Deep-penetrating-nevus-like melanoma arising in patients with familial adenomatous polyposis syndrome.

Deep penetrating nevi (DPN) are uncommon but distinctive melanocytic neoplasms that show an epithelioid to spindle cell morphology, prominent pigmentation with melanophages, and a plexiform growth pattern. Molecularly, most DPN are thought to be characterized by dual activation of the mitogen-activated protein kinase and the wingless-related integration site (Wnt) pathways, the latter being most commonly driven by activating ß-catenin mutations. DPN-like melanomas are very rare but can be recognized through their overlapping morphologic and architectural features with DPN. Familial adenomatous polyposis (FAP) is a hereditary cancer predisposition syndrome associated with multiple tumor types including colorectal carcinoma and desmoid fibromatosis. Like DPN, FAP is also driven by activation of the Wnt pathway, most commonly through loss of function mutations in APC, which is a major negative regulator of ß-catenin. Here we report two cases of DPN-like melanoma arising in FAP patients. While the small number of cases precludes definitive establishment of an etiologic link between these entities, the shared molecular pathogenesis of DPN-like lesions and FAP suggests that FAP patients may be at increased risk for this rare subtype of melanoma.

Primary cutaneous SMARCA4-deficient undifferentiated malignant neoplasm: first two cases with clinicopathologic and molecular comparison to eight visceral counterparts.

SMARCA4-deficient undifferentiated malignant neoplasms (SD-UMN) comprise a group of aggressive tumors with epithelioid morphology that are characterized by loss of function of SMARCA4, a component of the SWI/SNF chromatin remodeling complex. SD-UMN was first recognized in the thoracic cavity but is now appreciated to occur at multiple anatomic sites. A notable exception has been skin. Here we report the first two cases of primary cutaneous SD-UMN and compare their features to a cohort of eight visceral cases arising in lung, gastrointestinal tract, and gallbladder. Evidence for a bona fide cutaneous origin included extensive clinical, radiologic, and serologic analyses that failed to identify a metastatic source as well as the molecular identification of a UV-associated mutational pattern. The cutaneous cases showed strikingly similar morphologic, immunohistochemical, and molecular features to the visceral cases, strongly suggesting that they belong to this family of tumors. In addition to biallelic inactivation of SMARCA4, both cutaneous tumors also showed biallelic inactivation of TP53 and CDKN2A, findings which also appear common in visceral cases. One patient died of disease at 18 months after diagnosis, consistent with the aggressive nature of this tumor. Our results expand the anatomic spectrum of SD-UMN, adding this entity to an already challenging differential diagnosis that includes melanoma, squamous cell carcinoma, Merkel cell carcinoma, epithelioid sarcoma, and others. Given the potentially aggressive nature of SD-UMN, the timely and accurate diagnosis of this entity may have implications for prognosis and therapy.

Hedgehog Pathway Alterations Downstream of Patched-1 Are Common in Infundibulocystic Basal Cell Carcinoma.

ABSTRACT: The infundibulocystic variant of basal cell carcinoma (BCC) is characterized histologically by anastamosing strands of basaloid epithelium with associated small infundibular-type cysts. Since its first description in 1987, this rare entity has generated considerable controversy with some authors classifying it as a benign follicular neoplasm rather than a BCC subtype. Prior studies aiming to settle this issue using immunohistochemical analysis reached opposite conclusions. The defining feature of BCC is activation of the Hedgehog signaling pathway, and mutations in Patched-1 (PTCH1) are the most common molecular finding in both sporadic and inherited forms of BCC. Mutations in other downstream components including Smoothened (SMO) and Suppressor of Fused (SUFU) also occur, but are much less common. Here, we report a molecular genetic analysis of a small series of infundibulocystic BCC using a next-generation DNA sequencing platform. All 4 cases harbored mutations or other genetic alterations in components of the Hedgehog pathway, supporting the classification of this entity as a BCC variant. Interestingly, these tumors were enriched for genetic alterations downstream of PTCH1, involving SUFU, SMO, GLI1, and GLI2. This observation was of particular interest given that rare kindreds of the Multiple Hereditary Infundibulocystic BCC syndrome (MHIBCC), which is related, but possibly distinct from the nevoid BCC syndrome, harbored mutations in SUFU. Our results support the classification of the infundibulocystic variant as a subtype of BCC, and suggest that the level at which genetic alterations occur within the Hedgehog pathway may be an important determinant of the morphologic features in BCC.

