RESUMO
As the second most common malignant bone tumor in children and adolescents, Ewing sarcoma is initiated and exacerbated by a chimeric oncoprotein, most commonly, EWS-FLI1. In this study, we apply epigenomic analysis to characterize the transcription dysregulation in this cancer, focusing on the investigation of super-enhancer and its associated transcriptional regulatory mechanisms. We demonstrate that super-enhancer-associated transcripts are significantly enriched in EWS-FLI1 target genes, contribute to the aberrant transcriptional network of the disease, and mediate the exceptional sensitivity of Ewing sarcoma to transcriptional inhibition. Through integrative analysis, we identify MEIS1 as a super-enhancer-driven oncogene, which co-operates with EWS-FLI1 in transcriptional regulation, and plays a key pro-survival role in Ewing sarcoma. Moreover, APCDD1, another super-enhancer-associated gene, acting as a downstream target of both MEIS1 and EWS-FLI1, is also characterized as a novel tumor-promoting factor in this malignancy. These data delineate super-enhancer-mediated transcriptional deregulation in Ewing sarcoma, and uncover numerous candidate oncogenes which can be exploited for further understanding of the molecular pathogenesis for this disease.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Proteína Meis1/genética , Sarcoma de Ewing/genética , Transcrição Gênica , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Humanos , Motivos de Nucleotídeos/genética , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/patologia , Transdução de Sinais/genéticaRESUMO
The amplitude of transforming growth factor-ß (TGF-ß) signal is tightly regulated to ensure appropriate physiological responses. As part of negative feedback loop SMAD7, a direct transcriptional target of downstream TGF-ß signaling acts as a scaffold to recruit the E3 ligase SMURF2 to target the TGF-ß receptor complex for ubiquitin-mediated degradation. Here, we identify the deubiquitinating enzyme USP26 as a novel integral component of this negative feedback loop. We demonstrate that TGF-ß rapidly enhances the expression of USP26 and reinforces SMAD7 stability by limiting the ubiquitin-mediated turnover of SMAD7. Conversely, knockdown of USP26 rapidly degrades SMAD7 resulting in TGF-ß receptor stabilization and enhanced levels of p-SMAD2. Clinically, loss of USP26 correlates with high TGF-ß activity and confers poor prognosis in glioblastoma. Our data identify USP26 as a novel negative regulator of the TGF-ß pathway and suggest that loss of USP26 expression may be an important factor in glioblastoma pathogenesis.
Assuntos
Cisteína Endopeptidases/metabolismo , Enzimas Desubiquitinantes/metabolismo , Proteína Smad7/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina/metabolismo , Cisteína Endopeptidases/deficiência , Cisteína Endopeptidases/genética , Proteínas de Ligação a DNA , Glioblastoma/genética , Glioblastoma/fisiopatologia , Humanos , Prognóstico , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad7/genética , Transativadores , Fator de Crescimento Transformador beta/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The development of molecular testing for identifying somatic mutations and immune checkpoint biomarkers has directed treatment towards personalized medicine for patients with non-small cell lung cancer. The choice of molecular testing in a clinical setting is influenced by cost, expertise in the technology, instrumentation setup and sample type availability. The molecular techniques described in this review include immunohistochemistry (IHC), fluorescent in situ hybridization, direct sequencing, real-time polymerase chain reaction (PCR), denaturing high-performance liquid chromatography, matrix-assisted laser desorption/ionization time of flight mass spectrometry and next-generation sequencing (NGS). IHC is routinely used in clinical practice for the classification, differentiation, histology and identification of targetable alterations of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK) and programmed death ligand-1 (PD-L1). Recently, the PD-L1 pathway was identified as being exploited by tumour cells, allowing immune resistance and tumour evasion. The development of immune checkpoint inhibitors as treatment for tumours expressing checkpoints has highlighted the need for standardized IHC assays to inform treatment decisions for patients. Direct sequencing was historically the gold standard for mutation testing for EGFR, KRAS (Kirsten rat sarcoma viral oncogene homologue) and BRAF (v-Raf murine sarcoma viral oncogene homologue B1) requiring a high ratio of tumour to normal cells, but this has been superseded by more sensitive methods. NGS is a new emerging technique, which allows high-throughput coverage of frequently mutated genes, including less common BRAF and MET mutations and alterations in tumour suppressor genes. When an NGS platform is unavailable, PCR-based technologies offer an efficient and cost-effective single gene test to guide patient treatment. This article will review these techniques and discuss the future of molecular platforms underpinning clinical management decisions.