Eleni Galani

Biomarkers of benefit from cetuximab-based therapy in metastatic colorectal cancer: interaction of EGFR ligand expression with RAS/RAF, PIK3CA genotypes

BackgroundMore 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.MethodsPreviously 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.ResultsOnly 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).ConclusionsBRAF 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.

Vascular endothelial growth factor polymorphisms and clinical outcome in colorectal cancer patients treated with irinotecan-based chemotherapy and bevacizumab

The aim of the study was to evaluate the association of vascular endothelial growth factor (VEGF) genotypes with treatment efficacy in a randomized trial. This study compared two chemotherapy regimens (FOLFIRI versus XELIRI) combined with bevacizumab, as first-line treatment for metastatic colorectal cancer. DNA was extracted from blood samples of 173 patients participating in the trial. Genotyping was performed for selected SNPs (VEGF−1154, +936, −634, −2578 and −1498). All candidate genotypes were evaluated for associations with overall survival (OS), progression-free survival (PFS) and response rate (RR). There were no significant differences with respect to the distribution of genotypes in the treatment groups. The VEGF−1154 GG genotype was more frequent in patients not responding to treatment compared with responders (65.5 versus 39.8%, P=0.032). Furthermore, the VEGF−1154 GG genotype was associated with inferior median OS compared with GA (hazards ratio=1.68; 95% confidence interval: 1.10–2.57; P=0.016) or with the alternative genotypes (GA and AA) combined (hazards ratio=1.62; 95% confidence interval: 1.09–2.40; P=0.017). In multivariate analysis, the VEGF−1154 GG genotype remained a significant adverse factor for OS. Our results support the potential predictive ability of VEGF genotypes in patients with metastatic colorectal cancer receiving irinotecan-based chemotherapy plus bevacizumab, in terms of RR and OS. However, current results should be validated prospectively, in larger cohorts.

Tumors with high-density tumor infiltrating lymphocytes constitute a favorable entity in breast cancer: a pooled analysis of four prospective adjuvant trials

Background Tumor infiltrating lymphocytes (TILs) are considered in the prognosis of breast cancer (BC) patients. Here, we investigated the prognostic/predictive effect of TILs in patients treated in the frame of four prospective trials with adjuvant anthracycline-based chemotherapy in the pre- and post-trastuzumab era. Methods TILs density was histologically assessed as percentage of stromal area on whole routine sections of 2613 BC (1563 Luminal A/B; 477 Luminal HER2; 246 HER2-enriched; 327 triple negative [TNBC]) and were evaluated as high/low at three cut-offs (c/o; 50% [lymphocytic predominance, LP], 35% and 25%), in separate training and validation sets. Results High TILs were present in 3.5%, 6.5% and 11.5% of all tumors, using the 50%, 35% and 25% c/o, respectively. TILs status did not interact with BC subtypes or trastuzumab treatment. LPBC patient outcome was not affected by nodal status, while high TILs were favorable in TNBC with unfavorable nodal status. When adjusted for standard clinicopathological parameters and treatment, high TILs independently predicted for favorable outcome, e.g., disease-free survival with the 35% c/o in the entire cohort (HR = 0.44, 95% CI 0.28-0.69, p < 0.001) and in specific subtypes. Conclusions High TILs tumors, especially LPBC seem worthy validating as a separate entity of favorable prognosis in breast cancer.

Pegfilgrastim Administered on the Same Day with Dose-Dense Adjuvant Chemotherapy for Breast Cancer Is Associated with a Higher Incidence of Febrile Neutropenia as Compared to Conventional Growth Factor Support: Matched Case-Control Study of the Hellenic Cooperative Oncology Group