Targeted Genomic Profiling of Female Adnexal Tumors of Probable Wolffian Origin (FATWO).

Female adnexal tumor of probable Wolffian origin (FATWO) is a rare gynecologic neoplasm of low-malignant potential presumed to be derived from mesonephric remnants in the upper female genital tract. Similarly, mesonephric remnants in the lower female genital tract are thought to be the origin for mesonephric carcinoma. Although the molecular alterations in mesonephric carcinoma have been recently reported, the pathogenesis of and molecular alterations in FATWO are not well understood. The aims of this study were to examine the molecular alterations in FATWO and to establish whether these neoplasms are molecularly similar to mesonephric carcinoma. Eight FATWOs underwent massively parallel sequencing to detect single nucleotide variations, copy number variations, and structural variants by surveying exonic DNA sequences of 300 cancer genes and 113 introns across 35 genes. Good quality DNA was isolated from 7 of 8 cases. Novel KMT2D variants (1 frameshift, 3 missense) were identified in 4 of 7 cases (57%), but were variants of uncertain biologic significance. STK11 mutations (both frameshift) were identified in 2 of 7 cases (29%); one of these was in a patient with a known history of Peutz-Jeghers syndrome. A mutation in the chromatin remodeling gene ARID1B was identified in 1 of 7 cases (14%). No cases harbored KRAS, NRAS, TP53, PIK3CA, PTEN, or DICER1 mutations. There were relatively low numbers of copy number variations, and no recurrent copy number variations were identified. One case demonstrated moderate copy gain of CCND1. No structural variants were identified. In summary, FATWO is characterized molecularly by the absence of KRAS/NRAS mutations (characteristic of mesonephric carcinoma), absence of DICER1 mutations (characteristic of Sertoli-Leydig cell tumor) and frequent KMT2D mutations of unknown biologic significance. FATWOs exhibit a limited number of molecular aberrations that are significantly different from those reported in tumors in the differential diagnosis, and our results question the relationship of mesonephric carcinoma with FATWO. Disease-defining molecular alterations for FATWO have yet to be discovered.

Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing.

BACKGROUND: Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk. RESULTS: Cross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative. CONCLUSIONS: For sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. These results suggest that use of such adapters is critical to reduce false positive rates in assays that aim to identify low allele frequency events, and strongly indicate that dual-matched adapters be implemented for all sensitive MPS applications.

SMARCA4-deficient undifferentiated uterine sarcoma (malignant rhabdoid tumor of the uterus): a clinicopathologic entity distinct from undifferentiated carcinoma.

Small cell carcinoma of the ovary, hypercalcemic type is a rare, aggressive malignancy which usually occurs in young women and is characterized by mutations in SMARCA4, with few other alterations. We recently encountered uterine tumors with morphologic, immunohistochemical, and genetic similarities to small cell carcinoma of the ovary, hypercalcemic type. Herein we report the clinicopathologic and molecular features (using a targeted massively parallel sequencing [MPS] assay) of these tumors. The cases were diagnosed on cervical and endometrial biopsies (n = 2, 34, and 29 years) or hysterectomy and bilateral salpingo-oophorectomy (n = 3, 25, 33, and 58 years). The tumors were composed of sheets of large atypical epithelioid cells with prominent rhabdoid morphology, indistinguishable from the "large cell" variant of small cell carcinoma of the ovary, hypercalcemic type. In three cases, the ovaries were pathologically examined to exclude a primary ovarian malignancy. Immunohistochemically, four of four cases showed SMARCA4 loss, and were negative or only focally positive for keratins, EMA, and claudin-4. One of three cases was positive for WT-1. Targeted MPS was successfully performed on 4 of 5 tumors, and showed recurrent mutations in SMARCA4, with few other alterations. Of the cases diagnosed on hysterectomy, all had extensive lymphovascular invasion, extra-uterine spread, and marked infiltrative growth. These tumors were uniformly aggressive; all patients died of disease (median survival 7 months, range 1-43 months). We propose this entity be called "SMARCA4-deficient undifferentiated uterine sarcoma (malignant rhabdoid tumor of the uterus)", a term which describes both the tumor's underlying molecular abnormality and its morphology. Its unique clinicopathologic and molecular features differentiate it from other related malignancies, including undifferentiated endometrial carcinoma, small cell carcinoma of the ovary (hypercalcemic type), and epithelioid sarcoma. We review and discuss previously reported "rhabdoid tumors of the uterus;" while they are a heterogenous group of tumors, some of them are likely examples of this entity. Correctly identifying cases of SMARCA4-deficient uterine sarcoma from histologic mimics is important as it may have prognostic, predictive, and germline implications.

