Hyperplastic Germinal Centers Research Articles

The Sentient Propagation-Reactive B Cell-Rich Lymphoid Proliferation

Reactive B cell-rich lymphoid proliferation is a heterogeneous group of non-neoplastic lymphoid cell proliferation recapitulating diverse B cell lymphomas. The lesion is commonly categorized as nodal and extra-nodal follicular proliferations, nodal and extra-nodal nodular proliferations or nodal and extra-nodal immunoblastic proliferations. Florid follicular hyperplasia is comprised of quantifiably enhanced, disseminated primary and secondary lymphoid follicles with irregular outlines. Hyperplastic Germinal Centres (GCs) are comprised of admixed centroblasts and centrocytes, reactive T cells, Follicular Dendritic Cells (FDCs) and tingible body macrophages. Lymph node architecture depicts lymphoid follicles comprised of cells expressing B cell antigens wherein primary follicles are pre-eminently constituted of small lymphocytes BCL2+, BCL6- and CD10- immune reactivity. Progressive Transformation of Germinal Centre (PTGC) is constituted of singular or few enlarged lymphoid follicles of 4x to 5x magnitude wherein mantle zone cells display extensive invagination into adjacent germinal centres. Hyaline Vascular Castleman’s Disease (HVCD) delineates innumerable lymphoid follicles confined to the lymph node cortex and medulla or diverse extramedullary sites.

Progressively transformed germinal center-like follicular T-cell lymphoma:a clinicopathological analysis of 14 cases

Objective: To investigate the clinicopathologic features of progressively transformed germinal center-like follicular T-cell lymphoma (PTGC-like FTCL). Methods: The clinicopathologic data of 14 PTGC-like FTCL cases that were diagnosed at the Beijing Friendship Hospital Affiliated to the Capital Medical University from January 2017 to January 2022 were retrospectively collected. Clinicopathological features, immunophenotype, and Epstein-Barr virus (EBV) infection status were analyzed in these cases. Polymerase chain reaction (PCR) was performed to detect the clonal gene rearrangements of T cell receptor (TCR) and the immunoglobulin (Ig) in 10 and 8 cases, respectively. Results: The male to female ratio was 5∶2. The median age was 61 years (range 32-70 years). All patients had lymphadenopathy at the time of diagnosis. By using the Ann Arbor system staging, seven cases were classified as stage Ⅰ-Ⅱ, and seven cases as stage Ⅲ-Ⅳ. Seven cases had B symptoms, four cases had splenomegaly, and two cases had skin rash and pruritus. Previously, three cases were diagnosed as classic Hodgkin's lymphoma, three cases as small B-cell lymphoma, two cases as atypical lymphoid hyperplasia unable to exclude angioimmunoblastic T-cell lymphoma (AITL), one case as EBV-associated lymphoproliferative disorder, and one case as peripheral T-cell lymphoma (PTCL) associated with the proliferation of B cells. All the 14 cases showed that the large nodules were composed of mature CD20+, IgD+B lymphocytes admixed with small aggregates of neoplastic cells with pale to clear cytoplasm. Moreover, hyperplastic germinal centers (GCs) and Hodgkin/Reed-Sternberg-like (HRS-like) cells were seen within these nodules in two and five cases, respectively. The neoplastic cells expressed CD3 (14/14), CD4 (14/14), PD1 (14/14), ICOS (14/14), CD10 (9/14), bcl-6 (12/14), CXCL13 (10/14), and CD30 (10/14). The HRS-like cells in five cases expressed CD20 (2/5), PAX5 (5/5), CD30 (5/5), CD15 (2/5), LCA (0/5), OCT2 (5/5) and BOB1 (2/5). Moreover, neoplastic T cells formed rosettes around HRS-like cells. EBV-encoded RNA (EBER) in situ hybridization showed scattered, small, positive bystander B lymphocytes in 8/14 cases, including 3/5 cases containing HRS-like cells. All tested cases (including five with HRS-like cells) showed monoclonal TCR gene rearrangement and polyclonal Ig gene rearrangement. Conclusions: PTGC-like FTCL is a rare tumor originated from T-follicular helper cells. It could be distinguished from angioimmunoblastic T-cell lymphoma by the formation of follicular structure, and lack of follicular dendritic cell proliferation outside the follicles and the polymorphous inflammatory background. In addition, it should be differentiated from lymphocyte-rich classical Hodgkin's lymphoma and low-grade B cell lymphoma.

