ng/dL by LC-MS.

Participants and methods: Morning serum was obtained from 20 pre- (YW, age 20-40 years) and 20 post-menoapausal women (OW, age 60-80 years), who were not taking any hormonal therapy. We quantified 21 steroids by liquid chromatography-tandem mass spectrometry. To understand the adrenal transformations that occur with aging and might impact its androgen synthetic potential, we performed double immunofluorescence for the key androgenic enzyme and cofactor 3b-hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b₅ (CYB5A) in adrenal glands from 4 YW and 4 OW. The nonparametric Mann-Whitney Utest was used for between-groups comparison.

Results: DHEA, DHEAS and AD decreased significantly with aging (1.3-2.3 fold higher in YW, p<0.001) in accordance with previously published results. In contrast, 11KT and T did not decline, and 11OHAD, 11KAD and 11OHT increasedin OW (1.4-2 fold, p<0.02). The 11KT/T ratio remained relatively constant across ages (median 2.2). As observed previously, adrenal glands from YW displayed segregation of HSD3B2 and CYB5A to the zona fasciculata and zona reticularis, respectively. In contrast, the adrenal tissue from OW displayed areas of HSD3B2 and CYB5A co-expression. These findings suggest that with aging, the adrenal gland attains a higher synthetic capacity of active androgens, at the expense of the major precursors DHEA and DHEAS, which have trivial androgenic activity.

Conclusion: These results support the hypothesis that the adrenal gland assumes a major role in the synthesis of active androgens in menopausal women.

To study whether neutrophils can alter local TH levels we measured TH concentrations in cerebral spinal fluid (CSF) from patients with bacterial meningitis versus controls using LC-MSMS. Bacterial meningitis is a severe infection characterized by high neutrophil infiltration. To determine the role of D3 in neutrophil function we analyzed neutrophils derived from D3KO mice and WT controls. Neutrophil count, survival, phagocytosis and hydrogen peroxide production in response to an activating stimulus were measured. Finally, we determined the effect of D3 knockdown (KD) on mortality and neutrophil-microbe interaction in vivousing a zebrafish embryo meningitis model [4]. Transgenic zebrafish embryos with green fluorescent neutrophils were infected with S. pneumoniae in the hindbrain ventricle. Morpholino technology was used to modulate TH availability by knocking down D3 [5]. Zebrafish survival was quantified and neutrophil recruitment over time was assessed with confocal microscopy.

Both T4 and reverse T3 (rT3) levels were strongly increased in CSF from bacterial meningitis patients compared to control CSF. Increased permeability of the blood brain barrier during meningitis could allow for leakage of T4 from the circulation to the CSF. As CSF rT3 levels are too high to be explained by leakage from the blood, this suggests that T4 is locally converted to rT3 by D3 in infiltrating neutrophils. Neutrophils from D3KO mice exhibited impaired hydrogen peroxidase production upon activation compared to WT cells indicating decreased NADPH-oxidase activity, an important component of bacterial killing. Neutrophil amount, phagocytosis and spontaneous apoptosis were not affected by D3 deficiency. Finally, the mortality rate was higher in D3KD zebrafish embryos than in controls and confocal imaging revealed reduced neutrophil recruitment over time in infected D3KD zebrafish embryos.

In summary, the altered TH profile of human CSF during bacterial meningitis is consistent with elevated D3 activity in infiltrating neutrophils. In addition, the lack of D3 in mouse and zebrafish leads in vitro to impaired neutrophil function, as evidenced by decreased NADPH oxidase activity, and in vivo to decreased neutrophil recruitment and increased mortality during bacterial meningitis. These consistent findings across experimental models suggest a critical role for intracellular TH metabolism in neutrophil function during infection, a mechanism for which D3 appears essential.

Fine needle aspiration cytology plays a pivotal role in the evaluation of thyroid nodules. However, ~25% of the aspirates render an indeterminate diagnosis. A gene expression classifier (GEC) marketed as Afirma is utilized to select patients with Bethesda III or IV thyroid nodules appropriate for observation rather than immediate surgery. However, several reports of the assay performance results have been divergent. We sought to evaluate the GEC performance through a systematic review of the literature.

Methods:

We searched in PubMed for “gene expression classifier or Afirma or GEC” AND “thyroid”, and retrieved 87 articles that were screened by two independent reviewers. 34 articles were excluded by title; and 30 more after abstract review. After reviewing the full text of 23 articles, we excluded 5 reports with overlap of cohorts with the original validation study or other larger published series; 3 reports in which analysis was performed in a specific subset of patients, rather than in all consecutively tested nodules; and 3 reports in which the prevalence of malignancy and test metrics could not be calculated on resected nodules. Two authors independently extracted the data for each of the 12 remaining articles selected for the final analysis. Tests metrics were calculated with 95% confidence intervals. 

Results:

A total of 1502 nodules with B-III or IV cytology and informative GEC result were included. 646 (43%) were GEC-benign; and 856 (57%) were GEC-suspicious. A total of 842 nodules (56%) were resected with an overall prevalence of malignancy of 39% (36%-42%). Nodules with GEC-benign result had significantly lower rates of resection than GEC-suspicious nodules: 25 % (n=159) vs. 80 % (n=683), respectively (p<0.0001). Among nodules with histological follow-up, 312 were true positive, 369 false positive, 16 false negative and 143 true negative results. The overall performance of the test on resected nodules was: sensitivity 95% (92%-97%), specificity 28% (24%-32%), positive predictive value (PPV) 46% (42%-50%) and NPV 90% (84%-94%). Excluding the 210 nodules of the original validation study, the resection rate was 13% for GEC-benign nodules and 76% for GEC-suspicious; and the prevalence of malignancy amongst resected nodules 44%. The sensitivity, specificity PPV and NPV for the independent validation studies were 96% (93%-98%), 17% (14%-22%), 48% (43%-52%) and 85% (74%-92%), respectively.

Conclusion:

The NPV of the GEC has been poorly validated as only 13% of GEC-benign nodules had histological follow-up in independent validation series. This selection bias may be also responsible for the significantly higher prevalence of malignancy and lower specificity observed in independent studies in comparison to the original validation study. The NPV of GEC may be lower than expected and still needs independent validation.

Methods We conducted a retrospective chart review to evaluate whether, in a subset of patients with metastatic RAIR DTC, Ki therapy increased RAI uptake. Patients who were deemed to be RAIR and had diagnostic whole body scans (WBS) while on Ki therapy were included. If WBS demonstrated meaningful RAI uptake, a therapeutic dose of I131 was delivered. Post-treatment scans, thyroglobulin (TG) levels and serial CT scans were reviewed.

Results Thirteen patients with a median age of 56 (45-75) years were included. 7 (54%) were men. 10 (77%) had papillary thyroid cancer, 2 (15%) had poorly differentiated thyroid cancer, and 1 (8%) had follicular thyroid cancer. Molecular profiling of these tumors revealed 9 (70%) BRAF V600E mutations, 2 (15%) NRAS mutations, 1 (7.5%) KRAS mutation, and 1 (7.5%) with no identified mutation. Based on their mutations, they were treated with either a BRAF or a MEK inhibitor. The patient without a BRAF or RAS mutation was treated with a MEK inhibitor. Ki therapy was started for progressive disease, except in 2 patients in whom it was started for RAI resensitization. Ki therapy was continued for a median duration of 14.3 (1-76.4) months prior to diagnostic WBS.

Of the 13, 10 (77%) patients had documented RAI uptake on WBS, and 9 of these had clinically meaningful uptake to warrant therapy. The median delivered dose of I131 was 204.4 (150-253) mCi, and Ki therapy was discontinued 2 days after treatment. The median follow up was 8.3 (0-17.4) months after I131 therapy. Of those treated, 7 had sufficient follow up and restaging CT scans, and all had either an objective partial response or stable disease accompanied by decreasing TG levels, including one patient whose TG became undetectable at 9 months post-therapy.

Two of the treated patients had pneumonitis thought to be related to the I131 therapy. One patient’s symptoms are resolving and the other is undergoing evaluation.

Conclusions Use of targeted Ki therapy in BRAF- or RAS- mutated RAIR DTC can resensitize these tumors to iodine uptake and facilitate retreatment with I131 in the majority of cases. Clinically and radiographically meaningful responses are observed in appropriately selected patients. Further studies are needed to determine predictors of response and longer follow-up is required to assess the safety and long-term outcomes of this approach to treating RAIR DTC.

We reviewed genetic testing records of patients evaluated at a large academic medical center from 2005-2015. We estimated the number of at-risk relatives tested per positive proband by the proxy measure of ratio of number of site-specific genetic tests ordered over the total number of positive tests resulting from comprehensive full gene analysis.

A total of 91 individuals were found to carry mutations in SDHx, VHL, RET, NF1, TMEM127 and MAX by full gene sequencing analyses and 211 additional individuals had site-specific genetic tests, resulting in 2.3 tested relatives per index patient with a Hereditary PCC/PGL syndrome. Uptake of cascade testing varied by syndrome. For SDHx, a total of 50 positive full sequence analyses and 152 total site-specific tests were performed, resulting in 3 relatives tested per index carrier. For MEN2, a total of 10 positive full sequence analyses and 23 site-specific tests were performed, resulting in 2.3 relatives tested per index carrier. For VHL, a total of 28 positive full sequence analyses and 34 total site-specific tests were performed, resulting in 1.2 relatives tested per index carrier. Overall uptake of genetic testing in families with PCC/PGL related syndromes was 2.3 relatives tested per index mutation carrier, a rate similar to that observed in families with Lynch syndrome (2.2) and Li-Fraumeni syndrome (2.1), and higher than in families with Familial Adenomatous Polyposis (1.6), Hereditary Leiomyomatosis and Renal Cell Cancer (0.6), Birt-Hogg-Dubé syndrome (1.1), and Cowden’s disease (0.7).

We found that uptake of predictive genetic testing was higher in PCC/PGL families, especially those with SDHx mutations, compared to other hereditary cancer syndromes. Patient education regarding the benefits of asymptomatic screening for PCC/PGL and prompt access to genetic testing coordinated through a specialized Endocrine Oncology Clinic likely played a role. However, despite increased availability of genetic testing, the number of tested relatives per index patient remains suboptimal. These data highlight the importance of determining factors that influence the decision of family members to undergo genetic testing in order to increase the detection of genetic mutation carriers.

Pheochromocytomas (PHEOs) are neuroendocrine tumors arising from the adrenal medulla. They are frequently unilateral and sporadic. Bilateral PHEOs are typically associated with hereditary syndromes.

Objectives:

Our aim was to describe the clinical presentation, genetics, treatment and outcomes of patients with bilateral PHEOs.

Methods:

Medical records of patients with bilateral PHEOs at Mayo Clinic, Rochester, MN from 1951to 2015 were retrospectively reviewed. Diagnosis of PHEO was based on biochemical, imaging, or histological findings.

Results:

Out of 1161 patients with PHEO, 94 (7.9%) patients (48 women, 51%) were diagnosed with bilateral PHEOs. Seventy-six (81%) patients presented with synchronous tumors, while 18 (19%) patients developed metachronous tumors. The median age at the time of surgery for the first PHEO was 31 yrs (range, 4-70). In metachronous tumors, the second PHEO was detected at a median time of 4.5 yrs after the initial surgery (range, 1-38). Median tumor size of the first PHEO was 31 mm (range, 6-120). The second PHEO measured 23 mm (range, 4-87). In 32 (34%) patients the tumors were multicentric. A genetic syndrome was documented in 75 (80%) patients and in 16 (17%) patients very limited or no genetic testing was performed. Of the patients who had a detected genetic syndrome, 41 (43.6%) had MEN 2A, 18 (19%) VHL, 8 (8.5%) MEN 2B, and 8 (8.5%) NF1.

PHEO was discovered because of symptoms of catecholamine excess in 47 (50%) patients, genetic case detection testing in 26 (28%), an incidentaloma imaging finding in 17 (18%), mass effect symptoms in 1 (1%), and in 3 (3%) patients the method of discovery was not known. Pre-operative biochemical information was available for 69 patients and showed catecholamine excess in 67 (97%) patients. Fifty (53%) patients underwent adrenalectomy by the open approach; laparoscopic in 22 (23%), and unknown type surgical approach in 21 (22%) patients who had surgery performed elsewhere. Sixteen of 72 (22%) patients had cortical sparing surgery and of these 7 (44%) required permanent glucocorticoid replacement. One patient was not operated.

Patients were followed for a median of 10 yrs (range, 0-64). Recurrent PHEO within the adrenal gland occurred in 8 (8.5%) patients. Malignant PHEO occurred in 8 (8.5%) patients. Of 20 (23%) patients who died during follow-up, 2 (10%) died from PHEO.

Conclusion:

Bilateral PHEO was diagnosed in 94 (7.9%) of 1161 patients with PHEO over 64 years. MEN 2A and VHL were the two most common syndromes associated with bilateral PHEO. At the time of diagnosis, most bilateral PHEOs are likely to be synchronous. However, in metachronous PHEOs the second tumor may be detected anywhere from 1 to 38 yrs (median, 4.5) after the initial tumor resection.

Methods: Forty-five patients with PBMAH were evaluated at the National Institutes of Health (NIH) under an IRB-approved protocol. Clinical, biochemical and radiographic characteristics were assessed. Biochemical diagnosis of hypercortisolemia was confirmed with loss of diurnal variation of cortisol and/or elevated 24-h urinary free cortisol (UFC) and 17-hydroxysteroids (17OHS). Criteria for radiographic diagnosis included bilateral macronodules with hyperplasia. CT scans were used to create 3-dimensional volumetric models by contouring each adrenal gland, in each slice, using Vitrea Core Fx v6.3 software (Vital Images, Minnetonka, Minnesota).

Results: Forty-five patients with PBMAH (32 females, 52.3±11.9 years) were evaluated. Their midnight cortisol concentration, UFC and 17OHS were 8.3±4.9 μg/dL, 54.6±61.9 μg/24h and 9.3±4.7 μg/24h, respectively. Their mean adrenal volumes were; right 18.5cc±10.18, left 26.6cc±19.3, and total 45.1cc±24.4. In our mixed cohort, total adrenal volume positively correlated with 17OHS (r=0.4, p=0.01, n=41). The other biochemical parameters did not significantly correlate with adrenal volume. However, in the African American cohort (n=10), total adrenal volume positively correlated with midnight cortisol concentration (r=0.8, p=0.03, n=8), UFC (r=0.8, p=0.004, n=9) and 17OHS (r=0.9, p=0.004, n=8).

Conclusion: 3-dimensional volumetric modeling, in combination with clinical and biochemical measures, may be used as a marker of severity in patients with PBMAH. This appears to be particularly useful in African American patients with PBMAH, which may reflect their previously described increased prevalence of ARMC5-variant carrier state.

Polycystic ovary syndrome (PCOS) is a complex heterogeneous endocrine disorder in women, with poorly understood etiology. Insulin is an important regulator of glucose and lipid metabolism, and abnormal insulin signaling is a key feature of PCOS.

Hypothesis:

We investigated the gene expression profiles in unstimulated peripheral blood leucocytes of women with PCOS women vs. control women, using insulin-signaling pathway-specific PCR.

Experimental Design and Methods:

Study subjects included 12 women with PCOS, and 12 age- and BMI-matched normal ovulatory women who served as controls. Gene expression profiling was done using Qiagen RT² Profiler™ PCR Array Human Insulin Signaling Pathway. Microarray results for selected genes were confirmed by real-time quantitative PCR.

Results:

Of the 84 pathway-specific genes included in Human Insulin Signaling RT² Profiler, 13 genes were differentially expressed in women with PCOS compared to age- and BMI-matched controls. Those genes were found to be involved in lipid metabolism, insulin action, intracellular signaling, and in inflammation and immunity. Profiling results confirmed higher expression of MAP kinase 3 (5.12 fold), JAK2 (7.86-fold), UCP1 (6.82-fold), and SLC27A4 (8.73-fold). In addition, lipoprotein lipase (5.55-fold), VLDL receptor (6.54-fold), FABP4 (8.71-fold), resistin (8.34-fold), and PPARg (5.40-fold) were also differentially expressed in cases and controls. Furthermore, IGF1 receptor (4.00-fold), insulin receptor (6.48-fold), PEPCK (6.12-fold), and IL-18 receptor 1 (4.95-fold) were also differentially expressed in the two groups. The differential expression of MAP kinase 3 (P <0.001), lipoprotein lipase (P <0.001), resistin (P <0.001), SLC27A4 (P <0.001), IGF1 receptor (P <0.001), IL-18 receptor 1 (P <0.001), and PEPCK (P <0.001) were subsequently confirmed by qPCR in both PCOS and control groups.

CONCLUSION

PCR array-profiling analysis demonstrated differential expression of genes linked to insulin signaling, lipid metabolism, intracellular signaling, and inflammation in obese women with PCOS women compared to control women. It is very likely that these genes may be involved in altered metabolism associated with the different PCOS phenotypes, which in turn might provide novel drug targets and potential biological markers for PCOS.

Design/Participants/Main Outcome Measure: A 12-week prospective randomized open-label study was conducted with 40 infertile obese PCOS patients (aged 30,8± 3,7 years, BMI 36,7±3,5 kg/m2, mean ± SD) who had been previously poor responders to lifestyle intervention regarding weight loss. Before IVF they were assigned to metformin (MET) 1000 mg BID or combined MET 1000 mg BID and liraglutide 1.2 mg QD s.c. (COMBI) or to the control group (CON). CON directly proceeded with ovarian stimulation protocol, whereas MET and COMBI started with stimulation after 4 weeks medication free period.

Results: 11 women on MET, 13 on COMBI and 11 CON completed the study according to the protocol. Patients in MET lost on average 6,70±6,70 kg (P˂0.001) compared with 7,68± 3,74 kg loss in COMBI group (P˂0.001), with no significant between-treatment difference (P=NS). In addition, COMBI resulted in a significant reduction of visceral adipose tissue (VAT) area (-20,65± 7,40 cm2; P=0,028,). More than 5 % of weight reduction was achieved in 76,9% of patients in COMBI and 45,5% of patients in MET. In high responders who lost more than 5% of initial body weight numbers of blastocysts/patient were greater in both treatment arms than in controls (3,67±4,82 in COMBI; 3,60±6,95 in MET; vs 2,09±2,07 in CON). High responders in COMBI also had the highest number of oocytes/patient 14,67±9,59 and the highest percentage of mature oocyte 11,22±9,27 among all three groups, although the between treatment differences were not statistically significant yet. Furthermore, in COMBI one patient become spontaneously pregnant before IVF and give a birth after non-complicated pregnancy.

Conclusion: A subset of obese PCOS women who lost more than 5% of body weight reduction before IVF with short-term intervention with metformin alone or in combination with liraglutide had increased fertility potential. COMBI resulted in the highest number of women who lost more than 5% of weight and was associated with the highest number of blastocysts/patient.

The aim of the study was to evaluate the efficacy of testosterone treatment before controlled ovarian stimulation (COS) on clinical pregnancy and live birth rate in poor responders undergoing IVF. Materials and Methods: 149 women with poor ovarian response, defined according to ESHRE consensus/the Bologna criteria of low ovarian response were included. Patients were randomly divided into 2 groups: Testosterone undecanoate (TU) treatment group (98 women) or control group (51 women). For TU group Testosterone undecanoate oral form 40 mg was given daily for at least 40 days (median 54 days) preceding COS for IVF.

Primary outcome measures were clinical pregnancy (PR) and live birth (LR) rates in different age groups. Statistical research was made using a software package statistics (StatSoft Inc. U.S., version 12). Quantitative data is presented as median and quartile range. When comparing the quantitative data of two independent groups Mann Whitney U-test and Fisher exact two-tailed test were used. Values were considered statistically significant if p less than 0.05.

