Niklas Gebauer

TP53 mutations are frequent events in double-hit B-cell lymphomas with MYC and BCL2 but not MYC and BCL6 translocations

Abstract Double-hit lymphomas (DHL) with MYC and either BCL2 or BCL6 rearrangements are rare neoplasms with an aggressive clinical presentation and grim prognosis. Moreover, molecular characterization of DHL remains insufficient, and especially the role of TP53 pathway disruption is unknown. We employed a next-generation sequencing approach to investigate the mutational status of TP53 in DHL and correlated genomic data with immunohistochemical reactivity for p53. We identified TP53 mutations in MYC+/BCL2+ lymphomas at a frequency intermediate between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma. Remarkably, TP53 mutations were particularly scarce in MYC+/BCL6+ lymphomas. Our findings indicate a significant difference between these two types of DHL at a molecular level with pathogenetic implications, as arguably, TP53 mutations inhibiting p53 mediated promotion of apoptosis pose a synergistic advantage in clonal evolution of cells with malignantly enforced overexpression of BCL2. Immunohistochemical staining appears to be a sensitive surrogate of TP53 mutation status with moderate specificity.

Prevalence of targetable oncogenic mutations and genomic alterations in Epstein–Barr virus-associated diffuse large B-cell lymphoma of the elderly

Abstract Epstein–Barr virus (EBV)-associated diffuse large B-cell lymphoma (DLBCL) of the elderly constitutes a provisional clinicopathological entity in the current World Health Organization (WHO) classification and its genomic features remain sparsely characterized. We investigated a cohort of 26 cases of untreated de novo EBV-positive DLBCL of the elderly by high-resolution array-based comparative genomic profiling and fluorescence in situ hybridization (FISH). Moreover, we screened for activating mutations affecting nuclear factor (NF)-κB pathway signaling and chromatin remodeling (EZH2, CD79B, CARD11 and MYD88) due to their impact of gene expression signatures and postulated upcoming therapeutic targetability. We identified an overlap between genomic aberrations previously described to be exclusive features of plasmablastic lymphoma (PL), post-transplant lymphoproliferative disorder (PTLD) and DLBCL, respectively, indicating a close cytogenetic relationship between these entities. Few mutations affecting CD79B and CARD11 and no MYD88 mutations were detectable, hinting at EBV-mediated activation of NF-κB as an alternative to pathologically enforced B-cell receptor signaling in this rare entity.

MicroRNA expression and JAK2 allele burden in bone marrow trephine biopsies of polycythemia vera, essential thrombocythemia and early primary myelofibrosis.

Background/Aims: MicroRNAs (miRNAs) play an important role in cellular differentiation and cancer pathogenesis. However, their role in promoting the malignant phenotype of myeloproliferative diseases and their importance for differential diagnosis of early-stage chronic myeloproliferative diseases (CMPDs) remains widely obscure. Methods: In this study, we systematically evaluated the differential expression of miRNAs previously described to be associated with myelopoiesis and myeloproliferative pathogenesis by quantitative RT-PCR in polycythemia vera, essential thrombocythemia, early primary myelofibrosis (PMF) and normal hematopoiesis. Our goal was to establish certain miRNAs as potential markers for CMPDs to facilitate the differentiation between these diseases and to further investigate molecular differences between the subtypes of myeloproliferative neoplasia. Results: An aberrant expression of miRNAs 10a and 150 could be demonstrated for essential thrombocythemia and PMF as well as for polycythemia vera and PMF, respectively. The expression of miR-150 could further be shown to correlate with both JAK2 allele burden and peripheral blood counts. Conclusion: Thus, the miRNAs investigated in this study seem to be potential marker oncomiRs in the differential diagnosis of CMPDs and possibly hold potential for the elucidation of a JAK2-independent mechanism of pathogenesis.

