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Purpose Angiogenesis is a crucial phenomenon in the development and progression of breast cancer (BC), but the clinical significance of angiogenesis-related proteins in metastatic BC remains unknown. This study investigates the prognostic value of vascular endothelial growth factor receptors 1, 2, 3 (VEGFR1, VEGFR2, VEGFR3) as well as vascular endothelial growth factors A and C (VEGFA and VEGFC) in metastatic BC patients treated with trastuzumab-based regimens.Materials and Methods Two hundred female patients were included. Protein and mRNA expression of the studied angiogenesis-related factors were evaluated by immunohistochemistry and quantitative polymerase chain reaction, respectively.Results High expression of VEGFA, VEGFC, VEGFR1, VEGFR2, and VEGFR3 in the tumor cells was observed in 43.5%, 24.2%, 36%, 29.5%, and 43%, respectively. Stromal elements expressed high levels of VEGFA, VEGFC, VEGFR1, VEGFR2, and VEGFR3 in 78.9%, 93.3%, 90.7%, 90.2%, and 74.8% of tumors with available data. High tumor cell expression of VEGFR1 was a favorable prognosticator for survival among patients with human epidermal growth factor receptor 2 (HER2)–positive tumors (hazard ratio [HR], 0.55; p=0.013). A trend towards longer progression-free survival was detected univariately for patients with HER2-negative tumors and high expression of VEGFR2 (HR, 0.60; p=0.059).Conclusion VEGFR1 and VEGFR2 seem to have significant prognostic value in BC patients with metastatic disease treated with trastuzumab-based regimens.

Background: Dose-dense sequential chemotherapy based on anthracyclines and taxanes has achieved an 18% reduction of recurrence risk in the adjuvant setting of early breast cancer (BC). However, the optimal chemotherapy schedule and the preferable interval between cycles are still under investigation. Methods: A total of 990 eligible BC patients were randomized to receive either 3 cycles of epirubicin (E,110 mg/m2) every 2 weeks followed by 3 cycles of paclitaxel (T, 200 mg/m2) every 2 weeks followed by 3 cycles of intensified CMF (Control Arm A, E-T-CMF; 333 patients) or three cycles of epirubicin followed by 3 cycles of CMF followed by 9 consecutive weekly cycles of docetaxel (wD) 35 mg/m2 (Experimental Arm B, E-CMF-wD; 331) or 9 consecutive weekly cycles of paclitaxel (wT) 80 mg/m2 (Experimental Arm C, E-CMF-wT; 326). Trastuzumab was administered for HER2-positive disease. The primary endpoint was disease-free survival (DFS). Results: At a median follow-up of 13.3 years, 330 DFS events (33.3%) had been reported. DFS and overall survival (OS) did not differ between patients in the combined B and C arms versus control arm A either in the entire cohort (HR=0.90, p=0.38 and HR=0.85, p=0.20) or among HER2-positive, trastuzumab-treated patients (HR=0.69, p=0.13 and HR=0.67, p=0.13). Thirty-four patients (3.4%) developed secondary neoplasms. Conclusions: Overall, no significant differences in DFS or OS were found amongst the studied regimens after a long-term observational period. Trial registration: Australian New Zealand Clinical Trials Registry ACTRN12610000151033 Citation Format: Flora Zagouri, Georgia-Angeliki Koliou, Foteinos Dimitrakopoulos, Christos Papadimitriou, Ioannis Binas, Angelos Koutras, Pavlos Papakostas, Christos Markopoulos, Vasileios Venizelos, Grigorios Xepapadakis, Charisios Karanikiotis, Amanda Psyrri, Dimitrios Bafaloukos, Paris Kosmidis, Gerasimos Aravantinos, Eleni Res, Davide Mauri, Anna Koumarianou, Kalliopi Petraki, Anna Tsipoura, Dimitrios Pectasides, Helen Gogas, George Fountzilas. Dose-dense sequential adjuvant chemotherapy in the trastuzumab era: Final long-term results of the hellenic cooperative oncology group phase III HE10/05 trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-12-01.

Although primary colorectal carcinomas (CRCs) frequently share genetic alterations with their metastases, morphologic surrogates reflecting the genotype contexture of metastases remain largely unknown. We investigated phenotype/genotype associations in paired primary and metastatic colorectal adenocarcinomas from 75 patients. Thirty-three (44%) metastatic lesions were synchronous and 42 (56%) were metachronous. Tumor budding, micronecrosis, and tumor-infiltrating lymphocyte (TIL) density were compared with matched next-generation sequencing genotypes. Micronecrosis in the primary were significantly associated with nodal status (P = 0.0054) and with micronecrosis in metastatic sites (P = 0.0216), particularly in metachronous metastases (P = 0.0033). With a 57-gene panel, one or more mutations were identified in 64 (85.3%) cases. In metastases, high (brisk) TILs were associated with overall mutational burden (P = 0.0058) and with mutations in EGF (P = 0.0325), RAS genes (P = 0.0043), and MMR genes (P = 0.0069), whereas high-level micronecrosis correlated with mutations in APC (P = 0.0004) and MSH6 (P = 0.0385) genes. Genomic alterations were shared in 90.1% of primary/metastatic pairs, but clonality of the same mutation was shared in only 57.1% of paired lesions. Compared with synchronous, metachronous metastases had more private clonal alterations (P = 0.0291); in this group, clonal alterations coincided with brisk TILs (P = 0.0334) and high micronecrosis (P = 0.0133). High TILs in metastatic lesions were predictive of favorable overall survival (log-rank P = 0.044). The observed phenotype/genotype associations favor the clonal evolution model in CRC metastases that seems accompanied by intense host immune response. If the role of micronecrosis and brisk TILs in metachronous metastases is validated in larger studies, these histologic parameters will be worth adding in the armamentarium for the evaluation of metastatic CRC.

BACKGROUND: Dose-dense sequential chemotherapy with anthracyclines and taxanes achieved an 18% reduction of recurrence risk in early breast cancer (BC). The optimal chemotherapy schedule and interval between cycles remain under investigation. METHODS: Overall, 990 patients were randomised to receive either three cycles of epirubicin (E, 110 mg/m(2)) every 2 weeks followed by 3 cycles of paclitaxel (T, 200 mg/m(2)) every 2 weeks followed by three cycles of intensified CMF (Control Arm A, E-T-CMF) that was previously used in BC or three cycles of epirubicin followed by three cycles of CMF followed by nine consecutive weekly cycles of docetaxel (wD) 35 mg/m(2) (Arm B, E-CMF-wD) or nine consecutive weekly cycles of paclitaxel (wT) 80 mg/m(2) (Arm C, E-CMF-wT). Trastuzumab was administered for HER2-positive disease. RESULTS: At a median follow-up of 13.3 years, 330 disease-free survival (DFS) events (33.3%) were reported. DFS and overall survival (OS) did not differ between patients in the combined B and C arms versus arm A either in the entire cohort (HR = 0.90, P = 0.38 and HR = 0.85, P = 0.20) or among trastuzumab-treated patients (HR = 0.69, P = 0.13 and HR = 0.67, P = 0.13). Thirty-four patients (3.4%) developed secondary neoplasms. CONCLUSIONS: Overall, no significant differences in survival were found amongst the studied regimens after a long-term observational period. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12610000151033.

