HER-2 MLPA in patients with aberrant FISH patterns Lesley M. McMahon, Colin Purdie, Alastair Thompson, Norman R. Pratt Today I would like to present my A-grade project, entitled exploration of HER-2 gene status by MLPA in patients with aberrant FISH patterns. ACC Spring conference Liverpool 2008

Introduction HER-2 and breast cancer HER-2 amplification in ~25-30% of human breast cancers. Aggressive disease/poorer prognosis. The oncogene HER-2 encodes a RPTK receptor protein tyrosine kinase, belonging to the human epidermal growth factor receptor (HER) family of proteins. HER-2 proteins are expressed in various tissues and are involved in cell proliferation, differentiation and survival pathways. HER-2 gene amplification has been demonstrated in approximately 30% of human breast cancers. …and is associated with more aggressive tumour behaviour and a poorer prognosis. However, patients with HER-2 positive breast cancer have responded well to treatment with the humanised monoclonal Ab, Herceptin®. Therefore, accurate determination of the HER-2 status of breast tumours is a crucial step in management and treatment of patients with breast cancer. HER-2 +ve patients respond well to Herceptin. Tumour HER-2 status crucial for patient management.

Introduction HER-2 testing strategy followed by UK HER-2 reference laboratories Standardised, validated IHC assay IHC score 0 and 1+ IHC score 2+ IHC score 3+ = IHC Negative = IHC Borderline = IHC Positive HERCEPTIN = NO HERCEPTIN = YES HER-2 testing of all breast cancers in the UK is undertaken following the guidance and recommendations provided by three reference laboratories (Ellis, 2000; Ellis, 2004). Adopting a two-tier approach, HER-2 protein expression levels are first assessed by immunohistochemistry (IHC). Those with IHC 0 or 1+ scores are regarded as unequivocally negative whereas those with IHC 3+ are unequivocally positive and elegible for Herceptin treatment. HER-2 FISH testing is recommended when results from IHC tests prove equivocal ie. Those samples falling into the IHC2+ categories. FISH ratios < 2.0 are considered not amplified and not elegible for Herceptin whereas those > 2.0 …. For FISH assay FISH ratio <2.0 Not amplified Negative FISH ratio >2.0 Amplified Positive

Standard HER-2 FISH patterns HER-2 not amplified HER-2 amplified  Spectrum Green = CEP 17 Spectrum Orange = HER-2

“Variant” HER-2 FISH patterns unclassifiable co-amp HER-2 and CEP17 “polysomy” CEP17 single CEP17

Aims and objectives To evaluate HER-2 gene amplification by MLPA To compare tumours with borderline, low level and high level HER-2 Overall, I had three aims and objectives that included.. …evaluating MLPA as an alternative tool for the detection of HER-2 gene amplification and copy number change in breast cancer tumours …comparing the differences between tumours with borderline, low and high level HER-2 gene amplification previously determined by FISH and.. …and investigating the use of MLPA in cases with variant FISH patterns To investigate MLPA in cases with variant FISH patterns

Materials and methods (1) Patient cohort 60 samples Paraffin-embedded tissue sections (PETs) HER-2/CEP17 ratios previously determined by FISH Retrospectively grouped into 6 FISH categories In total I had 60 samples from 59 patients of which 18 were core biopsies and 42 were tissue sections. These samples were selected at random from the pathology archives. All samples had undergone diagnostic HER-2 FISH testing and so all had known HER-2/CEP17 ratios previously determined by FISH. For purposes of this project, these samples were grouped into 6 FISH amplification categories based on their HER-2/CEP17 ratios.

Materials and methods (2) FISH amplification status HER-2/CEP 17 ratio (i) not amplified < = 1.8 (ii) borderline > 1.8 < = 2.2 (iii) low > 2.2 < = 5.9 (iv) moderate >5.9 < =20 (v) high > 20 (vi) variant ratio undetermined due to variant FISH patterns 42 samples were tested that had normal FISH patterns and ..

Materials and methods (3) DNA extraction Sigma GenElute miniprep kit MLPA SALSA MLPA Kit P004 HER-2/neu (MRC Holland) MLPA data analysis MLPA data analysis software (coffalyser) DNA extraction was first optimised, before using the Sigma Gen Elute miniprep kit for extraction of all samples … MLPA was carried out according to the manufacturers instructions using the SALSA MLPA Kit P004 HER-2/neu (MRC Holland).   This kit contains thirty-nine probe pairs, of which three recognise different sequences of the human ERBB2/HER-2 gene. In addition eleven probes on chromosome 17 are included with two probes for TP53, TOP2A and BRCA1 (Table 2). The remaining twenty-five probes recognise single copy sequences on other chromosomes throughout the genome. All patient samples were assayed blind and for each MLPA run, one blank sample (1x TE buffer), DNA from five control samples (reduction mammoplast patients), one negative (MCF-7) and one positive (BT-474) sample were included. For each MLPA reaction, 150-250ng control patient DNA, 115ng MCF-7 or BT-474 DNA and 50-1000ng of patient DNA was used per reaction.

Results Standard FISH patterns Variant FISH patterns not amplified FISH amplification status HER-2 FISH Positive (%) HER-2 MLPA not amplified n = 9 borderline n = 2 100 low level n = 7 14 moderate n = 11 high variant n = 0 - Variant FISH patterns FISH amplification status HER-2 FISH Positive (%) HER-2 MLPA not amplified n = 2 50 borderline n = 0 - low level n = 4 100 moderate n = 5 high n = 3 variant n = 1 Overall… These results also show that low level amplified samples with variant FISH patterns are more likely to be amplified by MLPA than those with normal FISH patterns in this category. Therefore in cases where the HER-2/CEP17 ratio is obscured or difficult to score due to these variant patterns, MLPA may prove to be an alternative tool in the interpretation of HER-2 copy number changes in these tumours.

Conclusions MLPA unsuitable for borderline or low-level amplified samples with standard FISH patterns. BUT…….MLPA can identify HER-2 gene amplification in low level cases with “variant” FISH patterns. AND…..MLPA can detect HER-2 gene amplification in cases with undefined HER-2/CEP17 ratios due to variant FISH patterns. So to conclude… MLPA may provide an adjunct to existing FISH service in problematic cases.

Summary and Further work Clinical consequence of low level amplification Benefit of Herceptin – awaiting clinical audit. MLPA cheaper alternative At present the jury is out on the clinical significance and consequence of low level HER-2 FISH amplification and response to Herceptin therapy. In the future it may only be those with high level FISH amplification that would benefit from this treatment and in this case MLPA would certainly be a cheaper alternative to FISH for the detection of gene amplification. However, FISH remains the gold standard at present in the determination of HER-2 gene amplification and as we have seen here, is necessary for the detection of HER-2 gene amplification in borderline and low level amplified cases. FISH remains gold standard

Acknowledgements Ninewells Hospital Tumour Bank Sheila Sharp, University of Dundee John Hands, Molecular Genetics department Dr. Colin Purdie, Consultant Clinical Pathologist Miss Chris Maliszewska, Deputy Head of clinical cytogenetics Dr. Norman Pratt, Head of clinical cytogenetics NHS Tayside Human Genetics unit Finally, I would like to thank the following people for their help and support and also you for listening…especially as its 5.30pm….