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References Publications referenced by this paper. Clinical impact of molecular diagnostics in low-grade lymphoma. Concurrent detection of minimal residual disease MRD in childhood acute lymphoblastic leukaemia by flow cytometry and real-time PCR. Meanwhile, several groups have reported the analysis of chimerism in specific subsets to detect reappearing leukemic cells [27, 34, 58—61] or to monitor the effect of treatment . Lamb et al. Zetterquist et al. Mixed chimerism in the B cell compartment was found in 5 patients who also showed persistence of the clonality marker in the PCR. Mixed chimerism in the B cells was detected 2.
No relapse was observed in those seven cases with complete donor chimerism in the B cell compartment. Mattson et al. They used immunomagnetic labeling with antibodies against CD33, CD7 or CD45 to enrich the specific subpopulations from the peripheral blood or the bone marrow, achieving a final sensitivity between 2 and 4! Mixed chimerism in these populations 1 month after transplantation was observed in 14 of the 30 patients. The basic idea behind this was that the CD34 antigen is expressed on a very small population of normal hematopoietic progenitor cells, but can be frequently detected on blasts of different leukemias [62, 63].
After a median follow-up of days range 28—1, days , a total of 22 relapses were observed in the 84 patients showing engraftment. Since this assay can be performed with peripheral blood, the investigations can be done at short intervals. These data inspired us to start a randomized prospective multicenter trial comparing chimerism from the peripheral blood and subset chimerism within the CD34 compartment in patients with AML, ALL or MDS whose leukemia blasts express the CD34 antigen.
Thus taken together, these data clearly show that subset analysis is a very sensitive technique, with a limit of detection comparable to nested PCR. This technique adds important information, since it is able to clarify whether reappearing host cells are of leukemic origin or are T cells or other nonmalignant cells. In this article we have not focused specially on the use of chimerism in the setting of dose-reduced conditioning, since the detection of MRD is not substantially different after this form of transplantation. However, since mixed chimerism, especially in T cells, is much more common after dose-reduced preparative regimens, subset analysis is even more important to differentiate persistent mixed T cell chimerism from reappearing leukemic cells.
These reports are mainly focused on technical issues. As discussed above these questions are certainly important. However, especially for the detection of MRD, the use of the appropriate methods according to the clinical situation is most important. Based on our own experience and the literature data, our current recommendations for chimerism analyses are as follows: During engraftment and during the entire period of mixed macrochimerism i. When real-time PCR indicates that the level of residual host cells further declines follow-up monitoring with real-time PCR is recommended at regular intervals.
When this method indicates a level of residual host cells below 0. The length of the intervals should be chosen according to the tendency of relapse of the primary disease and the time after transplantation, with more frequent analyses weekly to every 2 weeks performed in patients with high risk disease like AML or ALL and early after transplantation.
This very tight schedule should be followed for the first 2 years after transplantation, since the majority of relapses occur during this period. Based on this information and the clinical situation i. If there is a genetic marker translocation, IgVH rearrangement which can be used for real-time PCR, this is certainly the first choice for MRD detection, since these markers are specific for leukemia. If not, chimerism analysis using the most sensitive and quantitative method available should be used.
We believe that a strategy like this will allow for a more accurate and reliable assessment of chimerism and might help to identify patients at risk of a reappearance of leukemia. However, prospective trials will clearly have to show whether these strategies can be used to achieve a longer leukemia-free survival after transplantation. Oxford, Blackwell Science, , pp — Science ; — Bone Marrow Transplant ;— Cancer Detect Prev ;— J Clin Oncol ; — Semin Hematol ;— Haematologica ;— J Hematother Stem Cell Res ;— Nature ;— Bone Marrow Transplant ; — Leuk Lymphoma ;— Haematologica ; — Bone Marrow Transplant ;— London, Academic Press, , pp 81— Leukemia ;18 2 — Am J Hum Genet ;— Bone Marrow Transplant ;31 suppl 1 :S23—A J Exp Med ;— Ann Hematol ;81 suppl 2 :S27—S Morphological analysis of small populations of cells related either to malignancy or recipient-associated markers may improve the accuracy of chimerism and MRD testing and delineate their clinical significance.
The detection of MRD at any stage of therapy may have a powerful implication for and impact on the clinical management. New international therapeutic protocols base treatment decisions on the level of MRD [1, 2]. The past decade has brought new technologies to the study of MRD in malignancies because the currently used methodologies are far from accurate and sensitive.
Epub Minimal Residual Disease In Hematologic Malignancies (Acta Haematologica 2004)
In addition, it is often difficult to differentiate a small leukemia blast population from normal hematopoietic blasts. When the malignant disease is associated with a specific chromosomal rearrangement conventional cytogenetic analysis and fluorescence in situ hybridization FISH are used to identify the MRD population. Both methods have a low sensitivity of approximately 10 —2 that is not significantly better than the sensitivity of a classical morphological analysis. The quantitative accuracy of FISH depends on the observed frequency and the number of cells scored, and thus is improved by scoring more cells .
