Publications by authors named "Anja Mottok"

81 Publications

A T-Cell Surface Marker Panel Predicts Murine Acute Graft-Versus-Host Disease.

Front Immunol 2020 29;11:593321. Epub 2021 Jan 29.

Department of Medicine II, University Hospital of Würzburg, Würzburg, Germany.

Acute graft-versus-host disease (aGvHD) is a severe and often life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). AGvHD is mediated by alloreactive donor T-cells targeting predominantly the gastrointestinal tract, liver, and skin. Recent work in mice and patients undergoing allo-HCT showed that alloreactive T-cells can be identified by the expression of α4β7 integrin on T-cells even before manifestation of an aGvHD. Here, we investigated whether the detection of a combination of the expression of T-cell surface markers on peripheral blood (PB) CD8 T-cells would improve the ability to predict aGvHD. To this end, we employed two independent preclinical models of minor histocompatibility antigen mismatched allo-HCT following myeloablative conditioning. Expression profiles of integrins, selectins, chemokine receptors, and activation markers of PB donor T-cells were measured with multiparameter flow cytometry at multiple time points before the onset of clinical aGvHD symptoms. In both allo-HCT models, we demonstrated a significant upregulation of α4β7 integrin, CD162E, CD162P, and conversely, a downregulation of CD62L on donor T-cells, which could be correlated with the development of aGvHD. Other surface markers, such as CD25, CD69, and CC-chemokine receptors were not found to be predictive markers. Based on these preclinical data from mouse models, we propose a surface marker panel on peripheral blood T-cells after allo-HCT combining α4β7 integrin with CD62L, CD162E, and CD162P (cutaneous lymphocyte antigens, CLA, in humans) to identify patients at risk for developing aGvHD early after allo-HCT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.593321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880247PMC
January 2021

Elotuzumab for the treatment of extramedullary myeloma: a retrospective analysis of clinical efficacy and SLAMF7 expression patterns.

Ann Hematol 2021 Feb 11. Epub 2021 Feb 11.

Department of Pathology, University of Würzburg, Würzburg, Germany.

Extramedullary disease (EMD) represents a high-risk state of multiple myeloma (MM) associated with poor prognosis. While most anti-myeloma therapeutics demonstrate limited efficacy in this setting, some studies exploring the utility of chimeric antigen receptor (CAR)-modified T cells reported promising results. We have recently designed SLAMF7-directed CAR T cells for the treatment of MM. SLAMF7 is a transmembrane receptor expressed on myeloma cells that plays a role in myeloma cell homing to the bone marrow. Currently, the only approved anti-SLAMF7 therapeutic is the monoclonal antibody elotuzumab, but its efficacy in EMD has not been investigated thoroughly. Thus, we retrospectively analyzed the efficacy of elotuzumab-based combination therapy in a cohort of 15 patients with EMD. Moreover, since the presence of the target antigen is an indispensable prerequisite for effective targeted therapy, we investigated the SLAMF7 expression on extramedullary located tumor cells before and after treatment. We observed limited efficacy of elotuzumab-based combination therapies, with an overall response rate of 40% and a progression-free and overall survival of 3.8 and 12.9 months, respectively. Before treatment initiation, all available EMD tissue specimens (n = 3) demonstrated a strong and consistent SLAMF7 surface expression by immunohistochemistry. Furthermore, to investigate a potential antigen reduction under therapeutic selection pressure, we analyzed samples of de novo EMD (n = 3) outgrown during elotuzumab treatment. Again, immunohistochemistry documented strong and consistent SLAMF7 expression in all samples. In aggregate, our data point towards a retained expression of SLAMF7 in EMD and encourage the development of more potent SLAMF7-directed immunotherapies, such as CAR T cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00277-021-04447-6DOI Listing
February 2021

BCL2 Expression in First-Line Diffuse Large B-Cell Lymphoma Identifies a Patient Population With Poor Prognosis.

Clin Lymphoma Myeloma Leuk 2020 Nov 10. Epub 2020 Nov 10.

Multidisciplinary Oncology Outpatient Clinic, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Turin, Italy.

Introduction: The prognostic value of B-cell lymphoma 2 (BCL2) expression in de novo diffuse large B-cell lymphoma (DLBCL) treated with immunochemotherapy is of interest to define a target patient population for clinical development of BCL2 inhibitors. We aimed to develop a reproducible immunohistochemistry algorithm and assay to determine BCL2 protein expression and assess the prognostic value of BCL2 in newly diagnosed DLBCL cohorts.

Patients And Methods: The prospectively defined algorithm incorporated BCL2 staining intensity and percentage of BCL2-positive cells. Functionally relevant cutoffs were based on the sensitivity of lymphoma cell lines to venetoclax. This assay was highly reproducible across laboratories. The prognostic impact of BCL2 expression was assessed in DLBCL patients from the phase 3 MAIN (n = 230) and GOYA (n = 366) trials, and a population-based registry (n = 310).

Results: Approximately 50% of tumors were BCL2 positive, with a higher frequency in high International Prognostic Index (IPI) and activated B-cell-like DLBCL subgroups. BCL2 expression was associated with poorer progression-free survival in the MAIN study (hazard ratio [HR], 1.66; 95% confidence interval [CI], 0.81-3.40; multivariate Cox regression adjusted for IPI and cell of origin). This trend was confirmed in the GOYA and registry cohorts in adjusted multivariate analyses (GOYA: HR, 1.72; 95% CI, 1.05-2.82; registry: HR, 1.89; 95% CI, 1.29-2.78). Patients with BCL2 immunohistochemistry-positive and IPI-high disease had the poorest prognosis: 3-year progression-free survival rates were 51% (GOYA) and 37% (registry).

