Abstract: 〈 b 〉 〈 i 〉 Introduction: 〈 /i 〉 〈 /b 〉 Interferon regulatory factor 4 (IRF4) is a transcriptional factor with a key role in the modulation of inflammation and immune surveillance. The 〈 i 〉 IRF4 〈 /i 〉 gene is downregulated in Philadelphia-negative myeloproliferative neoplasms, and its expression is associated with prognosis and response to treatment. 〈 b 〉 〈 i 〉 Methods: 〈 /i 〉 〈 /b 〉 We evaluated the 〈 i 〉 IRF4 〈 /i 〉 expression kinetics during tyrosine kinase inhibitor (TKI) treatment in a cohort of 116 chronic myeloid leukemia (CML) patients to elucidate its role in the disease course. 〈 b 〉 〈 i 〉 Results: 〈 /i 〉 〈 /b 〉 A relationship between the 〈 i 〉 IRF4 〈 /i 〉 expression and the disease burden was observed at various disease stages. A correlation analysis between the International Scale (IS) and 〈 i 〉 IRF4 〈 /i 〉 values confirmed this close association. A significant increase is detected after 3 months of TKI treatment. Patients achieving an early molecular response (EMR) had higher 〈 i 〉 IRF4 〈 /i 〉 values at both diagnosis and after 3 months of therapy as compared to those failing the EMR target. Patients achieving treatment-free remission did not show 〈 i 〉 IRF4 〈 /i 〉 fluctuations during monitoring, while a decreased 〈 i 〉 IRF4 〈 /i 〉 expression emerged at the time of molecular relapse. 〈 b 〉 〈 i 〉 Conclusion: 〈 /i 〉 〈 /b 〉 Our data seem to confirm the relevance of 〈 i 〉 IRF4 〈 /i 〉 in the pathogenesis of CML 〈 i 〉 , 〈 /i 〉 suggesting a pivotal role at the disease onset and a predictive value during the CML course.

Abstract: Erythrocytosis is a clinical condition characterized by increased red cell mass, hemoglobin, and hematocrit values. A significant fraction of patients is described as having idiopathic erythrocytosis. We have previously demonstrated an association between erythrocytosis and the JAK2 GGCC_46/1 haplotype and CALR rs1049481_G allele. In the present study, we investigated genomic and clinical features of 80 erythrocytosis patients with the aim to provide useful information in clinical practice. Patients with idiopathic erythrocytosis could have a genomic germline background, eventually associated with somatic variants. Through association analysis, we show that male patients presenting with idiopathic erythrocytosis, and normal EPO levels could be the best candidates for the search for the JAK2 GGCC_46/1 haplotype and CALR rs1049481_G allele. Further studies are needed to confirm these findings and to depict detailed genomic and phenotypical characteristics of these patients.

Abstract: Patients with hematological malignancies (HM) undergoing hematopoietic stem cell transplantation (HSCT) have an increased vulnerability to SARS-Cov-2 (Sharma et al, Lancet Haematology 2020; Ljungman et al, Leukemia 2021), the reason why international guidelines strongly support the need for a protective vaccination for these subjects. The most relevant data currently available on the response to a complete anti-SARS-Cov-2 vaccination cycle in HM patients after HSCT refer to 314 patients reported in a Lithuanian national survey (Maneikis et al, Lancet Haematol 2021). In this study, the median titers of antibodies against SARS-Cov-2, determined 7-21 days after the second vaccination, were comparable to that of healthy controls (HC) in both autologous and allogeneic groups, with no patient found below the protective threshold of 50 arbitrary units (AU)/ml. Notably, the large majority of patients had received the transplant more than 1 year before vaccination. In a prospective, cohort study, we compared 114 patients, who had received an autologous or allogeneic HSCT at least three months before the first dose of vaccination, to 107 HC, matched for age and sex. Study population and HC received two doses of BNT162b2 anti-SARS-Cov-2 mRNA vaccine on days 1 and 21, between April and May 