Stratification of chemotherapy-treated stage III colorectal cancer patients using multiplexed imaging and single-cell analysis of T-cell populations

Abstract

Colorectal cancer (CRC) has one of the highest cancer incidences and mortality rates. In stage III, postoperative chemotherapy benefits <20% of patients, while more than 50% will develop distant metastases. Biomarkers for identification of patients at increased risk of disease recurrence following adjuvant chemotherapy are currently lacking. In this study, we assessed immune signatures in the tumor and tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single-cell analysis technology (Cell DIVETM) and evaluated their correlations with patient outcomes. Tissue microarrays (TMAs) with up to three 1 mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin (FOLFOX) chemotherapy. Single sections underwent multiplexed immunofluorescence staining for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and tumor/cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase, and S6). We used annotations and a probabilistic classification algorithm to build statistical models of immune cell types. Images were also qualitatively assessed independently by a Pathologist as ‘high’, ‘moderate’ or ‘low’, for stromal and total immune cell content. Excellent agreement was found between manual assessment and total automated scores (p < 0.0001). Moreover, compared to single markers, a multi-marker classification of regulatory T cells (Tregs: CD3+/CD4+FOXP3+/PD1−) was significantly associated with disease-free survival (DFS) and overall survival (OS) (p = 0.049 and 0.032) of FOLFOX-treated patients. Our results also showed that PD1− Tregs rather than PD1+ Tregs were associated with improved survival. These findings were supported by results from an independent FOLFOX-treated cohort of 191 stage III CRC patients, where higher PD1− Tregs were associated with an increase overall survival (p = 0.015) for CD3+/CD4+/FOXP3+/PD1−. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cell types with stronger correlations with outcomes.

Are patients living far from hospital at higher risk of late adjuvant chemotherapy for colon cancer?

“…Our comparatively small sample size is explained by the specificity of the population targeted by our study, and is similar to the sample size of other surveys recently published on this particular population 35,36 . However, our restrictive selection criteria had the advantage of providing a homogenous sample, which limits potential confounding bias.…”

“…Heeg et al 34 population. 35,36 However, our restrictive selection criteria had the advantage of providing a homogenous sample, which limits potential confounding bias.…”

Using single cell atlas data to reconstruct regulatory networks

“…Recent advances of single cell technologies have led to a surge of studies that generate tissue specific genomic profiles at atlas scale. The HuBMAP consortium is one such effort generating several tissue-specific single cell genomic data sets ( 91–93 ).…”

The association between the tumor immune microenvironments and clinical outcome in low‐grade, early‐stage endometrial cancer patients

“…The fact that previous studies suggested a positive association between regulatory T‐cells and worse outcome [ 39 ] should be interpreted taking into account differences in the clinical characteristics of the cohorts studied and the methods used to quantify regulatory T‐cells. Further, one study analyzing chemotherapy‐treated stage III colorectal carcinomas found improved outcomes for regulatory T‐cell tumors [ 40 ].…”