Colorectal Cancer Surgical Specimens Research Articles

Abstract 276: Artificial intelligence-powered analysis of tumor-stroma ratio and fibroblast density as prognostic indicators in colorectal cancer

Abstract Introduction: The tumor microenvironment (TME) plays a critical role in cancer prognosis. In colorectal cancer (CRC), the tumor-stroma ratio (TSR) and cancer-associated fibroblast (CAF) density are emerging as prognostic markers. However, accurately quantifying them remains challenging. Recent studies have shown that artificial intelligence (AI)-powered analyzers can quantify features in the TME including these. Methods: Lunit SCOPE IO, an AI-powered H&E whole-slide image analyzer that detects fibroblasts and segments cancer area and stromal area, was applied to evaluate TSR and CAF density in treatment-naive, stage II or III CRC surgical specimens (N = 207) from Severance Hospital. TSR was calculated as the ratio of the area of the cancer area (CA) to the sum of the CA and the cancer stromal area (CS) within a slide. CAF density was evaluated as the fibroblast density within the CA or CS, or the ratio between them. Cox survival analysis determined the hazard ratios (HRs) in progression-free survival (PFS) between the high/low TSR/CAF groups, defining high/low groups based on optimized hazard ratio (HR) cutoffs. Results: The study found a mean TSR of 0.481±0.126 and CAF densities of 69.96±37.53/mm² (cancer area) and 1844.29±440.31/mm² (stromal area). Higher TSR, lower CAF density in CA, and higher CAF density in CS were linked to better prognosis (Table 1). The mean CAF ratio (CAF density of CS divided by CAF density of CA) was 33.92±25.35, and a group with high values showed a favorable prognosis. Dividing patients based on TSR and CAF ratio, we found that high levels in either or both metrics predicted better outcomes compared to low levels in both. Table 1. Prognosis by tumor-stroma ratio (TSR) and cancer-associated fibroblast (CAF) density Factor Group Hazard ratio (95% confidence interval) Mean progression-free survival (months) p-value TSR (cutoff: 0.351) Low (N = 34) Reference 43.7 <0.001 High (N = 173) 0.32 (0.19 - 0.56) 67.2 CAF density of cancer area (CA) (cutoff: 46.8) Low (N = 62) Reference 69.3 0.056 High (N = 145) 1.86 (0.98 - 3.50) 60.7 CAF density of cancer stromal area (CS) (cutoff:1,901) Low (N = 116) Reference 58.2 0.015 High (N = 91) 0.50 (0.29 - 0.88) 69.9 The ratio of CAF density of CS to CA (CAF ratio) (cutoff: 53.1) Low (N = 187) Reference 61.5 0.043 High (N = 20) 0.13 (0.02 - 0.94) 80.9 Combination of TSR and CAF ratio Both low (N = 32) Reference 41.2 <0.001 TSR high, CAF ratio low (N = 155) 0.32 (0.19 - 0.56) 65.8 TSR low, CAF ratio high (N = 2) 0.00 (0.00 - ∞) 83.4 Both high (N = 18) 0.06 (0.01 - 0.45) 80.2 Conclusions: In this study, we found a favorable prognosis in the group with higher TSR or higher density of CAF in CS compared to CA. This result suggests that AI-enhanced analysis of TSR and CAF can effectively predict prognosis in CRC, highlighting the potential roles of these markers in cancer prognosis. Citation Format: Minsun Jung, Jun Yong Kim, Mingu Kang, Jinhee Lee, Sanghoon Song, Taebum Lee, Wonkyung Jung, Soo Ick Cho. Artificial intelligence-powered analysis of tumor-stroma ratio and fibroblast density as prognostic indicators in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 276.

Profile of matrix‐entrapped extracellular vesicles of microenvironmental and infiltrating cell origin in decellularized colorectal cancer and adjacent mucosa

AbstractCellular elements that infiltrate and surround tumours and pre‐metastatic tissues have a prominent role in tumour invasion and growth. The extracellular vesicles specifically entrapped and stored within the extracellular matrix (ECM‐EVs) may reflect the different populations of the tumour microenvironment and their change during tumour progression. However, their profile is at present unknown. To elucidate this aspect, we isolated and characterized EVs from decellularized surgical specimens of colorectal cancer and adjacent colon mucosa and analyzed their surface marker profile. ECM‐EVs in tumours and surrounding mucosa mainly expressed markers of lymphocytes, natural killer cells, antigen‐presenting cells, and platelets, as well as epithelial cells, representing a multicellular microenvironment. No difference in surface marker expression was observed between tumour and mucosa ECM‐EVs in stage II‐III tumours. At variance, in the colon mucosa adjacent to stage IV carcinomas, ECM‐EV profile showed a significantly increased level of immune, epithelial and platelet markers in comparison to the matrix of the corresponding tumour. The increase of EVs from immune cells and platelets was not observed in the mucosa adjacent to low‐stage tumours. In addition, CD25, a T‐lymphocyte marker, resulted specifically overexpressed by ECM‐EVs from stage IV carcinomas, possibly correlated with the pro‐tolerogenic environment found in the corresponding tumour tissue. These results outline the tissue microenvironmental profile of EVs in colorectal carcinoma‐derived ECM and unveil a profound change in the healthy mucosa adjacent to high‐stage tumours.

