Detection of SARS-CoV-2 neutralizing antibodies: reliability of serological tests

Serological assays allow to explore the synthesis of antibodies against SARS-CoV-2 proteins. The gained knowledge finds applications in sero-epidemiology studies, to develop accurate forecasts of protective immunity spreading within a population. Indeed, last February, the European Centre for Disease prevention and Control (ECDC) recommended the use of serological tests for population surveys, in the context of epidemiological studies. The up-to-date reference method to determine the amount of protective antibodies is the in vitro neutralization test, which is expensive, timeconsuming and requires biosafety level 3 laboratories. The current study aims at highlighting the correlation between the results of the reference method and four different serological assays. Three immunoassays (Elecsys Anti SARS-CoV-2 S, MAGLUMI SARSCoV- 2 S-RBD IgG and MAGLUMI 2019-nCoV IgG test) and an ELISA surrogate viral neutralization test (EUROIMMUN SARS-CoV-2 NeutraLISA assay) have been studied on 83 patients, previously infected by SARS-CoV-2. The correlation between each serological test and the reference method is reported, as well as the predictive performances to distinguish serum samples with neutralizing antibody titers higher than 160. All the assays achieved good performances in terms of correlation with the reference method, as well as concerning the predictive ability over NAb titers > 160. The best correlation (Spearman coefficient = 0.784) and predictive features (area under curve = 0.921) have been observed for EUROIMMUN SARS-CoV-2 NeutraLISA assay, as displayed by ROC curve analyses.

Second versus first wave of covid-19 in patients with mpn

Introduction. MPN-COVID is a European LeukemiaNet cohort study, launched in March 2020 in patients with myeloproliferative neoplasms (MPN) with COVID-19. The first cohort of 175 cases was analyzed at the end of first wave (July 2020) and results provided estimates and risk factors of overall mortality (Barbui T. Leukemia, 2021), thrombosis incidence (Barbui T. Blood Cancer J, 2021), and post-COVID outcomes (Barbui T. Blood Cancer J, 2021). In the second wave of pandemic (June 2020 to June 2021), case-fatality risk in the general population has been found variable across different countries, and no information is available in MPN patients with COVID-19 diagnosed during the second wave in comparison with those of the first wave. Methods. In an electronic case report form, we registered a total of 479 cases of ET (n=161, 34%), PV (n=135, 28%), pre-PMF (n=49, 10%) and overt MF (n=134, 28%), from 39 European hematology units (Italy, Spain, Germany, France, UK, Poland, Croatia). Of these, 304 were diagnosed COVID-19 during the second wave. Results. Patients in the second wave were significantly different from those in the first wave, including parameters such as age (median: 63 vs. 71 years, p<.001), sex (females: 52% vs. 42%, p=0.037), MPN category (MF 24% vs. 34%, p=0.020), comorbidity (at least one comorbidity 63% vs. 74%, p=0.012), disposition (home: 68% vs. 23%, regular ward: 29% vs. 66%, ICU: 3% vs. 11%, p<.001), need of respiratory support (28% vs. 59%, p<.001) and degree of systemic inflammation (C-Reactive Protein: 51% vs. 74%, p=0.008;Neutrophil to Lymphocyte Ratio: 4.1 vs. 5.4, p=0.038). In regard to COVID-19-directed therapy, in the second wave steroids were more frequently prescribed (28% vs. 40%, p=0.007), while the use of antibiotics, antivirals, hydroxychloroquine and experimental therapies was significantly less frequent (p<.001 for all the differences). Interestingly, only 4 out of 46 patients (8.7%) discontinued Ruxolitinib during second-wave acute COVID (all MF admitted to regular ward). In the two waves, distribution probability of COVID-19 incidence by Kernel method showed a substantially similar shape, whereas the two incidence peaks were associated with very different mortality, as reported in Fig. 1A. The difference between the probability of death was highly significant during the first (n=175) vs. second (n=304): 31% vs. 9% at 60 days from COVID-19 diagnosis, respectively (p<.001) (Fig. 1B). Of note, among 26 deaths, 4 (15%) occurred at home, 19 (73%) on regular wards and 3 (12%) in the ICU, and death more frequently afflicted patients with (n=17, 65%) than ET (n=5, 19%) and PV (n=4. 15%) (p<.001). Independent risk factors for death in a multivariate Cox regression model fitted on the whole cohort and adjusted for the wave to which patients belonged, were age over 70 years (HR=5.2, 95% CI 1.8-15.1, p=0.002), male sex (HR=1.9, 95% CI 1.1-3.1, p=0.016), COVID-19 severity revealed by the need for respiratory support (HR=4.5, 95% CI 1.9-10.7, p=0.001), and Ruxolitinib discontinuation (HR=3.0, 95% CI 1.3-6.9, p=0.011). Conversely, in patients who continued this drug, no risk was documented (HR=1.21, p=0.566). Taking into account death as competing event, the second outcome of interest was the incidence of thrombosis, wich occurred in a significantly lower proportion of patients in the second wave compared to the first one (n=5 [1.6%] vs. n=14 [8.0%] at +60 days, respectively, SHR=0.20, p=0.002) (Fig. 1C). All the events, but one (n=4/5) were venous and were reported in patients with ET (SHR=4.4, 95% CI 1.8-10.7, p=0.001). Conclusions. This is the largest series of MPN patients who incurred COVID-19 from June 2020 onward, namely during the 'second COVID-19 wave'. Compared to the first wave, the second one recorded a lower overall COVID-19 severity, but Ruxolitinib discontinuation still remained a risk factor for a dismal outcome. Greater vulnerability of ET than PV in developing venous thrombosis was confirmed also during the second wave. This finding suggests that ET warrants a specific antithrombo ic prophylaxis in addition to heparin.

