Search

Papillary intralymphatic angioendothelioma (PILA) is an extremely rare vascular tumor and its pathogenesis is unknown. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-related overgrowth spectrum (PROS) is a heterogeneous group of disorders caused by mosaicism for activating mutations of PIK3CA and characterized by asymmetric overgrowth, skeletal anomalies, skin lesions, and vascular malformations. An association between PILA and PROS has not been known. We report a case of PILA involving the spleen of a young girl with the clinical and molecular diagnosis of PROS. Sequencing of the patient’s germ-line DNA detected a pathogenic PIK3CA variant c.1357G>A in 10.6% of alleles. Splenectomy revealed a 4-cm tumor composed of ectatic lymphatics with intraluminal papillary projections, consistent with PILA. The tumor cells showed immunohistochemical expression of CD31, CD34, ERG, FLI-1, PROX1, and caldesmon, while D2-40 was negative. The latter may suggest that the tumor derived from an endothelial precursor arrested in the final steps of lymphothelial differentiation, in keeping with the known role of the PIK3CA-governed molecular pathway in the progression of vascular progenitors to mature endothelial cells. The data implicates PIK3CA in the pathogenesis of PILA and broadens the spectrum of phenotypic expressions of PROS.

Targetable NTRK gene fusions can be detected across tumor types using methodologies such as pan-TRK IHC, DNA or RNA NGS testing, or FISH. Challenges for implementation of clinical testing for NTRK fusions may arise due to the range in NTRK fusion prevalence across tumors, endogenous levels of TRK expression in tissues, and the large number of potential fusion partners. In this study, we examined our experience evaluating driver mutation negative lung, urothelial or cholangiocarcinoma cases, in addition to cases with positive, equivocal, or weak staining by pan-TRK IHC for NTRK fusions. 63/127 (49.6%) of these cases were positive for pan-TRK IHC, of which 71.4% showed weak or focal staining, potentially due to physiologic or non-specific TRK expression. Of these 127 cases, 4 harbored a NTRK fusion (1 fusion was seen in two separate samples from the same patient) as confirmed by RNA fusion panel testing. Pan-TRK IHC was positive in 1 case with TPM3-NTRK1 fusion, equivocal in 1 case with GOLGA4-NTRK3 fusion, and negative in 2 samples with ADAM19-NTRK3 fusion. Our findings show that we were able to successfully identify NTRK fusions that resulted in targeted therapy. However, our results suggest limited sensitivity of pan-TRK IHC for NTRK3 fusions, and that the reduced specificity for pan-TRK IHC in tumors with physiologic or non-specific TRK expression, results in false positive samples that require confirmatory testing by RNA based NGS.

While molecular testing of hematologic malignancies is now standard of care, there is variability in practice and testing capabilities between different academic laboratories, with common questions arising on how to best meet clinical expectations. A survey was sent to hematopathology subgroup members of the Genomics Organization for Academic Laboratories (GOAL) consortium to assess current and future practice and potentially establish a reference for peer institutions. Responses were received from eighteen academic tertiary-care laboratories regarding next-generation sequencing (NGS) panel design, sequencing protocols and metrics, assay characteristics, laboratory operations, case reimbursement, and development plans. Differences in NGS panel size, use, and gene content were reported. Gene content for myeloid processes was reported to be generally excellent while genes for lymphoid processes were less well covered. Turnaround time (TAT) for acute cases including acute myeloid leukemia was reported to range from 2-7 to 15-21 calendar days with different approaches to achieving rapid TAT described. To help guide NGS panel design and standardize gene content, consensus gene lists based on current and future NGS panels in development were generated. Most survey respondents expected molecular testing at academic laboratories to continue to be viable in the future with rapid TAT for acute cases likely to remain an important factor. Molecular testing reimbursement was reported to be a major concern. The results of this survey and subsequent discussions improve the shared understanding of differences in testing practices for hematologic malignancies between institutions and will help provide a more consistent level of patient care.

AimsRefractory 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.MethodsWe 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.ResultsClonal 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.ConclusionsClonal 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.

