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Purpose: Gastric cancer is the fifth most common neoplasm worldwide with high rates of mortality. Afatinib, a low molecular, irreversible potent inhibitor of ErbB trans-membrane receptor family, has shown promising results according to preclinical and phase I clinical trial data when combined with chemotherapy. We aimed at evaluating the safety and efficacy of the combination of cisplatin, 5FU with afatinib in molecularly unselected patients with advanced gastric cancer. Methods: Patients with locally advanced or metastatic gastric/gastroesophageal junction adenocarcinoma received first line combination therapy of cisplatin, 5FU and afatinib every 21 days, followed by afatinib maintenance monotherapy. The primary endpoint was the Objective Response Rate (ORR); secondary endpoints included Overall Survival (OS), Progression Free Survival (PFS) and the safety profile. Unplanned exploratory analysis of HER2 and tumor mutational profile was performed. Results: Among 55 patients (ITT population) enrolled, 19 (34.5%) achieved an objective tumor response; stable disease was observed in 16 patients (29.1%) and progressive disease in 10 patients (18.2%). The ORR in the per protocol population (PP) was 42.9%. Within a median follow-up of 56 months, the median PFS and OS in the ITT population was 5.0 and 8.7 months, respectively. Seven of the 47 HER2 informative cases carried HER2 positive tumors while TP53, BRCA2 and SMAD4 were the most frequently mutated genes. The most common toxicities were neutrophil count and white blood cell decrease occurring in 56.4% of patients, followed by anemia (50.9%), hyperglycemia (40%), and diarrhea (38.2%). Conclusions: The combination of cisplatin/5FU with afatinib did not surpass the benchmarks of efficacy of the contemporary therapeutic regimens that are being applied for the treatment of patients with advanced gastric cancer. However, the observed efficacy and the improved safety profile support that our administration schedule may be further investigated to overcome toxicity problems when integrating afatinib to cytotoxic chemotherapy. Clinical trial registration: NCT01743365. © 2021 Acta Oncologica Foundation.

Background/Aim: Age may pertain to different tumor genotype characteristics which may interfere with treatment efficacy and prognosis. We investigated the distribution and prognostic effect of mutations and tumor infiltrating lymphocyte (stromal TIL density) in young (≤35 years) and elderly (>65 years) early breast cancer patients. Materials and Methods: Paraffin tumor genotypes of all clinical subtypes from 345 patients were examined. Results: A total of 638 mutations were detected in 221 patients (64.1%). Compared to young, elderly patients presented with lower TIL density (p

Background/Aim: Early-stage gastric cancer has a high risk of recurrence, despite trimodality therapy with surgery, chemotherapy and radiation. To improve patient selection for adjuvant chemoradiotherapy, we evaluated the prognostic significance of immunohistochemical and genetic biomarkers in patients with resected gastric adenocarcinoma. Patients and Methods: Tumors from 119 patients were subjected to immunohistochemistry for 12 protein biomarkers, as well as next-generation sequencing. Clinical and biomarker data were available for 91 patients. Results: EBV-positive tumors and tumors with mutations had higher intratumoral CD8 tumor-infiltrating lymphocyte density (p=0.009 and p=0.017, respectively). PIK3CA mutations were correlated with VEGFA overexpression (p=0.042), while KRAS mutations and HER2 expression were mutually exclusive (p=0.036). PTEN expression univariately confirmed longer overall survival (HR=0.27; p=0.046), while there was a trend between the presence of KRAS mutations and inferior disease-free and overall survival. Conclusion: PTEN protein expression and KRAS mutations may predict disease outcome in early-stage gastric cancer. These results need to be further validated in larger cohorts. © 2020 International Institute of Anticancer Research. All rights reserved.

Background/Aim: PTEN-loss and PIK3CA mutations have been addressed as markers of PI3K activation in breast cancer. We evaluated these markers in early high-risk breast cancer (EBC) focusing on PTEN immunohistochemistry (IHC) issues, particularly in HER2-positive disease. Materials and Methods: We examined PTEN-loss and PIK3CA mutations in 1265 EBC patients treated with adjuvant chemotherapy within two clinical trials. Two different methods for the evaluation of PTEN IHC were used, one upfront binary (loss; no-loss) and the other initially multi-scale allowing for the classification of “grey zone” tumors with low and very low PTEN protein expression. Results: PTEN-loss (33.4% and 22.1%, depending on the IHC method) and PIK3CA mutations (29.6%) were associated with ER/PgR/HER2-negative and ER/PgR-positive disease, respectively. Concordance of the two IHC methods was moderate (Cohen’s kappa 0.624). PTEN-loss discrepancy and intra-tumor heterogeneity concerned “grey zone” tumors that were prevalent among HER2-positive cancers. PTEN-loss independently conferred higher risk for relapse and death. Compared to single PIK3CA mutations, single PTEN-loss was independently associated with increased risk for relapse and death. Depending on the evaluation method, in HER2-positive cancer, PTEN-loss was without- or of marginal unfavorable prognostic significance. Conclusion: In EBC, PTEN-loss is an independent predictor of poor outcome. When occurring singly, PTEN-loss and PIK3CA mutations have opposite prognostic impact. In HER2-positive disease, assessment of PTEN-loss by IHC appears unreliable and the marker is without clear prognostic significance. © 2019 International Institute of Anticancer Research. All rights reserved.