Genomic landscape of pancreatic neuroendocrine tumors

AIM To investigate the prognostic role of genomic stability and copy number alterations (CNAs) pancreatic neuroendocrine tumors (PanNETs). METHODS A high-resolution array-based comparative genomic hybridization approach was utilized in order to investigate and quantify chromosomal aberrations in a panel of 37 primary PanNET and 11 metastatic samples. DNA samples were extracted from formalin-fixed and paraffin-embedded tumor specimen. Genomic findings were correlated with histopathological and immunohistochemical data. Moreover, the dataset was subjected to employing an unsupervised hierarchical clustering analysis approach utilizing Euclidean distance and average linkage and associations between genomically defined tumor groups and recurrent CNAs or clinicopathological features of the study group were assessed. RESULTS Numerous chromosomal aberrations were recurrently detected in both, primary tumor samples and metastases. Copy number gains were most frequently observed at 06p22.2-p22.1 (27.1%), 17p13.1 (20.8%), 07p21.3-p21.2 (18.8%), 09q34.11 (18.8%). Genomic losses were significantly less frequent and the only recurrent aberration affected 08q24.3 (6.3%). Moreover, we detected a high degree of genomic heterogeneity between primary tumors and metastatic lesions. Unsupervised hierarchical clustering of loci affected by CNAs in more than 3 primary tumor samples revealed two genetically distinct tumor groups as well as two chromosomal clusters of genomic imbalances indicating a small subset of tumors with common molecular features (13.5%). Aberrations affecting 6p22.2-22.1, 8q24.3, 9q34.11 and 17p13.1 (P = 0.011; 0.003; 0.003; 0.001), were significantly associated with a poorer survival prognosis. CONCLUSION This study suggests that several frequent CNAs in numerous candidate regions are involved in the pathogenesis and metastatic progression of PanNET.

Oncogenic mutations and chromosomal aberrations in primary extranodal diffuse large B-cell lymphomas of the thyroid--a study of 21 cases.

CONTEXT Primary extranodal diffuse large B-cell lymphomas of the thyroid (ptDLBCL) constitute a rare entity, which until now was not fully explored. OBJECTIVE Due to recently published data genetically linking ptDLBCL to a subset of thyroid carcinoma, we assessed the occurrence of oncogenic mutations and copy number alterations. DESIGN A high-resolution array-based comparative genomic hybridization approach was applied to quantify genomic aberrations in a study population of 21 ptDLBCL patients. In addition, we investigated the frequency of mutations involving the BRAF, NRAS, and MYD88 genes in correlation with immunohistochemical data. RESULTS Chromosomal gains were recurrently detected at 6p21.33-p21.31, 6p22.2, 12p13.31, 14q31.1, 14q32.33, 19p13.3, and 22q11.22; numeric losses were most frequently observed at 6p21.3-p21.31, 10q26.3, 19p13.3, 20q13.33, and 21q11.2. Aberrations affecting 6p22.2 and 14q32.33 as well as 22q11.22 differed slightly between germinal center B-cell (GCB) and non-GCB groups. Statistically significant deviations were detected at 20q13.33 and 21q11.2. These specific alterations do not seem to occur in thyroid carcinomas or other DLBCL, according to previously published literature. Analysis of BRAF and NRAS showed mutation frequencies of 4.8 and 9.5%, respectively. No MYD88 mutations could be detected in any of the analyzed cases. Fluorescence in situ hybridization demonstrated breakage events involving the BCL2, BCL6, and cMYC locus in 14.3, 9.5, and 9.5%, respectively. CONCLUSIONS Our study revealed ptDLBCL to be predominantly composed of the GCB type, harboring no MYD88 mutations and showing infrequent mutations in the BRAF and NRAS genes. Additionally, array comparative genomic hybridization showed no overlapping alterations between ptDLBCL and thyroid carcinomas or other nodal or extranodal DLBCL.

Intravascular natural killer cell lymphoma mimicking mycosis fungoides: a case report and review of the literature.

Intravascular lymphoma is a rare entity. Most cases constitute a variant of extranodal diffuse large B-cell lymphoma, and only 10% of the published cases are of T-cell or histiocytic origin. Even fewer cases of intravascular natural killer (NK) cell lymphoma have been reported. To date, only the intravascular lymphoma of B-cell linage is recognized as a distinct entity by the WHO Classification. Here, we report the clinical, morphological, immunohistochemical, and molecular findings of a 72-year-old male patient with intravascular NK-cell lymphoma of the skin who initially presented with red skin efflorescences suspicious of mycosis fungoides. A skin biopsy revealed large cell infiltrates of NK/T-cell phenotype (CD3ε, CD4, CD8, CD56, and TIA-1), which were localized strictly intravascularly and which were positive for Epstein-Barr virus nucleic acid EBER (Epstein-Barr virus-encoded small RNA). Molecular studies revealed a germline configuration for the T-cell receptor consistent with the possibility of an NK-cell origin. At the beginning, the disease appeared to be limited to the skin with no sign of bone marrow involvement or leukemic dissemination. Chemotherapy was initiated; however, the patient subsequently developed meningiosis lymphomatosa with recurrent epileptic episodes and bone marrow infiltration with pancytopenia 7 months after primary admission. Finally, the patient passed away in a septic shock.

