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Clinical Cancer Research Vol. 6, 4043-4048, October 2000
© 2000 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Human Breast Cancer Susceptibility to Paclitaxel Therapy Is Independent of Bcl-2 Expression1

Susan M. Poelman, Moses O. Adeyanju, Maria-Antonia Robertson, Wendy M. Recant, Theodore Karrison, Gini F. Fleming, Olufunmilayo I. Olopade and Suzanne D. Conzen2

Departments of Medicine [S. M. P., G. F. F., O. I. O., S. D. C.], Health Studies [M-A. R., T. K.], and Pathology [W. M. R.], University of Chicago, Chicago, Illinois 60637, and LabCorp Inc., Elmhurst, Illinois 60126 [M. O. A.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In laboratory studies, ectopic overexpression of the antiapoptotic protein Bcl-2 has been shown to result in resistance to the cytotoxic effects of many chemotherapeutic drugs. Furthermore, posttranslational modification of moderately expressed endogenous Bcl-2 has been correlated with susceptibility to paclitaxel treatment in vitro. To determine whether tumor expression of Bcl-2 protein correlates with response and ultimate outcome in vivo, we quantified Bcl-2 expression by immunohistochemical analysis of archived biopsy specimens from metastatic breast cancer patients treated with single-agent paclitaxel. The statistical association between the degree of Bcl-2 expression, objective tumor response, and clinical outcome was then determined. In patients (n = 39) whose tumors had low (<=10% cells positive) Bcl-2 levels by immunohistochemical analysis, the overall response (complete response + partial response) rate was 21% versus an overall response rate of 22% in patients (n = 36) with high (>10% cells positive) Bcl-2 expression (P = 0.92). In patients with low Bcl-2 expression, the median time to progression was 126 days [95% confidence interval (CI), 63–160 days]. This was not significantly different than the 105 days for patients with high tumor Bcl-2 expression (95% CI, 84–214 days). The median survival time from initiation of paclitaxel therapy for patients with low Bcl-2 expression was 663 days (95% CI, 456-1119 days) and was not significantly different than the 450 days (95% CI, 239-1058 days) observed for patients with high Bcl-2 expression. In conclusion, we found that in metastatic breast cancer, there is no significant association between tumor Bcl-2 expression and response to paclitaxel, median time to progression, or survival, suggesting that the main mechanism of paclitaxel-induced cytotoxicity in breast tumors is independent of Bcl-2 expression.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Paclitaxel (Taxol®) is an effective cytotoxic drug for the treatment of a wide range of cancers. In breast cancer therapy, the addition of paclitaxel to standard adjuvant chemotherapy has recently been shown to provide a significant survival benefit over therapy with doxorubicin and cyclophosphamide alone (1) . In metastatic breast cancer, response rates to single-agent paclitaxel therapy have been reported to vary from 20–35% (2) . Because susceptibility to chemotherapy is believed to reflect the ability of a tumor cell to undergo apoptosis, overexpression of antiapoptotic proteins such as Bcl-2 has been postulated to promote chemotherapy resistance in solid tumors. This hypothesis is supported by data from experimental systems, where ectopic overexpression of Bcl-2 can inhibit apoptosis in many different tumor cell lines derived from non-Hodgkin’s lymphoma, breast, lung, glioma, pancreatic, and ovarian cancers (3, 4, 5, 6, 7, 8) . In primary human breast cancer specimens, however, high Bcl-2 expression is associated with markers of good prognosis, including the expression of ER3 and progesterone receptor and low tumor grade (9, 10, 11) . Furthermore, within the subset of patients with ER-positive tumors, high Bcl-2 expression is associated with relatively indolent disease and predicts a good response to tamoxifen independent of the degree of ER positivity (12) . In contrast, in patients with metastatic breast cancer treated with various combinations of non-taxane chemotherapy, high Bcl-2 expression has been associated with resistance to therapy (13) or has been shown to have no effect (14) . The relationship between Bcl-2 expression and response to paclitaxel chemotherapy in patients with metastatic breast cancer has not been studied previously.

