Publications by authors named "Christian Steidl"

133 Publications

Single-cell profiles and prognostic impact of tumor-infiltrating lymphocytes co-expressing CD39, CD103, and PD-1 in ovarian cancer.

Clin Cancer Res 2021 May 7. Epub 2021 May 7.

Deeley Research Centre, BC Cancer

Purpose: Tumor-infiltrating lymphocytes (TIL) are strongly associated with survival in most cancers; however, the tumor-reactive subset that drives this prognostic effect remains poorly defined. CD39, CD103, and PD-1 have been independently proposed as markers of tumor-reactive CD8+ TIL in various cancers. We evaluated the phenotype, clonality and prognostic significance of TIL expressing various combinations of these markers in high-grade serous ovarian cancer (HGSC), a malignancy in need of more effective immunotherapeutic approaches.

Experimental Design: Expression of CD39, CD103, PD-1, and other immune markers was assessed by high-dimensional flow cytometry, single-cell sequencing, and multiplex immunofluorescence of primary and matched pre/post-chemotherapy HGSC specimens.

Results: Co-expression of CD39, CD103, and PD-1 ("triple-positive" phenotype) demarcated subsets of CD8+ TIL and CD4+ regulatory T cells (Tregs) with a highly activated/exhausted phenotype. Triple-positive CD8+ TIL exhibited reduced TCR diversity and expressed genes involved in both cytolytic and humoral immunity. Triple-positive Tregs exhibited higher TCR diversity and a tumor-resident phenotype. Triple-positive TIL showed superior prognostic impact relative to TIL expressing other combinations of these markers. TIGIT was uniquely upregulated on triple-positive CD8+ effector cells relative to their CD4+ Treg counterparts.

Conclusions: Co-expression of CD39, CD103, and PD-1 demarcates highly activated CD8+ and CD4+ TIL with inferred roles in cytolytic, humoral and regulatory immune functions. Triple-positive TIL demonstrate exceptional prognostic significance and express compelling targets for combination immunotherapy, including PD-1, CD39 and TIGIT.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-4394DOI Listing
May 2021

The contrasting role of nasopharyngeal angiotensin converting enzyme 2 (ACE2) transcription in SARS-CoV-2 infection: A cross-sectional study of people tested for COVID-19 in British Columbia, Canada.

EBioMedicine 2021 Apr 2;66:103316. Epub 2021 Apr 2.

British Columbia Centre for Disease Control, Vancouver, BC V5Z 4R4, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 1Z4, Canada. Electronic address:

Background: Angiotensin converting enzyme 2 (ACE2) protein serves as the host receptor for SARS-CoV-2, with a critical role in viral infection. We aim to understand population level variation of nasopharyngeal ACE2 transcription in people tested for COVID-19 and the relationship between ACE2 transcription and SARS-CoV-2 viral load, while adjusting for expression of: (i) the complementary protease, Transmembrane serine protease 2 (TMPRSS2), (ii) soluble ACE2, (iii) age, and (iv) biological sex. The ACE2 gene was targeted to measure expression of transmembrane and soluble transcripts.

Methods: A cross-sectional study of n = 424 "participants" aged 1-104 years referred for COVID-19 testing was performed in British Columbia, Canada. Patients who tested positive for COVID-19 were matched by age and biological sex to patients who tested negative. Viral load and host gene expression were assessed by quantitative reverse-transcriptase polymerase chain reaction. Bivariate analysis and multiple linear regression were performed to understand the role of nasopharyngeal ACE2 expression in SARS-CoV-2 infection.

Findings: Analysis showed no association between age and nasopharyngeal ACE2 transcription in those who tested negative for COVID-19 (P = 0•092). Mean relative transcription of transmembrane (P = 0•00012) and soluble (P<0•0001) ACE2 isoforms, as well as TMPRSS2 (P<0•0001) was higher in COVID-19-negative participants than COVID--19 positive ones, yielding a negative correlation between targeted host gene expression and positive COVID-19 diagnosis. In bivariate analysis of COVID-19-positive participants, transcription of transmembrane ACE2 positively correlated with SARS-CoV-2 viral RNA load (B = 0•49, R=0•14, P<0•0001), transcription of soluble ACE2 negatively correlated (B= -0•85, R= 0•26, P<0•0001), and no correlation was found with TMPRSS2 transcription (B= -0•042, R=<0•10, P = 0•69). Multivariable analysis showed that the greatest viral RNA loads were observed in participants with high transmembrane ACE2 transcription (Β= 0•89, 95%CI: [0•59 to 1•18]), while transcription of the soluble isoform appears to protect against high viral RNA load in the upper respiratory tract (Β= -0•099, 95%CI: [-0•18 to -0•022]).

