Colorectal Cancer Cohort Research Articles

Abstract 1250: Activating PIK3CA mutations and hedgehog signaling may confer resistance to KRAS inhibition in colorectal cancer

Abstract Background: Treatment with KRASG12C inhibitors results in an objective response in only small fraction of patients with KRASG12C mutant colorectal cancer (CRC) (7.1% and 22% objective response rates (ORR) in two prospective trials), in contrast to lung cancer where the ORR exceeds 35%. We hypothesize that co-mutation of other oncogenes and/or transcriptomic factors may contribute to the resistance of KRASG12C CRC to KRAS inhibitors. Methods: Mutation profiles of CRC patients were obtained from the AACR Project-GENIE v14 CRC cohort (N = 9441). The impact of KRAS knockout in 59 CRC cell lines was queried from the DepMap database. KRASG12C mutant CRC cell lines SW837 and SW1463 were treated with Sotorasib (KRASG12C inhibitor), Vismodegib and Taladegib (Hedgehog inhibitors), and cell viability was measured up to 96 hours after treatment. Results: In the AACR Project-GENIE CRC (N = 9441) cohort, KRASG12C tumors (2.6%, 245/9441) were significantly co-mutated with PIK3CAmut (19.2%, 47/245; Odds ratio = 1.7 (95% CI: 1.2 - 2.4), Chi-Square test, p-value = 0.002). Dependence on KRAS in CRISPR knockout viability assays was higher in KRASmut vs. wildtype CRC cell lines (median CERES score -1.3 vs -0.5, p-value < 0.001). Interestingly, KRASmut cell lines with PIK3CA co-mutation (n=10) were more resistant to KRAS knockout than PIK3CAwt cells (n=22) (median -1.0 vs -1.4, p-value = 0.03). The SW837 and SW1463 cell lines initially displayed high sensitivity to Sotorasib (IC50: 0.92 µM and 0.90 µM). However, overexpression of PIK3CAE545K mutant induced resistance to the same treatment (IC50: 5.18 µM and 6.45 µM). Furthermore, gene set enrichment analysis (GSEA) showed that Hedgehog signaling was significantly enriched in KRAS knockout resistant KRASmut CRC cell lines. SW837 cell line treated with Sotorasib (S) and a fixed dosage of 25 µM of Vismodegib (V) or Taladegib (T) showed higher sensitivities in combination treatment assays (IC50 decreased: ~11 folds in S+V vs S; and ~15 folds in S+T vs S) as compared to Sotorasib (S) treatment alone. Similar results were seen for the SW1463 cell line (IC50 decreased: ~18 folds in S+V vs S; and ~15 folds in S+T vs S). Conclusion: Resistance in CRC to KRASG12C inhibitors may be attributed to activated PIK3CA mutations and the Hedgehog signaling pathway. The future study will focus on detailed investigations involving comprehensive transcriptome and exome profiles in tumors and PDX models treated with anti-KRAS drugs in monotherapy and combination therapy. Citation Format: Saikat Chowdhury, Jibran Ahmed, Valsala Haridas, David S. Hong, Scott Kopetz, John Paul Shen. Activating PIK3CA mutations and hedgehog signaling may confer resistance to KRAS inhibition 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 1250.

Abstract 3163: (Phospho)proteomic analysis reveals vulnerabilities in refractory metastatic colorectal cancer

Abstract Proteomic networks are crucial for cellular homeostasis maintenance and, indeed, their aberrant regulation is implicated in cancer development and progression. Understanding alterations underlying these pathways, provides a way to design therapeutic interventions in human cancer. Improvements in mass spectrometry analysis have unlocked our potential to unveil cancer vulnerabilities. Here, we performed (phospho)proteomic analysis and ex-vivo drug testing on a cohort of refractory metastatic colorectal cancer (mCRC) tissues and matched patients-Derived Organoids (PDO) to identify new putative druggable biomarkers. The comparison between the proteome and phosphoproteome of 10 mCRCs tissues with their matched PDOs normalized to a pool of 6 colon mucosa tissues highlighted a strong overall correlation (R= 0.57 and R=0.6, respectively) suggesting a good level of similarity. Applying GSEA on our proteomic dataset, we identified up-regulation in MYC, cell cycle and MTORC1 signaling pathways in mCRC tissues and PDO, while kinase enrichment analysis highlighted an increase in Casein Kinase II (CKII) activity, an important cell cycle regulator. Our ex-vivo experiments showed that silmitasertib-mediated CKII inhibition reduced the cell viability in our PDO models, but with an overall modest effect. Analysis revealed that, even though not statistically significant, high Wnt signaling score correlated with an higher resistance to silmitasertib treatment (R=0.61; p=0.08). Previous works highlighted that MAPK activity is increased after silmitasertib treatment and responsible for resistance mechanisms to CKII inhibition. Strikingly, the combined trametinib-mediated MEK blockade and CKII blockade resulted in a synergistic interaction leading to enhanced antitumor activity, especially in PDO harboring high Wnt (R=0.74; p=0.022) and cell cycle (R=0.69; p=0.04) pathway activity. Moreover, the combined treatment strongly reduced S6 activity and RAPTOR expression, overcoming the significant effect exerted by the single agents. Surprisingly, none of them altered baseline MAPK activity, suggesting that silmitasertib and trametinib synergize their activity via inactivating the MTORC1-S6 axis in an ERK-independent manner. We are currently integrating genomic and transcriptomic data to better characterize the observed drug synergy. Taken together, our findings propose the silmitasertib and trametinib combination as a new therapeutic opportunity for refractory mCRC patients and strengthen the use of PDO as a suitable model for drug screening. Citation Format: Mattia Marinucci, Gina Faye Boot, Lara Zaidi, Cinzia Esposito, Mairene Coto Llerena, Savas Soysal, Otto Kolmar, Charlotte K Y Ng, Salvatore Piscuoglio. (Phospho)proteomic analysis reveals vulnerabilities in refractory metastatic 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 3163.

