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Biallelic germ line excision repair cross-complementing 6 like 2 (ERCC6L2) variants strongly predispose to bone marrow failure (BMF) and myeloid malignancies, characterized by somatic TP53-mutated clones and erythroid predominance. We present a series of 52 subjects (35 families) with ERCC6L2 biallelic germ line variants collected retrospectively from 11 centers globally, with a follow-up of 1165 person-years. At initial investigations, 32 individuals were diagnosed with BMF and 15 with a hematological malignancy (HM). The subjects presented with 19 different variants of ERCC6L2, and we identified a founder mutation, c.1424delT, in Finnish patients. The median age of the subjects at baseline was 18 years (range, 2-65 years). Changes in the complete blood count were mild despite severe bone marrow (BM) hypoplasia and somatic TP53 mutations, with no significant difference between subjects with or without HMs. Signs of progressive disease included increasing TP53 variant allele frequency, dysplasia in megakaryocytes and/or erythroid lineage, and erythroid predominance in the BM morphology. The median age at the onset of HM was 37.0 years (95% CI, 31.5-42.5; range, 12-65 years). The overall survival (OS) at 3 years was 95% (95% CI, 85-100) and 19% (95% CI, 0-39) for patients with BMF and HM, respectively. Patients with myelodysplastic syndrome or acute myeloid leukemia with mutated TP53 undergoing hematopoietic stem cell transplantation had a poor outcome with a 3-year OS of 28% (95% CI, 0-61). Our results demonstrated the importance of early recognition and active surveillance in patients with biallelic germ line ERCC6L2 variants., (© 2023 by The American Society of Hematology.)

Although hematologic malignancies (HM) are no longer considered exclusively sporadic, additional awareness of familial cases has yet to be created. Individuals carrying a (likely) pathogenic germline variant (e.g., in ETV6, GATA2, SAMD9, SAMD9L, or RUNX1) are at an increased risk for developing HM. Given the clinical and psychological impact associated with the diagnosis of a genetic predisposition to HM, it is of utmost importance to provide high-quality, standardized patient care. To address these issues and harmonize care across Europe, the Familial Leukemia Subnetwork within the ERN PaedCan has been assigned to draft an European Standard Clinical Practice (ESCP) document reflecting current best practices for pediatric patients and (healthy) relatives with (suspected) familial leukemia. The group was supported by members of the German network for rare diseases MyPred, of the Host Genome Working Group of SIOPE, and of the COST action LEGEND. The ESCP on familial leukemia is proposed by an interdisciplinary team of experts including hematologists, oncologists, and human geneticists. It is intended to provide general recommendations in areas where disease-specific recommendations do not yet exist. Here, we describe key issues for the medical care of familial leukemia that shall pave the way for a future consensus guideline: (i) identification of individuals with or suggestive of familial leukemia, (ii) genetic analysis and variant interpretation, (iii) genetic counseling and patient education, and (iv) surveillance and (psychological) support. To address the question on how to proceed with individuals suggestive of or at risk of familial leukemia, we developed an algorithm covering four different, partially linked clinical scenarios, and additionally a decision tree to guide clinicians in their considerations regarding familial leukemia in minors with HM. Our recommendations cover, not only patients but also relatives that both should have access to adequate medical care. We illustrate the importance of natural history studies and the need for respective registries for future evidence-based recommendations that shall be updated as new evidence-based standards are established., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Cytogenetics has long represented a critical component in the clinical evaluation of hematologic malignancies. Chromosome banding studies provide a simultaneous snapshot of genome-wide copy number and structural variation, which have been shown to drive tumorigenesis, define diseases, and guide treatment. Technological innovations in sequencing have ushered in our present-day clinical genomics era. With recent publications highlighting novel sequencing technologies as alternatives to conventional cytogenetic approaches, we, an international consortium of laboratory geneticists, pathologists, and oncologists, describe herein the advantages and limitations of both conventional chromosome banding and novel sequencing technologies and share our considerations on crucial next steps to implement these novel technologies in the global clinical setting for a more accurate cytogenetic evaluation, which may provide improved diagnosis and treatment management. Considering the clinical, logistic, technical, and financial implications, we provide points to consider for the global evolution of cytogenetic testing., (© 2022 by The American Society of Hematology.)

Acute leukemias (ALs) of ambiguous lineage are a heterogeneous group of high-risk leukemias characterized by coexpression of myeloid and lymphoid markers. In this study, we identified a distinct subgroup of immature acute leukemias characterized by a broadly variable phenotype, covering acute myeloid leukemia (AML, M0 or M1), T/myeloid mixed-phenotype acute leukemia (T/M MPAL), and early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Rearrangements at 14q32/BCL11B are the cytogenetic hallmark of this entity. In our screening of 915 hematological malignancies, there were 202 AML and 333 T-cell acute lymphoblastic leukemias (T-ALL: 58, ETP; 178, non-ETP; 8, T/M MPAL; 89, not otherwise specified). We identified 20 cases of immature leukemias (4% of AML and 3.6% of T-ALL), harboring 4 types of 14q32/BCL11B translocations: t(2,14)(q22.3;q32) (n = 7), t(6;14)(q25.3;q32) (n = 9), t(7;14)(q21.2;q32) (n = 2), and t(8;14)(q24.2;q32) (n = 2). The t(2;14) produced a ZEB2-BCL11B fusion transcript, whereas the other 3 rearrangements displaced transcriptionally active enhancer sequences close to BCL11B without producing fusion genes. All translocations resulted in the activation of BCL11B, a regulator of T-cell differentiation associated with transcriptional corepressor complexes in mammalian cells. The expression of BCL11B behaved as a disease biomarker that was present at diagnosis, but not in remission. Deregulation of BCL11B co-occurred with variants at FLT3 and at epigenetic modulators, most frequently the DNMT3A, TET2, and/or WT1 genes. Transcriptome analysis identified a specific expression signature, with significant downregulation of BCL11B targets, and clearly separating BCL11B AL from AML, T-ALL, and ETP-ALL. Remarkably, an ex vivo drug-sensitivity profile identified a panel of compounds with effective antileukemic activity., (© 2021 by The American Society of Hematology.)

