IMMUNOREACT 10: INFLUENCE OF BMI IN THE IMMUNOLOGICAL MICROENVIRONMENT OF PERITUMORAL HEALTHY MUCOSA IN RECTAL ADENOCARCINOMA

Introduction: Due to its rarity, anal adenocarcinoma (AA) does not have a standardized staging system. The tumor (T) stage of an adenocarcinoma arising from the anal canal could be based on depth of invasion, as for rectal adenocarcinoma, or on size, as in anal squamous cell carcinoma. We hypothesized that staging by adenocarcinoma histology, rather than anal location, would be most accurate in predicting survival of AA patients.

Methods: Adults with AA were identified in the Surveillance, Epidemiology, and End Results database between 2004 and 2019. Exclusion criteria were overlapping lesions of the anus/rectum, >1 lifetime diagnosis of cancer, and missing tumor size or extension. All patient stages were categorized according to the American Joint Committee on Cancer (AJCC) classifications of rectal adenocarcinoma (AJCC-rectum) and anal squamous cell carcinoma (AJCC-anus). Kaplan-Meier curves were used to determine 5-year overall (OS) and disease-specific survival (DSS) rates. Cox proportional hazard regressions, adjusting for age, gender, race, marital status, tumor grade, and treatment received, analyzed the association between prognosis and both overall and T stages.

Results: Of 424 patients, 55% were male, 63% were Caucasian, and 23% had distant metastases. Median age was 65 (interquartile ratio 53-75) years. Thirty-four percent of patients underwent chemoradiation (CRT) and abdominoperineal resection (APR), 15% CRT with local excision (LE), 26% CRT alone, 11% upfront APR, and 15% LE only. When patients were classified by AJCC-rectum staging, 36% had stage I disease, 25% stage II, and 23% stage III. Conversely, according to AJCC-anus, there were 19% stage I, 38% stage II, and 20% stage III patients. Both classifications included the same 97 patients (23%) with stage IV disease. The unadjusted 5-year DSS and OS were similar for early stages, while both classifications similarly differentiated more advanced disease (Figure 1). After adjustment, stages I and II maintained similar OS and DSS in both staging systems, while stages III and IV had significantly worse prognosis (Table 1). In contrast, when analyzing T stage alone (node-negative, non-metastatic patients), multivariable analyses demonstrated significant, incrementally lower OS and DSS for stage T1 to T4 for AJCC-rectum, but not for AJCC-anus (Table 1).

Conclusion: In this population-based cohort, neither staging system was able to accurately risk stratify stage I and II tumors, although both classifications appropriately differentiated the prognosis of more advanced disease. These observations suggest that a 3-tier localized, regional, and distant system could provide a more pragmatic option for prognostication. However, AJCC-rectum staging better discriminated between T stages, indicating that providers should consider utilizing this system in the clinical assessment of AA.

Introduction:
Improved oncological response of rectal cancer to neoadjuvant radiation therapy correlates with improved health outcomes. This response ranges from a complete response, or 0 on the AJCC-TRG scale, to little or no response, or a 3 on the scale. We previously demonstrated an association between increased SPATA20 expression and higher AJCC scores in a patient cohort. We hypothesize that knocking down SPATA20 in in-vitro rectal cell lines will increase their radiosensitivity.

Methods
HRT18 rectal cancer cell lines were transfected with a lentiviral construct containing small interfering RNAs (siRNA) targeting SPATA20 mRNA to create knockdown cell lines. Western blot was used for knockdown validation. Wildtype HRT18 and knockdown HRT18 cell lines were plated and irradiated with 10 Gy. Cell viability was assessed at the 48-hours interval using Annexin V/Propidium Iodide (PI) cell viability assay for both irradiated and non-irradiated controls.

Results
Decreased expression was confirmed in knockdown HRT18 cell lines compared to wildtype HRT18 using Western blot. In the non-radiated controls, Annexin V/PI demonstrated no statistical difference (p=0.63) in cell death when comparing wildtype HRT18 (28.3%) to knockdown HRT18 cell lines (30.6%). In the radiated samples on the other hand, there was a statistically significant increase (p=0.015) in cell death when comparing wildtype HRT18 (33.9%) to knockdown cell lines (52.87%). Comparing non-radiated to radiated knockdown HRT18, there was also a statistically significant increase (p=0.022) in cell death (30.6% vs 52.87%).

Conclusion
Manipulation of SPATA20 correlates with radiation response, signifying it as a possible modulator of radiosensitivity in rectal cancer. Further investigation is warranted to validate this observation in animal models and explore potential pharmacological inhibitors.

