del(20q) can be observed in hematologic neoplasms, including chronic myelogenous leukemia (CML), and has been reported in patients undergoing blast transformation. We describe 10 patients with CML in hematologic and cytogenetic remission with del(20q) detected by conventional cytogenetics. There were 6 men and 4 women with a median age of 56 years. All patients initially had BCR-ABL1 and t(9;22) (q34;q11.2) and achieved morphologic and cytogenetic remission after therapy. del(20q) was identified before (2/10 [20%]), at the time of (3/10 [30%]), or after (5/10 [50%]) cytogenetic remission and was not associated with morphologic evidence of dysplasia. At last follow-up, no patients had a myelodysplastic syndrome (MDS). Leukocyte and platelet counts were normal; 4 of 10 patients had mild anemia. Nine patients have remained in morphologic and cytogenetic remission with stable del(20q). BCR-ABL1 fusion transcript levels were absent or low (median, 0.01%). Recently, in 1 patient, recurrent CML developed and del(20q) was lost. We conclude that del(20q) in the setting of CML in remission is not predictive of MDS or blast transformation.
Chronic myelogenous leukemia
Recurrent cytogenetic abnormality
Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasm (MPN) originating from a clonal, pluripotent hematopoietic stem cell defined by the presence of the BCR-ABL1 fusion gene, usually as a result of t(9;22) (q34;q11.2).1 In a small subset of patients, more complex chromosomal abnormalities resulting in the BCR-ABL1 fusion gene are present.2 The BCR-ABL1 fusion protein has constitutive tyrosine kinase activity, promotes hematopoietic transformation,3 and provides a specific target for the treatment of CML.3 Current first-line therapy includes imatinib, a potent tyrosine kinase inhibitor (TKI), with a high response rate and favorable tolerance profile. Alternatively, other TKIs such as dasatinib and nilotinib can be used in patients with imatinib-resistant CML.4
The natural history of CML is characterized by an initial indolent chronic phase (CP), followed by an accelerated phase (AP) and/or a blast phase (BP). The onset of AP or BP indicates disease progression and is often associated with additional cytogenetic abnormalities and resistance to therapy.5–9 In most patients with CML, the diagnosis is made when patients are in CP and treatment usually consists of a TKI and chemotherapeutic agents with or without stem cell transplantation. Complete cytogenetic response is achieved commonly, in up to 87% of patients in 1 study.4 The patients no longer show detectable t(9;22)(q34;q11.2) or BCR-ABL1 gene rearrangement by conventional karyotyping or fluorescence in situ hybridization (FISH) studies.
Unbalanced chromosomal abnormalities are frequently found in hematologic neoplasms, such as MPN, myelodysplastic syndrome (MDS), and acute myeloid leukemia, and are considered potential locations of genes responsible for pathogenesis and/or disease progression. del(20q) is one of the most common recurrent cytogenetic abnormalities in myeloid neoplasms and a known marker for MDS.1,10 In CML, del(20q) has been detected during blast transformation and is considered a potential cytogenetic marker for clonal evolution.11 However, rare CML cases in cytogenetic and morphologic remission in which del(20q) has been detected are reported in the literature.12,13 The clinicopathologic and follow-up data on these cases are not available. Herein we describe the clinical, morphologic, and molecular genetic characteristics of 10 patients with CML with del(20q) as the sole cytogenetic abnormality during morphologic and cytogenetic remission.
Materials and Methods
We searched the files of the Clinical Cytogenetics Laboratory, Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, for cases of CML in cytogenetic remission with del(20q) after treatment from 1997 to 2009, and identified 10 cases. For comparison, cases of CML in AP or BP with del(20q) were also obtained from the same database to assess the possible role of del(20q) in disease development. The diagnosis of CML, including AP and BP, was based on the World Heath Organization classification of hematopoietic tumors.1 Complete cytogenetic remission was defined as the absence of BCR-ABL1 gene rearrangement by conventional cytogenetics and FISH analyses. A clone for del(20q) was defined when 2 or more metaphases showed an identical deletion in the long arm of chromosome 20 followed by FISH confirmation, if needed.
Bone Marrow Examination and Clinical Data Collection
We reviewed Wright-Giemsa–stained peripheral blood (PB) and bone marrow (BM) aspirate smears and H&E-stained sections of BM aspirate clot and core biopsy specimens. Clinical data were abstracted from the medical records.
