Upregulated Expression of ERBB2/HER2 in Multiple Myeloma as a Predictor of Poor Survival Outcomes.

The main goal of the present study was to examine if the RNA-sequencing (RNAseq)-based ERBB2/HER2 expression level in malignant plasma cells from multiple myeloma (MM) patients has clinical significance for treatment outcomes and survival. We examined the relationship between the RNAseq-based ERBB2 messenger ribonucleic acid (mRNA) levels in malignant plasma cells and survival outcomes in 787 MM patients treated on contemporary standard regimens. ERBB2 was expressed at significantly higher levels than ERBB1 as well as ERBB3 across all three stages of the disease. Upregulated expression of ERBB2 mRNA in MM cells was correlated with amplified expression of mRNAs for transcription factors (TF) that recognize the ERBB2 gene promoter sites. Patients with higher levels of ERBB2 mRNA in their malignant plasma cells experienced significantly increased cancer mortality, shorter progression-free survival, and worse overall survival than other patients. The adverse impact of high ERBB2 expression on patient survival outcomes remained significant in multivariate Cox proportional hazards models that accounted for the effects of other prognostic factors. To the best of our knowledge, this is the first demonstration of an adverse prognostic impact of high-level ERBB2 expression in MM patients. Our results encourage further evaluation of the prognostic significance of high-level ERBB2 mRNA expression and the clinical potential of ERBB2-targeting therapeutics as personalized medicines to overcome cancer drug resistance in high-risk as well as relapsed/refractory MM.

Impact of Therapy in Patients with Hematologic Malignancies on Seroconversion Rates After SARS-CoV-2 Vaccination.

INTRODUCTION: The leading professional organizations in the field of hematology have recommended severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) vaccination for all patients with hematologic malignancies notwithstanding efficacy concerns. Here we report a systematic literature review regarding the antibody response to SARS-CoV-2 vaccination in patients with hematologic malignancies and its key determinants. METHODS: We conducted a systematic search of original articles evaluating the seroconversion rates with SARS-CoV-2 vaccines in hematological malignancies from the PubMed database published between April 1, 2021 and December 4, 2021. Calculated risk differences (RD) and 95% confidence intervals (CI) to compare seroconversion rates between patients with hematologic malignancies versus healthy control subjects used the Review Manager software, version 5.3. RESULTS: In our meta-analysis, we included 26 studies with control arms. After the first dose of vaccination, patients with hematologic malignancies had significantly lower seroconversion rates than controls (33.3% vs 74.9%; RD: -0.48%, 95% CI: -0.60%, -0.36%, P < .001). The seroconversion rates increased after the second dose, although a significant difference remained between these 2 groups (65.3% vs 97.8%; RD: -0.35%, 95% CI: -0.42%, -0.28%, P < .001). This difference in seroconversion rates was particularly pronounced for Chronic Lymphocytic Leukemia (CLL) patients (RD: -0.46%, 95% CI: -0.56, -0.37, P < .001), and for patients with B-lineage leukemia/lymphoma treated with anti-CD20 antibodies (RD: -0.70%, 95% CI: -0.88%, -0.51%, P < .001) or Bruton Tyrosine Kinase Inhibitors (BTKi; RD: -0.63%, 95% CI: -0.85%, -0.41%, P < .001). The RD was lower for patients under remission (RD: -0.10%, 95% CI: -0.18%, -0.02%, P = .01). CONCLUSION: The seroconversion rates following SARS-CoV-2 vaccination in patients with hematologic malignancies, especially in CLL patients and patients treated with anti-CD20 antibodies or BTKi, were significantly lower than the seroconversion rates in healthy control subjects. Effective strategies capable of improving vaccine efficacy in these vulnerable patient populations are urgently needed.

Data-Driven identification of chemopreventive agents for breast cancer

Preclinical animal models of breast cancer provide the opportunity to identify chemopreventive drugs with single-agent activity as well as effective multi-modality regimens for primary as well as secondary prevention in high-risk persons. Our group has used the 7,12-dimethylbenz(a)anthracene (DMBA) mouse model of carcinogen-induced breast cancer to explore the clinical potential of two tyrosine kinase inhibitors and a nucleoside analog as chemopreventive agents. All three agents exhibited promising preclinical activity both as monotherapy and as components of combination therapy with the standard chemotherapy drug paclitaxel. The tumors developing despite chemoprevention were not only small and grew slowly, but they also displayed a uniquely more pro-apoptotic protein expression profile. Hence, our experimental chemopreventive drugs were capable of preventing the development of aggressive mammary gland tumors with an apoptosis-resistant protein expression profile.