Validation of a targeted next-generation sequencing approach to detect mismatch repair deficiency in colorectal adenocarcinoma.

Mismatch repair protein deficiency is a hallmark of cancers associated with Lynch syndrome and is a biomarker for response to immunotherapy. With the increasing adoption of cancer next-generation sequencing, there has been a movement to develop screening approaches that take advantage of the unique mutational signatures of mismatch repair-deficient tumors. Here, we develop a sequencing-based metric that distinguishes mismatch repair-deficient from mismatch repair-proficient colorectal adenocarcinomas with comparison to immunohistochemical staining. We find that a single criterion of three or more single base pair insertion or deletion mutations per megabase sequenced, occurring in mononucleotide repeat regions of four or more nucleotides, is sufficient to detect mismatch repair deficiency with 96% sensitivity and 100% specificity in a training set of 241 cancers and 96% sensitivity and 99% specificity in a validation set of 436 additional cancers. Using data from the same cohort, we also find that sequencing information from only three genes-ARID1A, KMT2D, and SOX9-is sufficient to detect mismatch repair-deficient colorectal adenocarcinomas with 76% sensitivity and 98% specificity in the validation set. These findings support the notion that targeted next-generation sequencing already being performed for clinical or research purposes can also be used to accurately detect mismatch repair deficiency in colorectal adenocarcinomas.

The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.

The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of 'personalized' therapeutic regimens.

Differentiated exophytic vulvar intraepithelial lesions are genetically distinct from keratinizing squamous cell carcinomas and contain mutations in PIK3CA.

Human papillomavirus-negative keratinizing vulvar cancers typically harbor TP53 mutations as do their precursors, differentiated vulvar intraepithelial neoplasia. However, atypical verruciform proliferations are also associated with these malignancies and their pathogenesis is poorly understood. This study compared 11 atypical verruciform lesions, including atypical verruciform hyperplasia, vulvar acanthosis with altered differentiation, and verruciform lichen simplex chronicus, with 14 human papillomavirus-negative keratinizing squamous cell carcinomas. Extracted tissue DNA was subjected to targeted massively parallel sequencing of the exonic regions of 300 genes. Eight (73%) and six (55%) of eleven atypical verruciform lesions contained mutations in PIK3CA and ARID2, respectively. No TP53 mutations were identified. Eleven (79%) and five (36%) of fourteen keratinizing squamous cell carcinomas tested contained TP53 and CDKN2A mutations, respectively. Keratinizing squamous cell carcinomas displayed the majority of copy number variations with some variations (7p gain and 8p loss) shared by some cases in both groups. One patient developed atypical verruciform lesions with PIK3CA mutations followed by a keratinizing carcinoma with mutations in both PIK3CA and TP53. This study, for the first time segregates atypical verruciform lesions by virtue of a unique genotype (PIK3CA mutant/TP53 wild type) illustrating an example of progression to a TP53-mutated keratinizing carcinoma. The findings indicate that although PIK3CA mutations are found in <10% of vulvar squamous cell carcinomas, they may be specific for a particular pathway involving atypical verruciform lesions, which could function as either a direct precursor or a risk factor for vulvar squamous cell carcinoma. Given the presence of a molecular signature, we propose the term 'differentiated exophytic vulvar intraepithelial lesion' for this group. Whether they function as direct precursors to a less common form of squamous cell carcinoma will require further study, but carcinomas associated with these lesions might warrant testing for PIK3CA mutations to address this question.

