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BACKGROUND: Leptomeningeal metastases occur across multiple solid and lymphoid cancers, and patients typically undergo cytopathologic assessment of cerebrospinal fluid (CSF) in this setting. For patients diagnosed with metastatic cancer, the detection of actionable somatic mutations in CSF can provide clinically valuable information for treatment without the need for additional tissue collection. METHODS: The authors validated a targeted next-generation sequencing assay for the detection of somatic variants in cancer (OncoPanel) on cell-free DNA (cfDNA) isolated from archival CSF specimens in a cohort of 25 patients who had undergone molecular testing of a prior tumor specimen. RESULTS: CSF storage time and volume had no impact on cfDNA concentration or mean target coverage of the assay. Previously identified somatic variants in CSF cfDNA were detected in 88%, 50%, and 27% of specimens diagnosed cytologically as positive, suspicious/atypical, and negative for malignancy, respectively. Somatic variants were identified in 81% of CSF specimens from patients who had leptomeningeal enhancement on magnetic resonance imaging compared with 31% from patients without such enhancement. CONCLUSIONS: These data highlight the stability of cfDNA in CSF, which allows for cytopathologic evaluation before triage for next-generation sequencing assays. For a subset of cases in which clinical suspicion is high but cytologic or radiographic studies are inconclusive, the detection of pathogenic somatic variants in CSF cfDNA may aid in the diagnosis of leptomeningeal metastases.
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Ácidos Nucleicos Livres , Neoplasias , Humanos , Ácidos Nucleicos Livres/genética , Mutação , Neoplasias/diagnóstico por imagem , Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
CONTEXT.: Droplet digital polymerase chain reaction (ddPCR) is a sensitive method to detect common pathogenic EGFR mutations in non-small cell lung cancer. Although targeted assays have not been specifically designed to detect them, uncommon EGFR mutations have been linked to response to targeted therapy. OBJECTIVE.: To describe atypical ddPCR patterns that correspond to uncommon but clinically actionable EGFR mutations. DESIGN.: A cohort of 1134 consecutive non-small cell lung cancers that underwent targeted next-generation sequencing was reviewed. Uncommon EGFR mutations involving probe binding sites were evaluated by ddPCR. RESULTS.: Two hundred fifty-five of 1134 cancers (22.5%) harbored pathogenic EGFR mutations. One hundred eighty-six of 255 (72.9%) had canonical EGFR exon 19 deletion or exon 21 p.L858R variants designed for detection by ddPCR. An additional 25 of 255 cases (9.8%) had uncommon EGFR mutations within the probe-binding site, including one case with concurrent uncommon mutations in both exon 19 and exon 21. These mutations included uncommon EGFR exon 19 deletions (n = 6), EGFR exon 19 substitutions p.L747P (n = 3) and p.L747A (n = 1), dinucleotide substitutions leading to EGFR p.L858R (n = 5), EGFR exon 21 substitutions p.K860I (n = 1) and p.L861Q (n = 9), and EGFR p.[L858R;K860I] (n = 1). Droplet digital polymerase chain reaction generated atypical but reproducible signal for each of these uncommon variants. CONCLUSIONS.: Droplet digital polymerase chain reaction analysis of uncommon pathogenic EGFR variants can yield unique and reproducible results. Recognition of atypical patterns in EGFR ddPCR testing can prompt confirmatory molecular testing and aid appropriate targeted therapy selection for patients with non-small cell lung cancer.
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Vulvar melanoma is a rare and aggressive cancer with a poor prognosis. The etiology of mucosal melanoma remains largely uncharacterized and no hereditary risk factors are established for this rare disease. While the germline variant MITF p.E318K confers an increased risk for cutaneous melanoma, this variant has not been associated with risk of non-cutaneous melanoma. Herein, we describe the presence of a germline MITF p.E318K pathogenic variant in a 47-year-old woman with vulvar melanoma and a family history of cutaneous melanoma in a first-degree relative. To our knowledge, this is the first reported case of MITF p.E318K in vulvar melanoma. This finding highlights the potential involvement of MITF p.E318K in risk assessment and clinical management of patients with vulvar melanoma. Further study of this observation is needed to inform appropriate identification of patients with non-cutaneous melanoma for MITF germline genomic evaluation and to potentially guide management for early detection of vulvar melanoma.
