Background:Patient-reported outcomes (PROs) are increasingly recognized in hematology, yet clinician awareness remains limited. In immune thrombocytopenia (ITP), an autoimmune disease, symptoms like fatigue impact quality of life (QoL). Design and Methods:This Italian survey evaluated hematologists' knowledge of PROs within the Italian ITP NET network. Between April 4-20, 2023, 63 hematologists from 49 centers were invited to complete a 63-item online questionnaire on demographics, QoL understanding, and attitudes toward clinical use. Results:Thirty-nine responded, treating a median of 50 ITP patients in the prior year. PROs were used in research by 51% and applied clinically by 56%. Greater PRO knowledge was found among hematologists with over 17 years' experience (72%) and extensive ITP exposure (64%) (p=0.033 and p=0.037). Larger centers reported more trial involvement (p=0.035) and regular QoL tool use. Conclusion:Results highlight knowledge gaps and emphasize how clinical experience and exposure support effective PRO implementation in ITP.

This study aimed to define the incidence and risk factors for diffuse large B cell lymphoma variant of RT (DLBCL-RT) in 976 patients with CLL who received ibrutinib therapy. DLBCL-RT was recorded in 83 (8.5%) patients, with a 7-year 15.6% rate. Most patients exhibited clinical signs of aggressive lymphoma, enlarged lymph nodes in 83%, cytopenia in 60%, and Suvmax values ≥ 5 at CT/PET in 98%. Among patients for whom the data was available, 83% had unmutated IGHV, 60% TP53 disruption, 26% mutated NOTCH1, 10% were categorized in subset #8 and 82% had a clonally-related lymphoma. Response to chemoimmunotherapy was achieved by 32% of patients. Median OS was 4.7 months, with cytopenia at DLBCL-RT diagnosis being the only significant factor for inferior survival (HR, 1.68). In multivariable analysis, factors predictive for increased risk of DLBCL-RT were age <70 years (HR: 1.98, p = 0.019), TP53 disruption (HR: 1.72, p = 0.044), with a trend to significance for prior treatment (HR: 1.91, p = 0.065). According to the number of these risk factors, DLBCL-RT rate varied from 4% to 22.6% (p < 0.0001). In conclusion, patients with CLL receiving ibrutinib with age <70 years, TP53 disruption and previously treated are at increased risk for developing DLBCL-RT and deserve close monitoring.

Chronic lymphocytic leukaemia (CLL) is characterised by the expansion of a neoplastic mature B cell clone. CLL clinical outcome is very heterogeneous, with some subjects never requiring therapy and some showing an aggressive disease. Genetic and epigenetic alterations and pro-inflammatory microenvironment influence CLL progression and prognosis. The involvement of immune-mediated mechanisms in CLL control needs to be investigated. We analyse the activation profile of innate and adaptive cytotoxic immune effectors in a cohort of 26 CLL patients with stable disease, as key elements for immune-mediated control of cancer progression. We observed an increase in CD54 expression and interferon (IFN)-γ production by cytotoxic T cells (CTL). CTL ability to recognise tumour-targets depends on human leukocyte antigens (HLA)-class I expression. We observed a decreased expression of HLA-A and HLA-BC on B cells of CLL subjects, associated with a significant reduction in intracellular calnexin that is relevant for HLA surface expression. Natural killer (NK) cells and CTL from CLL subjects show an increased expression of the activating receptor KIR2DS2 and a reduction of 3DL1 and NKG2A inhibiting molecules. Therefore, an activation profile characterises CTL and NK cells of CLL subjects with stable disease. This profile is conceivable with the functional involvement of cytotoxic effectors in CLL control.

The peer review history for this article is available at https://publons.com/publon/10.1002/hon.3047. The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Clinical or biological parameters useful to predict progression during treatment in real-life setting with ibrutinib, idelalisib and venetoclax in relapsed/refractory chronic lymphocytic leukemia (CLL) are still debated. We conducted a multi-center retrospective study on CLL patients treated with ibrutinib and/or idelalisib who were switched to venetoclax for progression or due to adverse events to identify any clinical and/or biological parameters useful to predict progression during treatment with venetoclax. Of all the 128 evaluable patients, 81 had received ibrutinib prior to switching to venetoclax, 35 had received idelalisib and 12 both. When comparing the three subgroups, we did not notice any statistical difference in terms of clinical or biological features. No variable at baseline and at different time points during the follow-up (at 6, 12, 18 and 24 months) was found to predict progression nor to have significance for Progression Free Survival (PFS) in the ibrutinib group and in the idelalisib group and in subgroups according to the line of treatment. Analyzing the data of the venetoclax treatment, after a median follow up of 14.3 months, median PFS was not reached and estimated 3-year PFS was 54%. Of the 128 patients treated with venetoclax, 28 (22%) experienced progressive disease. At multivariate analysis for predictive factors for progression, lymph node diameter >56.5 mm before starting treatment emerged as an independent risk factor for progression. The lymph node predictive role for progression during venetoclax treatment could be a new parameter that deserves to be investigate in future studies.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, whose presentation and clinical course are highly variable. Identification of novel prognostic factors may contribute to improving the CLL classification and providing indications for treatment options. The zinc finger protein ZNF224 plays a key role in cell transformation, through the control of apoptotic and survival pathways. In this study, we evaluated the potential application of ZNF224 as a novel marker of CLL progression and therapy responsiveness. To this aim, we analyzed ZNF224 expression levels in B lymphocytes from CLL patients at different stages of the disease and in patients showing different treatment outcomes. The expression of ZNF224 was significantly increased in disease progression and dramatically decreased in patients in complete remission after chemotherapy. Gene expression correlation analysis performed on datasets of CLL patients revealed that ZNF224 expression was well correlated with that of some prognostic and predictive markers. Moreover, bioinformatic analysis coupled ZNF224 to NF-κB pathway, and experimental data demonstrated that RNA interference of ZNF224 reduced the activity of the NF-κB survival pathway in CLL cells. Consistently with a pro-survival role, ZNF224 knockdown raised spontaneous and drug-induced apoptosis and inhibited the proliferation of peripheral blood mononuclear cells from CLL patients. Our findings provide evidence for the involvement of ZNF224 in the survival of CLL cells via NF-κB pathway modulation, and also suggest ZNF224 as a prognostic and predictive molecular marker of CLL disease.

