Identification and validation of heterotypic cell-in-cell structure as an adverse prognostic predictor for young patients of resectable pancreatic ductal adenocarcinoma

OBJECTIVES A proportion of resectable pancreatic ductal adenocarcinoma (PDAC) patients display poorer survival due to profound local immune suppression. However, a pathological/morphological parameter that could functionally read out immune evasion and predict patient survival has not been defined. This study investigated the feasibility of heterotypic cell-in-cell (CIC) structures for immune cell cannibalism by tumor cells to serve as a parameter for survival prediction in resectable PDAC patients. METHODS A total of 410 samples from PDAC patients were examined using the methods of “EML” multiplex staining or immunohistochemistry (IHC). Prognostic CIC candidates were initially identified in samples plotted in tissue microarray (n=300), then independently validated in specimens from the First Affiliated Hospital of Sun Yat-Sen University (n=110). The Kaplan–Meier estimator and/or the Cox regression model were used for univariate and multivariate analysis. A nomogram was made using the Regression Modeling Strategies. RESULTS CICs were prevalent in cancerous (203/235) but not non-malignant tissues (15/147). Among the 4 CIC subtypes identified, 2 heterotypic subtypes with tumor cells internalizing CD45 + lymphocytes (LiT, mOS = 8 vs. 14.5 months, p = 0.008) or CD68 + monocytes (MiT, mOS = 7.5 vs. 15 months, p = 0.001), and overall CICs (oCIC, mOS = 10 vs. 27 months, p = 0.021), but not homotypic CICs (TiT, p = 0.089), were identified in univariate analysis as adverse prognostic factors of overall survival (OS) of PDAC. Notably, through cannibalism of immune cells by tumor cells, heterotypic CICs (L/MiT: LiT plus MiT) could independently predict shorter OS (HR = 1.85, p = 0.008) in multivariate analysis, with a performance comparable or even superior to traditional clinicopathological parameters such as histological grade (HR = 1.78, p = 0.012) and TNM stage (HR=1.64, p = 0.108). This was confirmed in the validation cohort, where L/MiT (HR = 1.71, p = 0.02) and tumor–node–metastasis (TNM) stage (HR = 1.66, p = 0.04) were shown to be independent adverse prognostic factors. Moreover, L/MiT stood out as the most prominent contributor in nomogram models constructed for survival prediction (area under the curve = 0.696 at 14 months), the dropout of which compromised prediction performance (area under the curve = 0.661 at 14 months). Furthermore, stratification analysis indicated that L/MiT tended preferentially to impact young and female patients (HR = 11.61, p < 0.0001, and HR = 9.55, p = 0.0008, respectively) in particular with early-stage and low-grade PDAC (HR = 2.37, p < 0.0001, and HR = 2.19, p < 0.0001, respectively), while TNM stage demonstrated little preference. CONCLUSION This was the first CIC profiling to be performed in PDAC, and is currently largest for human tumors. Subtyped CICs, as a valuable input to the traditional variables such as TNM stage, represent a novel type of prognostic factor. The formation of heterotypic L/MiT may be a surrogate for local immune evasion and predict poor survival, particularly in young female patients of resectable PDAC. Study Highlights Prior knowledge The post-operation survival periods of resectable pancreatic ductal adenocarcinoma (PDAC) patients range widely, and the search for reliable prognostic biomarkers is warranted. Although profound local immune suppression is implicated in PDAC progression and poor patient survival, a prognostic marker to read immune evasion in situ is not yet available. The impact of subtyped cell-in-cell (CIC) structures, which target either tumor or immune cells for internalization and death, on PDAC patient survival is not clear. Novelty of study This study presents the first CIC subtype profiling in PDAC, which is currently the largest of its type for human cancers. Subtyped CIC structures were identified and confirmed independently as a valuable prognostic factor for PDAC patients, with a performance comparable or superior to traditional variables such as tumor–node–metastasis (TNM) stage. The L/MiT heterotypic CIC subtype, surrogating a type of cellular immune evasion, could independently predict poor survival, particularly for young female patients of resectable PDAC.