Added Value of HER-2 Amplification Testing by Multiplex Ligation-Dependent Probe Amplification in Invasive Breast Cancer

Abstract

BackgroundHER-2 is a prognostic and predictive marker, but as yet no technique is perfectly able to identify patients likely to benefit from HER-2 targeted therapies. We aimed to prospectively assess the added value of first-line co-testing by IHC, and multiplex ligation-dependent probe amplification (MLPA) and chromogenic in situ hybridization (CISH).MethodsAs local validation, HER-2 MLPA and CISH were compared in 99 breast cancers. Next, we reviewed 937 invasive breast cancers, from 4 Dutch pathology laboratories, that were prospectively assessed for HER-2 by IHC and MLPA (and CISH in selected cases).ResultsThe validation study demonstrated 100% concordance between CISH and MLPA, if both methods were assessable and conclusive (81.8% of cases). Significant variation regarding percentages IHC 0/1+ and 2+ cases was observed between the laboratories (p<0.0001). Overall concordance between IHC and MLPA/CISH was 98.1% (575/586) (Kappa = 0.94). Of the IHC 3+ cases, 6.7% failed to reveal gene amplification, whereas 0.8% of the IHC 0/1+ cases demonstrated gene amplification. Results remained discordant after retrospective review in 3/11 discordant cases. In the remaining 8 cases the original IHC score was incorrect or adapted after repeated IHC staining.ConclusionsMLPA is a low-cost and quantitative high-throughput technique with near perfect concordance with CISH. The use of MLPA in routinely co-testing all breast cancers may reduce HER-2 testing variation between laboratories, may serve as quality control for IHC, will reveal IHC 0/1+ patients with gene amplification, likely responsive to trastuzumab, and identify IHC 3+ cases without gene amplification that may respond less well.

Breast carcinomas with low amplified/equivocal HER2 by Ish: potential supporting role of multiplex ligation-dependent probe amplification

“…It is of paramount clinical importance to carefully define the HER2 status in these uncommon subsets of BC in which the mean CEP17 copy number drives in the final classification based on the ISH algorithm proposed by the guidelines. To this end, we evaluated whether MLPA, a quantitative molecular method [ 26 , 28 , 29 , 40 ] may be a helpful reflex test to define the HER2 status in 54 BC presenting a non classical HER2 ISH results (4.0–5.9 HER2 gene s/n). One hundred and sixteen BC including 55 HER2 clearly amplified (>6.0 gene s/n) and 61 clearly non amplified (less than 4.0 gene s/n, ratio < 2.0) BC represented our control series.…”

“…Several recent studies which compared MLPA and ISH techniques, were broadly concordant with our results. The data reported so far have demonstrated that there are specific scenarios in which MLPA may be of particular value in HER2 testing when selecting patients to undergo anti HER2 treatment [ 26 – 28 , 40 ]. In this context, BC patients displaying a mean of the HER2 gene s/n ranging between 4.0–5.9, including both low amplified and equivocal tumors, could benefit most from MLPA.…”

“…Nevertheless, the optimum HER2/CEP17 ratio cut-off for tumor response in BC patients treated with trastuzumab both in the (neo)-adjuvant and metastatic setting is still widely debated [ 15 – 19 ]. In view of this, novel emerging techniques are described and aimed to more accurately and more objectively estimate the HER2 status [ 20 – 26 ]. Although the majority of these novel techniques demonstrate a good overall correlation with each other in comparative studies, each assay has its own advantages and disadvantages and, up to now, there is neither real gold standard nor validated test to better define the equivocal subset.…”

“…In this context, Multiplex Ligation-dependent Probe Amplification (MLPA), a high-throughput PCR-based technique, seems to provide an alternative valuable and cheap tool to more objectively define HER2 status. Aimed to validate its use in clinical practice, several authors [ 26 – 29 ] compared MLPA with conventional techniques [ 30 , 31 ], obtaining optimal results mainly in clearly amplified and non amplified breast cancer. Conversely, limited MLPA data are available in the grey zone represented by BC with 4.0–5.9 HER2 gene s/n.…”

Quantification of HER family receptors in breast cancer

“…Third, they can be multiplexed, allowing simultaneous interrogation of multiple genes or different parts of genes, representing an ideal and low cost prescreening tool. Head-to-head comparisons between IHC, FISH, and CISH have shown good correlation among technologies [ 106 - 109 ]. The main weaknesses of PCR-based assays are that they do not preserve tissue morphology, may require sample macro- or microdissection to enrich for tumor content, heterogeneity can be missed and contamination with normal or ductal carcinoma in situ may lead to both false-negative and false-positive results.…”