Alison Marker

Microarray, qPCR, and KCNJ5 Sequencing of Aldosterone-Producing Adenomas Reveal Differences in Genotype and Phenotype between Zona Glomerulosa- and Zona Fasciculata-Like Tumors

CONTEXT Aldosterone-producing adenomas (APA) are heterogeneous. The recent finding of somatic KCNJ5 mutations suggests a genetic explanation. OBJECTIVES The objectives of this study were the following: 1) to compare transcriptional profiles in APA and adjacent adrenal gland (AAG); 2) to test whether gene expression profile clusters with different cell histology; and 3) to measure the frequency of KCNJ5 mutations and determine the genotype-phenotype relationship. DESIGN/SETTING The design of the study included laboratory analyses of 46 unselected APA. PATIENTS The patients in this study had primary hyperaldosteronism with unilateral APA. INTERVENTIONS The objectives of this study were the following: 1) Illumina beadchip analysis of RNA from eight paired APA-AAG; 2) a blinded review of cell histology for 46 APA; 3) laser capture microdissection of zona glomerulosa (ZG) and zona fasciculata (ZF) cells; and 4) sequencing of KCNJ5 in 46 APA. MAIN OUTCOME MEASURES The main outcome measures of this study were the following: 1) a difference in gene expression profile and a correlation with histological markers of ZF; 2) a frequency of KCNJ5 mutations and phenotypic comparisons of wild type with mutant APA. RESULTS The results of the study were the following: 1) a cluster analysis of microarray data separated APA from AAG. APA at opposite ends of the APA cluster had an approximately 800-fold difference in CYP17A1 mRNA expression, whereas histology showed 0% ZF-like cells in one vs. 100% in the other. A heat map ranking APA by CYP17A1 expression correctly predicted several genes (e.g. KCNK1, SLC24A3) to be enriched in laser capture microdissection samples of ZG; 2) known or novel mutations of KCNJ5 were found in 20 of 46 consecutive APA [43% (95% confidence interval [CI] (29, 58)%)]. The APA with KCNJ5 gene mutations were larger compared with tumors harboring the wild type, 1.63 [95% CI (1.37, 1.88)] vs. 1.14 [0.97, 1.30] cm (P = 0.0013), had predominantly ZF-like cells, and their CYP17A1 (log(2)-fold change) was higher than in wild type: -0.96 [95% CI (-0.07, -1.85)] vs. -2.54 [-1.61, -3.46], (P = 0.017). CONCLUSIONS KCNJ5 mutations are common in APA, particularly those arising from ZF. The long-recognized heterogeneity among APA may have a genetic basis.

Pregnancy, Primary Aldosteronism, and Adrenal CTNNB1 Mutations.

Recent discoveries of somatic mutations permit the recognition of subtypes of aldosterone-producing adenomas with distinct clinical presentations and pathological features. Here we describe three women with hyperaldosteronism, two who presented in pregnancy and one who presented after menopause. Their aldosterone-producing adenomas harbored activating mutations of CTNNB1, encoding β-catenin in the Wnt cell-differentiation pathway, and expressed LHCGR and GNRHR, encoding gonadal receptors, at levels that were more than 100 times as high as the levels in other aldosterone-producing adenomas. The mutations stimulate Wnt activation and cause adrenocortical cells to de-differentiate toward their common adrenal-gonadal precursor cell type. (Funded by grants from the National Institute for Health Research Cambridge Biomedical Research Centre and others.).

Familial adrenocortical carcinoma in association with Lynch syndrome

Context: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a poor prognosis. Although the majority of childhood ACC arises in the context of inherited cancer susceptibility syndromes, it remains less clear whether a hereditary tumor predisposition exists for the development of ACC in adults. Here, we report the first occurrence of familial ACC in a kindred with Lynch syndrome resulting from a pathogenic germline MSH2 mutation. Case: A 54-year-old female with a history of ovarian and colorectal malignancy was found to have an ACC. A detailed family history revealed her mother had died of ACC and her sister had previously been diagnosed with endometrial and colorectal cancers. A unifying diagnosis of Lynch syndrome was considered, and immunohistochemical analyses demonstrated loss of MSH2 and MSH6 expression in both AACs (proband and her mother) and in the endometrial carcinoma of her sister. Subsequent genetic screening confirmed the presence of a germline MSH2 mutation (resulting in deletions of exons 1–3) in the proband and her sister. Conclusion: Our findings provide strong support for the recent proposal that ACC should be considered a Lynch syndrome-associated tumor and included in the Amsterdam II clinical diagnostic criteria. We also suggest that screening for ACC should be considered in cancer surveillance strategies directed at individuals with germline mutations in DNA mismatch repair genes.

