Comparison of Kras Mutation Status Between Primary Tumors and Circulating Tumor Cells in Patients with Metastatic Colorectal Cancer (MCRC)
Authors: Aristea Kalikaki, Helen Politaki, Maria Tzardi, Elisavet Papadimitraki, Demitris Mavroudis, John Souglakos, Alexandra Voutsina, Vassilis Georgoulias
Journal: Annals of Oncology (2013)

Background: In metastatic colorectal cancer (mCRC), circulating tumor cell (CTC) analysis could represent a novel biopsy more representative of the current disease state and might be used as a biomarker of either sensitivity or acquired resistance to EGFR targeted therapy. The primary objective of this study was to investigate the feasibility of detecting KRAS mutations in CTC-enriched fractions isolated by the CellSearch system in patients with mCRC. Secondary objectives were to evaluate whether KRAS mutation status of CTCs correlates with that of corresponding primary tumors and examine the genetic heterogeneity of CTCs in respect to KRAS mutation status during treatment.Methods: Detection of the seven more common (codons 12 and 13) KRAS mutations was performed by the combination of the peptide nucleic acid (PNA) with allelic specific PCR in DNA isolated from the recovered cells following CTC enumeration using the CellSearch system. The technical feasibility to determine accurately the KRAS mutation status from a limited number of CTCs was validated by spiking tumor cells into healthy donors' blood. Consistent detection of KRAS mutations was achieved from samples containing an input of 20 cells.Results: A total of 48 blood samples were obtained from 17 patients with wild type and 14 with mutant KRAS primary tumors. All patients had primary tumors wild type for PIK3CA and BRAF. In 19 (61%) patients, blood samples were available from one time point and in 12 (39%) from at least 2 time points. Thirteen (42%) patients were chemotherapy-naïve at the time of first blood draw. Thirteen (27%) blood samples were collected prior to, 22 (46%) during the first-line chemotherapy, 10 (21%) at the time of disease progression on first-line treatment and 3 (6%) during the second-line chemotherapy. One or more CTCs were detected in 23 of 48 (48%) blood samples obtained from 15 of 31 (48%) patients; the average number of CTCs per 7.5 ml of blood was 96 (range 1-865). KRAS mutations were detected in 5 of the 13 (38%) CTC positive samples obtained from 3 of the 7 (43%) patients with KRAS mutant primary tumors and in 3 of the 10 (30%) CTC positive samples obtained from 2 of the 8 (25%) patients with KRAS wild type primary tumors. Analysis of serial blood samples revealed that the KRAS mutational status of CTCs may change during treatment and may be different from that of the corresponding primary tumors irrespective of treatment administration.Conclusion: These findings support the rationale for reassessing KRAS status on CTCs in order to predict more accurately, the response and evaluate the secondary resistance to anti-EGFR therapy of metastatic colorectal cancer patients. Background: In metastatic colorectal cancer (mCRC), circulating tumor cell (CTC) analysis could represent a novel biopsy more representative of the current disease state and might be used as a biomarker of either sensitivity or acquired resistance to EGFR targeted therapy. The primary objective of this study was to investigate the feasibility of detecting KRAS mutations in CTC-enriched fractions isolated by the CellSearch system in patients with mCRC. Secondary objectives were to evaluate whether KRAS mutation status of CTCs correlates with that of corresponding primary tumors and examine the genetic heterogeneity of CTCs in respect to KRAS mutation status during treatment. Methods: Detection of the seven more common (codons 12 and 13) KRAS mutations was performed by the combination of the peptide nucleic acid (PNA) with allelic specific PCR in DNA isolated from the recovered cells following CTC enumeration using the CellSearch system. The technical feasibility to determine accurately the KRAS mutation status from a limited number of CTCs was validated by spiking tumor cells into healthy donors' blood. Consistent detection of KRAS mutations was achieved from samples containing an input of 20 cells. Results: A total of 48 blood samples were obtained from 17 patients with wild type and 14 with mutant KRAS primary tumors. All patients had primary tumors wild type for PIK3CA and BRAF. In 19 (61%) patients, blood samples were available from one time point and in 12 (39%) from at least 2 time points. Thirteen (42%) patients were chemotherapy-naïve at the time of first blood draw. Thirteen (27%) blood samples were collected prior to, 22 (46%) during the first-line chemotherapy, 10 (21%) at the time of disease progression on first-line treatment and 3 (6%) during the second-line chemotherapy. One or more CTCs were detected in 23 of 48 (48%) blood samples obtained from 15 of 31 (48%) patients; the average number of CTCs per 7.5 ml of blood was 96 (range 1-865). KRAS mutations were detected in 5 of the 13 (38%) CTC positive samples obtained from 3 of the 7 (43%) patients with KRAS mutant primary tumors and in 3 of the 10 (30%) CTC positive samples obtained from 2 of the 8 (25%) patients with KRAS wild type primary tumors. Analysis of serial blood samples revealed that the KRAS mutational status of CTCs may change during treatment and may be different from that of the corresponding primary tumors irrespective of treatment administration. Conclusion: These findings support the rationale for reassessing KRAS status on CTCs in order to predict more accurately, the response and evaluate the secondary resistance to anti-EGFR therapy of metastatic colorectal cancer patients.