Qualitative RTPCR

Qualitative RT-PCR has several attributes that make it well suited for initial diagnosis. Bone marrow aspirate and peripheral blood are both suitable and comparable speci-mens,27 obviating the need for frequent invasive procedures, while sensitivity and rapid turnaround facilitate initiation of therapy.

Methodologically, qualitative RT-PCR can be performed with a simple, nested, or multiplex approach. However, nested methods are suboptimal due to a significant risk of PCR contamination and consequent false-positive results. With nonnested, simplex RT-PCR, one assay is performed using a single pair of primers. Primers for BCR exon 13 (b2) and ABL1 exon 2 (a2) identify both the e13a2 (b2a2) and e14a2 (b3a2) fusion transcripts that differ in size by 75 base pairs (bp) (Figure 35-5a, lanes 1 and 6, respectively). Using PCR primers to these regions allows for the molecular detection of CML in nearly all cases (~99%); however, variant breakpoints occur. When variant breakpoints fall outside of the region recognized by the primers, a false-negative RT-PCR result occurs, whereas if the variant breakpoints fall within the primer region, an RT-PCR product of unexpected size results. Variant breakpoints are diverse and often complex and cannot be globally assessed with any one pair of PCR primers; however, they are quite rare and are thus not a major cause of false-negative results. The incorporation of an ABL1 exon 3 (a3) primer in place of an exon 2 primer permits the routine detection of rare ABL1 intron 3 breakpoints and has been recom mended by some for routine evaluation.28 However, there have been fewer than ten patients with CML reported with this variant breakpoint, which is similarly rare in ALL.

Multiple fusion transcripts may be detectable with a single BCR intron 14 breakpoint due to alternative splicing of the primary transcript. With this breakpoint, the resultant chimeric gene is always directly transcribed to produce the e14a2 (b3a2) transcript as described above; however, concomitant alternative splicing may yield an accompanying e13a2 (b2a2) (Figure 35-5a, lane 2) and even an e1a2 (Figure 35-5a and b,lane 6) transcript.29,30 e1a2 alternative transcripts may also be seen with breakpoints following e13 (b2). Identification ofthe e1a2 transcript requires a separate BCR primer targeting BCR exon 1 (e1) (Figure 35-5b, lane 6). Although the e1a2 primer set usually is not essential for the diagnosis of CML, some studies suggest that the presence of this alternatively spliced transcript is associated with a poor prognosis and transformation to accelerated phase.8

In the diagnostic setting, qualitative RT-PCR serves several functions. The presence or absence of BCR-ABL1 transcripts distinguishes CML from leukemoid reactions

Diagnosis

Tool

Rationale

Cytogenetic karyotyping

Documentation of:

• Philadelphia chromosome

• Other chromosomal abnormalities a

Documentation of: • Specific fusion transcript

Monitoring

Therapy

Tool

ASCT 1

Interferon-a/imatinib mesylate 1

Chemotherapy

ASCT 1

Tool

Interferon-a/imatinib mesylate 1

All therapies

Chemotherapy

All therapies

Figure 35-4. Specific laboratory approaches to the (a) diagnosis and (b) therapeutic monitoring of CML. See also Figure 35-7. ASCT, allogeneic stem cell transplantation; FISH,fluorescence in situ hybridization; RT-PCR, qualitative reverse transcription-polymerase chain reaction; Q-PCR,quan-titative reverse transcription-polymerase chain reaction. Recent recommendations propose that FISH is no longer an appropriate method for monitoring patients in complete cytogenetic remission.78

Periodic cytogenetic karyotyping: ? clonal evolution

Figure 35-5. Conventional qualitative RT-PCR for detection of the most common BCR-ABL1 fusion transcripts using gel electrophoresis. (a) BCR exon 13 (b2) and ABL1 exon 2 (a2) primers. (b) BCR exon 1 (el) and ABL1 exon 2 (a2) primers. (c) ß2-microglobulin primers. Lanes: (1) BCR intron 13 breakpoint, (2) BCR intron 14 breakpoint with alternative splicing to BCR exon 13,(3) possibly novel breakpoint or nonspecific band, (4) BCR intron 13 breakpoint, (5) BCR intron 13 breakpoint, (6) BCR intron 14 breakpoint with alternative splicing to BCR exon 1. See text for details. (Used with permission from Vergilio JA and Bagg A. Chronic myeloid leukaemia—Molecular diagnosis and monitoring. In: Encyclopedia of Medical Genomics and Proteomics (EMGP). Jürgen Fuchs and Maurizio Podda,(eds.) Marcel Dekker,2005:252-258.)

Figure 35-5. Conventional qualitative RT-PCR for detection of the most common BCR-ABL1 fusion transcripts using gel electrophoresis. (a) BCR exon 13 (b2) and ABL1 exon 2 (a2) primers. (b) BCR exon 1 (el) and ABL1 exon 2 (a2) primers. (c) ß2-microglobulin primers. Lanes: (1) BCR intron 13 breakpoint, (2) BCR intron 14 breakpoint with alternative splicing to BCR exon 13,(3) possibly novel breakpoint or nonspecific band, (4) BCR intron 13 breakpoint, (5) BCR intron 13 breakpoint, (6) BCR intron 14 breakpoint with alternative splicing to BCR exon 1. See text for details. (Used with permission from Vergilio JA and Bagg A. Chronic myeloid leukaemia—Molecular diagnosis and monitoring. In: Encyclopedia of Medical Genomics and Proteomics (EMGP). Jürgen Fuchs and Maurizio Podda,(eds.) Marcel Dekker,2005:252-258.)

or other myeloproliferative disorders. A positive qualitative result is critical for the diagnosis of CML, particularly in those patients lacking the Philadelphia chromosome on routine karyotypic analysis. The presence of BCR-ABL1 transcripts validates the implementation of targeted therapy with imatinib mesylate, while identification of the type of fusion transcript that is specific to the neoplastic clone is important for subsequent monitoring. Although the majority of patients with CML possess the e13a2 (b2a2) or e14a2 (b3a2) transcript, identification of the e1a2 or e19a2 forms may explain unusual hematologic phenotypes (Figure 35-2c).

Thus, detection of the most common BCR breakpoints (e1, e13, and e14) and the common (a2) as well as variant (a3) breakpoints involving ABL1 can be accomplished with two parallel conventional RT-PCR assays that use BCR-exon 1/ABL1-exon 3 and BCR-exon 13/ABL1-exon 3 primers. An independent RT-PCR assay for an unrelated mRNA (e.g.,B2M,GAPDH,BCR,or ABL1) is also necessary as a quality control to assess the integrity of the RNA and the presence of RT-PCR inhibitors (Figure 35-5c). These three reactions may be multiplexed to allow for the simultaneous detection of the various transcripts, but multiplexing may reduce the sensitivity of the individual reactions. While reduced sensitivity is not relevant for diagnostic testing, it is very relevant for monitoring residual disease.

In addition to its role at diagnosis, qualitative RT-PCR had been the mainstay of minimal residual disease assessment. As noted, however, a single positive (or negative) qualitative result is of little clinical predictive value. Consequently, quantitative RT-PCR has emerged as a preferred modality for posttherapeutic monitoring and has further refined the prognostic significance of standard qualitative results.

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