The current study was conducted to compare simultaneously obtained bone marrow (BM) cytogenetics (CTG), peripheral blood (PB) and BM fluorescence in situ hybridization (FISH), and quantitative real-time polymerase chain reaction (Q-PCR) for BCR-ABL1 in monitoring response to treatment with tyrosine kinase inhibitors and homoharringtonine (HHT) in patients with chronic myeloid leukemia (CML).
PB and BM FISH (n = 112 samples) and/or Q-PCR (n = 132 samples) for BCR-ABL1 were simultaneously obtained in 70 patients with Philadelphia chromosome-positive (Ph+) CML in chronic (68%), accelerated (16%), and blast phase (16%) before the initiation of therapy and during the course of treatment with imatinib (IM) (n = 40 patients), dasatinib (n = 20 patients), nilotinib (n = 4 patients), bosutinib (n = 18 patients), or HHT (n = 4 patients) for patients with newly diagnosed (n = 13 patients), IM-sensitive (n = 34 patients), IM-resistant (n = 30 patients), or IM-intolerant (n = 9 patients) disease. Eighteen patients were found to have Ph+ variants or karyotypic abnormalities in addition to the Ph+.
Excellent correlations (r) were observed between PB and BM FISH (r = 0.95) and PB and BM Q-PCR (r = 0.87), as well as BM CTG and PB FISH (r = 0.89) and PB Q-PCR (r = 0.82). This correlation was not affected by the presence of the Ph+ variant or additional chromosomal abnormalities, the presence of ABL1 kinase domain mutations, phase of the disease, or treatment.
Targeting BCR-ABL1 using the specific tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) (Gleevec, Novartis, East Hanover, NJ) dramatically improved outcomes in patients with chronic myeloid leukemia (CML).1, 2 Similar to what was observed during treatment with interferon (IFN), the achievement of cytogenetic (CTG) remission is associated with improved disease-free and overall survivals with IM therapy.1 Therefore, tracking Philadelphia chromosome positivity (Ph+) yields valuable prognostic information. In addition, and with the availability of second- and third-generation TKIs, early detection, while patients are receiving IM, of suboptimal responses, loss of response, or disease progression permits treatment modifications that may favorably impact outcomes.3, 4
Assessing the number of bone marrow (BM)-derived Ph+ metaphases has been the gold standard for monitoring response to therapy in CML. However, more sensitive methods, such as fluorescence in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (Q-PCR), are not only more sensitive for detecting minimal residual disease, but are easily measurable on peripheral blood (PB) samples.5 Given the need for frequent monitoring in patients with CML, interest in noninvasive methods has increased over the years. PB FISH correlated with BM CTG and BM FISH during treatment with IFN, and excellent correlations have been reported between BM and PB PCR in patients treated with IM.6-12 Correlations between PB and BM assays to detect BCR-ABL1 during therapy with second-line agents and beyond have not been reported to the best of our knowledge, Therefore, we herein report results in simultaneously obtained PB and BM FISH, Q-PCR, and BM CTG in this setting.
MATERIALS AND METHODS
A computer database search retrospectively identified all FISH and Q-PCR for BCR-ABL1 performed on PB and/or BM samples between February 2004 and February 2009. Patients with Ph+ acute lymphoblastic leukemia were excluded. Ph+ CML patients with simultaneous (same day) PB and BM FISH and/or Q-PCR were selected for this analysis. Demographics, disease characteristics, treatment, response, and outcomes were extracted onto study-specific case report forms. This study was approved by the Emory University Institutional Review Board.
