Colorectal Carcinoma Samples Research Articles

Chromosome instability functions as a potential therapeutic reference by enhancing chemosensitivity to BCL-XL inhibitors in colorectal carcinoma.

Chromosome instability (CIN) and subsequent aneuploidy are prevalent in various human malignancies, influencing tumor progression such as metastases and relapses. Extensive studies demonstrate the development of chemoresistance in high-CIN tumors, which poses significant therapeutic challenges. Given the association of CIN with poorer prognosis and suppressed immune microenvironment observed in colorectal carcinoma (CRC), here we aimed to discover chemotherapeutic drugs exhibiting increased inhibition against high-CIN CRC cells. By using machine learning methods, we screened out two BCL-XL inhibitors Navitoclax and WEHI-539 as CIN-sensitive reagents in CRC. Subsequent analyses using a CIN-aneuploidy cell model confirmed the vulnerability of high-CIN CRC cells to these drugs. We further revealed the critical role of BCL-XL in the viability of high-CIN CRC cells. In addition, to ease the evaluation of CIN levels in clinic, we developed a three-gene signature as a CIN surrogate to predict prognosis, chemotherapeutic and immune responses in CRC samples. Our results demonstrate the potential value of CIN as a therapeutic target in CRC treatment and the importance of BCL-XL in regulating survival of high-CIN CRC cells, therefore representing a valuable attempt to translate a common trait of heterogeneous tumor cells into an effective therapeutic target.

The genomic landscape of 2,023 colorectal cancers

Colorectal carcinoma (CRC) is a common cause of mortality1, but a comprehensive description of its genomic landscape is lacking2–9. Here we perform whole-genome sequencing of 2,023 CRC samples from participants in the UK 100,000 Genomes Project, thereby providing a highly detailed somatic mutational landscape of this cancer. Integrated analyses identify more than 250 putative CRC driver genes, many not previously implicated in CRC or other cancers, including several recurrent changes outside the coding genome. We extend the molecular pathways involved in CRC development, define four new common subgroups of microsatellite-stable CRC based on genomic features and show that these groups have independent prognostic associations. We also characterize several rare molecular CRC subgroups, some with potential clinical relevance, including cancers with both microsatellite and chromosomal instability. We demonstrate a spectrum of mutational profiles across the colorectum, which reflect aetiological differences. These include the role of Escherichiacolipks+ colibactin in rectal cancers10 and the importance of the SBS93 signature11–13, which suggests that diet or smoking is a risk factor. Immune-escape driver mutations14 are near-ubiquitous in hypermutant tumours and occur in about half of microsatellite-stable CRCs, often in the form of HLA copy number changes. Many driver mutations are actionable, including those associated with rare subgroups (for example, BRCA1 and IDH1), highlighting the role of whole-genome sequencing in optimizing patient care.

Pokemon inhibits Bim transcription to promote the proliferation, anti-anoikis, invasion, histological grade, and dukes stage of colorectal neoplasms

PurposeThis study aims to determine whether Pokemon regulates Bim activity in colorectal carcinoma (CRC) carcinogenesis.MethodsClinical tissue samples were analyzed to detect the expression and clinicopathological significance of Pokemon and Bim in CRC. Proliferation, apoptosis, and invasion assays were conducted to identify the regulatory effect of Pokemon on Bim. The combined treatment effects of Pokemon knockdown and diamminedichloroplatinum (DDP) were also examined.ResultsImmunohistochemical analysis of 80 samples of colorectal epithelia (CRE), 80 cases of colorectal adenoma (CRA), and 160 of CRC samples revealed protein expression rates of 23.8%, 38.8%, and 70.6% for Pokemon, and 88.8%, 73.8%, and 31.9% for Bim, respectively. A significant negative correlation was observed between Pokemon and Bim expression across the CRE, CRA, and CRC lesion stages. In CRC, higher Pokemon and lower Bim expression correlated with higher histological grades, advanced Dukes stages, and increased cancer invasion. In both LoVo and HCT116 cells, overexpression of Pokemon significantly reduced Bim expression, leading to increased proliferation, resistance to anoikis, and cell invasion. Additionally, Pokemon overexpression significantly decreased DDP-induced Bim expression, reduction of anti-apoptosis and invasion, whereas Pokemon knockdown resulted in the opposite effects.ConclusionThese findings suggest that Pokemon inhibits Bim transcription, thereby promoting CRC proliferation, resistance to apoptosis, invasion, and advancing histological grade and Dukes staging. Pokemon knockdown enhances the therapeutic efficacy of DDP in the treatment of CRC.