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , DNA de Neoplasias/análise , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase/métodos , Quinase do Linfoma Anaplásico/genética , Antígeno B7-H1/genética , Biomarcadores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cromatografia Líquida de Alta Pressão/métodos , Receptores ErbB/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The value of precision oncology initiatives in Asian contexts remains unresolved. Here, we review the institutional implementation of prospective molecular screening to facilitate accrual of patients into biomarker-driven clinical trials, and to explore the mutational landscape of advanced tumors occurring in a prospective cohort of Asian patients (n = 396) with diverse cancer types. Next-generation sequencing (NGS) and routine clinicopathological assays, such as immunohistochemistry, copy number analysis and in situ hybridization tests, were performed on tumor samples. Actionable biomarker results were used to identify eligibility for early-phase, biomarker-driven clinical trials. Overall, NGS was successful in 365 of 396 patients (92%), achieving a mean depth of 1,943× and coverage uniformity of 96%. The median turnaround time from sample receipt to return of genomic results was 26.0 days (IQR, 19.0-39.0 days). Reportable mutations were found in 300 of 365 patients (82%). Ninety-one percent of patients at study enrollment indicated consent to receive incidental findings and willingness to undergo genetic counseling if required. The most commonly mutated oncogenes included KRAS (19%), PIK3CA (16%), EGFR (5%), BRAF (3%) and KIT (3%); while the most frequently mutated tumor suppressor genes included TP53 (40%), SMARCB1 (12%), APC (8%), PTEN (6%) and SMAD4 (5%). Among 23 patients enrolled in genotype-matched trials, median progression-free survival was 2.9 months (IQR, 1.5-4.0 months). Nine of 20 evaluable patients (45%; 95% CI, 23.1-68.5%) derived clinical benefit, including 3 partial responses and 6 with stable disease lasting ≥ 8 weeks.
Assuntos
Povo Asiático/genética , Biomarcadores Tumorais/genética , Ensaios Clínicos como Assunto/métodos , Neoplasias/genética , Neoplasias/terapia , Idoso , Biomarcadores Tumorais/metabolismo , Detecção Precoce de Câncer/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/metabolismo , Medicina de Precisão/métodos , Intervalo Livre de ProgressãoRESUMO
In the context of solid tumours, the evolution of cancer therapies to more targeted and nuanced approaches has led to the impetus for personalised medicine. The targets for these therapies are largely based on the driving genetic mutations of the tumours. To track these multiple driving mutations the use of next generation sequencing (NGS) coupled with a morphomolecular approach to tumours, has the potential to deliver on the promises of personalised medicine. A review of NGS and its application in a universal healthcare (UHC) setting is undertaken as the technology has a wide appeal and utility in diagnostic, clinical trial and research paradigms. Furthermore, we suggest that these can be accommodated with a unified integromic approach. Challenges remain in bringing NGS to routine clinical use and these include validation, handling of the large amounts of information flow and production of a clinically useful report. These challenges are particularly acute in the setting of UHC where tests are not reimbursed and there are finite resources available. It is our opinion that the challenges faced in applying NGS in a UHC setting are surmountable and we outline our approach for its routine application in diagnostic, clinical trial and research paradigms.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/diagnóstico , Análise de Sequência de DNA/métodos , Ensaios Clínicos como Assunto , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Seguro Saúde , Mutação , Medicina de Precisão , Análise de Sequência de DNA/economiaRESUMO
BACKGROUND: Signet ring cell colorectal cancer (SRCCa) has a bleak prognosis. Employing molecular pathology techniques we investigated the potential of precision medicine in this disease. METHODS: Using test (n=26) and validation (n=18) cohorts, analysis of mutations, DNA methylation and transcriptome was carried out. Microsatellite instability (MSI) status was established and immunohistochemistry (IHC) was used to test for adaptive immunity (CD3) and the immune checkpoint PDL1. RESULTS: DNA methylation data split the cohorts into hypermethylated (n=18, 41%) and hypomethylated groups (n=26, 59%). The hypermethylated group predominant in the proximal colon was enriched for CpG island methylator phenotype (CIMP), BRAF V600E mutation and MSI (P<0.001). These cases also had a high CD3+ immune infiltrate (P<0.001) and expressed PDL1 (P=0.03 in intra-tumoural lymphoid cells). The hypomethylated group predominant in the distal colon did not show any characteristic molecular features. We also detected a common targetable KIT mutation (c.1621A>C) across both groups. No statistically significant difference in outcome was observed between the two groups. CONCLUSIONS: Our data show that SRCCa phenotype comprises two distinct genotypes. The MSI+/CIMP+/BRAF V600E+/CD3+/PDL1+ hypermethylated genotype is an ideal candidate for immune checkpoint inhibitor therapy. In addition, one fourth of SRCCa cases can potentially be targeted by KIT inhibitors.