In vitro, paclitaxel treatment of tumor cell lines can result in the increased phosphorylation of Bcl-2, perhaps through a G2-M phase-dependent activation of an unidentified microtubule-associated kinase (15 , 16) . In a panel of tumor cell lines, Bcl-2 phosphorylation, in turn, has been shown to correlate with paclitaxel-induced apoptosis, prompting some investigators to speculate that both the level of Bcl-2 expression and its phosphorylation state may modulate the apoptotic response to paclitaxel. In a variety of cell lines, ectopic overexpression of Bcl-2 is associated with an increased resistance to a wide spectrum of chemotherapeutic drugs, including vincristine, cyclophosphamide, doxorubicin, and cisplatin (3, 4, 5, 6, 7, 8) . However, most studies examining primary tumor samples from non-Hodgkin’s lymphoma and breast, ovarian, brain, and aerodigestive cancers have not found a correlation between response to cytotoxic treatment and level of Bcl-2 expression (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) . This includes a study examining apoptosis in histocultures of surgical specimens from patients with head and neck cancers 24 h after paclitaxel therapy (29) . One exception to this finding is a study that reports an association between tumor Bcl-2 expression and response to combination chemotherapy in patients with metastatic breast cancer treated with CAF or CMF. In this study of 55 patients, it was found that tumors with >= 40% of cells positive for Bcl-2 were less likely to respond to chemotherapy than tumors with <40% of cells positive for Bcl-2 expression (13) . However, to our knowledge, no previous study has addressed the issue of whether tumor Bcl-2 expression is associated with tumor response to paclitaxel in vivo. Therefore, we undertook an IHC study of Bcl-2 expression in breast cancer specimens and evaluated the correlation between Bcl-2 expression and tumor response to paclitaxel therapy, time to progression, and overall survival.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Tumor Specimens.
This retrospective chart review evaluated 87 patients with metastatic breast cancer treated with single-agent paclitaxel therapy at the University of Chicago from 1996–1999. Eligible patients included those who received paclitaxel as either a first-line (n = 57), second-line (n = 13), or third-line (n = 5) therapy for measurable or evaluable metastatic disease. Patients were also required to have a primary or metastatic tumor biopsy available for IHC analysis and clinical follow-up information that was accessible to the investigators. Twelve of 87 patients were not eligible (no measurable disease, lack of adequate archived material, or chemotherapy administered in addition to paclitaxel). Of the remaining 75 patients, 73 received paclitaxel every 21 days, and 2 received weekly paclitaxel. Tumor response was determined retrospectively based on radiological review of computed tomography scans. Evaluable disease response was determined for patients with local or bone disease based on reported physical exam and review of skeletal imaging (see below for response criteria). When possible, formalin-fixed paraffin-embedded blocks were collected from metastatic tumors resected before paclitaxel treatment (n = 32). If no biopsy for metastasis was performed (n = 43), tumor blocks from the primary or most recent recurrent breast cancer were analyzed. We allowed primary tumors to be used for analysis based on the results of a previous study (31) and unpublished data4 that suggest that levels of Bcl-2 expression remain constant between a primary tumor and a subsequent metastasis. In the study of Sjogren et al. (31) , the authors analyzed Bcl-2 expression in asynchronous primary and metastatic tumors from 28 patients with breast cancer. The results revealed that only one patient developed a metastasis with a significant change in Bcl-2 expression compared with the primary breast cancer (a change from high Bcl-2 expression to low expression).

Response Criteria.
Patients were evaluated for response by studies performed 4–6 weeks after the sixth cycle of paclitaxel therapy (or earlier if PD was evident). Computed tomography was used to evaluate tumor dimensions in patients with visceral disease (n = 55), physical examination was used to assess evaluable local disease (n = 17), and a combination of bone scan, plain films, and clinical assessment was used to evaluate patients with osseous disease only (n = 3). In patients with nonosseous disease, CR was defined as the disappearance of all evidence of disease. For patients with disease involving the bone, CR was defined as the disappearance of all nonosseous cancer, bone scans or skeletal radiographs without evidence of progression or new lesions, and the disappearance of bone pain. PR in patients without osseous disease was defined as a reduction of more than 50% in the sum of cross-sectional areas of all measured lesions in nonosseous sites. In patients with bony disease, bone scans or skeletal radiographs were required to show no progression or new lesions, and patients had to exhibit an improvement of bone pain. Stable disease was defined as a steady state (<=25% increase in tumor dimension) or a response of <50%. PD was defined as any new site of disease or a >25% increase in any measurable or evaluable disease. Time to progression was defined as the time from initial treatment with paclitaxel to the first occurrence of PD, discontinuation of treatment, or death.