Results: There were no differences in patients’ characteristics between the two groups. There was no significant difference in starting FSH dose or total dose of gonadotropins administered between the groups. There was a significant increase in number of oocytes retrieved 2 [1; 3] vs 1 [1;2] in TU group (p=0.04). Сlinical pregnancy rate (per cycle) was significantly higher in TU group (24.5%) than in control group (7.8%), p=0.015. Live birth rate was higher in TU group than in control group 17.3% vs 5.9%, respectively, though the difference was not statistically significant (p=0.07).

The treatment and control groups were divided into 2 subgroups each according to age: 1 subgroup less than 40 years old and 2 subgroup – 40 years and older.

There was a significant increase in clinical pregnancy and live birth rate in TU 1 subgroup (less than 40 years old) compared to the control 1 subgroup: 38% vs 8.7% (p<0.001) and 29.3% vs 6.5% (p<0.01), respectively. There was no difference neither in clinical pregnancy nor in live birth rate between TU and control 2 subgroups.

Conclusions: Testosterone undecanoate treatment may increase pregnancy and live birth rate women with diminished ovarian response undergoing IVF. PR and LR rate are severely decreased in women 40 years and older, they may need more prolonged pretreatment with TU for better success rate.

Material and Methods: Skeletal muscle biopsies were obtained from 17 women with PCOS and 14 controls matched for age, weight and BMI. For analyses of gene expression and DNA methylation, Illuminas Human HT-12 Expression BeadChip and Infinium HumanMethylation450 BeadChip were used.

Results: Women with PCOS had more antral follicles, larger ovaries and higher circulating testosterone, and LH and LH/FSH ratio than controls. Fasting triglycerides, C-peptide, calculated C-peptide index, and HOMA-B were higher in women with PCOS than in controls indicating metabolic aberration and insulin resistance. Adipocyte size was enlarged and the sex steroid content were higher in women with PCOS compared with controls. After FDR-correction, 85 unique genes were found to be differentially expressed in skeletal muscle from PCOS women versus controls (q<0.05). 66% of the genes were up-regulated and 34% were down-regulated in PCOS women and the absolute expression differences were 21-186%. A large number of the identified genes are suggested to play a role in muscle function and metabolism, including DYRK1A, SYNPO2, KLF10, SCP2 and NAMPT. According to a gene set enrichment analysis, there was a significant gene expression disruption to pathways involved in the immune system in PCOS women. Also, several GWAS candidate genes for insulin resistance (e.g. APOE and PPARG) were differentially expressed in PCOS women versus controls. 21 of the significantly differentially expressed genes had one or more CpG site(s) in which the DNA methylation differed between PCOS women and controls. In addition, experiments where we quantify the expression of selected genes in isolated and cultured skeletal muscle cells derived from female donors in the presence of testosterone and insulin are ongoing.

Conclusion: We demonstrate that PCOS is associated with gene expression and DNA methylation aberrations in skeletal muscle which may explain some of the metabolic abnormalities seen in these women.

At approximately 35 sites in the US and the UK, approximately 220 women with PCOS and oligo-/amenorrhea will be randomized in a double-blind fashion 1:1:1:1 to receive MLE4901 40 mg, 60 mg, or 80 mg or placebo orally twice per day for 28 weeks following a progestin challenge. The primary objective is to evaluate the efficacy of MLE4901 in improving menstrual regularity in women with oligo-/amenorrhea due to PCOS. Secondary objectives include changes in ovulation regularity, each subject’s self-identified most bothersome symptom of PCOS, hormone levels, clinical signs of hyperandrogenism, metabolic syndrome-related parameters, quality of life, and work productivity. Menstrual period frequency will be assessed using a daily menstrual diary. Hirsutism will be assessed using the modified Ferriman-Gallwey score. Quality of life, work productivity, and the bothersomeness and severity of PCOS symptoms will be assessed using validated questionnaires. Once completed, this Phase 2b study will be one of the largest, most robustly designed studies conducted to date in PCOS.

Hepato-visceral fat excess is a feature of Polycystic Ovary Syndrome (PCOS). Risk factors for such excess include a reduced prenatal weight gain and an augmented postnatal weight gain. We studied whether PCOS in adolescent girls was preceded by a relatively low birth weight and/or a relatively high BMI at PCOS diagnosis.

Methods

The adolescent study population consisted of 298 non-obese and 169 obese girls with PCOS diagnosed respectively in Barcelona/Spain and Datteln/Germany; 87 healthy girls served as controls. Z-scores for weight-at-birth and for BMI-at-PCOS-diagnosis were derived from country-, age- and sex-specific references; individual changes between these Z-scores were calculated.

Results

Non-obese Spanish and obese German PCOS girls had mean birth weight Z-scores of -0.7 and 0.0, and mean BMI Z-scores of +0.4 and +2.7, so that mean Z-score increments amounted to +1.1 and +2.6 (P<0.001 versus controls).

Interpretation

PCOS in adolescent girls was found to be preceded by a marked Z-score rise between weight-at-birth and BMI-at-PCOS-diagnosis, thus corroborating the notion that prenatal and postnatal weight gain have opposing influences on PCOS development, as they have on adrenarche and on age at menarche. PCOS is not a gynecological syndrome, but an outcome of a metabolically unfavorable sequence of early weight gains.

An initial PCOS diagnosis is often the result of a purposeful interaction by the patient with her physician expressly seeking a diagnosis of her symptoms. In our survey, the most common symptom driving this interaction was menstrual dysfunction (i.e., menstrual irregularity, heavy bleeding or pelvic pain), reported by 57% of patients. Infertility was the next most common driver for women to seek a diagnosis, at approximately 15% of patients. In contrast to the most common drivers of diagnostic interactions, hyperandrogenism (e.g., facial/body hair and alopecia) and mood changes (e.g., anxiety and depression) were the two of the three most frequently reported bothersome symptoms by women with an established diagnosis of PCOS. Sixty-five percent (65%) of patients who suffer from excessive hair growth or alopecia rank these symptoms as “extremely bothersome” (≥6 on a 7-point scale) and 85% of respondents reported them as at least somewhat bothersome. However, symptoms of hyperandrogenism only drove the diagnostic interaction for 6% of patients. Mood changes such as anxiety and depression were reported in 60% of patients, and were rated as highly bothersome, to 48% of those patients. Our findings shed light on the changing burden of PCOS to patients from diagnosis through treatment. While the initial constellation of menstrual dysfunction symptoms generally respond to treatment, signs of hyperandrogenism tend to show much less patient reported improvement and remain burdensome during treatment. Our findings are consistent with the known poly-symptomatic nature of PCOS, and highlight the need for comprehensive treatment for all PCOS symptoms, beyond just menstrual dysfunction.

Conclusions: Our follow up data suggested that the metformin treatment might not reduce the risk for developing diabetes in obese Brazilian women with polycystic ovary syndrome. Also, this drug might reduce the risk of metabolic complications.

Clinical Case:  A 33 years old lady, referred to endocrinology in the context of left adrenal incidentaloma detected in CT chest performed to evaluate chronic cough. she had no history of fever, night sweat or weight loss, no history of abdominal pain, patient is not hypertensive and no symptoms to suggest Cushing's Syndrome, pheochromacytoma or hyperandrogenism. Family history was irrelevant and physical examination showed normal blood pressure, BMI of 18 and no sign suggestive of adrenal hyperfunctioning. CT scan showed, two left adrenal masses, the larger one is heterogeneous with thick peripheral calcification, mild peripheral enhancement and large central non enhancing component likely representing necrosis measured 7.7x6.2x 7.3 cm, there was no invasion to the adjacent structures. The other lesion was located inferior and medial to the former one and showed mild peripheral enhancement with central area of necrosis measured 3.5x3.3x3.3 cm. Scan through both lungs show multiple branching nodular densities representing tree in bud appearance. Laboratory investigations showed normal dexamethasone suppression test and plasma metanephrins, serologic test for Epstein- barr virus and HIV were negative. regarding her chronic cough patient was evaluated by pulmonologist and was diagnosed as hypersensitivity pneumonitis. she underwent uneventful left adrenalectomy. Gross pathologic evaluation indicated a grey lobulated mass weighed 262 grams and measured 15x9x5 cm. with pale tan whorly cut surface and focal classification. Microscopic evaluation showed a well –circumscribed and encapsulated spindle cells arranged in fascicles and whorls, no features of mitosis or pleomorphism , residual normal adrenal tissue was noted at the periphery. The immunohistochemical (IHC) studies were positive for smooth muscle actin (SMA) and desmin which support the diagnosis of lieomyoma. Additional IHC studies included Melan-A, human melanocyte black 45 ( HMB45) , and Ebestien –Barr virus latent membrane protein (EBV-LMP ) were all negative.

Conclusion: Adrenal liomyoma is very rare and should be considered in the differential diagnosis of huge unilateral non-functioning incidentally detected adrenal lesions.

Case: A 7-day old baby boy (BW 3.49 kg), infant of diabetic mother treated with diet, was found to have possible cystic kidneys on prenatal ultrasound (U/S). Physical exam revealed no focal abnormalities and normal male genitalia with bilateral palpable testes. A postnatal U/S showed an enlarged left adrenal gland without focal mass, a right adrenal mass (2.9 x 3.3 x 4 cm), normal appearing kidneys, and possible UPJ obstruction. His 17-OHP newborn screen was elevated at 229 ng/mL (extracted value, 68 ng/ml). Diagnostic studies showed elevated 17-OHP, 14,800 ng/dL (80-420 ng/dl) and elevated plasma renin activity 159.59 ng/ml/h (0.25-5.82)] confirming the diagnosis of CAH due to 21-hydroxylase deficiency. Additional studies showed elevated urinary homovanillic acid (HVA) and vanillylmandelic acid (VMA) consistent with the diagnosis of congenital neuroblastoma.

After confirmation of the CAH diagnosis, hydrocortisone and fludrocortisone replacement therapies were initiated. Subsequent MRI and MIBG scans were consistent with the diagnosis of neuroblastoma. Serial adrenal imaging has shown continued decrease in the size of the adrenal mass (now 2.3 x 2.2 x 2.0 cm). HVA and VMA excretion have decreased.

Genetic analyses showed normal male 46,XY karyotype. Genetic mutation analysis of CYP21A2 revealed two mutations, an intron 2G splicing mutation (c.293-13A/C>G) and a novel mutation, T>C at nucleotide c.527 in exon 4, which is predicted to generate a missense mutation, p.L175P. Based on bioinformatics tools, p.L175P is predicted to be deleterious. Amino acid alignment with other mammalian CYP21A2 genes indicates that this is a highly conserved amino acid (1).

Conclusion: CAH was diagnosed prior to neuroblastoma among the previously reported patients with concurrent CAH and neuroblastoma (2,3). The natural history of congenital NB is typically spontaneous resolution. The NB in our patient was likely detected because of the prenatal U/S findings and diagnosis of CAH. A novel CYP21A2 mutation was identified in our patient. Although the adrenal vein provides cortisol to the adrenal medulla, likely no causal relationship exists between CAH and NB. Rather the NB represents an incidental finding in a patient with CAH.

Introduction

Predominantly considered the diagnostic domain of the pediatrician, congenital adrenal hyperplasia (CAH) is most frequently associated with 21-hydroxylase deficiency. However, a less common form of CAH, 17-α-hydroxylase deficiency, can stay asymptomatic and undiagnosed until adulthood.

A case

A 32-year old female was referred to endocrinology department with complaints of mild hypertension and hypokalemia in order to investigate causes of secondary hypertension.

Her past medical history included presentation to gynecologist at 15 years of age with primary amenorrhea. A karyotype analysis revealed 46XY and after that her ovaries were surgically removed, and she was started on life time estrogen replacement therapy. Her blood pressure was normal at that time.

However, last few months before presentation, she started to have headaches every day and blood pressure was measured 150/90mmHg and above.

Height 174 cm and BMI: 25.8 kg/m2. Pubic and axillary hair were poor, and breast development was minimal (Tanner stage 3).

On given the patient’s information, an upstream block in steroid synthesis was suspected. Potassium: 2.79 mmol/L(3.5-4.5), serum cortisol: 0.89mcg/dl (6-23), estradiol <20pg/ml and testosterone <10ng/dl (14-76) and ACTH: 62.4pg/ml (0-46), FSH:80, LH:36. Aldosterone: 552pg/ml(30-300)) and renin: 2pg/ml/h (0.2-27). Synacten stimulation test showed cortisol peak 1.5 mcg/dl at 30’. These results were consistent with 17-α-hydroxylase deficiency.

An abdominal CT showed 27x32mm hypodense lesion on the right adrenal gland. 24x12mm hypodense-heterogeneous lesions on confluence point and 12x9mm on the lateral crus of the left adrenal gland. These lesions were <10 HU density.

Dynamic pituitary MRI showed no pathology of the gland.

Sequencing of CYP17A1 demonstrated heterozygosity for c.1283C>T on exon 8. Her mother, but not father, showed heterozygosity for the same genetic location.

The patient was commenced on prednisolone 10 mg/d. Hypokalemia and hypertension resolved rapidly.

Year later repeated abdominal CT demonstrated 59 % reduction of the lesion on the right and 29 % reduction on confluence point and 66 % reduction on lateral crus of the left adrenal gland.

Blood pressure is in normal range with no medications. She is on 5mg/day prednisolone.

Discussion

To date, there have been only two case reports with 17-α-hydroxylase deficiency in children from Turkey. This is the first case of the adult patient with undiagnosed 17-α-hydroxylase deficiency so far.

The condition should be considered in young women, who fail to enter puberty and have concomitant hypertension.

Although, our patient had a complete 17-α-hydroxylase enzyme deficiency, she had a mild hypertension, which may be the reason in the delay of the diagnosis. This case is a really challenge for the adult endocrinologists, as these conditions are barely seen in adults.

Case report: A 35-ys man, affected by adrenal hyperplasia due to 21-hydroxylase deficiency, diagnosed at age of 10 ys for anticipated adrenarche and microorchidism, came to our observation for a clinical control. His sister was also affected by the same syndrome, diagnosed by genetic studies. He had not been previously treated with suppressive or replacement therapy, resulting a chronic exposure to high T levels.

The patient showed an elevated 17-OH-Progesterone (P): basal 9.1, after ACTH 22 ng/ml (electro-chemiluminesce method or ECLIA, nomal range 0.2-0.8); T levels were 15.9 ng/ml (ECLIA, n.r. 2.5-8.4). At abdomen Magnetic Resonance Imaging, performed for adrenal investigation, a 18 mm large nodular lesion was discovered in the liver, together with increased serum α-fetoprotein level. Computed Tomography and Contrast Enhanced Ultrasound suggested increased arterial vascularity of the lesion; on the basis of needle biopsy assessment (showing an hyperplastic-adenomatous lesion with focal inflammatory infiltration and biliary metaplasia of hepatocytes) and of the high cancerous risk, he underwent surgical removal of the lesion. Histological findings showed HCC with moderate differentiation (G1/G2). We showed an unexpected lowering of T levels. One year later recurrence of HCC, localized in a different liver segment, was diasgnosed and treated by percutaneous radiofrequency ablation. Two other HCC recurrences were detected during the subsequent follow up and were treated with the same technique. Every HCC recurrence was accompanied by elevation of T, but also SHBG; therefore we hypothesized a facilitating effect of chronic androgen elevation, but also a possible production of SHBG by tumor itself.

Conclusion: The prevalence of HCC in adrenal hyperplasia is not well described in literature; our report suggests the need for screening of liver lesions in males affected by this syndrome; the role of T in inducing or facilitating the neoplasia and the possible involvement of SHBG secretion remain to be established.

Clinical Case: A 21 year old woman from Ecuador initially seen in pediatrics endocrinology at the age of 18years for primary amenorrhea. Her initial physical exam showed height of 154.5 cm, normal blood pressure of 113/74mmHg, sexual characteristics tanner 1. Initial blood work were as follows: Cortisol 19.3 nmol/L (110-606), ACTH 56.1 pmol/L (1.32-11), corticosterone >10000 nmol/dL ( 1.7-37.3) 11 Deoxycortisol 4.21nmol/L (<3.09), testosterone <0.0347nmol/L( 0.07-1.56) estradiol <7.3pmol/L (follicular phase 143-1376, luteal phase 176-1615) FSH 30.9 IU/L ( follicular phase 2.5-10.2 mid cycle peak 3.1-17.7, luteal phase 1.5-9.1) LH 24.5 IU/L (follicular phase 1.9-12.5, mid cycle peak 8.7-76.3, luteal phase 10.0-54.7) progesterone 2.0nmol/L (follicular phase<3.2, luteal phase8.3-68.3) Aldosterone <0.02nmol/L ( <0.77)

pelvic sonogram : no ovaries seen, small uterus

Karyotype 46 XX. Genetic evaluation: homozygous mRNA.C334-336:3 bp duplication of AATC. Codon 113. (This has not been previously reported as a mutation causing CAH due to CYP17A1 deficiency)

She was started on low dose estradiol with plans to titrate up. Once there is sonographic evidence of significant uterine growth, the plan is to treat the CAH with methylprednisolone as a means of reducing the high level of adrenal-derived progesterone and therefore induce menses. Current physical exam shows secondary sexual characteristics tanner 2.

Conclusion: this is the first case reported with CYP17A1 with a mutation on the codon 113 that can cause CAH due to CYP17A1 deficiency without hypertension or potassium abnormalities.

Congenital adrenal hyperplasia (CAH) is an inherited disorder of adrenal steroid synthesis, the majority of which is caused by 21-hydroxylase deficiency. Multiple alleles could be mutated with each allele being associated with classical or nonclassical CAH. In untreated compound heterozygous patients, this can still cause complications such as testicular adrenal rest tumors (TARTs) and benign adrenal tumors such as myelolipomas. These can be easily missed or misinterpreted on routine imaging.  

CLINICAL CASE:

37 year old male presented for evaluation after incidental imaging revealed bilateral adrenal enlargement. He was told he had adrenal cancer and should see an endocrinologist. At the office visit, he stated he was diagnosed with CAH as a child (type unknown) and started on fludrocortisone by a pediatric endocrinologist, which was stopped 12 years ago. He had never been on glucocorticoids, nor was he ever hospitalized for adrenal insufficiency. Prior to the visit, a CT abdomen/pelvis with contrast was performed for abdominal pain and showed enlargement of both adrenal glands: 7.8x9.1x6.5cm on the left, 5.1x10x7.1cm on the right. Multiple masses with fat density on the right anterior limb were also noted, the largest being 3.1x3.7x3.9cm, along with small hypodense lesions posteriorly. Additionally, a testicular ultrasound was performed after he noticed his testicles felt firm, and bilateral testicular tissue replaced with solid, hypoechoic lobulated masses were noted. The right was 4.1x1.8x2.4cm and 3.8x2.5x2.5cm on the left; both were surrounded by normal testicular tissue. While his adrenal and testicular abnormalities fit classical CAH with presumed myelolipomas and TARTs, respectively, the lack of prior glucocorticoid therapy pointed towards a non-classical etiology. Further work-up to establish diagnosis was then obtained. The 17-hydroxyprogesterone level was 34526 ng/dl (ref 42-196) and the CYP21A2 gene analysis showed P453S mutation and 30kb deletion. Plasma free metanephrines were negative: metanephrines <25pg/ml (ref<57), normetanephrines 78 pg/ml (ref<148), and total metanephrines 78 pg/ml (ref <205). The DHEA-S level was not elevated at 61 mcg/dl (ref 106-464). Initially, dexamethasone 0.75mg at bedtime was started as the patient also took carbamazepine, a CYP3A4 inducer; this was eventually decreased to 0.5mg. On follow up, the patient did well without cushingoid symptoms.

CONCLUSION:

Clinicians should consider a diagnosis of compounded heterozygous CAH in patients with findings of myelolipomas and TARTs. Failure to understand this entity can result in misdiagnosis, and the patient may undergo improper treatment which can be detrimental to their health. Currently, there is a lack of literature in regards to compounded heterozygous patients and the prevalence of TARTS and myelolipomas. 

