Eamonn J. O'Brien

Development and Independent Validation of a Prognostic Assay for Stage II Colon Cancer Using Formalin-Fixed Paraffin-Embedded Tissue

PURPOSE Current prognostic factors are poor at identifying patients at risk of disease recurrence after surgery for stage II colon cancer. Here we describe a DNA microarray-based prognostic assay using clinically relevant formalin-fixed paraffin-embedded (FFPE) samples. PATIENTS AND METHODS A gene signature was developed from a balanced set of 73 patients with recurrent disease (high risk) and 142 patients with no recurrence (low risk) within 5 years of surgery. RESULTS The 634-probe set signature identified high-risk patients with a hazard ratio (HR) of 2.62 (P < .001) during cross validation of the training set. In an independent validation set of 144 samples, the signature identified high-risk patients with an HR of 2.53 (P < .001) for recurrence and an HR of 2.21 (P = .0084) for cancer-related death. Additionally, the signature was shown to perform independently from known prognostic factors (P < .001). CONCLUSION This gene signature represents a novel prognostic biomarker for patients with stage II colon cancer that can be applied to FFPE tumor samples.

BRCA1 is both a prognostic and predictive biomarker of response to chemotherapy in sporadic epithelial ovarian cancer

OBJECTIVES We investigated the relationship between BRCA1 protein expression by immunohistochemistry (IHC) and clinical outcome following platinum and platinum/taxane chemotherapy in sporadic epithelial ovarian cancer (EOC). METHODS BRCA1 IHC was performed on a cohort of 292 ovarian tumours from two UK oncology centres. BRCA1 protein expression levels were correlated with overall survival (OS), progression free survival (PFS) and clinical response to chemotherapy by multivariate analysis. RESULTS EOC patients with absent/low BRCA1 protein expression (41%) had a better chance of clinical response following chemotherapy as compared to patients with high BRCA1 expression (odds ratio 2.47: 95%CI 1.10-5.55, p=0.029). Patients with absent/low BRCA1 had a higher probability of clinical response following single agent platinum compared to high BRCA1 expressing patients (68.5% vs. 46.8%), while addition of a taxane increased response rates independent of BRCA1. Overall, patients with absent/low BRCA1 had a better clinical outcome compared to patients with high BRCA1 protein expression in terms of both OS (HR=0.65: 95%CI 0.48-0.88, p=0.006) and PFS (HR=0.74, 95%CI 0.55-0.98, p=0.040). CONCLUSIONS We confirm that absent/low BRCA1 protein expression is a favourable prognostic marker. However, we also provide the first evidence that absent/low BRCA1 protein expression in sporadic EOC patients predicts for an improved clinical response to chemotherapy.

Association Between Results of a Gene Expression Signature Assay and Recurrence-Free Interval in Patients With Stage II Colon Cancer in Cancer and Leukemia Group B 9581 (Alliance)

PURPOSE Conventional staging methods are inadequate to identify patients with stage II colon cancer (CC) who are at high risk of recurrence after surgery with curative intent. ColDx is a gene expression, microarray-based assay shown to be independently prognostic for recurrence-free interval (RFI) and overall survival in CC. The objective of this study was to further validate ColDx using formalin-fixed, paraffin-embedded specimens collected as part of the Alliance phase III trial, C9581. PATIENTS AND METHODS C9581 evaluated edrecolomab versus observation in patients with stage II CC and reported no survival benefit. Under an initial case-cohort sampling design, a randomly selected subcohort (RS) comprised 514 patients from 901 eligible patients with available tissue. Forty-nine additional patients with recurrence events were included in the analysis. Final analysis comprised 393 patients: 360 RS (58 events) and 33 non-RS events. Risk status was determined for each patient by ColDx. The Self-Prentice method was used to test the association between the resulting ColDx risk score and RFI adjusting for standard prognostic variables. RESULTS Fifty-five percent of patients (216 of 393) were classified as high risk. After adjustment for prognostic variables that included mismatch repair (MMR) deficiency, ColDx high-risk patients exhibited significantly worse RFI (multivariable hazard ratio, 2.13; 95% CI, 1.3 to 3.5; P < .01). Age and MMR status were marginally significant. RFI at 5 years for patients classified as high risk was 82% (95% CI, 79% to 85%), compared with 91% (95% CI, 89% to 93%) for patients classified as low risk. CONCLUSION ColDx is associated with RFI in the C9581 subsample in the presence of other prognostic factors, including MMR deficiency. ColDx could be incorporated with the traditional clinical markers of risk to refine patient prognosis.

