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The sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) has been demonstrated to predict the response to high-dose cytarabine consolidation treatment in acute myeloid leukemia patients. Here, we evaluated SAMHD1 as potential biomarker for the response to high-dose cytarabine in mantle cell lymphoma (MCL) patients. We quantified SAMHD1 protein expression and determined the mutation status in patients of the MCL Younger and Elderly trials (n = 189), who had received high-dose cytarabine- or fludarabine-based polychemotherapy. Additionally, we quantified SAMHD1 expression in B cell lymphoma cell lines and exposed them to cytarabine, fludarabine, and clinically relevant combinations. Across both trials investigated, SAMHD1 mutations had a frequency of 7.1% (n = 13) and did not significantly affect the failure-free survival (FFS, P = .47). In patients treated with high-dose cytarabine- or fludarabine-containing regimes, SAMHD1 expression was not significantly associated with FFS or complete remission rate. SAMHD1 expression in B cell lymphoma cell lines, however, inversely correlated with their in vitro response to cytarabine as single agent (R = .65, P = .0065). This correlation could be reversed by combining cytarabine with other chemotherapeutics, such as oxaliplatin and vincristine, similar to the treatment regime of the MCL Younger trial. We conclude that this might explain why we did not observe a significant association between SAMHD1 protein expression and the outcome of MCL patients upon cytarabine-based treatment.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Biomarcadores de Tumor/genética , Resistencia a Antineoplásicos/genética , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células del Manto/tratamiento farmacológico , Proteína 1 que Contiene Dominios SAM y HD/genética , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Ciclofosfamida/farmacología , Ciclofosfamida/uso terapéutico , Citarabina/farmacología , Citarabina/uso terapéutico , Análisis Mutacional de ADN , Supervivencia sin Enfermedad , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Linfoma de Células B/genética , Linfoma de Células B/patología , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/mortalidad , Masculino , Persona de Mediana Edad , Mutación , Oxaliplatino/farmacología , Oxaliplatino/uso terapéutico , Prednisona/farmacología , Prednisona/uso terapéutico , Cultivo Primario de Células , Rituximab/farmacología , Rituximab/uso terapéutico , Análisis de Matrices Tisulares , Vidarabina/análogos & derivados , Vidarabina/farmacología , Vidarabina/uso terapéutico , Vincristina/farmacología , Vincristina/uso terapéuticoRESUMEN
Ovarian cancer patients with germline or somatic pathogenic variants benefit from treatment with poly ADP ribose polymerase (PARP) inhibitors. Tumor BRCA1/2 testing is more challenging than germline testing as the majority of samples are formalin-fixed paraffin embedded (FFPE), the tumor genome is complex, and the allelic fraction of somatic variants can be low. We collaborated with 10 laboratories testing BRCA1/2 in tumors to compare different approaches to identify clinically important variants within FFPE tumor DNA samples. This was not a proficiency study but an inter-laboratory comparison to identify common issues. Each laboratory received the same tumor DNA samples ranging in genotype, quantity, quality, and variant allele frequency (VAF). Each laboratory performed their preferred next-generation sequencing method to report on the variants. No false positive results were reported in this small study and the majority of methods detected the low VAF variants. A number of variants were not detected due to the bioinformatics analysis, variant classification, or insufficient DNA. The use of hybridization capture or short amplicon methods are recommended based on a bioinformatic assessment of the data. The study highlights the importance of establishing standards and standardization for tBRCA testing particularly when the test results dictate clinical decisions regarding life extending therapies.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Pruebas Genéticas/métodos , Neoplasias/genética , Pautas de la Práctica en Medicina , Biología Computacional , Variaciones en el Número de Copia de ADN/genética , Exones/genética , Frecuencia de los Genes/genética , Genotipo , HumanosRESUMEN
BACKGROUND: Following chemo-radiotherapy (CCRT) for human papilloma virus positive (HPV+) locally advanced head and neck cancer, patients frequently undergo unnecessary neck dissection (ND) and/or repeated biopsies for abnormal PET-CT, which causes significant morbidity. We assessed the role of circulating HPV DNA in identifying 'true' residual disease. METHODS: We prospectively recruited test (n=55) and validation (n=33) cohorts. HPV status was confirmed by E7 RT-PCR. We developed a novel amplicon-based next generation sequencing assay (HPV16-detect) to detect circulating HPV DNA. Circulating HPV DNA levels post-CCRT were correlated to disease response (PET-CT). RESULTS: In pre-CCRT plasma, HPV-detect demonstrated 100% sensitivity and 93% specificity, and 90% sensitivity and 100% specificity for the test (27 HPV+) and validation (20 HPV+) cohorts, respectively. Thirty-six out of 37 patients (test and validation cohort) with complete samples-set had negative HPV-detect at end of treatment. Six patients underwent ND (3) and repeat primary site biopsies (3) for positive PET-CT but had no viable tumour. One patient had positive HPV-detect and positive PET-CT and liver biopsy, indicating 100% agreement for HPV-detect and residual cancer. CONCLUSIONS: We demonstrate that HPV16-detect is a highly sensitive and specific test for identification of HPV DNA in plasma at diagnosis. HPV DNA post-treatment correlates with clinical response.
