Co-occurrence of mutations in NF1 and other susceptibility genes in pheochromocytoma and paraganglioma.

Introduction: The percentage of patients diagnosed with pheochromocytoma and paraganglioma (altogether PPGL) carrying known germline mutations in one of the over fifteen susceptibility genes identified to date has dramatically increased during the last two decades, accounting for up to 35-40% of PPGL patients. Moreover, the application of NGS to the diagnosis of PPGL detects unexpected co-occurrences of pathogenic allelic variants in different susceptibility genes. Methods: Herein we uncover several cases with dual mutations in NF1 and other PPGL genes by targeted sequencing. We studied the molecular characteristics of the tumours with co-occurrent mutations, using omic tools to gain insight into the role of these events in tumour development. Results: Amongst 23 patients carrying germline NF1 mutations, targeted sequencing revealed additional pathogenic germline variants in DLST (n=1) and MDH2 (n=2), and two somatic mutations in H3-3A and PRKAR1A. Three additional patients, with somatic mutations in NF1 were found carrying germline pathogenic mutations in SDHB or DLST, and a somatic truncating mutation in ATRX. Two of the cases with dual germline mutations showed multiple pheochromocytomas or extra-adrenal paragangliomas - an extremely rare clinical finding in NF1 patients. Transcriptional and methylation profiling and metabolite assessment showed an "intermediate signature" to suggest that both variants had a pathological role in tumour development. Discussion: In conclusion, mutations affecting genes involved in different pathways (pseudohypoxic and receptor tyrosine kinase signalling) co-occurring in the same patient could provide a selective advantage for the development of PPGL, and explain the variable expressivity and incomplete penetrance observed in some patients.

Recurrent Germline DLST Mutations in Individuals with Multiple Pheochromocytomas and Paragangliomas.

Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and 13C5-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (∼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.

Metastatic clear cell renal cell carcinoma to the thyroid gland: A clinico-pathological and immunohistochemical study of 8 cases and review of the literature.

When a patient with a previous history of neoplasm presents with a thyroid lesion, the possibility of it being metastatic should always be considered. In this series, we present the clinicopathological and immunohistochemical features of the thyroid metastases diagnosed in our department over the past 30 years. Here we present eight thyroidal metastases from clear cell renal cell carcinoma (ccRCCC), including a tumor to tumor metastasis, the patients being 2 men and 6 women with a median age of 62 years. The majority had a past history of goiter and a single and palpable metastasis. In one patient the thyroid metastases were the first sign of the ccRCCC. In the available cases, the metastasis showed positivity to PAX8 and CAIX and negativity to TTF1 and thyroglobulin. The median time from the detection of the primary renal tumor to thyroid metastasis and from thyroidectomy to last follow up were 84.17 and 54.50 months, respectively. After a median follow up of 158.50 months none of the patients had died from ccRCCC. Renal cell carcinoma (RCC) is the most frequent malignant neoplasm of the kidney and its incidence has increased over recent decades. In a clinical series, up to 1-3% of the oncologic thyroidectomies were due to thyroid metastases and the most frequent metastasizing tumor was RCC, followed by lung and breast cancer.

Gain-of-function mutations in DNMT3A in patients with paraganglioma.

PURPOSE: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors. METHODS: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed. RESULTS: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma. CONCLUSION: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.

The role of EZH2 in overall survival of colorectal cancer: a meta-analysis.

Enhancer of zeste homolog 2 (EZH2) is the catalitic subunit of polycomb repressive complex 2 and mediates gene silencing. EZH2 is overexpressed in many cancers and correlates with poor prognosis. The role of the gene EZH2 in colorectal cancer survival is uncertainly, the aim of this study is clear this relationship. Relevant literaure was searched from electronic databases. A meta-analysis was performed with elegible studies which quantitatively evaluated the relationship between EZH2 overexpression and survival of patients with colorectal cancer. Survival data were aggregated and quantitatively analyzed. We performed a meta-analysis of 8 studies (n = 1059 patients) that evaluated the correlation between EZH2 overexpression and survival in patients with colorectal cancer. Combined hazard ratios suggested that EZH2 overexpression was associated with better prognosis of overall survival (OS) HR(hazard ratio) = 0.61 95% CI (0.38-0.84) We performed bias analysis according Egger and Begg,s test and we did not find publication bias. EZH2 overexpression indicates a better prognosis for colorectal cancer.

Targeted Exome Sequencing of Krebs Cycle Genes Reveals Candidate Cancer-Predisposing Mutations in Pheochromocytomas and Paragangliomas.

