Breast Cancer Biomarkers: From Serendipity to Rational Discovery

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1 // Breast Cancer Biomarkers: From Serendipity to Rational Discovery Partha S. Ray, MD Clinical Assistant Professor Department of Surgery University of Illinois College of Medicine at Urbana Champaign Carle Cancer Center & Carle Foundation Hospital Disclosures Named inventor on patents related to in cancer Stock ownership in Oracle Biosciences Cancer Biomarkers Serum biomarkers Tissue biomarkers Screening biomarkers Diagnostic biomarkers Prognostic biomarkers Predictive biomarkers Theranostic biomarkers

2 // Utility of Cancer Biomarkers Diagnostic Biomarker Prognostic Biomarker Predictive Biomarker Theranostic Biomarker Identify patients Clinical outcomes Personalize therapy All of the above Historical Timeline of Breast Cancer Biomarker Discovery Anti ER therapy (Tamoxifen ) Anti HER therapy (Herceptin ) ER reported HER reported

3 // patients who all underwent mastectomy patients who all underwent mastectomy Estrogen receptor (ER) Diagnostic for a specific group of breast cancers that when advanced, tended to metastasize to bone Prognostic for predicting better disease-free, diseasespecific and overall survival compared to patients with ER- breast cancer Predictive of therapeutic efficacy of Tamoxifen and other anti-estrogen agents such as Arimidex, etc. Therefore is truly, in hindsight, a Theranostic Biomarker in breast cancer

4 // 89 patients were evaluated Human Epidermal Growth Factor Receptor (HER) Diagnostic for a specific group of breast cancers that when advanced, tended to metastasize to lung and brain Prognostic for predicting worse disease-free, diseasespecific and overall survival compared to patients with HER negative breast cancer Predictive of therapeutic efficacy of Herceptin and other anti-estrogen agents such as Lapatinib, etc. Therefore is truly, in hindsight, a Theranostic Biomarker in breast cancer Breast Cancer Classification Normal Breast Cancer Normal ER+ HER+ Triple- Negative

5 // Breast Cancer Diagnostic Biomarker ER HER Triple Negative Clinical Outcome Indolent Aggressive Very aggressive Personalized Therapy Tamoxifen Arimidex Herceptin Lapatinib? Delineated the existence of distinct molecular subtypes of breast cancer:. A. B. HER. Basal-like. Normal-like

6 // Intrinsic molecular subtypes Derived from gene expression analysis-transcriptomics Prognostic Predictive Distinct subtypes overlap with ER and HER Surviving Fraction MOLECULAR SUBTYPE P<. Basal MOLECULAR SUBTYPE HER All Patients Surviving Fraction.. HER. Basal P<.. Lymph Node - Negative Patients Breast Cancer Classification Normal Breast Cancer Normal ER+ subtype HER+ HER subtype? Basal-like subtype Breast Cancer Molecular Subtype HER Basal like Diagnostic Biomarker ER HER? Clinical Outcome Indolent Aggressive Very aggressive Personalized Therapy Tamoxifen Arimidex Herceptin Lapatinib?

7 // Triple negative breast cancer - % of all breast cancers No biomarker (ER PR HER ) Very heterogeneous patient population with variable prognosis and responses to drugs % patients develop metastasis; % die in years No effective way to stratify patients at risk of metastasis No effective way to ID patients in need of either more aggressive or less aggressive therapy No targeted therapy Need specific biomarker to identify/stratify patients and drive therapy Level of Information High Throughput Platform Cost effective Assay DNA RNA Protein DNA methylation Genomics Transcriptomics Proteomics Epigenomics Single / panel gene mutations qrt-pcr Immunohistochemistry Single / panel gene methylation Non-coding RNA Short Non-coding RNA Long Non-coding RNA Transcriptomics-driven Cancer Tissue Biomarker Discovery Surgical Biopsy/Resection Tumor Microdissection RNA Extraction Candidate biomarker selection Data Analysis Gene expression Microarray Assessment of Diagnostic Significance Assessment of Functional Significance Assessment of Prognostic Significance Clinical validation