However, scoring a large number of cells is not practical with routine FISH. Multiparametric fluorescence-activated cell sorter FACS - and polymerase chain reaction PCR -based techniques have a higher sensitivity of 10 —4 to 10 —6, respectively, for the detection of MRD. However, their false-positive rate is still very high .
Moreover, the detection of MRD with tumorspecific tests may not always be correlated with the risk of relapse. The relevance of the maturational stage of a given cell and the specific signs it presents e. Partial differentiation to more mature cells can occur and these cells may lack the potential to proliferate. It can be suggested that a full morphological analysis of the MRD cells carrying the chromosomal rearrangement would reveal that there are subclasses of cells which are not relevant to disease progression.
These methods as well as bright-field and fluorescence scanning and the combined analysis of morphology and FISH have previously been described in detail .
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Briefly, slides are prepared from PB or BM samples by density gradient centrifugation and cytospin of the final cell suspension containing , cells. Louis, Mo. In a series of experiments it was shown that no white-blood-cell subset is lost or reduced by the preparative procedure. The slides are then scanned by the bright-field mode of the system, based on a dual mode, fully automated microscope Axioplan2, Carl Zeiss, Jena, Germany , an XYmotorized stage with an accuracy of 0. It also classifies the cells into six categories according to their morphology: polymorphonuclears and band cells, lymphocytes, normoblasts, myelocytes, blasts and plasma cells PC.
An experienced morphology technician can observe the images and correct the system errors. The system enables observation of the fluorescent signals in the specific cells using a! Slides with FISH signals are searched either manually or automatically for target cells. When a target cell is identified, the system saves its morphological and FISH images, as well as its coordinates. Therefore, the system can locate and then characterize these cells for the determination of their potential as MRD by the combination of all these characteristics. Cells can be observed by any combination of morphology, FISH or immunohistochemistry.
For example, a large number of cells can be searched for blast morphology or according to CD34 positivity and then the FISH genotype of the cells can be elucidated. The hybridization signals can be removed by incubation in 2! In this way the target cells can be characterized by several cytogenetic markers.
It was suggested that using the system MRD can be accurately detected in hematological diseases because scanning of a large number of cells enables rapid and efficient identification of rare residual leukemia cells, thus increasing sensitivity. The multiple parameter analysis provides phenotypic and genotypic information on the suspected cell, thereby enhancing the specificity. However, if immature, proliferating cells are found to be positive, there is a high probability for relapse.
This assumption was confirmed by the study of Bielorai et al. Consecutive PCR tests were positive for 2. It is therefore possible that a small fraction of long-living lymphocytes originating from the leukemic clone survived despite the cytotoxic treatment. Because mature lymphocytes have a long life span in the circulation, this finding supports the fact that the patient was in remission. Moreover, since mature differentiated cells have a low proliferate capacity, there is a low risk of relapse. It can be suggested that the detection of the leukemic marker in differentiated cells made the probability of relapse very low and had an important clinical application.
Several investigations are focused on the use of the multiparametric system for chimerism testing and the detection of MRD after allogeneic BM transplantation [14—19]. Chimerism testing is used for routine documentation of engraftment and posttransplant follow-up of allogeneic transplant recipients. In sex-mismatched transplants when the recipient and donor are of different gen- Fig.
Combined analysis of morphology or cytochemistry and FISH on the same cell. The blast cells show these trisomies, while the PMN cells are normal two green and two red signals. The aim of these studies was to evaluate the notion that a full morphological analysis of the residual host cells would contribute to the accuracy of the detection of MRD. For this purpose 49 examinations of 31 patients were studied that were either transplanted from a sex-mismatched donor or had a defined chromosomal rearrangement.
Results were retrospectively correlated with outcome. It was shown that in sex-mismatched transplants the system could detect very small populations of recipient cells not detected by routine FISH. Thus the clinical significance of MRD detection was improved by identifying the morphology of recipient cells. In 10 patients, the recipient cells were mostly blasts and 7 of them relapsed 2—8 weeks after the examination 3 died of treatment, before clinical relapse could be documented.
Among the patients with mature hematopoietic morphology 1 died early during treatment and none of the others relapsed. It was concluded that identification of residual recipient-type cells as blasts predicts imminent relapse and these patients need additional therapy. The authors applied further prospectively the combined analyses for clinical management: when a minute recipient blast population was detected, immune suppression was rapidly withdrawn.
Conversely, when residual recipient cells displayed mature hematopoietic morphology, patients were followed by serial testing and it was not necessary to expose them to the hazards of unnecessary treatment. The authors noted that for detection of MRD tumorspecific markers probes are probably superior and more accurate than sex mismatch.