Purpose: Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes.Experimental Design: We used downregulation and methylation of RBP1, as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases.Results: One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in IDH1 A variant in GOT2, c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in SDHC in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline IDH3B mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio.Conclusions: This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. Clin Cancer Res; 23(20); 6315-24. ©2017 AACR.

Perineurial cells in granular cell tumors and neoplasms with perineural invasion: an immunohistochemical study.

Granular cell tumors (GCT) are nerve sheath neoplasms composed of Schwann cells with granular cytoplasm. Perineurial cells are the cellular component of the perineurium and of perineuriomas, neoplasms supposedly derived from perineurial cells. However, perineurial cells have also been found in other Schwann cell-derived tumors. These cells have not been well studied in GCTs. We studied the presence of perineurial cells in a series of 24 GCTs with EMA, claudin-1, and Glut-1, which are immunohistochemical markers for perineurial cells. Three cases lacked nerve fascicles. Three cases showed no perineurial proliferation (grade 0), 7 showed grade 1 proliferation, and 11 showed grade 2 proliferation. For comparison, we studied 17 cases of neoplasms with perineural invasion (PNI): 7 cutaneous neoplasms [squamous cell carcinomas (n = 3), cutaneous lymphoma, malignant melanoma, eccrine carcinoma, congenital neurotropic nevus (n = 1 each)] and 10 noncutaneous tumors [prostatic (n = 2), gastric (n = 2), and colonic (n = 2) adenocarcinomas; invasive ductal carcinoma of breast (n = 2); urothelial carcinoma of bladder (n = 1); and oral squamous cell carcinoma (n = 1)] with the same antibodies for perineurial cells. We found perineurial cell proliferation in 10 cases, 6 grade 1, and 4 grade 2. These perineurial cells were limited to the areas around the nerve fascicles. Most of the tumor was devoid of perineurial cells. Thus, it was interpreted more as a hyperplastic or reactive phenomenon than a neoplastic component. Claudin-1 was the most sensitive of the 3 markers that we used. Such proliferation was less intense in non-GCTs. In conclusion, proliferation of perineurial cells in GCTs and neoplasms with PNI is a common finding that had not been previously studied. It seems to be a non-neoplastic phenomenon.

MAX mutations cause hereditary and sporadic pheochromocytoma and paraganglioma.

PURPOSE: Pheochromocytomas (PCC) and paragangliomas (PGL) are genetically heterogeneous neural crest-derived neoplasms. Recently we identified germline mutations in a new tumor suppressor susceptibility gene, MAX (MYC-associated factor X), which predisposes carriers to PCC. How MAX mutations contribute to PCC/PGL and associated phenotypes remain unclear. This study aimed to examine the prevalence and associated phenotypic features of germline and somatic MAX mutations in PCC/PGL. DESIGN: We sequenced MAX in 1,694 patients with PCC or PGL (without mutations in other major susceptibility genes) from 17 independent referral centers. We screened for large deletions/duplications in 1,535 patients using a multiplex PCR-based method. Somatic mutations were searched for in tumors from an additional 245 patients. The frequency and type of MAX mutation was assessed overall and by clinical characteristics. RESULTS: Sixteen MAX pathogenic mutations were identified in 23 index patients. All had adrenal tumors, including 13 bilateral or multiple PCCs within the same gland (P < 0.001), 15.8% developed additional tumors at thoracoabdominal sites, and 37% had familial antecedents. Age at diagnosis was lower (P = 0.001) in MAX mutation carriers compared with nonmutated cases. Two patients (10.5%) developed metastatic disease. A mutation affecting MAX was found in five tumors, four of them confirmed as somatic (1.65%). MAX tumors were characterized by substantial increases in normetanephrine, associated with normal or minor increases in metanephrine. CONCLUSIONS: Germline mutations in MAX are responsible for 1.12% of PCC/PGL in patients without evidence of other known mutations and should be considered in the genetic work-up of these patients.

Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma.

Hereditary pheochromocytoma (PCC) is often caused by germline mutations in one of nine susceptibility genes described to date, but there are familial cases without mutations in these known genes. We sequenced the exomes of three unrelated individuals with hereditary PCC (cases) and identified mutations in MAX, the MYC associated factor X gene. Absence of MAX protein in the tumors and loss of heterozygosity caused by uniparental disomy supported the involvement of MAX alterations in the disease. A follow-up study of a selected series of 59 cases with PCC identified five additional MAX mutations and suggested an association with malignant outcome and preferential paternal transmission of MAX mutations. The involvement of the MYC-MAX-MXD1 network in the development and progression of neural crest cell tumors is further supported by the lack of functional MAX in rat PCC (PC12) cells and by the amplification of MYCN in neuroblastoma and suggests that loss of MAX function is correlated with metastatic potential.

