Sara Santos; Cláudia S. Baptista; Rui M. V. Abreu; Estela Bastos; Irina Amorim; Ivo G. Gut; Fátima Gärtner; Raquel Chaves

“ In recent years, a systematic investigation of the potential role of inherited germline variants of ERBB2 in breast cancer risk has been conducted using single nucleotide variants and haplotype-based analyses [21] – [22] . The most notable overall observation in this investigation is the lack of evidence to support a significant association between ERBB2 genomic sequence variants (SVs) and HBC initiation, despite the wealth of information supporting its role in breast cancer progression [12] , [23] , [22] . ”

“ This proximity indicates a potential effect on the affinity of the cat erbB-2 variant protein for trastuzumab.
In conclusion, our results concerning sequence variation detection in cat mammary non-neoplastic and neoplastic lesions, are an important contribution to genomic DNA characterisation in cancer, corroborating the idea fomented by Tao and collaborators (2009) for human cancers that large epidemiological studies of predisposed gene polymorphisms can provide new insights into the in vivo relationships between genes of interest and cancer risk [38] . ”

“ With respect to our IHC erbB-2 analysis, the results for the normal samples are in accordance with the work of Burrai and collaborators (2010) , which also found erbB-2 protein expression in normal cat mammary epithelium with strong, complete membrane staining [4] . These findings are contrary to those of other studies that reported no immunoreactivity or a faint, barely perceptible signal in part of the cell membrane in normal cat mammary ducts and acini [31] – [32] , [50] , and they are also contrary to what has been mainly observed in humans [51] . ”

“ We hypothesise that amino acid variant interactions could have incremental effects on the cat erbB-2 protein structure, function, and therapeutics, as it is known that the CR2 domain plays an important role in human breast cancer treatment. ”

“ Here, we demonstrate the prognostic value of the ERBB2 gene in cat mammary tumours, identifying this gene into a potential therapeutic target, similar to the results demonstrated for HBC. Additionally, the recurrent occurrence of low erbB-2 expression levels in cat mammary tumours suggests that cat mammary neoplasias could be a valuable model for erbB-2 negative human breast cancer. ”

“ The results of our ERBB2 gene expression analysis suggest the presence of ERBB2 gene post-transcriptional regulation and the occurrence of proteins with truncations and single point mutations in cat mammary neoplastic lesions. We should emphasise that our IHC and RT-qPCR results assigned the analysed cat mammary primary malignant lesions to a subtype of cat mammary carcinomas that are characterised by ERBB2 RNA and erbB-2 protein underexpression. ”

“ A basic single allelic model of association demonstrated that five variant alleles, which were only detected in CMLs, showed significant values of association with mammary lesion samples group (JQ284376: g.355G>A, g.1914G>C, g.2037G>C, g.2041A>C, and g.2065T>C) . The determination of the haplotype frequency for cat ERBB2 using the linkage disequilibrium test uncovered a divergent genotypic haplotype characterisation (nine different frequent haplotypes) between the normal samples and mammary lesion samples (table 1) . ”

“ The pathogenesis and progression of invasive breast cancer have been related to a large variety of growth factors, and the proteins of the epithelial growth factor receptor family have been the most investigated in this heterogeneous disease [8] , [9] . ”

“ Thus, it is clear that technical factors can affect the immunohistochemical assessment of erbB-2 expression in cat mammary gland samples [50] . Concordantly, several authors reported that despite the fact that IHC is currently used for human breast cancer erbB-2 protein categorisation, the application of different, common, commercially available erbB-2 antibodies produces variability in the IHC results [52] – [53] . ”

“ We constructed a variant genomic sequence (ERBB2_10-15_SV) in silico that comprised all of the detected variant alleles (Vector NTI 10.3.0 software; Invitrogen/Life Technologies) . Coding region sequences (CDSs) and protein sequences were determined based on the wild-type and variant DNA sequences. This intricate analysis involved the validation of the gDNA, DNA coding region and protein sequences obtained from the genome and protein databases (Ensembl, UCSC Genome Browser, UniProtKB/TrEMBL and NCBI) by alignment. ”

