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Prognostic Value of Histologic Grade and Proliferative Activity in Axillary Node–Positive Breast Cancer: Results From the Eastern Cooperative Oncology Group Companion Study, EST 4189

From the Department of Pathology, Vanderbilt University Medical Center, Nashville, TN; Dana Farber Cancer Institute, Boston, MA; University of North Carolina, Chapel Hill, NC; Flower Memorial Hospital, Sylvania, OH; University of Pretoria, Pretoria, South Africa; University of Wisconsin Comprehensive Cancer Center, Madison, WI; University of Rochester Cancer Center, Rochester, NY; and Fairfax Hospital, Falls Church, VA.

Address reprint requests to Jean F. Simpson, MD, Department of Pathology, MCN C3321, Vanderbilt University Medical Center, Nashville, TN 37232.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: The identification of a subset of patients with axillary lymph node–positive breast cancer with an improved prognosis would be clinically useful. We report the prognostic importance of histologic grading and proliferative activity in a cohort of patients with axillary lymph node–positive breast cancer and compare these parameters with other established prognostic factors.

PATIENTS AND METHODS: This Eastern Cooperative Oncology Group laboratory companion study (E4189) centered on 560 axillary lymph node–positive patients registered onto one of six eligible clinical protocols. Flow cytometric (ploidy and S-phase fraction [SPF]) and histopathologic analyses (Nottingham Combined Histologic Grade and mitotic index) were performed on paraffin-embedded tissue from 368 patients.

RESULTS: Disease recurred in 208 patients; in 161 (77%), within the first 5 years. Mitotic index and grade were associated with both ploidy and SPF (P .01). Within the first 5 years of follow-up, mitotic index (P = .004), grade (P = .004), ploidy (P = .006), and SPF (P = .05) were associated with time to recurrence; there was also a significant association with survival. The effect of mitotic index was largely a result of the difference between 0 to 2 mitoses/10 high-power fields (HPF; 5-year recurrence of 31%) and more than 2 mitoses/10 HPF (5-year recurrence of 52%). The 0 to 2 mitoses/10 HPF group was independently associated with improved prognosis at 5 years (P = .002) in regression models that included other standard prognostic factors.

CONCLUSION: A subset of axillary lymph node–positive patients with improved prognosis may be identified using a lower (< 3 mitoses/10 HPF) mitotic count than is usually performed.

	INTRODUCTION

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BREAST CANCER prognostication is largely driven by relevance to therapy. Although much of the recent emphasis of prognostication in breast cancer patients has been related to node-negative disease,^1,2 predicting clinical behavior and response to therapy may be relevant for patients with axillary metastases. When there is a lack of consensus regarding appropriate therapy in this group of patients, resolution should rely on dependable prediction of survival. Even though nodal status is the single most important predictor of distant metastasis and overall survival, this factor is not always sufficient to allow treatment decisions to be made. For those who present with involved axillary lymph nodes (approximately one quarter of all patients with breast cancer), therapeutic options range from tamoxifen only to high-dose adriamycin-based protocols. Within lymph node–positive breast cancer, patients who have fewer than four nodes involved have a better prognosis than those with four or more involved nodes. Refinement of prognostication is needed within these two groups to identify women with minimal node involvement who have a poor prognosis, as well as those with more than four nodes involved who may survive longer than expected based on lymph node staging.

Predicting treatment response, as well as behavior, is also useful in the search for prognostic factors in node-positive breast cancer. Recently, the relative importance of prognostic factors within different subsets of invasive carcinoma, including stratification by therapy, has been recognized. Thus, although the importance of the oncogene c-erbB-2 in breast cancer has been viewed by some as an independent prognostic indicator, there are conflicting reports of its ability to predict therapeutic responsiveness. It may indeed, however, be important within patients with lymph node–positive cancer.^3,4 For example, it has been shown that the expression of the c-erbB-2 gene may be important as a predictor of therapeutic response in adriamycin-containing regimens. In addition, c-erbB-2–expressing tumors may be amenable to immunotherapy (trastuzumab; Herceptin, Genentech, San Francisco, CA). Thus, analyzing prognostic factors within carefully defined subsets of patients may refine their predictive ability.