We herein report an innovative antisense approach based on Peptide Nucleic Acids (PNAs) to down-modulate CD5 expression levels in chronic lymphocytic leukemia (CLL). Using bioinformatics tools, we selected a 12-mer tract of the CD5 mRNA as the molecular target and synthesized the complementary and control PNA strands bearing a serine phosphate dipeptide tail to enhance their water solubility and bioavailability. The specific recognition of the 12-mer DNA strand, corresponding to the target mRNA sequence by the complementary PNA strand, was confirmed by non-denaturing polyacrylamide gel electrophoresis, thermal difference spectroscopy, circular dichroism (CD), and CD melting studies. Cytofluorimetric assays and real-time PCR analysis demonstrated the downregulation of CD5 expression due to incubation with the anti-CD5 PNA at RNA and protein levels in Jurkat cell line and peripheral blood mononuclear cells from B-CLL patients. Interestingly, we also observed that transfection with the anti-CD5 PNA increases apoptotic response induced by fludarabine in B-CLL cells. The herein reported results suggest that PNAs could represent a potential candidate for the development of antisense therapeutic agents in CLL.

Heterophilic antibodies can interfere with laboratory tests causing erroneous results. We report the case of a 55-year-old man with Mantle cell myeloma treated with Rituximab that caused false positive results of HIV serologic test. The cause of this interference was investigated, and very high levels (741 ng/ml) of human anti-mouse antibodies (HAMA) were demonstrated. Polyethylene glycol (PEG) precipitation of intact immunoglobulins was used to cross-check the antibody-related interference. The PEG treatment negativized the HIV test outcome, confirming that the cause of the false positive serologic HIV result was due to interference caused by HAMA. In conclusion, we suggest to cautiously interpreting laboratory findings in patients treated with Rituximab because of the possibility that HAMA may cause interference in serological assays.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease characterized by variable clinical courses among different patients. This notion was supported by the possible coexistence of two or more independent CLL clones within the same patients, identified by the characterization of the B cell receptor immunoglobulin (BcR IG) idiotypic sequence. By using the antigen-binding site of the BcR IG as bait, the identification and isolation of aggressive and drug-resistance leukemic B-cell clones could allow a deeper biological and molecular investigation. Indeed, by the screening of phage display libraries, we previously selected a peptide binder of the idiotypic region of CLL BCR IGs expressing the unmutated rearrangement IGHV1-69 and used it as a probe to perform a peptide-based cell sorting by flow cytometry in peripheral blood samples from patients with CLL. Since the IGHV1-69 clones persisted during the follow-up time in both patients, we explored the possibility of these clones having acquired an evolutive advantage compared to the other coexisting clones in terms of a higher expression of genes involved in the survival and apoptosis escape processes. To this end, we studied the expression patterns of a panel of genes involved in apoptosis regulation and in NF-kB-dependent pro-survival signals by comparative qRT-PCR assays. According to the results, IGHV1-69 clones showed a higher expression of pro-survival and anti-apoptotic genes as compared to the other CLL clones with different immunogenetic characteristics. Moreover, these IGHV1-69 clones did not carry any characteristic genetic lesions, indicating the relevance of our approach in performing a comprehensive molecular characterization of single tumor clones, as well as for designing new personalized therapeutic approaches for the most aggressive and persistent tumor clones.

Abstract Background Ibrutinib is a Bruton tyrosine kinase inhibitor, approved in the last few years for treatment as primary option for all subsets of chronic lymphocytic leukemia (CLL). Its use is associated with increased incidence of atrial fibrillation (AF). Objectives Aim of this study is to determine whether there are echocardiographic parameters that could identify patients at major risk of developing ibrutinib-related atrial fibrillation (IRAF). Methods We performed a retrospective review of 26 patients (mean age 70,34 ± 11,69; 27% females), admitted at our EchoLab in the last year, who underwent echocardiogram prior to Ibrutinib treatment. Echo-Doppler assessment was realized according to the standards of the European Association of Cardiovascular Imaging (EACVI) standardization of the echo report. Left atrial (LA) strain was measured with EchoPAC, obtaining peak atrial longitudinal strain (PALS) and peak atrial contraction strain (PACS) on 4-chambers and 2-chambers views. Continuous normally distributed variables were compared by using the Student t-test. A probability value < 0,05 was considered statistically significant. Analyses were performed with SPSS version 25 (IBM Corporation, Somers, New York). Results Six patients developed IRAF (23%). There weren't differences of clinical characteristics between the two groups (age, body mass index, arterial blood pressure, heart rate and diabetes, hypertension and cardiovascular diseases prevalence). It was noticed that IRAF's group had lower ejection fraction (EF) (54,67 ± 1,96 vs 60,90 ± 4,35, p-value < 0,0001), higher left atrium volume index (48,95 ± 16,31 vs 34,18 ± 9,07, p-value: 0,009), higher pulmonary arterial pressure values (PAPs) (44,16 ± 11,26 vs 32,89 ± 8,44, p-value: 0,015). Furthermore, it was noticed that peak atrial longitudinal strain (PALS) and peak atrial contraction strain (PACS) were reduced in patients who developed IRAF (PALS 4Ch: 18,89 ± 6,90 vs 29,40 ± 9,79, p-value 0,06; PACS 4Ch: 10,56 ± 5,66 vs 14,31 ± 5,72, p-value: 0,25; PALS 2Ch: 23,36 ± 5,02 vs 32,88 ± 16,57, p-value: 0,28; PACS 2Ch: 13,61 ± 8,16 vs 18,33 ± 9,87, p-value: 0,39), but not statistically significant, probably due to the sample size. Conclusions This is a preliminary pilot study which confirms the data already present in the literature. The importance of baseline evaluation by echocardiogram including measurement of atrial strain of patients before starting treatment with Ibrutinib is emphasized since, given the same anthropometric characteristics and risk factors, there are echocardiographic parameters that help us to identify patients at major risk of developing IRAF. Pharmacological intervention tailored on this type of basal echocardiographic evaluation could allow the reduction of IRAF's onset and improve patient outcomes in the long term. Certainly, further follow-up studies will be needed to confirm this hypothesis.

