Abstract A32: Prognostic stratification of stage I lung adenocarcinoma patients by HOXA9 promoter methylation ddPCR and blood vessel invasion analysis in FFPE tissues
Authors: Lissa Delphine , Ishigame Teruhide , Noro Rintaro , Tucker Marguerite J. , Bowman Elise D. , Harris Curtis C. , Robles Ana I. 
Published: February 2019

Abstract
Surgery with curative intent is the standard of care for stage I lung adenocarcinoma. However, disease recurrence eventually occurs in about a third of the patients. Consequently, prognostic biomarkers are urgently needed to predict recurrence following surgery and potentially guide the decision to administer adjuvant chemotherapy for high-risk patients. The type of biospecimens and the choice of assay platform are key issues in the process of cancer biomarker clinical development. Here, we demonstrate the application of a droplet digital PCR (ddPCR)-based assay to analyze DNA methylation in formalin-fixed, paraffin-embedded (FFPE) biospecimens. In recent years, ddPCR has become increasingly used clinically due to its ability to reliably detect and quantitate rare alleles, as well as its technical simplicity, rapidity, and cost-effectiveness. In addition, FFPE tissues are generated during routine pathologic assessment of resected patients, and are generally available for testing molecular markers. We developed a ddPCR assay to quantify HOXA9 promoter methylation in FFPE samples, with high sensitivity, specificity, and reproducibility. High HOXA9 promoter methylation has been associated with worse outcome of resected stage I lung adenocarcinoma patients in multiple studies. Here, the prognostic value of HOXA9 promoter methylation alone or in combination with blood vessel invasion (BVI), another well-described prognostic biomarker for patients with early stage lung adenocarcinoma, was evaluated by Kaplan-Meier survival analyses and Cox regression modeling, using 188 FFPE biospecimens, in two independent sets. We replicated previous observations that HOXA9 promoter is methylated de novo in stage I tumors (P &lt;0.0001), and that high methylation is associated with worse prognosis (Hazard Ratio [HR], 3.37; P=0.0002). HOXA9 methylation was associated with a transcriptome signature enriched in genes marked by Polycomb in Embryonic Stem Cells, a signature previously associated with poor differentiation and worse overall patient survival. Addition of this molecular marker improved a risk model comprising clinical and pathologic parameters (age, sex, smoking status, and tumor stage and size; P=0.003, nested likelihood ratio test). As expected, BVI was independently associated with poor outcome (HR, 2.62; P=0.054). A score that combined BVI with HOXA9 promoter methylation further stratified high-risk patients (Trend P=0.0001 comparing 0, 1 or 2 positive markers). Collectively, our results support the use of ddPCR to quantify HOXA9 promoter methylation and the analysis of BVI from routine pathology FFPE specimens to identify patients at high risk of recurrence. Our findings pave the way for future clinical evaluation of these promising prognostic markers. If validated in a larger independent study, these biomarkers should be considered in prospective trials to evaluate the benefit of adjuvant chemotherapy in high-risk early-stage lung adenocarcinoma patients.
Citation Format: Delphine Lissa, Teruhide Ishigame, Rintaro Noro, Marguerite J. Tucker, Elise D. Bowman, Curtis C. Harris, Ana I. Robles. Prognostic stratification of stage I lung adenocarcinoma patients by HOXA9 promoter methylation ddPCR and blood vessel invasion analysis in FFPE tissues [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A32.