Classical Hodgkin lymphoma (cHL) and mediastinal large B-cell lymphoma (MLBCL) are lymphoid malignancies with certain shared clinical, histologic, and molecular features. Primary cHLs and MLBCLs include variable numbers of malignant cells within an inflammatory infiltrate, suggesting that these tumors escape immune surveillance. Herein, we integrate high-resolution copy number data with transcriptional profiles and identify the immunoregulatory genes, PD-L1 and PD-L2, as key targets at the 9p24.1 amplification peak in HL and MLBCL cell lines. We extend these findings to lasercapture microdissected primary Hodgkin Reed-Sternberg cells and primary MLBCLs and find that programmed cell death-1 (PD-1) ligand/9p24.1 amplification is restricted to nodular sclerosing HL, the cHL subtype most closely related to MLBCL. Using quantitative immunohistochemical methods, we document the association between 9p24.1 copy number and PD-1 ligand expression in primary tumors. In cHL and MLBCL, the extended 9p24.1 amplification region also included the Janus kinase 2 (JAK2) locus. Of note, JAK2 amplification increased protein expression and activity, specifically induced PD-1 ligand transcription and enhanced sensitivity to JAK2 inhibition. Therefore, 9p24.1 amplification is a disease-specific structural alteration that increases both the gene dosage IntroductionClassical Hodgkin lymphoma (cHL) is a B-cell malignancy that occurs frequently in Western countries and commonly affects young adults. 1 These tumors are characterized by small numbers of neoplastic Reed-Sternberg (RS) cells within an extensive inflammatory/immune cell infiltrate. There are 4 subtypes of cHL, 2 of which comprise Ϸ 90% of cases: nodular sclerosing Hodgkin lymphoma (NSHL; 60% of cases) and mixed cellularity Hodgkin lymphoma (MCHL; 30% of cases). cHLs lack surface immunoglobulin expression and B-cell receptor-mediated signals and rely on alternative survival pathways, including aberrant nuclear factorB signaling. 1 In previous studies, we and others have defined shared molecular features of cHL and a specific subtype of diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (MLBCL). 2,3 Like cHL, MLBCLs have a T-helper cell type 2 (Th2)-skewed cytokine profile, decreased expression of B-cell receptor signaling pathway components, and constitutive activation of nuclear factorB. 2 MLBCL also exhibits certain clinical and histologic similarities to cHL, particularly the NSHL subtype. 4,5 For example, both diseases are most common in young adults and often present as an anterior mediastinal or localized nodal mass. 2,4,5 In addition, both MLBCLs and NSHLs include bands of sclerotic tissue and immune/inflammatory cell infiltrates. 4,5 However, the inflammatory infiltrate is less prominent in MLBCLs, which have a more diffuse growth pattern. 4 Although cHLs have an extensive polymorphous inflammatory infiltrate, there is little evidence of an effective host antitumor immune response. In fact, recent studies indicate that Hodgkin RS cells pro...

Purpose Programmed cell death ligand 1 (PD-L1) is a molecule expressed on antigen-presenting cells that engages the PD-1 receptor on T cells and inhibits T-cell receptor signaling. The PD-1 axis can be exploited by tumor cells to dampen host anti-tumor immune responses and foster tumor cell survival. PD-1 blockade has shown promise in multiple malignancies but should be directed towards patients in whom it will be most effective. In recent studies, we found that the chromosome 9p24.1 amplification increased the gene dosage of PD-L1 and its induction by JAK2 in a subset of patients with classical Hodgkin lymphoma (cHL). However, cHLs with normal 9p24.1 copy numbers also expressed detectable PD-L1, prompting analyses of additional PD-L1 regulatory mechanisms. Experimental Design Herein, we utilized immunohistochemical, genomic and functional analyses to define alternative mechanisms of PD-L1 activation in cHL and additional EBV+ lymphoproliferative disorders. Results We identified an AP-1-responsive enhancer in the PD-L1 gene. In cHL Reed Sternberg cells, which exhibit constitutive AP-1 activation, the PD-L1 enhancer binds AP-1 components and increases PD-L1 promoter activity. In addition, we defined EBV infection as an alternative mechanism for PD-L1 induction in cHLs with diploid 9p24.1. PD-L1 was also expressed by EBV-transformed lymphoblastoid cell lines as a result of latent membrane protein 1-mediated, JAK/STAT-dependent promoter and AP-1-associated enhancer activity. In addition, over 70% of EBV+ post-transplant lymphoproliferative disorders expressed detectable PD-L1. Conclusions AP-1 signaling and EBV infection represent alternative mechanisms of PD-L1 induction and extend the spectrum of tumors in which to consider PD-1 blockade.