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Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/terapia , Quimioradioterapia , ADN Viral/sangre , Neoplasias de Cabeza y Cuello/sangre , Neoplasias de Cabeza y Cuello/terapia , Papillomavirus Humano 16/genética , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Humanos , Neoplasias Hipofaríngeas/sangre , Neoplasias Hipofaríngeas/patología , Neoplasias Hipofaríngeas/cirugía , Neoplasias Laríngeas/sangre , Neoplasias Laríngeas/patología , Neoplasias Laríngeas/terapia , Disección del Cuello , Neoplasia Residual , Neoplasias Orofaríngeas/sangre , Neoplasias Orofaríngeas/patología , Neoplasias Orofaríngeas/terapia , Estudios Prospectivos , Sensibilidad y Especificidad , Resultado del TratamientoRESUMEN
Lung cancer diagnostics have progressed greatly in the previous decade. Development of molecular testing to identify an increasing number of potentially clinically actionable genetic variants, using smaller samples obtained via minimally invasive techniques, is a huge challenge. Tumour heterogeneity and cancer evolution in response to therapy means that repeat biopsies or circulating biomarkers are likely to be increasingly useful to adapt treatment as resistance develops. We highlight some of the current challenges faced in clinical practice for molecular testing of EGFR, ALK, and new biomarkers such as PDL1. Implementation of next generation sequencing platforms for molecular diagnostics in non-small-cell lung cancer is increasingly common, allowing testing of multiple genetic variants from a single sample. The use of next generation sequencing to recruit for molecularly stratified clinical trials is discussed in the context of the UK Stratified Medicine Programme and The UK National Lung Matrix Trial.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias Pulmonares/patología , Quinasa de Linfoma Anaplásico , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/análisis , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/genética , Mutación , Proteínas Tirosina Quinasas Receptoras/genética , Índice de Severidad de la EnfermedadRESUMEN
Single nucleotide polymorphisms (SNPs) in microRNA genes have been associated with colorectal cancer (CRC) risk, survival and response to treatment. Conflicting results are available on the association between rs4919510, a SNP in mature miR-608 and clinical outcome in CRC. Here, we analyzed the association between rs4919510 and benefit from perioperative treatment in a randomised phase II trial of neoadjuvant Capecitabine and Oxaliplatin (CAPOX) followed by chemo-radiotherapy, surgery and adjuvant CAPOX ± Cetuximab in high-risk locally advanced rectal cancer (LARC). A total of 155/164 (94.5%) patients were assessable. 95 (61.3%) were homozygous for CC, 55 (35.5%) heterozygous (CG) and 5 (3.2%) homozygous for GG. Median follow-up was 64.9 months. In the CAPOX arm the 5-year progression-free survival (PFS) and overall survival (OS) rates were 54.6% and 60.7% for CC and 82.0% and 82.1% for CG/GG, respectively (HR PFS 0.13, 95% CI: 0.12-0.83, P = 0.02; HR OS 0.38, 95% CI: 0.14-1.01, P = 0.05). In the CAPOX-C arm PFS and OS were 73.2 and 82.2%, respectively for CC carriers and 64.6 and 73.1% for CG/GG carriers (HR PFS 1.38, 95% CI: 0.61-3.13, P = 0.44; HR OS 1.34, 95% CI: 0.52-3.48, P = 0.55). An interaction was found between study treatment and rs4919510 genotype for both PFS (P = 0.02) and OS (P = 0.07). This is the first study investigating rs4919510 in LARC. The CC genotype appeared to be associated with worse prognosis compared to the CG/GG genotype in patients treated with chemotherapy and chemo-radiotherapy alone. Addition of Cetuximab to chemotherapy and chemo-radiotherapy in CC carriers appeared to improve clinical outcome.
