Revisiting Early Stages of Chronic Myeloid Leukemia (CML): Evidence That Leukemic Progenitors Appear First in Peripheral Blood during Molecular Recurrence

The use of tyrosine kinase inhibitors (TKI) has dramatically changed the natural history and the prognosis of CML with a major improvement of survival in patients responding to therapy. However, tyrosine kinase inhibitor (TKI) discontinuation trials in patients with deep molecular responses (DMR), show that 50-60% of patients will have a molecular recurrence requiring to resume the TKI therapy. The great majority of these relapses occur during the first 6 months of TKI discontinuation. Although the sustained persistence of leukemic stem cells (LSC) is clearly the cause of the recurrent disease, cellular dynamics of these early relapses and the pattern of cells giving rise to them are not determined. We had the opportunity to study this pattern in 4 patients (pts) who presented molecular recurrence after Imatinib mesylate (IM) discontinuation (n= 3) or on TKI therapy with Dasatinib (n= 1) after obtaining a deep molecular response de grade MR 4.5. The total duration of TKI therapy before TKI discontinuation was 14 years (UPN1), 15 years (UPN2) and 30 months (UPN3). All three pts (Aged 47, 66, 87 respectively) had CML-CP treated with IM 400 mg as a first line therapy with induction of DMR in all three. The duration of DMR (MR4.5- was for 6, 5 and 2 years for UPN1, 2 and 3 respectively. The reasons for TKI discontinuation were the TKI-discontinuation protocol in French observatory (UPN1, UPN2) and suspicion of interstitial pneumonia (UPN3). The patient UPN4 was on Dasatinib as a second line therapy and in DMR for 3 years. At the time of detection of molecular recurrence, all 4 pts had normal clinical examination with normal complete blood counts (CBC) with no evidence of circulating immature blood cells. After IM-discontinuation, molecular relapses occurred at 2 months (BCR-ABL IS 27%) 3 months (BCR-ABL IS transcript level at 13.1%) and at 9 months (BCR-ABL IS transcript level at 2.7%.) in patients UPN1, 2 and 3 respectively. In UPN 4, the molecular recurrence (BCR-ABL IS transcript 10%) was documented at + 4 months. In all 4 patients, colony forming cell (CFC) assays were performed from the same blood samples in which the molecular recurrence was documented. At day + 14, clonogenic cells (CFU-GM, BFU-E, CFU-Mixed) were plucked and BCR-ABL expression in each individual or pooled (2-4) CFC was determined. For 5.10⁴PBMC plated in methycellulose assays in culture, the mean numbers of CFC obtained varied between 12 and 29 CFC / dish. Despite the normal CBC, the presence of BCR-ABL-expressing CFC in blood was demonstrated in all 4 pts. In UPN1, all CFC (20/20) obtained from peripheral blood were positive for BCR-ABL with a heterogeneous expression amongst CFC (BCR-ABL /ABL ratio range 2- 25%, mean 33%). Interestingly in this patient, CFC and LTC-IC assays performed in a bone marrow aspirate prior to IM discontinuation showed no evidence of BCR-ABL-expressing progenitors, suggesting a rapid and massive expansion of a dormant LSC clone migrating to blood at the time of molecular relapse. In pt UPN2, 9 individual CFC were found to express BCR-ABL with a significant heterogeneity (BCR-ABL/ABL ratio 0.2% - 56% , mean = 15 %). In Pt UPN3, with the lowest levels of BCR-ABL during molecular relapse, 4/73 CFC expressed BCR-ABL. All three pts had a favorable response after resuming Imatinib (UPN1, 2, 3) or after switching therapy to Bosutinib (UPN4). On UPN4, 2 pools of 4 CFC/20 expressed BCR-ABL. In two pts with 100% clonogenic relapse (UPN 1 and UPN2) the estimated circulating leukemic CFC numbers were 607 / ml and 468 / ml of blood respectively, in the presence of normal appearing CBC. In UPN 3 and 4, with approximately 10% of CFC being BCR-ABL +, these numbers were estimated to be 507 and 45 per ml of blood, respectively. Thus, the study of these four patients demonstrates that circulating leukemic CFC are the first detectable abnormal cellular components during molecular recurrence and that the clonogenic relapse in peripheral blood precedes myeloid expansion. These results show also for the first time to our knowledge, that leukemic CFC egress from bone marrow to peripheral blood is an early event during early stages of CML and the CFC expansion takes place not only in bone marrow but also in peripheral blood. Besides shedding light into the pathophysiology of CML, these findings could be of major interest to predict relapse parameters and could lead to the design of novel biological follow-up protocols after TKI discontinuation.