RESUMO
Acute myeloid leukemia (AML) with mutated NPM1 is a newly recognized separate entity in the revised 2016 World Health Organization classification and is associated with a favorable prognosis. Although previous studies have evaluated NPM1 in a binary fashion, little is known about the significance of its mutant allele burden at diagnosis, nor has the effect of comutations (other than FLT3) been extensively evaluated. We retrospectively used targeted sequencing data from 109 patients with de novo AML with mutated NPM1 to evaluate the potential significance of NPM1 variant allele frequency (VAF), comutations, and clinical parameters with regard to patient outcomes. We observed that high NPM1 VAF (uppermost quartile) correlated with shortened overall survival (median, 12.1 months vs not reached; P < .0001) as well as event-free survival (median, 7.5 vs 65.44 months; P < .0001) compared with the other NPM1-mutated cases. In both univariate and multivariable analyses, high NPM1 VAF had a particularly adverse prognostic effect in the subset of patients treated with stem-cell transplantation in first remission (P = .0004) and in patients with mutated DNMT3A (P < .0001). Our findings indicate that the prognostic effect of NPM1 mutation in de novo AML may be influenced by the relative abundance of the mutated allele.
Assuntos
Frequência do Gene , Leucemia Mieloide Aguda/genética , Mutação , Proteínas Nucleares/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Intervalo Livre de Doença , Feminino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Acúmulo de Mutações , Nucleofosmina , Prognóstico , Estudos Retrospectivos , Transplante de Células-Tronco , Análise de Sobrevida , Adulto JovemRESUMO
Advances in technology that have transpired over the past 2 decades have enabled the analysis of cancer samples for genomic alterations to understand their biologic function and to translate that knowledge into clinical practice. With the power to analyze entire genomes in a clinically relevant time frame and with manageable costs comes the question of whether we ought to and when. This review focuses on the relative merits of 3 approaches to molecular diagnostics in hematologic malignancies: indication-specific single gene assays, gene panel assays that test for genes selected for their roles in cancer, and genome-wide assays that broadly analyze the tumor exomes or genomes. After addressing these in general terms, we review specific use cases in myeloid and lymphoid malignancies to highlight the utility of single gene testing and/or larger panels.
Assuntos
Exoma , Estudo de Associação Genômica Ampla/métodos , Neoplasias Hematológicas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , HumanosRESUMO
Recent work has identified distinct molecular subgroups of acute myeloid leukemia (AML) with implications for disease classification and prognosis. NPM1 is one of the most common recurrently mutated genes in AML. NPM1 mutations often co-occur with FLT3-ITDs and mutations in genes regulating DNA methylation, such as DNMT3A, TET2, and IDH1/2. It remains unclear whether these genetic alterations are associated with distinct immunophenotypic findings or affect prognosis. We identified 133 cases of NPM1-mutated AML and correlated sequencing data with immunophenotypic and clinical findings. Of 84 cases (63%) that lacked monocytic differentiation ("myeloid AML"), 40 (48%) demonstrated an acute promyelocytic leukemia-like (APL-like) immunophenotype by flow cytometry, with absence of CD34 and HLA-DR and strong myeloperoxidase expression, in the absence of a PML-RARA translocation. Pathologic variants in TET2, IDH1, or IDH2 were identified in 39/40 APL-like cases. This subset of NPM1-mutated AML was associated with longer relapse-free and overall survival, when compared with cases that were positive for CD34 and/or HLA-DR. The combination of NPM1 and TET2 or IDH1/2 mutations along with an APL-like immunophenotype identifies a distinct subtype of AML. Further studies addressing its biology and clinical significance may be especially relevant in the era of IDH inhibitors and recent work showing efficacy of ATRA therapy in NPM1 and IDH1-mutated AML.