Refining the predictive utility of tumor stage is an active area of research, and additional prognostic information may be derived from careful histopathologic assessment of invasive carcinoma. By recognizing particular patterns (special types) of invasive carcinoma and assignment of histologic grade, subsets with a good prognosis may be identified.⁵ The Nottingham Combined Histologic Grade (NCHG) system is a modification of the Scarff-Bloom-Richardson grading system⁶ and reflects combined analysis of glandular differentiation, nuclear grade, and mitotic activity. The NCHG has been shown to be predictive⁷ and reproducible⁸ and is in widespread use.

Measurements of proliferative activity can yield additional prognostic information and may be ascertained in several ways (in addition to the mitotic activity component of NCHG), including expression of the cell proliferation antigen Ki-67 (using MIB-1 immunohistochemical analysis) and DNA flow cytometry. These measures of proliferation are usually demonstrated to be clearly correlated,^9,10 but comparative studies of clinical outcome in the same patient group are rare.

The impact of histopathologic evaluation has been difficult to ascertain because of its frequent exclusion in univariate as well as multivariate analysis of potentially useful new prognostic factors. For example, proliferative capacity as determined by flow cytometric analysis of S-phase fraction (SPF) has been explored as a predictor,¹¹ but because histopathologic analysis is often not included, recognizing an independent contribution of SPF has been difficult.

In this study, we explored the interaction between specific histopathologic variables and flow cytometrically derived estimates of proliferation and ploidy. Our purpose was to examine histologic grade and proliferative activity to identify patients with an improved outcome despite the presence of axillary lymph node metastasis.

	PATIENTS AND METHODS

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This laboratory companion study (E4189) focused on 560 patients with node-positive breast cancer who had been previously registered to one of six eligible Eastern Cooperative Oncology Group breast cancer clinical protocols (Table 1), which comprised a variety of treatment regimens for pre- and postmenopausal women. Paraffin-embedded material was available from 398 primary tumors and 363 corresponding axillary node metastases. Clinical and pathologic characteristics, including flow cytometric and histopathologic analysis, were available from a total of 368 patients (Table 2). The analysis herein focuses on these 368 patients and directly compares these two parameters. The characteristics (type of therapy, number of involved lymph nodes, estrogen receptor [ER] status, tumor size, age at entry, and menopausal status) and survival data of patients entered onto E4189 were not different from those of patients not entered onto this ancillary trial.

Other parameters evaluated include number of involved lymph nodes, ER status, tumor size, menopausal status, and age at registration to clinical trial. The number of involved lymph nodes and tumor size were determined by the pathology report. ER content was analyzed by the charcoal-dextran method.

DNA Flow Cytometric Analysis
DNA flow cytometry was performed on formalin-fixed paraffin-embedded tissue using a modification of Hedley’s technique as previously described.¹⁴ All measurements were performed in a single laboratory on an EPICS 753 flow cytometer (Coulter Electronics, Hialeah, FL). Histograms were gated on peak versus integrated red fluorescence to minimize doublet contribution, and 25,000 gated events were collected for each histogram. Cell cycle analysis was performed using the Verify Modfit (Coulter Electronics) program, which incorporates models as previously described.¹⁵ The median S-phase value, 6.7%, was used to define high and low SPF. Criteria for the assessability of ploidy and SPF included coefficient of variation 7.00% for DNA diploid and aneuploid G0/G1 populations, confirmation of DNA diploid position (external control-negative lymph node sample), and 5% to 10% minimum events to define a peak as real versus artifact.¹⁶

Statistical Methods
Because of the heterogeneity in the E4189 patient population, the analysis was limited to evaluating the average effect of the prognostic factors across the study population. To avoid possible confounding with study and treatment effects, tests that compared prognostic groups were stratified on study and treatment. Comparisons were made within each stratum, and the contributions were summed over the strata to give an overall test. Time to relapse (TTR) was measured from date of entry onto the parent protocol until first evidence of recurrent disease or the development of new primary carcinoma in the contralateral breast and was censored at the date the patient was last known to be disease-free. Patients who died without recurrence were censored at the date of death. Survival was measured from date of entry onto the parent protocol to the date of death (from any cause). The main analysis follow-up was censored at 5 years for all patients to counteract difficulties for modeling effects that may be seen with prognostic factors at longer follow-up intervals. Separate analysis was performed at later time points for patients who survived and were disease-free at 5 years.