Background The clinical relevance of mismatch repair (MMR) status in patients with nonmetastatic cancer across tumour types remains unclear. Our goal was to investigate the prognostic role of MMR deficiency in patients with stage I-III colorectal and endometrial cancer. Methods Patients with nonmetastatic colorectal and endometrial cancer with tumour tissue available for analysis were identified through the Hellenic Cooperative Oncology Group (HeCOG)'s tumour repository. Patients had been referred to Departments of Medical Oncology affiliated with HeCOG. MMR protein expression was evaluated by immunohistochemistry. The primary outcome measure was overall survival (OS). Results From May 1990 to September 2012, 1158 patients with nonmetastatic colorectal (N = 991) and endometrial cancer (N = 167) were identified (median age: 64 years, men: 544). All patients with colorectal and 109 (65%) with endometrial cancer had received adjuvant treatment. MMR deficiency was observed in 114 (11.5%) of colorectal and 80 (47.9%) of endometrial tumours. More commonly deficient proteins were PMS2 (69 patients, 7%) and MLH1 (63 patients, 6.5%) in colorectal cancer and MSH2 (58 patients, 34.7%) in endometrial cancer. Colorectal MMR-deficient (dMMR) tumours were more likely to be right sided (65 % dMMR vs 27 % proficient MMR, pMMR; p < 0.001), high grade (31% vs 15%, Ï ‡ 2, p < 0.001) and with a mucinous component (64% vs 42%, p < 0.001). Endometrial dMMR tumours were more often of endometrioid histology (51.4 % endometrioid vs 20 % serous/clear cell, p = 0.020). Compared with MMR proficiency, MMR deficiency was associated with improved OS in patients with endometrial cancer (HR = 0.38, 95% CI 0.20 to 0.76, p = 0.006), but not in patients with colorectal cancer (HR = 0.73, 95% CI 0.49 to 1.09, p = 0.130). After adjusting for age, stage and grade, MMR deficiency maintained its favourable prognostic significance in patients with endometrial cancer (HR = 0.42, 95% CI 0.20 to 0.88, p = 0.021). Conclusions DMMR was associated with improved outcomes in patients with nonmetastatic endometrial cancer, but not in patients with nonmetastatic colorectal cancer who received adjuvant chemotherapy. © 2019 BMJ Publishing Group Limited. No commercial re-use. See rights and permissions. Published by BMJ.

The prognostic/predictive value of aberrant MYC gene copies and protein expression is not clear. It was therefore evaluated in 1060 early breast cancer patients treated with anthracycline-containing chemotherapy. MYC gene amplification in the absence of polysomy-8 was associated with adverse disease-free and overall survival, whereas nuclear Myc protein expression benefitted patients treated with paclitaxel. These data might aid in the interpretation of relevant findings from large clinical trials. Background: The prognostic/predictive value of aberrant MYC gene copies and protein expression is not clear in breast cancer. Patients and Methods: Early breast cancer patients were treated with anthracycline-containing chemotherapy within 2 randomized adjuvant trials. MYC gene and centromere-8 status, as well as Myc protein expression were investigated on 1060 paraffin tumors with fluorescence in situ hybridization and immunohistochemistry, respectively. Results: MYC amplification was present in 45% and polysomy-8 in 23% of the tumors. Cytoplasmic staining was observed in 60% and nuclear staining in 26% of the tumors, strongly correlating with each other but not with MYC gene status. MYC gene amplification in the absence of polysomy-8 was associated with adverse disease-free survival (DFS) and overall survival (OS), and remained as an independent unfavorable prognostic factor in multivariate analysis (Wald P =.022 for DFS; P =.032 for OS), whereas patients with MYC amplification and polysomy-8, with polysomy-8 only, and with normal MYC without polysomy-8 performed significantly better compared with those with MYC gene amplification only. Nuclear Myc protein expression benefitted patients treated with paclitaxel (interaction P =.052 for DFS; P =.049 for OS). This interaction remained independently significant in multivariate analysis for OS (overall P =.028). Conclusion: The effect of MYC gene status on breast cancer patient outcome seems to depend on the underlying chromosomal instability and appears unfavorable for tumors with MYC amplification without polysomy. Nuclear Myc protein expression seems predictive for benefit from adjuvant paclitaxel. These data might aid in the interpretation of relevant findings from large clinical trials. © 2017 Elsevier Inc.