Assuntos
Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ligação a DNA/genética , Dioxigenases , Intervalo Livre de Doença , Feminino , Humanos , Imunofenotipagem , Isocitrato Desidrogenase/genética , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/fisiologia , Nucleofosmina , Proteínas Proto-Oncogênicas/genética , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: The incidence of hematologic cancers increases with age. These cancers are associated with recurrent somatic mutations in specific genes. We hypothesized that such mutations would be detectable in the blood of some persons who are not known to have hematologic disorders. METHODS: We analyzed whole-exome sequencing data from DNA in the peripheral-blood cells of 17,182 persons who were unselected for hematologic phenotypes. We looked for somatic mutations by identifying previously characterized single-nucleotide variants and small insertions or deletions in 160 genes that are recurrently mutated in hematologic cancers. The presence of mutations was analyzed for an association with hematologic phenotypes, survival, and cardiovascular events. RESULTS: Detectable somatic mutations were rare in persons younger than 40 years of age but rose appreciably in frequency with age. Among persons 70 to 79 years of age, 80 to 89 years of age, and 90 to 108 years of age, these clonal mutations were observed in 9.5% (219 of 2300 persons), 11.7% (37 of 317), and 18.4% (19 of 103), respectively. The majority of the variants occurred in three genes: DNMT3A, TET2, and ASXL1. The presence of a somatic mutation was associated with an increase in the risk of hematologic cancer (hazard ratio, 11.1; 95% confidence interval [CI], 3.9 to 32.6), an increase in all-cause mortality (hazard ratio, 1.4; 95% CI, 1.1 to 1.8), and increases in the risks of incident coronary heart disease (hazard ratio, 2.0; 95% CI, 1.2 to 3.4) and ischemic stroke (hazard ratio, 2.6; 95% CI, 1.4 to 4.8). CONCLUSIONS: Age-related clonal hematopoiesis is a common condition that is associated with increases in the risk of hematologic cancer and in all-cause mortality, with the latter possibly due to an increased risk of cardiovascular disease. (Funded by the National Institutes of Health and others.).
Assuntos
Sangue , Transformação Celular Neoplásica/genética , Neoplasias Hematológicas/genética , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Mutação , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Células Clonais , Análise Mutacional de DNA , Exoma , Humanos , Pessoa de Meia-Idade , Fatores de Risco , Adulto JovemAssuntos
Medula Óssea/patologia , Leucemia Neutrofílica Crônica/patologia , Leucocitose/etiologia , Receptores de Fator Estimulador de Colônias/genética , Idoso de 80 Anos ou mais , Células Sanguíneas/patologia , Diagnóstico Diferencial , Fadiga/etiologia , Feminino , Humanos , Leucemia Neutrofílica Crônica/complicações , Leucemia Neutrofílica Crônica/genética , Mutação , Neutrófilos , Esplenomegalia/etiologia , SudoreseAssuntos
Células Clonais/transplante , Doenças Hematológicas/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Células-Tronco Hematopoéticas/patologia , Adolescente , Adulto , Idoso , Aloenxertos , Feminino , Marcadores Genéticos , Sobrevivência de Enxerto , Neoplasias Hematológicas/etiologia , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/terapia , Humanos , Leucopenia , Masculino , Pessoa de Meia-Idade , Mutação , Análise de Sequência de DNA , Doadores de Tecidos , Adulto JovemRESUMO
BACKGROUND: Obesity results from an imbalance between energy intake and expenditure. In rodents and newborn humans, brown adipose tissue helps regulate energy expenditure by thermogenesis mediated by the expression of uncoupling protein 1 (UCP1), but brown adipose tissue has been considered to have no physiologic relevance in adult humans. METHODS: We analyzed 3640 consecutive (18)F-fluorodeoxyglucose ((18)F-FDG) positron-emission tomographic and computed tomographic (PET-CT) scans performed for various diagnostic reasons in 1972 patients for the presence of substantial depots of putative brown adipose tissue. Such depots were defined as collections of tissue that were more than 4 mm in diameter, had the density of adipose tissue according to CT, and had maximal standardized uptake values of (18)F-FDG of at least 2.0 g per milliliter, indicating high metabolic activity. Clinical indexes were recorded and compared with those of date-matched controls. Immunostaining for UCP1 was performed on biopsy specimens from the neck and supraclavicular regions in patients undergoing surgery. RESULTS: Substantial depots of brown adipose tissue were identified by PET-CT in a region extending from the anterior neck to the thorax. Tissue from this region had UCP1-immunopositive, multilocular adipocytes indicating brown adipose tissue. Positive scans were seen in 76 of 1013 women (7.5%) and 30 of 959 men (3.1%), corresponding to a female:male ratio greater than 2:1 (P<0.001). Women also had a greater mass of brown adipose tissue and higher (18)F-FDG uptake activity. The probability of the detection of brown adipose tissue was inversely correlated with years of age (P<0.001), outdoor temperature at the time of the scan (P=0.02), beta-blocker use (P<0.001), and among older patients, body-mass index (P=0.007). CONCLUSIONS: Defined regions of functionally active brown adipose tissue are present in adult humans, are more frequent in women than in men, and may be quantified noninvasively with the use of (18)F-FDG PET-CT. Most important, the amount of brown adipose tissue is inversely correlated with body-mass index, especially in older people, suggesting a potential role of brown adipose tissue in adult human metabolism.
