Thomas Menter

PIM kinases are progression markers and emerging therapeutic targets in diffuse large B-cell lymphoma

Background:PIM serine/threonine kinases are often highly expressed in haematological malignancies. We have shown that PIM inhibitors reduced the survival and migration of leukaemic cells. Here, we investigated PIM kinases in diffuse large B-cell lymphoma (DLBCL) biopsy samples and DLBCL cell lines.Methods:Immunohistochemical staining for PIM kinases and CXCR4 was performed on tissue microarrays from a cohort of 101 DLBCL cases, and the effects of PIM inhibitors on the survival and migration of DLBCL cell lines were determined.Results:PIM1 expression significantly correlated with the activation of signal transducer and activator of transcription (STAT) 3 and 5, P-glycoprotein expression, CXCR4-S339 phosphorylation, and cell proliferation. Whereas most cases exhibited cytoplasmic or cytoplasmic and nuclear PIM1 and PIM2 expression, 12 cases (10 of the non-germinal centre DLBCL type) expressed PIM1 predominately in the nucleus. Interestingly, nuclear expression of PIM1 significantly correlated with disease stage. Exposure of DLBCL cell lines to PIM inhibitors modestly impaired cellular proliferation and CXCR4-mediated migration.Conclusion:This work demonstrates that PIM expression in DLBCL is associated with activation of the JAK/STAT signalling pathway and with the proliferative activity. The correlation of nuclear PIM1 expression with disease stage and the modest response to small-molecule inhibitors suggests that PIM kinases are progression markers rather than primary therapeutic targets in DLBCL.

In situ RHAMM protein expression in acute myeloid leukemia blasts suggests poor overall survival

Treatment options for patients with high-risk acute myeloid leukemia (AML) include high-dose chemotherapy regimens in combination with allogeneic hematopoietic stem cell transplantation, which takes advantage of the donor T-cell-mediated graft-versus-leukemia effect. Together with beneficial responses observed in assays targeted at leukemia-associated antigens (LAA), this encouraged research on cancer vaccines and adoptive cellular therapies in AML. The receptor for hyaluronic acid-mediated motility (RHAMM, CD168) was identified as one of the most promising LAA in AML. Thus far, little is known about in situ expression in leukemic bone marrow blasts or the prognostic role of RHAMM and its interaction partners in AML. We immunohistochemically analyzed the expression and prognostic significance of RHAMM on trephine bone marrow biopsies from 71 AML cases that had been evaluated for cytogenetics and presence of FLT3-internal tandem duplications and NPM1 mutations. Fifty-five patients (77%) were treated with curative intent, while 16 (23%) received the most appropriate supportive care. Twenty of 71 (28%) AML cases were considered RHAMM+. Receiver operating characteristic curves showed significant discriminatory power considering overall survival (OS) in AML patients treated curatively for RHAMM (p = 0.015). Multivariable analysis revealed that expression of RHAMM in >5% of leukemic blasts identifies a subgroup of curatively treated cases with adverse OS independent of failures to achieve complete remission. RHAMM not only represents a promising LAA with specific T-cell responses in AML but, if assessed in situ on blasts, also a probable prognostic factor.

Phenotype profiling of primary testicular diffuse large B-cell lymphomas

Primary testicular diffuse large B‐cell lymphomas (tDLBCL) are rare neoplasms with few comprehensive studies conducted so far. We aimed to systematically characterize the phenotype of tDLBCL.

LEF1: a highly specific marker for the diagnosis of chronic lymphocytic B cell leukaemia/small lymphocytic B cell lymphoma.

Aims Chronic lymphocytic B cell leukaemia (CLL)/small lymphocytic B cell lymphoma (SLL) has proven to be not a uniform entity but to consist of various disease subtypes. CLL might also pose diagnostic challenges by demonstrating an uncommon immunohistochemical profile. Recently, the role of lymphocyte enhancer-binding factor 1 (LEF1) in CLL was elucidated being highly expressed and seeming to have a prognostic value. Our aim was to test the applicability of LEF1 as marker for CLL in a diagnostic setting. Methods We investigated LEF1 expression in lymphomas by immunohistochemistry on tissue microarrays containing several lymphoma entities (altogether 720 cases, including 61 CLL cases). We also separated CLL cases by zeta-chain-associated protein kinase 70 (ZAP70) and CD38 stainings and fluorescence in situ hybridisation analyses for TP53 deletions and trisomy 12 into respective groups and correlated data with LEF1 expression. Results The area under the receiver operating characteristic curve for LEF1 as a diagnostic marker for CLL was 0.815 (95% CI 0.742 to 0.888). The relevant diagnostic cut-off value for LEF1 positivity determined by the Youdens index was 10% (specificity 92%, sensitivity 70%). The majority of CLL cases (70%) expressed LEF1. Eighteen per cent of (transformed) diffuse large B cell lymphoma cases also expressed LEF1. In most other lymphoma entities, LEF1 was negative. There was a positive correlation of LEF1 staining with ZAP70 expression (Spearmans rho: 0.438, p<0.001), but not with CD38 expression, TP53 deletions or trisomy 12. Conclusions LEF1 is a useful marker in the differential diagnosis of CLL in difficult cases. It shows a high specificity (92%) and a reasonable sensitivity (70%) for this entity.

