Metabolomics in the Diagnosis of Pheochromocytoma and Paraganglioma.

Metabolomics refers to the detection and measurement of small molecules (metabolites) within biological systems, and is therefore a powerful tool for identifying dysfunctional cellular physiologies. For pheochromocytomas and paragangliomas (PPGLs), metabolomics has the potential to become a routine addition to histology and genomics for precise diagnostic evaluation. Initial metabolomic studies of ex vivo tumors confirmed, as expected, succinate accumulation in PPGLs associated with pathogenic variants in genes encoding succinate dehydrogenase subunits or their assembly factors (SDHx). Metabolomics has now shown utility in clarifying SDHx variants of uncertain significance, as well as the accurate diagnosis of PPGLs associated with fumarate hydratase (FH), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH2) and aspartate transaminase (GOT2). The emergence of metabolomics resembles the advent of genetic testing in this field, which began with single-gene discoveries in research laboratories but is now done by standardized massively parallel sequencing (targeted panel/exome/genome testing) in pathology laboratories governed by strict credentialing and governance requirements. In this setting, metabolomics is poised for rapid translation as it can utilize existing infrastructure, namely liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the measurement of catecholamine metabolites. Metabolomics has also proven tractable to in vivo diagnosis of SDH-deficient PPGLs using magnetic resonance spectroscopy (MRS). The future of metabolomics - embedded as a diagnostic tool - will require adoption by pathologists to shepherd development of standardized assays and sample preparation, reference ranges, gold standards, and credentialing.

Bayesian approach to determining penetrance of pathogenic SDH variants.

BACKGROUND: Until recently, determining penetrance required large observational cohort studies. Data from the Exome Aggregate Consortium (ExAC) allows a Bayesian approach to calculate penetrance, in that population frequencies of pathogenic germline variants should be inversely proportional to their penetrance for disease. We tested this hypothesis using data from two cohorts for succinate dehydrogenase subunits A, B and C (SDHA-C) genetic variants associated with hereditary pheochromocytoma/paraganglioma (PC/PGL). METHODS: Two cohorts were 575 unrelated Australian subjects and 1240 unrelated UK subjects, respectively, with PC/PGL in whom genetic testing had been performed. Penetrance of pathogenic SDHA-C variants was calculated by comparing allelic frequencies in cases versus controls from ExAC (removing those variants contributed by The Cancer Genome Atlas). RESULTS: Pathogenic SDHA-C variants were identified in 106 subjects (18.4%) in cohort 1 and 317 subjects (25.6%) in cohort 2. Of 94 different pathogenic variants from both cohorts (seven in SDHA, 75 in SDHB and 12 in SDHC), 13 are reported in ExAC (two in SDHA, nine in SDHB and two in SDHC) accounting for 21% of subjects with SDHA-C variants. Combining data from both cohorts, estimated lifetime disease penetrance was 22.0% (95% CI 15.2% to 30.9%) for SDHB variants, 8.3% (95% CI 3.5% to 18.5%) for SDHC variants and 1.7% (95% CI 0.8% to 3.8%) for SDHA variants. CONCLUSION: Pathogenic variants in SDHB are more penetrant than those in SDHC and SDHA. Our findings have important implications for counselling and surveillance of subjects carrying these pathogenic variants.

Cousins not twins: intratumoural and intertumoural heterogeneity in syndromic neuroendocrine tumours.