Introduction:
Cytoreductive surgery followed by HIPEC is a treatment option for peritoneal carcinomatosis for colorectal cancer; however, there is a lack of consensus on the benefit and type of chemotherapy agent utilized. We propose a novel epigenetic agent Mithramycin A (MA), as an alternative treatment to Mitomycin C as a cytotoxic agent with enhanced capability to remodel the tumor genetic landscape.
Methods:
Utilizing two colon cancer cell lines, HT-29 and CaCO2, we performed cell proliferation assays after treatment with MA (750nM) or MC (4.48uM) incubated at 37° or 42° Celsius for 90 min. RNA Sequencing using triplicate samples of the HT-29 cell line was performed. Resultant data were organized into 50 hallmark gene sets, normalized enrichment scores, log fold changes, and adjusted p-values. The top 6 hallmark gene sets with the greatest change in expression measured by adjusted p-value were cross-referenced against the Catalog of Somatic Mutations in Cancer (COSMIC) list of 733 known cancer-associated genes and the Tumor Suppressor Gene Database (TSGD) list of 535 known tumor suppressor genes in colon cancer.
Results:
Treatment of CaCo2 and HT-29 at 42° Celsius demonstrated comparable cell viability (CaCo2: MA 48.9% vs. MC 40.9%, p=0.23) (HT-29: MA 38.1% vs. MC 25.1%, p=0.18). RNA sequencing results showed MA treatment had dramatic global effects on gene expression, with 3,523 (26.1%) genes upregulated and 4,128 (30.6%) downregulated compared to MC, which had only 369 (2.7%) upregulated and 947 (7.0%) downregulated. Compared to the COSMIC data set, the top six hallmark pathways contained 57 unique gene expressions versus control with statistical significance. Two genes whose upregulation has been previously implicated in colon cancer progression saw significant downregulation when treated with MA versus control: MYB (Log fold D= -3.45, adj p-value= 3.11x10^-5) and MYH9 (Log fold D= -3.96, adj p-value= 4.65x10^-7). Top six hallmark pathways were compared to the TSGD database and 21 genes with a significant positive log fold change were found, including CDO1 (log fold D= 5.07, adj p-value=2.01x10^-4), GPX3 (log fold D=5.44, adj p-value 5.34x10^-7), DNAJB4 (log fold D= 2.43, adj p-value= 5.44x10^-4), CDKN1C (log fold D= 1.97, adj p-value= 0.002) CDKN1A (log fold D=2.28, adj p-value= 1.88x10^-5) and CAV1 (log fold D= 3.24, adj p-value= 7.66x10^-4).
Conclusion:
While Mithramycin A shows comparable efficacy in vitro against colon cancer cell lines to Mitomycin C, MA genetically remodels tumorigenesis by epigenetic modulation. Further investigation of this novel agent is warranted as a potential agent to reduce tumor recurrence by upregulation of previously repressed tumor suppressor genes.

Introduction
The peritumoral microenvironment is known to act to suppress the immune response against cancer cells, and tumor-infiltrating lymphocytes have a crucial role in immune surveillance. Obesity leads to an imbalance in adipokines, gut dysbiosis, and endotoxemia, as well as IGF-1 activation pathways and free fatty acids release that can influence the immune microenvironment. The cross-talk between tumor cells and the immune microenvironment can be detected in the normal “healthy” mucosa surrounding cancer, according to the concept of the field of cancerization. The aim of this study is to analyze the healthy rectal mucosa surrounding rectal cancer in overweight/obese patients who underwent surgery to evaluate the potential alteration of immune surveillance mechanisms of healthy rectal mucosal
Methods
This study is a sub-analysis of data from the IMMUNOREACT 1 and 2 trials (NCT04915326 and NCT04917263). In this multicentric study, we collected healthy mucosa surrounding rectal cancer. A panel of immune markers was retrospectively investigated at immunohistochemistry: CD3, CD4, CD8, CD8beta, Tbet, FoxP3, PD-L1, MSH6, and PMS2 and CD80. A prospective analysis was performed with fluorescence-activated cell sorting to determine the proportion of epithelial cells expressing CD80, CD86, CD40, HLA ABC or HLA DR and the proportion of activated CD8+ T cells, CD4+ Th1 cell, and T reg. Immune markers of healthy rectal mucosa were compared between patients under and over the BMI 25, between infiltrated margins, and between complete response or not, respectively.
Results
A total of 213 patients with rectal cancer, whose data on body mass index were available, were analyzed: 103 in the retrospective cohort and 110 in the prospective cohort. In our study group, 88 patients were normal -weight while 125 were overweight or obese (BMI>25). Overweight patients with rectal cancer had a lower expression of HLA-ABC on the surface of their epithelial cells than those with BMI under 25 (CK+HLA-abc+ MFI (p= 0.069). In particular, in patients undergoing neoadjuvant therapy, overweight ones had a lower frequency of high expression of HLA-ABC on epithelial cells than normal-weight patients. Moreover, overweight patients had a lower infiltration of CD8beta+ T cells within the healthy mucosa surrounding the cancers than those with BMI under 25 (p=0.04). Finally, the infiltration of CD8+ T-cells in the healthy mucosa inversely correlated with BMI (rho=-0.34, p=0.03).
Conclusions
Our findings suggest that in patients with rectal cancer, those who are overweight have a lower activation of epithelial cells as antigen-presenting cells and a lower activation of cytotoxic T-cells in their healthy mucosa surrounding rectal cancer. These data could be useful to plan a tailored approach to overweight/obese patients with a rectal cancer diagnosis.