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell acute lymphoblastic lymphoma (T-LBL) are aggressive hematological malignancies that are currently treated with high-dose chemotherapy. Over the last several years, the search toward novel and less-toxic therapeutic strategies for T-ALL/T-LBL patients has largely focused on the identification of cell-intrinsic properties of the tumor cell. However, non-cell-autonomous activation of specific oncogenic pathways might also offer opportunities that could be exploited at the therapeutic level. In line with this, we here show that endogenous interleukin 7 (IL7) can increase the expression of the oncogenic kinase proviral integration site for Moloney-murine leukemia 1 (PIM1) in CD127+ T-ALL/T-LBL, thereby rendering these tumor cells sensitive to in vivo PIM inhibition. In addition, using different CD127+ T-ALL/T-LBL xenograft models, we also reveal that residual tumor cells, which remain present after short-term in vivo chemotherapy, display consistent upregulation of PIM1 as compared with bulk nontreated tumor cells. Notably, this effect was transient as increased PIM1 levels were not observed in reestablished disease after abrogation of the initial chemotherapy. Furthermore, we uncover that this phenomenon is, at least in part, mediated by the ability of glucocorticoids to cause transcriptional upregulation of IL7RA in T-ALL/T-LBL patient-derived xenograft (PDX) cells, ultimately resulting in non-cell-autonomous PIM1 upregulation by endogenous IL7. Finally, we confirm in vivo that chemotherapy in combination with a pan-PIM inhibitor can improve leukemia survival in a PDX model of CD127+ T-ALL. Altogether, our work reveals that IL7 and glucocorticoids coordinately drive aberrant activation of PIM1 and suggests that IL7-responsive CD127+ T-ALL and T-LBL patients could benefit from PIM inhibition during induction chemotherapy., (© 2020 by The American Society of Hematology.)

T-cell acute lymphoblastic leukemia (T-ALL) results from deregulation of a number of genes via multiple genomic mechanisms. We designed a comprehensive fluorescence in situ hybridization (CI-FISH) assay that consists of genomic probes to simultaneously investigate oncogenes and oncosuppressors recurrently involved in chromosome rearrangements in T-ALL, which was applied to 338 T-ALL cases. CI-FISH provided genetic classification into one of the well-defined genetic subgroups (ie, TAL/LMO, HOXA, TLX3, TLX1, NKX2-1/2-2, or MEF2C) in 80% of cases. Two patients with translocations of the LMO3 transcription factor were identified, suggesting that LMO3 activation may serve as an alternative to LMO1/LMO2 activation in the pathogenesis of this disease. Moreover, intrachromosomal rearrangements that involved the 10q24 locus were found as a new mechanism of TLX1 activation. An unequal distribution of cooperating genetic defects was found among the six genetic subgroups. Interestingly, deletions that targeted TCF7 or TP53 were exclusively found in HOXA T-ALL, LEF1 defects were prevalent in NKX2-1 rearranged patients, CASP8AP2 and PTEN alterations were significantly enriched in TAL/LMO leukemias, and PTPN2 and NUP214-ABL1 abnormalities occurred in TLX1/TLX3. This work convincingly shows that CI-FISH is a powerful tool to define genetic heterogeneity of T-ALL, which may be applied as a rapid and accurate diagnostic test., (Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Dissecting the pathogenesis of classical Hodgkin lymphoma (cHL), a common cancer in young adults, remains challenging because of the rarity of tumor cells in involved tissues (usually <5%). Here, we analyzed the coding genome of cHL by microdissecting tumor and normal cells from 34 patient biopsies for a total of ∼50 000 singly isolated lymphoma cells. We uncovered several recurrently mutated genes, namely, STAT6 (32% of cases), GNA13 (24%), XPO1 (18%), and ITPKB (16%), and document the functional role of mutant STAT6 in sustaining tumor cell viability. Mutations of STAT6 genetically and functionally cooperated with disruption of SOCS1 , a JAK-STAT pathway inhibitor, to promote cHL growth. Overall, 87% of cases showed dysregulation of the JAK-STAT pathway by genetic alterations in multiple genes (also including STAT3 , STAT5B , JAK1 , JAK2 , and PTPN1 ), attesting to the pivotal role of this pathway in cHL pathogenesis and highlighting its potential as a new therapeutic target in this disease., (© 2018 by The American Society of Hematology.)

Epithelial-to-mesenchymal-transition (EMT) is critical for normal embryogenesis and effective postnatal wound healing, but is also associated with cancer metastasis. SNAIL, ZEB, and TWIST families of transcription factors are key modulators of the EMT process, but their precise roles in adult hematopoietic development and homeostasis remain unclear. Here we report that genetic inactivation of Zeb2 results in increased frequency of stem and progenitor subpopulations within the bone marrow (BM) and spleen and that these changes accompany differentiation defects in multiple hematopoietic cell lineages. We found no evidence that Zeb2 is critical for hematopoietic stem cell self-renewal capacity. However, knocking out Zeb2 in the BM promoted a phenotype with several features that resemble human myeloproliferative disorders, such as BM fibrosis, splenomegaly, and extramedullary hematopoiesis. Global gene expression and intracellular signal transduction analysis revealed perturbations in specific cytokine and cytokine receptor-related signaling pathways following Zeb2 loss, especially the JAK-STAT and extracellular signal-regulated kinase pathways. Moreover, we detected some previously unknown mutations within the human Zeb2 gene (ZFX1B locus) from patients with myeloid disease. Collectively, our results demonstrate that Zeb2 controls adult hematopoietic differentiation and lineage fidelity through widespread modulation of dominant signaling pathways that may contribute to blood disorders., (© 2017 by The American Society of Hematology.)