IHC Analysis.
Each paraffin block submitted was cut into 5-mm sections, the first of which was stained with H&E to ensure adequate quality of fixation and ensure that the number of invasive tumor cells was sufficient for IHC analysis. The remaining 5-mm sections were mounted on positively charged slides and then heated to 60°C for 1 h, cooled, deparaffinized, and hydrated through three changes of xylene and graded alcohols. Slides were then washed with PBS three times, steamed with citric buffer in a rice cooker for 20 min, cooled, rinsed with PBS, quenched in 0.3% H202 in PBS for 30 min and blocked with 10% horse normal serum for 30 min. Specimens were incubated overnight in Bcl-2 monoclonal antibody 122 (Dako, Carpinteria, CA) using a stock concentration of 2.25 mg/ml at a 1:80 dilution. After rinsing with PBS, a secondary biotinylated antimouse IgG antibody at a final concentration of 30 µg/ml (Vector Laboratories, Burlingame, CA) was applied for 30 min, and slides were rinsed with PBS and exposed to diaminobenzidine tetrahydrochloride chromogen (Vector kit #SK-4100) to develop the color reaction product. To avoid color reduction, slides were washed with tap water, counterstained in hematoxylin for 2 min, washed, dehydrated in a series of ethanols, cleared in xylene, and mounted. Negative controls for this study were performed for each sample using an isotype-specific mouse antibody instead of the anti-Bcl-2 antibody. Human tonsil tissue was used as a positive control for Bcl-2 expression, as were tumor-infiltrating lymphocytes that were present in most samples.

IHC Scoring.
Slides were scored for Bcl-2 expression with all patient identifiers removed, according to the percentage of tumor cells that stained positively. Staining intensity revealed that the vast majority of samples (>80%) had moderate to strong Bcl-2 intensity when compared with lymphocyte Bcl-2 expression. Thus, the percentage of positive cells rather than staining intensity was used for analysis of data. For every sample, at least 200 tumor cells were analyzed. To determine the distribution of Bcl-2 staining in patients’ tumors, we divided the results into six IHC scoring categories depending on the percentage of Bcl-2-positive cells: (a) 0, none; (b) 1, <1%; (c) 2, 1–10%; (d) 3, 11–30%; (e) 4, 31–75%; and (f) 5, >75%. Based on prior studies of breast cancer (12 , 14 , 32) , lung (19) , and head and neck cancers (28) examining Bcl-2 expression and patient tumor response, >10% positively stained cells was used to represent positive Bcl-2 expression for this report. Because an association between Bcl-2 expression in >40% of breast tumor cells and resistance to chemotherapy had been demonstrated previously (13) , we also analyzed our data using a cutoff point of 40%. To ensure that there was no difference between tumors that were strongly positive and those that were less positive, we also analyzed our data using a cutoff of 75% positivity.

Statistical Analysis.
{chi}2 analysis was used to compare Bcl-2 IHC results with an objective tumor response (CR or PR) to paclitaxel. Estimation of progression-free and overall survival curves for patients with Bcl-2-positive or -negative tumors was determined by the Kaplan-Meier method (33) . The log-rank test was used to test the null hypothesis that the underlying survivor curves for Bcl-2-positive and Bcl-2-negative patients are equivalent (34) . {chi}2 analysis and Fisher’s exact test were used to assess the significance of an association of Bcl-2 positivity with known tumor characteristics such as ER status, grade, and site of metastasis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Seventy-five female patients were evaluable in this study with the following patient and tumor characteristics (median age, 49 years; age range, 25–77 years). Overall, 36 of 75 (48%) patients showed high levels of Bcl-2 expression (Fig. 1)Citation , as defined by >10% of cells with detectable IHC staining for Bcl-2 (a score of 3 or more; Table 1Citation ). The remaining 39 patients (52%) exhibited minimal staining for Bcl-2 (<=10% of cells, scores of 2 or less). Tissue analyzed was from the following anatomical sites: (a) breast, n = 54; (b) lung, n = 2; (c) chest wall, n = 4; (d) lymph node, n = 8; (e) ovary, n = 4; (f) liver, n = 2; and (g) brain, n = 1. Bcl-2 expression was determined by staining performed either on the primary tumors (43 patients; 57%) or at the site of relapse (32 patients; 43%). The distribution of Bcl-2 expression in primary versus relapsed tumor samples was similar, with 23 of 43 (53%) primary tumors and 13 of 32 (41%) metastatic tumors exhibiting high Bcl-2 expression (P = 0.38; Table 2Citation ).



