Mismatch Repair Deficiency Research Articles

Comprehensive analysis of gene mutations and mismatch repair in Chinese colorectal cancer patients.

RAS, BRAF, and mismatch repair (MMR)/microsatellite instability (MSI) are crucial biomarkers recommended by clinical practice guidelines for colorectal cancer (CRC). However, their characteristics and influencing factors in Chinese patients have not been thoroughly described. To analyze the clinicopathological features of KRAS, NRAS, BRAF, and PIK3CA mutations and the DNA MMR status in CRC. We enrolled 2271 Chinese CRC patients at the China-Japan Friendship Hospital. MMR proteins were tested using immunohistochemical analysis, and the KRAS/NRAS/BRAF/PIK3CA mutations were determined using quantitative polymerase chain reaction. Microsatellite status was determined using an MSI detection kit. Statistical analyses were conducted using SPSS software and logistic regression. The KRAS, NRAS, BRAF, and PIK3CA mutations were detected in 44.6%, 3.4%, 3.7%, and 3.9% of CRC patients, respectively. KRAS mutations were more likely to occur in patients with moderate-to-high differentiation. BRAF mutations were more likely to occur in patients with right-sided CRC, poorly differentiated, or no perineural invasion. Deficient MMR (dMMR) was detected in 7.9% of all patients and 16.8% of those with mucinous adenocarcinomas. KRAS, NRAS, BRAF, and PIK3CA mutations were detected in 29.6%, 1.1%, 8.1%, and 22.3% of patients with dMMR, respectively. The dMMR was more likely to occur in patients with a family history of CRC, aged < 50 years, right-sided CRC, poorly differentiated histology, no perineural invasion, and with carcinoma in situ, stage I, or stage II tumors. This study analyzed the molecular profiles of KRAS, NRAS, BRAF, PIK3CA, and MMR/MSI in CRC, identifying key influencing factors, with implications for clinical management of CRC.

Gastric SMARCA4-deficient undifferentiated tumor (SMARCA4-UT): a clinicopathological analysis of four rare cases

BackgroundSMARCA4, as one of the subunits of the SWI/SNF chromatin remodeling complex, drives SMARCA4-deficient tumors. Gastric SMARCA4-deficient tumors may include gastric SMARCA4-deficient carcinoma and gastric SMARCA4-deficient undifferentiated tumor (SMARCA4-UT). Gastric SMARCA4-UT is rare and challenging to diagnose in clinical practice. The present report aims to provide insight into the clinicopathological characteristics and genetic alterations of gastric SMARCA4-UTs.ResultsWe retrospectively reported four rare cases of gastric SMARCA4-UTs. All four cases were male, aged between 61 and 82 years. These tumors presented as ulcerated and transmural masses with infiltration, staged as TNM IV in cases 1, 2 and 4, and TNM IIIA in case 3. Pathologically, four cases presented solid architecture with undifferentiated morphology. Cases 2 and 3 showed focal necrosis and focal rhabdoid morphology. Immunohistochemical staining showed negative expression of epithelial markers and deficient expression of SMARCA4. Furthermore, positivity for Syn (cases 1, 2 and 3) and SALL4 (cases 1 and 2) were observed. Mutant p53 expression occurred in four cases, resulting in strong and diffuse staining of p53 expression in cases 1, 2 and 4, and complete loss in case 3. The Ki67 proliferative index exceeded 80%. 25% (1/4, case 4) of cases had mismatch repair deficiency (dMMR). Two available cases (cases 1 and 3) were detected with SMRACA4 gene alterations. The response to neoadjuvant therapy was ineffective in case 1.ConclusionsGastric SMARCA4-UT is a rare entity of gastric cancer with a poor prognosis, predominantly occurs in male patients. The tumors are typically diagnosed at advanced stages and shows a solid architecture with undifferentiated morphology. Negative expression of epithelial markers and complete loss of SMARCA4 immunoexpression are emerging as a useful diagnostic tool for rare gastric SMARCA4-UTs.

Comparison of PD-L1, VISTA, LAG-3, and GAL-3 Expressions and Their Relationships to Mismatch Repair Protein and p53 Expression in 529 Cases of Endometrial Carcinoma.

