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Background: Hereditary cancer predisposition syndromes are responsible for approximately 5-10% of all diagnosed cancer cases. In the past, single-gene analysis of specific high risk genes was used for the determination of the genetic cause of cancer heritability in certain families. The application of Next Generation Sequencing (NGS) technology has facilitated multigene panel analysis and is widely used in clinical practice, for the identification of individuals with cancer predisposing gene variants. The purpose of this study was to investigate the extent and nature of variants in genes implicated in hereditary cancer predisposition in individuals referred for testing in our laboratory., Methods: In total, 1197 individuals from Greece, Romania and Turkey were referred to our laboratory for genetic testing in the past 4 years. The majority of referrals included individuals with personal of family history of breast and/or ovarian cancer. The analysis of genes involved in hereditary cancer predisposition was performed using a NGS approach. Genomic DNA was enriched for targeted regions of 36 genes and sequencing was carried out using the Illumina NGS technology. The presence of large genomic rearrangements (LGRs) was investigated by computational analysis and Multiplex Ligation-dependent Probe Amplification (MLPA)., Results: A pathogenic variant was identified in 264 of 1197 individuals (22.1%) analyzed while a variant of uncertain significance (VUS) was identified in 34.8% of cases. Clinically significant variants were identified in 29 of the 36 genes analyzed. Concerning the mutation distribution among individuals with positive findings, 43.6% were located in the BRCA1/2 genes whereas 21.6, 19.9, and 15.0% in other high, moderate and low risk genes respectively. Notably, 25 of the 264 positive individuals (9.5%) carried clinically significant variants in two different genes and 6.1% had a LGR., Conclusions: In our cohort, analysis of all the genes in the panel allowed the identification of 4.3 and 8.1% additional pathogenic variants in other high or moderate/low risk genes, respectively, enabling personalized management decisions for these individuals and supporting the clinical significance of multigene panel analysis in hereditary cancer predisposition.

Background: There is an unmet need for more efficient patient stratification for receiving trastuzumab in the metastatic breast cancer (mBC) setting, since only part of such patients benefit from the addition of this agent to chemotherapy. The aim of this study was to investigate the prognostic value of biomarkers including MYC and MET in mBC patients treated with trastuzumab-based regimens., Methods: mBC patients, locally tested as HER2-positive, treated with trastuzumab and chemotherapy between 1998 and 2010 were evaluated. Paraffin tumors (n = 229) were retrospectively centrally assessed by immunohistochemistry (IHC) for HER2, ER, PgR and Ki67; fluorescence in situ hybridization (FISH) for HER2, TOP2A and centromere (CEN) 17, MYC and CEN8, MET and CEN7; qPCR for MYC, MET copy number (CN); and, for PI3K activation (PIK3CA mutations; PTEN and phospho-mTOR protein expression). Increased CEN CN was assessed based on normal cut-offs. Time to progression (TTP) and survival were evaluated from the initiation of trastuzumab as first line treatment., Results: Among all tumors, 90 were HER2-negative upon central testing (ambiguous HER2) and the rest were true HER2-positive. Further, 156 patients presented with mBC upon relapse of pre-treated disease (R-mBC) and 65 were diagnosed at stage IV (de novo mBC). Concordance between FISH and qPCR on gene CN status was fair for MYC (Kappa = 0.458) and absent for MET. The presence of MYC CN gain with qPCR and the absence of PI3K activation were infrequent events (7 and 8 % of evaluable tumors, respectively), while 41 % of tumors had increased CEN CN in one or more chromosomes, indicative of chromosomal instability. The most consistent finding in the entire cohort and in the above patient subgroups with respect to outcome was the unfavourable effect of MYC CN gain, which was retained upon multivariable analysis (e.g., survival in the entire cohort, HR 6.02; 95 % CI 2.67-13.6; p < 0.001). Further unfavourable prognosticators were increased CEN CN in one chromosome in R-mBC but not in de novo mBC (multivariable interaction p = 0.048), PI3K activation in R-mBC (multivariable p = 0.004) and increased Ki67 for patient TTP., Conclusions: MYC gene copies, centromere status and PI3K activation may adversely impact trastuzumab treated mBC patient outcome and seem worthy validating in larger series.