Assuntos
Carcinoma de Células em Anel de Sinete/genética , Neoplasias Colorretais/genética , Metilação de DNA/genética , Proteínas Proto-Oncogênicas B-raf/genética , Carcinoma de Células em Anel de Sinete/patologia , Neoplasias Colorretais/patologia , Ilhas de CpG/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genômica , Genótipo , Humanos , Masculino , Instabilidade de Microssatélites , Mutação , Proteínas de Neoplasias/biossíntese , Transcriptoma/genéticaRESUMO
BACKGROUND: Regorafenib, a multi-kinase inhibitor, is used in the treatment of patients with metastatic colorectal cancer refractory to standard therapy. However, this benefit was limited to 1.4 months improvement in overall survival, with more than half of patients experiencing grade 3 to 4 adverse events. We aim to elucidate the pharmacodynamic effects of regorafenib in metastatic colorectal cancer and discover potential biomarkers that may predict clinical benefit. METHODS: Patients with metastatic colorectal adenocarcinoma refractory to standard therapy with tumours amenable to biopsy were eligible for the study. Regorafenib was administered orally at 160 mg daily for 3 out of 4 weeks with tumour assessment every 2 cycles. Metabolic response was assessed by FDG PET-CT scans (pre-treatment and day 15); paired tumour biopsies (pre-treatment and day 21 post-treatment) were sampled for immunohistochemistry and proteomic profiling analyses. Plasma circulating cell free DNA was quantified serially before and after treatment. RESULTS: There were 2(6%) partial responses out of 35 patients, and 8(23%) patients had stable disease for more than 7 months. Adverse event profile was similar to reported data. Recurrent somatic mutations in K-RAS, PIK3CA and BRAF were detected in plasma circulating cell free DNA in 14 patients; some mutations were not found in archival tumour. Total plasma circulating cell free DNA inversely correlated with progression free survival (PFS), and presence of KRAS mutations associated with shorter PFS. Immunohistochemistry of pre- and post- treatment biopsies showed majority of patients had downregulation of phosphorylated-VEGFR2, podoplanin, phosphorylated-AKT, Ki-67 and upregulation of the MEK-ERK axis, phosphorylated-C-MET, phosphorylated-SRC, phosphorylated-STAT3 and phosphorylated-JUN. Proteomic analysis of fine needle tumour aspirates showed down-regulation of PI3K was associated with longer PFS. CONCLUSION: Plasma circulating cell free DNA may yield potential predictive biomarkers of regorafenib treatment. Downregulation of the PI3K-AKT axis may be an important predictor of clinical benefit.
Assuntos
Neoplasias Colorretais/sangue , Neoplasias Colorretais/tratamento farmacológico , DNA de Neoplasias/sangue , Compostos de Fenilureia/uso terapêutico , Piridinas/uso terapêutico , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Sistema Livre de Células , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA , Demografia , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Compostos de Fenilureia/efeitos adversos , Fosfatidilinositol 3-Quinases/metabolismo , Proteômica , Piridinas/efeitos adversos , Resultado do TratamentoRESUMO
Angiomatoid fibrous histiocytoma is a rare neoplasm of intermediate malignant potential that usually occurs in the dermis or subcutaneous tissues of the extremities in children or young adults. It is characterized by a nodular growth of spindled, histiocytic, or epithelioid cells and blood-filled spaces, surrounded by a fibrous pseudocapsule that contains a lymphocytic cuff. The histological spectrum of this condition has expanded to include cases that contain prominent myxoid stroma. We herein present another instance of myxoid angiomatoid fibrous histiocytoma and review the clinical and histological features, immunohistochemical profile, and molecular genetics of this uncommon variant. We also discuss the diagnostic mimics of this condition, including benign myxoid soft tissue tumors and sarcomas, to illustrate the potential pitfalls in arriving at the diagnosis.
