Despina Televantou

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.

Volumetric and MGMT parameters in glioblastoma patients: Survival analysis

BackgroundIn this study several tumor-related volumes were assessed by means of a computer-based application and a survival analysis was conducted to evaluate the prognostic significance of pre- and postoperative volumetric data in patients harboring glioblastomas. In addition, MGMT (O6-methylguanine methyltransferase) related parameters were compared with those of volumetry in order to observe possible relevance of this molecule in tumor development.MethodsWe prospectively analyzed 65 patients suffering from glioblastoma (GBM) who underwent radiotherapy with concomitant adjuvant temozolomide. For the purpose of volumetry T1 and T2-weighted magnetic resonance (MR) sequences were used, acquired both pre- and postoperatively (pre-radiochemotherapy). The volumes measured on preoperative MR images were necrosis, enhancing tumor and edema (including the tumor) and on postoperative ones, net-enhancing tumor. Age, sex, performance status (PS) and type of operation were also included in the multivariate analysis. MGMT was assessed for promoter methylation with Multiplex Ligation-dependent Probe Amplification (MLPA), for RNA expression with real time PCR, and for protein expression with immunohistochemistry in a total of 44 cases with available histologic material.ResultsIn the multivariate analysis a negative impact was shown for pre-radiochemotherapy net-enhancing tumor on the overall survival (OS) (p = 0.023) and for preoperative necrosis on progression-free survival (PFS) (p = 0.030). Furthermore, the multivariate analysis confirmed the importance of PS in PFS and OS of patients. MGMT promoter methylation was observed in 13/23 (43.5%) evaluable tumors; complete methylation was observed in 3/13 methylated tumors only. High rate of MGMT protein positivity (> 20% positive neoplastic nuclei) was inversely associated with pre-operative tumor necrosis (p = 0.021).ConclusionsOur findings implicate that volumetric parameters may have a significant role in the prognosis of GBM patients. Furthermore, volumetry could help not only to improve the prediction of outcome but also the outcome itself by identifying patients at high risk of treatment failure and, thus, seek alternative treatment for these patients. In this small series, MGMT protein was associated with less aggressive tumor characteristics.

Differential Response of Immunohistochemically Defined Breast Cancer Subtypes to Anthracycline-Based Adjuvant Chemotherapy with or without Paclitaxel

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

Improved outcome of high-risk early HER2 positive breast cancer with high CXCL13-CXCR5 messenger RNA expression.

UNLABELLED The CXCL13-CXCR5 is a chemokine axis that is activated in some breast cancers. A total of 321 tissue blocks from a group of patients who received adjuvant, dose-dense chemotherapy for high-risk early breast cancer were examined. Activation of this axis was found to be associated with determinants of poor prognosis but also with improved outcome in the human epidermal growth factor receptor 2 overexpressing subpopulation. BACKGROUND Chemokines are important in cell migration and are thought to play a key role in metastasis. We explored the prognostic significance of C-X-C ligand-motif (CXCL) 12, CXCL13, and receptor (CXCR) 5 on disease-free survival (DFS) and overall survival (OS) in early breast cancer. METHODS A total of 595 patients with high risk, [corrected] early breast cancer were treated in a 2-arm trial (HE10/97) with dose-dense sequential epirubicin followed by cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) with or without paclitaxel. RNA was extracted from 321 formalin-fixed paraffin-embedded primary tumor tissue samples and quantitative reverse-transcriptase polymerase chain reaction was used to assess messenger RNA (mRNA) expression of CXCL12, CXCL13, and CXCR5; estrogen receptor; progesterone receptor (PgR); microtubule-associated protein tau and human epidermal growth factor receptor 2 (HER2). RESULTS CXCL13 and CXCR5 were found to be negatively associated with estrogen receptor and microtubule-associated protein tau mRNA expression and with dense lymphocytic infiltration, and were positively associated with nuclear grade. Only CXCL13 was positively associated with HER2. Multivariate analysis revealed an association between high CXCL13 mRNA expression and improved DFS (hazard ratio [HR] 0.48 [95% CI, 0.25-0.90]; Wald, P = .023) but not OS; whereas high CXCL12 expression was significantly associated with increased OS (HR 0.53 [95% CI, 0.33-0.85]; Wald, P = .009). In the HER2 mRNA overexpressing subgroup, high CXCL13 mRNA expression was associated with improved DFS (P < .001), whereas high CXCR5 was associated with increased DFS and OS (P = .004 and P = .049, respectively). CONCLUSIONS The CXCL13-CXCR5 axis is associated with classic determinants of poor prognosis, such as high grade, hormone receptor negativity, and axillary node involvement. Interestingly, this chemokine axis seems to be strongly associated with improved outcome in patients with HER2(+) disease.

