Case study 5.2
A 37-year-old white female with normal karyotype CD1a−, CD8−, CD5weak, early T-ALL has completed course I, module A, of the hyper-CVAD regimen. She is asking you about her prognosis.
- You would quote the known literature about intermediate-risk T-ALL that achieves CR after one course of induction
- You would recommend measuring MRD by T-cell gene rearrangement at this point
- You would explain that measurement of MRD, by either T-cell gene rearrangement or flow cytometry, is not sensitive enough at the end of induction
- You would recommend measuring MRD by flow cytometry at this point
The first method to separate leukemic blasts from the normal constituents of the marrow was the measurement of nuclear terminal deoxynucleotidyl transferase and T-cell markers. This was followed by the development of monoclonal antibodies and clinical flow cytometers that led to the current use of multiparameter flow cytometry (MFC). Simultaneously, polymerase chain reaction (PCR) was developed initially to measure fusion transcripts such as BCR–ABL or MLL rearrangements and later to measure antigen receptor genes. These methods became the current mainstay of measuring MRD.
MFC can be used to detect three different leukemia-associated immunophenotypes. The first set relates to proteins that are tissue restricted, such as T-cell markers that are thymus restricted. Those are obviously limited to T-lineage ALL. The second set of leukemia-associated immunophenotypes is expressed by fusion proteins. One such example is the expression of high-molecular-weight melanoma-associated antigen, the human homolog of the rat NG2, on the surface of 11q23-positive ALL. The third set of leukemia-associated immunophenotypes is exemplified by an abnormal combination of markers usually present during lymphohematopoiesis.
PCR to measure fusion transcripts can distinguish leukemic blasts from normal cells in cases that harbor such transcripts; those are present in approximately 50% of adult ALL. Alternatively, PCR can measure clonal rearrangement of immunoglobulin and T-cell receptor genes. The first step in such an assay is to screen for clonal rearrangement by using PCR primers that match the opposite ends of various V and J regions of the immunoglobulin and T-cell receptor genes. The product is then sequenced, and the results can be used to design patient-specific oligonucleotides. Of note, T-cell receptor gene rearrangements are present in up to 95% of B-lineage ALL, and immunoglobulin gene rearrangements are present in approximately 20% of T-lineage ALL.
The benefit of MFC is that the leukemia-specific immunophenotype can be readily defined at diagnosis and used to detect MRD at a level of 0.01%. A limiting factor for the reliability of the assay is the number of cells to be assayed. For example, to detect one leukemic cell in 10,000, at least 100,000 cells have to be evaluated because 10 leukemic events are the minimum required for results interpretation. Finally, immunophenotype shifts at relapse have been described, decreasing the assay sensitivity.
The strength of the PCR technique to evaluate fusion transcripts is the association between the molecular aberration and the leukemic clone. However, the amount of transcript per leukemic cell may vary among patients with the same genetic aberration. Recently next generation sequencing emerged as a sensitive method for MRD detecting one cell in a million mononuclear cells.
The advantage of the PCR technique to evaluate antigen receptor genes is that the rearranged gene is present in one copy per cell, allowing quantitative PCR to accurately measure MRD. However, these genes may undergo secondary recombination events during the disease course, resulting in oligoclonality. This will complicate the ability to detect MRD because it cannot be predicted which subclone will cause relapse. Therefore, most will recommend measuring at least two markers.
The timing of measuring MRD is not yet standardized. In the largest study for de novo ALL, the Associazione Italiana di Ematologia Pediatrica (AIEOP)-BFM-ALL 2000 study, children were evaluated for MRD by PCR for antigen receptor gene expression at the end of induction (day 33) and at the end of induction consolidation (day 78). Interestingly, for the 3184 pre-B-ALL children, measuring MRD at the end of induction was highly predictive of relapse. However, for the 464 children with T-ALL, measuring MRD at the end of induction consolidation was the most important predictive factor of relapse. Specifically, the outcome of patients who were MRD-negative at day 78 was independent of their MRD status at day 33. No such studies exist in adult ALL.
Finally, a comparison of MFC and antigen receptor gene expression by PCR was recently conducted in 102 children and 136 adult ALL cases. Good concordance was detected between the two methods. Specifically, 13 samples, out of a total of 598 samples, were positive by the antigen receptor assay but negative by flow cytometry, and nine were vice versa. The conclusion was that if standardization and good quality control are maintained, both techniques are equal for MRD measurement.
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13: Hematopoietic Cell Transplantation in Acute Myeloid Leukemia
11: Minimal Residual Disease in Acute Myeloid Leukemia
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