Assessment of Breast Cancer with Borderline HER2 Status Using MIP Microarray
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1 Assessment of Breast Cancer with Borderline HER2 Status Using MIP Microarray Hui Chen, Aysegul A Sahin, Xinyan Lu, Lei Huo, Rajesh R Singh, Ronald Abraham, Shumaila Virani, Bal Mukund Mishra, Russell Broaddus, and Rajyalakshmi Luthra
2 HER2 Amplification in Breast Cancer HER2 overexpression/amplification: 15-20% of breast cancer Associated with aggressive clinical course Predicts a better response to anti-her2 targeted therapy Accurate assessment of HER2 status in a reproducible manner is essential to guide clinicians on HER2-targeted therapy HER2 status correlates with histology Lobular HER2 negative: invasive tubular, cribriform, mucinous, low grade lobular, and metaplastic carcinoma HER2 positive: carcinoma with apocrine differentiation Ductal
3 Current HER2 Evaluation Methods HER2 Overexpression IHC (3+) HER2 Amplification FISH ASCO/CAP and NCCN guidelines recommend testing for HER2 on all primary invasive breast cancers (and on metastatic site, if stage IV and if specimen available). Standardization of HER2 testing in clinical practice for patients with breast cancer Some degree of discordance between IHC and FISH results
4 ASCO/CAP 2013 Guideline for HER2 ISH FDA HER2/CEP17 Ratio ASCO/CAP 2007 HER2 signals ASCO/CAP 2013 Wolff et al., J Clin Oncology, 2013
5 FISH Assay Advantage Include invasive tumor cells Exclude DCIS Exclude normal cells Genetic heterogeneity Assessment of ploidy Limitation Inter-observer subjectivity Non-reproducibility Partial nuclear signals from FFPE Mimic monosomy Pseudo amplified (missing CEP17)
6 Technologies for Copy Number Analysis Fluorescent in situ hybridization (FISH) Recommended by ASCO/CAP for HER2 testing in breast cancer Individual probe hybridizing to focused and targeted sequence Single-probe, dual-probe Reverse transcription quantitative polymerase chain reaction (RT-qPCR) Laser-captured microdissection to capture invasive tumor cells Detect relative HER2 mrna levels Concordance with FISH and IHC Next generation sequencing (NGS) Normalized coverage depth to approximate copy number change Relative low sensitivity, not able to detect low level of amplification in some platforms SNP microarray Genome-wide analysis using SNP probes Detect allelic imbalance Polysomy versus segmental amplification Chromothripsis
7 Molecular Inversion Probes (MIP) High resolution in ~900 cancer genes and 220,000 SNP probes. DNA input at 60-80ng (OncoScan, Affymetrix). Courtesy of Affymetrix, Inc
8 Allelic peaks BAF Log2 ratio Data Visualization Normal (2N) Gain (3N) Loss (1N) LOH (2N) 50% mosaicism with CN LOH B allele frequency (BAF) = (B signal)/(a signal +B signal) A allele (AT channel) B allele (GC channel) Allele peaks =A - B Each allele has a value of 0.5
9 Allelic Peak BAF Log2 Ratio Segmental Amplification Chr 17 HER2
10 Allelic Peak BAF Log2 Ratio HER2 Amplification and Associated Chromothripsis Chr 17 HER2
11 HER2 Copy Number by FISH and MIP MIP HER2 high Amplified by ISH N=16 HER2 Notamplified by ISH N=18 HER2 CN>10 16 (100%) 0 HER2 amp FISH HER2 amp MIP HER2 neg FISH HER2 neg MIP HER2 CN< (100%) Chromothripsis at 17q12 8 (50%) 0 Excellent correlation of HER2 interpretation between FISH and MIP microarray; HER2 amplification is associated with chromothripsis involving 17q12. Borderline cases and one case with genetic heterogeneity were excluded.
12 Study Objective We hypothesized that MIP microarray can provide reproducible, accurate and quantitative HER2 assessment of breast cancer with borderline results. MIP microarray can objectively assign these borderline cases into appropriate categories to guide clinicians on anti-her2 targeted therapy.
