Loss of BRCA2 Correlates with Reduced Long-Term Survival of Sporadic Breast Cancer Patients

ANTICANCER RESEARCH 24: 281-290 (2004)

Loss of BRCA2 Correlates with Reduced Long-term Survival of Sporadic Breast Cancer Patients

J.A. HAMPL1*, M. HAMPL2*, G. REIß3, R. KOCH4, H.-D. SAEGER5 and H.K. SCHACKERT6

1Department of Neurosurgery, University of Cologne, 50931 Cologne; 2Department of Obstetrics and Gynecology, Heinrich Heine University of Düsseldorf, 40225 Düsseldorf; 3Department of Neurosurgery, 4Institute for Medical Informatics and Biometrics, 5Department of General/Thoracic and Vascular Surgery and 6Department of Surgical Research, Technical University of Dresden,01307 Dresden, Germany

Abstract. Background: The present study was undertaken to BRCA2 locus may be of use as a negative predictor for analyze the prognostic value of loss of heterozygosity (LOH) at metastases-free and overall survival. 13q12-13, 17q21 and 17p13, harboring BRCA2, BRCA1 and p53 to predict the clinical course of sporadic breast cancer Breast cancer is a complex disease in which numerous patients. Materials and Methods: LOH analysis was performed genetic alterations occur and accumulate. The clinical by PCR amplification of genomic DNA using nine course of breast cancer patients after first diagnosis is microsatellite markers. Fifty-three sporadic breast cancer individually variable, ranging from complete disease-free patients were followed clinically for a median of 55 months. survival for decades to rapid progressive disease within Disease-free and overall survival was documented as the months and short overall survival. These differences in endpoint for statistical evaluation. Results: Patients presenting clinical outcome may be explained by differences in the with LOH in their tumor samples at at least one of the loci pattern of genetic alterations acquired during lifetime and examined were found to have a reduced overall survival time being causative for breast carcinogenesis. compared to those retaining heterozygosity (61% versus 48%). Multiple treatment regimens have been evaluated and Focusing on the three target regions, patients with LOH at the established as postoperative adjuvant treatment for breast BRCA2 locus died earlier compared to patients retaining cancer patients and numerous new regimens and clinical heterozygosity (69% versus 50%) and, in addition, BRCA2 protocols are currently under investigation. In order to LOH-positive patients showed a shorter metastasis-free interval choose the best possible treatment schedule for each (30 versus 37 months). In a multivariate analysis, LOH at the individual patient, prediction of postoperative prognosis and 13q12-13 locus was found to be a significant predictor for clinical course on the basis of reliable prognostic markers is reduced long-term survival (risk ratio 2.33, 95% C.I., 1.0-5.3; of increasing importance. Reliable prognostic markers may p<0.05) and earlier metastases manifestation (risk ratio 2.32, assist the clinician in identifying patients who are at risk of 95% C.I., 1.0-5.3, p<0.05). Conclusion: Allelic loss at the relapsing early after surgery and who may benefit from a more aggressive course of adjuvant treatment. On the other hand, identifying low risk patients will help to avoid unnecessary, inconvenient and side-effect-rich adjuvant *These two authors contributed equally to the work. treatment. So far, clinicians have based their decision Abbreviations: LOH, loss of heterozygosity; PCR, polymerase chain regarding treatment regimen for individual patients on reaction; CI, confidence interval; TSG, tumor suppressor gene. traditional tumor characteristics like tumor size, grading, lymph node status, hormone receptor status and metastases. Correspondence to: J.A. Hampl, M.D., Department of Neurosurgery, Among the various types of genetic alterations involved University of Cologne, Joseph-Stelzmannstr. 9, 50931 Cologne, in the development and progression of breast cancer, allelic Germany. Tel: ++-49-221-478 4577, Fax: ++-49-221-478 7993, e- loss of a particular chromosomal region may indicate the mail: [email protected] existence of a tumor suppressor gene which has been Key Words: BRCA1, BRCA2, p53, LOH analysis, long-term deleted in this tumor (1, 2), thus playing a putative role in survival, prognostic marker, sporadic breast cancer. tumorigenesis. LOH in specific tumor suppressor gene