The current COVID-19 pandemic requires revisiting our current approach to major blood disorders, including ITP (Immune Thrombocytopenia), stirring up the production of several disease-specific practical guidelines. This report describes an updated version of consensus-based practical guidelines on the management of ITP, adapted to the Italian health system and social context. It highlights the role of the hematologist in offering guidance for choosing differentiated approaches in relation to specific circumstances and is intended to provide them with a useful tool for sharing the decision-making process with their patients. Probably, the greatest risk to avoid for a patient with suspected, ongoing or relapsed ITP - that is not severe enough to place him or her at risk for major bleeding - is to be infected in non-hospital and hospital healthcare settings. This risk must be carefully considered when adapting the diagnostic and therapeutic approach. More in detail, the document first addresses the appropriate management for COVID-19 negative patients with newly diagnosed ITP or who experience a relapse of previous ITP, according to first and second lines of treatment and then the management of COVID-19 positive patients according to their severity, from paucisymptomatic to those requiring admission to Intensive Cure Units (ICU). The pros and cons of the different treatments required to correct platelet count are discussed, as are some specific situations, including chronic ITP, splenectomy, thromboembolic complication and anti COVID-19 vaccination.

INTRODUCTION:In patients with primary immune thrombocytopenia (ITP), a short course of steroids is routinely given as first-line therapy. However, the response is often transient and additional therapy is usually needed. Thrombopoietin receptor agonists (TPO-RAs) are frequently used as second-line therapy, although there is little clinical guidance on the timing of their administration and on tapering/discontinuation of the drug. To provide clinical recommendations, we used the Delphi technique to obtain consensus for statements regarding administration and on tapering/discontinuation of second-line TPO-RAs among a group of Italian clinicians with expertise in management of ITP.METHODS:The Delphi process was used to obtain agreement on five statements regarding initiation and on tapering/discontinuation of second-line TPO-RAs. Agreement was considered when 75% of participants approved the statement. Eleven experts participated in the voting.RESULTS:Full consensus was reached for three of the five statements. The experts held that an early switch from corticosteroids to a TPO-RA has the dual advantage of sparing patients from corticosteroid abuse and improve long-term clinical outcomes. All felt that dose reduction of TPO-RAs can be considered in patients with a stable response and platelet count >100 × 109/L that is maintained for at least 6 months in the absence of concomitant treatments, although there was less agreement in patients with a platelet count >50 × 109/L. Near consensus was reached regarding the statement that early treatment with a TPO-RA is associated with an increase in clinically significant partial or complete response. The experts also agreed that optimization of tapering and discontinuation of TPO-RA therapy in selected patients can improve the quality of life.CONCLUSION:The present consensus can help to provide guidance on use of TPO-RAs in daily practice in patients with ITP.PLAIN LANGUAGE SUMMARY:Second-line administration of thrombopoietin receptor agonists in immune thrombocytopenia There is little guidance on the timing of administration and tapering/discontinuation of thrombopoietin receptor agonists (TPO-RAs) in patients with primary immune thrombocytopenia (ITP).The Delphi technique was used to obtain consensus for five statements.The present consensus among Italian clinicians aims to provide guidance on second-line use of TPO-RAs for patients with ITP in daily practice.

The direct antiglobulin test (DAT) detects immunoglobulin or complement bound in vivo to red blood cells (RBCs) and is widely used to diagnose immune-mediated hemolytic anemias. Positive DAT results, with or without clinically evident anemia, have been reported in a subset of patients with various viral infections. 1 Very recently, a few cases with simultaneous onset of SARS-CoV-2 infection and autoimmune hemolytic anemia (AIHA) have been described. 2,3

Inhibitors directed against factor V (FV) occur seldom, pri-marily in the elderly, and the clinical presentation varies from asymptomatic laboratory abnormalities to life-threatening bleedings. 1,2 Acquired FV inhibitors usually are immu-noglobulin of the G class (IgG) directed to the binding site for phosphatidylserine (present in the second C-type domain of the light chain of FV). Infections, drugs, surgical procedures, blood transfusions, solid and hematological cancers, and autoimmune disorders are associated with the risk of devel-oping acquired FV inhibitors. 3,4 According to the speci fi c targetedepitope (i.e., domains of lightchain orheavychain of FV), anti-FV antibodies affect the balance between pro- and anticoagulant functions of FV. 2,5 No de fi nitive treatment strategy has been established. In approximately 50% of cases, the inhibitor is eliminated spontaneously. 1 – 3 A 62-year-old Caucasian woman presented to the Hematology Department because of recurrent episodes of hematuria from 1 week, and bleeding from the sites of venous sampling. Coagulation tests showed that