Posttransplant lymphoproliferative disorders (PTLDs) are potentially fatal, EBVdriven B-cell malignancies that develop in immunocompromised solid organ or hematopoietic stem cell recipients. In PTLD, the expression of EBV proteins, including latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance foster the proliferation of EBV-transformed B cells. Current PTLD treatment strategies include reduction of immunosuppression, which increases the risk of graft rejection, anti-CD20 treatment, combination chemotherapy, and administration of EBV-specific cytotoxic T cells. In the present study, we report that EBV-transformed lymphoblastoid Bcell lines (LCLs) and primary PTLDs overexpress galectin-1 (Gal1), a carbohydratebinding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4 ؉ Th1 and Th17 cells and cytotoxic T cells. In transcriptional reporter assays, LMP2A and LMP1 each increased Gal1-driven luciferase expression, and the combination of LMP2A and LMP1 was additive. In addition, small interfering RNA (siRNA)-mediated depletion of LMP2A decreased Gal1 protein abundance in EBV-transformed LCLs. Gal1 expression in LCLs was dependent on both activating protein 1 (AP-1) and PI3K. A newly developed neutralizing Gal1 mAb selectively inhibited Gal1-mediated apoptosis of EBV-specific CD8 ؉ T cells. Given the tolerogenic and immunosuppressive function of Gal1, antibody-mediated Gal1 neutralization may represent a novel immunotherapeutic strategy for PTLD and other Gal1-expressing tumors. IntroductionGalectin-1 (Gal1) is a member of a highly conserved family of carbohydrate-binding proteins that modulate innate and adaptive immune responses and foster tumor immune escape. [1][2][3][4] Through the selective recognition of specific cell-surface glycans (Gal-␤1-4-NAcGlc [N-acetyllactosamine] units on the branches of N-or O-linked glycans) on receptors such as CD45, CD43, and CD7, Gal1 induces the apoptosis of specific T-cell subtypes: Th1, Th17, and cytotoxic T cells. 5 Th2 cells have different patterns of sialylation of cell-surface glycoproteins, lack Gal1 ligands, and resist Gal1-induced cell death. 5 Gal1 also instructs dendritic cells to become tolerogenic, further limiting the magnitude of an effective immune response. 6 Primary classic Hodgkin lymphomas (cHLs) include small numbers of malignant Reed-Sternberg cells within a Th2-and regulatory T cell-skewed inflammatory infiltrate. In previous studies, we found that Reed-Sternberg cells selectively overexpress Gal1, which promotes the immunosuppressive Th2/regulatory T cell-predominant microenvironment in cHL. 4 Hodgkin ReedSternberg cells exhibit constitutive activating protein 1 (AP-1) activation and express high levels of the AP-1 components, cJun and JunB. 7 In cHL, the overexpression of Gal1 is driven in large part by an AP-1-dependent enhancer. 4 A significant percentage of cHLs are associated with Epstein-Barr virus (EBV) infection.EBV is a B-lymphotropic ␥ herpes vir...

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Tonic B-cell receptor (BCR) signaling is a key survival pathway during normal B-cell ontogenesis and in a subset of diffuse large B-cell lymphomas (DLBCLs).We previously demonstrated that BCRdependent DLBCL cell lines and primary tumors underwent apoptosis after treatment with an ATP-competitive inhibitor of the BCR-associated spleen tyrosine kinase (SYK). These "BCR-type" tumors also have more abundant expression of the transcriptional repressor, BCL6, and increased sensitivity to BCL6 inhibition. IntroductionEmerging data highlight the important role of B-cell receptor (BCR)-mediated survival signals in B-cell lymphomas. BCR engagement induces the phosphorylation of Ig␣ and ␤ immunoreceptor tyrosine-based activation motifs (ITAMS) by SRC family kinases and the subsequent recruitment and activation of the protein tyrosine kinase (PTK), SYK, and downstream pathways. [1][2][3] Although BCR signaling is generally thought to be triggered by antigen binding, recent studies highlight the role of "tonic" BCR survival signals in the absence of receptor engagement. For example, in murine models, the inducible loss of the BCR or the selective excision of the Ig␣ ITAM led to the death of peripheral B cells. 4,5 The SYK PTK plays a central role in tonic BCR signaling, both transmitting downstream events and amplifying the original signal. 2,3,6,7 SYK activity is tightly regulated by BCR-associated phosphorylation and protein tyrosine phosphatase (PTP)-mediated inhibition. 8 We recently found that SYK is a major substrate of a tissue-specific and developmentally regulated PTP, PTPROt. 6 PTPROt specifically inhibited BCR-triggered SYK tyrosyl phosphorylation, activation of associated adaptor proteins, such as BLNK, and downstream signaling events. 6 In BCR-dependent lymphomas, PTPROt overexpression decreased cellular proliferation and induced apoptosis in the absence of BCR cross-linking, indicating that the phosphatase modulates SYK-dependent tonic BCR signaling. 6 PTPROt is a member of the PTPRO family (also designated GLEPP, PTP-, PTP-OC, and PTPu2), a group of highly conserved receptor-type PTPs with a single catalytic domain and transmembrane region and a variably sized extracellular sequence. 9,10 PTPRO includes an extended extracellular domain, whereas PTPROt contains a truncated extracellular region. The PTPROt 5Ј untranslated region also functions as an intron that is spliced out of the larger PTPRO cDNA. 11 These 2 isoforms have tissue-specific patterns of expression-PTPRO predominantly in epithelial cells and PTPROt primarily in B cells and macrophages. 12 Initial studies suggest that PTPROt is developmentally regulated and decreased in abundance in normal germinal center (GC) B cells and a subset of B-cell lymphomas. 12 We recently found that a subset of diffuse large B-cell lymphomas (DLBCLs) relies upon tonic BCR signaling as a survival mechanism and that PTPROt modulates SYK-dependent BCR signaling in these tumors. 6,13 DLBCLs that were dependent upon BCR signaling had a notable transcriptional profile with i...

