Determining Tumor Load and Biallelic Mutation in Patients with CALR Mutation Using Peripheral Blood Plasma

Background: CALR gene is frequently mutated in patients with myeloproliferative neoplasmas (MPN). Almost all mutations are indel - some with large (>50 bp) deletion. Detecting this type of mutations with acceptable sensitivity is difficult by sequencing. Fragment length analysis (FLA) is a reliable technique in detecting this type of mutations. Furthermore, FLA allows quantifying the mutant DNA and better evaluation of tumor load. Determining tumor load can be confusing and difficult when the mutation is biallelic. Distinguishing between patients with single allele mutation from those with biallelic mutations might add another dimension in predicting clinical behavior and determining the tumor load. We explored using cell free DNA in peripheral blood plasma to test for CALR mutations and determining tumor load.

Methods: Using Direct bidirectional sequencing and FLA, we detected CALR indel mutations in 71 of 522 (14%) patients suspected of having MPN and referred for testing for CALR mutation. No sample showed point mutation. DNA from cells and cell free DNA in plasma was available from 31 of the 71 cases. The mutant DNA peak was quantified and the relative percentage of mutant DNA was calculated in both cellular DNA and cell free plasma DNA. FLA and Sanger sequencing data were compared between cellular DNA and cell free plasma DNA. Any ratio >55% was considered as evidence of biallelic mutation.

Results: As expected all positive samples by cellular DNA testing also showed the indel mutation in cell free plasma by FLA. However, four of the 31 (13%) positive samples by FLA failed to show the mutation on Sanger sequencing. The most likely cause for failing to detect the mutation by Sanger is low level mutation load and the lower sensitivity of Sanger sequencing. When we compared ratios of mutant peak (tumor load) between cellular DNA and cell free DNA in plasma, mutant CALR DNA was significantly higher (P=0.0002, Wilcoxon matched pairs test) in cell free DNA in plasma than in cellular DNA. More importantly, we were able to determine the presence of homozygous mutation (>55% mutant DNA) in 5 of 31 (16%) patients when cell free DNA in plasma is used. In contrast, only 1 of 31 (3%) patients showed evidence of biallelic mutation when cellular DNA is used. Most of the mutations (25 of 31, 81%) were deletions. As deletions results in smaller size amplicon on the FLA and better amplification efficiency, we set 55% as a cut-off for biallelic mutation to account for the more efficient amplification of the deleted peak.

Conclusion: Cell free DNA in plasma is more reliable than cellular DNA for the detection of CALR mutations and for determining tumor load. Testing for CALR must include fragment length analysis. More importantly, biallelic CALR mutation is frequent and the clinical relevance of biallelic CALR mutation needs to be investigated.