Blinatumomab Redirects Donor Lymphocytes against CD19+ Acute Lymphoblastic Leukemia without Relevant Bystander Alloreactivity after Haploidentical Hematopoietic Stem Cell Transplantation.

Blinatumomab alone or with donor leukocyte infusions (DLI) has been used after allogeneic hematopoietic stem cell transplantation (HSCT) as a salvage therapy in relapsing patients with CD19+ hematological malignancies. It was effective in a fraction of them, with low incidence of Graft-versus-Host Disease (GvHD). Immunosuppressive drugs used as GvHD prophylaxis hinder T cell function and reduce the efficacy of the treatment. Because T cell-depleted haploidentical HSCT with donor regulatory and conventional T cells (Treg/Tcon haploidentical HSCT) does not require post-transplant immunosuppression, it is an ideal platform for the concomitant use of blinatumomab and DLI. However, the risk of GvHD is high because the donor is haploidentical. We treated two patients with CD19+ acute lymphoblastic leukemia (ALL) who had relapsed after Treg/Tcon haploidentical HSCT with blinatumomab and DLI. Despite the mismatch for one HLA haplotype, they did not develop GvHD and achieved complete remission with negative minimal residual disease. Consistently, we found that blinatumomab did not enhance T cell alloreactivity in vitro. Eventually, the two patients relapsed again because of their high disease risk. This study suggests that treatment with blinatumomab and DLI can be feasible to treat relapse after haploidentical transplantation, and its pre-emptive use should be considered to improve efficacy.

Clinical-Grade Expanded Regulatory T Cells Are Enriched with Highly Suppressive Cells Producing IL-10, Granzyme B, and IL-35.

In the setting of T cell-depleted, full-haplotype mismatched transplantation, adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) can prevent graft-versus-host disease (GVHD) and improve post-transplantation immunologic reconstitution and is associated with a powerful graft-versus-leukemia effect. To improve the purity and the quantity of the infused Tregs, good manufacturing practices (GMP)-compatible expansion protocols are needed. Here we expanded Tregs using an automated, clinical-grade protocol. Cells were extensively characterized in vitro, and their efficiency was tested in vivo in a mouse model. Tregs were selected by CliniMacs (CD4+CD25+, 94.5 ± 6.3%; FoxP3+, 63.7 ± 11.5%; CD127+, 20 ± 3%; suppressive activity, 60 ± 7%), and an aliquot of 100 × 106 was expanded for 14 days using the CliniMACS Prodigy System, obtaining 684 ± 279 × 106 cells (CD4+CD25+, 99.6 ± 0.2%; FoxP3+, 82 ± 8%; CD127+, 1.1 ± 0.8%; suppressive activity, 75 ± 12%). CD39 and CTLA4 expression levels increased from 22.4 ± 12% to 58.1 ± 13.3% (P < .05) and from 20.4 ± 6.7% to 85.4 ± 9.8% (P < .01), respectively. TIM3 levels increased from .4 ± .05% to 29 ± 16% (P < .05). Memory Tregs were the prevalent population, whereas naive Tregs almost disappeared at the end of the culture. mRNA analysis displayed significant increases in CD39, IL-10, granzyme B, and IL-35 levels at the end of culture period (P < .05). Conversely, IFNγ expression decreased significantly by day +14. Expanded Tregs were sorted according to TIM3, CD39, and CD62L expression levels (purity >95%). When sorted populations were analyzed, TIM3+ cells showed significant increases in IL-10 and granzyme B (P < .01) .When expanded Tregs were infused in an NSG murine model, mice that received Tcons only died of GVHD, whereas mice that received both Tcons and Tregs survived without GVHD. GMP grade expanded cells that display phenotypic and functional Treg characteristics can be obtained using a fully automated system. Treg suppression is mediated by multiple overlapping mechanisms (eg, CTLA-4, CD39, IL-10, IL-35, TGF-ß, granzyme B). TIM3+ cells emerge as a potentially highly suppressive population. © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Chemoradiotherapy with temozolomide after high-dose methotrexate for primary CNS lymphoma: a multicenter phase I study of a response-adapted strategy.