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Table I. Definition of a score system, summarizing age, tumor stage and cancer, were obtained from 53 patients treated at the University hormone receptor status to facilitate statistical analysis. Hospital of Dresden, Germany, between 1985 and 1998. Data on histological type, tumor grading, tumor stage (including tumor size, Variable Points lymph node status, distant metastases, TNM), estrogen and progesterone receptors and family history were obtained from Age > 50 y 1 patients or patient’s charts. Informed consent in the formal style < 50 y 2 approved by the Ethics Committee of the Hospital was obtained from Tumor staging (TNM) Stage I 2 all patients. To facilitate statistical evaluation of the clinical data, an Stage II 4 additional score (Table I) was defined for each individual case, where Stage III 6 age, staging and receptor status were summarized to a prognostic Stage IV 8 score, ranging from 4 (favorable prognosis) to 12 (worst case). Receptor ER/PR neg/neg 2 pos/neg 1 At the end of the evaluation period (May 2001), data on time to pos/pos 1 progression (diagnosis of recurrence or metastases) and overall not available 2 survival (time to death) were obtained for all patients either by contacting the primary care physicians or follow-up of the patients Sum of points = final score ™ in the breast cancer outpatient clinic at the Department of Surgery, (range 4-12) University Hospital of Dresden. The mean follow-up time of the 53 patients was 55 months (range 6-180 months). The mean age at diagnosis was 53.5 years (range 33-87 years). Four breast tumors were classified as stage I (8%), 34 tumors stage II (64%), 10 tumors stage III (19%) and 5 tumors stage IV (9%). Fourteen patients presented with negative lymph nodes (26%), 31 patients with lymph regions may therefore be useful as a new prognostic marker node status I (58%), 4 with lymph node status II and 4 with lymph in breast cancer therapy. LOH is a common event in node status III (8% each). Forty-seven tumors (88%) were sporadic breast cancers. Multiple studies have been diagnosed as invasive ductal breast carcinomas; 3 tumors (9%) were performed to identify regions that are commonly deleted in classified as grade I, 11 (32%) as grade II and 20 (59%) as grade breast tumors and have shown the correlation between III. Twenty tumors were estrogen receptor (ER)-positive (47%), allelic loss at certain regions with certain clinicopathological while 18 tumors were progesterone receptor (PR)-positive (43%). Patients underwent different adjuvant hormone, radiation and markers or poor outcome and prognosis of patients. We and chemotherapy regimens according to standard treatment guidelines. others have studied the prognostic value of LOH in the chromosomal regions 13q12-13, 17q21 and 17p13 where the DNA extraction. Immediately after surgery, microscopically breast cancer susceptibility