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Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is caused by de novo loss-of-function variants in the SON gene (MIM #617140). This multisystemic disorder is characterized by intellectual disability, seizures, abnormal brain imaging, variable dysmorphic features, and various congenital anomalies. The wide application and increasing accessibility of whole exome sequencing (WES) has helped to identify new cases of ZTTK syndrome over the last few years. To date, there have been approximately 45 cases reported in the literature. Here, we describe 15 additional individuals with variants in the SON gene, including those with missense variants bringing the total number of known cases to 60. We have reviewed the clinical and molecular data of these new cases and all previously reported cases to further delineate the most common as well as emerging clinical findings related to this syndrome. Furthermore, we aim to delineate any genotype-phenotype correlations specifically for a recurring pathogenic four base pair deletion (c.5753_5756del) along with discussing the impact of missense variants seen in the SON gene., (© 2021 Wiley Periodicals LLC.)

Resected lesions from the pineal region are rare specimens encountered by surgical pathologists, and their heterogeneity can pose significant diagnostic challenges. Here, we reviewed 221 pineal region lesions resected at New York-Presbyterian Hospital/Columbia University Irving Medical Center from 1994 to 2019 and found the most common entities to be pineal parenchymal tumors (25.3%), glial neoplasms (18.6%), and germ cell tumors (17.6%) in this predominantly adult cohort of patients. Six cases of a rare midline entity usually found exclusively in the fourth ventricle, the rosette-forming glioneuronal tumor, were identified. These tumors exhibit biphasic morphology, with a component resembling pilocytic astrocytoma admixed with variable numbers of small cells forming compact rosettes and perivascular pseudorosettes. Targeted sequencing revealed a 100% co-occurrence of novel and previously described genetic alterations in the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathways, suggesting a synergistic role in tumor formation. The most common recurrent mutation, PIK3CA H1047R, was identified in tumor cells forming rosettes and perivascular pseudorosettes. A review of the literature revealed 16 additional cases of rosette-forming glioneuronal tumors in the pineal region. Although rare, this distinctive low-grade tumor warrants consideration in the differential diagnosis of pineal region lesions., (© 2021 American Association of Neuropathologists, Inc. All rights reserved.)

Introduction: The 2018 updated molecular testing guidelines for patients with advanced lung cancer incorporated ALK immunohistochemistry (IHC) analysis as an equivalent to fluorescence in situ hybridization (FISH) method recommended in 2013. Nevertheless, no specific recommendation for alternative methods was proposed owing to insufficient data. The aim of this study was to compare the results of ALK IHC, FISH, RNA next-generation sequencing (NGS), and RNA in situ hybridization (ISH) with available clinical data., Methods: A search for lung carcinomas with ALK testing by greater than or equal to one modality (i.e., ALK IHC, FISH, NGS) was performed; a subset underwent RNA ISH. When available, clinical data were recorded., Results: The results were concordant among all performed testing modalities in 86 of 90 cases (95.6%). Of the four discordant cases, two were ALK positive by FISH but negative by IHC, RNA NGS, and RNA ISH. The remaining two cases failed RNA NGS testing, one was IHC negative, FISH positive, RNA ISH negative and the second was IHC positive, FISH positive, RNA ISH equivocal. RNA NGS identified one rare and one novel ALK fusion. Sufficient therapy data were available in 10 cases treated with tyrosine kinase inhibitors; three had disease progression, including one with discordant results (FISH positive, RNA NGS negative, IHC negative, RNA ISH negative) and two with concordant ALK positivity among all modalities., Conclusions: Our results reveal high concordance among IHC, RNA NGS, and RNA ISH. In cases of discordance with available RNA NGS, FISH result was positive whereas IHC and ISH results were negative. On the basis of our data, multimodality testing is recommended to identify discrepant results and patients (un)likely to respond to tyrosine kinase inhibitors., (© 2021 The Authors.)

