pan-B Cell Marker Research Articles

The Genetic Landscape of Primary Breast Marginal Zone Lymphoma Identifies a Mutational-driven Disease With Similarities to Ocular Adnexal Lymphoma.

Extranodal marginal zone lymphomas (eMZL) can occur in any organ and site of the body. Recent research has shown that they differ from organ to organ in terms of their mutational profile. In this study, we investigated a cohort of primary breast marginal zone lymphomas (PBMZL) to get a better insight into their morphologic and molecular profile. A cohort of 15 cases (14 female and 1 male) was characterized by immunohistochemistry (IHC) for 19 markers, fluorescence in situ hybridization (FISH), and high throughput sequencing (HTS) using a lymphoma panel comprising 172 genes. In addition, PCR for the specific detection of Borrelia spp. and metagenomics whole genome sequencing were performed for infectious agent profiling. Follicular colonization was observed in most cases, while lymphoepithelial lesions, though seen in many cases, were not striking. All 15 cases were negative for CD5, CD11c, and CD21 and positive for BCL2 and pan B-cell markers. There were no cases with BCL2 , BCL10 , IRF4 , MALT1 , or MYC translocation; only 1 had a BCL6 rearrangement. HTS highlighted TNFAIP3 (n=4), KMT2D (n=2), and SPEN (n=2) as the most frequently mutated genes. There were no Borrelia spp. , and no other pathogens detected in our cohort. One patient had a clinical history of erythema chronicum migrans affecting the same breast. PBMZL is a mutation-driven disease rather than fusion-driven. It exhibits mutations in genes encoding components affecting the NF-κB pathway, chromatin modifier-encoding genes, and NOTCH pathway-related genes. Its mutational profile shares similarities with ocular adnexal and nodal MZL.

Survival Trends in Diffuse Large B-Cell Lymphoma (DLBCL) in the CAR-T Cell Therapy Era Vs Pre-CAR-T Cell Therapy Era in the United States: A Population-Based Study

Background Chimeric Antigen Receptors (CARs) are novel engineered proteins that alter T-cell antigen specificity. CAR-T cell therapy is a novel form of immunotherapy that uses T-cells engineered with CARs to target tumor-associated antigens. The Food and Drug Administration (FDA) granted approval to the first CAR-T therapeutic agents in 2017. Axicabtagene ciloleucel (axicel) is one such therapy currently in use that was developed to target CD19 - a pan-B cell marker for the treatment of Diffuse Large B-Cell Lymphoma (DLBCL). 1 Previous population-based studies have shown improvements in Overall Survival (OS) after the introduction of rituximab (anti-CD20 monoclonal antibody) for the treatment of patients with DLBCL. 2 However, there is limited data on survival after the introduction of CAR-T cell therapy at the population level. With this study, we hypothesized that improvements in survival have been made in recent years with the introduction of CAR-T cell therapy for the treatment of DLBC and aim to demonstrate these outcomes. Methods We used the Surveillance, Epidemiology and End-Results (SEER-17) research program data to conduct a retrospective study of patients diagnosed with DLBCL between 2013-2020. This period was divided into two cohorts: pre-CAR-T approval (2013-2016) and post-CAR-T approval eras (2017-2020). Statistical analysis was performed using SPSS version 25. Patient characteristics were expressed as number (N) and percentages (%). Categorical variables were compared using the Chi-Square Test. The Kaplan Meier method was used to compare OS of both eras. OS was defined as the time from diagnosis to death. Cox proportional hazard regression was used to compare both cohorts and expressed as hazard ratios. A p-value ≤0.05 was considered statistically significant. Results All results are highlighted in Table 1. A total of 41219 DLBCL patients were identified between 2013 and 2020. Splitting by cohorts, 20139 DLBCL patients were identified in the pre-CAR-T cohort and 21080 DLBCL patients were identified in the post-CAR-T approval cohort. Patients in the post-CAR-T era demonstrated a higher proportion of patients ≥65 years (56.6% vs 53.7%) and distant stage disease (53.4% vs 52.1%) at the end of the time period.. The pre-CAR-T era demonstrated a higher proportion of people dead attributable to DLBCL (33% vs 23.5%). Figure 1 depicts OS for each group, with the post-CAR-T era (2017-2020) demonstrating a statistically significant higher OS (0.031). The post-CAR-T era cohort was associated with an improvement in overall survival (HR = 0.951, 95% CI: 0.913-0.990, p-value ≤0.014). Other independent prognostic variables found in our study were, ≥age 65 (HR = 2.351 95% CI: 2.251-2.455, p-value ≤0.001), male sex (HR = 1.260, 95% CI: 1.212-1.311, p-value ≤0.001), distant stage disease (HR = 1.653, 95% CI: 1.573-1.737, p-value ≤0.001), which indicated worse prognosis. Chemotherapy (HR = 0.331, 95% CI: 0.314-0.349, p-value ≤0.001) was found to be an independent prognostic factor for improvement of OS in DLBCL. Conclusion: To the best of our knowledge, our study is the first US population-based study to evaluate survival outcomes in DLBCL patients after the introduction of CAR-T cell therapy. The survival in patients in the CAR-T era improved compared with the pre-CAR-T era. A limitation of our study is that the SEER database does not contain data on the type of chemotherapy given to patients. Also, due to the relatively recent introduction of CAR-T cell therapy as a treatment modality further studies over longer time periods are required to better characterize its association with improvements in survival.

