Challenges of new discoveries of clinical applications into the management of cancer patients

Transcription

1 Challenges of new discoveries of clinical applications into the management of cancer patients Tomáš Zima, Veronika Mikulová Institute of Clinical Biochemistry and Laboratory Diagnostics, General University Hospital and First Faculty of Medicine in Prague Czech Republic

2 TUMOR MARKERS Tumor markers are defined as qualitative or quantitative alteration or deviation from normal of a molecule, substances, or process that can be detected by some type of assay above and beyond routine clinical and pathological evaluation. Tumor markers may be detected within malignant cells or surrounding stroma of a primary cancer, or in metastases in local (such as lymph nodes) or distant tissues, or either cellular-based or as soluble products in blood, secretions or excretions.

3 Tumor markers have a very long research history.. Discoveries of the best known tumor markers Ascheim- Zondek hcg Gutman PAP Bjorklund TPA Abelev AFP Gold CEA Koprowski CA Wang PSA Best CA Kufe CA 15-3

4 .. with many thousands of publications in the last four decades.. DEMONSTRATION OF TUMOR-SPECIFIC ANTIGENS IN HUMAN COLONIC CARCINOMATA BY IMMUNOLOGICAL TOLERANCE AND ABSORPTION TECHNIQUES* BY PHIL GOLD,$ M.D., AND SAMUEL O. FREEDMAN, M.D. (From the McGill University Medical Clinic, Montreal General Hospital, and the Department of Physiology, McGill University, Montreal, Canada) PLATES 35 TO 39 (Received for publication, November 16, 1964) ONLY A HANDFUL HAVE MOVED INTO CLINICAL PRACTICE TO DATE

5 CA 15-3 and CA p53 proteomic analysis upa and PAI-1 Cyclin E fragments CEA HER-2 IHC based markers of proliferation Cathepsin D CTC ER and PgR multiparameter gene expression analysis Oncotype DX ECD of HER-2 bone marraow micrometastases DNA flow cytometry-based parameters

6 REQUIREMENTS ESSENTIAL FOR ACCEPTANCE OF A TUMOR MARKER determine utility of marker evaluate magnitude of effect analyze reliability of marker technical issues (assay) analytical issues ( cutoff points, test/validation sets, multivariate analysis) trial design issues (appropriate patient population)

7 WHEN IS A TUMOR MARKER USEFUL (USE)?

8 MAGNITUDE OF THE TUMOR MARKER

9 PURE PROGNOSTIC FACTOR PURE PREDICTIVE FACTOR MIXED FACTOR

10 PRECISION AND ACCURACY OF THE TUMOR MARKER identifying a difference in outcomes for patients in two different states defined by marker results is insuffiecient to introduce in into the clinic many investigators conclude that their marker of interest has clinical utility if in their study the difference in outcomes between marker positive and marker negative patients is less then p<0,05 THIS CONCLUSION MAY BE MISTAKEN

11 TECHNICAL FACTORS AFFECTING MEASUREMENT OF TUMOR MARKERS difficulties arise because of poor sensitivity and/or specificity of the assay for the analyte poorly reproducible assays differences between assays that use different reagents for measurements of the same marker primary technical considerations are critical: 1.which type of assay should be used 2.reproducibility of the chosen assay

12 Pauletti, G. et al. J Clin Oncol; 18: , 2000

13 Comparison between FISH and IHC in predicting OS Pauletti, G. et al. J Clin Oncol; 18: , 2000

14 ANALYTICAL ISSUE CONSIDERATIONS assay interpretation visual assays intra- and interobserver variability automated and semiautomated systems appear to be highly accurate and are likely to be more reproducible cut-off point determination cut-off point validation initial evaluation should be performed using test set of patients the utility of the cut-off point should then be confirmed using a validation set

15 CIRCULATING TUMOR MARKERS proteins autoantibodies free somatic DNA mirna CTC GermLine DNA susceptibility pharmacogenetics

16 WHAT APPROACHES ARE AVAILABLE single gene assays somtatic mutations and other alterations (gene amplificatition) germline alterations multigene arrays and signitures classification prognostication prediction of treatment benefit next generation sequencing

17 Single gene assay germline BRCA1 mutations single gene with many different mutations dramatically increases risk of breast and ovarian cancer predisposition to triple negative breast cancer may impact response to treatment

18 BRCA1 BRCA1 mutation 80% risk of developing breast cancer increased risk of ovarian cancer Copyright 2005, Department of Biology, Davidson College, Davidson, NC 28036

19 BRCA2 BRCA2 mutation the later manifestation of breast cancer increased risk for digestive tract, prostate and melanoma cancer RCSB Protein Data Bank

20 Multigene arrays combination of expressed and repressed genes within a tumor information about: global state of the tumor, revealing information pertaining to cellular metabolic rates, proliferative status molecular interactions between malignant epithelial cells and surrounding cells

