RESUMEN
The value of genome-wide over targeted driver analyses for predicting clinical outcomes of cancer patients is debated. Here, we report the whole-genome sequencing of 485 chronic lymphocytic leukemia patients enrolled in clinical trials as part of the United Kingdom's 100,000 Genomes Project. We identify an extended catalog of recurrent coding and noncoding genetic mutations that represents a source for future studies and provide the most complete high-resolution map of structural variants, copy number changes and global genome features including telomere length, mutational signatures and genomic complexity. We demonstrate the relationship of these features with clinical outcome and show that integration of 186 distinct recurrent genomic alterations defines five genomic subgroups that associate with response to therapy, refining conventional outcome prediction. While requiring independent validation, our findings highlight the potential of whole-genome sequencing to inform future risk stratification in chronic lymphocytic leukemia.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Secuenciación Completa del Genoma , Mutación , Genómica , PronósticoAsunto(s)
Leucemia Linfocítica Crónica de Células B , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ciclofosfamida , Estudios de Seguimiento , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Pronóstico , Rituximab , Resultado del Tratamiento , Vidarabina/análogos & derivadosRESUMEN
Prognostication in patients with chronic lymphocytic leukemia (CLL) is challenging due to heterogeneity in clinical course. We hypothesize that constitutional genetic variation affects disease progression and could aid prognostication. Pooling data from seven studies incorporating 842 cases identifies two genomic locations associated with time from diagnosis to treatment, including 10q26.13 (rs736456, hazard ratio (HR) = 1.78, 95% confidence interval (CI) = 1.47-2.15; P = 2.71 × 10-9) and 6p (rs3778076, HR = 1.99, 95% CI = 1.55-2.55; P = 5.08 × 10-8), which are particularly powerful prognostic markers in patients with early stage CLL otherwise characterized by low-risk features. Expression quantitative trait loci analysis identifies putative functional genes implicated in modulating B-cell receptor or innate immune responses, key pathways in CLL pathogenesis. In this work we identify rs736456 and rs3778076 as prognostic in CLL, demonstrating that disease progression is determined by constitutional genetic variation as well as known somatic drivers.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo/métodos , Leucemia Linfocítica Crónica de Células B/genética , Polimorfismo de Nucleótido Simple , Anciano , Progresión de la Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , Sitios de Carácter Cuantitativo/genéticaRESUMEN
The transformation of chronic lymphocytic leukemia (CLL) to high-grade B-cell lymphoma is known as Richter syndrome (RS), a rare event with dismal prognosis. In this study, we conducted whole-genome sequencing (WGS) of paired circulating CLL (PB-CLL) and RS biopsies (tissue-RS) from 17 patients recruited into a clinical trial (CHOP-O). We found that tissue-RS was enriched for mutations in poor-risk CLL drivers and genes in the DNA damage response (DDR) pathway. In addition, we identified genomic aberrations not previously implicated in RS, including the protein tyrosine phosphatase receptor (PTPRD) and tumor necrosis factor receptor-associated factor 3 (TRAF3). In the noncoding genome, we discovered activation-induced cytidine deaminase-related and unrelated kataegis in tissue-RS affecting regulatory regions of key immune-regulatory genes. These include BTG2, CXCR4, NFATC1, PAX5, NOTCH-1, SLC44A5, FCRL3, SELL, TNIP2, and TRIM13. Furthermore, differences between the global mutation signatures of pairs of PB-CLL and tissue-RS samples implicate DDR as the dominant mechanism driving transformation. Pathway-based clonal deconvolution analysis showed that genes in the MAPK and DDR pathways demonstrate high clonal-expansion probability. Direct comparison of nodal-CLL and tissue-RS pairs from an independent cohort confirmed differential expression of the same pathways by RNA expression profiling. Our integrated analysis of WGS and RNA expression data significantly extends previous targeted approaches, which were limited by the lack of germline samples, and it facilitates the identification of novel genomic correlates implicated in RS transformation, which could be targeted therapeutically. Our results inform the future selection of investigative agents for a UK clinical platform study. This trial was registered at www.clinicaltrials.gov as #NCT03899337.