The efficacy and safety of thrombopoietin receptor agonists (TRAs) in older patients with primary immune thrombocytopenia (ITP) are unknown. We investigated TRA response and switch, thrombotic/hemorrhagic risk, and sustained responses off-treatment (SROTs) in 384 patients with ITP aged ≥60 years. After 3 months, 82.5% and 74.3% of eltrombopag- and romiplostim-treated patients, respectively, achieved a response; 66.7% maintained the response (median follow-up, 2.7 years). Eighty-five (22.2%) patients switched to the alternative TRA; although no cross-toxicity was observed, 83.3% of resistant patients had a response after the switch. Thirty-four major thromboses (3 fatal) and 14 major hemorrhages (none fatal) occurred in 18 and 10 patients, respectively, while on TRAs and were associated with thrombosis history (subdistribution hazard ratio, 2.04, P = .05) and platelet count <20 × 109/L (subdistribution hazard ratio, 1.69; P = .04), respectively, at TRA start. A recurrent event occurred in 15.6% of patients surviving thrombosis, in all cases but 1 during persisting TRA treatment (incidence rate, 7.7 per 100 patient-years). All recurrences occurred in the absence of adequate antithrombotic secondary prophylaxis. Sixty-two (16.5%) responding patients discontinued TRAs; 53 (13.8%) patients maintained SROTs, which were associated with TRA discontinuation in complete response (P < .001). Very old age (≥75 years; 41.1%) was associated with the more frequent start of TRAs in the persistent/acute phase but not with response or thrombotic/hemorrhagic risk. TRAs are effective in older patients with ITP, with no fatal hemorrhages and with SROTs in a significant portion of patients. Caution is warranted in patients with a history of thrombosis, and a careful risk/benefit balance should be considered.

The immunoglobulin B cell receptor (IgBCR) expressed by chronic lymphocytic leukemia (CLL) B cells plays a pivotal role in tumorigenesis, supporting neoplastic transformation, survival, and expansion of tumor clones. We demonstrated that in the same patient, two or more CLL clones could coexist, recognized by the expression of different variable regions of the heavy chain of IgBCR, composing the antigen-binding site. In this regard, phage display screening could be considered the easier and most advantageous methodology for the identification of small peptide molecules able to mimic the natural antigen of the tumor IgBCRs. These molecules, properly functionalized, could be used as a probe to specifically identify and isolate single CLL subpopulations, for a deeper analysis in terms of drug resistance, phenotype, and gene expression. Furthermore, CLL cells express another surface membrane receptor, the CD5, which is commonly expressed by normal T cells. Piece of evidence supports a possible contribution of CD5 to the selection and maintenance of autoreactivity in B cells and the constitutive expression of CD5 on CLL cells could induce pro-survival stimuli. In this brief research report, we describe a peptide-based single-cell sorting using as bait the IgBCR of tumor cells; in the next step, we performed a quantitative analysis of CD5 expression by qRT-PCR related to the expressed IgBCR. Our approach could open a new perspective for the identification, isolation, and investigation of all subsets of IgBCR-related CLL clones, with particular attention to the more aggressive clones.

The B cell receptor (BCR) is an immunoglobulin (Ig) expressed on the membrane surface of mature B cells [1].The IgBCR has two heavy and two light chains, each one made by a constant and a variable region.The variable sequences of the IgBCR are generated during B-cell differentiation through somatic recombination, so called VDJ recombination, and somatic hypermutation [2].Once assembled on the B-cell surface, the IgBCR recognizes a specific antigen through its binding to the variable regions.This event triggers the B-cell immune response against the antigen.Since the variable regions are B cell specific, the sequence of IgBCR allows the identification of single B-cell clones [3].In B-lymphoproliferative disorders, the IgBCR plays a key role in the development, proliferation, and survival of tumor B cells [4], by an antigen-driven process that triggers a molecular cascade of events that lead to transcriptional activation of proliferative and antiapoptotic genes [5][6][7].Chronic lymphocytic leukemia (CLL) is a Bproliferative disorder characterized by a clonal expansion and accumulation of neoplastic CD19/CD5/CD23/ CD20-positive B-lymphocytes in blood, bone marrow, and other tissues [8].Several studies support the hypothesis that a common pool of environmental antigens or self-antigens drives the selection of tumor B cells through the persistent triggering of IgBCR [9, 10].Consistently, the sequence analysis of VDJ rearrangement of tumor IgBCRs revealed a high level of homology in more than 30% of CLL patients, defined as stereotyped IgBCR, with the prevalence of VH1, VH3, and VH4 families [11,12].CLL are defined as mutated (M-CLL) or unmutated (U-CLL) depending on the mutational rate of the IgBCR variable regions, being more or less than 2% respect to the germline, respectively [11,12].U-CLL cells expressing the VH1-69 rearrangement usually have an inducible IgBCR and show an aggressive behavior [11,12].In this regard, it would be useful to develop new molecular tools for rapid detection of aggressive CLL clones in peripheral blood.We previously used phage display for identifying peptide ligands of B-lymphoma, multiple myeloma, and CLL IgBCRs [3,[13][14][15].In CLL patients, we documented the co-existence of different CLL clones in a single patient as detected by phage-expressed peptide ligands of the tumor IgBCRs [3,15].In this study, we used the phage display for identifying a peptide sequence that was commonly recognized by VH1-69 U-CLL clones of two CLL patients.These patients, named CLL#1 and CLL#5, were randomly referred to the Hematology Unit-University Federico II of Naples and initially diagnosed as CLL Binet stage A. At month 8, CLL#1 worsen to Binet stage C and returned to Binet stage A after 6 months of

