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Clinical Cancer Research 14, 2341, April 15, 2008. doi: 10.1158/1078-0432.CCR-07-4214
© 2008 American Association for Cancer Research
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Imaging, Diagnosis, Prognosis

Vascularization in Primary Breast Carcinomas: Its Prognostic Significance and Relationship with Tumor Cell Dissemination

Hari Prasad Dhakal1, Bjørn Naume2, Marit Synnestvedt2, Elin Borgen1, Rolf Kaaresen4, Ellen Schlichting4, Gro Wiedswang4, Assia Bassarova1, Karl-Erik Giercksky3 and Jahn M. Nesland1

Authors' Affiliations: 1 Pathology Laboratories, 2 Cancer Clinic, and 3 Surgical Clinic, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Montebello, Oslo, Norway, and 4 Surgical Department, Ullevål University Hospital, Kirkeveien, Oslo, Norway

Requests for reprints: Jahn M. Nesland, Pathology Laboratories, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Montebello 0310, Oslo, Norway. Phone: 47-2293-5620; Fax: 47-2273-0164; E-mail: j.m.nesland{at}medisin.uio.no.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The interaction between tumor cells, stroma, and endothelial cells is important for the dissemination of tumor cells. The aim of the present study is to examine vascularity in primary breast carcinomas and its prognostic significance and relationship with tumor cell dissemination.

Experimental Design: A total of 498 invasive breast carcinomas were analyzed. Representative tumor sections were stained for CD34 and CD105, and vascularity was quantified by the Chalkley method. The relationship between Chalkley counts, vascular invasion, disseminated tumor cells (DTC) in the bone marrow, other clinicopathologic variables, and clinical outcome was evaluated.

Results: High vascular grades determined by Chalkley counts were significantly associated with shorter distant disease–free survival and breast cancer–specific survival in all patients (P < 0.001, log-rank) and in node-negative patients not receiving adjuvant systemic therapy (P < 0.05). In multivariate analysis, both CD34 and CD105 Chalkley counts showed prognostic significance for distant disease–free survival (P = 0.014 and P = 0.026), whereas CD34 also showed prognostic significance for breast cancer–specific survival (P = 0.007). Vascular invasion and DTCs in the bone marrow showed independent prognostic significance. DTC did not discriminate survival for CD34 low Chalkley counts, whereas a very poor prognosis was observed for DTC-positive patients with high CD34 counts. In node-negative patients not receiving systemic chemotherapy, high CD34 and high CD105 counts in combination identified patients with unfavorable outcome, as opposed to all other CD34/CD105 combinations.

Conclusions: Improved identification of risk groups could be obtained by adding CD34 and CD105 vascular analysis to DTC, vascular invasion, and other primary tumor factors. This may facilitate the selection of candidates for adjuvant systemic therapy.

Breast cancer is one of the main causes of cancer death among women worldwide, with ~411,000 deaths per year (1). Stage, tumor size, histologic grade, hormone receptor status, and c-erbB2 status are still the most important determinants for risk stratification and adjuvant treatment decision (2, 3). Of these, lymph node status remains the most important prognostic factor. Despite a node-negative status, 20% to 30% of patients with breast cancer develop distant metastases (4).

The tumor cells can directly enter the circulation and disseminate to distant organs, bypassing lymph nodes, and causing tumor cell dissemination at an early stage (5). The presence of vascular invasion, representing invasion of tumor cells into lymphatic and/or blood vessels, has a marked effect on the risk of future metastasis (6, 7). Thus, methods for identifying circulating tumor cells in peripheral blood and disseminated tumor cells (DTC) in bone marrow in localized breast cancer have also been developed (8), and the presence of DTC in bone marrow has been confirmed as an independent prognostic factor (9–14). Smaller studies indicate the same association for circulating tumor cells (15, 16).

Angiogenesis is essential for cancer progression. It is required to meet the metabolic demands of a growing tumor mass (17). It may also be important for tumor cell escape from the primary site, and thus, potentially plays an important role in the metastatic process (18). The prognostic significance of angiogenesis in breast carcinomas has been shown in various studies (19–25). Assessment of intratumoral microvessel density using immunohistochemistry with panendothelial markers like factor VIII–related antigen, CD31, CD34, and the activated endothelial cell marker CD105 is a commonly used technique for quantifying tumor angiogenesis in breast cancer (19, 26). The Chalkley method is less subjective, fast, and easy to use (26).

