Type Of B-cell non-Hodgkin Lymphoma Research Articles

Pathology and Diagnosis of Mantle Cell Lymphoma

Mantle Cell Lymphoma (MCL) is a rare type of B-cell non-Hodgkin lymphoma (NHL), characterised by an aggressive clinical course and a poor prognosis that remains incurable for the majority of patients. Cyclin D1 overexpression, which results from t(11; 14)(q13; q32), is the pathogenic hallmark in MCL disease and causing cell cycle disruption. MCL has been categorized based on lymphoid malignancies in the WHO update into two significant subgroups, nodal and leukemic non-nodal MCL; each type has a particular clinical presentation and distinct molecular features. SOX11 is overexpressed in nodal MCL subtype, while the leukemic non-nodal sub-type is associated with SOX11 negativity. MCL has a wide range of differential diagnoses, including other types of low-grade lymphoma, most notably chronic lymphocytic leukaemia (CLL), follicular lymphoma (FL), marginal zone lymphoma (MZL), and lymphoblastic lymphoma (LBL). Therefore, accurate histological biopsy diagnosis is paramount in this rare subtype of NHL. MCL has a distinctive clinical presentation and particular morphological and immunophenotypic features with specific cytogenetic abnormalities. The recent advances in molecular and cytogenetic analysis have improved the accuracy of MCL diagnosis and enhanced disease prognosis. Furthermore, B cell receptor inhibitors have revolutionized MCL treatment. Therefore, an accurate diagnosis of MCL is very important since this may require an aggressive and novel targeted therapy.

Abstract A14: Detection of a biomarker for B-cell non-Hodgkin lymphomas or leukemias using circulating tumor DNA without the use of PCR or next-gen sequencing

Abstract There is a critical need for the development of cheap, safe, and accurate tools for cancer surveillance. Patients diagnosed with cancer require an accurate assessment of tumor burden through the course of their treatment, and the need for tumor surveillance is even more acute for many indolent B-cell malignancies such as follicular lymphoma and chronic lymphocytic leukemia. Over the past several years, we have developed an assay that quantifies a tumor biomarker for many different types of B-cell non-Hodgkin lymphomas or leukemias (B-NHLs). This biomarker, uracils in DNA, originates from the activity of a B-cell maturation enzyme, activation-induced deaminase (AID), which converts cytosines in DNA to uracil. AID is normally not expressed in circulating B cells or any other human tissue and hence this marker is not present in any other types of cells in the human body. We have developed an ELISA-like assay using chemicals with a fluorescence readout that can detect this biomarker in DNA with high sensitivity without PCR or NGS. We will present applications of this technology to genomic DNA from B-NHL tumors and circulating tumor DNA in blood plasma of such patients. Our data show we can detect the presence of this marker in DNA of patients with follicular lymphoma, diffuse large B-cell lymphoma, and chronic lymphocytic leukemia. Citation Format: Jessica A. Stewart, Ashok S. Bhagwat. Detection of a biomarker for B-cell non-Hodgkin lymphomas or leukemias using circulating tumor DNA without the use of PCR or next-gen sequencing [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A14.

B-cell non-Hodgkin lymphoma: importance of angiogenesis and antiangiogenic therapy

Angiogenesis is critical for the initiation and progression of solid tumors, as well as hematological malignancies. While angiogenesis in solid tumors has been well characterized, a large body of investigation is devoted to clarify the impact of angiogenesis on lymphoma development. B-cell non-Hodgkin lymphoma (B-NHL) is the most common lymphoid malignancy with a highly heterogeneity. The malignancy remains incurable despite that the addition of rituximab to conventional chemotherapies provides substantial improvements. Several angiogenesis-related parameters, such as proangiogenic factors, circulating endothelial cells, microvessel density, and tumor microenvironment, have been identified as prognostic indicators in different types of B-NHL. A better understanding of how these factors work together to facilitate lymphoma-specific angiogenesis will help to design better antiangiogenic strategies. So far, VEGF-A monoclonal antibodies, receptor tyrosine kinase inhibitors targeting VEGF receptors, and immunomodulatory drugs with antiangiogenic activities are being tested in preclinical and clinical studies. This review summarizes recent advances in the understanding of the role of angiogenesis in B-NHL, and discusses the applications of antiangiogenic therapies.

BCL-2 Proteins in Pathogenesis and Therapy of B-Cell Non-Hodgkin Lymphomas.