Conclusion: Findings support use of our BCL2 immunohistochemistry scoring system and assay to select patients with BCL2-positive tumors for future studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.clml.2020.11.004DOI Listing
November 2020

A Diagnostic Approach to the Identification of Burkitt-like Lymphoma With 11q Aberration in Aggressive B-Cell Lymphomas.

Am J Surg Pathol 2021 Mar;45(3):356-364

Department of Clinical Pathology, Robert-Bosch-Krankenhaus, Stuttgart.

Rare cases of aggressive B-cell lymphomas with a morphology similar to Burkitt lymphoma (BL) present with the BL-typical immunophenotype, but lacked MYC translocation (MYC-negative Burkitt-like lymphoma: mnBLL). A proportion of those with an imbalance pattern in chromosome 11q has been designated Burkitt-like lymphoma with 11q aberration in the recent update of the World Health Organization (WHO) classification. Because of the problems in the identification of Burkitt-like lymphoma with 11q aberration, our goal was to retrospectively analyze their frequency in a cohort of "candidate" aggressive lymphomas (cohort 1, n=35) such as mnBLL (n=16), diffuse large B-cell lymphoma with similarities to Burkitt lymphoma (DLBCL-BL; n=3), high-grade B-cell lymphomas, not otherwise specified (NOS) (n=16), as well as in a cohort of MYC-negative diffuse large B-cell lymphoma NOS (cohort 2, n=62). In total, 17/33 cohort 1 cases (52%) harbored the typical 11q aberration pattern, predominantly those that had been classified as mnBLL (12/16, 75%), but also as DLBCL-BL (2/3, 67%) and high-grade B-cell lymphomas, NOS (3/14; 21%). The specimens with this typical 11q aberration pattern were usually negative for the BCL2 protein. Of interest and as a new finding, samples harboring the 11q aberration pattern were often characterized by strikingly coarse apoptotic debris within starry sky macrophages facilitating their recognition. In contrast, only 1 of 62 garden variety DLBCL, NOS was positive for the 11q aberration pattern. In 2 DLBCL-BL, a dual MYC translocation/11q aberration pattern was detected. As a diagnostic algorithm, we, therefore, propose analysis of 11q status in MYC-negative high-grade lymphomas with features of BL, especially showing BCL2 negativity and a conspicuous coarse apoptotic debris in starry sky macrophages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/PAS.0000000000001613DOI Listing
March 2021

Impact of MYC and BCL2 structural variants in tumors of DLBCL morphology and mechanisms of false-negative MYC IHC.

Blood 2020 Oct 29. Epub 2020 Oct 29.

BC Cancer, Vancouver, Canada.

When the WHO defined high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) as a clinical category, rearrangements were the only structural variant (SV) incorporated. An "atypical double-hit" entity has been proposed, encompassing tumors with concurrent MYC and BCL2 SVs other than co-occurring translocations - i.e. copy number variations (CNVs). While the identification of a gene expression signature (DHITsig) shared among tumors harboring MYC and BCL2 rearrangements (HGBL-DH/TH-BCL2) has confirmed a shared underlying biology, the biological implication of MYC and BCL2 CNVs requires further elucidation. We performed a comprehensive analysis of MYC and BCL2 SVs, as determined by fluorescent in situ hybridization (FISH), in a cohort of 802 de novo tumors with diffuse large B-cell lymphoma (DLBCL) morphology. While BCL2 CNVs were associated with increased expression, MYC CNVs were not. Furthermore, MYC and BCL2 CNVs, in the context of atypical double-hit, did not confer a similar gene expression profile as HGBL-DH/TH-BCL2. Finally, while MYC IHC has been proposed as a screening tool for FISH testing, two mechanisms were observed that uncoupled MYC rearrangement from IHC positivity. 1) low MYC mRNA expression and 2) false-negative immunohistochemistry (IHC) staining mediated by a single nucleotide polymorphism resulting in an asparagine to serine substitution at the 11th amino acid residue of MYC (MYC-N11S). Taken together, these results support the current exclusion of MYC and BCL2 CNVs from HGBL-DH/TH and highlight the ability of a molecular based classification system to identify tumors with shared biology that FISH and IHC fail to fully capture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2020007193DOI Listing
October 2020

RAL GTPases mediate multiple myeloma cell survival and are activated independently of oncogenic RAS.

Haematologica 2020 09 1;105(9):2316-2326. Epub 2020 Sep 1.

Department of Medicine II, University Hospital of Würzburg, Germany.

