| Background and Objective:Renal cell carcinoma (RCC) has the highest mortality rate of all genitourinary carcinomas and includes several distinct clinicopathological entities, such as clear cell RCC, papillary RCC, chromophobe RCC and collecting duct carcinoma, as well as the recently recognized MiTF/TFE family renal translocation carcinoma. Each RCC type differs in morphology, genetics, behaviour and management. The most common histological type is clear cell RCC, also called conventional RCC, which represents 75%-80% of RCCs. Chromosome 3p deletion and inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene are the most common genetic alterations observed in this subtype. Adult RCCs can harbour poorly differentiated components, and in most cases these tumours are regarded as conventional RCCs with poorly differentiated areas or as composite tumours. However, the potential molecular mechanisms associated with features of poor differentiation such as anaplastic, rhabdoid or sarcomatoid cytological phenotypes, and the aggressive biological behaviour of these tumours, have not yet been elucidated. Whether or not this subset of RCCs has a distinct immunophenotype and molecular genetic features remains unclear.The yeast switch in the mating type (SWI)/sucrose non-fermentable (SNF) complex is one of several chromatin-remodelling complexes, and includes INI1 (also known as SMARCB1, SNF5 and BAF47), ARID1A (also known as BAF250A and SMARCF1), BAF180 (also known as PBRM1) and BRM (also known as SMARCA2)/BRG1 (also known as SMARCA4) subunits. The SWI/SNF complex is critical for growth control and cancer development, and complete loss of a SWI/SNF subunit can promote cancer formation. INI1, a core member of the complex, is a tumour suppressor gene. Loss of INI1 expression has been described in paediatric renal and extrarenal malignant rhabdoid tumours, atypical teratoid/rhabdoid tumours of the central nervous system, epithelioid sarcoma and renal medullary carcinoma. More recently, mutations in BAF180 were identified in 41% of RCCs, making BAF180 the second most common cancer gene in clear cell RCCs. The ARID1A subunit of SWI/SNF complexes was recently found to be mutated specifically in 50% of ovar-ian clear cell carcinomas and 30% of endometrioid carcinomas. In the recent prognostic study by Lichner et al., decreased ARID1A expression at both the protein and mRNA levels was also found to correlate with high tumour stage and nuclear grade in clear cell RCC. Mammalian BRM and BRG1 share approximately 75% identity at the amino acid level. BRG1 mutations and loss of expression have been identified in primary lung cancers. Moreover, BRM, a key SWI/SNF complex subunit, has been found to be inactivated in 10-20% of many solid tumour types, including lung, breast, colon, oesophageal, ovarian, bladder, prostate, gastric and head/neck tumours, suggesting that BRM is involved in human cancer. However, BRM status and function in renal epithelial tumours have not been analysed. It remains unclear if a deficiency in the BRM subunit of SWI/SNF complexes is linked to tumour progression and the poor prognosis of RCC.The aim of our study was to examine the status of BRM in clear cell RCCs, and to analyse the histopathology, immunophenotype, molecular features and prognosis of the BRM-negative cases. Futher more, we would like to explore the mechanism of loss of BRM expression in clear cell RCC using DNA sequencing, FISH, methylation-specific PCR or other molecular methods.Materials and Methods:625 clear cell RCCs were reviewed and examined for BRM immunostaining. BRM negative tumors were selected as the experimental group, and their clinicopathological and immunohistochemical features were recorded. Gene sequencing and FISH analysis of the von Hippel-Lindau (VHL) gene region were carried out to characterize the molecular feature of BRM-negative group. DNA sequencing, FISH, methylation-specific PCR of BRM gene were also performed to figure out the mechanism of loss of BRM expression in clear cell RCC.Results:We identified 19 cases of grade 4 tumours lacking BRM expression among 625 clear cell RCCs. All 19 cases exhibited features of poor differentiation:13 cases showed pure poorly differentiated morphology, while 6 were identified composite tumours with an admixed typical low-grade component.Staining for BRM was negative in the poorly differentiated regions in all 19 cases. In contrast, positive BRM nuclear staining was observed in stromal cells and the low-grade clear cell RCC component of all 6 composite RCCs.All 19 cases were positive for CKpan, CA-IX and vimentin and negative for SMA, TFEB, TFE3, HMB45, cathepsin K, CK7 and CD117. The expression of CD10, P504S and Ksp-cadherin in different cases, or in different components of the same case were inconsistent. The immunophenotype of these cases was similar to clear cell RCC.To further determine the molecular alterations and origins of these tumours, we analysed VHL gene status in all 19 BRM-negative RCCs (cases 1-19). For comparison, seven composite RCCs with BRM positive staining (cases 20-26) and 10 pure poorly differentiated BRM-positive RCCs (cases 27-36) were also included as control groups. We found 24 distinct intragenic mutations in nine of the 19 BRM-negative cases (47%) and nine of the 17 BRM-positive RCCs (53%). Remarkably, the different components of the composite tumours exhibited identical VHL gene status in every case.The 36 cases selected for VHL sequencing were also subjected to FISH analysis. FISH analysis was unsuccessful in two composite tumours (cases 17 and 26). Of the remaining cases, chromosome 3p deletion was detected in 20 pure poorly differentiated cases and in both areas of the 11 composite tumours. All poorly differentiated areas of the 34 tumours analysed showed polysomy of chromosome 3. In contrast, no chromosomal losses or gains of chromosome 3 were observed in the clear cell RCC areas of the 11 composite RCCsWe also explore the mechanism of loss of BRM expression in clear cell RCC using DNA sequencing, FISH of chromosome 9 and methylation-specific PCR.78.9% BRM-negative tumors harbored BRM coding regeion mutations,43.8% existed 9p deletion or haploidy of chromosome 9,42.8% exhibited BRM promoter hypermethylation,89.5% had at least one BRM gene abnormity while 47.4% had two or more. The positive findings could at least partly explain the mechanisms of BRM negative staining in these tumors.During the exploration of composite tumors, we found that BRM mutations and 9p deletions only exist in poorly differentiated areas, suggesting BRM is an attractive candidate for being the’second hit’in clear cell RCCs and may play an important role during tumour progression.Conclusions:1. We identified loss of BRM expression as a frequently observed event (39.6%) in poorly differentiated clear cell carcinoma. The negative immunostaining of BRM was highly associated with a tumor nuclear grade.2. The immunophenotype and molecular feature of the BRM-negative group were generally similar to clear cell RCC.3. These BRM-negative RCCs demonstrated anaplastic features and poor prognosis. Whether they presented an independent subtype require further investigation.4. Compared with BRM inactivation, VHL gene mutation and chromosome 3p deletion were early events during clear cell RCC development.5. In the composite RCCs, both clear cell RCC areas and poorly differentiated regions had identical VHL gene status, suggesting a clonal origin. While poorly differentiated regions were identified extra BRM gene changes.6. BRM gene mutation, chromosome 9p deletion and hypermethylation of BRM promoter in BRM-negative tumors coule at least partly explain the mechanisms of BRM inactivation.7. BRM is an attractive candidate for being the ’second hit’ in clear cell RCCs and may play an important role during tumour progression. |
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