COVID-19 in Patients Receiving CD20-depleting Immunochemotherapy for B-cell Lymphoma

The clinical and immunological impact of B-cell depletion in the context of coronavirus disease 2019 (COVID-19) is unclear. We conducted a prospectively planned analysis of COVID-19 in patients who received B-cell depleting anti-CD20 antibodies and chemotherapy for B-cell lymphomas. The control cohort consisted of age- and sex-matched patients without lymphoma who were hospitalized because of COVID-19. We performed detailed clinical analyses, in-depth cellular and molecular immune profiling, and comprehensive virological studies in 12 patients with available biospecimens. B-cell depleted lymphoma patients had more severe and protracted clinical course (median hospitalization 88 versus 17 d). All patients actively receiving immunochemotherapy (n = 5) required ICU support including long-term mechanical ventilation. Neutrophil recovery following granulocyte colony stimulating factor stimulation coincided with hyperinflammation and clinical deterioration in 4 of the 5 patients. Immune cell profiling and gene expression analysis of peripheral blood mononuclear cells revealed early activation of monocytes/macrophages, neutrophils, and the complement system in B-cell depleted lymphoma patients, with subsequent exacerbation of the inflammatory response and dysfunctional interferon signaling at the time of clinical deterioration of COVID-19. Longitudinal immune cell profiling and functional in vitro assays showed SARS-CoV-2-specific CD8+ and CD4+ T-effector cell responses. Finally, we observed long-term detection of SARS-CoV-2 in respiratory specimens (median 84 versus 12 d) and an inability to mount lasting SARS-CoV-2 antibody responses in B-cell depleted lymphoma patients. In summary, we identified clinically relevant particularities of COVID-19 in lymphoma patients receiving B-cell depleting immunochemotherapies.


Introduction
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has led to a rapidly unfolding pandemic with extensive morbidity and mortality. [1][2][3] The clinical presentation of COVID-19 is highly variable, ranging from asymptomatic infection to respiratory failure with fatal outcome. [3][4][5] Several risk factors for severe clinical course have been described, including malignancies and immunocompromised state due to anticancer therapies. 2,[6][7][8][9] Therefore, clinicians now have to balance the risks and benefit of providing state-of-the-art anticancer therapies and the possibility of more severe clinical course of SARS-CoV-2 infections. 10,11 However, currently available guidelines are primarily based on expert opinion as data on specific malignancies and treatments is still limited. 12,13 Standard treatment of patients with B-cell non-Hodgkin lymphomas (B-NHL) frequently includes monoclonal anti-CD20 antibodies (eg, rituximab and obinutuzumab), which deplete B-cells for at least 6-9 months. 14,15 B-cell depletion could compromise adaptive antiviral immune responses, delay viral clearance, and prolong viral shedding [16][17][18] and lead to more severe disease course, higher risk of re-infection, and prolonged infectivity. [19][20][21][22][23] Here, we report a prospectively planned analysis of COVID-19 in patients from two university hospital registries who received B-cell depleting immunochemotherapies for B-NHL during the first wave of the COVID-19 pandemic in Europe. We provide a detailed analysis of the clinical course with a long-term follow-up along with in-depth profiling of the humoral, cellular and molecular immune responses in comparison to an age-and sexmatched cohort of COVID-19 patients without B-NHL.

