Abstract PD02-03: Added value of HER-2 amplification testing by multiplex ligation-dependent probe amplification (MLPA)

Introduction Accurate determination of HER-2 status is critical for selection of breast cancer (BC) patients for trastuzumab treatment. According to the most commonly used testing protocol, patients with 3+ immunohistochemistry (IHC) are considered for trastuzumab. In addition, 2+ IHC cases are tested for gene amplification and, when amplified, these patients are considered for trastuzumab treatment as well. Because in our experience IHC results have been proven to be sensitive to pre-analytical variation and inter- and intra-observer variation, we apply amplification testing not solely to 2+, but also to 1+ and 3+ cases. At Symbiant Pathology Expert Centre, a validation study (n = 99) comparing MLPA with chromogenic in situ hybridization (CISH) showed 100% concordance. Starting from April 2011, MLPA has been used as the method for HER-2 amplification testing, because in our hands this technique is more time- and cost-effective than CISH for analyzing large sample numbers. CISH was performed when MLPA was inconclusive, IHC score was heterogeneous or when invasive carcinoma could not be physically separated from DCIS. Here we report the results of a comparison between HER-2 amplification tests and IHC in a cohort of 928 BC patients. Methods Pathology reports of 928 BC patients from 4 pathology laboratories were retrospectively reviewed. 728 patients were diagnosed between April 2011 and February 2012 in one of three laboratories of Symbiant Pathology Expert Centre. The other 200 patients were diagnosed in University Medical Centre Utrecht between January 2010 and December 2011. 18 patients had multiple tumors that were included as separate cases. In total, 949 tumors were reviewed. Results With IHC, major differences are observed between the 4 pathology laboratories concerning the percentage of cases scored as 0–1+ and 2+ (Table 1). A considerable number of 3+ cases (6.4%) failed to show gene amplification by MLPA and/or CISH. A smaller percentage of 0–1+ cases (1.8%) did show gene amplification (Table 2). 90 equivocal cases were double tested by CISH and MLPA. In 52/90 of these cases (58%) at least one test was inconclusive. The other 38 (42%) cases showed 100% concordance between MLPA and CISH. Conclusion MLPA is an accurate and cost-effective method for the determination of HER-2 status. We recommend the use of a gene amplification test to confirm HER-2 status in all IHC scores, not just the 2+ cases. However, it is still unclear whether the small subgroups of patients showing only HER-2 protein overexpression or only HER-2 gene amplification would benefit from trastuzumab. Follow-up data are therefore needed to determine the clinical value of HER-2 status assessed by each technique in order to improve the current standard of care for HER-2 testing. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD02-03.