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1.
J Transl Med ; 21(1): 725, 2023 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-37845764

RESUMO

BACKGROUND: Molecular Tumor Boards (MTB) operating in real-world have generated limited consensus on good practices for accrual, actionable alteration mapping, and outcome metrics. These topics are addressed herein in 124 MTB patients, all real-world accrued at progression, and lacking approved therapy options. METHODS: Actionable genomic alterations identified by tumor DNA (tDNA) and circulating tumor DNA (ctDNA) profiling were mapped by customized OncoKB criteria to reflect diagnostic/therapeutic indications as approved in Europe. Alterations were considered non-SoC when mapped at either OncoKB level 3, regardless of tDNA/ctDNA origin, or at OncoKB levels 1/2, provided they were undetectable in matched tDNA, and had not been exploited in previous therapy lines. RESULTS: Altogether, actionable alterations were detected in 54/124 (43.5%) MTB patients, but only in 39 cases (31%) were these alterations (25 from tDNA, 14 from ctDNA) actionable/unexploited, e.g. they had not resulted in the assignment of pre-MTB treatments. Interestingly, actionable and actionable/unexploited alterations both decreased (37.5% and 22.7% respectively) in a subset of 88 MTB patients profiled by tDNA-only, but increased considerably (77.7% and 66.7%) in 18 distinct patients undergoing combined tDNA/ctDNA testing, approaching the potential treatment opportunities (76.9%) in 147 treatment-naïve patients undergoing routine tDNA profiling for the first time. Non-SoC therapy was MTB-recommended to all 39 patients with actionable/unexploited alterations, but only 22 (56%) accessed the applicable drug, mainly due to clinical deterioration, lengthy drug-gathering procedures, and geographical distance from recruiting clinical trials. Partial response and stable disease were recorded in 8 and 7 of 19 evaluable patients, respectively. The time to progression (TTP) ratio (MTB-recommended treatment vs last pre-MTB treatment) exceeded the conventional Von Hoff 1.3 cut-off in 9/19 cases, high absolute TTP and Von Hoff values coinciding in 3 cases. Retrospectively, 8 patients receiving post-MTB treatment(s) as per physician's choice were noted to have a much longer overall survival from MTB accrual than 11 patients who had received no further treatment (35.09 vs 6.67 months, p = 0.006). CONCLUSIONS: MTB-recommended/non-SoC treatments are effective, including those assigned by ctDNA-only alterations. However, real-world MTBs may inadvertently recruit patients electively susceptible to diverse and/or multiple treatments.


Assuntos
Neoplasias , Estados Unidos , Humanos , National Cancer Institute (U.S.) , Estudos Retrospectivos , Mutação , Neoplasias/genética , DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biomarcadores Tumorais/genética
2.
J Biomed Inform ; 142: 104394, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37209976

RESUMO

The Biomedical Research field is currently advancing to develop Clinical Trials and translational projects based on Real World Evidence. To make this transition feasible, clinical centers need to work toward Data Accessibility and Interoperability. This task is particularly challenging when applied to Genomics, that entered in routinary screening in the last years via mostly amplicon-based Next-Generation Sequencing panels. Said experiments produce up to hundreds of features per patient, and their summarized results are often stored in static clinical reports, making critical information inaccessible to automated access and Federated Search consortia. In this study, we present a reanalysis of 4620 solid tumor sequencing samples in five different histology settings. Furthermore, we describe all the Bioinformatics and Data Engineering processes that were put in place in order to create a Somatic Variant Registry able to deal with the large biotechnological variability of routinary Genomics Profiling.


Assuntos
Pesquisa Biomédica , Neoplasias , Humanos , Genômica , Biologia Computacional/métodos , Sistema de Registros , Neoplasias/diagnóstico , Neoplasias/genética
3.
Cancers (Basel) ; 14(12)2022 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-35740637

RESUMO

The approval of osimertinib for adjuvant treatment of stage I-II-III EGFR-mutated NSCLC (early stage) represents a paradigm shift, raising the question of whether other genotype-matched therapeutics approved for advanced-stage NSCLC can also provide clinical benefit in the adjuvant setting. However, there is a paucity of real-world data on the prevalence of actionable genomic alterations (GAs) in early-stage NSCLC. We used next-generation sequencing, complemented by immunohistochemistry and fluorescence in situ hybridization, to screen our single-institution cohort of 1961 NSCLC consecutive cases for actionable molecular targets. The prevalence of actionable GAs was comparable in early versus advanced-stage NSCLC, the only exception being KRAS mutations (more frequent in early-stage cases). Consistent with advanced-stage tumors being more aggressive, co-occurrence of TP53 and EGFR GAs as well as copy number gains were less frequent in early-stage tumors. EGFR mutations and high expression of PD-L1 were inversely associated, whereas KRAS mutations and high PD-L1 reactivity showed positive association. Recapitulating advanced-stage tumors, early-stage NSCLC had the highest share of EGFR mutations in lepidic and acinar subtypes. Resected lepidic tumors contained the highest proportion of the KRAS G12C actionable variant. These results, obtained with routine diagnostic technologies in an unselected clinical setting, provide a significant addition of real-world data in early-stage NSCLC.