“ From the prepared wt cat erbB-2 model, different homology models of the 5 detected variants were established by incorporating the respective mutation. Using the Protein Preparation Wizard protocol available in the Maestro software program [74] , the extracellular domains of the homology models of cat ERBB2 wt and the 5 variants were prepared, with hydrogens added and water molecules removed. The wt and variant 3D models were aligned using Pymol software, and a preliminary comparative analysis was performed. ”

“ Of the 4 normal mammary tissues studied, 3 were obtained from 2 cats with hyperplasia in a different mammary gland (Table S1: BeL5 and BeL6) . Additionally, 8 residual normal mammary glands peripheral to affected mammary tissues (Table S1: MaLs6, 10, 14, 17, 18, 20, 21 and MeL4) were used in the present study. Moderate (erbB-2 ++) or strong (erbB-2 +++) IHC membrane labelling was observed in 66.7% of normal samples for the CBE356/ext antibody and in 91.7% of normal samples for the CB11/int antibody (Figure 3a, b and Figure 4) . ”

“ Immunohistochemistry (IHC) erbB-2 protein quantification
For each anti-human erbB-2 antibody used, one specific for the internal domain (CB11/int) and one specific for the external domain (CBE356/ext) , the immunoreactivity was assessed according to the Hercep-Test guidelines as described in ASCO recommendations [34] . The two anti-human erbB-2 antibodies used were validated for cat species by western blotting onto cat mammary protein extracts from frozen samples (in RNAlatter solution) (Figure S4) . Both antibodies recognized the expected erbB-2 oncoprotein of 185 kDa. ”

“ Different approaches were employed, such as comparative studies of the cat ERBB2 DNA sequence with its human counterpart, sequence variant characterisation, DNA and mRNA status evaluation by qRT-PCR, protein level quantification by immunohistochemistry (with two different antibodies) , in silico coding sequence and respective translated protein construction, and determination of the consequences of the identified exonic non-synonymous sequence variants (nsSVs) in the cat erbB-2 3D structure. ”

“ ERBB2_10-15 sequence variants detection in normal and mammary lesion samples
To perform gDNA sequence variant detection in normal and CML samples, gDNA fragments were amplified with two sets of primers: E10/E14 (from exon 10 to 14) and E12/E15 (from exon 12 to 15) . The positive PCR reaction products were purified, and sequencing was performed in both directions. The sequences were edited directly with the chromatograms in the ContigExpress Vector NTI module (Invitrogen/Life Technologies) , and a final consensus sequence was established for each sample. ”

“ Interestingly, in this work, we found lower levels of CBE356/ext labelling in all tissue sample types, including the normal samples, suggesting the presence of this erbB-2 isoform. Moreover, another mechanism of ERBB2 gene expression regulation could be involved, such as specific promoter regulation [59] , defective RNAs, and mutations in ERBB2 [60] . Camp and collaborators (2003) speculated that tumours might overexpress another growth factor receptor that promotes tumour aggression via a ligand-dependent or -independent mechanism [29] , [60] , [61] . ”

“ In order to amplify and sequence the ERBB2_10-15 gene fragment, the primer set E10/E15 was applied using gDNA obtained from two blood samples from two cats with no history of mammary disease. The PCR fragments were excised from the agarose gel, purified (Geneclean II kit; BioGene) , and cloned into the pCR 2.1-TOPO® vector (TOPO TA cloning® kit; Invitrogen/Life Technologies) . The plasmid DNA was extracted (Quickgene DNA plasmid kit; Fujifilm/Life Sciences) , and four positive clones were selected for sequencing. ”