Detection of recipientderived cells does not necessarily correlate with the risk of relapse as recipient cells contributing to mixed chimerism may belong to the malignant clone, may belong to normal hematopoiesis, or may be stromal cells. During the first few weeks after standard allogeneic transplantation, recipient cells can still be detected when sensitive tests are used. These are mostly T cells surviving the conditioning regimen. Host cells may be detected at low levels! Hardan et al. Del 13q is the best-studied chromosomal Acta Haematol ;—29 27 aberration in MM, associated with a lower response rate, progression-free survival and overall survival.
The FISH technology can detect cryptic chromosome 13 abnormalities not detected by conventional cytogenetic analysis and therefore it is strongly recommended for use in MM patients [22—24]. This cutoff level is specifically problematic in BM samples with a low proportion of PC due to the patchy pattern of PC distribution in the BM, difficulty in the BM aspiration and dilution of the sample by blood. In addition, patients with MM and del 13q carry a substantial number of PC without this abnormality . This del 13q -positive population varies in its size and may explain the high false-positive rate.
These data were used as a basis for the determination of chromosome 13 status in the PC population: the percentage of the del 13q -positive cells in the PC population was counted, as well as the percentage of the del 13q -positive cells in the non-PC population. The authors introduced a new index for the characterization of the chromosome 13q status in PC population of the BM: the ratio of del 13q -positive cells in the PC population to del 13q -positive cells in the non-PC population was defined as del 13q index. This del 13q index is high when most of the deletion-carrying cells are PC and low when most of the cells with deletion are non-PC.
In conclusion, combined simultaneous morphological and cytogenetic multiparametric analysis offers advantages that may help disclose the relevance of MRD detection. Adding the morphological analysis of small populations of cells to malignancy or recipient-associated markers may improve the accuracy of chimerism and MRD testing, and delineate their clinical significance.
It seems that the detection of MRD, preceding the appearance of overt leukemia, in such a small subpopulation of cells could only be accomplished by a combined morphological and FISH analysis. This technology merits further study in larger-scale trials. Acknowledgment We thank Ms. Bella Weismann and Dr. Galina Ishuev for excellent technical assistance. References 1 Campana D, Pui CH: Detection of minimal residual disease in acute lymphoblastic leukemia: Methodological advances and clinical significance. Arch Pathol Lab Med ;— Cancer Genet Cytogenet ;— Blood ;86 suppl 1 Br J Haematol ;— Genes Chromosomes Cancer ; — N Engl J Med ; — Israel abstract Biol Bone Marrow Transplant ; Hematol J ;4:S Cancer Res ;— Ann Hematol ;— Am J Pathol ;— Factors which affect the sensitivity and consequently the validity of MRD results are reviewed.
RNA degradation in unstabilized peripheral blood PB samples does not play a major role in samples being processed on the day of blood collection. However, the simulation of sample shipping at room temperature with a delay of sample processing of up to 3 days causes a dramatic loss of intact RNA. Additionally, the stabilizing procedure is capable of keeping real-time quantitative polymerase chain reaction RQ-PCR results comparable whether the sample is processed immediately or with a delay of up to 3 days.
Consistent quantitative data cannot be obtained with unstabilized blood samples. Ten milliliters of PB is sufficient for processing on the day of blood collection whereas the use of only 5 ml PB may result in false-negative results. Standardization of preanalytical and analytical factors is necessary to provide a comparability of RQ-PCR results from different laboratories within multicenter studies. However, investigators are using different protocols which is important when it comes to interpretation of minimal residual disease MRD data.
The drawback of this variety of techniques is the noncomparability of the results caused by nonstandardized procedures concerning the volume of used peripheral blood PB , transit time from patient to laboratory, temperature during transport, RNA extraction meth- Dr. Martin C.
Since most of the PB or bone marrow samples are not processed at the center of blood collection RNA degrades time-dependently during shipment to a specialized laboratory. The use of suboptimal starting template material leads to a random, unpredictable amplification of detectable products, which raises questions about the reliability of single negative PCR tests.
To determine the volume of PB necessary for a sensitive MRD analysis, we evaluated two RNA extraction methods using 5 or 10 ml of freshly drawn PB, each in patients with a complete cytogenetic response on imatinib therapy 11 male, 4 female, median age 65 years, range 44—67 years. The laborious but very efficient CsCl gradient ultracentrifugation method was compared to the PAXgene extraction method.
PAXgene 0. Comparison of 5 vs. CsCl gradient ultracentrifugation. Considering that this trial was performed using immediately processed PB after only 2 h of incubation at room temperature, the problem of RNA degradation could be neglected. Thus, one might conclude that at least 10 ml of PB are needed for MRD analysis provided that early processing of the material is guaranteed. Higher volumes of PB should be used for longer transfer times without stabilization. At least 10 ml of PB seem to be sufficient in CML patients with an excellent response to therapy complete cytogenetic response when processed on the same day.
