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Response to Cyclophosphamide, Methotrexate, and Fluorouracil in Lymph Node–Positive Breast Cancer According to HER2 Overexpression and Other Tumor Biologic Variables

From the Molecular Targeting Unit, Department of Experimental Oncology, Pathology Department, Medical Oncology Unit, Scientific Direction, Istituto Nazionale Tumori; Institute of Medical Statistics and Biometry, Università degli Studi di Milano, Milan, Italy.

Address reprint requests to Sylvie Ménard, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milano, Italy; email menard@ istitutotumori.mi.it.

	ABSTRACT

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PURPOSE: There is considerable interest in biologic markers able to predict the response of cancer patients to therapy. HER2 overexpression is a potential indicator of responsiveness to doxorubicin and paclitaxel and of unresponsiveness to tamoxifen in breast carcinoma patients. However, the significance of HER2 overexpression in responsiveness to cyclophosphamide, methotrexate, and fluorouracil (CMF) has remained unclear. In this study, we investigated this issue in the 386 breast cancer patients in the first CMF controlled clinical trial with a 20-year follow-up.

PATIENTS AND METHODS: Node-positive breast carcinoma patients were randomly assigned to receive either no further treatment after radical mastectomy (179 women) or 12 monthly cycles of adjuvant CMF chemotherapy (207 women). Overexpression of HER2 and the status of other tumor variables was assessed by immunohistochemistry in at least 324 (84%) of the 386 patients. Statistical analyses were performed to assess the efficacy of CMF treatment for the subgroups defined by HER2 and the status of other variables using a Bayesian approach. The end points considered were relapse-free survival (RFS) and cause-specific survival (CSS).

RESULTS: Bayesian analysis of the treatment effect for HER2 and other variables indicated a clinical benefit from CMF treatment in all subgroups defined according to variables status. In particular regarding HER2 status, Bayesian estimates of RFS hazard ratios were equal to 0.484 and 0.641 and estimates of CSS hazard ratios were equal to 0.495 and 0.730 for HER2-positive and -negative tumors, respectively.

CONCLUSION: CMF treatment showed a clinical benefit in the considered subgroups, defined according to HER2 and other tumor variables status. Patients with HER2-positive or HER2-negative tumors benefit from CMF treatment, and the poor prognosis associated with the HER2 overexpression in the untreated group could be completely overcome by the chemotherapy treatment.

	INTRODUCTION

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RANDOMIZED CLINICAL trials have clearly demonstrated the efficacy of systemic adjuvant chemotherapy or hormone therapy in women with breast carcinoma, although the advantage is of a modest magnitude. Such an advantage is the result of the unselective application of adjuvant therapy to all patients within a broad category of risk. In view of the potential benefit of identifying those patients who will actually get a therapeutic advantage from a specific treatment, the investigation of biologic markers able to predict response has become one of the major goals in oncobiology. A better knowledge of the biology of breast carcinoma indicates a heterogeneous expression of molecules that are likely involved in the sensitivity of the neoplastic cells to therapy, offers an explanation for the differential response of tumors to different therapeutic procedures, and provides important tools for investigating this aspect in the clinic.

So far, the biologic classification of tumors as responsive or not responsive to medical treatment has been difficult and disputed. To date, the only accepted predictor of response is the hormone receptor expression for endocrine therapy. Several retrospective studies have suggested the predictive value of proliferation index as well as tumor grade in the response to chemotherapy, but no prospective clinical trials have validated these markers as predictors of response to specific drugs. More recently, retrospective studies have pointed to the importance of HER2 overexpression in the response to doxorubicin^1-3 or to paclitaxel,⁴ whereas, tamoxifen was found to be ineffective⁵ and possibly detrimental in patients with HER2+ tumors,⁶ even though recent new data indicates that tamoxifen is effective among estrogen receptor (ER)-positive patients independent of HER2 status.⁷ Prediction of the response to the cyclophosphamide, methotrexate, and fluorouracil (CMF) regimen is more controversial. Some studies suggested that ER-negative tumors had a better outcome after CMF chemotherapy.⁸ Other reports suggested that CMF is effective only in HER2- tumor patients, with no benefit to patients with HER2+ tumors.^9,10 Another study reported a good response only in HER2+ tumor patients,¹¹ whereas Miles et al⁸ found response independent of HER2 status.

