RT-qPCR analysis of laser capture micro-dissected material from CD31 stained FFPE tissue sections

Transcription

1 RT-qPCR analysis of laser capture micro-dissected material from CD31 stained FFPE tissue sections Julian Schuster, Beatrix Bahle, Andrea Herold, Sabine Lohmann Roche Innovation Center Penzberg, Germany 4 th Munich Biomarker Conference, November 25 th 2014

2 RT-qPCR analysis of laser capture micro-dissected material from CD31 stained FFPE tissue sections Scientific Rationale Method Development: Combining histological staining of FFPET with LCM & RT-qPCR Introducing additional complexity: CD31 IHC staining Summary

3 Scientific Rationale Complementary roles of Ang2 & VEGF-A in tumor angiogenesis 3

4 Schematic representation of Ang2-VEGF-A CrossMab Blocking VEGF-A and Ang-2 function simultaneously VEGF-A VEGF-A Ang-2 Ang-2 Ang-2 Ang-2 CH1 Ck cross anti-vegf-a = Avastin anti-ang-2 = LC06 Ang-2 crossed (CH1-Cκ crossover) Fab domain knobs into holes original bevacizumab (Avastin) light chain Kiennast et al. 2013; Clin Cancer Res Dec 15;19(24): doi: / CCR

5 Hypothesis/Rationale for Biomarker Study: Ang-2 & Tie 2 Expression Differential Ang2 & Tie2 Gene Expression is described in literature for various tumor entities and/or tumor-associated endothelial cells (EC): Cellular model systems 1 Xenograft model systems (Colorectal cancer & melanoma) 2 Glioblastoma 3 Melanoma 4 Breast Cancer 5 Hypothesis for prediction of response to anti-angiogenic therapy described by Helfrich et al. 6 Low angiogenic tumors Reduced levels of pro-angiogenic factors & their receptors in endothelial cells Low-vascularized tumors Association with resistance to α-vegf therapy High angiogenic tumors High levels of pro-angiogenic factors & their receptors in endothelial cells High-vascularized tumors Enhanced sensitivity of intra-tumoral micro-vessels to α-vegf therapy 1) Scharpfenecker et al. J Cell Sci 2005; Fiedler et al. Blood ) Fathers et al. AJP ) Stratmann et. al. Am. J Pathol 1998; Zadeh et al. AJP ) Helfrich et al., Clin Cancer Res ) Dales et al.,int. J Oncol ) Helfrich et. al. JEM 2010, 5

6 RT-qPCR analysis of laser capture micro-dissected material from CD31 stained FFPE tissue sections Scientific Rationale Method Development: Combining histological staining of FFPET with LCM & RT-qPCR Introducing additional complexity: CD31 IHC staining Summary

7 Tumor Heterogeneity Whole FFPET Slide versus micro-dissected material Separation of vessels and tumor cell nests from CD31 stained tissue NSCLC CD31 stained Tumor cell nests [1mm 2 to be collected] Vessels

8 Separation of Tumor Cells and Vessels via LCM* Prior to analysis of Ang-2 and Tie2 gene expression by RT-qPCR NSCLC FFPET section CD31 stained *Laser Capture Microdisscection

9 Workflow Description Isolation of tumor cells & vessels from FFPET tissue samples by LCM followed RT-qPCR analysis 9 Sectioning Deparaffinization Laser Capture RNA Isolation cdna Synthesis qpcr & Staining Microdissection (LCM) Microtome Xylene NSCLC FFPE tissue samples 5 µm sections on membrane slides Staining with Methyl green Dissect region of interest (tumor, vessel) High Pure FFPET RNA Isolation Kit Specific Multiplex multiplex cdna cdna synthesis priming (rev Primer) Transcriptor 1. strand cdna synthesis Kit Rel. Quantification Ang2,Tie2/HPRT (target/reference) Dedicated RealTime ready Assays

10 Ang2 & Tie2 are predominantly expressed in Vessels rather than in Tumor Cells Methyl green staining Ang2 expression Tie2 expression Gene expression in isolated vessels (RED) and tumor cells (BLUE) after cell separation via LCM from NSCLC samples 10

