Clonal chromosomal abnormalities in CD34⁺/CD38⁻ hematopoietic cells from cytogenetically normal chronic myeloid leukemia patients with a complete cytogenetic response to tyrosine kinase inhibitors

Letter to the Editor

Clonal chromosomal abnormalities in Ph-negative cells (CCA/Ph-) have been identified in 3-15% of chronic myeloid leukemia (CML) patients with a partial or complete cytogenetic response (CCyR) to imatinib(1). Trisomy 8 and deletions of chromosome 7 [either monosomy 7 or del 7(q)] account for the majority of cases, although a range of other karyotypic changes were seen at lower frequency. A recent study reported CCA/Ph- in patients on dasatinib, indicating that the phenomenon is not limited to imatinib therapy. In some patients, CCA/Ph is associated with a myelodysplastic syndrome (MDS), and progression to acute myeloid leukemia has been reported(1, 2). A single center report suggested inferior survival in newly diagnosed patients who develop CCA/Ph-(3). However, a larger study found that CCA/Ph- does not adversely affect the prognosis of patients with a major cytogenetic response (MCyR) to imatinib, indicating that this is a mostly benign condition(4). The reported incidence of CCA/Ph- is based on metaphase karyotyping, which is limited by the small number of cells analyzed and by the fact that only cells are assayed that can be induced to divide within the culture period. We thus hypothesized that conventional karyotyoping may underestimate the incidence of CCA/Ph- and decided to screen CD34⁺/CD38⁻ cells from a cohort of CML patients with a CCyR to tyrosine kinase inhibitor (TKI) therapy for abnormalities of chromosomes 7 and 8. This primitive cell compartment is known to be enriched for hematopoietic progenitor and stem cells(5). We find CCA/Ph- in CD34⁺/CD38⁻ cells from 4/19 patients, suggesting CCA/Ph- is more common than previously appreciated.

PATIENTS AND METHODS

Patients

Samples were from consecutive CML patients on TKI therapy who were undergoing bone marrow aspirates at Oregon Health&Science University as part of their clinical care. The only selection criterion was a normal karyotype on the previous biopsy. At the start of TKI therapy one patient (#14) was in the accelerated phase, while all others were in the chronic phase. All patients provided informed consent to an IRB-approved protocol. Control samples (normal bone marrow mononuclear cells, MNC) were purchased from a commercial supplier.

Cell selection

MNC were separated from bone marrow by density gradient centrifugation using Ficoll (Nycomed, Oslo, Norway) and depleted of lineage-positive cells using an antibody cocktail and magnetic beads (Stem Cell Technologies, Vancouver, Canada). CD34⁺/CD38⁻ (and in some experiments CD34⁺/CD38⁺) cells were sorted on a BD FACSARIA after staining with FITC-conjugated anti-CD34 and PE-conjugated anti-CD38 monoclonal antibodies (BD). Sorted cells were sedimented at 1000g and resuspended in 1.2 ml 3:1 methanol: acetic acid. Cells were then dropped onto glass slides and allowed to dry one drop at a time.

Fluorescence in situ hybridization (FISH)

Interphase FISH (I-FISH) for BCR-ABL was performed on 200 unselected bone marrow cells as part of routine diagnostics, using the Vysis ABL (9q34, red), ASS (9q34, aqua) and BCR (22q11.2, green) probes (Vysis, Downer's Grove, IL). FACS-sorted cells were subjected to I-FISH for chromosomes 7 and 8 abnormalities, using the Vysis D7S522 (7q31, red), CEP7 (green) and CEP8 (aqua) probes in a single co-hybridization assay (Figure 1A). Samples were analyzed under a Nikon Eclipse E800 photoscope, and representative photographs were taken using CytoVision software from Applied Imaging. We aimed to analyze 100 cells, the coordinates of which were recorded. In case of ambiguous results, additional 100 cells were analyzed if possible. To assess the BCR-ABL status of cells with chromosome 7/8 abnormalities, the slides were ‘stripped’ using 2xSSC/0.3% NP-40 and re-hybridized with the BCR-ABL probe. Individual cells were identified by their previously recorded coordinates and analyzed for co-localization of BCR and ABL signals. All samples were analyzed by 2 independent observers.

figure 1.

