BACKGROUND

small B-cell neoplasms can show plasmacytic differentiation and may potentially progress to aggressive lymphoma (DLBCL). Epstein-Barr virus (EBV) infection may cause the transformation of malignant cells in vitro.

METHODS

we established VR09 cell line with plasmacytic differentiation, obtained from a case of atypical, non-CLL B-cell chronic lymphoproliferative disease with plasmacytic features. We used flow cytometry, immunohistochemistry, polymerase chain reaction, cytogenetic analysis and florescence in situ hybridization in the attempt at thoroughly characterizing the cell line. We showed VR09 tumorigenic potential in vivo, leading to the development of activated DLBCL with plasmacytic features.

RESULTS

VR09 cells displayed plasmacytic appearance and grew as spherical tumors when inoculated subcutaneously into immunodeficient Rag2(-/-) γ-chain(-/-) mice. VR09 cell line and tumors displayed the phenotype of activated stage of B cell maturation, with secretory differentiation (CD19+ CD20+ CD79a+ CD79b+/- CD138+ cyclin D1- Ki67 80% IgM+ IgD+ MUM1+ MNDA+ CD10- CD22+ CD23+ CD43+ K+, λ- Bcl2+ Bcl6-) and they presented episomal EBV genome, chromosome 12 trisomy, lack of c-MYC rearrangement and Myd88 gene mutation, presence of somatic hypermutation in the VH region, and wild-type p53.

CONCLUSIONS

This new EBV-positive cell line may be useful to further characterize in vivo activated DLBCL with plasmacytic features.

According to World Health Organization (WHO) classification (2008), B-cell neoplasms are classified into precursor B-cell or a mature B-cell phenotype and this classification was also kept in the latest WHO revision (2016). We are reporting a male patient in his fifties, with tonsillar swelling diagnosed as diffuse large B-cell lymphoma (DLBCL), germinal center. He received 6 cycles of RCHOP and showed complete metabolic response. Two months later, he presented with severe CNS symptoms. Flow cytometry on bone marrow (BM) showed infiltration by CD10-positive Kappa-restricted B-cells with loss of CD20 and CD19, and downregulation of CD79b. Moreover, the malignant population showed Tdt expression. BM Cytogenetics revealed t(8;14)(q24;q32) within a complex karyotype. Retrospectively, MYC and Tdt immunostains performed on original diagnostic tissue and came negative for Tdt and positive for MYC. It has been rarely reported that mature B-cell neoplasms present with features of immaturity; however the significance of Tdt acquisition during disease course was not addressed before. What is unique in this case is that the emerging disease has acquired an immaturity marker while retaining some features of the original mature clone. No definitive WHO category would adopt high-grade neoplasms that exhibit significant overlapping features between mature and immature phenotypes.

Occasional marginal zone lymphomas have extensive plasmacytic differentiation (P-MZL). We present an argument based on our data and previous published observations that P-MZL represent neoplasms of precursor plasma cells. Five P-MZL were analyzed by flow cytometry and the phenotype was compared with conventional MZL, plasma cell myeloma (PCM), reactive plasmacytoses (RP), and normal marrow plasma cells. The clonal cells in four of five P-MZL were CD19+, CD20(dim/⁻), CD38(bright), CD45++, CD56⁻, CD138⁻, and surface light chain(dim/⁻). One case was CD56+, CD138+, and CD19⁻. A separate clonal mature B cell population [CD20+, CD79b+] was not detected in any of these tumors. Hierarchical clustering demonstrated similarity between the neoplastic cells in four of the five P-MZL with plasma cells in RP comprised of precursor plasma cells. Analysis of normal bone marrow plasma cells revealed a CD45++CD38(bright)CD19+CD138⁻/+ precursor plasma cell population similar to the plasma cells in RP and P-MZL and a very small population of mature CD45+/⁻CD38(bright)CD138+CD19⁻ plasma cellsresembling the neoplastic cells in PCM. The findings suggest that P-MZL represents a malignancy of a plasma cell stage of differentiation distinct from the plasma cell stage corresponding to PCM. We propose the term precursor plasma cell neoplasm for these tumors.

