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Evaluation of the Predictive Value of Her-2/neu Overexpression and p53 Mutations in High-Risk Primary Breast Cancer Patients Treated With High-Dose Chemotherapy and Autologous Stem-Cell Transplantation

From the University of Colorado Bone Marrow Transplant Program and Departments of Pathology and Biostatistics, University of Colorado, Denver, CO.

Address reprint requests to Yago Nieto, MD, University of Colorado Health Sciences Center, B# 190, 4200 East Ninth Avenue, Denver, CO 80262; email yago.nieto{at}uchsc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
PURPOSE: To ascertain the predictive value of Her-2/neu overexpression and p53 mutations, assessed by immunohistochemistry, in high-risk primary breast cancer (HRPBC) treated with high-dose chemotherapy (HDCT).

PATIENTS AND METHODS: We obtained paraffin-embedded tumor blocks from 146 HRPBC patients previously enrolled at our program onto clinical trials of HDCT for four to nine involved axillary lymph nodes, 10 involved axillary nodes, or inflammatory carcinoma. All patients received the same HDCT regimen, with cyclophosphamide, cisplatin, and carmustine (STAMP-I), followed by autologous stem-cell transplantation. Median follow-up was 42 months (range, 5 to 90 months). The same pathologist, blinded to clinical outcome, reviewed all immunostained slides.

RESULTS: Positive results for Her-2/neu and p53 were found in 44.5% and 34% of the patients, respectively. Positivity for Her-2/neu was significantly associated with increased risk of relapse and death. No correlation was found between p53 mutations and relapse-free survival (RFS) or overall survival (OS). Multivariate analyses included Her-2/neu overexpression and the following variables previously identified as independent predictors of outcome in this population: tumor size, nodal ratio (number of involved nodes/number of dissected nodes), and hormone receptor status. All four variables had independent value.

CONCLUSION: Her-2/neu overexpression is an independent negative predictor of RFS and OS in HRPBC treated with HDCT. Its inclusion in our previously described predictive model increases the predictive capacity of this model for the low-risk subgroup. In contrast, p53 mutations lack predictive value in this setting.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
OVEREXPRESSION OF the Her-2/neu oncogene and mutations of the tumor suppressor gene p53 are important events in breast cancer tumorigenesis. The Her-2/neu receptor is a member of the epidermal growth factor receptor family of receptor tyrosine kinases, which are considered to be important mediators of cell proliferation and differentiation.¹ It is activated in 20% to 30% of cases through amplification and overexpression of the oncogene. Overexpression of Her-2/neu reflects an increased proliferative activity of the tumor. Her-2/neu positivity has been reported to be a negative predictor of response to hormonal therapy, adjuvant radiotherapy, and adjuvant chemotherapy with cyclophosphamide, methotrexate, and fluorouracil (CMF).² In contrast, it seems to increase tumor sensitivity to doxorubicin^3,4 and to dose increments of this drug when included in the adjuvant therapy for node-positive disease.^5,6 An association of Her-2/neu overexpression with increased responsiveness to paclitaxel in metastatic breast cancer (MBC) has been suspected.^7,8

Wild-type p53 plays two major roles in cell function: first, it regulates the checkpoints G1 to S and G2 to M of the cell cycle, through regulation of transcription of p21 and other genes; second, it induces apoptosis after genotoxic damage.⁹ p53 mutations are detected in 18% to 45% of breast tumors. Studies that evaluated their prognostic value in node-negative and node-positive breast cancer have offered conflicting results.¹⁰ In addition, p53 mutations may be negative predictors of response to doxorubicin¹¹ and CMF.¹² Response to tamoxifen in p53-positive patients has been shown to be decreased¹³ or unchanged.¹⁴

High-dose chemotherapy (HDCT) with autologous stem-cell transplantation attempts to maximally capitalize on the dose-response effect of certain drugs used in the treatment of breast cancer. Phase II trials of HDCT as part of adjuvant therapy for patients with high-risk primary breast cancer (HRPBC), which is defined by 10 or more involved axillary lymph nodes,^15,16 four to nine involved nodes,¹⁷ or inflammatory breast carcinoma (IBC),^18,19 have reported 57% to 71% relapse-free survival (RFS) rates at 2 to 5 years.

