Microsatellite Instability Predicts Poor Short-Term Survival in Patients with Advanced Breast Cancer after High-Dose Chemotherapy and Autologous Stem-Cell Transplantation

Throughout the 1990s, the focus in breast cancer research was on high-dose chemotherapy (HDCT) and dose-intensive regimens. But the treatment of breast cancer patients with extensive lymph node involvement or systemic metastases using HDCT and autologous stem cell transplantation (ASCT) is still subject of controversial scientific dispute. HDCT is based on the hypothesis that increased dosage will overcome drug resistance, eradicate metastatic disease, and increase cure rates. Continuous negative evidence for the benefit of HDCT/ASCT (1) contrasts thousands of HDCT/ASCT procedures performed in breast cancer patients. Over the course of the last decade, there has been tremendous interest in identifying more tolerable regimens that may also be more effective, with a focus on targeted therapies.

There are also no definitive prognostic makers for patient outcome in HDCT/ASCT. Identifying such markers could facilitate the definition of a subgroup of patients with advanced breast cancer that will most likely respond to this aggressive treatment. A plethora of molecular markers are currently evaluated in the treatment of breast cancer, but none of these markers are routinely used in clinical practice (2). Accepted prognostic factors for patients with metastatic breast cancer undergoing standard therapy regimens are hormone receptor status (estrogen receptor and progesterone receptor), menopausal status, localization of metastases, interval between initial diagnosis and appearance of metastases, and response to chemotherapy. On the basis of these clinical findings, Possinger et al. (3) have developed a prognostic score to classify patients with metastatic breast cancer into three different risk groups (high, intermediate, and low): metastatic pattern, receptor status, and disease-free survival were considered qualitative parameters. Combining these parameters provides a reliable indicator of tumor progression, but more precise prognostic factors are needed that reflect both the phenotype and the genetic alterations of the tumor. Standardized molecular analyses might help to select patients who would benefit from differential chemotherapeutic approaches. The cohort designated “chemoresistant” could then be offered alternative therapy modalities, which in turn could be derived from molecular characteristics of the tumor.

The aim of this study was to define molecular alterations that could predict treatment response and/or clinical outcome of breast cancer patients undergoing HDCT/ASCT.

Thirty-nine patients with primary invasive carcinoma of the breast were studied as part of a prospective Phase II-trial of sequential HDCT/ASCT (Department of Hematology and Oncology, University of Regensburg, Regensburg, Germany). Informed consent was obtained from all patients, and the study was approved by the Institutional Review Board. Patients had either advanced local breast cancer with level III lymph node metastases or systemic metastases. After initial chemotherapy in standard dosage (vasoactive intestinal peptide-E; 500 mg/m³ etoposid, 4 g/m² ifosfamid, 50 mg/m² cisplatin, and 50 mg/m² epirubicin) to test for chemosensitivity and to mobilize stem cells, patients received two cycles of HDCT (VIC; 1500 mg/m² etoposid, 12 g/m² ifosfamid, and 1500 mg/m² carboplatin) followed by ASCT. Clinical follow-up was available for all patients, none of which had a family history of breast cancer. Clinical and histopathological data are summarized in Table 1. Sixteen patients (41%) had received standard adjuvant chemotherapy before enrollment into the HDCT/ASCT trial. Tissue samples of the primary tumor and corresponding additional histopathological data were obtained from 11 different pathology laboratories. In 14 cases, only archival H&E-stained tissue sections were available for microdissection, precluding additional immunohistochemical analyses. For the remaining cases, formalin-fixed, paraffin-embedded tissue (n = 25) and additional frozen tumor tissue (n = 4) were available. Nonneoplastic breast parenchyma distant from the tumor, skin, bone marrow, or gastric mucosa were used as a source for reference DNA in subsequent microsatellite analyses.

Genomic DNA was isolated from 5-μm tissue sections in cases for which paraffin blocks were available. Pure tumor cell populations (>80% of tumor cells) were obtained using a PALM Robot-Microbeam (Wolfratshausen, Germany) laser microdissection device as described previously (6). H&E-stained archival slides were incubated in xylene overnight (cases for which paraffin blocks were not available). After removal of the coverslip, the slides were rehydrated in 96% ethylene but not exposed to 70% ethanol to preserve H&E staining. In 4 cases, frozen tissue was available for frozen sections and touch preparations from the partially thawed tissue surface as described previously (7). At least 200 tumor cells were isolated from each specimen to prevent preferential monoallelic amplification.