TH1 and TH17 cells promote crescent formation in experimental autoimmune glomerulonephritis

Autoimmunity against the Goodpasture antigen α3IV‐NC1 results in crescentic glomerulonephritis (GN). Both antibodies and T cells directed against α3IV‐NC1 have been implicated in disease development and progression. Using the model of experimental autoimmune glomerulonephritis (EAG) in DBA/1 mice, we aimed to characterize the frequency and function of α3IV‐NC1‐specific CD4+ T cells in the kidneys. DBA/1 mice repeatedly immunized with human α3IV‐NC1 developed necrotizing/crescentic GN. Kidneys with crescentic GN contained CD4+ cells responding to α3IV‐NC1 with the production of IFN‐γ or IL‐17A, demonstrating the accumulation of both α3IV‐NC1‐specific TH1 and TH17 cells. To test the functional relevance of TH1 and TH17 cells, EAG was induced in DBA/1 mice deficient in IFN‐γR, IL‐17A or IL‐23p19. Mice of all knockout groups mounted α3IV‐NC1 IgG, developed nephrotic range proteinuria, and IgG deposition to the glomerular basement membranes at levels similar to immunized wild‐type mice. However, all knockout groups showed significantly fewer glomerular crescents and attenuated tubulointerstitial damage. Our results suggest that both α3IV‐NC1‐specific TH1 and TH17 cells accumulate in the kidneys and are crucial for the development of necrotizing/crescentic GN. Copyright

Diagnostic Utility of the Germinal Center-associated Markers GCET1, HGAL, and LMO2 in Hematolymphoid Neoplasms.

Implementation of new phenotypic markers in routine diagnostics of hematolymphoid neoplasms is a challenging task with a plethora of potentially relevant proteins. We investigated 3 recently discovered proteins expressed in the germinal centers of lymph nodes (LMO2, GCET1, and HGAL) in a compilation of leukemia, lymphoma, and thymic tumor entities. Altogether, 1590 cases (1519 on tissue microarrays, 71 on conventional slides) were included. Expressions of LMO2, GCET1, and HGAL were investigated by immunohistochemistry, evaluated for their differential diagnostic relevance, and correlated with the clinical outcome of patients. In Hodgkin lymphoma (HL), the expression of LMO2, GCET1, and HGAL could be largely seen in tumor cells of nodular lymphocyte predominant HL (NLPHL) but only occasionally in classic HL. The majority of B-cell lymphoma cases was positive for LMO2 [except for Burkitt lymphoma (BL)] and HGAL with weaker to moderate staining intensity, compared with the intensely staining follicular lymphomas (FL). Except for FL (60% of cases) and diffuse large B-cell lymphomas (DLBCL, 36% of cases), all other B-cell lymphomas expressed little or no GCET1. In thymomas, the non-neoplastic immature T-cells were LMO2-negative, whereas the neoplastic lymphoblasts were LMO2-positive in more than half of the lymphoblastic lymphomas (LBL). Our findings provide new potential assistance in the differential diagnosis of FL to marginal zone lymphoma, classic HL to NLPHL and primary mediastinal B-cell lymphoma, DLBCL to BL, and thymoma to LBL. Finally, HGAL proved to be a prognostic marker for classic HL regarding the background population and in DLBCL regarding the tumor cells.

Clonal relationship of classical hodgkin lymphoma and its recurrences.

Purpose: We investigated whether the first and all subsequent manifestations of Hodgkin lymphoma (HL) in a patient are clonally related. Experimental Design: We identified a collective of 20 patients with sometimes multiple HL recurrences. Relapses were classified as early, that is, within twelve months (eight events in seven patients) or as late, that is, later than one year after the previous neoplasm (24 events in 17 patients). Hodgkin and Reed–Sternberg cells were microdissected after CD30 staining using laser capture technique. Immunoglobulin heavy chain (IgH) gene fragment lengths were analyzed after DNA preamplification, applying consensus FR3 and J primers by ABI 310 Genetic Analyzer. Sequencing of the amplified IgH products was carried out by ABI 3130 and 3730XL Genetic Analyzer. Epstein-Barr virus (EBV) association was assessed by EBV early RNA and LMP1. Results: Three cases with early relapses after a first HL diagnosis were clonally related to the initial tumor, whereas three of four patients with early relapses after a first or second relapse were not, which was accompanied by change of EBV association in one case. Six patients presenting with late relapses were clonally unrelated, which was accompanied by change of phenotype in two cases and change of EBV association in one case. Two samples from recurrent tumors of the same patient could be successfully sequenced. These two late relapses were clonally unrelated by IgH fragment length and sequencing analysis. Conclusions: Recurrent HL, especially those accompanied by an EBV-association switch or after a relapse, can represent an unrelated novel neoplasm. Our finding might play a role in clinical decision making. Clin Cancer Res; 17(16); 5268–74. ©2011 AACR.