Background: We evaluated the association between pathogenic mutations and overall survival (OS) in patients with cancer referred to Hellenic Cooperative Oncology Group-affiliated Departments. Patients and methods: Patients referred from 12/1980 to 1/2017 had molecular testing (for research) of archival tumor tissue collected at the time of first diagnosis (non-metastatic, 81%; metastatic, 19%). Tumor-specific gene panels (16-101 genes) were used to identify pathogenic mutations in clinically relevant genes. NGS genotyping was performed at the Laboratory of Molecular Oncology, Aristotle University of Thessaloniki. Annotation of mutations was performed at MD Anderson Cancer Center. Results: We analyzed 3,084 patients (median age, 57 years; men, 22%) with sequencing data. Overall, 1,775 (58% of 3,084) patients had pathogenic mutations. The median follow-up was 7.52 years (95% CI, 7.39-7.61). In patients with non-metastatic tumors, after stratification by tumor type, increasing age, higher grade, and histology other than adenocarcinoma were associated with shorter OS. OS was also shorter in patients with pathogenic TP53 (HR=1.36; p

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 Cancer-associated thrombosis (CAT), the second leading cause of death in patients with cancer can be treated with low molecular weight heparin (LMWH) according to guidelines. Patients and methods A multicenter prospective observational study was carried out to record anti-thrombotic treatment practice, assess thrombosis recurrence and bleeding, and identify potential risk factors. Adult patients from 18 Oncology Departments throughout Greece were followed-up for 12 months. Results A total of 120 patients with CAT receiving anticoagulant treatment were enrolled (35% incidental); 85% were treated for more than 6 months, 95.8% were treated with tinzaparin and smaller percentages with other agents. Thrombosis recurred in three patients and there was minor bleeding in four patients. Bleeding was associated with high body mass index (>35 kg/m2), trauma history, renal insufficiency and bevacizumab use. Conclusion Incidental thrombosis contributes significantly to CAT burden. Long-term use of LMWH seems to be effective and safe. Several risk factors associated with bleeding should be considered during anti-coagulation therapy planning.

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 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.

Prognostic and predictive biomarkers are being studied for the diagnosis and treatment of breast cancer. The present study retrospectively assessed the mRNA expression of HER family receptor ligands and of other potential prognostic biomarkers and their association with time to progression (TTP), survival and clinicopathological characteristics in patients with metastatic breast cancer (MBC) treated with trastuzumab. A total of 145 tumour tissue samples were analysed. mRNA expression analysis of the transcripts of interest was performed and the association of these markers with selected clinicopathological parameters was examined. HER2 status was centrally re-evaluated. Only 67.6% of patients were truly HER2-positive according to the central HER2 re-evaluation. Heparin binding epidermal growth factor (EGF)-like growth factor, transforming growth factor β1 (TGFB1) and thyroid hormone receptor α (THRA) mRNA expression was higher in HER2-positive patients (P=0.026, P

Thromboprophylaxis, as a preventive measure for cancer-associated thrombosis (CAT), may be beneficial for patients with active cancer and high-risk for thrombosis. The present post hoc analysis include a total of 407 patients enrolled in the Greek Management of Thrombosis study, who received thromboprophylaxis with tinzaparin. The objectives of the present analysis were: i) To obtain sufficient evidence for the administration of prophylaxis in patients with active cancer, irrespective of Khorana risk assessment model score; ii) to identify the selection criteria for both dose and duration of tinzaparin; and iii) to evaluate the efficacy and safety of tinzaparin administered for CAT prophylaxis. The main tumor types for the patients included in the present study were as follows: Lung (25.1%), pancreatic (14.3%), breast (9.1%), stomach (8.4%), colorectal (7.9%) and ovarian (7.6%). Furthermore, metastatic disease was observed in 69.5% of the patients. High thrombotic burden agents (HTBAs) were administered to 66.3% of the patients, and 17.4% received erythropoietin. A total of 43.7% of the patients exhibited a Khorana score2. The results of the present study demonstrated that both the presence of metastatic disease and the use of HTBAs seemed to influence oncologists' decisions for the use of thromboprophylaxis in patients with active cancer, regardless of Khorana score. Tinzaparin, in dose expressed in the standard notation for heparins, i.e., anti-Xa factor international units (Anti-Xa IU), was administered at an intermediate dose (InterD; 8,000-12,000 Anti-Xa IU; once daily) to 52.4% of patients, while the remaining patients received a prophylactic dose (ProD; ≤4,500 Anti-Xa IU; once daily). The average duration of thromoprophylaxis was 5 months. Furthermore, a total of 14 (3.4%) thrombotic events and 6 (1.5%) minor bleeding events were recorded. A total of four thrombotic events were observed following an InterD treatment of tinzaparin, while 10 thrombotic events were observed following ProD treatment. The present study also demonstrated that an InterD of tinzaparin was administered more frequently to patients with a body mass index30 kg/m

BACKGROUND: Src, CDKN1B, and JAK2 play a crucial role in the coordination of cell signaling pathways. In the present study, we aim to investigate the prognostic significance of these biomarkers in HER2-positive metastatic breast cancer (MBC) patients treated with trastuzumab (T). METHODS: Formalin-fixed paraffin-embedded tumor tissue samples from 197 patients with HER2-positive MBC treated with T were retrospectively collected. All tissue samples were centrally assessed for ER, PgR, Ki67, HER2, and PTEN protein expression; EGFR gene amplification; PI3KCA mutational status; and tumor-infiltrating lympocytes density. Src, CDKN1B, and JAK2 mRNA expression was evaluated using quantitative reverse transcription-polymerase chain reaction. RESULTS: Only 133 of the 197 patients (67.5%) were found to be HER2-positive by central assessment. CDKN1B mRNA expression was strongly correlated with Src (rho = 0.71) and JAK2 (rho = 0.54). In HER2-positive patients, low CDKN1B conferred higher risk for progression [hazard ratio (HR) = 1.58, 95% confidence interval (CI) 1.08-2.32, P = .018]. In HER2-negative patients, low Src was associated with longer survival (HR = 0.56, 95% CI 0.32-0.99, P = .045). Upon multivariate analyses, only low CDKN1B and JAK2 mRNA expression remained unfavorable factors for PFS in de novo and relapsed (R)-MBC patients, respectively (HR = 2.36, 95% CI 1.01-5.48, P = .046 and HR = 1.76, 95% CI 1.01-3.06, P = .047, respectively). CONCLUSIONS: Low CDKN1B and JAK2 mRNA expressions were unfavorable prognosticators in a cohort of T-treated MBC patients. Our results suggest that CDKN1B and JAK2, if validated, may serve as prognostic factors potentially implicated in T resistance, which seems to be associated with distinct pathways in de novo and R-MBC.

Background-aim: SRC, CDKN1B and JAK2 play a crucial role in the coordination and facilitation of cell-signaling pathways controlling a wide range of cellular functions. In the present study, we investigated the prognostic significance and clinical utlity of these biomarkers in metastatic breast cancer (MBC) patients treated with trastuzumab (T). Methods: We assessed SRC, CDKN1B and JAK2 mRNA expression with qRT-PCR (Taqman-MGB assays) on 197 paraffin tumors. PIK3CA mutation status was previously assessed. Relapsed (RMBC) and de novo MBC (dnMBC) patients had received T for metastatic disease only. Tumors were centrally re-assessed for HER2 status. Results: Only 133/197 patients (67.5%) were found to be truly HER2(+). CDKN1B mRNA expression strongly correlated with SRC (rho = 0.71) and JAK2 (rho = 0.54); high CDKN1B was more frequent in RMBC compared to dnMBC (p = 0.001) and in PIK3CA wild-type tumors (p = 0.005). In HER2(+) patients, low CDKN1B conferred higher risk for progression (HR 1.58, 95% CI 1.08-2.32, p = 0.018). In HER2(-) patients, low SRC was associated with longer survival (HR 0.56, 95% CI 0.32-0.99, p = 0.045) and, as a trend, with increased progression-free survival (PFS) (p = 0.067). For PFS, in RMBC, we observed trends for unfavorable low CDKN1B (p = 0.068) and JAK2 (p = 0.086); similarly, in dnMBC for unfavorable low CDKN1B (p = 0.072). Low SRC showed a trend for better survival in RMBC (p = 0.087). Upon multivariable analyses, only PIK3CA mutations strongly predicted for unfavorable PFS in HER2(+) patients (HR 3.37, 95% CI 1.98-5.73, p < 0.001). Low CDKN1B and JAK2 mRNA expression remained unfavorable factors for PFS in dnMBC and RMBC patients (HR 2.36, 95% CI 1.01-5.48, p = 0.046 and HR 1.76, 95% CI 1.01-3.06, p = 0.047, respectively). Conclusions: Low CDKN1B and JAK2 mRNA expression were unfavorable prognosticators in a cohort of T-treated MBC patients previously unexposed to this agent, with distinct impact in de novo and RMBC. Our results highlight biological and clinical differences between de novo and RMBC and suggest that CDKN1B and JAK2, if validated, may serve as prognostic factors potentially implicated in T-resistance, which seems to be associated with distinct pathways in the two MBC settings. Citation Format: Economopoulou P, Kotoula V, Koliou G-A, Papadopoulou K, Christodoulou C, Pentheroudakis G, Koutras A, Bafaloukos D, Papakostas P, Pectasides D, Kotsakis A, Razis E, Samantas E, Kalogeras KT, Economopoulos T, Fountzilas G. Prognostic impact of SRC, CDKN1B and JAK2 expression in metastatic breast cancer patients treated with trastuzumab [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-20.