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  		Fig. 1. Microscopic appearance of invasive breast cancers (x400). Anti-Bcl-2 IHC staining (A) and H&E staining (B) of a Bcl-2-positive tumor and IHC staining (C) and H&E staining (D) of a Bcl-2-negative tumor.

 

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  		Table 1 %Summary of Bcl-2 immunostaining

 

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  		Table 2 %Analysis of association of Bcl-2 immunohistochemical score with patient or tumor characteristics (n = 75)

 
To determine whether the Bcl-2-positive and -negative patient groups were similar with respect to established prognostic characteristics, the two groups were compared. There was no significant association between Bcl-2 expression and age, grade, number of prior regimens, or location of metastases (Table 2)Citation . There was, however, evidence of an association between Bcl-2 expression (>10% of cells) and positive ER status (P = 0.084), although it did not reach statistical significance. It should be noted, however, that this association approached statistical significance when a cutoff of 75% positively stained cells was used (P = 0.052).

Because the low Bcl-2- and high Bcl-2-expressing groups were similar in terms of tumor characteristics, we next examined overall response. The overall response rate to chemotherapy was 21% (95% CI, 12–32%). In Bcl-2-positive patients, 8 of 36 (22%) responded (0 CRs and 8 PRs). In Bcl-2-negative patients, 8 of 39 patients responded [1 CR and 7 PRs (21%); see Table 3Citation ]. Thus, there was no significant difference between response to paclitaxel in patients with either Bcl-2-positive or -negative tumors (P = 0.92). If a cutoff of 40% was used for Bcl-2 positivity [as was used in the report of Bonetti et al. (13) , which found a correlation between CMF or CAF response to chemotherapy and Bcl-2 positivity], there was still no significant difference in response rate (21% for Bcl-2-negative patients versus 22% for Bcl-2-positive patients, P = 0.85). Moreover, we analyzed the relationship between those tumors that expressed very high levels of Bcl-2 (using a cutoff of 75% positively stained cells) and response rates and still found no significant association with response (P = 0.64).


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  		Table 3 %Outcome according to Bcl-2 expression using cutoffs of 10%, 40%, and 75% of tumor cells staining positively for Bcl-2

 
Time to progression in the two Bcl-2 groups (using our original cutoff of 10% of cells to determine positivity) is shown in Fig. 2Citation A. There was no significant difference between the two curves as measured by the log-rank test (P = 0.98). Median progression-free survival times were 126 days (95% CI, 63–160 days) in the Bcl-2-negative group and 105 days (95% CI, 88–214 days) in the Bcl-2-positive group. Similarly, overall survival rates (Fig. 2Citation B) were not significantly different (log rank P = 0.37) with a median survival time of 663 days (95% CI, 456-1119 days) in the Bcl-2-negative group and 450 days (95% CI, 239-1058) in the Bcl-2-positive group. Thus, tumor Bcl-2 expression did not correlate with either response to paclitaxel, time to progression, or time to death.



