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ALK‐fused Spitz melanocytic neoplasms are a distinct subgroup of melanocytic lesions exhibiting unique histopathologic characteristics. These lesions often manifest as exophytic or polypoid tumors, characterized by fusiform‐to‐epithelioid melanocytes arranged in a nested, fascicular, or plexiform growth pattern. Several fusion partners of the ALK gene have been identified in spitzoid melanocytic neoplasms, with TPM3 and DCTN1 being the most prevalent. Less common fusion partners include NPM1, TPR, CLIP1, GTF3C2, EEF2, MYO5A, KANK1, and EHBP1. The MLPH gene, which encodes melanophilin (MLPH), playing a crucial role in regulating skin pigmentation by acting as a linker between RAB27A and myosin Va during melanosome transport, has also recently been recognized as a rare fusion partner of ALK in Spitz melanocytic neoplasms. Currently, there exists a sparse documentation within English literature, illustrating a limited number of cases featuring MLPH::ALK fusion in Spitz melanocytic neoplasms. In this report, we present two additional cases, including a previously unreported instance of Spitz melanoma, contributing to the expanding knowledge on ALK‐fused Spitz melanocytic neoplasms. In addition, we provide a comprehensive review of the clinical, histopathologic, and molecular features observed in documented cases with this novel fusion. [ABSTRACT FROM AUTHOR]

Background: Genomic profiling is needed to identify actionable alterations in non–small cell lung cancer (NSCLC). Panel‐based testing such as next‐generation sequencing (NGS) is often preferred to interrogate multiple alterations simultaneously. In this study, we evaluate the utility of an RNA‐based NGS assay to detect genomic alterations in NSCLC cytology specimens and compare these results to fluorescence in situ hybridization (FISH) testing. Methods: A retrospective review was performed of 264 NSCLC cytology specimens that were concurrently tested for gene fusions by RNA‐based NGS and ALK, RET, and/or ROS1 by FISH. Results: Genomic alterations were detected in 29 cases by NGS, including ALK, RET, ROS1, NTRK, NUTM1, and FGFR3 fusions and MET exon 14 skipping alterations. Of the 20 cases with ALK, RET, and ROS1 fusions detected by NGS, 16 (80%) were concordant with the corresponding FISH results. Three cases showed discordance, where EML4::ALK (n = 2) and SLC34A2::ROS1 (n = 1) fusions were not detected by the corresponding FISH assay; one case with EZR::ROS1 was inadequate for FISH. No gene fusions were detected in 181 cases by NGS and 54 cases failed testing. The concordance rates for detecting ALK, RET, and ROS1 fusions using NGS and FISH were 97%, 100%, and 99.5%, respectively. Conclusion: RNA‐based NGS can be used to detect gene fusions in NSCLC cytology cases with high concordance with FISH results. However, RNA‐based NGS may have high failure rates and therefore a low threshold for reflexing inadequate cases to an orthogonal testing method is essential for comprehensive genomic profiling. This study explores the use of an RNA‐based next‐generation sequencing assay for detecting genomic alterations in non–small cell lung cancer cytology specimens in comparison to fluorescence in situ hybridization testing. The results have important implications for clinical oncology practice, including selection of biomarkers and biomarker testing workflow, in addition to specimen collection and optimizing the use of cytology samples. [ABSTRACT FROM AUTHOR]