BreaKmer: detection of structural variation in targeted massively parallel sequencing data using kmers.

Genomic structural variation (SV), a common hallmark of cancer, has important predictive and therapeutic implications. However, accurately detecting SV using high-throughput sequencing data remains challenging, especially for 'targeted' resequencing efforts. This is critically important in the clinical setting where targeted resequencing is frequently being applied to rapidly assess clinically actionable mutations in tumor biopsies in a cost-effective manner. We present BreaKmer, a novel approach that uses a 'kmer' strategy to assemble misaligned sequence reads for predicting insertions, deletions, inversions, tandem duplications and translocations at base-pair resolution in targeted resequencing data. Variants are predicted by realigning an assembled consensus sequence created from sequence reads that were abnormally aligned to the reference genome. Using targeted resequencing data from tumor specimens with orthogonally validated SV, non-tumor samples and whole-genome sequencing data, BreaKmer had a 97.4% overall sensitivity for known events and predicted 17 positively validated, novel variants. Relative to four publically available algorithms, BreaKmer detected SV with increased sensitivity and limited calls in non-tumor samples, key features for variant analysis of tumor specimens in both the clinical and research settings.

Morphologic correlates of molecular alterations in extrauterine Müllerian carcinomas.

Extrauterine high-grade serous carcinomas can exhibit various histologic patterns including (1) classic architecture that is papillary, micropapillary and infiltrative and (2) solid, endometrioid, and transitional (ie, SET) patterns. Although the SET pattern has been associated with germline BRCA mutations, potential molecular underpinnings have not been fully investigated. DNA was isolated from 174 carcinomas of the fallopian tube, ovary, or peritoneum. Targeted next-generation sequencing was performed and single-nucleotide and copy number variants were correlated with morphologic subtype. Overall, 79% of tumors were classified as high-grade serous carcinoma (n=138), and the most common mutations in high-grade serous carcinomas were TP53 (94%), BRCA1 (25%), BRCA2 (11%), and ATM (7%). Among chemotherapy-naive high-grade serous carcinomas, 40 cases exhibited classic morphology and 40 cases had non-classic morphology (SET or ambiguous features). Mutations in homologous recombination pathways were seen across all tumor histotypes. High-grade serous carcinomas with homologous recombination mutations were six times more likely to be associated with non-classic histology (P=0.002) and were significantly more likely to be platinum sensitive and have improved progression-free survival (PFS) (P=0.007 and P=0.004, respectively). In a multivariate analysis adjusted for age, homologous recombination mutation status and increased copy number variants were independently associated with improved PFS (P=0.008 and P=0.005, respectively). These findings underscore the potential significance of variant morphologic patterns and comprehensive genomic analysis in high-grade serous carcinomas with potential implications for pathogenesis, as well as response to targeted therapies.

Somatic activating ARAF mutations in Langerhans cell histiocytosis.

The extracellular signal-regulated kinase (ERK) signaling pathway is activated in Langerhans cell histiocytosis (LCH) histiocytes, but only 60% of cases carry somatic activating mutations of BRAF. To identify other genetic causes of ERK pathway activation, we performed whole exome sequencing on purified LCH cells in 3 cases. One patient with wild-type BRAF alleles in his histiocytes had compound mutations in the kinase domain of ARAF. Unlike wild-type ARAF, this mutant was a highly active mitogen-activated protein kinase kinase in vitro and was capable of transforming mouse embryo fibroblasts. Mutant ARAF activity was inhibited by vemurafenib, a BRAF inhibitor, indicating the importance of fully evaluating ERK pathway abnormalities in selecting LCH patients for targeted inhibitor therapy.

Targeted genomic analysis of Müllerian adenosarcoma.

