Instability In Colorectal Cancer Research Articles

Lymphocytic infiltration in stage II microsatellite stable colorectal tumors: A retrospective prognosis biomarker analysis.

BackgroundIdentifying stage II patients with colorectal cancer (CRC) at higher risk of progression is a clinical priority in order to optimize the advantages of adjuvant chemotherapy while avoiding unnecessary toxicity. Recently, the intensity and the quality of the host immune response in the tumor microenvironment have been reported to have an important role in tumorigenesis and an inverse association with tumor progression. This association is well established in microsatellite instable CRC. In this work, we aim to assess the usefulness of measures of T-cell infiltration as prognostic biomarkers in 640 stage II, CRC tumors, 582 of them confirmed microsatellite stable.Methods and findingsWe measured both the quantity and clonality index of T cells by means of T-cell receptor (TCR) immunosequencing in a discovery dataset (95 patients with colon cancer diagnosed at stage II and microsatellite stable, median age 67, 30% women) and replicated the results in 3 additional series of stage II patients from 2 countries. Series 1 and 2 were recruited in Barcelona, Spain and included 112 fresh frozen (FF, median age 69, 44% women) and 163 formalin-fixed paraffin-embedded (FFPE, median age 67, 39% women) samples, respectively. Series 3 included 270 FFPE samples from patients recruited in Haifa, Northern Israel, as part of a large case-control study of CRC (median age 73, 46% women). Median follow-up time was 81.1 months. Cox regression models were fitted to evaluate the prognostic value of T-cell abundance and Simpson clonality of TCR variants adjusting by sex, age, tumor location, and stage (IIA and IIB). In the discovery dataset, higher TCR abundance was associated with better prognosis (hazard ratio [HR] for ≥Q1 = 0.25, 95% CI 0.10–0.63, P = 0.003). A functional analysis of gene expression on these tumors revealed enrichment in pathways related to immune response. Higher values of clonality index (lower diversity) were not associated with worse disease-free survival, though the HR for ≥Q3 was 2.32 (95% CI 0.90–5.97, P = 0.08). These results were replicated in an independent FF dataset (TCR abundance: HR = 0.30, 95% CI 0.12–0.72, P = 0.007; clonality: HR = 3.32, 95% CI 1.38–7.94, P = 0.007). Also, the association with prognosis was tested in 2 independent FFPE datasets. The same association was observed with TCR abundance (HR = 0.41, 95% CI 0.18–0.93, P = 0.03 and HR = 0.56, 95% CI 0.31–1, P = 0.042, respectively, for each FFPE dataset). However, the clonality index was associated with prognosis only in the FFPE dataset from Israel (HR = 2.45, 95% CI 1.39–4.32, P = 0.002). Finally, a combined analysis combining all microsatellite stable (MSS) samples demonstrated a clear prognosis value both for TCR abundance (HR = 0.39, 95% CI 0.26–0.57, P = 1.3e-06) and the clonality index (HR = 2.13, 95% CI 1.44–3.15, P = 0.0002). These associations were also observed when variables were considered continuous in the models (HR per log2 of TCR abundance = 0.85, 95% CI 0.78–0.93, P = 0.0002; HR per log2 or clonality index = 1.16, 95% CI 1.03–1.31, P = 0.016).LimitationsThis is a retrospective study, and samples had been preserved with different methods. Validation series lack complete information about microsatellite instability (MSI) status and pathology assessment. The Molecular Epidemiology of Colorectal Cancer (MECC) study had information about overall survival instead of progression-free survival.ConclusionResults from this study demonstrate that tumor lymphocytes, assessed by TCR repertoire quantification based on a sequencing method, are an independent prognostic factor in microsatellite stable stage II CRC.

Age-dependent performance of BRAF mutation testing in Lynch syndrome diagnostics.

BRAF V600E mutations have been reported as a marker of sporadic microsatellite instability (MSI) colorectal cancer (CRC). Current international diagnostic guidelines recommend BRAF mutation testing in MSI CRC patients to predict low risk of Lynch syndrome (LS). We evaluated the age-specific performance of BRAF testing in LS diagnostics. We systematically compared the prevalence of BRAF mutations in LS-associated CRCs and unselected MSI CRCs in different age groups as available from published studies, databases and population-based patient cohorts. Sensitivity/specificity analysis of BRAF testing for exclusion of LS and cost calculations were performed. Among 969 MSI CRCs from LS carriers in the literature and German HNPCC Consortium, 15 (1.6%) harbored BRAF mutations. Six of seven LS patients with BRAF-mutant CRC and reported age were <50 years. Among 339 of 756 (44.8%) of BRAF mutations detected in unselected MSI CRC, only 2 of 339 (0.6%) BRAF mutations were detected in patients <50 years. The inclusion of BRAF testing led to high risk of missing LS patients and increased costs at age <50 years. BRAF testing in patients <50 years carries a high risk of missing a hereditary cancer predisposition and is cost-inefficient. We suggest direct referral of MSI CRC patients <50 years to genetic counseling without BRAF testing.

