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We report the case of a patient with a BRAFV600E mutant mCRC, with evidence of acquired 'NeoBRAF wild-type' (wt) state. The patient, longitudinally assessed by liquid biopsy, obtained a remarkable clinical outcome with a multimodal approach including surgery, systemic treatment and targeted therapy. In patients with newly diagnosed RAS and BRAFV600E mutant mCRC, longitudinal assessment with liquid biopsy is not routinely used in clinical practice. We report the case of a patient with a BRAFV600E mutant mCRC, with evidence of acquired 'neoBRAF wild-type' (wt) state. The patient obtained a remarkable clinical outcome and has been longitudinally assessed by liquid biopsy.
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Epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) patients treated with EGFR-tyrosine kinase inhibitors (TKIs) inevitably develop resistance through several biological mechanisms. However, little is known on the molecular mechanisms underlying acquired resistance to suboptimal EGFR-TKI doses, due to pharmacodynamics leading to inadequate drug exposure. To evaluate the effects of suboptimal EGFR-TKI exposure on resistance in NSCLC, we obtained HCC827 and PC9 cell lines resistant to suboptimal fixed and intermittent doses of gefitinib and compared them to cells exposed to higher doses of the drug. We analyzed the differences in terms of EGFR signaling activation and the expression of epithelial-mesenchymal transition (EMT) markers, whole transcriptomes byRNA sequencing, and cell motility. We observed that the exposure to low doses of gefitinib more frequently induced a partial EMT associated with an induced migratory ability, and an enhanced transcription of cancer stem cell markers, particularly in the HCC827 gefitinib-resistant cells. Finally, the HCC827 gefitinib-resistant cells showed increased secretion of the EMT inducer transforming growth factor (TGF)-ß1, whose inhibition was able to partially restore gefitinib sensitivity. These data provide evidence that different levels of exposure to EGFR-TKIs in tumor masses might promote different mechanisms of acquired resistance.
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Carcinoma Pulmonar de Células não Pequenas , Movimento Celular , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Receptores ErbB , Gefitinibe , Neoplasias Pulmonares , Inibidores de Proteínas Quinases , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Gefitinibe/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antineoplásicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
MOTIVATION: Assessment of genetic mutations is an essential element in the modern era of personalized cancer treatment. Our strategy is focused on 'multiple network analysis' in which we try to improve cancer diagnostics by using biological networks. Genetic alterations in some important hubs or in driver genes such as BRAF and TP53 play a critical role in regulating many important molecular processes. Most of the studies are focused on the analysis of the effects of single mutations, while tumors often carry mutations of multiple driver genes. The aim of this work is to define an innovative bioinformatics pipeline focused on the design and analysis of networks (such as biomedical and molecular networks), in order to: (1) improve the disease diagnosis; (2) identify the patients that could better respond to a given drug treatment; and (3) predict what are the primary and secondary effects of gene mutations involved in human diseases. RESULTS: By using our pipeline based on a multiple network approach, it has been possible to demonstrate and validate what are the joint effects and changes of the molecular profile that occur in patients with metastatic colorectal carcinoma (mCRC) carrying mutations in multiple genes. In this way, we can identify the most suitable drugs for the therapy for the individual patient. This information is useful to improve precision medicine in cancer patients. As an application of our pipeline, the clinically significant case studies of a cohort of mCRC patients with the BRAF V600E-TP53 I195N missense combined mutation were considered. AVAILABILITY: The procedures used in this paper are part of the Cytoscape Core, available at (www.cytoscape.org). Data used here on mCRC patients have been published in [55]. SUPPLEMENTARY INFORMATION: A supplementary file containing a more detailed discussion of this case study and other cases is available at the journal site as Supplementary Data.