Conventional Cytogenetic and FISH Analyses
Conventional cytogenetic analysis was performed on metaphase cells prepared from BM aspirates cultured for 24 or 48 hours without mitogens, using standard techniques as described previously.14 After hypotonic treatment and fixation with 3:1 methanol acetic acid solution, cell suspensions were dropped on clean slides. G-banding was performed after the slides were dried at 60°C overnight. Chromosomal analysis was performed according to a standard protocol used in the Clinical Cytogenetics Laboratory, and the results were reported according to the 2005 International System for Human Cytogenetic Nomenclature.15
FISH analysis was performed on interphase nuclei from matched samples according to the standard protocol used in the Clinical Cytogenetics Laboratory.9 The LSI D20S108 probe (Vysis/Abbott Molecular, Downers Grove, IL) was used to detect deletion in the long arm of chromosome 20. Assessment of BCR-ABL1 rearrangement associated with the t(9;22)(q34;q11.2) was performed using the LSI ES BCR-ABL1 probe (Vysis/Abbott Molecular), as described previously.7 The cutoffs for positive BCR-ABL1 rearrangement and D20S108 deletion in 20q used in the Clinical Cytogenetics Laboratory are 1.5% and 4.4%, respectively.
Levels of BCR-ABL1 fusion transcripts were quantified using a multiplex real-time reverse transcriptase–polymerase chain reaction (RT-PCR) assay that simultaneously detects b2a2, b3a2, and e1a2 transcript types. The RNA was extracted from PB or BM samples using Trizol reagent (Gibco-BRL, Gaithersburg, MD) according to the manufacturer’s instructions. Reverse transcription was performed on total RNA (1 μg) using random hexamers and SuperScript II reverse transcriptase (Gibco-BRL) as described previously.16 The resulting complementary DNA was subjected to PCR to amplify BCR-ABL1 fusion transcripts on an ABI PRISM 7700 sequence detector (Perkin Elmer-Applied Biosystems, Foster City, CA) using primers and conditions described previously.16 Quantitative BCR-ABL1 levels were normalized to total ABL1 transcript levels and reported as a ratio, BCR-ABL1/ABL1, multiplied by 100 to yield a percentage.
Clinical Characteristics at Time of Initial Diagnosis of CML
We identified 10 patients with CML in morphologic and cytogenetic remission with an isolated del(20q). There were 6 men and 4 women, with a median age of 56 years (range, 33 to 72 years). For the patients in whom leukocyte or platelet counts at time of initial diagnosis of CML were available, all patients had leukocytosis (n = 8); 1 patient had thrombocytosis. The diagnosis of CML was made initially at other institutions before the patients came to our hospital. Data related to the initial presentation were available for 6 patients. Three patients had a clinical picture suggesting infection, 1 patient had epistaxis, 1 patient complained of dizziness, and 1 patient was asymptomatic with the diagnosis first detected during routine physical examination.
At the time of admission to our institution, 0 to 19 months after initial diagnosis of CML, 6 patients had leukocytosis (WBC count, median, 14,700/μL [14.7 × 109/L]; range, 6,400–118,200/μL [6.4–118.2 × 109/L]; reference range, 4,000–11,000/μL [4.0–11.0 × 109/L]); 6 patients had anemia (hemoglobin level, median, 12.7 g/dL [127 g/L]; range, 7.3–14.4 g/dL [733–144 g/L]; reference range, 14–18 g/dL [140–180 g/L] for men and 12–16 g/dL [120–160 g/L] for women); and 4 patients had thrombocytosis (platelet count, median, 413 × 103/μL [413 × 109/L]; range, 83–822 × 103/μL [83–822 × 109/L]; reference range, 140–440 × 103/μL [140–440 × 109/L]). Serum lactate dehydrogenase (median, 628 U/L [10.5 μkat/L]; range, 389–2,439 U/L [6.5–40.7 μkat/L]; reference range, 313–618 U/L [5.2–10.3 μkat/L]) and β2-microglobulin (n = 6, median, 2.4 mg/L; range, 1.2–4.5 mg/L; reference range, 0.6–2.0mg/L) levels were elevated in 5 and 4 patients, respectively Table 1. All patients were treated with imatinib or nilotinib, with or without interferon alfa, cytarabine, homoharringtonine, and/or hematopoietic stem cell transplantation, and all achieved morphologic and cytogenetic remission.
In all patients, BM specimens at initial diagnosis were hypercellular (range, 65%–100%; median, 88%) with granulocytic and atypical megakaryocytic hyperplasia, basophilia, and/or eosinophilia consistent with CML. Subsequent BM biopsy specimens, when patients were in remission, showed normocellular BM with trilineage hematopoiesis and no morphologic evidence of CML.
