The KMT2A recombinome of acute leukemias in 2023.

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5'-KMT2A, two patients had a 5'-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.

Complex MLL rearrangements in t(4;11) leukemia patients with absent AF4.MLL fusion allele.

The human mixed lineage leukemia (MLL) gene is frequently involved in genetic rearrangements with more than 55 different translocation partner genes, all associated with acute leukemia. Reciprocal chromosomal translocations generate two MLL fusion alleles, where 5'- and 3'-portions of MLL are fused to gene segments of given fusion partners. In case of t(4;11) patients, about 80% of all patients exhibit both reciprocal fusion alleles, MLL.AF4 and AF4.MLL, respectively. By contrast, 20% of all t(4;11) patients seem to encode only the MLL.AF4 fusion allele. Here, we analyzed these 'MLL.AF4(+)/AF4.MLL(-)' patients at the genomic DNA level to unravel their genetic situation. Cryptic translocations and three-way translocations were found in this group of t(4;11) patients. Reciprocal MLL fusions with novel translocation partner genes, for example NF-KB1 and RABGAP1L, were identified and actively transcribed in leukemic cells. In other patients, the reciprocal 3'-MLL gene segment was fused out-of-frame to PBX1, ELF2, DSCAML1 and FXYD6. The latter rearrangements caused haploinsufficiency of genes that are normally expressed in hematopoietic cells. Finally, patients were identified that encode only solitary 3'-MLL gene segments on the reciprocal allele. Based on these data, we propose that all t(4;11) patients exhibit reciprocal MLL alleles, but due to the individual recombination events, provide different pathological disease mechanisms.

[Saccharomyces cerevisiae fungemia associated with multifocal pneumonia in a patient with alcohol-related hepatic cirrhosis]. / Fungemia da Saccharomyces cerevisiae con polmonite a focolai multipli in paziente con cirrosi epatica alcool correlata.

Saccharomyces cerevisiae is usually considered non-pathogenic and has rarely been reported as a cause of fungemia in immunocompromised patients, especially those admitted to an intensive care unit or those affected by acquired immune deficiency syndrome or under immunosuppressive treatment. In all described cases the use of probiotic yeast has been given as the main risk factor. We report a case of S. cerevisiae sepsis complicated by pneumonia in a patient affected by alcohol-related cirrhosis with no evidence of probiotic drug intake. In this case recovery was obtained after a treatment course with liposomal amphotericin B. S. cerevisiae should be taken into consideration when sepsis lacks to isolate any aetiological agent.

The MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.

Genetic polymorphism of NAD(P)H:quinone oxidoreductase is associated with an increased risk of infant acute lymphoblastic leukemia without MLL gene rearrangements.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a detoxification enzyme that protects cells against oxidative stress and toxic quinones. A polymorphism (C609T) in the gene produces in the heterozygous individuals (C/T) a reduction and in those homozygous for the variant allele (T/T) the abolishment of NQO1 protein activity. To assess whether NQO1 inactivating polymorphism (CT/TT) was a possible risk factor for infant acute lymphoblastic leukemia (iALL), we investigated the distribution of NQO1 genotype in 50 iALL patients, 32 with MLL gene rearrangements (MLL+) and 18 without (MLL-). As controls, 106 cases of pediatric ALL (pALL), and 147 healthy subjects were also studied. Compared to normal controls, the frequency of the low/null activity NQO1 genotypes was significantly higher in the iALL MLL- (72 vs 38%, P=0.006; odds ratio (OR) 4.22, 95% confidence interval (CI) 1.43-12.49), while no differences were observed in iALL MLL+ (44 vs 38%, P=0.553; OR 1.26, 95% CI 0.58-2.74). Similar results were observed when pALL were used as control. Our results indicate that only the iALL patients without MLL rearrangements had a significantly higher frequency of NQO1 genotypes associated with low/null activity enzyme, suggesting a possible role for NQO1 gene as an MLL-independent risk factor, in the leukemogenic process of this subtype of iALL.

Levels of L-asparagine in CSF after intramuscular administration of asparaginase from Erwinia in children with acute lymphoblastic leukemia.

