A New Hierarchy of Research Evidence for Tumor Pathology: A Delphi Study to Define Levels of Evidence in Tumor Pathology.

The hierarchy of evidence is a fundamental concept in evidence-based medicine, but existing models can be challenging to apply in laboratory-based health care disciplines, such as pathology, where the types of evidence and contexts are significantly different from interventional medicine. This project aimed to define a comprehensive and complementary framework of new levels of evidence for evaluating research in tumor pathology-introducing a novel Hierarchy of Research Evidence for Tumor Pathology collaboratively designed by pathologists with help from epidemiologists, public health professionals, oncologists, and scientists, specifically tailored for use by pathologists-and to aid in the production of the World Health Organization Classification of Tumors (WCT) evidence gap maps. To achieve this, we adopted a modified Delphi approach, encompassing iterative online surveys, expert oversight, and external peer review, to establish the criteria for evidence in tumor pathology, determine the optimal structure for the new hierarchy, and ascertain the levels of confidence for each type of evidence. Over a span of 4 months and 3 survey rounds, we collected 1104 survey responses, culminating in a 3-day hybrid meeting in 2023, where a new hierarchy was unanimously agreed upon. The hierarchy is organized into 5 research theme groupings closely aligned with the subheadings of the WCT, and it consists of 5 levels of evidence-level P1 representing evidence types that merit the greatest level of confidence and level P5 reflecting the greatest risk of bias. For the first time, an international collaboration of pathology experts, supported by the International Agency for Research on Cancer, has successfully united to establish a standardized approach for evaluating evidence in tumor pathology. We intend to implement this novel Hierarchy of Research Evidence for Tumor Pathology to map the available evidence, thereby enriching and informing the WCT effectively.

Moving Forward on Tumor Pathology Research Reporting: A Guide for Pathologists From the World Health Organization Classification of Tumors Living Evidence Gap Map by Tumour Type Group.

Evidence-based medicine (EBM) can be an unfamiliar territory for those working in tumor pathology research, and there is a great deal of uncertainty about how to undertake an EBM approach to planning and reporting histopathology-based studies. In this article, reviewed and endorsed by the Word Health Organization International Agency for Research on Cancer's International Collaboration for Cancer Classification and Research, we aim to help pathologists and researchers understand the basics of planning an evidence-based tumor pathology research study, as well as our recommendations on how to report the findings from these. We introduce some basic EBM concepts, a framework for research questions, and thoughts on study design and emphasize the concept of reporting standards. There are many study-specific reporting guidelines available, and we provide an overview of these. However, existing reporting guidelines perhaps do not always fit tumor pathology research papers, and hence, here, we collate the key reporting data set together into one generic checklist that we think will simplify the task for pathologists. The article aims to complement our recent hierarchy of evidence for tumor pathology and glossary of evidence (study) types in tumor pathology. Together, these articles should help any researcher get to grips with the basics of EBM for planning and publishing research in tumor pathology, as well as encourage an improved standard of the reports available to us all in the literature.

Safety and efficacy of induction immunochemotherapy with rituximab, methotrexate, ifosfamide, and vincristine (R-MIV) in patients with primary CNS lymphoma including recent COVID-19 pandemic experience.

Clinical data on primary central nervous system (CNS) lymphoma (PCNSL) patients is mostly generated from prospective studies, and many frail real-world patients are not included. Recently,the diagnosis and treatment of PCNSL patients was confounded by the COVID-19 pandemic. In particular, treatment with high-dose cytarabine was linked to increased risk of pneumonia and virus persistence. We report on outcome of the induction regimen R-MIV (rituximab, methotrexate, ifosfamide, and vincristine) involving intensive administration of high-dose methotrexate (3.5 g/m2 ) with ifosfamide, every 2 weeks and rituximab once per week for six doses. The median age and performance status (PS) for 64 patients was 58 years and 2 (PS 3; 22%) respectively. The overall response rate by magnetic resonance imaging/computed tomography (MRI/CT) was 73% (n = 46/63), with an additional 17.5% (n = 11/63) patients without measurable disease at baseline. Grade 3-4 haematological toxicity was low for R-MIV (neutropenia: 25% and thrombocytopenia: 1%). Three patients (4.7%) died from treatment-related toxicity. Co-existence of SARS-CoV-2 infection with cytomegalovirus reactivation and the varicella-zoster virus in two patients was fatal. Fifty patients (78%) were eligible for consolidation. Median progression-free and overall survival were not reached (median follow-up: 44 months). In conclusion, the R-MIV regimen is feasible in routine practice, effective and safe, even during the COVID-19 pandemic.

