MSI-H Tumors Research Articles

Neoadjuvant botensilimab (BOT) plus balstilimab (BAL) in resectable mismatch repair proficient (pMMR) and deficient (dMMR) colorectal cancer (CRC): NEST clinical trial update.

207 Background: Checkpoint inhibitors (CPI) have been transformative for localized dMMR CRC (microsatellite instability-high [MSI-H]), but not for localized pMMR CRC (microsatellite stable [MSS]). The combination of BOT, a novel Fc-enhanced multifunctional CTLA-4 antibody, and BAL, an anti-PD-1 antibody, has demonstrated significant activity in metastatic CRC. We examined this novel dual CPI therapy in patients with MSI-H and MSS localized CRC. Methods: This is a single arm trial of neoadjuvant BOT 75 mg/m 2 (day 1) and BAL 240 mg/m 2 every 2 weeks x2 (NEST1) or x4 (NEST2) in patients with localized CRC who were candidates for surgery. The primary endpoint was major pathologic response (MPR), defined as ≥90% pathologic tumor regression. The 95% confidence interval (CI) was computed based on exact method for binomial distribution. Results: NEST1 (n=12: 9 MSS / 3 MSI-H) accrued from 3/21/23 to 9/25/23 and NEST2 (n=14: 13 MSS / 1 MSI-H; 2 MSS patients had synchronous CRC) accrued from 2/15/24 to 5/6/24; 2 patients in NEST1 were not evaluable (1 with rectal cancer not resected [ypT0N0 on local excision] and 1 patient with occult lung metastasis). In total, 7 MSS CRC tumors were evaluable from NEST1 and 15 from NEST2; and 3 MSI-H tumors were evaluable from NEST1 and 1 from NEST2, leaving a final evaluable study population of 24 patients (20 MSS / 4 MSI-H) and 26 tumors (22 MSS / 4 MSI-H). The Table provides pathologic response rates across NEST1, NEST2, and MSI-H. With a median follow-up of 13.1 months (NEST1) and 4.8 months (NEST2), no patients have recurred and ctDNA testing remains negative. No grade 4 toxicities were observed and the only treatment-related grade 3 toxicity was diarrhea/colitis in 15% and fever in 4%. Treatment-related grade 2 diarrhea/colitis occurred in 8%, grade 2 fever in 15%, and grade 2 fatigue in 4% of patients. Diarrhea/colitis resolved in all cases with steroids ± anti-TNFα therapy. All patients underwent laparoscopic resection, with 1 conversion to open. No surgeries were delayed due to treatment-related adverse events. Median length of stay was 2 days (range 1-14). Translational data including pathologic evaluation of tumor infiltrate and response will be presented. Conclusions: Neoadjuvant BOT/BAL is safe, with no delays to surgery, and effective. We observed high MPR rates in both MSS and MSI-H CRC with no recurrences to date. The MPR and pCR rate improved with extended time to surgery. Clinical trial information: NCT05571293 . Pathologic response to neoadjuvant BOT/BAL in localized CRC. Pathologic Response NEST 1n=7 MSS tumors NEST 2n=15 MSS tumors MSI-H (3 NEST1, 1 NEST2) 100% (CR%, 95%CI) 1 (14%, 0.4-58%) 6* (40%, 16-68%) 3** (75%, 19-99%) ≥ 90% (MPR%, 95%CI) 2 (29%, 4-71%) 7 (47%, 21-73%) 4^ (100%, 40-100%) ≥ 50% 4 (57%) 9 (60%) 4 (100%) Median days to Surgery (range) 29 (21-37) 57 (45-104) 46 (34-78) *2 with carcinoma in situ , no tumor bed. ^MPR was in NEST1. **1 was rectal.

A retrospective study of colorectal cancer CT imaging features involving microsatellite instability.

265 Background: This study aimed to identify correlations between CT imaging characteristics accessible to the radiologist with microsatellite instability (MSI) status among colorectal cancer. Noninvasive identification of MSI status can decrease time to treatment if pathology can be bypassed. Methods: 109 colorectal cancer patients were identified retrospectively from 2011 to 2018 with an average age of 61 years (58 male, 51 female) and had MSI pathological assessment. These subjects all had CT abdomen and pelvis obtained at the time of initial diagnosis. Imaging features of both the primary and metastatic lesions were assessed. Primary lesion features included: location, size, stage, attenuation compared to the liver, growth pattern, tumor margin, primary mass area, and the presence/absence of mesenteric infiltration. Metastatic lesion features included: size, attenuation relative to liver, and enhancement pattern. Patient age, sex, and tumor staging were obtained from patient medical records. Statistical analysis was performed using various methods, including chi-square, Mann-Whitney and Kruskal-Wallis tests. Results: 5 tumor characteristics displayed a statistically significant relationship with MSI status. MSI-H lesions were more likely to be clinical Stage 2, and MSI-L lesions were more likely to be Clinical Stage 3 (p=0.012). Tumors with distant metastasis were more likely MSI-L versus regional metastasis were more likely MSI-H (p<0.001). Primary mass area was larger in the MSI-H than MSI-L group (p<0.001). Tumors in the left colon are more likely to be MSI-L versus CRC tumors in the right colon are more likely to be MSI-H (p<0.001). MSI-H tumors had a lower primary tumor Hounsfield Unit standard deviation (SD) than MSI-L tumors (p=0.002). The remaining features were not statistically significant. For primary lesion, these include mesenteric infiltration (p=0.189), tumor margin (p=1.000), enhancement pattern (p=0.498), growth pattern (p=0.127), tumor CT density (p=0.162), and liver density (p=0.105). Enhancement of metastasis also had no difference (p=0.376). Conclusions: Multiple CT imaging characteristics were predictive of MSI classification in CRC tumors, holding potential clinical relevance that can help guide individualized therapy. While these results were statistically significant, more research must be done to validate these findings and to derive useful nomograms.