Consistency and heterogeneity of microsatellite instability (MSI) status in paired biopsy and surgical specimens of colorectal cancer: A necessity for MSI reassessment after treatment?

149 Background: Microsatellite instability (MSI) plays a crucial role as a cancer immunotherapy and prognosis biomarker, making accurate MSI detection essential. However, currently, there is still a lack of available data on the impact of neoadjuvant therapy on MSI status, as well as validated data on standardized methods for MSI testing in small biopsy samples. The study aimed to investigate the concordance of MSI status between paired biopsy and surgical samples, as well as the impact of neoadjuvant therapy on MSI status using a novel MSI next-generation sequencing (MSI-NGS) detection panel. Methods: A total of 137 colorectal cancer (CRC) patients were enrolled for this study, of whom 116 with paired biopsy and surgical samples were analyzed. A custom MSI-NGS panel was employed and its performance was compared to MSI polymerase chain reaction (MSI-PCR), which served as the gold standard. Results: Out of the 116 cases, 112 patients showed consistent MSI status between biopsy and surgical samples, with an overall accuracy of 97% regardless of the detection method used. In the cases with MSI discrepancy (6 cases), 83% (5/6) of the patients showed a transition from MSS to MSI-H from biopsy to surgery. Interestingly, all eight patients who received neoadjuvant chemotherapy maintained an unchanged MSI status. However, in one patient who received adjuvant treatment and underwent a repeat biopsy after surgery, the MSI status exhibited alterations. Further analysis of clonal evolution revealed that this heterogeneity stemmed from the disappearance of the original clones and the emergence of new clones. Additionally, the NGS panel exhibited strong concordance with MSI-PCR and high accuracy, sensitivity and specificity with an AUC of 0.942 after rigorous validation. Conclusions: The study revealed a high concordance in MSI status between paired biopsy and surgical samples by employing a custom MSI-NGS panel for MSI status detection, providing reliable basis for further research in this field. Moreover, neoadjuvant chemotherapy seemed to have no impact on MSI status in our study, whereas postoperative adjuvant chemotherapy may influence changes in MSI status, highlighting the necessity of reevaluating MSI status after surgery. The findings also underscore the potential of NGS-based MSI detection as a valuable tool for clinical decision-making.

Outcomes for patients with Lynch Syndrome in Manitoba.

e22548 Background: Lynch Syndrome (LS) is the leading cause of hereditary colorectal cancer (CRC) and is also associated with an increased risk of extracolonic cancers including endometrial, ovarian, upper gastrointestinal tract and genitourinary malignancies. Since 2013 in Manitoba, Canada, all CRC surgical specimens in patients ≤70 undergo reflex screening for the mismatch repair (MMR) proteins (MLH1, MSH2, MSH6, PMS2) via immunohistochemistry. Since 2016, all endometrial cancers (EC) in patients ≤60 undergo similar reflex screening. The aim of this study was to examine the demographics, treatments and outcomes of patients with LS in Manitoba who have had a cancer diagnosis. Methods: Patients with pathogenic/likely pathogenic (P/LP) LS gene variants in Manitoba from 1999 were identified using records from the Program of Genetics and Metabolism. Those with a cancer diagnosis were identified using the Manitoba Cancer Registry (MCR). Non-melanomatous skin cancers and in-situ cancers were excluded. Descriptive statistics were used to report patient characteristics, LS gene variants, cancer diagnoses, treatments and outcomes. A survival analysis was undertaken using a matched cohort of patients with a diagnosis of CRC from 2004 to 2021 to compare overall survival (OS) between those with and without LS gene variants. A landmark survival analysis was performed to compare OS between those who had CRC before or after LS diagnosis. Results: 311 individuals with P/LP LS gene variants (96% pathogenic) and a record in the MCR were identified. The most common gene was MLH1 (33%), followed by MSH2 (29%), MSH6 (20%), PMS2 (16%) and EPCAM (2%). Most (72%) LS diagnoses occurred after 2014. There were 310 cancer diagnoses. The most common cancers amongst patients with LS were CRC (56%), EC (21%), urinary tract (5%), and ovarian (4%). Most cancer diagnoses (56%) occurred between 40-59 years old; 52% had stage I-II disease; 89% underwent surgery, 17% radiation & 40% received systemic therapy. Of 12 patients diagnosed with cancer at < 30 years old, 7 (58%) carried an MLH1 variant and 5 (42%) an MSH2 variant. 75 patients had ≥2 cancer diagnoses, 27 with an MLH1 variant (range 2-4); 30 MSH2 (2-5), 7 MSH6 (2-3), 9 PMS2 (2-3), 2 EPCAM (2-3). Using a matched CRC cohort to compare OS between those with and without LS, controlling for age, stage, sex, year of diagnosis, income quintile and treatment received, LS diagnosis was associated with a trend towards lower risk of death (HR 0.456, 95% CI 0.205-1.012, p = 0.053). There was no difference in OS according to whether a CRC diagnosis occurred pre- or post- LS diagnosis (p = 0.120). Conclusions: In this population-based study, the most common cancer diagnoses in patients with LS were CRC and EC. Many patients were diagnosed at a young age & experienced multiple cancer diagnoses. More LS diagnoses occurred after 2014, supporting the role of reflex tumor testing. Amongst those diagnosed with CRC, LS is associated with a trend towards improved OS.

