Diffuse large B cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy that can be divided in two major subgroups, germinal center B-cell-like (GCB) and activated B-cell-like (ABC). Activating mutations of genes involved in the BCR and NF-κB pathways (CD79A, CD79B, MYD88, and CARD11) or in epigenetic regulation (EZH2) have been recently reported, preferentially in one of the two DLBCL subtypes. We analyzed the mutational status of these five recurrently mutated genes in a cohort of 161 untreated de novo DLBCL. Overall, 93 mutations were detected, in 61 (38%) of the patients. The L265P MYD88 mutation was the most frequent MYD88 variant (n = 18), observed exclusively in the ABC subtype. CD79A/CD79B ITAM domains were targeted in ABC DLBCL (12/77; 16%), whereas CARD11 mutations were equally distributed in the two subtypes. The EZH2 Y641 substitution was found almost exclusively in the GCB subgroup (15/62; 24%). Twenty cases (12%) displayed two activating mutations, including the most frequent CD79/MYD88 variants combination (n = 8) which is observed exclusively in the ABC subtype. When considering only ABC DLBCL patients treated by rituximab plus chemotherapy, the presence of an activating NF-κB mutation was associated with an unfavorable outcome (3-years OS 26% for mutated cases versus 67% for the cases without mutations, P = 0.0337). Our study demonstrates that activating and targetable mutations are observed at a very high frequency in DLBCL at the time of diagnosis, indicating that sequencing of a limited number of genes could help tailor an optimal treatment strategy in DLBCL.

IGHV3-21-using chronic lymphocytic leukemia (CLL) is a distinct entity with restricted immunoglobulin gene features and poor prognosis and is more frequently encountered in Northern than Southern Europe. To further investigate this subset and its geographic distribution in the context of a country (Italy) with both continental and Mediterranean areas, 37 IGHV3-21 CLLs were collected out of 1076 cases enrolled by different institutions from Northern or Central Southern Italy. Of the 37 cases, 18 were identified as homologous (hom)HCDR3-IGHV3-21 CLLs and were found almost exclusively (16 of 18) in Northern Italy; in contrast, 19 nonhomHCDR3-IGHV3-21 cases were evenly distributed throughout Italy. Clinically, poor survivals were documented for IGHV3-21 CLLs as well as for subgroups of mutated and homHCDR3-IGHV3-21 CLLs. Negative prognosticators CD38, ZAP-70, CD49d, and CD79b were expressed at higher levels in homHCDR3 than nonhomHCDR3-IGHV3-21 cases. Differential gene expression profiling (GEP) of 13 IGHV3-21 versus 52 non-IGHV3-21 CLLs identified, among 122 best-correlated genes, TGFB2 and VIPR1 as down- and up-regulated in IGHV3-21 CLL cases, respectively. Moreover, GEP of 7 homHCDR3 versus 6 nonhomHCDR3-IGHV3-21 CLLs yielded 20 differentially expressed genes, with WNT-16 being that expressed at the highest levels in homHCDR3-IGHV3-21 CLLs. Altogether, IGHV3-21 CLLs, including those with homHCDR3, had a peculiar global phenotype in part explaining their worse clinical outcome.

We have investigated whether the quantitative flow cytometry is an useful tool to better characterize B-cell chronic lymphoproliferative disorders (CLDs). Peripheral blood samples from 104 patients with leukemic B-cell disorders and 20 healthy donors were analyzed. Directly phycoerythrin-conjugated CD19, CD20, CD22, CD23, CD79b, and CD5 monoclonal antibodies (MoAbs) and QuantiBRITE pre-calibrated beads were used to calculate the number of antigen molecules per cell, expressed as antibody binding capacity (ABC). As compared to normal controls, in chronic lymphocytic leukemias (CLL) all MoAbs tested, with the exception of CD23 and CD5, showed lower ABC levels. In prolymphocytic leukemias (PL), CD5 and CD23 antigens were constantly absent while lower CD19 and CD22 ABC levels were observed. Hairy cell leukemias (HCL) displayed very high levels of CD20 and CD22. Of interest, splenic lymphomas with villous lymphocytes (SLVL) could be discriminated from HCL for higher CD79b and lower CD19 ABC values. Finally, higher CD20 levels were detected in follicular lymphomas (FL), whereas higher CD79b and CD5 levels characterized mantle cell lymphomas (MCL). Seven out of 61 CLL cases were defined as morphologically atypical. When compared with typical forms, lower levels of CD19 and CD23 and higher CD20 and CD22 ABC values were detected. However, we failed to demonstrate quantitative differences between atypical CLL and MCL. Our results suggest that quantitative flow cytometry may be a useful additional tool to better identify some types of B-cell CLDs.

