bluuebunny/crossref_metadata_2025 huggingface dataset:
Racial/ethnic differences of germline pathogenic variants in cancer susceptibility genes among patients with early-onset colorectal cancer.
Authors: Holowatyj Andreana N , Seagle Hannah M , Keller Samantha , Tavtigian Sean V , Goldgar David E , Horton Carolyn
Published: January 2023

19

Background: The burden of early-onset colorectal cancer (CRC diagnosed before age 50 years) differs across racial/ethnic groups. Yet the distinct prevalence and spectrum of germline pathogenic variants associated with early-onset CRC by race/ethnicity is uncharacterized. Methods: We identified 3,980 early-onset CRC patients unselected for family CRC history who underwent clinical multi-gene panel testing of 14 CRC susceptibility genes ( MLH1, MSH2, MSH6, PMS2, SMAD4, APC, BMPR1, CHEK2, EPCAM, MUTYH, PTEN, STK11, CDH1, and TP53) performed by a nationwide clinical testing laboratory from 2012 to 2016 and identified as Ashkenazi Jewish, Asian, Black, Hispanic, or White. Multi-gene panel testing was performed by targeted custom capture and sequencing and chromosome rearrangement analysis. Clinical and individual/family histories were ascertained from clinician-completed test requisition forms. Germline variant prevalence and spectrum in early-onset CRC was determined by race/ethnicity. Pathogenic variant comparisons by race/ethnicity were evaluated using chi-square tests and multivariable logistic regression adjusted for sex, age, CRC site and sequence. Results: Among 3,980 early-onset CRC patients, a total of 503 germline genetic variants were identified in 481 individuals (12.1%). By race/ethnicity, 12.7% of Ashkenazi Jewish, 8.6% of Asian, 10.3% of Black, 14.0% of Hispanic, and 12.3% of White individuals presented with a pathogenic variant in a CRC susceptibility gene. Nearly 7% of early-onset CRC patients (n = 268) presented with a germline pathogenic mutation in a mismatch repair gene—with the prevalence of Lynch Syndrome in early-onset CRC differing across racial/ethnic groups ( P= 0.037). The prevalence of germline APC, CHEK2, MLH1, monoallelic MUTYH, and PTEN variants also varied by race/ethnicity (all P< 0.02). Overall, Asian individuals with early-onset CRC had 49% lower odds of carrying a pathogenic germline variant in a CRC susceptibility gene versus White individuals in adjusted models (Odds Ratio [OR] = 0.51, 95% Confidence Interval [CI]: 0.26-0.99, P= 0.046). Ashkenazim and Hispanic individuals had higher odds of presenting with pathogenic APC and MLH1 variants, respectively, compared with White individuals in adjusted models ( APC: OR = 8.69, 95% CI: 2.68-28.21, P= 0.0003; MLH1: OR = 2.67, 95% CI: 1.30-5.49, P= 0.007). Conclusions: Of 3,980 patients with early-onset CRC unselected for family history, the prevalence and spectrum of pathogenic germline variants differed across racial/ethnic populations. Beyond multigene panel testing for all early-onset CRC patients, further study is needed to optimize genes selected for germline genetic testing in early-onset CRC across diverse groups—which would have clinical implications for disease surveillance and intervention.
Metagenomic analysis of gut microbiota of patients with colorectal cancer at the Federal Medical Centre (FMC), Abeokuta, Ogun State, Nigeria.
Authors: Sulaimon Lateef Adegboyega , Joel Ireoluwa Yinka , Sanusi Olalekan Sabur , Olanipekun Quadri Arise-kola , Haliru Amatullah Mahe , Ajanaku Zainab Adedolapo , Hassan Aaliyah Eniola , Zakariyah Ikimot Fehintola , Ajibade Faidat Bukola , Olajide Monsurat , Lawal Modinat Olasunkanmi , Ajibare Abosede Christiana
Published: January 2023

195

Background: It has been suggested that the intestinal microbiome plays a vital role in the development of colorectal cancer (CRC), but the changes in intestinal bacteria in healthy and CRC subjects have not yet been examined in the Nigerian population. The study sought to investigate changes in intestinal bacteria in healthy subjects and patients with CRC. Methods: Fecal samples were collected from healthy and CRC subjects at the Federal Medical Centre, Abeokuta with the study ethical approval number FMCA/47O/HREC/01/2021/23. We extracted genomic DNA with magnetic bead extraction and sequenced the whole genome using a Nanopore MinION sequencer. We performed metagenomic analysis on fecal samples from 20 healthy subjects and 20 patients with colorectal cancer (CRC) to study the bacterial community structure, relative abundance, differentially abundant bacteria, association networks, taxonomic profiling, and specialty gene expression. Results: CRC patients have significantly different bacterial community structures compared to healthy subjects. Compared to healthy subjects, CRC patients had low intestinal bacterial diversity. Only CRCs contain Gammaproteobacteria at the phylum level. Compared to CRC, Bacteroidia and Actinobacteria were significantly more abundant in healthy subjects, while Bacilli and Negativicutes were significantly less abundant. Bacteroides and Prevotella are more abundant in healthy subjects than in CRC at the genus level. There was a greater abundance of Escherichia and Pseudomonas in CRC as compared with healthy subjects. It is noteworthy that Escherichia coli, which co-occurred in both healthy and CRC subjects, was significantly elevated only in CRC, while Pseudomonas aeruginosa only occurs in CRC. Based on phylogenetic analysis, there were notable relationships between six strains of E. coli and four strains of Shigella sp. identified in CRC. There were several pathogenic-associated virulent and antimicrobial resistant genes expressed in these strains. These genes conferred resistance via antibiotic inactivation, efflux pump activity, alteration of cell wall proteins, and regulation of antibiotic permeability. Conclusions: The present study demonstrated that the taxonomic composition and functional genes of intestinal bacteria were significantly altered in CRC. Also, E. coli and P. aeruginosa are at least partially involved in the pathogenesis of CRC.
Tumor mutations associated with outcomes in colorectal peritoneal metastases.
Authors: Uppal Abhineet , White Michael , Chang George J. , Zeineddine Fadl A. , Zeineddine Mohammad A. , Yousef Abdelrahman M.G. , Scally Christopher , Fournier Keith F. , Newhook Timothy E. , You Y. Nancy , Willis Jason , Kopetz Scott , Shen John Paul Y.C.
Published: January 2023

