Summary We hypothesized that Wilms tumour 1 gene (WT1) expression levels in acute myeloid leukaemia (AML) patients might have predictive value and reveal molecular relapse kinetics. WT1 level was determined at diagnosis, during therapy and post‐therapy follow‐up in 89 patients who reached first complete remission (CR1) (952 samples, median 8 samples/patient, range 2–38). CR1 bone marrow (BM) WT1 level above normal (based on 39 healthy donors) was an independent adverse prognostic factor regarding both disease‐free survival [hazard ratio (HR) 4·46, P = 0·001] and overall survival (HR 2·62, P = 0·019). By grouping 34 BM and 99 peripheral blood (PB) complete remission samples in monthly intervals prior to clinical relapse, disease development was delineated and a simple mathematical model constructed, that allowed for the prediction of relapse detection rates (RDRs) as well as median times [tms] from WT1 positivity to clinical relapse. BM sampling was required to obtain RDRs above 93% and tms above 67 d. Acceptable RDRs and tms (81% and 44 d, respectively) could be acquired by bimonthly PB sampling. In conclusion, CR1 WT1 expression is an independent prognostic factor in AML. According to our model, BM is superior for relapse prediction, but PB samples are useful when shorter sampling intervals are possible.
treatment, marrow blasts increased to 40%. FISH for BCR-ABL remained negative (also negative with the sorted CD34 þ / CD38 þ and CD34 þ /CD38-population) whereas À7 was present in 82% of the marrow cells. Quantitative PCR analysis was not performed. However, with nested RT-PCR using sets of BIOMED primers 2 the e1a2 transcript was still detectable suggesting presence of a very small and quiescent population of imatinib-resistant BCR-ABL-positive leukemic stem cells. There are only a few reports on the use of imatinib in Ph þ AML. 3-7 Most cases reporting molecular remission with imatinib had short follow-up and long-term clinical and molecular data is not available. Our case of AML with 45,XX,inv(3)(q21q26),À7, t(9;22)(q34;q11.2) shows two recognized secondary cytogenetic anomalies in advanced CML, that is, monosomy 7 and chromosome aberrations involving band 3q21or 3q26, 8 which initially would have suggested a case of CML blast crisis. However, simultaneous presence of À7 and inv(3)(q21q26) in blastic CML is rare (http://cgap.nci.nih.gov/Chromosomes). We propose that owing to the secondary nature of the Ph chromosome, the rarity in which these three cytogenetic changes are found together in blast crisis, the lack of p210 BCR-ABL protein transcript and the absence of splenomegaly, our case is more in keeping with de novo Ph þ AML over myeloid blastic CML. This is confirmed by FISH studies, which revealed the Ph chromosome is acquired as a late event during clonal evolution. It is salutary to note that combination of FISH and RT-PCR analysis was needed to avoid the erroneous conclusion of imatinib resistance as the size of the BCR-ABL clone went down which is not compatible with BCR-ABL-dependent imatinib resistance.
In acute myeloid leukemia (AML) mouse models, the RUNX1-RUNX1T1 fusion protein has failed to produce leukemia by itself, but alternative splicing of exon 9a of the RUNX1-RUNX1T1 fusion transcript (FT) has recently been shown to enhance the leukemogenic potential. We have analyzed 138 diagnosis and follow-up samples from 13 RUNX1-RUNX1T1+ patients as well as diagnosis samples from 13 RUNX1-RUNX1T1- AML patients and 26 healthy donors. Levels of native RUNX1T1 mRNA were low in both healthy and RUNX1-RUNX1T1-negative AML samples. Likewise, the ratio between RUNX1T1 mRNA harboring exon 9a and lacking exon 9a was low and tightly regulated (0.017-0.11). In contrast, 11/13 RUNX1-RUNX1T1-positive AML patients displayed high and variable ratios of FT ranging from 0.05 to 0.46 (P < 0.001, Wilcoxon rank-sum test), indicating altered exon 9a splicing in these patients. Importantly, patients who remained in continuous complete remission displayed a faster disappearance of the RUNX1-RUNX1T1 exon 9a splice variant compared to patients bound to relapse (P = 0.02). In conclusion, alternative splicing seems to be part of the leukemogenic process in the majority of RUNX1-RUNX1T1-positive AML patients, and splice variant kinetics under cytoreduction may be a predictor for patients prone to relapse.