Furthermore the amount of usable RNA can be significantly increased by the use of the PAXgene stabilization method in case the arrival at the laboratory is not guaranteed on the day of blood collection. The PAXgene system represents an easy to use procedure which immediately stabilizes PB after phlebotomy and makes possible a standardized RNA extraction. Investigators of future clinical studies will need to agree on common preanalytical and analytical procedures such as cDNA synthesis and PCR protocols to achieve comparable MRD results. Our conclusions are derived from the experience with CML patients.
However, the proven prognostic significance of PCR monitoring in other leukemic disorders calls for the use of optimized and standardized protocols regardless of the molecular target. However, best experiences have been made by using random hexamer priming. Summary and Future Directions Optimal sample quality is indispensable for molecular monitoring of leukemic fusion transcripts after therapy. However, the amount of usable RNA after arrival in the specialized laboratory depends on a multitude of factors. Clin Chem ;— Low levels of residual disease are associated with continuous remission.
Leuk Lymphoma ;11 suppl 1 — Blood ;a. Recent technological advancements enable detection of submicroscopic leukemic cells. The extent of reduction in the level of minimal residual disease MRD during the first phase of therapy can be exploited for improved risk classification of children with ALL. If treatment is discontinued at this stage all children will eventually relapse. These facts indicate that at the end of remission induction chemotherapy not all clonogenic malignant lymphoblasts are killed although most of the patients are in clinical and morphological remission.
Thus a low level of clonogenic malignant cells remains in a substantial proportion of the patients even after completion of chemotherapy. Therefore more sensitive techniques for the detection of rare leukemic cells are required. This is the rationale behind the recent incorporation of modern techniques of detection of minimal residual disease MRD into treatment protocols of childhood ALL. For a more detailed information about methodologies and clinical trials the interested readers are referred to several excellent recent reviews and to other papers in this special edition of Acta Haematologica [4—7].
This is not trivial since leukemia is characterized by arrest in the normal developmental program. Thus, leukemic blasts are similar in many respects to normal lymphoid precursors. Therefore, the threshold for detection should be at least one leukemic cell among 1, normal cells i. Since malignant cells are naturally genetically unstable, some potential markers present at the time of diagnosis may be lost later in the disease. A search for such an unstable marker later in the disease may lead, therefore, to false-negative result.
This is essential for conduction of large multicenter studies. Ideally, the techniques should be simple and reproducible enough to be eventually incorporated into the routine clinical laboratory, similarly to cytogenetic and immunophenotyping techniques that are currently routinely used in most large hematological clinical centers. The currently available approaches can be divided into two groups: methodologies that are based on the identification of abnormal phenotypes and techniques that follow aberrant genotypes of the leukemic cell.
The specific phenotype of a leukemic cell is manifested by its morphology and by the aberrant expression of surface and intracellular proteins. An updated review of this technology by Campana and Coustan-Smith  who pioneered this approach for MRD detection can be found in this issue. The advantages of this technology are as follows: 1 Adequate sensitivity one leukemic cell can be detected within normal cells.
Thus potentially this technique can become routine and be less dependent on centrally placed sophisticated laboratories. There are, however, a few disadvantages that currently delay the implementation of FACS-MRD into the clinical routine: 1 The analysis is quite complex and depends on the expertise of the operator. This problem may be overcome by modern communication technology.
In the near future it may be possible to analyze centrally results obtained in the local centers. Thus expert operators have to continuously be available locally for the analysis, which is a major limitation for multicenter studies. This problem may be solved by the approach delineated in 1. This problem can be solved by choosing several leukemia-specific antigens for follow-up of MRD.
There are a few technical issues with these two nucleic acids that may be of clinical importance and thus deserve mentioning here. Genomic DNA is relatively stable and easy to obtain. It may be shipped at room temperature, a big advantage for multicenter studies. The amount of DNA is equal in all somatic cells. Therefore the actual quantity of leukemia-specific DNA correlates with the number of leukemic cells.
Because of its stability, however, it is impossible to distinguish between DNA that originates in living and dying leukemic cells. These enzymes are ubiquitous and stable. These limitations also hold for shipment of RNA. These are major limitations for routine clinical applications. In the near future, however, thanks to significant efforts made by the biotechnology industry, it may be possible to keep and send RNA at room temperature.
Thus, theoretically the same quantity of a leukemia-specific RNA may originate in few cells possessing a lot of this RNA species or in many more cells each producing a small quantity of this transcript. Such studies are in progress . This approach exploits the physiologic process of somatic rearrangement of Ig and TcR gene loci that occur during early differentiation of any lymphocyte.
Thus, any single lymphocyte carries a unique rearrangement that is not shared by any other lymphoid cell. This process ensures the level of diversity of the immune response against an unlimited number of antigens. Since leukemia is clonal, i. The details of the techniques are described elsewhere [6, 12]. Its biggest disadvantages are the costs and complexity. Currently the technique is well standardized. Unlike conventional cytogenetics it does not require dividing cells and can be performed on smears of bone marrow or peripheral blood.
myelodysplastic syndromes | bloodjournal
New promising automated methodologies that combine morphological and FISH examinations are being developed [13—15]. Detection and quantification of leukemia-specific mRNA are rapidly gaining popularity as a reliable, sensitive method at relatively low costs. They con- 36 Acta Haematol ;—39 sist of either fusion mRNA resulting from chromosomal translocations e.