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Biomarcadores Tumorais , Biologia Computacional/métodos , Suscetibilidade a Doenças , Neoplasias/etiologia , Medicina de Precisão/métodos , Redes Reguladoras de Genes , Humanos , Redes e Vias Metabólicas , Neoplasias/metabolismo , Mapas de Interação de Proteínas , Transdução de SinaisRESUMO
Increasing evidence suggests that liquid biopsy might play a relevant role in the management of metastatic non-small cell lung cancer (NSCLC) patients. Here, we show how the Molecular Tumor Board (MTB) in our cancer center employed liquid biopsy to support therapeutic decisions in a patient with NSCLC carrying a rare EGFR mutation. A 44-year-old woman, never-smoker with an EGFR, ALK, and ROS1-negative lung adenocarcinoma and multiple brain metastases received systemic therapy and surgery before being referred to our Institute. The MTB suggested NGS testing of tumor biopsy that revealed a rare exon-20 EGFR insertion (p.His773dup; c.2315_2316insCCA) and EGFR amplification. The MTB recommended treatment with erlotinib and follow-up with liquid biopsy, by using both cell-free DNA (cfDNA) and circulating tumor cells (CTCs). An increase of EGFR mutation levels in cfDNA revealed resistance to treatment about 6 months before clinical progression. Extremely low levels of EGFR p.T790M were detected at progression. Based on preclinical data suggesting activity of osimertinib against EGFR exon-20 insertions, the MTB recommended treatment with brain and bone radiotherapy and osimertinib. A dramatic reduction of EGFR mutation levels in the cfDNA was observed after 4 weeks of treatment. The PET scan demonstrated a metabolic partial remission that was maintained for 9 months. This case supports the evidence that liquid biopsy can aid in the management of metastatic NSCLC. It also suggests that treatment with osimertinib might be a therapeutic option in patients with EGFR exon-20 insertions when a clinical trial is not available.
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Carcinoma Pulmonar de Células não Pequenas , Ácidos Nucleicos Livres , Neoplasias Pulmonares , Adulto , Compostos de Anilina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/genética , Receptores ErbB/uso terapêutico , Éxons/genética , Feminino , Humanos , Biópsia Líquida , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genéticaRESUMO
BACKGROUND: Since the first complete genome sequencing of SARS-CoV-2 in December 2019, more than 550,000 genomes have been submitted into the GISAID database. Sequencing of the SARS-CoV-2 genome might allow identification of variants with increased contagiousness, different clinical patterns and/or different response to vaccines. A highly automated next generation sequencing (NGS)-based method might facilitate an active genomic surveillance of the virus. METHODS: RNA was extracted from 27 nasopharyngeal swabs obtained from citizens of the Italian Campania region in March-April 2020 who tested positive for SARS-CoV-2. Following viral RNA quantification, sequencing was performed using the Ion AmpliSeq SARS-CoV-2 Research Panel on the Genexus Integrated Sequencer, an automated technology for library preparation and sequencing. The SARS-CoV-2 complete genomes were built using the pipeline SARS-CoV-2 RECoVERY (REconstruction of COronaVirus gEnomes & Rapid analYsis) and analysed by IQ-TREE software. RESULTS: The complete genome (100%) of SARS-CoV-2 was successfully obtained for 21/27 samples. In particular, the complete genome was fully sequenced for all 15 samples with high viral titer (> 200 copies/µl), for the two samples with a viral genome copy number < 200 but greater than 20, and for 4/10 samples with a viral load < 20 viral copies. The complete genome sequences classified into the B.1 and B.1.1 SARS-CoV-2 lineages. In comparison to the reference strain Wuhan-Hu-1, 48 total nucleotide variants were observed with 26 non-synonymous substitutions, 18 synonymous and 4 reported in untranslated regions (UTRs). Ten of the 26 non-synonymous variants were observed in ORF1ab, 7 in S, 1 in ORF3a, 2 in M and 6 in N genes. CONCLUSIONS: The Genexus system resulted successful for SARS-CoV-2 complete genome sequencing, also in cases with low viral copies. The use of this highly automated system might facilitate the standardization of SARS-CoV-2 sequencing protocols and make faster the identification of novel variants during the pandemic.
