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Context: Polymerase epsilon (POLE) gene missense hotspot mutations can generate pathogenic (p) proofreading defects resulting in hypermutated genomic profiles. Aim: Determine the prevalence, genomic consequences and immunotherapy sensitivity of advanced POLE mutated tumors according to mutation site, primary tumor and tumor mutational burden (TMB). Results: Pan-Cancer TCGA & MSKCC databases genomic analyses found a prevalence of non-pathogenic POLE mutations (POLEnp) of 3.4% with median TMB of 11 mutations/Megabase (mt/Mb, IQR 3-34). Pathogenic POLE mutations (POLEp) prevalence was 0.4% with median TMB of 215 mt/Mb (IQR 107-324), predominantly in colorectal and endometrial cancers. Prevalence dropped to 0.1% in metastatic cancers. We assessed prospectively the efficacy of PD-1 blockade in mismatch repair proficient advanced solid tumors harboring POLE missense mutations (phase II ASCe Nivolumab trial; NCT03012581). Variants were categorized prospectively by a molecular board as POLEp, POLEnp or Variants with Unknown significance (VUS). The primary endpoint was the Overall Response Rate (ORR) at 12 weeks according to RECIST 1.1, and secondary endpoints included survival analyses according to POLE variants pathogenicity. Among 61 screened patients, 21 were eligible and 20 received Nivolumab and 19 were assessable for response (table 1). The 12-week ORR was 37% for patients harboring POLEp and VUS and resulted in major survival improvement compared to POLEnp patients (HR=0.1 ; CI95% 0.02-0.7); see results in Table 1. Among patients POLEp tumors, while higher TMB was not predictive of response, higher proportion of POLE-related mutational signature correlated with improved benefit. In silico exonucleasic POLE domain analyses confirmed that all POLEp and 2 VUS clustered in the DNA binding or the Catalytic site. Recategorizing the VUS according to the location within the exonucleasic domain improved the prediction of survival outcomes. Impact: This study gives new insights on how DNA repair defects, mutational burden and signatures sensitize to PD-1 blockade and may offer emerging tumor agnostic biomarkers for benefit to checkpoint blockade. POLE variant pathogenicity All(N=21) POLEnp(N=5) VUS(N=4) POLEp(N=12) Age, years ± SD 57 ± 16 64 ± 10 56 ± 16 54 ± 17 Sex, Male (%) 12 (57) 5 (100) 2 (50) 5 (42) PS (ECOG)=1 (%) 16 (75) 4 (80) 2 (50) 10 (83) Primary tumor Colorectal 9 (43) 2 (40) 2 (50) 5 (42) Endometrial 6 (29) 0 (0) 0 (0) 6 (50) Gastric 2 (9) 2 (40) 0 (0) 0 (0) Glial 1 (5) 0 (0) 0 (0) 1 (8) Biliary tract 1 (5) 0 (0) 1 (25) 0 (0) Pancreas 2 (9) 1 (20) 1 (25) 0 (0) Number of previous treatments 2.4 ± 2 5 ± 2 1.8 ± 1 1.5 ± 1 TMB (mt/Mb, Min-Max)(N=16) 36.2 (2-385) 5 (4-9) 3 (2-4) 114 (25-385) ORR at 12 weeks (CR+PR) 37%(N=7/19) 0%(N=0/5) 50%(N=2/4) 46%(5/10) DCR at 12 weeks (CR+PR+SD) 58%(N=11/19) 0%(N=0/5) 75%(N=3/4) 80%(8/10) Median Progresssion-Free survival (months) 5.6 2.3 10.3vs POLEnp: HR=0.2 IC95% 0.1-0.7 Median Overall Survival (months) 9.1 5.0 Not Reachedvs POLEnp:HR=0.1 IC95% 0.02-0.7 Citation Format: Benoît Rousseau, Ivan Bieche, Eric Pasmant, Nadim Hamzaoui, Nicolas Leulliot, Lucas Michon, Aurelien de Reynies, Mike Foote, Julien Masliah-Planchon, Magali Svrcek, Romain Cohen, Victor Simmet, Paule Augereau, David Malka, Antoine Hollebecque, Damien Pouessel, Carlos Gomez-Roca, Rosine Guimbaud, Amandine Bruyas, Marielle Guillet, Muriel Duluc, Sophie Cousin, Christelle de la Fourchardiere, Frederic Rolland, Sandrine Hiret, Esma Saada-Bouzid, Olivier Bouche, Thierry Andre, Diane Pannier, Farid El Hajbi, Stephane Oudard, Christophe Tournigand, Jean-Charles Soria, Drew Gerber, Dennis Stephens, Michelle Lamandola-Essel, Steven B Maron, Bill Diplas, Guillem Argiles, Asha Krishnan, Neil Segal, Andrea Cercek, Nathalie Hoog-Labouret, Frederic Legrand, Clotide Simon, Assia Lamrani-Ghaouti, Luis A. Diaz, Pierre Saintigny, Sylvie Chevret, Aurelien Marabelle. PD-1 blockade in solid tumors with defects in polymerase epsilon [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT021.