Genotypic and phenotypic comparisons of tumors in multiple tissue samples from the same patient are important for understanding disease evolution and treatment possibilities. Panel NGS genotyping is currently widely used in this context, whereby NGS variant filtering and final evaluation constitute the basis for meaningful comparisons. Here, we present the genotype data used for genotype / phenotype comparisons between matched primary / metastatic colorectal tumors in the work by Chatzopoulos et al (doi: 10.1016/j.humpath.2020.10.009), as well as the process followed for obtaining these data. We describe key issues while processing routinely formalin-fixed paraffin-embedded (FFPE) tumors for genotyping, NGS application (Ion Torrent), a stringent variant filtering algorithm for genotype analyses in FFPE tissues and particularly in matched tumor samples, and provide the respective datasets. Apart from research, tumor NGS genotyping is currently applied for clinical diagnostic purposes in Oncology. The datasets and method description provided herein (a) are important for comprehending the peculiarities of FFPE tumor genotyping, which is still mostly based on principles of germline DNA genotyping; (b) can be used in pooled analyses, e.g., of primary / metastatic tumors for the investigation of tumor evolution.

Background/aim Human epidermal growth factor receptor 2 (HER2) P95-isoform could be involved in trastuzumab resistance in HER2 metastatic breast cancer. Materials and methods A total of 114 metastatic breast cancer patients treated with trastuzumab were evaluated retrospectively. HER2 was centrally reviewed. P95 was evaluated along with other markers possibly affecting trastuzumab efficacy in regards to progression-free survival and overall survival. Results HER2 was centrally negative in 54 cases. P95 expression was significantly higher in HER2-positive tumors. High p95 was associated with gain of HER2 copy number variations (CNVs), high pHER2Tyr877, Ki67 and HER2 mRNA. P95 as a continuous variable was positively correlated with mRNA expression of HER2 and negatively correlated with HER4 and IGF1. HER2-negative p95-high patients had a marginally higher risk for death (HR=2.15, p=0.055). Conclusion p95 was associated with higher HER2 CNVs and mRNA expression, pHER2Tyr877 expression and high Ki67, indicating a more aggressive phenotype.

Simple Summary Data on the prognostic and predictive value of germline cancer predisposing pathogenic/likely pathogenic variants (P/LPVs) in patients with pancreatic ductal adenocarcinoma is limited. This research was a study of germline testing of 62 cancer susceptibility genes in 549 unselected patients with pancreatic cancer. We reported a significant proportion of European patients with pancreatic cancer carrying P/LPVs in clinically significant genes, irrespectively of age, family history or disease stage. Importantly, P/LPVs were identified in pancreatic cancer-associated genes and in homologous recombination repair genes in 4.0% and 7.7% of patients, respectively. The presence of any P/LPVs was associated with improved overall survival univariately; however, it did not retain its independent prognostic significance in multivariate analysis. The presence of P/LPVs in homologous recombination repair genes did not predict benefit from platinum-based treatment. These results should be prospectively validated through universal genetic testing of patients with pancreatic cancer, taking into consideration the administration of newer treatments. Abstract Our aim was to determine the prevalence, prognostic and predictive role of germline pathogenic/likely pathogenic variants (P/LPVs) in cancer predisposing genes in patients with pancreatic ductal adenocarcinoma (PDAC). Germline testing of 62 cancer susceptibility genes was performed on unselected patients diagnosed from 02/2003 to 01/2020 with PDAC, treated at Hellenic Cooperative Oncology Group (HeCOG)-affiliated Centers. The main endpoints were prevalence of P/LPVs and overall survival (OS). P/LPVs in PDAC-associated and homologous recombination repair (HRR) genes were identified in 22 (4.0%) and 42 (7.7%) of 549 patients, respectively. P/LPVs were identified in 16 genes, including ATM (11, 2.0%) and BRCA2 (6, 1.1%), while 19 patients (3.5%) were heterozygotes for MUTYH P/LPVs and 9 (1.6%) carried the low-risk allele, CHEK2 p.(Ile157Thr). Patients carrying P/LPVs had improved OS compared to non-carriers (22.6 vs. 13.9 months, p = 0.006). In multivariate analysis, there was a trend for improved OS in P/LPV carriers (p = 0.063). The interaction term between platinum exposure and mutational status of HRR genes was not significant (p-value = 0.35). A significant proportion of patients with PDAC carries clinically relevant germline P/LPVs, irrespectively of age, family history or disease stage. The predictive role of these P/LPVs has yet to be defined. ClinicalTrials.gov Identifier: NCT03982446.

Background: Cancer patients are at high risk for cancer-associated thrombosis (CAT). CAT is the second leading cause of death in these patients but it can be preventable with thromboprophylaxis. Patients and Methods: An observational, prospective, multicenter study aiming to record CAT management in clinical practice was conducted by the Hellenic Society of Medical Oncology (HeSMO). Results: A total of 426 active cancer patients (mean age 65.3 years, mean BMI: 26.1 kg/m2) who received thromboprophylaxis, were included from 18 oncology units. Tumor types were lung 25.1%, pancreas 13.9%, breast 8.7%, stomach 8.5%, ovarian 7.8%, and others 36%, while 69% had metastases. A total of 71% had a Khorana score &le, 2 and 61% received High Thrombotic Risk Chemotherapy Agents (HTRCAs, e.g., platinum). For thromboprophylaxis patients received mainly Low Molecular Weight Heparins (LMWHs), on higher than prophylactic doses in 50% of cases. Overall, 16 (3.8%) thrombotic events and 6 (1.4%) bleeding events were recorded. Notably, patients on higher doses of LMWHs compared to patients who received standard prophylactic doses had 70% lower odds to develop thrombotic events (OR: 0.3, 95% CI: 0.10&ndash, 1.0, p = 0.04). Conclusion: CAT is an important issue in oncology. Along with the Khorana score, factors as metastasis and use of HTRCAs should also be taken into consideration. Thromboprophylaxis for active cancer patients with LMWHs, even on higher doses is safe and efficient.

