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Minimal residual disease diagnostics in myeloid malignancies in the post transplant period

Abstract

Allogeneic SCT is important in myelodysplastic syndrome, the BCR-ABL-negative chronic myeloproliferative diseases (CMPDs) and in poor-risk AML. Techniques to monitor the minimal residual disease, for example, by PCR or immunophenotyping gain increasing importance in the post transplantation period as basis for improved and earlier therapeutic interventions in impending relapse. Recent markers such as the NPM1 mutations in AML or the JAK2V617F mutation in the CMPD can be exactly quantified by real-time PCR and were evaluated for their prognostic value in the post transplantation phase and for their utility to plan adoptive immunotherapy in case of molecular relapse. With respect to chimerism, new and very sensitive methods were introduced, for example, quantitative assessment of genetic polymorphisms by real-time PCR, but also methods here are still highly individualized. Only in CML, where SCT focuses now on poor-risk cases or cases of tyrosine kinase inhibitor failure, follow-up schedules are standardized. Standardization of the different diagnostic techniques and of the intervals in the post transplantation period is urgently needed also in other myeloid malignancies and should be focus of future studies.

Introduction

In contrast to CML with its rather homogeneous profile due to the BCR-ABL fusion all other myeloid malignancies, AML, myelodysplastic syndrome (MDS) and the BCR-ABL-negative chronic myeloproliferative diseases (CMPDs) represent very heterogeneous disorders on the basis of diverse chromosomal or molecular aberrations.1, 2, 3 The clinical courses are very variable and the response to therapy varies even within the distinct subentities as defined by specific genetic markers. This clinical variability is seen also after allogeneic SCT (allo-SCT), which is important in MDS, the CMPD and also in AML,4 as reduced intensity conditioning (RIC) allows the inclusion of elderly patients or patients with comorbidities.5

However, 30–80% of all patients with myeloid malignancies relapse after SCT depending on the subentity, the genetically based risk profile, and other parameters such as the remission status at the time of SCT. Withdrawal of immunosuppression or use of donor lymphocyte infusions (DLIs) are interesting options for impending relapse after SCT6, 7 that justify further evaluation. Therefore, optimization of the diagnostic schedules in the post transplant period aiming at the earliest possible therapeutic interventions has gained increasing attention.

In CML, follow-up strategies after SCT are standardized,8, 9 and adoptive immunotherapy by donor lymphocytes represents a well-established strategy in case of molecular or clinical relapse after SCT.10, 11 However, the introduction of tyrosine kinase inhibitor (TKI) treatment in recent years induced a shift of allo-SCT toward poor-risk cases with advanced disease or TKI failure.4, 12, 13 This selection of adverse-risk cases is a specific challenge for diagnostics after SCT, and new elements such as molecular screening for TKI resistance-conferring mutations has been included in diagnostics in the post transplantation period.14, 15 Finally, cases of TKI-associated MDS or AML due to Ph-negative clonal evolution16, 17 are new indications for allo-SCT and require diligent follow-up after SCT.

A broad panel of diverse diagnostic methods of cyto- and histomorphology, classical cytogenetics and fluorescence in situ hybridization (FISH), molecular analyses and immunophenotyping is available for monitoring myeloid malignancies after SCT. The value of quantitative PCR for the determination of the minimal residual disease (MRD) load was confirmed in numerous previous studies,18, 19, 20 and recent molecular markers such as the NPM1 mutations in AML21, 22, 23 and the JAK2V617F in the CMPD24, 25, 26, 27 might further contribute to the spectrum of follow-up markers in the post transplant period.6, 28 Novel molecular methods—for example, on the basis of quantitative PCR—provide the opportunity of higher sensitivity for chimerism analyses.29, 30, 31

However, the choice of the techniques and the intervals of follow-up diagnostics in the post transplant period are still very variable between individual centers. Only in CML with its homogeneous genetic profile has an optimized integration of molecular diagnostics with therapy during the follow-up32, 33, 34 so far been achieved and the international standardization of the BCR-ABL quantification is an ongoing process.8, 9 Due to the heterogeneous genetic profiles in all other myeloid malignancies, standardization of diagnostic schedules in the post transplant period is highly demanding and requires first an evaluation of the optimal intervals and of the most sensitive methods for follow-up analyses in this specific context. This paper gives an overview on the available diagnostic techniques in the diverse myeloid malignancies (Table 1) and summarizes recent results in each entity trying to further draw attention to development of diagnostics in the myeloid malignancies in the post transplant period.