ACUTE Lymphoblastic Leukemia (ALL) and COVID-19 Infection. a Campus ALL Report

[Formula presented] Introduction. The recent spread of the COVID-19 infection has represented an important challenge in the management of acute lymphoblastic leukemia (ALL) patients. Aims and methods. To investigate the incidence, features, source of contagion and outcome of patients with ALL who developed a COVID-19 infection, a survey was conducted among 34 hematology centers throughout Italy within the Campus ALL network. The period covered by the survey spanned from February 2020 to April 2021 and included 756 adult ALL patients actively followed during this time period. Results. Sixty-three of the 756 ALL patients (8.3%) developed a COVID-19 infection, with an equal distribution among the various regions. The majority of cases (90.5%) was recorded during the second wave of the pandemic, between September 2020 and April 2021. The source of the infection was nosocomial in 26 cases (41.3%), familial in 23 (36.5%), unknown in 13 (20.6%) and work-related in 1 (1.6%). The infected patients were prevalently male (n=43, 68.2%) with a similar distribution among age groups: 21 patients aged 18-35 years, 17 35-50, 15 50-65 and 10 older than 65. Seventeen patients (27%) had a diagnosis of T-ALL, 28 (44.4%) of Ph- B-ALL and 18 (28.6%) of Ph+ ALL. Thirty-six (57.1%) of the infected patients had no concomitant comorbidities, whereas 27 (42.9%) had one or more comorbidities. The infection was documented at the onset of the disease in 4 patients (6.3%), during induction in 10 (15.9%), consolidation in 13 (20.6%), chemotherapy maintenance in 11 (17.5%), after allogenic transplant in 15 (23.8%), during maintenance with tyrosine kinase inhibitors (TKI) treatment or off-treatment in 8 (12.7%) and at relapse in 2 (3.2%). Of the infected patients, 9 were asymptomatic, 10 had only isolated fever, 36 had respiratory symptoms and 8 presented other symptoms, including - but not limited to - ageusia and anosmia. As a consequence, management of the infection was variable: 29 (46%) patients did not require hospitalization, 28 (44.4%) were hospitalized in a COVID ward and 13 of them required respiratory assistance;finally, 6 (9.5%) patients were transferred to an ICU. Importantly, in 54 patients (85.7%) there were no sequelae, in 1 patient a pulmonary fibrosis was documented and in 1 patient the delay in treatment led to a relapse of the disease, while 7 (11.1%) succumbed to the infection. Finally, in 6 cases (9.5%) the infection was still ongoing at the time of the survey, and at the last update (July 2021) it had resolved in all. Since a key aspect in the management of ALL is the adherence to the timing of treatment, we also investigated if COVID-19+ patients stopped treatment during the infection. Out of the 42 evaluable patients (patients who had undergone an allogeneic transplant or were off-treatment were excluded from this analysis), ALL treatment was suspended in 28 (66.6%). Importantly, while in Ph+ ALL only very few patients stopped treatment (3/12), in Ph- B-ALL the majority did interrupt it (18/22, p<0.001);likewise, also in T-ALL most patients suspended treatment (7/8). Conclusions. The incidence of SARS-CoV-2 infection in adult ALL patients in Italy over a 15 month period has been similar to that observed in the general population and has been recorded mostly during the second wave of the pandemic. The contagion was mainly nosocomial, suggesting that outward care should be pursued as much as possible in ALL. The infection was manageable, with 46% of patients not requiring any medical intervention and an overall death rate of 11%. Strikingly, in line with previous reports 1, it appears that Ph+ ALL patients were more manageable, with less treatment interruptions. These findings underline the advantage of the TKI-based induction/consolidation strategy without systemic chemotherapy in Ph+ ALL used in the GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) protocols and further point to a possible protective role of TKIs in COVID-19-infected patients. 1. Foà R et al, Br J Haematol. 2020;190(1):e3-e5 Disclosures: Chiarett : Incyte: Consultancy;novartis: Consultancy;pfizer: Consultancy;amgen: Consultancy. Bonifacio: Bristol Myers Squibb: Honoraria;Pfizer: Honoraria;Novartis: Honoraria;Amgen: Honoraria. Marco: Jazz: Consultancy;Insight,: Consultancy;Janssen: Consultancy. Curti: Novartis: Membership on an entity's Board of Directors or advisory committees;Abbvie: Membership on an entity's Board of Directors or advisory committees;Pfizer: Membership on an entity's Board of Directors or advisory committees;Jazz Pharma: Membership on an entity's Board of Directors or advisory committees. Delia: Gilead: Consultancy;Amgen: Consultancy;abbvie: Consultancy;Jazz pharmaceuticals: Consultancy. Forghieri: Jannsen: Membership on an entity's Board of Directors or advisory committees;Novartis: Speakers Bureau;Jazz: Honoraria. Lussana: Amgen: Honoraria;Astellas Pharma: Honoraria;Pfizer: Honoraria;Incyte: Honoraria.

COVID-19 Infection in chronic myeloid leukemia after 1 year of the pandemic in Italy. A campus CML analysis