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MicroARNs/genética , Neoplasias del Recto/genética , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Quimioradioterapia , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante , Polimorfismo de Nucleótido Simple , Neoplasias del Recto/mortalidad , Neoplasias del Recto/terapia , Estudios RetrospectivosRESUMEN
Rare cases of possible materno-fetal transmission of cancer have been recorded over the past 100 years but evidence for a shared cancer clone has been very limited. We provide genetic evidence for mother to offspring transmission, in utero, of a leukemic cell clone. Maternal and infant cancer clones shared the same unique BCR-ABL1 genomic fusion sequence, indicating a shared, single-cell origin. Microsatellite markers in the infant cancer were all of maternal origin. Additionally, the infant, maternally-derived cancer cells had a major deletion on one copy of chromosome 6p that included deletion of HLA alleles that were not inherited by the infant (i.e., foreign to the infant), suggesting a possible mechanism for immune evasion.
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Intercambio Materno-Fetal/genética , Intercambio Materno-Fetal/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/complicaciones , Complicaciones Neoplásicas del Embarazo/genética , Complicaciones Neoplásicas del Embarazo/inmunología , Adulto , Secuencia de Bases , ADN de Neoplasias/genética , Femenino , Genes abl , Antígenos HLA/genética , Humanos , Lactante , Pérdida de Heterocigocidad , Repeticiones de Microsatélite , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Embarazo , ARN Mensajero/genética , ARN Neoplásico/genéticaRESUMEN
Limited data exists to show the correlation of (tumour protein 53) TP53 mutation detected by Next generation sequencing (NGS) and the presence/absence of deletions of 17p13 detected by FISH. The study which is the largest series to date includes 2332 CLL patients referred for analysis of del(17p) by FISH and TP53 mutations by NGS before treatment. Using a 10% variant allele frequency (VAF) threshold, cases were segregated into high burden mutations (≥10%) and low burden mutations (<10%). TP53 aberrations (17p [del(17p)] and/or TP53 mutation) were detected in 320/2332 patients (13.7%). Using NGS analysis, 429 TP53 mutations were identified in 303 patients (13%). Of these 238 (79%) and 65 (21%) were cases with high burden and low burden mutations respectively. In our cohort, 2012 cases did not demonstrate a TP53 aberration (86.3%). A total of 159 cases showed TP53 mutations in the absence of del(17p) (49/159 with low burden TP53 mutations) and 144 cases had both TP53 mutation and del(17p) (16/144 with low burden mutations). Only 17/2332 (0.7%) cases demonstrated del(17p) with no TP53 mutation. Validated NGS protocols should be used in clinical decision making to avoid missing low-burden TP53 mutations and can detect the vast majority of TP53 aberrations.
RESUMEN
Molecular testing for the BCR-ABL1 fusion gene by real time quantitative polymerase chain reaction (RT-qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT-qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society.
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Proteínas de Fusión bcr-abl/biosíntesis , Leucemia Mielógena Crónica BCR-ABL Positiva , Monitoreo Fisiológico/métodos , Inhibidores de Proteínas Quinasas/uso terapéutico , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Humanos , Irlanda , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Biología Molecular , Guías de Práctica Clínica como Asunto , Sociedades Médicas , Reino UnidoRESUMEN
While most testicular germ cell tumours (TGCTs) exhibit exquisite sensitivity to platinum chemotherapy, ~10% are platinum resistant. To gain insight into the underlying mechanisms, we undertake whole exome sequencing and copy number analysis in 40 tumours from 26 cases with platinum-resistant TGCT, and combine this with published genomic data on an additional 624 TGCTs. We integrate analyses for driver mutations, mutational burden, global, arm-level and focal copy number (CN) events, and SNV and CN signatures. Albeit preliminary and observational in nature, these analyses provide support for a possible mechanistic link between early driver mutations in RAS and KIT and the widespread copy number events by which TGCT is characterised.