Cytology Smears: An Enhanced Alternative Method for Colorectal Cancer pN Stage-A Multicentre Study.

Stage II colorectal cancer (CRC) recurrence remains a clinical problem. Some of these patients are true stage III CRC with a pN0 pathology stage. This large prospective multicentre cohort study aimed at evaluating the diagnostic ability of lymph node (LN) cytology smears to perform the pN stage and compare it with the conventional haematoxylin and eosin (H&E) pathology pN stage. Additionally, we used the One-Step Nucleic Acid Amplification (OSNA), a high-sensitive molecular method of LN staging. A total of 3936 fresh LNs from 217 CRC surgical specimens were examined by three methods, H&E, LN cytology smears, and OSNA. H&E detected 29% of patients with positive LNs, cytology smears 35%, and OSNA 33.2% (p < 0.0001). H&E and cytology concordantly classified 92.2% of tumours, and 88.5% between OSNA and H&E. Cytology had 96.8% sensitivity and 90.3% specificity to discriminate positive/negative patients compared to H&E (p = 0.004), and 87.3% sensitivity and 89% specificity when compared to OSNA (p = 0.56). Patients with positive LNs detected by any of the three methods had significantly worse disease-free and overall survival. We conclude that pN stage accuracy for detecting positive LNs is superior with LN cytological smears than with conventional H&E, which would enable a better pN stage and management of early-stage CRC patients.

Abstract 2887: Three dimensional colon cancer organoids model the response to CEA CD3 T cell engagers

Abstract Rationale: The CEA-CD3 T cell bispecific antibody cibisatamab (CEA-TCB) is currently undergoing clinical trials. Here we study its performance against three-dimensional tumor organoids in cocultures with T cells as compared to a higher affinity CEACAM5-CD3 (CEACAM5-TCB) bispecific antibody using time-lapse confocal microscopy. Methods: Pre-labelled spheroids derived from colon cancer cell lines and primary organoids derived from four colorectal cancer surgical specimens, which expressed different graded levels of CEA, were exposed in cocultures to T lymphocytes. Cocultures were treated with CEA-CD3 T-cell engagers and were followed by live confocal microscopy. Caspase 3 activation detected in real-time was used as an indicator of tumor cell death. Co-cultures were also set up with autologous tumor-associated fibroblasts to test the co-stimulatory effect of a fibroblast activated protein (FAP)- targeted 4-1BBL bispecific antibody fusion protein currently undergoing clinical trials. Results: Tumor-cell killing of 3D colon carcinoma cultures was dependent on the levels of surface CEA expression, in such a way that the lower affinity agent (CEA-TCB) did not mediate killing by human preactivated T cells below a certain CEA expression threshold, while the high affinity construct (CEACAM5-TCB) remained active on the low CEA expressing organoids. Modelling heterogeneity in the levels of CEA expression by coculturing CEA high and low organoids showed measurable but weak bystander killing. Cocultures of tumor organoids, autologous fibroblasts and T cells allowed to observe a costimulatory effect of anti-FAP-4-1BBL both to release IFNγ and to attain more efficacious tumor cell killing. Conclusion: Three-dimensional tumor cocultures with T cells using live confocal microscopy provide suitable models to test the requirements for colon-cancer redirected killing as elicited by CEA-targeted T-cell engagers undergoing clinical trials and treatment allow combinations to be tested in a relevant preclinical system. Citation Format: Alvaro Teijeira, Itziar Migueliz, Saray Garasa, Marina Bacac, Christian Klein, Ignacio Melero. Three dimensional colon cancer organoids model the response to CEA CD3 T cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2887.

Three-dimensional colon cancer organoids model the response to CEA-CD3 T-cell engagers.