The aim of this study is to evaluate the expressions of programmed death-ligand 1 (PD-L1), V-domain Ig suppressor of T-cell activation (VISTA), lymphocyte activation gene-3 (LAG-3), and galectin-3 (GAL-3), in mismatch repair-deficient (MMRd)/MMR-proficient and abnormal p53 expressing endometrial carcinomas and their relationship with clinical-histopathological features. Patients who underwent surgery for endometrial carcinoma between January 2008 and December 2018 were included in the study. Immunohistochemical analysis of MLH1, PMS2, MSH2, MSH6, p53, PD-L1, VISTA, LAG-3, and GAL-3 was performed on the tissue samples of microarray. A total of 529 patients were included. MMRd and p53-mutant tumors accounted for 31.5% and 11.5% of cases, respectively. PD-L1 and LAG-3 expressions in the MMRd and p53-mutant groups were higher than in the MMR-proficient group (P < 0.001). GAL-3 expression in the MMR-proficient group was statistically higher than in the MMRd and p53-mutant groups (P < 0.001). Mean age, grade, International Federation of Gynecology and Obstetrics stage, lymphovascular invasion, and lymph node metastasis were significantly higher in the p53-mutant group (P < 0.001). In the group with PD-L1 expression, nonendometrioid histologic type, tumor grade, and lymphovascular invasion were significantly higher (P < 0.001). Tumor grade, lymphovascular invasion, lymph node metastasis, and microcystic, elongated and fragmented pattern of invasion were significantly higher in the group with high VISTA expression (P < 0.05). Tumor grade was significantly higher in the group with LAG-3 expression (P < 0.001). Immunohistochemically determined subgroups and PD-L1, VISTA, LAG-3, and GAL-3 expression levels may be useful indicators of molecular features, and clinical outcomes also may have important implications for the development of targeted therapies in endometrial carcinoma.

A Subset of Microsatellite Unstable Cancer Genomes Prone to Short Insertions over Deletions Is Associated with Elevated Anticancer Immunity.

Deficiencies in DNA mismatch repair (MMRd) leave characteristic footprints of microsatellite instability (MSI) in cancer genomes. We used data from the Cancer Genome Atlas and International Cancer Genome Consortium to conduct a comprehensive analysis of MSI-associated cancers, focusing on indel mutational signatures. We classified MSI-high genomes into two subtypes based on their indel profiles: deletion-dominant (MMRd-del) and insertion-dominant (MMRd-ins). Compared with MMRd-del genomes, MMRd-ins genomes exhibit distinct mutational and transcriptomic features, including a higher prevalence of T>C substitutions and related mutation signatures. Short insertions and deletions in MMRd-ins and MMRd-del genomes target different sets of genes, resulting in distinct indel profiles between the two subtypes. In addition, indels in the MMRd-ins genomes are enriched with subclonal alterations that provide clues about a distinct evolutionary relationship between the MMRd-ins and MMRd-del genomes. Notably, the transcriptome analysis indicated that MMRd-ins cancers upregulate immune-related genes, show a high level of immune cell infiltration, and display an elevated neoantigen burden. The genomic and transcriptomic distinctions between the two types of MMRd genomes highlight the heterogeneity of genetic mechanisms and resulting genomic footprints and transcriptomic changes in cancers, which has potential clinical implications.

Mismatch repair deficiency: how reliable is the two-antibody approach? A national real-life study.

Traditionally, mismatch repair (MMR) status is determined by a panel of four antibodies (MLH1, PMS2, MSH2, MSH6). If all proteins are retained, cases are MMR proficient (pMMR), while loss of one or more proteins is indicative of MMR deficiency (dMMR). This approach has been challenged in favour of a two-antibody approach, using PMS2 and MSH6 as a first screening. Their retainment is deemed sufficient to declare cases pMMR. In this study we aim to verify the validity of the two-antibody approach. We performed a nationwide study in colorectal cancer (CRC) and endometrial cancer (EC) diagnosed between 2016 and 2023, including 47,657 patients to evaluate the two-antibody approach. In 0.17% and 0.4% of cases of CRC and EC, respectively, dMMR cases would be missed with the two-antibody approach. Subgroup analyses pointed towards slightly increased miss rates in younger patients (under the age of 50 years) in both groups and identified special subtypes (signet ring cell carcinoma, medullary carcinoma, and mucinous carcinoma in CRC and clear cell carcinoma in EC) with increased miss rates. For these specific subgroups, a low threshold should be used for further testing. In case of ambiguous or heterogeneous staining patterns, four antibodies should be used. In general, the application of a two-antibody MMR testing strategy does not lead to considerable failure of dMMR identification and saves costs.

Multiple primary tumors in children: a clinicopathological analysis of four cases

Objective: To investigate the clinicopathological features of children with metachronous or synchronous primary tumors and to identify related genetic tumor syndromes. Methods: The clinicopathological data of 4 children with multiple primary tumors diagnosed in the Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China from 2011 to 2023 were collected. The histological, immunophenotypic and molecular characteristics were examined using H&E staining, immunohistochemical staining, PCR, Sanger sequencing and next-generation sequencing (NGS). The patients were followed up. Results: Case 1 was an 8-year-old boy with the adrenal cortical carcinoma, and 5 years later a poorly differentiated gastric adenocarcinoma was detected. Case 2 was a 2-year-old boy, presented with a left ventricular choroid plexus carcinoma, and a hepatoblastoma was detected 8 months later. Case 3 was a 9-month-old girl, diagnosed with renal rhabdoid tumor first and intracranial atypical teratoid/rhabdoid tumor (AT/RT) 3 months later. Case 4 was a 7-year-old boy and had a sigmoid colon adenocarcinoma 3 years after the diagnosis of a glioblastoma. The morphology and immunohistochemical features of the metachronous or synchronous primary tumors in the 4 cases were similar to the corresponding symptom-presenting/first-diagnosed tumors. No characteristic germ line mutations were detected in cases 1 and 2 by relevant molecular detection, and the rhabdoid tumor predisposition syndrome was confirmed in case 3 using NGS. Case 4 was clearly related to constitutional mismatch repair deficiency as shown by the molecular testing and clinical features. Conclusions: Childhood multiple primary tumors are a rare disease with histological morphology and immunophenotype similar to the symptom-presenting tumors. They are either sporadic or associated with a genetic (tumor) syndrome. The development of both tumors can occur simultaneously (synchronously) or at different times (metachronously). Early identification of the children associated with genetic tumor syndromes can facilitate routine tumor screening and early treatment.

