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Studies have shown that fluorescence in situ hybridization (FISH) testing increases lung cancer detection on cytology specimens in peripheral nodules. The goal of this study was to determine whether a predictive model using clinical features and routine cytology with FISH results could predict lung malignancy after a nondiagnostic bronchoscopic evaluation. Patients with an indeterminate peripheral lung nodule that had a nondiagnostic bronchoscopic evaluation were included in this study (N = 220). FISH was performed on residual bronchial brushing cytology specimens diagnosed as negative (n = 195), atypical (n = 16), or suspicious (n = 9). FISH results included hypertetrasomy (n = 30) and negative (n = 190). Primary study end points included lung cancer status along with time to diagnosis of lung cancer or date of last clinical follow-up. Hazard ratios (HRs) were calculated using Cox proportional hazards regression model analyses, and P values b .05 were considered statistically significant. The mean age of the 220 patients was 66.7 years (range, 35-91), and most (58%) were men. Most patients (79%) were current or former smokers with a mean pack year history of 43.2 years (median, 40; range, 1-200). After multivariate analysis, hypertetrasomy FISH (HR = 2.96, P b .001), pack years (HR = 1.03 per pack year up to 50, P = .001), age (HR = 1.04 per year, P = .02), atypical or suspicious cytology (HR = 2.02, P = .04), and nodule spiculation (HR = 2.36, P = .003) were independent predictors of malignancy over time and were used to create a prediction model (C-statistic = 0.78). These results suggest that this multivariate model including test results and clinical features may be useful following a nondiagnostic bronchoscopic examination. [ABSTRACT FROM AUTHOR]

OBJECTIVES:A polysomy fluorescence in situ hybridization (FISH) result in a pancreatobiliary brushing from a patient with primary sclerosing cholangitis (PSC) is very worrisome for carcinoma. However, treatment is not recommended unless verified by corroborative evidence of malignancy because of less than perfect test specificity in this population. The aim of this study was to evaluate the clinical outcome of PSC patients with serial polysomy FISH results.METHODS:Patients with PSC underwent endoscopic retrograde cholangiopancreatography with brushings when clinically indicated per standard practice. Brushings were evaluated by routine cytology and FISH. Retrospective review identified patients with a polysomy FISH result without definitive imaging or pathological evidence of malignancy at the time of the first polysomy, who underwent follow-up examinations including subsequent FISH testing (n=30). Patient records were reviewed to determine clinical outcome.RESULTS:In all, 9 of 13 patients (69%) with a subsequent polysomy result (i.e., serial polysomy) were diagnosed with cholangiocarcinoma (CCA) compared with 3 of 17 patients (18%) with subsequent non-polysomy results (P=0.008). There was a significant difference in time to a diagnosis of CCA between PSC patients with serial polysomy compared with those with subsequent non-polysomy (P=0.01). In four patients with serial polysomy results, imaging/pathological evidence of CCA was not found until 1-2.7 years after the initial polysomy FISH result.CONCLUSIONS:FISH may detect polysomic cells in pancreatobiliary brushings before other pathological or imaging techniques identify CCA. Patients with serial polysomy FISH results are at higher risk for having CCA than those with subsequent non-polysomy FISH results. [ABSTRACT FROM AUTHOR]

Background & Aims Cellular and nuclear material from tumors disseminates into the bloodstream (tumoremia), but it is not clear whether medical procedures cause release of this material or contribute to formation of metastases. We performed a prospective study of blood samples from patients with pancreatic adenocarcinoma (PDAC) to determine whether endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) associates with markers of tumoremia. Methods We obtained peripheral blood from 104 patients (35 with PDAC) before and after EUS-FNA of primary tumors; blood samples from 69 healthy individuals were used as controls. Plasma concentrations of cell-free DNA (cfDNA) were measured, and cfDNA and primary tumor samples were analyzed to detect activating mutations in KRAS. Potential development of tumoremia was defined by an increase in cfDNA of 2-fold or more, and/or detection of mutant KRAS in samples collected after FNA from patients whose blood samples did not contain detectable mutant KRAS before FNA. Results Peripheral blood concentrations of cfDNA were 1200 ng/ml (500–3300 ng/ml) before FNA vs 1400 ng/ml (900–4000 ng/ml) after FNA (P =.391). Tumoremia was detected in 10/35 patients (28.6%): 7 patients had a ≥2-fold increase in cfDNA concentration (20.6%) and 3 patients had circulating tumor DNA with KRAS mutations after FNA that were not detected before FNA (8.8%). New distant metastases were detected in 1.3 ± 0.82 patients with tumoremia vs 0.64 ± 0.81 without (P =.0375). Overall mortality did not differ significantly between patients with tumoremia (10/10 deaths, 100%) vs those without (19/25 deaths, 76%) nor did survival times of deceased patients (13.3 months for patients with tumoremia; range, 5.8–14.9 months vs 11.1 months for patients without tumoremia; range, 5.5–14.5 months). However, 6 patients without tumoremia were alive at a mean 23.9 months after EUS-FNA (range, 19.9–25 months after EUS-FNA) vs none of the patients with tumoremia. Conclusion In patients with PDAC, EUS-FNA associates with increased plasma concentration of cfDNA and increased detection of mutant KRAS after the procedure (markers of tumoremia and possible new distant metastasis). Although levels of cfDNA and activating mutations in KRAS are logical markers of tumoremia, they may not serve as the ideal biomarkers of this process. These findings are preliminary and do not indicate a need to modify current practice, yet further studies are needed. [ABSTRACT FROM AUTHOR]