Ofatumumab with iphosphamide, etoposide and cytarabine for patients with transplantation-ineligible relapsed and refractory diffuse large B-cell lymphoma.

The efficacy of salvage treatment of diffuse large B-cell lymphoma (DLBCL) patients who relapse or progress (rrDLBCL) after initial therapy is limited. Efficacy and safety of ofatumumab with iphosphamide, etoposide and cytarabine (O-IVAC) was evaluated in a single-arm study. Dosing was modified for elderly patients. Patients received up to six cycles of treatment. The primary end-point was the overall response rate (ORR). Patients were evaluated every two cycles and then six and 12 months after treatment. Other end-points included progression-free survival (PFS), event-free survival (EFS), overall survival (OS) and safety. Seventy-seven patients received salvage treatment with O-IVAC. The average age was 56.8 years; 39% had an Eastern Cooperative Oncology Group (ECOG) performance status of at least 3; 78% had disease of Ann Arbor stage 3 or 4; 58% received one or more prior salvage therapies. The ORR for O-IVAC was 54.5%. The median duration of study follow-up was 70 months. The median PFS and EFS were 16.3 months each. The median OS was 22.7 months. Age, ECOG performance status and the number of prior therapy lines were independent predictors of survival. Treatment-related mortality was 15.5%. O-IVAC showed a high response rate in a difficult-to-treat population and is an attractive treatment to bridge to potentially curative therapies.

Rituximab and Chemotherapy for Newly Diagnosed Follicular Lymphoma: Real-World Report of Polish Lymphoma Research Group.

INTRODUCTION: Follicular lymphoma (FL) is the most common type of indolent B-cell lymphoma with a favorable prognosis in the majority of patients. The induction treatment is still based on rituximab and chemotherapy, though new anti-CD20 antibody and chemo-free regimen have been recently introduced. The aim of the study was to analyze the management, outcomes, and determinants of prognosis of newly diagnosed patients with FL in real-world experience. METHODS: Data of consecutive patients diagnosed with FL in 5 years period (2011-2015) in three oncohematological centers were reviewed. Variables were compared using Mann-Whitney or χ2 test as appropriate, survival outpoints were calculated using Kaplan-Meier method. RESULTS: One hundred eighty-one patients were included in the study. The median patients' age at diagnosis was 56.6 years. Low histological grade (G1-G2) was found in 62.1% of patients and advanced clinical stage in 77.0% of patients. ECOG 0 performance status was observed in 57.1% of patients. The median follow-up was 5.91 years. Initially, 31.5% of the patients were qualified to watch-and-wait (W&W) strategy, and 84.0% of the whole patients' group received systemic treatment during the observation period. As induction treatment, 53.9% and 41.4% of patients received RCVP and RCHOP regimens, respectively; 39.8% received rituximab maintenance (RM) after first-line therapy. During follow-up, transformation to aggressive lymphoma occurred in 7.2% of patients. Median overall survival (OS) was not achieved, and median progression-free survival (PFS) was 8.28 years (95% CI; 7.35, NA), 19.6% of patients relapsed during 24 months from the start of the treatment (POD24). Median PFS for POD24 group was 1.1 years (95% CI; 0.56, 1.45) with a median OS longer than 8 years. ECOG 0, low PRIMA PI, and no POD24 were found as determinants of longer PFS and OS. CONCLUSIONS: Our data from clinical practice showed that rituximab and chemotherapy is still an effective method of FL treatment resulting in survival more than 8 years from diagnosis in most patients. RCVP protocol followed with RM is a reasonable choice for the first-line therapy especially in low/intermediate group of patients. The prognosis was significantly worse in patients with POD24. Therefore, searching for precise initial clinical and biological markers is warranted and development therapies to improve prognosis of POD24 patients.