2021. Serological tests were performed by a commercially available immunoassay for the quantitative determination of anti-spike IgG antibodies to SARS-Cov-2. The cut-off for defining responders was 50 or greater AU/ml. Patients and HC samples were collected four weeks after the second dose of the vaccine. Table 1 reports the main clinical characteristics of patients and HC. Eighteen of 114 patients (16%) did not respond (24% in the allogeneic group, 6% in autologous recipients). Overall, median antibodies titers did not differ between HC and the entire cohort of transplanted patients, recipients of allogeneic HSCT, all patients responding to the vaccine or responders in the autologous subgroup (Figure 1A). All autologous HSCT recipients had significantly lower titers of antibodies than HC, while higher levels were found in responders who had received allogeneic HSCT (Figure 1A). Responders in the allogeneic subgroup showed antibodies titers significantly higher than responders in the autologous subgroup (Figure 1B). We further stratified patients in three groups, according to the time elapsed from transplant to vaccination: G1: & lt;1 year; G2:1-5 years; G3: & gt;5 years. Higher antibodies titers were observed in HC compared to all transplanted patients in G1 (Figure 1C), including both allogeneic (Figure 1D) and autologous (Figure 1E) HSCT recipients. No differences emerged in G2 between HC and all patients (Figure 1C), allogeneic (Figure 1D) or autologous (Figure 1E) HSCT recipients. Finally, no differences were found in G3 when comparing HC with all patients (Figure 1C) or allogeneic recipients (Figure 1D), whereas patients in the autologous subgroup showed significantly lower titers than HC (Figure 1E). Myeloma patients with controlled disease showed higher titers than patients with active disease (Figure 1F). According to median age, autologous HSCT recipients older than 57 years had significantly lower antibody levels than younger patients (Figure 1G). Autologous vs allogeneic HSCT, age of all patients and of allogeneic HSCT recipients, sex, type of allogeneic HSCT, conditioning regimen, age and sex of donor, occurrence of GVHD, disease type and single vs double autologous HSCT did not significantly impact on antibody levels (data not shown). No relevant side effects were recorded after vaccination. With a median follow up of 12 weeks, no case of COVID19 occurred among vaccinated patients. In our single center study, patients with a previous history of HSCT tolerated well BNT162b2 vaccine and mounted a potentially protective immune response in the majority of cases one month after two doses of vaccine. However, lack of response was not rare, especially in the allogeneic setting. The main factor associated with the quality of response was the time from HSCT, with lower responses within the first year from transplant and differences between autologous and allogeneic groups transplanted more than five years before vaccination. Here, a consolidated, complete immune reconstitution in allogeneic HSCT recipients, as well as age and a still active disease in the autologous setting, could have played opposite pivotal roles. Figure 1 Figure 1. Disclosures Delia: Gilead: Consultancy; Amgen: Consultancy; abbvie: Consultancy; Jazz pharmaceuticals: Consultancy.