The value of HDCT compared with conventional chemotherapy in HRPBC is currently under evaluation in randomized phase III trials and remains questionable. The United States Intergroup CALGB 9082 study compared HDCT that included cyclophosphamide, cisplatin, and carmustine (BCNU) (STAMP-I regimen) with intermediate doses of the same drugs, in patients with 10 or more involved nodes. A recently reported preliminary analysis shows a higher relapse rate in the control arm with nonoverlapping confidence intervals, a higher toxic death rate in the HDCT arm, and no significant differences in RFS and overall survival (OS) between both arms at a follow-up of 37 months.²⁰ Preliminary analysis of a Scandinavian trial, which compared high-dose cyclophosphamide, thiotepa, and carboplatin to a tailored dose-intensified combination of cyclophosphamide, epirubicin, and fluorouracil, shows no difference in outcome at a median follow-up of 24 months.²¹

Although definitive results of most randomized trials were still pending at the time of this writing, it is nonetheless important to identify subgroups of HRPBC patients who might benefit from HDCT, as currently given, and those for whom new approaches need to be explored. Little is known about predictive factors in this setting, in contrast to the conventional chemotherapy scenario. Somlo et al²² identified progesterone receptor (PR) negativity as the only independent predictor of relapse in their series of HRPBC patients treated with two different HDCT combinations. In our own series of HRPBC patients treated with HDCT, we identified tumor size, estrogen receptor (ER)/PR status, and the axillary nodal ratio (number of involved nodes/number of sampled nodes) as independent predictors.²³ The resulting predictive model, based on these three factors, was subsequently validated in an independent patient set treated with the same HDCT regimen. In this study, we analyzed the predictive value of Her-2/neu overexpression and mutations of p53, as determined by immunohistochemistry (IHC) of the primary tumor, in 146 HRPBC patients included in clinical trials of HDCT, using STAMP-I.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
University of Colorado HDCT Clinical Trials
We reviewed 146 patients treated with HDCT between August 1991 and November 1996 at the University of Colorado (Table 1), who were enrolled onto Institutional Review Board–approved clinical trials that had the following inclusion criteria, respectively: 10 involved axillary lymph nodes (n = 66), four to nine involved axillary nodes (n = 56), and IBC (n = 24). Nine patients who died of treatment-related complications in the first 100 days after HDCT (3.8% of the total accrual in these trials at our institution) were excluded from this analysis. Median follow-up for the whole group was 42 months (range, 5 to 90 months). Median follow-up was 44 months (range, 5 to 90 months) for all living patients and 44 months (range, 24 to 90 months) for those patients alive with no evidence of relapse.

Immunohistochemical Analysis
Paraffin-embedded tumor blocks were obtained from the pathology departments of the referring hospitals. Histologic sections were processed routinely, sectioned at a 4-micron thickness, and stained with hematoxylin and eosin. Immunostaining was performed on diagnostic sections using mouse monoclonal antibodies for Her-2/neu (clone CB 11, Vantana Medical Systems, Tucson, AZ) and p53 (clone DO7, BioGenex, San Ramon, CA).

Freshly cut sections were mounted on positively charged slides (Fisher Scientific, Pittsburgh, PA) and dried overnight at 60°C, deparaffinized in xylene, and rehydrated through decreasing concentrations of ethanol to distilled water. Epitope retrieval was accomplished by simmering at 100% power in 20 nmol/L of citrate buffer for p53, or BioGenex buffer 10x for Her-2/neu, at pH 6.0 for 30 minutes after coming to a full boil (7.5 minutes) in a 800-W microwave oven. Slides were cooled at room temperature for 30 minutes in the citrate buffer before proceeding. Slides were then microwaved in 0.01 mol of citrate buffer, pH 6.0, using a standard technique. IHC staining was performed using an indirect biotin-avidin method on a Vantana 320ES automated immuno-stainer (Vantana Medical Systems). The stained sections were lightly counterstained with hematoxylin. Negative control reactions were performed by omitting the primary antibody and were included in each run. Two positive controls were included in each staining run. One was provided by DAKO (DAKO Corporation, Carpintaria, CA) and contained three pelleted formalin-fixed, paraffin-embedded human breast cancer lines with staining intensities of 0, 1+, 2+, and 3+. The other positive control was breast cancer tissue from a known Her-2/neu–positive staining fresh surgical specimen that was fixed, processed, and embedded in the same way as the study samples.