BACKGROUND: Wilms’ Tumor gene 1 (WT1) is highly expressed in 80% of AML patients and has been proposed as a valuable tool for minimal residual disease (MRD) detection. Though pilot studies have shown the value of MRD monitoring using WT1 as marker, particularly during therapy, no single center study has addressed the value of close monitoring both during therapy and post-therapy follow-up or developed models to reliably use this information in clinical decision-making. AIMS: To determine whether high residual WT1 expression at the time where complete remission is first diagnosed (CR1) is an adverse prognostic factor, to evaluate the value of WT1 expression levels in peripheral blood (PB) and bone marrow (BM) for prediction of disease relapse, and to develop mathematical models that will enable quantification of the central parameters related to prediction of clinical relapse. METHODS: WT1 levels were quantified using real-time quantitative RT-PCR by normalization to the control genes B2M and ABL, and in follow-up samples expressed as a fraction of the BM diagnostic level. In a cohort of 165 adults and 18 children, treated at the Departments of Hematology and Pediatrics, Aarhus University Hospital, BM and PB samples from 90 patients (76 adults and 14 children) were analyzed at diagnosis and during therapy and follow-up (952 samples, median 8 samples/patient, range 2-38). Prognostic difference between groups was determined using the Cox Proportional Hazards statistical model including age, sex, cytogenetics, de novo/secondary leukemia, FLT3-ITD, and white blood cell counts. RESULTS: WT1 levels in BM and PB at CR1 above that of the average plus two standard deviations of 39 BM or 30 PB of healthy donors were found to be an independent adverse prognostic factor regarding both disease-free survival (BM: Hazard ratio (HR) 4.91, P=0.001, PB: HR 4.97, P=0.008) and overall survival (BM: HR 2.65, P=0.047, PB: HR 3.55, P=0.025). We were able to describe relapse kinetics in 29/37 relapses observed in the 90 patient cohort. By grouping 34 BM and 99 PB samples in monthly intervals prior to clinical relapse, disease development was delineated (Figure). Based on mathematical considerations involving proportions of the integrals of the graphs shown in the figure we were able to predict how sampling intervals influence relapse detection rates (RDRs) as well as the median time from WT1 positivity to clinical relapse (t(m)). For example, a BM sampling interval of 4 months will lead to a RDR of 93% and a t(m) of 74 days. A PB sampling interval of 2 months will lead to a RDR of 81% and a t(m) of 44 days. CONCLUSIONS: CR1 WT1 expression levels in both BM and PB are independent prognostic factors in AML. Though relapse was seen earlier in BM than in PB our models demonstrates that frequent PB sampling provides an acceptable window of opportunity for initiation of preemptive cytoreduction as a consequence of molecular relapse. Relapse prediction in monthly intervals; x-axis: time, relapse at t=0, y-axis: samples with WT1 level above threshold as a fraction of all samples. Median number of relapses in each interval; BM: 7(range 6–9), PB: 9(range 6–13) Relapse prediction in monthly intervals; x-axis: time, relapse at t=0, y-axis: samples with WT1 level above threshold as a fraction of all samples. Median number of relapses in each interval; BM: 7(range 6–9), PB: 9(range 6–13)
Aims: Myelodysplastic syndromes (MDS) constitute a heterogeneous group of disorders with hematopoiesis in disarray and with preponderance to transformation to AML. In contrast to the situation in de novo AML patients, the hematopoiesis in MDS patients is often though to encompass most cell lineages with few non-malignant cells in the bone marrow due to displacement by the MDS clone. While the involvement of the myeloid sublineages has been relatively easy to verify in semi-solid CFU assays, that of the lymphoid lineages has required advanced flow sorting techniques in combination with FISH assays in the minority of MDS patients with a well-known genetic aberration such as 5q-, -7 or +8. Moreover, the latter studies have usually been restricted to the description of the involvement of the B-cell lineage. Transcriptional silencing of tumor suppressor genes by promoter hypermethylation is associated with hematological malignancy, including MDS. In a patient with a very immature MDS that was found to be hypermethylated in the p15, HIC, E-cadherin, and Estrogen receptor genes, we wanted to elucidate the methylation status in both myeloid and lymphoid cells. Material and methods: Mononuclear cells from a 69-year-old female MDS patient with RAEB was analyzed. Using magnetic cell sorting, CD34 and CD3 positive cells were isolated from BM-MNC and PB-MNC, respectively. The purity of the isolated cell fractions was determined by flow cytometry (CD34+: 95% and CD3+: 84%). DNA isolated from the cells was treated with sodium bisulfite and analyzed for promoter hypermethylation in the p15, HIC, E-cadherin, and Estrogen receptor genes by means of Bisulfite-Denaturing Gradient Gel Electrophoresis (Bisulfite-DGGE). Results: All cell fractions were found to be hypermethylated in the four genes analyzed. Since the Bisulfite-DGGE allows for the detection of heterogeneous methylation patterns, we were able to compare these patterns. Similar Bisulfite-DGGE band patterns were observed in total BM-MNC, total PB-MNC, CD34+ cells and CD3+ cells. Conclusion: Because the methylation band patterns were the same in all cell fractions, it seems unlikely that methylation has occurred independently in progenitors committed for myeloid and lymphoid maturation, respectively. We therefore conclude that promoter hypermethylation in this patient has occurred in a stem cell with potential for both myeloid and lymphoid maturation. This methodology should provide an easy way to assess the extent of clonality of hematopoiesis in MDS patients.
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