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1.
Diagnostics (Basel) ; 11(6)2021 Jun 07.
Article in English | MEDLINE | ID: mdl-34200245

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer, with most patients diagnosed at advanced stages. First-line treatment based on a combined chemotherapy (FOLFIRINOX or gemcitabine plus nab-paclitaxel) provides limited benefits. Olaparib, a PARP inhibitor, has been approved as maintenance for PDAC patients harboring germline BRCA1/2 pathogenic mutations and previously treated with a platinum-based chemotherapy. BRCA1/2 germline testing is recommended, but also somatic mutations could predict responses to PARP inhibitors. Analysis of tumor tissues can detect both germline and somatic mutations and potential resistance alterations. Few data are available about BRCA1/2 testing on pancreatic tumor tissues, which often include limited biological material. We performed BRCA1/2 testing, by an amplicon-based Next Generation Sequencing (NGS) panel, on 37 consecutive PDAC clinical samples: 86.5% of cases were adequate for NGS analysis, with a success rate of 81.2% (median DNA input: 10 nanograms). Three BRCA2 mutations were detected (11.5%). Failed samples were all from tissue macrosections, which had higher fragmented DNA than standard sections, biopsies and fine-needle aspirations, likely due to fixation procedures. BRCA1/2 testing on pancreatic tumor tissues can also be feasible on small biopsies, but more cases must be analyzed to define its role and value in the PDAC diagnostic algorithm.

2.
J Mol Diagn ; 22(5): 685-698, 2020 05.
Article in English | MEDLINE | ID: mdl-32173570

ABSTRACT

To understand the immune landscape of deficient mismatch repair colorectal cancer (dMMR CRC) tumor microenvironment, gene expression profiling was performed by the nCounter PanCancer Immune Profiling Panel. This study was conducted retrospectively on 89 dMMR-CRC samples. The expression of CD3, CD8, programmed death-1, and programmed death ligand-1 protein was evaluated on a subset of samples by immunohistochemistry, and lymphocyte density was calculated. A subset of deregulated genes was identified. Functional clustering analysis performed on these genes generated four main factors: antigen processing and presentation, with its major histocompatibility complex-II-related genes; genes correlated with the cytotoxic activity of immune system; T-cell chemotaxis/cell adhesion genes; and T-CD4+ regulator cell-related genes. A deregulation score (DS) was calculated for each sample. On the basis of their DS, tumors were then classified as COLD (DS ≤ -3) to select the samples with a strong down-regulation of the immune system and NOT COLD (DS ≥ -2). The COLD group of patients showed a worse prognosis in terms of survival considering all patients (P = 0.0172) and patients with metastatic disease (P = 0.0031). These results confirm that dMMR-CRCs do not constitute a homogeneous group as concerns the immune system activity of tumor microenvironment. In particular, the distinction between COLD and NOT COLD tumors may improve the management of these two subsets of patients.


Subject(s)
Colorectal Neoplasms/etiology , DNA Mismatch Repair , Immune System/immunology , Immune System/metabolism , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology , Alleles , Biomarkers, Tumor , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Computational Biology/methods , Disease Susceptibility , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genotype , Humans , Kaplan-Meier Estimate , Lymphocyte Count , Male , Matrix Metalloproteinases/genetics , Neoplasm Staging , Prognosis
3.
Eur J Cancer ; 110: 32-41, 2019 03.
Article in English | MEDLINE | ID: mdl-30739838

ABSTRACT

BACKGROUND: Neoadjuvant chemoradiotherapy (CRT) in locally advanced rectal cancer (LARC) does not achieve effective control of distant metastases. Induction chemotherapy is a promising strategy, and bevacizumab (BV) could improve the results of CRT. 5-Fluorouracil, oxaliplatin and irinotecan (FOLFOXIRI) plus BV is a treatment option in metastatic colorectal cancer. We evaluate feasibility and efficacy of neoadjuvant treatment comprising induction FOLFOXIRI plus BV followed by CRT with fluoropyrimidines plus BV. METHODS: In this phase II single-arm trial, patients node-positive or clinical T4 or high-risk T3 LARC underwent 6 cycles of induction FOLFOXIRI plus BV, followed by CRT (50.4 Gy plus concomitant capecitabine) and BV (5 mg/kg on days 1, 15 and 28). Surgery was planned 8 weeks after completion of CRT. Primary end-point was 2-year disease-free survival (DFS). RESULTS: We enrolled 49 patients: All but one (withdrewing consent after enrolment) were included in the per-protocol analyses. The study met its primary end-point: 36 patients were free of recurrence at 2 years (2-y DFS: 80.45%, 95% confidence interval [CI]: 78.79-82.10). Forty-four patients underwent surgery; pathologic complete response rate was 36.4%. Forty-six patients completed induction: neutropenia (41.6%) and diarrhoea (12.5%) were main G3/4 toxicities. Forty-five patients received CRT, but the protocol was amended and the capecitabine schedule during CRT was slightly modified after 13 patients due to the incidence of G3 hand-foot syndrome and proctitis (23.1%). After amendment, no severe events during CRT were reported. CONCLUSIONS: FOLFOXIRI plus BV followed by CRT plus BV is feasible and active. Results in terms of DFS suggest that this strategy may improve distant disease control in LARC.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Rectal Neoplasms/therapy , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Bevacizumab/administration & dosage , Bevacizumab/adverse effects , Camptothecin/administration & dosage , Camptothecin/adverse effects , Camptothecin/analogs & derivatives , Chemoradiotherapy/methods , Chemoradiotherapy/mortality , Disease-Free Survival , Female , Fluorouracil/administration & dosage , Fluorouracil/adverse effects , Humans , Leucovorin/administration & dosage , Leucovorin/adverse effects , Male , Middle Aged , Neoadjuvant Therapy/methods , Neoadjuvant Therapy/mortality , Organoplatinum Compounds/administration & dosage , Organoplatinum Compounds/adverse effects , Rectal Neoplasms/mortality , Treatment Outcome
4.
Eur J Cancer ; 109: 175-182, 2019 03.
Article in English | MEDLINE | ID: mdl-30735919