B-cell chronic lymphocytic leukaemia (B-CLL) cells fail to undergo apoptosis. The mechanism underlying this resistance to cell death is still largely unknown. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) effectively kills tumour cells but not normal cells, and thus represents an attractive tool for the treatment of cancer. Unfortunately, lymphocytes from B-CLL patients are resistant to TRAIL-mediated apoptosis. Thus, we aimed to study the involvement of PED, a DED-family member with a broad antiapoptotic action, in this resistance. We demonstrate that B lymphocytes obtained from patients with B-CLL express high levels of PED. Treatment of B-CLL cells with specific PED antisense oligonucleotides, a protein synthesis inhibitor or HDAC inhibitors, induced a significant downregulation of PED and sensitized these cells to TRAIL-induced cell death. These findings suggest a direct involvement of PED in resistance to TRAIL-induced apoptosis in B-CLL. It also identifies this DED-family member as a potential therapeutic target for this form of leukaemia. (c) 2006 Wiley-Liss, Inc.

American Journal of HematologyVolume 93, Issue 9 p. E243-E246 E-ONLY ARTICLEFree Access Predictors of refractoriness to therapy and healthcare resource utilization in 378 patients with primary autoimmune hemolytic anemia from eight Italian reference centers Wilma Barcellini MD, Corresponding Author Wilma Barcellini MD wbarcel@policlinico.mi.it orcid.org/0000-0003-1428-9944 UO Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy Correspondence Wilma Barcellini, UOC Ematologia, UOS Fisiopatologia delle Anemie - Padiglione Granelli, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via F. Sforza 35 - 20122 Milano, Italy. Email: wbarcel@policlinico.mi.itSearch for more papers by this authorAnna Zaninoni BS, Anna Zaninoni BS orcid.org/0000-0002-8614-3904 UO Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, ItalySearch for more papers by this authorBruno Fattizzo MD, Bruno Fattizzo MD orcid.org/0000-0003-0857-8379 UO Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, ItalySearch for more papers by this authorJuri Alessandro Giannotta MD, Juri Alessandro Giannotta MD UO Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, ItalySearch for more papers by this authorMonia Lunghi MD, Monia Lunghi MD SCDU Ematologia, Dipartimento di Medicina Traslazionale, Università del Piemonte Orientale Amedeo Avogadro, Novara, ItalySearch for more papers by this authorAntonella Ferrari MD, Antonella Ferrari MD UO Ematologia, Ospedale Sant'Andrea, Facoltà di Medicina e Psicologia, Università "Sapienza", Rome, ItalySearch for more papers by this authorAnna Paola Leporace MD, Anna Paola Leporace MD UO Ematologia, Ospedale Sant'Andrea, Facoltà di Medicina e Psicologia, Università "Sapienza", Rome, ItalySearch for more papers by this authorNilla Maschio MD, Nilla Maschio MD UO Trasfusionale e immunologia, Centro Regionale Malattie del Sangue, Castelfranco Veneto, ItalySearch for more papers by this authorLaura Scaramucci MD, Laura Scaramucci MD UO Ematologia, Ospedale S. Eugenio, Rome, ItalySearch for more papers by this authorSilvia Cantoni MD, Silvia Cantoni MD SC Ematologia, Niguarda Cancer Center, ASST Niguarda, Milan, ItalySearch for more papers by this authorFederico Chiurazzi MD, Federico Chiurazzi MD Ematologia, Università degli Studi di Napoli Federico II, Napoli, ItalySearch for more papers by this authorDario Consonni MD, Dario Consonni MD UO Epidemiologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, ItalySearch for more papers by this authorGiuseppe Rossi MD, Giuseppe Rossi MD SC Ematologia, Spedali Civili di Brescia, Brescia, ItalySearch for more papers by this authorPaolo De Fabritiis MD, Paolo De Fabritiis MD UO Ematologia, Ospedale S. Eugenio, Rome, ItalySearch for more papers by this authorGianluca Gaidano MD, Gianluca Gaidano MD SCDU Ematologia, Dipartimento di Medicina Traslazionale, Università del Piemonte Orientale Amedeo Avogadro, Novara, ItalySearch for more papers by this authorAlberto Zanella MD, Alberto Zanella MD UO Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, ItalySearch for more papers by this authorAgostino Cortelezzi MD, Agostino Cortelezzi MD UO Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy Università degli Studi di Milano, Milan, ItalySearch for more papers by this author Wilma Barcellini MD, Corresponding Author Wilma Barcellini MD wbarcel@policlinico.mi.it orcid.org/0000-0003-1428-9944 UO Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy Correspondence Wilma Barcellini, UOC Ematologia, UOS Fisiopatologia delle Anemie - Padiglione Granelli, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via F. Sforza 35 - 20122 Milano, Italy. Email: wbarcel@policlinico.mi.itSearch for more papers by this authorAnna Zaninoni BS, Anna Zaninoni BS orcid.org/0000-0002-8614-3904 UO Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, ItalySearch for more papers by this authorBruno Fattizzo MD, Bruno Fattizzo MD orcid.org/0000-0003-0857-8379 UO Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, ItalySearch for more papers by this authorJuri Alessandro Giannotta MD, Juri Alessandro Giannotta MD UO Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, ItalySearch for more papers by this authorMonia Lunghi MD, Monia Lunghi MD SCDU Ematologia, Dipartimento di Medicina Traslazionale, Università del Piemonte Orientale Amedeo Avogadro, Novara, ItalySearch for more papers by this authorAntonella Ferrari MD, Antonella Ferrari MD UO Ematologia, Ospedale Sant'Andrea, Facoltà di Medicina e Psicologia, Università "Sapienza", Rome, ItalySearch for more papers by this authorAnna Paola Leporace MD, Anna Paola Leporace MD UO Ematologia, Ospedale Sant'Andrea, Facoltà di Medicina e Psicologia, Università "Sapienza", Rome, ItalySearch for more papers by this authorNilla Maschio MD, Nilla Maschio MD UO Trasfusionale e immunologia, Centro Regionale Malattie del Sangue, Castelfranco Veneto, ItalySearch for more papers by this authorLaura Scaramucci MD, Laura Scaramucci MD UO Ematologia, Ospedale S. Eugenio, Rome, ItalySearch for more papers by this authorSilvia Cantoni MD, Silvia Cantoni MD SC Ematologia, Niguarda Cancer Center, ASST Niguarda, Milan, ItalySearch for more papers by this authorFederico Chiurazzi MD, Federico Chiurazzi MD Ematologia, Università degli Studi di Napoli Federico II, Napoli, ItalySearch for more papers by this authorDario Consonni MD, Dario Consonni MD UO Epidemiologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, ItalySearch for more papers by this authorGiuseppe Rossi MD, Giuseppe Rossi MD SC Ematologia, Spedali Civili di Brescia, Brescia, ItalySearch for more papers by this authorPaolo De Fabritiis MD, Paolo De Fabritiis MD UO Ematologia, Ospedale S. Eugenio, Rome, ItalySearch for more papers by this