497P Investigating the potential relationship between KMT2C/KMT2D mutations and immune checkpoint inhibitor (ICI) in colorectal cancer (CRC)

KMT2C and KMT2D usually forms a complex and are responsible for methylation at H3K4. According to our previous results, KMT2C/2D mutations in CRC were usually accompanied by DNA damage repair genes mutations and microsatellite instability high (MSI-H), both of which reported have tightly related with immunotherapy. We hypothesized that KMT2C/2D mutations may cause genomic instability in CRC and may be a predictor of ICI efficacy. Therefore, we investigated the relationship between the mutations of KMT2C/2D and immunotherapy, and analyzed the potential clinical benefit from ICI for patients with KMT2C/2D mutations.

Abstract 2825: High-plex, spatial RNA profiling of tumor infiltrating leukocytes and the tumor microenvironment of microsatellite instable colorectal cancer using GeoMx™ Digital Spatial Profiler

Abstract Immunotherapeutic intervention has revolutionized cancer treatment but improved understanding of immunomodulation in tumors is still necessary to expand the reach of these therapies and identify rational combination approaches. An important aspect of this process will be characterizing the molecular differences between tumor-infiltrating leukocytes (TILs) and stromal leukocytes (non-TILs) surrounding the same tissues. Most studies to date have focused on dissociated tissues, which means identifying the origin of the profiled leukocytes is only possible with post-hoc inference. High-plex profiling that retains spatial orientation has proven difficult in fixed tissues, preventing direct understanding of TIL localization beyond a handful of pre-selected targets. To explore the transcriptional profile of TILs in situ, we leveraged a high-plex (1,400+ gene) mRNA panel for the GeoMxTM Digital Spatial Profiler (DSP) to profile microsatellite instable (MSI) colorectal cancer (CRC) samples noted to have a high abundance of CD3+ TILs by 4-color immunofluorescence (IF). In this study, more than 5,000 unique probes (3 to 10 probes per target mRNA) with coupled photocleavable oligonucleotide tags were hybridized to formalin-fixed paraffin-embedded (FFPE) tissue sections from these CRC samples. Regions of interest were selected inside (n = 6, per tumor) and outside (n = 6, per tumor) the tumor invasive margin focusing on tumor or stromal regions with high numbers of CD3+ cells. Within each region of interest, we created a custom segmentation strategy to specifically illuminate CD3+ cells, and then sequentially illuminate regions neighboring those cells. These additional custom masks (n = 47) were defined by extending multiple contours around the initially selected TILs and non-TILs to determine differences in the local environment of each population. Collected photocleaved oligonucleotide tags were sequenced using the GeoMxTM NGS workflow. Targets included in the 1,400+ gene panel represent immune cell markers, checkpoint molecules, cytokines and chemokines, canonical cancer pathways, and biological signatures. We found that regions neighboring TILs express higher levels of known oncogenic pathways and stromal regions neighboring non-TILs were noted to have higher expression of ECM genes, confirming the specificity of the profiling approach. Furthermore, we found that TILs specifically up-regulate expression of cytolytic pathway genes, as well as several coinhibitory and costimulatory checkpoint genes. We also observe dysregulation of members of the adenosine metabolism pathway within the tumor regions profiled and TILs, but not in regions adjacent to the tumor itself. Together, our results demonstrate the feasibility of profiling specific cell populations with a high plex mRNA panel in situ in FFPE tissue, thus enabling pathway level differential expression analyses and exploration of key interactions between neighboring cell types while retaining their spatial context. For research use only. Not for use in diagnostic procedures. Citation Format: Zachary Norgaard, Dan Zollinger, Jason Reeves, Zoey Zhou, Michelle Kriner, Jill McKay-Fleisch, Arya Bahrami, Sarah Warren, Sarah Church, Chris Merritt, Margaret Hoang, Joseph Beechem. High-plex, spatial RNA profiling of tumor infiltrating leukocytes and the tumor microenvironment of microsatellite instable colorectal cancer using GeoMx™ Digital Spatial Profiler [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2825.