Background: Tumor-infiltrating lymphocytes (TILs) have been shown to be of prognostic value in several cancer types. In early breast cancer, TILs have a prognostic utility, as well, especially in HER2-positive and triple-negative breast cancer (TNBC). TILs presence is broadly associated with improved survival, however there is controversy regarding TILs subpopulations. In general, T cell infiltration is higher in non-luminal and more aggressive tumors, like the basal-like subtype. Among TILs subpopulations, CD8-positive T cell infiltration is associated with better outcome, whereas high numbers of FOXP3-positive T regulatory cells are associated with worse outcome in ER-positive tumors and better outcome in HER2-positive and TNBC tumors. Patients and Methods: Early breast cancer patients, treated with anthracycline-based chemotherapy within two randomized trials (HE10/97 and HE10/00) were included in the study. We evaluated, by qRT-PCR, 826 macrodissected formalin-fixed paraffin-embedded tumor tissue samples for mRNA expression of CD3, CD8 and FOXP3for potential prognostic significance in terms of disease-free survival (DFS) and overall survival (OS). TILs were evaluated in whole sections as percent of total cells. Results: Median age was 52.7 years, while 54.2% of the patients were postmenopausal and 79.0% ER/PgR-positive. After a median follow-up of 133.0 months, 255 patients (30.9%) had died and 314 (38.0%) had disease progression. All three mRNA markers were positively correlated with TILs (Spearman's r=0.52 for CD3, 0.41 for CD8 and 0.47 for FOXP3, all p-values Conclusions: High CD3 and CD8 mRNA expression in early breast cancer patients is of prognostic value for decreased risk for relapse and, in the future, could potentially be of importance in deciding the most appropriate therapeutic strategy in light of the recent immune-related treatment developments. Citation Format: Tsiatas M, Kalogeras KT, Manousou K, Wirtz RM, Gogas H, Veltrup E, Zagouri F, Lazaridis G, Koutras A, Christodoulou C, Pentheroudakis G, Petraki C, Bafaloukos D, Pectasides D, Kosmidis P, Samantas E, Karanikiotis C, Papakostas P, Dimopoulos M-A, Fountzilas G. Evaluation of the prognostic value of CD3, CD8 and FOXP3 mRNA expression in early breast cancer patients treated with anthracycline-based adjuvant chemotherapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-07-03.

Background-aim: Metastatic breast cancer (MBC) is an incurable disease. Trastuzumab, a recombinant humanized monoclonal antibody, was found to significantly prolongsurvival of patients with metastatic HER2 over-expressing and/or amplified breast cancer. In the current study, we performed a complete analysis, with four different methods, of all four HER family receptors, in a series of patients with metastatic breast cancer treated with trastuzumab-based regimens and evaluated their prognostic value. Methods: Formalin-fixed paraffin-embedded tumor tissue samples were collected from 227 patients, considered to be HER2-positive when assessed at the local laboratories. We evaluated gene amplification, copy number variations (CNVs), transcriptional profiling and protein expression of all four HER family members. In addition, our analysis included the evaluation of several other factors by immunohistochemistry (IHC), such as pHER2Tyr1221/1222, pHER2Tyr877 and PTEN. Results: Central review of HER2 status by IHC and fluorescence in situ hybridization revealed that of the 227 patients, only 139 (61.2%) were truly HER2-positive. Regarding the 191 patients treated with trastuzumab as first-line therapy, median time to progression (TTP) was 15.3 months and 10.4 months for HER2-positive and HER2-negative participants, respectively. Median survival was 50.4 months for HER2-positive and 38.1 months for HER2-negative patients. In HER2-positive patients, high HER3 mRNA expression was of favorable prognostic significance for TTP and survival (HR=0.43, 95% CI 0.21-0.88, Wald's p=0.022 and HR=0.43, 95% CI 0.21-0.88, p=0.021, respectively), while EGFR copy gain and EGFR protein expression were associated with higher risk for disease progression in HER2-negative patients (HR=3.53, 95% CI 1.19-10.50, p=0.023 and HR=3.37, 95% CI 1.12-10.17, p=0.031, respectively). Positive HER3 protein expression was a favorable factor for TTP in HER2-negative patients (HR=0.43, 95% CI 0.22-0.84, p=0.014). In the multivariate analysis, only EGFR copy gain retained its prognostic significance for TTP in the HER2-negative population (HR=4.81, 95% CI 1.52-15.82, p=0.008), while none of the examined factorsretained their prognostic significance for TTP or survival in the HER2-positive subgroup. Conclusions: The present study suggests that EGFR copy gain represents a negative prognostic factor for TTP in HER2-negative patients with metastatic breast cancer. In addition, high HER3 mRNA expression appears to be of favorable prognostic significance for TTP and survival in HER2-positive patients. Given the small number of patients included in the current analysis and the retrospective nature of the study, our findings should be validated in larger cohorts. Citation Format: Koutras A, Kotoula V, Koliou G-A, Kouvatseas G, Christodoulou C, Pectasides D, Batistatou A, Bobos M, Tsolaki E, Papadopoulou K, Pentheroudakis G, Papakostas P, Pervana S, Petraki K, Chrisafi S, Razis E, Psyrri A, Bafaloukos D, Kalogeras KT, Kalofonos HP, Fountzilas G. Evaluation of the prognostic value of all four HER family receptors in patients with metastatic breast cancer treated with trastuzumab: A hellenic cooperative oncology group (HeCOG) study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-07-24.

Background Trastuzumab is a monoclonal antibody against HER2-positive breast cancer. Despite improving the natural history of the disease, there is a number of patients who are resistant to it, whereas all patients will eventually develop resistance and disease will progress. Inconsistent preclinical data show that the IGF-R pathway may contribute to either de novo or acquired resistance to trastuzumab. Materials and methods In total, 227 trastuzumab-treated metastatic breast cancer patients were evaluated for IGF-1, IGF-1R, GLP-1R, Akt1, Akt2 Akt3 mRNA expression, and IGF-1Rα, IGF-1Rβ, IGF-2R protein expression. Results Only 139 patients were truly HER2-positive by central assessment. Among HER2-positive patients, high Akt2 and GLP-1R mRNA expression showed a trend towards higher and lower risk of progression, respectively (HR=1.83, 95%CI=0.90-3.72, p=0.094 and HR=0.62, 95%CI=0.36-1.06, p=0.079), while high Akt1 and GLP-1R mRNA expression presented a trend towards unfavorable survival (HR=1.67, 95%CI=0.93-2.99, p=0.086 and HR=1.67, 95%CI=0.94-2.96, p=0.080). Among HER2-negative patients, high GLP-1R mRNA expression and negative stromal IGF-1Rβ protein expression showed a trend towards worse survival (HR=2.31, 95%CI=0.87-6.13, p=0.094 and HR=2.03, 95%CI=0.94-4.35, p=0.071, respectively). In the multivariate analyses, HER2-positive patients with high Akt1 and GLP-1R mRNA expression had a worse survival (HR=1.86, 95%CI=1.01-3.43, p=0.045 and HR=1.83, 95%CI=0.99-3.41, p=0.055, respectively). Conclusion This study revealed a crosstalk between the IGF-R pathway and HER2. There was evidence that high Akt1 and GLP-1R mRNA expression might affect survival among HER2-positive metastatic breast cancer patients treated with trastuzumab.

