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Original Article

c-erbB-2 Expression and Response to Adjuvant Therapy in Women with Node-Positive Early Breast Cancer

Hyman B. Muss, Ann D. Thor, Donald A. Berry, Timothy Kute, Edison T. Liu, Frederick Koerner, Constance T. Cirrincione, Daniel R. Budman, William C. Wood, Maurice Barcos, and I. Craig Henderson

N Engl J Med 1994; 330:1260-1266May 5, 1994

Abstract

Background

The role of molecular markers in predicting the response to treatment of breast cancer is poorly defined. The Cancer and Leukemia Group B (CALGB) conducted a randomized adjuvant-chemotherapy trial (CALGB 8541) comparing three doses (high, moderate, and low) of cyclophosphamide, doxorubicin, and fluorouracil in 1572 women with node-positive breast cancer. This study (CALGB 8869) was designed to determine whether the DNA index, the S-phase fraction, c-erbB-2 expression, or p53 accumulation could be used as a marker to identify a subgroup of patients more likely than others to benefit from high doses of chemotherapy.

Methods

Tissue blocks were obtained from 442 patients randomly selected from the larger CALGB trial. Paraffin sections from the primary lesions were analyzed for DNA content, S-phase fraction, c-erbB-2 expression, and p53 accumulation.

Results

Patients randomly assigned to the high-dose regimen of adjuvant chemotherapy had significantly longer disease-free and overall survival if their tumors had c-erbB-2 overexpression. No further information was gained by adding the data on S-phase fraction or p53 accumulation to the analysis. There was no clear evidence of a dose-response effect in patients with minimal or no c-erbB-2 expression.

Conclusions

There is a significant dose-response effect of adjuvant chemotherapy with cyclophosphamide, doxorubicin, and fluorouracil in patients with overexpression of c-erbB-2 but not in patients with no c-erbB-2 expression or minimal c-erbB-2 expression. Overexpression of c-erbB-2 may be a useful marker to identify the patients who are most likely to benefit from high doses of adjuvant chemotherapy.

Media in This Article

Figure 1Disease-free and Overall Survival According to Treatment Group (High-Dose, Moderate-Dose, or Low-Dose Chemotherapy) and Level of c-erbB-2 Expression.
Table 1Characteristics of Patients with Node-Positive Early Breast Cancer.
Article

Several molecular markers have been associated with a poor prognosis in patients with breast cancer, and it is widely assumed that the presence of these markers is an indication for adjuvant therapy. Many physicians and patients believe that it is better to try chemotherapy in such cases, even if it may be ineffective, than to do nothing and let the patient die. However, it is likely that some prognostic factors can be used to identify a tumor that is intrinsically resistant to moderate doses of chemotherapy, in which case such therapy is likely to compromise substantially the patient's quality of life without prolonging it. This issue is particularly important with such treatments as high-dose chemotherapy and autologous bone marrow transplantation, since the associated toxic effects and financial costs are substantial.

Prognostic factors that may help predict a recurrence of breast cancer include the size of the tumor,1 number of lymph nodes involved,1,2 histologic grade,3 and estrogen and progesterone status4. New molecular prognostic factors may also aid in the selection of a treatment5. Measurements of tumor DNA content (ploidy), cell proliferation (S-phase fraction), and oncogene expression have been reported to help predict relapse and survival, especially in patients with node-negative breast cancer. Flow cytometry is a rapid means of assessing cellular DNA content and cell proliferation6 and can be performed with paraffin-embedded tumor sections, thus facilitating a retrospective analysis7,8. The c-erbB-2 (HER-2/neu) oncogene is a potentially useful prognostic marker that encodes a transmembrane glycoprotein whose extracellular region is structurally similar to that of the epidermal-growth-factor receptor9-11. Like the epidermal growth factor, c-erbB-2 expression reflects an increase in the proliferative activity of a tumor12,13. Overexpression of c-erbB-2 has been demonstrated in 15 to 30 percent of patients with breast cancer and has been found by most but not all investigators to be associated with shorter survival, particularly in patients with positive nodes14-18. Another molecular marker is the tumor suppressor gene p5319-22. Mutations of this gene, which have been found in 13 to 49 percent of patients with breast carcinomas,23 may have prognostic importance, particularly in patients with node-negative disease24-26.