Background and Aims In an era of precision medicine, customized genotyping of GI stromal tumors by screening for driver mutations will become the standard of care. The fidelity of genotype concordance between paired cytology smears and surgical pathology specimens is unknown. In patients with either primary or metastatic sporadic disease, we sought to determine the frequency of KIT and PDGFRA pathogenic alterations within such specimens, imatinib sensitivity, and the concordance of pathogenic alterations between paired specimens. Methods DNA obtained from cytology smears from 36 patients, 24 of whom had paired surgical pathology specimens, underwent targeted next-generation sequencing by using a custom panel to evaluate somatic mutations within KIT (exon 2, 9, 10, 11, 13, 14, 15, 17, 18) and PDGFRA (exon 12, 14, 15, 18) genes. Patients with KIT and PDGRFA wild-type genes completed the Qiagen Human Comprehensive Cancer GeneRead DNAseq Targeted Array V2. Results Genotyping revealed KIT and PDGFRA mutations in 68% and 15% of patients. The wild-type population did not harbor mutations in BRAF, RAS family, SDHB, SETD2, or NF1. Imatinib sensitivity based on the oncogenic kinase mutation prevalence was estimated to be 68%. Mutational concordance between paired cytology and surgical pathology specimens was 96%. Conclusions Our data have demonstrated the ability to stratify either primary or metastatic gastrointestinal stromal tumors by mutational subtype using a targeted next-generation sequencing 2 gene mutation panel. We highlight the ability to use cytology specimens obtained via minimally invasive techniques as a surrogate to surgical specimens given the high mutational landscape concordance between paired specimens. [ABSTRACT FROM AUTHOR]

An abstract of the article "Routine Cytology and Reflex Fluorescence In-Situ Hybridization Analysis of Bronchial Brushing Specimens for Central and Peripheral Lung Lesions," by Jesse S. Voss, Benjamin R. Kipp, Amy C. Clayton, Otis B. Rickman, James R. Jett, Michael R. Henry, and Kevin C. Halling is presented.

Background & Aims Pancreatobiliary cancer is detected by fluorescence in situ hybridization (FISH) of pancreatobiliary brush samples with UroVysion probes, originally designed to detect bladder cancer. We designed a set of new probes to detect pancreatobiliary cancer and compared its performance with that of UroVysion and routine cytology analysis. Methods We tested a set of FISH probes on tumor tissues (cholangiocarcinoma or pancreatic carcinoma) and non-tumor tissues from 29 patients. We identified 4 probes that had high specificity for tumor vs non-tumor tissues; we called this set of probes pancreatobiliary FISH. We performed a retrospective analysis of brush samples from 272 patients who underwent endoscopic retrograde cholangiopancreatography for evaluation of malignancy at the Mayo Clinic; results were available from routine cytology and FISH with UroVysion probes. Archived residual specimens were retrieved and used to evaluate the pancreatobiliary FISH probes. Cutoff values for FISH with the pancreatobiliary probes were determined using 89 samples and validated in the remaining 183 samples. Clinical and pathologic evidence of malignancy in the pancreatobiliary tract within 2 years of brush sample collection was used as the standard; samples from patients without malignancies were used as negative controls. The validation cohort included 85 patients with malignancies (46.4%) and 114 patients with primary sclerosing cholangitis (62.3%). Samples containing cells above the cutoff for polysomy (copy number gain of ≥2 probes) were classified as positive in FISH with the UroVysion and pancreatobiliary probes. Multivariable logistic regression was used to estimate associations between clinical and pathology findings and results from FISH. Results The combination of FISH probes 1q21, 7p12, 8q24, and 9p21 identified cancer cells with 93% sensitivity and 100% specificity in pancreatobiliary tissue samples and were therefore included in the pancreatobiliary probe set. In the validation cohort of brush samples, pancreatobiliary FISH identified samples from patients with malignancy with a significantly higher level of sensitivity (64.7%) than the UroVysion probes (45.9%) ( P < .001) or routine cytology analysis (18.8%) ( P < .001), but similar specificity (92.9%, 90.8%, and 100.0% respectively). Factors significantly associated with detection of carcinoma, in adjusted analyses, included detection of polysomy by pancreatobiliary FISH ( P < .001), a mass by cross-sectional imaging ( P < .001), cancer cells by routine cytology (overall P = .003), as well as absence of primary sclerosing cholangitis ( P = .011). Conclusions We identified a set of FISH probes that detects cancer cells in pancreatobiliary brush samples from patients with and without primary sclerosing cholangitis with higher levels of sensitivity than UroVysion probes. Cytologic brushing test results and clinical features were independently associated with detection of cancer and might be used to identify patients with pancreatobiliary cancers. [ABSTRACT FROM AUTHOR]