Interpretation: Nasopharyngeal ACE2 transcription plays a dual, contrasting role in SARS-CoV-2 infection of the upper respiratory tract. Transcription of the transmembrane ACE2 isoform positively correlates, while transcription of the soluble isoform negatively correlates with viral RNA load after adjusting for age, biological sex, and transcription of TMPRSS2.

Funding: This project (COV-55) was funded by Genome British Columbia as part of their COVID-19 rapid response initiative.
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http://dx.doi.org/10.1016/j.ebiom.2021.103316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016616PMC
April 2021

Characterization of DLBCL with a PMBL gene expression signature.

Blood 2021 Mar 8. Epub 2021 Mar 8.

BC Cancer, Vancouver, British Columbia, Canada.

Primary mediastinal large B-cell lymphoma (PMBL) is a type of aggressive B-cell lymphoma that typically affects young adults, characterized by presence of a bulky anterior mediastinal mass. Lymphomas with gene expression features of PMBL have been described in non-mediastinal sites, raising questions about how these tumors should be classified. Here, we investigated whether these "non-mediastinal PMBLs" are indeed PMBLs or instead represent a distinct group within DLBCL. From a cohort of 325 de novo DLBCL cases, we identified tumors from patients without evidence of anterior mediastinal involvement that expressed a PMBL expression signature (nm-PMBLsig-pos, n=16, 5%). The majority of these tumors expressed MAL and CD23 - proteins typically observed in bona fide PMBL (bf-PMBL). Evaluation of clinical features of nm-PMBLsig-pos cases revealed close associations with DLBCL, and the majority displayed a germinal center B-cell-like cell-of-origin (GCB). In contrast to bf-PMBL, nm-PMBLsig-pos patients presented at an older age, did not show pleural disease, and bone/bone marrow involvement was observed in three cases. However, while clinically distinct from bf-PMBL, nm-PMBL-sig-pos tumors resembled bf-PMBL at the molecular level with upregulation of immune response, JAK-STAT, and NF-kB signatures. Mutational analysis revealed frequent somatic gene mutations in SOCS1, IL4R, ITPKB and STAT6, as well as CD83 and BIRC3, with the latter genes being significantly more frequently affected than in GCB-DLBCL and bf-PMBL. Our data establish nm-PMBLsig-pos lymphomas as a group of DLBCL with distinct phenotypic and genetic features, and potential implications for gene expression- and mutation-based subtyping of aggressive B-cell lymphoma and related targeted therapies.
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http://dx.doi.org/10.1182/blood.2020007683DOI Listing
March 2021

Apoptotic Blocks in Primary Non-Hodgkin B Cell Lymphomas Identified by BH3 Profiling.

Cancers (Basel) 2021 Feb 28;13(5). Epub 2021 Feb 28.

Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC H3T 1E2, Canada.

To determine causes of apoptotic resistance, we analyzed 124 primary B cell NHL samples using BH3 profiling, a technique that measures the mitochondrial permeabilization upon exposure to synthetic BH3 peptides. Our cohort included samples from chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), high-grade B cell lymphoma with translocations in and (HGBL-DH), mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). While a large number of our samples displayed appropriate responses to apoptosis-inducing peptides, pro-apoptotic functional defects, implicating BAX, BAK, BIM or BID, were seen in 32.4% of high-grade NHLs (12/37) and in 3.4% of low-grade NHLs (3/87, < 0.0001). The inhibition of single anti-apoptotic proteins induced apoptosis in only a few samples, however, the dual inhibition of BCL2 and MCL1 was effective in 83% of samples, indicating MCL1 was the most common cause of lack of response to the BCL2 inhibitor, venetoclax. We then profiled Toledo and OCI-Ly8 high-grade lymphoma cell lines to determine which drugs could reduce MCL1 expression and potentiate venetoclax responses. Doxorubicin and vincristine decreased levels of MCL1 and increased venetoclax-induced apoptosis (all < 0.05). Overall, in primary NHLs expressing BCL2 that have no defects in pro-apoptotic signaling, a poor response to venetoclax is primarily due to the presence of MCL1, which may be overcome by combining venetoclax with doxorubicin and vincristine-based chemotherapy or with other anti-microtubule inhibitors.
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http://dx.doi.org/10.3390/cancers13051002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957722PMC
February 2021

Biology in Practice: Harnessing the Curative Potential of the Immune System in Lymphoid Cancers.