AimMicrosatellite instability (MSI), a hallmark of DNA mismatch repair deficiency, is a key molecular biomarker with multiple clinical implications including the selection of patients for immunotherapy, identifying patients who may have Lynch syndrome and predicting prognosis in patients with colorectal tumours. Next-generation sequencing (NGS) provides the opportunity to interrogate large numbers of microsatellite loci concurrently with genomic variants. We sought to develop a method to detect MSI that would not require paired normal tissue and would leverage the sequence data obtained from a broad range of tumours tested using our 467-gene NGS Columbia Combined Cancer Panel (CCCP).MethodsAltered mononucleotide and dinucleotide microsatellite loci across the CCCP region of interest were evaluated in clinical samples encompassing a diverse range of tumour types. The number of altered loci was used to develop a decision tree classifier model trained on the retrospectively collected cohort of 107 clinical cases sequenced by the CCCP assay.ResultsThe classifier was able to correctly classify all cases and was then used to analyse a test set of clinical cases (n=112) and was able to correctly predict their MSI status with 100% sensitivity and specificity. Analysis of recurrently altered loci identified alterations in genes involved in DNA repair, signalling and transcriptional regulation pathways, many of which have been implicated in MSI tumours.ConclusionThis study highlights the utility of this approach, which should be applicable to laboratories performing similar testing.

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.

Large cancer panels are being increasingly used in the practice of precision medicine to generate genomic profiles of tumors with the goal of identifying targetable variants and guiding eligibility for clinical trials. To facilitate identification of mutations in a broad range of solid and hematological malignancies, a 467-gene oncology panel (Columbia Combined Cancer Panel) was developed in collaboration with pathologists and oncologists and is currently available and in use for clinical diagnostics. Herein, we share our experience with this testing in an academic medical center. Of 255 submitted specimens, which encompassed a diverse range of tumor types, we were able to successfully sequence 92%. The Columbia Combined Cancer Panel assay led to the detection of a targetable variant in 48.7% of cases. However, although we show good clinical performance and diagnostic yield, third-party reimbursement has been poor. Reimbursement from government and third-party payers using the 81455 Current Procedural Terminology code was at 19.4% of billed costs, and 55% of cases were rejected on first submission. Likely contributing factors to this low level of reimbursement are the delays in valuation of the 81455 Current Procedural Terminology code and in establishing national or local coverage determinations. In the absence of additional demonstrations of clinical utility and improved patient outcomes, we expect the reimbursement environment will continue to limit the availability of this testing more broadly.

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.

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.

Mutant allele specific imbalance (MASI) was initially coined to describe copy number alterations associated with the mutant allele of an oncogene. The copy number gain (CNG) specific to the mutant allele can be readily observed in electropherograms. With the development of genome-wide analyses at base-pair resolution with copy number counts, we can now further differentiate MASI into those with CNG, with copy neutral alteration (also termed acquired uniparental disomy; UPD), or with loss of heterozygosity (LOH) due to the loss of the wild-type (WT) allele. Here we summarize the occurrence of MASI with CNG, aUPD, or MASI with LOH in some major oncogenes (such as EGFR, KRAS, PIK3CA, and BRAF). We also discuss how these various classifications of MASI have been demonstrated to impact tumorigenesis, progression, metastasis, prognosis, and potentially therapeutic responses in cancer, notably in lung, colorectal, and pancreatic cancers.

