Impact of viral hepatitis therapy in multiple myeloma and other monoclonal gammopathies linked to hepatitis B or C viruses.

Subsets of multiple myeloma (MM) and monoclonal gammopathies of undetermined significance (MGUS) present with a monoclonal immunoglobulin specific for hepatitis C virus (HCV), thus are presumably HCV-driven, and antiviral treatment can lead to the disappearance of antigen stimulation and improved control of clonal plasma cells. Here we studied the role of hepatitis B virus (HBV) in the pathogenesis of MGUS and MM in 45 HBV-infected patients with monoclonal gammopathy. We analyzed the specificity of recognition of the monoclonal immunoglobulin of these patients and validated the efficacy of antiviral treatment (AVT). For 18 of 45 (40%) HBV-infected patients, the target of the monoclonal immunoglobulin was identified: the most frequent target was HBV (n=11), followed by other infectious pathogens (n=6) and glucosylsphingosine (n=1). Two patients whose monoclonal immunoglobulin targeted HBV (HBx and HBcAg), implying that their gammopathy was HBV-driven, received AVT and the gammopathy did not progress. AVT efficacy was then investigated in a large cohort of HBV-infected MM patients (n=1367) who received or did not receive anti-HBV treatments and compared to a cohort of HCV-infected MM patients (n=1220). AVT significantly improved patient probability of overall survival (P=0.016 for the HBV-positive cohort, P=0.005 for the HCV-positive cohort). Altogether, MGUS and MM disease can be HBV- or HCV-driven in infected patients, and the study demonstrates the importance of AVT in such patients.

Determination of the target of monoclonal immunoglobulins: a novel diagnostic tool for individualized MGUS therapy, and prevention and therapy of smoldering and multiple myeloma.

Subsets of patients diagnosed with a monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM) or multiple myeloma (MM), present with a monoclonal immunoglobulin (Ig) specific for an infectious pathogen, including hepatitis C and B viruses (HCV, HBV), Helicobacter pylori and several Herpesviruses. Such cases are likely initiated by infection, since in the context of HCV- or HBV-infected patients, antiviral therapy can lead to the disappearance of antigenic stimulation, control of clonal plasma cells, and reduced or suppressed monoclonal Ig production. Complete remission has been obtained with anti-HCV therapy in refractory MM with a HCV-specific monoclonal Ig, and antiviral treatments significantly improved the probability of survival of MM patients infected with HCV or HBV prior to the diagnosis of MM. Monoclonal Igs may also target glucolipids, particularly glucosylsphingosine (GlcSph), and GlcSph-reducing therapy can lead to complete remission in SMM and MM patients presenting with a GlcSph-specific monoclonal Ig. The present review describes the importance of determining the target of the monoclonal Ig of MGUS, SMM and MM patients, and discusses the efficacy of target-reducing treatments in the management of MGUS, SMM and MM cases who present with a monoclonal Ig reactive against a treatable infectious pathogen or GlcSph.

Mutations, inflammation and phenotype of myeloproliferative neoplasms.

Knowledge on the myeloproliferative neoplasms (MPNs) - polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) - has accumulated since the discovery of the JAK/STAT-activating mutations associated with MPNs: JAK2V617F, observed in PV, ET and PMF; and the MPL and CALR mutations, found in ET and PMF. The intriguing lack of disease specificity of these mutations, and of the chronic inflammation associated with MPNs, triggered a quest for finding what precisely determines that MPN patients develop a PV, ET or PMF phenoptype. The mechanisms of action of MPN-driving mutations, and concomitant mutations (ASXL1, DNMT3A, TET2, others), have been extensively studied, as well as the role played by these mutations in inflammation, and several pathogenic models have been proposed. In parallel, different types of drugs have been tested in MPNs (JAK inhibitors, interferons, hydroxyurea, anagrelide, azacytidine, combinations of those), some acting on both JAK2 and inflammation. Yet MPNs remain incurable diseases. This review aims to present current, detailed knowledge on the pathogenic mechanisms specifically associated with PV, ET or PMF that may pave the way for the development of novel, curative therapies.

