Silencing of the DNA damage repair regulator PPP1R15A sensitizes acute myeloid leukemia cells to chemotherapy.

Acute Myeloid Leukemia (AML) is a life-threatening disease whose induction treatment consists of combination chemotherapy with Idarubicin and Cytarabine for fit patients. Treatment failures are frequent, urging the need for novel treatments for this disease. The DNA Damage Response Mechanism (DDR) comprises numerous molecules and pathways intended to arrest the cell cycle until DNA damage is repaired or else drive the cell to apoptosis. AML-derived cell lines after treatment with Idarubicin and Cytarabine were used for studying the expression profile of 84 DDR genes, through PCR arrays. Utilizing de novo AML patient and control samples we studied the expression of PPP1R15A, CDKN1A, GADD45A, GADD45G, and EXO1. Next, we performed PPP1R15A silencing in AML cell lines in two separate experiments using siRNA and CRISPR-cas9, respectively. Our findings highlight that DDR regulators demonstrate increased expression in patients with high cytogenetic risk possibly reflecting increased genotoxic stress. Especially, PPP1R15A is mainly involved in the recovery of the cells from stress and it was the only DDR gene upregulated in AML patients. The PPP1R15A silencing resulted in decreased viability of Idarubicin and Cytarabine-treated cell lines, in contrast to untreated cells. These findings shed light on new strategies to enhance chemotherapy efficacy and demonstrate that PPP1R15A is an important DDR regulator in AML and its downregulation might be a safe and effective way to increase sensitivity to chemotherapy in this disease.

Real-Life Multicenter Experience of Venetoclax in Combination with Hypomethylating Agents in Previously Untreated Adult Patients with Acute Myeloid Leukemia in Greece.

BACKGROUND: The landscape of first-line treatment for acute myeloid leukemia (AML) patients ineligible for intensive chemotherapy has changed remarkably after venetoclax approval. Accumulating real-world data further apprises us with more knowledgeable use. To assess the efficacy and safety challenges in the real-life setting of the combination of hypomethylated agent (HMA) and venetoclax, we conducted a multi-center retrospective study. METHODS: Forty adult AML patients treated with the combination of HMA and venetoclax as a first-line treatment after full approval (2020) were included. To confirm VIALE-A results, this group was compared to a historical cohort of 17 chemotherapy-ineligible AML patients treated with HMA monotherapy before 2020. RESULTS: The combination of HMA-venetoclax achieved a composite complete response rate of 86.8% (p < 0.001), median overall survival, and event-free survival of 33.8 and 19.7 months, respectively, in a median follow-up of 17.8 months (pos < 0.001, HR = 0.276, CI: 0.132-0.575, pEFS = 0.004, HR = 0.367, CI: 0.174-0.773). High rates of neutropenia (90%) and consequent infection rates (57.5%) were noted. Only 55% of our patients received antifungal prophylaxis, as its use remains controversial, and invasive fungal infections were presented in 7.5%. CONCLUSIONS: Evidently, venetoclax-HMA yields high response rates and profound survival benefits in real life and has changed our approach to alternative chemotherapy options.

Primary Adrenal Lymphomas with Cushing's Syndrome: Two Cases with Evidence of Endogeneous Cortisol Production by the Neoplastic Lymphoid Cells.

Primary adrenal lymphoma (PAL) is a rare entity that presents as unilateral or bilateral rapidly growing adrenal masses, with signs and symptoms most commonly related to adrenal insufficiency due to the mass effect on the surrounding tissues. Although steroeidogenesis has not been previously described in PAL, we herein report two cases of PAL presenting as adrenal incidentalomas (AIs) that demonstrated autonomous cortisol production. A 52-year-old woman presented with lumbar pain; a computed tomography (CT) scan demonstrated a left AI measuring 8.5 × 15 × 10 cm. Similarly, an 80-year-old woman presented with lumbar pain, demonstrating in a CT scan a bilateral AI (right: 9 × 6.5 cm, left: 3.6 × 3.2 cm). Both cases underwent a full hormonal evaluation according to the algorithm for the investigation of AIs, demonstrating increased 24-h cortisol excretion, suppressed fasting adrenocorticotropic hormone (ACTH) levels, and non-suppressed serum cortisol levels in both the overnight and the low-dose dexamethasone suppression tests, indicating autonomous cortisol secretion and Cushing's syndrome. In a relatively short time, both patients developed night sweats, and their clinical picture deteriorated, while the CT scans showed increased dimensions of the masses with radiological characteristics compatible to lymphoma. Both patients underwent ultrasound-guided biopsies (FNBs), revealing infiltration of the left adrenal by diffuse large B-cell lymphoma in the first case, whereas bilateral adrenal infiltration from the same histological type was noted in the second case. Subsequently, they were treated with immunochemotherapy, but the second patient died from an infection shortly after the initiation of the treatment. To our knowledge, this is the first report of PAL presenting with Cushing's syndrome due to autonomous cortisol production, indicating that neoplastic lymphoid cells in PAL might acquire the potential for steroidogenesis; therefore, more cases of PAL should be analyzed so as to further elucidate the complex pathogenesis and the natural course of this entity.