BACKGROUND

Accurate diagnosis of chronic lymphocytic leukemia (CLL) acquires immunophenotyping by flow cytometry in order to facilitate differential diagnosis between CLL and other mature B-cell neoplasms (MBCN).

OBJECTIVE

The aim of this study was to define immunological profile of CLL cells.

METHODS

Immunophenotyping by flow cytometry was performed on peripheral blood specimens at diagnosis in the group of 211 patients with de novo MBCN.

RESULTS

Absolute count of B-cells was significantly increased in all MBCN patients comparing to healthy control group (p < 0.05). B-cell monoclonality was detected in 96% of all MBCN patients, by using surface immunoglobulin (sIg) light chain restriction. B-cell antigens, CD19, CD20, CD22, were expressed with very high frequency in CLL and other MBCN. In comparison with other MBCN, in CLL group, the frequency of expression was higher for CD5 and CD23 (p < 0.0001), though lower for FMC7 antigen (p < 0.0001). CLL patients were characterized by lower expression patterns of CD20, CD22, CD79b, and sIg (p < 0.0001) as well as higher expression pattern of CD5 antigen (p < 0.05). Correlation between the final diagnosis of MBCN and values of CLL scoring system showed that the majority of CLL patients (97%) had higher values (5 or 4) whereas the majority of other MBCN patients (96%) had lower score values (0-3).

CONCLUSIONS

Our results have shown that characteristic immunophenotype which differentiates CLL from other MBCN is defined by following marker combination--CD19+ CD20(+Iow) CD22(+low) CD5(+high) CD23+ FMC7-CD79b(+Iow) sIg(+Iow). CLL score values of 5 or 4 points are highly suggestive for diagnosis of CLL.

Stem cell antigen 2 (SCA2) is a Ly6 family member whose function is largely unknown. To characterize biological properties and tissue distribution of chicken SCA2, SCA2 was expressed in E. coli, purified, and a polyclonal antibody developed. Utilizing the polyclonal antibody, SCA2 is a 13 kDa cell surface protein anchored by a glycosyl-phosphatidylinositol (GPI) moiety. SCA2 is expressed in connective tissues of thymus and bursa based on immunohistochemistry, immunoprecipitation, and western blots. In bursal follicles, SCA2 is specifically expressed on the cortical-medullary epithelial cells (CMEC) surrounded by MHC class II presenting cells. Expression profiles of bursal cells induced by contact with SCA2-expressing cells shows down-regulation of numerous genes including CD79B, B cell linker (BLNK), spleen tyrosine kinase (SYK), and gamma 2-phospholipase C (PLCG2) that are involved in the B cell receptor (BCR) and immune response signaling pathways. These results suggest chicken SCA2 plays a role in regulating B lymphocytes.

Three hundred and seventy six monoclonal antibodies (mAbs) raised against human leukocyte surface antigens were analyzed by flow cytometry for cross reactivities against mink leukocytes. We found 53 mAbs (14%) to cross react. This study defined cross reactions to the following human markers: CD1a, CD9 (4 mAbs), CD10, CD11a (2 mAbs), CD14 (3 mAbs), CD18 (5 mAbs), CD20 (atypical reaction), CD21, CD25 (atypical reaction), CD29 (3 mAbs), CD32, CD41, CD42a, CD44 (4 mAbs), CD45, CD45RO, CD47 (2 mAbs), CD49d (3 mAbs), CD61 (2 mAbs), CD62P, CD66abcd, CD71, CD75s, CD79b (2 mAbs), CD86, CD88, CD104 (atypical reaction), CD172a, CD236R (glycophorin C, (atypical reaction)), Xg(a) carbohydrate antigen, Rhesus antigen and two unspecified PAN-reactive mAbs. In order to characterize the molecular mass of the corresponding cross reacting mink markers, the mAbs were used to immunoprecipitate the surface antigens. Fourteen mAbs out of the 53 mAbs reactive with mink leukocytes gave reproducible IP findings. The masses of the precipitated antigens were generally in good agreement with those of the homologous human markers. We also performed immunohistochemical staining analyses on formalin fixed, paraffin embedded mink tissue from lymph node and spleen, and found 7 out of 22 mAbs to give a positive signal. Generally, the immunohistological analyses resulted in expected staining patterns.

