Mapping loss of heterozygosity at chromosome 13q: loss at 13q12-q13 is associated with breast tumour progression and poor prognosis

Several chromosome regions exhibit loss of heterozygosity (LOH) in human breast carcinoma and are thought to harbour tumour suppressor genes (TSG). At chromosome 13q, two TSGs have been identified, RB1 at 13q14 and BRCA2 at 13q12-q13. In this study, 139 sporadic breast tumours were analysed with 18 polymorphic microsatellite markers for detailed mapping of LOH at chromosome 13q and evaluation of an association with known progression factors. LOH with at least one marker was observed in 71 (51%) of the tumours analysed. The deletion mapping indicated three LOH target regions, 13q12-q13, 13q14 and 13q31-q34. LOH at chromosome 13q12-q13 was associated with low progesterone receptor content, a high S phase fraction and aneuploidy. Multivariate analysis adjusting for lymph node involvement and S phase fraction showed that patients with tumours exhibiting LOH at 13q12-q13 have a 3-4-fold increased risk of recurrence and death compared with other patients. Our results suggest there are at least three separate LOH target regions at chromosome 13q and inactivation of one or more genes at chromosome 13q12-q13 results in poor prognosis for breast cancer patients.     

Loss of heterozygosity in sporadic breast tumours at the BRCA2 locus on chromosome 13q12-q13.

Loss of heterozygosity (LOH) on chromosome 13 occurs on 25-30% of breast tumours. This may reflect the inactivation of the retinoblastoma susceptibility gene RB1. However, recently another candidate tumour-suppressor gene has been identified on chromosome 13 by linkage analysis, the breast cancer susceptibility gene BRCA2. To investigate the involvement of BRCA2 in sporadic breast cancer 200 breast tumours were tested for LOH on chromosome band 13q12-q14, using 11 highly polymorphic microsatellite markers. LOH was found in 65 tumours, which all showed simultaneously loss of BRCA2 and RB1. Of 12 breast tumour cell lines tested with polymorphic microsatellite markers, seven showed a contiguous region of homozygosity on 13q12-q14, suggesting LOH in the tumour from which the cell line had been derived. One cell line showed homozygosity in the BRCA2 region and heterozygosity at RB1. This is the only indication that BRCA2 is a distinct target for LOH on chromosome 13 in addition to RB1.     

Allelic imbalance at 11q23-q24 chromosome associated with estrogen and radiation-induced breast cancer progression

Multiple genetic alterations are common in cancers including those of the breast. The mechanisms leading to these alterations such as point mutations, gene amplifications, deletions and replication error are often associated with frequent and consistent loss of heterozygosity (LOH) or microsatellite instability (MSI). Several cytological and molecular studies have shown high frequency loss of genetic information on the long arm of chromosome 11 (i.e., 11q) in various primary breast cancers. In the present study allelic alterations in a refined position on the long arm of chromosome 11 were studied to identify the spectrum of induced damage at different stages of malignant transformation of MCF-10F cell lines after exposure to high-LET radiation using alpha-particles and exposure to estradiol by using PCR-single strand conformation polymorphism (SSCP) and fluorescence in situ hybridization (FISH) analysis. Microsatellite markers were selected from chromosome 11 (11q23-q24 loci) and it was found that frequency of allelic imbalance occurs at different stages of tumor progression with a range of 15-45% depending on the marker studied. These results strongly suggested the presence of several tumor suppressor genes in this critical region of chromosome 11 (11q23-q24). It also represents the first indication of allele loss at these loci in human breast epithelial cells induced by radiation and estrogen treatment suggesting a potential interventional target in breast carcinogenesis.     

Allelic loss on distal chromosome 17p is associated with poor prognosis in a group of Brazilian breast cancer patients.

We examined loss of heterozygosity (LOH) for two loci on chromosome 17p (D17S5 and TP53), and erbB-2 gene amplification, in primary breast cancers from 67 Brazilian patients. We identified two distinct regions of LOH on chromosome 17p, one spanning TP53 and the other a more telomeric region (D17S5). Based on a short-term follow-up, Kaplan-Meier analyses of patients'' disease-free survival showed that patients with LOH for D17S5, but retaining heterozygosity for TP53, were at higher risk of recurrence (P = 0.007) than those who retained heterozygosity for D17S5. Bivariate analyses indicated that patients with LOH for D17S5 alone had an increased risk of recurrence (hazard ratio = 7.2) over patients with erbB-2 amplification (hazard ratio = 3.7), when compared with patients with neither alteration (hazard ratio = 1.0). Further, lymph node-positive patients whose tumours had both LOH for D17S5 and erbB-2 gene amplification had a higher risk of recurrence than patients whose tumours had neither of these genetic alterations. Our data confirm previous reports of a putative tumour-suppressor gene, distinct from TP53, on distal chromosome 17p which is associated with breast cancer. They further suggest that LOH for loci in this region may provide an independent indicator to identify patients with poor prognosis.     

