Short tandem repeat genotyping using identifilerTM is a reproducible and cost-effective method for establishing and monitoring identity of pediatric cancer cell lines

403 Cell line identity is reportedly mistaken in an alarmingly high percentage of cases (16-30%). Short tandem repeat (STR) genotyping (microsatellite DNA fingerprinting) is the standard in forensics, theoretically providing unique profiles for every person on the planet. However, inherent genetic instability and/or selective pressures can sometimes cause slight alterations to STR profiles of cell lines, perhaps inhibiting wider use of the method in cancer research. We genotyped a large panel of pediatric cancer cell lines to assess the utility of STRs in identifying cell cultures and to determine when and to what degree microsatellite alterations (MSA) typically occur. We monitored costs and the occurrence of mistaken cell line identity to address the feasibility and necessity of the method. Polymerase chain reaction with the Applied Biosystems Ampf l STR TM Identifiler TM kit (16 loci amplified) was performed to obtain STR profiles for 124 cell lines we established and 51 lines from other investigators (175 total): 102 neuroblastoma (NB), 30 leukemia or lymphoma, 10 retinoblastoma, 10 Ewing’s family tumor (ESFT), 8 rhabdomyosarcoma, 5 brain tumor, 5 carcinoma, 1 prostate, and 4 EBV-transformed lymphoblastoid cell lines. We genotyped 525 total samples, including patient material. Comparison to patient tissue or cell lines from the same patient with separate establishment dates verified identity of 57 lines. All other lines showed unique profiles. Cell lines showed 1-3 MSA compared to blood cells from the same patients in 5 of 12 NB and 1 leukemia. We found no variation between cell lines established from different anatomic sites in the same patient at the same time in 5 NB and 1 rhabdomyosarcoma. Cell lines established after therapy and/or progression of disease showed MSA compared to lines established at diagnosis from the same patients in 5 of 8 NB, 1 leukemia, and 1 ESFT. Leukemia and ESFT lines showed greater MSA than NB lines. We also compared cell line profiles before and after in vitro manipulations. Two of 3 NB cell lines showed 1-2 MSA after selection for resistance to retinoic acid. Two of 5 NB lines showed 1-2 MSA after retroviral transfection and marker selection. Two of 4 NB lines showed 3 MSA in relation to corresponding EBV-transformed lymphoblastoid lines. Of 65 cell lines with 2 or more samples at different passages in vitro, 21 showed MSA in at least 1 sample; several lines showed high MSA. Despite MSA, unambiguous identification was achieved in all 525 samples. Cell line identity was mistaken in 9% of samples (36 cross-contaminated/mislabeled, 12 mixtures). Profiles of mixtures were composites of two known cell line profiles in all cases and were distinguishable from lines with high MSA. Genotyping with Identifiler TM costs ~$30/sample. In conclusion, Identifiler TM can be feasibly used to reliably prevent artifacts of misidentification.