Phase II preoperative pembrolizumab for MSI high or MSS/PD-L1 gastric cancer followed by surgery and adjuvant therapy with pembrolizumab (NCT03257163): Results of a multicenter study.

441 Background: Data suggest that patients with tumors with microsatellite instability (MSI-H), EBV expression, or positive PD-L1 expression can benefit from immunotherapy. This trial evaluated PD-1 immunotherapy in this subset of patients with operable gastric cancer. Methods: We present results of a phase 2 multi-institutional clinical trial (NCT03257163). Patients with cT2-T4, N0-N3, M0 gastric adenocarcinoma with MSI-H, PDL1 CPS > 1%, or EBV+ were included. Patients received 2 cycles preop pembrolizumab 200 mg IV q3 weeks followed by surgery. Pembrolizumab was given for 16 additional cycles postoperatively, concurrently with adjuvant chemoradiation (45 Gy) and 5 cycles of capecitabine (825 mg/m2 days 1-14 during cycles 3, 6 and 7; 625 mg/m2 BID days 1-14 during cycles 4 and 5 with radiation). Patients were defined as evaluable if they received > one cycle of postop pembrolizumab. Primary endpoint is DFS, powered to detect 3-yr DFS of 70% with 89% power. Results: All enrolled patients received two cycles of pembrolizumab preoperatively (n=45). Eight patients did not undergo surgery (1 due to frailty, 2 with distant progression on preoperative imaging, 2 with peritoneal disease on surgical exploration and 3 with locally advanced, unresectable tumors). Six patients did not receive adjuvant therapy (2 due to surgical complications, 2 for failure to thrive, 1 refusal and 1 pending adjuvant therapy). 31 patients received adjuvant therapy and are included in the predetermined, evaluable analytic cohort. These 31 patients had a median age of 67 years (range 44 – 85) with 22.6% Hispanic, 29.0% Asian, 25.8% Black and 25.8% White. Median follow-up was 31.1 months (range 3.45-63.8). In these patients, 14 (45.2%) had MSI-H, 2 (6.5%) EBV+, and 29 (93.5%) had PDL1 expression >1% tumors. Most tumors were advanced stage with 81% ≥ cT3 and 58% cN+. In the 31 evaluable patients, the 3-year DFS was 79.4%; 3-year OS was 78.9%. For MSI-H patients, 3-year DFS was 81.8%. For patients with MSS/PD-L1+ tumors, 3-year DFS was 77.8%. For the 45-patient cohort, 3-year DFS was 70%; 3-year OS was 65%. Three patients had pCR, two with MSI-H tumors and one with PD-L1 CPS of 4%. Downstaging occurred in 62.2% of patients, and 40% of surgical specimens had pT0/T1 tumors consistent with significant pathologic responses. Conclusions: A biomarker-driven preoperative treatment strategy for MSI-H, EBV+, and MSS/PDL1+ operable gastric adenocarcinoma patients is promising and warrants additional evaluation. This treatment strategy is novel and effective, and it incorporates immune checkpoint immunotherapy, low dose chemotherapy, and radiation thus avoiding more intensive combination chemotherapy regimens for patients with these biomarkers. Clinical trial information: NCT03257163 .

GOBLET study: Results of the safety run-in for first-line metastatic pancreatic ductal adenocarcinoma (PDAC) patients treated with pelareorep + modified FOLFIRINOX +/- atezolizumab.

730 Background: Immunotherapies are effective in only the small subset of metastatic PDAC patients with MSI-H or dMMR tumors. Pelareorep (pela) is a non-genetically modified, intravenously administered reovirus that selectively infects cancer cells. It stimulates the expansion of pre-existing tumor-infiltrating lymphocyte (TIL) clones, and it makes tumors visible to the immune system by upregulating interferon-induced gene expression including PD-L1, CXCL9, CXCL10, and CXCL11. PD-L1 upregulation also provides a basis for potential synergy between pela and immune checkpoint inhibitors. Pela combined with gemcitabine/nab-paclitaxel/atezolizumab showed promising tumor responses in mPDAC. Here, we are assessing the safety and preliminary efficacy of pela + mFOLFIRINOX +/- atezolizumab in a new cohort in the ongoing GOBLET study. Methods: GOBLET is a phase 1/2, open-label, Simon two-stage study in patients with advanced or metastatic GI cancers. In this new GOBLET cohort (Cohort 5), patients with newly diagnosed mPDAC are randomized to receive either pela + mFOLFIRINOX or pela + mFOLFIRINOX + atezolizumab. The primary endpoints are safety and objective response rate (ORR). In Stage 1, 15 evaluable patients will be enrolled in each arm. Both arms include a 3-6 patient safety run-in that must be successfully completed prior to opening the study to full enrollment. If pre-specified ORR success criteria are met, one or both arms may be advanced to Stage 2 during which 17 additional evaluable patients/per arm will be enrolled. Results: The 3-patient safety run-in for both arms of Cohorts 5 (6 patients total) have been enrolled, and all patients have completed the required 1-month evaluation period. Cohort 5 safety data have been reviewed by the independent Data Safety Monitoring Board (DSMB). The DSMB identified no safety signals attributable to the combination of pela and mFOLFIRINOX +/- atezolizumab and recommended that enrollment into these arms of Cohort 5 continue without modification. Reported adverse events are consistent with the known toxicities of the components of the treatment regimen. Tumor response results are pending. Conclusions: The results of the GOBLET Cohort 5 safety run-in indicate that pela can be given safely in combination with mFOLFIRINOX +/- atezolizumab to newly diagnosed mPDAC patients. Safety and efficacy of these combination therapies will continue to be monitored (Eudra-CT: 2020-003996-16). Clinical trial information: 2020-003996-16 .