authorGianluca Gaidano MD, Gianluca Gaidano MD SCDU Ematologia, Dipartimento di Medicina Traslazionale, Università del Piemonte Orientale Amedeo Avogadro, Novara, ItalySearch for more papers by this authorAlberto Zanella MD, Alberto Zanella MD UO Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, ItalySearch for more papers by this authorAgostino Cortelezzi MD, Agostino Cortelezzi MD UO Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy Università degli Studi di Milano, Milan, ItalySearch for more papers by this author First published: 07 July 2018 https://doi.org/10.1002/ajh.25212Citations: 31AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat To the Editor: Autoimmune hemolytic anemia (AIHA) is a rare and heterogeneous disease with a presentation/phenotype ranging from mild/compensated to life-threatening.1, 2 Moreover, some cases show refractoriness to/relapse after first and further therapy lines, leading to significant and underestimated healthcare burden. Here, we retrospectively analyzed 378 primary AIHA cases (135 M and 243 F, median age 61 years, range 5-94) from eight Italian hematological centers and followed for a median of 4.3 years (range 1-27) focusing on predictors of multiple refractoriness to second and further therapy lines and healthcare resource utilization. Figure 1A shows clinical and laboratory characteristics of AIHA patients at onset, divided according to the serological type in warm (direct antiglobulin test-[DAT]-positive for IgG or IgG + C), cold (CAD, usually IgM driven, DAT-positive for C), mixed (both IgG and high titer cold agglutinins), and atypical forms (DAT negative, IgA only or warm IgM). Hemoglobin values were significantly lower and LDH higher in IgG + C wAIHA, mixed and atypical cases (P < .001 and P = .0034, respectively), and hemoglobin and LDH values were negatively correlated (P < .001). Absolute reticulocytes were reduced in CAD and mixed forms (P = .0013), and the reticulocyte index was lower in CAD (P = .023) and in cases with Hb ≤ 6 g/dL (53 vs 86 P < .001) (Figure 1B). Overall, inadequate reticulocytosis was observed in more than half of patients. Second therapy line was mostly administered in mixed, IgG + C wAIHA, and in CAD (P = .011), and the ultrarefractory cases requiring four or more lines of therapy were mainly CAD, mixed, and atypical AIHA. Infections were observed in 14% of cases, mostly wAIHA and mixed forms (P = .02). Thrombosis occurred in 15% mostly IgG + C wAIHA and atypical AIHA (P = .04). Evans' syndrome was more frequent in mixed AIHA and, to a lesser extent, in atypical (P = .033) and in severe forms (74% with Hb < 8 g/dL vs 26%, P = .005). Acute renal failure was reported in 3% of patients, with no relationship with AIHA type/Hb values. Figure 1Open in figure viewerPowerPoint (A) Clinical and laboratory characteristics of patients at onset, therapy lines, complications and death, divided according to AIHA serological type. (B) Reticulocyte counts as a function of Hb at onset: AIHA cases are grouped in mild (Hb > 10 g/dL), moderate (Hb 8.1-10 g/dL), severe (Hb 6.1-8 g/dL), and very severe (Hb ≤ 6 g/dL) (left). Individual values of Hb are shown in relation to reticulocyte index (absolute reticulocyte count × patient' Hb/normal Hb), with the corresponding equation line (ret(× 1000) = 119 + 29.2 × Hb - 2.4 × Hb2) (right). Both panels A and B indicate that compensatory reticulocytosis increases until Hb values of about 8 g/dL, but more severe cases are characterized by reticulocytopenia/inadequate reticulocytosis. (C) Healthcare resource utilization: Percentage of patients receiving transfusions, highly expensive drugs (rituximab, bortezomib, eculizumab), and splenectomized, grouped according to AIHA serological type Predictors of relapse/refractoriness were analyzed in a total of 304 relapses, of whom 211 (69%) after first-line, 69 (23%) after second-line, 19 (6%) after third-line, and 5 (2%) after further therapy lines. Multivariate Cox regression analysis demonstrated that anemia severity at onset was associated with an increased risk of relapse, with the following hazard ratios: 1.98 (95%CI 1.22-3.21) for patients with Hb ≤ 6 g/dL, 1.74 (95%CI 1.09-2.77) for cases with Hb 6.1-8 g/dL, and 1.61 (95%CI 0.99-2.62) for those with Hb 8.1-10 g/dL. Even considering hemoglobin as a continuous variable, each gram of reduction yielded a 7% greater risk of relapse (95%CI 2-13, P < .013). In addition, analysis showed increased hazard risks for forms other than wAIHA (1.21, 95%CI 0.95-1.54), and for Evans association (1.84, 95%CI 1.24-2.74). Notably, the presentation of an AIHA other than wAIHA together with Evans syndrome or with Hb < 8 g/dL resulted in a 2-fold higher risk of relapse; the presence of both Hb < 8 g/dL and Evans syndrome gave a 3-fold increased risk, and the association of the three conditions led to a ~4-fold increased risk of multiple relapses. Seventy-five patients died during the follow-up, of whom 13 because of AIHA. Overall mortality was higher in more severe cases (24% for cases with Hb <6 g/dL vs 18% for those with Hb >6 g/dL, P = .04), with the following hazard risks: Evans syndrome (8, 95% CI 2.5-26, P = .001), acute renal failure (6.3, 95% CI 1.4-29, P = .016), and infections (4.8, 95% CI 1.5-15, P = .007). Thrombotic events did not result in increased risk of death. Response to treatment is shown in supplementary material. In summary, ~80% of cases displayed an overall response (OR) after first line treatment with glucocorticoids, however only 25% had a sustained response. Rituximab and immunosuppressants were comparably used in second line (31% and 26%), with better responses for the former (88% OR, 53% complete responses, CR); immunosuppressants gave mainly partial responses (PR, 46%) and were generally administered together with glucocorticoids. Rituximab had a longer relapse free survival compared with immunosuppressants (22 months, range 2-71 vs 12 months, range 2-43), and a greater proportion of sustained responses (23/35, 51% vs 13/31, 42%). Splenectomy (n = 38, ~10% of the whole series) had been performed mostly in second line (63%), particularly before 2000 (8/9), with very good responses (85%). Regarding third line therapy, rituximab was preferred compared to immunosuppressants (46% vs 15%). Importantly, OR and CR to rituximab were comparable to those observed in second line (92% and 41%). Splenectomy had become a third line option in most recent years (11/24, 46%) and confirmed its excellent performance. Further lines were required in 29 multirefractory patients (8% of cases), and included glucocorticoids/rituximab, glucocorticoids/immunosuppressants, regular transfusions, bortezomib (1.3 mg/m2 iv single agent on days 1, 4, 8, 11, n = 8), plasma-exchange (N = 5), eculizumab (N = 2), erythropoietin (N = 12), high dose