13 Patient Population Patient Characteristics Age: 44-85yr (median 66yr) Histology Invasive ductal carcinoma: 10 Invasive lobular carcinoma: 1 IHC Equivocal (2+): 8 Negative (1+): 3 FISH HER2 copy (4-6) and ratio>2: 5 HER2 copy (4-5) and ratio<2: 5 HER2 copy (3.9) and ratio<2: 1 Inclusion Criteria Female Primary invasive breast cancer Mastectomy without neoadjuvant chemotherapy Borderline HER2 status 1+ or 2+ by IHC HER2 copy of by FISH HER2/CEP17 ratio <3 by FISH Equivocal/conflicting HER2 status Between biopsy and excision Between IHC and FISH
14 Materials and Methods We selected archived tumor blocks to prepare H&E and unstained slides; Circled the area enriched for invasive carcinoma and estimated tumor percentage; Manual microdissection to extract genomic DNA; We used OncoScan FFPE Assay kit, OncoScan Console (Affymetrix) and Nexus TuScan for data analysis (Biodiscovery).
15 Estimate Copy Number of Tumor Cells % Aberr. Cell Reported Reported copy number (CN) is the actual CN for tumor cells % Aberr. Cell Not reported Reported CN is averaged CN for tumor and normal cells Calculate the actual CN for tumor cells on the basis of visual estimation of tumor fraction determined by pathologist CN reported - (1 - %Tumor) x 2 CN tumor = %Tumor
16 Allelic Peak BAF Log2 ratio HER2 Copy Number Gain HER X
17 Allelic Peak BAF Log2 ratio HER2 Copy Number Gain in Tetraploidy HER X
18 Allelic peak BAF Log2 ratio HER2 Amplification and Chromothripsis involving 17q12 HER
19 Breast Cancer with Borderline HER2 Status ISH Positive Equivocal MIP HER2 low Amplified N=5 HER2 Equivocal N=5 HER2 border Negative N=1 Negative HER2 CN 3-4 HER2 CN (60%) 4 (80%) 0 2 (40%) 1 (20%) 1 (100%)* HER2 amp FISH HER2 amp MIP HER2 Eq FISH HER2 Eq MIP HER2 neg FISH HER2 neg MIP Most HER2 borderline cases by FISH showed HER2 copy number gain (3-4) by MIP. *One HER2 borderline negative case by FISH showed HER2 copy number of 7 and chromothripsis at 17q12 by MIP.
20 Chromosome View HER2 HER2+ (16) HER2- (18) HER2 Eq (5) HER2 low + (5) HER2 border - (1) HER2 copy number of 3-4 by MIP was observed in cases with gains in 17q (n=5), 17q11-q21 (n=1), or tetraploidy (n=1); HER2 copy number of 5-7 by MIP was associated with amplification of a short segment in 17q12-q21 (n=3) or chromothripsis involving 17q12 (n=1).
21 Conclusions With minimal DNA requirements using FFPE tissue, MIP microarray provided quantitative and objective assessment of HER2 copy number of breast cancer. Excellent correlation between FISH and MIP microarray in HER2 highly amplified and HER2 non-amplified tumors. Chromothripsis involving 17q12 was observed in 50% of HER2 highly amplified tumors, but not in tumors without HER2 amplification. Breast cancer with borderline and conflicting HER2 results can be objectively assessed and classified by MIP on the basis of HER2 copy number and its associated genomic alterations in chromosome arm and chromosomal segments. MIP technology may potentially be critical in guiding clinical decisions regarding targeted therapy.
22 Acknowledgement Molecular diagnostic laboratory Raja Luthra, PhD Rajesh Singh, PhD Keyur Patel, MD, PhD Mark Routbort, MD, PhD Ronald Abraham Shumaila Virani Bal Mukund Mishra Hui Yao, PhD Pathology Russell Broaddus, MD, PhD Aysegul Sahin, MD Nour Sneige, MD Kenneth Aldape, MD, PhD Alex Bousamra, MD Lei Huo, MD, PhD Michael Gilcrease, MD, PhD Cytogenetic laboratory Xinyan Lu, MD Melissa Robinson Ricardo Fernandez Smita Trivedi
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