Fluorescence in situ hybridization (FISH) of FACS-sorted cells from CML patients with a complete cytogenetic response. (A) Schematic of the probes used to detect abnormalities of chromosomes 7 (D7S522 [red]/CEP 7 [green]) and 8 (CEP 8 [aqua]). (B) (left panel) Detection of trisomy 8 in a CD34⁺/C38⁺ cell from a patient with CCyR and trisomy 8 by conventional karyotyping. (right panel) The coordinates of the cell were recorded. The slide was stripped and rehybridized with a BCR-ABL/ASS probe with BCR in green, ABL in red, and ASS in aqua. The same cell was identified using the recorded coordinates. The presence of two red, two green and two aqua signals indicates the presence of two normal copies of chromosomes 9 and 22 and the absence of BCR-ABL fusion signals (juxtaposed red and green). (C) (left panel) Detection of del(7q) in a CD34⁺/CD38⁻ cell. (right panel) Rehybridization with a BCR-ABL probe revealed a normal pattern.

Statistical analysis

Categorical variables were analyzed by χ2 test and non-categorical variables with the Mann-Whitney U-test.

RESULTS AND DISCUSSION

In initial experiments we analyzed CD34⁺/CD38⁺ cells from 2 patients with known trisomy 8 in Ph-negative cells and one patient with a normal karyotype. In both trisomy 8 patients metaphase karyotyping confirmed the previously detected abnormality in 9/20 cells (45%), and I-FISH on unselected marrow was positive in 29 and 15%, respectively. Of the CD34⁺/CD38⁺ cells, 35/58 (60%) and 15/34 (44%) exhibited trisomy 8, suggesting a concordance between metaphase karyotyping and I-FISH of CD34⁺/CD38⁺ cells. No abnormal interphases were seen in the normal control sample. To interrogate a more primitive cell compartment we FACS-sorted CD34⁺/CD38⁻ cells from additional 19 patients and 4 normal controls (Table 1). Analyzing 100 cells as planned succeeded in only 10 of 20 CML samples and on average, 66±40 cells were available for scoring by I-FISH. Abnormal signal patterns were identified in 4/19 CML patients (21%) but in none of the normal controls (P=0.456). Abnormalities were trisomy 8, monosomy 7 and del(7q) (2 patients). One additional patient (#3) had trisomy 8 in 1/84 analyzable interphases; this is technically above the normal cutoff of 1%, but no additional cells were available for analysis to verify the finding. There was no significant difference between samples with and without abnormalities with respect to the number of cells available for analysis (49±42 vs. 81±64, p=0.36). Re-hybridization of the same slides with a BCR-ABL probe did not detect BCR-ABL in any of the cells with abnormalities (Figure 1B). None of the biopsies showed dysplasia; however in patient #11 marrow infiltration with a low grade B-cell lymphoma (positive for CD19, 20, 22, 45; negative for CD34) was found. Follow-up karyotyping was normal for patients #2 (26 months) and #5 (2, 5, 8, 20 months). Patient #15 showed 3/20, 4/20 and 4/20 metaphases lacking the Y chromosome at 6, 12 and 24 months. There were no dysplastic changes in any of these subsequent biopsies. No follow-up is available for patients #8 and #11. We analyzed potential correlations between abnormalities in BCR-ABL-negative CD34⁺/CD38⁻ cells and sex, age, disease duration and duration of TKI therapy. Patients with abnormalities were significantly older (median 72 years, range 64-80) than patients without abnormalities (median 57 years, range 40-74, p = 0.02), while no other significant differences were seen.

Table 1.

Patient characteristics and results of fluorescence in situ hybridization (FISH)