Refractory celiac disease type II (RCD II), also referred to as "cryptic" enteropathy-associated T-cell lymphoma (EATL) or "intraepithelial T-cell lymphoma," is a rare clonal lymphoproliferative disorder that arises from innate intraepithelial lymphocytes. RCD II has a poor prognosis and frequently evolves to EATL. The pathogenesis of RCD II is not well understood and data regarding the immunophenotypic spectrum of this disease and underlying genetic alterations are limited. To gain further biological insights, we performed comprehensive immunophenotypic, targeted next-generation sequencing, and chromosome microarray analyses of 11 RCD II cases: CD4-/CD8- (n=6), CD8+ (n=4), and CD4+ (n=1), and 2 of 3 ensuing EATLs. Genetic alterations were identified in 9/11 (82%) of the RCD II cases. All 9 displayed mutations in members of the JAK-STAT signaling pathway, including frequent, recurrent STAT3 (7/9, 78%) and JAK1 (4/9, 44%) mutations, and 9/10 evaluable cases expressed phospho-STAT3. The mutated cases also harbored recurrent alterations in epigenetic regulators (TET2, n=5 and KMT2D, n=5), nuclear factor-κB (TNFAIP3, n=4), DNA damage repair (POT1, n=3), and immune evasion (CD58, n=2) pathway genes. The CD4-/CD8- and other immunophenotypic subtypes of RCD II exhibited similar molecular features. Longitudinal genetic analyses of 4 RCD II cases revealed stable mutation profiles, however, additional mutations were detected in the EATLs, which occurred at extraintestinal sites and were clonally related to antecedent RCD II. Chromosome microarray analysis demonstrated copy number changes in 3/6 RCD II cases, and 1 transformed EATL with sufficient neoplastic burden for informative analysis. Our findings provide novel information about the immunophenotypic and genomic characteristics of RCD II, elucidate early genetic events in EATL pathogenesis, and reveal potential therapeutic targets., Competing Interests: Conflicts of Interest and Source of Funding: S.H. received honoraria from Illumina, Loxo Oncology, and Medscape; research funding from Bristol Myers Squibb. A.A.G. is a co-investigator with Seattle Genetics, Innate Pharma. Investigator and consultant with StemLine. P.H.G. is on the Advisory board of Johnson & Johnson/Janssen, ImmunogenX, ImusanT. The remaining authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The availability and cost of next-generation sequencing (NSG) now allow testing large numbers of genes simultaneously. However, the gold standard for predictive testing has been to test only for a known family mutation or confirmed family disease. The goal of this study was to investigate the psychological impact of predictive testing for autosomal dominant neurodegenerative diseases without a known family mutation using next-generation sequencing panels compared to single-gene testing of a known family mutation. Fourteen individuals from families with a known mutation and 10 individuals with unknown family mutations participated. Participants completed questionnaires on demographics, genetic knowledge, and psychological measures of anxiety, depression, perceived personal control, rumination, and intolerance to uncertainty at baseline and 1 and 6 months after receiving results. Decision regret was measured 1 and 6 months after receiving results. Participants completed a modified Huntington disease genetic testing protocol with genetic counseling and neurological and psychological evaluation. Genetic testing of either the known family mutation or an NGS panel of neurodegenerative disease genes was performed. Semi-structured interviews were performed at 6 months post-results about their experience. Two-sample t tests were performed on data collected at each time point to identify significant between-group differences in demographic variables, baseline psychological scores, and baseline genetic knowledge scores. Within-group change over time was assessed by a mixed-effects model. Results of this study indicate that NGS panels for predictive testing for neurodegenerative disease are safe and beneficial to participants when performed within a modified HD protocol. Though significant differences in psychological outcomes were found, these differences may have been driven by genetic results and baseline psychological differences between individuals within the groups. Participants did not regret their decision to test and were largely pleased with the testing protocol., (© 2020 National Society of Genetic Counselors.)

We report a case of a slow-growing, diffuse, infiltrating glioma in the right brainstem of a 9-yr-old boy. The tumor was negative by immunohistochemical staining for histone H3 K27M, BRAF V600E, and IDH1 R132H mutations. Fluorescence in situ hybridization did not reveal a BRAF duplication. Genomic profiling of the tumor, by DNA methylation array and cancer whole-exome and transcriptome sequencing, was performed. This analysis showed copy-number alterations, including gains of several chromosomes. In addition, a novel fusion involving the first 17 exons of FGFR2 fused to exon 2 of VPS35 was identified. This novel fusion is predicted to result in activation of fibroblast growth factor receptor (FGFR) signaling and is potentially targetable using FGFR inhibitors. This tumor expands the spectrum of pediatric diffuse gliomas., (© 2020 Zanazzi et al.; Published by Cold Spring Harbor Laboratory Press.)

Indolent T-cell lymphoproliferative disorders of the gastrointestinal tract are rare clonal T-cell diseases that more commonly occur in the intestines and have a protracted clinical course. Different immunophenotypic subsets have been described, but the molecular pathogenesis and cell of origin of these lymphocytic proliferations is poorly understood. Hence, we performed targeted next-generation sequencing and comprehensive immunophenotypic analysis of ten indolent T-cell lymphoproliferative disorders of the gastrointestinal tract, which comprised CD4 + (n=4), CD8 + (n=4), CD4 + /CD8 + (n=1) and CD4 - /CD8 - (n=1) cases. Genetic alterations, including recurrent mutations and novel rearrangements, were identified in 8/10 (80%) of these lymphoproliferative disorders. The CD4 + , CD4 + /CD8 + , and CD4 - /CD8 - cases harbored frequent alterations of JAK-STAT pathway genes (5/6, 82%); STAT3 mutations (n=3), SOCS1 deletion (n=1) and STAT3-JAK2 rearrangement (n=1), and 4/6 (67%) had concomitant mutations in epigenetic modifier genes ( TET2 , DNMT3A , KMT2D ). Conversely, 2/4 (50%) of the CD8 + cases exhibited structural alterations involving the 3' untranslated region of the IL2 gene. Longitudinal genetic analysis revealed stable mutational profiles in 4/5 (80%) cases and acquisition of mutations in one case was a harbinger of disease transformation. The CD4 + and CD4 + /CD8 + lymphoproliferative disorders displayed heterogeneous Th1 (T-bet + ), Th2 (GATA3 + ) or hybrid Th1/Th2 (T-bet + /GATA3 + ) profiles, while the majority of CD8 + disorders and the CD4 - /CD8 - disease showed a type-2 polarized (GATA3 + ) effector T-cell (Tc2) phenotype. Additionally, CD103 expression was noted in 2/4 CD8 + cases. Our findings provide insights into the pathogenetic bases of indolent T-cell lymphoproliferative disorders of the gastrointestinal tract and confirm the heterogeneous nature of these diseases. Detection of shared and distinct genetic alterations of the JAK-STAT pathway in certain immunophenotypic subsets warrants further mechanistic studies to determine whether therapeutic targeting of this signaling cascade is efficacious for a proportion of patients with these recalcitrant diseases., (Copyright© 2020 Ferrata Storti Foundation.)