To analyze the cellular antigens of human B-cell lineage, a monoclonal antibody, NU-B1, was raised using the acute lymphoblastic leukemia (ALL) cell line NALM-16 as the immunogen. NU-B1 reacted with 7.7+/-3.9% of the healthy adult peripheral blood mononuclear cells but not with neutrophils, monocytes, red blood cells or thymocytes. In order to distinguish the reaction specificity of NU-B1, two-color immunofluorescence staining using tonsillar cells was performed, and it was demonstrated that NU-B1-positive cells coexpressed CD20, which is a representative B-cell antigen. The expression of NU-B1 was highly restricted to cells of B-cell lineage when a panel of hematopoietic cell lines was examined. In a pathoimmunohistological study using human lymph node tissue, NU-B1-positive cells were localized in the mantle and marginal zones. In a clinical study, NU-B1 reacted specifically with leukemias/lymphomas of B-cell lineage: all 43 cases of ALL including common ALL and biphenotypic leukemia, all 4 cases of B-cell ALL, 6/7 B-cell type malignant lymphomas and 2/4 B-cell chronic lymphocytic leukemias. NU-B1 did not react with multiple myeloma, T-cell or myeloid leukemias/lymphomas. Immunoprecipitation of NU-B1 revealed two clear bands at 50 kDa and 42 kDa under either reducing or nonreducing conditions. Although anti-IgM treatment induced dramatic down modulation of CD79b, the NU-B1 antigen was also down modulated, but only slightly. However, crosslinking of NU-B1 did not induce tyrosine phosphorylation of intracellular proteins or the mobilization of calcium in NALM-16. The present results revealed that the antigenic determinant recognized by NU-B1 is not surface immunoglobulin chains, HLA-DR, a receptor for C3, Fc for immunoglobulin chains or any known CD molecule. We conclude that monoclonal antibody NU-B1 recognizes a novel human B-cell restricted antigen distinct from known CD molecules, and that it is a useful antibody in the immunophenotyping and classification of leukemias/lymphomas.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous subtype of non-Hodgkin lymphoma. In addition to clinical and immunophenotypic characteristics, recurrent gene mutations have recently been identified in patients with DLBCL using next-generation sequencing technologies. The aim of this study is to investigate the clinical relevance of B-cell function gene mutations in DLBCL. Clinical analysis was performed on 680 Chinese DLBCL patients (146 non-CR and 534 CR cases) treated with six cycles of 21-day R-CHOP (Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), alone or followed by two additional doses of rituximab consolidation on patients' own intention. Somatic mutations of B-cell function genes were further screened on 275 (71 non-CR and 204 CR) cases with available tumor samples by targeted sequencing, including genes involved in B-cell receptors (BCRs) pathway (CARD11, LYN, CD79A, and CD79B), Toll-like receptors (TLRs) pathway (MYD88), and tumor necrotic factor receptor (TNFR) pathway (TRAF2 and TNFAIP3). B-cell function gene mutations occurred in 44.0% (121/275) of DLBCL patients. The TLRs and TNFR related gene mutations were more frequently observed in non-CR patients (p=0.019 and p=0.032, respectively). BCRs related gene mutations, as well as revised IPI (R-IPI) and double BCL-2/MYC expression, were independently related to short progression-free survival in DLBCL after CR. The adverse prognostic effect of BCRs related gene mutations could be overcome by two additional doses of rituximab consolidation. These results highlight the molecular heterogeneity of DLBCL and identify a significant role of B-cell function gene mutations on lymphoma progression and response to rituximab in DLBCL.