Rituximab bij auto-immune blaarziekten

Rituximab for autoimmune bullous diseases For a long time, the treatment of autoimmune blistering diseases consisted of classical immunosuppressants. Recently, biologicals, including rituximab, directed against the pan B-cell marker CD20, are used. Currently, practical guidelines for the optimal use of rituximab in the treatment of autoimmune blistering diseases are still under discussion. This literature review provides an overview of the efficacy, side effects, indication, optimal dosage and administration interval of rituximab in this population. In total, 37 publications were included, based on a systematic search strategy and selection procedure. The reported efficacy of rituximab in pemphigoid, expressed as complete remission without further therapy, ranges from 20% to 79%. For pemphigus, this amounts to 89% in the RITUX-3 study, which formed the basis for approval as first-line therapy. Side effects occur in 24% to 85% of the cases. The most important are infusion-related reactions and infections. In general, the safety profile corresponds to the known side effects of rituximab in other indications. The evidence for the practical guidelines is mainly built around pemphigus. Currently, the ‘high-dose rheumatoid arthritis protocol’ is preferred, although randomised, comparative studies are lacking. The high relapse rate justifies the need for repeated administrations. Further research should clarify whether repeated administration should be done systematically or based on different parameters. Rituximab is an effective and relatively safe therapy in autoimmune blistering diseases in both first and second line. In terms of practical use, further research is recommended.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Burkitt lymphoma (BL) is a rare but highly aggressive B-cell non-Hodgkin lymphoma. BL exhibits a characteristic immunophenotype that is positive for pan-B cell markers and CD10. The genetic hallmark of BL is the translocation of the MYC oncogene under the regulation of an immunoglobulin (IG) heavy or light chain enhancer, resulting in MYC protein overexpression. Notably, MYC translocation by itself is not sufficient for BL oncogenesis and a variety of cancer genes are recurrently mutated. BL can be divided epidemiologically but also, more recently, genetically based on driver mutations including DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Despite high survival rates, pediatric BL patients suffer from long-term side effects and relapses are usually fatal. In order to develop more targeted and less toxic therapies, a better understanding is needed of the ethology of the disease. Therefore, our aim is to characterise the cell-of-origin (COO) and dissect the life history of BL subtypes, by pinpointing when during tumorigenesis somatic mutations play a role and by identifying the rate-limiting steps of malignant transformation. Method: We collected single-cell suspensions of BL patient samples, including lymph node biopsies, bone marrow aspirates, as well as ascites and pleural fluid. To perform flow cytometry, single cells were stained with DAPI and a panel of antibodies: CD10/CD45/CD19 or CD10/CD3/CD20. Live B cells were separated with the DAPI-CD45+ CD19+ or DAPI-CD3-CD20+ phenotype. Subsequently, CD10+ B cells and CD10- B cells were sorted in 96-well plates (Figure 1). To confirm the presence or absence of the IG::MYC translocation a PCR was perfomed using patient-specific primers that flank the translocation locus. Whole genome amplification was carried out on the DNA of single cells using the Primary Template-directed Amplification (PTA) technique that was subsequently whole genome sequenced. Results: From a bone marrow sample of a 4-year-old female BL patient we found that 1% of immune cells were CD10+ B cells, while 17% of B cells were negative for the CD10 marker (Figure 2A). In the lymph node sample from the same patient, we found that 87% of B cells were CD10+ and 9% were CD10- (Figure 2B). PCR confirmed the MYC translocation in 2/6 CD10+ and 0/6 CD10- bone marrow-derived B cells (Figure 3). Keywords: Genomics, Epigenomics, and Other -Omics, Non-Hodgkin (Pediatric, Adolescent, and Young Adult), Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

