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© 2002 American Society for Clinical Oncology Association of Angiogenesis and Disease Outcome in Node-Positive Breast Cancer Patients Treated With Adjuvant Cyclophosphamide, Doxorubicin, and Fluorouracil: A Cancer and Leukemia Group B Correlative Science Study From Protocols 8541/8869ByFrom the North Shore Medical Center, Salem, MA; University of Texas, M.D. Anderson Cancer Center, Houston, TX; Cancer and Leukemia Group B Statistical Center, Durham, NC; Mt Sinai Medical Center, and Memorial Sloan Kettering Cancer Center, New York; North Shore University Hospital, Manhasset, NY; University of California at San Francisco, San Francisco, CA; and University of Michigan Comprehensive Cancer Center, Ann Arbor, MI. Address reprint requests to Daniel F. Hayes, MD, University of Michigan Comprehensive Cancer Center, CCGC 6312, 1500 E Medical Center Dr, Ann Arbor, MI 48109-0942; email: hayesdf@ umich.edu.
PURPOSE: Increased microvessel density (MVD), a reflection of tumor angiogenesis, is associated with diminished relapse-free and overall survival (OS) in several subsets of breast cancer patients. However, the utility of this assay in node-positive patients treated with adjuvant cyclophosphamide, doxorubicin, and fluorouracil (CAF) has not been well studied. PATIENTS AND METHODS: Immunostaining for factor VIII–related antigen was performed on tissue sections from a subset of node-positive patients who received one of three dose/schedule regimens of CAF during participation in Cancer and Leukemia Group B protocol 8541. Sections from 577 cancers exhibited acceptable tumor and immunostaining quality and were included in the study. Each section was examined quantitatively for MVD as well as nonquantitatively by scoring the presence or absence of a prominent vascular pattern. RESULTS: MVD counts were not associated with relapse-free or OS in univariate analysis. The presence of a prominent plexiform vascular pattern was correlated with decreased OS (P = .0085) in univariate analysis, but this pattern was not an independent prognostic indicator of survival in multivariate analysis. No apparent clinically important interactions between measures of angiogenesis, other prognostic factors, administration of tamoxifen, and chemotherapy dose were observed. CONCLUSION: Assessment of angiogenesis does not provide useful information regarding prognosis in node-positive breast cancer patients treated with adjuvant CAF, nor do these measures predict which patients will benefit from dose intensification or addition of tamoxifen.
ANGIOGENESIS IS a critical requirement for tumor progression at all stages of breast cancer development. Prior studies have examined the prognostic utility of microvessel density (MVD) counts, a quantitative assessment of tumor angiogenesis, for patients with breast cancer.1-22 However, not all published series suggest that MVD in primary breast cancers is a significant prognostic factor for patients with and without regional lymph node metastases.16-21 These studies are difficult to compare, because of differences in the study populations, the number of patients examined, the length of follow-up, the use of different types of adjuvant endocrine and/or chemotherapy, the methods of microvessel quantitation, and the statistical methods used.23 In Cancer and Leukemia Group B (CALGB) trial 8541, 1,572 women with node-positive breast cancer were randomly assigned to one of three doses of cyclophosphamide, doxorubicin, and fluorouracil (CAF). Overall, higher dose and schedule regimens of CAF were most effective in reducing recurrences and mortality than lower doses of CAF.24,25 The randomized trial design permitted us to examine whether angiogenesis, as measured by MVD and/or the presence of a plexiform vascular pattern (PVP), might either be prognostic in the setting of adjuvant chemotherapy or even predictive of which patients might be most likely to benefit from the higher doses of CAF.
The study population represents a subset of patients enrolled in CALGB protocol 8541. In this clinical study, 1,572 women with stage II, node-positive breast cancer were randomly assigned to one of three dose and schedule regimens for CAF: 300/30/300 mg/m2 administered intravenously (IV) every 4 weeks for four cycles, 400/40/400 mg/m2 IV every 4 weeks for six cycles, and 600/60/600 mg/m2 IV every 4 weeks for four cycles. In each cycle, cyclophosphamide and doxorubicin were given on day 1, and fluorouracil was given on days 1 and 8. The overall results of this trial have been previously published.24,25 In a companion study (CALGB 8869), 944 formalin-fixed, paraffin-embedded tissue blocks containing primary cancer tissue from patients who participated in CALGB 8541 were collected and used to evaluate a number of biologic markers, including DNA content, S-phase fraction, c-erbB2, and p53 expression. Details regarding the various methodologies used for these assays and the results from these studies have been previously published.26,27 For the current study, multiple additional 5-µm tissue sections were prepared from each paraffin block and were stored at room temperature for variable intervals (years) before staining and analysis.