4.
Biosens Bioelectron ; 170: 112648, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33010708

RESUMO

RAS mutations in the blood of colorectal cancer (CRC) patients are emerging as biomarkers of acquired resistance to Epidermal Growth Factor Receptor therapy. Unfortunately, reliable assays granting fast, real-time monitoring of treatment response, capable of refining retrospective, tissue-based analysis, are still needed. Recently, several methods for detecting blood RAS mutations have been proposed, generally relying on multi-step and PCR-based, time-consuming and cost-ineffective procedures. By exploiting a liquid biopsy approach, we developed an ultrasensitive nanoparticle-enhanced plasmonic method for detecting ~1 aM RAS single nucleotide variants (SNVs) in the plasma of CRC patients. The assay does not require the extraction of tumor DNA from plasma and detects it in volumes as low as 40 µL of plasma, which is at least an order of magnitude smaller than that required by state of the art liquid biopsy technologies. The most prevalent RAS mutations are detected in DNA from tumor tissue with 100% sensitivity and 83.33% specificity. Spike-in experiments in human plasma further encouraged assay application on clinical specimens. The assay was proven in plasma from CRC patients and healthy donors, and full discrimination between mutated DNA from patients over wild-type DNA from healthy volunteers was obtained thus demonstrating its promising avenue for cancer monitoring based on liquid biopsy.


Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos Livres/isolamento & purificação , Neoplasias Colorretais , Proteínas ras/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , DNA de Neoplasias/genética , Humanos , Mutação , Reação em Cadeia da Polimerase , Estudos Retrospectivos
5.
Oncoimmunology ; 7(8): e1457602, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30221053

RESUMO

Whether PD-L1 expression is associated with survival outcomes in gastric cancer (GC) is controversial. The inhibition of the PD-1/PD-L1 pathway is effective against genomically unstable tumors. Hypothesizing that also the clinical significance of PD-L1 might be dependent on the activation of molecular circuits ensuring genomic stability, we evaluated PD-L1 expression in tissue samples from 72 advanced GC patients treated with first-line chemotherapy. Samples were already characterized for DNA damage repair (DDR) component expression (pATM, pChk1, pWee1, γ-H2AX and pRPA2) along with mutations in DDR-linked genes (TP53 and ARID1A). Overall, PD-L1 expression was not associated with progression-free survival (PFS) and overall survival (OS), independently on whether we considered its expression in tumor cells (PD-L1-TCs) or in the immune infiltrate (PD-L1-TILs). In subgroup analysis, positive PD-L1-TC immunostaining was associated with better PFS in patients whose tumors did not carry DDR activation (multivariate Cox: HR 0.34, 95%CI: 0.15-0.76, p = 0.008). This subset (DDRoff) was characterized by negative pATM expression or the presence of ARID1A mutations. Conversely, the relationship between PD-L1-TC expression and PFS was lost in a molecular scenario denoting DDR activation (DDRon), as defined by concomitant pATM expression and ARID1A wild-type form. Surprisingly, while PD-L1-TC expression was associated with better OS in the DDRoff subset (multivariate Cox: HR 0.41, 95% CI: 0.17-0.96, p = 0.039), in the DDRon subgroup we observed an opposite impact on OS (multivariate Cox: HR 2.56, 95%CI: 1.06-6.16, p = 0.036). Thus, PD-L1-TC expression may impact survival outcomes in GC on the basis of the activation/inactivation of genome-safeguarding pathways.