Comparison of Monoclonal Gammopathies Linked to Poliovirus or Coxsackievirus vs. Other Infectious Pathogens.

Chronic stimulation by infectious pathogens or self-antigen glucosylsphingosine (GlcSph) can lead to monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). Novel assays such as the multiplex infectious antigen microarray (MIAA) and GlcSph assays, permit identification of targets for >60% purified monoclonal immunoglobulins (Igs). Searching for additional targets, we selected 28 purified monoclonal Igs whose antigen was not represented on the MIAA and GlcSph assays; their specificity of recognition was then analyzed using microarrays consisting of 3760 B-cell epitopes from 196 pathogens. The peptide sequences PALTAVETG and PALTAAETG of the VP1 coat proteins of human poliovirus 1/3 and coxsackievirus B1/B3, respectively, were specifically recognized by 6/28 monoclonal Igs. Re-analysis of patient cohorts showed that purified monoclonal Igs from 10/155 MGUS/SM (6.5%) and 3/147 MM (2.0%) bound to the PALTAVETG or PALTAAETG epitopes. Altogether, PALTAV/AETG-initiated MGUS are not rare and few seem to evolve toward myeloma.

Efficacy of Antiviral Treatment in Hepatitis C Virus (HCV)-Driven Monoclonal Gammopathies Including Myeloma.

Multiple myeloma (MM) remains an incurable plasma cell malignancy. While its origin is enigmatic, an association with infectious pathogens including hepatitis C virus (HCV) has been suggested. Here we report nine patients with monoclonal gammopathy of undetermined significance (MGUS) or MM with previous HCV infection, six of whom received antiviral treatment. We studied the evolution of the gammopathy disease, according to anti-HCV treatment and antigen specificity of purified monoclonal immunoglobulin, determined using the INNO-LIA™ HCV Score assay, dot-blot assays, and a multiplex infectious antigen microarray. The monoclonal immunoglobulin from 6/9 patients reacted against HCV. Four of these patients received antiviral treatment and had a better evolution than untreated patients. Following antiviral treatment, one patient with MM in third relapse achieved complete remission with minimal residual disease negativity. For two patients who did not receive antiviral treatment, disease progressed. For the two patients whose monoclonal immunoglobulin did not react against HCV, antiviral treatment was not effective for MGUS or MM disease. Our results suggest a causal relationship between HCV infection and MGUS and MM progression. When HCV was eliminated, chronic antigen-stimulation disappeared, allowing control of clonal plasma cells. This opens new possibilities of treatment for MGUS and myeloma.

Anti-Glucosylsphingosine Autoimmunity, JAK2V617F-Dependent Interleukin-1ß and JAK2V617F-Independent Cytokines in Myeloproliferative Neoplasms.

Inflammatory cytokines play a major role in myeloproliferative neoplasms (MPNs) as regulators of the MPN clone and as mediators of clinical symptoms and complications. Firstly, we investigated the effect of JAK2V617F on 42 molecules linked to inflammation. For JAK2V617F-mutated patients, the JAK2V617F allele burden (%JAK2V617F) correlated with the levels of IL-1ß, IL-1Rα, IP-10 and leptin in polycythemia vera (PV), and with IL-33 in ET; for all other molecules, no correlation was found. Cytokine production was also studied in the human megakaryocytic cell line UT-7. Wild-type UT-7 cells secreted 27/42 cytokines measured. UT-7 clones expressing 50% or 75% JAK2V617F were generated, in which the production of IL-1ß, IP-10 and RANTES was increased; other cytokines were not affected. Secondly, we searched for causes of chronic inflammation in MPNs other than driver mutations. Since antigen-driven selection is increasingly implicated in the pathogenesis of blood malignancies, we investigated whether proinflammatory glucosylsphingosine (GlcSph) may play a role in MPNs. We report that 20% (15/75) of MPN patients presented with anti-GlcSph IgGs, distinguished by elevated levels of 11 cytokines. In summary, only IL-1ß and IP-10 were linked to JAK2V617F both in patients and in UT-7 cells; other inflammation-linked cytokines in excess in MPNs were not. For subsets of MPN patients, a possible cause of inflammation may be auto-immunity against glucolipids.

