FISH Adequate to Follow CML after Response
Abstract & Commentary
By Andrew S. Artz, MD, Division of Hematology/Oncology, University of Chicago. Dr. Artz reports no financial relationships relevant to this field of study.
Synopsis: Chromosome banding analysis (CBA) of marrow metaphase cells is the standard method to assess response in CML. The authors compared CBA to interphase fluorescence in situ hybridization (I-FISH) in 664 samples where both methods were performed. Of the 537 samples showing complete cytogenetic remission (CCyR) by CBA, I-FISH was < 1% in 82.7%. Of 451 cases with less than 1% positive cells by I-FISH, 444 (98.4%) were in CCyR by CBA. PCR for BCR/ABL showed major molecular responses were more likely for I-FISH less than 1% relative to those with I-FISH 1% to 5% (66.8% vs. 51.6%, p < .001). This difference by I-FISH was also found when restricted to samples showing a CCyR by CBA. A cutoff-value of 1% for I-FISH was established. I-FISH is more sensitive than CBA and could be used to monitor once a CCyR is established by conventional cytogenetics.
Source: Testoni N, et al. Chronic myeloid leukemia: A prospective comparison of interphase fluorescence in situ hybridization and chromosome banding analysis for the definition of complete cytogenetic response: A study of the GIMEMA CML WP. Blood. 2009;114:4939-4943.
Chronic Myeloid Leukemia (CML) is characterized by the Philadelphia chromosome (i.e., 9;22 translocation) created by the fusion of the ABL gene on chromosome 9 and the BCR gene on chromosome 22. As a consequence, activation of protein tyrosine kinase drives leukemogenesis and also enables accurate diagnosis and disease monitoring. The traditional method for monitoring chromosome abnormalities has been cytogenetic banding. For known translocations, fluorescence in situ hybridization (FISH) can be used. In addition, the BCR-ABL may be detected and quantified by reverse transcriptase polymerase chain reaction (PCR).1
Imatinib inhibits the aberrant tyrosine kinase activity of the Bcr-Abl fusion protein and has considerable activity.2,3 Achieving a complete cytogenetic response (CCyR), as measured by chromosome banding analysis (CBA) of marrow cells, defines response to therapy. However, CBA analysis is time consuming and may be hampered by inadequate marrow samples. Increasingly, FISH is being used to determine CCyR, but prospective data on outcomes are lacking based on I-FISH.
Testoni et al pooled data from three CML trials evaluating various doses of imatinib. CBA and FISH studies were prospectively performed on marrow samples at six months and one year to assess response. The studies evaluated 567 patients, and each patient may have had several marrow examinations. Of these, 90% had evaluable cytogenetics. Among the 537 cases of CCyR by CBA, 71 (13.2%) had 1% to 5% positive cells by I-FISH and 22 (4.1%) had more than 5% positive cells. For the 77 cases of partial CyR by CBA, I-FISH revealed seven (9.1%) had less than 1% of positive cells, 32 (41.6%) had 1% to 5%, and 38 (49.3%) had greater than 5% positive cells. Alternatively, of the 451 cases of < 1% of cells by I-FISH, 98.4% had 0% (CCyR) by CBA. For I-FISH with 1% to 5% or more than 5%, CBA showed 68.9% and 36.7%, respectively.
PCR for BCR-ABL showed that molecular responses were similar for CCyR by CBA or < 1% positive cells by I-FISH. Among CBA with no Philadelphia chromosomes detected (CCyR), the molecular response was 66.1% when I-FISH was < 1%, compared to 49.4% for those where I-FISH was greater (p = 0.004).
Ironically, the advent of highly effective therapy for CML also has introduced a welcome challenge the need to accurately measure complete responses. Data from the large imatinib versus interferon trial confirmed the essential value of achieving a complete cytogenetic remission (CCyR)4 as a gold standard, indicating a very low risk of disease progression. The standard assessment requires metaphase cytogenetics analyzed by chromosome banding analysis (CBA). Not infrequently, samples are inadequate. Further, outside of large university centers, most facilities transport samples to reference laboratories, leading to problems and delays which may further reduce yields. FISH allows rapid analysis, fewer inadequate samples, and analyzes a larger number of cells. By using dual-fusion probes with FISH, false positives are drastically reduced, leading to lower thresholds for cutoff values to around 1%. Still, lack of data for FISH has led to uncertainty in interpreting FISH results, especially when discordant with CBA.
In this prospective study, Testoni et al show interphase FISH may be superior to standard CBA of marrow metaphase cells. Most samples (98.4%) showed a CCyR by CBA (i.e., no Philadelphia chromosome-positive cells). Interestingly, for samples showing CCyR by CBA, I-FISH detected 13.2% of samples with 1% to 5% positive cells and 4.1% of samples with more than 5% positive cells. PCR for BCR/ABL confirmed that greater numbers of cells by I-FISH translated into reduced chances of major molecular responses. The added sensitivity of FISH over standard CBA for detecting low-level disease among those with a CCyR by CBA was not surprising. CBA typically analyzes 20 cells and, sometimes, fewer cells are available. The utility of FISH likely would be even greater if one accounted for samples with failed CBA but where FISH could still be performed.
As Testoni et al point out, CBA still retains value. If I-FISH becomes positive, CBA also may be needed. Importantly, only CBA can detect non-Philadelphia chromosome-acquired abnormalities, which is particularly relevant if loss of response occurs. For now, metaphase cytogenetics by CBA should be performed on subjects receiving tyrosine kinase inhibitors for CML. However, once a CCyR is reached, I-FISH may have additional value, or could be substituted. Other techniques of monitoring, such as I-FISH or molecular monitoring from the blood, hold promise, particularly once a cytogenetic remission is confirmed from the marrow.
In conclusion, interphase FISH is more sensitive to detect residual Philadelphia chromosome-positive cells once in complete cytogenetic remission by standard marrow CBA. FISH is an option to confirm continued CCyR previously documented by CBA.
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2. Druker BJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001;344: 1031-1037.
3. O'Brien SG, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med. 2003; 348:994-1004.
4. Hochhaus A, et al. Six-year follow-up of patients receiving imatinib for the first-line treatment of chronic myeloid leukemia. Leukemia. 2009;23: 1054-1061.