The relation between early tumor dissemination and vascularization has not been extensively studied. In two smaller studies, tumor vascularity has been reported to be associated with bone marrow micrometastasis (27, 28). In the present study, we have examined primary tumor vascularity, its prognostic significance and association with vascular invasion, and the presence of isolated tumor cells in the bone marrow from 498 patients with breast carcinoma.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients, tumors, and DTC. We have examined vascularity in 498 primary invasive breast carcinomas from the Oslo Micrometastasis Project, which enrolled a total of 920 patients in the period from 1995 to 1998. The patients were selected on the basis of availability and adequacy of primary tumor material for angiogenesis analysis. Studies of DTC's prognostic significance and relationship with clinicopathologic variables have been reported previously (9, 29).

At primary surgery (immediately prior to), a total of 40 mL of bone marrow (BM) was aspirated from the anterior and posterior iliac crests bilaterally (10 mL per site), and processed as described previously (29). After separation by density centrifugation, mononuclear cells were collected and cytospins prepared (5 x 105 mononuclear cells/slide). Four slides (2 x 106 bone marrow mononuclear cells) were incubated with the anticytokeratin monoclonal antibodies AE1 and AE3 (Sanbio), and the same number of slides were incubated with an isotype-specific irrelevant control monoclonal antibody. The visualization step included the alkaline phosphatase/antialkaline phosphatase reaction, and the slides were counterstained with hematoxylin to visualize nuclear morphology. The cytospins were manually screened by light microscopy using the x10 lens. Immunostained cells present in cell clusters or with nuclear size clearly enlarged, as compared with surrounding hematopoietic cells, were scored as DTC. Cells lacking these signs could be scored as DTC if presenting no recognizable hematopoietic cell characteristics, these DTC typically had strong and/or irregular cytoplasmic staining partially covering the nucleus. The presence of positive cells classified as tumor cells both in AE1/AE3-stained slides and in the corresponding negative controls resulted in the exclusion of the sample from diagnostic conclusions.

The median follow-up time was 85 months, ranging from 1 to 125 months. Relevant clinicopathologic data were extracted from the database of the Oslo Micrometastasis Study Project. The study was approved by the Regional Ethical Committee. Written consent was obtained from all patients.

In 457 cases, we had available information regarding surgical treatment. Three hundred and twenty-six (71%) had breast conservation surgery and 131 (29%) had undergone modified radical mastectomy.

Of the 478 patients with information regarding nonsurgical treatment, 224 (47%) had received radiation therapy and 254 (53%) had postoperative systemic adjuvant therapy (chemotherapy and/or tamoxifen) according to national guidelines. The standard adjuvant chemotherapy regimen consisted of six cycles every three weeks of i.v. cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, and fluorouracil 600 mg/m2. Patients who had received preoperative chemotherapy or who had metastases within 1 month after operation were not included in our present study.

Morphology. Tumors were classified according to WHO recommendations (7). Tumor grading was done in accordance with Elston and Ellis (30) and showed the following distribution: GI108 (21.7%), GII 251 (50.4%), and GIII139 (27.9%). The presence of tumor cells in vessels was recorded from H&E-stained slides. Tumor size was obtained from the original pathology report. Necrosis and inflammatory cell infiltrates in the primary tumor, including tumor margins, were noted. The inflammatory cellular infiltrate was categorized subjectively into two groups: minimal/mild in one and moderate/marked in another. The relationship between tumor cell mass and tumor stroma (tumor/stroma ratio) was subjectively evaluated and divided into two categories: tumor cells with more than tumor stroma, and tumor cells with less than tumor stroma. All cases were evaluated without knowledge of clinical data and bone marrow status.

Immunohistochemistry. Four-micrometer-thick sections with representative tumor tissue were cut from the formalin-fixed paraffin-embedded blocks and stained for CD34 and CD105. The primary monoclonal murine antibody (IgG1) against CD34, QBEND-10 (Monosan) in 1:200 dilution and mouse monoclonal antibody IgG2a (Novocastra) against CD105 in 1:40 dilution were applied, using the Dako EnVision+ System Peroxidase (DAB, K4007; Dako Corporation) and Dako Autostainer.