21 Molecular heterogeneity of breast cancer has prognostic implication Sorlie T., Proc. Natl. Acad Sci, 2001

22 Gene signatures Oncotype DX 21 Gene Recurrence Score assay Proliferation Ki67 STK15 Survivin Cyclin B1 MYBL3 Invasion Stromolysin 3 Cathepsin L2 GSTM1 BAG2 HER2 GRB7 HER2 Estrogen ER PR Bcl2 SCUBE2 CD68 Reference Beta-actin GAPDH RPLPO GUS TFRC Category RS (0-100) LOW RISK RS < 18 MODERATE RISK HIGH RISK RS RS 31

23 RFS and OS among the 295 patients using four distinct gene signatures each of the signatures used different gene sets with minimal overlap the signatures showed significant agreement in the outcome predictions for individual patients and are probably tracking a common set of biologic phenotypes implications: many genes track together a high proportion of the genes may have limited biologic significance and are probably not targetable

24 Next generation sequencing (massively parallel sequencing) technology has revolutionised our ability to characterize cancers at the genomic, transcriptomic and epigenetic levels is cataloguing all mutations, copy number aberrations and somatic rearrangements at base pair resolution can be used as a means for unbiased transcriptomic analysis of mrnas, small RNAs and noncoding RNAs, genome-wide methylation assays and high-throughput chromatin immunoprecipitation assays

25 Next generation sequencing - methods

26 Next generation sequencing - methods

27 Next generation sequencing - applications

28 Crucial role of CTCs in the metastatic cascade tumor cells must invade the basement membrane and surrounding tissue and enter the bloodstream or lymphatics tumor dissemination changes in cell-to-cell adhesion and extracellular matrix (ECM) adhesion switch in cadherin expression (E-cadherin, N-cadherin) degradation of the ECM MMPs, upa system (poor prognosis) tumor progression epithelial-mesenchymal transition (EMT) process of self-seeding Klaus Pantel & Ruud H. Brakenhoff Nature Reviews Cancer 4, (June 2004)

29 Factors affecting CTCs count Mego M:Nature Reviews Clinical Oncology 7, (December 2010).

30 characterization of EMT epithelial cells: lose cell-to-cell contacts lose cell polarity downregulate epithelialassociated genes acquire mesenchymalgene expression undergo changes in their cytoskeleton EMT process acquire mesenchymal appearance with increased motility and invasiveness Iwatsuki, M: Cancer Science, 101: (2010)

31 EMT and CTCs EMT must have a role in the generation of at least a fraction of CTCs EMT associated markers on CTCs TWIST1, AKT2, PI3Kα EMT has also been associated with the stem-cell phenotype and resistance to apoptotic signals CTCs markers linked to cancer stem cells NOTCHI1 (gene associated with self-renewing) more than 60% of CTCs ALDH1 almost 70% of CTCs Mego M:Nature Reviews Clinical Oncology 7, (December 2010).

32 Why are we interested in CTC detection?

33 Gene expression profiling in cancer circulating cells (CTCs) in breast carcinoma patients - a tool for early metastasis detection and therapy individualisation. Targets 1. Introducing gene expression testing of CTCc by the AdnaGen diagnostic system and implementing standard operating procedure (SOP) for the tests 2. Reducing the costs of the overall testing process by increasing the efficiency of gene expression testing by the new real-time PCR expression profiling system BIOMARK from Fluidigm Inc. ( (one of only two instruments in Europe are available to us for this project) 3. Establishing disease classification methods including predictive scores based on the measurements of CTC oncomarkers using GenEx software in collaboration with MultiD. 4. Individualizing therapy based on the obtained information, thereby increasing the quality and efficiency of the treatment.

34 Trial population number of enrolled patients *419 tests have been done in total

35 Study flowchart

36 Circulating tumor cells testing

37 Patients characteristic s Study results Total CTC-positive Total 197 Tumor size stadium (22%) stadium (44%) stadium (39%) stadium (12%) Nodal status node-negative (31%) node-positive (35%) Histology ductal (25%) lobular 10 2 (20%) others 46 5 (10%)

38 Patients characteristic s Study results Total CTC-positive Grading G (60%) G (30%) G (32%) unknown ER status ER-negative (29%) ER-positive (33%) PR status PR-negative (26%) PR-positive (33%) HER-2 status HER2-positive (29%) HER2-negative (28%) Triple negative (32%)

39 Study results CTC positivity rate The CTC positivity has been described in 33 % in patients with an early breast cancer (M0) undergoing adjuvant chemotherapy. In the metastatic patients the CTCs have been described in 43% of patients at least in one sampling. After treatment (CHT, RT) the positivity rate decreased to the 12%. In the group of neoadjuvant patients 35% samples have been positive before therapy, after 2 CHT- cycles only 5% remained positive. Comparing the dynamics of CTC count within the adjuvant treatment the CTC-positivity rate decreased after completing CHT from 26% down to 13%.