Analysis of the Targets and Glycosylation of Monoclonal IgAs From MGUS and Myeloma Patients.

Previous studies showed that monoclonal immunoglobulins G (IgGs) of "monoclonal gammopathy of undetermined significance" (MGUS) and myeloma were hyposialylated, thus presumably pro-inflammatory, and for about half of patients, the target of the monoclonal IgG was either a virus-Epstein-Barr virus (EBV), other herpes viruses, hepatitis C virus (HCV)-or a glucolipid, lysoglucosylceramide (LGL1), suggesting antigen-driven disease in these patients. In the present study, we show that monoclonal IgAs share these characteristics. We collected 35 sera of patients with a monoclonal IgA (6 MGUS, 29 myeloma), and we were able to purify 25 of the 35 monoclonal IgAs (6 MGUS, 19 myeloma). Monoclonal IgAs from MGUS and myeloma patients were significantly less sialylated than IgAs from healthy volunteers. When purified monoclonal IgAs were tested against infectious pathogens and LGL1, five myeloma patients had a monoclonal IgA that specifically recognized viral proteins: the core protein of HCV in one case, EBV nuclear antigen 1 (EBNA-1) in four cases (21.1% of IgA myeloma). Monoclonal IgAs from three myeloma patients reacted against LGL1. In summary, monoclonal IgAs are hyposialylated and as described for IgG myeloma, significant subsets (8/19, or 42%) of patients with IgA myeloma may have viral or self (LGL1) antigen-driven disease.

Characteristics of MGUS and Multiple Myeloma According to the Target of Monoclonal Immunoglobulins, Glucosylsphingosine, or Epstein-Barr Virus EBNA-1.

: Chronic stimulation by infectious or self-antigens initiates subsets of monoclonal gammopathies of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or multiple myeloma (MM). Recently, glucosylsphingosine (GlcSph) was reported to be the target of one third of monoclonal immunoglobulins (Igs). In this study of 233 patients (137 MGUS, 6 SMM, 90 MM), we analyzed the GlcSph-reactivity of monoclonal Igs and non-clonal Igs. The presence of GlcSph-reactive Igs in serum was unexpectedly frequent, detected for 103/233 (44.2%) patients. However, GlcSph was targeted by the patient's monoclonal Ig for only 37 patients (15.9%); for other patients (44 MGUS, 22 MM), the GlcSph-reactive Igs were non-clonal. Then, the characteristics of patients were examined: compared to MM with an Epstein-Barr virus EBNA-1-reactive monoclonal Ig, MM patients with a GlcSph-reactive monoclonal Ig had a mild presentation. The inflammation profiles of patients were similar except for moderately elevated levels of 4 cytokines for patients with GlcSph-reactive Igs. In summary, our study highlights the importance of analyzing clonal Igs separately from non-clonal Igs and shows that, if autoimmune responses to GlcSph are frequent in MGUS/SMM and MM, GlcSph presumably represents the initial pathogenic event for ~16% cases. Importantly, GlcSph-initiated MM appears to be a mild form of MM disease.

International external quality assurance of JAK2 V617F quantification.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Epstein-Barr Virus and Astrocytoma.

Epstein-Barr virus (EBV) is an established pathogen linked to a wide range of lymphoproliferative disorders and solid tumors. Astrocytoma is one of the most frequent brain tumors in children, adolescents, and young adults. Astrocyte proliferation usually occurs after brain tissue aggression, which may be of different types, including viral infection. In particular, it has been suggested that EBV may play a role in astrocytoma pathogenesis. This article presents the summarized results of a systematic review of the literature on the relationship between EBV infection and astrocytoma pathophysiology. Although most studies detect the presence of EBV DNA in subsets of astrocytoma tumors, our conclusion is that currently, except for rare cases, there is no clear evidence that EBV plays a role in the development of astrocytoma.