Background Targeted next-generation sequencing has the potential to stratify a tumor by molecular subtype and aid the development of a biomarker profile for prognostic risk stratification and theranostic potential. Objective To assess the frequency and distribution of pathogenic alterations in malignant lymph node cytology specimens. Design Multigene molecular profiling of archived malignant EUS-FNA lymph node cytology specimens using the Ion Ampliseq Cancer Hotspot Panel v2, which targets at least 2855 possible mutations within 50 cancer-associated genes. Setting Single tertiary referral center. Patients Sporadic, treatment naive, locally advanced primary rectal cancer by EUS-FNA (n = 76) who subsequently completed neoadjuvant therapy with on-site oncologic surgery. Main Outcome Measurements The frequency and distribution of pathogenic alterations in malignant lymph node cytology specimens by the mitogen-activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling pathways, by KRAS or NRAS wild-type lymph node status, by extramesenteric lymph node status, and by a complete pathologic response status. Results Eleven patients (14.5%) were 50-gene panel wild-type. Sixty-five patients had 139 pathogenic alterations (2 [1-3] per patient) in 13 of 50 evaluated genes. The following represent a spectrum of identified alterations: TP53 (n = 52; 68.4%), APC (n = 36; 47.4%), KRAS (n = 22; 28.9%), FBXW7 (n = 8; 10.5%), NRAS (n = 6; 7.9%), PIK3CA (n = 4; 5.3%), SMAD4 (n = 3; 3.9%), and BRAF (n = 3; 3.9%). Pathogenic alterations were identified in the MAPK and PI3K signaling pathways in 41% and 5% of patients, respectively. Limitations Findings were limited to a 50 cancer-associated gene analysis. Conclusions Molecular EUS lymph node assessments using cancer “hotspot” panels can identify pathogenic alteration frequency and distribution and have theranostic potential for individualized patient care. [ABSTRACT FROM AUTHOR]

Context.--The need for appropriate specimen use for ancillary testing has become more commonplace in the practice of pathology. This, coupled with improvements in technology, often provides less invasive methods of testing, but presents new challenges to appropriate specimen collection and handling of these small specimens, including thoracic small biopsy and cytology samples. Objective.--To develop a clinical practice guideline including recommendations on how to obtain, handle, and process thoracic small biopsy and cytology tissue specimens for diagnostic testing and ancillary studies. Methods.--The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. Core needle biopsy, touch preparation, fine-needle aspiration, and effusion specimens with thoracic diseases including malignancy, granulomatous process/sarcoidosis, and infection (eg, tuberculosis) were deemed within scope. Ancillary studies included immunohistochemistry and immunocytochemistry, fluorescence in situ hybridization, mutational analysis, flow cytometry, cytogenetics, and microbiologic studies routinely performed in the clinical pathology laboratory. The use of rapid on-site evaluation was also covered. Results.--Sixteen guideline statements were developed to assist clinicians and pathologists in collecting and processing thoracic small biopsy and cytology tissue samples. Conclusions.--Based on the systematic review and expert panel consensus, thoracic small specimens can be handled and processed to perform downstream testing (eg, molecular markers, immunohistochemical biomarkers), core needle and fine-needle techniques can provide appropriate cytologic and histologic specimens for ancillary studies, and rapid on-site cytologic evaluation remains helpful in appropriate triage, handling, and processing of specimens. [ABSTRACT FROM AUTHOR]