This multi-institutional study was undertaken to evaluate interrater reliability of the 2017 Association for Molecular Pathology/American Society of Clinical Oncology/College of American Pathologists guidelines for interpretation and reporting of oncology sequence variants and to assess current practices and perceptions surrounding these guidelines. Fifty-one variants were distributed to 20 participants from 10 institutions for classification using the new guidelines. Agreement was assessed using chance-corrected agreement (Cohen κ). κ was 0.35. To evaluate if data sharing could help resolve disagreements, a summary of variant classifications and additional information about each variant were distributed to all participants. κ improved to 0.7 after the original classifications were revised. Participants were invited to take a web-based survey regarding their perceptions of the guidelines. Only 20% (n = 3) of the survey respondents had prior experience with the guidelines in clinical practice. The main perceived barriers to guideline implementation included the complexity of the guidelines, discordance between clinical actionability and pathobiologic relevance, lack of familiarity with the new classifications, and uncertainty when applying criteria to potential germline variants. This study demonstrates noteworthy discordances between pathologists for variant classification in solid tumors when using the 2017 Association for Molecular Pathology/American Society of Clinical Oncology/College of American Pathologists guidelines. These findings highlight potential areas for clarification/refinement before mainstream clinical adoption., (Copyright © 2020 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

The cytogenetic alterations in renal oncocytoma (RO) are poorly understood. We analyzed 130 consecutive RO for karyotypic alterations. Clonal chromosome abnormalities were identified in 63 (49%) cases, which could be categorized into three classes of mutually exclusive cytogenetic categories. Class 1 (N = 20) RO had diploid karyotypes with characteristic 11q13 rearrangement in balanced translocations with 10 or more different chromosome partners in all cases. We identified recurrent translocation partners at 5q35, 6p21, 9p24, 11p13-14, and 11q23, and confirmed that CCND1 gene rearrangement at 11q13 utilizing fluorescence in situ hybridization (FISH). Class 2 RO (N = 25) exhibited hypodiploid karyotypes with loss of chromosome 1 and/or losses of Y in males and X in females in all cases. The class 3 tumors comprising of 18 cases showed diverse types of abnormalities with the involvement of two or more chromosomes exclusive of abnormalities seen in classes 1 and 2 tumors. Furthermore, karyotypically uninformative cases were subjected to FISH analysis to identify classes 1 and 2 abnormalities. In this group, we found similar frequencies of CCND1 rearrangement, loss of chromosome 1 or Y as with karyotypically abnormal cases. We validated our results against 91 tumors from the Mitelman database. Correlation of clinical data with all the three classes of ROs showed no clear evidence of overall patient survival. Our findings support the hypothesis that RO exhibit three principal cytogenetic categories, which may have different roles in initiation and/or progression. These cytogenetic markers provide a key tool in the diagnostic evaluation of RO., (© 2019 Wiley Periodicals, Inc.)

The clinical management of pediatric liver tumors involves stratification into risk groups. One previously defined, high-risk group of hepatoblastomas is the small cell undifferentiated variant. In light of molecular studies showing SMARCB1 deletion in these tumors, it is now recognized that most small cell, undifferentiated liver tumors represent an aggressive unrelated tumor-the malignant rhabdoid tumor (MRT). SMARCB1 is a member of the chromatin remodeling SWI/SNF complex and encodes the INI1 protein. The histologic diagnosis of MRT is currently based on INI1 negative immunoreactivity and the presence of rhabdoid morphology. INI1-negative small cell liver tumors lacking classic rhabdoid morphology are often misclassified as small cell undifferentiated hepatoblastomas (SCUD-HB), according to the current classification. Pediatric liver tumors diagnosed between 2003-2017 as SCUD-HB (four cases) or MRT (two cases) were identified from the Columbia University Pathology Department Archives. All tumors were associated with normal or low serum alpha fetoprotein levels, and showed an absence of immunohistochemical staining of hepatocellular markers (Hep-par1, Arginase) and loss of INI1 staining. Two cases were initially diagnosed as MRT, one with prominent rhabdoid morphology, the other with predominant small cell morphology. The remaining four cases with small cell morphology were classified as SCUD-HB. Ancillary molecular studies confirmed the loss of SMARCB1 , supporting the diagnosis of MRT in all cases, proving morphology an unreliable criterion. It is critical to eliminate the term INI1-negative hepatoblastoma from the current classification scheme, and classify INI1-negative tumors as MRT, particularly since high-risk HB-chemotherapy regimens are not effective for treating MRT.