A Rare Case of Unilateral Lymphadenopathy: Unicentric Castleman Disease

Background Castleman Disease (CD) involves a group of disorders characterized by an enlargement of lymphoid follicles with capillary proliferation. The disorders are largely separated into unicentric (UCD) or multicentric (MCD) depending on the number of involved lymph nodes. MCD has been linked to HHV8 or immunosuppression secondary to HIV, while UCD has been thought to be driven by a clonal neoplastic process, likely from the follicular dendritic cell. Case Presentation A 14-year-old female with ADHD, OCD, and autism spectrum disorder (ASD) presents to clinic with a 2-year history of a right-sided neck mass. There was no history of trauma or illness around the time of the mass being noted and the mass has not changed significantly in size for the past two years. An excisional biopsy was attempted but, due to excessive vascularity around the mass, was ultimately deferred. Following subsequent MRI and CT scans, a core needle biopsy was performed, revealing lymphoid tissue with regressed follicles and focal hyalinization without signs of malignancy. PET/CT scans at this time showed increased metabolic activity confined to the right-sided neck mass without any increased metabolic activity in the other lymph nodes of the head and neck. Eventually, the 7 cm diameter mass was excised; pathology studies revealed distorted architecture with hyaline deposits in the germinal centers and sclerotic blood vessels penetrating the germinal centers consistent with the hypervascular variant of Castleman Disease. Pre- and post-operative labs such as the C-reactive protein, immunoglobulin counts, and protein electrophoresis panels were all within normal limits. Furthermore, post-operative PET/CT scans showed no metabolically active foci indicating that the lesion was indeed unicentric Castleman Disease. After the excision of the mass, the patient continues to do well without any further surgeries or complications. Discussion Differentiation between UCD and MCD requires imaging, often with a CT scan, to establish the number and locality of the affected lymph nodes. UCD often presents with one bulky lymph node or multiple enlarged lymph nodes within the same region, whereas MCD tends to be smaller lymph nodes spread over multiple regions. MCD is also more often associated with B symptoms and abnormal lab values such as elevated CRP or ESR along with abnormal protein levels. Other subtypes exist, thought to be driven primarily by cytokines like VEGF and IL-12 similar to other neoplasms. The hypervascular type, more commonly associated with UCD, consists of fibrous bands running through lymphoid follicles with regressed germinal centers while the plasmacytic type, found more often in MCD, consists of sheets of plasma cells and hyperplastic germinal centers. Lastly, mixed type has features of both the main types. Preferred treatment for resectable UCD is surgery with treatments for unresectable UCD involving observation (if asymptomatic) or a combination of rituximab and steroids with anti-IL6 monoclonal antibody treatment (such as tocilizumab) followed by surgery or radiation therapy. Similarly, MCD is often treated with rituximab, steroids, and tocilizumab. Conclusion Due to the presence or absence of other signs and symptoms, Castleman Disease is often difficult to diagnose without a biopsy. Further studies into the etiologies of CD, including possible associations with other neoplastic or immunosuppressed states, are needed to develop better criteria for diagnosing the disease. Developing standardized patient registries with information on disease progression as well as proper follow-up for patients after treatment are vital to diagnosing, treating, and providing surveillance for patients with Castleman Disease.

Idiopathic Multicentric Castleman Disease Interactive Disease Awareness and Educational Tool for Pathologists

Background: Idiopathic multicentric Castleman disease (iMCD) is a rare systemic lymphoproliferative disease that is challenging to diagnose. Diagnostic criteria require the identification of characteristic histopathology in the context of supportive clinical, radiologic, and serologic findings. However, the histologic features of iMCD can be subtle and nonspecific, which delays an accurate diagnosis. Timely diagnosis and early intervention are essential to reduce patient mortality. Developing a disease awareness and educational tool for pathologists may support the identification of future iMCD cases. Methods: International pathologists were identified to support the development of an interactive pathology tool for iMCD. To validate the tool, a selection of pathologists attended a virtual workshop to provide insight and help shape the content and format of the tool. Prior to workshop, attendees were asked to complete a 14-question survey on the diagnosis of iMCD, including exclusion of other diseases, conditions with similar histopathological features, and features specific to iMCD. Results: The educational tool is currently in development and takes into consideration the varied diagnostic journeys in different centers/countries. Histopathologic criteria for iMCD require evaluation of the degree of plasmacytosis, regressed germinal centers, follicular dendritic cell proliferation, vascular proliferation, and hyperplastic germinal centers. While the differences between high grade (grade 3) and normal (grade 0) findings are straightforward, differentiating grade 1 from grade 2 (the diagnostic cut-off) is challenging and subjective. The tool will focus on these diagnostic grey-zones to include points of differentiation between Grades 1 and 2 iMCD, disease exclusion criteria, and the use of diagnostic images that can be explored. Thus far, development of the tool has shown there is a need for standardized disease terminology between pathologists, that the use of template reports and language guidance may support the diagnostic process, and that patient case studies and the use of real-world examples support disease recognition. The educational tool is anticipated to be ready for release in September 2022. Conclusions: Diagnosis of iMCD is complex, and, despite the existence of well-established diagnostic criteria, pathological differentiation remains a challenge. Increasing awareness of iMCD-specifically the key diagnostic criteria-will assist with both timely diagnoses and the treatment of this disease.

Virome Capture Sequencing in Castleman Disease Identifies Associations with Herpesviridae Family Members but No Novel Viruses

