Dynamic changes of live/apoptotic circulating tumour cells as predictive marker of response to sunitinib in metastatic renal cancer

Abstract

Background: Recently, we developed an apoptotic assay for expanding the monitoring capabilities of the circulating tumour cells (CTC) test during therapy. An automated platform for computing CTCs was integrated with a mAb (M30) targeting a neoepitope disclosed by caspase cleavage at cytokeratin 18 in early apoptosis; we showed that live CTCs were associated with progression, consistent with enhanced cell migration and invasion. The test was first applied here to mRCC.

Methods: Live/apoptotic CTCs changes were measured in mRCC patients receiving first-line Sunitinib and compared with circulating endothelial cell (CEC) levels.

Results: The presence of EpCAM-positive, live CTCs predicts progression in individual mRCC patient, being associated with distant metastasis under first-line Sunitinib. Synchronous detection of CTCs and CEC levels discloses for the first time an association between their dynamic changes and outcome: a rapid increase of the CEC number as early as the first cycle of therapy is associated with CTC decrease in non-progressed patients, whereas a delayed response of CECs is related to higher CTC values in the progressed group indicating treatment failure.

Conclusion: We demonstrated that a delayed response to antiangiogenic treatment indicated by persistent detection of CECs correlates with persistent live CTCs and more aggressive disease.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

EpCAM expression in mRCC primary tumours, metastasis and CTC. (A) EpCAM expression was analysed by IHC in primary tumours (T, n=28) and metastasis (M, n=17) obtained from 34 patients, for comparing IHC data with CTC results. The white bars represent the EpCAM-negative IHC in T (19 out of 28) and in M (10 out of 17) and patients that were CTC-negative by CellSearch assay (5 out of 34). The light brown bars represent weak staining in T (6 out of 9) and M (4 out of 7); the dark brown bars represent Moderate staining in T (3 out of 9) and M (3 out of 7). The light blue bar symbolises patients that had 1–4 CTC (20 out of 29), the dark blue bar symbolises patients that had 5–10 CTC (5 out of 29), the deep blue bar symbolises patients that had more than 10 CTC (4 out of 29). (B) The EpCAM staining profile is consistent in secondary lesions and synchronous CTC detection; one representative case of 4 is shown. The primary renal lesion (patient number 58) does not feature any EpCAM immunostaining (Original magnification 20X). (C) Characteristic EpCAM membranous immunoreaction appears in the tumour biopsy obtained from the surrenalic metastasis (patient number 58). (Original magnification × 20). (D) Analysis of three rare cells in baseline blood sample of patient number 58 using an Analyzer II device (Veridex, Raritan, NJ, USA). Horizontally, the photos show the same cell stained for the combination (Comp) of CK (green) and DAPI (violet); CK PE only; DAPI only; CD45 APC only; and M30 FITC only. The red squares indicate positively stained cells: events #1 and #7 are live CTC, exhibiting strong and irregular CK staining, respectively; in the photo #19 based on M30 staining profile (sufficient signal relative to background) the event on the right is classified as apoptotic CTC; the event on the left is a leukocyte (CD45-positive).

Figure 2

Progression-free survival (PFS) for patients with negative or equal 0 vs positive ΔAUC. Progression-free survival was measured as the time between the baseline CTC assessment and documentation of first radiographic evidence of disease progression or death. The ΔAUC criterion represents the difference between live and apoptotic CTC concentration-time area and was calculated in all patients according to the following formula:

Figure 3

CEC and CTC changes in mRCC patients under Sunitinib. (A) CEC and CTC values (mean +1 s.d., white and black symbols, respectively) synchronously determined in 30 mRCC patients receiving Sunitinib treatment are plotted in relation to time. (B) The bars represent the number of CEC and CTC samples (white and black symbols, respectively) collected at each time point. (C) CEC and CTC longitudinal graphs (white and black symbols, respectively) are separately plotted for non-progressed and progressed patients (○ and ∇ symbols, respectively). Time points with n ⩾14 were showed.