Background: Non–small cell lung carcinoma (NSCLC) patients with BRAF V600E–mutated tumors respond to targeted therapy. Testing for BRAF V600E is commonly performed with molecular methods; however, a mutation‐specific VE1 antibody clone can provide an alternative testing option using immunohistochemistry (IHC) for practices using single‐gene testing and in situations when the specimen is inadequate for molecular testing. This study evaluates the usefulness of VE1 IHC in screening for BRAF V600E mutations in NSCLC cytology specimens. Methods: The authors retrospectively identified cytology cases with a diagnosis of NSCLC that had BRAF V600E IHC performed on cell block sections with the monoclonal VE1 antibody clone. The BRAF V600E IHC results were compared with those of molecular testing performed with an amplicon‐based next‐generation sequencing assay. Results: There were 201 NSCLC cases evaluated. The VE1 IHC was positive in seven of seven BRAF V600E–mutated tumors (100%) and was negative in 158 of 158 nonmutated BRAF V600E tumors (100%). Thirty cases did not undergo molecular testing, primarily because of insufficient tissue or because molecular testing was performed on an alternative specimen. Six cases showed equivocal weak/focal staining: Two cases demonstrated BRAF V600E mutations, and four cases were negative by molecular testing. Conclusions: This study suggests that BRAF V600E IHC can be used reliably to screen NSCLC cytology specimens, and negative results strongly indicate the absence of a BRAF V600E mutation. Having a low threshold for equivocal staining is recommended with molecular confirmation of BRAF V600E for any cases demonstrating weak and/or focal cytoplasmic staining. BRAF V600E immunohistochemistry can be used reliably to screen cytologic specimens of non–small cell lung carcinoma, and negative results strongly indicate the absence of a BRAF V600E mutation. Having a low threshold for equivocal staining is recommended with molecular confirmation of BRAF V600E for any cases demonstrating weak and/or focal cytoplasmic staining. [ABSTRACT FROM AUTHOR]

Simple Summary: There are limited studies that incorporate genetic/epigenetic alterations into the assessment of the microsatellite instability (MSI) and mismatch repair (MMR) determination of tumors. While MSI and MMR testing are part of the screening for the eligibility to employ immune checkpoint inhibitor (ICI) therapy, data from next-generation sequencing (NGS) are not used in the current practice. For most neoplasms, IHC- and PCR-based MSI testing results are concordant. However, for neoplasms with major discordance in IHC and MSI testing, the addition and integration of next-generation sequencing (NGS) results and MLH1 promoter methylation analyses can be beneficial for resolving borderline cases, thereby facilitating patient management. Background: A deficiency in DNA mismatch repair function in neoplasms can be assessed by an immunohistochemical (IHC) analysis of the deficiency/loss of the mismatch repair proteins (dMMR) or by PCR-based methods to assess high microsatellite instability (MSI-H). In some cases, however, there is a discrepancy between the IHC and MSI analyses. Several studies have addressed the issue of discrepancy between IHC and MSI deficiency assessment, but there are limited studies that also incorporate genetic/epigenetic alterations. Methods: In this single-institution retrospective chart-review study, we reviewed 706 neoplasms assessed between 2015 and 2021. All eligible neoplasms were assessed by IHC testing, MSI analysis by PCR-based assay, and tumor-normal paired next-generation sequencing (NGS) analysis. Eighty percent of neoplasms with MLH1 protein loss had a concurrent MLH1 promoter methylation analysis. Mutation data for MMR genes, IHC, MSI analysis, and tumor histology were correlated with each other. Results: Fifty-eight (8.2%) of 706 neoplasms had MSI-H by PCR and/or dMMR by IHC. Of the 706 analyzed neoplasms, 688 neoplasms (98%) had concordant results: MSI-H/dMMR (n = 44), microsatellite-stable (MSS)/proficient MMR (pMMR) (n = 625), and MSI-Low (L)/pMMR (n = 19). Of the remaining 18 neoplasms, 9 had a major discordance: MSS/loss of MSH2 and MSH6 (n = 3), MSS/loss of MSH6 (n = 2), MSS/Loss of MLH1 and PMS2 (n = 1), and MSI-High/pMMR (n = 3). In total, 57% of cases with dMMR and 61% of cases with MSI-H had a null mutation of an MMR gene mutation (or methylation of the MLH1 promoter), whereas this figure was 1% for neoplasms with a normal IHC or MSI pattern (p < 0.001). Among 9 cases with major discordance between MSI and IHC, only 3 cases (33%) had an underlying genetic/epigenetic etiology, whereas 37 (76%) of 49 cases with MSI-H and/or dMMR and without major discordance had an underlying genetic abnormality (p = 0.02). Discussion: For most neoplasms, IHC and PCR-based MSI testing results are concordant. In addition, an underlying genetic abnormality (a null mutation of an MMR gene or MLH1 promoter methylation) was attributable to dMMR and/or MSI-H findings. For neoplasms with major discordance in IHC and MSI testing, the addition and integration of NGS results and MLH1 promoter methylation analyses can be beneficial for resolving borderline cases, thereby facilitating patient management. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the gene mutational profile of urachal carcinoma in correlation with its clinicopathologic features.Methods: We analyzed genetic mutations in 30 cases of urachal carcinoma by next-generation sequencing (NGS) test. Histologic slides and clinical data were reviewed.Results: The patients included 21 men and 9 women, with a mean age of 53 years (range, 24-75 years). The urachal carcinomas included mucinous (11), enteric (10), signet ring cell (8), and high-grade neuroendocrine (1) subtypes. Targeted NGS analysis demonstrated genetic mutations in all the urachal tumors (mean, 2; range, 1-4). TP53 was the most mutated gene (25), followed by KRAS (9) and GNAS (8) genes. TP53 mutations were more common in the signet ring cell subtype (7/8), and GNAS mutations were present only in the mucinous (5/11) and signet ring cell subtypes (3/8) but not in the enteric subtype (0/10). KRAS mutations were significantly associated with cancer stage IV (P = .02) and younger patient age (P = .046). Furthermore, the presence of KRAS mutations in urachal carcinoma portended a poorer overall survival (P = .006).Conclusions: Urachal carcinoma demonstrates frequent gene mutations that are associated with distinct clinicopathologic features. Gene mutation may underlie the development and progression of this aggressive disease. [ABSTRACT FROM AUTHOR]