Discussion: In summary, this is a case of 45-year-old phenotypically and (likely) karyotypically female, but who identifies socially and psychologically as male. He most likely has deficiency in CYP21A2 activity due to the high levels of plasma 17-hydroxyprogesterone, physical exam findings and medical history. In spite of childhood rearing as a female, his social and psychological preference is to identify as a male. In review of the literature, between 5-15% of patients with CAH, karyotypically female, will identify as male. This is felt to be related to the concept of androgen imprinting where elevated levels of androgens on the developing brain influence gender preference over karyotype and sexual assignment(1). It is important to note that the androgen elevation is not from testicular or ovarian production but from the adrenal glands. These lifelong elevated androgen levels in this patient likely contributed to his gender identity.

Conclusion: In cases of congenital adrenal hyperplasia it is important to consider the role of androgens in gender identity which may have more influence than childhood sexual assignment.

The patient is a 56 year-old African-American man who was referred to Endocrine in 2002 for hypertension with a 3.0x2.8x2.1 cm, well circumscribed, ovoid, hypodense, soft tissue mass in the medial limb of the right adrenal first noted in 2001. Endocrine evaluation was negative for primary hyperaldosteronism, Cushing’s syndrome, and pheochromocytoma, however plasma renin activity (PRA) by liquid chromatography tandem mass spectrometry (LC/MS/MS) was low at 0.52 ng/ml/hr., at least suggesting the possibility of a deoxycorticosterone-secreting adenoma (DOComa). Unstimulated serum deoxycorticosterone by LC/MS/MS on 9/20/11 was 38 ng/dl (3.5-11.5). Both hypo- and hyperkalemia were occasionally noted, but not at DOC or aldosterone sampling times. Unstimulated serum 11-deoxycortisol by LC/MS/MS on 1/31/08 was 130 ng/dl (<76). Serum 17-OH-pregnenolone was normal and serum 17-OH-progesterone by LC/MS/MS was low at 43 ng/dl (61-334). Metformin 500 mg daily after supper was started on 5/6/08, following which serum 11-deoxycortisol fell to 84 ng/dl. After this the patient was lost to follow-up for several years and ran out of metformin. On 6/13/12 his serum 25-OH-Vitamin D by immunoassay (IA) was 6 ng/ml (30-100). Between 1/8/08 and 6/8/12 the patient had lost 2.3 kg, after which he underwent right below knee amputation due to peripheral arterial disease with gangrene. On 7/15/16 serum 11-deoxycortisol fell to 53 ng/dl (<42), serum deoxycorticosterone normalized to 6ng/dl (2-19), serum 25-OH-Vitamin D was 14.4 ng/ml (30.0-100.0). On 8/5/16 serum 11-deoxycortisol fell to 47 ng/dl, DOC was 5.2 ng/dl, and 25-OH-Vitamin D was 36.3 ng/ml. The right adrenal mass has not changed in size or appearance since 2001. This case illustrates the importance of considering NCAH in the differential diagnosis of adrenal incidentaloma and again illustrates the potential benefit of improvement of insulin sensitivity by both weight loss and correction of hypovitaminosis D in NCAH.

Adrenal cysts are rare, around 600 cases have been reported in the Medical literature. They are usually non-functional, asymptomatic and less than 10 cm in diameter when discovered incidentally. They may occur at any age, but most of them are seen in the 3rd to 4th decades of life with a higher preponderance in females. Traditionally, adrenal cysts are divided into neoplastic and nonneoplastic groups. Non-neoplastic adrenal cysts may be further categorized as any of the 4 major types: pseudocyst(39%), epithelial (9%), parasitic (7% generally echinococcal), or endothelial cysts (45%). Symptoms occur when adrenal cysts become large enough to cause pain or as a consequence of intracystic bleeding or infection. Less frequent presentations include hypertension or spontaneous rupture of the cyst. Radiologic evaluation is helpful in identification benign versus malignant lesions (7 % of adrenal cyst are potentially malignant). Any functional lesions, potentially malignant lesions, or benign lesions more than 5 cm in diameter deserves surgical treatment. For small, benign lesions, conservative management is a viable option, although no surveillance protocols have been described.

Clinical case:

A 41 years old male referred to endocrinology in the context of left adrenal mass found in CT abdomen performed to evaluate right loin pain, patient is normotensive and no history of adrenal hyper functioning. physical examination showed normal blood pressure with no signs of adrenal hyper functioning.

CT scan showed mass originating from the lateral limb of the left adrenal gland measuring 6 X 5 cm and showing CT characteristic of clear fluid with attenuation value 13 HU consistent with adrenal cyst. No evidence of solid component or septations. No evidence of calcifications. There is no infiltration or invasion of surrounding structure.The right adrenal gland is normal.

MRI adrenals showed thin walled well defined rounded 6 x 5 cm lesion involves the lateral limb of the left adrenal gland. It depicts high T2 and low T1 signal intensity and no change of signal in the out of phase sequence. No septa or hemorrhagic component.

Laboratory investigations showed negative hydatid antibody, normal DST and normal urinary metanephrines.

Patient offered left adrenalectomy but he elects to wait with follow up images.

CT abdomen a year later showed increase in the size of the cyst to 6.7X6.8 cm, 2 years later the size increase to 8.2x7.4 cm, and 3 years later the size increase to 10x9 cm no change in the radiological characteristic. Patient remain asymptomatic. Repeated hormonal screen was again normal. Patient finally agree for adrenalectomy which will be done in 2 months.

Conclusion:

 With the increasing use of imaging modalities in patient evaluation, an increase in incidentally detected adrenal cysts is expected. Surgical excision of these cysts is an acceptable method of treatment where the indications are met.

Case: A 34 year old male with history of hypertension diagnosed four years before presentation was brought to the emergency department after falling approximately 12 feet from his deer stand to the ground, sustaining a fracture of the left posterior acetabulum. Computed tomography (CT) of the abdomen revealed a 10 cm right adrenal cyst with peripheral calcifications but no septations, hemorrhage, or solid component. Blood pressure (BP, 197/121 mm Hg) and heart rate (HR, 110-120 bpm) were significantly elevated, and the patient reported he had stopped BP lowering medications approximately three years before admission. The magnitude of BP and HR elevations raised concern for a cystic pheochromocytoma, though plasma free metanephrines drawn while the patient was still in the emergency room were unremarkable. Morning cortisol suppressed to < 1.8 mg/dL on an overnight dexamethasone suppression test. Echinococcal serologies were obtained due to a military deployment to Iraq, with no significant titer of Echinococcus IgG antibody detected. Hypertension and tachycardia were controlled with diltiazem and prazosin, and the patient did well after open reduction and internal fixation of his fracture. Unfortunately, he did not come to endocrinology clinic after hospital discharge.

Conclusions. Historically, adrenal cysts have presented with abdominal pain and a palpable flank mass, though a large number are now discovered incidentally on abdominal imaging studies. They are generally classified into four major histopathological types: endothelial, pseudocyst, epithelial, and parasitic. Endothelial cysts and pseudocysts are most common, though the distinction may be insignificant as both appear to be variants of vascular cysts. In most series, a small number (≤ 5%) of cysts are associated with pheochromocytomas or adrenocortical carcinomas. Surgery is recommended for symptomatic patients, cysts > 5 cm, functional lesions, hemorrhagic cysts, cysts with evidence of mass effect, or suspected carcinoma. Adrenalectomy is preferred by most authors, though in some case series laparoscopic aspirations and gland sparing surgeries have been performed. Asymptomatic patients with cysts < 5 cm and no evidence of hormonal secretion or carcinoma can be followed prospectively with abdominal CT imaging, though there is no consensus on the timing and duration of surveillance due to the low incidence of cases. Surgery was indicated for our patient due to the size of his cyst, though he was unfortunately lost to follow up.

Adrenocortical cancer (ACC) is a rare and aggressive malignancy with metastatic potential in 40% of patients within 2 years despite complete resection. The pathologic differentiation of ACC from benign neoplasms is challenging.

We present the case of a 49-year-old woman with recurrent masses despite complete left adrenalectomy 4 years prior for what was deemed benign adrenocortical hyperplasia.

Case report:

A 49-year-old presented in 11/2011 for evaluation of an incidental adrenal mass. She was noted to have a 6.7cm left adrenal mass with Hounsfield units<10. Hormonal evaluation was negative. She underwent left adrenalectomy in 4/2012. Pathology was notable for a modified Weiss score positive for 3/9 criteria, consistent with ACC. However, given the size of the neoplasm, mitotic activity and invasiveness, ultimately she was classified to have adrenocortical hyperplasia.

She was lost to follow up for 4 years finally returning to clinic in 6/2016. She complained of weight gain, unusual facial hair growth and constant left upper quadrant, abdominal pain. Her physical exam was negative for Cushingoid features, or hirsutism.

Her biochemical evaluation was significant for: aldosterone 8.7 (4.0-31ng/dl), renin:0.7 (0.2-1.6ng/ml/hr), DHEA-S: 94(32-240 ug/dl), 24 hr urine Cortisol: 15(0-50ug/24hr) for a creatinine of 2.2g/24h, TSH: 1.31(0.5-5 IUI/ml), 24hr urine epinephrine, norepinephrine, metanephrine were within normal limits. Repeat CT revealed new lesions in the left adrenal bed, anterior to the left kidney and adjacent to the diaphragm concerning for recurrent or metastatic disease.

CT guided FNA of the diaphragmatic lesion was performed. Cytology revealed similar features to the patient’s previously resected adrenal lesion. According to the modified Weiss score these features are consistent with a malignant lesion of uncertain potential.

Repeat CT scan in 10/2016 revealed further enlargement of patient’s left adrenalectomy bed nodule. She is awaiting surgical resection of the nodules.

Discussion:

ACC is a rare but aggressive malignancy that can lead to multiple endocrine disorders. Five-year survival is approximately 45 to 60% for early stage disease, and 10 to 25% for advanced stage disease. A modified Weiss scoring system is used for pathologic diagnosis. This case highlights the difficulties in pathologic diagnosis of ACC and the importance of strict adherence to the Weiss scoring system. Patients diagnosed with a malignant lesion or lesions with uncertain potential should be treated aggressively and followed closely.

Clinical Case: A 24 years-old man presented with acute onset of right abdominal pain while lying in bed. Past medical history was significant only for hepatitis A at age 15. Physical exam showed a healthy-looking male who was afebrile, with heart rate 106, BP 115/74, and BMI 27 kg/m2. He had mild bilateral gynecomastia but no other unusual signs. Initial labs showed elevated WBC 20K and normal liver function tests. Abdominal ultrasound showed a large cystic adrenal/liver mass. CT scan showed a 15cm x 11cm x 10cm well-circumscribed hypodense cystic right adrenal mass abutting the liver and kidney. The mass was relatively homogenous except for a fluid-layering effect, with the nondependent upper half measuring 18 HU and the dependent lower half measuring 28 HU, suggesting recent bleeding settling in the dependent areas. The parenchyma of the right adrenal gland appeared to be compressed by the mass and displaced anteromedially.

The patient was admitted for pain control and workup of the adrenal mass. 24-hour urine collection (2070 mL) showed elevated free cortisol, measuring 282 mcg/24 hrs (normal 0-50). Plasma and 24-hour urine metanephrines and normetanephrines were normal. Plasma aldosterone was <1.0 ng/dL, and plasma renin activity was 1.39 ng/mL/hr, with A-R ratio <1. Random ACTH was suppressed at 3.0 pg/mL (normal 7-63). DHEA-S was low at 87 mcg/dL (normal 164-530). The remaining laboratory workup was notable for low albumin 3.6 g/dL (3.7-5.2 g/dL), low total protein 6.0 g/dL (normal 6.4-8.5), anemia with Hgb 11.8 g/dL (normal 13.3-17.2), and hypocalcemia of 8.4 mg/dL (normal 8.9-10.7). Although his WBC was elevated at presentation, it decreased to normal spontaneously.

He underwent open right adrenalectomy uneventfully with perioperative stress dose hydrocortisone. Histological examination showed a benign adrenal gland associated with a hemorrhagic pseudocyst. No adrenal cortical or medullary tumor was seen on extensive pathologic sectioning. The adrenal cyst consisted of a thick fibrous wall with organized internal contents of fibrin clot and degenerating blood. There was chronic periadrenal inflammation. The normal appearing adrenal gland was intimately associated with the fibrous wall of the pseudocyst. The patient was discharged home with hydrocortisone taper and currently doing well on low dose replacement 20mg/5 mg hydrocortisone.

Conclusion: Adrenal pseudocysts are rare and can present with nonspecific abdominal pain, but hormonal excess due to adrenal cysts is rare, with pheochromocytoma the most common. Some case reports have documented spontaneous hemorrhage or infection in adrenal cysts. Based on literature review, this may be the first case of a hemorrhagic adrenal pseudocyst causing Cushing’s syndrome.

Clinical Case: A 45-year-old Japanese woman with 2 drug hypertension was recently found to be hypokalemic (serum K = 2.0 mEq/L). Laboratory testing showed: plasma renin activity (PRA) = 0.1 ng/ml/h, plasma aldosterone concentration (PAC) = 43.9 ng/dL, and urine aldosterone excretion = 44 mcg/d (urine Na 188 mEq/d). The PAC was not suppressed with the captopril challenge test or the saline infusion test, and PA was confirmed. CT scan demonstrated a low density right adrenal nodule measuring 11 x 6 mm. The right adrenal vein was visualized with multidetector CT and AVS with cosyntropin stimulation was performed. However, the serum cortisol levels in IVC and the right and the left adrenal veins (17.1, 15.1 and 122.6 mcg/dL, respectively), showed unsuccessful cannulation of the right adrenal vein. PAC levels in IVC and the right and the left adrenal veins were 63.2, 10.9 and 313.1 ng/dL, respectively. Instead of repeating AVS, a NP-59 SPECT/CT scan with dexamethasone (DEX)-suppression was obtained. The patient took oral DEX (3 mg/day for 4 days before and 2 mg/day for 7 days after NP-59 intravenous injection) to suppress cortisol production in normal adrenal gland. The patient was also treated with iodine tablets for 9 days to block thyroidal uptake of free 131-I. SPECT/CT images were obtained at 5 and 7 days after NP-59 administration. NP-59 SPECT/CT revealed intense uptake within the right adrenal gland without significant uptake in left adrenal gland. After laparoscopic right adrenalectomy, serum K, PAC and PRA were 4.3 mEq/L, 9.2 ng/dL, and 1.0 ng/mL/hr, respectively. Her blood pressure normalized without antihypertensive medications.

Conclusion: The role of NP-59 planar scintigraphy has been thought to be limited for subtype classification of PA because tracer uptake is poor in APAs smaller than 1.5 cm in diameter (sensitivity <50%). However, with the development of SPECT technology the resolution of scintigraphy has been improved. NP-59 SPECT/CT with DEX-suppression should be reconsidered as an effective test to determine the clinical significance of CT-detected adrenal nodules in patients with PA.

Takotsubo cardiomyopathy is believed to be induced by excess catecholamine release and is frequently associated with emotional stress. We report a case of this condition associated with an Aldosterone Producing Adenoma (APA).

Case Presentation:

A 47-year-old African American female with hypertension since age 28 presented with an acute MI. Her history was significant for right renal artery fibromuscular dysplasia which was treated with angioplasty. Her blood pressure had been controlled with calcium channel blockers until age 45 when an ARB was added. Four months prior to presentation she had chest pain and was diagnosed clinically with perimyocarditis. A cardiac catheterization showed normal coronary and a chest CT revealed a 1.6 x 1.8 cm left adrenal incidentaloma (0 Hounsfield units). Subsequent laboratory evaluation showed a serum aldosterone of 42 ng/dl (normal <3.0 – 23.2 ng/dl), and a renin <0.15 mg/ml/hr. Epleronone therapy was started, and four days later she presented with rapidly escalating chest pain associated with headache, high blood pressure, and diapherosis. The ECG showed an acute inferior wall MI and the troponin level rose to 16 ng/dl. she denied emotional stress. A cardiac catheterization showed apical hypokinesis, no evidence of ASCVD, and normal renal arteries. The ejection fraction was 55%. An adrenal MRI showed a 2.0 x 1.3 x 1.8 cm left adrenal nodule which was consistent with a lipid rich adenoma. A 24 hour urine collection for catecholamines, metanephrenes , normetanephrines, and cortisol was normal. The epleronone dose was adjusted and surgical consultation was obtained.

Discussion:

The effect of hypealdosteronism on cardiac morbidity could be from HTN alone and/or the direct effects of aldosterone on myocardial and vascular cells. It is known that high levels of aldosterone stimulate renal sodium retention by increasing expression of the thiazide-sensitive sodium-chloride cotransporter and the amiloride-sensitive epithelial sodium channel (ENaC) leading to plasma volume expansion and HTN. The role of aldosterone in eliciting acute vascular effects through nongenomic signaling pathways has been increasingly recognized. High levels of aldosterone increase markers of oxidative stress in plasma and heart tissue and alter intracellular Mg and Ca concentrations in smooth muscle cells and lead to coronary artery dysfunction. Acute aldosterone exposure causes dose-dependent myosin light-chain phosphorylation, a mechanism by which it can mediate vasoconstriction. These rapid effects of aldosterone can be inhibited by both spironolactone and eplerenone.

Conclusion:

To our knowledge, this is the first report of Takotsubo’s cardiomyopathy associated with an APA. It suggests that non-genomic effects of aldosterone may enhance the risk of acute coronary vasoconstriction and can lead to myocardial ischemia in susceptible patients.

Case Description. A 48-year-old man with severe bilateral PA refractory to medical therapy underwent unilateral adrenalectomy of the dominant adrenal. Although computed tomography (CT) showed three left-sided cortical adenomas, post-surgical histopathology and genetic analysis revealed five different adrenocortical adenomas. Two zona fasciculata (ZF)-like aldosterone producing adenomas (APAs) each harbored distinct known somatic KCNJ5 mutations (L168R and T158A). A zona glomerulosa (ZG)-like APA harbored a known CACNA1D G403R somatic mutation, while a zona reticularis (ZR)-like adenoma, which was a so-called black adenoma (pigmented appearance) with histologic characteristics more associated cortisol-producing adenomas, expressed CYP11B2, CYP17 and DHEA-ST by immunohistochemistry (IHC) and harbored no known somatic mutations. The fifth tumor was ZF-type, negative for CYP11B2 and CYP17 IHC, and harbored no known somatic mutations.

Conclusions. This case highlights complex intra-patient heterogeneity in histology, steroidogenesis and somatic mutations in multiple adrenocortical adenomas arising in a patient with PA. The extent of multiclonal adenoma involvement in PA, including apparent unifcoal tumors by morphology, warrants additional study.

Primary aldosteronism is mainly caused by either unilateral aldosterone-producing adenoma (APA) or bilateral adrenal hyperplasia (BAH). Usual diagnostic approaches in patients with primary aldosteronism are focused on differentiating unilateral APA and BAH. Bilateral adrenal cortical adenomas in the setting of primary aldosteronism are very rare, but possible.

