Bozena Fender

Apelin: A putative novel predictive biomarker for bevacizumab response in colorectal cancer

Bevacizumab (bvz) is currently employed as an anti-angiogenic therapy across several cancer indications. Bvz response heterogeneity has been well documented, with only 10-15% of colorectal cancer (CRC) patients benefitting in general. For other patients, clinical efficacy is limited and side effects are significant. This reinforces the need for a robust predictive biomarker of response. To identify such a biomarker, we performed a DNA microarray-based transcriptional profiling screen with primary endothelial cells (ECs) isolated from normal and tumour colon tissues. Thirteen separate populations of tumour-associated ECs and 10 of normal ECs were isolated using fluorescence-activated cell sorting. We hypothesised that VEGF-induced genes were overexpressed in tumour ECs; these genes could relate to bvz response and serve as potential predictive biomarkers. Transcriptional profiling revealed a total of 2,610 differentially expressed genes when tumour and normal ECs were compared. To explore their relation to bvz response, the mRNA expression levels of top-ranked genes were examined using quantitative PCR in 30 independent tumour tissues from CRC patients that received bvz in the adjuvant setting. These analyses revealed that the expression of MMP12 and APLN mRNA was significantly higher in bvz non-responders compared to responders. At the protein level, high APLN expression was correlated with poor progression-free survival in bvz-treated patients. Thus, high APLN expression may represent a novel predictive biomarker for bvz unresponsiveness.

Loss of Chromosome 18q11.2-q12.1 Is Predictive for Survival in Patients With Metastatic Colorectal Cancer Treated With Bevacizumab

Purpose Patients with metastatic colorectal cancer (mCRC) have limited benefit from the addition of bevacizumab to standard chemotherapy. However, a subset probably benefits substantially, highlighting an unmet clinical need for a biomarker of response to bevacizumab. Previously, we demonstrated that losses of chromosomes 5q34, 17q12, and 18q11.2-q12.1 had a significant correlation with progression-free survival (PFS) in patients with mCRC treated with bevacizumab in the CAIRO2 clinical trial but not in patients who did not receive bevacizumab in the CAIRO trial. This study was designed to validate these findings. Materials and Methods Primary mCRC samples were analyzed from two cohorts of patients who received bevacizumab as first-line treatment; 96 samples from the European multicenter study Angiopredict (APD) and 81 samples from the Italian multicenter study, MOMA. A third cohort of 90 samples from patients with mCRC who did not receive bevacizumab was analyzed. Copy number aberrations of tumor biopsy specimens were measured by shallow whole-genome sequencing and were correlated with PFS, overall survival (OS), and response. Results Loss of chromosome 18q11.2-q12.1 was associated with prolonged PFS most significantly in both the cohorts that received bevacizumab (APD: hazard ratio, 0.54; P = .01; PFS difference, 65 days; MOMA: hazard ratio, 0.55; P = .019; PFS difference, 49 days). A similar association was found for OS and overall response rate in these two cohorts, which became significant when combined with the CAIRO2 cohort. Median PFS in the cohort of patients with mCRC who did not receive bevacizumab and in the CAIRO cohort was similar to that of the APD, MOMA, and CAIRO2 patients without an 18q11.2-q12.1 loss. Conclusion We conclude that the loss of chromosome 18q11.2-q12.1 is consistently predictive for prolonged PFS in patients receiving bevacizumab. The predictive value of this loss is substantiated by a significant gain in OS and overall response rate.

Assessment of concordance between fresh-frozen and formalin-fixed paraffin embedded tumor DNA methylation using a targeted sequencing approach

DNA methylation is altered in many types of disease, including metastatic colorectal cancer. However, the methylome has not yet been fully described in archival formalin-fixed paraffin embedded (FFPE) samples in the context of matched fresh-frozen (FF) tumor material at base-pair resolution using a targeted approach. Using next-generation sequencing, we investigated three pairs of matched FFPE and FF samples to determine the extent of their similarity. We identified a ‘bowing’ pattern specific to FFPE samples categorized by a lower CG proportion at the start of sequence reads. We have found no evidence that this affected methylation calling, nor concordance of results. We also found no significant increase in deamination, measured by C>T transitions, previously considered a result of crosslinking DNA by formalin fixation and a barrier to the use of FFPE in methylation studies. The methods used in this study have shown sensitivity of between 60-70% based on positions also methylated in colorectal cancer cell lines. We demonstrate that FFPE material is a useful source of tumor material for methylation studies using targeted sequencing.

