p53 gene mRNA expression and chromosome 17p allele loss in breast cancer

p53 messenger RNA expression was examined using a cDNA probe in 76 fresh primary breast tumour specimens, 15 of which came from patients treated with toxoxifen prior to surgery. A 2.8 kb mRNA for p53 was expressed in 43 of the 76 specimens. In 19 tumours the levels were similar to those seen in non-malignant (reduction mammoplasty) breast tissue, but in 24 tumours over-expression of mRNA for p53, approaching that seen in three breast cancer cell lines, was found. The cell lines MCF-7, T-47D and MDA-MB-231 expressed three p53 mRNA species of about 2.8 kb and a forth of 1.6 kb. Increased mRNA expression for p53 correlated (P less than 0.05) with loss of genetic material from the short arm of chromosome 17 as demonstrated by allele loss with the VNTR probe YNZ 22.1. There was also statistically significant correlation between increased p53 mRNA expression and low oestrogen receptor protein content in the tumours (P less than 0.05), but not with other clinical parameters. The findings support the view that p53 is involved in breast tumour biology, and suggest that its role may be complex.     

Allelotype of breast cancer: cumulative allele losses promote tumor progression in primary breast cancer

Allele loss on a specific chromosome has implied the existence of a tumor suppressor gene such as the p53 gene and the RB gene. In order to determine which chromosome(s) carries a tumor suppressor gene(s) that contributes to tumor progression in primary breast cancer, we analyzed the loss of heterozygosity for each autosomal chromosome arm by using 39 restriction fragment length polymorphism markers including 25 variable numbers of tandem repeat probes. In 79 primary breast cancers, we found the frequent loss on the long arm of chromosome 13 (21%), the long arm of chromosome 16 (45%), and the short arm of chromosome 17 (56%). Interestingly, breast cancers in which loss of both chromosomes 13q and 17p was detected showed more malignant histopathological features, and a group of the tumors in which chromosome 16q loss was detected presented with frequent lymph node metastasis. Furthermore, the result of the deletion mapping on chromosome 17p implied the existence of a tumor suppressor gene distal to the p53 gene as well as the p53 gene itself for primary breast cancer. These results suggest that at least 4 tumor suppressor genes exist on chromosomes 13q, 16q, and 17p for primary breast cancer.     

Multiple regions of chromosome 4 demonstrating allelic losses in breast carcinomas.

Allelotyping studies suggest that allelic losses at one or both arms of chromosome 4 are frequent in several tumor types, but information about breast cancer is scant. A recent comparative genomic hybridization analysis revealed frequent losses of chromosome 4 in breast carcinomas. In an effort to more precisely locate the putative tumor suppressor gene(s) on chromosome 4 involved in the pathogenesis of breast carcinomas, we performed loss of heterozygosity studies using 19 polymorphic microsatellite markers. After precise microdissection of archival surgical cases, we analyzed DNA obtained from 44 breast carcinomas for loss of heterozygosity. In addition, DNA from tumor cell lines derived from 14 of these 44 breast carcinomas were also analyzed. We observed deletions of chromosome 4 at multiple sites in both tumor cell lines and breast carcinomas. The deletions in cell lines and their corresponding tumors were extensive in nature, whereas they were more localized in noncultured breast carcinomas. The localized deletions in the noncultured breast carcinomas clearly defined four nonoverlapping regions of frequent deletions: 4q33-34 (76%); 4q25-26 (63%); 4p15.1-15.3 (57%); and 4p16.3 (50%). Our results suggest that there may be multiple putative tumor suppressor genes, located on both arms of chromosome 4, whose inactivation is important in the pathogenesis of breast cancer.     

