1. 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

2. 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.

3. 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

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

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

6. 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

7. 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.

8. 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 ..

9. 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, ng control patient DNA, 115ng MCF-7 or BT-474 DNA and ng of patient DNA was used per reaction.

10. 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.

11. 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.

12. 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

13. 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….