J Clin Oncol 2021 Feb 12;39(5):346-360. Epub 2021 Jan 12.

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

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http://dx.doi.org/10.1200/JCO.20.01761DOI Listing
February 2021

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

Blood 2021 Apr;137(16):2196-2208

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

When the World Health Organization 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" category has been proposed, encompassing tumors with concurrent MYC and BCL2 SVs other than cooccurring translocations (ie, copy number variations [CNVs]). Although the identification of a gene expression signature (DHITsig) shared among tumors harboring MYC and BCL2 rearrangements (HGBL-DH/TH-BCL2) has confirmed a common 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 morphology. Although 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, although MYC immunohistochemistry (IHC) has been proposed as a screening tool for FISH testing, 2 mechanisms were observed that uncoupled MYC rearrangement from IHC positivity: (1) low MYC messenger RNA expression; and (2) false-negative 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.
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http://dx.doi.org/10.1182/blood.2020007193DOI Listing
April 2021

Activation-induced cytidine deaminase localizes to G-quadruplex motifs at mutation hotspots in lymphoma.

NAR Cancer 2020 Dec 13;2(4):zcaa029. Epub 2020 Oct 13.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, , , ,  and , along with the established and structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent and oncogene amplification and mutations. Ninety-seven percent of the mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified and , supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis.
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http://dx.doi.org/10.1093/narcan/zcaa029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556405PMC
December 2020

Establishment and characterization of VOA1066 cells: An undifferentiated endometrial carcinoma cell line.

PLoS One 2020 14;15(10):e0240412. Epub 2020 Oct 14.

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

Dedifferentiated endometrial carcinoma (DDEC) is a rare but highly aggressive type of endometrial cancer, in which an undifferentiated carcinoma arises from a low-grade endometrioid endometrial carcinoma. The low-grade component is often eclipsed, likely due to an outgrowth of the undifferentiated component, and the tumor may appear as a pure undifferentiated endometrial carcinoma (UEC). We and others have recently identified inactivating mutations of SMARCA4, SMARCB1 or ARID1B, subunits of the SWI/SNF chromatin-remodeling complex, that are unique to the undifferentiated component and are present in a large portion of DDEC and UEC. However, the understanding of whether and how these mutations drive cancer progression and histologic dedifferentiation is hindered by lack of cell line models of DDEC or UEC. Here, we established the first UEC cell line, VOA1066, which is highly tumorigenic in vivo. This cell line has a stable genome with very few somatic mutations, which do include inactivating mutations of ARID1A and ARID1B (2 mutations each), and a heterozygous hotspot DICER1 mutation in its RNase IIIb domain. Immunohistochemistry staining confirmed the loss of ARID1B, but ARID1A staining was retained due to the presence of a truncating non-functional ARID1A protein. The heterozygous DICER1 hotspot mutation has little effect on microRNA biogenesis. No additional DICER1 hotspot mutations have been identified in a cohort of 33 primary tumors. Therefore, we have established the first UEC cell line with dual inactivation of both ARID1A and ARID1B as the main genomic feature. This cell line will be useful for studying the roles of ARID1A and ARID1B mutations in the development of UEC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240412PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556492PMC
December 2020

Mutational landscape of gray zone lymphoma.

Blood 2021 Apr;137(13):1765-1776

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

The mutational landscape of gray zone lymphoma (GZL) has not yet been established, and differences from related entities are largely unknown. Here, we studied coding sequence mutations of 50 Epstein-Barr virus (EBV)-negative GZLs and 20 polymorphic EBV+ diffuse large B-cell lymphoma (DLBCL) not otherwise specified (poly-EBV-L) in comparison with classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), and DLBCL. Exomes of 21 GZL and 7 poly-EBV-L cases, along with paired constitutional DNA, 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) exhibited 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 that was enriched in mutations related to apoptosis defects (TP53 [39%], BCL2 [28%], BIRC6 [22%]) and depleted in GNA13, XPO1, or NF-κB signaling pathway mutations (TNFAIP3, NFKBIE, IKBKB, NFKBIA). They also exhibited more BCL2/BCL6 rearrangements compared with thymic GZL. Poly-EBV-L cases presented a distinct mutational profile, including STAT3 mutations and a significantly lower coding mutation load in comparison with EBV- GZL. Our study highlights characteristic mutational patterns in GZL associated with presentation in the thymic niche, suggesting a common cell of origin and disease evolution overlapping with related anterior mediastinal lymphomas.
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http://dx.doi.org/10.1182/blood.2020007507DOI Listing
April 2021

Hodgkin lymphoma.

