Immunohistochemistry Successfully Uncovers Intratumoral Heterogeneity and Widespread Co-Losses of Chromatin Regulators in Clear Cell Renal Cell Carcinoma

Abstract

Recent studies have shown that intratumoral heterogeneity (ITH) is prevalent in clear cell renal cell carcinoma (ccRCC), based on DNA sequencing and chromosome aberration analysis of multiple regions from the same tumor. VHL mutations were found to be universal throughout individual tumors when it occurred (ubiquitous), while the mutations in other tumor suppressor genes tended to be detected only in parts of the tumors (subclonal). ITH has been studied mostly by DNA sequencing in limited numbers of samples, either by whole genome sequencing or by targeted sequencing. It is not known whether immunohistochemistry (IHC) can be used as a tool to study ITH. To address this question, we examined the protein expression of PBRM1, and PBRM1-related proteins such as ARID1A, SETD2, BRG1, and BRM. Altogether, 160 ccRCC (40 per stage) were used to generate a tissue microarray (TMA), with four foci from each tumor included. Loss of expression was defined as 0–5% of tumor cells with positive nuclear staining in an individual focus. We found that 49/160 (31%), 81/160 (51%), 23/160 (14%), 24/160 (15%), and 61/160 (38%) of ccRCC showed loss of expression of PBRM1, ARID1A, SETD2, BRG1, and BRM, respectively, and that IHC could successfully detect a high prevalence of ITH. Phylogenetic trees were constructed that reflected the ITH. Striking co-losses among proteins were also observed. For instance, ARID1A loss almost always accompanied PBRM1 loss, whereas BRM loss accompanied loss of BRG1, PBRM1 or ARID1A. SETD2 loss frequently occurred with loss of one or more of the other four proteins. Finally, in order to learn the impact of combined losses, we compared the tumor growth after cells acquired losses of ARID1A, PBRM1, or both in a xenograft model. The results suggest that ARID1A loss has a greater tumor-promoting effect than PBRM1 loss, indicating that xenograft analysis is a useful tool to investigate how these losses impact on tumor behavior, either alone or in combination.

Intratumoural heterogeneity may hinder precision medicine strategies in patients with clear cell renal cell carcinoma

“…Our study highlights the need of developing specific sampling strategies for molecular analysis of RCCs, both in case of tumour biopsies and also for surgical specimens since, as seen in our study, wide sampling might identify foci in which mutations are present. These results are in agreement with the paper by Ruiz-Cerda12 who has suggested that results change significantly with the number of samples analysed and by Jiang13 who has observed that intratumoural heterogeneity is prevalent in ccRCC.…”

“…The most frequently documented genetic aberration in ccRCC is an inactivating somatic alteration on VHL gene and loss of heterozygosity of chromosome 3p harbouring the second copy of the gene 16 17. According to the Cancer Genome Atlas Database, the PI3K pathway, as observed in our present case report, is frequently mutated in RCC, a fact that supports this pathway as a potential therapeutic target in ccRCC 13. The PI3K pathway, a critical signal transduction system linking oncogenes and multiple receptor classes to many essential cellular functions, is perhaps the most commonly activated signalling pathway in human cancer, and therefore it has been suggested to play a role in cancer therapy.…”

Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

“…The detailed tally of the protein expression loss was described before [14]. We found that 31% of tumors lost expression of PBRM1.…”

Solid‐type poorly differentiated adenocarcinoma of the stomach: Deficiency of mismatch repair and SWI/SNF complex

“…A previous study reported that ARID1A knockdown promotes tumor growth in xenograft models . In this study, solid‐type PDAs showed larger size, more frequent expansive growth, and losses of SWI/SNF family members compared to GC in the control group.…”

“…In our assessment of ARID1A, INI1, BRM, BRG1, BAF155, and BAF170, only unequivocally clearly absent staining in the nuclei of viable tumor tissue (away from necrotic areas) was considered “loss.” As a control, the presence of homogenous strong nuclear staining of stromal fibroblasts, inflammatory cells, vascular endothelial cells, or normal epithelial cells in the background was a prerequisite for assessable staining in the tumor. Staining proportion was graded as complete loss (0%‐4%), partial loss (5%‐49%), or retained (50%‐100%) based on a described labeling index …”