Tissue microarray (TMA) has completely revolutionized biomedical research. The ability to rapidly test new molecular markers on hundreds of tumor samples incorporated in a single slide, has considerably increased the knowledge of diagnostic, prognostic and predictive value of these molecules. However, for certain proteins, like cancer stem cells markers (CSCs), this technology should be used in a selective manner. Appropriate checks should be also randomly carried out on the corresponding whole sections coming from paraffin blocks of the same samples included in TMA. In fact, CSCs can be heterogeneously distributed within tumor area. Our personal experience, based on the employment of a specific breast cancer TMA, showed a non-perfect correlation with the expression analysis carried out for the same markers on the corresponding whole sections.
Background
Tissue microarray (TMA) could be considered as the corresponding morpho-pathologic version of gene-array. In fact, it is a powerful tool able to quickly analyze molecular markers in hundreds of tumors through a single experiment. Samples preparation procedure involves a series of steps: from the automatic coring of the samples embedded in paraffin blocks, to the cutting for the fitting of the sections, from the immunostaining, to the acquisition and interpretation of the slides.
TMA can be used for: testing new diagnostic antibodies, identifying new prognostic markers and determining applications in developmental biology Citation[1].
There are also several types of TMA: normal tissue array: used for identifying the topographic distribution of a specific antigen in various tissues; multi-tissue array: mainly used in quali-quantitative screening of the distribution of specific targets in different tumors, with the opportunity of analyzing the deviation of the distribution of the targets compared with normal; progression TMA: useful in identifying probable morphological or molecular alterations in tumor progression; prognostic TMA: commonly used to identify markers responsible for tumor progression and therefore correlated to a worse prognosis Citation[1]. It is associated with a database containing all the follow-up data of the patient.
Several technologies have been developed for CSCs detection in human cancers, including tumorisphere formation, flow cytometry, invasion assay system, multimarker immunohistochemistry (IHC) and gene expression analysis through real-time PCR Citation[3].
However, IHC is commonly used as it offers the possibility to associate stem cell niches with morphology. In fact, it is very important to define the location of these stem clones within the tumor mass. For this reason, a series of high specific antibodies for IHC have been designed. The use of TMA for the analysis of these markers, however, presents fundamental limits.
CSCs markers in solid tumors
Human solid tumors are extremely heterogeneous in terms of cell morphology, and their cancer stem phenotype can be also very variegated. In recent years, a series of molecular markers have been identified which are able to characterize tumor stem cells within different neoplasias. In most cases, the association of more CSC markers expression correctly defines the stem clones within the neoplasm. In some human tumors, the markers panel of stemness seems to be the same and in many cases these same markers represent important prognostic factors too. Transmembrane proteins CD44 and CD24 are among the most studied breast cancer stem cells markers (BCSCs).
High levels of CD44 associated with low levels of CD24 (CD44(+)/CD24(-/low)) would characterize stem populations in breast cancer Citation[4]. The clinical impact of CD24 and CD44 expression in breast tumors remains unclear even if the absence/low expression of CD24 associated with CD44 high expression in BCSCs was recently related to an invasive phenotype, poor prognosis and low survival Citation[5]. Moreover, CD44(+)/CD24(-/low) breast cancer stem-like cells play an important role in the clinical behavior of triple-negative breast cancer Citation[6].
Recently, CD44 has been associated with stem cell-like phenotype in some lung cancer cell lines Citation[7] and in vivo in primary lung cancer, where it seems to also play a prognostic role Citation[8]. CD133 was not only reported as a marker of tumorigenic brain cancer cells, but CD133+ tumor cells also represent the cellular population that confers glioma radioresistance and could be the source of tumor recurrence after radiation Citation[9]. Moreover, CD133 is also described as CSCs marker in other solid tumors, including invasive breast cancer triple negative, with very low levels of expression compared with other CSCs markers previously reported. The employment of this tumor marker of stemness in breast cancers has become popular very recently and its expression is often described as associated with a worse prognosis. The expression of CD133 is able to predict the prognosis in patients with triple-negative invasive ductal breast carcinoma, correlating with tumor size, metastasis of the axillary lymph nodes and the clinical stage Citation[10]. Moreover, in a multivariate analysis, CD133 showed an independent prognostic value in pN0 BC patients with a highly significant value regarding bone metastasis development, while no significant relationship was found between CD133 expression and age, histotype, tumor grade/stage and hormone receptor expression Citation[10].