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive leukemia for which knowledge on disease mechanisms and effective therapies are currently lacking. Only a handful of recurring genetic mutations have been identified and none is specific to BPDCN. In this study, through molecular cloning in an index case that presented a balanced t(3;5)(q21;q31) and molecular cytogenetic analyses in a further 46 cases, we identify monoallelic deletion of NR3C1 (5q31), encoding the glucocorticoid receptor (GCR), in 13 of 47 (28%) BPDCN patients. Targeted deep sequencing in 36 BPDCN cases, including 10 with NR3C1 deletion, did not reveal NR3C1 point mutations or indels. Haploinsufficiency for NR3C1 defined a subset of BPDCN with lowered GCR expression and extremely poor overall survival (P = .0006). Consistent with a role for GCR in tumor suppression, functional analyses coupled with gene expression profiling identified corticoresistance and loss-of-EZH2 function as major downstream consequences of NR3C1 deletion in BPDCN. Subsequently, more detailed analyses of the t(3;5)(q21;q31) revealed fusion of NR3C1 to a long noncoding RNA (lncRNA) gene (lincRNA-3q) that encodes a novel, nuclear, noncoding RNA involved in the regulation of leukemia stem cell programs and G1/S transition, via E2F. Overexpression of lincRNA-3q was a consistent feature of malignant cells and could be abrogated by bromodomain and extraterminal domain (BET) protein inhibition. Taken together, this work points to NR3C1 as a haploinsufficient tumor suppressor in a subset of BPDCN and identifies BET inhibition, acting at least partially via lncRNA blockade, as a novel treatment option in BPDCN., (© 2016 by The American Society of Hematology.)

MYC translocations represent a genetic subtype of T-lineage acute lymphoblastic leukemia (T-ALL), which occurs at an incidence of ∼6%, assessed within a cohort of 196 T-ALL patients (64 adults and 132 children). The translocations were of 2 types; those rearranged with the T-cell receptor loci and those with other partners. MYC translocations were significantly associated with the TAL/LMO subtype of T-ALL (P = .018) and trisomies 6 (P < .001) and 7 (P < .001). Within the TAL/LMO subtype, gene expression profiling identified 148 differentially expressed genes between patients with and without MYC translocations; specifically, 77 were upregulated and 71 downregulated in those with MYC translocations. The poor prognostic marker, CD44, was among the upregulated genes. MYC translocations occurred as secondary abnormalities, present in subclones in one-half of the cases. Longitudinal studies indicated an association with induction failure and relapse., (© 2014 by The American Society of Hematology.)

To provide new insights into the genomic profile of desmoplastic round cell tumors (DSRCT), we applied fluorescence in situ hybridization (FISH) and metaphase comparative genomic hybridization (M-CGH) to two newly diagnosed cases. FISH detected multiple subclones bearing one to three copies of der(11)t(11;22)(p13;q12) and/or der(22)t(11;22)(p13;q12) in both patients. This peculiar genomic imbalance might result from derivative chromosome duplication due to non-disjunction and/or mitotic recombination between normal and derivative chromosomes 11 and 22. Concomitant loss of normal chromosomes (i.e., 11 in patient 1 and 22 in patient 2) caused loss of the WT1 or EWSR1 wild-type allele. M-CGH identified other genomic imbalances: gain at chromosome 3 in both cases and chromosome 5 polysomy in patient 1. Common genomic events (i.e., trisomy 3 and extra EWSR1-WT1 and WT1-EWSR1 copies) probably contributed to disease pathogenesis and/or evolution of DSRCT. Our study demonstrated that an integrated molecular cytogenetic approach identified EWSR1-WT1 cooperating molecular events and genetic markers for prognosis. Thus, FISH and M-CGH might well be applied in a large series of patients to elucidate the genomic background of DSRCT., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The MLLT10 gene, located at 10p13, is a known partner of MLL and PICALM in specific leukemic fusions generated from recurrent 11q23 and 11q14 chromosome translocations. Deep sequencing recently identified NAP1L1/12q21 as another MLLT10 partner in T-cell acute lymphoblastic leukemia (T-ALL). In pediatric T-ALL, we have identified 2 RNA processing genes, that is, HNRNPH1/5q35 and DDX3X/Xp11.3 as new MLLT10 fusion partners. Gene expression profile signatures of the HNRNPH1- and DDX3X-MLLT10 fusions placed them in the HOXA subgroup. Remarkably, they were highly similar only to PICALM-MLLT10-positive cases. The present study showed MLLT10 promiscuity in pediatric T-ALL and identified a specific MLLT10 signature within the HOXA subgroup.

We set up a diagnostic double-color double-fusion fluorescence in situ hybridization (DCDF-FISH) assay to investigate a case of a de novo acute myeloid leukemia (AML)-M4 bearing an inv(11)(p15q22). DCDF-FISH detected the NUP98-DDX10 rearrangement as two fusion signals, at the short and the long arms of the inv(11). Reverse transcription-polymerase chain reaction (RT-PCR) and cloning experiments confirmed the NUP98-DDX10 fusion and identified two splicing fusion isoforms: the known "type II fusion," originating from the fusion of NUP98 exon 14 to DDX10 exon 7 and a new in-frame fusion transcript between NUP98 exon 15 and DDX10 exon 7, which we termed "type III fusion.", (Copyright © 2013 Elsevier Inc. All rights reserved.)

In an adult case of B-cell acute lymphoblastic leukemia (B-ALL) with a complex karyotype, both chromosomes 14 were involved in unbalanced rearrangements, specifically, der(14)t(13;14)(q21;q21) and dup(14)(q11q32). Fluorescence in situ hybridization (FISH) detected two CEBPE-IGH rearrangements at the dup(14). One was found at the duplication breakpoint and the other derived from insertion of CEBPE into an apparently normal IGH locus. Hypotheses to account for these unusual chromosomal rearrangements are discussed. This case provides the first evidence that chromosome duplication and cryptic insertion produce the CEBPE-IGH fusion and that more than one CEBPE-IGH recombination can occur in a leukemic cell. Our findings confirm that deregulated CEBPE plays a crucial role in the pathogenesis of CEBPE-IGH positive B-ALL., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Acute myeloid leukemia (AML) with mutated NPM1 usually carries normal karyotype (NK), but it may harbor chromosomal aberrations whose significance remains unclear. We addressed this question in 631 AML patients with mutated/cytoplasmic NPM1. An abnormal karyotype (AK) was present in 93 of 631 cases (14.7%), the most frequent abnormalities being +8, +4, -Y, del(9q), +21. Chromosome aberrations in NPM1-mutated AML were similar to, but occurred less frequently than additional chromosome changes found in other AML with recurrent cytogenetic abnormalities according to WHO classification. Four of the 31 NPM1-mutated AML patients karyotyped at different time points had NK at diagnosis but AK at relapse: del(9q) (n = 2), t(2;11) (n = 1), inv(12) (n = 1). NPM1-mutated AML with NK or AK showed overlapping morphologic, immunophenotypic (CD34 negativity), and gene expression profile (down-regulation of CD34 and up-regulation of HOX genes). No difference in survival was observed among NPM1-mutated AML patients independently of whether they carried a NK or an AK, the NPM1-mutated/FLT3-ITD negative cases showing the better prognosis. Findings in our patients point to chromosomal aberrations as secondary events, reinforce the concept that NPM1 mutation is a founder genetic lesion, and indicate that NPM1-mutated AML should be clinically handled as one entity, irrespective of the karyotype.