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Resistencia a Antineoplásicos/efectos de los fármacos , Genómica/métodos , Neoplasias de Células Germinales y Embrionarias/tratamiento farmacológico , Platino (Metal)/uso terapéutico , Neoplasias Testiculares/tratamiento farmacológico , Variaciones en el Número de Copia de ADN , Predisposición Genética a la Enfermedad/genética , Humanos , Masculino , Mutación , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de Células Germinales y Embrionarias/metabolismo , Compuestos Organoplatinos/uso terapéutico , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Transducción de Señal/genética , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Secuenciación del Exoma/métodos , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
There are limited data on circulating, cell-free, tumour (ct)DNA analysis in locally advanced rectal cancer (LARC). Digital droplet (dd)PCR was used to investigate KRAS/BRAF mutations in ctDNA from baseline blood samples of 97 LARC patients who were treated with CAPOX followed by chemoradiotherapy, surgery and adjuvant CAPOX ± cetuximab in a randomised phase II trial. KRAS mutation in G12D, G12V or G13D was detected in the ctDNA of 43% and 35% of patients with tumours that were mutant and wild-type for these hotspot mutations, respectively, according to standard PCR-based analyses on tissue. The detection rate in the ctDNA of 10 patients with less common mutations was 50%. In 26 cases ctDNA analysis revealed KRAS mutations that were not previously found in tissue. Twenty-two of these (84.6%) were detected following repeat tissue testing by ddPCR. Overall, the ctDNA detection rate in the KRAS mutant population was 66%. Detection of KRAS mutation in ctDNA failed to predict prognosis or refine patient selection for cetuximab. While this study confirms the feasibility of ctDNA analysis in LARC and the high sensitivity of ddPCR, larger series are needed to better address the role of ctDNA as a prognostic or predictive tool in this setting.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , ADN Tumoral Circulante/genética , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias del Recto/terapia , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Capecitabina/administración & dosificación , Capecitabina/uso terapéutico , Cetuximab/administración & dosificación , Cetuximab/uso terapéutico , Quimioradioterapia Adyuvante , Procedimientos Quirúrgicos del Sistema Digestivo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Compuestos Organoplatinos/administración & dosificación , Compuestos Organoplatinos/uso terapéutico , Oxaliplatino , Pronóstico , Neoplasias del Recto/genética , Resultado del TratamientoRESUMEN
The approval, in 2015, of the first poly (adenosine diphosphate-ribose) polymerase inhibitor (PARPi; olaparib, Lynparza) for platinum-sensitive relapsed high-grade ovarian cancer with either germline or somatic BRCA1/2 deleterious mutations is changing the way that BRCA1/2 testing services are offered to patients with ovarian cancer. Ovarian cancer patients are now being referred for BRCA1/2 genetic testing for treatment decisions, in addition to familial risk estimation, and irrespective of a family history of breast or ovarian cancer. Furthermore, testing of tumor samples to identify the estimated 3%-9% of patients with somatic BRCA1/2 mutations who, in addition to germline carriers, could benefit from PARPi therapy is also now being considered. This new testing paradigm poses some challenges, in particular the technical and analytical difficulties of analyzing chemically challenged DNA derived from formalin-fixed, paraffin-embedded specimens. The current manuscript reviews some of these challenges and technical recommendations to consider when undertaking BRCA1/2 testing in tumor tissue samples to detect both germline and somatic BRCA1/2 mutations. Also provided are considerations for incorporating genetic analysis of ovarian tumor samples into the patient pathway and ethical requirements.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Pruebas Genéticas , Síndrome de Cáncer de Mama y Ovario Hereditario/diagnóstico , Neoplasias Ováricas/genética , Femenino , Mutación de Línea Germinal , Síndrome de Cáncer de Mama y Ovario Hereditario/genética , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Guías de Práctica Clínica como AsuntoRESUMEN
The implementation of personalised medicine in childhood cancers has been limited by a lack of clinically validated multi-target sequencing approaches specific for paediatric solid tumours. In order to support innovative clinical trials in high-risk patients with unmet need, we have developed a clinically relevant targeted sequencing panel spanning 311 kb and comprising 78 genes involved in childhood cancers. A total of 132 samples were used for the validation of the panel, including Horizon Discovery cell blends (n=4), cell lines (n=15), formalin-fixed paraffin embedded (FFPE, n=83) and fresh frozen tissue (FF, n=30) patient samples. Cell blends containing known single nucleotide variants (SNVs, n=528) and small insertion-deletions (indels n=108) were used to define panel sensitivities of ≥98% for SNVs and ≥83% for indels [95% CI] and panel specificity of ≥98% [95% CI] for SNVs. FFPE samples performed comparably to FF samples (n=15 paired). Of 95 well-characterised genetic abnormalities in 33 clinical specimens and 13 cell lines (including SNVs, indels, amplifications, rearrangements and chromosome losses), 94 (98.9%) were detected by our approach. We have validated a robust and practical methodology to guide clinical management of children with solid tumours based on their molecular profiles. Our work demonstrates the value of targeted gene sequencing in the development of precision medicine strategies in paediatric oncology.
