Oscar Nappi

BRAF/NRAS Mutation Frequencies Among Primary Tumors and Metastases in Patients With Melanoma

PURPOSEnThe prevalence of BRAF, NRAS, and p16CDKN2A mutations during melanoma progression remains inconclusive. We investigated the prevalence and distribution of mutations in these genes in different melanoma tissues.nnnPATIENTS AND METHODSnIn all, 291 tumor tissues from 132 patients with melanoma were screened. Paired samples of primary melanomas (n = 102) and synchronous or asynchronous metastases from the same patients (n = 165) were included. Tissue samples underwent mutation analysis (automated DNA sequencing). Secondary lesions included lymph nodes (n = 84), and skin (n = 36), visceral (n = 25), and brain (n = 44) sites.nnnRESULTSnBRAF/NRAS mutations were identified in 58% of primary melanomas (43% BRAF; 15% NRAS); 62% in lymph nodes, 61% subcutaneous, 56% visceral, and 70% in brain sites. Mutations were observed in 63% of metastases (48% BRAF; 15% NRAS), a nonsignificant increase in mutation frequency after progression from primary melanoma. Of the paired samples, lymph nodes (93% consistency) and visceral metastases (96% consistency) presented a highly similar distribution of BRAF/NRAS mutations versus primary melanomas, with a significantly less consistent pattern in brain (80%) and skin metastases (75%). This suggests that independent subclones are generated in some patients. p16CDKN2A mutations were identified in 7% and 14% of primary melanomas and metastases, with a low consistency (31%) between secondary and primary tumor samples.nnnCONCLUSIONnIn the era of targeted therapies, assessment of the spectrum and distribution of alterations in molecular targets among patients with melanoma is needed. Our findings about the prevalence of BRAF/NRAS/p16CDKN2A mutations in paired tumor lesions from patients with melanoma may be useful in the management of this disease.

Detection of KRAS mutations in colorectal carcinoma patients with an integrated PCR/sequencing and real-time PCR approach

AIMSnPatients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit from treatment with anti-EGF receptor monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting to aid in the choice of appropriate therapy.nnnMATERIALS & METHODSnWe developed a cost-effective approach for the determination of KRAS mutations in codons 12 and 13 in clinical practice based on a sensitive PCR/sequencing technique and the commercially available real-time PCR-based Therascreen kit (DxS Ltd).nnnRESULTS & CONCLUSIONnThe PCR/sequencing test was able to detect 10% mutant DNA in a background of wild-type DNA. By using this assay, we determined the mutational status of KRAS in 527 out of 540 (97.6%) formalin-fixed paraffin-embedded tissues from mCRC patients. PCR/sequencing was not conclusive in 13 cases, in which low-intensity peaks suggestive of potential mutations were identified. The DxS assay, which showed a sensitivity of 1%, identified mutations in 11 out of 13 inconclusive cases. Interestingly, five of these 11 cases showed high levels of DNA fragmentation. No significant difference was found in the ability of PCR/sequencing and DxS to identify KRAS mutations within 160 cases with more than 30% tumor cells. However, in 24 samples with less than 30% tumor cells, DxS showed an higher sensitivity. In conclusion, our findings suggest that PCR/sequencing can be used for mutational analysis of the majority of tumor samples that have more than 30% tumor cell content, whereas more sensitive and expensive tests should be reserved for inconclusive cases and for samples with a low amount of tumor cells.

KRAS Mutations Testing in Colorectal Carcinoma Patients in Italy: From Guidelines to External Quality Assessment

Background Monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been approved for the treatment of patients with metastatic colorectal carcinoma (mCRC) that do not carry KRAS mutations. Therefore, KRAS testing has become mandatory to chose the most appropriate therapy for these patients. Methodology/Principal Findings In order to guarantee the possibility for mCRC patients to receive an high quality KRAS testing in every Italian region, the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytopathology -Italian division of the International Academy of Pathology (SIAPEC-IAP) started a program to improve KRAS testing. AIOM and SIAPEC identified a large panel of Italian medical oncologists, pathologists and molecular biologists that outlined guidelines for KRAS testing in mCRC patients. These guidelines include specific information on the target patient population, the biological material for molecular analysis, the extraction of DNA, and the methods for the mutational analysis that are summarized in this paper. Following the publication of the guidelines, the scientific societies started an external quality assessment scheme for KRAS testing. Five CRC specimens with known KRAS mutation status were sent to the 59 centers that participated to the program. The samples were validated by three referral laboratories. The participating laboratories were allowed to use their own preferred method for DNA extraction and mutational analysis and were asked to report the results within 4 weeks. The limit to pass the quality assessment was set at 100% of true responses. In the first round, only two centers did not pass (3%). The two centers were offered to participate to a second round and both centers failed again to pass. Conclusions The results of this first Italian quality assessment for KRAS testing suggest that KRAS mutational analysis is performed with good quality in the majority of Italian centers.

High CXCR4 expression correlates with sunitinib poor response in metastatic renal cancer.

