RESUMEN
Phosphorylated biomarkers are crucial for our understanding of drug mechanism of action and dose selection during clinical trials, particularly for drugs that target protein kinases, such as DNA-damage-response (DDR) inhibitors. However, tissue fixation conditions needed to preserve DDR-specific phospho-biomarkers have not been previously investigated. Using xenograft tissues and tightly controlled formalin fixation conditions, we assessed how preanalytical factors affect phosphorylated DDR biomarkers pRAD50(Ser635), ɣH2AX(Ser139), pKAP1(Ser824), and non-phosphorylated biomarkers cMYC and ATM. Cold ischemia times ranged from 15 min to 6 hr, and the fixation duration ranged from 24 hr to 4 weeks. Epitopes pRAD50 and pKAP1 appeared the most labile assessed with staining loss after just 15 min of cold ischemia time, while ATM was more robust showing consistent expression up to 1 hr of cold ischemia. Notably, ɣH2AX expression was lost with formalin fixation over 48 hr. The use of core needle biopsies where possible and novel fixation methods such as the 2-step temperature-controlled formalin approach may improve phosphorylated biomarker preservation; however, practical challenges may affect wider clinical application. The most essential tissue-processing step when downstream analysis includes DDR phosphorylated biomarkers is immediate tissue submersion in formalin, without delay, upon excision from the patient, followed by room temperature fixation for 24 hr.
Asunto(s)
Daño del ADN , Formaldehído , Humanos , Epítopos , Biomarcadores , Fijación del Tejido/métodosRESUMEN
Identification of somatic variants in cancer by high-throughput sequencing has become common clinical practice, largely because many of these variants may be predictive biomarkers for targeted therapies. However, there can be high sample quality control (QC) failure rates for some tests that prevent the return of results. Stem-loop inhibition mediated amplification (SLIMamp) is a patented technology that has been incorporated into commercially available cancer next-generation sequencing testing kits. The claimed advantage is that these kits can interrogate challenging formalin-fixed, paraffin-embedded tissue samples with low tumor purity, poor-quality DNA, and/or low-input DNA, resulting in a high sample QC pass rate. The study aimed to substantiate that claim using Pillar Biosciences oncoReveal Solid Tumor Panel. Forty-eight samples that had failed one or more preanalytical QC sample parameters for whole-exome sequencing from the Australian Translational Genomics Centre's ISO15189-accredited diagnostic genomics laboratory were acquired. XING Genomic Services performed an exploratory data analysis to characterize the samples and then tested the samples in their ISO15189-accredited laboratory. Clinical reports could be generated for 37 (77%) samples, of which 29 (60%) contained clinically actionable or significant variants that would not otherwise have been identified. Eleven samples were deemed unreportable, and the sequencing data were likely dominated by artifacts. A novel postsequencing QC metric was developed that can discriminate between clinically reportable and unreportable samples.
Asunto(s)
Formaldehído , Neoplasias , Humanos , Fijación del Tejido , Australia , Neoplasias/diagnóstico , Neoplasias/genética , ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biomarcadores de Tumor/genética , Mutación , Adhesión en ParafinaRESUMEN
Basal-like breast cancer (BLBC) has a greater overlap in molecular features with high-grade serous ovarian cancer (HGSOC) than with other breast cancer subtypes. Similarities include BRCA1 mutation, high frequency of TP53 mutation, and amplification of CCNE1 (encoding the cyclin E1 protein) in 6-34% of cases, and these features can be used to group patients for targeted therapies in clinical trials. In HGSOC, we previously reported two subsets with high levels of cyclin E1: those in which CCNE1 is amplified, have intact homologous recombination (HR), and very poor prognosis; and a CCNE1 non-amplified subset, with more prevalent HR defects. Here, we investigate whether similar subsets are identifiable in BLBC that may allow alignment of patient grouping in clinical trials of agents targeting cyclin E1 overexpression. We examined cyclin E1 protein and CCNE1 amplification in a cohort of 76 BLBCs and validated the findings in additional breast cancer datasets. Compared to HGSOC, CCNE1 amplified BLBC had a lower level of amplification (3.5 versus 5.2 copies) and lower relative cyclin E1 protein, a lack of correlation of amplification with expression, and no association with polyploidy. BLBC with elevated cyclin E1 protein also had prevalent HR defects, and high-level expression of the cyclin E1 deubiquitinase ubiquitin-specific protease 28 (USP28). Using a meta-analysis across multiple studies, we determined that cyclin E1 protein overexpression but not amplification is prognostic in BLBC, while both cyclin E1 overexpression and amplification are prognostic in HGSOC. Overall CCNE1 gene amplification is not equivalent between BLBC and HGSOC. However, high cyclin E1 protein expression can co-occur with HR defects in both BLBC and HGSOC, and is associated with poor prognosis in BLBC.
