Serum HER2 levels are increased in cats with mammary carcinomas and predict tissue HER2 status

Abstract

HER2 is overexpressed in about 30% of feline mammary carcinomas (FMC) and in 15-30% of breast cancers. Women with HER2-positive breast tumors are associated with shorter survival. This study aimed to optimize the detection and quantification of serum HER2 (sHER2) in cats and to evaluate its potential in diagnosing cats with mammary carcinomas (MC) overexpressing HER2. A prospective study was conducted in 60 queens showing MC and 20 healthy animals. Pre-operative serum samples were collected for sHER2 quantification using two immunoassays: ELISA and Dot blot assay. sHER2 levels were compared with tissue HER2 status assessed by immunohistochemistry. Queens with FMC showed significantly higher mean levels of sHER2 by both ELISA and Dot blot assay. A significant difference in the sHER2 levels was also found between cats with HER2-positive MC and those with low-expressing HER2 MC. A significant correlation between sHER2 levels and tumor HER2 status was also found, particularly when ELISA was used (r = 0.58, p < 0.0001). The value of 10 ng/ml was proposed as the optimal cutoff for both immunoassays by ROC analysis. Like in humans, sHER2 levels are increased in cats with MC HER2-positive, strongly suggesting that evaluation of sHER2 levels can be very useful in feline oncology. The results show that ELISA and Dot blot assay can replace the immunohistochemistry technique, due to their efficacy and lower costs for diagnostic purposes and for monitoring the response to anti-HER2 therapies in cats.

Keywords: ELISA; HER2; dot blot assay; feline mammary carcinomas; serum HER2 levels.

Figure 1. Standard curve for sHER2 measurements using a commercial ELISA kit

The intra and inter-assay coefficients of variation were < 10%.

Figure 2. Box plot diagrams representing the sHER2 levels in control cats (healthy group) and in cats with mammary carcinoma (cancer group) determined by ELISA (A) and Dot blot assay (B)

Cats with mammary carcinomas had significantly higher mean of sHER2 levels than healthy cats both by ELISA (p = 0.04) and Dot blot assay (p = 0.01). Outliers are indicated by open circles.

Figure 3. Box plot diagrams showing that tumor HER2 status correlates with sHER2 levels as assessed by both ELISA (A) and Dot blot assay (B)

A. A significant difference was found (p = 0.001) between the sHER2 levels of cats with MC overexpressing HER2 (IHC-positive group: mean 42.6 ng/ml [range of values 0-147.05 ng/ml]) and the sHER2 levels of cats with HER2-negative MC (IHC-negative group: mean 8.8 ng/ml [range of values 0-73.89 ng/ml]), using ELISA (p = 0.001). B. The Dot blot assay also showed a significant difference between the sHER2 levels measured in these two studied groups, with a p-value of 0.03 (IHC-positive group: mean 24.4 ng/ml [range of values 0-75 ng/ml]; IHC-negative group: mean 8 ng/ml [range of values 0-20 ng/ml]).

Figure 4. Soluble truncated HER2 forms carry a portion of the ICD and are quantifiable by Dot blot assay

A. The anti-HER2 SP3 antibody specifically recognizes a protein band of about 185 kDa in human and feline whole cell extracts (SKBR3 and FMCp, lanes 1 and 2), the purified rHER2-ECD (lane 3, ∼75 kDa), and a protein with a molecular weight that ranges from 100 to 140 kDa in cat serum samples (lanes 4 and 5). B. A representative image of an immunoblot performed with serum samples collected from healthy cats (line 2) and cats with mammary carcinoma (lines 3-6). sHER2 levels were semi-quantified by comparing the intensity of each serum dot with the intensities of the dots obtained from rHER2-ECD dilutions (line 1).

Figure 5. Receiver-operating characteristic (ROC) curve for sHER2 levels for ELISA (A) and Dot blot assay (B)

The optimal sHER2 cutoff value (10 ng/ml) was chosen to maximize the sum of the sensitivity and specificity on the Youden index (sensivity+specifity-1). The AUC was 0.70 (95% CI: 0.55-0.85) for ELISA A. and 0.73 (95% CI: 0.58-0.88) for Dot blot assay B., demonstrating that the sHER2 levels are a good biomarker to differentiate cats with MC overexpressing HER2 from cats with MC non-expressing HER2 or healthy cats. When the cutoff value of 10 ng/ml was set, the sensibility was 69% and the specificity was 67% for ELISA, whilst for Dot blot assay the sensibility and the specificity were 53% and 78%, respectively.