Objectives Validation of RNA-based NGS assays for the detection of therapeutically targetable gene fusions is challenging. Here, we report systematic validation and quality control monitoring of our targeted fusion panel for the detection of 17 clinically relevant fusion transcripts across several tumor types. We implemented this RNA Fusion Panel as a reflex test for tumors lacking DNA driver mutations. Design Forty-four formalin-fixed, paraffin-embedded (FFPE) or fresh-frozen lung, brain, soft tissue and skin tumors were used to determine the accuracy of the assay. Additional fusion-positive specimens and a calibrated reference standard were used to establish the precision, reproducibility and sensitivity of the assay. All aspects of the validation, including quality control metrics, were performed according to New York State guidelines. Results For the RNA fusion panel, accuracy, reproducibility and precision studies were above 99%. Reproducibility and sensitivity studies with the reference standard were helpful in identifying inconsistencies. The limit of detection for most RNA fusion transcripts was 50 copies. Application of the RNA fusion assay as a reflex test to 450 tumor samples lacking DNA driver mutations resulted in a 10% increase in diagnostic yield with minimal additional processing time. Conclusions The validated RNA fusion panel provides clinical utility in therapy selection for patients with solid tumors. By using a sequential testing approach, the RNA fusion assay complements the DNA hotspot assay in identifying clinically relevant variants across many tumor types with minimal additional increase in processing time., Highlights • RNA fusion NGS panel uses anchored multiplex PCR to detect known and novel fusions. • Pre- and post-sequencing quality control increases accuracy of results. • Sequential testing of RNA, on samples without DNA variants is practical and cost-effective. • Detection of novel RNA fusions expands the therapeutic landscape of tumors.

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.

9036 Background: Exon 14 skipping mutations in the mesenchymal-epithelial transition ( MET) gene are reported in 2-5% of lung adenocarcinomas and are mutually exclusive of other driver mutations. Small-molecule MET tyrosine kinase inhibitors, capmatinib and tepotinib, showed durable responses in previously treated and treatment-naïve patients harboring MET-exon-14 skipping mutations. Studies suggest that for detection of MET-ex14 mutations, DNA-based assays alone may be sub-optimal when compared to RNA-based NGS assays. We compared the performance of DNA and RNA-based assays for detection of MET-ex14 variants. Methods: We examined NGS-based profiling data of lung adenocarcinomas (or when this diagnosis could not be excluded) to identify MET-ex14 mutations missed by DNA but identified by RNA analysis. The carcinomas were profiled by a DNA-based NGS panel that targets MET exons 2, 14, 16, 18 and 19. Cases without driver mutations were reflexed to an NGS-based RNA fusion panel (Archer’s Anchored Multiplex PCR). Results: Over a 21-month period, MET-ex14 skipping events were detected in 16/644 (2.5%) lung carcinomas by DNA profiling. RNA analysis on driver-negative cases identified 9 additional MET-ex14 mutations. All 16 MET-ex14 DNA variants occurred at or around the intron 14 splice donor site, as the assay did not include the intron 13 splice acceptor site. Clinical characteristics of the MET positive cohort include a male to female ratio of 0.8:1.0, an average age of 76.5 years and 52% non-smoker status. All tumors were adenocarcinomas (including one with a < 10% spindle/pleomorphic component) with the exception of 3 adenosquamous carcinomas and 1 squamous cell carcinoma. Conclusions: DNA based NGS-panels can potentially miss MET-ex14 skipping events in lung carcinomas, when the primers do not target both 3′ splice site of intron 13, and the 5′ splice site of intron 14. A reflex work flow interrogating RNA fusions can potentially capture such events. The clinical and molecular characterization of the variants detected only by RNA NGS assays warrants further exploration.

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.

Classically, granuloma annulare (GA) is a cutaneous disorder localized to the dorsum of the hands and/or feet in children and young adults. Very rarely it can present on the face and rarer still on periorbital structures such as the eyelid and orbital rim. Diagnosis hinges on clinical presentation and histological features, such as palisading granulomas with central destruction of collagen, presence of mucin and lymphohistiocytic infiltration. The etiology of this condition remains unknown, but may involve a delayed-type hypersensitivity reaction, malignancy and/or infection. Herein is the first reported case of an intraorbital GA in an 86-year-old male patient who presented with right eye proptosis.

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.

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.

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.

Lung cancer is a rare event in the pediatric and adolescent population. To date, only a few case reports and small case series have been published, and little is known about the risk factors associated with this entity in children and adolescents. We describe a case of adenocarcinoma in situ in a 15-year-old adolescent girl with previous surgical treatment for malignant melanoma. We provide a detailed genomic characterization of this neoplasm by comparative genomic hybridization, genome-wide single-nucleotide polymorphism array, and fluorescence in situ hybridization analyses. We identify chromosomal regions with copy number changes and correlate the corresponding genes within these regions with the available literature in the area.