Nat Rev Dis Primers 2020 07 23;6(1):61. Epub 2020 Jul 23.

Division of Oncology, Washington University School of Medicine, St Louis, MO, USA.

Hodgkin lymphoma (HL) is a B cell lymphoma characterized by few malignant cells and numerous immune effector cells in the tumour microenvironment. The incidence of HL is highest in adolescents and young adults, although HL can affect elderly individuals. Diagnosis is based on histological and immunohistochemical analyses of tissue from a lymph node biopsy; the tissue morphology and antigen expression profile enable classification into one of the four types of classic HL (nodular sclerosis, mixed cellularity, lymphocyte-depleted or lymphocyte-rich HL), which account for the majority of cases, or nodular lymphocyte-predominant HL. Although uncommon, HL remains a crucial test case for progress in cancer treatment. HL was among the first systemic neoplasms shown to be curable with radiation therapy and multiagent chemotherapy. The goal of multimodality therapy is to minimize lifelong residual treatment-associated toxicity while maintaining high levels of effectiveness. Recurrent or refractory disease can be effectively treated or cured with high-dose chemotherapy followed by autologous haematopoietic stem cell transplantation, and prospective trials have demonstrated the potency of immunotherapeutic approaches with antibody-drug conjugates and immune checkpoint inhibitors. This Primer explores the wealth of information that has been assembled to understand HL; these updated observations verify that HL investigation and treatment remain at the leading edge of oncological research.
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http://dx.doi.org/10.1038/s41572-020-0189-6DOI Listing
July 2020

TBL1XR1 Mutations Drive Extranodal Lymphoma by Inducing a Pro-tumorigenic Memory Fate.

Cell 2020 07 2;182(2):297-316.e27. Epub 2020 Jul 2.

Division of Hematology/Oncology, Department of Medicine, Weill Cornell Medicine, Cornell University, New York, NY 10021, USA. Electronic address:

The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics the human disease. Both human and murine lymphomas feature expanded MB-like cell populations, consistent with a MB-cell origin and delineating an unforeseen pathway for malignant transformation of the immune system.
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http://dx.doi.org/10.1016/j.cell.2020.05.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384961PMC
July 2020

Toward a New Molecular Taxonomy of Diffuse Large B-cell Lymphoma.

Cancer Discov 2020 Sep 2;10(9):1267-1281. Epub 2020 Jul 2.

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

Diffuse large B-cell lymphoma (DLBCL) represents a grouping of clinically and biologically heterogeneous tumors. Application of advanced molecular technology has significantly expanded our knowledge of DLBCL pathobiology, allowing identification of subgroups with common, potentially targetable, biological themes. Here, we review the recent molecular analyses that could provide a paradigm shift to a new taxonomy, foundational to the rational transition to precision medicine. We discuss how classification systems may be synthesized into a common taxonomy, drawing strength from the relationships between genetic alterations, gene expression, and tumor microenvironment. Finally, challenges to translating such a taxonomy to the clinic will be outlined.
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http://dx.doi.org/10.1158/2159-8290.CD-20-0174DOI Listing
September 2020

Genetic and evolutionary patterns of treatment resistance in relapsed B-cell lymphoma.