Background: Endoscopic ultrasound–guided tissue acquisition (EUS‐TA), especially endoscopic ultrasound–guided fine‐needle aspiration (EUS‐FNA), is the mainstay of tissue acquisition for the diagnosis of pancreatic ductal adenocarcinoma (PDAC). Recently, endoscopic ultrasound–guided fine‐needle biopsy (EUS‐FNB) using flexible biopsy needles has been used for patients with PDAC in an effort to increase diagnostic yields and biomarker testing. However, the role of EUS‐TA in biomarker testing for personalized therapy or precise chemotherapy for PDAC is not well established. Methods: PDAC cases with specimens acquired through concurrent EUS‐FNA and EUS‐FNB were identified retrospectively. Smears were prepared from EUS‐FNA sampling, and cell blocks (CBs) were prepared from EUS‐FNB sampling. Rapid onsite evaluation was conducted for all cases for diagnostic adequacy. The adequacy for biomarker testing, including next‐generation sequencing (NGS) and immunohistochemistry (IHC) assays, was evaluated, and cases with smears and CBs adequate for NGS were processed for targeted NGS. Results: There were 26 PDAC cases concurrently sampled by EUS‐FNA and EUS‐FNB. EUS‐FNA smears for all 26 cases and EUS‐FNB CBs for 20 cases (77%) were diagnostic for PDAC. Twenty‐one smears (81%) and 11 CBs (42%) were adequate for NGS. Nine cases with both smears and CBs adequate for NGS underwent NGS, which identified clinically significant gene mutation variants, including KRAS, TP53, and SMAD4 mutations. Conclusions: Both EUS‐FNA and EUS‐FNB can provide optimal material for targeted NGS for PDACs. In PDAC cases subjected to concurrent EUS‐FNA and EUS‐FNB, EUS‐FNA specimens had greater diagnostic yields and more adequate material for NGS than EUS‐FNB specimens, whereas EUS‐FNB was more suitable for IHC‐based biomarker testing. Samples acquired via either endoscopic ultrasound–guided fine‐needle aspiration or endoscopic ultrasound–guided fine‐needle biopsy are suitable for next‐generation sequencing testing, whereas endoscopic ultrasound–guided fine‐needle biopsy could provide material for immunohistochemistry‐based biomarker testing. [ABSTRACT FROM AUTHOR]

