# Loss of Argininosuccinate Synthetase-1 (ASS1) Occurs in Esophageal Adenocarcinoma and Represents a Promising Biomarker for Therapy with Pegargiminase

## TL;DR

A significant portion of esophageal adenocarcinoma patients lack a key enzyme (ASS1), making them potential candidates for a therapy that deprives tumors of arginine.

## Contribution

This study identifies a subset of esophageal adenocarcinoma patients who may benefit from arginine deprivation therapy due to ASS1 loss.

## Key findings

- 6.2% of esophageal adenocarcinoma tumors showed complete loss of ASS1 expression.
- An additional 6.2% exhibited low ASS1 expression, totaling 12.4% potentially eligible for arginine deprivation therapy.
- Patients with ASS1 loss or low expression were younger and did not respond to neoadjuvant therapy.

## Abstract

Downregulation of Argininosuccinate Synthetase-1 (ASS1) supports tumor progression. Following the downregulation of ASS1, tumor cells rely on an external arginine delivery–a potential “Achilles heel” of these tumors. The treatment of patients with ASS1-deficient malignant pleural mesothelioma with pegargiminase, an arginine deprivation therapy agent, has shown prolonged survival. We aimed to assess the frequency of ASS1 downregulation in patients with esophageal adenocarcinoma. In this study, we performed ASS1 immunohistochemical stainings in 97 tumors. Among the patients included in this analysis, 6.2% exhibited an ASS1 loss, and a further 6.2% showed low ASS1 expression. Our results indicate that a significant number of patients with esophageal adenocarcinoma (12.4%) could potentially be eligible for arginine deprivation therapy. These findings underscore the need for clinical trials investigating the efficacy of pegargiminase treatment in patients with esophageal adenocarcinoma.

Background/Objectives: Despite the introduction of targeted therapies such as Nivolumab, survival outcomes for patients with esophageal adenocarcinoma remain poor. During tumorigenesis, some tumors develop auxotrophy by downregulation of Argininosuccinate Synthetase-1 (ASS1), making them reliant on external arginine supply and thus potentially susceptible to arginine deprivation therapy. Arginine deprivation therapy with agents such as pegargiminase has shown improved survival in patients with pleural mesothelioma exhibiting ASS1 loss in tumor cells. Therefore, we investigated the prevalence of ASS1 loss in esophageal adenocarcinoma. Methods: First, we compared the staining patterns of three antibodies for ASS1 with RNA in situ Scope analysis results to identify the most reliable antibody for ASS1 immunohistochemical staining in esophageal adenocarcinoma. Subsequently, we performed ASS1 immunohistochemical staining on samples from 97 patients who underwent curative resection. The staining results were classified into three categories based on expression levels: negative, low-positive, and positive. Results: Among all included patients, 6.2% exhibited an ASS1 loss, and 6.2% showed low ASS1 expression. Notably, patients with an ASS1 loss did not demonstrate a response to neoadjuvant therapy. Patients with ASS1 loss or low expression were significantly younger. Conclusions: Our findings indicate that approximately 12.4% of patients with esophageal adenocarcinoma may be eligible and could potentially benefit from arginine deprivation therapy. This underscores the urgent need for clinical trials evaluating the efficacy of pegargiminase in this patient population. Additionally, incorporating ASS1 immunohistochemical staining into pre-neoadjuvant biopsy assessments should be considered to optimize neoadjuvant treatment strategies and advance the implementation of personalized cancer therapy.