The Pilot Study "Metastatic renal cancer: CTC determination in first-line Sunitinib
treated patients (CTC/Sun)", IOV-IRCCS, Padova (Italy)
The purpose of this pilot study was to investigate the feasibility of an automated
immunocytometrical assay to quantify CTC in patients affected by mRCC receiving firstline Sunitinib. Considering the cost of the CTC assay and the little already known about
this new method in renal cancer (prognosticator) and in the antiangiogenic treatment
(predictor) [1] the proposed sample size was fifty patients.
CTC/Sun inclusion criteria were advanced/metastatic renal cancer, any hystotype
excluding collecting duct carcinoma (CDC) and urothelial carcinoma (UC), measurable or
not measurable, candidate to Sunitinib administration according to Italian Drug Agency
[Agenzia Italiana del FArmaco (AIFA)] indication. Exclusion criteria included CDC and UC
hystotype,
prior
sunitinib,
sorafenib,
bevacizumab,
interferon,
interleukin-2
and
chemotherapy, lack of consensus to undergo multiple CTC tests.
Primary objectives of the CTC/Sun study were: (a) to quantify total and apoptotic CTC by
the CellSearch system, (b) to demonstrate whether their number is influenced by the
delivered treatment and (c) to identify progression earlier than using traditional
instrumental investigations. Synchronous CTC and CEC enumeration were compared.
Serial blood samples were collected at day 1 and 28 of the first two cycles, and
sequentially at day 1 of the III, V and VII cycles of treatment or at disease progression, for
a total of eight additional evaluations. Radiographic studies were performed at I and III
cycles as well as at progression. On disease progression, all patients were offered the
opportunity to continue CTC testing with future therapy. CTC results were provided to
clinicians and patients with the understanding that decisions regarding patient care would
be based on standard clinical and radiographic evaluations. The study was independently
designed by the lead investigator (R.Z.).
Immunohistochemistry (IHC)
Overall, forty-five formalin-fixed, paraffin-embedded tissue samples obtained from 34
patients with metastatic renal cancers (primary = 28 cases; metastatic = 17 cases) were
considered. The expression of EpCam was immunohystochemical assessed by applying
monoclonal antibodies against EpCam antigen (clone MOC-31; Cell Marque, Rocklin, CAUSA). The immunostaining was automatically performed (Ventana Benchmark XT system,
Touchstone, AZ) [20] according to the manufacturer’s instructions. Appropriate positive
and negative controls were run concurrently. In primary tumor samples, normal renal
parenchyma (i.e. distal tubules and collecting ducts) served as internal positive control.
EpCam expression was jointly scored by two pathologists (M.R. & M.F.), according to
previous experiences with minor modifications [14]. Only positive membranous
immunostaining was considered and semi-quantitatively scored in a four-tier scale which
combined intensity (score-0, score-1, score-2, and score-3) and prevalence (%) of positive
immunostaining. Results were grouped as following: Negative= no stain; Weak= score-1 in
< 60%, or score-2 in < 30% of tumor cells; Moderate= score-1 in ≥ 60%, or score-2 in
30%-80%, or score-3 in <30% of tumor cells; Strong= score-2 in ≥ 80%, or score-3 in ≥
30% of tumor cells (no “Strong” cases were faced in the present series).
References
[1] Peter Bacchetti, Charles E. McCulloch, Mark R. Segal, Richard Simon, Peter Muller,
Gary L. Rosner, James A. Hanley, and Stan Shapiro, "Simple, Defensible Sample Sizes
Based on Cost Efficiency -- With Discussion and Rejoinder" (June 2009). COBRA Preprint
Series. Article 55.
http://biostats.bepress.com/cobra/ps/art55