A robust method for the rapid detection of microsatellite instability in colorectal cancer

Although several computational tools using next-generation sequencing (NGS) data have been proposed to detect microsatellite instability (MSI) status, they still have limitations and need improvement. We developed a NovoPM-MSI method to detect MSI status based on NGS data. This method evaluated target mononucleotide microsatellite loci that were sequenced during targeted gene enrichment analysis and reported sample instability score as the fraction of unstable loci within the target set after assessing locus instability by comparing length distribution in paired tumor-normal samples. We validated this method against the conventional MSI-PCR method in 113 paired colorectal cancer (CRC) specimens and compared the performance of NovoPM-MSI to that of mSINGS and MANTIS in accuracy and runtime efficiency. By using the MSI status from MSI-PCR as the gold standard, the three computational methods showed the same sensitivity of 88.9% but different specificities (NovoPM-MSI 97.1%, MANTIS 86.5% and mSINGS 99.0%). Only NovoPM-MSI could greatly improve both the sensitivity and specificity by setting an ambiguous interval. MANTIS had the shortest average runtime (16.3 sec), followed by NovoPM-MSI (18.3 sec) and mSINGS (109.0 sec). In short, the NovoPM-MSI method provides a fast and reliable MSI detection method with accuracy comparable to MSI-PCR in paired CRC samples.

Integrin-Linked-Kinase Overexpression Is Implicated in Mechanisms of Genomic Instability in Human Colorectal Cancer.

Genomic instability is a hallmark of cancer cells contributing to tumor development and progression. Integrin-linked kinase (ILK) is a focal adhesion protein with well-established role in carcinogenesis. We have previously shown that ILK overexpression is critically implicated in human colorectal cancer (CRC) progression. In light of the recent findings that ILK regulates centrosomes and mitotic spindle formation, we aimed to determine its implication in mechanisms of genomic instability in human CRC. Association of ILK expression with markers of genomic instability (micronuclei formation, nucleus size, and intensity) was investigated in diploid human colon cancer cells HCT116 upon ectopic ILK overexpression, by immunofluorescence and in human CRC samples by Feulgen staining. We also evaluated the role of ILK in mitotic spindle formation, by immunofluorescence, in HCT116 cells upon inhibition and overexpression of ILK. Finally, we evaluated association of ILK overexpression with markers of DNA damage (p-H2AX, p-ATM/ATR) in human CRC tissue samples by immunohistochemistry and in ILK-overexpressing cells by immunofluorescence. We showed that ILK overexpression is associated with genomic instability markers in human colon cancer cells and tissues samples. Aberrant mitotic spindles were observed in cells treated with specific ILK inhibitor (QLT0267), while ILK-overexpressing cells failed to undergo nocodazole-induced mitotic arrest. ILK overexpression was also associated with markers of DNA damage in HCT116 cells and human CRC tissue samples. The above findings indicate that overexpression of ILK is implicated in mechanisms of genomic instability in CRC suggesting a novel role of this protein in cancer.

MLH1 Deficiency Induces Cetuximab Resistance in Colon Cancer via Her-2/PI3K/AKT Signaling.

The rapid onset of resistance to cetuximab (CTX) limits its clinical utility in colorectal cancer (CRC) patients. This study aims to understand a potential role of mismatch repair gene mutL homolog 1 (MLH1) in CTX response. Functional analysis of MLH1 in Her‐2/phosphoinositide 3‐kinases (PI3K)/PKB protein kinase (AKT)‐regulated CTX sensitivity is performed using human CRC specimens, CRC cell lines with different MLH1 expression levels, and a subcutaneous xenograft model. Overexpression, knockdown, small interfering RNA, and inhibitors are used to examine the role of MLH1 and HER‐2 downstream signaling and apoptotic targets in CTX sensitivity. Reduced MLH1 expression is correlated with unfavorable prognosis in cetuximab‐treated patients. MLH1 loss decreases CTX sensitivity through Her‐2/PI3K/AKT signaling and apoptosis resistance in culture and in xenografts, while MLH1 overexpression increases CTX sensitivity. Blocking Her‐2 signaling increases CTX sensitivity of microsatellite instability CRC in vitro and in vivo. MLH1 loss induces activation of Her‐2/PI3K/AKT signaling and leads to cetuximab resistance in colon cancer.