Novel circular RNAs of the apoptosis-related BAX and BCL2L12 genes identified in a chronic lymphocytic leukemia cell line using nanopore sequencing.

Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key regulators of gene expression, with deregulated expression and established involvement in leukemia. The products of BCL2 and its homologs, including BAX and BCL2L12, are implicated in chronic lymphocytic leukemia (CLL). However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their role in CLL. We sought to further elucidate the contribution of BAX and BCL2L12 in CLL by unraveling the identity, localization, and potential role of their circRNAs. Therefore, total RNA from the EHEB cell line and peripheral blood mononuclear cells (PBMCs) of CLL patients and non-leukemic blood donors was extracted and reverse-transcribed using random hexamers. Next, nested PCRs with divergent primers were performed and the purified PCR products were subjected to 3rd generation nanopore sequencing. Nested PCRs were also applied to first-strand cDNAs synthesized from total RNA extracts of PBMCs from CLL patients and non-leukemic blood donors. Lastly, a single-molecule resolution fluorescent in situ hybridization method called circFISH was used to visualize the circRNA distribution in EHEB cells. We discovered several novel circRNAs produced by BAX and BCL2L12, which were characterized by great exon structure diversity. In addition, intriguing findings regarding their formation emerged. Interestingly, visualization of the most abundant circRNAs showed distinct intracellular localization. Moreover, a complex BAX and BCL2L12 circRNA expression pattern was revealed in CLL patients and non-leukemic blood donors. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL.

Mast cell leukemia: clinical and molecular features and survival outcomes of patients in the ECNM Registry.

Mast cell leukemia (MCL) is a rare subtype of systemic mastocytosis defined by ≥20% mast cells (MC) on a bone marrow aspirate. We evaluated 92 patients with MCL from the European Competence Network on Mastocytosis registry. Thirty-one (34%) patients had a diagnosis of MCL with an associated hematologic neoplasm (MCL-AHN). Chronic MCL (lack of C-findings) comprised 14% of patients, and only 4.5% had "leukemic MCL" (≥10% circulating MCs). KIT D816V was found in 62/85 (73%) evaluable patients; 9 (11%) individuals exhibited alternative KIT mutations, and no KIT variants were detected in 14 (17%) subjects. Ten evaluable patients (17%) had an abnormal karyotype and the poor-risk SRSF2, ASXL1, and RUNX1 (S/A/R) mutations were identified in 16/36 (44%) patients who underwent next-generation sequencing. Midostaurin was the most common therapy administered to 65% of patients and 45% as first-line therapy. The median overall survival (OS) was 1.6 years. In multivariate analysis (S/A/R mutations excluded owing to low event rates), a diagnosis of MCL-AHN (hazard ratio [HR], 4.7; 95% confidence interval [CI], 1.7-13.0; P = .001) and abnormal karyotype (HR, 5.6; 95% CI, 1.4-13.3; P = .02) were associated with inferior OS; KIT D816V positivity (HR, 0.33; 95% CI, 0.11-0.98; P = .04) and midostaurin treatment (HR, 0.32; 95% CI, 0.08-0.72; P = .008) were associated with superior OS. These data provide the most comprehensive snapshot of the clinicopathologic, molecular, and treatment landscape of MCL to date, and should help further inform subtyping and prognostication of MCL.

Incidence and risk factors for central nervous system relapse in patients with primary mediastinal large B-cell lymphoma in the rituximab era.

