Summary The standardized EuroFlow protocol, including CD19 as primary B‐cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B‐cell precursor acute lymphoblastic leukaemia (BCP‐ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP‐ALL patients treated with CD19‐targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19‐positive, whereas this was 81% in patients that became (partially) CD19‐negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.
Background: CD20 expression is a controversial issue regarding response prediction to anti-CD20 therapy in chronic lymphocytic leukemia (CLL). Methods: Median fluorescence intensities (MFIs) of standard fluorescence beads from the daily calibration of flow cytometers according to EuroFlow protocols were used to establish a normalization approach to study CD20 expression on CLL cells. CD20 MFI was retrospectively assessed prior to and during treatment from flow cytometric measurements of peripheral blood in patients with different depths of molecular response in the four phase-II CLL2-BXX trials (BIG; BAG; BIO; BCG; N = 194) administering either Obinutuzumab or Ofatumumab in combination with targeted agents. Results: No significant difference was observed between the normalized and measured MFIs of CD19 and CD20 on CLL cells. During treatment, CD20 expression levels on CLL cells did not significantly differ between the four investigated different treatment schemes, but a strong molecular response to Ofatumumab seemed to correlate with higher CD20 expression prior to therapy. Conclusions: Standardized staining and instrument monitoring enable a robust assessment of longitudinal biological variations of marker expression based on MFI values. Obinutuzumab showed a higher proportion of patients with a strong MRD response independent from initial CD20 expression, whereas high pre-therapeutic CD20 expression levels seem to correlate with a profound response to Ofatumumab.
In murine abdominal sepsis by colon ascendens stent peritonitis (CASP), a strong increase in serum IgM and IgG antibodies was observed, which reached maximum values 14 days following sepsis induction. The specificity of this antibody response was studied in serum and at the single cell level using a broad panel of bacterial, sepsis-unrelated as well as self-antigens. Whereas an antibacterial IgM/IgG response was rarely observed, studies at the single-cell level revealed that IgM antibodies, in particular, were largely polyreactive. Interestingly, at least 16% of the IgM mAbs and 20% of the IgG mAbs derived from post-septic mice showed specificity for oxidation-specific epitopes (OSEs), which are known targets of the innate/adaptive immune response. This identifies those self-antigens as the main target of B cell responses in sepsis.
Patients with B cell precursor acute lymphoblastic leukemia (BCP-ALL) have a favorable prognosis, yet current treatment protocols are based on intensive cytotoxic chemotherapy and therapy options are limited when patients relapse. Novel immunotherapy approaches are therefore needed. We recently reported that the preB cell receptor signaling unit CD79a (known as Igα) is crucial for BCP-ALL engraftment in vivo, particularly in the central nervous system (CNS) employing BCR-ABL + and E2A-PBX1 + patient derived xenograft (PDX) models (Lenk et al., Communications Biology, 2021). CD79a forms a heterodimer with CD79b (known as Igß), which is also expressed on the surface of mature B cells as part of the B cell antigen receptor. Accordingly, the CD79b antibody drug conjugate (ADC) Polatuzumab Vedotin (PolVed) has shown therapeutic efficacy in the treatment of refractory/relapsed (r/r) diffuse large B cell lymphoma. Moreover, CD79a/CD79b may also be present on the cell surface at the pro/pre B cell stage. We therefore hypothesize that CD79b can serve as a therapeutic target in BCP-ALL. First, to substantiate that CD79b is important for BCP-ALL engraftment in vivo, a murine/murine transplantation model was applied. B cell precursors were isolated from mice harboring CD79b with a non-functional signaling domain (CD79b-ITAM-KO) or wildtype mice, malignantly transformed with a BCR-ABL fusion construct and transplanted into NSG-mice. Whereas control cells caused overt leukemia in all animals within 25 days, no animal injected with CD79b-ITAM-KO cells had developed leukemia by the time of sacrifice at 162 days (P<0.001). To investigate the frequency of surface (s)CD79b expression in BCP-ALL patients, we measured sCD79b levels via flow cytometry in diagnostic samples of pediatric BCP-ALL patients with different cytogenetic backgrounds. We detected sCD79b-positivity (defined as ≥10% sCD79b + BCP-ALL cells) in 23/94 patients including BCR-ABL +, E2A-PBX1 +, MLL-rearranged (MLLr), TEL-AML1 + and B-other BCP-ALL patients indicating a population of sCD79b + patients within different cytogenetic BCP-ALL subgroups (Figure 1a). To validate CD79b as a therapeutic target, we applied an unconjugated monoclonal CD79b antibody (CD79b-mAB) to PDX mice bearing either pediatric BCR-ABL + or E2A-PBX1 + PDX samples with high sCD79b expression (49.6% sCD79b + cells and 37.7% sCD79b + cells, respectively). Treatment was initiated one day after BCP-ALL injection, modelling a minimal residual disease (MRD) situation. Therapy with CD79b-mAB (1mg/kg) resulted in a mild reduction of leukemia burden in the spleen (SP) and bone marrow (BM) of PDX mice and a significant reduction of CNS-involvement in both PDX-models as compared to control treated mice (P<0.05 and P<0.01, respectively). To test the hypothesis that treatment of BCP-ALL with a CD79b-ADC outperforms CD79b-mAB, we applied PolVed (1mg/kg) in NSG mice bearing the same E2A-PBX1 + and BCR-ABL + PDX cells. PolVed therapy resulted in a significant reduction of BCP-ALL engraftment in SP, BM and CNS (P<0.01, respectively) and a significant survival prolongation compared to control treated mice in both models (P<0.01, respectively). Of note, 4/5 PolVed-treated E2A-PBX1-PDX animals were free of leukemia by the time of sacrifice (236 days) (Figure 1B). To test the efficacy of PolVed on different BCP-ALL subgroups, we conducted a preclinical phase II-like PDX study, using sCD79b high and sCD79b low PDX samples (defined by sCD79b-expression above or below the median) (5E2A-PBX1 +, 3 BCR-ABL +, 2 MLLr, 1 E2A-HLF + and 1 ETV6-NTRK3 +). Two NSG mice per patient were injected with PDX cells, randomly assigned into treatment groups and PolVed therapy was initiated when 1% PDX-cells were detected in the peripheral blood, modelling an overt leukemia situation. sCD79b low PDX mice did not respond to PolVed treatment (Figure 1c), but we detected a response to therapy and a significant survival prolongation in 5/6 sCD79b high PDX samples irrespective of the cytogenetic background (P<0.05) (Figure 1d). Taken together, our data indicate that a subgroup of BCP-ALL patients is sCD79 + positive and may respond to PolVed treatment. Therefore, we suggest CD79b as a novel therapeutic target in BCP-ALL and propose PolVed as a potential therapeutic agent in r/r disease. *LL and DW contributed equally to this work Figure 1 Figure 1. Disclosures Lenk: OSE Immunotherapeutics: Research Funding. Richter: HTG Molecular Diagnostics, Inc.: Current Employment, Research Funding. Schrappe: SHIRE: Other: research support; JazzPharma: Honoraria, Other: research support; Servier: Honoraria, Other: research support; SigmaTau: Other: research support; Novartis: Honoraria; JazzPharma: Honoraria; Novartis: Honoraria, Other: research support; Servier: Honoraria; Amgen: Other: research support. Cario: Novartis: Other: Lecture Fee. Brüggemann: Incyte: Other: Advisory Board; Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau; Janssen: Speakers Bureau. Schewe: Jazz Pharmaceuticals: Other: Advisory Board; SOBI: Other: Advisory Board; Bayer: Other: Advisory Board; OSE Immunotherapeutics: Research Funding.
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