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Salivary gland neoplasms may pose diagnostic difficulties due to overlapping morphologic features. Recently, specific gene fusions have been discovered that correspond to particular tumor types, and can aid in accurate diagnosis. Gene rearrangements are commonly assessed by fluorescence in situ hybridization (FISH), although use of next-generation sequencing is increasing. However, there is no "gold standard" for fusion detection. We determined the concordance between FISH and a targeted RNA sequencing panel in gene fusion detection across twenty-two salivary gland tumors, including five mucoepidermoid carcinomas, four acinic cell carcinomas, four pleomorphic adenomas, two adenoid cystic carcinomas, two NUT carcinomas, and one each of basal cell adenoma, salivary duct carcinoma ex-pleomorphic adenoma, salivary duct carcinoma, clear cell carcinoma, and secretory carcinoma. Directed FISH testing based on the diagnosis was performed on cases that did not already have FISH conducted during clinical workup. Targeted RNA sequencing of 507 genes and their partners (using the Illumina TruSight Fusion Panel) was completed. Six of twenty-two (27.3%) cases had discordant results. In three cases, FISH results were negative while RNA sequencing results found fusion transcripts, which were all confirmed with RT-PCR and Sanger sequencing. In three cases, RNA sequencing results were negative while FISH results were positive for a gene rearrangement. Thus, if fusion analysis results are conflicting with the morphologic impression, a second mode of fusion detection may be warranted. Although both methods have advantages and drawbacks, RNA sequencing provides additional information about novel fusion partners and fusions that may not have been originally considered.

Characterizing the genomic landscape of cancers is a routine part of clinical care that began with the discovery of the Philadelphia chromosome and has since coevolved with genomic technologies. Genomic analysis of tumors at the nucleotide level using DNA sequencing has revolutionized the understanding of cancer biology and identified new molecular drivers of disease that have led to therapeutic advances and improved patient outcomes. However, the application of next-generation sequencing in the clinical laboratory has generally been limited until very recently to targeted analysis of selected genes. Recent technological innovations and reductions in sequencing costs are now able to deliver the long-promised goal of tumor whole-genome sequencing as a practical clinical assay.

The classification of myeloid neoplasms and acute leukemias was last updated in 2016 within a collaboration between the World Health Organization (WHO), the Society for Hematopathology, and the European Association for Haematopathology. This collaboration was primarily based on input from a clinical advisory committees (CACs) composed of pathologists, hematologists, oncologists, geneticists, and bioinformaticians from around the world. The recent advances in our understanding of the biology of hematologic malignancies, the experience with the use of the 2016 WHO classification in clinical practice, and the results of clinical trials have indicated the need for further revising and updating the classification. As a continuation of this CAC-based process, the authors, a group with expertise in the clinical, pathologic, and genetic aspects of these disorders, developed the International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias. Using a multiparameter approach, the main objective of the consensus process was the definition of real disease entities, including the introduction of new entities and refined criteria for existing diagnostic categories, based on accumulated data. The ICC is aimed at facilitating diagnosis and prognostication of these neoplasms, improving treatment of affected patients, and allowing the design of innovative clinical trials.

Myeloid neoplasms and acute leukemias derive from the clonal expansion of hematopoietic cells driven by somatic gene mutations. Although assessment of morphology plays a crucial role in the diagnostic evaluation of patients with these malignancies, genomic characterization has become increasingly important for accurate diagnosis, risk assessment, and therapeutic decision making. Conventional cytogenetics, a comprehensive and unbiased method for assessing chromosomal abnormalities, has been the mainstay of genomic testing over the past several decades and remains relevant today. However, more recent advances in sequencing technology have increased our ability to detect somatic mutations through the use of targeted gene panels, whole-exome sequencing, whole-genome sequencing, and whole-transcriptome sequencing or RNA sequencing. In patients with myeloid neoplasms, whole-genome sequencing represents a potential replacement for both conventional cytogenetic and sequencing approaches, providing rapid and accurate comprehensive genomic profiling. DNA sequencing methods are used not only for detecting somatically acquired gene mutations but also for identifying germline gene mutations associated with inherited predisposition to hematologic neoplasms. The 2022 International Consensus Classification of myeloid neoplasms and acute leukemias makes extensive use of genomic data. The aim of this report is to help physicians and laboratorians implement genomic testing for diagnosis, risk stratification, and clinical decision making and illustrates the potential of genomic profiling for enabling personalized medicine in patients with hematologic neoplasms.

Objectives The 2019 Workshop of the Society for Hematopathology/European Association for Haematopathology received and reviewed cases covering the spectrum of mastocytosis and related diseases, including morphologic mimics, focusing on recent updates and relevant findings for pathologists. Methods The workshop panel reviewed 99 cases of cutaneous and systemic mastocytosis (SM) and SM and associated hematologic neoplasms (SM-AHN). Results Despite a common theme of KIT mutation (particularly D816V), mastocytosis is a heterogeneous neoplasm with a wide variety of presentations. This spectrum, including rare subtypes and extramedullary organ involvement, is discussed and illustrated by representative cases. Conclusions In the age of targeted treatment aimed at KIT, the accurate diagnosis and classification of mastocytosis has major implications for therapy and further interventions. Understanding the clinical, pathologic, and genetic findings of mastocytosis is crucial for selecting the proper tests to perform and subsequent arrival at a correct diagnosis in this rare disease.