Table 1 Overview of parameters, sensitivities of methods and recommendations for MRD diagnostics in myeloid malignancies in the post transplantation period

Cytomorphology and histomorphology

In general, the first BM control is performed between 14 and 30 days following SCT. At this time, BM cyto- and histomorphology show the first signs of engraftment especially of granulopoiesis often triggered also by G-CSF application and further with slow regeneration of erythropoiesis being followed by the megakaryocytic lineage. In this early period, signs of dysplasia, for example, megaloblastic erythropoiesis, can be normal and should not be mistaken as early MDS. At one month from SCT, the cellularity and quantity of lymphocytes should be normalized as indicators of normal engraftment,35, 36 whereas an acellular or hypocellular BM, a high degree of fibrosis, and large clusters of macrophages at this time point indicate late or failing engraftment or toxicity due to drugs or virus infection.35, 36 The speed of hematopoietic recovery depends on a variety of parameters: conditioning with reduced intensity is associated with faster BM recovery than myeloablative regimens,35 whereas HLA disparity is associated with slower kinetics of this regeneration process.37 Thus, it has to be discussed individually if both techniques, that is, cytomorphology plus trephine biopsy, are needed and give independent information.

Classical and molecular cytogenetics

Cytogenetic follow-up in the post transplant period requires the knowledge of the presence of an aberrant karytoype before SCT. In practice, the frequency of aberrant karyotypes is highly variable in the myeloid malignancies and depends strongly on the subentity, the stage of the disorder and the history of disease. In AML, cytogenetic aberrations are found in 55% of de novo cases,1, 38, 39 but have a higher frequency and more complexity in therapy-associated cases or in relapse. In MDS, the rate of karyotype aberrations ranges from 35% in the initial stages such as refractory anemia or RA with ring sideroblasts (with a minor role in the transplantation setting) to 60% in the advanced stages, for example, refractory anemia with excess of blasts to 90% in cases of therapy-associated cases.40, 41 Within the different BCR-ABL-negative CMPD, the rate of aberrant karyotypes is highest in primary myelofibrosis (PMF) with 40%, followed by polycythemia vera (PV) in 30–35% of all cases, whereas in essential thrombocythemia (ET) cytogenetic abnormalities are rare—but the indication for SCT in ET is an exception.42, 43, 44 During the transformation process of the CMPD to secondary AML the frequency of cytogenetic abnormalities increases to 80% of cases.

The selection of patients at advanced stages or at relapse of myeloid malignancies in the transplantation setting will probably be associated with higher rates of cytogenetic aberrations when compared to standard cohorts, which again emphasizes the importance of cytogenetic diagnostics before SCT.

Chromosome banding analyses are limited to the detection of microscopically visible abnormalities and can be hampered by poor quality BM samples, for example, in myelofibrosis or after TBI as live cells are required for metaphase cultures. Diverse techniques of FISH are available for the detection of submicroscopic alterations, for the clarification of complex alterations and also for MRD purposes by interphase FISH.45 The selection of the appropriate probes is very valuable also for the post transplant period.46 Interphase FISH provides a higher sensitivity as 100–200 cells can be evaluated without problems in comparison to 20–25 metaphases by chromosomal banding and does not require live cells.47

Molecular techniques and immunophenotyping

The majority of all patients with myeloid malignancies can be further characterized using PCR techniques. In AML, this applies mainly to the 25% of cases with the reciprocal t(15;17)/PML-RARA, t(8;21)/AML1-ETO, inv(16)/CBFB-MYH11 and 11q23/MLL rearrangements, but also to 85% of cases with normal karyotype.2 In the CMPD, the JAK2V617 activating mutation shows frequencies as high as >95% in PV and >55% in PMF and ET.24, 25, 26, 27 When compared to the situation in AML and the CMPD, in MDS the evaluation of molecular mutations is only at the beginning, but recently some mutations, for example, in the AML1 gene were described in 10–25% of all patients.48, 49