CRISPR-Cas9 Genome Editing of Primary Chronic Lymphocytic Leukemia Cells

Chronic lymphocytic leukemia (CLL) is a common B-cell neoplasia that exhibits a very heterogeneous course. Over the last decade, multiple molecular studies have described the genomic, epigenomic and transcriptional landscape of the disease. These studies have led to the identification of more than 60 recurrently mutated genes as well as a large number of deregulated genes and regulatory elements. While the prognostic significance of some of these alterations is well described, their specific contributions to the pathogenesis of the disease remains largely unknown. Attempts to address this experimentally have employed genetically engineered mouse models and human cell lines. Although mouse models have been helpful to recapitulate disease aspects that can be only properly studied in vivo, significant limitations exist that prevent extrapolation of data from mice to human. Moreover, cell lines are commonly obtained from patients with end-stage, refractory diseases and are frequently EBV-positive, showing great discrepancies with primary cells, including high proliferation rate and absence of spontaneous apoptosis. In addition, they do not faithfully recapitulate the transcriptional and chromatin landscape of CLL primary cases. Thus, there is a need of better cellular models that recapitulate these disease aspects. An attractive solution to this problem would be the use of primary human CLL cells as a platform for ex vivo genetic manipulation, applying CRISPR-Cas9 genome editing technology. However, this is technically challenging, as (i) CLL cells are critically dependent on stimulatory signals coming from the tumor microenvironment, particularly in the lymph nodes (LN), and (ii) primary cells are resistant to most gene transfer methods. In order to overcome these limitations, we took advantage of a cell culture system that is reminiscent of a LN environment and induces vigorous proliferation of CLL primary cells for several weeks, allowing retroviral gene transfer with high efficiency and minimal toxicity (Mangolini, M. et al., 2022, Nat Commun, in press). To avoid undesired off-target effects induced by the constitutive expression of a CRISPR-Cas9 viral plasmid, we used transient expression systems. As an initial proof of concept, we designed sgRNA targeting the CD19 gene, a pan-B cell marker highly expressed in CLL cells, and assessed the transfection efficiency and cell viability when introducing the Cas9-CD19 sgRNA ribonucleoprotein complex using the Neon Transfection System (Thermo Fisher Scientific). Cryopreserved CLL primary cells (from peripheral blood samples) were expanded for 3 days in vitro and were subsequently electroporated with optimized conditions. Cell viability and electroporation efficiency were assessed 24 hours later and demonstrated a remarkable low percentage of apoptotic cells, with only 10% of cell death compared to non-electroporated cells (measured by flow cytometry using the LIVE/DEAD fixable Aqua dead cell stain, n= 6 cases); while high transfection efficiency was accomplished, with over 80% of cells containing the fluorescent Cas9-ATTO550 sgRNA ribonucleoprotein. Finally, 72 hours after electroporation we observed a downregulation greater than 85% of CD19 levels by flow cytometry (n=6 cases). After successful CD19 downregulation, we aimed at targeting the cell cycle regulatory protein Cyclin D2 (CCND2), which is upregulated by CLL cells at proliferation centers and the most upregulated cyclin in our cell culture system. Two independent sgRNAs were electroporated with the Cas9 protein into 3 CLL primary cases, with a minimum effect on cell viability after 24 hours. CCND2 knock-down (KD) efficiency was of 70% and 60% respectively in all cases studied, assessed by qRT-PCR. Interestingly, despite being exposed to cell culture conditions promoting vigorous proliferation, CCND2 KD blocked cell proliferation, and a cell growth reduction of 50-70% was obtained with both sgRNAs compared with non-targeting controls already at 48 hours. Altogether, these results demonstrate the feasibility to genetically modify primary CLL cells using the CRISPR-Cas9 system. This strategy overcomes the current dependency on cell lines or mouse models and represents a powerful tool to unveil the role of candidate genes involved in CLL pathogenesis.

Investigation of canine extracellular vesicles in diffuse large B-cell lymphomas.

Diffuse large B-cell lymphomas (DLBCLs) are the most common lymphoproliferative diseases in dogs. DLBCL diagnosis to date has relied on histopathological analysis; however liquid biopsies have gained attention in recent years as a source of diagnostic and prognostic information. Liquid biopsies can be a source of circulating DNA, miRNA, circulating tumour cells or extracellular vesicles (EVs). In this study EVs were isolated from the plasma of healthy dogs, and dogs with lymphoma, and adenocarcinoma by iodixanol density gradient centrifugation. These EVs were positive for the EV markers CD63 and TSG101 and the pan-B cell markers CD79a, CD21, CD45, CD20. NTA analysis revealed that the DLBCL and adenocarcinoma dogs had elevated plasma EVs relative to the healthy dogs. Furthermore, the modal size of lymphoma EVs had decreased relative to healthy dogs while adenocarcinoma EVs were unchanged. This study demonstrates that the plasma EV population is altered in canine lymphoma patients in a manner similar to previous studies on human lymphomas. The similar changes to the EV population in dogs, together with the similar pathological features and treatment protocols in canine and human non-Hodgkin lymphomas would make dogs a good comparative model for studying the role of EVs in DLBCL development and progression.

The protective effects of estrogen on biliary and liver damage are independent of ER‐β signaling in female Mdr2‐/‐ mice

