Sperman regression pointed out valid correlations between isolated CD24 versus CD24−/CD44+ expression (P=0.0018, r=0.35) and isolated CD133 versus CD133+/ESA+ expression (P<0.0001; r=0.08). These correlations were just expected, because isolated antibodies were part of the CSC profiles.
The just known impacts of the other classic clinicopathologic variables on relapse, death risk, and survival were confirmed in this study. Thus, smoking habit was the most impactful variable on relapse risk: on univariate analysis, increased 14.5 times this possibility (P=0.006; 95% CI, 2.11-100), what was confirmed on multivariate analysis, that revealed a 15.6 times greater risk of tumor relapse (P=0.006; 95% CI, 2.1-100.7). Smokers were also under on greater risk to death than nonsmokers, both on univariate analysis (P=0.03; 95% CI, 1.14-15.29, 4.1 times) and multivariate analysis (P=0.004; 95% CI, 1.9-36.4, 8.3 times). Smoking habit impacted both free of disease and overall survival of patients. The former was diminished by 40% in up 30 months of follow-up and the last reduced by 40% in up 25 months of follow-up (P=0.03).
“T” stage was the most impactful variable on death risk. Tumors classified as T3/T4 stages were associated with 12.8 times increased risk of death in the univariate analysis (P=0.002; 95% CI, 2.45-67.15) and with 10.4 times increased risk of relapse in the multivariate analysis (P=0.024; 95% CI, 1.94-55.91). Advanced “T” stages and breast skin invasion were other important variables which negatively influenced similarly free of disease and overall survival.
Other important variable was breast skin invasion. When vascular emboli was observed in the skin, relapse risk increased 4 times in the univariate analysis (P=0.039; 95% CI, 1.07-14.86). When any type of breast skin invasion (vascular or direct) was present, the death risk were 10.5 times greater in the univariate analysis (P=0.006; 95% CI, 1.92-56.58). These results were confirmed in the multivariate analysis: vascular emboli in the skin and direct skin invasion, respectively, increased the death risk by 10.4 times (P=0.006; 95% CI, 1.9-56.6) and by 7.3 times (P=0.007; 95% CI, 1.7-30.9).
Other variables associated with increased relapse risk in the univariate analysis were the “N” stage, familial history of cancer, and tumor volume. Tumor multifocality and tumor volume were the other variables associated with death risk in the univariate analysis. Positive axillary lymph nodes (increased risk of 37.2 times, P=0.0004; 95% CI, 4.9-279.6) and familial history of cancer (increased risk of 13.7 times, P=0.0009; 95% CI, 2.9-64.1) were confirmed in the multivariate analysis as relapse risk associations.
This study aimed to characterize immunohistochemical expression of putative CSC antibodies in paraffin-embedded tissue of breast tumors and to discuss their possible clinical and prognostic values. In neoplastic tissue, CSC are very scarce and heterogenously distributed and numerical counting of these cells did not follow a normal distribution. Thus, in the literature, CSC frequency were reported as <2% of the total cell population in neoplastic tissue and the identification of these cells depends on the sampled tumor area.3 For these reasons, CSC counting was categorized simply as positive or negative in this study. So, the cases were considered positive even when an isolated cell was stained.
The sampling of a small area of the tumor in TMA can impair the CSC detection, but minimal invasive procedures are increasing nowadays. So, small surgical specimens in diagnostic routine are frequent. The choice of microarray is consistent with this tendency, as it is an easy procedure to do in the daily routine. Beside to this choice, some studies in the literature considered that TMA can sample satisfactorily the tumors, adding some economic advantages, such the less quantity of antibodies needed on these samples.22,23
In this study, the features of the selected population were in agreement with the epidemiological profiles observed in other studies in the literature, especially the ones involving high socioeconomic levels of population.17–19
The new findings of this study were related mainly with the CSC profiles antibodies (CD24−/CD44+ and CD133+/ESA+). Thus, these profiles were positive in the majority of the cases and were associated with some important clinicopathologic variables and poor prognosis.
In this regard, it was observed that CSC-1 was more frequently positive in early “T” stages (T1/T2) in this study. This finding can be related to another result of this study: CSC-1 was more frequently positive in patients older than 40 years. Even though not confirmed in other studies in the literature, breast cancer is assumed to grow slower in older groups than in younger groups. The cut-off on 40 year old was established in this study for several reasons, mainly for breast cancer epidemiologic features, as that is the recommended age group to begin the screening of this cancer.