Staging and Response Criteria
Patients were classified according to World Health Organization criteria and assigned to the worst phase of the disease they ever reached. Ph+ patients in chronic phase had <10% PB and/or BM blasts and a platelet count ≥100 ×109/L; patients in accelerated phase had ≥10% to 19% PB and/or BM blasts, thrombocytopenia (platelet count <100 ×109/L) or thrombocytosis (platelet count >1000 ×109/L), and the presence of chromosomal abnormalities in addition to the Ph+ variant.13 Blast phase was defined by the presence of >20% PB and/or BM blasts or the presence of extramedullary disease. CTG responses were complete if Ph+ metaphases were 0%; partial if they were between 1% and 34%, and minor if they were between 35% to 90%.14
Fluorescence In Situ Hybridization
Fluorescence in situ hybridization (FISH) was performed using the protocol provided by the manufacturer for the dual fusion/dual color probe set of BCR-ABL1 (Abbott Molecular, Inc, Des Plaines, Ill) on fixed cells from both PB and/or BM. At least 2 technologists scored analyzable interphase cells of each specimen, recording all patterns observed, for a total of 200 nuclei. Results were considered clonal when the percentage of cells containing the BCR-ABL1 rearrangement exceeded the established, validated normal cutoff value of >1.0%.
Real-Time Quantitative PCR
Red cells were lysed by Ammonium chloride lysis and white cells were stored in RNALater (Applied Biosystems/Ambion, Austin, Tex) at a concentration of 5 million cells/500 μL. RNA was extracted either using the Ambion RNAqueous-4PCR kit (Applied Biosystems/Ambion) (before September 15, 2008) or the Qiagen RNeasy Mini Kit on the QIAcube (Qiagen, Valencia, Calif) (after September 15, 2008). Reverse transcription and real-time PCR were performed using the Light Cycler- t(9;22) Quantification Kit and the Light Cycler 1.2 with Light*Cycler software (Roche Diagnostics, Indianapolis, Ind). This kit detects products of b3a2 (e14a2), b3a3 (e14a3), b2a2 (e13a2), b2a3 (e13a3), and e1a2 translocations; but does not distinguish between different transcripts. Specific breakpoints were not determined in this study; however, the method used detects the common breakpoints observed in CML (e13a2 and e14a2). Relative quantitation was performed using the delta Ct method. Results were reported as a ratio of BCR-ABL1 to glucose-6-phosphate dehydrogenase (G6PDH) and also as log-10 change from a normalized baseline.
ABL1 Kinase Domain Mutation Analysis
Mutation analyses were performed by PCR and sequencing of the entire ABL1 kinase domain at independent commercial laboratories (Genzyme Genetics [Westborough, Mass] and ARUP Laboratories [Salt Lake City, Utah]).
BM mononuclear cells were cultured according to standard methods; 20 metaphase cells were analyzed for numerical and structural chromosome abnormalities by G-banding with trypsin-Giemsa staining. Karyotype designation was performed using the International System for Cytogenetic Nomenclature criteria.15 For the correlation analyses, CTG findings were expressed as a percentage by dividing the number of Ph+ metaphases by the total number of metaphases examined.
The Pearson correlation statistic was used to analyze the relation and generate the correlation coefficient between pairs of continuous variables. One-way analysis of variance was used to compare the differences between mean CTG responses.
Patient characteristics are summarized in Table 1. A total of 70 patients who had throughout the course of their treatment with 1 or ≥1 TKI and/or homoharringtonine (HHT) simultaneous BM CTG, PB FISH, and BM FISH (n = 53 patients and 91 samples) or BM CTG, PB Q-PCR, and BM Q-PCR (n = 58 patients and 115 samples) were identified. Fifty-two patients had all 5 tests (BM CTG, BM Q-PCR, BM FISH, PB FISH, and PB Q-PCR) performed simultaneously at 84 various time points. Patients in chronic phase (n = 48 patients), accelerated phase (n = 11 patients), or blast phase (n = 11 patients) had, at any given time, newly diagnosed (n = 13 patients), IM-sensitive (n = 34 patients), IM-resistant (n = 30 patients), or IM-intolerant (n = 9 patients) CML, and received IM (n = 40 patients), dasatinib (Sprycel; Bristol-Myers Squibb, Hillside, NJ; n = 20 patients), nilotinib (Tasigna, Novartis; n = 4 patients), bosutinib (SKI-606, Pfizer, New York, NY; n = 18 patients), and/or HHT (CGX-635, Chemgenex, Menlo Park, Calif; n = 4 patients). Samples were collected at diagnosis and after the initiation of treatment. Approximately 68% of patients had samples collected at >1 time point, and 15 patients had samples collected during sequential treatments with different drugs. The median age of the patients was 51 years (range, 18-79 years). Forty-nine patients with IM-resistant CML were tested for the presence of ABL1 kinase domain mutations. Eighteen patients had Ph+ variant or chromosomal abnormalities in addition to the Ph+. Sixteen had a detectable mutation, and 5 had >1 mutation detected during the course of their therapies.
