DEVELOPMENT AND VALIDATION OF THE ILEOANAL POUCH SYNDROME DISTRESS INVENTORY

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.

Background:
Inflammatory bowel disease and disorders of gastrointestinal motility overlap significantly in the clinical setting. While ileus is commonly seen, the mechanisms involved remain poorly understood, in part due to a lack of robust in vivo models. Prior studies have shown that serotonergic signaling plays a role in control of intestinal motility, although the precise mechanisms by which colonic and ileal motility are linked during inflammatory processes remain unclear. We hypothesize that the small intestinal dysmotility seen in a colonic inflammation model is precipitated through aberrant changes in serotonergic signaling from the inflamed colon to the small intestine.

Methods:
Four-week-old C57BL/6 mice were randomized into control and DSS groups. DSS groups received 7-day exposure to DSS (MW 40,000–50,000 kDa) ad libitum at concentrations of 2.5% and 5% in 0.5% sucrose drinking water. Mice were sacrificed on day 7. 70kda fluorescein-dextran was administered and animals were sacrificed 30 minutes later; the whole intestine was divided into 2cm sections. Sections were homogenized, and FITC-fluorescence measured in the supernatant. The small intestinal transit time was derived from the position of the geometric (Geom) center (GC) of FITC-dextran. Colon length was measured, and samples of stomach, ileum, and colon were taken. mRNA expression of interleukin 6 (IL-6), interleukin 1ß (IL1ß), and lipocalin-2 (LCN2) as well as serotonergic pathway genes tryptophan hydroxylase (Tph1), and serotonin transporter (5’HTT) were normalized to ribosomal protein large P0 (Rplp0).

Results:
Compared to controls, mice receiving 2.5% or 5% DSS gained less percent weight (Ctrl=24.50 vs 2.5%DSS=5.18 vs 5%DSS= -7.41; p<0.0001), had shorter colons (Ctrl=59.35mm vs 2.5%DSS=50.57mm vs 5%DSS=41.00mm; p<0.05) and showed significant upregulation of antimicrobial peptide, LCN2 (Ctrl=38.50 vs 2.5%DSS= 218.40; p<0.01) and pro-inflammatory cytokines IL6 (Ctrl=0.94 vs 2.5%DSS=4.38; p<0.05) and IL1ß (Ctrl=9.61 vs 2.5%DSS=69.95; p<0.01). DSS significantly reduced intestinal motility (Ctrl, GC=10.5 vs 2.5%DSS, GC=8 vs 5%DSS, GC=6; p<0.01). Ileal serotonergic markers showed significant alteration in DSS groups with Tph1 gene expression upregulated (Ctrl=4.01 vs 2.5%DSS=6.25; p<0.05) and serotonin transporter (5’HTT) expression downregulated (Ctrl=91.68 vs 2.5%DSS=52.04; p<0.05). However, no significant changes in key pro-inflammatory cytokines (IL6, IL1β and TNFα) were found in the terminal ileum.

Conclusions:
Colonic inflammation induced significant intestinal dysmotility that was associated with changes in the expression of genes that regulate serotonergic signaling. These findings suggest the potential for novel approaches for the control of colitis associated ileus through modulation of the serotonergic signaling system.

Background
Restorative proctocolectomy (TPC) with ileal pouch-anal anastomosis (IPAA) is the gold standard surgical treatment of ulcerative colitis (UC), offering complete removal of the diseased colon and effective prevention and treatment of colorectal cancer. Segmental colectomy (SC) is rarely performed, though considered in select patients such as those with quiescent disease, or elderly with comorbidities who are high risk for postoperative morbidity and poor function following IPAA surgery. We aimed to assess postoperative and long-term outcomes of SC in UC
Methods
Retrospective chart review of UC patients who underwent SC at our tertiary care center from 1999 to 2022 was performed. Primary outcomes were postoperative complication, early flare, cancer recurrence, & subsequent total abdominal colectomy (TAC)/TPC rate. Data is presented as mean (standard deviation), median [25-75 percentiles] or frequency (percent)
Results
61 patients [21 (34.4%) female] were identified. Median age at the time of diagnosis and surgery were 41.5 (30-59) and 67.7 (56.8-76.5) years, respectively. Median body mass index was 27.9 (24.6-31.7) kg/m². 36 (59%) patients had American Society of Anesthesiologists score of III. 53 (86.9%) patients had at least one comorbidity. 51 (83.6%) patients had Mayo score of 0-1 at the time of surgery. Patients underwent: Right hemicolectomy (n=30, 49.2%), left hemicolectomy (n=6, 9.8%), sigmoidectomy (n=19, 31.1%), low anterior resection (n=4, 6.6%), and segmental resection (n=2, 9.8%) for cancer (n=15, 24.6%), dysplasia/polyps (n=21, 34.4%), diverticular disease (n=13, 21.3%), stricture (n=6, 9.8%), fistula (n=3, 4.9%), bleeding (n=1, 1.6%), and megacolon (n=1, 1.6%). The median duration of operation and estimated blood loss were 168.5 (130-210) min and 100 (40-150) ml, respectively. 18 (29.5%) patients had postoperative complications, most commonly ileus (n=7, 11.5%). 7 (11.5%) patients had complications of Clavien-Dindo Class III-V. Median time to start of diet and return of bowel function were 1.5 (1-4) and 3 (2-4) days, respectively. One (1.6%) patient had anastomotic leak, while sepsis occurred in 1 (1.6%) patient. Median length of hospital stay was 5 (3-8.5) days. Early postoperative flare (within 3 months of surgery) occured in 5 (8.2%) patients. Among 36 patients with dysplasia/cancer, 1 had metachronous colon cancer and 1 developed distant recurrence at 19.5 and 23 months after surgery, respectively. 4 (6.6%) patients underwent subsequent TAC/TPC. Median follow-up time was 35.5 (5.5-63.9) months. 2-year and 5-year overall survival rates were 92% and 78.7%, respectively
Conclusion
In selected UC patients, such as elderly patients with comorbidities who have no-minimal disease activity, SC can be a safe and feasible option providing low postoperative complication, early flare, cancer recurrence and need for subsequent TAC/TPC rates

