ATBF1 and NQO1 as candidate targets for allelic loss at chromosome arm 16q in breast cancer: absence of somatic ATBF1 mutations and no role for the C609T NQO1 polymorphism.

BACKGROUND: Loss of heterozygosity (LOH) at chromosome arm 16q is frequently observed in human breast cancer, suggesting that one or more target tumor suppressor genes (TSGs) are located there. However, detailed mapping of the smallest region of LOH has not yet resulted in the identification of a TSG at 16q. Therefore, the present study attempted to identify TSGs using an approach based on mRNA expression. METHODS: A cDNA microarray for the 16q region was constructed and analyzed using RNA samples from 39 breast tumors with known LOH status at 16q. RESULTS: Five genes were identified to show lower expression in tumors with LOH at 16q compared to tumors without LOH. The genes for NAD(P)H dehydrogenase quinone (NQO1) and AT-binding transcription factor 1 (ATBF1) were further investigated given their functions as potential TSGs. NQO1 has been implicated in carcinogenesis due to its role in quinone detoxification and in stabilization of p53. One inactive polymorphic variant of NQO1 encodes a product showing reduced enzymatic activity. However, we did not find preferential targeting of the active NQO1 allele in tumors with LOH at 16q. Immunohistochemical analysis of 354 invasive breast tumors revealed that NQO1 protein expression in a subset of breast tumors is higher than in normal epithelium, which contradicts its proposed role as a tumor suppressor gene.ATBF1 has been suggested as a target for LOH at 16q in prostate cancer. We analyzed the entire coding sequence in 48 breast tumors, but did not identify somatic sequence changes. We did find several in-frame insertions and deletions, two variants of which were reported to be somatic pathogenic mutations in prostate cancer. Here, we show that these variants are also present in the germline in 2.5% of 550 breast cancer patients and 2.9% of 175 healthy controls. This indicates that the frequency of these variants is not increased in breast cancer patients. Moreover, there is no preferential LOH of the wildtype allele in breast tumors. CONCLUSION: Two likely candidate TSGs at 16q in breast cancer, NQO1 and ATBF1, were identified here as showing reduced expression in tumors with 16q LOH, but further analysis indicated that they are not target genes of LOH. Furthermore, our results call into question the validity of the previously reported pathogenic variants of the ATBF1 gene.

Expression analysis of candidate breast tumour suppressor genes on chromosome 16q.

INTRODUCTION: Chromosome arm 16q is the second most frequent target of loss of heterozygosity in breast cancer and is, therefore, a candidate to contain one or more classic tumour suppressor genes (TSGs). E-cadherin at 16q22 was identified as a TSG in lobular breast cancer, but TSGs in ductal breast cancer remain elusive. Several genes have been suggested as potential candidates (e.g. CBFA2T3, CTCF and WWOX) but no inactivating mutations could be identified in these genes and they thus fail to fit the classic two-hit model for a TSG. With the completion of the human transcriptome, new candidate genes can be distinguished. Besides mutational inactivation, a TSG could, at least in a subset of the tumours, be transcriptionally suppressed or even inactivated. Studying candidate genes for expression and somatic mutations could thus identify the TSGs. METHODS: Possible candidates CBFA2T3, TERF2 and TERF2IP, FBXL8 and LRRC29 and FANCA were studied for insertion and deletion mutations and for expression differences using quantitative RT-PCR in a panel of tumour cell lines and primary tumours with and without loss of 16q. RESULTS: None of the genes showed mutations or obvious expression differences. FANCA expression increased with tumour grade. CONCLUSION: Apparently, the underlying genetics at chromosome 16q are complex or the TSGs remain to be identified. Multiple mechanisms, such as mutations, promoter hypermethylation or haploinsufficiency, might lead to the inactivation of a TSG.

Different mechanisms of chromosome 16 loss of heterozygosity in well- versus poorly differentiated ductal breast cancer.

Loss of heterozygosity (LOH) at the long arm of chromosome 16 is a frequent genetic alteration in breast cancer. It can occur by physical loss of part of or the entire chromosomal arm, resulting in a decrease in copy number or loss followed by mitotic recombination. Comparative genomic hybridization (CGH) demonstrated that well-differentiated breast tumors showed significantly more physical loss of 16q than did poorly differentiated ones and that this difference was already discernable in the preinvasive stage. However, polymorphic markers detected no difference in the frequency of 16q LOH between invasive tumors of different histological grade. Here, by combining data on LOH (n=52), fluorescence in situ hybridization (n=18) with chromosome 16-specific probes, and CGH (n=34), we show a preference in well-differentiated grade I tumors for physical loss of chromosome arm 16q, whereas in poorly differentiated grade III tumors LOH is accompanied by mitotic recombination. This clarifies the discrepancies observed between CGH and LOH for 16q in breast cancer. These different somatic genetic mechanisms may reflect the presence of multiple tumor suppressor genes that are the target of LOH at chromosome arm 16q.

Infiltrating leukocytes confound the detection of E-cadherin promoter methylation in tumors.