Correlation between immunohistochemical staining and clinicopathological findings in endometrial carcinoma.

To analyze the immunohistochemical staining pattern of mismatch repair (MMR) proteins and p53 in endometrial carcinoma cases, including different subtypes and stages, to gain insights into their role in the pathogenesis and clinical behaviour of this malignancy. In this study, we investigate the association between MMR deficiency, p53 mutational status, and clinical outcomes in various subtypes of endometrial carcinoma. The immunohistochemical staining pattern of MMR proteins in 96 cases of endometrial carcinoma have been analyzed, including 72 endometrioid, 14 papillary serous, 5 clear cell, and 5 mixed Müllerian tumor. The results showed that 36 cases were MMR deficient, with the majority being of endometrioid subtype. The p53 immunostain showed a mutational pattern in a subset of cases, with a documented dismal prognosis. However, aforementioned stains failed to predict synchronous or metachronous cancers in 5 patients. These findings highlight the importance of MMR and p53 immunohistochemical staining in the classification, and prognosis of endometrial carcinoma.

Incidence and potential prognostic implications of fibroblast growth factor receptor gene alterations and coexisting mutations in colorectal cancer.

e15558 Background: The fibroblast growth factor receptor (FGFR) family comprises four tyrosine kinase receptors (FGFR1-4). FGFR1 amplification (6.3%) as well as overexpression of FGFR1 (10.1%), FGFR2 (5.5%) and FGFR3 (16.2%) have been demonstrated in primary colorectal cancer (CRC, incidence in brackets). The prognostic utility of testing FGFR alterations and association with response to FGFR-inhibitors are still emerging. Here, we report FGFR alteration data with clinicopathological correlations and survival outcomes from a Sydney based Local Health District in Australia. Methods: Clinicopathological data was analysed from 341 ethnically diverse patients with CRC treated at Southwestern Sydney Local Health District from 2014-2021. Next generation sequencing for point mutations, deletions, and amplifications was performed using the Qiagen 30-gene panel. Mismatch repair (MMR) status was determined using immunohistochemical staining. Demographic characteristics, histological features and survival outcomes were recorded from electronic medical records. Results: Of the 341 patients, 42 (12.3%) had FGFR alterations of which 52% were men and 48% women. 32 patients were Caucasian, 4 Middle Eastern, 5 Asian and 1 South American. Median age at diagnosis was 66 years. Of the 42 patients, FGFR1 mutations were present in 26.2%, FGFR1 amplifications in 4.7% and FGFR1 deletions in 4.7%. FGFR2 mutations were present in 16.7%, and 50% had FGFR3 mutations. One patient had concurrent FGFR1 and FGFR3 mutations. The vast majority were adenocarcinoma (81%), followed by mucinous adenocarcinoma (17%) and adenocarcinoma with neuroendocrine features (2%). 21.4% of patients with FGFR alterations had concurrent KRAS mutations, 28.6% had concurrent BRAF mutations (91.6% V600E) and MMR-deficiency was present in 19%. Median overall survival of patients with FGFR alterations was 44 months, for those who also had deficient MMR 48 months, concurrent BRAF V600E mutations 51 months and concurrent KRAS mutations 71 months. 43% (18 patients) were stage 4 at presentation. Of these 18 patients, 7 had FGFR1 mutations, 1 had a FGFR2 mutation and 10 had FGFR3 mutations. The median overall survival of patients with stage 4 disease was 11 months and those with concurrent BRAF V600E mutations 17 months. Two patients had concurrent KRAS mutations (both G12V), with median overall survival of 5.5 months and 1 patient had MMR deficiency, with an overall survival of 9 months. Conclusions: FGFR alterations were identified in 12.3% of patients with colorectal cancer in an ethnically diverse cohort in Australia. Concurrent KRAS and BRAF mutations appear to affect survival outcomes. Further multivariate analyses are underway.

Perioperative chemotherapy for patients with gastric cancer with microsatellite instability or deficient mismatch repair: A systematic review and meta-analysis.