40 Study results Comparison of HER2, MUC1 and EpCAM expression (ng/ul) in early and metastatic breast cancer patients evaluated on 2100 Bioanalyzer (Agilent) HER2 positive CTCs have been detected in 35% of patients with HER2 negative primary tumor. In HER2-positive primary tumors the concordance of HER2 expression was 68,2% on primary tumor and CTC.

41 Study conclusions CTC presence or absence monitoring could help to predict the therapy efficacy in the group of BC patients undergoing neodjuvant chemotherapy treatment CTC could be prognostically important CTC presence prompt about an increased risk of disease progression in the group of BC patients with generalization HER-2 status monitoring on CTCs could help to indicate a need to change the therapeutical attempt

42 HER2 assessment in circulating tumor cells (CTC) in patients with first relapse of HER2-negative breast cancer ML Purpose The detection and characterization of CTC in breast cancer patients with HER2 negative primary tumor and comparison of its HER2 status with the HER2 status in metastasis. This survey has direct implications for minimal invasive tumor characterization and personalized therapy optimization Study design Multicenter, international, non-therapeutic, epidemiological survey

43 Primary objectives to determine CTC count in breast cancer patients with first relapse that were originally HER2 negative in the primary tumor to determine HER2 status (positive/negative) in the CTCs to test for concordance of HER2 status in CTCs and in the biopsy of the metastasis or locoregional relapse collected from the same patient.

44 Human Epidermal Growth Factor 2 (HER2) Cell surface-bound receptor tyrosine kinase Involved in the signal transduction pathways leading to cell growth and differentiation. Encoded within the genome by HER2/neu = proto-oncogene. Over-expression or amplification of the HER2/neu gene in breast cancer is associated with more aggressiveness (15-20% of patients) Trastuzumab (Herceptin) = monoclonal antibody against HER2 Herceptin is effective only in HER2+ tumors normal expression over-expression

45 HER2 signaling pathway

46 Trial population number of subjects total required size of the recruited samples calculated: H0: concordance =<70% vs. H1: concordance =>90% 29% of all patients with HER2 negative primary tumor show HER2+ CTC Minimal required number of patients 352

47 Inclusion and exclusion criteria INCLUSION CRITERIA signed written informed consent female patients aged 18 Histologicaly confirmed HER2 negative primary tumor first documented relapse (locoregional relapse and/or metastatic disease) EXCLUSION CRITERIA previous treatment for breast cancer relapse HER2 positive primary disease contralateral breast cancer clinically indicated for biopsy of the metastasis or loco-regional recurrance

48 Study flowchart Breast cancer patient with first relaps primary tumor HER2 YES [80%] NO Excluded from study YES [50%] Included in study CTC + CTC HER2+[29%] HER2-[71%]

49 Co-operating centers Czech Republic Prague General University Hospital Faculty Hospital Kralovske Vinohrady Thomayer General Hospital and Polyclinic Pilzen Faculty Hospital Pilsen Olomouc Faculty Hospital Olomouc

50 Co-operating centers Slovak Republic Bratislava Oncology Institute of St. Elizabeth National Oncology Institute Trnava Faculty Hospital Trnava Kosice Institute of Oncology in Eastern Slovakia

51 Study preliminary results Total 19* Total Total Tumor size HER-2 status primary tumor pt1 13 HER2-negative 19 pt2 T4 6 Nodal status HER-2 status rebiopsy node-negative 13 HER-negative 17 node-positive 6 HER-positive 2 Histology ER status ductal 16 ER-negative 8 lobular 2 ER-positive 11 others 1 PR status Grading PR-negative 10 I 5 PR-positive 9 II 8 III 6 * 3 patients have been excluded from the study

52 Study preliminary results CTC were found in 4 out of 19 patients (21%) HER2 positive CTC were found in 3 out of 4 CTC positive patients HER2 negative CTC were found in 1 out of 4 CTC positive patients (CTC were positive only for MUC-1 and EpCAM) concordance of HER2 positive status in CTC and the biopsy of the metastasis or locoregional relapse was observed only in 1 out 3 HER2 positive CTC (5% of all included patients) HER2 positive status on CTC in 2 out of 3 patients do not correspond to HER2 status of biopsy (HER2 status was negative)

53 Take home messages There is an urgent need for biomarkers for realtime monitoring of the efficacy of systemic therapy in individual patients. CTC monitoring could provide new insights for appropriate biological therapy selection based on the identification of tumor cells. HER2-positive CTCs can be detected in a relevant number of patients with HER2 negative primary tumors. Therefore, it will be mandatory to correlate the assay-dependent HER2 status of CTCs to the clinical response on HER2-targeted therapies.

54