This study aimed to define the maximum tolerated dose (MTD) of temozolomide (TMZ) concurrent with radiotherapy (RT) after high-dose methotrexate (HD-MTX) for newly diagnosed primary central nervous system lymphoma (PCNSL). Adult patients with PCNSL were treated according to a response-adapted strategy. HD-MTX (3.5 g/m2) was followed by concomitant RT and escalating TMZ (50-60-75 mg/m2/day, 5 days/week). The total radiation dose was modulated according to the patient's response to HD-MTX. All patients received 30 Gy to the whole brain plus leptomeninges to C2, including the third posterior of the orbital cavity (clinical target volume 2; CTV2), plus 6, 10, or 16 Gy to the primary site, including the residual mass (CTV1), if a complete response (CR), partial response (PR)/stable disease (SD), or progressive disease (PD) was observed, respectively. Acute toxicities were graded according to the RTOG-EORTC criteria. Dose-limiting toxicity (DLT) was defined as grade 4 hematological toxicity or grade 3-4 hepatic toxicity, although 75 mg/m2/day was the maximum dose regardless of DLT. Neurocognitive function was evaluated using the Mini-Mental State Examination. Three patients were enrolled at each TMZ dose level (total = 9 patients). Twelve lesions were treated. Six patients received 2 cycles of HD-MTX, while 3 received only 1 cycle because of hepatic or renal toxicity. All patients completed chemoradiotherapy without interruptions. No DLT events were recorded. TMZ appears to be tolerable at a dose of 75 mg/m2/day when administered concomitantly with radiotherapy and after HD-MTX.

Allogeneic hematopoietic stem cell transplantation in patients with diffuse large B cell lymphoma relapsed after autologous stem cell transplantation: a GITMO study.

Patients who relapse after an autologous hematopoietic stem cell transplantation (SCT) have a very poor prognosis. We have retrospectively analyzed diffuse large B cell lymphoma patients who underwent an allo-SCT after an auto-SCT relapse reported in the Gruppo Italiano Trapianto di Midollo Osseo (GITMO) database. From 1995 to 2008, 3449 autologous transplants were reported in the GITMO database. Eight hundred eighty-four patients relapsed or progressed after transplant; 165 patients, 19% of the relapsed patients, were treated with allo-transplant. The stem cell donor was related to the patient in 108 cases. A reduced intensity conditioning regimen was used in 116. After allo-SCT, 72 patients (43%) obtained a complete response and 9 obtained a partial response with an overall response rate of 49%; 84 patients (51%) experienced rapid progression of disease. Ninety-one patients died, 45 due to disease and 46 due to treatment-related mortality. Acute graft-versus-host disease was recorded in 57 patients and a chronic GvHD in 38 patients. With a median follow-up of 24 months (2-144) after allo, overall survival (OS) was 39%, and after a median of 21 months (2-138) after allo, progression-free survival (PFS) was 32%. Multivariate analysis indicated that the only factors affecting OS were status at allo-SCT, and those affecting PFS were status at allo-SCT and stem cell donor. This retrospective analysis shows that about one-fifth of patients with diffuse large B cell lymphoma who experience relapse after autologous transplantation may be treated with allogeneic transplantation. Moreover, the only parameter affecting either OS or PFS was the response status at the time of allo-SCT.

Impact of total marrow/lymphoid irradiation dose to the intestine on graft-versus-host disease in allogeneic hematopoietic stem cell transplantation for hematologic malignancies.

Background and purpose: Graft-versus-host disease (GvHD) is a leading cause of non-relapse mortality in patients undergoing allogeneic hematopoietic stem cell transplantation. The Perugia Bone Marrow Transplantation Unit designed a new conditioning regimen with total marrow/lymphoid irradiation (TMLI) and adaptive immunotherapy. The present study investigated the impact of radiotherapy (RT) doses on the intestine on the incidence of acute GvHD (aGvHD) in transplant recipients, analyzing the main dosimetric parameters. Materials and methods: Between August 2015 and April 2021, 50 patients with hematologic malignancies were enrolled. All patients underwent conditioning with TMLI. Dosimetric parameters (for the whole intestine and its segments) were assessed as risk factors for aGvHD. The RT dose that was received by each intestinal area with aGvHD was extrapolated from the treatment plan for each patient. Doses were compared with those of the whole intestine minus the affected area. Results: Eighteen patients (36%) developed grade ≥2 aGvHD (G2 in 5, G3 in 11, and G4 in 2). Median time to onset was 41 days (range 23-69 days). The skin was involved in 11 patients, the intestine in 16, and the liver in 5. In all 50 TMLI patients, the mean dose to the whole intestine was 7.1 Gy (range 5.07-10.92 Gy). No patient developed chronic GvHD (cGvHD). No dosimetric variable emerged as a significant risk factor for aGvHD. No dosimetric parameter of the intestinal areas with aGvHD was associated with the disease. Conclusion: In our clinical setting and data sample, we have found no clear evidence that current TMLI dosages to the intestine were linked to the development of aGvHD. However, due to some study limitations, this investigation should be considered as a preliminary assessment. Findings need to be confirmed in a larger cohort and in preclinical models.