BACKGROUND & AIMCholestatic liver diseases present with ductular reaction, biliary senescence, peribiliary inflammation and bridging fibrosis, and sex‐dependent differences in liver injury exist in different pathologies. The multidrug resistance 2 knockout (Mdr2‐/‐) mouse is a model of toxic bile‐induced cholangitis which show a higher degree of injury in female mice. Estrogen signals through its receptors, ER‐α and ER‐β, that form homodimers and act as transcription factors. Interestingly, ER‐α and ER‐β can have differing downstream responses. In a model of autoimmune cholestasis, inhibition of ER‐β reduces biliary damage, inflammation, immune cell infiltration and liver fibrosis in both male and female mice. We aimed to determine the role of estrogen/ER‐β signaling on biliary and liver damage in female Mdr2‐/‐ mice.METHODSStudies were performed in female wild‐type (WT) and Mdr2‐/‐ mice at 12 wks of age, with a subset of Mdr2‐/‐ mice subjected to ovariectomy (OVX, to reduce estrogen levels) or treated with PHTPP (ER‐β antagonist, 15 mg/kg BW/day by I.P. implanted minipump) for 1 wk. All studies utilized n=4 mice per group. We evaluated liver damage by H&E staining. Ductular reaction was measured by CK‐19 staining and semi‐quantification, and biliary senescence was imaged using p16 co‐staining with CK‐19 (to mark bile ducts). Peribiliary inflammation was evaluated using F4/80 (macrophage marker) staining and semi‐quantification, and qPCR for IL‐6 and TNF‐α in total liver samples. Immune cell infiltration was visualized by staining for CD3 (pan‐T cell marker) and CD20 (pan‐B cell marker). We determined liver fibrosis by (i) Sirius red staining and semi‐quantification, (ii) qPCR for TIMP1, Col1a1 and αSMA in total liver samples and (iii) co‐staining for desmin (hepatic stellate cell marker) with CK‐19. Human female control (avg. age 24.5 yr) and late‐stage, untreated PSC patient (avg. age 51.8 yr) serum samples were evaluated for estrogen levels by EIA.RESULTSOVX exacerbated liver damage, ductular reaction, biliary senescence, inflammation, immune response and liver fibrosis in female Mdr2‐/‐ mice, demonstrating a protective effect of estrogen signaling during cholangitis. Interestingly, female Mdr2‐/‐ mice treated with PHTPP (blocks ER‐β activity) had no change in any parameters of biliary damage or liver injury when compared to controls suggesting that estrogen’s protective effects are not mediated through ER‐β. Serum estrogen levels increased in female PSC samples versus control, potentially as a compensatory response or abnormal dysregulation as a consequence of injury.CONCLUSIONEstrogen may have a protective effect against biliary damage, inflammation, immune response and liver fibrosis during cholestasis. However, the protective effects are not dependent upon ER‐β signaling in female Mdr2‐/‐ mice and future work is ongoing to understand the role or ER‐α in this setting. Alteration of specific estrogen signaling mechanisms may be important for targeting cholestatic liver diseases in female patients.

Basic immunohistochemistry for lymphoma diagnosis.

Immunohistochemistry is a technique that uses antigen-antibody interactions to detect specific proteins in cells. This technique has several essential applications in lymphoma diagnosis, including identifying the cell lineage and phase of maturation, detecting specific genetic alterations, visualizing the degree of cell proliferation, and identifying therapeutic targets. CD3 is a pan T-cell marker expressed on most of the mature T/NK-cell lymphomas, except for anaplastic large cell lymphoma, whereas CD20 is a pan B-cell marker that is expressed on most of the mature B-cell lymphomas. CD79a may be a good alternative to CD20, compensating for its loss owing to the plasmocytic differentiation of tumor cells or history of rituximab administration. CD56, a neuroendocrine marker, is used as an NK cell marker in lymphoma diagnosis. Characteristic translocations occurring in follicular lymphoma (BCL2) and mantle cell lymphoma (CCND1) can be detected by the overexpression of Bcl-2 and cyclin D-1 in immunohistochemistry, respectively. Ki-67 reflects the degree of tumor cell proliferation by indicating cells in cell cycle phases other than G0. With the development of immunotherapy, several antibodies against markers such as programmed death-ligand 1 (PD-L1), CD19, and CD30 have been used as biomarkers to identify therapeutic targets. It is critical to properly fix the specimens to obtain accurate immunohistochemical results. Therefore, all processes, from tissue collection to the final pathological diagnosis, must be performed appropriately for accurate lymphoma diagnosis.

Lymphoplasmacytic lymphoma: a clinicopathological and prognostic analysis of 27 cases

Objective: To study the clinical manifestations, pathologic features, diagnosis and differential diagnosis, treatment and prognosis of lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia (LPL/WM). Methods: Twenty-seven cases of LPL from January 2016 to December 2020 at Guangdong Provincial People's Hospital were collected. The clinical data, histomorphology, immunophenotype, MYD88 L265P mutation, treatment and prognosis were analyzed retrospectively. Results: There were 19 males and 8 female patients, with median age of 63 years. The most common initial symptoms were fatigue related to anemia. Bone marrow was involved in all cases, lymphadenopathy was seen in 11 cases and splenomegaly in 10 cases. Monoclonal IgM type protein was detected in 25 cases, meeting the diagnostic criteria of WM. Microscopically, bone marrow and lymph nodes were infiltrated by small lymphocytes, plasmacytoid lymphocytes or plasma cells. The cells expressed pan B-cell markers and showed immunoglobulin light chain restriction. There was no expression of CD5, and low expression of CD23 and CD10; Ki-67 index was usually low. The positive rate of MYD88 L265P mutation was 73.9% (17/23). Most of the patients were treated with rituximab combined with alkylating agents, nucleoside analogues or immunomodulators, and the few patients with relapse or progression were treated with Ibutinib. During the 3-168 months' follow-up period, recurrence or progression were seen in nine cases. Thrombocytopenia, elevated β2-microglobulin and high-risk group were associated with recurrence or progression of the disease (P<0.05). The overall survival (OS) and progression-free survival (PFS) of the high-risk patients were significantly lower than those of the low-medium risk patients (P<0.05). Conclusions: LPL/WM is an exclusive diagnosis; the detection of MYD88 L265P mutation has high diagnostic value, but it is not specific. These cases should be assessed comprehensively for their clinical manifestation, serum IgM protein level and immunophenotype. The overall prognosis of LPL/WM is good, but there are still a small number of high-risk patients with rapid progress, and so the symptomatic patients should be diagnosed accurately and treated in a timely manner.