Association of PD-L1 positivity with Epstein Barr virus infection and microsatellite instability in gastric carcinomas with lymphoid stroma

Gastric carcinoma with lymphoid stroma (GCLS) is characterized by dense intra-and peritumoral lymphocytic infiltration and a high rate of Epstein Barr Virus (EBV) infection, suggesting being a promising candidate for immunotherapy. We investigated correlations between PD-L1 expression and clinicopathologic factors, including EBV positivity and microsatellite instability (MSI) status in GCLSs. The study included resected 214 GCLSs and 300 gastric adenocarcinomas (GACs) for control. Epstein Barr Virus encoding region in situ hybridization (EBER ISH), immunohistochemistry for PD-L1 and HER2, dual-colored in situ hybridization for HER2, and MSI analysis were performed. EBV positivity was found in 181 (85%) of 214 GCLSs. MSI analysis demonstrated that 0.6% of EBV + GCLSs and 54.5% of EBV-GCLSs were MSI-high compared to 7% of EBV-GACs. Approximately 3% and 3.9% of HER2 amplifications were found in EBV- and EBV + GCLSs compared to 13% of EBV-GACs. PD-L1 expression with ≥ 1, ≥ 5, and ≥ 10 combined positive scores (CPS) were observed in 81.8%, 70.2%, and 55.3% of EBV + GCLSs. PD-L1 expression with ≥ 10 CPS was observed in 21.2% of EBV-GCLSs, predominantly in MSI-H tumors (85.7%). EBV positivity and MSI are associated with PD-L1 positivity rates in patients with GCLS who may respond better to PD-1/PD-L1 inhibitors but not anti-HER2 inhibitors.

Exploring the molecular profile of localized colon cancer: insights from the AIO Colopredict Plus registry.

Understanding the mutational landscape of colon cancer (CC) is crucial for targeted therapy development. Microsatellite instability (MSI-H), rat sarcoma (RAS), and B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutations (MT) are pivotal markers. Further investigation into clinicopathological features of RAS and BRAF MT in microsatellite stable (MSS) and MSI-H tumors is warranted. A retrospective analysis of 4883 localized CC patients (pts.) was conducted. Molecular profiling assessed MSI, KRAS, NRAS, and BRAF MT. Correlation with clinicopathological data employed ANOVA and Chi-square tests. Disease-free survival (DFS) and overall survival (OS) were analyzed adjusting for age, gender, sidedness, UICC stage, Charlson Comorbidity Index (CCI). A Cox model incorporated all variables as covariates. This analysis included 4883 pts. (2302 female/2572 male, 3865 (79.2%) MSS, 1018 (20.8%) MSI-H). MSS pts. had more All-Wild Type (WT), KRAS MT, and NRAS MT tumors vs. MSI-H pts. (42.1% vs. 21.1%; 39.8% vs. 15.4%; 3.6% vs. 0.7%; p<0.001 for each). BRAF MT tumors (95.5% BRAF V600E MT) were more prevalent in MSI-H individuals (62.8% vs. 8.1%, p<0.001). KRAS and BRAF MT tumors were more frequently right-sided, while BRAF MT tumors were associated with female gender, advanced disease stage, lymph node positivity, and poorer differentiation in the MSS subset (p<0.001). Common KRAS mutations included p.G12D (30.44%) and p.G12V (21.3%) in MSS and p.G13D (28.9%) and p.G12D (22.37%) in MSI-H. NRAS MT tumors were dominated by codon 61 mutations (51.7%). Survival analysis revealed worst prognosis in BRAF MT MSS tumors (DFS: HR 1.74 (95% CI 1.15-2.62, p=0.009; OS: HR 1.61 (95% CI 0.99-2.6), p=0.055). The 3-years DFS and 5-years OS rates were lowest in this subset (61.6% and 57.7% respectively). These findings highlight the complex interplay between molecular subtypes, clinicopathological features, and survival outcomes in early CC. Further research is needed to elucidate underlying mechanisms and develop personalized treatment strategies.

A comparison of the clinicopathological features and genetic alterations in stage II/III gastric cancer with no recurrence, early recurrence and late recurrence after curative surgery.