Because the CB-11 antibody has been shown to have 100% specificity,²⁴ staining of any intensity observed on the membrane of cancer cells in any percentage was considered positive. A semiquantitated scoring system was also used, which was based on the estimated fraction of positively stained cells. These scoring criteria were as follows: 0% stained cells = 0; 1% to 33% = 1+; 34% to 66% = 2+; and 67% to 100% = 3+. All immunostained slides were reviewed by the same pathologist (S.N.), who remained blinded to patient outcome.

Statistical Analysis
The associations between Her-2/neu overexpression and p53 mutations with dichotomous and continuous parameters were evaluated using the ² test and t test, respectively. RFS and OS were measured from the start of HDC and estimated using the Kaplan-Meier product-limit method.²⁵ Univariate analyses of Her-2/neu and p53 positivity with RFS and OS were performed using the two-sided log-rank test.²⁶

Multivariate analyses of significant variables were performed with the proportional-hazards regression method.²⁷ In a first analysis, we included factors that we previously identified as independent predictors of outcome in this patient population: pathologic tumor size, nodal ratio (number of involved nodes/number of dissected nodes), and the combined ER/PR status (considered negative if both ER and PR were negative and positive if either or both were positive).²³ In a second proportional-hazards regression analysis, patient score was substituted for size, nodal ratio, and ER/PR status. This score, which prospectively assigns to each patient a low or high risk for relapse, results from a mathematical combination of these three variables²³:

In this formula, tumor size is entered in centimeters and ER/PR is replaced by 1 if negative and by 0 if positive. Scores 2.41 and less than 2.41 assign high and low probabilities of relapse, respectively. This score-based prognostic model was validated in an independent HRPBC data set.²³ All statistical analyses used the Statistica software package (StatSoft, Inc, Tulsa, OK).

	RESULTS

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IHC Analysis
Her-2/neu positivity was found in 65 (44.5%) of 146 of patients. The proportion of Her-2/neu–positive patients in the three patient groups was as follows: four to nine positive nodes, 41%; 10 positive nodes, 48%; and IBC, 48%. Figure 1 shows an IHC slide with membrane staining for Her-2/neu.

View larger version (136K): [in this window] [in a new window]   Fig 1. Immunohistochemical analysis for Her-2/neu. Positive staining of tumor cell membranes (400x).

View larger version (108K): [in this window] [in a new window]   Fig 2. Immunohistochemical analysis for p53. Positive staining of tumor cell nuclei (400x).

Univariate Prognostic Analyses
Her-2/neu overexpression, considered as a dichotomous variable (negative or positive), correlated with a higher incidence of relapse after HDCT (P = .0005) and was a significant negative predictor of RFS (P = .002) (Fig 3A) and OS (P = .02) (Fig 3B). Considering Her-2/neu overexpression as an ordinal variable estimated by IHC semiquantitated scoring (from 0 to 3+), it correlated significantly with RFS (P < .05). The difference in RFS between Her-2neu 2+ and 3+ tumors was not statistically significant (P = .47). The correlation between the IHC scoring of Her-2/neu and OS did not reach statistical significance (P = .15). In contrast, p53 mutations were not associated with any of the following parameters: post-HDC recurrence (P = .22), RFS (P = .36), and OS (P = .42) (Fig 4).

View larger version (21K): [in this window] [in a new window]   Fig 3. (A) RFS according to Her-2/neu status and (B) OS according to Her-2/neu status.

View larger version (22K): [in this window] [in a new window]   Fig 4. (A) RFS according to p53 status and (B) OS according to p53 status.