Abstract 4344: Comprehensive analysis of LINE-1 transposable elements in colorectal cancer

Abstract Background: Colorectal cancer (CRC) is the third most frequent cancer worldwide, and approximately a third of patients die from the disease. There is therefore an urgent need to better understand CRC biology. A particular feature in (colorectal) cancer is the activation of LINE-1 (L1) retrotransposons: DNA fragments which propagate to other places in the genome through an RNA intermediate. Per genome, about 100-150 genomic L1 copies are still retrotransposition-competent, but repressed in most somatic tissues. Their role in tumor development is currently unknown. Recently, the PCAWG consortium characterized the landscape of somatic L1 retrotranspositions in a series of primary cancers of different origin, and identified 124 recurrently active L1 source regions. However, only a limited number of these concerned CRC. Aim: This study aims to perform a comprehensive analysis of L1 activity in a large cohort of metastatic colorectal cancer. Methods: To this end, we retrieved data from the Hartwig Medical Foundation, comprising deep (106x) whole genome sequencing (WGS) data from metastatic CRC (n=745), to compare to breast (n=888) and lung cancer samples (n=640). We determined L1 activity as the number of somatic L1 insertions reported by the HMF DNA analysis pipeline and used the activity of each of the 124 L1 sources as previously reported by the PCAWG consortium. Results: 99% of mCRC samples (n=741) displayed at least 1 somatic L1 retrotransposition, with at least 10 retrotranspositions in 89% of samples. L1 retrotransposition is more prevalent in mCRC than in metastatic lung and breast cancer, in which only 40% and 20% had at least 10 retrotranspositions per sample, respectively. Each cancer type showed a unique profile of active L1 sources. We did not observe a clear preference of L1 insertions for a single genomic region. However, in CRC, the gene most frequently affected in an exon was APC (n=13), an important tumor-suppressor gene. An analysis of somatic SNVs and SVs confirmed an association of mutations in some known L1 regulators such as TP53 with increased L1 activity, whereas mutations in others showed no change in the number of somatic retrotranspositions. Conclusions: We conclude that somatic L1 retrotranspositions are highly prevalent in metastatic colorectal cancer. Presence of L1 insertions in APC suggests retrotranspositions start very early in CRC development and prompts further investigation into earlier CRC stages. Citation Format: Renske de Wit, Soufyan Lakbir, Caterina Buranelli, Gerrit A. Meijer, Sanne Abeln, Remond J. Fijneman. Comprehensive analysis of LINE-1 transposable elements 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 4344.

Abstract 6827: Myocardin and myocardin-related transcription factor as potential drivers of the myofibroblastic cancer-associated fibroblast phenotype in colorectal cancer

Abstract Background: Cancer-associated fibroblasts (CAFs) play a key role in modifying the tumor microenvironment (TME) and could be a future target for treatment. CAFs are typically categorized based on function: myofibroblastic CAFs (myCAFs) amplify extracellular matrix production that blocks therapy and immune cell infiltration while inflammatory CAFs (iCAFs) produce immuno-suppressive cytokines and chemokines. Here we sought to better understand the role and mechanisms of myCAFs in altering the TME by identifying their unique markers and potential drivers. Methods: Z-scores of ACTA2/TAGLN or ICAM1/PDPN were used to create datasets high in myCAF and iCAF markers, respectively, from TCGA data. A gene set enrichment analysis (GSEA) was performed on the resulting differential expression. Samples of patients were collected to create a cohort of colorectal cancers that were stained via immunohistochemistry (IHC) for alpha-smooth muscle actin (αSMA), transgelin (TAGLN), myocardin (MYOCD) and myocardin-related transcription factor A (MRTFA). Slides were evaluated for collagen using Masson’s trichrome stain. MYOCD and MRTFA nuclear expression was expressed as positively stained nuclei per high powered field (HPF; 400x). TAGLN, αSMA, and collagen were scored on a scale of 0-3+. Results: Of the 592 TCGA patients compiled across all disease stages, 23 (median age 63) formed the myCAF-high group (7 males, 16 females). Within this group, GSEA indicated an upregulation of myogenesis (q <0.001) with the following genes positively associated with myCAF markers: MYLK (q < 0.001, log2 fold change [FC] 1.52), DES (q < 0.001, 5.32), and MYH11 (q < 0.001, 3.57). The following were also upregulated in the myCAF high cohort: CNN1 (q < 0.001, 2.95) and MYOCD (q< 0.001, 2.27). These genes are targets of MYOCD and MRTFA transcription factors potentially driving the myCAF phenotype. To assess this potential relationship, we evaluated 306 tumor samples from 153 patients with CRC. The cohort consisted of 56 female patients and 97 male patients aged 13-90 years old across all disease stages. High amounts of nuclear MRTFA were associated with high scores for myCAF markers aSMA (average score 1.54 when MRTFA+ nuclei < 5 per HPF vs 2.06 when MRTFA + nuclei > 5 per HPF, p<0.001, Wilcoxon Rank Sum Test), TAGLN (1.37 vs 1.62, p = 0.043), and collagen (1.73 vs 2.10, p = 0.009). Likewise, for MYOCD, αSMA (1.79 vs 2.14, p = 0.001) and collagen (1.87 vs 2.15, p = 0.017) were positively correlated. Conclusions: Increased nuclear expression of MYOCD and MRTFA in stromal cells of CRC are positively correlated with myCAF gene expression, supporting our initial hypothesis of these being drivers of the phenotype. Our findings now motivate further analysis as to whether the inhibition of these transcription factors could lead to a reduction in the immune regulatory myCAF phenotype. Citation Format: Alec B. Cornelio, Katherine A. Johnson, Yousef Gadalla, Sharanya Nath, Cheri A. Pasch, Wei Zhang, Kristina A. Matkowskyj, Dustin A. Deming. Myocardin and myocardin-related transcription factor as potential drivers of the myofibroblastic cancer-associated fibroblast phenotype 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 6827.

Abstract 5165: Development and validation of an assay for the characterization of SSTR2 expression on CTCs from liquid biopsies