Castleman disease (CD) describes multiple distinct and heterogeneous disorders defined by common lymph node histopathology, including atrophic or hyperplastic germinal centers, prominent follicular dendritic cells, hypervascularization, polyclonal lymphoproliferation, and/or polytypic plasmacytosis. Unicentric CD (UCD) involves a solitary lymph node station that displays CD histopathology, whereas multicentric CD (MCD) involves multiple regions of enlarged nodes. Most UCD patients do not experience systemic symptoms and are successfully treated by removal of the enlarged node. In contrast, MCD involves constitutional symptoms, cytopenia, hepatosplenomegaly, fluid accumulation, cytokine storm-associated multiple organ system dysfunction, and is often fatal. In 1994, Kaposi sarcoma-associated herpesvirus/human herpesvirus-8 (HHV-8) was identified as the etiological driver of MCD in immunocompromised individuals. However, approximately 50% of MCD patients are HHV-8 negative (idiopathic MCD (iMCD)). Its idiopathic nature limits targeted treatment for iMCD patients who do not respond to anti-Interleukin-6 therapy. iMCD is itself heterogeneous, and recent work has identified at least one distinct sub-population of iMCD patients with a severe clinical presentation featuring thrombocytopenia, anasarca, myelofibrosis, renal dysfunction, and organomegaly (iMCD-TAFRO). Several hypotheses have been proposed to explain the pathophysiology of iMCD and UCD, all of which remain untested. One notable hypothesis entertains that iMCD and UCD are in fact driven by viral infection–either HHV-8 that is not detected by current diagnostic methods, a novel virus, or a known but unanticipated virus. In support of the last possibility, there are several case reports of associations with Epstein-Barr virus (EBV), HHV-6 and Hepatitis B virus (HBV); however, neither a systematic study of known viruses, nor a search for novel viruses in CD has been reported.To this end, we employed Virome Capture Sequencing for Vertebrate Viruses (VirCapSeq-VERT),-a RNA-hybrid capture, deep sequencing and bioinformatics pipeline designed for viral discovery. In accordance with the Treaty of Helsinki, we obtained freshly frozen lymph node tissue from patients with iMCD (n=11) and UCD (n=12) as experimental conditions. HHV-8-positive MCD (n=2) was included as a positive control for HHV-8 infection. Lymph nodes from patients with lymphoma without clinical suspicion of HHV-8 infection (n=6) and an additional reactive lymph node were included as a negative control. HHV-8 was only detected in HHV-8-positive MCD and not in iMCD or UCD, consistent with prior clinical testing. Additionally, we did not identify any previously unknown viruses in iMCD or UCD lymph node tissue. Members of the herpesviridae family, including EBV, HHV-6 and HHV-7, were found in all conditions. Interestingly, in iMCD, the identification of herpesviridae family members was associated with relapsing disease and the more clinically severe iMCD-TAFRO variant. Additional numerous associations of unclear significance were made with viruses such as Human Endogenous Retrovirus Type K (HERV-K), transfusion transmitted virus/Torque teno virus (TTV), and Pegivirus.To investigate the significance of the EBV infection, we analyzed the number of EBV positive cells by in situ hybridization on lymph node tissue from a separate cohort of patients with CD and related disorders. The mean number of Epstein-Barr virus-encoded small RNAs (EBER) positive cells/high-powered fields (HPF) across 10 HPF was significantly lower in UCD (2 ± 2.3; n=9) and iMCD (1.7 ± 2.8; n=12) than in HHV-8 positive MCD (106.5 ± 75.6; n=2) and EBV-associated lymphoproliferation (63.5 ± 54.4; n=2) (p < 0.005 for all comparisons). The only iMCD-TAFRO case in this cohort had 4 EBER-positive cells/HPF. Further study is needed with larger cohorts of patients to validate the prognostic and potentially pathogenic significance of these associations, and in particular, the association of EBV and other herpesviridae family members with iMCD-TAFRO. DisclosuresFajgenbaum:Janssen Pharmaceuticals: Research Funding.

Primarily VEGF-Driven Etiopathogenesis of Tafro Syndrome and Fibroblastic Reticular Cells As a Probable Castleman Cell - Qualitative Metasynthesis

Introduction: Idiopathic multicentric Castleman disease (iMCD) is a heterogenous group of lymphoproliferative disorders believed to be driven by IL-6 with the cell(s) of origin not yet identified (Fajgenbaum et al. 2019). One clinical subtype with thrombocytopenia, anasarca, fever, renal dysfunction, and organomegaly (TAFRO) is especially aggressive. We encountered a case of TAFRO syndrome that prompted a systematic literature review focusing on the role of VEGF in the pathogenesis of TAFRO syndrome. Interim findings support a role of VEGF in the etiopathogenesis of TAFRO syndrome, with fibroblastic reticular cells (FRCs) as the pathological cell. Methodology: We started with a focused review (step 1, Table 1), followed by empirical review (step 2, Table 1) of published articles on TAFRO syndrome, using methods of qualitative review (Campbell et al. 2011, Levitt 2018) and discussed interim results from phase 1 review (Table 2). The process of reciprocally translating the findings from each study into those from all the other studies in the synthesis, if applied rigorously, provides a method for combining qualitative data in a structured manner. Interim Results: During phase 1 step 1, we reviewed 97 articles published between 2012 and 2020. Majority were case reports. The interim data comprised a total of 34 male and 32 female patients with age ranging from 3 to 85 (median age of 50.5). 26 patients were identified as severe disease and 25 were non-severe per iMCD diagnostic criteria. At the time of presentation, VEGF levels ranged from 44-7520 pg/ml (median 360), IL-6 ranged from 5-4800 pg/ml (median 39.3), and sIL-2Rα ranged from 448-8944 (median 1250). Histologically, hyaline vascular subtype was most common as seen in 24 lymph nodes compared to 4 plasmacytic and 14 mixed. Most common bone marrow cellularity was hypercellular in 19 cases, compared to 16 normocellular, and 4 hypocellular (Table 2). During phase 1 step 2, we explored the fundamental relationship between IL-6 and VEGF. In the lymph node, IL-6 is secreted from endothelial cells and FRCs. IL-6 is further augmented due to stimulation of bone marrow stromal cells by VEGF secreted from FRCs (Tanaka, Narazaki, and Kishimoto 2014). FRCs are the principal cells facilitating entry of dendritic cells and present a cognate antigen by mediating interaction between dendritic cells and T-cells (Fletcher, Acton, and Knoblich 2015, Brown and Turley 2015). Persistent antigenic stimulation leads to the release of VEGF by FRCs in the lymph node and its potentiation through release of IL-6 through VEGF-mediated stimulation of bone marrow stromal cells (Novotny et al. 2008). This reciprocal stimulation between lymph nodes and bone marrow mediate growth effects driven by VEGF, while autocrine-paracrine stimulation of VEGF and IL-6 within lymph nodes and bone marrow drives IL-6 induced flares. Discussion: IL-6 is considered a main driver of pathogenesis underlying TAFRO syndrome, but in the only randomized controlled trial of IL-6 blockade in iMCD, 66% patients did not respond to therapy. IL-6 blockade targets PI3K/Akt/mTOR signaling, an underlying pathway for the pathogenesis of TAFRO syndrome. Our empirical review of literature into TAFRO syndrome reveals elevated VEGF and CD8+ T cells as the hallmarks of TAFRO flares. FRCs are the principal VEGF mRNA-expressing cells in lymph nodes and also contribute to IL-6 production. Bone marrow stromal cells are the principal cells that drive IL-6 release due to potent stimulation from VEGF. This intertwined loop of IL-6 and VEGF becomes a vicious cycle due to chronic antigenic persistence from infections to inflammations. The data from Phase 1 review provide evidence to explain the clinical and pathologic findings in TAFRO syndrome. Atretic germinal centers, characteristic of MCD, result from the mass recruitment of centroblasts and their differentiation into plasmablasts due to chronic VEGF stimulation. IL-6 promotes differentiation of plasmablasts into plasma cells as well as maintaining their survival. We hypothesize that hyperplastic germinal centers and plasmacytosis of plasmacytic lymph node reflect predominant pathologic features during early stages of pathogenesis, while regressed germinal centers and prominent FDCs due to hyalinization of the hypervascular lymph node represent advanced pathologic features. Our findings could potentially be translated into distinguishing clinical stages of MCD. Disclosures No relevant conflicts of interest to declare.