For molecular analysis, normal and tumor DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s specifications. Elution with 2 × 100 μl of 70°C preheated water was performed to increase yield. Each elution step included a 5-min incubation of the QIAamp spin column with the preheated water at 70°C before centrifugation. PCR template concentration was additionally increased by reducing the elution volume to 50 μl (SpeedVac SC110; Savant, Farmingdale, NY) and by performing an improved primer extension preamplification PCR (whole genome amplification) as described previously (8).

Fifteen microsatellite markers on chromosomes 1, 2, 4, 5, 6, 11, 17, 18, 19, and 21 were selected for sensitive detection of microsatellite instability (MSI) in breast cancer according to literature review. Characteristics of microsatellite primers have been reported previously (6). PCR amplification reactions were performed using 50 ng of purified genomic DNA in a final volume of 20 μl in a MJ Research Thermocycler (PRC100; MJ Research, Watertown, MA; Ref. 9). PCR products were subsequently analyzed by 6.7% polyacrylamide/50% urea gel electrophoresis (1 h, 1500 V, 55°C) in a SequiGen sequencing gel chamber (Bio-Rad, Hercules, CA) followed by silver nitrate staining as described previously (10).

MSI was defined as the presence of novel bands after PCR amplification of tumor DNA that were not present in PCR products of the corresponding normal DNA. All gels were evaluated independently by three different investigators (P. J. W., W. D., and A. H). According to recommendations of the 1997 Bethesda Consensus Conference (11), MSI was diagnosed if at least 3 of 15 markers (≥20%) were unstable, samples with instability in only one or two markers were classified as MSI low. Microsatellite-stable samples (MSS) did not show instability in any of the investigated markers. Loss of heterozygosity was defined as a decrease in signal intensity of the tumor sample allele to at least 50% relative to the matched normal DNA allele. Chromosomal instability (CIN) was assumed if at least 40% (≥6 of 15 investigated markers) revealed loss of heterozygosity. All samples testing positive for MSI or loss of heterozygosity were reevaluated twice to exclude the possibility of false-positive results because of preferential monoallelic PCR amplification (8).

Exons 5–9 of the P53tumor suppressor gene were directly sequenced in 34 tumors; the methodology has been described in detail previously (12).

Expression analyses of the mismatch repair proteins hMSH2 (n = 25) and hMLH1 (n = 26) were performed as described previously (13). Immunohistochemical studies for the expression of P53 (n = 26) and HER-2/neu (n = 26) used an avidin-biotin peroxidase method with a 3,3′-diaminobenzidine chromatogen. After antigen retrieval (microwave oven for 30 min at 250 W), immunohistochemistry was carried out in a NEXES immunostainer (Ventana, Tucson, AZ) following manufacturer’s instructions. The following antibodies were used: P53 (mouse monoclonal Bp53-12, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; dilution 1:1000) and HER-2/neu (rabbit polyclonal A0485, DAKO, Glostrup, Denmark; dilution 1:400). One histopathologist performed a blinded immunohistochemical evaluation (A. H.), without knowledge of the molecular data. P53 positivity was defined as strong nuclear staining in at least 10% of the tumor cells. HER-2/neu expression was scored according to the DAKO HercepTest (14).

Metastatic pattern, hormone receptor status and disease-free survival before HDCT/ASCT were scored quantitatively as previously described by Possinger et al. (3), assigning each patient to one of three different risk groups: high, intermediate, or low.

Differences were considered statistically significant when Ps were <0.05. A statistical correlation between clinicopathological and molecular parameters was tested, using a two-sided Fisher’s exact test. A logistic regression was performed to study the correlation between age and categorical variables. Progression-free survival (PFS) and overall survival (OS) time were calculated according to the Kaplan-Meier survival curves (15). PFS and OS were measured from the start of HDCT and compared between women with or without any of the clinical, pathological, or molecular risk factors by two-sided log-rank statistics (16). A stepwise multivariable Cox regression model (17) was adjusted, testing the independent prognostic relevance of MSI. The limit for reverse selection procedures was P = 0.2. The proportionality assumption for all variables was assessed with log-negative-log survival distribution functions. The characteristics of all variables are shown in Table 2.