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COVID-19 , SARS-CoV-2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Itália , Sequenciamento Completo do GenomaRESUMO
Fibroblast growth factor receptors (FGFRs) are tyrosine kinase receptors involved in many biological processes. Deregulated FGFR signaling plays an important role in tumor development and progression in different cancer types. FGFR genomic alterations, including FGFR gene fusions that originate by chromosomal rearrangements, represent a promising therapeutic target. Next-generation-sequencing (NGS) approaches have significantly improved the discovery of FGFR gene fusions and their detection in clinical samples. A variety of FGFR inhibitors have been developed, and several studies are trying to evaluate the efficacy of these agents in molecularly selected patients carrying FGFR genomic alterations. In this review, we describe the most frequent FGFR aberrations in human cancer. We also discuss the different approaches employed for the detection of FGFR fusions and the potential role of these genomic alterations as prognostic/predictive biomarkers.
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Terapia de Alvo Molecular/métodos , Neoplasias/metabolismo , Neoplasias/terapia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genética , Progressão da Doença , Fusão Gênica , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Neoplasias/diagnóstico , Neoplasias/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Combination of chemotherapies (fluoropirimidines, oxaliplatin and irinotecan) with biologic drugs (bevacizumab, panitumumab, cetuximab) have improved clinical responses and survival of metastatic colorectal cancer (mCRC). However, patients' selection thorough the identification of predictive factors still represent a challange. Cetuximab (Erbitux®), a chimeric monoclonal antibody binding to the Epidermal Growth Factor Receptor (EGFR), belongs to the Immunoglobulins (Ig) grade 1 subclass able to elicite both in vitro and in vivo the Antibody-Dependent Cell-mediated Cytotoxicity (ADCC). ADCC is the cytotoxic killing of antibody-coated target cells by immunologic effectors. The effector cells express a receptor for the Fc portion of these antibodies (FcγR); genetic polymorphisms of FcγR modify the binding affinity with the Fc of IgG1. Interestingly, the high-affinity FcγRIIIa V/V is associated with increased ADCC in vitro and in vivo. Thus, ADCC could partially account for cetuximab activity. METHODS/DESIGN: CIFRA is a single arm, open-label, phase II study assessing the activity of cetuximab in combination with irinotecan and fluorouracile in FcγRIIIa V/V patients with KRAS, NRAS, BRAF wild type mCRC. The study is designed with a two-stage Simon model based on a hypothetical higher response rate (+ 10%) of FcγRIIIa V/V patients as compared to previous trials (about 60%) assuming ADCC as one of the possible mechanisms of cetuximab action. The test power is 95%, the alpha value of the I-type error is 5%. With these assumptions the sample for passing the first stage is 14 patients with > 6 responses and the final sample is 34 patients with > 18 responses to draw positive conclusions. Secondary objectives include toxicity, responses' duration, progression-free and overall survival. Furthermore, an associated translational study will assess the patients' cetuximab-mediated ADCC and characterize the tumor microenvironment. DISCUSSION: The CIFRA study will determine whether ADCC contributes to cetuximab activity in mCRC patients selected on an innovative immunological screening. Data from the translational study will support results' interpretation as well as provide new insights in host-tumor interactions and cetuximab activity. TRIAL REGISTRATION: The CIFRA trial (version 0.0, June 21, 2018) has been registered into the NIH-US National Library of Medicine, ClinicalTrials.gov database with the identifier number ( NCT03874062 ).
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Cetuximab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/uso terapêutico , Irinotecano/uso terapêutico , Receptores de IgG/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Humanos , Polimorfismo Genético , Resultado do TratamentoRESUMO
BACKGROUND: Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material. METHODS: We describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples. RESULTS: Reproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies. CONCLUSION: This methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10 ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.