Sample	Patient #	Sex	Age	Disease duration (month)	Prior treatment	Time of TKI therapy (months)	Karyotype	34+/38− cells scored	I-FISH
07/028	1	m	59	228	IFN, IM, autologous SCT	77	46XY[20]	100	normal
07/031	2	f	64	36	IM, DAS	34	46XX[20]	67	del(7q) (9%)
07/061	5	m	47	96	IFN, IM, DAS, allogeneic SCT	84	46XY[20]	84	[+8 (1.2%)]
07/231	6	m	74	109	IM	89	46XY[20]	100	normal
07/246	7	f	66	29	IM, DAS	26	46XX[20]	21	normal
07/248	8	m	74	108	IM	89	46XY[20]	100	del(7q) (3%)
07/257	10	f	56	35	IM, DAS	30	46XX[20]	100	normal
07/263	11	f	80	166	IM	89	46XX[20]	17	+8 (76.5%)
07/279	12	m	72	84	IM	74	46XY[20]	100	normal
07/294	13	m	48	84	IM	80	46XY[20]	22	normal
07/299	14	m	51	84	IM	34	46XY[20]	100	normal
07/300	15	m	70	17	IM	75	XY,t(9;22;17)(q34;q11.2;q21)(1)/45,X,-Y(3)/46,XY(16)	12	−7 (16.7%)
07/315	16	m	57	96	IM, DAS	13	46XY[20]	100	normal
07/328	17	f	41	94	IM	86	46XX[20]	61	normal
07/329	18	f	49	34	IM, DAS	82	46XX[20]	103	normal
07/330	16	m	40	93	IM	30	46XY[20]	11	normal
07/344	17	f	63	54	IM, Ara-C	81	46XX[20]	6	normal
07/349	18	m	61	24	IM, NIL	60	46XY[20]	200	normal
08/001	19	m	72	70	IM, DAS, Ara-C, Arsenic trioxide	17	46XY[20]	15	normal

Ara-C – cytosine arabinoside; DAS – dasatinib; IFN – interferon-alpha; IM – imatinib; NIL – nilotinib; SCT – stem cell transplant

Our data show that clonal chromosomal abnormalities in Ph-negative primitive (CD34⁺/CD38⁻) cells from CML patients with a complete CCyR and a normal karyotype are fairly common. Given that we were limited by the number of analyzable interphases and that we restricted our analysis to screening for abnormalities of chromosome 8 and 7, it is possible that the frequency is even higher than evident from our data. None of the patients with chromosome 7/8 abnormalities had dysplastic morphologic features, consistent with the overall low incidence of MDS in CML patients with CCA/Ph-(4). Interestingly, patient #11 was found to have a marrow infiltration with a low grade B-cell lymphoma. Although the lymphoma cells did not express CD34 it is possible that the CD34⁺/CD38-cells with trisomy 8 that were present in this patient are part of the NHL clone. Unfortunately no follow-up material is available to test this. Patients with abnormalities were significantly older than patients without, suggesting that CCA/Ph- may be acquired over a lifetime. Alternatively, the number of residual normal stem cells in older patients may be smaller, which could place these cells under enhanced pressure to re-establish non-leukemic hematopoiesis in the presence of a TKI, resulting in enhanced genetic instability. We did not detect chromosome 7/8 abnormalities in 4 normal controls. However, this number is too small for a reliable comparison and it is possible that abnormalities would be detected in a larger and age-matched control group.

The question whether the acquisition of Ph is preceded by an as yet unidentified genetic event has attracted considerable attention ever since the first report of a bias of glucose-6 phosphate dehydrogenase isoenzyme expression in Ph-negative EBV-transformed B cells lines toward the phenotype of the Ph+ CML cells(6). CCA/Ph-, with rare exceptions, does not fit this model, given that the same specific cytogenetic abnormalities are not usually present in Ph-negative as well as Ph-positive cells. At face value, the fact that Ph-negative hematopoiesis of patients with CCyR to IFN-alpha or imatinib is polyclonal(7, 8) argues against a Ph- pathologic state. However, one must bear in mind that a hematopoietic stem cell could acquire a mutation that confers genetic instability, but not a competitive advantage over normal cells. This hypothetical parent cell could give rise to multiple related abnormal cell clones, one being the BCR-ABL-positive CML clone, and the others CCA/Ph-clones that become visible only upon CCyR induction by TKIs. Alternatively, multiple unrelated abnormal clones could result from simultaneous or successive damage to multiple hematopoietic stem cells. In CML patients, this could occur during the re-establishment of Ph-negative hematopoiesis in the presence of tyrosine kinase inhibitors, which may place considerable stress on a small pool of residual normal cells, perhaps enhanced by TKI inhibition of KIT or other critical pathways. To distinguish between the two fundamental possibilities it will be necessary to establish the clonal relationship between the Ph-positive and abnormal Ph-negative cell clones. Recent data from patients with Ph-negative myeloproliferative neoplasms suggest that both scenarios are possible(9). Despite the fact that trisomy 8 and chromosome 7 deletions are associated with MDS and AML, most CML with CCA/Ph- have a favorable outcome, suggesting a requirement for cooperating mutations to produce these phenotypes. In CML patients on TKI therapy, the risk of such additional mutations must be small, and therefore their prognosis is usually favorable.