So, CSC-1 expression in early stage of breast tumors arising in older patients can explain this unusual decreasing of overall survival in these cases, even if have not been observed associations with other known prognostic factors in breast cancer, as expression of hormonal receptors and overexpression of HER-2/neu oncogen. This finding is unedited in the literature and it was not confirmed in any revised study.6,8,15,26–30,33,38 Immunohistochemical search of this profile can be valid in diagnostic routine, as it seems to be an independent prognostic variable.
Whereas CSC-1 was associated with early “T” stages, isolated CD24+ cells were frequently related with advanced “T” stages (T3/T4) tumors in this study. In other studies, isolated CD24+ cells were associated with earlier breast tumor relapse.31,32 Although, CD24+ cells were supposed to be more prevalent in this present study because of luminal molecular subtypes predominance.15 Moreover, advanced “T” stages are themselves a known worst prognostic factor. Therefore, isolated CD24 cells were not considered an independent factor of bad prognosis.
Another unedited finding of this study was that a greater frequency of different positive CSC markers in a same case was associated to some clinicopathologic factors of advanced tumors (P=0.004), as T3/T4 stages and less differentiated tumors (final histologic grades of Nottingham 2 and 3), reinforcing the association of CSC with clinicopathologic factors of worse prognosis. Thus, isolated positive CSC markers were not related with any prognostic or clinicopathologic relevant variable neither in this study nor in other studies.6,24
However, isolated CD133+ cells were an exception in this study. Interestingly, there were observed that isolated CD133+ cells were more frequently associated with tumors arising in patients in menopause. In breast cancer, this finding opposed the concept that hormones do not influence the CSC, because these cells do not express hormonal receptors.34,35 Actually, hormones could regulate stem-cell phenotype expression.35 In other revised study, the authors found CD133 expression associated to hormonal receptors expression in uterine carcinosarcomas, due to a possible common origin of this tumor from a CD133+ cell, from a mesenchymal müllerian progenitor.36 Moreover, speculation is that CD133 expression can be related to worst therapeutic response in luminal subtypes of breast cancer. Although, additional studies are necessary to investigate this possible association.
Finally, CSC-2 (CD133+/ESA+) was an important factor of bad prognosis in this study, raising the risk of relapse and diminishing free of disease survival, but with no association with any clinicopathologic variable, on compliance to other studies.8 ,15,26–30,33,37
The other single stained antibodies—OCT 3/4, C-KIT, CD10, CD34, p63, SOX-2, and ALDH-1—were not associated with any clinical or pathologic variables of practical value, confirming other studies findings. For these reasons, they cannot be considered true CSC markers, especially when interpreted in an isolated context.6,24,25
The associations between classical clinicopathologic variables in breast cancer, as smoking habit, breast skin invasion, “T” and “N” stages, with strong relapse and risk of death only were confirmed in this present study and were not new findings.
There were attested 2 CSC immunohistochemical profiles (CD24−/CD44+ and CD133+/ESA+), respectively, by the negative impact on overall survival and free of disease survival. The absence of associations with known prognostic factors strengthens these CSC profiles as independent prognostic factors in breast cancer in this study.
Some of these putative CSC antibodies were irrefutably associated with important clinicopathologic factors in this study, justifying immunohistochemical search of these antibodies in diagnostic routine.
Thus, CSC-1 were associated with early “T” stages and with patients older than 40 years, a greater number of different positive antibodies in a same case were related with advanced “T” stages and with less differentiated tumors (final histologic grades of Nottingham 2 and 3) and isolated CD133 was related with tumors arising in patients in menopause.
The therapeutic resistance of some of breast cancers is probably caused by these cancer stem cells, which may become an important therapeutic target in the future, to improve survival on breast cancer.
The authors thank specially to Marina França de Resende, MD (A.C. Camargo Cancer Center, São Paulo-SP, Brazil), who performed the IHC technique in the cases and to all this service staff, that supported with the other techniques and materials needed for this study. The authors thank to the statistical service of Medical Sciences Faculty of the State University of Campinas (FCM-UNICAMP, Campinas-SP, Brazil), which helped to analyze the obtained data.
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Keywords:Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved.
breast cancer; cancer stem cell; immunohistochemistry