The most common forms of cancer and oncogenic papillomaviruses

Breast, prostate, lung as well as colorectal carcinomas belong to leading positions in the world cancer incidence and mortality rankings. They make up about 40 % of newly diagnosed cancers. Connection of these cancers with oncogenic human papilloma viruses (HPVs) is being examined intensively, however it remains subject for discussion. Basing on case-control meta-analyses data were examined on oncogenic HPV detection in clinical samples of breast, prostate, lung and colorectal carcinomas. Findings on HPV genome activities were summarized. The results available prove to oncogenic HPVs as risk factors of the four enumerated above carcinomas.

Comparison of genomic profiling of patient-matched primary colorectal and surgical resected distant metastatic (stage IV) colorectal carcinoma for drug actionability

It is often difficult to obtain adequate tissue for genomic study from distant metastases for assessment of targeted therapy in colorectal carcinomas. The study aims to explore the genomic differences between matched distant metastatic colorectal carcinomas (mCRC) and primary carcinoma using surgical specimens of both with adequate tissue. Thirty-four paired primary and distant metastatic colorectal carcinoma samples (liver, ovary, and lung) were obtained from surgical excisions (not small biopsies) and are microsatellite stable. They were subjected to DNA sequencing using comprehensive next-generation sequencing. This included mutation concordance analysis and mutational signature analysis. The mutation concordance analysis showed 49.6% shared mutations between primary and metastatic tumours, with 23.0% mutations exclusive to primary tumours and 27.4% mutations exclusive to distant metastases. While many patients with KRAS/BRAF mutations had shared mutations, two cases had unique KRAS mutations in the primary tumours only. Additionally, TMB (tumour mutational burden) analysis revealed that half of the TMB-high (≥7.5 mutations/Mb) metastatic colorectal carcinomas had a low TMB (<7.5 mutations/Mb) in the primary tumours. The mutational signature analysis identified de novo signatures consistent with known single base substitution patterns such as SBS11 (alkylation agents) and SBS30 (base excision repair deficiency) post-chemotherapy. To conclude, this study demonstrates significant genomic variations in resected distant metastasis when compared to primary colorectal carcinomas when adequate tissue is available. This finding underscores the importance of considering these differences and selecting tissue for mutation analysis in planning targeted and effective treatment strategies for mCRC.

Abstract LB252: iCellSight: An interactive tool for analyzing and visualizing in-situ high-plex cellular protein data

Abstract Introduction The increased use of tissue-based multiplex immunohistochemistry (mIHC) in cancer research has significantly enhanced the generation of spatial hyper-plex images, crucial for understanding complex tumor-immune microenvironments. This advancement has led to a growing demand for spatial data analysis tools that enable quick and insightful evaluation. While existing spatial analysis software like Steinbock and SPIAT offer foundational support, effectively managing the high-dimensional and voluminous cellular data to derive meaningful biological insights remains challenging. This is especially true for researchers lacking bioinformatics expertise who seek to conduct quick preliminary evaluations. To address this gap, we introduce iCellSight: a user-friendly, code-free interactive analysis and visualization tool tailored to empower researchers with rapid and intuitive interpretation of in-situ cellular data. Material and methods A formalin-fixed, paraffin-embedded colorectal carcinoma (CRC) patient tissue section was subjected to staining using COMET (Lunaphore, Switzerland), which automates sequential immunofluorescence staining and imaging of up to 40 protein markers. The stacked image file produced was analyzed with HALO (Indica Labs, USA), which generated cell-level data like cell coordinates, stain intensity values and user-defined cell types. We developed a Shiny App tool, using the Shiny R package, with key features for cell data interpretation: (1) user-input cell number subsampling; (2) dimensional reduction plots with PhenoGraph for unsupervised clustering, allowing a choice of UMAP or t-SNE, and neighbor number selection of 30, 50 or 150; (3) 2D tissue space mapping of PhenoGraph clusters; and (4) heatmap visualizations displaying cluster-wise biomarker expression (with customizable color schemes and scaling), biomarker co-expression, and cluster pair-wise distance. Results Using iCellSight, 297,000 cells were detected from the CRC sample, where 27 clusters were identified. We observed 3 distinct clusters from the t-SNE plot that consist of macrophages, granulocytes, and NK cells and monocytes respectively. From the cluster projection in 2D tissue space, the cluster of NK cells and monocytes was found to be densely populated in a particular area of the tumor nest. Conclusion We presented iCellSight, a code-free visualization and analysis tool which streamlines the semi-automated analysis of spatial hyper-plex protein data. Designed with an intuitive user interface, this app enables researchers to efficiently navigate its features without requiring any prior coding expertise. We anticipate that the visualizations and analysis provided by this tool will significantly assist researchers in their data analyses and facilitate the validation of experimental hypotheses. Citation Format: Yancun Zhu, Felicia Wee, Menaka Priyadharsani Rajapakse, Esther Gek Teo, Nicholas Ang, Solomonraj Wilson, Zheng Yi Ho, Willa Wen-You Yim, Li Yen Chong, Craig Ryan Joseph, Jeffrey Chun Lim, Zhen Wei Neo, Chwee Ming Lim, Bernett Lee, Olaf Rotzschke, Joe Yeong, Mai Chan Lau. iCellSight: An interactive tool for analyzing and visualizing in-situ high-plex cellular protein data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB252.