Allelic loss at 1p34-36 predicts poor prognosis in node-negative breast cancer.

Allelic losses of specific chromosomal regions in the DNA of tumor cells, which imply loss of tumor suppressor genes normally resident at those loci, may become useful postoperative prognostic indicators for breast cancers that have not yet metastasized to lymph nodes. To examine whether specific allelic losses might correlate with postoperative disease-free survival, we tested tumors from a cohort of 228 node-negative breast cancer patients for allelic losses at 18 microsatellite loci chosen to represent either a known tumor suppressor gene or a region where genetic alterations are frequent in breast tumors. We followed the patients clinically for 5 years or until death (if patient death occurred before completion of 5 years of follow-up). Patients whose tumors had lost an allele at 1p34-36 bore significantly higher risks of postoperative recurrence than those whose tumors retained both alleles of the markers in that region [the 5-year recurrence rate was 15% among patients with losses versus 2% among patients with retention (P = 0.001)]. Multivariate analysis demonstrated that allelic loss at 1p34-36 was an independent postoperative predictor of shorter disease-free survival (hazard ratio, 5.8; P = 0.0117). Thus, allelic losses at 1p34-36 in a tumor might have a potential to serve as a negative prognostic indicator to guide postoperative management of breast cancer patients, especially in the selection of high-risk women who will benefit from adjuvant chemotherapy and endocrine therapy.     

Amplification of genes within the chromosome 11q13 region is indicative of poor prognosis in patients with operable breast cancer.

Amplification of the chromosome 11q13 region, which harbors the BCL1 region and the PRAD1, EMS1, HSTF1, and INT2 genes, was found in 36 (16%) of a series of 226 breast carcinomas. In the 153 patients with stage I-IIIa disease who had received no therapy prior to surgery and who were treated with curative intent, 11q13 amplification was associated with the presence of lymph node metastases (P less than 0.002). The presence of an 11q13 amplification was associated with a significantly shorter relapse-free survival (P less than 0.002) and a higher breast cancer-specific mortality (P less than 0.003). Stepwise multivariate analysis showed that, in addition to lymph node status, 11q13 amplification was the best predictor for short survival. Stratified log-rank analysis indicated that, within the group of lymph node-positive breast cancer patients, 11q13 amplification identifies a subgroup at high risk.     

Arpin downregulation in breast cancer is associated with poor prognosis

Background:

The Arp2/3 complex is required for cell migration and invasion. The Arp2/3 complex and its activators, such as the WAVE complex, are deregulated in diverse cancers. Here we investigate the expression of Arpin, the Arp2/3 inhibitory protein that antagonises the WAVE complex.

Methods:

We used qRT–PCR and reverse phase protein arrays in a patient cohort with known clinical parameters and outcome, immunofluorescence in breast biopsy cryosections and breast cancer cell lines.

Results:

Arpin was downregulated at the mRNA and protein levels in mammary carcinoma cells. Arpin mRNA downregulation was associated with poor metastasis-free survival (MFS) on univariate analysis (P=0.022). High expression of the NCKAP1 gene that encodes a WAVE complex subunit was also associated with poor MFS on univariate analysis (P=0.0037) and was mutually exclusive with Arpin low. Arpin low or NCKAP1 high was an independent prognosis factor on multivariate analysis (P=0.0012) and was strongly associated with poor MFS (P=0.000064).

Conclusions:

Loss of the Arp2/3 inhibitory protein Arpin produces a similar poor outcome in breast cancer as high expression of the NCKAP1 subunit of the Arp2/3 activatory WAVE complex.     

Lymph node metastasis is associated with allelic loss on chromosome 13q12-13 in esophageal squamous cell carcinoma.