Clinical case

A 48-year-old man was referred to our hospital with hypokalemia and poorly controlled hypertension despite four antihypertensive medications. Physical examination showed blood pressure of 180/100 mmHg. The serum potassium level was 3.0 mEq/L, arterial blood gas analysis revealed metabolic alkalosis. The Plasma aldosterone concentration (PAC) was 57.62 ng/dL (normal 1.3 to 14.5), and the Plasma rennin activity (PRA) was 0.26 ng/mL/hr (normal 0.32 to 1.84) with a calculated aldosterone to renin ratio was 221.6. Intravenous saline infusion test for confirmation failed to suppress plasma aldosterone level significantly (pre-infusion PAC: 35.9 ng/dL, post-infusion PAC: 58.5 ng/dL), consistent with a primary aldosteronism. Upright posture for 240 minutes suppressed plasma aldosterone concentration. (Basal PAC: 50.1 ng/dL, after upright PAC: 38.7 ng/dL). Abdomen computed tomography (CT) demonstrated a 1.5 cm sized right adrenal mass and a 1.2 cm sized left adrenal mass both with less than 10 Hounsfield unit (HU) on pre-contrast image. The adrenal venous sampling was performed and revealed the following adrenal vein - inferior vena cava (IVC) ratio of cortisol was greater than 10, right - left adrenal vein ratio of aldosterone - cortisol ratio was 5.56, left adrenal vein - IVC ratio of aldosterone - cortisol ratio was 0.64. Although the right-sided hyper-functioning mass was suspected in the adrenal vein sampling, we planned to explore both adrenal glands due to discrete masses were existed on both adrenal glands on CT. Right total adrenalectomy and left partial adrenalectomy were performed by laparoscopic approach. The pathology confirmed the bilateral adrenal cortical adenomas with marked cytologic atypia and the sections from both sides shared a nearly identical appearance. 7 days after surgery, laboratory finding showed the normal basal PAC of 0.68 ng/dL, after intravenous saline infusion PAC was 0.23 ng/Dl and PRA was 0.2 ng/ml/hr. 14 Days after surgery, the patient was normotensive (130/80 mmHg) on a calcium channel blocker and a angiotensin ll receptor blocker (Nefidipine 90 mg, fimasartan 30 mg a day), and the serum potassium was 4.5 mEq/L without any potassium supplement.

Conclusion

In this case, unilateral hyperfunction was suspected in the adrenal vein sampling, but bilateral adrenal adenomas was presented on the CT and then confirmed by pathology. Clinicians should consider the possibility of bilateral adenomas in diagnosis and treatment with primary aldosteronism.

This case highlights the many difficulties associated with investigations and management of suspected primary hyperaldosteronism in pregnancy. Outside of pregnancy, the accepted screening test is the aldosterone to renin ratio, with confirmatory tests including a saline suppression test, captopril challenge or salt/fludrocortisone load. However, these tests lack validated pregnancy reference ranges or are contraindicated. Pregnancy has significant effects on the renin-angiotensin-aldosterone pathway leading to physiologic elevations in both aldosterone and renin: progesterone competitively binds to aldosterone receptors; ovaries and placental tissues secrete renin; estrogen stimulates angiotensinogen production. While primary hyperaldosteronism has been associated with poor pregnancy outcomes, optimal management in pregnancy is not clearly established. Conservative treatment with traditional antihypertensives, cautious use of mineralocorticoid receptor antagonists, or surgical adrenalectomy are all possible treatment options reported in the literature.

Clinical case: A 39 y.o. male with Congenital Adrenal Hyperplasia (CAH) (simple virilizing form, that was diagnosed as a neonate) was studied; his twin with the same diagnosis, died before 5 y.o.

He was treated with oral Hydrocortisone in different schedules. At 29 y.o., high blood pressure was detected and dose is reduced to 10 mg/day. Finally, he discontinued Hydrocortisone 6 months before admission. In the last 2 years, abdominal and left lumbar pain appeared. Abdominal ultrasound reported enlarged adrenals and the CT-scan showed bilateral adrenal masses of 16 cm at right and 24 cm at left (large diameter), with features of myelolipomas. He was admitted in our center for surgical resolution. At physical exam he was dehydrated with low blood pressure and tachycardia. Height: 147cm, pigmented skin, palpable masses in the upper abdomen. Clinically, impressed like adrenal insufficiency, serum sodium: 131 mEq/L and potassium: 4.7 mEq/L. The early morning Cortisol was 61.6 ug/dl. After that, we continued the study performing adrenal steroids determinations. Free urinary Cortisol: 924 µg/24h, plasma ACTH: 1019 pg/ml, Aldosterone: 100 ng/ml, plasma renin activity: 36 ng/ml/h, 17 OH Progesterone: >20 µg/dl, Total Testosterone: 1740 ng/dl.

These results suggested GCRS. Then, an evening dose of 1mg oral Dexamethasone was started. The morning cortisol decreased to 19.9 ug/dl and ACTH to 116 pg/ml. His general condition improved, serum sodium and potassium normalized. Sellar MRI was normal and testicular ultrasound showed bilateral adrenal rests. Bilateral adrenalectomy was performed. The histology confirmed bilateral myelolipomas. Cortisol at one week was undetectable.

Molecular analysis did not find a mutation in the GR but, a variant in theNF1 gene (NF1 (NF1) ADp.P678A c.2032_2034delCCGinsGCA). Nevertheless, there were not obvious manifestations of neurofibromatosis.

Conclusions: GCRS is very rare. It can be confused with CAH. It should be suspected in all patients with elevated cortisol and ACTH without signs of hypercortisolism. We do not know what role if any the NF1 gene mutation played in this case, but there are several non-GR-mutant cases of GCRS.

Bilateral adrenal macronodular hyperplasia (BAMH) is a rare form of Cushing's syndrome characterized by the presence of bilateral cortisol-secreting adrenal nodules and hypercortisolism. Familial Adenomatous polyposis(FAP) is an autosomal dominant disease resulting from an inactivating germline mutation of the tumor suppressor gene APC. The association of these two syndromes is rare and it is possible that they share common pathophysiologic pathways.

Case Presentation.

A 68 yo female presented with plethora, lower extremity edema, progressive proximal myopathy, centripetal weight gain, recent diagnosis of type 2 DM, hypertension and metabolic alkalosis with hypokalemia. She had severe osteoporosis with multiple thoracic vertebral compression fractures. Laboratory evaluation revealed elevated 24 hour urine cortisol ranging from 177 – 227(4 - 50 mcg/24h), cortisol level 20.6 mcg/dL after administration of 1 mg of dexamethasone, with ACTH level < 5 pg/ml. There was no evidence of hyperaldosteronism. Abdominal CT revealed enlarged adrenal glands with multinodular appearance with maximum diameters of 6.5 x 2.6 cm on the right side and 6.9 x 3.4 cm on the left side, pre-contrast attenuation of -3 and 10HU. The patient underwent bilateral adrenalectomy confirming a multinodular cortical hyperplasia with a left adrenal gland weighing 76 grams and right adrenal gland weighing 42 grams. Four weeks following surgery, she underwent a colonoscopy for evaluation of hematochezia, revealing 10 to 15 pediculated polyps averaging 5 to 20 mm and involving the recto-sigmoid area. A fungating, polypoid non-obstructing large mass was biopsied. Histopathology reported tubulovillous adenoma with high-grade dysplasia with immunohistochemistry positive for MLH1, MSH2, MSH6, PMS2 compatible with microsatellite stability. Discussion

BAMH is a rare cause of Cushing Syndrome(CS), accounting for less than 1% of adrenal CS.(1) Cushing syndrome in BAMH may result from intra-adrenal corticotropin signaling, from aberrant adrenal expression of ectopic receptors or increased activity of eutopic peptide hormone receptors(2). BAMH has been reported in patients with hereditary leiomyomatosis, renal cell cancer, MEN-1 and McCune–Albright syndrome (3). The medical literature reveals 3 other cases of BMAH associated with familial adenomatous polyposis. Yamakita et al reported a case of BAMH associated with multiple colon carcinomas. Gaujoux et al isolated a heterozygous germ line deletion (c.1863_1866del4/p.Thr621fsX628) in a patient with BAMH and FAP, while Hsiao et al described a heterozygous APC gene mutation(4393_4394delAG)in a patient with BAMH and colonic polyps. Our case illustrates the association between colonic polyposis and BAMH. Physicians should strongly consider systematic familial screening with genetic testing for APC in similar clinical presentations.

Clinical Case: 51-year-old male presented to the emergency room after a motor vehicle accident with left lower quadrant abdominal pain. On computed tomography (CT) scan of abdomen and pelvis it was noted that he had a contusion of the sigmoid colon as well bilateral lobular heterogeneously enhancing right greater than left adrenal masses, with patchy and rim-like areas of calcifications in both adrenals. The size of the right measured 9.5 x 5.1

x 6.1 cm and left measured 3.2 x 2.8 x 3.2 cm.

Biochemical work up revealed normal cortisol dynamics, no evidence of hyperaldosteronism or pheochromocytoma. However he was found to have slight elevation in urine dopamine levels that were less than two times the upper limit of normal.

CT guided biopsy of the right adrenal mass showed mature neural stroma with mixed ganglion cells consistent with a ganglioneuroma. After discussion with the patient and in tumor board, conservative management was chosen. A follow up CT scan three months later showed stability of the adrenal masses.

Although rare, adrenal ganglioneuromas can be found incidentally on imaging studies. Diagnosis is proven by tissue pathology, as there is a vast differential diagnosis once an adrenal mass is found on radiographic imaging. AGN are often hormonally silent or may sometimes secrete hormones. Such hormones include testosterone or if it is a composite tumor with pheochromocytoma, it may secrete catecholamines. In such cases, the diagnosis is challenging.

Conclusion: AGN needs to be considered in the differential diagnosis of an adrenal incidentaloma. Careful history, physical exam, biochemical testing, along with pathological examination is fundamental in making a diagnosis. AGN are benign and usually asymptomatic but need to be monitored for changes in size.

Clinical Case: A 60 year old woman with hypothyroidism presented to her primary care provider with abdominal pain. Abdominal exam revealed distension with discomfort on palpation. No organomegaly or masses were identified. A CAT scan of the abdomen and pelvis with contrast was done which revealed ascites, peritoneal masses and a large left ovarian mass suspicious for ovarian malignancy. Incidentally, a left adrenal 4.3 cm mass was also identified. She underwent total hysterectomy and bilateral oophorectomy. An omentectomy, peritoneal mass biopsy, and lymph node sampling were also performed. The ovarian mass was diagnosed to be a fibroma. However, the peritoneal biopsies, revealed poorly differentiated carcinoma consistent with ACC.

She was found to have normal plasma metanephrines and normetanephrines at 0.15 and 0.88 nmol/L respectively. Urine metanephrines and normetanephrines were elevated mildly at 0.91 and 0.71mg/day perhaps due to the stress of the diagnosis. Morning cortisol was normal at 18 μg/dL and the ACTH was also normal at 49 pg/mL. Her electrolytes were normal except for mild hyponatremia. Sodium was 132mmol/L and potassium was 4.2 mmol/L. The patient was started on chemotherapy with etoposide, cisplatin, and doxorubicin along with mitotane. The patient was not able to tolerate the side effects of mitotane and chose to only be on platinum based chemotherapy agents.

Conclusion: This case highlights that patients with non-functioning ACC can present with clinical manifestations of tumor growth such as abdominal or flank pain or with constitutional symptoms like weight loss, anorexia. They may even be detected incidentally on imaging performed for a different reason. One needs to have a high index of suspicion to diagnose ACC given the low incidence rate. A careful history and physical examination should be performed to exclude pheochromocytoma, hyperaldosteronism, hyperandrogenism, and Cushing's syndrome. Complete surgical resection is the treatment of choice, but in cases with metastasis, chemotherapy and mitotane is needed. Five-year survival is approximately 45 to 60 percent for early stage disease, and 10 to 25 percent for advanced stage disease. As a result early diagnosis is key to better outcomes.

Clinical case: A 57-year-old man was referred to our endocrinology clinic for persistent hypertension and hypokalemia. Laboratory tests showed serum sodium levels varied between high normal and high with persistently low serum potassium (in the absence of potassium depleting diuretics), plasma renin activity (PRA) of 0.28 ng/ml/hour, plasma aldosterone concentration (PAC) of 36.6 ng/dl and markedly elevated PAC/PRA ratio of 130.71 ng/dl per ng/ml/hr. CT abdomen/pelvis showed a 4.3 cm mass in the left adrenal gland. Repeated CT scans showed marked interval enlargement, suggestive of an adrenal carcinoma. The patient underwent left adrenalectomy. Pathology showed a 7 cm organ-confined, necrotic adrenocortical carcinoma with clear margin, large vessel (muscular venous) vascular invasion and Ki-67 of <10%. No evidence of regional lymph node or distant metastasis was identified. Plasma aldosterone became undetectable after the operation. Adjuvant therapy was not started as it was categorized as stage II, Ki-67 was < 10% and resection margin was negative. However, a year following surgery the patient developed a nodule in the bed of the resected left adrenal gland. Another resection was performed and pathology showed recurrent adrenocortical carcinoma partly evident within a distended muscular vein. Mitotane was started since then.

Conclusion: Therefore, we propose that vascular invasion and necrotic state of the tumor might be associated with higher risk of recurrence and should therefore be considered in determining the need for adjuvant chemotherapy, in addition to the criteria based on the international consensus. This case provides evidence to support Weiss criteria, which takes into account mitotic count, tumor necrosis and vascular invasion status in predicting recurrence.

Clinical case: A 19 year-old man presented to the ER complaining of abdominal pain and nausea. Initial CT abdomen/pelvis showed a large right adrenal mass measuring 11x10x16cm with calcifications. Initial endocrine work up was remarkable for slightly elevated plasma and urinary normetanephrines, 1.20 nmol/L (0-0.89) and 673 μg/d (95-379) respectively, with normal metanephrines. Morning baseline cortisol was 22 mcg/dL (7.0-22.0) with ACTH of 31 pg/mL (7-69). A 1mg-dexamethasone suppression test revealed a morning cortisol of 20.2 mcg/dL (dexamethasone level of 427 ng/dL). PRA and aldosterone, potassium, total testosterone and SHBG were within normal limits. DHEA-S value was highly elevated at 1168 μg/dL (88-483). Patient also had normal TSH, FT4, calcium, iPTH, and creatinine. Calcitonin and chromogranin A were negative. Repeat CT abdomen/pelvis confirmed a large right adrenal mass with invasion of the inferior vena cava, hepatic veins and right atrium. CTA chest revealed bilateral pulmonary emboli and innumerable nodules suggestive for metastasis. Echocardiogram revealed a 4.7cm non-mobile mass obstructing the right atrium. Considering the stage of the disease, no surgery was recommended by surgical oncology, cardiothoracic and vascular surgery. CT-guided biopsy of the adrenal mass was performed after alpha and beta blockade. Pathology showed poorly differentiated ACC. Immunohistochemichal profile was positive for MelanA, and focally positive for Synaptophysin and Inhibin, negative for Calretinin and S-100. Patient was initiated on adjuvant mitotane monotherapy. After 1 week of mitotane, DHEA-S level went down to 166 μg/dL. Cortisol levels also decreased due to the adrenolytic effect of mitotane. Patient was initiated on glucocorticoid replacement with 30mg daily. PRA was in the normal range and there was no need for mineralocorticoid replacement. Palliative chemotherapy with cisplatin/etoposide/doxorubincin was also offered to the patient but was decilined.

Conclusion: ACC is an aggressive, rare and heterogeneous tumor with poor prognosis, particularly if it occurs in children/young adults. It is essential to differentiate it from benign adenomas by correlating with clinical, biochemical, imaging and histological features, as the outcome can vary greatly. Also because of the increased incidence of genetic mutations in this population with ACC, genetic testing for TP53 germline mutation should be considered.

A 66-year-old female presented to the emergency department with acute onset chest pain, vomiting and back pain. She was hypotensive and tachycardic. Electrocardiogram showed sinus tachycardia and new anterior ST segment elevations. Laboratory evaluation revealed markedly elevated troponin. She also had acute kidney injury, acute liver injury with elevated transaminases >1000 IU/L (reference <52 IU/L) and elevated lactate. She underwent emergent cardiac catheterization showing normal coronary arteries. Echocardiogram revealed reduced ejection fraction (EF) of 13% with severe hypokinesis of the anterior, lateral and inferior wall. Takotsubo cardiomyopathy was diagnosed although she did not report any acute physical or emotional stress. Inotropic support and afterload reduction led to clinical improvement. Cardiac imaging 6 days later demonstrated dramatic improvement of EF to 64%. An abdominal ultrasound performed on presentation to evaluate the abnormal liver function revealed a right upper quadrant mass of uncertain origin. This prompted abdominal computed tomography which showed an 8.5 x 8 x 7.5 cm heterogeneous adrenal mass. On further questioning, the patient reported a 3-year history of debilitating episodes of substernal pressure associated with belching, vomiting, and palpitations. Her symptoms had been diagnosed as gastroesophageal reflux disease. PCC had not been considered since she was always normotensive. Hormonal testing to evaluate this mass revealed elevated total plasma free metanephrines 20,878 pg/ml (<205 pg/ml) with normetanephrine 17,279 pg/ml and metanephrines 3599 pg/ml. Catecholamine excess from PCC was considered to be the cause of her TC. Doxazosin was titrated over 3 weeks for adequate alpha blockade and propranolol was added prior to surgery to control reflex tachycardia. She underwent a successful open adrenalectomy. Pathology confirmed PCC.

Pheochromocytoma is a rare disease with variable presentation. Takotsubo cardiomyopathy is an increasingly recognized, yet still uncommon, presentation of PCC. In our patient, the diagnosis of PCC was entertained only after an adrenal mass was incidentally found on imaging. We suggest evaluation for PCC in patients with TC even in the absence of hypertension, as a missed diagnosis can have fatal consequences.

Pheochromocytoma is a rare tumor from chromaffin cells of the adrenal medulla first described in 1884 by Felix Fränkel. This tumor has an annual incidence of 0.8 per 100,000 person year.

CASE PRESENTATION

A 56-year-old Caucasian man went to the emergency room because of sudden left lower extremity pain radiated to the chest and back. The patient’s physical examination showed blood pressure (BP) of 110/70, heart rate of 112 bpm and diaphoresis, the rest was unremarkable. The EKG did not show signs of ischemia although troponin was elevated. Patient was sent to coronary catheterization which revealed normal coronaries and severe left ventricular dysfunction and ejection fraction (EF) of 20%. Twelve hours after the procedure the patient developed hypotension and shock requiring vasopressor support. Hemoglobin dropped from 15 g/dl to 9.4 g/dl. CT abdomen was done to rule out bleed and showed a large left retroperitoneal hematoma and a retroperitoneal mass (12x10x13 cm) displacing the kidney into the pelvis. Patient's clinical course progressed to severe acute kidney injury and anuria requiring temporal hemodialysis. An embolization of the left middle adrenal artery was done to stop the bleeding. Endocrine work-up of retroperitoneal mass resulted in normal values of 7-OH progesterone, androstenedione, DHEA sulfate, testosterone and cortisol. Urine and plasma metanephrines were consistently elevated in multiple occasions and the diagnosis of pheochromocytoma was made.

For preoperative treatment patient was started on alpha blocker (doxazosin 1mg daily) but patient had multiple episodes of hypotension. Given intolerance to alpha blocker pre-operative treatment consisted mainly of metyrosine 250mg every 6 hours before surgery. After 14 days of pre-op treatment he was taken to surgery to remove the mass. The pathology showed a benign pheochromocytoma. Patient improved clinically and his EF increased to 60%.

DISCUSSION

This case highlights a unique presentation and complication of pheochromocytoma and opens the possibility of different treatment modalities when patients cannot tolerate alpha blocker. Our patient presented with lower extremity and back pain with retroperitoneal hematoma and did not have any of the classic symptoms of episodic headache, sweating, tachycardia and hypertension. About 5-15% of patients can present with normal blood pressure like is the case with our patient. He also had a rare complication of pheochromocytoma which is dilated cardiomyopathy and it is due to catecholamine production by the tumor. Lastly our patient was not able to be treated with alpha blocker given recurrent episodes of hypotension so treatment was started with metyrosine which inhibits the production of catecholamines and is an uncommon treatment modality.