Abstract 6357: Establishing a reverse translational platform to dissect mode of action and mechanisms of resistance to GUCY2C-CD3 T cell engager

Abstract PF-07062119 (GUCY2C x CD3), a bispecific T cell engager (TCE) targeting Guanylyl Cyclase C (GUCY2C), a GI tumor associated antigen, is currently being evaluated in a Phase 1 clinical trial in colorectal cancer (CRC) and other GI malignancies. Improved pre-clinical understanding of the correlates of PF-07062119 resistance mechanisms and PD in a human disease relevant context is critical to better inform drug combination and patient selection strategy. Due to limited translatability of mouse syngeneic tumor models, establishing a more human disease-relevant platform leveraging tumor patient material to better inform PF-07062119 clinical strategy is needed. Here, we will discuss our efforts to perform immune profiling of T cell and myeloid tumor infiltrates, and to evaluate tumor cell depletion and cytokine production of colorectal carcinoma (CRC) samples treated ex vivo with PF-07062119. This platform may be leveraged to 1) provide clinically relevant disease insights to the effect of TCEs across a range of tumor indications; 2) determine impact of different combinatorial partners on tumor cell killing to inform clinical decisions; 3) address questions related to the minimal efficacy of TCEs in the solid tumor space; and 4) provide potential solutions for future combination partners to overcome immunosuppression in the tumor microenvironment. Citation Format: Joana Borlido, Jade Lolarga, Rachael Whaley, Michael A. Damore, Cen Guo, Keith Abayasiriwardana, Xiaojun Xu, Maggie Liu, Gosia Nocula-Lugowska, Sam Stoner, Christopher Kraser, Ioanna Cheronis, Maria Delioukina, Lee Clark, Raj Bhadra, Murali Gururajan. Establishing a reverse translational platform to dissect mode of action and mechanisms of resistance to GUCY2C-CD3 T cell engager [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 6357.

Quantitative Analysis of NKX2-3 Expression in Human Colon: An Immunohistochemical Study.

In mice, Nkx2-3 homeodomain transcription factor defines the vascular specification of secondary and tertiary lymphoid tissues of the intestines. In human studies, polymorphisms in NKX2-3 have been identified as a susceptibility factor in inflammatory bowel diseases, whereas in mice, its absence is associated with protection against experimental colitis and enhanced intestinal epithelial proliferation. Here, we investigated the expression of NKX2-3 in normal, polyp, and adenocarcinoma human colon samples using immunohistochemistry and quantitative morphometry, correlating its expression with endothelial and mesenchymal stromal markers. Our results revealed that the expression of NKX2-3 is regionally confined to the lamina propria and lamina muscularis mucosae, and its production is restricted mostly to endothelial cells and smooth muscle cells with variable co-expression of CD34, alpha smooth muscle antigen (αSMA), and vascular adhesion protein-1 (VAP-1). The frequency of NKX2-3-positive cells and intensity of expression correlated inversely with aging. Furthermore, in most colorectal carcinoma samples, we observed a significant reduction of NKX2-3 expression. These findings indicate that the NKX2-3 transcription factor is produced by both endothelial and non-endothelial tissue constituents in the colon, and its expression changes during aging and in colorectal malignancies. (J Histochem Cytochem XX: XXX-XXX, XXXX).

