Targetable alterations in primary extranodal diffuse large B-cell lymphoma.

Primary extranodal diffuse large B-cell lymphoma (PE-DLBCL) is a heterogeneous subgroup of DLBCL. We investigated the prevalence and prognostic value of surface expression of PD-L1, PD1, and CD30, copy number of 9p24.1 (PD-L1 region), and mutations in MYD88, CD79B, CARD11, and BTK in a cohort of 116 patients, localized in the mediastinum (PMBL, n = 12), ear, nose and throat (ENT, n = 28), central nervous system (n = 29), testis (n = 7), breast (n = 4), stomach (n = 10), bone (n = 8), spleen (n = 2), and skin (n = 16). PD-L1 expression is most frequent in PMBL (92%), followed by lymphomas originating in the stomach (57%), ENT (23%), and skin (18%). PD1 was expressed at low levels in less than 13% of PE-DLBCL, while CD30 expression was found in 58% of PMBL. Mutation analysis revealed an unexpectedly high frequency of MYD88 and CD79B mutations in ENT lymphomas (46% and 50%, respectively). CARD11 mutations are rare but more frequently found in gastric lymphomas (30%), suggesting BTK resistance. Thirty-four of 113 (30%) of the lymphomas harbored both MYD88 and CD79B mutations. Lower overall and progression-free survival rates were found for cases with MYD88, CD79B, and BTK mutations. These data confirm the biologic singularity of PE-DLBCLs and provide some suggestions for targeted therapies.

Combination of two monoclonal antibodies with SOCS1 N- and C-terminal binding sites to address SOCS1 status in B cells and B-cell lymphoma.

INTRODUCTION: Accumulating studies show that the tumour suppressor SOCS1 is one of the most frequently mutated genes in lymphomas, often affecting the coding sequence of SOCS1 protein. Depending on the type of mutation and lymphoma concerned, SOCS1 mutations have different impacts on progression-free and overall survival. Two antibodies binding the N and C terminals of SOCS1 would be a suitable 'test pair' to identify truncated versions of SOCS1. We, therefore, compared the C-terminal antibody 424C with the N-terminal antibody 4H1. MATERIALS AND METHODS: As 424C has already been characterised, we performed a comparative analysis of anti-SOCS1 antibody 4H1 using immunohistochemistry on human tonsil tissue and chamber slides, immunoblots on SOCS1 wildtype and mutated transfected HEK293T cells and lymphoma cell lines and cross-reactivity analysis and epitope mapping with protein microarrays. RESULTS: Compared with 424C, anti-SOCS1 antibody 4H1 showed various cross-reactions with other proteins resulting in a 'pancellular' immunohistochemical staining pattern in FFPE lymphoid tissue. Like 424C, 4H1 identified SOCS1 wildtype and SOCS1 mutations in immunoblot experiments but also bound an unknown protein with high intensity. CONCLUSION: Anti-SOCS1 antibody 4H1 may be useful in a molecular setting but is disqualified as an immunohistochemical diagnostic tool due to its very broad non-specific binding.

Usefulness of BATF3 Immunohistochemistry in Diagnosing Classical Hodgkin Lymphoma.

It is well recognized that the AP-1 transcription factor BATF3 is constitutively expressed in Hodgkin/Reed-Sternberg (HRS) cells, but its potential as a diagnostic marker for classical Hodgkin lymphoma (cHL) has not yet been addressed. In this study, we performed immunohistochemistry and analyzed the BATF3 expression in lymphoma cells on 218 lymphoma samples belonging to 14 different lymphoma entities. We observed varying degrees of BATF3 expression in nearly half of the cases (n = 100) with BATF3 expression being a constitutive feature of cHL (n = 53) and anaplastic large cell lymphoma (ALCL). By scoring BATF3 expression (BATF3-score) we observed constitutively high BATF3-scores in cHL and ALCL and low to moderate BATF3-scores in all other entities examined. Western blot analysis confirmed BATF3 protein expression in cell lysates from cHL cell lines (n = 7). Thus, BATF3 can be considered a useful IHC marker for the diagnosis of cHL as it is highly sensitive and sufficiently specific when analyzed by BATF3-scoring.

A new reliable and highly specific monoclonal antibody to detect the C-terminal region of silencer of cytokine signaling 1.