Central nervous system (CNS) involvement is rare in primary mediastinal large B-cell lymphoma (PMLBCL). We aimed to evaluate the incidence of CNS relapse as first treatment failure event and the effect of the induction chemotherapy regimen, central nervous system - international prognostic index (CNS-IPI) and other clinical and laboratory variables on the risk of CNS relapse in 564 PMLBCL patients treated with immunochemotherapy. Only 17 patients (3.0%) received CNS prophylaxis. During a 55-month median follow-up only 8 patients experienced CNS relapse as first event, always isolated. The 2-year cumulative incidence of CNS relapse (CI-CNSR) was 1.47% and remained unchanged thereafter. The CI-CNSR was not affected by the chemotherapy regimen (R-CHOP or R-da-EPOCH). None of the established International Prognostic Index factors for aggressive lymphomas predicted CNS relapse in PMLBCL. The 2-year CI-CNSR in patients with versus without kidney involvement was 13.3% versus 0.96% (p < 0.001); 14.3% versus 1.13% with versus without adrenal involvement (p < 0.001); and 10.2% versus 0.97% with versus without either kidney or adrenal involvement. CNS-IPI was also predictive (2-year CI-CNSR in high-risk vs. intermediate/low-risk: 10.37% vs. 0.84%, p < 0.001). However, this association may be driven mainly by kidney and/or adrenal involvement. In conclusion, in PMLBCL, CNS relapse is rare and appears to be strongly associated with kidney and/or adrenal involvement.

Chronic Neutrophilic Leukemia: A Comprehensive Review of Clinical Characteristics, Genetic Landscape and Management.

Chronic neutrophilic leukemia (CNL) represents a rare disease, that has been classified among the BCR/ABL-negative myeloproliferative neoplasms. The disease is characterized by marked leukocytosis with absolute neutrophilia and its clinical presentation may vary from asymptomatic to highly symptomatic with massive splenomegaly and constitutional symptoms. CNL prognosis remains relatively poor, as most patients succumb to disease complications or transform to acute myeloid leukemia. Recent studies have demonstrated that CSF3R mutations drive the disease, albeit the presence of other secondary mutations perplex the genetic landscape of the disease. Notably, the presence of CSF3R mutations has been adopted as a criterion for diagnosis of CNL. Despite the vigorous research, the management of the disease remains suboptimal. Allogeneic stem cell transplantation represents the only treatment that could lead to cure; however, it is accompanied by high rates of treatment-related mortality. Recently, ruxolitinib has shown significant responses in patients with CNL; however, emergence of resistance might perturbate long-term management of the disease. The aim of this review is to summarize the clinical course and laboratory findings of CNL, highlight its pathogenesis and complex genetic landscape, and provide the context for the appropriate management of patients with CNL.

Real-life Experience With Rituximab-CHOP Every 21 or 14 Days in Primary Mediastinal Large B-cell Lymphoma.

BACKGROUND/AIM: Primary mediastinal large B-cell lymphoma (PMLBCL) is an aggressive B-cell non-Hodgkin lymphoma (NHL), whose prognosis has greatly improved since the incorporation of the anti-CD20 monoclonal antibody rituximab into current therapeutic regimens. Evidence, however, on the optimal time interval between consecutive chemoimmunotherapy (CIT) cycles is still scarce. This study aimed to evaluate the efficacy outcomes of the more commonly administered 3-weekly regimens to the biweekly ones in a PMLBCL patients' population, who were mostly treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone every 21 days (R-CHOP-21) or R-CHOP-14. PATIENTS AND METHODS: We retrospectively studied our cohort of consecutively treated PMLBCL patients, focusing on their treatment density, in order to determine possible differences in treatment outcomes. RESULTS: CIT, in the form of both R-CHOP-21 as well as R-CHOP-14 (or similar regimens), is highly active in PMLBCL, with low rates of early treatment failure. In our cohort of patients, R-CHOP-14 did not result in a meaningful improvement of freedom from progression (FFP) or overall survival (OS). CONCLUSION: Both R-CHOP-14 and R-CHOP-21 are probably equally effective in PMLBCL, yet further, prospective, randomized studies are warranted to clarify whether dose-dense regimens can be associated with better disease control and long-term results.

Monoclonal Antibodies in the Treatment of Diffuse Large B-Cell Lymphoma: Moving beyond Rituximab.