Asunto(s)
Evolución Clonal/genética , Regulación Neoplásica de la Expresión Génica/genética , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células B Grandes Difuso/patología , ARN Neoplásico/genética , Transcriptoma , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Secuencia de Bases , Células Clonales/patología , Terapia Combinada , Ciclofosfamida/administración & dosificación , Reparación del ADN , Progresión de la Enfermedad , Doxorrubicina/administración & dosificación , Femenino , Redes Reguladoras de Genes , Genes Relacionados con las Neoplasias , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Masculino , Persona de Mediana Edad , Mutación , Proteínas de Neoplasias/genética , Prednisona/administración & dosificación , Estudios Prospectivos , ARN Neoplásico/biosíntesis , Síndrome , Vincristina/administración & dosificación , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artefacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same tumor. METHODS: Four variant callers (i.e. Strelka2, Mutect2, VarScan2 and Shimmer) were used to call somatic variants on matching FF and FFPE WGS samples from a metastatic prostate tumor. Using the variants identified by these callers, we developed a heuristic to maximize the overlap between the FF and its FFPE counterpart in terms of sensitivity and precision. The proposed variant calling approach was then validated on nine matched primary samples. Finally, we assessed what fraction of the discrepancy could be attributed to intra-tumor heterogeneity (ITH), by comparing the overlap in clonal and subclonal somatic variants. RESULTS: We first compared variants between an FF and an FFPE sample from a metastatic prostate tumor, showing that on average 50% of the calls in the FF are recovered in the FFPE sample, with notable differences between callers. Combining the variants of the different callers using a simple heuristic, increases both the precision and the sensitivity of the variant calling. Validating the heuristic on nine additional matched FF-FFPE samples, resulted in an average F1-score of 0.58 and an outperformance of any of the individual callers. In addition, we could show that part of the discrepancy between the FF and the FFPE samples can be attributed to ITH. CONCLUSION: This study illustrates that when using the correct variant calling strategy, the majority of clonal SNVs can be recovered in an FFPE sample with high precision and sensitivity. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studies.
Asunto(s)
Biomarcadores de Tumor/genética , ADN de Neoplasias/genética , Neoplasias Pulmonares/genética , Mutación , Recurrencia Local de Neoplasia/genética , Neoplasias de la Próstata/genética , Fijación del Tejido/métodos , ADN de Neoplasias/análisis , Estudios de Factibilidad , Formaldehído/química , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/secundario , Masculino , Recurrencia Local de Neoplasia/patología , Adhesión en Parafina/métodos , Pronóstico , Neoplasias de la Próstata/patología , Manejo de Especímenes , Secuenciación Completa del GenomaRESUMEN
Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore disease-related/genetic features contributing to long-term outcomes. In 499 LRF CLL4 cases, a trial with >12 years follow-up, we employed targeted resequencing of 22 genes, identifying 623 mutations. After background mutation rate correction, 11/22 genes were recurrently mutated at frequencies between 3.6% (NFKBIE) and 24% (SF3B1). Mutations beyond Sanger resolution (<12% VAF) were observed in all genes, with KRAS mutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm <12% VAF TP53 mutations, we assessed the clinical impact of TP53 clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between <12% VAF TP53 mutations and either wild type or ≥12% VAF TP53mut cases. Secondly, we identified biallelic BIRC3 lesions (mutation and deletion) as an independent marker of inferior PFS and OS. Finally, we observed that mutated MAPK-ERK genes were independent markers of poor OS in multivariate survival analysis. In conclusion, our study supports using targeted resequencing of expanded gene panels to elucidate the prognostic impact of gene mutations.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Biomarcadores de Tumor/genética , Leucemia Linfocítica Crónica de Células B/mortalidad , Sistema de Señalización de MAP Quinasas/genética , Mutación , Proteína p53 Supresora de Tumor/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Estudios de Cohortes , Ciclofosfamida/administración & dosificación , Quinasas MAP Reguladas por Señal Extracelular/genética , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Pronóstico , Tasa de Supervivencia , Vidarabina/administración & dosificación , Vidarabina/análogos & derivadosRESUMEN
Chronic lymphocytic leukemia patients with mutated immunoglobulin heavy-chain genes (IGHV-M), particularly those lacking poor-risk genomic lesions, often respond well to chemoimmunotherapy (CIT). DNA methylation profiling can subdivide early-stage patients into naive B-cell-like CLL (n-CLL), memory B-cell-like CLL (m-CLL), and intermediate CLL (i-CLL), with differing times to first treatment and overall survival. However, whether DNA methylation can identify patients destined to respond favorably to CIT has not been ascertained. We classified treatment-naive patients (n = 605) from 3 UK chemo and CIT clinical trials into the 3 epigenetic subgroups, using pyrosequencing and microarray analysis, and performed expansive survival analysis. The n-CLL, i-CLL, and m-CLL signatures were found in 80% (n = 245/305), 17% (53/305), and 2% (7/305) of IGHV-unmutated (IGHV-U) cases, respectively, and in 9%, (19/216), 50% (108/216), and 41% (89/216) of IGHV-M cases, respectively. Multivariate Cox proportional analysis identified m-CLL as an independent prognostic factor for overall survival (hazard ratio [HR], 0.46; 95% confidence interval [CI], 0.24-0.87; P = .018) in CLL4, and for progression-free survival (HR, 0.25; 95% CI, 0.10-0.57; P = .002) in ARCTIC and ADMIRE patients. The analysis of epigenetic subgroups in patients entered into 3 first-line UK CLL trials identifies m-CLL as an independent marker of prolonged survival and may aid in the identification of patients destined to demonstrate prolonged survival after CIT.