This clinical trial assessed the efficacy and toxicity of panitumumab combined with oxaliplatin and capecitabine as first-line treatment in KRAS exon 2 wild-type metastatic colorectal cancer (mCRC) patients. Patients with exon 2 KRAS wild-type mCRC received panitumumab 9 mg/Kg, oxaliplatin 130 mg/m2, and capecitabine 2000 mg/m2 repeated every 3 weeks. The primary endpoint was objective response rate (ORR, minimum 42 responses). We retrospectively assessed mutations in genes implicated in CRC with massively parallel sequencing; ERBB2 and EGFR amplification with fluorescence in situ hybridization, and tumor-infiltrating lymphocyte density. Among 78 patients enrolled, 45 (57.7%) completed 6 cycles. Most common grade 3–4 toxicities were skin rash (19.2%), diarrhea (18%), and neuropathy (6.4%). Among 5 (6.4%) potentially treatment-related deaths, 2 (2.6%) were characterized toxic. Objective response occurred in 43 (55.1%) of the patients (complete 6.4% and partial response 48.7%; stable 17.9% and progressive disease 7.7%), while 3.8% were non-evaluable and 15% discontinued their treatment early. Additional mutations in KRAS/NRAS/BRAF were found in 11/62 assessable (18%) tumors. After 51 months median follow-up, median progression-free (PFS) was 8.1 and overall survival 20.2 months, independently of KRAS/NRAS/BRAF or PI3K-pathway mutation status. Patients with TP53 mutations (n = 34; 55%), as well as those with left colon primary tumors (n = 66; 85%), had significantly better PFS, also confirmed in multivariate analysis. Although the clinical trial met its primary endpoint, according to the current standards, the efficacy and tolerability of the drug combination are considered insufficient. Extended genotyping yielded interesting results regarding the significance of TP53 mutations. ClinicalTrials.gov identifier: NCT01215539, Registration date: Sep 29, 2010. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.

Small molecules, mainly tyrosine kinase inhibitors, are currently used in various malignancies. Lapatinib, a dual inhibitor of EGFR/HER2 tyrosine kinases, has demonstrated effectiveness in brain metastases from HER2-overexpressing breast cancer. It also appears to sensitize EGFR-expressing cell lines to radiation. To evaluate the efficacy of lapatinib in combination with whole brain radiotherapy (WBRT) in patients with brain metastases from non-small cell lung cancer (NSCLC) and breast cancer, as assessed by volumetric analysis by MRI. 81 patients were treated with WBRT (30 Gy in ten fractions) in combination with lapatinib 1250 mg once daily, followed by lapatinib 1500 mg once daily for a total 6 weeks. 21 patients had primary breast cancer and 60 patients NSCLC. Pre- and post-treatment MRI scans in a compact disk for central volumetric assessment were available for 43 patients. 27 patients (62.8%) achieved partial response, 15 patients (34.9%) had stable disease and only one patient (2.3%) had disease progression. Response was not associated to EGFR protein expression. All 81 patients were assessed for safety. The large majority of the adverse events were mild. Eight deaths occurred, four of which were considered related to the study drugs but there were also other contributing factors. Nine cases of serious infections were observed in eight patients, but they were also receiving dexamethasone. Lapatinib in combination with WBRT in patients with brain metastases from breast cancer and NSCLC is a feasible approach that can be further studied in larger clinical trials. © 2017, Springer Science+Business Media, LLC.

Background-Aim: To evaluate the prognostic role of elaborate molecular clusters encompassing cyclin D1, cyclin E1, p21, p27 and p53 in the context of various breast cancer subtypes. Methods: Cyclin E1, cyclin D1, p53, p21 and p27 were evaluated with immunohistochemistry in 1077 formalin-fixed paraffin-embedded tissues from breast cancer patients who had been treated within clinical trials. Jaccard distances were computed for the markers and the resulted matrix was used for conducting unsupervised hierarchical clustering, in order to identify distinct groups correlating with prognosis. Results: Luminal B and triple-negative (TNBC) tumors presented with the highest and lowest levels of cyclin D1 expression, respectively. By contrast, TNBC frequently expressed Cyclin E1, whereas ER-positive tumors did not. Absence of Cyclin D1 predicted for worse OS, while absence of Cyclin E1 for poorer DFS. The expression patterns of all examined proteins yielded 3 distinct clusters; (1) Cyclin D1 and/or E1 positive with moderate p21 expression; (2) Cyclin D1 and/or E1, and p27 positive, p53 protein negative; and, (3) Cyclin D1 or E1 positive, p53 positive, p21 and p27 negative or moderately positive. The 5-year DFS rates for clusters 1, 2 and 3 were 70.0%, 79.1%, 67.4% and OS 88.4%, 90.4%, 78.9%, respectively. Conclusions: It seems that the expression of cell cycle regulators in the absence of p53 protein is associated with favorable prognosis in operable breast cancer. © 2017 Zagouri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Purpose: RACGAP1 is a Rac GTPase-activating protein involved in cell growth regulation, cell transformation and metastasis. The aim of the present study was to explore the prognostic and/or predictive significance of RACGAP1 mRNA expression on disease-free survival (DFS) and overall survival (OS) in high-risk early breast cancer patients and compare it to that of Ki67 protein expression and to the Nottingham prognostic index (NPI). Methods: A total of 595 high-risk breast cancer patients were treated in a two-arm trial evaluating postoperative dose-dense sequential chemotherapy with epirubicin followed by CMF with or without paclitaxel. RNA was extracted from 314 formalin-fixed paraffin-embedded primary tumor tissue samples followed by one-step quantitative RT-PCR for assessing RACGAP1 mRNA expression. Results: High RACGAP1 mRNA expression (above the median) was associated with poor DFS (log-rank, p = 0.002) and OS (p < 0.001). High histological grade, as well as high Ki67 protein expression, was more frequent in the high-expression group of RACGAP1. Results of the Cox multivariate regression analysis revealed that high RACGAP1 mRNA expression independently predicted poor overall survival (Wald's p = 0.008). High Ki67 protein expression was also an adverse prognostic factor for death (p = 0.016), while high NPI score values were not. Conclusions: High RACGAP1 mRNA expression, as assessed by qRT-PCR, was found to be of adverse prognostic significance in high-risk early breast cancer patients treated with dose-dense sequential chemotherapy. The utility of RACGAP1 mRNA expression in patient selection for treatment with aggressive chemotherapy regimens should be further explored and validated in larger cohorts. © 2012 Springer-Verlag Berlin Heidelberg. 71 1 245 255