genes BRCA2, BRCA1 and p53 identified tumor tissue was snap-frozen in liquid nitrogen and stored are located (3-22). We have previously found that, in a at -80ÆC (n=43). Histopathological diagnosis was determined by a subgroup of patients with brain metastases, accumulation of pathologist according to the WHO criteria. Corresponding normal genetic alterations in these regions is associated with tissue or peripheral blood samples were obtained from each patient. reduced survival after occurrence of distant metastasis (23) When no fresh material was available, paraffin-embedded tumor and that LOH accumulation in primary tumors and material (n=10) was used for DNA extraction, performing microdissection. Extraction of genomic DNA from fresh or paraffin- additionally in the corresponding distant metastases is embedded tumor samples and peripheral blood leukocytes was associated with poor outcome (24). performed using the QIAamp blood and tissue kit (QIAGEN, Because of the interactive role of inactivation of BRCA1, Valencia, CA, USA) according to the manufacturer’s instructions. BRCA2 and p53 in breast carcinogenesis, we hypothesized that single or combined allelic loss of the regions harboring Microsatellite analysis. DNA samples from 53 tumors and these breast cancer susceptibility genes might be useful as corresponding normal tissues were analyzed for genetic alterations prognostic factors to predict the clinical course of breast in 3 different gene loci (harboring BRCA1, BRCA2 and p53) using 9 microsatellite markers selected on the basis of reported cancer patients after first diagnosis. We examined sporadic, heterozygosity and located in or tightly linked to the breast cancer primary breast carcinomas for LOH in 13q12-13, 17q21 and genes. Oligonucleotide primer sequences were retrieved from 17p13 using nine polymorphic microsatellite markers and Genome Data Base (GDB). The following microsatellite markers attempted to correlate the LOH data with the overall were used: D17S855, D17S1322, D17S1323, D17S1327 for the survival and/or time to progression in a long-term (up to 13 17q21 chromosomal region, D13S289, D13S260, D13S171, D13S267 years) follow-up study of 53 patients. for 13q12-13 and the marker TP53 for 17p13.1. PCR amplifications were performed with 50 ng genomic DNA in a reaction containing 2.5 Ìl of 10 x PCR buffer (Perkin-Elmer Cetus, Foster City, CA, Materials and Methods USA), 2.0 mM MgCl2, 200 ÌM dNTP, 0.1 ÌM primer each and 1.5 units of Taq polymerase (Perkin-Elmer Cetus) in a final volume of Patients and tumor samples. Fresh (n=43) or paraffin-embedded 25 Ìl. Target DNA was amplified in a Gene Amp 9600 tumor samples (n=10), including corresponding normal tissue (blood thermocycler (Perkin-Elmer Cetus) with an initial denaturation or normal breast tissue) of patients with primary, sporadic breast step (94ÆC for 5 min) and a total of 27 cycles of denaturing (94ÆC

