Chronic myeloid leukaemia presenting with isolated thrombocythaemia, a case revealing its stem cell biology

Chronic myeloid leukaemia (CML) is the result of the t(9;22) balanced translocation developing from primitive haematopoietic stem cells, and typically presents with marked leucocytosis dominated by myeloid cells of all stages of maturation. However, in rare instances Philadelphia positive (Ph+) CML can present with isolated thrombocythaemia (CML-T) with no, or only slight elevation of granulocyte counts (Michiels et al, 2004). This special presentation of CML, previously also referred to as Ph+ Essential Thrombocytosis, is seen almost exclusively in women and is characterized by absence of splenomegaly and presence of atypical, small mononuclear megakaryocytes in the bone marrow (BM) (Michiels et al, 2004; Girodon et al, 2005). We speculated whether this extraordinary presentation could represent a distinct subgroup where the initiating leukaemic hit has taken place in a more lineage-committed cell than in classical CML, and undertook for the first time to delineate the stem cell biology of a such case. In December 2010, a 66-year-old woman 3 years into a follow-up for follicular lymphoma in remission unexpectedly presented with a platelet count of 2062 × 109/l, haemoglobin concentration 132 g/l, white blood cell (WBC) count 8·0 × 109/l, with a neutrophil count of 3·9 × 109/l and basophil count of 0·94 × 109/l. All haematological values had been within normal limits since she finished treatment (6 series of rituximab-cyclophosphamide-vincristine-prednisolone) more than 2·5 years previously. While the patient was negative for the JAK2 V617F mutation, she was found Ph+ in all of 25 metaphases by standard karyotyping as well as by quantitative polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH). In concordance with previous reports on CML-T (Michiels et al, 2004), the patient had no splenomegaly and a BM biopsy showed light to moderate hyperplasia with a marked increase in small and hypolobulated megakaryocytes. Treatment with hydroxycarbamide and imatinib 400 mg was initiated, and haematological remission and a major molecular response (MMolR) was attained (Fig 1). Unfortunately, the patient reacted with severe side effects (dyspnoea, acrocyanosis, fatigue, itching, headaches, abdominal pains) to imatinib and also to subsequent attempts of treatment with both nilotinib 300 mg and dasatinib 50 mg, and these drugs had to be terminated after a total of 26 weeks of therapy. Currently, the patient is on pegylated interferon α-2a and BCR-ABL1 expression has stabilized approximately one log below the initial level. BM aspirates were obtained at 0, 3 and 9 months after the diagnosis of CML after informed consent from the patient and approval by the local ethical committee. Thawed mononuclear cells or CD34+ enriched (STEMCELL Technologies, Vancouver, Canada) samples were stained with monoclonal antibodies and sorted by fluorescence-activated cell sorting (FACS) on a BD FACSAria™ III (BD Biosciences, San Jose, CA, USA). Colony-forming cell (CFC) assay was performed using Methocult H4434 medium (STEMCELL Technologies) and 14-d colonies scored as erythroid (BFU-E, CFU-E), myeloid (CFU-GM, CFU-G, CFU-M) or multi-lineage (CFU-GEMM). Interphase FISH analysis was done according to manufacturers instructions using BCR FISH DNA split signal probe (DAKO, Glostrup, Denmark) evaluating 200 nuclei by two independent observers. Quantitative PCR was performed as previously described (Stentoft et al, 2001) on a Mx3000P™ system (Stratagene, Santa Clara, CA, USA), using RNeasy micro kit or QIAmp RNA Blood mini kit (QIAGEN, Sollentuna, Sweden) for RNA purification. Performance of the assay was verified by standard curves of each gene based on serial dilutions of the cell line K562. No signs of Ph+ cells by FISH or qPCR were found in the original material (lung biopsy and trephine biopsy) from FL diagnosis. At CML diagnosis, both CD34+/CD38− stem cells and CD34+/CD38+ progenitors contained large fractions of Ph+ cells (74% and 78% respectively) and displayed high expression of the BCR-ABL1 fusion gene (Fig 2 A–B). Moreover, at 9 months after diagnosis, CML cells were identified in neutrophils (CD66+), B-cell precursors (CD45low/CD19+/CD10+), and mature B-cells (CD19+) and T-cells (CD3+) (Fig 2C). In the CFC assay, colonies derived from CD34+ cells at the time of diagnosis were almost entirely (94%) of erythroid lineage, and only 4% myeloid colonies and 2% multi-lineage colonies appeared. All colonies were BCR-ABL1 positive by qPCR (Fig 2D, Supporting Information Table SI). Despite the achievement of MMolR at 3 months after initiating imatinib treatment, both FISH positivity and high levels of BCR-ABL1 expression were retained in the progenitor compartment, especially among CD34+/CD38− stem cells (Fig 2B). On the other hand, the treatment resulted in reappearance of myeloid lineage CFCs and loss of BCR-ABL1 expression in all colonies (Fig 2D). This is the first report to detail the biological characteristics of a patient with the rare variant of CML presenting solely with thrombocythaemia. In reviewing the present literature, three different scenarios have been presented regarding the borderline territory between Ph+ CML and myeloproliferative neoplasms (MPN). (i) Development of Ph+ CML in an already known MPN, which may either be negative (Mizutani et al, 2010; Weng & Shih, 2011) or positive (Jallades et al, 2008) for the JAK2 V617F mutation, (ii) Diagnosis of Ph+ CML followed by the unmasking of an already present occult MPN after the start of tyrosine-kinase inhibitor (TKI) treatment (Krämer et al, 2007) and (iii) CML-T as a solitary disorder (Michiels et al, 2004). Our patient adhered to the previously described phenotype of CML-T, and the uniclonal nature of the disease was confirmed after the start of the treatment where monotherapy with TKI sufficed in controlling platelet count. Our data demonstrated the presence of CML cells within CD34+/CD38− stem cells and committed cells of erythroid, myeloid, and lymphoid lineages. This points towards an origin of the disease within the most primitive haematopoietic stem cells, similar to CML presenting in the classical manner. In addition, the disease seems to respond to TKI treatment in a comparable fashion, with rapid suppression of differentiated leukaemic cells and resistance in the progenitor fraction. The lack of myeloid colony forming capacity shown here was more pronounced than previously reported for classical CML (Chávez-González et al, 2004) and suggests that identification of the mechanisms responsible for the selective expansion of megakaryocytic lineage cells will require direct studies of the very earliest progenitors. In the meantime, we suggest that these patients should not be treated differently from other CML patients. The authors would like to thank the patient for her kind participation in the study, and Mariann Christensen, Institute of Pathology, Aarhus University Hospital, Aarhus for specific laboratory assistance. This work was supported by grants from The Danish Cancer Society, the Danish MRC, and the Karen Elise Jensen Foundation. PvKN and PH designed the study and wrote the first draft. PvKN, ASR and CCP performed the laboratory work. EK performed the cytogenetic analyses. KB performed the pathologic analyses. CGN designed the molecular analyses. All authors contributed to revising the paper. The authors declare no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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