The ability to inhibit mitochondrial apoptosis is a hallmark of B-cell non-Hodgkin lymphomas (B-NHL). Activation of mitochondrial apoptosis is tightly controlled by members of B-cell leukemia/lymphoma-2 (BCL-2) family proteins via protein-protein interactions. Altering the balance between anti-apoptotic and pro-apoptotic BCL-2 proteins leads to apoptosis evasion and extended survival of malignant cells. The pro-survival BCL-2 proteins: B-cell leukemia/lymphoma-2 (BCL-2/BCL2), myeloid cell leukemia-1 (MCL-1/MCL1) and B-cell lymphoma-extra large (BCL-XL/BCL2L1) are frequently (over)expressed in B-NHL, which plays a crucial role in lymphoma pathogenesis, disease progression, and drug resistance. The efforts to develop inhibitors of anti-apoptotic BCL-2 proteins have been underway for several decades and molecules targeting anti-apoptotic BCL-2 proteins are in various stages of clinical testing. Venetoclax is a highly specific BCL-2 inhibitor, which has been approved by the US Food and Drug Agency (FDA) for the treatment of patients with chronic lymphocytic leukemia (CLL) and is in advanced clinical testing in other types of B-NHL. In this review, we summarize the biology of BCL-2 proteins and the mechanisms of how these proteins are deregulated in distinct B-NHL subtypes. We describe the mechanism of action of BH3-mimetics and the status of their clinical development in B-NHL. Finally, we summarize the mechanisms of sensitivity/resistance to venetoclax.

Mantle Zone Cell Lymphomas. New Opportunities for Diagnosis and Treatment (Epidemiological Research)

Introduction. Mantle cell lymphoma is a rare type of B-cell non-Hodgkin lymphoma. According to statistics the incidence of this disorder amounts to 2-3 per 100,000 people; this is about 6% of all non-Hodgkin lymphomas. It has been established that various molecular genetic characteristics of mantle cell lymphoma patients may present opportunities for a patient-specific approach to the disease prognosis and treatment strategy.Materials and methods. The paper presents a retrospective analysis of 45 mantle cell lymphoma patients treated at the GAUZ RKOD of the Ministry of Healthcare from 2015 until now. The data used in the analysis included clinical examination, lab panels, PET CT, tumour and bone marrow biopsy specimen cytology, histology and immunohistochemistry. We analysed the epidemiological data, the patients’ clinical presentation characteristics, treatment approaches, immediate and long-term outcomes.Results and discussion. We have established that the pathological process most frequently involves bone marrow (44%) and spleen (41%). The MIPI scores distribution was as follows: high in 14 (30%), medium in 20 (45%), low in 11 (41%). Ki67 was recorded at under 30% in four cases, in others it amounted to over 30%. In 2015–2017 patients were treated with the R-CHOP protocol with the following support with rituximab. PFS averaged at 20 months, 8 (17%) of patients remain in lasting remission (since 2015). In 2017 the R-BAC (high dose cytarabine for SCT candidates) and R-B (for the elderly and comorbid patients) became protocols of first line. Since 2018 eight patients have undergone auto-SCT (at the first late recurrence) as a treatment consolidation stage. As of now 13 patients have been examined in federal centres for del17p and the TP53 mutation.Conclusion. We have demonstrated the need for and the option of treatment depending on the results of molecular genetic testing of mantle cell lymphomas.

Intravascular lymphoma presenting with paraneoplastic syndrome

Intravascular lymphomatosis (IVL) is a rare type of B-cell non-Hodgkin lymphoma (BCNHL), which can mimic many other diseases. Fever, neurological symptoms, and skin findings are the most frequent clinical findings. Intravascular lymphomatosis may be associated with genetic factors and infection with human immunodeficiency virus (HIV), human herpesvirus 8 (HHV-8), human T-lymphotropic virus 1 (HTLV1), Epstein-Barr virus (EBV), and hepatitis B virus (HBV). A 50-year-old man was hospitalized with recalcitrant hyponatremia of unknown cause. He had also telangiectatic, indurated, slightly erythematous plaques on his trunk for the last 10 days. His past medical history was unremarkable, although he was a carrier of hepatitis B. Multiple skin biopsies were performed and were considered to be diagnostic of IVL. The hyponatremia was unresponsive to water restriction and hypertonic solution support but it resolved with B cell directed chemotherapy. A final diagnosis was made as syndrome of inappropriate antidiuretic hormone (SIADH) in the setting of IVL. This case had a relatively early diagnosis with just 10-days of skin lesions. Intravascular lymphomatosis is a very rare disease and is usually difficult to diagnose. An even more uncommon presentation is IVL complicated by a paraneoplastic syndrome. There are prior reported cases of SIADH in the setting of IVL. However, this case underscores the importance of evaluating patients with SIADH for potential IVL.

