title: 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.
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Department of Pathology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.
authors.forename: Essel
authors.initials: E
authors.lastname: Dulaimi
authors.affiliation: Biostatistics and Bioinformatics Facility, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.
authors.forename: Karthik
authors.initials: K
authors.lastname: Devarajan
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Theodore
authors.initials: T
authors.lastname: Parsons
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Lili
authors.initials: L
authors.lastname: Liao
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Eun-Ah
authors.initials: EA
authors.lastname: Cho
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Raymond
authors.initials: R
authors.lastname: O'Neill
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Charalambos
authors.initials: C
authors.lastname: Solomides
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Peiper
authors.affiliation: Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Testa
authors.affiliation: Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.|Department of Surgical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Uzzo
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Yang
mesh_terms: D000818:Animals; D002292:Carcinoma, Renal Cell / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D018740:Genetic Heterogeneity; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007680:Kidney Neoplasms / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010802:Phylogeny; D046888:Tissue Array Analysis / Q000379:methods*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C112476:ARID1A protein, human; D002843:Chromatin; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C102195:PBRM1 protein, human; C084011:SMARCA2 protein, human; D014157:Transcription Factors; D011495:Histone-Lysine N-Methyltransferase; C521636:SETD2 protein, human; C084108:SMARCA4 protein, human; D004265:DNA Helicases