Oncogenic RAS provides crucial survival signaling for up to half of multiple myeloma cases, but has so far remained a clinically undruggable target. RAL is a member of the RAS superfamily of small GTPases and is considered to be a potential mediator of oncogenic RAS signaling. In primary multiple myeloma, we found RAL to be overexpressed in the vast majority of samples when compared with pre-malignant monoclonal gammopathy of undetermined significance or normal plasma cells. We analyzed the functional effects of RAL abrogation in myeloma cell lines and found that RAL is a critical mediator of survival. RNAi-mediated knockdown of RAL resulted in rapid induction of tumor cell death, an effect which was independent from signaling via mitogen-activated protein kinase, but appears to be partially dependent on Akt activity. Notably, RAL activation was not correlated with the presence of activating RAS mutations and remained unaffected by knockdown of oncogenic RAS. Furthermore, transcriptome analysis yielded distinct RNA expression signatures after knockdown of either RAS or RAL. Combining RAL depletion with clinically relevant anti-myeloma agents led to enhanced rates of cell death. Our data demonstrate that RAL promotes multiple myeloma cell survival independently of oncogenic RAS and, thus, this pathway represents a potential therapeutic target in its own right.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2019.223024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556628PMC
September 2020

Lack of NFATc1 SUMOylation prevents autoimmunity and alloreactivity.

J Exp Med 2021 Jan;218(1)

Institute of Pathology, University of Wuerzburg, Wuerzburg, Germany.

Posttranslational modification with SUMO is known to regulate the activity of transcription factors, but how SUMOylation of individual proteins might influence immunity is largely unexplored. The NFAT transcription factors play an essential role in antigen receptor-mediated gene regulation. SUMOylation of NFATc1 represses IL-2 in vitro, but its role in T cell-mediated immune responses in vivo is unclear. To this end, we generated a novel transgenic mouse in which SUMO modification of NFATc1 is prevented. Avoidance of NFATc1 SUMOylation ameliorated experimental autoimmune encephalomyelitis as well as graft-versus-host disease. Elevated IL-2 production in T cells promoted T reg expansion and suppressed autoreactive or alloreactive immune responses. Mechanistically, increased IL-2 secretion counteracted IL-17 and IFN-γ expression through STAT5 and Blimp-1 induction. Then, Blimp-1 repressed IL-2 itself, as well as the induced, proliferation-associated survival factor Bcl2A1. Collectively, these data demonstrate that prevention of NFATc1 SUMOylation fine-tunes T cell responses toward lasting tolerance. Thus, targeting NFATc1 SUMOylation presents a novel and promising strategy to treat T cell-mediated inflammatory diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20181853DOI Listing
January 2021

Mutational Landscape of Grey Zone Lymphoma.

Blood 2020 Sep 22. Epub 2020 Sep 22.

British Columbia Cancer, Vancouver, British Columbia, Canada.

The mutational landscape of grey zone lymphoma (GZL) has not yet been established and differences to related entities are largely unknown. Here, we studied coding sequence mutations of 50 EBV-negative GZL and 20 polymorphic EBV-positive DLBCL NOS (poly-EBV-L) in comparison to classical Hodgkin lymphoma (cHL), primary mediastinal large B cell lymphoma (PMBCL), and diffuse large B cell lymphoma (DLBCL). Exomes of 21 GZL and 7 poly-EBV-L cases along with paired normals were analyzed as a discovery cohort followed by targeted sequencing of 217 genes in an extension cohort of 29 GZL and 13 poly-EBV-L cases. GZL cases with thymic niche involvement (anterior mediastinal mass) displayed a mutation profile closely resembling cHL and PMBCL, with SOCS1 (45%), B2M (45%), TNFAIP3 (35%), GNA13 (35%), LRRN3 (32%) and NFKBIA (29%) being the most recurrently mutated genes. In contrast, GZL cases without thymic niche involvement (N=18) had a significantly distinct pattern, enriched in mutations related to apoptosis defects (TP53 (39%), BCL2 (28%), BIRC6 (22%)) and depleted in GNA13, XPO1or NFKB signaling pathway mutations (TNFAIP3, NFKBIE, IKBKB, NFKBIA). They also presented more BCL2/BCL6 rearrangements as opposed to thymic GZL. Poly-EBV-L cases presented a distinct mutational profile including STAT3 mutations and a significantly lower coding-mutation load in comparison to EBV-negative GZL. Our study highlights characteristic mutational patterns in GZL associated with presentation in the thymic niche suggesting a common cell of origin with disease evolution overlapping with related anterior mediastinal lymphomas.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2020007507DOI Listing
September 2020

Identification of the atypically modified autoantigen Ars2 as the target of B-cell receptors from activated B cell-type diffuse large B-cell lymphoma.

Haematologica 2020 07 16. Epub 2020 Jul 16.

Saarland Medical School, Internal Medicine I, Homburg/Saar, Germany.

It has been suggested that B-cell receptor (BCRs) stimulation by specific antigens plays a pathogenic role in diffuse large B-cell lymphoma (DLBCL). Here, it was the aim to screen for specific reactivities of DLBCL-BCRs in the spectrum of autoantigens and antigens of infectious origin. Arsenite resistance protein 2 (Ars2) was identified as the BCR target of 3/5 ABC-type DLBCL cell lines and 2/11 primary DLBCL cases. Compared to controls, Ars2 was hypo-phosphorylated exclusively in cases and cell lines with Ars2-specific BCRs. In a validation cohort, hypo-phosphorylated Ars2 was found in 8/31 ABC-type, but only 1/20 germinal center B cell (GBC)-like type DLBCL. Incubation with Ars2 induced BCR-pathway activation and increased proliferation, while an Ars2/ETA-toxin conjugate induced killing of cell lines with Ars2-reactive BCRs. Ars2 appears to play a role in a subgroup of ABC-type DLBCLs. Moreover, transformed DLBCL lines with Ars2-reactive BCRs still show growth advantage after incubation with Ars2. These results provide knowledge about the pathogenic role of a specific antigen stimulating the BCR pathway in DLCBL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2019.241653DOI Listing
July 2020

Gene expression profiling of gray zone lymphoma.