196

Background: The association between tumor mutations beyond KRAS and BRAF for patients with peritoneal metastases (PM) from colorectal cancer (CRC) are poorly understood. Here we describe these associations with survival after diagnosis of PM. Methods: CRC patients with PM with or without synchronous liver or lung metastases treated at a single institution from 2016 to 2020 were retrospectively studied. Standard of care next-generation sequencing was performed on tissue specimens with a targeted panel of genes frequently mutated in cancer. Clinical data, including survival and last contact, were obtained from medical records. Multivariate Cox regression was used to identify associations between overall survival after diagnosis of PM (OS) with clinical, histopathologic and somatic mutation data. Results: Of 526 PM patients, 179 (34.2%) also had synchronous liver metastases, and these patients had worse OS compared to PM alone (HR 1.67, [1.26-2.22], p=0.0003). Lung metastases were identified in 57 (10.8%) and were not associated with worse OS (HR 1.34, [0.90-1.97], p=0.15). Poorly differentiated grade (HR 2.08, [1.42-3.04], p=0.0002), mucinous histology (HR 1.44, [1.07-1.94], p=0.016), male gender (HR 1.37, [1.05-1.78), p=0.019), and age (HR 1.02, [1.01-1.04], p=0.004) were independently associated with OS. Tumor sequencing data was available for 442 (87%) patients. TP53 was the most common mutation (61.5% of patients) and was associated with decreased OS (HR=1.51 [1.12-2.02], p=0.005). BRAF mutation (85% of which were V600E) was identified in 9.7% of patients and was also associated with decreased OS (HR=1.88 [1.23-2.86], p=0.003). In contrast, PTEN mutation (4.3%) was associated with improved OS (0.41 (0.17-0.99), p=0.049), while both KRAS (51.4%) and SMAD4 mutations (16%) were not (KRAS: HR 0.94, [0.72-1.24], p=0.68; SMAD4: HR 0.78, [0.53-1.16], p=0.21). Conclusions: Mutational data for PM patients has a distinct profile and unique associations with OS when compared to other sites of metastatic disease. TP53 and BRAF mutations were associated with worse survival, while PTEN mutation was associated with improved OS. KRAS mutation was more prevalent than expected in an unselected cohort, and not associated with OS. A better understanding of PM-specific associations with survival may influence treatment strategies for this difficult disease site.
Concordance of genomic alterations in anal cancer by next-generation sequencing in tissue and circulating tumor DNA.
Authors: LaPelusa Michael Brian , Cann Christopher G , Eng Cathy
Published: January 2023

2

Background: The feasibility of identifying genomic alterations via next-generation sequencing (NGS) of peripheral blood (PB) to spare patients an invasive biopsy is an active area of investigation. High concordance of mutations between tissue and PB has been observed in several cancer types. However, the concordance between tissue and PB in patients with SCCA is unknown. Methods: We identified 11 patients with SCCA who had NGS (TEMPUS) performed on tissue and PB between 2017 - 2022. The IRB at Vanderbilt-Ingram Cancer Center approved this study. Results: See Table. Conclusions: In 6 of the 11 patients, concordant genomic alterations found in tissue were also identified in PB. More patients with anal cancer should be studied to better understand this phenomenon and the impact of therapy on ctDNA kinetics.[Table: see text]
Comparison between interval and sporadic colon cancer in terms of clinicopathologic and molecular features.
Authors: Huh Kyu Chan , Kim Dae Sung , Shin Jeong Eun , Lee Yoo Jin , Kim Kyeong Ok , Kim Jae Hyun , Kang Gyeong Hoon
Published: January 2023

205

Background: Interval colon cancer occurs due to either limitations of colonoscopy or rapidly developing new tumors, possibly reflecting molecular and environmental differences in tumorigenesis. This study aimed to compare the clinicopathologic and molecular features of interval colon cancer with those of sporadic colon cancer in Asia. Methods: This prospective, multicenter, cross‐sectional study was conducted from May 2017 to December 2021 at six university hospitals in South Korea. Interval colon cancer was defined as colon cancer diagnosed within 5 years of complete colonoscopy. We compared the clinicopathologic and molecular biological characteristics, including CpG island methylator phenotype (CIMP), KRAS, BRAF, and microsatellite instability (MSI), between patients with interval and sporadic colon cancers. Results: We compared and analyzed 28 patients with interval colon cancer and 72 patients with sporadic cancer. There was no difference in terms of sex, BMI, underlying disease, and family history in the clinical comparison between the interval and sporadic cancer groups. There was no difference in the location, shape, or stage of cancer including metastasis, between the two groups. In molecular biological analysis, CIMP was 5.1% positive for sporadic cancer and 17.9% positive for interval colon cancer, indicating an approximately 13% greater frequency in interval colon cancer (p = .036). KRAR mutation was observed in 36.7% of patients with sporadic cancers and 17.9% of those with interval colon, which was about 18% higher in sporadic cancer, but there was no statistically significant difference (p = .066). There was no difference between MSI and BRAF genotype between the sporadic and interval cancer groups. Conclusions: Unlike the findings of western studies, there was no clinical difference between sporadic and interval colon cancer. In molecular biological comparison, the CIMP positivity rate was significantly higher in patients with interval colon cancer.
Differences in tumor-associated T cell receptor repertoires between young-onset and average-onset colorectal cancer.
Authors: Schmit Stephanie , Tsai Ya-Yu , Nair Kanika G. , Barot Shimoli V , Xiang Shao , Kamath Suneel Deepak , Melas Marilena , Walker Christopher P , Srivastava Raghvendra , Chan Timothy An-thy , Bonner Joseph D. , McDonnell Kevin , Idos Gregory , Greenson Joel K , Rennert Hedy S , Rennert Gad , Gruber Stephen B. , Khorana Alok A. , Liska David
Published: January 2023

