Culture and Characterization of Circulating Tumor Cells (CTC) From Patients With Malignant Melanoma and Other Cancers
A strong correlation has been established between CTC and the progression of breast, colon
and prostate cancers. The number of CTC appears to act as a prognostic marker for relapse
and survival in a number of epithelial cancers. Unfortunately, current CTC assays are not
effective in reliably detecting circulating melanoma cells and the assay procedures damage
cells prohibiting further study.
This study is designed to evaluate the sensitivity and specificity of the TrueCells assay, a
novel approach to isolate and preserve cells for further research.
- Evaluate feasibility and reproducibility of isolation of CTC using TrueCells
- Optimize quantitative CTC recovery
- Provide preliminary data concerning correlation of these measurements with other
methods of CTC recovery such as immunofluorescence, lipid-based staining.
- Explore correlation of immunofluorescent CTC assay and TrueCells CTC cultures with
response to treatment by objective and immune response criteria, progression free
survival, 1 and 2-year survival landmarks, and overall survival in cancer patients.
- Attempt to isolate long-term tumor cell lines for evaluation of somatic gene mutations,
and gene and protein expression patterns related to individual tumors (not intended to
identify familial or inherited mutations), and to test drug and biologic sensitivity.
- Isolate plasma RNA and DNA to assess expression tumor-specific markers (e.g.,
tyrosinase, B-FRAF, V600E, etc.) and metastasis-associated genes (e.g., MSH-1,
thymidylate synthase, etc.).
- Perform ongoing developmental testing to continue to develop and optimize novel
technical approaches to facilitate isolation of intact CTC.
Blood samples will be collected from patients with melanoma (200), prostate cancer (200),
other solid tumor cancers (up to 400), benign hematologic conditions (100), and healthy
volunteers (up to 100). Sequential samples will be obtained from selected patients during
disease progression or treatment response to allow exploration of potential correlation of
CTC with response to treatment, progression-free survival and overall survival. Samples will
be immediately de-identified and assigned a numerical code, dated and sent to TrueCells,
LLC. Once donated, sample ownership passes to TrueCells, LLC.
Research studies include:
- CTC independently counted using immunofluorescent staining of thick smear prepared from
entire buffy coat of two tubes of blood (tubes 1 & 2), stained with appropriate primary
pan-tumor monoclonal antibody or lipid markers with confirmatory secondary antibodies
to exclude inadvertent inclusion of leukocyte and endothelial subsets and verify
recognition of the patient's cancer.
- Leukocytes (containing CTC) isolated by density gradient centrifugation. The buffy coat
is plated into TrueCells proprietary cell culture medium and tumor cells grown and
propagated (tubes 3 & 4). Cell colonies enumerated and compared to number of colonies
identified by immunofluorescent staining.
- In some cases CTC may be able to be grown as long-term cell lines. These cells may be
subject to further analysis such as evaluation of somatic gene mutations as well as
gene and protein expression patterns to identify characteristics of individual tumors
as well as drug and biologic agent sensitivity. There may be additional future in vitro
experimental applications for these de-identified early-passage cell lines that have
not yet been identified or anticipated in this application.
- Plasma (byproduct of leukocyte isolation process from tubes 3 & 4) will be collected
and temporarily frozen for DNA and RNA isolation for molecular assays of melanoma
molecular markers and metastatic gene expression. These samples are consumed in the
Clinical information will be abstracted from medical records and de-identified and stored in
a password-protected spreadsheet. Information to be collected includes age, gender, T, N, M
status, other health conditions, serum LDH, any specific tumor markers (e.g. PSA),
circulating tumor cells (if analyzed by commercial assay), somatic genetic mutations present
in cancer (e.g. B-RAF V600E in melanoma patients) mitotic rate of tumor, date of tumor
diagnosis, treatment history, date of regional and metastatic progression and date of death
Observational Model: Case Control, Time Perspective: Prospective
Circulating tumor cell (CTC) isolation and colony counts
Wolfram Samlowski, MD
Comprehensive Cancer Centers of Nevada
United States: Institutional Review Board
|Comprehensive Cancer Centers of Nevada||Las Vegas, Nevada 89109|