Lack of correlation of stem cell markers in breast cancer stem cells

BACKGROUND: Various markers are used to identify the unique sub-population of breast cancer cells with stem cell properties. Whether these markers are expressed in all breast cancers, identify the same population of cells, or equate to therapeutic response is controversial. METHODS: We investigated the expression of multiple cancer stem cell markers in human breast cancer samples and cell lines in vitro and in vivo, comparing across and within samples and relating expression with growth and therapeutic response to doxorubicin, docetaxol and radiotherapy. RESULTS: CD24, CD44, ALDH and SOX2 expression, the ability to form mammospheres and side-population cells are variably present in human cancers and cell lines. Each marker identifies a unique rather than common population of cancer cells. In vivo, cells expressing these markers are not specifically localized to the presumptive stem cell niche at the tumour/stroma interface. Repeated therapy does not consistently enrich cells expressing these markers, although ER-negative cells accumulate. CONCLUSIONS: Commonly employed methods identify different cancer cell sub-populations with no consistent therapeutic implications, rather than a single population of cells. The relationships of breast cancer stem cells to clinical parameters will require identification of specific markers or panels for the individual cancer

Cancer stem cell markers in breast cancer: pathological, clinical and prognostic significance

INTRODUCTION: The cancer stem cell (CSC) hypothesis states that tumours consist of a cellular hierarchy with CSCs at the apex driving tumour recurrence and metastasis. Hence, CSCs are potentially of profound clinical importance. We set out to establish the clinical relevance of breast CSC markers by profiling a large cohort of breast tumours in tissue microarrays (TMAs) using immunohistochemistry (IHC). METHODS: We included 4, 125 patients enrolled in the SEARCH population-based study with tumours represented in TMAs and classified into molecular subtype according to a validated IHC-based five-marker scheme. IHC was used to detect CD44/CD24, ALDH1A1, aldehyde dehydrogenase family 1 member A3 (ALDH1A3) and integrin alpha-6 (ITGA6). A 'Total CSC' score representing expression of all four CSC markers was also investigated. Association with breast cancer specific survival (BCSS) at 10 years was assessed using a Cox proportional-hazards model. This study was complied with REMARK criteria. RESULTS: In ER negative cases, multivariate analysis showed that ITGA6 was an independent prognostic factor with a time-dependent effect restricted to the first two years of follow-up (hazard ratio (HR) for 0 to 2 years follow-up, 2.4; 95% confidence interval (95% CI), 1.2 to 4.8; P = 0.009). The composite 'Total CSC' score carried independent prognostic significance in ER negative cases for the first four years of follow-up (HR for 0 to 4 years follow-up, 1.3; 95% CI, 1.1 to 1.6; P = 0.006). CONCLUSIONS: Breast CSC markers do not identify identical subpopulations in primary tumours. Both ITGA6 and a composite Total CSC score show independent prognostic significance in ER negative disease. The use of multiple markers to identify tumours enriched for CSCs has the greatest prognostic value. In the absence of more specific markers, we propose that the effective translation of the CSC hypothesis into patient benefit will necessitate the use of a panel of markers to robustly identify tumours enriched for CSCs

Interobserver variability in upfront dichotomous histopathological assessment of ductal carcinoma in situ of the breast: the DCISion study

Histopathological assessment of ductal carcinoma in situ, a nonobligate precursor of invasive breast cancer, is characterized by considerable interobserver variability. Previously, post hoc dichotomization of multicategorical variables was used to determine the “ideal” cutoffs for dichotomous assessment. The present international multicenter study evaluated interobserver variability among 39 pathologists who performed upfront dichotomous evaluation of 149 consecutive ductal carcinomas in situ. All pathologists independently assessed nuclear atypia, necrosis, solid ductal carcinoma in situ architecture, calcifications, stromal architecture, and lobular cancerization in one digital slide per lesion. Stromal inflammation was assessed semiquantitatively. Tumor-infiltrating lymphocytes were quantified as percentages and dichotomously assessed with a cutoff at 50%. Krippendorff’s alpha (KA), Cohen’s kappa and intraclass correlation coefficient were calculated for the appropriate variables. Lobular cancerization (KA = 0.396), nuclear atypia (KA = 0.422), and stromal architecture (KA = 0.450) showed the highest interobserver variability. Stromal inflammation (KA = 0.564), dichotomously assessed tumor-infiltrating lymphocytes (KA = 0.520), and comedonecrosis (KA = 0.539) showed slightly lower interobserver disagreement. Solid ductal carcinoma in situ architecture (KA = 0.602) and calcifications (KA = 0.676) presented with the lowest interobserver variability. Semiquantitative assessment of stromal inflammation resulted in a slightly higher interobserver concordance than upfront dichotomous tumor-infiltrating lymphocytes assessment (KA = 0.564 versus KA = 0.520). High stromal inflammation corresponded best with dichotomously assessed tumor-infiltrating lymphocytes when the cutoff was set at 10% (kappa = 0.881). Nevertheless, a post hoc tumor-infiltrating lymphocytes cutoff set at 20% resulted in the highest interobserver agreement (KA = 0.669). Despite upfront dichotomous evaluation, the interobserver variability remains considerable and is at most acceptable, although it varies among the different histopathological features. Future studies should investigate its impact on ductal carcinoma in situ prognostication. Forthcoming machine learning algorithms may be useful to tackle this substantial diagnostic challenge