The mutational landscape of Burkitt-like lymphoma with 11q aberration is distinct from that of Burkitt lymphoma.

The new recently described provisional lymphoma category Burkitt-like lymphoma with 11q aberration comprises cases similar to Burkitt lymphoma (BL) on morphological, immunophenotypic and gene-expression levels but lacking the IG-MYC translocation. They are characterized by a peculiar imbalance pattern on chromosome 11, but the landscape of mutations is not yet described. Thus, we investigated 15 MYC-negative Burkitt-like lymphoma with 11q aberration (mnBLL,11q,) cases by copy-number analysis and whole-exome sequencing. We refined the regions of 11q imbalance and identified the INO80 complex-associated gene NFRKB as a positional candidate in 11q24.3. Next to recurrent gains in 12q13.11-q24.32 and 7q34-qter as well as losses in 13q32.3-q34, we identified 47 genes recurrently affected by protein-changing mutations (each ≥3 of 15 cases). Strikingly, we did not detect recurrent mutations in genes of the ID3-TCF3 axis or the SWI/SNF complex that are frequently altered in BL, or in genes frequently mutated in germinal center-derived B-cell lymphomas like KMT2D or CREBBP An exception is GNA13, which was mutated in 7 of 15 cases. We conclude that the genomic landscape of mnBLL,11q, differs from that of BL both at the chromosomal and mutational levels. Our findings implicate that mnBLL,11q, is a lymphoma category distinct from BL at the molecular level.

CCND2 and CCND3 hijack immunoglobulin light-chain enhancers in cyclin D1- mantle cell lymphoma.

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation resulting in overexpression of cyclin D1. However, a small subset of cyclin D1- MCL has been recognized, and approximately one-half of them harbor CCND2 translocations while the primary event in cyclin D1-/D2- MCL remains elusive. To identify other potential mechanisms driving MCL pathogenesis, we investigated 56 cyclin D1-/SOX11+ MCL by fluorescence in situ hybridization (FISH), whole-genome/exome sequencing, and gene-expression and copy-number arrays. FISH with break-apart probes identified CCND2 rearrangements in 39 cases (70%) but not CCND3 rearrangements. We analyzed 3 of these negative cases by whole-genome/exome sequencing and identified IGK (n = 2) and IGL (n = 1) enhancer hijackings near CCND3 that were associated with cyclin D3 overexpression. By specific FISH probes, including the IGK enhancer region, we detected 10 additional cryptic IGK juxtapositions to CCND3 (6 cases) and CCND2 (4 cases) in MCL that overexpressed, respectively, these cyclins. A minor subset of 4 cyclin D1- MCL cases lacked cyclin D rearrangements and showed upregulation of CCNE1 and CCNE2. These cases had blastoid morphology, high genomic complexity, and CDKN2A and RB1 deletions. Both genomic and gene-expression profiles of cyclin D1- MCL cases were indistinguishable from cyclin D1+ MCL. In conclusion, virtually all cyclin D1- MCLs carry CCND2/CCND3 rearrangements with immunoglobulin genes, including a novel IGK/L enhancer hijacking mechanism. A subset of cyclin D1-/D2-/D3- MCL with aggressive features has cyclin E dysregulation. Specific FISH probes may allow the molecular identification and diagnosis of cyclin D1- MCL.

The whole-genome landscape of Burkitt lymphoma subtypes.