Deletion of three distinct regions on chromosome 13q in human non-small-cell lung cancer

We examined loss of heterozygosity (LOH) at the retinoblastoma susceptibility gene (RBI) locus on chromosome 13q14 in 20 non-small-cell lung cancers (NSCLCs) using polymorphic markers. The expression of RB protein was examined by immunohistochemical analysis of paraffin-embedded specimens of the same tumors. The results revealed that 10 of 16 informative cases showed an LOH at the RBI locus, whereas only 2 of the 10 tumors lost expression of the RB protein. These 2 tumors had mutations in the remaining RBI allele. Thus, inactivation of the RBI gene appears to be involved in a small subset of NSCLCs only. To elucidate the presence of tumor-suppressor genes other than RBI on 13q, heterozygosity at 15 different loci was investigated. Of 20 tumors analyzed, 15 showed an LOH at least at one locus, and the regions 13q12.1-qter, 13q12.2–14.2 and 13q14.1–q14.3, including the RBI locus, were deleted in significant numbers of the tumors. Our results suggest that, in addition to the RBI gene, abnormalities of other tumor-suppressor genes on chromosome 13q are involved in the development of human NSCLCs. Int. J. Cancer 74:45–49. © 1997 Wiley-Liss, Inc.     

Allele losses on chromosome 17 in human epithelial ovarian carcinoma

16 epithelial ovarian carcinomas (12 serous, 2 mucinous and 2 endometrioid) and 2 benign ovarian adenomas were studied using recombinant DNA technology to compare loci on chromosome 17 in germ-line and tumour DNA. Four polymorphic probes mapping to 17p and one mapping to 17q were used. There was a high frequency of allele loss on 17q/(77%) and a slightly lower frequency on 17p (69%). These findings probably indicate loss of tumour suppressor genes which are of fundamental importance in the genesis or progression of epithelial ovarian carcinomas.     

Evidence of chromosome regions and gene involvement in inflammatory breast cancer

Inflammatory breast cancer (IBC) is a rare but particularly aggressive form of primary breast cancer. In contrast to noninflammatory breast cancer (non IBC), the molecular alterations underlying IBC are poorly known. We postulated that the kind and frequency of these alterations might differ between IBC and non IBC and account for its particular aggressiveness. We investigated allelic losses associated with primary breast cancer (on chromosome arms 1p, 3p, 6p, 6q, 7q, 8p, 9p, 11p, 11q, 16q, 17p and 17q) by analyzing 71 microsatellite markers in 66 cases of IBC. Loss of heterozygosity (LOH) was frequent, with a mean fractional allelic loss (FAL) index of 52%. Relative to published data on non IBC, allelic loss was particularly frequent at 3p21-p14, 6p, 8p22, 11q, 13q14 and 17q21, suggesting the presence of genes that are markedly altered in IBC. In contrast, the DNA amplification levels of ERBB2, MYC and CCND1, as measured by real-time quantitative PCR, did not differ between IBC and non IBC.     

Genetic profiling of chromosome 1 in breast cancer: mapping of regions of gains and losses and identification of candidate genes on 1q

Chromosome 1 is involved in quantitative anomalies in 50-60% of breast tumours. However, the structure of these anomalies and the identity of the affected genes remain to be determined. To characterise these anomalies and define their consequences on gene expression, we undertook a study combining array-CGH analysis and expression profiling using specialised arrays. Array-CGH data showed that 1p was predominantly involved in losses and 1q almost exclusively in gains. Noticeably, high magnitude amplification was infrequent. In an attempt to fine map regions of copy number changes, we defined 19 shortest regions of overlap (SROs) for gains (one at 1p and 18 at 1q) and of 20 SROs for losses (all at 1p). These SROs, whose sizes ranged from 170 kb to 3.2 Mb, represented the smallest genomic intervals possible based on the resolution of our array. The elevated incidence of gains at 1q, added to the well-established concordance between DNA copy increase and augmented RNA expression, made us focus on gene expression changes at this chromosomal arm. To identify candidate oncogenes, we studied the RNA expression profiles of 307 genes located at 1q using a home-made built cDNA array. We identified 30 candidate genes showing significant overexpression correlated to copy number increase. In order to substantiate their involvement, RNA expression levels of these candidate genes were measured by quantitative (Q)-RT-PCR in a panel of 25 breast cancer cell lines previously typed by array-CGH. Q-PCR showed that 11 genes were significantly overexpressed in the presence of a genomic gain in these cell lines, and 20 overexpressed when compared to normal breast.     