ABSTRACT

BACKGROUND: Alternating induction and maintenance phases is a common strategy in metastatic colorectal cancer (mCRC). Metronomic chemotherapy (metroCT) may represent a well-tolerated chemotherapy backbone for maximising bevacizumab effect during maintenance. The MOMA trial was designed to compare metroCT plus bevacizumab versus bevacizumab alone as maintenance following 4 months of induction with FOLFOXIRI plus bevacizumab. PATIENTS AND METHODS: In this phase II study, patients with unresectable mCRC were randomised to receive up to 8 cycles of FOLFOXIRI plus bevacizumab, followed by bevacizumab (arm A) or the same regimen followed by bevacizumab plus metroCT (capecitabine 500 mg/three times per day and cyclophosphamide 50 mg/die, arm B) until disease progression. The primary end-point was progression-free survival (PFS). According to the Rubinstein and Korn's design, to detect a hazard ratio[HR] of 0.75 favouring arm B, with 1 sided-alpha and beta errors of 15% and 80%, 173 events and 222 patients were required. RESULTS: Between May 2012 and March 2015, 232 patients, mostly with RAS (65%) or BRAF (9%) mutant tumours, were randomised in 16 Italian centres. At a median follow-up of 47.8 months, 210 and 164 progression and death events were registered. The primary end-point was not met. Median PFS was 10.3 and 9.4 months in arm B and A, respectively (HR: 0.94 [70% confidence interval {CI}: 0.82-1.09], p = 0.680). No significant differences were reported in terms of overall survival (OS) (median OS arm B/A: 22.5/28 months; HR: 1.16 [95%CI: 0.99-1.37], p = 0.336). Response rate with FOLFOXIRI plus bevacizumab was 63% (arm B/A: 58%/68%). In the liver-limited subgroup, the secondary resection rate was 49% (arm B/A: 45%/55%). CONCLUSIONS: The addition of metroCT to maintenance with bevacizumab does not significantly improve PFS of mCRC patients. The activity of FOLFOXIRI plus bevacizumab is confirmed in a population with high prevalence of RAS/BRAF mutations treated with a 4-months induction. TRIAL REGISTRATION: www.clinicaltrials.gov NCT02271464.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Colorectal Neoplasms/drug therapy , Adolescent , Adult , Aged , Bevacizumab/administration & dosage , Capecitabine/administration & dosage , Colorectal Neoplasms/secondary , Cyclophosphamide/administration & dosage , Female , Fluorouracil/administration & dosage , Follow-Up Studies , Humans , Irinotecan/administration & dosage , Leucovorin/administration & dosage , Male , Middle Aged , Prognosis , Prospective Studies , Survival Rate , Young Adult
5.
Front Oncol ; 9: 1390, 2019.
Article in English | MEDLINE | ID: mdl-31921647

ABSTRACT

The diagnosis of the primary origin of metastases to the thyroid gland is not easy, in particular in case of concomitant lung adenocarcinoma which shares several immunophenotypical features. Although rare, these tumors should be completely characterized in order to set up specific therapies. This is the case of a 64-years-old woman referred to our institution for a very advanced neoplastic disease diagnosed both as poorly differentiated/anaplastic thyroid cancer (PDTC/ATC) for the huge involvement of the neck and concomitant lung adenocarcinoma (LA). Neither the clinical features and the imaging evaluation nor the tumor markers allowed a well-defined diagnosis. Moreover, the histologic features of the thyroid and lung biopsies confirmed the synchronous occurrence of two different tumors. The molecular analysis showed a c.34G>T (p.G12C) mutation in the codon 12 of K-RAS gene, in both tissues. Since, this mutation is highly prevalent in LA and virtually absent in PDTC/ATC the lung origin of the malignancy was assumed, and the patient was addressed to the correct therapeutic strategy.

6.
Genes Cancer ; 8(1-2): 438-452, 2017 Jan.
Article in English | MEDLINE | ID: mdl-28435517

ABSTRACT

Malignant pleural mesothelioma (MPM) is a cancer of the pleural cavity resistant to chemotherapy. The identification of novel therapeutic targets is needed to improve its poor prognosis. Following a review of literature and a screening of specimens we found that platelet-derived growth factor receptor beta (PDGFRB) is over-expressed, but not somatically mutated, in MPM tissues. We aimed to ascertain whether PDGFRB is a MPM-cancer driver gene. The approaches employed included the use of gene silencing and the administration of small molecules, such as crenolanib and imatinib (PDGFR inhibitors) on MPM cell lines (IstMes2, Mero-14, Mero-25). Met5A cells were used as non-malignant mesothelial cell line. PDGFRB-silencing caused a decrease in the proliferation rate, and a reduced colony formation capacity, as well as an increase of the share of cells in sub-G1 and in G2 phase, and increased apoptotic rate of MPM cell lines. Loss of migration ability was also observed. Similar, or even further enhanced, results were obtained with crenolanib. Imatinib showed the least effective activity on the phenotype. In conclusion, our study highlights PDGFRB as target with a clear role in MPM tumorigenesis and provided a rationale to explore further the efficacy of crenolanib in MPM patients, with promising results.