Burkitt lymphoma (BL) is an aggressive, MYC-driven lymphoma comprising 3 distinct clinical subtypes: sporadic BLs that occur worldwide, endemic BLs that occur predominantly in sub-Saharan Africa, and immunodeficiency-associated BLs that occur primarily in the setting of HIV. In this study, we comprehensively delineated the genomic basis of BL through whole-genome sequencing (WGS) of 101 tumors representing all 3 subtypes of BL to identify 72 driver genes. These data were additionally informed by CRISPR screens in BL cell lines to functionally annotate the role of oncogenic drivers. Nearly every driver gene was found to have both coding and non-coding mutations, highlighting the importance of WGS for identifying driver events. Our data implicate coding and non-coding mutations in IGLL5, BACH2, SIN3A, and DNMT1. Epstein-Barr virus (EBV) infection was associated with higher mutation load, with type 1 EBV showing a higher mutational burden than type 2 EBV. Although sporadic and immunodeficiency-associated BLs had similar genetic profiles, endemic BLs manifested more frequent mutations in BCL7A and BCL6 and fewer genetic alterations in DNMT1, SNTB2, and CTCF. Silencing mutations in ID3 were a common feature of all 3 subtypes of BL. In vitro, mass spectrometry-based proteomics demonstrated that the ID3 protein binds primarily to TCF3 and TCF4. In vivo knockout of ID3 potentiated the effects of MYC, leading to rapid tumorigenesis and tumor phenotypes consistent with those observed in the human disease.

First-line R-CVP versus R-CHOP induction immunochemotherapy for indolent lymphoma with rituximab maintenance. A multicentre, phase III randomized study by the Polish Lymphoma Research Group PLRG4.

R-CVP (cyclophosphamide, vincristine, prednisone) and R-CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone + rituximab) are immunochemotherapy regimens frequently used for remission induction of indolent non-Hodgkin lymphomas (iNHLs). Rituximab maintenance (RM) significantly improves progression-free survival (PFS) in patients with complete/partial remission (CR/PR). Here we report the final results of a randomized study comparing R-CVP to R-CHOP both followed by RM. Untreated patients in need of systemic therapy with symptomatic and progressive iNHLs including follicular (FL) and marginal zone lymphoma (MZL), mucosa-associated lymphoid tissue (MALT), small lymphocytic (SLL), and lymphoplasmacytic (LPL) lymphoma were eligible. Patients were randomized to receive R-CVP or R-CHOP for eight cycles or until complete response (CR). All patients with CR/PR (partial response) received RM 375 mg/m2 q 2 months for 12 cycles. Primary endpoint was event-free survival (EFS). Two-hundred and fifty patients [FL 42%, MZL/MALT 38%, LPL/ Waldenström Macroglobulinaemia (WM) 11%, SLL 9%] were enrolled and randomized (R-CHOP: 127, R-CVP: 123). Median age was 56 years (21-85), 44% were male, 90% were in stage III-IV, 43% of FL patients had a Follicular Lymphoma International Prognostic Index (FLIPI) score ≥3, and 33·4% of all patients had an IPI score ≥3. At the end of induction treatment, the CR/PR rate was 43·6/50·9% and 36·3/60·8% in the R-CHOP and R-CVP groups (P = 0·218) respectively. After a median follow-up of 67, 66, and 70 months, five-year EFS was 61% vs. 56% (not significant), progression-free survival (PFS) was 71% vs. 69% (not significant) and overall survival (OS) was 84% vs. 89% in the R-CHOP vs. the R-CVP arm respectively. Grade III/IV adverse events (65 vs. 22) occurred in 40 (33·1%) and 18 (15·3%) patients, P = 0·001; neutropenia in 16 (11·6%) and 4 (3·4%) patients, P = 0·017; infection in 14 (10·7%) and 3 (2·5%) patients,; P = 0·011; and a second neoplasm in three versus seven patients., in the R-CHOP and the R-CVP groups respectively. This multicentre randomized study with >five-year follow-up shows similar outcome in patients with indolent lymphoma in need of systemic therapy treated with R-CVP or R-CHOP immunochemotherapy and rituximab maintenance in both arms. The minor toxicity of the R-CVP regimen makes it a reasonable choice for induction treatment, leaving other active agents like doxorubicin or bendamustin for second-line therapy.