Deparaffinized sections were microwaved in Tris/EDTA (pH 9.0) to unmask the epitopes, followed by treatment with 0.03% hydrogen peroxide for 5 min to block the endogenous peroxidase. The sections were incubated with the monoclonal antibodies for 30 min at room temperature and then with peroxidase-labeled polymer conjugated to goat anti-mouse antibody for 30 min, and finally stained for 10 min with 3'3-diaminobenzidine tetrahydrochloride. All sections were counterstained with hematoxylin.

Positive controls with tissues known to be positive for the markers were included. Negative controls included substitution of the monoclonal antibody with mouse myeloma proteins of the same subclasses and concentrations as with the primary monoclonal antibodies. All controls gave satisfactory results.

Quantification of tumor vascularity. We used the Chalkley method for estimation of tumor vascularity as proposed in a recent international consensus meeting (31). The reproducibility and prognostic value have been previously reported (24, 25, 32). Tumor sections were scanned carefully at x40 and then at x100 magnification. The most intense vascular areas (hotspots) were selected subjectively from each tumor section as described by Weidner (33) at x100 magnification. A 25-point Chalkley eyepiece graticule was applied to each hotspot area at x200 magnification with a Chalkley grid area of 0.1886 mm2 (Eclipse E400, Nikon microscope). Counts were done in areas with carcinoma invasion, including the tumor periphery. Sclerotic and necrotic areas were avoided. All Chalkley counts were done by one pathologist (H.P. Dhakal) without knowledge of clinical data, bone marrow status or patient's prognostic outcome similar to earlier reports (24, 34). The graticule was oriented to permit the maximum number of points to hit on or within the areas of immunohistochemically stained microvessels. The number of points hitting the stained vessels and endothelial cells were counted in each hotspot. The highest score among the three hotspot counts was taken for further analyses.

We used the same procedure for both CD34 and CD105. As the basis for further analysis, a preselected cutoff, the high tertile or 67th percentile based on an earlier report (25), was used to stratify patients into high and low vascular groups. This was 7 for CD34 and 6 for CD105 Chalkley counts. Ninety-two cases were re-evaluated by the same pathologist (H.P. Dhakal) for both CD34 and CD105 quantification using the same method, and we did find a very good correlation (Pearson's r, 0.82; P < 0.001), similar to that reported by Hansen et al. for intraobserver correlation (with r = 0.85, P < 0.001; n = 40; ref. 32), and intraclass correlation coefficient (0.90; P < 0.001) for CD34. Similar results were obtained for CD105 (Pearson's r = 0.79; intraclass correlation coefficient, 0.88; P < 0.001).

Statistical analysis. We used a preselected cutoff point of ≥7 for CD34 and ≥6 for the highest CD105 Chalkley counts, respectively, to categorize into high and low vascular categories for further statistical analyses. The primary end point for the survival analyses was breast cancer–specific survival (BCSS), measured from the date of surgery to breast cancer–related death, or otherwise censored at the time of the last follow-up visit, or at non–cancer-related death. Secondary end points were time to locoregional and systemic relapse, and were measured in the same way. Metastases in the skeleton, liver, lungs, or central nervous system were recorded as systemic relapse. Kaplan-Meier survival curves for time to distant disease–free survival (DDFS), and BCSS were constructed. P values were computed by the log-rank test. Cox proportional hazard regression was used for multivariate (stepwise backward elimination) analyses of prognostic effect of relevant variables. The number of variables included in the multivariable analyses was restricted to ~10% of the number of events (systemic relapses/breast cancer deaths) within the population analyzed. The Pearson's {chi}2 test was used to test the association between angiogenesis and clinicopathologic variables. Intraobserver correlation was tested by intraclass correlation coefficient and Pearson's correlation. All P values were two-sided and P < 0.05 values were considered significant. For statistical analysis, the SPSS software (version 13.1) was used.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Patients and tumor characteristics. Patient age ranged from 30 to 90 years (mean, 59 years). Of the 498 cases, 54.4% were pT1, 38.2% were pT2, 5% were pT3-4, and 2.4% were pTX. The tumors were classified as infiltrating ductal carcinoma (71.5%), invasive lobular carcinoma (18.7%), mucinous carcinoma (1.6%), mixed carcinoma (4.2%), neuroendocrine carcinoma (1.2%), and other subtypes (2.8%). Three hundred and eight patients (63.1%) were lymph node–negative and 180 (36.9%) were node positive (Table 1 ).