Detection of Fusobacterium nucleatum in Patients with Colitis-Associated Colorectal Cancer

Fusobacterium nucleatum is supposed to play a critical role in the development of colorectal cancer. The species has also been associated with ulcerative colitis (UC) that can progress into colorectal cancer, however, the involvement of bacteria in this process remains unclear. We analysed 177 colon biopsies obtained from patients during screening, including 20 healthy controls, 56 UC cases and 69 cases at different stages of progression to colitis-associated cancer (CAC); 32 samples of sporadic colorectal carcinoma (sCRC) were also included. The presence of F. nucleatum was detected by quantitative real-time PCR (qPCR). Our data show an association between the presence of the bacteria and the progression of carcinogenesis in UC patients. In 39.5% of CAC samples F. nucleatum was detected, compared to only 1.8% in UC cases. The bacteria were detected in 6.3% of samples with initial neoplastic transformation, so-called low-grade dysplasia (LGD), whereas high-grade dysplasia (HGD) resulted in 33.3% of samples positive for F. nucleatum. The fraction of F. nucleatum-positive samples from sCRC cases was 56.3%, which was not significantly different to the CAC group. We conclude that F. nucleatum is associated with the occurrence and progression of colon carcinogenesis, rather than with UC itself.

STING is a cell-intrinsic metabolic checkpoint restricting aerobic glycolysis by targeting HK2.

Evasion of antitumour immunity is a hallmark of cancer. STING, a putative innate immune signalling adaptor, has a pivotal role in mounting antitumour immunity by coordinating innate sensing and adaptive immune surveillance in myeloid cells. STING is markedly silenced in various human malignancies and acts as a cell-intrinsic tumour suppressor. How STING exerts intrinsic antitumour activity remains unclear. Here, we report that STING restricts aerobic glycolysis independent of its innate immune function. Mechanistically, STING targets hexokinase II (HK2) to block its hexokinase activity. As such, STING inhibits HK2 to restrict tumour aerobic glycolysis and promote antitumour immunity in vivo. In human colorectal carcinoma samples, lactate, which can be used as a surrogate for aerobic glycolysis, is negatively correlated with STING expression level and antitumour immunity. Taken together, this study reveals that STING functions as a cell-intrinsic metabolic checkpoint that restricts aerobic glycolysis to promote antitumour immunity. These findings have important implications for the development of STING-based therapeutic modalities to improve antitumour immunotherapy.

A lipid metabolism-related gene signature reveals dynamic immune infiltration of the colorectal adenoma-carcinoma sequence

BackgroundLipid metabolism-related genes (LMRGs) have been reported to be correlated with the immune infiltration of colorectal cancer (CRC). This study aimed to investigate the immune infiltration characteristics along the colorectal adenoma-carcinoma sequence (ACS) based on LMRGs.MethodsGene expression data of colorectal adenoma and carcinoma samples were obtained from the public databases. The “limma” package was applied to determine the differentially expressed LMRGs. Unsupervised consensus clustering was used to cluster colorectal samples. The features of the tumor microenvironment were analyzed by the “ESTIMATE”, “GSVA”, and “TIDE” algorithms.ResultsThe expression of 149 differentially expressed LMRGs was defined as the LMRG signature. Based on this signature, the adenoma and carcinoma samples were divided into three clusters. Unexpectedly, these sequential clusters showed a directional relationship and collectively constituted the progressive course of colorectal ACS. Interestingly, the LMRG signature revealed that adenoma progression was accompanied by a progressive loss of immune infiltration and a stepwise establishment of a cold microenvironment, but carcinoma progression was characterized by a progressive gain of immune infiltration and a gradual establishment of a hot microenvironment.ConclusionsThe LMRG signature reveals dynamic immune infiltration along the colorectal ACS, which substantially changes the understanding of the tumor microenvironment of CRC carcinogenesis and provides novel insight into the role of lipid metabolism in this process.