Age determines the prognostic role of the cancer stem cell marker aldehyde dehydrogenase-1 in breast cancer

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to compare the expression and the prognostic effect of the breast cancer stem cell marker aldehyde dehydrogenase-1 (ALDH1) in young and elderly breast cancer patients.</p> <p>Methods</p> <p>The study population (N = 574) consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Median follow-up was 17.9 years (range: 0.1 to 23.5). Tissue microarray slides were immunohistochemically stained for ALDH1 expression and quantified by two independent observers who were blinded to clinical outcome. Assessment of the prognostic effect of ALDH1 expression was stratified according to age and systemic treatment.</p> <p>Results</p> <p>Complete lack of expression of ALDH1 was found in 40% of tumors. With increasing age more tumors showed complete absence of ALDH1 expression (<it>P </it>< .001). In patients aged > 65 years, ALDH1 status was not associated with any clinical outcome. Conversely, in patients aged < 65 years, ALDH1 positivity was an independent risk factor of worse outcome for relapse free period (hazard ratio = 1.71 (95% CI, 1.09 to 2.68); <it>P </it>= .021) and relative survival (relative excess risks of death = 2.36 (95% CI, 1.22 to 3.68); <it>P </it>= .016). Ten-year relative survival risk was 57% in ALDH1-positive patients compared to 83% in ALDH1-negative patients.</p> <p>Conclusion</p> <p>ALDH1 expression and its prognostic effect are age-dependent. Our results support the hypothesis that breast cancer biology is different in elderly patients compared to their younger counterparts and emphasizes the importance of taking into consideration age-specific interactions in breast cancer research.</p

Interobserver variability in the assessment of stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative invasive breast carcinoma influences the association with pathological complete response: the IVITA study

High stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC) are associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). Histopathological assessment of sTILs in TNBC biopsies is characterized by substantial interobserver variability, but it is unknown whether this affects its association with pCR. Here, we aimed to investigate the degree of interobserver variability in an international study, and its impact on the relationship between sTILs and pCR. Forty pathologists assessed sTILs as a percentage in digitalized biopsy slides, originating from 41 TNBC patients who were treated with NAC followed by surgery. Pathological response was quantified by the MD Anderson Residual Cancer Burden (RCB) score. Intraclass correlation coefficients (ICCs) were calculated per pathologist duo and Bland–Altman plots were constructed. The relation between sTILs and pCR or RCB class was investigated. The ICCs ranged from −0.376 to 0.947 (mean: 0.659), indicating substantial interobserver variability. Nevertheless, high sTILs scores were significantly associated with pCR for 36 participants (90%), and with RCB class for eight participants (20%). Post hoc sTILs cutoffs at 20% and 40% resulted in variable associations with pCR. The sTILs in TNBC with RCB-II and RCB-III were intermediate to those of RCB-0 and RCB-I, with lowest sTILs observed in RCB-I. However, the limited number of RCB-I cases precludes any definite conclusions due to lack of power, and this observation therefore requires further investigation. In conclusion, sTILs are a robust marker for pCR at the group level. However, if sTILs are to be used to guide the NAC scheme for individual patients, the observed interobserver variability might substantially affect the chance of obtaining a pCR. Future studies should determine the ‘ideal’ sTILs threshold, and attempt to fine-tune the patient selection for sTILs-based de-escalation of NAC regimens. At present, there is insufficient evidence for robust and reproducible sTILs-guided therapeutic decisions

Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer

Abstract Introduction In breast cancer, gene expression analyses have defined five tumor subtypes (luminal A, luminal B, HER2-enriched, basal-like and claudin-low), each of which has unique biologic and prognostic features. Here, we comprehensively characterize the recently identified claudin-low tumor subtype. Methods The clinical, pathological and biological features of claudin-low tumors were compared to the other tumor subtypes using an updated human tumor database and multiple independent data sets. These main features of claudin-low tumors were also evaluated in a panel of breast cancer cell lines and genetically engineered mouse models. Results Claudin-low tumors are characterized by the low to absent expression of luminal differentiation markers, high enrichment for epithelial-to-mesenchymal transition markers, immune response genes and cancer stem cell-like features. Clinically, the majority of claudin-low tumors are poor prognosis estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and epidermal growth factor receptor 2 (HER2)-negative (triple negative) invasive ductal carcinomas with a high frequency of metaplastic and medullary differentiation. They also have a response rate to standard preoperative chemotherapy that is intermediate between that of basal-like and luminal tumors. Interestingly, we show that a group of highly utilized breast cancer cell lines, and several genetically engineered mouse models, express the claudin-low phenotype. Finally, we confirm that a prognostically relevant differentiation hierarchy exists across all breast cancers in which the claudin-low subtype most closely resembles the mammary epithelial stem cell. Conclusions These results should help to improve our understanding of the biologic heterogeneity of breast cancer and provide tools for the further evaluation of the unique biology of claudin-low tumors and cell lines