MiR-146a in Myasthenia Gravis Thymus Bridges Innate Immunity With Autoimmunity and Is Linked to Therapeutic Effects of Corticosteroids.

Toll-like receptor (TLR)-mediated innate immune responses are critically involved in the pathogenesis of myasthenia gravis (MG), an autoimmune disorder affecting neuromuscular junction mainly mediated by antiacetylcholine receptor antibodies. Considerable evidence indicate that uncontrolled TLR activation and chronic inflammation significantly contribute to hyperplastic changes and germinal center (GC) formation in the MG thymus, ultimately leading to autoantibody production and autoimmunity. miR-146a is a key modulator of innate immunity, whose dysregulation has been associated with autoimmune diseases. It acts as inhibitor of TLR pathways, mainly by targeting the nuclear factor kappa B (NF-κB) signaling transducers, interleukin 1 receptor associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6); miR-146a is also able to target c-REL, inducible T-cell costimulator (ICOS), and Fas cell surface death receptor (FAS), known to regulate B-cell function and GC response. Herein, we investigated the miR-146a contribution to the intrathymic MG pathogenesis. By real-time PCR, we found that miR-146a expression was significantly downregulated in hyperplastic MG compared to control thymuses; contrariwise, IRAK1, TRAF6, c-REL, and ICOS messenger RNA (mRNA) levels were upregulated and negatively correlated with miR-146a levels. Microdissection experiments revealed that miR-146a deficiency in hyperplastic MG thymuses was not due to GCs, but restricted to the GC-surrounding medulla, characterized by IRAK1 overexpression. We also showed higher c-REL and ICOS mRNA levels, and lower FAS mRNA levels, in GCs than in the remaining medulla, according to the contribution of these molecules in GC formation. By double immunofluorescence, an increased proportion of IRAK1-expressing dendritic cells and macrophages was found in hyperplastic MG compared to control thymuses, along with GC immunoreactivity for c-REL. Interestingly, in corticosteroid-treated MG patients intrathymic miR-146a and mRNA target levels were comparable to those of controls, suggesting that immunosuppressive therapy may restore the microRNA (miRNA) levels. Indeed, an effect of prednisone on miR-146a expression was demonstrated in vitro on peripheral blood cells. Serum miR-146a levels were lower in MG patients compared to controls, indicating dysregulation of the circulating miRNA. Our overall findings strongly suggest that defective miR-146a expression could contribute to persistent TLR activation, lack of inflammation resolution, and hyperplastic changes in MG thymuses, thus linking TLR-mediated innate immunity to B-cell-mediated autoimmunity. Furthermore, they unraveled a new mechanism of action of corticosteroids in inducing control of autoimmunity in MG via miR-146a.

Histologic and Laboratory Characteristics of Symptomatic and Asymptomatic Castleman Disease in the Pediatric Population.

Compare the morphologic, laboratory, and clinical features of asymptomatic and symptomatic Castleman disease in the pediatric population. We reviewed clinical records and histopathology of patients with Castleman disease from 2 pediatric institutions. Of 39 patients with pediatric Castleman disease, 37 had unicentric disease, all classified with the hyaline vascular variant of Castleman disease, 8 of which were clinically symptomatic. These 8 patients demonstrated abnormal laboratory findings, including microcytic anemia, elevated erythrocyte sedimentation rate and C-reactive protein, and hypoalbuminemia. In addition, histopathologic evaluation showed that the 8 symptomatic cases had more hyperplastic germinal centers, fewer atrophic or regressed germinal centers, fewer mantle zones containing multiple germinal centers, reduced "onion skinning" of mantle zones, and fewer "lollipop" formations compared with the asymptomatic cases. This series of pediatric Castleman disease showed that lymph nodes from asymptomatic patients generally demonstrated the more classic hyaline vascular histology, whereas those with symptoms could lack or have only focal classic findings. As such, reactive lymph nodes with subtle Castleman-like features should prompt clinical correlation to ensure proper diagnosis.

