Annelies Debucquoy

Molecular Response to Cetuximab and Efficacy of Preoperative Cetuximab-Based Chemoradiation in Rectal Cancer

PURPOSE To characterize the molecular pathways activated or inhibited by cetuximab when combined with chemoradiotherapy (CRT) in rectal cancer and to identify molecular profiles and biomarkers that might improve patient selection for such treatments. PATIENTS AND METHODS Forty-one patients with rectal cancer (T3-4 and/or N+) received preoperative radiotherapy (1.8 Gy, 5 days/wk, 45 Gy) in combination with capecitabine and cetuximab (400 mg/m2 as initial dose 1 week before CRT followed by 250 mg/m2 /wk for 5 weeks). Biopsies and plasma samples were taken before treatment, after cetuximab but before CRT, and at the time of surgery. Proteomics and microarrays were used to monitor the molecular response to cetuximab and to identify profiles and biomarkers to predict treatment efficacy. RESULTS Cetuximab on its own downregulated genes involved in proliferation and invasion and upregulated inflammatory gene expression, with 16 genes being significantly influenced in microarray analysis. The decrease in proliferation was confirmed by immunohistochemistry for Ki67 (P = .01) and was accompanied by an increase in transforming growth factor-alpha in plasma samples (P < .001). Disease-free survival (DFS) was better in patients if epidermal growth factor receptor expression was upregulated in the tumor after the initial cetuximab dose (P = .02) and when fibro-inflammatory changes were present in the surgical specimen (P = .03). Microarray and proteomic profiles were predictive of DFS. CONCLUSION Our study showed that a single dose of cetuximab has a significant impact on the expression of genes involved in tumor proliferation and inflammation. We identified potential biomarkers that might predict response to cetuximab-based CRT.

T-Cell Responses to Survivin in Cancer Patients Undergoing Radiation Therapy

Purpose: The goal of this study was to determine if radiation therapy (RT) of human cancer enhances or diminishes tumor-specific T-cell reactivity. This is important if immunotherapy is to be harnessed to improve the outcome of cancer radiotherapy. Experimental Design: Lymphocytes were isolated from colorectal cancer (CRC) patients before, during, and after presurgical chemoradiotherapy. Similar samples were taken from prostate cancer patients receiving standard RT. The level of CD8+ T cells capable of binding tetramers for the tumor-associated antigen survivin, which is overexpressed in both cancer types, was enumerated in HLA-A*0201 patient samples. CD4+, CD25high, Foxp3+ cells were also enumerated to evaluate therapy-induced changes in Tregulatory cells. For CRC patients, most of whom were enrolled in a clinical trial, pathologic response data were available, as well as biopsy and resection specimens, which were stained for cytoplasmic and intranuclear survivin. Results: Survivin-specific CD8+ T lymphocytes were detected in the peripheral blood of CRC and prostate cancer patients and increased after therapy in some, but not all, patients. Increases were more common in CRC patients whose tumor was downstaged after chemoradiotherapy. Biopsy specimens from this cohort generally had higher nuclear to cytoplasmic survivin expression. Tregulatory cells generally increased in the circulation following therapy but only in CRC patients. Conclusion: This study indicates that RT may increase the likelihood of some cancer patients responding to immunotherapy and lays a basis for future investigations aimed at combining radiation and immunotherapy.