Splenic Marginal Zone Lymphoma (SMZL) is a rare B-cell neoplasm comprising less than 2% of non-Hodgkin lymphomas. We hereby report a case of SMZL in a 66-year-old female who presented with fever and massive splenomegaly. Peripheral blood smear examination showed atypical lymphoid cells showing variable cytoplasmic processes. Flowcytometric immunophenotyping of peripheral blood showed tumour cells which were found to be positive for CD19, CD79b and showing kappa light chain restriction along with lack of expression for CD5, CD10, CD23, CD103 and lambda. These findings were suggestive of B cell chronic lymphoproliferative disorder. Various differential diagnoses considered in this case were analysed by using different diagnostic clues to arrive at the diagnosis. Bone marrow examination and Immunohistochemical (IHC) analysis showed tumour cells in nodular, interstitial and intrasinusoidal pattern of infiltration which were positive for CD20 and CD79b with kappa light chain restriction and lack of expression of CD5, CD10, CD23 and CD103 which further corroborated the flowcytometric immunophenotyping. The diagnosis of SMZL is arrived at by a combination of diagnostic clues like clinical features, peripheral smear findings, flowcytometric immunophenotyping, morphological and IHC findings in bone marrow biopsy. This case highlights the significance of flowcytometric immunophenotyping and bone marrow biopsy with immunohistochemistry to arrive at a diagnosis of SMZL even in absence of splenic histopathology.

B cell responses and their concomitant signal transduction pathways are not well understood in marsupial mammals, despite the availability of gene expression data for key immunoglobulin genes and for elements of the CD79a/CD79b heterodimer signalling complex for two model marsupials. Broader studies of factors that influence B cell responses are still hampered by a lack of species-specific reagents and there are few reports of other factors that influence gene expression such as the potential for splice variants in BCR components, which may influence immune signalling pathways. In this study, we characterise CD79a and CD79b genes in the endangered macropod marsupial, Onychogalea fraenata (the bridled nailtail wallaby) and show that domains and residues important for the structural and functional integrity of both monomers are conserved in this species, consistent with results previously reported for the closely-related macropod, Macropus eugenii (the tammar wallaby). We extend this work to report the detection of splice variants for CD79a and CD79b in wallaby species; three CD79a isoforms and one CD79b isoform. Of these, two CD79a isoforms and the CD79b isoform have not been reported in any other mammalian species.

The role of microenvironment-mediated biophysical forces in human lymphomas remains elusive. Diffuse large B cell lymphomas (DLBCLs) are heterogeneous tumors, which originate from highly proliferative germinal center B cells. These tumors, their associated neo-vessels, and lymphatics presumably expose cells to particular fluid flow and survival signals. Here, we show that fluid flow enhances proliferation and modulates response of DLBCLs to specific therapeutic agents. Fluid flow upregulates surface expression of B cell receptors (BCRs) and integrin receptors in subsets of ABC-DLBCLs with either CD79A/B mutations or WT BCRs, similar to what is observed with xenografted human tumors in mice. Fluid flow differentially upregulates signaling targets, such as SYK and p70S6K, in ABC-DLBCLs. By selective knockdown of CD79B and inhibition of signaling targets, we provide mechanistic insights into how fluid flow mechanomodulates BCRs and integrins in ABC-DLBCLs. These findings redefine microenvironment factors that regulate lymphoma-drug interactions and will be critical for testing targeted therapies.

Refractory/relapsed diffuse large B-cell lymphoma remains a major unmet medical need with poor outcome, especially for patients considered ineligible for stem cell transplant. Polatuzumab vedotin (PV) is a first-in-class anti-CD79b antibody-drug conjugate that contains the microtubule inhibitor monomethyl auristatin E. The development of PV is currently very active. This drug was US FDA approved in 2019 in combination with bendamustine and rituximab for the treatment of refractory/relapsed diffuse large B-cell lymphoma in third line and more, after demonstrating relevant efficacy and acceptable safety in a pivotal randomized Phase II trial. This review summarizes the features of this new drug with the primary focus on the clinical work supporting efficacy, relevance and tolerability of PV.

Keywords: CD79b; antibody–drug conjugate; diffuse large B-cell lymphoma; polatuzumab vedotin.

