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 CD38 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 CD38 and the lack of CD16/CD56 with CD38 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.

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.

Primary central nervous system lymphoma (PCNSL) is a rare, highly aggressive, extranodal form of non-Hodgkin lymphoma, predominantly diagnosed as primary diffuse large B-cell lymphoma of the central nervous system (CNS DLBCL). Fast and precise diagnosis of PCNSL is critical yet challenging. microRNAs, important regulators in physiology and pathology are potential biomarkers. In 131 patients with CNS DLBCL and with non-malignant brain lesions (n-ML), miR-21, miR-19b and miR-92a, miR-155, miR-196b, miR-let-7b, miR-125b, and miR-9 were examined by RT-qPCR in brain biopsy samples (formalin-fixed paraffin-embedded tissues, FFPET; CNS DLBCL, n = 52; n-ML, n = 42) and cerebrospinal fluid samples (CSF; CNS DLBCL, n = 30; n-ML, n = 23) taken for routine diagnosis. FFPET samples were split into study and validation sets. Significantly higher CSF levels of miR-21, miR-19b, and miR-92a were identified in PCNSL but not in n-ML, and differentiated PCNSL from n-ML with 63.33% sensitivity and 80.77% specificity. In FFPETs, miR-155 and miR-196b were significantly overexpressed and miR-let-7b, miR-125b, and miR-9 were downregulated in PCNSL as compared to n-ML. Combined miR-155 and miR-let-7b expression levels in FFPETs discriminated PCNSL and n-ML with a 97% accuracy. In conclusion, tissue miR-155, miR-196b, miR-9, miR-125b, and miR-let-7b expression profiles differentiate PCNSL from n-ML. PCNSL CSFs and the relevant biopsy samples are characterized by specific, different microRNA profiles. A logistic regression model is proposed to discriminate between PCNSL and non-malignant brain lesions. None of the examined microRNAs influenced overall survival of PCNSL patients. Further ongoing developments involve next generation sequencing-based profiling of biopsy and CSF samples.

Background: Burkitt lymphoma (BL) is characterized by a non-specific morphology and immunophenotype, a high proliferation rate, MYC rearrangements (MYC +), and by a simple karyotype. However, 5% of BL cases have no MYC rearrangements (MYC -) detectable by FISH. It is a matter of debate whether a true MYC (-) BL does exist.The WHO 2008 classification does not clearly define MYC (-) BL cases, and such cases are often misdiagnosed and treated as diffuse large B-cell lymphoma (DLBCL). We have previously described a provisional category of aggressive B-cell lymphoma unclassifiable (BCLU) with recurrent chromosome 11q aberrations, referred to as B-NHL(11q), with clinical, pathomorphological, and gene expression profile features typicalof BL,but MYC (-). B-NHLs(11q) carry proximal gains and telomeric losses of 11q. Karyotyping (CC) and FISH defined the gain region as dup(11)(q23q13) involving CCND1, ATM and KMT2A. As we have recently shown, BL and B-NHL(11q) express different levels of CD38 and CD16&CD56, and both have lower levels of miRNA-155, -21 and -26a than DLBCL. Here we describe a series of BL patients with a set of critical 11q aberrations and propose a diagnostic algorithm for a rapid work-up. Methods: Within a group of 82 BL cases diagnosed and treated with the BL protocol at our institution, we identified 15 cases of B-NHL(11q) with BL features and 11q aberrations: MYC (-) in 11(male/female 10/1, median age [range] 24 [18-62]) and MYC (+) in 4 cases (male/female 3/1, median age [range] 36.5 [20-82]). In MYC (-) pts, the disease was confined to a single site in 82%, was bulky (>7 cm) in 64% with diameter >20 cm in 45% of cases. BL, BCLU and DLBCL diagnosis according to WHO 2008 classification was based on histopathological/immunohistochemical examination (HP/IHC), CC, FISH, and clinical characteristics in all pts. For the final evaluation, the flow cytometry (FCM) immunophenotype, array comparative genomic hybridization (aCGH) data, and miRNA expression was assessed on samples obtained by the fine needle aspiration biopsy (FNAB). In the B-NHL(11q) cases we identified 11q duplication, dup(11q), with an inversion (inv) of the duplicated region and a deletion of its telomeric region, referred to as critical set of 11q aberrations, as opposed to non-critical aberration set that did not involve all three changes. B-NHL(11q) cells were evaluated with the panel of antibodies by IHC (CD20/CD10/BCL6/ BCL2/MUM1/MYC/Ki-67/CD43/CD44), and by FCM with CD (19, 20, 22, 23, 52, 79β, 81, 5, 25, 38, 43, 44, 45, 16&56, 56, 52, 62L, 71, 200), FMC7, HLADR, and BCL2. All B-NHL(11q) cases were evaluated by CC, FISH (MYC, BCL2, BCL6, CCND1, ATM, KMT2A and telomeric 11q) and aCGH. The relative positions of CCND1, ATM, and KMT2A within a duplicated region on the aberrant chromosome 11 were used to identify inversions. Results: A median follow-up of MYC (-) B-NHL(11q) pts treated with the BL regimen was 30 months, and 2-yr OS was 72% (95% CI: 45%, 99%). In 53% of B-NHL(11q) pts tingible body macrophages were less pronounced than in classic BL. All MYC (-) and MYC (+) B-NHLs(11q) cases presented the same phenotype and Ki-67 index of 100% and met the IHC criteria for BL. In MYC (-) and MYC (+) B-NHLs(11q) pts, all with a simple or less simple karyotype, we confirmed a critical or non-critical 11q aberrations in 10 and 5 pts, respectively. In 87% of pts we identified dup(11q), of two types: the larger part between 11q12.1 and 11q24.3 bands, and the smaller part between 11q22.3 and 11q24.1, with an additional multiplication of KMT2A inside the duplication region. In 13% of cases an inv without dup(11q) was detected. We found an inv of dup(11q) region in 73% of all cases, and no inv in dup(11q) in 2 MYC (+) cases only. Bulky tumors of >20 cm correlated with increased KMT2A copy number in B-NHLs(11q)cases. Conclusions: B-NHL(11q) cases are clinically homogenous while 11q aberrations are heterogeneous. We believe that BLs MYC (-) do exist. Combination of HP/IHC with FNAB/FCM/CC/FISH is a reliable method for credible diagnosis of BLMYC (-). BLMYC (-) should only be diagnosed in cases where critical 11q aberrations and a simple karyotype are identified. BCLU(11q) or DLBCL(11q) cases should be diagnosed if there are more complex karyotypes accompanied by 11q aberrations of any type. We hypothesize that in BLMYC (-) and other aggressive B-NHL(11q), 11q aberrations may determine clinical and pathomorphological features equivalent to those resulting from MYC rearrangements. Disclosures Walewski: Gilead: Consultancy, Honoraria, Other: travel, accommodation; Seattle: Other: travel, accommodation; GSK/Novartis: Research Funding; Genetics: Other: travel, accommodation; Celgene: Honoraria, Other: travel, accommodation, Research Funding; Teva: Consultancy, Honoraria; Servier: Consultancy; Karyopharm: Consultancy; Boehringer Ingelheim: Consultancy; Ariad: Consultancy; Takeda: Consultancy, Honoraria, Other; Roche: Consultancy, Honoraria, Other: travel, accommodation, Research Funding; Sanofi: Honoraria, Other: travel, accommodation; Mundipharma: Consultancy, Honoraria, Research Funding; Janssen-Cilag: Consultancy.

