The aim of this work is to assess if cumulative dose (CD) and dose intensity (DI) of everolimus may affect survival of advanced pancreatic neuroendocrine tumors (PNETs) patients. One hundred and sixteen patients (62 males and 54 females, median age 55 years) with advanced PNETs were treated with everolimus for ≥3 months. According to a Receiver operating characteristics (ROC) analysis, patients were stratified into two groups, with CD ≤ 3000 mg (Group A; n = 68) and CD > 3000 mg (Group B; n = 48). The response rate and toxicity were comparable in the two groups. However, patients in group A experienced more dose modifications than patients in group B. Median OS was 24 months in Group A while in Group B it was not reached (HR: 26.9; 95% CI: 11.0–76.7; P < 0.0001). Patients who maintained a DI higher than 9 mg/day experienced a significantly longer OS and experienced a trend to higher response rate. Overall, our study results showed that both CD and DI of everolimus play a prognostic role for patients with advanced PNETs treated with everolimus. This should prompt efforts to continue everolimus administration in responsive patients up to at least 3000 mg despite delays or temporary interruptions.

Introduction KRAS oncogene mutations (MUTKRAS) drive resistance to EGFR inhibition by providing alternative signaling as demonstrated in colo-rectal cancer. In non-small cell lung cancer (NSCLC), the efficacy of treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) depends on activating EGFR mutations (MUTEGFR). However, inhibition of EGFR may select resistant cells displaying alternative signaling, i.e., KRAS, or restoration of EGFR activity due to additional MUTEGFR, i.e., the c.2369C > T (p.T790MEGFR). Aim The aim of this study was to investigate the appearance of MUTKRAS during EGFR-TKI treatment and their contribution to drug resistance. Methods This study used cell-free circulating tumor DNA (cftDNA) to evaluate the appearance of codon 12 MUTKRAS and p.T790MEGFR mutations in 33 advanced NSCLC patients progressing after an EGFR-TKI. Results p.T790MEGFR was detected in 11 (33.3%) patients, MUTKRAS at codon 12 in 3 (9.1%) while both p.T790MEGFR and MUTKRAS codon 12 were found in 13 (39.4%) patients. Six patients (18.2%) were KRAS wild-type (WTKRAS) and negative for p.T790MEGFR. In 8 subjects paired tumor re-biopsy/plasma samples were available; the percent concordance of tissue/plasma was 62.5% for p.T790MEGFR and 37.5% for MUTKRAS. The analysis of time to progression (TTP) and overall survival (OS) in WTKRAS vs. MUTKRAS were not statistically different, even if there was a better survival with WTKRAS vs. MUTKRAS, i.e., TTP 14.4 vs. 11.4 months (p = 0.97) and OS 40.2 vs. 35.0 months (p = 0.56), respectively. Conclusions MUTKRAS could be an additional mechanism of escape from EGFR-TKI inhibition and cftDNA is a feasible approach to monitor the molecular development of drug resistance.

