Statistical Parametric Mapping to detect the effects of the secondary jump direction on landing kinematics.

Anterior cruciate ligament (ACL) injuries in sports often occur with non-contact mechanisms, such as landing and cutting. Previous studies explored the ACL injury biomechanical risk factors through drop-jumps combined with secondary jumps. This study aimed to investigate the effect of the secondary jump direction on first landing kinematic temporal series. Fifty-seven participants (29 males, 28 females) performed three single-leg drop-jumps followed by secondary jumps in vertical (single-planar), 45°-medial and 45°-lateral direction (multi-planar). Lower limb and trunk landing kinematics was recorded using a 9-camera motion capture system and analysed with a One-way ANOVA through Statistical Parametric Mapping (SPM), from initial contact to maximum knee flexion. All variables were affected by the secondary jump direction, except trunk rotation. In sagittal plane, kinematic main differences were found between single- and multi-planar tasks. The latter elicited higher trunk, hip, and knee flexion. Frontal plane kinematics was more influenced by medio-lateral components of secondary jumps. Our results could underline how a single task may be insufficient for ACL injury risk assessment. Single- and multi-planar tasks including a secondary jump should be considered for more comprehensive evaluations in prevention and rehabilitation programs, but caution should be used when comparing results of studies adopting different tasks.

Soccer and volleyball players do not land differently: implications for anterior cruciate ligament injury risk.

BACKGROUND: Non-contact anterior cruciate ligament injuries are common in soccer and volleyball, occurring during changes of direction and landings. This study aimed to investigate kinematic differences between soccer and volleyball players in single-planar and multiplanar landing tasks, simulating sport-specific injury mechanisms. Since the anterior cruciate ligament injury rate in soccer is higher than in volleyball, we hypothesized that volleyball players would adopt safer landing strategies, especially in single-planar landing tasks. METHODS: Twenty-two soccer and 19 volleyball players performed single-leg drop landing, drop jump in vertical, 45°-medial and 45°-lateral directions. Box height and jump length were adapted to the subject's height and performance level, respectively. A 9-camera motion capture system provided lower limb kinematics. Two mixed multivariate analyses of covariance (sport, task, sex as covariate) were used to compare soccer and volleyball players' initial contact and peak kinematics (α=0.05). RESULTS: Task had significant effects on lower limb initial contact and peak angles, as expected. Sport and task × sport interaction had no significant effects on kinematics. CONCLUSIONS: Soccer and volleyball players' landing strategies were thus similar in each task, in opposition to initial hypotheses. We might speculate that the higher anterior cruciate ligament injury rate in soccer may be more related to non-predictable factors than the isolated landing kinematics.

Customized Landing Task for ACL Injury Risk Assessment: Kinematic Sex-Related Differences.

BACKGROUND: Women present a higher anterior cruciate ligament (ACL) injury rate than men, suggesting sex-related biomechanical differences. Task characteristics are often fixed for both sexes, possibly affecting the perceived difficulty. We investigated kinematic sex differences across landing tasks for ACL injury risk assessment, adjusted to participants' anthropometrics/performance, and whether different tasks affect kinematic sex comparisons. HYPOTHESIS: Female subjects would exhibit motion patterns more associated with ACL injury risk, and sex-related differences may depend on task type. STUDY DESIGN: Descriptive laboratory study. LEVEL OF EVIDENCE: Level 3. METHODS: A total of 27 female and 29 male amateur players (18-30 years) executed horizontal hop, drop jump (DJ), and DJ followed by vertical or forward jump (length, proportional to maximal forward jump; box, 20% participant's height). An optoelectronic system provided lower limb kinematics at initial contact and peaks until maximum knee flexion (KF), analyzed separately by multivariate analysis of variance (MANOVA) (α = 0.05). RESULTS: At initial contact, the interaction term had significant effects on hip adduction (P < 0.01) and knee abduction (KAb) (P = 0.04); female participants demonstrated higher KAb (P < 0.01) and knee internal rotation (P = 0.05). For peaks analysis, the interaction term had no significant effects on any individual variable, although significant in MANOVA; female participants had higher KAb (P = 0.01) and lower KF (P = 0.04). Task type affected hip flexion and knee angles in both analyses. CONCLUSION: All variables in which significant sex-related differences were found are potential ACL injury risk factors, and all findings indicate that the analyzed female sample exhibited higher injury-related patterns. Although customized, male and female participants showed different landing strategies depending on the task. CLINICAL RELEVANCE: The findings underline how female participants adopted potentially harmful kinematics while executing customized landing tasks (adjusted by subject's anthropometrics/performance), which may enhance risk of ACL injury.