The transmembrane signaling subunits (TSSs) bearing the immunoreceptor tyrosine-based activation motif (ITAM) play a crucial role in triggering the effector functions of mammalian leukocytes. The involvement in key immune reactions and obvious extension through duplication events make TSSs valuable markers of the evolution of the immune system. We surveyed the genomic sequences of the teleostean fish Fugu rubripes for the presence of genes encoding these accessory molecules. Automatic gene prediction was not efficient because of the poor ability of the programs used to recognize the short exons encoding the intracellular regions of TSSs. However, the unique compactness of the Fugu genome and the conservation of the exon/intron arrangements of the TSS genes facilitated their recognition by visual inspection of the candidate genomic sequences. Evidence for the presence of the CD3epsilon, CD3gamma/delta, CD79a, CD79b, TCRzeta, FcRgamma, DAP12 and DAP10 genes in the Fugu genome was obtained. Furthermore, conserved synteny for the short regions including the TSS genes was revealed by comparison of the Fugu and human genomes. The data demonstrate that the set of TSSs arose before the teleost-tetrapod split and provide a starting point for experimental investigation of the molecular evolution of the leukocyte-activating receptor complexes from fish species to mammals.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease defined by transcriptional classifications, specific signaling and survival pathways, and multiple low-frequency genetic alterations. Preclinical model systems that capture the genetic and functional heterogeneity of DLBCL are urgently needed. Here, we generated and characterized a panel of large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) models, including 8 that reflect the immunophenotypic, transcriptional, genetic, and functional heterogeneity of primary DLBCL and 1 that is a plasmablastic lymphoma. All LBCL PDX models were subjected to whole-transcriptome sequencing to classify cell of origin and consensus clustering classification (CCC) subtypes. Mutations and chromosomal rearrangements were evaluated by whole-exome sequencing with an extended bait set. Six of the 8 DLBCL models were activated B-cell (ABC)-type tumors that exhibited ABC-associated mutations such as MYD88, CD79B, CARD11, and PIM1. The remaining 2 DLBCL models were germinal B-cell type, with characteristic alterations of GNA13, CREBBP, and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC Only 25% of the DLBCL PDX models harbored inactivating TP53 mutations, whereas 75% exhibited copy number alterations of TP53 or its upstream modifier, CDKN2A, consistent with the reported incidence and type of p53 pathway alterations in primary DLBCL. By CCC criteria, 6 of 8 DLBCL PDX models were B-cell receptor (BCR)-type tumors that exhibited selective surface immunoglobulin expression and sensitivity to entospletinib, a recently developed spleen tyrosine kinase inhibitor. In summary, we have established and characterized faithful PDX models of DLBCL and demonstrated their usefulness in functional analyses of proximal BCR pathway inhibition.

A hallmark of classical Hodgkin lymphoma (cHL) is that the B-cell-derived Hodgkin and Reed-Sternberg (HRS) tumor cells have largely lost the B-cell-typical gene expression program. The factors causing this 'reprogramming' of HRS cells are only partly understood. As early B-cell factor 1 (EBF1), a major B-cell transcription factor, is downregulated in HRS cells, we analyzed whether this downregulation contributes to the lost B-cell phenotype and tested the consequences of EBF1 re-expression in cHL cell lines. EBF1 re-expression caused an upregulation of B-cell genes, such as CD19, CD79A and CD79B, although the B-cell genes FOXO1 and PAX5 remained lowly expressed. The re-expression of CD19, CD79A and CD79B occurred largely without demethylation of promoter CpG motifs of these genes. In the cHL cell line L-1236 fitness decreased after EBF1 re-expression. These data show that EBF1 has the ability to reintroduce part of the B-cell signature in cHL cell lines. Loss of EBF1 expression in HRS cells therefore contributes to their lost B-cell phenotype. Notably, in the cHL cell line KM-H2 destructive mutations were found in one allele of EBF1, indicating that genetic lesions may sometimes have a role in impairing EBF1 expression.

OBJECTIVE

The aim of this study was to analyze the hepatic differentiation potential of human umbilical cord blood-derived mesenchymal stem cells (hUCBMSCs) after transplantation into severe combined immune deficiency (SCID) mice with liver injury induced by D-galactosamine/lipopolysaccharide (GalN/LPS) and to explore the possibility that cells can partially repair GalN/LPS-induced hepatic damage.