Assuntos
Tecido Adiposo Marrom , Índice de Massa Corporal , Metabolismo Energético , Adipócitos Marrons , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/diagnóstico por imagem , Tecido Adiposo Marrom/metabolismo , Adiposidade , Antagonistas Adrenérgicos beta/uso terapêutico , Adulto , Fatores Etários , Idoso , Glicemia/análise , Feminino , Fluordesoxiglucose F18/farmacocinética , Humanos , Imuno-Histoquímica , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Pescoço , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/farmacocinética , Caracteres Sexuais , Estatísticas não Paramétricas , Temperatura , Adulto JovemRESUMO
Langerhans cell histiocytosis (LCH) has a broad spectrum of clinical behaviors; some cases are self-limited, whereas others involve multiple organs and cause significant mortality. Although Langerhans cells in LCH are clonal, their benign morphology and their lack (to date) of reported recurrent genomic abnormalities have suggested that LCH may not be a neoplasm. Here, using 2 orthogonal technologies for detecting cancer-associated mutations in formalin-fixed, paraffin-embedded material, we identified the oncogenic BRAF V600E mutation in 35 of 61 archived specimens (57%). TP53 and MET mutations were also observed in one sample each. BRAF V600E tended to appear in younger patients but was not associated with disease site or stage. Langerhans cells stained for phospho-mitogen-activated protein kinase kinase (phospho-MEK) and phospho-extracellular signal-regulated kinase (phospho-ERK) regardless of mutation status. High prevalence, recurrent BRAF mutations in LCH indicate that it is a neoplastic disease that may respond to RAF pathway inhibitors.
Assuntos
Predisposição Genética para Doença , Histiocitose de Células de Langerhans/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Adolescente , Adulto , Substituição de Aminoácidos , Antígenos CD1/metabolismo , Criança , Pré-Escolar , Análise Mutacional de DNA , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Imunofluorescência , Genótipo , Histiocitose de Células de Langerhans/metabolismo , Histiocitose de Células de Langerhans/patologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Adulto JovemRESUMO
Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy, with varying prognosis after the gold standard rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Several prognostic models have been established by focusing primarily on characteristics of lymphoma cells themselves, including cell-of-origin (COO), genomic alterations, and gene/protein expressions. However, the prognostic impact of the lymphoma microenvironment and its association with characteristics of lymphoma cells are not fully understood. Using the nCounter-based gene expression profiling of untreated DLBCL tissues, we assess the clinical impact of lymphoma microenvironment on the clinical outcomes and pathophysiological, molecular signatures in DLBCL. The presence of normal germinal center (GC)-microenvironmental cells, including follicular T cells, macrophage/dendritic cells, and stromal cells in lymphoma tissue indicates a positive therapeutic response. Our prognostic model, based on quantitation of transcripts from distinct GC-microenvironmental cell markers, clearly identified patients with graded prognosis independently of existing prognostic models. We observed increased incidences of genomic alterations and aberrant gene expression associated with poor prognosis in DLBCL tissues lacking GC-microenvironmental cells relative to those containing these cells. These data suggest that the loss of GC-associated microenvironmental signature dictates clinical outcomes of DLBCL patients reflecting the accumulation of "unfavorable" molecular signatures.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Linfoma Difuso de Grandes Células B , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Centro Germinativo/metabolismo , Humanos , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Fenótipo , Prednisona/uso terapêutico , Rituximab/uso terapêutico , Microambiente Tumoral , Vincristina/uso terapêuticoRESUMO