Head-centric computing for vestibular stimulation under head-free conditions.

Background: The vestibular end organs (semicircular canals, saccule and utricle) monitor head orientation and motion. Vestibular stimulation by means of controlled translations, rotations or tilts of the head represents a routine manoeuvre to test the vestibular apparatus in a laboratory or clinical setting. In diagnostics, it is used to assess oculomotor postural or perceptual responses, whose abnormalities can reveal subclinical vestibular dysfunctions due to pathology, aging or drugs. Objective: The assessment of the vestibular function requires the alignment of the motion stimuli as close as possible with reference axes of the head, for instance the cardinal axes naso-occipital, interaural, cranio-caudal. This is often achieved by using a head restraint, such as a helmet or strap holding the head tightly in a predefined posture that guarantees the alignment described above. However, such restraints may be quite uncomfortable, especially for elderly or claustrophobic patients. Moreover, it might be desirable to test the vestibular function under the more natural conditions in which the head is free to move, as when subjects are tracking a visual target or they are standing erect on the moving platform. Here, we document algorithms that allow delivering motion stimuli aligned with head-fixed axes under head-free conditions. Methods: We implemented and validated these algorithms using a MOOG-6DOF motion platform in two different conditions. 1) The participant kept the head in a resting, fully unrestrained posture, while inter-aural, naso-occipital or cranio-caudal translations were applied. 2) The participant moved the head continuously while a naso-occipital translation was applied. Head and platform motion were monitored in real-time using Vicon. Results: The results for both conditions showed excellent agreement between the theoretical spatio-temporal profile of the motion stimuli and the corresponding profile of actual motion as measured in real-time. Conclusion: We propose our approach as a safe, non-intrusive method to test the vestibular system under the natural head-free conditions required by the experiential perspective of the patients.

The Effects of FIFA 11+ Kids Prevention Program on Kinematic Risk Factors for ACL Injury in Preadolescent Female Soccer Players: A Randomized Controlled Trial.

This study aimed to investigate the effects of the 8-week FIFA 11+ Kids program on kinematic risk factors for ACL injury in preadolescent female soccer players during single-leg drop landing. For this, 36 preadolescent female soccer players (10-12 years old) were randomly allocated to the FIFA 11+ Kids program and control groups (18 players per group). The intervention group performed the FIFA 11+ Kids warm-up program twice per week for 8 weeks, while the control group continued with regular warm-up. Trunk, hip, and knee peak angles (from initial ground contact to peak knee flexion) were collected during the single-leg drop landing using a 3D motion capture system. A repeated measure ANOVA was used to analyze groups over time. Significant group × time interactions were found for the peak knee flexion, with a medium effect size (p = 0.05; effect size = 0.11), and peak hip internal rotation angles, with a large effect size (p < 0.01; effect size = 0.28). We found that the FIFA 11+ Kids program was effective in improving knee flexion and hip internal rotation, likely resulting in reducing ACL stress during single-leg drop landing in young soccer players.

Animal conflicts escalate in a warmer world.

The potential for climate change to affect animal behaviour is widely recognized, yet its possible consequences on aggressiveness are still unclear. If warming and drought limit the availability of food resources, climate change may elicit an increase of intraspecific conflicts stemming from resource competition. By measuring aggressivity indices in a group-living, herbivorous mammal (the Apennine chamois Rupicapra pyrenaica ornata) in two sites differing in habitat quality, and coupling them with estimates of plant productivity, we investigated whether harsh climatic conditions accumulated during the growing season influenced agonistic contests at feeding via vegetation-mediated effects, and their interaction with the site-specific habitat quality. We focused on females, which exhibit intra-group contest competition to access nutritious food patches. Accounting for confounding variables, we found that (1) the aggression rate between foraging individuals increased with the warming accumulated over previous weeks; (2) the probability to deliver more aggressive behaviour patterns toward contestants increased with decreasing rainfall recorded in previous weeks; (3) the effects of cumulative warming and drought on aggressivity indices occurred at time windows spanning 15-30 days, matching those found on vegetation productivity; (4) the effects of unfavourable climatic conditions via vegetation growth on aggressivity were independent of the site-specific habitat quality. Simulations conducted on our model species predict a ~50 % increase in aggression rate following the warming projected over the next 60 years. Where primary productivity will be impacted by warming and drought, our findings suggest that the anticipated climate change scenarios may trigger bottom-up consequences on intraspecific animal conflicts. This study opens the doors for a better understanding of the multifactorial origin of aggression in group-living foragers, emphasising how the escalation of agonistic contests could emerge as a novel response of animal societies to ongoing global warming.

