Impaired response to first <scp>SARS‐CoV</scp>‐2 dose vaccination in myeloproliferative neoplasm patients receiving ruxolitinib

Abstract

Covid-19, the disease caused by pandemic SARS-CoV-2 infection, had significant impact on patients with hematologic conditions1; a meta-analysis involving 3377 patients with hematologic malignancies who were affected by Covid-19 reported a mortality rate of 34%.2 A similarly dismal outcome was documented among 175 patients with chronic myeloproliferative neoplasms (MPN), collected in a European observational study, where mortality rate was 30% for the entire cohort, reaching 48% in primary overt myelofibrosis (MF).3 Covid-19 was also associated with higher incidence of thrombosis in patients with essential thrombocythemia (ET), compared to MF and polycythemia vera (PV) (20% vs 5% for both, respectively).4 Finally, MPN patients surviving the acute phase may suffer from additional long-term sequelae from Covid-19, that furtherly increase mortality and morbidity.5 The JAK1 and JAK2 inhibitor (JAKi) ruxolitinib is approved for the treatment of patients with MF and hydroxyurea resistant/refractory PV.6 By inhibiting JAK–STAT signaling, ruxolitinib has profound effects on different cell compartments of the immune system, including T cells, natural killer and dendritic cells, in addition to potently dampening inflammatory cytokine production.7 These properties have been mechanistically linked to the increased rate of infections in MPN patients receiving ruxolitinib, and, conversely, were explored successfully in the setting of steroid-refractory acute graft vs host disease following allogeneic stem cell transplantation.8 In the above cited European study in MPN, rapid discontinuation of the drug was implicated in 75% of deaths occurring in the ruxolitinib-treated cohort; these were ascribed to a previously described “discontinuation syndrome”, a potentially fatal complication due to a cytokine storm that follows the abrupt suspension of ruxolitinib.9, 10 In fact, observational studies support the effectiveness of ruxolitinib to quench the hyper inflammatory reaction accompanying Covid-19 in the general population.11, 12 Due to the immunomodulatory properties of ruxolitinib, the question arises whether response to vaccination for SARS-CoV-2 in patients under stable ruxolitinib therapy might be impaired. We prospectively assessed serologic response following the first injection of SARS-CoV-2 mRNA vaccine in 30 consecutive patients with PV, ET and MF who were referred to the Center of Research and Innovation of Myeloproliferative Neoplasms (CRIMM), Florence. Patients signed an informed consent to participate in the study, that was approved by the local Ethical Committee of Azienda Ospedaliera Careggi, Florence. Patients were eligible if they had a diagnosis of MPN according to the 2016 WHO criteria and all the following at the time of study entry: no history of positivity for SARS-CoV-2 by PCR on swab; negativity of serum anti-nucleoprotein antibodies; no clinical suspicion of Covid-19. A cohort of 14 healthy volunteers without prior SARS-CoV-2 infection was used as a reference group. The vaccines used were the Moderna and Pfizer vaccine in 25 (83%) and five (17%) MPN patients, and 10 (71%) and four (29%) healthy controls, respectively. Blood samples were collected before first vaccination (T0) and right before the second dose administration (T1, day 21 for Pifzer, day 28 for Moderna). Serologic tests for SARS-CoV-2 antibodies were performed to demonstrate presence of IgG antibodies against spike (S) protein, receptor binding domain (RBD) and neutralizing antibodies. A cut-off value of test positivity was established for each antibody type according to manufacturer's instructions; patients above the upper cut-off level were considered as “responders”, and those below as “non responders”. Categorical variables were expressed as frequency and percentage. Chi-square test was used to compare categorical variables. Clinical characteristics of the patients are outlined in Table S1. There were 10, seven and 13 patients with PV, ET and MF (two and three patients were post-PV and post-ET MF, respectively). Of these, 18 were on a stable dose of ruxolitinib since at least 3 months (ruxo-patients), while 12 were not currently treated, nor had received before, ruxolitinib (no-ruxo patients), including five patients under watch-and-wait and five under hydroxyurea therapy since at least 3 years. The current median dose of ruxolitinib was 20 mg daily (range, 10–50 mg), and the median duration of ruxolitinib therapy was 7.3 years (range, 0.8–13.8 years). Figure 1 shows the levels of individual anti-SARS-CoV-2 antibodies at T0 and T1. Anti-S IgG, anti-RBD IgG and neutralizing Ab were not detected before vaccination in any of the three groups, accordingly to predefined cut-off levels. In general, the extent of specific antibody response after first dose vaccination (T1 time point), measured as binding antibody unit (BAU)/mL for anti-S and anti-RBD immunoglobulin, and relative index for neutralizing antibodies (Figure 1(A)–(C)), was significantly lower in ruxo-patients compared to healthy controls and the no-ruxo group. The latter conversely did not differ significantly from controls, suggesting that the potentiality to mount adequate immune response is maintained in most MPN patients who were not receiving ruxolitinib. In detail, mean anti-S BAU levels/ml were 111.4, 513.4 and 510.8 for ruxo, no-ruxo and controls, respectively (Figure 1(A)); corresponding anti-RBD BAU levels/mL were 65.2, 631.7 and 254.2 (Figure 1(B)); and, for neutralizing antibodies, the mean relative index was 1.1, 3.5 and 3.8 (Figure 1(C)). All these values were statistically different when comparing ruxo patients vs no-ruxo and controls, but not between controls and no-ruxo patients (p values are reported in Figure 1(A)–(C)). According to the predefined cut-off levels, all the 14 healthy volunteers were considered as responders to vaccination for the anti-S IgG, anti RBD IgG and neutralizing antibodies (100% each), compared to 38.8%, 33.3% and 33.3%, respectively, for the ruxo-patients (p < 0.001 vs controls for each antibody type), and 91.6% (p = not significant), 91.6% (p = not significant) and 58.3% (p = < 0.01) in the no-ruxo group (Figure 1(D)–(F)). In summary, these findings, with the limitation of the small number of subjects included, make a strong and urgent argument for an impaired early response to SARS-CoV-2 vaccine in patients receiving ruxolitinib. Further and future studies are needed to address whether such unresponsive status persists after the second dose of vaccine, as suggested by a study performed in Israel, where the rate of seropositivity (anti-S1/S2 IgG) after complete vaccination in patients with MPN was 42% for those using JAKi.13 It will be important to address whether also responses mediated by T-cell and other myeloid cells are impaired by ruxolitinib treatment, owing to their key role in SARS-CoV-2 infection.14-16 Although clear-cut relationships between specific anti-SARS-CoV-2 immunoglobulin titers and protection against the virus has not been unequivocally established in the general population, MPN patients receiving ruxolitinib should be urged to continue to adopt the best preventive measures against Covid-19 even after receiving vaccination, in the light of the evidences presented herein. Furthermore, it is also suggested that, since MPN patients not receiving ruxolitinib overall developed antibody titers that were comparable to healthy volunteers, but a proportion of them not did actually produce neutralizing antibodies, initiation of ruxolitinib therapy in a naïve patient might be prudentially delayed after completion of vaccination, unless urgently needed. This notwithstanding, we reinforce that patients with MPN, as for any hematologic malignancies, should be vaccinated, since the possibility of protection at any extent outweighs minor risks,2 as supported also by recommendations from the American Society of Hematology (https://www.hematology.org/covid-19). Open Access Funding provided by Universita degli Studi di Firenze within the CRUI-CARE Agreement. A.M.V., membership on Advisory Board, Novartis; P.G., membership on Advisory Board, Novartis. Other authors declare no conflict of interest. Paola Guglielmelli, Alessandro M Vannucchi, Francesco Annunziato designed research Paola Guglielmelli, Alessio Mazzoni, Laura Maggi, Seble Tekle Kiros, Lorenzo Zammarchi, Sofia Pilerci, Arianna Rocca, Michele Spinicci, Miriam Borella, Alessandro Bartoloni, Gian Maria Rossolini, Francesco Annunziato, Alessandro M Vannucchi collected patients and performed research. Paola Guglielmelli, Alessio Mazzoni, Francesco Annunziato, Alessandro M Vannucchi wrote the manuscript. All authors read and approved the final version. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions Table S1 Patients' characteristics. 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.

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