Assuntos
Histiocitoma Fibroso Maligno/genética , Histiocitoma Fibroso Maligno/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Adolescente , Proteínas de Ligação a Calmodulina/genética , Histiocitoma Fibroso Maligno/química , Humanos , Imageamento por Ressonância Magnética , Masculino , Proteína EWS de Ligação a RNA , Proteínas de Ligação a RNA/genética , Neoplasias Cutâneas/químicaRESUMO
Inhibition of the phosphatidylinositol-3-kinase (PI3K) signaling pathway is a cancer treatment strategy that has entered into clinical trials. We performed a meta-analysis on the frequency of prominent genetic (PIK3CA mutation, PIK3CA amplification and PTEN deletion) and protein expression (high PI3K, PTEN loss and high pAkt) aberrations in the PI3K pathway in gastric cancer (GC) and colorectal cancer (CRC). We also performed laboratory analysis to investigate the co-occurrence of these aberrations. The meta-analysis indicated that East Asian and Caucasian GC patients differ significantly for the frequencies of PIK3CA Exon 9 and 20 mutations (7% vs. 15%, respectively), PTEN deletion (21% vs. 4%) and PTEN loss (47% vs. 78%), while CRC patients differed for PTEN loss (57% vs. 26%). High study heterogeneity (I(2) > 80) was observed for all aberrations except PIK3CA mutations. Laboratory analysis of tumors from East Asian patients revealed significant differences between GC (n = 79) and CRC (n = 116) for the frequencies of PIK3CA amplification (46% vs. 4%) and PTEN loss (54% vs. 78%). The incidence of GC cases with 0, 1, 2 and 3 concurrent aberrations was 14%, 52%, 27% and 8%, respectively, while for CRC it was 10%, 60%, 25% and 4%, respectively. Our study consolidates knowledge on the frequency, co-occurrence and clinical relevance of PI3K pathway aberrations in GC and CRC. Up to 86% of GC and 90% of CRC have at least one aberration in the PI3K pathway, and there are significant differences in the frequencies of these aberrations according to cancer type and ethnicity.
Assuntos
Neoplasias Colorretais/genética , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais/fisiologia , Neoplasias Gástricas/genética , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/etnologia , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Humanos , Mutação , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/fisiologia , Neoplasias Gástricas/etnologia , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologiaRESUMO
BACKGROUND: Next generation sequencing (NGS) promises many benefits for clinical diagnostics. However, current barriers to its adoption include suboptimal amenability for low clinical throughputs and uncertainty over data accuracy and analytical procedures. We assessed the feasibility and performance of low-throughput NGS for detecting germline mutations for Lynch syndrome (LS). METHODS: Sequencing depth, time, and cost of 6 formats on the MiSeq and Personal Genome Machine platforms at 1-12 samples/run were calculated. Analytical performance was assessed from 3 runs of 3 DNA samples annotated for 7500 nucleotides by BeadChip arrays. The clinical performance of low-throughput NGS and 9 analytical processes were assessed through blinded analysis of DNA samples from 12 LS cases confirmed by Sanger sequencing, and 3 control cases. RESULTS: The feasibility analysis revealed different formats were optimal at different throughputs. Detection was reproducible for 2619/2635 (99.39%) replicate variants, and sensitivity and specificity to array annotation were 99.42% and 99.99% respectively. Eleven of 16 inconsistently detected variants could be specifically identified by having allele frequencies ≤ 0.15, strand biases >-35, or genotype quality scores ≤ 80. Positive selection for variants in the Human Genome Mutation Database (colorectal cancer, nonpolyposis) and variants with ≤ 5% frequency in the Asian population gave the best clinical performance (92% sensitivity, 67% specificity). CONCLUSIONS: Low-throughput NGS can be a cost-efficient and reliable approach for screening germline variants; however, its clinical utility is subject to the quality of annotation of clinically relevant variants.