6.
J Transl Med ; 16(1): 247, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30180862

RESUMO

We have previously reported that nuclear expression of the Hippo transducer TAZ in association with Wnt pathway mutations negatively impacts survival outcomes in advanced gastric cancer (GC) patients. Here, we extended these previous findings by investigating another oncogenic cooperation, namely, the interplay between YAP, the TAZ paralogue, and p53. The molecular output of the YAP-p53 cooperation is dependent on TP53 mutational status. In the absence of mutations, the YAP-p53 crosstalk elicits a pro-apoptotic response, whereas in the presence of TP53 mutations it activates a pro-proliferative transcriptional program. In order to study this phenomenon, we re-analyzed data from 83 advanced GC patients treated with chemotherapy whose tissue samples had been characterized for YAP expression (immunohistochemistry, IHC) and TP53 mutations (deep sequencing). In doing so, we generated a molecular model combining nuclear YAP expression in association with TP53 missense variants (YAP+/TP53mut(mv)). Surprisingly, this signature was associated with a decreased risk of disease progression (multivariate Cox for progression-free survival: HR 0.53, 95% CI 0.30-0.91, p = 0.022). The YAP+/TP53mut(mv) model was also associated with better OS in the subgroup of patients who received chemotherapy beyond the first-line setting (multivariate Cox: HR 0.36, 95% CI 0.16-0.81, p = 0.013). Collectively, our findings suggest that the oncogenic cooperation between YAP and mutant p53 may translate into better survival outcomes. This apparent paradox can be explained by the pro-proliferative program triggered by YAP and mutant p53, that supposedly renders cancer cells more vulnerable to cytotoxic therapies.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Mutação de Sentido Incorreto , Fosfoproteínas/genética , Neoplasias Gástricas/genética , Proteína Supressora de Tumor p53/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Antineoplásicos/uso terapêutico , Proliferação de Células , Progressão da Doença , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Fosfoproteínas/metabolismo , Modelos de Riscos Proporcionais , Análise de Regressão , Neoplasias Gástricas/mortalidade , Fatores de Transcrição , Resultado do Tratamento , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Sinalização YAP
7.
Oncogenesis ; 7(7): 55, 2018 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-30032163

RESUMO

Genomic technologies are reshaping the molecular landscape of colorectal cancer (CRC), revealing that oncogenic driver mutations (APC and TP53) coexist with still underappreciated genetic events. We hypothesized that mutational analysis of CRC-linked genes may provide novel information on the connection between genetically-deregulated pathways and clinical outcomes. We performed next-generation sequencing (NGS) analysis of 16 recurrently mutated genes in CRC exploiting tissue specimens from 98 advanced CRC patients. Multiple correspondence analysis (MCA) was used to identify gene sets characterizing negative and positive outliers (patients in the lowest and highest quartile of progression-free survival, PFS). Variables potentially affecting PFS and overall survival (OS) were tested in univariate and multivariate Cox proportional hazard models. Sensitivity analyses and resampling were used to assess the robustness of genomic predictors. MCA revealed that APC and TP53 mutations were close to the negative outlier group, whereas mutations in other WNT pathway genes were in proximity of the positive outliers. Reasoning that genetic alterations interact epistatically, producing greater or weaker consequences in combination than when individually considered, we tested whether patients whose tumors carried a genetic background characterized by APC and TP53 mutations without coexisting mutations in other WNT genes (AMER1, FBXW7, TCF7L2, CTNNB1, SOX9) had adverse survival outcomes. With this approach, we identified two oncodriver signatures (ODS1 and ODS2) associated with shorter PFS (ODS1 multivariate Cox for PFS: HR 2.16, 95%CI: 1.28-3.64, p = 0.004; ODS2 multivariate Cox for PFS: HR 2.61, 95%CI: 1.49-4.58, p = 0.001). Clinically-focused and molecularly-focused sensitivity analyses, resampling, and reclassification of mutations confirmed the stability of ODS1/2. Moreover, ODS1/2 negatively impacted OS. Collectively, our results point to co-occurring driver mutations as an adverse molecular factor in advanced CRC. This relationship depends on a broader genetic context highlighting the importance of genetic interactions.

8.
Breast Cancer Res ; 20(1): 59, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29921315

RESUMO

BACKGROUND: As crucial regulators of the immune response against pathogens, macrophages have been extensively shown also to be important players in several diseases, including cancer. Specifically, breast cancer macrophages tightly control the angiogenic switch and progression to malignancy. ID4, a member of the ID (inhibitors of differentiation) family of proteins, is associated with a stem-like phenotype and poor prognosis in basal-like breast cancer. Moreover, ID4 favours angiogenesis by enhancing the expression of pro-angiogenic cytokines interleukin-8, CXCL1 and vascular endothelial growth factor. In the present study, we investigated whether ID4 protein exerts its pro-angiogenic function while also modulating the activity of tumour-associated macrophages in breast cancer. METHODS: We performed IHC analysis of ID4 protein and macrophage marker CD68 in a triple-negative breast cancer series. Next, we used cell migration assays to evaluate the effect of ID4 expression modulation in breast cancer cells on the motility of co-cultured macrophages. The analysis of breast cancer gene expression data repositories allowed us to evaluate the ability of ID4 to predict survival in subsets of tumours showing high or low macrophage infiltration. By culturing macrophages in conditioned media obtained from breast cancer cells in which ID4 expression was modulated by overexpression or depletion, we identified changes in the expression of ID4-dependent angiogenesis-related transcripts and microRNAs (miRNAs, miRs) in macrophages by RT-qPCR. RESULTS: We determined that ID4 and macrophage marker CD68 protein expression were significantly associated in a series of triple-negative breast tumours. Interestingly, ID4 messenger RNA (mRNA) levels robustly predicted survival, specifically in the subset of tumours showing high macrophage infiltration. In vitro and in vivo migration assays demonstrated that expression of ID4 in breast cancer cells stimulates macrophage motility. At the molecular level, ID4 protein expression in breast cancer cells controls, through paracrine signalling, the activation of an angiogenic programme in macrophages. This programme includes both the increase of angiogenesis-related mRNAs and the decrease of members of the anti-angiogenic miR-15b/107 group. Intriguingly, these miRNAs control the expression of the cytokine granulin, whose enhanced expression in macrophages confers increased angiogenic potential. CONCLUSIONS: These results uncover a key role for ID4 in dictating the behaviour of tumour-associated macrophages in breast cancer.