Asunto(s)
Neoplasias de la Mama , Ciclina E , Proteínas Oncogénicas , Neoplasias Ováricas , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patología , Ciclina E/genética , Ciclina E/metabolismo , Femenino , Amplificación de Genes , Humanos , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Pronóstico , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismoRESUMEN
BACKGROUND: The absence of the putative DNA/RNA helicase Schlafen11 (SLFN11) is thought to cause resistance to DNA-damaging agents (DDAs) and PARP inhibitors. METHODS: We developed and validated a clinically applicable SLFN11 immunohistochemistry assay and retrospectively correlated SLFN11 tumour levels to patient outcome to the standard of care therapies and olaparib maintenance. RESULTS: High SLFN11 associated with improved prognosis to the first-line treatment with DDAs platinum-plus-etoposide in SCLC patients, but was not strongly linked to paclitaxel-platinum response in ovarian cancer patients. Multivariate analysis of patients with relapsed platinum-sensitive ovarian cancer from the randomised, placebo-controlled Phase II olaparib maintenance Study19 showed SLFN11 tumour levels associated with sensitivity to olaparib. Study19 patients with high SLFN11 had a lower progression-free survival (PFS) hazard ratio compared to patients with low SLFN11, although both groups had the benefit of olaparib over placebo. Whilst caveated by small sample size, this trend was maintained for PFS, but not overall survival, when adjusting for BRCA status across the olaparib and placebo treatment groups, a key driver of PARP inhibitor sensitivity. CONCLUSION: We provide clinical evidence supporting the role of SLFN11 as a DDA therapy selection biomarker in SCLC and highlight the need for further clinical investigation into SLFN11 as a PARP inhibitor predictive biomarker.
Asunto(s)
Daño del ADN/genética , Proteínas Nucleares/metabolismo , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Basal-like breast cancers (BLBC) are aggressive breast cancers that respond poorly to targeted therapies and chemotherapies. In order to define therapeutically targetable subsets of BLBC we examined two markers: cyclin E1 and BRCA1 loss. In high grade serous ovarian cancer (HGSOC) these markers are mutually exclusive, and define therapeutic subsets. We tested the same hypothesis for BLBC. Using a BLBC cohort enriched for BRCA1 loss, we identified convergence between BRCA1 loss and high cyclin E1 protein expression, in contrast to HGSOC in which CCNE1 amplification drives increased cyclin E1. In cell lines, BRCA1 loss was associated with stabilized cyclin E1 during the cell cycle, and BRCA1 siRNA led to increased cyclin E1 in association with reduced phospho-cyclin E1 T62. Mutation of cyclin E1 T62 to alanine increased cyclin E1 stability. We showed that tumors with high cyclin E1/BRCA1 mutation in the BLBC cohort also had decreased phospho-T62, supporting this hypothesis. Since cyclin E1/CDK2 protects cells from DNA damage and cyclin E1 is elevated in BRCA1 mutant cancers, we hypothesized that CDK2 inhibition would sensitize these cancers to PARP inhibition. CDK2 inhibition induced DNA damage and synergized with PARP inhibitors to reduce cell viability in cell lines with homologous recombination deficiency, including BRCA1 mutated cell lines. Treatment of BRCA1 mutant BLBC patient-derived xenograft models with combination PARP and CDK2 inhibition led to tumor regression and increased survival. We conclude that BRCA1 status and high cyclin E1 have potential as predictive biomarkers to dictate the therapeutic use of combination CDK inhibitors/PARP inhibitors in BLBC.