The association between grouped or categorical factors was determined by Mantel-Haenszel tests. TTR and survival curves were estimated using the method of Kaplan and Meier.¹⁷ Comparisons of TTR and survival distributions among groups were made using stratified log-rank tests.¹⁸ The joint effect of multiple factors was modeled using the partial likelihood analysis of the proportional hazards model.¹⁹ These models were stratified on study and on treatment arm. Partial likelihood ratio tests were used to assess the significance of effects. Martingale residual plots²⁰ were used to check the adequacy of models for continuous variables.

	RESULTS

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Patients Available for Study
During the time that E4189 was open to accrual (July 1990 to March 1994), of the 2,643 eligible patients, 560 were registered, with 97% enlisted by the end of 1992. The numbers of patients entered by parent protocol and treatment arm are listed in Table 1. There were no differences in age, tumor size, number of positive nodes, ER status, TTR, and survival between patients from the parent protocol who were entered onto E4189 and those who were not (data not shown). Thus, the registered patients seem representative of the patients from the parent protocols.

There were 368 patients who were suitable for histologic grading and who had been analyzed by flow cytometric analysis. Of these, SPF was assessable in 257. There were no differences in age, tumor size, number of positive nodes, ER status, TTR, and survival between E4189 patients included in the main analysis and the patients for whom grade or flow cytometry were not analyzed (data not shown). Thus, the 368 patients seem representative of the patients registered for E4189.

The clinical and pathologic characteristics of the patients are presented in Table 2. Because all but one of the Eastern Cooperative Oncology Group clinical protocols were restricted to pre- or postmenopausal patients (Table 1), little information remained regarding the effect of menopause after adjusting the analysis for study effects; therefore, this variable is not further considered in the analysis. ER status was known for all but three patients, each an elderly patient. Because breast cancer in the elderly is usually ER-positive, these patients were considered with those who were ER-positive for the purposes of analysis.

Histologic Grade and Mitotic Index
The vast majority of patients had invasive carcinomas of no special type. There were too few special-type carcinomas to evaluate separately for prognostic significance. The only special-type carcinoma that would confound a grading study is medullary carcinoma, which does so because it often has a good prognosis despite high-grade features. There were four patients with medullary or medullary variant carcinoma, and these patients were not included in the outcome analysis. Indeed, all four were alive and disease-free at follow-up times from 8.6 to 11.6 years.

The distribution of the NCHG is presented in Table 3. Note that if patients were evaluated by architectural assessment only, 79% of all patients would be classified as poorly differentiated (< 10% of tumor-forming tubules). Similarly, if only nuclear grade were assessed, 36% of patients would be high grade (marker nuclear pleomorphism) and only 13% would qualify as low grade. Yet by the NCHG, the three different grades are more evenly distributed, with 22% grade 1, 45% grade 2, and 33% grade 3 carcinomas.

Flow Cytometric Analysis
Three quarters of the patients were aneuploid. Of the total 368 patients analyzed, SPF was assessable in 257 (70%). The median value was 6.88%. Patients were divided into three groups according to SPF: less than 4.55, 4.56 to 9.34, and more than 9.34.

Associations of Variables
Of the factors listed in Table 4, ER status, DNA ploidy, and SPF were significantly associated with the NCHG (P < .001, P = .0036, and P < .0001, respectively) (Table 4). These same factors were also significantly associated with mitoses/10 HPF (P < .0001, P = .013, and P < .0001, respectively). None of the factors (grade, mitotic index, ploidy, or SPF) showed significant associations with age, tumor size, or number of positive nodes (Table 4). All except ploidy showed a significant association with ER status (P < .003). The association of S phase with mitotic index and with grade was highly significant, and all three variables were associated with ploidy (Table 4).