We aimed to assess whether endometrial cancer (EC) can be detected in shed DNA collected with vaginal tampon by analyzing copy number, methylation markers, and mutations. Tampons were collected prior to hysterectomy from 38 EC patients and 28 women with benign indications. Extracted tampon DNA underwent the following: 1) low-coverage whole genome sequencing (LC-WGS) to assess copy number, 2) pyrosequencing to measure percent promotor methylation of HOXA9, RASSF1 , and CDH13 and 3) next generation sequencing (NGS) to identify mutations in 19 genes associated with EC identified through The Cancer Genome Atlas. Sensitivity and specificity for each test and test combinations were calculated. Methylation analysis yielded the highest specificities but lowest sensitivities (37–40% sensitivity; 100% specificity for HOXA9 , RASSF1 and HTR1B) while mutation analysis had improved sensitivity (50% sensitivity; 83% specificity). Only one "false positive" result for copy number variants was identified among women with benign surgical indications, which was based on detection of copy number changes, and associated with a leiomyosarcoma that was only recognized at hysterectomy. Considering any of the 3 biomarker classes as a positive, resulted in a sensitivity of 92% and specificity of 86%. Mutation analysis did not add sensitivity to the combination of analysis of copy number and methylation. This study demonstrates a proof-of-principle for non-invasive yet precise detection of endometrial cancer. We propose that with improved biomarker testing, it may be possible to develop a clinically useful test for detecting EC. • A combined CNV, methylation, and mutation panel in tampon samples is sensitive and specific for endometrial cancer. • This sensitivity and specificity is preserved with the combination of CNV and methylation alone. • One patient with a clinically unsuspected leiomyosarcoma diagnosed at hysterectomy yielded a false positive via CNV. [ABSTRACT FROM AUTHOR]

Background: Mucinous ovarian tumors represent a distinct histotype of epithelial ovarian cancer. The rarest (2-4 % of ovarian carcinomas) of the five major histotypes, their genomic landscape remains poorly described. We undertook hotspot sequencing of 50 genes commonly mutated in human cancer across 69 mucinous ovarian tumors. Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be "RAS-pathway alteration negative", using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations. Methods: Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel. Regions of disparate ERBB2-amplification status were sequenced independently for two mucinous carcinoma (MC) cases, previously established as showing ERBB2 amplification/overexpression heterogeneity, to assess the hypothesis of subclonal populations containing either KRAS mutation or ERBB2 amplification independently or simultaneously. Results: We detected mutations in KRAS, TP53, CDKN2A, PIK3CA, PTEN, BRAF, FGFR2, STK11, CTNNB1, SRC, SMAD4, GNA11 and ERBB2. KRAS mutations remain the most frequently observed alteration among MC (64.9 %) and mucinous borderline tumors (MBOT) (92.3 %). TP53 mutation occurred more frequently in carcinomas than borderline tumors (56.8 % and 11.5 %, respectively), and combined IHC and mutation data suggest alterations occur in approximately 68 % of MC and as many as 20 % of MBOT. Proven and potential RAS-pathway activating changes were observed in all but one MC. Concurrent ERBB2 amplification and KRAS mutation were observed in a substantial number of cases (7/63 total), as was co-occurrence of KRAS and BRAF mutations (one case). Microdissection of ERBB2-amplified regions of tumors harboring KRAS mutation suggests these alterations are occurring in the same cell populations, while consistency of KRAS allelic frequency in both ERBB2 amplified and non-amplified regions suggests this mutation occurred in advance of the amplification event. Conclusions: Overall, the prevalence of RAS-alteration and striking co-occurrence of pathway "double-hits" supports a critical role for tumor progression in this ovarian malignancy. Given the spectrum of RAS-activating mutations, it is clear that targeting this pathway may be a viable therapeutic option for patients with recurrent or advanced stage mucinous ovarian carcinoma, however caution should be exercised in selecting one or more personalized therapeutics given the frequency of non-redundant RAS-activating alterations. [ABSTRACT FROM AUTHOR]

