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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.

We investigated the predictive and prognostic significance of tumoral messenger RNA levels of vascular endothelial growth factor A (VEGFS) splice variants in metastatic colorectal cancer (mCRC) patients treated with bevacizumab. VEGFA145b had negative predictive significance predominantly in those patients with right-sided primary tumors. All VEGFAxxxb variants were negative prognosticators for patients with right-sided mCRC, whereas VEGFA165b was of favorable prognostic significance in patients with left-sided tumors. © 2019 Elsevier Inc. Background: Alternative splicing of vascular endothelial growth factor A (VEGFA) results in VEGFAxxxb antiangiogenic isoforms that fail to activate angiogenesis. Bevacizumab, widely used in patients with metastatic colorectal cancer (CRC), binds both VEGFA and VEGFAxxxb isoforms. Patients and Methods: Formalin-fixed, paraffin-embedded primary tumors from metastatic CRC patients treated with first-line FOLFIRI (leucovorin, 5-fluorouracil, irinotecan, and oxaliplatin) + bevacizumab (n = 285) or FOLFIRI only (n = 75) were collected. The relative expression of VEGFA121a, 121b, 145a, 145b, 165a, and 165b was assessed with custom TaqMan-MGB assays and quantitative PCR. Results: At a median follow-up of 101.5 months, left-sided primary CRC was a favorable prognosticator (median survival, 29.2 vs. 18.2 months; P = .015). Positive high VEGFA145b was an unfavorable factor for progression-free survival (PFS; hazard ratio [HR] = 1.66; 95% confidence interval [CI], 1.13-2.44; P = .009) in patients who received FOLFIRI + bevacizumab, without prognostic significance in FOLFIRI-only patients (HR = 0.70; 95% CI, 0.34-1.44; P = .33). The adverse effect on PFS of 145b was more pronounced in patients with right-sided colon cancer (HR = 2.62; 95% CI, 1.35-5.12; P = .005), especially in those who received bevacizumab (HR = 2.85; 95% CI, 1.31-6.21; P = .008). In patients with right-sided colon primary tumors, isoform 121b correlated with inferior PFS (HR = 1.73; 95% CI, 0.94-3.18; P = .076) and overall survival (OS; HR = 2.0; 95% CI, 1.08-3.72; P = .028). In patients with left-sided primary tumors, positive high 165b correlated with superior PFS (HR = 0.76; 95% CI, 0.59-0.99; P = .044) and OS (HR = 0.68; 95% CI, 0.52-0.90; P = .006). At multivariate analysis, right-sided primary tumor was associated with inferior PFS (HR = 1.28; 95% CI, 1.00-1.64), while 145b consistently retained predictive significance for lack of benefit in PFS with bevacizumab (HR = 1.71; 95% CI, 1.16-2.53). Multivariate analysis for OS showed that VEGFA165b expression was favorable in patients with left-sided but unfavorable in patients with right-sided primary tumors (Pinteraction < .001). Conclusion: The antiangiogenic isoform VEGFA145b messenger RNA may predict resistance to bevacizumab. Differences in biological relevance and prognostic significance of various VEGFA isoforms were found for right- versus left-sided primary tumors. © 2019 Elsevier Inc.

Background/Aim: KRAS mutations are reported in 20-25% of non-small cell lung cancer (NSCLC) and their prognostic role is unclear. We studied KRAS and EGFR genotyping in Greek NSCLC patients. Patients and Methods: KRAS and EGFR genotypes were centrally evaluated in 421 NSCLC patients (diagnosed September 1998 -June 2013) and associated with clinicopathological parameters. Outcome comparisons were performed in 288 patients receiving first line treatment. Results: Most patients were male (78.6%), >60 years old (63.9%), current smokers (51.1%), with adenocarcinoma histology (63.9%). EGFR and KRAS mutations were found in 10.7% and 16.6% of all histologies, respectively, and in 14.9% and 21.9% of adenocarcinomas. At 4.5 years median follow-up, KRAS status was an independent negative prognostic factor for overall survival (OS, p=0.016). KRAS mutations conferred 80% increased risk of death in patients receiving first-line treatment (p=0.002). Conclusion: The presence of KRAS mutations is an independent negative prognosticator among Greek NSCLC patients and an independent response predictor to first line treatment. © 2019 International Institute of Anticancer Research. All rights reserved.