A new approach to the validation of tissue microarrays

Although tissue microarrays (TMA) have been widely used for a number of years, it is still not clear how many core biopsies should be taken to determine a reliable value for percentage positivity or how much heterogeneity in marker expression influences this number. The first aim of this study was to validate the human visual semi‐quantitative scoring system for positive staining of tumour tissue with the exact values determined from computer‐generated images. The second aim was to determine the minimum number of core biopsies needed to estimate percentage positivity reliably when the immunohistochemical staining pattern is heterogeneous and scored in a non‐binary way. Tissue sections from ten colorectal cancer specimens were stained for carbonic anhydrase IX (CA IX). The staining patterns were digitized and 400 artificial computer‐generated images were generated to test the accuracy of the human scoring system. To determine the minimal number of core biopsies needed to account for tumour heterogeneity, 50 (artificial) core biopsies per section were taken from the tumoural region of the ten digitally recorded full tissue sections. Based on the semi‐quantitative scores from the 50 core biopsies per section, 2500 × n (n = 1–10 core biopsies) experimental core biopsies were then generated and scores recorded. After comparison with field‐by‐field analysis from the tumoural region of the whole tissue section, the number of core biopsies that need to be taken to minimize the influence of heterogeneity could be determined. In conclusion, visual scoring accurately estimated the percentage positivity and the percentage tumour present in a section, as judged by comparison with the artificial images. The exact number of core biopsies that has to be examined to determine tumour marker positivity using TMAs is affected by the degree of heterogeneity in the expression pattern of the protein, but for most purposes at least four is recommended. Copyright

A kernel-based integration of genome-wide data for clinical decision support.

BackgroundAlthough microarray technology allows the investigation of the transcriptomic make-up of a tumor in one experiment, the transcriptome does not completely reflect the underlying biology due to alternative splicing, post-translational modifications, as well as the influence of pathological conditions (for example, cancer) on transcription and translation. This increases the importance of fusing more than one source of genome-wide data, such as the genome, transcriptome, proteome, and epigenome. The current increase in the amount of available omics data emphasizes the need for a methodological integration framework.MethodsWe propose a kernel-based approach for clinical decision support in which many genome-wide data sources are combined. Integration occurs within the patient domain at the level of kernel matrices before building the classifier. As supervised classification algorithm, a weighted least squares support vector machine is used. We apply this framework to two cancer cases, namely, a rectal cancer data set containing microarray and proteomics data and a prostate cancer data set containing microarray and genomics data. For both cases, multiple outcomes are predicted.ResultsFor the rectal cancer outcomes, the highest leave-one-out (LOO) areas under the receiver operating characteristic curves (AUC) were obtained when combining microarray and proteomics data gathered during therapy and ranged from 0.927 to 0.987. For prostate cancer, all four outcomes had a better LOO AUC when combining microarray and genomics data, ranging from 0.786 for recurrence to 0.987 for metastasis.ConclusionsFor both cancer sites the prediction of all outcomes improved when more than one genome-wide data set was considered. This suggests that integrating multiple genome-wide data sources increases the predictive performance of clinical decision support models. This emphasizes the need for comprehensive multi-modal data. We acknowledge that, in a first phase, this will substantially increase costs; however, this is a necessary investment to ultimately obtain cost-efficient models usable in patient tailored therapy.

Biomarkers for Cetuximab-Based Neoadjuvant Radiochemotherapy in Locally Advanced Rectal Cancer

Purpose: Phase II trials in locally advanced rectal cancer have shown that cetuximab-based neoadjuvant radiochemotherapy is feasible but without an improvement in complete pathologic response rates. Our goal was to identify patients who would benefit from cetuximab-based neoadjuvant chemoradiation measuring gene expression levels of proteins involved in tumor growth [endothelial growth factor receptor (EGFR)], angiogenesis [VEGF, VEGF receptors 1 and 2 (VEGFR1, VEGFR2)], DNA repair [excision repair cross-complementing 1 (ERCC1)], and drug metabolism [thymidylate synthetase (TS)]. We also determined mutation status of KRAS and BRAF. Experimental Design: This study was carried out on 130 patients with locally advanced rectal cancer who were enrolled in 4 different phase II clinical trials, using cetuximab-based chemoradiation. Tumor tissues were obtained before neoadjuvant and at surgical therapy. After microdissection, intratumoral gene expression levels and KRAS/BRAF mutation status were analyzed. Results: A significant decrease of TS, VEGFR1, and VEGFR2 gene expression was seen following neoadjuvant therapy (P < 0.03). High pretreatment VEGF gene expression levels were associated with nonresponse (P = 0.070). KRAS mutations were found in 42% and mutant KRAS (KRAS mt) was significantly associated with pathologic nonresponse (P = 0.037). In patients with wild-type KRAS (KRAS wt), low EGFR was significantly associated with higher nonresponse and VEGF mRNA expressions were associated with complete pathologic response (P = 0.012; P = 0.06). KRAS transversion (KRAS tv) was associated with tumor regression: nonresponse was more common in patients with KRAS tv than with KRAS wt (P = 0.007). BRAF V600E mutations were not detected in any of the patients. Conclusion: This study suggests that pretreatment intratumoral EGFR and VEGF mRNA expression levels as well as KRAS mutation status are predictive markers of pathologic response to neoadjuvant cetuximab-based chemoradiation in locally advanced rectal cancer. Clin Cancer Res; 17(10); 3469–77. ©2011 AACR.