OBJECTIVES: Potentially targetable genomic alterations have been identified in lung squamous cell carcinoma (LUSC), but none have yet translated into effective therapy. We examined potential benefits of next generation sequencing (NGS) in a cohort of consecutive LUSC patients with emphasis on distinctions between smokers and light/never smokers and implications for clinical trial enrollment. METHODS: We retrospectively evaluated results from an internally developed NGS assay (OncoPanel) targeting â¼300 genes with a mean overall target coverage of >200x for consecutive LUSC seen at our institution over 30 months. RESULTS: Tissue was obtained from 172 patients for targeted NGS. 42 (24 %) samples were insufficient for testing. Median age of tested patients was 66, including 87 % moderate/heavy versus 13 % light/never smokers; 66 % were stage IIIB or IV. Of 130 patients with evaluable NGS results, 49 (38 %) had at least 1 alteration qualifying for enrollment to a LungMAP treatment arm (PIK3CA, MET, FGFR family, cell cycle, or homologous recombination pathways) or for an approved therapy or other clinical trial (e.g. EGFR sensitizing mutations, MET exon 14 splice mutations, TSC1/2 mutation, or microsatellite instability). Therapeutic targets were enriched in light/never smokers (47 % vs 35 % moderate/heavy smokers). Unexpectedly, genomic features suggested an alternative diagnosis (metastatic cutaneous squamous carcinoma; mesothelioma) in 7 patients, including 35 % of never/light smokers. CONCLUSION: NGS in a real-world LUSC cohort yields potentially targetable genomic alterations informing clinical trial enrollment and approved therapies and critical diagnostic insights. Our findings strongly support current guidelines recommending mutational profiling of LUSC arising in light/never smoking patients; the utility of sequencing in smokers with LUSC appears to be limited to identification of research targets.
Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Pulmonares/diagnóstico , Mutação , Adenocarcinoma de Pulmão/genética , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
BACKGROUND: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin caused by either the integration of Merkel cell polyomavirus (MCPyV) and expression of viral T antigens or by ultraviolet-induced damage to the tumor genome from excessive sunlight exposure. An increasing number of deep sequencing studies of MCC have identified significant differences between the number and types of point mutations, copy number alterations, and structural variants between virus-positive and virus-negative tumors. However, it has been challenging to reliably distinguish between virus positive and UV damaged MCC. METHODS: In this study, we assembled a cohort of 71 MCC patients and performed deep sequencing with OncoPanel, a clinically implemented, next-generation sequencing assay targeting over 400 cancer-associated genes. To improve the accuracy and sensitivity for virus detection compared to traditional PCR and IHC methods, we developed a hybrid capture baitset against the entire MCPyV genome and software to detect integration sites and structure. RESULTS: Sequencing from this approach revealed distinct integration junctions in the tumor genome and generated assemblies that strongly support a model of microhomology-initiated hybrid, virus-host, circular DNA intermediate that promotes focal amplification of host and viral DNA. Using the clear delineation between virus-positive and virus-negative tumors from this method, we identified recurrent somatic alterations common across MCC and alterations specific to each class of tumor, associated with differences in overall survival. Finally, comparing the molecular and clinical data from these patients revealed a surprising association of immunosuppression with virus-negative MCC and significantly shortened overall survival. CONCLUSIONS: These results demonstrate the value of high-confidence virus detection for identifying molecular mechanisms of UV and viral oncogenesis in MCC. Furthermore, integrating these data with clinical data revealed features that could impact patient outcome and improve our understanding of MCC risk factors.