Identification of two distinct regions of allelic imbalance on chromosome 18Q in metastatic prostate cancer

Like most cancers, prostate cancer (CaP) is believed to be the result of the accumulation of genetic alterations within cells. Previous studies have implicated numerous chromosomal regions with elevated rates of allelic imbalance (AI), using mostly primary CaPs with an unknown disease outcome. These regions of AI are proposed sites for tumor suppressor genes. One of the regions previously implicated as coding for at least one tumor suppressor gene is the long arm of chromosome 18 (18q). To confirm this observation, as well as to narrow the critical region for this putative tumor suppressor, we analyzed 32 metastatic CaP specimens for AI on chromosome 18q. Thirty-one of these 32 specimens (96.8%) exhibited AI at one or more loci on chromosome 18q. Our analysis using 17 polymorphic markers revealed statistically significant AI on chromosome 18q at 3 markers, D18S35, D18S64 and D18S461. Using these markers as a guide, we have been able to identify 2 distinct minimum regions of AI on 18q. The first region is between the genetic markers D18S1119 and D18S64. The second region lies more distal on the long arm of the chromosome and is between the genetic markers D18S848 and D18S58. To determine if 18q loss is a late event in the progression of CaP, we also examined prostatic intraepithelial neoplasia (PIN) and primary prostate tumors from 17 patients for AI with a subset of 18q markers. We found significantly higher AI in the metastatic samples. Our results are consistent with 18q losses occurring late in CaP progression.     

Microsatellite instability of D17S513 on chromosome 17 is associated with progression of breast cancer

On 21 patients with T1b-T3b tumours subjected to external radiotherapy and brachytherapy, the expression of P53 and glutathione S-transferase pi [GST-pi], immunohistochemically detected, the S-phase cells fraction (H-3-thymidine labeling index, TLI) and DNA content evaluated by image analysis were determined on biopsies before and after the first 10 Gy. P53 accumulation was reduced in 60% of P53-overexpressing tumours and not induced in P53-negative tumours, GST-pi was induced in about 40% of pretreatment GST-pi-negative cases, TLI was reduced in 70% of the cases regardless of pretreatment values, and DNA profiles remained unchanged in two-thirds of the cases. P53 accumulation was a predictor of 3-year relapse-free survival after radiotherapy, followed by GST-pi expression, whereas TLI did not influence prognosis.     

Molecular-cytogenetic analysis of fragmentation of chromosome 17 in the breast cancer cell line EFM-19

Because a previous study by conventional cytogenetics had revealed a nullisomy 17 in the breast cancer cell line EFM-19, we analysed that cell line by SKY-FISH and by FISH using different probes derived from chromosome 17. A bicolor FISH using a HER2-specific probe and a chromosome 17 centromeric probe showed five HER2 and six centromeric signals all appearing on different chromosomes A further bicolor FISH using a chromosome 17-specific painting probe and a HER2-specific probe revealed that the HER2 signals were always localized within chromosome 17 segments constituting part of structurally altered chromosomes as deduced from their G-banding. Further FISH analyses using single-locus probes of chromosome 17, i.e., for MDS, p53, SMS and RARA, showed that all five chromosome 17 painting segments contained material from the long arm but only two painting segments had additional material from the short arm. A SKY-FISH confirmed the results of the chromosome 17 painting by FISH, except for one structurally altered chromosome showing additional chromosome 17 material detected by the SKY experiment. These results allow us to conclude that, in this cell line, polysomy 17 has preceeded the fragmentation of chromosome 17 leading to amplification of small parts of that chromosome as well as to extended losses. As to a general mechanism, polysomy 17 and a fragility of this, chromosome in breast cancer cells may not only account for part of the cases with HER2 amplification but, at the same time, may further support malignant progression due to the loss of tumor suppressor genes as e.g. p53.     