As immuno-oncology drugs grow more popular in the treatment of cancer, better methods are needed to quantify the tumor immune cell component to determine which patients are most likely to benefit from treatment. Methods such as flow cytometry can accurately assess the composition of infiltrating immune cells; however, they show limited use in formalin-fixed, paraffin-embedded (FFPE) specimens. This article describes a novel hybrid-capture RNA sequencing assay, ImmunoPrism, that estimates the relative percentage abundance of eight immune cell types in FFPE solid tumors. Immune health expression models were generated using machine learning methods and used to uniquely identify each immune cell type using the most discriminatively expressed genes. The analytical performance of the assay was assessed using 101 libraries from 40 FFPE and 32 fresh-frozen samples. With defined samples, ImmunoPrism had a precision of ±2.72%, a total error of 2.75%, and a strong correlation (r2 = 0.81; P

DNMT3A Overgrowth Syndrome (DOS, also known as Tatton-Brown Rahman Syndrome/TBRS) is one of several overgrowth syndromes with complex phenotypes caused by constitutional mutations in genes encoding epigenetic regulators. The clinical features of DOS are variable but include overgrowth (tall stature and/or obesity) and intellectual disability. DNMT3A is essential for de novo DNA methylation and plays an important role in hematopoiesis. Somatic mutations in DNMT3A are among the most common initiating mutations in normal karyotype acute myeloid leukemia (AML) patients and in elderly people with clonal hematopoiesis. The natural history of DOS has not been fully explored since the first description of this rare condition in 2014. Because of the association of somatic DNMT3A mutations and leukemia development, we assessed information from the ~200 known DOS patients world-wide and were able to document eight with hematologic malignancies. Based on this prevalence, we suggest DOS is a cancer predisposition syndrome, especially for hematologic malignancies. Using recommendations from an expert panel, we suggest DOS patients should be prospectively monitored for hematologic malignancies, which may allow for early intervention and permit its natural history to be better defined.

Background The most common B-cell cancers, chronic lymphocytic leukemia/lymphoma (CLL), follicular and diffuse large B-cell (FL, DLBCL) lymphomas, have distinct clinical courses, yet overlapping “cell-of-origin”. Dynamic changes to the epigenome are essential regulators of B-cell differentiation. Therefore, we reasoned that these distinct cancers may be driven by shared mechanisms of disruption in transcriptional circuitry. Methods We compared purified malignant B-cells from 52 patients with normal B-cell subsets (germinal center centrocytes and centroblasts, naive and memory B-cells) from 36 donor tonsils using >325 high-resolution molecular profiling assays for histone modifications, open chromatin (ChIP-, FAIRE-seq), transcriptome (RNA-seq), transcription factor (TF) binding, and genome copy number (microarrays). Findings From the resulting data, we identified gains in active chromatin in enhancers/super-enhancers that likely promote unchecked B-cell receptor signaling, including one we validated near the immunoglobulin superfamily receptors FCMR and PIGR. More striking and pervasive was the profound loss of key B-cell identity TFs, tumor suppressors and their super-enhancers, including EBF1, OCT2(POU2F2), and RUNX3. Using a novel approach to identify transcriptional feedback, we showed that these core transcriptional circuitries are self-regulating. Their selective gain and loss form a complex, iterative, and interactive process that likely curbs B-cell maturation and spurs proliferation. Interpretation Our study is the first to map the transcriptional circuitry of the most common blood cancers. We demonstrate that a critical subset of B-cell TFs and their cognate enhancers form self-regulatory transcriptional feedback loops whose disruption is a shared mechanism underlying these diverse subtypes of B-cell lymphoma. Funding National Institute of Health, Siteman Cancer Center, Barnes-Jewish Hospital Foundation, Doris Duke Foundation.

Breast implant–associated anaplastic large T-cell lymphoma (BIA-ALCL) was first recognized by the World Health Organization in 2016. The total number of cases worldwide continues to increase, with >800 cases confirmed through a combination of Food and Drug Administration data, verified reports, and registries. To date, 33 deaths have been reported. Typical presentation includes a late seroma containing monoclonal T cells that are CD30 positive and anaplastic lymphoma kinase negative. We present a review of the current literature and report on 3 cases of BIA-ALCL at our institution, which serve to illustrate our approach to diagnosis and management of this disease. In 2 cases, the diagnosis of BIA-ALCL was not initially confirmed due to an incomplete workup but was recognized upon explantation. The seroma fluid was sent for flow cytometry. Initially, the cells were reported as morphologically suspicious for malignancy with phenotypically normal T cells based on standard CD3+ T-cell gating. Subsequent cytology specimens were reported as consistent with recurrent adenocarcinoma. However, upon regating of flow-cytometry data, a population of CD30+, CD3– T cells was noted and the diagnosis of BIA-ALCL was confirmed by immunohistochemical stains of the excised breast capsule specimen. Given the increasing incidence of this disease, as plastic surgeons we must stay informed to order the correct workup to avoid misdiagnosis and be prepared to appropriately refer affected patients to centers with multidisciplinary teams experienced in the management of BIA-ALCL. Level of Evidence: 4

Progression from myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (AML) is associated with the acquisition and expansion of subclones. Our understanding of subclone evolution during progression, including the frequency and preferred order of gene mutation acquisition, remains incomplete. Sequencing of 43 paired MDS and secondary AML samples identified at least one signaling gene mutation in 44% of MDS and 60% of secondary AML samples, often below the level of standard sequencing detection. In addition, 19% of MDS and 47% of secondary AML patients harbored more than one signaling gene mutation, almost always in separate, coexisting subclones. Signaling gene mutations demonstrated diverse patterns of clonal evolution during disease progression, including acquisition, expansion, persistence, and loss of mutations, with multiple patterns often coexisting in the same patient. Multivariate analysis revealed that MDS patients who had a signaling gene mutation had a higher risk of AML progression, potentially providing a biomarker for progression.Significance:Subclone expansion is a hallmark of progression from MDS to secondary AML. Subclonal signaling gene mutations are common at MDS (often at low levels), show complex and convergent patterns of clonal evolution, and are associated with future progression to secondary AML.See related article by Guess et al., p. 316 (33).See related commentary by Romine and van Galen, p. 270.This article is highlighted in the In This Issue feature, p. 265

Myeloproliferative neoplasms (MPNs) are associated with significant alterations in the bone marrow microenvironment that include decreased expression of key niche factors and myelofibrosis. Here, we explored the contribution of TGF-β to these alterations by abrogating TGF-β signaling in bone marrow mesenchymal stromal cells. Loss of TGF-β signaling in Osx-Cre-targeted MSCs prevented the development of myelofibrosis in both MPLW515L and Jak2V617F models of MPNs. In contrast, despite the absence of myelofibrosis, loss of TGF-β signaling in mesenchymal stromal cells did not rescue the defective hematopoietic niche induced by MPLW515L, as evidenced by decreased bone marrow cellularity, hematopoietic stem/progenitor cell number, and Cxcl12 and Kitlg expression, and the presence of splenic extramedullary hematopoiesis. Induction of myelofibrosis by MPLW515L was intact in Osx-Cre Smad4fl/fl recipients, demonstrating that SMAD4-independent TGF-β signaling mediates the myelofibrosis phenotype. Indeed, treatment with a c-Jun N-terminal kinase (JNK) inhibitor prevented the development of myelofibrosis induced by MPLW515L. Together, these data show that JNK-dependent TGF-β signaling in mesenchymal stromal cells is responsible for the development of myelofibrosis but not hematopoietic niche disruption in MPNs, suggesting that the signals that regulate niche gene expression in bone marrow mesenchymal stromal cells are distinct from those that induce a fibrogenic program.