Assuntos
Neoplasias da Mama/genética , Proteínas Inibidoras de Diferenciação/genética , Neovascularização Patológica/genética , Neoplasias de Mama Triplo Negativas/genética , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Reprogramação Celular/genética , Citocinas/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Interleucina-8/genética , Macrófagos/patologia , MicroRNAs/genética , Neovascularização Patológica/patologia , Neoplasias de Mama Triplo Negativas/patologia , Fator A de Crescimento do Endotélio Vascular/genética
9.
J Exp Clin Cancer Res ; 37(1): 124, 2018 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-29941002

RESUMO

BACKGROUND: Circulating tumor DNA (ctDNA) and miRNA (ctmiRNA) are promising biomarkers for early tumor diagnosis, prognosis and monitoring, and to predict therapeutic response. However, a clear understanding of the fine control on their circulating levels is still lacking. METHODS: Three human colorectal carcinoma cell lines were grown in culture and as tumor xenograft models in nude mice. Chip-based and droplet digital PCR platforms were used to systematically and quantitatively assess the levels of DNAs and miRNAs released into the culture supernatants and mouse blood plasma. RESULTS: Strikingly, mutated DNAs from the same (KRAS) and different (PIK3CA and FBWX7) genomic loci were differentially detected in culture supernatants and blood, with LS174T releasing 25 to 60 times less DNA in culture, but giving rise to 7 to 8 times more DNA in blood than LoVo cells. Greater LS174T ctDNA accumulation occurred in spite of similar CD31 immunostaining (micro-vascularization) and lesser proliferation and tissue necrosis as compared to LoVo. As to the three selected miRNAs (miR-221, miR-222 and miR-141), all of them were constitutively present in the plasma of tumor-free mice. Micro-RNA miR-141 was released into HT-29 cell supernatants 10 and 6.5 times less abundantly with respect to LoVo and LS174T, respectively; on the contrary, release of miR-141 in blood of HT-29 xenografted mice was found similar to that observed in LoVo and LS174T mice. CONCLUSIONS: Taken together, our results support the existence of multiple, finely tuned (non-housekeeping) control gateways that selectively regulate the release/accumulation of distinct ctDNA and miRNA species in culture and tumor xenograft models. Different xenografts (proxies of different patients) considerably differ in gateway usage, adding several layers of complexity to the well-known idea of molecular heterogeneity. We predict that even high tissue representation of mutated DNA and miRNA may result in insufficient diagnostic analyte representation in blood. In this respect, our data show that careful modeling in mice may considerably help to alleviate complexity, for instance by pre-screening for the most abundant circulating analytes in enlarged sets of tumor xenografts.


Assuntos
MicroRNA Circulante , DNA Tumoral Circulante , Neoplasias Colorretais/genética , Biópsia Líquida , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Neoplasias Colorretais/sangue , Neoplasias Colorretais/diagnóstico , Modelos Animais de Doenças , Xenoenxertos , Humanos , Camundongos , Reação em Cadeia da Polimerase , Fluxo de Trabalho
10.
Ther Adv Med Oncol ; 10: 1758835918774337, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29899761

RESUMO

BACKGROUND: Limited information is available on the applicative value of liquid biopsy (LB) in rare tumors, including Ewing's sarcoma (ES). The accepted precision diagnostics standards would greatly benefit from a non-invasive LB test monitoring pathognomonic gene rearrangements in the bloodstream. METHODS: Tissue and blood samples were collected from six and four ES patients, respectively. Plasma was cleared by two successive rounds of centrifugation and stored frozen until RNA extraction by the QIAmp CNA kit. RNA was retro-transcribed and subjected to real-time quantitative polymerase chain reaction (RT-qPCR) and digital polymerase chain reaction (dPCR). Reactions were set up using two custom primer sets identifying types 1 and 2 EWS-FLI1 fusion transcripts. RESULTS: The two prevalent types of EWS-FLI1 rearrangements could be identified using only two sets of polymerase chain reaction primers, regardless of patient-specific EWS-FLI1 DNA breakpoints. RT-qPCR and dPCR discriminated the two variants in five tumor tissue RNAs and in four circulating tumor RNAs (ctRNAs). Of note, EWS-FLI1 molecular diagnosis was possible using blood samples even when tumor tissue was not available. ctRNA levels correlated (p < 0.05) with volume-based positron emission tomography (PET) parameters (metabolic tumor volume and total lesion glycolysis), and allowed the fine tracking of disease course after surgery, during adjuvant as well as neoadjuvant chemotherapy, and at follow up in one patient. CONCLUSIONS: To our knowledge, this is one of the few single-marker LB assays in solid tumors specifically designed to detect rearranged RNAs in blood, and the first study describing EWS circulating tumor RNAs in ES patients. Altogether, our results support the idea that LB may have a considerable impact on ES patient monitoring and management.

