miR-15a targets the HSP90 co-chaperone Morgana in chronic myeloid leukemia.

Morgana is a ubiquitous HSP90 co-chaperone protein coded by the CHORDC1 gene. Morgana heterozygous mice develop with age a myeloid malignancy resembling human atypical myeloid leukemia (aCML), now renamed MDS/MPN with neutrophilia. Patients affected by this pathology exhibit low Morgana levels in the bone marrow (BM), suggesting that Morgana downregulation plays a causative role in the human malignancy. A decrease in Morgana expression levels is also evident in the BM of a subgroup of Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) patients showing resistance or an incomplete response to imatinib. Despite the relevance of these data, the mechanism through which Morgana expression is downregulated in patients' bone marrow remains unclear. In this study, we investigated the possibility that Morgana expression is regulated by miRNAs and we demonstrated that Morgana is under the control of four miRNAs (miR-15a/b and miR-26a/b) and that miR-15a may account for Morgana downregulation in CML patients.

Comprehensive Molecular Profiling of NPM1-Mutated Acute Myeloid Leukemia Using RNAseq Approach.

Acute myeloid leukemia (AML) is a complex hematologic malignancy with high morbidity and mortality. Nucleophosmin 1 (NPM1) mutations occur in approximately 30% of AML cases, and NPM1-mutated AML is classified as a distinct entity. NPM1-mutated AML patients without additional genetic abnormalities have a favorable prognosis. Despite this, 30-50% of them experience relapse. This study aimed to investigate the potential of total RNAseq in improving the characterization of NPM1-mutated AML patients. We explored genetic variations independently of myeloid stratification, revealing a complex molecular scenario. We showed that total RNAseq enables the uncovering of different genetic alterations and clonal subtypes, allowing for a comprehensive evaluation of the real expression of exome transcripts in leukemic clones and the identification of aberrant fusion transcripts. This characterization may enhance understanding and guide improved treatment strategies for NPM1mut AML patients, contributing to better outcomes. Our findings underscore the complexity of NPM1-mutated AML, supporting the incorporation of advanced technologies for precise risk stratification and personalized therapeutic strategies. The study provides a foundation for future investigations into the clinical implications of identified genetic variations and highlights the importance of evolving diagnostic approaches in leukemia management.

Patient Position in Operative Endoscopy.

It is well known by surgeons that patient positioning is fundamental to exposing the organs when performing an operation via laparoscopy, as gravity can help move the organs and facilitate the exposure of the surgical site. But is it also important for endoscopic procedures? This paper examines various types of endoscopic operations and addresses the issue of the patient's position. The patient's position can be changed not only by rotating the patient along the head-toe axis but also by tilting the surgical bed, as is undertaken during laparoscopic surgical procedures. In particular, it is useful to take into account the effect of gravity on lesion exposure, tumour traction during dissection, crushing by body weight, risk of sample drop, risk of damage to adjacent organs, and anatomical exposure for procedures with radiological support. The endoscopist should always keep in mind the patient's anatomy and the position of the endoscope during operative procedures, not limited to considering only intraluminal vision.

Diode Laser Therapy for Radiation-Induced Vascular Ectasia: Long-Term Results and Cost Analysis.

BACKGROUND: Collateral damage to surrounding healthy tissues has been reported in patients who undergo radiation therapy for pelvic malignancies. This study aimed to evaluate the safety, efficacy and cost efficiency of endoscopic diode laser therapy in patients diagnosed with chronic radiation proctitis (CRP). METHODS: The data of 24 patients (median age 78, range 67-90 years) who presented rectal bleeding and were diagnosed with CRP after undergoing high-dose radiotherapy for prostatic cancer and underwent diode laser therapy were evaluated retrospectively. Non-contact fibers were used in the patients who underwent the procedure without sedation in an outpatient setting. RESULTS: The patients underwent a median of two sessions; overall, a mean of 1591 J of laser energy per session was used. No complications were noted during or after the procedures. Bleeding was completely resolved in 21/24 (88%) patients, and two patients showed improvement (96%). It was not necessary to suspend antiplatelet (six patients) or anticoagulant (four patients) therapy during the treatment course. The mean cost per session was EUR 473.4. CONCLUSIONS: The study findings demonstrated that endoscopic non-contact diode laser treatment in CRP patients is safe, effective and cost efficient. For this procedure, antiplatelet and anticoagulant therapy suspension, intraprocedural sedation and hospital admission are not required.

