| In addition to being a diagnostic tool, FCM analysis has also been used for prognostic purposes. The main caveat, when determining the prognostic significance of biological parameters of neoplastic cells, is that the validity of the results is influenced by various factors such as laboratory methodologies, clinical staging procedures, and therapeutic protocols. Despite such drawbacks, studies have shown that the DNA index may be of prognostic significance in childhood ALL and the S-phase fraction is useful in grading a lymphoproliferative disorder/non-Hodgkin lymphoma (LPD/NHL). The current classification of LPD/NHL according to the WHO scheme is partly based on cellular ontogeny and differentiation rather than on the biological behavior of the tumor. The choice of therapeutic regimens in lymphomas is still based on the grade of the tumor, however, because cell cycle-dependent drugs continue to feature prominently in the arsenal of chemotherapy for LPD/NHL. Therefore, it would be helpful that FCM testing on lymphomas includes DNA and cell cycle analysis, as the S-fraction gives an indication as to whether the tumor has a high growth-fraction (i.e., aggressive, high grade) or a low growth-fraction (i.e., indolent, low grade), which in turn influences the patient’s response to therapy and survival. Determination of the S-fraction by multiparameter DNA analysis is preferable to paraffin-based immunostaining for Ki67 or PCNA (proliferating cell nuclear antigen), where it is not always possible to distinguish proliferating lymphoma cells from the intimately admixed proliferating benign T-cells. The value of DNA ploidy in LPD/NHL as a prognostic factor still remains controversial. The presence of DNA aneuploidy is helpful, however, to identify those cases of suspected peripheral T-cell malignancies in which the hematologic and immunophenotyping data reveal no abnormalities. In Sézary syndrome, DNA ploidy by FCM has been shown recently to be useful for diagnosis and minimal residual disease (MRD) monitoring. Furthermore, the presence of aneuploidy is associated with increased numbers of large cells in the involved tissues. In addition to cell cycle analysis, there have also been attempts to correlate certain antigenic features with the patient’s response to therapy or survival. While there is only limited evidence that the expression of any particular antigen could serve as a reliable predictor of prognosis, it appears that the intensity of CD45 expression affects the outcome in pediatric ALL, and CD38 positivity in CLL is associated with an unfavorable clinical course. More recently, the advent of gene expression profiling by DNA microarray analysis has led to the identification of genes that may discriminate the subtypes of CLL. Most notably, the differential expression of the Zap-70 gene has been shown to correlate with the mutational status of immunoglobulin heavy-chain variable-region (IgVH) genes, the latter being an important prognostic factor in CLL. Because Zap-70 protein can be readily determined by FCM, in contrast with the more labor intensive and costly DNA sequencing analysis for IgVH mutations, Zap-70 is currently viewed as the best surrogate marker of IgVH mutational status in CLL. According to recent studies, the expression of Zap-70 protein identifies a subgroup of CLL with a more rapidly progressive clinical course and poorer outcome. Note that testing for Zap-70 is limited to peripheral blood samples only, however. An important application of FCM analysis is MRD monitoring. With the emergence of new treatment protocols combining high-dose chemotherapy, autologous stem cell transplants and immunotherapy, MRD detection is becoming necessary in clinical laboratories. The applications of MRD monitoring are multifold, depending on the particular hematologic malignancy. For instance, in childhood ALL, the patient’s MRD status during the initial phase of therapy is a powerful prognostic indicator based on which patients can be stratified into different risk groups. Routine monitoring of MRD during clinical remission in acute leukemias also facilitates early therapeutic intervention, so as to reduce the morbidity/mortality associated with overt clinical relapse. Furthermore, MRD assay is a helpful tool for assessingtumor response to new treatment modalities, such as humanized anti-CD33 conjugated to calicheamicin (gemtuzumab ozogamicin) for AML, and the tyrosine kinase inhibitor, imatinib, in Ph1-CML. The use of MRD detection in mature lymphoid malignancies differs from that in acute leukemia, in that it is usually not applicable at the time of front-line treatment because most of the patients, especially those with low-grade disease, still harbor MRD while in “complete” clinical remission after conventional therapy. MRD detection is therefore applied mainly to patients who, because of relapse, receive high-dose chemotherapy with stem cell transplant, and/or immunotherapy such as anti-CD20 (rituximab) or, in the case of CLL, anti-CD52 (alemtuzumab). FCM was thought not to be as sensitive a technique for MRD detection as polymerase chain reaction (PCR)-based methodologies. However, this apparent lack of sensitivity is most likely due to the fact that the number of cells acquired in a standard FCM clinical assay is far less than that used in PCR analysis. Studies have shown that one leukemic cell in 104 to 105 bone marrow mononuclear cells can be detected by FCM when a large number of cells are analyzed, thus achieving a sensitivity level comparable with that of molecular analysis. These two techniques complement each other and are best applied in tandem to reduce any potential false-negative results. The FCM approach has the advantage of being less labor intensive and achieving a faster turnaround time. Furthermore, the ability of FCM to separate viable from dying cells permits a more accurate quantitation of MRD levels. Irrespective of the methodology, it appears that the clinically significant MRD level is 0.01% (i.e., 10–4). The presence of residual leukemic cells above this level at the end of therapy or an increase in MRD levels in consecutive bone marrow samples during clinical remission has been shown to be associated with a higher risk of relapse and a poorer overall survival, and it tends to correlate with adverse cytogenetic abnormalities. MRD studies have not been feasible in all patients with acute leukemia, however. Oligoclonality, clonal evolution, lack of specific leukemia sequences or absence of nonrandom genetic abnormalities are some of the limiting factors to PCR-based MRD detection. Similarly, monitoring MRD status by FCM analysis can only be achieved if the leukemic blasts exhibit specific antigenic features differentiating them from their normal counterparts. In precursor B-ALL, it has been shown recently by DNA microarray analysis that a significant number of genes are overexpressed in leukemic cells in comparison with normal B-cell progenitors in the bone marrow. Of the several proteins encoded by the overexpressed genes, CD58 has become the marker of choice for MRD monitoring by FCM analysis because the protein is consistently overexpressed in a large number of patients with precursor B-ALL, and fluorochrome-conjugated anti-CD58 antibodies are commercially readily available. |
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