Haploidentical age-adapted myeloablative transplant and regulatory and effector T cells for acute myeloid leukemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the most effective treatment in eradicating high-risk acute myeloid leukemia (AML). Here, we present data from a novel HLA-haploidentical HSCT protocol that addressed the 2 remaining major unmet medical needs: leukemia relapse and chronic graft-versus-host disease (cGVHD). Fifty AML patients were enrolled in the study. The conditioning regimen included total body irradiation for patients up to age 50 years and total marrow/lymphoid irradiation for patients age 51 to 65 years. Irradiation was followed by thiotepa, fludarabine, and cyclophosphamide. Patients received an infusion of 2 × 106/kg donor regulatory T cells on day -4 followed by 1 × 106/kg donor conventional T cells on day -1 and a mean of 10.7 × 106 ± 3.4 × 106/kgpurified CD34+ hematopoietic progenitor cells on day 0. No pharmacological GVHD prophylaxis was administered posttransplantation. Patients achieved full donor-type engraftment. Fifteen patients developed grade ≥2 acute GVHD (aGVHD). Twelve of the 15 patients with aGVHD were alive and no longer receiving immunosuppressive therapy. Moderate/severe cGVHD occurred in only 1 patient. Nonrelapse mortality occurred in 10 patients. Only 2 patients relapsed. Consequently, at a median follow-up of 29 months, the probability of moderate/severe cGVHD/relapse-free survival was 75% (95% confidence interval, 71%-78%). A novel HLA-haploidentical HSCT strategy that combines an age-adapted myeloablative conditioning regimen with regulatory and conventional T-cell adoptive immunotherapy resulted in an unprecedented cGVHD/relapse-free survival rate in 50 AML patients with a median age of 53 years. This trial was registered with the Umbria Region Institutional Review Board Public Registry as identification code 02/14 and public registry #2384/14 and at www.clinicaltrials.gov as #NCT03977103.

Combined interphase fluorescence in situ hybridization elucidates the genetic heterogeneity of T-cell acute lymphoblastic leukemia in adults.

BACKGROUND: Molecular lesions in T-cell acute lymphoblastic leukemias affect regulators of cell cycle, proliferation, differentiation, survival and apoptosis in multi-step pathogenic pathways. Full genetic characterization is needed to identify events concurring in the development of these leukemias. DESIGN AND METHODS: We designed a combined interphase fluorescence in situ hybridization strategy to study 25 oncogenes/tumor suppressor genes in T-cell acute lymphoblastic leukemias and applied it in 23 adult patients for whom immunophenotyping, karyotyping, molecular studies, and gene expression profiling data were available. The results were confirmed and integrated with those of multiplex-polymerase chain reaction analysis and gene expression profiling in another 129 adults with T-cell acute lymphoblastic leukemias. RESULTS: The combined hybridization was abnormal in 21/23 patients (91%), and revealed multiple genomic changes in 13 (56%). It found abnormalities known to be associated with T-cell acute lymphoblastic leukemias, i.e. CDKN2A-B/9p21 and GRIK2/6q16 deletions, TCR and TLX3 rearrangements, SIL-TAL1, CALM-AF10, MLL-translocations, del(17)(q12)/NF1 and other cryptic genomic imbalances, i.e. 9q34, 11p, 12p, and 17q11 duplication, del(5)(q35), del(7)(q34), del(9)(q34), del(12)(p13), and del(14)(q11). It revealed new cytogenetic mechanisms for TCRB-driven oncogene activation and C-MYB duplication. In two cases with cryptic del(9)(q34), fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction detected the TAF_INUP214 fusion and gene expression profiling identified a signature characterized by HOXA and NUP214 upregulation and TAF_I, FNBP1, C9orf78, and USP20 down-regulation. Multiplex-polymerase chain reaction analysis and gene expression profiling of 129 further cases found five additional cases of TAF_I-NUP214-positive T-cell acute lymphoblastic leukemia. CONCLUSIONS: Our combined interphase fluorescence in situ hybridization strategy greatly improved the detection of genetic abnormalities in adult T-cell acute lymphoblastic leukemias. It identified new tumor suppressor genes/oncogenes involved in leukemogenesis and highlighted concurrent involvement of genes. The estimated incidence of TAF_I-NUP214, a new recurrent fusion in adult T-cell acute lymphoblastic leukemias, was 4.6% (7/152).