Naquotinib exerts antitumor activity in activated B-cell-like diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL), the most common type of B-cell non-Hodgkin lymphoma (NHL), is categorized into two major subtypes, activated B-cell-like (ABC) and germinal center B-cell-like (GCB). The ABC subtype is associated with worse prognosis than the GCB subtype using currently available therapies such as combination treatment with rituximab plus standard cytotoxic chemotherapy. The B-cell receptor (BCR) pathway is activated in ABC DLBCL, suggesting that inhibition of this pathway could provide an alternative strategy for treatment. Naquotinib is an irreversible tyrosine kinase inhibitor (TKI) originally designed to target the epidermal growth factor receptor (EGFR). As sequence alignment analysis indicates that irreversible EGFR-TKIs also inhibit Bruton's tyrosine kinase (BTK), here, we characterized the inhibitory effects of naquotinib against BTK in comparison to ibrutinib, acalabrutinib, tirabrutinib and spebrutinib. Naquotinib inhibited BTK kinase activity with similar potency to that for EGFR activating mutations. In vivo, naquotinib induced tumor regression and suppressed tumor recurrence in TMD8 and OCI-Ly10, ABC DLBCL cell line xenograft models, at a lower dose than the clinically relevant dose. Compared to other BTK inhibitors, naquotinib showed faster onset and comparable inhibition of BTK following incubation with cell lines for 3 and 20 h. In addition, naquotinib showed longer continuous inhibition of BTK following removal of the compound, lasting for at least 26 h after removal. Pharmacokinetics studies in the TMD8 xenograft model showed higher concentration and slower elimination of naquotinib in tumors than other BTK inhibitors. These data suggest that naquotinib may have therapeutic potential in ABC DLBCL patients.

Role of Angiogenesis and Anti-Angiogenic Treatment Strategies in Mantle Cell Lymphoma