Objectives Acute myeloid leukemia (AML) with t(8;16)(p11;p13) abnormalities is a rare, aggressive, and diagnostically challenging subtype that results in KAT6A-CREBBP gene fusion. Methods To investigate their immunophenotype and genomic features, we identified 5 cases of AML with t(8;16) through a retrospective review of the databases at Northwestern Memorial Hospital in Chicago, IL, and Washington University Medical Center, in St Louis, MO. Results In all, 4 of 5 cases were therapy related and 1 was possibly therapy related. The leukemic blasts showed distinctive features, including bright CD45 expression and remarkably high side scatter that overlapped with maturing myeloid elements, making the blasts difficult to identify on initial examination. They were positive for CD13, CD33, and CD64 and negative for CD34 and CD117. Next-generation sequencing profiling of 4 cases revealed pathogenic ASXL1 (2 cases), FLT3-tyrosine kinase domain (TKD) mutations (2 cases), and other pathogenic mutations. In 3 patients, t(8;16) was the sole cytogenetic abnormality; additional aberrations were found in 2 patients. Single nucleotide polymorphism microarray revealed 1 case with 7q deletion as a secondary clone. Conclusions Our data highlight the distinctive immunophenotypic profile of AML with t(8;16), which, along with its unique morphology, often presents a diagnostic challenge. We showed that mutations of either ASXL1 or FLT3-TKD are seen in most cases of this leukemia.

BACKGROUND: Genomic analysis is essential for risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Whole-genome sequencing is a potential replacement for conventional cytogenetic and sequencing approaches, but its accuracy, feasibility, and clinical utility have not been demonstrated. METHODS: We used a streamlined whole-genome sequencing approach to obtain genomic profiles for 263 patients with myeloid cancers, including 235 patients who had undergone successful cytogenetic analysis. We adapted sample preparation, sequencing, and analysis to detect mutations for risk stratification using existing European Leukemia Network (ELN) guidelines and to minimize turnaround time. We analyzed the performance of whole-genome sequencing by comparing our results with findings from cytogenetic analysis and targeted sequencing. RESULTS: Whole-genome sequencing detected all 40 recurrent translocations and 91 copy-number alterations that had been identified by cytogenetic analysis. In addition, we identified new clinically reportable genomic events in 40 of 235 patients (17.0%). Prospective sequencing of samples obtained from 117 consecutive patients was performed in a median of 5 days and provided new genetic information in 29 patients (24.8%), which changed the risk category for 19 patients (16.2%). Standard AML risk groups, as defined by sequencing results instead of cytogenetic analysis, correlated with clinical outcomes. Whole-genome sequencing was also used to stratify patients who had inconclusive results by cytogenetic analysis into risk groups in which clinical outcomes were measurably different. CONCLUSIONS: In our study, we found that whole-genome sequencing provided rapid and accurate genomic profiling in patients with AML or MDS. Such sequencing also provided a greater diagnostic yield than conventional cytogenetic analysis and more efficient risk stratification on the basis of standard risk categories. (Funded by the Siteman Cancer Research Fund and others.)

The detection of recurrent gene fusions can help confirm diagnoses in solid tumors, particularly when the morphology and staining are unusual or nonspecific, and can guide therapeutic decisions. Although fluorescence in situ hybridization and PCR are often used to identify fusions, the rearrangement must be suspected, with only a few prioritized probes run. It was hypothesized that the Illumina TruSight RNA Fusion Panel, which detects fusions of 507 genes and their partners, would uncover fusions with greater sensitivity than other approaches, leading to changes in diagnosis, prognosis, or therapy. Targeted RNA sequencing was performed on formalin-fixed, paraffin-embedded sarcoma and carcinoma cases in which fluorescence in situ hybridization, RT-PCR, or DNA-based sequencing was conducted during the diagnostic workup. Of the 153 cases, 138 (90%) were sequenced with adequate quality control metrics. A total of 101 of 138 (73%) cases were concordant by RNA sequencing and the prior test method. RNA sequencing identified an additional 30 cases (22%) with fusions that were not detected by conventional methods. In seven cases (5%), the additional fusion information provided by RNA sequencing would have altered diagnosis and management. A total of 19 novel fusion pairs (not previously described in the literature) were discovered (14%). Overall, the findings show that a targeted RNA-sequencing method can detect gene fusions in formalin-fixed, paraffin-embedded specimens with high sensitivity.

PURPOSE Physicians treating hematologic malignancies increasingly order targeted sequencing panels to interrogate recurrently mutated genes. The precise impact of these panels on clinical decision making is not well understood. METHODS Here, we report our institutional experience with a targeted 40-gene panel (MyeloSeq) that is used to generate a report for both genetic variants and variant allele frequencies for the treating physician (the limit of mutation detection is approximately one AML cell in 50). RESULTS In total, 346 sequencing reports were generated for 325 patients with suspected hematologic malignancies over an 8-month period (August 2018 to April 2019). To determine the influence of genomic data on clinical care for patients with acute myeloid leukemia (AML), we analyzed 122 consecutive reports from 109 patients diagnosed with AML and surveyed the treating physicians with a standardized questionnaire. The panel was ordered most commonly at diagnosis (61.5%), but was also used to assess response to therapy (22.9%) and to detect suspected relapse (15.6%). The panel was ordered at multiple timepoints during the disease course for 11% of patients. Physicians self-reported that 50 of 114 sequencing reports (44%) influenced clinical care decisions in 44 individual patients. Influences were often nuanced and extended beyond identifying actionable genetic variants with US Food and Drug Administration–approved drugs. CONCLUSION This study provides insights into how physicians are currently using multigene panels capable of detecting relatively rare AML cells. The most influential way to integrate these tools into clinical practice will be to perform prospective clinical trials that assess patient outcomes in response to genomically driven interventions.

