Combination of mutations in genes controlling DNA repair and high mutational load plays a prognostic role in pancreatic ductal adenocarcinoma (PDAC): a retrospective real-life study in Sardinian population.

BACKGROUND: Patients with pancreatic ductal adenocarcinoma (PDCA) carrying impaired mismatch repair mechanisms seem to have an outcome advantage under treatment with conventional chemotherapy, whereas the role for the tumor mutation burden on prognosis is controversial. In this study, we evaluated the prognostic role of the mutated genes involved in genome damage repair in a real-life series of PDAC patients in a hospital-based manner from the main Institution deputed to surgically treat such a disease in North Sardinia. METHODS: A cohort of fifty-five consecutive PDAC patients with potentially resectable/border line resectable PDAC (stage IIB-III) or oligometastatic disease (stage IV) and tumor tissue availability underwent next-generation sequencing (NGS)-based analysis using a panel containing driver oncogenes and tumor suppressor genes as well as genes controlling DNA repair mechanisms. RESULTS: Genes involved in the both genome damage repair (DR) and DNA mismatch repair (MMR) were found mutated in 17 (31%) and 15 (27%) cases, respectively. One fourth of PDAC cases (14/55; 25.5%) carried tumors presenting a combination of mutations in repair genes (DR and MMR) and the highest mutation load rates (MLR-H). After correction for confounders (surgery, adjuvant therapy, stage T, and metastasis), multivariate Cox regression analysis indicated that mutations in DR genes (HR = 3.0126, 95% CI 1.0707 to 8.4764, p = 0.0367) and the MLR (HR = 1.0018, 95%CI 1.0005 to 1.0032, p = 0.009) were significantly related to worse survival. CONCLUSIONS: The combination of mutated repair genes and MLR-H, which is associated with a worse survival in our series of PDAC patients treated with conventional chemotherapy protocols, might become a predictive biomarker of response to immunotherapy in addition to its prognostic role in predicting survival.

Liquid Biopsy-Driven Cetuximab Rechallenge Strategy in Molecularly Selected Metastatic Colorectal Cancer Patients.

Background: Rechallenge with EGFR inhibitors represents a promising strategy for patients with RAS wild type (WT) colorectal cancer (CRC) but definitive selection criteria are lacking. Recently, the RAS WT status on circulating tumor DNA (ct-DNA) emerged as a potential watershed for this strategy. Our study explored the liquid biopsy-driven cetuximab rechallenge in a RAS and BRAF WT selected population. Methods: CRC patients with RAS and BRAF WT both on tumor tissue and on ct-DNA at baseline receiving rechallenge with cetuximab were eligible for our analysis. Ct-DNA was analyzed for RAS-BRAF mutations with pyro-sequencing and nucleotide sequencing assays. Real-time PCR and droplet digital PCR were performed to confirm the RAS-BRAF mutational status. Results: A total of 26 patients were included in our analysis. In the global population, RR was 25.0%, median overall survival (mOS) was 5.0 months, and median progression-free survival (mPFS) was 3.5 months. Previous response to anti-EGFR was associated with improved mPFS (5.0 vs. 2.0 months, HR: 0.26, p = 0.048); anti-EGFR free interval > 14 months and anti-EGFR free interval > 16 months were associated with improved mPFS (respectively 7.0 vs. 3.0 months, HR: 0.27, p = 0.013 and not reached vs. 3.0 months, HR: 0.20, p = 0.002) and with improved mOS (respectively 13.0 vs. 5.0 months, HR: 0.27, p = 0.013 and 13.0 vs. 5.0 months, HR: 0.20, p = 0.002). Previous lines >2 were correlated with improved mPFS (4.0 vs. 1.0 month, HR: 0.05, p = 0.041) and with improved mOS (7.0 vs. 1.0 month, HR: 0.045, p = 0.034). In a multiple logistic regression model, only the anti-EGFR free interval was confirmed to be a significant predictor for mOS and mPFS. Conclusions: Liquid biopsy-driven cetuximab rechallenge was confirmed to be effective. The clinical outcome was consistent with available results from phase II studies. In addition to the molecular selection through the analysis of ct-DNA for RAS, the long anti-EGFR free interval is confirmed as a prospective selection criterion for this therapeutic option.

Global prognostic impact of driver genetic alterations in patients with lung adenocarcinoma: a real-life study.