Downregulation of GLYR1 contributes to microsatellite instability colorectal cancer by targeting p21 via the p38MAPK and PI3K/AKT pathways

BackgroundGLYR1 has a high mutation frequency in microsatellite instability colorectal cancer (MSI CRC) and is presumed to be a novel tumor suppressor. However, the role of GLYR1 in tumors has never been studied. In particular, the downregulation of GLYR1 in MSI CRC is worthy of further investigation.MethodsWestern blot and immunohistochemistry analyses were used to detect GLYR1 protein expression in CRC tissues and cell lines, and the clinical significance of GLYR1 was also analyzed. The relationship between GLYR1 and MLH1 was validated by immunofluorescence, immunoprecipitation and bioinformatics analyses. Western blotting, qRT-PCR, CCK-8 assays, colony formation assays, flow cytometry and Hoechst 33258 staining assays were used to assess the effect of GLYR1 on the cell cycle progression, proliferation, differentiation and apoptosis of CRC cells in vitro. The related mechanisms were initially investigated by Western blotting.ResultsGLYR1 was significantly downregulated in MSI CRC and its expression was negatively correlated with tumor size and positively correlated with tumor differentiation in CRC patients. In addition, GLYR1 interacted with MLH1 to regulate its nuclear import and expression. Moreover, downregulation of GLYR1 accelerated G1/S phase transition, promoted proliferation and inhibited differentiation of SW480 and SW620 cells in vitro. Furthermore, downregulation of GLYR1 decreased the sensitivity to 5-fluorouracil (5-FU) by inhibiting the mitochondrial apoptosis pathway in CRC cells. Inhibition of the p38 mitogen-activated protein kinase (p38MAPK) and activation of the phosphatidyl 3-kinase/protein kinase B (PI3K/Akt) signaling pathways were involved in the mechanism by which GLYR1 downregulated p21.ConclusionsOurs is the first study to elucidate the role of GLYR1 in tumors and provide evidence for GLYR1 as a biological marker that reflects the degree of malignancy and sensitivity to 5-FU in MSI CRC.

Development of a Humanized 3D Tumor Spheroid Models and Discovery of Effective Immunotherapy Strategies Against Resistant Microsatellite Stable Colorectal Cancers

Abstract Despite the current success of immunotherapies, only microsatellite instable (MSI) Colorectal Cancer (CRC) typically respond to T cell-driven immunotherapies while microsatellite stable (MSS) CRC displays poor response. Our goal is to identify resistance mechanisms in MSS-CRC to uncover novel therapeutic targets. Current syngeneic CRC models are largely immunogenic and lack complex structure. Therefore, we developed novel 3D humanized CRC mouse models which form complex human-like tumors which are typically absent in syngeneic systems. We investigated molecular profiles of human MSI-versus MSS-CRC tumor xenografts following Keytruda immunotherapy and identified an immunosuppressive profile in MSS models that was not observed in MSI-CRC. We evaluated the therapeutic potential of blocking inflammation in combination with other immuno-modulatory therapies in both cold and hot CRC models and identified a significant regression of tumor burden in MSS-CRC mice. Our studies provide a novel CRC in vivo model and a unique opportunity to better define how immune cells interact in human MSS- and MSI-CRC microenvironments, to enable clinical therapeutic implementation.

CAT25 defines microsatellite instability in colorectal cancer by high-resolution melting PCR

ABSTRACT Background CAT25 (T25mononucleotide repeat of the Caspase 2 gene), is a promising DNA marker for detecting microsatellite instability (MSI) in colorectal cancer. CAT25 has the potential to be incorporated into the Bethesda panel, a commonly used panel of DNA microsatellites, or replace it in its entirety. We aimed to develop and validate a high-resolution melting-PCR (HRM-PCR) method for CAT25 instability detection in clinical samples. Methods The instability of CAT25, BAT25 (a poly(A) tract occurring in c‐kit) and BAT26 (a poly(A) tract localized in hMSH2) microsatellites were assessed in DNA from tumour and peripheral blood obtained from 110 patients with colorectal cancer using HRM-PCR and capillary electrophoresis. Immunohistochemistry (IHC) staining for MSH2, MSH6, MLH1, and PMS2 enzymes was performed on tumours with jigj MSI. Allelic size variation of CAT25 was analysed on peripheral blood DNA from 208 healthy volunteers. Results The HRM-PCR for CAT25 was validated in clinical samples. CAT25 showed a tight range of 64–66 base pairs. Of 110 tumours, 11 had High MSI, later confirmed by IHC. CAT25 defines MSI alone as well as when used together with BAT25 and BAT26. CAT25 results provided 100% predictive values and p < 0.0001 to classify a tumour as having high MSI. Conclusions We developed and validated a new HRM-PCR assay to detect CAT25 instability. Our findings showed a limited allelic size variation of CAT25 and highlighted to CAT25 as a promising marker for MSI analysis.