Diverse molecular techniques allow detection of molecular mutations with high specificity and make possible semiquantitative or exact monitoring of the respective mutations, for example, by real-time quantitative PCR (RQ-PCR). With these techniques sensitivities as high as 1:105–1:106 can be achieved. The sensitivity provided by nested PCR is in the same range.50 These techniques gain increasing importance also in the post transplant period. Molecular analysis before SCT is not only essential for risk stratification,51 but is also useful for exact definition of the mutational subtypes and of the breakpoints for later follow-up diagnostics.47

However, molecular markers are not yet available for all patients with advanced MDS or AML before SCT. Additionally, for some markers, for example, for mutations within the loop of the FLT3 tyrosine kinase domain (TKD), assays for quantitative monitoring52 are being developed but are not available in most laboratories at this time. Thus, immunophenotyping by multiparameter flow cytometry with sensitivities up to 1:102–1:104 provides an additional suitable method for the post transplant period. The definition of leukemia-associated immunophenotypes (LAIPs) of the blasts is possible in >95% of patients53, 54, 55, 56 and should be performed in AML and advanced MDS at diagnosis and before SCT as basis for MRD diagnostics within the post transplant period.

Chimerism

Frequent monitoring of chimerism can provide an early indication of incipient relapse of the underlying malignancy.57, 58 The clinical value of regular monitoring of chimerism in myeloid malignancies after SCT is demonstrated by reports on the successful withdrawal of immunosuppression and adoptive immunotherapeutic intervention by DLI in case of decreasing chimerism as reported by Bader et al.59 in five pediatric patients with AML and MDS. The introduction of RIC has further increased the clinical importance of chimerism as patients show more frequently mixed chimerism (MC) when compared to standard conditioning regimens.60 Thus, the interpretation of chimerism results should always include consideration of the conditioning strategies.61

Recent years showed a shift in the methods, as restriction fragment length polymorphisms now play a minor role. For gender-mismatch transplants, interphase FISH is commonly performed and offers many advantages, such as the easy commercial availability and standardization,62, 63 and allows sensitivities of up to 1%.64 The evaluation of higher numbers of cells permits higher sensitivity, but evaluation of 200–500 cells is the upper limit and the method is available for gender-mismatch transplantation cases only. Other common approaches include microsatellite analysis of short tandem repeats (STRs) or of variable number of tandem repeats by PCR.57, 65 These methods require minimal amounts of material and are not dependent on sex mismatch. However, they provide qualitative or semiquantitative assessment only and require extensive pretesting of the individual donor and recipient pairs.66 The sensitivity of these approaches is 0.1–1%, but can be increased considerably by lineage-specific chimerism after fractionation of cells.60, 67 At this time, the most sensitive approaches are quantitative real-time PCR for genetic polymorphisms (10−4) and for Y-specific sequences (10−5).29, 31, 61, 67, 68, 69 However, all assays are very much individualized between different centers, and the interpretation of findings should always include the different levels of sensitivity as achieved by the diverse methods.70, 71 The combination of different chimerism methods, for example, of interphase FISH and STR analyses, was recommended to improve the safety.71

The definitions of chimerism results are variable. Recipient chimerism is defined by most authors as <1% of donor alleles as detected by PCR or as <1% of donor cells by interphase FISH. In case of MC, recipient and donor cells are each detectable by FISH or PCR at >1%. Full donor chimerism (DC) means that no recipient allelic signals are detectable by PCR or <1% of recipient cells by interphase FISH.72 According to other suggestions, full chimerism is defined by a threshold of 95% of donor cells, and loss of chimerism by <4% of donor cells.61 The intervals of chimerism analyses after SCT also vary between different centers. Some investigators recommend assessment on days 30 and 100 post-SCT to provide comparable results,71 whereas others recommend weekly monitoring.69

Specific enrichment of single leukocyte subsets after flow-sorting (FACS), for example, for lymphocyte populations or magnetic beads-based techniques, can further improve the sensitivity as a basis for earlier treatment decisions.57, 73 This approach is time- and labor intensive, but allows a two-log higher sensitivity and therefore can be helpful in unclear cases.60, 74 Following SCT with RIC, Mohty et al.75 found that patients with myeloid malignancies with delayed achievement of full donor CD3+ T-cell chimerism at day 30 had a relapse risk of 40% in comparison to 0% in patients with full T-cell DC at this time point.