RESUMEN
PURPOSE: Expanded RAS/BRAF mutations have not been assessed as predictive for single-agent cetuximab in metastatic colorectal cancer (mCRC), and low mutant allele frequency (MAF) mutations are of unclear significance. We aimed to establish cetuximab efficacy in optimally selected patients using highly sensitive beads, emulsion, amplification, and magnetics (BEAMing) analysis, capable of detecting alterations below standard clinical assays. PATIENTS AND METHODS: CO.17 trial compared cetuximab versus best supportive care (BSC) in RAS/BRAF-unselected mCRC. We performed RAS/BRAF analysis on microdissected tissue of 242 patients in CO.17 trial using BEAMing for KRAS/NRAS (codons 12/13/59/61/117/146) and BRAF V600E. Patients without BEAMing but with previous Sanger sequencing-detected mutations were included. RESULTS: KRAS, NRAS, and BRAF mutations were present in 53%, 4%, and 3% of tumors, respectively. Cetuximab improved overall survival [OS; HR, 0.51; 95% confidence interval (CI), 0.32-0.81; P = 0.004] and progression-free survival (PFS; HR, 0.25; 95% CI, 0.15-0.41; P < 0.0001) compared with BSC in RAS/BRAF wild-type patients. Cetuximab did not improve OS/PFS for KRAS-, NRAS-, or BRAF-mutated tumors, and tests of interaction confirmed expanded KRAS (P = 0.0002) and NRAS (P = 0.006) as predictive, while BRAF mutations were not (P = 0.089). BEAMing identified 14% more tumors as RAS mutant than Sanger sequencing, and cetuximab lacked activity in these patients. Mutations at MAF < 5% were noted in 6 of 242 patients (2%). One patient with a KRAS A59T mutation (MAF = 2%) responded to cetuximab. More NRAS than KRAS mutations were low MAF (OR, 20.50; 95% CI, 3.88-96.85; P = 0.0038). CONCLUSIONS: We establish single-agent cetuximab efficacy in optimally selected patients and show that subclonal RAS/BRAF alterations are uncommon and remain of indeterminate significance.
Asunto(s)
Biomarcadores de Tumor/genética , Cetuximab/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Cetuximab/farmacología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Análisis Mutacional de ADN , Resistencia a Antineoplásicos/genética , Femenino , GTP Fosfohidrolasas/genética , Frecuencia de los Genes , Humanos , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Mutación , Supervivencia sin Progresión , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially.
