A New Valuable Technique in Diagnosis and Treatment Monitoring of Myeloproliferative Neoplasms

Introduction: Tumor-derived fragmentary DNA, known as circulating tumor DNA (ctDNA), is applied in the diagnosis and treatment of solid tumors maturely now, and also great progress has been achieved in the application of ctDNA to hematological malignancies such as lymphoma and multiple myeloma in recent years, which is considered to be important in the diagnosis, prognosis and treatment monitoring of diseases. The diagnosis and prognosis of myeloproliferative neoplasms (MPNs) depend on molecular biology examination. Mutational characterization of MPNs currently depend on bone marrow (BM) aspirates, which is an invasive technique. CtDNA has replaced bone marrow biopsy as a new blood-based biomarker for early diagnosis and treatment monitoring of lymphoma and multiple myeloma, which is non-invasive and easily accessible. In this study, we aimed to prove the mutational status of MPNs patients by using ctDNA and using BM cell DNA as control. We tried to assess whether ctDNA could replace BM cell DNA.

Methods: Paired BM cell DNA and ctDNA were collected from 19 MPNs patients with JAK2V617F mutation in Department of Hematology, Zhongnan Hospital of Wuhan University from September 30, 2018 to June 13, 2019 . All patients gave a written informed conset for sample collection and analysis.We analysed samples for JAK2V617F mutation that is the driven mutation of MPNs by droplet digital polymerase chain reaction (DDPCR) which can achieve absolute quantification. BM cell DNA was extracted from bone marrow cells and ctDNA was obtained from peripheral blood plasma. All these patients met the 2016 WHO diagnosed criteria and was positive for JAK2V617F by Sanger Squencing. Mutation rates of JAK2V617F respectively from paired BM cells DNA and ctDNA detected by DDPCR were analysed using SPSS21.0, then the relevance between them was determined.Limit of detection for DDPCR was 0.01%.

Results: The present study enrolled 19 patients (6 males, 13 females), with 13 first visit and 6 no first visit. The subtypes included polycythemia vera (PV, N=7), essential thrombocythemia (ET, N=10) and primary myelofibrosis (PMF, N=2). The median age of the cohort was 58.5 years (range 32-74). The median hemoglobin in PV patients was 194.7g/L (range116.6-211.4g/L). The median number of platelets in ET patients was 687x10⁹/L (range 240-1304x10⁹/L). The median number of white blood cells in PMF patients was 6.3x10⁹/L (range 2.6- 10.0x10⁹/L). All patients were detected positive for JAK2V617F mutation both in BM cell DNA and ctDNA by DDPCR. The mean mutated allele frequency of JAK2V617F was 50.2%+27.4% (range 0.13-97.4%) in ctDNA and 47.0%+27.4% (range 0.12-90.6%) in BM cell DNA. Moreover, there was a significant correlation between ctDNA and BM cell DNA (Pearson Correlation, r=0.959, p<0.001). One follow-up male patients with ET came to call back after 3 months' hydroxyurea treatment. He conducted Sanger Squencingfor JAK2V617F using PB cell DNA that indicated negative. CtDNA was also extracted from the same peripheral blood. But DDPCR examination detected for JAK2V617F using ctDNA showed 2.44% mutated allele frequency above the limit of detection. The initial mutation rate was 26% in ctDNA by DDPCR. His blood routine examination return back to normal: the count of platelets decreased from 675x10⁹/L when first visited to 344x10⁹/L.

Conclusion: CtDNA shows high sensitivity and a high degree of correlation with BM cell DNA . Therefore, ctDNA analysis may serve as an alternative to BM cell DNA . As ctDNA can be abtained by venous blood sampling which is non-invasive and convenient, ctDNA might be a newly valuable technique in diagnosis and treatment monitoring of MPNs.