Assessment of ALK gene rearrangements is strongly recommended by the Molecular Testing Guideline for Selection of Lung Cancer Patients proposed by IASLC, AMP, and CAP at the time of diagnosis for patients with advanced stage disease. Non-small-cell lung cancer (NSCLC) with ALK gene rearrangements or the resulting fusion proteins have been, for the most part, successfully targeted with ALK tyrosine kinase inhibitors (TKIs). The most frequent rearrangement, the EML4-ALK oncogenic fusion, has more than 10 distinct variants, each with a discrete breakpoint in EML4 Recent studies have suggested that EML4-ALK variants may have differential responses to TKIs. Additionally, non-EML4-ALK fusions that result from ALK rearrangements with diverse 5' partners could possibly have varied biologic and clinical implications in their therapeutic responses and outcomes of patients with NSCLC. Existing literature documents at least 20 non-EML4 fusion partners for ALK, and the clinical responsiveness to crizotinib ranges from increased sensitivity to resistance. This underscores the importance of identifying the precise 5' fusion partner to ALK before initiation of therapy. Herein we report the identification of a novel SLMAP-ALK fusion in a patient with NSCLC., (© 2019 Pagan et al.; Published by Cold Spring Harbor Laboratory Press.)

The incorporation of tumor-normal genomic testing into oncology can identify somatic mutations that inform therapeutic measures but also germline variants associated with unsuspected cancer predisposition. We describe a case in which a RET variant was identified in a 3-yr-old male with relapsed leukemia. Sanger sequencing revealed the patient's father and three siblings carried the same variant, associated with multiple endocrine neoplasia 2A (MEN2A). Evaluation of the father led to the diagnosis and treatment of metastatic medullary thyroid carcinoma. Detection of RET mutations in families with hereditary MTC allows for genetic risk stratification and disease surveillance to reduce morbidity and mortality., (© 2019 Pendrick et al.; Published by Cold Spring Harbor Laboratory Press.)

We developed and validated a clinical whole-genome and transcriptome sequencing (WGTS) assay that provides a comprehensive genomic profile of a patient's tumor. The ability to fully capture the mappable genome with sufficient sequencing coverage to precisely call DNA somatic single nucleotide variants, insertions/deletions, copy number variants, structural variants, and RNA gene fusions was analyzed. New York State's Department of Health next-generation DNA sequencing guidelines were expanded for establishing performance validation applicable to whole-genome and transcriptome sequencing. Whole-genome sequencing laboratory protocols were validated for the Illumina HiSeq X Ten platform and RNA sequencing for Illumina HiSeq2500 platform for fresh or frozen and formalin-fixed, paraffin-embedded tumor samples. Various bioinformatics tools were also tested, and CIs for sensitivity and specificity thresholds in calling clinically significant somatic aberrations were determined. The validation was performed on a set of 125 tumor normal pairs. RNA sequencing was performed to call fusions and to confirm the DNA variants or exonic alterations. Here, we present our results and WGTS standards for variant allele frequency, reproducibility, analytical sensitivity, and present limit of detection analysis for single nucleotide variant calling, copy number identification, and structural variants. We show that The New York Genome Center WGTS clinical assay can provide a comprehensive patient variant discovery approach suitable for directed oncologic therapeutic applications., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Aims: Refractory coeliac disease type II (RCDII), a rare complication of coeliac disease (CD) associated with high morbidity, requires identification of a clonal population of phenotypically aberrant intraepithelial lymphocytes (IELs) for diagnosis. However, data regarding the frequency and significance of clonal T cell receptor (TCR) gene rearrangements (TCR-GRs) in small bowel (SB) biopsies of patients without RCDII are limited., Methods: We analysed results of TCR-GR analyses performed on SB biopsies at our institution over a 3-year period, which were obtained from eight active CD, 172 CD on gluten-free diet (GFD), 33 RCDI, and three RCDII patients and 14 patients without CD. TCR-GR patterns were divided into clonal, polyclonal and prominent clonal peaks (PCPs) and these patterns were correlated with clinical and pathological features., Results: Clonal TCR-GR products were detected in biopsies from 67% of patients with RCDII, 17% of patients with RCDI and 6% of patients with GFD. PCPs were observed in all disease phases (range 12%-33%). There was no significant difference in the TCR-GR patterns between the non-RCDII disease categories (p=0.39). A higher frequency of surface CD3(-) IELs was noted in cases with clonal TCR-GR, but the PCP pattern did not show associations with any clinical or pathological feature. Persistence of clonal or PCP pattern on repeat biopsy was seen for up to 2 years without evidence of RCDII., Conclusions: Clonal TCR-GRs are not infrequent in cases lacking features of RCDII, while PCPs are frequent in all disease phases. TCR-GR results should be assessed in conjunction with immunophenotypic, histological and clinical findings for appropriate diagnosis and classification of RCD., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Changes in coding and coverage generate an uncertain reimbursement environment for molecular pathology laboratories. We analyzed our experience with two representative molecular oncology tests: a T-cell receptor (TCR) β rearrangement test and a large (467-gene) cancer next-generation sequencing panel, the Columbia Combined Cancer Panel (CCCP). Before 2013, the TCR β test was coded using stacked current procedural terminology codes and subsequently transitioned to a tier 1 code. CCCP was coded using a combination of tier 1 and 2 codes until 2015, when a new Genomic Sequencing Procedure code was adopted. A decrease in reimbursement of 61% was observed for the TCR β test on moving from stacking to tier 1 codes. No initial increase in total rejection rate was observed, but a subsequent increase in rejection rates in 2015 and 2016 was noted. The CCCP test showed a similar decrease (48%) in reimbursement after adoption of the new Genomic Sequencing Procedure code and was accompanied by a sharp increase in rejection rates both on implementation of the new code and over time. Changes in coding can result in substantial decreases in reimbursement. This may be a barrier to patient access because of the high cost of molecular diagnostics. Revisions to the molecular code set will continue. These findings help laboratories and manufacturers prepare for the financial impact and advocate appropriately., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Objective: The staging of multiple pulmonary adenocarcinomas requires the distinction of intrapulmonary metastasis (IPM) from multiple primary lung cancers (MPLCs). This can be challenging in some patients, and the addition of data from oncogenic driver mutations in these tumors may be helpful in this determination., Methods: As a proof of principle, molecular driver results from primary tumors and their metastases in 45 patients were compared (cohort 1). Then, 69 patients with a total of 154 synchronous or metachronous lung carcinomas were identified, and the pathologic findings were compared with oncogenic driver mutation. Each patient was assigned a highest potential T or M category on the basis of clinical, histopathologic, and molecular findings (cohort 2)., Results: The concordance rate of EGFR, KRAS, BRAF, and ALK receptor tyrosine kinase gene (ALK) mutations was 96% in cohort 1. In cohort 2, 36% of multiple same-lobe nodules were MPLCs, 40% were IPM, and 24% were noninformative by molecular findings. Of nodules with multiple lobe involvement, 81.5% were MPLCs and 7.4% were IPM, with 11% noninformative. Of metachronous tumors, 52.9% were MPLCs. Overall survival was 100% at 2 years, 95% at 3 years, and 80% at 4 years in patients with available follow-up., Conclusions: Oncogenic driver mutations are concordant between primary tumors and metastasis. The largest proportion of MPLCs was seen in tumors of multiple lobes, but with a substantial proportion of MPLCs among single-lobe nodules and with metachronous tumors. Overall survival was higher than expected for the respective highest T or M category, which is in support of the high frequency of MPLC., (Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Ataxia-telangiectasia (A-T) is an autosomal recessive chromosome breakage disorder caused by mutations in the ATM gene. Typically, it presents in early childhood with progressive cerebellar dysfunction along with immunodeficiency and oculocutaneous telangiectasia. An increased risk of malignancy is also associated with the syndrome and, rarely, may be the presenting feature in small children. We describe a 17-year-old boy with slurred speech, mild motor delays and learning disability diagnosed with atypical A-T in the setting of T-cell acute lymphoblastic leukemia. Suspicion for A-T was raised after review of a peripheral blood karyotype demonstrating rearrangements involving chromosomes 7 and/or 14. The diagnosis was confirmed after molecular testing identified a novel homozygous missense variant in ATM (c.5585T>A; p.Leu1862His) that resulted in protein instability and abolished serine/threonine protein kinase activity. To our knowledge, this is the first report of concurrent A-T and lymphoid malignancy diagnoses in an older child or adult with only mild neurological disease. Our experience suggests that screening for the disorder should be considered in any individual with lymphoid malignancy and neurological findings, especially as radiation and certain chemotherapy protocols are contraindicated in A-T.