Colon tumors with deficient DNA mismatch repair (dMMR) are infiltrated more densely by T cells than tumors with proficient mismatch repair (pMMR). However, high tumor-infiltrating lymphocytes (TIL) are found in a subset of pMMR tumors and low TIL are seen in a subset of dMMR colon cancer. In this study, we compared T-cell repertoires in 20 pMMR and 27 dMMR colon tumors with high and low TIL counts. CDR3 regions of the TCR beta chain were sequenced by ImmunoSEQ Assay (Adaptive Biotechnologies, Seattle, WA). The fraction T cells was calculated by normalizing TCR beta template counts to the total DNA. Sample richness was calculated as the number of unique productive rearrangements in a sample after computationally down-sampling to a common number of T cells to control for variation in sample depth. Simpson clonality was computed as a measure of how evenly productive rearrangements are distributed amongst the total number of T cells. Statistical analyses were performed using Prism 8.0 (GraphPad, La Jolla, CA). The mean TIL counts per high power field (HPF) for TIL-high and TIL-low dMMR tumors were 17.4 (range, 5-37) and 1.8 (range, 0-4). In pMMR tumors, they were 12.8 (range, 6-37) for TIL-high and 0.3 (range, 0-1) for TIL-low groups. The number of T cells measured by TCR sequencing correlated positively with the TIL count (Spearman rho = 0.391; P = 0.007). When comparing by MMR status, we found that dMMR tumors exhibited a trend towards higher Simpson clonality index (P = 0.09), with significantly lower repertoire richness compared to pMMR tumors (P = 0.030). Within dMMR tumors, TIL-high dMMR tumors had higher density (P = 0.017), higher clonality (P = 0.003), and a less rich repertoire (P = 0.003) compared to TIL-low dMMR tumors. Within pMMR tumors, while the T cell density was higher in the TIL-high tumors, we did not observe differences in T cell clonality or repertoire richness between TIL-high vs TIL-low pMMR tumors. Our study found that T cells in dMMR tumors are more clonal and their repertoire is less rich compared with T cells in pMMR tumors. In the dMMR group, T cells in TIL-high tumors were more clonal and their repertoire was less rich compared with T cells in TIL-low tumors, but in the pMMR group, T-cell diversity in TIL-high tumors was comparable to T-cell diversity in TIL-low tumors. These findings suggest that T cells clonally expand in dMMR tumors, possibly in response to MMR deficiency-induced tumor neoantigens. Citation Format: Jin K. Kim, Chin-Tung Chen, Asama Khan, Neil Segal, Zsofia Stadler, Jinru Shia, Vinod P. Balachandran. Comparison of intratumoral T-cell repertoires in DNA mismatch repair proficient and deficient colon adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1382.

PURPOSE: Microsatellite instability-high (MSI-H) colorectal carcinomas (CRCs) show high rates of response to immune checkpoint inhibitors (IOs). B2M mutations and protein loss have been proposed as causes of resistance to IOs, yet they are enriched in MSI-H CRC. We aimed to characterize B2M-mutant, IO-naive CRC. PATIENTS AND METHODS: All CRCs with results for Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets, a next-generation sequencing assay that interrogates > 400 genes for mutations as well as MSI status, were surveyed for B2M mutations. All B2M-mutant CRCs were assessed for expression of B2M, major histocompatibility complex class I, and programmed death-1 ligand (PD-L1) via immunohistochemistry and average CD3(+) and CD8(+) tumor-infiltrating lymphocyte counts against a control group of MSI-H B2M wild-type CRCs. RESULTS: Fifty-nine (3.4%) of 1,751 patients with CRC harbored B2M mutations, with 84% (77 of 92) of the mutations predicted to be truncating. B2M mutations were significantly enriched in MSI-H CRCs, with 44 (24%) of 182 MSI-H CRCs harboring B2M mutations (P < .001). Thirty-two of 44 B2M-mutant CRCs with available material (73%) had complete loss of B2M expression, whereas all 26 CRCs with wild-type B2M retained expression (P < .001). B2M mutation status was not associated with major histocompatibility complex class I expression, KRAS or BRAF mutation, tumor-infiltrating lymphocyte level, or PD-L1 expression after adjustment for MSI status. Of 13 patients with B2M-mutant CRC who received programmed death-1 or PD-L1 IOs, 11 (85%) achieved clinical benefit, defined as stable disease or partial response using Response Evaluation Criteria in Solid Tumors criteria. CONCLUSION: B2M mutations occur in approximately 24% of MSI-H CRCs and are usually associated with loss of B2M expression. Most patients with B2M-mutant MSI-H CRC with loss of protein expression obtain clinical benefit from IOs.