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  		Table 1. Clinicopathologic characteristics of patients and their relation with angiogenesis

 
Angiogenesis. The CD34 Chalkley counts for primary tumor vascularity ranged from 2 to 13 (mean, 6.02; median, 6; SD, 1.97). As a dichotomized variable, predefined with 7 as cutoff value, 201 (40.4%) had high vascularity and 297 (59.6%) had low vascularity.

The CD105 Chalkley counts ranged from 2 to 11 (mean, 4.84; median, 5; SD, 1.77). One hundred and sixty-three (32.7%) were classified into the high vascular group and 335 (67.3%) into the low vascular group when dichotomized at a cutoff point of 6. Table 1 lists the results of the angiogenesis compared with clinicopathologic characteristics.

CD34 and CD105 Chalkley counts and relation with other variables. As shown in Table 1, high CD34 Chalkley counts as well as high CD105 Chalkley counts were strongly associated with higher tumor grade, presence of necrosis, presence of moderate/marked inflammatory cell infiltrates, hormone receptor negativity (estrogen receptor and progesterone receptor), and relatively more tumor cells than tumor stroma (P ≤ 0.003, {chi}2 test). High vascularity was more frequent among infiltrating ductal carcinomas compared with lobular and other histologic types (CD34, P < 0.001; CD105, P = 0.008), and was also associated with higher pT status (P < 0.001), vascular invasion (CD34 Chalkley count, P = 0.006; CD105, P = 0.014), and with positive p53 immunostaining (P ≤ 0.005). Her2 status and lymph node status did not show any association with CD34 or CD105 Chalkley counts (P ≥ 0.25). CD105 Chalkley estimates showed good correlation with CD34 Chalkley estimates as continuous variables with Pearson's correlation coefficient (0.61; P < 0.001). CD105 and CD34 Chalkley counts as binary variables using cutoffs of 6 and 7, respectively, showed significant association with each other (P < 0.001, {chi}2; see Table 1). Of the tumors with CD34-high or CD105-high counts, 45% were categorized as high for both variables. Among 472 of 498 patients with interpretable bone marrow status, 13.1% were DTC positive. No significant correlation was observed between the presence of DTC and CD34 or CD105 Chalkley counts (Table 1). Separate analysis of lobular carcinoma (n = 88) showed a high Chalkley count to be associated with DTC [CD34 (P = 0.026, {chi}2) and at borderline significance CD105 (P = 0.065)]. For infiltrating ductal carcinoma (n = 338) and other subtypes (n = 46), no such association was observed.

Clinical outcome. During the follow-up period (median, 85 months; range, 1-125 months), 112 of 491 (22.8%) patients with available information for systemic relapse experienced distant metastases. According to vascularity, the CD34-high vascular group was associated with markedly higher relapse rate (32.2%) than what was observed in the CD34-low vascular group (16.4%; P < 0.001, log-rank; Table 2 ). Similarly, 30.6% of patients in the CD105-high and 19% of patients in the CD105-low vascular group (P < 0.001, log-rank) experienced systemic relapse. Local recurrence was observed in 50 cases, with significant correlation to high CD105 Chalkley estimates (Table 2).


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  		Table 2. Systemic relapse (n = 491), local recurrence (n = 491), and survival status (n = 498) data comparing CD34 and CD105 Chalkley counts

 
Eighty-six of the 498 patients (17.3%) died of breast cancer, 28.4% of patients within the CD34-high vascular group and 9.8% in the CD34-low vascular group (P < 0.001, log-rank). For CD105, 25.8% of the high and 13.1% of the low vascular group died of breast cancer (P < 0.001, log-rank).

Sixty-one of 410 patients died of breast cancer disease in the bone marrow–negative group (14.9%), whereas 22 of 62 (35.5%) patients died in the bone marrow–positive group (P = 0.001, log-rank). Furthermore, 84 of 403 (20.8%) experienced systemic relapse in the bone marrow–negative group, and 23 of 62 (37.1%) patients in the bone marrow–positive group (P = 0.001, log-rank) experiencing systemic relapse. Local recurrence did not show any association with the presence of tumor cells in the bone marrow (P = 0.882, log-rank).