Objective We demonstrate the feasibility of detecting EC by combining minimally-invasive specimen collection techniques with sensitive molecular testing. Methods Prior to hysterectomy for EC or benign indications, women collected vaginal pool samples with intravaginal tampons and underwent endometrial brushing. Specimens underwent pyrosequencing for DNA methylation of genes reported to be hypermethylated in gynecologic cancers and recently identified markers discovered by profiling over 200 ECs. Methylation was evaluated individually across CpGs and averaged across genes. Differences between EC and benign endometrium (BE) were assessed using two-sample t-tests and area under the curve (AUC). Results Thirty-eight ECs and 28 BEs were included. We evaluated 97 CpGs within 12 genes, including previously reported markers (RASSF1, HSP2A, HOXA9, CDH13, HAAO, and GTF2A1) and those identified in discovery work (ASCL2, HTR1B, NPY, HS3ST2, MME, ADCYAP1, and additional CDH13 CpG sites). Mean methylation was higher in tampon specimens from EC v. BE for 9 of 12 genes (ADCYAP1, ASCL2, CDH13, HS3ST2, HTR1B, MME, HAAO, HOXA9, and RASSF1) (all p < 0.05). Among these genes, relative hypermethylation was observed in EC v. BE across CpGs. Endometrial brush and tampon results were similar. Within tampon specimens, AUC was highest for HTR1B (0.82), RASSF1 (0.75), and HOXA9 (0.74). This is the first report of HOXA9 hypermethylation in EC. Conclusion DNA hypermethylation in EC tissues can also be identified in vaginal pool DNA collected via intravaginal tampon. Identification of additional EC biomarkers and refined collection methods are needed to develop an early detection tool for EC. [ABSTRACT FROM AUTHOR]

An amplicon-based targeted next-generation sequencing (NGS) assay for the detection of gene fusions in sarcomas was developed, validated, and implemented. This assay can detect fusions in targeted regions of 138 genes and BCOR internal tandem duplications. This study reviews our experience with testing on the first 652 patients analyzed. Gene fusions were detected in 238 (36.5%) of 652 cases, including 83 distinct fusions in the 238 fusion-positive cases, 10 of which had not been previously described. Among the 238 fusion-positive cases, the results assisted in establishing a diagnosis for 137 (58%) cases, confirmed a suspected diagnosis in 66 (28%) cases, changed a suspected diagnosis in 25 (10%) cases, and were novel fusions with unknown clinical significance in 10 (4%) cases. Twenty-six cases had gene fusions (ALK, ROS1, NTRK1, NTRK3, and COL1A1::PDGFB) for which there are targetable therapies. BCOR internal tandem duplications were identified in 6 (1.2%) of 485 patients. Among the 138 genes in the panel, 66 were involved in one or more fusions, and 72 were not involved in any fusions. There was little overlap between the genes involved as 5'-partners (31 different genes) and 3'-partners (37 different genes). This study shows the clinical utility of a next-generation sequencing gene fusion detection assay for the diagnosis and treatment of sarcomas., Competing Interests: Disclosure Statement None declared., (Copyright © 2025 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: The COVID-19 pandemic emphasized an urgent need for devices used in the self-collection of biospecimens in an evolving patient care system. The mailing of biospecimen self-collection kits to patients, with samples returned via mail, provides a more convenient testing regimen, but could also impart patient sampling variabilities. User compliance with device directions is central to downstream testing of collected biospecimens and clear instructions are central to this goal., Methods: Here, we performed an evaluation of 10 oral DNA collection devices involving either swab or saliva self-collection and analyzed ease of use and comfort level with a device, as well as DNA recovery quantity/quality and sample stability., Results: We show that while these DNA quality/quantity metrics are comparable between devices, users prefer direct saliva collection over swab-based devices., Conclusions: This information is useful in guiding future experiments including their use in human RNA, microbial, or viral sample collection/recovery and their use in clinical testing., (© Association for Diagnostics & Laboratory Medicine 2024.)