Promoter hypermethylation is known to result in transcriptional downregulation of many genes including the CDH1 gene. In this study we set out to determine CDH1 promoter methylation in breast tumors with decreased or absent E-cadherin protein expression and without CDH1 gene mutations by methylation-specific PCR (MSP). Interestingly, some tumor samples with normal E-cadherin expression yielded a methylation-specific PCR product. We hypothesized that other cells than tumor cells contribute to these products. Since in normal breast tissue no CDH1 promoter methylation is detected, infiltrating leukocytes, often present in tumors, might account for these methylation-specific fragments. Indeed, a methylation-specific fragment is found in all twelve leukocyte samples tested. Furthermore, activated T-cells also yielded a methylation-specific fragment. Sequencing of these fragments reveals two distinct methylation profiles. Leukocytes have only partial methylation of some CpGs, while the tumor-associated methylation profile shows complete methylation of most CpGs. Therefore, to assess whether CDH1 methylation is tumor associated, sequencing of MSP products is a prerequisite. Here we show that out of six lobular tumors lacking E-cadherin protein expression, three have tumor-associated CDH1 promoter methylation while in three other tumors no methylation is detected.

Mutations in the HLA class II genes leading to loss of expression of HLA-DR and HLA-DQ in diffuse large B-cell lymphoma.

Loss of expression of human leukocyte antigen (HLA) class II molecules on tumor cells affects the onset and modulation of the immune response through lack of activation of CD4+ T lymphocytes. Previously, we showed that the frequent loss of expression of HLA class II in diffuse large B-cell lymphoma (DLBCL) of the testis and the central nervous system (CNS) is mainly due to homozygous deletions in the HLA region on chromosome band 6p21.3. A minority of cases showed hemizygous deletions or mitotic recombination, implying that mutation of the remaining copy of the class II genes might be involved. Here, we studied three DLBCLs with loss of HLA-DQ expression for mutations in the DQB1 and DQA1 genes and three tumors with loss of HLA-DR expression for mutations in the DRB1 and DRA genes. In one case, a point mutation in exon 2 of the DQB1 gene, leading to the formation of a stop codon, was detected at position 47. In a second case, a stop codon was found at position 11 due to a deletion of 19 bp in exon 1 of the DRA gene. No mutations were found in the promoter sequences of the DRA, DQA1 and DQB1 genes. We conclude that both homozygous deletions and hemizygous deletions or mitotic recombination with mutations of the remaining allele may lead to loss of expression of the HLA class II genes, which is comparable to the mechanisms affecting HLA class I expression in solid cancers.

Beta2-microglobulin aberrations in diffuse large B-cell lymphoma of the testis and the central nervous system.

Human leukocyte antigen (HLA) class I molecules are expressed on the surface of all nucleated cells and present antigenic peptides to cytotoxic T cells, thereby playing an important role in initiating the cellular anti-tumor immune response. We previously reported that loss of HLA class I expression in diffuse large B-cell lymphoma (DLBCL) of the central nervous system (CNS) and the testis is a common event. Loss of expression and mutations of the light chain of the HLA class I molecule, beta(2)-microglobulin (beta(2)m) have been described in a variety of human tumors and cell lines. In our study, we screened 15 DLBCL cases with a combined loss of HLA class I and beta(2)m expression for mutations in the latter gene by direct sequencing. Frame shift mutations in repetitive sequences within the beta(2)m gene leading to loss of functional beta(2)m were detected in 2 cases. Loss of heterozygosity (LOH) and fluorescent in situ hybridization (FISH) analysis for chromosome 15 exhibited loss of the remaining copy of the beta(2)m gene in both cases but also hemizygous deletions and monosomies in 6 additional cases. Since similar mutations in the beta(2)m gene have been associated with microsatellite instability (MSI), we used 8 markers to study MSI involvement in DLBCL. Low MSI was more frequent (33%) as compared to nodal DLBCL (n=15) but did not correlate with the beta(2)m mutations. Our data indicate that multiple mechanisms lead to downregulation of beta(2)m and concomitant loss of HLA class I expression in DLBCL.

Hemizygous deletions in the HLA region account for loss of heterozygosity in the majority of diffuse large B-cell lymphomas of the testis and the central nervous system.

Loss of heterozygosity (LOH) is a major mechanism for inactivation of tumor-suppressor genes and has been observed in various solid tumors and lymphomas. The human leukocyte antigen (HLA) region is located at chromosome band 6p21.3, and loss or alteration of this region may provide tumor cells with a mechanism to escape from the immune system. We previously identified small homozygous deletions within the HLA class II region in many of the diffuse large B-cell lymphomas (DLCLs) of the central nervous system (CNS) and the testis. In the present study, we focused on the mechanism leading to LOH in the HLA region. Twenty microsatellite markers, of which 12 were specific for HLA, were applied on 11 extranodal DLCLs of the CNS and 28 of the testis. Additionally, fluorescence in situ hybridization with seven HLA-specific probes and a centromere 6-specific probe was performed on 20 cases to study the mechanism of LOH. In contrast to previously published data on spontaneously mutated lymphoblastoid cell lines, intrachromosomal hemizygous deletion, not mitotic recombination, was the major cause of LOH of the HLA region in these lymphomas. However, opposed to data in colorectal cancer, these deletions were rarely (one of nine cases) associated with an interchromosomal rearrangement such as a translocation.