7.
Exp Hematol Oncol ; 6: 6, 2017.
Article in English | MEDLINE | ID: mdl-28344857

ABSTRACT

BACKGROUND: The BRAF K601E mutation occurs in 5% of patients with melanoma, and is the third most common type of BRAF mutation. However, treatment with BRAF and mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors is only approved in patients with BRAF V600-positive melanoma, and patients with K601E-mutated melanoma do not have access to such drugs. CASE PRESENTATION: A female patient was diagnosed with high tumor burden metastatic melanoma harboring the BRAF K601E mutation. After chemotherapy failure, she underwent compassionate treatment with trametinib. Trametinib showed good activity and efficacy, with 48% shrinkage of a metastatic lymphadenopathy after 4 months' treatment. However, the patient reported treatment-related skin toxicity that required dosage reduction and a personalized intermittent trametinib dosing schedule. After over 36 months from the first trametinib administration, and resection of a metastatic lymphadenopathy, the patient experienced complete response. CONCLUSIONS: This case report shows that trametinib could be a valid therapeutic option in patients with metastatic melanoma harboring the rare BRAF K601E mutation.

8.
Eur J Cancer ; 73: 74-84, 2017 03.
Article in English | MEDLINE | ID: mdl-27986363

ABSTRACT

Secondary resection is a chance of cure for a subgroup of metastatic colorectal cancer (mCRC) patients with unresectable liver-limited disease. Medical treatment has a dual goal: to induce tumour shrinkage and to prevent disease relapse. The aims of the present analysis were to assess the efficacy of FOLFOXIRI plus bevacizumab in this setting, and to investigate whether this regimen could revert the poor prognosis of high-risk patients defined by clinical and molecular factors. We performed a pooled analysis of patients with unresectable and liver-limited mCRC, treated with first-line FOLFOXIRI plus bevacizumab in three prospective clinical trials by Gruppo Oncologico del Nord Ovest. 205 (37.9%) patients with liver-limited disease were selected, out of 541 treated patients. Liver metastases were synchronous, ≥4 and bilobar in 90%, 61%, and 79% of cases, respectively. The largest diameter was >5 cm in 42% of cases, and ≥6 segments were involved in 25%. Seventy-four patients (36.1%) underwent R0 or R1 resection of metastases. R2 resections were performed in 17 cases (8.3%). Having <6 involved segments (p < 0.001) and achieving RECIST response (p = 0.019) were associated with higher chances of resection. R0/R1 resected patients had significantly longer median progression-free survival (PFS) (18.1 versus 10.7 months, HR: 0.48 [0.35-0.66], p < 0.001) and overall survival (OS) (44.3 versus 24.4 months, HR: 0.32 [0.22-0.48], p < 0.001) compared with other patients, both in the univariate and multivariate analyses (PFS p = 0.025; OS p < 0.001). The 5-year PFS and OS rate in R0 resected patients were 12% and 43%, respectively. Neither negative baseline characteristics nor high clinical risk scores or RAS/BRAF mutations were associated with poor post-resection outcomes. In conclusion, FOLFOXIRI plus bevacizumab demonstrates efficacy in the conversion setting with considerable long-term outcome results independent of clinical and molecular prognostic factors (NCT00719797, NCT01163396 and NCT02271464).


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bevacizumab/therapeutic use , Colorectal Neoplasms/pathology , Liver Neoplasms/drug therapy , Liver Neoplasms/secondary , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Camptothecin/administration & dosage , Camptothecin/analogs & derivatives , Disease-Free Survival , Female , Fluorouracil/administration & dosage , Humans , Kaplan-Meier Estimate , Leucovorin/administration & dosage , Male , Middle Aged , Multivariate Analysis , Organoplatinum Compounds/administration & dosage , Prospective Studies , Young Adult
9.
J Clin Endocrinol Metab ; 101(11): 4413-4420, 2016 11.
Article in English | MEDLINE | ID: mdl-27571181

ABSTRACT

CONTEXT: Approximately 40% of papillary thyroid carcinomas (PTCs) harbor the BRAF V600E mutation, which is significantly associated with the advanced clinicopathological features of PTC at diagnosis, a higher recurrence rate, and disease-related mortality. BRAF alterations other than V600E are less common in PTC, and their clinical significance remains to be established. OBJECTIVE: The aim of the study was to describe a large cohort of rare exon 15 BRAF alterations (r-BRAF) and the clinicopathological features of PTC harboring these alterations and to clarify their clinical significance. METHODS: A total of 2961 PTCs were collected from 2006 to 2013 and screened for exon 15 BRAF alterations. RESULTS: Exon 15 BRAF alterations were found in 1186 of 2961 PTC cases (40.0%). In particular, we found the BRAF V600E mutation in 95.3% (1131 of 1186) and r-BRAF in 4.7% (55 of 1186) of the cases. r-BRAF were found in 18 microcarcinomas, 33 follicular variants, one classic variant, and one trabecular/solid variant. The most frequent r-BRAF was BRAF K601E (35 of 55; 63.6%), followed by BRAF V600_K601delinsE (seven of 55; 12.7%) and BRAF T599I-V600_R603del (two of 55; 3.6%). The remaining 11 alterations were found in one case only. The large majority of these tumors were unifocal (34 of 55; 61.8%), completely encapsulated (46 of 55; 83.6%), and intrathyroidal (53 of 55; 96.4%) with a low prevalence of lymph node metastases (one of 55; 1.8%) and a less advanced tumor stage at diagnosis (American Joint Commission on Cancer stage I/II, 51 of 55; 92.7%). CONCLUSIONS: r-BRAF are very uncommon in PTC and are found almost exclusively in PTC with low-risk clinicopathological features.