Among the variables analyzed, grade, mitoses/10 HPF, and SPF were associated with TTR (P = .004, P = .004, and P = .04, respectively). Figures 1 to 3 show time-to-recurrence curves based on these variables. As seen in Fig 1, the proportion of women who had recurrence was significantly greater for those with higher grade carcinomas, compared with low-grade tumors. The effect of proliferative activity on time to recurrence is illustrated in Figs 2 and 3. Stratifying by mitoses/10 HPF demonstrates an improved time to recurrence for those women whose tumors were less mitotically active (< 10 mitoses/10 HPF) than those who had more than 10 mitoses/10 HPF. As seen in Fig 2B, the group of women whose tumors had low proliferative activity (< 3 mitoses/10 HPF) were responsible for this improved time to recurrence (P = .0004). Although SPF was marginally associated with TTR overall (Fig 3), analysis within patients with diploid only and those with aneuploid only was not significant (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig 1. Kaplan-Meier curves. 5-year time to recurrence of axillary lymph node–positive breast carcinoma, stratified by combined histologic grade.

View larger version (23K): [in this window] [in a new window]   Fig 2. 5-year time to recurrence of node-positive breast carcinoma, by mitotic activity. (A) Lowest group was less than 10 mitoses/10 HPF. (B) Most of the improved survival in the lowest group was a result of patients with less than 3 mitoses/10 HPF.

View larger version (17K): [in this window] [in a new window]   Fig 3. Time to recurrence (censored at 5 years) based on SPF.

Models that combined SPF with grade and mitoses/10 HPF are given in Tables 6 and 7. The effect of SPF was not significant if either mitoses/10 HPF or grade was included in the model. Mitoses/10 HPF was significant combined with SPF, whereas grade was not. From these models, the preferred combination contained mitoses/10 HPF because the mitotic index contributed significantly (Table 7). In this model, both age and ER status had greater significance when mitoses/10 HPF was included.

Time Effects
Analysis within the 5-year interval, as well as after 5 years, was also performed. Within the 5-year interval, the proportional hazards assumption was tested by allowing hazard ratios to change at 2 years, and the estimates in the two subintervals were compared. There were 85 failures in the first 2 years and 76 in years 2 to 5. When each factor was compared between the two subintervals, only ER status showed marginally significant differences (P = .04). In the data beyond 5 years, only nodes and tumor size were significant (data not shown).

5-Year Survival
Among the 368 patients included in the primary analysis, there were 189 (51%) deaths. Within the first 5 years, there were 108 deaths, and 81 patients died more than 5 years after follow-up. Information on cause of death was not complete at the time of this writing, but of the deaths during the first 5 years, all but eight were after disease recurrence, and of the deaths after the first 5 years, all but 15 were after disease recurrence.

Among the variables analyzed, grade, mitoses/10 HPF, and SPF were associated with survival (P = .0001, P = .00004, and P = .026, respectively). Survival curves based on these variables are shown in Figs 4 to 6. As with TTR, dividing patients into two groups based on mitoses/10 HPF, 0 to 2 mitoses/10 HPF and 3 or greater mitoses/10 HPF, allowed for a striking separation in 5-year survival (Fig 5). NCHG (Fig 4) and SPF (Fig 6) also predicted survival.

View larger version (16K): [in this window] [in a new window]   Fig 4. Kaplan-Meier survival curves. Overall survival based on combined histologic grade.

View larger version (18K): [in this window] [in a new window]   Fig 5. Survival curves based on mitotic activity. The group of patients with less than 3 mitoses/10 HPF had an improved 5-year survival.

View larger version (16K): [in this window] [in a new window]   Fig 6. Overall survival based on SPF. The separation in this group of node-positive patients was not as striking as for grade or mitotic activity.

As with time to recurrence, extending follow-up beyond 5 years decreased the significance of these factors. After 5 years, only nodal status remained significant (data not shown).

