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

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.

Tumor markers have a very long research history …..

Discoveries of the best known tumor markers 1928 - Ascheim- Zondek hCG 1936 - Gutman PAP 1957 - Bjorklund TPA 1963 - Abelev AFP 1965 - Gold CEA 1979 - Koprowski CA 19-9 1979 - Wang PSA 1981 - Best CA 125 1983 - Kufe CA 15-3

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)

….. with many thousands of publications in the last four decades …..

ONLY A HANDFUL HAVE MOVED INTO CLINICAL PRACTICE TO DATE

CA 15-3 and CA 27.29

CEA

ER and PgR

DNA flow cytometry-based parameters

IHC based markers of proliferation

HER-2

ECD of HER-2 p53

uPA and PAI-1

Cathepsin D Cyclin E fragments

proteomic analysis

multiparameter gene expression analysis Oncotype DX

bone marraow micrometastases

CTC

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)

WHEN IS A TUMOR MARKER USEFUL (USE)?

MAGNITUDE OF THE TUMOR MARKER

PURE PROGNOSTIC FACTOR

PURE PREDICTIVE FACTOR

MIXED FACTOR

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

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

Pauletti, G. et al. J Clin Oncol; 18:3651-3664, 2000

Pauletti, G. et al. J Clin Oncol; 18:3651-3664, 2000

Comparison between FISH and IHC in predicting OS

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“

CIRCULATING TUMOR MARKERS

proteins autoantibodies free somatic DNA miRNA CTC GermLine DNA

susceptibility pharmacogenetics

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

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

BRCA1

• BRCA1 mutation ▫ 80% risk of developing breast

cancer ▫ increased risk of ovarian

cancer

© Copyright 2005, Department of Biology, Davidson College, Davidson, NC 28036

BRCA2

• BRCA2 mutation ▫ the later manifestation of

breast cancer ▫ increased risk for digestive

tract, prostate and melanoma cancer

© RCSB Protein Data Bank

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

Molecular heterogeneity of breast cancer has prognostic implication

Sorlie T., Proc. Natl. Acad Sci, 2001

Gene signatures

Oncotype DX 21 Gene Recurrence Score assay

Proliferation Ki67 STK15 Survivin Cyclin B1 MYBL3

Estrogen ER PR Bcl2 SCUBE2

Invasion Stromolysin 3 Cathepsin L2

HER2 GRB7 HER2

GSTM1

BAG2

CD68

Reference Beta-actin GAPDH RPLPO GUS TFRC

Category RS (0-100)

LOW RISK RS < 18

MODERATE RISK

RS 19 – 30

HIGH RISK RS ≥31

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

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

Next generation sequencing - methods

Next generation sequencing - methods

Next generation sequencing - applications

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, 448-456 (June 2004)

Factors affecting CTCs count

Mego M:Nature Reviews Clinical Oncology 7, 693-701 (December 2010).

EMT process • characterization of EMT ▫ epithelial cells:

lose cell-to-cell contacts lose cell polarity downregulate epithelial-associated genes acquire mesenchymal-gene expression undergo changes in their cytoskeleton

acquire mesenchymal appearance with increased motility and invasiveness

Iwatsuki, M: Cancer Science, 101: 293–299 (2010)

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, 693-701 (December 2010).

Why are we interested in CTC detection ?

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. (www.fluidigm.com) (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.

Trial population – number of enrolled patients

*419 tests have been done in total

Study flowchart

Circulating tumor cells testing

Study results • Patients characteristic´s

Total CTC-positive

Total 197

Tumor size

stadium 1 45 20 (22%)

stadium 2 80 40 (44%)

stadium 3 34 29 (39%)

stadium 4 11 2 (12%)

Nodal status

node-negative 76 24 (31%)

node-positive 111 39 (35%)

Histology

ductal 141 36 (25%)

lobular 10 2 (20%)

others 46 5 (10%)

Study results • Patients characteristic´s

Total CTC-positive

Grading

G1 15 9 (60%)

G2 54 16 (30%)

G3 84 27 (32%)

unknown 45 ---

ER status

ER-negative 118 34 (29%)

ER-positive 64 21 (33%)

PR status

PR-negative 96 24 (26%)

PR-positive 83 27 (33%)

HER-2 status

HER2-positive 42 12 (29%)

HER2-negative 134 37 (28%)

Triple negative 44 14 (32%)

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%.

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.

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

HER2 assessment in circulating tumor cells (CTC) in patients with first relapse of HER2-negative breast cancer – ML 25147

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

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.

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

HER2 signaling pathway

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

Inclusion and exclusion criteria INCLUSION CRITERIA • signed written informed consent • female patients aged ≥ 18 • Histologicaly confirmed HER2

negative primary tumor • first documented relapse (loco-

regional relapse and/or metastatic disease)

• clinically indicated for biopsy of

the metastasis or loco-regional recurrance

EXCLUSION CRITERIA • previous treatment for breast

cancer relapse • HER2 positive primary disease • contralateral breast cancer

Study flowchart

 

Breast cancer patient with first relaps 

 primary tumor HER2‐ 

Excluded from study NO

YES [80%]

Included in study 

CTC +  CTC ‐ 

YES [50%]

HER2+[29%] HER2-[71%]

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

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

Study preliminary results Total Total

Total 19*

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

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)

Take home messages • There is an urgent need for biomarkers for real-

time 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.