8 // expression in basal-like subtype Partial gene list of IGS overexpressed in Basal-like Cluster Expression profiles of genes coordinately up-regulated with up-regulation Expression profiles of genes coordinately down-regulated with up-regulation Expression profiles of the whole gene signature 9 patient human breast cancer gene expression microarray dataset from Ivshina et. al. Cancer Res, 9- (). Comparative Analysis of basal-like gene markers 8 8 CK 8 CK 8 CK 8 Bcrystallin 8 Moesin 8 EGFR 8 CD9 8 C-Kit 8 ITGB 8 p-cadherin 8 FOXC Normal Breast A / B HER- Basal -like Bcrystallin Moesin CD9 P-cadherin EGFR CK CK CK C-Kit ITGB FOXC fold change in basal-like subtype P <. Normal HER Basal like Breast A / B N= dataset Richardson et. al. Cancer Cell 9, - ().. P <..... HER Basal like A / B N=9 dataset Ivshina et. al. Cancer Res, 9- ().. P <..... HER Basal like A / B N= dataset Miller et. al. PNAS, - ().. P <... HER Basal like. A / B N=9 dataset van de Vijver et. al. N Engl J Med, (). 8

9 // Quantitative ranking of proposed biomarkers Gene A Mean±SD (Median) B Mean ± SD (Median) HER Mean ± SD (Median) Basal like Mean ± SD (Median) Normal like Mean ± SD (Median) Univariate Wilcoxon Rank Sum Test (Basal like vs. Pooled non Basallike) P value Multivariate Logistic Regression P value CRYAB KRT KIT CDH MSN KRT EGFR KRT ITGB.9±.9 (.8).±. (.).9±. (.).±. (.).±.9 (.).±. (.).±.9 (.).±.9 (.).8±. (.9).±. (.).±. (.).8±. (.8).±.8 (.8).8±. (.8).±.9 (.).±.9 (.).±. (.).±. (.).±. (.).±. (.).±. (.).9±.9 (.9).±. (.8).±. (.9).±.8 (.).±. (.).±.9 (.).±. (.).8±. (.).±. (.8).9±. (.8).±. (.8).±. (.).±. (.).±. (.).±. (.).±. (.).±. (.).8±.(.).±.9 (.).±.9 (.).±. (.).±. (.).±. (.).±. (.).±. (.).±.9 (.).±.9 (.).9±. (.).±. (.) <. <. <.. <. <. <. <..8 < patient human breast cancer gene expression microarray dataset from van de Vijver et. al. N Engl J Med, (). Is of cancer cell origin? BT MX HCC9 BT-9 MDA-MB- HCC9 Hs8T MDA-MB-s MDA-MB- CAL- CAL- DU SW HCC8 CAL8- HDQ-P JIMT- HCC9 HCC HCC8 MDA-MB-8 HCC HCC9 KPL AU SKBR EVSA-T CAL-8 MDA-MB-VI CAMA- TD HCC8 MFM- BT MDA-MB- HCC9 BT8 UACC-8 HCC MDA-MB-VII ZR- HCC8 ZR- MDA-MB- HCC UACC-89 EFM-9A MDA-MB- MCF KPL EFM9 Basal-like Non-basal HER+ Basal -like (triple -negative) ER-PR- HMEC MCF- TD ZR MDA-MB- MDA-MB- MDA-MB- MDA-MB-8 HCC-8 HCC-9 BT-9 HS8T HCC SKBR ß-actin Validating proposed biomarker 9