Patients with tumors that are positive for estrogen receptors derive the greatest benefit from adjuvant endocrine therapy10,27. However, the relation between prognostic factors and the response to adjuvant chemotherapy is less clear28-31. In patients with tumors of unfavorable grade28,29 or a high thymidine-labeling index,30,31 adjuvant chemotherapy has improved disease-free and overall survival, but the value of flow cytometry and c-erbB-2 and p53 studies to predict therapeutic responses in patients with node-positive breast cancer is uncertain. Since the DNA content, S-phase fraction, and c-erbB-2 and p53 expression are indirect measures of proliferative activity, we hypothesized that they might help predict the outcome of treatment. We tested our hypothesis with biopsy material from a randomized trial, reported elsewhere in this issue,32 comparing three doses of adjuvant chemotherapy to determine whether dose and dose intensity were related to survival in women with node-positive, stage II breast cancer.

Methods

Subjects

The patients in this study (CALGB 8869) were drawn from the larger trial of adjuvant chemotherapy (CALGB 8541). They were randomly selected from the 12 strata defined by the year of enrollment in the study (1985, 1986, 1987, or 1988) and the treatment received (a low-, moderate-, or high-dose regimen of cyclophosphamide, doxorubicin, and fluorouracil) in the adjuvant trial. Eligibility requirements for enrollment in the larger trial included a radical mastectomy, modified radical mastectomy, or breast-conservation therapy within six weeks before initiation of the chemotherapy protocol; no prior chemotherapy or irradiation; a CALGB performance score of 0 or 1 (no symptoms or minimal symptoms); an age of at least 16 years; a white-cell count of 3500 or more per cubic millimeter and a platelet count of 100,000 or more per cubic millimeter; a hemoglobin level of 100 g or more per liter; blood urea nitrogen, serum creatinine, and bilirubin levels less than 1.5 times normal values; no concomitant cancer; and informed consent. Information on estrogen-receptor status was recorded at the time of enrollment in the study; standardization of estrogen- and progesterone-receptor assays was not required, but almost all participating institutions have certified laboratories meeting the requirements of the College of American Pathologists. For patients treated with lumpectomy or segmental mastectomy, a standardized irradiation protocol was administered after the completion of chemotherapy. All patients were followed in a standardized fashion after treatment to determine the frequency and rate of recurrent disease and overall survival.

Treatment

Patients were randomly assigned to receive cyclophosphamide, doxorubicin, and fluorouracil at one of three levels of dose intensity: 600 mg, 60 mg, and 600 mg per square meter of body-surface area, respectively, every four weeks for four cycles (group 1); 400 mg, 40 mg, and 400 mg per square meter every four weeks for six cycles (group 2); or 300 mg, 30 mg, and 300 mg per square meter every four weeks for four cycles (group 3). Fluorouracil was repeated on day 8 of each cycle. The cumulative doses of cyclophosphamide, doxorubicin, and fluorouracil were identical in groups 1 and 2 and 50 percent lower in group 3. The protocol was amended in April 1988 to require tamoxifen therapy (10 mg orally twice a day for five years) for all disease-free perimenopausal or postmenopausal patients who were positive for estrogen or progesterone receptors (≥ 7 fmol per milligram of protein). Tamoxifen was instituted after the completion of chemotherapy.

Specimen Preparation

Formalin-fixed, paraffin-embedded blocks from the primary breast lesions were obtained from participating CALGB institutions. A slide stained with hematoxylin and eosin was prepared from each block and used for pathological confirmation of breast cancer. From the same block, 4-micrometer tissue sections were prepared for immunohistochemical studies, and 50-micrometer sections for flow-cytometric analysis. All tumors were graded according to a nuclear grading system modified from that described by Black et al.33 and were histologically categorized by a reference pathologist without knowledge of the case.

