Subclones with variants of uncertain clinical significance might contribute to ineffective hemopoiesis and leukemia predisposition.

BACKGROUND: Splicing modifications, genomic instability, and hypomethylation are central mechanisms promoting myelodysplasia and acute myeloid leukemia (AML). In this real-life retrospective study, to elucidate pathophysiology of clonal hemopoiesis in hematological malignancies, we investigated clinical significance of mutations in leukemia-related genes of known pathogenetic significance and of variants of uncertain clinical significance (VUS) in a cohort of patients with MDS and AML. METHODS: A total of 59 consecutive subjects diagnosed with MDS, 48 with AML, and 17 with clonal cytopenia with unknown significance were screened for somatic mutations in AML-related genes by next-generation sequencing. RESULTS: We showed that TET2, SETBP1, ASXL1, EZH2, RUNX1, SRSF2, DNMT3A, and IDH1/2 were commonly mutated. MDS patients also showed a high genetic complexity, especially for SETBP1. Moreover, the presence of SETBP1 wild-type or two or more simultaneous VUS variants identified a subgroup of AML and MDS patients with better outcome, while the presence of single SETBP1 VUS variant was related to a worse prognosis, regardless TET2 mutational status. CONCLUSIONS: In conclusions, we linked both pathogenic and VUS variants in AML-related genes to clonal hematopoiesis; therefore, we proposed to consider those variants as prognostic markers in leukemia and myelodysplasia. However, further studies in larger prospective cohorts are required to validate our results.

Giant plasma cells with multiple immature nuclei in a young woman with newly diagnosed multiple myeloma.

BACKGROUND: Multiple myeloma with giant multinucleated plasma cells is a very rare entity and mostly reported cases are dated. This plasma cell morphology has been observed after monoclonal antibody treatments, such as daratumumab, and patients have experienced a worse prognosis with partial responses and a high rate of relapse. RESULTS: Here, we showed a newly diagnosed multiple myeloma with giant plasma cells with multiple (up to 13) immature nuclei who achieved a complete remission after a first line therapy and underwent to autologous hematopoietic stem cell transplantation, as per international guidelines.

Axitinib in Ponatinib-Resistant B-Cell Acute Lymphoblastic Leukemia Harboring a T315L Mutation.

Adult acute lymphoblastic leukemia (ALL) with BCR-ABL1 rearrangement (Philadelphia chromosome, Ph) is a hematological aggressive disease with a fatal outcome in more than 50% of cases. Tyrosine kinase inhibitors (TKIs) targeting the activity of BCR-ABL1 protein have improved the prognosis; however, relapses are frequent because of acquired somatic mutations in the BCR-ABL1 kinase domain causing resistance to first, second and third generation TKIs. Axitinib has shown in vitro and ex vivo activity in blocking ABL1; however, clinical trials exploring its efficacy in ALL are missing. Here, we presented a 77-year-old male with a diagnosis of Ph positive ALL resistant to ponatinib and carrying a rare threonine to leucine (T315L) mutation on BCR-ABL1 gene. The patient was treated with axitinib at 5 mg/twice daily as salvage therapy showing an immediate although transient benefit with an overall survival of 9.3 months. Further dose-finding and randomized clinical trials are required to assess the real efficacy of axitinib for adult Ph positive ALL resistant to third generation TKIs.

A Multicenter Phase II Study of Twice-Weekly Bortezomib plus Rituximab in Patients with Relapsed Follicular Lymphoma: Long-Term Follow-Up.

Single-agent bortezomib (B) has shown activity in heavily pretreated patients with relapsed/refractory indolent lymphoma. On the basis of these findings, we performed a phase II study of B combined with rituximab (R) in patients with relapsed follicular lymphoma (FL). Forty-five patients with fairly good prognostic profiles were enrolled from 2007 to 2011 and received a total of 6 cycles of the B+R combination. The endpoints were the overall response rate (ORR), progression-free survival (PFS), duration of remission (DoR), overall survival (OS), and toxicity evaluation. When considering all the enrolled patients the ORR was 64%. At 5 years, the estimated PFS, DoR, and OS were 34, 49, and 70%, respectively. After excluding the 7 R-naïve patients, the ORR was 58%, with a PFS of 19 months. The most common grade >2 toxicities were thrombocytopenia (18%), peripheral neuropathy (13%), and neutropenia (2%). Our study shows the feasibility, long-term efficacy, and excellent tolerability of the B+R combination. We are aware that our study has specific limitations, such as the small sample size consisting of patients with a relatively good prognostic profile. However, because FL patients will be treated with subsequent chemotherapy regimens, a well-tolerated and effective chemotherapy-free therapy could be considered an additional tool for long-term disease control.