Translating In Vivo Metabolomic Analysis of Succinate Dehydrogenase–Deficient Tumors Into Clinical Utility

Purpose Mutations in the mitochondrial enzyme succinate dehydrogenase (SDH) subunit genes are associated with a wide spectrum of tumours including phaeochromocytoma and paraganglioma (PPGL) 1, 2, gastrointestinal stromal tumours (GIST) 3, renal cell carcinoma (RCC) 4 and pituitary adenomas5. SDH-related tumorigenesis is believed to be secondary to accumulation of the oncometabolite succinate. Our aim was to investigate the potential clinical applications of MRI spectroscopy (1H-MRS) in a range of suspected SDH-related tumours. Patients and methods Fifteen patients were recruited to this study. Respiratory-gated single-voxel 1H-MRS was performed at 3T to quantify the content of succinate at 2.4 ppm and choline at 3.22 ppm. Results A succinate peak was seen in six patients, all of whom had a germline SDHx mutation or loss of SDHB by immunohistochemistry. A succinate peak was also detected in two patients with a metastatic wild-type GIST (wtGIST) and no detectable germline SDHx mutation but a somatic epimutation in SDHC. Three patients without a tumour succinate peak retained SDHB expression, consistent with SDH functionality. In six cases with a borderline or absent peak, technical difficulties such as motion artefact rendered 1H-MRS difficult to interpret. Sequential imaging in a patient with a metastatic abdominal paraganglioma demonstrated loss of the succinate peak after four cycles of [177Lu]-DOTATATE, with a corresponding biochemical response in normetanephrine. Conclusions This study has demonstrated the translation into clinical practice of in vivo metabolomic analysis using 1H-MRS in patients with SDH-deficient tumours. Potential applications include non-invasive diagnosis and disease stratification, as well as monitoring of tumour response to targeted treatments.

Rapid disease progression in a patient with mismatch repair-deficient and cortisol secreting adrenocortical carcinoma treated with pembrolizumab

Context Metastatic adrenocortical carcinoma (ACC) is an aggressive malignancy with a poor prognosis and limited therapeutic options. A subset of ACC is due to Lynch syndrome, an inherited tumor syndrome resulting from germline mutations in mismatch repair (MMR) genes. It has been demonstrated that several cancers characterized by MMR deficiency are sensitive to immune checkpoint inhibitors that target PD-1. Here, we provide the first report of PD-1 blockade with pembrolizumab in a patient with Lynch syndrome and progressive cortisol-secreting metastatic ACC. Case report A 58-year-old female with known Lynch syndrome presented with severe Cushings syndrome and was diagnosed with a cortisol-secreting ACC. Three months following surgical resection and adjuvant mitotane therapy the patient developed metastatic disease and persistent hypercortisolemia. She commenced pembrolizumab, but her second cycle was delayed due to a transient transaminitis. Computed tomography performed after 12 weeks and 2 cycles of pembrolizumab administration revealed significant disease progression and treatment was discontinued. After 7 weeks, the patient became jaundiced and soon died due to fulminant liver failure. Conclusion Treatment of MMR-deficient cortisol-secreting ACC with pembrolizumab may be ineffective due to supraphysiological levels of circulating corticosteroids, which may in turn mask severe drug-induced organ damage.

802 SOMATIC MUTATIONS OF KCNJ5 ARE COMMON IN ALDOSTERONE-PRODUCING ADENOMAS OF THE ADRENAL, AND DISTINGUISH A PHENOTYPE OF YOUNGER WOMEN WITH ZF-LIKE LARGE ADENOMAS FROM OLDER MEN WITH ZG-LIKE SMALL ADENOMAS

Background: Among aldosterone-producing adenomas (APAs), heterogeneity of phenotype is well recognized. Chois finding of KCNJ5 mutations in 8/22 APAs [1], offered a possible explanation for heterogeneity and hence prompted the question whether there are genotype:phenotype correlations. Method: The K+-selectivity region of KCNJ5 was sequenced in 46 APAs. Each was blindly histologically graded. A microarray comparison of ZF- vs ZG-like APAs suggested putative ZG genes, whose expression was compared between ZG and ZF cells from normal adrenal using laser capture microdissection (LCM). Confirmed ZG genes were compared between mutant and wild-type APA. Immunohistochemistry for KCNJ5 was undertaken in adrenals with and without the mutation. Result: 20/46 APAs (43%(95%CI[29,58]%)) had KCNJ5 somatic mutations. Mutant APAs were larger, 2.27 vs 0.78cm3, (p = 0.001). Regression analysis distinguished: [i] younger women, with mainly mutant, larger APAs, from [ii] older men with mainly wild-type smaller APAs (Figure). APAs with KCNJ5 mutation had histological appearance of ZF cells, in particular the classical feature of higher cytoplasm to nucleus ratio. Genes confirmed as ZG-selective on LCM were less abundant in mutant APAs while the ZF-gene CYP17A1 was higher (0.51 vs 0.15, P = 0.011). KCNJ5 staining was observed in all APAs regardless of genotype. Figure. No caption available. Conclusion: The mutant and wild-type phenotypes differ by age, gender, size, gene expression profile and histology, and may arise respectively from ZF and ZG. Small ZG-like APAs may elude diagnosis at a young age and progress to cause resistant hypertension. ReferenceChoi M et al. K+-channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension. Science 2011;331:768-772.