Development and interpretation of a pathomics-based model for the prediction of microsatellite instability in Colorectal Cancer.

Microsatellite instability (MSI) has been approved as a pan-cancer biomarker for immune checkpoint blockade (ICB) therapy. However, current MSI identification methods are not available for all patients. We proposed an ensemble multiple instance deep learning model to predict microsatellite status based on histopathology images, and interpreted the pathomics-based model with multi-omics correlation.Methods: Two cohorts of patients were collected, including 429 from The Cancer Genome Atlas (TCGA-COAD) and 785 from an Asian colorectal cancer (CRC) cohort (Asian-CRC). We established the pathomics model, named Ensembled Patch Likelihood Aggregation (EPLA), based on two consecutive stages: patch-level prediction and WSI-level prediction. The initial model was developed and validated in TCGA-COAD, and then generalized in Asian-CRC through transfer learning. The pathological signatures extracted from the model were analyzed with genomic and transcriptomic profiles for model interpretation.Results: The EPLA model achieved an area-under-the-curve (AUC) of 0.8848 (95% CI: 0.8185-0.9512) in the TCGA-COAD test set and an AUC of 0.8504 (95% CI: 0.7591-0.9323) in the external validation set Asian-CRC after transfer learning. Notably, EPLA captured the relationship between pathological phenotype of poor differentiation and MSI (P < 0.001). Furthermore, the five pathological imaging signatures identified from the EPLA model were associated with mutation burden and DNA damage repair related genotype in the genomic profiles, and antitumor immunity activated pathway in the transcriptomic profiles.Conclusions: Our pathomics-based deep learning model can effectively predict MSI from histopathology images and is transferable to a new patient cohort. The interpretability of our model by association with pathological, genomic and transcriptomic phenotypes lays the foundation for prospective clinical trials of the application of this artificial intelligence (AI) platform in ICB therapy.

Genetic and epigenetic analysis of the beta-2-microglobulin gene in microsatellite instable colorectal cancer.

One of the most common mechanisms of immune evasion in MSI colorectal cancers (CRCs) is loss of HLA class I expression due to mutations in B2M gene which can become a negative predictor for checkpoint blockade therapy. The aim of this study was the determination of prevalence of B2M somatic mutations in MSI CRC patients and relationship between B2M mutations and lymphocytes infiltration and other clinicopathological features as well as detection of methylation changes in B2M promoter region which can be another mechanism of immune escape. In our study, 37 MSI-H and 5 MSI-L patients were selected for screening of B2M mutational and methylation status. The characterization of patients was based on standard histopathological diagnosis and TNM classification; BRAF, KRAS mutations, tumor-infiltrating lymphocytes and peritumoral lymphoid reaction were also determined. MSI analysis was performed using fragment analysis. B2M mutations were identified by Sanger sequencing, and methylation of CpG islands in promoter region was detected by methylation-specific PCR. Heterozygous mutations in the B2M gene were detected in five MSI-H patients (13.5%), while the mutation c.45_48delTTCT was determined in four patients and mutation c.276delC was found in two patients. One of these five patients was compound heterozygote harboring both mutations. Methylation of the promoter region of the B2M gene was observed in one patient with MSI-H colorectal cancer. Detection of genetic and epigenetic changes in B2M gene could be important in personalized therapy for CRC patients as these changes may be one of the mechanisms of secondary resistance of MSI positive tumors to immunotherapy.

Immune Checkpoint Inhibition in Colorectal Cancer: Microsatellite Instability and Beyond.

Immune checkpoints inhibitors (ICIs) have been a breakthrough, with unique response and survival patterns compared with chemotherapy for patients with advanced Mismatch Repair-deficient/Microsatellite instable (dMMR/MSI) colorectal cancer, but have shown disappointing results in Mismatch Repair-proficient/Microsatellite stable (pMMR/MSS) colorectal cancer. As up to 50% of patients harboring dMMR/MSI advanced cancers will ultimately progress after PD-1 blockade, biomarkers are needed to predict response/resistance to immunotherapy and to select patients for immunomodulating combination therapies. Patients with pMMR/MSS colorectal cancer present with distinct immune profiles compared to dMMR/MSI tumors, giving evidence of different immune escape mechanisms, which could be overcome through individualized immunotherapeutic strategies. In this review we discuss the latest developments in the field of immunotherapy for dMMR/MSI and pMMR/MSS colorectal cancers, and unresolved questions and considerations concerning the use of ICI therapies in this population. Future immunomodulation strategies based on biomarker selection (tumor mutational burden, Immunoscore®, mutational profile) are discussed.

