Circulating biomarkers for prediction of treatment response

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1 Cremona, October 5-7, 2013 Circulating biomarkers for prediction of treatment response Maria Grazia Daidone Department of Experimental Oncology & Molecular Medicine Milan, Italy

Big challenges in the development of biomarkers for personalized medicine Intratumor heterogeneity and branched evolution: one or two accessible metastases may not be representative of biologically

2 Big challenges in the development of biomarkers for personalized medicine Intratumor heterogeneity and branched evolution: one or two accessible metastases may not be representative of biologically and clinically relevant tumor features. Gerlinger et al., NEJM2012 Impact of the microenvironment on tumor progression Liquid biopsy as a window for therapeutic intervention between initial dissemination and eventual metastatic dissemination Primary tumor or metastasis, and their microenvironment

3 Blood as a real-time liquid biopsy? (for CTCs, ct-dna, ct-mirna) The liquid biopsy concept Peripheral blood contains CTCs, nucleic acids, proteins, microvescicleand exosomes, etc., derived from primary and metastatic lesions Easy accessibility, with minimally invasive approaches Dynamic and real-time monitoring feasible and reliable for: therapeutic targets drug-resistance conferring mutations new actionable mutations early detection of disease progression

4 Workflow for primary systemic trials Medical Oncology Dept. treatment baseline 1st cycle of treatment 2nd cycle of treatment BLOOD WITHDRAWAL Pre-surgery Post-surgery Experimental Oncology Dept. Circulating microrna BLOOD PROCESSING Circulating Tumor Cells(CTC) enumeration molecular characterization Mutations in cell-free DNA

5 CTCs as a real-time liquid biopsy C. Alix-Panabieres, K. Pantel, Clin Chem, 2013

6 CTCs: not only enumeration Towards the rapid assessment of therapy efficacy CIRCULATING TUMOR CELLS: NOT ALL DETECTED CELLS ARE BAD AND NOT ALL BAD CELLS ARE DETECTED, HAYESDF, WICHAM, JCO 2011

7 Summary of currently used CTC characterization methods METHOD ADVANTAGES DISADVANTAGES PROTEIN EXPRESSION (immunofluorescence) CTC count obtainable in the same assay Assessment of inter-cell heterogeneity Cut-off defined at the cell-level by comparison with cell lines Assessment of protein-protein interactions No cut-off at the sample level due to CTC heterogeneity within one sample Limitations for multi-plexing mrna EXPRESSION (qrt-pcr ) Multi-plexing possible up to a large number of genes Small reaction volume required No information on CTC count in a sample No information on heterogeneity Hampered by leukocyte contamination CHROMOSOMAL ABNORMALITIES (FISH) CTC count obtainable in the same assay Assessment of inter-cell heterogeneity Lower sensitivity for small genes due to large size of FISH probes Knowledge needed of possibly altered genes for probe design Limitations for multi-plexing

8 Clinical relevance of CTC characterization in breast cancer: treatment response Author Xenidis (2007) Xenidis (2009) Saloustros (2011) Georgoulias (2012) Xenidis (2013) Muller (2012) Method for CTC characterization qrt-pcr for CK-19 mrna qrt-pcr for CK-19 mrna qrt-pcr for CK-19 mrna qrt-pcr for CK-19 mrna qrt-pcr for CK-19 mrna CellSearch vs ADNA Test (EPCAM, ERBB2, MUC1) # Patients Setting Treatment Clinical relevance 119 Early, ER+/PR+ Tamoxifen 437 Early Epirubicin+ Docetaxel (ET) Presence of CK-19 mrna+ cells duringtreatment associatedwith lowerdfs and OS, alsoin multivariate Presence of CK-19 mrna+ cells after adjuvanttreatment associatedwith lowerdfs and OS, alsoin multivariate 312 Early ET Presence of CK-19 mrna+ cells after treatment associatedwith riskof relapse 75 Early, HER2-545 Early FEC or EC vs ETC or ET Trastuzumab Presence of CK-19 mrna+ cells after treatment associated with lower DFS and risk of recurrence Absence of CK-19 mrna+ cells after treatment associatedwith treatment response and favourable clinical outcome 254 Metastatic Various The clinicalrelevancedependson the test methods 8