207

Background: The incidence of colorectal cancer (CRC) among individuals younger than age 50 (young-onset CRC; YOCRC) has increased, yet the etiology and molecular mechanisms underlying this alarming rise remain unclear. Some studies suggest that YOCRCs have more aggressive biology. Here, we compare tumor-associated T cell repertoires between patients with YOCRC and average-onset CRC (AOCRC). A robust, focused T cell response is a positive prognostic indicator; therefore, we hypothesized that YOCRCs demonstrate lower T cell abundance and greater T cell diversity than AOCRCs. Methods: The discovery cohort included 242 patients with histologically confirmed Stage I-IV CRC who underwent surgical resection at Cleveland Clinic from 2000 to 2020 and consented to a biorepository. YOCRC was defined as < 50 years at diagnosis (N = 126), and AOCRC as > 60 years (N = 116). DNA was extracted from frozen tumors, and T Cell Receptor (TCR) abundance and clonality were measured using immunoSEQ (Adaptive Biotechnologies). Following quality control, logistic regression models were used to evaluate the associations between TCR repertoire features and age of onset, adjusting for sex, race, tumor location, and stage. Findings were replicated in 152 YOCRC and 1,984 AOCRC cases from the Molecular Epidemiology of Colorectal Cancer Study with adjustment for sex, Jewish ethnicity, tumor location, and stage. Results: YOCRC patients were more likely to have left-sided tumors and be diagnosed at advanced stages in both cohorts. In the discovery cohort, YOCRC tumors had significantly lower TCR clonality (higher T cell diversity) compared to AOCRC tumors in a multivariable model adjusting for sex, race, tumor location and stage (OR: 0.38, 95% Confidence Interval (CI): 0.25-0.56, p < 0.0001). This association was also observed in the replication cohort (OR: 0.74, 95% CI: 0.62-0.89, p = 0.0013). In the replication cohort, further adjustment for microsatellite instability status did not substantially change the association (OR: 0.73, 95% CI: 0.61-0.88 p = 0.0012). When restricting to microsatellite stable tumors, clonality remained statistically significant in both the discovery and replication cohorts. No significant difference in TCR abundance was observed between YOCRC and AOCRC in either cohort. Conclusions: Higher T cell repertoire diversity, indicating a less focused intratumoral T cell response, is more frequently observed in YOCRC. Further studies are warranted to investigate the role of T cell diversity in the etiology and outcomes of YOCRC.
Clinical outcomes and healthcare utilization among young adults with colon cancer on Medicaid vs private insurance.
Authors: Deenadayalan Vaishali , Solipuram Vinod , Sequeira Gross Harry Guillermo , Gupta Shweta
Published: January 2023

22

Background: The incidence of colon cancer in young adults has been increasing. The role of insurance on the outcomes of young adults diagnosed with colon cancer is unclear. In this study we sought to study the impact of Private insurance vs Medicaid on the outcomes of colon cancer in young adults (<50 years). Methods: Healthcare Cost and Utilization Project National Inpatient Sample (HCUP-NIS) 2016-2019 was queried to identify young adult patients (Age <50 years) admitted for colon cancer using the ICD-10 (International Classification of Diseases) codes. Study population was stratified based on the insurance (private vs Medicaid). Primary outcomes included mortality, length of stay (LOS), and total hospitalization charges (THC). Secondary outcomes included prevalence of pulmonary embolism (PE), ICU admission, acute kidney injury (AKI), acute respiratory failure (ARF), blood transfusion, and sepsis. Statistics were performed using multivariate linear and logistic regression using STATA v17. Results: 59,065 young adult admissions were identified with a principal diagnosis of colon cancer between 2016-2019. Among them 36,765 (62.2%) were on private insurance while 13,480 (22.8%) were on Medicaid. Medicaid patients were significantly younger, female, Black and Hispanic with a higher Charlson comorbidity index (p<0.05). Of the 870 (1.5%) patients that died during the admission, 46.5% (N= 405) had private insurance whereas 26.4% (N= 230) had Medicaid. There was no significant difference in the odds of mortality between private insurance and Medicaid (adjusted OR 1.07, 95% CI 0.69-1.67; p=0.77). The LOS was increased by 1.5 days for Medicaid patients compared to private insurance (95% CI 1.15-1.90; p<0.001). There was no significant difference in total hospitalization charges among both groups. Medicaid patients had higher odds of sepsis (OR 1.5, 95% CI 1.09-1.94; p=0.012), requiring blood transfusion (OR 1.6, 95% CI 1.32-1.83; p<0.000) and less likely to undergo colectomy (OR 0.61, 95% CI 0.55-0.67; p<0.000). There were no significant differences in ICU admission, AKI and ARF. Conclusions: Compared to private insurance, Medicaid patients had similar healthcare outcomes and hospital charges despite a longer LOS. In an era where the country explores options for healthcare reform including single payor system, our study provides insight into similar effectiveness of Medicaid in caring for young colon cancer patients compared to Private Insurance despite higher comorbidities in Medicaid patients.
Tumor mutation burden in colorectal cancers with POLE exonuclease and non-exonuclease domain variants.
Authors: Ringenbach Salena , Shah Shreya M. , Demidova Elena , Faezov Bulat , Andrake Mark D. , Mur Pilar , Xiu Joanne , Korn Wolfgang Michael , Goel Sanjay , Lenz Heinz-Josef , Marshall John , Shields Anthony Frank , Oberley Matthew James , Spetzler David , Valle Laura , Dunbrack Roland , Hall Michael J. , Arora Sanjeevani
Published: January 2023