Favorable prognostic impact of RAS mutation status in multiple myeloma treated with high-dose melphalan and autologous stem cell support in the era of novel agents: a single center perspective

Oncogenic mutations affecting members of the RAS gene family (NRAS, KRAS, HRAS) have been recurrently described in many solid tumors and hematological malignancies including multiple myeloma in a variety of frequencies showing entity-dependent patterns of distribution [1]. In the context of multiple myeloma, there appear to be high and approximately equal rates (10–20%) of KRAS and NRAS mutations with a significant predominance over mutations affecting other members of the RAS gene family [2]. While RAS mutant myeloma cells are traditionally considered to be highly refractory to establish treatment approaches including reduced cytotoxicity of nitrogen mustard alkylating agent melphalan, recent in vitro assays revealed a dominant addiction of RAS mutants to an operational proteasome [3,4]. Moreover, no in vivo data on the association of RAS mutations with chemoresistance towards melphalan have been reported so far. A recently published randomized trial comparing chemotherapy plus lenalidomide with autologous stem cell transplantation (ASCT) followed by maintenance with lenalidomide-prednisone or lenalidomide alone in patients with newly diagnosed myeloma demonstrated significantly prolonged progression-free survival (PFS) in the transplant arm (60% vs 38% at 3 years), thus widely settling the question whether ASCT still has a role in myeloma treatment in the age of novel agents [5]. Novel agent-based (e.g. thalidomide, lenalidomide and bortezomib) combination therapies have emerged as the contemporary standard of care for induction therapy prior to ASCT in multiple myeloma; moreover, they represent viable options for treatment at relapse, maintenance therapy or in transplant ineligible patients [5–7]. Contemporary transplant trials involving novel agents for induction therapy revealed that responses could be deepened to the effect that the number of patients achieving complete response (CR) and very good partial response (VGPR) is increased between induction and the posttransplant consolidation [8]. Investigations pre-dating the introduction of novel agents reported especially KRAS mutations to be associated with unfavorable prognosis, whilst recent studies focusing on bortezomib-based treatment for relapsed and refractory myeloma identified NRAS rather then KRAS mutations to herald inferior response in refractory and relapsed multiple myeloma [2,9,10]. Pathogenetic, prognostic and therapeutic implications of RAS mutations in multiple myeloma constitute a widely unresolved matter and no profound consideration has been given to the role of ASCT in this context, especially in the era of proteasome inhibition and immunomodulatory treatment. To our knowledge, this retrospective study is the first assessment of the prognostic impact of RAS mutations on clinical outcome of multiple myeloma patients treated with high-dose melphalan followed by autologous stem cell transplantation in the era of novel agents, especially in the context of post-relapse survival and maintenance therapy with thalidomide, lenalidomide and bortezomib. Ninety consecutive transplant eligible patients at a single institution who were treated for multiple myeloma with highdose melphalan followed by autologous stem cell support were primarily included in the current study. Representative bone marrow mononuclear cell samples were available in 70 cases. Of the patients included in the current study, 29 received planned tandem ASCT. Patients received a mean of 1.4 lines of therapy prior to ASCT. All patients were subsequently analysed for RAS mutation status, which was correlated with clinical characteristics at diagnosis and therapeutic outcome. Table I summarizes the baseline and treatment characteristics for all patients included in the study.

MicroRNA profiling of low-grade and transformed nodal marginal zone lymphoma reveals a similar signature pattern distinct from diffuse large B cell lymphoma.

Background/Aims: As critical post-transcriptional regulators of gene expression, microRNAs are involved in several cellular processes of vital impact including cell growth and apoptosis. Many hematologic malignancies exhibit distinct microRNA signatures. MicroRNA implication in the pathogenesis of nodal marginal zone lymphoma (NMZL), however, remains widely elusive. Methods: Comprehensive morphologic, immunophenotypic and cytogenetic studies were carried out on a cohort of NMZL (n = 30) incorporating indolent as well as transformed MZL. In addition, microRNA signatures were generated, employing a quantitative real-time polymerase chain reaction approach. These were then compared to signatures from cases of diffuse large B cell lymphoma (DLBCL) alongside reactive lymph node controls. Results: While microRNA signatures of low-grade and transformed NMZL did not differ significantly, several microRNAs were differentially expressed between transformed NMZL and DLBCL, hinting at molecularly distinct mechanisms of lymphomagenesis and indicating the biological disparity of transformed NMZL from DLBCL. Conclusion: In the light of the unresolved issue regarding the classification of marginal zone-derived transformed B-cell neoplasms, microRNAs may be a valuable aid in discriminating NMZL from DLBCL.