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The purpose of this study was to determine the association between time from the diagnosis of rotator cuff tear to repair and the rate of subsequent revision surgery for re-tear. A national insurance database was queried from 2007 to 2016 for patients who underwent arthroscopic rotator cuff repair after a diagnosis of rotator cuff tear with minimum 5-year follow-up. On the basis of time from diagnosis to repair, patients were stratified into an early (<6 weeks), a routine (between 6 weeks and 12 months), or a delayed (>12 months) repair cohort. The rates of subsequent revision rotator cuff repair were compared pairwise between cohorts with Pearson's chi-square tests. Multivariate logistic regression was used to adjust for patient demographics and comorbidity burden. A total of 2759 patients were included, with 1510 (54.7%) undergoing early repair, 1104 (40.0%) undergoing routine repair, and 145 (5.3%) having delayed repair. The overall revision rate at 5-year follow-up was 9.6%. The revision rate was higher in the delayed group (15.2%) relative to the early (9.9%) and routine (8.3%) groups ( P =.048 and P =.007, respectively). On multivariate analysis, delayed repair was associated with increased odds of revision surgery (odds ratio, 1.97; P =.009) compared with routine repair. Delayed rotator cuff repair beyond 12 months of diagnosis was associated with an increased risk of undergoing subsequent revision rotator cuff repair while controlling for age and comorbidity burden. [ Orthopedics . 2020;43(6):340–344.]

Purpose: Patients with mixed lineage leukemia (MLL)-rearranged B-lymphoblastic leukemias (B-ALL) have an unfavorable prognosis and require intensified treatment. Multiple MLL fusion partners have been identified, complicating the diagnostic evaluation of MLL rearrangements. We analyzed molecular markers of MLL rearrangement for use in rapid diagnostic assays and found the immunomodulatory protein, Galectin-1 (Gal-1), to be selectively expressed in MLL-rearranged B-ALL.Experimental Design: Transcriptional profiling of ALL subtypes revealed selective overexpression of Gal-1 in MLL-rearranged ALLs. For this reason, we analyzed Gal-1 protein expression in MLL-germline and MLL-rearranged adult and infant pediatric B-ALLs and cell lines by immunoblotting, immunohistochemistry, and intracellular flow cytometry of viable tumor cell suspensions. Because deregulated gene expression in MLL-rearranged leukemias may be related to the altered histone methyltransferase activity of the MLL fusion protein complex, we also analyzed histone H3 lysine 79 (H3K79) dimethylation in the LGALS1 promoter region using chromatin immunoprecipitation.Results: Gal-1 transcripts were significantly more abundant in MLL-rearranged B-ALLs. All 32 primary MLLrearranged B-ALLs exhibited abundant Gal-1 immunostaining, regardless of the translocation partner, whereas only 2 of 81 germline-MLL B-ALLs expressed Gal-1. In addition, Gal-1 was selectively detected in newly diagnosed MLL-rearranged B-ALLs by intracellular flow cytometry. The LGALS1 promoter H3K79 was significantly hypermethylated in MLL-rearranged B-ALLs compared with MLL-germline B-ALLs and normal pre-B cells.Conclusion: In B-ALL, Gal-1 is a highly sensitive and specific biomarker of MLL rearrangement that is likely induced by a MLL-dependent epigenetic modification.