Dimeric drug polymeric nanoparticles with exceptionally high drug loading and quantitative loading efficiency.

Encapsulation of small-molecule drugs in hydrophobic polymers or amphiphilic copolymers has been extensively used for preparing polymeric nanoparticles (NPs). The loadings and loading efficiencies of a wide range of drugs in polymeric NPs, however, tend to be very low. In this Communication, we report a strategy to prepare polymeric NPs with exceptionally high drug loading (>50%) and quantitative loading efficiency. Specifically, a dimeric drug conjugate bearing a trigger-responsive domain was designed and used as the core-constructing unit of the NPs. Upon co-precipitation of the dimeric drug and methoxypoly(ethylene glycol)-block-polylactide (mPEG-PLA), NPs with a dimeric drug core and a polymer shell were formed. The high-drug-loading NPs showed excellent stability in physiological conditions. No premature drug or prodrug release was observed in PBS solution without triggering, while external triggering led to controlled release of drug in its authentic form.

CD22ΔE12 as a molecular target for RNAi therapy.

B-precursor acute lymphoblastic leukaemia (BPL) is the most common form of cancer in children and adolescents. Our recent studies have demonstrated that CD22ΔE12 is a characteristic genetic defect of therapy-refractory clones in paediatric BPL and implicated the CD22ΔE12 genetic defect in the aggressive biology of relapsed or therapy-refractory paediatric BPL. The purpose of the present study is to evaluate the biological significance of the CD22ΔE12 molecular lesion in BPL and determine if it could serve as a molecular target for RNA interference (RNAi) therapy. Here we report a previously unrecognized causal link between CD22ΔE12 and aggressive biology of human BPL cells by demonstrating that siRNA-mediated knockdown of CD22ΔE12 in primary leukaemic B-cell precursors is associated with a marked inhibition of their clonogenicity. Additionally, we report a nanoscale liposomal formulation of CD22ΔE12-specific siRNA with potent in vitro and in vivo anti-leukaemic activity against primary human BPL cells as a first-in-class RNAi therapeutic candidate targeting CD22ΔE12.

Nanoscale liposomal formulation of a SYK P-site inhibitor against B-precursor leukemia.

We report preclinical proof of principle for effective treatment of B-precursor acute lymphoblastic leukemia (ALL) by targeting the spleen tyrosine kinase (SYK)-dependent antiapoptotic blast cell survival machinery with a unique nanoscale pharmaceutical composition. This nanoscale liposomal formulation (NLF) contains the pentapeptide mimic 1,4-Bis (9-O dihydroquinidinyl) phthalazine/hydroquinidine 1,4-phathalazinediyl diether (C61) as the first and only selective inhibitor of the substrate binding P-site of SYK. The C61 NLF exhibited a very favorable pharmacokinetic and safety profile in mice, induced apoptosis in primary B-precursor ALL blast cells taken directly from patients as well as in vivo clonogenic ALL xenograft cells, destroyed the in vivo clonogenic fraction of ALL blast cells, and, at nontoxic dose levels, exhibited potent in vivo antileukemic activity against patient-derived ALL cells in xenograft models of aggressive B-precursor ALL. Our findings establish SYK as an attractive molecular target for therapy of B-precursor ALL. Further development of the C61 NLF may provide the foundation for therapeutic innovation against therapy-refractory B-precursor ALL.

Serine phosphorylation by SYK is critical for nuclear localization and transcription factor function of Ikaros.

Ikaros is a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis through transcriptional regulation of the earliest stages of lymphocyte ontogeny and differentiation. Functional deficiency of Ikaros has been implicated in the pathogenesis of acute lymphoblastic leukemia, the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros activity is considered of paramount importance, but the operative molecular mechanisms responsible for its regulation remain largely unknown. Here we provide multifaceted genetic and biochemical evidence for a previously unknown function of spleen tyrosine kinase (SYK) as a partner and posttranslational regulator of Ikaros. We demonstrate that SYK phoshorylates Ikaros at unique C-terminal serine phosphorylation sites S358 and S361, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Mechanistically, we establish that SYK-induced Ikaros activation is essential for its nuclear localization and optimal transcription factor function.