Clinical Case: The patient was seen in the emergency room prior to the index pregnancy for nausea, vomiting and intermittent, left lower quadrant abdominal pain. A CT scan of the abdomen and pelvis showed a 4.8 x 4.3 cm heterogeneously enhancing mass near the left adrenal gland and renal hilum. She reported episodic sweating for one year, palpitations weekly, and feeling anxious. Laboratory results were concerning for a norepinephrine secreting tumor. She had normal plasma metanephrines of 0.46 nmol/L (normal <0.49 nmol/L), but markedly elevated plasma normetanephrines of 7.12 nmol/L (normal <0.89). 24-hour urine epinephrine and dopamine were normal (<6 mcg/24 hrs and 218 mcg/24 hrs, respectively). Urine norepinephrine was elevated to 604 mcg/24 hrs (normal <90 mcg/24 hrs) and urine catecholamines were elevated to 610 mcg/24 hrs (normal <115 mcg/24 hrs). Chromogranin A level was elevated to 940 ng/mL (normal 0-95 ng/mL). Cortisol, DHEAS, renin, and aldosterone levels were normal. Soon after evaluation, the patient became pregnant. Her systolic and diastolic blood pressure were elevated during this time to 140 mmHg systolic and 100 mmHg diastolic. She was referred to endocrine surgery for resection. At 12 weeks of gestation, doxazosin was started. The mass was thought to be a paraganglioma, and removal was felt to be safest during the second trimester. Doxazosin dose was up titrated prior to surgery for blood pressure control. At 18 weeks of pregnancy, she underwent a robotic left retroperitoneal paraganglioma resection with excellent maternal and fetal outcomes.

Conclusion: Catecholamine secreting tumors in pregnancy are rare but must be recognized and treated to reduce maternal and fetal complications. Biochemical testing with 24-hour urine metanephrines and catecholamines is the same as for non-pregnant patients (1). Despite no formal guidelines for management of these patients, it is generally recommended tumors be removed either before 24 weeks of gestation or after delivery. Second trimester operative intervention is considered safest due to decreased risk of spontaneous abortion and less anatomic distortion (1). Medical management prior to surgery is approached similarly to non-pregnant patients. 

History of presenting illness: At the age of 53 years old, she presented to the gynaecological outpatient clinic with two weeks of left upper abdomen fullness, fatigue and weight loss. No reported history of headache, hypertension or sweating.

Past Medical/Surgical History: Right lobe thyroidectomy in 1983 for cosmetic reasons. Dilation and curettage procedure for investigation of infertility

Social/Family History: She was a non-smoker and had minimal C2H5OH intake. There was no family history of neuroendocrine tumours.

Medications (+ allergies): Eltroxin 100mcg OD and no drug allergies

Physical Examination: She was not tachycardic or hypertensive. Only positive findings were previous partial thyroidectomy scar and splenomegaly.

Report of laboratory and imaging studies: 

Summary of Relevant investigations

• CT TAP - 18cm adrenal mass with calcification at the centre appearing to arise from the left adrenal gland. No evidence of lymphovascular invasion or distal metastatis
• MRI Abdomen - 18cm left adrenal mass with left kidney pushed inferiomedially by the mass. Lesion encapsulated with no local extension. Suggestive of adrenal carcinoma
• 24 hour urinary catecholamine collection - adrenalin 24 nmol/24 hours (0-230nmol/24housr), noradrenalin 160nmol/24 hours (0-290nmol/24 hours) and VMA 9.6 umol/24 hours (2.5-40 umol/24 hours)

Interventions and outcome: Left adrenalectomy with nephrectomy 2006. No intra-operative hypertension or complications. Histology confirmed phaeochromocytoma with malignant potential. It showed tumour composed of epithelioid appearing cells with abundant pink cytoplasm. Individual cells showed severe nuclear pleomorphism including numerous giant forms with prominent nucleoli. Occasional mitotic figures are identified. In addition, there is extensive necrosis. There is evidence of definite lymphovascular space invasion and capsule invasion. A rim of normal residual adrenal gland is identified. These appearances favor a malignant pheochromocytoma.The submitted kidney unremarkable. Immunochemistry testing confirmed neuroendocrine histogenesis/pheochromocytoma. MIB 1 proliferation index of 30-40%. is extremely high and strongly supports a diagnosis of malignancy Subsequent follow up over ten years has revealed negative MIBG scans, CT TAPs, urinary and plasma catecholamines and genetic screening tests. This case raises the issues of management and radiological and biochemical follow up of non functioning and malignant phaeochromocytomas and the role of genetic screening for such tumours. It also highlights the advances in diagnostics that have occurred over the last decade.

Clinical Case:A 59 year old woman with a history of hypertension (controlled with a thiazide and ACE inhibitor) and prediabetes presented after a motor vehicle collision. She was incidentally found to have a 4.5 x 3.8 x 3.4 cm heterogeneous, enhancing left adrenal mass on the CT scan of the abdomen. She endorsed occasional palpitations but denied headaches, tremors, and diaphoresis.

Blood pressure (119/73 mmHg) and heart rate (76 bpm) were normal. Troponin was elevated at 1.830 ng/ml (reference range: < 0.060 ng/ml) with concern for an anterolateral infarct with ST elevations on EKG. Medical therapy was initiated to treat for possible ischemic heart disease. Transthoracic echocardiogram showed a moderately reduced EF of 31-35% and findings suggestive of takotsubo cardiomyopathy. Cardiology did not perform coronary angiography as the patient was in traction due to C6 and C7 vertebral fractures from the MVC. Additional laboratory studies included plasma fractionated normetanephrines that were elevated at 27 nmol/L (reference range: 0.00 to 0.89 nmol/L) and 24h urine normetanephrines at 7088 ug/d (reference range: 109 to 393 ug/d). Alpha and beta blockade was initiated in anticipation of laparoscopic adrenalectomy. However, the surgery was delayed as the patient required cervical fixation of the vertebral fractures. Repeat transthoracic echocardiogram after 2 weeks of medical therapy showed an improved EF of 70% with resolution of the takotsubo cardiomyopathy. The patient subsequently underwent a left laparoscopic adrenalectomy with surgical pathology confirming a 6.5 cm pheochromocytoma.

Discussion: Takotsubo cardiomyopathy was first described in 1990 in Japan and occurs in approximately 1 to 2 percent of patients presenting with troponin-positive acute coronary syndrome (ACS) or ST-elevation myocardial infarction. This syndrome is characterized by transient regional systolic dysfunction of the left ventricle (LV), mimicking myocardial infarction, but in the absence of angiographic evidence of obstructive coronary artery disease or acute plaque rupture. In this case report we will review the literature describing the occurrence of takotsubo cardiomyopathy and pheochromocytoma as well as the treatment considerations of these two clinical conditions.

Oncocytic Adrenocortical neoplasms(OAN) are extremely rare. Only 150 cases of OAN have been reported in the literature. It is most frequently found as an incidental finding between the ages 27–72 years. It is more common in women and in the left gland [1-3]. We report a case of OAN in a 25 year old man with a 6.7 cm right adrenal incidentaloma producing dopamine and a positive MIBG scan.

Case Report:

25 year old Hispanic male with PMH of alcoholism and polysubstance abuse was referred to Endocrinology for palpitation, sweating and elevated BP of 169/111mmHg. ROS was negative. Family history was negative for pheochromocytoma. Vital signs and physical examination was normal. In 2012, CT abdomen showed an incidental 2.1 x 1.6 cm right adrenal lesion. In 6/2015 CT showed a 5.5 x 4 cm solid heterogeneous right adrenal mass. A 24 hour urine study showed elevated dopamine levels of 1276µg/24h(52-480). MRI in 10/2015 showed a 6.7 x 4.7 x 4.5 cm well circumscribed mass of the right adrenal gland with several irregular foci with T2 hyperintense signal compatible with necrosis. MIBG scan showed a focus of increased tracer uptake in region corresponding to lesion seen on MRI. After premedication with α blocker, he underwent right adrenalectomy. The pathology report showed predominant cells with eosinophilic and granular cytoplasm, high nuclear grade and diffuse architectural pattern consistent with OAN favoring an adenoma. Immnunohistochemistry was positive for MART-1, inhibin and calretinin, negative for S-100, Chromogranin, Oct4 and SF-1 consistent with adrenocortical origin. 24 hour urine for dopamine normalized 1 month after surgery.

Discussion:

Only 17% of OAN are functional adrenal masses [1-3]. Only 3 case reports of OAN mimicking pheochromocytoma have been reported. (4-6). Our patient presentation was unique with the tumor on the right side, with an isolated elevation of dopamine and false positive MIBG scan. OAN are classified according to Weiss criteria(7) which include major criteria (mitotic rate of >5 mitoses per 50 high power fields, atypical mitoses and venous invasion), minor criteria (large size >10 cm and huge weight > 200 g, necrosis, capsular invasion, sinusoidal invasion) and definitional criteria(predominantly cells with eosinophilic and granular cytoplasm, high nuclear grade, diffuse architectural pattern). The presence of any one of the major criteria indicates malignancy, one to four minor criteria indicates uncertain potential and the absence of all major and minor criteria indicates benign behavior. Routine imaging studies cannot be used to differentiate benign versus malignant oncocytic neoplasms. Microscopic criteria are able to identify precise histology and clinical behavior, so adrenalectomy is the mainstay of therapy.

CT imaging of pheochromocytomas almost always reveals precontrast attenuation of greater than 10 HU, are heterogeneous on venous phase imaging, and <50% washout on delayed phase. In fact, some authorities suggest that biochemical evaluation for pheochromocytomas is unnecessary when precontrast HU are <10.² We report a patient with a pheochromocytoma with precontrast HU of 2.

Clinical Case:

A 36 year old female was referred for an incidentally discovered left adrenal mass during a workup for groin pain and an enlarged lymph node. She reported an increasing frequency of paroxysmal episodes consisting of diaphoresis, palpitations, flushing and increased systolic hypertension (up to 160 mm Hg) that lasted for 1 hour and spontaneously resolved. These episodes started 2-3 years ago and increased in frequency to 2-3 times per week over the past year. On exam her BP was 140/90 mm Hg with no orthostasis. Her past medical history included anxiety and hypertension, diagnosed at 21 years of age treated with lisinopril.

24 hour urine normetanephrines were 3412 ug/24h (82-500), metanephrines 160 ug/24h (45-290), norepinephrine 447 ug/24h (0-135). On CT imaging at our facility the left adrenal mass measured 4.8 cm x 1.8 cm x 4.2 cm with homogeneous non-contrast attenuation of 2HU, heterogeneous attenuation of 80HU in portal venous phase while 15 minute delayed attenuation of 20HU. Absolute washout was 76%.

History, and biochemical assessment confirmed a pheochromocytoma despite imaging of the left adrenal mass with HU <10. She was optimized on alpha- and beta-adrenergic blockade prior to left adrenalectomy. Pathology showed an 8 cm x 2.5 cm x 2 cm pheochromocytoma extending into the periadrenal adipose tissue with invasion into the capsule. In the immediate postoperative period the patient did not require anti-hypertensive medications.

Conclusion:

Clinical suspicion and laboratory assessment indicated our patient had a pheochromocytoma, while the diagnosis of a pheochromocytoma was less likely based on non-contrast attenuation data published in the peer reviewed medical literature. This is a reminder that while 87-100% of pheochromocytomas exhibit precontrast attenuation HU of >10¹, rare cases present with HU<10.

Pemphigus vulgaris is an epidermal disease affecting the skin and mucous membranes caused by autoantibodies to the desmosomal protein desmoglein ultimately resulting in acantholysis and blistering with complement activation and release of inflammatory mediators [1]. Multiple studies suggest that non-neuronal adrenergic/cholinergic systems play a pathophysiologic role in several dermatoses, including pemphigus [2]. Pheochormocytomas are catecholamine-secreting tumors that arise from chromaffin cells of the adrenal medulla leading to a hyperadrenergic state.

Case Presentation

The patient is a 39 year-old female with new onset hypertension, palpitations panic attacks, anxiety and headaches. She had history of pemphigus vulgaris diagnosed by skin biopsy revealing intraepidermal acantholysis and linear/granular IgG and C3 antibody epithelial surface deposits on immunofluorescence with no IgA or IgM antibodies. She was treated with mycophenolate mofetil without adequate response. She underwent a CT of the abdomen for evaluation of nephrolithiasis  that revealed a 3.4 cm left adrenal tumor with no signal dropout on out of face imaging and high T2 weighted intensity strongly suggesting a pheochromocytoma. Hormonal evaluation revealed elevated 24 hour urine metanephrine 841 mcg/24h (normal <190 mcg/24h), normetanephrine 1192 mcg/24h (normal < 482 mcg/24h) and total metanephrines 2033 mcg/24h (normal < 695 mcg/24h). Aldosterone, renin, TSH, comprehensive metabolic profile and urine free cortisol where unrevealing. Her PTH was elevated at 81 pg/mL, calcium of 10.1 mg/dL, with normal phosphorus, albumin and vitamin D level. The diagnosis of normocalcemic hyperparathyroidism was entertained raising the question of a multi-gland neoplastic syndrome currently under investigation.

The patient underwent successful laparoscopic left adrenalectomy. Pathology revealed a left 3.8 cm pheochromocytoma. Within a week, her blood pressure normalized and surprisingly, her pemphigus spontaneously resolved.

Discussion and conclusions

Multiple studies suggest that the epidermal-adrenergic/cholinergic signal pathway controls calcium homeostasis, cell growth, differentiation, motility and pigmentation via B2 and a1 adrenoreceptors [2]. In addition, patients with may also have anti acetylcholine receptor antibodies [3]. Research in mice has shown that these non-desmoglein antibodies can induce pemphigus-like lesions [4].

These findings, along with the striking clinical improvement seen in our patient suggest that catecholamines may play an important role in the pathophysiology of pemphigus vulgaris. Paraneoplastic pemphigus has been described associated with multiple types of tumors [5-7]. To the best of our knowledge this is the first case report of refractory pemphigus spontaneously resolving after resection of a pheochromocytoma.

Case: A 41 year old African-American male with past history of polycythemia vera, hyperlipidemia and CAD was incidentally found to have a palpable non-tender left-sided abdominal mass in setting of normotension by his hematologist during a routine follow up visit. A CT abdomen/pelvis revealed a left retroperitoneal 14.8 x 12.0 x 17.0 cm mixed cystic and solid mass of uncertain origin with calcific focus, displacing the surrounding organs. An FNA biopsy was done without complication, which showed neuroendocrine tumor consistent with pheo/paraganglioma with atypical features. Biopsy sample stained positive for chromogranin A, synaptophysin, CD56, GATA3 and focal positivity for S100. There was no increase in mitotic activity or necrosis and Ki-67 proliferation index was <5%. At this point, the patient was referred to endocrinology and endocrine surgery services. Home medications included carvedilol 3.125mg BID and Lisinopril 2.5mg daily for cardioprotection in setting of CAD. The patient denied ever having hypertension, dizzy spells, sweating, palpitations, headaches, chest pain, fainting, or vertigo. No family history of pheo/para. Biochemical work up revealed 24-hour urine: metanephrines 2410 (52-341 mcg/24hr), normetanephrines 3670 (88-444 mcg/24hr), norepinephrine 38 (15-80 mcg/24hr), epinephrine 25 (0-20 mcg/24hr) and dopamine 7874 (65-400 mcg/24hr); plasma: metanephrine 750 (<57 pg/mL), normetanephrines 3519 (<148 pg/mL), norepinephrine 751 (0-600 pg/mL), epinephrine 86 (0-90 pg/mL), and dopamine <20 (0-35 pg/mL). He was started on phenoxybenzamine for blockage and underwent adrenalectomy without complications. Pathology revealed a 20 cm pheochromocytoma without extension into surrounding soft tissue or lymph node involvement.

Conclusion: This case illustrates the rare presentation of a giant asymptomatic hormone-secreting pheo. While large pheochromocytomas can be asymptomatic, they are usually known to have lower secretory function. There is very little literature on any pheos greater than 20 cm with elevated hormone levels that are asymptomatic. Interestingly, this patient did not go into catecholamine storm while on beta blocker therapy without simultaneous alpha blockade or while the mass was manipulated during initial biopsy, prior to its classification as a pheo. The extremely large size of the tumor, degree of metabolic activity and absence of end organ damage or metastatic disease, as seen in this patient, may be attributable to biochemically inactive hormone production.

Pheochromocytoma can cause hypertension and other non-specific symptoms. The associated clinical picture depends on the amount and pattern of catecholamine release. Most pheochromocytomas present as adrenal incidentalomas with biochemical evidence of catecholamine excess. The typical management of these tumors has been surgical resection.

We present a case of pheochromocytoma with low secretory activity in a hypertensive elderly patient in whom surgical resection of the tumor did not have any clinical impact on patient’s symptoms or hypertension.

Clinical case:

A 73-year-old African American male was referred to the endocrinology clinic for evaluation of a 3.2 cm left adrenal incidentaloma. He did not have any complaints of episodic headaches, palpitations, sweating or chest pain. He had hypertension for 10 years, which was well controlled on Lisinopril 10 mg daily, Carvedilol 6.25 mg twice daily and Amlodipine 10 mg daily. There was no history of weight gain, excess hair growth, acne or proximal muscle weakness.

On examination, pulse was 81 beats/min and blood pressure was 109/74 mmHg. The rest of his physical examination was unremarkable.

MRI showed a 3.2 cm lipid poor, left adrenal mass.

Biochemical tests showed: plasma aldosterone (15.4 ng/dl), plasma renin (0.4 ng/ml/hr), aldosterone/renin ratio (38.5), DHEA-S (77 ug/dl, n 28-175 ug/dl), low-dose dexamethasone suppression test (cortisol after 1 mg dexamethasone 1.9 ug/dl), plasma metanephrine (0.24 nmol/l, n <0.49 nmol/l), plasma normetanephrine (5.3 nmol/l, n <0.89 nmol/l), 24-hour urinary metanephrine (218 ug/d, n 30-350 ug/d) and 24-hour urinary normetanephrine (1780 ug/d, n 50-650 ug/d).

Further testing showed: plasma aldosterone (13.4 ng/dl), plasma renin (0.4 ng/ml/hr), aldosterone/renin ratio (33.5), saline infusion test (aldosterone level after infusion of 2 liters of 0.9% saline IV over 4 hours was 5.6 ng/dl), late night salivary cortisol (0.134 ug/dl, n < 0.228 ug/dl), plasma metanephrine (0.15 nmol/l, n <0.49 nmol/l), plasma normetanephrine (3.56 nmol/l, n <0.89 nmol/l). Repeated plasma normetanephrine level at different occasions was (3.42, 3.51 nmol/l).

Although being asymptomatic, patient elected to proceed with surgery. He was prescribed doxazosin 2 mg and carvedilol 12.5 mg twice a day prior to the surgery. He underwent left adrenalectomy with no complications. Surgical pathology showed pheochromocytoma.

After surgery, he continued to require carvedilol 6.25 mg twice daily, amlodipine 10 mg and lisinopril 10 mg daily to control his blood pressure. Biochemical testing after surgery showed: plasma normetanephrine (0.99 nmol/l, nl <0.89 nmol/l).

Conclusion:

Pheochromocytoma with steady low catecholamine release may not be the cause of hypertension. Close monitoring of such cases with controlled hypertension might be an alternative option to surgery in otherwise asymptomatic patients.

Pheochromocytoma (PC) is a neuroendocrine catecholamine secreting tumor that originates from the adrenal medulla. We present a case of pheochromocytoma presenting initially with abdominal pain with extreme elevation of plasma normetanephrines. This case highlights the challenges in preoperative management of a patient with pheochromocytoma and associated with low normal blood pressure.