Comprehensive characterization of the DNA amplification at 13q34 in human breast cancer reveals TFDP1 and CUL4A as likely candidate target genes.

INTRODUCTION: Breast cancer subtypes exhibit different genomic aberration patterns with a tendency for high-level amplifications in distinct chromosomal regions. These genomic aberrations may drive carcinogenesis through the upregulation of proto-oncogenes. We have characterized DNA amplification at the human chromosomal region 13q34 in breast cancer. METHODS: A set of 414 familial and sporadic breast cancer cases was studied for amplification at region 13q34 by fluorescence in situ hybridization (FISH) analysis on tissue microarrays. Defining the minimal common region of amplification in those cases with amplification at 13q34 was carried out using an array-based comparative genomic hybridization platform. We performed a quantitative real-time - polymerase chain reaction (qRT-PCR) gene expression analysis of 11 candidate genes located within the minimal common region of amplification. Protein expression levels of two of these genes (TFDP1 and CUL4A) were assessed by immunohistochemical assays on the same tissue microarrays used for FISH studies, and correlated with the expression of a panel of 33 antibodies previously analyzed. RESULTS: We have found 13q34 amplification in 4.5% of breast cancer samples, but the frequency increased to 8.1% in BRCA1-associated tumors and to 20% in basal-like tumors. Tumors with 13q34 amplification were associated with high grade, estrogen receptor negativity, and expression of EGFR, CCNE, CK5, and P-Cadherin, among other basal cell markers. We have defined a 1.83 megabases minimal common region of genomic amplification and carried out mRNA expression analyses of candidate genes located therein, identifying CUL4A and TFDP1 as the most likely target genes. Moreover, we have confirmed that tumors with 13q34 amplification significantly overexpress CUL4A and TFDP1 proteins. Tumors overexpressing either CUL4A or TFDP1 were associated with tumor proliferation and cell cycle progression markers. CONCLUSIONS: We conclude that 13q34 amplification may be of relevance in tumor progression of basal-like breast cancers by inducing overexpression of CUL4A and TFDP1, which are both important in cell cycle regulation. Alternatively, as these genes were also overexpressed in non-basal-like tumor samples, they could play a wider role in cancer development by inducing tumor proliferation.

[Sarcomatoid carcinoma in a patient with Sjögren's syndrome]. / Carcinoma sarcomatoide en paciente diagnosticado de síndrome de Sjögren.

Sarcomatoid carcinoma is an extremely rare small bowel tumor whose clinical manifestations are insidious and nonspecific, ranging from diffuse abdominal pain to gastrointestinal bleeding or intestinal occlusion. Thus, diagnostic delay is highly common with poor treatment outcome and prognosis. To date, only 20 cases have been reported in the literature. We describe the case of a small bowel sarcomatoid carcinoma localized in the jejunum, with emphasis on the clinical and pathological features of this entity. The hypothetical association with Sjögren's syndrome, an autoimmune disease, is also discussed.

Estrogen receptor status could modulate the genomic pattern in familial and sporadic breast cancer.

PURPOSE: Familial breast cancer represents 5% to 10% of all breast tumors. Mutations in the two known major breast cancer susceptibility genes, BRCA1 and BRCA2, account for a minority of familial breast cancer, whereas families without mutations in these genes (BRCAX group) account for 70% of familial breast cancer cases. EXPERIMENTAL DESIGN: To better characterize and define the genomic differences between the three classes of familial tumors and sporadic malignancies, we have analyzed 19 BRCA1, 24 BRCA2, and 31 BRCAX samples from familial breast cancer patients and 19 sporadic breast tumors using a 1-Mb resolution bacterial artificial chromosome array-based comparative genomic hybridization. RESULTS: We found that BRCA1/2 tumors showed a higher genomic instability than BRCAX and sporadic cancers. There were common genomic alterations present in all breast cancer groups, such as gains of 1q and 16p or losses of 8ptel-p12 and 16q. We found that the presence/absence of the estrogen receptor (ER) may play a crucial role in driving tumor development through distinct genomic pathways independently of the tumor type (sporadic or familial) and mutation status (BRCA1 or BRCA2). ER(-) tumors presented higher genomic instability and different altered regions than ER+ ones. CONCLUSIONS: According to our results, the BRCA gene mutation status (mainly BRCA1) would contribute to the genomic profile of abnormalities by increasing or modulating the genome instability.

Immunohistochemical classification of non-BRCA1/2 tumors identifies different groups that demonstrate the heterogeneity of BRCAX families.