Patients and data
Patients are part of the prospective COVID-19 registries of the University Hospital of the Ludwig-Maximilians-University (CORKUM, WHO Trial ID DRKS00021225) or the University Hospital of Technical University of Munich (COMRI). Patients were eligible for this study if they had SARS-CoV-2 infection confirmed by specific real-time polymerase chain reaction (RT-PCR) in a respiratory specimen. The study cohort consisted of patients who received B-cell depleting anti-CD20 antibody-containing immunochemotherapies (ICT) for B-NHL. The control cohort consisted of age-and sex-matched patients without B-NHL who were hospitalized because of symptomatic COVID-19 (See Supplemental Digital Figure 1, http://links.lww.com/HS/A169, CONSORT diagram). We only included patients with available peripheral blood samples (collected centrally up to twice weekly as an optional part of the registries) to determine humoral, cellular and molecular immune profiles (See Supplemental Digital Figure 2, http://links.lww.com/HS/A169). Clinical and routine laboratory data were prospectively collected within the COVID-19 registries and verified and complemented by individual chart review. Clinical deterioration was defined as the need of ICU support. Respiratory deterioration was defined as the need of mechanical ventilation.
Patient data were pseudonymized for analysis, and the study was approved by the local ethics committees (No: #20-245 and #221/20 S). Written informed consent was obtained from each patient or authorized by proxy before any study-related procedure.

SARS-CoV-2 RT-PCR and serology
RT-PCR from respiratory specimen and blood serum used targets in the N and E genes as previously described. 24 IgG against SARS-CoV-2 was detected in serum samples using Anti-SARS-CoV-2-ELISA targeting the spike domain S1, according to manufacturers' protocols (Euroimmune and Roche). 25 Additional technical details are provided in Supplemental Methods section, http://links.lww.com/HS/A169.

Virus culture
Culture for SARS-CoV-2 was performed as previously described. 26 In brief, Vero E6 cells were inoculated with patient respiratory samples (nasopharygeal swap or tracheal aspirate as indicated) and observed daily for a cytopathic effect up to day 7. If negative, an additional passage was performed and observed for another 7 days. On observation of a cytopathogenic effect, the supernatant was harvested and further analyzed for the presence of SARS-CoV-2 RNA by specific real-time PCR detecting the N gene.

Flow cytometry of peripheral blood mononuclear cells
Immune profiling of peripheral blood mononuclear cells (PBMCs) was done by 32-parameter flow cytometry according to previously published guidelines. 27 Flow cytometry measurements were performed on a CytoFLEX LX flow cytometer (Beckman Coulter) and data were analyzed using FlowJo, v10 (Tree Star Inc., Ashland, OR, USA). The commercial fluorochrome conjugated antibodies are listed in the Supplemental Methods, http://links.lww.com/HS/A169.

Digital multiplexed gene expression profiling of PBMCs
Digital multiplexed gene expression profiling of PBMCs was performed as previously described. 28,29 Briefly, RNA from PBMCs was isolated using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA concentration was measured by NanoDrop 1000 (ThermoFisher, Waltham, MA, USA) and 100 ng were assayed with the PanCancer Human IO 360 Panel according to the manufacturer's protocol (NanoString, Seattle, WA, USA). Normalization was performed by subtracting the mean + 2 SDs of negative control as a cutoff and adjustment for geometric mean of defined positive controls and defined housekeeping genes. Housekeeping gene normalization threshold was set at 0.1-to 10-fold.
All data and protocols are available to other investigators. For original data, please contact oliver.weigert@med.unimuenchen.de.
We compared the known clinical risk factors for severe COVID-19 manifestation between B-cell depleted lymphoma patients and controls (Table 1): 1 and 4 patients had arterial hypertension, 2 each had diabetes, 0 and 1 had chronic pulmonary disease, and 1 each was obese, respectively.
All but 1 lymphoma patient (83%) required mechanical ventilation as compared to 2 control patients (33%). Respiratory failure developed in all lymphoma patients on active ICT (no. 1-5) within a median of 14 days (range 11-26 d) after the diagnosis of COVID-19, while patient no. 6 (who had completed ICT before her SARS-CoV-2 infection) had a remarkably mild clinical course. The median time on mechanical ventilation was 62 versus 10 days, the median ICU stay 68 versus 14 days. ICU management of B-cell depleted lymphoma patients was highly complex including extracorporeal membrane oxygenation (n = 1), continuous veno-venous hemodialysis (n = 3), and extracorporeal cytokine adsorption (CytoSorb, n = 1). After a median follow-up of 169 days (range 81-217), 5 patients were alive and Neutrophil recovery frequently coincided with hyperinflammation and clinical deterioration Overall, lymphoma patients had rising inflammatory markers in routine laboratory testing at the time of clinical deterioration, including C-reactive protein (CRP) and IL-6 serum levels ( Fig. 2A, B). Respiratory deterioration coincided with leukocyte and neutrophil recovery (Fig. 2C, D) and a trend toward lower lymphocyte counts (Fig. 2E), resulting in a massively increased neutrophil-lymphocyte-ratio (NLR, Fig. 2F). Of note, 4 of 5 patients on active ICT had received granulocyte colony stimulating factor (G-CSF) injections to accelerate neutrophil recovery, there of 3 within 1 week before ICU admission. G-CSF was given as part of the R-CHOP14 protocol 30 in 2 patients (no. 2 and 4) or to treat chemotherapy-induced