We are interested in developing a second generation of antibody-drug conjugates (ADCs) for the treatment of non-Hodgkin's lymphoma (NHL) that could provide a longer duration of response and be more effective in indolent NHL than the microtubule inhibiting ADCs pinatuzumab vedotin (anti-CD22-vc-MMAE) and polatuzumab vedotin (anti-CD79b-vc-MMAE). Pinatuzumab vedotin (anti-CD22-vc-MMAE) and polatuzumab vedotin (anti-CD79b-vc-MMAE) are ADCs that contain the microtubule inhibitor monomethyl auristatin E (MMAE). Clinical trial data suggest that these ADCs have promising efficacy for the treatment of non-Hodgkin's lymphoma (NHL), however, some patients do not respond or become resistant to the ADCs. We tested an anti-CD22 ADC with a seco-CBI-dimer payload, thio Hu-anti-CD22-(LC:K149C)-SN36248, and compared it to pinatuzumab vedotin for its efficacy and duration of response in xenograft models and its ability to deplete normal B cells in cynomologus monkeys. We found that anti-CD22-(LC:K149C)-SN36248 was effective in xenograft models resistant to pinatuzumab vedotin, gave a longer duration of response, had a different mechanism of resistance and was able to deplete normal B cells better than pinatuzumab vedotin. These studies provide evidence that anti-CD22-(LC:K149C)-SN36248 has the potential for longer duration of response and more efficacy in indolent NHL than MMAE ADCs and may provide the opportunity to improve outcomes for NHL patients.

Canine atopic dermatitis (CAD) has a hereditary basis that is modified by interactions with the environment, including diet. Differentially expressed genes in non-lesional skin, determined by RNA sequencing before and after a dietary intervention, were compared between dogs with naturally occurring CAD (n = 4) and healthy dogs (n = 4). The dogs were fed either a common commercial heat-processed high carbohydrate food (kibble diet) (n = 4), or a non-processed high fat food (raw meat-based diet) (n = 4). At the end of the diet intervention, 149 differentially expressed transcripts were found between the atopic and healthy dogs. The main canonical pathways altered by the dysregulation of these genes were angiopoietin signaling, epidermal growth factor signaling, activation of angiogenesis, and alterations in keratinocyte proliferation and lipid metabolism. On the other hand, 33 differently expressed transcripts were found between the two diet groups, of which 8 encode genes that are annotated in the current version of the dog genome: immunoglobulin heavy constant mu (IGHM), immunoglobulin lambda-like polypeptide 5 (IGLL5), B-cell antigen receptor complex-associated protein beta chain (CD79B), polymeric immunoglobulin receptor (PIGR), cystathionine β-synthase (CBS), argininosuccinate synthase 1 (ASS1), secretory leukocyte peptidase inhibitor (SLPI), and mitochondrial ribosome recycling factor (MRRF). All genes were upregulated in the raw diet group. In conclusion the findings of this study suggest alterations in lipid and keratinocyte metabolism as well as angiogenesis in the skin of atopic dogs. Additionally, a possible enhancement of innate immunity and decrease in oxidative stress was seen in raw food fed dogs, which could have an important role in preventing hypersensitivities and disturbed immunity at young age.

Keywords: RNAseq; atopic dermatitis; canine; diet; gene expression; kibble diet; raw meat-based diet; skin.

