Efficacy control of therapy using circulating epithelial tumor cells (CETC) as "liquid biopsy": trastuzumab in HER2/neu-positive breast carcinoma

Abstract

Purpose: The majority of targeted personalized cancer therapies are effective only in part of the patients, and most of these drugs are excessively expensive. Therefore, methods are urgently required, which reveal already early during treatment, whether the therapy is effective. In the present report, monitoring of circulating epithelial tumor cells (CETC) was used as a timely control of trastuzumab therapy in patients with HER2/neu-positive breast cancer.

Methods: Seventy-nine sequential HER2/neu-positive breast cancer patients, 35 without trastuzumab, and 36 treated with 1 year of trastuzumab treatment were included. CETC from unseparated white blood cells stained with FITC-anti-EpCAM were analyzed repeatedly during chemotherapy and between 2 and 10 times during 1 year of maintenance treatment or observation.

Results: Patients treated with trastuzumab had a better relapse-free survival than patients without trastuzumab treatment during the first 2-4 years of follow-up. Decrease in numbers or no change versus highly variable numbers or increase (fivefold or more) allowed to discriminate highly significantly and clearly (P < 0.0001, hazard ratio 5.5) between patients with a low or high risk of relapse. An increase in CETC was accompanied by an increasing portion of cells containing a very high number of HER2/neu gene amplificates.

Conclusions: Analysis of the behavior of CETC can, in the future, contribute to evaluate the efficacy of targeted therapy early during the course of the disease, sparing patients unnecessary treatment but also to reduce the costs for the health system and to downsize the extent and length of clinical studies.

Fig. 1

a Fluoromicrograph of a typical group of four epithelial antigen-positive cells (taken from Darb-Esfahani et al. 2009). Three of the cells display tiny caps, whereas the fourth exhibits strong staining, which was considered as intracellular staining. b Hematoxylin–eosin staining (HE) reveals that the intracellularly stained cell is a dying cell. In addition, a normal epithelial antigen-negative leukocyte is visible only in HE staining. c Fluoromicrograph of epithelial cells with green fluorescence, exclusively surface located including two vital epithelial antigen-positive cells with typical green caps (green arrows), adjacent to two faintly CD45-stained granulocytes (white arrows) and a bright CD45 stained vital lymphocyte (red arrow) in addition to a dead PI-positive cell (orange arrow). d Overlay of a fluoromicrograph with transmitted light. Note, the red 7AAD-positive fluorescing nucleus of a dead normal leukocyte in the neighborhood of the green fluorescing CETC and a red and green fluorescing dying CETC. e Two CETC from the same patient using the green and red filter, respectively. In both cells, the typical green cap of the EpCam, and in addition, the two green signals of the CEP17 alpha DNA probe can be identified. The upper cell carries only two HER2/neu signals, whereas in the lower cell more than 10 signals can be detected. f Fluoromicrograph overlay of the HER2/neu FISH analysis of a group of tumor cells from a breast cancer patient: besides the normal blood cells (nuclear stain was DAPI, only the nuclei are visible), one can see a group of tumor cells staining with typical green caps with the FITC-labeled anti-EpCAM. These cells carry a varying quantity of HER2/neu amplifications (pink-red dots in the cell nucleus)

Fig. 2

Results from 36 HER2/neu-positive patients treated with 1 year sequential trastuzumab after chemotherapy (green line) as compared to a historical patient sample of 35 patients not receiving trastuzumab therapy (red line)

Fig. 3

Typical courses of CETC numbers from 3 individual patients each. Values on the negative x-axis are cell values during chemotherapy, while on the positive x-axis, the values during trastuzumab therapy are plotted. a Typical courses for three individual patients during chemotherapy followed by 1 year of trastuzumab decrease under trastuzumab with decreasing CETC. b Typical courses of CETC in patients during chemotherapy followed by 1 year of trastuzumab with highly variable or increasing CETC during maintenance therapy with trastuzumab CETC. c, d Typical CETC courses in patients without trastuzumab treatment. All three of the patients with increasing CETC have already relapsed. e, f Two patients with decrease as well as increase in CETC during trastuzumab and increase or decrease in the fraction of cells with different amplificates of the HER2/neu gene. The fraction of cells with different amplificates of the HER2/neu gene is given on the left y-axis

Fig. 4

a Kaplan–Meier relapse-free survival curves for patients without trastuzumab (green line decreasing/stable CETC, red line increasing/variable CETC). b Kaplan–Meier relapse-free survival curves for patients with trastuzumab (green line decreasing/stable CETC, red line increasing/variable CETC). c Combined Kaplan–Meier relapse-free survival curves for patients with increasing and decreasing CETC in the untreated, the trastuzumab-treated and the primarily metastasized patients receiving trastuzumab together with chemotherapy (dashed lines patients without trastuzumab treatment; red line patients with increasing or variable CETC numbers; green line patients with decreasing or stable CETC numbers). Solid lines patients with trastuzumab treatment; red line patients with increasing or variable CETC numbers; green line patients with decreasing or stable CETC numbers. Dotted lines primarily metastasized patients; red line patients with increasing or variable CETC numbers; green line patients with decreasing or stable CETC numbers. Fat lines all patients combined independent of treatment; red line patients with increasing or variable CETC numbers; green line patients with decreasing or stable CETC numbers