BACKGROUND: Advanced lung adenocarcinoma (LAC) is one of the most lethal malignancies worldwide. The aim of this study was to evaluate the global survival in a real-life cohort of patients with LAC harboring driver genetic alterations. METHODS: A series of 1282 consecutive Sardinian LAC patients who underwent genetic testing from January 2011 through July 2016 was collected. Molecular tests were based on the clinical needs of each single case (EGFR-exon18/19/21, ALK, and, more recently, BRAF-exon15), and the availability of tissue (KRAS, MET, and presence of low-frequency EGFR-T790M mutated alleles at baseline). RESULTS: The mean follow-up time of the patients was 46 months. EGFR, KRAS, and BRAF mutations were detected in 13.7%, 21.3%, and 3% of tested cases, respectively; ALK rearrangements and MET amplifications were found respectively in 4.7% and 2% of tested cases. As expected, cases with mutations in exons 18-21 of EGFR, sensitizing to anti-EGFR tyrosine kinase inhibitors (TKIs) agents, had a significantly longer survival in comparison to those without (p < 0.0001); conversely, KRAS mutations were associated with a significantly lower survival (p = 0.0058). Among LAC patients with additional tissue section available for next-generation sequencing (NGS)-based analysis, 26/193 (13.5%) patients found positive for even low-rate EGFR-T790M mutated alleles at baseline were associated with a highly significant lower survival in comparison to those without (8.7 vs. 47.4 months, p < 0.0001). CONCLUSIONS: In addition to its predictive value for addressing targeted therapy approaches, the assessment of as more inclusive mutation analysis at baseline may provide clues about factors significantly impacting on global survival in advanced LAC patients.

KIT and PDGFRa mutational patterns in Sardinian patients with gastrointestinal stromal tumors.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal malignancy of the gastrointestinal tract. We provide in the present article the molecular characterization of a series of primary GISTs in a cohort of Sardinian patients (Italy), with the aim to describe the patterns of KIT and PDGFRa mutations and the corresponding clinical features. Ninety-nine Sardinian patients with histologically-proven diagnosis of GIST were included in the study. Medical records and pathology reports were used to assess the demographic and clinical features of the patients and the disease at the time of the diagnosis. Formalin-fixed, paraffin-embedded tissue samples were retrieved for each case, and mutation analysis of the KIT and PDGFRa genes was performed. KIT and PDGFRa mutations were detected in 81.8% and 5% of the cases, respectively. The most common KIT mutation was W557_K558del in exon 11, while D842V in exon 18 was the most common PDGFRa genetic alteration; V561D was the only PDGFRa mutation found in exon 12. The global "wild-type" cases, with no mutations in either the KIT or PDGFRa genes, were 13 (13.1%). The mean survival of those patients was approximately 46.9 (±43.9) months. Globally, 86.9% of Sardinian patients with GIST had a KIT or PDGFRa mutation; the former were more frequent in comparison with other Italian cohorts, while PDGFRa mutations were rare. No statistical differences in survival between mutated and wild-type cases, and between KIT and PDGFRa mutated cases were detected in our study.

Comparison of BRAF Mutation Screening Strategies in a Large Real-Life Series of Advanced Melanoma Patients.

Malignant melanoma (MM) is one of the deadliest skin cancers. BRAF mutation status plays a predominant role in the management of MM patients. The aim of this study was to compare BRAF mutational testing performed by conventional nucleotide sequencing approaches with either real-time polymerase chain reaction (rtPCR) or next-generation sequencing (NGS) assays in a real-life, hospital-based series of advanced MM patients. Consecutive patients with AJCC (American Joint Committee on Cancer) stage IIIC and IV MM from Sardinia, Italy, who were referred for molecular testing, were enrolled into the study. Initial screening was performed to assess the mutational status of the BRAF and NRAS genes, using the conventional methodologies recognized by the nationwide guidelines, at the time of the molecular classification, required by clinicians: at the beginning, Sanger-based sequencing (SS) and, after, pyrosequencing. The present study was then focused on BRAF mutation detecting approaches only. BRAF wild-type cases with available tissue and adequate DNA were further tested with rtPCR (Idylla™) and NGS assays. Globally, 319 patients were included in the study; pathogenic BRAF mutations were found in 144 (45.1%) cases examined with initial screening. The rtPCR detected 11 (16.2%) and 3 (4.8%) additional BRAF mutations after SS and pyrosequencing, respectively. NGS detected one additional BRAF-mutated case (2.1%) among 48 wild-type cases previously tested with pyrosequencing and rtPCR. Our study evidenced that rtPCR and NGS were able to detect additional BRAF mutant cases in comparison with conventional sequencing methods; therefore, we argue for the preferential utilization of the aforementioned assays (NGS and rtPCR) in clinical practice, to eradicate false-negative cases and improve the accuracy of BRAF detection.