Müllerian adenosarcoma (MA) is a rare mixed mesenchymal tumour of the female genital tract, composed of malignant stroma and benign-appearing epithelium. Sarcomatous overgrowth (SO) is the only established histological variable associated with higher stage and shorter survival. Specific molecular or immunohistochemistry (IHC) tools for the diagnosis of MA are lacking. Our goal was to study genomic mutations and copy number variations (CNVs) in MA to understand better its pathobiology, and develop specific diagnostic and prognostic tools. DNA was extracted from 20 samples of MA from 18 subjects (12 without SO and 6 with SO), including two in which areas of both typical MA histology and SO were independently tested. Samples were analysed using a targeted next-generation sequencing assay interrogating exonic sequences of 275 cancer genes for mutations and CNVs as well as 91 introns across 30 genes for cancer-associated rearrangements. The mean number of mutations in MA with SO (mean 9.7; range 3-14) did not differ significantly from that in MA without SO (mean 9.6; range 5-16). MA with SO had significantly higher mean numbers of gene-level CNVs (24.6) compared to MA without SO (5; p = 0.0002). The most frequent amplification involved MDM2 and CDK4 (5/18; 28%), accompanied by focal CDK4 and MDM2 and diffuse HMGA2 expression using immunohistochemistry. MYBL1 amplification was seen in 4/18 (22%), predominantly in SO. Alterations in PIK3CA/AKT/PTEN pathway members were seen in 13/18 (72%). Notably, TP53 mutations were uncommon, present in only two cases with SO. Three out of 18 (17%) had mutations in ATRX, all associated with SO. No chromosomal rearrangements were identified. We have identified a number of recurrent genomic alterations in MA, including some associated with SO. Although further investigation of these findings is needed, confirmation of one or more may lead to new mechanistic insights and novel markers for this often difficult-to-diagnose tumour.

Genomic analysis of diffuse pediatric low-grade gliomas identifies recurrent oncogenic truncating rearrangements in the transcription factor MYBL1.

Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from BRAF kinase mutations or duplications in specific subclasses, few genetic driver events are known. Diffuse PLGGs comprise a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. We performed high-resolution copy-number analysis on 44 formalin-fixed, paraffin-embedded diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gain, was observed in 28% of diffuse astrocytoma grade IIs and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene v-myb avian myeloblastosis viral oncogene homolog (MYB) on 6q23.3. Whole-genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.

High-throughput oncogene mutation profiling in human cancer.

Systematic efforts are underway to decipher the genetic changes associated with tumor initiation and progression. However, widespread clinical application of this information is hampered by an inability to identify critical genetic events across the spectrum of human tumors with adequate sensitivity and scalability. Here, we have adapted high-throughput genotyping to query 238 known oncogene mutations across 1,000 human tumor samples. This approach established robust mutation distributions spanning 17 cancer types. Of 17 oncogenes analyzed, we found 14 to be mutated at least once, and 298 (30%) samples carried at least one mutation. Moreover, we identified previously unrecognized oncogene mutations in several tumor types and observed an unexpectedly high number of co-occurring mutations. These results offer a new dimension in tumor genetics, where mutations involving multiple cancer genes may be interrogated simultaneously and in 'real time' to guide cancer classification and rational therapeutic intervention.

MAP2K1 and MAP3K1 mutations in Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is now understood to be a neoplastic disease in which over 50% of cases have somatic activating mutations of BRAF. However, the extracellular signal-related (ERK) pathway is activated in all cases including those with wild type BRAF alleles. Here, we applied a targeted massively parallel sequencing panel to 30 LCH samples to test for the presence of additional genetic alterations that might cause ERK pathway activation. In 20 BRAF wild type samples, we found 3 somatic mutations in MAP2K1 (MEK1) including C121S and C121S/G128D in the kinase domain, and 56_61QKQKVG>R, an in-frame deletion in the N-terminal regulatory domain. All three variant proteins constitutively phosphorylated ERK in in vitro kinase assays. The C121S/G128D and 56_61QKQKVG>R variants were resistant to the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib in vitro. Within the entire sample set, we found 3 specimens with mutations in MAP3K1 (MEKK1), including two truncation mutants, T779fs and T1481fs; T1481fs encoded an unstable and nonfunctional protein when expressed in vitro. T779fs was present in a specimen carrying BRAF V600E. The third variant was a single nucleotide substitution, E1286V, which was fully functional and is likely a germline polymorphism. These results indicate that LCH cells can harbor additional genetic alterations in the RAS-RAF-MEK pathway which, in the case of MAP2K1, may be responsible for ERK activation in a wild type BRAF setting. The resistance of some of these variants to trametinib may also have clinical implications for the combined use of RAF and MEK inhibitors in LCH.