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Biomarcadores Tumorais/genética , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase Multiplex , Proteínas de Fusão Oncogênica/genética , Quinase do Linfoma Anaplásico , Biópsia , Linhagem Celular Tumoral , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/patologia , Linfonodos/patologia , Masculino , Glicoproteínas de Membrana/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret/genética , Receptores Proteína Tirosina Quinases/genética , Receptor trkB/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: The number of predictive biomarkers that will be necessary to assess in clinical practice will increase with the availability of drugs that target specific molecular alterations. Therefore, diagnostic laboratories are confronted with new challenges: costs, turn-around-time and the amount of material required for testing will increase with the number of tests performed on a sample. Our consortium of European clinical research laboratories set out to test if semi-conductor sequencing provides a solution for these challenges. METHODS: We designed a multiplex PCR targeting 87 hotspot regions in 22 genes that are of clinical interest for lung and/or colorectal cancer. The gene-panel was tested by 7 different labs in their own clinical setting using ion-semiconductor sequencing. RESULTS: We analyzed 155 samples containing 112 previously identified mutations in the KRAS, EGFR en BRAF genes. Only 1 sample failed analysis due to poor quality of the DNA. All other samples were correctly genotyped for the known mutations, even as low as 2%, but also revealed other mutations. Optimization of the primers used in the multiplex PCR resulted in a uniform coverage distribution over the amplicons that allows for efficient pooling of samples in a sequencing run. CONCLUSIONS: We show that a semi-conductor based sequencing approach to stratify colon and lung cancer patients is feasible in a clinical setting.
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Neoplasias do Colo/genética , Técnicas de Genotipagem , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Pulmonares/genética , Análise Mutacional de DNA , Humanos , Reação em Cadeia da Polimerase Multiplex , Mutação , Taxa de Mutação , Reprodutibilidade dos TestesRESUMO
Increasing evidence demonstrates that target-based agents are active only in molecularly selected populations of patients. Therefore, the identification of predictive biomarkers has become mandatory to improve the clinical development of these novel drugs. Mutations of the epidermal growth factor receptor (EGFR) or rearrangements of the ALK gene in non-small-cell lung cancer, and BRAF mutations in melanoma are clear examples of driver mutations and predictive biomarkers of response to treatment with specific inhibitors. Predictive biomarkers might also identify subgroups of patients that are not likely to respond to specific drugs, as shown for KRAS mutations and anti-EGFR monoclonal antibodies in colorectal carcinoma. The discovery of novel driver molecular alterations and the availability of drugs capable to selectively block such oncogenic mechanisms are leading to a rapid increase in the number of putative biomarkers that need to be assessed in each single patient. In this respect, two different approaches are being developed to introduce a comprehensive molecular characterization in clinical practice: high throughput genotyping platforms, which allow the detection of recognized genetic aberrations in clinical samples, and next generation sequencing that can provide information on all the different types of cancer-causing alterations. The introduction of these techniques in clinical practice will increase the possibility to identify molecular targets in each individual patient, and will also allow to follow the molecular evolution of the disease during the treatment. By using these approaches, the development of personalized medicine for patients with cancer will finally become possible.