PURPOSE: As part of a study on the pharmacokinetics associated with the administration of asparaginase (ASNase) from Erwinia to the CNS, we determined the levels of asparagine in the CSF of children with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Twenty children received eight standard doses of intramuscular ASNase (10,000 IU/m2) every 3 days as part of induction therapy. In the postremission phase of therapy, the children were randomized to receive either 20 courses of high-dose intramuscular ASNase (25,000 IU/m2) weekly (n = 8) or four courses of standard-dose intramuscular ASNase (10,000 IU/m2) every 3 days (n = 12). RESULTS: All patients had detectable levels of L-asparagine in the CSF at the time of diagnosis. The levels of L-asparagine in CSF were undetectable in 15 of 20 (75%) and in seven of 19 (36.8%) children 3 and 5 days, respectively, after administration of standard-dose ASNase. After administration of high-dose ASNase, the levels of L-asparagine in the CSF were undetectable in five (62.5%) and two (25%) of eight children after 3 and 5 days, respectively. CONCLUSION: In this study 60% to 70% and 25% to 35% of children had complete depletion of L-asparagine from the CSF after 3 and 5 days, respectively, after administration of both schedules of ASNase from Erwinia. In the remaining patients, administration of ASNase may have resulted in a suboptimal antileukemic effect at the CNS level.

Effect of protracted high-dose L-asparaginase given as a second exposure in a Berlin-Frankfurt-Münster-based treatment: results of the randomized 9102 intermediate-risk childhood acute lymphoblastic leukemia study--a report from the Associazione Italiana Ematologia Oncologia Pediatrica.

PURPOSE: To assess in a randomized study the therapeutic effect of the addition of high-dose L-asparaginase (HD ASP) in the context of a Berlin-Frankfurt-Münster (BFM)-based chemotherapy regimen for intermediate risk (IR) childhood acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: From March 1991 to April 1995, a total of 705 patients, with 59% of the cohort of patients fewer than 15 years old, with newly diagnosed non-B ALL, enrolled onto the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) ALL-91 study, were assigned to the IR group. Patients in remission at the beginning of the reinduction phase were randomized either to the standard treatment (SD ASP arm) or the experimental treatment (HD ASP arm; weekly intramuscular administration of HD ASP 25,000 IU/m(2) repeated for a total of 20 weeks). Most of the patients (90%) were treated with Erwinia chrysanthemi L-asparaginase product. RESULTS: Among the 610 patients randomized to the SD ASP arm (n = 322) or to the HD ASP arm (n = 288), relapse occurred at a median time of 24 months after randomization in 76 (24%) and in 64 children (22%), respectively. Most of the relapses occurred in the marrow (100 isolated, 21 combined). There was no significant difference between the disease-free survival in the two treatment arms (P =.64), with estimated values at 7 years from randomization of 72.4% (SE 3.1) v 75.7% (SE 2.6) in the SD ASP and HD ASP arms, respectively. CONCLUSION: No advantage was observed for IR ALL children treated with BFM-based intensive chemotherapy who received protracted E chrysanthemi HD ASP during reinduction and the early continuation phase.

Is there selection on RFLP differences in mitochondrial DNA?

Experimental populations of Drosophila simulans were established for the purpose of detecting the presence or absence of selection on a restriction fragment length polymorphism in mitochondrial DNA (mtDNA). It was then discovered that the founding strains differed with respect to the Rickettsia-mediated incompatibility system in this species, which is maternally transmitted together with the mtDNA differences. A population model was constructed using the known fitness effects of the incompatibility system, with the result that the population trajectories can be completely explained by the effects of the microorganism with no need to invoke selection on mtDNA. The strong conclusion is that in this case we can rule out the strong selection proposed by MacRae and Anderson to explain the "dramatic mtDNA changes" in their Drosophila pseudoobscura populations. The population theory used for the experiments is discussed in the context of natural populations. Estimated parameters include the possibility that with two populations, one with the organism and one without it, there may be no bias as to which will invade the other, which in turn suggests no global tendency for the infection to spread or decline.

Large number of replacement polymorphisms in rapidly evolving genes of Drosophila. Implications for genome-wide surveys of DNA polymorphism.