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&mdash, 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&ndash, 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.

6593 Background: The routine use of large next generation sequencing (NGS) cancer panels is required to identify the increasing number of, but often uncommon actionable alterations present across multiple tumor histologies to guide therapy. Inconsistent coverage and variable payment is hindering adoption of these tests into clinical practice. A review of clinical utility, coverage and reimbursement was conducted in a cohort of adult oncology patients who received expanded genomic panel testing as part of their clinical care. Methods: The Columbia Combined Cancer Panel (CCCP), a 467 gene panel designed to detect single nucleotide variants, indels, and copy number variations in solid and liquid tumors was performed in a CLIA-certified laboratory at Columbia University Irving Medical Center. Clinical utility categories included: immediate change in management; informed future treatment options; provided diagnostic/prognostic information; and other impact. Claims were submitted between 1/1/17 and 4/30/18. Carriers were categorized into commercial, managed-government, and government plans. Results: 300 tumors underwent NGS. Reimbursement data were available for 258 cases. 57% of testing was performed for a treatment-resistant, recurrent, or high stage cancer, or for a cancer of rare/mixed histology (21%). Findings were clinically actionable in 183 cases (61%). Results led to an immediate change in management (n = 6, 2%), informed future treatment options (n = 140, 47%), and provided diagnostic/prognostic information (n = 29, 10%). Only 57 tests (22%) received coverage. In 59% of denials (118/201), a clinically-actionable result was found. Commercial plans reimbursed 29/119 tests (24%) and managed-government plans reimbursed 28/54 tests (52%). Government plans provided no coverage for 85 tests. On average, insurers reimbursed 10% of the total CCCP service charges: 12.5% for commercial and 22% for managed-government plans. Conclusions: Expanded genomic testing identified clinically-impactful alterations in 61% of cases. Limited coverage and low reimbursement remain a barrier and broader reimbursement policies are needed to adopt expanded genomic testing that benefits patients into clinical practice.

TPS3154 Background: Assessing treatment response in castrate resistant prostate cancer (CRPC), remains a challenge due to the limited sensitivity and specificity of existing imaging modalities. Understanding prostate cancer biology with tumor biopsies does not address the issue of tumor heterogeneity or cellular degradation during the decalcification process of bone biopsies. Next generation positron emission tomography (PET) imaging and circulating biomarkers might provide additional insights on treatment responses and inform clinical decision-making earlier in therapy. 18F-DCFPyL (PyL) is a second-generation fluorinated PSMA PET tracer that has superior sensitivity and specificity to detect prostate cancer compared to standard imaging. Its role in assessing tumor response to therapy has not been evaluated. Circulating tumor DNA (ctDNA) in blood can provide tumor genomic information, while exosomes in serum and urine may provide data on the proteomic landscape of tumors. Methods: We are conducting a prospective study of 15 men with metastatic CRPC who are scheduled to start a new systemic therapy for their disease. Upon enrollment, subjects will have baseline assessments with standard cross-sectional imaging, 99mTc bone scan, and blood work. Standard scans will be performed every 8-12 weeks until progression of disease. PyL PET/CT scans and liquid biopsies (ctDNA and exosomes) will occur at baseline, 6 weeks after starting their new therapy, and at disease progression. Lesions seen on PET/CT images will be identified by a certified reader. The maximum standardized uptake value (SUVmax) will be measured and recorded in up to the five hottest lesions and normalized to a background SUVmean measured in the liver, spleen, kidney, mediastinum, and parotid glands. Changes in the normalized SUV from baseline to the 6-week PyL PET scan will be correlated to PSA response by the Pearson’s correlation coefficient. The Kaplan-Meier method will be used to evaluate progression free survival and overall survival dichotomized by the median value of SUV change. Blood for ctDNA and exosomes will be stored for future analysis. The study is open with two patients enrolled at the time of submission. One patient has completed his initial PyL PET/CT scan. Clinical trial information: NCT03585114.