Rationale: The CEA-CD3 T cell bispecific antibody cibisatamab (CEA-TCB) is currently undergoing clinical trials. Here we study its performance against three-dimensional tumor organoids in cocultures with T cells as compared to a higher affinity CEACAM5-CD3 (CEACAM5-TCB) bispecific antibody using time-lapse confocal microscopy.Methods: Pre-labelled spheroids derived from colon cancer cell lines and primary organoids derived from four colorectal cancer surgical specimens, which expressed different graded levels of CEA, were exposed in cocultures to T lymphocytes. Cocultures were treated with CEA-CD3 T-cell engagers and were followed by live confocal microscopy. Caspase 3 activation detected in real-time was used as an indicator of tumor cell death. Co-cultures were also set up with autologous tumor-associated fibroblasts to test the co-stimulatory effect of a fibroblast activated protein (FAP)- targeted 4-1BBL bispecific antibody fusion protein currently undergoing clinical trials.Results: Tumor-cell killing of 3D colon carcinoma cultures was dependent on the levels of surface CEA expression, in such a way that the lower affinity agent (CEA-TCB) did not mediate killing by human preactivated T cells below a certain CEA expression threshold, while the high affinity construct (CEACAM5-TCB) remained active on the low CEA expressing organoids. Modelling heterogeneity in the levels of CEA expression by coculturing CEA high and low organoids showed measurable but weak bystander killing. Cocultures of tumor organoids, autologous fibroblasts and T cells allowed to observe a costimulatory effect of anti-FAP-4-1BBL both to release IFNγ and to attain more efficacious tumor cell killing.Conclusion: Three-dimensional tumor cocultures with T cells using live confocal microscopy provide suitable models to test the requirements for colon-cancer redirected killing as elicited by CEA-targeted T-cell engagers undergoing clinical trials and treatment allow combinations to be tested in a relevant preclinical system.

The Prognostic Impact of Tumor Border Configuration, Tumor Budding and Tumor Stroma Ratio in Colorectal Carcinoma.

Tumor border configuration, tumor budding and tumor stroma ratio are reliable histopathological parameters that play a central role in the invasion-metastasis cascade. This study aimed to investigate the prognostic impact of these parameters and a new combined score in colorectal cancer. A cohort of 103 colorectal cancer surgical specimens was retrospectively evaluated for tumor border configuration, tumor budding and tumor stroma ratio using H&E sections. A combined risk score was then constructed to divide cases into low risk-tumors and high risk-tumors. Infiltrating tumor border, high tumor budding, low tumor stroma ratio and high combined risk score were associated with positive lymph node involvement, presence of metastasis, high tumor grade, lymphovascular invasion, poor overall survival and short recurrence-free survival. Infiltrating tumor border, high tumor budding and high combined risk score were associated with advanced T stage. High tumor budding, and low tumor stroma ratio were associated with perineural invasion. Infiltrating tumor border was associated with increased tumor size and conventional adenocarcinoma, high tumor budding and low tumor stroma ratio. Low tumor stroma ratio was associated with high tumor budding. On multivariate survival analysis, tumor stroma ratio was found to be an independent predictor for overall survival and recurrence-free survival. Tumor border configuration, tumor budding, tumor stroma ratio and the newly constructed combined risk score are potential predictors of outcome in colorectal cancer patients, suggesting that their incorporation in the routine histopathological evaluation could be useful in determining the prognosis of colorectal cancer cases.

Dataset for Pathology Reporting of Colorectal Cancer: Recommendations From the International Collaboration on Cancer Reporting (ICCR).

Objective:The aim of this study to describe a new international dataset for pathology reporting of colorectal cancer surgical specimens, produced under the auspices of the International Collaboration on Cancer Reporting (ICCR).Background:Quality of pathology reporting and mutual understanding between colorectal surgeon, pathologist and oncologist are vital to patient management. Some pathology parameters are prone to variable interpretation, resulting in differing positions adopted by existing national datasets.Methods:The ICCR, a global alliance of major pathology institutions with links to international cancer organizations, has developed and ratified a rigorous and efficient process for the development of evidence-based, structured datasets for pathology reporting of common cancers. Here we describe the production of a dataset for colorectal cancer resection specimens by a multidisciplinary panel of internationally recognized experts.Results:The agreed dataset comprises eighteen core (essential) and seven non-core (recommended) elements identified from a review of current evidence. Areas of contention are addressed, some highly relevant to surgical practice, with the aim of standardizing multidisciplinary discussion. The summation of all core elements is considered to be the minimum reporting standard for individual cases. Commentary is provided, explaining each element's clinical relevance, definitions to be applied where appropriate for the agreed list of value options and the rationale for considering the element as core or non-core.Conclusions:This first internationally agreed dataset for colorectal cancer pathology reporting promotes standardization of pathology reporting and enhanced clinicopathological communication. Widespread adoption will facilitate international comparisons, multinational clinical trials and help to improve the management of colorectal cancer globally.