BACKGROUND: Prevention of bone metastases is a major issue for breast cancer patients, as it would improve quality of life in a population where long survival is anticipated. PATIENTS AND METHODS: Early breast cancer patients, who had been treated with anthracycline-based chemotherapy within two randomized trials, were included in the study. We evaluated, by quantitative reverse transcription–polymerase chain reaction, 819 formalin-fixed paraffin-embedded tumor tissue samples for mRNA expression of RANK, OPG, and RANKL, as well as their ratios, for potential prognostic significance for the development of bone metastases and also for disease-free survival (DFS) and overall survival. RESULTS: Median age was 52.7 years, whereas 54.2% of the patients were postmenopausal and 78.3% estrogen receptor/progesterone receptor positive. After a median follow-up of 119.9 months, 226 patients (27.6%) had died and 291 patients (35.5%) had disease progression. Low mRNA expression of RANKL was associated with postmenopausal status and greater number of positive lymph nodes (P = .002 and P

Background-aim: HER2-positive breast cancer (BC) features high rates of tumor infiltrating lymphocytes (TILs) and mutations (mut) in various genes, more frequently in TP53. We investigated associations between TILs and mutations in HER2-positive BC and their impact on patient outcome in early and metastatic BC (EBC and MBC, respectively), which remain largely unexplored. Methods:In 352 primary paraffin tumors from patients with HER2-positive disease, we examined amino acid changing mutations (20% variant frequency). Study groups were: (A) 218 EBC, including 117 patients treated with adjuvant chemotherapy only (CT) and 101 patients treated with CT and trastuzumab (CTT); (B) 134 MBC, including 95 patients who relapsed upon adjuvant CT without trastuzumab (R-MBC) and 39 patients who were first diagnosed with metastatic disease (de novo MBC). TILs were assessed as percentage of stromal tumor area. Clinical endpoints were disease-free survival in 5 years (5yr DFS) for EBC, and time-to-progression (TTP) from 1st line CTT treatment start for MBC. Results: 243/352 tumors (69%) carried at least one mut; 27/352 (8%) of tumors >10 up to 150 mut (hypermut); 192/352 (54%) at least one possibly clonal mut. Mean mut number and TP53 mut in particular were highest in R-MBC and lowest in EBC; mean TILs density followed the opposite pattern (all p3 compared to 0-3 positive nodes (OR 3.83, CI 1.76-8.34, p=0.001); lower risk for relapse was observed for higher TILs irrespectively of treatment (OR 0.93, CI 0.90-0.97, p=0.001), for TP53 mut (OR 0.39, CI 0.18-0.87, p=0.022) and for clonal TP53 mut in CTT-treated patients (OR 0.10, CI 0.02-0.58) but not in CT-treated patients (interaction p=0.084). The presence of any clonal mut (hazard ratio [HR] 2.77, CI 1.42-5.38) and of clonal TP53 mut (HR 2.24, CI 1.20-4.17) conferred worse TTP in de novo but not in R-MBC; these interactions remained significant upon multivariate analysis (interaction p=0.007 and p=0.061, respectively). Higher TILs in the absence of clonal mut conferred longer TTP (HR 0.75, CI 0.56-0.99) but no such effect was observed for tumors with clonal mut (multivariate interaction p=0.052). Classic independent predictors of unfavorable TTP in MBC were younger age (p=0.002), absence of hormone receptors (p=0.001) and poor performance status (p=0.044). PIK3CA mut did not remain significant in any of the examined settings. Conclusions: The expected pattern of higher TILs associated with mutation number and clonality was not observed in HER2-positive BC; the favorable effect of TILs only in the absence of clonal mut in MBC may imply exhausted immune response. Clonal TP53 mut may serve as a predictor for trastuzumab benefit in EBC but as an adverse prognosticator in trastuzumab-treated de novo MBC, which, if further validated, is of potential clinical relevance.Background-aim: HER2-positive breast cancer (BC) features high rates of tumor infiltrating lymphocytes (TILs) and mutations (mut) in various genes, more frequently in TP53. We investigated associations between TILs and mutations in HER2-positive BC and their impact on patient outcome in early and metastatic BC (EBC and MBC, respectively), which remain largely unexplored. Methods:In 352 primary paraffin tumors from patients with HER2-positive disease, we examined amino acid changing mutations (20% variant frequency). Study groups were: (A) 218 EBC, including 117 patients treated with adjuvant chemotherapy only (CT) and 101 patients treated with CT and trastuzumab (CTT); (B) 134 MBC, including 95 patients who relapsed upon adjuvant CT without trastuzumab (R-MBC) and 39 patients who were first diagnosed with metastatic disease (de novo MBC). TILs were assessed as percentage of stromal tumor area. Clinical endpoints were disease-free survival in 5 years (5yr DFS) for EBC, and time-to-progression (TTP) from 1st line CTT treatment start for MBC. Results: 243/352 tumors (69%) carried at least one mut; 27/352 (8%) of tumors >10 up to 150 mut (hypermut); 192/352 (54%) at least one possibly clonal mut. Mean mut number and TP53 mut in particular were highest in R-MBC and lowest in EBC; mean TILs density followed the opposite pattern (all p3 compared to 0-3 positive nodes (OR 3.83, CI 1.76-8.34, p=0.001); lower risk for relapse was observed for higher TILs irrespectively of treatment (OR 0.93, CI 0.90-0.97, p=0.001), for TP53 mut (OR 0.39, CI 0.18-0.87, p=0.022) and for clonal TP53 mut in CTT-treated patients (OR 0.10, CI 0.02-0.58) but not in CT-treated patients (interaction p=0.084). The presence of any clonal mut (hazard ratio [HR] 2.77, CI 1.42-5.38) and of clonal TP53 mut (HR 2.24, CI 1.20-4.17) conferred worse TTP in de novo but not in R-MBC; these interactions remained significant upon multivariate analysis (interaction p=0.007 and p=0.061, respectively). Higher TILs in the absence of clonal mut conferred longer TTP (HR 0.75, CI 0.56-0.99) but no such effect was observed for tumors with clonal mut (multivariate interaction p=0.052). Classic independent predictors of unfavorable TTP in MBC were younger age (p=0.002), absence of hormone receptors (p=0.001) and poor performance status (p=0.044). PIK3CA mut did not remain significant in any of the examined settings. Conclusions: The expected pattern of higher TILs associated with mutation number and clonality was not observed in HER2-positive BC; the favorable effect of TILs only in the absence of clonal mut in MBC may imply exhausted immune response. Clonal TP53 mut may serve as a predictor for trastuzumab benefit in EBC but as an adverse prognosticator in trastuzumab-treated de novo MBC, which, if further validated, is of potential clinical relevance. Citation Format: Kotoula V, Giannoulatou E, Kouvatseas G, Tikas I, Lazaridis G, Charalambous E, Efstratiou I, Bobos M, Tsolaki E, Zagouri F, Christodoulou C, Pentheroudakis G, Koutras A, Papakostas P, Kosmidis PA, Pectasides D, Fountzilas G. Mutation characteristics and tumor infiltrating lymphocytes in early and metastatic HER2-positive breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-07.

Since syndecan-1 is an adhesion molecule involved in tumor invasion and metastasis, we evaluated the relationship between syndecan-1 expression and histopathological features of gastric carcinomas.Syndecan-1 expression was evaluated in 104 gastric carcinomas using immunohistochemistry.High, moderate and low syndecan-1 expression in carcinoma cells was observed in 17/104, 25/104 and 62/104 cases, respectively. High, moderate and low syndecan-1 expression in stromal cells was observed in 5/104, 22/104 and 77/104 cases, respectively. Low epithelial syndecan-1 expression was significantly associated with increased depth of invasion (p=0.034) and lymph vessel invasion (p=0.035). Low stromal syndecan-1 expression was significantly associated with histologic type (intestinal vs diffuse/mixed; p=0.04), increased histologic grade (p=0.04) and large tumor size (p=0.026).Low levels of tumor and stromal syndecan- 1 expression were associated with adverse histopathological parameters in gastric carcinoma. This suggests that syndecan-1 expression may be helpful for assessing the aggressiveness of gastric carcinomas.

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%, χ2, 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.