Geographic Variability of Nodular Lymphocyte-Predominant Hodgkin Lymphoma.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) differs from classic Hodgkin lymphoma (CHL) in terms of clinicopathologic features, including Epstein-Barr virus (EBV) association. CHL geographic variability is well known, with higher frequencies of mixed-cellularity subtype and EBV positivity in low/middle-income countries (LMICs), but there are few well-characterized series of NLPHL from LMICs. We detail clinicopathologic findings of 21 NLPHL cases received in consultation from Kenya and summarize reports of NLPHL with EBV testing published since 2000. Median age of consultation cases was 36 years, and male/female ratio was 3.2. All cases involved peripheral lymph nodes and showed at least some B-cell-rich nodular immunoarchitecture, with prominent extranodular lymphocyte-predominant (LP) cells and T-cell-rich variant patterns most commonly seen. LP cells expressed pan-B-cell markers, including strong OCT2; lacked CD30 and CD15 expression in most cases; and were in a background of expanded/disrupted follicular dendritic cell meshworks and increased T-follicular helper cells. LP cells were EBV negative in 18 cases. Historical cases showed a low rate of EBV positivity with no significant difference between LMICs and high-income countries. Unlike CHL, NLPHL shows few geographic differences in terms of clinicopathologic features and EBV association. These findings have implications for diagnosis, prognostication, and treatment of patients with NLPHL in LMICs.

A Deep Insight Into CAR-T Cell Therapy in Non-Hodgkin Lymphoma: Application, Opportunities, and Future Directions.

Non-Hodgkin’s lymphoma (NHL) is a cancer that starts in the lymphatic system. In NHL, the important part of the immune system, a type of white blood cells called lymphocytes become cancerous. NHL subtypes include marginal zone lymphoma, small lymphocytic lymphoma, follicular lymphoma (FL), and lymphoplasmacytic lymphoma. The disease can emerge in either aggressive or indolent form. 5-year survival duration after diagnosis is poor among patients with aggressive/relapsing form of NHL. Therefore, it is necessary to understand the molecular mechanisms of pathogenesis involved in NHL establishment and progression. In the next step, we can develop innovative therapies for NHL based on our knowledge in signaling pathways, surface antigens, and tumor milieu of NHL. In the recent few decades, several treatment solutions of NHL mainly based on targeted/directed therapies have been evaluated. These approaches include B-cell receptor (BCR) signaling inhibitors, immunomodulatory agents, monoclonal antibodies (mAbs), epigenetic modulators, Bcl-2 inhibitors, checkpoint inhibitors, and T-cell therapy. In recent years, methods based on T cell immunotherapy have been considered as a novel promising anti-cancer strategy in the treatment of various types of cancers, and particularly in blood cancers. These methods could significantly increase the capacity of the immune system to induce durable anti-cancer responses in patients with chemotherapy-resistant lymphoma. One of the promising therapy methods involved in the triumph of immunotherapy is the chimeric antigen receptor (CAR) T cells with dramatically improved killing activity against tumor cells. The CAR-T cell-based anti-cancer therapy targeting a pan–B-cell marker, CD19 is recently approved by the US Food and Drug Administration (FDA) for the treatment of chemotherapy-resistant B-cell NHL. In this review, we will discuss the structure, molecular mechanisms, results of clinical trials, and the toxicity of CAR-T cell-based therapies. Also, we will criticize the clinical aspects, the treatment considerations, and the challenges and possible drawbacks of the application of CAR-T cells in the treatment of NHL.

CAR T-Cells for CNS Lymphoma: Driving into New Terrain?