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Melanoma , Neoplasias Cutâneas , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Melanoma/genética , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/genética , Pessoa de Meia-Idade , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Melanoma Maligno CutâneoRESUMO
Activating mutations in EGFR predict benefit from tyrosine kinase inhibitor therapy for patients with advanced non-small cell lung cancer. Directing patients to appropriate therapy depends on accurate and timely EGFR assessment in the molecular pathology laboratory. This article describes the analytical design, performance characteristics, and clinical implementation of an assay for the rapid detection of EGFR L858R and exon 19 deletion mutations. A droplet digital polymerase chain reaction (ddPCR) assay was implemented with probe hydrolysis-dependent signal detection. A mutation-specific probe was used to detect EGFR L858R. A loss of signal design was used to detect EGFR exon 19 deletion mutations. Analytical sensitivity was dependent on DNA input and was as low as 0.01% variant allele fraction for the EGFR L858R assay and 0.1% variant allele fraction for the EGFR exon 19 deletion assay. Correlation of 20 clinical specimens tested by ddPCR and next generation sequencing showed 100% concordance. ddPCR showed 53% clinical sensitivity in the detection of EGFR mutations in plasma cell-free DNA from patients with lung cancer. The median clinical turnaround time was 5 days for ddPCR compared to 13 days for next generation sequencing. The findings show that ddPCR is an accurate and rapid method for detecting EGFR mutations in patients with non-small cell lung cancer.
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Carcinoma Pulmonar de Células não Pequenas/genética , Análise Mutacional de DNA/métodos , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase/métodos , Receptores ErbB/genética , Humanos , Mutação , Sensibilidade e EspecificidadeRESUMO
The molecular mechanisms leading to resistance to PD-1 blockade are largely unknown. Here, we characterize tumor biopsies from a patient with melanoma who displayed heterogeneous responses to anti-PD-1 therapy. We observe that a resistant tumor exhibited a loss-of-function mutation in the tumor suppressor gene FBXW7, whereas a sensitive tumor from the same patient did not. Consistent with a functional role in immunotherapy response, inactivation of Fbxw7 in murine tumor cell lines caused resistance to anti-PD-1 in immunocompetent animals. Loss of Fbxw7 was associated with altered immune microenvironment, decreased tumor-intrinsic expression of the double-stranded RNA (dsRNA) sensors MDA5 and RIG1, and diminished induction of type I IFN and MHC-I expression. In contrast, restoration of dsRNA sensing in Fbxw7-deficient cells was sufficient to sensitize them to anti-PD-1. Our results thus establish a new role for the commonly inactivated tumor suppressor FBXW7 in viral sensing and sensitivity to immunotherapy. SIGNIFICANCE: Our findings establish a role of the commonly inactivated tumor suppressor FBXW7 as a genomic driver of response to anti-PD-1 therapy. Fbxw7 loss promotes resistance to anti-PD-1 through the downregulation of viral sensing pathways, suggesting that therapeutic reactivation of these pathways could improve clinical responses to checkpoint inhibitors in genomically defined cancer patient populations.This article is highlighted in the In This Issue feature, p. 1241.
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Resistencia a Medicamentos Antineoplásicos/genética , Proteína 7 com Repetições F-Box-WD/genética , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Idoso , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Linhagem Celular Tumoral/transplante , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Modelos Animais de Doenças , Proteína 7 com Repetições F-Box-WD/metabolismo , Regulação Neoplásica da Expressão Gênica/imunologia , Células HeLa , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Mutação com Perda de Função , Masculino , Camundongos , Mutagênese Sítio-Dirigida , Receptor de Morte Celular Programada 1/antagonistas & inibidores , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
OBJECTIVES: 16S ribosomal RNA (rRNA) sequencing is a powerful but expensive tool for the identification of bacteria in culture-negative endocarditis. Histologic criteria to screen formalin-fixed, paraffin-embedded (FFPE) specimens for testing are evaluated. METHODS: Sixty-eight cases of infective endocarditis and controls were histologically reviewed and analyzed by 16S rRNA gene sequencing. RESULTS: Sequencing identified a specific pathogenic organism in 33 (49%) of 68 cases with acute inflammation and in 0 of 10 controls (P = .004). Visualization of organisms by Gram or Grocott methenamine silver stains had the strongest association with positive sequencing, while antibiotic treatment effect and acid decalcification decreased sensitivity. Molecular identifications were concordant with blood culture results in 90% of the cases, and a positive sequencing result was obtained in approximately half of the cases with negative valve cultures. CONCLUSIONS: Histologic screening criteria are extremely helpful for identifying cases likely to be positive by molecular testing and can provide significant cost savings in filtering out low-yield specimens.