RESUMEN
AIMS: Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. METHODS: 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. RESULTS: Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p<0.0001), and yield variation from engineered samples was not significant (p=0.3782). Two laboratories failed DNA extraction from samples that may be attributed to operator error. DNA extraction protocols themselves were not found to contribute significant variation. 10/13 labs reported yields averaging 235.8 ng (95% CI 90.7 to 380.9) from cell-negative samples, which was attributed to issues with spectrophotometry. DNA measurements using Qubit Fluorometry demonstrated a median fivefold overestimation of DNA quantity by Nanodrop Spectrophotometry. DNA integrity and PCR inhibition were factors not found to contribute significant variation. CONCLUSIONS: In this study, we provide evidence demonstrating that variation in pre-PCR steps is prevalent and may detrimentally affect the patient's ability to receive critical therapy. We provide recommendations for preanalytical workflow optimisation that may reduce errors in down-stream sequencing and for next-generation sequencing library generation.
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Análisis Mutacional de ADN/normas , Receptores ErbB/genética , Fijadores/normas , Formaldehído/normas , Ensayos de Aptitud de Laboratorios , Mutación , Adhesión en Parafina/normas , Reacción en Cadena de la Polimerasa/normas , Proteínas Proto-Oncogénicas B-raf/genética , Fijación del Tejido/normas , Línea Celular Tumoral , Análisis Mutacional de ADN/métodos , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Errores Diagnósticos/prevención & control , Fluorometría/normas , Humanos , Variaciones Dependientes del Observador , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Espectrofotometría/normas , Fijación del Tejido/métodos , Transfección , Reino Unido , Estados Unidos , Flujo de TrabajoRESUMEN
PURPOSE: Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies. EXPERIMENTAL DESIGN: We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL. RESULTS: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P = 0.01). In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02-4.4; P = 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05-4.55; P = 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08-5.2; P = 0.03). CONCLUSIONS: We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively.
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Linfoma de Células B de la Zona Marginal/diagnóstico , Linfoma de Células B de la Zona Marginal/genética , Mutación , Neoplasias del Bazo/diagnóstico , Neoplasias del Bazo/genética , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Femenino , Marcadores Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Persona de Mediana Edad , Análisis Multivariante , Factor 88 de Diferenciación Mieloide/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Pronóstico , Receptor Notch2/genética , Factores de Riesgo , Factores de Transcripción/genética , Resultado del Tratamiento , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Proteína p53 Supresora de Tumor/genéticaRESUMEN
INTRODUCTION: Detection of the ALK rearrangement in a solid tumor gives these patients the option of crizotinib as an oral form of anticancer treatment. The current test of choice is fluorescence in situ hybridization (FISH), but various cheaper and more convenient immunohistochemical (IHC) assays have been proposed as alternatives. METHODS: Fifteen FISH-positive cases from patients, seven with data on crizotinib therapy and clinical response, were evaluated for the presence of ALK protein using three different commercially available antibodies: D5F3, using the proprietary automated system (Ventana), ALK1 (Dako), and 5A4 (Abcam). A further 14 FISH-negative and three uncertain (<15% rearrangement detected) cases were also retrieved. Of the total 32 specimens, 17 were excisions and 15 were computed tomography-guided biopsies or cytological specimens. All three antibodies were applied to all cases. Antibodies were semiquantitatively scored on intensity, and the proportion of malignant cells stained was documented. Cutoffs were set by receiver operating curve analysis for positivity to optimize correct classification. RESULTS: All three IHC assays were 100% specific but sensitivity did vary: D5F3 86%, ALK 79%, 5A4 71%. Intensity was the most discriminating measure overall, with a combination of proportion and intensity not improving the test. No FISH-negative IHC-positive cases were seen. Two FISH-positive cases were negative with all three IHC assays. One of these had been treated with crizotinib and had failed to show clinical response. The other harbored a second driving mutation in the EGFR gene. CONCLUSIONS: IHC with all three antibodies is especially highly specific (100%) although variably sensitive (71%-86%), specifically in cases with scanty material. D5F3 assay was most sensitive in these latter cases. Occasional cases are IHC-positive but FISH-negative, suggesting either inaccuracy of one assay or occasional tumors with ALK rearrangement that do not express high levels of ALK protein.