Evaluation of the prognostic role of centromere 17 gain and HER2/topoisomerase II alpha gene status and protein expression in patients with breast cancer treated with anthracycline-containing adjuvant chemotherapy: pooled analysis of two Hellenic Cooperative Oncology Group (HeCOG) phase III trials

BackgroundThe HER2 gene has been established as a valid biological marker for the treatment of breast cancer patients with trastuzumab and probably other agents, such as paclitaxel and anthracyclines. The TOP2A gene has been associated with response to anthracyclines. Limited information exists on the relationship of HER2/TOP2A gene status in the presence of centromere 17 (CEP17) gain with outcome of patients treated with anthracycline-containing adjuvant chemotherapy.MethodsFormalin-fixed paraffin-embedded tumor tissue samples from 1031 patients with high-risk operable breast cancer, enrolled in two consecutive phase III trials, were assessed in a central laboratory by fluorescence in situ hybridization for HER2/TOP2A gene amplification and CEP17 gain (CEP17 probe). Amplification of HER2 and TOP2A were defined as a gene/CEP17 ratio of >2.2 and ≥2.0, respectively, or gene copy number higher than 6. Additionally, HER2, TopoIIa, ER/PgR and Ki67 protein expression was assessed by immunohistochemistry (IHC) and patients were classified according to their IHC phenotype. Treatment consisted of epirubicin-based adjuvant chemotherapy followed by hormonal therapy and radiation, as indicated.ResultsHER2 amplification was found in 23.7% of the patients and TOP2A amplification in 10.1%. In total, 41.8% of HER2-amplified tumors demonstrated TOP2A co-amplification. The median (range) of HER2, TOP2A and CEP17 gain was 2.55 (0.70-45.15), 2.20 (0.70-26.15) and 2.00 (0.70-26.55), respectively. Forty percent of the tumors had CEP17 gain (51% of those with HER2 amplification). Adjusting for treatment groups in the Cox model, HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with time to relapse or time to death.ConclusionHER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with outcome in high-risk breast cancer patients treated with anthracycline-based adjuvant chemotherapy.Trial registrationAustralian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12611000506998 and ACTRN12609001036202

Prognostic significance of the Wnt pathway in squamous cell laryngeal cancer

OBJECTIVES We sought to determine the prognostic significance of the Wnt signaling pathway in operable squamous cell carcinoma of the larynx. MATERIALS AND METHODS In an annotated cohort of 289 operable laryngeal cancers we evaluated the prognostic impact of E-cadherin, P-cadherin and β-catenin protein expression with immunohistochemistry, as well as the mRNA expression of 7 key effectors of the Wnt pathway including secreted frizzled-related protein 4 (SFRP4), SNAI2 (SLUG) and WNT5A with qPCR (relative quantification [RQ]). RESULTS Using median immunoreactive scores as a pre-defined cut-off, patients whose tumors overexpressed both cytoplasmic E-cadherin and β-catenin experienced longer median OS as compared to those whose tumors overexpressed β-catenin only (median OS 124 vs. 72 months, p=0.0301) and patients whose tumors overexpressed both cytoplasmic and membranous E-cadherin experienced longer DFS as compared to those whose tumors overexpressed cytoplasmic E-cadherin only (median 118 vs. 91 months, p=0.0106). Upon hierarchical clustering of SFRP4, SNAI2 and WNT5A RQ values, profiles including co-expression of all 3 genes but also profiles with under-expression of SNAI2 and WNT5A were associated with worse outcome as compared to profiles not related to the Wnt pathway. In multivariate analysis, clustering was an independent predictor for DFS (p=0.0221) and OS (p=0.0077). CONCLUSION We identified gene expression profiles and IHC patterns associated with aberrant Wnt signaling conferring aggressive clinical behavior in operable squamous cell carcinoma of the larynx. Prospective validation of these results will determine whether targeting the Wnt pathway merits investigation in this disease.

