Multi-omic profiling of follicular lymphoma reveals changes in tissue architecture and enhanced stromal remodeling in high-risk patients.

Follicular lymphoma (FL) is a generally incurable malignancy that evolves from developmentally blocked germinal center (GC) B cells. To promote survival and immune escape, tumor B cells undergo significant genetic changes and extensively remodel the lymphoid microenvironment. Dynamic interactions between tumor B cells and the tumor microenvironment (TME) are hypothesized to contribute to the broad spectrum of clinical behaviors observed among FL patients. Despite the urgent need, existing clinical tools do not reliably predict disease behavior. Using a multi-modal strategy, we examined cell-intrinsic and -extrinsic factors governing progression and therapeutic outcomes in FL patients enrolled onto a prospective clinical trial. By leveraging the strengths of each platform, we identify several tumor-specific features and microenvironmental patterns enriched in individuals who experience early relapse, the most high-risk FL patients. These features include stromal desmoplasia and changes to the follicular growth pattern present 20 months before first progression and first relapse.

Positional influence on cellular transcriptional identity revealed through spatially segmented single-cell transcriptomics.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for describing cell states. Identifying the spatial arrangement of these states in tissues remains challenging, with the existing methods requiring niche methodologies and expertise. Here, we describe segmentation by exogenous perfusion (SEEP), a rapid and integrated method to link surface proximity and environment accessibility to transcriptional identity within three-dimensional (3D) disease models. The method utilizes the steady-state diffusion kinetics of a fluorescent dye to establish a gradient along the radial axis of disease models. Classification of sample layers based on dye accessibility enables dissociated and sorted cells to be characterized by transcriptomic and regional identities. Using SEEP, we analyze spheroid, organoid, and in vivo tumor models of high-grade serous ovarian cancer (HGSOC). The results validate long-standing beliefs about the relationship between cell state and position while revealing new concepts regarding how spatially unique microenvironments influence the identity of individual cells within tumors.

The immune microenvironment shapes transcriptional and genetic heterogeneity in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), B-cell receptor signaling, tumor-microenvironment interactions, and somatic mutations drive disease progression. To better understand the intersection between the microenvironment and molecular events in CLL pathogenesis, we integrated bulk transcriptome profiling of paired peripheral blood (PB) and lymph node (LN) samples from 34 patients. Oncogenic processes were upregulated in LN compared with PB and in immunoglobulin heavy-chain variable (IGHV) region unmutated compared with mutated cases. Single-cell RNA sequencing (scRNA-seq) distinguished 3 major cell states: quiescent, activated, and proliferating. The activated subpopulation comprised only 2.2% to 4.3% of the total tumor bulk in LN samples. RNA velocity analysis found that CLL cell fate in LN is unidirectional, starts in the proliferating state, transitions to the activated state, and ends in the quiescent state. A 10-gene signature derived from activated tumor cells was associated with inferior treatment-free survival (TFS) and positively correlated with the proportion of activated CD4+ memory T cells and M2 macrophages in LN. Whole exome sequencing (WES) of paired PB and LN samples showed subclonal expansion in LN in approximately half of the patients. Since mouse models have implicated activation-induced cytidine deaminase in mutagenesis, we compared AICDA expression between cases with and without clonal evolution but did not find a difference. In contrast, the presence of a T-cell inflamed microenvironment in LN was associated with clonal stability. In summary, a distinct minor tumor subpopulation underlies CLL pathogenesis and drives the clinical outcome. Clonal trajectories are shaped by the LN milieu, where T-cell immunity may contribute to suppressing clonal outgrowth. The clinical study is registered at clinicaltrials.gov as NCT00923507.

Clinicopathologic and Molecular Characterization of Epstein-Barr Virus-positive Plasmacytoma.