Subject(s)
Carcinoma/genetics , Carcinoma/pathology , Carcinoma/therapy , Proto-Oncogene Proteins B-raf/genetics , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathology , Thyroid Neoplasms/therapy , Adult , Carcinoma, Papillary , Exons , Female , Humans , Male , Middle Aged , Thyroid Cancer, Papillary , Treatment Outcome
10.
Oncol Rep ; 36(2): 1166-72, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27373829

ABSTRACT

Activating EGFR mutations are important genetic alterations that have strong therapeutic implications for non-small cell lung cancer (NSCLC) patients. However, the role of KRAS mutations in this process is still under evaluation. Here, we report on the feasibility of a large­scale EGFR and KRAS mutation analysis in the daily routine of a single center. NSCLCs from 2,387 patients were screened for EGFR and KRAS mutations from January 2010 to September 2015. Mutational analyses were performed in a single laboratory using single strand conformation polymorphism (SSCP)-Sanger sequencing and matrix­assisted laser desorption ionization­time of flight (MALDI­TOF) on Sequenom platform for EGFR and pyrosequencing for KRAS. Activating EGFR mutations were found in 14.1% of all tumors, whereas KRAS mutations were found in 30.5% of all tumors. Direct sequencing showed analyzable cytological, small biopsy and surgical specimen percentages of 90.3, 90.9 and 98.1%, respectively, whereas the MALDI­TOF platform showed analyzable cytological samples, small biopsies and surgical specimens percentages of 94.6, 95.7 and 96.9%, respectively. The mean analytical turnaround times (TAT) were 4 and 3 days for direct sequencing and the MALDI­TOF platform, respectively. Our results confirm that small biopsy or cytological samples can be used for reliable EGFR and KRAS mutation testing and indicate that adopting the MALDI­TOF platform reduces the rate of missed samples among the samples. Moreover, the 3-day analytical TAT of the MALDI-TOF multi-target technique is appropriate for clinical management and reduces the overall treatment decision time.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , ErbB Receptors/genetics , Lung Neoplasms/genetics , Mutation/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Adult , Aged , Aged, 80 and over , Biopsy, Fine-Needle/methods , DNA Mutational Analysis/methods , Female , Humans , Italy , Male , Middle Aged
11.
Cancer Cytopathol ; 124(5): 340-9, 2016 May.
Article in English | MEDLINE | ID: mdl-26749005

ABSTRACT

Fine-needle aspiration (FNA) is routinely used in the preoperative evaluation of thyroid nodules. However, 15% to 30% of aspirations yield indeterminate cytologic findings. Because the assessment of BRAF mutations seems to improve the diagnostic accuracy, this study evaluated BRAF mutations with Sanger sequencing and real-time methods in 650 consecutive thyroid aspirates. In addition, the expression of a large number of genes involved in basement membrane remodeling, extracellular matrix proteolysis, and cell adhesion was studied in both benign and malignant nodules to identify new diagnostic tools. In this prospective series, despite the use of a very sensitive BRAF mutational testing method, the frequency of a BRAF alteration being identified in indeterminate FNA samples was 3 of 68. Expression analysis revealed several genes that were differentially expressed between benign and malignant nodules (transforming growth factor, cadherin 1, collagen α1, catenin α1, integrin α3, and fibronectin 1 [FN1]), between follicular adenomas and follicular variant of papillary thyroid carcinoma (FN1, laminin γ1, integrin ß2, connective tissue growth factor, catenin δ1, and integrin αV), and between BRAF-wild-type and BRAF-mutated papillary thyroid carcinomas (TIMP metallopeptidase inhibitor 1; catenin α1; secreted phosphoprotein 1; FN1; ADAM metallopeptidase with thrombospondin type 1 motif, 1; and selectin L). These data were partially confirmed with real-time polymerase chain reaction analysis and immunohistochemistry. When the cost/benefit ratio of the procedures was taken into account, BRAF mutational testing failed to increase diagnostic accuracy in cytologically indeterminate nodules. However, the additional analysis of the expression of specific molecular markers could have possible utility as a diagnostic tool, although further evidence based on a large series of samples is needed before definitive conclusions can be drawn. Cancer Cytopathol 2016;124:340-9. © 2015 American Cancer Society.