4039 Background: Currently, the efficacy of perioperative chemotherapy for gastric cancer (GC) patients with deficient mismatch repair or microsatellite instability-high (dMMR/MSI-H) remains controversial. It is still inconclusive whether chemotherapy can be spared for dMMR/MSI-H GC patients. Meanwhile, in the different therapeutic settings (e.g. neoadjuvant, adjuvant, or both), the efficacy of chemotherapy remains to be clarified. Therefore, a systematic review and meta-analysis in this regard was conducted. Methods: Studies comparing perioperative chemotherapy with surgery alone in resectable dMMR/MSI-H GCs (up to December 1, 2023) were included. The hazard ratio (HR) and its 95% confidence interval (CI) of survival outcomes were extracted from the original research or recalculated using Kaplan-Meier curves if the number of patients at risk was provided. In the pooled analysis, a random-effects model was employed. Subgroup analyses (perioperative chemotherapy in stage II and stage III diseases) and sensitivity analysis including studies reporting the results from multivariable analyses were conducted. This study was previously registered on PROSPERO platform (CRD42023494276). Results: Nineteen studies, encompassing over 1500 dMMR/MSI-H GC patients, were included in this study. The results revealed that perioperative chemotherapy (including neoadjuvant and adjuvant chemotherapy) did not significantly improve the overall survival (OS) (HR 0.86, 95% CI 0.57–1.31) and disease-free survival (DFS) (HR 0.70, 95% CI 0.46–1.05) in dMMR/MSI-H GCs. Furthermore, adjuvant chemotherapy did not confer a significant survival advantage for dMMR/MSI-H GCs (OS, HR 0.83, 95% CI 0.50–1.37) and (DFS, HR 0.67, 95% CI 0.41–1.07). Similar results were observed for neoadjuvant chemotherapy (OS, HR 1.09, 95% CI 0.55–2.15). In addition, stage stratification analysis demonstrated no significant survival benefit of adjuvant chemotherapy for stage II (OS, HR 0.77, 95% CI 0.31–1.90) or stage III (OS, HR 0.72, 95% CI 0.36–1.46) dMMR/MSI-H GCs. In the sensitivity analysis, the results remained consistent. Conclusions: Perioperative (including adjuvant and/or neoadjuvant) chemotherapy does not significantly improve survival in resectable dMMR/MSI-H GC patients. Immunotherapy may be better suitable for these patients in the future.

BRCA functional domains associated with PARP-inhibitors in patients with prostate cancer: The PROGRESS study.

10543 Background: Homologous recombination repair (HRR) defects are driver mutational imprints and actionable biomarkers in prostate cancer (PrC) patients. However, recent research provides evidence of PrC onset also in the context of mismatch repair deficiency (MMRd). In the current report, we investigated whether the position of pathogenic variants (PVs) in the functional domains (FDs) of HRR genes could be associated with reduced or increased sensitivity to PARP-inhibitors (PARPis). We also questioned whether the HRR FDs had a prognostic significance and affected the development of second primary tumor (SPT) in males with a genetic predisposition. Methods: This was a real-world, observational, study including PrC patients undergoing germline, somatic, and liquid biopsy testing between January 2020 and December 2023. Prognostic factors, SPT history, and PARPi benefit were evaluated according to mutated genes ( BRCA1, BRCA2,No- BRCAHRR), PV location within the FDs, and germline (g) or somatic (s) PV. The BRCA1FDs were RING, DNA-binding domain (DBD), BRCA1 C terminus (BRCT), or other location, and the BRCA2 FDs were RAD51-BD, DBD, or other location. Results: A total of 833 metastatic PrC patients, aging 40 to 91, were included in this study; 169 (20.3%) were carriers of germline (n.48, 5.8%) or somatic (n.121, 14.5%) PVs in HRR genes: 17 in BRCA1 (g BRCA1: n.4, 2.4%; s BRCA1: n.13, 7.7% ), 102 in BRCA2 (g BRCA2: n.33, 19.5%; s BRCA2: n.69, 40.8% ), and 53 in no- BRCA HRR genes (gHRR: n.11, 6.5%; sHRR: n.42, 24.8%). CHEK2was the HRR gene most frequently affected by germline PVs (27.3%), while the most frequent somatic PVs were in the ATM (40.5%). Liquid biopsy identified PVs in 33 patients out of 185 tested, including n.10 (5.4%) not detected through somatic and germline testing, considerably expanding the therapeutic window for the use of PARPi. Notably, differences in PFS rates at 48 months were observed when comparing sub-groups according to PV location in the FDs of BRCA2.Surprisingly, when we investigated the SPT in the cohorts of patients with and without germline PVs, the difference was not significant (14.5% vs 12.4%, respectively) and, in the subgroup without germline PVs, the sites of SPT were predominantly colorectal, gastric, bladder or other tumors classically dominated by MMRd and recognized as Lynch Syndrome-spectrum tumors. This observation may result in unexplored forms of hypermutable tumor phenotypes, potentially impacting cancer risk management and increasing therapeutic opportunities. Conclusions: Our results suggest preliminary evidence that not all BRCA2-mutated PrC are equally sensitive to PARPis, and the response could depend on the PV location within FDs. The study of SPT in patients with no-HRR-associated PrC highlights the need to expand the genes in multigene panel testing, including MMR along with HRR genes, to capture unrecognized constitutional predisposition.