Asunto(s)
Metilación de ADN , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/genética , Adulto , Anciano , Anciano de 80 o más Años , Aberraciones Cromosómicas , Biología Computacional/métodos , Epigénesis Genética , Epigenómica/métodos , Femenino , Perfilación de la Expresión Génica , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/mortalidad , Leucemia Linfocítica Crónica de Células B/terapia , Masculino , Persona de Mediana Edad , Mutación , Estadificación de Neoplasias , Pronóstico , Modelos de Riesgos ProporcionalesRESUMEN
Purpose: Unmutated (UM) immunoglobulin heavy chain variable region (IgHV) status or IgHV3-21 gene usage is associated with poor prognosis in chronic lymphocytic leukemia (CLL) patients. Interestingly, IgHV3-21 is often co-expressed with light chain IgLV3-21, which is potentially able to trigger cell-autonomous BCR-mediated signaling. However, this light chain has never been characterized independently of the heavy chain IgHV3-21.Experimental Design: We performed total RNA sequencing in 32 patients and investigated IgLV3-21 prognostic impact in terms of treatment-free survival (TFS) and overall survival (OS) in 3 other independent cohorts for a total of 813 patients. IgLV3-21 presence was tested by real-time PCR and confirmed by Sanger sequencing.Results: Using total RNA sequencing to characterize 32 patients with high-risk CLL, we found a high frequency (28%) of IgLV3-21 rearrangements. Gene set enrichment analysis revealed that these patients express higher levels of genes responsible for ribosome biogenesis and translation initiation (P < 0.0001) as well as MYC target genes (P = 0.0003). Patients with IgLV3-21 rearrangements displayed a significantly shorter TFS and OS (P < 0.05), particularly those with IgHV mutation. In each of the three independent validation cohorts, we showed that IgLV3-21 rearrangements-similar to UM IgHV status-conferred poor prognosis compared with mutated IgHV (P < 0.0001). Importantly, we confirmed by multivariate analysis that this was independent of IgHV mutational status or subset #2 stereotyped receptor (P < 0.0001).Conclusions: We have demonstrated for the first time that a light chain can affect CLL prognosis and that IgLV3-21 light chain usage defines a new subgroup of CLL patients with poor prognosis. Clin Cancer Res; 24(20); 5048-57. ©2018 AACR.
Asunto(s)
Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/mortalidad , Péptidos/genética , Biomarcadores de Tumor , Aberraciones Cromosómicas , Ensayos Clínicos como Asunto , Biología Computacional/métodos , Femenino , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Ontología de Genes , Humanos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Masculino , Mutación , Pronóstico , Análisis de Secuencia de ADN , TranscriptomaRESUMEN
The 100 000 Genome Project aims to develop a diagnostics platform by introducing whole genome sequencing (WGS) into clinical practice. Samples from patients with chronic lymphocytic leukaemia were subjected to WGS. WGS detection of single nucleotide variants and insertion/deletions were validated by targeted next generation sequencing showing high concordance (96·3%), also for detection of sub-clonal variants and low-frequency TP53 variants. Copy number alteration detection was verified by fluorescent in situ hybridisation and genome-wide single nucleotide polymorphism array (concordances of 86·7% and 92·9%, respectively), confirming adequate sensitivity by WGS. Our results confirm that WGS can provide comprehensive genomic characterisation for clinical trials, drug discovery and, ultimately, precision medicine.