Composite plasma cell neoplasm (PCN) and low grade B-cell lymphoma (B-NHL) in the bone marrow are uncommon and raise the differential diagnosis of B-NHL with plasmacytic differentiation and PCN with lymphoplasmacytic morphology. This can be a challenging differential diagnosis, and the distinctions are important because of differences in management. We report five cases of composite PCN with B-NHL or clonal B-cell infiltrates involving the bone marrow. By using multiple different diagnostic modalities, including immunophenotyping by flow cytometry and immunohistochemistry, cytogenetic analysis and IGH gene rearrangement studies by polymerase chain reaction, we were able to distinguish two distinct clonally unrelated neoplasms in all cases. We describe the utility and pitfalls of these different diagnostic modalities. Flow cytometric analysis with a panel of antibodies that includes CD19, CD56, CD138, CD45 and other aberrant markers commonly expressed by PCN will allow identification of clonally unrelated PCN and B-NHL in a composite neoplasm, and distinguish them from B-NHL with plasmacytic differentiation and PCN with lymphoplasmacytic morphology. Cytogenetic and molecular analyses can give false-negative or false-positive results. In summary, a multimodal approach utilizing these different tools, including clinical data, should be used to arrive at the correct diagnosis.

We report a rare case of peripheral T-cell lymphoma arising in a 52-year-old man with biopsy-proven aggressive polymyositis, who had cardiac involvement, progressive bulbar symptoms, and died 11 months post diagnosis due to multiorgan failure. Using a multimodality approach including immunohistochemistry, genome-wide single nucleotide polymorphism (SNP)-array analysis, and high-throughput sequencing of the complementary determining region 3 (CDR3) of T-cell receptor beta (TCRβ) genes, our study demonstrates a molecular link between polymyositis and T-cell lymphoma, and provides evidence of the rapid and possibly late occurrence of genomic instability during neoplastic transformation of an oligoclonal T-cell population. Immunohistochemical analysis revealed loss of CD5, CD7, and CD8 antigen expression in autopsy tissue samples, as well as the occurrence of aberrant CD56 expression, not seen in pre-mortem biopsies, supporting the emergence of a neoplastic T-cell population. Multiplex polymerase chain reaction and next-generation sequencing of the TCRβ CDR3 region displayed two unique T-cell clones in both the diagnostic biopsy confirming polymyositis and the autopsy muscle tissue exhibiting T-cell lymphoma, linking the two pathological processes. SNP-array analysis revealed complex genomic abnormalities at autopsy but not in the pre-mortem muscle biopsies displaying polymyositis, confirming malignant transformation of the oligoclonal T-cell infiltrate. Our findings raise the possibility that clinically aggressive polymyositis might represent a preneoplastic condition in some instances, similar to certain other autoimmune and inflammatory disorders.

Although monosomy X is the most common karyotype in patients with Turner syndrome, the presence of Y chromosome material has been observed in about 10% of patients. Y chromosome material in patients with Turner syndrome poses an increased risk of gonadoblastoma and malignant transformation. We report a woman with a diagnosis of Turner syndrome at 12 years of age, without signs of virilization, and karyotype reported as 46,X,del(X)(q13). At 26 years, cytogenetic studies indicated the patient to be mosaic for monosomy X and a cell line that contained a du­plicated Yq chromosome. Bilateral gonadectomy was performed and revealed streak gonads, without evidence of gonadoblastoma. Histological analysis showed ovarian stromal cells with few primordial tubal structures. FISH performed on streak gonadal tissue showed a heterogeneous distribution of SRY, with exclusive localization to the primordial tubal structures. DNA extraction from the gonadal tissue showed a 6.5% prevalence of SRY by microarray analysis, contrasting the 86% prevalence in the peripheral blood sample. This indicates that the overall gonadal sex appears to be determined by the majority gonosome complement in gonadal tissue in cases of sex chromosome mosaicism. This case also raises questions regarding malignancy risk associated with Y prevalence and tubal structures in gonadal tissue.

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.