The influence of task type and movement speed on lower limb kinematics during single-leg tasks.

BACKGROUND: Single-leg squats and step-downs are commonly used to assess kinematic variables that may be linked to injuries. Task type and movement speed may influence the outcomes of interest because of different balance requirements. RESEARCH QUESTION: What is the influence of task type and movement speed on lower limb kinematics? METHODS: This is a cross-sectional within-subjects study where 22 physically active females performed three single-leg functional tasks (Squat, Anterior step-down, and Lateral step-down) at three movement speeds (slow [5 s], fast [2 s], and self-selected), while three-dimensional kinematic variables were recorded. Displacement values from the initial position in single-leg support until 60° or peak knee flexion were calculated. Two-way repeated measures ANOVA was used to compare tasks and speeds, and Cohen's d effect size (ES) was calculated for significant pairwise comparisons. RESULTS: At 60°, lateral step-down presented the greatest hip adduction (large ES) and internal rotation (small ES). The anterior step-down had the lowest knee abduction displacement while the squat had the greatest (small to medium ES). At peak knee flexion, values increased but differences between tasks followed a similar pattern. Slow speed induced smaller displacement angles at the knee and hip (trivial to small ES). SIGNIFICANCE: When knee abduction is the variable of interest, the SLS may be the best test since it elicits the greatest displacement, but when evaluating hip motion, SDLAT might be best. Knee abduction and internal rotation were lowest in the slow condition, suggesting that faster speed may be more appropriate to detect abnormal movement patterns. However, the small difference in absolute values (i.e., degrees of movement) may indicate that the differences are not clinically significant, particularly for speed comparisons. Researchers and clinicians should take this into consideration when choosing the most appropriate task and the instruction to give during its execution.

The mitogenome of the true bug Nysius cymoides (Insecta, Heteroptera) and the phylogeny of Lygaeoidea.

The complete mitochondrial genome of the true bug (Homoptera) Nysius cymoides (Spinola, 1837) is herein described and used for phylogenetic comparison with other species of Lygaeoidea. The mtDNA has a gene order and other molecular features typically observed in hexapods, and a long A + T-rich region, due to the occurrence of several repeat units. The phylogenetic analyses support the monophyly of all families except Rhyparochromidae.

Fatigue Induced by Repeated Changes of Direction in Élite Female Football (Soccer) Players: Impact on Lower Limb Biomechanics and Implications for ACL Injury Prevention.

BACKGROUND: The etiology of Anterior Cruciate Ligament (ACL) injury in women football results from the interaction of several extrinsic and intrinsic risk factors. Extrinsic factors change dynamically, also due to fatigue. However, existing biomechanical findings concerning the impact of fatigue on the risk of ACL injuries remains inconsistent. We hypothesized that fatigue induced by acute workload in short and intense game periods, might in either of two ways: by pushing lower limbs mechanics toward a pattern close to injury mechanism, or alternatively by inducing opposed protective compensatory adjustments. AIM: In this study, we aimed at assessing the extent to which fatigue impact on joints kinematics and kinetics while performing repeated changes of direction (CoDs) in the light of the ACL risk factors. METHODS: This was an observational, cross-sectional associative study. Twenty female players (age: 20-31 years, 1st-2nd Italian division) performed a continuous shuttle run test (5-m) involving repeated 180°-CoDs until exhaustion. During the whole test, 3D kinematics and ground reaction forces were used to compute lower limb joints angles and internal moments. Measures of exercise internal load were: peak post-exercise blood lactate concentration, heart rate (HR) and perceived exertion. Continuous linear correlations between kinematics/kinetics waveforms (during the ground contact phase of the pivoting limb) and the number of consecutive CoD were computed during the exercise using a Statistical Parametric Mapping (SPM) approach. RESULTS: The test lasted 153 ± 72 s, with a rate of 14 ± 2 CoDs/min. Participants reached 95% of maximum HR and a peak lactate concentration of 11.2 ± 2.8 mmol/L. Exercise duration was inversely related to lactate concentration (r = -0.517, p < 0.01), while neither%HR max nor [La-] b nor RPE were correlated with test duration before exhaustion (p > 0.05). Alterations in lower limb kinematics were found in 100%, and in lower limb kinetics in 85% of the players. The most common kinematic pattern was a concurrent progressive reduction in hip and knee flexion angle at initial contact (10 players); 5 of them also showed a significantly more adducted hip. Knee extension moment decreased in 8, knee valgus moment increased in 5 players. A subset of participants showed a drift of pivoting limb kinematics that matches the known ACL injury mechanism; other players displayed less definite or even opposed behaviors. DISCUSSION: Players exhibited different strategies to cope with repeated CoDs, ranging from protective to potentially dangerous behaviors. While the latter was not a univocal effect, it reinforces the importance of individual biomechanical assessment when coping with fatigue.

