Search

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.

Classical Hodgkin lymphoma (cHL) is composed of rare malignant Hodgkin Reed-Sternberg (HRS) cells within an extensive, but ineffective, inflammatory/immune cell infiltrate. HRS cells exhibit near-universal somatic copy gains of chromosome 9p/9p24.1, which increase expression of the programmed cell death protein 1 (PD-1) ligands. To define genetic mechanisms of response and resistance to PD-1 blockade and identify complementary treatment targets, we performed whole-exome sequencing of flow cytometry–sorted HRS cells from 23 excisional biopsies of newly diagnosed cHLs, including 8 Epstein-Barr virus–positive (EBV(+)) tumors. We identified significantly mutated cancer candidate genes (CCGs) as well as somatic copy number alterations and structural variations and characterized their contribution to disease-defining immune evasion mechanisms and nuclear factor κB (NF-κB), JAK/STAT, and PI3K signaling pathways. EBV(–) cHLs had a higher prevalence of genetic alterations in the NF-κB and major histocompatibility complex class I antigen presentation pathways. In this young cHL cohort (median age, 26 years), we identified a predominant mutational signature of spontaneous deamination of cytosine- phosphate-guanines (“Aging”), in addition to apolipoprotein B mRNA editing catalytic polypeptide-like, activation-induced cytidine deaminase, and microsatellite instability (MSI)–associated hypermutation. In particular, the mutational burden in EBV(–) cHLs was among the highest reported, similar to that of carcinogen-induced tumors. Together, the overall high mutational burden, MSI-associated hypermutation, and newly identified genetic alterations represent additional potential bases for the efficacy of PD-1 blockade in cHL. Of note, recurrent cHL alterations, including B2M, TNFAIP3, STAT6, GNA13, and XPO1 mutations and 2p/2p15, 6p21.32, 6q23.3, and 9p/9p24.1 copy number alterations, were also identified in >20% of primary mediastinal B-cell lymphomas, highlighting shared pathogenetic mechanisms in these diseases.

Chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplant reflects a complex immune response resulting in chronic damage to multiple tissues. Previous studies indicated that donor B cells and the antibodies they produce play an important role in the development of cGVHD. To understand the pathogenic role of antibodies in cGVHD, we focused our studies on posttransplant production of immunoglobulin G antibodies targeting cell surface antigens expressed in multiple cGVHD affected tissues, due to their potential functional impact on living cells in vivo. Using plate-bound cell membrane proteins as targets, we detected a significantly higher level of antibodies reactive with these membrane antigens in patients who developed cGVHD, compared with those who did not and healthy donors. Plasma-reactive antibody levels increased significantly prior to the clinical diagnosis of cGVHD and were reduced following cGVHD therapies including prednisone, interleukin-2, or extracorporeal photophoresis. Using cell-based immunoprecipitation with plasma from cGVHD patients and mass spectrometry, we identified 43 membrane proteins targeted by these antibodies. The presence of antibodies in cGVHD patients’ plasma that specifically target 6 of these proteins was validated. Antibodies reactive with these 6 antigens were more frequently detected in patients with cGVHD compared with patients without cGVHD and healthy donors. These results indicate that antibodies that target membrane antigens of living cells frequently develop in cGVHD patients and further support a role for B cells and antibodies in the development of cGVHD.