Factor VIII Immunohistochemistry
Assessment of Neovascularization Both quantitative and nonquantitative assessments of neovascularization were performed. MVD counts were performed using the method of Weidner et al.1 A minimum of five x200 fields (x20 objective lens and x10 ocular lens; 0.74 mm2 per field) were counted in areas of most intense neovascularization (ie, vascular hot spots), that were identified by low-power scanning. If multiple hot spots were present, MVD counts were performed in each hot spot. If vascular hot spots were not obvious at low power, a careful high-magnification scanning was necessary to identify the areas of highest MVD. In these cases it was often necessary to count more than five fields to assure that the most vascular field was included in the analysis. Microvessels were defined as any discrete factor VIII–positive endothelial cell or endothelial cell cluster, with or without definable lumens. To improve the accuracy of MVD counts, a four-quadrant crosshair eyepiece was used. The highest MVD count was used in the analysis.
In addition to the quantitative assessment of MVD, a nonquantitative assessment was performed that focused on the presence or absence of a pattern of neovascularization that has not been well described in prior studies. In most tumors, vascular hot spots were characterized by numerous discrete vessels that could be easily counted (Fig 1A). In a minority of cases, hot spots were characterized by a complex arborizing network of vessels that completely or nearly completely encircled tumor cells in a plexiform pattern (Fig 1B). The plexiform pattern of increased neovascularization often resulted in relatively low MVD counts, despite the fact that these tumors were richly vascular, because of the paucity of individual quantifiable vessels. Plexiform neovascularization was scored as present if
Assessment of Intra- and Interobserver Reproducibility To assess intraobserver reproducibility, 50 study cases were randomly selected from the larger cohort and were reanalyzed by the same pathologist (A.J.G.), who was blinded to the results of the original assessment. A second quantitative assessment of MVD was performed, and each case was recategorized with respect to the presence or absence of a prominent plexiform vascular pattern. To assess interobserver reproducibility, a second pathologist with experience in MVD quantitation (B.H.) recounted each of the 50 randomly selected cases (200x magnification, 0.79 mm2 per field). In addition, two pathologists (B.H., I.J.B.) simultaneously recategorized each of the 50 cases with respect to the presence or absence of a prominent plexiform vascular pattern, using the 20% cutoff.
Statistical Analysis The Wilcoxon sum rank test was used to determine differences in medians or proportions of various factors between two groups (MVD high or low, plexiform vascular patter present or absent). For this analysis, tumor size, number of positive nodes, and S-phase fraction were analyzed as continuous variables, and vascular invasion, ER, PgR, aneuploidy, erbB-2, and p53 were examined as dichotomous variables.
Intraobserver reproducibility for MVD counts was assessed using regression analysis. Differences in MVD counts by plexiform pattern were tested by Wilcoxon rank sum test. P values ER and PgR information was provided by participating institutions using their own reference laboratories (a combination of immunohistochemical and biochemical assays). c-erbB-2 and p53 assays were performed by immunohistochemical assays, as previously described.27
Tissue blocks or unstained slides were collected retrospectively from 944 of the 1,572 patients who participated in clinical trial 8541. Tissue blocks from the other 628 patients were not available. Of the 944 tissue sections immunostained for factor VIII, 367 cases were excluded from the study. Reasons for exclusion included 167 cases with insufficient primary invasive tumor present on recut sections, 57 cases exhibiting histologic changes consistent with prior biopsy site (which precluded an analysis of true tumor MVD because of the presence of neovascularization secondary to wound healing), and 143 cases with poor immunostaining quality, including overstained cases and cases with unacceptably low factor VIII immunoreactivity. When extra sections could be obtained, these cases were restained for factor VIII and reevaluated, and were still found to exhibit unacceptable factor VIII immunoreactivity. This latter phenomenon was probably in part due to fixative effects and/or the prolonged period of slide storage.28 In total, therefore, 577 cases exhibited sufficient invasive tumor with acceptable immunostaining quality and were included in the data analysis. With median follow-up of 9.5 years, 248 patients (43%) have suffered a recurrence, and 205 patients (36%) have died. These 577 patients did not differ in terms of mean age, tumor size, and mean number of positive lymph nodes from the remaining 995 patients enrolled in protocol 8541 but for whom blocks were not available or for whom adequate factor VIII staining could not be obtained. The two groups (those included in this study and those not included) did differ significantly in regard to menstrual status (premenopausal, 40% v 45%; P = .05), hormone receptor status (ER and/or PgR positive, 77% v 69%; P = .0006), and treatment with adjuvant tamoxifen (treated, 38% v 29%; P = .0003) (Table 1). The two groups (included and not included) did not differ in regard to RFS (P = .16) or OS (P = .11).