Blood Adv 2020 06;4(11):2523-2535

Centre for Lymphoid Cancer, British Columbia Cancer, Vancouver, BC, Canada.

Gray zone lymphoma (GZL), a B-cell lymphoma with features intermediate between large B-cell lymphoma (LBCL) and classic Hodgkin lymphoma (cHL), is a rare and poorly defined entity. Alongside GZL, a subset of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) has been described with polymorphic/GZL-like morphology (polymorphic-EBV-L). To fill the important gap in our understanding of the pathogenic process underlying these entities, we performed a gene expression study of a large international cohort of GZL and polymorphic-EBV-L, combined with cHL and primary mediastinal large B-cell lymphoma (PMBCL) cases. In an unsupervised principal component analysis, GZL cases presented with intermediate scores in a spectrum between cHL and PMBCL, whereas polymorphic-EBV-L clustered distinctly. The main biological pathways underlying the GZL spectrum were related to cell cycle, reflecting tumor cell content, and extracellular matrix signatures related to the cellular tumor microenvironment. Differential expression analysis and phenotypic characterization of the tumor microenvironment highlighted the predominance of regulatory macrophages in GZL compared with cHL and PMBCL. Two distinct subtypes of GZL were distinguishable that were phenotypically reminiscent of PMBCL and DLBCL, and we observed an association of PMBCL-type GZL with clinical presentation in the "thymic" anatomic niche. In summary, gene expression profiling (GEP) enabled us to add precision to the GZL spectrum, describe the biological distinction compared with polymorphic-EBV-L, and distinguish cases with and without thymic involvement as 2 subgroups of GZL, namely PMBCL-like and DLBCL-like GZL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2020001923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284085PMC
June 2020

Cathepsin S Regulates Antigen Processing and T Cell Activity in Non-Hodgkin Lymphoma.

Cancer Cell 2020 05 23;37(5):674-689.e12. Epub 2020 Apr 23.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, EPFL, Lausanne, 1015 Switzerland; Swiss Cancer Center Leman (SCCL), Lausanne, 1015 Switzerland. Electronic address:

Genomic alterations in cancer cells can influence the immune system to favor tumor growth. In non-Hodgkin lymphoma, physiological interactions between B cells and the germinal center microenvironment are coopted to sustain cancer cell proliferation. We found that follicular lymphoma patients harbor a recurrent hotspot mutation targeting tyrosine 132 (Y132D) in cathepsin S (CTSS) that enhances protein activity. CTSS regulates antigen processing and CD4 and CD8 T cell-mediated immune responses. Loss of CTSS activity reduces lymphoma growth by limiting communication with CD4 T follicular helper cells while inducing antigen diversification and activation of CD8 T cells. Overall, our results suggest that CTSS inhibition has non-redundant therapeutic potential to enhance anti-tumor immune responses in indolent and aggressive lymphomas.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccell.2020.03.016DOI Listing
May 2020

[Microenvironment in classical Hodgkin lymphoma].

Authors:
Anja Mottok

Pathologe 2020 May;41(3):254-260

Institut für Humangenetik, Universitätsklinikum Ulm, Albert-Einstein-Allee 11, 89081, Ulm, Deutschland.

Classical Hodgkin lymphoma (cHL) is histologically characterized by a quantitatively dominating immune cell infiltrate. Its composition differs depending on the histological subtype and EBV (Epstein-Barr-Virus) status. Current pathogenic concepts postulate that the malignant cells, the so-called Hodgkin and Reed-Sternberg (HRS) cells, act as master recruiters, thereby actively shaping the microenvironment to support their proliferation and outgrowth. This view on the pathogenesis of cHL is further solidified by genetic studies, which have identified important mechanisms by which the HRS cells are enabled to escape immune surveillance. Besides an insufficient antigen presentation mediated by mutations and structural chromosomal changes in key components or regulators of major histocompatibility class I and II molecules, copy number gains of the 9p24.1 genomic locus encompassing JAK2 and the ligands of the programmed cell death protein 1 (PD-1), PD-L1 and PD-L2, play an important role in the pathogenesis of this disease as the engagement of those ligands with their cognate receptor leads to suppression of the immune response. Of importance, the reversibility of this inhibitory receptor-ligand interaction is key to the clinical success that checkpoint inhibitors had and continue to have in cHL patients, especially in the relapse setting. In addition, comprehensive assessment of microenvironment composition, integration with results from genetic studies, and correlation with clinical outcomes have led to the development of prognostic models, which may assist in an improved risk stratification, informed selection of treatment regimens, and therefore better outcomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00292-020-00774-zDOI Listing
May 2020

TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma.

Nat Med 2020 04 24;26(4):577-588. Epub 2020 Feb 24.

Centre for Lymphoid Cancer, British Columbia Cancer, Vancouver, British Columbia, Canada.

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-020-0757-zDOI Listing
April 2020

Selective Inhibition of HDAC3 Targets Synthetic Vulnerabilities and Activates Immune Surveillance in Lymphoma.