F-18 FDG PET/CT quantization parameters as predictors of outcome in patients with diffuse large B-cell lymphoma.

AIM: We evaluated the prognostic significance of standardized uptake value (SUVmax), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) obtained by F-18 FDG PET/CT (PET/CT) in patients with diffuse large B-cell Lymphomas (DLBCL) presenting intermediate IPI score. MATERIAL AND METHODS: Fifty-two patients (61 ± 13 yr) underwent PET/CT before the first-line chemotherapy. The mean SUVmax value, the summed MTV (cm(3) ; 42% threshold), and the cumulative TLG (g) were registered. The patients were followed up 18 months thereafter (range 3-41 months). The PET/CT results were compared to the event-free survival (EFS). RESULTS: At univariate analysis, SUVmax and lactate dehydrogenase (LDH) levels were predictive, but discordantly. The Kaplan-Meier survival analysis for SUVmax showed a significant better EFS in patients presenting higher values as compared to those having lesser (P = 0.0002, HR 0.13). Summed MTV and cumulative TLG were not suitable for predicting EFS. CONCLUSION: Despite the availability of new tools for the quantitative assessment of disease activity on PET/CT, the SUVmax rather than MTV and TLG remains the only predictor for EFS in DLBCL patients. The magnitude of glycolytic activity rather than the amount of metabolically active burden holds a predominant value for determining the response to chemotherapy in DLBCL.

Post-Transplant Cyclophosphamide versus Anti-Thymocyte Globulin in Patients with Hematological Malignancies Treated with Allogeneic Hematopoietic Stem Cell Transplantation from Haploidentical and Matched Unrelated Donors: A Real-Life Experience.

Background: Post-transplant cyclophosphamide (PTCY) is widely used as graft versus host disease (GvHD) prophylaxis in allogeneic hematopoietic stem cell transplant (HSCT) recipients, with reported clinical benefits in patients who underwent transplant from a matched unrelated donor (MUD). However, real-life data on clinical efficacy and safety of PTCY in haploidentical and MUD transplantations are still poor. Methods: In our real-life retrospective observational study, we included a total of 40 consecutive adult patients who underwent haploidentical or MUD HSCT for various hematological malignancies and who received PTCY (n = 24) or ATG (n = 16) as GvHD prophylaxis at Hematology Units from hospitals of Salerno and Avellino, Italy, and clinical outcomes were compared. Results: We showed protective effects of PTCY against disease relapse with the relapse rate after transplantation of 16% versus 50% in the ATG arm (p = 0.02). All-cause mortality was lower (36% vs. 75%; p = 0.02) and the 2-year overall survival was slightly superior in patients administered PTCY (61% vs. 42%; p = 0.26). Conclusions: We support the use of PTCY, even in a real-life setting; however, the optimization of this protocol should be further investigated to better balance relapse prevention and GvHD prophylaxis.

Autoimmune cytopenias in chronic lymphocytic leukemia.

The clinical course of chronic lymphocytic leukemia (CLL) may be complicated at any time by autoimmune phenomena.The most common ones are hematologic disorders, such as autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP). Pure red cell aplasia (PRCA) and autoimmune agranulocytosis (AG) are, indeed, more rarely seen. However, they are probably underestimated due to the possible misleading presence of cytopenias secondary to leukemic bone marrow involvement or to chemotherapy cytotoxicity. The source of autoantibodies is still uncertain, despite the most convincing data are in favor of the involvement of resting normal B-cells. In general, excluding the specific treatment of underlying CLL, the managementof these complications is not different from that of idiopathic autoimmune cytopenias or of those associated to other causes. Among different therapeutic approaches, monoclonal antibody rituximab, given alone or in combination, has shown to be very effective.

Effective Neutralizing Antibody Response Against SARS-CoV-2 Virus and Its Omicron BA.1 Variant in Fully Vaccinated Hematological Patients.