Relationships Among Indicators of Angiogenesis, Clinical Features, and Biologic Features of the Tumor Table 2 provides distribution of various pathologic features and biologic markers within categories of MVD (high v low) and plexiform vascular patterns (some v none). A significant difference was detected in the presence of lymphatic invasion by tumor between patients with high versus low MVD (percentage of patients with vascular invasion for MVD high, 53%; for MVD low, 38%; P = .0006). Significant differences in the following features were observed between patients whose tumors exhibited PVP and those that did not: tumor size (median tumor size for PVP present, 3.0 cm; for PVP absent, 2.5 cm; P = .003), lymphatic vascular invasion (percentage of patients with vascular invasion for PVP present, 52%; for PVP absent, 41%; P = .01), ER expression (percentage of ER-positive patients for PVP present, 62%; for PVP absent, 77%; P = .0008), PgR expression (percentage of PgR-positive patients for PVP present, 51%; for PVP absent 70%; P = .0001), increased S-phase fraction (median S-phase fraction for PVP present, 16%; for PVP absent, 10%; P = .0001), aneuploidy (percentage of patients with aneuploid tumors for PVP positive, 70%; for PVP negative, 56%; P = .002), c-erbB2 expression (percentage of patients erbB-2 positive for PVP present, 30%; for PVP absent, 23%; P = .05), and p53 expression (percentage of patients with p53 expression for PVP present, 39%; for PVP absent, 30%; P = .02).
Angiogenesis and Outcome: Prognosis Independent of Therapy The median MVD count was 102 vessels/field (range, 23 to 314 vessels/field). When all patients were considered, independent of CAF dose, no association was observed between MVD counts and RFS or OS by univariate analysis (P = .55 and .89, respectively) (Fig 2A and 2B).
A prominent PVP was observed in 153 (27%) of 577 cases. A trend was observed between the presence of PVP and decreased RFS by univariate analysis, but the results were not statistically significant (P = .072) (Fig 3A). A significant association was observed between the presence of PVP and decreased OS by univariate analysis (P = .0085) (Fig 3B). However, in multivariate analysis, PVP was not an independent prognostic factor because of its association with a number of other poor prognostic features (Tables 3 and 4).
Tumors with a PVP did not exhibit significantly higher median MVD counts than tumors without a PVP (median MVD in presence of PVP, 109 vessels/field; range, 23 to 264; MVD in absence of PVP, 105 vessels/field; range, 31 to 314; P = .24). Furthermore, a subset of highly vascular tumors with a prominent plexiform pattern yielded relatively low MVD counts because of the paucity of discrete countable vessels. This observation was particularly evident when the majority of the tumor exhibited a plexiform vascular pattern. For this reason, we examined survival data for patients who had tumors exhibiting either a prominent plexiform pattern or an MVD count above the median value (100 vessels/field). Moreover, we examined survival data for patients who had tumors exhibiting both a prominent plexiform pattern and an MVD count above the median value. Patients with tumors showing either a plexiform vascular pattern or high MVD counts exhibited a trend towards decreased RFS compared with patients with neither feature, but the results were not statistically significant (P = .25). Likewise, a nonstatistically significant trend was observed in OS between these two groups (P = .068). Patients with tumors showing both a prominent plexiform vascular pattern and high MVD counts had a decreased RFS and a decreased OS compared with all other patients, but again the results were either marginally or not statistically significant (P = .07 and P = .05, respectively).