EGF61 polymorphism predicts complete pathologic response to cetuximab-based chemoradiation independent of KRAS status in locally advanced rectal cancer patients

Background: Cetuximab has shown significant clinical activity in metastatic colon cancer. However, cetuximab-containing neoadjuvant chemoradiation has not been shown to improve tumor response in locally advanced rectal cancer patients in recent phase I/II trials. We evaluated functional germline polymorphisms of genes involved in epidermal growth factor receptor pathway, angiogenesis, antibody-dependent cell-mediated cytotoxicity, DNA repair, and drug metabolism, for their potential role as molecular predictors for clinical outcome in locally advanced rectal cancer patients treated with preoperative cetuximab-based chemoradiation. Methods: 130 patients (74 men and 56 women) with locally advanced rectal cancer (4 with stage II, 109 with stage III, and 15 with stage IV, 2 unknown) who were enrolled in phase I/II clinical trials treated with cetuximab-based chemoradiation in European cancer centers were included. Genomic DNA was extracted from formalin-fixed paraffin-embedded tumor samples and genotyping was done by using PCR-RFLP assays. Fishers exact test was used to examine associations between polymorphisms and complete pathologic response (pCR) that was determined by a modified Dworak classification system (grade III vs. grade IV: complete response). Results: Patients with the epidermal growth factor (EGF) 61 G/G genotype had pCR of 45% (5/11), compared with 21% (11/53) in patients heterozygous, and 2% (1/54) in patients homozygous for the A/A allele (P < 0.001). In addition, this association between EGF 61 G allele and pCR remained significant (P = 0.019) in the 59 patients with wild-type KRAS. Conclusion: This study suggested EGF A+61G polymorphism to be a predictive marker for pCR, independent of KRAS mutation status, to cetuximab-based neoadjuvant chemoradiation of patients with locally advanced rectal cancer. Clin Cancer Res; 17(15); 5161–9. ©2011 AACR.

Integration of Epidermal Growth Factor Receptor Inhibitors with Preoperative Chemoradiation

In many different cancer cell types, the epidermal growth factor receptor (EGFR) pathway becomes hyperactivated because of overproduction of the ligand, overproduction of the receptor, or constitutive activation of the receptor. The overproduction of EGFR and its ligands correlates with poor prognosis in several solid tumors such as lung, colon, and ovary. These observations led to the development of EGFR inhibitors for anticancer treatment. In the last few years, promising results have been obtained in several tumor types, with EGFR inhibitors given as monotherapy or in combined treatments. In particular, cetuximab in combination with curative-intent radiotherapy in head and neck cancer increases median survival over radiation alone. Similarly, the same approach might benefit patients with locally advanced rectal cancer. Unfortunately, the first clinical studies combining chemoradiation with cetuximab in rectal cancer gave disappointing results. Translational research suggested that the low response rate observed might have been due to the strong antiproliferative effect of cetuximab that may have compromised the activity of chemotherapeutics that target proliferating cells. This result indicates the need for more translational research to unravel how the molecular mechanisms might be manipulated to optimize the combined treatment regimen and to identify biomarkers that can select those patients who will derive most benefit. Clin Cancer Res; 16(10); 2709–14. ©2010 AACR.

Molecular and clinico-pathological markers in rectal cancer: a tissue micro-array study.