Harmonization of Next-Generation Sequencing Procedure in Italian Laboratories: A Multi-Institutional Evaluation of the SiRe® Panel.

Background: Next-generation sequencing (NGS) needs to be validated and standardized to ensure that cancer patients are reliably selected for target treatments. In Italy, NGS is performed in several institutions and harmonization of wet and dry procedures is needed. To this end, a consortium of five different laboratories, covering the most part of the Italian peninsula, was constituted. A narrow gene panel (SiRe®) covering 568 clinically relevant mutations in six different genes (EGFR, KRAS, NRAS, BRAF, cKIT, and PDGFRα) with a predictive role for therapy selection in non-small cell lung cancer (NSCLC), gastrointestinal stromal tumor, colorectal carcinoma (CRC), and melanoma was evaluated in each participating laboratory. Methods: To assess the NGS inter-laboratory concordance, the SiRe® panel, with a related kit and protocol for library preparation, was used in each center to analyze a common set of 20 NSCLC and CRC routine samples. Concordance rate, in terms of mutation detected and relative allelic frequencies, was assessed. Then, each institution prospectively analyzed an additional set of 40 routine samples (for a total of 160 specimens) to assess the reproducibility of the NGS run parameters in each institution. Results: An inter-laboratory agreement of 100% was reached in analyzing the data obtained from the 20 common sample sets; the concordance rate of allelic frequencies distribution was 0.989. The prospective analysis of the run metric parameters obtained by each center locally showed that the analytical performance of the SiRe® panel in the different institutions was highly reproducible. Conclusions: The SiRe® panel represents a robust diagnostic tool to harmonize the NGS procedure in different Italian laboratories.

Genetic Instability Markers in Cancer.

High frequency of mutations seems to determine a higher occurrence of neoepitope formation and, thus, tumor immunogenicity. A somatic hypermutated status could thus act as a predictive biomarker of responsiveness to immunotherapy with recent immune checkpoint inhibitors. Among several factors involved in determining the hypermutated status, such as inactivating mutations in the DNA polymerases as well as exposure to external (cigarette smoke, UV radiation, chemicals) and endogenous (reactive oxygen species) mutagens, a defective DNA mismatch repair system may give rise to genetic instability and, particularly, to microsatellite instability (MSI). The occurrence of MSI has been associated with increased load of mutations and expression of abundant peptides that serve as neoantigens to elicit an immune response within a context of a favorable tumor microenvironment. Here we describe methodological strategies to investigate for the presence of the MSI phenotype in cancer samples, through a combination of molecular approaches performed on paraffin-embedded tissues.

EGFR, KRAS, BRAF, ALK, and cMET genetic alterations in 1440 Sardinian patients with lung adenocarcinoma.

BACKGROUND: Lung cancer is one of the most incident neoplastic diseases, and a leading cause of death for cancer worldwide. Knowledge of the incidence of druggable genetic alterations, their correlation with clinical and pathological features of the disease, and their interplay in cases of co-occurrence is crucial for selecting the best therapeutic strategies of patients with non-small cell lung cancer. In this real-life study, we describe the molecular epidemiology of genetic alterations in five driver genes and their correlations with the demographic and clinical characteristics of Sardinian patients with lung adenocarcinoma. METHODS: Data from 1440 consecutive Sardinian patients with a histologically proven diagnosis of lung adenocarcinoma from January 2011 through July 2016 were prospectively investigated. EGFR mutation analysis was performed for all of them, while KRAS and BRAF mutations were searched in 1047 cases; ALK alterations were determined with fluorescence in situ hybridization in 899 cases, and cMET amplifications in 788 cases. RESULTS: KRAS mutations were the most common genetic alterations involving 22.1% of the cases and being mutually exclusive with the EGFR mutations, which were found in 12.6% of them. BRAF mutations, ALK rearrangements, and cMET amplifications were detected in 3.2, 5.3, and 2.1% of the cases, respectively. Concomitant mutations were detected only in a few cases. CONCLUSIONS: Almost all the genetic alterations studied showed a similar incidence in comparison with other Caucasian populations. Concomitant mutations were rare, and they probably have a scarce impact on the clinical management of Sardinians with lung adenocarcinoma. The low incidence of concomitant cMET amplifications at diagnosis suggests that these alterations are acquired in subsequent phases of the disease, often during treatment with TKIs.