Angiogenesis and Outcome: Prediction of Benefit From Different Doses of CAF
Independent Prognostic Features in Multivariate Analysis When MVD was included in the model, the following factors retained independent prognostic significance for RFS in multivariate analysis: number of positive lymph nodes (P = .0001), tumor size (P = .005), chemotherapy dose (P = .007), hormone receptor status (P = .03), and interaction of c-erbB2 expression and chemotherapy dose (P = .04) (Table 3). Other factors that were included in the multivariate model but that did not achieve statistical significance included age, body-surface area, erbB-2 (by immunohistochemical staining when not considered as an interaction term with chemotherapy dose), and MVD (Table 3). In a model for OS that contained the same variables, the following factors retained independent prognostic significance: the number of positive lymph nodes (P = .0001), tumor size (P = .001), CAF dose (P = .05), hormone receptor status (P = .007), and interaction of c-erbB2 expression and chemotherapy dose (P = .02). Again, MVD failed to achieve statistical significance. A second multivariate model was performed that included plexiform pattern rather than MVD, with very similar results (Table 4). As with MVD, plexiform pattern alone was not statistically significantly associated with worse prognosis, and no interaction between plexiform pattern and CAF dose was identified.
Interactions Among Angiogenesis, Prognostic Factors, and Outcome Because both MVD and plexiform patterns were associated with various other prognostic factors (Table 2), we tested for the existence of interactions between these factors and outcomes (RFS, OS) (Table 5). No statistically significant interactions between MVD and menopause status, hormone receptor status, or administration of tamoxifen and either RFS or OS were observed. Likewise, none was detected between the presence of plexiform pattern and menopausal status, hormone receptor status, and the administration of tamoxifen and RFS or OS. Finally, no statistically significant interaction was observed when MVD, tamoxifen, and dose of a CAF were tested for either RFS (P = .94) or OS (P = .99). In addition, no significant interaction was detectable among plexiform pattern, tamoxifen, and dose of CAF for RFS (P = .84) or OS (P = .86). These data suggest that there was no subgroup of patients for which either MVD or plexiform pattern predicted better outcomes from higher doses of CAF. Rather, all subgroups of patients benefited from higher doses of CAF, regardless of angiogenic status.
Intra- and Interobserver Reproducibility Fifty randomly selected cases from the 577 study cases were reanalyzed by the original pathologist (A.J.G.) to assess intraobserver reproducibility. Regression analysis performed on both sets of MVD counts yielded a correlation coefficient of 0.91 (r2 = 0.83; P < .0001). In addition, there was agreement in the original classification of plexiform vascular pattern in 48 (96%) of the 50 cases. Two of the 15 cases originally classified as having more than 20% plexiform pattern were reclassified as having less than 20% plexiform pattern on review. Interobserver reproducibility for MVD counts performed by two pathologists (A.J.G. and B.H.) for the same 50 cases yielded a correlation coefficient of 0.76 (P = .0001). In addition, a review of the 50 cases by two pathologists (B.H. and I.B.) resulted in agreement on the presence of a prominent plexiform vascular pattern with the original pathologist in 42 (84%) of the 50 cases.