Totipotent stem cells bearing del(20q) maintain multipotential differentiation in Shwachman Diamond syndrome.

SBDS/7q11 gene mutations underlie the congenital Shwachman Diamond syndrome (SDS), characterized by bone marrow failure and high risk of haematological malignancies. In two cases of SDS with bone marrow failure and isolated del(20q) interphase fluorescence in situ hybridization (I-FISH) found no abnormalities in FHIT/3p14.2, IKZF1/7p13, D7S486/7q31, PTEN/10q23.3, WT1/11p13, ATM/11q23, D13S25/13q14, TP53/17p13, NF1/17q11, SMAD2/18q21, RUNX1/21q22. Fluorescence immunophenotype combined with I-FISH found del(20q) in a totipotent haematopoietic stem cell (CD34(+), CD133(+)) and downstream myelocyte (CD33(+), CD14(+), CD13(+)), erythrocyte (Glycophorin A(+)) and lymphocyte lineages (CD19(+), CD20(+), CD3(+), CD7(+)). These findings and clinical follow-ups confirm the benign course of SDS with isolated del(20q).

The Evolution of T Cell Depleted Haploidentical Transplantation.

Work on bone marrow transplantation from haploidentical donor has been proceeding for over 20 years all over the world and new transplant procedures have been developed. To control both graft rejection and graft vs. host disease, some centers have preferred to enhance the intensity of the conditioning regimens and the post-transplant immune suppression in the absence of graft manipulation; others have concentrated on manipulating the graft in the absence of any additional post-transplant immune suppressive agent. Due to the current high engraftment rates, the low incidence of graft-vs.-host disease and regimen related mortality, transplantation from haploidentical donors have been progressively offered even to elderly patients. Overall, survivals compare favorably with reports on transplants from unrelated donors. Further improvements will come with successful implementation of strategies to enhance post-transplant immune reconstitution and to prevent leukemia relapse.

T cell depletion and no post transplant immune suppression allow separation of graft versus leukemia from graft versus host disease.

Allogeneic hematopoietic cell transplantation from a human leukocyte antigen (HLA) haplotype mismatched donor (haploidentical transplantation) was not feasible for the treatment of hematologic malignancies until the early 1990s, due to the high risk of rejection and graft-versus-host disease (GVHD). The first successful protocol of haploidentical transplantation was based on a highly myeloablative and immunosuppressive conditioning regimen and the infusion of a "mega-dose" of T-cell-depleted hematopoietic stem cells. More than 90% of patients engrafted and <10% developed GVHD. The protocol did not include post-transplant immunosuppression, which favored the graft-versus-tumor effect mediated by alloreactive NK cells and residual alloreactive T cells. However, donor post-transplant immune reconstitution was slow with a high risk of infection-related mortality. More recently, T-cell-depleted haploidentical transplantation has become the platform for innovative cell therapies that aim to enhance T-cell immunity while preventing adverse reactions against host tissues. One strategy is adoptive immunotherapy with conventional T cells and regulatory T cells. Preclinical studies and clinical trials have proven that regulatory T cells control GVHD caused by co-infused conventional T cells while the graft-versus-tumor effect is retained. The use of regulatory T cells in the absence of any other form of immune suppression allowed for a conventional T cell-mediated full eradication of disease in the vast majority of high-risk acute leukemia patients.

Cryptic chromosome 9q34 deletion generates TAF-Ialpha/CAN and TAF-Ibeta/CAN fusion transcripts in acute myeloid leukemia.

In hematologic malignancies chromosome aberrations generating fusion genes include cryptic deletions. In a patient with acute myeloid leukemia and normal karyo-type we discovered a new cryptic 9q34 deletion and here report the cytogenetic and molecular findings. The 9q34 deletion extends 2.5 megabases and juxtaposes the 5' TAF-I to the 3' CAN producing a TAF-I/CAN fusion gene. TAF-I/CAN transcribes into two fusion proteins bearing either TAF-Ialpha or TAF-Ibeta moieties. We set up molecular assays to monitor the chimeric TAF-Ialpha/CAN and TAF-Ibeta/CAN transcripts which, after hematopoietic stem cell transplantation from an HLA-identical sibling, were no longer detected.