Copy number load predicts outcome of metastatic colorectal cancer patients receiving bevacizumab combination therapy

Increased copy number alterations (CNAs) indicative of chromosomal instability (CIN) have been associated with poor cancer outcome. Here, we study CNAs as potential biomarkers of bevacizumab (BVZ) response in metastatic colorectal cancer (mCRC). We cluster 409 mCRCs in three subclusters characterized by different degrees of CIN. Tumors belonging to intermediate-to-high instability clusters have improved outcome following chemotherapy plus BVZ versus chemotherapy alone. In contrast, low instability tumors, which amongst others consist of POLE-mutated and microsatellite-instable tumors, derive no further benefit from BVZ. This is confirmed in 81 mCRC tumors from the phase 2 MoMa study involving BVZ. CNA clusters overlap with CRC consensus molecular subtypes (CMS); CMS2/4 xenografts correspond to intermediate-to-high instability clusters and respond to FOLFOX chemotherapy plus mouse avastin (B20), while CMS1/3 xenografts match with low instability clusters and fail to respond. Overall, we identify copy number load as a novel potential predictive biomarker of BVZ combination therapy.Increased copy number alterations, indicative of chromosomal instability, is associated with poor cancer outcome. Here, metastatic colorectal cancer patients displaying intermediate-high CIN associate with improved outcome following chemotherapy and bevacizumab treatment, suggesting CIN as a predictive biomarker.

Chromosome 18q11.2 loss as a predictive marker for response to bevacizumab in metastatic colorectal cancer

28 EORTC – NCI – AACR Symposium on Molecular Targets and Cancer Therapeutics 29 November 2016 - 02 December 2016

Abstract 4529: Tailoring approaches for global epigenome analysis from archival formalin-fixed paraffin-embedded tissue samples

Novel DNA extraction methodologies allow the use of archival material from formalin fixed paraffin embedded (FFPE) tissue samples in genomic studies. A major limitation of this source of DNA is the fragmented nature and low overall yield generally obtained from clinical materials, making downstream applications such as epigenetic analysis challenging. Previous published attempts have focussed on smaller regions of CpG islands, such as the use of Illumina9s 450k arrays (which measure methylation at approximately 485,000 sites). The objective of this study was to optimize experimental and analytical workflows that allow effective interrogation of global DNA methylation profiles from FFPE samples. Methylation capture was conducted on DNA from matched FFPE and fresh frozen samples from the same metastatic colorectal cancer patient as well as two colorectal cancer cell lines, using the SeqCap Epi (Roche) methyl capture system. The custom capture designed includes 5.5 million CpG sites across the genome, a greater than 10-fold increase compared to previously published studies. The wet-lab protocol was robustly optimized for several parameters, such as overall yield and bisulphite conversion efficiency (measured by shallow-read next generation sequencing). A data analysis pipeline composed of the bisulfite-converted DNA aligner BWA-meth, as well as in-house Perl and R scripts, was used to generate detailed methylation maps for individual sample types in order to identify differentially methylated regions (DMRs), which were further validated using targeted bisulphite sequencing for selected loci. Preliminary analysis of the data revealed 98% bisulphite conversion efficiency and low PCR duplicate rate (4-5%) across both sample types. Intriguingly, we observe an 80% concordance between the overall DNA methylation profiles between FFPE and fresh frozen samples, with a 60% overlap between the FFPE and fresh frozen samples in terms of direction of methylation i.e. hyper or hypomethylation. Mapping overall methylation levels to CpG ‘resorts’ (+/- 4kb of CpG islands) indicated ∼10% of methylation occurred in these regions totalling 245MB. Known genomic features, including exons, promoters of coding/non-coding genes and enhancers, contained ∼30% (1.5 million) of the methylated positions. In addition, this methodology also allowed us to identify C to T transitions across all samples, a common artefact of formalin fixed samples. The analysis revealed that a significantly low proportion of C to T transitions were detected across all samples, the levels of the transitions being consistent between the two sample types. Taken together, these results demonstrate a robust and novel approach to generate DNA methylation profiles from difficult-to-handle, but frequently available material, thus establishing a suitable platform for a whole methylome profiling from archival samples. Citation Format: Sudipto Das, Bruce Moran, Dominiek Smeets, Gillian Peuteman, Rut Klinger, Bozena Fender, Kate Connor, Matthias Ebert, Timo Gaiser, JHM Prehn, Orna Bacon, Elaine Kay, Bryan Hennessy, Verena Murphy, William Gallagher, Annette Byrne, Diether Lambrechts, Darran O’Connor. Tailoring approaches for global epigenome analysis from archival formalin-fixed paraffin-embedded tissue samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4529.