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 first-line 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, CA-USA). 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