METHODS

Mononuclear cells (MNCs) were isolated from fresh human umbilical cord blood, characterized by flow cytometry, and then transplanted into GalN/LPS-injured mice. Specimens were collected at 7, 14, 21, and 28 days after hUCBMSC transplantation. Histopathological changes were analyzed by hematoxylin and eosin staining. Polymerase chain reaction (PCR) for a specific marker of human cells, the human Alu sequence, was performed to locate exogenous hUCBMSCs in mouse livers. Expression of human hepatocyte-specific markers such as human albumin (hALB), human alpha-fetoprotein (hAFP), human cytokeratin 18 (hCK18), and human cytokeratin 19 (hCK19) were analyzed by reverse transcriptase (RT)-PCR and immunohistochemical staining.

RESULTS

The hUCBMSCs were positive for the human MSC-specific markers CD271, CD29, CD90, CD105, and CD73, but negative for CD31, CD79b, CD133, CD34, and CD45. Histological findings showed that the hepatic damage in mice was attenuated after hMSC administration, and liver architecture was much better preserved. Human cells in the injured liver of recipient mice were detected by PCR for the human Alu sequence. In addition, expression of markers of hepatic lineage, including hALB, hAFP, hCK18, and hCK19, was detected by immunohistochemistry and RT-PCR in mouse livers after hUCBMSC transplantation, suggesting the formation of hepatocyte-like cells in vivo.

CONCLUSIONS

MSCs from hUCB exhibit the potential to differentiate into hepatocyte-like cells in the livers of hUCB-transplanted mice as well as partially repair the liver damage induced by GalN/LPS.

Leukemic B cells in chronic lymphocytic leukemia (B-CLL) typically exhibit low or undetectable surface Ig. Because the B29 (CD79b and Ig beta) and mb-1 (CD79a and Ig alpha) gene products are required for surface Ig display in the B-cell receptor complex (BCR), we analyzed the expression of these genes in B-CLL cells. The majority (83%) of the randomly selected B-CLL patient samples analyzed exhibited low or undetectable surface BCR measured by mu heavy chain and B29 expression. Levels of mb-1 mRNA in these B-CLL samples with low surface BCR were similar to those in normal B cells. Among those with decreased surface expression, B29 mRNA was not detected in half of these B-CLL samples. The remaining B-CLL samples with diminished surface BCR contained normal levels of B29 mRNA. Further analysis of cDNA clones from the majority of these latter samples contained point mutations, insertions, or deletions that were largely located in the B29 transmembrane and cytoplasmic domains. These results indicate the occurrence of somatic mutations predicted to affect B29 expression and/or function in the majority of B-CLL and suggest that these aberrations underlie the diminished surface BCR display and loss of BCR signaling characteristic of this leukemia.

BACKGROUND

It is well known that HIV-1 infection induces profound alterations in the immune system, including hyperactivation of B cells. TNF-alpha induces HIV-1 replication and immunodysregulation, including polyclonal B-cell activation.

OBJECTIVE

We sought to determine the effects of surface-binding HIV-1 envelope glycoprotein (gp120) and TNF-alpha on human B-cell function.

METHODS

HIV-1 seronegative peripheral blood human B cells were purified and activated by CD40 mAb and IL-4. In vitro studies of B-cell proliferation, cyclic AMP (cAMP) generation, receptor expression, and Ig production were performed.

RESULTS

gp120, an Ig superantigen, stimulated HIV-1 seronegative and HIV-1 seropositive human B-cell cAMP generation, proliferation, and Ig production. These gp120-induced B-cell responses were demonstrated to be specific as evidenced by the abrogation of the stimulatory response in the presence of anti-gp120 mAb, blocking of CD4 resulting in no change on gp120-induced B-cell responses, and the binding of gp120 in these B cells. TNF-alpha also stimulated cAMP generation, proliferation, and Ig production in B cells, and the binding of gp120 to these B cells stimulated by TNF-alpha further enhanced cell proliferation, cAMP generation, and Ig production. Antigenic expression of the B-cell receptor CD79b was down-regulated by gp120 but was not altered by the addition of TNF-alpha.

CONCLUSIONS

gp120 modulation of TNF-alpha-induced B-cell receptor- and cAMP-mediated signal transduction events may be involved in the B-cell abnormalities observed in HIV-1 infection.