Few studies have explored the genetic changes and clinicopathological features of stage II/III gastric cancer (GC) patients with no tumor recurrence, early recurrence, or late recurrence after curative surgery. In this study, 376 patients who underwent curative surgery for stage II/III GC were analyzed. The clinical and genetic features of patients with no recurrence, early recurrence (<2 years), and late recurrence (≥2 years) were compared. Among the 376 patients, 155 experienced tumor recurrence, including 116 with early recurrence and 39 with late recurrence. Patients with early recurrence had larger tumors, fewer superficial tumors, increased lymphovascular invasion, advanced T and N categories, higher TNM stages, and poorer 5-year survival (3.4% vs. 38.5% vs. 63.5%, p<0.001) than those with late recurrence or no recurrence. For intestinal-type GC, patients with early recurrence had more MSI-H tumors than those with late recurrence or no recurrence. For diffuse-type or node-positive GC, patients with early recurrence had more PIK3CA amplifications than did those with late recurrence or with no recurrence. Peritoneal dissemination and lung metastasis were associated with PIK3CA amplifications, whereas liver metastasis was associated with larger numbers of MSI-H tumors and PI3K/Akt pathway mutations. Multivariate analysis revealed that age, tumor recurrence, and pathological T and N categories were independent prognostic factors for both overall survival and disease-free survival. Distinct clinical features and genetic alterations were observed in specific groups of stage II/III GC patients with differences in time to recurrence and recurrence patterns, and targeted therapy and immunotherapy may benefit these groups of patients.

Analysis of Concordance Between Next-Generation Sequencing Assessment of Microsatellite Instability and Immunohistochemistry-Mismatch Repair From Solid Tumors.

The new CAP guideline published in August 2022 recommends using immunohistochemistry (IHC) to test for mismatch repair defects in gastroesophageal (GE), small bowel (SB), or endometrial carcinoma (EC) cancers over next-generation sequencing assessment of microsatellite instability (NGS-MSI) for immune checkpoint inhibitor (ICI) therapy eligibility and states there is a preference to use IHC over NGS-MSI in colorectal carcinoma (CRC). We assessed the concordance of NGS-MSI and IHC-MMR from a very large cohort across the spectrum of solid tumors. Of the over 190,000 samples with both NGS-MSI and IHC-MMR about 1,160 were initially flagged as discordant. Of those samples initially flagged as discordant, 50.9% remained discordant after being reviewed by an additional pathologist. This resulted in a final discordance rate of 0.31% (590/191,767). Among CRC, GE, SB and EC, 55.4% of mismatch repair proficient/MSI high (MMRp/MSI-H) tumors had at least one somatic pathogenic mutation in an MMR gene or POLE. Mismatch repair deficient/microsatellite stable (MMRd/MSS) tumors had a significantly lower rate of high tumor mutational burden than MMRp/MSI-H tumors. Across all solid tumors, MMRd/MSI-H tumors had significantly longer overall survival (OS; hazard ratio [HR], 1.47, P < .001) and post-ICI survival (HR, 1.82, P < .001) as compared with MMRp/MSS tumors. The OS for the MMRd/MSS group was slightly worse compared to the MMRp/MSI-H tumors, but this difference was not statistically significant (HR, 0.73, P = .058), with a similar pattern when looking at post-ICI survival (HR, 0.43, P = .155). This study demonstrates that NGS-MSI is noninferior to IHC-MMR and can identify MSI-H tumors that IHC-MMR is unable to detect and conversely IHC-MMR can identify MMRd tumors that NGS-MSI misses.

356 (PB344): Preclinical characterization of NTX-452, a potent, selective and highly efficacious WRN inhibitor for the treatment of MSI-H tumors

356 (PB344): Preclinical characterization of NTX-452, a potent, selective and highly efficacious WRN inhibitor for the treatment of MSI-H tumors

A pilot study on the detection of microsatellite instability using long mononucleotide repeats in solid tumors.

Microsatellite instability (MSI) status is a prognostic biomarker for immunotherapy in certain types of cancers, such as colorectal cancers (CRCs) and endometrial cancers (ECs). Tumors that are categorized as having high MSI (MSI-H) express high levels of neoantigens for immune recognition. The typical MSI test measures the length of short mononucleotide repeats (SMR) poly(A) 21-27; however, a limitation of this test is the difficulty in determining the shift size, particularly in endometrial cancer. To investigate an MSI detection assay with improved performance, the present study analyzed the use of poly(A) 40-44 mononucleotide repeats to detect the MSI status of 100 patients with either CRC (n=50) or EC (n=50). Capillary electrophoresis was used to evaluate five long mononucleotide repeat (LMR) markers, including poly(A) 40-A, 40-B, 40-C, 40-D and 44. The concordance rate of the LMR-MSI assay compared with an immunohistochemistry MSI detection assay was 96.0 and 95.1% for CRCs and ECs respectively, with the detection limit of the LMR-MSI assay demonstrated to be 2.5% MSI-H in HCT116 colorectal carcinoma cell lines. The LMR-MSI assay yielded a 95.1% concordance rate in ECs compared with that in the SMR-MSI test (87.8%). The LMR-MSI test identified a significantly higher mean shift size (13 bp) in MSI-H tumors compared with the SMR-MSI test (10 bp), in both EC and CRC tissue samples. Together, the present study suggested that the LMR-MSI test could potentially be a sensitive and practical technology for molecular laboratory testing, particularly in the use of immunotherapy for patients with CRCs and ECs.