EZmito: a simple and fast tool for multiple mitogenome analyses.

Complete mitochondrial genome data are frequently applied to address phylogenetic/phylogeographic issues at different taxonomic levels in ecology and evolution. While sample preparation/sequencing is becoming more and more straightforward thanks to dropping costs for next-generation sequencing (NGS), data preparation and visualization remains a manually intensive step that may lead to errors if improperly conducted. We have elaborated, and here introduce, EZmito, a simple and intuitive, freely accessible Web Server aimed at automating some of these tasks. EZmito is divided into three main tools: EZpipe that assembles DNA matrices for phylo-mitogenomic analyses; EZskew that calculates genome, strand, and codon nucleotide compositional skews and EZcodon which computes Relative Synonymous Codon Usage statistics as well as amino acid usage frequency over multiple mitogenomes. Output is produced in tabular format as well as publication-quality graphics.

Characterization of the complete mitochondrial genome of Neoasterolepisma foreli (Insecta: Zygentoma: Lepismatidae) and the phylogeny of basal Ectognatha.

The silverfish Neoasterolepisma foreli belongs to the family Lepismatidae within Zygentoma and is well known for the peculiar habit of living in strict association with ant nests (myrmecophily). In this study, we describe its mitochondrial genome, a circular molecule of 15,398 bp including the canonical 13 PCGs, 22 tRNAs, 2 rRNAs, as well as a 403 bp AT-rich region. A phylomitogenomic analysis of the new sequence, alongside basal hexapod mtDNAs, confirmed the monophyly of all orders, with some uncertainty over the position of the enigmatic Tricholepidion gertschi that would make Zygentoma paraphyletic. Neoasterolepisma foreli is recovered in a basal position within family Lepismatidae, at odd with our current understanding of the group that would, in turn, suggest a closer relationship with the genus Lepisma (Mendes, 1991).

Temporal variation in foraging activity and grouping patterns in a mountain-dwelling herbivore: Environmental and endogenous drivers.

In temperate ecosystems, seasonality influences animal behaviour. Food availability, weather, photoperiod and endogenous factors relevant to the biological cycle of individuals have been shown as major drivers of temporal changes in activity rhythms and group size/structure of herbivorous species. We evaluated how diurnal female foraging activity and grouping patterns of a mountain herbivore, the Apennine chamois Rupicapra pyrenaica ornata, varied during a decreasing gradient of pasture availability along the summer-autumn progression (July-October), a crucial period for the life cycle of mountain ungulates. Females increased diurnal foraging activity, possibly because of constrains elicited by variation in environmental factors. Size of mixed groups did not vary, in contrast with the hypothesis that groups should be smaller when pasture availability is lower. Proportion of females in groups increased, possibly suggesting that they concentrated on patchily distributed nutritious forbs. Occurrence of yearlings in groups decreased, which may have depended on dispersal of chamois in this age class. Presence of kids in groups did not show variation through summer-autumn, suggesting a close mother-juvenile relationship even at the end of weaning and/or, possibly, low summer mortality. Both endogenous and environmental factors contribute to shape variation in foraging activity and grouping behaviour in mountain-dwelling herbivores.

The mitogenome of the jumping bristletail Trigoniophthalmus alternatus (Insecta, Microcoryphia) and the phylogeny of insect early-divergent lineages.

The complete mitochondrial genome of the machilid Trigoniophthalmus alternatus (Silvestri 1904) is herein described and applied to phylogenetic analyses, inclusive of the most early-divergent lineages of hexapods. Both gene content and order generally conform with the organization of the arthropods' mitochondrial genome. One gene translocation involving trnA is the autapomorphic character observed in this species. Another peculiar molecular feature is the long size of the A + T-rich region, due to the occurrence of repeat units. The phylogenetic analyses support the typical placement, along the hexapods' tree, of Ectognatha, Monocondylia and Dicondylia, with Diplura as the adelphotaxon of all true insects.