Pleomorphic xanthoastrocytoma (PXA) is a World Health Organization (WHO) Grade II glioma occurring primarily in children and young adults. Most PXAs harbor the known activating mutation BRAF V600E. We report a case of locally recurrent PXA with anaplastic features in a 10-yr-old female. The PXA was negative by immunohistochemical (IHC) staining for BRAF V600E mutation. Whole-exome and transcriptome sequencing of the tumor confirmed the absence of BRAF V600E, but identified copy-number alterations (including loss of the tumor suppressor CDKN2A ) and a novel TMEM106B - BRAF fusion. Based on similar BRAF fusion proteins, this novel fusion is predicted to result in activation of BRAF signaling. Demonstration of positive IHC for phospho-ERK1/2 and phospho-MEK1/2 supported this prediction, and implicated MEK inhibitors as a potential therapeutic strategy.

Anorectal melanoma is a rare disease that carries a poor prognosis. To date, limited genetic analyses confirmed KIT mutations as a recurrent genetic event similar to other mucosal melanomas, occurring in up to 30% of anorectal melanomas. Importantly, a subset of tumors harboring activating KIT mutations have been found to respond to c-Kit inhibitor-based therapy, with improved patient survival at advanced tumor stages. We performed comprehensive targeted exon sequencing analysis of 467 cancer-related genes in a larger series of 15 anorectal melanomas, focusing on potentially actionable variants based on gain- and loss-of-function mutations. We report the identification of oncogenic driver events in the majority (93%) of anorectal melanomas. These included variants in canonical MAPK pathway effectors rarely observed in cutaneous melanomas (including an HRAS mutation, as well as a BRAF mutation resulting in duplication of threonine 599), and recurrent mutations in the tumor suppressor NF1 in 20% of cases, which represented the second-most frequently mutated gene after KIT in our series. Furthermore, we identify SF3B1 mutations as a recurrent genetic event in mucosal melanomas. Our findings provide an insight into the genetic diversity of anorectal melanomas, and suggest significant potential for alternative targeted therapeutics in addition to c-Kit inhibitors for this melanoma subtype.