Abstract Background: Expression of Somatostatin Receptor subtype 2 (SSTR2) is observed in multiple cancers, including breast and colorectal cancer. Its role as a prognostic or predictive marker is cancer-type dependent. Recently, there has been renewed interest in assessing this biomarker since it plays a critical role as a target for radiotherapeutics (peptide receptor radionuclide therapy - PRRT) in patients with neuroendocrine tumors (NETs). Blood-based liquid biopsies are a non-invasive tool with proven effectiveness in characterizing cancer biomarker expression in patients. In this study, we describe the development and validation of a novel assay to analyze circulating tumor cells (CTCs) for expression of SSTR2 using the RareCyte platform. Experimental Procedures: Using AccuCyte®, an unbiased density-based method for collecting nucleated cells from whole blood, we transferred nucleated cells to slides and stained for SSTR2, CD45, and CK/EpCAM to identify CTCs (CK+/EpCAM+, CD45-), and evaluate SSTR2 expression. Slides were imaged with CyteFinder®, an automated multiparameter immunofluorescent (IF) microscopy system that applies machine learning algorithms for rare cell identification. Both clinical and spike-in samples were used for assay validation. Single-cell SSTR2 mean fluorescence intensities (MFI) were analyzed to determine protein expression levels. Colorectal cancer with neuroendocrine features and Merkel cell cancer (neuroendocrine tumor of the skin) patient samples were evaluated for CTC enumeration and SSTR2 expression. Results: The assay’s performance metrics for SSTR2 expression were 90+% accuracy, 85% sensitivity, 100% specificity with repeatability and intermediate precision CVs of 14.4% and 16.4%, respectively. A small cohort of colorectal cancer (1) and Merkel cell cancer (2) patient blood samples were tested using the assay. All 3 patients had high CTC levels (300+ per 7.5 mL). The percentage of SSTR2-positive CTCs ranged from 20-60%, reflective of tumor heterogeneity. Conclusions: SSTR2 is an important target now in the advent of theranostics - PRRT. Prognostic and therapeutic implications of SSTR2 expression is an area of active clinical research. SSTR2 targeted therapeutics like 177Lu-dotatate illustrate the possible utility of SSTR2 as a predictive biomarker of treatment response. To date, most of the work has been focused on assessing SSTR2 on tissue. Herein, we show how a CTC platform can be adapted to assess a clinically relevant cell surface protein biomarker. This is now included as an important correlative in a clinical trial for Merkel Cell Cancer patients called iPRRT that is actively accruing (NCT05583708). Citation Format: Erin G. Bayer, Rachel N. Ponting, Ciara M. Kelly, Areeb Lutfi, Maaz K. Afghan, Arturo B. Ramirez, Pashtoon M. Kasi. Development and validation of an assay for the characterization of SSTR2 expression on CTCs from liquid biopsies [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 5165.

Abstract 4634: Inhibitory-κB kinase alpha as a biomarker and therapeutic target in colorectal cancer

Abstract Introduction/Aims: Colorectal cancer (CRC) remains a leading cause of cancer-related mortality. The canonical NF-κB pathway driven by IKKβ has been implicated in CRC development and progression, however less research has focused on non-canonical NF-κB signaling, regulated by inhibitory-κB kinase alpha (IKKα). This project aimed to assess IKKα, phospho-IKKα (pIKKαs176) and IKKβ expression in a retrospective CRC cohort to establish association with survival outcomes. Subsequently, this project aimed to determine the effect of abrogating IKKα activity without perturbing the canonical NF-κB pathway controlled by IKKβ using a first-in-class selective IKKα inhibitor in vitro/ex vivo. Methods: Immunohistochemical staining for IKKα/pIKKα s176/IKKβ was performed using tissue microarrays from a CRC patient cohort (n=787). QuPath® software was used to semi-quantitively assess expression levels and scores were analyzed for association with cancer-specific survival (CSS). CRC cell lines (n=3), patient-derived organoids (PDOs; n=5) and patient-derived explants (n=5) were treated with novel IKKα inhibitor SU1644 and assessed for cell viability and proliferation. Results: Positive staining for IKKα, pIKKαs176 and IKKβ was detected in tumor specimens. High cytoplasmic expression of IKKα within tumor cells was associated with reduced CSS in patients with right-sided colon cancer (HR=3.091, 95%CI; 1.128-9.467, log rank p=0.021). Conversely, high IKKβ expression was a marker of good prognosis (HR=0.627, 95%CI; 0.457-0.861, log rank p=0.004). A high ratio of IKKα to IKKβ was associated with reduced CSS in the full cohort (HR=1.404, 95%CI; 1.050-1.879, log rank p=0.021) and this was potentiated in right-sided colon cases (HR=1.772, 95%CI; 1.107-2.837, log rank p=0.016). Selective inhibition of IKKα using 1µM SU1644 in vitro caused a significant reduction in HT29 colony formation (p=0.012) and cell viability (p=0.039). PDOs showed altered cell morphology, reduced cell viability and proliferation when treated with SU1644. In the patient-derived explant model, Ki67 expression was reduced in tumor explants treated with SU1644. Conclusions: These data establish high cytoplasmic IKKα expression within tumor cells as a marker of poor prognosis in CRC and conversely high IKKβ expression as a marker of good prognosis. This highlights the importance of development of selective IKKα inhibitor SU1644. IKKα inhibition using SU1644 demonstrated anti-cancer activity in recapitulative CRC disease models. Citation Format: Kathryn A. Pennel, Molly McKenzie, Guang-Yu Lian, Jean A. Quinn, Sara Samir Foad Al-Badran, Campbell S. Roxburgh, Simon P. Mackay, Joanna Birch, Joanne Edwards. Inhibitory-κB kinase alpha as a biomarker and therapeutic target 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 4634.

Abstract 5059: Robust single sample consensus molecular subtype classification for primary and metastatic colorectal cancer

Abstract Introduction Consensus Molecular Subtypes (CMS) represent a well-established molecular stratification framework for colorectal cancer (CRC). Existing methods for CMS classification rely on a representative input cohort as a preprocessing step (CMScaller) or have difficulty generalizing to metastatic samples (CMSclassifier). We developed Tempus CMS to overcome these limitations. Our method normalizes gene expression data from input samples to a static reference cohort to enable single sample classification of both primary and metastatic tumors. We evaluated the performance of this classifier on a large, de-identified CRC cohort comprising 8,489 samples from primary and metastatic sites. Methods To normalize input data, our method shifts and scales each gene expression value based on the mean and standard deviation of the expression of that gene in the reference cohort (n=2,787 primary and metastatic CRC). CMS calls are then generated via nearest template prediction similar to CMScaller (Eide et al., 2017). The analysis cohort was selected from clinical biopsies within 30 days of primary or metastatic diagnosis excluding reference cohort samples. CMS calls were assessed by comparing subtype prevalence to reported rates and by testing for known enrichments of pathways and genomic markers. Results Subtype prevalences for lower gastrointestinal (GI) biopsies and resections (n=5,090) were comparable to reported rates for primary CRC tumors: CMS1 - 12%, CMS2 - 28%, CMS3 - 19%, and CMS4 - 33% with 7.6% no calls. Biopsies from metastatic tissue showed site-specific changes in prevalence: CMS2 increased to 43% in liver samples (n=1,715) and CMS4 was identified in 49% of samples from metastatic sites excluding the liver (n=1,684). In addition, we found that purity had an effect on prevalence: in lower GI tumors with ≥ 50% tumor purity rates of CMS2 and CMS4 were 34% and 26%, respectively, while rates in tumors with purity between 20% and 50% were 20% CMS2 and 41% CMS4. Across all tissue groups, pathway enrichment showed significant associations, including TGF-ꞵ signaling in CMS4 (adj P ≤ 4.4e-15) and cell differentiation in CMS3 (adj P ≤ 3.0e-27). Expected enrichments of CMS markers were also observed: BRAF mutations and microsatellite instability in CMS1 and KRAS mutations in CMS3. In liver tissue, higher KRAS mutation rates were noted in CMS1 (57%) and CMS3 (83%) compared to GI tissue (35% and 67%, respectively). Conclusions In this study, we developed an enhanced, single sample CMS-calling algorithm optimized for primary and metastatic sites. Application of this algorithm in a large cohort recapitulated known biology, which suggests that the tool can be used to support clinical studies requiring robust molecular stratification. Citation Format: Andrew J. Sedgewick, Tushar Chandra, Timothy Taxter, Justin Guinney. Robust single sample consensus molecular subtype classification for primary and metastatic 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 5059.