Absolute Quantitation of Glycoforms of Two Human IgG Subclasses Using Synthetic Fc Peptides and Glycopeptides.

Immunoglobulins, such as immunoglobulin G (IgG), are of prime importance in the immune system. Polyclonal human IgG comprises four subclasses, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitative method for these subclasses and their Fc N-glycoforms, (glyco)peptides were synthesized using a solid-phase approach and used as internal standards. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR standards, respectively. For IgG1, a similar glycopeptide where tyrosine (Y) was isotopically labelled was used to quantify monoclonal IgG1 that had been treated with the enzyme Endo-F2, i.e., yielding tryptic glycopeptide EEQYN(GlcNAc)STYR. The next step was to quantify single subclasses within polyclonal human IgG samples. Although ion abundances in the MALDI spectra often showed higher signals for IgG2 than IgG1, depending on the spotting solvent used, determination of amounts using the newly developed quantitative method allowed to obtain accurate concentrations where IgG1 species were predominant. It was observed that simultaneous analysis of IgG1 and IgG2 yielded non-quantitative results and that more success was obtained when subclasses were quantified one by one. More experiments served to assess the respective extraction and ionization efficiencies of EEQYNSTYR/EEQFNSTFR and EEQYN(GlcNAc)STYR/EEQFN(GlcNAc)STFR mixtures under different solvent and concentration conditions. Graphical Abstract ᅟ.

Pro-inflammatory State in Monoclonal Gammopathy of Undetermined Significance and in Multiple Myeloma Is Characterized by Low Sialylation of Pathogen-Specific and Other Monoclonal Immunoglobulins.

Multiple myeloma (MM) and its pre-cancerous stage monoclonal gammopathy of undetermined significance (MGUS) allow to study immune responses and the chronology of inflammation in the context of blood malignancies. Both diseases are characterized by the production of a monoclonal immunoglobulin (mc Ig) which for subsets of MGUS and MM patients targets pathogens known to cause latent infection, a major cause of inflammation. Inflammation may influence the structure of both polyclonal (pc) Ig and mc Ig produced by malignant plasma cells via the sialylation of Ig Fc fragment. Here, we characterized the sialylation of purified mc and pc IgGs from 148 MGUS and MM patients, in comparison to pc IgGs from 46 healthy volunteers. The inflammatory state of patients was assessed by the quantification in serum of 40 inflammation-linked cytokines, using Luminex technology. While pc IgGs from MGUS and MM patients showed heterogeneity in sialylation level, mc IgGs from both MGUS and MM patients exhibited a very low level of sialylation. Furthermore, mc IgGs from MM patients were less sialylated than mc IgGs from MGUS patients (p < 0.01), and mc IgGs found to target an infectious pathogen showed a lower level of sialylation than mc IgGs of undetermined specificity (p = 0.048). Regarding inflammation, 14 cytokines were similarly elevated with a p value < 0.0001 in MGUS and in MM compared to healthy controls. MM differed from MGUS by higher levels of HGF, IL-11, RANTES and SDF-1-α (p < 0.05). MGUS and MM patients presenting with hyposialylated pc IgGs had significantly higher levels of HGF, IL-6, tumor necrosis factor-α, TGF-ß1, IL-17, and IL-33 compared to patients with hyper-sialylated pc IgGs (p < 0.05). In MGUS and in MM, the degree of sialylation of mc and pc IgGs and the levels of four cytokines important for the anti-microbial response were correlated, either positively (IFN-α2, IL-13) or negatively (IL-17, IL-33). Thus in MGUS as in MM, hyposialylation of mc IgGs is concomitant with increased levels of cytokines that play a major role in inflammation and anti-microbial response, which implies that infection, inflammation, and abnormal immune response contribute to the pathogenesis of MGUS and MM.

Monoclonal IgG in MGUS and multiple myeloma targets infectious pathogens.

