Non-invasive prenatal test identifies circulating cell-free DNA chromosomal abnormalities derived from clonal hematopoiesis in aggressive hematological malignancies.

Liquid biopsy is a minimally invasive diagnostic tool for identification of tumor-related mutations in circulating cell-free DNA (cfDNA). The aim of this study was to investigate feasibility, sensitivity, and specificity of non-invasive prenatal test (NIPT) for identification of chromosomal abnormalities in cfDNA from a total of 77 consecutive patients with non-Hodgkin B-cell lymphomas, Hodgkin lymphoma (HL), or plasma cell dyscrasia. In this case series, half of patients had at least one alteration, more frequently in chromosome 6 (23.1%), chromosome 9 (20.5%), and chromosomes 3 and 18 (16.7%), with losses of chromosome 6 and gains of chromosome 7 negatively impacting on overall survival (OS), with a 5-year OS of 26.9% and a median OS of 14.6 months, respectively (P = 0.0009 and P = 0.0004). Moreover, B-cell lymphomas had the highest NIPT positivity, especially those with aggressive lymphomas, while patients with plasma cell dyscrasia with extramedullary disease had a higher NIPT positivity compared to conventional cytogenetics analysis and a worse outcome. Therefore, we proposed a NIPT-based liquid biopsy a complementary minimally invasive tool for chromosomal abnormality detection in hematological malignancies. However, prospective studies on larger cohorts are needed to validate clinical utility of NIPT-based liquid biopsy in routinely clinical practice.

Real-life study on the use of response adapted therapy in patients with Hodgkin Lymphoma: Results from a multicenter experience.

Few data are known regarding the use of interim positron emission tomography (iPET) after the first two cycles (iPET2) of chemotherapy in treatment-naïve classical Hodgkin lymphoma (cHL) in routine clinical practice, and about the real-life adoption of intensification strategies for iPET positive patients. We conducted a multicenter retrospective study on cHL to investigate the use of iPET in the real-life setting, its prognostic role and outcomes of patients early shifted to intensification. Six hundreds and forty-one patients were enrolled (62% had advanced stage). iPET2 was positive in 89 patients (14%) including 8.7% and 17% early and advanced stage patients, respectively (p = 0.003). Among iPET 2 positive cases treatment was immediately modified in 19 cases; in 14 cases treatment was modified after an additional positive iPET4. Overall 56 iPET2 positive patients never received intensified therapies. Most frequently used intensified therapy was autologous stem cell transplantation followed by BEACOPP. After a median follow-up of 72 months, the 5-year progression-free survival (PFS) was 82% with iPET2 positive patients showing a worse PFS compared with iPET2 negative cases: 31% versus 85%. Focusing on advanced stage patients with a positive iPET2, the 5-year PFS was 59% for patients shifted to intensified therapy at any time point versus 61% for patients who never received intensified therapy. Our study confirmed the higher curability of naïve cHL patients in a real-world setting, and the prognostic role of iPET2 in this setting. A poor adherence to response-adapted strategy which however did not translate into a difference in patient outcomes.

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.

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.

Hodgkin Lymphoma and Hairy Cell Leukemia Arising from Chronic Lymphocytic Leukemia: Case Reports and Literature Review.

Richter's syndrome represents the progression of chronic lymphocytic leukemia (CLL) to more aggressive diseases, most frequently diffuse large B-cell lymphoma, while Hodgkin's lymphoma (HL) and hairy cell leukemia (HCL) are rarely described. The first case involved a 67-year-old man with a diagnosis of a high-risk stage-II CLL treated with rituximab and ibrutinib, developed a HL nodular sclerosis variant after three months of therapy for CLL. After achieving a complete remission for HL and ibrutinib cessation because of drug-related cardiotoxicity, the patient relapsed after five months off-therapy and died due to disease progression after two cycles of brentuximab-vedotin. The second case involved an 83-year-old female with a diagnosis of stage-IV CLL treated with rituximab plus bendamustine who developed a HCL eight years later. Pentostatin was unsuccessfully employed as upfront HCL therapy, and the patient was then switched to rituximab while in remission for CLL. In conclusion, Richter's transformation risk rate might be higher in patients treated with novel targeted therapies, and multiparametric flow cytometry and lymph node biopsy at relapse could help in early identifying small clones. The treatment of predominant neoplasia is mandatory, and disease-specific drugs are administered; however, clinical efficacy might be lower in these patients.

Implementation of International Prognostic Index with flow cytometry immunophenotyping for better risk stratification of chronic lymphocytic leukemia.

BACKGROUND: Current chronic lymphocytic leukemia (CLL) International Prognostic Index (IPI) stratifies patients based on clinical, molecular, and biochemical features; however, B-cell markers also influence CLL outcomes. Here, prognostic roles of CD11c, CD38, and CD49d were first evaluated, and then an immunophenotypic score was combined with CLL-IPI for risk stratification of CLL patients. METHODS: A total of 171 CLL subjects were included, and surface marker expression was assessed by flow cytometry. Levels ≥30% were chosen as cut-off of positivity to a marker; then values of 1 (for CD11c and CD38) or 3 (for CD49d) were assigned and scores determined for each patient's clone immunophenotype. RESULTS: CD49d positivity was significantly associated with simultaneous expression of CD11c and/or CD38, unmutated IGHV status, and higher ß2-microglobulin levels compared to those with CD49d negativity. Moreover, CD49d+ patients experienced a shorter progression-free survival and time to treatment. When the immunophenotypic score was combined with CLL-IPI, patients with high-risk immunophenotype had a significantly lower time-to-treatment regardless CLL-IPI. CONCLUSIONS: Our results suggested clinical utility of an integrated prognostic score for better risk stratification of CLL patients. These results require further validation in prospective larger studies.