Background/aim Early-stage colorectal cancer (CRC) carries a wide range of survival probabilities. Novel biomarkers in this setting are eagerly awaited. Cancer stem cells (CSCs) are considered one of the reasons for treatment failure. This study sought to determine whether activation of pathways governing the function of CSC's could correlate with treatment outcomes. Materials and methods Tumor specimens from 325 patients were analyzed with immunohistochemistry (IHC) for Hedgehog and Notch pathway activation and results were correlated with prognosis. Results Positive Notch3 protein expression was an unfavorable prognostic factor for disease-free survival (DFS) and overall survival (OS) (HR=2.43, p=0.024 and HR=2.56, p=0.028, respectively). Activation of the Shh pathway showed univariately longer DFS (HR=0.49, p=0.032). Possible crosstalk between the two pathways was indicated. No further associations between pathway activation and outcome were evident. Conclusion Apart from Notch 3, activation of the pathways, as indicated by IHC expression of their components, did not result in differences in terms of DFS or OS.

There is discrepancy and failure to adhere to current international guidelines for the management of metastatic colorectal cancer (CRC) in hospitals in Greece and Cyprus. The aim of the present document is to provide a consensus on the multidisciplinary management of metastastic CRC, considering both special characteristics of our Healthcare System and international guidelines. Following discussion and online communication among the members of an executive team chosen by the Hellenic Society of Medical Oncology (HeSMO), a consensus for metastastic CRC disease was developed. Statements were subjected to the Delphi methodology on two voting rounds by invited multidisciplinary international experts on CRC. Statements reaching level of agreement by ≥80% were considered as having achieved large consensus, whereas statements reaching 60-80% moderate consensus. One hundred and nine statements were developed. Ninety experts voted for those statements. The median rate of abstain per statement was 18.5% (range: 0-54%). In the end of the process, all statements achieved a large consensus. The importance of centralization, care by a multidisciplinary team, adherence to guidelines, and personalization is emphasized. R0 resection is the only intervention that may offer substantial improvement in the oncological outcomes.

Background-Aim: There is an unmet need for de-selecting HER2-positive patients with advanced breast cancer (ABC), since only some of those patients benefit from the addition of anti-HER2 agents to chemotherapy. The aim of this study was to investigate candidate biomarkers, including MYC and MET, in parallel with an extended array of biomarkers previously associated with trastuzumab (T) resistance. Patients and Methods: Two hundred and twenty-nine ABC patients treated with T and chemotherapy over a period of 13 years were included in the study. Paraffin tumors were retrospectively centrally assessed with immunohistochemistry (IHC) for breast cancer subtypes; fluorescence in situ hybridization (FISH) for HER2, TOP2A and centromere (CEN) 17, MYC and CEN8, MET and CEN7; qPCR for MYC and MET copy number (CN); and, for PI3K activation (PIK3CA mutations, PTEN and phospho-mTOR IHC). Patterns of CEN CN aberrations corresponding to chromosome "polysomy" were also evaluated, with cut-offs based on normal tissue. Time to progression (TTP) and survival were evaluated from the initiation of T as first-line treatment. Results: Median follow-up was 70 months. Of the 229 patients treated with T as HER2-positive, central analysis identified 90 cases being HER2-negative, as per current guidelines (39.3% of the total cohort). HER2-positive patients showed a trend for survival benefit over HER2-negative patients (median 50.7 vs. 38.1 months, respectively, p=0.118). HER2-positive tumors were subtyped as Luminal-HER2 (n=77) and HER2-enriched (n=53); 156 patients presented with ABC and 65 with disease initially diagnosed at stage IV (de novo ABC). MET and MYC CN gains (≥2.5 copies) were found in 40 (25%) and 15 (9%) cases with qPCR, while MET and MYC amplification with FISH was present in 4 (2.5%) and 31 (18%) cases, respectively. Concordance between FISH and qPCR was low for MYC (kappa value 0.46) and absent for MET. Polysomy was collectively observed in 70 cases, in 54 of them (32% of all tumors) concerning any 1 of the 3 examined chromosomes. This condition, called restricted polysomy, interacted with ABC presentation, conferring decreased survival to patients with ABC (HR=2.32, 95% CI 1.43-3.76, Wald's p=0.001) but not to those with de novo ABC (interaction p=0.077). MYC CN gain was the only marker significantly associated with increased risk for progression (HR=3.22, 95% CI 1.66-6.24, p Conclusions: MYC CN gain is a strong unfavorable prognosticator in T-treated ABC patients. Distinguishing between HER2-positive subtypes seems important for identifing T benefit in ABC. Chromosomal polysomy may distinctly affect T benefit in patients with pre-treated and de novo ABC. Citation Format: Gogas H, Kotoula V, Alexopoulou Z, Christodoulou C, Kostopoulos I, Bobos M, Raptou G, Charalambous E, Tsolaki E, Xanthakis I, Pentheroudakis G, Koutras A, Bafaloukos D, Papakostas P, Aravantinos G, Psyrri A, Petraki K, Kalogeras KT, Fountzilas G, Pectasides D. Genomic parameters affecting the outcome of patients with advanced breast cancer treated with trastuzumab. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-14-05.

Several studies support an important role of angiogenesis in breast cancer growth and metastasis. The main objectives of the study were to investigate the immunohistochemical expression of vascular endothelial growth factor (VEGF) family ligands (VEGF-A and VEGF-C) and receptors (VEGFR1, VEGFR2 and VEGFR3) in breast cancer and their associations with clinicopathological parameters, cancer subtypes/subgroups and patient outcome. Formalin-fixed paraffin-embedded tumor tissue samples were collected from early-stage breast cancer patients treated with anthracycline-based chemotherapy within a randomized trial. Immunohistochemistry was performed on serial 2.5 μm thick tissue sections from tissue microarray blocks. High VEGF-A, VEGF-C, VEGFR1, VEGFR2 and VEGFR3 protein expression was observed in 11.8% (N = 87), 80.8% (N = 585), 28.1% (N = 202), 64.6% (N = 359) and 71.8% (N = 517) of the cases, respectively. Significant associations were observed among all proteins (all p-values

Background The retinoblastoma (RB) gene is a tumor-suppressor gene that plays a central role in regulating the cell cycle. Inactivation of this gene is involved in breast cancer. Patients and methods A total of 827 patients with breast cancer treated with taxane-based adjuvant chemotherapy were included in the study. Protein expression of RB, phosphorylated RB (pRB), p16, cyclin D1 and p53 was evaluated by immunohistochemistry. Results Neither of the retinoblastoma markers (RB and pRB) reached statistical significance in terms of their association with disease-free or overall survival. Nevertheless, when clustering analysis was performed, patients with tumors featuring low levels of p16, cyclin D1 and p53 with concomitantly high levels of pRB had reduced risk for relapse (Wald's p=0.015). Conclusion The p53-mediated sensitivity of breast cancer cells to chemotherapeutic agents appears to be driven mostly by pRB. Using agents that enhance RB phosphorylation might possibly increase the chemosensitivity of breast cancer cells.

Background: Epidermal growth factor receptor (EGFR) aberrations have been implicated in the pathogenesis of triple-negative breast cancer (TNBC) but their impact on prognosis and, therefore, druggability, remain controversial. Herein, we studied EGFR aberrations at different molecular levels and assessed their prognostic impact in patients with operable TNBC treated with adjuvant anthracycline-based chemotherapy. Materials and Methods: We evaluated the prognostic impact of EGFR gene status by fluorescent in situ hybridization (FISH), EGFR coding mutations by Sanger and next-generation sequencing, relative EGFR messenger RNA (mRNA) levels by qPCR (upper quartile) and EGFR and p53 protein expression by immunohistochemistry (IHC), in 352 centrally-assessed tumors from an equal number of TNBC patients. Results: Approximately 53.5% of the tumors expressed EGFR, 59.3% p53 and 35.9% both EGFR and p53 proteins; 4.1% showed EGFR gene amplification and 4.4% carried EGFR mutations. The latter were located outside the druggable kinase domain region and presented at low frequencies. Amplification and mutations overlapped only in one case of glycogen-rich carcinoma. EGFR and CEN7 copies were higher in tumors from older patients (p=0.002 and p=0.003, respectively). Patients with amplified tumors (n=11) had excellent prognosis (0 relapses and deaths). Upon multivariate analysis, high EGFR copies conferred significantly favorable disease-free survival (HR=0.57, 95% CI 0.36-0.90, Wald’s p=0.017) and high CEN7 copies favorable overall survival (HR=0.49, 95% CI=0.29-0.83, Wald’s p=0.008). Patients with EGFR–/p53+ and EGFR+/p53– tumors had significantly higher risk for relapse than those with EGFR–/p53– and EGFR+/p53+ tumors (HR=1.73, 95% CI=1.12-2.67, Wald’s p=0.013). Conclusion: EGFR gene amplification and mutations are rare in TNBC, the latter of no apparent clinical relevance. Surrogate markers of EGFR-related chromosomal aberrations and combined EGFR/p53 IHC phenotypes appear to be associated with favorable prognosis in patients with operable TNBC receiving conventional adjuvant chemotherapy.