Background: We investigated the impact of PIK3CA and TP53 mutations and p53 protein status on the outcome of patients who had been treated with adjuvant anthracycline-taxane chemotherapy within clinical trials in the pre- and post-trastuzumab era. Results: TP53 and PIK3CA mutations were found in 380 (21.5%) and 458 (25.9%) cases, respectively, including 104 (5.9%) co-mutated tumors; p53 immunopositivity was observed in 848 tumors (53.5%). TP53 mutations (p < 0.001) and p53 protein positivity (p = 0.001) were more frequent in HER2-positive and triple negative (TNBC) tumors, while PIK3CA mutations were more frequent in Luminal A/B tumors (p < 0.001). TP53 mutation status and p53 protein expression but not PIK3CA mutation status interacted with trastuzumab treatment for disease-free survival; patients with tumors bearing TP53 mutations or immunopositive for p53 protein fared better when treated with trastuzumab, while among patients treated with trastuzumab those with the above characteristics fared best (interaction p = 0.017 for mutations; p = 0.015 for IHC). Upon multivariate analysis the above interactions remained significant in HER2-positive patients; in the entire cohort, TP53 mutations were unfavorable in patients with Luminal A/B (p = 0.003) and TNBC (p = 0.025); p53 immunopositivity was strongly favorable in patients treated with trastuzumab (p = 0.009). Materials and Methods: TP53 and PIK3CA mutation status was examined in 1766 paraffin tumor DNA samples with informative semiconductor sequencing results. Among these, 1585 cases were also informative for p53 protein status assessed by immunohistochemistry (IHC; 10% positivity cut-off). Conclusions: TP53 mutations confer unfavorable prognosis in patients with Luminal A/B and TNBC tumors, while p53 immunopositivity may predict for trastuzumab benefit in the adjuvant setting.

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. © 2016 The Author(s).

The purpose of this study is to investigate whether the outcome of breast cancer (BC) patients treated with adjuvant chemotherapy is affected by co-mutated TP53 and PIK3CA according to stromal tumor-infiltrating lymphocytes (TILs). Paraffin tumors of all clinical subtypes from 1661 patients with operable breast cancer who were treated within 4 adjuvant trials with anthracycline–taxanes chemotherapy were informative for TP53 and PIK3CA mutation status (semiconductor sequencing genotyping) and for stromal TILs density. Disease-free survival (DFS) was examined. TP53 mutations were associated with higher (p

Background: Cancer of unknown primary (CUP) possesses distinct biology and peculiar natural history, in which the roles of the winged and hedgehog signalling pathways are unclear. Materials and methods: We constructed tissue microarrays and studied the immunohistochemical (IHC) expression of β-catenin, smoothened (SMO) and the transcription factors TCF, LEF, GLI1 in 87 CUP cases for prognostic significance. Results: A low rate of IHC expression of proteins was seen, the cut-off used being any expression in ≥ 1% of tumour cells. At univariate analysis, only nuclear IHC SMO expression displayed a statistically significant association with favourable outcome [median Overall survival (OS) of 19 months in SMO-positive vs. 12 months in SMO-negative cases, P = 0·01]. An activated Wnt pathway, defined as IHC expression of any of nuclear β-catenin, TCF and LEF, was significantly associated with favourable progression free survival (median 9 vs. 5 months, P = 0·037) and OS (median 19 vs. 13 months, P = 0·04). This prognostic impact on OS was mainly driven by nuclear expression of TCF and/or LEF (P = 0·03). No prognostic significance of the hedgehog pathway activation status, defined as IHC expression of SMO or nuclear GLI1, could be established. A favourable prognostic impact of the concurrent activation of both pathways was observed. A trend for association of activated Wnt with response to chemotherapy (responders 67% among activated Wnt cases vs. 35% among nonactivated Wnt cases, P = 0·07) was observed in CUP adenocarcinomas. Conclusions: Activation of the Wnt pathway was a positive prognostic factor in a small CUP series, possibly via enhanced chemosensitivity. Independent validation is warranted. © 2015 Stichting European Society for Clinical Investigation Journal Foundation. 45 11 1145 1152