Abstract 1040: Development of a novel RNA-based microsatellite stable predictive response signature (MSS-PRS) to identify MSS colorectal cancer (CRC) patients with a microsatellite instability-high (MSI-H) molecular phenotype

Abstract Introduction: MSI-H and mismatch repair deficient (dMMR) tumors are associated with favorable immune checkpoint inhibitor (ICI) responses (PMID: 3326454). However, up to 95% of CRC tumors are MSS/proficient MMR (pMMR) leading to poorer prognosis and treatment outcomes than MSI-H/dMMR patients (PMID: 12867608, 26028255). In MSS/pMMR metastatic CRC, multiple combination therapies are being investigated despite a lack of biomarkers to guide therapy. Therefore, we developed an RNA-based MSS-PRS with a primary objective to identify MSS tumors that have MSI-H/dMMR molecular features indicative of ICI response. Experimental Procedures: The MSS-PRS was developed using the TCGA CRC cohort (COAD; n=268). Training labels were assigned using the MSI Mantis score and mutation status from 8 genes with reported MSI association. Samples called “activated” had both a gene mutation and an MSI Mantis score > 0.4 (i.e., MSI-H), and samples called “non-activated” were wild type for all genes and had MSI Mantis score < 0.4. All other samples were designated “ambiguous” and excluded from training. Two thirds of the non-ambiguous samples were assigned to the classifier training set and all remaining samples were assigned to the test set. Using ClaNC software (PMID: 16269418) and cross-validation in the training set, a nearest centroid classifier was developed from a set of high mean, high variance candidate genes to select an optimal gene set to separate activated and non-activated groups. The classifier performance was evaluated in the test set. Results: The MSS-PRS contained 112-genes enriched for mismatched DNA binding, DNA damage repair, PD-L1, innate and adaptive immune response, cellular immunity, and cytokines. In the test set, the classifier called 44 of 46 MSI-H samples activated; of the remaining 77 samples that were MSS tumors, 38 were called activated and 39 were called not activated. Visual inspection of heat maps of tumor by MSI/MSS-PRS status and their association with TMB and CRC-related mutations suggest greater differences between MSS tumors called activated and MSI-H tumors than between MSS tumors called activated and MSS tumors called not activated. In contrast, immune marker expression profiles in MSS tumors called activated were markedly more similar to MSI-H tumors compared to MSS tumors called non-activated. Summary and Conclusions: Herein we described the development of a novel MSS-PRS that captures an MSI-H/dMMR-like molecular phenotype in a subset of patients with MSS tumors despite their lack of high TMB or overt MSI defects. Based upon these initial findings, further development of the MSS-PRS and its clinical validation as a tool to select patients with MSS tumors who may benefit from ICI-containing treatment regimens is warranted. Citation Format: Kirk Pappan, Gregory Mayhew, Jonathan Shepherd, Yuelong Guo, Kirk Beebe, Joel Eisner, Michael Milburn. Development of a novel RNA-based microsatellite stable predictive response signature (MSS-PRS) to identify MSS colorectal cancer (CRC) patients with a microsatellite instability-high (MSI-H) molecular phenotype [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 1040.

Abstract 1756: Characterizing the mutational spectrum of metastatic mucinous adenocarcinoma of the appendix

Abstract Background: Appendiceal cancer (AC) is an orphan malignancy with only 1-2 cases per 100,000 people in the US. The genomic composition of metastatic, mucinous AC is yet to be resolved at the whole exome level and few groups have assessed the clinical actionability of target genes. Here we performed whole-exome sequencing (WES) on AC specimens to 1) elucidate the mutational spectrum of AC, 2) identify recurrent mutations at frequencies unique to AC or in common with colorectal cancer (CRC), and 3) uncover recurrent AC mutations that are associated with patient survival outcomes. Methodology: Twenty-four FFPE samples, one from each of 24 AC patients treated at our institution with cytoreductive surgery (CRS) followed by heated intraperitoneal chemotherapy (HIPEC), were collected from our tumor bank with IRB approval. DNA from tumor and corresponding normal tissue (PBMCs) was extracted, assessed for integrity and quantity, then analyzed by WES using the Illumina NextSeq 6000 platform. The resulting data were processed using both GATK and DRAGEN pipelines. The frequencies of recurrent mutations in AC were compared with those of CRC from cBioPortal and statistically compared by Fisher’s Exact Test. Logrank test was performed to estimate survival associations based off mutation status. Mutational signatures derived from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium were cross-compared to our cohort to identify previously unknown mutational processes in AC. Results: The most frequent mutations were found in MUC6 (63%), KRAS (58%), HLA-B (54%), PABPC1 (50%), and MUC5AC (50%). Distinct from a 534-patient CRC cohort, AC had lower rates of alteration in APC (4% vs 73% CRC, p<0.001) and TP53 (21% vs. 62%, p<0.001), with higher rates of alteration in HLA-B (54% vs 3%, p<0.001), GNAS (38% vs 5%, p<0.001), and ABCA1 (29% vs 7%, p=0.001). Notably, ABCA1 mutation frequency was higher in mucinous CRC (n=56) when compared to non-mucinous CRC (n=478, 20% vs 7%; p<0.001), suggesting that ABCA1 may be uniquely altered in bowel cancers of mucinous phenotype. Alterations in Rho-A were a statistically significant predictor of poor survival in both AC (p<0.001) and CRC (p=0.05). PCAWG analysis showed that the predominant mutational signature in AC most resembles SBS5, suggesting that the mutational etiology of AC may be linked to age and/or smoking status. Discussion: Here we present the largest whole-exome sequencing analysis of AC analyzed to date and identify previously undescribed recurrently mutated genes unique to AC as compared to CRC. We demonstrate novel associations between altered genes and patient survival, which opens discussion regarding the clinical utility of molecular markers in AC for assisting treatment decision making. Citation Format: Daniel J. Gironda, Ming Leung, Guangxu Jin, Liang Liu, Konstantinos I. Votanopoulos, Edward A. Levine, Lance D. Miller. Characterizing the mutational spectrum of metastatic mucinous adenocarcinoma of the appendix [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 1756.