Activated B-cell (ABC)-Diffuse large B-cell lymphomas (DLBCLs) are clinically aggressive and phenotypically complex malignancies, whose transformation mechanisms remain unclear. Partially differentiated antigen-secreting cells (plasmablasts) have long been regarded as cells-of-origin for these tumors, despite lack of definitive experimental evidence. Recent DLBCL re-classification based on mutational landscapes identified "MCD/C5" tumors as specific ABC-DLBCLs with unfavorable clinical outcome, activating mutations in the signaling adaptors MYD88 and CD79B, and immune evasion through mutation of antigen presenting genes. MCD/C5s manifest prominent extranodal dissemination and similarities with primary extranodal lymphomas (PENLs). In this regard, recent studies on TBL1XR1, a gene recurrently mutated in MCD/C5s and PENLs, suggest that aberrant memory B-cells (MBs), and not plasmablasts, are the true cells-of-origin for these tumors. Moreover, transcriptional and phenotypic profiling suggest that MCD/C5s, as a class, represent bonafide MB tumors. Based on emerging findings we propose herein a generalized stepwise model for MCD/C5 and PENLs pathogenesis, whereby acquisition of "founder" mutations in activated B-cells favors the development of aberrant MBs prone to avoid plasmacytic differentiation upon recall and undergo systemic dissemination. Cyclic reactivation of these MBs through persistent antigen exposure favors their clonal expansion and accumulation of mutations, which further facilitate their activation. As a result, MB-like clonal precursors become trapped in an oscillatory state of semi-permanent activation and phenotypic sway, that facilitates ulterior transformation, and accounts for the extranodal clinical presentation and biology of these tumors. In addition, we discuss diagnostic and therapeutic implications of a MB cell-of-origin for these lymphomas.

Influenza has frequently been epidemic in recent years. However, the mechanisms of severe pneumonia with postinfluenza Streptococcus pneumoniae (SP) secondary infection have not been fully understood. In this study, we explored the mechanisms of pneumonia in postinfluenza A virus (IAV) infection via a mouse model. Mice were intranasally inoculated with SP three days after IAV inoculation. We then collected samples at three time points to dynamically observe the pathological progression. In IAV infection alone, lymphocyte infiltration and widened alveolar intervals were observed. In the blood, levels of the CD19+, CD19+CD21+ and CD19+CD79β+B lymphocyte subpopulations were reduced, and IFN-γ and IL-10 were elevated. Slight atrophy was seen in the spleen, which was due to splenic B lymphocyteinitiated apoptosis through the mitochondrial pathway. When SP infection occurred after IAV infection, the pulmonary inflammation was significantly aggravated; a fair number of lymphocytes and neutrophils infiltrated simultaneously with exfoliated bronchial epithelial cells, vascular endothelial cells, widened alveolar septum and hemorrhaging. Increasing edema fluid and bacteria accumulated in the alveolar cavity. Decreased CD19+, CD19+CD21+ and CD19+CD79β+B lymphocyte subpopulations and increased interferon gamma (IFN-γ) or interleukin 10 (IL-10) were more prominent compared to those with viral infection alone. Spleen atrophy resulting from coinfection was more obvious because of massive splenic B lymphocyte apoptosis through the mitochondrial pathway compared to viral infection alone. This study shows that although inflammation caused by SP infection alone was temporary, preceding IAV infection provided favorable conditions for SP colonization and multiplication by destroying lung structure and suppressing humoral immunity. Synergistic IAV-SP coinfection is likely to facilitate more SP colonization and promote B lymphocyte-suppression and reduction. Eventually, the pneumonia worsened.

Background: Low T3 syndrome is frequent in patients admitted to intensive care units for critical illness and pneumonia. It has been reported also in patients with COVID-19, Hodgkin disease and chronic lymphocytic leukemia. We analyzed the clinical relevance of Low T3 syndrome in COVID-19 patients and, in particular, in those with associated hematological malignancies.

Methods: Sixty-two consecutive patients, hospitalized during the first wave of SARS-CoV-2 outbreak in Sant'Andrea University Hospital in Rome, were subdivided in 38 patients (Group A), showing low levels of FT3, and in 24 patients (Group B), with normal FT3 serum values. During the acute phase of the disease, we measured serum, radiologic and clinical disease severity markers and scores, in search of possible correlations with FT3 serum values. In addition, in 6 COVID-19 patients, 4 with Low T3 syndrome, including 2 with a hematological malignancy, and 2 with normal FT3 values, we performed, high-dimensional single-cell analysis by mass cytometry, multiplex cytokine assay and gene expression profiling in peripheral blood mononuclear cells (PBMC).