Assuntos
Carcinoma de Célula de Merkel/genética , Mutação , Infecções por Polyomavirus/genética , Neoplasias Cutâneas/genética , Infecções Tumorais por Vírus/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Célula de Merkel/patologia , Carcinoma de Célula de Merkel/virologia , Criança , DNA de Neoplasias/genética , DNA Viral/genética , Feminino , Testes Genéticos/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Polyomavirus/genética , Polyomavirus/patogenicidade , Infecções por Polyomavirus/patologia , Infecções por Polyomavirus/virologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Análise de Sobrevida , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/virologiaRESUMO
BACKGROUND: Increased aurora A kinase (AAK) expression occurs in acute myeloid leukaemia; AAK inhibition is a promising therapeutic target in this disease. We therefore aimed to assess the activity of alisertib combined with 7â+â3 induction chemotherapy in previously untreated patients with high-risk acute myeloid leukaemia. METHODS: We did a single-arm, phase 2 trial of patients recruited from the Dana-Farber/Harvard Cancer Center in the USA. Eligible patients had previously untreated acute myeloid leukaemia, an Eastern Cooperative Oncology Group performance status of 0-2, and were at high risk of disease as defined by the presence of an adverse-risk karyotype, the presence of secondary acute myeloid leukaemia arising from previous myelodysplastic syndrome or myeloproliferative neoplasm, the presence of therapy-related acute myeloid leukaemia, or being 65 years or older. Enrolled patients received 7â+â3 induction chemotherapy of continuous infusion of cytarabine (100 mg/m2 per day on days 1-7) and intravenous bolus of idarubicin (12 mg/m2 per day on days 1-3). Oral alisertib (30 mg) was given twice per day on days 8-15. Patients could receive up to four consolidation cycles with cytarabine and alisertib, and alisertib maintenance for 12 months. The primary endpoint was a composite including the proportion of patients achieving complete remission and those with a complete remission with incomplete neutrophil or platelet count recovery. Analyses were per-protocol. This study is registered with Clinicaltrials.gov, number NCT02560025, and has completed enrolment. FINDINGS: Between Dec 31, 2015, and Aug 1, 2017, we enrolled a total of 39 eligible patients. 19 (49%) of 39 patients had secondary acute myeloid leukaemia and three (8%) had therapy-related acute myeloid leukaemia. At mid-induction, 33 (85%) of 39 patients showed marrow aplasia, six (15%) received re-induction. The median follow-up was 13·7 months (IQR 12·7-14·4). Composite remission was 64% (two-stage 95% CI 48-79), with 20 (51%) of 39 patients achieving complete remission and five (13%) achieving complete remission with incomplete neutrophil or platelet count recovery. The most common grade 3 or 4 adverse events included febrile neutropenia (16 [41%] of 39), neutropenia (12 [31%]), thrombocytopenia (13 [33%]), anaemia (11 [28%]), anorexia (nine [23%]), and oral mucositis (four [10%]). No treatment-related deaths were observed. INTERPRETATION: These results suggest that alisertib combined with induction chemotherapy is active and safe in previously untreated patients with high-risk acute myeloid leukaemia. This study met criteria to move forward to a future randomised trial. FUNDING: Millennium Pharmaceuticals.
Assuntos
Azepinas/administração & dosagem , Quimioterapia de Indução , Leucemia Mieloide Aguda/tratamento farmacológico , Pirimidinas/administração & dosagem , Idoso , Azepinas/efeitos adversos , Citarabina/administração & dosagem , Citarabina/efeitos adversos , Feminino , Seguimentos , Humanos , Idarubicina/administração & dosagem , Idarubicina/efeitos adversos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Pirimidinas/efeitos adversos , Fatores de RiscoRESUMO
Mast cells participate in pathophysiological processes that range from antimicrobial defense to anaphylaxis and inflammatory arthritis. Much of the groundwork for the understanding of mast cells was established in mice that lacked mast cells through defects in either stem cell factor or its receptor, Kit. Among available strains, C57BL/6-Kit(W-sh) (W(sh)) mice are experimentally advantageous because of their background strain and fertility. However, the genetic inversion responsible for the W(sh) phenotype remains poorly defined, and its effects beyond the mast cell have been incompletely characterized. We report that W(sh) animals exhibit splenomegaly with expanded myeloid and megakaryocyte populations. Hematopoietic abnormalities extend to the bone marrow and are reflected by neutrophilia and thrombocytosis. In contrast, mast cell-deficient WBB6F1-Kit(W)/Kit(W-v) (W/W(v)) mice display mild neutropenia, but no changes in circulating platelet numbers. To help define the basis for the W(sh) phenotype, a "DNA walking" strategy was used to identify the precise location of the 3' breakpoint, which was found to reside 67.5 kb upstream of Kit. The 5' breakpoint disrupts corin, a cardiac protease responsible for the activation of atrial natriuretic peptide. Consistent with this result, transcription of full-length corin is ablated and W(sh) mice develop symptoms of cardiomegaly. Studies performed using mast cell-deficient strains must consider the capacity of associated abnormalities to either expose or compensate for the missing mast cell lineage.