To Correlate the Expression of KRAS and BRAF V600e with Histological Grades and Variants in Tissue Samples of Colorectal Carcinoma

OBJECTIVES This study aimed to correlate the expression of KRAS and BRAF V600E with histological grades and variants in tissue samples of colorectal carcinoma.METHODOLOGY In this cross-sectional study total of 51 cases of Colorectal cancer (CRC) were analyzed for immunohistochemical staining using KRAS and BRAF antibodies on representative tissue blocks. Clinical and pathological records were retrieved for the collection of data. The results of the immunohistochemical analysis were correlated with the recorded clinicopathological parameters.RESULTSFifty-one cases of CRC were analyzed for immunoexpression of KRAS and BRAF V600E. The age of the patients ranged from 14 to 85 years, with a mean age of 60.96 years. Among the 51 cases, 37(72.5%) cases were males and 14(27.4%) were females. 37(72.5%) were localized to left side colon and 14(27.4%) were found in the right colon. For KRAS immunostaining, 41(80.3%) out of 51 cases showed overexpression, while the remaining 10(19.6%) cases revealed negative expression. In the case of BRAF V600E, positive expression was seen in 20(39.2%) cases, whereas 31(60.7%) cases showed negative expression of BRAFV600E. A significant association was seen between KRAS overexpression and histological variants, i.e. glandular carcinomas. CONCLUSIONOver-expression of KRAS was observed in advanced tumors. The presence of BRAF V600E mutation in the present study signifies the importance of BRAF V600E inhibitors as a potential alternate therapeutic tool in EGFR inhibitors and chemotherapy-resistant tumors.

KRAS, NRAS, BRAF, HER2 and MSI Status in a Large Consecutive Series of Colorectal Carcinomas

This study aimed to analyze clinical and regional factors influencing the distribution of actionable genetic alterations in a large consecutive series of colorectal carcinomas (CRCs). KRAS, NRAS and BRAF mutations, HER2 amplification and overexpression, and microsatellite instability (MSI) were tested in 8355 CRC samples. KRAS mutations were detected in 4137/8355 (49.5%) CRCs, with 3913 belonging to 10 common substitutions affecting codons 12/13/61/146, 174 being represented by 21 rare hot-spot variants, and 35 located outside the "hot" codons. KRAS Q61K substitution, which leads to the aberrant splicing of the gene, was accompanied by the second function-rescuing mutation in all 19 tumors analyzed. NRAS mutations were detected in 389/8355 (4.7%) CRCs (379 hot-spot and 10 non-hot-spot substitutions). BRAF mutations were identified in 556/8355 (6.7%) CRCs (codon 600: 510; codons 594-596: 38; codons 597-602: 8). The frequency of HER2 activation and MSI was 99/8008 (1.2%) and 432/8355 (5.2%), respectively. Some of the above events demonstrated differences in distribution according to patients' age and gender. In contrast to other genetic alterations, BRAF mutation frequencies were subject to geographic variation, with a relatively low incidence in areas with an apparently warmer climate (83/1726 (4.8%) in Southern Russia and North Caucasus vs. 473/6629 (7.1%) in other regions of Russia, p = 0.0007). The simultaneous presence of two drug targets, BRAF mutation and MSI, was observed in 117/8355 cases (1.4%). Combined alterations of two driver genes were detected in 28/8355 (0.3%) tumors (KRAS/NRAS: 8; KRAS/BRAF: 4; KRAS/HER2: 12; NRAS/HER2: 4). This study demonstrates that a substantial portion of RAS alterations is represented by atypical mutations, KRAS Q61K substitution is always accompanied by the second gene-rescuing mutation, BRAF mutation frequency is a subject to geographical variations, and a small fraction of CRCs has simultaneous alterations in more than one driver gene.

A DNA methylation signature for the prediction of tumour recurrence in stage II colorectal cancer.