Targeted Treatment against Cancer Stem Cells in Colorectal Cancer.

The cancer stem cell (SC) theory proposes that a population of SCs serves as the driving force behind fundamental tumor processes, including metastasis, recurrence, and resistance to therapy. The standard of care for patients with stage III and high-risk stage II colorectal cancer (CRC) includes surgery and adjuvant chemotherapy. Fluoropyrimidines and their combination with oxaliplatin increased the cure rates, being able to eradicate the occult metastatic SC in a fraction of patients. The treatment for unresectable metastatic CRC is based on chemotherapy, antibodies to VEGF and EGFR, and tyrosine-kinase inhibitors. Immunotherapy is used in MSI-H tumors. Currently used drugs target dividing cells and, while often effective at debulking tumor mass, these agents have largely failed to cure metastatic disease. SCs are generated either due to genetic and epigenetic alterations in stem/progenitor cells or to the dedifferentiation of somatic cells where diverse signaling pathways such as Wnt/β-catenin, Hedgehog, Notch, TGF-β/SMAD, PI3K/Akt/mTOR, NF-κB, JAK/STAT, DNA damage response, and Hippo-YAP play a key role. Anti-neoplastic treatments could be improved by elimination of SCs, becoming an attractive target for the design of novel agents. Here, we present a review of clinical trials assessing the efficacy of targeted treatment focusing on these pathways in CRC.

Comprehensive genomic profiling and therapeutic implications for Taiwanese patients with treatment-naïve breast cancer.

Breast cancer is a heterogeneous disease categorized based on molecular characteristics, including hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) expression levels. The emergence of profiling technology has revealed multiple driver genomic alterations within each breast cancer subtype, serving as biomarkers to predict treatment outcomes. This study aimed to explore the genomic landscape of breast cancer in the Taiwanese population through comprehensive genomic profiling (CGP) and identify diagnostic and predictive biomarkers. Targeted next-generation sequencing-based CGP was performed on 116 archived Taiwanese breast cancer specimens, assessing genomic alterations (GAs), including single nucleotide variants, copy number variants, fusion genes, tumor mutation burden (TMB), and microsatellite instability (MSI) status. Predictive variants for FDA-approved therapies were evaluated within each subtype. In the cohort, frequent mutations included PIK3CA (39.7%), TP53 (36.2%), KMT2C (9.5%), GATA3 (8.6%), and SF3B1 (6.9%). All subtypes had low TMB, with no MSI-H tumors. Among HR + HER2- patients, 42% (27/65) harbored activating PIK3CA mutations, implying potential sensitivity to PI3K inhibitors and resistance to endocrine therapies. HR + HER2- patients exhibited intrinsic hormonal resistance via FGFR1 gene gain/amplification (15%), exclusive of PI3K/AKT pathway alterations. Aberrations in the PI3K/AKT/mTOR and FGFR pathways were implicated in chemoresistance, with a 52.9% involvement in triple-negative breast cancer. In HER2+ tumors, 50% harbored GAs potentially conferring resistance to anti-HER2 therapies, including PIK3CA mutations (32%), MAP3K1 (2.9%), NF1 (2.9%), and copy number gain/amplification of FGFR1 (18%), FGFR3 (2.9%), EGFR (2.9%), and AKT2 (2.9%). This study presents CGP findings for treatment-naïve Taiwanese breast cancer, emphasizing its value in routine breast cancer management, disease classification, and treatment selection.

Characterization of mutational signatures demonstrating adaptive mutability post anti-EGFR therapy in a real-world metastatic colorectal cancer cohort.

3545 Background: Clinical benefit from anti-EGFR therapy (EGFRi) for RAS/RAF WT metastatic colorectal cancer (mCRC) is limited by secondary mutations in mitogen-activated protein kinases. The source of these new clones has been debated. In contrast to the outgrowth of pre-existing rare subclones, a new model, termed adaptive mutability, proposes the transient loss of DNA repair genes and concurrent induction of error-prone polymerase repair mechanisms (Russo et al Science, ’19). However, clinical evidence for this effect in a large patient cohort is lacking. Using longitudinal circulating tumor DNA (ctDNA) in a real-world cohort, we sought to explore mutational signatures and assess the presence of adaptive mutability in mCRC patients after EGFRi treatment. Methods: 288 patients from Guardant Health’s GuardantINFORM database with mCRC who received EGFRi and underwent longitudinal ctDNA NGS testing by Guardant360 (Redwood City, CA) between December 2014 and March 2023 were included. We extracted de novo single-nucleotide variant mutational signatures from the somatic ctDNA mutations per-cohort level, comparing pre- to post-treatment mutational profiles. Extracted mutational signatures were compared against COSMIC catalogue. Results: Of the 288 patients identified, 99.7% (287/288) were MSS/MSI-L prior to treatment. Pre-treatment, patients had a mean and median of 3 reportable variants per sample, with 30.2% (87/288) samples with &gt;5 variants. Post-treatment, patients had a mean of 5 and a median of 4 reportable variants, with 64.6% (186/288) of patients with &gt;5 variants. Overall mean shift in reportable variants between pre- and post-EGFRi was 5 variants, with a median of 4 changes. In comparing pre- and post-EGFRi cohorts for new mutations, three mutational signatures emerged: SBS17b, SBS15, and SBS3. SBS15 is associated with mismatch repair (MMR) deficiency and classically seen in MSI-H tumors, though notably this cohort was overwhelmingly MSS. SBS15 has been associated with polymerase epsilon exonuclease (POLE) defects and likely reflects repair by alternate error-prone polymerases such as Polι, Polλ, and Polκ. SBS3 has been associated with homologous recombination (HR) deficiency. SBS17b has been associated with exposure to 5-FU based chemotherapy in CRC patients, which was co-administered in many of the EGFRi treated patients. Conclusions: New mutations arising with EGFR inhibition demonstrate strong evidence for adaptive mutability, showcasing loss of mismatch repair and HR fidelity, and reliance on error-prone polymerases. This is the first large cohort to indirectly demonstrate evidence for this genomic mechanism underlying secondary acquired resistance. Further validation of the adaptive mutability signatures may support therapeutic strategies leveraging this vulnerability to extend benefit from targeted therapies.