Case:

A 40 year old female with no medical history presented with complaints of vague right-sided abdominal pain of 2 weeks associated with hot flashes. Physical exam showed blood pressure of 115/85 mmHg and heart rate of 75 beats per minute. A Computed Tomography (CT) of abdomen without contrast showed a 6 cm soft tissue attenuation on the right adrenal. A follow up CT abdomen with contrast confirmed a 6 cm right adrenal mass with marked hypervascularity and no significant washout consistant with PC. A MIBG scan showed large solitary right adrenal lesion consistent with PC with no other abnormal MIBG activity seen. Lab evaluation revealed elevated plasma metanephrine of 0.78 nmol/L (0.00-0.49 nmol/L), plasma normetanephrine >50 nmol/L (0.00-0.89 nmol/L), urine metanephrine of 449 mcg/24hr (52-341 mcg/24h), urine normetanephrine 18233 mcg/24hr (88-444 mcg/24h), and urine epinephrine of 9 mcg/24hr (2-24 mcg/24hr). Blood pressure remained normal to low with a systolic blood pressure of 110-80 mmHg and diastolic blood pressure of 80-55 mmHg. Surgical resection of PC was contemplated with preoperative preparation with alpha blockade. As alpha blockers could further worsen the hypotension, the patient was observed in the hospital for 24-hours and therapy initiated with phenoxybenzamine 10 mg twice daily. Liberal fluid and dietary salt intake was encouraged. She was given sodium chloride tablets 1 gm three times daily. After one week of phenoxybenzamine, beta blockade was initiated with metoprolol 12.5 mg daily for tachycardia. The patient underwent laparoscopic right adrenalectomy without complications. Phenoxybenzamine and metoprolol were stopped after surgery and the patient was discharged 2 days later in stable condition. After 6 weeks, repeated work up showed low plasma metanephrine < 0.10 nmol/L (0.00-0.49 nmol/L) and plasma normetanephrine 0.30 nmol/L (0.00-0.89 nmol/L).

Conclusion:

We present a rare case of normotensive pheochromocytoma with extreme elevation of plasma normetanephrines and normal metanephrines which was managed successfully with pre-operative alpha blockade using phenoxybenzamine and beta blockers. This case highlights the challenges in the preoperative management of normotensive PC and demonstrates successful management with alpha and beta blockers without complications. In addition this case reinforces the need for prospective clinical trials to address the optimum management of normotensive pheochromocytoma.

Clinical case:
A 51-year old male presented on a weekend with chest pain. He had an 18 month history of headaches and flushing episodes. He is on treatment for hypertension and had no other significant past medical or family history. On examination he was in overt heart failure.

Results:
His troponin was 23,488 ng/L (0-39ng/L). Transthoracic echocardiogram showed a global left ventricular systolic dysfunction, ejection fraction of 10%. All walls were almost akinetic. Cardiac MRI excluded a myocardial infarction. CT aortogram excluded dissection, however an incidental indeterminate, lipid poor right adrenal lesion was found.
The adrenal lesion along with the presentation gave the clinical appearance of a pheochromocytoma. Plasma and urine metanephrines were requested, although they can take up to a week to be reported. Urine metadrenaline which were reported at a later stage were markedly elevated at 89,408 nmol/24h (0-2000 nmol/24h).

Management:
Acute heart failure was treated with diuretics. His case was discussed with the cardiac transplant centre in case he failed to respond to medical treatment. As the patient was in extremis, we had to proceed with coronary angiography without metanephrines results. In this setting, as a life saving measure, we started an alpha blocker and proceeded cautiously, expecting worsening of crisis and considered other agents (eg-nitroprusside infusion if needed). The coronary angiogram was uneventful and revealed normal coronary arteries.

Discussion:
This case highlights:
1. The necessity of multidisciplinary approach when dealing with possible pheochromocytoma. This case required the direct interaction of the consultant cardiologist, endocrinologist, radiologist and clinical biochemist to discuss the findings and plan the necessary investigations.
2. The need to carefully consider the pros and cons of an intensive intervention (coronary angiogram) in a patient with possible pheochromocytoma. This can be a lifesaving measure, but needed to be done before the results of the urinary/plasma metanephrines, especially as the patient presented over a weekend.
3. Urinary metanephrines require a full 24 hour accurate collection with restrictions. The analysis is only done once per week in our centre. Plasma metanephrines are often sent to a reference lab and therefore take more than 4 weeks to come back. This adds pressure on the handling of acute presentations like our case. We needed to ensure multi-disciplinary interaction and full engagement with patient, family to appreciate the uncertainties and impact on decision making.

 Prior records revealed admissions in 2008 and 2012 for left pleuritic chest pain. EKGs, cardiac enzymes and nuclear stress tests were negative. Echocardiogram revealed small pericardial effusion, and he also had a pleural effusion on chest X-ray. At that time, the patient had no signs of adrenal insufficiency with normal blood pressure and electrolytes. Adrenal insufficiency was diagnosed in 2013, when he presented with headache, hypotension, hyperpigmentation, hyponatremia and hyperkalemia. Plasma ACTH was 1250 pg/mL (9-52) and baseline cortisol <1.9 mcg/dL (normal 8am cortisol: 8-19) with no response to cosyntropin stimulation. The patient was started on hydrocortisone and fludrocortisones replacement, but he stopped these after several months.

 Autoimmune polyglandular syndrome (APS) type 2, is defined by the co-occurence of autoimmune adrenal insufficiency, thyroid disease and/or type 1 diabetes mellitus. Other manifestations include hypogonadism, vitiligo and pernicious anemia. So far, the definition does not include pericarditis. In the literature, there have been few reported cases of pericarditis associated with Addison’s disease. In APS, the occurrence of pericarditis prior to the development of adrenal insufficiency, as in this patient who only had type 1 diabetes when he presented with the initial two episodes of pericarditis, has only been reported once before. We propose that pericarditis can be the initial manifestation of APS type 2, and that the mechanism and pathophysiology of this is not currently understood.

Currently employed hydrocortisone replacement regimens for patients with adrenal failure insufficiently mimic the circadian rhythm of cortisol secretion, resulting in temporary hypo- and hypercortisolism. Robust biomarkers measuring cortisol exposure over weeks or months are lacking.

FKBP5 is a co-chaperone of hsp90 which regulates glucocorticoid receptor sensitivity. Previous studies on mice showed that chronic exposure to glucocorticoids (GC) results in loss of DNA methylation in the introns 1 and 5 of the FKBP5 gene in blood and other tissues. Four weeks of GC exposure reduce FKBP5 methylation in a dose-dependent manner. Moreover, demethylation of the FKBP5promoter was recently shown in human trabecular cells after dexamethasone treatment.

The aim of this pilot study was to evaluate the relevance of CpG methylation within the FKBP5gene as a possible long-term biomarker of cortisol exposure.

Materials and Methods

Twenty-one healthy adults (6 females, 15 males, median age: 24 years) were recruited. A high dose (250 mcg) ACTH stimulation test was performed to evaluate adrenal function in all subjects. Serum cortisol was measured prior to and 1h following the stimulation. In addition, blood samples were taken prior and 24h following ACTH-exposure. FKBP5 methylation in leukocytes was measured using pyrosequencing. The CpG-site analysed was located in the promoter region of FKBP5(chr6: 35,729,046; hg38).

Results

Methylation values ranged from 17.72% to 29,79% (mean = 24.14%) prior to the ACTH-test and 19.30% to 31.45% (mean = 25.73%) following the ACTH-test, meaning no significant change in average methylation was found. Basal FKBP5methylation before ACTH stimulation was negatively correlated with basal serum cortisol levels (r = -0.509; p = 0.04). Furthermore, a negative correlation was found between serum cortisol 1h and methylation 24h following ACTH-exposure (r = -0.588; p = 0.005) as well as between the post ACTH-test methylation status and the delta of serum cortisol (r = -0.626; p = 0.002). In addition, delta methylation and delta serum cortisol were negatively correlated as well (r = -0.537; p = 0.012).

Discussion

The negative correlation observed between FKBP5 methylation and serum cortisol supports previous studies, showing that FKBP5 methylation is altered by GC exposure. Average FKBP5 promoter methylation remained unchanged. Considering previous studies showing demethylation of FKBP5 after 2-4 weeks of GC stimulation, our findings suggest that FKBP5 methylation could be used as a a robust biomarker that reacts stronger to long term GC exposure than to a singular ACTH stimulus as used in our study. This hypothesis will be evaluated in future studies.

Methods: Retrospective review of hydrocortisone loading curves performed in adrenal insufficient patients between 2008 and 2015 in our institute. Serum cortisol levels were measured prior and 30, 60, 120, 180, 240 and 300 minutes following administration of the patient`s regular morning hydrocortisone dose. Dose adjustments were performed by the treating physician based on cortisol profile results. Metabolic data, including weight, blood pressure and lipid profile were documented before and after performance of the curve.

Results: Sixty five hydrocortisone loading curves were performed in 51 patients during the study period. Pertinent clinical data was available for 28 patients. Mean age 44.8±13.7 years, 43% males, average weight was 82.9±21.9kg, 36% were hypertensive and 25% had diabetes. Mean daily hydrocortisone dose was 23.1±7.3 mg. Based on cortisol curve results, the dose was decreased in 54% of patients, unchanged in 36% and increased in 11%. There were no statistically significant between-group differences in age, sex, starting dose, starting weight, diabetes and hypertension prevalence. The average dose change was -8.2mg in the dose reduction group and +1.5mg in the rest of the study population (p<0.05). The average change in blood pressure was -11.38/-9.13 in the dose reduction group as compared to +0.8/+2.9 in the rest of the study population (p<0.05). A statistically non-significant reduction in weight, LDL and total cholesterol levels was also noted in the dose reduction group.

Discussion: Our findings suggest that cortisol curves are an effective tool for hydrocortisone dose titration, leading to a significant reduction in blood pressure in this cohort.

Methods: 28 healthy adults, mean age 55 yr, 16 men, were subjected to a randomized, double blind, placebo-controlled cross-over study. They were made hypogonadal with leuprolide, and randomized to placebo or gender-specific sex steroid addback. Eucortisolemia was accomplished by ketoconazole and continuous cortisol infusion. Peptide infusion sessions and 10-min blood sampling (2200 h overnight to 1200 h the next day) comprised 4 separate randomly ordered double-blind continuous i.v. infusions of CRH, AVP, the combination or saline at maximally stimulatory doses. At the end of the 10 h infusion, a submaximal dose of the non-infused hormone was injected i.v. and blood sampling continued for 2.5 h. Data are mean ± SEM.

Results. Mean 10-h ACTH concentrations (ng/L) in the gender-combined analysis were: saline 32±4.6, AVP 29±4.6, CRH 67±6.2 and CRH-AVP 67±8.8: saline vs AVP P=NS; CRH vs CRH-AVP P=NS; CRH vsAVP P<0.0001. Secretagogues induced stable ACTH concentrations after 4-6 h. Comparable contrasts were obtained by deconvolution analysis. Secretory burst regularity and burst mode were similar among groups. Cortisol levels were higher in women than men (P<0.0001), and higher during CRH than AVP infusion (P<0.0001). ApEn was increased by AVP and CRH (P<0.0001). Pattern synchrony of ACTH and cortisol was diminished by CRH and CRH-AVP, but not by AVP. Gender and sex-hormone administration were not significant categorical variables. AVP injection after exposure to CRH yielded mean 2.5-h ACTH concentrations of 46±4.3, exceeding that after CRH or saline injection (26±3.3 and 24±3.6, respectively; P=0.002 and 0.001).

Conclusions. CRH infusion is more potent than AVP. Exogenous single and dual-peptide clamping does not interfere with pulsatile ACTH secretion, but yields higher secretory randomness putatively by deleting endogenous peptide feedforward control. Conversely, clamping with AVP, an inferably desensitizing ACTH secretagogue in this setting, resulted in endogenously alleged CRH pulses’ driving lesser ACTH secretion approaching saline infusion. As expected the increased forward drive caused increase of ApEn and cross-ApEn. Bolus AVP injection induced greater ACTH release, likely caused by the sensitization of the corticotrope by CRH infused in the preceding 10 h. Accordingly, this paradigm generates a framework for investigating AVP’s putative desensitization of CRH action, and CRH’s inferred potentiation of AVP action. These interactions may explain unexpected effects of certain neuropsychiatric stressors.

338 subjects in 4 groups were studied: healthy controls (HC)(n=243), oral contraceptive users (OCP)(n=31), patients with cirrhosis (n=38) and intensive care unit patients (ICU)(n=26). FC and TC were measured by LC-MS/MS and albumin by spectrophotometry (Quest Diagnostics Nichols Institute, San Juan Capistrano, CA), and CBG by ELISA (Biovendor, Ashville, NC).

Mean age was 48±17 y and mean BMI 28±7 kg/m². Mean albumin (g/dL) and CBG (μg/mL) were higher in OCP and HC than in cirrhosis and ICU [(albumin: OCP 4.6±0.3, HC 4.7±0.3 versus cirrhosis 3.5±0.9 and ICU 3.1±0.7, p<0.001) and (CBG: OCP 57±18 and HC 28±7 versus ICU 22±4 and cirrhosis 21±7, p<0.0001)]. CBG correlated weakly with albumin in HC (R=0.32, p<0.0001) and cirrhosis (R=0.46, p<0.02) but not in OCP or ICU. Mean random TC (μg/dL) was 18.9±8.5 in OCP, 14.7±6.6 in ICU, 11.5±7.3 in HC and 9.9±5.7 in cirrhosis. Mean FC (μg/dL) and % FC were highest in ICU subjects (FC: ICU 1.29±1.08, cirrhosis 0.59±0.47, HC 0.58±0.67 and OCP 0.33±0.15, p<0.01 vs. ICU; % FC: ICU 7.7±4.8%, cirrhosis 5.4±2.7%, HC 4.2±2.2% and OCP 1.8±0.8%, p<0.05 vs. ICU). TC strongly correlated with FC in the ICU, HC and cirrhosis groups and more weakly in the OCP group (ICU R=0.91, HC R=0.90, cirrhosis R=0.86, OCP R=0.70, all p<0.0001). Consistent with Hamrahian et al, ICU subjects with albumin <2.5 gm/dL had higher FC despite similar TC. In receiver operator curve (ROC) analysis in the HC group, an FC cutoff of 1.0 μg/dL predicted a TC of ≥18 μg/dL with 91% sensitivity and 98% specificity (AUC 0.98, p<0.0001). No HC subject with an FC ≥ 1.6 μg/dL had a TC < 18 μg/dL (67% sensitivity; 100% specificity). An FC cutoff of ≥1.0 μg/dL predicted a TC of ≥15.1 mcg/dL in ICU and ≥13.4 mcg/dL in cirrhosis with 100% sensitivity but only 92% and 83% specificity, respectively. In a separate group of 8 HC who underwent ACTH-stimulation testing, all had stimulated TC ≥ 18 μg/dL and FC levels ≥1.0 μg/dL, except one, who had a TC of 16.7 μg/dL with an FC of 1.4 μg/dl.

In conclusion, an FC of ≥ 1.0 μg/dL in this LC-MS/MS assay predicts a TC level of ≥ 18 μg/dL with maximum sensitivity and specificity. Clinicians should use caution in interpreting TC levels in patient groups with altered CBG levels, particularly in women on OCPs, given the variable resulting increase in CBG.

Subjects and Methods: The pegvisomant integrated clinical trial database includes data from 550 unique (non-duplicate) subjects (56% males) who received at least one dose of PEGV and 47 unique placebo subjects (51% males), all of whom were included in the safety analysis. Because subjects may have participated in one or more studies and received more than one dosage of PEGV, a total of 827 subjects (456 (55%) males, mean age 47 yrs., range 20 – 84) enrolled in 13 phase 2b, 3 and 4 clinical trials were included in the efficacy analysis. Studies were of variable length and only the studies with daily dosing were included. IGF-I normalization rates were defined as the percent of subjects who achieved an IGF-I value < 1.2 X ULN at least once at any time during the study, at the end of study, or based on last observation carried forward.

Results:Treatment duration ranged from 6 to 84.7 weeks and average PEGV dose was 7.8 mg to 22.6 mg/day. IGF-I normalization rates ranged from 19.4 to 94.7%, with the highest rate using daily therapy, longer study drug exposure and appropriate dose titration. Over 50% of acromegaly subjects had normalization of IGF-I within 2 weeks. IGF-I normalization was achieved in >50% of subjects in 9 studies, >75% of subjects in 7 studies, and >90% of subjects in 2 studies. Normalization of IGF-I was found to persist throughout 84.7 weeks of treatment. Average duration of the treatment, average dose, and proper PEGV titration appear to be related to the proportion of subjects with IGF-I normalization; adequate titration of PEGV was key for efficacy, as ~95% of subjects had IGF-I normalization with proper PEGV dose titration. Among PEGV patients, 83.8% reported Treatment Emergent Adverse Events (TEAE), and 8.4% discontinued due to AEs. Among PEGV female patient, 89.3% reported TEAE, and 11.6% discontinued due to AEs. Among PEGV male patient, 79.3% reported TEAE, and 5.8% discontinued due to AE. Common adverse events reported by > 10% of PEGV subjects included headache, nasopharyngitis, arthralgia, diarrhea, fatigue and back pain.

Conclusions: PEGV is found to be highly effective in normalizing IGF-I levels in subjects with acromegaly. The integrated data shows that full efficacy of PEGV is contingent upon adequate duration of exposure and suitable dose titration, which ultimately results in IGF-I control in the majority of subjects without evidence of drug tolerance. Review of safety data supports the positive benefit-risk profile.

Objective: To study the prevalence of adrenal changes in patients with CD and to correlate the findings with clinical characteristics at diagnosis and impact on postoperative outcome.

Methods: From 2007 to 2016, were evaluated 163 patients with ACTH-dependent Cushing's syndrome. Of these, we selected 89 cases with confirmed diagnosis of CD, who had CT (93%) or MRI of the abdomen/adrenal with imaging available for reassessment by a single radiologist with experience in adrenal diseases. Cases were excluded when imaging was not available for the re-evaluation or in cases with exams done out of our Service.

Results: There was a prevalence of 30% (27/89) of adrenal abnormal findings: 16 cases (18%) had hyperplasia of the adrenal glands (Hyperplasia Group: HPG) and 11 cases (12%) had unilateral or bilateral nodules (Nodules Group: NDG). In NDG, seven cases were unilateral with an average diameter of 16.6 ± 5.1 mm and 12.7 ± 13.5 Hounsfield unit (HU) and four cases were bilateral with a diameter of 17.6 mm ± 4.9 and 1.3 ± 6.9 HU. All nodules had sharp edges and 85% had a homogeneous texture. In relation to the hormonal profile, higher cortisol levels were found in HPG patients. Late-night salivary cortisol (LNSC): HPG: 2270.3 ± 4818.6 ng/dL (Reference [R]: <120 ng/dL); normal group (NG): 644.6 ± 660.4 ng/dL; NDG: 574.5 ± 453.0 ng/dL. 24h urinary cortisol (UC): HPG: 2994.7 ± 8041.3 µg/24h (R: 50-310 µg/24h); NG: 876.4 ± 864.4 µg/24h; NDG: 808.6 ± 856.9 µg/24h. Overall, remission rate after transsphenoidal surgery was 68% (59/87), similar in all subgroups of adrenal imaging. Recurrence was detected in 24% of cases in a median follow-up of 36 months: 12 cases in NG, two cases in HPG and one case in NG. Adrenal imaging (CT) was reassessed in six patients of NG (55%, 3 uni- and 3 bilateral): unilateral: one nodule reduced (21 to 9 mm), one described as hyperplasia and one case did not show nodule; bilateral (six nodes in three cases): four reduced (18.0 ± 4.5 to 14.3 ± 5.9 mm) and two (one patient without remission) increased in size (15.0 to 27.5 mm). Hyperaldosteronism and pheochromocytoma were excluded in the researched cases.

Conclusion: There was a higher prevalence of adrenal abnormalities in patients with CD, greater than observed in the normal population. Concentrations of LNSC and UC denote greater severity of Cushing's syndrome in the HPG. There was no impact on remission and recurrence rates compared to adrenal findings. The partial re-evaluation of these nodules during follow-up suggests that chronic stimulation of ACTH is responsible for the appearance of these changes.

Objective: The purpose of our study was to study BMI and its relationship with cardiovascular risk factors before and after surgical treatment in pediatric patients with CS.