A major challenge in stage II colorectal carcinoma is to identify patients with increased risk of recurrence. Biomarkers that distinguish patients with poor prognosis from patients without recurrence are currently lacking. This study aims to develop a robust DNA methylation classifier that allows the prediction of recurrence and chemotherapy benefit in patients with stage II colorectal cancer. We performed a genome-wide DNA methylation capture sequencing in 243 stage II colorectal carcinoma samples and identified a relapse-specific DNA methylation signature consisting of eight CpG sites. Two hundred and forty-three patients with stage II CRC were enrolled in this study. In order to select differential methylation sites among recurrence and non-recurrence stage II CRC samples, DNA methylation profiles of 62 tumour samples including 31 recurrence and 31 nonrecurrence samples were analysed using the Agilent SureSelectXT Human Methyl-Seq, a comprehensive target enrichment system to analyse CpG methylation. Pyrosequencing was applied to quantify the methylation level of candidate DNA methylation sites in 243 patients. Least absolute shrinkage and selection operator (LASSO) method was employed to build the disease recurrence prediction classifier. We identified a relapse-related DNA methylation signature consisting of eight CpG sites in stage II CRC by DNA methylation capture sequencing. The classifier showed significantly higher prognostic accuracy than any clinicopathological risk factors. The Kaplan-Meier survival curve showed an association of high-risk score with poor prognosis. In multivariate analysis, the signature was the most significant prognosis factor, with an HR of 2.80 (95% CI, 1.71-4.58, P < 0.001). The signature could identify patients who are suitable candidates for adjuvant chemotherapy. An eight-CpG DNA methylation signature is a reliable prognostic and predictive tool for disease recurrence in patients with stage II CRC.

Somatic mutations in FAT cadherin family members constitute an underrecognized subtype of colorectal adenocarcinoma with unique clinicopathologic features.

The FAT cadherin family members (FAT1, FAT2, FAT3 and FAT4) are conserved tumor suppressors that are recurrently mutated in several types of human cancers, including colorectal carcinoma (CRC). To characterize the clinicopathologic features of CRC patients with somatic mutations in FAT cadherin family members. We analyzed 526 CRC cases from The Cancer Genome Atlas PanCancer Atlas dataset. CRC samples were subclassified into 2 groups based on the presence or absence of somatic mutations in FAT1, FAT2, FAT3 and FAT4. Individual clinicopathological data were collected after digital slide review. Statistical analysis was performed using t tests and chi-square tests. This CRC study cohort had frequent mutations in the FAT1 (10.5%), FAT2 (11.2%), FAT3 (15.4%) and FAT4 (23.4%) genes. Two hundred CRC patients (38.0%) harbored somatic mutations in one or more of the FAT family genes and were grouped into the FAT mutated CRC subtype. The FAT-mutated CRC subtype was more commonly located on the right side of the colon (51.0%) than in the rest of the cohort (30.1%, P < 0.001). It showed favorable clinicopathologic features, including a lower rate of positive lymph nodes (pN1-2: 33.5% vs 46.4%, P = 0.005), a lower rate of metastasis to another site or organ (pM1: 7.5% vs 16.3%, P = 0.006), and a trend toward an early tumor stage (pT1-2: 25.0% vs 18.7%, P = 0.093). FAT somatic mutations were significantly enriched in microsatellite instability CRC (28.0% vs 2.1%, P < 0.001). However, FAT somatic mutations in microsatellite stable CRC demonstrated similar clinicopathologic behaviors, as well as a trend of a better disease-free survival rate (hazard ratio = 0.539; 95% confidence interval: 0.301-0.967; log-rank P = 0.073). FAT cadherin family genes are frequently mutated in CRC, and their mutation profile defines a subtype of CRC with favorable clinicopathologic characteristics.