Aberrant Platelet Aggregation as Initial Presentation of Essential Thrombocythemia: Failure of Entero-Coated Aspirin to Reduce Platelet Hyperactivation.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm variant characterized by excessive production of platelets. Since the most common cause of mortality and morbidity in ET patients is thrombosis, the excessive production of platelets may cause thrombotic events. However, little is known about the function of platelets in ET. We report a female patient who presented as asymptomatic, without a remarkable medical history, and ET was diagnosed after an incidental finding of moderate thrombocytosis. Notably, together with thrombocytosis, an abnormal platelet phenotype was found for the presence of a massive, rapid and spontaneous formation of aggregates and platelet hypersensitivity to subthreshold concentrations of aggregating agonists. Bone marrow histopathological examination and genetic analysis with the JAK2 (V617F) gene mutation findings confirmed the initial suspicion of ET. Although the ET patient was placed on aspirin, the persistence of the platelet hyperactivation and hyperaggregability prompted a switch in antiplatelet medication from entero-coated (EC) to plain aspirin. As result, platelet hypersensitivity to agonists and spontaneous aggregation were no longer found. Collectively, our study demonstrates that platelet function analysis could be a reliable predictor of ET and that plain aspirin should be preferred over EC aspirin to attenuate platelet hyperreactivity.

Endoscopic sleeve gastroplasty: results from a single surgical bariatric centre.

The aim of this study was to evaluate the safety and efficacy of the endoscopic sleeve gastroplasty (ESG) procedure. Patients ineligible for bariatric surgery due to comorbidities or low Body Mass Index (BMI) were offered ESG. Gastric tubularization was carried out via multiple multi-bite sutures across the greater curvature of the stomach. The patients underwent a water-soluble swallow test on post-operative day 1 (POD-1) to assess gastric emptying and were placed on a soft diet if upper GI tract function was confirmed. From January 2019 to March 2022, 27 patients underwent ESG: 14 for severe obesity with comorbidities, including liver transplant, end-stage kidney disease, severe cardiovascular and respiratory diseases. The mean BMI before treatment was 36 ± 9 kg/m2. Two patients (7%) who developed gastric bleeding were successfully treated with packed red blood cells (PRBC) transfusions. After a mean follow-up of 18 months, the percentage of total body weight loss (%TBWL) and the percentage of excess weight (%EWL) were 11 ± 7 and 39 ± 27, respectively. The latter was significantly higher in the patients with an initial BMI < 40 kg/m2 (50 vs 22, p < 0.05). The patients whose gastric sleeve extended for more than a third of the length of the stomach (p < 0.05) had better results. ESG was found to be effective and safe in high-risk surgical patients whose initial BMI was (< 40). Studies characterized by larger number of patients and longer follow-up periods will be able to confirm these results.

Detection of SF3B1 p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms.

Mutations in SF3B1 are found in 20% of myelodysplastic syndromes and 5-10% of myeloproliferative neoplasms, where they are considered important for diagnosis and therapy decisions. Sanger sequencing and NGS are the currently available methods to identify SF3B1 mutations, but both are time-consuming and expensive techniques that are not practicable in most small-/medium-sized laboratories. To identify the most frequent SF3B1 mutation, p.Lys700Glu, we developed a novel fast and cheap assay based on PNA-PCR clamping. After setting the optimal PCR conditions, the limit of detection of PNA-PCR clamping was evaluated, and the method allowed up to 0.1% of mutated SF3B1 to be identified. Successively, PNA-PCR clamping and Sanger sequencing were used to blind test 90 DNA from patients affected by myelodysplastic syndromes and myeloproliferative neoplasms for the SF3B1 p.Lys700Glu mutation. PNA-PCR clamping and Sanger sequencing congruently identified 75 negative and 13 positive patients. Two patients identified as positive by PNA-PCR clamping were missed by Sanger analysis. The discordant samples were analyzed by NGS, which confirmed the PNA-PCR clamping result, indicating that these samples contained the SF3B1 p.Lys700Glu mutation. This approach could easily increase the characterization of myelodysplastic syndromes and myeloproliferative neoplasms in small-/medium-sized laboratories, and guide patients towards more appropriate therapy.

Droplet Digital PCR for BCR-ABL1 Monitoring in Diagnostic Routine: Ready to Start?