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  		Fig. 2. Kaplan-Meier estimates of the distribution of (A) time to disease progression and (B) time to death for Bcl-2 negative (---) and positive (——) patients.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Failure to undergo apoptosis is considered a major mechanism of chemoresistance. Bcl-2, a potent antiapoptotic protein, is highly expressed in up to 70% of low-grade breast cancers and in 50% of breast cancers overall (35) . In vitro studies performed using cell lines with genetically engineered overexpression of Bcl-2 have consistently shown a strong positive correlation between Bcl-2 expression and resistance to cytotoxic chemotherapy. However, the degree of Bcl-2 overexpression in genetically modified cell lines is typically much greater than even strong endogenous expression (36) . Indeed, most studies performed with a variety of clinical human tumor specimens have not found a significant association between Bcl-2 expression and chemotherapy resistance (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) . One exception to this finding is a study by Bonetti et al. (13) in women receiving CMF or CAF for metastatic breast cancer in which a correlation was found between resistance to CMF or CAF therapy and tumor Bcl-2 expression. In contrast, Sjostrom et al. (14) did not find a correlation between Bcl-2 expression and response to 5-fluorouracil, epirubicin, and cyclophosphamide in 103 patients with metastatic breast cancer. In the current study, we investigated whether an association exists between tumor Bcl-2 expression and cytotoxic response to paclitaxel therapy in patients with metastatic breast cancer, and we found that response to paclitaxel therapy is independent of the degree of Bcl-2 expression in the patient’s tumor.

Because paclitaxel treatment can result either directly or indirectly in Bcl-2 hyperphosphorylation and consequent functional inactivation, one might alternatively hypothesize that tumors with moderate Bcl-2 expression (in contrast to tumors having little or no expression) might be more susceptible to paclitaxel-induced apoptosis. However, we did not find a correlation with moderate Bcl-2 expression (11–75% of cells) and response in this group of patients (data not shown). This indicates that Bcl-2 is unlikely to directly mediate paclitaxel-induced cytotoxicity in metastatic breast cancer. The relationship between tumor Bcl-2 phosphorylation status and response to paclitaxel must await the development of a phospho-specific anti-Bcl-2 antibody. Low levels of Bax, a proapoptotic protein, have been associated with resistance to chemotherapy and shortened survival rates in patients with metastatic breast cancer treated with combination chemotherapy (32) . Further investigation is ongoing to examine the relationships that might exist between Bax and paclitaxel sensitivity.

In early-stage breast cancer, Bcl-2 expression has been correlated with an improved prognosis, ER positivity, and low tumor grade (37) . Although we found a trend toward an association with ER positivity, the lack of association between Bcl-2 and tumor grade may be a result of the aggressive metastatic nature of the tumors studied in this report, none of which were grade 1.

In summary, to our knowledge, this is the first study to examine the relationship between tumor Bcl-2 expression and the corresponding clinical response to paclitaxel. Contrary to what might be expected based on results obtained in defined tissue culture systems, there is no evidence to support a correlation between tumor Bcl-2 expression levels and resistance to paclitaxel treatment for metastatic breast cancer. It remains possible, however, that earlier stage or lower grade tumors (presumably with fewer accumulated genetic changes) might show a relationship between Bcl-2 expression and susceptibility to paclitaxel treatment. The results from this study, however, suggest that Bcl-2 expression does not influence breast cancer susceptibility to treatment with paclitaxel.


   ACKNOWLEDGMENTS
 
We thank Terry Li and Julie Auger for excellent technical assistance and advice with IHC analysis.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grant R21 CA66132 and NIH Grant CA14599-25-SI (to the University of Chicago Cancer Center), The Entertainment Industry Foundation’s National Women’s Cancer Research Alliance, The Falk Medical Research Trust, and an unrestricted research grant from Bristol-Myers Squibb. Back

2 To whom requests for reprints should be addressed, at Section of Hematology/Oncology, Department of Medicine, MC 2115, University of Chicago, Chicago IL 60637. Phone: (773) 834-2604; Fax: (773) 834-0188; E-mail: sconzen{at}medicine.bsd.uchicago.edu Back

3 The abbreviations used are: ER, estrogen receptor; IHC, immunohistochemical; CR, complete response; PR, partial response; CI, confidence interval; CAF, cyclophosphamide, doxorubicin (Adriamycin), and 5-fluorouracil; CMF, cytoxan, methotrexate, and 5-fluorouracil; PD, progressive disease. Back

4 S. M. Poelman and S. D. Conzen, unpublished data. Back

Received 4/24/00; revised 6/30/00; accepted 7/12/00.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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