Renal neoplasia occurring as a second malignancy following childhood cancer has been most closely associated with neuroblastoma and Wilms tumor. While some cases have been associated with a genetic predisposition, nearly all are thought to result from "late effects" of therapy-related toxicity that involves chemotherapy or radiation. It is unclear if these tumors are enriched for specific molecular or morphologic characteristics. A query of our institutional nephrectomy registry of 8295 patients for renal neoplasia occurring post-treatment for childhood cancer revealed 6 patients with Wilms tumor, 4 with neuroblastoma, and 1 with acute lymphoblastic leukemia (ALL). Three additional cases of MiT family translocation renal cell carcinoma (RCC), from 2 patients, following chemotherapy for neuroblastoma and systemic lupus erythematosus and another of clear cell RCC post-ALL were included. The most common tumor type was clear cell RCC: 9/19 cases (47.4%), followed by metanephric adenoma and MiT family translocation RCC (3/19, 15.8%). There were no characteristic features to indicate a unique renal neoplasia subtype. Potential syndromic renal neoplasia occurred in 2 patients, metanephric adenomas and oncocytoma in a patient with hyperparathyroidism-jaw tumor syndrome post-treatment of Wilms tumor and a fumarate hydratase-deficient RCC in a patient post-treatment for ALL. The mean age at diagnosis of childhood neoplasia or treatment with chemotherapy or radiation was 4.7 years, and the average time to subsequent renal neoplasia was 31 years. Five (of 14) patients developed metastatic RCC, and there were 2 RCC-related deaths. These results indicate the need for extended clinical follow-up of these patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Despite the current classification of high-grade serous carcinoma (HGSCA) and low-grade serous carcinoma (LGSCA) as mutually exclusive diseases based on morphology and molecular pathogenesis, cases with mixed morphologic features of HGSCA and LGSCA have been reported. Herein we assess the clinicopathologic, immunohistochemical (IHC), and molecular genetic characteristics of a group of these cases, which we termed indeterminate grade serous carcinoma (IGSCA) in comparison with groups of HGSCA and LGSCA. Using the World Health Organization (WHO) classification criteria, we selected 27 LGSCA and 19 IGSCA for detailed morphologic study. Thirteen classic HGSCA, 19 classic LGSCA, and 19 IGSCA were selected for p53 and BRAF V600E IHC and molecular genetic testing by next-generation sequencing. IGSCA showed the architectural patterns of invasion of LGSCA, but with higher grade nuclear features focally and a mitotic index intermediate between LGSCA and HGSCA. Few cases in the IGSCA group showed mutant TP53 by IHC or sequencing (4/18, 22.2%), 1 case had mutant BRAF non-V600E by sequencing, and 1 had an NRAS mutation. When present, the mutations were identical in the low-grade and high-grade areas. The IGSCA group had a long-term survival similar to the classic HGSCA group. IGSCA with mixed morphologic features of HGSCA and LGSCA is a rare and potentially clinically aggressive variant of serous carcinoma. Their distinct morphologic, but heterogenous molecular features, including low frequency of TP53 and BRAF mutations suggest that these rare tumors may have a different pathogenesis pathway compared with classic HGSCA and classic LGSCA.

Anaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK) + or ALK - , based on the presence or absence of ALK rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, MSC E116K , exclusively in ALK - ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of MSC E116K for ALK - ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting DUSP22 rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin-bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSC E116K acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of MYC and cell cycle inhibition. Chromatin immunoprecipitation-sequencing and transcriptome analysis identified the cell cycle regulatory gene E2F2 as a direct transcriptional target of musculin. MSC E116K reversed E2F2-induced cell cycle arrest and promoted expression of the CD30-IRF4-MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the MSC promoter. Finally, ALCL cells expressing MSC E116K were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent MSC mutation as a key driver of the CD30-IRF4-MYC axis and cell cycle progression in a unique subset of ALCLs., (© 2019 by The American Society of Hematology.)

