Does cytogenetic mosaicism in CD34⁺CD38^low cells reflect the persistence of normal primitive hematopoietic progenitors in myeloid metaplasia with myelofibrosis?

Myeloid metaplasia with myelofibrosis (MMM) is a rare chronic myeloproliferative disorder characterized by myelofibrosis, extramedullary hematopoiesis, and absence of BCR-ABL rearrangement.^1,2 

Myeloproliferation is considered clonal and fibrosis, reactive.²  Hierarchic level, primary mechanism, and/or gene alteration responsible for the malignant clone remain unknown. Here, we analyzed the clonality of CD34⁺ cells (CD34⁺), and questioned the hierarchic level of the disease and the origin of karyotypically normal CD34⁺.

Karyotypes were performed on white blood cells (WBCs) and on immunomagnetically selected circulating CD34⁺ (purity, ≥ 97%) from 23 patients as described.^3-5  According to previous reports,^1,6  34.8% (8/23) of patients exhibited a high proportion of cytogenetic abnormal WBCs (nearly 100%). CD34⁺ carried the same cytogenetic aberrations as WBCs in patients 13, 17, 19, and 33 (Table 1), but CD34⁺ abnormal cell percentages were heterogeneous: 100% abnormal metaphases in patients 13, 17, and 19; 33% in patient 33; and 0% in patient 57.

This mosaicism could be due to normal residual CD34⁺ whose proliferation is inhibited by unknown mechanism(s).⁴  Normal WBC and CD34⁺ karyotypes in the other 14 patients strengthen the alternative hypothesis that the primitive genetic lesion remains cryptic and that karyotypic alterations occur secondarily. Mosaicism present in several CD34⁺ karyotypes and absent in WBCs also raises the question of the hierarchic level of the clonal event in MMM. The 6-fold higher proportion of CD34^highCD38^low cells in MMM than in normal blood (25% vs 4%) suggested that the clonal myeloproliferation derives from primitive hematopoietic progenitors.³  Therefore, cytogenetic and fluorescence in situ hybridization (FISH) studies were performed on CD34⁺CD38^low, CD34⁺CD38^high, CD3⁺, and CD19⁺ sorted cells (99% pure) in patient 19. FISH confirmed the reciprocal translocation with monoallelic 13q14 deletion (13q14–) (lsi D13S319 probe; Vysis, Downers Grove, IL).

Interestingly, in patient 19, 13q14–(FISH) was detected only in about 80% of freshly CD34⁺CD38^low- and CD34⁺CD38^high-sorted subpopulations (Figure 1A-B). This percentage increased after 7-day and 14-day cultures in the progeny of CD34⁺CD38^low and CD38^highCD34⁺, suggesting a growth advantage of 13q14–cells. CD3⁺ (T lymphocytes) and CD19⁺ (B lymphocytes) compartments did not show the genetic marker (Figure 1C). These results contrast with those of Reeder et al⁸  showing a heterogeneous clonal involvement (13q–,20q–) of immunomagnetically selected T and B lymphocytes from 4 patients. Spleen fibroblast karyotypes performed in patients 19 and 33 were normal, confirming that, in MMM, fibroblasts are polyclonal reactive cells.

In conclusion, we demonstrate that circulating primitive CD34⁺CD38^low are clonal in MMM, at least in some patients. The absence of a cytogenetic marker in a variable percentage of CD34⁺, T and B cells could reflect residual normal hematopoiesis. Alternatively, karyotypically normal cells could derive from primitive progenitors/stem cells whose gene alteration(s) is cryptic.

Investigating these not mutually exclusive hypotheses would allow a better understanding of MMM pathogenesis and would improve its therapy.