Abstract B13: Development of a low-cost method for collecting fecal samples in clinical trials
Authors: Peloza Kimberly E. , Gills Joell J. , Shaikh Fyza Y. , White James R. , Glass Sara , Lansiquot Carisse , Stevens Courtney , Assan William , Sharfman William H. , Le Dung T. , Naidoo Jarushka , Lipson Evan J. , Pardoll Drew M. , Sears Cynthia L. 
Published: December 2020

Abstract
Although immune checkpoint inhibitors (ICIs) have shown promise in treating various cancers, fewer than half of patients with most tumor types experience a durable response. Thus, there is a need for biomarkers to better predict outcomes. Recent studies suggest that the presence of a handful of microbial species and greater alpha-diversity in the gut may serve as a biomarker for and might facilitate ICI responses. However, the specific bacteria, or bacterial communities, that associate with improved ICI responses vary across study populations, and the factors that contribute to these discrepant findings remain elusive. Thus, a standardized method by which biospecimens may be collected, transported, and stored for gut microbiome studies in the context of ICI therapy is needed. In this study, we evaluated a method for shipping fecal samples using a low-cost (&lt;$3.00) ThinPrep Pap Test® methanol-based preservative kit. Under an IRB-approved protocol, we recruited patients with melanoma, Merkel cell carcinoma, endometrial cancer, and non-small cell lung cancer who had experienced &gt;1 year of durable tumor response after ICI therapy. Patients were provided with stool collection kits and either asked to collect fecal samples at home within 48 hours of their clinic visit and store at 4°C (fresh) or asked to place stool in preservative and ship at ambient temperature to our laboratory (fixed). For fresh samples, a portion of each sample was frozen and another portion placed into preservative as a paired control. DNA was extracted using Zymo Quick-DNA™ Fecal/Soil Microbe kit. For fixed samples, methanol was removed by evaporation prior to DNA extraction. Microbial composition was analyzed with 16S rRNA amplicon sequencing with V1-V2 primers with 150bp paired-end sequencing using an Illumina platform. Among n=10 samples collected in clinic, fixed portions demonstrated decreased alpha-diversity and a uniform shift in beta-diversity compared to the paired frozen portions. In all fixed samples, Faecalibacterium and Roseburia relative abundance decreased with a corresponding increase in Bacteroides. Among a second set of n=11 samples that were placed in preservative by patients and then shipped to our laboratory, we analyzed the effects of shipping by comparing fresh stool samples and shipped fixed samples collected by the same patient within one month. The data revealed similar trends, suggesting that fixation, rather than shipping, drives the overall effect. We are currently verifying the relative abundance of specific bacterial species in both sets of samples using quantitative RT-PCR. Our data show that methanol-based preservation must be optimized prior to clinical utilization for accurate assessment. If optimized, we have identified a low-cost method to collect fecal samples that could be adopted in community practices and low-income areas. Ongoing work from our group includes optimization of processing procedures, ratio of fecal matter to preservative, and storage conditions.
Citation Format: Kimberly E. Peloza, Joell J. Gills, Fyza Y. Shaikh, James R. White, Sara Glass, Carisse Lansiquot, Courtney Stevens, William Assan, William H. Sharfman, Dung T. Le, Jarushka Naidoo, Evan J. Lipson, Drew M. Pardoll, Cynthia L. Sears. Development of a low-cost method for collecting fecal samples in clinical trials [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B13.