We present a survey of nucleotide polymorphism of three novel, rapidly evolving genes in populations of Drosophila melanogaster and D. simulans. Levels of silent polymorphism are comparable to other loci, but the number of replacement polymorphisms is higher than that in most other genes surveyed in D. melanogaster and D. simulans. Tests of neutrality fail to reject neutral evolution with one exception. This concerns a gene located in a region of high recombination rate in D. simulans and in a region of low recombination rate in D. melanogaster, due to an inversion. In the latter case it shows a very low number of polymorphisms, presumably due to selective sweeps in the region. Patterns of nucleotide polymorphism suggest that most substitutions are neutral or nearly neutral and that weak (positive and purifying) selection plays a significant role in the evolution of these genes. At all three loci, purifying selection of slightly deleterious replacement mutations appears to be more efficient in D. simulans than in D. melanogaster, presumably due to different effective population sizes. Our analysis suggests that current knowledge about genome-wide patterns of nucleotide polymorphism is far from complete with respect to the types and range of nucleotide substitutions and that further analysis of differences between local populations will be required to understand the forces more completely. We note that rapidly diverging and nearly neutrally evolving genes cannot be expected only in the genome of Drosophila, but are likely to occur in large numbers also in other organisms and that their function and evolution are little understood so far.

Rates of DNA evolution in Drosophila depend on function and developmental stage of expression.

DNA-sequence divergence of genes expressed in the embryonic stage was compared with the divergence of genes expressed in adults for 13 species of Drosophila representing various degrees of relatedness. DNA-DNA hybridization experiments were conducted using as tracers complementary DNA (cDNA) reversed transcribed from poly(A)+ mRNA isolated from different developmental stages. The results indicate: (1) cDNA is less diverged than total single-copy DNA; (2) cDNA sequences are not in the rapidly evolving fraction of the single-copy genome of Drosophila; (3) early in evolutionary divergence embryonic messages are about half as diverged as adult messages; sequence data from some of the species compared indicate this is likely due to differences in rates of silent substitutions in genes expressed at different stages of development; and (4) at greater evolutionary distance, the differences in embryonic and adult messages disappear; this could be due to lineage-specific shifts in codon usage.

Clonal stability in children with acute lymphoblastic leukemia (ALL) who relapsed five or more years after diagnosis.

Although most relapses of childhood acute lymphoblastic leukemia (ALL) occur 24-36 months after first CR has been achieved, few patients relapse 5 or more years after CR achievement. The assessment of clonality has proved to be useful in determining whether even those very late events represent the reoccurrence of the original clone or alternatively a secondary leukemia. To gain further information on clonal stability in such late relapse, we performed detailed comparative Southern blotting and PCR analyses of TcRdelta and TcRgamma gene rearrangements in five ALL at presentation and subsequent relapse which occurred more than 5 years after diagnosis. At least one stable rearranged allele of the TcRdelta and TcRgamma loci was traced in all cases at presentation and clinical relapse despite a wide heterogeneity of the pattern of rearrangements. Our study extends to a larger series of patients previous findings which have sought to analyze the phenomenon of clonal evolution in children relapsed after more than 5 years of CCR. With respect to the potential pitfalls in monitoring minimal residual disease in childhood ALL for the presence of clonal evolution, our results highlight the combination of two target genes (such as TcRgamma and TcRdelta) as a tool to reduce false negative MRD results.

Clonal variegation and dynamic competition of leukemia-initiating cells in infant acute lymphoblastic leukemia with MLL rearrangement.

Distinct from other forms of acute lymphoblastic leukemia (ALL), infant ALL with mixed lineage leukemia (MLL) gene rearrangement, the most common leukemia occurring within the first year of life, might arise without the need for cooperating genetic lesions. Through Ig/TCR rearrangement analysis of MLL-AF4+ infant ALL at diagnosis and xenograft leukemias from mice transplanted with the same diagnostic samples, we established that MLL-AF4+ infant ALL is composed of a branching subclonal architecture already at diagnosis, frequently driven by an Ig/TCR-rearranged founder clone. Some MLL-AF4+ clones appear to be largely quiescent at diagnosis but can reactivate and dominate when serially transplanted into immunodeficient mice, whereas other dominant clones at diagnosis can become more quiescent, suggesting a dynamic competition between actively proliferating and quiescent subclones. Investigation of paired diagnostic and relapse samples suggested that relapses often occur from subclones already present but more quiescent at diagnosis. Copy-number alterations identified at relapse might contribute to the activation and expansion of previously quiescent subclones. Finally, each of the identified subclones is able to contribute to the diverse phenotypic pool of MLL-AF4+ leukemia-propagating cells. Unraveling of the subclonal architecture and dynamics in MLL+ infant ALL may provide possible explanations for the therapy resistance and frequent relapses observed in this group of poor prognosis ALL.