Background: alphaB-crystallin is a small heat shock protein that has recently been characterized as an oncoprotein correlating with the basal core phenotype and with negative prognostic factors in breast carcinomas. The purpose of this study was to evaluate alphaB-crystallin with respect to clinicopathological parameters and the outcome of patients with operable high-risk breast cancer. Methods. A total of 940 tumors were examined, derived from an equal number of patients who had participated in two randomized clinical trials (paclitaxel-containing regimen in 793 cases). Immunohistochemistry for ER, PgR, HER2, Ki67, CK5, CK14, CK17, EGFR, alphaB-crystallin, BRCA1 and p53 was performed. BRCA1 mutation data were available in 89 cases. Results: alphaβ-crystallin was expressed in 170 cases (18.1%) and more frequently in triple-negative breast carcinomas (TNBC) (45% vs. 14.5% non-TNBC, p < 0.001). alphaB-crystallin protein expression was significantly associated with high Ki67 (Pearson chi-square test, p < 0.001), p53 (p = 0.002) and basal cytokeratin protein expression (p < 0.001), BRCA1 mutations (p = 0.045) and negative ER (p < 0.001) and PgR (p < 0.001). Its overexpression, defined as >30% positive neoplastic cells, was associated with adverse overall survival (Wald's p = 0.046). However, alphaB-crystallin was not an independent prognostic factor upon multivariate analysis. No interaction between taxane-based therapy and aβ-crystallin expression was observed. Conclusions: In operable high-risk breast cancer, alphaB-crystallin protein expression is associated with poor prognostic features indicating aggressive tumor behavior, but it does not seem to have an independent impact on patient survival or to interfere with taxane-based therapy. Trial registrations. ACTRN12611000506998 (HE10/97 trial) and ACTRN12609001036202 (HE10/00 trial). © 2014 Koletsa et al.; licensee BioMed Central Ltd.

Background: We sought to determine the predictive value of in situ mRNA measurement compared to traditional methods on a cohort of trastuzumab-treated metastatic breast cancer patients. Methods: A tissue microarray composed of 149, classified as HER2-positive, metastatic breast cancers treated with various trastuzumab-containing chemotherapy regimens was constructed. HER2 intracellular domain(ICD), HER2 extracellular domain(ECD) and HER2 mRNA were assessed using AQUA. For HER2 protein evaluation, CB11 was used to measure ICD and SP3 to measure ECD of the HER2 receptor. In addition, HER2 mRNA status was assessed using RNAscope assay ERRB2 probe. Kaplan - Meier estimates were used for depicting time-to-event endpoints. Multivariate Cox regression models with backward elimination were used to assess the performance of markers as predictors of TTP and OS, after adjusting for important covariates. Results: HER2 mRNA was correlated with ICD HER2, as measured by CB11 HER2, with ECD HER2 as measured by SP3 (Pearson's Correlation Coefficient, r = 0.66 and 0.51 respectively) and with FISH HER2 (Spearman's Correlation Coefficient, r = 0.75). All markers, HER2 mRNA, ICD HER2 and ECD HER2, along with FISH HER2, were found prognostic for OS (Log-rank p = 0.007, 0.005, 0.009 and 0.043 respectively), and except for FISH HER2, they were also prognostic for TTP Log-rank p = 0.036, 0.068 and 0.066 respectively) in this trastuzumab-treated cohort. Multivariate analysis showed that in the presence of pre-specified set of prognostic factors, among all biomarkers only ECD HER2, as measured by SP3, is strong prognostic factor for both TTP (HR = 0.54, 95% CI: 0.31-0.93, p = 0.027) and OS (HR = 0.39, 95%CI: 0.22-0.70, p = 0.002). Conclusions: The expression of HER2 ICD and ECD as well as HER2 mRNA levels was significantly associated with TTP and OS in this trastuzumab-treated metastatic cohort. In situ assessment of HER2 mRNA has the potential to identify breast cancer patients who derive benefit from Trastuzumab treatment. © 2014 Vassilakopoulou et al.

Background: HER2 and TOP2A gene status are assessed for diagnostic and research purposes in breast cancer with fluorescence in situ hybridization (FISH). However, FISH probes do not target only the annotated gene, while chromosome 17 (chr17) is among the most unstable chromosomes in breast cancer. Here we asked whether the status of specifically targeted genes on chr17 might help in refining prognosis of early high-risk breast cancer patients. Methods: Copy numbers (CN) for 14 genes on chr17, 4 of which were within and 10 outside the core HER2 amplicon (HER2- and non-HER2-genes, respectively) were assessed with qPCR in 485 paraffin-embedded tumor tissue samples from breast cancer patients treated with adjuvant chemotherapy in the frame of two randomized phase III trials. Principal Findings: HER2-genes CN strongly correlated to each other (Spearman's rho >0.6) and were concordant with FISH HER2 status (Kappa 0.6697 for ERBB2 CN). TOP2A CN were not concordant with TOP2A FISH status (Kappa 0.1154). CN hierarchical clustering revealed distinct patterns of gains, losses and complex alterations in HER2- and non-HER2-genes associated with IHC4 breast cancer subtypes. Upon multivariate analysis, non-HER2-gene gains independently predicted for shorter disease-free survival (DFS) and overall survival (OS) in patients with triple-negative cancer, as compared to luminal and HER2-positive tumors (interaction p = 0.007 for DFS and p = 0.011 for OS). Similarly, non-HER2-gene gains were associated with worse prognosis in patients who had undergone breast-conserving surgery as compared to modified radical mastectomy (p = 0.004 for both DFS and OS). Non-HER2-gene losses were unfavorable prognosticators in patients with 1-3 metastatic nodes, as compared to those with 4 or more nodes (p = 0.017 for DFS and p = 0.001 for OS). Conclusions: TOP2A FISH and qPCR may not identify the same pathology on chr17q. Non-HER2 chr17 CN patterns may further predict outcome in breast cancer patients with known favorable and unfavorable prognosis. © 2014 Kotoula et al.