This issue has not been resolved yet. Direct comparisons between these methodologies in a single institution  have shown them to be largely equivocal. For example, the St. Until comparative multicenter standardized studies are published, IgTcR-PCR remains the benchmark, to which any method should be compared. Clinical Significance of MRD Monitoring Several large retrospective analyses of samples collected during prospective clinical trials yielded strikingly similar results table 1 [7, 8, 19—23].
It appears that the pace of reduction in leukemic burden during the first few weeks of therapy is the strongest prognostic indicator that overrides any other conventional prognostic factors. These results strengthen and refine previous observations made by the international BFM group that the extent of early response to therapy is the most important prognostic factor. Still most of the relapses occur in the majority of patients that are good prednisone responders. Monitoring of molecular MRD has refined this group. Thus, for the first time it is possible to prospectively identify those patients who may benefit from reduction in therapy.
This assumption requires some caution, however. The excellent prognosis of the rapid responders may actually indicate that the type of therapy given is effective and suitable. Therefore, a reduction in treatment intensity may result in an increased rate of relapse. Patients receive the same induction therapy and, according to MRD assessment at two time points and some clinical features [presence of t 4;11 or t 9;22 , response to prednisone in the first week, clinical remission after induction], patients are stratified into three different risk groups with tailored therapy.
Intensification of treatment of the high risk group and reduction of treatment of the low risk group is tested in a randomized fashion. The long-term results of this study and comparable studies are likely to show whether this more sensitive and specific evaluation of remission and early response to treatment could speed up further improvement in the cure rates of children with ALL. The value of MRD monitoring during maintenance therapy and off therapy is less clear [20, 23, 25].
Most patients become MRD negative.
The absence of residual disease after remission induction is associated with a good prognosis. If multiple bone marrow samples are analyzed during follow-up, a steady decrease of MRD levels to becoming undetectable is observed in childhood ALL patients. The persistence of residual blasts beyond 4—6 months or the reemergence of residual disease, even at the level of 1! Finally, the value of MRD monitoring has also been shown in the follow-up of relapsed patients. Pretransplant levels highly correlate with the outcome of stem cell transplantation . Questions and Challenges Over the last decade sensitive methodologies for MRD detection have been developed and the prognostic power of MRD monitoring during early phases of therapy has clearly been proven.
The availability of these technologies provides now the opportunity to answer few critical questions: 1 Would tailoring treatment by risk classification based on MRD improve outcome of children with ALL? In particular is it appropriate to reduce toxic treatment to the large group of patients, in which MRD levels drop below 0. Is 10 —4 enough or is it too much? Clearly, if a reduction in therapy proves futile, MRD methodologies do not need to be so sensitive. In addition, since leukemia may be a patchy dis- ease, an extremely sensitive determination of MRD in one bone marrow sample may not reflect the total body burden of leukemia.
Indeed a real challenge is developing new methodologies that will make it possible to estimate the whole body burden and distribution of leukemia during remission. How can MRD quantification be made more cost-effective? Should the definition of relapse be changed ? Is it useful to diagnose molecular relapse based on rising levels of MRD? If yes, what is their significance? The answers to these questions will determine whether the amazing technological achievements will also translate into improved outcome of children with ALL. Acknowledgments We wish to thank Drs.
Gianni Cazzaniga and Andrea Biondi for sharing an unpublished manuscript with us. Supported in part by the Israel Cancer Association. Rev Clin Exp Hematol ;—, — Best Pract Res Clin Haematol ;— A case control study of the International BFM study group. Acta Haematol ;— Acta Haematol ; — Acta Haematol ;— Leukemia ;6: — Raanani I. Nevertheless, approximately two thirds of patients relapse due to persisting leukemic blasts. The persistence of these cells, below the threshold of morphological detection, is termed minimal residual disease MRD and various methods are used for its detection.
These methods include classical cytogenetics, fluorescence in situ hybridization, qualitative and quantitative RT-PCR and multiparametric flow cytometry. The kinetics of disappearance of molecular markers in AML differs between the various types of leukemias, although at least a 2 log reduction of transcript after induction chemotherapy is necessary for long-term remission in all types. Conversely, the change of PCR from negativity to positivity is highly predictive of relapse. Whereas in acute lymphoblastic leukemia, multiparametric flow cytometry is an established method for MRD detection, this is less so in AML.
The reason is the absence of well-characterized leukemia-specific antigens and the existence of phenotypic changes at relapse. On the other hand, this method is convenient due to its simplicity and universal applicability. Several issues still remain to be settled including the choice of the best method and the timing for MRD monitoring and above all the practical clinical implications of MRD in the various types of AML.