The complete amino acid sequence (582 residues), positions of the twelve disulfide bridges and three carbohydrate substituents, position and identity of the ten vitamin Independent γ-carboxyglutamic acid (Gla) residues has been determined. A synthetic substrate specific for Factor Xa has been developed on the basis of the prothrombin structure. The primary structure of hirudin has been nearly completed and suggests a mechanism for its thrombin specificity.The pro-part of prothrombin contains two 83 residue regions with identical disulfide-bridge pattern and 31 sequence identities. These “kringle” structures show sequence similarities with the pancreatic secretory trypsin inhibitor and hirudin. A chymotryptic digest of CM-plasminogen was investigated to find the heavy chain-light chain overlap region. This was isolated and sequenced (28 from h.c., 10 from l.c.) to study the specificity of plasminogen activators. From the same digest sequence evidence has been obtained which indicates that the heavy chain part of plasmin consists mainly of four kringle structures like the two in prothrombin, suggesting a common general structure and evolutionary origin of these two large zymogen structures.

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The diagnosis of primary central nervous system (CNS) lymphoma, which is predominantly of the diffuse large B-cell lymphoma type (CNS DLBCL), is challenging. MicroRNAs (miRs) are gene expression-regulating non-coding RNAs that are potential biomarkers. We aimed to distinguish miR expression patterns differentiating CNS DLBCL and non-malignant CNS diseases with tumor presentation (n-ML). Next generation sequencing-based miR profiling of cerebrospinal fluids (CSFs) and brain tumors was performed. Sample source-specific (CSF vs. brain tumor) miR patterns were revealed. Even so, a set of 17 miRs differentiating CNS DLBCL from n-ML, no matter if assessed in CSF or in a tumor, was identified. Along with the results of pathway analyses, this suggests their pathogenic role in CNS DLBCL. A combination of just four of those miRs (miR-16-5p, miR-21-5p, miR-92a-3p, and miR-423-5p), assessed in CSFs, discriminated CNS DLBCL from n-ML samples with 100% specificity and 67.0% sensitivity. Analyses of paired CSF-tumor samples from patients with CNS DLBCL showed significantly lower CSF levels of miR-26a, and higher CSF levels of miR-15a-5p, miR-15b-5p, miR-19a-3p, miR-106b-3p, miR-221-3p, and miR-423-5p. Noteworthy, the same miRs belonged to the abovementioned set differentiating CNS DLBCL from non-malignant CNS diseases. Our results not only add to the basic knowledge, but also hold significant translational potential.

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