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Background/Aim: Cancers with a microsatellite instability-high (MSI-H) or deficient mismatch repair (dMMR) status respond to immune checkpoint inhibition (ICI). Regardless of the tumor type, MSI-H/dMMR status is a reliable biomarker for ICI responsiveness. This study aimed at determining the MSI-H status in precursor lesions to esophageal adenocarcinoma (EAC) such as Barrett’s esophagus (BE) and BE with either low-grade dysplasia (LGD) or high-grade dysplasia (HGD). Patients and Methods: We performed immunohistochemical staining (IHC) for PMS2, MSH6, PD1, and PD-L1. Results: All cases of BE (50), LGD (48), and HGD (50) had intact PMS2 and MSH6 nuclear expression; were negative for PD1; and had a PD-L1 combined positive score (CPS) score 1, and one EAC case (2%) was MSI-H. MSI-H tumors usually show PD-L1 expression, although the MSI-H EAC in this study had a PD-L1 CPS score of

Additional file 3: Supplementary Figure 3.PHGDH knockdown does not affect liver function. shPHGDH (N = 10) and shREN (N = 10) mouse serum was collected and analyzed by IDEXX (Liver Panel). (A) ALP – Alkaline phosphatase. (B) AST – aspartate transaminase. (C) ALT – alanine transaminase. (D) CK – creatine kinase. (E) – Total albumin. (E) Total bilirubin.

Additional file 2: Supplementary Figure 2. shPHGDH mice have poor knockdown in the spleen. Western blot analysis of PHGDH, GFP and HSP90 protein levels in spleen of shPHGDH and shREN mice. Mice were placed on a 200 ppm doxycycline diet for 8 months. ns, non-specific band.

Additional file 4: Supplementary Figure 4.PHGDH knockdown does not affect brain serine and lipids. (A) Brain serine quantities of 5- to 9-month-old shREN (N = 15) and shPHGDH (N = 14) mice. Quantities were normalized to mg of tissue. (B) Quantity of individual ceramides in the brain of 5- to 9-month-old shREN (N = 14) and shPHGDH (N = 15) mice. Quantities were normalized to mg of tissue. (C) Volcano plot of lipidomics analysis of shPHGDH (N = 15) brain compared to shREN (N = 15). Significant metabolites are in bold. Triacylglycerol species are indicated in red. (D) Individual TAG species in the brain of shPHGDH mice compared to shREN. Levels are normalized to shREN.

Additional file 1: Supplementary Figure 1.Validation of shRNAs for model development. NIH3T3 cells expressing Renilla or PHGDH-targeting shRNAs (#1-10) were treated with 1 μg/mL doxycycline for 6 days and PHGDH expression determined by western blot. β-actin is used as a loading control.

PURPOSE: Histone deacetylase inhibitors (HDACi) enhance tumor immunogenicity through several mechanisms and may improve response to immune checkpoint inhibitors (ICIs). In a phase 1/1b trial, we tested the oral HDACi vorinostat combined with the programmed cell death protein 1 inhibitor pembrolizumab in advanced/metastatic non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Patients received intravenous pembrolizumab (200 mg every 3 weeks) plus oral vorinostat (200 or 400 mg/day). Primary endpoint was safety/tolerability. Secondary endpoints included response rate, progression-free survival, disease control rate (DCR), and overall survival. Tumor gene expression changes, T-cell density, and myeloid cell levels were studied in serial tissue specimens. RESULTS: Thirty-three patients were treated (13 in phase 1, 20 in phase 1b). In phase 1, both ICI-naive and ICI-pretreated patients were enrolled to determine dose-limiting toxicities (DLTs). No DLTs were observed, and the recommended phase 2 dose was pembrolizumab 200 mg and vorinostat 400 mg. Any-grade adverse events were mainly fatigue (33%) and nausea/vomiting (27%). Of 6 ICI-naive and 24 ICI-pretreated patients evaluable for response, 4 (13%) had partial response (2 confirmed, 1 unconfirmed with subsequent prolonged stable disease [SD], 1 unconfirmed with subsequent progressive disease [PD]), 16 (53%) had SD, and 10 (33%) had PD for a DCR of 67%. In the ICI-pretreated cohort, 3 patients (1 confirmed, 2 unconfirmed) had partial response and 10 had SD. Pretreatment CD8(+) T-cell presence in tumor stromal regions was associated with treatment benefit. CONCLUSIONS: Pembrolizumab plus vorinostat was well tolerated and demonstrated preliminary anti-tumor activity despite progression on prior ICI treatment.