On the basis of responses to surface IgM (sIgM) cross-linking, B cells from 41 patients with B-chronic lymphocytic leukemia were categorized as 15 nonresponders (group I) and 26 responders (group II). The latter cases were subclassified as those seven where proliferation was induced (subgroup IIa) and the remaining 19 in whom apoptosis occurred (subgroup IIa). Signal disruption in group I was confirmed by the absence of Ca2+ mobilization. Activation of PI3K was constitutive in subgroup IIa, but not in subgroup IIb, and that of Akt induced by anti-mu in subgroup IIa, but not in subgroup IIb. Among the MAPK, ERK was more highly activated relative to p38 in subgroup IIa, whereas activation of p38 predominated over that of ERK in subgroup IIb. For subgroup IIb cells, based on tyrosine phosphorylation and translocation into lipid rafts, sIgM signaling was shown to be enhanced by Zap70. The different consequences of signaling through sIgM were associated with biological prognosis indicators. These included high levels of CD38, lack of mutations in the IgVH chain genes, preferential usage of full-length CD79b, and severe clinical stage. Thus, modification of sIgM-induced signaling could be a therapeutic approach.

Viral infection induces potent cellular immunity and activated intracellular signaling, which may dictate the driver events involved in immune escape and clonal selection of virus-associated cancers, including Epstein-Barr virus (EBV)-positive lymphomas. Here, we thoroughly interrogated PD-L1/PD-L2-involving somatic aberrations in 384 samples from various lymphoma subtypes using high-throughput sequencing, particularly focusing on virus-associated lymphomas. A high frequency of PD-L1/PD-L2-involving genetic aberrations was observed in EBV-positive lymphomas [33 (22%) of 148 cases], including extranodal NK/T-cell lymphoma (ENKTL, 23%), aggressive NK-cell leukemia (57%), systemic EBV-positive T-cell lymphoproliferative disorder (17%) as well as EBV-positive diffuse large B-cell lymphoma (DLBCL, 19%) and peripheral T-cell lymphoma-not otherwise specified (15%). Predominantly causing a truncation of the 3'-untranslated region, these alterations represented the most prevalent somatic lesions in ENKTL. By contrast, the frequency was much lower in EBV-negative lymphomas regardless of histology type [12 (5%) of 236 cases]. Besides PD-L1/PD-L2 alterations, EBV-positive DLBCL exhibited a genetic profile distinct from EBV-negative one, characterized by frequent TET2 and DNMT3A mutations and the paucity of CD79B, MYD88, CDKN2A, and FAS alterations. Our findings illustrate unique genetic features of EBV-associated lymphomas, also suggesting a potential role of detecting PD-L1/PD-L2-involving lesions for these lymphomas to be effectively targeted by immune checkpoint blockade.

OBJECTIVE

Whole-genome sequencing has revealed MYD88 L265P and CXCR4 mutations (CXCR4(mut)) as the most prevalent somatic mutations in Waldenström macroglobulinemia. CXCR4 mutation has proved to be of critical importance in Waldenström macroglobulinemia, in part due to its role as a mechanism of resistance to several agents. We have therefore sought to unravel the different aspects of CXCR4 mutations in Waldenström macroglobulinemia.

METHODS

We have scanned the two coding exons of CXCR4 in Waldenström macroglobulinemia using deep next-generation sequencing and Sanger sequencing in 98 patients with Waldenström macroglobulinemia and correlated with SNP array landscape and mutational spectrum of eight candidate genes involved in TLR, RAS, and BCR pathway in an integrative study.

RESULTS

We found all mutations to be heterozygous, somatic, and located in the C-terminal domain of CXCR4 in 25% of the Waldenström macroglobulinemia. CXCR4 mutations led to a truncated receptor protein associated with a higher expression of CXCR4. CXCR4 mutations pertain to the same clone as to MYD88 L265P mutations but were mutually exclusive to CD79A/CD79B mutations (BCR pathway). We identified a genomic signature in CXCR4(mut) Waldenström macroglobulinemia traducing a more complex genome. CXCR4 mutations were also associated with gain of chromosome 4, gain of Xq, and deletion 6q.

CONCLUSIONS

Our study panned out new CXCR4 mutations in Waldenström macroglobulinemia and identified a specific signature associated to CXCR4(mut), characterized with complex genomic aberrations among MYD88L265P Waldenström macroglobulinemia. Our results suggest the existence of various genomic subgroups in Waldenström macroglobulinemia.