Objective: Recombinant human granulocyte-colony-stimulating factors such as filgrastim and pegfilgrastim have been employed as primary and secondary prophylaxis against neutropenia in cancer patients receiving chemotherapy. This study was conducted to evaluate the rate of febrile neutropenia in patients with high-risk early breast cancer receiving dose-dense chemotherapy and, as primary prophylaxis, either pegfilgrastim 6 mg fixed dose on the same day as chemotherapy or filgrastim on days 2–10 of each cycle. Secondary objectives included the rate of severe neutropenia, treatment delays and dose reductions. Methods: This was a nonrandomized matched case-control study with 214 patients receiving dose-dense chemotherapy. Each group receiving supportive therapy included 107 patients (pegfilgrastim and filgrastim groups). Results: Fourteen patients (13%) in the pegfilgrastim group developed febrile neutropenia as compared to 1 patient (1%) in the filgrastim group (p = 0.001). No statistically significant differences regarding the rate of severe neutropenia, treatment delays and dose reductions were observed. Conclusion: The results demonstrate that pegfilgrastim administered as primary prophylaxis on the same day as dose-dense chemotherapy is less efficacious than filgrastim administered on days 2–10 of each chemotherapy cycle. For the particular regimens given in this retrospective matched case-control study, the current recommendation for administering pegfilgrastim at least 24 h after chemotherapy completion seems justified. However, further randomized controlled trials are needed to clarify this finding.

The prognostic role of ephrin A2 and endothelial growth factor receptor pathway mediators in patients with advanced colorectal cancer treated with cetuximab.

BACKGROUND Patients with colorectal cancer (CRC) with wild-type KRAS mutations are often treated with the endothelial growth factor receptor (EGFR) monoclonal antibody cetuximab. Despite the presence of a specific molecular target, most patients still do not derive benefit from this biological treatment. Our study explores the role of ephrin A2 (EphA2) receptor expression and of EGFR pathway mediators as predictors of cetuximab benefit. PATIENTS AND METHODS Formalin-fixed paraffin-embedded (FFPE) tumor biopsy samples from 226 cetuximab-treated patients with CRC were studied for mRNA expression of insulin growth factor binding protein 2 (IGFBP2), insulin growth factor receptor 1 (IGF1R), cMET, EphA2, human epidermal growth factor receptor 2 (HER2), HER3, and HER4 by means of TaqMan reverse-transcribed polymerase chain reaction (RT-PCR). RESULTS Of the 226 patients evaluable for exploratory analysis, 222 had complete data from follow-up visits. The univariate analysis revealed the following significant adverse prognostic factors for risk of death: high EphA2 mRNA levels (hazard ratio [HR], 1.61; P = .015), high HER2 mRNA levels (HR, 1.51; P = .045), and high IGF1R mRNA levels (HR, 1.56; P = .021). Low EphA2 tumor expression was significantly associated with objective response to cetuximab therapy. In multivariate analysis of a broad biomarker panel, factors with independent prognostic value included EphA2 mRNA levels (HR, 1.67; P = .029), high amphiregulin (AREG) mRNA levels in KRAS wild-type tumors (HR, 0.17; P < .0001), and high epiregulin (EREG) mRNA levels (HR, 0.38; P = .006). CONCLUSION High EphA2 receptor expression in CRC was associated with a worse outcome in patients treated with cetuximab-based therapy. Prospective validation in treated and control patients is required to dissect the predictive from prognostic role in advanced CRC.

Immune response gene expression in colorectal cancer carries distinct prognostic implications according to tissue, stage and site: a prospective retrospective translational study in the context of a hellenic cooperative oncology group randomised trial.