Epstein-Barr virus (EBV)-positive plasmacytoma is a rare plasma cell neoplasm. It remains unclear whether EBV-positive plasmacytoma represents a distinct entity or a variant of plasmacytoma. It shares morphologic features with plasmablastic lymphoma (PBL) and may cause diagnostic uncertainty. To better understand EBV-positive plasmacytoma and explore diagnostic criteria, this study describes 19 cases of EBV-positive plasmacytoma, compared with 27 cases of EBV-negative plasmacytoma and 48 cases of EBV-positive PBL. We reviewed the clinicopathologic findings and performed immunohistochemistry, in situ hybridization for EBV, fluorescence in situ hybridization for MYC , and next-generation sequencing. We found that 63.2% of patients with EBV-positive plasmacytoma were immunocompromised. Anaplastic features were observed in 7/19 cases. MYC rearrangement was found in 25.0% of them, and extra copies of MYC in 81.3%. EBV-positive and EBV-negative plasmacytomas possessed similar clinicopathologic features, except more frequent cytologic atypia, bone involvement and MYC aberrations in the former group. The survival rate of patients with EBV-positive plasmacytoma was comparable to that of patients with EBV-negative plasmacytoma. In comparison to PBL, EBV-positive plasmacytoma is less commonly associated with a "starry-sky" appearance, necrosis, absence of light chain expression, and a high Ki67 index (>75%). The most recurrently mutated genes/signaling pathways in EBV-positive plasmacytoma are epigenetic regulators, MAPK pathway, and DNA damage response, while the most frequently reported mutations in PBL are not observed. Collectively, EBV-positive plasmacytoma should be regarded as a biological variant of plasmacytoma. Thorough morphologic examination remains the cornerstone for distinguishing EBV-positive plasmacytoma and PBL, and molecular studies can be a valuable complementary tool.

Neutrophil and Eosinophil Extracellular Traps in Hodgkin Lymphoma.

Classic Hodgkin lymphoma (cHL), nodular sclerosis (NS) subtype, is characterized by the presence of Hodgkin/Reed-Sternberg (HRS) cells in an inflammatory background containing neutrophils and/or eosinophils. Both types of granulocytes release extracellular traps (ETs), web-like DNA structures decorated with histones, enzymes, and coagulation factors that promote inflammation, thrombosis, and tumor growth. We investigated whether ETs from neutrophils (NETs) or eosinophils (EETs) are detected in cHL, and evaluated their association with fibrosis. We also studied expression of protease-activated receptor-2 (PAR-2) and phospho-extracellular signal-related kinase (p-ERK), potential targets/effectors of ETs-associated elastase, in HRS cells. Expression of tissue factor (TF) was evaluated, given the procoagulant properties of ETs. We analyzed 32 HL cases, subclassified as 12 NS, 5 mixed-cellularity, 5 lymphocyte-rich, 1 lymphocyte-depleted, 4 nodular lymphocyte-predominant HL (NLPHL), and 5 reactive nodes. Notably, a majority of NS cHL cases exhibited NET formation by immunohistochemistry for citrullinated histones, with 1 case revealing abundant EETs. All other cHL subtypes as well as NLPHL were negative. Immunofluorescence microscopy confirmed NETs with filamentous/delobulated morphology. Moreover, ETs formation correlates with concurrent fibrosis (r = 0.7999; 95% CI, 0.6192-0.9002; P ≤ 0.0001). Results also showed that HRS cells in NS cHL expressed PAR-2 with nuclear p-ERK staining, indicating a neoplastic or inflammatory phenotype. Remarkably, TF was consistently detected in the endothelium of NS cHL cases compared with other subtypes, in keeping with a procoagulant status. A picture emerges whereby the release of ETs and resultant immunothrombosis contribute to the inflammatory tumor microenvironment of NS cHL. This is the first description of NETs in cHL.

Expanding the Spectrum of EBV-positive Marginal Zone Lymphomas: A Lesion Associated With Diverse Immunodeficiency Settings.