10 // Validating proposed biomarker Examination of immunoreactivity of hybridoma subclones. MCF- breast cancer cells were transfected with vector or. Cell lysates were subjected to immunoblotting with cell culture supernatant (with a : dilution) from hybridoma subclones. Results from representative clones are shown. The top band is the full-length and other bands are truncated. In vitro characterization of anti- monoclonal antibody. Left, immunoblotting of MCF- cells transfected with either vector or with the purified anti- antibody (:). Right, immunofluorescence staining of the same cells. The blue signal represents the nuclear DNA staining by DAPI. The green signal represents the staining by the antibody. Immunohistochemistry of on human breast cancer samples. Images of Three representative triple-negative (ER-PR-HER-) samples are shown. Verifying prognostic significance of using transcriptomics - I. MOLECULAR SUBTYPE. GENE SIGNATURE. mrna EXPRESSION Surviving Fraction. HER.. Basal P<.. Surviving Fraction P=.. Surviving Fraction... High P=.. Low All Patients. MOLECULAR SUBTYPE. GENE SIGNATURE. mrna EXPRESSION Surviving Fraction.. HER. Basal P<.. Surviving Fraction P=.. Surviving Fraction.... P=. High Low Lymph Node - Negative Patients Verifying prognostic significance of using transcriptomics - II BRAIN METASTASIS FREE SURVIVAL BONE METASTASIS FREE SURVIVAL Metastasis -free Fraction. HER Basal -like. P=.9. Metastasis -free Fraction. Basal -like HER. P=.. MOLECULAR SUBTYPE A / B HER Basal -like BRAIN METASTASIS FREE SURVIVAL BONE METASTASIS FREE SURVIVAL Metastasis -free Fraction Low. High. P <.. Metastasis -free Fraction High. Low. P=.. mrna LEVEL < 9 th percentile > 9 th percentile

11 // Validating prognostic significance of using IHC - I. N=8. N=8. N= Survival distribution.8... P=. N=8 Survival distribution.8... P=. N= Survival distribution.8... P=. N=. Survival time (years) All Others ER- HER-. Survival time (years) All Others ER- HER-Basal +. Survival time (years) All others ER- HER- + Validating prognostic significance of using IHC - II Validating prognostic significance of using IHC - II

12 // Predictive significance of Optimizing assay for clinical use FFPE Tissue Block FFPE Section Y Y Y YYY IHC grade Monoclonal antibody Y Y Y YY Y Detector system Subjective scoring Digital Image Analysis FFPE Section RNA extraction qrt PCR Data Analysis Prognostic Summary Score Report What is s functional significance? Cell proliferation (MDA-MB-) Migratory cells Invasive cells Days Cell proliferation (MCF-) Days Migratory cells Invasive cells Soft agar

13 // What is s functional significance? MMP (ng/ml) MCF MDA MB MMP9 (ng/ml) MDA MB MCF MDA-MB- Cyclin D β actin Vimentin Fibronectin α SMA β actin Relative protein levels ( FACS) 8 MCF BT9 T β actin MCF-A E cadherin P cadherin β actin What is s functional significance? Cell proliferation Control shrna shrna shrna (T) Days Migratory cells 8 Control shrna Invasive cells 8 shrna shrna Control shrna shrna shrna Control shrna shrna (T) shrna β-actin Cell proliferation. Control shrna shrna shrna. (BT9). Days Migratory cells 8 Control shrna shrna shrna Invasive cells 8 Control shrna shrna shrna Control shrna shrna shrna (BT9) β-actin What is s functional significance? Control shrna shrna shrna D Matrigel Soft agar shrna shrna Ctrl shrna

14 // Breast Cancer Classification Normal Breast Cancer Normal HER+ ER+ HER subtype subtype Basal-like subtype Breast Cancer Molecular Subtype HER Basal like Diagnostic Biomarker ER HER Clinical Outcome Indolent Aggressive Very aggressive Personalized Therapy Tamoxifen Arimidex Herceptin Lapatinib? Historical Timeline of Breast Cancer Biomarker Discovery Anti ER therapy (Tamoxifen ) Anti HER therapy (Herceptin ) Anti therapy? ? ER reported HER reported reported

15 // Conclusions appears to be a powerful surrogate tissue biomarker of BLBC with high diagnostic and prognostic significance, and emerging predictive significance. is functionally important for maintenance of the basal-like phenotype. may also serve as a potential molecular therapeutic target for BLBC. Current findings support conducting a large-scale prospective validation trial of as a theranostic biomarker of basal-like triple negative breast cancer. expression in conjunction with other markers may also help elucidate hybrid subtypes such as basal-her. Questions???

16 //

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