Around 25% of hereditary breast and ovarian cancer families have mutations in the BRCA1 and BRCA2 genes. The search for other genes has until now failed, probably because there is not one single BRCAX gene, but rather various genes that may each be responsible for a small number of breast cancer families and/or may interact according to a polygenic model. We have studied 50 tumors from probands belonging to non-BRCA1/2 breast cancer families (BRCAX), using 25 immunohistochemical markers. The objective was to classify these tumors and confirm that they are heterogeneous. Unsupervised cluster analysis showed the existence of the following two main groups of tumors: high-grade and estrogen receptor (ER)-negative tumors (50%), and low-grade and ER-positive tumors (50%). In addition we identified five subgroups, three among the high-grade and two among the low-grade groups; one overexpressing HER-2 (18%); one with a basal-like phenotype (14%); one with a normal breast-like phenotype (18%); a luminal A subgroup (36%), and a luminal B subgroup (14%). Hypermethylation of the BRCA1 gene was observed in 42% of the cases, spread across all five subgroups, but only 37% of those had loss of heterozygosity as well. These latter cases were all clustered in the high-grade group and the majority of them in the basal-like subgroup. Our results show that familial non-BRCA1/2 tumors are heterogeneous and suggest a polygenic model for explaining the majority of BRCAX families. In addition we have defined a subset of them that have somatic inactivation of the BRCA1 gene.

Identification of a proliferation signature related to survival in nodal peripheral T-cell lymphomas.

PURPOSE: Nodal peripheral T-cell lymphomas (PTCLs) constitute a heterogeneous group of neoplasms, suggesting the existence of molecular differences contributing to their histologic and clinical variability. Initial expression profiling studies of T-cell lymphomas have been inconclusive in yielding clinically relevant insights. We applied DNA microarrays to gain insight into the molecular signatures associated with prognosis. MATERIALS AND METHODS: We analyzed the expression profiles of 35 nodal PTCLs (23 PTCLs unspecified and 12 angioimmunoblastic) using two different microarray platforms, the cDNA microarray developed at the Spanish National Cancer Centre and an oligonucleotide microarray. RESULTS: We identified five clusters of genes, the expression of which varied significantly among the samples. Genes in these clusters seemed to be functionally related to different cellular processes such as proliferation, inflammatory response, and T-cell or B-cell lineages. Regardless of the microarray platform used, overexpression of genes in the proliferation signature was associated significantly with shorter survival of patients. This proliferation signature included genes commonly associated with the cell cycle, such as CCNA, CCNB, TOP2A, and PCNA. Moreover the PTCL proliferation signature showed a statistically significant inverse correlation with clusters of the inflammatory response (P < .0001), as well as with the percentage of CD68(+) cells. CONCLUSION: Our findings indicate that proliferation could be an important factor in evaluating nodal PTCL outcome and may help to define a more aggressive phenotype.

Vimentin and laminin expression is associated with basal-like phenotype in both sporadic and BRCA1-associated breast carcinomas.

AIMS: To determine whether basal-like phenotype and vimentin and/or laminin are related in both sporadic/familial (BRCA1 or BRCA2 mutated) tumours. METHODS: 230 non-familial and 28 hereditary node-negative invasive breast carcinomas were immunohistochemically analysed for oestrogen receptors (ER), progesterone receptors (PR), cytokeratin 5/6 (CK5/6), epidermal growth factor receptors (EGFR), Ki67, p53, vimentin and laminin, using tissue microarrays. Tumours were considered to have basal-like phenotype if they were ER negative and HER2 negative, but positive for CK5/6 and/or EGFR. RESULTS: In sporadic tumours, vimentin expression was found in 77.8% cases with basal-like phenotype and 15.5% of non-basal cases (p<0.001). In familial cases, vimentin was expressed in 83.3% basal-like cancers and 16.7% of non-basal tumours (p<0.001). Vimentin expression was more frequent in BRCA1 than BRCA2 mutation carriers. Vimentin expressing tumours were associated with poor prognosis (p = 0.012) among patients not receiving adjuvant chemotherapy and showed a trend for local recurrence or visceral but not bone metastasis (p = 0.021). Laminin expression was also related to basal-like phenotype in both sporadic/familial cases (p<0.001 and p = 0.007, respectively), but neither with prognosis nor recurrence pattern in sporadic cancers. CONCLUSIONS: Vimentin and laminin expression is associated with basal-like phenotype in breast cancer. Expression of vimentin and laminin is characteristic of BRCA1 associated tumours. Since vimentin and laminin staining is widely used by pathologists for diagnostic purposes, thus demonstrating the robustness of their specific antibodies, the immunohistochemical evaluation of these two molecules could be used in identification of basal-like breast tumours in both sporadic/familial cases.