Hereditary endocrine neoplasias, including phaeochromocytoma/paraganglioma and medullary thyroid cancer, are caused by autosomal dominant mutations in several familial cancer genes. A common feature of these diseases is the presentation of multiple primary tumours, or multifocal disease representing independent tumour clones that have arisen from the same initiating genetic lesion, but have undergone independent clonal evolution. Such tumours provide an opportunity to discover common cooperative changes required for tumourigenesis, while controlling for the genetic background of the individual. We performed genomic analysis of synchronous and metachronous tumours from five patients bearing germline mutations in the genes SDHB, RET, and MAX. Using whole exome sequencing and high-density single-nucleotide polymorphism arrays, we analysed two to four primary tumours from each patient. We also applied multi-region sampling, to assess intratumoural heterogeneity and clonal evolution, in two cases involving paraganglioma and medullary thyroid cancer, respectively. Heterogeneous patterns of genomic change existed between synchronous or metachronous tumours, with evidence of branching evolution. We observed striking examples of evolutionary convergence involving the same rare somatic copy-number events in synchronous primary phaeochromocytoma/paraganglioma. Convergent events also occurred during clonal evolution of metastatic medullary thyroid cancer. These observations suggest that genetic or epigenetic changes acquired early within precursor cells, or pre-existing within the genetic background of the individual, create contingencies that determine the evolutionary trajectory of the tumour. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Analysis of SDHAF3 in familial and sporadic pheochromocytoma and paraganglioma.

BACKGROUND: Germline mutations in genes encoding subunits of succinate dehydrogenase (SDH) are associated with the development of pheochromocytoma (PC) and/or paraganglioma (PGL). As assembly factors have been identified as playing a role in maturation of individual SDH subunits and assembly of the functioning SDH complex, we hypothesized that SDHAF3 variants may be associated with PC/PGL and functionality of SDH. METHODS: DNA was extracted from the blood of 37 individuals (from 23 families) with germline SDH mutations and 18 PC/PGL (15 sporadic, 3 familial) and screened for mutations using a custom gene panel, containing SDHAF3 (SDH assembly factor 3) as well as eight known PC/PGL susceptibility genes. Molecular and functional consequences of an identified sequence variant of SDHAF3 were assessed in yeast and mammalian cells (HEK293). RESULTS: Using massively parallel sequencing, we identified a variant in SDHAF3, c.157 T > C (p.Phe53Leu), associated with increased prevalence in familial and sporadic PC/PGL (6.6%) when compared to normal populations (1.2% [1000 Genomes], p = 0.003; 2.1% [Exome Aggregation Consortium], p = 0.0063). In silico prediction tools suggest this variant is probably damaging to protein function, hence we assessed molecular and functional consequences of the resulting amino acid change (p.Phe53Leu) in yeast and human cells. We showed that introduction of SDHAF3 p.Phe53Leu into Sdh7 (ortholog of SDHAF3 in humans) null yeast resulted in impaired function, as observed by its failure to restore SDH activity when expressed in Sdh7 null yeast relative to WT SDHAF3. As SDHAF3 is involved in maturation of SDHB, we tested the functional impact of SDHAF3 c.157 T > C and various clinically relevant SDHB mutations on this interaction. Our in vitro studies in human cells show that SDHAF3 interacts with SDHB (residues 46 and 242), with impaired interaction observed in the presence of the SDHAF3 c.157 T > C variant. CONCLUSIONS: Our studies reveal novel insights into the biogenesis of SDH, uncovering a vital interaction between SDHAF3 and SDHB. We have shown that SDHAF3 interacts directly with SDHB (residue 242 being key to this interaction), and that a variant in SDHAF3 (c.157 T > C [p.Phe53Leu]) may be more prevalent in individuals with PC/PGL, and is hypomorphic via impaired interaction with SDHB.

A Clinicopathologic and Molecular Analysis of Fumarate Hydratase-deficient Pheochromocytoma and Paraganglioma.