BACKGROUND

Prostate stromal cells can be readily cultured in vitro. Are these proliferating cells representative of stromal cells in situ? Since the expression of cell surface molecules, like the cluster of differentiation (CD) antigens, can be affected by changes in physiological conditions cultured stromal cells may differ from uncultured stromal cells in their complement of CD molecules.

METHODS

Prostate stromal cells were prepared from tissue specimens and cultured. Flow cytometry was used to analyze the expression of 107 CD molecules in the resultant cells. Expression of the CD molecules by prostate cells in situ was done by immunohistochemistry.

RESULTS

The expression of a number of cell surface molecules such as CD10, CD13, CD26, and CD44 is elevated in prostatic stromal cells cultured in vitro. These are markers of epithelial cells in tissue. Other molecules expressed by the cultured stromal cells include CD29, CD49a, CD49b, CD49d, CD49f, CD51/61, CD54, CD55, CD56, CD58, CD59, CD61, CD71, CD79b, CD81, CD82, CD90, CD95, CD107a, CD130, and CD147. Among these are stromal, epithelial, and nonstromal/nonepithelial markers as defined by tissue immunohistochemistry.

CONCLUSIONS

Cultured stromal cells express a number of CD molecules normally found in other cell types of the prostate. Cells can express different CD molecules under different conditions.

Low levels of B-cell-receptor (BCR) expression are the hallmark of tumoral B lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL). These cells also respond inadequately to stimulation through the BCR. This receptor consists of a surface immunoglobulin associated with a CD79a/CD79b heterodimer. We previously showed that the intracellular synthesis of BCR components, from transcription onward, is normal. Here, we investigated the glycosylation status and cellular localization of mu, CD79a, and CD79b chains in 10 CLL patients differing in surface immunoglobulin M (IgM) expression. We reported a severe impairment of the glycosylation and folding of mu and CD79a. These defects were associated with the retention of both chains in the endoplasmic reticulum and lower levels of surface IgM expression. In contrast, no clear impairment of glycosylation and folding was observed for CD79b. No sequence defects were identified for BCR components and for the chaperone proteins involved in BCR folding processes. These data show, for the first time, that lower levels of BCR surface expression observed in CLL are accounted for by an impaired glycosylation and folding of the mu and CD79a chains.

New technology allows highly sensitive flow cytometric detection and quantitative analysis of intracellular antigens in normal and malignant hemopoietic cells. With this technology, the earliest stages of myeloid and lymphoid differentiation can easily and reliably be identified using antibodies directed against (pro-)myeloperoxidase/MPO, CD22 and CD3 antigens, respectively. Particularly for the analysis of undifferentiated acute myeloblastic leukemia (AML) cells, the immunological demonstration of intracellular MPO or its enzymatically inactive proforms is highly relevant, since other myeloid marker molecules such as CD33, CD13, or CDw65 are either not restricted to the granulomonocytic lineage or appear later in differentiation. By combining MPO staining with staining for lactoferrin (LF), undifferentiated cells can be distinguished from the granulomonocytic maturation compartment in bone marrow, since LF is selectively expressed from the myelocyte stage of differentiation onward. The list of informative intracellular antigens to be used in leukemia cell analysis will certainly expand in the near future. One candidate, intracellular CD68, has already been tested by us, and results are presented. Also dealt within this article are surface marker molecules not (as yet) widely used in leukemia cell analysis but with the potential to provide important additional information. Among them are the surface structures CD15, CD15s, CDw65, CD79a (MB-1), CD79b (B29), CD87 (uPA-R), and CD117 (c-kit).

We have previously identified 12 surface antigens whose differential expression represented the signature of B-cell chronic lymphocytic leukemia (B-CLL) subsets with different prognosis. In the present study, expression data for these antigens, as determined in 137 B-CLL cases, all with survivals, were utilized to devise a comprehensive immunophenotypic scoring system of prognostic relevance for B-CLL patients. In particular, univariate z score was employed to identify the markers with greater prognostic impact, while maximally selected log-rank statistics were chosen to define the optimal cut-off points capable to split patients into two groups with different survivals. A weighted immunophenotypic scoring system was developed by integrating results from these analyses. Six antigens were selected: three positive prognosticators (CD62L, CD54, CD49c) and three negative prognosticators (CD49d, CD38, CD79b), with cut-off values ranging from 30% to 50% of positive cells. By weighing the expression of each marker according to its statistical power, a complete scoring system, with point values comprised between 0 (complete absence of phenotypic conditions associated with good prognosis) and 9 (all the phenotypic conditions associated with good prognosis fulfilled), allowed to split the whole set of B-CLL patients, into three distinctive prognostic groups (P = 4.78 x 10(-11)) with high- (score 0-3), intermediate- (score 4-6), and low- (score 7-9) risk of death. The three risk groups showed different distribution of cases as for Rai's stages, IgVH mutations, and ZAP-70 expression. The proposed immunophenotypic scoring system may be an additional useful tool in routine diagnostic/prognostic procedures for B-CLL.