Pi3k/Akt/Mtor Pathway Activity Is Increased in Lymph Node Tissue from Idiopathic Multicentric Castleman Disease Patients with Tafro Syndrome

Castleman disease (CD) describes a group of heterogeneous diseases defined by shared lymph node histopathology, including atrophic or hyperplastic germinal centers, prominent follicular dendritic cells, hypervascularization, polyclonal lymphoproliferation, and/or polytypic plasmacytosis. Unicentric CD (UCD) involves a solitary enlarged lymph node that displays CD histopathology, and patients rarely experience systemic symptoms. In contrast, multicentric CD (MCD) involves multiple regions of enlarged lymph nodes, systemic inflammation, cytopenias, and vital organ dysfunction due to a cytokine storm often including interleukin-6. MCD is caused by uncontrolled infection with Kaposi sarcoma-associated/human herpesvirus-8 (HHV-8) in ~50% of cases. The etiology of the remaining HHV-8-negative MCD cases is idiopathic (iMCD). In iMCD patients, blockade of IL-6 signaling with siltuximab, the only FDA-approved iMCD treatment, induced responses in 34% of cases in the phase II registrational trial. The large proportion of non-responders suggest that alternative pathways are responsible for driving disease pathogenesis in some patients. For these individuals, identification of molecular and cellular abnormalities for therapeutic targeting is urgently needed, particularly for those with the most severe clinical presentations. In fact, a clinical subgroup of iMCD was recently described with a very severe presentation: thrombocytopenia, anasarca, myelofibrosis, renal dysfunction, and organomegaly (iMCD-TAFRO). We previously reported increased phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling–a central pathway downstream of multiple cell surface receptors, implicated in both autoimmune and oncologic disorders–in a treatment refractory iMCD-TAFRO case that experienced an extended remission on treatment with an mTOR inhibitor. To extend these findings, herein we report immunohistochemistry for phosphorylated ribosomal protein S6 (phospho-S6), a marker of mTOR activation, in lymph node tissue from additional iMCD-TAFRO cases (n=10) and sentinel lymph nodes from breast cancer patients without evidence of metastasis (n=5). Anti-phospho-S6 (Ser235/236, Clone D57.2.2E) was used following standard protocols, and Aperio ImageScope and Image Analysis Toolkit software (color deconvolution v9 algorithm) were used to quantify pixel staining intensity in the germinal center, mantle zone, follicular and interfollicular regions. This analysis identified an increased number of pixels staining weak, medium, and strong for phospho-S6 in the interfollicular region of iMCD-TAFRO cases (p<0.005 for all comparisons) and an increased number of pixels staining weak for phospho-S6 in the germinal center (p<0.05) compared to control cases. Given that T cells are largely represented in the interfollicular region and mTOR signaling is critical to T cell proliferation, we hypothesized that the observed increase in phospho-S6 signal would occur in CD3+ cells. However, co-immunofluorescence assays for phospho-S6 and CD3 (Dako, A0452) across iMCD-TAFRO cases identified 0.08 ± 0.16% (mean ± standard deviation; n = 4 cases) of phospho-S6-positive cells as expressing CD3. In contrast, co-immunofluorescence for phospho-S6 and CD138 (Dako, Clone MI15) revealed 17.89 ± 11.26% of phospho-S6-positive cells as plasma cells. iMCD is considered an IL-6 driven disorder; however, anti-IL-6 therapy is effective in only a portion of cases. Alternative signaling pathways driving CD pathogenesis are poorly understood. This study provides the largest quantification to-date of aberrant PI3K/Akt/mTOR activity in iMCD-TAFRO, the first systematic study demonstrating increased mTOR activation in iMCD-TAFRO, and the first to identify a cell type, plasma cells. These findings are key to advancing our understanding of the pathological cell types and disrupted signaling pathways in iMCD. DisclosuresFajgenbaum:Janssen Pharmaceuticals, Inc.: Research Funding.

IL-21 Biased Alemtuzumab Induced Chronic Antibody-Mediated Rejection Is Reversed by LFA-1 Costimulation Blockade.

Despite its excellent efficacy in controlling T cell mediated acute rejection, lymphocyte depletion may promote a humoral response. While T cell repopulation after depletion has been evaluated in many aspects, the B cell response has not been fully elucidated. We tested the hypothesis that the mechanisms also involve skewed T helper phenotype after lymphocytic depletion. Post-transplant immune response was measured from alemtuzumab treated hCD52Tg cardiac allograft recipients with or without anti-LFA-1 mAb. Alemtuzumab induction promoted serum DSA, allo-B cells, and CAV in humanized CD52 transgenic (hCD52Tg) mice after heterotopic heart transplantation. Additional anti-LFA-1 mAb treatment resulted in reduced DSA (Fold increase 4.75 ± 6.9 vs. 0.7 ± 0.5; p < 0.01), allo-specific B cells (0.07 ± 0.06 vs. 0.006 ± 0.002 %; p < 0.01), neo-intimal hyperplasia (56 ± 14% vs. 23 ± 13%; p < 0.05), arterial disease (77.8 ± 14.2 vs. 25.8 ± 20.1%; p < 0.05), and fibrosis (15 ± 23.3 vs. 4.3 ± 1.65%; p < 0.05) in this alemtuzumab-induced chronic antibody-mediated rejection (CAMR) model. Surprisingly, elevated serum IL-21 levels in alemtuzumab-treated mice was reduced with LFA-1 blockade. In accordance with the increased serum IL-21 level, alemtuzumab treated mice showed hyperplastic germinal center (GC) development, while the supplemental anti-LFA-1 mAb significantly reduced the GC frequency and size. We report that the incomplete T cell depletion inside of the GC leads to a systemic IL-21 dominant milieu with hyperplastic GC formation and CAMR. Conventional immunosuppression, such as tacrolimus and rapamycin, failed to reverse AMR, while co-stimulation blockade with LFA-1 corrected the GC hyperplastic response. The identification of IL-21 driven chronic AMR elucidates a novel mechanism that suggests a therapeutic approach with cytolytic induction.