Results: Low FT3 serum values were correlated with increased Absolute Neutrophil Count, NLR and dNLR ratios and with reduced total count of CD3+, CD4+ and CD8+ T cells. Low FT3 values correlated also with increased levels of inflammation, tissue damage and coagulation serum markers as well as with SOFA, LIPI and TSS scores. The CyTOF analysis demonstrated reduction of the effector memory and terminal effector subtypes of the CD4+ T lymphocytes. Multiplex cytokine assay indicates that mainly IL-6, IP-10 and MCAF changes are associated with FT3 serum levels, particularly in patients with coexistent hematological malignancies. Gene expression analysis using Nanostring identified four genes differently expressed involved in host immune response, namely CD38, CD79B, IFIT3 and NLRP3.

Conclusions: Our study demonstrates that low FT3 serum levels are associated with severe COVID-19. Our multi-omics approach suggests that T3 is involved in the immune response in COVID-19 and coexistent hematological malignancy and new possible T3 target genes in these patients have been identified.

Keywords: Coronavirus disease (COVID-19); CyTOF; Differentially expressed genes; Hematological malignancies; Low T3 syndrome; NanoString; Thyroid function.

PAX5 and EBF1 work synergistically to regulate genes that are involved in B lymphocyte differentiation. We used the KIS-1 diffuse large B cell lymphoma cell line, which is reported to have elevated levels of PAX5 expression, to investigate the mechanism of EBF1- and PAX5-regulated gene expression. We demonstrate the lack of expression of hallmark B cell genes, including CD19, CD79b, and EBF1, in the KIS-1 cell line. Upon restoration of EBF1 expression we observed activation of CD19, CD79b and other genes with critical roles in B cell differentiation. Mass spectrometry analyses of proteins co-immunoprecipitated with PAX5 in KIS-1 identified components of the MLL H3K4 methylation complex, which drives histone modifications associated with transcription activation. Immunoblotting showed a stronger association of this complex with PAX5 in the presence of EBF1. Silencing of KMT2A, the catalytic component of MLL, repressed the ability of exogenous EBF1 to activate transcription of both CD19 and CD79b in KIS-1 cells. We also find association of PAX5 with the MLL complex and decreased CD19 expression following silencing of KMT2A in other human B cell lines. These data support an important role for the MLL complex in PAX5-mediated transcription regulation.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease whose prognosis is associated with clinical features, cell-of-origin and genetic aberrations. Recent integrative, multi-omic analyses had led to identifying overlapping genetic DLBCL subtypes. We used targeted massive sequencing to analyze 84 diagnostic samples from a multicenter cohort of patients with DLBCL treated with rituximab-containing therapies and a median follow-up of 6 years. The most frequently mutated genes were IGLL5 (43%), KMT2D (33.3%), CREBBP (28.6%), PIM1 (26.2%), and CARD11 (22.6%). Mutations in CD79B were associated with a higher risk of relapse after treatment, whereas patients with mutations in CD79B, ETS1, and CD58 had a significantly shorter survival. Based on the new genetic DLBCL classifications, we tested and validated a simplified method to classify samples in five genetic subtypes analyzing the mutational status of 26 genes and BCL2 and BCL6 translocations. We propose a two-step genetic DLBCL classifier (2-S), integrating the most significant features from previous algorithms, to classify the samples as N1^2-S, EZB^2-S, MCD^2-S, BN2^2-S, and ST2^2-S groups. We determined its sensitivity and specificity, compared with the other established algorithms, and evaluated its clinical impact. The results showed that ST2^2-S is the group with the best clinical outcome and N1^2-S, the more aggressive one. EZB^2-S identified a subgroup with a worse prognosis among GCB-DLBLC cases.