9 EPCAM is mainly represented in CTCs from metastatic breast cancers compared to CTCs from early-stage disease positivity percentage neoadjuvant metastatic 10 0 EPCAM MUC1 ERBB2 PIK3CA AKT2 TWIST1 ALDH1 markers

5 ml blood CTC capture : AdnaGEN Lysis: Agencourt lysis buffer RNA extraction : Agencourt RNA Advance

10 CTCs: Not only enumeration Gene Expression Profiling collaboration between INT and Cremona Hospital Breast Unit Patients: women with metastatic breast cancer CTC counts: Cell Search on 7.5 ml blood CTC capture : AdnaGEN Lysis: Agencourt lysis buffer RNA extraction : Agencourt RNA Advance cell v2 kit Gene expression profiling: DASL Illumina (29,000 genes) Bioinformatics analysis: Experimental Oncology Department at INT

leukocyte genes Sample with 300 CTCs: excellent GEP Samples with 10-100

11 Samples clustered by PAM50 genes Towards the rapid assessment of therapy efficacy Gene Expression Profiling of CTCs Clustering with 24 selected leukocyte genes Sample with 300 CTCs: excellent GEP Samples with CTCs: technical failure or CTC Technical failure negativity? True CTC -ve samples

12 Gene expression profiling of CTCs INT experience Experiments with spiked cells have demonstrated the technical and biological reliability of GEPs obtained from at least 25 tumor cells A pilot study with the Cremona Breast Unit demonstrated (in the metastatic setting) the feasibility of a multicentric CTC gene profiling study Studies in the advanced breast cancer setting are crucial for validating the hypothesis that CTCs may represent the cell population that drives disease progression and that their features (rather than the primary tumor) guide prognosis and treatment response If succesfull such a paradigm can be exploited also for NGS studies at DNA level to identify possible druggable targets directly on CTCs as surrogate of the metastatic cell population

13 Hurdles and solutions in CTC research (Plaks, Koopman & Werb, Science 2013) Biophysical factors that maydiminishctc detection Biological factors likely complicatingisolation& detection of clinically relevant CTC populations Future research 13

mirnas were found to be stable and detectable in many biological fluids Towards the rapid assessment

from tumor cells to periferal circulation due to a passive or active release

presence of RNA-binding proteins(i.e., Ago-2) or into microvesicles shed during membrane blebbing

cancer and many other diseases (Shen et al. Cancer letter 2013 and Weilandet al.

14 mirnas were found to be stable and detectable in many biological fluids Towards the rapid assessment of therapy efficacy Blood as a real-time liquid biopsy: circulating microrna mirnas can disseminate from tumor cells to periferal circulation due to a passive or active release mirnashavebeenfoundpackagedin exosomesderivedfrom multivesicularbodies, or to be exported in the presence of RNA-binding proteins(i.e., Ago-2) or into microvesicles shed during membrane blebbing Recent studies demonstrated that circulating mirnas have diagnostic and prognostic significance in cancer and many other diseases (Shen et al. Cancer letter 2013 and Weilandet al. RNA Biol 2013) mirna extraction from plasma/serum mirna mirna signature test Diagnosis, prognosis, etc. Potentially novel non-invasive biomarkers for cancer 14

15 MicroRNAs for cancer: therapeutic applications (modified from William C.S. Cho, BBA, 2010) association with drug sensitivity association with endocrine sensitivity association with anti-her2 treatments

16 Different studies, different results Poor overlap/reproducibility of results Several studies using different approaches Towards the rapid assessment of therapy efficacy Outcome of studies on cell-free mirnas in breast cancer: diagnosis & prognosis Author Body fluid Method Normalization # Pts(#HDs) Candidate mir, #(DE) Zhao(2010) Schrauder (2011) Cuk(2012) Plasma Whole blood Plasma Arrays (Illumina) Arrays, qrt-pcr TaqMan LD-arrays, qrt-pcr Quantile 20(20) 1145 (mir-589, mir-425, let7c) VSN 72 (81) Quantile 127(80) VanSchoone-veld (2012) Serum qrt-pcr mir (20) Schwarzen-bach(2010) Serum qrt-pcr mir (53) 1100 (16 up-and 46 downregulated mirs) 667(miR-148b, mir-376c, mir p, mir-801) 4 (mir-215, mir-299-5p, mir- 411, mir-452) 4 (mir-19a, mir-20a, mir-21, mir-214) Heneghan (2010) Whole blood qrt-pcr mir (44) 7(miR-195, let7a) Cookson (2012) Plasma qrt-pcr mir-16, mean mir level (miR-18b, let-7b, let-7c) Roth (2010) Serum qrt-pcr mir (29) 4 (mir-10b, mir-34a, mir-155) Asaga (2011) Serum qrt-pcr mir (20) 1 (mir-21) Madhavan (2012) Plasma TaqManLD-arrays, qrt-pcr Quantile, cel-mir (76) 667(miR-141, mir-200a,b,c, mir-210, mir-375, mir-203, mir-801) 16