Duplication of two distinct regions on chromosome 5Q in non-papillary renal-cell carcinomas

RFLP studies have indicated a duplication of DNA sequences at the chromosome 5q22 region and showed a breakpoint cluster between the apc and mcc genes in non-papillary renal-cell carcinoma (RCC). We have now made a high-density fluorescent microsatellite assay to investigate the allelic status and determine the smallest duplication at this region in 62 sporadic non-papillary RCCs. Duplication at each informative locus was found in 27 cases. Partial duplications in 3 tumors delineated 2 distinct regions. One was found at loci D5S659, D5S1720 and w2005 at chromosome band 5q22; this region partially overlaps with the smallest duplicated region found by previous RFLP analysis. Another small duplication was marked by loci D5S816 and D5S476 at chromosome band 5q31.1 and included the α-catenin gene (ctnna1). Int. J. Cancer 76:337–340, 1998.© 1998 Wiley-Liss, Inc.     

Frequent allelic losses on chromosome 13q in human male breast carcinomas

Loss of genetic material on chromosomes 13q and 17 has been suggested to be of importance in the initiation and progression of female breast cancer, but their involvement is less well illustrated in male breast carcinomas. The present study was designed to investigate the incidence of allelic loss and microsatellite instability for chromosomes 13q, 17p and 17q in 13 sporadic male breast carcinomas using matched normal-tumour DNA samples and seven polymorphic microsatellite markers. Genetic imbalance was found in one or more informative markers in 85% of the patients, with more frequent loss of heterozygosity and microsatellite instability at loci on chromosome 13q. Thus, a high incidence of allelic losses was observed at the retinoblastoma gene (4/6) and likewise at the D13S263 locus (7/12), which also exhibited the highest frequency of microsatellite instability. The intragenic microsatellite in intron 1 of the TP53 gene on chromosome 17p revealed loss of heterozygosity in 3 of 8 informative patients. The investigated proximal region of chromosome 13q is postulated to harbour several potential tumour suppressor genes associated with female breast cancer. The high incidence of allelic losses at the D13S263 microsatellite, located distal to both the BRCA2 and the Brush-1 loci but proximal to the retinoblastoma gene, possibly indicates the presence of an additional tumour suppressor gene which may be involved in male breast carcinomas. However, this hypothesis needs verification in an extended study of male breast carcinomas.     

HER-2, TOP2A and chromosome 17 alterations in breast cancer

HER-2 amplification is a biomarker for identifying patients who respond to trastuzumab and has been evaluated as a factor predicting the response to anthracyclines. The relationship between HER-2 and response to anthracycline therapy may also be the result of the close localization of TOP2A on 17q. It has been a matter of debate whether these two genes, HER-2 and TOP2A, behave separately on different amplicons or act together thus making it possible to predict the TOP2A status from the HER-2 status. In this study TOP2A, HER-2 and chromosome 17 aneusomy were investigated by fluorescent in situ hybridization (FISH) in 50 consecutive breast cancer patients. HER-2 amplification was detected in 11 patients (22%) and TOP2A changes were seen in 6 patients (12%); two amplifications and two deletions were observed in HER-2-amplified cases and two deletions in HER-2-nonamplified cases. Three of the TOP2A-deleted cases had polysomy 17. HER-2 copy number was higher than the TOP2A copy number in one patient with co-amplification. Polysomy was observed in 9 cases (18%) and monosomy in 6 cases (12%). Aneusomy was the sole anomaly in 11 patients (22%). We conclude that the TOP2A status cannot be predicted from the HER-2 status and evaluation of the TOP2A status only in patients with HER-2 overexpression may lead to missing cases with TOP2A deletion with possible resistance to therapy. Other factors modulating topo IIα activity may also affect the response to therapy. Studies evaluating different parameters that can modulate topo IIα activity and the response to the drugs targeting the enzyme are necessary.     