Although rituximab has revolutionized the treatment of diffuse large B-cell lymphoma (DLBCL), a significant proportion of patients experience refractory disease or relapse early after the end of treatment. The lack of effective treatment options in the relapsed/refractory (R/R) setting had made the prognosis of these patients dismal. The initial enthusiasm for novel anti-CD20 antibodies had been short-lived as they failed to prove their superiority to rituximab. Therefore, research has focused on developing novel agents with a unique mechanism of action. Among them, two antibody-drug conjugates, namely polatuzumab vedotin (PolaV) and loncastuximab tesirine, along with tafasitamab, an anti-CD19 bioengineered antibody, have been approved for the treatment of R/R DLBCL. Whereas PolaV has been FDA and EMA approved, EMA has not approved loncastuximab tesirine and tafasitamab yet. Results from randomized trials, as well as real-life data for PolaV have been promising. Novel agents as bispecific antibodies bridging CD3 on T-cells to CD20 have shown very promising results in clinical trials and are expected to gain approval for treatment of R/R DLBCL soon. As the therapeutic armamentarium against DLBCL is expanding, an improvement in survival of patients with R/R and higher cure rates might soon become evident.

Kinetics of Nucleocapsid, Spike and Neutralizing Antibodies, and Viral Load in Patients with Severe COVID-19 Treated with Convalescent Plasma.

COVID-19 is an ongoing pandemic with high morbidity and mortality. Despite meticulous research, only dexamethasone has shown consistent mortality reduction. Convalescent plasma (CP) infusion might also develop into a safe and effective treatment modality on the basis of recent studies and meta-analyses; however, little is known regarding the kinetics of antibodies in CP recipients. To evaluate the kinetics, we followed 31 CP recipients longitudinally enrolled at a median of 3 days post symptom onset for changes in binding and neutralizing antibody titers and viral loads. Antibodies against the complete trimeric Spike protein and the receptor-binding domain (Spike-RBD), as well as against the complete Nucleocapsid protein and the RNA binding domain (N-RBD) were determined at baseline and weekly following CP infusion. Neutralizing antibody (pseudotype NAb) titers were determined at the same time points. Viral loads were determined semi-quantitatively by SARS-CoV-2 PCR. Patients with low humoral responses at entry showed a robust increase of antibodies to all SARS-CoV-2 proteins and Nab, reaching peak levels within 2 weeks. The rapid increase in binding and neutralizing antibodies was paralleled by a concomitant clearance of the virus within the same timeframe. Patients with high humoral responses at entry demonstrated low or no further increases; however, virus clearance followed the same trajectory as in patients with low antibody response at baseline. Together, the sequential immunological and virological analysis of this well-defined cohort of patients early in infection shows the presence of high levels of binding and neutralizing antibodies and potent clearance of the virus.

Predicting outcome in higher-risk myelodysplastic syndrome patients treated with azacitidine.

5-Azacitidine (5-AZA) is widely used for the treatment of higher-risk myelodysplastic syndromes. However, response and survival rates vary considerably, while indicated treatment duration remains undefined. For these reasons, factors determining response and survival are of major importance. Clinical, morphological, flow cytometry, cytogenetic and molecular factors are discussed in this review. Biomarkers predictive of response and prognosis, as well as their link to the mode of action of 5-AZA are also addressed, shifting the focus from clinical practice to investigational research. Their use could further improve prognostic classification of 5-AZA treated higher-risk myelodysplastic syndromes in the near future.

Lay abstract Myelodysplastic syndrome is a disorder in which patients have dysfunctional blood cells. The only chance of curing patients with high-risk myelodysplastic syndrome (HR-MDS) is allogeneic stem cell transplantation. However, most HR-MDS patients are not young or fit enough to receive such a toxic treatment. For these patients, hypomethylating drugs such as 5-azacitidine (5-AZA) and decitabine are preferable treatments. These drugs work by reducing the number of molecules known as methyl groups that are attached to DNA, which can lead to cell death. About half of patients respond to it, and those who do respond, survive longer than those who do not. In addition, 5-AZA delays disease progression from HR-MDS to acute myeloid leukemia, a type of blood cancer, and may help patients who do not improve to live longer. However, 5-AZA has side effects that can be hard to bear, such as an increased need for red blood cells and/or platelet transfusions. For this reason, it would be good to predict the clinical and biological factors associated with either response or final outcome following treatment. In this manuscript, we attempt to summarize current knowledge on this topic.

Prognostic molecular biomarkers in diffuse large B-cell lymphoma in the rituximab era and their therapeutic implications.