A new practical classification of desmoplastic reaction in endoscopic forceps biopsy of colorectal cancer.

The histopathological discrepancy between endoscopic forceps biopsy (EFB) and post-resection specimens is considered a practical clinical problem. This retrospective study aimed to determine the current diagnostic concordance between the EFB and surgical specimens of colorectal cancer (CRC) and then investigated the useful factors in EFB diagnosis. We used the representative pathological data of 2188 CRCs. The comparison of histopathological discrepancy between EFB and the related surgical specimens was performed. Furthermore, 418 biopsy specimen slides in our hospital were reviewed to determine the classification of intratumor desmoplastic reaction (DR). Among the 2188 patients, the positive sensitivity of EFB for adenocarcinoma was 82.7%. The discrepancy rate between the EFB and surgical specimens was 10.8-40.0% corresponding to different T stages. On the basis of DR classification, 32, 131, and 255 tumors were categorized as little, moderate and extensive, respectively. The correlation between DR classification and tumor invasion based on T stage was significant (Spearman's rho= 0.112; p<0.05). The extensive DR provided better estimates for advanced tumors than the little and moderate DR (χ²= 3.977, p=0.046). Besides DR, factors including deeper cutting the slides and histological types were significantly associated with "adenocarcinoma" diagnosis in EFB of CRCs (p<0.05). To the best of our knowledge, this is the first time that a DR classification specifically for EFB specimens was proposed. It might contribute to improve the accuracy of biopsy-based diagnosis of CRC.

Circulating let-7e-5p, miR-106a-5p, miR-28-3p, and miR-542-5p as a Promising microRNA Signature for the Detection of Colorectal Cancer.

Simple SummaryThe detection of early-stage colorectal cancer increases the chance to prevent tumor progression and death by the disease. Colonoscopy is one sensitive screening test to detect malignant or potentially malignant lesions in the intestines. However, it has some disadvantages, including sedation requirements, increased risk of colon perforation, and bleeding. Circulating microRNAs (miRNAs) in plasma or serum from cancer patients have been investigated and described as potential diagnostic or prognostic markers. We conducted an miRNAs screening test in plasma samples from colorectal cancer patients and subjects without cancer, aiming to identify markers for the early detection of the disease. We identified and validated four miRNAs capable of distinguishing cancer from non-cancer cases. Our non-invasive diagnostic biomarkers presented high performance and are easily applicable to clinical practice.Colorectal cancer (CRC) is a disease with high incidence and mortality. Colonoscopy is a gold standard among tests used for CRC traceability. However, serious complications, such as colon perforation, may occur. Non-invasive diagnostic procedures are an unmet need. We aimed to identify a plasma microRNA (miRNA) signature for CRC detection. Plasma samples were obtained from subjects (n = 109) at different stages of colorectal carcinogenesis. The patients were stratified into a non-cancer (27 healthy volunteers, 17 patients with hyperplastic polyps, 24 with adenomas), and a cancer group (20 CRC and 21 metastatic CRC). miRNAs (381) were screened by TaqMan Low-Density Array. A classifier based on four differentially expressed miRNAs (miR-28-3p, let-7e-5p, miR-106a-5p, and miR-542-5p) was able to discriminate cancer versus non-cancer cases. The overexpression of these miRNAs was confirmed by RT-qPCR, and a cross-study validation step was implemented using eight data series retrieved from Gene Expression Omnibus (GEO). In addition, another external data validation using CRC surgical specimens from The Cancer Genome Atlas (TCGA) was carried out. The predictive model’s performance in the validation set was 76.5% accuracy, 59.4% sensitivity, and 86.8% specificity (area under the curve, AUC = 0.716). The employment of our model in the independent publicly available datasets confirmed a good discrimination performance in five of eight datasets (median AUC = 0.823). Applying this algorithm to the TCGA cohort, we found 99.5% accuracy, 99.7% sensitivity, and 90.9% specificity (AUC = 0.998) when the model was applied to solid colorectal tissues. Overall, we suggest a novel signature of four circulating miRNAs, i.e., miR-28-3p, let-7e-5p, miR-106a-5p, and miR-542-5p, as a predictive tool for the detection of CRC.

Lymph Node Tumor Burden Correlates With Tumor Budding and Poorly Differentiated Clusters: A New Prognostic Factor in Colorectal Carcinoma?