Severe congenital neutropenia (SCN) is a collection of diverse disorders characterized by chronically low absolute neutrophil count in the peripheral blood, increased susceptibility to infection, and a significant predisposition to the development of myeloid malignancies. SCN can be acquired or inherited. Inherited forms have been linked to variants in a group of diverse genes involved in the neutrophil-differentiation process. Variants that promote resistance to treatment have also been identified. Thus, genetic testing is important for the diagnosis, prognosis, and management of SCN. Herein we describe clinically validated assay developed for assessing patients with suspected SCN. The assay is performed from a whole-exome backbone. Variants are called across all coding exons, and results are filtered to focus on 48 genes that are clinically relevant to SCN. Validation results indicated 100% analytical sensitivity and specificity for the detection of constitutional variants among the 48 reportable genes. To date, 34 individuals have been referred for testing (age range: birth to 67 years). Several pathogenic and likely pathogenic variants have been identified, including one in a patient with late-onset disease. The pattern of cases referred for testing suggests that this assay has clinical utility in a broader spectrum of patients beyond those in the pediatric population who have classic early-onset symptoms characteristic of SCN.

A subset of endometrial adenocarcinomas (EACs) exhibit microsatellite instability and have deficient DNA mismatch repair (dMMR). The overall aim of the study was to compare the spectrum of mutations in endometrioid-type EAC with and without dMMR by using a clinically validated next-generation sequencing assay. We retrospectively identified 19 EACs with known mismatch repair status that had undergone targeted sequencing of a panel of cancer-related genes. The mismatch repair status was ascertained by immunohistochemistry against MLH1, PMS2, MSH2, and MSH6 mismatch proteins. Somatic mutations in EAC with dMMR were compared against those in cases with proficient MMR (pMMR). The dMMR EAC showed a normalized mean of 66.6 mutations/Mb per case compared with pMMR EAC with a mean of 26.2 (P

In myelodysplastic syndromes (MDS), somatic mutations occur in five major categories: RNA splicing, DNA methylation, activated cell signaling, myeloid transcription factors, and chromatin modifiers. Although many MDS cases harbor more than one somatic mutation, in general, there is mutual exclusivity of mutated genes within a class. In addition to the prognostic significance of individual somatic mutations, more somatic mutations in MDS have been associated with poor prognosis. Prognostic assessment remains a critical component of the personalization of care for patient with MDS because treatment is highly risk adapted. Multiple methods for risk stratification are available with the revised International Prognostic Scoring System (IPSS-R), currently considered the gold standard. Increasing access to myeloid gene panels and greater evidence for the diagnostic and predictive value of somatic mutations will soon make sequencing part of the standard evaluation of patients with MDS. In the absence of formal guidelines for their prognostic use, well-validated mutations can still refine estimates of risk made with the IPSS-R. Not only are somatic gene mutations advantageous in understanding the biology of MDS and prognosis, they also offer potential as biomarkers and targets for the treatment of patients with MDS. Examples include deletion 5q, spliceosome complex gene mutations, and TP53 mutations.

To the editor: Quantitative response evaluation in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDSs) relies on the morphologic quantification of bone marrow (BM) blasts. This process is subject to the operator-dependent quality of BM collection and the interobserver variability

PCR amplification, a key step in next-generation sequencing (NGS) library construction, can generate an unlimited amount of product from limited input; however, it cannot create more information than was present in the original template. Thus, NGS libraries can be made from very little DNA, but reducing the input may compromise assay sensitivity in ways that are difficult to ascertain unless library complexity (ie, the number of unique DNA molecules represented in the library) and depth of coverage with unique sequence reads (those derived from input DNA molecules) versus duplicate sequence reads (those resulting from overamplification of particular molecules) are discretely measured. A series of experiments was performed to explore the impact of low DNA input on an amplicon-based NGS assay using unique molecular identifiers to track unique versus duplicate reads. At high sequencing depths, unique and total (unique plus duplicate) read coverage are not well correlated, so increasing the number of sequenced reads does not necessarily improve sensitivity. Unique coverage depth tends to improve with more input, but improvements are not consistent. Fluctuations in library complexity complicated variant detection using both standardized and clinical specimens, often resulting in technical replicates with vastly different estimates of variant allelic fraction. In conclusion, depth of coverage with unique reads must be tracked in clinical NGS to ensure that sensitivity and accuracy are maintained.

Salivary high-grade neuroendocrine carcinomas (NECs) are rare, occur predominantly in the parotid gland, and are difficult to differentiate from metastatic cutaneous Merkel cell carcinomas (MCCs), which have overlapping morphologic, immunophenotypic, and molecular profiles. Oncogenic Merkel cell polyomavirus (MCPyV), found in 70% to 80% of MCCs, has also been reported in a few salivary NECs, but this is controversial. A promising biomarker to distinguish the 2 tumor types are UV signature mutations. UV signature mutations indicate a sun damage-induced mechanism of pathogenesis and recently have been found to be highly prevalent in MCPyV-negative MCCs but would be inconsistent with salivary origin. Here, we examine UV signature mutations as a molecular marker to distinguish primary salivary high-grade NEC from MCC. Whole exome DNA sequencing was performed on matched tumor-normal tissue from 4 MCPyV-negative high-grade salivary NECs with no other primary source identified, as well as 3 melanomas and 3 lung NECs as positive and negative controls, respectively. UV signature mutations were found in all salivary NECs, when defined as ≥60% of total mutations being C-to-T transitions at dipyrimidine sites, and when compared with known human cancer-related mutational signatures. The presence of UV signature mutations in salivary high-grade NECs strongly favors these to be occult metastatic MCCs. True salivary primary NECs are likely exceedingly rare. When a high-grade NEC is encountered in the salivary gland, the presence of UV signature mutations or MCPyV may be useful to exclude occult unknown primary MCC.