Background: Molecular testing is recommended as an adjunct to improve the preoperative diagnosis of fine‐needle aspiration (FNA) of thyroid nodules. Centrifuged supernatants from FNA samples, which are typically discarded, have recently emerged as a novel liquid‐based biopsy for molecular testing. This study evaluates the use of thyroid FNA supernatants for detecting clinically relevant mutations. Methods: Supernatants from thyroid FNA samples (n = 156) were evaluated. A 50‐gene next‐generation sequencing (NGS) assay was used, and mutation analysis results from a subset of samples were further compared with those of paired FNA smears and/or cell blocks. Results: All 156 samples yielded adequate DNA (median, 135 ng; range, 11‐3180 ng), and 129 of these samples (83%) were successfully sequenced by NGS. The most frequently detected somatic mutations included BRAF and RAS mutations, which were followed by RET, TP53, PTEN, CDKN2A, and PIK3CA mutations. Eleven of 31 cases with an indeterminate cytologic diagnosis and 9 of 12 cases that were suspicious for malignancy had somatic mutations, including the BRAF V600E mutation, which is highly definitive for papillary thyroid carcinoma (PTC). Seven of the 9 indeterminate and suspicious cases with the BRAF V600E mutation had surgical follow‐up, and they were all confirmed to be PTC. A comparison of the mutation profiles derived from supernatants with those of paired smears and/or cell blocks in a small subset of cases (n = 8) showed 100% concordance. Conclusions: This study provides evidence that FNA supernatants can be used as a surrogate for thyroid molecular testing to improve diagnostic accuracy in indeterminate nodules, provide prognostic/predictive information, and improve overall patient management. Centrifuged supernatants from fine‐needle aspiration (FNA) samples, which are typically discarded, have recently emerged as a novel liquid‐based biopsy for molecular testing. This study provides evidence that FNA supernatants can be used as a surrogate for thyroid molecular testing to improve diagnostic accuracy in indeterminate thyroid nodules, provide prognostic/predictive information, and improve overall patient management. [ABSTRACT FROM AUTHOR]

The rapid adoption of next-generation sequencing (NGS) in clinical molecular laboratories has redefined the practice of cytopathology. Instead of simply being used as a diagnostic tool, cytopathology has evolved into a practice providing important genomic information that guides clinical management. The recent emphasis on maximizing limited-volume cytology samples for ancillary molecular studies, including NGS, requires cytopathologists not only to be more involved in specimen collection and processing techniques but also to be aware of downstream testing and informatics issues. For the integration of molecular informatics into the clinical workflow, it is important to understand the computational components of the NGS workflow by which raw sequence data are transformed into clinically actionable genomic information and to address the challenges of having a robust and sustainable informatics infrastructure for NGS-based testing in a clinical environment. Adapting to needs ranging from specimen procurement to report delivery is crucial for the optimal utilization of cytology specimens to accommodate requests from clinicians to improve patient care. This review presents a broad overview of the various aspects of informatics in the context of NGS-based testing of cytology specimens. Cancer Cytopathol 2017;125:236-244. © 2016 American Cancer Society. [ABSTRACT FROM AUTHOR]

Objectives: Clinical laboratories are rapidly implementing next-generation sequencing (NGS) tests for mutation analysis, but there are few guidelines regarding sample quality for successful results.Methods: We aimed to establish tissue quality parameters for successful NGS in solid tumors and to improve NGS performance.Results: Analysis of 614 clinical cases tested in 2013 using a 50-gene hotspot mutation panel identified the major cause for unsuccessful NGS analysis was DNA less than 10 ng (91%, 67/74) associated with extremely small and low cellularity samples. High success rates were associated with resection procedures (333/342, 97%) and biopsied tumor larger than 10 mm(2) (77/77, 100%). NGS can be successfully performed on bone specimens processed with formic acid-based decalcification procedures (8/11, 73%). Tumor type and paraffin block age did not affect success. We demonstrated that NGS can be carried out on samples with less than 10 ng DNA. Analysis of 408 cases tested in 2014 using an optimized workflow showed improved NGS success rates from 88% to 95% (387/408) with pronounced improvement among tiny (<10 mm(2)) samples (from 76% to 94%) as well as cytology samples (from 58% to 87%).Conclusions: Identifying preanalytical tissue factors allows us to improve NGS performance and to successfully test tumors obtained from minimally invasive procedures. [ABSTRACT FROM AUTHOR]