AimsThe aims of the study were to study the effect of pre-operative treatment on the expression of tumour-related proteins and to correlate the expression of these proteins with response and survival of patients with advanced rectal cancer.Materials and methodsTissue micro-arrays from pre- and post-treatment biopsies of 99 patients with rectal cancer treated with pre-operative (chemo)radiotherapy were stained for epidermal growth factor receptor (EGFR), carbonic anhydrase IX, Ki67, vascular endothelial growth factor, cyclo-oxygenase 2 (COX-2) and cleaved cytokeratin 18 (c-CK18). Also, fibro-inflammatory alterations after treatment were evaluated.ResultsPre-operative (chemo)radiotherapy caused fibro-inflammatory changes, a downregulation of proliferation (Ki67) and EGFR and an upregulation of apoptosis (cleaved CK18). Patients with a good regression during pre-operative treatment showed less proliferating and apoptotic cells in the resection specimen. Multivariate analysis showed that T downstaging, fibro-inflammatory changes in the resection specimen and COX-2 expression in the biopsy correlated with overall survival.ConclusionsPre-operative treatment has an effect on proliferation, apoptosis, inflammation and EGFR expression. The classical clinical parameters as well as fibro-inflammatory changes and COX-2 expression seem most valuable as predictors for survival.

Morphological features and molecular markers in rectal cancer from 95 patients included in the European Organisation for Research and Treatment of Cancer 22921 trial: prognostic value and effects of preoperative radio (chemo) therapy.

In this study, the prognostic and/or predictive value of different proteins (cyclo-oxygenase 2 (COX-2), Ki67 and cleaved cytokeratin (CK) 18) and fibro-inflammatory changes which might be of importance for the response to treatment were evaluated using tissue micro arrays. Samples were obtained from a subset of 95 patients included in the European Organisation for Research and Treatment of Cancer 22921 clinical trial, which randomised patients with rectal cancer to one of four arms treated with preoperative radiotherapy with or without pre- and/or postoperative chemotherapy. From our results, we can conclude that the addition of preoperative chemotherapy to radiotherapy led to significantly less COX-2 upregulation, less proliferation and more inflammation, as was seen in the resection specimen as well as less invasion and metastasis. For COX-2, Ki67 or cleaved CK18, no predictive or prognostic value could be identified. However, the fibro-inflammatory reaction after preoperative radiochemotherapy correlated with T-downstaging and seems to be an important factor for response.

Double blind randomized phase II study with radiation+5-fluorouracil+/-celecoxib for resectable rectal cancer

PURPOSE To assess the feasibility and efficacy of the COX-2 inhibitor celecoxib in conjunction with preoperative chemoradiation for patients with locally advanced rectal cancer in a double blind randomized phase II study. MATERIALS AND METHODS Thirty-five patients of the initially planned 80 patients with locally advanced rectal cancer were treated with preoperative radiation (45 Gy; 1.8 Gy/fraction, 5 days/week) combined with 5-fluorouracil (continuous infusion, 225 mg/m(2)/day) and celecoxib (2 x 400 mg/day) or placebo. Pathological response and toxicity of study treatment were evaluated, as well as expression of COX-2 and Ki67 in tumor tissue and IL-6 in plasma as possible molecular correlates and predictors of response to treatment. RESULTS Patients treated with celecoxib tended to show a better response (61%) when compared to those treated with placebo (35%), although not significant (p=0.13). T-downstaging and N-downstaging were also slightly higher with celecoxib. Plasma IL-6 levels and intratumoral COX2 or Ki67 were altered by chemoradiation, but were not further altered by celecoxib treatment and therefore not useful for prediction of treatment benefit. Celecoxib therapy in conjunction with chemoradiation was not associated with additional toxicity and seemed to help mitigate therapy-related pain. CONCLUSIONS Addition of celecoxib to preoperative chemoradiation is feasible for patients with locally advanced rectal cancer. To study the individual effect of COX-2 inhibitors on pathological response phase III studies are required.