Comparison of fresh frozen vs. formalin-fixed, paraffin-embedded specimens and the expression profiling of 16 target genes in neoplastic and non-neoplastic canine mammary tissues using a multiplex branched-DNA assay (2016)

Mammary gland tumors are one of the most
 common neoplasms in female dogs and certain breeds are prone to develop the
 disease. The use of markers in human BC has been widely studied and some
 genes are routinely used as predictive, diagnostic and prognostic factors as
 well as for treatment reasons. The use of biomarkers
 in CMT is, however, still restricted to research purposes only. Therefore,
 gene profiles in different canine mammary entities were analyzed in the
 present study in order to evaluate the strength of potential markers serving
 as future prognostic and predictive factors.
 For this study, a branched-DNA assay was
 utilized to analyze the gene expression in FF and FFPE samples of canine
 mammary tissues. The b-DNA assay permits to analyze specimens with small
 amounts of RNA (and therefore suitable to analyze valuable FFPE specimens) as
 well as several target genes within a single sample. The reasons for the
 study were: (i) to analyze if valuable FFPE specimens, which represent a
 unique source of archived biological material, could be measured with the
 branched DNA assay; (ii) to discuss the advantages offered by the referred
 method when analyzing gene expression in comparison to techniques such as IHC
 and qPCR. Therefore, it was of interest to determine whether the gene expression between FFPE and FF specimens is
 comparable when applying the referred technique and whether the different
 storage times have any influence on the outcomes. Moreover, the opportunity
 to evaluate the performance of three different housekeepers in mammary canine
 tissues was taken.
 Overall, the technique revealed to be
 feasible to analyze FF and FFPE samples of canine mammary tissues. The
 relative gene expression of the two different origins of samples showed an
 agreement of 63% when normalizing the data to the average of the three HKG.
 Still, care should be taken as FFPE specimens showed, in general, lower
 expression of the analyzed targets when compared to FF samples. It was hypothesized that this finding is caused by the effect of storage
 time. The subsequent comparison among three different storage times of FFPE
 revealed a clearly higher expression of target genes in the short storage
 time group with a decrease in the expression in the other groups over time. Regarding the different HKG, data
 normalized against ACTB was found to present the lowest significant
 difference between the FF and FFPE samples when comparing the histological
 groups followed by HPRT1 and GAPDH.
 Bearing in mind that the technique showed
 to be feasible for the analysis of FF and FFPE samples in canine mammary
 tissues, it enabled us to analyze the gene expressions of different target
 genes in neoplastic (benign and malignant) and non-neoplastic tissues with the
 intention of finding new potential tumor markers. 16 onco- and
 suppressor-genes (BRCA1; BRCA2; FOXO3; GATA4; HER2; HMGA1; HMGA2; HMGB1;
 MAPK1; MAPK3; MCL1; MYC; PFDN5; PIK3CA; PTEN and TP53) regarded as
 being involved in neoplasm development were analyzed
 in the present study using a multiplex b-DNA assay.
 As hormone receptors also play an important role in human BC, the opportunity
 was taken to concomitantly analyze the gene expressions of ER, PR,
 PRLR and GHR herein. The results and discussion about them are,
 however, described in another work.
 Considering that an agreement of 63% was
 revealed between FF and FFPE, the samples were analyzed separately.
 The significant reduction in the
 expression of the tumor suppressors TP53, FOXO3, PTEN and PFDN5
 in malignant tumors found in the present study confirms previous reports,
 underlining their role in cellular growth. This is the first study which
 analyzed the gene expression of FOXO3 in CMT.  Hence, these genes
 could represent potential markers for CMT. On the contrary to what is
 normally reported in humans, the genes HMGA1, HMGB1, MCL1,
 MAPK3 and PIK3CA, which are genes known to stimulate cellular
 growth, revealed to be consistently lower expressed in malignant tumor.
 Therefore, a hypothesis is raised whether those genes play a different role
 in canine mammary tumor.
 The multiplex b-DNA technology is certainly
 a method which enables the analysis of several samples which present small
 amounts of RNA of FF and FFPE tissue. Still, care should be taken considering
 that FFPE specimens generally present a lower expression of the analyzed
 target genes when compared to FF samples, fact which is related to the
 storage time. In this work, a considerably larger number of samples was
 analyzed when compared to other studies performed in this field so far,
 permitting, consequently, a more reliable outcome. Based on the outcomes of the present
 work, the suppressor genes TP53, FOXO3, PTEN and PFDN5
 are considered as potential markers for the behavior of canine mammary
 tumors. The comparison of the
 expression patterns of the candidate genes in neoplastic (benign and
 malignant) and non-neoplastic tissues herein described might serve to gain a
 better understanding of the tumor pathogenesis of different canine mammary
 tumor subtypes in further studies. Moreover, potential target genes might
 assist to determine whether certain breeds are prone to develop CMT. The
 identification of suitable target genes offers the prerequisite to search for
 novel therapeutic approaches in the future, just like in the human
 counterpart.