Analysis of Axillary Lymph Node Metastasis
For the purposes of this study, tissue was collected from primary carcinomas as well as positive axillary lymph nodes. A total of 227 patients had lymph nodes available for histologic grading, assessment of mitotic activity, and DNA analysis (Table 9). The primary and metastatic carcinoma correlated in NCHG and in mitotic counts in 78% of patients. Analysis of grade and mitotic activity did not predict 5-year time to recurrence (data not shown). DNA ploidy was determined for axillary lymph node metastasis in 278 patients, and there was 70% agreement between primary and metastasis (diploid v aneuploid). There were 140 patients in whom S-phase analysis was determined both in the primary and axillary lymph node metastasis. Analysis of the nodal metastasis resulted in three groups of equal size, which were defined by SPF less than 2.25%, 2.25% to 6.98%, and more than 6.98%. These cutoff values (2.25 and 6.98) were lower than those of the primary carcinoma (4.55 and 9.34), which reflects a lower SPF in the nodes in general compared with the primary carcinoma. Table 9 relates the SPF in the primary carcinoma to that in the metastasis using 25^th, 50^th, and 75^th percentiles for the 140 patients. The agreement between these lower, intermediate, and upper groups defined in this manner was 44%. Neither ploidy nor SPF of metastasis predicted 5-year time to recurrence.

	DISCUSSION

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In contrast, the vast majority of patients with node-positive breast cancer can expect to receive adjuvant chemotherapy routinely.²² Prognostic factors in this group have received less attention, in large part because of the definite benefit derived from adjuvant chemotherapy and the belief that additional measures would have little impact on the prognosis predicted by the presence of positive axillary lymph nodes.²³ It is accepted, however, that the subset of patients with fewer than four involved lymph nodes have a better prognosis than those with metastases in four or more nodes,^22,24 although both groups of patients are candidates for systemic chemotherapy.

Despite the fact that studies dating back to 1920 have shown the value of histologic grading,²⁵ only recently has there been an acceptance of grade as an important prognostic factor for breast cancer. As with any prognostic factor, verification of histologic grade in formal studies has been necessary before widespread adoption. In the majority of studies, grading breast cancer has added prognostic information, despite the fact that there has not been a generally accepted method of grading.²⁶ Although the Scarff-Bloom-Richardson grading system has been in use since the 1950s and is the grading system of the World Health Organization, modifications by Elston and Ellis (NCHG)^6,27 have resulted in a widely accepted, reproducible⁸ grading system that remains significant in multivariate analysis in the face of other factors.^7,22

The three parameters assessed in the NCHG include differentiation (glandular formation), nuclear pleomorphism, and an assessment of mitotic activity. In an analysis of these individual parameters, it has been shown that the presence of mitotic activity is the strongest predictor of decreased survival.²⁸ Other investigators have analyzed mitotic activity alone and have shown that it independently predicts survival^29-31 and adds information beyond that obtained with histologic grade.

In addition to mitotic activity, other methods to assess proliferative activity include flow cytometric analysis of the fraction of cells in the S phase and immunohistochemical analysis of proliferation antigens (ie, Ki-67). Both of the last two methods have been shown to predict outcome for both patients with node-negative and those with node-positive breast cancer.^32-34

In the current study, histologic grade, mitotic activity, and SPF all were associated with time to recurrence. In multivariate analysis that included ER status, age at entry, tumor size, and number of lymph nodes involved, either histologic grade or mitotic activity was significant. In the presence of both histologic parameters, mitotic activity remained significant, whereas grade did not. This is not unexpected considering the studies of Dupont et al,³⁵ which are noted above regarding the contribution of mitotic activity to the predictive power of histologic grade. By stratifying the lowest category of mitotic activity (< 10 mitoses/10 HPF) into two groups, we identified a group (less than 3 mitoses/10 HPF) of patients with node-positive breast cancer with a significantly improved prognosis.

SPF has been shown to be an important estimator of cellular proliferation and, in many studies, has been a predictor of outcome. Unfortunately, careful histologic assessment, including grade, is rarely presented in these studies; thus, the true independent nature of SPF is difficult to assess. One study of node-negative breast cancer from Guy’s Hospital included both SPF and histologic grade in outcome analysis.³⁶ Whereas SPF was an important predictor of outcome in carcinomas larger than 1 cm, it was not significant when histologic grade was included in the analysis. In the current study, the effect of SPF became insignificant when grade or mitotic activity was included in the outcome model, and the preferred model included mitoses/10 HPF because of its significant contribution.