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Terapia de Alvo Molecular , Neoplasias/diagnóstico , Neoplasias/terapia , Patologia Molecular , Medicina de Precisão , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , PrognósticoRESUMO
Analysis of plasma-derived cell-free DNA (cfDNA) might allow for the early identification of resistance in metastatic colorectal carcinoma (mCRC) patients receiving anti-EGFR monoclonal antibodies. We tested plasma samples from the Erbitux Metastatic Colorectal Cancer Strategy (ERMES) phase III trial of FOLFIRI+Cetuximab in first-line treatment of RAS/BRAF wild-type mCRC. Samples were collected at baseline (n = 37), at 8 weeks of treatment (n = 32), progressive disease (PD; n = 36) and 3 months after PD (n = 21). cfDNA testing was performed using the Idylla™ ctKRAS and ctNRAS-BRAF tests and the Oncomine Pan-Cancer Cell-Free Assay. Analysis of basal samples revealed RAS/BRAF mutations in 6/37 cases. A transient RAS positivity not associated with PD was observed at 8 weeks in five cases that showed no mutations at baseline and PD. The frequency of mutant cases increased at PD (33.3%) and decreased again at 3 months after PD (9.5%). The median progression-free survival (mPFS) of patients RAS/BRAF mutant at PD was 7.13 months versus 7.71 months in wild-type patients (p = 0.3892). These data confirm that the occurrence of RAS/BRAF mutations in mCRC patients receiving anti-EGFR agents is relatively frequent. However, the cfDNA dynamics of RAS mutations in patients treated with anti-EGFR agents plus polychemotherapy are complex and might not be directly associated with resistance to treatment.
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Triple negative breast cancer is a subtype of breast cancer that lacks expression of an estrogen receptor (ER), a progesterone receptor (PR), and HER2. It is characterized by its unique molecular profile, aggressive behavior, and distinct pattern of metastasis. Epidemiological studies show a high prevalence of triple negative breast cancer among younger women and those of African descent. Although sensitive to chemotherapy, early relapse is common, and a predilection for visceral metastasis, including brain metastasis, has been described. Gene-expression profiling approaches demonstrated that triple negative breast cancer is a heterogeneous group of diseases composed of different, molecularly distinct subtypes. Although not synonymous, the majority of triple negative breast cancers carry the "basal-like" molecular profile on gene-expression arrays. However, several studies have shown that triple negative breast cancer includes tumors with a non-basal expression profile and, in particular, the "normal-breast," the "multiple marker negative," and the recently identified "claudin-negative" subtypes. Target-based agents, including epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and poly-ADP-ribose polymerase (PARP) inhibitors, are currently in clinical trials and hold promise in the treatment of this aggressive disease.
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Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Receptor ErbB-2/deficiência , Receptores de Estrogênio/deficiência , Receptores de Progesterona/deficiência , Neoplasias da Mama/patologia , Mapeamento Cromossômico , Hibridização Genômica Comparativa , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes erbB-2 , Humanos , Células-Tronco Neoplásicas/patologia , Receptor ErbB-2/genética , Receptores de Estrogênio/genética , Receptores de Progesterona/genéticaRESUMO
Treatment of breast cancer cells with a combination of the EGFR-tyrosine kinase inhibitor (EGFR-TKI) gefitinib and the anti-ErbB-2 monoclonal antibody trastuzumab results in a synergistic antitumor effect. In this study, we addressed the mechanisms involved in this phenomenon. The activation of signaling pathways and the expression of cell cycle regulatory proteins were studied in SK-Br-3 and BT-474 breast cancer cells, following treatment with EGFR and/or ErbB-2 inhibitors. Treatment with the gefitinib/trastuzumab combination produced, as compared with a single agent, a more prolonged blockade of AKT and MAPK activation, a more pronounced accumulation of cells in the G0/G1 phase of the cell cycle, a more significant increase in the levels of p27(kip1) and of hypophosphorylated pRb2, and a decrease in the levels of Cyclin D1 and survivin. Similar findings were observed with the EGFR/ErbB-2 inhibitor lapatinib. Gefitinib, trastuzumab, and their combination increased the stability of p27(kip1), with the combination showing the highest effects. Blockade of both receptors with gefitinib/trastuzumab or lapatinib induced a significant increase in the levels of p27(kip1) mRNA and in the nuclear levels of the p27(kip1) transcription factor FKHRL-1. Inhibition of PI3K signaling also produced a significant raise in p27(kip1) mRNA. Finally, down-modulation of FKHRL-1 with siRNAs prevented the lapatinib-induced increase of p27(kip1) mRNA. The synergism deriving from EGFR and ErbB-2 blockade is mediated by several different alterations in the activation of signaling proteins and in the expression of cell cycle regulatory proteins, including transcriptional and posttranscriptional regulation of p27(kip1) expression.