Assuntos
Inversão Cromossômica , Cardiopatias Congênitas/genética , Coração/fisiologia , Sistema Hematopoético , Mastócitos/fisiologia , Serina Endopeptidases/metabolismo , Animais , Sistema Hematopoético/anormalidades , Sistema Hematopoético/imunologia , Masculino , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Serina Endopeptidases/genética , Baço/anormalidades , Baço/citologia , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
Large scale sequencing projects over the past 2 decades have led to the identification of many common genomic alterations in hematolymphoid neoplasms, some of which with diagnostic, therapeutic, and prognostic implications [1-3]. Although these alterations can be tested individually with high sensitivity and specificity using dedicated single gene tests, it is increasingly impractical and costly to test them separately as the number of alterations grows. Instead, multiplex testing platforms that can test multiple targets in a specimen have been developed. Among these platforms, massively parallel sequencing technologies (so-called next-generation sequencing [NGS] [4]) prove to be most versatile and is increasingly being used to build tests to meet the clinical testing need. In hematolymphoid neoplasms, the early incorporation of molecular findings into the diagnostic criteria by WHO [5] has further accelerated the adoption of NGS-based tests in routine clinical practice. This article focuses on what is used in the clinical diagnostic laboratories today and is not intended to be a review of the NGS technology or its future direction. Following discussion of the 2 families of sequencing instruments from Illumina and Thermo Fisher and 3 target enrichment methods, aspects of the analysis and report of NGS results that is clinically relevant are discussed.
Assuntos
Testes Genéticos/métodos , Genômica/métodos , Neoplasias Hematológicas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Hematológicas/patologia , Humanos , PrognósticoRESUMO
OBJECTIVES: The 2017 Workshop of the Society for Hematopathology/European Association for Hematopathology aimed to review premalignant clonal hematopoietic proliferations. METHODS: The workshop panel reviewed 27 cases of clonal proliferations of indeterminate significance or potential (18 myeloid, nine lymphoid) and rendered consensus diagnoses. RESULTS: Immunophenotyping and genetic studies on peripheral blood, bone marrow, and lymph node samples have led to the incidental detection of small clonal populations in asymptomatic individuals. These premalignant clonal myeloid and lymphoid proliferations include monoclonal gammopathy of uncertain significance, monoclonal B-cell lymphocytosis, in situ follicular neoplasia, in situ mantle cell neoplasia, clonal hematopoiesis of indeterminate potential, and clonal cytopenia of undetermined significance. CONCLUSIONS: Current diagnostic criteria for the diagnoses of premalignant clonal hematopoietic proliferations are reviewed and discussed in the context of the cases presented at the workshop.
Assuntos
Neoplasias Hematológicas/genética , Gamopatia Monoclonal de Significância Indeterminada/genética , Síndromes Mielodisplásicas/genética , Lesões Pré-Cancerosas/genética , Neoplasias Hematológicas/patologia , Humanos , Gamopatia Monoclonal de Significância Indeterminada/patologia , Síndromes Mielodisplásicas/patologia , Lesões Pré-Cancerosas/patologiaRESUMO
Programmed cell death protein 1 (PD-1) inhibitors have efficacy in treating squamous cell carcinoma of the head and neck (SCCHN), but objective response rates are low. PD-1 ligand (PD-L1) expression alone is not considered a robust predictor of response and additional biomarkers are needed. This 3-year observational cohort followed 126 SCCHN patients treated with anti-PD-1/L1 therapy. Prior to treatment, 81 (64%) had targeted massively parallel tumor sequencing. Of these, 42 (52%) underwent fluorescence-activated cell sorting and PD-L1 immunohistochemistry for tumor immunoprofiling. Six (5%) complete responses (CRs) and 11 (9%) partial responses (PRs) were observed. Those treated with prior chemotherapy (98, 78%) versus only surgery and/or radiation had longer overall survival (OS) (10 vs. 3 months, P = 0.02). Smokers had a higher total mutational burden (TMB) (P = 0.01). Virus-positive patients had a lower TMB (P < 0.01) and improved OS (P = 0.02). Among virus-negative responders, NOTCH1 and SMARCA4 were more frequently mutated and frameshift events in tumor suppressor genes occurred more frequently (P = 0.03). Higher TMB and CD8+ T cell infiltrates predicted anti-PD-1/L1 benefit (P < 0.01, P < 0.01, respectively) among virus-negative tumors. TIM-3/LAG-3 coexpression with PD-1 was higher on T cells among nonresponders (P = 0.03 and 0.02, respectively). Somatic frameshift events in tumor suppressor genes and higher TMB among virus-negative SCCHN tumors predict anti-PD-1/L1 response.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Genes Supressores de Tumor , Neoplasias de Cabeça e Pescoço/terapia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/genética , Biópsia , Análise Mutacional de DNA , Feminino , Mutação da Fase de Leitura , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Terapia Neoadjuvante/métodos , Procedimentos Cirúrgicos Otorrinolaringológicos , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Estudos Prospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Análise de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