Some studies have suggested that high-grade (high proliferative rate) carcinomas may have a better therapeutic response to chemotherapy.^37,38 The current study was heterogeneous, including patients with node-positive breast cancer from several different treatment arms. Because the number of recovered paraffin tissue blocks was less than initially anticipated, analysis within individual treatment arms was not possible.

Although this study was somewhat hampered by its retrospective design within this multicenter cooperative trial and relatively small size of tumor samples analyzed, the utility of grade and mitotic activity in determining time to recurrence is well demonstrated. This is an important point, because the greatest utility of these histologic assessments has been demonstrated when tissue is uniformly fixed and processed and when the peripheral portion of the tumor is analyzed. This is the area where the greatest amount of proliferative activity is observed and in which grading and assessing mitotic activity is recommended.^25,39 Rather than detracting from the value of histologic assessment, the fact that histologic measures were so predictive even though they were derived from less than ideally sampled material underscores its strength. In prospective studies in which tissue was sectioned and processed in a uniform manner, histologic grade and mitotic activity were even stronger in their predictive power.³⁹

By separately analyzing events within the first 5 years after diagnosis, we have shown, as have other investigators, that the effect of histologic grade and mitotic activity are greatest in this initial interval and become less important after 5 years.⁴⁰ However, most of the recurrences are observed within the first 5 years, and it may be that an early survival advantage is maintained, whereas early recurrence is indicative of a poor prognosis in this group presenting with axillary metastases.

One might expect that analysis of the primary carcinoma would add little in the setting of axillary lymph node metastasis, the most relevant biologic measure of a tumor’s capacity for distant spread. After having shown that, indeed, additional prognostic information could be obtained in node-positive breast cancer by histologic and flow cytometric analysis, we analyzed the actual lymph node metastasis. Although the analysis of lymph node metastasis was somewhat limited by the number of patients in whom this information was obtainable, there was good correlation in the measures of grade, mitotic activity, and SPF between primary and metastatic carcinomas. Significant correlation between primary and axillary lymph node metastasis has been shown when nuclear morphometry, including mitotic activity,⁴¹ and proliferative activity measured by thymidine analog uptake have been measured.⁴² Some investigators have suggested that the proliferative activity of lymph node metastases may predict distant metastases,⁴³ although we did not find separate analysis of lymph node metastases to be predictive of 5-year recurrence or overall survival.

Prognostic information can also be obtained by recognizing special histologic types of breast cancer.^44,45 In the current study, there were too few special-type carcinomas to evaluate separately for prognostic significance. The only special-type carcinoma that would confound a grading study is medullary carcinoma, because it often has a good prognosis despite high-grade features. There were four patients with medullary or medullary variant carcinoma, and they were not included in the outcome analysis. Indeed, all four were alive and disease-free at follow-up times from 8.6 to 11.6 years.

We conclude that, for patients with axillary lymph node–positive breast carcinoma, proliferative activity, specifically mitotic activity, is able to identify a group with an improved disease-free and overall survival. In this cooperative group series, approximately one half of patients had mitotic activity of less than 10 mitoses/10 HPF. Refinement of prognostication in this group was obtained by further stratification, with an improved outcome identified for patients whose primary carcinomas had less than 3 mitoses/10 HPF, compared with patients having 3 or more mitoses/10 HPF. Further study of these histopathologic parameters is needed in a homogeneously treated group of patients to refine the ability of these parameters to predict response to a particular therapy.

	NOTES

 
Conducted by the Eastern Cooperative Oncology Group (Robert L. Comis, MD, Chair) and supported in part by Public Health Service grants no. CA49957, CA23318, CA59307, CA21076, CA11083, CA66636, and CA21115 from the National Cancer Institute, National Institutes of Health, and the Department of Health and Human Services, Bethesda, MD.

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Submitted May 6, 1999; accepted February 3, 2000.

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