BCR-ABL1 mRNA levels represent the key molecular marker for the evaluation of minimal residual disease (MRD) in chronic myeloid leukemia (CML) patients and real-time quantitative PCR (RT-qPCR) is currently the standard method to monitor it. In the era of tyrosine kinase inhibitors (TKIs) discontinuation, droplet digital PCR (ddPCR) has emerged to provide a more precise detection of MRD. To hypothesize the use of ddPCR in clinical practice, we designed a multicentric study to evaluate the potential value of ddPCR in the diagnostic routine. Thirty-seven RNA samples from CML patients and five from healthy donors were analyzed using both ddPCR QXDxTMBCR-ABL %IS Kit and LabNet-approved RT-qPCR methodologies in three different Italian laboratories. Our results show that ddPCR has a good agreement with RT-qPCR, but it is more precise to quantify BCR-ABL1 transcript levels. Furthermore, we did not find differences between duplicate or quadruplicate analysis in terms of BCR-ABL1% IS values. Droplet digital PCR could be confidently introduced into the diagnostic routine as a complement to the RT-qPCR.

Alignment of Qx100/Qx200 Droplet Digital (Bio-Rad) and QuantStudio 3D (Thermofisher) Digital PCR for Quantification of BCR-ABL1 in Ph+ Chronic Myeloid Leukemia.

In recent years, the digital polymerase chain reaction has received increasing interest as it has emerged as a tool to provide more sensitive and accurate detection of minimal residual disease. In order to start the process of data alignment, we assessed the consistency of the BCR-ABL1 quantification results of the analysis of 16 RNA samples at different levels of disease. The results were obtained by two different laboratories that relied on The Qx100/Qx200 Droplet Digital PCR System (Bio-Rad) and Quant Studio 3D dPCR System (Thermofisher) platforms. We assessed the compatibility between the estimated values by linear regression, Bland-Altman bias-plot, and Mann-Whitney nonparametric test. The results confirmed the compatibility of the measures, allowing us tocompute an 'alignment factor' (AF), equal to 1.41, which was further validated by a different series of experiments. We conclude that the performed measurements by the two laboratories are comparable, and also equalized through the introduction of an alignment factor.

Standardization of BCR-ABL1 p210 Monitoring: From Nested to Digital PCR.

The introduction of tyrosine kinase inhibitors in 2001 as a targeted anticancer therapy has significantly improved the quality of life and survival of patients with chronic myeloid leukemia. At the same time, with the introduction of tyrosine kinase inhibitors, the need for precise monitoring of the molecular response to therapy has emerged. Starting with a qualitative polymerase chain reaction, followed by the introduction of a quantitative polymerase chain reaction to determine the exact quantity of the transcript of interest-p210 BCR-ABL1, molecular monitoring in patients with chronic myeloid leukemia was internationally standardized. This enabled precise monitoring of the therapeutic response, unification of therapeutic protocols, and comparison of results between different laboratories. This review aims to summarize the steps in the diagnosis and molecular monitoring of p210 BCR-ABL1, as well as to consider the possible future application of a more sophisticated method such as digital polymerase chain reaction.

Sustained Complete Remission with Incomplete Hematologic Recovery (CRi) in a Patient with Relapsed AML and Concurrent BCR-ABL1 and CBFB Rearrangement Treated with a Combination of Venetoclax and 5-Azacytidine.

The co-occurrence of BCR-ABL1 fusion and core-binding factor (CBF) rearrangements is uncommonly reported in AML. Although CBF rearrangements carry a favorable prognosis, the coexistence of BCR-ABL1 is associated with aggressive disease suggesting a potential advantage of high-intensity chemotherapy in association with tyrosine kinase inhibitors. Herein, we describe a refractory AML patient harboring BCR-ABL1 fusion and CBFB rearrangement that was successfully treated with a combination of venetoclax and hypomethylating agent.

Philadelphia-positive lymphoblastic lymphoma: a case report and review of the literature.

Philadelphia positive acute lymphoblastic leukemia is well documented nowadays but very little is known about Philadelphia positive lymphoblastic lymphoma (LBL). Only two cases are available in literature and both of them died during treatment whereas the patient treated in our center is still alive 3 years after the initial diagnosis. A chemo-free regimen was used in induction with dasatinib plus steroids with local radiotherapy on the mass, and then the patient underwent bone marrow transplant. Philadelphia positive lymphoblastic lymphoma is a difficult diagnosis to make and the management of this extremely rare disease is very challenging.

