Development of a reliable and accurate algorithm to quantify the tumor immune stroma (QTiS) across tumor types

Abstract

The tumor microenvironment plays an important role in the tumor biology. Overall survival of tumor patients after resection is influenced by tumor-infiltrating lymphocytes (TILs) as a component of the tumor stroma. However, it is not clear how to assess TILs in the tumor stroma due to heterogeneous methods in different cancer types. Therefore, we present a novel Quantification of the Tumor immune Stroma (QTiS) Algorithm to reliably and accurately quantify cells in the tumor stroma. Immunohistochemical staining of CD3 and CD8 cells in sections of metastatic colorectal cancer (mCRC), ovarian cancer (OvCa), hepatocellular carcinoma (HCC), and pancreatic ductal adenocarcinoma (PDAC), alltogether N = 80, was performed. Hot spots of infiltrating immune cells are reported in the literature. Reliability of the hot spot identification of TILs was examined by two blinded observers. Accuracy was tested in 1 and 3 hot spots using computed counting methods (ZEN 2 software counting (ZC), ImageJ software with subjective threshold (ISC) and ImageJ with color deconvolution (IAC)) and compared to manual counting. All tumor types investigated showed an accumulation of TILs in the tumor stroma (peri- and intratumoral). Reliability between observers indicated a high level consistency. Accuracy for CD8+/CD3+ ratio and absolute cell count required 1 and 3 hot spots, respectively. ISC was found to be the best for paraffin sections, whereas IAC was ideal for frozen sections. ImageJ software is cost-effective and yielded the best results. In conclusion, an algorithm for quantification of tumoral stroma could be established. With this QTiS Algorithm counting of tumor stromal cells is reliable, accurate, and cost-effective.

Prognostic Impact of Tumor-Infiltrating Lymphocytes and Neutrophils on Survival of Patients with Upfront Resection of Pancreatic Cancer

“…Finally, we showed that the QTiS algorithm for different tumor types described previously [39,69] retrieved relevant results to highlight the importance of peritumoral TILs in patients with PDAC. Results with colorectal cancer indicate that quantification of immune cell infiltration has the potential to become an integral part of the TNM classification of PDAC [70].…”

Fibroblasts in urothelial bladder cancer define stroma phenotypes that are associated with clinical outcome

“…The PDGFRa and -b antibodies have been validated and described previously and cross-detection of the two related receptors has been excluded 33,36 . The ASMA 39,[63][64][65] , CD90 66,67 , FAP 54,68 and CD8a 65,69,70 antibodies are widely used in IHC by several groups and have been published previously. Cross-reactivity of these antibodies has been validated by Western Blot analysis by the manufacturers and all antibodies are recommended for IHC according to the manufacturer's datasheet.…”

Promising abscopal effect of combination therapy with thermal tumour ablation and intratumoural OK-432 injection in the rat osteosarcoma model

“…Five tumours/groups were assessed. The number of positively-stained cells was counted via microscopy in three selected fields of tumour sections at ×200 magnification using ImageJ Macro (National Institutes of Health, USA) 14,15 . The greatest count from the chosen fields was selected, and the absolute cell number obtained on five slides per tumour was calculated.…”