Mitochondrial DNA sequences in prehistoric human remains from the Alps.

The spread of agriculture that started in the Near East about 10 000 years ago caused a dramatic change in the European archaeological record. It is still unclear if that change was caused mostly by movement of people or by cultural transformations. In particular, there is disagreement on what proportion of the current European gene pool is derived either from the pre-agricultural, paleolithic and mesolithic people, or from neolithic farmers immigrating from the south-east. To begin to characterise the mtDNA gene pool of prehistoric Europe we examined five human remains from the Eastern Italian Alps, dated between 14 000 and 3000 years ago. Three of them yielded sufficient amount of mtDNA for analysis. DNA extracts were prepared in two independent laboratories, and PCR products from the first hypervariable segment of the mtDNA control region were cloned and sequenced. Together with the 5200 year old 'ice man', these DNA sequences show that European mtDNA diversity was already high at the beginning of the neolithic period. All the neolithic sequences have been observed in contemporary Europeans, suggesting genealogical continuity between the neolithic and present-day European mtDNA gene pool. The mtDNA sequence from a 14 000 year-old specimen was not observed in any contemporary Europeans, raising the possibility of a lack of continuity between the mesolithic and present-day European gene pools.

Mitochondrial lineages in Ladin-speaking communities of the eastern Alps.

European mitochondrial alleles cluster into five haplogroups. Haplogroup 2 is rare in general, but represents more than half of the few known sequences among Ladin speakers of the Alps. Here we describe DNA diversity in control region I of the hypervariable D-loop in 43 Ladins, and in 25 Italian speakers. Analysis of these data, and of previously published sequences, confirms a high degree of differentiation among Ladins and their geographical neighbours. This cannot be regarded as a simple effect of isolating factors, geographic or linguistic, as diversity is high within Ladin communities too. Rather, allele genealogies, population trees, and principal component analysis suggest a relationship between Ladin and Near Eastern samples. Two evolutionary hypotheses seem compatible with these findings. The view whereby Ladins could be descended from Palaeolithic inhabitants of the Alps is supported by the identification, in this study, of the probable ancestral haplotype of group 2, never previously observed in central Europe. Alternatively, a comparatively recent, Neolithic immigration of the ancestors of current Ladin speakers seems consistent with recent linguistic theories. In both cases, the number of lineages present, and their extensive diversity, are not compatible with a serious bottleneck in the Ladin population's history.

HCV infection in HBsAg positive chronic liver disease.

The prevalence of anti-HCV antibodies was determined for a group of 68 patients with various forms of chronic liver disease. All patients that were anti-HCV positive but did not show signs of HBV replication had severe liver disease. We therefore suggest that HCV may be responsible for liver damage in HBsAg positive subjects when there are no evident signs of HBV replication.

Preliminary investigation on intrafamilial spread of hepatitis C virus (HCV).

To determine the risk of cohabitant HCV infection, we investigated the sera of 101 family members of 53 anti-HCV antibody positive chronic liver disease patients. Altogether 14.8% of the cohabitants were also anti-HCV antibody positive, compared to a prevalence of 1.4% in the general population. These results suggest that hepatitis-C-virus may spread by person-to-person infection.

In vitro production of type 1 and type 2 cytokines by peripheral blood mononuclear cells from subjects coinfected with human immunodeficiency virus and Leishmania infantum.

To explore the type 1 and type 2 cytokine profile in cases coinfected with human immunodeficiency virus (HIV) and Leishmania infantum, production of interleukin-4 (IL-4), interleukin-10 (IL-10), interferon-gamma (IFN-gamma), and interleukin-2 receptor (IL-2R) was investigated in mitogen-stimulated and unstimulated peripheral blood mononuclear cell cultures from eight HIV/Leishmania coinfected subjects matched with eight anti-HIV-positive subjects with no evidence of Leishmania coinfection. Levels of IL-4 and IL-2R increased significantly from the baseline levels in the peripheral blood mononuclear cell supernatants of HIV/Leishmania coinfected subjects following stimulation with phytohemoagglutin, whereas the postchallenge concentration of IFN-gamma was significantly increased in the HIV-infected group. The levels of IL-4 and IL-10 were significantly higher in the HIV/Leishmania group throughout evaluation. Post-stimulation IFN-gamma production was significantly higher in the HIV-positive group in comparison with that of the HIV-Leishmania coinfected subjects. These observations support the notion that a Th2 cytokine response is present during a Leishmania infection, even among HIV-coinfected individuals.

