21 patients with chronic myelocytic leukaemia (CML) have been followed closely during and after development of blastic transformation (BT). Severe fatigue in previously asymptomatic patients was the most frequently observed prodrome of transformation. Most significant clinical findings were anaemia, rapidly increasing WBC and increasing size of the spleen in patients with previously well-controlled CML. 11 out of 24 patients responded to chemotherapy and had a median survival of 3 months, 6 obtained a complete remission (CR). Median survival time for all patients was 2 months from BT. Quality of life after BT was in most cases inferior. Thus, only 5 patients spent more than 1 month outside hospital. Cytomorphological classification of blasts in marrow as granular or agranular showed no correlation to the results of chemotherapy or to survival.

Abnormalities of chromosomes 5 and 7, the most frequent cytogenetic aberrations in t-MDS and t-AML, are associated with therapy with alkylating agents and have poor prognostic impact. However, cytogenetic and molecular studies of patients with t-MDS and t-AML show that abnormalities of chromosomes 5 and 7 clearly define different genetic pathways. Thus, cases with -5/5q- with or without abnormalities of chromosome 7 are characterised by mutations of the TP53 gene, a complex karyotype, deletion or loss of 17p, and gain of chromosome band 11q23 with amplification of the MLL gene. Cases with -7/7q- and normal chromosomes 5 on the other hand are characterised by a simpler karyotype, somatic mutations of the AML1/RUNX1 gene and frequent methylation of the p15 promoter. The origin of hypoploidy, a frequent phenomenon in t-MDS and t-AML, will be discussed.

Frameshift mutations of the nucleophosmin gene ( NPM1 ) were recently reported as a frequently occurring abnormality in patients with de novo acute myeloid leukemia (AML). To evaluate the frequency of NPM1 mutations in patients with therapy-related myelodysplasia (t-MDS) and therapy-related AML (t-AML), and their possible association to type of previous therapy and to other gene mutations, 140 patients with t-MDS or t-AML were analyzed for mutations of NPM1 . NPM1 mutations were observed in 7 of 51 patients presenting as overt t-AML, as compared to only 3 of 89 patients presenting as t-MDS ( P =0.037). The mutations were not related to any specific type of previous therapy, but they were significantly associated with a normal karyotype and mutations of FLT3 ( P =0.0002 for both comparisons). Only 1 of 10 patients with NPM1 mutations presented chromosome aberrations characteristic of therapy-related disease, and 7q−/−7, the most frequent abnormalities of t-MDS/t-AML, were not observed ( P =0.002). This raises the question whether some of the cases presenting NPM1 mutations were in fact cases of de novo leukemia. The close association to class I mutations and the inverse association to class II mutations suggest mutations of NPM1 as representing a class II mutation-like abnormality in AML.

Myelodysplasia (MDS) and acute myeloid leukemia (AML) are heterogeneous, closely associated diseases arising de novo or following chemotherapy with alkylating agents, topoisomerase II inhibitors, or after radiotherapy. Whereas de novo MDS and AML are almost always subclassified according to cytogenetic characteristics, therapy-related MDS (t-MDS) and therapy-related AML (t-AML) are often considered as separate entities and are not subdivided. Alternative genetic pathways were previously proposed in t-MDS and t-AML based on cytogenetic characteristics. An increasing number of gene mutations are now observed to cluster differently in these pathways with an identical pattern in de novo and in t-MDS and t-AML. An association is observed between activating mutations of genes in the tyrosine kinase RAS–BRAF signal-transduction pathway (Class I mutations) and inactivating mutations of genes encoding hematopoietic transcription factors (Class II mutations). Point mutations of AML1 and RAS seem to cooperate and predispose to progression from t-MDS to t-AML. Recently, critical genetic effects underlying 5q−/−5 and 7q−/−7 have been proposed. Their association and cooperation with point mutations of p53 and AML1, respectively, extend the scenario of cooperating genetic abnormalities in MDS and AML. As de novo and t-MDS and t-AML are biologically identical diseases, they ought to be subclassified and treated similarly.

Alternative genetic pathways were previously outlined in the pathogenesis of therapy-related myelodysplasia (t-MDS) and acute myeloid leukemia (t-AML) based on cytogenetic characteristics. Some of the chromosome aberrations, the recurrent balanced translocations or inversions, directly result in chimeric rearrangement of genes for hematopoietic transcription factors ( class II mutations) which disturb cellular differentiation. Other genetic abnormalities in t-MDS and t-AML comprise activating point mutations or internal tandem duplications of genes involved in signal transduction as tyrosine kinase receptors or genes more downstream in the RAS-BRAF pathway ( class I mutations). The alternative genetic pathways of t-MDS and t-AML can now be further characterized by a different clustering of six individual class I mutations and mutations of AML1 and p53 in the various pathways. In addition, there is a significant association between class I and class II mutations possibly indicating cooperation in leukemogenesis, and between mutations of AML1 and RAS related to subsequent progression from t-MDS to t-AML. Therapy-related and de novo myelodysplasia and acute myeloid leukemia seem to share genetic pathways, and surprisingly gene mutations were in general not more frequent in patients with t-MDS or t-AML as compared to similar cases of de novo MDS and AML studied previously.