Allelotype analysis of whole chromosomes showed that loss of heterozygosity (LOH) on 13q was exclusively associated with lymph node metastasis and poor prognosis in esophageal squamous cell carcinoma (ESC). To identify a locus responsible for lymph node metastasis, we performed fine deletion mapping on 13q by analyzing 60 ESCs with 18 polymorphic markers. Allelic loss was observed with at least one marker in 34 tumors (56.7%), and lymph node metastasis was significantly correlated with LOH (P = 0.0053). We found frequent loss at D13S260 (43.7%), D13S171 (38.6%), and D13S267 (43.6%) on 13q12-13. Among these markers, LOH at D13S171 showed a significant correlation with lymph node metastasis (P = 0.0441). Because these markers flank the BRCA2 gene, we intensively examined a mutation of the gene through all coding exons and exon-intron junctions by PCR-single-strand conformational polymorphism analysis under two different assays. We found only seven nucleotide substitutions as normal polymorphic changes; tumor-specific mutations were not detected, and loss of expression was not observed, indicating that the BRCA2 gene might not be a target of allelic loss in this region. Relatively frequent LOH was also found at the RB1 locus (34.7%), but a significant correlation with lymph node metastasis was not observed (P = 0.7430). Protein expression of RB1 was examined in 31 ESC cell lines, and loss of expression was infrequent (6.5%), indicating that inactivation of the RB1 gene might not be responsible for lymph node metastasis. Taken together, allelic loss at 13q12-13 of the primary ESC was closely associated with lymph node metastasis, and unidentified tumor suppressor gene(s) in this region might be involved.     

Gain of distal chromosome arm 17q is not associated with poor prognosis in neuroblastoma.

PURPOSE: In several studies, gain of the distal long arm of chromosome 17 was shown to be a frequent and prognostically relevant factor in neuroblastoma, in addition to MYCN amplification (MNA) or 1p deletion. We asked whether this observation could be confirmed in a German cohort. EXPERIMENTAL DESIGN: To evaluate the frequency and prognostic impact of 17q gain, we investigated tissue samples from 193 neuroblastoma patients by the use of fluorescence in situ hybridization. The DNA probe (MPO) was located in distal 17q in the region of interest as used by several groups. To analyze the association of patients' outcome with the breakpoint position within 17q, we used the more proximal DNA probe ERBB2 in 17q21 on a selected number of cases. Gain was defined as an excess of 17q material compared with the chromosome 17 centromere in at least 50% of the analyzed tumor cells. In addition, alterations in chromosomes 1p, 3p, and 11q, as well as MYCN status, were determined to describe the interrelationship between the different parameters and to evaluate an independent prognostic influence. RESULTS: Gain of 17q was found in 61% of the investigated tumors. An additional 23% displayed an excess of 17q in less than half of all cells. Gain correlated with stage 4 disease (P = 0.003) and with other chromosomal alterations, such as 1p (P < 0.001), 3p (P = 0.01), 11q (P = 0.001), and MNA (P = 0.016), as well as with increased patient age (P = 0.01). Outcome was not different between patients with 17q gain compared with those without, however. A prognostic influence could not be delineated in all stages or in localized or in stage 4 subgroups or in the MYCN nonamplified patient cohort. Outcome did not differ between patients with additional 17q material in <10% of the cells or in >70%. Patients showing a breakpoint in the more proximal part of 17q could not be described as having a more favorable outcome compared with patients with a more distal breakpoint. Prognosis was poor in patients with MNA and/or 11q loss either with or without 17q gain. A multivariate analysis including the chromosomal parameters 17q, 11q, and MYCN status, as well as stage, showed MYCN and 11q status (P < 0.001), but not 17q or stage as significant prognostic factors. CONCLUSION: Although the most frequent aberration in neuroblastoma to date, we found no association between 17q gain and an inferior prognosis in our cohort; the status of MYCN and 11q, however, allowed reliable prediction of patients' outcomes.     

Three independently deleted regions at chromosome arm 16q in human prostate cancer: allelic loss at 16q24.1-q24.2 is associated with aggressive behaviour of the disease, recurrent growth, poor differentiation of the tumour and poor prognosis for the patient.

Loss of heterozygosity at chromosome arm 16q is a frequent event in human prostate cancer. In this study, loss of heterozygosity at 16q was studied in 44 prostate cancer patients exhibiting various clinical features. Fifteen polymorphic polymerase chain reaction (PCR) markers were used to identify the separately deleted areas and the findings were compared with clinicopathological variables and 5-year survival of the patients. The results indicated that there are at least three independently deleted regions at 16q. Allelic losses at the central and distal areas were associated significantly with aggressive behaviour of the disease (16q24.1-q24.2, P < 0.01, and 16q24.3-qter, P < 0.05), and the central area of deletion was further significantly associated with poorly differentiated tumour cells (P < 0.05) and with recurrent (P < 0.01) growth of the tumour. During the follow-up period, 28% of the patients initially with M0 disease developed distant metastases. Of the patients showing allelic loss at 16q24.1-q24.2, distant metastasis were found in 45% during the 5-year follow-up period, and 31% of the patients showing loss at 16q21.1 also developed distant metastases. After the 5-year follow-up period, 14 (32%) of the patients remained alive, whereas 19 (43%) had died because of their prostate cancer. The overall survival rate of the patients showing allelic loss at 16q21.1 or 16q24.1-q24.2 was significantly lower than that of the patients with retained heterozygosity.     