282 Hampl et al: LOH of BRCA2 and Long-term Survival

Table II. Clinicopathological parameters and LOH data of 53 patients with primary sporadic breast cancer

Patient Age a Histology b Stage c Receptor d Score e 17q21 13q12-13 17p13 (years) (ER/PR) BRCA1 BRCA2 P53

MT 7 67 inv.lob. 3 pos/neg 8 LOH het n.i. MT 13 57 inv.duct. 2 n.a. 7 het het het MT 33 50 inv.duct. 2 n.a. 7 het n.i. LOH MT 41 55 inv.duct. 3 neg/neg 9 het het het MT 48 50 inv.duct. 4 neg/neg 12 LOH het het MT 54 41 inv.duct. 2 neg/pos 7 LOH LOH LOH MT 65 40 inv.duct. 4 neg/neg 12 het het het MT 93 45 inv.duct. 3 neg/neg 11 LOH n.i. LOH MT 100 55 inv.duct. 2 neg/neg 7 het LOH LOH MT 112 55 inv.duct. 2 pos/pos 6 het LOH het MT 117 50 inv.duct./inv.lob. 1 neg/pos 4 n.i. het LOH MT 119 46 inv.duct. 2 neg/pos 7 het LOH het MT 122 71 inv.duct. 4 pos/neg 10 het het het MT 124 66 inv.duct. 2 neg/neg 7 LOH LOH het MT 126 87 inv.duct./multifoc. 3 neg/pos 8 het LOH het MT 139 50 inv.duct. 2 pos/pos 6 het het het MT 143 79 inv.duct./inv.lob. 1 n.a. 4 n.i. LOH het MT 146 86 inv.duct. 2 neg/neg 7 het het het MT 147 46 inv.duct. 2 n.a. 8 het het het MT150 60 inv.duct. 3 n.a. 9 het het het MT 152 72 inv.duct. 2 pos/pos 6 het het het MT 157 52 inv.duct. 2 pos/pos 6 het het n.i. MT 181 59 inv.duct. 2 pos/pos 6 n.i. het het MT 189 45 inv.duct. 2 neg/neg 8 het het het MT 199 56 inv.duct. 2 pos/neg 6 het LOH het MT 207 55 inv.duct. 2 pos/neg 6 het het LOH MT 208 42 inv.duct./lymph. 2 pos/pos 7 het het het MT 215 50 inv.lob./multifoc./lymph. 3 neg/neg 9 LOH het n.i. MT 265 33 inv.duct. 2 n.a. 8 het het LOH MT 286 46 inv.duct. 3 neg/pos 9 LOH het LOH MT 289 33 inv.duct./lymph. 2 neg/neg 8 LOH LOH LOH MT 302 75 inv.duct. 2 pos/pos 6 het LOH het MT 307 64 inv.duct. 2 neg/pos 6 het LOH LOH MT 308 58 inv.duct. 2 pos/neg 6 het het het MT 325 53 inv.duct. 1 pos/pos 4 het het het MT 330 54 inv.duct. 2 n.a. 7 het het het MT 333 69 intraductal 2 n.a. 7 n.i. het LOH MT 336 46 inv.duct./multifoc. 3 neg/neg 10 het LOH het MT 342 49 inv.duct. 3 pos/pos 9 het het het MT 427 70 inv.duct. 2 pos/pos 6 het het LOH MT 482 50 inv.duct. 2 pos/neg 6 het het het MT 548 46 inv.duct. 2 n.a. 8 het het het MT 581 45 inv.duct. 3 neg/neg 10 het het het MT 620 50 inv.duct. 4 pos/pos 10 het LOH het MT 629 47 inv.duct. 2 neg/neg 8 LOH het het MT 660 43 inv.duct. 1 pos/neg 5 het het het MT 677 36 inv.duct. 2 n.a. 8 het het LOH MT 692 54 inv.duct. 4 pos/neg 10 LOH het het MT 832 46 inv.duct. 2 neg/neg 8 LOH het LOH MT 976 44 inv.duct/inv.lob. 2 neg/neg 8 het het het MT 1137 41 inv.duct 2 pos/pos 7 het het n.i. MT1401 50 inv.duct 2 neg/neg 7 n.i. het het MT 1702 46 inv.duct. 2 neg/neg 8 n.i. het het a Age of first diagnosis of breast cancer; b Histology of breast carcinomas: inv. duct.=invasive ductal carcinoma; inv. lob.=invasive lobular carcinoma, multifoc.=multifocal, lymph.=lymphangiosis carcinomatosa. c primary breast tumors were staged at the time of first surgery according to TNM classification. d Hormone receptor status: listed are the estrogen/progesterone receptors, pos=positive, neg=negative, n.a.=not available. e The score system is defined in Table I. LOH=loss of heterozygosity, het=heterozygous, n.i.=not informative