INTRODUCTION:Molecular lymph node (LN) staging in early colorectal cancer (CRC) has demonstrated to be more precise than conventional histopathology pN staging. Tumor budding (TB) and poorly differentiated clusters (PDCs) are associated with LN metastases, recurrences, and lower survival in CRC. We evaluated the correlation between the total tumor load (TTL) in LNs from CRC surgical specimens with patient outcome, TB, and PDC.METHODS:In this retrospective multicentre study, 5,931 LNs from 342 stage I–III CRC were analyzed by both hematoxylin and eosin and molecular detection of tumor cytokeratin 19 mRNA by one-step nucleic acid amplification. TB and PDC were evaluated by hematoxylin and eosin and cytokeratin 19 immunohistochemistry.Results:One-step nucleic acid was positive in 38.3% patients (n = 131). Tumor Budding was low in 45% cases, intermediate in 25%, and high in 30%. Poorly Differentiated Clusters were low-grade G1 in 53%, G2 in 32%, and G3 in 15%. TB and PDC correlated with TTL, high-grade, lymphovascular and perineural invasion, pT, pN and stage (P < 0.001). TB, PDC, and TTL ≥ 6,000 copies/µL were associated with worse overall survival (P = 0.002, P = 0.013, and P = 0.046) and disease-free survival (P < 0.001).Discussion:The implementation of more sensitive molecular methods to assess LN status is a promising alternative approach to pN staging, which could be integrated to other factors to help risk stratification and management of patients with early-stage CRC. This study demonstrates the correlation of the amount of LN tumor burden with TB and PDCs. TTL is related to the outcome and could be used as a new prognostic factor in CRC (see Visual , Supplementary Digital Content 2, http://links.lww.com/CTG/A512).

Predictors of lymph-node metastasis in surgically resected T1 colorectal cancer in Western populations.

BackgroundThe risk of lymph-node metastasis (LNM) in T1 colorectal cancer (CRC) has not been well documented in heterogeneous Western populations. This study investigated the predictors of LNM and the long-term outcomes of patients by analysing T1 CRC surgical specimens and patients’ demographic data.MethodsPatients with surgically resected T1 CRC between 2004 and 2014 were identified from the Surveillance, Epidemiology, and End Results (SEER) database. Patients with multiple primary cancers, with neoadjuvant therapy, or without a confirmed histopathological diagnosis were excluded. Multivariate logistic-regression analysis was used to identify the predictors of LNM.ResultsOf the 22,319 patients, 10.6% had a positive lymph-node status based on the final pathology (nodal category: N1 9.6%, N2 1.0%). Younger age, female sex, Asian or African-American ethnicity, poor differentiation, and tumor site outside the rectum were significantly associated with LNM. Subgroup analyses for patients stratified by tumor site suggested that the rate of positive lymph-node status was the lowest in the rectum (hazard ratio: 0.74; 95% confidence interval: 0.63–0.86).ConclusionThe risk of LNM was potentially lower in Caucasian patients than in API or African-American patients with surgically resected T1 CRC. Regarding the T1 CRC site, the rectum was associated with a lower risk of LNM.

The meiosis-specific cohesin component stromal antigen 3 promotes cell migration and chemotherapeutic resistance in colorectal cancer.

Chromosome instability is one of the hallmarks of cancer. Stromal antigen (STAG) 3 is a core component of the meiosis-specific cohesin complex, which regulates sister chromatid cohesion. Although aberrantly activated genes encoding the cohesin complex have been identified in cancers, little is known about the role of STAG3 in colorectal cancer (CRC). Here, we evaluated the prognostic impact and role of STAG3 in CRC. Analysis of 172 CRC surgical specimens revealed that high STAG3 expression was associated with poor prognosis. STAG3 knockdown inhibited cell migration and increased drug sensitivity to oxaliplatin, 5-fluorouracil, irinotecan hydrochloride hydrate, and BRAF inhibitor in CRC cell lines. The enhanced drug sensitivity was also confirmed in a human organoid established from a CRC specimen. Moreover, suppression of STAG3 increased γH2AX foci. Particularly, in BRAF-mutant CRC cells, STAG3 silencing suppressed the expression of snail family transcriptional repressor 1 and phosphorylation of extracellular signal-regulated kinase via upregulation of dual-specificity phosphatase 6. Our findings suggest that STAG3 is related to poor clinical outcomes and promotes metastasis and chemotherapeutic resistance in CRC. STAG3 may be a novel prognostic marker and potential therapeutic target for CRC.