Based on in vitro analysis, the role of HER2 overexpression in sensitivity of cancer cells to drugs is indirect,¹² suggesting that the in vivo response is also indirect and perhaps rests on the association of HER2 with other factors involved in drug response. Indeed, HER2 overexpression is associated with tumor grade, high number of mitoses, topoisomerase II expression, and decreased hormone receptor.¹³

Therefore, in investigating the independent contribution of HER2 and other tumor variables on response to therapy, a case series with a suitable control group must be available, and the statistical analysis must be performed with suitable multivariate procedures that make allowance for the possible interrelationships between biologic and clinical variables, avoiding the drawbacks of conventional subgroups analyses. In the present study, we analyzed the impact of HER2 status, jointly with other clinical and pathologic variables, on the therapeutic response of node-positive breast cancer patients enrolled onto a randomized trial of CMF versus no treatment after radical mastectomy.

	PATIENTS AND METHODS

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Patients
The study group consisted of patients enrolled between June 1973 and September 1975 in a prospective randomized trial carried out at the Istituto Nazionale Tumori in Milan, Italy.¹⁴ All women 75 years of age or younger who had had a radical mastectomy for unilateral carcinoma of the breast and who had histologic evidence of involvement of one or more axillary nodes were considered for inclusion in the study. The protocol design was approved by the members of the institute’s research and ethics committees.

After stratification for age ( 49 years and 50 to 75 years), number of axillary nodes involved (one to three and four or more), and type of radical mastectomy (conventional or extended), patients were randomly assigned to receive either CMF (cyclophosphamide orally 100 mg/m² days 1 to 14, methotrexate intravenously 40 mg/m² days 1 and 8, and fluorouracil intravenously 600 mg/m² days 1 and 8) for 12 monthly cycles or no further treatment. No additional therapy was planned beyond that allowed in the protocol without documented evidence of treatment failure. In particular, no postoperative irradiation and no adjuvant endocrine therapy were administered. Of the original group of 391 patients, five patients could not be evaluated,¹⁰ thus leaving 386 patients (control, n = 179; CMF, n = 207) fully assessable.

For the relapse-free survival (RFS), treatment failure was considered to have occurred with the first documented evidence of new manifestations of disease in locoregional areas (including homolateral supraclavicular adenopathy), distant sites, the contralateral breast, or any combination of these sites. Among the total case series of 386 women, 270 patients experienced new manifestation of disease; 53 in locoregional areas, 192 in distant sites (of which 25 in distant plus locoregional), and 25 in contralateral breast. Neither second primary cancers nor deaths as a result of causes other than breast cancer were considered treatment failures.

On a total of 282 deaths, 238 patients died from breast cancer, and 44 patients died from other causes (14 for a tumor in other sites and 30 for nonneoplastic causes). Causes of death were ascertained through medical records, death certificates, family doctors, and, when available, autopsy records. For cause-specific survival (CSS) only deaths related to breast cancer were considered treatment failures. It is worth mentioning that timings of non–breast cancer deaths were fairly well distributed between the two treatment groups.

Before surgery, all patients underwent a complete physical examination, x-ray study of the chest and skeleton (skull, spine, pelvis, and upper third of femurs), bilateral mammography, a differential blood count with platelet count, and biochemical tests. In the absence of symptoms, physical examination was performed every 4 weeks during the first year, every 6 months for the next 4 years, and every 12 months for the following 10 years. Biochemical tests, chest roentgenography, and bone roentgenography or bone scanning were performed every 6 to 8 months during the first 5 years and once a year thereafter. Mammography was planned once a year. After the 15th year of follow-up, the patients were examined every 12 to 18 months. In patients with suspicious or controversial radiologic findings, examinations were performed more often. Liver ultrasonography was performed only if there were suspicious clinical or biochemical findings. Median follow-up for the entire case series was 20 years at the time of present analysis.