Assuntos
Análise Mutacional de DNA/métodos , Mutação em Linhagem Germinativa/genética , Idoso , Estudos de Viabilidade , Humanos , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Methylation-induced silencing of promoter CpG islands in tumor suppressor genes plays an important role in human carcinogenesis. In colorectal cancer, the CpG island methylator phenotype (CIMP) is defined as widespread and elevated levels of DNA methylation and CIMP+ tumors have distinctive clinicopathological and molecular features. In contrast, the existence of a comparable CIMP subtype in gastric cancer (GC) has not been clearly established. To further investigate this issue, in the present study we performed comprehensive DNA methylation profiling of a well-characterised series of primary GC. METHODS: The methylation status of 1,421 autosomal CpG sites located within 768 cancer-related genes was investigated using the Illumina GoldenGate Methylation Panel I assay on DNA extracted from 60 gastric tumors and matched tumor-adjacent gastric tissue pairs. Methylation data was analysed using a recursively partitioned mixture model and investigated for associations with clinicopathological and molecular features including age, Helicobacter pylori status, tumor site, patient survival, microsatellite instability and BRAF and KRAS mutations. RESULTS: A total of 147 genes were differentially methylated between tumor and matched tumor-adjacent gastric tissue, with HOXA5 and hedgehog signalling being the top-ranked gene and signalling pathway, respectively. Unsupervised clustering of methylation data revealed the existence of 6 subgroups under two main clusters, referred to as L (low methylation; 28% of cases) and H (high methylation; 72%). Female patients were over-represented in the H tumor group compared to L group (36% vs 6%; P = 0.024), however no other significant differences in clinicopathological or molecular features were apparent. CpG sites that were hypermethylated in group H were more frequently located in CpG islands and marked for polycomb occupancy. CONCLUSIONS: High-throughput methylation analysis implicates genes involved in embryonic development and hedgehog signaling in gastric tumorigenesis. GC is comprised of two major methylation subtypes, with the highly methylated group showing some features consistent with a CpG island methylator phenotype.
Assuntos
Adenocarcinoma/genética , Ilhas de CpG , Metilação de DNA , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Gástricas/genética , Adenocarcinoma/complicações , Adenocarcinoma/metabolismo , Fatores Etários , Idoso , Estudos de Casos e Controles , Ciclina A1/genética , Feminino , Infecções por Helicobacter/complicações , Helicobacter pylori , Proteínas de Homeodomínio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Proteínas de Membrana/genética , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Fenótipo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Fatores Sexuais , Transdução de Sinais , Neoplasias Gástricas/complicações , Neoplasias Gástricas/metabolismo , Proteínas ras/genéticaRESUMO
INTRODUCTION: We developed an analytic strategy that correlates gene expression and clinical outcomes as a means to identify novel candidate oncogenes operative in breast cancer. This analysis, followed by functional characterization, resulted in the identification of Jumonji Domain Containing 6 (JMJD6) protein as a novel driver of oncogenic properties in breast cancer. METHODS: Through microarray informatics, Cox proportional hazards regression was used to analyze the correlation between gene expression and distant metastasis-free survival (DMFS) of patients in 14 independent breast cancer cohorts. JMJD6 emerged as a top candidate gene robustly associated with poor patient survival. Immunohistochemistry, siRNA-mediated silencing, and forced overexpression of JMJD6 in cell-based assays elucidated molecular mechanisms of JMJD6 action in breast cancer progression and shed light on the clinical breast cancer subtypes relevant to JMJD6 action. RESULTS: JMJD6 was expressed at highest levels in tumors associated with worse outcomes, including ER- and basal-like, Claudin-low, Her2-enriched, and ER+ Luminal B tumors. High nuclear JMJD6 protein was associated with ER negativity, advanced grade, and poor differentiation in tissue microarrays. Separation of ER+/LN- patients that received endocrine monotherapy indicated that JMJD6 is predictive of poor outcome in treatment-specific subgroups. In breast cancer cell lines, loss of JMJD6 consistently resulted in suppressed proliferation but not apoptosis, whereas forced stable overexpression increased growth. In addition, knockdown of JMJD6 in invasive cell lines, such as MDA-MB231, decreased motility and invasion, whereas overexpression in MCF-7 cells slightly promoted motility but did not confer invasive growth. Microarray analysis showed that the most significant transcriptional changes occurred in cell-proliferation genes and genes of the TGF-ß tumor-suppressor pathway. High proliferation was characterized by constitutively high cyclin E protein levels. The inverse relation of JMJD6 expression with TGF-ß2 could be extrapolated to the breast cancer cohorts, suggesting that JMJD6 may affect similar pathways in primary breast cancer. CONCLUSIONS: JMJD6 is a novel biomarker of tumor aggressiveness with functional implications in breast cancer growth and migration.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ciclina E/biossíntese , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Prognóstico , Interferência de RNA , RNA Interferente Pequeno , Fator de Crescimento Transformador beta2/genéticaRESUMO
