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.

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.

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.

Cryptic MYC insertions in Burkitt lymphoma: New data and a review of the literature.

The occurrence of MYC-negative Burkitt lymphoma (BL) has been discussed for many years. The real frequency of the MYC insertion in MYC-negative BL is still unknown. Fine-needle aspiration biopsies of 108 consecutive patients with clinicopathologically suspected BL (suspBL) were evaluated by flow cytometry, classical cytogenetics, and fluorescence in situ hybridization (FISH). We found 12 cases (11%) without the MYC rearrangement by FISH with a MYC breakapart probe: two patients (1.9%) with cryptic MYC/IGH fusion (finally diagnosed as BL) and 10 patients (9.3%) with 11q gain/loss (finally diagnosed as Burkitt-like lymphoma with 11q aberration). The exact breakpoints of the cryptic MYC/IGH were investigated by next-generation sequencing. The MYC insertions' breakpoints were identified in PVT1 in the first case, and 42 kb upstream of 5'MYC in the second case. To date, a molecular characterization of the MYC insertion in BL has only been reported in one case. Detailed descriptions of our MYC insertions in a routinely and consecutively diagnosed suspBL cohort will contribute to resolving the issue of MYC negativity in BL. In our opinion, the presence of the MYC insertions in BL and other lymphomas might be underestimated, because routine genetic diagnostics are usually based on FISH only, without karyotyping.

A Set of 17 microRNAs Common for Brain and Cerebrospinal Fluid Differentiates Primary Central Nervous System Lymphoma from Non-Malignant Brain Tumors.

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.

Prognostic Value of the Immunological Subtypes of Adolescent and Adult T-Cell Lymphoblastic Lymphoma; an Ultra-High-Risk Pro-T/CD2(-) Subtype.

(1) Background: T-cell lymphoblastic lymphoma (T-LBL) is extremely rare and highly aggressive, with no practical risk model defined yet. The prognostic value of T-LBL immunological subtypes is still a matter of controversy. (2) Methods: We re-evaluated 49 subsequent adult T-LBL patients treated according to the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL) protocols, 05/93 (n = 20) and T-LBL 1/2004 (n = 29), 85.7% of which achieved complete remission (CR). (3) Results: The 5/10-year overall survival (OS) and event-free survival (EFS) were 62%/59% and 48%/43%, respectively. In 96% of patients, flow cytometry analyses defining the WHO 2008 immunophenotypes were available. Cortical, early/pro-T/CD2(-), early/pre-T/CD2(+), and mature subtypes were identified in 59.5%, 19%, 15%, and 6.5% of patients, respectively. Overall, 20% of patients had the early T-cell precursor (ETP)-LBL immunophenotype, as proposed by the WHO 2017 classification. For the early/pro-T/CD2(-) subtype, the five-year OS and EFS were 13% and 13%, while for all the other, non-pro-T subtypes, they were 69% and 67%. By multivariate analysis, only CD2(-) status and age > 35 years emerged as strong, independent factors influencing OS and EFS, while the risk of CR failure was influenced by age only (>35 years). (4) Conclusions: ETP was non-significant for OS, unless an ultra-high-risk pro-T/CD2(-) subtype was concerned.

Genetic progression of post-transplant Burkitt-like lymphoma case with 11q-Gain/Loss and MYC amplification.

"Burkitt-like lymphoma with 11q aberration" is a new provisional entity in the latest revision of lymphoma's World Health Organization classification described as carrying the specific 11q-gain/loss aberration and lacking MYC rearrangement. Morphologically, phenotypically and by gene and microRNA expression profiling these lymphomas resemble Burkitt lymphoma. The 11q-gain/loss was also found in post-transplant patients with molecular Burkitt lymphoma signature without MYC rearrangement. Recent reports describe aggressive lymphomas with coexistence of 11q-gain/loss and MYC rearrangement. In this report we describe post-transplant Burkitt-like lymphoma with 11q aberration and MYC amplification. Genetic studies were conducted at two time points: before therapy and during progression. In both cytogenetic examinations, peculiar 11q-gain/loss was detected. Percentage of cells carrying MYC amplification increased from 2% at initial diagnosis to 97% during progression. The MYC amplification can functionally correspond to MYC translocation and to MYC overexpression. The presence of MYC amplification seems to increase the aggressiveness of the reported disease course, so even a small clone with this change should be indicated in cytogenetic result.

Significance of CD10 protein expression in the diagnostics of follicular lymphoma: A comparison of conventional immunohistochemistry with flow cytometry supported by the establishment of BCL2 and BCL6 rearrangements.