Recurrent genomic mutations in uterine and non-uterine leiomyosarcomas have not been well established. Using a next generation sequencing (NGS) panel of common cancer-associated genes, 25 leiomyosarcomas arising from multiple sites were examined to explore genetic alterations, including single nucleotide variants (SNV), small insertions/deletions (indels), and copy number alterations (CNA). Sequencing showed 86 non-synonymous, coding region somatic variants within 151 gene targets in 21 cases, with a mean of 4.1 variants per case; 4 cases had no putative mutations in the panel of genes assayed. The most frequently altered genes were TP53 (36%), ATM and ATRX (16%), and EGFR and RB1 (12%). CNA were identified in 85% of cases, with the most frequent copy number losses observed in chromosomes 10 and 13 including PTEN and RB1; the most frequent gains were seen in chromosomes 7 and 17. Our data show that deletions in canonical cancer-related genes are common in leiomyosarcomas. Further, the spectrum of gene mutations observed shows that defects in DNA repair and chromosomal maintenance are central to the biology of leiomyosarcomas, and that activating mutations observed in other common cancer types are rare in leiomyosarcomas.

Quality assurance for clinical next-generation sequencing (NGS)-based assays is difficult given the complex methods and the range of sequence variants such assays can detect. As the number and range of mutations detected by clinical NGS assays has increased, it is difficult to apply standard analyte-specific proficiency testing (PT). Most current proficiency testing challenges for NGS are methods-based PT surveys that use DNA from reference samples engineered to harbor specific mutations that test both sequence generation and bioinformatics analysis. These methods-based PTs are limited by the number and types of mutations that can be physically introduced into a single DNA sample. In silico proficiency testing, which evaluates only the bioinformatics component of NGS assays, is a recently introduced PT method that allows for evaluation of numerous mutations spanning a range of variant classes. In silico PT data sets can be generated from simulated or actual sequencing data and are used to test alignment through variant detection and annotation steps. In silico PT has several advantages over the use of physical samples, including greater flexibility in tested variants, the ability to design laboratory-specific challenges, and lower costs. Herein, we review the use of in silico PT as an alternative to traditional methods-based PT as it is evolving in oncology applications and discuss how the approach is applicable more broadly.

Traditional response criteria in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are based on bone marrow morphology and may not accurately reflect clonal tumor burden in patients treated with non-cytotoxic chemotherapy. We used next-generation sequencing of serial bone marrow samples to monitor MDS and AML tumor burden during treatment with epigenetic therapy (decitabine and panobinostat). Serial bone marrow samples (and skin as a source of normal DNA) from 25 MDS and AML patients were sequenced (exome or 285 gene panel). We observed that responders, including those in complete remission (CR), can have persistent measurable tumor burden (that is, mutations) for at least 1 year without disease progression. Using an ultrasensitive sequencing approach, we detected extremely rare mutations (equivalent to 1 heterozygous mutant cell in 2000 non-mutant cells) months to years before their expansion at disease relapse. While patients can live with persistent clonal hematopoiesis in a CR or stable disease, ultimately we find evidence that expansion of a rare subclone occurs at relapse or progression. Here we demonstrate that sequencing of serial samples provides an alternative measure of tumor burden in MDS or AML patients and augments traditional response criteria that rely on bone marrow blast percentage.

Summary How specific genetic lesions contribute to transformation of non-malignant myeloproliferative neoplasms (MPNs) and myelodysplastic syndromes (MDSs) to secondary acute myeloid leukemia (sAML) are poorly understood. JARID2 is lost by chromosomal deletions in a proportion of MPN/MDS cases that progress to sAML. In this study, genetic mouse models and patient-derived xenografts demonstrated that JARID2 acts as a tumor suppressor in chronic myeloid disorders. Genetic deletion of Jarid2 either reduced overall survival of animals with MPNs or drove transformation to sAML, depending on the timing and context of co-operating mutations. Mechanistically, JARID2 recruits PRC2 to epigenetically repress self-renewal pathways in hematopoietic progenitor cells. These studies establish JARID2 as a bona fide hematopoietic tumor suppressor and highlight potential therapeutic targets.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation is the only curative treatment for patients with myelodysplastic syndrome (MDS). The molecular predictors of disease progression after transplantation are unclear. METHODS: We sequenced bone marrow and skin samples from 90 adults with MDS who underwent allogeneic hematopoietic stem-cell transplantation after a myeloablative or reduced-intensity conditioning regimen. We detected mutations before transplantation using enhanced exome sequencing, and we evaluated mutation clearance by using error-corrected sequencing to genotype mutations in bone marrow samples obtained 30 days after transplantation. In this exploratory study, we evaluated the association of a mutation detected after transplantation with disease progression and survival. RESULTS: Sequencing identified at least one validated somatic mutation before transplantation in 86 of 90 patients (96%); 32 of these patients (37%) had at least one mutation with a maximum variant allele frequency of at least 0.5% (equivalent to 1 heterozygous mutant cell in 100 cells) 30 days after transplantation. Patients with disease progression had mutations with a higher maximum variant allele frequency at 30 days than those who did not (median maximum variant allele frequency, 0.9% vs. 0%; P

Salivary neuroendocrine carcinomas are rare and the overwhelming majority is high-grade. The parotid gland is the most commonly involved site followed by the submandibular gland. Most arise de novo but rare examples occurring as a high-grade transformation of another type of salivary gland neoplasm exist. There is significant morphologic and immunophenotypic overlap with neuroendocrine carcinomas of other sites, especially the skin. Like cutaneous neuroendocrine (or Merkel cell) carcinomas, approximately three-fourths are cytokeratin 20 positive. Cytokeratin 20 positive salivary neuroendocrine carcinomas are often referred to as being of the ‘Merkel cell type’ since most other non-cutaneous neuroendocrine carcinomas are cytokeratin 20 negative. Salivary neuroendocrine carcinomas may be challenging to separate from Merkel cell carcinomas of the head and neck on pathologic grounds because the latter often metastasize to the parotid gland. Clinical history is often relied upon to separate primary salivary tumors from cutaneous metastases but may not be helpful in all cases. Here we review the clinical, pathologic and molecular features of salivary neuroendocrine carcinomas focusing on high-grade major salivary gland tumors. The difficulty in separating salivary tumors from metastatic Merkel cell carcinoma will be highlighted.