BRAF Mutations and Dysregulation of the MAP Kinase Pathway Associated to Sinonasal Mucosal Melanomas.

Sinonasal mucosal melanoma (SNM) is a rare and aggressive type of melanoma, and because of this, we currently have a limited understanding of its genetic and molecular constitution. The incidence among SNMs of somatic mutations in the genes involved in the main molecular pathways, which have been largely associated with cutaneous melanoma, is not yet fully understood. Through a next-generation sequencing (NGS) approach using a panel of 25 genes involved in melanoma pathogenesis customized by our group, we performed a mutation analysis in a cohort of 25 SNM patients. Results showed that pathogenic mutations were found in more than 60% of SNM cases at a somatic level, with strikingly 32% of them carrying deleterious mutations in the BRAF gene. The identified mutations mostly lack the typical UV signature associated with cutaneous melanomas and showed no significant association with any histopathological parameter. Oncogenic activation of the BRAF-depending pathway, which may induce immune tolerance into the tumour microenvironment (i.e., by increasing the VEGF production) was poorly associated with mutations in genes that have been related to diminished clinical benefit of the treatment with BRAF inhibitors. Screening for mutations in BRAF and other MAPK genes should be included in the routine diagnostic test for a better classification of SNM patients.

Mutational concordance between primary and metastatic melanoma: a next-generation sequencing approach.

BACKGROUND: Cutaneous malignant melanoma (CMM) is one of the most common skin cancers worldwide. Limited information is available in the current scientific literature on the concordance of genetic alterations between primary and metastatic CMM. In the present study, we performed next-generation sequencing (NGS) analysis of the main genes participating in melanoma pathogenesis and progression, among paired primary and metastatic lesions of CMM patients, with the aim to evaluate levels of discrepancies in mutational patterns. METHODS: Paraffin-embedded tumor tissues of the paired lesions were retrieved from the archives of the institutions participating in the study. NGS was performed using a specific multiple-gene panel constructed by the Italian Melanoma Intergroup (IMI) to explore the mutational status of selected regions (343 amplicons; amplicon range: 125-175 bp; coverage 100%) within the main 25 genes involved in CMM pathogenesis; sequencing was performed with the Ion Torrent PGM System. RESULTS: A discovery cohort encompassing 30 cases, and a validation cohort including eleven Sardinian patients with tissue availability from both the primary and metachronous metastatic lesions were identified; the global number of analyzed tissue specimens was 90. A total of 829 genetic non-synonymous variants were detected: 101 (12.2%) were pathogenic/likely pathogenic, 131 (15.8%) were benign/likely benign, and the remaining 597 (72%) were uncertain/unknown significance variants. Considering the global cohort, the consistency in pathogenic/pathogenic like mutations was 76%. Consistency for BRAF and NRAS mutations was 95.2% and 85.7% respectively, without statistically significant differences between the discovery and validation cohort. CONCLUSIONS: Our study showed a high level of concordance in mutational patterns between primary and metastatic CMM, especially when pathogenic mutations in driver genes were considered.

Dietary compounds and cutaneous malignant melanoma: recent advances from a biological perspective.

Cutaneous malignant melanoma is a heterogeneous disease, being the consequence of specific genetic alterations along several molecular pathways. Despite the increased knowledge about the biology and pathogenesis of melanoma, the incidence has grown markedly worldwide, making it extremely important to develop preventive measures. The beneficial role of correct nutrition and of some natural dietary compounds in preventing malignant melanoma has been widely demonstrated. This led to numerous studies investigating the role of several dietary attitudes, patterns, and supplements in the prevention of melanoma, and ongoing research investigates their impact in the clinical management and outcomes of patients diagnosed with the disease. This article is an overview of recent scientific advances regarding specific dietary compounds and their impact on melanoma development and treatment.

Female Adnexal Tumors of Probable Wolffian Origin (FATWO): A Case Series With Next-Generation Sequencing Mutation Analysis.

