Introduction: The proliferation of literature regarding the COVID-19 pandemic has served to highlight a wide spectrum of disease manifestations and complications, such as thrombotic microangiopathies. Our review with a brief case presentation highlights the increasing recognition of TTP in COVID-19 and describes its salient characteristics. Methods: We screened the available literature in PubMed, EMBASE, and Cochrane databases from inception until April 2022 of articles mentioning COVID-19-associated TTP in English language. Results: From 404 records, we included 8 articles mentioning data of 11 patients in our review. TTP was predominantly reported in females (72%) with a mean age of 48.2 years (SD 15.1). Dyspnea was the most common symptom in one third of patients (36.6%). Neurological symptoms were reported in 27.3% of cases. The time to diagnosis of TTP was 10 days (SD 5.8) from onset of COVID-19. All 11 cases underwent plasma exchange (PLEX), with a mean of 12 sessions per patient, whereas 6 cases received Rituximab (54.5%), and 3 received Caplacizumab (27.3%). One patient died from the illness. Conclusion: This review of available literature highlights the atypical and refractory nature of COVID-19-associated TTP. It required longer sessions of PLEX, with half of the patients receiving at least one immunosuppressant.
Background: Aggressive non-Hodgkin lymphoma (NHL) represents >90% of all NHL that occur in children and adolescents. Among all NHLs, Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5 yrs, EFS) following short but intense multi-agent chemotherapy (Cairo et al. Blood, 2007). Patients who relapse with CD20+ B-NHL and B cell Acute lymphoblastic leukemia (B-ALL) have a dismal prognosis, often associated with chemotherapy resistance and may require alternative therapeutic strategies (Cairo et al. JCO, 2012, Barth/Cairo et al. BJH, 2013). Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe and well-tolerated and is associated with >90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Goldman/Cairo et al. Leukemia, 2013). Resistance to RTX, however, may predispose patients with CD20+ B-NHL/ALL to an increase risk of relapse and/or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012,). Obinutuzumab, a novel glycoengineered type II CD20 antibody, has been shown to enhance cell death and ADCC vs. RTX (Herter et al, Clinc Canc Res, 2013), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate anti-tumor activity of obinutuzumab vs RTX against RTX resistant and sensitive BL and pre-B-ALL tumor targets in-vitro and in-vivo in xenografted NSG mice. Methods: Raji (CD20+) and Loucy (T-ALL, CD20-), (ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs. Cell death was evaluated by staining with AnnexinV/7AAD and analysis by flow-cytometry. Loucy cells (CD20-) were used as the negative control. ADCC were performed with K562-IL-15-41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) at 20:1 effector: target ratio (E: T, n=3) using an europium release assay (Perkin-Elmer).The lentiviral construct, pSico PolII-eGFP-Luc2, was transfected into Raji, Raji 4RH (RTX resistant), U698M and Loucy for in vivo evaluation by BLI. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), mice, bred in-house under pathogen free conditions, were divided into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab 10 mg/kg, obinutuzumab (30 mg/kg), and RTX (30 mg/kg). Mice were xenografted with intravenous injections of Luc+ Raji, Raji4RH, U698M and Loucy cells at 5x106 tumor cells/mouse. 6-8 days after tumor cell injection, mice were then injected every 7 days with the respective therapy for 8 weeks. Mice were monitored for tumor burden and survival for up to 12 weeks ( approx. 80 days) via bioluminescent imaging (BLI) using the IVIS Spectrum system. Results: Obinutuzumab compared to RTX (100 mg/ml, 24hrs), significantly enhanced cell death in Raji 45.1±3.3% vs 32.7±6.8%, (p=0.005), Raji4RH 15.8±2.2% vs 2.1±1.5% (p=0.001) and U698-M 40.5±2.9 % vs 26.36±2.6% (p=0.001) n=6. Obinutuzumab vs RTX also elicited a significant increase ADCC with K562-IL15-41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% (p=0.001), Raji-4RH 40.0±1.6% vs 0.5±1.1%, (p=0.001), and U-698-M 70.0±6 % vs. 45.56± 0.1% (p=0.001) n=3. Further, we found that, in vivo, obinutuzumab was significantly more effective than RTX when administered at the same doses in BL (RTX resistant/sensitive) and pre-B-ALL xenografts. Overall survival in mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to mice receiving 30 mg/kg of RTX in BL; Raji (p=0.05), Raji4RH (p=0.024) and U698-M (p=0.03) (Figure1: A, B and C). Conclusion: Obinutuzumab significantly enhances cell death and NK mediates ADCC in sensitive and RTX resistant CD20+ B-NHL and B-ALL compared to RTX. These preliminary studies also demonstrate that RTX sensitive/resistant BL and pre-B-ALL xenografted mice display significantly increased survival when given 30 mg/kg of obinutuzumab and decreased tumor burden in BL and Pre-B-ALL xenografts compared to an equal dose of RTX. Obinutuzumab may be a novel agent to investigate as adjuvant therapy in patients with relapsed refractory CD20+ B-NHL and/or B-ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
A 76-year-old man with hypogammaglobulinemia on monthly intravenous immunoglobulin infusions presented to the hospital with fever, cough, and shortness of breath and was diagnosed with COVID-19 pneumonia requiring intensive care unit admission but not intubation. He was treated with convalescent plasma, remdesivir and corticosteroids. Sixteen days into his hospitalisation he began to report weakness without sensory symptoms and was found on biopsy to have a necrotising myopathy.