A machine learning algorithm based on multi-omics biomarkers for the detection of tumor microsatellite instability.

1554 Background: Microsatellite instability (MSI) occurs via defects in mismatch repair (MMR) and is characterized by high tumor mutational burden (TMB) and a robust inflammatory response. Accurate MSI-high classification of immune-evasive tumors is vital due to the effectiveness of immune checkpoint inhibitor (ICI) therapy, such as pembrolizumab, which is FDA approved for all MSI-high tumors irrespective of anatomic origin. Detection methods of MSI-status vary widely, including immunohistochemistry (IHC), polymerase chain reaction (PCR), and next-generation sequencing (NGS). We developed a machine learning (ML) model to predict MSI status in patients with solid tumors using comprehensive genomic and immune profiling (CGIP), independent of direct sequencing data from microsatellite sites. Methods: We analyzed samples from 1,838 patients with colorectal cancer (CRC) by CGIP, which included DNA panel testing (523 genes) for pathogenic single nucleotide variants (pSNV) and determination of TMB (mut/Mb), and RNA sequencing (397 genes) for gene expression (GEX). The Boruta algorithm was used to select key genomic and GEX changes associated with MSI status. A distributed gradient boosting algorithm created a predictive model that was trained on 70% of the cohort. The model was tested on the remaining CRC cohort (Test), and in the PanCancer Atlas CRCs (TCGA), endometrial adenocarcinomas (EMCA), and CRC with indeterminate MSI testing by CGIP. Performance of the trained model was assessed using sensitivity, specificity, and positive (PPV) and negative (NPV) predictive value. Results: Feature selection identified 79 genes with pSNVs, 63 GEX changes, and TMB as informative for MSI prediction. The model showed high predictive accuracy in differentiating MSI-H tumors. Of the 39 cases that failed MSI component of CGIP (Indeterminate), 17 had MMR IHC results available, of which 1 demonstrated loss of MLH1/PMS2 and the other 16 showed intact (normal) expression. Testing of this cohort identified 3 cases as MSI-high, at least one of which was confirmed by IHC. No cases with intact MMR protein IHC were identified as MSI-high by the algorithm. Conclusions: Our ML-driven approach accurately assessed MSI status of CRC and EMCA using CGIP data. This approach also identified potential cases with MSI-high status where direct sequencing of microsatellites failed. This study highlights a method to identify patients with potential MSI-high status for orthogonal screening with the MSI component of a test fails. [Table: see text]

A phase II, multicenter, open-label study of polyPEPI1018 in combination with atezolizumab in participants with relapsed or refractory microsatellite-stable metastatic colorectal (MSS mCRC) cancer (Oberto-301).

3594 Background: The efficacy of checkpoint inhibitor immunotherapy in MSI-H mCRC has not been replicated in MSS mCRC. Therefore, additional interventions are needed to convert immunologically “cold” MSS CRC to “hot” tumors resembling MSI-H tumors. PolyPEPI1018 is an off-the-shelf, multi-peptide vaccine containing 12 immunogenic epitopes derived from 7 shared tumor antigens which demonstrated early evidence of clinical activity in first-line MSS mCRC. Methods: Patients with MSS mCRC who have progressed on 2-3 lines of prior chemotherapy regimen received PolyPEPI1018 (1.2 mg, sc) and atezolizumab (1,200 mg, iv) Q3W until disease progression or unacceptable toxicity. The primary endpoint was safety. A Simon 2-stage design was applied. Data on objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and correlation studies will be presented. Results: 18 patients (66 % female) were enrolled for stage 1 of the study. Median age was 54 years (range 38–79), 50% had liver metastases and 67% received at least 3 lines of prior therapies. PD-L1 expression was &lt;1% for 85% of patients. The combination was well-tolerated; most common side effect related to treatment was Grade (Gr) 1-2 local skin reaction (n=22). Gr 2 events (n=2) at least possibly related to treatment were nausea, pyrexia and increased blood alkaline phosphatase. There were no Gr 3-5 events or study discontinuation due to treatment AE. The ORR was 0% and the DCR was 61%. The mPFS was 2.7 months (95%CI 1.4-4.0) and the mOS was not reached (95%CI 8.75 – NE months) at the data cut-off date. Ex-vivo FluoroSpot identified 13/16 subjects with robust, vaccine-specific CD8+ and/or CD4+ T cell responses detected against multiple vaccine antigens. For patients with available biopsy pairs (n=8), both the average PD-L1 expression (IC%, p=0.029) and the density of CD8+ tumor-infiltrating lymphocytes (TILs, p=0.019) were significantly increased, post-treatment. Patients with increased PFS (&gt; 12 weeks) had both higher levels of post-treatment PD-L1 (p=0.007) and TILs (p=0.016) than patients with PFS ≤ 12 weeks. Conclusions: PolyPEPI1018 in combination with atezolizumab was well-tolerated with no Grade 3 AEs observed. Despite no objective tumor responses could be detected, correlative data suggest contribution of PolyPEPI1018-induced immune responses to the modulation of tumor microenvironment and to improved disease control. The study did not proceed to stage 2; it is on-going for the collection of survival data. Clinical trial information: NCT05243862 .