Characterization of the inflammatory infiltrate in Streptococcus pneumoniae pneumonia in young and elderly patients.

There is an increased susceptibility and mortality in the elderly due to pneumonia caused by Streptococcus pneumoniae. We aimed to assess the inflammatory cell composition with respect to age in pneumococcal pneumonia patients. Neutrophilic granulocytes and various lymphocyte and macrophage subpopulations were immunohistochemically quantified on lung tissue specimens of young (n = 5; mean age 8.4 years), middle-aged (n = 8; mean age 55.9 years) and elderly (n = 9; mean age 86.6 years) pneumonia patients with microbiologically proven S. pneumoniae pneumonia. We discovered a higher percentage of neutrophilic granulocytes in elderly as opposed to young patients (95 vs. 75%, p = 0.012). Conversely, young patients versus elderly patients had more alveolar macrophages (CD11c+: 20 vs. 9%, p = 0.029) and M1 macrophages (CD14+: 30 vs. 10%, p = 0.012 and HLA-DR+: 52 vs. 11%, p = 0.019). There was no significant difference concerning M2 macrophages and lymphocytes. Comparison of young patients with middle-aged patients showed similar significant results for alveolar macrophages (p = 0.019) and subsignificant results for M1 macrophages and neutrophilic granulocytes (p < 0.08). This is the first study characterizing the inflammatory infiltrate of pneumococcal pneumonia in situ. Our observations improve the understanding of the innate immune mechanisms of pneumococcal lung infection and point at the potential of therapies for restoring macrophage function and decreasing neutrophilic influx in order to help prevent or cure pneumonia.

Mechanisms of Immune Evasion and Immune Modulation by Lymphoma Cells

Purpose Targeting cancer cells by modulating the immune system has become an important new therapeutic option in many different malignancies. Inhibition of CTLA4/B7 and PD1/PDL1 signaling is now also being investigated and already successfully applied to various hematologic malignancies. Methods A literature review of PubMed and results of our own studies were compiled in order to give a comprehensive overview on this topic. Results We elucidate the pathophysiological role of immunosuppressive networks in lymphomas, ranging from changes in the cellular microenvironment composition to distinct signaling pathways such as PD1/PDL1 or CTLA4/B7/CD28. The prototypical example of a lymphoma manipulating and thereby silencing the immune system is Hodgkin lymphoma. Also other lymphomas, e.g., primary mediastinal B-cell lymphoma and some Epstein–Barr virus (EBV)-driven malignancies, use analogous survival strategies, while diffuse large B-cell lymphoma of the activated B-cell type, follicular lymphoma and angioimmunoblastic T-cell lymphoma to name a few, exert further immune escape strategies each. These insights have already led to new treatment opportunities and results of the most important clinical trials based on this concept are briefly summarized. Immune checkpoint inhibition might also have severe side effects; the mechanisms of the rather un(der)recognized hematological side effects of this treatment approach are discussed. Conclusion Silencing the host’s immune system is an important feature of various lymphomas. Achieving a better understanding of distinct pathways of interactions between lymphomas and different immunological microenvironment compounds yields substantial potential for new treatment concepts.

Mutational landscape of B-cell post-transplant lymphoproliferative disorders

It is currently unclear whether post‐transplant diffuse large B‐cell lymphomas (PT‐DLBCL) display a similar genomic landscape as DLBCL in immunocompetent patients (IC‐DLBCL). We investigated 50 post‐transplant lymphoproliferative disorders (PTLDs) including 37 PT‐DLBCL samples for somatic mutations frequently observed in IC‐DLBCL. Targeted Next Generation Sequencing (NGS) using the Ion Torrent platform and a customized panel of 68 genes was performed on genomic DNA. Non‐tumoural tissue was sequenced to exclude germline variants in cases where available. A control cohort of 76 IC‐DLBCL was available for comparative analyses. In comparison to IC‐DLBCLs, PT‐DLBCL showed more frequent mutations of TP53 (P = 0·004), and absence of ATM and B2M mutations (P = 0·004 and P = 0·016, respectively). In comparison to IC‐DLBCLs, Epstein–Barr virus (EBV)+ PT‐DLBCL had fewer mutated genes (P = 0·007) and particularly fewer mutations in nuclear factor‐κB pathway‐related genes (P = 0·044). TP53 mutations were more frequent in EBV‐ PT‐DLBCL as compared to IC‐DLBCL (P = 0·001). Germinal centre B cell (GCB) subtype of PT‐DLBCL had fewer mutations and mutated genes than GCB‐IC‐DLBCLs (P = 0·048 and 0·04 respectively). Polymorphic PTLD displayed fewer mutations as compared to PT‐DLBCL (P = 0·001). PT‐DLBCL differs from IC‐DLBCL with respect to mutations in genes related to DNA damage control and immune‐surveillance, and EBV association is likely to have a bearing on the mutational pattern.