Introduction: Role of angiogenesis in growth, spread and survival of malignant lymphomas remains largely unexplored. Bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), has been approved for treatment of several types of solid tumors including colorectal carcinoma or breast carcinoma, in all cases in combination with chemotherapy. Mantle cell lymphoma (MCL) is a type of B-cell non-Hodgkin lymphoma characterized by frequent extranodal involvement. Two agents approved for the therapy of MCL including lenalidomide and temsirolimus exert their anti-lymphoma activity at least partially by inhibition of angiogenesis. Our aim was to investigate role of VEGF-dependent angiogenesis on growth and spread of MCL in vivo using different murine models of MCL including patient-derived xenografts (PDXs). We also analyzed a potential anti-lymphoma synergy of a multi-level inhibition of angiogenesis achieved by bevacizumab and temsirolimus.Methods: Experimental therapies were implemented using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice purchased from Jackson Laboratory (Bar Harbor, Maine, USA). Therapies were initiated three to five days after subcutaneous (SC) or intravenous (IV) injection of MCL cells (= day 1, D1). Therapies were terminated when SC tumor reached 2 cm, or when mice developed signs of terminal disease. Mice were xenografted either with MCL cell lines (JEKO-1, HBL2) or with PDX cells (VFN-M1, VFN-M2) derived in our laboratory from patients with relapsed MCL. Both PDXs were confirmed by NGS to keep majority of somatic mutations with the primary MCL cells. Growth curves of calculated tumor volumes and overall survivals were compared in case of SC and IV-xenografted animals, respectiverly, in the treated mice compared to untreated controls. VEGF levels were analyzed in different tissues (blood, tumor lysate, spleen lysate) by commercially available ELISA kit. TaqMan Angiogenesis Array was used to compare gene expression changes associated with bevacizumab failure.Results: Bevacizumab significantly inhibited growth of SC MCL tumors all tested models (Figure 1) (JEKO-1: 0.71g ±0.24g vs 3.22g ±0.65g, p=0.0061; HBL-2: 0.95g ±0.11g vs 4.2g ±0.28g, p<0.0001; VFN-M1: 0.49g ±0.04g vs 2.18g ±0.13g, p<0.0001; VFN-M2: 0.77g ±0.07g vs 1.15g ±0.18g, p=0.0012). Unexpectedly, bevacizumab failed to prolong overall survival of IV-xenografted mice in three out of four murine models (HBL-2, JEKO-1, VFN-M1) compared to controls (p=0.89, 0.87 and 0.09, respectively), and only prolonged survival of bevacizumab-treated mice in VFN-M2-xenografted mice by as few as 3 days (Figure 2). Analysis of VEGF levels in lysates of SC tumors revealed a positive correlation between the tumor size and the VEGF level. In contrast, analysis of murine blood and spleen did not detect measureable amounts of VEGF in any case. We hypothesize that SC growth of MCL cells (in form of a lymphoma mass) is associated with hypoxia, which induces secretion of VEGF that stimulates angiogenesis and functions as a paracrine growth factor for MCL cells. SC tumor growth is therefore VEGF-stimulated and sensitive to inhibition by bevacizumab. Indeed, murine (but not human) vessels were detected by immunohistochemistry in SC tumors in all MCL models including both PDX models. In sharp contrast, systemic engraftment and spread of MCL cells (e.g. infiltration of the bone marrow or involvement of the spleen) are not associated with hypoxia, which would explain why bevacizumab failed to prolong overall survival of IV-xenografted mice. Combination of temsirolimus and bevacizumab did not lead to anti-lymphoma synergy, but was associated with additive effect only. Failure of bevacizumab monotherapy was associated with higher expression of several proangiogenic genes including angiogenin, transcription factor FOXC2 or interferon-beta I suggesting activation of alternate pro-angiogenic pathways after blockage of VEGF.Conclusions: Our data clearly indicate that all anti-angiogenic treatment approaches in malignant lymphomas have to be validated in vivo not only using SC xenografts, but also using systemic (IV) models. Our data also indicate that anti-angiogenic therapies might have auxiliary role (in combination with chemotherapy or targeted agents) in the treatment of malignant lymphomas. The gene expression data after failure of bevacizumab suggest activation of alternate pro-angiogenic pathways. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An Unusual Case of Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a rare and aggressive type of B-cell non-Hodgkin lymphoma (NHL). MCL accounts for 7% of all cases of NHL. Typically, 75% of MCL cases present with lymphadenopathy and systemic symptoms, while only 25% of cases have extranodal presentation, and of these, 15-30% involve the GI tract. Reports of MCL involving the pancreas are even less common. Herein we describe an aggressive and diffuse MCL with rare findings of both gastric and pancreatic involvement in a patient with few physical manifestations. An 84 year old male presented with generalized weakness. His medical history was significant for hereditary spherocytosis treated with splenectomy. Physical exam was notable for generalized abdominal tenderness. Lab work revealed WBC count 65.25 k/uL, hemoglobin 12.8 g/dL, MCV 101.3 fL, and calcium 12.3 mg/dL. CT imaging was remarkable for thickening of the stomach wall, gastrohepatic and peripancreatic lymphadenopathy, and a mass-like enlargement of the pancreas with edema and inflammatory changes. Endoscopic ultrasound revealed hypoechogenicity and a general enlargement of the pancreas rather than a specific mass (Fig 1). Fine needle aspiration of the pancreatic head correlated with MCL. Esophagogastroduodenoscopy confirmed diffusely thickened gastric folds (Fig 2A). Immunohistochemistry (IHC) of gastric biopsies revealed lymphocytic infiltrates (Fig 2B) positive for cyclin-D1 (Fig 2C) and SOX-11, consistent with MCL. IHC of bone marrow and fluorescence in situ hybridization showing t(11;14) were also consistent with MCL. Since diagnosis, the patient has successfully completed 5 cycles of bendamustine and rituximab chemotherapy. A restaging positron emission tomography showed near complete resolution of all activity. MCL is commonly associated with t(11;14) chromosomal translocation that results in overexpression of cyclin-D1. The SOX-11 mutation has been linked with a more aggressive form of MCL and is a poor prognostic indicator. Stomach and pancreas are two unusual extranodal sites of MCL presentation. This case describes a positive patient outcome after diagnosis of an aggressive and advanced MCL involving these rare sites. A greater understanding of the molecular pathogenesis of MCL has led to advances in targeted therapies; however, its diverse presentation remains an obstacle to establishing a standard of care. It may be that GI tract involvement in MCL is underreported and endoscopic evaluation after diagnosis is warranted.Figure: Endoscopic ultrasound (EUS) of pancreas. EUS showed hypoechogenicity, enlargement, and diffuse infiltration of pancreas without a focal mass.Figure: Gastric biopsy. (A) Diffusely thickened gastric folds seen with EGD. (B) Hematoxylin and eosin staining showing dense lymphomatous infiltrate (x400). (C) Cyclin D1 immunostaining (x200).

Venetoclax Synergizes with Radiotherapy for Treatment of B-cell Lymphomas.