Abstract 1063: IsomiR relative proportion (IRP) within miRNA family as an efficient biomarker for noninvasive colorectal cancer detection

Abstract Background: Cell free miRNA has been widely used for disease diagnosis, particularly in cancers. The latest research has also demonstrated that isomiRs (isoform miRNAs) can serve as superior markers, displaying enhanced diagnostic performance in cancer diagnosis. However, the low detection sensitivities of miRNA/isomiRs in previous methods in plasma have limited their broad implementations for screening purpose. In this study, we propose a new isomiRs feature of isomiR relative proportion (IRP), which was validated with high performance in Colorectal Cancer (CRC) early diagnosis. Methods: A total of 1036 participants were enrolled from the National Colorectal Cancer Cohort (NCRCC), and 836 participants were eligible for plasma collection, including 238 controls, 373 CRC patients, 41 inflammatory bowel disease (IBD), 5 intramucosal carcinoma (Tis) and 179 advanced precancerous lesions (APL). Total circulating small RNA was extracted, followed by library preparation using UMI-based approach. Subsequently, small RNA sequencing was conducted using DNB-seq, and bioinformatic analysis was performed thereafter. The samples were randomly assigned into training (49%), test (30%) and validation (21%) sets. Models based on isomiR relative proportion (IRP), isomiR relative expression (IRE) or both features were constructed by beam search and random forest algorithms in training and test sets, and results were verified in validation set. Results: Differential IRP or IRE features were identified between cancer and controls. IRP identified 132 specific differential expressed isomiRs. Models constructed using either IRP or IRE features showed high performance (both AUC > 0.9). However, there was improvement in IRP feature model in the validation set (sensitivity of IRP: 0.923, sensitivity of IRE: 0.859) compared with IRE model. We then found that there may be complementarities between IRP and IRE features, and the combination model (34 IRP+IRE features) improved cancer detection performance (AUC: 0.953). Finally, the risk scores predicted by this combination model increased gradually across CRC progression (R=0.921), and the sensitivity of APL was 0.626. Conclusion: We improved the small-RNA sequencing method and developed an IRP method, and present that IRP as a new feature not only demonstrate the ability of CRC early detection, but also revealed the CRC progressing process. Besides, the combination of IRP and IRE improved the performance of diagnosis. These results highlight the potential value of the IRP feature in early cancer detection. Citation Format: Yeting Hu, Wei Lu, Chengcheng Liu, Xiaozhuan Dai, Jiasheng Xu, Xiangxing Kong, Yunfeng Zhu, Yao Ye, Lihao Chen, Xiaolong An, Fan Wu, Shiqing Ma, Nana Wang, Qian Xiao, Jun Liu, Kefeng Ding. IsomiR relative proportion (IRP) within miRNA family as an efficient biomarker for noninvasive colorectal cancer detection [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 1063.

Abstract 3673: Monitoring treatment response in patients with metastatic colorectal cancer using cfDNA fragmentomics testing: The DOLPHIN trial

Abstract Background: Accurate monitoring of treatment response in patients with metastatic colorectal cancer (mCRC) is important to decide when to adjust treatment regimen or to proceed to local therapy of metastases. At present, clinical response is determined by computed tomography (CT) imaging-based assessment of changes in tumor size. However, this monitoring approach is constrained by limited sensitivity in detecting lymph node and peritoneal metastases, as well as inter-reader variability. Detection of circulating tumor DNA (ctDNA) is indicative of the amount of neoplastic cells and may have added clinical value to CT imaging for assessment of treatment response and guidance in treatment decision making in mCRC patients. We recently developed a tumor mutation-independent ctDNA test that utilizes low-coverage whole genome sequencing to analyze the plasma cell-free DNA (cfDNA) fragmentome to measure the ctDNA tumor fraction: the DELFI-tumor fraction (DELFI-TF) score. Aim: The DOLPHIN study aims to investigate whether the DELFI-TF ctDNA assay can provide a sensitive, affordable and broadly applicable ctDNA test to monitor treatment response of mCRC patients. Methods: The prospective, observational, multi-center DOLPHIN study is a substudy of the Prospective Dutch ColoRectal Cancer cohort (PLCRC). Clinical data, images and blood samples from 400 patients receiving systemic therapy in 8-10 Dutch hospitals will be collected. Blood samples will be drawn longitudinally in conjunction with regularly scheduled CT imaging. Plasma cfDNA will be analyzed for tumor-specific fragmentation patterns using the DELFI-TF score. Droplet digital PCR ctDNA-testing of RAS/RAF hotspot mutations will be performed as reference, when feasible. Primary endpoint is the association between ctDNA changes and clinical response. Secondary endpoints are the association between ctDNA changes and radiological response according to RECIST and to serum carcinoembryonic antigen (CEA) at various time points during systemic treatment, lead time of ctDNA-testing compared to CT imaging to detect progressive disease, and the prognostic value of longitudinal ctDNA-testing. Results: Since the inclusion of the first patient in March 2023, 117 patients have been included in the DOLPHIN study (November 2023). Seven hospitals throughout the Netherlands are currently open for patient inclusion and more sites have been approached to participate. Discussion: The DOLPHIN study will assess the added clinical value of longitudinal ctDNA-testing in treatment response monitoring of mCRC patients and whether imaging can be complemented and/or partly replaced by ctDNA-testing. The results of this study may lead to novel strategies for monitoring treatment response and to ctDNA-guided treatment decision making in mCRC patients. Citation Format: Denise E. van Steijn, Remond J. Fijneman, Geraldine R. Vink, Jeanine M. Roodhart, Miriam Koopman, Max J. Lahaye, Manon N.G. Braat, Veerle M. Coupé, Daan A. van den Broek, Gerrit A. Meijer, Haoyue Wang, Marjolein J. Greuter, Birgit I. Lissenberg-Witte, Lorenzo Rinaldi, E Peters, Alissa Konicki, Nicholas C. Dracopoli, Niels F. Kok, Victor E. Velculescu. Monitoring treatment response in patients with metastatic colorectal cancer using cfDNA fragmentomics testing: The DOLPHIN trial [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 3673.