We have studied a patient with acute myeloid leukemia (AML) and t(10;11)(q23;p15) as the sole cytogenetic abnormality. Molecular analysis revealed a translocation involving nucleoporin 98 (NUP98) fused to the DNA-binding domain of the hematopoietically expressed homeobox gene (HHEX). Expression of NUP98/HHEX in murine bone marrow cells leads to aberrant self-renewal and a block in normal differentiation that depends on the integrity of the NUP98 GFLG repeats and the HHEX homeodomain. Transplantation of bone marrow cells expressing NUP98/HHEX leads to transplantable acute leukemia characterized by extensive infiltration of leukemic blasts expressing myeloid markers (Gr1(+)) as well as markers of the B-cell lineage (B220(+)). A latency period of 9 months and its clonal character suggest that NUP98/HHEX is necessary but not sufficient for disease induction. Expression of EGFP-NUP98/HHEX fusions showed a highly similar nuclear localization pattern as for other NUP98/homeodomain fusions, such as NUP98/HOXA9. Comparative gene expression profiling in primary bone marrow cells provided evidence for the presence of common targets in cells expressing NUP98/HOXA9 or NUP98/HHEX. Some of these genes (Hoxa5, Hoxa9, Flt3) are deregulated in NUP98/HHEX-induced murine leukemia as well as in human blasts carrying this fusion and might represent bona fide therapeutic targets.

The nucleophosmin (NPM1) gene encodes for a multifunctional nucleocytoplasmic shuttling protein that is localized mainly in the nucleolus. NPM1 mutations occur in 50% to 60% of adult acute myeloid leukemia with normal karyotype (AML-NK) and generate NPM mutants that localize aberrantly in the leukemic-cell cytoplasm, hence the term NPM-cytoplasmic positive (NPMc+ AML). Cytoplasmic NPM accumulation is caused by the concerted action of 2 alterations at mutant C-terminus, that is, changes of tryptophan(s) 288 and 290 (or only 290) and creation of an additional nuclear export signal (NES) motif. NPMc+ AML shows increased frequency in adults and females, wide morphologic spectrum, multilineage involvement, high frequency of FLT3-ITD, CD34 negativity, and a distinct gene-expression profile. Analysis of mutated NPM has important clinical and pathologic applications. Immunohistochemical detection of cytoplasmic NPM predicts NPM1 mutations and helps rationalize cytogenetic/molecular studies in AML. NPM1 mutations in absence of FLT3-ITD identify a prognostically favorable subgroup in the heterogeneous AML-NK category. Due to their frequency and stability, NPM1 mutations may become a new tool for monitoring minimal residual disease in AML-NK. Future studies should focus on clarifying how NPM mutants promote leukemia, integrating NPMc+ AML in the upcoming World Health Organization leukemia classification, and eventually developing specific antileukemic drugs.

Because of a lack of specific clonality markers, information on lineage involvement and cell of origin of acute myeloid leukemia with normal karyotype (AML-NK), is missing. Because Nucleophosmin (NPM) gene is frequently mutated in AML-NK and causes aberrant NPM cytoplasmic localization (NPMc+), it was used as an AML lineage clonality marker. Clonal NPM exon 12 mutations were detected in myeloid, monocytic, erythroid, and megakaryocytic cells but not in fibroblasts or endothelia that were laser-microdissected from 3 patients with NPMc+ AML. Aberrant cytoplasmic expression of mutated NPM proteins was identified with anti-NPM antibodies in 2 or more myeloid hemopoietic cell lineages in 99 (61.5%) of 161 of NPMc+ AML paraffin-embedded bone marrow biopsies; lymphoid involvement was excluded in 3 investigated cases. These findings suggest that NPMc+ AML derives from either a common myeloid or earlier progenitor. Immunohistochemical studies show that varying combinations and ratios of NPMc+ leukemic cells from distinct lineages are responsible for heterogeneity within each French-American-British (FAB) classification type and for NPMc+ AML falling into different FAB categories. These findings question the value of FAB criteria in subdividing the WHO category of "AML not otherwise characterized" and suggest that, for clinical use, NPMc+ AML be provisionally regarded as a separate AML with prognostic significance.

Nucleophosmin (NPM) exon-12 mutations occur in 50% to 60% of adult acute myeloid leukemia (AML) with normal karyotype and are predictors of favorable prognosis. We evaluated bone marrow or peripheral blood samples from 450 adult patients with AML of the GIMEMA (Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto)/AML12 EORTC (European Organization for Research and Treatment of Cancer) trial to (1) search for new exon-12 NPM mutations; (2) determine whether NPM immunostaining on paraffin-embedded biopsies predicts NPM mutations; and (3) investigate altered nucleocytoplasmic NPM traffic in primary AML cells. Fourteen NPM mutations, including 8 new variants, were identified. All 200 AML cases expressing cytoplasmic NPM (NPMc(+) AML) carried NPM mutations. None of the 250 cases with nucleus-restricted NPM (NPMc(-) AML) was mutated. At the C-terminus, NPM leukemic mutants carried mutations of only tryptophan 290 or of both tryptophans 288 and 290 and a new nuclear export signal (NES) motif, which appear to underlie their nuclear export. The specific Crm1/exportin-1 inhibitor leptomycin-B relocated NPM mutants from cytoplasm to nucleus of primary NPMc(+) AML cells, demonstrating that nuclear export is NES dependent. NPM mutants bound and recruited wild-type NPM into leukemic cell cytoplasm. Because alterations at C-terminus of leukemic NPM mutants are similar, immunohistochemistry detects all exon-12 NPM mutations and is a valuable, inexpensive tool in the diagnostic-prognostic work-up of patients with AML with normal karyotype.

We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AML-associated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus.