Multiple chromosomal regions are affected by deletions in cervical cancer (CC) genomes, but their consequence and target gene involvement remains unknown. Our single nucleotide polymorphism (SNP) array identified 8p copy number losses localized to an 8.4 Mb minimal deleted region (MDR) in 36% of CC. The 8p MDR was associated with tumor size, treatment outcome, and with multiple HPV infections. Genetic, epigenetic, and expression analyses of candidate genes at MDR identified promoter hypermethylation and/or inactivation of decoy receptors TNFRSF10C and TNFRSF10D in the majority of CC patients. TNFRSF10C methylation was also detected in precancerous lesions suggesting that this change is an early event in cervical tumorigenesis. We further demonstrate here that CC cell lines exhibiting downregulated expression of TNFRSF10C and/or TNFRSF10D effectively respond to TRAIL-induced apoptosis and this affect was synergistic in combination with DNA damaging chemotherapeutic drugs. We show that the CC cell lines harboring epigenetic inactivation of TRAIL decoy receptors effectively activate downstream caspases suggesting a critical role of inactivation of these genes in efficient execution of extrinsic apoptotic pathway and therapy response. Therefore, these findings shed new light on the role of genetic/epigenetic defects in TRAIL decoy receptor genes in the pathogenesis of CC and provide an opportunity to explore strategies to test decoy receptor gene inactivation as a biomarker of response to Apo2L/TRAIL-combination therapy., (© 2015 Wiley Periodicals, Inc.)

Background: Correlation between histology and genotype has been described in lung adenocarcinomas. For example, studies have demonstrated that adenocarcinomas with an anaplastic lymphoma kinase (ALK) gene rearrangement may have mucinous features. The objective of the current study was to determine whether a similar association can be identified in cytological specimens., Methods: A retrospective search for ALK-rearranged cytopathology (CP) and surgical pathology (SP) lung carcinomas was conducted. Additional ALK-negative (-) lung adenocarcinomas served as controls. For CP and SP cases, the clinical data (i.e., age, sex, and smoking history), architecture, nuclear features, presence of mucin-containing cells (including signet ring cells), and any additional salient characteristics were evaluated., Results: The search yielded 20 ALK-positive (+) adenocarcinomas. Compared with patients with ALK(-) lung adenocarcinomas (33 patients; 12 with epidermal growth factor receptor [EGFR]-mutation, 11 with Kristen rat sarcoma [KRAS]-mutation, and 10 wild-type adenocarcinomas), patients with ALK(+) adenocarcinoma presented at a younger age; and there was no correlation noted with sex or smoking status. The most common histological pattern in SP was papillary/micropapillary. Mucinous features were associated with ALK rearrangement in SP specimens. Signet ring cells and psammoma bodies were evident in and significantly associated with ALK(+) SP and CP specimens. However, psammoma bodies were observed in rare adenocarcinomas with an EGFR mutation. Both the ALK(+) and ALK(-) groups had mostly high nuclear grade., Conclusions: Salient features, including signet ring cells and psammoma bodies, were found to be significantly associated with ALK(+) lung adenocarcinomas and are identifiable on CP specimens. Recognizing these may be especially helpful in the molecular triage of scant CP samples., (© 2014 American Cancer Society.)

Background: Coexistence of complete mole and a live fetus is uncommon (1:22,000-100,000), more so with euploidy., Case: We present a case of a molar pregnancy with a euploid fetus who had close fetal evaluation for second trimester bleeding. The patient presented at 29 weeks' pregnancy with decreased fetal movements, a result of fetomaternal hemorrhage. She underwent cesarean section and delivered a live infant. By close follow-up and a multidisciplinary approach, the appropriate diagnosis and a favorable outcome were achieved. Both mother and the child at 5 years of age are doing well., Conclusion: Detailed anatomic and molecular studies demonstrated a complete mole resulting from confined placental mosaicism, with molar tissue showing a single paternal allele at 8/8 informative loci, all shared with the fetus, thus this coexistent molar pregnancy was not that of a separate conceptus.