Patients with tumor cell vascular invasion showed a strong association with breast cancer–related death (38%, 41 of 108) and systemic relapse (42.9%, 45 of 105) compared with patients without vascular invasion (11.5%, 45 of 390 and 17.4%, 67 of 386, respectively). Vascular invasion was associated with local recurrence (P < 0.001, log-rank).

Survival analyses. Kaplan-Meier survival analyses (Fig. 1A-B, E-F ) showed reduced DDFS and BCSS among patients with tumors within the high vascular group for both CD34 and CD105 Chalkley counts. The same was evident for the presence of vascular invasion and positive DTC status (Fig. 1C-D, G-H). In subgroup analysis of the patients with node-negative status not receiving adjuvant systemic treatment, DDFS and BCSS were clearly reduced on CD34-high and CD105-high vascular groups (Fig. 1I-J, M-N). The presence of vascular invasion, but not bone marrow status, also discriminated for survival (Fig. 1K-L, O-P).


Figure 1
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  		Fig. 1. Kaplan-Meier survival curves for DDFS (A-D) in all patients and (I-L) in node negative no-adjuvant therapy groups. BCSS (E-H) in all and (M-P) in node-negative no–systemic therapy patients when assessed for CD34 and CD105 Chalkley counts (CD34, CD105), bone marrow (BM) status, and vascular invasion (Vascular inv), respectively.

 
All the variables listed in Table 1 were analyzed for their association with clinical outcome in univariate analysis, for selection of significant variables (by log-rank test) to include in the multivariable Cox regression analysis. We chose to perform separate analyses for CD34 and CD105 because of their close association. The variables included were tumor status (pT), vascular invasion, histologic grade, nodal status, hormone receptor status, presence of necrosis, presence of moderate/marked inflammatory infiltrates, and DTC status as these variables were significantly associated with BCSS in log-rank tests in Kaplan-Meier survival analyses (data not shown).

Systemic therapy was also included in the variable list. Although significant in the univariate analysis, p53 and c-erbB2 were not included because of statistical restrictions in the number of markers to be included in the calculations. However, p53 and c-erbB2 were tested in the multivariate analysis in the entire cohort of patients, and did not reach statistical significance (9).

As shown in Table 3 , CD34 Chalkley count, vascular invasion, hormone receptor status, bone marrow status, and nodal status were significantly associated with both BCSS and DDFS. CD105 Chalkley count and histologic grade were significantly associated with DDFS and necrosis, along with pT status with BCSS. The omission of necrosis and inflammatory infiltrates from the analysis (not widely used for therapy planning) resulted in a CD105 Chalkley count that was significantly associated with BCSS [relative risk, 1.77 (1.12-2.8), P = 0.015], but otherwise, did not affect results (data not shown).


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  		Table 3. Multivariate analysis by Cox regression for BCSS (n = 451) and DDFS (n = 432) in patients with complete data sets for CD34 and CD105 Chalkley counts

 
The association between CD34, CD105, and DTC was also analyzed. The survival analysis (Fig. 2A-D ) revealed that in the CD34-low vascular group, DTC status did not discriminate outcome, whereas for the CD34-high group, DTC-positive patients experienced a markedly reduced outcome (P < 0.001, log-rank). In contrast, the presence of DTC was associated with reduced survival for both CD105-low and CD105-high groups (Fig. 2E-H).


Figure 2
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  		Fig. 2. Kaplan-Meier survival curves (A) DDFS and (B) BCSS in the low CD34 vascular group; (C) DDFS and (D) BCSS in the high CD34 vascular group with disseminated tumor cells (DTC– and DTC+); (E) DDFS and (F) BCSS in low and (G) DDFS and (H) BCSS in high CD105 vascular groups with DTC – and DTC+; (I) DDFS and (J) BCSS with combined CD34/CD105 vascular groups in all patients. (K) DDFS and (L) BCSS with combined CD34/CD105 Chalkley counts in node-negative no–systemic therapy group. I–L, low (L) and high (H) CD34/CD105 levels. M and N, combined CD34/CD105 for DDFS and BCSS in node-negative no systemic therapy group of patients with presence of vascular invasion and O and P, in node-negative no systemic therapy group of patients with absence of vascular invasion, respectively.