Toxicity associated with PD-1 blockade after allogeneic haematopoietic cell transplantation.

Cancer immunotherapy with immune checkpoint inhibitors (ICIs) such as programmed death ligand-1 (PD-L1) blockers offers pronounced clinical benefit with durable responses and a manageable safety profile. Patients with a high risk of immune-related adverse events are generally excluded from clinical trials testing ICI therapy. Thus, only a little information on the safety and clinical outcome of patients treated with an ICI after allogeneic haematopoietic cell transplantation (HCT) is currently available. Here, we report the characteristics and outcomes of six patients with, respectively, clear cell renal carcinoma, diffuse large cell B-cell lymphoma, Hodgkin lymphoma, a microsatellite instable colorectal cancer and melanoma who were treated with PD-1 blocking antibodies. All patients had previously undergone allogeneic HCT. Severe grade 3&ndash;5 immune-related adverse events were observed in three of five patients who received full-dose ICI therapy. One patient received a lower dose of PD-1 blocking antibody. Only one patient had an objective response, whereas all the other patients had progressive disease. The high toxicity of a full- dose anti-PD-1 treatment regimen suggests that other treatment approaches for patients after allogeneic HCT are needed outside of the context of relapsed Hodgkin disease. In cases where ICI therapy is the only treatment option, reduced dosing should be explored.

Consensus on the detection of microsatellite instability in colorectal cancer and other related solid tumors in China

Microsatellite instability (MSI) which resulted from the deficiency of DNA mismatch repair (MMR), is an important clinical significance in the related solid tumors, such as colorectal cancer and endometrial cancer. There are several methods to detect MSI status, including immunohistochemistry for MMR protein, multiplex fluorescent polymerase chain reaction (PCR) for microsatellite site and MSI algorithm based on next generation sequencing (NGS). The consensus elaborates the definition and clinical significance of MSI as well as the advantages and disadvantages of the three detection methods. Through this expert consensus, we hope to promote the screening which based on MSI status in malignant tumors and improve the acknowledge of clinicians about various testing methods. Thereby, they could interpret the results more accurately and provide better clinical services to patients.

125P - Tumour mutation burden and microsatellite instability in colorectal cancer

BackgroundThe role of tumour mutation burden (TMB) as predictive biomarker of response to immune checkpoint inhibitors (ICI) is being explored in colorectal cancer (CRC). Microsatellite instability status (MSI) is currently used to identify CRC patients who may benefit from ICI. However, TMB values vary significantly among MSI CRC. In addition, selected microsatellite stable (MSS) with high TMB might also benefit treatment with ICI. MethodsTMB evaluation was performed with the Oncomine Tumor Mutation Load Assay (OTML, Thermofisher) on the Ion S5XL platform. Data analysis was carried out using Ion Reporter Software v5.10. TMB was calculated as the total number of non-synonymous somatic single nucleotide variants (SNVs) and indels divided by number of bases sequenced. MSI status was analysed by means of the Idylla MSI assay (Biocartis), which evaluates the presence of mutations in 7 novel MSI loci. ResultsTMB analysis was performed on 106 formalin-fixed paraffin embedded CRC samples and the data were compared with the MSI results. The Idylla assay classified 68 samples as MSS and 38 samples as microsatellite instable (MSI-H). The TMB values ranged from zero to 21.22 (median: 5.005) in the MSS and from 13.42 to 204.8 in the MSI-H group (median: 25.66), with a significant difference in median values (P<0.0001). A significant difference in TMB values was also observed when the number of mutated MSI-associated loci was ≥2 versus <2. We next correlated the presence of mutations in a group of driver genes with the TMB values. Significantly different median TMB values were registered when a BRAF (Mann Whitney p value: 0.0023) or PIK3CA mutation (p value: 0.0082) was present, but not when KRAS alterations were detected. ConclusionsThe TMB values assessed with the OTML assay strongly correlated with MSI status in CRC. However, significant heterogeneity in TMB levels were detected among both MSI and MSS tumors, suggesting that TMB testing might provide additional information on sensitivity to ICI in CRC. The correlations with driver gene alterations might help in selecting tumors to be tested for TMB. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Transcriptome and Network Dissection of Microsatellite Stable and Highly Instable Colorectal Cancer.

