Study of nucleophosmin (NPM) gene mutation in patients with acute myeloid leukemia and myelodysplastic syndromes

Objective To investigate nucleophosmin (NPM) gene mutations in patients with de novo acute myeloid leukemia (AML) with normal cytogenetics and primary myelodysplastic syndromes (MDS). Methods Genomic DNA corresponding to exon 12 of NPM gene was amplified by polymerase chain reaction (PCR) in 40 AML patients (28 case untreated and 12 in first remission) and     

Pericentric chromosome 8 inversion associated with the 5′RUNX1/3′CBFA2T1 gene in acute myeloid leukemia cases

In the present paper we report pericentric chromosome 8 inversions in two (2.4%) of 82 acute myeloid leukemia (AML) cases characterized by the 5RUNX1/3CBFA2T1 fusion gene. Molecular cytogenetic characterization was achieved using appropriate bacterial artificial chromosome (BAC) and P1 artificial chromosome (PAC) probes in fluorescence in situ hybridization (FISH) experiments. In these two cases the fusion gene was detected on the der(8) short arm, resulting from a pericentric chromosome 8 inversion followed by a t(8;21) rearrangement. These results suggest that heterogeneous mechanisms can lead to the generation of the 5RUNX1/3CBFA2T1chimeric gene.     

TET2 gene mutation is unfavorable prognostic factor in cytogenetically normal acute myeloid leukemia patients with NPM1+ and FLT3-ITD− mutations

Cytogenetically normal acute myeloid leukemia (cn-AML) is a group of heterogeneous diseases. Gene mutations are increasingly used to assess the prognosis of cn-AML patients and guide risk-adapted treatment. In the present study, we analyzed the molecular genetics characteristics of 373 adult cn-AML patients and explored the relationship between TET2 gene mutations or different genetic mutation patterns and prognosis. We found that 16.1 % of patients had TET2 mutations, 31.6 % had FLT3 internal tandem duplications (ITDs), 6.2 % had FLT3 tyrosine kinase domain mutations, 2.4 % had c-KIT mutations, 37.8 % had NPM1 mutations, 11.3 % had WT1 mutations, 5.9 % had RUNX1 mutations, 11.5 % had ASXL1 mutations, 3.8 % had MLL-PTDs, 7.8 % had IDH1 mutations, 7.8 % had NRAS mutations, 12.3 % had IDH2 mutations, 1.6 % had EZH2 mutations, and 14.7 % had DNMT3A mutations, while none had CBL mutations. Gene mutations were detected in 76.94 % (287/373) of all patients. In the NPM1m⁺ patients, those with TET2 mutations were associated with a shorter median overall survival (OS) as compared to TET2 wild-type (wt) patients (9.9 vs. 27.0 months, respectively; P = 0.023); Interestingly, the TET2 mutation was identified as an unfavorable prognostic factor and was closely associated with a shorter median OS as compared to TET2-wt (9.5 vs. 32.2 months, respectively; P = 0.013) in the NPM1m⁺/FLT3-ITDm^? patient group. Thus, identification of TET2 combined with classic NPM1 and FLT3-ITD mutations allowed us to stratify cn-AML into distinct subtypes.     

A polymorphism in the 3′-untranslated region of the NPM1 gene causes illegitimate regulation by microRNA-337-5p and correlates with adverse outcome in acute myeloid leukemia

Nucleophosmin, encoded by NPM1, is a haploinsufficient suppressor in hematologic malignancies. NPM1 mutations are mostly found in acute myeloid leukemia patients with normal karyotype and associated with favorable prognosis. A polymorphic nucleotide T deletion with unknown significance is present in the NPM1 3′-untranslated region. Here, we showed that the homozygous nucleotide T deletion was associated with adverse outcomes and could independently predict shortened survival in patients with de novo acute myeloid leukemia. Mechanistically, we demonstrated that the nucleotide T deletion created an illegitimate binding NPM1 for miR-337-5p, which was widely expressed in different acute myeloid leukemia subtypes and inhibited NPM1 expression. Accordingly, NPM1 levels were found to be significantly reduced and correlated with miR-337-5p levels in patients carrying a homozygous nucleotide T-deletion genotype. Together, our findings uncover a microRNA-mediated control of NPM1 expression that contributes to disease heterogeneity and suggest additional prognostic values of NPM1 in acute myeloid leukemia.     

How predictive is the finding of clonal hematopoiesis for the development of myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML)?