Background The principles of dose-dense (dd) and sequential (s) chemotherapy (CT) were introduced in the adjuvant setting by HeCOG in 1997. We aimed to explore the long-term clinical benefit and safety of treatment of adjuvant dds-CT in patients (pts) with high-risk BC. Methods We performed a pooled analysis from 6 HeCOG-initiated clinical trials (3 randomized and 3 observational). Patients with operable, early-stage breast cancer received adjuvant treatment with epirubicin, taxane and cyclophosphamide/methotrexate/ fluorouracil (CMF), except from 297 patients included in one study who did not receive taxanes. Granulocyte colony-stimulating factor was given prophylactically with all treatment regimens. The primary study endpoint was 10-year disease-free survival (DFS) rate. Results Between 06/1997 and 04/2013, 4,767 pooled pts were treated with dds-CT (median age 52.9, range 20.9-82.7, postmenopausal: 54%). Patients presented with Hormone receptor (HR)-positive/HER2-negative tumors (56%), HER2-positive tumors (29%) or triple negative breast cancer (TNBC, 15%). Overall, 92.2% of pts completed CT according to study protocol. Grade 4 adverse events were recorded in 356 (8%) pts, mostly neutropenia (81%). Grade 3-4 cardiac adverse events were reported in 6 (0.1%) pts. Six pts succumbed from toxicity during CT including febrile neutropenia (2 pts), acute myocardial infarction (1 pt), infection (1 pt), pulmonary embolism (1 pt) and acute respiratory failure (1 pt). In total, 134 (3%) pts were diagnosed with a secondary cancer; 126 (2.8%) with solid tumors and 16 (0.4%) with a hematologic malignancy. Median follow-up time was 8.9 years (95% CI 8.8-9.1). The 10-year DFS rate was 76% for pts with HR-positive/HER2-negative tumors, 73% with TNBC and 74% for those with HER2-positive tumors (62% and 82% in the pre- and post-trastuzumab era, respectively (p Conclusions dds-CT with epirubicin, taxane and CMF in the adjuvant setting was well-tolerated and was associated with favorable clinical outcomes. Legal entity responsible for the study Hellenic Cooperative Oncology Group (HeCOG). Funding Hellenic Cooperative Oncology Group (HeCOG). Disclosure E. Fountzilas: Travel / Accommodation / Expenses: K.A.M ONCOLOGY / HEMATOLOGY; Travel / Accommodation / Expenses: Merck; Shareholder / Stockholder / Stock options: Deciphera. G. Pentheroudakis: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Leadership role, Research grant / Funding (institution): Roche; Advisory / Consultancy, Leadership role, Research grant / Funding (institution): Amgen; Leadership role, Research grant / Funding (institution): Boehringer; Advisory / Consultancy, Leadership role, Research grant / Funding (institution): Merck; Advisory / Consultancy, Leadership role, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy, Leadership role, Research grant / Funding (institution): Lilly; Advisory / Consultancy, Leadership role: Bristol-Myers Squibb; Advisory / Consultancy: MSD; Leadership role: Enorasis. C. Christodoulou: Advisory / Consultancy: Merck; Advisory / Consultancy: Genesis Pharmaceuticals; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy: Roche; Advisory / Consultancy, Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy: Bristol-Myers Squibb; Travel / Accommodation / Expenses: Sanofi. A. Koutras: Advisory / Consultancy: Roche; Advisory / Consultancy, Travel / Accommodation / Expenses: Genesis; Advisory / Consultancy: AstraZeneca; Travel / Accommodation / Expenses: Novartis; Travel / Accommodation / Expenses: Bristol-Myers Squibb; Travel / Accommodation / Expenses: Pfizer; Travel / Accommodation / Expenses: Merck Serono; Travel / Accommodation / Expenses: Astellas; Travel / Accommodation / Expenses: Amgen. E. Samantas: Advisory / Consultancy: Merck; Advisory / Consultancy: MSD; Advisory / Consultancy: Asta-Zeneca; Advisory / Consultancy: Roche; Advisory / Consultancy: Amgen; Advisory / Consultancy: Genesis. G. Aravantinos: Advisory / Consultancy: Novartis; Advisory / Consultancy: BMS; Advisory / Consultancy: Roche Hellas; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Amgen; Advisory / Consultancy: Genesis Pharma; Advisory / Consultancy: Merck; Advisory / Consultancy: Pfizer. P. Papakostas: Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Merck; Advisory / Consultancy: Genesis Pharmaceuticals. A. Psyrri: Honoraria (institution), Advisory / Consultancy: Amgen; Honoraria (institution), Advisory / Consultancy: Merck Serono; Honoraria (institution), Advisory / Consultancy: Roche; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): BMS; Honoraria (institution), Advisory / Consultancy: AstraZeneca; Honoraria (institution), Advisory / Consultancy: MSD; Research grant / Funding (institution): Kura. P.A. Kosmidis: Honoraria (self): Novartis; Honoraria (self), Travel / Accommodation / Expenses: MSD; Honoraria (self), Travel / Accommodation / Expenses: Pfizer; Travel / Accommodation / Expenses: Genesis. A. Koumarianou: Advisory / Consultancy: Genesis Pharma; Honoraria (self): Pfizer; Speaker Bureau / Expert testimony: Roche; Research grant / Funding (self): Merck; Travel / Accommodation / Expenses: MSD. E. Razis: Honoraria (self), Advisory / Consultancy: Novartis; Travel / Accommodation / Expenses: Genesis Pharmaceuticals; Travel / Accommodation / Expenses: Pfizer; Travel / Accommodation / Expenses: Roche; Travel / Accommodation / Expenses: Bristol-Myers Squibb; Travel / Accommodation / Expenses: Genekor. H. Gogas: Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Bristol-Myers Squibb; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): MSD Oncology; Honoraria (institution), Advisory / Consultancy: Amgen; Honoraria (institution), Advisory / Consultancy: Novartis; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Advisory / Consultancy: Pierre-Fabre. G. Fountzilas: Advisory / Consultancy: Pfizer; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Roche; Honoraria (self): AstraZeneca. All other authors have declared no conflicts of interest.