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Leucemia Linfocítica Crónica de Células B/genética , Secuenciación Completa del Genoma/normas , Adulto , Anciano , Variaciones en el Número de Copia de ADN/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación INDEL/genética , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
PURPOSE: Fresh-frozen (FF) tissue is the optimal source of DNA for whole-genome sequencing (WGS) of cancer patients. However, it is not always available, limiting the widespread application of WGS in clinical practice. We explored the viability of using formalin-fixed, paraffin-embedded (FFPE) tissues, available routinely for cancer patients, as a source of DNA for clinical WGS. METHODS: We conducted a prospective study using DNAs from matched FF, FFPE, and peripheral blood germ-line specimens collected from 52 cancer patients (156 samples) following routine diagnostic protocols. We compared somatic variants detected in FFPE and matching FF samples. RESULTS: We found the single-nucleotide variant agreement reached 71% across the genome and somatic copy-number alterations (CNAs) detection from FFPE samples was suboptimal (0.44 median correlation with FF) due to nonuniform coverage. CNA detection was improved significantly with lower reverse crosslinking temperature in FFPE DNA extraction (80 °C or 65 °C depending on the methods). Our final data showed somatic variant detection from FFPE for clinical decision making is possible. We detected 98% of clinically actionable variants (including 30/31 CNAs). CONCLUSION: We present the first prospective WGS study of cancer patients using FFPE specimens collected in a routine clinical environment proving WGS can be applied in the clinic.
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Variaciones en el Número de Copia de ADN/genética , Genoma Humano/genética , Neoplasias/genética , Secuenciación Completa del Genoma/métodos , Toma de Decisiones , Femenino , Humanos , Masculino , Neoplasias/sangre , Neoplasias/patología , Adhesión en Parafina , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
BACKGROUND: Genomic tests may improve the stratification of patients to receive new therapies in several disease areas. However, the use of expensive targeted therapies can impact on the cost effectiveness of these tests. This study presents an economic evaluation of genomic testing in chronic lymphocytic leukaemia in the context of the UK National Health Service. METHODS: Cost-effectiveness and cost-utility analyses (using life-years and quality-adjusted life-years) were undertaken from a National Health Service and societal perspective. Five strategies were evaluated across several age groups using Markov modelling: three strategies that reflected varying current genetic testing practice and two configurations of genomic testing (including ibrutinib treatment). RESULTS: Genomic testing strategies yielded the most life-years/quality-adjusted life-years per patient, but were not cost effective compared with a threshold of £30,000 per life-year/quality-adjusted life-year gained. Cost-effectiveness acceptability curves indicated that there was some uncertainty surrounding this result. A genomic testing strategy becomes the most cost-effective option if a higher end-of-life cost-effectiveness threshold of £50,000 is applied, if a societal costing perspective is considered in 25-year-old patients or if the cost of ibrutinib treatment falls. CONCLUSION: Stratifying patients with chronic lymphocytic leukaemia to targeted treatment using genomic testing improves health outcomes, but will likely only represent a cost-effective use of limited National Health Service resources if a higher cost-effectiveness threshold or societal costing perspective is applied, or if the price of ibrutinib treatment is reduced. This result may be broadly indicative of the likely cost effectiveness of other genomic tests that inform the stratification of patients to high cost-targeted therapies.
Asunto(s)
Antineoplásicos/uso terapéutico , Genómica/métodos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Adenina/análogos & derivados , Adulto , Antineoplásicos/economía , Toma de Decisiones Clínicas , Análisis Costo-Beneficio , Humanos , Leucemia Linfocítica Crónica de Células B/economía , Leucemia Linfocítica Crónica de Células B/genética , Cadenas de Markov , Piperidinas , Pirazoles/economía , Pirimidinas/economía , Años de Vida Ajustados por Calidad de Vida , Incertidumbre , Reino UnidoRESUMEN
Although TP53, NOTCH1, and SF3B1 mutations may impair prognosis of patients with chronic lymphocytic leukemia (CLL) receiving frontline therapy, the impact of these mutations or any other, alone or in combination, remains unclear at relapse. The genome of 114 relapsed/refractory patients included in prospective trials was screened using targeted next-generation sequencing of the TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3, and MYD88 genes. We performed clustering according to both number and combinations of recurrent gene mutations. The number of genes affected by mutation was ≥ 2, 1, and 0 in 43 (38%), 49 (43%), and 22 (19%) respectively. Recurrent combinations of ≥ 2 mutations of TP53, SF3B1, and ATM were found in 22 (19%) patients. This multiple-hit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P = .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcome.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/terapia , Mutación , Recurrencia Local de Neoplasia/genética , Terapia Recuperativa/métodos , Proteínas de la Ataxia Telangiectasia Mutada/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Leucemia Linfocítica Crónica de Células B/diagnóstico , Recurrencia Local de Neoplasia/diagnóstico , Fosfoproteínas/genética , Pronóstico , Estudios Prospectivos , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U2/genética , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/genéticaRESUMEN
SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development.