Asunto(s)
Anemia/prevención & control , Riñón/efectos de la radiación , Proteína bcl-X/metabolismo , Proteína bcl-X/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2/genética , Daño del ADN , Femenino , Rayos gamma , Neoplasias Hematológicas/patología , Inflamación , Riñón/metabolismo , Riñón/patología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transcriptoma , Proteínas Supresoras de Tumor/genética , Proteína bcl-X/deficiencia , Proteína bcl-X/genéticaRESUMEN
PURPOSE: Metastatic colorectal cancers (mCRCs) assigned to the transit-amplifying (TA) CRCAssigner subtype are more sensitive to anti-epidermal growth factor receptor (EGFR) therapy. We evaluated the association between the intratumoral presence of TA signature (TA-high/TA-low, dubbed as TA-ness classification) and outcomes in CRCs treated with anti-EGFR therapy. PATIENTS AND METHODS: The TA-ness classes were defined in a discovery cohort (n = 84) and independently validated in a clinical trial (CO.20; cetuximab monotherapy arm; n = 121) and other samples using an established NanoString-based gene expression assay. Progression-free survival (PFS), overall survival (OS), and disease control rate (DCR) according to TA-ness classification were assessed by univariate and multivariate analyses. RESULTS: The TA-ness was measured in 772 samples from 712 patients. Patients (treated with anti-EGFR therapy) with TA-high tumors had significantly longer PFS (discovery hazard ratio [HR], 0.40; 95% CI, 0.25 to 0.64; P < .001; validation HR, 0.65; 95% CI, 0.45 to 0.93; P = .018), longer OS (discovery HR, 0.48; 95% CI, 0.29 to 0.78; P = .003; validation HR, 0.67; 95% CI, 0.46 to 0.98; P = .04), and higher DCR (discovery odds ratio [OR]; 14.8; 95% CI, 4.30 to 59.54; P < .001; validation OR, 4.35; 95% CI, 2.00 to 9.09; P < .001). TA-ness classification and its association with anti-EGFR therapy outcomes were further confirmed using publicly available data (n = 80) from metastatic samples (PFS P < .001) and patient-derived xenografts (P = .042). In an exploratory analysis of 55 patients with RAS/BRAF wild-type and left-sided tumors, TA-high class was significantly associated with longer PFS and trend toward higher response rate (PFS HR, 0.53; 95% CI, 0.28 to 1.00; P = .049; OR, 5.88; 95% CI, 0.71 to 4.55; P = .09; response rate 33% in TA-high and 7.7% in TA-low). CONCLUSION: TA-ness classification is associated with prognosis in patients with mCRC treated with anti-EGFR therapy and may further help understanding the value of sidedness in patients with RAS/BRAF wild-type tumors.
RESUMEN
PURPOSE: The ALLOCATE study was designed as a pilot to demonstrate the feasibility and clinical utility of real-time targeted molecular profiling of patients with recurrent or advanced ovarian cancer for identification of potential targeted therapies. PATIENTS AND METHODS: A total of 113 patients with ovarian cancer of varying histologies were recruited from two tertiary hospitals, with 99 patient cases suitable for prospective analysis. Targeted molecular and methylation profiling of fresh biopsy and archived tumor samples were performed by screening for mutations or copy-number variations in 44 genes and for promoter methylation of BRCA1 and RAD51C. RESULTS: Somatic genomic or methylation events were identified in 85% of all patient cases, with potentially actionable events with defined targeted therapies (including four resistance events) detected in 60% of all patient cases. On the basis of these findings, six patients received molecularly guided therapy, three patients had unsuspected germline cancer-associated BRCA1/2 mutations and were referred for genetic counseling, and two intermediate differentiated (grade 2) serous ovarian carcinomas were reclassified as low grade, leading to changes in clinical management. Additionally, secondary reversion mutations in BRCA1/2 were identified in fresh biopsy samples of two patients, consistent with clinical platinum/poly (ADP-ribose) polymerase inhibitor resistance. Timely reporting of results if molecular testing is done at disease recurrence, as well as early referral for patients with platinum-resistant cancers, were identified as factors that could improve the clinical utility of molecular profiling. CONCLUSION: ALLOCATE molecular profiling identified known genomic and methylation alterations of the different ovarian cancer subtypes and was deemed feasible and useful in routine clinical practice. Better patient selection and access to a wider range of targeted therapies or clinical trials will further enhance the clinical utility of molecular profiling.