Background: Dose-dense sequential chemotherapy including anthracyclines and taxanes has been established in the adjuvant setting of high-risk operable breast cancer. However, the preferable taxane and optimal schedule of administration in a dose-dense regimen have not been defined yet., Methods: From July 2005 to November 2008, 1001 patients (990 eligible) were randomized to receive, every 2 weeks, 3 cycles of epirubicin 110 mg/m2 followed by 3 cycles of paclitaxel 200 mg/m2 followed by 3 cycles of intensified CMF (Arm A; 333 patients), or 3 cycles of epirubicin followed by 3 cycles of CMF, as in Arm A, followed 3 weeks later by 9 weekly cycles of docetaxel 35 mg/m2 (Arm B; 331), or 9 weekly cycles of paclitaxel 80 mg/m2 (Arm C; 326). Trastuzumab was administered for one year to HER2-positive patients post-radiation., Results: At a median follow-up of 60.5 months, the 3-year disease-free survival (DFS) rate was 86%, 90% and 88%, for Arms A, B and C, respectively, while the 3-year overall survival (OS) rate was 96% in all arms. No differences were found in DFS or OS between the combined B and C Arms versus Arm A (DFS: HR = 0.81, 95% CI: 0.59-1.11, P = 0.20; OS: HR = 0.84, 95% CI: 0.55-1.30, P = 0.43). Among the 255 patients who received trastuzumab, 189 patients (74%) completed 1 year of treatment uneventfully. In all arms, the most frequently reported severe adverse events were neutropenia (30% vs. 27% vs. 26%) and leucopenia (12% vs. 13% vs. 12%), while febrile neutropenia occurred in fifty-one patients (6% vs. 4% vs. 5%). Patients in Arm A experienced more often severe pain (P = 0.002), neurological complications (P = 0.004) and allergic reactions (P = 0.004), while patients in Arm B suffered more often from severe skin reactions (P = 0.020)., Conclusions: No significant differences in survival between the regimens were found in the present phase III trial. Taxane scheduling influenced the type of severe toxicities. HER2-positive patients demonstrated comparable 3-year DFS and OS rates with those reported in other similar studies., Trial Registration: Australian New Zealand Clinical Trials Registry ACTRN12610000151033.

Background: Metronomic chemotherapy is considered an anti-angiogenic therapy that involves chronic administration of low-dose chemotherapy at regular short intervals. We investigated the optimal metronomic dose of oral vinorelbine when given as monotherapy in patients with metastatic cancer., Methods: Patients with recurrent metastatic breast (BC), prostate (PC) or non-small cell lung cancer (NSCLC) and adequate organ functions were randomly assigned to 30, 40 or 50 mg vinorelbine, taken orally three times a week. Treatment continued until disease progression, unacceptable toxicity, withdrawal of consent or maximum 24 months. Primary endpoint was time-to-treatment failure (TTF) and secondary were progression-free survival (PFS), toxicity, changes in blood concentrations of angiogenesis-associated biomarkers and pharmacokinetics., Results: Seventy-three patients were enrolled. Four-month TTF rate did not differ between the three arms: 25.9% (11.1%-46.2% 95% Confidence Interval), 33.3% (15.6%-55.3%) and 18.2% (5.2%-40.3%) for the 30 mg, 40 mg and 50 mg arms (p-value = 0.56). Objective response was seen in 2 patients with NSCLC (treated at 30 and 50 mg respectively), one with BC (at 40 m g) and one with PC (at 50 mg) and lasted from 4 to 100 weeks, with maximum response duration achieved at 50 mg. Adverse events were mild and negligible and did not differ between the three arms. Blood levels of vinorelbine reached steady state from the second week of treatment and mean values for the 30, 40 and 50 mg were respectively 1.8 ng/ml (SD 1.10), 2.2 ng/ml (SD 1.87) and 2.6 ng/ml (SD 0.69). Low pre-treatment blood concentrations of FGF2 and IL8 predicted favorable response to therapy (p values 0.02 and 0.006, respectively), while high levels of TEK gene transcript predicted treatment resistance., Conclusions: Considering the antitumor activity and response duration, the negligible toxicity of the highest dose investigated and the lack of drug accumulation over time, we suggest that 50 mg given three times a week is the optimal dose for metronomic oral vinorelbine. Further investigation of metronomic oral vinorelbine (MOVIN) at this dose is warranted in combination with conventional chemotherapy regimens and targeted therapies., Trial Registration: Clinicaltrials.gov NCT00278070.