Flow-Cytometric Analyses

All flow-cytometric analyses were performed in a single reference laboratory. The procedure of Hedley et al.,34 as modified by Kute et al.,8 was used to determine DNA content and cell-cycle kinetics. The 50-micrometer sections were deparaffinized, and flow cytometry was performed on malignant areas dissected from the specimen with the help of a tissue map (tumor enrichment). A diploid DNA standard was obtained from the nonmalignant tissue in the block, and the histogram for the DNA standard was compared with the histogram for the tumor-enriched area of the same block to correct for artifacts in fixation and preparation. The DNA index was obtained by comparing the ratio of the G1 peak channel of the malignant cells to that of the nonmalignant cells. Samples in which the G0G1 peaks of the diploid standard and the tumor did not match were not used for analysis. Three methods were used to analyze cell kinetics: the rectangular-fit model,35 a computer modeling procedure (Modfit)8 that determines G1, S, and G2 activity and corrects for variability in the coefficient of variation and the location of the peak channel, and an area-fit procedure. Only the Modfit model corrects for debris. The results of the three methods were closely correlated (data not shown); the rectangular-fit model was used in this analysis because the S-phase differences computed with this method provided the best correlation with prognosis. The relatively high median value of S-phase activity in the analysis is most likely related to the use of the rectangular-fit model. Flow-cytometric studies were performed without knowledge of information about the patients.

Immunohistochemical Analysis of c-erbB-2 Expression

All immunohistochemical analyses were performed in a single reference laboratory with the use of previously described methods31. Briefly, a polyclonal antibody (OA-11-854; Cambridge Research Biochemicals, Wilmington, Del.) reactive with the cytoplasmic domain of c-erbB-2 was used with avidin-biotin-peroxidase immunohistochemical methods. All slides were evaluated for c-erbB-2 overexpression by two investigators without knowledge of patient information. The percentage of stained invasive malignant cells was estimated, and tissue was considered to be positive for c-erbB-2 expression if any membranous activity was found in these cells at a magnification of 100. All malignant cells on each slide were evaluated. The two investigators had generally similar estimates of the frequency of stained cells on the slides (less than a 5 percent discrepancy between estimates). In a previous study, an immunohistochemical analysis of c-erbB-2 expression was shown to be closely correlated with HER-2/neu gene amplification36.

Immunohistochemical Analysis of p53 Expression

Expression of p53 was evaluated according to previously published methods24. Briefly, a monoclonal anti-p53 antibody (anti-p53 PAb1801; Cambridge Research Biochemicals) diluted 1:4000 was applied after the sections had been incubated overnight with diluted normal horse serum at 4 °C. The slides were rinsed and sequentially incubated with biotin-horse-antimouse and streptavidin-horseradish peroxidase (Zymed Laboratories, San Francisco). Diaminobenzidine (Sigma Chemical, St. Louis) was used to visualize antibody binding. Slides were counterstained with 1 percent aqueous methyl green, dehydrated, cleared, and mounted. For each assay, slides from fixed, embedded cell pellets from MDA-MB-231 (positive control) and HTB5 (negative control) (both from the American Type Culture Collection, Rockville, Md.) were included to ensure interassay consistency24. Two investigators evaluated each slide by light microscopy. If invasive tumor cells displayed nuclear brown staining, the slide was considered to be positive for p53 overexpression. An estimate was made of the percentage of positive invasive tumor cells visible at a magnification of 100.

Study Design and Statistical Analysis

At the time this trial was initiated, DNA content appeared to be the strongest flow-cytometric predictor of recurrent disease in patients with early-stage breast cancer37. A sample size of 134 patients per treatment group was estimated to have the capacity to detect a 25 percent difference in disease-free survival within each treatment group for patients whose tumors had aneuploid as compared with diploid DNA content (a power of 0.8 and a two-sided P value of 0.05). Blocks from a total of 442 patients were obtained for analysis. The demographic, clinicopathological, and laboratory variables we analyzed included treatment group, age at the time of enrollment in the study, menopausal status, tumor size, number of positive nodes, histologic type and grade, estrogen- and progesterone-receptor status, tamoxifen therapy, DNA content, S-phase fraction, c-erbB-2 expression, and p53 accumulation. We analyzed tumor size as a dichotomous variable ( ≤ 2 cm or >2 cm), as a continuous (linear) variable, and with square-root and logarithmic transformations. In both univariate and multivariate analyses, the dichotomous variables predicted overall and disease-free survival better than the other variables. Some variables were transformed to increase their predictive value. For example, we used the square root of the number of positive nodes, which performed better than the number of positive nodes in both linear and logarithmic scales. With the square-root transformation, an increase in the number of positive nodes from 1 to 4 carried about the same incremental risk as an increase in the number of positive nodes from 4 to 9 or from 9 to 16.