“ Primers were designed and probes were selected against conserved cat and human cDNA sequences corresponding to the ERBB2 sequence that encodes the internal region of the cat erbB-2 protein. Primers (F, 5′-CTTTTGGGGCCAAACCTTAT-3′ and R, 5′-CTAGTGGGACGCGGACAT-3′) were designed using Roche LightCycler Probe Design Software (version 2.0) . The concentration of the working solution was 20 μM for all oligomers. The hybridisation probe was selected from the Human Universal Probe Library from Roche Applied Science (UPL58) . The GAPDH gene was used as a housekeeping gene. ”

“ The concentration of the plasmid with the insert was measured using the NanoDrop ND-1000 (NanoDrop Technologies) equipment and the corresponding plasmid copy number was calculated
using the following equation: DNA (copy number) = [6.023×1023 (copy number/mol) × DNA amount (g) ] / [DNA length (bp) ×660 (g/mol/bp) ] , where Avogadro number is 6.023×1023 (copy number) /1mol and the average molecular weight of a double-stranded DNA molecule is 660 g/mol/bp. In the respective formula the recombinant plasmid DNA length is 4650 bp (pCR 2.1-TOPO vector 3900 bp and the insert 750 bp) . ”

“ Also, when we compared the CBE356/ext with CB11/int results, the staining intensities concordance revealed was 50%.
For both antibodies, the CMLs showed a lower percentage of erbB-2 positive tissues than the normal samples. The percentage of positive CMLs was lower for CBE356/ext than for CB11/int labelling (Figure 4 and Table S5) . When the IHC results were compared, the scores concordance was higher for the erbB-2 negative (48.48%) than for the positive (27.27%) CMLs samples. ”

“ Multi-alignment between wild-type (Cat erbB-2 10–15 protein wt) and variant cat erbB-2 protein sequence and with the corresponding Human erbB-2 partial protein sequence. The Human sequence correspond to part of the chain A from the 3BE1 model. The amino-acid changes detected are indicated in red. Adobe (.PDF) ; paper size 21×15 cm. (DOC) (data file pone.0083673.s003.doc)
Figure S4 Western blot with the CBE356 and CB11 anti-erbB2 antibodies. Reactivity observed by western immunoblot with the CBE356 and CB11 anti-erbB2 antibodies, in 3 feline mammary tissues. ”

“ The output includes a probability score that classifies the mutation as possibly damaging (score <0.15) , probably damaging (score > 0.85) or benign (score 0.15–0.85) .
In order to complete the homology modelling and molecular dynamics studies, a cat erbB-2 wild-type homology model was built using the SWISS-MODEL online homology modelling server [72] . The complete human erbB-2 structure was kindly supplied by Dr. ”

“ Table 2 "Resume of the significant two-tailed bivariate correlation results between ERBB2 RNA, erbB-2 protein expression levels, and clinicopathological features of mammary lesions. (10.1371/journal.pone.0083673.t002) "File:Pone.0083673.t002The significant values of the Pearson's correlation analysis were confirmed by the Spearman's test. (#) Significant correlation only by Spearman's test. (*) Correlation is significant at the 0.05 level. (**) Correlation is significant at the 0.01 level. (p: positive; n: negative) Indication of the direction of the relationship between variables. ”

“ We are grateful to: DVM. Liliana Martins, DVM. Joana Santos, DVM Jorge Ribeiro, Technician Carlos Frias of the UPVet Small Animal Clinic (ICBAS-UP) , for the help in follow-up clinical evaluation and assistance ”

“ Amsbaugh, SC & King, CR, “Overexpression of the EGF receptor-related proto-oncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms”, EMBO J 6: 605–610, [ [pmid:3034598 KrausMHPopescuNCAmsbaughSCKingCR] ] ↑ Tao, W; Wang, C; Han, R & Jiang, H, “HER2 codon 655 polymorphism and breast cancer risk: a meta-analysis”, Breast Cancer Res Treat 114: 371–376, [ [pmid:18438707 TaoWWangCHanRJiangH] ] ↑ Cuadros, M ”