DETERMINATION OF HER2 STATUS IN BREAST CANCER PATIENTS BY TWO METHODS: REAL-TIME QUANTITATIVE PCR (QPCR) AND IMMUNOENZIMATIC ASSAY (EIA)
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1 DETERMINATION OF HER2 STATUS IN BREAST CANCER PATIENTS BY TWO METHODS: REAL-TIME QUANTITATIVE PCR (QPCR) AND IMMUNOENZIMATIC ASSAY (EIA) Maria Savino, Paola Parrella, Massimiliano Copetti, Raffaela Barbano, Roberto Murgo, Vito M. Fazio, Vanna Maria Valori, Massimo Carella, Maria Garrubba, Stefano Angelo Santini Laboratorio Analisi Cliniche, IRCCS Ospedale Casa Sollievo della Sofferenza San Giovanni Rotondo (FG) 1 HER2 and Cancer In 30% of breast cancer patients the Her2 protein is over expressed Overexpression of HER2 is associated with poor prognosis and advanced-stage cancers Overexpressed HER2 protein still responds to regulation, making them attractive targets for anticancer therapies 2
2 ErbB Family of Tyrosine Kinase Receptors Family of evolutionarily conserved type I receptor tyrosine kinases Four members: ErbB-1 (EGFR/HER1) ErbB-2 (HER2) ErbB-3 (HER3) ErbB-4 (HER4) Extracellular Domain (Binds Ligand) TM Domain Cytoplasmic Domain (Kinase Activity) 3 Target therapy MoAb Herceptin 4
3 Evaluation of HER2 status IHC Protein FISH DNA CISH IHC = immunohistochemistry FISH = fluorescence in-situ hybridisation CISH = chromogenic in-situ hybridisation 5 HER2 serum determination in breast cancer patients 6
4 HER2 mrna expression by RT PCR in breast cancer 7 Aim of the project Determination of HER2 expression by QPCR in the blood of breast cancer patients HER2 serum protein determination by ELISA Results from these analyses were correlated with HER2 status in the tissue as determined by IHC 8
5 Matherials Tumor dimension Limph node status, no.of patients (%) T1 39 (46%) Posistive 36 (42%) T2 35 (42%) Negative 41 (48%) T3 2 (2%) Unknown 8 (9%) T4 9 (10%) Grade, no. Of patients % Estrogen receptor, no. Of patients (%) I 4 (5%) Positive 38 (45%) II 39 (46%) Negative 32 (38%) III 26 (31%) Unknown 15 (18%) Unknown 16 (19%) HER2 Immunoistochemistry Progesteron receptor, no. Of patients (%) IHC (58%) Positive 29 (34%) IHC 0/1+ 36 (42%) Negative 49 (47%) Total 85 Unknown 16 (19%) 9 HER2 serum levels by EIA 10
6 RT-QPCR RT QPCR RNA extraction cdna synthesis Peripheral blood collection Quantificatio n Taqman chemistry based 11 QPCR Box plot for the HER2 serum levels determined by EIA HER2 serum concentration Range median IHC IHC 0/ Healthy Her2 serum concentrations P<0.001 (Dunn Test) 0 Healthy Neg IHC Pos IHC 12
7 Box plot for the HER2 28EC copy number ratios determined by QPCR HER2 copy number Range median IHC IHC 0/ Healthy Her2/28SEC copy number ratio P<0.001 Dunn Test 0 Healthy Neg IHC Pos IHC 13 EIA QPCR True positive rate True positive rate False positive rate Cut off : 22 ng/ml Sensibility : 59% Specificity : 95% False positive rate Cut off : 4.74 copy number Sensibility : 78% Specificity : 91% 14
8 Scatter plot for HER2/28SEC copy number ratio and HER2 serum concentrations Her2 serum concentrations ng/ml IHC Neg >22 n=5 (14%) <22 n=31 (86%) IHC Pos sensitivity >22 n=27 (55%) <22 n=22 (45%) Her2/28SEC copy number ratio IHC Neg >4.74 n=8 (22%) <4.74 n=28 (78%) IHC Pos sensitivity >4.74 n=37 (76%) <4.74 n=12 (24%) specificity specificity 15 Comparison of ELISA and QPCR 16
9 Correlation with limph node status 17 Logistic Regression Model Predicted probability of being limph node positive Her2 blood mrna copy number Elevated blood mrna levels of HER2 were associated with a higher probability of a positive lymph node status 18
10 Take Home Messages QPCR is suitable alternative method for the determination of HER2 status in the blood of breast cancer patients QPCR is more sensitive than ELISA in discriminating HER2 IHC positive tumours from both healthy individuals and IHC negative breast cancers QPCR could be used as diagnostic tool when primary tumour samples are unavailable to monitor the outcome of the disease and the response to therapy during follow up of breast cancer patients. 19 Acknowledgements Laboratorio Analisi Cliniche Stefano Angelo Santini Maria Garrubba Laboratorio di Oncologia Paola Parrella Laboratorio di Genetica Medica Massimo Carella Dipartimento di onco-ematolgia Vanna Maria Valori Dipartimento di Chirurgia Roberto Murgo Istituto di Metodi QuantitativiUniversità Foggia Massimiliano Copetti 20
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