Pathologic Determinations
Primary tumor diameter and axillary nodal status were obtained from histopathologic reports. Hematoxylin and eosin-stained slides of all patients were retrospectively reviewed for diagnostic reassessment of the histotype of the tumor, histologic grade, presence of cell necrosis, and lymphocytic infiltration. Histologic grade was determined and scored as described by Elston and Ellis¹⁵ and defined as Nottingham/Tenovus grade. The method used involves semiquantitative evaluation of the percentage of tubule formation, the degree of nuclear pleomorphism (nuclear grade), and the mitotic count per 10 high power field (x40 magnification). The density of lymphocyte infiltration was classified semiquantitatively as mild, moderate, or marked, when present or absent.

Biologic Determinations
At the same time of retrospective pathologic review, a panel of immunocytochemical stains was performed on paraffin-embedded tissue. The following panel of monoclonal antibodies was applied: anti c-erbB-2 CB11 (1:10 diluted, Ylem, Avezzano, Aquila, Italy), anti-p53 MAb DO7 (1:500 diluted, Novocastra, Newcastle-on Tyne, United Kingdom), anti bcl2 MAb 100 (1:20 diluted, a kind gift from David Mason), anti-ER (clone 1D5; 1:200 diluted, DBA, Segrate, Milan, Italy), anti–progesterone receptor (PgR) MAb1A6 (1:100 diluted, DBA, Segrate, Milan, Italy), anti–laminin receptor MLuC5,¹⁶ and epidermal growth factor receptor.

Immunostaining was performed by a sensitive peroxidase-streptavidin method on Bouin-fixed, paraffin-embedded material. Consecutive sections were processed (cut, deparaffinized, and rehydrated) and pretreated with heat-induced epitope retrieval method.¹⁷ After washes in phosphate-buffered saline, immunostaining was performed using an automated immunostainer (Dako TechMate 1,000, Milan, Italy). The primary antibody was replaced with a nonimmune serum, from the same species in which the primary antibody was produced, as negative control. Appropriate cases with known reactivity for each antibody applied were used as positive controls. Sections were scored positive when more than approximately 10% of the tumor cells were labeled except HER2, which was scored as positive when a strong or intermediate membrane labeling was observed.

Statistical Analysis
Association between HER2 status and other ordinal variables was measured by Kendall’s _b exact tests, and pertinent 95% confidence limits are provided. This statistic ranges between -1 (total inverse association) and 1 (total direct association). For the special case of 2 x 2 tables the absolute value of _b is identical to the Phi and Tschuprov contingency coefficients.¹⁸ Because of the multiple testing procedure, to account for the inflation of type I statistical error, the Bonferroni correction was adopted.

Survival curves for the four groups defined by the combination of treatment and HER2 status were estimated by a stratified product limit estimator proposed by Chen et al,¹⁹ which, for each group, takes into account the presence of unequal survival functions among strata defined by the other variables.

Different adjuvant treatment effects in subsets identified by the covariate modalities were investigated by the Bayesian subset analysis method proposed by Simon et al.²⁰ This approach was adopted to limit the risks one takes when analyses investigating the interaction between treatment and several covariates are carried out in a frequentist frame. This method works under the assumption that the interaction terms are exchangeable, ie, no interaction is considered a priori more likely than any other and the interactions in one direction are no more likely than those in the opposite direction. For the working hypotheses proposed by the clinical staff, this was reasonable because the available information about possible differential effect of chemotherapy in patient subgroups defined by the considered covariates was consistent with the above assumptions. The procedure shrinks the estimated interaction effects toward zero, with a resulting control of the multiplicity of tests on the interactions, avoiding the fact that significant results will emerge only on the basis of chance alone.

Briefly, a Cox regression model with treatment, clinical, pathologic, and biologic covariates and the interaction between treatment and covariates was applied. In this instance, only binary covariates were adopted in the model, and the continuous variables were categorized according to conventional clinical criteria. The proportional hazard assumption, underlying the application of the Cox model, was tested according to the method proposed by Grambsch and Therneau.²¹ Following Simon et a,l²⁰ a skeptical prior distribution for interaction might correspond to assuming a null average (absence of interaction) and to calculate variance under a specific hypothesis on the probability of reducing the hazard for the treatment in one subset, given no treatment effect in an adjacent subset is 0.05. The reduction of 50% of the hazard was the smallest treatment difference considered clinically relevant. The untreated group of patients was taken as the reference group; the results on the posterior distribution are reported as hazard ratios for CMF treatment, together with their 95% highest posterior density (HPD) intervals and the probability of the reversion of the treatment effect. HPD corresponds to the estimated posterior distribution given prior assumptions and sample information. Ninety-five percent HPD intervals are simply the posterior mean, plus or minus 1.96 times the posterior SD.