Clonal hematopoiesis (CH) – a biological state in which one or a small number of hematopoietic stem or progenitor cells contribute disproportionately to blood cell production, usually as a result of somatic gene mutations in the stem cells – is often considered to be a precursor to myeloid neoplasia, especially myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). However, the majority of people with CH never develop an overt myeloid neoplasm, and CH can be a precursor to lymphoid cancers as well as myeloid neoplasms. In addition, CH increases all-cause mortality and augments the risk of several non-neoplastic medical conditions, including atherosclerotic cardiovascular disease. CH can arise during aging, or in the context of an inherited marrow failure syndrome, aplastic anemia, or hematopoietic cell transplantation. Risk factors for progression of CH to myeloid neoplasia include larger clone size; the presence of a TP53, IDH1/2, or splicing mutation; multiple mutations; and associated cytopenias or abnormal red blood cell indices. The receipt of genotoxic chemotherapy or radiation, which can promote clonal expansion of mutant clones at the expense of healthy progenitor cells, may result in therapy-related MDS/AML.     

Five-day 4′-(9-acridinylamino)methanesulphon-m-anisidide and intermediate-dose cytosine arabinoside in high-risk relapsing or refractory acute myeloid leukemia

Summary Twenty-two patients with acute myeloid leukemia (AML), having a median age of 48.3 years (range 26–70; 10 male, 12 female), were treated with 4-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA) 100 mg/m² and cytosine arabinoside (AraC) 2×1000 mg/m² i.v. on days 1–5. There were 2 M1, 8 M2, 9 M4, 2 M4 Eo, and 1 M5a. Of these, 12 achieved a complete remission, 3 a partial remission and 6 did not respond. The median remission duration was 9.0 months and the median overall survival 8.1 months. Side-effects of induction consisted mainly of haematological toxicity and infections with a median duration of WHO-grade-4 granulopenia and thrombopenia of 20 and 28 days respectively. Organ toxicity was mild with mucositis and cutaneous and liver toxicity being experienced by only a few patients. There was one treatment-related death. Five-day m-AMSA and intermediate-dose AraC is an easy-to-handle condensed treatment schedule with tolerable toxicity. Its effectiveness in relapsed and refractory AML is comparable to combinations of high-dose AraC with m-AMSA, anthracyclines or etoposide.Abbreviations AraC cytosine arabinoside - m-AMSA 4-(9-acridinylamino)methanesulphon-m-anisidide     

A germ-line insertion in the Birt-Hogg-Dubé (BHD) gene gives rise to the Nihon rat model of inherited renal cancer

A rat model of hereditary renal carcinoma (RC) was found in a rat colony of the Sprague-Dawley strain in Japan and named the "Nihon" rat. In heterozygotes, RCs, predominantly the clear cell type, develop from early preneoplastic lesions, which began to appear as early as 3 weeks of age, to adenocarcinomas by the age of 6 months. The Nihon rat is an example of a Mendelian dominantly inherited predisposition for development of RCs like the Eker (Tsc2 gene mutant) rat. We have previously shown that the Nihon mutation was tightly linked to genes that are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2 gene (human 16p13.3)-Il3 gene-Nihon gene-Llgl1 locus- Myhse gene. We now describe a germ-line mutation in the Birt-Hogg-Dubé gene (Bhd) (human 17p11.2) caused by the insertion of a single nucleotide in the Nihon rat, resulting in a frameshift and producing a stop codon 26 aa downstream. We found that the homozygous mutant condition was lethal at an early stage of fetal life in the rat. We detected a high frequency of loss of heterozygosity (LOH) in primary RCs (10/11) at the Bhd locus and found a point mutation (nonsense) in one LOH-negative case, fitting Knudson's "two-hit" model. The Nihon rat may therefore provide insights into a tumor-suppressor gene that is related to renal carcinogenesis and an animal model of human BHD syndrome.     