Translocations and mutations involving the nucleophosmin (NPM1) gene in lymphomas and leukemias.

Nucleophosmin (NPM) is a ubiquitously expressed nucleolar phoshoprotein which shuttles continuously between the nucleus and cytoplasm. Many findings have revealed a complex scenario of NPM functions and interactions, pointing to proliferative and growth-suppressive roles of this molecule. The gene NPM1 that encodes for nucleophosmin (NPM1) is translocated or mutated in various lymphomas and leukemias, forming fusion proteins (NPM-ALK, NPM-RARalpha, NPM-MLF1) or NPM mutant products. Here, we review the structure and functions of NPM, as well as the biological, clinical and pathological features of human hematologic malignancies with NPM1 gene alterations. NPM-ALK indentifies a new category of T/Null lymphomas with distinctive molecular and clinico-pathological features, that is going to be included as a novel disease entity (ALK+ anaplastic large cell lymphoma) in the new WHO classification of lymphoid neoplasms. NPM1 mutations occur specifically in about 30% of adult de novo AML and cause aberrant cytoplasmic expression of NPM (hence the term NPMc+ AML). NPMc+ AML associates with normal karyotpe, and shows wide morphological spectrum, multilineage involvement, a unique gene expression signature, a high frequency of FLT3-internal tandem duplications, and distinctive clinical and prognostic features. The availability of specific antibodies and molecular techniques for the detection of NPM1 gene alterations has an enormous impact in the biological study diagnosis, prognostic stratification, and monitoring of minimal residual disease of various lymphomas and leukemias. The discovery of NPM1 gene alterations also represents the rationale basis for development of molecular targeted drugs.

A common 93-kb duplicated DNA sequence at 1q21.2 in acute lymphoblastic leukemia and Burkitt lymphoma.

In three patients with acute lymphoblastic leukemia (ALL) and in another with Burkitt lymphoma (BL), conventional cytogenetics and fluorescence in situ hybridization (FISH), applied singly or in combination, showed 1q duplication in two cases of ALL with hyperdiploid karyotypes, 1q duplication resulting from an unbalanced translocation in a third case of ALL, and inv dup(1)(q) in a patient with BL. Centromeric or telomeric breakpoints and extension of the 1q duplicons varied in each case. FISH defined a minimal, common duplicated region of 93kb at band 1q21.2 corresponding to clone RP11-212K13. In this region three putative oncogenes or tumor suppressor genes have been mapped: SF3B4 (splicing factor 3b, subunit 4), OTUD7B (OTU domain containing 7B), and MTMR11 (myotubularin related protein 11). For the first time, a minimal common 1q21.2 duplicated sequence has been identified in lymphoid malignancies in a region where putative oncogenes or suppressor genes have been mapped. This finding elucidates the genomic background of ALL and BL with 1q duplication and provides the basis for molecular studies investigating which genes are involved in leukemogenesis or disease progression in these cases.

Denaturing high-performance liquid chromatography: a valid approach for identifying NPM1 mutations in acute myeloid leukemia.

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.

Prevalence, incidence and clinical outcome of hepatitis B virus and hepatitis C virus hepatitis in patients undergoing allogeneic hematopoietic stem cell transplantation between 2001 and 2004.

The prevalence, incidence and clinical course of viral hepatitis were prospectively determined in consecutive recipients of T-cell depleted hematopoieic stem cell transplants (49 mismatched, 60 matched, mean age 38 years; range 11-65). The prevalence of hepatitis B virus (HBV) was 15.6% and that of hepatitis C virus was 3.7% (HCV). HBV reactivated in one patient. Another developed ex novo acute hepatitis B which progressed to chronic hepatitis. There were no new cases of hepatitis C or worsening of pre-transplant HCV infections. HBV and HBC did not affect the outcome of T-cell depleted hematopoieic stem cell transplantation. Surveillance is important given the risk of HCV and HBV infection and/or reactivation and the efficacy of the new anti-hepatitis drugs.

The Total Body Irradiation Schedule Affects Acute Leukemia Relapse After Matched T Cell-Depleted Hematopoietic Stem Cell Transplantation.