Constitutive B-cell receptor signaling leads to overexpression of the antiapoptotic BCL-2 protein and is implicated in the pathogenesis of many types of B-cell non-Hodgkin lymphoma (B-NHL). The BCL-2 small-molecule inhibitor venetoclax shows promising clinical response rates in several lymphomas, but is not curative as monotherapy. Radiotherapy is a rational candidate for combining with BCL-2 inhibition, as DNA damage caused by radiotherapy increases the activity of pro-apoptotic BCL-2 pathway proteins, and lymphomas are exquisitely sensitive to radiation. We tested B-NHL responses to venetoclax combined with either external beam radiotherapy or radioimmunotherapy (RIT), which joins the selectivity of antibody targeting with the effectiveness of irradiation. We first tested cytotoxicity of cesium-137 irradiation plus venetoclax in 14 B-NHL cell lines representing five lymphoma subtypes. Combination treatment synergistically increased cell death in 10 of 14 lines. Lack of synergy was predicted by resistance to single-agent venetoclax and high BCL-XL expression. We then assessed the efficacy of external beam radiotherapy plus venetoclax in murine xenograft models of mantle cell (MCL), germinal-center diffuse large B-cell (GCB-DLBCL), and activated B-cell (ABC-DLBCL) lymphomas. In each model, external beam radiotherapy plus venetoclax synergistically increased mouse survival time, curing up to 10%. We finally combined venetoclax treatment of MCL and ABC-DLBCL xenografts with a pretargeted RIT (PRIT) system directed against the CD20 antigen. Optimal dosing of PRIT plus venetoclax cured 100% of mice with no detectable toxicity. Venetoclax combined with radiotherapy may be a promising treatment for a wide range of lymphomas Cancer Res; 77(14); 3885-93. ©2017 AACR.

A clinical prediction model for infusion-related reactions to rituximab in patients with B cell lymphomas.

Background Infusion-related reactions (IRRs) are a major adverse event of rituximab. Objective To develop a prediction model for IRRs to rituximab among patients with B cell non- Hodgkin's lymphomas (B-NHL). Setting A 1000-bed university hospital in Tokyo. Methods Patients with B-NHL treated with rituximab at our institution from 2004 to 2014 were retrospectively analysed. Chills, fever, rash, nausea, asthenia, headache, cardiovascular symptoms, and respiratory symptoms of any grade, in association with rituximab infusion, were identified as IRRs. Risk factors for IRRs to rituximab found in the intergroup analysis were subsequently evaluated by using multivariate analysis. Main outcome measure Occurrence of IRRs to rituximab. Results A total of 140 patients with various types of B-NHL, including 74% with diffuse large Bcell lymphoma, were analysed. Among them, 55 and 85 patients were assigned to the IRR group and the non-IRR group, respectively. Indolent histological subtypes, bulky disease (>10cm), B symptoms, higher serum soluble interleukin-2 receptor concentration, and bone marrow involvement were more common in the IRR group. The multivariate logistic regression analysis identified low-grade lymphomas [odds ratio (OR) 2.81, p=0.017] and bulky disease (OR 2.52, p=0.037) as independent risk factors for IRRs to rituximab. The incidence rates of IRRs to rituximab among patients with neither, one, or both of these risk factors were 26, 54, and 78%, respectively (χ2=16.4, p<0.001). Conclusions A simple combination of histopathological subtype and bulkiness of disease could predict the risk of IRRs to rituximab among patients with B-NHL.

Primary Mantle Cell Lymphoma of the Colon

Background: Mantle cell lymphoma (MCL) is an aggressive type of B-cell Non-Hodgkin lymphoma that originates from small to medium sized lymphocytes located in the mantle zone of the lymph node. Extra nodal involvement is present in the majority of cases, with a peculiar tendency to invade the gastrointestinal tract in the form of multiple lymphomatous polyposis. It has a reported frequency of between 4-9% of all gastrointestinal B-cell MCLs can be accurately diagnosed with the use of the highly specific marker Cyclin D1. The number of cases reported of primary mantle cell lymphoma of the GI tract is very limited, and even rarer in the colon. Here we present a case of primary malignant multifocal polypoid mantle cell lymphoma of the colon presenting with periumbilical pain. Case presentation: A 75-year-old female with a history of hypertension and renal artery stenosis who presented with abdominal pain worsening constipation and decreased appetite over the course of a week. She had a colonoscopy five years ago that showed a tubular adenoma in the cecum and diverticulosis. Exam showed some mild periumbilical tenderness. Colonoscopy revealed multiple atypical appearing polypoid mass lesions in the sigmoid, descending and transverse colon. The largest lesion was in rectum and had an umbilicated center. It measured 3 cm in size. Biopsies of the lesions showed primary malignant mantle cell lymphoma. The histology and immuno-histochemistry revealed prominent lymphoid infiltrate, involving the lamina propria and focally into the submucosa with positivity to CD20 B cells with co-expression of Cyclin D1 marker and CD5. The patient is currently doing well on her third cycle of VR-CAP (Bortezomib plus Rituximab, Cyclophosphamide, Doxorubicin and Prednisone) therapy.Figure 1Figure 2Figure 3Conclusion: This is a rare case of primary multifocal colonic mantle cell lymphoma presenting with constipation, abdominal pain and a relatively recent benign colonoscopy. Our case highlights the fact that this rare cancer can present with little specific physical findings and the polyps have an atypical polypoid fleshy multifocal appearance. It also highlights the importance of pattern recognition and differentiation from other polypoid lesions during colonoscopy.