283 ANTICANCER RESEARCH 24: 281-290 (2004)

Figure 2. Overall survival as a function of 13q12-13 LOH in 53 patients with sporadic breast cancer and a median observation time of 55 months. Figure 1. Illustration of LOH. Example of a computer printout of a polyacrylamide gel analysis of PCR products by an automated DNA Sequencer. Shown are PCR products from a selected breast tumor (second row) and corresponding blood samples (first row), amplified with microsatellite primers. The microsatellite alleles are represented by two Results (heterozygote) peaks, LOH at one allele (arrow) corresponds to a significant reduction in one of the two peaks (imbalance factor I.F. ≥ 1.5). Calculation Clinical data. Sporadic breast tumors were screened for of allelic loss was determined semi-quantitatively using Fragment Manager LOH at 13q12-13, 17q21 and 17p13 using 9 polymorphic software (Pharmacia Biotech) as described previously (25). microsatellite markers. In addition to the conventional clinicopathological parameters for statistical evaluation, we defined a prognostic score for each individual summarizing the individual data of age, tumor stage and receptor status for 1 min), annealing (54-65ÆC for 1 min, depending on primers) (Table I). Forty patients presented with a score ≤ 8, 13 and extension (72ÆC for 1 min), followed by 10 min at 72ÆC (final patients with a prognostic unfavorable score of ≥ 8. The extension). One primer of each pair of PCR primers was Cy5- clinicopathological data of patients are summarized in labeled. Table II. A total of 53 female patients were analyzed, Gel electrophoresis of amplified PCR products was performed focusing on metastasis-free interval and survival within the on denaturing Long Ranger gels and detected on an automated observation time. The average time of follow-up was 55 laser fluorescence (ALF) sequencer (Pharmacia Biotech, Sweden). months, ranging from 6 up to 180 months. From the 53 In heterozygous cases, two peaks were obtained according to the cases, 30 patients had died at the end of the study, 23 were different migration patterns of the two alleles. Calculation of allelic loss was determined semi-quantitatively using Fragment Manager still alive. The average metastasis-free interval was 35 software (Pharmacia Biotech) as described previously (25). months, ranging from 0 to 121 months. Microsatellite markers were informative for heterozygosity or LOH, whereas homozygosity or microsatellite instability was Incidence of allelic losses in primary breast tumors. The LOH considered non-informative. frequencies at the three chromosomal regions in 53 primary breast tumors are listed in Table II and an illustrative Statistical methods. The main outcomes of this study were overall sample for LOH is shown in Figure 1. LOH was detected in survival and time to progression/disease-free interval after diagnosis of breast cancer. The overall survival was calculated from 11 out of 47 (23%) informative cases at the 17q21 locus, 13 the time of first diagnosis of breast cancer to death or the date of out of 51 patients (26%) at 13q12-13 and 14 out of 49 cases last follow-up. Disease-free survival was calculated from the date (29%) at the TP53 locus on 17p13. LOH rates of paraffin- of surgery until relapse or the last date of our follow-up where embedded and fresh tumor material were comparable in the patients were known to be disease-free. Survival distributions were BRCA loci, but higher in p53 (for 17q21 20% versus 24%, generated according to the Kaplan-Meier method and the for 13q12-13 22% versus 26%, whereas for p53 50% versus significance of differences in survival rate was tested by the log- 23%). Loss of at least one of the three chromosomal regions rank test for censored survival data. Cox proportional-hazard model for the risk ratio was used to assess the simultaneous was observed in 28 out of 53 informative cases (53%), contribution of each covariate in the multivariate analysis. P values whereas 15% of our patients presented with loss of two loci. <0.05 were considered statistically significant. Only two patients had lost all 3 regions (4 %). Combined