Blood Adv 2020 07;4(13):2886-2898

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

Diffuse large B-cell lymphoma (DLBCL) patients are typically treated with immunochemotherapy containing rituximab (rituximab, cyclophosphamide, hydroxydaunorubicin-vincristine (Oncovin), and prednisone [R-CHOP]); however, prognosis is extremely poor if R-CHOP fails. To identify genetic mechanisms contributing to primary or acquired R-CHOP resistance, we performed target-panel sequencing of 135 relapsed/refractory DLBCLs (rrDLBCLs), primarily comprising circulating tumor DNA from patients on clinical trials. Comparison with a metacohort of 1670 diagnostic DLBCLs identified 6 genes significantly enriched for mutations upon relapse. TP53 and KMT2D were mutated in the majority of rrDLBCLs, and these mutations remained clonally persistent throughout treatment in paired diagnostic-relapse samples, suggesting a role in primary treatment resistance. Nonsense and missense mutations affecting MS4A1, which encodes CD20, are exceedingly rare in diagnostic samples but show recurrent patterns of clonal expansion following rituximab-based therapy. MS4A1 missense mutations within the transmembrane domains lead to loss of CD20 in vitro, and patient tumors harboring these mutations lacked CD20 protein expression. In a time series from a patient treated with multiple rounds of therapy, tumor heterogeneity and minor MS4A1-harboring subclones contributed to rapid disease recurrence, with MS4A1 mutations as founding events for these subclones. TP53 and KMT2D mutation status, in combination with other prognostic factors, may be used to identify high-risk patients prior to R-CHOP for posttreatment monitoring. Using liquid biopsies, we show the potential to identify tumors with loss of CD20 surface expression stemming from MS4A1 mutations. Implementation of noninvasive assays to detect such features of acquired treatment resistance may allow timely transition to more effective treatment regimens.
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http://dx.doi.org/10.1182/bloodadvances.2020001696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362366PMC
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.
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http://dx.doi.org/10.1182/bloodadvances.2020001923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284085PMC
June 2020

Validation of the RHL30 digital gene expression assay as a prognostic biomarker for relapsed Hodgkin lymphoma.

Br J Haematol 2020 09 8;190(6):864-868. Epub 2020 Jun 8.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.

Despite continuing improvements in the management of classical Hodgkin lymphoma (cHL), relapse remains associated with a risk of lymphoma-related mortality. The biological composition of relapse tumour biopsies shows interpatient variability, which can be leveraged to design prognostic biomarkers. Here, we validated the RHL30 assay, a previously reported gene expression model in an independent cohort of 41 patients with relapsed cHL. Patients classified as high-risk by the RHL30 assay had inferior failure-free survival (FFS) after autologous stem cell transplantation (2-year FFS 41% vs. 92%, P = 0·035). The RHL30 model is a robust biomarker that risk-stratifies patients considered for autologous stem cell transplantation.
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http://dx.doi.org/10.1111/bjh.16777DOI Listing
September 2020

Brentuximab vedotin in combination with rituximab, cyclophosphamide, doxorubicin, and prednisone as frontline treatment for patients with CD30-positive B-cell lymphomas.

Haematologica 2020 05 15. Epub 2020 May 15.

University of Pennsylvania, Philadelphia, PA, USA.

We conducted a phase I/II multicenter trial using 6 cycles of brentuximab vedotin (BV) in combination with rituximab, cyclophosphamide, doxorubicin, and prednisone (R-CHP) for treatment of patients with CD30-positive (+) B-cell lymphomas. Thirty-one patients were evaluable for toxicity and 29 for efficacy including 22 with primary mediastinal B-cell lymphoma (PMBCL), 5 with diffuse large B-cell lymphoma (DLBCL), and 2 with gray zone lymphoma (GZL). There were no treatment-related deaths; 32% of patients had non-hematological grade 3/4 toxicities. The overall response rate was 100% (95% CI: 88-100) with 86% (95% CI: 68-96) of patients achieving complete response at the end of systemic treatment. Consolidative radiation following end of treatment response assessment was permissible and used in 52% of all patients including 59% of patients with PMBCL. With a median follow-up of 30 months, the 2-year progression-free survival (PFS) and overall survival (OS) were 85% (95% CI: 66-94) and 100%, respectively. In the PMBCL cohort, 2-year PFS was 86% (95% CI: 62-95). In summary, BV-R-CHP with or without consolidative radiation is a feasible and active frontline regimen for CD30+ B-cell lymphomas (NCT01994850).
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http://dx.doi.org/10.3324/haematol.2019.238675DOI Listing
May 2020

Mutant EZH2 Induces a Pre-malignant Lymphoma Niche by Reprogramming the Immune Response.