A phase I–II study of plerixafor in combination with fludarabine,idarubicin, cytarabine,and G-CSF (PLERIFLAG regimen) for the treatment of patients with the first early-relapsed or refractory acute myeloid leukemia

Clinical outcomes of patients with acute myeloid leukemia (AML) showing the first primary refractory or early-relapsed disease remain very poor. The Programa Español de Tratamientos en Hematología (PETHEMA) group designed a phase I–II trial using FLAG-Ida (fludarabine, idarubicin, cytarabine, and G-CSF) plus high-dose intravenous plerixafor, a molecule inducing mobilization of blasts through the SDF-1α-CXCR4 axis blockade and potentially leading to chemosensitization of the leukemic cells. We aimed to establish a recommended phase 2 dose (RP2D) of plerixafor plus FLAG-Ida, as well as the efficacy and safety of this combination for early-relapsed (first complete remission (CR/CRi)?<?12 months) or primary refractory AML. Between 2012 and 2015, 57 patients were enrolled, and 41 received the RP2D (median age 52 years [range, 18–64]). Among these patients, 20 (49%) achieved CR/CRi, and 3 (7%) died during induction. CR/CRi rate was 50% (13/26) among primary refractory and 47% (7/15) among early relapse. Overall, 25 patients (61%) were allografted. Median overall and disease-free survivals were 9.9 and 13 months, respectively. In summary, the combination of plerixafor plus FLAG-Ida resulted in a relatively high CR/CRi rate in adult patients with primary refractory or early relapsed AML, with an acceptable toxicity profile and induction mortality rate, bridging the majority of patients to allogeneic stem cell transplantation. ClinicalTrials.gov Identifier: NCT01435343     

Impact of cytogenetics on the outcome of autotransplantation for acute myeloid leukemia in first remission: is the benefit of intensive pretransplant therapy limited to patients with good karyotypes?

A total of 81 adults with acute myeloid leukemia (AML) (47% favorable karyotypes) were autografted in first remission after melphalan-total body irradiation, having received 0 (n=7), 1 (n=19), 2 (n=51), or 3 (n=4) consolidation chemotherapy cycles before harvest. The cumulative 5-year incidences of relapse and transplant-related mortality were 37 and 17%, respectively. The actuarial 5-year probability of disease-free survival (DFS) was 46%. In Cox analysis, favorable karyotypes, increasing numbers of consolidation cycles (0 vs > or =1 or 1 vs >1), and higher nucleated cell doses were associated with lower relapse rates and higher DFS. Patients with favorable karyotypes benefited from every additional cycle of consolidation therapy (0 vs > or =1 as well as 1 vs >1). Among patients with other karyotypes, while the benefit of one cycle of consolidation was clear (0 vs > or =1), there was no obvious beneficial impact of further consolidation therapy (1 vs >1). Administration of consolidation chemotherapy prior to harvest is essential in AML. While it is possible to enhance the benefit of consolidation with favorable karyotypes by delivering two cycles, its usefulness is limited in others. In them, it may be worthwhile exploring alternatives not normally used in AML (eg high-dose cyclophosphamide) that could have antileukemic effects while permitting mobilization of stem cells.     

Daunorubicin,cytarabine and fludarabine (DAF) for remission induction in relapsed or refractory acute myeloid leukemia. Evaluation of safety,tolerance and early outcome—Polish Adult Leukemia Group (PALG) pilot study

In 1992–1993, synergistic interaction of ribonucleotide reductase inhibitors (fludarabine, cladribine) and cytarabine (Ara-C) increasing Ara-CTP concentration in myeloblasts was proved. Based on these findings and encouraging results of the addition of cladribine to standard daunorubicin+Ara-C induction regimen (DAC) in acute myeloid leukemia (AML), the Polish Adult Leukemia Group (PALG) conducted a pilot study on the administration of cytarabine, daunorubicin, and fludarabine (DAF) as a reinduction treatment of AML to assess tolerance, toxicity, and early outcome. The DAF regimen consisted of daunorubicine 60 mg m⁻² day⁻¹ iv on days 1–3 and fludarabine 25 mg m⁻² day⁻¹ iv on days 1–5 given before cytarabine 200 mg m⁻² day⁻¹ in ci on days 1–7. Thirty-four AML patients with median age 39, 24% relapsed and 76% refractory, were included into the study between September 2003 and August 2004. Achieved response rate in the whole study population was 56%; n = 16 patients with complete remission (CR), and n = 3 patients with partial remission (PR). Fifteen of 16 patients achieved CR after the first course of therapy. Only 9% of total population died before the assessment of remission. All patients developed severe neutropenia. Serious infections were observed in 47% of the cases. Severe thrombocytopenia was observed in 72% of the patients. All patients required substitution of platelet concentrates (median 4), and PRBC (median 5). Severe alopecia, mucositis, vomiting were of low frequency. Liver, kidney, or circulatory failure, diarrhea, or polyneuropathy were not observed. The probability of overall survival (OS) for 1 year for the whole study population (34 patients) and the group of 16 patients in CR was: 44% (95% confidence interval [CI] 36–52%) and 69% (95% CI 55–83%), respectively. The probability of leukemia-free survival (LFS) for 1 year was 38% (95% CI 22–54%). Summarizing, DAF regimen used as the induction therapy in relapsed/refractory AML was well tolerated with acceptable toxicity and early efficacy.     