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/enzimologia , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptor ErbB-2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27 , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Gefitinibe , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lapatinib , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/farmacologia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/metabolismo , Proteína p130 Retinoblastoma-Like/metabolismo , Fatores de Tempo , TrastuzumabRESUMO
Colorectal carcinoma (CRC) is an heterogeneous disease in which different genetic alterations play a role in its pathogenesis and progression and offer potential for therapeutic intervention. The research on predictive biomarkers in metastatic CRC (mCRC) mainly focused on the identification of biomarkers of response or resistance to anti-epidermal growth factor receptor monoclonal antibodies. In this respect, international guidelines suggest testing mCRC patients only for KRAS, NRAS and BRAF mutations and for microsatellite instability. However, the use of novel testing methods is raising relevant issue related to these biomarkers, such as the presence of sub-clonal RAS mutations or the clinical interpretation of rare no-V600 BRAF variants. In addition, a number of novel biomarkers is emerging from recent studies including amplification of ERBB2, mutations in ERBB2, MAP2K1 and NF1 and rearrangements of ALK, ROS1, NTRK and RET. Mutations in POLE and the levels of tumor mutation burden also appear as possible biomarkers of response to immunotherapy in CRC. Finally, the consensus molecular subtypes classification of CRC based on gene expression profiling has prognostic and predictive implications. Integration of all these information will be likely necessary in the next future in order to improve precision/personalized medicine in mCRC patients.
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BACKGROUND: Colorectal cancer (CRC) is the second most common cause of cancer-specific death in both sexes in Western countries. KRAS mutations occur in about 50% of metastatic CRCs (mCRCs). The prognostic value of specific KRAS mutations still remains unexplored and unclear. METHODS: Two hundred and forty KRAS wild-type and 206 KRAS/NRAS mutant consecutive unresectable mCRC patients with PS Eastern Cooperative Oncology Group (ECOG) 0 or 1, aged < 80 years, and with a life expectancy >3 months entered into this study. DNA was extracted from paraffin-embedded formalin-fixed tumour tissues, and it was sequenced with the Oncomine Solid Tumour DNA kit (Thermo Fisher Scientific, Waltham, MA, USA). Data were analysed using the Torrent Suite Software v5.0 (Thermo Fisher Scientific). The primary outcome was the analysis of the prognostic role of different KRAS mutations in terms of overall survival (OS). RESULTS: There were no significant differences among the most prevalent mutations (p.G12D, p.G12V, p.G13D, p.G12A, p.G12C, and p.G12S) in terms of age (<65 vs. ≥65 years), gender (male vs. female), grading (G1/G2 vs. G3), side of primary tumour (left vs. right), pT, and pN. At the median follow-up of 25.6 months, there were 77 deaths in KRAS-mutated patients and 94 in wild-type patients. Three homogeneous prognostic groups were identified: wild-type patients (group A, median survival: 27.5 months), p.G13D/p.G12A/p.G12V/p.G12D mutants (group B, median survival: 17.3 months), and p.G12C/p.G12S mutants (group C, median survival: 5.0 months, p < 0.0001 according to Log Rank test). Upon multivariate analysis, metastatic involvement and p.G12C/p.G12S KRAS mutation group C (vs. other mutations) emerged as independent prognostic variables for survival. CONCLUSIONS: We show that mutant KRAS is a negative prognostic factor and that p.G12C/p.G12S variants present the worst clinical courses. This information suggests a clear difference among KRAS mutations, and it will be useful to test potentiated and/or innovative therapeutic strategies in p.G12C/p.G12S metastatic CRC patients.