Tumor progression is critically dependent on the ability of malignant cells to induce a vascular stroma. In breast cancer, angiogenesis begins at the earliest stages of in situ disease.29 After invasion occurs, neovascularization is required for tumor growth and metastasis.30,31 Nearly a decade ago, Weidner et al1 were the first to demonstrate that quantifying tumor neovascularization yields prognostically useful information in patients with invasive breast cancer. Subsequently, a large number of studies have examined the prognostic utility of MVD counts in a variety of human tumors, including breast cancers. In general, an inverse relationship has been observed between MVD counts in primary breast cancers2-10,13-15 or axillary lymph node metastases21 and RFS and OS, although not all studies have demonstrated this association.16-22 These prior studies have differed in many respects, including the type and number of patients studied, the length of follow-up, the treatment modalities used, the methods of microvessel quantitation, and the statistical methods used in data analysis. These differences have made it difficult to reach firm conclusions regarding the prognostic utility of MVD counts in specific subgroups of breast cancer patients. One important factor that frequently confounds tumor marker studies is the effect of treatment.32-35 Angiogenesis might be purely prognostic. In other words, high levels of MVD might be associated with worse outcome independently of treatment effects.33-35 Alternatively, neovascularization might interact with treatment effects, either positively or negatively, and MVD would therefore serve as a predictive factor. Finally, the utility of MVD might be mixed, serving as both a prognostic and predictive factor. In the current study, we have examined angiogenesis in a large, randomized, prospective study that involves a narrowly defined patient population (ie, node-positive patients treated with one of three dose schedules of CAF) for whom long-term follow-up data are available. Because of the trial design of protocol 8541, we could address both prognosis and prediction for angiogenesis in node-positive breast cancer patients who received doxorubicin-based adjuvant chemotherapy. In this population, MVD was not prognostic. Therefore, one cannot use the presence or absence of high levels of MVD in the primary tumor to select patients who might be candidates for further treatment after four to six cycles of doxorubicin-based chemotherapy (such as very high-dose chemotherapy requiring bone marrow stem-cell support or additional cycles of paclitaxel).36,37 Moreover, MVD did not seem to be predictive of relative benefit across the three doses of CAF studied in this clinical trial. Other investigators have examined the prognostic or predictive utilities of angiogenesis in patients who have received standard, postoperative, or neoadjuvant doxorubicin-based chemotherapy and the results are mixed.10,15,38-40 Surprisingly, in one of the largest studies that suggests that angiogenesis retains prognostic utility in the setting of doxorubicin, neither size of primary tumor nor number of positive lymph nodes was prognostic.38 This enigmatic finding suggests that other, unknown features may have influenced outcome in this patient population.15,38 In summary, we conclude that the higher-dose regimen of adjuvant CAF is preferred for node-positive breast cancer patients, independent of angiogenic subgroup. Preclinical studies have suggested that neovascularization may occur in normal and/or malignant tissue as a response to hypoxic stress, perhaps through p53 protein expression and membrane-bound growth factor receptor/tyrosine-kinase pathways.41-45 Results from a recently reported investigation have suggested that mutated p53 is associated with high intratumoral levels of vascular endothelial growth factor and worse prognosis in patients with breast cancer.46 In that regard, previous studies using tissues from CALGB protocol 8541 have suggested an interaction between CAF dose and p53 protein expression and erbB-2 protein overexpression and/or gene amplification.26,27 Indeed, in the current study, measures of angiogenesis (in particular, the plexiform vascular pattern) were significantly associated with each of these factors, although not with CAF dose. These observations might deserve further investigation regarding the biologic pathways controlled by these important genes. One can speculate that angiogenesis may have failed to be prognostic in this study because of methodologic considerations. A number of different objective methods for quantifying angiogenesis have been described (see review in Vermeulen et al47). For the current study, we chose the method of Weidner et al.1 This method is the most commonly used in published reports, and it has been shown to provide prognostic information in a number of different patient populations by different investigators. In addition, we were able to demonstrate acceptable intra- and interobserver reproducibility using this method. Other quantitative methods that have more recently been shown to provide prognostic information in breast cancer patients include Chalkley counts and image analysis.22,47-49 Whether these methods prove superior to the method of Weidner remains to be determined. A second important methodologic consideration is the choice of a