Background:Colorectal cancer (CRC) is one of the most common cancers worldwide with high number of mortality every year. Microsatellite instability (MSI) is a considerable feature of CRC which affects prognosis and treatment. High level of MSI or MSI-high (MSI-H) colorectal cancer has better prognosis and immunotherapy response, while microsatellite stable (MSS) CRC has better response to 5-fluorouracil (5-FU)-based chemotherapy. More studies are needed, specifically on MSS CRC which has worse prognosis, to further reveal biological differences and similarities between MSS and MSI colorectal cancer, which may equip us with the knowledge to develop more promising therapeutic approaches to target both types or be more effective for each type. Methods:We aimed to find affected biological processes and their regulators in both type, MSS and MSI-H, of CRC; as well as reveal specific ones in each type. We applied meta- and network analysis on freely available transcriptome data in MSS and MSI-H colorectal cancer from gene expression omnibus (GEO) database to detect common differentially expressed genes (DEGs) and critical biological processes and predict their most significant regulators. Results:Our results demonstrate considerable up and downregulation in cell cycle and lipid catabolism processes, respectively; and introduced MYC and FOXM1 as two central and up-stream regulators of DEGs in both type of CRC. Chemokine-mediated processes displayed up-regulation in MSI-H type, while metastasis-related processes showed more activation in MSS CRC. Additionally, DACH1 and TP53 were detected as two important transcription factors that differentially expressed just in MSS and MSI-H, respectively. Conclusion:Our results can explain why MSI and MSS CRC display different immunotherapy response, prognosis, and metastasis feature. Moreover, our predicted up-stream regulators in the regulatory networks may be promising therapeutic targets.

Spatial UBE2N protein expression indicates genomic instability in colorectal cancers

BackgroundOne major hallmark of colorectal cancers (CRC) is genomic instability with its contribution to tumor heterogeneity and therapy resistance. To facilitate the investigation of intra-sample phenotypes and the de novo identification of tumor sub-populations, imaging mass spectrometry (IMS) provides a powerful technique to elucidate the spatial distribution patterns of peptides and proteins in tissue sections.MethodsIn the present study, we analyzed an in-house compiled tissue microarray (n = 60) comprising CRCs and control tissues by IMS. After obtaining protein profiles through direct analysis of tissue sections, two validation sets were used for immunohistochemical evaluation.ResultsA total of 28 m/z values in the mass range 800–3500 Da distinguished euploid from aneuploid CRCs (p < 0.001, ROC AUC values < 0.385 or > 0.635). After liquid chromatograph-mass spectrometry identification, UBE2N could be successfully validated by immunohistochemistry in the initial sample cohort (p = 0.0274, ROC AUC = 0.7937) and in an independent sample set of 90 clinical specimens (p = 0.0070, ROC AUC = 0.6957).ConclusionsThe results showed that FFPE protein expression profiling of surgically resected CRC tissue extracts by MALDI-TOF MS has potential value for improved molecular classification. Particularly, the protein expression of UBE2N was validated in an independent clinical cohort to distinguish euploid from aneuploid CRCs.

Abstract 3975: Tumor mutation burden and microsatellite instability in colorectal cancer

