INVASIVE FRONT OR TUMOR CORE: CANCER STEM CELL LOCATION AND CORRELATIONS WITH CELLULAR BEHAVIOR AND PATIENT OUTCOME

Presentation: C034
Topic: Oral Cancer
Type: Poster
Date: Thursday, April 19, 2018
Session: 9:00 AM - 7:00 PM
Authors: Farshad N Chowdhury, MD1, Stephen B Keysar, PhD2, Tugy Chimed, MS2, Julie Reisinger, CVT2, Hilary Somerset, MD3, John I Song, MD1, Antonio Jimeno, MD, PhD4
Institution(s): 1Department of Otolaryngology, University of Colorado Anschutz Medical Campus, 2Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, 3Department of Pathology, University of Colorado Anschutz Medical Campus, 4Division of Medical Oncology, Department of Medicine and Department of Otolaryngology, University of Colorado Anschutz Medical Campus

Introduction: Frequent locoregional recurrence and poor five-year survival of head and neck squamous cell cancer (HNSCC) persist as treatment challenges. Disease-free margins are critical in surgical management of HNSCC, but tumor behavior at the margin is ill-defined. Investigations into molecular markers at the surgical margin have drawn attention to the role of cancer stem cells (CSCs) in disease aggression and recurrence. Previously, our group characterized a CSC population (ALDH+/CD44high) with enhanced tumorigenicity, chemoresistance, and radioresistance in patient-derived xenograft (PDX) tumors. This study seeks to understand the biological and clinical impact of CSCs at the tumor invasive front.

Methods:

CSC Distribution by FACS: Representative PDXs were sectioned into “leading edge (LE)” (outer 2-3mm) and “core” compartments. The CSC percentage (ALDH+/CD44high) by compartment was measured.

Determination of SOX2 Staining Pattern by IHC: Oral cavity (OC) and oropharynx (OP) HNSCC patients with three years of follow-up were selected from a prospective database (n=74). SOX2 staining architecture was classified as “leading-edge enriched (LEE)” or “diffuse staining (DS)” and compared with FACS CSC distribution.

Functional Analysis of Stemness by Tissue Compartment in a Leading-Edge Enriched PDX Case:  PDX tumors were sectioned into “LE” and “core” compartments. CSCs (ALDH+/CD44high) from each compartment were identically sorted via FACS.  Sorted CSCs were seeded (5,000 cells/well) and cultured in suspension in serum-free media, and the resulting spheres were counted.

Patient Outcomes Analysis:  Kaplan-Meier curves were generated for disease-free and overall survival for “LEE” and “DS” SOX2 expression groups. Subset analysis was performed on HPV negative cases. Curves were compared via the log-rank method.

Results: To assess CSC distribution,  three PDX cases underwent FACS analysis for CSC enrichment by tumor compartment. CUHN111 and CUHN109 demonstrated CSC enrichment of the LE (0.26% LE vs 0.04% core, p=0.004; 0.18% LE vs 0.07% Core, p = 0.034) while one PDX, CUHN013, demonstrated no difference (0.712 ± 0.3087 LE vs 3.56 ± 3.164 Core, p = 0.4). The CSC distribution in these PDXs mirrored the “LEE” or “DS” SOX2 staining patterns found in their respective originating patient resection specimens. All 74 OC/OP patient specimens were stained for SOX2 by IHC. 22 of 74 were SOX2+, 9/22 were “LEE”, and 13/22 were “DS.” 

Functional differences in the CSCs isolated from the LE vs the Core were assessed using a sphere formation assay. In the LE-enriched case CUHN111, LE CSCs demonstrated greater sphere-forming efficiency, with a LE:Core efficiency ratio of 1.8:1 (n=5, p = 0.011).

Outcomes were assessed in SOX2 expressing cases. HPV-negative, “LEE” cases demonstrated improved survival vs “DS” cases (HR 3.2, p < 0.05).  No other statistically significant differences were identified.

Conclusions: Here, we demonstrate that the distribution of SOX2+ cells by IHC mirrors that of ALDH+/CD44high CSCs by FACS.  CSC behavior varies by tissue compartment, as LE CSCs demonstrate greater proliferative potential.  Core CSCs may play a distinct functional role, as non-LE SOX cases demonstrated poorer overall survival. These data support further investigation into the impact of CSC localization and CSC-microenvironment interactions on CSC proliferation, CSC treatment resistance, and patient survival.