primary antibody to highlight microvessels. Several antibodies are currently available, but the most commonly used are antifactor VIII, anti-CD34, and anti-CD31. For the current study, we chose antifactor VIII, the antibody used by Weidner et al in their original study.1 Although anti-CD31 has been shown by some investigators to be more sensitive in the detection of endothelial cells,4 we have found this antibody to be less reliable in our archival material (data not shown). Moreover, in our experience, anti-CD31 is associated with nonspecific staining of inflammatory cells. Similar observations have been reported by others.47,50 It is possible that had we chosen an alternate quantitative method or a different primary antibody, we might have observed a significant association between angiogenesis and survival in this patient population. However, studies comparing various quantitative methods have shown that they tend to correlate with each other.50 This fact, coupled with the lack of any major trends toward increased MVD counts and decreased survival in our study population, argues that it is unlikely that other methodologies would have significantly altered our results. It is of interest that Axelsson et al18 also failed to observe a relationship between tumor MVD and survival in node-positive and node-negative breast cancer patients using the Weidner method of microvessel quantitation. Indeed, reanalysis of these same cases using the Chalkley method again failed to demonstrate a significant association between angiogenesis and survival.51 In addition to a quantitative analysis of angiogenesis, we also scored each tumor for the presence or absence of a PVP of neovascularization. The plexiform pattern has not been well described in prior studies. However, identification of this pattern might be important because tumors with a PVP may yield artificially low MVD counts by the Weidner method. The underlying mechanisms responsible for the PVP are unknown, but it may reflect a two-dimensional representation of vascular patterns previously described in animal studies.52 The presence or absence of this pattern may reflect regional differences in the type, concentration, and/or relative proportion of angiogenesis-stimulatory and inhibitory cytokines associated with these tumors. Unlike MVD counts, the presence of the PVP correlated with decreased OS in univariate analysis in our patient population. Furthermore, the presence of a plexiform pattern correlated with a number of other aggressive tumor characteristics. The biologic significance of these associations is unclear, but the practical consequence is that the plexiform pattern fails to provide independent prognostic information in multivariate analysis, which limits its use as a prognostic factor in this patient population. It is also possible that our results are not more positive because of the selection of the population we studied. For example, other investigators have reported that angiogenesis is prognostic and perhaps predictive in patients who have received no systemic therapy or in patients who have been treated with adjuvant tamoxifen or non–anthracycline-containing chemotherapy regimens.11,12,53 As discussed, CAF chemotherapy may negate the prognostic effects of angiogenesis. Otherwise, we were unable to detect important differences between the patients for whom tissues were available for this study and the larger population of participants in clinical trial 8541, either in regards to known prognostic factors or outcomes. Furthermore, tests for interaction between these factors, the dose of CAF, and the presence or absence of tamoxifen failed to identify subgroups in which measures of angiogeneses might be clinically useful. Like most other investigations of angiogenesis and prognosis, the studies described in this report were performed in primary breast cancer tissues. We have previously reported a preliminary, hypothesis-generating study in which the angiogenic phenotype in axillary lymph node metastases was more prognostic than that in primary cancer.21 Unfortunately, tissue blocks from axillary lymph nodes were unavailable for patients who participated in CALGB protocol 8541, and so we were unable to address this hypothesis directly. Perhaps other means of evaluating angiogenesis besides counting vessels after staining with antifactor VIII, CD31, or CD34 would provide more impressive results. Proposed novel methods have included staining for the transforming growth factor-beta receptor (endoglin), the vascular cell adhesion molecule, or for various angiogenic factors, such as vascular endothelial growth factor, platelet-derived endothelial cell growth factor, or one of the many members of the fibroblast growth factor family.54-56 Yet another consideration regards assessment of the maturity of the vessels, as can be evaluated by an antibody that recognizes the lamina lucida of mature small vessels and capillaries but not of new vessels.57 Although each is intriguing, none of these methods has been experimentally proven superior to another, or to older methods such as were used in the present study, in regard to correlation with outcomes, especially in node-positive patients who have received CAF chemotherapy. In conclusion, in this study, we were unable to demonstrate independent prognostic utility of two different assessments of neovascularization in