B-cell chronic lymphocytic leukemia (B-CLL) is a well-defined clinical entity with heterogeneous molecular and cytogenetic features. Here, we analyze the impact of trisomy 12, del(13q), del(17p), and del(11q) as determined by interphase fluorescence in situ hybridization analysis of purified neoplastic B-CLL cells on their immunophenotype, DNA ploidy status and proliferative rate.Overall, 111 of 180 (62%) B-CLL cases studied displayed one (50%) or more (12%) genetic abnormalities, del(13q) (35%) being more frequently detected than trisomy 12 (23%) followed by del(11q) (9%) and del(17p) (8%). Trisomy 12 was associated with a higher frequency of DNA aneuploidy, stronger expression of CD19, CD20, CD22, CD24, CD27, CD79b, CD38, and sIg and lower reactivity for CD43 with respect to cytogenetically nonaltered cases. In turn, cases with del(13q) displayed greater reactivity for CD20, FMC7, CD27, CD22, CD5, and bcl2, while del(11q) was associated with brighter expression of CD38, FMC7, CD25, and sIg. Hierarchical clustering analysis of the immunophenotype of B-CLL cases with cytogenetic abnormalities allowed the identification of three different groups of patients with increasing frequencies of trisomy 12, del(11q), and del(13q). Remarkably, none of the cytogenetic abnormalities analyzed except coexistence of 13q- and 17p- had a clear impact on the proliferative index of B-CLL cells.

Mutations in 2 upstream components of the nuclear factor κB (NF-κB) pathway, CD79B and MYD88, are important information for new target therapy in malignant lymphoma. We examined the prevalence and clinicopathologic characteristics of CD79B and MYD88 mutation in a cohort of Asian diffuse large B cell lymphoma (DLBCL) patients. CD79B and MYD88 mutations were analyzed by Sanger sequencing in 187 DLBCL tissue samples. CD79B immunoreceptor tyrosine-based activation motif spanning exon 5 and 6 and MYD88 TIR domain spanning exons 3, 4 and 5 were amplified and sequenced. The cell-of-origin was determined based on immunohistochemical stains for CD10, BCL-6 and MUM-1 by Hans' algorithm. CD79B was mutated in 16 cases (8.5%), mostly involving the first tyrosine (Y196) of immunoreceptor tyrosine-based activation motif. For MYD88, L265P mutation was found in 31 cases (out of 161, 19.3%). In 11 of these, a CD79B mutation coexisted, which constituted 69% of CD79B mutants and 36% of MYD88 L265P cases. Clinicopathologic comparison between the mutant and the wild-type group showed that the mean age was older for both CD79B (66 versus 58 years) and MYD88 L265P mutant groups (64 versus 58 years). Survival analyses showed that neither CD79B mutation nor MYD88 L265P was a significant prognostic indicator. In conclusion, CD79B and MYD88 mutations are associated with an older age at onset in DLBCL with a significant overlap, which did not affect the outcome of the disease.

Tumor-associated antigens (TAA) or cancer biomarkers are major targets for cancer therapies. Antibody- based agents targeting the cancer biomarkers include monoclonal antibodies (MoAbs), radiolabeled MoAbs, bispecific T cell engagers, and antibody-drug conjugates. Antibodies targeting CD19, CD20, CD22, CD30, CD33, CD38, CD79B and SLAMF7 are in clinical applications for hematological malignancies. CD123, CLL-1, B cell maturation antigen, and CD138 are targets for cancer immunotherapeutic agents, including the chimeric antigen receptor - engineered T cells. Immune checkpoint inhibitors (ICIs) against PD-1, PD-L1, and CTLA-4 have led to the revolution of cancer immunotherapy. More ICIs targeting IDO, LAG3, TIM-3, TIGIT, SIGLECs, VISTA and CD47 are being explored. Small molecule inhibitors (SMIs) against tyrosine kinase oncoproteins such as BCR-ABL, JAK2, Bruton tyrosine kinase, FLT3, EGFR, ALK, HER2, VEGFR, FGFR, MEK, and MET have fundamentally changed the landscape of cancer therapy. SMIs against BCL-2, IDHs, BRAF, PI3 kinase, mTOR, PARP, and CDKs have become the mainstay in the treatment of a variety of cancer types. To reduce and avoid off-tumor toxicities, cancer-specific TAAs such as CD33 are being manufactured through systems biology approach. Search for novel biomarkers and new designs as well as delivery methods of targeted agents are fueling the next wave of advances in cancer therapy.