Traditionally low-grade B-cell lymphomas have been excluded from the category of monomorphic posttransplant lymphoproliferative disorders. However, recent reports identified Epstein-Barr virus-positive (EBV) extranodal marginal zone lymphomas (MZL), almost exclusively seen in the posttransplant setting. Some reported cases responded to reduced immunosuppression, suggesting that they should be considered as a form of posttransplant lymphoproliferative disorders. We identified 10 cases of EBV MZL, 9 in extranodal sites and 1 presenting in lymph node. Two cases arose following solid organ transplantation, but other settings included iatrogenic immunosuppression for rheumatoid arthritis (2); prior chemotherapy (2); congenital immune deficiency (1); and increased age (3), as the only potential cause of immune dysfunction. There were 4 males and 6 females; age range 18 to 86. The atypical plasmacytoid and/or monocytoid B cells were positive for EBV in all cases, with either latency I or II in all cases tested. Monotypic light chain expression was shown in all with 6 cases positive for IgG, and 2 for IgM, undetermined in 2. Clonal immunoglobulin gene rearrangement was positive in all cases with successful amplification. MYD88 L265P was wild type in the 6 cases tested. We show that EBV MZLs can arise in a variety of clinical settings, and are most often extranodal. Treatment varied, but most patients had clinically indolent disease with response to reduction of immune suppression, or immunochemotherapy.

A multiprotein supercomplex controlling oncogenic signalling in lymphoma.

B cell receptor (BCR) signalling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients1. Gene expression profiling identified two major subtypes of DLBCL, known as germinal centre B cell-like and activated B cell-like (ABC)2,3, that show poor outcomes after immunochemotherapy in ABC. Autoantigens drive BCR-dependent activation of NF-κB in ABC DLBCL through a kinase signalling cascade of SYK, BTK and PKCß to promote the assembly of the CARD11-BCL10-MALT1 adaptor complex, which recruits and activates IκB kinase4-6. Genome sequencing revealed gain-of-function mutations that target the CD79A and CD79B BCR subunits and the Toll-like receptor signalling adaptor MYD885,7, with MYD88(L265P) being the most prevalent isoform. In a clinical trial, the BTK inhibitor ibrutinib produced responses in 37% of cases of ABC1. The most striking response rate (80%) was observed in tumours with both CD79B and MYD88(L265P) mutations, but how these mutations cooperate to promote dependence on BCR signalling remains unclear. Here we used genome-wide CRISPR-Cas9 screening and functional proteomics to determine the molecular basis of exceptional clinical responses to ibrutinib. We discovered a new mode of oncogenic BCR signalling in ibrutinib-responsive cell lines and biopsies, coordinated by a multiprotein supercomplex formed by MYD88, TLR9 and the BCR (hereafter termed the My-T-BCR supercomplex). The My-T-BCR supercomplex co-localizes with mTOR on endolysosomes, where it drives pro-survival NF-κB and mTOR signalling. Inhibitors of BCR and mTOR signalling cooperatively decreased the formation and function of the My-T-BCR supercomplex, providing mechanistic insight into their synergistic toxicity for My-T-BCR+ DLBCL cells. My-T-BCR supercomplexes characterized ibrutinib-responsive malignancies and distinguished ibrutinib responders from non-responders. Our data provide a framework for the rational design of oncogenic signalling inhibitors in molecularly defined subsets of DLBCL.

Hodgkin lymphoma variant of Richter transformation: morphology, Epstein-Barr virus status, clonality, and survival analysis-with comparison to Hodgkin-like lesion.

Hodgkin/Reed-Sternberg (HRS) cells in the setting of chronic lymphocytic leukemia (CLL) exist in 2 forms: type I with isolated HRS cells in a CLL background (Hodgkin-like lesion) and type II with typical classic Hodgkin lymphoma, a variant of Richter transformation (CHL-RT). The clinical significance of the 2 morphological patterns is unclear, and their biological features have not been compared. We retrospectively reviewed 77 cases: 26 of type I and 51 of type II CHL-RT; 3 cases progressed from type I to type II. We examined clinical features, Epstein-Barr virus (EBV) status, and clonal relatedness after microdissection. Median age for type I was 62 years versus 73 years for type II (P=.01); 27% (type I) versus 73% (type II) had a history of CLL. HRS cells were positive for EBV in 71% (55/77), similar in types I and II. Clonality analysis was performed in 33 cases (type I and type II combined): HRS cells were clonally related to the underlying CLL in 14 and unrelated in 19. ZAP-70 expression of the CLL cells but not EBV status or morphological pattern was correlated with clonal relatedness: all 14 clonally related cases were ZAP-70 negative, whereas 74% (14/19) of clonally unrelated cases were ZAP-70 positive. Overall median survival (types I and II) after diagnosis was 44 months. Advanced age was an adverse risk factor for survival, but not histologic pattern, type I versus type II. HRS-like cells in a background of CLL carries a similar clinical risk to that of CHL-RT and may progress to classic Hodgkin lymphoma in some cases.