Background: An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) started in December 2019 in the province of Hubei in China. Italy was one of the most affected countries with many cases diagnosed already in February 2020 and a lockdown was declared on March 9th. Limited information has been reported with regard to the impact of the pandemic on chronic myeloid leukemia (CML) patients. Aims: To observe the temporal course of Covid-19 infection and the characteristics of positive patients. Methods: The Campus CML Italian group carried out a first survey on the management of CML patients during the lockdown. We launched a second survey during the pandemic phases 2 and 3, between May 2020 and January 2021. Results: We collected retrospective information on 8665 CML patients followed at 46 centers throughout the country. Within this cohort, we recorded 217 Covid-19-positive symptomatic patients (2.5%). Most patients (57%) were diagnosed as having Covid-19 infection between September 2020 and January 2021;30% were diagnosed in phase 1 (March-April 2020) and only 13% between May and August. Most of the positive patients were between 50 and 65 years (35%), while 26% had less than 50 years, 18.8% were between 65 and 75 years, and 11% had more than 75 years. A male prevalence was observed (73%). The median time from CML diagnosis to Covid-19 infection was 6 years (3 months-18 years). Fifty-six percent of patients presented concomitant comorbidities at the time of infection. When Covid-19 was diagnosed, 27% of patients were receiving imatinib, 26% nilotinib, 18% dasatinib, 8% ponatinib, 8% bosutinib, 2% asciminib, while 11% were not receive treatment. At the time of the infection, 74% of patients were in molecular remission, 6% in complete cytogenetic remission, 3% in partial cytogenetic remission, 6% in complete hematological response and 11% in treatment-free remission. At diagnosis, 28% of patients presented fever and respiratory symptoms, 13% cough, 10% isolated fever, 13% ageusia, 12% anosmia, 4% had more than 1 symptom, while 20% were completely asymptomatic. Twenty-one patients (9.6%) required hospitalization without the need of respiratory assistance, 18 (8.2%) were hospitalized for respiratory assistance, 8 (3.6%) were admitted to an ICU, while 150 patients (69%) were only quarantined. Twenty-three percent of patients discontinued TKI therapy during the infection. The source of contagion was familiar in 49% of patients, 18% due to work, 3% in healthcare professionals, whereas in 30% was not known. Twelve patients died due to Covid-19 infection with a mortality rate of 5.5% in the positive cohort and of 0.13% in the whole cohort. Five patients reported consequences post-infection: 1 patient reported a Guillan-Barrè syndrome, 1 patient a maculopapular rash, 1 patient a pulmonary fibrosis, 1 patient a bacterial endocarditis and 1 patient was diagnosed as having alterations of the microcirculation. Summary/Conclusion: This study reports the 1-year of data on the Covid- 19 infection in a specific hematological malignancy in the European country first hit by the pandemic. A longer follow-up is needed to further define the impact of Covid-19 infection sequelae in CML patients.

Long-term follow-up of recovered mpn patients with covid-19