Abstract Introduction In colorectal cancer (CRC), the use of immunotherapy is currently based on the evaluation of microsatellite instability (MSI) for the identification of patients who may benefit from this treatment. Recently, tumour mutation burden (TMB) has been evaluated as a useful biomarker to predict response to immune checkpoint (IO) therapy. TMB can be measured by means of targeted next generation sequencing with panels covering approximately 1 Mb of the human genome. We performed TMB analysis on tumor cell lines and CRC samples and compared the results with MSI status to explore the relationship between these two biomarkers. Materials and methods MSI status was evaluated by means of routine immunohistochemistry (IHC), the Idylla MSI assay (Biocartis) and with fluorescence capillary electrophoresis-based DNA fragment size analysis of a T17 mononucleotide sequences within intron 8 of heat shock protein 110 kDa HSP110 (17 polythymine, T17). TMB evaluation was performed with the Oncomine Tumor Mutation Load Assay (Thermofisher) on the Ion S5XL platform. Data analysis was carried out using Ion Reporter Software v5.6. TMB was calculated as the total number of somatic single nucleotide variants (SNVs) divided by number of bases with sufficient coverage. Results We first evaluated the TMB in 13 tumor cell lines and correlated the results with massively parallel sequencing data from &amp;gt;1600 genes for the same cell lines available on cBioPortal, demonstrating an high correlation between the Oncomine Tumor Mutation Load Assay and the TMB value obtained by wide genetic profiling (R² = 0.976). We next analyzed a cohort of 23 MMR deficient (MMR-D) and 32 MMR-proficient (MMR-P) formalin fixed paraffin embedded (FFPE) CRC, as classified by IHC. The MMR data were compared to TMB and revealed TMB values not in line with the expected results, since some samples in the MMR-D group had low TMB values (TMB range: 5.5-134.4). We re-evaluated the 23 MMR-D samples by means of the Idylla System. The Idylla MSI assay re-classified 7 samples as microsatellite stable (MSS) and categorized the remaining 16 samples as MSI-High (MSI-H). The results were confirmed by the T17 analysis. In the MSS group (n. 39), the mean and median TMB value were 11.48 and 10.39, with TMB ranging from 4.88 to 33.29. The mean and median TMB for the MSI-H group (n. 16) were 43.89 and 38.63, and the TMB values ranged from 19.63 to 134.4. While the medians of the two groups were significantly different (Mann-Whitney test P&amp;lt;0.0001), these data confirm a high heterogeneity of TMB within MSI-H CRC. One MSS case displayed a TMB close to the median TMB of the MSI-H group (TMB: 33.29). The MSS status of this sample was confirmed by the Idylla System. Conclusions These data suggest that routine IHC methods for MSI evaluation can lead to a wrong classification of CRC. On the other hand, TMB assessment might better identify patients who may benefit from IOs within both the MSI-H and the MSS subgroups. Citation Format: Francesca Fenizia, Raffaella Pasquale, Cristin Roma, Francesca Bergantino, Anna Maria Rachiglio, Nicoletta Chicchinelli, Paolo Graziano, Gerardo Botti, Fabiana Tatangelo, Giosuè Scognamiglio, Christopher Allen, Matilde Lambiase, Alessia Iannaccone, Nicola Normanno. Tumor mutation burden and microsatellite instability in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3975.

Abstract 1743: Evaluating the effect of USP22 depletion on chromosome instability in colorectal cancer

Abstract Chromosome instability (CIN) is an aberrant phenotype defined as an increase in the rate at which whole chromosomes or large chromosomal fragments are gained or lost, and is observed in virtually all cancer types. Although CIN is associated with aggressive tumors, multi-drug resistance and poor patient prognosis, the genetic defects underlying CIN remain poorly understood. Preliminary data from our laboratory suggest that diminished USP22 (Ubiquitin-Specific Peptidase 22) expression induces CIN. Importantly, USP22 deletions and reduced USP22 expression are frequently observed in multiple cancer types, including colorectal cancer, suggesting diminished USP22 expression/function may be a pathogenic event underlying oncogenesis. Therefore, the present study seeks to evaluate the impact diminished USP22 expression has on CIN by determining whether karyotypically stable colorectal cancer (HCT116) and immortalized fibroblast (hTERT) cell lines can be converted into karyotypically unstable lines following the reduction of USP22 expression. Diminished USP22 expression was achieved by siRNA-based silencing or CRISPR-Cas9-mediated gene knockout (KO) and quantitative image-based single-cell analyses were employed to monitor changes in surrogate markers for CIN, including nuclear areas and chromosome numbers. USP22 silencing induced significant changes in nuclear area as well as increased frequencies of chromosomes gains and losses, which suggest USP22 is a CIN gene. Next, to assess the long-term impact diminished USP22 expression has on CIN, two HCT116 USP22-KO clones were monitored for 10 weeks and revealed dynamic changes in nuclear areas and chromosome numbers over time relative to controls. Collectively, these data identify USP22 as a novel CIN gene and indicate that USP22 deletions in cancer may have long-term impact on CIN and may thereby promote oncogenesis. Further analysis is ongoing to investigate the mechanism through which USP22 depletion induces CIN, and in particular its effect on H2B monoubiquitination. Characterizing the effect diminished USP22 expression has on CIN is the first step towards the development of therapeutic strategies targeting USP22-deficient cancers. Citation Format: Lucile M. Jeusset, Kirk J. McManus. Evaluating the effect of USP22 depletion on chromosome instability in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1743.