Introduction: The proliferation of literature regarding COVID-19 pandemic has served to highlight a wide spectrum of disease manifestations and complications like thrombotic microangiopathies. Our review with a brief case presentation highlights the increasing recognition of TTP in COVID-19 and describes its salient characteristics. Methods: We screened the available literature in Pubmed, EMBASE and Cochrane database from inception till April 2022 of articles mentioning COVID-19 associated TTP in English Language. Results: From 404 records, we included 8 articles mentioning data of 11 patients in our review. TTP was predominantly reported in females (72%) with a mean age of 48.2 years (SD 15.1). Dyspnea was the most common symptom in 1/3rd of patients (36.6%). Neurological symptoms were reported in 27.3% of cases. The time to diagnosis of TTP was 10 days (SD: 5.8) from onset of Covid-19. All 11 cases underwent plasma exchange (PLEX), with a mean of 12 sessions per patient, whereas six cases received Rituximab (54.5%), and three received Caplacizumab (27.3%). One patient died from the illness. Conclusion: This review of available literature highlights the atypical and refractory nature of COVID-19 associated TTP. It required longer sessions of PLEX with half of the patients receiving at least one immunosuppressant.
Background: Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007). The prognosis has improved with the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013, Cairo et al. JCO, 2012). However, a subset of patients with chemoimmunotherapy-resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however, the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab, mediates enhanced cell death & ADCC against diffuse B-cell lymphoma vs. RTX (Mössner et al. Blood, 2010), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathway proteins altered differentially after obinutuzumab or RTX treatment against RTX sensitive/ resistant BL cell lines. Methods: Raji (CD20+, ATCC, Manhass, VA) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs . Cell death was evaluated by staining with AnnexinV/7AAD and analyzed by flow-cytometry. ADCC were performed with K562-IL-15-41BBL expanded NK cells at 20:1 effector: target ratio (E: T, n=3) using an europium release assay (Perkin-Elmer). For Phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji, /Raji4RH in the presence/absence of obinutuzumab or rituximab (100µg/ml for 24h) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides were subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation. An LTQ Orbitrap XL in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data that were searched with the Swissprot Human taxonomic protein database. Results: Obinutuzumab, compared to RTX, significantly enhanced cell death in Raji 45.1±3.3% vs. 32.7±6.8%, (p=0.005) & Raji4RH 15.8±2.2% vs. 2.1±1.5% (p=0.001), respectively. Overall survival of mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to mice receiving 30 mg/kg of RTX in Raji (p=0.05) & Raji4RH (p=0.024), respectively. In Raji, 329 proteins were differentially phosphorylated (>1.5-fold change) between obinutuzumab vs. RTX. Of these proteins, 171 were expressed at higher levels in obinutuzumab than in RTX. Proteins differentially phosphorylated in response to obinutuzumab and RTX treatment were involved in the B-cell receptor (BCR) signaling pathway (LYN, BTK, CD19, PLCG2, INPP5D, NFATC1 and PIK3AP1), the spliceosome (TRA2A, DDX46 and PRPF31), and the cell cycle signaling pathway (WEE1, MMC3, GSK3B and CCNH). (Fig.1A) Obinutuzumab and RTX also resulted in a differential phosphorylation of 606 proteins in Raji4RH. These proteins were involved in the spliceosome (CDC42, TRA2A and DDX42), tight junction (HCLS1, PRKCD, EPB41 and MYH2) and nucleotide excision repair (POLD3, CCNH and LIG1) pathways. (Fig.1B) Differential phosphorylation of BCR signaling pathways proteins (BTK, PLCG2 and GSK3B) was validated by western blot studies after incubation with obinutuzumab vs. RTX in Raji/Raji4RH cell lines, reveled up regulation of BTK and PLCY2 after obinutuzumab treatment vs. RTX treatment in Raji BL cell line. Conclusions: These data suggest that obinutuzumab vs. rituximab treatment result in global changes in BL proteins involved in BCR, spliceosome, cell cycle, nucleotide excision repair & tight junction signaling pathway. Furthermore, BCR signaling pathways appear more affected by obinutuzumab compared to RTX in Raji cell lines as compared to Raji4RH. Further, these data revealed the utility of unbiased phosphoproteome interrogation of obinutuzumab vs. rituximab mediated signaling events as well as characterizing signaling networks that may provide insights into pathogenetic mechanisms of rituximab resistance. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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