Background:Discrepant data have been published on the incidence and prognostic significance of ESR1 gene amplification in early breast cancer.Patients and Methods:Formalin-fixed paraffin-embedded tumor blocks were collected from women with early breast cancer participating in two HeCOG adjuvant trials. Messenger RNA was studied by quantitative PCR, ER protein expression was centrally assessed using immunohistochemistry (IHC) and ESR1 gene copy number by dual fluorescent in situ hybridization probes.Results:In a total of 1010 women with resected node-positive early breast adenocarcinoma, the tumoral ESR1/CEP6 gene ratio was suggestive of deletion in 159 (15.7%), gene gain in 551 (54.6%) and amplification in 42 cases (4.2%), with only 30 tumors (3%) harboring five or more ESR1 copies. Gene copy number ratio showed a significant, though weak correlation to mRNA and protein expression (Spearman's Rho

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. © 2013 Fountzilas et al.; licensee BioMed Central Ltd.

RACGAP1 is a Rac GTPase-activating protein involved in cell growth regulation, cell transformation and metastasis. The aim of the present study was to explore the prognostic and/or predictive significance of RACGAP1 mRNA expression on disease-free survival (DFS) and overall survival (OS) in high-risk early breast cancer patients and compare it to that of Ki67 protein expression and to the Nottingham prognostic index (NPI). A total of 595 high-risk breast cancer patients were treated in a two-arm trial evaluating postoperative dose-dense sequential chemotherapy with epirubicin followed by CMF with or without paclitaxel. RNA was extracted from 314 formalin-fixed paraffin-embedded primary tumor tissue samples followed by one-step quantitative RT-PCR for assessing RACGAP1 mRNA expression. High RACGAP1 mRNA expression (above the median) was associated with poor DFS (log-rank, p = 0.002) and OS (p < 0.001). High histological grade, as well as high Ki67 protein expression, was more frequent in the high-expression group of RACGAP1. Results of the Cox multivariate regression analysis revealed that high RACGAP1 mRNA expression independently predicted poor overall survival (Wald’s p = 0.008). High Ki67 protein expression was also an adverse prognostic factor for death (p = 0.016), while high NPI score values were not. High RACGAP1 mRNA expression, as assessed by qRT-PCR, was found to be of adverse prognostic significance in high-risk early breast cancer patients treated with dose-dense sequential chemotherapy. The utility of RACGAP1 mRNA expression in patient selection for treatment with aggressive chemotherapy regimens should be further explored and validated in larger cohorts.

To explore the impact of dose intensity (DI) in the adjuvant setting of breast cancer, a randomized phase III trial was conducted comparing postoperative dose-dense sequential chemotherapy with epirubicin, paclitaxel, and cyclophosphamide, methotrexate and fluorouracil (CMF)in high-risk breast cancer patients. From Oct 2000 to June 2005, 1,121 node-positive patients were randomized to dose-dense sequential epirubicin 110 mg/m 2 and paclitaxel (Taxol®, Bristol Myers-Squibb, Princeton, NJ) 250 mg/m 2 (group A), or concurrent epirubicin 83 mg/m 2 and paclitaxel 187 mg/m 2 (group B), both followed by three cycles of "intensified" combination chemotherapy with CMF. By protocol design total cumulative dose and duration of treatment were identical in both groups. Dose intensity of epirubicin and paclitaxel was double in the dose-dense arm. Prophylactic treatment with granulocyte colony-stimulating factor was given with the dose-dense treatments. Disease-free survival (DFS) was the primary endpoint. At a median follow-up of 76 months, 253 patients (23%) had documented disease relapse (123 vs. 130 in groups A and B, respectively) and 208 deaths (101, group A and 107, group B) had been observed. The 5-year DFS rate of 74 and 74% and OS rate of 86 and 85% were observed for group A and group B, respectively. No differences were found in DFS or OS between the two treatment groups (P = 0.78 and P = 0.45 for DFS and OS, respectively). Safety analysis results showing that both regimens were well tolerated and safe have been previously published (Fountzilas et al. Ann Oncol 2008). No DFS or OS benefit from the dose-dense sequential epirubicin and paclitaxel was detected when compared to the concurrent administration of the same drugs. No additional safety issues were raised with long-term follow-up. © 2011 Springer Science+Business Media, LLC.

