Multiplex imaging reveals spatially resolved DNA-damage response neighborhoods in TP53-mutated myelodysplastic neoplasms.

While increased DNA damage is a well-described feature of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), it is unclear whether all lineages and all regions of the marrow are homogeneously affected. In this study, we performed immunohistochemistry on formalin-fixed, paraffin-embedded whole-section bone marrow biopsies using a well-established antibody to detect pH2A.X (phosphorylated histone variant H2A.X) that recognizes DNA double-strand breaks. Focusing on TP53-mutated and complex karyotype MDS/AML, we find a greater pH2A.X+ DNA damage burden compared to TP53 wild-type neoplastic cases and non-neoplastic controls. To understand how double-strand breaks vary between lineages and spatially in TP53-mutated specimens, we applied a low-multiplex immunofluorescence staining and spatial analysis protocol to visualize pH2A.X+ cells with p53 protein staining and lineage markers. pH2A.X marked predominantly mid- to late-stage erythroids, whereas early erythroids and CD34+ blasts were relatively spared. In a prototypical example, these pH2A.X+ erythroids were organized locally as distinct colonies, and each colony displayed pH2A.X+ puncta at a synchronous level. This highly coordinated immunophenotypic expression was also seen for p53 protein staining and among presumed early myeloid colonies. Neighborhood clustering analysis showed distinct marrow regions differentially enriched in pH2A.X+/p53+ erythroid or myeloid colonies, indicating spatial heterogeneity of DNA-damage response and p53 protein expression. The lineage and architectural context within which DNA damage phenotype and oncogenic protein are expressed is relevant to current therapeutic developments that leverage macrophage phagocytosis to remove leukemic cells in part due to irreparable DNA damage. © 2024 The Pathological Society of Great Britain and Ireland.

Long-Lived Plasma Cells Are Contained within the CD19(-)CD38(hi)CD138(+) Subset in Human Bone Marrow.

Antibody responses to viral infections are sustained for decades by long-lived plasma cells (LLPCs). However, LLPCs have yet to be characterized in humans. Here we used CD19, CD38, and CD138 to identify four PC subsets in human bone marrow (BM). We found that the CD19(-)CD38(hi)CD138(+) subset was morphologically distinct, differentially expressed PC-associated genes, and exclusively contained PCs specific for viral antigens to which the subjects had not been exposed for more than 40 years. Protein sequences of measles- and mumps-specific circulating antibodies were encoded for by CD19(-)CD38(hi)CD138(+) PCs in the BM. Finally, we found that CD19(-)CD38(hi)CD138(+) PCs had a distinct RNA transcriptome signature and human immunoglobulin heavy chain (VH) repertoire that was relatively uncoupled from other BM PC subsets and probably represents the B cell response's "historical record" of antigenic exposure. Thus, our studies define human LLPCs and provide a mechanism for the life-long maintenance of anti-viral antibodies in the serum.

Insertion of atypical glycans into the tumor antigen-binding site identifies DLBCLs with distinct origin and behavior.

Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma-associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell-like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell-like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN-expressing M2-polarized macrophages.

Glycoprotein V is a relevant immune target in patients with immune thrombocytopenia.

Platelet autoantibody-induced platelet clearance represents a major pathomechanism in immune thrombocytopenia (ITP). There is growing evidence for clinical differences between anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib/IX mediated ITP. Glycoprotein V is a well characterized target antigen in Varicella-associated and drug-induced thrombocytopenia. We conducted a systematic study assessing the prevalence and functional capacity of autoantibodies against glycoprotein V. A total of 1140 patients were included. In one-third of patients, platelet-bound autoantibodies against glycoproteins Ib/IX, IIb/IIIa, or V were detected in a monoclonal antibody immobilization of platelet antigen assay; platelet-bound autoantiglycoprotein V was present in the majority of samples (222 out of 343, 64.7%). Investigation of patient sera revealed the presence of free autoantibodies against glycoprotein V in 13.5% of these patients by an indirect monoclonal antibody immobilization of platelet antigen assay, but in 39.6% by surface plasmon resonance technology. These antibodies showed significantly lower avidity (association/dissociation ratio 0.32±0.13 vs 0.73±0.14; P<0.001). High- and low-avidity antibodies induced comparable amounts of platelet uptake in a phagocytosis assay using CD14+ positively-selected human macrophages [mean phagocytic index, 6.81 (range, 4.75-9.86) vs 6.01 (range, 5.00-6.98); P=0.954]. In a NOD/SCID mouse model, IgG prepared from both types of anti-glycoprotein V autoantibodies eliminated human platelets with no detectable difference between the groups from the murine circulation [mean platelet survival at 300 minutes, 40% (range, 27-55) vs 35% (16-46); P=0.025]. Our data establish glycoprotein V as a relevant immune target in immune thrombocytopenia. We would suggest that further studies including glycoprotein V will be required before ITP treatment can be tailored according to platelet autoantibody specificity.