Cytogenetic and Molecular Findings
The results of conventional cytogenetic and molecular analyses at the time of initial diagnosis of CML showed the t(9;22)(q34;q11.2) with no evidence of del(20q). RT-PCR revealed high BCR-ABL1 levels. The results of conventional cytogenetic analysis at the time of hematologic and cytogenetic remission are shown Image 1 and Image 2 and summarized in Table 2. del(20q) was the sole abnormality in all cases. The breakpoint within del(20q) was at q11.2 in all cases, and 6 cases (60%) showed interstitial deletion involving q11.2q13. FISH analyses were negative for BCR-ABL1 in all 10 cases at the time of hematologic and cytogenetic remission and confirmed the presence of del(20q) in all cases. Molecular analysis of BCR-ABL1 levels by RT-PCR revealed BCR-ABL1/ABL1 levels ranging from less than 0.01% to 0.0559% (median, 0.01) at the time of the last follow-up.
The detection of del(20q) occurred at different time points in the 10 patients with CML. In 2 patients, del(20q) was identified during CP, 25 months and 8.9 months, respectively, before clinical and cytogenetic remission. In 3 patients, del(20q) was detected at the same time as CML remission, and in 5 patients, del(20q) was observed 3.5 to 37.5 months after first remission Table 3. The patients were followed up for a median of 98.6 months (range, 45.2 to 165.4 months) after the diagnosis of CML and 49 months (range, 0 to 103) after detection of del(20q). None of the patients exhibited morphologic evidence of dysplasia in PB or BM at the time of detection of del(20q), nor have MDS or other hematologic malignancies developed at time of most recent follow-up. At last follow-up, the WBC (median, 7,300/μL [7.3 × 109/L]; range, 3,300–10,300/μL [3.3–10.3 × 109/L]) and platelet (median, 183 × 103/μL [183 × 109/L]; range, 151–281 × 103/μL [151–281 × 109/L]) counts were normal. Of 10 patients, 4 (40%) had mild anemia (hemoglobin range, 11.0–14.8 g/dL [110–148 g/L]). del(20q) persisted in all patients except 1 who progressed to CML-BP, at which time del(20q) was lost, 55 months after initial diagnosis and 50 months after first CML remission (case 1). Patients in the remaining 9 cases remained in cytogenetic and morphologic remission of CML at the last follow-up.
In our search of cases with CML in AP or BP, we identified 4 cases that also carried del(20q) as a part of the secondary cytogenetic changes. No case with an isolated del(20q) was found. The detailed clinical status and cytogenetic changes in these patients are listed in Table 4. In case 1, a patient with CML-CP for approximately 5 years, del(20q) developed at the time of CML-BP. This abnormality disappeared in 2 months at the time of the next cytogenetic examination when the patient had a very complex karyotype. In case 2, a patient with CML-CP for approximately 4 years, del(20q) developed, and there was no evidence of MDS for 24 months before AP developed. Case 4 was similar to case 2 in that the patient had CML-CP for approximately 4 years before del(20q) developed. He did not have evidence of MDS for 86 months before BP developed. In case 3, a patient with CML-CP followed by AP for more than 7 years then developed del(20q) with both clonal evolution and clonal divergence detected during the course of BP.
Conventional cytogenetic analysis shows the presence of del(20q) in a female karyotype from a patient with chronic myelogenous leukemia in cytogenetic remission (left). An idiogram for normal chromosome 20 shows the location of breakpoints (arrows) that commonly occur in del(20q) (right).
Fluorescence in situ hybridization (FISH) confirmation in 2 interphase cells indicating del(20q) (arrows) (A) in a patient with chronic myelogenous leukemia in cytogenetic remission. FISH was negative for BCR-ABL1 rearrangement in a metaphase cell (B).
In most patients with CML in AP or BP, secondary chromosomal aberrations are present and signify clonal evolution.17 An interstitial deletion in the long arm of chromosome 20 has been rarely described in this setting and implicated as a marker for disease progression.11 It has been postulated that deletion of genetic material on chromosome 20q may result in the loss of one or several tumor suppressor genes, causing haploinsufficiency of target proteins.18 Schnittger et al13 recently reported a case of CML that developed an aberrant Philadelphia chromosome (Ph)-negative clone that contained del(20q) during cytogenetic and molecular remission. In this report, the authors stated that no secondary disease had developed at last follow-up; however, the duration of the follow-up was not provided. This is, to the best of our knowledge, the only case in the literature describing del(20q) in patients with CML during hematologic and cytogenetic remission. Herein we describe 10 patients with CML in remission in whom an isolated del(20q) was detected and provide detailed clinical, morphologic, cytogenetic, and molecular information to better understand this phenomenon.