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Endocardite Bacteriana/diagnóstico , RNA Ribossômico 16S/análise , DNA Bacteriano , Endocardite Bacteriana/microbiologia , Humanos , Inclusão em Parafina , Análise de Sequência de DNARESUMO
OBJECTIVES: Flow cytometry immunophenotyping is limited by poor resolution of T-cell clones. A newly described antibody was recently used to distinguish normal peripheral blood T cells from malignant T-cell clones. Here, we evaluate this antibody as a new diagnostic tool for detecting T-cell clonality in mature peripheral T-cell lymphomas. METHODS: Immunostaining for the T-cell receptor ß chain constant region 1 (TRBC1) along with routine T-cell markers was performed on 51 peripheral blood and two bone marrow samples submitted to the flow cytometry laboratory for suspected T-cell malignancy. RESULTS: TRBC immunophenotyping identified malignant T-cell clones with 97% sensitivity and 91% specificity. Findings correlated with molecular T-cell clonality testing. In cases with equivocal molecular results, TRBC1 immunophenotyping provided additional diagnostic information. CONCLUSIONS: TRBC1 flow cytometric immunophenotyping is a robust and inexpensive method for identifying T-cell clonality that could easily be incorporated into routine flow cytometric practice.
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Citometria de Fluxo/métodos , Linfoma de Células T Periférico/diagnóstico , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imunofenotipagem , Linfoma de Células T Periférico/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T gama-delta/análise , Valores de ReferênciaRESUMO
PURPOSE: Implementing cancer precision medicine in the clinic requires assessing the therapeutic relevance of genomic alterations. A main challenge is the systematic interpretation of whole-exome sequencing (WES) data for clinical care. METHODS: One hundred sixty-five adults with metastatic colorectal and lung adenocarcinomas were prospectively enrolled in the CanSeq study. WES was performed on DNA extracted from formalin-fixed paraffin-embedded tumor biopsy samples and matched blood samples. Somatic and germ-line alterations were ranked according to therapeutic or clinical relevance. Results were interpreted using an integrated somatic and germ-line framework and returned in accordance with patient preferences. RESULTS: At the time of this analysis, WES had been performed and results returned to the clinical team for 165 participants. Of 768 curated somatic alterations, only 31% were associated with clinical evidence and 69% with preclinical or inferential evidence. Of 806 curated germ-line variants, 5% were clinically relevant and 56% were classified as variants of unknown significance. The variant review and decision-making processes were effective when the process was changed from that of a Molecular Tumor Board to a protocol-based approach. CONCLUSION: The development of novel interpretive and decision-support tools that draw from scientific and clinical evidence will be crucial for the success of cancer precision medicine in WES studies.Genet Med advance online publication 26 January 2017.
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Sequenciamento do Exoma/métodos , Exoma/genética , Medicina de Precisão/métodos , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Adulto , Neoplasias Colorretais/genética , Bases de Dados Genéticas , Genômica/métodos , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/genética , Mutação/genética , Estudos Prospectivos , Análise de Sequência de DNA/métodosRESUMO
Mismatch repair protein deficiency (MMR-D) and high microsatellite instability (MSI-H) are features of Lynch syndrome-associated colorectal carcinomas and have implications in clinical management. We evaluate the ability of a targeted next-generation sequencing panel to detect MMR-D and MSI-H based on mutational phenotype. Using a criterion of >40 total mutations per megabase or >5 single-base insertion or deletion mutations in repeats per megabase, sequencing achieves 92% sensitivity and 100% specificity for MMR-D by immunohistochemistry in a training cohort of 149 colorectal carcinomas and 91% sensitivity and 98% specificity for MMR-D in a validation cohort of 94 additional colorectal carcinomas. False-negative samples are attributable to tumor heterogeneity, and next-generation sequencing results are concordant with analysis of microsatellite loci by PCR. In a subset of 95 carcinomas with microsatellite analysis, sequencing achieves 100% sensitivity and 99% specificity for MSI-H in the combined training and validation set. False-positive results for MMR-D and MSI-H are attributable to ultramutated cancers with POLE mutations, which are confirmed by direct sequencing of the POLE gene and are detected by mutational signature analysis. These findings provide a framework for a targeted tumor sequencing panel to accurately detect MMR-D and MSI-H in colorectal carcinomas.