	RESULTS

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Association Between HER2 Overexpression and Patient and Tumor Characteristics
HER2 determination was available for 337 (87.3%) of 386 patients. The frequency of HER2 overexpression was similar in the two arms (22 [14.6%] of 151 patients in the control group and 32 [17.2%] of 186 patients in the CMF-treated group). The clinical, pathologic, and biologic characteristics and the distribution of HER2 overexpression are listed in Table 1. Considering Bonferroni’s correction for multiple testing procedures and in keeping with previous reports, HER2 overexpression was found inversely associated with the expression of ER and PgR.

View larger version (16K): [in this window] [in a new window]   Fig 1. RFS relative to HER2 status. CMF-treated cases (dashed line), untreated cases (solid line).

View larger version (16K): [in this window] [in a new window]   Fig 2. CSS relative to HER2 status. CMF-treated cases (dashed line), untreated cases (solid line).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

The analysis took into account the HER2 status and several classical prognostic factors (such as age, number of positive axillary lymph nodes, mitosis, and ER and PgR status), as well as more recent biologic markers of tumor aggressiveness (such as p53 and laminin receptor). The results of the Bayesian analysis on the effect of treatment showed that CMF exerted a protective effect for both HER2+ and HER- tumors as well as for the other groups defined by the other variables considered (Table 2). Present findings, which are in contrast to some published data, warrant some comments on the reported resistance, complete or partial, to chemotherapy for tumors that overexpress her2 protein.

Early studies suggested that HER2-positive tumors do not benefit from CMF-based chemotherapy that, by contrast, has a considerable effect in tumors with no or minimal expression of this oncogene. However, caution is necessary in accepting the conclusions reported on the divergent effect of CMF-based regimens. Allred et al¹⁰ has suggested a role for HER2 in resistance to CMF adjuvant therapy. However, in that study of node-negative patients, the HER2+ group displayed a particularly good prognosis, much better than that in the HER2- group, and it is unlikely that any treatment would have improved this good overall survival. In addition, in that study the outcome analysis for women randomized to observation only was restricted to the subset of patients with small, ER-positive and predominantly invasive tumors. In the study by Gusterson et al,⁹ in node-positive breast cancers, the more prolonged and effective regimen consisted in the association of CMF plus low-dose prednisone, and tamoxifen was also given to postmenopausal women, whereas CMF alone, given for one single cycle in the perioperative period, was taken as the control group. In light of a potential detrimental effect of tamoxifen in some subsets of HER2+ tumors,⁶ results from this case series are not adequate to conclusively assess prediction of responsiveness to CMF. Similarly, in node-negative patients, the administration of one single cycle of chemotherapy may be insufficient to test drug resistance. In a report by Stal et al,²³ which compared survival according to radiotherapy with CMF therapy, the authors concluded that patients with HER2+ tumors benefited more from radiotherapy than from chemotherapy. However, no conclusions concerning the intrinsic responsiveness to CMF of these tumors can be drawn from these data, in that this report included only 28% of the total population enrolled onto the original randomized study. Moreover, that study included only 12 HER2+ patients treated with CMF who experienced a particularly poor survival. So far, only Klijn et al¹¹ have reported a preferential sensitivity to CMF in HER2+ tumors from patients with metastatic breast cancer, citing a response rate of 75% in these patients versus 45% in patients with HER2- tumors; whereas, in keeping with our findings, Miles et al⁸ reported a similar sensitivity of HER2+ and HER2- tumors to CMF. Similarly, also the hormone receptor status is not predictive of the response to CMF, as suggested recently.⁸

Our case series has the unique feature of being derived from a randomized study, with a follow-up of a median of 20 years and with a concomitant control group treated solely with radical mastectomy. Despite the fact that sample size was more limited compared with other series,^1-3 the probability of a reversion of treatment effect is almost negligible (Table 2), thus confuting the conclusions reported in the above studies and confirming the benefit of CMF regardless of HER2 expression.