miRNA Expression Correlated with Morphological Findings in Chronic Myeloid Leukemia Treated with Imatinib Mesylate

response to therapy and resistance to TK inhibitor treatment [4–6] . The role of miRNAs and secondary deregulated mRNAs in CML at diagnosis and during treatment with IM with various degrees of therapeutic response has not been assessed so far. In this study, we used qRT-PCR to investigate miRNA and mRNA expression in bone marrow trephine biopsies and correlated our findings with morphological, immunohistochemical, and clinical parameters. Formalin-fixed and paraffin-embedded bone marrow trephines were retrieved from the registry of the Institute for Pathology, University Hospital of Schleswig-Holstein, Campus Luebeck [CML at diagnosis, prior to any treatment n = 34, CML treated with IM for 18 months n = 32 (independent from the group prior to treatment), reactive nonneoplastic controls n = 15]. All studies were approved by the local Ethics Committee and are in accordance with the Declaration of Helsinki. Diagnosis and therapeutic response were evaluated by two experienced hematopathologists (A.C.F. and H.M.) according to WHO criteria and the morphological score established by Lugli et al. [7] for CML treated with IM [8] . Total RNA for miRNA experiments was isolated using the Recently miRNAs have been identified as a novel epigenetic mechanism in regulation of gene expression [1] . It has been shown that these small noncoding RNAs contribute to numerous biological processes including cell growth, differentiation, apoptosis, and pathogenesis of malignant neoplasia by downregulating one or several genes via translational repression and target degradation. Expression levels of miRNAs appear to differ between normal and malignant tissues and it is believed that specific miRNA profiles exist for all types of tissues and tumors. Additionally miRNAs are valuable as prognostic factors in various types of malignancies [2] . Chronic myeloid leukemia (CML) is characterized by the expression of the BCR-ABL fusion gene resulting from the t(9; 22) (q34;q11) translocation and displaying constitutive tyrosine kinase (TK) activity. The TK inhibitor imatinib mesylate (IM) is currently the first-line therapy for CML patients, inducing remission in most cases. A subset of patients, however, does not respond to IM treatment, owing to intolerance or drug resistance [3] . Aberrant miRNA expression in CML has been analyzed, revealing characteristic aberrations of miRNA expression patterns associated with phases of disease and Received: April 3, 2013 Accepted after revision: May 20, 2013 Published online: September 5, 2013

Indolent lymphoma with composite histology and simultaneous transformation at initial diagnosis exhibit clinical features similar to de novo diffuse large B-cell lymphoma

While various studies characterized clinical and prognostic properties of de novo diffuse large B-Cell lymphoma (DLBCL) and transformed indolent lymphomas, the clinicopathological features of indolent lymphoma and simultaneous secondary transformation upon initial diagnosis (ssDLBCL) are insufficiently established. Between 2010 and 2017, 247 consecutive patients admitted to our institution and treated for DLBCL were investigated for composite histology of ssDLBCL-type. Upon systematical histopathological evaluation composite histology was identified in 22/247 cases (8.9%). The predominant histology of the underlying indolent lymphoma was follicular lymphoma of variable grading (I-IIIA; 81.8%) whereas marginal zone lymphoma represented a minor sub group (18.2%). Clinicopathological investigation revealed a high degree of concordance between ssDLBCL and de novo DLBCL upon initial diagnosis and clinical courses were shown to be strikingly similar. The predominant fraction of ssDLBCL were germinal center derived lymphomas (GCB-type) with a trend towards a superior outcome compared with non-GCB-type ssDLBCL. Additionally, we demonstrate a significant adverse prognostic impact of an underlying indolent lymphoma component other than follicular-type lymphoma (e.g. marginal zone lymphoma). Moreover, the frequency of double-hit (DHL) or double-expressor lymphomas (DEL) appears to be low. Our findings provide substantial insight into the behavior of ssDLBCL, highlight the ramifications of the concurrent high-grade fraction within indolent lymphomas and underline therapeutic efficacy of R-CHOP type immunochemotherapy in the majority of ssDLBCL patients.