Validation of the multiplex ligation-dependent probe amplification (MLPA) technique for the determination of HER2 gene amplification in breast cancer.

BACKGROUND Multiplex ligation-dependent probe amplification (MLPA) is a polymerase chain reaction-based assay that can assess HER2 gene copy number relative to control genes. DESIGN Institutional ethics board approval was obtained. Using commercially available kits, 208 consecutive invasive breast cancers undergoing routine in situ hybridization testing (chromogenic in situ hybridization and fluorescence in situ hybridization) were also tested independently by MLPA. The American Society of Clinical Oncology/College of American Pathologists guidelines were applied for the reporting of ISH results. In accordance with earlier studies, MLPA results were reported as amplified when the HER2 gene copy number per cell was ≥ 4.0. RESULTS At the conclusion of all ISH testing 25 of 208 cases (12.0%) were regarded as amplified, 182 (87.5%) as nonamplified, and 1 case (0.5%) as undetermined owing to insufficient tissue. This case is excluded from further analysis. Of the 182 cases categorized as not amplified by ISH, all were also negative by MLPA. Of the 25 cases amplified by ISH, 23 (92.0%) were amplified by MLPA, but the 2 remaining ISH-amplified cases were negative by MLPA. Using ISH as the reference test, MLPA has the following diagnostic indices: sensitivity 92.0%, specificity 100%, positive predictive value 100%, negative predictive value 98.9%, and overall accuracy 99.0%. CONCLUSIONS The high level of concordance between MLPA and ISH results, exceeding the American Society of Clinical Oncology requirements for validation of a new test, supports the use of MLPA for assessment of HER2-amplification. These results merit further consideration of MLPA as a possible alternative or additional platform for HER2 testing.