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.

Expression of DNA repair and replication genes in non-small cell lung cancer (NSCLC): a role for thymidylate synthetase (TYMS)

BackgroundBRCA1 (B), ERCC1 (E), RRM1 (R) and TYMS (T) mRNA expression has been extensively studied with respect to NSCLC patient outcome upon various chemotherapy agents. However, these markers have not been introduced into clinical practice yet. One of the reasons seems to be lack of a standard approach for the classification of the reported high/low mRNA expression. The aim of this study was to determine the prognostic/predictive impact of B, E, R, T in routinely-treated NSCLC patients by taking into account the expression of these genes in the normal lung parenchyma.MethodsB, E, R, T mRNA expression was examined in 276 NSCLC samples (real-time PCR). The normal range of B, E, R, T transcript levels was first determined in matched tumor – normal pairs and then applied to the entire tumor series. Four main chemotherapy categories were examined: taxanes-without-platinum (Tax); platinum-without-taxanes (Plat); taxanes/platinum doublets (Tax/Plat); and, all-other combinations.ResultsIn comparison to remotely located normal lung parenchyma, B, E, R, T mRNA expression was generally increased in matched tumors, as well as in the entire tumor series. Therefore, tumors were classified as expressing normal or aberrant B, E, R, T mRNA. In general, no marker was associated with overall and progression free survival (OS, PFS). Upon multivariate analysis, aberrant intratumoral TYMS predicted for shorter PFS than normal TYMS in 1st line chemo-naïve treated patients (p = 0.012). In the same setting, specific interactions were observed for aberrant TYMS with Plat and Tax/Plat (p = 0.003 and p = 0.006, respectively). Corresponding patients had longer PFS in comparison to those treated with Tax (Plat: HR = 0.234, 95% CI:0.108-0.506, Wald’s p < 0.0001; Tax/Plat: HR = 0.242, 95% CI:0.131-0.447, Wald’s p < 0.0001). Similar results were obtained for PFS in 1st line chemo-naïve and (neo)adjuvant pre-treated patients. Adenocarcinoma, early disease stage, and treatment with Tax/Plat doublets independently predicted for prolonged OS in patients who received only one line of treatment (adjuvant or 1st line).ConclusionClassifying intratumoral B, E, R, T mRNA expression in comparison to normal lung may facilitate standardization of these parameters for prospective studies. With this approach, NSCLC patients with aberrant intratumoral TYMS expression will probably fare better with platinum-based treatments.

Abstract 5554: Tumor tissue dissection for gene expression assessments in translational research studies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background - aim: Translational research studies increasingly employ routine histologic material (FFPE) for the investigation of gene expression with extract-based methods, like quantitative real time PCR (QPCR). Statistically significant differences in the expression levels of mRNA transcripts in tumor vs neighbouring non-tumor tissues have led to the consensus of including as high as possible tumor cell rates in the extract. Herein, we investigated whether the same mRNA markers yield different clinically relevant values when examined in macrodissected (MD) and non-macrodissected (NMD) FFPE breast carcinoma tissues. Methods: Matched NMD and MD samples from primary tumors (P, n=98 sample pairs) and from metastatic lymph nodes (LN, n=72, overlapping but not matching P samples) were recruited from a cohort of 357 early high-risk breast cancer patients that had been adjuvantly treated with epirubicin-CMF regimens with or without paclitaxel. FFPE sections were evaluated for tumor cell content (TCC) and were used as whole sections and upon manual macrodissection for the same tumor. All samples were assessed for ESR1, ERBB2, MAPT, MMP7 and RACGAP1 mRNA expression with QPCR, and the obtained relative quantification (RQ) values were compared as continuous variables and upon hierarchical clustering in matched sample series with respect to patient outcome. Results: Mean, median (±SD) values for TCC% in P-NMD / P-MD samples were 27.0, 25.0 (±14.8) / 68.1, 67.5 (±20.5) and in LN-NMD / LN-MD samples 30.1, 35.0 (±18.0) / 82.0, 90.0 (±18.2), respectively. Minimum TCC% was 2.5 in P-NMD and in LN-NMD samples, 35.0 in P-MD and 27.5 in LN-MD. In comparison to matched NMD samples, mRNA expression of ESR1 (p<0.001) and MMP7 (p=0.016) was significantly lower in P-MD samples; MMP7 was also lower (p=0.006) but ESR1 (p=0.008), ERBB2 (p<0.001), MAPT (p=0.005) and RACGAP1 (p<0.001) were higher in LN-MD samples. A three-cluster approach yielded 68% concordance between P-NMD and P-MD, and 61% concordance between LN-NMD and LN-MD samples. Upon testing in each one of the four sample series, significant associations were observed for the above markers individually and in clusters with respect to patient disease-free and overall survival. However, the results obtained for P-NMD and LN-NMD samples did not differ from the ones in P-MD and in LN-MD samples, respectively. Conclusions: Although TCC% largely affects the obtained RQ values reflecting mRNA expression, the obtained results for ESR1, ERBB2, MAPT, MMP7 and RACGAP1 mRNA expression in MD samples did not practically add to, nor did they alter the evaluation of these markers with respect to patient outcome. For the five markers examined in this study, and probably for additional markers as well, the necessity of performing tissue macrodissection for enrichment in tumor cells in translational research and, potentially, diagnostics may need to be revisited. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5554. doi:1538-7445.AM2012-5554