MicroRNA Expression of Primary and Metastatic Colorectal and Breast Carcinoma

Background: Colorectal and breast carcinoma are frequently diagnosed cancers. At advanced stages, cancers metastasize to certain organs resulting in loss of function of these organs, and eventually death. Therefore, there is a specific need for the prognosis of these cancers. Currently, microRNAs (miRNAs), have emerged as a new target of cancer-specific biomarker. Objective: To examine the expression of miRNA in primary and metastatic breast and colorectal cancers. Methods: This study investigated the expression of 6 miRNAs (miR-10b, miR-21, miR-145, miR-155, miR-200c, and miR-373) in formalin fixed paraffin embed tissues from pairs of normal tissues with primary and metastatic tumor samples of breast and colorectal carcinoma cases in Ramathibodi Hospital, Thailand by real-time RT-PCR. Results: Among 6 miRNAs, miR-145 decreased significantly in all samples of primary and metastatic colorectal and breast carcinoma. There was significantly decreased expression of miR-145 in metastatic colorectal carcinoma compared to their primary colorectal carcinoma (P &lt; .05). Whereas miR-10b, miR-155, and miR-200c showed a decreased expression; miR-21, and miR-373 showed an increased expression in the majority of cases. Unlike miR-145, other miRNAs showed no significant difference of expression (P &gt; .05). Conclusions: This finding indicates that miR-145 may be the potential metastatic biomarker. Decrease of miR-145 could be applied to the prognosis and target for therapy of breast and colorectal carcinoma.

Abstract 1705: Spatial interrogation of tertiary lymphoid structures (TLS) in colorectal carcinoma (CRC) tumor microenvironment (TME) using the MultiOmyx࣪ assay

Abstract Immune checkpoint blockade (ICB) therapy has revolutionized the landscape of cancer therapy in multiple tumor types since Ipilimumab, the first ICB agent, was approved for the treatment of metastatic melanoma is 2011. Current predictive biomarkers for therapy response include PD-L1 expression, tumor mutational burden (TMB) and microsatellite instability (MSI) status. However, responses to ICB vary widely and these predictive markers have demonstrated imperfect predictive power to ICB therapy. For instance, half of microsatellite instability-high (MSI-H) colorectal carcinoma (CRC) patients do not respond to ICB immunotherapy. There is still a crucial need to identify and develop biomarkers predictive of outcome to ICB therapy. Tertiary lymphoid structures (TLS) have been observed in a variety of solid tumors in humans, and their presence is a favorable prognostic indicator for survival in a variety of solid cancers including CRC. Further, it was recently shown that the presence of mature TLS was associated with improved response rate to ICB and overall survival in patients with advanced tumors in a large retrospective study. In this study, we used a 17-plex MultiOmyx panel to detect TLS, explore TLS maturation stage and characterize the tumor microenvironment (TME) in 40 CRC patients with known MSI status. The 17-plex includes CD3, CD4, CD8, CD45RO, FOXP3, CD20, CD56, CD68, CTLA-4, PD1, PD-L1, CXCL13, PNAd, CD21, CD23, DC-LAMP and PanCK. Using this panel, we successfully detected different stages of TLS in these CRC samples. Using proprietary deep-learning-based image analysis algorithm (NeoLYTX), we quantified TLS’s in the CRC samples and classified them by maturation stage based on biomarker expression and spatial organization of immune cells. While there was no difference in the number of early TLS (E-TLS; characterized by CD3, CD20 and pNAd) between MSI-H and microsatellite-stable (MSS) CRC samples, more primary follicle-like TLS (P-TLS; defined by CD3, CD20 and CD21) and secondary follicle-like TLS (S-TLS; defined by CD3, CD20 and CD23) were observed in the MSI-H group than the MSS group. In particular, the abundance of mature S-TLS was significantly higher in MSI-H CRC samples. This 17-plex MultiOmyx assay provides a powerful tool to characterize the cellular composition and spatial organization of the tumor microenvironment. The panel enables quantification of TLS, PD-L1 expression and abundance of tumor infiltrating lymphocytes from one single FFPE slide. The rich datasets generated by the MultiOmyx assay can provide greater understanding of the immune contexture within the TME and deeper insights into the correlations between biomarkers. These findings may have broad application and help identify biomarker signatures with improved predictive performance to immune checkpoint inhibition efficacy in solid tumors. Citation Format: Harry Nunns, Erinn A. Parnell, Michael Lazare, Judy Kuo, Eric Leones, Flora Sahafi, Josette William, Qingyan Au. Spatial interrogation of tertiary lymphoid structures (TLS) in colorectal carcinoma (CRC) tumor microenvironment (TME) using the MultiOmyx࣪ assay [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 1705.