Overall survival was defined as the time from enrollment in the study to death; data on survivors were censored at the last follow-up visit. Disease-free survival was defined as the time from enrollment to a documented relapse or death without a relapse. Data on patients who did not have a relapse were censored at the last follow-up visit. To date, 98 of the 442 patients in this study have died, 95 of recurrent breast cancer. Survival curves were drawn according to the Kaplan-Meier product-limit method38-40. Two or more survival distributions were compared with the log-rank test. We used the Cox proportional-hazards model to relate the various covariables to disease-free and overall survival41. We used this method in univariate analyses to screen for prognostic variables and in multivariate analyses to identify sets of prognostic variables while controlling for the effects of other variables. This model assumes a constant ratio of hazard rates for different levels of therapy with prognostic variables. Categorical variables were compared with the chi-square test or Fisher's exact test. We used the Kruskal-Wallis test to compare variables across dose levels. Correlation coefficients were calculated to measure the association between pairs of variables that were potential predictors of survival.

Results

Patient Sample and Clinical Variables

Tissue blocks were obtained from 442 patients randomly selected from the 1572 patients enrolled in the adjuvant-chemotherapy trial. Of these 442 blocks, 397 (90 percent) were technically satisfactory for analysis of DNA content (ploidy) and c-erbB-2 expression, 394 (89 percent) for analysis of p53 expression, and 302 (68 percent) for analysis of the S-phase fraction. Aneuploid tumors were found in 59 percent of the patients, and the median S-phase fraction was 11 percent. At least some c-erbB-2 expression was found in 59 percent of the samples, and in 29 percent of the samples at least 50 percent of the cells stained for c-erbB-2. Some p53 expression was found in 42 percent of the samples, and in 17 percent of the samples 10 percent or more of the cells displayed p53 expression. This report includes follow-up data as of January 1, 1992. The median follow-up for all patients was 38.5 months, with a range of 1 week to 80 months.

Table 1Table 1Characteristics of Patients with Node-Positive Early Breast Cancer. provides clinical data for the 442 patients in the three treatment groups, as well as for the total patient population. These data indicate that our sample was representative of the overall patient population. Patients in the three treatment groups had similar clinicopathological features except for histologic type; there were fewer infiltrating ductal carcinomas in group 3 than in groups 1 and 2 (87 percent vs. 95 and 97 percent, respectively). Although significant (P<0.01), this difference did not affect the outcome in either univariate or multivariate analyses. DNA content, c-erbB-2 expression, and p53 expression were similar among the three treatment groups (Table 2Table 2DNA Content, S-Phase Fraction, and c-erbB-2 and p53 Expression.). There was a correlation between the S-phase fraction and c-erbB-2 expression (r = 0.13, P = 0.03) and between the S-phase fraction and p53 accumulation (r = 0.25, P<0.01).

Disease-free and Overall Survival

The data from the larger CALGB trial at 3.2 years of follow-up showed that disease-free survival and overall survival in groups 1 and 2 (patients treated with high- or moderate-dose chemotherapy) were significantly longer than in group 3 (patients treated with low-dose chemotherapy)32. The data from the 442 patients in this analysis show a similar trend: a moderate- or high-dose regimen was associated with significantly longer disease-free survival (P<0.01) and overall survival (P = 0.13) (data not shown).

A univariate analysis demonstrated that the well-established clinical prognostic factors -- larger tumor size, higher number of positive nodes, higher tumor grade, and lack of estrogen and progesterone receptors -- were associated with shorter survival (Table 3Table 3Results of Univariate Analyses of Overall and Disease-free Survival.). In contrast, older age and postmenopausal status were significantly associated with longer survival, and patients who received tamoxifen did better than those who did not receive this drug; tamoxifen, however, was administered to a select group of patients.

Factors that were found to be significant in the univariate analysis (P<0.05), as well as c-erbB-2 expression, p53 accumulation, and S-phase fraction, were examined in the multivariate Cox model for their relation to disease-free and overall survival. Initially, we analyzed the results for the 269 patients whose tumor blocks were technically satisfactory for analyses of c-erbB-2 expression, p53 accumulation, S-phase fraction, and ploidy; the pretreatment characteristics of these patients and the 442 in the entire sample were similar (data not shown). This multivariate analysis revealed that a larger number of positive lymph nodes, a larger tumor size, premenopausal status, and greater c-erbB-2 expression were significant predictors of shorter disease-free and overall survival. In contrast, p53 accumulation was of marginal significance as a predictor of survival. Chemotherapy dose, age, histologic type and grade, estrogen- and progesterone-receptor status, and S-phase fraction were not predictors of either disease-free or overall survival.