Background: Activating mutations of KRAS oncogene drive resistance to EGFR inhibition by providing an alternative signal transduction pathway [1]. In non-small cell lung cancer (NSCLC), the efficacy of treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) depends on activating EGFR mutations that are mutually exclusive with KRAS mutations. However, pharmacological inhibition of EGFR signaling has the potential to select cells whose growth may depend, at least in part, on alternative proliferation pathways or continued EGFR signaling due to the c.2369C > T (p.T790M) gatekeeper mutation within the ATP-binding pocket of EGFR. NSCLC heterogeneity can drive the therapeutic decisions; therefore, tissue availability is increasingly recognized as a crucial issue [2]. Unfortunately, the location of the tumor and the risk of complications are serious limitations to re-biopsies in NSCLC. Alternatively, the detection of somatic mutations in cell-free tumor DNA (cftDNA) released in plasma could be instrumental for a better understanding of the genetic modifications driven by the selective pressure of drug treatments on NSCLC [3]. Material and methods: This study used cell-free circulating tumor DNA (cftDNA) to evaluate the appearance of codon 12 KRAS and p.T790M EGFR mutations in 33 advanced NSCLC patients that progressed after an EGFR-TKI. Six ml of blood samples were drawn from patients at disease progression and cftDNA was extracted by Circulating Nucleic Acid extraction kit (Qiagen) and analysed by digital droplet PCR (BioRad). Results:KRAS mutation at codon 12 alone or in combination with p.T790M was demonstrated in 3 (9.1%) and 13 patients (39.4%), respectively. p.T790M was detected in 11 subjects (33.3%) alone and in 13 patients (39.4%) with mutant KRAS. Six patients (18.2%) were negative for both KRAS and p.T790M. In 8 subjects paired tumor re-biopsy/plasma samples were available; the percent concordance of tissue/plasma was 62,5% for p.T790M and 37,5% for KRAS. Conclusions: In conclusion, mutation of KRAS could be an additional mechanism of escape to EGFR-TKI and cftDNA is a feasible approach to monitor the molecular development of drug resistance. Therefore, the clinical relevance of this finding, expecially for what concerns mutKRAS, needs to be evaluated prospectively. References 1Han SW, et al Clin Cancer Res 2006;12:2538–44. 2Bosc C, et al. Target Oncol 2014 [DOI 10.1007/s11523-014-0332-y]. 3Del Re M, et al. Ex Rev Mol Diagn 2014;14:453–68

Aim: Resistance to EGFR tyrosine kinase inhibitors (TKI) is a clinically relevant problem that needs to be addressed by the use of appropriate technological platforms to discover the early appearance of mutations during treatment and before clinical disease progression. However, there is a lack of standardised approaches to monitor the molecular evolution of the disease under the selective pressure exerted by targeted treatments. One potential, not yet fully validated approach, is the analysis of gene mutations conferring drug resistance on cell-free circulating tumor DNA (cctDNA) released into the peripheral blood from primary tumor and metastatic sites. Periodic monitoring of the genetic evolution of the tumor, unfeasible with repeated biopsies, will greatly contribute to a better understanding and clinical management of drug resistance in cancer patients.

4084 Background: Studies on Asian, US, and German patients have moved some criticisms on the validity of the 7th edition of the AJCC classification to discriminate outcome of gastric cancer stages. We investigated the effect of this AJCC classification in a high-quality surgical populations of patients receiving D2 lymphadenectomy. Methods: From the prospective database at San Salvatore Hospital, Pesaro, we identified 515 patientswith gastroesophageal junction (Siewert II and III) or stomach adenocarcinoma who underwent gastrectomy with curative intent from 1998 to 2010. Lymphadenectomy extended to the 3rd level 12p/b nodes (D2/D3) was performed in all patients. Overall survival (OS) probabilities, calculated from the date of surgery to the date of death, from any cause, were estimated using the Kaplan-Meier method and compared using the log-rank test. Results: 58% of patients were male,median age was 73 years (range 36-96). Median number of examined lymph nodes was 32 (range, 1-89), and only 8.9% of patients had less than 15 examined lymph nodes; 96 patients received adjuvant chemo- or chemoradiotherapy. As shown in the table, we proposed a revised staging system (Pesaro Staging System, PSS), which performs better than the 7th edition of AJCC classification in terms of survival differences between stages. Conclusions: This study confirms once again that the 7th edition of the AJCC classification does not discriminate adequately the outcome from stage to stage. In a European population of patients undergoing gastrectomy plus at least D2 lymphadenectomy, the revised staging system, PSS, better defines patient prognosis. [Table: see text]

AIMS:HtrA1 is a member of the HtrA (high-temperature requirement factor A) family of serine proteases. HtrA1 plays a protective role in various malignancies due to its tumour suppressive properties. The aim of this study was to determine HtrA1 expression as a predictor of chemoresponse in patients with advanced gastric cancer.METHODS AND RESULTS:HtrA1 expression was determined by immunohistochemistry on specimens of primary gastric cancer from 80 patients treated consecutively with cisplatin-based combination chemotherapy. Response to chemotherapy was assessed according to Response Evaluation Criteria in Solid Tumours (RECIST) criteria. Our population consisted of males/females [51/29; median age 64 years (range 32-82)]. A complete or partial response was observed in 71.4% [95% confidence interval (CI) 54.7-88.2], 66.7% (95% CI 47.8-85.5) and 28.6% (95 CI 11.8-45.3) of tumours showing high, medium and low HtrA1 expression, respectively. A statistically significant association between HtrA1 expression and the clinical response was observed (P = 0.002). The median overall survival for patients with high/medium expression was 17 months compared to 9.5 months for patients with low HtrA1 expression (P = 0.037).CONCLUSIONS:Identification of HtrA1 in gastric cancer prior to chemotherapy indicates that levels of HtrA1 could be used to predict response to platinum-based combination therapies. Further assessment of HtrA1 expression is highly warranted in large, prospective studies.