Size-dependent tumor penetration and in vivo efficacy of monodisperse drug-silica nanoconjugates.

The size of a nanomedicine strongly correlates with its biodistribution, tissue penetration, and cell uptake. However, there is limited understanding how the size of nanomedicine impacts the overall antitumor efficacy. We designed and synthesized camptothecin-silica nanoconjugates (Cpt-NCs) with monodisperse particle sizes of 50 and 200 nm, two representative sizes commonly used in drug delivery, and evaluated their antitumor efficacy in murine tumor models. Our studies revealed that the 50 nm Cpt-NC showed higher anticancer efficacy than the larger analogue, due presumably to its faster cellular internalization and more efficient tumor accumulation and penetration. Our findings suggest that nanomedicine with smaller sizes holds great promise for improved cancer therapy.

CD22 EXON 12 deletion as a pathogenic mechanism of human B-precursor leukemia.

Here, we report that primary leukemic cells from infants with newly diagnosed B-precursor leukemia express a truncated and functionally defective CD22 coreceptor protein that is unable to transmit apoptotic signals because it lacks most of the intracellular domain, including the key regulatory signal transduction elements and all of the cytoplasmic tyrosine residues. Expression of this structurally and functionally abnormal CD22 protein is associated with a very aggressive in vivo growth of patients' primary leukemia cells causing disseminated overt leukemia in SCID mice. The abnormal CD22 coreceptor is encoded by a profoundly aberrant mRNA arising from a splicing defect that causes the deletion of exon 12 (c.2208-c.2327) (CD22ΔE12) and results in a truncating frameshift mutation. The splicing defect is associated with multiple homozygous mutations within a 132-bp segment of the intronic sequence between exons 12 and 13. These mutations cause marked changes in the predicted secondary structures of the mutant CD22 pre-mRNA sequences that affect the target motifs for the splicing factors hnRNP-L, PTB, and PCBP that are up-regulated in infant leukemia cells. Forced expression of the mutant CD22ΔE12 protein in transgenic mice perturbs B-cell development, as evidenced by B-precursor/B-cell hyperplasia, and corrupts the regulation of gene expression, causing reduced expression levels of several genes with a tumor suppressor function. We further show that CD22ΔE12-associated unique gene expression signature is a discriminating feature of newly diagnosed infant leukemia patients. These striking findings implicate CD22ΔE12 as a previously undescribed pathogenic mechanism in human B-precursor leukemia.

STAT3 is a substrate of SYK tyrosine kinase in B-lineage leukemia/lymphoma cells exposed to oxidative stress.

We provide unprecedented genetic and biochemical evidence that the antiapoptotic transcription factor STAT3 serves as a substrate for SYK tyrosine kinase both in vitro and in vivo. Induction of SYK in an ecdysone-inducible mammalian expression system results in STAT3 activation, as documented by tyrosine phosphorylation and nuclear translocation of STAT3, as well as amplified expression of several STAT3 target genes. STAT3 activation after oxidative stress (OS) is strongly diminished in DT40 chicken B-lineage lymphoma cells rendered SYK-deficient by targeted disruption of the syk gene. Introduction of a wild-type, C-terminal or N-terminal SH2 domain-mutated, but not a kinase domain-mutated, syk gene into SYK-deficient DT40 cells restores OS-induced enhancement of STAT-3 activity. Thus, SYK plays an important and indispensable role in OS-induced STAT3 activation and its catalytic SH1 domain is critical for this previously unknown regulatory function. These results provide evidence for the existence of a novel mode of cytokine-independent cross-talk that operates between SYK and STAT3 pathways and regulates apoptosis during OS. We further provide experimental evidence that SYK is capable of associating with and phosphorylating STAT3 in human B-lineage leukemia/lymphoma cells challenged with OS. In agreement with a prerequisite role of SYK in OS-induced STAT3 activation, OS does not induce tyrosine phosphorylation of STAT3 in SYK-deficient human proB leukemia cells. Notably, inhibition of SYK with a small molecule drug candidate prevents OS-induced activation of STAT3 and overcomes the resistance of human B-lineage leukemia/lymphoma cells to OS-induced apoptosis.