Abstract 2496: Molecular characterization of stage III colon cancer patients with recurrence after adjuvant chemotherapy

Abstract Introduction: Patients with stage III colon cancer are routinely treated with resection followed by adjuvant chemotherapy (ACT) with a fluoropyrimidine and oxaliplatin. This one-size-fits-all approach does not account for heterogeneity in tumor biology and fails to cure ~30% of patients, highlighting the urgency for biomarkers to better understand and predict resistance to ACT or to offer alternative targeted treatment. Aim: To identify molecular characteristics predictive of resistance to standard ACT in stage III colon cancer. Methods: Patients with resected stage III colon cancer who received ACT (93% CAPOX, 7% CAP) were selected from the Prospective Dutch Colorectal Cancer cohort (PLCRC). RNA exome capture sequencing was performed on resected FFPE tumor tissue to compare differential gene expression, hallmark pathway enrichment, immune cell deconvolution and consensus molecular subtypes (CMS) between patients with versus without recurrence after surgery and ACT. Postsurgery circulating tumor DNA (ctDNA) detected in PLCRC-PROVENC3 (separate abstract) was used to select a subgroup with minimal residual disease (MRD) postsurgery, as sensitivity analysis of patients with recurrence despite ACT (chemoresistant) versus curation by ACT (chemosensitive). Results: For 264 out of 278 patients (95%), tumor-derived RNA sequencing libraries were of sufficient quality. 67 patients (25%) with recurrence (median after 13 months [IQR 7-23]) were compared to 178 patients with at least 2 year recurrence-free follow-up (median 42 months [IQR 30-53]). A total of 320 genes were differentially expressed between patients with versus without recurrence. Genesets defining epithelial-mesenchymal transition (p = 0.002) and angiogenesis (p < 0.001) were enriched in the primary tumor of patients with recurrence, in part explained by the estimated larger fraction of stroma (Wilcoxon effect size r 0.18, p < 0.001) and cancer-associated fibroblasts (CAFs) (r 0.19, p = 0.003). In contrast, the fraction of CD4+ T cells was lower (r 0.14, p = 0.001). Consistently, CMS4 was found more frequently in tumors from patients with versus without recurrence (57% versus 34%, p < 0.001). Of the subgroup with ctDNA-based MRD postsurgery (26 out of 171 assessed patients), 19 patients (73%) with recurrence (median after 12 months [IQR 7-17]) were compared to 7 patients without recurrence (minimum follow-up 32 months). This sensitivity analysis showed similar trends in pathways, immune cells and CMS as the main analysis, with a more pronounced effect size for CAFs (r 0.27). Conclusion: Stage III colon tumors of patients who experience recurrence after resection and ACT are enriched for stroma and CAFs, while displaying less CD4+ T cells. The observed trends are sustained in the patients with MRD postsurgery. These findings may help to better predict resistance to standard ACT and elucidate targets for alternative personalized treatment. Citation Format: Ingrid Franken, Steven Ketelaars, Carmen Rubio-Alarcón, Sietske van Nassau, Suzanna Schraa, Gerrit Meijer, Mark Sausen, Miriam Koopman, Geraldine Vink, Sanne Abeln, Remond Fijneman, Jeanine Roodhart, on behalf of the PLCRC-MEDOCC group. Molecular characterization of stage III colon cancer patients with recurrence after adjuvant chemotherapy [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 2496.

Abstract 5022: MEDOCC-CrEATE trial: Feasibility of measuring circulating tumor DNA after surgery to guide adjuvant chemotherapy in stage II colon cancer patients

Abstract Introduction: Patients with stage II colon cancer not classified as high risk (pT4 microsatellite stable) do not receive adjuvant chemotherapy (ACT) according to Dutch guidelines. However, 15-20% of patients with stage II colon cancer experience a disease recurrence, indicating that there is an unmet clinical need to identify patients who could benefit from adjuvant treatment. Observational studies demonstrate that postoperative circulating tumor DNA (ctDNA) is indicative of minimal residual disease (MRD) and a strong prognostic biomarker for disease recurrence. Aim: The MEDOCC-CrEATE trial aims to assess 1) the proportion of stage II colon cancer patients with detectable postoperative ctDNA accepting ACT and 2) whether ctDNA-guided ACT reduces 2-year recurrence rate (RR). Methods: MEDOCC-CrEATE is an interventional trial following the ‘trial within cohorts’ design. Participants of the Prospective Dutch Colorectal Cancer cohort with stage II colon cancer and no indication for ACT randomized to the intervention arm undergo postoperative tumor-informed ctDNA testing using next-generation sequencing of 33 genes through the PGDx elio™ liquid biopsy assay. If ctDNA is detected, they are offered 4 cycles of adjuvant capecitabine plus oxaliplatin. Patients in the intervention arm who test ctDNA negative and patients in the control arm receive standard of care follow-up. For all patients, blood is collected every 6 months for 3 years to monitor disease recurrence. The primary endpoint is the proportion of patients with detectable postoperative ctDNA willing to receive ACT. Secondary endpoints include 2-year RR, disease-free and overall survival, quality of life and cost-effectiveness of ctDNA-guided ACT. The study is powered for 2-year RR, randomizing 660 patients to each study arm in order to treat 30 patients in the intervention arm with detectable postoperative ctDNA, assuming 5% ctDNA detection and 10% noncompliance. Results: Logistics for timely multicenter collection of tumor tissue and blood have been optimized across 28 Dutch hospitals. At present, 240 patients have been randomized. Of the 119 patients in the intervention arm, 98 provided consent for immediate postoperative ctDNA analysis (82%). Of the 92 currently available results, 5 (5 %) had detectable ctDNA. The median time from surgery to blood collection was 15 days (IQR 11-19), with a median turnaround time from surgery to ctDNA result of 47 days (IQR 39-54). Conclusion: Multicenter postoperative tumor-informed ctDNA testing for MRD is operationally and technically feasible within the clinically relevant 8-12 week window to start ACT. The observed ctDNA detection rate is in accordance with expectations and the study design. Upon study finalization, the results will be used for health technology assessment to demonstrate the putative clinical utility of ctDNA-guided ACT in stage II colon cancer. Citation Format: Ingrid Franken, Suzanna Schraa, Karlijn van Rooijen, Dave van der Kruijssen, Carmen Rubio-Alarcón, Steven Ketelaars, Sietske van Nassau, Teunise Snetselaar, Bregje Adriaans, Jillian Phallen, Sam Angiuoli, Amy Greer, Ellen Verner, Veerle Coupé, Helena Verkooijen, Miranda van Dongen, Linda Bosch, Mirthe Lanfermeijer, Daan van den Broek, Gerrit Meijer, Victor Velculescu, Mark Sausen, Miriam Koopman, Remond Fijneman, Geraldine Vink, on behalf of the PLCRC-MEDOCC group. MEDOCC-CrEATE trial: Feasibility of measuring circulating tumor DNA after surgery to guide adjuvant chemotherapy in stage II colon cancer patients [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 5022.