Mutations of the FLT3 , c-KIT , c-FMS , KRAS , NRAS , BRAF and CEBPA genes in the receptor tyrosine kinase (RTK)/RAS-BRAF signal-transduction pathway are frequent in acute myeloid leukemia (AML). We examined 140 patients with therapy-related myelodysplasia or AML (t-MDS/t-AML) for point mutations of these seven genes. In all, 11 FLT3 , two c-KIT , seven KRAS , eight NRAS and three BRAF mutations were identified in 29 patients (21%). All but one patient with a FLT3 mutation presented with t-AML ( P =0.0002). Furthermore, FLT3 mutations were significantly associated with previous radiotherapy without chemotherapy ( P =0.03), and with a normal karyotype ( P =0.004), but inversely associated with previous therapy with alkylating agents ( P =0.003) and with −7/7q− ( P =0.001). RAS mutations were associated with AML1 point mutations ( P =0.046) and with progression from t-MDS to t-AML ( P =0.008). Noteworthy, all three patients with BRAF mutations presented as t-AML of M5 subtype with t(9;11)(p22;q23) and MLL -rearrangement ( P =0.01). In t-AML RAS / BRAF mutations were significantly associated with a very short survival ( P =0.017). Half of the patients with a mutation in the RTK/RAS-BRAF signal-transduction pathway (denoted ‘class-I’ mutations) simultaneously disclosed mutation of a hematopoietic transcription factor (denoted ‘class-II’ mutations) ( P =0.046) suggesting their cooperation in leukemogenesis.

The AML1 transcription factor is essential for normal hematopoiesis and is the target of several chromosomal translocations in acute leukemia. Acquired somatic AML1 mutations were recently demonstrated sporadically in de novo myelodysplasia (MDS) and acute myeloid leukemia (AML) including a few cases of therapy-related disease (t-MDS/t-AML). We examined 140 patients with t-MDS or t-AML for AML1 mutations by direct sequencing. We identified 9 missense, 3 nonsense, and 10 frameshift mutations, all heterozygous, in 22 patients (15.7%). Thirteen mutations were located in the N-terminal Runt homology domain (RHD), whereas 9 mutations were located in the C-terminal region including the transactivation domain (TAD). Nineteen patients with AML1 mutations had previously received alkylating agents whereas 2 patients had received radiotherapy only. AML1 mutations were highly significantly associated with presentation of the disease as t-MDS (P = .003), with deletion or loss of chromosome arm 7q (P = .001) and with subsequent transformation to overt t-AML (P = .0001). Patients with missense mutations presented a shorter survival compared with patients with nonsense/frameshift mutations (P = .03). Our results suggest that AML1 mutations and deletion of genes on chromosome arm 7q cooperate in leukemogenesis and predispose to leukemic transformation.

Amplification or duplication of the AML1 gene at chromosome band 21q22 was detected by FISH using a locus-specific probe in three out of 171 unselected patients with therapy-related myelodysplasia (t-MDS) or t-AML (1.7%). In two patients AML1 signals were located tandemly on derivative chromosomes, in one patient on a dic(9; 21) and in the the other patient on a derivative chromosome 18 made up of interchanging layers of material from chromosomes 9, 14, 18, and 21. In the third patient three single supernumerary copies of AML1 were located on derivatives of chromosomes 19 and 21. All three patients were older, had previously received therapy with alkylating agents without topoisomerase II inhibitors, had complex karyotypes including abnormalities of chromosomes 5 or 7, and presented acquired point mutations of the TP53 gene. No point mutations of the AML1 gene were observed. The results support a pivotal role of impaired TP53 function in the development of gene amplification or duplication in t-MDS and t-AML.