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INTRODUCTION: Circulating cell-free DNA (cfDNA) testing has emerged as an alternative to tumor tissue analyses for the management of metastatic non-small-cell lung cancer (NSCLC) patients. Analysis of cfDNA is a minimally invasive procedure that might better reflect tumor heterogeneity and allows repeated testing over the time. Areas covered: This review article covers the different applications of cfDNA testing in NSCLC: early diagnosis of the disease; detection of minimal residual disease in early lung cancer; identification of predictive and prognostic markers in advanced NSCLC patients; monitoring the response to therapy; assessment of tumor mutation burden. Expert commentary: The use of liquid biopsy is rapidly expanding to different applications. The combination of different circulating biomarkers (cfDNA, protein, miRNA) might improve the sensitivity and specificity of this approach in patients with low tumor burden. cfDNA testing is representing a valid source for molecular profiling in management of metastatic NSCLC patients and is providing important knowledge on tumor heterogeneity. Clinical trials are needed in order to transfer the information deriving from liquid biopsy testing in new therapeutic strategies.
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Carcinoma Pulmonar de Células não Pequenas/diagnóstico , DNA Tumoral Circulante/genética , Neoplasias Pulmonares/diagnóstico , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Detecção Precoce de Câncer/métodos , Humanos , Biópsia Líquida/métodos , Neoplasias Pulmonares/genética , Prognóstico , Sensibilidade e EspecificidadeRESUMO
Previous findings suggest that metastatic colorectal carcinoma (mCRC) patients with KRAS/NRAS/BRAF/PIK3CA wild-type (quadruple-wt) tumors are highly sensitive to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs). However, additional molecular alterations might be involved in the de novo resistance to these drugs. We performed a comprehensive molecular profiling of 21 quadruple-wt tumors from mCRC patients enrolled in the "Cetuximab After Progression in KRAS wild-type colorectal cancer patients" (CAPRI-GOIM) trial of first line FOLFIRI plus cetuximab. Tumor samples were analyzed with a targeted sequencing panel covering single nucleotide variants (SNVs), insertions/deletions (Indels), copy number variations (CNVs), and gene fusions in 143 cancer-related genes. The analysis revealed in all 21 patients the presence of at least one SNV/Indel and in 10/21 cases (48%) the presence of at least one CNV. Furthermore, 17/21 (81%) patients had co-existing SNVs/Indels in different genes. Quadruple-wt mCRC from patients with the shorter progression free survival (PFS) were enriched with peculiar genetic alterations in KRAS, FBXW7, MAP2K1, and NF1 genes as compared with patients with longer PFS. These data suggest that a wide genetic profiling of quadruple-wt mCRC patients might help to identify novel markers of de novo resistance to anti-EGFR MoAbs.
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Recent findings suggest that a fraction of EGFR-mutant non-small-cell lung cancers (NSCLC) carry additional driver mutations that could potentially affect the activity of EGFR tyrosine kinase inhibitors (TKIs). We investigated the role of concomitant KRAS, NRAS, BRAF, PIK3CA, MET and ERBB2 mutations (other mutations) on the outcome of 133 EGFR mutant patients, who received first-line therapy with EGFR TKIs between June 2008 and December 2014. Analysis of genomic DNA by Next Generation Sequencing (NGS) revealed the presence of hotspot mutations in genes other than the EGFR, including KRAS, NRAS, BRAF, ERBB2, PIK3CA, or MET, in 29/133 cases (21.8%). A p.T790M mutation was found in 9/133 tumour samples (6.8%). The progression free survival (PFS) of patients without other mutations was 11.3 months vs. 7 months in patients with other mutations (log-rank test univariate: p = 0.047). In a multivariate Cox regression model including the presence of other mutations, age, performance status, smoking status, and the presence of p.T790M mutations, the presence of other mutations was the only factor significantly associated with PFS (Hazard Ratio 1.63, 95% CI 1.04â»2.58; p = 0.035). In contrast, no correlation was found between TP53 mutations and patients' outcome. These data suggest that a subgroup of EGFR mutant tumours have concomitant driver mutations that might affect the activity of first-line EGFR TKIs.