Up to 40% of pheochromocytomas (PCCs) and paragangliomas (PGLs) are hereditary. Germline mutations/deletions in fumarate hydratase ( FH ) cause hereditary leiomyomatosis and renal cell carcinoma syndrome which manifests predominantly with FH-deficient uterine/cutaneous leiomyomas and renal cell carcinomas (RCCs)-tumors characterized by loss of immunohistochemical (IHC) expression of FH and/or positive staining for S-(2-succino)-cysteine. Occasional patients develop PCC/PGL. We investigated the incidence, morphologic, and clinical features of FH-deficient PCC/PGL. We identified 589 patients with PCC/PGLs that underwent IHC screening for FH and/or S-(2-succino)-cysteine. Eight (1.4%) PCC/PGLs were FH deficient (1.1% in an unselected population). The median age for FH-deficient cases was 55 (range: 30 to 77 y) with 50% arising in the adrenal. All 4 with biochemical data were noradrenergic. Two (25%) metastasized, 1 dying of disease after 174 months. Germline testing was performed on 7 patients, 6 of whom had FH missense mutations. None were known to have a significant family history before presentation or developed cutaneous leiomyomas, or FH-deficient RCC at extended follow-up. The patient wild-type for FH on germline testing was demonstrated to have somatic FH mutation and loss of heterozygosity corresponding to areas of subclonal FH deficiency in her tumor. One patient did not undergo germline testing, but FH mutation was demonstrated in his tumor. We conclude that FH-deficient PCC/PGL are underrecognized but can be identified by IHC. FH-deficient PCC/PGL are strongly associated with germline missense mutations but are infrequently associated with leiomyoma or RCC, suggesting there may be a genotype-phenotype correlation. FH-deficient PCC/PGL may have a higher metastatic risk.

Functional significance of germline EPAS1 variants.

Mosaic or somatic EPAS1 mutations are associated with a range of phenotypes including pheochromocytoma and/or paraganglioma (PPGL), polycythemia and somatostatinoma. The pathogenic potential of germline EPAS1 variants however is not well understood. We report a number of germline EPAS1 variants occurring in patients with PPGL, including a novel variant c.739C>A (p.Arg247Ser); a previously described variant c.1121T>A (p.Phe374Tyr); several rare variants, c.581A>G (p.His194Arg), c.2353C>A (p.Pro785Thr) and c.2365A>G (p.Ile789Val); a common variant c.2296A>C (p.Thr766Pro). We performed detailed functional studies to understand their pathogenic role in PPGL. In transient transfection studies, EPAS1/HIF-2α p.Arg247Ser, p.Phe374Tyr and p.Pro785Thr were all stable in normoxia. In co-immunoprecipitation assays, only the novel variant p.Arg247Ser showed diminished interaction with pVHL. A direct interaction between HIF-2α Arg247 and pVHL was confirmed in structural models. Transactivation was assessed by means of a HRE-containing reporter gene in transiently transfected cells, and significantly higher reporter activity was only observed with EPAS1/HIF-2α p.Phe374Tyr and p.Pro785Thr. In conclusion, three germline EPAS1 variants (c.739C>A (p.Arg247Ser), c.1121T>A (p.Phe374Tyr) and c.2353C>A (p.Pro785Thr)) all have some functional features in common with somatic activating mutations. Our findings suggest that these three germline variants are hypermorphic alleles that may act as modifiers to the expression of PPGLs.

Multiple Endocrine Tumors Associated with Germline MAX Mutations: Multiple Endocrine Neoplasia Type 5?