Thymidylate synthase expression is known to be higher in squamous cell carcinoma than in adenocarcinoma of the lung. It is thought that this is the reason for the poor efficacy of pemetrexed in squamous cell carcinoma. However, there is limited data on thymidylate synthase expression in adenosquamous carcinoma, a distinct subtype of lung cancer containing both squamous and glandular differentiation. Furthermore, molecular alterations like epidermal growth factor receptor and Kirsten rat sarcoma 2 viral oncogene homolog mutations, which are seen in adenocarcinomas, are not well understood in mixed histology tumors such as adenosquamous carcinoma. In our study, we sought to better characterize adenosquamous tumors of the lung. Using immunohistochemistry to evaluate thymidylate synthase protein levels, we found that the expression of thymidylate synthase in these mixed tumors roughly parallel that of squamous cell carcinoma, instead of falling in between squamous cell and adenocarcinoma. Of note, in adenosquamous samples, the expression of thymidylate synthase was more closely correlated within the two components than would be expected by random chance alone. Also, we had a relatively high rate of epidermal growth factor receptor (11%) and Kirsten rat sarcoma 2 viral oncogene homolog (33%) mutations in these specimens, with the mutations showing convergence in both the glandular and squamous components upon microdissection. Our results indicate that adenosquamous carcinomas are not simple mixtures of their two histological components; they rather behave as their own entity, and it is important to further understand their behavior. Given the similarity of thymidylate synthase expression between squamous cell and adenosquamous carcinoma, and that thymidylate synthase is the main target of pemetrexed, we extrapolate that pemetrexed may also have inferior clinical activity in adenosquamous carcinoma.

Background Molecular characterization has the potential to advance the management of pediatric cancer and high-risk hematologic disease. The clinical integration of genome sequencing into standard clinical practice has been limited and the potential utility of genome sequencing to identify clinically impactful information beyond targetable alterations has been underestimated. Methods The Precision in Pediatric Sequencing (PIPseq) Program at Columbia University Medical Center instituted prospective clinical next generation sequencing (NGS) for pediatric cancer and hematologic disorders at risk for treatment failure. We performed cancer whole exome sequencing (WES) of patient-matched tumor-normal samples and RNA sequencing (RNA-seq) of tumor to identify sequence variants, fusion transcripts, relative gene expression, and copy number variation (CNV). A directed cancer gene panel assay was used when sample adequacy was a concern. Constitutional WES of patients and parents was performed when a constitutionally encoded disease was suspected. Results were initially reviewed by a molecular pathologist and subsequently by a multi-disciplinary molecular tumor board. Clinical reports were issued to the ordering physician and posted to the patient’s electronic medical record. Results NGS was performed on tumor and/or normal tissue from 101 high-risk pediatric patients. Potentially actionable alterations were identified in 38% of patients, of which only 16% subsequently received matched therapy. In an additional 38% of patients, the genomic data provided clinically relevant information of diagnostic, prognostic, or pharmacogenomic significance. RNA-seq was clinically impactful in 37/65 patients (57%) providing diagnostic and/or prognostic information for 17 patients (26%) and identified therapeutic targets in 15 patients (23%). Known or likely pathogenic germline alterations were discovered in 18/90 patients (20%) with 14% having germline alternations in cancer predisposition genes. American College of Medical Genetics (ACMG) secondary findings were identified in six patients. Conclusions Our results demonstrate the feasibility of incorporating clinical NGS into pediatric hematology-oncology practice. Beyond the identification of actionable alterations, the ability to avoid ineffective/inappropriate therapies, make a definitive diagnosis, and identify pharmacogenomic modifiers is clinically impactful. Taking a more inclusive view of potential clinical utility, 66% of cases tested through our program had clinically impactful findings and samples interrogated with both WES and RNA-seq resulted in data that impacted clinical decisions in 75% of cases. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0389-6) contains supplementary material, which is available to authorized users.

