Somatic third site Kras mutations in T-ALL. In cell lines E1 (A) and E3 (B), the single amino acid substitution T50I was present at ∼50% frequency based on relative abundance of sequence reads. It was detected at a similar frequency in the spleens of the secondary recipients used to generate these cell lines and in a single primary recipient. (C) The sequence GAGACC was inserted between amino acids 69 and 70 of Kras in T-ALL cell line Y4. This mutation is present in ∼50% of Kras transcripts in cell line Y4, at a lower frequency in the spleen of the secondary recipient used to generate this cell line, and is not seen in the primary leukemia.