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Adenocarcinoma/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Análise Mutacional de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Adenocarcinoma/diagnóstico , Sequência de Bases , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Reparo de Erro de Pareamento de DNA , Genes Neoplásicos , Humanos , Instabilidade de Microssatélites , Repetições de Microssatélites , MutaçãoRESUMO
BACKGROUND. Comprehensive genomic profiling of a patient's cancer can be used to diagnose, monitor, and recommend treatment. Clinical implementation of tumor profiling in an enterprise-wide, unselected cancer patient population has yet to be reported. METHODS. We deployed a hybrid-capture and massively parallel sequencing assay (OncoPanel) for all adult and pediatric patients at our combined cancer centers. Results were categorized by pathologists based on actionability. We report the results for the first 3,727 patients tested. RESULTS. Our cohort consists of cancer patients unrestricted by disease site or stage. Across all consented patients, half had sufficient and available (>20% tumor) material for profiling; once specimens were received in the laboratory for pathology review, 73% were scored as adequate for genomic testing. When sufficient DNA was obtained, OncoPanel yielded a result in 96% of cases. 73% of patients harbored an actionable or informative alteration; only 19% of these represented a current standard of care for therapeutic stratification. The findings recapitulate those of previous studies of common cancers but also identify alterations, including in AXL and EGFR, associated with response to targeted therapies. In rare cancers, potentially actionable alterations suggest the utility of a "cancer-agnostic" approach in genomic profiling. Retrospective analyses uncovered contextual genomic features that may inform therapeutic response and examples where diagnoses revised by genomic profiling markedly changed clinical management. CONCLUSIONS. Broad sequencing-based testing deployed across an unselected cancer cohort is feasible. Genomic results may alter management in diverse scenarios; however, additional barriers must be overcome to enable precision cancer medicine on a large scale. FUNDING. This work was supported by DFCI, BWH, and the National Cancer Institute (5R33CA155554 and 5K23CA157631).
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Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias/genética , Análise Mutacional de DNA , Humanos , Mutação , Medicina de Precisão , Estudos RetrospectivosRESUMO
BACKGROUND: The diversity of clinical tumor profiling approaches (small panels to whole exomes with matched or unmatched germline analysis) may engender uncertainty about their benefits and liabilities, particularly in light of reported germline false positives in tumor-only profiling and use of global mutational and/or neoantigen data. The goal of this study was to determine the impact of genomic analysis strategies on error rates and data interpretation across contexts and ancestries. METHODS: We modeled common tumor profiling modalities-large (n = 300 genes), medium (n = 48 genes), and small (n = 15 genes) panels-using clinical whole exomes (WES) from 157 patients with lung or colon adenocarcinoma. We created a tumor-only analysis algorithm to assess germline false positive rates, the impact of patient ancestry on tumor-only results, and neoantigen detection. RESULTS: After optimizing a germline filtering strategy, the germline false positive rate with tumor-only large panel sequencing was 14 % (144/1012 variants). For patients whose tumor-only results underwent molecular pathologist review (n = 91), 50/54 (93 %) false positives were correctly interpreted as uncertain variants. Increased germline false positives were observed in tumor-only sequencing of non-European compared with European ancestry patients (p < 0.001; Fisher's exact) when basic germline filtering approaches were used; however, the ExAC database (60,706 germline exomes) mitigated this disparity (p = 0.53). Matched and unmatched large panel mutational load correlated with WES mutational load (r(2) = 0.99 and 0.93, respectively; p < 0.001). Neoantigen load also correlated (r(2) = 0.80; p < 0.001), though WES identified a broader spectrum of neoantigens. Small panels did not predict mutational or neoantigen load. CONCLUSIONS: Large tumor-only targeted panels are sufficient for most somatic variant identification and mutational load prediction if paired with expanded germline analysis strategies and molecular pathologist review. Paired germline sequencing reduced overall false positive mutation calls and WES provided the most neoantigens. Without patient-matched germline data, large germline databases are needed to minimize false positive mutation calling and mitigate ethnic disparities.