Syntenin-1 promotes colorectal cancer stem cell expansion and chemoresistance by regulating prostaglandin E2 receptor

BackgroundThe protein syntenin-1 is expressed by a variety of cell types, and is upregulated in various malignancies, including melanoma, breast cancer and glioma. Although the mechanism by which elevated syntenin-1 expression contributes to cancer has been described, the exact pathway has not been elucidated.MethodsTo investigate the involvement of syntenin-1 in colorectal cancer (CRC), we performed immunohistochemical analysis of 139 CRC surgical specimens. We also examined syntenin-1 knockdown in CRC cell lines.ResultsHigh syntenin-1 expression was associated with less differentiated histologic grade and poor prognosis, and was an independent prognostic indicator in CRC. Syntenin-1 knockdown in CRC cells reduced the presence of cancer stem cells (CSCs), oxaliplatin chemoresistance and migration. DNA microarray analysis and quantitative real-time polymerase chain reaction showed decreased prostaglandin E2 receptor 2 (PTGER2) expression in syntenin-1-knockdown cells. PTGER2 knockdown in CRC cells yielded the same phenotype as syntenin-1 knockdown. Celecoxib, which has anti-inflammatory effects by targeting cyclooxygenase-2, reduced CSCs and decreased chemoresistance, while prostaglandin E2 (PGE2) had the opposite effect.ConclusionsOur findings suggested that syntenin-1 enhanced CSC expansion, oxaliplatin chemoresistance and migration capability through regulation of PTGER2 expression. Syntenin-1 may be a promising new prognostic factor and target for anti-cancer therapies.

Band 3 ectopic expression in colorectal cancer induces an increase in erythrocyte membrane-bound IgG and may cause immune-related anemia.

Autoimmune hemolytic anemia (AIHA) is a rare comorbidity in colorectal cancer (CRC) and has an unknown etiology. Previously, we described an AIHA case secondary to CRC with ectopic band 3 expression. Herein, we investigated ectopic band 3 expression and erythrocyte membrane-bound IgG in a CRC cohort. Between September 2016 and August 2018, 50 patients with CRC and 26 healthy controls were enrolled in the present study. The expression of band 3 and SLC4A1 mRNA was observed in 97% of CRC surgical specimens. Although clinical AIHA was not observed in any patient with CRC, a direct antiglobulin test was positive in 10 of the patients in the CRC group (p = 0.01). Flow cytometry revealed significantly increased erythrocyte membrane-bound IgG among patients with CRC compared to healthy controls (mean ± standard deviation; 38.8 ± 4.7 vs. 29.9 ± 15.6, p = 0.012). Normocytic anemia was observed, including in cases negative for fecal occult blood, suggesting a shortened erythrocyte life-span due to increased membrane-bound IgG. Immunoprecipitation revealed increased anti-band 3 autoantibodies in patients' sera. Mouse experiments recapitulated this phenomenon. We also confirmed that band 3 expression is controlled by 5'AMP-activated protein kinase under hypoxic conditions. These findings increase our understanding of the etiology of cancer-related anemia.

Promotion of the Warburg effect is associated with poor benefit from adjuvant chemotherapy in colorectal cancer.

Metabolic reprogramming, including the Warburg effect, is a hallmark of cancer. Indeed, the diversity of cancer metabolism leads to cancer heterogeneity, but accurate assessment of metabolic properties in tumors has not yet been undertaken. Here, we performed absolute quantification of the expression levels of 113 proteins related to carbohydrate metabolism and antioxidant pathways, in stage III colorectal cancer surgical specimens from 70 patients. The Warburg effect appeared in absolute protein levels between tumor and normal mucosa specimens demonstrated. Notably, the levels of proteins associated with the tricarboxylic citric acid cycle were remarkably reduced in the malignant tumors which had relapsed after surgery and treatment with 5‐fluorouracil‐based adjuvant therapy. In addition, the efficacy of 5‐fluorouracil also decreased in the cultured cancer cell lines with promotion of the Warburg effect. We further identified nine and eight important proteins, which are closely related to the Warburg effect, for relapse risk and 5‐fluorouracil benefit, respectively, using a biomarker exploration procedure. These results provide us a clue for bridging between metabolic protein expression profiles and benefit from 5‐fluorouracil adjuvant chemotherapy.

Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

BackgroundLong non-coding RNAs (lncRNAs) are tumor-associated biological molecules and have been found to be implicated in the progression of colorectal cancer (CRC). This study aims to examine the effects of lncRNA RP11-468E2.5 and its target genes (STAT5 and STAT6) on the biological activities of CRC cells via the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway.MethodsWe initially screened the GEO database for differentially expressed lncRNAs related to CRC and then made a prediction of the implicated target genes. Then we collected CRC tissues and adjacent normal tissues from 169 CRC patients. Human CRC HCT116 and SW480 cells were treated with small interference RNA (siRNA) against RP11-468E2.5, AG490 (an inhibitor of the JAK/STAT signaling pathway), or both in combination. Next, we measured the effects of RP11-468E2.5 treatment on cellular activities such as cell viability, cycle distribution and cell apoptosis, and studied interactions among RP11-468E2.5, STAT5/STAT6, and the JAK/STAT signaling pathway. Finally, an in vivo tumor formation assay was performed to observe the effect of RP11-468E2.5 on tumor growth.ResultsThe CRC-related gene microarray data showed low expression of RP11-468E2.5 in CRC surgical specimens. However, RP11-468E2.5 was confirmed to target STAT5 and STAT6, which participate in the JAK/STAT signaling pathway. CRC tissues showed lower expression of RP11-468E2.5, higher expression of STAT5, STAT6 and of the cell cycle marker Cyclin D1 (CCND1), compared to the findings in adjacent normal tissues. The treatment of siRNA against RP11-468E2.5 increased expression of JAK2, STAT3, STAT5, STAT6, CCND1 and Bcl-2 along with the extent of STAT3, STAT5 and STAT6 phosphorylation, while lowering expression of P21 and P27. Treatment with AG490 exhibited approximately opposite effects, whereas siRNA against RP11-468E2.5 treatment stimulated CRC cell proliferation and reduced cell apoptosis, while promoting cell cycle entry; AG490 treatment reversed these results.ConclusionsAltogether, we conclude that up-regulation of RP11-468E2.5 inhibits the JAK/STAT signaling pathway by targeting STAT5 and STAT6, thereby suppressing cell proliferation and promoting cell apoptosis in CRC.