No established second-line chemotherapy is available for patients with advanced gastric cancer failing to respond or progressing to first-line chemotherapy. However, 20–40% of these patients commonly receive second-line chemotherapy. We evaluated the influence of clinico-pathologic factors on the survival of 175 advanced gastric cancer patients, who received second-line chemotherapy at three oncology departments. Univariate and multivariate analyses found five factors which were independently associated with poor overall survival: performance status 2 (hazard ratio (HR), 1.79; 95% CI, 1.16–2.77; P =0.008), haemoglobin ⩽11.5 g l −1 (HR, 1.48; 95% CI, 1.06–2.05; P =0.019), CEA level >50 ng ml −1 (HR, 1.86; 95% CI, 1.21–2.88; P =0.004), the presence of greater than or equal to three metastatic sites of disease (HR, 1.72; 95% CI, 1.16–2.53; P =0.006), and time-to-progression under first-line chemotherapy ⩽6 months (HR, 1.97; 95% CI, 1.39–2.80; P <0.0001). A prognostic index was constructed dividing patients into low- (no risk factor), intermediate- (one to two risk factors), or high- (three to five risk factors) risk groups, and median survival times for each group were 12.7 months, 7.1 months, and 3.3 months, respectively ( P <0.001). In the absence of data deriving from randomised trials, this analysis suggests that some easily available clinical factors may help to select patients with advanced gastric cancer who could derive more benefit from second-line chemotherapy.

PURPOSE:The objective is to investigate whether polymorphisms with putative influence on fluorouracil/oxaliplatin activity are associated with clinical outcomes of patients with advanced colorectal cancer treated with first-line oxaliplatin, folinic acid, and fluorouracil palliative chemotherapy.MATERIALS AND METHODS:Consecutive patients were prospectively enrolled onto medical oncology units in Central Italy. Patients were required to have cytologically/histologically confirmed metastatic disease with at least one measurable lesion. Peripheral blood samples were used for genotyping 12 polymorphisms in thymidylate synthase, methylenetetrahydrofolate reductase, xeroderma pigmentosum group D (XPD), excision repair cross complementing group 1 (ERCC1), x-ray cross complementing group 1, x-ray cross complementing protein 3, glutathione S-transferases (GSTs) genes. The primary end point of the study was to investigate the association between genotypes and progression-free survival (PFS).RESULTS:In 166 patients, ERCC1-118 T/T, XPD-751 A/C, and XPD-751 C/C genotypes were independently associated with adverse PFS. The presence of two risk genotypes (ERCC1-118 T/T combined with either XPD-751 A/C or XPD-751 C/C) occurred in 50 patients (31%). This profiling showed an independent role for unfavorable PFS with a hazard ratio of 2.84% and 95% CI of 1.47 to 5.45 (P = .002). Neurotoxicity was significantly associated with GSTP1-105 A/G. Carriers of the GSTP1-105 G/G genotype were more prone to suffer from grade 3 neurotoxicity than carriers of GSTP1-105 A/G and GSTP1-105 A/A genotypes.CONCLUSION:A pharmacogenetic approach may be an innovative strategy for optimizing palliative chemotherapy in patients with advanced colorectal cancer. These findings deserve confirmation in additional prospective studies.