Simple SummaryPrimary or secondary central nervous system (CNS) lymphoma is frequently associated with a poor prognosis. CAR T-cells are being established as a relevant treatment approach in hematological B-cell malignancies. Unfortunately, most clinical studies on chimeric antigen-receptor (CAR) T-cells have excluded patients with CNS involvement but several clinical trials on CAR T-cell therapy in CNS lymphoma patients are currently ongoing. Preclinical and preliminary clinical data suggest an overall acceptable safety profile and considerable anti-tumor effects might be extrapolated for CAR T-cell therapy in CNS lymphoma.Primary CNS lymphomas (PCNSL) represent a group of extranodal non-Hodgkin lymphomas and secondary CNS lymphomas refer to secondary involvement of the neuroaxis by systemic disease. CNS lymphomas are associated with limited prognosis even after aggressive multimodal therapy. Chimeric antigen receptor (CAR) T-cells have proven as a promising therapeutic avenue in hematological B-cell malignancies including diffuse large B-cell lymphoma, B-cell acute lymphoblastic leukemia, and mantle-cell lymphoma. CARs endow an autologous T-cell population with MHC-unrestricted effectivity against tumor target antigens such as the pan B-cell marker CD19. In PCNSL, compelling and long-lasting anti-tumor effects of such therapy have been shown in murine immunocompromised models. In clinical studies on CAR T-cells for CNS lymphoma, only limited data are available and often include both patients with PCNSL but also patients with secondary CNS lymphoma. Several clinical trials on CAR T-cell therapy for primary and secondary CNS lymphoma are currently ongoing. Extrapolated from the available preliminary data, an overall acceptable safety profile with considerable anti-tumor effects might be expected. Whether these beneficial anti-tumor effects are as long-lasting as in animal models is currently in doubt; and the immunosuppressive tumor microenvironment of the brain may be among the most pivotal factors limiting efficacy of CAR T-cell therapy in CNS lymphoma. Based on an increasing understanding of CAR T-cell interactions with the tumor cells as well as the cerebral tissue, modifications of CAR design or the combination of CAR T-cell therapy with other therapeutic approaches may aid to release the full therapeutic efficiency of CAR T-cells. CAR T-cells may therefore emerge as a novel treatment strategy in primary and secondary CNS lymphoma.

Monomorphic Epitheliotropic Intestinal T-Cell Lymphoma (MEITL) Presenting As An Obstructive Pancreaticoduodenal Mass: A Case Report

Abstract Casestudy: A 74-year-old woman of Scottish descent presented with obstructive jaundice and weight loss. Imaging revealed a head of pancreas mass with extrahepatic bile duct dilation. Fine needle aspiration cytology of the mass was non-diagnostic, therefore the patient underwent a pancreaticoduodenectomy. Gross examination revealed a circumferential, necrotic lesion involving the periampullary duodenum with invasion into the pancreas and peri-pancreatic soft tissue. Microscopically, the lesion was composed of diffuse sheets of monomorphic medium-sized cells with fine chromatin, inconspicuous nucleoli, and scant pale cytoplasm. The duodenal mucosa overlying the lesion was ulcerated. Epitheliotropism was noted in the duodenal villi, crypt epithelium and pancreatic duct epithelium. The tumor showed extensive areas of necrosis and conspicuous perineural invasion. The duodenal mucosa uninvolved by the tumor showed no evidence of an underlying enteropathy, such as Celiac disease. By immunohistochemistry, the neoplastic cells were positive for CD45, CD3, CD8, CD56, TIA-1, granzyme B, TCR-BF1, and TCR-gamma, and were negative pan B-cell markers, CD4, CD5, CD25, CD30, EBER, HHV-8, and TCL-1. Molecular studies for T cell clonality revealed clonal TCR-alpha-beta and gamma-delta gene rearrangements. Given the morphologic and immunophenotypic findings, the diagnosis of monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) was rendered. This case is unique with features not typical of MEITL, namely mass-like pancreaticoduodenal presentation resulting in a Whipple resection, extensive necrosis, and perineural invasion.

Immunohistochemical Assessment of the Diagnostic Utility of PD-L1 (Clone SP142) for Methotrexate-Associated Lymphoproliferative Disorders With an Emphasis of Neoplastic PD-L1 (Clone SP142)-Positive Classic Hodgkin Lymphoma Type.

We describe results of programmed death ligand 1 (PD-L1) immunohistochemical assessment in methotrexate (MTX)-associated lymphoproliferative disorders (LPDs) and highlight the characteristics of classic Hodgkin lymphoma (CHL) type MTX-LPD. Fifty cases of MTX-LPD, including CHL type (n = 9), diffuse large B-cell lymphoma type (n = 15), and polymorphic B-cell LPD (n = 21), were investigated. Staining with anti-PD-L1 clone SP142 was exclusively found in CHL type (89%) but not in the others. Cases of CHL type MTX-LPD involved nodal disease and were associated with Epstein-Barr virus. They were histopathologically characterized by a vaguely nodular pattern, predominance of mononuclear cells, and strong expression of at least one pan-B-cell marker. Their clinical course was variable, with spontaneous regression in 5 patients, relapse in 2, and a fatal course in 1. The PD-L1 (clone SP142) workup aids the diagnostic approach to patients with MTX-LPD. CHL type MTX-LPD appears to represent a unique morphologic variant of CHL.