Follicular lymphoma (FL) in situ (FLIS) was first described and proposed as a distinct entity associated with an indolent clinical course in 2002. To gain further insight into the biology of this enigmatic lymphoproliferation, we analyzed morphologic, phenotypic, cytogenetic and molecular features of tissue specimens manifesting a pattern of follicular colonization by Bcl-2(bright+)CD10(+) B-cells and associated lymphomas from 13 adults and evaluated their clinical outcomes. We observed this immunoarchitectural pattern in lymph nodes (n = 8), at extranodal sites (n = 4), or at both locations (n = 1) at diagnosis. All except 3 cases showed concomitant bright CD10 expression. Six (46%) patients had synchronous and 2 (15%) developed metachronous B-cell lymphomas, with 5 representing high-grade lymphomas. The Bcl-2(bright+)CD10(+) B-cells colonizing reactive follicles and synchronous lymphomas were clonally related in 4/5 (80%) cases analyzed and 5/6 (83%) displayed BCL2 translocations. Two cases exhibited complex karyotypes in both components; a genetic "triple hit" was detected in one instance and 2 copies of t(14,18) were observed in a lymph node biopsy lacking evidence of lymphoma from an individual with stage 4 disease, suspected on imaging, who subsequently displayed a mantle zone/perifollicular infiltrate of Bcl-2(bright+)CD10(+) B-cells in the adenoids. Our findings suggest that bright Bcl-2, and often bright CD10 expression, by B-cells colonizing reactive follicles might represent a phenomenon related to follicular homing of lymphoma, rather than being an attribute of preneoplastic FL precursors. Furthermore, due to the relatively high frequency of overt lymphomas observed, complete staging workup is recommended for patients exhibiting a Bcl-2(bright+)CD10(+) B-cell follicular colonization pattern on biopsy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Primary effusion lymphoma (PEL) is a rare type of B-cell non-Hodgkin lymphoma (NHL), which predominantly occurs in HIV-infected individuals, and is pathogenetically linked with Kaposi sarcoma (KS)-associated herpes virus/human herpes virus-8 (KSHV/HHV-8) infection with or without evidence of Epstein-Barr virus (EBV) co-infection. Although uncommon, PELs have been reported in immunocompetent patients and recipients of solid organ allografts. Rare cases of KSHV(-) EBV(+) post-transplant effusion lymphomas resembling PEL have also been described, as have KSHV(-) EBV(-) effusion lymphomas, the latter including those arising in individuals with chronic liver disease. We report a unique KSHV(-) EBV(-) post-transplant effusion lymphoma associated with serum paraproteins, occurring in an HIV(-) individual, which had cytologic features and phenotype similar to PEL, and displayed a complex karyotype including isochromosome 12p and translocation t(8;22), resulting in rearrangement of c-MYC., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Background and Objectives: In early and polymorphic post-transplant lymphoproliferative disorders (PTLD) Epstein-Barr virus (EBV), through its latency proteins, drives the proliferation of B lymphocytes, a process which in immunocompetent individuals leads to the establishment of latently infected memory B cells., Design and Methods: We analyzed 11 cases, which included early and polymorphic PTLD, and 12 controls for latency of EBV infection and their antigenic profile., Results: We identified a minority of terminally differentiated EBER+ IRTA1+ memory B cells and EBER+ CD138+ PRDM1+ plasma cells in these samples. These elements were identified both in PTLD and in tumor-free tonsils from post-transplant patients but not in EBV- control tonsils. The expression of EBV latency proteins is heterogeneous, and is associated with activation of the NF-kB pathway. EBV signaling (through EBNA2, LMP1 and LMP2A) and NF-kB activation correlated with upregulation of target proteins: cMYC, JunB, CCL22, TRAF1 and IRF4. EBV-infected lymphocytes in early and polymorphic PTLDs represent a mixture of latencies II, III and, in at least 1/3 of infected cells, of latency 0., Interpretation and Conclusions: EBV infection correlates with NF-kB activation, with EBV-dependent cell signaling, and lastly, with the presence of EBV-infected plasma cells and memory cells.

Nephrogenic adenoma is a rare lesion of the urinary tract. The diagnosis usually is straightforward when characteristic microscopic and clinical findings are present, and the entity is familiar. However, misdiagnosis, in particular of adenocarcinoma of the prostate gland, may occur. Immunohistochemical stains often are needed to make such a distinction, but currently available markers offered only partial help. It recently was demonstrated that nephrogenic adenoma in renal transplant patients originated from the renal tubular epithelium. This newly proved, but long sought information may be helpful in the differential diagnosis of nephrogenic adenoma. In this study, we investigated the expression of a renal transcription factor, PAX2, in 39 nonrenal transplant-related nephrogenic adenomas, 100 adenocarcinomas of the prostate gland, and 47 urothelial carcinomas of the urinary tract. A strong and distinct nuclear staining of PAX2 was found in all 39 cases of nephrogenic adenoma (100%), but not in normal prostate tissue, normal urothelium, adenocarcinomas of the prostate gland, and invasive urothelial carcinomas. Focal CD10 was detected in six of 13 nephrogenic adenomas in the superficial papillary component and in normal prostate epithelium, normal urothelium, lymphocytes, adenocarcinoma of the prostate gland, and urothelial carcinoma. There was no uroplakins detected in nephrogenic adenoma. Therefore, these findings are suggesting that nephrogenic adenoma in nonrenal transplant patients may also arise from the renal epithelium, as did the comparable lesions after transplantation. PAX2 is a specific and sensitive immunohistochemical marker in identification and differential diagnosis of nephrogenic adenoma.

Cervical/endocervical cytology screening has decreased morbidity and mortality, and implementing adjunctive human papilloma virus (HPV) DNA testing for atypical squamous cells of undetermined significance has improved the specificity for detecting premalignant squamous lesions. Currently, there are no guidelines to perform HPV DNA testing on cervical/endocervical ThinPreps with atypical glandular cells (AGC). To assess the potential role of HPV DNA testing on AGC cases, Hybrid Capture 2 (Digene Corp.) testing was performed on 144 cervical/endocervical AGC specimens. One hundred three of 144 cases had follow-up; 60/103 (58.3%) were high-risk HPV negative and 43/103 (42.3%) were high-risk HPV positive. Of 43 HPV-positive patients, 37 had adenocarcinoma in situ (AIS), atypical squamous cells of undetermined significance (ASCUS), or cervical squamous intraepithelial neoplasia, while only one patient without high-risk HPV had a squamous intraepithelial neoplasia. Furthermore, most high-risk HPV positive AGC cases harbored high-grade squamous intraepithelial lesion (HSIL) rather than AIS. Our data support HPV DNA testing of all AGC specimens to detect cervical, especially squamous, neoplasia., (2006 Wiley-Liss, Inc.)