Background: The aim of the present study was to investigate the efficacy of adjuvant dose-dense sequential chemotherapy with epirubicin, paclitaxel, and CMF in subgroups of patients with high-risk operable breast cancer, according to tumor subtypes defined by immunohistochemistry (IHC). Materials and Methods: Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 1,039 patients participating in two adjuvant dose-dense sequential chemotherapy phase III trials were centrally assessed in tissue micro-arrays by IHC for 6 biological markers, that is, estrogen receptor (ER), progesterone receptor (PgR), HER2, Ki67, cytokeratin 5 (CK5), and EGFR. The majority of the cases were further evaluated for HER2 amplification by FISH. Patients were classified as: luminal A (ER/PgR-positive, HER2-negative, Ki67low); luminal B (ER/PgR-positive, HER2-negative, Ki67high); luminal-HER2 (ER/PgR-positive, HER2-positive); HER2-enriched (ER-negative, PgR-negative, HER2-positive); triple-negative (TNBC) (ER-negative, PgR-negative, HER2-negative); and basal core phenotype (BCP) (TNBC, CK5-positive and/or EGFR-positive). Results: After a median follow-up time of 105.4 months the 5-year disease-free survival (DFS) and overall survival (OS) rates were 73.1% and 86.1%, respectively. Among patients with HER2-enriched tumors there was a significant benefit in both DFS and OS (log-rank test; p = 0.021 and p = 0.006, respectively) for those treated with paclitaxel. The subtype classification was found to be of both predictive and prognostic value. Setting luminal A as the referent category, the adjusted for prognostic factors HR for relapse for patients with TNBC was 1.91 (95% CI: 1.31-2.80, Wald's p = 0.001) and for death 2.53 (95% CI: 1.62-3.60, p

In spite the close association of the triple-negative breast cancer immunophenotype with hereditary breast cancers and the BRCA1 pathway, there is a lack of population studies that determine the frequency of BRCA1 mutations among triple-negative breast cancer patients. To address this, we have screened a large sample of 403 women diagnosed with triple-negative invasive breast cancer, independently of their age or family history, for germline BRCA1 mutations. Median age at diagnosis was 50 years (range 20-83). The overall prevalence of triple-negative cases among the initial patient group with invasive breast cancer was 8 %. BRCA1 was screened by direct DNA sequencing in all patients, including all exons where a mutation was previously found in the Greek population (exons 5, 11, 12, 16, 20, 21, 22, 23, 24-77 % of the BRCA1 coding region), including diagnostic PCRs to detect the three Greek founder large genomic rearrangements. Sixty-five deleterious BRCA1 mutations were identified among the 403 triple-negative breast cancer patients (16 %). Median age of onset for mutation carriers was 39 years. Among a total of 106 women with early-onset triple-negative breast cancer (

Cancer of unknown primary (CUP) is a heterogeneous entity, managed on the basis of "one size fits all" therapeutic concepts; insights into the molecular biology of CUP are urgently needed. We retrospectively examined the immunohistochemical (IHC) expression of Notch1, 2, 3, Jagged1, cMET, and pMAPK biomolecules in 100 CUP tumors using tissue microarrays, aiming to study their correlation to clinicopathologic characteristics and prognostic utility for patient outcome. Notch3 and pMAPK were most frequently expressed (97 and 91 %, respectively). A linear correlation of Notch3 and cMET expression was found (p = 0.001), while pMAPK emerged as the major adverse prognostic factor (median overall survival OS 9 vs. 17 months, p = 0.016), carrying also a significantly positive predictive value (p = 0.02). Our study indicated a favorable prognostic impact of cMET expression in CUP, both in univariate (median OS 15 vs. 9 months, p = 0.05) and in multivariate analysis (Relative Risk RR for death 0.48, p = 0.025). cMET and Notch3 expression were found to be statistically more frequent in squamous carcinomas (positive in 90 % of cases), associated with a unique metastatic IHC pattern (cMET-high in soft tissue/lymph node metastases, p < 0.001, Notch3-high in visceral, peritoneal/pleural and soft tissue/lymph node metastases, p < 0.001). Our study points to the MAPK and cMET axes as crucial in defining cancer progression and outcome in CUP patients and, if validated, could justify attempts at their therapeutic modulation. © 2012 Springer Science+Business Media, LLC. 29 6 603 614