CONTEXT: Pathogenic germline MAX variants are associated with pheochromocytoma and paraganglioma (PPGL), pituitary neuroendocrine tumors and, possibly, other endocrine and nonendocrine tumors. OBJECTIVE: To report 2 families with germline MAX variants, pheochromocytomas (PCs) and multiple other tumors. METHODS: Clinical, genetic, immunohistochemical, and functional studies at University hospitals in Australia on 2 families with germline MAX variants undergoing usual clinical care. The main outcome measures were phenotyping; germline and tumor sequencing; immunohistochemistry of PC and other tumors; functional studies of MAX variants. RESULTS: Family A has multiple individuals with PC (including bilateral and metastatic disease) and 2 children (to date, without PC) with neuroendocrine tumors (paravertebral ganglioneuroma and abdominal neuroblastoma, respectively). One individual has acromegaly; immunohistochemistry of PC tissue showed positive growth hormone-releasing hormone staining. Another individual with previously resected PCs has pituitary enlargement and elevated insulin-like growth factor (IGF-1). A germline MAX variant (c.200C>A, p.Ala67Asp) was identified in all individuals with PC and both children, with loss of heterozygosity in PC tissue. Immunohistochemistry showed loss of MAX staining in PCs and other neural crest tumors. In vitro studies confirmed the variant as loss of function. In Family B, the proband has bilateral and metastatic PC, prolactin-producing pituitary tumor, multigland parathyroid adenomas, chondrosarcoma, and multifocal pulmonary adenocarcinomas. A truncating germline MAX variant (c.22G>T, p.Glu8*) was identified. CONCLUSION: Germline MAX mutations are associated with PCs, ganglioneuromas, neuroblastomas, pituitary neuroendocrine tumors, and, possibly, parathyroid adenomas, as well as nonendocrine tumors of chondrosarcoma and lung adenocarcinoma, suggesting MAX is a novel multiple endocrine neoplasia gene.

Genotype-Phenotype Features of Germline Variants of the TMEM127 Pheochromocytoma Susceptibility Gene: A 10-Year Update.

PURPOSE: This work aimed to evaluate genotype-phenotype associations in individuals carrying germline variants of transmembrane protein 127 gene (TMEM127), a poorly known gene that confers susceptibility to pheochromocytoma (PHEO) and paraganglioma (PGL). DESIGN: Data were collected from a registry of probands with TMEM127 variants, published reports, and public databases. MAIN OUTCOME ANALYSIS: Clinical, genetic, and functional associations were determined. RESULTS: The cohort comprised 110 index patients (111 variants) with a mean age of 45 years (range, 21-84 years). Females were predominant (76 vs 34, P < .001). Most patients had PHEO (n = 94; 85.5%), although PGL (n = 10; 9%) and renal cell carcinoma (RCC, n = 6; 5.4%) were also detected, either alone or in combination with PHEO. One-third of the cases had multiple tumors, and known family history was reported in 15.4%. Metastatic PHEO/PGL was rare (2.8%). Epinephrine alone, or combined with norepinephrine, accounted for 82% of the catecholamine profiles of PHEO/PGLs. Most variants (n = 63) occurred only once and 13 were recurrent (2-12 times). Although nontruncating variants were less frequent than truncating changes overall, they were predominant in non-PHEO clinical presentations (36% PHEO-only vs 69% other, P < .001) and clustered disproportionately within transmembrane regions (P < .01), underscoring the relevance of these domains for TMEM127 function. Integration of clinical and previous experimental data supported classification of variants into 4 groups based on mutation type, localization, and predicted disruption. CONCLUSIONS: Patients with TMEM127 variants often resemble sporadic nonmetastatic PHEOs. PGL and RCC may also co-occur, although their causal link requires further evaluation. We propose a new classification to predict variant pathogenicity and assist with carrier surveillance.

Primary hyperparathyroidism as first manifestation in multiple endocrine neoplasia type 2A: an international multicenter study.