In this study, all 10 patients were diagnosed in CML-CP, treated with at least 1 TKI, and achieved hematologic and cytogenetic remission. Although del(20q) occurred at different time points during the clinical course, in all patients, the deletion occurred after the initiation of TKI therapy. Others have described previously that clonal cytogenetic abnormalities emerge in Ph-negative clones in some patients with CML treated with imatinib,12,19 although the underlying mechanisms are not clear. One possibility is that genomic instability leads to the production of multiple abnormal clones, including a clone with t(9;22) and other Ph-negative clones. The TKI-induced cytogenetic remission eliminates the proliferative advantage of the t(9;22) clone and unmasks the Ph-negative clones.12 In the 10 cases we report, because del(20q) occurred after TKI therapy was initiated, it is possible that the TKI had a role in selecting a del(20q) Ph-negative clone. However, by conventional cytogenetics, there was no evidence of del(20q) before TKI was initiated. One possible explanation is that del(20q) was present before TKI therapy at a low level, below the threshold of detection by conventional cytogenetics.
A second hypothesis for the acquisition of aberrant Ph-negative clones is that the TKI treatment itself induced or favored the emergence of the Ph-negative clones by involvement of DNA damage repair pathways.20ABL1 is known to interact with a number of proteins involved in the response to DNA damage and DNA repair.20,21 Inhibition of ABL1 by TKIs may contribute to genetic mutations that fail to be repaired and accumulate in the Ph-negative stem cells. This is of particular interest as the BM replenishes poly-clonal hematopoiesis with a relatively limited Ph-negative stem cell repertoire during CML remission. Since abnormal Ph-negative clones occur in only a small subset of patients, genetic susceptibility in the patients also may have an important role in addition to the TKI effect in the accumulation of these aberrant clones.
For comparison, we searched for CML cases in AP or BP that developed del(20q), and we identified 4 cases. The clinical course of the patients suggests that del(20q) is likely not clinically important in disease progression. Two patients (cases 2 and 4) showed no morphologic changes in the bone marrow when a clonal del(20q) was observed, which disappeared before disease progression in 1 patient (case 4) or was accompanied by additional clonal abnormalities when disease progressed (case 2). In 1 patient (case 1), del(20q) was transiently present for 2 months during the course of BP. The presence of clonal divergence with 3 separate clones in this patient suggests that the role of del(20q) in disease development was not important. In case 3, the i(9)(q10) observed is usually considered an unfavorable recurrent marker.17 In combination with the occurrence of clonal divergence with numerous abnormalities, including an extra Philadelphia chromosome, the role of del(20q) in disease development in case 3 was also likely to be minimal.
del(20q) is a well-known cytogenetic marker for MDS and MPN.1 It is interesting that none of the 10 cases in this study showed significant morphologic evidence of dysplasia or cytopenia at the time of last follow-up, up to 103 months since the detection of del(20q), although del(20q) has been present stably in most patients. The underlying mechanism is not known; however, del(20q) does not seem to cause MDS-related changes in this group of patients, nor does it indicate CML clonal divergence or disease progression. Long-term follow-up and continued monitoring of cytogenetic composition of these cases would be essential to elucidate the prognostic impact of this finding.
In summary, we describe the clinical, morphologic, cytogenetic, and molecular characteristics of 10 patients with CML in hematologic and cytogenetic remission who carried del(20q) as the sole abnormality in the Ph-negative clones. The presence of del(20q) as a Ph-negative clone in this group of patients is a rare occurrence. Our data suggest that the aberrant Ph-negative clones with isolated del(20q) do not indicate CML progression or myelodysplasia in this clinical setting.
Emergence of clonal cytogenetic abnormalities in Ph– cells in some CML patients in cytogenetic remission to imatinib but restoration of polyclonal hematopoiesis in the majority. Blood. 2003;101:1941–1949.
RQ-PCR based WT1 expression in comparison to BCR-ABL quantification can predict Philadelphia negative clonal evolution in patients with imatinib-treated chronic myeloid leukaemia. Br J Haematol. 2009;146:665–668.