New Tools For Cancer Diagnostics: Circulating Tumor Cells and Novel Molecular Targets

SvetIana Pack, PhD Chromosome Pathology Unit

Primary Research Areas

IDENTIFYING NOVEL MOLECULAR DRIVERS AS THERAPEUTIC TARGETS IN PEDIATRIC CANCERS: Sarcoma - Ewings, Rhabdomyosarcoma (RMS), and GIST
Glioma - Diffuse Intrinsic Pontine Glioma, DIPG

DEVELOPMENT OF NOVEL CLINICAL DIAGNOSTICS:

Circulating Tumor Cells (CTCs)
Future non-invasive tool to diagnose, study and treat metastatic cancer. Development of a quick diagnostic method for isolation of CTCs in the of patients blood for cancer diagnosis, tumor profiling for targeted therapy selection, residual disease monitoring. (Lung and breast cancer as model systems)

Automated Methods for decoding cell morphology from microscopy images with applications to pathology and cytology.
High-precision H&E image segmentation and new mathematical and statistical data analysis methods can be used to automatically differentiate and grade malignancies based on nuclear structure (size, shape, chromatin distribution, as well as other cellular organelles and stains).

Molecular Beacons a method for a quick, high-sensitive allele-specific mutation and RNA
expression analyses. It can be combined with DNA FISH to simultaneously detect DNA Copy Number Variations (CNV), gene rearrangements and RNA expression.

Example 1: Isolation and Characterization of Circulating Tumor Cells

HER2
CEP17 FISH

Control

SKBR3 Breast Cancer Cells in donor blood (500 cells in 5ml whole peripheral blood healthy donor)

Lung Cancer, NSCLC

ALK BA FISH

UroVysion FISH

negative
Isolation of CTCs by size exclusion from patient peripheral blood by CellSieve TM microfilters with dense, uniform 8m pores.

Example 2: Automated Methods for tumor diagnosis, staging, prognosis:

Breast- Ductal Carcinoma In Situ vs Intraductal Carcinoma with early infiltrate Class D
IDC

H&E Class C
DCIS

Classification results

For alpha = 0.0025 (visualizations next slide) Predicted Class C vs. Class D Class C Class D Class C 11 0 Actual Class D 0 8 Accuracy 100%

The new methods can be effective in automatically uncovering hidden and visually interpretable differences in cell morphology between disease states. These methods could be used to grade malignancies based on nuclear structure as well as other cellular organelles and stains.

Research Implications
Characterization of CTC
Non-invasive fast and effective method for assessment of metastatic disease Finding best assay for characterization of isolated CTC DNA FISH (gene rearrangements, deletions, amplifications-tumor fingerprinting); PCR, sequencing mutation analysis RNA ViewRNA (Affymetrix), Molecular Beacons for gene expression Protein immunohistochemistry, mass-spectrometry

Insight into mechanisms of metastasis

detect differential molecular markers expressed in a primary tumor vs metastatic cells (CTC)

Clinical application Targeted therapy for cancer patients

patient-specific detection of the molecular targets for treatment decision
development of better tools to monitor tumor residual disease and metastasis in most frequent types of cancer. CTC detection assay can be a strong prognostic marker for overall survival in patients with metastatic disease.

Future Direction
Clinical Application
Development of clinical assay for detection of CTCs New diagnostics development that combine DNA and RNA analyses

Basic Research
Production of patient-specific metastatic tumors from CTCs in mice (Xenograft assay) Therapeutic Target/Drug validation on xenografts

What are the current challenges & obstacles? Availability of the clinical material acquired through NIH protocol
(identify barriers and limitations beyond time, money, people, travel, and space)

Collaborators
Laboratory of Pathology - CCR
Frederic Barr, M.D., Ph.D. Stephen Hewitt, M.D., Ph.D. Elaine Jaffe, M.D. Maria Merino, M.D. Markku Miettinen, M.D. Carl Oberholtzer, M.D., Ph.D. Martha Quezado, M.D. Mark Raffeld, M.D. Maria Tsokos, M.D.

Georgetown University
Giuseppe Giaccone, MD

Carnegie Mellon University

Gustavo Rhode, Ph.D.

NCI, CCR
Udayan Guha, Medical Oncology Branch, CCR Marston Linehan, Urologic Oncology Branch, CCR Lauren Wood, Vaccine Branch, CCR Alexander Gorbach, Ph.D.

NIBIB