Sixty-one patients had 112 FISH analyses performed simultaneously on PB and BM samples. BM CTG results were available in 54 of those patients. As shown in Figure 1, a very strong correlation (r) was found between PB FISH and BM CTG (r = 0.89; P < .0001) (Fig. 1a) and PB FISH and BM FISH (r = 0.95; P < .0001) (Fig. 1b), as well as BM FISH and BM CTG (r = 0.95; P < .0001) (data not shown). When the analysis was restricted to patients with detectable Ph+ by BM CTG, the correlation between BM CTG and both PB FISH (r = 0.82; P < .0001) (data not shown) and BM FISH (r = 0.92; P < .0001) (data not shown) remained very strong.
Sixty-four patients had 132 Q-PCR analyses performed simultaneously on PB and BM samples. BM CTG results were available in 58 of those patients. As shown in Figure 2, a very strong correlation was found between PB Q-PCR and BM CTG (r = 0.82; P < .0001) (Fig. 2a) and PB Q-PCR and BM Q-PCR (r = 0.87; P < .0001) (Fig. 2b), as well as BM Q-PCR and BM CTG (r = 0.78; P < .0001) (data not shown). Twelve patients in complete CTG remission and with negative FISH had 37 detectable BCR-ABL1 by Q-PCR on simultaneously obtained BM and PB samples. The correlation between BM and PB Q-PCR was very strong (r = 0.96) (data not shown).
FISH and Q-PCR in Patients With Ph+ Variants and Additional Chromosomal Abnormalities
We assessed correlations between PB and BM assays in the presence of the Ph+ variant or additional chromosomal abnormalities to the Ph+ variant;. Eighteen patients had a Ph+ variant (n = 2) or additional chromosomal abnormalities (n = 16). The Ph+ variants included: t(9;12) and t(9;19;22), and the chromosomal abnormalities were der(9)t(9;22) (1 case), der(17) (1 case), der(22)t(9;22) (1 case), +8 (1 case), t(15;22) (1 case), and complex abnormalities (11 cases). Correlations between BM CTG and both PB FISH (r = 0.71; P = .01) (Fig. 3a) and PB Q-PCR (r = 0.75; P = .001) (data not shown) were strong. Very strong correlations were found between BM FISH and BM CTG (r = 0.84; P < .0001) (data not shown) and PB FISH and BM FISH (r = 0.92; P < .0001) (Fig. 3b), as well as BM Q-PCR and PB Q-PCR (r = 0.89; P < .0001) (data not shown). However, a weak correlation between BM Q-PCR and BM CTG was observed (r = 0.54; P = .026) (data not shown).
FISH and Q-PCR in Patients With Detectable ABL1 Kinase Mutations
Sixteen patients with IM-resistant CML with a detectable ABL1 kinase domain mutation had 17 simultaneous PB and BM FISH, and 15 PB and BM Q-PCR. Correlations between BM CTG and both PB FISH (r = 0.79; P = .002) (data not shown) and PB Q-PCR (r = 0.86; P < .0001) (data not shown) were very strong. A very strong correlation was also observed between BM and PB FISH (r = 0.95; P < .0001) (data not shown) and PB Q-PCR and BM Q-PCR (r = 0.95; P < .0001) (data not shown). In addition, a very strong correlation was also observed between BM FISH and BM CTG (r = 0.93; P < .0001) (data not shown) as well as BM Q-PCR and BM CTG (r = 0.84; P < .0001) (data not shown). Correlations were not affected by stage of CML or the type of TKI or HHT (data not shown).