EBV-positive large B-cell lymphomas in young patients: a nodal lymphoma with evidence for a tolerogenic immune environment.

Few studies have reported Epstein-Barr virus-positive (EBV(+)) large B-cell lymphomas (LBCLs) in young patients without immunodeficiency. We identified 46 such cases in patients ≤45 years of age and analyzed the clinical and pathological characteristics. EBV(+) LBCLs affected predominantly males (male:female = 3.6:1), with a median age of 23 years (range, 4-45 years). All patients presented with lymphadenopathy and 11% also had extranodal disease. Morphologically, 3 patterns were identified: T-cell/histiocyte-rich large B-cell lymphoma-like (n = 36), gray zone lymphoma (n = 7), and diffuse LBCL-not otherwise specified (n = 3). Tumor cells (EBV(+) in >90% of cells) expressed B-cell antigens, were often CD30 and PD-L1 positive, and showed a nongerminal center immunophenotype. A total of 93% expressed EBV latency type II and 7% latency type III. Indoleamine 2,3-dioxygenase was expressed on background accessory cells. The most common treatment regimen was rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (58%), with local radiation therapy added in 21%. With a median follow-up of 22 months, 82% of patients are in clinical remission and only 8% died of disease. Younger patients achieved a significantly higher overall survival than prior series of EBV(+) LBCLs reported in the elderly (P < .0001). In conclusion, EBV(+) LBCLs are not restricted to the elderly. Young patients present with nodal disease and have a good prognosis.

Lymphomatoid granulomatosis--a single institute experience: pathologic findings and clinical correlations.

Lymphomatoid granulomatosis (LYG) is a rare angiocentric and angiodestructive Epstein-Barr virus (EBV)-associated B-cell lymphoproliferative disorder. It is hypothesized that these patients have dysregulated immune surveillance of EBV. We reviewed the biopsies of 55 patients with LYG who were referred for a prospective trial at the National Cancer Institute (1995 to 2010) and evaluated the histologic, immunohistochemical, in situ hybridization, and molecular findings of these biopsies in conjunction with clinical information. Grading of the lesions was based on morphologic features and the number of EBV-positive B cells. The median age was 46 years (M:F 2.2:1). Clinically, all patients had lung involvement (100%), with the next most common site being the central nervous system (38%). No patient had nodal or bone marrow disease. All patients had past EBV exposure by serology but with a low median EBV viral load. We reviewed 122 biopsies; the most common site was lung (73%), followed by skin/subcutaneous tissue (17%); other sites included kidney, nasal cavity, gastrointestinal tract, conjunctiva, liver, and adrenal gland. Histologically, the lesions showed angiocentricity, were rich in T cells, had large atypical B cells, and were positive for EBV. Grading was performed predominantly on the lung biopsy at diagnosis; they were distributed as follows: LYG grade 1 (30%), grade 2 (22%), and grade 3 (48%). Necrosis was seen in all grades, with a greater degree in high-grade lesions. Immunoglobulin gene rearrangement studies were performed, and a higher percentage of clonal rearrangements were seen in LYG grade 2 (50%) and grade 3 (69%) as compared with grade 1 (8%). LYG is a distinct entity that can usually be differentiated from other EBV-associated B-cell lymphoproliferative disorders on the basis of the combination of clinical presentation, histology, and EBV studies. Grading of these lesions is important because it dictates the treatment choice.