Heme oxygenase-1 & 2 and their potential contribution in heme induced colorectal carcinogenesis

BackgroundLiterature suggests Heme oxygenase (HO) system to be a double-edged sword which can promote both cytoprotection as well as carcinogenicity. The aim of this study was to investigate the role of heme in HO-1 and HO-2 induced colorectal carcinogenesis and the clinicopathological significance of their expressions in patients with colorectal carcinoma (CRC). MethodsHO-1 and HO-2 expression alterations in normal colonic epithelial (FHC) and colon cancer cells (SW480) were explored following treatment with 0 µM, 25 µM, 100 µM and 250 µM concentrations of hemin, using qPCR. Fifty paired CRC and adjacent non-neoplastic samples were subjected to qPCR to determine the HO-1 and HO-2 expression. Clinicopathological associations of HO-1 and HO-2 expression levels were determined. ResultsLow concentrations of hemin caused upregulation and high concentration caused downregulation of HO-1 expression, whereas HO-2 expression was significantly downregulated with all hemin concentrations in FHC. HO-1 expression in SW480 was increased with all hemin concentrations and HO-2 expression was downregulated at the highest hemin concentration. HO-1 and HO-2 expressions in adjacent non-neoplastic tissue was significantly higher than that of CRC. Expression of HO-1 was significantly higher than HO-2, in both CRC and adjacent non-neoplastic tissue. Sex, HFE expression and lymph-vascular invasion were significantly correlated with HO-1 expression. HO-2 expression showed significant associations with staging, local spread and recurrence of tumour. ConclusionHO-1 and HO-2 expression is respectively induced and repressed by exogenous hemin in normal colon and colon cancer cells. HO-1 and HO-2 expression profiles in CRC are correlated with the assessed clinicopathological features of CRC, suggesting the possible implications of HO expression status in CRC pathogenesis.

Integration of multiple lineage measurements from the same cell reconstructs parallel tumor evolution

Organoid evolution models complemented with integrated single-cell sequencing technology provide a powerful platform to characterize intra-tumor heterogeneity (ITH) and tumor evolution. Here, we conduct a parallel evolution experiment to mimic the tumor evolution process by evolving a colon cancer organoid model over 100 generations, spanning 6months in time. We use single-cell whole-genome sequencing (WGS) in combination with viral lineage tracing at 12 time points to simultaneously monitor clone size, CNV states, SNV states, and viral lineage barcodes for 1,641 single cells. We integrate these measurements to construct clonal evolution trees with high resolution. We characterize the order of events in which chromosomal aberrations occur and identify aberrations that recur multiple times within the same tumor sub-population. We observe recurrent sequential loss of chromosome 4 after loss of chromosome 18 in four unique tumor clones. SNVs and CNVs identified in our organoid experiments are also frequently reported in colorectal carcinoma samples, and out of 334 patients with chromosome 18 loss in a Memorial Sloan Kettering colorectal cancer cohort, 99 (29.6%) also harbor chromosome 4 loss. Our study reconstructs tumor evolution in a colon cancer organoid model at high resolution, demonstrating an approach to identify potentially clinically relevant genomic aberrations in tumor evolution.

Expression of KRAS in tissue samples of colorectal carcinoma and its correlation with various histo-pathological parameters.

Objective: To assess the expression of KRAS in tissue samples of colorectal carcinoma and to correlate it with histopathological parameters. Study Design: Cross Sectional study. Setting: Department of Pathology, PNS Shifa Hospital Karachi. Period: March 2016 to February 2019. Material &amp; Methods: A total of 51 cases of CRC were analyzed for immunohistochemical staining using KRAS antibody on representative tissue blocks. Clinical and pathological records were retrieved for collection of data. The results of immunohistochemical analysis were correlated with the recorded clinico-pathological parameters. Results: In this study 51 cases of CRC were analyzed for immunoexpression of KRAS. The age of the patients ranged from 14 to 85 years with the mean age of 60.96 years. Among the 51 cases, 37(72.5%) cases were males and 14(27.4%) were females. 37(72.5%) were localized to left side colon and 14(27.4%) were found in the right colon. For KRAS immunostaining, 41(80.3%) out of 51 cases showed overexpression. Significant association was seen between KRAS overexpression and histological variants i.e. glandular carcinomas. Conclusion: In the present study over expression of KRAS was observed in advanced tumors. Majority of these cases were adenocarcinomas with few showed mucinous histology. The present study signifies that established KRAS expression is usually seen in rapidly dividing cells with association of advanced tumors.