For some malignancies, disease-specific methods were developed: in a study on 87 patients with AML, decrease of chimerism in CD34+-selected cells was detected earlier than the clinical manifestation of relapse in >90% of cases as shown by Thiede et al.76 Another AML case was reported where study of unseparated cells still showed 90% of DC on day 28 post transplant, whereas CD34+ chimerism from the BM already indicated 28% of recipient cells. In this case, withdrawal of CsA at this early time point could prevent relapse although the patient developed severe graft-versus-host-reaction.77 This sensitive approach is useful especially for AML patients without specific cyto- or molecular genetic markers for other MRD techniques.76

AML

Morphology

In AML performance of the BM cytomorphology control 16–30 days following SCT allows assessment of early blast clearance and graft composition. A reduction of blasts <10% soon after the first induction course (‘day 16 blasts’) was proven in previous large studies as a strong prognostic favorable parameter.78, 79 In patients who are not in CR at the time of SCT, some centers perform an additional BM control after the first days of the conditioning schedule to gain an early impression of the response to therapy.

Cytogenetics

Revised criteria of remission in AML are not solely based on morphology but include as well the absence of previous cytogenetic abnormalities (‘cytogenetic remission’) or of an aberrant immunophenotype as previously defined by multiparameter flow cytometry (‘immunologic remission’).80 This novel concept should also be integrated in the assessment of remission criteria in the post transplantation period. Due to the limited sensitivity of chromosomal banding,81 interphase FISH seems to be a more suitable parameter for the assessment of the cytogenetic remission status. There are only few studies on the prognostic value of interphase FISH after AML therapy,45 and even fewer studies addressing this issue in the post transplant period. In pediatric cases with MDS or AML, Fuehrer et al.46 showed that clonal markers as assessed by interphase FISH (for example, monosomy 7) reappeared at relapse after SCT and could be followed also after a second SCT. Also, metaphase FISH proved suitable for MRD diagnostics following standard chemotherapy or allo-SCT in an analysis of 22 AML patients performed by El-Rifai et al.82, as all but one of the patients with persisting or increasing levels of abnormal cells relapsed.

Molecular genetics

The highest sensitivity for detection of MRD in AML is certainly provided by molecular techniques such as real-time quantitative PCR with the TaqMan or LightCycler technology or nested PCR. Quantitative PCR monitoring in AML is best validated for the reciprocal gene fusions t(15;17)/PML-RARA, inv(16)/CBFB-MYH11 and t(8;21)/AML1-ETO with sensitivities ranging from 10−4 to 10−6,20, 83, 84, 85 as the score of the aberrant gene expression compared after consolidation therapy and at diagnosis has a significant prognostic impact,19 and distinct thresholds of transcript copy numbers were determined to correlate with an increased relapse risk.84, 85 The interval between the increase of the respective fusion transcripts and the clinical manifestation of relapse can be as long as 3–6 months. Due to the favorable prognosis with standard chemotherapy today allo-SCT is performed in these genetic subtypes only in case of relapse or poor response to therapy.86, 87, 88 Molecular assessment in these rearrangements is helpful also after SCT, as stable remissions were associated in all analyzed patients with the CBFB-MYH1189 or AML1-ETO subtypes90 associated with the achievement of PCR negativity.

Increasing proportions of AML cases with a normal karyotype can be further characterized and monitored by molecular methods. The NPM1 mutations and the FLT3-LM/ITD (internal tandem duplications in the FLT3-gene) have central roles in this aspect. The prognostically adverse FLT3-LM/ITD in 40% of normal karyotype cases91, 92 are controversially discussed concerning their validity for MRD purposes as instability rates as high as 17% in relapse of AML were reported.93 However, some studies suggest that loss of the mutation occurs in <5% of cases only and that accumulation of the mutation with a higher ratio of mutated to unmutated alleles is associated with an increased relapse risk.94 Thus, some have suggested including the FLT3-LM in post transplantation follow-up strategies,95 whereas others doubt its validity for this purpose.50 Quantitative follow-up of the FLT3-LM and the FLT3-TKD in four mutated patients in the post transplant period by real-time quantitative PCR showed a significant association of relapse with PCR positivity whereas disease-free survival was associated with the achievement of PCR negativity.52 This small series indicates that the FLT3-mutations warrant further evaluation as MRD parameters after SCT. However, the FLT3-LM require the design of patient-specific primers for follow-up by quantitative PCR as their length and starting points are very variable, which makes MRD techniques labor intensive.96 Further the stability of the FLT3-LM/ITD might be limited,97 so combination with other MRD methods, for example, other molecular mutations in double-mutated cases or with multiparameter flow cytometry, is strongly recommended.