EBV may be expressed in the LP cells of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) in both children and adults.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) and classical Hodgkin lymphoma (CHL) are classified separately because of their distinct clinical and pathologic features. Whereas Epstein-Barr virus (EBV) is detected in the neoplastic cells of 25% to 70% of CHL, NLPHL is generally considered to be EBV(-). We assessed EBV status in 302 pediatric and adult cases of NLPHL. A total of 145 pediatric (age 18 y or younger) and 157 adult cases of NLPHL were retrieved from 3 North American centers and tested for EBV by in situ hybridization (EBV-encoded small RNA). Clinical and pathologic features were analyzed. Five (3.4%) pediatric and 7 (4.5%) adult NLPHL cases contained EBV(+) lymphocyte-predominant (LP) cells. Although all 12 cases met the criteria for diagnosis of NLPHL, atypical features were present, including capsular fibrosis, atrophic germinal centers, and pleomorphic or atypical LP cells. CD20 and OCT-2 were strongly and diffusely positive in all except 1 case. However, PAX5 and CD79a were weak and/or variable in 7/8 and 6/6 cases tested, respectively. EBV(+) cases were more likely to be CD30(+) (75%) compared with EBV(-) cases (25%) (P=0.0007); CD15 was negative in all cases. Our results show that EBV(+) LP cells may occur in NLPHL. Distinguishing EBV(+) NLPHL from CHL can be challenging, as EBV(+) NLPHL can have partial expression of CD30 and weak PAX5 staining as well as pleomorphic-appearing LP cells. However, the overall appearance and maintenance of B-cell phenotype, with strong and diffuse CD20 and OCT-2 expression, support the diagnosis of NLPHL in these cases.

MDM2 antagonist nutlin-3 displays antiproliferative and proapoptotic activity in mantle cell lymphoma.

PURPOSE: Mantle cell lymphoma (MCL) has one of the poorest prognoses of the non-Hodgkin's lymphomas, and novel therapeutic approaches are needed. We wished to determine whether Nutlin-3, a novel small-molecule murine double minute 2 (MDM2) antagonist that efficiently activates TP53, might be effective in inducing cell death in MCL. EXPERIMENTAL DESIGN: MCL cell lines with known TP53 status were treated with Nutlin-3, and biological and biochemical consequences were studied. Synergies with the prototypic genotoxic agent doxorubicin and the novel proteasome inhibitor bortezomib were assessed. RESULTS: Nutlin-3 resulted in a reduction in cell proliferation/viability (IC50 < 10 micromol/L), an increase in the apoptotic fraction, and cell cycle arrest in wild-type (wt) TP53 Z-138 and Granta 519 cells. These effects were accompanied by TP53 accumulation and induction of TP53-dependent proteins p21, MDM2, Puma, and Noxa. Cell cycle arrest was characterized by suppression of S phase and an increase in the G0-G1 and G2-M fractions and accompanied by suppression of total and phosphorylated retinoblastoma protein and a decrease in G2-M-associated proteins cyclin B and CDC2. The combination of Nutlin-3 with doxorubicin or bortezomib was synergistic in wt-TP53 MCL cells. Nutlin-3 also induced cell cycle arrest and reduced cell viability in the mutant TP53 MINO cells but at a significantly higher IC50 (22.5 micromol/L). These effects were associated with induction of the TP53 homologue p73, slight increases in p21 and Noxa, and caspase activation. Nutlin-3 and bortezomib synergistically inhibited cell growth of MINO. CONCLUSION: These findings suggest that the MDM2 antagonist Nutlin-3 may be an effective agent in the treatment of MCL with or without wt-TP53.

Expression of the interferon regulatory factor 8/ICSBP-1 in human reactive lymphoid tissues and B-cell lymphomas: a novel germinal center marker.