Expression of TP53 is associated with the outcome of MCL independent of MIPI and Ki-67 in trials of the European MCL Network.

Currently, prediction of time to treatment failure (TTF) and overall survival (OS) in mantle cell lymphoma (MCL) is based on the clinical factors included in the Mantle Cell Lymphoma International Prognostic Index (MIPI), and proliferation is assessed by Ki67. However, TP53 and SOX11 immunohistochemistry might improve risk stratification. We performed SOX11 and TP53 immunohistochemistry on the so far largest published cohort of lymphoma specimens (n = 365). All patients were treated in prospective trials of the European MCL Network. In multivariate analyses, including MIPI and Ki67, SOX11 expression was not associated with TTF, but patients with low SOX11 expression had shorter OS. On the contrary, high TP53 expression was a strong predictor of TTF and inferior OS compared with low TP53 expression in univariate and multivariate analyses adjusting for MIPI score and Ki-67 index (hazard ratio [HR], 2.0; P = .0054 for TTF, and HR, 2.1; P = .068 for OS). In particular, patients with high TP53 expression (>50% positive lymphoma cells) had a shorter TTF and poor OS independent of both MIPI score and Ki-67 index. Thus, TP53 immunohistochemistry is a suitable test for routine diagnostic practice to assess MCL prognosis.

Validation of the MCL35 gene expression proliferation assay in randomized trials of the European Mantle Cell Lymphoma Network.

Mantle cell lymphoma (MCL) is still considered incurable and the course of the disease is highly variable. Established risk factors include the Mantle Cell Lymphoma International Prognostic Index (MIPI) and the quantification of the proliferation rate of the tumour cells, e.g. by Ki-67 immunohistochemistry. In this study, we aimed to validate the prognostic value of the gene expression-based MCL35 proliferation assay in patient cohorts from randomized trials of the European Mantle Cell Lymphoma Network. Using this assay, we analysed the gene expression proliferation signature in routine diagnostic lymph node specimens from MCL Younger and MCL Elderly trial patients, and the calculated MCL35 score was used to assign MCL patients to low (61%), standard (27%) or high (12%) risk groups with significantly different outcomes. We confirm here in our prospective clinical trial cohort of MCL patients, that the MCL35 assay is strongly prognostic, providing additional information to the Ki-67 index and the MIPI. Thus, this robust assay may assist in making treatment decisions or in devising risk-adapted prospective clinical trials in the future.

A comprehensive flow-cytometry-based immunophenotypic characterization of Burkitt-like lymphoma with 11q aberration.

We previously described a subset of MYC translocation-negative aggressive B-cell lymphomas resembling Burkitt lymphoma, characterized by proximal gains and distal losses in chromosome 11. In the 2016 WHO classification, these MYC-negative lymphomas were recognized as a new provisional entity, 'Burkitt-like lymphoma with 11q aberration'. Here we present an immunophenotype analysis of Burkitt-like lymphomas with 11q aberration. Cells were acquired by fine needle aspiration biopsy from 10 young adult patients, 80% of whom presented recurrence-free 5-year survival. Twenty-three MYC-positive Burkitt lymphomas, including three carrying both MYC rearrangement and 11q aberration, served as controls. By immunohistochemistry, all Burkitt-like lymphomas with 11q aberration were CD20+/CD10+/BCL6+/BCL2-/MUM1-/MYC+/EBV-, usually LMO2+/CD44-/CD43- and sometimes CD56+, and showed high proliferation rate. By flow cytometry, Burkitt-like lymphoma with 11q aberration immunophenotypically resembled MYC-positive Burkitt lymphoma, except for significantly (adjusted P<0.001) more frequent CD38higher expression in Burkitt lymphoma (91% MYC-positive Burkitt lymphoma vs 10% Burkitt-like lymphoma with 11q aberration), more frequently diminished CD45 expression in Burkitt lymphoma (74% vs 10%), an exclusive CD16/CD56 and highly restricted CD8 expression in Burkitt-like lymphoma with 11q aberration (60% vs 0% and 40% vs 4%, respectively). We showed high diagnostic accuracy and effectiveness of flow cytometry in Burkitt lymphoma. CD16/CD56 expression without CD38higher and the lack of CD16/CD56 with CD38higher expression proves to be a reliable, fast, and cost-effective method for diagnosing 11q aberration and MYC rearrangements in CD10(+) aggressive lymphomas, respectively. In addition, we confirmed a pattern of an inverted duplication with telomeric loss of 11q, as a recurrent 11q abnormality, but one case presented alternative changes, possibly resulting in an equivalent molecular effect. Our findings reveal similarities along with subtle but essential differences in the immunophenotype of Burkitt-like lymphoma with 11q aberration and MYC-positive Burkitt lymphoma, important for the differential diagnosis, but also for understanding the pathogenesis of Burkitt-like lymphoma with 11q aberration.