INTRODUCTION: Histopathological examination and immunohistochemistry (IHC) with a crucial role of CD10 expression remain a standard diagnostic tool in follicular lymphoma (FL). The results of IHC CD10 detection with different primary antibodies are not fully reproducible, but some reports show that flow cytometry (FCM) can be a reliable method of CD10 identification. METHODS: The aim of the study was to compare results of CD10 expression in FL by IHC and FCM including immunophenotypic features in the context of the BCL2 and BCL6 alterations. RESULTS: Out of 76 histopathologically diagnosed FL, a group of 25 cases had simultaneously FCM. Immunohistochemically 77.6% of cases were CD10-positive with comparable and reproducible results to FCM. Differences between the FCM expression of CD5/CD10/CD11c/CD25/CD43 and BCL2 overexpression (BCL2(+)higher ) correlated with the BCL2 and BCL6 rearrangements (R) status. Lack of CD10 expression corresponded with the absence of BCL2R and higher MUM1 expression by IHC results but had no clinical impact on the long-time outcomes. CONCLUSIONS: Immunohistochemistry staining is a comparable method to FCM assessment in the evaluation of CD10 expression and diagnosis of FL. Fine-needle aspiration biopsy/FCM (FNAB/FCM) could be a useful tool for verifying FL diagnosis and CD10 detection. Despite its heterogeneity, FL has a characteristic immunophenotype. BCL2R and BCL6R FL cases differ mainly in levels of BCL2 and CD10 with CD43 co-expression; BCL2(+)higher by FCM correlates with BCL2R. Moreover, FNAB plays an important role in material provision for supportive karyotyping and BCL2R, BCL6R assessed by FISH.

Differentiation of single lymphoma primary cells and normal B-cells based on their adhesion to mesenchymal stromal cells in optical tweezers.

We have adapted a non-invasive method based on optical tweezers technology to differentiate between the normal B-cells and the B-cell non-Hodgkin lymphoma (B-NHL) cells derived from clinical samples. Our approach bases on the nascent adhesion between an individual B-cell and a mesenchymal stromal cell. In this study, a single B-cell was trapped and optically seeded on a mesenchymal stromal cell and kept in a direct contact with it until a stable connection between the cells was formed in time scale. This approach allowed us to avoid the introduction of any exogenous beads or chemicals into the experimental setup which would have affected the cell-to-cell adhesion. Here, we have provided new evidence that aberrant adhesive properties found in transformed B-cells are related to malignant neoplasia. We have demonstrated that the mean time required for establishing adhesive interactions between an individual normal B-cell and a mesenchymal stromal cell was 26.7 ± 16.6 s, while for lymphoma cell it was 208.8 ± 102.3 s, p < 0.001. The contact time for adhesion to occur ranged from 5 to 90 s and from 60 to 480 s for normal B-cells and lymphoma cells, respectively. This method for optically controlled cell-to-cell adhesion in time scale is beneficial to the successful differentiation of pathological cells from normal B-cells within the fine needle aspiration biopsy of a clinical sample. Additionally, variations in time-dependent adhesion among subtypes of B-NHL, established here by the optical trapping, confirm earlier results pertaining to cell heterogeneity.

Tumor and Cerebrospinal Fluid microRNAs in Primary Central Nervous System Lymphomas.

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.

The 11q-Gain/Loss Aberration Occurs Recurrently in MYC-Negative Burkitt-like Lymphoma With 11q Aberration, as Well as MYC-Positive Burkitt Lymphoma and MYC-Positive High-Grade B-Cell Lymphoma, NOS.

OBJECTIVES: The latest revision of lymphoma's World Health Organization classification describes the new provisional entity "Burkitt-like lymphoma with 11q aberration" (BLL, 11q) as lacking MYC rearrangement, but harboring the specific11q-gain/loss aberration. We report genetic characteristics of 11 lymphoma cases with this aberration. METHODS: Classical cytogenetics, fluorescence in situ hybridization (FISH), and single nucleotide polymorphism/array comparative genomic hybridization. RESULTS: The 11q aberrations were described as duplication, inversion, and deletion. Array comparative genomic hybridization showed two types of duplication: bigger than 50 megabase pairs (Mbp) and smaller than 20 Mbp, which were associated with bulky tumor larger than 20 cm and amplification of the 11q23.3 region, including KMT2A. Six cases revealed a normal FISH status of MYC and were diagnosed as BLL,11q. Five cases showed MYC rearrangement and were diagnosed as Burkitt lymphoma (BL) or high-grade B-cell lymphoma, not otherwise specified (HGBL, NOS). CONCLUSIONS: The 11q-gain/loss is not specific for BLL, 11q, but occurs recurrently in MYC-positive BL and MYC-positive HGBL.

Human regulatory T cells suppress proliferation of B lymphoma cells.

Activated regulatory T cells (Tregs) suppress proliferation and differentiation of normal B cells. In our study, allogeneic polyclonal CD4 (+) CD25 (+) Tregs and CD4 (+) CD25 (+) CD127(lo)Tregs expanded in vitro in the presence of rapamycin and low dose IL-2 suppressed proliferation of 11 out of 12 established lymphoma B-cell lines. The effect of expanded CD4 (+) CD25 (+) Tregs on survival of freshly isolated lymphoma B cells maintained in culture with soluble multimeric CD40L and IL-4 was variable across lymphoma entities. The survival of freshly isolated follicular lymphoma cells usually decreased in cocultures with CD4 (+) CD25 (+) Tregs. Treg effect on chronic lymphocytic leukemia/small lymphocytic lymphoma cells ranged from suppression to help in individual patients. CD4 (+) CD25 (+) Tregs or CD4 (+) CD25 (+) CD127(lo)Tregs expanded ex vivo with rapamycin could be used to suppress regrowth of residual lymphoma after autologous hematopoietic cell transplantation (HCT), and to counteract both graft-versus-host disease and lymphoma re-growth after allogeneic HCT in select patients with lymphoma susceptible to the regulation by Tregs.