INTRODUCTION: SOCS1, a negative regulator of JAK/STAT signaling, is among the most frequently mutated genes in DLBCL and classical Hodgkin lymphoma. The C-terminal SOCS box domain, mediating the degradation of phospho-JAK2, is often affected or even lacking. The analysis of such variants is hampered by the lack of a SOCS1-specific monoclonal antibody recognizing the C-terminus of SOCS1. As this C-terminus is often lost or mutated in B-cell lymphomas, staining with amino-terminal targeting antibodies in a lymphoma setting might be misleading. METHODS: BALB/c mice were immunized with a truncated SOCS1 C-terminal protein. The supernatant of generated hybridoma cells was screened by ELISA and, immunohistochemically, on formalin-fixed and paraffin-embedded tonsil. After antibody purification by affinity chromatography, epitope mapping and cross-reactivity check followed via substitution scans. SOCS1 protein expression was investigated on cell cultures and cytoblocks of SOCS1WT stably transfected HEK293T cells, lymphoma cell lines and lymphoid tissues. RESULTS: Procedures resulted in one monoclonal IgG1 anti-SOCS1 antibody, 424C, that recognizes and strongly binds to the C-terminal region of SOCS1 in immunoblot and immunohistochemistry analyses. CONCLUSION: This new anti-SOCS1 monoclonal antibody is a valuable tool to detect SOCS1 expression dependent on an existing SOCS1 box and, therefore, indicating a full-length SOCS1 protein.

The PTP1B mutant PTP1B∆2-4 is a positive regulator of the JAK/STAT signalling pathway in Hodgkin lymphoma.

The neoplastic Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) depend on chronic activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathways to maintain survival and proliferation. Accumulating reports highlight the importance of the inactivation or reduced expression of negative JAK/STAT regulators such as the protein-tyrosine phosphatase 1B (PTP1B/PTPN1) in this process. Various PTPN1 mRNA variants as well as truncated PTP1B proteins were identified in cHL cell lines and primary cHL tumour samples. These PTPN1 mRNA variants lack either one or several exon sequences and therefore render these PTP1B variants catalytically inactive. Here, we show that one of these mutants, PTP1B∆2-4, is not only a catalytically inactive variant, but also augmented the IL-4-induced JAK/STAT activity similar to the recently reported PTP1B∆6 splice variant. Moreover, while PTP1B∆6 diminished the activity and protein levels of PTP1BWT, PTP1BWT remained unaffected by PTP1B∆2-4, arguing for different molecular mechanisms of JAK/STAT modulation by PTP1B∆6 and PTP1B∆2-4. Collectively, these data indicate that PTPN1 variants missing one or more exon sequences originated either from alternative splicing or from gene mutation, create PTP1B gain-of-function variants with oncogenic potential by augmenting JAK/STAT signalling in cHL.

A novel PTPN1 splice variant upregulates JAK/STAT activity in classical Hodgkin lymphoma cells.

Chronic activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways is a hallmark of a variety of B-cell lymphomas, including classical Hodgkin lymphoma (cHL). Constitutive JAK/STAT signaling is crucial for survival and proliferation of Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of cHL. Although the molecular basis of this constitutive JAK/STAT signaling in cHL has not been completely understood, accumulating reports highlight the role of an inactivation or reduced expression of negative JAK/STAT regulators such as silencer of cell signaling 1 (SOCS1) or protein-tyrosine phosphatase 1B (PTP1B) in this process. Here, we report the expression of truncated PTP1B mRNA variants identified in cHL cell lines and primary cHL tumor samples lacking either 1 or several exon sequences. One of these novel PTP1B variants, a splice variant lacking exon 6 (PTP1BΔ6), was found expressed at low levels in cHL cell lines. However, serum stimulation of cHL augmented the expression of PTP1BΔ6 significantly. Functional characterization of PTP1BΔ6 revealed a positive effect on interferon-γ- and interleukin-4-induced JAK/STAT activity in HEK293 or HEK293-STAT6 cells, and on the basal STAT activity in stably transfected L-428 and U-HO1 cHL cell lines. Furthermore, PTP1BΔ6 expression increased the proliferation of L-428 and U-HO1 cells and reduced cytotoxic effects of the chemotherapeutical agents gemcitabine and etoposide distinctively. Collectively, these data indicate that PTP1BΔ6 is a positive regulator of JAK/STAT signaling in cHL.