Purpose: It is well recognized that breast cancer is a heterogeneous disease. The purpose of the current study was to classify patients according to the immunohistochemical phenotype of their tumors in an effort to evaluate the outcome of the respective groups of patients and specifically of those with triple-negative breast cancer (TNBC) following dose-dense sequential adjuvant chemotherapy. Methods: A total of 595 patients with high-risk breast cancer were treated with adjuvant anthracycline-based dose-dense sequential chemotherapy with or without paclitaxel in the context of a randomized study. ER, PgR, HER2, Ki67, EGFR, and CK5 protein expression were evaluated in 298 formalin-fixed paraffin-embedded tumor samples by immunohistochemistry (IHC). HER2 was also evaluated by chromogen in situ hybridization (CISH). HER2 status and Ki67 protein expression differentiated luminal IHC subtypes (luminal B tumors being HER2 and/or Ki67-positive). Results: Among the 298 tumors, the immunohistochemical panel classified 37 (12%) as luminal A, 198 (66%) as luminal B, 27 (9%) as HER2 enriched, and 36 (12%) as TNBC. The median follow-up time was 97 months. Patients with luminal A tumors had the best prognosis, with improved disease-free survival (log-rank, P = 0.033) and overall survival (P = 0.006) compared with the other three tumor subtypes. The three subtypes had an increased risk for relapse and death compared with luminal A in multivariate analysis, as well. No benefit from paclitaxel treatment was detected in any of the four subtypes or the total cohort. Hierarchical clustering based on mRNA expression of ER, PgR, and HER2 by quantitative RT-PCR identified patient groups that were comparable to the subtypes identified by IHC. Conclusions: The results of this study confirm that triple negative, luminal B and HER2-enriched phenotypes identified by IHC are of adverse prognostic value in high-risk breast cancer patients treated with dose-dense sequential adjuvant chemotherapy. © 2011 Springer-Verlag.

Background: The epithelial to mesenchymal transition (EMT) has been associated with metastatic dissemination and poor outcome in several solid tumour types. Our aim was to study its incidence and its prognostic significance in cancer of unknown primary (CUP). Patients and Methods: One hundred tumour samples of CUP were loaded in tissue microarrays and were studied for immunohistochemical (IHC) expression of E-cadherin, N-cadherin, vimentin, the EMT transcription factor (SNAIL) and the stem cell marker octamer-binding transcription marker 4(OCT4). An EMT phenotype was defined as low expression of E-cadherin, expression of N-cadherin with/without vimentin with concomitant expression of SNAIL, as assessed by percentage of tumour cell staining. Results: Among 100 CUP cases, the histological diagnosis was adenocarcinoma in 55, squamous carcinoma in 20 and undifferentiated carcinoma in 15, with a high grade seen in 46. Therapy consisted of palliative chemotherapy, mostly platinum based. The median progression-free survival and overall survival (OS) were 7 and 12 months respectively. Distributional studies resulted in selection of IHC cut-offs for E-cadherin (negative when expressed in

BACKGROUND: Randomized studies have shown that bevacizumab combined with taxane-based regimens increases response rates and prolongs progression-free survival (PFS) of patients with metastatic breast cancer (MBC). However predictive or prognostic biological markers that identify the appropriate target population, thus improving the cost-effectiveness ratio of this treatment, are still needed. PATIENTS AND METHODS: Retrospectively, 124 patients with MBC treated either with paclitaxel 90 mg/m(2) weekly x12 plus bevacizumab 10 mug/kg every 2 weeks or 15 mug/kg every 3 weeks (85 patients) or paclitaxel 175 mg/m(2) plus bevacizumab 15 mug/kg every 3 weeks for 6 cycles (36 patients) were identified. Additionally, the prognostic significance of a panel of key biological markers was evaluated centrally by immunohistochemistry (IHC) in 88 evaluable patients. RESULTS: More than two thirds of the patients completed chemotherapy, as planned. The response rate was almost identical (55.3% vs. 55.6%) in the patients treated with weekly or 3-weekly paclitaxel, respectively. After a median follow-up time of 23 months, the median PFS of the study population was 13 months, while median survival had not yet been reached. Common severe adverse events were neutropenia (33%), neuropathy (18.6%) and metabolic disturbances (17.6%). The incidence of hypertension of all grades was 28.1%. High expression of vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3) was associated with clinical response, while high expression of VEGFR1 was associated with poor survival. CONCLUSION: The safety and activity of the combination of bevacizumab with paclitaxel given either weekly or 3-weekly in patients with MBC is confirmed. Anticancer Res

We evaluated the prognostic and predictive utility of beta-tubulin isotype III (TUBB3) tumour gene transcription in early breast cancer patients enrolled in a randomised study. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied for assessment of TUBB3, ER, PgR, HER2 and MAPT messenger RNA and immunohistochemistry (IHC) for protein expression in 314 patients enrolled in trial HE10/97, evaluating epirubicin-alkylator adjuvant chemotherapy with or without paclitaxel. High TUBB3 mRNA status was associated with advanced T stage, high histological grade, low mRNA and protein levels of ER, PgR and MAPT, and high levels of HER2 (p < 0.001). At a median follow-up of 98 months, multivariate analysis showed high TUBB3 mRNA status to have prognostic significance for DFS (HR = 1.83, 95% CI 1.25-2.68, p = 0.002) and OS (HR = 1.71, 95% CI 1.03-2.83, p = 0.038), along with the number of involved axillary nodes, PgR mRNA status and tumour grade. TUBB3 mRNA levels did not predict benefit from inclusion of paclitaxel in adjuvant chemotherapy (test for interaction p = 0.96 for OS, p = 0.46 for DFS). Transcriptional activity of beta-tubulin isotype III in early breast cancer is an adverse prognostic factor, though not a predictive one for taxane efficacy. Breast Cancer Res Treat

European Journal of Cancer