Multivariate Analyses
Regression analyses of Her-2/neu overexpression combined with either tumor size, nodal ratio, and ER/PR status (Table 2) or with patient score (Table 3) showed that all variables analyzed were independent predictors of both RFS and OS. The addition of Her-2/neu overexpression to patient score can allocate patients to one of four different categories: (A) low score, Her-2/neu–negative (n = 65); (B) low score, Her-2/neu–positive (n = 45); (C) high score, Her-2/neu–negative (n = 16); and (D) high score, Her-2/neu–positive (n = 20). These four categories had statistically significant RFS curves (P < .000001), with differences between A and B (P = .002) and between B and C (P = .03) but not between C and D (P = .88) (Fig 5).

View larger version (17K): [in this window] [in a new window]   Fig 5. RFS curves based on patient score and Her-2/neu status. Group A (n = 65): low score, Her-2/neu–negative; B (n = 45): low score, Her-2/neu–positive; C (n = 16): high score, Her-2/neu–negative; D (n = 20): high score, Her-2/neu–positive. Differences between A and B: P = .002; between B and C: P = .03; between C and D: P = .88.

View larger version (27K): [in this window] [in a new window]   Fig 6. (A) RFS curves of the final model: group A (good risk; n = 65)—low score, Her-2/neu–negative; B (intermediate risk; n = 45)—low score, Her-2/neu–positive; C (poor risk; n = 36)—high score, any Her-2/neu. (B) OS curves of the final model, with the same patient groups as in (A).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

A previous analysis of patients who underwent transplantation at our program using STAMP-I showed that tumor size, nodal ratio (number of involved nodes/number of sampled nodes), and the combined ER/PR status were independent predictors of risk of relapse after transplantation.²³ A scoring system based on these three variables assigns high and low probabilities of relapse after HDCT. In the current study, we found that Her-2/neu overexpression, determined by IHC, was an additional independent negative predictor of RFS and OS. In contrast, p53 mutations lacked predictive significance. The addition of Her-2/neu overexpression to our model increased its predictive ability for the low-risk subset, whereas it had no impact on the high-risk category.

Although there is a wide variety of methods to evaluate Her-2/neu, studies that used paraffin-fixed material have used two of them: fluorescent in situ hybridization (FISH) to assess the amplification of the gene and IHC to demonstrate the overexpression of the protein. IHC for Her-2/neu has not been standardized yet, and assay variability, particularly concerning the types of anti–Her-2/neu antibodies used, constitutes a major problem when comparing results across studies that used IHC. Press et al²⁴ analyzed a panel of 28 anti–Her-2/neu antibodies, seven of them polyclonal and 21 monoclonal, in tumor blocks with known Her-2/neu amplification and overexpression. A great variability in sensitivity and specificity was observed, with monoclonal antibodies having greater specificity than polyclonal antibodies. The CB-11 monoclonal antibody showed 53% sensitivity and 100% specificity. Of note, that study did not use epitope retrieving methods or avidin-biotin complexes as detection systems. Both steps, used in our study, have been shown to substantially increase the sensitivity of the assay.^28,29 Thus, in all likelihood, our CB-11 assay has greater sensitivity than that in the study by Press et al.

The prevalence of Her-2/neu overexpression in our study (44.5%) is significantly higher than that of pooled data (with more than 800 patients) from published series that used CB-11 (18%)^24,30-33 (² test; P < 1 x 10^-14). Although these studies used the same antibody, differences in other details of the assay or in the arbitrary definition of positive staining might account for the difference. However, the fact that the frequency of Her-2/neu positivity seems comparable across those studies, with the exception of ours, makes this explanation unlikely. Because our HDCT trials targeted patients with high axillary tumor burden or IBC, a more aggressive tumor phenotype, possibly correlating with a higher prevalence of Her-2/neu overexpression, is an alternative explanation.

FISH has been claimed to be the most accurate technique to evaluate Her-2/neu.² However, in a recently reported comparison between IHC, using the Food and Drug Administration–approved DAKO polyclonal antibody, and FISH, a high (91%) concordance rate was observed between both methods.³⁴ Positivity by FISH and by IHC was found in 26% and 23% of the tumors, respectively. Because FISH is more time-consuming and expensive than IHC, these data do not support its consideration as the routine technique to evaluate Her-2/neu.