The prognostically favorable NPM1 mutations that lead to an aberrant cytoplasmic localization of the nucleophosmin-1 (NPM) protein are identified in 55% of normal karyotype cases21, 22, 98, 99, 100 and are promising parameters for MRD strategies due to their homogeneous structure. Quantitative assessment of the mutation by real-time PCR before and after chemotherapy correlated with prognosis in two studies.101, 102 The sensitivity of real-time PCR for NPM1 mutational level assessment is as high as 10−4. In our own retrospective analysis of 14 NPM1-mutated patients the achievement of PCR negativity after SCT was a precondition for stable remissions,28 whereas relapse of AML after SCT was preceded by an increase of the NPM1 mutational level in 90% of cases. In all MRD studies performed so far, the NPM1 mutations showed high stability which suggested that this parameter might be ideal for MRD studies.101, 102 However, this has to be confirmed by additional studies as the number of analyzed cases is so far very limited.

Expression of the WT1 (Wilms' tumor) gene is another promising marker for the follow-up period after SCT.50, 103 Quantitative monitoring by reverse transcription–PCR in 50 patients with AML after SCT allowed determination of thresholds above which relapse within 40 days was predictable for all patients. There was a 100% relapse probability at an expression level of 1.0–5.0 × 10−2, whereas in patients with an expression level of <4.0 × 10−4 the relapse risk was 0.8% only. In addition, those patients at relapse who responded to donor lymphocytes or withdrawal of immunosuppression showed a significantly longer doubling time of the WT1-expression level than those who were refractory to adoptive immunotherapy, and a short doubling time of WT1 transcripts <13 days was predictive for failure of these immunomodulatory approaches.104 However, although sensitivity of WT1 measurement ranges as high as 10−4, it has to be considered that WT1 expression is found at a certain level also in healthy individuals, which can complicate the assessment for MRD purposes due to a certain background level.97 Therefore, at this time MRD studies solely on the basis of WT1 measurement in AML cannot be recommended; they should always be combined with another MRD parameter.

Multiparameter flow cytometry

The value of immunophenotyping by multiparameter flow cytometry for assessment of remission status in AML was confirmed in many studies.80 The decrease of cells with a specific LAIP when compared at diagnosis and shortly after initial therapy correlates significantly with the achievement of CR and with survival,53, 54, 55, 56 and the reduction of aberrant cells to a threshold of 0.1% after chemotherapy was assessed as predictor for long-time survival in another study.105

However, in the post transplantation setting the use of multiparameter flow cytometry still has to be validated as changes of the immunophenotype might be more frequent in relapse following SCT than after standard therapy where complete loss of the previous LAIP is seen in 25%.106 In one case an increase of cells with the AML-specific immunophenotype 2 months from transplantation was an indication for the withdrawal of immunosuppression that was followed by a reduction of aberrant cells.107 So far, only very few studies108 addressed the value of MRD by immunophenotyping in AML specifically in the post transplantation period, but it was shown that leukemic blasts can be separated from regenerating blasts by multiparameter flow cytometry after SCT.109 In the study of Perez-Simon et al.110 that included only six patients with AML and seven with MDS after RIC, a positive MRD status as assessed by multiparameter flow cytometry within 21–56 days after SCT was associated with a relapse risk of 58%, whereas all patients with a negative MRD status in this interval achieved stable remissions. Although there are few studies for the post transplant period available, it might be reasonable to include multiparameter flow cytometry in MRD strategies after SCT, as in most AML patients LAIPs can be defined. Immunophenotyping further might be ideal in combination with other MRD parameters, for example, molecular markers.

