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The Value of the Bone Marrow Aspirate, Biopsy: Part 3

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Vol. 13 •Issue 4 • Page 22
PERSPECTIVES IN PATHOLOGY

The Value of the Bone Marrow Aspirate, Biopsy: Part 3

(Editor's note: This is the conclusion of a three-part series that reviews methods to obtain adequate bone marrow specimens and assure the optimal utilization of this technique in patient care.)

The use of conventional chromosome banding for monitoring the patient response to treatment and the early detection of disease relapse has been supplemented in recent years by molecular analysis of bone marrow specimens.

Molecular Techniques

Fluorescence in situ hybridization (FISH) is an alternative to chromosome banding that uses fluorochrome-labeled DNA probes specific for structural and numeric chromosomal abnormalities, including microdeletions and microduplications. FISH is technically rapid and easy to perform and does not require dividing cells. Multiple myeloma is an example of a disease that has greatly benefited from FISH and other molecular techniques of analysis. Not only have new drugs with novel mechanisms of action (thalidomide, arsenic trioxide and the proteasome inhibitor PS-341) been introduced into clinical trials, but the unraveling of the genetics of the disease has led to more accurate prognostic markers. Since plasma cells are difficult to assay with conventional cytogenetics, interphase FISH for immunoglobulin gene translocations (i.e., deletion of 13q14, t(11;14)(p16;q32), t(11;14)(q13;q32), t(14;16)(q32;q23), etc.) has been used for prognostic stratification and minimal residual disease (MRD) detection after bone marrow transplantation. In the future, these discoveries may lead to targeted chemotherapy, such as the use of tyrosine kinase inhibitors to target the ectopic expression of receptor tyrosine kinase fibroblast growth factor receptor 3 associated with the t(4;14) translocation.65

PCR

Besides FISH and cytogenetics, many of the other molecular assays are based on amplification, specifically the polymerase chain reaction (PCR). The chief advantage of such molecular analysis is sensitivity, but other virtues of PCR include minimal tissue requirements, shortened laboratory turnaround time, detecting submicroscopic abnormalities, and the fact that such assays do not require fresh or dividing cells.66 PCR requires strategic use of nucleotide sequences that flank the genetic region of interest specific to the particular lesion. Exploiting fully automated and repeated cycles of DNA denaturation, primer annealing and extension with a thermostable DNA-polymerase, PCR serves to generate an exponential amount of amplified product of a targeted genetic sequence. Such amplified product can then be detected through a variety of methods, including gel electrophoresis or by solution-based means such as fluorescent or chemiluminescent systems.

As the World Health Organization has recently demonstrated in its classifications, genetic analysis of lymphomas and leukemias has prognostic and therapeutic implications.67 The molecular techniques in use today, and those being developed in the future, demonstrate either rearrangements in antigen receptors or translocations specific to hematologic or lymphoid disease (Fig. 3).66 Immunoglobulin (Ig) and T-cell receptor (TCR) genes undergo considerable rearrangement during immunologic maturation. Monoclonal expression of Ig and/or TCR is sometimes, but not always, expressed in lymphoid malignancies and is commonly detected by well-characterized PCR methods.

Similarly, there is a continuously expanding menu of assays using either PCR or reverse transcriptase PCR (RT-PCR) to detect chromosomal translocations described in hematologic malignancies. Examples from this list are t(11;14), t(9;22) and t(15;17) of mantle cell lymphoma, chronic myelogenous leukemia and acute promyelocytic leukemia, respectively. Both FISH and various types of PCR have proven highly sensitive for monitoring the BCR-ABL gene in patients receiving imatinib mesylate (Gleevec) chemotherapy or bone marrow transplantation.68-70 In childhood ALL, the presence of clonal antigen receptor gene rearrangements (IgH or TCR) at 12 or 24 months from diagnosis are highly predictive of relapse and were reported to have independent prognostic significance.71

Most PCR methods are qualitative and are most useful for the diagnosis of disease. However, a variety of methods now allow PCR quantitation.72 Monitoring MRD is made possible by quantitative PCR and its virtues of highest sensitivity, specificity and reproducibility compared to non-PCR molecular methods. For example, RT-PCR for the detection of BCR-ABL mRNA is up to four orders of magnitude more sensitive than cytogenetics or FISH (Fig. 4).73 Past studies have shown the utility of minimizing submicroscopic ALL, as detected by PCR, and that eradication of all leukemia cells may not be necessary for cure.74 Detection of 10-5 levels of the BCR-ABL transcript using RT-PCR for allogeneic bone marrow transplant recipients for treatment of CML over a six- to 12-month window is predictive of future relapse.75

In the future, monitoring of patients over time intervals or measuring more than one lesion at a time with PCR assays may be necessary in patient scenarios.76 A recent study has demonstrated that simultaneous detection of seven common leukemia translocations, using a multiplex RT-PCR system from a single patient specimen, is extremely valuable for clinical practice.77 Other studies have demonstrated the use of peripheral blood for monitoring disease by PCR may be used instead of bone marrow.78,79 Thus, the noninvasive use of peripheral blood may be used for molecular methods in the future in lieu of bone marrow specimens for certain diseases or translocations. The future of PCR-based tests will bring on an increased number of assays and more sensitive tests. What must be kept in mind is that false-negative PCR results do occur. Thus, the molecular-based assays in the future will still be used in combination with bone marrow morphology and other diagnostic tools.

Cytogenetics

Immunosuppressive and myeloablative chemotherapy and pre-transplant conditioning regimens may allow residual recipient hematopoietic cells to coexist with reconstituted hemopoietic tissue of donor origin following bone marrow transplantation. This extraordinary immunogenetic state, known as mixed chimerism, exerts a significant influence on transplant outcome, including the rate of relapse, severity of graft vs. host disease and antileukemic effect.80-83 Polymorphic markers of the donor enable differentiation from the host.

Drs. Riley, Idowu, Chesney, Clark, Massey and Ben-Ezra are with the Department of Pathology, Medical College of Virginia Hospitals of Virginia Commonwealth University, Richmond.

For a list of references and related graphics go to www.advanceweb.com and click through to ADVANCE for Administrators of the Laboratory.




     

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