Progesterone Receptor Isoform Analysis by Quantitative Real-Time Polymerase Chain Reaction in Formalin-Fixed, Paraffin-Embedded Canine Mammary Dysplasias and Tumors

Abstract

Cloning and sequencing of the progesterone receptor gene in dogs have revealed 2 isoforms, A and B, transcribed from a single gene. Distribution of isoforms A and B in canine mammary lesions has hitherto been investigated only by Western blot analysis. This study analyzed progesterone receptor and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign tumors, and 59% of carcinomas. Carcinomas, and particularly simple epithelial-type carcinomas, displayed the lowest levels of expression. A high rate of agreement was recorded between RT-qPCR and immunohistochemical labeling. Isoforms A and B were successfully amplified, with correlation coefficients of 0.99 and amplification efficiencies close to 2, and were expressed in all lesion types analyzed. Predominance of A over B expression was observed in carcinomas and complex adenomas. Low-grade tumors exhibited higher progesterone receptor messenger RNA (mRNA) levels, but no difference was observed in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors of the canine mammary gland. These findings will facilitate future research into the role of progesterone receptor isoforms in the progression of canine mammary tumors.

Progesterone receptor isoform A may regulate the effects of neoadjuvant aglepristone in canine mammary carcinoma

“…It was then amplified and melting-curve analysis was carried out using the LightCycler® 480 Real-Time PCR System. One-step RT-qPCR was performed using the QuantiFast® SYBR® Green RT-PCR (Qiagen, Copenhagen, Denmark), following a previously-described protocol [8]. Canine-specific primers for PR gene were designed specifically to target the region of canine isoform B and the region common to both isoforms (total PR) using Primer3Plus and based on the reported canine PR sequence (NM_001003074) [4].…”

“…Canine-specific primers for PR gene were designed specifically to target the region of canine isoform B and the region common to both isoforms (total PR) using Primer3Plus and based on the reported canine PR sequence (NM_001003074) [4]. Primers flanking one intron were chosen wherever possible and their specificity was checked by performing a BLAST® search showing 100% homology to target genes [8]. Moreover, primers were designed to produce an amplicon smaller than 100 bp in order to ensure that the sequences were unique for the template (Table 1, Additional file 1).…”

“…For PRB, however, melting-curve analysis revealed one major sharp peak but also additional extra minor peaks at lower Tm consistent with the agarose gel [8]. Concordance between data of immunohistochemistry and gene expression data was calculated using Cohen’s Kappa statistics.…”

“…Accordingly, it has been suggested that the relative proportion of PR isoforms A and B might affect the prognosis and thus influence therapeutic decisions [5]. We have previously shown that 1) neoadjuvant treatment with aglepristone decreases cell proliferation in PR positive carcinomas [3], and 2) PRA and PRB mRNA expression can be analysed in formalin-fixed, paraffin-embedded canine mammary gland tissue samples by RT-qPCR [8]. This study sought to examine the link between the effects of aglepristone on the proliferation index and mRNA expression of PRA and PRB in canine mammary carcinomas.…”

Prognostic impact of neoadjuvant aglepristone treatment in clinicopathological parameters of progesterone receptor‐positive canine mammary carcinomas

“…MIB‐1 labelling (proliferation index, PI) was evaluated quantitatively in digital pictures of tissue sections taken at a ×40 magnification from 10 randomly selected neighbouring, non‐overlapping fields per sample. The number of positive and negative cells was counted with ImageJ software . PI was expressed as the percentage of positive cells related to the total number of cells.…”