First Case Report of Primary Testicular Diffuse Large B-Cell Lymphoma from the Western Region of Saudi Arabia

Primary testicular lymphoma (PTL) represents 1-2% of all types of non-Hodgkin lymphomas (NHLs) and 1-10% of testicular neoplasms. Up to the best of my knowledge, this is the first case of PTL of the diffuse large B-cell lymphoma (DLBCL) in a 60-year-old man presented with a painless mass in the left testis as revealed by physical examination in a tertiary care hospital in Al-Madinah Al-Munwarah in the western region of the Kingdom of Saudi Arabia (KSA). Radiological examination revealed a large well-defined heterogeneous predominantly hypo-echoic mass with increased vascularity in the upper portion of the testis. On the other hand, histopathological examination revealed a tumor involving the whole left testis, which was large (measuring 6 3.5 3.3 cm), solid and dark red with focal areas of hemorrhage and epididymal infiltration. Immunohistochemistry showed positivity of leucocyte common antigen (LCA), pan B-cell marker (CD20) and negativity of pan T-cell marker (CD3). Other immunohistochemical markers such as CD10, placental alkaline phosphatase (PLAP), cytokeratin, vimentin, desmin and S100 protein were also negative. However, there was a marked expression of Ki67 and Bcl2 markers. Accordingly, the diagnosis of DLBCL was established. The tumor was classified as stage I according to the Ann Arbor system. The case was treated by orchiectomy followed by prophylactic anthracycline-based chemotherapy and irradiation of the contralateral testis and central nervous system.

Clinical Experience with Bispecific T Cell Engagers.

Bispecific T cell engagers are antibody constructs directed to a tumor-specific target on the one hand and to CD3-positive T cells on the other hand. Blinatumomab is a compound with specificity for the pan-B cell marker CD19. Clinical activity was tested in relapsed and refractory (R/R) non-Hodgkin's Lymphoma (NHL), R/R acute lymphoblastic leukemia (ALL), and ALL patients with minimal residual disease. Trials have already been started in de novo ALL. The most clinically relevant toxicities are neurologic events and cytokine release syndrome as with other T cell-activating therapies. The mechanisms of resistance are not fully understood. Higher leukemia load and later stage disease represent unfavorable factors. Besides, an upregulation of regulatory T cells and inhibitory molecules like PD-1/PD-L1 may have a role as the loss of target by several mechanisms. The future will show whether the use of bispecifics in ALL can change the standard treatment algorithms and whether bispecific T cell engagers will also be successfully used in other malignant entities.

Profiling the Atlantic Salmon IgM+ B Cell Surface Proteome: Novel Information on Teleost Fish B Cell Protein Repertoire and Identification of Potential B Cell Markers.

Fish immunology research is at a pivotal point with the increasing availability of functional immunoassays and major advances in omics approaches. However, studies on fish B cells and their distinct subsets remain a challenge due to the limited availability of differentially expressed surface markers. To address this constraint, cell surface proteome of Atlantic salmon IgM+ B cells were analyzed by mass spectrometry and compared to surface proteins detected from two adherent salmon head kidney cell lines, ASK and SSP-9. Out of 21 cluster of differentiation (CD) molecules identified on salmon IgM+ B cells, CD22 and CD79A were shortlisted as potential markers based on the reported B cell-specific surface expression of their mammalian homologs. Subsequent RT-qPCR analyses of flow cytometry-sorted subpopulations from head kidney leukocytes confirmed that both cd22 and cd79a genes were highly expressed in IgM+ lymphoid cells but were observed in barely detectable levels in IgM− non-lymphoid suspension and adherent cells. Similarly, significantly high cd22 and cd79a mRNA levels were observed in IgM+ or IgT+ lymphoid cells from the spleen and peritoneal cavity, but not in their corresponding IgM− IgT− non-lymphoid fractions. This suggests that the B cell restrictive expression of CD22 and CD79A extend down to the transcription level, which was consistent across different lymphoid compartments and immunoglobulin isotypes, thus strongly supporting the potential of CD22 and CD79A as pan-B cell markers for salmon. In addition, this study provides novel information on the salmon B cell surface protein repertoire, as well as insights on B cell evolution. Further investigation of the identified salmon CD molecules, including development of immunological tools for detection, will help advance our understanding of the dynamics of salmon B cell responses such as during infection, vaccination, or immunostimulation.