Myelodysplastic syndromes

There are only very few studies addressing the problem of follow-up diagnostics in MDS after SCT. Cytomorphological follow-up is limited by the frequent occurrence of signs of dysplasia during the normal regeneration process following SCT. One more option is provided by cytogenetic monitoring in primarily aberrant cases.111 Major cytogenetic remission is defined as disappearance of a cytogenetic abnormality and minor cytogenetic response by a 50% reduction of abnormal metaphases.80 Follow-up procedures in MDS might further be optimized by interphase FISH analyses (for example, for chromosome 5 or 7 aberrations) due to the higher sensitivity, but this needs evaluation in the post transplant setting.

Whether molecular MRD strategies are convenient for MDS still has to be evaluated. Mutations in the AML1/RUNX gene occur in 10–25%,48, 49 within the NRAS protooncogene in 6–10%, and the FLT3-LM/ITD are found in 3–5% of all MDS cases.112, 113, 114 These mutations all increase during leukemic transformation, so it is possible that in the transplantation setting with a selection of high-risk cases the frequency of these markers might be overrepresented when compared to standard cohorts. However, the validity of these markers for MRD purposes in MDS has still not been evaluated.

Another option for MRD studies in MDS might be offered by measurement of expression of the above-mentioned WT1 gene,115 as its expression was shown to increase during leukemic transformation of MDS and to decrease following chemotherapy or allo-SCT.116 In a pediatric case with MDS successful withdrawal of immunosuppression due to an increase of WT1 was described: on day 263 after SCT the patient had an increase of the WT1 gene-expression level in the BM (16 000 copies per mg RNA) and in peripheral blood (3700 copies per mg RNA). Cytogenetics confirmed continuing complete cytogenetic remission (he had a trisomy 8 before SCT), and there was full DC. Immunosuppression by tacrolimus was tapered rapidly, which was followed by a short period of self-limiting GVHD (degree II) of the skin. Following discontinuation of immunosuppression, WT1 expression gradually decreased in the peripheral blood and was not detectable any more after 2 months.117

Further, flow cytometric scores to detect aberrant antigen patterns as assessed by multiparameter flow cytometry in MDS cases before SCT were proven predictable for the outcome of the transplantation procedure.118 Therefore, immunophenotyping for follow-up of MDS patients after SCT needs further evaluation, but such studies have not so far been performed.

CML

In CML, hematologic, cytogenetic and molecular methods offer diverse levels of sensitivity for the evaluation of remission status after SCT. Cytogenetic response criteria have a central role in the evaluation of the response to therapy,8, 119 but sensitivity is limited in most cases to a maximum of 25 metaphases. The combination with interphase FISH allows evaluation of 100–500 cells, but molecular techniques offer the most sensitive quantification of the residual BCR-ABL load.9, 120, 121 Quantification by real-time PCR is internationally standardized by normalization with the ABL gene.8, 9 Before SCT, the specific BCR-breakpoint should be evaluated, as most cases show the b2a2 or b3a2 transcript types corresponding to the M-BCR breakpoint, but 1% of all patients are observed with other breakpoints, for example, e1a2.122, 123

In primary treatment of CML, the European Leukemia Net (ELN) recommends chromosome banding analyses of BM at least every 6 months and suggests that after the achievement of complete cytogenetic remission intervals of 12 months are sufficient, whereas molecular monitoring is being recommended every 3 months.8 However, in the post transplantation period, quantitative PCR for BCR-ABL should be performed at shorter intervals, at least every 4 weeks, to allow early intervention by adoptive immunotherapy or TKI in case of impending relapse. Dazzi et al.11 defined thresholds of a BCR-ABL/ABL ratio of >0.02% in three samples or of >0.05% in two samples as criteria for donor lymphocyte intervention. Quantitative real-time PCR was found reliable to monitor the response to these interventions in several studies.16, 17, 124, 125 Both real-time PCR and nested PCR allow comparable sensitivities of 10−4–10−6,8, 126 to evaluate the response to imatinib127 or donor lymphocytes11 in relapse after SCT. However, the kinetics of disease can only be monitored by quantitative real-time PCR whereas nested PCR is able to confirm the achievement of complete molecular remission. Quantitative real-time PCR and nested PCR can also be used to compare the efficacy of different approaches, for example, by definition of the time to molecular remission or the molecular remission rates as achieved by imatinib or donor lymphocytes post transplant.128 Major molecular remission (MMR) is defined as a BCR-ABL/ABL ratio of 0.1%.8, 126 Complete molecular remission is defined by negative results with nested PCR.126