Abstract: Abstract 2294 Background. The TKIs Nilotinib and Dasatinib offer additional therapeutic options for patients with CML who are resistant or intolerant to Imatinib. These agents, active against the majority of Imatinib resistant BCR-ABL mutated clones, have a different pattern of kinase target selectivity, pharmacokinetics parameters, cell uptake, efflux properties and adverse events profiles. Preliminary results suggest that some patients may respond to a second TKI used as third line therapy, but little is known about the long term benefit of such an approach.Aim of this collaborative Italian study was to verify the response (rate and duration) and the clinical outcome in patients with CML treated with a third TKI after sequential failure of the previous ones. Methods. We evaluated 66 patients with CML, resistant/intolerant to Imatinib and treated with Dasatinib or Nilotinib, then switched to a third- line TKI after treatment failure. Of these, 29 patients were treated with dasatinib after imatinib/nilotinib failure and 37 with nilotinib after imatinib/dasatinib failure. Patients were monitored with complete blood counts, cytogenetic analysis, bone marrow aspiration RT-PCR and mutational analysis. Results. A total of 66 patients (median age 63 years, range, 33–85 years) were treated with sequential TKIs; 40 (61%) patients had received interferon-a before starting Imatinib; 26 (39%) patients received imatinib as first line therapy. The median time on imatinib therapy was 47.5 months (range 4–101 months). At the start of nilotinib as second line, 27/29 (93%) patients were in CP, 1 (3.5%) in AP, and 1 (3.5%) in BP. 9 patients (31%) had developed mutations before starting treatment. The median time on second line TKI was 8 months (range 2–36 months). In the resistant patients 4 new mutations were identified (F359V in two patients, T315I, Y253H+F359V). At the start of dasatinib as second line, 33/37 (89.2%) patients were in CP, 4 (10.8%) in AP. 7 patients (18.9%) had developed mutations before starting treatment. The median time on second line TKI was 14 months (range 4–59 months).In the resistant patients 5 new mutations were identified (F137L in three pts, M318T, M244V+F317L). At the start of the third TKI, 60/66 (90.9%) patients were in CP, 5 (7.6%) in AP, and 1 (1.5%) in BP. Of these, 7 patients (18.9%) on dasatinib and 7 (24.1%) on nilotinib had mutations before starting treatment. The best response to the third line treatment with TKI was 10 (15.2%) MMR, 10 (15.2%) CCyR, 8 PcyR (12.1%), 5 (7.5%) mCyR, 24 (36.4%) CHR and 9 (13.6%) No Response (NR). In the dasatinib group, 9 (31%) patients discontinued treatment because of toxicity versus 17 (45.9%) patients in the nilotinib group.Two new mutations (F317L, E255V) emerged with dasatinib as third line therapy.After a median follow up of 13 months (range 2–37 months) 50 patients (48 CP, 2 AP) are continuing therapy (33 on nilotinib, 17 on dasatinib).Since the start of the third TKI, 61 patients (92.4%) are still alive for a median overall survival of 110 months (range 15–300) (52 CP, 7 AP, 2 NA); the 5 deaths (7.6%) were caused by disease progression and spread of the gene mutation T315I. Discussion. In our study, about one third of patients derived benefit from the use of three sequential TKIs; patients with better, longer response (28.7%) to third TKI were the same patients with a better response to the Imatinib and 2TKIs therapy. All these patients had taken interferon therapy before the Imatinib. In this subset of patients (good responders: CCyR and MMR) 5 patients developed mutations that were sensitive to the sequential treatment.The lack of a durable cytogenetic remission could be explained by the emergence of new kinase domain mutations as patients are exposed to sequential TKI; a change of therapy resulted in an adequate response. In our series, patients with poor prognosis showed mutations not sensitive to the TKIs treatment. Conclusions. Although allogeneic SCT is the treatment of choice in all patients failing 2 TKIs who are suitable candidates for this approach, alternative strategies are required for ineligible patients. The use of a third TKI after failure of two previous TKIs induces response in some patients. Longer follow up of a larger series of patients is needed to determine the long term impact of the response. Disclosures: No relevant conflicts of interest to declare.