In a larger group of patients and with the use of a monoclonal antibody, our results confirm previous data from Bitran et al,³⁵ who determined Her-2/neu overexpression using a polyclonal rabbit antibody in 25 patients with more than 10 involved nodes treated with high-dose cyclophosphamide and thiotepa, which suggests a negative impact of this molecular alteration on freedom from relapse. In our study, the timing of relapses was different in Her-2/neu–negative and –positive patients. Only Her-2/neu–positive patients experienced a relapse 13 months after transplantation. This observation is intriguing and might be related to unique biologic features associated with the overexpression of this oncogene.

In contrast to the prognostic value of Her-2/neu overexpression in the HRPBC setting, its value in patients with MBC who are receiving HDCT is unclear. Doroshow et al³⁶ reported that Her-2/neu positivity was an independent negative predictor of survival in 55 patients treated with two different high-dose regimens. In contrast, in the randomized trial conducted by Bezwoda³⁷ that compared standard-dose to HDCT as front-line therapy for MBC, Her-2/neu positivity correlated with a worse outcome in the control arm but not in the high-dose arm.

In recent years, the anti–Her-2/neu monoclonal antibody trastuzumab has emerged as an active treatment against Her-2/neu–positive breast cancer.^38,39 Preclinical studies have shown pharmacologic synergy between trastuzumab and the following drugs: cisplatin, carboplatin, docetaxel, etoposide, and thiotepa.⁴⁰ Slamon et al⁴¹ demonstrated that trastuzumab plus chemotherapy, using either paclitaxel or adriamycin-cyclophosphamide, is superior to chemotherapy alone in MBC. These data provide a rationale for combining HDCT with trastuzumab in Her-2/neu–positive HRPBC patients.

We did not find a prognostic value for p53 mutations in our analysis. As with Her-2/neu, the study method and the type of antibody used constitute a major source of variability in results. Bonsing et al⁴² compared the sensitivity and specificity of a panel of seven monoclonal antibodies in tumor lines with previously known p53 mutations and concluded that two of them, DO1 and DO7, seemed superior to the rest.

The 34% prevalence of p53 mutations in our study was not significantly different from the 30% prevalence in the pooled results of published series that used DO7 monoclonal antibody (> 1,700 patients)^43-47 (P = .35), despite the inclusion of patients with greater numbers of positive nodes in our trials. This confirms previous reports that suggested that the prevalence of p53 mutations, contrary to that of Her-2/neu overexpression, does not vary significantly according to axillary status.^44,47

In contrast to our results, Somlo et al⁴⁸ reported in abstract form that p53 mutations, analyzed by IHC with a polyclonal antibody, were an adverse prognostic factor in 93 HRPBC patients treated with HDCT, using cyclophosphamide, etoposide, and either doxorubicin (n = 48) or cisplatin (n = 45). Previous studies have shown that the specificity of the antibody could be critical in the correlation of p53 status with outcome, with a significant association observed with antibodies with low but not high specificity.^45,47 Differences in the specificity of the polyclonal antibody used in the study by Somlo et al and our monoclonal DO7 antibody or in the drug composition between their regimens and STAMP-I might account for the different results.

The widely held belief that tumor cells die from apoptosis after anticancer therapy, which requires presence of wild-type p53, has been recently called into question in the case of solid tumors.⁴⁹ Preclinical studies that used clonogenic assays, which assess overall cell kill, instead of short-term assays, which focus on immediate apoptosis, show results contrary to the established tenet that tumor cells with mutations in p53 that make them resistant to apoptosis are also resistant to DNA-damaging agents. Such long-term studies show that the loss of functioning p53 does not alter sensitivity to doxorubicin,⁵⁰ camptothecin,⁵¹ paclitaxel, or vincristine⁵² and that it increases sensitivity to drugs whose toxicity is modulated by nucleotide excision repair, such as nitrogen mustards or platinum compounds.^53,54 Bunz et al⁵⁵ recently showed that p53 mutations simultaneously conferred resistance to fluorouracil and sensitivity to doxorubicin and radiotherapy in a human colon cancer cell line. It has been speculated that, depending on which p53-modulated activity predominates in a particular cell line, different observations might be expected after treatment of cells with p53 mutations.⁵⁶ Thus, in cells in which the control of S phase entry predominates, the loss of wild-type p53 would sensitize the cell to DNA damage. Conversely, if apoptosis induction predominates, loss of functioning p53 would confer resistance.