NPM1 gene mutations are the most frequent genetic lesion in the 60% of adult acute myeloid leukemias (AMLs) with normal karyotype and no evidence of typical fusion genes (BCR/ABL1, PML/RARA, AML1/ETO, CBFB/MYH11, DEK/CAN). Using direct sequencing we previously identified six different heterozygous mutants within exon 12 encoding the nucleophosmin C-terminus. Because of these mutations the shuttling protein nucleophosmin is aberrantly delocalized in the cytoplasm of leukemic cells (NPMc+). Here, we designed and tested a denaturing high-performance liquid chromatography (DHPLC) assay to detect NPM1 mutated variants. To assess specificity, sensitivity, reliability, and reproducibility, we analyzed DNA from 120 primary adult AMLs and compared DHPLC results with immunohistochemistry and sequencing. All electropherogram profiles in the 26 NPMc+ leukemias were different from the wild type, indicating 100% sensitivity. Sequencing categorized mutations A, B, and D, and all mutation A cases gave identical elution profiles. The other mutations showed typical chromatograms, with mutations B and D differing for one nucleotide. Elution profiles and sequencing also identified four new variants. Our results suggest that DHPLC detects NPM1mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML.

Between 1996 and 2000, 90 newly diagnosed adult patients with T-acute lymphoblastic leukemia (T-ALL) were registered in the Gruppo Italiano Malattie Ematologiche dell'Adulto (GIMEMA) Leucemia Acuta Limfoide (LAL) 0496 protocol. Cases were centrally processed for morphology, immunophenotype, cytogenetics, molecular biology, and multidrug resistance (MDR). Twenty-two patients were females and 68 were males. Four percent of cases were pro-T, 47% pre-T, 39% cortical T, and 10% mature T-ALL. Fifty-six percent of patients with pro-T + pre-T-ALL achieved complete remission (CR) compared with 91% for cortical + mature cases (P = .002). CD34 expression was associated with a significantly lower CR rate: 54% versus 84% (P = .009). Thirty-one (36.5%) of 85 patients had an abnormal karyotype, the most common abnormality (15%) being a partial del(6q). The cytogenetic profile did not impact on CR achievement. MDR1 function, present in 26% of cases, correlated significantly with CR achievement (P = .004). A highly significant (P = .001) difference in CR rate was observed between patients who did not express the CD13/CD33/CD34 antigens and were MDR functionally negative (96%) compared with patients positive for at least one of these markers (57%). Multivariate analysis showed an impact on CR achievement for CD33 expression and MDR1 function. An extensive biologic workup of adult T-ALL cases at presentation is recommended in order to design tailored therapeutic strategies aimed at improving CR rates.

Nucleophosmin (NPM1) exon-12 gene mutations are the hallmark of a large acute myelogenous leukemia (AML) subgroup with normal karyotype, but their prognostic value in this AML subset has not yet been determined. We screened 401 AML patients with normal karyotype treated within the German AML Cooperative Group Protocol 99 (AMLCG99) study for NPM1 mutations. Results were related with partial tandem duplications within the MLL gene (MLL-PTD), Fms-like tyrosine kinase 3-length mutations (FLT3-LM), the tyrosine kinase domain of FLT3 (FLT3-TKD), NRAS, KIT, and CEBPA mutations and with clinical characteristics and outcome. NPM1 mutations were detected in 212 (52.9%) of 401 patients. Fourteen mutations, including 8 new variants, were identified. NPM1-mutated cases associated frequently with FLT3 mutations but rarely with other mutations. The NPM1-mutated group had a higher complete remission (CR) rate (70.5% vs 54.7%, P = .003), a trend to a longer overall survival (OS; median 1012 vs 549 days, P = .076), and significantly longer event-free survival (EFS; median 428 vs 336 days; P = .012). The favorable impact of NPM1 mutations on OS and EFS clearly emerged in the large group (264 [66.8%] of 395 cases) of normal-karyotype AML without FLT3-LM. This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM1+/FLT3-LM+ double positive was similar to NPM1-/FLT3-LM+ cases. In conclusion, this study demonstrates that NPM1+/FLT3-LM- mutations are an independent predictor for a favorable outcome in AML with normal karyotype.

Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein involved in leukemia-associated chromosomal translocations, and it regulates the alternate reading frame (ARF)-p53 tumor-suppressor pathway. Recently, it has been demonstrated that mutations of the NPM1 gene alter the protein at its C-terminal, causing its cytoplasmic localization. Cytoplasmic NPM was detected in 35% of adult patients with primary non-French-American-British (FAB) classification M3 acute myeloid leukemia (AML), associated mainly with normal karyotype. We evaluated the prevalence of the NPM1 gene mutation in non-M3 childhood AML patients enrolled in the ongoing Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP-AML02) protocol in Italy. NPM1 mutations were found in 7 (6.5%) of 107 successfully analyzed patients. NPM1-mutated patients carried a normal karyotype (7/26, 27.1%) and were older in age. Thus, the NPM1 mutation is a frequent abnormality in AML patients without known genetic marker; the mutation may represent a new target to monitor minimal residual disease in AML and a potential candidate for alternative and targeted treatments.

Approximately one third of acute myeloid leukemias (AMLs) are characterized by aberrant cytoplasmic localization of nucleophosmin (NPMc+ AML), consequent to mutations in the NPM putative nucleolar localization signal. These events are mutually exclusive with the major AML-associated chromosomal rearrangements, and are frequently associated with normal karyotype, FLT3 mutations, and multilineage involvement. We report the gene expression profiles of 78 de novo AMLs (72 with normal karyotype; 6 without major chromosomal abnormalities) that were characterized for the subcellular localization and mutation status of NPM. Unsupervised clustering clearly separated NPMc+ from NPMc- AMLs, regardless of the presence of FLT3 mutations or non-major chromosomal rearrangements, supporting the concept that NPMc+ AML represents a distinct entity. The molecular signature of NPMc+ AML includes up-regulation of several genes putatively involved in the maintenance of a stem-cell phenotype, suggesting that NPMc+ AML may derive from a multipotent hematopoietic progenitor.