Cancer Discov 2020 03 8;10(3):440-459. Epub 2020 Jan 8.

Department of Lymphoma & Myeloma, The University of Texas MD Anderson Cancer Center, Houston, Texas.

mutations are highly recurrent in B-cell lymphomas and either inactivate its histone acetyltransferase (HAT) domain or truncate the protein. Herein, we show that these two classes of mutations yield different degrees of disruption of the epigenome, with HAT mutations being more severe and associated with inferior clinical outcome. Genes perturbed by mutation are direct targets of the BCL6-HDAC3 onco-repressor complex. Accordingly, we show that HDAC3-selective inhibitors reverse -mutant aberrant epigenetic programming, resulting in: (i) growth inhibition of lymphoma cells through induction of BCL6 target genes such as and (ii) restoration of immune surveillance due to induction of BCL6-repressed IFN pathway and antigen-presenting genes. By reactivating these genes, exposure to HDAC3 inhibitors restored the ability of tumor-infiltrating lymphocytes to kill DLBCL cells in an MHC class I and II-dependent manner, and synergized with PD-L1 blockade in a syngeneic model . Hence, HDAC3 inhibition represents a novel mechanism-based immune epigenetic therapy for -mutant lymphomas. SIGNIFICANCE: We have leveraged the molecular characterization of different types of mutations to define a rational approach for targeting these mutations through selective inhibition of HDAC3. This represents an attractive therapeutic avenue for targeting synthetic vulnerabilities in -mutant cells in tandem with promoting antitumor immunity..
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-19-0116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275250PMC
March 2020

Single-Cell Transcriptome Analysis Reveals Disease-Defining T-cell Subsets in the Tumor Microenvironment of Classic Hodgkin Lymphoma.

Cancer Discov 2020 03 19;10(3):406-421. Epub 2019 Dec 19.

Centre for Lymphoid Cancer, British Columbia Cancer, Vancouver, British Columbia, Canada.

Hodgkin lymphoma is characterized by an extensively dominant tumor microenvironment (TME) composed of different types of noncancerous immune cells with rare malignant cells. Characterization of the cellular components and their spatial relationship is crucial to understanding cross-talk and therapeutic targeting in the TME. We performed single-cell RNA sequencing of more than 127,000 cells from 22 Hodgkin lymphoma tissue specimens and 5 reactive lymph nodes, profiling for the first time the phenotype of the Hodgkin lymphoma-specific immune microenvironment at single-cell resolution. Single-cell expression profiling identified a novel Hodgkin lymphoma-associated subset of T cells with prominent expression of the inhibitory receptor LAG3, and functional analyses established this LAG3 T-cell population as a mediator of immunosuppression. Multiplexed spatial assessment of immune cells in the microenvironment also revealed increased LAG3 T cells in the direct vicinity of MHC class II-deficient tumor cells. Our findings provide novel insights into TME biology and suggest new approaches to immune-checkpoint targeting in Hodgkin lymphoma. SIGNIFICANCE: We provide detailed functional and spatial characteristics of immune cells in classic Hodgkin lymphoma at single-cell resolution. Specifically, we identified a regulatory T-cell-like immunosuppressive subset of LAG3 T cells contributing to the immune-escape phenotype. Our insights aid in the development of novel biomarkers and combination treatment strategies targeting immune checkpoints...
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-19-0680DOI Listing
March 2020

Reproducing the molecular subclassification of peripheral T-cell lymphoma-NOS by immunohistochemistry.

Blood 2019 12;134(24):2159-2170

Department of Pathology and Microbiology and.

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of mature T-cell malignancies; approximately one-third of cases are designated as PTCL-not otherwise specified (PTCL-NOS). Using gene-expression profiling (GEP), we have previously defined 2 major molecular subtypes of PTCL-NOS, PTCL-GATA3 and PTCL-TBX21, which have distinct biological differences in oncogenic pathways and prognosis. In the current study, we generated an immunohistochemistry (IHC) algorithm to identify the 2 subtypes in paraffin tissue using antibodies to key transcriptional factors (GATA3 and TBX21) and their target proteins (CCR4 and CXCR3). In a training cohort of 49 cases of PTCL-NOS with corresponding GEP data, the 2 subtypes identified by the IHC algorithm matched the GEP results with high sensitivity (85%) and showed a significant difference in overall survival (OS) (P = .03). The IHC algorithm classification showed high interobserver reproducibility among pathologists and was validated in a second PTCL-NOS cohort (n = 124), where a significant difference in OS between the PTCL-GATA3 and PTCL-TBX21 subtypes was confirmed (P = .003). In multivariate analysis, a high International Prognostic Index score (3-5) and the PTCL-GATA3 subtype identified by IHC were independent adverse predictors of OS (P = .0015). Additionally, the 2 IHC-defined subtypes were significantly associated with distinct morphological features (P < .001), and there was a significant enrichment of an activated CD8+ cytotoxic phenotype in the PTCL-TBX21 subtype (P = .03). The IHC algorithm will aid in identifying the 2 subtypes in clinical practice, which will aid the future clinical management of patients and facilitate risk stratification in clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2019000779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908831PMC
December 2019

Probabilistic cell-type assignment of single-cell RNA-seq for tumor microenvironment profiling.