 
We further studied the combination of CD34 and CD105 categories as pure prognostic factors, as well as the interrelation between these factors and vascular invasion and tumor dissemination. Kaplan-Meier survival analyses were done for the node-negative, no adjuvant subgroup. The vascularity groups constructed for this analysis were CD34low/CD105low, CD34low/CD105high, CD34high/CD105low, and CD34high and CD105high. As shown in Fig. 2I-L, no prognostic information was observed if none or only one of the vascular factors had high counts. In contrast, high Chalkley counts for both CD34 and CD105 identified patients with a markedly reduced survival (P ≤ 0.01). When vascular invasion was taken into account, no certain prognostic information of vascularity was detected for the vascular invasion–positive group (Fig. 2M-N). However, the small number of cases in each subgroup restricts the interpretation. Interestingly, in the larger vascular invasion–absent group (n = 177), CD34high/CD105high was clearly associated with reduced DDFS and BCSS (Fig. 2O-P). Bone marrow status alone did not significantly discriminate prognosis in the node-negative no-adjuvant group (Fig. 1L, P). However, in the CD34high/CD105high group, bone marrow status was associated with reduced survival (P = 0.003 for BCSS, P = 0.121 for DDFS), and all three relapses/deaths among bone marrow–positive patients were in the CD34high/CD105high group (five bone marrow–positive patients in this group). Furthermore, among patients with a combined absence of vascular invasion and CD34high/CD105high vascularity, a positive bone marrow status identified a small number of patients with extremely poor prognosis, as three out of three patients showed metastases and subsequent death (P < 0.001, log-rank; compared with bone marrow–negative patients).


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The development of metastasis in patients with breast cancer is a complex, multistep process in which angiogenesis, vascular invasion, and the settlement of single tumor cells in distant organs play a role. The presence of tumor cells in the bone marrow indicates that tumor cells have detached from the primary site, invaded the stroma and vessels, and entered into the circulation before settling in the bone marrow (5, 8). These DTCs could signal future metastasis, as has been shown in several studies (10), but may also reside as dormant cells. Also, previously published studies show that far from all patients at risk for future metastasis are identified with the current DTC (or circulating tumor cells) detection methods, emphasizing the importance of searching for alternative or complementary indicators directly related to tumor dissemination within the primary tumors. In this study, the presence of vascular invasion consistently showed strong prognostic significance in the whole as well as in different subgroups of patients, similar to earlier reports (6, 35). This reconfirms the prognostic importance of the assessment of vascular invasion in routine staining. In our study, we have shown that the combination of CD34 and CD105 Chalkley counts and DTC status add valuable prognostic information to vascular invasion. Thus, with the help of these analyses in concert, groups at high risk, otherwise not detected, could be identified.

By using various types of endothelial markers, different methods for the quantification of breast tumor vascularity have been applied, and their prognostic significances have been reported (19, 23–25, 36–39). CD34, a pan-endothelial marker widely used in angiogenesis research, is expressed in almost all endothelial cells without discriminating between vascular endothelial cells in normal or tumor tissues (19, 26, 39). CD105, a marker strongly expressed in activated vascular endothelial cells of both peritumoral and intratumoral blood vessels, is not expressed or is expressed at low levels in inactive vascular endothelial cells in normal tissue blood vessels (39, 40). We analyzed both CD34 and CD105 as markers for primary tumor vascularity using the Chalkley method. A clear association between high vascularity and unfavorable prognosis was found in node-negative patients who did not receive systemic chemotherapy. In the multivariate analysis, both CD34 and CD105 Chalkley counts retained prognostic significance for DDFS, whereas CD34 counts were also significant for BCSS. Our findings are comparable to other reports using the Chalkley method for vascular quantification (23–25, 34, 39). Interestingly, in the present study, the correlation to clinical outcome in the node-negative no-adjuvant group required high vascularity, as determined by both CD34 and CD105. A group of patients at high-risk was identified, which could be considered for adjuvant systemic therapy. Also, a markedly reduced survival in the CD34-high group with the presence of DTC and lack of DTC survival discrimination in the CD34-low group, clearly showed that these analyses in concert give higher prognostic precision. These results may indicate that vascular density by CD34 Chalkley count can select patients in postoperative phase for whom DTC analysis may be of value for optimal therapy-related risk stratification.