By omitting the S-phase fraction from the model, we increased the sample size from 269 to 388 patients. Analysis of this group with the Cox model showed that all variables were significant predictors of survival except p53 accumulation and that menopausal status was no longer a significant factor for overall survival. A third multivariate analysis omitted both p53 accumulation and S-phase fraction (Table 4Table 4Multivariate Analysis of Overall and Disease-free Survival (Cox Regression Model).). Again, the number of positive lymph nodes, tumor size, and c-erbB-2 expression were significantly related to both disease-free and overall survival. Moreover, there was a highly significant interaction between chemotherapy dose and c-erbB-2 expression for both disease-free and overall survival. Once the interaction between chemotherapy dose and c-erbB-2 expression had been accounted for, the interactions between the dose and the other variables did not provide additional predictive information. Replacing the S-phase fraction with histologic grade in the model did not give additional information. The dose of chemotherapy was not independently related to disease-free or overall survival in any of the models used in the multivariate analysis.

Overexpression of c-erbB-2 as a Predictive Factor for Survival

Although a cutoff point is occasionally used to define a high- or low-risk group,37 this approach tends to oversimplify and even distort the relations between variables and outcomes. In the statistical modeling, we analyzed c-erbB-2 overexpression as a continuous variable. To portray these data graphically, however, we used a 50 percent cutoff point (Figure 1Figure 1Disease-free and Overall Survival According to Treatment Group (High-Dose, Moderate-Dose, or Low-Dose Chemotherapy) and Level of c-erbB-2 Expression.). Patients whose tumors had 50 percent or more c-erbB-2 overexpression constituted 29 percent of the study population. Separation of the curves for both disease-free and overall survival in this group was both substantial and significant, indicating a dose-response effect (Figure 1C and Figure 1D). Notably, of the patients who were enrolled in group 1 of the adjuvant trial, those whose tumors overexpressed c-erbB-2 had longer disease-free and overall survival than those whose tumors did not overexpress c-erbB-2. In contrast, no dose-response effect was seen when the 71 percent of patients with no or low c-erbB-2 overexpression were grouped together. This relation between the dose-response effect and the c-erbB-2 level was relatively independent of other risk factors, such as the number of nodes involved. There was little difference in disease-free survival among the three treatment groups for patients whose tumors had no or low c-erbB-2 expression, regardless of the nodal status (Figure 1A and Figure 1B). Among those with a high level of overexpression, there was a significant dose-response effect for all nodal groups (data not shown).

Discussion

This analysis of molecular markers in breast cancer suggests that increasing the dose intensity of adjuvant chemotherapy may not result in a similar benefit for all patients with positive nodes. It appears that patients whose tumors overexpress c-erbB-2 may derive the greatest benefit from higher doses of chemotherapy. In contrast, no benefit from dose intensification was observed in patients with no or low c-erbB-2 expression. These results should not be misconstrued to mean that lower doses of adjuvant therapy are not beneficial in patients with node-positive breast cancer. On the contrary, the results of an overview analysis suggest that combination chemotherapy significantly improves survival in such patients as compared with similar patients not receiving chemotherapy27.

Adjuvant chemotherapy appears to be more effective in patients whose tumors have a high thymidine-labeling index than in those whose tumors have a lower thymidine-labeling index30. Adjuvant chemotherapy has also been associated with improved disease-free and overall survival in patients with a higher tumor grade than in those with a lower tumor grade28,29. In a study of preoperative chemotherapy in patients with primary breast cancer, a high S-phase fraction was associated with a better response rate than a low S-phase fraction42. Similar correlations between the response to chemotherapy and the rate of tumor proliferation have been found in small cohorts of patients with locally advanced breast cancer43 or distant metastases44.