Single-agent cytarabine is insufficient for the treatment of human mantle cell lymphoma in mouse xenograft model.

Mantle cell lymphoma is an aggressive type of B-cell non-Hodgkin lymphoma with adverse prognosis. It was demonstrated that alternation of CHOP and DHAP chemotherapy improved outcome of mantle cell lymphoma patients. However, which components of DHAP, cisplatin, cytarabine, or both, were responsible for the improved outcome remained unclear. To answer this question, antitumor efficacies of equally toxic doses of cytarabine, cisplatin, and three different combinations were compared in vivo using mouse xenograft models of mantle cell lymphoma. We demonstrated that cisplatin, alone or with cytarabine, is significantly superior to single-agent cytarabine in both eliminating lymphoma cells and suppressing their proliferation rate.

Level of PAX5 in differential diagnosis of non-Hodgkin's lymphoma.

Background & objectives:The PAX5, a paired box transcription factor and B-cell activator protein (BSAP), activates B-cell commitment genes and represses non-B-cell lineage genes. About 14 transcript variants of PAX5 have been observed in human. Any alteration in its expression pattern leads to lymphogenesis or associated diseases and carcinogenesis in non-lymphoid tissues. Its mechanisms of function in pathophysiology of non-Hodgkin's lymphoma (NHL) are unclear. This study was intended to explore influence of PAX5 in cascade of NHL pathogenesis and diagnosis.Methods:Samples of 65 patients were evaluated by immunohistochemical staining for cellular localization of PAX5, CD19, CD3, cABL, p53, Ras and Raf and by TUNEL assay, RNA-isolation and reverse transcriptase (RT)-PCR, Western blot analysis, and lactate dehydrogenase (LDH) specific staining.Results:B-cell type NHL patients were positive for PAX5, p53, Ras, CD19, Raf and CD3. All of them showed TUNEL-positive cells. The differential expression pattern of PAX5, CD19, p53, CD3, ZAP70, HIF1α, Ras, Raf and MAPK (mitogen-activated protein kinase) at the levels of transcripts and proteins was observed. The LDH assay showed modulation of LDH4 and LDH5 isoforms in the lymph nodes of NHL patients.Interpretation & conclusions:The histological observations suggested that the patients represent diverse cases of NHL like mature B-cell type, mature T-cell type and high grade diffuse B-cell type NHL. The findings indicate that patients with NHL may also be analyzed for status of PAX5, CD19 and ZAP70, and their transcriptional and post-translational variants for the differential diagnosis of NHL and therapy.

Clinical features of patients with nodal marginal zone lymphoma compared to follicular lymphoma: similar presentation, but differences in prognostic factors and rate of transformation

Nodal marginal zone lymphoma (NMZL) is a rare type of B-cell non-Hodgkin lymphoma. This study assessed the clinical features of 56 patients with NMZL in comparison to 46 patients with follicular lymphoma (FL). Patients with NMZL and FL had a largely similar clinical presentation, but patients with FL had a higher disease stage at presentation, more frequent abdominal lymphadenopathy and bone marrow involvement, and showed more common transformation into diffuse large B-cell lymphoma (DLBCL) during the course of disease. Overall survival and event-free survival were similar for patients with NMZL and FL, but factors associated with worse prognosis differed between the two groups. Transformation into DLBCL was associated with a significantly poorer outcome in both groups, but the phenotypes were different: DLBCL arising in FL was mainly of germinal center B-cell phenotype, whereas DLBCL arising in NMZL was mainly of non-germinal center B-cell phenotype.

Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells.

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.

Observational Study Alternating R-CHOP21 and R-Cytarabine (3+3 Cycles) for Patients with Newly Diagnosed Mantle Cell Lymphoma Not Eligible for High-Dose Therapy: A Czech Lymphoma Study Group Trial Preliminary Results