284 Hampl et al: LOH of BRCA2 and Long-term Survival

Although statistically not significant, early recurrence was found to be more common in patients with tumors exhibiting allelic loss at the 13q12-13 region than in patients without LOH (Figure 3). The metastasis-free interval was 30 months in patients with LOH at the BRCA2 locus compared to 37 months for patients retaining heterozygosity. Time to progression and LOH at the BRCA1 locus and p53 locus was statistically not significant.

Multivariate analysis. Univariate analysis of the conventional parameters like age at diagnosis (< or > 50 years), staging (tumor stage I-IV), hormone receptor status and our specific prognostic "score" as well as data on LOH at BRCA1, BRCA2 or p53 revealed that "score" (p<0.01) and receptor status (p<0.05) had significant correlation with long-term survival. Including score as well as LOH at the three critical Figure 3. Disease-free survival as a function of 13q12-13 LOH in 53 regions as additional variables for multivariate analysis using patients with sporadic breast cancer and a median disease-free interval of 35 months. Cox proportional-hazard regression, we found that allelic loss only at the 13q12-13 region was significantly correlated with reduced overall survival of the patients after breast surgery (p<0.05). Concomitant LOH of two regions, e.g. BRCA2 and p53 (p<0.05), was not identified as a stronger loss of the BRCA1 and BRCA2 locus was found in 3 predictive factor in comparison to LOH of the BRCA2 patients, BRCA1 and p53 was concomitantly lost in 5 region alone. The hazard ratio for "score" was 1.5 (95% CI, patients, the BRCA2 and p53 loci in 4 patients. 1.2-1.9; p<0.01) and for 13q12-13 LOH 2.3 (95% CI, 1.0-5.3; p<0.05). Entering the variable BRCA2 LOH into the Overall survival and time to metastasis analysis. To identify multivariate analysis, the significance of p53/BRCA2 LOH, single or combined LOH events that might predict outcome which was primarily visible, was lost; therefore a risk ratio and survival of patients with primary invasive breast cancer, for this variable was not available. None of the additional we attempted to correlate clinical and long-term follow-up entered parameters proved to be predictors for survival in data as well as analysis of allelic loss at the three breast this model. cancer susceptibility regions with metastases-free and overall A second Cox proportional-hazard regression analysis survival of 53 patients. Patients with LOH in their tumor with the identical variables was performed to test the samples had a worse overall survival compared to patients prognostic value of LOH in predicting time to progression retaining heterozygosity: at the end of our follow-up period, (disease-free survival). In the univariate analysis, again 17 patients of the 28 patients (61%) presenting with LOH in "score" (p<0.01), hormone receptor status (p<0.05) and at least one locus had died, whereas from the 23 heterozygous staging (p<0.01) proved to be independent prognostic patients only 11 deceased (48%, p>0.05). The metastasis-free parameters. In the multivariate analysis, again LOH of interval was not significantly different between LOH-positive BRCA2 was found to be a significant predictor of earlier and -negative tumors (27.6 versus 30 months). manifestation of metastases. The hazard ratio for "score" This analysis was followed by a separate analysis of the was 1.8 (95% CI, 1.3-2.4; p<0.01) and in addition, patients three target regions BRCA1, BRCA2 and p53 in a Kaplan with LOH at 13q12-13 had an approximately 2.3-fold Meier analysis of survival and log rank test. From the 13 increase in risk of recurrence (p<0.05, 95% CI, 1.0-5.3). patients revealing LOH at the 13q12-13 region (BRCA2), 9 had died (69%) at the end of our follow-up period, whereas Discussion only 50% of the heterozygous patients were dead. Figure 2 documents the results of Kaplan-Meier analysis of survival The decision on postoperative treatment of breast cancer with regard to LOH status in the 13q12-13 region in which patients currently relies on conventional parameters like a remarkable, although not significant (p≥0.05), survival age, tumor stage (TNM, grading) and hormone receptor disadvantage of LOH-positive patients could be seen. status. However, the clinical course of individual patients is Concerning LOH at 17q21 and 17p13, no significant variable and identification of subsets of patients with a more association between survival and LOH could be or less aggressive course would be helpful. Research is demonstrated (p≥0.05). therefore focused on the identification of reliable additional