SARS-CoV-2 and its variants cause CoronaVIrus Disease 19 (COVID-19), a pandemic disease. Hematological malignancies increase susceptibility to severe COVID-19 due to immunosuppression. Anti-SARS-CoV-2 neutralizing antibodies protect against severe COVID-19. This retrospective real-life study aimed to evaluate seropositivity and neutralizing antibody rates against SARS-CoV-2 and its Omicron BA.1 variant in hematological patients. A total of 106 patients with different hematologic malignancies, who have mostly received three or more vaccine doses (73%), were included in this study. Serum was collected between May and June 2022. The primary endpoint was anti-SARS-CoV-2 antibody response against ancestral (wild type; wt) and Omicron BA.1 virus, defined as a neutralizing antibody titer ≥ 1:10. Adequate neutralizing antibody response was observed in 75 (71%) and 87 (82%) of patients for wt and Omicron BA.1 variants, respectively.However, patients with B-cell lymphoproliferative disorders and/or those treated with anti-CD20 monoclonal antibodies in the prior 12 months showed a lower seropositivity rate compared to other patients against both Omicron BA.1 variant (73% vs 91%; P = 0.02) and wt virus (64% vs 78%; P = 0.16). Our real-life experience confirmed that full vaccination against SARS-CoV-2 induces adequate neutralizing antibody protection for both the wt virus and Omicron BA.1 variants, even in hematological frail patients. However, protective measures should be maintained in hematological patients, especially those with B-cell lymphoproliferative diseases treated with anti-CD20 monoclonal antibodies, because these subjects could have a reduced neutralizing antibody production.

A shorter time to the first treatment may be predicted by the absolute number of regulatory T-cells in patients with Rai stage 0 chronic lymphocytic leukemia.

Regulatory T-cells (Tregs) are increased in chronic lymphocytic leukemia(CLL) and correlates with clinical and biological features of active/progressive disease. However, little is known about their ability to predict the time to first treatment (TFT). We evaluated 75 patients with Rai stage 0 CLL, in whom the absolute number of Tregs was determined at diagnosis, and correlated to main clinical and biological features, as well as to the need of receiving any specific therapy during the course of the disease. After a median follow-up of 30 months, 12 patients(16%) required therapy at some time from the diagnosis. Treated patients showed a significant higher number of peripheral white blood cells and B-lymphocytes, platelet count, cases with unmutated immunoglobulin heavy chain status, and high-risk cytogenetic abnormalities,as well as lower hemoglobin values, than patients who did not need therapy. A greater number of circulating Tregs was detected in treated patients (P < 0.001). Multivariate analysis confirmed that the absolute number of Tregs was an independent predictor of TFT in these patients, the best predictive cut-off being 41/mL. These data show that the absolute Tregs cell number is able to identify Rai stage 0 CLL patients at higher risk of requiring therapy.

Efficacy of Decitabine and Venetoclax as Salvage and Bridge Therapy to Haploidentical Hematopoietic Stem Cell Transplantation in a Multiresistant Acute Myeloid Leukemia Patient.

Treatment of relapsed/refractory or elderly unfit acute myeloid leukemia (AML) is still challenging, and hypomethylating agents in combination with venetoclax, an oral selective BCL2 inhibitor, might be successfully used as salvage therapy. However, clinical trials evaluating the efficacy and safety of this combination in the setting of multiresistant AML treatment also as a bridge to transplant are still ongoing. Here, we reported a 50-year-old male diagnosed with AML with normal cytogenetics and wild type for fms-like tyrosine kinase 3, nucleophosmin 1, and KIT, and treated with decitabine and venetoclax as the fifth line of therapy and after a relapse post-allogeneic transplant. The patient achieved a complete remission and successfully underwent a haploidentical transplant with an overall survival of 48.6 months.

Serum Free Light-Chain Ratio at Diagnosis Is Associated with Early Renal Damage in Multiple Myeloma: A Case Series Real-World Study.