Background Although host immune response is an emerging prognostic factor for colorectal cancer, there is no consensus on the optimal methodology, surrogate markers or tissue for study. Patients and Methods Tumour blocks were prospectively collected from 344 patients with stage II/III colorectal cancer (CRC) treated with adjuvant chemotherapy. Whole section lymphocytic infiltration was studied along with mRNA expression of CD3Z, CD8, CD4, CXCL9, CXCL13, IGHM, FOXP3, SNAI2 and ESR1 by qRT-qPCR in tissue microarray (TMA) cores from the centre of tumour, invasive margin and adjacent normal mucosa. Results Lymphocytic infiltration, deficient MMR (10.9%), KRAS (40.7%) and BRAF (4.9%) mutations or single mRNA gene expression were not prognostic. Tumour ESR1 gene expression (Hazard Ratio [HR] for relapse 2.33, 95% CI 1.35-4.02; HR for death 1.74, 95% CI 1.02-2.97) and absence of necrosis (HR for relapse 1.71, 95% CI 1.05-2.71; HR for death 1.98, 95% CI 1.14-3.43) were adverse prognostic features. We used CD3Z and CD8 expression in order to devise the mRNA-based Immune Score (mIS) and proceeded to partitioning analysis in 267 patients, with age, stage, tumour site (Right vs Left CRC), KRAS mutation and tumour mIS as input factors. Only in patients with stage III right-sided colon cancer, a low immune response was associated with inferior disease-free survival (mIS-low, HR for relapse 2.28, 95% CI 1.05-8.02). No prognostic significance was seen for tumour mIS in any other stage or site of CRC, or for a similar mIS score derived from adjacent normal mucosa. Independent adverse prognostic significance was retained in multivariable analysis for absence of necrosis, tumour ESR1 expression in all patients and low tumour mIS in stage III right-sided CRC. Conclusions In localised CRC, mRNA-based CD3Z/CD8 profiling of tumour immune response may have stage, site and tissue-specific prognostic significance, along with ESR1 expression. Trial Registration ANZCTR.org.au ACTRN12610000509066

Economic evaluation of taxane-based first-line chemotherapy in the treatment of patients with metastatic breast cancer in Greece: an analysis alongside a multicenter, randomized phase III clinical trial.

BACKGROUND An economic evaluation was undertaken alongside a randomized phase III trial comparing three regimens for metastatic breast cancer (MBC). MATERIALS AND METHODS Trial resource utilization and unit price data were combined to evaluate the cost of chemotherapy, concomitant medications, hospitalizations, diagnostic and laboratory tests. Treatment cost was combined with survival to estimate the incremental cost per life year saved. Quality-of-life data were used to estimate cost per quality-adjusted life year saved. Sensitivity analysis was used to compute results for various subgroups and for discounting cost and effects. RESULTS The combination of gemcitabine (Gemzar, Eli Lilly, Indianapolis, USA) with docetaxel (Taxotere, Aventis Pharma, Dagenham, UK) (GDoc) is the least costly but least effective treatment. The combination of paclitaxel (Taxol) with carboplatin (Paraplatin, Bristol-Myers Squibb, Princeton, USA) is associated with higher cost and effectiveness compared with GDoc, while weekly paclitaxel (Pw), associated with the highest cost, is the most effective option. The incremental cost per life year saved of Pw versus GDoc was 3660 Euros (95% uncertainty interval dominance-9261). This result remained fairly constant in sensitivity analysis. CONCLUSIONS The corresponding economic evaluation indicates that Pw represents an attractive treatment option for patients with MBC from an economic perspective in the context of the Greek National Health Service.

Differential Expression of the Insulin-Like Growth Factor Receptor among Early Breast Cancer Subtypes

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<0.001 for both comparisons). IGF2R was overexpressed significantly more frequently in HR negative tumors (p = 0.001) and had an inverse correlation with all other biomarkers. Patients with luminal A and B tumors with high IGF1R-alpha and negative EGFR expression (N = 190) had significantly higher 4-year survival rates, as compared to the rest (log-rank p = 0.046), as did patients with luminal A and B tumors with high IGF1R-alpha and low IGF2R expression, as compared to the rest (N = 91), (log-rank p = 0.035). After adjustment for significant variables, patients in the latter group had a relative 45% reduction in the risk of death, as compared to the rest (p = 0.035). Conclusion Aberrant expression of components of the IGF1R pathway is associated with better clinical outcomes in women with luminal A and B, node positive, early breast cancer.