Furthermore, CD133 antigen was described as a potential lung cancer stem/initiating-cell marker. The presence of CD133+ cells both in fresh biopsies of non-small cell lung cancer (NSCLC) and in stabilized cell lines was identified. This population was able to give rise to spheres and could act as tumor-initiating cells even if no statistically significance correlation was found with clinical parameters Citation[11].
Aldehyde dehydrogenase-1 (ALDH1) is a cytosolic enzyme, primarily responsible for oxidizing a variety of intracellular aldehydes. ALDH1 is highly expressed in hematopoietic progenitors and in intestinal crypt cells. However, ALDH1 has now been successfully employed as a stem cell marker in head and neck squamous cell carcinoma, lung, prostate, pancreas and breast cancer Citation[12].
Many other markers have been described in human cancers, although their prognostic value remains to be defined. Likely, several combinations of these markers would allow a more appropriate characterization of CSCs in different tumor subtypes.
Immunohistochemical evaluation of CSCs markers in breast & lung tumors: our experience
A few studies have reported the use of TMA for CSCs detection in solid tumors. An IHC assay was performed for CD44 on TMA of gastric adenocarcinoma, showing an increased expression in intestinal metaplasia Citation[13]. A TMA was also used for CD44/CD24 detection in pancreatic cancer Citation[14]. The low expression of ALDH1 represents an important prognostic marker as reported by a study on pancreatic cancer TMA Citation[15]. ALDH1 expression was also detected on breast cancer TMA, where its prognostic role is correlated to patient's age. Furthermore, the same marker on triple negative/basal-like breast cancer TMA has shown an aberrant expression in the stromal component of the tumors and it has been associated with a better prognosis Citation[16]. Again, CD133 and CD44 markers were detected in pediatric solid tumors through TMA employment, highlighting their potential prognostic clinic value Citation[17]. Moreover, validation studies were performed in ovarian carcinoma Citation[18], and lung cancer Citation[19].
Our experience is particularly based on the evaluation of a series of CSCs markers, specifically CD133, ALDH1 and CD44, in tissues like breast and lung, where these markers are thoroughly described to have an important prognostic value.
Several studies have shown a good correlation between tumor TMA spots and whole sections, examining known markers expression such as Ki67, p53 and pRB, with the use of at least three cores of tumor tissue Citation[20].
However, we have recently had the opportunity to compare the expression of these markers on our breast and lung cancers case series. We have compared the expression of the three CSCs markers on TMA cores and on single/whole sections of the same samples. This has allowed us to re-evaluate, in most of the cases, the expression of the markers assigning values to the percentage of positive cells which were completely different from those values assigned to TMA cores. Moreover, some cases proved positive where the spots were originally negative for CSCs markers. In detail, we have analyzed CD133, CD44 and ALDH1 expression on a triple negative breast cancer TMA, including 159 patients and using two spots of the most representative areas from each single case. All tumors and controls were reviewed by two experienced pathologists (MDB/RF), detecting CD133+ cells in 44% (70/159) of patients; CD44+ cells in 36% (58/159) and ALDH1 in 47% (76/159) of BC patients. All negative cases for three markers expression were selected for whole sections setting and for reproducing the IHC analysis. For CD133 marker, 14% (13/89) of negative samples showed positive cells; for ALDH1 marker, 9% (8/83) of negative samples showed positive cells; while for CD44 marker, only 3% (3/101) of negative samples showed positive cells.
Based upon these data, CD133 appeared to be the most problematic marker because the percentage of positive cells described in tumors is generally always very low.
This has obviously led to a statistical re-evaluation of the association between IHC CSCs expression and all clinico-pathological and follow-up parameters, with the definition of new important correlations [Manuscript in Preparation].
Besides, in our experience, we had the opportunity of using the whole sections of the same samples to purify RNA in order to investigate the gene expression of CSCs markers. In this case too, RT PCR analysis has often shown a clear expression of the markers in samples with either high or low IHC expression (data not shown). It seems to be important to highlight our previous work, in which a good overlap between the data of gene and protein expression has been shown, in particular for CD133.
Conclusion
Our personal experience in TMAs employment for CSCs detection in some solid human tumors has allowed us to define the inadequacy of this technique for the detection of stem cells niches. This elucidation can supply useful information for future investigations to establish the real prognostic value of these markers. In order to identify stem clones, it is necessary to analyze the whole area of the tumor, as in many cases TMA cores cannot be adequately representative.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.
No writing assistance was utilized in the production of this manuscript.
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