PB FISH and Q-PCR Values at the Time of Complete BM CTG Remission
Patients in whom sequential quarterly monitoring was available from the time of diagnosis or CTG recurrence to complete CTG remission were selected. No patient had a positive PB FISH and a complete CTG remission. Three patients had a transient detectable BM Ph+ (2 of 20, 3 of 20, and 1 of 40 Ph+ metaphases/total metaphases analyzed, respectively) in the absence of a detectable BCR-ABL1 by PB FISH. After normalization of the Q-PCR data using log-2 transformation, a median log-2 Q-PCR (± the standard deviation) was determined at the time of various CTG responses. As expected and shown in Figure 4, the highest values were found in patients with newly diagnosed CML, patients with CTG recurrences, and/or patients with no CTG responses, and the lowest values were noted in patients with complete CTG response (P<.01). There was no overlap in the confidence intervals between patients with no/partial CTG responses and patients in complete CTG response.
A cost analysis was performed to compare standard monitoring using BM CTG with PB monitoring using FISH and PCR. The costs for monitoring with BM biopsies was calculated by including professional and technical fees, as well as fees associated with conscious sedation. The costs to monitor patients using PB FISH and Q-PCR were estimated by including professional and technical fees, as well as fees associated with phlebotomies. Costs for standard semi-annual monitoring using BM biopsies for CTG analyses in the first 18 months after the diagnosis of CML was US$11,104, whereas PB monitoring was US$2592 for a difference of US$8512.
Recent years have witnessed major advances in the treatment of patients with CML.16 Treatment of IM-resistant or IM-intolerant CML with second-generation TKIs led to significant and durable responses.17-19 Monitoring for the presence of the balanced translocation between chromosome 9 and 22 remains the gold standard in evaluating CML response to treatment,20 because major reductions in the numbers and the disappearance of Ph+ metaphases are associated with improved survival.1, 21 In addition to predicting outcomes, tracking BCR-ABL1 permits the early detection of loss of CTG response, and a switch in therapy to at least a second-line agent or allogeneic stem cell transplantation. Early intervention may be relevant. In a recent report, patients who were switched from IM to dasatinib at the time of a loss of CTG response had better outcomes compared with those switched during loss of hematologic response.4 In addition, and given that CML is a stem cell disease that will likely not be completely eradicated with TKIs,22, 23 long-term monitoring will remain an essential tool in the management of these patients. Therefore, reliable noninvasive and convenient monitoring methods are desirable.
Reliability of PB FISH and/or Q-PCR to monitor for the presence of BCR-ABL1 has previously been investigated and is summarized in Table 2.6-12 Strong correlations were observed between PB Q-PCR and BM CTG7, 9, 10 or BM Q-PCR7, 9-11 after transplantation, IFN combinations, and IM. Strong correlations were also reported between BM FISH and BM CTG and/or PB FISH during therapy with IFN combinations and after transplantion.6, 8, 12, 24 To the best of our knowledge, the current study is the first report to describe correlations between PB and BM Q-PCR, FISH, and BM CTG in simultaneous samples obtained from patients treated with 1 or more than 1 TKI and/or HHT. These correlations were found to be strong and not affected by the stage of the disease, the type of TKI, and the presence of additional chromosomal abnormalities, Ph+ variants, or ABL1 kinase domain mutations.
Table 2. Summary of Studies Comparing PB and BM CTG, FISH and/or Q-PCR in CML
No. of Patients/ Total Samples/ Paired Samples
Correlation r Value
PB FISH Versus BM FISH
PB FISH Versus BM CTG
BM FISH Versus BM CTG
PB Q-PCR Versus BM Q-PCR
PB Q-PCR Versus BM CTG
BM Q-PCR Versus BM CTG
PB indicates peripheral blood; BM, bone marrow; CTG, cytogenetics; FISH, fluorescence in situ hybridization; Q-PCR, quantitative real-time polymerase chain reaction; CML, chronic myeloid leukemia; IFN, interferon; Ara C, cytosine arabinoside; BU, busulfan; ICE, ifosfamide, carboplatin, and etoposide; BMT, bone marrow transplantation; NA, not available, IM, Imatinib; HHT, homoharringtonine.