Upregulated Expression of ErbB1 in Diffuse Large B-Cell Lymphoma as a Predictor of Poor Overall Survival Outcome.

We examined the transcript-level expression of ErbB family protein tyrosine kinases, including ERBB1, in primary malignant lymphoma cells from 498 adult patients with diffuse large B-cell lymphoma (DLBCL). ERBB1 expression in DLBCL cells was significantly higher than in normal B-lineage lymphoid cells. An upregulated expression of ERBB1 mRNA in DLBCL cells was correlated with an amplified expression of mRNAs for transcription factors that recognized ERBB1 gene promoter sites. Notably, amplified ERBB1 expression in DLBCL and its subtypes were associated with significantly worse overall survival (OS). Our results encourage the further evaluation of the prognostic significance of high-level ERBB1 mRNA expression and the clinical potential of ERBB1-targeting therapeutics as personalized medicines in high-risk DLBCL.

CD22 Exon 12 Deletion as an Independent Predictor of Poor Treatment Outcomes in B-ALL.

We previously reported a splicing defect (CD22ΔE12) associated with the deletion of exon 12 of the inhibitory co-receptor CD22 (Siglec-2) in leukemia cells from patients with CD19+ B-precursor acute lymphoblastic leukemia (B-ALL). CD22ΔE12 causes a truncating frameshift mutation and yields a dysfunctional CD22 protein that lacks most of the cytoplasmic domain required for its inhibitory function, and it is associated with aggressive in vivo growth of human B-ALL cells in mouse xenograft models. Although CD22ΔE12 with selective reduction of CD22 exon 12 (CD22E12) levels was detected in a high percentage of newly diagnosed as well as relapsed B-ALL patients, its clinical significance remains unknown. We hypothesized that B-ALL patients with very low levels of wildtype CD22 would exhibit a more aggressive disease with a worse prognosis because the missing inhibitory function of the truncated CD22 molecules could not be adequately compensated by competing wildtype CD22. Here, we demonstrate that newly diagnosed B-ALL patients with very low levels of residual wildtype CD22 ("CD22E12low"), as measured by RNAseq-based CD22E12 mRNA levels, have significantly worse leukemia-free survival (LFS) as well as overall survival (OS) than other B-ALL patients. CD22E12low status was identified as a poor prognostic indicator in both univariate and multivariate Cox proportional hazards models. CD22E12low status at presentation shows clinical potential as a poor prognostic biomarker that may guide the early allocation of risk-adjusted, patient-tailored treatment regimens and refine risk classification in high-risk B-ALL.

High Intra-Tumor Transforming Growth Factor Beta 2 Level as a Predictor of Poor Treatment Outcomes in Pediatric Diffuse Intrinsic Pontine Glioma.

Here, we report that tumor samples from newly diagnosed pediatric diffuse intrinsic pontine glioma (DIPG) patients express significantly higher levels of transforming growth factor beta 2 (TGFB2) messenger ribonucleic acid (mRNA) than control pons samples, which correlated with augmented expression of transcription factors that upregulate TGFB2 gene expression. Our study also demonstrated that RNA sequencing (RNAseq)-based high TGFB2 mRNA level is an indicator of poor prognosis for DIPG patients, but not for pediatric glioblastoma (GBM) patients or pediatric diffuse midline glioma (DMG) patients with tumor locations outside of the pons/brainstem. Notably, DIPG patients with high levels of TGFB2 mRNA expression in their tumor samples had significantly worse overall survival (OS) and progression-free survival (PFS). By comparison, high levels of transforming growth factor beta 3 (TGFB3) mRNA expression in tumor samples was associated with significantly better survival outcomes of DIPG patients, whereas high levels of transforming growth factor beta 1 (TGFB1) expression was not prognostic. Our study fills a significant gap in our understanding of the clinical significance of high TGFB2 expression in pediatric high-grade gliomas.

CD22 Exon 12 deletion is a characteristic genetic defect of therapy-refractory clones in paediatric acute lymphoblastic leukaemia.