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Invasive mucinous adenocarcinoma is a variant of lung adenocarcinoma, which may be mixed with nonmucinous adenocarcinoma. KRAS mutations are common, but other clinical and genetic features are not clearly established. Lung adenocarcinomas (n=760) with ≥5 years of follow-up comprised 3 nonoverlapping cohorts for survival analysis. Mucinous tumors were evaluated with Ion AmpliSeq Cancer Hotspot Panel v2. Cases without detected mutations were tested for ALK and ROS1 and by OncoScan array. Fifty-seven invasive mucinous adenocarcinomas and 54 mixed mucinous/nonmucinous adenocarcinomas were identified. Mucinous tumors constituted 27 of 218 nonselected patients (12.4%), 23 of 268 never-smokers (8.6%), and 61 of 274 in a smokers cohort enriched for lepidic growth (22.3%). In the lepidic-enriched smokers, patients with mucinous tumors experienced worse overall survival (P=.006) and progression-free survival (P=.024), which persisted on multivariable analysis. No survival differences were observed in the other cohorts. KRAS mutations were common (76% of invasive mucinous adenocarcinomas, 68% of mixed mucinous/nonmucinous), and 38% of KRAS mutations occurred with other mutations, especially STK11. Six cases had potentially targetable mutations (3 ALK, 2 EGFR, 1 BRAF V600E). All ALK-rearranged tumors were mixed mucinous/nonmucinous. Four of 6 cases without hotspot mutations showed complex copy number/structural abnormalities. Pulmonary invasive mucinous adenocarcinomas and mixed nonmucinous/mucinous adenocarcinomas are clinically and genetically similar, except for a higher rate of ALK rearrangement in mixed tumors. Survival for mucinous tumors is similar to that for nonmucinous tumors in a nonselected cohort, although worse survival was seen in a cohort of smokers enriched for lepidic growth., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sporadic lymphangioleiomyomatosis is a progressive pulmonary cystic disease resulting from the infiltration of smooth muscle-like lymphangioleiomyomatosis cells into the lung. The migratory/metastasizing properties of the lymphangioleiomyomatosis cell together with the presence of somatic mutations, primarily in the tuberous sclerosis complex gene (TSC2), lead many to consider this a low-grade malignancy. As malignant tumors characteristically accumulate somatic structural variations, which have not been well studied in sporadic lymphangioleiomyomatosis, we utilized mate pair sequencing to define structural variations within laser capture microdissected enriched lymphangioleiomyomatosis cell populations from five sporadic lymphangioleiomyomatosis patients. Lymphangioleiomyomatosis cells were confirmed in each tissue by hematoxylin eosin stain review and by HMB-45 immunohistochemistry in four cases. A mutation panel demonstrated characteristic TSC2 driver mutations in three cases. Genomic profiles demonstrated normal diploid coverage across all chromosomes, with no aneuploidy or detectable gains/losses of whole chromosomal arms typical of neoplastic diseases. However, somatic rearrangements and smaller deletions were validated in the two cases which lacked TSC2 driver mutations. Most significantly, one of these sporadic lymphangioleiomyomatosis cases contained two different size deletions encompassing the entire TSC1 locus. The detection of a homozygous deletion of TSC1 driving a predicted case of sporadic lymphangioleiomyomatosis, consistent with the common two-hit TSC2 mutation model, has never been reported for sporadic lymphangioleiomyomatosis. However, while no evidence of the hereditary tuberous sclerosis complex disease was reported for this patient, the potential for mosaicism and sub-clinical phenotype cannot be ruled out. Nevertheless, this study demonstrates that somatic structural rearrangements are present in lymphangioleiomyomatosis disease and provides a novel method of genomic characterization of sporadic lymphangioleiomyomatosis cells, aiding in defining cases with no detected mutations by conventional methodologies. These structural rearrangements could represent additional pathogenic mechanisms in sporadic lymphangioleiomyomatosis disease, potentially affecting response to therapy and adding to the complex genetic story of this rare disease.

Purpose Molecular diagnostic testing has become an integral part of the evaluation of patients with metastatic colorectal cancer (CRC). Expanded mutational testing, such as next-generation sequencing (NGS), often identifies mutations with unclear clinical or prognostic implications. One such example is BRAF mutations that occur outside of codon 600 ( non-V600 BRAF mutations). Methods We conducted this multicenter, retrospective cohort study to characterize the clinical, pathologic, and survival implications of non-V600 BRAF mutations in metastatic CRC. We pooled patients in whom non-V600 BRAF mutations were identified from NGS databases at three large molecular genetics reference laboratories. Results A total of 9,643 patients with metastatic CRC underwent NGS testing. We identified 208 patients with non-V600 BRAF mutations, which occurred in 2.2% of all patients tested and accounted for 22% of all BRAF mutations identified. Cancers with non-V600 BRAF mutations, compared with cancers with V600E BRAF ( V600E BRAF) mutations, were found in patients who were significantly younger (58 v 68 years, respectively), fewer female patients (46% v 65%, respectively), and patients who had fewer high-grade tumors (13% v 64%, respectively) or right-sided primary tumors (36% v 81%, respectively). Median overall survival was significantly longer in patients with non-V600 BRAF-mutant metastatic CRC compared with those with both V600E BRAF-mutant and wild-type BRAF metastatic CRC (60.7 v 11.4 v 43.0 months, respectively; P < .001). In multivariable analysis, non-V600 BRAF mutation was independently associated with improved overall survival (hazard ratio, 0.18; P < .001). Conclusion Non-V600 BRAF mutations occur in approximately 2.2% of patients with metastatic CRC and define a clinically distinct subtype of CRC with an excellent prognosis.