Subject(s)
Biomarkers, Tumor/genetics , Gene Expression Profiling/methods , Mutation/genetics , Proto-Oncogene Proteins B-raf/genetics , Thyroid Neoplasms/diagnosis , Thyroid Nodule/diagnosis , Adenocarcinoma, Follicular/diagnosis , Adenocarcinoma, Follicular/genetics , Adenoma/diagnosis , Adenoma/genetics , Carcinoma, Papillary/diagnosis , Carcinoma, Papillary/genetics , DNA Mutational Analysis , Humans , Prospective Studies , Real-Time Polymerase Chain Reaction , Thyroid Neoplasms/genetics , Thyroid Nodule/genetics
12.
BMC Cancer ; 15: 874, 2015 Nov 09.
Article in English | MEDLINE | ID: mdl-26553291

ABSTRACT

BACKGROUND: The enhancer of zeste-homolog 2 (EZH2) is involved in cancer development through gene silencing by trimethylation of lysine 27 of histone 3 (H3K27me3). The C/C genotype for the EZH2 rs3757441 single-nucleotide polymorphism (SNP) is linked with poor prognosis in metastatic colorectal cancer (CRC), but molecular and pathological characterization of this SNP is lacking. METHODS: 119 primary CRCs were analyzed. SNP was evaluated by real-time PCR from colonic healthy tissue, while EZH2 and H3K27me3 expression were studied by immunohistochemistry. We primarily looked for correlation between EZH2 rs3757441 genotypes and EZH2/H3K27me3 expression. Potential associations between EZH2/H3K27me3 expression and clinico-pathological features or KRAS exon 2 and BRAF exon 15 mutations were secondary endpoints. Statistical analysis was performed by chi-square test, T-test or ANOVA. RESULTS: The C/C genotype was significantly associated with higher EZH2 (100 vs. 44 %; P = 0.019) and H3K27me3 (100 vs. 38 %; P = 0.009) staining intensity compared with C/T and T/T. EZH2 3+ staining significantly correlated with stronger H3K27me3 expression (P = 0.039). KRAS and BRAF mutations were not associated with EZH2 or H3K27me3 expression. CONCLUSION: EZH2 rs3757441 C/C genotype is associated with stronger EZH2 and H3K27me3 immunoreactivity in primary CRC: this SNP may serve as a promising biomarker for EZH2-targeting agents and may add independent information to KRAS and BRAF testing.


Subject(s)
Biomarkers, Tumor/genetics , Colorectal Neoplasms/genetics , Polycomb Repressive Complex 2/genetics , Prognosis , Biomarkers, Tumor/biosynthesis , Colorectal Neoplasms/pathology , Enhancer of Zeste Homolog 2 Protein , Female , Gene Expression Regulation, Neoplastic , Genotype , Histones/genetics , Humans , Male , Mutation , Neoplasm Staging , Polycomb Repressive Complex 2/biosynthesis , Polymorphism, Single Nucleotide , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras)/genetics
13.
Lancet Oncol ; 16(13): 1306-15, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26338525

ABSTRACT

BACKGROUND: In the TRIBE study, FOLFOXIRI (fluorouracil, leucovorin, oxaliplatin, and irinotecan) plus bevacizumab significantly improved progression-free survival of patients with metastatic colorectal cancer compared with FOLFIRI (fluorouracil, leucovorin, and irinotecan) plus bevacizumab. In this updated analysis, we aimed to provide mature results for overall survival-a secondary endpoint-and report treatment efficacy in RAS and BRAF molecular subgroups. METHODS: TRIBE was an open-label, multicentre, phase 3 randomised study of patients (aged 18-70 years with Eastern Cooperative Oncology Group [ECOG] performance status of 2 or less and aged 71-75 years with an ECOG performance status of 0) with unresectable metastatic colorectal cancer who were recruited from 34 Italian oncology units. Patients were randomly assigned (1:1) via a web-based procedure to receive FOLFIRI plus bevacizumab or FOLFOXIRI plus bevacizumab. Bevacizumab was given as a 5 mg/kg intravenous dose. FOLFIRI consisted of a 180 mg/m(2) intravenous infusion of irinotecan for 60 min followed by a 200 mg/m(2) intravenous infusion of leucovorin for 120 min, a 400 mg/m(2) intravenous bolus of fluorouracil, and a 2400 mg/m(2) continuous infusion of fluorouracil for 46 h. FOLFOXIRI consisted of a 165 mg/m(2) intravenous infusion of irinotecan for 60 min, followed by an 85 mg/m(2) intravenous infusion of oxaliplatin given concurrently with 200 mg/m(2) leucovorin for 120 min, followed by a 3200 mg/m(2) continuous infusion of fluorouracil for 48 h. Tissue samples for RAS and BRAF mutational status analyses were centrally collected. In this updated analysis, we assessed the secondary endpoint of overall survival in the main cohort and treatment efficacy in RAS and BRAF molecular subgroups. All analyses were by intention to treat. TRIBE was concluded on Nov 30, 2014. The trial is registered with ClinicalTrials.gov, number NCT00719797. FINDINGS: Between July 17, 2008, and May 31, 2011, 508 patients were randomly assigned. At a median follow-up of 48·1 months (IQR 41·7-55·6), median overall survival was 29·8 months (95% CI 26·0-34·3) in the FOLFOXIRI plus bevacizumab group compared with 25·8 months (22·5-29·1) in the FOLFIRI plus bevacizumab group (hazard ratio [HR] 0·80, 95% CI 0·65-0·98; p=0·03). Median overall survival was 37·1 months (95% CI 29·7-42·7) in the RAS and BRAF wild-type subgroup compared with 25·6 months (22·4-28·6) in the RAS-mutation-positive subgroup (HR 1·49, 95% CI 1·11-1·99) and 13·4 months (8·2-24·1) in the BRAF-mutation-positive subgroup (HR 2·79, 95% CI 1·75-4·46; likelihood-ratio test p<0·0001). Treatment effect was not significantly different across molecular subgroups (pinteraction=0·52). INTERPRETATION: FOLFOXIRI plus bevacizumab is a feasible treatment option for those patients who meet the inclusion criteria of the present study, irrespective of baseline clinical characteristics and RAS or BRAF mutational status.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bevacizumab/therapeutic use , Colorectal Neoplasms/drug therapy , Aged , Angiogenesis Inhibitors/administration & dosage , Angiogenesis Inhibitors/adverse effects , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Bevacizumab/administration & dosage , Bevacizumab/adverse effects , Camptothecin/administration & dosage , Camptothecin/analogs & derivatives , Colorectal Neoplasms/genetics , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , DNA Mutational Analysis , Disease-Free Survival , Drug Administration Schedule , Female , Fluorouracil/administration & dosage , Humans , Infusions, Intravenous , Intention to Treat Analysis , Irinotecan , Italy , Kaplan-Meier Estimate , Leucovorin/administration & dosage , Male , Middle Aged , Mutation , Neoplasm Metastasis , Organoplatinum Compounds/administration & dosage , Oxaliplatin , Proportional Hazards Models , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras) , Time Factors , Treatment Outcome , ras Proteins/genetics
14.
Mod Pathol ; 28(4): 487-97, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25341653