A high number of IgG4-positive plasma cells rules out nodular lymphocyte predominant Hodgkin lymphoma.

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is a subtype of Hodgkin lymphoma that frequently shows a nodal growth pattern with abundant reactive B cells in the microenvironment. Early NLPHL cases can be particularly difficult to differentiate from progressively transformed germinal centers (PTGC). Since PTGC have been described to be IgG4 associated in a relatively high proportion of cases, the aim of the present study was to determine if IgG4 immunostaining can be helpful in the differential diagnosis between NLPHL and PTGC. We furthermore aimed to learn if LP cells can express IgG4. For this purpose, 58 cases of PTGC and 56 cases of NLPHL were assessed using IgG4 immunostaining. We could confirm that a significant number of PTGC cases showed high numbers of IgG4-positive plasma cells (22/58, 38%), whereas hot spot areas of IgG4-positive plasma cells were not found in any of the NLPHL cases. In lymph node areas with the differential diagnosis of NLPHL and PTGC, IgG4 immunostaining can therefore provide a helpful diagnostic tool to rule out NLPHL when a high number of IgG4-positive plasma cells are encountered. We also assessed 13 cases with a combination of NLPHL and PTGC in the same lymph node. Five of these cases presented hot spot areas of IgG4-positive plasma cells in the PTGC regions, while no significant numbers of IgG4-positive plasma cells were observed in the NLPHL part of the lymph node. LP cells were never IgG4 positive. Furthermore, immunoglobulin heavy chain rearrangements of single IgG4-positive plasma cells were analyzed, revealing a polyclonal plasma cell population. In summary, our data suggest that IgG4 immunostaining can provide additional information in the diagnostic workup of cases with the differential diagnosis of NLPHL and PTGC. IgG4's inefficiency in clearing antigens may explain why lymph nodes with PTGC are usually strongly enlarged and develop a high number of hyperplastic germinal centers. Polyclonal immunoglobulin heavy chain rearrangements in IgG4-positive plasma cells further support the hypothesis that PTGC represent a misled immune reaction.

Characteristic Histological Findings of Asymptomatic EBV-associated Lymphoproliferative Disorders in Tonsils

Recently, an in situ hybridization (ISH) and immunohistochemical study demonstrated that Epstein-Barr virus (EBV) infection may be involved in tonsillar hypertrophy and recurrent tonsillitis in children and young adolescents. The present study was based on 630 consecutive specimens from tonsillectomies performed at the Dokkyo University School of Medicine between 2002 and May 2017. Clinical findings were obtained from hospital records. Histologically, a “pale clear zone” was characterized by hyperplastic germinal centers with ill-defined borders and interfollicular expansion. Immunohistologically, the majority of immunoblasts were CD20-positive, whereas medium to large lymphoid cells usually expressed CD3. Among 14 lesions, numerous EBV-encoded small RNA (EBER)-positive cells were detected in 10. In 7 of these 10 lesions, EBER-positive cells were detected in germinal centers as well as in the interfollicular area. Based on our results, the “pale clear zone” suggests asymptomatic EBV infection of the tonsil. The present study demonstrated that “pale clear zones” should be taken into consideration when diagnosing asymptomatic EBV-associated LPDs in the tonsils of children and young adolescents as well as in middle-aged patients.

An Activating Janus Kinase-3 Mutation Is Associated with Cytotoxic T Lymphocyte Antigen-4-Dependent Immune Dysregulation Syndrome.

Heterozygous mutations in the cytotoxic T lymphocyte antigen-4 (CTLA-4) are associated with lymphadenopathy, autoimmunity, immune dysregulation, and hypogammaglobulinemia in about 70% of the carriers. So far, the incomplete penetrance of CTLA-4 haploinsufficiency has been attributed to unknown genetic modifiers, epigenetic changes, or environmental effects. We sought to identify potential genetic modifiers in a family with differential clinical penetrance of CTLA-4 haploinsufficiency. Here, we report on a rare heterozygous gain-of-function mutation in Janus kinase-3 (JAK3) (p.R840C), which is associated with the clinical manifestation of CTLA-4 haploinsufficiency in a patient carrying a novel loss-of-function mutation in CTLA-4 (p.Y139C). While the asymptomatic parents carry either the CTLA-4 mutation or the JAK3 variant, their son has inherited both heterozygous mutations and suffers from hypogammaglobulinemia combined with autoimmunity and lymphoid hyperplasia. Although the patient’s lymph node and spleen contained many hyperplastic germinal centers with follicular helper T (TFH) cells and immunoglobulin (Ig) G-positive B cells, plasma cell, and memory B cell development was impaired. CXCR5+PD-1+TIGIT+ TFH cells contributed to a large part of circulating T cells, but they produced only very low amounts of interleukin (IL)-4, IL-10, and IL-21 required for the development of memory B cells and plasma cells. We, therefore, suggest that the combination of the loss-of-function mutation in CTLA-4 with the gain-of-function mutation in JAK3 directs the differentiation of CD4 T cells into dysfunctional TFH cells supporting the development of lymphadenopathy, hypogammaglobulinemia, and immunodeficiency. Thus, the combination of rare genetic heterozygous variants that remain clinically unnoticed individually may lead to T cell hyperactivity, impaired memory B cell, and plasma cell development resulting finally in combined immunodeficiency.

Follicular helper T cells in the pathogenesis of IgG4-related disease.