RESUMEN
BACKGROUND: The Irinotecan Cetuximab Evaluation and Cetuximab Response Evaluation (ICECREAM) study assessed the efficacy of cetuximab monotherapy compared with cetuximab combined with chemotherapy for quadruple wild-type (KRAS, NRAS, BRAF, or P13KCA exon 20) metastatic colorectal cancer. PATIENTS AND METHODS: Patients were enrolled in an open-label, multicenter, phase II trial and randomly assigned to cetuximab 400 mg/m2, then 250 mg/m2 cetuximab weekly, with or without irinotecan 180 mg/m2 every 2 weeks. The primary endpoint was 6-month progression-free survival; secondary endpoints were response rate, overall survival, toxicity, and quality of life. RESULTS: From 2012 to 2016, 48 patients were recruited. Two were ineligible, and 2 were not evaluable for response. Characteristics were balanced, except gender (male, 62% vs. 72%) and primary sidedness (left, 95% vs. 68%). For cetuximab compared with cetuximab-irinotecan, progression-free survival was 14% versus 41% (hazard ratio, 0.39; 95% confidence interval, 0.20-0.78; P = .008); response rate was 10% (2 partial responses) versus 38% (1 complete, 8 partial); P = .04. Grade 3 to 4 toxicities were less with cetuximab monotherapy (23% vs. 50%); global and specific quality of life scores did not differ. CONCLUSION: In comparison with cetuximab alone, cetuximab plus irinotecan increases the response rate and delays progression in irinotecan-resistant RAS wild-type colorectal cancer. This echoes data from molecularly unselected patients.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Adulto , Anciano , Cetuximab/administración & dosificación , Fosfatidilinositol 3-Quinasa Clase I/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Estudios de Seguimiento , GTP Fosfohidrolasas/genética , Humanos , Irinotecán/administración & dosificación , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Mutación , Pronóstico , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Tasa de SupervivenciaRESUMEN
OBJECTIVES: Readily apparent cyclin E1 expression occurs in 50% of HGSOC, but only half are linked to 19q12 locus amplification. The amplified/cyclin E1hi subset has intact BRCA1/2, unfavorable outcome, and is potentially therapeutically targetable. We studied whether non-amplified/cyclin E1hi HGSOC has similar characteristics. We also assessed the expression of cyclin E1 degradation-associated proteins, FBXW7 and USP28, as potential drivers of high cyclin E1 expression in both subsets. METHODS: 262 HGSOC cases were analyzed by in situ hybridization for 19q12 locus amplification and immunohistochemistry for cyclin E1, URI1 (another protein encoded by the 19q12 locus), FBXW7 and USP28 expression. Tumors were classified by 19q12 amplification status and correlated to cyclin E1 and URI1 expression, BRCA1/2 germline mutation, FBXW7 and USP28 expression, and clinical outcomes. Additionally, we assessed the relative genomic instability of amplified/cyclin E1hi and non-amplified/cyclin E1hi groups of HGSOC datasets from The Cancer Genome Atlas. RESULTS: Of the 82 cyclin E1hi cases, 43 (52%) were amplified and 39 (48%) were non-amplified. Unlike amplified tumors, non-amplified/cyclin E1hi tumor status was not mutually exclusive with gBRCA1/2 mutation. The non-amplified/cyclin E1hi group had significantly increased USP28, while the amplified/cyclin E1hi cancers had significantly lower FBXW7 expression consistent with a role for both in stabilizing cyclin E1. Notably, only the amplified/cyclin E1hi subset was associated with genomic instability and had a worse outcome than non-amplified/cyclin E1hi group. CONCLUSIONS: Amplified/cyclin E1hi and non-amplified/cyclin E1hi tumors have different pathological and biological characteristics and clinical outcomes indicating that they are separate subsets of cyclin E1hi HGSOC.
