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.
Highlights
For early and locally advanced colorectal cancer (CRC), the standard treatment of choice for low-risk patients is surgical resection
The tissue microarray (TMA) cores from the patients were assessed, showed that nearly 50% of patients were by a Pathologist (M.B.L.) and, after exclusion criteria, 62 patients low in all T-cell subtypes; Kaplan–Meier analyses showed had 3 assessable cores, 99 had 2 assessable cores, whereas 7 that their prognosis was similar to patients with higher level of patients had only 1 assessable core
A large number of multigene signatures using tumor gene expression profiles have emerged in the last decade, such as Consensus Molecular Subgroups (CMS) and CRC Intrinsic Subtypes (CRIS), which classify patients into molecular subtypes for risk prediction[19,20,21]
Summary
For early and locally advanced (stage I and II) colorectal cancer (CRC), the standard treatment of choice for low-risk patients is surgical resection. Subsequent oncological treatment decisions for non-metastatic CRC are based largely on the anatomical AJCC/ UICC TNM staging classification[1]. After the MOSAIC study in 2004, patients with stage III CRC commonly receive oxaliplatin/ fluoropyrimidine/leucovorin (5-fluorouracil (5FU), FOLFOX; or xeloda/capecitabine, XELOX) as standard adjuvant treatment[2]. Of patients with stage III CRC treated with adjuvant chemotherapy, only ~20% will benefit from adjuvant FOLFOX, and 30% relapse within 2–3 years after surgery. Improvements in the understanding of CRC heterogeneity are paving the way for more personalized approaches that combine both histological and molecular data for patient stratification and therapy selection, including selecting which patients will benefit from adjuvant chemotherapy[4,5]
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