IgG4-related disease (IgG4-RD) is a recently recognized disease entity characterized by high serum IgG4 concentrations and infiltration of IgG4+ plasma cells with hyperplastic ectopic germinal centres at affected sites. Although the underlying immune mechanism of this disease remains unclear, T cells are abundantly present at affected sites and key players in IgG4-RD pathogenesis. The role of T cell subsets has been investigated thoroughly in this disease. Recent advances in this field have clarified the importance of T follicular helper cells. In this review, we describe the role of T follicular helper cells in the disease process of IgG4-RD, in particular, for IgG4 class-switching, plasmablast and plasma cell differentiation, and germinal centre formation.

International, evidence-based consensus diagnostic criteria for HHV-8–negative/idiopathic multicentric Castleman disease

AbstractHuman herpesvirus-8 (HHV-8)–negative, idiopathic multicentric Castleman disease (iMCD) is a rare and life-threatening disorder involving systemic inflammatory symptoms, polyclonal lymphoproliferation, cytopenias, and multiple organ system dysfunction caused by a cytokine storm often including interleukin-6. iMCD accounts for one third to one half of all cases of MCD and can occur in individuals of any age. Accurate diagnosis is challenging, because no standard diagnostic criteria or diagnostic biomarkers currently exist, and there is significant overlap with malignant, autoimmune, and infectious disorders. An international working group comprising 34 pediatric and adult pathology and clinical experts in iMCD and related disorders from 8 countries, including 2 physicians that are also iMCD patients, was convened to establish iMCD diagnostic criteria. The working group reviewed data from 244 cases, met twice, and refined criteria over 15 months (June 2015 to September 2016). The proposed consensus criteria require both Major Criteria (characteristic lymph node histopathology and multicentric lymphadenopathy), at least 2 of 11 Minor Criteria with at least 1 laboratory abnormality, and exclusion of infectious, malignant, and autoimmune disorders that can mimic iMCD. Characteristic histopathologic features may include a constellation of regressed or hyperplastic germinal centers, follicular dendritic cell prominence, hypervascularization, and polytypic plasmacytosis. Laboratory and clinical Minor Criteria include elevated C-reactive protein or erythrocyte sedimentation rate, anemia, thrombocytopenia or thrombocytosis, hypoalbuminemia, renal dysfunction or proteinuria, polyclonal hypergammaglobulinemia, constitutional symptoms, hepatosplenomegaly, effusions or edema, eruptive cherry hemangiomatosis or violaceous papules, and lymphocytic interstitial pneumonitis. iMCD consensus diagnostic criteria will facilitate consistent diagnosis, appropriate treatment, and collaborative research.

Follicular dendritic cell pattern in early lymphomas involving follicles.

This article reviews the most typical follicular dendritic cell (FDC) patterns displayed by early lymphomas involving follicles. In in situ follicular lymphoma, FDCs form a well-developed round (spherical) dendritic meshwork with a sharp outline. Other patterns that can be seen include contracted/distorted/disintegrated FDC meshworks. In early mantle cell lymphoma with mantle zone pattern, FDCs compose an ill-outlined and loosely arranged "centrifugal" meshwork. In marginal zone lymphoma, the FDC meshwork forms a relatively well-developed nodular meshwork with irregular outlines. In nodular lymphocyte predominant (LP) Hodgkin lymphoma, LP tumor cells are localized within an environment with prominent FDC meshwork and rare or absent LP cells outside the nodules. In angioimmunoblastic T-cell lymphoma (AITL), scattered regressed germinal centers are usually noted, although hyperplastic germinal centers are seen during the early stages of the disease (EAITL). Identifying the FDC meshwork, mainly in association with blood vessels, constitutes one of the most typical findings of the disease. In conclusion, the morphologic pattern of the FDC meshwork in early lymphomas of follicular origin is heterogenous and differs according to the lymphoma subtypes. These FDC patterns are recognizable in the most typical cases when the lymphoma is in its early stage and still maintains a follicular/nodular pattern of growth. The FDC patterns seen in the CD21 and CD23 stains contribute to the recognition of early stages of lymphomas involving follicles. Conversely, tumor cell immunostains for BCL2, cyclin D1, and other stains form the basis for the definition of lymphoma subtypes.

Atypical interfollicular hyperplasia of tonsils resembling mucosa-associated lymphoid tissue lymphoma: a clinicopathological, immunohistochemical study and epstein-barr virus findings in 12 cases.

This study attempted to clarify the clinicopathological and immunohistochemical findings and presence or absence of Epstein-Barr virus (EBV) in tonsillar atypical interfollicular hyperplasia (AIFH). A total of 597 consecutive specimens from tonsillectomies performed in Dokkyo University School of Medicine between 1999 and July 2013 were reexamined. Using formalin-fixed, paraffin-embedded sections, histological and immunohistochemical analyses, and in situ hybridization (ISH) were performed. AIFH was identified in the tonsils in 12 (2.0%) cases. These included 7 males and 5 females, aged 3 to 19 years (mean, 7). Histologically, there was expansion of the interfollicular areas by polymorphous infiltration resulting in distortion, but not obliteration of the normal tonsillar architecture. In some areas, the lymphoid follicles had hyperplastic germinal centers with ill-defined borders surrounded by sheet-like proliferation of polymorphous infiltrate showing a marginal zone distribution pattern. The infiltrate was composed of small to medium-sized (transformed) lymphocytes and immunoblasts accompanied by numerous plasma cells and plasmacytoid cells, and resembling monocytoid B cells. The numerous immunoblasts were MUM1(+), CD10(-), BCL-6(-). An ISH study demonstrated EBV-encoded small RNA-1 (EBER-1)(+) cells in 9 lesions. Moreover, in 3 lesions, numerous EBER-1(+) cells were present in germinal centers as well as in interfollicular areas. The present study indicated that EBV may cause at least a portion of tonsillar AIFH in children and adolescents. In conclusion, an atypical lymphoid infiltration resulting in distortion of tonsillar architecture with numerous MUM1(+), CD10(-), BCL-6(-) immunoblasts should raise the suspicion of a reactive process.