Phase II trial of domatinostat (4SC-202) in combination with avelumab in patients with previously treated advanced mismatch repair proficient oesophagogastric and colorectal adenocarcinoma: EMERGE

Most oesophagogastric adenocarcinomas (OGAs) and colorectal cancers (CRCs) are mismatch repair proficient (MMRp), responding poorly to immune checkpoint inhibition. We evaluated the safety and efficacy of domatinostat (histone deacetylase inhibitor) plus avelumab (anti-PD-L1 antibody) in patients with previously treated inoperable, advanced/metastatic MMRp OGA and CRC. Eligible patients were evaluated in a multicentre, open-label dose escalation/dose expansion phase II trial. In the escalation phase, patients received escalating doses of domatinostat [100 mg once daily (OD), 200 mg OD, 200 mg twice daily (BD)] orally for 14 days followed by continuous dosing plus avelumab 10 mg/kg administered intravenously 2-weekly (2qw) to determine the recommended phase II dose (RP2D). The trial expansion phase evaluated the best objective response rate (ORR) during 6 months by RECIST version 1.1 using a Simon two-stage optimal design with 2/9 and 1/10 responses required to proceed to stage 2 in the OGA and CRC cohorts, respectively. Patients (n= 40) were registered between February 2019 and October 2021. Patients in the dose escalation phase (n= 12) were evaluated to confirm the RP2D of domatinostat 200 mg BD plus avelumab 10 mg/kg. No dose-limiting toxicities were observed. Twenty-one patients were treated at the RP2D, 19 (9 OGA and 10 CRC) were assessable for the best ORR; 2 patients with CRC did not receive combination treatment and were not assessable for the primary endpoint analysis. Six patients were evaluated in the dose escalation and expansion phases. In the OGA cohort, the best ORR was 22.2% (95% one-sided confidence interval lower bound 4.1) and the median duration of disease control was 11.3 months (range 9.9-12.7 months). No responses were observed in the CRC cohort. No treatment-related grade 3-4 adverse events were reported at the RP2D. Responses in the OGA cohort met the criteria to expand to stage 2 of recruitment with an acceptable safety profile. There was insufficient signal in the CRC cohort to progress to stage2. NCT03812796 (registered 23rd January 2019).

A comprehensive multi-omics analysis identifies a robust scoring system for cancer-associated fibroblasts and intervention targets in colorectal cancer

BackgroundCancer-associated fibroblasts (CAF) play a critical role in promoting tumor growth, metastasis, and immune evasion. While numerous studies have investigated CAF, there remains a paucity of research on their clinical application in colorectal cancer (CRC).MethodsIn this study, we collected differentially expressed genes between CAF and normal fibroblasts (NF) from previous CRC studies, and utilized machine learning analysis to differentiate two distinct subtypes of CAF in CRC. To enable practical application, a CAF-related genes (CAFGs) scoring system was developed based on multivariate Cox regression. We then conducted functional enrichment analysis, Kaplan–Meier plot, consensus molecular subtypes (CMS) classification, and Tumor Immune Dysfunction and Exclusion (TIDE) algorithm to investigate the relationship between the CAFGs scoring system and various biological mechanisms, prognostic value, tumor microenvironment, and response to immune checkpoint blockade (ICB) therapy. Moreover, single-cell transcriptomics and proteomics analyses have been employed to validate the significance of scoring system-related molecules in the identity and function of CAF.ResultsWe unveiled significant distinctions in tumor immune status and prognosis not only between the CAF clusters, but also across high and low CAFGs groups. Specifically, patients in CAF cluster 2 or with high CAFGs scores exhibited higher CAF markers and were enriched for CAF-related biological pathways such as epithelial–mesenchymal transition (EMT) and angiogenesis. In addition, CAFGs score was identified as a risk index and correlated with poor overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), and recurrence-free survival (RFS). High CAFGs scores were observed in patients with advanced stages, CMS4, as well as lymphatic invasion. Furthermore, elevated CAFG scores in patients signified a suppressive tumor microenvironment characterized by the upregulation of programmed death-ligand 1 (PD-L1), T-cell dysfunction, exclusion, and TIDE score. And high CAFGs scores can differentiate patients with lower response rates and poor prognosis under ICB therapy. Notably, single-cell transcriptomics and proteomics analyses identified several molecules related to CAF identity and function, such as FSTL1, IGFBP7, and FBN1.ConclusionWe constructed a robust CAFGs score system with clinical significance using multiple CRC cohorts. In addition, we identified several molecules related to CAF identity and function that could be potential intervention targets for CRC patients.

DR6 Augments Colorectal Cancer Cell Growth, Invasion, and Stemness by Activating AKT/NF-κB Pathway.

This study aims to elucidate the role and mechanisms of Death Receptor 6 (DR6), a member of the tumor necrosis factor receptor superfamily, in the malignant progression of colorectal cancer (CRC). The association of DR6 expression levels and CRC patient survival was examined using the CRC cohort data from GEPIA database. The functional role of DR6 in CRC cells was investigated by performing loss-of-function and gain-of-function experiments based on CCK-8 proliferation assay, transwell migration and invasion assay, and sphere-forming assays. Xenograft model of CRC cells in nude mouse was established to evaluate the impact of DR6 knockdown on CRC tumorigenesis. Elevated expression of DR6 was correlated with an unfavorable prognosis in CRC patients. In vitro functional assays demonstrated that silencing DR6 considerably suppressed the proliferation, migration, invasion, and stemness of CRC cells, whereas its overexpression showed an opposite effect. DR6 knockdown also attenuated tumor formation of CRC cells in the nude mice. Mechanistically, silencing DR6 reduced the phosphorylation of AKT and NF-κB in CRC cells, and the treatment with an AKT activator (SC79) abrogated the inhibitory effects of DR6 knockdown on the malignant features of CRC cells. Our data suggest that DR6 contributes to the malignant progression of CRC by activating AKT/NF-κB pathway, indicating its clinical potential as a prognostic marker and therapeutic target for CRC.