The Gruppo Italiano Malattie Ematologiche dell'Adulto (GIMEMA) 0496 protocol, through the central handling of bone marrow samples at presentation, allowed us to combine cytogenetic and molecular information on a large series of adults with acute lymphoblastic leukemia (ALL) treated homogeneously, enabling us to define as broadly as possible their genetic profile and to determine the impact on outcome of the cytogenetic-molecular signature. Of 414 patients centrally processed, 325 were considered for the categorization into the following cytogenetic-molecular subgroups: normal, t(9;22)/BCR-ABL, t(4;11)/MLL-AF4, t(1;19)/E2A-PBX1, 9p/p15-p16 deletions, 6q deletions, miscellaneous structural abnormalities, and hyperdiploid. The inclusion into each subgroup was based on a hierarchical approach: molecular abnormalities with adverse prognosis had precedence over karyotypic changes with less-defined prognosis and the latter over ploidy. Patients without abnormalities and those with isolated 9p/p15-p16 deletions showed a relatively favorable outcome (median disease-free survival [DFS], > 3 years). The t(9;22)/BCR-ABL, t(4;11)/MLL-AF4, t(1; 19)/E2A-PBX1 defined a group with dismal prognosis (median DFS, 7 months), whereas 6q deletions, miscellaneous aberrations, and hyperdiploidy predicted an intermediate prognosis (median DFS, 19 months). This study highlights the importance of a combined cytogenetic-molecular profiling of adult ALL at presentation as a critical independent determinant of their outcome, providing further evidence of the necessity of a risk-adapted therapeutic algorithm for an optimal management of these patients.

To elucidate the biologic and clinical heterogeneity of adult pro-B acute lymphoblastic leukemia (ALL) (ie, terminal deoxynucletidyl-transferase-positive[TdT+], CD19+, CD10-, surface immunoglobulin-negative [SIg-]), we evaluated 66 patients enrolled in the Italian multicentric Gruppo Italiano Malattie Ematologiche dell'Adulto (GIMEMA) 0496 study between October 1996 and December 1999. The ALL1/AF4 fusion transcript, originating from the t(4;11) translocation, was detected in 24 patients (36.4%), and the BCR/ABL chimeric product was found in 6 patients (9%), while the remaining 36 cases (54.6%) were ALL1/AF4-BCR/ABL-negative. A white blood cell (WBC) count higher than 50 x 109/L was found in 13 of 24, 2 of 6, and 6 of 36 of the ALL1/AF4-positive, BCR/ABL-positive, and ALL1/AF4-BCR/AB-negative patients, respectively (P =.007). None of the 24 ALL1/AF4-positive patients coexpressed the CD13 and/or CD33 myeloid antigens. By contrast, CD13 and CD33 molecules were detected, respectively, in 3 of 6 and in 14 of 33 cases of the BCR/ABL-positive patient group, and in 2 of 6 and 9 of 35 cases of the ALL1/AF4-BCR/ABL-negative patient group. These differences still remained statistically significant even if the BCR/ABL-positive patients were excluded from the analysis. A complete remission (CR) was achieved in 52 (83.4%) of the 62 patients with ALL evaluable for response to treatment. CR rates were similar in the 3 genotypic groups. By contrast, comparing patients with or without the ALL1/AF4 gene the probability of remaining in continuous complete remission (CCR) at 3.5 years was 16% and 49.8%, respectively (P =.005). Our data demonstrate that in adult pro-B-ALL a distinction should be made between pro-B-ALL cases with and without the ALL1/AF4 or the BCR/ABL chimeric genes, since the absence of both of these fusion genes correlates with a significantly better clinical outcome after intensive polychemotherapy treatment without hematopoietic stem cell transplantation.

Little is known about the prognostic role of multidrug resistance (MDR) in adults with newly diagnosed acute lymphoblastic leukemia (ALL). In the context of the GIMEMA ALL0496 protocol, we evaluated the impact of MDR1 (protein expression and function) on the achievement of complete remission (CR) and clinical outcome. Flow cytometric analysis of MDR1 expression (D) and function (rhodamine-123 efflux) was obtained in 203 and 158 patients, respectively. MDR1 expression was detected in 44 (21.7%) of 203 patients, and function was found in 23 (14.6%) of 158 (14.6%) patients. Expression of the multidrug resistance-associated protein 1 (MRP1) and lung-resistance protein (LRP) evaluated in 43 samples was found in 13 and 26 patients, respectively. Among the 200 patients evaluable for the clinical correlation study, 125 (79.6%) of 157 without MDR1 expression achieved CR compared with 23 (53.5%) of 43 with MDR1 expression (P =.001). At univariate analysis, MDR1 expression was significantly associated with CR when considered as a dichotomized (P =.001) or continuous (P =.01) variable. At multivariate analysis, dichotomized evaluation of MDR1 expression independently predicted CR (P =.004) with age (P =.03) and CD34 (P =.03); as a continuous variable, MDR1 expression (P =.03) was the only significant factor other than CD34 (P =.01). MDR1 function failed to predict achievement of CR or of MRP1 and LRP expression. MDR1 expression did not correlate with CR duration, nor did it predict for survival duration. These results demonstrate that MDR1 expression in de novo adult ALL is an independent predictor of CR achievement.

Fusion between the NUP98 and NSD3 genes in a patient with acute myeloid leukemia associated with t(8;11)(p11.2;p15), is reported for the first time. The t(8;11)(p11.2;p15) was identified by classical cytogenetics. Fluorescence in situ hybridization (FISH) analysis revealed a split signal with a mix of BAC 118H17 and 290A12, indicating the translocation disrupted NUP98. FISH restriction at 8p11-12 showed a split of BAC 350N15. Molecular investigations into candidate genes in this BAC showed the NUP98 fusion partner at 8p11.2 was the NSD3 gene. To date the NSD3 gene has never been implicated in hematologic malignancies.

A patient with type II cryoglobulinemic vasculitis and hepatitis C virus (HCV) infection presented with a leukemiclike proliferation of B cells bearing marginal zone B-cell phenotypic markers. A partial trisomy 3 (bands 3q11-29) and overexpression of Bcl-2 without t(14;18) translocation was detected in the monoclonal B cells that were classic rheumatoid factor-producing B cells bearing the WA cross-idiotype. Treatment with interferon-alpha produced a complete clinical remission and synchronous marked decreases in viremia and monoclonal B-cell prevalence. This is the first report of partial trisomy 3 and Bcl-2 overexpression in type II cryoglobulinemic vasculitis associated with HCV infection. Further studies of HCV-infected patients with and without type II cryoglobulinemia are required to determine the prevalence and possible physiologic and/or pathophysiologic significance of these findings.