Cancer Cell 2020 05;37(5):655-673.e11

Division of Hematology/Oncology, Department of Medicine, Weill Cornell Medicine, Cornell University, New York, NY 10021, USA. Electronic address:

Follicular lymphomas (FLs) are slow-growing, indolent tumors containing extensive follicular dendritic cell (FDC) networks and recurrent EZH2 gain-of-function mutations. Paradoxically, FLs originate from highly proliferative germinal center (GC) B cells with proliferation strictly dependent on interactions with T follicular helper cells. Herein, we show that EZH2 mutations initiate FL by attenuating GC B cell requirement for T cell help and driving slow expansion of GC centrocytes that become enmeshed with and dependent on FDCs. By impairing T cell help, mutant EZH2 prevents induction of proliferative MYC programs. Thus, EZH2 mutation fosters malignant transformation by epigenetically reprograming B cells to form an aberrant immunological niche that reflects characteristic features of human FLs, explaining how indolent tumors arise from GC B cells.
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http://dx.doi.org/10.1016/j.ccell.2020.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298875PMC
May 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.
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http://dx.doi.org/10.1016/j.ccell.2020.03.016DOI Listing
May 2020

Cathepsin S Alterations Induce a Tumor-Promoting Immune Microenvironment in Follicular Lymphoma.

Cell Rep 2020 05 23;31(5):107522. Epub 2020 Apr 23.

Blood Cancer Research Group, Mater Research, University of Queensland, Translational Research Institute, Brisbane, QLD 4101, Australia.

Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4 T cells in vitro. Tumors with hyperactive CTSS showed increased CD4 T cell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment.
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http://dx.doi.org/10.1016/j.celrep.2020.107522DOI Listing
May 2020

Coding and noncoding drivers of mantle cell lymphoma identified through exome and genome sequencing.

Blood 2020 07;136(5):572-584

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

Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.
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http://dx.doi.org/10.1182/blood.2019002385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7440974PMC
July 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.
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http://dx.doi.org/10.1038/s41591-020-0757-zDOI Listing
April 2020

Ultrasensitive Detection of NOTCH1 c.7544_7545delCT Mutations in Chronic Lymphocytic Leukemia by Droplet Digital PCR Reveals High Frequency of Subclonal Mutations and Predicts Clinical Outcome in Cases with Trisomy 12.

J Mol Diagn 2020 04 6;22(4):571-578. Epub 2020 Feb 6.

Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada; Department of Pathology, British Columbia Cancer Agency, Vancouver, British Columbia, Canada. Electronic address:

NOTCH1 is recurrently mutated in chronic lymphocytic leukemia (CLL), most commonly as a 2-bp frameshift deletion (c.7541_7542delCT). This mutated allele encodes a truncated form of the receptor (p.P2514Rfs∗4) lacking the C-terminal proline, glutamic acid, serine, and threonine (PEST) degradation domain that increases NOTCH1 signaling duration. NOTCH1 mutation has been associated with poor clinical outcomes in CLL. We validated a highly sensitive and quantitative droplet digital PCR assay for the NOTCH1 delCT mutation, which was anticipated to perform well compared with Sanger sequencing and allele-specific PCR. Performance characteristics of this assay were tested on 126 samples from an unselected CLL cohort and a separate cohort of 85 samples from patients with trisomy 12 CLL. The delCT mutation was detected at allele frequencies as low as 0.024%; 25% of unselected cases and 55% of trisomy 12 cases were positive at the 0.024% detection threshold. Mutational burdens ≥1% were significantly associated with shorter overall survival (OS) in patients with trisomy 12+ disease in multivariate analysis (median OS, 9.1 versus 13 years, with hazard ratio of 2.34; P = 0.031). Mutational burdens <1% correlated with shorter OS in univariate, but not multivariate, analyses. These results suggest that droplet digital PCR testing for NOTCH1 delCT mutation may aid in risk stratification and/or disease monitoring in certain subsets of patients with CLL.
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http://dx.doi.org/10.1016/j.jmoldx.2020.01.008DOI Listing
April 2020

Objective quantification of BCL2 protein by multiplex immunofluorescence in routine biopsy samples of diffuse large B-cell lymphoma demonstrates associations with survival and gene alterations.

Leuk Lymphoma 2020 06 16;61(6):1334-1344. Epub 2020 Jan 16.

Department of Pathology and Molecular Medicine, Queen's University, Kingston, Canada.