Background: Stromal tumor infiltrating lymphocytes (TILs) density is an outcome predictor in triple-negative breast cancer (TNBC). Herein we asked whether TILs are related to coding mutation load and to the chemical class of the resulting mutated amino acids, i.e., charged, polar, and hydrophobic mutations. Methods: We examined paraffin tumors from TNBC patients who had been treated with adjuvant chemotherapy mostly within clinical trials (training cohort, N = 133; validation, N = 190) for phenotype concordance; TILs density; mutation load and types. Results: Concordance of TNBC phenotypes was 42.1% upon local / central, and 72% upon central / central pathology assessment. TILs were not associated with mutation load, type and class of mutated amino acids. Polar and charged mutation patterns differed between TP53 and PIK3CA (p50% TILs tumors (training p = 0.003; validation p = 0.015). Conclusions: TILs density is unrelated to mutation load in TNBC, which may be regarded as an unstable phenotype. If further validated, hydrophobic mutations along with TILs density may help identifying TNBC patients in higher risk for relapse. © 2016 Kotoula 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.

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: 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.

Introduction: We sought to determine the level of protein expression of the critical components of the insulin-like growth factor receptor (IGFR) pathway and to evaluate their prognostic significance across the different early breast cancer subtypes. Patients and Methods: Archival tumor tissue from 1,021 women with early, node positive breast cancer, who were prospectively evaluated within two randomized clinical trials, was used to construct tissue microarrays that were stained for hormone receptors (HR), Ki67, HER2, epidermal growth factor receptor (EGFR) and cytokeratins 5/6, to classify tumors into five immunophenotypical subgroups. Immunohistochemical (IHC) expression of IGF1R-alpha and beta subunits, IGF2R and IGF-binding protein 2 (IGFBP2) was assessed using the immunoreactive score (IRS). Repeated internal cross-validation was performed to examine the statistical validity of the cut off points for all biomarkers. Results: After a median follow-up time of 105.4 months, overall 370 women (36.2%) had relapsed and 270 (26.4%) had died. Tumors expressing IGF1R-alpha above the median IRS were significantly more frequently HR positive (luminal A+B+HER2), as compared to HER2-enriched and triple negative ones (p

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: More than half of patients with KRAS-wild type advanced colorectal cancer (CRC) fail anti-EGFR monoclonal antibodies. We studied EGFR-axis messenger RNA (mRNA) expression and RAS, RAF, PIK3CA mutations in order to identify additional biomarkers of cetuximab efficacy. Methods: Previously genotyped (KRAS, NRAS, BRAF, PIK3CA mutations) formalin-fixed paraffin-embedded tumour biopsies of 226 cetuximab-treated CRC patients (1st to 3rd line therapy) were assessed for mRNA expression of epidermal growth factor receptor (EGFR) and its ligands EGF, Transofrming Growth Factor-a (TGFA), Amphiregulin (AREG) and Epiregulin (EREG) with real time quantitative PCR. Mutations were detected in 72 (31.9%) tumours for KRAS, in 6 (2.65%) for BRAF, in 7 (3.1%) for NRAS and in 37 (16.4%) for PIK3CA. Results: Only PIK3CA mutations occasionally coexisted with other gene mutations. In univariate analysis, prognostic significance for survival ( from metastases until death) was seen for BRAF mutations (Hazard Ratio HR 8.1, 95% CI 3.4-19), codon 12-only KRAS mutations (HR 1.62, 95% CI 1.1-2.4), high AREG mRNA expression only in KRAS wild type CRC (HR 0.47, 95% CI 0.3-0.7) and high EREG mRNA expression irrespective of KRAS mutation status (HR 0.45, 95% CI 0.28-0.7). EREG tumoural mRNA expression was significantly associated with a 2.26-fold increased likelihood of objective response to cetuximab therapy (RECIST 1.1). In multivariate analysis, favourable predictive factors were high AREG mRNA in KRAS wild type tumours, high EREG mRNA, low Ephrin A2 receptor mRNA. Cetuximab-treated patients with AREG-low KRAS wild type CRC fared very poorly, their survival being similar to KRAS mutant CRC. Patients with KRAS codon 13 or other non-codon 12 mutations had a median survival (30 months, 95% CI 20-35) similar to that of patients with KRAS wild-type (median survival 29 months, 95% CI 25-35), in contrast to patients with KRAS codon 12 mutations who fared worse (median survival 19 months, 95% CI 15-26). Conclusions: BRAF and codon 12 KRAS mutations predict for adverse outcome of CRC patients receiving cetuximab. AREG mRNA reflects EGFR signalling in KRAS wild type tumours, predicting for cetuximab efficacy when high and failure when low. EREG may have a prognostic role independent of KRAS mutation. © 2013 Pentheroudakis et al; licensee BioMed Central Ltd. 13

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

Hamartomas of the spleen (splenomas) are very rare benign tumors composed of an aberrant mixture of normal splenic elements. Herein we present a unique case of a symptomatic non-palpable splenoma in a 64-year-old female patient presented with anemia and thrombocytopenia and we describe imaging findings in ultrasound, computed tomography and magnetic resonance imaging. To our knowledge, this is the first case of a relatively small splenic hamartoma (35 mm at histopathology) associated with thrombocytopenia and anemia that resolved completely several months after splenectomy.