cyclophosphamyde/vincristine (N = 1), and autologous peripheral stem cell transplant (N = 1). Response to treatments is difficult to assess, due to their combinations/overlap. Notably, six cases underwent splenectomy as forth or further line, all obtaining a response. Considering all the 32 cases that respond to splenectomy (either performed in second, third, or further lines), 13 (41%) relapsed after a median of 16 months (range 2-147), while 19 (59%) cases are still on remission at the time of the study. Regarding response to treatment according to AIHA type, a CR to glucocorticoids was mostly observed in wAIHA, mixed and atypical cases (~50%), whereas CR rate was lower in CAD (24%) (P = .0015), and generally observed at high dosages. A complete response to rituximab was mainly observed in IgG wAIHA (62%), whereas the drug was less effective in IgG + C wAIHA, CAD, and mixed forms (44%-54%). Figure 1C and Supporting Information show healthcare utilization in a subgroup of 190 cases comparable to the remaining 188 in terms of demographics, hematological parameters, number, and type of therapy lines. One hundred twenty-one patients (64%) required at least one hospital admission, with 9% needing three or more. The median admission duration was 15 days, with 13% of cases needing more than 30 days, and admissions length was negatively correlated to Hb levels (r = −0.26, P < .001). The higher frequency was observed in IgG + C wAIHA, mixed and atypical cases (P = .006). Concerning outpatients, median number of visits per year was five, but 42 cases (22%) required more than a monthly visit. As regards therapeutic procedures, 103 patients (54%) required at least one transfusion, with 11/103 patients (11%) needing more than 20 transfusions (the established threshold for chelation), and six cases more than 50. Transfusion need was higher in IgG + C wAIHA, mixed and atypical cases (P = .004), and correlated with a more severe disease, that is, anemia, hemolysis and inadequate bone marrow compensation (Hb r = −.26, P < .001; LDH r = .21, P < .01; BMRI r = .15, P < .05). Considering transfusions as a function of follow-up length, the median was 2 per year, with 27 cases necessitating 4 or more (one subject about a weekly support). Highly expensive drugs were administered in 65 cases (34%, 56 rituximab, 8 bortezomib, and 2 eculizumab), and splenectomy performed in 20 patients (11%). The administration of highly expensive drugs was higher in CAD and mixed cases (P = .01). In conclusion, this is the largest study aimed at identifying predictors of refractoriness to multiple therapy lines and healthcare resource utilization in primary AIHA in the real-life and outside clinical trials. We found that hemoglobin level at onset is the only laboratory parameter associated with an increased risk of refractoriness, from 2-fold for moderate to 3-fold for severe cases. Anemia severity was associated with reticulocytopenia/inadequate reticulocytosis, present in more than a half of patients, typically CAD and mixed forms. Thrombocytopenia (Evans syndrome) resulted in a 2-fold increased relapse risk, highlighting the detrimental effect of a more complex immune dysregulation involving multiple hematological lineages. Moreover, refractory/relapsing AIHAs were those with complement involvement (warm IgG + C, mixed, and CAD), suggesting the primary role of this powerful amplification system in the degree of AIHA immune dysregulation. Regarding therapy, known to be mainly based on experts opinion,3-5 rituximab had a longer relapse free survival (~2 years), and was effective in ~50% of cases in first and further lines. The drug was used as first or early-second line in very severe cases, particularly in CAD cases that are known to respond to unacceptable high glucocorticoid doses only. At variance, immunosuppressants induced partial responses with decreasing efficacy after first line. Splenectomy had high response rates (~85%) and was curative in ~60% of warm AIHA cases, even when performed as third or further lines. This option, although marked by infectious and thrombotic complications, and contraindicated in CAD, should not be forgotten, given its potential curative ability, as recently suggested for immune thrombocytopenia.6 Healthcare resource utilization was important in AIHA, with ~2/3 of patients requiring hospital admissions, 1/2 transfusions, and ~1/3 highly expensive drugs. In a small group of severe/ultrarefractory cases (10%-15%, mainly CAD, IgG + C wAIHA, mixed and atypical forms) admissions lasted more than 30 days, transfusion need exceeded 20 units per year, the established threshold for chelation, and several highly expensive drugs were administered, representing a significant burden on the patient and the medical system. ACKNOWLEDGMENTS This work was supported by research funding from Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, grant number RC 2016-17. Conflict of Interests All the Authors disclose no commercial affiliation as well as consultancies, stock or equity interests, and patent licensing that could be considered to pose a conflict of interest regarding the submitted article. Author Contributions Study design: WB Patients follow-up: JAG, ML, AF, APL, NM, LS, SC, FC, PDF Data collection: WB, AZ, BF, JAG, ML, AF, APL, NM, LS, SC, FC, PDF Data analysis: WB, AZ, BF, DC Data interpretation: WB, DC Manuscript writing: WB, AZ, BF Revised manuscript: JAG, ML, AF, APL, NM, LS, SC, FC, PDF, GR, DC, JAG, AZ, AC Supporting Information Filename Description ajh2512-supp-0001-Supinfo.docWord document, 145.5 KB Supporting Information Table S1. Response to treatments according to therapy line and AIHA type. Supporting Information Table S2. Healthcare resource utilization in AIHA Supporting Information Figure S1. Response to treatments according to therapy line and AIHA type Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES 1Barcellini W, Fattizzo B, Zaninoni A, et al. Clinical heterogeneity and predictors of outcome in primary autoimmune hemolytic anemia: a GIMEMA study of 308 patients. Blood. 2014; 124(19): 2930- 2936. 2Fattizzo B, Zaninoni A, Nesa F, et al. Lessons from very severe, refractory, and fatal primary autoimmune hemolytic anemias. Am J Hematol. 2015; 90: E149- E151. 3Barcellini W. Current treatment strategies in autoimmune hemolytic disorders. Expert Rev Hematol. 2015; 8(5): 681- 691. 4Go RS, Winters JL, Kay NE. How I treat autoimmune hemolytic anemia. Blood. 2017; 129(22): 2971- 2979. 5Berentsen S. How I manage patients with cold agglutinin disease. Br J Haematol. 2018 May; 181(3): 320- 330. 6Chaturvedi S, Arnold DM, McCrae KR. Splenectomy for immune thrombocytopenia: down but not out. Blood. 2018; 131(11): 1172- 1182. Citing Literature Volume93, Issue9September 2018Pages E243-E246 FiguresReferencesRelatedInformation