Comprehensive histopathological diagnostics of aggressive B-cell lymphomas based on the updated criteria of the World Health Organisation's 2017 classification.

Revision of the fourth edition of the World Health Organisation (WHO) Classification of Haematopoietic and Lymphatic Tissues, which was published in 2017, introduced important changes updating the biology, pathology, genetics, and clinical presentation of aggressive B-cell lymphomas. High grade B-cell lymphomas (HGBLs) replaced B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, the new provisional entity Burkitt-like lymphoma with 11q aberration was identified, and some categories were upgraded, e.g. EBV-positive diffuse large B-cell lymphoma, not otherwise specified. Still the histopathological diagnostics is based on morphology and immunoprofile, but to define the HGBLs evaluation of MYC, BCL2, and BCL6 gene statuses is required. According to the presented WHO criteria, in the comprehensive histopathological diagnostics of aggressive B-cell lymphomas a highly specialised diagnostic team including a pathologist, a molecular biologist, a geneticist, a haematologist, and immunophenotyping technicians is needed.

Two neoplasms rich in small lymphocytes, B1B2 thymoma and small lymphocytic lymphoma, intermingled in one tumor mass. A case report.

We present a case of a 52-year-old man with myasthenia gravis and a mediastinal tumor who was admitted to our hospital for surgical treatment. The pathologic examination of the resected tumor revealed a very rare case of a collision tumor: a B1B2 thymoma and a small lymphocytic lymphoma. Flow cytometry of the peripheral blood revealed the presence of a small number of leukemic cells. After postoperative irradiation of the mediastinum and chemotherapy a complete response in both diseases was achieved. The case confirms that a pathologist should always be aware that two different neoplasms can coexist in rare cases.

A recurrent 11q aberration pattern characterizes a subset of MYC-negative high-grade B-cell lymphomas resembling Burkitt lymphoma.

The genetic hallmark of Burkitt lymphoma (BL) is the t(8;14)(q24;q32) and its variants leading to activation of the MYC oncogene. It is a matter of debate whether true BL without MYC translocation exists. Here, we identified 59 lymphomas concordantly called BL by 2 gene expression classifiers among 753 B-cell lymphomas. Only 2 (3%) of these 59 molecular BL lacked a MYC translocation, which both shared a peculiar pattern of chromosome 11q aberration characterized by interstitial gains including 11q23.2-q23.3 and telomeric losses of 11q24.1-qter. We extended our analysis to 17 MYC-negative high-grade B-cell lymphomas with a similar 11q aberration and showed this aberration to be recurrently associated with morphologic and clinical features of BL. The minimal region of gain was defined by high-level amplifications in 11q23.3 and associated with overexpression of genes including PAFAH1B2 on a transcriptional and protein level. The recurrent region of loss contained a focal homozygous deletion in 11q24.2-q24.3 including the ETS1 gene, which was shown to be mutated in 4 of 16 investigated cases. These findings indicate the existence of a molecularly distinct subset of B-cell lymphomas reminiscent of BL, which is characterized by deregulation of genes in 11q.