17 Outcome of studies on cell-free mirnas in breast cancer: treatment response Author Body fluid source Method Normalization # Pts (#HDs) Stage Candidate mir, #(DE) Relation with: Zhao (2011) Plasma qrt-pcr mir II, III mir-221 Overall response to NAC, ER- Wang(2012) Serum qrt-pcr mir II, III 4(miR-125b) FECresponse, high PCNA, low AI Wu(2012) Serum Deep sequencing (Illumina), qrt-pcr edger algorithm Jung(2012) Plasma qrt-pcr U6-RNA 29(28) II, III >800(miR-122, mir-375, mir- 184, mir-1299, mir-196a, mir- 381,miR-41, mir- 1246) I-III I-III 4(miR-126, mir- 21, mir-210, mir-29a) pcrtoac+/- Tx+trastuzumab, distant metastasis post-treatment, response to Trastuzumab Sun(2012) Serum qrt-pcr mir III mir-155 post-treatment, responseto chemotherapy 17

18 Technical/biological aspects challenging circulating mirna studies Feature Challenge Outcome Towards the rapid assessment of therapy efficacy Hemolysis in plasma/serum samples affects mirna profile Identification hemolyzed samples of Developed a hemolysis score able to identify low level of hemolysis independently from lipema. Heparin could be used as anticoagulant Plasma or serum samples mirna are short, have variable GC content and high homology between family members, are present in low concentration in circulation Established reference mirnas are missing Heparin inhibits amplification reaction (RT-PCR) Is mirna profile equivalent? Are conventional available techniques (such as RT-PCR, mirna microarray) suitable for circulating mirna profiling? Identification of appropriate normalization methods If adequately treated, heparinized plasma samples could be suitable for mirna expression analysis, without affecting mirna detection performance. P Tiberio, et al. The Journal of Molecular Diagnostics, 2013;15: Ongoing Agilent microarray is suitable for measuring mirna expression in human tissues and in archival(>20 years old) plasma samples. M Callari, et al. Analytical Biochemistry 2013;437: CallariM,etal. PLoS One.2013May14;8(5). Ongoing (three normalization methods adopted: raw data, normalization approaches ratio-based or using housekeeping mirnas)

19 micrornasasindicatorsof sensitivity/resistanceto specific treatments In vitro studieson cell coltures In vivo correlative studies within PST/NAC trials Identification of: 1. mirnaassociatedto drug sensitivity/ resistance 2. treatment-induced mirna changes End point: identificationof novel biomarkers/ pathways Pre-clinical in vitro/in vivo functional validation Identification of: 1. mirnaassociatedwith clinical outcome 2. mirnas as monitoring tools: Prospective clinical validation PST/NAC trials integrating clinico-pathologic& molecular findings 19

Blood as a real-time liquid biopsy: mutations in

cancer cells ( ) my be released into the bloodstream,

integrity and microsatellite alterations.

useful for: early detection prognosis monitoring

20 Blood as a real-time liquid biopsy: mutations in circulating DNA Primary tumor or metastasis DNA from cancer cells ( ) my be released into the bloodstream, enabling the detection of mutations, methylation, DNA integrity and microsatellite alterations. Identification of cancer mutations in plasma may be useful for: early detection prognosis monitoring tumor dynamics over time detection of minimal disease monitoring recurrence

21 Identification of treatment-associated mutational changes from exome sequencing of serial plasma samples M Murtaza et al. 2013

22 Monitoring Circulating Tumor DNA Early detection of relapse and/or of disease progression NGS of tumor sample Selection of DNA alterations (tumor barcodes ) Tracking of mutations in serial plasma samples Breast cancer at surgery (relapse detection) Early detection of relapse?