11.
J Transl Med ; 16(1): 22, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29402328

RESUMO

BACKGROUND: An extensive crosstalk co-regulates the Hippo and Wnt pathway. Preclinical studies revealed that the Hippo transducers YAP/TAZ mediate a number of oncogenic functions in gastric cancer (GC). Moreover, comprehensive characterization of GC demonstrated that the Wnt pathway is targeted by oncogenic mutations. On this ground, we hypothesized that YAP/TAZ- and Wnt-related biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. METHODS: In the present study, we included 86 patients with advanced GC treated with first-line chemotherapy in prospective phase II trials or in routine clinical practice. Tissue samples were immunostained to evaluate the expression of YAP/TAZ. Mutational status of key Wnt pathway genes (CTNNB1, APC and FBXW7) was assessed by targeted DNA next-generation sequencing (NGS). Survival curves were estimated and compared by the Kaplan-Meier product-limit method and the log-rank test, respectively. Variables potentially affecting progression-free survival (PFS) were verified in univariate Cox proportional hazard models. The final multivariate Cox models were obtained with variables testing significant at the univariate analysis, and by adjusting for all plausible predictors of the outcome of interest (PFS). RESULTS: We observed a significant association between TAZ expression and Wnt mutations (Chi-squared p = 0.008). Combined TAZ expression and Wnt mutations (TAZpos/WNTmut) was more frequently observed in patients with the shortest progression-free survival (negative outliers) (Fisher p = 0.021). Uni-and multivariate Cox regression analyses revealed that patients whose tumors harbored the TAZpos/WNTmut signature had an increased risk of disease progression (univariate Cox: HR 2.27, 95% CI 1.27-4.05, p = 0.006; multivariate Cox: HR 2.73, 95% CI 1.41-5.29, p = 0.003). Finally, the TAZpos/WNTmut signature negatively impacted overall survival. CONCLUSIONS: Collectively, our findings indicate that the oncogenic YAP/TAZ-Wnt crosstalk may be active in GC, conferring chemoresistant traits that translate into adverse survival outcomes.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Via de Sinalização Wnt/genética , Idoso , Biomarcadores Tumorais/metabolismo , Feminino , Via de Sinalização Hippo , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Análise de Sobrevida , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Resultado do Tratamento
12.
Int J Cancer ; 140(11): 2587-2595, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28233295

RESUMO

The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM-Chk2 and ATR-Chk1-Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G1 -S transition (e.g., TP53) and ATM/ATR-initiated DNA repair (e.g., ARID1A). We hypothesized that DDR-linked biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Immunohistochemical assessment of DDR kinases (pATM, pChk2, pChk1 and pWee1) and DNA damage markers (γ-H2AX and pRPA32) was performed in biological samples from 110 advanced GC patients treated with first-line chemotherapy, either in phase II trials or in routine clinical practice. In 90 patients, this characterization was integrated with targeted ultra-deep sequencing for evaluating the mutational status of TP53 and ARID1A. We recorded a positive association between the investigated biomarkers. The combination of two biomarkers (γ-H2AXhigh /pATMhigh ) was an adverse factor for both progression-free survival (multivariate Cox: HR 2.23, 95%CI: 1.47-3.40) and overall survival (multivariate Cox: HR: 2.07, 95%CI: 1.20-3.58). The relationship between the γ-H2AXhigh /pATMhigh model and progression-free survival was consistent across the different TP53 backgrounds and was maintained in the ARID1A wild-type setting. Conversely, this association was no longer observed in an ARID1A-mutated subgroup. The γ-H2AXhigh /pATMhigh model negatively impacted survival outcomes in GC patients treated with chemotherapy. The mutational status of ARID1A, but apparently not TP53 mutations, affects its predictive significance.