Gastric gastrointestinal stromal tumors (GISTs) usually contain the mast/stem cell growth factor receptor Kit gene (KIT) or platelet-derived growth factor receptor A (PDGFRA) mutations that can be targeted by, or mediate resistance to, imatinib. Diagnostic material often is obtained by endoscopic ultrasound-guided fine-needle aspiration, which often is unsuitable for molecular analysis. We investigated whether targeted next-generation sequencing (NGS) can be used in multiplex genotype analysis of cytology samples collected by endoscopic ultrasound-guided fine-needle aspiration. We used the Ion AmpliSeq V2 Cancer Hotspot NGS Panel (Life Technologies, Carlsbad, CA) to identify mutations in more than 2800 exons from 50 cancer-associated genes in GIST samples from 20 patients. We identified KIT mutations in 58% of samples (91% in exon 11 and 9% in exon 17) and PDGFRA mutations in 26% (60% in exon 18 and 40% in exon 12); 16% of samples had no mutations in KIT or PDGFRA. No pathogenic alterations were found in PIK3CA, BRAF, KRAS, NRAS, or FGFR3. We predicted that 32% of patients would have primary resistance to imatinib, based on mutations in exon 17 of KIT, exon 18 of PDGFRA (D842V), or no mutation in either gene. Targeted NGS of cytology samples from GISTs is feasible and provides clinically relevant data about kinase genotypes that can help guide individualized therapy., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Determination of tumor genetic architecture based on tissue analysis yields important information on signaling pathways involved in cancer pathogenesis and plays a growing role in choosing the optimal medical management of malignancies. Specifically, the advent of next-generation sequencing has led to a rapidly evolving era of relatively inexpensive, high-throughput DNA sequencing of tumors. One such example is multiplexed tumor genotyping (ie, panel testing) of more than 2800 mutations across 50 commonly mutated cancer-associated genes. This resulting mutational landscape shows medically actionable pathogenic alterations to optimize antitumor therapy. We recently assessed the performance and outcome of targeted next-generation sequencing with archived endoscopic ultrasound fine-needle aspirates across a broad range of primary and metastatic sites with encouraging accuracy. As a result, endoscopic ultrasound has the potential to move from a test for diagnosis or confirmation of malignancy, to one in which it could facilitate the personalization of cancer-directed therapy., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Grading criteria for biliary dysplasia associated with primary sclerosing cholangitis (PSC) have been recently described. Although a dysplasia to cholangiocarcinoma (CCA) sequence is implied, supportive data are lacking. Seventeen liver explants with biliary dysplasia from patients with PSC were selected. Formalin-fixed, paraffin-embedded blocks from each patient were evaluated to identify areas of normal/reactive biliary epithelium, intestinal metaplasia, low-grade dysplasia, high-grade dysplasia, and CCA. Areas of interest were assessed for chromosomal alteration with fluorescence in situ hybridization using probes directed to locus 9p21 and centromeres 3, 7, and 17. The cutoffs for calling probe copy number abnormalities for polysomy, single locus gain, and homozygous 9p21 loss were established by receiver operating characteristic curve analysis. Of 4 areas of intestinal metaplasia, 19 low-grade dysplasias, 19 high-grade dysplasias, and 5 CCAs, 0%, 11%, 58%, and 40% displayed polysomy and 0%, 0%, 16%, and 40% exhibited homozygous 9p21 loss as the most severe abnormality, respectively. Patients with prior or current CCA were more likely to display polysomy in dysplasia than patients without CCA (70% versus 14%; P = .05); however, high-grade dysplasia was proportionally more common in the CCA-associated dysplasia group. Polysomy and homozygous 9p21 loss are detected in biliary dysplasia and CCA. These findings support a dysplasia-carcinoma sequence in PSC patients and suggest that fluorescence in situ hybridization analysis could help refine the grading of biliary dysplasia in these patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Introduction: Recent discoveries of inherited glioma risk loci and acquired IDH mutations are providing new insights into glioma etiology. IDH mutations are common in lower grade gliomas and secondary glioblastomas and uncommon in primary glioblastomas. Because the inherited variant in 11q23 has been associated with risk of lower grade glioma and not with glioblastomas, we hypothesized that this variant increases susceptibility to IDH-mutated gliomas, but not to IDH-wild-type gliomas., Methods: We tested this hypothesis in patients with glioma and controls from the San Francisco Adult Glioma Study, the Mayo Clinic, and Illumina controls (1102 total patients, 5299 total controls). Case-control additive associations of 11q23 risk alleles (rs498872, T allele) were calculated using logistic regression, stratified by tumor IDH status (mutated or wild-type) and by histology and grade. We also adjusted for the recently discovered 8q24 glioma risk locus rs55705857 G allele., Results: The 11q23 glioma risk locus was associated with increased risk of IDH-mutated gliomas of all histologies and grades (odds ratio [OR] = 1.50; 95% confidence interval [CI] = 1.29-1.74; P = 1.3X10(-7)) but not with IDH-wild-type gliomas of any histology or grade (OR = 0.91; 95% CI = 0.81-1.03; P = 0.14). The associations were independent of the rs55705857 G allele., Conclusion: A variant at the 11q23 locus increases risk for IDH-mutated but not IDH-wild-type gliomas, regardless of grade or histology.