To assess the effect of an insole with subtalar strapping on patients with medial compartment osteoarthritis (OA) of the knee.Novel lateral wedged insoles with elastic subtalar strapping (the subtalar strapping support group) and ankle supporters with a lateral wedged heel insert (the sock-type ankle support group) were prepared. Eighty-eight female outpatients with knee OA were treated with 1 of the 2 insoles for 8 weeks. Femorotibial angle was assessed by standing radiographs with and without unilateral insole use for each subject. Symptoms of knee OA were evaluated according to the severity index of Lequesne et al at baseline and at the final assessment.Participants wearing the insole with subtalar strapping (n = 42) demonstrated significantly decreased femorotibial angle (an average of change: -3.1 degrees +/- 2.5 degrees, P0.0001), but a significant difference was not found in the sock-type ankle support group (n = 46; -0.4 degrees +/- 1.1 degrees, P0.05). In the subtalar strapping support group, pain during bed rest with full extension of the knee (P0.0001), pain after getting up (P = 0.04), pain on getting up from a seated position (P = 0.021), maximum distance walked (P = 0.009), and aggregate severity score (P0.0001) were significantly improved compared with baseline. In contrast, significant symptomatic improvement was detected only in the aggregate score (P = 0.016) in the sock-type ankle support group, but not in any of the 10 specific categories.The lateral wedged insole with subtalar strapping induces correction of the femorotibial angle and symptomatic relief in patients with varus-deformity knee OA.

Canine malignant melanoma (CMM) is a spontaneous, aggressive, and metastatic neoplasm. Preclinical mouse studies have shown that xenogeneic DNA vaccination with genes encoding tyrosinase family members can induce antibody and cytotoxic T-cell responses, resulting in tumor rejection. These studies provided the rationale for a trial of xenogeneic DNA vaccination in CMM using the human tyrosinase gene.Three cohorts of three dogs each with advanced (WHO stage II, III, or IV) CMM received four biweekly i.m. injections (dose levels 100, 500, or 1500 micro g, respectively/vaccination) of human tyrosinase plasmid DNA i.m. via the Biojector2000 delivery device.Mild local reactions at injection sites were the only toxicities observed, with no signs of autoimmunity. One dog with stage IV disease had a complete clinical response in multiple lung metastases for 329 days. Two dogs with stage IV disease had long-term survivals (421 and 588+ days) in the face of significant bulky metastatic disease, and two other dogs with locally controlled stage II/III disease had long-term survivals (501 and 496 days) with no evidence of melanoma on necropsy. Four other dogs were euthanized because of progression of the primary tumor. The Kaplan-Meier median survival time for all nine dogs was 389 days.The results of this trial demonstrate that xenogeneic DNA vaccination of dogs with advanced malignant melanoma is a safe and potentially therapeutic modality. On the basis of these results, additional evaluation of this novel therapeutic is warranted in locally controlled CMM and advanced human melanoma.

To investigate anthropometric measures that closely correlate with symptomatic relief of osteoarthritis (OA) of the knee in response to lateral wedged insole use.Seventy-one patients with medial compartment knee OA were treated with insoles with subtalar strapping or insoles with talonavicular strapping. Randomization was performed according to birth date. The following variables were evaluated: age, disease duration, Kellgren-Lawrence radiographic stage, body mass index, percent body fat, waist to hip ratio, lower extremity lean body mass (L-LBM) per body weight, and radiographic femorotibial angle at baseline. The trial lasted 8 weeks. The correlation between each variable and the remission score (delta score) using the Lequesne index of severity was analyzed.In the subtalar strapping group (n = 34), delta score of knee OA was more strongly associated with age (p = 0.004, r = 0.48) than other variables. A significant correlation was also observed between L-LBM per body weight and delta score (p = 0.041, r = -0.36) in the subtalar strapping group. No other variables significantly correlated with the delta score in the subtalar strapping groups. No variable significantly correlated with the ? score in the talonavicular strapping group (n = 37).We previously reported that use of insoles with subtalar strapping leads to valgus realignment of the femorotibial angle in patients with knee OA with varus deformity, and it may have a similar therapeutic effect to that of high tibial osteotomy. These data suggest that the insole with subtalar strapping is more efficacious for younger patients and those with a higher L-LBM per body weight, and less efficacious for older patients with sarcopenia.