A digital pathology AI model to predict immune-oncology biomarker status and pembrolizumab response in a real-world cohort of patients with colorectal and breast cancer.

e13617 Background: Deep learning (DL) algorithms have garnered significant attention for prediction of biomarker status in digitized tissue slides. Here, we trained and validated a digital pathology algorithm to predict microsatellite instability (MSI)/mismatch repair deficiency (MMRd) in colorectal cancer (CRC) and PD-L1 status in breast cancer (BC) using a large real-world patient cohort. Methods: H&amp;E and immunohistochemistry (IHC) slides of CRC and BC from Caris Life Sciences (Phoenix, AZ) with known biomarker status were digitally scanned (CRC: N=27,494, 6.6% MSI-high by next-generation sequencing [NGS]; N=25,788, 6.4% MMRd by IHC; BC: N=16,069, 15.8% PD-L1+ by IHC). A transformer-based DL model (Wagner, Sophia J., et al., 2023) was trained to predict biomarker status, with 5-fold cross-validation. Accuracy, F1 score, area under the curve (AUC), and other metrics were calculated comparing the model to NGS/IHC “truth”. Risk stratification was performed on holdout datasets of pembrolizumab-treated CRC (N=422-454) and triple negative breast cancer (TNBC; N=513) pts. Post-treatment overall survival (OS) was calculated from start of pembrolizumab to last contact, per insurance claims data. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated by Cox-Proportional hazards and compared between model and “truth”. Results: When applied to holdout datasets for biomarker prediction, the validated model achieved AUC of 0.78-0.95. In CRC and BC, performance was better among primary specimens than metastatic. For MSI-H/MMRd risk stratification in pembrolizumab-treated CRC, HR for OS were comparable between model and “truth” for primary and metastatic cohorts. When applied to the TNBC holdout dataset, the model performed better than “truth” for PD-L1 risk stratification in pembrolizumab-treated pts. Conclusions: A digital pathology DL model predicts biomarker status with high accuracy and is non-inferior to NGS/IHC for risk stratification of pembrolizumab-treated CRC and TNBC pts. These results support further investigation of AI algorithms developed from large datasets as possible rapid screening tools for biomarker detection in molecular testing programs. [Table: see text]

517P Real-world efficacy and safety of immune checkpoint inhibitors (ICI) in gastrointestinal tumors with mismatch repair deficiency or microsatellite instability (dMMR/MSI-H)

517P Real-world efficacy and safety of immune checkpoint inhibitors (ICI) in gastrointestinal tumors with mismatch repair deficiency or microsatellite instability (dMMR/MSI-H)

511P Testing patterns for microsatellite instability & mismatch repair deficiency in gastrointestinal cancers: Real-world evidence from a tertiary care cancer center

511P Testing patterns for microsatellite instability & mismatch repair deficiency in gastrointestinal cancers: Real-world evidence from a tertiary care cancer center

30P Immunological markers of pathological and clinical and outcomes in mismatch repair deficient (MMRd) colorectal cancer (CRC)

30P Immunological markers of pathological and clinical and outcomes in mismatch repair deficient (MMRd) colorectal cancer (CRC)

Clinical outcomes and impact of mismatch repair deficiency (dMMR) in patients with pT4N0 colon cancer (CC): Data from a large, consecutive, multicenter, real-world cohort.

