ASCO 2015 maintrac® circulating tumor cells

S

Vemurafenib to eliminate BRAF-mutated circulating

melanoma tumor cells from blood of patients with malignant melanoma

D.Zimon, M.Pizon, E.L.Stein, U.Pachmann, K.Pachmann Transfusion Center Bayreuth, Germany Email: dzimon@simfo.de

Background:

Almost 50 % of melanomas harbor mutations in BRAF, mainly V600E. The mutations are usually identified in the primary tumor. However, the primary tumor is often no longer available and metastases not always reflect the characteristics of the primary tumor. In melanoma it is not clear at what stage epithelial antigen is expressed. CETCs are present in peripheral blood in a significant proportion of patients with malignant melanoma. The aim of the present study was to analyze whether these cells belong to melanoma clone due to the presence of the BRAF gene mutation in these cells. For this reason, the analysis of multiple isolated CETCs from individual patients for BRAF gene mutations was performed.

Methods:

Blood from patients with malignant melanoma was analyzed for cells positive for EpCAM and Melan A using the maintrac® approach, avoiding cell selection and using an image analysis system for detection. Subsequently, between 8-20 EpCAM and Melan A positive cells from each patient were isolated individually using a semi-automated capillary approach and deposited one by one into micro cups. The DNA of individual cells was amplified by whole genome amplification and assayed using the cobas® BRAF V600 mutation test. Furthermore, we performed mutation analysis of cells after magnetic bead enrichment which is known to contain a mixture of CETCs and leukocytes.

Semi-automated capillary device

S  Semi-automated capillary device for aspiration of individual cells from cell suspensions (slides in the front) and deposition in individual cups (slides in the background)

Methods (1):

S  Blood samples (2-7 ml) from patients with malignant melanoma were drawn into normal blood count tubes with EDTA as an anticoagulant. For determining the number of circulating epithelial tumor cells (CETCs), tumor cells were stained with FITC-conjugated epithelial cell adhesion molecule antibody (EpCAM) and PE-conjugated Melan A antibody and enumerated using image analysis in a Scan-R microscope. Circulating melanoma cells were defined as EpCAM and Melan A positive cells with intact morphology.

Procedure of Single Cell Isolation

Methods (2):

S  In a further step 8-20 vital cells stained with EpCAM and Melan A among the white blood cells were selected individually using a semi-automated capillary approach (CellEctor Plus, MMI) and deposited one by one into micro cups. For mutation analysis the DNA was subsequently amplified using REPLI-g® Single Cell Kit (Qiagen) and assayed using the cobas® BRAF V600 Mutation Test Kit (Roche) and cobas z 480 Light Cycler (Roche).

Results:

S  DNA could be amplified from all individually isolated cells. In addition, we analyzed the presence of V600 mutation after magnetic bead enrichment. A BRAF mutation was detected in 20-75 % of evaluable cells in patients with BRAF mutation in primary tumor. In advanced stage or metastatic patients under Vemurafenib therapy, we were not able to find mutated CETCs.

Figure 1 Illustrative pictures of double antibody staining

for EpCAM (green) and Melan A (red)

S  BRAF mutation analysis was performed in patients with metastatic disease

S  Number of CETCs ranged from 20 – 310/100 μl of cell suspension

S  Double antibody staining enabled identification of three subpopulations of circulating melanoma cells:

S  only EpCAM positive cells (mean value 23 %)

S  only Melan A positive cells (mean value 7 %)

S  EpCAM and Melan A positive cells (mean value 70 %, Figure 1)

S  In spiking experiments 250 mutated cells among 160 000 white blood cells were sufficient to detect the respective mutation (1.56 %) (Table 1)

S  In all examined patients with BRAF-mutated primary tumor, we detected also BRAF-mutated circulating melanoma cells with a high heterogeneity in individual patients

S  In contrast, we were not able to detect BRAF-mutated CETCs in patients under Vemurafenib treatment

Table 1

Number of spiked tumor cells (%)

BRAF mutation result

16 000 (100) Mutation detected

12 000 (75) Mutation detected

8 000 (50) Mutation detected

4 000 (25) Mutation detected

2 000 (12.5) Mutation detected

1 000 (6.25) Mutation detected

500 (3.125) Mutation detected

250 (1.56) Mutation detected

125 (0,78) Mutation not detected

Spiking experiments

Table 2 Exemplary patient and control results

Number of isolated cells with wild type

(%)

Number of isolated cells with detected mutation

(%)

Invalid samples (%)

Lymphocytes (negative control): • 4 isolated cells 4/4 (100) - -

SK-MEL-28 cell line (positive control): • 4 isolated cells - 4/4 (100) -

Patient 1 (without Vemurafenib treatment): • 8 isolated cells • MACS

3/8 (37.5)

3/8 (37.5 mutant

2/8 (25)

Patient 2 (under Vemurafenib treatment): • 4 isolated cells • MACS

4/4 (100) wild type

-

-

Patient 3 (without Vemurafenib treatment): • 8 isolated cells • MACS

3/8 (37.5)

5/8 (62.5) mutant

-

Patient 4 (under Vemurafenib treatment): • 6 isolated cells • MACS

5/6 (83.3)

-

1/6 (16.7)

Conclusion

S  Individually isolated CETCs from the peripheral blood from patients with melanoma allow not only to detect mutations but also to determine the frequency of mutated cells. This proves that at least part of the CETCs is originated from the primary tumor. Furthermore, detection of BRAF mutation in CETCs may be crucial for a successful molecular-targeted therapy.

Transfusion Center Bayreuth - TZB

SIMFO Specialized Immunology Research + Development GmbH & Laboratory Dr. Ulrich Pachmann

Laboratory Dr. Pachmann Kurpromenade 2 95448 Bayreuth Germany

www.maintrac.com