Asunto(s)
Cromosomas Humanos Par 19 , Ciclina E/genética , Cistadenocarcinoma Seroso/genética , Proteínas Oncogénicas/genética , Neoplasias Ováricas/genética , Adulto , Anciano , Anciano de 80 o más Años , Proteína BRCA1/biosíntesis , Proteína BRCA1/genética , Proteína BRCA2/biosíntesis , Proteína BRCA2/genética , Ciclina E/biosíntesis , Cistadenocarcinoma Seroso/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/biosíntesis , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Femenino , Amplificación de Genes , Inestabilidad Genómica , Humanos , Persona de Mediana Edad , Proteínas Oncogénicas/biosíntesis , Neoplasias Ováricas/metabolismo , Ubiquitina Tiolesterasa/biosíntesis , Ubiquitina Tiolesterasa/genéticaRESUMEN
Background: Hyperactivation of phosphoinositide 3-kinase (PI3K) signaling is common in cancers, but the precise role of the pathway in glioma biology remains to be determined. Some understanding of PI3K signaling mechanisms in brain cancer comes from studies on neural stem/progenitor cells (NSPCs), where signals transmitted via the PI3K pathway cooperate with other intracellular pathways and downstream transcription factors to regulate critical cell functions. Methods: To investigate the role of the PI3K pathway in glioma initiation and development, we generated a mouse model targeting the inducible expression of a PIK3CAH1047A oncogenic mutant and deletion of the PI3K negative regulator, phosphatase and tensin homolog (PTEN), to NSPCs. Results: Expression of a Pik3caH1047A was sufficient to generate tumors with oligodendroglial features, but simultaneous loss of PTEN was required for the development of invasive, high-grade glioma. Pik3caH1047A-PTEN mutant NSPCs exhibited enhanced neurosphere formation which correlated with increased Wnt signaling, while loss of cAMP response element binding protein (CREB) in Pik3caH1047A-Pten mutant tumors led to longer symptom-free survival in mice. Conclusion: Taken together, our findings present a novel mouse model for glioma demonstrating that the PI3K pathway is important for initiation of tumorigenesis and that disruption of downstream CREB signaling attenuates tumor expansion.
Asunto(s)
Neoplasias Encefálicas/patología , Carcinogénesis/patología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Células-Madre Neurales/patología , Fosfohidrolasa PTEN/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , Ciclo Celular , Movimiento Celular , Proliferación Celular , Fosfatidilinositol 3-Quinasa Clase I , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Ratones , Ratones Noqueados , Células-Madre Neurales/metabolismo , Fosforilación , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Technology has progressed from single gene panel to large-scale genomic sequencing. This is raising expectations from clinicians and patients alike. The utility and performance of this technology in a clinical setting needs to be evaluated. AIM: This pilot study investigated the feasibility of using exome-scale sequencing (ESS) to identify molecular drivers within cancers in real-time for Precision Oncology in the clinic. METHODS: Between March 2014 and March 2015, the Victorian Comprehensive Cancer Centre Alliance explored the feasibility and utility of ESS in a pilot study. DNA extracted from the tumour specimens underwent both ESS and targeted 'hotspot' sequencing (TS). Blood was taken for germline analysis. A multi-disciplinary molecular tumour board determined the clinical relevance of identified mutations; in particular, whether they were 'actionable' and/or 'druggable'. RESULTS: Of 23 patients screened, 15 (65%) met the tissue requirements for genomic analysis. TS and ESS were successful in all cases. ESS identified pathogenic somatic variants in 73% (11/15 cases) versus 53% (8/15 cases) using TS. Clinically focused ESS identified 63 variants, consisting of 30 somatic variants (including all 13 identified by TS) and 33 germline variants. Overall, there were 48 unique variants. ESS had a clinical impact in 53% (8/15 cases); 47% (7/15 cases) were referred to the familial cancer clinic, and 'druggable' targets were identified in 53% (8/15 cases). CONCLUSION: ESS of tumour DNA impacted clinical decision-making in 53%, with 20% more pathogenic variants identified through ESS than TS. The identification of germline variants in 47% was an unexpected finding.
