Pancreatic cancer: Circulating Tumor Cells and Primary Tumors show Heterogeneous KRAS Mutations

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Circulating tumor cells (CTC) in the blood are hypothesized as the means of systemic tumor spread. Blood obtained from healthy donors and patients with PDAC was therefore subject to size-based CTC-isolation. We additionally compared Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in pancreatic CTC and corresponding tumors, and evaluated their significance as prognostic markers. Samples from 68 individuals (58 PDAC patients, 10 healthy donors) were analyzed; CTCs were present in patients with UICC stage IA-IV tumors and none of the controls (p < 0.001). Patients with >3 CTC/ml had a trend for worse median overall survival (OS) than patients with 0.3–3 CTC/ml (P = 0.12). Surprisingly, CTCs harbored various KRAS mutations in codon 12 and 13. Patients with a KRAS G12V mutation in their CTC (n = 14) had a trend to better median OS (24.5 months) compared to patients with other (10 months), or no detectable KRAS mutations (8 months; P = 0.04). KRAS mutations in CTC and corresponding tumor were discordant in 11 of 26 “tumor-CTC-pairs” (42%), while 15 (58%) had a matching mutation; survival was similar in both groups (P = 0.36). Genetic characterization, including mutations such as KRAS, may prove useful for prognosis and understanding of tumor biology.

Figure 1

(a) Isolation method of CTC. White arrows point at filter pores, black arrows at “CTC” grey arrow points at the metal rim of the filter. Examples of CTC in patients with PDAC. (b) CTC cluster (patient 24), the scale bar represents 50 µm C single CTC (patient 23) the scale bar represents 10 µm. Arrows point at CTC, arrowheads at filter pores.

Figure 2

CTC and overall survival. (a) Survival analysis of PDAC patients with 0.3–3 CTC in the blood (blue, N = 23, MS 20 months) versus those with >3 CTC/ml blood (red dots, N = 16, MS 11.5 months; P = 0.12). (b) Survival analysis of PDAC patients with CTC containing a KRAS ^G12V mutation (MS 24.5 months) and other KRAS mutations (MS 10 months; P = 0.21).

Figure 3

(a) Example Chromatogram (antisense strand) wild-type KRAS in a negative control sample. (b) Example Chromatogram (antisense strand) KRAS c.35G > A (pG12D) in a CTC sample. (c) Example ddPCR result (Patient 45) for KRAS ^G12D. The number of events is very low which represents a very low copy of mutant KRAS ^G12D molecules in the specimen. The circle encircles the area of interest and the approximately 6 events.

Figure 4

Distribution of KRAS mutations in CTC and the primary tumor. (a) KRAS mutations in CTC and the primary tumor: Several patients (n = 13) had multiple mutations in the CTC or primary tumor, in 20 cases no tissue was available. (b) Substantial discordance of KRAS mutations in CTC and the primary tumor (n = 9): those with no available tissue samples were excluded (n = 20); 5 were CTC negative. Cyto pos = cytology positive; MS = median survival (in months).