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Background: Colorectal cancer (CRC) is a complex and heterogeneous disease characterized by distinct molecular features such as RAS and BRAF mutations. Assessing these molecular aberrations provides clinical guidance for selecting and predicting response with therapeutic modalities. Few reports have suggested that patient symptom burden at diagnosis differs across molecular subtypes of CRC. However, the association of RAS and BRAF mutations and symptom burden post-diagnosis in CRC patients has yet to be examined in a prospective cohort. Methods: We assessed CRC patients, stage I-IV, enrolled in the Moffitt ColoCare Study site with clinical molecular testing results and who completed a six-month questionnaire. Tumor mutation (MT) status (RAS/BRAF/Wildtype (WT)) was abstracted from the medical record. The MD Anderson Symptom Inventory (MDASI) was used to assess the severity of 13 cancer-related symptoms on a 1-to-10 scale. Mean scores in mutation subgroups were compared with one-way ANOVA. Linear regression analysis evaluated the association between symptom burden and mutation status. Multivariable regression models were adjusted for age, sex, stage at diagnosis, treatment, and microsatellite instability (MSI). Results: Among the 158 patients, the mean age at diagnosis was 60.9 ± 11.58 years. The prevalence of RAS and BRAF mutations was 30.4% (N=48) and 6.3% (N=10), respectively. Statistically significant mean differences between the mutation subgroups were distress (p=0.005), difficulty remembering (p=0.015), lack of appetite (p=0.042), and sadness (p=0.007). Patients with a BRAF-MT reported significantly more distress (p=0.001), difficulty remembering (p=0.029), and sadness (p=0.002) at six months compared to RAS-MT or RAS/BRAF-WT. Patients with a RAS-MT reported an increased lack of appetite (p=0.038) compared to BRAF-MT or RAS/BRAF-WT. Patients with a BRAF-MT reported higher severity of symptoms interfering with the enjoyment of life (p Conclusions: Tumors with BRAF-MT are molecularly classified into the serrated pathway, which has distinct clinical characteristics and risk factor profile. In this study, patients with tumors harboring BRAF-MT reported a higher symptom burden six months post-diagnosis than those with RAS-MT or RAS/BRAF-WT tumors. While the prevalence of BRAF-MT was low in this population, these findings warrant further investigation in a larger cohort. Citation Format: Gazelle Rouhani, Amanda M. Bloomer, Maria F. Gomez, Gillian K. Trujillo, Devon N. Conant, Seth I. Felder, Cornelia M. Ulrich, Christopher I. Li, Jane C. Figueiredo, Adetunji T. Toriola, Biljana Gigic, Martin Schneider, David Shibata, Erin M. Siegel. Profiling symptom burden of RAS and BRAF mutations in patients with colorectal cancer: Results from the ColoCare Study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4205.

Background: The gut microbiome (GM) and the metabolites it produces and regulates, such as bile acids (BAs), may act individually or interact to influence colorectal cancer development. We used data from a multi-center screening study to investigate the associations of the normal colon tissue microbiome and circulating BAs with colorectal adenoma. Methods: We used data from the multi-center Colorectal Neoplasia Screening with Colonoscopy in Average-Risk Women Regional Navy/Army Medical Centers study (CONCeRN), comprising women undergoing complete colonoscopies. We individually matched women with adenoma (n=165 cases) to those without adenoma (n=311 controls). We extracted DNA using the Animal Tissue DNA Extraction Kit (AutoGene) and sequenced the V4 region of the 16S rRNA gene to characterize normal colonic mucosal bacteria, including alpha and beta diversity, taxonomic abundance, and co-abundance groups (CAGs). Fasting serum collected before colonoscopy underwent targeted quantitative liquid chromatography-tandem mass spectrometry to measure 13 primary and secondary BAs. We used multivariable conditional logistic regression, partial Pearson’s correlations, and linear regression to estimate interrelationships among the GM, BAs, and adenoma. Results: Multiple genera were statistically significantly associated with adenomas. For example, for the centered log-ratio transformed relative abundance of Phascolarctobacterium, Parvimonas, and Porphymonas, the ORs (95% CIs) were 0.75 (0.60-0.95, P=0.02), 0.71 (0.55-0.92; P=0.01), and 0.67 (0.49-0.93; P=0.02), respectively. A CAG comprising phylum Firmicutes, Actinobacteria, Proteobacteria, and Fusobacteria was less abundant among cases than controls (log2folddiff=-0.80, P=0.002). Alpha and beta diversity associations were close to null and not statistically significant. However, alpha diversity metrics were inversely associated with primary BAs [e.g., for Shannon (R2=-0.13, P=0.01)] and positively associated with secondary BAs [e.g., for Shannon (R2=0.16, P=0.001)]. Though no circulating BAs were statistically significantly associated with adenomas, those in the highest relative to lowest tertile of taurine-conjugated (TC) BAs, primary BAs, and secondary BAs had a 44% (95% CI: 0.85-2.46), 24% (95% CI: 0.73-2.12), and 11% (95% CI: 0.61-2.01) higher odds of adenoma, respectively. We found that TCBAs, primary BAs, and secondary BAs were inversely associated with the abundance of Parvimonas [e.g., for TCBAs (R2=-0.11, P=0.02)] and Porphyromonas [e.g., for TCBAs (R2=-0.11, P=0.02)]. Conclusions: We found short-chain fatty acid-producing and oral-originating bacteria were inversely associated with adenomas. Our oral bacteria findings are contrary to findings from prior studies. Our findings suggest potential interrelationships among the GM and BAs in adenoma development. Citation Format: Maria F. Gomez, Doratha A. Byrd, Stephanie R. Hogue, Jessica R. Burns, Nathan Smith, Joshua Sampson, Erikka Loftfield, Andrew Warner, Casey Dagnall, Kristine Jones, Belynda Hicks, Yunhu Wan, Youngchul Kim, Jin Xu, Rashmi Sinha, Emily Vogtmann. Associations of the mucosal microbiome and circulating bile acids with colorectal adenoma among average-risk women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3055.