Abstract: Introduction Tyrosine kinase inhibitors (TKIs) have dramatically changed the outcome of chronic phase chronic myeloid leukemia patients (CP-CML),improving the long-term outcome; indeed, life expectancy is now very close to that of age matched individuals in the general population. Imatinib (IMA) the first generation TKI, increased the overall survival(OS)by more than 80%. Second-generation TKIs, (2gen TKIs) used in first line dasatinib and nilotinib, induced faster molecular responses, rapidly reducing the disease burden, but did not change the OS of CP-CML newly diagnosed patients. Most of the available data reported were extrapolated from sponsored clinical trials. The aim of this analysis is to detail and analyze the prognostic features influencing the OS in a large Italian CML cohort of patients prospectively enrolled in the GIMEMA CML Italian study. Methods Relevant clinical, demographic, biological and treatment-related information were web-based collected during a multicenter,observational Italian study that enrolled consecutive patients in each disease phase, at 68 Italian hematologic centers belonging to the GIMEMA network from January 2013 to June 2020. We analyzed prognostic factors influencing the OS by Kaplan Meier method and Cox multivariable models. Results A cohort of 1206 patients was prospectively analyzed, 608 of them received frontline IMA and 598 2genTKIs. Median age in the IMA cohort was 69 years (range 58-77) vs 52 years in the 2genTKIs cohort (range 41-63). The male/female ratio was 1.7 in the IMA group and 1.35 in the 2genTKIs cohort. Ninety-eight percent of patients were in CP. Results of molecular analysis of the BCR-ABL transcript at baseline showed: b2a2 in 33.1 % of patients and b3a2 in 59.9%, while an atypical transcript was found in 7%. The cytogenetic analysis at baseline showed major and minor additional aberrations in 5.7% and 1.6% of patients respectively. In the IMA cohort,according to the Sokal score, 27.7%, 57.3% and 15% of patients were stratified as low, intermediate and high risk, whereas according to the ELTS score 51.3%, 35.5% and 13.3%, of patients were classified as low, intermediate and high risk. In the 2genTKIs cohort, according to the Sokal score, 44.8%, 34.5% and 20.8%, were low, intermediate and high risk, respectively, whereas according to the ELTS score, 66.9%, 22% and 11% were assigned to the respective risk groups.The Charlson comorbidity index in the IMA cohort was 2-3 and 4-5 in 74% and 26% of patients respectively; in the 2genTKIs cohort the score was 2-3 in 89% and 4-5 only in 10% of patients. Overall, median follow-up of the whole population was 24.7 months (range 13.3-39.3).Seventy-three patients (6.1%) in the overall population died, the majority of them in the IMA cohort: 56 patients (9.2%), at median age of 80.5 years,11/608 (1.8%) due to CML-related causes. Conversely,in the 2genTKIs cohort only 17 patients (2.8%) died, at a median age of 62 years, 10/598 (1.7%) for CML-related causes. Estimated 60-months OS of the overall population was 86.4% (95% CI 81.3-90.2): 75.8% (95% CI 64.5-84) in the IMA cohort and 93.8% (95% CI 87.5-97) in the 2genTKIs group (p & lt;0.0001). The ELTS score provides a better stratification of 60 months OS both in the IMA (OS 60-months 91.4%, 65.2%, 48.7% in low, intermediate and high risk, p & lt;0.0001) (Fig.1a) and the 2genTKIs subgroups (OS 60-months OS 95.4%, 92%, 87.9% in low, intermediate and high risk, p=0.0013)(Fig.1b). An adjusted Cox model on the entire population showed that prognostic factors influencing OS are: ELTS score (high risk vs low HR= 5.2, 95%CI 2.7-10.03, p & lt;0.0001),the type of TKI (2genTKIs vs IMA HR= 0.46; 95% CI 0.24-0.87, p=0.018), age (HR=1.03 per year, 95%CI 1.00-1.05, p=0.025) and the Charlson index (4-5 vs 2-3, HR= 1.75, 95%CI 1.43-2.1, p & lt;0.0001). Conclusions In this first analysis of our study different clinical behaviors were observed among Italian hematologists, who prevalently prescribed IMA to older patients,with more comorbidities, as compared to 2genTKIs.These differences explain a better OS for patients treated with 2genTKIs vs IMA, however, the risk of death for CML related causes is quite similar between the two groups, all the differences being attributable to other causes of death.Prognostic baseline features associated to an increased OS confirmed that, in addition to age, the ELTS score and the comorbidities are the main clinical factors that independently influence the long-term OS Disclosures Pregno: Incyte-Italy,: Membership on an entity's Board of Directors or advisory committees, Other: conference reports; Novartis-Italy: Membership on an entity's Board of Directors or advisory committees, Other: conference reports; Pfizer-Italy: Membership on an entity's Board of Directors or advisory committees, Other: conference reports. Breccia:Bristol-Myers Squibb/Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Abbvie: Consultancy. Castagnetti:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Galimberti:Novartis: Speakers Bureau; Incyte: Honoraria. Bocchia:CELGENE: Honoraria; Incyte: Honoraria. Abruzzese:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bms: Honoraria; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Levato:Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rosti:Pfizer: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau; Incyte: Speakers Bureau. Di Raimondo:GILEAD, Incyte: Research Funding; Amgen, Takeda, Novartis: Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; GSK: Consultancy, Honoraria. Pane:Janssen: Other: Travel Expenses; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis pharma SAS: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Other: travel expenses, Speakers Bureau; Daiichi Sankyo: Consultancy, Other: Travel Expenses; Amgen: Consultancy, Other: Travel Expenses, Speakers Bureau; AbbVie: Consultancy, Other: Travel Expenses, Speakers Bureau. Foà:Incyte: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Saglio:Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Incyte: Research Funding; Roche: Research Funding; Novartis: Research Funding; Ariad: Research Funding.

