Validation of the Multiplex Ligand-Dependent Probe Amplification (MLPA) Technique for the Determination of HER2 Gene Amplification in Breast Cancer
G Farshid, G Cheetham, R Davies, S Moore, B Rudzki. IMVS, South Australian Pathology, Adelaide, SA, Australia
Background: Randomized clinical trials have demonstrated significantly improved outcomes for women with HER2 positive breast cancer treated with traztuzumab in both the adjuvant and metastatic settings. Consequently, it is now mandatory to establish the HER2 status of all breast cancers at diagnosis. The range of testing platforms available includes immunohistochemistry to detect over-expression of the receptor and in situ hybridization (ISH) to establish gene amplification. Given the case volume, PCR based tests are attractive in that they offer the advantages of automation, high throughput and low cost. MLPA is a PCR based assay that quantifies gene copy number, allowing assessment of amplification of the HER2 gene relative to control genes located on chromosome 17 and on other chromosomes. This study aims to compare the results of HER2 gene amplification for breast cancer as assessed by MLPA with CISH and FISH as gold standards.
Design: Institutional ethics board approval was obtained. Using commercially available kits, 208 consecutive invasive breast cancers undergoing routine CISH testing at our laboratory were also tested independently using MLPA. The ACSO/CAP guidelines were applied for the reporting of ISH results. FISH at a central laboratory assessed cases with no signal or equivocal CISH results. Supplementary in-house FISH was carried out as required for the study. In accordance with prior studies, MLPA results were reported as amplified when the HER2 gene copy number per cell was 4.0 or above in any 2 of 3 HER2 probes included in the kit.
Results: At the conclusion of all ISH testing (CISH & FISH) 25 of 208 cases (12.0%) were regarded as amplified, 182 (87.5%) as non-amplified and one case (0.5%) as undetermined due to insufficient tissue. This case is excluded from further analysis. Of the 25 cases amplified by ISH, 23 (92.0%) were classified as amplified by MLPA, the remaining 2 ISH amplified cases were classified as negative by MLPA. Of the 182 cases categorized as not amplified by ISH, all were also classified as negative by MLPA. Using ISH as the gold standard, the sensitivity of MLPA is 92.0%, its specificity 100%, positive predictive value 100%, negative predictive value 98.9% and overall accuracy 99.0%.
Conclusions: The high levels of concordance between MLPA and ISH results merit further consideration of MLPA as a possible additional platform for HER2 testing.
Tuesday, March 10, 2009 9:30 AM
Poster Session III # 57, Tuesday Morning