CSF1R inhibition reprograms tumor-associated macrophages to potentiate anti-PD-1 therapy efficacy against colorectal cancer

PD-1 blockade therapy has made great breakthroughs in treatment of multiple solid tumors. However, patients with microsatellite-stable (MSS) colorectal cancer (CRC) respond poorly to anti-PD-1 immunotherapy. Although CRC patients with microstatellite instability (MSI) or microsatellite instability-high (MSI-H) can benefit from PD-1 blockade therapy, there are still some problems such as tumor recurrence. Tumor-associated macrophages (TAMs), most abundant immune components in tumor microenvironment (TME), largely limit the therapeutic efficacy of anti-PD-1 against CRC. The CSF1/CSF1R pathway plays a key role in regulating macrophage polarization, and blocking CSF1R signaling transduction may be a potential strategy to effectively reprogram macrophages and remodel TME. Here, we found that increasing expression of CSF1R in macrophages predicted poor prognosis in CRC cohort. Furthermore, we discovered a novel potent CSF1R inhibitor, PXB17, which significantly reprogramed M2 macrophages to M1 phenotype. Mechanically, PXB17 significantly blocked activation of PI3K/AKT/mTORC1 signaling, resulting in inhibition of cholesterol biosynthesis. Results from 3D co-culture system suggested that PXB17-repolarized macrophages could induce infiltration of CD8+ T lymphocytes in tumors and improve the immunosuppressive microenvironment. In vivo, PXB17 significantly halted CRC growth, with a stronger effect than PLX3397. In particular, PXB17 potently enhanced therapeutic activity of PD-1 mAb in CT-26 (MSS) model and prevented tumor recurrence in MC-38 (MSI-H) model by promoting formation of long-term memory immunity. Our study opens a new avenue for CSF1R in tumor innate and adaptive anti-tumor immunomodulatory activity and suggests that PXB17 is a promising immunotherapy molecule for enhancing the efficacy of PD-1 mAb or reducing tumor recurrence of CRC.

Identification of enterotype and its predictive value for patients with colorectal cancer

BackgroundGut microbiota dysbiosis involved in the pathogenesis of colorectal cancer (CRC). The characteristics of enterotypes in CRC development have not been determined.ObjectiveTo characterize the gut microbiota of healthy, adenoma, and CRC subjects based on enterotype.MethodsThe 16 S rRNA sequencing data from 315 newly sequenced individuals and three previously published datasets were collected, providing total data for 367 healthy, 320 adenomas, and 415 CRC subjects. Enterotypes were analyzed for all samples, and differences in microbiota composition across subjects with different disease states in each enterotype were determined. The predictive values of a random forest classifier based on enterotype in distinguishing healthy, adenoma, and CRC subjects were evaluated and validated.ResultsSubjects were classified into one of three enterotypes, namely, Bacteroide- (BA_E), Blautia- (BL_E), and Streptococcus- (S_E) dominated clusters. The taxonomic profiles of these three enterotypes differed among the healthy, adenoma, and CRC cohorts. BA_E group was enriched with Bacteroides and Blautia; BL_E group was enriched by Blautia and Coprococcus; S_E was enriched by Streptococcus and Ruminococcus. Relative abundances of these genera varying among the three human cohorts. In training and validation sets, the S_E cluster showed better performance in distinguishing among CRC patients, adenoma patients, and healthy controls, as well as between CRC and non-CRC individuals, than the other two clusters.ConclusionThis study provides the first evidence to indicate that changes in the microbial composition of enterotypes are associated with disease status, thereby highlighting the diagnostic potential of enterotypes in the treatment of adenoma and CRC.

Classification of molecular subtypes for colorectal cancer and development of a prognostic model based on necroptosis-related genes

BackgroundNecroptosis could regulate immunity in cancers, and stratification of colorectal cancer (CRC) subtypes based on key genes related to necroptosis might be a novel strategy for CRC treatment. MethodThe RNA-sequencing data of CRC and other 31 types of cancers were obtained from The Cancer Genome Atlas (TCGA) database. Consensus clustering was performed based on protein-coding genes (PCGs) related to necroptosis score calculated by single sample gene set enrichment analysis (ssGSEA). Module genes showing a significant positive correlation with the necroptosis score were identified by weighted correlation network analysis (WGCNA) and further used to develop a risk stratification model applying least absolute shrinkage and selection operator (LASSO) and Cox regression analysis. The risks score for each sample in CRC cohorts, immunotherapy cohorts and pan-cancer study cohorts was calculated. ResultTwo subgroups (C1 cluster and C2 cluster) of CRC were identified based on the necroptosis score. Compared with C1 cluster, the survival possibility of C2 cluster was greatly reduced, the levels of necroptosis score, immune cell infiltration, immune score and expression of immune checkpoint molecules were significantly increased and immunotherapy response was less active. Low-risk patients defined by the risk model had a significant survival advantage than high-risk counterparts in both CRC and the other 31 cancer types. Furthermore, the risk model was also more efficient than the Tumor Immune Dysfunction and Exclusion (TIDE) tool in predicting OS and immunotherapy response for the samples in the immunotherapy cohort. ConclusionCRC patients were classified by necroptosis score-related PCGs, and a risk model was designed to evaluate the immunotherapy and prognosis of patients with CRC. The current molecular subtype and prognostic model could help stratify patients with different risks and predict their prognosis and immunotherapy sensitivity.

Younger patients with colorectal cancer may have better long-term survival after surgery: a retrospective study based on propensity score matching analysis

BackgroundThe relationship between postoperative long-term prognosis and age in colorectal cancer patients remains controversial. The purpose of this study based on a Chinese CRC cohort is to determine the disparity in long-term survival outcomes between younger and older colorectal cancer (CRC) patients after surgery using a propensity score matching (PSM).MethodsData for this study was derived from the CRC cohort of the Database from Colorectal Cancer (DACCA) at West China Hospital of Sichuan University from January 2007 to September 2022. The long‑term prognoses were compared between younger and older groups.ResultsA total of 2374 CRC patients were evaluated in this study, including 1039 older patients and 1335 younger ones. After 1:1 ratio PSM, each group contained 784 CRC patients. There was no significant difference in baseline information after PSM (p < 0.05). Multivariate analysis showed that younger age was an independent predictor of better overall survival (OS) (p < 0.001, HR = 1.750, 95% CI = 1.407–2.177) and disease-specific survival (DSS) (p < 0.001, HR = 1.718, 95% CI = 1.369–2.157). In terms of different tumor pathological stages after PSM, in comparison to older group, younger group had better OS in stage II (p < 0.001), stage III (p = 0.0085), and stage IV (p = 0.0014) and better DSS in stage II (p = 0.0035), stage III (p = 0.0081), and stage IV (p < 0.001).ConclusionYounger CRC patients have better prognosis than older CRC patients after surgery, especially, and have better OS and DSS in stages II, III, and IV CRC. Younger CRC patient may gain greater benefit from CRC resection and combined therapy. As for the cut-off age, it may be determined by a specific model suitable for local patients.