A total of 39 patients with advanced breast cancer was studied using 15 microsatellite markers on 10 chromosomes. Instability in at least 3 of the 15 markers was found in 13 of 39 patients (33%). All MSI-positive tumors showed normal expression of the mismatch repair proteins hMSH2 and hMLH1. Nine patients (23%) were scored as chromosomal unstable (CIN), with at least 6 markers revealing loss of heterozygosity. The negative correlation of MSI and CIN reported for colorectal carcinoma (18) could not be detected in this series of breast cancers. Sequencing of exons 5–9 of the P53gene, a region that contains 90% of all mutations (19), revealed a P53missense mutation in 6 of 34 cases (18%) in which a complete sequence analysis of exons 5–9 could be performed. Expression of P53 and HER-2/neu was evaluated by immunohistochemistry. Overexpression of HER-2/neu was found in 7 of 27 (27%) and of P53 in 12 of 26 (46%) evaluated patients, respectively. As expected (20), P53 protein expression and mutation analysis correlated significantly (P = 0.001). All tumors with a missense mutation revealed nuclear P53 protein expression because of the prolonged half-life of mutated P53.

Patients were followed for a median of 24.4 months. Of 39 patients, 4 (10%) achieved at least a partial remission, 22 patients (56%) were in complete remission after HDCT. Mean OS time was 40.1 months with a 95% confidence interval (CI) of 33.67–46.45. The median OS time could not be reached; at the qualifying date (March 1, 2000) 26 patients (67%) were still alive. No therapy-associated deaths were observed, but 2 patients progressed rapidly (Nos. 17 and 39). Mean and median PFS was 21.6 and 13.2 months, respectively. The 2-year survival rates and 95% CIs concerning PFS and OS were 0.23 (0.099–0.363) and 0.54 (0.382–0.695).

Applying the Possinger score for metastatic breast cancer (3), 12 patients (31%) were estimated to have a poor prognosis, another 12 patients to have an intermediate prognosis, and the remaining 15 patients (38%) were considered to have a favorable prognosis. However, PFS and OS were not significantly different between the three groups.

For descriptive data analysis, all relevant variables were correlated with the MSI status (Table 2), but no significant association was seen. Cases showing MSI (≥3 of 15 markers) were compared with microsatellite stable (MSS and MSI low) cases regarding PFS and OS by univariate log-rank statistics. MSI was highly associated with shorter PFS (P = 0.02) and OS (P = 0.0004; Figs. 1, A and B). Microsatellite stable cases (MSS and MSI low) had a median PFS of 28.4 months compared with 2.5 months in patients with MSI tumors. Median OS for MSI-positive cases was 22.3 months, whereas the median OS time for patients with MSS and low MSI could not be estimated because <50% of patients died within the time of follow-up. Initial tumor size (P = 0.008) and a history of adjuvant chemotherapy (P = 0.003) were the only other variables which correlated significantly with PFS. Menopausal status (P = 0.04) and previous standard adjuvant chemotherapy (P = 0.02) were associated with shorter OS. None of the other clinical, pathological, or molecular factors correlated significantly with OS (Table 2). Interestingly, younger patients showed a tendency to have microsatellite unstable tumors (P = 0.07; odds ratio = 1.11; 95% CI, 0.99–1.23).

In a multivariate analysis, three different Cox regression models were developed for assessment of the OS rate. Characteristics of variables are shown in Table 3. Because of model assumptions (noninformative censoring, proportional hazards), only MSI, age at diagnosis, CIN, estrogen receptor status, and Possinger score were considered.

In the raw model, only MSI was correlated with poor outcome (P = 0.002). With reverse selection, the hazard ratio for death from breast cancer concerning MSI was 7.18 (95% CI, 2.07–24.88); accordingly, in cases with MSI, the probability of tumor-related death was seven times higher than that in stable cases (MSI low and MSS). Because of the assumption of proportional hazards, the probability of tumor-related death was consistently valid during the entire observation period. None of the other variables had a significant effect on OS.