In the current study, we performed a complete analysis, with four different methods, of all four HER family receptors, in a series of patients with metastatic breast cancer treated with trastuzumab-based regimens and evaluated their prognostic value. Formalin-fixed paraffin-embedded tumor tissue samples were collected from 227 patients, considered to be HER2-positive when assessed at the local laboratories. We evaluated gene amplification, copy number variations (CNVs), mRNA and protein expression of all four HER family members. In addition, our analysis included the evaluation of several other factors by immunohistochemistry (IHC), such as pHER2Tyr1221/1222, pHER2Tyr877 and PTEN. Central review of HER2 status by IHC and fluorescence in situ hybridization revealed that of the 227 patients, only 139 (61.2%) were truly HER2-positive. Regarding the 191 patients treated with trastuzumab as first-line therapy, median time to progression (TTP) was 15.3 and 10.4 months for HER2-positive and HER2-negative participants, respectively, whereas median survival was 50.4 and 38.1 months, respectively. In HER2-positive patients, high HER3 mRNA expression was of favorable prognostic significance for TTP and survival (HR = 0.43, 95% CI 0.21-0.88, Wald's p = 0.022 and HR = 0.43, 95% CI 0.21-0.88, p = 0.021, respectively), while EGFR copy gain and EGFR protein expression were associated with higher risk for disease progression in HER2-negative patients (HR = 3.53, 95% CI 1.19-10.50, p = 0.023 and HR = 3.37, 95% CI 1.12-10.17, p = 0.031, respectively). Positive HER3 protein expression was a favorable factor for TTP in HER2-negative patients (HR = 0.43, 95% CI 0.22-0.84, p = 0.014). In the multivariate analysis, only EGFR copy gain retained its prognostic significance for TTP in the HER2-negative population (HR = 3.96, 95% CI 1.29-12.16, p = 0.016), while high HER3 mRNA expression retained its favorable prognostic significance for TTP in the HER2-positive subgroup (HR = 0.47, 95% CI 0.23-0.99, p = 0.048). The present study suggests that EGFR copy gain represents a negative prognostic factor for TTP in HER2-negative patients with metastatic breast cancer treated with trastuzumab. In addition, high HER3 mRNA expression appears to be of favorable prognostic significance for TTP in HER2-positive patients. Given the small number of patients included in the current analysis and the retrospective nature of the study, our findings should be validated in larger cohorts.

Background The shift towards an earlier diagnosis of breast cancer (BC) highlights the need for biomarkers that would identify patients at risk for relapse and metastatic spread and indicate the potential value of additional treatment strategies. Osteopontin (OPN) is a matricellular protein that has been suggested to be a potential biomarker in BC. In the present study, we used archived BC patient samples to assess the clinical utility of OPN. Methods Formalin-fixed paraffin-embedded tumor tissue samples from 975 patients were collected from two large phase III randomized adjuvant chemotherapy trials (HE10/97 and HE10/00) that included patients with high risk BC. All tissue samples were assessed for ER, PgR, Ki67 and HER2 protein expression. OPN protein and mRNA expression was evaluated using immunohistochemistry and quantitative reverse transcription-polymerase chain reaction, respectively. Results OPN mRNA expression data were available for 814 patients, whereas OPN protein expression data were available for 546 patients. The majority of patients were ER/PgR-positive (78.3%), HER2-negative (76.5%) and Ki67-positive (55.2%) and had received adjuvant radiation therapy (76.8%) and hormonal therapy (81.1%). OPN mRNA expression was significantly associated with age (60.9% in high OPN tumors vs. 54.1% in low OPN tumors, p = 0.047), ER/PgR-negative status (25.7 vs. 17.2%, p = 0.004) and BC subtypes (p = 0.021). In addition, high OPN mRNA expression was significantly associated with reduced DFS (HR 1.26, 95% CI 1.00–1.59, Wald’s p = 0.050) and OS (HR 1.37, 95% CI 1.05–1.78, p = 0.019), while it retained its prognostic significance for both DFS (HR 1.39, 95% CI 1.10–1.77, p = 0.007) and OS (HR 1.54, 95% CI 1.61–2.05, p = 0.003) in the multivariate analysis. Conclusions We showed that high OPN mRNA expression is associated with decreased DFS and OS in a large cohort of BC patients treated with adjuvant chemotherapy in a clinical trial setting. Our results suggest that OPN may serve as a prognostic factor and a potential target for therapy. Trial registration Australian New Zealand Clinical Trials Registry; HE10/97 ACTRN12611000506998; HE10/00 ACTRN12609001036202

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.

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

Gastric cancer is a relatively common malignancy. Recently, the presence of the human epidermal growth factor receptor 2 (HER2) was identified as a molecular target in a proportion of patients who benefited from the addition of appropriate anti-HER2 treatments. Our study explored the clinical and prognostic role of known HER family members, human epidermal growth factor receptor 1 (EGFR or HER1), HER2, HER3 and HER4.Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 249 gastric cancer patients were studied by immunohistochemistry for protein expression of EGFR, HER2, HER3 and HER4.Of the 249 evaluable patients, 32 did not have complete data of treatment details and/or follow-up and were excluded from the survival analyses. Of the 217 patients with complete treatment and follow-up data, 178 were operated and treated for early disease (group 1), while 39 for advanced disease (group 2). The frequency of positive EGFR, HER2, HER3 and HER4 protein expression in all patients was 17.5%, 11.8%, 14.8% and 32.9%, respectively. There were no differences in protein expression of any of the markers between the two groups. There were, however, statistically significant associations between HER4 and all other HER family members, as well as between HER2 and HER3 expression. Of note, EGFR-positive membranous protein expression was significantly associated with the presence of lymphovascular invasion (p=0.027) and HER3 and HER4 negative cytoplasmic protein expression with well/moderately-differentiated tumors (p=0.030 and p=0.014, respectively). None of the HER family members were of prognostic value for OS in univariate analysis.The present study confirmed the known protein expression frequencies of HER family members in gastric cancer in a Greek population. Several associations were observed among the HER family members and between clinicopathological characteristics and HER family members. Further research is needed on their exact prognostic role, as well as their therapeutic targeting.

Background-Aim Early breast cancer is a heterogeneous disease, and, therefore, prognostic tools have been developed to evaluate the risk for distant recurrence. In the present study, we sought to develop a risk for recurrence score (RRS) based on mRNA expression of three proliferation markers in high-risk early breast cancer patients and evaluate its ability to predict risk for relapse and death. In addition the Adjuvant! Online score (AOS) was also determined for each patient, providing a 10-year estimate of relapse and mortality risk. We then evaluated whether RRS or AOS might possibly improve the prognostic information of the clinical treatment score (CTS), a model derived from clinicopathological variables. Methods A total of 1,681 patients, enrolled in two prospective phase III trials, were treated with anthracycline-based adjuvant chemotherapy. Sufficient RNA was extracted from 875 samples followed by multiplex quantitative reverse transcription-polymerase chain reaction for assessing RACGAP1, TOP2A and Ki67 mRNA expression. The CTS, slightly modified to fit our cohort, integrated the prognostic information from age, nodal status, tumor size, histological grade and treatment. Patients were also classified to breast cancer subtypes defined by immunohistochemistry. Likelihood ratio (LR) tests and concordance indices were used to estimate the relative increase in the amount of information provided when either RRS or AOS is added to CTS. Results The optimal RRS, in terms of disease-free survival (DFS) and overall survival (OS), was based on the co-expression of two of the three evaluated genes (RACGAP1 and TOP2A). CTS was prognostic for DFS (p3 positive nodes (LR-Δχ2 23.9, p3 positive nodes.

In rectal cancer management, accurate staging by magnetic resonance imaging, neo-adjuvant treatment with the use of radiotherapy, and total mesorectal excision have resulted in remarkable improvement in the oncological outcomes. However, there is substantial discrepancy in the therapeutic approach and failure to adhere to international guidelines among different Greek-Cypriot hospitals. The present guidelines aim to aid the multidisciplinary management of rectal cancer, considering both the local special characteristics of our healthcare system and the international relevant agreements (ESMO, EURECCA). Following background discussion and online communication sessions for feedback among the members of an executive team, a consensus rectal cancer management was obtained. Statements were subjected to the Delphi methodology voting system on two rounds to achieve further consensus by invited multidisciplinary international experts on colorectal cancer. Statements were considered of high, moderate or low consensus if they were voted by ≥80%, 60-80%, or