224

Background: Driver mutations in the exonuclease domain (ExoD) of DNA polymerase epsilon ( POLE) have been associated with high tumor mutation burden (TMB). High TMB is an important marker for immunotherapy, and thus understanding the mechanisms that lead to high TMB is an area of active investigation. Many tumors with POLE ExoD driver mutations have additional POLE variants of uncertain significance (VUS). Some of these VUS lie within the ExoD, while most lie outside the ExoD. This study investigates the role of such POLE VUS on TMB and POLE functionality of ExoD-mutated tumors. Methods: Retrospective colorectal cancer (CRC) genomic profiles from Caris Life Sciences (n=1,870) were separated into groups based on the presence of POLE ExoD mutations and the TMB status. Groups were defined as: ‘TMB-high (TMB-H) POLE ExoD driver without additional POLE VUS’, ‘TMB-H POLE ExoD driver plus POLE VUS’, and ‘TMB-low (TMB-L) POLE variant(s) without ExoD driver’. An additional group with ‘TMB-H POLE variant(s) without ExoD driver’ was identified as potentially novel drivers for future study. The microsatellite instability status (MSI or MSS) was also considered. The level of TMB, the presence of polymerase proofreading-associated mutational signatures and POLE stability/functionality analyzed using AlphaFold2 models and Rosetta Molecular Modeling Suite, were evaluated. Results: POLE variants were identified in 4.9% of the CRCs studied (92/1870). In all, 52.2% (48/92) of those tumors were TMB-H, and 38.0% (35/92) of them contained POLE ExoD drivers. The highest median TMB (mTMB) was found in the ‘TMB-H POLE ExoD driver plus POLE VUS’ group, and this was significantly different from the ‘TMB-H POLE ExoD driver without additional POLE VUS’ and the ‘TMB-L POLE variant without ExoD driver’ group, even when MSI tumors were excluded (p<0.001, Mann-Whitney). These results were validated in TCGA CRC tumors and in other tumor types. Within the ‘TMB-H POLE ExoD driver plus POLE VUS’ group, the mTMB increased with the number of accumulated POLE VUS (p<0.001, Mann Whitney). The nucleotide sequence context of 77.8% of the POLE VUS matched mutational spectra associated with POLE ExoD defects, suggesting that most of the POLE VUS are secondary to the ExoD driver mutation. Finally, the majority of POLE VUS within ‘TMB-H POLE ExoD driver plus POLE VUS’ group have a predicted effect on POLE stability and/or function. Conclusions: Our results suggest that the presence of secondary POLE non-driver variants increase TMB in POLE ExoD-mutated tumors. Although still preliminary, these data could impact prognosis and response to immune checkpoint inhibitors in patients. [Table: see text]
Survival of patients with colorectal cancer (CRC) with low expression of homologous recombination proficient (HRP) genes.
Authors: Walden Daniel , Deshmukh Sachin , Batalini Felipe , Zheng-Lin Binbin , Wu Sharon , Xiu Joanne , Lou Emil , Ahn Daniel H. , Wu Christina , Goel Sanjay , Shields Anthony Frank , Spetzler David , Oberley Matthew James , Korn Wolfgang Michael , Bekaii-Saab Tanios S.
Published: January 2023

225

Background: HR deficient (HRD) CRC has improved outcomes following exposure to DNA damaging agent (DDA) (irinotecan, IR; oxaliplatin, OX) compared to HRP CRC. Low expression of wild type (WT) BRCA1 mRNA is associated with prolonged OS in ovarian cancer; however, this finding has not been investigated in CRC or outside of BRCA. Here, we examine the effect of low expression of HR genes in HRP CRC on post-DDA survival. Methods: 12,860 CRC samples were analyzed by NGS (592, NextSeq; WES, NovaSeq) and WTS (NovaSeq) at Caris Life Sciences (Phoenix, AZ). Samples were classified by RNA expression percentiles. Real world OS was extracted from insurance claims and calculated using Kaplan-Meier estimates for molecularly defined cohorts from first of OX or IR to last contact. Results: Post-IR survival was prolonged with low expression of ATM, CHEK2 and PALB2 in WT ATM, PALB2, and CHEK2 WT CRC, respectively (bottom vs. top 10%, bottom vs. top 25%; p<0.05). Notably, low PALB2 expression (bottom 10%) showed a 14.5-month post-IR benefit compared to high PALB2 expression (top 10%) in WT PALB2 CRC (p=0.003). Post-OX survival was not significantly prolonged with low expression of CHEK2 in WT CHEK2 CRC (bottom 10% vs top 10%, bottom 25% vs top 25%) but was with low expression of ATM (+9.7 months, p=0.02) and PALB2 (+5.6 months, p=0.03) in WT ATM and PALB2 CRC, respectively (all bottom 25% vs top 25%). Conclusions: Here we report the first findings to suggest a novel subclass of CRC defined as the low expression of mRNA of non-mutated HRD genes that exhibit sensitivity to DDA. Low expression of WT ATM, CHEK2, and PALB2 correlates with prolonged OS following IR, post-OX survival was prolonged with low expression of ATM and PALB2. Further characterization defining sensitivity of low expressing HRP genes to DDA may help guide treatment considerations in HRP CRC. [Table: see text]
Comprehensive characterization of KRAS mutations and inter-relation with primary tumor location in colorectal cancers.
Authors: Salem Mohamed E. , Kopetz Scott , Tabernero Josep , Sinicrope Frank A. , Chalabi Myriam , Tie Jeanne , Kadakia Kunal C. , George Thomas J. , Mauer Elizabeth , Macera Lisa , Chao Calvin Y. , Lonardi Sara , Van Cutsem Eric , Andre Thierry , Overman Michael J.
Published: January 2023