CD20 and CD37 antibodies synergize to activate complement by Fc-mediated clustering.

CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.

Antibodies That Efficiently Form Hexamers upon Antigen Binding Can Induce Complement-Dependent Cytotoxicity under Complement-Limiting Conditions.

Recently, we demonstrated that IgG Abs can organize into ordered hexamers after binding their cognate Ags expressed on cell surfaces. This process is dependent on Fc:Fc interactions, which promote C1q binding, the first step in classical pathway complement activation. We went on to engineer point mutations that stimulated IgG hexamer formation and complement-dependent cytotoxicity (CDC). The hexamer formation-enhanced (HexaBody) CD20 and CD38 mAbs support faster, more robust CDC than their wild-type counterparts. To further investigate the CDC potential of these mAbs, we used flow cytometry, high-resolution digital imaging, and four-color confocal microscopy to examine their activity against B cell lines and primary chronic lymphocytic leukemia cells in sera depleted of single complement components. We also examined the CDC activity of alemtuzumab (anti-CD52) and mAb W6/32 (anti-HLA), which bind at high density to cells and promote substantial complement activation. Although we observed little CDC for mAb-opsonized cells reacted with sera depleted of early complement components, we were surprised to discover that the Hexabody mAbs, as well as ALM and W6/32, were all quite effective at promoting CDC in sera depleted of individual complement components C6 to C9. However, neutralization studies conducted with an anti-C9 mAb verified that C9 is required for CDC activity against cell lines. These highly effective complement-activating mAbs efficiently focus activated complement components on the cell, including C3b and C9, and promote CDC with a very low threshold of MAC binding, thus providing additional insight into their enhanced efficacy in promoting CDC.

Hexamerization-enhanced CD20 antibody mediates complement-dependent cytotoxicity in serum genetically deficient in C9.

We examined complement-dependent cytotoxicity (CDC) by hexamer formation-enhanced CD20 mAb Hx-7D8 of patient-derived chronic lymphocytic leukemia (CLL) cells that are relatively resistant to CDC. CDC was analyzed in normal human serum (NHS) and serum from an individual genetically deficient for C9. Hx-7D8 was able to kill up to 80% of CLL cells in complete absence of C9. We conclude that the narrow C5b-8 pores formed without C9 are sufficient for CDC due to efficient antibody-mediated hexamer formation. In the absence of C9, we observed transient intracellular increases of Ca2+ during CDC (as assessed with FLUO-4) that were extended in time. This suggests that small C5b-8 pores allow Ca2+ to enter the cell, while dissipation of the fluorescent signal accompanying cell disintegration is delayed. The Ca2+ signal is retained concomitantly with TOPRO-3 (viability dye) staining, thereby confirming that Ca2+ influx represents the most proximate mediator of cell death by CDC.

Increased numbers of CD23(+) CD21(hi) Bin-like B cells in human reactive and rheumatoid arthritis lymph nodes.