PURPOSE: We sought to determine whether the total body irradiation (TBI) schedule affected outcome in patients with acute leukemia in complete remission who received T cell-depleted allogeneic hematopoietic stem cell transplantation from HLA identical siblings. METHODS AND MATERIALS: The study recruited 55 patients (median age, 48 years; age range, 20-66 years; 30 men and 25 women; 34 with acute myeloid leukemia and 21 with acute lymphoid leukemia). Hyperfractionated TBI (HTBI) (1.2 Gy thrice daily for 4 days [for a total dose of 14.4 Gy] from day -12 to day -9) was administered to 29 patients. Single-dose TBI (STBI) (8 Gy, at a median dose rate of 10.7 cGy/min on day -9) was given to 26 patients. RESULTS: All patients achieved primary, sustained engraftment with full donor-type chimerism. At 10 years, the overall cumulative incidence of transplant-related mortality was 11% (SE, ±0.1%). It was 7% (SE, ±0.2%) after HTBI and 15% (SE, ±0.5%) after STBI (P=.3). The overall cumulative incidence of relapse was 33% (SE, ±0.5). It was 13% (SE, ±0.5%) after HTBI and 46% (SE, ±1%) after STBI (P=.02). The overall probability of disease-free survival (DFS) was 59% (SE, ±7%). It was 67% (SE, ±0.84%) after HTBI and 37% (SE, ±1.4%) after STBI (P=.01). Multivariate analyses showed the TBI schedule was the only risk factor that significantly affected relapse and DFS (P=.01 and P=.03, respectively). CONCLUSIONS: In patients with acute leukemia, HTBI is more efficacious than STBI in eradicating minimal residual disease after HLA-matched T cell-depleted hematopoietic stem cell transplantation, thus affecting DFS.

Partial duplication of the MLL oncogene in patients with aggressive acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: MLL translocations generate a fusion gene between the 5' end of MLL and the 3' end of different partner genes. Several chromosomal mechanisms including complex and cryptic changes lead to these recombinations. Our objective was to analyze the molecular composition of chromosomes in complex karyotypes with specific MLL translocations. DESIGN AND METHODS: Fluorescence in situ hybridization (FISH) was performed in two acute leukemias (AL), one acute myeloid leukemia (AML) M5a, and one treatment-related-AL (t-AL), to investigate the nature of complex changes accompanying the respective t(9;11)(p22;q23)-MLL/AF9 and t(11;16)(q23;p13.3)-MLL/CBP. RESULTS: In the case with the MLL/AF9 chimeric transcript, duplication of a der(1) originated from an additional unbalanced translocation between the der(9)t(9;11) and a chromosome 1. The 5'AF9/3'MLL chimeric gene was present on both der(1). In the second case, there was a t(11;16)(q23;p13.3) producing one der(11) and two der(16) which derived from both homologs. One der(16) was present in multiple copies all containing the 5'CBP/3'MLL fusion gene. INTERPRETATION AND CONCLUSIONS: In both cases the 3' end of MLL was present in multiple copies. Mitotic recombination and non-disjunction may underlie the extra derivatives in both cases. In this genomic imbalance not only the 5'MLL but also the 3'end of MLL could play a critical role in the leukemic process.

Submicroscopic deletions in 5q- associated malignancies.

BACKGROUND AND OBJECTIVES: The deletion of the long arm of chromosome 5 is common in myelodysplastic syndromes (MDS) but is not limited to the 5q- syndrome as it is also seen in acute myeloid leukemia (AML), where it is often associated with other karyotypic aberrations. The aim of this study was to investigate whether deletions of known suppressor sequences occur in myeloid malignancies associated with 5q-. DESIGN AND METHODS: Thirty patients with MDS or AML were selected for the presence of a 5q karyotypic deletion, either isolated (19 cases) or associated with other chromosome changes (11 cases). Multiple fluorescent in situ hybridization (FISH) in interphase nuclei was applied in all cases using a panel of eleven probes for known suppressor genes or loci deleted in MDS/AML. Metaphase FISH was also performed to clarify discrepancies between conventional and molecular cytogenetics. RESULTS: No additional deletions were found in nineteen cases with an isolated 5q-. Mono-allelic deletions where found in 9/11 cases, 3 of which were related to monosomies by conventional cytogenetics. Interphase-FISH showed p53, AML1, D13S25, NF1, or Ikaros in six out of nine (66%) patients with 5q- and additional karyotypic changes. Metaphase FISH was helpful in assigning some of these cryptic events to non-proliferating cells. INTERPRETATION AND CONCLUSIONS: Our study emphasizes that isolated 5q- is the marker of a highly stable clone in both MDS and AML. AML with isolated 5q- are molecularly closer to 5q- syndrome than to AML with complex changes. Interphase-FISH data strongly support a mutator phenotype underlying complex karyotypes with a 5q deletion.