Nat Methods 2019 10 9;16(10):1007-1015. Epub 2019 Sep 9.

Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada.

Single-cell RNA sequencing has enabled the decomposition of complex tissues into functionally distinct cell types. Often, investigators wish to assign cells to cell types through unsupervised clustering followed by manual annotation or via 'mapping' to existing data. However, manual interpretation scales poorly to large datasets, mapping approaches require purified or pre-annotated data and both are prone to batch effects. To overcome these issues, we present CellAssign, a probabilistic model that leverages prior knowledge of cell-type marker genes to annotate single-cell RNA sequencing data into predefined or de novo cell types. CellAssign automates the process of assigning cells in a highly scalable manner across large datasets while controlling for batch and sample effects. We demonstrate the advantages of CellAssign through extensive simulations and analysis of tumor microenvironment composition in high-grade serous ovarian cancer and follicular lymphoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41592-019-0529-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485597PMC
October 2019

The time to relapse correlates with the histopathological growth pattern in nodular lymphocyte predominant Hodgkin lymphoma.

Am J Hematol 2019 11 21;94(11):1208-1213. Epub 2019 Aug 21.

First Department of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Dusseldorf, University of Cologne, Cologne, Germany.

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) can present with different histopathological growth patterns. The impact of these histopathological growth patterns on relapse characteristics is unknown. We therefore analyzed paired biopsies obtained at initial diagnosis and relapse from 33 NLPHL patients who had received first-line treatment within German Hodgkin Study Group (GHSG) trial protocols, and from a second cohort of 41 relapsed NLPHL patients who had been treated outside GHSG studies. Among the 33 GHSG patients, 21 patients presented with a typical growth pattern at initial diagnosis, whereas 12 patients had a variant histology. The histopathological growth patterns at initial diagnosis and at relapse were consistent in 67% of cases. A variant histology at initial diagnosis was associated with a shorter median time to lymphoma recurrence (2.8 vs 5.2 years; P = .0219). A similar tendency towards a shorter median time to lymphoma recurrence was observed for patients presenting with a variant histology at relapse, irrespective of the growth pattern at initial diagnosis. Results obtained from the 41 NLPHL patients who had been treated outside GHSG studies were comparable (median time to lymphoma recurrence for variant histology vs typical growth pattern at initial diagnosis: 1.5 vs 7.0 years). In conclusion, the histopathological growth pattern remains consistent at relapse in the majority of NLPHL cases, and has major impact on the time of relapse.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ajh.25607DOI Listing
November 2019

Integrative genomic analysis identifies key pathogenic mechanisms in primary mediastinal large B-cell lymphoma.

Blood 2019 09 10;134(10):802-813. Epub 2019 Jul 10.

British Columbia Cancer, Centre for Lymphoid Cancer, Vancouver, BC, Canada.

Primary mediastinal large B-cell lymphoma (PMBL) represents a clinically and pathologically distinct subtype of large B-cell lymphomas. Furthermore, molecular studies, including global gene expression profiling, have provided evidence that PMBL is more closely related to classical Hodgkin lymphoma (cHL). Although targeted sequencing studies have revealed a number of mutations involved in PMBL pathogenesis, a comprehensive description of disease-associated genetic alterations and perturbed pathways is still lacking. Here, we performed whole-exome sequencing of 95 PMBL tumors to inform on oncogenic driver genes and recurrent copy number alterations. The integration of somatic gene mutations with gene expression signatures provides further insights into genotype-phenotype interrelation in PMBL. We identified highly recurrent oncogenic mutations in the Janus kinase-signal transducer and activator of transcription and nuclear factor κB pathways, and provide additional evidence of the importance of immune evasion in PMBL ( and ). Our analyses highlight the interferon response factor (IRF) pathway as a putative novel hallmark with frequent alterations in multiple pathway members ( and ). In addition, our integrative analysis illustrates the importance of and mutations driving oncogenic signaling. The identified driver genes were significantly more frequently mutated in PMBL compared with diffuse large B-cell lymphoma, whereas only a limited number of genes were significantly different between PMBL and cHL, emphasizing the close relation between these entities. Our study, performed on a large cohort of PMBL, highlights the importance of distinctive genetic alterations for disease taxonomy with relevance for diagnostic evaluation and therapeutic decision-making.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2019001126DOI Listing
September 2019

Molecular and Genetic Characterization of MHC Deficiency Identifies EZH2 as Therapeutic Target for Enhancing Immune Recognition.

Cancer Discov 2019 04 31;9(4):546-563. Epub 2019 Jan 31.

Centre for Lymphoid Cancer, British Columbia Cancer, Vancouver, British Columbia, Canada.