Prognostic significance of CD105 immunostaining in breast carcinoma has been reported in both fresh-frozen tumor tissue (39, 41) and in paraffin-embedded tissue (42, 43). Kumar et al. applied Chalkley counts at x400 magnification in 106 invasive breast carcinomas for both CD34 and CD105 markers, and found only CD105 to be prognostically significant (39). In a study of 905 invasive breast carcinoma cases by Dales et al., microvessel count was reported to be of prognostic significance in all cases, as well as in node-negative patients (41). In another study of 122 cases, CD105 marker in paraffin-embedded tumor tissue showed independent prognostic significance (43).

In our present study using paraffin-embedded tissue, we found prognostic significance of CD105 Chalkley estimates for both BCSS and DDFS. Prognostic significance for DDFS was retained in multivariate analysis. Thus, CD105 immunoexpression in paraffin-embedded tissue estimated by the Chalkley method seems to be useful in stratifying breast cancer cases into different prognostic groups and is particularly useful for the node-negative group, adding important prognostic information to CD34.

With an extended median follow-up period of 85 months, bone marrow status continued to show an independent prognostic significance for both BCSS and DDFS. Previous reports have suggested an association between primary tumor vascularity and DTC in the bone marrow (27, 28). We did not find a statistically significant association between high Chalkley counts of tumor vascularity and bone marrow micrometastasis, despite the higher percentage of DTC in the high vascular group. When analyzed separately for different histologic types, a high Chalkley count was only positively associated with DTC in bone marrow in invasive lobular carcinomas.

In our series, we had a lower detection rate (13.1%) of DTCs in the bone marrow compared with what was reported in the two previous studies (20% and 53%, respectively; refs. 27, 28). The possible reasons of low detection rate could partly be different stage distributions, differences in the frequency of markers for aggressiveness, and to some extent, methodology, including the type of antibody applied and differences in study population (9).

The frequency of pT1 and pN0 tumors was higher in the present study compared with that previously reported (27, 28). Also, traditional markers for aggressiveness, such as hormone receptor negativity and histologic grade 3 were less frequent in our series.

High motility and increased transendothelial invasiveness are crucial for metastasis to occur and have recently been shown in a subgroup of breast cancer cell lines coexpressing Her2 and early placenta insulin-like growth factor (44), indicating the possibility of an early tumor cell dissemination before a significant increase in vascularity. Higher vascularity provides a larger endothelial surface area to interact for tumor cells, and chances for dissemination logically should be higher (27, 28). However, high vessel count alone is not sufficient for tumor cell dissemination (27). Incomplete and immature newly formed microvessels in the beginning of angiogenesis (18) and tumor cell characteristics are important for invasion and dissemination (44–47).

In conclusion, our study confirms the prognostic significance of Chalkley estimates of primary breast carcinoma vascularity using both CD34 and CD105 endothelial markers, vascular invasion, and DTC in the bone marrow. Combined CD34/CD105 Chalkley counts analysis gives important additional information to vascular invasion and DTC status, improving the ability to identify relevant risk groups for the development of metastasis in breast cancer. This may improve the future possibility for individualization of adjuvant systemic treatment.


   Acknowledgments
 
We are grateful to Ellen Hellesylt and Mette Førsund for the high-quality immunohistochemistry, Berit Sandstad for multivariate analysis, and the Oslo Breast Cancer Micrometastasis Project for permitting the use of data.


   Footnotes
 
Grant support: The Norwegian Cancer Society.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/13/07; revised 11/27/07; accepted 12/17/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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B. D. Robinson, G. L. Sica, Y.-F. Liu, T. E. Rohan, F. B. Gertler, J. S. Condeelis, and J. G. Jones
Tumor Microenvironment of Metastasis in Human Breast Carcinoma: A Potential Prognostic Marker Linked to Hematogenous Dissemination
Clin. Cancer Res., April 1, 2009; 15(7): 2433 - 2441.
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