Allogeneic hematopoietic cell transplantation (alloHCT) is a potentially curative treatment for myelodysplastic syndromes (MDS), but patients who relapse after transplant have poor outcomes. In order to understand the contribution of tumor clonal evolution to disease progression,we applied exome and error-corrected targeted sequencing coupled with copy number analysis to comprehensively define changes in the clonal architecture of MDS in response to therapy using 51 serially acquired tumor samples from 9 patients who progressed after an alloHCT. We show that small subclones before alloHCT can drive progression after alloHCT. Notably, at least one subclone expanded or emerged at progression in all patients. Newly acquired structural variants (SVs) were present in an emergent/expanding subclone in 8 of 9 patients at progression, implicating the acquisition of SVs as important late subclonal progression events. In addition, pretransplant therapy with azacitidine likely influenced the mutation spectrum and evolution of emergent subclones after alloHCT. Although subclone evolution is common, founding clone mutations are always present at progression and could be detected in the bone marrow as early as 30 and/or 100 days after alloHCT in 6 of 8 (75%) patients, often prior to clinical progression. In conclusion, MDS progression after alloHCT is characterized by subclonal expansion and evolution, which can be influenced by pretransplant therapy.

With the advent of large-scale genomic analysis, the genetic landscape of glioblastoma (GBM) has become more clear, including characteristic genetic alterations in EGFR. In routine clinical practice, genetic alterations in GBMs are identified using several disparate techniques that consume already limited amounts of tissue and add to overall testing costs. In this study, we sought to determine if the full spectrum of EGFR mutations in GBMs could be detected using a single next generation sequencing (NGS) based oncology assay in 34 consecutive cases. Using a battery of informatics tools to identify single nucleotide variants, insertions and deletions, and amplification (including variants EGFRvIII and EGFRvV), twenty-one of the 34 (62%) individuals had had at least one alteration in EGFR by sequencing, consistent with published datasets. Mutations detected include several single nucleotide variants, amplification (confirmed by fluorescence in situ hybridization), and the variants EGFRvIII and EGFRvV (confirmed by multiplex ligation-dependent probe amplification). Here we show that a single NGS assay can identify the full spectrum of relevant EGFR mutations. Overall, sequencing based diagnostics have the potential to maximize the amount of genetic information obtained from GBMs and simultaneously reduce the total time, required specimen material, and costs associated with current multimodality studies.

Authors' Reply to the Letter to the Editor by Montgomery et al (Identification of Germline Variants in Tumor Genomic Sequencing Analysis. J Mol Diagn 2017, 19:XXXX-XXXX).

In lung adenocarcinoma, canonical EML4-ALK inversion results in a fusion protein with a constitutively active ALK kinase domain. Evidence of ALK rearrangement occurs in a minority (2-7%) of lung adenocarcinoma, and only ~60% of these patients will respond to targeted ALK inhibition by drugs such as crizotinib and ceritinib. Clinically, targeted anti-ALK therapy is often initiated based on evidence of an ALK genomic rearrangement detected by fluorescence in situ hybridization (FISH) of interphase cells in formalin-fixed, paraffin-embedded tissue sections. At the genomic level, however, ALK rearrangements are heterogeneous, with multiple potential breakpoints in EML4, and alternate fusion partners. Using next-generation sequencing of DNA and RNA together with ALK immunohistochemistry, we comprehensively characterized genomic breakpoints in 33 FISH-positive lung adenocarcinomas. Of these 33 cases, 29 (88%) had detectable DNA level ALK rearrangements involving EML4, KIF5B, or non-canonical partners including ASXL2, ATP6V1B1, PRKAR1A, and SPDYA. A subset of 12 cases had material available for RNA-Seq. Of these, eight of eight (100%) cases with DNA rearrangements showed ALK fusion transcripts from RNA-Seq; three of four cases (75%) without detectable DNA rearrangements were similarly negative by RNA-Seq, and one case was positive by RNA-Seq but negative by DNA next-generation sequencing. By immunohistochemistry, 17 of 19 (89%) tested cases were clearly positive for ALK protein expression; the remaining cases had no detectable DNA level rearrangement or had a non-canonical rearrangement not predicted to form a fusion protein. Survival analysis of patients treated with targeted ALK inhibitors demonstrates a significant difference in mean survival between patients with next-generation sequencing confirmed EML4-ALK rearrangements, and those without (20.6 months vs 5.4 months, P

Context Diffuse large B-cell lymphoma and chronic lymphocytic leukemia are 2 of the most common B-cell lymphomas in adults. Both diffuse large B-cell lymphoma and chronic lymphocytic leukemia share heterogeneous outcomes, and the use of prognostic biomarkers to better stratify risk in these patients has now become commonplace. Objective To review chronic lymphocytic leukemia and diffuse large B-cell lymphoma biomarkers commonly used in the clinical laboratory, which can be divided into the following 3 main groups by testing methodology: chromosomal based (including fluorescence in situ hybridization and cytogenetics), expression based (including immunohistochemistry and flow cytometry), and DNA based (including gene sequencing for somatic mutations and IGVH mutational status). Data Sources Review of recent literature. Conclusions In chronic lymphocytic leukemia, important biomarkers include expression of CD38 and ZAP-70, IGVH mutational status, somatic mutations in TP53 and NOTCH1, and abnormalities in chromosomes 11, 12, 13q, and 17. In diffuse large B-cell lymphoma, important biomarkers include chromosomal rearrangement of BCL2, BCL6, and MYC and expression of CD5, BCL2, and CD43, as well as somatic mutations in TP53 and BCL6.