Kinases downstream of B-cell antigen receptor (BCR) represent attractive targets for therapy in non-Hodgkin lymphoma (NHL). As clinical responses vary, improved knowledge regarding activation and regulation of BCR signaling in individual patients is needed. Here, using phosphospecific flow cytometry to obtain malignant B-cell signaling profiles from 95 patients representing 4 types of NHL revealed a striking contrast between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) tumors. Lymphoma cells from diffuse large B-cell lymphoma patients had high basal phosphorylation levels of most measured signaling nodes, whereas follicular lymphoma cells represented the opposite pattern with no or very low basal levels. MCL showed large interpatient variability in basal levels, and elevated levels for the phosphorylated forms of AKT, extracellular signal-regulated kinase, p38, STAT1, and STAT5 were associated with poor outcome. CLL tumors had elevated basal levels for the phosphorylated forms of BCR-signaling nodes (Src family tyrosine kinase, spleen tyrosine kinase [SYK], phospholipase Cγ), but had low α-BCR-induced signaling. This contrasted MCL tumors, where α-BCR-induced signaling was variable, but significantly potentiated as compared with the other types. Overexpression of CD79B, combined with a gating strategy whereby signaling output was directly quantified per cell as a function of CD79B levels, confirmed a direct relationship between surface CD79B, immunoglobulin M (IgM), and IgM-induced signaling levels. Furthermore, α-BCR-induced signaling strength was variable across patient samples and correlated with BCR subunit CD79B expression, but was inversely correlated with susceptibility to Bruton tyrosine kinase (BTK) and SYK inhibitors in MCL. These individual differences in BCR levels and signaling might relate to differences in therapy responses to BCR-pathway inhibitors.

Chronic lymphocytic leukemia (CLL) is a phenotypically distinguishable form of B-lymphoid leukemias. The regularity of surface membrane antigen expression patterns, their interrelationships as well as the effects of the three frequent chromosomal aberrations, ie 11q deletion, 13q deletion and trisomy 12, were investigated in 35 classic CLL cases by flow cytometry. The two-way cluster analysis of 31 individual antigens revealed three expression patterns: (1) most cells in most cases positive (CD5, CD19, CD20, CD23, CD27, CD40, CD45, CD45RA); (2) most cells in most cases negative (CD10, CD14, CD34, CD122, CD154, mIgG); and (3) a mixed pattern with a variable number of positive cases and a variable percentage of positive cells in individual cases (CD11c, CD21, CD22, CD25, CD38, CD45RO, CD79b, CD80, CD95, CD124, CD126, CD130, FMC7, mIgD, mIgkappa, mIglambda, mIgM). The expressions of several antigens were strongly interdependent, even when antigens belonged to entirely different gene families. Such antigen pairs were: CD11c/CD21; CD19/CD45; CD19/CD79b; CD22/CD45RA; CD23/Igkappa; CD25/mIgM; CD27/CD45; CD45/CD79b; CD45RA/Igkappa. In contrast, the expression of some antigens was mutually exclusive, the best examples being CD45RA/CD45RO, CD38/CD80 and CD45RA/CD80. Deletion of chromosome arm 11q attenuated expression of splicing variant CD45RA, but enhanced CD45RO expression. In contrast, cases of trisomy 12 were associated with enhanced CD45RA and attenuated CD45RO expression. Similarly, trisomy 12 was associated with enhanced CD27 and mIgkappa expression. The variable levels of signaling surface membrane antigens, their interactions and interference by genetic aberrations are likely to affect the clinical progression and drug response of CLL.