Allelic imbalance at chromosome 17p13.3 (YNZ22) in breast cancer is independent of p53 mutation or p53 overexpression and is associated with poor prognosis at medium-term follow-up.

Molecular and immunohistochemical studies of genetic events on chromosome 17p were prospectively compared with conventional clinical and pathological parameters and disease behaviour at a minimum of 72 months follow-up. In a series of 91 patients with primary operable breast cancer, 37 out of 91 (41%) patients had disease relapse and 23 out of 91 (25%) had died during the follow-up period. Allelic imbalance at the YNZ22 locus (17p13.3), demonstrated in 33 out of 63 (52%) informative patients, was significantly associated with disease recurrence (P < 0.01, 2 d.f. Cox analysis) and showed a trend towards impaired survival (P = 0.08, 2 d.f. Cox analysis) after a mean follow-up of 84 months for survivors. By contrast, p53 mutation (in 10 out of 60, 17% of cancers), p53 allelic imbalance (in 23 out of 56, 41% informative patients), p53 mRNA expression (in 47 out of 87, 54% patients), p53 mRNA overexpression (in 24 out of 87, 28%) or p53 protein expression (detected in 25/76, 32%) were not associated with disease behaviour. There was no significant association between allelic imbalance at YNZ22 and any abnormality of p53 DNA, RNA or protein. Allelic imbalance at 17p13.3 (YNZ22) serves as a marker of poor prognosis in breast cancer. As yet unidentified genes on 17p13.3, distinct from and telomeric to p53, are therefore likely to be of clinical importance in breast cancer.     

Vimentin expression is not associated with poor prognosis in breast cancer

The clinical significance of vimentin intermediate filament (VIF) expression was studied in relation to other established prognostic parameters in primary breast cancer. Archival tumour samples embedded in paraffin were examined by immunohistochemistry with monoclonal antibodies (MAbs) to VIF, p53 protein and cell proliferation marker MIB-1. The vimentin staining pattern was heterogeneous, but in vimentin-positive areas > 80% of the tumour cells were positive. There was no association between vimentin expression and tumour size or the number of axillary lymph nodes involved. Vimentin expression was significantly associated with high-grade tumours, absence of hormone receptors, increased p53 expression and high tumour proliferation fraction as estimated by MIB-1 count. Despite these associations with several recognised features of tumour aggressiveness, vimentin expression was not associated with increases in risk of relapse or death from breast cancer. © 1996 Wiley-Liss, Inc.     

A loss of c-kit expression is associated with an advanced stage and poor prognosis in breast cancer

To evaluate the c-kit expression in breast cancer, 217 invasive ductal carcinomas of the breast were immunohistochemically stained for c-kit protein. The c-kit expression was positive in 59 (27%) of 217 tumours, while the c-kit expression was negative in 158 (73%) of 217 tumours. There was a significant correlation between a negative expression of the c-kit protein and lymph node metastasis (P < 0.0001), and the incidence of a negative expression of the c-kit protein increased as the number of the metastatic lymph nodes increased (P = 0.0003). The c-kit expression did not significantly correlate with the tumour size, nuclear grade, oestrogen receptor status, MIB-1 counts and p53 protein expression. A univariate analysis indicated the patients with the negative c-kit expression to have a worse disease-free survival (DFS) than those with the positive c-kit expression (P = 0.0041), while a multivariate analysis determined lymph node metastases and the MIB-1 counts to be independently significant factors for DFS. In conclusion, a loss of the c-kit expression was found in about three-fourth of invasive ductal carcinoma of the breast and was associated with lymph node metastases. The prognostic implications of the c-kit expression seem to be due to fact that a loss of the c-kit expression is associated with an advanced stage of breast cancer.     

Frequent loss of heterozygosity on chromosome 17 at 17q11.2-q12 in Barrett's adenocarcinoma.

