Clinicopathologic And Molecularpathologic Study Of Tumors Associated With Deficient INI1,a Core Subunit Of SWI/SNF Complex

BACKGROUND AND OBJECTIVE:SWI/SNF complex (switch in mating type/sucrose non fermentation, SWI/SNF) is a member of ATP -dependent chromatin remodellers.Using ATP hydrolysis energy to mobilize nucleosomes, the SWI/SNF complex modulates the accessibility of promoters to transcription factors, and play an important role in regulating cellular processes including differentiation, proliferation, and DNA repair. Recent reports suggested that this complex plays a key role in suppressing cancer development. In humans, SWI/SNF complex contains one of two mutually exclusive ATPase subunits, BRG1 (SMARCA4) or BRM (SMARCA2). The complex also includes core subunits: INI1 (SMARCB1, SNF5, and BAF47), BAF155 (SMARCC1), and BAF170 (SMARCC2). Lastly, the complex has accessory subunits that are associated with all versions of the complex:PBRM1 (which encodes BAF180), ARID1A (BAF250A), along with others.The INI1 (also known as SMARCB1, BAF47, and hSNF5) gene, located on chromosomal position 22q11.23, encodes a core subunit of the SWI/SNF complex. SWI/SNF complex could initiate transcription of a gene by INI1 binding c-Myc. Homozygous deletion of INI1 gene can result in embryo death during gestation of 3.5-5.5 day. The deletion of INI1 gene and loss of INI1 were first observed in malignant rhabdoid tumor, and became its diagnostic marker. Since then the loss of INI1 was found in different tumors:atypical teratoid/rhabdoid tumor (AT/RT), epithelioid sarcoma (ES), epithelioid malignant peripheral nerve sheath tumor, carcinoma of renal medulla, and so on.In INI1 deficient tumors, AT/RT and ES were the most common. They not only lost INI1 expression, but also have more or less rhabdoid tumor cells. Recent studies have showed that INI1 genetic inactivation is responsible for tumorigenic properties of AT/RT and ES. However, they were different in tumor biological behaviour, epidemiology and histology. AT/RT was most common in infants less than 3 years of age, has a rapidly fatal course. ES occured in the distal extremities of young adults, frequent local recurrence, but has a slow course of disease. Through cytogenetic and molecular pathologic study of AT/RT and ES, frequent loss of chromosome 22q often could be found in the two tumors, but they have different changes. Chromosome abnormalities in AT/RT were simple, except for 22q, other chromosomes were rarely involved. However, ES often has complex karyotypes, in addition to chromosome 22q, several chromosomes could be involved. Although the deletion and mutation of INI1 gene have been reported in these two tumors, the status of INI1 and the role of INI 1 in tumorigenesis may be different. For the rarity of AT/RT and ES, there were only a few reports about it and the results of some studies were conflicting. The molecular mechanisms resulting in loss of INI 1 in AT/RT and ES were still unclear.Understanding status of INI1 gene is not only beneficial to make diagnosis, and will be help for the tumor treatment. INI1 was tumor suppressor gene, inactivation of biallelic gene play a key role in tumorigenesis of related tumors. Except germline mutation of INI1 was found only in a few AT/RT and malignant rhabdoid tumor, the biallelic inactivation of INI in most of AT/RT and all of ES (germline mutation of INI1 has never be reported in ES) was caused by mutation, deletion and epigenetics.Epigenetic changes may explain hallmark features of cancers and may produce the same effect as genetic damages. Epigenetic changes contain DNA methylation, microRNA and chromatin modification. As well known that hypermethylation of DNA promoter region of suppressor genes can silence gene and extensively investigated causes of carcinogenesis and sarcomagenesis. CpG islands are quite large in the promoter region of the INI1 gene, suggesting a possible hypermethylation mechanism for gene silencing. In our study, inactivation DNA damage of INI1 were not observed in 87.5% of ES and 25% of AT/RT, suggested epigenetic changes may play an important role in loss of INI1. So we wanted to determine whether methylation of the promoter region of INI1 could result in nuclear INI1 protein loss in epithelioid sarcoma and AT/RT using methylation-specific PCR.Recently, with the emergence of the second generation of gene sequencing technology, whole-exome surveys of human cancers have showed that components of SWI/SNF to be mutated at high requency. And mutations of several subunits of SWI/SNF had been reported in single cancer type. Concurrent mutaions of PBRM1 and ARID1A have been found in renal clear cell carcinoma, and indicated that certain SWI/SNF complex components might act synergistically in the pathogenesis of cancer. Whether alterations of other core members of the SWI/SNF complex might play roles in AT/RT and ES pathogenesis remains unclear. In this study, we investigated the expression of the SWI/SNF complex subunits INI1, BRG1, BRM, PBRM1, and BAF155 in AT/RT and ES, and this study has never been reported.We, therefore, retrospectively analyzed clinicopathologic and molecularpathologic feathers of 9 AT/RT and 22 ES. The purposes of this study were (1) to delineate the clinical, morphological, and immunohistochemical features of AT/RT and ES; (2) to detect the expression of INI1 and other subunits of SWI/SNF complex PBRM1, BRM, BAF155 and BRG1 in AT/RT and ES, and their role in diagnosis and differential