Abstract 791: Low DARPP32 and related profiles in glioblastomas

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC DARPP32 (PPP1R1B, an inhibitor of protein phosphatase-1) plays a central role in dopamine signalling in the brain and has been associated with a number of diverse conditions, ranging from nicotine dependence to cancer drug resistance, while its overexpression has been reported as pro-oncogenic in various epithelial cancers. Since data on this molecule in astrocytic carcinogenesis are missing, this study focused on DARPP32 expression and related profiles in astrocytic gliomas. In a panel of 157 routinely diagnosed astrocytic glioma tissues we assessed (i) relative expression of DARPP32 along with 21 additional transcript targets from key regulators in signalling pathways commonly disturbed in glioblastomas (GBM), (ii) genomic status of selected genes and (iii) activation status of the Akt and Stat pathways. Normal brain tissues expressed DARPP32 at considerable levels. In comparison, the expression of this molecule was strongly decreased (low = decrease of >1 order of magnitude) according to histological grade, more frequently in grade IV (GBM, 89/116 [77%]) than in grade III (11/22 [50%]) and grade II (4/19 [21%]) tumors (p<0.0001). Low DARPP32 was associated with EGFR gene amplification (p=0.0006) and mRNA/protein overexpression; high pro-angiogenic VEGF isoform (p=0.0002) and hTERT expression (p=0.0008); low SRC/STAT expression (STAT3, 5A and 5B ps<0.0001) but common Stat3 (p=0.0003) and Akt/PKB phosphorylation (p=0.0036). Two specific Akt deactivating phosphatases, PHLPP2 and PHLPP1 were also identified to be downregulated/absent in GBM (63% and 23%, respectively), in line with their proposed tumor suppressor role. Strikingly, low expression of these two genes almost coincided with low DARPP32, with strongly decreased DARPP32/PHLPP2 and DARPP32/PHLPP2/PHLPP1 in 54% and 17.2% of GBMs, respectively. In 66 out of the 116 GBM patients with known follow-up, this latter profile was associated with a slightly better survival (log rank, p=0.0496); all corresponding tumors had activated Akt-Thr308 but lacked EGFR/EGFRvIII, MET, SRC, STATs, TERT (12/12), and VEGF isoform (8/12) overexpression, and 12q gene amplification (10/12). In conclusion, DARPP32 expression is strongly decreased in high grade astrocytic tumors where, in contrast to what has been described for common epithelial cancers, low DARPP32 seems to be related to pro-oncogenic and adverse prognostic parameters. This study also demonstrates that, except for PTEN, additional phosphatases regulating Akt signalling are commonly missing in GBM, mostly in parallel with low DARPP32. Further dissecting the molecular environment accompanying the decreased expression of these genes seems important for understanding the behaviour of this highly heterogeneous group of tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 791.