Jens Pedersen-Bjergaard, Debes H. Christiansen, Mette K. Andersen. Dept. Clinical Genetics, University Hospital Rigshospitalet, Copenhagen, Denmark. 140 unselected patients with t-MDS (n=90) or overt t-AML (n=50) were studied for chromosome aberrations, for mutations of 6 different genes and 69 patients were examined for methylation of the p15 promotor. Cytogenetically, 5q−/ −5: n=34; 7q−/ −7 but normal chromosomes 5: n=39; balanced translocations: n=23; normal karyotype: n=24; other abnormalities: n=20. Mutations of p53 : n=34; AML1 : n=22; FLT3 : n=11; c-KIT : n=2; K - or N - RAS : n=14; BRAF : n=2; methylation of the p15 promotor: 55/69 cases. Abnormalities of chromosomes 5 and 7 were significantly associated with t-MDS (p=0.001), whereas balanced translocations and a normal karyotype were significantly associated with overt t-AML, (p=0.0001 and p=0.006 respectively). Mutations of p53 were significantly associated with 5q−/ −5 (p=0.001), with 17p−/−17 (p=0.0004), with a complex karyotype (p=0.001), and with MLL and AML1 amplifications. Mutations of AML1 were significantly associated with 7q−/−7 )(p=0.001) and with t-MDS progressing to t-AML (p=0.0001). Mutations of FLT3 were significantly associated with presentation as overt t-AML (p=0.0002), with a normal karyotype (p=0.004) and with previously radiotherapy only (p=0.03). RAS mutations were significantly associated with t-MDS progressing to overt t-AML (p=0.008) and borderline with a normal karyotype (p=0.07). Methylation of p15 was significantly associated with 7q−/−7 (p=0.0006). Mutations of FLT3 , RAS and c-KIT were mutually exclusive, and a synergism between AML1 and RAS mutations (22/5/14, p=0.046) and between p53 and AML1 mutations in patients with 7q−/−7 is suggested. Based on these findings a revised model of the genetic pathways of t-MDS and t-AML (Blood2002;99: 1909–1912) is discussed.

The p14 ARF , p15 INK4B , and p16 INK4A genes are important negative cell-cycle regulators often inactivated by deletions, mutations, or hypermethylation in malignancy. Hypermethylation of the three genes was studied in 81 patients with therapy-related myelodysplasia (t-MDS) or acute myeloid leukemia (t-AML) by methylation-specific PCR, and p15 methylation additionally by bisulfite genomic sequencing. In all, 55 patients disclosed p15 methylation, five patients showed p16 methylation, whereas p14 methylation was not observed. Methylation of p15 was closely associated with deletion or loss of chromosome arm 7q ( P =0.0006). In t-MDS, the p15 methylation frequency and the p15 methylation density both increased significantly by stage ( P =0.004 and 0.0002), and p15 methylation frequency increased with an increasing percentage of myeloblasts in the bone marrow ( P =0.006). In a two-variable Cox model including the percentage of myeloblasts, p15 methylation was an independent prognostic factor ( P =0.005). Methylation of p15 was less common in t-AML of subtype M5 than in other FAB subtypes ( P =0.03). Methylation of p15 was unrelated to type of previous therapy, to latent period from start of therapy, to platelet count, and to p53 mutations. Inactivation of p15 and deletion of genes on chromosome arm 7q possibly cooperate in leukemogenesis.

New insights into causative factors for the development of myelodysplasia (MDS) and acute myeloid leukemia (AML), with associations to specific cytogenetic and genetic abnormalities have been obtained primarily from studies of patients with the therapy-related subsets of the two diseases. Current knowledge now makes it possible to distinguish between at least seven major genetic subgroups of MDS and AML, and has directed research towards more specific causative factors also for de novo MDS and AML.

The Workshop identified 48 unselected patients with therapy‐related myelodysplastic syndrome or acute myeloid leukemia (t‐MDS/t‐AML) and inv(16), and 41 patients with t(15;17) after chemotherapy (CT) and/or radiotherapy (RT) for a malignant or nonmalignant disease. The primary diseases were: breast cancer, 33 patients; lymphomas, 24 patients; various other solid tumors, 30 patients; and nonmalignant diseases, 2 patients. The general type of previous therapy was RT only in 10 patients with an inv(16) and in 12 patients with a t(15;17), alkylating agents plus topoisomerase II inhibitors in 24 patients with an inv(16) and in 18 patients with a t(15;17), topoisomerase II inhibitors only in 5 patients with an inv(16) and in 2 patients with a t(15;17), alkylating agents only in 6 patients in each subgroup, and other types of chemotherapy in 3 patients in each subgroup. Most CT‐treated patients (69%) also received RT. The latency period to development of t‐MDS/t‐AML was short: a median of 22 months in patients with inv(16) and 29 months in patients with t(15;17). Twenty‐six patients (54%) with an inv(16) and 17 patients (41%) with a t(15;17) had additional cytogenetic abnormalities, which were unrelated to age and survival in both subgroups. Trisomy of chromosomes 8, 21, and 22 and del(7q) were the most frequent additional abnormalities in the inv(16) subgroup, whereas +8, −5, and del(16q) were most frequent in the t(15;17) subgroup. The disease was overt t‐AML in 38/48 patients (79%) with an inv(16) and in 38/41 patients (93%) with a t(15;17). Thirty‐three of 39 intensively treated patients (85%) with an inv(16) obtained a complete remission, whereas 24 of 35 intensively treated patients (69%) with a t(15;17) obtained a complete remission. The median overall survival of intensively treated patients was 29 months in both cytogenetic subgroups. In the inv(16) subgroup, patients younger than 55 years of age had a longer survival when compared with older patients ( P = 0.006). The study supports the observation that t‐MDS/t‐AML with inv(16) and t(15;17) is often associated with prior therapy with topoisomerase II inhibitors; however, a notable finding was the high frequency of treatment with only radiotherapy, 29% of t(15;17) and 21% of inv(16). Response rates to intensive chemotherapy in this study were comparable to those of de novo disease. © 2002 Wiley‐Liss, Inc.