diagnosis. (3) Using fluorescence in situ hybridization (FISH) and direct sequencing to detect INI1 gene abnormality in formalin-fixed, paraffin-embedded (FFPE) tissues. (4) Design primers for the methylation-specific PCR to detect the status of INI1 promoter methylation in AT/RT and ES, and to explore new avenue for future treatment.MATERIALS AND METHODS:Between 1997 and 2012,173 caes including central nervous system neoplasms, soft tissue tumors and metastatic carcinoma with rhabdoid features in Nanjing Military Hospital and the First Affiliated Hospital of Anhui Medical University were collected. According to the WHO classification of tumors of the central nervous system, and tumors of soft tissue,9 AT/RT and 22 ES cases were diagnosed and the controls were established. The tumors were morphologically and immunohistochemically characterized with follow-up. FISH and direct sequencing used to detect INI1 gene abnormality in INI1 dificient cases. And the status of INI1 promoter methylation was detected by the methylation-specific PCR.RESULTS:1. The clinical features and follow-up:There were 12 males and 10 females in 22 ES, the age at diagnosis ranged from 16-75 years (mean,40.2years). CES mostly occurred in the extremities, PES cases were often in the perianal and genital areas. In 17 cases with follow-up,9 patients had local recurrences, and 2 patients died of the disese after metastasis to the lung.9 AT/RT, the age of at diagnosis ranged from 4 month-14 years (mean,7.5years). The most common site was the cerebral lobar (6 cases), followed by left cerebellar lobar (1 case), left cerebellopontine angle (1 case) and cervical spinal (1 case). The most common symptom was lethargy, headache, vomiting, and specific symptoms including facial paralysis, abnormal limb muscle strength and paralysis. The AT/RT has nonspecific performances on imaging, and it is difficult to distinguish AT/RT from CNS PNET and medulloblastoma by CT and MRI.8 cases with follow-up,7 caes died within 1-2 years.2. Morphological features:Upon microscopic examination, classic ES is characterized by pseudogranulomatous and multinodular growths. PES samples showed multinodular and sheet-like patterns consisting predominantly of epithelioid and rhabdoid cells with minor spindle-cell components. The fibroma-like variant is composed mainly of spindle cells with a storiform growth pattern. Case 11 had an angiomatoid growth pattern. Rather than CES, PES contained more rhabdoid tumor cells. Of the seven PES samples, two tumors exhibited features similar to rhabdoid tumors.AT/RT showed a wide range of histomorphological features. Rhabdoid regions were created by medium-large, jumbled cells including rhabdoid cell, arranged in incohesive growth pattern. Sometimes, the tumor was completely composed by rhabdoid cells. Undifferentiated small cell regions had the similar histological features to primitive neuroectodermal (PNET) and medulloblastoma.3. Immunohistochemical features:Of 22 ES,17 cases showed a complete absence of INI1 expression.18 cases showed the loss of PBRM1. Concurrent loss of INI 1 and PBRM1 expression was observed in most (16/22) cases. Two cases which exhibited classic ES features expressed ENI1, but lost PBRM1 expression. In contrast,1 case was positive for PBRM1 but negative for INI1. It is interesting to note that BRM was negative in two PESs with pure rhabdoid tumor features and negative INI1 and PBRM1. All cases were positive for BRG1 and BAF155. Except for rhabdoid tumor of kidney, INI1 and PBRM1 were retained in nearly all controls.The loss of PBRM1 and INI1 was observed in all AT/RT, BRM was lost in 5 cases. Different from the expression of INI1, loss of BRM was in rhabdoid regions, and positive in sarcomatous areas. BRG1 and BAF155 were positive in all cases and the controls4. Direct sequencing of INI1 in INI1 negative casesTwo missense mutations of INI1 were found in only 1 case of 16 ES.Mutations of INI1 including nonsense mutation, frameshift mutation, synonymous mutation and missense mutation were found in 6 cases of 8 AT/RT.5. FISH of INI1 in INI 1negative casesIn 16 ES, monosomy, homozygous and heterozygous deletions were observed in 2cases,2 cases cases and 5 cases respectively. In 8 AT/RT, monosomy was observed in 5 cases and homozygous deletion in 1 case.6. Methylation-specific PCR in INI1 negative casesAlthough inactivation DNA damage of INI1 were not observed in 87.5% of ES and 25% of AT/RT, we find that INI1 promoter region methylation was not detected in AT/RT and ES by methylation-specific PCR.CONCLUSION:(1) INI1 and PBRM1 were frequently lost in AT/RT and ES, suggested that they might act synergistically in the pathogenesis of AT/RT and ES. By analysis with control, INI1 and PBRM1 should play an important role in diagnosis.(2) Loss of BRM in rhabdoid regions with lost INI1 and PBRM1 simultaneously, suggested that loss of BRM may be involved in the underlying mechanisms of rhabdoid feature and suggested several SWI/SNF comlex subunits lost successively in AT/RT and ES.(3) In ES, missense mutation of INI1 was found in only 1 case, the gene damages which could lead to loss of INI1 only were found in two ES with homozygous deletions, and suggested that loss of INI1 may be caused by epigenetics.(4) AT/RT was characterized by nonsense mutation, frameshift mutation and monosomy suggested that loss of INI1 was caused by gene damages.(5) Through methylation-specific PCR, INI1 promoter region methylation was not observed in AT/RT and ES.