Gene expression profiling (GEP) of primary leukaemic cells (PLC) from 157 paediatric B-lineage acute lymphoblastic leukaemia (ALL) patients, including a direct comparison of matched pair initial diagnosis versus first relapse leukaemic specimens, provided previously unknown evidence that relapse clones are characterized by significantly higher expression levels of a CD22 exon 12 deletion (CD22ΔE12)-associated signature transcriptome than the PLC from newly diagnosed patients. In agreement with and validating these GEP results, reverse transcription polymerase chain reaction and Western blot analysis of PLC from 19 of 19 paediatric ALL patients in first bone marrow relapse occurring within 12 months of the completion of primary therapy confirmed them to be CD22ΔE12(+). Likewise, PLC in diagnostic initial bone marrow specimens from seven of seven therapy-refractory newly diagnosed paediatric B-lineage ALL patients with <7 months event-free survival (EFS), including four patients with induction failures and three patients with early relapses, were CD22ΔE12(+), whereas PLC from only one of five newly diagnosed paediatric B-lineage ALL patients with >18 months EFS was CD22ΔE12(+). CD22ΔE12(+) could be detected in PLC of therapy-refractory patients both at the time of initial diagnosis as well as at the time of documented treatment failure. Our study implicates the CD22ΔE12 genetic defect in the aggressive biology of relapsed or therapy-refractory paediatric B-lineage ALL.

Tyrosine kinases in KMT2A/MLL-rearranged acute leukemias as potential therapeutic targets to overcome cancer drug resistance.

Aim: The main goal of this study was to elucidate at the transcript level the tyrosine kinase expression profiles of primary leukemia cells from mixed lineage leukemia 1 gene rearranged (KMT2A/MLL-R+) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Methods: We evaluated protein tyrosine kinase (PTK) gene expression profiles of primary leukemic cells in KMT2A/MLL-R+ AML and ALL patients using publicly available archived datasets. Results: Our studies provided unprecedented evidence that the genetic signatures of KMT2A/MLL-R+ AML and ALL cells are characterized by transcript-level overexpression of specific PTK. In infants, children and adults with KMT2A/MLL-R+ ALL, as well as pediatric patients with KMT2A/MLL-R+ AML, the gene expression levels for FLT3, BTK, SYK, JAK2/JAK3, as well as several SRC family PTK were differentially amplified. In adults with KMT2A/MLL-R+ AML, the gene expression levels for SYK, JAK family kinase TYK2, and the SRC family kinases FGR and HCK were differentially amplified. Conclusion: These results provide new insights regarding the clinical potential of small molecule inhibitors of these PTK, many of which are already FDA/EMA-approved for other indications, as components of innovative multi-modality treatment platforms against KMT2A/MLL-R+ acute leukemias.

ERBB1/EGFR and JAK3 Tyrosine Kinases as Potential Therapeutic Targets in High-Risk Multiple Myeloma.

Our main objective was to identify abundantly expressed tyrosine kinases in multiple myeloma (MM) as potential therapeutic targets. We first compared the transcriptomes of malignant plasma cells from newly diagnosed MM patients who were risk-categorized based on the patient-specific EMC-92/SKY-92 gene expression signature values vs. normal plasma cells from healthy volunteers using archived datasets from the HOVON65/GMMG-HD4 randomized Phase 3 study evaluating the clinical efficacy of bortezomib induction/maintenance versus classic cytotoxic drugs and thalidomide maintenance. In particular, ERBB1/EGFR was significantly overexpressed in MM cells in comparison to normal control plasma cells, and it was differentially overexpressed in MM cells from high-risk patients. Amplified expression of EGFR/ERBB1 mRNA in MM cells was positively correlated with increased expression levels of mRNAs for several DNA binding proteins and transcription factors with known upregulating activity on EGFR/ERBB1 gene expression. MM patients with the highest ERBB1/EGFR expression level had significantly shorter PFS and OS times than patients with the lowest ERBB1/EGFR expression level. High expression levels of EGFR/ERBB1 were associated with significantly increased hazard ratios for unfavorable PFS and OS outcomes in both univariate and multivariate Cox proportional hazards models. The impact of high EGFR/ERBB1 expression on the PFS and OS outcomes remained significant even after accounting for the prognostic effects of other covariates. These results regarding the prognostic effect of EGFR/ERBB1 expression were validated using the MMRF-CoMMpass RNAseq dataset generated in patients treated with more recently applied drug combinations included in contemporary induction regimens. Our findings provide new insights regarding the molecular mechanism and potential clinical significance of upregulated EGFR/ERBB1 expression in MM.