We read with great interest the report of Latagliata et al[1][1] on therapy-related myelodysplastic syndrome (t-MDS) and acute myelogenous leukemia (t-AML) following treatment of acute promyelocytic leukemia (APL). The authors observed 5 out of 77 patients with APL, who after intensive chemotherapy

Clonal Ph-negative hematopoiesis in CML after therapy with imatinib mesylate is frequently characterized by trisomy 8

Therapy-related acute myeloid leukemia (t-AML) in most cases develops after chemotherapy of other malignancies and shows characteristic chromosome aberrations. Two general types of t-AML have previously been identified. One type is observed after therapy with alkylating agents and characteristically presents as therapy-related myelodysplasia with deletions or loss of the long arms of chromosomes 5 and 7 or loss of the whole chromosomes. The other type is observed after therapy with topoisomerase II inhibitors and characteristically presents as overt t-AML with recurrent balanced chromosome aberrations. Recent research suggests that these 2 general types of t-AML can now be subdivided into at least 8 genetic pathways with a different etiology and different biologic characteristics.

Eighty-two unselected cases of therapy-related myelodysplasia (t-MDS) or acute myeloid leukemia (t-AML) were investigated for internal tandem duplications of the FLT3gene (FLT3/ITD), for internal tandem duplications of the MLL gene (MLL/ITD) and for mutations of the WT1 gene. FLT3/ITD were observed in three patients, another two patients presented MLL/ITD whereas mutations of the WT1 gene were not observed. All FLT3/ITD included the tyrosine-rich stretch between codons 589 and 599, and both MLL/ITD presented break points within Alu-repeats, as previously observed in de novo AML. The ITD were not related to any specific type of previous therapy, but three out of the five cases were observed among only six patients with overt t-AML and a normal karyotype (P = 0.0043). Interestingly, one of the patients with FLT3/ITD presented overt t-AML of subtype M1 with a normal karyotype after treatment with an alkylating agent. Complete remission was observed following treatment with daunorubicin and cytosine arabinoside, but after 37 months the patient relapsed with t-AML of subtype M3 with a t(15;17) and the same FLT3/ITD was still present. Thus FLT3/ITD may in this case represent a primary event in leukemogenesis, whereas the t(15;17) may represent a secondary event most likely induced by subsequent therapy. In conclusion, FLT3/ITD and MLL/ITD are mainly observed in uncharacteristic cases of t-AML with a normal karyotype and unrelated to previous therapy for which reason they could represent sporadic cases of de novoAML.

PURPOSE:To study mutations and loss of heterozygosity (LOH) of p53 in therapy-related myelodysplasia (t-MDS) and acute myeloid leukemia (t-AML).PATIENTS AND METHODS:Fifty-two unselected patients with t-MDS and 25 patients with t-AML were studied by polymerase chain reaction (PCR)-single-strand conformational polymorphism (SSCP) at the DNA level and by reverse transcriptase (RT)-PCR-SSCP at the mRNA level, and cases with aberrant SSCP patterns were sequenced.RESULTS:Somatically acquired mutations of p53 were observed in 21 of 77 cases of t-MDS or t-AML, and 19 of these 21 patients had received alkylating agents. Single-base substitutions at A:T pairs were more common in t-MDS and t-AML, whereas single-base substitutions at G:C pairs are most common in MDS and AML de novo and in solid tumors. Six patients demonstrated a cytogenetic loss of 17p13, and these six and an additional nine patients with p53 mutations demonstrated LOH of p53 at the DNA or mRNA level. This suggests a cytogenetic loss of the normal p53 allele in these nine cases combined with duplication of the homologous chromosome 17 carrying the mutated p53 allele. Mutations of p53 were significantly associated with deletion or loss of 5q (P <.0001) and a complex karyotype (P =.0001), but surprisingly were not associated with deletion or loss of 7q (P =.73), and were infrequent in patients with balanced chromosome translocations (P =.03). Mutations of p53 were more common in older patients (P =.036) and were associated with an extremely poor prognosis (P =.014), apparently restricted to the 15 cases with LOH of p53 ( P =.046).CONCLUSION:Mutations with loss of function of p53 are significantly associated with deletion or loss of 5q in t-MDS and t-AML after previous treatment with alkylating agents and are associated with genetic instability.