The application of next-generation sequencing (NGS) to characterize cancer genomes has resulted in the discovery of numerous genetic markers. Consequently, the number of markers that warrant routine screening in molecular diagnostic laboratories, often from limited tumor material, has increased. This increased demand has been difficult to manage by traditional low- and/or medium-throughput sequencing platforms. Massively parallel sequencing capabilities of NGS provide a much-needed alternative for mutation screening in multiple genes with a single low investment of DNA. However, implementation of NGS technologies, most of which are for research use only (RUO), in a diagnostic laboratory, needs extensive validation in order to establish Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP)-compliant performance characteristics. Here, we have reviewed approaches for validation of NGS technology for routine screening of tumors. We discuss the criteria for selecting gene markers to include in the NGS panel and the deciding factors for selecting target capture approaches and sequencing platforms. We also discuss challenges in result reporting, storage and retrieval of the voluminous sequencing data and the future potential of clinical NGS. [ABSTRACT FROM AUTHOR]

Objectives: The advent of next-generation sequencing (NGS) platforms in the realm of clinical molecular diagnostics provides multigene mutational profiling through massively parallel sequencing.Methods: We analyzed 415 breast carcinoma samples from 354 patients using NGS in known hotspots of 46 commonly known cancer-causing genes.Results: A total of 281 somatic nonsynonymous mutations were detected in 62.1% of patients. TP53 was most frequently mutated (38.8%), followed by PIK3CA (31.7%), AKT1 (6%), and ATM (3.9%), with other mutations detected at a lower frequency. When stratified into clinically relevant therapeutic groups (estrogen receptor [ER]/progesterone receptor [PR]+ human epidermal growth factor receptor 2 [HER2]-, ER/PR+HER2+, ER/PR-HER2+, ER/PR/HER2-), each group showed distinct mutational profiles. The ER/PR+HER2- tumors (n = 132) showed the highest frequency of PIK3CA mutations (38%), while the triple-negative tumors (n = 64) had a significantly higher number of TP53 mutations (62%). Of the 61 patients tested for both primary and metastatic tumors, concordant results were seen in 47 (77%) patients, while 13 patients showed additional mutations in the metastasis.Conclusions: Our results indicate that breast cancers may harbor potentially actionable mutations for targeted therapeutics. Therefore, NGS-based mutational profiling can provide useful information that can guide targeted cancer therapy. [ABSTRACT FROM AUTHOR]

BACKGROUND: The use of cytology specimens for next-generation sequencing (NGS) is particularly challenging because of the unconventional substrate of smears and the often limited sample volume. An analysis of factors affecting NGS testing in cytologic samples may help to increase the frequency of successful testing. METHODS: This study reviewed variables associated with all in-house cytology cases (n5207) that were analyzed by NGS with the Ion Torrent platform during a 10-month interval. A statistical analysis was performed to measure the effects of the DNA input threshold, specimen preparation, slide type, tumor fraction, DNA yield, and cytopathologist bias. RESULTS: One hundred sixty-four of 207 cases (79%) were successfully sequenced by NGS; 43 (21%) failed because of either a low DNA yield or a template/library preparation failure. The median estimated tumor fraction and DNA concentration for the successfully sequenced cases were 70% and 2.5 ng/lL, respectively, whereas they were 60% and 0.2 ng/μL, respectively, for NGS failures. Cell block sections were tested in 91 cases, and smears were used in 116 cases. NGS success positively correlated with the DNA yield but not the tumor fraction. Cell block preparations showed a higher success rate than smears. Frosted-tip slides yielded significantly more DNA than fully frosted slides. Lowering the input DNA concentration below the manufacturer's recommended threshold of 10 ng (>0.85 ng/μL) resulted in a marked increase in the NGS success rate from 58.6% to 89.8%. CONCLUSIONS: The failure of NGS with cytology samples is usually a result of suboptimal DNA due to multiple pre-analytical factors. Knowledge of these factors will allow better selection of cytology material for mutational analysis. [ABSTRACT FROM AUTHOR]