The serum free light-chain (FLC) ratio is a sensitive tool for the differential diagnosis of plasma cell disorders and is biomarker of multiple myeloma (MM) progression from premalignant conditions. Here, we investigate the potential role of FLC ratio at diagnosis in identifying early renal damage in MM patients and other correlations with clinical, laboratory, and molecular findings. A total of 34 MM patients who had undergone autologous stem cell transplantation were included in this retrospective case series study, and FLC quantification was performed with nephelometric assays. In our study, sFLC ratio was significantly associated with light-chain MM and ß-2 microglobulin levels, likely indicating a high disease burden at diagnosis, especially in patients without heavy chain M-protein at serum electrophoresis. Moreover, the sFLC ratio was inversely correlated with glomerular filtration rate, possibly identifying early renal damage in MM patients. Our preliminary results confirm the importance of early sFLC evaluation, especially in patients with the light-chain MM type and low disease burden, to minimize the risk of late renal failure.

Inter-intra instrument comparison and standardization of a 10-color immunophenotyping for B and T cell non-Hodgkin lymphoma diagnosis and monitoring.

Harmonization of flow cytometry protocols from instrument settings to antibody panel and reagents is highly encouraged for inter-laboratory data comparison in both research and clinical settings, especially for minimal residual disease monitoring evaluation in hematological diseases across centers. Here, we described inter-intra instrument comparison of two standardized 10-color staining dried tubes for B- and T-cell lymphoproliferative disorder diagnosis and monitoring on two different flow cytometers, a Beckman Coulter NaviosEx and a Beckman Coulter DxFlex. A total of 47 consecutive patients were enrolled, and 39 of them were evaluable for further studies. We show highly comparable results between the two cytometers for cell frequency and fluorescence intensity signals for both standardized 10-color staining dried tubes. For this latter, fluorescence of each antibody and subject was normalized on the mean value obtained from the entire study cohort thus reducing the effects of biological variability and allowing comparison between instruments with different detector sensitivity. In summary, dried tubes were confirmed as an optimal standardized diagnostic tool, especially when associated with EuroFlow standardized procedures by minimizing technical and biological variability. However, data analysis is still operator-dependent, and more efforts are needed to develop automated or semi-automated software for flow cytometry data analysis for diagnostic purposes.

WT1 Expression Levels Combined with Flow Cytometry Blast Counts for Risk Stratification of Acute Myeloid Leukemia and Myelodysplastic Syndromes.

Wilm's tumor 1 (WT1), a zinc-finger transcription factor and an epigenetic modifier, is frequently overexpressed in several hematologic disorders and solid tumors, and it has been proposed as diagnostic and prognostic marker of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, the exact role of WT1 in leukemogenesis and disease progression remains unclear. In this real-world evidence retrospective study, we investigated prognostic role of WT1-mRNA expression levels in AML and MDS patients and correlations with complete blood counts, flow cytometry counts, and molecular features. A total of 71 patients (AML, n = 46; and MDS, n = 25) were included in this study, and WT1 levels were assessed at diagnosis, during treatment and follow-up. We showed that WT1 expression levels were inversely correlated with normal hemopoiesis in both AML and MDS, and positively associated with blast counts. Flow cytometry was more sensitive and specific in distinguishing normal myeloid cells from neoplastic counterpart even just using linear parameters and CD45 expression. Moreover, we showed that a simple integrated approach combining blast counts by flow cytometry, FLT3 mutational status, and WT1 expression levels might be a useful tool for a better prognostic definition in both AML and MDS patients.

Real-world evidence of cytomegalovirus reactivation in non-Hodgkin lymphomas treated with bendamustine-containing regimens.

Cytomegalovirus (CMV) reactivation during chemotherapy or after organ or hematopoietic stem cell transplantation is a major cause of morbidity and mortality, and the risk of reactivation increases with patients' age. Bendamustine, an alkylating agent currently used for treatment of indolent and aggressive non-Hodgkin lymphomas, can augment the risk of secondary infections including CMV reactivation. In this real-world study, we described an increased incidence of CMV reactivation in older adults (age >60 years old) with newly diagnosed and relapsed/refractory indolent and aggressive diseases treated with bendamustine-containing regimens. In particular, patients who received bendamustine plus rituximab and dexamethasone were at higher risk of CMV reactivation, especially when administered as first-line therapy and after the third course of bendamustine. In addition, patients with CMV reactivation showed a significant depression of circulating CD4+ T cell count and anti-CMV IgG levels during active infection, suggesting an impairment of immune system functions which are not able to properly face viral reactivation. Therefore, a close and early monitoring of clinical and laboratory findings might improve clinical management and outcome of non-Hodgkin lymphoma patients by preventing the development of CMV disease in a subgroup of subjects treated with bendamustine more susceptible to viral reactivation.