Can Achievement of Complete CTG Remission be Predicted Using PB FISH? In a recently reported GIMEMA study in which BM FISH was simultaneously obtained with BM CTG,25 BM FISH reliably predicted complete, but not partial, CTG responses during treatment with IM. In the current study, albeit in a smaller number of patients, a very strong correlation was observed between BM CTG and both BM and PB FISH in patients with detectable Ph+ when monitored on a quarterly basis. In addition and throughout treatment with different agents, no false-positive PB FISH was found when complete BM CTG remission was achieved, and only 5% of patients (3 of 56 patients) had transient low levels of detectable BM Ph+ in the absence of a detectable BCR-ABL1 as measured by PB FISH.
Limitations of the current study include the relatively small number of patients/samples, the retrospective nature of the analysis, and the heterogeneous patient population that ranged from those with newly diagnosed untreated CML to those receiving long-term treatment with IM, and IM-resistant or IM-intolerant patients receiving at least second-line agents. In addition, FISH and Q-PCR are selective assays for the specific detection of BCR-ABL1, and therefore are inadequate to detect emerging clonal abnormalities that may develop in the absence of the Ph+. The significance of these clonal abnormalities occurring in Ph-negative metaphases remains unclear.20, 26-29 Clinical context can help tailor suspicions and guide additional BM examinations. Indeed, these cytogenetic abnormalities tend to be relatively rare (3.4%), frequently transient, are observed in patients previously treated with IFN, are usually detected before CTG remission occurs, and are often associated with late-onset cytopenias.26-28, 30
Well-accepted recommendations for monitoring patients with chronic phase CML receiving IM therapy include BM CTG analyses at 3 months and 6 months, then every 6 months for first 18 months or until complete CTG response.14, 30, 31 Recommendations for the management of patients treated with dasatinib or nilotinib have not been established. The results of the current study, if independently confirmed, may reassure physicians and help reduce the number of routine BM biopsies in patients treated with at least second-line agents and to a certain degree patients receiving IM. In IM-treated patients, after a diagnostic BM has established the phase of the disease, an optimal BM CTG response at 3 months followed by a very low or negative PB FISH percentage at 6 months may spare those optimally responding patients subsequent BM biopsies. These patients can be monitored by PB FISH until FISH negativity, and subsequently by PB Q-PCR to assess molecular response. Restaging BM biopsies would be necessary at the time a rise in PB Q-PCR leads to FISH positivity. Given that interventions at the time of rising levels of Q-PCR without loss of CTG response remain unproven, and the excellent correlations between PB Q-PCR, PB FISH, and BM CTG reported herein, such approach is reasonable. As highlighted in the recent European LeukemiaNet guidelines,31 BM biopsies would still be required in at-risk patients who develop new late-onset cytopenia while receiving IM regardless of Q-PCR levels.21, 26-30 This strategy could also be acceptable for patients with IM-resistant or IM-intolerant CML receiving at least second-line agents, in whom definitions for suboptimal responses are not as well established. This proposed approach would also be financially cost effective in the United States, as suggested by our cost analysis. The approximate saving of US$8500 using PB analyses was based on monitoring during the first 18 months after the initiation of IM for newly diagnosed, chronic phase CML.32 This difference in costs would be magnified, should patients require more frequent monitoring during or beyond the first 18 months of therapy, as is often the case for patients with IM-resistant CML receiving at least a second-generation TKI or HHT.
In conclusion, the data from the current study suggest that PB FISH and Q-PCR are reliable methods with which to monitor CTG response to modern therapy in all phases of CML, and should cautiously be considered a positive step in sparing CML patients “routine” BM biopsies.
We thank Stephanie McMillan, RN; Lori Covais, RN; Marian Shepard, PAC; and Stacie Holloway, RN for their crucial role in securing the simultaneous collection of peripheral blood samples.