ABSTRACT

Testing for NRAS is now integral part in the assessment of metastatic melanoma patients because there is evidence that NRAS-mutated patients may be sensitive to MEK inhibitors, and RAS mutation is a common mechanism of acquired resistance during treatment with BRAF inhibitors. This study evaluated the sensitivity and specificity of immunohistochemical analysis using an N-Ras (Q61R) antibody to detect the presence of the NRASQ61R mutation in melanoma patients. A total of 98 primary cutaneous melanomas that have undergone examination of NRAS mutation were retrieved from a multicentric database. Formalin-fixed and paraffin-embedded melanoma tissues were analyzed for BRAF and NRAS mutations by independent, blinded observers using both conventional DNA molecular techniques and immunohistochemistry with the novel anti-human N-Ras (Q61R) monoclonal antibody (clone SP174). The antibody showed a sensitivity of 100% (14/14) and a specificity of 100% (83/83) for detecting the presence of an NRASQ61R mutation. Of the NRAS-mutated cases, none of the non-Q61R cases stained positive with the antibody (0/7). There were three cases with discordant NRAS mutational results. Additional molecular analysis confirmed the immunohistochemically obtained NRAS result in all cases, suggesting that a multiple analytical approach can be required to reach the correct sample classification. The reported immunohistochemical method is an accurate, rapid, and cost-effective method for detecting NRASQ61R mutation in melanoma patients, and represents a valuable supplement to traditional mutation testing. If validated in further studies, genetic testing would only be required for immunohistochemistry-negative patients to detect non-Q61R mutations.


Subject(s)
GTP Phosphohydrolases/metabolism , Immunohistochemistry , Melanoma/metabolism , Membrane Proteins/metabolism , Mutation , Skin Neoplasms/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Female , GTP Phosphohydrolases/genetics , Humans , Male , Melanoma/genetics , Melanoma/pathology , Membrane Proteins/genetics , Middle Aged , Sensitivity and Specificity , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Young Adult
15.
Int J Cancer ; 136(1): 83-90, 2015 Jan 01.
Article in English | MEDLINE | ID: mdl-24806288

ABSTRACT

NRAS mutations occur in 3-5% of colorectal cancer. Differently from KRAS and BRAF mutations, the role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer (mCRC) has been investigated to a lesser extent. A retrospective series suggested the role of NRAS mutations as predictors of resistance to anti-EGFR monoclonal antibodies (MoAbs) in chemo-refractory patients with mCRC. In our study, KRAS codons 12, 13, 61 and BRAF codon 600 mutational status were evaluated in mCRCs referred to our Institution from 2009 to 2012. NRAS codons 12, 13 and 61 mutational status was analyzed in KRAS/BRAF wt patients. We collected pathological and clinical features in the overall population and outcome data in a subset of NRAS mutated chemo-refractory patients treated with anti-EGFR MoAbs in advanced lines. NRAS was mutated in 47/786 (6%) mCRCs. NRAS and KRAS mutated tumors did not show significant differences in terms of clinical and pathological characteristics, except for a lower prevalence of mucinous histology (p = 0.012) and lung metastases (p = 0.012) among NRAS mutated tumors. In the uni- and multivariate model, NRAS mutations were associated with shorter overall survival (OS) compared to all wt patients (median OS 25.6 vs 42.7 months; univ: HR = 1.91, 95% CI 1.39-3.86, p = 0.0013; multiv: HR = 1.75, 95% CI 1.1.3-2.72, p = 0.013). None of the chemo-refractory NRAS mutated patients evaluable for response to anti-EGFRs achieved response. In conclusion, NRAS mutations have a relevant incidence in patients with mCRC and showed an association with specific clinical and pathological features. NRAS mutations affect mCRC patients' prognosis and predict lack of response to anti-EGFRs.