Background: The HER2 gene has been established as a valid biological marker for the treatment of breast cancer patients with trastuzumab and probably other agents, such as paclitaxel and anthracyclines. The TOP2A gene has been associated with response to anthracyclines. Limited information exists on the relationship of HER2/TOP2A gene status in the presence of centromere 17 (CEP17) gain with outcome of patients treated with anthracycline-containing adjuvant chemotherapy., Methods: Formalin-fixed paraffin-embedded tumor tissue samples from 1031 patients with high-risk operable breast cancer, enrolled in two consecutive phase III trials, were assessed in a central laboratory by fluorescence in situ hybridization for HER2/TOP2A gene amplification and CEP17 gain (CEP17 probe). Amplification of HER2 and TOP2A were defined as a gene/CEP17 ratio of >2.2 and ≥2.0, respectively, or gene copy number higher than 6. Additionally, HER2, TopoIIa, ER/PgR and Ki67 protein expression was assessed by immunohistochemistry (IHC) and patients were classified according to their IHC phenotype. Treatment consisted of epirubicin-based adjuvant chemotherapy followed by hormonal therapy and radiation, as indicated., Results: HER2 amplification was found in 23.7% of the patients and TOP2A amplification in 10.1%. In total, 41.8% of HER2-amplified tumors demonstrated TOP2A co-amplification. The median (range) of HER2, TOP2A and CEP17 gain was 2.55 (0.70-45.15), 2.20 (0.70-26.15) and 2.00 (0.70-26.55), respectively. Forty percent of the tumors had CEP17 gain (51% of those with HER2 amplification). Adjusting for treatment groups in the Cox model, HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with time to relapse or time to death., Conclusion: HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with outcome in high-risk breast cancer patients treated with anthracycline-based adjuvant chemotherapy., Trial Registration: Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12611000506998 and ACTRN12609001036202.

Background: The vast majority of patients with HER2-positive metastatic breast cancer (MBC) treated with trastuzumab eventually develop resistance to this agent. There is an unmet need therefore, for identifying biological markers with possible prognostic/predictive value in such patients. The aim of this study was to investigate the prognostic role of topoisomerase II alpha gene (TOP2A) amplification and protein (TopoIIa) expression in patients treated with trastuzumab-containing regimens., Methods: Formalin-fixed paraffin-embedded tumor tissue samples were retrospectively collected from 225 eligible patients treated with trastuzumab. Protein expression of ER, PgR, Ki67, PTEN, HER2 and TopoIIa were centrally assessed by immunohistochemistry. HER2 and TOP2A gene amplification was evaluated by fluorescence in situ hybridization. PIK3CA mutations were identified by single nucleotide polymorphism genotyping. Survival was evaluated from the initiation of trastuzumab as 1st line treatment to the date of last follow-up or death., Results: Among the 225 samples analyzed, only 137 (61%) were found to be HER2-positive. TOP2A was amplified in 41% and deleted in 16% of such tumors. TOP2A gene amplification was more frequent in ER-negative tumors. TopoIIa protein expression was observed in the majority (65%) of the samples and was associated with ER-positive status, high Ki67 expression, presence of PTEN protein and PIK3CA mutations. Median follow-up for patients treated in the 1st line was 51 months. Survival was more prolonged with trastuzumab-containing treatment in HER2-positive patients (50 months, log-rank, p=0.007). TOP2A non-amplified or deleted tumors were associated with increased risk for death compared to TOP2A amplified tumors (HR=2.16, Wald's p=0.010 and HR=2.67, p=0.009, respectively). In multivariate analysis, a significant interaction of TOP2A with anthracycline treatment (either in the adjuvant or the 1st line setting) was observed for survival (Wald's p=0.015). Among the TOP2A amplified subgroup, anthracycline-treated patients were associated with decreased risk for death., Conclusions: TOP2A gene amplification was shown to be a favorable prognostic marker in HER2-positive MBC patients treated with trastuzumab, such an effect however, appears to rather be related to treatment with anthracyclines (predictive marker for benefit from anthracyclines). The results of the present retrospective study warrant validation in larger cohorts of patients treated in the context of randomized trials.