3619 Background: T4 is widely recognized as one of the most important and independent prognostic factors in adjuvant setting for CC patients. However, the optimal management in terms of adjuvant treatment in stage II (pT4N0) as well as the impact of DNA mismatch repair deficiency (dMMR) in this subset of patients remain unclear. Here, we present a large, multicenter, real-world analysis of pT4N0 CC patients. Methods: A real-world database including pts treated at 9 Italian institutions from April 2010 to November 2023 was queried. Clinico-pathological characteristics of pts diagnosed with a stage II pT4N0 CC were analyzed. Pts were stratified according to duration of treatment, DNA mismatch repair (MMR) status, type of adjuvant chemotherapy (ACT). The primary endpoints were median relapse-free survival (RFS) and overall survival (OS). Results: A total of 380 pts was included, and outcomes data were available for 288 pts.Median age was 73 years and 49% of pts were male. 47 pts (16%) had dMMR CC, among which Lynch Syndrome was diagnosed in 19%. 10% of pts had BRAF mutant tumor, and 38% KRAS mutant tumor. After a median follow-up of 40.6 months (37.9-49.3), 3-yr RFS and 5-yr RFS were 70% and 55%, respectively, regardless treatment; 3-yr OS was 88% and 5-yr OS 73%. 154 pts (53%) received ACT: oxaliplatin (oxa)-based CT was given to 103 pts (67%), compared to 51 pts who received monotherapy (33%). Six-month ACT was given to 38 pts (74.5%) and 75 pts (72.8%) in the monotherapy and oxa-based group, respectively. Pts receiving 6-month ACT had a significant longer OS compared to those who did not receive ACT (HR 0.17; 0.08-0.34; p &lt;.001). A trend towards improved OS was observed in pts receiving 6-month ACT versus pts receiving 3-month ACT (HR 0.46; 0.16-1.29; p 0.129). Similar results were observed in terms of RFS: 6-month ACT showed a longer RFS compared to 3-month ACT (HR 0.58; 0.32-1.05; p .068), and a significantly longer RFS compared to no-ACT (HR 0.50; 0.32-0.77; p .001). Oxa-based ACT was associated with a significant improvement in OS compared to monotherapy (HR 0.30; 0.11-0.85; p .016). Overall, pts with dMMR CC did not show a statistically significant difference in terms of OS and RFS (p .66 and .19 respectively) compared to proficient MMR pts. Conclusions: In this large real-world dataset, T4 was confirmed being a key poor prognostic factor, independently to MMR status. We observed that ACT provides large benefit in this population, and oxa-based ACT may be better than monotherapy. In addition, 6-month oxa-based ACT showed a trend towards a better outcome in comparison to 3-month ACT, even if not statistically significant. Given the retrospective nature of the study, these data should be validated in larger and prospective cohorts; however, this work may help oncologists in shared decision making in this specific subgroup of patients.

Evaluating reflexive NGS and testing rates in community oncology.

e23302 Background: In oncology, NGS allows for the identification of DNA sequence variations within a cancer. The goal of testing is to identify/define therapeutic targets &amp; improve patient outcomes, employing specific therapies directed at those targets. In breast cancer, reflex tumor testing (ER/PR/Her2neu) has been performed for &gt; 20 yrs. In CRC, as published by ASCO, guideline-adherent biomarker testing rates for RAS, BRAF, &amp; MSI/MMR-d were 41%, 43%, and 51%, respectively1. In lung cancer, recent data has shown overall testing rates minimally better at 58%2. Methods: The study was conducted on historical data of lung, colorectal, and breast cancer patients seen in Verdi community oncology clinics between 4/1/2022-7/31/2023. Clinical &amp; social health data were collected &amp; abstracted from patient medical history (Doctor’s notes, Pathology studies, NGS results). The database contains demographic &amp; clinical data features for each patient. Results: 153 lung cancer patients were identified, median age 71(44 – 96). 82 F, 71 M. 85% of patients were smokers (130 smokers, 22 non-smokers, 1 unknown). Self-identified race: Asian 1, Black 22, white 106, 24 patients declined to identify. 61.9% (65) of later stage (≥IIB) patients had NGS testing. Of 258 colon/rectal cancer patients (anal cancer was excluded), median age 67(29-96), 122 M,146 F. Self-identified race: Asian 3, Black 26, 181 white, 34 patients declined to identify, 14 were unknown. Overall, 34.5% (89) of patients had NGS testing, and 76.2% (34) of Stage IV patients. There were 511 breast cancer patients, median age 63(25-85), 506 F, 5 M. Self-identified race: Asian/HPI 2, Black 137, white 299, 56 patients declined to identify, 17 were unknown. 100% had NGS, with minimum testing of ER/PR/Her2neu status. In the lung &amp; colorectal patients, statistically significant differences in rates of testing were seen in males (p &lt; 0.0001), older age (p &lt; 0.0001), and non-whites (p = 0.002). Conclusions: Testing at Verdi clinics were higher than reported testing. We propose that to improve testing rates, NGS should be reflexively ordered in other tumor types, as is the standard of care in breast cancer. This would eliminate many issues, including waiting time patients must undergo when testing is not ordered until the patient is seen by the medical oncologist. Proactive testing allows potential targeted treatments to be delivered in real-time and thus improve patient outcomes. 1Genomic Profiling for KRAS, NRAS, BRAF, Microsatellite Instability, and Mismatch Repair Deficiency Among Patients With Metastatic Colon Cancer. JCO Precision Oncology (2019)3(3). 2Improving biomarker testing in advanced non-small cell lung cancer and metastatic colorectal cancer: experience from a large community oncology network in the USA. Future Oncology (2023)19(20),1397-1414.

Population-Level Identification of Patients With Lynch Syndrome for Clinical Care, Quality Improvement, and Research.

