HER-2 MLPA in patients with
aberrant FISH patterns
Lesley M. McMahon, Colin Purdie,
Alastair Thompson, Norman R. Pratt
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.
 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 Negative
HERCEPTIN = NO
IHC score
2+
= IHC
Borderline
IHC score
3+
= IHC Positive
HERCEPTIN = YES
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
“polysomy” CEP17
single CEP17
co-amp HER-2 and CEP17
unclassifiable
Aims and objectives
 To evaluate HER-2 gene amplification by MLPA
 To compare tumours with borderline, low level
and high level HER-2
 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
Materials and methods (2)
FISH
amplification
status
(i) not amplified
FISH
HER-2/CEP 17 ratio
< = 1.8
(ii) borderline
> 1.8 < = 2.2
(iii) low
> 2.2 < = 5.9
(iv) moderate
(v) high
(vi) variant
>5.9 < =20
> 20
ratio undetermined
due to variant FISH
patterns
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)
Results
Standard FISH patterns
Variant FISH patterns
HER-2
FISH
Positive
(%)
HER-2
MLPA
Positive
(%)
not amplified
n=2
0
50
0
borderline
n=0
-
-
100
14
low level
n=4
100
100
moderate
n = 11
100
100
moderate
n=5
100
100
high
n=7
100
100
high
n=3
100
100
-
-
variant
n=1
100
100
FISH
amplification
status
HER-2
FISH
Positive
(%)
HER-2
MLPA
Positive
(%)
0
0
borderline
n=2
100
low level
n=7
not amplified
n=9
variant
n=0
FISH
amplification
status
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.
 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
 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
cytogenetics
Maliszewska,
Deputy
Head
• Dr. Norman Pratt, Head of clinical cytogenetics
• NHS Tayside Human Genetics unit
of
clinical