Down-Regulation of CD25 Antigen in Hairy Cell Leukemia Patients after Treatment

Background:Hairy Cell Leukemia (HCL) is a rare hematological malignancy, comprising only of 2% of all leukemias, with an estimated 900 new cases diagnosed each year in the United States. HCL displays a characteristic immunophenotypic profile that include pan-B cell markers including CD103, CD11c, and CD25. World Health Organization guidelines defines two forms of HCL, classic HCL (cHCL) and variant HCL (HCLv) as two distinct clinical entities. Patients with cHCL have a distinct immunophenotypic profile on their malignant leukemic cells including CD20+, CD19+, CD11c+, CD25+, CD103+, and CD123+, while the leukemic cells from patients with HCLv show CD11c+, CD20+ and CD19+, while lacking CD25 and CD123 expression. Some patients with cHCL will retain CD25 positivity while demonstrating negativity for other typical markers, herein termed atypical HCL (aHCL). Presence or absence of CD25 is an important determinant in classifying patients into cHCL and HCLv. Although it has previously been reported that CD25 expression may be lost during treatment with the targeted agent vemurafenib, we sought to identify whether this immunophenotypic change occurs following other treatment types, including standard purine nucleoside analog therapy and with targeted BTK inhibition.Methods:Adult patients (≥18 years) with a diagnosis of HCL whom had immunophenotype data collected before and after treatment between 2010 and 2018 were included in the study. Immunophenotype and morphological characteristics of initial and follow-up peripheral blood, bone marrow aspirate, and core biopsy specimens were reviewed and correlated with the treatment received.Results:We evaluated 30 HCL patients who underwent different therapies. All available specimens were reviewed and showed morphologic features characteristic for cHCL (n=26, 86.7%), and aHCL (n=4, 13.3%). The median age at HCL diagnosis was 50 years (44-76 years) with male predominance (76%). Patients with aHCL were treated with ibrutinib (n=2) and pentostatin (n=2). Patients with cHCL were treated with pentostatin (n=12), ibrutinib (n=8), vemurafenib (n=4), dabrafenib (n=1), and cladribine (n=1). Bone marrow analyses showed that all the patients had leukemic B-lymphocyte co-expression of CD19, CD20, CD103, CD11c, CD25, and CD123 prior to treatment. Some patients also had a smaller percentage of lymphocytes lacking CD25 expression along with the CD25 positive lymphocytes. Follow-up bone marrow and peripheral blood analysis showed that almost half (n=14, 46%) of treated patients had a partial or complete loss of CD25 expression regardless of the treatment type. Leukemic cells continued to express other HCL signature markers.Conclusion:Our study indicates that during the course of disease some patients display a loss of CD25 expression after therapy. This phenomenon was observed across different therapies and is not specific to the type of treatment. This is the first study to show treatment-dependent CD25 variability with pentostatin, ibrutinib and dabrafenib. Our results advocate for caution when using CD25 for the differential diagnosis of cHCL versus HCLv in treated patients. Future studies are needed in larger patient cohorts to determine the overall role and utility of CD25 in the diagnosis of cHCL and HCLv. DisclosuresLozanski:Genentech: Research Funding; Stem Line: Research Funding; BI: Research Funding; Novartis: Research Funding; Beckman: Research Funding; Coulter: Research Funding. Andritsos:Astra Zeneca: Consultancy; HCLF: Membership on an entity's Board of Directors or advisory committees.

Epstein-Barr Virus–Positive CD20- and CD45-Negative Diffuse Large B-Cell Lymphoma: A Diagnostic Challenge

Diffuse large B-cell lymphoma (DLBCL) is characterized by medium- to large-sized neoplastic cells that express a wide range of B-cell markers including CD19, CD20, CD22, and CD79a. Also, as this is a hematopoietic malignancy, there is expression of the leukocyte common antigen CD45. Lack of CD20 expression occurs in a specific rare heterogeneous subgroup of DLBCL including primary effusion lymphoma, plasmablastic lymphoma, ALK-positive large B-cell lymphoma, and large B-cell lymphoma arising in HHV8+ multicentric Castleman disease. In this article, we report a rare case of CD20- and CD45-negative Epstein-Barr virus-positive DLBCL in which the entities listed above were ruled out, thereby posing a significant diagnostic challenge. Arriving at the correct diagnosis of Epstein-Barr virus-positive DLBCL was supported by immunoreactivity for the B-cell transcription factor Oct-2 and the pan-B cell marker CD79a.

Contribution of Immunocytochemistry to the Diagnosis of Usual and Unusual Lymphoma Cases.

Some of the limitations of fine needle aspiration (FNA) in the cytodiagnosis of lymphoma include problems encountered in differentiating reactive hyperplasia from low-grade non-Hodgkin lymphoma (NHL), lower cytodiagnostic accuracy for NHL with a follicular (nodular) pattern and nodular sclerosis type of classical Hodgkin lymphoma (HL), and overlapping morphological features between T-cell-rich B-cell lymphoma (TCRBCL), anaplastic large cell lymphoma (ALCL), and HL. Immunocytochemistry may be of help in such situations. The B-cell lymphomas such as small lymphocytic lymphoma/CLL, follicular lymphoma (FL), mantle cell lymphoma (MCL), MALT lymphoma, Burkitt lymphoma (BL), and diffuse large B-cell lymphoma (DLBCL) have pan-B-cell markers (CD19, CD20, CD22, CD23, and CD79a). The FL (centrocytic), MCL, and MALT lymphoma can be differentiated with the use of a panel consisting of CD5, CD10, and CD23. In addition, FL is BCL2+ and MCL is BCL2+ as well as cyclin D1+. The DLBCL is BCL6+ in 60–90% cases. Besides pan B-cell marker, the immunocytochemical profile of BL includes CD10+, BCl6+, EBV±, and Ki67+ (100% cells). TCRBCL, a rare variant of DLBCL can be immunocytochemically differentiated from anaplastic large cell lymphoma (CD45+, CD30+, CD15‒, T±, B‒, EMA+, ALK1±) and classical HL (CD30+, CD15+, CD45‒, B‒, T‒, EMA‒). Unlike classical HL, the nodular lymphocytic predominant HL has a phenotype that includes LCA+, CD20+, CD79a+, CD15‒, and CD30‒. Whereas the immature neoplastic cells of T-lymphoblastic lymphoma (LBL) are CD3+, CD20‒, and Tdt+, the rarely encountered mature T-CLL/T-PLL are immunophenotypically CD3+, CD4+, CD5+, CD7+, CD8‒, CD20‒, CD23‒, and Tdt‒.