OBJECTIVE: Multiple endocrine neoplasia type 2A (MEN 2A) is a rare syndrome caused by RET germline mutations and has been associated with primary hyperparathyroidism (PHPT) in up to 30% of cases. Recommendations on RET screening in patients with apparently sporadic PHPT are unclear. We aimed to estimate the prevalence of cases presenting with PHPT as first manifestation among MEN 2A index cases and to characterize the former cases. DESIGN AND METHODS: An international retrospective multicenter study of 1085 MEN 2A index cases. Experts from MEN 2 centers all over the world were invited to participate. A total of 19 centers in 17 different countries provided registry data of index cases followed from 1974 to 2017. RESULTS: Ten cases presented with PHPT as their first manifestation of MEN 2A, yielding a prevalence of 0.9% (95% CI: 0.4-1.6). 9/10 cases were diagnosed with medullary thyroid carcinoma (MTC) in relation to parathyroid surgery and 1/10 was diagnosed 15 years after parathyroid surgery. 7/9 cases with full TNM data were node-positive at MTC diagnosis. CONCLUSIONS: Our data suggest that the prevalence of MEN 2A index cases that present with PHPT as their first manifestation is very low. The majority of index cases presenting with PHPT as first manifestation have synchronous MTC and are often node-positive. Thus, our observations suggest that not performing RET mutation analysis in patients with apparently sporadic PHPT would result in an extremely low false-negative rate, if no other MEN 2A component, specifically MTC, are found during work-up or resection of PHPT.

Glioblastoma Recurrence Correlates With Increased APE1 and Polarization Toward an Immuno-Suppressive Microenvironment.

While treatment with surgery, radiotherapy and/or chemotherapy may prolong life for patients with glioblastoma, recurrence is inevitable. What is still being discovered is how much these treatments and recurrence of disease affect the molecular profiles of these tumors and how these tumors adapt to withstand these treatment pressures. Understanding such changes will uncover pathways used by the tumor to evade destruction and will elucidate new targets for treatment development. Nineteen matched pre-treatment and post-treatment glioblastoma tumors were subjected to gene expression profiling (Fluidigm, TaqMan assays), MGMT promoter methylation analysis (pyrosequencing) and protein expression analysis of the DNA repair pathways, known to be involved in temozolomide resistance (immunohistochemistry). Gene expression profiling to molecularly subtype tumors revealed that 26% of recurrent post-treatment specimens did not match their primary diagnostic specimen subtype. Post-treatment specimens had molecular changes which correlated with known resistance mechanisms including increased expression of APEX1 (p < 0.05) and altered MGMT methylation status. In addition, genes associated with immune suppression, invasion and aggression (GPNMB, CCL5, and KLRC1) and polarization toward an M2 phenotype (CD163 and MSR1) were up-regulated in post-treatment tumors, demonstrating an overall change in the tumor microenvironment favoring aggressive tumor growth and disease recurrence. This was confirmed by in vitro studies that determined that glioma cell migration was enhanced in the presence of M2 polarized macrophage conditioned media. Further, M2 macrophage-modulated migration was markedly enhanced in post-treatment (temozolomide resistant) glioma cells. These findings highlight the ability of glioblastomas to evade not only the toxic onslaught of therapy but also to evade the immune system suggesting that immune-altering therapies may be of value in treating this terrible disease.

TERT structural rearrangements in metastatic pheochromocytomas.

Pheochromocytomas (PC) and paragangliomas (PGL) are endocrine tumors for which the genetic and clinicopathological features of metastatic progression remain incompletely understood. As a result, the risk of metastasis from a primary tumor cannot be predicted. Early diagnosis of individuals at high risk of developing metastases is clinically important and the identification of new biomarkers that are predictive of metastatic potential is of high value. Activation of TERT has been associated with a number of malignant tumors, including PC/PGL. However, the mechanism of TERT activation in the majority of PC/PGL remains unclear. As TERT promoter mutations occur rarely in PC/PGL, we hypothesized that other mechanisms - such as structural variations - may underlie TERT activation in these tumors. From 35 PC and four PGL, we identified three primary PCs that developed metastases with elevated TERT expression, each of which lacked TERT promoter mutations and promoter DNA methylation. Using whole genome sequencing, we identified somatic structural alterations proximal to the TERT locus in two of these tumors. In both tumors, the genomic rearrangements led to the positioning of super-enhancers proximal to the TERT promoter, that are likely responsible for the activation of the normally tightly repressed TERT expression in chromaffin cells.

Treatment preferences in men with erectile dysfunction: an open label study in Korean men switching from sildenafil citrate to tadalafil.