The molecular cloning of the t(5;10)(q33;q22) associated with atypical chronic myeloid leukemia (CML) is reported. Fluorescence in situ hybridization (FISH), Southern blot, and reverse transcriptase- polymerase chain reaction analysis demonstrated that the translocation resulted in an H4/platelet-derived growth factor receptor betaR (PDGFbetaR) fusion transcript that incorporated 5' sequences from H4 fused in frame to 3' PDGFbetaR sequences encoding the transmembrane, WW-like, and tyrosine kinase domains. FISH combined with immunophenotype analysis showed that t(5;10)(q33;q22) was present in CD13(+) and CD14(+) cells but was not observed in CD3(+) or CD19(+) cells. H4 has previously been implicated in pathogenesis of papillary thyroid carcinoma as a fusion partner of RET. The H4/RET fusion incorporates 101 amino acids of H4, predicted to encode a leucine zipper dimerization domain, whereas the H4/PDGFbetaR fusion incorporated an additional 267 amino acids of H4. Retroviral transduction of H4/PDGFbetaR, but not a kinase-inactive mutant, conferred factor-independent growth to Ba/F3 cells and caused a T-cell lymphoblastic lymphoma in a murine bone marrow transplantation assay of transformation. Mutational analysis showed that the amino-terminal H4 leucine zipper domain (amino acids 55-93), as well as H4 amino acids 101 to 386, was required for efficient induction of factor-independent growth of Ba/F3 cells. Tryptophan-to-alanine substitutions in the PDGFbetaR WW-like domain at positions 566/593, or tyrosine-to-phenylalanine substitutions at PDGFbetaR positions 579/581 impaired factor-independent growth of Ba/F3 cells. H4/PDGFbetaR is an oncoprotein expressed in t(5;10)(q33;q22) atypical CML and requires dimerization motifs in the H4 moiety, as well as residues implicated in signal transduction by PDGFbetaR, for efficient induction of factor-independent growth of Ba/F3 cells. (Blood. 2001;97:3910-3918)

Twenty-five patients (22 adults and 3 infants) with ALL1/AF4-positive acute lymphoblastic leukemia (ALL) were prospectively monitored by reverse transcriptase-polymerase chain reaction (RT-PCR) between January 1992 and July 1999. After high-dose induction and consolidation chemotherapy without bone marrow transplantation, all patients had a complete hematologic remission. Using nested RT-PCR (sensitivity 10(-4)), we observed conversion to PCR negativity in 11 (44%) of the patients. Thirteen of the 14 patients who did not have a molecular remission had a relapse at a median time of 4 months (range, 1 - 20 months). Of the 11 patients who had a conversion to PCR negativity, 5 reconverted to PCR positivity within 1 to 14 months. These 5 patients all progressed to hematologic relapse after 2, 3, 4, 4, and 7 months, respectively. Of the remaining 6 patients, 4 are in persistent hematologic and molecular remission at 12, 14, 88, and 96 months, whereas 2 are early in their follow-up. Actuarial probabilities of relapse and overall survival were 100% and 0% at 14 and 24 months and 67% and 43% at 96 and 100 months, respectively, in patients who had persistent RT-PCR positivity and in those who had a molecular remission. For both relapse and survival, the differences observed between the two groups were significant (P =.003 and P <.005, respectively). This study, which represents the first prospective analysis of residual-disease monitoring carried out in a substantial series of patients with t(4;11)-positive ALL, emphasizes the clinical relevance of RT-PCR-based methods to monitor minimal residual disease in this leukemia subset. (Blood. 2000;95:96-101)

The ETV6 (also known as TEL) gene on chromosome 12p13 is the target of a number of translocations associated with various hematologic malignancies. The contribution of ETV6 to leukemogenesis occurs through different mechanisms that involve either its helix-loop-helix dimerization domain or its E26 transformation-specific (ETS) DNA-binding domain. Using fluorescence in situ hybridization we characterized seven new ETV6 rearrangements in chronic myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and non-Hodgkin's lymphoma. These aberrations, not always discernible at the cytogenetic level, include a t(5;12)(q31;p13), t(6;12;17)(p21;p13;q25), t(7;12)(p15;p13), t(7;12)(p12;p13), t(7;12)(q36;p13), t(12;13)(p13;q12), and a not completely defined t(12;?)(p13;?). Loss or disruption of the second ETV6 allele by a del(12)(p12p13) or by an intragenic ETV6 deletion was detected in two cases. In six cases the 12p13 breakpoint occurred in the 5' end of ETV6, upstream to exons encoding the HLH domain, whereas the remaining case had a breakpoint between the exons coding for the HLH domain and the exons coding for the ETS domain of ETV6. These observations provide further evidence for the multiple contributions of ETV6 in the pathogenesis of a wide range of hematologic malignancies.

Fluorescent in situ hybridization (FISH) analysis with a panel of DNA probes for 13q13.1-q14.3 was performed on 20 cases of myeloid malignancies, of which 17 showed a del(13)(q) and three had translocations affecting 13q. By chromosome morphology, deletions consistently involved bands q14 and q21. In addition to confirming the chromosome data, FISH allowed us to delineate a commonly deleted region that was flanked by YAC 833A2 and YAC 854D4. Three cases with 13q translocations unexpectedly showed accompanying cryptic microdeletions of 13q, and in one case the commonly deleted region could be narrowed to a genomic segment, which includes YAC 937C7, RB1, and YAC 745E3. Homozygous deletions were not detected. This region overlaps with the smallest deleted region of 13q14 in chronic lymphocytic leukemia.

Translocation t(5;12)(q33;p13), resulting in an ETV6/PDGFRB gene fusion, is a recurrent chromosomal abnormality associated with chronic myelomonocytic leukemia (CMML). An analogous translocation was also found in four cell lines with features of pre-B acute lymphoblastic leukemia (ALL). Using fluorescence in situ hybridization (FISH) we show here that in three of these cell lines identical complex rearrangements occurred. However, the regions involved on 5q and 12p are different from the breakpoints in CMML, and the translocation is accompanied by seemingly identical cryptic deletions of both 5q and 12p chromosome sequences in all analyzed pre-B ALL cell lines. The similar cytogenetic, FISH, and immunophenotyping findings in the three cell lines suggest that the t(5;12)(q31q33;p12) defines a new entity of pre-B ALL.