Karger AG, Basel Dr. For many years, counting cells and identifying them by microscopic inspection have determined the number of normal or abnormal cells in the bone marrow BM. These traditional methods have very limited sensitivity but recently developed, more sensitive techniques are providing new information of significant value . At diagnosis patients with acute leukemia may have a total of approximately malignant cells. In quantitative terms: a 12 log leukemic cell kill is induced leaving a total leukemic burden of neoplastic cells.
From that point until overt clinical relapse the level of leukemic cells in the body is largely unknown, resulting in clinical management strategies that do not discriminate among patients by their levels of residual disease. Thus, patients with leukemic cells are treated with the same regimen as those with much lower levels or, perhaps, with no leukemia at all [2, 3]. The persistence of residual malignant cells below the threshold of conventional morphological findings is termed minimal residual disease MRD and may identify patients at a higher risk of relapse .
The final goal of detecting low numbers of residual leukemic cells is to obtain a more precise evaluation of the effectiveness of treatment in order to give information on disease prognosis, design patient-adapted post-remission therapies, assess information on the response to chemotherapy, predict impending relapses prior to clinical manifestations, make a better assessment of the quality of the stem cells harvested for autologous transplant and the efficacy of purging methods and facilitate early therapeutic interventions i.
The study of MRD uses modern technical methods to identify disease far below the detection threshold of routine pathology. Methodologies for Detecting MRD in AML If the original leukemic cell carries a molecular or antigenic marker that distinguishes it from nonleukemic cells, then all cells of the leukemic clone will exhibit the same marker. This property allows the application of sensitive new techniques that use either the polymerase chain reaction PCR or antibody to detect or quantify leukemic cells . Each method has relative advantages, disadvantages and sensitivity for detecting MRD .
Conversely, the other methods including conventional cytogenetics, FISH, multiparametric flow cytometry and PCR are much more specific and sensitive [14—17]. Conventional Cytogenetics Karyotype analysis at diagnosis is essential and allows the detection of clonal abnormalities including structural and numerical changes specific to the leukemic clone. Cytogenetics remains one of the most important prognostic factors in AML and can be applied during remission to detect residual leukemia.
An additional benefit of this approach is its ability to detect the acquisition of new clonal aberrations with disease progression . Fluorescence in situ Hybridization FISH can increase sensitivity by screening nondividing interphase cells using chromosome-specific or locus-specific probes. The sensitivity of FISH is between 10 —1 and 10 —3 and the detection of numerical losses or gains of chromosomes is generally more sensitive than the detection of translocations . FISH also allows the identification of cryptic translocations that may be clinically relevant.
PCR is highly sensitive up to 1! The common finding for all molecular subtypes in AML is that the transcript levels are at least 1 log higher in BM than in peripheral blood PB [14—17, 19]. While qualitative RT-PCR methods have been clinically useful in monitoring MRD in specific leukemias, in certain types of leukemia the qualitative detection of the respective fusion gene transcripts did not distinguish patients in durable remission from those at high risk of relapse. For this reason quantifying target genes as a measure of residual leukemia provides a better means for monitoring MRD .
Quantification of the level of transcripts of a target gene can be carried out either by the end point competitive RT-PCR or the cycle-cycle real-time techniques. Competitive RT-PCR assays are labor intensive and use manual protocols that may require up to 48 h to produce results. They require exponential amplification for precise quantifications and the final amount of the PCR product is very sensitive to slight variations in the reaction components that need to be rigorously controlled.
To overcome these shortcomings, real-time RT-PCR techniques for quantification of target sequences have been developed. The principle behind this technique is to estimate the level of PCR products as they accumulate rather than estimating the level of the final products. Consequently, during each subsequent PCR cycle 42 Acta Haematol ;—54 more fluorescence signal will be detected. In the hydrolysis probe technique, the hydrolysis probe is conjugated with a quencher fluorochrome, which absorbs the fluorescence of the reporter fluorochrome as long as the probe is intact.
As Taq polymerase has also the activity of endonuclease upon amplification of the target sequence, the hydrolysis probe is displaced and subsequently hydrolyzed by the Taq polymerase. This displaces the reporter from the quencher fluorochrome and thus the fluorescence of the reporter fluorochrome becomes detectable. Consequently, during each subsequent PCR cycle more fluorescence signal will be detected. Reactions are characterized by the number of cycles after which the fluorescent signal is first detected due to the accumulation of enough reporter molecules released — CT.
In the third method, the hybridization probes technique, one probe is labeled with a donor fluorochrome at the 3 end and a second probe is labeled with an acceptor fluorochrome. When the two fluorochromes are in close vicinity, the emitted light of the donor fluorochrome will excite the acceptor fluorochrome. This results in the emission of fluorescence, which subsequently can be detected.
Consequently, during each subsequent PCR cycle more fluorescence signal will be detected [19, 20]. Although a recent comparison of real-time RT-PCR with competitive RT-PCR using samples from t 8;21 AML patients showed that these two methodologies are comparable for sensitivity, linearity and reproducibility, the former method of analysis appears to offer technical advantages by providing absolute quantification of the target sequence, expanding the dynamic range of quantification to over six orders of magnitude, eliminating the post-PCR processing, and reducing labor and carry-over contamination .