Phase 1 with expansion cohorts in a study of NEO-201 in adults with chemo-resistant solid tumors.

TPS2645 Background: NEO-201 is a novel humanized IgG1 monoclonal antibody (mAb) generated against the Hollinshead allogenic colorectal cancer vaccine platform. Briefly, tumor-associated antigens (TAA) derived from tumor membrane fractions pooled from colorectal cancer surgical specimens were screened for delayed-type hypersensitivity and evaluated in clinical trials. The original vaccine was used to generate monoclonal antibodies, one of which is NEO-201. In preclinical data generated in our laboratory, we have demonstrated that NEO-201 exert anti-tumor activity by natural killer (NK)-mediated antibody-dependent cytotoxicity (ADCC) against several tumor type including colorectal and pancreatic cancer models (Fantini, et al. 2018). We have identified NEO-201 antigen as a glycosylated form of CEACAM-5 and -6, which is expressed by tumor tissue but is not present in the surrounding healthy tissue (David, et al. 2018). This could result in a specific anti-tumor activity without significant normal tissue toxicity. Nevertheless, toxicity was further assessed in non-human primates and transient neutropenia was the only adverse event observed. Based on this data we designed a first in human phase I trial to evaluate the safety, maximum-tolerated dose (MTD), pharmacokinetics (PK) and pharmacodynamics (PD) of the humanized monoclonal antibody NEO-201. Methods: This is a first-in-human phase 1 study with expansion cohort to determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of NEO-201 in adults with advanced solid tumors that have high likelihood pof expression NEO201 antigen and have progressed to standard of treatments and have a PS0-2 ECOG. Study design is a classic Fibonacci (3+3) dose escalation, with a cohort expansion at the MTD. NEO-201 is administered intravenously every two weeks, and four different dose levels will be explored (DL1 = 1mg/kg, DL2 = 2mg/kg, DL3 = 4mg/kg and DL4 = 6mg/kg). No intra-patient dose escalation is allowed. Patients will be evaluated for safety every two weeks, with weekly laboratory testing, according to CTCAEv4.0. and with a DLT window of 28 days (cycle 1). Response will be assessed every 8 weeks (2 cycles of treatment) according to RECISTv1.1. Additionally, biological samples will be collected to understand NEO-201 pharmacokinetic, the effect on the immune process and their correlation with treatment toxicity and response. As of February 2019 we have completed enrollment in the first DL and are evaluating for DLT. Clinical trial information: NCT03476681.

Involvement of MAF1homolog, negative regulator of RNA polymeraseIII in colorectal cancer progression.

Polymerase (Pol) III dependent transcription controls the abundance of transfer RNAs, 5S ribosomal RNA and small non coding RNAs within cells, and is known to serve an essential role in the maintenance of intracellular homeostasis. However, its contribution to cancer progression has not been extensively explored. The present study demonstrated that the evolutionarily conserved MAF1 homolog, negative regulator of RNA Pol III (MAF1) may be closely associated with malignant potential and poor prognosis in colorectal cancer (CRC). Notably, immunohistochemical analysis of 146 CRC surgical specimens revealed that high expression levels of MAF1 were associated with advanced tumor depth, lymph node metastasis, distant metastasis and poor prognosis. In vitro loss of function assays revealed that MAF1 knockdown suppressed chemoresistance and migration of CRC cancer cells. Furthermore, detailed analysis of an independent CRC dataset (n=615) demonstrated that the prognostic impact of MAF1 gene expression was particularly marked in microsatellite instability (MSI)-positive patients, who benefit from immune checkpoint blockade. High expression levels of MAF1 were revealed to be an independent prognostic indicator in MSI-positive CRC. These findings suggested that MAF1 may have an essential role in CRC progression, particularly in MSI positive cases.