Association of genetic alterations on chromosome 17 and loss of hormone receptors in breast cancer.

To investigate possible relationships between genetic alterations and hormonal deregulation during breast cancer development and/or progression, we examined 616 primary breast cancers for loss of heterozygosity (LOH) at chromosomal regions 16q24, 17p13.3 and 17q21, and for amplifications of the ERBB2 and c-MYC loci. A comparison of oestrogen receptor (ER) and progesterone receptor (PgR) status in tumour cells with data concerning these genetic alterations revealed that LOH at 17q21 was significantly correlated with absence of oestrogen receptors (ER) (P < 0.0003) or progesterone receptors (PgR) (P < 0.0001), and with the absence of both (P < 0.0001). Similarly, a significant association was observed between amplification of ERBB2 and the absence of either ER or PgR. LOH at 17p13.3 was associated with the absence of PgR (P < 0.01). These data suggest a possible relationship between specific genetic changes on chromosome 17 and hormonal deregulation in the progression of breast cancer.     

Identification of distinct regions of allelic loss on chromosome 13q in nasopharyngeal cancer from paraffin embedded tissues.

Our main purpose was to identify tumor suppressor gene loci on chromosome 13 responsible for nasopharyngeal cancer (NPC) development by analyzing loss of heterozygosity (LOH) and RB protein expression in paraffin embedded tissues. Normal and tumor DNA were extracted from microdissected samples, and their whole genomes were amplified using degenerate oligonucleotide primers. The polymerase chain reaction (PCR) products were analyzed by repeated amplification using primers derived from 16 microsatellite regions spanning the long arm of this chromosome. Among 50 informative cases, LOH was observed in 44 tumors. Thirty-one tumors displayed partial loss and provided an informative basis for detailed deletion mapping. Three minimal regions of loss were delineated; the first flanked by D13S120 and D13S219, the second by D13S126 and D13S119, and the third by D13S137 and 13qter. These 3 regions were linked to BRCA2 on 13q12, RB1 on 13q14, and 13q14.3-ter, respectively. Seven and 4 cases showed LOH either on 13q12 or 13q14, respectively. Nineteen cases showed LOH of both loci separately. One NPC displayed 13q12 and 13q14.3-ter LOH. RB protein expression was detectable in 76% of the cases. Ten out of 15 cases with the allelic losses limited to 13q14 showed RB protein expression. Contrasting that, 6 out of 7 cases devoid of RB protein expressions showed 13q14LOH. In conclusion, 13qLOH, involving 3 tumor suppressor gene loci, appears to be a frequent genetic event occurring during NPC development. However, other tumor suppressor genes besides RB1, may be responsible for the majority of 13q14LOH.     

Monosomy of chromosome 17 in breast cancer during interpretation of HER2 gene amplification

Monosomy of chromosome 17 may affect the assessment of HER2 amplification. Notably, the prevalence ranges from 1% up to 49% due to lack of consensus in recognition. We sought to investigate the impact of monosomy of chromosome 17 to interpretation of HER2 gene status. 201 breast carcinoma were reviewed for HER2 gene amplification and chromosome 17 status. FISH analysis was performed by using double probes (LSI/CEP). Absolute gene copy number was also scored per each probe. HER2 FISH test was repeated on serial tissue sections, ranging in thickness from 3 to 20 µm. Ratio was scored and subsequently corrected by monosomy after gold control test using the aCGH method to overcome false interpretation due to artefactual nuclear truncation. HER2 immunotests was performed on all cases. 26/201 cases were amplified (13%). Single signals per CEP17 were revealed in 7/201 (3.5%) cases. Five out of 7 cases appeared monosomic with aCGH (overall, 5/201, 2.5%) and evidenced single signals in >60% of nuclei after second-look on FISH when matching both techniques. Among 5, one case showed amplification with a pattern 7/1 (HER2/CEP17>2) of copies (3+ at immunotest); three cases revealed single signals per both probes (LSI/CEP=1) and one case revealed a 3:1 ratio; all last 4 cases showed 0/1+ immunoscore. We concluded that: 1) monosomy of chromosome 17 may be observed in 2.5% of breast carcinoma; 2) monosomy of chromosome 17 due to biological reasons rather than nuclear truncation was observed when using the cut-off of 60% of nuclei harboring single signals; 3) the skewing of the ratio due to single centromeric 17 probe may lead to false positive evaluation; 4) breast carcinomas showing a 3:1 ratio (HER2/CEP17) usually show negative 0/1+ immunoscore and <6 gene copy number at FISH.     