A unique population of CD23(+) CD21(high) B cells in inflamed nodes (Bin) has been shown to accumulate in lymph nodes (LNs) draining inflamed joints of TNF-transgenic (TNF-tg) mice. Bin cells contribute to arthritis flare in mice by distorting node architecture and hampering lymphatic flow, but their existence in human inflamed LNs has not yet been described. Here, we report the characterization of resident B-cell populations in fresh popliteal lymph nodes (PLNs) from patients with severe lower limb diseases (non-RA) and rheumatoid arthritis (RA) patients, and from banked, cryopreserved reactive and normal human LN single cell suspension samples. Bin-like B cells were shown to be significantly increased in reactive LNs, and strikingly elevated (>30% of total) in RA samples. Histopathology and immunofluorescence analyses were consistent with B follicular hyperplasia and histological alterations in RA vs. non-RA PLNs. This is the first description of Bin-like B cells in human inflamed LNs. Consistent with published mouse data, this population appears to be associated with inflammatory arthritis and distortion of LN architecture. Further analyses are necessary to assess the role of CD23(+) CD21(hi) Bin-like B cells in RA pathogenesis and arthritic flare.

Referral to and receipt of allogeneic hematopoietic stem cell transplantation in older adults with acute myeloid leukemia.

INTRODUCTION: Recent data have shown improved outcomes in selected older adults with acute myeloid leukemia (AML) following allogeneic hematopoietic stem cell transplantation (HSCT). Nonetheless, practice patterns for referring and performing HSCT vary. We aimed to evaluate referral, utilization, and reasons for not referring/proceeding to HSCT in older adults with AML. MATERIALS AND METHODS: This is a single center retrospective analysis of patients aged ≥60 years diagnosed with AML evaluating rates of HSCT referral and utilization. Fisher's exact test was used to compare rates of referral and utilization across age groups and years of diagnosis. RESULTS: Median age of the 97 patients was 70 years (range 61-95); 30% (29/97) were referred for HSCT and of these, 69% (20/29) received HSCT. Common documented reasons (can be multiple) for not referring were performance status (n = 21), advanced age (n = 16), patient refusal (n = 15), refractory disease (n = 14), and prohibitive comorbidity (n = 6). Among patients who were referred but did not receive HSCT (n = 9/29), documented reasons for not proceeding with HSCT were refractory disease (n = 5), advanced age (n = 2), and prohibitive comorbidity (n = 2). HSCT referral and utilization rates significantly decreased with age (p < 0.01) but were generally stable over time from 2014 to 2017 (p = 0.40 for referral and p = 0.56 for utilization). DISCUSSION: Despite improvements in supportive care and HSCT techniques, HSCT referral and utilization rates remained low among older adults with AML but stable over time.

DC-SIGN binding to mannosylated B-cell receptors in follicular lymphoma down-modulates receptor signaling capacity.

In follicular lymphoma (FL), surface immunoglobulin (sIg) carries mandatory N-glycosylation sites in the variable regions, inserted during somatic hypermutation. These glycosylation sites are tumor-specific, indicating a critical function in FL. Added glycan unexpectedly terminates at high mannose (Mann) and confers capability for sIg-mediated interaction with local macrophage-expressed DC-SIGN lectin resulting in low-level activation of upstream B-cell receptor signaling responses. Here we show that despite being of low-level, DC-SIGN induces a similar downstream transcriptional response to anti-IgM in primary FL cells, characterized by activation of pathways associated with B-cell survival, proliferation and cell-cell communication. Lectin binding was also able to engage post-transcriptional receptor cross-talk pathways since, like anti-IgM, DC-SIGN down-modulated cell surface expression of CXCR4. Importantly, pre-exposure of a FL-derived cell line expressing sIgM-Mann or primary FL cells to DC-SIGN, which does not block anti-IgM binding, reversibly paralyzed the subsequent Ca2+ response to anti-IgM. These novel findings indicate that modulation of sIg function occurs in FL via lectin binding to acquired mannoses. The B-cell receptor alternative engagement described here provides two advantages to lymphoma cells: (i) activation of signaling, which, albeit of low-level, is sufficient to trigger canonical lymphoma-promoting responses, and (ii) protection from exogenous antigen by paralyzing anti-IgM-induced signaling. Blockade of this alternative engagement could offer a new therapeutic strategy.

Musashi 2 influences chronic lymphocytic leukemia cell survival and growth making it a potential therapeutic target.