285 ANTICANCER RESEARCH 24: 281-290 (2004) prognostic markers that could determine the grade of role in maintaining genomic stability, acting as a cell cycle malignancy of the individual tumor and help to predict the checkpoint and either allowing the repair of damaged DNA patient’s individual risk of recurrence. by inducing a transient G1 arrest or inducing cell apoptosis Amplification of the c-myc proto-oncogene (26-30), (for review see 74, 76, 77). p53 abnormalities are found in mutations and overexpression of p53 (31-36) and level of 20-40% of sporadic breast cancers (10, 13, 35, 78-80). plasminogen activator type I inhibitor (37) have been Furthermore, recent studies have shown an interaction of evaluated in preclinical studies, whereas determination of p53 and BRCA proteins in breast tumor carcinogenesis by the expression status of the HER-2/erbB2 gene (38-42) has coordinately regulating gene transcription (81-84) and a entered the clinic worldwide recently. concomitant role in DNA damage repair (85, 86). In Analysis of loss of chromosomal material in a specific addition, p53 is more frequently inactivated in BRCA1- region occurring frequently in a certain tumor type may associated tumors (21, 87-91). This fact suggests an indicate the existence of a TSG in this region. In case of association between p53 and breast cancer susceptibility mutational or regulatory inactivation of one allele, the emphasizing the concomitant function of BRCA and p53 additional loss of the second allele will unmask the protein in tumorigenesis. underlying alteration and inactivation of the TSG occurs. In Due to the suspected concomitant role of BRCA1 breast cancer, two chromosomal regions with frequent BRCA2 and p53 inactivation in breast carcinogenesis, we allelic loss have been studied extensively, leading to the initiated a study of long-term follow-up of 53 breast cancer identification of the breast cancer susceptibility genes patients and screened their tumor material for allelic loss in BRCA1 and BRCA2, located at 17q21 (43) and 13q12-13 17q21, 13q12-13 and 17p13 to evaluate the prognostic (44), respectively. significance of single or combined LOH in predicting the Recent data have shown that BRCA1 and BRCA2 are risk of recurrence and overall postoperative survival. We two proteins with similar functions, which are involved in used LOH analysis because it is a fast and easy method (if cell cycle progression and play an important role in DNA using automated screening on a sequencer) useful in the damage repair and gene transcription (45, 46). Mutations in clinic to screen a large number of patients in a moderate these genes (also called caretakers) predispose to genetic time period, having in mind that LOH analysis only instability, but do not initiate tumor growth directly. Cells identifies loss of one allele, whereas the second allele has to that lack BRCA1 or BRCA2 accumulate chromosomal be either mutated or otherwise impaired in function in abnormalities, like chromosomal breaks, aneuploidy and order to knock out the gene completely. centrosome amplification (46, 47), which may be the So far, most studies have focused on the correlation of pathogenic basis for breast tumor formation. Loss of one LOH at BRCA1 and/or BRCA2 and/or p53 with allele of BRCA1 or BRCA2 is a very common event (30- clinicopathological data (3-7, 9-11, 14, 15, 17, 18, 50), but 70%) in sporadic breast tumors (3, 8, 10, 12-14, 20, 22, 48- long-term survival studies are less frequent (19-22) with only 50), suggesting that somatic loss of BRCA genes may be one study revealing that LOH at these three regions associated with breast tumor carcinogenesis, but somatic predicts reduced postoperative mortality (49). point mutations in the BRCA genes are rarely found in In our group of 53 patients with primary sporadic breast sporadic breast tumors (51-57, own data). Recent studies cancer, we detected LOH in the 17q21 region in 23 %, in have shown that, in a majority of sporadic breast tumors, the 13q12-13 region in 26 % and in the 17p13 chromosomal BRCA1 message and protein are reduced or absent (58-62), region in 29 % of the cases. These LOH rates are in whereas BRCA2 mRNA/protein levels seem to be elevated accordance with the reported data in the literature ranging (63-65) as a result of BRCA2 up-regulation in highly from 20 - 63 % in BRCA1 (3, 6, 7, 92) and 20-74 % in proliferating tumors. Probably mechanisms other than BRCA2 (3, 5-10, 12, 15, 18, 22, 49, 92, 93). LOH mutation are responsible for the inactivation of the second frequencies in the p53 gene region at the short arm of allele after LOH like haplo-insufficiency, transcriptional chromosome 17 range from 14-60 % (17). Previous data silencing, incorrect subcellular localization or promotor have shown that allelic loss at these regions are associated hypermethylation, which has been shown to be a common with breast tumors, revealing features that are linked to mechanism in BRCA1 (60, 66-71). The apparent more aggressive clinical behavior such as large size, high contradictory results of high BRCA2 mRNA expression (63, grade and lack of estrogen receptors (6, 9, 17, 92). 65) and frequent LOH may be due to failure of post- Univariate analysis of our data set indicated that "score" and translational modification (45, 72, 73). receptor status are significant predictors for overall survival. The p53 gene located at 17p13 has been identified as In addition, high score, negative receptor status and guardian of the genome (gatekeeper) (74) and is known to advanced tumor stage are predictors for early disease play a critical role in breast cancer carcinogenesis as well recurrence. Since our "score" system summarizes the clinical (for review see 75). The wild-type p53 protein plays a crucial parameters age, tumor stage (TNM) and receptor status in