Subsequently, the trichotomous variables age at diagnosis and Possinger score were dichotomized by the introduction of dummy variables (AGEDUM1, AGEDUM2, POSSDUM1, and POSSDUM2). Again, only MSI was significant in the global model. After reverse selection, AGEDUM1, CIN, and MSI (P = 0.0009; hazard ratio = 10.10; 95% CI, 2.57–39.68) remained in the model.

Finally, multiplicative terms of interaction (INTER1–6) were considered, representing interactions between MSI and dichotomous covariables. Because of the multitude of variables compared with the small number of cases, interpretation was difficult. The global model included POSSDUM2, INTER3, and INTER6 as significant variables. After reverse selection, a model containing MSI, AGEDUM1, POSSDUM2, INTER3, and INTER6 was found (P < 0.01).

Assuming different model constructs, the following conclusions can be drawn: (a) MSI was a prognostic factor for the OS probability of advanced breast cancer patients undergoing HDCT/ASCT; (b) other factors were relevant as well, particularly age at diagnosis (≤40 years) and Possinger score (score = 3 versus score = 1); and (c) potential interactions between the MSI status and OS were influenced by CIN and Possinger score (score = 3 versus score = 1).

This is the first study showing MSI of the primary tumor to be predictive of PFS and OS in patients with advanced breast cancer undergoing HDCT/ASCT. MSI, i.e., small deletions or insertions in short repetitive genomic sequences, has been investigated in numerous breast cancer studies (reviewed in Ref. 21), but large disparities in the percentage of microsatellite unstable tumors are reported. The range of MSI varied from 0% (22) to 100% (23), likely because of heterogeneous patient cohorts, suboptimal choices of MSI markers, and ambiguous diagnostic criteria for MSI. There are no well-defined criteria for the assessment of MSI in breast and other noncolorectal cancers, contrasting the rigorous diagnostic guidelines established in the hereditary nonpolyposis colorectal cancer model (11). We demonstrated MSI (defined as ≥3 of 15 markers unstable) in 33% (13 of 39) of cases, a frequency that is comparatively high. Possible explanations would include the use of laser-assisted tissue microdissection (6) and a selection of a sensitive MSI marker panel, but the highly selected patient cohort with advanced breast cancers remains a strong potential bias.

In colorectal cancer, MSI was shown to be independently predictive of a relatively favorable patient outcome (24), reduced likelihood of metastases (25), and better survival after adjuvant chemotherapy with fluorouracil-based regimens (26). However, Ribic et al. (27) have recently shown that adjuvant chemotherapy with fluorouracil benefited patients with colon tumors exhibiting CIN but not those with tumors exhibiting MSI. The data suggest that fluorouracil-based adjuvant therapy actually decreased the rate of survival among patients with tumors showing MSI. This is apparently an inconsistent finding in view of the fact that colon carcinomas with MSI were associated with an overall better survival than those with CIN. Patients with colorectal cancers that exhibit high-frequency MSI have longer survival than stage-matched patients with cancers revealing MSI (24, 26).

Several groups have reported a poorer prognosis for MSI-positive breast carcinomas (28, 29, 30, 31), but MSI has never been correlated with patient outcome in HDCT studies. Testing various multivariate Cox regression models, we could demonstrate that MSI is a highly significant, independent, and adverse risk factor for OS of breast cancer patients receiving HDCT/ASCT with a 7-fold increase in the relative risk of tumor-related death. MSI of the primary tumor is the most significant prognostic factor characterized in HDCT/ASCT studies thus far. On the analogy of Ribic et al. (27) for colon cancer, HDCT/ASCT was only beneficial for breast cancer patients exhibiting microsatellite stability or low instability (<3 of 15 markers). As expected for advanced tumor stages, lymph node status was of less prognostic importance for OS (P = 0.2) and was excluded from subsequent multivariate analyses. Event rates for tumor progression and tumor-related death were too small to meet the proportional hazards assumption regarding lymph node status.