The mitochondrial genome of the springtail Bourletiella arvalis (Symphypleona, Collembola).

The complete mitochondrial genome of the springtail Bourletiella arvalis (Fitch, 1863) is herein described and applied to a Bayesian phylogenetic analysis, inclusive of all the Collembola mitochondrial DNAs sequenced so far. The gene content and order, as well as the nucleotide composition, conform with the well-known features of hexapods' mitochondrial genomes. The phylogenetic analysis supports the monophyly of Collembola, Poduromorpha, Entomobryomorpha and Symphypleona. However, no mtDNA from Neelipleona is available to date, therefore limiting the application of mitochondrial genomes to further investigate springtail systematics.

Droplet digital PCR for the quantification of Alu methylation status in hematological malignancies.

BACKGROUND: Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. Alu sequences lie in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu methylation has been associated with tumor aggressiveness, and also previously discussed in hematological malignancies, by applying different approaches. Moreover, today different techniques designed to measure global DNA methylation are focused on the methylation level of specific repeat elements. In this work we propose a new method of investigating Alu differential methylation, based on droplet digital PCR (ddPCR) technology. METHODS: Forty-six patients with hematological neoplasms were included in the study: 30 patients affected by chronic lymphocytic leukemia, 7 patients with myelodysplastic syndromes at intermediate/high risk, according with the International Prognostic Scoring System, and 9 patients with myelomonocytic leukemia. Ten healthy donors were included as controls. Acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with decitabine (DEC) hypomethylating agent, was also analyzed. DNA samples were investigated for Alu methylation level by digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation, followed by ddPCR. RESULTS: Using ddPCR, a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, was observed. Moreover, comparing the global Alu methylation levels at diagnosis and after azacytidine (AZA) treatment in MDS patients, a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis was evident. We also observed a significant decrease of the Alu methylation level in CLL patients compared to HD, and, finally, for CMML patients, a decrease of Alu sequences methylation was observed in patients harboring the SRSF2 hotspot gene mutation c.284C>D. CONCLUSIONS: In our work, we propose a method to investigate Alu differential methylation based on ddPCR technology. This assay introduces ddPCR as a more sensitive and immediate technique for Alu methylation analysis. To date, this is the first application of ddPCR to study DNA repetitive elements. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose.

Impact of Bone Marrow Aspirate Tregs on the Response Rate of Younger Newly Diagnosed Acute Myeloid Leukemia Patients.

Acute myeloid leukemia (AML) is widely considered a distinct clinical entity with a well-defined molecular and genetics-based prognosis. Particularly in a younger patient, the therapeutic approach depends largely on diagnostic risk stratification, which has an impact on the outcome after therapy. We added Treg evaluation to the usual molecular and cytogenetics profile in the AML younger patients' diagnostic bone marrow aspirate (dBMA) in order to search for any correlation between Tregs and overall response (OR) as well as survival (OS) rates. We studied 23 AML young patients, all treated with standard induction chemotherapy: OR (complete remission (CR) + CR incomplete (CRi)) was documented in 10 of 23 patients (44%); there were two partial responder patients. The optimal dBMA Treg cut-off value for predicting response to treatment (≥21/µL) was obtained by ROC curve analysis. However, in multivariate analysis, apart from the expected impact of the molecular/cytogenetic risk (p = 0.049) and NPM mutation (p = 0.001), dBMA Tregs ≥ 21/µL was not correlated with OR. Actually, higher dBMA Tregs were associated with the good intermediate molecular/cytogenetic risk group (p = 0.02), whose median OS was confirmed to be better as compared with that of the poor risk group (18 versus 5 months, p = 0.05) and equal to the dBMA Tregs ≥ 21/µL group (5 versus 5 months, p = 0.902), respectively. The possible prognostic value of such an immunological player as BMA Tregs in the diagnostic and successive phases of AML needs to be confirmed in larger patient numbers.

Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia.