Up-regulation of in cases of diffuse large B-cell lymphoma (DLBCL) can confer treatment resistance. Quantitative immunofluorescence (QIF) histology allows objective quantification of protein-based biomarkers. We investigated the utility of QIF for evaluating BCL2 as a biomarker in DLBCL by quantifying BCL2 selectively in CD20-expressing lymphoma cells in biopsy samples from 116 cases of DLBCL in two cohorts one of which consisted of relapsed/refractory cases from a clinical trial. BCL2 protein by QIF correlated with mRNA abundance and was associated with both translocation and copy number gain of the gene. Elevated BCL2 protein expression by QIF, but not immunohistochemistry or mRNA quantification, was associated with inferior overall and relapse-free survival in the relapsed/refractory cohort. QIF is an effective means of quantifying BCL2 protein objectively in routine cancer biopsy specimens and shows promise for identifying relapsed/refractory DLBCL patients at risk of inferior outcomes after salvage therapy.
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http://dx.doi.org/10.1080/10428194.2020.1713318DOI Listing
June 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.

Deeley Research Centre, 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...
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http://dx.doi.org/10.1158/2159-8290.CD-19-0680DOI Listing
March 2020

Molecular features of a large cohort of primary central nervous system lymphoma using tissue microarray.

Blood Adv 2019 12;3(23):3953-3961

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

The objective of this study was to evaluate the distribution and prognostic impact of a broad range of molecular attributes in a large cohort of immunocompetent patients with primary central nervous system lymphoma (PCNSL) by using tissue microarray. Patients diagnosed with PCNSL were initially identified in the BC Cancer Lymphoid Cancer clinical and pathology databases. Tissue microarrays were constructed by using archival formalin-fixed paraffin-embedded diagnostic biopsy tissue. Immunohistochemistry and fluorescent in situ hybridization studies were performed. A total of 115 patients with PCNSL with diffuse large B-cell lymphoma (DLBCL) histology were identified. The majority of cases (≥75%) had a non-germinal center B-cell phenotype according to immunohistochemistry algorithms, but cell of origin did not affect progression-free or overall survival. MYC (40%), BCL2 (75%), and programmed death-ligand 1 (29%) protein expression were common, but their corresponding gene rearrangements were rare (≤1% each), suggesting that alternate mechanisms were driving expression. There were no dual rearrangements involving MYC and BCL2. Only 22% of cases had membranous expression of major histocompatibility complex class II, suggesting a mechanism for escape from immune surveillance. Epstein-Barr virus-encoded RNA was positive in 1 immunocompetent patient. BCL6 protein expression (77%) and BCL6 rearrangements (31%) were frequent; the latter was the only factor associated with a poor prognosis in the overall cohort and in the subgroup of 52 patients treated with high-dose methotrexate-based regimens. This large population-based study shows that prominent molecular features of PCNSL are unique and different from those of systemic DLBCL. These results may better inform drug development in PCNSL.
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http://dx.doi.org/10.1182/bloodadvances.2019000989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963243PMC
December 2019

Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Cell 2019 Nov;179(5):1207-1221.e22

Department of Molecular Oncology, BC Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada.

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.
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http://dx.doi.org/10.1016/j.cell.2019.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912164PMC
November 2019

Single Cell Phenotypic Profiling of 27 DLBCL Cases Reveals Marked Intertumoral and Intratumoral Heterogeneity.

Cytometry A 2020 06 22;97(6):620-629. Epub 2019 Oct 22.

Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada.

Diffuse large B-cell lymphoma (DLBCL) is the most common histologic subtype of non-Hodgkin lymphoma and is notorious for its clinical heterogeneity. Patient outcomes can be predicted by cell-of-origin (COO) classification, demonstrating that the underlying transcriptional signature of malignant B-cells informs biological behavior in the context of standard combination chemotherapy regimens. In the current study, we used mass cytometry (CyTOF) to examine tumor phenotypes at the protein level with single cell resolution in a collection of 27 diagnostic DLBCL biopsy specimens from treatment naïve patients. We found that malignant B-cells from each patient occupied unique regions in 37-dimensional phenotypic space with no apparent clustering of samples into discrete subtypes. Interestingly, variable MHC class II expression was found to be the greatest contributor to phenotypic diversity. Within individual tumors, a subset of cases showed multiple phenotypic subpopulations, and in one case, we were able to demonstrate direct correspondence between protein-level phenotypic subsets and DNA mutation-defined subclones. In summary, CyTOF analysis can resolve both intertumoral and intratumoral heterogeneity among primary samples and reveals that each case of DLBCL is unique and may be comprised of multiple, genetically distinct subclones. © 2019 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.23919DOI Listing
June 2020

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