B-cell depleted patients can mount a SARS-CoV-2-specific T-effector cell response
We next investigated the adaptive immune response in B-cell depleted SARS-CoV-2-infected patients (Fig. 3A). Flow cytometry of PBMCs confirmed complete B-cell depletion in all lymphoma patients (Fig. 3B). At the time of clinical deterioration, absolute lymphocyte counts dropped further in these patients (Fig. 3C), but the fraction of CD8 + T-cells increased (Fig. 3D). Furthermore, we noticed a shift from naïve phenotype to an effector memory (EM) subtype (Fig. 3E). A similar predominance of EM as well as T-effector memory cells re-expressing CD45RA (TEMRA) was also seen in CD8 + T-cells from the B-cell depleted lymphoma patient with uncomplicated COVID-19 (Patient no. 6) and in CD8 + T-cells from control patients, all collected at later time points of the disease (Fig. 3E). In CD4 + T-cells, the central memory (CM) phenotype fraction was generally larger compared to CD8 + T-cells, but we still observed a similar shift from naive T-cells to EM phenotype in B-cell depleted lymphoma patients (Fig. 3F, G).
To investigate whether the expanding EM populations comprise bona fide virus-specific T-cells, we probed CD8 + and CD4 + T-cells from serially collected peripheral blood samples of B-cell depleted patients for their specificity towards SARS-CoV-2 spike protein.
Virus-specific T-cells were not detectable at early time points but became detectable during the course of the disease in all 3 evaluable cases. Time to first measurable SARS-CoV-2 reactive T-cells was highly variable, ranging from 35 to 77 days for CD8 + T-cells (Fig. 3H, I) and from 17 to 40 days for CD4 + T-cells (Fig. 3J, K), respectively.

Monocyte hyperactivation in B-cell depleted patients
As activation of the monocyte-macrophage axis has been described before in severe COVID-19, 31 we determined the shift of monocyte phenotypes during the course of the disease (Fig. 3L, M), distinguishing classical (CD14 + CD16 − ), intermediate (CD14 + CD16 + ), and nonclassical (CD14 dim CD16 + ) monocytes (Fig. 3N). 32 While classical monocytes are critical for the initial inflammatory response, intermediate monocytes express higher levels of proinflammatory cytokines (including IL-1, IL-6, and IL-10), 33 and nonclassical monocytes exhibit distinct functional properties, including promotion of neutrophil adhesion to inflamed endothelium. 34 Interestingly, we observed    (Fig. 3M, N), which was more pronounced compared to controls.