Reply to L. Casadaban et al

We presented univariable results according to the REMARK guidelines for associations between ColDx score and prognostic factors for recurrence-free interval (RFI; Appendix Table A1 [online only] in our article). As Casadaban et al point out, ColDx is associated with T-stage and lymphovascular invasion but not the number of nodes examined, perineural invasion, or tumor grade. It is not clear why such a relationship would be expected. The assay was designed to be independent from other known prognostic clinical factors and to add new prognostic information. As Casadaban et al suggest, we considered the subgroup of high-risk patients who we defined as exhibiting any one of the following clinical characteristics: obstruction or perforation (six patients), lymphovascular invasion (42 patients), fewer than 12 nodes sampled (176 patients), or microsatellite instability low or stable (283 patients; n 5 317; 80 RFI events). RFI was then compared between highrisk patients and low-risk patients as determined by ColDx score. Results were significant at P5 .05, with a hazard ratio of 1.62 (95%CI, 0.99 to 2.68). Thus, ColDx provides further discrimination in this higher-risk subgroup. The number of events was too small to make this comparison in the low-risk subgroup. In the parent trial, Alliance C9581, investigators sought to determine whether the use of edrecolomab—a relatively nontoxic adjuvant therapy—would demonstrate an overall survival benefit in a cohort of patients with resected, stage II colon cancer that excluded patients with high-risk factors. Patients were considered disease-free postsurgery. Thus, tumor response was not a study end point. Patient samples were obtained before treatment with edrecolomab. Overall survival and disease-free survival between treated and untreated patients were essentially equivalent (Fig 2A in our article). Nonetheless, under the case-cohort design in our validation study, we randomly selected patients stratified by assigned treatment and accounted for stratification in the analysis. Overall, toxicity was low. A maximum of grade 3 toxicity was reported for 242 (29.4%) of 823 participants who reported adverse events with edrecolomab treatment, and 48 patients (5.8%) experienced a maximum grade 4 toxicity. No individual adverse event was reported in . 5% of patients, the most prevalent of which was diarrhea. One death occurred within 30 days of completing edrecolomab therapy and was not attributed to treatment. This validation study used the same primary end point on which the gene signature was developed. Among patients who were studied in the Alliance C9581 trial, we found that it is important to distinguish between disease-related death and other causes of death in this low-risk, older patient population with stage II disease. We found large differences in outcome by sex and age for all-cause mortality that were primarily caused by association of these factors with death as a result of other causes. Including deaths as a result of other causes as an event may unduly bias results. Regarding sample insufficiency, in clinical testing, the quality control fail rate that was observed for the study is not unusual considering the average age of the formalin-fixed, paraffinembedded tissue used in the validation study (average age, 13.2 years). This limitation is acknowledged in the manuscript. In addition, average quality control fail rate within fresh formalinfixed, paraffin-embedded tissue is 5%. We stated the reason for the different prognostic score cut points in our article, which was “migration of the ColDx assay from the Affymetrix GeneChip System 3000 7G scanner to the Affymetrix microarray platform GeneChip System 3000Dx v.2.” With respect to the association with lymphocyte proliferation and activation of biologic functions with recurrence-free survival in colorectal liver metastases, the validation study was performed within primary tissuematerial. Biologic signaling within metastatic tissue is inherently different from that found within primary tissue material. That said, the most significant molecular pathways measured by the ColDx assay are detailed by Kennedy et al, among which are TGF-b and chemokine signaling, and both are associated with lymphocyte proliferation and recruitment. It is not unusual that two assays, such as the 12-gene recurrence score and ColDx, have good discrimination and calibration but do not agree with one another in individual probability predictions. It is more relevant to determine which assay is better calibrated and has better discrimination—that is, which assay is better at generating estimates that are closer to observed values. We agree with Casadaban et al that further studies are needed to demonstrate the ability of the gene expression signature to predict treatment benefit. Despite its limitations, our study was prospectively planned and used specimens and clinical data from a cohesive, well-conducted clinical trial. The results demonstrate the additive prognostic value of the measure.

Abstract MIP-055: IDENTIFICATION OF A MOLECULAR SUBTYPE OF HIGH GRADE SEROUS OVARIAN CANCER REPRESENTING MAPK PATHWAY ACTIVATION AND PLATINUM RESISTANCE

BACKGROUND: We previously defined 3 molecular subgroups of High Grade Serous Ovarian Cancer (HGSOC), using gene expression data from 265 FFPE samples obtained from treatment naive patients, who received platinum based treatment following surgical resection. The 3 molecular subgroups were Angio: characterised by upregulation of angiogenesis genes; Immune: characterised by upregulation of immune genes and AngioImmune: characterised by upregulation of angiogenesis and immune genes. Patients within these 3 subgroups respond differently to standard of care treatment The Immune subgroup have the best prognosis and the Angio and AngioImmune subgroups have similar worse prognosis. A weighted gene signature to identify each of the molecular subgroups was developed. This dataset was used as a reference to investigate the effect of chemotherapy on molecular subgroup designation. METHODS: To investigate the effect of chemotherapy on predefined molecular subgroups, we analysed 35 matched pre- and post- chemotherapy samples by gene expression. The molecular subgroup assignment for each of the paired samples was determined using the gene expression signatures for each subgroup. Novel cisplatin resistant HGSOC cell lines were generated to study the mechanisms of acquired cisplatin resistance. RESULTS: 40% of the treatment naive samples that were aligned with the AngioImmune subgroup and this increased to 67.5% post-chemotherapy. 10/15 (67%) treatment naive tumours that were initially assigned to the good prognostic Immune molecular subgroup shifted to the bad prognostic AngioImmune molecular subgroup post chemotherapy. Hence platinum chemotherapy selects for the AngioImmune subgroup, suggesting that this subgroup represents tumours which are innately platinum resistant but also provides a mechanism of acquired resistance. Additionally we demonstrate that the AngioImmune subgroup is driven by activation of the MAPK pathway and shows that cisplatin resistant HGSOC cell lines are specifically sensitive to MEK inhibitors. CONCLUSIONS: The MAPK pathway is a mechanism of innate and acquired platinum resistance in HGSOC. Furthermore the data suggests that original pre-treatment surgical/biopsy samples may fall within a different molecular subgroup to samples taken post-platinum therapy. Citation Format: Aya El-Helali, Nuala McCabe, Charlie Gourley, Andrena McCavigan, Caroline O. Michie, Bethanie Price, Niamh McGivern, Michael Churchman, Aya El-Helai, Eamonn J. O9Brien, Laura Hill, Timothy S Davison, Alistair Williams, W Glenn McCluggage, Katherine E Keating, Denis P Harkin, and Richard Kennedy. IDENTIFICATION OF A MOLECULAR SUBTYPE OF HIGH GRADE SEROUS OVARIAN CANCER REPRESENTING MAPK PATHWAY ACTIVATION AND PLATINUM RESISTANCE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-055.