Asunto(s)
Exoma/genética , Neoplasias/genética , Análisis de Secuencia de ADN , Adolescente , Adulto , Anciano , ADN de Neoplasias/análisis , Femenino , Marcadores Genéticos , Mutación de Línea Germinal , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Masculino , Persona de Mediana Edad , Proyectos Piloto , Medicina de Precisión , Adulto JovenRESUMEN
Genetic alterations that potentiate PI3K signaling are frequent in prostate cancer, yet how different genetic drivers of the PI3K cascade contribute to prostate cancer is unclear. Here, we report PIK3CA mutation/amplification correlates with poor survival of patients with prostate cancer. To interrogate the requirement of different PI3K genetic drivers in prostate cancer, we employed a genetic approach to mutate Pik3ca in mouse prostate epithelium. We show Pik3caH1047R mutation causes p110α-dependent invasive prostate carcinoma in vivo Furthermore, we report that PIK3CA mutation and PTEN loss coexist in patients with prostate cancer and can cooperate in vivo to accelerate disease progression via AKT-mTORC1/2 hyperactivation. Contrasting single mutants that slowly acquire castration-resistant prostate cancer (CRPC), concomitant Pik3ca mutation and Pten loss caused de novo CRPC. Thus, Pik3ca mutation and Pten deletion are not functionally redundant. Our findings indicate that PIK3CA mutation is an attractive prognostic indicator for prostate cancer that may cooperate with PTEN loss to facilitate CRPC in patients.Significance: We show PIK3CA mutation correlates with poor prostate cancer prognosis and causes prostate cancer in mice. Moreover, PIK3CA mutation and PTEN loss coexist in prostate cancer and can cooperate in vivo to accelerate tumorigenesis and facilitate CRPC. Delineating this synergistic relationship may present new therapeutic/prognostic approaches to overcome castration/PI3K-AKT-mTORC1/2 inhibitor resistance. Cancer Discov; 8(6); 764-79. ©2018 AACR.See related commentary by Triscott and Rubin, p. 682This article is highlighted in the In This Issue feature, p. 663.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/genética , Mutación , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Animales , Línea Celular Tumoral , Progresión de la Enfermedad , Amplificación de Genes , Eliminación de Gen , Humanos , Masculino , Ratones , Invasividad Neoplásica , Neoplasias Experimentales , Pronóstico , Análisis de SupervivenciaRESUMEN
Polymorphisms in NFKB1 that diminish its expression have been linked to human inflammatory diseases and increased risk for epithelial cancers. The underlying mechanisms are unknown, and the link is perplexing given that NF-κB signaling reportedly typically exerts pro-tumorigenic activity. Here we have shown that NF-κB1 deficiency, even loss of a single allele, resulted in spontaneous invasive gastric cancer (GC) in mice that mirrored the histopathological progression of human intestinal-type gastric adenocarcinoma. Bone marrow chimeras revealed that NF-κB1 exerted tumor suppressive functions in both epithelial and hematopoietic cells. RNA-seq analysis showed that NF-κB1 deficiency resulted in aberrant JAK-STAT signaling, which dysregulated expression of effectors of inflammation, antigen presentation, and immune checkpoints. Concomitant loss of STAT1 prevented these immune abnormalities and GC development. These findings provide mechanistic insight into how polymorphisms that attenuate NFKB1 expression predispose humans to epithelial cancers, highlighting the pro-tumorigenic activity of STAT1 and identifying targetable vulnerabilities in GC.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Inflamación/genética , Inflamación/metabolismo , FN-kappa B/deficiencia , Factor de Transcripción STAT1/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Animales , Presentación de Antígeno/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Redes Reguladoras de Genes , Humanos , Inflamación/patología , Ratones , Ratones Noqueados , Factor de Transcripción STAT1/deficiencia , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/patologíaRESUMEN
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.
Asunto(s)
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
Organisations and governments seeking to implement genomics into clinical practice face numerous challenges across multiple, diverse aspects of the health care system. It is not sufficient to tackle any one aspect in isolation: to create a system that supports genomic medicine, they must be addressed simultaneously. The growing body of global knowledge can guide decision-making, but each jurisdiction or organisation needs a model for genomic (or personalised) medicine that is tailored to its unique context, its priorities and the funds available. Poor decisions could greatly reduce the benefits that could potentially arise from genomic medicine. Demonstration projects enable models to be tested, providing valuable evidence and experience for subsequent implementation. Here, we present the Melbourne Genomics Health Alliance demonstration project as an exemplar of a collaborative, holistic approach to phased implementation of genomics across multiple autonomous institutions. The approach and lessons learned may assist others in determining how best to integrate genomics into their healthcare system.
RESUMEN
[This corrects the article DOI: 10.1038/s41525-017-0017-4.].