Background: African-American (AA) breast cancer patients consistently show a shortened survival when compared with Caucasian patients. This worse prognosis is most likely due to a combination of socioeconomic factors and differences in tumor biology. Previous studies have demonstrated that cyclin D1 overexpression is strongly associated with estrogen receptor (ER) expression in breast cancer, but these series either included primarily Caucasian patients or did not specify race. MATERIALS AND METHODS. We analyzed 200 breast cancer cases obtained from AA and Caucasian patients who were matched on age, stage, ER status, and year of diagnosis. We examined expression levels of cyclin D1, p53, p27Kip1 (p27), and p21Cip1 (p21), and correlated their expression with ER status., Results: Cyclin D1, p53, p27, and p21 expression rates were similar in matched cases of AA and Caucasian breast cancer (p values>0.05). However, cyclin D1 overexpression was significantly associated with ER status in only the Caucasian (p=0.0005), and not the AA cases (p=0.07)., Conclusion: This finding suggests a novel biological difference, which may contribute to the more aggressive phenotype of AA breast cancer.

Sarcomas are rare but aggressive malignant tumors associated with high mortality, for which the efficacy of standard therapies remains limited. In order to develop immunotherapeutic approaches for the treatment of sarcoma, we studied the relevance of cancer/testis antigens (CTAs), a group of antigens whose expression is developmentally regulated and that is specifically found in some tumor types, as sarcoma vaccine targets. CTA expression was assessed by PCR and/or immunohistochemistry (IHC) in sarcoma tumor samples that included different histological subtypes and sarcoma cell lines. Expression of HLA class I was assessed by IHC in tumor samples and by FACS analysis in cell lines. More than 70% of the tumor samples expressed at least one CTA. The majority of tumors and cell lines expressed normal levels of HLA class I. HLA class I expression in cell lines was enhanced upon treatment with IFN-gamma. CTA expression was enhanced or induced by treatment with the demethylating agent 5-aza-2'-deoxycytidine, resulting in recognition by specific CTLs. Interestingly, a spontaneous humoral and CD8+ T cellular response to the CTA NY-ESO-1 was detected in a synovial sarcoma patient. Together, these findings strongly support the implementation of CTA-based immunotherapy of sarcoma as a means to improve the efficacy of the standard therapy.

Background: Male germ cell tumor (GCT) is a highly curable malignancy, which exhibits exquisite sensitivity to cisplatin treatment. The genetic pathway(s) that determine the chemotherapy sensitivity in GCT remain largely unknown., Results: We studied epigenetic changes in relation to cisplatin response by examining promoter hypermethylation in a cohort of resistant and sensitive GCTs. Here, we show that promoter hypermethylation of RASSF1A and HIC1 genes is associated with resistance. The promoter hypermethylation and/or the down-regulated expression of MGMT is seen in the majority of tumors. We hypothesize that these epigenetic alterations affecting MGMT play a major role in the exquisite sensitivity to cisplatin, characteristic of GCTs. We also demonstrate that cisplatin treatment induce de novo promoter hypermethylation in vivo. In addition, we show that the acquired cisplatin resistance in vitro alters the expression of specific genes and the highly resistant cells fail to reactivate gene expression after treatment to demethylating and histone deacetylase inhibiting agents., Conclusions: Our findings suggest that promoter hypermethylation of RASSF1A and HIC1 genes play a role in resistance of GCT, while the transcriptional inactivation of MGMT by epigenetic alterations confer exquisite sensitivity to cisplatin. These results also implicate defects in epigenetic pathways that regulate gene transcription in cisplatin resistant GCT.

Patients with advanced stage invasive cervical cancer (CC) exhibit highly complex genomic alterations and respond poorly to conventional treatment protocols. In our efforts to understand the molecular genetic basis of CC, we examined the role of Fanconi Anemia (FA)-BRCA pathway. Here, we show that FANCF gene is disrupted by either promoter hypermethylation and/or deregulated gene expression in a majority of CC. Inhibition of DNA methylation and histone deacetylases induces FANCF gene re-expression in CC cell lines. FANCF-deregulated CC cell lines also exhibit a chromosomal hypersensitivity phenotype after exposure to an alkylating agent, a characteristic of FA patients. We also show the involvement of BRCA1 gene by promoter hypermethylation or down-regulated expression in a small subset of CC patients. Thus, we have found inactivation of genes in the FA-BRCA pathway by epigenetic alterations in a high proportion of CC patients, suggesting a major role for this pathway in the development of cervical cancer. Thus, these results have important implications in understanding the molecular basis of CC tumorigenesis and clinical management in designing targeted experimental therapeutic protocols.