Large cancer panels are being increasingly used in the practice of precision medicine to generate genomic profiles of tumors with the goal of identifying targetable variants and guiding eligibility for clinical trials. To facilitate identification of mutations in a broad range of solid and hematological malignancies, a 467-gene oncology panel (Columbia Combined Cancer Panel) was developed in collaboration with pathologists and oncologists and is currently available and in use for clinical diagnostics. Herein, we share our experience with this testing in an academic medical center. Of 255 submitted specimens, which encompassed a diverse range of tumor types, we were able to successfully sequence 92%. The Columbia Combined Cancer Panel assay led to the detection of a targetable variant in 48.7% of cases. However, although we show good clinical performance and diagnostic yield, third-party reimbursement has been poor. Reimbursement from government and third-party payers using the 81455 Current Procedural Terminology code was at 19.4% of billed costs, and 55% of cases were rejected on first submission. Likely contributing factors to this low level of reimbursement are the delays in valuation of the 81455 Current Procedural Terminology code and in establishing national or local coverage determinations. In the absence of additional demonstrations of clinical utility and improved patient outcomes, we expect the reimbursement environment will continue to limit the availability of this testing more broadly.

BACKGROUND: Despite evidence supporting perioperative chemotherapy, few randomized studies compare neoadjuvant and adjuvant chemotherapy for bladder cancer. Consequently, the standard of care regarding the timing of chemotherapy for locally advanced bladder cancer remains controversial. We compared patient outcomes following neoadjuvant or adjuvant systemic chemotherapy for cT2-T4aN0-N2M0 bladder cancer. METHODS: In a retrospective review of a single institutional database from 1988 through 2009, we identified patients receiving neoadjuvant or adjuvant multiagent platinum-based systemic chemotherapy for locally advanced bladder cancer. Survival analysis was performed comparing disease-specific survival (DSS) and overall survival (OS). RESULTS: A total of 146 patients received systemic perioperative chemotherapy (73 neoadjuvant, 73 adjuvant). Of these, 84% (122/146) received cisplatin-based chemotherapy compared with carboplatin-based chemotherapy (24/146, 16.4%). Most patients receiving cisplatin-based chemotherapy were treated with methotrexate/vinblastine/adriamycin/cisplatin (79/122, 64.8%), whereas the remaining patients received gemcitabine/cisplatin (GC) (43/122, 35.2%). In multivariable analysis, there was no significant difference in DSS (P = .46) or OS (P = .76) between neoadjuvant or adjuvant chemotherapy groups. There was statistically significant improvement in DSS when patients received neoadjuvant GC rather than adjuvant GC (P = .049, hazard ratio, 10.6; 95% confidence interval, 1.01-112.2). CONCLUSION: In this study, there was no statistically significant difference in OS and DSS between patients receiving neoadjuvant versus adjuvant systemic platinum-based chemotherapy for locally advanced bladder cancer. In addition, there was no significant difference between neoadjuvant and adjuvant cisplatin- or carboplatin-based chemotherapy. Chemotherapy sequence relative to surgery appeared less important than whether or not a patient actually received perioperative chemotherapy. Cancer 2011;. © 2011 American Cancer Society.

2033Background: Depatuxizumab mafodotin (depatux-m, formerly ABT-414) is an EGFR-directed antibody-drug conjugate being developed for treatment of EGFR-amplified glioblastoma (GBM). As therapeutic ...