The occurrence of neurotrophic tyrosine receptor kinase (NTRK) gene fusions in a wide range of tumor types presents an attractive opportunity for using a tropomyosin receptor kinase (TRK) inhibitor as cancer therapy. Recent clinical studies have demonstrated highly efficacious outcomes associated with the use of TRK inhibitors, such as larotrectinib and entrectinib in NTRK fusion-bearing cancers, in both adult and pediatric populations. While NTRK gene fusions are commonly found in some uncommon adult and pediatric malignancies, they are also found, albeit rarely, in a wide range of more common malignancies. The potential value of testing for NTRK gene fusions in practically all advanced malignancies is underpinned by the remarkable therapeutic outcomes that TRK inhibitors offer. This requirement presents practical and financial challenges in real-world oncological practice. Furthermore, different testing platforms exist to detect NTRK gene fusions, each with its advantages and disadvantages. It is, therefore, imperative to develop strategies for NTRK gene fusion testing in an attempt to optimize the use of limited tissue specimen and financial resources, and to minimize the turnaround time. A multidisciplinary task force of Singapore medical experts in both public and private sectors was convened in late 2020 to propose testing algorithms for adult colorectal tumors, sarcomas, non-small cell lung cancer, and pediatric cancers, with particular adaptation to the Singapore oncological practice. The recommendations presented here highlight the heterogeneity of NTRK-fusion positive cancers, and emphasize the need to customize the testing methods to each tumor type to optimize the workflow.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Neoplasias , Adulto , Algoritmos , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Criança , Consenso , Fusão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/uso terapêutico , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/uso terapêutico , Receptor trkA/genética , Receptor trkA/uso terapêutico , Receptor trkB/genética , Receptor trkB/uso terapêutico , SingapuraRESUMO
IRF4 is a master regulator of immunity and is also frequently overexpressed in mature lymphoid neoplasms. Here, we demonstrate the oncogenicity of IRF4 in vivo, its potential effects on T-cell development and clonal evolution using a zebrafish model. IRF4-transgenic zebrafish develop aggressive tumors with massive infiltration of abnormal lymphocytes that spread to distal organs. Many late-stage tumors are mono- or oligoclonal, and tumor cells can expand in recipient animals after transplantation, demonstrating their malignancy. Mutation of p53 accelerates tumor onset, increases penetrance, and results in tumor heterogeneity. Surprisingly, single-cell RNA-sequencing reveals that the majority of tumor cells are double-negative T-cells, many of which express tcr-γ that became dominant as the tumors progress, whereas double-positive T-cells are largely diminished. Gene expression and epigenetic profiling demonstrates that gata3, mycb, lrrn1, patl1 and psip1 are specifically activated in tumors, while genes responsible for T-cell differentiation including id3 are repressed. IRF4-driven tumors are sensitive to the BRD inhibitor.
Assuntos
Linfoma de Células T , Linfoma , Animais , Diferenciação Celular , Evolução Clonal , Linfoma/genética , Peixe-Zebra/genéticaRESUMO
PURPOSE: Precision oncology, such as next generation sequencing (NGS) molecular analysis and bioinformatics are used to guide targeted therapies. The laboratory turnaround time (TAT) is a key performance indicator of laboratory performance. This study aims to formally apply statistical process control (SPC) methods such as CUSUM and EWMA to a precision medicine programme to analyze the learning curves of NGS and bioinformatics processes. PATIENTS AND METHODS: Trends in NGS and bioinformatics TAT were analyzed using simple regression models with TAT as the dependent variable and chronologically-ordered case number as the independent variable. The M-estimator "robust" regression and negative binomial regression were chosen to serve as sensitivity analyses to each other. Next, two popular statistical process control (SPC) approaches which are CUSUM and EWMA were utilized and the CUSUM log-likelihood ratio (LLR) charts were also generated. All statistical analyses were done in Stata version 16.0 (StataCorp), and nominal P < 0.05 was considered to be statistically significant. RESULTS: A total of 365 patients underwent successful molecular profiling. Both the robust linear model and negative binomial model showed statistically significant reductions in TAT with accumulating experience. The EWMA and CUSUM charts of overall TAT largely corresponded except that the EWMA chart consistently decreased while the CUSUM analyses indicated improvement only after a nadir at the 82nd case. CUSUM analysis found that the bioinformatics team took a lower number of cases (54 cases) to overcome the learning curve compared to the NGS team (85 cases). CONCLUSION: As NGS and bioinformatics lead precision oncology into the forefront of cancer management, characterizing the TAT of NGS and bioinformatics processes improves the timeliness of data output by potentially spotlighting problems early for rectification, thereby improving care delivery.