Detection of minimal residual disease: methods and relationship to outcome in T-lineage acute lymphoblastic leukemia.

The molecular basis of acute lymphoblastic leukemia (ALL) of both B-cell and T-cell lineages seems better understood using polymerase chain reaction (PCR) methods. The analysis of clone-specific junctional regions of rearranged genes for both Immunoglobulin (Ig H) and T-cell receptor (TcR) is the most sensitive tool for detection of minimal residual disease (MRD) in ALL. Because of the heterogeneity of all ALL patients examined in several studies, the detection of MRD at different times of treatment has not as yet been correlated with disease outcome. In contrast, T-ALL is a homogeneous disease characterized by expansion of a single clone showing a specific Rearranged junctional region of TcR delta and/or gamma genes. The use of a clone-specific probe allows detection of residual leukemia throughout treatment. However, 60 % of patients with T-ALL relapse during treatment or towards the end of therapy, with resurgence of the original leukemic clone. It is possible that the detection of MRD at a specific time-point after diagnosis, as well as at the beginning of maintenance, may help to identify a group of T-ALL patients at high risk of relapse. The correlation between detection of MRD and treatment phase may be used in the future to evaluate whether treatment regimens can be improved allowing for stratification, based on PCR-mediated detection of MRD.

Methadone maintenance and hepatitis C virus infection among injecting drug users.

Harm reduction strategies for the prevention of transmission of human immunodeficiency virus (HIV) transmission among injecting drug users (IDUs) have been widely implemented in Australia and are seen to have been effective in preventing the spread of HIV. A major strategy has been increasing the availability of and accessibility to methadone maintenance therapy (MMT) programmes. We have reviewed the experience of a major MMT general practice with hepatitis C virus (HCV) infection from 1991 to 1995. Of 1741 individuals tested for HCV antibodies at least once 66.7% were positive. Of 73 IDUs who were initially seronegative and were retested at least once, 19 were subsequently seropositive. Seroconverters to HCV were younger than non-seroconverters, and were more likely to have evidence of previous hepatitis B infection. The overall HCV incidence rate was 22 cases per 100 person-years, and this did not differ between those on MMT programs (continuous or interrupted) between HCV tests and those not on MMT. These findings suggest that the role of MMT in the control of the spread of HCV infection among IDUs needs further assessment, and that control of the current epidemic of HCV infection among IDUs in Australia will be very difficult.

Visceral leishmaniasis in HIV infected patients: treatment with high dose liposomal amphotericin B (AmBisome).

Visceral leishmaniasis (VL) in patients coinfected with human immunodeficiency virus (HIV) is often atypical, and characteristically relapses after treatment. We treated 10 HIV infected patients (9 men) with parasitologically confirmed VL with liposomal amphotericin B ("AmBisome': L-AMB) at a dose of 4 mg/kg/day on days 1, 2, 3, 4, 5, 10, 17, 24, 31, and 38. Patients were hospitalized for the first 5 days, and were monitored during, and 1 week and 1, 3 and 6 months after, L-AMB therapy. There were no serious adverse events, and L-AMB was well tolerated. 9/10 patients completed therapy, one patient defaulted at day 24. Clinical improvement was seen in all nine patients and the bone marrow aspirate was cleared of visible/culturable parasites in 8/9 patients. During follow-up, one patient defaulted. The seven remaining patients relapsed at 2, 3, 3, 5, 5, 6 and 7 months. Re-treatment with a variety of antileishmanial drugs was unsatisfactory. The time from first diagnosis of VL to death in six patients was 5-40 months (mean 18.8 months). Only one patient remained alive 26 months after treatment. L-AMB is safe and provides a good initial clinical response. Intermittent dosing enables a short period of hospitalization. However, relapse is probably inevitable.