Female adnexal tumors of probable Wolffian origin are rare gynecologic tumors with <90 cases reported in the current scientific literature. Their clinical features have been described extensively; less is known about the pathophysiological mechanisms and the molecular alterations underlying their development and growth. We performed a complete histopathologic examination and a systematic mutation analysis using a next-generation sequencing approach on 3 female adnexal tumors of probable Wolffian origin from the archives of our institution to detect possible genetic alterations and to explore their role in the development of these rare tumors. The 3 cases contained missense mutations in different genes belonging to distinct molecular pathways: CTNNB1 and MET mutations for the first case, PIK3CA for the second one, and BRAF and CDKN2A for the third one. Two variants with an unknown functional effect on the protein were found in KDR and TP53 genes. In conclusion, genetic heterogeneity was found in our series. No constant involvement of the most common pathways involved in tumorigenesis was found; nevertheless, further studies are necessary to confirm the results of this pilot study.

Long non-coding RNA CASC2 in human cancer.

Long non-coding RNAs cover large part of the non-coding information of the human DNA, which represents more than 90% of the whole genome. They constitute a wide and complex group of molecules with more than 200 nucleotides, which generally lack an open reading frame, and are involved in various ways in the pathophysiology of cancer. Their roles in the regulation of gene expression, imprinting, transcription, and post-translational processing have been described in several types of cancer. CASC2 was discovered in 2004 in patients with endometrial carcinoma as a potential tumor suppressor. Since then, additional studies in other types of neoplasia have been carried out, and both mechanisms and interactions of CASC2 in cancer have been better elucidated. In this review, we summarize the current knowledge on the role of CASC2 in the genesis, progression, and clinical management of human cancer.

Breast Nodular Fasciitis: A Comprehensive Review.

BACKGROUND: This article describes the demographic, clinical, pathological and prognostic features of breast nodular fasciitis through a comprehensive review of the cases reported in modern literature. MATERIAL AND METHODS: English-language articles published from January 1970 to October 2015 and related to breast nodular fasciitis were non-systematically retrieved using the PubMed database. Titles, abstracts and references were evaluated in order to include the most relevant studies. The demographic characteristics of the patients and the signs and symptoms of the disease were reviewed, as well as the results of the radiological and diagnostic procedures employed and of the treatments adopted. RESULTS: 26 papers reporting on 28 cases were enrolled; however, manuscripts published before 1990 were excluded because of old methodologies, technologies and clinical approaches. Thus, 22 cases included in 20 papers were analyzed in detail. CONCLUSION: The rarity and the unspecific clinical and radiological characteristics of breast nodular fasciitis make the differential diagnosis and management challenging. Knowledge of the clinical, pathological and prognostic aspects of this condition is crucial for breast care specialists in order to improve their diagnostic and therapeutic interventions.

Prognostic impact of KRAS, NRAS, BRAF, and PIK3CA mutations in primary colorectal carcinomas: a population-based study.

BACKGROUND: Activation of oncogenes downstream the EGFR gene contributes to colorectal tumorigenesis and determines the sensitivity to anti-EGFR treatments. The aim of this study was to evaluate the prognostic value of KRAS, BRAF, NRAS and PIK3CA mutations in a large collection of CRC patients from genetically-homogeneous Sardinian population. METHODS: A total of 1284 Sardinian patients with histologically-proven diagnosis of colorectal carcinoma (CRC) and presenting with metastatic disease were included into the study. Genomic DNA was isolated from formalin-fixed, paraffin-embedded primary tumour tissue samples of CRC patients and screened for mutations in RAS and BRAF genes, using pyrosequencing assays, and in PIK3CA gene, using automated DNA sequencing assays. RESULTS: Overall, mutation rates were 35.6 % for KRAS, 4.1 % for NRAS, and 2.1 % for BRAF. Among available DNA samples, 114/796 (14.3 %) primary CRCs were found to carry a mutation in the PIK3CA gene. In this subset of patients analysed in all four genes, a pathogenetic mutation of at least one gene was discovered in about half (378/796; 47.5 %) of CRC cases. A mutated BRAF gene was found to steadily act as a negative prognostic factor for either time to progression as metastatic disease (from detection of primary CRC to diagnosis of first distant metastasis; p = 0.009) or partial survival (from diagnosis of advanced disease to the time of death or last control; p = 0.006) or overall survival (p < 0.001). No significant impact on prognosis was observed for mutated KRAS, NRAS, and PIK3CA genes or combined RAS mutations (all RAS). CONCLUSIONS: Our study defines both prevalence and prognostic role of main activated oncogenes in a population-based large collection of CRC patients.