Could the results achieved by CMF in HER2+ tumors be improved by the use of doxorubicin-containing regimens? Despite the intriguing results from two case series^3,12 and the reported association between levels of her2 protein and topoisomerase II expression in breast cancer,¹² there are not enough data at present to positively answer this question. In fact, none of the two reports could evaluate the role of doxorubicin in comparison with the classical CMF adjuvant chemotherapy. The Cancer and Acute Leukemia Group B activated a study in node-positive patients randomly allocated to three dose levels of cyclophosphamide, doxorubicin, and fluorouracil (CAF), and a first analysis, performed on a subset of 397 patients reported a similar treatment outcome for all three dose levels in HER2- tumors but showed a significant dose-relationship in HER2+ tumors. The authors concluded that overexpression of this protein could be a useful marker to identify patients who are most likely to benefit from a high dose of the CAF regimen.¹ A more recent validation study included an additional 595 patients, a higher fraction of whom also received tamoxifen therapy. The updated analysis of data from the test series confirmed an interaction between HER2 expression and dose levels of CAF chemotherapy; whereas in the validation series, the significance was achieved only after adjustment for differences by use of a prognostic index to balance an apparent failure of randomization in the low-dose arm.² Paik et al,³ from the National Surgical Adjuvant Breast and Bowel Project, examined the effect of doxorubicin in node-positive, hormone receptor–negative breast cancer patients randomly allocated to receive L-phenylalanine mustard plus fluorouracil with or without the addition of the anthracycline. They reported that the administration of doxorubicin (albeit delivered at a low dose of 30 mg/m²) exhibited a statistically significant benefit in tumors that were positive for HER2, but no effect of the anthracycline was detected in HER2- tumors.

The work discussed above and the interest dedicated to the definition of factors predicting response to therapy is clearly based on the growing need to identify, within each broad category of patients at similar risk of relapse and death, specific subsets of cases who have a significant probability of response to therapy and eventually cure, as well as to sort out those women who have very little chance of response. The embedded risk of this search is that, based on controversial findings from studies designed for other purposes, patients are considered resistant to treatments that are indeed active. With this in mind, should contemporary adjuvant chemotherapy regimens be selected on the base of HER2? In our opinion, the inconclusive and controversial nature of the available retrospective studies^9,10 and the diametrically opposite results of the current work on CMF do not support the routine use of HER2 status in everyday practice to make a choice of drugs for adjuvant therapy. The findings from our study refute the hypothesis of resistance to non–doxorubicin-containing regimens for HER2-positive tumors. There are several reasons for the divergence of our data and previous reports, and all of them point to the risk of basing therapeutic decisions on soft ground. Published data are largely derived from randomized studies that were not designed for, and may not have sufficient statistical power to, appropriately testing interaction of treatments, content of her2 protein, and the role of other molecular markers in this interaction. In addition, laboratory techniques and scoring methods must be standardized so that results are reproducible across different case series. Once this standardization is achieved, then new prospective randomized trials comparing optimal conventional chemotherapy regimens versus newer and potentially more effective combinations (eg, taxane-based regimens) must enroll a number of patients sufficient to adequately test treatment-molecular marker interactions.

In conclusion, the data from the present analysis show that the negative prognostic value of HER2 overexpression in women who underwent radical mastectomy as the only therapeutic modality is overcome by the administration of adjuvant CMF. To date, there is no definite proof that overexpression of HER2 is a marker of resistance to CMF chemotherapy. This observation further supports the concept that appropriate analyses of controlled studies are needed to shed light on the complex interaction between marker expression such as HER2 status and hormone receptor status of the tumor and the therapeutic activity of hormonal and cytotoxic drugs before drawing conclusions that may negate active therapeutic options to women with breast cancer.

	ACKNOWLEDGMENTS

 
This work was partially supported by Associazione Italiana per la Ricerca sul Cancro.

	NOTES

 
The first two authors contributed equally to this work.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted October 27, 1999; accepted August 22, 2000.

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