RJX Improves Wound Healing in Diabetic Rats.

Background: We recently reported the clinical safety profile of RJX, a well-defined intravenous GMP-grade pharmaceutical formulation of anti-oxidant and anti-inflammatory vitamins as active ingredients, in a Phase 1 study in healthy volunteers (ClinicalTrials.gov Identifier: NCT03680105) (Uckun et al., Front. Pharmacol. 11, 594321. 10.3389/fphar.2020.594321). The primary objective of the present study was to examine the effects of GMP-grade RJX on wound and burn injury healing in diabetic rats. Methods: In the present study, a rat model of T2DM was used that employs HFD in combination with a single injection of STZ intraperitoneally (i.p) at a moderate dose level (45 mg/kg). Anesthetized diabetic rats underwent full-thickness skin excision on the back or were subjected to burn injury via a heated brass probe and then started on treatments with normal saline (NS = vehicle) or RJX administered via intraperitoneal injections for three weeks. Findings: Notably, diabetic rats treated with the 1.25 mL/kg or 2.5 mL/kg RJX (DM+RJX groups) rapidly healed their wounds as fast as non-diabetic control rats. Inflammatory cell infiltration in the dermis along with fibrin and cell debris on the epithelial layer persisted for up to 14 days in the DM+NS group but not in RJX-treated groups. The histopathological score of wound healing on days 7 and 14 was better in diabetic rats treated with RJX than diabetic rats treated with NS and comparable to the scores for non-diabetic healthy rats consistent with an accelerated healing process. The residual wound area of RJX-treated rats was significantly smaller than that of NS-treated diabetic rats at each evaluation time point (P<0.001). The accelerating effect of RJX on diabetic wound healing was dose-dependent. We obtained similar results in the burn injury model. Our results demonstrate that RJX - at a dose level >10-fold lower than its clinical maximum tolerated dose (MTD) - accelerates the healing of excision wounds as well burn injury in diabetic rats.

Evaluation of the potential of Rejuveinix plus dexamethasone against sepsis.

Aim: Our main objectives were to compare the effects of Rejuveinix (RJX), dexamethasone (DEX) and their combination on the severity of sepsis and survival outcome in an animal model of fatal sepsis. Methods: We used the LPS plus D-galactosamine mouse model of sepsis to compare the anti-inflammatory activities of RJX, dexamethasone and a combination of RJX plus DEX. Additionally, we examined the clinical feasibility and tolerability of combining RJX with DEX in COVID-19 patients in a clinical phase I study. Data were analyzed using standard methods. Results & conclusion: RJX exhibited potent anti-inflammatory activity in the murine sepsis model. The combination of RJX plus DEX was more effective than either agent alone, decreased the inflammatory cytokine responses and associated organ damage, and improved the survival outcome in mice. In the phase I clinical study, RJX plus DEX was well tolerated by COVID-19 patients.

Recombinant human CD19-ligand protein as a potent anti-leukaemic agent.

We report the cloning and characterization of a novel 54-kDa high-mobility group (HMG)-box protein as the ligand for the human pan-B cell co-receptor CD19 (CD19-L), which interacts with the extracellular domain of CD19 in trans. CD19-L is the first CD19-specific recombinant human protein with potent anti-leukaemic activity against B-lineage acute lymphoblastic leukaemia (ALL), the most common form of childhood cancer and the second most common form of acute leukaemia in adults. Soluble recombinant CD19-L protein exhibited exquisite specificity for the extracellular domain of CD19 and strong binding to the surface of B-lineage leukaemia/lymphoma cells. Engagement of CD19 co-receptor on B-lineage ALL cells with CD19-L perturbed the CD19-associated signalling network, altering the expression levels of multiple genes directly involved in regulation of apoptosis, and triggered rapid apoptotic cell death in a CD19-specific manner. The identification of human CD19-L may lead to therapeutic innovation for B-lineage ALL and other B-lineage lymphoid malignancies as well as B-cell lymphoproliferative states and systemic autoimmunity.