This method has been applied to a variety of molecular markers. For most markers, a lack of decline of transcript levels by less than 2 logs after chemotherapy has been established as a poor prognostic sign. Moreover, increases in transcript levels are almost invariably associated with relapse [14—17, 19]. Multiparametric Flow Cytometry Normal cells display a variety of cell surface antigens in a strictly defined sequence according to cell type and differentiation status. The malignant process often disturbs this programmed expression and the aberrant expression of cell surface antigens can thus be used to distinguish malignant cells from normal cells.
Flow cytometry is potentially quite rapid. However, to fully optimize its sensitivity for detecting the leukemic clones, combinations of several antibodies are needed to define the aberrant antigen expression, requiring considerable technical expertise . In 2- or 3-color immunofluorescence analysis the leukemic cells demonstrate multiple differences from their presumed physiological counterparts due to different light scattering properties and an aberrant or asynchronous antigen expression.
The most frequent abnormal immunophenotype found is an asynchronous myeloid antigen expression, and the most frequent lymphoid markers coexpressed with myeloid antigens are T cell-associated antigens, especially CD7 [12, 14—17, 22, 23]. The kinetics of disappearance of molecular markers in AML are influenced by the various therapeutic regimens but are mainly different between the various types; for example, while molecular remission is achieved within the first 6 months in patients with acute promyelocytic leukemia, PCR markers may persist for several years in apparently cured patients with CBF leukemias [19, 26, 36, 37].
CBF Leukemias AML with translocation t 8;21 q22;q22 and pericentric inversion of chromosome 16, inv 16 p13q22 share some unique phenotypic and clinical properties. In t 8;21 q22q22 or inv 16 p13q22 , failure to detect submicroscopic cryptic rearrangements of the involvedgenes leads to false-negative results.
Sensitive molecular methodologies such as RT-PCR have been successfully used to detect cryptic CBF abnormalities in diagnostic samples of AML patients with karyotypes that are otherwise negative for t 8;21 q22;q22 or inv 16 13q22 [41, 42]. Acta Haematol ;—54 43 t 8;21 q22;q22 The t 8;21 q22;q22 translocation was first described by Rowley  in These patients are generally of a younger age mostly!
The immunophenotype of patients with t 8;21 is characterized by an overexpression of the CD34 antigen, in some cases with an aberrant coexpression of the CD19 antigen. AML with t 8;21 is associated with a favorable response to chemotherapy with both a high remission rate and long-term disease-free survival [44, 45]. However, despite the relatively good prognosis relapse remains a major problem, especially in the first 2 years of remission.
Standard cytogenetic analysis is the most commonly used method to detect t 8; These findings indicate that the sensitivity for the detection of a t 8;21 can be increased by molecular screening for all AML patients . Moreover, persistence of residual disease has also been 44 Acta Haematol ;—54 described after autologous BM transplantation. The biological significance of this finding is uncertain, although it suggests that the t 8;21 is only one step in the multistep pathogenesis of AML. In prospectively studied patients, negative PCR results correlated with an absence of relapse.
Tobal et al. They identified threshold levels, termed relapse-increased risk threshold of to! Levels above the upper limit of these thresholds were indicative of hematological relapse within 3—6 months. Data published to date from real-time RT-PCR quantification of MRD in t 8;21 -positive AML are limited to small numbers of patients and are generally consistent with the findings obtained with competitive assays [18, 31, 58].
The decrease of transcripts after induction therapy varies between 2 and 4 logs [25, 28, 36, 50, 59]. There are indications that a slow decrease! A 1—3 log difference between BM and PB was observed in complete hematological remission [19, 28]. Viehmann et al. A linear decrease of 2—4 logs could be seen from the start of therapy until the beginning of consolidation in most of the children. Most cases remained positive at a low level during the consolidation treatments.
Recently, Schnittger et al. They established a new prognostic score based on the combination of transcript levels at two checkpoints: the 75 percentile of the expression ratio at diagnosis and the median expression ratio after consolidation therapy. Alternatively, epigenetic factors, such as cytokine exposure released from accessory cells, may promote or inhibit the outgrowth of dormant leukemia clones.
Lastly, perhaps the immune system controls the outgrowth of the malignancy. Another explanation is that the residual cells expressing the hybrid transcript are no longer clonogenic, i. Varella-Garcia et al. Remission samples from 29 patients were studied. An analysis of un- sorted marrow samples revealed that residual t 8;21 cells could be detected at levels of 0.