231

Background: The recent development of KRAS G12C inhibitors underscores the potential to target KRAS mutations. Right-sided and left-sided colon tumors (RT and LT) exhibit different molecular features. We characterize the prevalence of KRAS-variants, interrelation with primary tumor location, and association with immune biomarkers in CRC. Methods: We retrospectively reviewed CRC tumors of all stages (with known sidedness) that underwent NGS with the Tempus xT assay (DNA-seq of 648 genes at 500x coverage, full transcriptome RNA-seq). Bivariate analyses were performed to compare KRAS alterations, immune biomarkers, and co-mutations by tumor location. P-values comparing individual co-mutations between groups were adjusted for false discovery (FDR). Results: A total of 3,391 CRC were analyzed (RT: n = 442 [13%], transverse: n = 116 [3%], LT; n = 2,833 [84%]) of which 1486 (44%) tumors harbored KRAS mutations. Overall, KRAS mutations were more frequent in RT compared to transverse tumors and LT (52% vs 41% vs 43%, p<0.001, respectively). The most frequent KRAS mutation variants observed were G12D (29 %), G12V (22%), G13D (16%), and G12C (5.7%). There was no significant difference in the prevalence of KRAS variant types between LT and RT (p=0.5). Significant differences in genomic co-mutations with various KRAS variants were observed in the following genes: TP53, FBXW7, and NF1 (FDR- P<0.05). RT and transverse tumors were more likely have MSI-H and TMB-H (>10 mut/mb) status than LT (MSI-H: 18% vs 22% vs 2.2% and TMB-H (20% vs 22% vs 3%, P<0.001), respectively. CRC tumors harboring G13D variants were more likely to be associated with and MSI-H and TMB-H status (and 7.7% and 8.5%) compared to G12D (2.8 % and 3.9 %), G12V (1.8 % and 2.1%), and G12C (0% and 2.4%); P = 0.003 and 0.001. Conclusions: The most frequent KRAS mutation variants observed in CRC tumors were G12D, G12V, G13D, and G12C. There was no significant difference in the prevalence of KRAS variant types between tumors of the left vs right colon. CRC tumors that harbored G13D variants were significantly more likely to be associated with MSI-H and TMB-H status. [Table: see text]
Rectal cancer–specific subtyping using transcriptomics datasets derived from treatment-naive patients to assess novel prognostic signatures and key differences to CMS-based subtyping.
Authors: Prehn Jochen , Kisakol Batuhan , Matveeva Anna
Published: January 2023

235

Background: Molecular subtyping in colorectal cancer provided novel insights into tumour heterogeneity and delivered new prognostic signatures. Existing methods (such as CMS-based subtyping) were generated without much consideration of differences in expression profiles between colon and rectal cancer tissues. Moreover, stage 2 and 3 rectal cancer patients often receive neo-adjuvant radio-chemotherapy which may impact on gene expression when determined from resection samples. Methods: We investigated possible differences between treatment-naive colon and rectal cancers at the molecular level. We collected mRNA expression profiles for rectal and colon cancer samples available from Gene Expression Omnibus (GEO) database (n=2139) and applied batch-effect correction using ComBat. Results: To explore whether CMS subtypes changed after neoadjuvant chemoradiation in rectal cancers, we analysed rectal tumour samples from 40 patients where both pre-operative biopsies and resections after chemoradiotherapy were available. More than half of samples shifted from CMS1-3 to CMS4 after patients received chemoradiotherapy. We therefore focused our further analysis on treatment-naive rectal cancers. We observed that CMS-based subtyping showed different prognostic potential in treatment-naïve colon vs. rectal cancers. Rectal cancers had significantly better disease-free survival (DFS) when assigned to CMS 2 or 4 compared to colon cancers. This suggested differences in disease biology that were not captured by CMS subtyping. We next aimed to identify molecular subtypes in rectal cancers, and identified three prevalent rectal-specific subtypes (RSS) by applying hierarchical clustering in 182 untreated rectal cancer samples. We used gene co-expression network analysis to define functional gene modules to identify these subtypes. Based on the gene modules’ signatures, we built a classifier to predict our new subtypes in different transcriptomics datasets (n=692). We observed that RSS1 had better disease-free survival (DFS), whereas RSS2 exhibited worse short-term DFS (less than 3 years) and RSS3 showed worse long-term DFS (3+ years). We discovered the deactivation of EGFR, MAPK and WNT pathways on RSS3 as well as high CD8+ infiltration and low CD4+ counts in these tumours. RSS2 exhibits low plasma cell and regulatory T-cell abundance and high activations of TGF-β, NF-κB, and TNF-α. We also observed high EMT, angiogenesis and inflammatory response in this subtype. Lastly, RSS1 showed high expression on MYC target and low activity on angiogenesis genes. Moreover, the immune and inflammatory responses were also lower than in the other subtypes. Conclusions: Rectal-specific subtypes are of prognostic importance and provide new insight into molecular disease pathways and potential targets.
Pathological features associated with lymph node disease in patients with appendiceal neuroendocrine tumors.
Authors: Rodriguez Franco Salvador , Thomas Madeline , Khomiak Andrii , Roque Assumpcao Lia , Sugawara Toshitaka , Stewart Camille , Schulick Richard D. , Del Chiaro Marco , Ahrendt Steven , Gleisner Ana
Published: January 2023