We performed a genomic, transcriptomic, and immunophenotypic study of 347 patients with diffuse large B-cell lymphoma (DLBCL) to uncover the molecular basis underlying acquired deficiency of MHC expression. Low MHC-II expression defines tumors originating from the centroblast-rich dark zone of the germinal center (GC) that was associated with inferior prognosis. MHC-II-deficient tumors were characterized by somatically acquired gene mutations reducing MHC-II expression and a lower amount of tumor-infiltrating lymphocytes. In particular, we demonstrated a strong enrichment of mutations in both MHC-I- and MHC-II-negative primary lymphomas, and observed reduced MHC expression and T-cell infiltrates in murine lymphoma models expressing mutant . Of clinical relevance, EZH2 inhibitors significantly restored MHC expression in -mutated human DLBCL cell lines. Hence, our findings suggest a tumor progression model of acquired immune escape in GC-derived lymphomas and pave the way for development of complementary therapeutic approaches combining immunotherapy with epigenetic reprogramming. SIGNIFICANCE: We demonstrate how MHC-deficient lymphoid tumors evolve in a cell-of-origin-specific context. Specifically, mutations were identified as a genetic mechanism underlying acquired MHC deficiency. The paradigmatic restoration of MHC expression by EZH2 inhibitors provides the rationale for synergistic therapies combining immunotherapies with epigenetic reprogramming to enhance tumor recognition and elimination...
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-18-1090DOI Listing
April 2019

Double-Hit Gene Expression Signature Defines a Distinct Subgroup of Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma.

J Clin Oncol 2019 01 3;37(3):190-201. Epub 2018 Dec 3.

1 British Columbia Cancer Centre for Lymphoid Cancer, Vancouver, British Columbia, Canada.

Purpose: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) has a poor outcome after standard chemoimmunotherapy. We sought to understand the biologic underpinnings of HGBL-DH/TH with BCL2 rearrangements (HGBL-DH/TH- BCL2) and diffuse large B-cell lymphoma (DLBCL) morphology through examination of gene expression.

Patients And Methods: We analyzed RNA sequencing data from 157 de novo germinal center B-cell-like (GCB)-DLBCLs, including 25 with HGBL-DH/TH- BCL2, to define a gene expression signature that distinguishes HGBL-DH/TH- BCL2 from other GCB-DLBCLs. To assess the genetic, molecular, and phenotypic features associated with this signature, we analyzed targeted resequencing, whole-exome sequencing, RNA sequencing, and immunohistochemistry data.

Results: We developed a 104-gene double-hit signature (DHITsig) that assigned 27% of GCB-DLBCLs to the DHITsig-positive group, with only one half harboring MYC and BCL2 rearrangements (HGBL-DH/TH- BCL2). DHITsig-positive patients had inferior outcomes after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone immunochemotherapy compared with DHITsig-negative patients (5-year time to progression rate, 57% and 81%, respectively; P < .001), irrespective of HGBL-DH/TH- BCL2 status. The prognostic value of DHITsig was confirmed in an independent validation cohort. DHITsig-positive tumors are biologically characterized by a putative non-light zone germinal center cell of origin and a distinct mutational landscape that comprises genes associated with chromatin modification. A new NanoString assay (DLBCL90) recapitulated the prognostic significance and RNA sequencing assignments. Validating the association with HGBL-DH/TH- BCL2, 11 of 25 DHITsig-positive-transformed follicular lymphomas were classified as HGBL-DH/TH- BCL2 compared with zero of 50 in the DHITsig-negative group. Furthermore, the DHITsig was shared with the majority of B-cell lymphomas with high-grade morphology tested.

Conclusion: We have defined a clinically and biologically distinct subgroup of tumors within GCB-DLBCL characterized by a gene expression signature of HGBL-DH/TH- BCL2. This knowledge has been translated into an assay applicable to routinely available biopsy samples, which enables exploration of its utility to guide patient management.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1200/JCO.18.01583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804880PMC
January 2019

Gray-zone Lymphoma Between cHL and Large B-Cell Lymphoma: A Histopathologic Series From the LYSA.

Am J Surg Pathol 2019 03;43(3):341-351

INSERM1052, CNRS 5286, Lyon-Sud Charles Mérieux Lyon-1 Faculty, Claude Bernard University.

Gray-zone lymphoma (GZL) with features intermediate between classic Hodgkin lymphoma (cHL) and large B-cell lymphoma (LBCL) was introduced as a provisional entity into the World Health Organization classification in 2008. However, as diagnostic criteria are imprecise, reliable identification of GZL cases remains challenging. Here, we describe the histopathologic features of 139 GZL cases from a retrospective Lymphoma Study Association (LYSA) study with the goal to improve classification accuracy. Inclusion criteria were based on literature review and an expert consensus opinion of the LYSA hematopathologist panel. We observed 86 cases with a morphology more closely related to cHL, but with an LBCL immunophenotype based on strong and homogenous B-cell marker expression (CD20 and/or CD79a, OCT2, BOB1, PAX5) on all tumor cells (cHL-like GZL). Fifty-three cases were morphologically more closely related to LBCL but harbored a cHL immunophenotype (LBCL-like GZL). Importantly, we observed a continuous morphologic and immunophenotypic spectrum within these 2 GZL categories. The majority of cases presented genetic immune escape features with CD274/PDCD1LG2 and/or CIITA structural variants by fluorescence in situ hybridization. Patients without mediastinal involvement at diagnosis (17%) were older than those with mediastinal tumors (median: 56 vs. 39 y). Cases associated with Epstein-Barr virus (24%) presented with similar patient characteristics and outcome as Epstein-Barr virus negative cases. In summary, we provide refined diagnostic criteria that contribute to a more precise pathologic and clinical characterization of GZL within a broad spectrum from cHL-like to LBCL-like disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/PAS.0000000000001198DOI Listing
March 2019

High-resolution architecture and partner genes of rearrangements in lymphoma with DLBCL morphology.