Identification of those at risk of hereditary cancer syndromes using electronic health record (EHR) data sources is important for clinical care, quality improvement, and research. We describe diagnostic processes, previously seldom reported, for a common hereditary cancer syndrome, Lynch syndrome (LS), using EHR data within a community-based, multicenter, demographically diverse health system. Within a retrospective cohort enrolled between 2015 and 2020 at Kaiser Permanente Northern California, we assessed electronic diagnostic domains for LS including (1) family history of LS-associated cancer; (2) personal history of LS-associated cancer; (3) LS screening via mismatch repair deficiency (MMRD) testing of newly diagnosed malignancy; (4) germline genetic test results; and (5) clinician-entered diagnostic codes for LS. We calculated proportions and overlap for each diagnostic domain descriptively. Among 5.8 million individuals, (1) 28,492 (0.49%) had a family history of LS-associated cancer of whom 3,635 (13%) underwent genetic testing; (2) 100,046 (1.7%) had a personal history of a LS-associated cancer; and (3) 8,711 (0.1%) were diagnosed with colorectal cancer of whom 7,533 (86%) underwent MMRD screening and of the positive screens (486), 130 (27%) underwent germline testing. One thousand seven hundred and fifty-seven (0.03%) were diagnosed with endometrial cancer of whom 1,613 (92%) underwent MMRD screening and of the 195 who screened positive, 55 (28%) underwent genetic testing. (4) 30,790 (0.05%) had LS germline genetic testing with 707 (0.01%) testing positive; and (5) 1,273 (0.02%) had a clinician-entered diagnosis of LS. It is feasible to electronically characterize the diagnostic processes of LS. No single data source comprehensively identifies all LS carriers. There is underutilization of LS genetic testing for those eligible and underdiagnosis of LS. Our work informs similar efforts in other settings for hereditary cancer syndromes.

A multi-centre, single arm, phase 2 trial of pembrolizumab in treatment-naïve patients with carcinoma of unknown primary site.

2659 Background: Cancer of unknown primary (CUP) represents a heterogeneous group of cancers that present with metastatic epithelial disease with no identifiable primary lesion at the time of diagnosis. Patients with poor prognostic features have limited effective systemic therapy options. Researchers hypothesize that these primary lesions are occult, in part, due to anti-cancer immune surveillance. We hypothesize, pembrolizumab will have activity in this setting. Methods: This single arm phase 2 study evaluated the use of pembrolizumab in treatment naïve patients with poor prognosis CUP. Patients ≥18 years of age with ECOG PS 0-1 and histologically proven, measurable metastatic carcinoma with no primary site identified were eligible. Pembrolizumab was administered at 200 mg intravenously every 3-weeks for up to 24 months. The primary endpoint was safety and objective response rate (ORR) per RECIST 1.1. Secondary objectives included progression free survival (PFS) and overall survival (OS). The study used a Simon’s two stage design based on a one-sided test with a type 1 error rate of 0.1, null hypothesis for ORR ≤ 20% yielding a power of 0.8 with an alternate hypothesis of ≥40%. The null hypothesis will be rejected if 8 or more responses are observed in 25 patients. Mismatch repair (MMR) and microsatellite instability (MSI) testing was not mandatory for patients. Results: There were 27 evaluable and 6 unevaluable (no response assessment, included in adverse event analysis) patients. The median age was 64 (range 21-80) and the majority were male (57.6%) and ECOG 1 (84.8%). Four patients experienced grade 3 treatment related adverse events (pain, anorexia, fatigue, confusion, and anemia). There were no treatment related deaths or new signals of toxicity. Of the evaluable patients, the ORR was 18.5% (4 partial responses and 1 complete response), disease control rate was 51.9% (9 stable disease) and thus the null hypothesis could not be rejected. For evaluable patients, the median PFS and OS were 2.56 (95% CI 2.10 – 7.46) and 9.79 (95% CI 3.84 – 16.03) months respectively. The one patient who developed a CR had MMR deficiency and has completed 2 years of therapy. Of the 4 patients with a partial response, one had an MMR proficient tumour, while the remaining 3 had unknown MMR status. Conclusions: Although not meeting the primary endpoint, pembrolizumab has some activity in CUP patients. Patients with CUP should be screened for MMR or MSI. Future studies should evaluate combination therapies with the addition of pembrolizumab. Clinical trial information: NCT03391973 .

Spatially resolved multi-region proteomics for prediction of clinical outcome in deficient mismatch repair metastatic colorectal cancer treated with PD-1 blockade.