A pan-cancer analysis of SMARCA4 alterations and the unique clinicogenomic characteristics associated with SMARCA4 mutation types.

3073 Background: SWI/SNF complexes are regulators of cell lineage and alterations in these proteins occur in &gt;20% of solid tumors with SMARCA4being the most common. SMARCA4 mutations are associated with worse clinical outcomes in various cancer types but are also vulnerable to novel SMARCA2 inhibitors currently being evaluated in clinical trials. We examined SMARCA4 alterations across cancer types to understand their interplay with other genomic abnormalities and clinical consequences. Methods: We analyzed the Memorial Sloan Kettering (MSK) institutional cohort profiled by MSK-IMPACT tumor-normal sequencing. SMARCA4 mutations were annotated based on OncoKB and literature. FACETS resolved zygosity and genomic metrics of complexity including tumor mutational burden (TMB) and fraction genome altered (FGA). The pan-cancer landscape of somatic SMARCA4 mutations was mapped by both category (Class I vs II) and zygosity (mono- vs biallelic). DISCOVER was employed to evaluate mutational signatures and frequency-adjusted co-occurring and mutually exclusive mutations. We then extracted progression-free (PFS) and overall survival (OS) to systemic therapy using natural language processing. Results: We identified 3,385 tumors from 3,049 patients across cancer types with a total 3,965 oncogenic somatic SMARCA4 alterations. Copy number estimation was successful for 2,343 (69%). Non-small cell lung cancer (NSCLC) was the most abundant SMARCA4-altered histology ( n=493, 7.1% of all NSCLC), and the majority (87%) were biallelic. Other common histologies were colorectal ( n=163, 2.9%), bladder ( n=122, 5.5%), uterine ( n=88, 4.3%), pancreatic ( n=81, 3.2%) and esophagogastric ( n=92, 4%). SMARCA4biallelic mutated tumors had higher chromosomal instability compared to WT (FGA = 0.53 vs 0.47; p&lt;0.001), while monoallelic had lower (FGA = 0.3; p&lt;0.001). Monoallelic SMARCA4 altered tumors had a higher TMB (10.5) than both WT (4.4) and biallelic cancers (6.1; p&lt;0.001). Monoallelic and biallelic SMARCA4 cancers exhibited distinct mutational signatures in most tumor types. Patients with biallelic SMARCA4 mutations had worse outcomes with both immune checkpoint blockade (ICB) and platinum chemotherapy across in certain tumor types, whereas monoallelic uterine cancers were associated with improved outcomes with ICB. Conclusions: This pan-cancer clinicogenomic analysis demonstrates that monoallelic and biallelic SMARCA4-mutated tumors have distinguishable genomic features. In addition, biallelic SMARCA4 alterations correlated with poor outcomes across different cancer-types and classes of therapy, while improved outcomes to ICB in monoallelic SMARCA4alterations were potentially due to higher representation of MSI-H tumors in this subset. Our findings suggest SMARCA4 zygosity may be important biomarkers for ongoing clinical trials in SMARCA4-deficient tumors.

Mutations in Mismatch Repair Genes and Microsatellite Instability Status in Pancreatic Cancer.

Patients with pancreatic cancer (PC) showing mismatch repair (MMR) deficiency may benefit from immunotherapy. Microsatellite instability (MSI) is a hallmark of MMR deficiency (MMR-D). Here, we estimated the prevalence of MSI in PC, investigated germline and somatic mutations in the three MMR genes (MLH1, MSH2, and MSH6), and assessed the relationship between MMR genes mutations and MSI status in PC. Clinical specimens from PC patients were analyzed using targeted next-generation sequencing, including paired normal and tumor specimens from 155 patients, tumor-only specimens from 86 patients, and normal-only specimens from 379 patients. The MSI status of 235 PCs was assessed via PCR. Pathogenic/likely pathogenic (P/LP) germline variants in the MMR genes were identified in 1.1% of patients, while somatic variants were found in 2.6% of patients. No MSI-H tumors were detected. One patient carried two variants (P (VAF = 0.57) and LP (VAF = 0.25)) simultaneously; however, their germline/somatic status remains unknown due to the investigation focusing solely on the tumor and MSI analysis was not performed for this patient. MSI is rare in PC, even in tumors with MMR genes mutations. Our findings underscore the importance of assessing tumor MMR-D status in PC patients with confirmed Lynch syndrome when deciding whether to prescribe immunotherapy.