CCND2 rearrangements are the most frequent genetic events in cyclin D1(-) mantle cell lymphoma.

Cyclin D1(-) mantle cell lymphomas (MCLs) are not well characterized, in part because of the difficulties in their recognition. SOX11 has been identified recently as a reliable biomarker of MCL that is also expressed in the cyclin D1(-) variant. We investigated 40 lymphomas with MCL morphology and immunophenotype that were negative for cyclin D1 expression/t(11;14)(q13;q32) but positive for SOX11. These tumors presented clinically with generalized lymphadenopathy, advanced stage, and poor outcome (5-year overall survival, 48%). Chromosomal rearrangements of the CCND2 locus were detected in 55% of the cases, with an IG gene as partner in 18 of 22, in particular with light chains (10 IGK@ and 5 IGL@). No mutations in the phosphorylation motifs of CCND1, CCND2, or CCND3 were detected. The global genomic profile and the high complexity of the 32 cyclin D1(-) SOX11(+) MCL patients analyzed by copy number arrays were similar to the conventional cyclin D1/SOX11 MCL. 17p deletions and high Ki67 expression conferred a significantly worse outcome for the patients. This comprehensive characterization of a large series of cyclin D1(-) MCL patients indicates that these tumors are clinically and biologically similar to the conventional cyclin D1(+) MCL and provides a basis for the proper identification and clinical management of these patients.

miR expression in MYC-negative DLBCL/BL with partial trisomy 11 is similar to classical Burkitt lymphoma and different from diffuse large B-cell lymphoma.

Fast and reliable differential diagnosis of Burkitt lymphoma (BL) vs. diffuse large B cell lymphoma (DLBCL) is of major importance for therapeutic decisions and patient outcome. Aggressive B cell non-Hodgkin lymphomas (B-NHLs) that do not belong to the abovementioned entities were categorized by the current WHO lymphoma classification as "B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL" (DLBCL/BL). We have recently described a DLBCL/BL subgroup with recurrent chromosome 11q aberrations, resembling BL (B-NHLs[11q]). Here, we analyzed 102 prospectively collected fine needle aspirates from patients with aggressive B-NHLs in order to investigate the potential of microRNA (miR)-155, its precursor BIC, as well as miR-21 and miR-26a to differentiate BL from DLBCL, and from DLBCL/BL that include B-NHLs[11q]. Both BL and DLBCL/BL cases, including B-NHLs[11q], demonstrated significantly lower expression levels of miR-155/BIC, miR-21, and miR-26a compared to primary DLBCL. In conclusion, the miRs expression in B-NHLs[11q] provides a new suggestion, in addition to pathomorphological and clinical similarities between classical, i.e., MYC translocation-positive BL, and B-NHLs[11q], to recognize the B-NHLs[11q] subgroup of DLBCL/BL category as a MYC translocation-negative variant of BL in most cases, and points to the potential utility of miR-155/BIC/miR-21/miR-26a for the differential diagnosis of a heterogeneous category of DLBCL/BL.

Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue of the dura mimicking meningioma: a case report and literature review.

MALT lymphoma of the dura is a very rare type of low-grade B-cell lymphoma. Little more than 100 cases have been reported in the literature to date. We report a 43-year-old woman who was referred to hospital because of a series of three tonic-clonic seizures on the day of admission. Neurological examination revealed confusion and aphasia. Magnetic resonance imaging (MRI) showed a contrast-enhanced, broad-based lesion along the dura in the left parieto-occipital area. The suspicion of an en plaque meningioma was raised. The tumour invaded the brain parenchyma with visible extension into the brain sulci. There was a marked brain oedema surrounding the lesion and causing the midline shift 8 mm to the right. After stabilization of neurological condition (intravenous diuretics and steroids), the operation was performed. The diagnosis of dural MALT lymphoma was established. During the pathological examination, it was especially problematic to distinguish MALT lymphoma from follicular lymphoma, but the final diagnosis was MALT lymphoma. Surgical partial removal with additional R-CVP immunochemotherapy (rituximab, cyclophosphamide, vincristine and prednisone) resulted in complete remission. The follow-up period is 1 year. Our presented case of a MALT lymphoma highlights the fact that surgical partial removal with additional immunochemotherapy is an available option in these rare intracranial tumours.