566PPROGNOSTIC SIGNIFICANCE OF TUMOUR-ASSOCIATED IMMUNE RESPONSE GENE EXPRESSION, ESR1 AND CLINICOPATHOLOGIC PARAMETERS IN STAGE II/III COLORECTAL CANCER: A TRANSLATIONAL RESEARCH STUDY OF THE HELLENIC COOPERATIVE ONCOLOGY GROUP (HECOG)

ABSTRACT Aim: Tumour-associated immune response and colon cancer site emerge as parameters impacting on disease biology and patient outcome. Methods: mRNA gene expression of immune response (IR) genes (CD3Z, CD8, CXCL9, CXCL13, IGHM, FOXP3), SNAI2 and ESR1 were quantified by RTqPCR in formalin-fixed tumours of 408 oxaliplatin-treated patients with stage II/III colorectal cancer (trial ACTRN12610000509066). Results were submitted to hierarchical clustering and analysed. Results: We selected a mRNA gene signature (mRNA 2-cluster immunoscore, mIS2) based on CD3Z and CD8 expression (Cluster 2: upregulated CD3Z CD8 expression; Cluster 1: more heterogeneous but overall downregulated CD3Z CD8 expression). Partitioning analysis including age, stage, site, mIS2 and KRAS gene mutation, revealed that tumour stage, tumour site and mIS2 identified eight patient populations with distinct DFS (p MULTIVARIATE ANALYSIS HR for relapse P-value HR for death P-value STAGE_SITE_mIS2 0.0008 0.0008 Stage II Right mIS2-downreg 0.23 0.1702 0.17 0.09 Stage II Right mIS2-upreg 0.19 0.1188 0.00 0.98 Stage II Left mIS2-downreg 0.26 0.0466 0.22 0.02 Stage II Left mIS2-upreg 0.62 0.4803 0.46 0.25 Stage III Right mIS2-downreg 3.00 0.0152 2.62 0.02 Stage III Right mIS2-upreg 0.79 0.7355 1.50 0.43 Stage III Left mIS2-downreg 1.43 0.4001 1.04 0.91 Stage III Left mIS2-upreg 1 1 HIGH TUMOUR MRNA EXPRESSION OF ESR1 2.91 0.0003 1.95 0.02 PRESENCE OF NECROSIS 0.42 0.0068 0.39 0.002 Conclusions: The prognostic significance of tumour mRNA-based CD3 and CD8 immune response signature may be distinct in different stages and different sites of colon cancer. Disclosure: R. Wirtz: Stocks at Stratifyer Molecular Pathology, Cologne, Germany. All other authors have declared no conflicts of interest.

1497PLESSONS FROM THE PAST: LONG-TERM SAFETY AND EFFICACY OUTCOMES OF A PREMATURELY TERMINATED, RANDOMIZED PHASE III TRIAL OF PRECAUTIONARY VERSUS HEMOGLOBIN-BASED ERYTHROPOIETIN ADMINISTRATION FOR CHEMOTHERAPY-ASSOCIATED ANEMIA IN PATIENTS WITH SOLID TUMORS

ABSTRACT Aim: Prophylactic erythropoiesis-stimulating agent (ESA) administration for chemotherapy-induced anemia (CIA) is not supported by current guidelines. Long-term follow-up of patients that had been treated with EPO for CIA in the past may provide useful information. Methods: In 2002 we undertook a prospective, randomized phase III trial of prophylactic versus hemoglobin (Hgb)-based (threshold: 12 mg/dl) ESA administration in patients with solid tumors and CIA, which was permanently suspended in 2005 in the view of published data at that time, while patient surveillance continued. Herein we present safety and efficacy outcomes after more than a decade of follow-up. Results: Among 630 evaluable patients, 40.1% were male, 50.9% had advanced cancer at diagnosis, 39.2% had Hgb levels Conclusions: Prophylactic administration of ESA for CIA in patients with solid tumors was associated with increased incidence of a composite of thrombosis-related adverse events, especially in patients receiving adjuvant treatment, but did not have a detrimental impact on relapse/progression and survival rates. Disclosure: All authors have declared no conflicts of interest.