The findings of Varella-Garcia et al. In this context, it is possible that the development of overt leukemia requires additional mutations to express the transformed phenotype in t 8;21 AML. Detection of Minimal Residual Disease in Acute Myelogenous Leukemia Acta Haematol ;—54 inv 16 p13;q22 and t 16;16 p13;q22 AML-M4Eo according to the FAB classification is often associated with rearrangements of chromosome 16, mostly involving 16p13 and 16q22, leading either to pericentric inversion, inv 16 p13q22 or, less commonly, to a translocation between the homologous chromosomes, t 16;16 p13q Despite the favorable response to chemotherapy in this group, many patients relapse .
This finding might be caused by the diversity in the amount of inv 16 transcripts together with the presence of alternatively spliced transcripts or poor quality RNA . Nevertheless, a gradual decline of MRD occurs between 4 and 12 months after CR achievement with PCR negativity achieved with slow kinetics after 6—18 months in patients remaining in long-term CR, while persisting positivity indicates relapse. This indicates that even without further chemotherapy, a progressive clearing of residual leukemic cells occurs.
This observation is apparently in sharp contrast with the findings reported in t 8;21 leukemia, where long-term complete CR is usually associated with persistence of MRD [19, 38, 68]. Both competitive and real-time amplification technologies were employed in small patient series analyzed retrospectively.
Guerrasio et al. Furthermore, negative PCR status, using the qualitative assay, was strongly associated with prolonged CR in keeping with the results obtained by real-time amplification. Buonamici et al. These findings are in agreement with other studies where the degree of reduction of leukemic transcripts was predictive of clinical outcome . According to quantitative RT-PCR studies, a more than 2 log decline after induction therapy was found in patients achieving long-term CR [38, 72, 73].
Patients with a less than 2 log early reduction are at high risk of relapse . However, relapses also occur in patients with sufficient initial molecular responses and relapses from PCR negativity have also been observed [17, 21, 38]. Nevertheless, there is strong evidence that high copy numbers after the end of consolidation treatment are associated with hematological relapse. Increasing transcript levels predict relapse 2 months before hematological relapse in the BM [19, 21, 73, 74]. In conclusion, data suggests that RT-PCR positivity in rearrangements of chromosome 16 involving 16p13 and 16q22 gradually decreases over time, but most patients remain positive until their 1st year in CR, at least in their BM.
PCR positivity at this stage may be compatible with long-term remission or cure. However, the degree of reduction of leukemic transcripts over time is predictive of the clinical outcome. This represents an important achievement from the clinical point of view as it may allow treatment modification during CR according to risk group. Translocations involving the MLL gene are not associated with a specific lineage or subtype of leukemia and have been described in AML, acute lymphoblastic leukemia ALL , biphenotypic or mixed-lineage leukemia and even in cases of lymphomas.
The diversity of phenotypes might be caused by the great variety of chromosomal fusion partners . The most common translocations are t 4;11 q21;q23 , t 9;11 q22;q23 and t 11;19 q23;p13 [75, 79, 80]. Regardless of their association with other high-risk factors at presentation, 11q23 rearrangements are strongly predictive of a poor clinical outcome. Recently, Scholl et al. Normalized copy numbers were positive at diagnosis and relapse. Samples from only 2 of 7 patients collected at the time of CR became negative. The 5 cases in CR still had positive copy numbers.
In addition to rearrangements involving various partner genes, MLL rearrangements can be found within MLL as the result from PTD usually spanning exons 2 through 6 or exons 2 through 8 [82, 83]. The clinical outcome in this AML subgroup is also unfavorable [32, 79, 84, 85]. While MLL rearrangement assessment by molecular methods is difficult due to the high number of partner genes, flow cytometry can provide additional data on MLL status, establishing the abnormal immunophenotype that will be used in the MRD study .
Of particular interest is AML with t 6;9 that is associated with basophilia and carries a poor prognosis. Expression Markers An abnormal expression of several genes not involved in translocations has been noticed in AML. PRAME PRAME preferentially expressed antigen of melanoma has been identified to code for a tumor antigen consisting of amino acids recognized by cytotoxic T cells that was originally established from a melanoma patient .
It has been reported that this gene is expressed in a variety of cancer cells including leukemic cells using semiquantitative RT-PCR [90—93].
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Matsushita et al. An increased expression was detected in the 2 patients who relapsed, 1 of whom before cytological diagnosis. Larramendy et al. Their results revealed 50 differentially expressed genes in at least 3 out of 15 patients. Twenty-two genes were upregulated and 28 genes were downregulated. Schoch et al. They demonstrated an unequivocal association between disease-specific translocations and distinct gene expression profile. For each of the three subtypes of AML patterns of gene expression were identified that were homogenous within all samples of the respective subgroup and clearly differed between the three subgroups.
By using two different strategies for microarray data analyses, this study revealed a unique correlation between AML-specific cytogenetic aberrations and gene expression profiles. Levy-Nissenbaum et al. Focusing on genes that were more highly expressed by the leukemic- than by the remission-phase leukocytes in 3 AML patients, they showed that the expression of PYST2 was fold higher in the leukemic than in the remission phase. These results were verified in another 12 patients and in 8 leukemic cell lines [97, 98].