Dysregulated molecular immune responses in B-cell depleted lymphoma patients with COVID-19
To comprehensively characterize immune responses in B-cell depleted lymphoma patients versus controls on a transcriptional level, we performed immune gene expression profiling from PBMCs collected at early time points of COVID-19 (ie, before clinical deterioration) (Fig. 4A). Unsupervised clustering clearly separated lymphoma patients from controls (see Supplemental Figure 3A, http://links.lww.com/HS/A169). We inferred abundances of individual PBMC populations by marker gene expression. As expected, B-cell depleted lymphoma patients had lower B-cell scores as compared to controls (see Supplemental Figure  4B, http://links.lww.com/HS/A169), showing significant and large fold lower expression of B-cell genes, including MS4A1 (CD20), CD19, CD79A, and FCRL2 (Fig. 4B). Gene set analysis showed downregulation of the "lymphoid compartment" (Fig. 4C), including lower expression of IL7R, CCR4, and CXCR4 (Fig. 4D).
In contrast, macrophage and neutrophil scores were higher in B-cell depleted patients as compared to controls (see Supplemental Figure 4C, D, http://links.lww.com/HS/A169). Gene set analysis revealed that top differentially expressed genes were enriched within the biological pathways "cytokine and chemokine signaling," "interferon signaling," "myeloid compartment," and "cytotoxicity," including upregulation of IL10, IL18, CSF1R, and CSF3R (Fig. 4C, D). Furthermore, differential gene expression showed large fold higher expression of C1QA and C1QB (Fig. 4B), the first components of the classical serum complement cascade.
Longitudinal analysis of immune gene expression in PBMCs from B-cell depleted patients (Fig. 4E) showed a further increase in macrophage scores (Fig. 4F) as well as CD8 + T-cell scores (Fig.  4G) at the time of clinical deterioration. Gene set enrichment analysis (GSEA) over time showed significant enrichment of the GSEA MSigDB hallmark signature "inflammatory response," with further upregulation of IL10, IL1A, and CSF1 (Fig. 4H-K).  In summary, our immune gene expression indicates early hyperactivation of neutrophils and monocytes/macrophages, as well as the complement system in B-cell depleted patients with COVID-19, with subsequent exacerbation of the inflammatory response and dysfunctional interferon signaling at the time of clinical deterioration.
Long-term detection of replication-competent SARS-CoV-2 in B-cell depleted lymphoma patients Finally, we compared SARS-CoV-2 clearance kinetics in patients with and without B-cell depletion. B-cell depleted lymphoma patients had a markedly longer detectability of SARS-CoV-2 RNA in respiratory specimens compared to controls as assessed by RT-PCR (median 84 versus 12 d) (Fig. 5A-C). To determine whether patients shed replication-competent virus, we tested respiratory specimens from 3 lymphoma patients for the presence of virus in cell culture. Remarkably, SARS-CoV-2 could be cultured from specimen of all tested patients, one patient (no. 4) harboring infectious virus for >20 weeks (Fig. 5B). Two patients showed recurring viremia, 1 patient over a time period of >4 weeks. SARS-CoV-2-specific IgG antibodies (Fig. 5B) could only be detected transiently at low levels in 3 B-cell depleted patients shortly after the administration of convalescent plasma. Similar results were obtained with a different antibody assay (see Supplemental Digital Figure 9, http://links.lww.com/HS/ A169).