Subsets of mature B cell neoplasms are linked to infection with intracellular pathogens such as Epstein-Barr virus (EBV), hepatitis C virus (HCV), or Helicobacter pylori. However, the association between infection and the immunoglobulin-secreting (Ig-secreting) B proliferative disorders remains largely unresolved. We investigated whether the monoclonal IgG (mc IgG) produced by patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM) targets infectious pathogens. Antigen specificity of purified mc IgG from a large patient cohort (n = 244) was determined using a multiplex infectious-antigen array (MIAA), which screens for reactivity to purified antigens or lysates from 9 pathogens. Purified mc IgG from 23.4% of patients (57 of 244) specifically recognized 1 pathogen in the MIAA. EBV was the most frequent target (15.6%), with 36 of 38 mc IgGs recognizing EBV nuclear antigen-1 (EBNA-1). MM patients with EBNA-1-specific mc IgG (14.0%) showed substantially greater bone marrow plasma cell infiltration and higher ß2-microglobulin and inflammation/infection-linked cytokine levels compared with other smoldering myeloma/MM patients. Five other pathogens were the targets of mc IgG: herpes virus simplex-1 (2.9%), varicella zoster virus (1.6%), cytomegalovirus (0.8%), hepatitis C virus (1.2%), and H. pylori (1.2%). We conclude that a dysregulated immune response to infection may underlie disease onset and/or progression of MGUS and MM for subsets of patients.

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia.

Thrombopoietin (Tpo) and its receptor (Mpl) are the principal regulators of early and late thrombopoiesis and hematopoietic stem cell maintenance. Mutations in MPL can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is characterized by severe thrombocytopenia at birth, which progresses to bone marrow failure and pancytopenia. Here we report unique familial cases of CAMT that presented with a previously unreported MPL mutation: T814C (W272R) in the background of the activating MPL G117T (K39N or Baltimore) mutation. Confocal microscopy, proliferation and surface biotinylation assays, co-immunoprecipitation, and western blotting analysis were used to elucidate the function and trafficking of Mpl mutants. Results showed that Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, lacks detectable surface expression while being strongly colocalized with the endoplasmic reticulum (ER) marker calreticulin. Both WT and K39N-mutated Mpl were found to be signaling competent, but single or double mutants bearing W272R were unresponsive to Tpo. Function of the deficient Mpl receptor could be rescued by using 2 separate approaches: (1) GRASP55 overexpression, which partially restored Tpo-induced signaling of mutant Mpl by activating an autophagy-dependent secretory pathway and thus forcing ER-trapped immature receptors to traffic to the cell surface; and (2) CRISPR-Cas9 gene editing used to repair MPL T814C mutation in transfected cell lines and primary umbilical cord blood-derived CD34+ cells. We demonstrate proof of principle for rescue of mutant Mpl function by using gene editing of primary hematopoietic stem cells, which indicates direct therapeutic applications for CAMT patients.

Pathogenesis of Myeloproliferative Neoplasms: Role and Mechanisms of Chronic Inflammation.

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal diseases characterized by the excessive and chronic production of mature cells from one or several of the myeloid lineages. Recent advances in the biology of MPNs have greatly facilitated their molecular diagnosis since most patients present with mutation(s) in the JAK2, MPL, or CALR genes. Yet the roles played by these mutations in the pathogenesis and main complications of the different subtypes of MPNs are not fully elucidated. Importantly, chronic inflammation has long been associated with MPN disease and some of the symptoms and complications can be linked to inflammation. Moreover, the JAK inhibitor clinical trials showed that the reduction of symptoms linked to inflammation was beneficial to patients even in the absence of significant decrease in the JAK2-V617F mutant load. These observations suggested that part of the inflammation observed in patients with JAK2-mutated MPNs may not be the consequence of JAK2 mutation. The aim of this paper is to review the different aspects of inflammation in MPNs, the molecular mechanisms involved, the role of specific genetic defects, and the evidence that increased production of certain cytokines depends or not on MPN-associated mutations, and to discuss possible nongenetic causes of inflammation.