Abstract Background: Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma characterized by frequent relapses and adverse prognosis. Implementation of high-dose cytarabine (HDAC) and incorporation of anti-CD20 antibody rituximab (R) into induction and maintenance, and consolidation with high-dose therapy and autologous stem cell transplantation (HDT-ASCT) significantly improved pt´s outcome. Therapy of the elderly or comorbid (not eligible for HDT-ASCT) is largely based on R-CHOP or R-bendamustin induction with or without R maintenance. Minimal residual disease (MRD) assessment by quantitative PCR (qPCR) from peripheral blood or bone marrow (BM) has emerged as a powerful molecular marker of outcome. Based on the results of the younger pts some centers adopted the use of HDAC in elderly as well, but no data has been presented yet. Aim: We initiated the observational study as a non-intervention, multi-center trial with the primary objectives to prospectively evaluate efficacy of alternating R-CHOP21 and R-HDAC (1 or 2 g / m2, 2 doses a 24 hours) in newly diagnosed MCL pts not eligible for HDT-ASCT. Methods: Primary endpoints were overall response rate (ORR) by PET-CT, and MRD assessment by qPCR after completion of induction. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and toxicity. The choice between 1 and 2 g / m2 cytarabine was left at investigator´s discretion, as well as the rituximab maintenance. Patients with known cardiac co-morbidity could be treated with R-COEP (etoposid 50 mg / m2, D1-D3) instead of R-CHOP. Exclusion criteria included eligibility to HDT-ASCT, ECOG≥ 4, and CNS involvement. Results: Altogether 61 pts (38 men and 23 women, ratio 1.7:1) were enrolled into the study between 16-JUN-2011 and 18-MAR-2014. Median age was 70 years both for men and women. 91.5%, and 80% pts presented with stage 3/4 disease, and infiltration of BM, resp. According to the MIPI, 55.7%, 39.4%, and 4.9% pts had high, intermediate and low risk disease, resp. B-symptoms were recorded in 33.3% of pts. 76.8% and 24.2% pts were diagnosed from the lymph node, and trephine biopsy, resp. 62.8%, 27.9%, and 9.3% pts presented with classical, pleomorphic and blastoid variant MCL, resp. Ki67/MIB1 ≥ 30% was observed in 38% of pts. Bulky disease &gt;5cm, and &gt;10cm, was noticed in 31% and 13% pts. Spleen involvement was observed in 53% pts. Extra-nodal involvement other than BM was histologically confirmed in 11.5% pts. R-CHOP was used in 88% pts (12% pts received R-COEP), 86% pts received 2g / m2 cytarabine (14% pts 1g / m2). 86.3% pts with response were treated with R maintenace. Only one patient was excluded from the study due to unacceptable toxicity. 57.7% pts developed grade 3/4 hematologic toxicity (neutropenia, anemia or thrombocytopenia). Grade 3/4 non-hematologic toxicity occurred in 27.3% pts. All pts had PET-CT restaging after completion of induction. Overall response rate (CR+PR) reached 91.7%. CR and PR rate by PET was 76.7%, and 15%, resp. SD, and progression on therapy was noticed in 3.3%, and 5.0%, resp. Samples for MRD assessment were collected from 41 out of 54 pts, who completed induction and achieved response (CR or PR). At the time of abstract submission 21, 7, and 7 (out of 36 so far evaluated pts) were MRD negative, MRD positive-not quantifiable, and MRD positive-quantifiable, resp. With the median follow-up 19.1 month, there were 7 progressions and 6 deaths. 2-year PFS and OS probability were 83.9% and 88.1%. Conclusion: Alternation of R-CHOP and R-HDAC in newly diagnosed elderly or co-morbid MCL pts represents a promising, very effective and well-tolerated treatment approach that induces high ORR, and MRD negativity. Grant Support: IGA-MZ NT/13072-4, PRVOUK-27/LF1/1 Disclosures No relevant conflicts of interest to declare.

Abstract 647: CD20-specific Engineered Toxin Body demonstrates direct cell kill of multiple B-cell Non-Hodgkin's lymphoma types

Abstract The B-cell membrane protein CD20 is expressed by both benign and neoplastic lymphocytes. It has been a superb target for immunotherapies targeting B-cell derived diseases. There are several anti-CD20 antibodies which have been developed for B-cell lymphoma therapy. The most successful example of this targeted approach is rituximab, which is approved for front and second line therapy of Non-Hodgkin lymphoma (NHL). However, rituximab has limited direct cell-kill effects, acting primarily through indirect immune responses induced by antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC). While CD20-targeted radiotherapeutics have shown promise, their administrative challenges have curtailed their clinical utility. Thus, a therapeutic approach with direct cell-kill activity against CD20-expressing cells would be clinically useful. We have developed an engineered toxin body (ETB) comprising a CD20 scFv and a modified Shiga-like Toxin A (MT-3724) capable of specifically recognizing and killing CD20 expressing cells. MT-3724 is selectively cytotoxic against B-cell NHL cell lines and against NHL cells obtained from patients. It demonstrated potent cytotoxicity against Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma with IC (50) values ranging from 1.4 nM to 12 nM. Through immunofluorescent tracking, MT-3724 internalizes within one hour after binding to CD20 (+) B cells. To our knowledge, this is the first example of efficient internalization of an immunotoxin via CD20. Consistent with the in vitro activity, MT-3724 demonstrated anti-tumor efficacy in mice-bearing NHL cell line xenografts. These results show that MT-3724 is a promising targeted therapeutic agent against CD20 positive B-cell lymphomas and is currently under further development. Citation Format: Sangeetha Rajagopalan, Rachel Wirth, Jennifer Erdman, Kari Grant, Brigitte Brieschke, Rodney Flores, William Null, Jen-Sing Liu, Erin K. Willert, Jack P. Higgins. CD20-specific Engineered Toxin Body demonstrates direct cell kill of multiple B-cell Non-Hodgkin's lymphoma types. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 647. doi:10.1158/1538-7445.AM2014-647