Targeted next-generation sequencing (NGS) cancer panels have become a popular method for the identification of clinically predictive mutations in cancer. Such methods typically detect single nucleotide variants (SNVs) and small insertions/deletions (indels) in known cancer genes and can provide further information regarding diagnosis in challenging surgical pathology cases, as well as identify therapeutic targets and prognostically significant mutations. However, in addition to SNVs and indels, other mutation classes, including copy number variants (CNVs) and translocations, can be simultaneously detected from targeted NGS data. Here, as proof of methods, we present clinical data which demonstrate that targeted NGS panels can separate synchronous liver tumors based on CNV status, in the absence of distinct SNVs and indels. Such CNV-based analysis can be performed without additional cost using existing targeted cancer panel data and publically available software.

Merkel cell carcinoma is a highly aggressive cutaneous neuroendocrine tumor that has been associated with Merkel cell polyomavirus in up to 80% of cases. Merkel cell polyomavirus is believed to influence pathogenesis, at least in part, through expression of the large T antigen, which includes a retinoblastoma protein-binding domain. However, there appears to be significant clinical and morphological overlap between polyomavirus-positive and polyomavirus-negative Merkel cell carcinoma cases. Although much of the recent focus of Merkel cell carcinoma pathogenesis has been on polyomavirus, the pathogenesis of polyomavirus-negative cases is still poorly understood. We hypothesized that there are underlying human somatic mutations that unify Merkel cell carcinoma pathogenesis across polyomavirus status, and to investigate we performed whole exome sequencing on five polyomavirus-positive cases and three polyomavirus-negative cases. We found that there were no significant differences in the overall number of single-nucleotide variations, copy number variations, insertion/deletions, and chromosomal rearrangements when comparing polyomavirus-positive to polyomavirus-negative cases. However, we did find that the retinoblastoma pathway genes harbored a high number of mutations in Merkel cell carcinoma. Furthermore, the retinoblastoma gene (RB1) was found to have nonsense truncating protein mutations in all three polyomavirus-negative cases; no such mutations were found in the polyomavirus-positive cases. In all eight cases, the retinoblastoma pathway dysregulation was confirmed by immunohistochemistry. Although polyomavirus-positive Merkel cell carcinoma is believed to undergo retinoblastoma dysregulation through viral large T antigen expression, our findings demonstrate that somatic mutations in polyomavirus-negative Merkel cell carcinoma lead to retinoblastoma dysregulation through an alternative pathway. This novel finding suggests that the retinoblastoma pathway dysregulation leads to an overlapping Merkel cell carcinoma phenotype and that oncogenesis occurs through either a polyomavirus-dependent (viral large T antigen expression) or polyomavirus-independent (host somatic mutation) mechanism.

Introduction Desmoplastic infantile ganglioglioma is a rare intracranial neoplasm classified as World Health Organization grade I tumor under neuronal and mixed neuronal-glial tumors (2007 World Health Organization brain tumor classification). It is usually a good prognosis, but 40% of patients require further medical, radiation, and/or surgical intervention, and 15% develop leptomeningeal spread or die from desmoplastic infantile ganglioglioma. Transformation to malignant glioblastoma occurs, but the genetic alterations associated with the transformation are generally unknown. Methods We describe a desmoplastic infantile ganglioglioma in a 2-month-old boy, which showed aggressive behavior, requiring debulking at 2.5 months of age and chemotherapy at 10 months of age after tumor progression. At 8.5 years of age he developed malignant transformation to glioblastoma. Chromosome microarray analysis using oligo array and genomic sequencing was performed on the biopsy specimen from 2 months of age and on the subsequent transformed malignant glioblastoma. Results After being clinically stable for 7.5-years, transformation to glioblastoma transformation occurred. He did well for 1 year but subsequently died from tumor progression. Chromosome microarray analysis using oligo array performed on the biopsy specimen obtained at 2 months of age did not reveal significant abnormalities; but there were significant genomic deletions and duplications associated with the glioblastoma. These included multiple genomic losses involving 4q and Y, gains of 5q, and amplification of 12q14. Genomic sequencing revealed a single nucleotide variant, p.R248Q in exon 7 of TP53 , in the primary desmoplastic infantile ganglioglioma and the glioblastoma multiforme. Conclusions The nonsynonymous variant (p.R248Q in exon 7) of the TP53 gene is predicted to alter the structure of the L2/L3 motif of the DNA binding domain of p53 protein. It was detected in the primary desmoplastic infantile ganglioglioma and glioblastoma multiforme. This child illustrates the rare recurrence of desmoplastic infantile ganglioglioma with malignant transformation to glioblastoma caused by a nonsynonymous TP53 mutation, providing explanation for other rare benign tumor transformations. The TP53 gene is a known primary site of genetic alteration that predisposes to malignant tumors, and this case indicates that it might also be involved in the behavior and outcome of desmoplastic infantile ganglioglioma. Therefore more genetic testing is recommended on desmoplastic infantile ganglioglioma tumors, which may provide biologic prognostic markers.

Viral pathogens have been implicated in the development of certain cancers including human papillomavirus (HPV) in squamous cell carcinoma and Epstein-Barr virus (EBV) in Burkitt's lymphoma. The significance of viral pathogens in brain tumors is controversial, and human cytomegalovirus (HCMV) has been associated with glioblastoma (GBM) in some but not all studies, making the role of HCMV unclear. In this study we sought to determine if viral pathogen sequences could be identified in an unbiased manner from previously discarded, unmapped, non-human, next-generation sequencing (NGS) reads obtained from targeted oncology, panel-based sequencing of high grade gliomas (HGGs), including GBMs. Twenty one sequential HGG cases were analyzed by a targeted NGS clinical oncology panel containing 151 genes using DNA obtained from formalin-fixed, paraffin-embedded (FFPE) tissue. Sequencing reads that did not map to the human genome (average of 38,000 non-human reads/case (1.9%)) were filtered and low quality reads removed. Extracted high quality reads were then sequentially aligned to the National Center for Biotechnology Information (NCBI) non-redundant nucleotide (nt and nr) databases. Aligned reads were classified based on NCBI taxonomy database and all eukaryotic viral sequences were further classified into viral families. Two viral sequences (both herpesviruses), EBV and Roseolovirus were detected in 5/21 (24%) cases and in 1/21 (5%) cases, respectively. None of the cases had detectable HCMV. Of the five HGG cases with detectable EBV DNA, four had additional material for EBV in situ hybridization (ISH), all of which were negative for expressed viral sequence. Overall, a similar discovery approach using unmapped non-human NGS reads could be used to discover viral sequences in other cancer types.