11 RT-qPCR analysis of laser capture micro-dissected material from CD31 stained FFPE tissue sections Scientific Rationale Method Development: Combining histological staining of FFPET with LCM & PCR Introducing additional complexity: CD31 IHC staining Summary

12 High salt buffer stabilizes RNA during IHC staining spectral photometric concentration analysis of isolated RNA. 40,00 35,00 30,00 concentration [ng/µl] 25,00 20,00 15,00 10,00 5,00 not stained CD31 staining without NaCl CD31 staining with NaCl 0,00

13 High salt buffer during IHC staining stabilizes RNA RT-qPCR analysis for 2 reference genes

14 Key Steps in Methods Development 1) High salt buffer for IHC staining procedure: comprising sodium chloride in a concentration of M 2) Coating of membrane slides with poly-l-lysine poly-l-lysine coating to attach tissue to the membrane slide during low temperature heat induced epitope retrieval (LT-HIER)

15 Workflow Description RT-qPCR analysis of micro-dissected material from CD31 stained FFPET sections Microtome Sectioning NSCLC FFPE tissue samples 5 µm sections on membrane slides RNA Isolation trna Deparaffinization Xylene cdna Synthesis Random hexamer + Oligo dt IHC Staining compatible membrane slides & RNA isolation Pre-Amplification Specific primer pool Laser Capture Microdissection (LCM) From CD31 stained FFPET sections qpcr Rel. Quantification Dedicated RealTime ready Assays

16 Workflow Description RT-qPCR analysis of micro-dissected material from CD31 stained FFPET sections Microtome Sectioning NSCLC FFPE tissue samples 5 µm sections on membrane slides RNA Isolation trna Deparaffinization Xylene cdna Synthesis Random hexamer + Oligo dt IHC Staining compatible membrane slides & RNA isolation Pre-Amplification Specific primer pool Laser Capture Microdissection (LCM) From CD31 stained FFPET sections qpcr Rel. Quantification Ang2,Tie2/ALAS1, HPRT Dedicated RealTime ready Assays 16

17 Ang2 & Tie2 are predominantly expressed in Vessels rather than in Tumor Cells CD31 stained NSCLC samples Ratio ANG ANG2 - Tumor 0 0 0,52 ANG2 - Vessels 73,65 15, Ratio TIE2 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0, TIE2- Tumor TIE2 - Vessels 0,788 0,5703 0,68 The CD31 stained vessels were identified to express Ang2 and Tie2. The results from the previous study could be reproduced. We used the identical NSCLC samples as in the previous Ang2-VEGF study. 17

18 Molecular Pathology: Angiopoietin-2 and VEGF-A Two major players in tumor angiogenesis and growth Ang-2 Ang-2 is expressed by endothelial cells (vasculature) Tie2 VEGFR-2 VEGF-A VEGF-A is mainly produced by tumor cells Modified from Folkman J, Angiogenesis: an organizing principle for drug discovery?, Nature Reviews Drug Discovery 6, (April 2007) 18

19 RT-qPCR analysis of laser capture micro-dissected material from CD31 stained FFPE tissue sections Conclusions & Outlook A CD 31 staining procedure that can be applied for FFPE NSCLC tissue on membrane slides, that is compatible with subsequent LCM, RNA isolation & RT-qPCR has been developed. Limited sample number, no statistics Optimize IHC staining method for each marker and tumor tissue type Very labour-intensive workflow Not applicable for high sample numbers Suitable for Biomarker hypothesis generation Alternative/additional down stream application opportunities should be possible: - Profiling/arrays - Sequencing - qpcr/dna analysis

20 Thank you to Biomarker Team at Diagnostics Pharma Research Early Development Julian Schuster Beatrix Bahle Gisela Betzl Caroline Sirsch Viola Mayer-Hoyle Andrea Herold Sabine Moosmann Monika Singer Suzanna Vega-Haring Lisa Coulten Astrid Köhler

21 Doing now what patients need next