Analytical validation of CanAssist-Breast: an immunohistochemistry based prognostic test for hormone receptor positive breast cancer patients.

BACKGROUND: CanAssist-Breast is an immunohistochemistry based test that predicts risk of distant recurrence in early-stage hormone receptor positive breast cancer patients within first five years of diagnosis. Immunohistochemistry gradings for 5 biomarkers (CD44, ABCC4, ABCC11, N-Cadherin and pan-Cadherins) and 3 clinical parameters (tumor size, tumor grade and node status) of 298 patient cohort were used to develop a machine learning based statistical algorithm. The algorithm generates a risk score based on which patients are stratified into two groups, low- or high-risk for recurrence. The aim of the current study is to demonstrate the analytical performance with respect to repeatability and reproducibility of CanAssist-Breast. METHODS: All potential sources of variation in CanAssist-Breast testing involving operator, run and observer that could affect the immunohistochemistry performance were tested using appropriate statistical analysis methods for each of the CanAssist-Breast biomarkers using a total 309 samples. The cumulative effect of these variations in the immunohistochemistry gradings on the generation of CanAssist-Breast risk score and risk category were also evaluated. Intra-class Correlation Coefficient, Bland Altman plots and pair-wise agreement were performed to establish concordance on IHC gradings, risk score and risk categorization respectively. RESULTS: CanAssist-Breast test exhibited high levels of concordance on immunohistochemistry gradings for all biomarkers with Intra-class Correlation Coefficient of ≥0.75 across all reproducibility and repeatability experiments. Bland-Altman plots demonstrated that agreement on risk scores between the comparators was within acceptable limits. We also observed > 90% agreement on risk categorization (low- or high-risk) across all variables tested. CONCLUSIONS: The extensive analytical validation data for the CanAssist-Breast test, evaluating immunohistochemistry performance, risk score generation and risk categorization showed excellent agreement across variables, demonstrating that the test is robust.

Validation of CanAssist Breast immunohistochemistry biomarkers on an automated platform and its applicability in tissue microarray.

CanAssist Breast (CAB) is a prognostic test for early-stage hormone receptor-positive invasive breast cancer. The test involves performing immunohistochemical (IHC) analysis for five biomarkers, namely CD44, ABCC4, ABCC11, N-cadherin, and pan-cadherin. In addition to IHC grading information, three clinical features, i.e., tumor size, grade, and lymph node status, serve as input into the machine learning-based algorithm to generate the CAB risk score. CAB was developed and initially validated using manual IHC. This study's objectives included: i) automate CAB IHC on an autostainer and establish its performance equivalence with manual IHC ii) validate CAB test using samples in Tissue MicroArray (TMA) format. IHC for CAB biomarkers was standardized on Ventana BenchMark XT autostainer. Two IHC methods were compared for IHC gradings and corresponding CAB risk scores/risk categories. A concordance analysis was done using MedCalcTM software. The manual and automated IHC staining methods exhibited a high level of concordance on IHC gradings for 40 cases with an Intra-class Correlation Coefficient (ICC) of >0.85 for 4 of 5 biomarkers. 100% concordance was achieved in risk categorization (low- or high-risk), with very good agreement between the risk scores demonstrated by a kappa statistic of 0.83. TMA versus whole tissue section concordance was analyzed using 45 samples on an autostainer, and the data showed 92% concordance in terms of risk category. The results confirm the equivalence between manual and automated staining methods and demonstrate the utility of TMA as an acceptable format for CanAssist Breast testing.

Comparison of breast cancer prognostic tests CanAssist Breast and Oncotype DX.

BACKGROUND: CanAssist Breast (CAB) is a prognostic test for early stage hormone receptor-positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) breast cancer patients, validated on Indian and Caucasian patients. The 21-gene signature Oncotype DX (ODX) is the most widely used commercially available breast cancer prognostic test. In the current study, risk stratification of CAB is compared with that done with ODX along with the respective outcomes of these patients. METHODS: A cohort of 109 early stage breast cancer patients who had previously taken the ODX test were retested with CAB, and the results respectively compared with old cut-offs of ODX as well as cut-offs suggested by TAILORx, a prospective randomized trial of ODX. Distant metastasis-free survival after 5 years was taken as the end point. RESULTS: CanAssist Breast stratified 83.5% of the cohort into low-risk and 16.5% into high-risk. With the TAILORx cut-offs, ODX stratified the cohort into 89.9% low-risk and 10.1% into high-risk. The low, intermediate, and high-risk groups with ODX old cut-offs were 62.4%, 31.2%, and 6.4%, respectively. The overall concordance of CAB with ODX using both cut-offs is 75%-76%, with ~82%-83% concordance in the low-risk category of these tests. The NPV of the low-risk category of CAB was 93.4%, and of ODX with TAILORx cut-offs was 91.8% and 89.7% with old cut-offs. CONCLUSIONS: Compared to the concordance reported for other tests, CAB shows high concordance with ODX, and in addition shows comparable performance in the patient outcomes in this cohort. CAB is thus an excellent and cost-effective alternative to ODX.