BACKGROUND:The incidence of peripherally inserted central catheter (PICC)-related adverse events has been uncertain in the setting of acute myeloid leukemia (AML) compared with the incidence of centrally inserted central catheter (CICC) adverse events. PATIENTS AND METHODS:We conducted a monocentric, randomized trial of patients with previously untreated AML. Of the 93 patients, 46 had received a PICC and 47 had received a CICC as frontline intravascular device. Thereafter, all patients underwent intensive chemotherapy for hematologic remission induction. The primary endpoint was catheter-related (CR)-bloodstream infection (BSI) and venous thrombosis (VT) rate. The secondary endpoints catheter malfunction, catheter removal, and patient overall survival. RESULTS:The CR-BSI and CR-VT rate in the PICC and CICC groups was 13% and 49%, respectively, with a difference of 36 percentage points (relative risk for CR-BSI or CR-VT, 0.266; P = .0003). The CR-BSI incidence was 1.4 and 7.8 per 1000 catheters daily in the PICC and CICC groups, respectively. Among the CR thromboses, the symptomatic VT rate was 2.1% in the PICC group and 10.6% in the CICC group. In the CICC group, 16 of the 47 patients (34%) had the catheter removed for BSI (n = 5), septic thrombophlebitis (n = 4), VT (n = 2), or malfunction (n = 5) a median of 7 days after insertion. In the PICC group, only 6 of the 46 patients (13%) required catheter removal for VT (n = 2) or malfunction (n = 4). At a median follow-up of 30 days, 6 patients in the CICC group died of CR complications versus none of the patients in the PICC group (P = .012). Using PICCs, the reduction in BSI and symptomatic VT decreased mortality from CR infection and venous thromboembolism. In contrast, the CICC approach led to early catheter removal mostly for difficult-to-treat infectious pathogens. CONCLUSION:Our data have confirmed that BSI and symptomatic VT are the major complications affecting frontline central intravascular device-related morbidity in the leukemia setting. The use of a PICC is safer than that of a CICC and maintains the effectiveness for patients with AML undergoing chemotherapy, with an approximate fourfold lower combined risk of infection or thrombosis at 30 days.