Validation of circulating tumor cells (CTC) detection techniques in patients with advanced solid tumors

The development of metastasis in cancer reduces prognosis, increases morbidity
and marks a change in the focus of treatment since this is no longer aimed at
curation but at palliation. The pathophysiology of metastasis therefore is the
subject of extensive research. The process of metastasis involves different
stages: outgrowth of the primary tumor, angiogenesis, intravasation of tumor
cells into circulation, extravasation of tumor cells at distant organs,
proliferation into micrometastasis and outgrowth of metastasis.
Although they are rare, intravasated circulating tumor cells (CTC) can be
identified in whole blood and bone marrow of a cancer patient. In prospective
trials of breast cancer patients, the number of CTCs at baseline was found to
be a predictor of progression-free survival and overall survival and an
increase in number of CTCs during treatment indicated a deterioration of
prognosis. CTC detection assays could be used to monitor efficacy of therapy
and to detect disease progression in an early stage. Currently, CTC detection
techniques are being developed to serve in the clinic to improve diagnostics
and to predict prognosis.
Because CTCs are rare, isolation is mainly based on combined enrichment and
subsequent detection of cell-specific markers by, for example,
immunocytochemistry or semi-quantitative RT-PCR. Current enrichment techniques
include both immunomagnetic enrichment and depletion, filtration by size and a
recently described adapted flow chamber. Quantification techniques include
immunocytochemistry, immunofluorescence and semi-quantitative RT-PCR.
For breast cancer, by group van*t Veer, already a highly sensitive and specific
assay for whole blood was developed and optimized. CTCs were found in 87,5% of
patients with advanced breast cancer. This assay uses an immunomagnetic column
with both ErbB2 and CD326 antibodies followed by semi-quantitative RT-PCR with
selected multiple marker genes.

In our opinion, CTCs could serve as a pharmacodynamic marker to monitor
anti-cancer therapy in clinical trials. Encouraging results, that show that
CTCs have pharmacodynamic properties, were recently found in a prospective
trial in which CTC number was studied in whole blood in patients with adjuvant
chemotherapy for breast cancer. An increase or decrease in CTC number after
starting chemotherapy was associated with respectively a worsened and improved
prognosis (Pachmann et al 2008).
Such biomarkers are very much needed in developing drugs to assess anti-tumor
activity in an early stage of treatment, to identify patients that could
benefit from treatment and to target proper dosing. For example, for specific
anti-angiogenic therapy, circulating endothelial cells (CECs) is being studied
as such in (Bertolini et al 2006).
The focus of this study is to further optimize and to validate enrichment and
detection techniques for CTCs in whole blood. For this, CTC assays need to be
optimized in a well defined but heterogenic group of patients with metastasized
solid tumors.

Observational, laboratory experimental.

Written informed consent will be obtained from all patients and normal
volunteers. For every selected type of tumor, we will investigate a group of a
minimum of 5 patients and a maximum of 20 patients that are being treated at
the NKI-AVL. Normal volunteers will serve as control group.
From every patient, per visit, 3x8 ml of whole blood will be drawn
simultaneously. With these samples, 2 different techniques will be compared
(2x8 ml). The third (1x8 ml) will serve as duplicate or, in later analysis, for
storage analysis.
From every patient, sampling will be repeated with a total maximum of 6 times.
The inclusion period will be the same as the overall study time. This will be 5
months.

The burden of sampling includes veni-puncture under the skin and could consist
of well-known side-effects: discomfort, bruising and hematoma, infection.