Immunohistochemistry 22C3 pharmDx Research Articles

Abstract C106: New therapeutic strategy by integrative analysis of germline, somatic genetic alterations and tumor mutational burden (TMB) in deficient mismatch repair (dMMR) gastric cancer

Abstract Background: The prevalence of microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) and Lynch syndrome with germline MMR mutations in gastric cancer (GC), are 5% and 1.6%, respectively. According to TCGA data, BRCA mutations, one of DNA Damage Response (DDR) pathway genes, occur in about 10% of GC (BRCA1 2.7%, BRCA2 7%), but the incidence is increased in MSI-H GC (BRCA1 16%, BRCA2 34%). Our study focused on integrative analysis of germline and somatic genetic alterations according to dMMR status for new therapeutic strategy. Methods: Retrospective comparison analysis of advanced gastric cancer patients between 20 dMMR and 48 proficient MMR (pMMR) was performed using targeted next-generation sequencing (NGS) for somatic genetic alterations and Global Screening Array (Illumina©) to detect germline alterations. PD-L1 expression using immunohistochemistry 22C3 pharmDx was evaluated. TMB was calculated based on somatic nonsynonymous missense mutations and DDR genes were compared. DNA pyrosequencing was used to determine promoter methylation status of MLH1 gene. Results: dMMR was associated with higher TMB (median 64 mut/Mb, IQR 54 -73 vs. 42 mut/Mb, IQR 34 - 50, P<0.001), but not with PD-L1 overexpression (p=0.7). The promoter methylation of MLH1 gene was observed in 50% of total dMMR (n=10/20), and had higher TMB (73 mut/Mb, IQR 63 - 88) compared to MLH1 unmethylated dMMR group (57 mut/Mb, IQR 52 – 62) (P<0.001). Germline or somatic gene alterations associated with MMR expression were not different between dMMR and pMMR (p=0.7, p=0.3, respectively). Somatic DDR alterations were elevated in dMMR compared to pMMR (median 45 mut vs. 36 mut, P=0.004), and in high TMB group with > 50 mut/Mb (n=29, p<0.001). In dMMR group, highly mutated ATM (E166D), CDK12 (E711Q, E716Q, R712T, R713T), PARLB2 (Q559R), STAG2 (V343L) and SMARCA4 (A314P) were observed, implying dMMR GC patients’ sensitivity to PARP inhibitor. Conclusions: There were no differences in germline variations according to MMR status, suggesting no beneficial information from germline testing in advanced GC without family history. However, we observed elevated DDR gene alterations in dMMR GC with higher TMB. These findings may be applicable in strategies to combine immune checkpoint inhibitors with PARP inhibitors in patient with dMMR and/or higher TMB GC. Citation Format: Garden Lee, Jihyun Hwang, Sejung Park, Woo Sun Kwon, Jinseul Park, Jii Bum Lee, Choong-kun Lee, Hyun Cheol Chung, Sun Young Rha. New therapeutic strategy by integrative analysis of germline, somatic genetic alterations and tumor mutational burden (TMB) in deficient mismatch repair (dMMR) gastric cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C106. doi:10.1158/1535-7163.TARG-19-C106

Effects of Decalcifying Agents of Variable Duration on PD-L1 Immunohistochemistry.

To evaluate the effects of decalcifying agents on programmed cell death ligand 1 (PD-L1) immunohistochemistry (IHC). Fragments of 10 placentas (high PD-L1 expressor) and 10 lungs (lower PD-L1 expressor) were formalin-fixed and subjected to four decalcifying solutions (EDTA, formic acid/MasterCal IM Plus [FA/MC], 12% HCl, and Decal STAT/23% HCl) for 1, 2, 6, or 24 hours. H&E staining and PD-L1 using IHC 22C3 pharmDx were performed, and PD-L1 staining was assessed. Minimal to no change in staining intensity or proportion of stained cells was seen with EDTA or FA/MC at all decalcifying durations. Both HCl-based decalcifiers demonstrated a progressive decrease in percentage of positive cells and staining intensity with longer decalcifying duration, particularly with Decal STAT. EDTA and FA/MC have little effect on PD-L1 expression. 12% HCl causes a progressive decline in staining. Decal STAT dramatically reduced staining with all treatment durations, especially at 24 hours.

2023P - Evaluating the prevalence of the expression of PD-L1 in NSCLC specimens with short-duration formalin fixation using IHC 22C3 pharmDx

BackgroundImmune checkpoint inhibitors mediated by PD-1 and PD-L1 are promising treatments for various tumors. The PD-L1 expression in tumor cells was found to be correlated with the likelihood of a response to PD-1- or PD-L1-targeted therapy in non-small cell lung cancer (NSCLC). For PD-L1 immunohistochemistry (IHC), 22C3 pharmDx is the only companion diagnostic assay that can identify NSCLC patients suitable for pembrolizumab treatment. Specimens are scored and divided into 3 categories (<1%, 1-49%, ≥50%) based on the tumor proportion score (TPS). The PD-L1 prevalence in NSCLC patients screened for the KEYNOTE-024 trial was 31% with TPS <1%, 39% with TPS 1-49%, and 30% with TPS ≥50%. While specimens used for the IHC 22C3 assay are recommended to be formalin-fixed for 12-72h, the specimens were fixed in 10% neutral-buffered formalin for less than 6h in our institution. Thus, we retrospectively assessed the PD-L1 prevalence in NSCLC patients to determine whether the PD-L1 TPS differs according to the duration of formalin fixation. MethodsWe screened consecutive NSCLC patients who underwent tumor biopsy by bronchoscopy between January 2017 and June 2018 at National Hospital Organization Fukuokahigashi Medical Center. In the present study, we only included 70 patients whose tumors were formalin-fixed for less than 6h and whose PD-L1 expression had been evaluated by IHC with antibodies to human PD-L1 (22C3 pharmDx). ResultsThe PD-L1 prevalence in patients with NSCLC in our hospital was 26 (37%) with TPS<1%, 23 (33%) with TPS 1-49%, and 21 (30%) with TPS ≥50%, suggesting that small biopsy samples obtained by bronchoscopy that were formalin-fixed for <6h are suitable for PD-L1 IHC. We also performed analyzed in 35 surgically-resected NSCLC specimens that had been formalin-fixed for 1-7 days by PD-L1 IHC. The PD-L1 prevalence was 20 (57%) for TPS < 1%, 9 (26%) for TPS 1-49%, and 6 (17%) for TPS ≥50%. ConclusionsThe PD-L1 prevalence in specimens with short-duration formalin fixation was consistent with the results of the KEYNOTE-024 trial. We will present our conclusion regarding these results, including the characteristics of patients and the PD-L1 prevalence in specimens fixed for 12-72h in our institution, from July 2018 onward. Legal entity responsible for the studyNational Hospital Organization Fukuokahigashi Medical Center. FundingJSPS KAKENHI JP18K15927. DisclosureAll authors have declared no conflicts of interest.