We report a customized gene panel assay based on multiplex long-PCR followed by third generation sequencing on nanopore technology (MinION), designed to analyze five frequently mutated genes in chronic lymphocytic leukemia (CLL): TP53, NOTCH1, BIRC3, SF3B1 and MYD88. For this purpose, 12 patients were selected according to specific cytogenetic and molecular features significantly associated with their mutational status. In addition, simultaneous analysis of the targets genes was performed by molecular assays or Sanger Sequencing. Data analysis included mapping to the GRCh37 human reference genome, variant calling and annotation, and average sequencing depth/error rate analysis. The sequencing depth resulted on average higher for smaller amplicons, and the final breadth of coverage of the panel was 94.1%. The error rate was about 6% and 2% for insertions/deletions and single nucleotide variants, respectively. Our gene panel allows analysis of the prognostically relevant genes in CLL, with two PCRs per patient. This strategy offers an easy and affordable workflow, although further advances are required to improve the accuracy of the technology and its use in the clinical field. Nevertheless, the rapid and constant development of nanopore technology, in terms of chemistry advances, more accurate basecallers and analysis software, offers promise for a wide use of MinION in the future.

Droplet Digital PCR Is a Robust Tool for Monitoring Minimal Residual Disease in Adult Philadelphia-Positive Acute Lymphoblastic Leukemia.

The breakpoint cluster region-abelson 1 p190 fusion transcript is the most frequent variant observed in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). Qualitative-PCR and real-time quantitative PCR are the currently used methods to monitor minimal residual disease (MRD) in Ph+ ALL patients; for the latter, full standardization and an international quality validation are lacking. Here, we developed a droplet digital PCR (ddPCR) assay for MRD monitoring in p190+ ALL cases. The analytical performance was assessed by the limit-of-detection determination, showing a reliability, sensitivity, and precision of the assay of up to 0.001%. Comparison of results obtained with qualitative PCR and ddPCR in 117 follow-up samples from 16 of 26 Ph+ ALL patients showed discordant results in 27% of cases (32 of 117). Real-time quantitative PCR analysis of 19 ddPCR-positive samples with a low tumor burden failed to provide quantitative results in 63% of cases (12 of 19). These results highlight that in p190+ ALL the ddPCR method has a sufficient analytical performance for very low MRD monitoring and for predicting molecular relapse several months before hematologic relapse. In conclusion, MRD monitoring by ddPCR may better stratify Ph+ ALL patients at risk of disease progression.

Genomic BCR-ABL1 breakpoint characterization by a multi-strategy approach for "personalized monitoring" of residual disease in chronic myeloid leukemia patients.

For monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) the most recommended method is quantitative RT-PCR (RT-qPCR) for measuring BCR-ABL1 transcripts. Several studies reported that a DNA-based assay enhances the sensitivity of detection of the BCR-ABL1 genomic rearrangement, even if its characterization results difficult. We developed a DNA-based method for detecting and quantifying residual BCR-ABL1 positive leukemic stem cells in CML patients. We propose two alternative approaches: the first one is a fluorescence in situ hybridization (FISH)-based step followed by Sanger sequencing; the second one employs MinION, a single molecule sequencer based on nanopore technology. Finally, after defining the BCR-ABL1 genomic junction, we performed the target CML patient-specific quantification, using droplet digital PCR (ddPCR). FISH and MinION steps, respectively, together with ddPCR analysis, greatly reduce the complexity that has impeded the use of "personalized monitoring" of CML in clinical practice. Our report suggests a feasible pipeline, in terms of costs and reproducibility, aimed at characterizing and quantifying the genomic BCR-ABL1 rearrangement during MRD monitoring in CML patients.

RARA and RARG gene downregulation associated with EZH2 mutation in acute promyelocytic-like morphology leukemia.

Most acute promyelocytic leukemia (APL) patients express PML-RARA fusion; in rare cases, RARA is rearranged with partner genes other than PML. To date, only 2 patients presenting features similar to APL showing the RARG gene rearrangement have been described. We report an acute myeloid leukemia patient with morphology resembling APL without involvement of the RARA gene. Molecular and fluorescent in situ hybridization analyses excluded PML-RARA fusion and variant rearrangements involving RARA and RARG loci. Targeted next-generation sequencing showed EZH2- D185H mutation. As this mutation involved the region of interaction with DNA methyltransferases, we speculate an epigenetic alteration of genes involved in the APL-like phenotype. Expression analysis by droplet digital polymerase chain reaction revealed downregulation of the RARA and RARG genes. We hypothesize a novel mechanism of EZH2 function alteration, which may be responsible for an acute myeloid leukemia with APL-like phenotype featuring dysregulation of the RARA and RARG genes.