Early kinetics of the BCR-ABL fusion allow prognostic predictions after SCT: a slow reduction of the BCR-ABL transcripts on days +28 and +56 showed a significant correlation with higher relapse rates, whereas the achievement of PCR negativity after SCT was associated with stable long-term remissions in a study of Lange et al.129 Asnafi et al.130 determined a threshold of 10−4 of BCR-ABL/ABL on day +100 after SCT to predict the probability of relapse, and another study showed higher relapse rates when BCR-ABL fusion transcripts were detected within the first 3–5 months after SCT.131

Resistance to TKI can be mediated by different mechanisms, for example, point mutations in the ABL gene,132, 133, 134 which can be revealed by sequencing after screening by high performance liquid chromatography. The ELN formulated clear indications: screening is indicated in cases where patients fail to achieve major cytogenetic response (reduction of Ph-positive metaphases <35%) at 12 months or show loss of molecular response as defined by an increase in the BCR-ABL/ABL ratio of 1 on a logarithmic scale.8, 126 Screening for TKI resistance conferring mutations should already have been performed in the pretransplant period as it might influence the decision to perform SCT—for example, the T315I mutation confers resistance to imatinib and also to second generation TKIs and might be an indication for allo-SCT.126 In case of relapse of CML after SCT mutational screening might be considered again as there might be shifts within the mutation patterns during the course of disease.135, 136 Additionally, resistance can also be associated with cytogenetic evolution detected by chromosomal banding analyses.

Chronic myeloproliferative diseases

The increasing use of allo-SCT in the CMPD made more sensitive criteria for the assessment of remission in these entities necessary. On the basis of an international consensus, the revised criteria for response to treatment in PMF137 include cyto- and histomorphological, cytogenetic and molecular findings. The histologic remission criteria apply to cellularity, myeloblast percentage and signs of myelofibrosis in the BM. SCT with RIC was documented to induce complete or nearly complete regression of BM fibrosis in 60% of PMF patients within the first 100 days and in 90% within the first 6 months.138 Cytogenetic response criteria discriminate major response with an absence of preexisting chromosomal abnormalities from minor cytogenetic response defined by a 50% reduction of abnormal metaphases.

Further, the detection of the activating V617F point mutation in the JAK2 gene24, 25, 26, 27 and the development of different assays, for example, allele-specific PCR, real-time PCR or pyrosequencing139 provided new monitoring strategies for the CMPD: quantitative assessment of the JAK2V617F mutational level by real-time PCR showed significant correlations with prognosis in 21 patients with PMF after SCT.6 In total, 78% of patients achieved PCR negativity after a median of 89 days, whereas persistence of the mutation after SCT was always associated with relapse. Further, quantitative PCR was useful for the planning of adoptive immunotherapy by donor lymphocytes.6 Additional reports showed clearance of the JAK2V617F mutation in individual cases after SCT.140, 141 Quantification of the JAK2V617F can further be performed by mass spectrometry.142 Survival was always associated with complete clearance of the JAK2V617F mutational level as assessed by this technique in a study on 60 mutated patients with CMPD after allo-SCT.142 Also, residual JAK2V617F mutational loads after SCT were found to be significantly associated with MC.6, 143 Due to the favorable response of cases showing persistence or increase of the V617F mutational load to the use of donor lymphocytes,6 it might be suggested that JAK2 mutational monitoring be performed at frequent intervals in the post transplant period—for example, every 4 weeks—to plan adoptive immunotherapy early. However, although all studies so far showed excellent sensitivity and stability of the V617F for MRD purposes in the post transplant period, the number of studies and of analyzed cases remains limited, so more studies should follow to confirm these results.

The spectrum of activating molecular mutations in the CMPD is continuously expanding: 5% of PMF cases were recently observed to carry somatic gain-of-function mutations within the MPL gene (W515).144, 145, 146 Whether this mutational subtype might also qualify for MRD strategies in the post transplant period still has to be evaluated.