T-cell receptor gamma (TRG) gene rearrangement status is useful for the differential diagnosis of T-cell lesions. The BIOMED-2 protocol that uses two sets of Jgamma and four sets of Vgamma primers in a multiplex, two-tube reaction followed by capillary gel electrophoresis is emerging as a standard assay for this application. Here, we report a computer-aided method to evaluate the significance of a peak in this TRG clonality assay. A best-fit normal distribution (ND) curve and the chi(2) error for each peak are used to determine whether a peak is significantly taller than the background (cutoff for Vgamma(1-8) is 1). Eighty clinical samples that have been previously analyzed by a GC-clamped primer polymerase chain reaction/denaturing gradient gel electrophoresis assay were reanalyzed with the BIOMED-2 assay and scored by the ND method and four previously published methods: relative peak height (RPH), relative peak ratio (RPR), height ratio (HR), and peak height ratio (Rn). A greater than 90% concordance rate was observed between RPH and ND analysis, whereas RPR, Rn, and HR had a lower threshold to call a peak positive. The advantage of the ND method is that it is more objective, reproducible, and can be automated.
Assuntos
Eletroforese Capilar/métodos , Rearranjo Gênico/genética , Genes Codificadores da Cadeia gama de Receptores de Linfócitos T/genética , Linfoma de Células T/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Algoritmos , Southern Blotting , Estudos de Avaliação como Assunto , HumanosRESUMO
OBJECTIVES: To determine whether V H family usage in B-cell lymphoproliferative disorders can be deduced from polymerase chain reaction (PCR) product-length information obtained through the BIOMED-2 (Invivoscribe, San Diego, CA) clonality assay. METHODS: We develop an algorithm that uses the sizing information of the BIOMED-2 immunoglobulin heavy chain (IGH) clonality assay to deduce V H family usage. PCR with family-specific primers on 51 clinical samples containing 54 rearranged alleles were used to validate the algorithm. RESULTS: The clonal PCR products in different framework reactions contain the same NDN segment (because they are from the same allele). Subtracting the size of the framework III product from the size of the framework I and II products yields the relative position of the framework primer binding sites for the V H segment used. The V H family can be assigned with these relative positions because they are V H family specific in the BIOMED-2 assay. The V H family assigned by the algorithm was concordant with family-specific PCR results for 49 (96%) of the 51 specimens. CONCLUSIONS: We have developed an algorithm that can correctly assign V H family usage when all three BIOMED-2 framework reactions produced clonal products. Given the wide adoption of BIOMED-2 assay, the algorithm can facilitate collection of IGH V H usage data without additional cost to the laboratories.
Assuntos
Rearranjo Gênico de Cadeia Pesada de Linfócito B , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma de Células B/genética , Transtornos Linfoproliferativos/genética , Reação em Cadeia da Polimerase/métodos , Algoritmos , Alelos , Linfócitos B/imunologia , Células Clonais , Primers do DNA/genética , Humanos , Linfoma de Células B/diagnóstico , Linfoma de Células B/imunologia , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/imunologia , Técnicas de Diagnóstico Molecular , Sensibilidade e EspecificidadeRESUMO
The emergence of precision medicine has been predicated on significant recent advances in diagnostic technology, particularly the advent of next-generation sequencing (NGS). Although the chemical technology underlying NGS is complex, and the computational biology expertise required to build systems to facilely interpret the results is highly specialized, the variables involved in designing and deploying a genomic testing program for cancer can be readily understood and applied by understanding several basic considerations. In this review, we present key strategic decisions required to optimize a genomic testing program and summarize the technical aspects of different technologies that render those methods more or less suitable for different types of programs.