// Karthik A. Ganapathi 1 , Vaidehi Jobanputra 1 , Fabio Iwamoto 2 , Preti Jain 1 , Jinli Chen 1 , Luciano Cascione 3 , Odelia Nahum 1 , Brynn Levy 1 , Yi Xie 4 , Pallavi Khattar 5 , Daniela Hoehn 1 , Francesco Bertoni 3 , Vundavalli V. Murty 1 , Stefania Pittaluga 4 , Elaine S. Jaffe 4 , Bachir Alobeid 1 , Mahesh M. Mansukhani 1,* and Govind Bhagat 1,* 1 Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA 2 Department of Neurology, Columbia University Medical Center, New York, NY, USA 3 Institute of Oncology Research and Oncology Institute of Southern Switzerland, Bellinzona, Switzerland 4 Hematopathology Section, Laboratory of Pathology, National Cancer Institute, Bethesda, MD, USA 5 Department of Pathology, New York Medical College, Valhalla, NY, USA * These authors have contributed equally to this work Correspondence to: Govind Bhagat, email: // Mahesh M. Mansukhani, email: // Keywords : dural marginal zone lymphoma, genome, mutations, TNFAIP3, NOTCH2 Received : April 21, 2016 Accepted : May 14, 2016 Published : May 27, 2016 Abstract The dura is a rare site of involvement by marginal zone lymphoma (MZL) and the biology of dural MZL is not well understood. We performed genome-wide DNA copy number and targeted mutational analysis of 14 dural MZL to determine the genetic landscape of this entity. Monoallelic and biallelic inactivation of TNFAIP3 by mutation (n=5) or loss (n=1) was observed in 6/9 (67%) dural MZL exhibiting plasmacytic differentiation, including 3 IgG4+ cases. In contrast, activating NOTCH2 mutations were detected in 4/5 (80%) dural MZL displaying variable monocytoid morphology. Inactivating TBL1XR1 mutations were identified in all NOTCH2 mutated cases. Recurrent mutations in KLHL6 (n=2) and MLL2 (n=2) were also detected. Gains at 6p25.3 (n=2) and losses at 1p36.32 (n=3) were common chromosomal imbalances, with loss of heterozygosity (LOH) of these loci observed in a subset of cases. Translocations involving the IGH or MALT1 genes were not identified. Our results indicate genetic similarities between dural MZL and other MZL subtypes. However, recurrent and mutually exclusive genetic alterations of TNFAIP3 and NOTCH2 appear to be associated with distinct disease phenotypes in dural MZL.

Post-transplant lymphoproliferative disorders of T- or NK-cell origin (T/NK-PTLD) are rare entities and their genetic basis is unclear. We performed targeted sequencing of 465 cancer-related genes and high-resolution copy number analysis in 17 T-PTLD and 2 NK-PTLD cases. Overall, 377 variants were detected, with an average of 20 variants per case. Mutations of epigenetic modifier genes (TET2, KMT2C, KMT2D, DNMT3A, ARID1B, ARID2, KDM6B, n=11). and inactivation of TP53 by mutation and/ or deletion (n=6) were the most frequent alterations, seen across disease subtypes, followed by mutations of JAK/STAT pathway genes (n=5). Novel variants, including mutations in TBX3 (n=3), MED12 (n=3) and MTOR (n=1), were observed as well. High-level microsatellite instability was seen in 1 of 14 (7%) cases, which had a heterozygous PMS2 mutation. Complex copy number changes were detected in 8 of 16 (50%) cases and disease subtype-specific aberrations were also identified. In contrast to B-cell PTLDs, the molecular and genomic alterations observed in T/ NK-PTLD appear similar to those reported for peripheral T-cell lymphomas occurring in immunocompetent hosts, which may suggest common genetic mechanisms of lymphoma development.

Purpose To evaluate the toxicity and efficacy of individualized neoadjuvant androgen deprivation (AD) to maximal response followed by external beam radiotherapy (RT) with continued AD for a total of 9 months in a prospective phase II trial. Patients and Methods One hundred twenty-three patients received a total of 9 months of flutamide and luprolide combined with RT. RT initiation was individualized to begin after maximum response to AD as assessed by monthly digital rectal examination and prostate-specific antigen (PSA). The neoadjuvant phase was restricted to no more than 6 months. Results Median time to initiation of RT was 4.7 months. Indications to begin RT (and their rates) were undetectable PSA (28%), PSA unchanged from one month to the next (46%), PSA rising from one month to the next (10%), 6 months of AD (14%), and other (2%). Five-year outcomes were biochemical disease-free survival, (DFS) 63% ± 7%; clinical DFS, 75% ± 5%; cancer-specific survival, 99% ± 1%; and overall survival, 89% ± 3%. Patients initiating RT after 6 months of AD had significantly lower biochemical and clinical DFS. Those patients whose testosterone recovered to normal after completion of AD had a significantly superior survival rate. Of those patients potent before treatment, 65% remained so at last follow-up. Conclusion The combination of 9 months of AD and RT, with initiation of RT individualized on the basis of maximum response to AD, achieves disease control rates comparable with past studies, while preserving potency in many patients. Further studies are warranted to determine the optimal combination of AD and RT in this patient population.