Microsatellite instability in mismatch repair proficient colorectal cancer: clinical features and underlying molecular mechanisms

Both defects in mismatch repair (dMMR) and high microsatellite instability (MSI-H) have been recognised as crucial biomarkers that guide treatment strategies and disease management in colorectal cancer (CRC). As MMR and MSI tests are being widely conducted, an increasing number of MSI-H tumours have been identified in CRCs with mismatch repair proficiency (pMMR). The objective of this study was to assess the clinical features of patients with pMMR/MSI-H CRC and elucidate the underlying molecular mechanism in these cases. From January 2015 to December 2018, 1684 cases of pMMR and 401 dMMR CRCs were enrolled. Of those patients, 93 pMMR/MSI-H were identified. The clinical phenotypes and prognosis were analysed. Frozen and paraffin-embedded tissue were available in 35 patients with pMMR/MSI-H, for which comprehensive genomic and transcriptomic analyses were performed. In comparison to pMMR/MSS CRCs, pMMR/MSI-H CRCs exhibited significantly less tumour progression and better long-term prognosis. The pMMR/MSI-H cohorts displayed a higher presence of CD8+ T cells and NK cells when compared to the pMMR/MSS group. Mutational signature analysis revealed that nearly all samples exhibited deficiencies in MMR genes, and we also identified deleterious mutations in MSH3-K383fs. This study revealed pMMR/MSI-H CRC as a distinct subgroup within CRC, which manifests diverse clinicopathological features and long-term prognostic outcomes. Distinct features in the tumour immune-microenvironment were observed in pMMR/MSI-H CRCs. Pathogenic deleterious mutations in MSH3-K383fs were frequently detected, suggesting another potential biomarker for identifying MSI-H. This work was supported by the Science and Technology Commission of Shanghai Municipality (20DZ1100101).

Family history as a major prerequisite for microsatellite instability screening in colorectal cancer is a poor selection tool.

Deficient mismatch repair (MMR) leading to microsatellite instability (MSI) in tumors is thought to be present in over 15% of colorectal cancer (CRC) cases. Testing CRC for MSI has traditionally been recommended following the fulfillment of clinical criteria. However, the performance of clinical criteria, especially the family history, as a selection tool for MSI screening in CRC is questionable. We retrospectively investigated the incidence of high degree MSI (MSI-H) tumors in an unselected population of CRC patients and compared its prevalence between individuals with and without family history of cancers within the spectrum of MSI-H tumors as defined in the revised Bethesda criteria. The study population included 274 patients, 70 with positive and 204 without family history of MSI-H tumors with complete data including findings from MSI analysis. The overall incidence of MSI-H CRC was 18.98%. There was no statistically significant difference in the incidence of MSI-H CRC amongst both groups. The sensitivity and specificity of family history with regard to the presence of an MSI-H tumor in this collective was 36.5% and 77.5%, respectively. A relevant number of cases with high MSI-H CRC may be missed secondary to screening based on clinical criteria like family history alone. Thus, systematic screening independent of clinical characteristics, especially family history of cancer should be recommended in all cases with CRC.

Association between microsatellite status and characteristics and outcomes of early-onset compared to late-onset rectal cancer

BackgroundMicrosatellite instability (MSI) is an important prognosticator for colorectal cancer (CRC). The present study aimed to assess the impact of MSI status on the characteristics and outcomes of early-onset compared to late-onset rectal cancer.MethodsThis retrospective cohort study used data from the US National Cancer Database (2004–2019) to assess the baseline characteristics, treatment patterns, short-term outcomes, and overall survival (OS) of early-onset rectal adenocarcinoma affecting patients < 50 years compared to late-onset rectal adenocarcinoma according to the MSI status.ResultsThe present study included 48,407 patients (59.9% male) with rectal cancer, 17.3% of patients were < 50 years and 6.3% had MSI-H tumors. In the early-onset group, patients with MSI-H tumors had a lower mean age (41.5 vs 43 years, p < 0.001) and presented less often with stage IV disease (22.1% vs 17.7%, p = 0.03) and liver metastasis (9.1% vs 13.5%, p = 0.011) than patients with MSS tumors. In the late-onset group, patients with MSI-H and MSS tumors had similar demographics, disease stage, and metastatic pattern, yet MSI-H patients more often received neoadjuvant radiation therapy (58.9% vs 55.1%, p = 0.009) and neoadjuvant systemic therapy (40% vs 36.2%, p = 0.005). In both age groups, MSI-H tumors were associated with more pathologic T3-4 stage and were more likely mucinous and poorly differentiated carcinomas than MSS tumors. The median OS of MSI-H tumors was similar to MSS tumors (108.09 vs 102.31 months, p = 0.1), whether in the early-onset (139.5 vs 134.2 months, p = 0.821) or late-onset groups (106.1 vs 104.3 months, p = 0.236).ConclusionsIn both age groups, MSI-H rectal cancers were more often mucinous and poorly differentiated carcinomas and had pT3-4 stage more often than MSS cancers. MSI-H rectal cancers tend to present less often with distant metastases and nodal involvement than MSS cancers only in early-onset, but not in late-onset rectal cancers. The association between MSI status and survival was not notable in this study, whether in the early-onset or late-onset groups.