23 Most frequently mutated breast cancer genes (according to Baird & Caldas, BMC Medicine 2013) Approximate mutation frequency(%) Gene mutation Function Overall Luminal A Luminal B HER2- enriched Basallike PIK3CA TP53 GATA3 Catalytic subunit of PI3K, key signal transduction enzyme involvedin cellgrowth& survival, and insulin signaling Tumor suppressor, key regulatorof cellcycle, DNA repair and apoptosis Transcription factor regulating luminal epithelial cell differentiationin the mammary glad MAP3K1 KinaseactivatingERK and JNK pathways MLL3 CDH1 Histone-lysine N-methyltransferaseinvolvedin transcriptional co-activation Cell-cell adhesion glycoprotein: LoFmutationsin E-cadherinasa featureof lobular breast cancer

Dynamics of tumor-specific mutations in plasma of a metastatic BC

tumor material used to identifiy tumor mutations Selection of 10

Tracking of mutations in serial plasma samples Common pattern of

24 Dynamics of tumor-specific mutations in plasma of a metastatic BC patient undergoing two phases of chemotherapy Whole-genome seq of tumor material used to identifiy tumor mutations Selection of 10 mutations (tumor barcode ) Epirubicine paclitaxel CT scan Tracking of mutations in serial plasma samples Common pattern of sharp decline in AF upon chemotherapy and increase upon disease progression

26 Circulating tumor DNA to monitor metastatic breast cancer: Study Overview and Outcome This study evaluated the sensitivity of assaying tumor DNA circulating in the plasma. This assay is compared with three other approaches: radiographic imaging, assay of cancer antigen 15-3 (CA 15-3) levels, and CTC assay. Circulating tumor DNA successfully detected in 97% (29/30) of women in whom genomic alterations were identified (CA15.3 and CTCs detected in 78% (21/27) and 87% (26/30) of the cases, respectively) Circulating tumor DNA levels showed greated dynamic range, greater correlation with changes in tumor burden, earliest measure of treatment response (in 53% of women, 10/19) This proof-of-concept analysis showed that circulating tumor DNA is an informative, inherently specific, and highly sensitive biomarker of metastatic breast cancer. Now, VALIDATION!!!

27 Circulating biomarkers to monitor progression/treatment response Current points of strength CTC molecular profile mirnas Mutations in cell-free DNA Feasible on small sample amount (<1.5 ml) NO YES YES Independence from tissue heterogeneity YES YES YES Direct information on tumor biology, distinct subpopulations & heterogeneity Accounting for microenvironment interactions Dynamic time-related monitoring (feasibility for longitudinal studies) YES NO YES (only through WGS) NO YES NO YES YES YES Identification of clonal resistance YES NO YES Suitable to investigate druggable targets YES NO YES Suitable for retrospective studies NO YES YES Taylored for personalized monitoring NO NO YES

28 Circulating biomarkers to monitor progression/treatment response Current points of weakness CTC molecular profile mirnas Mutations in cellfree DNA Low feasibility Dependent on capture efficiency NO NO Contamination by other cells/other cell products YES YES (hemolysis issue) YES (possible dilution) Possible missing of clinically relevant populations YES NYI* NYI* Technical-related hurdles YES YES YES Data analysis-related issues NO YES YES Timing of blood withdrawal NO YES YES Few studies with clinical correlations Discordance among published results Tayloring for individual patients YES YES YES NO (single group studies) YES NO (single group studies) NO NO YES * NYI: Not Yet Investigated

29 The best preparation for tomorrow is to do today s work superbly well Sir William Osler Canadian Physician ( )

30 Thanks to Colleagues of the: Department of Medical Oncology Istituto Nazionale Milan Breast Unit Istituti Cremona Biomarkers Unit Dept. of Experimental Oncology Istituto Nazionale Milan 30

Disclosure. Summary. Circulating DNA and NGS technology 3/27/2017. Disclosure of Relevant Financial Relationships. JS Reis-Filho, MD, PhD, FRCPath

Disclosure. Summary. Circulating DNA and NGS technology 3/27/2017. Disclosure of Relevant Financial Relationships. JS Reis-Filho, MD, PhD, FRCPath Circulating DNA and NGS technology JS Reis-Filho, MD, PhD, FRCPath Director of Experimental Pathology, Department of Pathology Affiliate Member, Human Oncology and Pathogenesis Program Disclosure of Relevant