To our knowledge, there is no information on long-term follow-up of recovered patients with chronic myeloproliferative neoplasms (MPN) with COVID-19. It can be hypothesized that cytokine storm of the acute phase and the post-COVID persistence of a residual inflammatory state may contribute to elicit hematopoietic stem cell insults and continuous vascular endothelial damage, leading to MPN disease progression and persistent high risk of thrombosis. Aims: To describe sequelae of COVID-19 in surviving patients with MPN following COVID-19. Methods: MPN-COVID study involved 38 European blood centers, and accrued 180 patients with MPN diagnosed with COVID-19 from Feb to Jun 2020, assessing mortality and incidence of thrombosis and bleeding during the acute phase of the pandemic [Barbui T et al. Leukemia. 2021;35(2):485-493. Barbui T et al. Blood Cancer J. 2021;11(2):21]. One-hundred-twenty-five (69%) of these patients survived and were followed up for at least 6 months. Centers were asked to update symptoms, treatments, hematological changes, major outcomes (i.e., thrombosis, disease evolution and death). Results: Among the 125 surviving patients, all eligible for the follow-up update, with a median age 70 years (IQR: 58-79), the following phenotypes were registered: PV (n=38, 30%), ET (n=37, 30%), early PMF (n=14, 11%) and MF (n=36, 29%). During the acute phase of infection, 38 (30%) were managed at home, 80 (64%) in a regular ward and 7 (6%) in ICU. Symptoms (i)The 3 prevalent symptoms during the acute phase of the disease were fever (79%), cough (56%) and dyspnea (53%), while gastrointestinal, neurological, musculoskeletal symptoms, as well as fatigue and anosmia/ dysgeusia, were present in a minor proportion, ranging from 1.6% to 17%. (ii) In the post-acute COVID-19 phase, 36 of 125 patients (32%) declared the persistence of some of these symptoms, fatigue being the most frequent (19%), while none presented persistence of fever and only 10% of dyspnea. Major outcomes (i) Major thrombosis was documented in 5 patients and involved 3 patients with MF (one fatal intestinal ischemia, two non-fatal events: splenic infarction and peripheral artery thrombosis), one case in PV (acute myocardial infarction) and one with ET (DVT of the legs with pulmonary embolism). Age varied from 61 to 80 years. The first event occurred five months after COVID-19 recovery and the Kaplan Meier thrombosis-free survival probability after 9 months was 82%. (ii) Acute myelogenous leukemia (AML) was ascertained in 3 patients (1 in MF, 1 in early-PMF, 1 in ET);one was fatal and occurred in a 49-yearold patient, the other 2 in 78- and 82-year-old patients, respectively. One non-Hodgkin′s lymphoma (in ET) and one progression of a previous parotid carcinoma (in MF) were seen in two patients aged 60 and 77 years, respectively. (iii) Deaths were reported in 8 patients (6.4%), due to AML (n=1), thrombosis (n=1), progression to prior carcinoma (n=2, 1 suspected), multi organ failure (n=1) and heart failure (n=2);the cause was unknown in a single patient. Five deaths (63%) occurred in MF patients. (iv) Overall, the event-free survival pooling together thrombosis, disease evolution and death reached 66% after 9 months from COVID- 19 recovery, indicating that, during this time of observation, 1 out of 3 patients died or have experienced at least one of the other two severe events. Summary/Conclusion: These results indicate that MPN patients who have survived SARS-CoV-2 infection continue to experience severe events suggesting an increased vigilance in the post-COVID period.

