Title: 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
Abstract: 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.
Publication Year: 2016
Publication Date: 2016-01-01
Language: en
Type: dissertation
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