Assuntos
Antineoplásicos/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Idoso , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Intervalo Livre de Doença , Feminino , Mucosa Gástrica/metabolismo , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estômago/efeitos dos fármacos , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo
13.
Sci Rep ; 6: 34006, 2016 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-27666868

RESUMO

The circadian system is composed of a set of clock-genes including PERIOD, CLOCK, BMAL1 and CRY. Disrupting this system promotes cancer development and progression. The expression levels of miR-206, miR-219, miR-192, miR-194 and miR-132 regulating clock-genes and three functional polymorphisms rs11133373 C/G, rs1801260 T/C, rs11133391 T/C in CLOCK sequence were associated with the survival of 83 mCRC patients (50 males and 33 females). Longer overall survival (OS) was observed in women compared to men, 50 versus 31 months. This difference was associated with rs11133373 C/C genotype (p = 0.01), rs1801260 T/C+C/C genotype (p = 0.06) and rs11133391 T/T genotype (p = 0.06). Moreover women expressing high levels (H) of miR-192 (p = 0.03), miR-206 (p = 0.003), miR-194 (p = 0.02) and miR-219 (p = 0.002) had a longer OS compared to men. In women longer OS was reinforced by the simultaneous presence of two or more H-miR, 58 months versus 15 months (p = 0.0008); in this group of women an OS of 87 months was reached with the additional presence of rs11133391T/T genotype (p = 0.02). In this study we identified a subgroup of female patients who seems to have a better prognosis. Personalized medicine should prospectively take into account both genetic and gender differences.

14.
Oncotarget ; 7(28): 44113-44128, 2016 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-27286453

RESUMO

Colorectal cancer (CRC) is one of the most common and lethal cancers worldwide. Despite recent progress, the prognosis of advanced stage CRC remains poor, mainly because of cancer recurrence and metastasis. The high morbidity and mortality of CRC has been recently ascribed to a small population of tumor cells that hold the potential of tumor initiation, i.e. cancer stem cells (CSCs), which play a pivotal role in cancer recurrence and metastasis and are not eradicated by current therapy. We screened CRC-SCs in vitro with a library of protein kinase inhibitors and showed that CRC-SCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. Nonetheless, broad-spectrum inhibition by the staurosporin derivative UCN-01 blocks CRC-SC growth and potentiates the activity of irinotecan in vitro and in vivo CRC-SC-derived models. Reverse-Phase Protein Microarrays (RPPA) revealed that, albeit CRC-SCs display individual phospho-proteomic profiles, sensitivity of CRC-SCs to UCN-01 relies on the interference with the DNA damage response mediated by Chk1. Combination of LY2603618, a specific Chk1/2 inhibitor, with irinotecan resulted in a significant reduction of CRC-SC growth in vivo, confirming that irinotecan treatment coupled to inhibition of Chk1 represents a potentially effective therapeutic approach for CRC treatment.


Assuntos
Camptotecina/análogos & derivados , Neoplasias Colorretais/tratamento farmacológico , Dano ao DNA , Células-Tronco Neoplásicas/efeitos dos fármacos , Estaurosporina/análogos & derivados , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Camptotecina/administração & dosagem , Camptotecina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Sinergismo Farmacológico , Células HCT116 , Humanos , Irinotecano , Camundongos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Estaurosporina/administração & dosagem , Estaurosporina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
15.
Eur J Cancer ; 58: 52-61, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26963794

RESUMO

The ErbB tyrosine kinase receptors play a key role in regulating many cellular functions and human papillomaviruses (HPVs) may interact with transductional pathway of different growth factor receptors. Here, these interactions were analysed in W12 cell line carrying HPV 16 genome and in clinical samples. W12 cells, in which HPV16 becomes integrated during passages, were utilised to detect viral and ErbB family expression at early (W12E) and late passages (W12G). Interestingly, a strong reduction of ErbB-3 expression was observed in W12G. Loss of the E2 and E5 viral genes occurs in W12G and this may affect ErbB-3 receptor expression. E2 and E5 rescue experiments demonstrated that only E2 gene was able to restore ErbB-3 expression. E2 is a transcriptional factor but the expression levels of ErbB3 were unaffected and ErbB-3 promoter did not show any consensus sequence for E2, thus E2 may interact in another way with ErbB3. Indeed, HPV 16 E2 can modulate ErbB-3 by interacting with the ubiquitin ligase neuregulin receptor degradation protein 1 (Nrdp-1) that is involved in the regulation of this receptor, via ubiquitination and degradation. E2 co-immunoprecipitated in a complex with Nrdp-1 leading to hypothesise an involvement of this interaction in ErbB-3 regulation. In addition, 90% of the clinical samples with integrated virus and E2 loss showed no or low ErbB-3 positivity, confirming in vitro results. In conclusion, the new discovered interaction of HPV-16 E2 with Nrdp-1 may affect ErbB-3 expression.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/enzimologia , Receptor ErbB-3/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Displasia do Colo do Útero/enzimologia , Neoplasias do Colo do Útero/enzimologia , Adulto , Idoso , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Viral , Proteínas de Ligação a DNA/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Genoma Viral , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/genética , Humanos , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Ligação Proteica , Proteólise , Receptor ErbB-3/genética , Fatores de Tempo , Transfecção , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Integração Viral , Adulto Jovem , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/patologia , Displasia do Colo do Útero/virologia
16.
Oncotarget ; 5(21): 10382-92, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25071015