Allelic loss on chromosome 17 in 18 Barrett''s oesophageal tumours was analysed with 17 polymorphic microsatellite markers. Loss of heterozygosity (LOH) of one or more markers was seen in 72% (13 of 18) tumours on 17p and 56% (10 of 18) on 17q. The highest 17p losses were found at D17S799 (62%, five of eight) and D17S261 (55%, five of nine), while loss at the p53 locus was 31% (5 of 16). The highest loss on 17q was found at the TCF-2 (17q11.2-q12) locus with 66% (8 of 12) LOH. TCF-2 was the only marker lost in two of the tumour samples; furthermore, TCF-2 was lost in four other tumours which retained heterozygosity at the markers on either side of it, D17S261 and D17S740. Six markers were used to assess LOH at 17q11.2-q12, and five of eight of the tumour specimens which had LOH at TCF-2 had no other loss on 17q. No statistically significant correlations were found between loss on 17q or 17p and any clinicopathological parameters. We propose from these data that the 17q11.2-q12 region contains a novel predisposing gene in Barrett''s adenocarcinomas and may represent the site of a tumour-suppressor gene.     

Allelic loss of chromosome 13q14.3 in human oral cancer: Correlation with lymph node metastasis

To evaluate the role of chromosome 13 deletions in oral squamous cell carcinoma (SCC) progression and to define the precise localization of putative tumor suppressor genes, we studied tumors from 34 unrelated patients with oral SCC by the polymerase chain reaction (PCR)-loss of heterozygosity (LOH) assay, using 18 different polymorphic loci. Chromosome 13q allelic losses (LOH) were observed in 67.6% at 1 or more loci. These results enabled the identification of a putative minimal region of deletion mapped at 13q14.3. The commonly deleted region is located close, but telomeric to the RB1 locus. We also examined the same samples for inactivation of the RB1 gene by immunohistochemical analysis of paraffin-embedded samples, but no significant variation in RB protein expression was detected. In addition, we also performed PCR-single-strand conformational polymorphism (SSCP) analysis to detect any mutation of the RB1 gene using 52 primer pairs, which covers all exons of this gene. We found no mutations of the RB1 gene in our samples. Interestingly, we found significant correlation between LOH of 13q14.3 and lymph node metastasis. Our results indicate that LOH of 13q is a common event in oncogenesis and/or progression of oral SCC, and also suggest the existence of a new suppressor gene near D13S273-D13S176 loci which may play a role in these events. Int. J. Cancer (Pred. Oncol.) 79:312–317, 1998. © 1998 Wiley-Liss, Inc.     

Characterisation of amplification patterns and target genes at chromosome 11q13 in CCND1-amplified sporadic and familial breast tumours

Amplification of chromosomal region 11q13, containing the cell cycle regulatory gene CCND1, is frequently found in breast cancer and other malignancies. It is associated with the favourable oestrogen receptor (ER)-positive breast tumour phenotype, but also with poor prognosis and treatment failure. 11q13 spans almost 14 Mb and contains more than 200 genes and is affected by various patterns of copy number gains, suggesting complex mechanisms and selective pressure during tumour progression. In this study, we used 32 k tiling BAC array CGH to analyse 94 CCND1-amplified breast tumours from sporadic, hereditary, and familial breast cancers to fine map chromosome 11q13. A set containing 281 CCND1-non-amplified breast tumours was used for comparisons. We used gene expression data to further validate the functional effect of gene amplification. We identified six core regions covering 11q13.1-q14.1 that were amplified in different combinations. The major core contained CCND1, whereas two cores were found proximal of CCND1 and three distal. The majority of the CCND1-amplified tumours were ER-positive and classified as luminal B. Furthermore, we found that CCND1 amplification is associated with a more aggressive phenotype within histological grade 2 tumours and luminal A subtype tumours. Amplification was equally prevalent in familial and sporadic tumours, but strikingly rare in BRCA1- and BRCA2-mutated tumours. We conclude that 11q13 includes many potential target genes in addition to CCND1.     

Increased MTHFD2 expression is associated with poor prognosis in breast cancer

The aim of this study was to investigate the expression levels of methylenetetrahydrofolate dehydrogenase (NADP?+?-dependent) 2 (MTHFD2) and the associated clinical implications in breast cancer. MTHFD2 expression was measured by Western blot and immunohistochemistry in 698 tissue sections taken from breast cancer patients. The relationship between MTHFD2 expression, clinicopathological parameters, and the prognosis of breast cancer was subsequently determined. In comparison with para-carcinoma tissue specimens, an enhanced expression of MTHFD2 was observed in breast cancer tissue specimens (P?P?=?0.001, 0.002, 0.001, and 0.001, respectively). Furthermore, patients with MTHFD2-expressing tumors had a significantly poorer prognosis than those with no or low MTHFD2 expression. (P?=?0.002). Using the Cox regression test, MTHFD2 was identified as an independent prognostic factor (P?=?0.001). MTHFD2 was differentially expressed in breast cancer tissue. Therefore, this protein may be an independent prognostic factor and a potential therapeutic target for future breast cancer treatments.