Purpose: Oropharyngeal cancer (OPC) incidence continues to increase dramatically in the US. Accumulating evidence suggests that oral microbiota (communities of microorganisms that reside in the oral cavity) may influence cancer treatment-related toxicities. We sought to examine the composition and diversity of oral microbiota in OPC patients prior to treatment, and identify associations between oral microbiota and subsequent development of oral mucositis (inflammation of mucous membranes) and candidiasis (fungal infection with Candida yeast). Methods: 60 newly diagnosed, treatment naïve, OPC patients were recruited from Moffitt Cancer Center (2015-2017). Patients provided mouthwash-based oral gargles before starting chemoradiation or radiation. DNA was isolated using the QIAGEN DNeasy PowerSoil kit, and the V1-V3 region of the bacterial 16S rRNA gene was sequenced. An operational taxonomic unit table was generated and Ribosomal Database Project Classifier used to assign taxonomy. The development of oral mucositis (no/mild vs. severe) and candidiasis (no vs. yes) during treatment was abstracted from medical records. Comparisons of bacterial relative abundance (Mann Whitney U), alpha diversity (Chao1, Shannon, and Simpson), and beta diversity (Bray-Curtis) were performed in R and its extension (Phyloseq). Results: Of 60 OPC patients, 65% were stage IV, 48% tonsillar and 42% base of tongue, and 40% never smokers. Pre-treatment levels of genus Klebsiella and class Gammaproteobacteria were significantly greater in the oral cavity of patients who had no/mild mucositis vs. those who developed severe mucositis (p Conclusions: Microbiome profiling may hold promise as a prognostic biomarker of treatment-related toxicities. Several potentially predictive taxa were identified to be differentially abundant in OPC patients who subsequently developed oral mucositis or candidiasis. Future analyses will evaluate the role of oral microbial resilience (the rate of recovery after disturbance) using oral specimens collected 3 mo. after treatment. Translational research focused on understanding how oral microbiota influence local immune and inflammatory responses may aid in the development of microbiota-based interventions to minimize adverse events. Citation Format: Christine M. Pierce, Stephanie Hogue, Shirlene Paul, Bo-Young Hong, Wildson Vieira da Silva, Maria F. Gomez, Anna R. Giuliano, Jimmy J. Caudell, George M. Weinstock. Mucositis, candidiasis, and associations with the oral microbiome in treatment naive patients with oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3326.