238

Background: Appendiceal neuroendocrine tumors (ANETs) are well known for their benign and slow progression; nonetheless, the presence of lymph node metastasis has been described in up to 50% of all cases. The risk of metastasis is mainly associated with tumor size, and the role of other pathological features remains unclear. Hence, this study was designed to assess the pathologic features of ANETs associated with an increased risk of lymph node metastasis. Methods: Patients ≥18yrs of age with ANETs were identified in the National Cancer Database (NCDB) from 2004 to 2017. Groups were defined based on lymph node status as N(-) (all examined nodes were negative) and N(+) (one or more examined nodes were positive). Multivariable models using demographics and oncological features were used to identify factors independently associated with nodal metastasis (N+). Marginal effects, specifically for tumors <2 cm were also calculated. Results: A total of 2,465 cases were identified and included for data analysis. Among these, 23% had lymph node metastases, including 33 of 979 tumors <1 cm (3.4%), and 145 of 696 tumors ≥1 cm but ≤2 cm (20.8%). Tumor size, depth of tumor invasion, and presence of lymphovascular invasion (LVI) were identified as independent predictors of lymph node metastasis (p <0.001). When evaluating marginal effects, both the depth of tumor invasion and the presence of LVI were associated with increased risk of node metastasis in small tumors (<2 cm), comparable to the mere risk of node metastasis in big tumors (≥2cm). In addition, for small ANETs (<2cm), the true positive rates (TPR) of node metastasis vary according to the number of assessed lymph nodes, differing significantly between patients with at least 12 evaluated lymph nodes vs. those with less than 12 (TPR 4.7% vs. 1.6% for <1 cm [p =0.02], and 40.1% vs. 25.7% in ≤2 cm ANETs, [p <0.001]). Conclusions: In ANETs, the presence of high-risk pathological features, including LVI and tumor depth, increases the risk of node metastasis in addition to tumor size, and should be considered when selecting the optimum management and follow-up in these patients. [Table: see text]
Delta radiomic parameters for the evaluation of liver metastases in patients with colorectal cancer treated with cetuximab rechallenge plus avelumab in the CAVE mCRC study.
Authors: Martini Giulia , Nardone Valerio , Ciardiello Davide , De Chiara Marco , Troiani Teresa , D'ambrosio Luca , Napolitano Stefania , Cardone Claudia , Cremolini Chiara , Pietrantonio Filippo , Maiello Evaristo , Avallone Antonio , Cappabianca Salvatore , Ciardiello Fortunato , Reginelli Alfonso , Martinelli Erika
Published: January 2023

241

Background: We performed a retrospective analysis of metastatic colorectal cancer (mCRC) patients enrolled in CAVE trial. Cave mCRC is a single arm, academic, multicenter, phase II trial that evaluated the efficacy of cetuximab rechallenge plus avelumab in mCRC patients. Methods: CT imaging of patients were collected and retrospectively analysed. Inclusion criteria included evidence of liver metastases that could be measured and segmented, availability of an enhanced contrast phase at baseline and restaging CT imaging (within 3 months from baseline). The gross tumor volume (GTV) of liver metastases was evaluated at both baseline and first restaging CT. Multiple texture parameters were extracted with LifeX Software, and delta-texture analysis (D-TA) was calculated as percentage variations in the two time points. Patients were divided in a training cohort (coordinating center, University of Campania “L. Vanvitelli”) and validation cohort (other centers involved in CAVE mCRC trial). Patients were further divided in responders/non-responders according to median progression free survival (mPFS). ROC curve were calculated to obtain a cut-off. Survival analysis of progression free survival (PFS) and overall survival (OS) with Kaplan-Meier method was used in the two subgroups to test the cut-off found with the methods previously described. Results: The original dataset of CAVE mCRC protocol included 77 patients. Liver metastasis were present in 55 patients (71%); after application of inclusion and exclusion criteria, a total of 42 patients were included in the current analysis. The training cohort consisted of 22 patients (for a total of 32 liver metastasis) and the validation cohort of 20 patients (for a total of 28 liver metastasis). After Bonferroni correction, the only D-TA parameters that significantly correlated with mPFS in the training cohort were EntropyHistogram (p:0,021), HomogeneityGLCM (p<0,001) and Dissimilarity GLCM (p:0,002). At multivariate analysis only HomogeneityGLCM resulted significant both in training (p:0,001, OR 0,1) and validation cohort (p:0,021, OR 0,3). ROC curves were generated and the cut-off of HomogeneityGLCM was equal to 0. Patients who developed a reduction of HomogeneityGLCM at first restaging CT scan showed a better PFS and OS (in both training and validation cohorts). Conclusions: Our results suggest that D-TA parameter such as HomogeneityGLCM is able to discriminate better survival outcomes in patient treated with cetuximab rechallenge plus avelumab, further prospective investigations are needed. [Table: see text]
Pretreatment risk prediction for distant metastasis in locally advanced rectal cancer.
Authors: Song Changhoon , Kang Byung-Hee , Kim Jae-Sung
Published: January 2023