INTRODUCTION
Limited evidence exists regarding survival benefit of adjuvant chemotherapy for non-metastatic appendiceal adenocarcinoma patients, and many providers defer to colorectal adenocarcinoma guidelines for adjuvant therapy regimens. Decision-making regarding systemic therapy initiation is often based on nodal positivity, to which adequate staging is achieved via right hemicolectomy (RHC). However, treatment in the elderly patient population can present additional considerations given the potential morbidity of surgery and systemic therapy. We sought to analyze outcomes in elderly patients with node-positive appendiceal adenocarcinoma.

METHODS
Patients diagnosed with Stage III appendiceal adenocarcinoma who underwent a RHC with 12 or more examined lymph nodes were identified using the National Cancer Database (2004-2019). Elderly patients were defined as patients with age of diagnosis greater than 65 years old, while Non-elderly patients were defined as patients with age of diagnosis 65 years or younger. Propensity score matching (PSM) was performed between elderly and non-elderly patients adjusting for gender, race, Charlson-Deyo Comorbidity Index (CCI), number of positive lymph nodes, and grade. Chi-squared testing, Kaplan-Meier method with log-rank test, and Cox proportional hazards regression models were employed.

RESULTS
1475 patients were identified, with 58.2% (858) elderly patients and 41.8% (617) non-elderly patients. Elderly patients were more likely to present with a CCI ≥ 2 (9.7% v. 3.8%, p<0.001). Tumor grade or mucinous histology were similar between the groups. Elderly patients were less likely to receive chemotherapy (68.6% v. 85.3%, p<0.001) or multi-agent chemotherapy (48.4% v. 74.3%, p<0.001). On unadjusted Kaplan Meier analyses, elderly patients receiving single-agent chemotherapy had similar median survival (54.1 mo, 95% CI 11.5-31.6) compared to multi-agent (59.8 mo, 95% CI 49.0-70.6), while both conferred survival advantage compared to no chemotherapy (19.4 mo, 95% CI 13.0-25.7) (Figure 1A). On multivariate analyses after PSM, single-agent (HR 0.46) and multi-agent (HR 0.41) chemotherapy regimens conferred a survival advantage compared to elderly patients who did not receive chemotherapy. There was no survival advantage between single-agent versus multi-agent (p=0.55). Greater than 3 positive lymph nodes (HR 1.73) and grade (Grade 2 HR 2.32, Grade 3 HR 2.90, Grade 4 HR 4.02) were associated with decreased survival.

CONCLUSIONS
Elderly patients presenting with stage III appendiceal adenocarcinoma confer a survival benefit from adjuvant chemotherapy after right hemicolectomy, regardless of histological grade or CCI score. However, multiagent chemotherapeutic regimens demonstrate a diminished survival advantage, and toxicities of these regimens may outweigh benefit in elderly patients.

Background: The Patient Reported Outcomes after Pouch Surgery (PROPS) Delphi consensus study identified a list of seven bowel symptoms and seven consequences that were utilized to develop and validate the Ileoanal Pouch Syndrome Severity Index score. The aim of the present study was to develop a meaningful measure of bowel function that accounts for the possibility of patients perceiving the same symptoms differently over time.

Methods: Patients who had a proctocolectomy with ileoanal pouch for ulcerative colitis ≥ 12 months of restored intestinal continuity were recruited by a combination of mail/email to patients treated at 11 high volume IBD centers, and online advertisements through the Crohn’s and Colitis Foundation social media pages. After obtaining consent, questionnaires regarding bowel function were administered. Participants were asked to rate how “bothersome” each symptom was on a scale of 1-9. Participants also reported on quality of life. Individual question score values were designated to form the “Ileoanal Pouch Syndrome Distress Inventory”. Validity was tested by receiver operating characteristic (ROC) curve analyzing the association between distress inventory score and quality of life. Convergent validity was assessed by comparing scores to the LARS, FIQoL, MSK bowel function score, and Wexner incontinence score. Lastly, clinical validity was assessed by comparing scores between patients based on the absence of key clinical variables.

Results: Questionnaires were completed by 676 patients eligible for inclusion. Weighted scores based on symptom bother scores were computed on the basis of the questionnaire results. The range of possible scores was 0 to 64 (Interquartile range 12-38). As scores increased, the percentage of patients reporting good quality of life decreased and the percentage of patients reporting poor quality of life increased (Figure 1). The distress index score was then used to predict poor quality of life, where the ROC curve showed an area under the curve of 0.87. This is compared to an area under the curve of 0.85 using the IPS severity index score. The score showed excellent convergent validity with all tested bowel function scores (p<0.001) and was highly correlated with long term outcomes such as pouchitis, cuffitis, and other pouch complications.

Conclusions: This study developed a patient-centered scoring system that utilizes patients perception of how bothersome various symptoms are, and then correlates these reported perceptions with quality of life. This tool will ideally allow clinicians to follow individual patients as they potentially accommodate to their symptoms and learn to cope over time, even if the actual frequency/severity of symptoms remains unchanged, which may allow for a more detailed understanding of how various interventions affect the quality of life of patients with pouches.