AIM: To evaluate patient preferences for sildenafil citrate or tadalafil (PDE-5 inhibitors available for the treatment of erectile dysfunction [ED]) and assess potential reasons for these preferences. METHODS: This open-label study was conducted on Korean men taking sildenafil, at least 6 weeks prior to study entry, for ED. Following screening, patients continued sildenafil treatment for 4 weeks, then after a 1-week washout period, switched to tadalafil for 8 weeks. Patients then continued with their treatment of choice during an extension phase. Psychosocial factors (time concern, spontaneity, sexual self-confidence) were evaluated using Psychological and Interpersonal Relationship Scales (PAIRS), while timing of dose to sexual attempt patterns were assessed from patient diaries. RESULTS: The present study enrolled 160 Korean men (mean age 55 years) with prior median sildenafil use of 585 days. During the extension phase, 73.7% of patients elected to take tadalafil, whereas 26.3% chose sildenafil (P < 0.001). After switching from sildenafil to tadalafil, mean PAIRS time concern scores decreased from 2.54 to 2.42 (P = 0.002), with no statistically significant differences observed between the sildenafil and tadalafil assessment phases in sexual spontaneity and self-confidence scores. Sexual attempts made > 4 h to =/< 36 h post-dose occurred in 4.5% of patients during the sildenafil assessment phase compared with 17.5% during the tadalafil assessment phase. CONCLUSION: After experiencing both sildenafil and tadalafil, the majority of patients exhibited a preference for tadalafil. This preference might be influenced by psychosocial factors, such as decreased time concerns, and a broader window of opportunity available for sexual activity.

Comprehensive analyses of somatic TP53 mutation in tumors with variable mutant allele frequency.

Somatic mutation of the tumor suppressor gene TP53 is reported in at least 50% of human malignancies. Most high-grade serous ovarian cancers (HGSC) have a mutant TP53 allele. Accurate detection of these mutants in heterogeneous tumor tissue is paramount as therapies emerge to target mutant p53. We used a Fluidigm Access Array™ System with Massively Parallel Sequencing (MPS) to analyze DNA extracted from 76 serous ovarian tumors. This dataset has been made available to researchers through the European Genome-phenome Archive (EGA; EGAS00001002200). Herein, we present analyses of this dataset using HaplotypeCaller and MuTect2 through the Broad Institute's Genome Analysis Toolkit (GATK). We anticipate that this TP53 mutation dataset will be useful to researchers developing and testing new software to accurately determine high and low frequency variant alleles in heterogeneous aneuploid tumor tissue. Furthermore, the analysis pipeline we present provides a valuable framework for determining somatic variants more broadly in tumor tissue.

Utility of the succinate: Fumarate ratio for assessing SDH dysfunction in different tumor types.

OBJECTIVE: Mutations of genes encoding the four subunits of succinate dehydrogenase (SDH) have been associated with pheochromocytoma and paraganglioma (PPGLs), gastrointestinal stromal tumors (GISTs) and renal cell carcinomas (RCCs). These tumors have not been characterized in a way that reflects severity of SDH dysfunction. Mass spectrometric analysis now allows measurement of metabolites extracted from formalin fixed paraffin embedded (FFPE) specimens. We assess whether SDH deficiency in various tumor types characterized by loss of SDHB protein expression correlates with SDH dysfunction as assessed by the ratio of succinate:fumarate in FFPE specimens. PATIENTS AND METHODS: Sections of FFPE tumor specimens from 18 PPGL, 10 GIST and 11 RCC patients with known SDHx mutation status for SDH deficiency were collected for mass spectrometric analysis of succinate and fumarate. RESULTS: FFPE samples showed higher succinate:fumarate ratios in SDH-deficient PPGLs compared to SDH-sufficient PPGLs. Similarly, a higher succinate:fumarate ratio was able to distinguish SDH-deficient GISTs and RCCs from their SDH-sufficient counterparts with great selectivity. Interestingly, the cut-off value of the succinate:fumarate ratio was two-folds greater in RCCs than GISTs. CONCLUSION: Analyzing biochemical imbalances preserved in FFPE specimens with mass spectrometry expands the method and sample type repertoire available for characterisation of multiple neoplasias associated with SDH deficiency.