Asunto(s)
Inmunohistoquímica , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Proteínas Tirosina Quinasas Receptoras/genética , Adulto , Anciano , Anciano de 80 o más Años , Quinasa de Linfoma Anaplásico , Anticuerpos , Crizotinib , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Curva ROC , Proteínas Tirosina Quinasas Receptoras/análisis , Translocación GenéticaRESUMEN
BACKGROUND: The cobas 4800 BRAF V600 Mutation Test is a CE-marked and FDA-approved in vitro diagnostic assay used to select patients with metastatic melanoma for treatment with the selective BRAF inhibitor vemurafenib. We describe the pre-approval validation of this test in two external laboratories. METHODS: Melanoma specimens were tested for BRAF V600 mutations at two laboratories with the: cobas BRAF Mutation Test; ABI BRAF test; and bidirectional direct sequencing. Positive (PPA) and negative (NPA) percent agreements were determined between the cobas test and the other assays. Specimens with discordant results were tested with massively parallel pyrosequencing (454). DNA blends with 5% mutant alleles were tested to assess detection rates. RESULTS: Invalid results were observed in 8/116 specimens (6·9%) with Sanger, 10/116 (8·6%) with ABI BRAF, and 0/232 (0%) with the cobas BRAF test. PPA was 97·7% for V600E mutation for the cobas BRAF test and Sanger, and NPA was 95·3%. For the cobas BRAF test and ABI BRAF, PPA was 71·9% and NPA 83·7%. For 16 cobas BRAF test-negative/ABI BRAF-positive specimens, 454 sequencing detected no codon 600 mutations in 12 and variant codon 600 mutations in four. For eight cobas BRAF test-positive/ABI BRAF-negative specimens, four were V600E and four V600K by 454 sequencing. Detection rates for 5% mutation blends were 100% for the cobas BRAF test, 33% for Sanger, and 21% for the ABI BRAF. Reproducibility of the cobas BRAF test was 111/116 (96%) between the two sites. CONCLUSIONS: It is feasible to evaluate potential companion diagnostic tests in external laboratories simultaneously to the pivotal clinical trial validation. The health authority approved assay had substantially better performance characteristics than the two other methods. The overall success of the cobas BRAF test is a proof of concept for future biomarker development.
Asunto(s)
Análisis Mutacional de ADN/métodos , Pruebas Diagnósticas de Rutina/métodos , Indoles/uso terapéutico , Melanoma/tratamiento farmacológico , Melanoma/genética , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/genética , Sulfonamidas/uso terapéutico , Humanos , Indoles/farmacología , Melanoma/patología , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfonamidas/farmacología , VemurafenibRESUMEN
BACKGROUND: The clinical need to determine the presence of epidermal growth factor receptor (EGFR) gene mutations in non-small-cell lung cancers (NSCLC) in order to make informed decisions for patient treatment has seen the widespread introduction of EGFR molecular testing in many laboratories. To ensure high-quality molecular testing and allow laboratories to externally measure the standard of the service, an external quality assessment (EQA) scheme was provided to assess the whole testing process. METHODS: Formalin-fixed paraffin-embedded NSCLC tumour sections were distributed to laboratories for routine EGFR molecular testing, and the genotyping accuracy, interpretation of the result and clerical accuracy of the report were independently assessed. RESULTS: Three rounds of assessment have identified many genotyping errors and have highlighted the need for external assessment and education in many testing laboratories. The main issues raised were the importance of accurate genotyping, including the use of common mutation nomenclature, clear unambiguous interpretation of the result, the impact of tumour sample assessment regarding amount of tumour being analysed and the heterogeneity of the sample on the molecular test result. CONCLUSIONS: Improvements in all these areas were observed during the progression of the three EQA rounds, however, continuous unacceptably high genotyping error rates demonstrate the clear need for continual external assessment and education in this field.