Aberrant immunoarchitecture distinguishes hyperplastic germinal centres in pattern 1 angioimmunoblastic T‐cell lymphoma from reactive follicles

We compare 30 biopsies each of Pattern 1 angioimmunoblastic T-cell lymphoma (AITL1) and reactive lymphoid hyperplasia (RLH) by immunohistology, in-situ hybridization for Epstein-Barr virus-encoded RNA and T-cell receptor-γ (TRG)-clonality. AITL1 cases, more often than RLH controls, were older [median ages 61 (range 23-79) vs 46 (range 11-59) years, p < 10(-4)], non-Chinese [16/30 (53%) vs 8/28 (29%), p = 0.035], presented nodally [29/30 (97%) vs 23/30 (77%), p = 0.024], showed: pan-T cell antigen attenuation [25/29 (86%) vs 5/21 (24%), p = 1.0 × 10(-5)], CD4 predominance [25/28 (89%) vs 12/23 (52%), p = 3.4 × 10(-3)], interfollicular lymphoid CD10-positivity [16/30 (53%) vs 1/29 (3%), p = 1.5 × 10(-5)], TRG clonality [16/28 (57%) vs 1/20 (5%), p = 1.4 × 10(-4)], higher maximum number of Epstein-Barr virus-encoded RNA + nuclei per 0.5-mm high-power field [median 6 (range 0-70) vs 1 (range 0-40), p = 0.012] and interfollicular Ki-67 proliferation fraction [median 40% (range 10-80%) vs 20% (range 5-40), p < 10(-4)], whereas their germinal centres (GCs) more often showed attenuation of CD10 [30/30 (100%) vs 11/29 (38%), p = 5.3 × 10(-8)] and CD57 [18/25 (72%) vs 4/22 (18%), p = 2.4 × 10(-4)] (respectively). GC-predominant PD-1 and ICOS immunoreactivity were more often seen in RLH [20/22 and 9/19 controls (91% and 47%)] than AITL1 [9/25 and 3/19 cases (36% and 16%), p = 1.0 × 10(-4) and 0.033, respectively]. Significant independent predictors against AITL1 were: solid GC CD10 immunoreactivity {p = 0.023, odds ratio (OR) for AITL1 0.01 [95% confidence interval (CI): 0.0002-0.529]}; lower interfollicular proliferation fraction [p = 0.047, OR for AITL1 1.1 (95% CI: 1.001-1.209) per % rise in Ki-67]; younger presenting age [p = 0.028, OR for AITL1 1.136 (95% CI: 1.014-1.272) per year older]. Hence, GCs and perifollicular zones in AITL1 are distinct from those in RLH.

Clinical characteristics of Mikulicz’s disease as an IgG4-related disease

Mikulicz's disease (MD) was considered to be a subtype of Sjögren's syndrome (SS), based on histopathological similarities. However, recent studies have indicated that patients with MD show high serum IgG4 concentration, and suggested that MD is one of "IgG4-related disease" and distinguishable from SS. Therefore, we clinically and histopathologically examined the disease states of MD and SS in detail. Twenty patients with Mikulicz's disease and 18 with SS were comparatively studied to determine clinical characteristics in MD patients. Sialography in MD patients did not show the "apple-tree sign" typically seen in SS. Serologically, high serum IgG4 levels but not anti-SS-A or anti-SS-B antibodies were observed in MD. SS showed lymphocytic infiltration of various subsets with atrophy or severe destruction of the acini, while MD showed selective infiltration of IgG4+ plasma cells with hyperplastic germinal centers and mild acini destruction. Corticosteroid treatment of MD reduced IgG and IgG4 levels and improved salivary function. A negative correlation between disease duration and increasing rate of salivary flow was observed in MD. These results suggested that the pathogenesis of MD might be different from those of SS. early diagnosis and treatment of MD is important for the improvement of salivary function.

Angioimmunoblastic T‐cell lymphoma with hyperplastic germinal centres (pattern 1) shows superior survival to patterns 2 and 3: a meta‐analysis of 56 cases

Angioimmunoblastic T-cell lymphoma (AITL) may present in patterns 1, 2 or 3, representing those with hyperplastic, regressed or effaced germinal centres (GCs), respectively, but the prognostic utility of this subclassification has not been previously validated. Twenty-five cases of AITL were reviewed immunohistologically and with in-situ hybridization for Epstein-Barr virus-encoded RNA and polymerase chain reaction for T-cell receptor gamma and immunoglobulin heavy chain clonality and followed for up to 120 months. Four cases had conventional hyperplastic GCs, two had floral GCs, and one had progressively transformed GCs, consistent with pattern 1 and one additional case had hyalinized GCs, consistent with pattern 2. The remaining 17 (pattern 3) cases lacked morphologically discernible GCs. The Kaplan-Meier survival distribution of pattern 1 cases (5-year survival 83%) was superior to that of pattern 2 and 3 cases [5-year-survival 36% (P = 0.0417)] only when combined with the 31 cases, seven of which were pattern 1, that Attygalle et al. had followed for up to 247 months and previously published. Furthermore, the development of B-lineage (classical Hodgkin or diffuse large-cell) lymphoma was associated exclusively with pattern 3 (P = 0.0057). Pattern 1 represents an indolent phase/grade of AITL, unassociated with the development of secondary B-lineage lymphoma and uninfluenced by treatment regimen.