Expression profiling reveals a distinct transcription signature in follicular thyroid carcinomas with a PAX8-PPAR(gamma) fusion oncogene.

The demonstration of the PAX8-PPAR(gamma) fusion oncogene in a subset of follicular thyroid tumors provides a new and promising starting point to dissect the molecular genetic events involved in the development of this tumor form. In the present study, we compared the gene expression profiles of follicular thyroid carcinomas (FTCs) bearing a PAX8-PPAR(gamma) fusion against FTCs that lack this fusion. Using unsupervised clustering and multidimensional scaling analyses, we show that FTCs possessing a PAX8-PPAR(gamma) fusion have a highly uniform and distinct gene expression signature that clearly distinguishes them from FTCs without the fusion. The PAX8-PPAR(gamma)(+) FTCs grouped in a defined cluster, where highly ranked genes were mostly associated with signal transduction, cell growth and translation control. Notably, a large number of ribosomal protein and translation-associated genes were concurrently underexpressed in the FTCs with the fusion. Taken together, our findings further support that follicular carcinomas with a PAX8-PPAR(gamma) rearrangement constitute a distinct biological entity. The current data represent one step to elucidate the molecular pathways in the development of FTCs with the specific PAX8-PPAR(gamma) fusion.

Intratumoral heterogeneity identified at the epigenetic, genetic and transcriptional level in glioblastoma.

Heterogeneity is a hallmark of glioblastoma with intratumoral heterogeneity contributing to variability in responses and resistance to standard treatments. Promoter methylation status of the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) is the most important clinical biomarker in glioblastoma, predicting for therapeutic response. However, it does not always correlate with response. This may be due to intratumoral heterogeneity, with a single biopsy unlikely to represent the entire lesion. Aberrations in other DNA repair mechanisms may also contribute. This study investigated intratumoral heterogeneity in multiple glioblastoma tumors with a particular focus on the DNA repair pathways. Transcriptional intratumoral heterogeneity was identified in 40% of cases with variability in MGMT methylation status found in 14% of cases. As well as identifying intratumoral heterogeneity at the transcriptional and epigenetic levels, targeted next generation sequencing identified between 1 and 37 unique sequence variants per specimen. In-silico tools were then able to identify deleterious variants in both the base excision repair and the mismatch repair pathways that may contribute to therapeutic response. As these pathways have roles in temozolomide response, these findings may confound patient management and highlight the importance of assessing multiple tumor biopsies.

Assessing mutant p53 in primary high-grade serous ovarian cancer using immunohistochemistry and massively parallel sequencing.

The tumour suppressor p53 is mutated in cancer, including over 96% of high-grade serous ovarian cancer (HGSOC). Mutations cause loss of wild-type p53 function due to either gain of abnormal function of mutant p53 (mutp53), or absent to low mutp53. Massively parallel sequencing (MPS) enables increased accuracy of detection of somatic variants in heterogeneous tumours. We used MPS and immunohistochemistry (IHC) to characterise HGSOCs for TP53 mutation and p53 expression. TP53 mutation was identified in 94% (68/72) of HGSOCs, 62% of which were missense. Missense mutations demonstrated high p53 by IHC, as did 35% (9/26) of non-missense mutations. Low p53 was seen by IHC in 62% of HGSOC associated with non-missense mutations. Most wild-type TP53 tumours (75%, 6/8) displayed intermediate p53 levels. The overall sensitivity of detecting a TP53 mutation based on classification as 'Low', 'Intermediate' or 'High' for p53 IHC was 99%, with a specificity of 75%. We suggest p53 IHC can be used as a surrogate marker of TP53 mutation in HGSOC; however, this will result in misclassification of a proportion of TP53 wild-type and mutant tumours. Therapeutic targeting of mutp53 will require knowledge of both TP53 mutations and mutp53 expression.