Cytogenomic features of Richter transformation.

BACKGROUND: Richter transformation (RT) is the development of aggressive lymphoma in patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). This rare disease is characterised by dismal prognosis. In recent years, there has been a deeper understanding of RT molecular pathogenesis, and disruptions of apoptosis (TP53) and proliferation (CDKN2A, MYC, NOTCH1) has been described as typical aberrations in RT. RESULTS: A single-institution cohort of 33 RT patients were investigated by karyotyping, fluorescence in situ hybridization and single nucleotide polymorphism/copy number (CN) arrays. Most of RTs were typically manifested by diffuse large B-cell lymphoma, not otherwise specified, among the remaining cases one was classified as high-grade B-cell lymphoma with 11q aberrations. The most frequent alterations (40-60% of cases) were represented by MYC rearrangement/gain, deletions of TP53 and CDKN2A, IGH rearrangement and 13q14 deletion. Several other frequent lesions included losses of 14q24.1-q32.33, 7q31.33-q36.3, and gain of 5q35.2. Analysis of 13 CLL/SLL-RT pairs showed that RT arised from the CLL/SLL by acquiring of 10 ~ 12 cytogenetic or CN lesions/case, but without acquisition of loss of heterozygosity regions. Our result affirmed the higher genetic complexity in RT than CLL/SLL and confirmed the linear features of RT clonal evolution as predominant. CONCLUSIONS: Cytogenomic profile was concordant with the literature data, however the role of IGH rearrangement, 14q deletion and 5q35.2 gain need to be explored. We anticipate that further characterization of RT lesions will probably facilitate better understanding of the RT clonal evolution.

Development and Characterization of 3D Hybrid Spheroids for the Investigation of the Crosstalk Between B-Cell Non-Hodgkin Lymphomas and Mesenchymal Stromal Cells.

Purpose: B-cell non-Hodgkin lymphomas (B-NHLs) are the most common lymphoproliferative malignancy. Despite targeted therapies, the bone marrow involvement remains a challenge in treating aggressive B-NHLs, partly due to the protective interactions of lymphoma cells with mesenchymal stromal cells (MSCs). However, data elucidating the relationship between MSCs and B-NHLs are limited and inconclusive due to the lack of reproducible in vitro three-dimensional (3D) models. Here, we developed and described a size-controlled and stable 3D hybrid spheroids of Ri-1 (diffuse large B-cell lymphoma, DLBCL) and RAJI (Burkitt lymphoma, BL) cells with HS-5 fibroblasts to facilitate research on the crosstalk between B-NHL cells and MSCs. Materials and Methods: We applied the commercially available agarose hydrogel microwells for a fast, low-cost, and reproducible hybrid lymphoma/stromal spheroids formation. Standard histological automated procedures were used for formalin fixation and paraffin embedding (FFPE) of 3D models to produce good quality slides for histopathology and immunohistochemical staining. Next, we tested the effect of the anti-cancer drugs: doxorubicin (DOX) and ibrutinib (IBR) on mono-cultured and co-cultured B-NHLs with the use of alamarBlue and live/dead cell fluorescence based assays to confirm their relevancy for drug testing studies. Results: We optimized the conditions for B-NHLs spheroid formation in both: a cell line-specific and application-specific manner. Lymphoma cells aggregate into stable spheroids when co-cultured with stromal cells, of which internal architecture was driven by self-organization. Furthermore, we revealed that co-culturing of lymphoma cells with stromal cells significantly reduced IBR-induced apoptosis compared to the 3D mono-culture. Conclusion: This article provides details for generating 3D B-NHL spheroids for the studies on the lymphoma- stromal cells. This approach makes it suitable to assess in a relevant in vitro model the activity of new therapeutic agents in B-NHLs.