Prophylactic letermovir decreases cytomegalovirus reactivation after stem cell transplantation: a single-center real-world evidence study.

Cytomegalovirus (CMV) reactivation is a major cause of morbidity and mortality after organ or hematopoietic stem cell transplantation (HSCT). Letermovir (LTV) is a novel antiviral agent approved for CMV prophylaxis after allogeneic transplantation. In this single-center real-world study, we evidenced the efficacy and safety of LTV for CMV prophylaxis in allogeneic HSCT recipients. A total of 133 consecutive patients who underwent autologous or allogeneic HSCT were included in the study, and a subgroup of 13 allogeneic HSCT recipients received CMV prophylaxis with LTV 240 mg/daily from day +7 to +100 (allo-LTV cohort). All patients in the allo-LTV cohort were at moderate or high risk of reactivation based on donor/recipient serology status, and 62% also received haploidentical HSCT and cyclophosphamide which further increased the CMV reactivation risk. CMV infection rate was also compared to that observed in allogeneic HSCT patients without CMV prophylaxis and autologous recipients who have the lowest reported CMV infection incidence and were used as a control cohort. In our experience, patients receiving LTV showed a significant decline in CMV reactivation incidence to similar rates described in autologous HSCT recipients (7.7% of allogeneic LTV-treated vs 68% of allogeneic recipients without prophylaxis vs 15% of autologous patients; p> 0.0001). The only patient in the allo-LTV cohort with CMV reactivation was a 25-year-old female with a diagnosis of very high-risk acute lymphoblastic leukemia who received a haploidentical HSCT after ex vivo T cell depletion. CMV reactivation occurred beyond LTV course, at +187 days from transplantation. In addition, we confirmed efficacy and safety of valganciclovir 450 mg/daily as pre-emptive therapy or for treatment of CMV disease in allogeneic and autologous HSCT recipients who experienced CMV reactivation even after LTV prophylaxis. However, further clinical trials in larger populations and longer follow-up are required to confirm our preliminary results.

Peg-filgrastim versus filgrastim after autologous stem cell tranplantation: case-control study in patients with multiple myeloma and review of the literature.

We investigated the effects of a single s.c. injection of peg-filgrastim in 32 patients with multiple myeloma who underwent autologous stem cell transplantation (AuSCT) as first line treatment. For comparison, 32 myeloma patients with similar characteristics and receiving standard daily administration of filgrastim were matched. Overall, there were no statistically significant differences between peg-filgrastim and filgrastim in terms of tolerability, marrow recovery, severity of neutropenia, incidence and duration of febrile neutropenia, documented infections and transfusions. However, some favourable trends or effects in favour of peg-filgrastim were observed. This was confirmed by a review of the published papers about this topic.

Lipegfilgrastim in the management of chemotherapy-induced neutropenia of cancer patients.

Neutropenia and febrile neutropenia (FN) are frequent and potentially fatal toxicities of myelosuppressive anticancer treatments. The introduction of granulocyte colony-stimulating factors (G-CSFs) in clinical practice has remarkably reduced the duration and severity of neutropenia, as well as the incidence of FN, thus allowing the administration of chemotherapeutic agents at the optimal dose and time with lower risk. The current scenario of G-CSFs in Europe includes filgrastim, lenograstim, some G-CSF biosimilars, and pegfilgrastim. Recently, a novel long-acting G-CSF, lipegfilgrastim, became available. Lipegfilgrastim is a glycopegylated G-CSF, alternative to pegfilgrastim, and has shown in randomized trials, to be equivalent to pegfilgrastim in reducing the incidence of severe neutropenia and FN in patients with breast cancer receiving chemotherapy, with a similar safety profile. Furthermore, lipegfilgrastim was more effective than the placebo in reducing the incidence of severe neutropenia, its duration, and time to absolute neutrophil count recovery, in patients with non-small cell lung cancer receiving myelosuppressive therapy. Although the number of studies currently published is still limited, lipegfilgrastim seems to be a promising drug in the management of chemotherapy-induced neutropenia.