Patients and methods: We selected patients biochemically cured after surgery of CS and with at least 1 visit following cure. Data were collected from 73 patients, 40 males (58%), median age of 12 years (IQR: 9-15 years) and median follow up time of 14 months (IQR: 11-27 months). Cushing disease was diagnosed in 58 patients and primary adrenal CS in 15 patients according to pathological diagnosis.

Results: Mean BMI z-score at diagnosis was 2.0±0.7 (range: -0.4-3.1), 8 (11%) patients had a normal weight, 23 (31.5%) were overweight and 42 (57.5%) were obese. Hypertension (HTN) was diagnosed in 68.5% patients, dyslipidemia in 41.4% and DM2 in 4%. BMI z-score correlated positively with the duration of disease (r: 0.23, P=0.04), HOMA-IR (r:0.58, P<0.001), and negatively with age (r:-0.39, P=0.03). At the last follow up after cure HTN was present in 3.3%, dyslipidemia in 18.8%, whereas no patient had DM2. BMI z-score at last follow up was 1.2±1.1 (range:-1.5-2.8) and positively correlated with HOMA-IR (r: 0.54, P=0.01).Importantly, while overall the BMI z-score improved from pre-operative to post-cure values (2.0±0.7 to 1.2±1.1, P<0.001) a large percentage of patients remained overweight (24.6%) or obese (28.7%).

There were no differences in BMI, HTN, dyslipidemia and DM2 according to the etiology of CS or gender. The estimated cumulative frequency of achieving a normal weight by Kaplan-Meier curve reached 50% of patients at 16 months (95% CI: 3.93-28.06) and 62% at 25 months. Patients with normal weight at final follow up compared with those without it had shorter duration of hypercortisolism (2.1±2.2 vs 3.6±2.6 years, P=0.03), lower BMI z-score at diagnosis (2.0±0.4 vs 2.6±0.2, P<0.001), lower HOMA-IR (4.9±3.4 vs 36.6±9.7, P<0.006) and higher mean BMI z-score change at 6 months (0.8±0.7 vs 0.2±0.09, P<0.001) and at first visit after cure (1±0.6 vs 0.1±0.1, P<0.001). Cox regression analysis demonstrated that only the change of BMI z-score at 6 months was an independent predictor factor for reaching normal weight (HR: 3.73, 95% CI: 1.02- 12.85; P=0.03) with high accuracy when using a cutoff of 0.5 on ROC analysis (AUC= 0.87 (0.72-1, P<0.001).

Conclusions: Pediatric patients with CS had a great risk of remaining obese or overweight associated with an increase of insulin resistance despite improvements after cure of their CS. A decrease in BMI z-score of – 0.5 by 6 months post-operatively is highly predictive of reaching a normal BMI; thus, efforts should be focused in these early phases post surgery for healthy diet and lifestyle.

Methods: We retrospectively studied all patients undergoing surgical treatment of CS under our protocols at the NIH Clinical Center from 12/2011 through 9/2016. Ectopic ACTH syndrome (EAS), Adrenal adenoma (AA) or Cushing ’s disease (CD) was diagnosed based on surgical pathology. Pts were considered to have HTN based on medical history, physical examination or if they were on oral antihypertensive agents (AHAs). After surgery pts remained inpatient for 7-15 days. Hydrocortisone (10-12mg/m2) was started when remission was confirmed, usually by post-operative day 4. Resolution of HTN was defined as systolic BP ≤120 mmHg and diastolic BP ≤80 mmHg off AHAs. BP values were compared between 1) the first morning after pre-operative inpatient admission from 0600-0800h, 2) the morning of discharge (DC), and 3) the morning of follow up (f/u) clinic visit(s).

Results: At baseline (BL) 30 (25 female) of 51 patients had a diagnosis of HTN and all received AHAs (25=CD, 4=EAS, 1-AA); each achieved remission. By DC, 12 were on no AHAs, 15 had a decrease in the number or dose of AHAs, and 2 had no change in AHAs. The median systolic value decreased from 130 to 125 mmHg with no change in diastolic values (p>0.05). Three pts had no further f/u.

Of 25 patients with 6 month f/u, 13 were taking no AHAs, 10 had a decrease in AHA number from BL, and 2 had no change to medications. Of note, one patient with no change was on an increased dose of hydrocortisone due to severe fatigue. There was a decrease in systolic (p=0.012, median 124 vs 130 mmHg) and diastolic (p=0.016, median 72 vs 75 mmHg) values from BL.

23 pts had 12 month f/u, including 2 who were not seen at 6 mo. Of these, 15 were on no AHAs; 5 had a decrease in medication number or dose. Of 3 pts with no change from BL, 2 were on a stable antihypertensive regimen from BL to 12 month f/u. One pt, who was on a decreased regimen at DC, restarted BL AHAs 3 months post-surgery due to rising BP. There was a decrease in systolic (p=0.0026, median 116 mmHg) and diastolic (p=0.0015, median 66 mmHg) values from BL. HTN fully resolved in 11 of the 23 pts.

Conclusion: While many factors affect BP, surgical remission of CS can lead to rapid improvement of HTN, requiring adjustment of antihypertensive agents as early as the first week after surgery. We recommend close monitoring of BP, especially in the first 15 days post operatively, patient education, and long term follow up for optimal treatment of hypertension in this population.

IN this study, we examined changes in serum DHEA-S in patients with prolactinomas who had normal HPA function and others with impaired function before and after medical therapy with dopamine agonist.

Methods: We included 30 patients (20-60 years of age; 34±10.2) with prolactinomas who had normal HPA function (23F / 7M) as well as 5 males (51 ± 15.1 years of age) with prolactinomas and impaired HPA function (Am cortisol of 2.3 ±1.9 ug/dL). Serum levels of cortisol, PRL and DHEA-S were measured repeatedly after dopamine agonist therapy. None of the 30 patients were known to have other illnesses or were taking medications known to influence PRL levels or adrenal function. Patients with impaired HPA function had other pituitary hormone deficits whereas those with normal HPA function had only gonadal dysfunction.

Results:  In all patients with normal HPA function serum PRL levels decreased gradually from a baseline of 421.8±859 to 30.8±40.3 at 3-6 months (P<0.01) and to 12.2±12.8 ug/L at 12 months (P<0.01). Concurrently, their serum DHEA-S levels decreased from 231.8±191.9 to 170.2±137 at 3-6 months (P<0.001) and to 119.1±61 ug/dL at 12 months (P<0.001). Even though the decline in mean PRL concentration between 3-6 and 12 months was modest (from 30.8 to 12. 2 ug/L) and of borderline significance (P=0.06) it was still associated with significant (P<0.04) decrease in respective DHEA-S levels. Discontinuation of therapy in some patients was associated with a rise in serum PRL and a parallel increase in DHEA-S levels. Although younger patients had higher serum DHEA-S levels, the aforementioned parallel changes in PRL and DHEA-S levels were observed in all patients irrespective of their age. However, despite marked elevation in serum PRL 374±196 ug/L in patients with impaired HPA function, the respective serum DHEA-S levels remained very low (28.4±24 ug/dL).

Conclusion: The data indicate that hyperprolactinemia results in elevation of serum DHEA-S levels that decrease with medical therapy. However, despite markedly elevated serum PRL levels, patients with impaired HPA function had low serum DHEA-S concentrations that remained as low when PRL levels were lowered with dopamine agonists. This indicates that the modulating influence of PRL on serum DHEA-S levels requires normal ACTH secretion. Thus, a normal age and gender adjusted serum DHEA-S level implies normal HPA function even in patients with hyperprolactinemia.

Methods: Patients with CD treated in 2 neurosurgical tertiary referral centers in Germany and patients with CD/CS who are members of the U.S. based Cushing’s Support and Research Foundation (CRSF) completed a self-developed survey inquiring about disease burden, coping strategies and timepoints when support was needed the most. Additionally, the degree of interest in different offers, e.g. internet-based programs and seminars, was assessed. Data were analyzed using SPSS. Answers provided in free-text fields were clustered and counted.

Results: 84 U.S. and 71 German patients answered the questionnaire. There was no difference between groups with regard to sex and age (p>0.05). Patients in both countries indicated to suffer primarily from common Cushing-related symptoms (e.g. weight gain, buffalo hump, skin problems), reduced performance and psychological problems (e.g. depression, anxiety). 48.8% of patients from the U.S. and 44.4% of the German patients stated that good medical care and skilled doctors helped them the most in coping with the illness. Support was needed to a greater extent before therapy by the U.S patients (63.1%) than by the German patients (45.1%) with p=0.035. The U.S. patients were significantly more interested in support groups (51.2% vs. 33.8%, p=0.035) and in courses on illness coping than the German patients (48.8% vs. 26.8%, p=0.008), who stated to prefer brochures (45.1% vs 20.2%, p=0.001). 89.3% of U.S. patients would attend internet-based programs compared to 75.4% of German patients (p=0.040). There were no differences between groups for the duration of and the willingness to pay for such a program, but U.S. patients would be willing to travel longer distances to attend a support meeting (p=0.027).

Conclusion: Patients in both countries need skilled physicians and long-term medical care in dealing with the effects of CD/CS, whereas the interest in specific topics addressed in support programs differs between patients of both countries. The latter implies that not only disease-specific but also culture-specific training programs would need to be considered to satisfy the needs of patients in different countries.

Cushing’s syndrome results from the chronic effects of excessive glucocorticoids. Endogenous Cushing’s syndrome is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary tumors (i.e., Cushing’s disease), primary adrenal gland tumors, or ectopic (non-pituitary) ACTH-secreting tumors. Medical therapies for Cushing’s are generally used immediately prior to transsphenoidal surgery (for pituitary tumors) or following surgery if there is disease recurrence. Medical therapies can be broadly categorized as steroidogenesis inhibitors, cortisol receptor blockers or centrally-acting agents. In the United States (US), only mifepristone and pasireotide are labeled for the treatment of Cushing’s syndrome and Cushing’s disease, respectively; and both of these medications are limited to specific subsets of patients. Consequently, there is a need for additional medical therapies for Cushing’s syndrome.

ATR-101-301 is a Phase 2 randomized, double-blind, placebo-controlled clinical study that will be conducted at approximately 10 centers in the US and the United Kingdom (UK). Subjects with endogenous Cushing’s syndrome will first enter a 6-week open-label dose-titration period. All enrolled subjects will be started on ATR-101 250 mg orally BID. Every 14 days, the ATR-101 dose will be titrated to a maximum dose of 1000 mg BID as needed based on 24-h urinary free cortisol levels. After the open-label dose-titration period, responders will be randomized in a double-blind fashion either to continue their current dose of ATR-101 or be switched to a matching placebo for up to 4 weeks. The primary objective is to evaluate the efficacy and safety of ATR-101 in subjects with Cushing’s syndrome. Secondary objectives include changes in adrenal steroids and intermediates, and changes in ACTH. The study is open to adults with endogenous Cushing’s syndrome who are not considered to be surgical candidates and do not have cyclic Cushing’s syndrome. Approximately 16 subjects will be enrolled.

Ritonavir is known to cause inhibition of cytochrome P450 CYP3A4, a fact that is exploited in the various “Ritonavir boosted” regimens in patients with HIV. Though this works to our advantage in formulation of HAART (Highly Active Antiretroviral Therapy) regimens, it also results in altered metabolism of other medications, such as glucocorticoids.

Objective:

To describe iatrogenic Cushing’s (IC) in patients with HIV (on HAART) with exposure to intraarticular steroids and a review of the literature.

We present a series of 4 patients with HIV, on various “boosted” HAART regimens, who were diagnosed with secondary adrenal insufficiency. This is followed by a review of the literature and a comparison with our patient group, of the patient characteristics and the type of steroidal preparations that have been described to be associated with Cushing's.

Our patients were diagnosed with IC, with symptom onset related temporally to various intraarticular (IA) steroidal injections. Literature search suggests that though IC in HIV has been described, most of the cases reported were secondary to inhaled glucocorticoids. Ritonavir, the most commonly used medication to “boost” the levels of other protease inhibitors (PI), was associated with all the case reports that have been previously described. Although 3/4 patients in our cohort were on Ritonavir boosted therapies, one developed Cushing's while on treatment with a cobicistat boosted regimen.

The mean age of our patient population was 50, with an average of about 22 years on antiretroviral therapy. All patients received IA triamcinolone a minimum of 2 times within a span of 2-5 months. Although the steroid dosing and frequency were similar, the time to onset of symptoms varied widely, ranging from a minimum of 4 weeks to a maximum noted duration of 5 months. Recovery of their HPA axis occured within several months (~ 5.1). 48.8% of the patients described in literature required exogenous steroids, a finding similar to our cohort. All patients on presentation had undetectable serum cortisol which increased to an average 60 minute cortisol of 18.45mcg/dL at the time of resolution with complete symptom recovery.

Conclusion:

Awareness and strict pharmacovigilance is necessary when prescribing exogenous steroids to patients on HAART. Missing HPA axis suppression could have potentially disastrous consequences. Furthermore, the overlap of symptoms with PI associated lipodystrophy may sometimes delay diagnosis. We advocate a high clinical suspicion for IC in patients on HAART who receive any formulation of exogenous steroids. In tandem with the increasing life expectancy of patients living with HIV, age related health problems are also on a rise in this subgroup of patients. Hence, presentations similar to our cohort of patients are, now, more likely to be encountered in various clinical settings with steroid exposure.

Data was retrospectively collected from clinical records of patients followed up between June 1992 and May 2016. Tumor size (TS, MI < 10 mm, MA ≥ 10mm) and basal prolactin levels (bPRL, ng/ml) were registered. Biochemical and radiological responses were evaluated during the follow-up (F/U).

Response to dopaminergic agonists (DA) was defined as 50% reduction of TS and/or PRL. Resistance (R) was considered as the absence of response despite CBG dose > 3.5 mg/weekly (w). Statistical software corresponded to SPSS 20.0

Among 1040 patients with pituitary adenomas, 209 PA (19.8%) were identified: 60.2% MA (n=124) and 39.8% MI (n=82). Gender distribution according to TS was 70 F and 54 M in MA and 75 F and 7 M in MI (p<0.001). 99 patients were included for analysis of F/U: 71 F (29.4±12.3 yo) and 28 M (40.6±13.8 yo) (p<0.001 regarding gender and age); 68.7% were MA (23±12.8 mm) and 31.3% MI (5.7±1.8 mm). Mean bPRL was 2707.6±6564.4 (90-33000) in MA and 120.6±84.5 (54.4-349) in MI.

After age subgroup distinction (<25 vs ≥ 45 yo), significant differences were found regarding TS (p=0.02) and bPRL (p=0.03).

The most common initial complaints were menstrual abnormalities 50%, galactorrhea 30.4%, visual defects 27.9%, headache 19.6%, infertility 15.7% and decreased libido 8.8%.

As initial treatment (T) 81.8% received CBG and 18.2% BEC, with median dose of 1 (0.25-13) mg/w and 5 (1.25-20) mg/day, respectively. Mean duration of T was 41.9±36.4 months (6-204).

Treatment achieved PRL normalization in 87.8% (n=87) and TS reduction in 48.5% (n=48). Accordingly to age subgroups, no differences were present regarding T duration, complete tumor disappearance and PRL normalization.

R was found in 15.1% (n=15), without differences in relation to age, contrary to bPRL (5360.9±9236.5 vs 1239.1±4350; p<0.001) and TS (28.9±17 vs 15.6±11.6; p=0.002). A higher proportion corresponded to MA (p=0.002) and M (p=0.002). T withdrawal was reached in 36.4% (n=36), with MI preponderance and recurrence documentation in 33.3% (n=12).

In discrepancy with most reports, a fewer PA frequency was found in this cohort, with predominance of MA, since ours is a neurosurgical reference centre. As expected, the majority of patients achieved response with low CBG doses, regardless of TS, with PRL reduction and TS shrinkage. Younger patients presented with larger TS and higher bPRL; however no differences were established in T response. In concordance with most important series, CBG R was uncommon, prevailing in MA and M, associated with higher bPRL and larger TS. Recurrence observed in this cohort was 33.3%

In patients with Cushing’s syndrome, we have to use metyrapone frequently to control severe hypercortisolemia before surgery. Theoretically, plasma ACTH increases after metyrapone treatment in patients with Cushing’s disease (CD). However, we have experienced that plasma ACTH decreased after its treatment in some patients with CD. In this study, we investigated the background of those patients retrospectively.

[Methods]

Fifteen patients with Cushing’s disease (CD) were divided into two groups. Eight patients showed that plasma ACTH increased one week after metyraponse initiation (group A). On the other hand, seven patients showed that plasma ACTH decreased after metyrapone (group B). The clinical backgrounds were compared between two groups.

[Results]

The doses of metyrapone between in group A and B were not different (3000 ± 755 vs, 2000 ± 800 mg/day), respectively. Urinary free cortisol excretions between in both groups were not different (490 ± 618 vs. 382 ± 1368 μg/day). Plasma ACTH levels were greater in group B than in group A (138 ± 49 vs. 186 ± 149 pg/mL, P<0.05). Plasma ACTH responses to CRH were greater in group A than in group B (401± 304 vs. 39 ± 2.9% of basal levels, P< 0.03). The sizes of pituitary adenomas were significantly larger in group B than group A (25 ± 9.4 vs. 6 ± 4.0 mm in diameter, P<0.005).

[Discussion and Conclusion]

ACTH secretion from pituitary is negatively regulated by glucocorticoid. Therefore, metyrapone, a 11 beta hydroxylase inhibitor, theoretically decreases plasma cortisol and subsequently plasma ACTH level increases. From our results, CD with macroadenoma and low ACTH response to CRH may show the ACTH suppression by metyrapone. These suggest that glucocorticoid unexpectedly stimulate ACTH secretion in some patients with CD.

Objective: To show longitudinally the change in volume (Vol) and percentage of the gland occupied (POGO) of nonfunctioning PAs in children treated with GH.

PTS/Methods: Children identified to have a PA who underwent post-contrast MRI with 1-2mm spatial resolution at the Div. of Neuroradiology at NYMC between 2007-16 were considered for inclusion. 16 patients (PTS) were identified who met these criteria. We excluded 8 PTS without follow-up (f/u) MRIs, 3 with functioning adenomas, and 1 with hydrocephalus. The 4 included PTS had nonfunctioning incidental PAs and were treated for GHD with GH. For those included, ages ranged from 4-12 YRs at baseline (mean 9.23±3.32YRs, median (MD) 10.65 YRs). Time between scans ranged from .41-2.24 YRs (mean .98±.49 YRs, MD .96 YRs). Number of scans ranged from 2-9 (mean 5.5±2.89, MD 5.5). Pituitary (Pit) and PA Vols were calculated with the ellipsoid formula (LxWxH/2).The POGOs of the PAs were calculated as PA Vol/Pit Vol x 100. PAs with an initial width 10 mm were classified as a macroadenoma (MAAD), and those with a width <10 mm were classified as a microadenoma (MIAD).

Results: 2 PTS were found to have a MIAD, and 2 PTS were found to have a MAAD. PA Vol increased in 2 PTS and decreased in 2 PTS. One MIAD resolved. POGO decreased in 3/4 PTS. Change in PA Vol between f/u ranged from -52.0-68.4mm3 with a mean of 2.72±32.41mm3 (MD 3.69mm3). Rate of PA growth ranged from -114.05-66.74mm3/YR with a mean of 1.31±41.88mm3/YR (MD 3.46mm3/YR). Change in POGO between f/u ranged from -15.25-12.80% with a mean of -2.10±8.09% (MD -2.53%). Rate of change (RC) in POGO ranged from -27.25-14.58%/YR with a mean of -3.01±10.63%/YR (MD -2.85%/YR). For the MIADs, change in PA Vol between f/u ranged from -25.77-40.24mm3 with a mean of 5.35±28.78mm3 (MD -.26mm3), rate of PA growth ranged from -40.87-45.85mm3/YR with a mean of 2.64±35.98mm3/YR (MD 0.21mm3/YR), change in POGO between f/u ranged from -14.20-12.80% with a mean of -1.42±9.47% (MD -3.11%), and RC in POGO ranged from -17.21-14.58%/YR with a mean of -1.97±11.12%/YR (MD -3.65%/YR). For the MAADs, change in PA Vol between f/u ranged from -51.96-68.40mm3 with a mean of 1.40±35.23mm3(MD 4.48mm3), rate of PA growth ranged from -114.05-66.74mm3/YR with a mean of .64±46.06mm3/YR (MD 7.97mm3/YR), change in POGO between f/u ranged from -15.25-12.53% with a mean of -2.44±7.75% (MD -2.25%), and RC in POGO ranged from -27.25-12.22%/YR with a mean of -3.53±10.84%/YR (MD -2.85%/YR).