Prognostic role of KRAS mutations in Sardinian patients with colorectal carcinoma.

The presence of mutations in the KRAS gene is a predictor of a poor clinical response to EGFR-targeted agents in patients affected by colorectal cancer (CRC), but its significance as a global prognostic factor remains unclear. The aim of the present study was to evaluate the impact of the KRAS mutational status on time to first metastasis (TTM) and overall survival (OS) in a cohort of Sardinian CRC patients. A total of 551 patients with metastatic CRC at the time of enrolment were included. Clinical and pathological features of the disease, including follow-up information, were obtained from medical records and cancer registry data. For mutational analysis formalin-fixed paraffin-embedded tissue samples were processed using a standard protocol. The coding sequence and splice junctions of exons 2 and 3 of the KRAS gene were screened for mutations by direct automated sequencing. Overall, 186 KRAS mutations were detected in 183/551 (33%) patients: 125 (67%) were located in codon 12, 36 (19%) in codon 13, and 18 (10%) in codon 61. The remaining mutations (7; 4%) were detected in uncommonly-affected codons. No significant correlation between KRAS mutations and gender, age, anatomical location and stage of the disease at the time of diagnosis was identified. Furthermore, no prognostic value of KRAS mutations was found considering either TTM or OS. When patients were stratified by KRAS mutational status and gender, males were significantly associated with a longer TTM. The results of the present study indicate that KRAS mutation correlated with a slower metastatic progression in males with CRC from Sardinia, irrespective of the age at diagnosis and the codon of the mutation.

Genome-wide association study of susceptibility loci for breast cancer in Sardinian population.

BACKGROUND: Despite progress in identifying genes associated with breast cancer, many more risk loci exist. Genome-wide association analyses in genetically-homogeneous populations, such as that of Sardinia (Italy), could represent an additional approach to detect low penetrance alleles. METHODS: We performed a genome-wide association study comparing 1431 Sardinian patients with non-familial, BRCA1/2-mutation-negative breast cancer to 2171 healthy Sardinian blood donors. DNA was genotyped using GeneChip Human Mapping 500 K Arrays or Genome-Wide Human SNP Arrays 6.0. To increase genomic coverage, genotypes of additional SNPs were imputed using data from HapMap Phase II. After quality control filtering of genotype data, 1367 cases (9 men) and 1658 controls (1156 men) were analyzed on a total of 2,067,645 SNPs. RESULTS: Overall, 33 genomic regions (67 candidate SNPs) were associated with breast cancer risk at the p < 0(-6) level. Twenty of these regions contained defined genes, including one already associated with breast cancer risk: TOX3. With a lower threshold for preliminary significance to p < 10(-5), we identified 11 additional SNPs in FGFR2, a well-established breast cancer-associated gene. Ten candidate SNPs were selected, excluding those already associated with breast cancer, for technical validation as well as replication in 1668 samples from the same population. Only SNP rs345299, located in intron 1 of VAV3, remained suggestively associated (p-value, 1.16 x 10(-5)), but it did not associate with breast cancer risk in pooled data from two large, mixed-population cohorts. CONCLUSIONS: This study indicated the role of TOX3 and FGFR2 as breast cancer susceptibility genes in BRCA1/2-wild-type breast cancer patients from Sardinian population.

Impact of tissue type and content of neoplastic cells of samples on the quality of epidermal growth factor receptor mutation analysis among patients with lung adenocarcinoma.