There is growing evidence that MSI in breast cancer patients may represent a type of genetic instability different from that seen in colorectal cancer. All 13 MSI-positive breast tumors in our study showed normal expression of the mismatch repair proteins hMSH2 and hMLH1. Lack of MSI in breast cancers occurring in nonpolyposis colorectal cancer families has led to the exclusion of breast cancer as an integral tumor of the nonpolyposis colorectal cancer syndrome (32). MSI in breast cancer has typically been found in tri- and tetranucleotide repeats in this and other studies (30, 33, 34). In contrast, diagnosis of nonpolyposis colorectal cancer-associated replication errors is most easily determined by examination of a panel of simple mononucleotide (BAT25and BAT26) and dinucleotide (D5S346, D17S250, and D2S123) repeat sequences (11). A pattern of MSI similar to that found in the current study has been detected in non-small cell lung cancer, termed elevated MSI at selected tetranucleotide markers (35). This type of MSI was associated with P53mutations and displayed a phenotype inconsistent with defects in mismatch repair pathways. Similar to studies by Ahrendt et al. (35), our series of breast cancers revealed MSI predominantly in tetranucleotide repeats. Eighty-five percent of all MSI-positive tumors were unstable in at least one of the five tested tetranucleotide markers, but the mechanism causing these alterations still remains unclear.

In a randomized trial of HDCT in patients with operable breast cancer and extensive lymph node involvement with a median follow-up of 6.9 years, both the number of tumor-positive axillary lymph nodes after induction chemotherapy and the clinical T-stage before chemotherapy were significant factors for OS (36). Numerous novel molecular variables have been proposed as putative prognostic factors in breast cancer (2). However, only few studies investigated molecular alterations in the primary tumor in patients receiving HDCT/ASCT. In a cytogenetic analysis of 34 patients with lymph node-positive, high-risk breast cancer treated with HDCT/ASCT using comparative genomic hybridization, Climent et al. (37) showed that genomic loss of chromosome arm 18p predicted an adverse clinical outcome with shorter PFS.

Data on the importance of P53alterations as a predictor of poor outcome and chemoresistance in breast cancer are controversial (38), probably as a result of heterogeneity of studied patient cohorts and chemotherapeutic regimens, the different methods of assessing response (clinically versus histologically), and variations in the methods used to determine the P53status. In several studies, mutations in the P53gene correlated independently with poor patient outcome in breast cancer (20, 39, 40), likely reflecting an increased proliferative capacity of P53-mutant tumor cells and the greater resistance of such cells to the induction of apoptosis by a variety of chemotherapeutic agents (39). In a study of 149 high-risk primary breast cancer patients undergoing HDCT/ASCT, P53has been shown to be the strongest predictor of survival with a relative risk of 6.06 (41). However, in vitro results of an association of P53mutation and chemoresistance failed to translate into a significant correlation between immunohistochemically detected overexpression of P53 protein and clinical response to therapy in breast cancer patients (42, 43). Thus, the effect of P53mutations on the sensitivity of breast cancer to genotoxic drugs has been disputed. In high-grade, locally advanced breast cancers, inactivation of the P53pathway seemed to improve the response to HDCT (six cycles of 75 mg/m² epirubicin and 1200 mg/m² cyclophosphamide), whereas initial P53status and histological tumor response were strongly associated (P = 0.0001; Ref. 39).

HER-2/neu overexpression may represent yet another independent negative prognostic factor for patients with metastatic breast cancer who undergo HDCT/ASCT; two studies reported an association of Her-2/neu overexpression with generally shorter PFS and OS (44, 45). On the basis of these data, Nieto et al. (46) have proposed a prognostic model for relapse after HDCT in which HER-2/neu overexpression, number of tumor sites, and primary nodal ratio were independent predictors of long-term outcome (median follow-up 62 months) for stage IV oligometastatic breast cancer. In the present study, however, neither P53 nor HER-2/neu alterations correlated significantly with patient survival.

In summary, we demonstrated an independent negative correlation between MSI in the primary tumor and OS in advanced breast cancer patients treated with HDCT/ASCT. MSI may represent a potential molecular marker to predict differential patient’s responses to treatment and clinical outcomes. Larger prospective studies are currently conducted to validate our findings.

We thank Andrea Schneider, Monika Kerscher, and Rudolf Jung for excellent technical assistance, Holger Moch and Stefanie Meyer for carefully revising the manuscript, and Dr. Jürgen Löffler, medicomp mbH, Planegg, Germany, for excellent assistance with the complex statistical analyses.