BACKGROUNDnAlmost 30% of the sunitinib-treated patients for metastatic renal carcinoma (mRCC) do not receive a clinical benefit. Convincing evidences demonstrated a cross talk between the VEGF and CXCR4 pathways. It was hypothesized that CXCR4 expression in primary renal cancer could predict sunitinib responsiveness.nnnPATIENTS AND METHODSnIn this exploratory study sixty-two mRCC patients receiving sunitinib as first-line treatment were evaluated for CXCR4 expression through immunohistochemistry (IHC). Correlations between CXCR4 expression, baseline patients and tumour characteristics were studied by contingency tables and the chi-square test. Univariable analysis was performed with the log-rank test, and the Cox model was applied for multivariable analysis.nnnRESULTSnThe objective response rate of sunitinib first-line therapy was 35.5% (22/62) with a disease control rate (response and stable disease) of 62.9% (39/62). CXCR4 expression was absent/low in 30 (48.4%), moderate in 17 (27.4%), and high in 15 (24.2%) tumors respectively. Low or absent CXCR4 expression predicted response to sunitinib therapy. Moreover, Fuhrman grading and concomitant, CXCR4 and Fuhrman grading, strongly predicted sunitinib first line therapy responsiveness on progression-free survival and overall survival.nnnCONCLUSIONSnHigh CXCR4 expression correlates with sunitinib poor response in metastatic renal cancer.

Abstract 3677: Pattern and distribution of BRAF/NRAS and P16CDKN2A mutations among primary an secondary lesions in melanoma patients

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnBackground: Mutations of NRAS and BRAF genes have been identified with high frequency in nevi, cutaneous melanomas, and melanoma metastases. Prevalence of such mutations during the disease progression phases and among the different types of metastasis still remains inconclusive. Methods: A total of 275 tumour tissues from 116 melanoma patients were screened for mutations; among them, paired samples of microdissected primary melanomas (N=92) and synchronous or asynchronous metastases (N=156) from same patients were included. Tissue samples underwent mutation analysis by automated DNA sequencing. Secondary lesions were from: lymph nodes (LM; N=77), skin (SM; N=36), visceral (VM; N=23) and brain (BM; N= 44) sites. The full coding sequences and splice junctions of p16CDKN2A (exons 1, 2, and 3) and NRAS (exons 2 and 3) genes as well as the entire sequence at the exon 15 of the BRAF gene were screened for mutations by direct sequencing on automated fluorescence-cycle sequencer (ABIPRISM 3130, Applied Biosystems, Foster City, CA). Results: Overall, mutations were identified in 56/95 (59%) primary melanomas [43% BRAF - 16% NRAS], 49/77 (64%) lymph nodes [48% BRAF - 16% NRAS], 22/36 (61%) subcutaneous metastases [53% BRAF - 8% NRAS], 13/23 (57%) visceral metastases [43% BRAF - 13% NRAS], and 31/44 (70%) brain metastases [48% BRAF - 23% NRAS]. Overall, a slight and not significant increase in mutation frequency after progression from primary melanoma was observed in our series: 115/180 (64%) mutated metastases [48% BRAF - 16% NRAS]. Considering the paired samples from the same patients, LM (92% consistency) and VM (96% consistency) presented a highly similar prevalence and distribution of BRAF/NRAS mutations in comparison with primary melanomas, whereas a discontinuous pattern of mutations was detected in BM (80% consistency) or, mostly, SM (75% consistency). Occurrence of distinct mutation distribution between primary melanomas and correspondent metastases suggests that independent subclones have been generated in a limited fraction of patients. The rate of mutations in p16CDKN2A gene was found to steadily increase moving from primary melanomas (5/69; 7%) to melanoma metastases (21/144; 15%), with the highest prevalence of p16CDKN2A alterations (18/29; 62%) in melanoma cell lines Conclusions: Our results provide further clues about the impact of NRAS and BRAF mutations among the different stages of melanoma progression. Moreover, we confirmed that p16CDKN2A mutation rates are increasing during disease progression.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3677. doi:1538-7445.AM2012-3677

Neuroendocrine tumors diagnosed at the "Antonio Cardarelli" hospital (Naples, Campania, Italy) between 2006-2009: A single-institution analysis

Neuroendocrine tumors (NETs) are rare, with an incidence of about 5 per 100,000 inhabitants. As no study on NETs has ever been specifically conducted on the population of Campania, we performed a retrospective analysis of all newly diagnosed NETs at the “Antonio Cardarelli” hospital between 2006–2009. A search of the registry of the Pathology Department of the “Antonio Cardarelli” hospital was carried out to retrieve available data on all newly diagnosed NET cases. Two hundred and ninety-nine NET tumors were diagnosed at our Institution from January, 2006 to December, 2009. Globally, 121 patients (40% of the population) had a lung NET, while 92 patients (30% of the population) presented a GEP-NET. The most common primary tumor site varied by sex, with female patients being more likely to have a primary NET in the lung, breast or colon, and male patients being more likely to have a primary tumor in the lung. Also, twenty-three cases of breast NETs were identified, and clinical information regarding therapy and response was available for 22 patients. Our study represents a pioneering effort to provide the medical community in Campania with basic information on a large number of patients with different types of NETs. The “Antonio Cardarelli” hospital could greatly benefit from cooperation with other hospitals in order to become a highly specialized center for NETs in the region and Southern Italy.