Assessment of the epidermal growth factor receptor (EGFR) mutational status has become crucial in recent years in the molecular classification of patients with lung cancer. The impact of the type and quantity of malignant cells of the neoplastic specimen on the quality of mutation analysis remains to be elucidated, and only empirical and sporadic data are available. The aim of the present study was to investigate the impact of tissue type and content of neoplastic cells in the specimen on the quality of EGFR mutation analysis among patients with lung adenocarcinoma. A total of 515 patients with histologically-confirmed disease were included in the present study. Formalin-fixed paraffin embedded tissue samples were used for the mutation analysis and the content of the neoplastic cells was evaluated using light microscopy. Genomic DNA was isolated using a standard protocol. The coding sequences and splice junctions of exons 18, 19 and 21 in the EGFR gene were then screened for mutations by direct automated sequencing. The mean age of the patients examined was 64.9 years and 357 (69.3%) were male. A total of 429 tissue samples (83.3%) were obtained by biopsy and the remaining samples were obtained by surgery. A total of 456 samples (88.5%) were observed from primary lung adenocarcinomas, while 59 (11.5%) were from metastatic lesions. EGFR mutations occurred in 59 cases (11.5%); exon 18 mutations were detected in one case (1.7%), whereas exon 19 and 21 mutations were detected in 30 (51%) and 28 (47.3%) cases, respectively. EGFR mutations were more frequent in females and patients that had never smoked. The distribution of the mutations among primary and metastatic tissues exhibited no significant differences in the proportions of EGFR mutations detected. However, a statistically significant difference in the number of mutations detected was found between samples with at least 50% of neoplastic cells (450 cases-57 mutations; 12.7%) and those with <50% of neoplastic cells (65 cases-2 mutations; 3.1%).

ERCC1 polymorphisms as prognostic markers in T4 breast cancer patients treated with platinum-based chemotherapy.

BACKGROUND: Polymorphisms in the excision repair cross-complimentary group 1 (ERCC1) gene have been involved in the prognosis of various cancers. In the present study, we evaluated the prognostic role of the two most common ERCC1 polymorphisms in patients with T4 breast cancer receiving platinum-based chemotherapy. METHODS: A total of 47 patients with T4 breast cancer undergoing treatment with a platinum-based regimen were collected and followed up (median 159 months; range, 42-239 months). ERCC1 C8092A (rs3212986) and T19007C (rs11615) polymorphisms were genotyped, using an automated sequencing approach. The same series was screened for BRCA1/2 mutations by DHPLC analysis and DNA sequencing. RESULTS: Among the tested patients, 16 (34%) and 25 (53%) presented the 8092A (homo-zygosity A/A or heterozygosity A/C) and the 19007C (homozygosity C/C or heterozygosity C/T) genotypes, respectively. The 8092A and 19007C genotypes in ERCC1 were significantly associated with overall survival in T4 breast cancer patients treated with chemotherapy containing platinum (p-values = 0.036 and 0.004, respectively). Univariate and multivariate Cox regression analyses showed that combination of 8092A and 19007C genotypes acts as a significant prognostic factor in women with T4 breast cancer receiving platinum-based chemotherapy (p-values = 0.022 and 0.049, respectively). Two (4.3%) out of 47 cases were found to carry BRCA1/2 mutations; they presented the highest overall survival rates into the series. CONCLUSIONS: The ERCC1 8092A and 19007C genotypes or their combination may predict a favorable prognosis in T4 breast cancer patients undergoing a platinum-based treatment. Further large-scale, prospective studies are needed to validate our findings.

KRAS mutational concordance between primary and metastatic colorectal adenocarcinoma.

KRAS mutation analysis is commonly performed on tissue samples obtained from primary colorectal cancers (CRCs). The metastatic lesions of CRC are usually considered as qualitatively similar or even identical to the primary tumors. The aim of this study was to evaluate the spectrum and distribution of KRAS mutations in a large collection of CRCs, while also evaluating the concordance of primary and metastatic lesions among available paired specimens from the same patients. A total of 729 patients with histologically confirmed advanced CRC at the University Hospital and Local Health Unit (Sassari, Italy) were included. Clinical and pathological features were obtained from medical records and/or pathology reports. Formalin-fixed, paraffin-embedded tissue samples were used for mutation analysis. Genomic DNA was isolated using a standard protocol; the coding sequence and splice junctions of exons 2 and 3 in the KRAS gene were screened by direct automated sequencing. Overall, 219 (30%) KRAS mutations were found; 208 (30.1%) were identified in the 690 primary tumors and 11 (28.2%) in the 39 metastatic tissue samples. Among the 31 (4.3%) patients who had paired samples of primary CRC and synchronous or asynchronous metastases, 28 (90.3%) showed consistent mutation patterns between the primary tumors and metastatic lesions. In one case, an additive mutation (Q61L) was found in the metastatic tissue, while two other discrepant cases exhibited a different mutation distribution; Q61H in the primitive lesion and G13V in the metastatic lesion in one case, and a mutated primary tumor (Q61L) and wild-type metastasis in another case. The results of this study confirm that a high concordance exists between the results of KRAS mutation analysis performed in primitive and metastatic CRCs; independent subclones may be generated in a limited amount of patients.