Mounting evidence suggests that a prominent T cell response in the colorectal cancer (CRC) tumor microenvironment is a valuable prognostic indicator, independent of stage at diagnosis. However, the strength and quality of host immune responses are highly variable across patients, and the factors that influence this variability are not well understood. The unique microbial communities present in the CRC tumor microenvironment may contribute to the modulation of local host immune responses, but prior investigations have been limited and focused on candidate microbes. Here, we investigated the association between the CRC tumor bacterial microbiome and the quantity and clonality of the T cell infiltrate. This investigation included 51 CRC from the Molecular Epidemiology of Colorectal Cancer (MECC) study, a population-based case-control study in northern Israel. Genomic DNA was extracted from fresh frozen tumors using microbiome-optimized techniques and sequenced using a PCR amplicon-mediated workflow on the Illumina MiSeq. Sequences from the V1-V3 hypervariable regions of the bacterial 16S rRNA gene region were clustered into operational taxonomic units (OTUs), a proxy for species, and taxonomic identification was determined using the Ribosomal Database Project. In addition, T cell repertoires were measured using the DNA-based immunoSEQ assay (Adaptive Biotechnologies), and T cell fraction (i.e. proportion of rearranged T cells relative to all nucleated cells) and clonality (i.e. T cell receptor diversity) were calculated. Bacterial sequencing was successful in 48 (94%) of samples, with a total of 868 OTUs identified. Klebsiella and Escherichia/Shigella were the most prevalent genera, detected in approximately 90% of tumors, followed by Propionibacterium, Pelomonas, and Veillonella, with each detected in approximately 50% of tumors. Fusobacterium, a genus of oral anaerobic bacteria believed to accelerate tumor progression and enable immune evasion in CRC, was detected in 20% of tumors. When present, Bacteroides and Fusobacterium were the most abundant, comprising 10-15% of all bacteria within each tumor, on average. ImmunoSEQ analysis identified a mean productive T cell fraction of 0.08 (SD: 0.16) and a mean productive clonality of 0.14 (SD: 0.06). Analysis of molecular variance (AMOVA) of the Theta YC measure of dissimilarity showed a statistically significant difference in bacterial community structure (i.e. composition and relative abundance) between samples in the highest versus lower three quartiles of T cell fraction (P=0.03) and clonality (P=0.01). These preliminary findings suggest a potential relationship between microbial community structure and the quantity and diversity of the CRC T cell infiltrate. Additional analyses are underway to better understand this association as well as the independent contributions of the microbiome to CRC prognosis. Citation Format: Christine M. Pierce, Bo-young Hong, Hannah J. Hoehn, Maria F. Gomez, Marilena Melas, Kevin McDonnell, Youngchul Kim, Erica Sodergren, George Weinstock, Hedy S. Rennert, Thomas Giordano, Joel Greenson, Gad Rennert, Stephen B. Gruber, Stephanie L. Schmit. Microbes in the tumor microenvironment: Bacterial influences on host immunity in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4746.

PURPOSE: Despite advances in cancer treatment, patients with advanced head and neck cancer (HNC) have a median overall survival of METHODS: Fresh frozen tumor tissues from 30 newly diagnosed, treatment-naïve patients who received chemoradiation at Moffitt Cancer Center for squamous cell HNC were evaluated. Genomic DNA was extracted from 25mg of tissue. The V1-V3 hypervariable region of the bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) 1 region were sequenced using a PCR amplicon-mediated workflow on the Illumina MiSeq. 16S sequences were clustered into operational taxonomic units and taxonomic identification was determined using the RDP database and ITS sequences were classified using an in house reference database. Patients' charts were reviewed to ascertain clinical response 3, 6 and 12 months after treatment cessation. Response was dichotomized as complete (no evidence of disease) and incomplete (partial response, stable disease, and progressive disease). RESULTS: At the time of tumor collection, patients ranged in age from 20-79 years, and were predominantly men (83%) and current smokers (40%). The most common anatomic site was the oral cavity (70%) followed by the larynx (20%) and oropharynx (10%), and most were diagnosed at stage 4 (87%). Three, 6 and 12 months after treatment cessation, 4 (14%), 9 (36%), and 10 (40%) patients had an incomplete response to therapy, respectively. Bacterial sequencing was successful in 29 (97%) samples and fungal in 15 (50%). Clustering analyses of bacterial abundance profiles suggested that 4 types of communities existed, each dominated by Fusobacterium, Prevotella, Streptococcus, or Treponema. HNC patients with incomplete clinical responses (3, 6, or 12 months after treatment) were more likely to have treatment-naïve tumors dominated by Fusobacterium (cluster 1; 56%) than any other genus, whereas patients with complete clinical responses were more likely to have treatment-naïve tumors dominated by Prevotella (cluster 2; 40%) than any other genus. None of the patients with incomplete responses were dominated by Streptococcus (cluster 3) or Treponema (cluster 4). Fungal ITS profiling revealed that the tumor microbiome was largely dominated by the fungi Malassezia. CONCLUSIONS: The current study demonstrates that intratumoral bacterial profiles dominated by Prevotella may serve as a biomarker of improved prognosis, while profiles dominated by Fusobacterium may indicate a poor prognosis following chemoradiation in HNC patients. Citation Format: Christine M. Pierce Campbell, Bo-Young Hong, Maria F. Gomez, Blake M. Hanson, Erica Sodergren, Sybil T. Sha, Jeffery S. Russell, George M. Weinstock. Microbial profiling of the head and neck tumor microenvironment as a biomarker of clinical response to chemoradiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2252. doi:10.1158/1538-7445.AM2017-2252