Two previous studies suggest that overexpression of c-erbB-2 may be associated with resistance to chemotherapy45,46. Both these studies used conventional doses of cyclophosphamide, methotrexate, and fluorouracil. In one of the studies, patients with node-negative breast cancer were randomly assigned to receive either six months of adjuvant chemotherapy or no chemotherapy at all45. Chemotherapy resulted in significantly longer disease-free survival among patients whose tumors did not express c-erbB-2 than among those with c-erbB-2 overexpression. In the other study, patients with positive nodes were randomly assigned to six months or one month of adjuvant chemotherapy. The longer period of therapy was associated with longer disease-free and overall survival46. However, among the patients who received six months of chemotherapy, disease-free survival was significantly longer among those whose tumors did not express c-erbB-2 than among those whose tumors overexpressed the oncogene.

What accounts for the differences between the results of these studies and our results? An important distinction is that our adjuvant-chemotherapy regimen included doxorubicin, whereas the regimens in the previous trials did not. Furthermore, c-erbB-2 expression may indeed be a marker of relative resistance to chemotherapy, but an escalation of the dose may overcome that resistance. Patients whose tumors overexpress c-erbB-2 may thus benefit from higher doses of chemotherapy given in regimens containing anthracyclines. On the other hand, patients whose tumors do not overexpress c-erbB-2 may not need higher doses of chemotherapy regimens that include anthracyclines to obtain the maximal benefit from adjuvant chemotherapy.

Doxorubicin is generally recognized as the most effective agent against breast cancer. It is possible that its efficacy is due in part to its ability to overcome the relative resistance to chemotherapy associated with c-erbB-2 overexpression. In a recent in vitro study, breast-cancer cells overexpressing c-erbB-2 frequently overexpressed topoisomerase IIalpha, a target enzyme for doxorubicin47. We are planning to measure the expression of topoisomerase II in available tissue specimens from patients in this study to determine whether c-erbB-2 expression is a marker for topoisomerase II expression through linkage. Tsai and colleagues have recently reported that increased resistance to the cytotoxicity of doxorubicin was directly related to increased c-erbB-2 expression in non-small-cell lung-cancer cell lines48. These data suggest that a high dose of doxorubicin may be necessary to kill tumor cells in patients with a high level of c-erbB-2 expression.

Expression of c-erbB-2 was significantly associated with treatment outcome in this trial, but it is not necessarily the ideal marker for predicting sensitivity to chemotherapy. Estimation of c-erbB-2 overexpression is imprecise, and no standardized assay is available. Estimates of c-erbB-2 expression are currently provided by many laboratories as part of a prognostic profile, but further data are needed before this marker can be used in making clinical decisions. Moreover, differences in assay and reporting methods make comparisons of our data and those of others tenuous.

Adjuvant chemotherapy prolongs disease-free and overall survival in patients with node-positive early breast cancer. However, the majority of patients with clinically occult metastases at the time of diagnosis ultimately have a relapse and die in spite of receiving a moderate-dose regimen of cyclophosphamide, doxorubicin, and fluorouracil or cyclophosphamide, methotrexate, and fluorouracil27. Trials are under way to determine whether high-dose chemotherapy with autologous bone marrow support improves the outcome for women with node-positive breast cancer. Such treatment involves substantial toxicity as well as a high cost49. Therefore, measurements that help identify those patients most likely to benefit from intensive treatment will be important. Data from other prospective trials are needed to corroborate our observations and determine whether either c-erbB-2 expression or other biologic markers can identify tumors that are particularly responsive or resistant to specific chemotherapeutic agents or to escalated doses of such agents.

Supported by grants (CA-03927, CA-44768, CA-31946, CA-33601, CA-47559, CA-07968, CA-12449, CA-37207, and CA-32291) from the National Cancer Institute.

Source Information

From the Bowman Gray School of Medicine, Winston-Salem, N.C. (H.B.M., T.K.); Massachusetts General Hospital, Boston (A.D.T., F.K.); the Statistical Office of the Cancer and Leukemia Group B, Durham, N.C. (D.A.B., C.T.C.); the Department of Medicine, University of North Carolina School of Medicine, Chapel Hill (E.T.L.); North Shore University Hospital (New York Hospital), New York (D.R.B.); Emory University, Atlanta (W.C.W.); the Department of Medicine, Roswell Park Memorial Institute, Buffalo, N.Y. (M.B.); and the University of California, San Francisco (I.C.H.).

Address reprint requests to Dr. Thor at the Department of Pathology, University of Vermont School of Medicine, Medical Alumni Bldg., Burlington, VT 05405.

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