Clinical, histologic, cytogenetic, and molecular genetic data of 31 patients with extranodal, nodal, and splenic marginal zone B-cell lymphoma (MZBCL) are presented. Despite these variable clinical manifestations, a similar spectrum of morphologic features as well as distinctive immunophenotypic findings were noted. In all cases, a monotypic B-cell proliferation consistently negative for CD5, CD10, and CD23 was found expanding the marginal zone of the B follicle with and without colonization of the follicle centers. Clonal chromosomal abnormalities were detected in 23 of the 31 patients. Recurrent aberrations included whole or partial trisomy 3 (18 cases), trisomy 18 (9 cases), and structural rearrangements of chromosome 1 with breakpoints in 1q21 (9 cases) or 1p34 (6 cases), all of which were seen in extranodal, nodal, as well as splenic MZBCL. Abnormalities of the additional chromosome 3, such as +del(3)(p13),+i(3)(q10), or structural changes involving the distal part of the long arm, were evident in 9 of the 18 cases. All recurrent abnormalities were found in MZBCL more frequently than in other histologic entities of B-cell non-Hodgkin's lymphoma (B-NHL). None of the known lymphoma-associated chromosomal changes or rearrangements of the BCL1, BCL2, BCL3, BCL6, and CMYC genes were detected. We conclude that MZBCL represent a distinct entity of B-NHL with characteristic morphologic and immunophenotypic features and particular chromosomal abnormalities, and that a close histogenetic relationship between extranodal, nodal, and splenic MZBCL is likely, although the clinical presentation may vary.

A t(5;12)(q33;p13) translocation is a recurrent chromosome abnormality in a subgroup of myeloid malignancies with features of both myeloproliferative disorders and myelodysplastic syndromes (MDSs). The molecular consequence of a t(5;12) is a fusion between the platelet-derived growth factor receptor-B gene on chromosome 5 and a novel ETS-like gene, TEL, on chromosome 12. We report on three patients with a t(5;12)(q33;p13) diagnosed as chronic myelomonocytic leukemia, and one case of a t(10;12)(q24;p13) in a progressive MDS, with eosinophilia and monocytosis. Involvement of the TEL gene in these chromosome translocations was investigated by fluorescence in situ hybridization (FISH) with cosmid probes containing selectively the 5' end or 3' end of TEL. Hybridization of these cosmids to the der(5)/der(10) or a der(12), respectively, demonstrated a rearrangement of TEL in both translocations, showing that the t(10;12) is a variant translocation of the t(5;12). Cloning of the fusion cDNA of one case of t(5;12) showed that the breakpoint occurred at the RNA level at exactly the same position as reported by Golub et al (Cell 77:307, 1994). In addition, the TEL gene on chromosome 12 could be localized between two probes previously mapped to 12p13, namely PRB1 and D12S178, leading to a better definition of the position of TEL in this chromosome region. Moreover, in the case involving chromosome 10, the breakpoint occurred between cKTN206 and cKTN312/LYT-10 at 10q24. Clinicohematological data in these studies as well as the restriction mapping of chromosomal breakpoints strongly suggest that (1) common features in MDSs involving the TEL gene are monocytosis and eosinophilia, (2) chromosomes other than no. 5 may be involved and at least a t(10;12)(q24;p13) variant chromosome translocation does exist in these MDSs, and (3) both standard and variant 12p/TEL translocations may be identified by FISH with appropriate probes.

A 14q+ marker with extra material derived from chromosome 11 long arm, i.e. segment q13----qter, has been found in cells from a pleural effusion in a patient with highly malignant multiple myeloma. The segment 11q13----qter was trisomic because of the presence of both apparently normal homologous chromosomes 11.

We present four patients with acute nonlymphocytic leukemia, one M2 and three M4 French-American-British (FAB) types and one patient with refractory anemia with excess of blasts in transformation who at diagnosis had trisomy of chromosome 4 as the primary karyotypic anomaly. This chromosome anomaly probably defines a previously undescribed subset of acute nonlymphoid leukemias. Hematologic characteristics commonly found in these patients were dysplastic features of all bone marrow lineages, suggesting that trisomy 4-associated disorders involve the early myeloid hematopoietic stem cell.

We describe a case of Burkitt-type acute lymphoblastic leukemia (L3 according to the classification FAB) with a variant t(2;8)(p12;q24) and additional chromosomal abnormalities at diagnosis. The karyotype was 47,X,Xq+,t(2;8)(p12;q24),7q+,12p+,+mar. The literature on chromosome rearrangements associated with t(2;8) in L3 leukemias has been reviewed.

Cytologic and cytogenetic results obtained from patients fulfilling the FAB criteria for the diagnosis of acute nonlymphocytic leukemia (ANLL) of megakaryocytic lineage (ANLL-M7) are reported. Eleven cases were de novo ANLL-M7, of whom three presented with acute myelofibrosis. Four cases were megakaryoblastic transformations of chronic myelogenous leukemia (two cases), refractory anemia with excess of blasts (one case), and polycythemia vera (one case). Four patients showed a minority of granular blasts, with occasional Auer rods in one. Positive myeloperoxidase and/or sudan black-B stainings and CD13 positivity in these cases were consistent with the presence of a myeloid involvement. Morphologic evidence of associated myelodysplastic features was detected in all evaluable patients with de novo ANLL-M7. These cytologic findings indicate that ANLL-M7 may frequently represent a multilineage proliferation. Cytogenetic studies revealed -7/7q- and +8, alone or in combination with additional aberrations, in three cases each. Rearrangements involving bands 3q21 or 3q26 were seen in two patients and +21, as an additional aberration, in one. Other structural rearrangements all observed in a single patient were inv(16)(p13q22) at megakaryoblastic relapse with bone marrow eosinophilia, t(13;20)(q13 or 14;q11), del(20)(q11), and der(7)t(7;17)(p14;q22). Most breakpoints of these aberrations are located at bands frequently rearranged in malignant myeloid stem cell disorders. A review of 31 cases of the literature showed a frequent occurrence of -7/7q- and -5/5q- in ANLL-M7. Many of the chromosome aberrations so far described in ANLL-M7 appear to be shared by a spectrum of myeloid neoplasias and may be related to mechanisms conferring proliferative advantage to undifferentiated stem cells.