Abstract P6-11-20: Capacity-building for molecular analyses of formalin-fixed paraffin-embedded breast cancer samples from Rwanda
Authors: Chamberlin Mary Dickinson , Forbes Victoria , Dushimimana Germaine , Kettenbach Arminja , Muller Kristen , Musoni Emile , Rutaganda Eric , Wilkins Owen M. , Kolling Fred W. , Christensen Brock 
Published: March 2020

Background: The global burden of cancer is rising exponentially in countries with the least resources to manage it. Breast cancer mortality is higher in African women than European women, but neither race nor access to care completely account for this discrepancy. Molecular profiling and methylation analysis could be informative but is expensive and the feasibility of using archived samples from low-resource settings is not known. We present a retrospective tissue collection study of breast cancer patients diagnosed at the University of Rwanda to investigate the quality of DNA from FFPE.

Methods

IRB approval and Material Transfer Agreement were obtained. Fifty tumor blocks and eight sets of FFPE slides from 2014-2018 were transported from Rwanda to USA in 2 batches. Pathology was reviewed for concordance. DNA was extracted using the QIAamp DNA FFPE Tissue kit (Qiagen). Batch 1: DNA quality and quantity was assessed using the Qubit 3.0 fluorometer (Life Technologies). Batch 2: DNA quality was assessed using Kapa hgDNA pPCR QC Assay. Bisulfite conversion and array hybridization protocols for the Illumina EPIC methylation platform to detect 2.5% methylated DNA target were used.

DNA methylation data generated using the Infinum MethylationEPIC BeadChip (Illumina, Inc.) were pre-processed using minfi (v1.30.0), prior to quality control using Enmix (v1.20.2). A detection P-value threshold of 1E-06 was used to identify poorly detected probes, and a maximum threshold of 5% low quality data was used to identify poorly performing samples and CpG loci. Outliers based on beta-value distributions were also flagged as poor-quality samples. Mean bisulfite intensity values were calculated using Infinium type I and II control probes. DNA methylation beta-values were calculated to assess the proportion of methylated alleles at each individual CpG locus.

Results: Histology was 82% concordant. All samples in Batch 1 had &gt;5% low quality CpG’s. FFPE processing was reviewed and buffered formalin was not used. For Batch 2 10% neutral buffered formalin was transported and delivered to Central University Hospital of Kigali in March 2019. Eight additional patient samples were obtained and 6 out of 8 had &lt;5% low quality CpG’s.

Conclusion: Complex molecular studies are feasible with international collaboration. Optimal reagents and processing are crucial for successful analysis. Quality checkpoints must be done early in the process to maximize the potential for interpretable results. Capacity-building to improve DNA quality is the first step toward optimizing cancer treatment and minimizing disparities as molecular profiling increasingly directs cost-effective treatment.These results confirm methods appropriate for future molecular research on breast cancer in low resource settings.

Citation Format: Mary Dickinson Chamberlin, Victoria Forbes, Germaine Dushimimana, Arminja Kettenbach, Kristen Muller, Emile Musoni, Eric Rutaganda, Owen M. Wilkins, Fred W. Kolling, Brock Christensen. Capacity-building for molecular analyses of formalin-fixed paraffin-embedded breast cancer samples from Rwanda [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-11-20.