Mouse models of mantle cell lymphoma, complex changes in gene expression and phenotype of engrafted MCL cells: implications for preclinical research

Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma (NHL) associated with poor prognosis. Animal models of MCL are scarce. We established and characterized various in vivo models of metastatic human MCL by tail vein injection of either primary cells isolated from patients with MCL or established MCL cell lines (Jeko-1, Mino, Rec-1, Hbl-2, and Granta-519) into immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. MCL infiltration was assessed with immunohistochemistry (tissues) and flow cytometry (peripheral blood). Engraftment of primary MCL cells was observed in 7 out of 12 patient samples. The pattern of engraftment of primary MCL cells varied from isolated involvement of the spleen to multiorgan infiltration. On the other hand, tumor engraftment was achieved in all five MCL cell lines used and lymphoma involvement of murine bone marrow, spleen, liver, and brain was observed. Overall survival of xenografted mice ranged from 22±1 to 54±3 days depending on the cell line used. Subsequently, we compared the gene expression profile (GEP) and phenotype of the engrafted MCL cells compared with the original in vitro growing cell lines (controls). We demonstrated that engrafted MCL cells displayed complex changes of GEP, protein expression, and sensitivity to cytotoxic agents when compared with controls. We further demonstrated that our MCL mouse models could be used to test the therapeutic activity of systemic chemotherapy, monoclonal antibodies, or angiogenesis inhibitors. The characterization of MCL murine models is likely to aid in improving our knowledge in the disease biology and to assist scientists in the preclinical and clinical development of novel agents in relapsed/refractory MCL patients.

Downregulation of deoxycytidine kinase in cytarabine-resistant mantle cell lymphoma cells confers cross-resistance to nucleoside analogs gemcitabine, fludarabine and cladribine, but not to other classes of anti-lymphoma agents.

BackgroundMantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. Implementation of high-dose cytarabine (araC) into induction therapy became standard-of-care for all newly diagnosed younger MCL patients. However, many patients relapse even after araC-based regimen. Molecular mechanisms responsible for araC resistance in MCL are unknown and optimal treatment strategy for relapsed/refractory MCL patients remains elusive.MethodsFive araC-resistant (R) clones were derived by long-term culture of five MCL cell lines (CTRL) with increasing doses of araC up to 50 microM. Illumina BeadChip and 2-DE proteomic analysis were used to identify gene and protein expression changes associated with araC resistance in MCL. In vitro cytotoxicity assays and experimental therapy of MCL xenografts in immunodeficient mice were used to analyze their relative responsiveness to a set of clinically used anti-MCL drugs. Primary MCL samples were obtained from patients at diagnosis and after failure of araC-based therapies.ResultsMarked downregulation of deoxycytidine-kinase (DCK) mRNA and protein expression was identified as the single most important molecular event associated with araC-resistance in all tested MCL cell lines and in 50% primary MCL samples. All R clones were highly (20-1000x) cross-resistant to all tested nucleoside analogs including gemcitabine, fludarabine and cladribine. In vitro sensitivity of R clones to other classes of clinically used anti-MCL agents including genotoxic drugs (cisplatin, doxorubicin, bendamustine) and targeted agents (bortezomib, temsirolimus, rituximab) remained unaffected, or was even increased (ibrutinib). Experimental therapy of immunodeficient mice confirmed the anticipated loss of anti-tumor activity (as determined by overall survival) of the nucleoside analogs gemcitabine and fludarabine in mice transplanted with R clone compared to mice transplanted with CTRL cells, while the anti-tumor activity of cisplatin, temsirolimus, bortezomib, bendamustine, cyclophosphamide and rituximab remained comparable between the two cohorts.ConclusionsAcquired resistance of MCL cells to araC is associated with downregulation of DCK, enzyme of the nucleotide salvage pathway responsible for the first phosphorylation (=activation) of most nucleoside analogs used in anti-cancer therapy. The data suggest that nucleoside analogs should not be used in the therapy of MCL patients, who relapse after failure of araC-based therapies.