RESUMO
The EPHA2 tyrosine kinase receptor is implicated in tumor progression and targeted therapies resistance. We evaluated EPHA2 as a potential resistance marker to the antiepidermal growth factor receptor (EGFR) monoclonal antibody cetuximab in colorectal cancer. We studied activation of EPHA2 in a panel of human colorectal cancer cell lines sensitive or resistant to anti-EGFR drugs. The in vitro and in vivo effects of ALW-II-41-27 (an EPHA2 inhibitor) and/or cetuximab treatment were tested. Formalin-fixed paraffin-embedded tumor specimens from 82 RAS wild-type (WT) metastatic colorectal cancer patients treated with FOLFIRI + cetuximab as first-line therapy in the CAPRI-GOIM trial were assessed for EPHA2 expression by immunohistochemistry and correlated with treatment efficacy. EPHA2 was differentially activated in colorectal cancer cell lines. Combined treatment with ALW-II-41-27 plus cetuximab reverted primary and acquired resistance to cetuximab, causing cell growth inhibition, inducing apoptosis and cell-cycle G1-G2 arrest. In tumor xenograft models, upon progression to cetuximab, ALW-II-41-27 addition significantly inhibited tumor growth. EPHA2 protein expression was detected in 55 of 82 tumor samples, frequently expressed in less-differentiated and left-sided tumors. High levels of EPHA2 significantly correlated with worse progression-free survival [8.6 months; confidence interval (CI) 95%, 6.4-10.8; vs. 12.3 months; CI 95%, 10.4-14.2; P = 0.03] and with increased progression rate (29% vs. 9%, P = 0.02). A specific EPHA2 inhibitor reverts in vitro and in vivo primary and acquired resistance to anti-EGFR therapy. EPHA2 levels are significantly associated with worse outcome in patients treated with FOLFIRI + cetuximab. These results highlight EPHA2 as a potential therapeutic target in metastatic colorectal cancer.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Efrina-A2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos Imunológicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Biomarcadores Tumorais/metabolismo , Camptotecina/análogos & derivados , Camptotecina/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cetuximab/administração & dosagem , Neoplasias Colorretais/patologia , Efrina-A2/antagonistas & inibidores , Receptores ErbB/antagonistas & inibidores , Feminino , Fluoruracila/uso terapêutico , Humanos , Leucovorina/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Intervalo Livre de Progressão , Interferência de RNA , Receptor EphA2 , Transdução de Sinais/efeitos dos fármacos , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The adipocyte-derived peptide leptin acts through binding to specific membrane receptors, of which six isoforms (obRa-f) have been identified up to now. Binding of leptin to its receptor induces activation of different signaling pathways, including the JAK/STAT, MAPK, IRS1, and SOCS3 signaling pathways. Since the circulating levels of leptin are elevated in obese individuals, and excess body weight has been shown to increase breast cancer risk in postmenopausal women, several studies addressed the role of leptin in breast cancer. Expression of leptin and its receptors has been demonstrated to occur in breast cancer cell lines and in human primary breast carcinoma. Leptin is able to induce the growth of breast cancer cells through activation of the Jak/STAT3, ERK1/2, and/or PI3K pathways, and can mediate angiogenesis by inducing the expression of vascular endothelial growth factor (VEGF). In addition, leptin induces transactivation of ErbB-2, and interacts in triple negative breast cancer cells with insulin like growth factor-1 (IGF-1) to transactivate the epidermal growth factor receptor (EGFR), thus promoting invasion and migration. Leptin can also affect the growth of estrogen receptor (ER)-positive breast cancer cells, by stimulating aromatase expression and thereby increasing estrogen levels through the aromatization of androgens, and by inducing MAPK-dependent activation of ER. Taken together, these findings suggest that the leptin system might play an important role in breast cancer pathogenesis and progression, and that it might represent a novel target for therapeutic intervention in breast cancer.