Second-Line Bosutinib in Elderly CML Patients: Final Results of Best Study

Introduction: The median age of CML patients failing a first-line TKI because of resistance or intolerance is higher than 60 years. Bosutinib (BOS), dasatinib (DAS) and nilotinib (NIL) have similar second-line efficacy, but in elderly patients DAS and NIL toxicity is more frequent and more clinically relevant. BOS safety profile may be an added value in this setting, but the approved initial dose of 500 mg OAD may be higher than necessary. Aims: All TKIs have been tested in CML patients at a fixed initial dose, with dose reductions in case of toxicity. On the contrary, the aim of our study was to evaluate the efficacy and the tolerability of low-dose second-line BOS in elderly CML patients, using the molecular response at given timepoints to increase the dose only in selected patients, thus finding the minimum effective dose. Methods: A prospective phase 2 single-arm multicenter study has been designed by the GIMEMA CML Working Party (NCT02810990). Study design: All patients started BOS 200 mg OAD for 2 weeks (ârun-inâ period), then the dose was increased to 300 mg OAD;after 3 months, patients with BCR-ABLIS transcript ≤ 1% continued 300 mg OAD, while in patients with transcript > 1% the dose is furtherly increased to 400 mg OAD, in absence of relevant toxicity. The primary endpoint was the rate of MR3 at 12 months. Key inclusion criteria: > 60 yrs old, chronic phase CML, intolerance or failure of any first-line TKI (2013 ELN criteria), absence of T315I or V299L mutation. Results: Sixty-three patients have been enrolled. Median age: 73 yrs (range 60-90). Reasons for switching to BOS: Intolerance 63%, resistance 37%. First-line TKI: Imatinib 83%, DAS 11%, NIL 6%. All patients reached at least 1-year observation. Due to the emergency situation caused by SARS CoV2 spread in Italy, few data are still missing, but final results will be presented onsite. Maximum BOS dose: 400 mg OAD, 19%;300 mg OAD, 76%;200 mg OAD, 5%. At baseline, 17% of patients were already in MR3;MR3 rates at 3, 6 and 12 months were 44%, 54% and 59%, respectively. The cumulative rate of patients achieving or maintaining a MR3 by 12 months was 67%;patients achieving MR4 or MR4.5 by 12 months were 44% and 24%, respectively. Overall, 30%, 29% and 8% of patients had 1 log, 2 logs or > 3 logs reduction from baseline BCR-ABLIS transcript level (67% of patients had a molecular improvement from baseline). Selected adverse events: Acute coronary syndromes, 4 patients;pericarditis, 2 patients;peripheral arterial thrombosis, 1 patient;no pleural effusions were observed. Events leading to permanent treatment discontinuation: 2 unrelated deaths, 7 adverse events, 4 unsatisfactory responses (without progressions), 1 second neoplasia. Fourty-nine patients are still on BOS at the last contact: 10% of them on 400 mg OAD, 61% on 300 mg OAD, 29% on 200 mg OAD. Conclusions: These results trial showed that in elderly patients intolerant to or failing a first-line TKI BOS may be highly effective and better tolerated at a dose lower than 500 mg OAD, namely at 300 mg OAD.