Conclusion: Pediatric PAs demonstrate variable changes in size. We found minimal differences between the progression of MIADs and MAADs. POGO tended to decrease, so Pit growth outpaced PA growth. The variable growth during GH therapy suggests that GH plays no role in the evolution of PAs. We ask any investigator with similar PTS to contact us to develop a comprehensive database.

Methods: Medical charts of 2596 patients who were followed-up at University of Istanbul, Cerrahpasa Medical School, Endocrinology and Metabolism Outpatient Clinic between 2007-2016 with a diagnosis of pituitary adenoma, empty sella, and hypopituitarism were reviewed retrospectively. A total of 14 patients (9F/5 M) were diagnosed with hypophysitis.

Results: The mean age of patients at diagnosis was 39.14 ±17.7 years. Median follow-up time was 45 (IQR: 13.5-61.2) months. Four out 7 female patients were postmenauposal and three were nulliparous. All but one patient did not have an accompanying autoimmune disease. The most common symptoms and signs were headache (42.9%) and diabetes inspidus (42.9%), followed by hypopituitarism (35.7%). Four out of 14 patients had secondary hypophysitis (3 patients with histiocytosis X, 1 patient with neurosarcoidosis). The remaining 10 patients had primary hypophysitis (8 patients with lymphocytic hypophysitis, 2 patients with xanthomatous hypophysitis). A total of 9 patients were operated. Eight out of 9 patients had a presumptive diagnosis of pituitary macroadenoma. These patients had the histopathological evidence of hypophysitis. One patient was operated due to the mass effect of the lesion .The presumptive diagnosis for this case was lymphocytic hypophysitis which was confirmed by histopathology. Five patients were started on glucocorticoid therapy. The starting doses for metylprednisolone were 60 mg/d in three patients and 120mg/d in two patients. The glucocorticoid dose was tapered in 2 months. The clinical response was assessed in 4 patients . Half of the patients were good responders. Avascular necrosis of femoral head was the most common complication (40%) after glucocorticoid therapy. Two patients with primary hypophysitis required gama-knife or cyber-knife radiosurgery after failure of surgery and glucocorticoid therapy . Another patient with histiocytosis X received gama-knife radiosurgery after operation. The response to radiosurgery was good in all of the patients. No significant side effect was detected. Anterior and posterior pituitary hormone deficiencies were still present at the last follow-up visit in all of the patients who had one or more pituitary hormone deficiencies at the time of diagnosis.

Conclusisons: Hypophysitis, albeit rare, may cause severe pituitary dysfunction which tend to persist during the disease course. Misdiagnosis is frequent. Disease’s pathogenesis might involve mechanisms other than autoimmunity in some patients. A considerable number of patients may require combined therapies which might cause serious side effects.

Cyclical Cushing’s syndrome (CCS) can be an elusive diagnosis and may go unrecognized for years, even after appropriate endocrine evaluation. We illustrate a case in which serial salivary cortisol levels were used to make the diagnosis of CCS.

Clinical Case

A 36 year old woman presented with one year history of fifty pound weight gain, irregular menses, hirsuitism, irritability, fatigue and proximal muscle weakness. She had already undergone extensive testing, which collectively did not clearly establish Cushing’s. Five of twelve dexamethasone (Dex) suppression tests (DST) were abnormal, ranging from 0.6-6 µg/dL (<1.8). Fourteen of eighteen urine free cortisols (UFC) were elevated, ranging from 29-139.7 µg/24h (4-50). A Dex-CRH test showed a cortisol of 2.9 µg/dL (<1.8) after two days of Dex and 30-minute cortisol peak of 5.7 µg/dL (>20% rise is consistent with Cushing’s disease). However, 48-hr DST suppressed serum cortisol (1.1 µg/dL) and UFC (9.5 µg/24h). She was normotensive (111/55) but obese (81.6 kg, BMI 32.7), with facial plethora, rounded face, subtle cervical fat pads, proximal muscle weakness and mild hypertrichosis. There was no acanthosis nigricans, supraclavicular fat pads, edema, purple striae or hyperpigmentation.

Late night salivary cortisols over ten months were intermittently above normal, ranging from <0.04-0.97 µg/dL (0-0.1), but never long enough for localization testing. During testing, 31% of salivary cortisols were above normal. ACTH levels were never suppressed. MRI showed a 3 mm adenoma in the left inferior aspect of the pituitary gland. A 10 µg desmopressin stimulation test was also performed. ACTH increased by 131% and cortisol increased by 34%, consistent with Cushing’s disease.

At transsphenoidal exploration, intraoperative smear showed a monomorphic population of cells compatible with adenoma. However, final pathology showed adenohypophysis tissue with patchy areas of expanded acini. The cells in these areas stained with ACTH and CM5.2 with the final diagnosis of corticotroph hyperplasia. Post-op day one and two AM cortisols were 33 and 9 µg/dL respectively and she ultimately developed relative adrenal insufficiency which responded to hydrocortisone. Off hydrocortisone, salivary cortisols ranged from undetectable to occasionally elevated, in the range of 0.12-0.19 µg/dL. A repeat desmopressin stimulation test was essentially flat.

Conclusion

Here we demonstrate the effective use of serial salivary cortisols in diagnosing CCS. Second, we illustrate the utility of the desmopressin stimulation test in the work-up of ACTH-dependent Cushing’s syndrome. Although not necessary in the routine evaluation of Cushing’s syndrome, when faced with challenging diagnostic dilemmas such as that presented by CCS, it can be a valuable, additional tool to differentiate between Cushing’s disease and ectopic ACTH secretion.

This highly potent PTH1R receptor antagonist antibody has the potential to become a valuable therapeutic agent in a variety of indications including hyperparathyroidism, humoral hypercalcemia of malignancy, and, potentially, the PTHrP-mediated cachexia seen in some cancers.

Revathy Carnagarin^a,b,c, , Arun M. Dharmarajan^a,c, Crispin R. Dass^a,b

^aCurtin Health Innovation Research Institute, Bentley 6102, Australia

^bSchool of Pharmacy, Curtin University, Bentley 6102, Australia

^cSchool of Biomedical Sciences, Curtin University, Bentley 6102, Australia

Bone regeneration is a complex physiological process that is currently addressed by a plethora of different strategies and autologous bone grafting continues to remain the gold standard. However, massive defects arising from skeletal abnormalities post trauma, infection and tumour resection necessitates the identification of further strategies in the form tissue engineering, gene therapy and potent osteo-inductive agents that could enhance the bone repair process. PEDF has a signatory effect on osteogenesis. PEDF gene defect results in osteogenesis imperfecta IV characterised by bone mineralisation defects and PEDF restoration improved bone mineralisation in murine models of osteogenesis imperfecta VI and normalised mineralisation defects in pluripotent stem cells derived from PEDF null patients [Belinsky et al 2016]. PEDF regulates the expression of osteoblastic genes, enhanced mineralisation in murine and human MSCs [Elahy et al 2016] and suppressed inhibitors of bone formation [Li et al 2013, 2015]. Muscle plays a vital role in bone regeneration by contributing satellite cells, muscle stem cells and growth factors and the use of muscle flaps to cover bone defects and prevent infections could be further improvised to support bone healing directly by using a suitable osteokine such as PEDF.

To study the potency of PEDF in this perspective, we analysed PEDF-treated mice and human skeletal myoblasts for osteogenic properties. PEDF treatment of skeletal myoblasts at a physiological concentration of 100nM induced the expression of osteogenic markers such as osteocalcin and alkaline phosphatase. PEDF activated Erk1/2 MAPK signalling which governed the expression of osteogenic markers and mineralisation. Additional analysis demonstrated PEDF as a potent osteogenic signal that induced osteogenesis independent of osteogenic supplements to convert the C2C12 myogenic differentiation pathway into osteoblast lineage. PEDF also enhanced mineral deposition in human primary muscle myoblasts. These effects of PEDF were tested in vivo using Balbc mice. This study provides new insights into the signalling and molecular aspects of PEDF to modulate the differentiation commitment of skeletal myocytes, and we speculate that this could pave the way for new strategies that could overcome the limitations of existing therapies to accelerate bone regeneration, or even to address skeletal disorders.

Past studies have shown that parathyroid hormone (PTH) stimulates bone modeling via enhanced expression of the insulin-like growth factor-1 (IGF‐I) and the ephrinB2 and EphB4 in osteoblasts.

To define the interactions between PTH/IGF-1/Eph signaling pathways in bone, male mice at 4 weeks of age were injected 80 mcg/kg/day for 5 days. To our surprise we found 3 fold increase in the expression of Ephrin B1 in the femoral cortical shells from male mice, while the expression of ephrinB2 or EphB4 did not differ significantly between PTH treated and untreated groups. Thus, we used the osteoblast- and osteocyte-specific ephrinB1 knockout (KO) mice (using the osteocalcin-cre and the dentin matrix protein (DMP)-1-cre, respectively) to investigate their bone response to intermittent PTH treatment.

We used micro Computer Tomography (CT) to characterize the basal morphology of femurs dissected from male mice. We found decreases in tissue mineral density in cortical bone of the mid-shaft femur in both Osteocalcin-ephrinB1 (1.45 ± 0.05 g/cm³, p=0.01) and DMP-1-ephrinB1 KO mice (1.465 ± 0.05 mg/cm³, p=0.02) as compared to controls (1.64 ± 0.02 mg/cm³). Similarly, we found decreased bone mineral density in the trabecular bone assessed at the distal femur (Osteocalcin-eprinB1 0.147±0.005 p=0.004, DMP-1-ephrinB1 0.175 ± 0.02 p=0.06, and controls 0.248 ± 0.02 mg/cm³). However, cortical and trabecular bone morphology of males did not differ between the groups. Female DMP-ephrinB1 KO mice showed decreased total cross-sectional area (1.14 ± 0.09 vs 1.27±0.06 mm², p = 0.02), polar moment of inertia (0.12 ± 0.03 vs 0.14± 0.01 mm⁴, p= 0.03), and marrow area (0.76 ± 0.03 vs 0.86 ± 0.06 mm², p = 0.03) as compared to control mice with no significant difference in tissue mineral density. Female DMP-ephrinB1 KO mice did not show any trabecular bone phenotype.

Bone anabolic response to intermittent PTH treatment in Osteocalcin-eprinB1 and DMP-1-ephrinB1 KO mice is under investigation.

Background: FIO is a rare metabolic bone disease clinically characterized by generalized bone pain andfragility fractures in both the axial and appendicular skeleton.Bone biopsy shows extensive mineralization defect (hyperosteoidosis)and loss of collagen birefringence under polarized microscopy.Etiology of the diseaseis unknown and noeffective therapycurrently exists.

Methods: We performed global gene expression profiling in mRNA isolated from bone biopsy specimen from a 48yold man with FIO, which suggested a possible molecular basis for thepathogenesis of FIO. Based on this information he was treated with human growth hormone for one year with frequent biochemical monitoring and a repeat bone biopsy.

Result: The global gene expression profile revealed 8,916 differentially expressed genes (out of total 35,307). The genetic and functional analyses suggestedthat FIO is associated with defects in osteoblast maturation, collagen fibril arrangement, matrix organization and bone mineralization with a possible defect in paracrine action of IGF1. Following therapy with human growth hormone there was a marked clinical, radiological and histological improvement.

Conclusion: Gene profile analysis of the bone biopsy specimen from a patient with FIO provided crucial information underlying the bone abnormality and a therapeutic basis for clinical management. We suggest that growth hormone may be a potential novel therapy for FIO. 

Objective: In the present study, we investigated the mechanisms underlying the apoptosis of Hsp47-/- cells and involvement of autophagy and unfolded protein response (UPR) in the clearance of misfolded type I procollagen in the absence of Hsp47. We hypothesized that misfolded type I procollagen in the ER might cause ER stress, resulting in apoptosis of Hsp47-disrupted triple helix of collagens when autophagy isinhibited.

Methodology: Peripheral blood mononuclear cell (PBMC) derived osteoblasts were cultured. RNA and proteins were isolated from bone. Mutation analysis of Hsp47 gene was done through sequencing followed by restriction digestion. Hsp47 control- and mutant-gene were cloned in expression vector pEGFP-C1.Further,in-vitro analysis was performed to study the interaction between triple helix and mutant Hsp47 under confocal microscope. Immunoblot for mutant protein analysis was done, RT-PCR and quantitative RT-PCR were performed for expression and functional analyses.

Results and Conclusion: ER stress-induced apoptosis may underlie the clearance of collagen-producing osteoblast cells. Experimental findings suggest that Hsp47 plays a pivotal role in ER/cis-Golgi transport. The changes in triple helical structure could affect fibril assembly in extra cellular matrix and lead to irregular mineralization and an OI phenotype.

We show, through transfection of hVDR and hRXRα GFP-tagged constructs, that both hVDR and hVDR/ hRXRα complex are localized in the nucleus of MT1107 cells treated with either 1α,25(OH)₂D₃ or 25-hydroxyvitamin D₃ [25(OH)D₃]. In contrast, in CYP27B1 knockout AOH932 cells impaired nuclear localization of hVDR and hVDR/hRXRα is observed. Furthermore, we demonstrate using Fluorescence Resonance Energy Transfer (FRET) that hVDR/hRXRα interaction is also impaired in AOH931 cells. Lastly, we demonstrate using Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss in Photobleaching (FLIP) that the nucleocytoplasmic trafficking of VDR within the nucleus of CYP27B1 knockout AOH931 cells is reduced consistent with its binding to chromatin.

Our results show that CYP27B1 knockout disrupts VDR function by significantly impairing its nuclear localization, nucleocytoplasmic trafficking and hVDR/hRXR interaction consistent with the complex binding to DNA.

Natpara (PTH1-84) is approved for subcutaneous injection as an adjunct to vitamin D and calcium in patients with hypoparathyroidism but has not demonstrated the ability to reduce the incidence of hypercalcemia, hypocalcemia, or hypercalciuria relative to conventional therapy. Continuous infusion of teriparatide (PTH1-34) has been shown to be superior to twice daily injections in clinical studies. Thus, a PTH product that provides continuous exposure with daily dosing could represent a major step forward in addressing a large unmet medical need. TransCon PTH is a prodrug releasing unmodified teriparatide via non-enzymatic hydrolysis of the proprietary TransCon Linker for the treatment of hypoparathyroidism.

Objective

TransCon PTH has been designed to maintain a normal range, steady concentration of PTH in the blood stream. It is intended to address the fundamental limitation of short-acting PTH molecules by providing infusion-like PTH blood levels.

Design and methods

TransCon PTH was administered to rat and cynomolgus monkeys in single dose studies and in 28-day toxicology studies with daily dosing. The systemic concentrations of TransCon PTH and calcium were assessed at multiple time point following dosing.

Results

TransCon PTH exhibited in both rat and monkey long half-lives providing infusion-like pharmacokinetics. In both species, exposure to TransCon PTH increased in a dose proportional manner. Sex differences in systemic exposure of TransCon PTH and serum calcium were observed in rats, with females exhibiting increased exposures compared to males. No differences between sexes were observed for TransCon PTH or serum calcium in cynomolgus monkeys.

Conclusion

Nonclinical experiments in rats and cynomolgus monkeys demonstrated that TransCon PTH may provide full therapeutic coverage in contrast to available therapies; PTH(1-34) and PTH(1-84) both have half-lives on the order of minutes. The PK characteristics of TransCon PTH after steady state resemble an infusion like profile, suggesting that TransCon PTH can support continuous PTH exposure in hypoparathyroid patients. The substantial extension of PTH half-life may more closely mimic physiological levels of PTH observed in healthy individuals and therefore maintain blood calcium and phosphorous levels while normalizing urinary calcium excretion.

We expressed wildtype (WT) Arg60 and mutant Cys60 Gα11 in HEK293 cells stably expressing the CASR and measured the intracellular calcium response to changes in extracellular calcium concentrations. The leftward shift of the concentration-response curve in the cells expressing mutant Gα11 illustrated an increased sensitivity to calcium. We treated these cells with the calcilytic NPS 2143 and the EC₅₀of the mutant cells returned to that of WT cells.

With CRISPR/Cas9 technology, we created a mouse model harboring the Arg60Cys mutation. Compared to WT littermates, nine-week-old mice heterozygous and homozygous for Gα11 R60C were hypocalcemic. Compared to WT animals, PTH was inappropriate in heterozygous and homozygous animals. Serum phosphate was higher in homozygous mice. We also noted increased pigmentation of the tails and ears of mutant mice, implicating the activation of other GPCRs by the mutant Gα11. μCT analysis of 12-week old mice revealed lower bone mineral density (BMD) and bone volume/total volume (BV/TV) of female heterozygous/homozygous and male heterozygous mice. This finding was surprising given that humans with hypoparathyroidism typically have increased BMD. The urinary fractional excretion index of calcium (FECa) was not different in mutant mice, raising the possibility that Gα11 does not have a dominant effect on CASR function in the distal tubule cells.

We injected 30 mg/kg of NPS 2143 i.p.; 4 hours after injection, serum calcium was increased in mice WT, heterozygous and homozygous for R60C. Serum PTH increased in all animal groups, and serum phosphate increased in WT mice. The urinary FECa decreased in heterozygous and homozygous animals. We tested the effects of the specific Gα11/Gαq inhibitor YM-254890. A single dose of 0.15mg/kg YM-254890 i.p. increased the calcium in WT and heterozygous R60C mice. Published crystallography studies predict that YM-254890 inhibits WT Gα11, but not Gα11 R60C, consistent with our findings of no change in serum calcium in response to treatment of R60C homozygous animals.

We have used CRISPR/Cas9 to create a mouse with the p.Arg60Cys mutation found in humans with ADH2. Our mouse model mimics the biochemical findings of the human disease, i.e. hypocalcemia and inadequate PTH. Mutant mice also have decreased BMD, increased pigmentation, and unchanged urinary FECa. This mouse model has identified phenotypes, in addition to hypocalcemia, associated with Gα11 mutations.

Objectives and patients: Characterize the methylation pattern of the GNAS-AS2-DMR in patients with AD-PHP1B and LOM at the GNAS-A/B-DMR (AD+: n=10) and in patients without (AD-: n=4) STX16 deletion, sporPHP1B (n=10) and controls (n=10). STX16 and GNAS deletion were excluded in the AD- patients by MLPA and genomic multiplex and quantitative PCR of the GNAS and the STX16 regions.

Results:

1- AD- patients showed significantly higher methylation compared to controls (respectively 38% and 22,7%; P<0.05). GNAS-AS2 was unmethylated in AD+ and sporPHP1B patients (respectively 4.0% and 2.9%).

2- Subsequently, we performed bisulfite sequencing of the GNAS-AS2-DMR. First, we identified 2 CG-rich subdomains separated by 184 bp. Second, the AD- patients displayed a unique pattern of methylation (methylated on the 1^st subdomain, unmethylated on the 2^nd subdomain) that was absent in controls, AD+ and sporPHP1B patients.

Conclusion: We have better refined the DMR GNAS-AS2. We have identified a subgroup of PHP1B patients who have a specific pattern of methylation/signature at the GNAS-AS2 DMR.