Subject(s)
Biomarkers, Tumor/genetics , Colorectal Neoplasms/genetics , GTP Phosphohydrolases/genetics , Liver Neoplasms/genetics , Membrane Proteins/genetics , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal, Humanized/therapeutic use , Cetuximab , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , DNA Mutational Analysis , Disease-Free Survival , Drug Resistance, Neoplasm/genetics , ErbB Receptors/antagonists & inhibitors , Female , Genetic Association Studies , Humans , Kaplan-Meier Estimate , Liver Neoplasms/drug therapy , Liver Neoplasms/mortality , Liver Neoplasms/secondary , Male , Middle Aged , Mutation, Missense , Prognosis , Proportional Hazards Models , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras) , Retrospective Studies , ras Proteins/genetics
16.
J Exp Clin Cancer Res ; 33: 77, 2014 Oct 11.
Article in English | MEDLINE | ID: mdl-25300933

ABSTRACT

BACKGROUND: We aimed at investigating the outcomes of female patients with stage IIIB-IV adenocarcinoma of the lung according to EGFR and K-Ras mutational status. METHODS: One hundred and three consecutive female patients genotyped at a single Italian Institution were analyzed. Patients were planned to receive first-line platinum-based chemotherapy (CT) and a salvage treatment with anti-EGFR tyrosine-kinase inhibitors (TKIs) was proposed irrespective of tumor mutational status. EGFR (exons 18-21) and K-Ras (exon 2, codons 12-13) mutations were evaluated by real-time PCR and pyrosequencing. The association of mutational status with clinical variables and treatment benefit was investigated by chi-square test and log-rank test. RESULTS: EGFR and K-Ras mutations were found in 31 (30%) and 13 (15%) cases, respectively. Sixty-six patients received platinum CT: no correlation was observed between EGFR or K-Ras mutational status and response rate (RR) (p > 0.05). However, patients treated with first-line CT harboring EGFR activating mutations experienced a significantly reduced progression-free survival (PFS) in comparison with wild-type ones (4.4 vs. 6.4 months, respectively; HR 0.597, 95% CI 0.287-0.975; p = 0.048). Thirty-nine patients received salvage treatment with erlotinib: EGFR activating mutations were significantly correlated with RR (60% vs. 12.5%; p = 0.004) and PFS (11.4 vs. 4.5 months; HR 0.491, 95% CI 0.216-0.936; p = 0.044). Responses to erlotinib were not reported among women with K-Ras mutant tumors, while 50% of those with wild-type K-Ras achieved an objective remission (p = 0.296). Median PFS (3.5 vs. 8.8 months; HR 0.284, 95% CI 0.015-0.510; p = 0.010) and OS (3.9 vs. 19.8 months; HR 0.158, 95% CI 0.001-0.075; p < 0.001) were significantly shorter among K-Ras mutant patients treated with TKI. CONCLUSIONS: In our population of Caucasian women with advanced lung adenocarcinoma we observed that the presence of EGFR activating mutations correlates with a significant reduction in the benefit from first-line platinum-based CT, emphasizing the importance of an upfront use of anti-EGFR TKIs in this patient subset. K-Ras mutations seem to correlate with a detrimental effect from anti-EGFR TKI, but this finding deserves further investigation.


Subject(s)
Adenocarcinoma/genetics , ErbB Receptors/genetics , Lung Neoplasms/genetics , Mutation , Proto-Oncogene Proteins/genetics , ras Proteins/genetics , Adenocarcinoma/drug therapy , Adenocarcinoma/mortality , Adenocarcinoma/pathology , Adenocarcinoma of Lung , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Female , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Middle Aged , Neoplasm Metastasis , Neoplasm Staging , Prognosis , Proto-Oncogene Proteins p21(ras) , Retrospective Studies , Salvage Therapy
17.
Arch Pathol Lab Med ; 138(11): 1449-58, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24885803

ABSTRACT

CONTEXT: Echinoderm microtubule associated proteinlike 4-anaplastic lymphoma receptor tyrosine kinase (EML4-ALK) translocation has been described in a subset of patients with non-small cell lung cancer (NSCLC) and has been shown to have oncogenic activity. Fluorescence in situ hybridization (FISH) is used to detect ALK-positive NSCLC, but it is expensive, time-consuming, and difficult for routine application. OBJECTIVE: To evaluate the potential role of immunohistochemistry (IHC) as a screening tool to identify candidate cases for FISH analysis and for ALK inhibitor therapy in NSCLC. DESIGN: We performed FISH and IHC for ALK and mutational analysis for epidermal growth factor receptor (EGFR) and KRAS in 523 NSCLC specimens. We conducted IHC analysis with the monoclonal antibody D5F3 (Ventana Medical Systems, Tucson, Arizona) and a highly sensitive detection system. We also performed a MassARRAY-based analysis (Sequenom, San Diego, California) in a small subset of 11 samples to detect EML4-ALK rearrangement. RESULTS: Of the 523 NSCLC specimens, 20 (3.8%) were positive for ALK rearrangement by FISH analysis. EGFR and KRAS mutations were identified in 70 (13.4%) and 124 (23.7%) of the 523 tumor samples, respectively. ALK rearrangement and EGFR and KRAS mutations were mutually exclusive. Of 523 tumor samples analyzed, 18 (3.4%) were ALK(+) by IHC, 18 samples (3.4%) had concordant IHC and FISH results, and 2 ALK(+) cases (0.3%) by FISH failed to show ALK protein expression. In the 2 discrepant cases, we did not detect any mass peaks for the EML4-ALK variants by MassARRAY. CONCLUSIONS: Our results show that IHC may be a useful technique for selecting NSCLC cases to undergo ALK FISH analysis.


Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Gene Rearrangement , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Protein Kinase Inhibitors/therapeutic use , Pyrazoles/therapeutic use , Pyridines/therapeutic use , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Adult , Aged , Aged, 80 and over , Anaplastic Lymphoma Kinase , Carcinoma, Non-Small-Cell Lung/enzymology , Cell Cycle Proteins/genetics , Crizotinib , Female , Genes, erbB-1 , Genetic Testing , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Lung Neoplasms/enzymology , Male , Microtubule-Associated Proteins/genetics , Middle Aged , Mutation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Serine Endopeptidases/genetics , ras Proteins/genetics
18.
Oncol Lett ; 7(5): 1532-1536, 2014 May.
Article in English | MEDLINE | ID: mdl-24765171

ABSTRACT

v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) genotyping is required prior to anti-epidermal growth factor receptor monoclonal antibody therapy administered in cases of metastatic colorectal carcinoma (CRC). Thus, KRAS mutation screening is required for patient management. The present study reported the experience of KRAS/v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutational screening on synchronous CRC pairs from 26 patients, which were defined as index lesions (ILs) and concurrent lesions (CLs) on the basis of tumor grade and dimension and their respective lymph node and distant metastases. Overall, KRAS mutations were present in 38.4% of patients, whereas BRAF mutations were present at a frequency of 11.5%. The genotyping of paired synchronous carcinomas indicated that 11 patients (42.3%) exhibited discordant KRAS mutational statuses in terms of the presence of a mutation in only one lesion of the pair or of two different mutations harbored by each lesion. BRAF mutations were present in the synchronous tumors of two cases, whereas in two other cases, only the IL or CL harbored mutant BRAF. Overall, the mutational statuses of distant and lymph node metastases confirm the genetic heterogeneity of synchronous primary tumors. These results highlighted the fact that adequate sampling and comprehensive testing, when feasible, is likely to optimize the decision-making process for treatment approaches, even in the relatively rare event of multiple synchronous lesions.

19.
Cancer Cytopathol ; 122(6): 445-53, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24648382

ABSTRACT

BACKGROUND: Anaplastic lymphoma kinase (ALK) gene rearrangements are detected in approximately 5% of cases of non-small cell lung cancer (NSCLC). Patients who are positive for ALK rearrangements may be successfully treated with the ALK inhibitor crizotinib. Because advanced-stage lung cancers are not suitable for surgical resection, approximately 70% of patients are diagnosed via preoperative specimens. In the current study, the authors evaluated the suitability of stained cytologic direct smears and cell blocks for fluorescence in situ hybridization (FISH) to determine ALK status compared with small biopsies. METHODS: A total of 493 consecutive patients with NSCLC were analyzed by FISH for ALK gene rearrangements. The analyzed sample comprised 180 cytological samples, 94 direct smears, 86 cell blocks, and 313 preoperative small biopsies. Moreover, in the same series, 426 patients and 369 patients, respectively, were evaluated for epidermal growth factor receptor and KRAS mutations, respectively. RESULTS: Of the total of 493 patients, 18 patients who were positive for a gene rearrangement (4.4%) demonstrated ALK FISH rearrangements, whereas 387 patients (95.6%) were negative. All other cases were classified as inadequate (7.7%) and insufficient (10.1%). A strong statistical association was found between the cytology and the small biopsy with respect to ALK rearrangements (P=.0048). Fifty-three patients (12.4%) demonstrated an epidermal growth factor receptor mutation, whereas 90 patients (24.4%) were found to have KRAS mutations. None of these patients presented with ALK gene rearrangements. CONCLUSIONS: ALK gene rearrangements may be easily detected in cytological samples and particularly in direct smears, thereby yielding important improvements in the diagnostic approach to patients with advanced NSCLC. Cytological samples may be useful for ALK analysis when insufficient material is available in cell blocks or small biopsies.


Subject(s)
Adenocarcinoma/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Squamous Cell/genetics , Cytodiagnosis , Gene Rearrangement , Lung Neoplasms/genetics , Receptor Protein-Tyrosine Kinases/genetics , Adenocarcinoma/pathology , Aged , Anaplastic Lymphoma Kinase , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Squamous Cell/pathology , ErbB Receptors/genetics , Female , Follow-Up Studies , Humans , In Situ Hybridization, Fluorescence , Lung Neoplasms/pathology , Male , Mutation/genetics , Neoplasm Staging , Prognosis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , ras Proteins/genetics
20.
Epigenetics ; 9(4): 621-33, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24500500

ABSTRACT

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.


Subject(s)
Colon/metabolism , Colorectal Neoplasms/metabolism , DNA Methylation , Folic Acid/metabolism , Intestinal Mucosa/metabolism , Metabolic Networks and Pathways , Promoter Regions, Genetic , Rectum/metabolism , Aged , Aged, 80 and over , Biomarkers/metabolism , Case-Control Studies , Colon/pathology , Colorectal Neoplasms/pathology , Female , Homocysteine/blood , Humans , Intestinal Mucosa/pathology , Male , Middle Aged , Polymorphism, Genetic , Rectum/pathology , Vitamin B 12/blood
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