Kaposi's sarcoma associated with chronic myeloid leukemia and imatinib mesylate therapy.

Kaposi's sarcoma is associated with immunosuppression and human herpesvirus 8 infection, while rarely described in myeloid malignancies. Here, we illustrate a rare case of chronic myeloid leukemia treated with imatinib, a tyrosine kinase inhibitor, who developed a human herpesvirus 8-related Kaposi's sarcoma.

Real-world experience with the Sydney System on 1458 cases of lymph node fine needle aspiration cytology.

INTRODUCTION: Lymph node (LN) fine needle aspiration cytology (FNAC) is a safe, quick, inexpensive, reliable, and minimally invasive technique for the diagnosis of lymphadenopathies. Recently, an international committee of experts proposed guidelines for the performance, classification, and reporting of LN-FNAC: the Sydney System. We set out to analyse the diagnostic performance of the Sydney System in a retrospective study. METHODS: We retrieved 1458 LN-FNACs, reformulated the diagnoses according to the Sydney System, and compared them to the histological control where available (n = 551, 37.8%). RESULTS: The risk of malignancy for each of the five categories was 66.7% for inadequate/insufficient, 9.38% for benign (overall: 0.84%), 28.6% for atypical, 100% for suspicious and 99.8% for malignant. LN-FNAC showed a sensitivity of 97.94%, a specificity of 96.92%, a positive predictive value of 99.58%, and a negative predictive value of 86.30%. CONCLUSIONS: These data support the usage of LN-FNAC as an agile first-level technique in the diagnosis of lymphadenopathies. The Sydney System supports and enhances this role of LN-FNAC, and its adoption is encouraged. In negative cases, coupled with ancillary techniques, LN-FNAC can reassure the clinician regarding the benignity of a lymphadenopathy and indicate the need for clinical follow-up, which will catch possible false negatives. In positive cases, LN-FNAC can provide sufficient information, including predictive biomarkers, to initiate management and obviate the need for subsequent, more invasive procedures. Given its speed, minimal invasiveness, and low cost, LN-FNAC can be performed in most cases, even when more invasive techniques are not feasible.

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.

Adding hydroxyurea in combination with ruxolitinib improves clinical responses in hyperproliferative forms of myelofibrosis.

Ruxolitinib, an orally bioavailable and selective inhibitor of Janus kinase 1 (JAK1) and JAK2, significantly reduces splenomegaly and disease-related symptoms in patients with myelofibrosis (MF). However, no clear survival benefit has been demonstrated, which may in part reflect suboptimal drug exposure related to lower dosages needed to minimize hematological toxicity, specifically cytopenias. Furthermore, the optimal management of specific conditions such as leukocytosis or thrombocytosis in patients under ruxolitinib therapy is still undefined. In these cases, combining ruxolitinib with a cytoreductive agent like hydroxyurea might improve hematological response. This observational multi-center study enrolled 20 adult patients with intermediate- or high-risk primary MF, post- polycythemia vera MF, or postessential thrombocythemia MF with hyperproliferative manifestations of the disease and WBC and/or platelet counts not controlled by ruxolitinib therapy. The patients received treatment with a combination of ruxolitinib and hydroxyurea. A clinical response of any type was obtained in 8 patients (40%) during ruxolitinib monotherapy and in 17 patients (85%) during ruxolitinib-hydroxyurea combination (P = 0.003). After a median duration of 12.4 months of combination therapy, 16/20 patients had a hematological response; 14/17 patients who had started combination therapy to control WBC count and 2/3 who started in order to reduce platelets count. The number of patients requiring ruxolitinib dosage reduction or discontinuations was lower during combination therapy and, at the end of follow-up the median ruxolitinib dose was increased in 50% of patients. In conclusion, the combination of hydroxyurea with ruxolitinib yielded a high clinical response rate and increased ruxolitinib exposure in patients with hyperproliferative forms of MF.

Prognostic factors in noncirrhotic patients with splanchnic vein thromboses.

OBJECTIVES AND METHODS: Splanchnic vein thrombosis (SVT), not associated with cancer or liver cirrhosis, is a rare event and scanty data are available on its natural history, long-term prognosis, and treatment. In this study 121 SVT patients consecutively seen from January 1998 to December 2005 were included and 95 of them were followed up for a median time of 41 months. Screening for thrombophilic factors was performed in 104 patients. New thrombotic or bleeding episodes were registered and anticoagulant therapy was performed according to preestablished criteria. RESULTS: SVT was an incidental finding in 34 (28.1%) patients; 34 (28.1%) presented with abdominal infarction; 39 (32.2%) had bowel ischemia or acute portal vein thrombosis; 14 (11.6%) had bleeding from portal hypertensive sources. Survival rates at 1, 3, and 7 yr were 95%, 93.3%, and 89.6%, respectively; 87.5% of deaths occurred at onset of SVT as complications of intestinal infarction. Patients with isolated portal vein thromboses had symptoms and intestinal infarction in 16/41 (39%) and 0/41 (0%) of the cases, respectively, whereas superior mesenteric vein thromboses, isolated or not, were associated with symptoms and intestinal infarction in 69/75 (92%) and 34/75 (45%), respectively. During the follow-up 14 (14.7%) suffered from 39 episodes of gastrointestinal bleeding with no deaths. A previous gastrointestinal bleed was associated with new hemorrhagic events during follow-up. New venous thrombotic episodes occurred in 10 of 95 patients (10.5%), of which 73% were in the splanchnic area. Seven out of these 10 patients had a chronic myeloproliferative disease (MPD) and none was on anticoagulation. CONCLUSIONS: Anticoagulant therapy was effective to obtain recanalization of acute SVT in 45.4% of patients and preserved patients from recurrent thrombosis when given lifelong.