248

Background: Distant metastasis is the most common form of recurrence in locally advanced rectal cancer. This study aimed to identify pretreatment risk factors for distant metastases in patients with locally advanced rectal cancer. Methods: This single-institution retrospective study included 285 patients who underwent preoperative chemoradiotherapy followed by surgical resection between January 2016 and December 2020. Pretreatment clinical characteristics, MRI findings, and blood profiles were analyzed to investigate its association with distant metastasis. Results: With a median follow-up of 39 months (range: 6 - 78), 42 patients had confirmed distant metastasis (14.7%), and most distant metastasis occurred within three years (40 patients, 95.2%). In the multivariate analysis, pretreatment extramural venous invasion (EMVI, HR = 3.05, p < 0.001) and carcinoembryonic antigen (CEA) > 10 ng/mL (HR = 2.77, p = 0.002) were independent predictors of distant metastasis. Patients with no adverse factors had significantly better distant metastasis-free survival than patients with 1 or 2 adverse features (92% vs. 70% vs. 59% (p < 0.001) at 3 years). Conclusions: Pretreatment CEA and EMVI have predictive significance for distant metastasis in locally advanced rectal cancer. Our findings may help select patients who may benefit from intensified preoperative treatment, such as total neoadjuvant therapy.
A plasma-based analysis and genomic landscape of patients with high tumor mutational burden (TMB-H), microsatellite stable (MSS) colorectal cancer (CRC).
Authors: Saeed Anwaar , Barnett Reagan , Lou Emil , Madeira da Silva Luciana , Cho May Thet , Lieu Christopher Hanyoung , Fakih Marwan , Kopetz Scott , Loree Jonathan M. , Gholami Sepideh
Published: January 2023

249

Background: An estimated 85% of CRC are microsatellite stable (MSS) and this population has limited response to immune checkpoint inhibitors (ICIs). Biomarkers are needed to identify responders. Although pembrolizumab received FDA universal approval for chemorefractory cancers with tissue TMB-H > 10 mut/Mb, it is not effective across all TMB-H cancers. We aim to identify TMB-H/MSS CRC patients that derive benefit from ICIs through analysis of circulating tumor DNA (ctDNA). Methods: We retrospectively queried Guardant Health database (2020-2022) for patients with advanced CRC who had ctDNA NGS (Guardant360, Redwood City, CA) as part of routine clinical care. The assay covers a 1Mb panel for TMB that counts synonymous and non-synonymous somatic SNVs and indels to calculate a TMB score. Co-mutations were evaluated for MSS cancers with plasma TMB-H; ≥20mut/Mb vs those with low TMB (TMB-L; < 20mut/Mb). RAS- mutated signature was defined as those harboring an activating somatic alteration in KRAS, NRAS, or BRAF. Statistical significance was calculated using Fisher’s exact test. Results: We identified 6412 CRC patients: 11% (711) TMB-H/MSS (median age 64), 69% (4426) TMB-L/MSS (median age 62), 3% MSI-H, and 17% unevaluable. When compared to TMB-L/MSS, patients with TMB-H/MSS had a significantly higher frequency of select genetic mutations, including TP53, APC, KRAS, EGFR, and PIK3CA (p < 0.001). Treatment-naïve TMB-H/MSS cancers had a higher frequency of ARID1A (p = 0.05) and TERT (p = 0.02) alterations, while those post therapy progression had higher frequency of KRAS (p = 0.02), EGFR (p < 0.001), and MAP2K1 (p = 0.002) alterations. A total of 556 (78%) CRC patients in the TMB-H/MSS cohort harbored a RAS-mutated genetic signature. Of those, 432 (78%) belonged to the post therapy progression group, likely representing adaptive clones post anti-EGFR therapy. While RAS-mutated tumors had a higher prevalence of PIK3CA (p = 0.001) and SMAD4 (p = 0.007) co-alterations, RAS-wildtype ones carried a higher frequency of ERBB2 (p = 0.068) alterations. Conclusions: We describe the genomic landscape of patients with plasma TMB-H/MSS CRC. A clinical cohort of TMB-H/MSS CRC patients identified by ctDNA and treated with ICIs will be presented.
Effect of spatial transcriptomic-derived intrinsic epithelial signatures on immune cell recruitment and behaviour in the microenvironment of colorectal cancer.
Authors: Wood Colin , Hatthakarnkul Phimmada , Leslie Holly , Legrini Assya , Cameron Andrew , Pennel Kathryn AF , Sansom Owen J , Edwards Joanne , Jamieson Nigel Balfour , Steele Colin William
Published: January 2023

251

Background: Colorectal Cancer (CRC) remains a significant healthcare problem. Interest in the role of the Tumour Microenvironment (TME) in progression and metastasis is established, yet much remains to be learned about the mechanism of immune cell recruitment to the TME of primary and metastatic tumours. The Klintrup Makinen (KM) score measures the density of immune infiltrate to the invasive edge of tumours and is associated with good prognosis in primary CRC. Neutrophil infiltration to metastases is a poor prognostic feature suggesting phenotypic differences in the TME at primary and metastatic sites. We set out to examine epithelial determination of the TME in primary CRC. Methods: 180 patient’s primary CRC from a curated TMA underwent deep phenotyping using a multiplex-immunofluorescent panel (DAPI, Panck, CD3 (T cells), FOXP3 (T regulatory cells), CD68 (Macrophages), CD163 (Tumour associated macrophage), CD66b (Neutrophils), KI67). The Nanostring GeoMx Spatial Transcriptomic platform was used to obtain segmented (Tumour: Panck+; TME: Panck-) whole transcriptomic (18000 genes) readout from 54 matched cores from the same TMA. Results: No significant association was demonstrated between immune cell density of any cell types and clinicopathological variables including tumour side, MMR status, N-stage or survival. High KM grade was associated with high CD3 (P = 0.01) and FOXP3 (P = 0.002). Density of all immune cell types were highly corelated. Spatial transcriptomic analysis of the segmented epithelium of patients with high KM grade demonstrated over-expression of known neutrophil chemo-attractant CXCL5 (log2FC 3.76 Adj.P < 0.005). The TME compartments of cores with high CD68 and low CD163 counts were associated with adaptive immune co-ordination with high expression of CXCL13 (log2FC -2.97, Adj.P < 0.005) and CD74 (log2FC -1.42 Adj.P 0.007). IGF2 (log2FC 6.08 Adj.P < 0.005) and LCN15 (log2FC 4.96 Adj.P < 0.005) were globally over-expressed in both TME and epithelium of cores with generalised low immune infiltration. The epithelial compartment neutrophil dense cores also over-expressed IGF2 (log2FC -3.53 Adj.P 0.02). Conclusions: TMAs provide a unique opportunity for broad transcriptomic phenotyping of large numbers of patients simultaneously. The Nanostring GeoMx platform is a flexible tool which can be used in novel ways with other proteomic technologies to drive deeper analysis of the TME. CXCL5 expression in the epithelial compartment drives global immune infiltration to the TME and may be manipulated in different contexts therapeutically through CXCR2 signalling in future.
AI-powered multi-omic signature to predict treatment response of patients with colorectal cancer liver metastasis.
Authors: Kapelanski-Lamoureux Audrey , Tsamchoe Migmar , Krzywon Lucyna , Bloom Jessica , Cardin Julie , K Petrillo Stephanie , Gao Zu-hua , Jenna Sarah , Lazaris Anthoula , Metrakos Peter
Published: January 2023