Progression of chronic lymphocytic leukemia (CLL) results from the expansion of a small fraction of proliferating leukemic B cells. When comparing the global gene expression of recently divided CLL cells with that of previously divided cells, we found higher levels of genes involved in regulating gene expression. One of these was the oncogene Musashi 2 (MSI2), an RNA-binding protein that induces or represses translation. While there is an established role for MSI2 in normal and malignant stem cells, much less is known about its expression and role in CLL. Here we report for the first time ex vivo and in vitro experiments that MSI2 protein levels are higher in dividing and recently divided leukemic cells and that downregulating MSI2 expression or blocking its function eliminates primary human and murine CLL and mature myeloid cells. Notably, mature T cells and hematopoietic stem and progenitor cells are not affected. We also confirm that higher MSI2 levels correlate with poor outcome markers, shorter time-to-first-treatment, and overall survival. Thus, our data highlight an important role for MSI2 in CLL-cell survival and proliferation and associate MSI2 with poor prognosis in CLL patients. Collectively, these findings pinpoint MSI2 as a potentially valuable therapeutic target in CLL.

Clinicopathologic and genetic profile of intracranial marginal zone lymphoma: a primary low-grade CNS lymphoma that mimics meningioma.

PURPOSE: Although rare overall, marginal zone B-cell lymphoma (MZBCL) is the most common primary low-grade CNS lymphoma reported in the literature. The aim of this study is to elucidate the biology and genetic features of this unusual tumor. PATIENTS AND METHODS: Fifteen CNS MZBCLs were studied clinically, pathologically, and genetically, including fluorescent in situ hybridization analyses with commercially available MALT1 and IgH break-apart and centromere 3, 7, 12, and 18 probes. RESULTS: CNS MZBCLs preferentially affect middle-aged women (female-to-male ratio, 4:1), with 93% presenting as dural-based masses mimicking meningioma. Ten patients with 1 to 7.6 years of follow-up after diagnosis showed no evidence of disease after radiation and/or chemotherapy. Like MZBCLs outside of the CNS, they consisted of CD20+, CD3- small B lymphocytes with varying degrees of plasmacytic differentiation and predominantly kappa light-chain restriction (78%). Lymphoid follicles with follicular colonization were seen in three patients and deposition of amyloid was noted in samples from two patients, one of which was tumefactive. Neither Bcl-6 protein nor Epstein-Barr virus-encoded RNA was expressed. Trisomy 3 was detected in six of 12 patients, with no rearrangements of MALT1 or IgH and no trisomies of 7, 12, or 18 detected. CONCLUSION: Our data suggest that intracranial MZBCL is an indolent primary CNS lymphoma that typically presents as a meningioma-like dural-based mass. Trisomy 3, but not MALT1 or IgH translocation, is a common genetic abnormality that may contribute to the pathogenesis of this CNS lymphoma.

Ipilimumab-Induced Neutropenia in Melanoma.

Ipilimumab is a human monoclonal IgG1 antibody against CTLA-4 that has been shown to prolong the overall survival of advanced melanoma. The most common adverse events associated with ipilimumab are immune-related. Severe hematological toxicity is rare. We report a case of severe neutropenia following ipilimumab therapy that fully resolved after the administration of prednisone, cyclosporine, and anti-thymocyte globulin therapies.

Real-time analysis of the detailed sequence of cellular events in mAb-mediated complement-dependent cytotoxicity of B-cell lines and of chronic lymphocytic leukemia B-cells.