To assess the role of interferon regulatory factor (IRF) 8 in B-cell development and lymphomagenesis, we studied its expression in reactive lymphoid tissues, its relationship to other B-cell transcription factors, and its expression in a series of 232 B-cell tumors and 30 cell lines representing a variety of B-cell developmental stages. We found that although IRF8 was detectable in most reactive B-cells, its expression levels differed with developmental stage. Germinal center B cells contained the highest levels of IRF8, with lower levels seen in mantle and marginal zone B cells and none in plasma cells. IRF8 was coexpressed with PAX-5, Pu.1, and B-cell lymphoma (BCL)-6, and similar to BCL-6, was absent from the small population of IRF4-positive germinal center B cells thought to be committed to postgerminal center developmental programs. Similarly, IRF8 was most strongly expressed in lymphomas of germinal center origin with lower levels present in mantle cell lymphomas, chronic lymphocytic leukemia, and marginal zone lymphomas, and no expression observed in plasmacytic/plasmablastic neoplasms. The reciprocal expression pattern with IRF4 in reactive tissues was generally maintained in lymphomas with some exceptions. These results suggest an important role for IRF8 during germinal center B-cell development and in related lymphomas, and provide a new diagnostic marker helpful in distinguishing B-cell non-Hodgkin lymphoma subtypes.

NPM-ALK-dependent expression of the transcription factor CCAAT/enhancer binding protein beta in ALK-positive anaplastic large cell lymphoma.

CCAAT/enhancer binding protein beta (C/EBPbeta) is one of a 6-member family of C/EBPs. These transcription factors are involved in the regulation of various aspects of cellular growth and differentiation. Although C/EBPbeta has important functions in B- and T-cell differentiation, its expression has not been well studied in lymphoid tissues. We, therefore, analyzed its expression by immunohistochemistry and Western blot in normal lymphoid tissues and in 248 well-characterized lymphomas and lymphoma cell lines. Nonneoplastic lymphoid tissues and most B-cell, T-cell, and Hodgkin lymphomas lacked detectable levels of C/EBPbeta. In contrast, most (40 of 45; 88%) cases of ALK-positive anaplastic large cell lymphoma (ALCL) strongly expressed C/EBPbeta. Western blot analysis confirmed C/EBPbeta expression in the ALK-positive ALCLs and demonstrated elevated levels of the LIP isoform, which has been associated with increased proliferation and aggressiveness in carcinomas. Transfection of Ba/F3 and 32D cells with NPM-ALK and a kinase-inhibitable modified NPM-ALK resulted in the induction of C/EBPbeta and demonstrated dependence on NPM-ALK kinase activity. In conclusion, we report the constitutive expression of C/EBPbeta in ALK-positive ALCL and show its relationship to NPM-ALK. We suggest that C/EBPbeta is likely to play an important role in the pathogenesis and unique phenotype of this lymphoma.

Sequestration of p27Kip1 protein by cyclin D1 in typical and blastic variants of mantle cell lymphoma (MCL): implications for pathogenesis.

p27 is a cyclin-dependent kinase inhibitor that plays a critical role in regulating G(1)/S progression, and whose activity is, in part, regulated through interactions with D-type cyclins. Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation resulting in deregulated cyclin D1. We previously showed that p27 expression in MCL, as assessed by immunohistochemistry (IHC), does not show the usual inverse relationship to proliferate seen in most other lymphomas that do not overexpress cyclin D1. This suggested that the normal expression or control of p27 activity on cell growth might be altered through potential interactions with cyclin D1. Using Western blot and coimmunoprecipitation studies, we assessed the interrelationship between cyclin D1 and p27 in several cyclin D1(+) cell lines and primary MCL cases. Similar to our previous results by IHC, typical MCLs showed lower expression of p27 when compared to the more highly proliferative blastic cases or cell lines (mean arbitrary units: 58 versus 236 versus 120). Cyclin D1 was expressed at variable levels in both typical and blastic MCLs. p27 protein could be consistently coimmunoprecipitated with cyclin D1 from both cell lines and cases. Using techniques of exhaustive immunoprecipitation, we could demonstrate that most p27 protein was sequestered into complexes containing cyclin D1. We hypothesize that mantle cell lymphomagenesis results not only from direct consequences of inappropriate cyclin D1 expression, but also from the ability of overexpressed cyclin D1 to buffer physiologic changes in p27 levels, thereby rendering p27 ineffective as an inhibitor of cellular growth.