252

Background: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in North America with over 50% of CRC patients developing liver metastases (LM) and 90% will die from metastatic disease. CRCLM are categorized into two main histological growth patterns (HGP) lesions: Desmoplastic (DHGP) and Replacement (RHGP). We have previously published that HGPs display distinct patterns of vascularization, local invasion, growth and response to treatment. Resected CRCLM patients with predominantly DHGP metastasis (~45%) receiving anti-VEGF therapy and chemotherapy have more than double the 5-year overall survival compared to patients with RHGP (~55%) who have received the same treatment. We are therefore treating patients that will not respond and should in fact be managed differently. Currently, there are no available biomarkers or stratification tools that predict colorectal cancer liver metastasis response to therapy. Methods: In collaboration with My Intelligent Machines (MIMs’), we used a proprietary GI2 (Genetic Interaction Graph Inference) algorithm inferring genetic interactions. We developed an AI based pipeline to develop a multi-modality signature, based on blood test analysis, to identify CRCLM patients who will 1) respond to Angiogenic Inhibitor-based therapies and 2) monitor the development of drug resistance (non-responders). Results: We obtained transcriptomic (tumor tissue) as well as proteomic data (liquid biopsy: Extracellular vesicle cargo), linked to clinical data from 40 chemonaïve patients, and fed the data into MIMs platform (BioMark). The software was able to identify differentially expressed genes common to the top differentially enriched proteins leading to a robust liquid biopsy biomarker signature distinguishing DHGP and RHGP. Furthermore, our data demonstrates a unique multi-modality signature providing biological insight for therapeutic failures and/or success. Conclusions: We will use these signatures to not only predict response to treatment but also decipher the molecular mechanisms driving these two phenotypes and identify unique targets. It is expected that this knowledge will lead to optimization of current treatment strategies, allowing for a precision therapy approach to the management of metastatic disease and cost saving for the health care system.
Identifying targeted therapy combination for refractory colorectal cancer using micro-organospheres.
Authors: Ding Shengli , Kanikarla Marie Preeti , Zhang Ray , Baro Nicholas , Brown Daniel W , Bohn Alan , Pittman Kelly , Paduch Marcin , Hsu Shiaowen David , Kopetz Scott , Shen Xiling
Published: January 2023

27

Background: Metastatic colorectal cancer (CRC) patients who have become resistant to standard-of-care (SOC) treatments are often put on targeted therapies. However, patients with targeted mutations still often do not respond to the targeted therapies alone, hence it remains a critically unmet need to explore potential combinatorial regimens that will enhance the efficacy. Using micro-organospheres (MOS) derived from metastatic refractory patients who received targeted therapies, we performed a high-throughput combo screen and compared the response of these patient tumor avatars with their clinical responses. Methods: Biopsies from metastatic CRC patients who became resistant to SOC and were about to receive targeted therapies in clinical trials were molecularly profiled and implanted into immunodeficient mice to generate patient-derived xenografts (PDX). MOS were derived and established from PDX using droplet-based microfluidics for high-throughput 3D screening. Response of the MOS patient avatars to dose-titrated single and combination drug treatments including the available targeted therapies were measured by a fully automated robotic dispensing and imaging pipeline and quantified by AI-based live imaging analytical algorithm to generate drug response curves with IC50 and EC50. Results: MOS were successfully established in all PDX derived from refractory patients with targetable mutations including KRAS G12C and BRAF 600E as well as patients without targetable mutations. We tested both FDA-approved and experimental targeted therapies as single agent or in combination in 5-dose titrations. Longitudinal tracking of each MOS with Live/dead fluorescence dye signals was obtained for 5 days. MOS drug response curves and IC50s show correlation with clinical outcomes. Furthermore, this comprehensive dataset enabled us to assess the potential benefit of different combinations, help deconvolute the efficacy and synergy of individual drugs, and provide insight into patients who fail to response to targeted therapies despite possessing the targeted mutations. As the MOSdiagnostic assay can be completed from a patient biopsy within 10 days and is currently in a registered, multi-site clinical trial (ClinicalTrials.gov # NCT05189171), this study provides proof-of-principle for a potential clinical MOS-based assay to guide refractory patients to the optimal combination regimen. Conclusions: Refractory patients may benefit from a MOS-based high-throughput screen assay to select the optimal combination regimen containing targeted therapy.