Complement-dependent cytotoxicity is an important mechanism of action of certain mAbs used in cancer immunotherapy, including ofatumumab and rituximab. However, the detailed sequence of cellular changes that occur in nucleated cells attacked by mAb and complement has not been delineated. Recently developed CD20 mAbs, engineered to form hexamers on binding to cells, react with B-cells in serum, chelate C1q, and then activate complement and promote cell killing considerably more effectively than their wild-type precursors. We used these engineered mAbs as a model to investigate the sequence of events that occur when mAbs bind to B-cell lines and to primary cells from patients with chronic lymphocytic leukemia and then activate complement. Based on four-color confocal microscopy real-time movies and high resolution digital imaging, we find that after CD20 mAb binding and C1q uptake, C3b deposits on cells, followed by Ca(2+) influx, revealed by bright green signals generated on cells labeled with FLUO-4, a Ca(2+) indicator. The bright FLUO-4/Ca(2+) signal fades, replaced by punctate green signals in mitochondria, indicating Ca(2+) localization. This step leads to mitochondrial poisoning followed by cell death. The entire sequence is completed in <2 min for hexamerization-enhanced CD20 mAb-mediated killing. To our knowledge this is the first time the entire process has been characterized in detail in real time. By identifying multiple discrete steps in the cytotoxic pathway for nucleated cells our findings may inform future development and more effective application of complement-fixing mAbs to cancer treatment.

Immunohistochemical expression of mTOR in germinal center and nongerminal center group of diffuse large B-cell lymphoma: a clinicopathological study.

BACKGROUND: The mammalian target of rapamycin (mTOR) pathway regulates many major cellular processes and is implicated in an increasing number of neoplasms, including lymphoma. PATIENTS AND METHODS: We correlated immunohistochemical expression of mTOR with germinal center and nongerminal center phenotype, B cell lymphoma-2 (bcl-2) and cellular homolog of the retroviral v-myconcogene (c-myc) expression, and International Prognostic Index (IPI) score in 31 patients with diffuse large B-cell lymphoma (DLBCL). RESULTS: Virtually all patients in our study with high mTOR scores had a germinal center phenotype. Furthermore within the germinal center subgroup, patients with high mTOR scores were associated with higher IPI scores (P < .001). CONCLUSION: Based on our results we propose that within the category of germinal center phenotype of DLBCL, mTOR expression might help identify a subset of patients with potentially more aggressive tumors who might benefit from use of targeted therapy using mTOR inhibitors.

Inhibition of COP9-signalosome (CSN) deneddylating activity and tumor growth of diffuse large B-cell lymphomas by doxycycline.

In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes.

Phase II study of alisertib, a selective Aurora A kinase inhibitor, in relapsed and refractory aggressive B- and T-cell non-Hodgkin lymphomas.

PURPOSE: Aurora A kinase (AAK) is overexpressed in aggressive lymphomas and can correlate with more histologically aggressive forms of disease. We therefore designed a phase II study of alisertib, a selective AAK inhibitor, in patients with relapsed and refractory aggressive non-Hodgkin lymphomas. PATIENTS AND METHODS: Patients age ≥ 18 years were eligible if they had relapsed or refractory diffuse large B-cell lymphoma (DLBCL), mantle-cell lymphoma (MCL), transformed follicular lymphoma, Burkitt's lymphoma, or noncutaneous T-cell lymphoma. Alisertib was administered orally at 50 mg twice daily for 7 days in 21-day cycles. RESULTS: We enrolled 48 patients. Histologies included DLBCL (n = 21), MCL (n = 13), peripheral T-cell lymphoma (n = 8), transformed follicular lymphoma (n = 5), and Burkitt's (n = 1). Most common grade 3 to 4 adverse events were neutropenia (63%), leukopenia (54%), anemia (35%), thrombocytopenia (33%), stomatitis (15%), febrile neutropenia (13%), and fatigue (6%). Four deaths during the study were attributed to progressive non-Hodgkin lymphoma (n = 2), treatment-related sepsis (n = 1), and unknown cause (n = 1). The overall response rate was 27%, including responses in three of 21 patients with DLBCL, three of 13 with MCL, one of one with Burkitt's lymphoma, two of five with transformed follicular lymphoma, and four of eight with noncutaneous T-cell lymphoma. The alisertib steady-state trough concentration (n = 25) revealed the expected pharmacokinetic variability, with a trend for higher incidence of adverse event-related dose reductions at higher trough concentrations. Analysis for AAK gene amplification and total AAK protein revealed no differences between histologies or correlation with clinical response. CONCLUSION: The novel AAK inhibitor alisertib seems clinically active in both B- and T-cell aggressive lymphomas. On the basis of these results, confirmatory single-agent and combination studies have been initiated.