286 Hampl et al: LOH of BRCA2 and Long-term Survival a point system, our data are in accordance with previous In conclusion, our data suggest that LOH analysis at the results showing that LOH at the BRCA1, BRCA2 or p53 breast cancer susceptibility gene locus 13q12, if verified in a chromosomal region is correlated with indicators commonly larger patient population, may be used as a new molecular associated with poor disease prognosis like negative marker to identify patients with an unfavorable disease course receptor status (22, 92), large tumor size (14), high rate of and shortened recurrence-free and overall survival. This proliferation (7, 22), lymph node metastases (12, 15) or marker may be useful in addition to the conventional features higher grade (15). like age, TNM status and hormone receptor status to identify Multivariate analysis of our data set revealed that, in a subgroup of patients with poor prognosis, which may benefit addition to "score", there was a significant relationship from a more aggressive treatment. We think that LOH between LOH at the BRCA2 locus, as well as concomitant represents an easy and reliable technique, which permits loss of the BRCA2 and p53 gene region, and overall survival, routine screening of tumor samples of breast cancer patients which is in accordance with several previous studies. and may represent a practical diagnostic tool in the future. Remarkably, the significance of combined loss of p53 and BRCA2 was lost in the multivariate model when entering References LOH BRCA2 as a variable, indicating that LOH BRCA2 1 Knudson AG: Antioncogenes and human cancer. Proc Natl alone is such a strong predictor that addition of p53 LOH Acad Sci USA 90(23): 10914-21, 1993. does not give additional information. In addition, LOH of 2 Callahan R: Somatic mutations that contribute to breast cancer. BRCA2 was found to be a significant predictor of earlier Biochem Soc Symp 63: 211-21, 1998. manifestation of metastases and had an approximately 2.3- 3 Beckmann MW, Picard F, An HX, van Roeyen CR, Dominik fold increase in risk of recurrence. Van den Berg et al. (22) SI, Mosny DS, Schnurch HG, Bender HG and Niederacher D: reported a significant correlation between LOH in the Clinical impact of detection of loss of heterozygosity of BRCA1 and BRCA2 markers in sporadic breast cancer. Br J Cancer BRCA2 region and recurrence-free as well as overall survival. 73(10): 1220-6, 1996. In accordance with our data are the results of Eiriksdottir et 4 Bieche I, Nogues C, Rivoilan S, Khodja A, Latil A and Lidereau al. (21), with a 5-year survival rate of 60% among patients R: Prognostic value of loss of heterozygosity at BRCA2 in with tumors showing LOH at chromosome 13q and 78% human breast carcinoma. Br J Cancer 76(11): 1416-8, 1997. among patients whose tumors retained heterozygosity. A 5 Cleton Jansen AM, Collins N, Lakhani SR, Weissenbach J, further study focusing on LOH at 1p34, 13q12, 17p13.3 and Devilee P, Cornelisse CJ and Stratton MR: Loss of heterozygosity 17q21.1 (49) found a 3.1 times higher risk of postoperative in sporadic breast tumours at the BRCA2 locus on chromosome 13q12-q13. Br J Cancer 72(5): 1241-4, 1995. mortality in patients with LOH at 13q12. Likewise, allelic loss 6 Gonzalez R, Silva JM, Dominguez G, Garcia JM, Martinez G, of both regions 13q12 and 17p13 was associated with a 9.6 Vargas J, Provencio M, Espana P and Bonilla F: Detection of times greater risk of mortality, whereas in our study loss of heterozygosity at RAD51, RAD52, RAD54 and BRCA1 concomitant LOH of both regions BRCA2 and p53 and BRCA2 loci in breast cancer: pathological correlations. Br (p=0.0454) was not identified as a stronger predictive factor J Cancer 81(3): 503-9, 1999. in comparison to LOH of the BRCA2 region alone 7 Hanby AM, Kelsell DP, Potts HW, Gillett CE, Bishop DT, Spurr (p=0.0367). This result does not fit completely in the recent NK and Barnes DM: Association between loss of heterozygosity of BRCA1 and BRCA2 and morphological attributes of sporadic model of the function of the BRCA and p53 proteins in breast cancer. Int J Cancer 88(2): 204-8., 2000. tumorigenesis. According to the theory of Kinzler et al. (94), 8 Hamann U, Herbold C, Costa S, Solomayer EF, Kaufmann M, the BRCA proteins may be considered as caretakers. Bastert G, Ulmer HU, Frenzel H and Komitowski D: Allelic Inactivation of a caretaker does not promote tumor initiation imbalance on chromosome 13q: evidence for the involvement directly; it rather leads to genetic instabilities that result in an of BRCA2 and RB1 in sporadic breast cancer. Cancer Res increased mutation rate affecting all genes including 56(9): 1988-90, 1996. gatekeepers (for example p53), which directly regulate the 9 Johnson SM, Shaw JA and Walker RA: Sporadic breast cancer in young women: prevalence of loss of heterozygosity at p53, growth of tumors by inhibiting growth or promoting death. BRCA1 and BRCA2. Int J Cancer 98(2): 205-9., 2002. According to this theory, inactivation of BRCA2 alone may 10 Katsama A, Sourvinos G, Zachos G and Spandidos DA: Allelic not be enough for tumor initiation. 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