Interaction of methods

Of course each diagnostic method should always be seen in the context of other results and the clinical history—for example, the intensity of the conditioning regimen or the primary response to therapy. Thus, the interpretation of the remission status following SCT should primarily consider peripheral cell counts and BM cytomorphology and include eventually also BM histopathology, for example, in cases of CMPD. The cytogenetic results at diagnosis and before SCT allow the choice of the appropriate interphase probes for later follow-up diagnostics after SCT, for example, in case of numerical or structural abnormalities. If chromosomal analyses reveal a normal karytoype, molecular analyses, for example, for NPM1 and FLT3-LM, should be performed due to the high frequency of these molecular markers in the respective cytogenetic subgroup. In AML or advanced MDS multiparameter flow cytometry should be performed to determine the individual LAIP as basis for follow-up after SCT. Chimerism preferably by quantitative PCR also plays a central role in this panel of post transplantation diagnostics and should be frequently monitored.

Thus, the optimal schedule for the planning of diagnostic methods during the post transplant period should be individualized and consider patient-specific factors in parallel to the findings of initial diagnostics. At this time definite recommendations for the intervals between the diagnostic follow-ups are difficult to define, but the intervals of follow-up diagnostics should always consider the kinetics of relapse. In AML frequent monitoring of MRD and of chimerism is recommended60 to plan immunological intervention as early as possible,6, 7, 59 whereas in disorders with slower kinetics such as the CMPD longer intervals might be justified.

Conclusions

In CML monitoring of the BCR-ABL transcript levels is included in routine strategies in the post transplantation period to identify the need for adoptive immunotherapy or TKI treatment,34, 128 but in all other myeloid malignancies, AML, MDS and the BCR-ABL-negative CMPD, comparable approaches still have to be developed with regard to quantitative PCR,18, 20, 147 multiparameter flow cytometry,53, 54, 55 and interphase FISH.45 Studies that analyze these methods for their predictive value within the post transplantation period are still limited. There are reports on small series of cases where post transplant therapeutic strategies were planned and monitored by MRD methods, for example, by the results of immunophenotyping in AML,107 by molecular WT1 measurement in MDS or by quantitative assessment of the JAK2V617F-mutation in the CMPD.6

The validity of the single MRD parameters and the different levels of sensitivity still require evaluation specifically for the post transplant situation. Moreover, different assays and methods are used for the detection and the monitoring of these markers. As with CML,126 standardization of the diverse methods would facilitate the comparison of results between the diverse centers and also the performance of international studies.

Also, the validity of chimerism versus MRD methods has to be defined for the specific disorders. In CML, quantitative measurement of the BCR-ABL-expression level was found to be more suitable for the planning of therapeutic interventions than chimerism.60, 124 In patients relapsing with AML after SCT it was shown that the NPM1 mutations precede the decrease of molecular chimerism and the increase of the FLT3-LM in double-mutated cases by intervals of 2–3 weeks,28 but this has to be confirmed by additional studies and such comparisons are missing for other markers in myeloid malignancies. With respect to chimerism, the different levels of sensitivity as provided by different methods must be taken into account in the interpretation of studies.60

In conclusion, standardization of methods for MRD in the myeloid malignancies and of chimerism66 in the post transplant period is a high priority. The evaluation of the optimal time points and intervals for follow-up diagnostics is another major requirement. Also, the introduction of RIC strategies has increased the demands for chimerism diagnostics due to the higher frequencies of MC after SCT.60 New techniques such as quantitative real-time PCR for single nucleotide polymorphisms31 or for Y-specific sequences29, 67 provide the basis for more sensitive monitoring, but the choice of assays and the intervals remain highly variable between different centers. Standardization of these approaches would provide the basis for optimal therapeutic strategies in myeloid malignancies in the post transplant period and would facilitate the development of new treatment schedules for patients who relapse with these heterogeneous and complex disorders.

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Bacher, U., Zander, A., Haferlach, T. et al. Minimal residual disease diagnostics in myeloid malignancies in the post transplant period. Bone Marrow Transplant 42, 145–157 (2008). https://doi.org/10.1038/bmt.2008.185

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Keywords

  • allogeneic SCT
  • MRD
  • quantitative PCR
  • myeloid malignancies

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