CALR-positive myeloproliferative disorder in a patient with Ph-positive chronic myeloid leukemia in durable treatment-free remission: a case report.

Current diagnostic criteria for Philadelphia-negative myeloproliferative neoplasia (MPN) have been redefined by the discovery of Janus kinase 2 (JAK2), myeloproliferative leukemia (MPL) and calreticulin (CALR) genetic alterations. Only few cases of coexistence of CALR-mutated MPN and Philadelphia-positive chronic myeloid leukemia (CML) have been described so far. Here we report the case of a patient with CML diagnosed in 2001, treated with imatinib and pegylated interferon (IFN) frontline. She reached complete molecular remission (CMR) and discontinued imatinib, maintaining treatment free remission. Due to persistent thrombocytosis, we repeated bone marrow (BM) analysis and diagnosed CARL-mutated essential thrombocythemia (ET). A CALR-positive clone was found to be present since 2001, and was unaffected by imatinib treatment, possibly representing a molecular abnormality arising at stem cell level.

mTOR pathway activation in multiple myeloma cell lines and primary tumour cells: pomalidomide enhances cytoplasmic-nuclear shuttling of mTOR protein.

mTOR is a protein kinase that plays a central role in regulating critical cellular processes. We evaluated the activation and cellular localization of the mTOR pathway in multiple myeloma (MM) and analyzed the role of pomalidomide in regulating mTOR. By immunohistochemistry cytoplasmic p-mTOR stained positive in 57 out 101 (57.6%) cases with a nuclear p-mTOR localization in 14 out 101 samples (13.8%). In the 70 MM samples analyzed for the entire pathway, p-mTOR expression significantly correlated with p-AKT, p-P70S6K, and p-4E-BP1 suggesting that the AKT/mTOR pathway is activated in a subset of MM patients. Immunofluorescence assays demonstrated that mTOR protein is distributed throughout the cytoplasm and the nucleus at baseline in MM cell lines and in plasma cells of 13 MM patients and that pomalidomide facilitated the shift of the mTOR protein in the nucleus. By western blotting, treatment with pomalidomide increased nuclear mTOR and p-mTOR expression levels in the nucleus with a concomitant decrease of the cytoplasmic fractions while does not seem to affect significantly AKT phosphorylation status. In MM cells the anti-myeloma activity of pomalidomide may be mediated by the regulation of the mTOR pathway.

Morgana acts as an oncosuppressor in chronic myeloid leukemia.

We recently described morgana as an essential protein able to regulate centrosome duplication and genomic stability, by inhibiting ROCK. Here we show that morgana (+/-) mice spontaneously develop a lethal myeloproliferative disease resembling human atypical chronic myeloid leukemia (aCML), preceded by ROCK hyperactivation, centrosome amplification, and cytogenetic abnormalities in the bone marrow (BM). Moreover, we found that morgana is underexpressed in the BM of patients affected by atypical CML, a disorder of poorly understood molecular basis, characterized by nonrecurrent cytogenetic abnormalities. Morgana is also underexpressed in the BM of a portion of patients affected by Philadelphia-positive CML (Ph(+) CML) caused by the BCR-ABL oncogene, and in this condition, morgana underexpression predicts a worse response to imatinib, the standard treatment for Ph(+) CML. Thus, morgana acts as an oncosuppressor with different modalities: (1) Morgana underexpression induces centrosome amplification and cytogenetic abnormalities, and (2) in Ph(+) CML, it synergizes with BCR-ABL signaling, reducing the efficacy of imatinib treatment. Importantly, ROCK inhibition in the BM of patients underexpressing morgana restored the efficacy of imatinib to induce apoptosis, suggesting that ROCK inhibitors, combined with imatinib treatment, can overcome suboptimal responses in patients in which morgana is underexpressed.

FIP1L1-PDGFRα-positive hypereosinophilic syndrome in childhood: a case report and review of literature.