RESUMO

Mounting evidence supports the role of p53 in metabolic processes involved in breast carcinogenesis. We investigated whether p53 status affects the association of pre-treatment fasting glucose with treatment outcomes in 106 non diabetic, HER2 positive breast cancer patients treated with trastuzumab. p53 status was validated against gene sequencing of selected codons in 49 patients. The Kaplan-Meier method and log rank test were used to compare survival by categories of fasting glucose in the overall population and separate settings. Cox models included age and body mass index. Direct sequencing confirmed the lack of mutations in 73.7% of p53 negative patients and their presence in 53.3% of p53 positive cases. At 66 months, 88.3% of patients with glucose ≤ 89.0 mg/dl (median value) did not experiment disease progression compared with 70.0% in the highest category (p=0.034), with glucose being an independent predictor (p=0.046). Stratified analysis confirmed this association in p53 negative patients only (p=0.01). In the early setting, data suggested longer disease free survival in p53 negative patients in the lowest glucose category (p=0.053). In our study, p53 status acted as effect modifier of the investigated association. This may help differentiate target sub-groups and affect outcomes interpretation in similarly characterized patients.


Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/epidemiologia , Carcinoma/diagnóstico , Carcinoma/epidemiologia , Proteína Supressora de Tumor p53/genética , Adulto , Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos/administração & dosagem , Glicemia/análise , Neoplasias da Mama/mortalidade , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinoma/mortalidade , Análise Mutacional de DNA , Diagnóstico Diferencial , Progressão da Doença , Modificador do Efeito Epidemiológico , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Receptor ErbB-2/metabolismo , Análise de Sobrevida , Trastuzumab , Resultado do Tratamento
17.
J Exp Clin Cancer Res ; 32: 13, 2013 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-23497522

RESUMO

In a prospective multicenter phase II trial of radioembolization with yttrium-90 ((90)Y-RE) in chemorefractory liver-dominant metastatic colorectal cancer (mCRC), we showed that median survival was 12.6 months (95% CI 7.0-18.3) with 48% of 50 patients achieving disease control. In this extension retrospective study, we analyzed whether a panel of biomarkers, known to be associated to an adverse clinical outcome, underwent variations in CRC liver metastases pre and post (90)Y-RE.Of the 50 patients included in the study, 29 pre-(90)Y-RE therapy and 15 post-(90)Y-RE had liver biopsy specimens available. In these series we investigated survivin, p53, Bcl-2 and Ki-67 expression pre- and post-(90)Y-RE by immuhistochemistry (IHC). Our findings evidenced a decrease of survivin (77% vs 33%), p53 (93% vs 73%), Bcl-2 (37% vs 26%) expression as well as of Ki-67 proliferation index (62.5% vs 40%) on liver biopsies collected post-(90)Y-RE as compared to pre-(90)Y-RE. In the subset of 13 matched liver metastases we further confirmed the reduction of survivin (92.3% vs 53.8%; p = 0.06), p53 (100% vs 69.2%; p = 0.05) and Bcl-2 (69.2% vs 53.8%; p = 0.05) expression post-(90)Y-RE. This biomarker modulation was accompanied by morphological changes as steatohepatitis, hepatocyte necrosis, collagen deposition, proliferating and/or bile duct ectasia, focal sinusoidal dilatation and fibrosis.Although our analysis was conducted in a very limited number cases, these changes appear strictly related to the response to (90)Y-RE therapy and may deserve further investigation on a larger series of patients.


Assuntos
Neoplasias Colorretais/terapia , Embolização Terapêutica/métodos , Proteínas Inibidoras de Apoptose/biossíntese , Neoplasias Hepáticas/terapia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Radioisótopos de Ítrio/administração & dosagem , Adolescente , Adulto , Idoso , Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Resinas Compostas/administração & dosagem , Feminino , Genes bcl-2 , Genes p53 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/secundário , Masculino , Microesferas , Pessoa de Meia-Idade , Metástase Neoplásica , Estudos Prospectivos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Survivina , Proteína Supressora de Tumor p53/genética , Adulto Jovem
18.
Int J Cancer ; 132(5): 1022-31, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22847294