Abstract: Optical genome mapping (OGM) is a new genome-wide technology that can reveal both structural genomic variations (SVs) and copy number variations (CNVs) in a single assay. OGM was initially employed to perform genome assembly and genome research, but it is now more widely used to study chromosome aberrations in genetic disorders and in human cancer. One of the most useful OGM applications is in hematological malignancies, where chromosomal rearrangements are frequent and conventional cytogenetic analysis alone is insufficient, necessitating further confirmation using ancillary techniques such as fluorescence in situ hybridization, chromosomal microarrays, or multiple ligation-dependent probe amplification. The first studies tested OGM efficiency and sensitivity for SV and CNV detection, comparing heterogeneous groups of lymphoid and myeloid hematological sample data with those obtained using standard cytogenetic diagnostic tests. Most of the work based on this innovative technology was focused on myelodysplastic syndromes (MDSs), acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL), whereas little attention was paid to chronic lymphocytic leukemia (CLL) or multiple myeloma (MM), and none was paid to lymphomas. The studies showed that OGM can now be considered as a highly reliable method, concordant with standard cytogenetic techniques but able to detect novel clinically significant SVs, thus allowing better patient classification, prognostic stratification, and therapeutic choices in hematological malignancies.

Abstract: The connection between autoimmune disease (AID) and lymphoproliferative disorders is a complex bidirectional relationship that has long been a focus of attention by researchers and physicians. Although advances in pathobiology knowledge have ascertained an AID role in the development of lymphoproliferative diseases developing, results about AID influence on the prognosis of lymphoma are discordant. In this review, we collect the most relevant literature debating a direct or indirect link between immune-mediated diseases and lymphoma prognosis. We also consider the molecular, genetic, and microenvironmental factors involved in the pathobiology of these diseases in order to gain a deeper understanding of the nature of this link.

Abstract: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is a rare, distinct subtype of T-ALL characterized by genomic instability, a dismal prognosis and refractoriness to standard chemotherapy. Since its first description in 2009, the expanding knowledge of its intricate biology has led to the definition of a stem cell leukemia with a combined lymphoid-myeloid potential: the perfect trick. Several studies in the last decade aimed to better characterize this new disease, but it was recognized as a distinct entity only in 2016. We review current insights into the biology of ETP-ALL and discuss the pathogenesis, genomic features and their impact on the clinical course in the precision medicine era today.

Abstract: Smoldering multiple myeloma (SMM) is an asymptomatic disorder of clonal bone marrow (BM) plasma cells (PCs) in between the premalignant condition known as monoclonal gammopathy of undetermined significance and overt multiple myeloma (MM). It is characterized by a deep biological heterogeneity that is reflected in a markedly variable progression risk among patients. Recently proposed risk stratification models mainly rely on indirect markers of disease burden and are unable to identify cases in whom clonal PCs have already undergone the “malignant switch” but major clonal expansion has not occurred yet. In the last years, the application of next-generation sequencing (NGS) techniques has led to profound advances in the understanding of the molecular bases of SMM progression, and in all likelihood, it will contribute to the needed improvement of SMM prognostication. In this Review, we describe the recent advances in characterizing the genomic landscape of SMM and intrinsic determinants of its progression, highlighting their implications in terms of understanding of tumor evolution and prognostication. We also review the main studies investigating the role of the microenvironment in this early disease stage. Finally, we mention the results of the first randomized clinical trials and discuss the potential clinical translability of the genomic insights.