3520 Background: In patients with metastatic colorectal cancer (CRC) and deficient DNA mismatch repair (dMMR), we previously found transcriptomic signatures reflecting stromal compartment composition and proliferative capacity that can predict the efficacy of immune checkpoint blockade (ICB). To identify proteomic markers of response and/or resistance to ICB, we performed spatially resolved proteomic analysis of the tumor microenvironment. Methods: Patients with surgically resected, primary dMMR metastatic CRCs received pembrolizumab (Mayo Clinic, N=30). In FFPE tissues, Digital Spatial Profiling of 71 proteins was performed using panels covering immune cells, immune activation, cell death, PI3/AKT signaling, MAPK signaling, and pan-tumor markers (GeoMx nCounter). In ten regions of interest/slide (~600 µm2) at the tumor invasive margin, protein expression was determined in four cellular compartments: epithelium (panCK+), immune cells (CD45+; leucocyte common antigen), stroma (panCK-), and nonimmune stroma (panCK-/CD45-). After normalization, protein abundance per compartment was analyzed by response (RECIST 1.1) [OmicVerse Python]. Differentially expressed proteins (binary) were examined by progression-free survival (PFS) using multivariable Cox regression with covariate adjustment (Lifelines Python). Results: Pre-treatment panels including 71 proteins were analyzed by treatment response and PFS after PD-1 inhibition. Proteins found to be significantly associated with response (p&lt; 0.01) and PFS are described. Across all compartments, responders showed upregulation of NK cells and T cell subsets (CD3+, CD4+, CD8+), and ICOS; strongest association was found for CD4+: HRadj: 0.16; 95%CI: 0.08-0.31; p&lt; 0.001. Nonresponders showed higher phospho-JNK (HRadj: 2.09; 95%CI: 1.28-3.42; p=0.003) and SMA. In the epithelial compartment, upregulation of the Treg marker CD25 and reduced caspase-9 were observed in responders. Among responders, the stromal compartment was highly enriched in HLA-DR, dendritic cells (CD11c), PD-L1, immune activation markers (CD40, CD44, CD80, CD27), CD95/Fas, and GZMB. In the stromal compartment, downregulation of BCL6 and phospho-GSK3B was observed in responders. The immune compartment of responders showed increased beta2-microglobulin and Ki-67, while the non-immune stromal compartment showed increase in the macrophage marker CD68. Importantly, proteins found to be upregulated in responders were significantly and independently associated with longer PFS whereas the opposite was true for nonresponders. Conclusions: In metastatic dMMR CRCs, spatially defined protein markers were significantly associated with anti-PD-1 response, and independently associated with patient PFS (strongest predictive utility for CD4 in immune stroma and phospho-JNK in epithelium).

Preoperative (neoadjuvant) therapy with tislelizumab for locally advanced colorectal cancer with high microsatellite instability or deficient mismatch repair: An open-label, single-arm, multicenter phase II study.

3599 Background: Tislelizumab (TIS), an anti-programmed cell death protein 1 (PD-1) antibody, demonstrated improved anti-tumor response and tolerable safety in patients (pts) with previously treated, locally advanced unresectable or metastatic microsatellite instability high/deficient mismatch repair (MSI-H/dMMR) solid tumors, including colorectal cancer (CRC; NCT03736889). Recent studies of other anti-PD-1 antibodies indicate potential clinical benefit of neoadjuvant anti-PD-1 therapy in pts with resectable MSI-H/dMMR CRC. Here, we report efficacy and safety outcomes of neoadjuvant TIS from a multicenter phase II trial (NCT05116085) in this population. Methods: Pts aged ≥18 years with stage II/III MSI-H/dMMR CRC, ECOG PS 0-1, evaluable disease per RECIST v1.1, and eligible for R0 (complete) resection were enrolled. Pts received TIS 200 mg IV every 3 weeks for 3 cycles followed by surgical resection within 10 weeks of first TIS dose. The primary endpoint was major pathological response (MPR) rate (proportion of pts with ≤10% residual viable tumor in the surgically resected primary tumor). Secondary endpoints included pathological complete response (pCR) rate (proportion of pts with absence of residual tumor in the surgically resected specimen), and safety. The R0 resection rate was an exploratory endpoint. Results: Overall, 33 pts were enrolled across eight sites in China. The median age of enrolled pts was 52.0 years (range: 23-84), 14 (42.4%) pts were male. Ten (30.3%) pts had rectal cancer and 23 (69.7%) colon cancer. At study entry, clinical tumor (T) stage was reported as T2, T3, and T4 in 1 (3.0%), 7 (21.2%), and 25 (75.8%) pts, respectively, and node (N) stage as N0, N1, and N2 in 6 (18.2%), 14 (42.4%), and 13 (39.4%) pts, respectively. All pts had a metastasis (M) stage of M0. A total of 29 (87.9%) pts underwent surgery and were evaluable for efficacy endpoints. Surgery was not performed in 4 pts, 3 of whom were managed non-operatively (1 pt had progressive disease). MPR and pCR rates were 89.7% and 62.1%, respectively (Table). Any-grade and grade ≥3 TIS-related adverse events (TRAEs) were reported in 20 (60.6%) and 1 (3.0%) pts, respectively. Two (6.1%) pts experienced serious TRAEs; no TRAEs led to death or surgery cancellation. One pt (3.0%) experienced a TRAE (grade 2 hypothyroidism) leading to surgery delay. No grade ≥3 or serious TIS-related surgery-relevant adverse events were reported. Immune-mediated adverse events were reported in 10 (30.3%) pts (all grade 1-2). Conclusions: Neoadjuvant TIS was associated with high MPR and pCR rates and an acceptable safety profile, and did not compromise surgery in pts with resectable MSI-H/dMMR CRC. Clinical trial information: NCT05116085 . [Table: see text]