Background Elderly patients have been often excluded from or underrepresented in the study populations of combination chemotherapy trials. The primary end point of this study was to determine the response rate and the toxicity of the weekly oxaliplatin, 5-fluorouracil and folinic acid (OXALF) regimen in elderly patients with advanced gastric cancer. The secondary objective was to measure the time to disease progression and the survival time. Methods Chemotherapy-naive patients with advanced gastric cancer aged 70 or older were considered eligible for study entry. Patients received weekly oxaliplatin 40 mg/m2, fluorouracil 500 mg/m2 and folinic acid 250 mg/m2. All drugs were given intravenously on a day-1 schedule. Results A total of 42 elderly patients were enrolled. Median age was 73 years and all patients had metastatic disease. The response rate according to RECIST criteria was 45.2% (95% CIs: 30%–56%) with two complete responses, 17 partial responses, 13 stable diseases and 10 progressions, for an overall tumor rate control of 76.2% (32 patients). Toxicity was generally mild and only three patients discontinued treatment because of treatment related adverse events. The most common treatment-related grade 3/4 adverse events were fatigue (7.1%), diarrhoea (4.8%), mucositis (2.4%), neurotoxicity (2.4%) and neutropenia (4.8%). The median response duration was 5.3 months (95% CIs: 2.13 – 7.34), the median time to disease progression was 5.0 months (95% CIs: 3.75 – 6.25) and the median survival time was 9.0 months (95% CIs: 6.18 – 11.82). Conclusion OXALF represents an active and well-tolerated treatment modality for elderly patients with locally advanced and metastatic gastric cancer.

PURPOSE:To investigate whether polymorphisms with putative influence on fluorouracil/cisplatin activity are associated with clinical outcomes of patients with advanced gastric cancer (AGC).PATIENTS AND METHODS:Peripheral blood samples from 175 prospectively enrolled AGC patients treated with fluorouracil/cisplatin palliative chemotherapy were used for genotyping 13 polymorphisms in nine genes (TS, MTHFR, XPD, ERCC1, XRCC1, XRCC3, GSTPI, GSTTI, GSTMI). Genotypes were correlated to response and survival.RESULTS:The overall response rate was 41%, the median progression-free survival (PFS) was 24 weeks (range, 4 to 50 weeks), and the median overall survival (OS) was 39 weeks (range, 8 to 72+ weeks). Chemoresistance and poor survival were significantly associated with TS 5'-UTR 3G-genotype (2R/3G, 3C/3G, 3G/3G) and GSTP1 105 A/A homozygous genotype. Sixty-one patients (35%) did not show any of these risk genotypes (group 0), 57 patients (32.5%) showed one of the two risk genotypes (group 1), and 57 patients (32.5%) showed both risk genotypes (group 2). Median PFS and OS in group 0 patients were 32 weeks (range, 8 to 50 weeks) and 49 weeks (range, 18 to 72+ weeks), respectively. Group 1 and group 2 patients showed significantly worse PFS (median, 26 weeks [range, 6 to 44 weeks] and 14 weeks [range, 4 to 38 weeks], respectively) and worse OS (median, 39 weeks [range, 10 to 58 weeks] and 28 weeks [range, 8 to 56 weeks]), respectively, than group 0 patients. This adverse effect was retained in multivariate analysis.CONCLUSION:Specific polymorphisms may influence clinical outcomes of AGC patients. Selecting palliative chemotherapy on the basis of pretreatment genotyping may represent an innovative strategy that warrants prospective studies.

BACKGROUND:Polymorphisms in the interleukin 1beta gene (IL-1B-31T/C and IL-1B-511C/T single nucleotide changes) and in the interleukin 1 receptor anatagonist gene (IL-1RN2 variable number of tandem repeats) have been studied with respect to gastric cancer susceptibility. Available data support an aetiologic role of these genetic variants in the presence of concomitant Helicobacter pylori infection. Their contribution without H. pylori infection is still an open field of investigation.MATERIALS AND METHODS:IL-1B and IL-1RN polymorphisms were investigated in 138 H. pylori-negative Italian patients with sporadic gastric cancer and 100 H. pylori-negative controls. Unconditional regression with odd ratios (OR) and 95% confidence intervals (CI), haplotype and linkage disequilibrium analyses were used to investigate the association of the polymorphisms with disease.RESULTS:In all gastric cancer cases, carriers of the homozygous IL-1B-511T/T genotype showed a significant risk for the development of the disease (OR 3.2 with 95% CI 1.27-8.05). In cases with intestinal-type gastric cancer, however, both IL-1B-511T and IL-1RN2 alleles were associated with disease. In this subgroup, the odds ratio for carriers of both IL-1B-511T and IL-1RN2 was 6.49 (95% CI 2.07-20.4). Haplotype analysis supported the aetiologic contribution of these alleles in gastric cancer of the intestinal histotype.CONCLUSIONS:In conclusion, IL-1B-511T and IL-1RN2 may contribute to intestinal gastric cancer risk in the absence of concomitant H. pylori infection. In this setting, future epidemiologic studies should consider dietary habits and exposure to carcinogens interacting with pro-inflammatory host genotypes.