Blood Adv 2018 10;2(20):2755-2765

Centre for Lymphoid Cancer, BC Cancer, Vancouver, BC, Canada.

Genomic rearrangements in the locus occur in ∼12% of lymphomas with diffuse large B-cell lymphoma (DLBCL) morphology and are associated with inferior outcome. Previous studies exploring rearrangements have primarily used fluorescence in situ hybridization (FISH) assays to characterize break-apart status but have rarely examined breakpoint location, and in some cases have not examined partner identity. We performed targeted sequencing of , , , and the immunoglobulin () loci in 112 tumors with DLBCL morphology harboring rearrangement. We characterized the location of the rearrangement at base pair resolution and identified the partner in 88 cases. We observed a cluster of breakpoints upstream of the coding region and in intron 1 (the "genic cluster"). Genic cluster rearrangements were enriched for translocations involving (80%), whereas nongenic rearrangements occurred mostly downstream of the gene with a variety of partners, including and Other recurrent partners included , , and , which has not previously been described as a partner. We compared 2 commercially available FISH break-apart assays for the locus and observed discordant results in 32% of cases examined, including some with - and - rearrangements. In cases of high-grade B-cell lymphoma with and and/or rearrangement (HGBL-DH), so-called "double-hit" lymphomas, the majority of rearrangements had non- partners (65%), with breakpoints outside the genic cluster (72%). In patients with de novo HGBL-DH of DLBCL morphology, - rearrangements showed a trend toward inferior time to progression and overall survival compared with -non- rearrangements. Our data reveal clinically relevant architecture of rearrangements in lymphomas with DLBCL morphology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2018023572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199666PMC
October 2018

Genome-wide discovery of somatic regulatory variants in diffuse large B-cell lymphoma.

Nat Commun 2018 10 1;9(1):4001. Epub 2018 Oct 1.

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3' UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-06354-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167379PMC
October 2018

Molecular classification of primary mediastinal large B-cell lymphoma using routinely available tissue specimens.

Blood 2018 11 26;132(22):2401-2405. Epub 2018 Sep 26.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Scottsdale, AZ.

Primary mediastinal large B-cell lymphoma (PMBCL) is recognized as a distinct entity in the World Health Organization classification. Currently, the diagnosis relies on consensus of histopathology, clinical variables, and presentation, giving rise to diagnostic inaccuracy in routine practice. Previous studies have demonstrated that PMBCL can be distinguished from subtypes of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures. However, requirement of fresh-frozen biopsy material has precluded the transfer of gene expression-based assays to the clinic. Here, we developed a robust and accurate molecular classification assay (Lymph3Cx) for the distinction of PMBCL from DLBCL subtypes based on gene expression measurements in formalin-fixed, paraffin-embedded tissue. A probabilistic model accounting for classification error, comprising 58 gene features, was trained on 68 cases of PMBCL and DLBCL. Performance of the model was subsequently evaluated in an independent validation cohort of 158 cases and showed high agreement of the Lymph3Cx molecular classification with the clinicopathological diagnosis of an expert panel (frank misclassification rate, 3.8%). Furthermore, we demonstrate reproducibility of the assay with 100% concordance of subtype assignments at 2 independent laboratories. Future studies will determine Lymph3Cx's utility for routine diagnostic purposes and therapeutic decision making.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2018-05-851154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265647PMC
November 2018

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

Br J Haematol 2019 02 10;184(4):616-624. Epub 2018 Aug 10.

Institute of Pathology, University of Würzburg and Comprehensive Cancer Centre (CCC) Mainfranken, Würzburg, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bjh.15519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361690PMC
February 2019

Biology of classical Hodgkin lymphoma: implications for prognosis and novel therapies.

Blood 2018 04 2;131(15):1654-1665. Epub 2018 Mar 2.

Department for Lymphoid Cancer Research, British Columbia Cancer Agency, Vancouver, BC, Canada; and.

Hodgkin lymphoma is considered a prime example of treatment success, with cure rates exceeding 80% using modern combined modality therapies. However, especially in adolescents and young adults, treatment-related toxicity and long-term morbidity still represent persistent challenges. Moreover, outcomes in patients with relapsed or refractory disease remain unfavorable in the era of high-dose chemotherapy and stem-cell transplantation. Hence, there is a high demand for novel and innovative alternative treatment approaches. In recent years, many new therapeutic agents have emerged from preclinical and clinical studies that target molecular hallmarks of Hodgkin lymphoma, including the aberrant phenotype of the tumor cells, deregulated oncogenic pathways, and immune escape. The antibody-drug conjugate brentuximab vedotin and immune checkpoint inhibitors have already shown great success in patients with relapsed/refractory disease, leading to US Food and Drug Administration approval and new trials testing these agents in various clinical settings. The expanding knowledge and understanding of Hodgkin lymphoma biology and disease progression, as well as the development of robust tools for biomarker-driven risk stratification and therapeutic decision making, continue to be fundamentally important for the success of these and other novel agents. We anticipate that the availability and clinical implementation of novel molecular assays will be instrumental in an era of rapid shifts in the treatment landscape of this disease. Here, we review the current knowledge of Hodgkin lymphoma pathobiology, highlighting the related development of novel treatment strategies and prognostic models that hold the promise to continually challenge and change the current standard of care in classical Hodgkin lymphoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2017-09-772632DOI Listing
April 2018