Pheo-Type: A Diagnostic Gene-expression Assay for the Classification of Pheochromocytoma and Paraganglioma.

CONTEXT: Pheochromocytomas and paragangliomas (PPGLs) are heritable neoplasms that can be classified into gene-expression subtypes corresponding to their underlying specific genetic drivers. OBJECTIVE: This study aimed to develop a diagnostic and research tool (Pheo-type) capable of classifying PPGL tumors into gene-expression subtypes that could be used to guide and interpret genetic testing, determine surveillance programs, and aid in elucidation of PPGL biology. DESIGN: A compendium of published microarray data representing 205 PPGL tumors was used for the selection of subtype-specific genes that were then translated to the Nanostring gene-expression platform. A support vector machine was trained on the microarray dataset and then tested on an independent Nanostring dataset representing 38 familial and sporadic cases of PPGL of known genotype (RET, NF1, TMEM127, MAX, HRAS, VHL, and SDHx). Different classifier models involving between three and six subtypes were compared for their discrimination potential. RESULTS: A gene set of 46 genes and six endogenous controls was selected representing six known PPGL subtypes; RTK1-3 (RET, NF1, TMEM127, and HRAS), MAX-like, VHL, and SDHx. Of 38 test cases, 34 (90%) were correctly predicted to six subtypes based on the known genotype to gene-expression subtype association. Removal of the RTK2 subtype from training, characterized by an admixture of tumor and normal adrenal cortex, improved the classification accuracy (35/38). Consolidation of RTK and pseudohypoxic PPGL subtypes to four- and then three-class architectures improved the classification accuracy for clinical application. CONCLUSIONS: The Pheo-type gene-expression assay is a reliable method for predicting PPGL genotype using routine diagnostic tumor samples.

Fumarate Hydratase-deficient Uterine Leiomyomas Occur in Both the Syndromic and Sporadic Settings.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome secondary to germline fumarate hydratase (FH) mutation presents with cutaneous and uterine leiomyomas, and a distinctive aggressive renal carcinoma. Identification of HLRCC patients presenting first with uterine leiomyomas may allow early intervention for renal carcinoma. We reviewed the morphology and immunohistochemical (IHC) findings in patients with uterine leiomyomas and confirmed or presumed HLRCC. IHC was also performed on a tissue microarray of unselected uterine leiomyomas and leiomyosarcomas. FH-deficient leiomyomas underwent Sanger and massively parallel sequencing on formalin-fixed paraffin-embedded tissue. All 5 patients with HLRCC had at least 1 FH-deficient leiomyoma: defined as completely negative FH staining with positive internal controls. One percent (12/1152) of unselected uterine leiomyomas but 0 of 88 leiomyosarcomas were FH deficient. FH-deficient leiomyoma patients were younger (42.7 vs. 48.8 y, P=0.024) and commonly demonstrated a distinctive hemangiopericytomatous vasculature. Other features reported to be associated with FH-deficient leiomyomas (hypercellularity, nuclear atypia, inclusion-like nucleoli, stromal edema) were inconstantly present. Somatic FH mutations were identified in 6 of 10 informative unselected FH-deficient leiomyomas. None of these mutations were found in the germline. We conclude that, while the great majority of patients with HLRCC will have FH-deficient leiomyomas, 1% of all uterine leiomyomas are FH deficient usually due to somatic inactivation. Although IHC screening for FH may have a role in confirming patients at high risk for hereditary disease before genetic testing, prospective identification of FH-deficient leiomyomas is of limited clinical benefit in screening unselected patients because of the relatively high incidence of somatic mutations.