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Adenocarcinoma/genética , Antígenos de Neoplasias/genética , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Medicina de Precisão , Adenocarcinoma/diagnóstico , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/patologia , Bases de Dados Genéticas , Exoma , Reações Falso-Positivas , Perfilação da Expressão Gênica , Genômica/métodos , Mutação em Linhagem Germinativa , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Taxa de Mutação , Linhagem , Análise de Sequência de DNARESUMO
INTRODUCTION: Mucinous differentiation is observed in a subset of lung adenocarcinomas with unique clinical and pathological features, but the biology of these neoplasms is poorly understood. METHODS: We apply targeted next-generation sequencing to characterize the mutational profiles of 21 invasive mucinous adenocarcinomas, mixed mucinous/nonmucinous adenocarcinomas, and adenocarcinomas with mucinous features of the lung and validate key findings on 954 additional lung adenocarcinomas from our institution and 514 lung adenocarcinomas from The Cancer Genome Atlas. RESULTS: Sequencing identifies pathogenic mutations in the oncogenes Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), erb-b2 receptor tyrosine kinase 2 (ERBB2), and anaplastic lymphoma receptor tyrosine kinase (ALK) and recurrent mutations in tumor protein p53 (TP53), serine/threonine kinase 11 (STK11), NK2 homeobox 1 (NKX2-1), and SET domain containing 2 (SETD2). In the combined discovery and validation cohorts, we identify nine neoplasms with distinct molecular and pathological features. All are invasive mucinous adenocarcinomas or mixed mucinous/nonmucinous adenocarcinomas with mutations of KRAS and frameshift or nonsense mutations of NKX2-1. Immunohistochemical analysis shows that these neoplasms are associated with altered differentiation states, including loss of expression of the pulmonary marker thyroid transcription factor 1 (also called Nkx2.1) and expression of gastrointestinal markers. CONCLUSIONS: These findings describe recurrent NKX2-1 mutations in invasive mucinous adenocarcinomas of the lung and support NKX2-1 as a lineage-specific tumor suppressor gene in lung carcinogenesis.
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Adenocarcinoma Mucinoso/genética , Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Mutação , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patologia , Análise Mutacional de DNA , Genes ras , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Nucleares/metabolismo , Estudos Prospectivos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/metabolismoRESUMO
PD-1 immune checkpoint blockade occasionally results in durable clinical responses in advanced metastatic cancers. However, mechanism-based predictors of response to this immunotherapy remain incompletely characterized. We performed comprehensive genomic profiling on a tumor and germline sample from a patient with refractory lung adenocarcinoma who achieved marked long-term clinical benefit from anti-PD-L1 therapy. We discovered activating somatic and germline amino acid variants in JAK3 that promoted PD-L1 induction in lung cancer cells and in the tumor immune microenvironment. These findings suggest that genomic alterations that deregulate cytokine receptor signal transduction could contribute to PD-L1 activation and engagement of the PD-1 immune checkpoint in lung cancer.
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Antígeno B7-H1/antagonistas & inibidores , Janus Quinase 3/metabolismo , Neoplasias Pulmonares/metabolismo , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Expressão Gênica , Perfilação da Expressão Gênica , Genômica , Humanos , Janus Quinase 3/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Mutação , Metástase Neoplásica , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Genomic structural variation (SV), a common hallmark of cancer, has important predictive and therapeutic implications. However, accurately detecting SV using high-throughput sequencing data remains challenging, especially for 'targeted' resequencing efforts. This is critically important in the clinical setting where targeted resequencing is frequently being applied to rapidly assess clinically actionable mutations in tumor biopsies in a cost-effective manner. We present BreaKmer, a novel approach that uses a 'kmer' strategy to assemble misaligned sequence reads for predicting insertions, deletions, inversions, tandem duplications and translocations at base-pair resolution in targeted resequencing data. Variants are predicted by realigning an assembled consensus sequence created from sequence reads that were abnormally aligned to the reference genome. Using targeted resequencing data from tumor specimens with orthogonally validated SV, non-tumor samples and whole-genome sequencing data, BreaKmer had a 97.4% overall sensitivity for known events and predicted 17 positively validated, novel variants. Relative to four publically available algorithms, BreaKmer detected SV with increased sensitivity and limited calls in non-tumor samples, key features for variant analysis of tumor specimens in both the clinical and research settings.