The domestic ferret (Mustela putorius furo) serves as an animal model for the study of several viruses that cause human disease, most notably influenza. Despite the importance of this animal model, characterization of the immune response by flow cytometry (FCM) is severely hampered due to the limited number of commercially available reagents. To begin to address this unmet need and to facilitate more in-depth study of ferret B cells including the identification of antibody-secreting cells, eight unique murine monoclonal antibodies (mAb) with specificity for ferret immunoglobulin (Ig) were generated using conventional B cell hybridoma technology. These mAb were screened for reactivity against ferret peripheral blood mononuclear cells by FCM and demonstrate specificity for CD79β+ B cells. Several of these mAb are specific for the light chain of surface B cell receptor (BCR) and enable segregation of kappa and lambda B cells. Additionally, a mAb that yielded surface staining of nearly all surface BCR positive cells (i.e., pan ferret Ig) was generated. Collectively, these MαF-Ig mAb offer advancement compared to the existing portfolio of polyclonal anti-ferret Ig detection reagents and should be applicable to a wide array of immunologic assays including the identification of antibody-secreting cells by FCM.

Lymphoplasmacytic lymphoma (LPL) is usually associated with a serum IgM paraprotein, corresponding to Waldenström's Macroglobulinemia (WM). Cases presenting with IgG or IgA, or without a monoclonal protein are extremely rare. We analyzed clinical characteristics, frontline treatment, and the outcome of 45 patients with non-IgM LPL, and compared them with a control group of WM patients. The median age was similar, with significantly higher prevalence of females in non-IgM LPL, than in WM patients (60% vs 39%, P = .016). Patients with non-IgM LPL more frequently presented with lymphadenopathies (53% vs 15%, P < .001), splenomegaly (22% vs 8%, P = .015) or extranodal involvement (20% vs 8%, P = .05). In non-IgM LPL a serum monoclonal protein and bone marrow infiltration were less common than in WM patients (69% and 84% of cases respectively, P < .001 for both comparisons). The MYD88 (L265P) mutation was found in 8/19 patients using allele-specific polymerase chain reaction. A CXCR4 mutation was found in 4/17 cases using Sanger. In 16 patients we performed targeted next-generation sequencing of genes MYD88, CXCR4, ARID1-A, KMT2D, NOTCH2, TP53, PRDM1, CD79B, TRAF3, MYBBP1A, TNFAIP3. Seven patients (44%) had a MYD88 mutation (S219C in one), four (25%) a CXCR4 mutation, three (19%) a KMT2D mutation, one (6%) a TP53 mutation and one (6%) a TRAF3 mutation. With a median follow-up of 55.7 months, 36 non-IgM LPL patients (80%) were treated. Non-IgM LPL patients received more frequently anthracycline-containing regimens, as compared with WM patients, who mainly received alkylating-based therapies. Five-year overall survival (OS) was 84%, similar to that of WM patients.

Introduction: New agents for the management of B-cell non-Hodgkin lymphomas (NHLs) are needed, particularly for high-risk and relapsed or refractory patients. Antibody-drug conjugates (ADCs) provide targeted drug delivery to tumors with a broader therapeutic index of cytotoxic agent thus reducing their systemic toxicity while increasing intracellular concentrations. Among them, polatuzumab vedotin, an anti-CD79b conjugated to the microtubule inhibitor monomethyl auristatin E (MMAE) raises particular interest.

Areas covered: We discuss the pharmacological development of polatuzumab vedotin and offer insights into the challenges of designing a successful ADC. We summarize data from preclinical studies and recent trials, and the ongoing phase III POLARIX trial.

Expert opinion: Clinical data from phase Ib/II studies demonstrate significant promise for polatuzumab vedotin in association with rituximab and bendamustine in relapsed or refractory (R/R) DLBCL. In first line, in association with rituximab, cyclophosphamide, doxorubicin and prednisone (R-CHP), promising phase II results led to the development of the phase III POLARIX trial whose results are eagerly awaited. If dosing and scheduling are adequately managed to avoid the risk of peripheral neuropathy, polatuzumab vedotin might become a key component of DLBCL treatment and should be investigated in other lymphomas. This antibody drug conjugate offers new opportunities of combination with non-cytotoxic agents or immunological interventions; this may reshape the treatment of DLBCL.

Keywords: CD79a; antibody-drug conjugates; diffuse large B-cell lymphoma; polatuzumab vedotin.