The thymuses of 44 koalas, ranging from less than 30 d to more than 14 y of age, were examined histologically and immunohistologically. The thymuses from 17 of these koalas dying acutely through trauma were regarded as not being significantly affected by disease and formed the basis for study of the normal thymus. Most other koalas had chronic illness and, consequently, disease affected (involuted) thymuses. Histologically, thymuses showed obvious corticomedullary differentiation with small Hassall's corpuscles visible in koalas more than 8 mo of age. Most cortical and medullary lymphocytes stained for CD3 and CD5 (T lymphocyte markers) while some cells (predominantly medullary) stained for CD79b (B lymphocytes and plasma cells), IgG (plasma cells) or MHC class II (reticular epithelium, macrophages and possibly lymphocytes). Adults of up to 5-6 y of age which had died through trauma had little evidence of involution and had prominent Hassall's corpuscles and medullary epithelial thymocytes. Thymic eosinopoiesis was an inconsistent finding. In traumatised animals over this age, involution was obvious with fibrous replacement of lobules, loss of Hassall's corpuscles and the development of dilated ducts lined by nonciliated epithelium. However, loss of lymphocytes was gradual and pockets of lymphocytes, centrally located in lobules, were still present in the oldest koala examined. In these involuted thymuses, remaining lymphocytes stained for CD3 and lesser numbers of CD5 and CD79b. Plasma cells were common and often stained both for IgG and MHC class II. Thymuses of chronically diseased koalas showed accelerated involution when age matched with thymuses from traumatised koalas. Chronically ill koalas as young as 18-24 mo showed advanced involution, but the morphological and immunohistological characteristics of involuted thymus from diseased koalas could not be distinguished from those of involuted thymuses derived from traumatised koalas. It was concluded that development of the koala thymus is completed at 8 mo of age and that for normal koalas involution is a gradual process which starts not at but after sexual maturity. Immunohistological characterisation of the thymus was comparable to that reported for a variety of eutherian mammals.

The distribution of T- and B-cells in the developing lymphoid and immunohaematopoietic tissues of the tammar wallaby were investigated using antibodies to the mature cell surface markers, CD3, CD5 and CD79b. In the thymus, CD3- and CD5-positive T-cells were first observed at day 12 postpartum whilst rare B-cells were first detected at day 23. Both T- and B-lymphocytes were first stained on day 21 postpartum in the spleen and day 24 in lymph nodes. In one sample from a 7-day-old animal, rare CD79b-positive (CD79b+) lymphocytes were observed in the gut-associated lymphoid tissues. However, CD3+ cells were not apparent until day 12 and CD5+ cells were not detected until day 74 postpartum. No lymphocytes were detected in liver or bone marrow samples and no bronchus-associated lymphoid tissues were observed. The pattern of development and the distribution of T- and B-cells in the lymphoid and immunohaematopoietic tissues were similar to those observed in eutherian mammals and in limited studies of other metatherians. However, the detection of apparently mature T- and B-cells in the thymus and gut-associated lymphoid tissues (GALT) at the same postnatal age highlights the need for a more substantial study of the development of GALT. This is, at present, limited by availability of marsupial-specific antibodies.

B cell Ag receptor (BCR) signaling occurs via tyrosine phosphorylation of CD79a and CD79b ITAMs, leading to recruitment and activation of Lyn and Syk tyrosine kinases and subsequent downstream events. CD45 expression is required for BCR triggering of certain of these downstream events, such as calcium mobilization and p21ras activation. However, the site in the BCR signaling cascade at which CD45 impinges is poorly defined. To address this question, we have studied CD45 function in the CD45-deficient (CD45-) and CD45-reconstituted (CD45+) J558L mu m3 plasmacytoma. In both CD45+ and CD45- cells, Ag stimulation led to CD79a and CD79b tyrosine phosphorylation as well as Syk tyrosine phosphorylation, recruitment to the receptors, and activation. In contrast to CD45+ cells, Lyn exhibited high basal tyrosine phosphorylation in the CD45- cells and was not further phosphorylated upon Ag stimulation. Mapping studies indicated that the observed constitutive phosphorylation of Lyn reflects phosphorylation of its C-terminal tyrosine, Y508, at high stoichiometry. Constitutively Y508-phosphorylated Lyn was neither recruited to the BCR nor activated upon Ag stimulation. Moreover, CD79a-ITAM phosphopeptides failed to bind Lyn from the CD45- cells. Thus, Y508 phosphorylation of Lyn occurs in the absence of cellular CD45 expression and appears to render the kinase unable to associate with the phosphorylated receptor complex via its Src homology 2 domain and to participate in signal propagation. Surprisingly, in view of previous findings implicating Src family kinases in ITAM phosphorylation, the data indicate that Ag-induced CD79a and CD79b tyrosine phosphorylation and Syk recruitment and activation can occur in the absence of CD45 expression and, hence, Src-family kinase activation.