Hypereosinophilic syndromes in children are rare disorders traditionally characterized by an eosinophil count exceeding 1,500/mm³ on at least 2 occasions or evidence of tissue eosinophilia associated with symptoms and marked blood eosinophilia, lacking any secondary cause (such as infections, allergic disease, chemical-induced eosinophilia, hypoadrenalism, cancer). Until now there have only been 3 reported cases of pediatric FIP1L1-PDGFRα-positive hypereosinophilic syndromes. We describe a fourth patient, a white 14-year-old boy, the third treated with imatinib.

Chromosome abnormalities additional to the Philadelphia chromosome at the diagnosis of chronic myelogenous leukemia: pathogenetic and prognostic implications.

Additional chromosome abnormalities (ACAs) occur in less than 10% of cases at diagnosis of Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML). In some cases, on the basis of the persistence of the ACAs in Ph-negative cells after response to imatinib, a secondary origin of the Ph chromosome has been demonstrated. In this study, the possible prognostic value of this phenomenon was evaluated. Thirty-six Ph-positive CML patients were included in the study. In six patients, ACAs persisted after the disappearance of the Ph. A complete cytogenetic response (CCR) was obtained in five of these six patients, and five of six also had a high Sokal score. In all the other cases, ACAs disappeared together (in cases of response to therapy with imatinib) or persisted with the Ph (in cases of no response to imatinib). In the former cases, the primary origin of the Ph was demonstrated. CCR was obtained in 22 cases (17 with low to intermediate Sokal scores), while no response was observed in 8 patients (5 with a high Sokal score). Sokal score seems to maintain its prognostic value for patients in whom the Ph occurs as a primary event, but not in those in whom it occurs as a secondary one.

Sustained molecular remissions are achievable with tyrosine kinase inhibitor therapy in patients with chronic myeloid leukemia and additional cytogenetic clonal evolution.

Little is known regarding the activity of tyrosine kinase inhibitors (TKis) on chronic myeloid leukemia (CML) clonal evolution (CE). We treated 10 CE CML patients in either hematologic chronic (8 cases) or accelerated (2 cases) phase with imatinib or second generation TKi. Additional chromosomal abnormalities appeared during the course of disease in seven cases, being present at diagnosis in three. A total of 6/10 (60%) patients achieved complete cytogenetic remission (CCyR) with imatinib in 3-14 months. Major or complete molecular remission (CMR) was obtained in four CCyR patients after 21, 25, 22, and 12 months, as well as in a fifth patient who started nilotinib because of suboptimal response after 75 months of imatinib treatment. One patient received nilotinib due to imatinib intolerance after 56 months of therapy while on CMR, and maintained such status. After a median follow-up of 82 months (range, 3-116), six patients are alive, five of which are in continuous CCyR while one patient is in his third CCyR on dasatinib after relapsing on imatinib and nilotinib. Five patients are in complete (four) or major (one) molecular remission, ongoing at 3, 48, 61, 95, and 96 months, on imatinib (three) or nilotinib (two). Although a small number of patients was studied, our results suggest that long-term cytogenetic and molecular remission can be achieved in CML CE patients with TKis treatment.

Deletions of the derivative chromosome 9 do not influence the response and the outcome of chronic myeloid leukemia in early chronic phase treated with imatinib mesylate: GIMEMA CML Working Party analysis.

PURPOSE: Deletions of the derivative chromosome 9 [der(9)] have been associated with a poor prognosis in chronic myeloid leukemia (CML) across different treatment modalities. In the imatinib era, the prognostic impact of der(9) deletions has been evaluated mainly in patients with late chronic-phase (CP) CML, giving partially conflicting results. Few data are available in the early CP setting. For this reason, in 2006, the European LeukemiaNet recommendations still considered der(9) deletions as a candidate adverse prognostic factor and required a careful monitoring of the patient. PATIENTS AND METHODS: To investigate the prognostic value of der(9) deletions in early CP CML, we performed an analysis of three prospective imatinib trials of the Italian Group for Hematological Malignancies of the Adult (GIMEMA) CML Working Party. RESULTS: A fluorescent in situ hybridization (FISH) analysis of bone marrow cells was performed at diagnosis; der(9) deletions were detected in 60 (12%) of 521 evaluable patients. At 60 months, the cumulative incidence of complete cytogenetic response and major molecular response-and the probability of event-free survival, failure-free survival, progression-free survival, and overall survival-in patients with and without deletions were not statistically different. CONCLUSION: Our data strongly support the notion that, when investigated by FISH, der(9) deletions are not a poor prognostic factor in patients with early CP CML treated with imatinib.