Objectives: To design and evaluate a next-generation sequencing (NGS)–based method for T-cell receptor γ (TCRG) gene-based T-cell clonality testing on the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA) platform. Methods: We analyzed a series of peripheral blood, bone marrow, and formalin-fixed paraffin-embedded tissue specimens with NGS vs traditional capillary electrophoresis methods. Results: Using a custom analysis algorithm that we developed, our NGS assay identified between 2,215 and 48,222 unique TCRG rearrangements in a series of 48 samples. We established criteria for assigning clonality based on parameters derived from both the relative and absolute frequencies of reads. In a comparison with standard capillary electrophoresis, 19 of 19 polyclonal samples and 24 of 27 samples that appeared clonal were in agreement. The three discrepant samples demonstrated some of the pitfalls of amplicon length–based testing. Dilution studies with T-lymphoid cell lines demonstrated that a known clonal sequence could be routinely identified when present in as few as 0.1% of total cells demonstrating suitability in residual disease testing. A series of samples was also analyzed on a second NGS platform and yielded very similar results with respect to the frequency and sequence of the clonal rearrangement. Conclusions: In this proof-of-concept study, we describe an NGS-based T-cell clonality assay that is suitable for routine clinical testing either alone or as an adjunct to traditional methods.

Mutations in exon 2 of the MED12 gene have been reported in 50% to 70% of uterine leiomyomas. To determine the frequency of MED12 mutations in various types of smooth muscle tumors as well as normal uterine myometrium adjacent to a leiomyoma, we selected a total of 143 cases for analysis of MED12 exon 2 mutations by polymerase chain reaction and Sanger sequencing. MED12 mutations were detected in 54% of classical uterine leiomyomas (15/28) and in 15% of cases in myometrium adjacent to leiomyomas (2/13); 34% of leiomyoma/leiomyomatosis in pelvic/retroperitoneal sites (10/29); 0% of extrauterine leiomyomas (0/29); 8% of smooth muscle tumor of uncertain malignant potential (1/12); 30% of uterine leiomyosarcomas (6/20); and 4% of extrauterine leiomyosarcomas (1/25). Mutations were clustered around codons 44, 40, 41, and 36, and consisted primarily of single nucleotide substitutions and small in-frame deletions. Our results confirm the findings of similar recent studies and further show that pelvic and retroperitoneal leiomyomas harbor an increased frequency of MED12 mutations (34%) as compared with other extrauterine sites (0%; P = 0.0006), and that histologically unremarkable adjacent myometrium can harbor similar MED12 mutations. These findings suggest that smooth muscle tumors in pelvic/retroperitoneal sites are subject to the same mutational changes as those of uterine myometrium, and that these mutations may precede the gross or histological development of a leiomyoma.

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.

Background Mesenchymal hamartoma of the liver (MHL) is an uncommon benign primary liver tumor that typically occurs in the pediatric population, although cases have been described in adults. MHL is sometimes associated with the highly malignant undifferentiated embryonal sarcoma (UES), and the synchronous or metachronous occurrence of MHL and UES suggests they share a common genetic link. Although the exact mechanism of tumorigenesis has not been identified, MHL cases harbor recurring chromosomal rearrangements involving 19q13. Design In order to provide more details on the genetic events of MHL tumorigenesis, capture-based next generation sequencing (NGS) targeted to loci recently shown to be involved in a translocation in a case of UES arising in MHL (specifically, the MALAT1 gene on chromosome 11 and a gene poor region termed MHLB1 on chromosome 19) was performed on formalin fixed paraffin embedded tissue from seven cases of MHL. Results Chromosome rearrangements involving the MHLB1 locus were identified in three of the seven cases, including the translocation t(11,19)(q13.1;q13.42) involving the MALAT1 gene; the translocation t(2,19)(q31.1;q13.42) involving AK023515, an uncharacterized noncoding gene; and the inversion inv(19,19)(q13.42;q13.43) involving the PEG3 gene encoding a Kruppel-type zinc-finger protein. Rearrangements were exclusively identified in pediatric tumors. In each case, the presence of the rearrangement was confirmed by PCR and interphase FISH. Interphase FISH also demonstrated that the arrangements occur within the spindle cell component but not within the epithelial components of the tumor. Conclusions Since the MHLB1 locus contains a CpG-rich region whose methylation regulates C19MC miRNA genes, rearrangements that disrupt this region may contribute to MHL development through alteration of miRNA expression. The demonstration that the loose stromal cells harbor the rearrangements indicates that (some cases of) MHL are a neoplastic process due to a somatic genetic change and not a germline abnormality.

Next-generation sequencing (NGS) has emerged as a powerful technique for the detection of genetic variants in the clinical laboratory. NGS can be performed using DNA from FFPE tissue, but it is unknown whether such specimens are truly equivalent to unfixed tissue for NGS applications. To address this question, we performed hybridization-capture enrichment and multiplexed Illumina NGS for 27 cancer-related genes using DNA from 16 paired fresh-frozen and routine FFPE lung adenocarcinoma specimens and conducted extensive comparisons between the sequence data from each sample type. This analysis revealed small but detectable differences between FFPE and frozen samples. Compared with frozen samples, NGS data from FFPE samples had smaller library insert sizes, greater coverage variability, and an increase in C to T transitions that was most pronounced at CpG dinucleotides, suggesting interplay between DNA methylation and formalin-induced changes; however, the error rate, library complexity, enrichment performance, and coverage statistics were not significantly different. Comparison of base calls between paired samples demonstrated concordances of >99.99%, with 96.8% agreement in the single-nucleotide variants detected and >98% accuracy of NGS data when compared with genotypes from an orthogonal single-nucleotide polymorphism array platform. This study demonstrates that routine processing of FFPE samples has a detectable but negligible effect on NGS data and that these samples can be a reliable substrate for clinical NGS testing.