RESUMO
Early relapse after platinum chemotherapy in epithelial ovarian cancer (EOC) portends poor survival. A-priori identification of platinum resistance is therefore crucial to improve on standard first-line carboplatin-paclitaxel treatment. The DNA repair pathway homologous recombination (HR) repairs platinum-induced damage, and the HR recombinase RAD51 is overexpressed in cancer. We therefore designed a REMARK-compliant study of pre-treatment RAD51 expression in EOC, using fluorescent quantitative immunohistochemistry (qIHC) to overcome challenges in quantitation of protein expression in situ. In a discovery cohort (n = 284), RAD51-High tumours had shorter progression-free and overall survival compared to RAD51-Low cases in univariate and multivariate analyses. The association of RAD51 with relapse/survival was validated in a carboplatin monotherapy SCOTROC4 clinical trial cohort (n = 264) and was predominantly noted in HR-proficient cancers (Myriad HRDscore < 42). Interestingly, overexpression of RAD51 modified expression of immune-regulatory pathways in vitro, while RAD51-High tumours showed exclusion of cytotoxic T cells in situ. Our findings highlight RAD51 expression as a determinant of platinum resistance and suggest possible roles for therapy to overcome immune exclusion in RAD51-High EOC. The qIHC approach is generalizable to other proteins with a continuum instead of discrete/bimodal expression.
Assuntos
Neoplasias Ovarianas , Platina , Carcinoma Epitelial do Ovário/tratamento farmacológico , Feminino , Humanos , Recidiva Local de Neoplasia , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel , Rad51 Recombinase/genéticaRESUMO
BACKGROUND: Differentiating between malignant and normal cells within tissue samples is vital for molecular profiling of cancer using advances in genomics and transcriptomics. Cell-surface markers of tumour-normal discrimination have additional value in terms of translatability to diagnostic and therapeutic strategies. In gastric cancer (GC), previous studies have identified individual genes or proteins that are upregulated in cancer. However, a systematic analysis of cell-surface markers and development of a composite panel involving multiple candidates to differentiate tumour from normal has not been previously reported. METHODS: Whole transcriptome sequencing (WTS) of GC and matched normal samples from the Singapore Gastric Cancer Consortium (SGCC) was used as a discovery cohort to identify upregulated putative cell-surface proteins. Matched WTS data from the The Cancer Genome Atlas (TCGA) was used as a validation cohort. Promising candidates from this analysis were validated orthogonally using multispectral immunohistochemistry (mIHC) with automated quantitative analysis using the Vectra platform. mIHC was performed on a tissue microarray containing matched normal, marginal and tumour tissues. The receiver-operating characteristic (ROC) curves were analysed to identify markers with the highest diagnostic validity independently and in combination. RESULTS: Analysis of putative membrane protein transcripts from the SGCC discovery cohort WTS data (n=15 matched tumour and normal pairs) identified several differentially and highly expressed candidates in tumour compared with normal tissues. After validation with TCGA data (n=29 matched tumour and normal pairs), the following proteins were selected for mIHC analysis: CEACAM5, CEACAM6, CLDN4, CLDN7, and EpCAM. These were compared with established glycoprotein markers in GC, namely CA19-9 and CA72-4. Individual ROC curves yielded the best performance for CEACAM5 (area under the ROC curve (AUC)=0.80), CEACAM6 (AUC=0.82), EpCAM (AUC=0.83), and CA72-4 (AUC=0.76). Combined multiplexed imaging of these four markers revealed improved specificity and sensitivity for detection of tumour from normal tissue (AUC of 4-plex=0.91). CONCLUSION: CEAMCAM5, CEACAM6, EpCAM, and CA72-4 form a versatile set of markers for robust discrimination of GC from adjacent normal tissue. As cell-surface markers, they are compatible with both IHC and live imaging approaches. These candidates may be exploited to improve automated identification of tumour tissue in GC.