Discussion
Through detailed analysis of the clinical course and comprehensive laboratory, immunological and virological studies, we identify several clinically relevant particularities of COVID-19 in patients with lymphoma who have received B-cell depleting immunochemotherapies.
First, patients on active immunochemotherapy have long and severe clinical courses. This is in line with earlier reports of severe COVID-19 in B-cell depleted patients. 35,36 In contrast to the previously reported excess mortality, 37 only 1 patient in our series died from COVID-19. Of note, this patient presented late during the course of her disease to another hospital before being transferred to our ICU with rapid clinical deterioration, immediately requiring mechanical ventilation and extracorporeal membrane oxygenation support. This suggests that B-cell depleted patients with COVID-19 can benefit from close clinical observation to implement early and rigorous ICU support, including long-term mechanical ventilation.
Second, we observe a coincidence of rapid clinical deterioration and hematological recovery after immunochemotherapy, suggesting that particular close clinical monitoring is mandatory during this critical time period. Neutrophil recovery, further drop of lymphocyte counts, and especially an increasing neutrophil-to-lymphocyte ratio (NLR) 38,39 may be useful clinical indicators of impending respiratory decompensation.
In fact, the rapid rise of neutrophils and monocytes during hematological recovery could directly contribute to respiratory decompensation by facilitating myeloid cell recruitment to the lungs. 40 Of note, these cells are highly prevalent in inflammatory www.hemaspherejournal.com lung infiltrates in severe  This may also explain the remarkably indolent clinical course of COVID-19 in our B-cell depleted patient (no. 6) who was not on active lymphoma treatment, and hence, did not undergo this phase of hematologic recovery during her infection. On the other hand, G-CSF is frequently administered to accelerate neutrophil recovery after immunochemotherapy for lymphoma, 30,42,43 as was the case with 4 of our 5 patients on active treatment. Therefore, our study should at least raise concerns over the routine use of G-CSF in these patients, and justifies further investigations. 44 Third, our study provides novel insights into the particular immune reactions in response to COVID-19 in B-cell depleted patients. We notice an early activation of the myeloid compartment, with subsequent hyperactivation of monocytes and pathological inflammation with high levels of IL-6, IL-10, IL-18 45 and others ("cytokine storm") 46,47 at the time of hematological recovery and clinical deterioration. However, due to limited size of our study, we realize that this data is primarily hypothesis-generating at this point and requires validation in larger cohorts with longer follow-up.
Finally, our study shows that B-cell depleted patients are incapable of developing lasting SARS-CoV-2 antibody responses, with long-term detectable viral RNA in upper respiratory tract specimens despite receiving convalescent plasma. In fact, we documented shedding of replication-competent virus over months, providing evidence that these patients are potentially capable of transmitting SARS-CoV-2 beyond commonly used quarantine periods. Furthermore, these patients remain at increased risk of SARS-CoV-2 reinfections, as has recently been documented in a case report. 19 Interestingly, all but 1 B-cell depleted patient ultimately eliminated SARS-CoV-2, yet it took them >6 times longer compared to controls. This strongly suggests that rapid virus elimination critically depends on virus-specific antibody responses. [48][49][50] In our series, administration of convalescence plasma resulted only in transient and low antibody levels in some patients and are therefore unlikely to have substantially contributed to virus elimination. Instead, we notice SARS-CoV-2-specific T-effector cell responses over time that are comparable to controls in the resolution phase of COVID-19 (data not shown). It remains to be studied, to what extent these T-cell responses actually contribute to virus elimination and whether they could potentially provide protective immunity in these patients. 51 We realize that the small cohort size is a major limitation of our study. Also, we only studied patients infected with the SARS-CoV-2 lineages B, B.1, and B.1.1 (data not shown) who were hospitalized during the first wave of the COVID-19 pandemic in Europe. As such, it remains unclear if these results are representative of the clinical course with other SARS-CoV-2 lineages or subsequent pandemic waves in other areas of the world. Importantly, however, our data with longterm follow-up complements previous retrospective studies 9,10,12 by an in-depth analysis of a homogenous cohort of patients with B-NHL who received B-cell depleting frontline immunochemotherapy. Our major findings are highly consistent and clinically relevant. Based on this study, we now take particular precautions at our institutions in managing COVID-19 in patients who have received B-cell depleting immunochemotherapies: 1. We admit all of these patients to the hospital irrespective of the severity of COVID-19 at the time of presentation. 2. We provide particular close monitoring at the time of hematological recovery and avoid the routine use of G-CSF. 3. We recommend early transfer to an ICU with experience in treating patients with COVID-19, prepared to provide long-term life-sustaining treatment. 4. We monitor these patients for long-term virus excretion and inform patients and their contacts (including health care providers) about the possibility of long-term infectivity and the risk of reinfections.
Ultimately, whenever clinically feasible, 13 we aim to vaccinate these patients prior to initiating B-cell depleting immunochemotherapies.