p53 allele losses, mutations and expression in breast cancer and their relationship to clinico-pathological parameters.

The p53 locus on the short arm of chromosome 17 at 17p 13.1 was examined for loss of heterozygosity, mutation, mRNA and protein expression in 60 primary breast cancers. Allele loss around the p53 locus was detected in 19/45 informative tumours (42%). p53 mutations in the evolutionarily conserved exons 5 to 9 were detected in 17/60 (28%) by amplification mismatch and confirmed by direct DNA sequencing. p53 mRNA expression was detected by Northern blot in 36/59 (61%) of tumours, and p53 protein expression using antibody 1801 on frozen-tissue sections in 13/44 of the tumours examined. p53 mutation was significantly associated with oestrogen-receptor-poor tumours (p less than 0.01) and hence with poor prognosis, but not with other clinical or pathological parameters. There was no statistical correlation between loss of heterozygosity around the p53 locus at 17p13.1 and p53 mutation. Furthermore, p53 mutation was not associated with p53 expression detected by immunohistochemical staining with antibody 1801 or as p53 mRNA. In addition, events on 17p (allele losses, p53 mutation, p53 expression) were independent of c-erbB-2 expression. In breast cancer, by contrast with colorectal, lung and ovarian cancer, there appears to be no clear association between p53 DNA abnormalities and p53 expression.     

Allelic losses at chromosome 17p in human renal cell carcinoma are inversely related to allelic losses at chromosome 3p.

Recent studies have demonstrated that allelic losses at chromosome 17p are associated with the genesis of a wide variety of human cancers. In order to assess whether the rearrangement of chromosome 17p was responsible for the genesis of renal cell carcinoma (RCC), we used restriction fragment length polymorphism analysis of chromosome 17p. We studied 48 RCCs, including 6 metastatic RCCs, from 43 patients with 5 polymorphic probes to loci within or near the p53 gene. Allelic losses at chromosome 17p were detected in only 6 of the 36 informative cases (17%), and no definitive correlation was demonstrated between allelic losses at 17p and the tumor stages. The 6 RCCs with allelic losses at 17p were histopathologically classified as a clear cell type in one, a mixed cell type in one, and granular cell types in the other four cases. Allelic losses at 17p in the clear cell type of RCC were infrequent (6%, 1 of 18), and were not detected even in the metastatic tumor from a highly advanced case. This finding suggests that allelic losses at 17p could be random genetic rearrangements in the case of the clear cell type of RCC. On the other hand, allelic losses at 17p in the granular cell type of RCC were demonstrated with a significantly higher frequency (44%, 4 of 9). We previously reported that allelic losses at 3p were specific to the clear cell type of RCC (Ogawa et al., Cancer Res., 51:949-953, 1991). Examination of the association of allelic losses at 17p with those at 3p revealed that none of 5 informative RCCs with allelic losses at 17p showed allelic losses at 3p. Conversely, 17 of 25 informative RCCs with retention of 17p alleles lost alleles at 3p. Thus, an inverse relationship was demonstrated with statistical significance (P less than 0.01). These data suggest that the types of rearrangement on chromosome 17p and/or chromosome 3p can differentiate between the histopathological subtypes of RCC.