Microvessel Density and Status of p53 Protein as Potential Prognostic Factors for Adjuvant Anthracycline Chemotherapy in Retrospective Analysis of Early Breast Cancer Patients Group

In the present retrospective study, immunohistochemical staining was used to determine the prognostic significance of MVD and p53 status in a homogenous patient cohort (T1-T2, N1-N2, M0) who received anthracyclines as adjuvant chemotherapy. In this study we have shown, according to our best knowledge for the first time, that all patients with higher MVD tumors survived 5 years without any evidence of cancer disease, whereas DFS for patients with lower MVD cancers was 79.7 % (median follow-up 36 months). The role of MVD as independent prognostic parameter was also confirmed in multivariate Cox regression analysis. A significant difference in DFS was also observed between women with p53 negative tumors (88.3 %) and patients with p53 positive cancers (78.7 %), however, in multivariate Cox regression analysis p53 status did not reach statistical significance.

In this study, higher MVD level was a favourable prognostic factor for patients DFS after anthracyclines based adjuvant chemotherapy. This finding is in concordance with results obtained by several other authors. Ioachim et al. [16] have analyzed MVD, using Factor VIII as endothelial marker, in the group of 82 patients with T1-T3, N0-N+, M0 breast cancer. They have found, similarly to us, significant correlation between higher MVD and increased relapse free survival (RFS) and overall survival (OS). In turn, Protopapa et al. [17] in a small group of 26 premonopausal women with ductal invasive, 2–5 cm in diameter carcinomas, treated before or after mastectomy with anthracyclines, have shown longer survival in the group of patients with higher MVD (assessed by Masson’s Trichrome technique) tumors. Gunel et al. [18] in 42 early breast cancer patients (T1-2, N0-2, M0), revealed that anthracycline based adjuvant chemotherapy was particularly effective in lymph node positive breast cancer patients with increased angiogenesis. One of possible explanations of positive correlation between higher MVD and better results of anthracyclines treatment is related with the mechanisms of these drugs’ action. A principal mechanism of anthracycline cytotoxic effects is their ability to intercalate into DNA and to inhibit topoisomerase II activity. Along with topoisomerase inhibition, anthracyclines stimulate the formation of reactive oxygen species (ROS), which at high level, can significantly contribute to the cytotoxic activity of these drugs through induction of cell death, apoptosis and senescence [29]. Therefore, in well vascularized tumors, with good oxygen access, the production of ROS may be even 2-fold greater [30] and thus cytotoxicity may be also greater. It has been also suggested that ROS, as signalling molecules, stimulate angiogenesis process via induction of redox sensitive gene expression (VEGF, matrix metalloproteinases, urokinase plasminogen activator) [31]. Taking into account all these facts, it is a kind of positive feedback loop, in which high level of ROS (result of anthracycline acction) stimulates higher cytotoxicity and activates new vessel formation that is responsible for even higher ROS level. Higher antharcycline sensitivity in more vascularised tumors may be also related with increased cytotoxic agents access to tumor cells.

By contrast, there are three studies, in which reverse correlation between MVD and the patients survival after anthracycline based adjuvant chemotherapy was found. In the group of 215 women with T1-T3, N+, M0 breast cancer treated with four cycles of doxorubicin–containing therapy followed by high doses of cyclophosphamide/cisplatin/carmustine, Nieto et al. [8] have found significantly longer RFS and OS for patients with lower MVD tumors. Similar relation was found in the studies of Tas et al. [9] in the group of 120 breast cancer (T1-T3, N0-3, M0) patients treated with CAF or cyclophosphamide/methotrexate/fluorouracil (CMF) and of Viens et al. [10] who studied 135 women with breast cancer (T0-3, N1-3, M0) after CAF. The authors explain these results by important role of angiogenesis in tumor growth, invasion and metastasis [7, 32].

Moreover, there are several papers [11‐15], in which no significant relation between MVD level and treatment outcome after anthracyclines based adjuvant chemotherapy was obtained. The discordances in results concerning the MVD prognostic value may be a consequence of many reasons. First of all, in many studies, great heterogeneity in adjuvant chemotherapy schedule and tumor clinical stage was observed [9, 12, 13]. Besides, some studies, in which higher MVD level was related with worse treatment outcome, analyzed relatively long term follow-up (9 years in the study of Nieto et al. [8], and 5 years in Viens et al. [10]). As in our study the median follow-up was 36 months, it could be speculated that the prognostic significance of MVD may change during follow-up time and higher MVD level is related with good prognosis within 3 years after initial treatment. Another reason of contrary results respecting MVD prognostic potential is connected with differences in the methodology and criteria of MVD evaluation. These differences concern the use of different antibodies (Factor VIII [12, 15, 16], CD-31 [8, 10, 11, 14], CD-34 [9, 13], endoglin [11]) characterised by different specificity [33], the method applied to count vessels (hot spot method [8, 11, 12, 14‐16] or Chalkley count [13]) and “hot spot” technique modification concerning the number of analyzed hot spots, which can vary from 1 (first hot-spot method) [9], trough 3 [12‐14, 16] to 5 [10] or more [15]. Moreover, there are some discrepancies between authors concerning cutoff points used to distingiush lower and higher MVD tumor subgroups. We identified cutoff point at the level of 75th percentile which is quite often found as statistically most potent by other authors [8, 33]. In other reports median [9‐12, 14, 15], or tertiles [13, 18] were used as a cutoff point.

In the present study, MVD did not correlate with TOPOIIα expression, nevertheless on the basis of these two biomarkers we were able to identify a subgroup of patients without any evidence of early recurrence after anthracycline treatment [3]. As we have shown, higher MVD (in our study, “low risk” patient group) was significantly related with lower N stage, hormonal receptors positivity and cytokeratin 5/6 negativity. Previously we have shown that lower TOPOIIα expression in tumors (indicated also patients with good prognosis) was significantly related with lower histologial grade, hormonal receptor positivity, cytokeratin 5/6 negativity, p53 negativity, lower proliferation rate and luminal A immunophenotype [3]. All these observations may suggest that these two biomarkers: MVD and TOPOIIα expression, allow for identification of two different subgroups of patients, both characterised by good prognosis and hence confirm the hypothesis that different molecular mechanisms are responsible for sensitivity or resistance to chemotherapy [29].

In our study p53 negativity was significantly related to longer DFS. This finding is in accordance with the results of other authors [21‐24]. They have reported statistically significant association between p53 immunopositivity and lack of response to adjuvant anthracycline based chemotherapy. Also in experimental studies performed on cell lines [19] or on mice with p53 lacking tumors [20] this effect was seen. These results can be explained in part by the fact that certain p53 gene can up-regulate the expression of multidrug resistance gene (MDR1) via stimulation of gene promoter [34]. Moreover, p53 has the key role in apoptosis activation. However, some reports [25‐27] failed to show a p53 prognostic potential determined by immonohistochemistry for response to adjuvant chemotherapy with anthracyclines. Lack of association between p53 mutations and p53 immunoreactivity may explain, in part, these contradictionary results. Lack of immunostaining is particularly frequent in tumors with p53 nonsense mutations [35]. Mutations in p53 may have also different biological effects. Some studies have shown that mutations in L2 and L3 domains, which are critical regions responsible for DNA binding, were associated with poor prognosis in breast cancer and correlated with resistance to doxorubicin [26, 35]. In turn, Bug et al. [36] have shown that even closely related anthracyclines induce the synthesis of different, opposing transcripts from p53 locus. Therefore, the minor differences in treatment schedule can influence the results concerning p53 prognostic role.