Application of next-generation sequencing (NGS) technology to routine clinical practice has enabled characterization of personalized cancer genomes to identify patients likely to have a response to targeted therapy. The proper selection of tumor sample for downstream NGS based mutational analysis is critical to generate accurate results and to guide therapeutic intervention. However, multiple pre-analytic factors come into play in determining the success of NGS testing. In this review, we discuss pre-analytic requirements for AmpliSeq PCR-based sequencing using Ion Torrent Personal Genome Machine (PGM) (Life Technologies), a NGS sequencing platform that is often used by clinical laboratories for sequencing solid tumors because of its low input DNA requirement from formalin fixed and paraffin embedded tissue. The success of NGS mutational analysis is affected not only by the input DNA quantity but also by several other factors, including the specimen type, the DNA quality, and the tumor cellularity. Here, we review tissue requirements for solid tumor NGS based mutational analysis, including procedure types, tissue types, tumor volume and fraction, decalcification, and treatment effects. [ABSTRACT FROM AUTHOR]

• NTRK gene fusions are targetable oncogenic drivers independent of tumor type. • Selective TRK inhibitor larotrectinib is active in tumors with NTRK gene fusions. • A diverse array of methods can be used to detect NTRK gene fusions. • The most common testing approach was RNA-based next-generation sequencing. • 54 different NTRK fusion partners identified in 225 patients with TRK fusion cancer. NTRK gene fusions are targetable oncogenic drivers independent of tumor type. Prevalence varies from highly recurrent in certain rare tumors to <1% in common cancers. The selective TRK inhibitor larotrectinib was shown to be highly active in adult and pediatric patients with tumors harboring NTRK gene fusions. We examined the techniques used by local sites to detect tumor NTRK gene fusions in patients enrolled in clinical trials of larotrectinib. We also report the characteristics of the detected fusions in different tumor types. The analysis included 225 patients with 19 different tumor types. Testing methods used were next-generation sequencing (NGS) in 196 of 225 tumors (87%); this was RNA-based in 96 (43%); DNA-based in 53 (24%); DNA/RNA-based in 46 (20%) and unknown in 1 (<1%); FISH in 14 (6%) and PCR-based in 12 (5%). NanoString, Sanger sequencing and chromosome microarray were each utilized once (<1%). Fifty-four different fusion partners were identified, 39 (72%) of which were unique occurrences. The most common local testing approach was RNA-based NGS. Many different NTRK gene fusions were identified with most occurring at low frequency. This supports the need for validated and appropriate testing methodologies that work agnostic of fusion partners. [ABSTRACT FROM AUTHOR]

Background Understanding the biology of breast cancer is important for guiding treatment strategies and revealing resistance mechanisms. Our objectives were to investigate the relationship between previous systemic therapy exposure and mutational spectrum in metastatic breast cancer and to identify clinicopathological factors associated with identified frequent somatic mutations. Methods Archival tissues of patients with metastatic breast cancer were subjected to hotspot molecular testing by next-generation sequencing. The variables that significantly differed ( P < 0.05) in univariate analysis were selected to fit multivariate models. Logistic models were fit to estimate the association between mutation status and clinical variables of interest. Five-fold cross-validation was performed to estimate the prediction error of each model. Results A total of 922 patients were included in the analysis. In multivariate analysis, previous systemic treatment before molecular testing (N = 186) was associated with a significantly higher rate of TP53 and PIK3CA mutations compared with the lack of systemic treatment ( P < 0.001 for both). Conclusion Systemic treatment exposure is an independent risk factor for high rates of TP53 and PIK3CA mutation, which suggests the importance of testing samples after systemic therapy to accurately assess mutations. It is worth testing the gene profile when tumours become resistant to systemic treatments. [ABSTRACT FROM AUTHOR]