Diffuse large B-cell lymphoma (DLBCL) represents a group of tumors characterized by substantial heterogeneity in terms of their pathological and biological features, a causal factor of their varied clinical outcome. This variation has persisted despite the implementation of rituximab in treatment regimens over the last 20 years. In this context, prognostic biomarkers are of great importance in order to identify high-risk patients that might benefit from treatment intensification or the introduction of novel therapeutic agents. Herein, we review current knowledge on specific immunohistochemical or genetic biomarkers that might be useful in clinical practice. Gene-expression profiling is a tool of special consideration in this effort, as it has enriched our understanding of DLBCL biology and has allowed for the classification of DLBCL by cell-of-origin as well as by more elaborate molecular signatures based on distinct gene-expression profiles. These subgroups might outperform individual biomarkers in terms of prognostication; however, their use in clinical practice is still limited. Moreover, the underappreciated role of the tumor microenvironment in DLBCL prognosis is discussed in terms of prognostic gene-expression signatures, as well as in terms of individual biomarkers of prognostic significance. Finally, the efficacy of novel therapeutic agents for the treatment of DLBCL patients are discussed and an evidence-based therapeutic approach by specific genetic subgroup is suggested.

Solitary extramedullary plasmacytoma of the nasopharynx: The role of flow cytometry.

Extramedullary plasmacytoma (EMP) represents a distinct yet rare entity among the plasma cell neoplasms. Given its rarity, no therapeutic consensus has been met. We report the case of a 57-year-old man with a one-year history of nasal congestion and occasional dyspnoea. Imaging showed a hypermetabolic mass in the right nasopharynx extending backward towards the adjacent oropharynx, infiltrating the epiglottis. As incisional biopsy showed histologic and immunophenotypic features consistent with plasma cell neoplasm, whereas the possibility of a marginal zone lymphoma with plasmacytic differentiation was included in the differential diagnosis. A final diagnosis of EMP was reached by using flow cytometry (FC) of a cell suspension from the neoplastic tissue. The patient received local radiotherapy (RT) which resulted to complete remission. In conclusion, flow cytometry might serve as an auxiliary method in cases where immunohistochemistry cannot differentiate between a plasma cell dyscrasia and a B-non-Hodgkin lymphoma. In cases of an established diagnosis of solitary nasopharyngeal EMP RT represents an excellent treatment modality offering prolonged disease-free survival.

The Multifaceted Role and Utility of MicroRNAs in Indolent B-Cell Non-Hodgkin Lymphomas.

Normal B-cell development is a tightly regulated complex procedure, the deregulation of which can lead to lymphomagenesis. One common group of blood cancers is the B-cell non-Hodgkin lymphomas (NHLs), which can be categorized according to the proliferation and spread rate of cancer cells into indolent and aggressive ones. The most frequent indolent B-cell NHLs are follicular lymphoma and marginal zone lymphoma. MicroRNAs (miRNAs) are small non-coding RNAs that can greatly influence protein expression. Based on the multiple interactions among miRNAs and their targets, complex networks of gene expression regulation emerge, which normally are essential for proper B-cell development. Multiple miRNAs have been associated with B-cell lymphomas, as the deregulation of these complex networks can lead to such pathological states. The aim of the present review is to summarize the existing information regarding the multifaceted role of miRNAs in indolent B-cell NHLs, affecting the main B-cell subpopulations. We attempt to provide insight into their biological function, the complex miRNA-mRNA interactions, and their biomarker utility in these malignancies. Lastly, we address the limitations that hinder the investigation of the role of miRNAs in these lymphomas and discuss ways that these problems could be overcome in the future.

A Phase II Study on the Use of Convalescent Plasma for the Treatment of Severe COVID-19- A Propensity Score-Matched Control Analysis.

COVID-19 is a global pandemic associated with increased morbidity and mortality. Convalescent plasma (CP) infusion is a strategy of potential therapeutic benefit. We conducted a multicenter phase II study to evaluate the efficacy and safety of CP in patients with COVID-19, grade 4 or higher. To evaluate the efficacy of CP, a matched propensity score analysis was used comparing the intervention (n = 59) to a control group (n = 59). Sixty patients received CP within a median time of 7 days from symptom onset. During a median follow-up of 28.5 days, 56/60 patients fully recovered and 1 patient remained in the ICU. The death rate in the CP group was 3.4% vs. 13.6% in the control group. By multivariate analysis, CP recipients demonstrated a significantly reduced risk of death [HR: 0.04 (95% CI: 0.004-0.36), p: 0.005], significantly better overall survival by Kaplan-Meir analysis (p < 0.001), and increased probability of extubation [OR: 30.3 (95% CI: 2.64-348.9), p: 0.006]. Higher levels of antibodies in the CP were independently associated with significantly reduced risk of death. CP infusion was safe with only one grade 3 adverse event (AE), which easily resolved. CP used early may be a safe and effective treatment for patients with severe COVID-19 (trial number NCT04408209).