Bcrabl In Diagnosis And Monitoring Of Minimal Residual Disease

Cytogenetic studies detect the Ph translocation in more than 90 percent of patients with CML in chronic phase. However, the sensitivity of cytogenetic analysis is low (1 to 5 percent) due to the limited number of metaphases that are examined (usually 20 to 25). Cytogenetic analysis is nevertheless pivotal in two areas: 1. at diagnosis where almost 100 percent of CML progenitor cells in a sample express the Ph translocation; and 2. for the identification of additional karyotype abnormalities in the course of the disease (clonal evolution). Several molecular techniques have been developed that allow a more precise assessment of measuring subclinical disease and are therefore more suitable to monitor the levels of residual disease during therapy and their impact on disease relapse. Molecular studies are also pivotal at diagnosis in the 10 percent of patients who will not show a Ph chromosome by cytogenetic analysis. About two-thirds of these patients will be positive for BCR-ABL by either Southern blotting or polymerase chain reaction (PCR) and their clinical course and prognosis is identical to patients who are Ph-positive.

Fluorescence in situ hybridization (FISH) and PCR have become the two most important molecular tools that are used to monitor the response of patients under treatment. FISH detects specific DNA target sequences with fluorescent-color-tagged probes on interphase (nondividing) cells (interphase FISH, or I-FISH) and/or metaphase (dividing) cells (hyperme-taphase FISH, or H-FISH). In both cases, FISH permits analysis of a large number of cells (> 500) in a timely and efficient manner. Further advantages are that FISH can assess the status of BCR-ABL involvement in various cell lineages as much as in single cells, and that the results of FISH are easily quantifiable. I-FISH can be done on peripheral blood samples, but false-positive results occur in up to 10 percent of the patients. In contrast, no false-positive results are observed with H-FISH, but H-FISH cannot be done on peripheral blood.147-149 FISH techniques that use double-color probes have been introduced for the diagnosis and monitoring of Ph-positive leukemias. Although initial results are promising in view of its superior sensitivity and specificity, not enough data are available to validate its impact in the clinical practice.150

Most centers use PCR or one of its variants as their method of choice to detect residual disease on a molecular level. PCR has an unsurpassed sensitivity and can identify one malignant cell among 104 to 106 normal cells. PCR depends on recognition of a specific primer sequence followed by a target sequence of variable length that is exponentially amplified in a multistep, temperature-regulated reaction cycle. The target sequence may be a leukemia-specific cytogenetic abnormality such as the Ph translocation, or a clone-specific sequence of rearranged immunoglobulin or T-cell receptor genes. In many chromosomal translocations, breakpoints vary or occur within large intronic DNA sequences. In these cases, fusion mRNA (made up entirely of transcribed coding exons) is incubated with the enzyme reverse transcriptase (RT), and the resulting cDNA is amplified by PCR (RT-PCR).151,152

Cytogenetic remissions can be achieved by allogeneic stem cell transplantation in 50 to 80 percent of patients with CML in chronic phase, and in about 30 to 40 percent of patients who have been treated with interferon-alpha. Using PCR, BCR-ABL transcripts can still be detected in up to 30 percent of patients who are in a complete cytogenetic remission after stem cell transplantation and in almost 100 percent of patients who are treated with interferon-alpha.153 However, Kurzrock and colleagues154 have demonstrated that these patients may become PCR-negative if followed long enough.

Many studies have used PCR assays to detect residual disease and have correlated these findings with the outcome of stem cell transplantation and treatment with interferon-alpha. Some observations have emerged from these trials. Qualitative measurements, especially at single time points, are not suitable to predict relapse. Most studies have demonstrated a high incidence of PCR positivity up to 6 months after transplant without evidence of cytogenetic or hematologic relapse.155-157 In a multivariate analysis of 346 patients with CML who underwent allogeneic stem cell transplantation and were monitored by PCR, detection of BCR-ABL at 6 to 12 months after the transplant was associated with relapse.158 Serial and quantitative evaluations of the expression of BCR-ABL suggest that a consistent evolution pattern exists prior to relapse in that rising or persistently high levels of residual disease precede clinical relapse, whereas stable, low, or falling levels predict continued remission.159

Molecular diagnosis and monitoring in CML has also illuminated important biological aspects of residual disease and interactions between host and leukemic cells. The observation that patients who are in long-term clinical remission can still harbor BCR-ABL transcripts and not relapse offers several possibilities. A specific genetic abnormality may characterize the leukemic cells, but may not be more than a marker that by itself is not able to confer the residual CML cell with the leukemic phenotype. Residual clones of leukemic cells may also establish subclones that have lost the original molecular markers and thus evade detection by PCR or FISH. Tumor cells may survive in a state of tumor dormancy during which the disease burden is either so small or certain host immune mechanisms are so efficient that the residual cells are unable to establish the clinical manifestations of the disease. It should be emphasized, therefore, that a positive PCR reaction does not always equal relapse, and a negative PCR reaction is not always absence of any disease. Therefore, at least in some cases, cure can be understood as "functional" rather than "molecular", that is, the absence of all evidence of disease.160

In addition to biological characteristics of the disease, PCR is still faced with technical challenges that may lead to both false-negative and false-positive results (contamination of the samples, discrepancy of results between different laboratories, sensitivity too high). End-point measurements (analysis of the reaction product after amplification is completed) are commonly used for quantification. These measurements are based on multiple dilutions or coamplification of standards (internal or external to the reaction system) and are cumbersome, error prone, and technically demanding. Real-time PCR is a more recent kinetic quantification technique in which the data are acquired during the actual amplification process in a closed system by using fluorogenic probes or intercalating dyes. The kinetic PCR quantification method is more efficient, accurate, and less time-consuming than end-point PCR quan-tification.161

Molecular analysis by PCR has become a cornerstone technique for the diagnosis and monitoring of the response during therapy in patients with CML. Its future impact on clinical decision making for these patients will depend largely on the repro-ducibility of the results from PCR assays and its correlation to clinical response.160

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