In conclusion, we evaluated 146 HRPBC patients receiving HDCT and found that Her-2/neu overexpression determined by IHC but not p53 mutations is an independent negative predictor of RFS and OS in this setting. The addition of Her-2/neu overexpression to our predictive model, which is based on tumor size, nodal ratio, and ER/PR status, increased its predictive capacity in the low-risk category.

	APPENDIX

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
The following Pathology Departments submitted patient tumor blocks: Albert Einstein Medical Center, Philadelphia, PA; AnaPath Diagnostics, Inc, Cheyenne, WY; Aurora Pathology Associates, Denver, CO; Boulder Community Hospital, Boulder, CO; Central Suffolk Hospital, Riverhead, NY; Cunningham Pathology Associates, Birmingham, AL; Deaconess Medical Center, Billings, MT; Denver General Hospital, Denver, CO; Florida Hospital, Lake Mary, FL; Good Samaritan Regional Medical Center, Suffern, NY; Good Samaritan Medical Center, Phoenix, AZ; High Plains Baptist Hospital, Amarillo, TX; Humana Hospital, Chicago, IL; Ivinson Memorial Hospital, Laramie, WY; Johns Hopkins Hospital, Baltimore, MD; Lenox Hill Hospital, New York, NY; Littleton Hospital, Littleton, CO; Lourdes Hospital, Binghamton, NY; Lutheran Medical Center, Wheat Ridge, CO; Craig Memorial Hospital, Craig, CO; Memorial Sloan-Kettering Cancer Center, New York, NY; Mercy Medical Center, Durango, CO; Montrose Memorial Hospital, Montrose, CO; North Colorado Medical Center, Greeley, CO; North Shore University Hospital, Manhasset, NY; Northwestern Memorial Hospital, Chicago, IL; Norton Hospital, Louisville, KY; Orlando Medical Center, Orlando, FL; Parkview Episcopal Medical Center, Pueblo, CO; Pathology Services, P.C., Denver, CO; Penrose–St Francis Health Care Center, Colorado Springs, CO; Porter Memorial Hospital, Denver, CO; Poudre Valley Hospital, Fort Collins, CO; Presbyterian–St Luke Medical Center, Denver, CO; Rose Medical Center, Denver, CO; Rush-Presbyterian-St Luke Medical Center, Chicago, IL; San Juan Regional Medical Center, Farmington, NM; Memorial Hospital of Sheridan County, Sheridan, WY; Sinai Hospital, Detroit, MI; Southwest Hospital, Cortez, CO; St Anthony Hospital, Denver, CO; St Joseph Hospital, Denver, CO; St Luke’s Hospital of Kansas City, Kansas City, MO; St Mary-Corwin Hospital, Pueblo, CO; St Thomas More Hospital, Canyon City, CO; St Vincent Hospital, Billings, MT; Swedish Medical Center, Englewood, CO; Unipath, Denver, CO; University of Chicago Hospitals, Chicago, IL; University of Utah Medical Center, Salt Lake City, UT; Valley View Hospital, Glenwood Springs, CO; Wesley Medical Center, Wichita, KS; William Beaumont Hospital, Troy, MI; Wilson Memorial Regional Medical Center, Johnson City, TN; Wyoming Medical Center, Casper, WY.

	NOTES

 
Presented in part at the Thirty-Fifth Annual Meeting of the American Society of Clinical Oncology, Atlanta, GA, May 15-18, 1999. Y.N. is an American Society of Clinical Oncology Merit Award recipient.

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Submitted September 22, 1999; accepted January 28, 2000.

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