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. Predictive biomarkers for identification of patients with increased risk for disease recurrence are currently lacking, with progress in biomarker discovery hindered by the inherent heterogeneity of the disease. The immune profile of colorectal tumors has previously been found to have prognostic value. The aims of this study were to evaluate immune signatures in the tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single cell analysis technology (Cell DIVE™). Tissue microarrays (TMAs) with up to three 1mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin chemotherapy. Single sections underwent multilplexed immunofluorescence with Cy3- and Cy5-conjugated antibodies for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase and S6). We applied a probabilistic multi-class, multi-label classification algorithm based on multi-parametric models to build statistical models of protein expression to classify immune cells. Expert annotations of immune cell markers were made on a range of images, and Support Vector Machines (SVM) were used to derive a statistical model for cell classification. Images were also manually scored independently by a Pathologist as "high", "moderate" or "low", for stromal and total immune cell content. Excellent agreement was found between manual and total automated scores (p<0.0001). Higher levels of multi-marker classified regulatory T cells (CD3+CD4+FOXP3+PD1-) were significantly associated with disease-free survival (DFS) and overall-survival (OS) (p=0.049 and 0.032), compared to FOXP3 alone. Our results also showed that PD1- Tregs rather than PD1+ Tregs were associated with improved survival. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cells with greater potential for predicting patient 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.…”

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].…”