Is FLT3 internal tandem duplication significant indicator for allogeneic transplantation in acute myeloid leukemia? An analysis of patients from the Czech Acute Leukemia Clinical Register (ALERT)

To assess the prognostic relevance of activating mutations of FLT3 gene on outcome of allogeneic transplantations in AML patients, we performed an analysis of all patients with FLT3 mutations registered in the Czech Acute Leukemia Clinical Register (ALERT) from 2003 till the end of 2005. Within the mentioned period 170 patients with AML of median age 56 years (23-77) were investigated for FLT3 mutation, within them 36 cases (21%) with FLT3 mutations (32 FLT3 ITD and 4 FLT3 D835) were found.Out of FLT3 ITD positive patients 13 had allogeneic transplantation, 20 patients with mutations of FLT3 were treated with chemotherapy without transplantation. Results of the treatment of these patients were compared with the results of the group of patients without FLT3 mutation, which was according to other characteristics identical with the group of patients with FLT3 mutations (n=134). Median overall survival (OS) was significantly shorter for patients with FLT3 ITD (34.8 weeks) than for those without FLT3 mutations (67.7 weeks; P=0.028). Median OS of patients with FLT3 ITD who had allogeneic transplantation was 42.5 weeks; median OS of patients with FLT3 ITD treated only with chemotherapy was 29.6 weeks (P=0.362). After allogeneic transplantation, median OS of FLT3 mutations negative patients was similar to FLT3 ITD positive patients (46.7 versus 42.5 weeks; P=0.443). Our results suggest that at present there is no strong evidence that FLT3 status alone should influence the decision to proceed to allogeneic transplantation in AML patients. Decision to proceed to alogeneic transplantation should not be based on the FLT3 status only, but it should also consider other prognostic factors.     

Collagenase-3 (matrix metalloprotease 13) is preferentially localized in the deep layer of human arthritic cartilage in situ. In vitro mimicking effect by transforming growth factor β

Objective. To examine, by immunohistochemistry, the localization and distribution of human collagenase-3 in normal, osteoarthritis (OA), and rheumatoid arthritis (RA) cartilage, and to investigate the effects of interleukin-1β (IL-1β) and transforming growth factor β (TGFβ) on the synthesis and distribution of collagenase-3. Methods. Human cartilage specimens were obtained from tibial plateaus. In the first series of experiments, the OA specimens were excised from fibrillated and nonfibrillated areas of cartilage, and RA specimens were excised from lesional areas, including the cartilage-pannus junction when present. In the second series, full strips of cartilage were processed for culture in the presence or absence of IL-1β (100 units/ml) or TGFβ (150 ng/ml). Each specimen was processed for immunohistochemical analysis using a collagenase-3 monoclonal antibody. Results. The number of cells that stained for collagenase-3 in normal cartilage was very low (±3%). In OA cartilage, the percentage increased dramatically, and no difference was found between fibrillated and nonfibrillated areas. A statistically significant increase in the percentage of cells staining for collagenase-3 was found in the deep layer compared with the superficial layer. This finding was noted in both the fibrillated areas (mean ± SEM 58.4 ± 1.6% and 40.1 ± 3.9%, respectively; P < 0.007) and the nonfibrillated areas (55.4 ± 3.2% and 43.2 ± 2.7%; P < 0.01). Similarly, RA cartilage showed a statistically significant (P < 0.001) increase in the level of chondrocytes staining positive for collagenase-3 in the deep layers (46.4 ± 4.1%) compared with the superficial layers (26.2 ± 3.4%). In these RA specimens, the numbers of positively staining chondrocytes were similar both close to and at a distance from the pannus junction. Both IL-1β and TGFβ increased the number of chondrocytes producing collagenase-3. Interestingly, in normal specimens, TGFβ had a predominant effect in the deep layers, while IL-1β had a greater effect on the superficial layers. Conclusion. This study demonstrates that, in situ, the increase in the level of chondrocytes synthesizing collagenase-3 in arthritic cartilage is predominant in the deep layers. The results further indicate that TGFβ can up-regulate the level of this enzyme and, in normal cartilage in vitro, can cause a mimicking of the in situ distribution observed in arthritic cartilage.