PURPOSE:A high interleukin-1beta (IL-1B) and interleukin-1 receptor antagonist (IL-RN) ratio underlies an unfavorable proinflammatory status. Also, it seems to be involved in the mechanisms of cancer cachexia and tumor angiogenesis and metastasis. Two single nucleotide polymorphisms in IL-1B gene (IL-1B-511C/T,IL-1B-31T/C) and a variable number of tandem repeat polymorphisms in IL-RN gene (IL-1RNlong/2) enhance the circulating levels of the two cytokines. The prognostic role of IL-1B/IL-1RN genotypes was investigated in patients with relapsed and metastatic gastric cancer treated with palliative chemotherapy. PATIENTS AND METHODS:Before starting palliative chemotherapy, 123 prospectively enrolled patients supplied peripheral-blood samples for DNA extraction. Survival data were analyzed according to IL-1RN/IL-1B genotypes. RESULTS:Forty-two patients showed wild-type genotypes (IL-1RNlong/long, IL-1B-511C/C, and IL-1B-31T/T; group A). Forty-five patients showed the IL-1RN2 polymorphism, with wild-type IL-1B genotypes in seven patients and with IL-1B-511C/T and/or IL-1B-31T/C polymorphisms in 38 patients (group B). The remaining 36 patients demonstrated wild-type IL-1RN, with IL-1B-511C/T and/or IL-1B-31T/C polymorphisms (group C). In group A and B patients, the median progression-free survival (PFS) was 25 and 26 weeks, respectively, and median overall survival (OS) was 42 and 43 weeks, respectively. Group C patients showed worse PFS (median, 16 weeks) and OS (median, 28 weeks) than group A (P = .006 for PFS; P = .0001 for OS) and group B patients (P = .01 for PFS; P = .0001 for OS). The long/T/C haplotype was overrepresented in patients with shortened PFS (P = .001) and OS (P = .0005). CONCLUSION:In patients with advanced gastric cancer, IL-1B polymorphisms showed adverse prognostic influence when coupled with wild-type IL-1RN genotype. These findings deserve further investigation for potential anticancer activity of recombinant IL-RN.

The aim of our analysis was to assess retrospectively the effect on local relapse, overall survival (OS) and disease-free survival (DFS) of a limited or an extended lymphadenectomy in radically resected gastric cancer patients. This study was performed in order to identify a subgroup of patients possibly not benefiting from a therapeutic approach such as chemoradiation therapy. We divided our patients into two groups according to lymphadenectomy type: group A for limited (<25 resected lymph nodes) and group B for extended (>25 resected lymph nodes) lymph nodes resection. A total of 418 patients were analysed: tumour stage at diagnosis was pT2–3 pN1–3 M0 in 339 patients and pT3 N0 M0 in 79 patients. Median age at diagnosis was 68 years (range 30–92 years). A total of 306 patients (73.2%) were in group A and 112 (26.8%) in group B. The median survival time (OS) for patients in groups A and B was 58.8 and 84.8 months, respectively (P=0.0371); median DFS was 28.8 months in group A and 59.9 months in group B (P=0.0027). At multivariate analysis, extension within the gastric wall, nodal involvement and the number of resected lymph nodes appeared to affect both OS and DFS. An inadequate lymph nodes resection can affect survival and result in a higher incidence of local relapse, making the latter group of patients optimal candidates for adjuvant chemoradiation.