RESUMO

Phospholipase Cγ1 (PLCγ1) is highly expressed in several tumors. We have previously reported that both stable and inducible PLCγ1 down-regulation resulted in an almost complete inhibition of breast cancer-derived experimental lung metastasis formation. The aim of our study is to evaluate the association between the expression of PLCγ1 and of PLCγ1 phosphorylated at Tyr1253 (PLCγ1-pY1253) and at Tyr783 (PLCγ1-pY783) with the clinical outcome of patients with node negative, T1/T2 breast cancers. The study groups consisted of 292 (training set) and 122 (validation set) patients presenting with primary unilateral breast carcinoma (T1-T2), with no evidence of nodal involvement and distant metastases. PLCγ1, PLCγ1-pY1253 and PLCγ1-pY783 protein expression were assessed by immunohistochemistry on tissue microarrays and the results correlated with the clinical data using Kaplan-Meier curves and multivariate Cox regression analysis. Tumor cells while expressing variable proportions of cytoplasmic PLCγ1, express PLCγ1-pY1253 and PLCγ1-pY783 predominantly in the nucleus. High expression of PLCγ1, and of its activated forms, is associated with a worse clinical outcome in terms of incidence of distant metastases, and not of local relapse in T1-T2, N0 breast cancer patients undergone adjuvant chemotherapy. PLCγ1 over-expression appears to be a reliable predictive surrogate marker of development of metastases. Thus, targeting PLCγ1 pathways might represent a potential therapeutic approach for the prevention of metastatic disease in breast cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Fosfolipase C gama/biossíntese , Idoso , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Quimioterapia Adjuvante , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fatores de Risco
19.
Neoplasia ; 13(9): 822-30, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21969815

RESUMO

The nonclassic class I human leukocyte antigen E (HLA-E) molecule engages the inhibitory NKG2A receptor on several cytotoxic effectors, including natural killer (NK) cells. Its tissue distribution was claimed to be wider in normal than in neoplastic tissues, and surface HLA-E was undetectable in most tumor cell lines. Herein, these issues were reinvestigated taking advantage of HLA-E-specific antibodies, immunohistochemistry, and biochemical methods detecting intracellular and surface HLA-E regardless of conformation. Contrary to published evidence, HLA-E was detected in a few normal epithelia and in a large fraction (approximately 1/3) of solid tumors, including those derived from HLA-E-negative/low-normal counterparts. Remarkably, HLA-E was detected in 30 of 30 tumor cell lines representative of major lymphoid and nonlymphoid lineages, and in 11 of 11, it was surface-expressed, although in a conformation poorly reactive with commonly used antibodies. Coexpression of HLA-E and HLA class I ligand donors was not required for surface expression but was associated with NKG2A-mediated protection from lysis by the cytotoxic cell line NKL and polyclonal NK cells from healthy donors, as demonstrated by antibody-mediated relief of protection in 10% to 20% of the tested target-effector combinations. NKG2A-mediated protection of additional targets became evident on NK effector blocking with antibodies to activating receptors (DNAM-1, natural cytotoxicity receptors, and NKG2D). Thus, initial evidence that the long-elusive HLA-E molecule is enhanced by malignant transformation and is functional in tumor cells is presented here, although its importance and precise functional role remain to be addressed in the context of a general understanding of the NK ligand-receptor network.


Assuntos
Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Anticorpos Monoclonais , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Antígenos de Histocompatibilidade Classe I/química , Humanos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias/metabolismo , Receptores de Células Matadoras Naturais/metabolismo , Antígenos HLA-E
20.
Clin Cancer Res ; 17(7): 2055-64, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21307144

RESUMO

PURPOSE: To analyze HER2 status in primary breast cancer (PBC) compared with correspondent metachronous metastases and to investigate whether BC phenotype may be predictive of change in HER2 expression. EXPERIMENTAL DESIGN: HER2 was investigated by immunohistochemistry, silver in situ hybridization (SISH), and FISH, in a series of 137 tumors, building up a tissue microarray to concurrently analyze each single PBC and metastatic (MBC) on the same slide. RESULTS: HER2 status was discordant in 14 cases (10%): 12 negative in PBC and positive in metastases and two positive in PBC and negative in metastases (P = 0.04). These findings were confirmed by a PCR based test termed Multiplex Ligation-dependent Probe Amplification (MLPA). HER2 status changed in hormone receptor-positive BC more frequently than in negative ones (P = 0.002). In addition, we evaluated HER2 gene and chromosome 17 copy number by SISH in the 123 cases with unchanged HER2 status during progression. We found consistent HER2 gene copy number stability in the 100 nonamplified cases. Conversely, of the 23 amplified PBC, 13 (57%) demonstrated a significant increase in the HER2 gene and chromosome 17 copy number in their paired metastases (P = 0.01), as defined by SISH (k = 0.54, P < 0.0001) and MLPA. Patients who changed HER2 status from negative to positive, presented significant longer time to progression when treated with trastuzumab compared to those who were untreated (P = 0.04). CONCLUSIONS: When feasible, HER2 reassessment in metastatic lesions should be carefully taken into account, especially for metastases coming from primary hormone receptor-positive BC.


Assuntos
Neoplasias da Mama/patologia , Neoplasias da Mama/secundário , Receptor ErbB-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Cromossomos Humanos Par 17/genética , Variações do Número de Cópias de DNA , Intervalo Livre de Doença , Feminino , Amplificação de Genes , Humanos , Hibridização In Situ , Pessoa de Meia-Idade , Terapia Neoadjuvante , Metástase Neoplásica , Receptor ErbB-2/metabolismo , Trastuzumab , Resultado do Tratamento
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