Genomic aberrations in cervical adenocarcinomas in Hong Kong Chinese women.

Although the rates of cervical squamous cell carcinoma have been declining, the rates of cervical adenocarcinoma are increasing in some countries. Outcomes for advanced cervical adenocarcinoma remain poor. Precision mapping of genetic alterations in cervical adenocarcinoma may enable better selection of therapies and deliver improved outcomes when combined with new sequencing diagnostics. We present whole-exome sequencing results from 15 cervical adenocarcinomas and paired normal samples from Hong Kong Chinese women. These data revealed a heterogeneous mutation spectrum and identified several frequently altered genes including FAT1, ARID1A, ERBB2 and PIK3CA. Exome sequencing identified human papillomavirus (HPV) sequences in 13 tumors in which the HPV genome might have integrated into and hence disrupted the functions of certain exons, raising the possibility that HPV integration can alter pathways other than p53 and pRb. Together, these provisionary data suggest the potential for individualized therapies for cervical adenocarcinoma based on genomic information.

Targeted genomic profiling reveals recurrent KRAS mutations and gain of chromosome 1q in mesonephric carcinomas of the female genital tract.

Mesonephric carcinoma is a rare form of gynecologic cancer derived from mesonephric remnants usually located in the lateral wall of the uterine cervix. An analogous tumor occurs in the adnexa, female adnexal tumor of probable Wolffian origin. The pathogenesis and molecular events in mesonephric carcinoma are not known. The aim of this study was to examine the molecular alterations in mesonephric carcinoma to identify driver mutations and therapeutically targetable mutations. This study consisted of 19 tumors from 17 patients: 18 mesonephric carcinomas (15 primary tumors and three metastatic tumors) and 1 female adnexal tumor of probable Wolffian origin. In two patients, both primary and metastatic tumors were available. Genomic DNA was isolated and targeted next-generation sequencing was performed to detect mutations, copy number variations, and structural variants by surveying full exonic regions of 300 cancer genes and 113 selected intronic regions across 35 genes. Fluorescence in situ hybridization (FISH) for 1p and 1q was performed in two cases. Eighty-one percent (13/16) of mesonephric carcinomas had either a KRAS (n=12) or NRAS (n=1) mutation. Mutations in chromatin remodeling genes (ARID1A, ARID1B, or SMARCA4) were present in 62% of mesonephric carcinomas. All mesonephric carcinomas lacked mutations in PIK3CA and PTEN. The most common copy number alteration was 1q gain, found in 12 (75%) mesonephric carcinomas; this was confirmed by FISH in two cases. Mesonephric carcinoma is characterized by molecular alterations that differ from those of more common variants of cervical and endometrial adenocarcinoma, which harbor KRAS/NRAS mutations in 7% and 25% of cases, respectively. KRAS/NRAS mutations are common in mesonephric carcinoma and are often accompanied by gain of 1q and mutations in chromatin remodeling genes. Targeting inhibitors of the RAS/MAPK pathway may be useful in the treatment of mesonephric carcinoma.

Loss of ATRX, genome instability, and an altered DNA damage response are hallmarks of the alternative lengthening of telomeres pathway.

The Alternative Lengthening of Telomeres (ALT) pathway is a telomerase-independent pathway for telomere maintenance that is active in a significant subset of human cancers and in vitro immortalized cell lines. ALT is thought to involve templated extension of telomeres through homologous recombination, but the genetic or epigenetic changes that unleash ALT are not known. Recently, mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 were found to correlate with features of ALT in pancreatic neuroendocrine cancers, pediatric glioblastomas, and other tumors of the central nervous system, suggesting that these mutations might contribute to the activation of the ALT pathway in these cancers. We have taken a comprehensive approach to deciphering ALT by applying genomic, molecular biological, and cell biological approaches to a panel of 22 ALT cell lines, including cell lines derived in vitro. Here we show that loss of ATRX protein and mutations in the ATRX gene are hallmarks of ALT-immortalized cell lines. In addition, ALT is associated with extensive genome rearrangements, marked micronucleation, defects in the G2/M checkpoint, and altered double-strand break (DSB) repair. These attributes will facilitate the diagnosis and treatment of ALT positive human cancers.