node-positive breast cancer patients treated with different doses and schedules of adjuvant CAF. Furthermore, measures of tumor angiogenesis did not seem to help predict which patients would benefit from higher doses of CAF. Therefore, we conclude that neovascularization as determined by factor VIII immunostaining cannot be used to select node-positive patients who might receive additional adjuvant therapy beyond four to six cycles of CAF, nor can it be used to identify patients for whom lower doses of CAF (such as 300/30/300 mg/m2) might be as effective as higher doses (such as such as 600/60/600 mg/m2). The results of this study, however, do not refute the findings of other investigators who have shown that blood vessel enumeration provides prognostically useful information in other subsets of breast cancer patients, including patients without lymph node metastases, those who do not receive adjuvant chemotherapy, and patients who are treated with other types of adjuvant chemotherapy. It is also possible that, as new therapeutic modalities that specifically target tumor angiogenesis are developed and begin to be introduced into clinical use, an assessment of microvessel density and distribution may provide useful insights into which patients with breast cancer may best benefit from these therapies. Regardless of its potential role as a tumor prognostic or predictive factor, tumor angiogenesis remains a critical element in tumor biology.7,58,59
APPENDIX The following institutions participated in the study: CALGBStatistical Office, Durham, NC, Stephen George, PhD (grant no. CA33601); Christiana Care Health Services, Inc. Community Clinical Oncology Program (CCOP),Wilmington, DE, Irving M. Berkowitz, DO (grant no. CA45418); Community Hospital -Syracuse CCOP, Syracuse, NY, Jeffrey Kirshner, MD (grant no. CA45389); Dana Farber Cancer Institute, Boston, MA, George P Canellos, MD (grant no. CA32291); Dartmouth Medical School, Norris Cotton Cancer Center, Lebanon, NH, Marc S. Ernstoff, MD (grant no. CA04326); Duke University Medical Center, Durham, NC, Jeffrey Crawford, MD (grant no. CA47577); Eastern Maine Medical Center CCOP, Bangor, ME, Philip L. Brooks, MD (grant no. CA35406); Kaiser Permanente CCOP, San Diego, CA, Jonathan A. Polikoff, MD (grant no. CA45374); Massachusetts General Hospital, Boston, MA, Michael L. Grossbard, MD (grant no. CA12449); McGill Department of Oncology, Montreal, PQ, Brian Leyland-Jones, MD (grant no. CA31809); Memorial Sloan-Kettering Cancer Center, New York, NY, George Bosl, MD (grant no. CA77651); Mount Sinai Medical Center, Miami, FL, Enrique Davila, MD (grant no. CA45564); Mount Sinai School of Medicine, New York, NY, Lewis R. Silverman, MD (grant no. CA04457); North Shore-Long Island Jewish Health System, Manhasset, NY, Daniel Budman, MD (grant no. CA35279); Rhode Island Hospital, Providence, RI, Louis A. Leone, MD (grant no. CA08025); Roswell Park Cancer Institute, Buffalo, NY, Ellis Levine, MD (grant no. CA02599); South New Jersey CCOP, Camden, NJ, Jack Goldberg, MD (grant no. CA54697); Southeast Cancer Control Consortium Inc. CCOP, Goldsboro, NC, James N. Atkins, MD (grant no. CA45808); Southern Maine Medical Center, Scarborough, ME, Thomas J. Ervin, MD (grant no. CA37447); Southern Nevada Cancer Research Foundation CCOP, Las Vegas, NV, John Ellerton, MD (grant no. CA35421); State University of New York Maimonides Medical Center, Brooklyn, NY, Samuel Kopel, MD (grant no. CA25119); State University of New York Upstate Medical University, Syracuse, NY, Stephen L. Graziano, MD (grant no. CA21060); University of Alabama, Birmingham, AL, Robert Diasio, MD (grant no. CA47545); University of California at San Diego, San Diego, CA, Stephen L Seagren, MD (grant no. CA11789); University of Chicago Medical Center, Chicago, IL, Gini Fleming, MD (grant no. CA41287); University of Iowa Hospitals, Iowa City, IA, Gerald Clamon, MD (grant no. CA47642); University of Maryland Cancer Center, Baltimore, MD, David Van Echo, MD (grant no. CA31983); University of Massachusetts Medical Center, Worcester, MA, F. Marc Stewart, MD (grant no. CA37135); University of Missouri/Ellis Fischel Cancer Center, Columbia, MO, Michael C Perry, MD (grant no. CA12046); University of North Carolina at Chapel Hill, Chapel Hill, NC, Thomas C. Shea, MD (grant no. CA47559); University of Tennessee Memphis, Memphis, TN, Harvey B. Niell, MD (grant no. CA47555); Wake Forest University School of Medicine, Winston-Salem, NC, David D Hurd, MD (grant no. CA03927); Walter Reed Army Medical Center, Washington, DC, John C. Byrd, MD (grant no. CA26806); Washington University School of Medicine, St. Louis, MO, Nancy Bartlett, MD (grant no. CA77440); and Weill Medical College of Cornell University, New York, NY, Michael Schuster, MD (grant no. CA07968).
Supported by grants from the National Cancer Institute to the Cancer and Leukemia Group B (CA31946, U10-CA64061 Richard L. Schilsky, MD, Chairman), to I.C.H. (CA60138) and D.F.H. (U01 CA64507 and CA77597). Support was also provided by the Read Trust for Science (to A.J.G.) and QVC Presents the Fashion Footwear Association of New York Shoes-on-Sale (to D.F.H.).
The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute.
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ABSTRACT
INTRODUCTION
20% of the tumor displayed this pattern. 
.05 were considered statistically significant. 