Classical Hodgkin lymphoma (cHL) and primary mediastinal large B-cell lymphoma (PMBL) are aggressive tumors with distinct cells of origin and pathomorphological features. However, these lymphomas share certain transcriptional signatures and aberrant signaling pathways. CHLs and PMBLs both exhibit constitutive activation of NF-κB and JAK/STAT signaling and genetic bases of PD-1 mediated immune evasion including frequent 9p24.1/PD-L1/PD-L2 copy gains. In both lymphomas, PD-1 blockade is a FDA-approved therapy for relapsed/refractory disease. To characterize genetic bases of response to PD-1 blockade and identify complementary treatment targets in cHL and PMBL, we defined the comprehensive genetic signatures of both diseases. First, we obtained flow cytometry-sorted Hodgkin Reed Sternberg (HRS) cells from 23 biopsies of newly diagnosed cHLs and intact tumor biopsy specimens from 37 newly diagnosed PMBLs. The isolated HRS cells and paired normal DNAs and PMBL biopsy specimens were subjected to whole exome sequencing using an optimized workflow for low input samples and an expanded bait set to capture structural variants (SVs), including translocations. We used newly developed and established analytical pipelines to analyze tumor samples without paired normals (PMBLs) and identify significantly mutated genes (candidate cancer genes [CCGs], MutSig2CV, CLUMPS), SCNAs (GISTIC2.0) and SVs(4 algorithms) in both cHL and PMBL. In cHL, we identified 15 CCGs, 13 recurrent SCNAs, SVs in ETV6 and CIITA, complementary alterations of JAK/STAT, NF-κB and PI3K signaling pathway components and a median number of 11 genetic drivers per tumor. Previously unappreciated aspects of the cHL genetic signature included the increased incidence of driver mutational events in cHLs with ARID1A alterations (p=0.012). Analyses of co-occurring genetic events in EBV+ and EBV- cHLs confirmed that EBV- cHLs were significantly more likely to exhibit alterations of specific NF-κB signaling intermediaries (such as TNFAIP3 mutation and/or focal copy loss, p=0.006) and perturbations of MHC class I antigen presentation pathway components (inactivating B2M mutations, HLA-B mutations or focal copy loss of 6p21.32/HLA-B, p=0.008). The latter findings provide genetic bases for the reported differences in cell surface expression of MHC class I in EBV+ and EBV- cHLs. In PMBL, we defined 15 CCGs and more selective perturbations of specific epigenetic modifiers (ZNF217 and EZH2), transcription factors (PAX5 and IRF2BP2) and TP53, in comparison with cHL. The majority of these alterations were clonal supporting their role as early drivers. We identified 18 SCNAs and additional SVs in CIITA and PD-1 ligands, recurrent alterations of JAK/STAT and NF-κB signaling pathway components and a median of 9 genetic drivers per PMBL. Antigen presentation pathways in PMBL were perturbed by multiple recurrent alterations, including B2M mutations, focal copy losses of B2M and the MHCI/II loci, SVs of CTIIA and EZH2 mutations. There was a significant correlation between genetic perturbations of MHC class I pathway components and absence of MHC class I expression in PMBL, as previously described in cHL. Recurrent cHL alterations including B2M, TNFAIP3, STAT6, GNA13 and XPO1 CCGs and 2p/2p15/2p16.1, 6p21.32, 6q23.2 and 9p/9p24.1 SCNAs were also identified in >20% of PMBLs, highlighting shared pathogenetic mechanisms in these diseases. These tumors of predominantly young adults (median age: cHL 26 yrs; PMBL 34 yrs) both had a high rate of spontaneous deamination of CpGs, a clock-like mutational signature that is typically associated with aging. CHLs and PMBLs both exhibited previously uncharacterized molecular features that may increase sensitivity to PD-1 blockade, including high mutational burdens, in comparison with other lymphoid and solid tumors. In particular, the mutational burden in EBV- cHLs was among the highest reported, similar to that in carcinogen-induced cancers (melanoma and NSCLC). Additionally, both cHLs and PMBLs had an increased incidence of microsatellite instability and APOBEC mutational signatures, features associated with a more favorable response to PD-1 blockade. Taken together, these data define genetic similarities and differences in cHL and PMBL and establish a framework to comprehensively assess molecular bases of response to PD-1 blockade and develop rational combination therapies in these diseases. Disclosures Armand: Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Otsuka: Research Funding; Sigma Tau: Research Funding; Adaptive: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Affimed: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding; Pfizer: Consultancy; ADC Therapeutics: Consultancy; Infinity: Consultancy; Genentech: Research Funding; Tensha: Research Funding. Rodig:Merck: Research Funding; Affirmed: Research Funding; Kite, a Gilead Company: Research Funding; Bristol Myers Squib: Consultancy, Honoraria, Other: Travel Expenses, Speakers Bureau. Fromm:Merck, Inc.: Research Funding. Getz:Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Shipp:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Merck & Co.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Classical Hodgkin lymphoma (cHL) is composed of rare malignant Hodgkin Reed Sternberg (HRS) cells within an extensive, but ineffective, inflammatory/immune cell infiltrate. Emerging data suggests that cHLs use multiple genetic mechanisms to evade immune recognition. We previously found that HRS cells exhibit near-universal somatic copy number alterations (SCNAs) involving chromosome 9p24.1/PD-1-L1/PD-L2 and rare chromosomal rearrangements of PD-L1 or PD-L2. The 9p24.1 amplicon also includes JAK2, which increases JAK2 copy numbers, augments JAK2/STAT signaling and further induces PD-1 ligand expression. However, HRS cells also have inactivating mutations of B2M and decreased or absent MHC class I expression. In cHL, clinical responses to PD-1 blockade are unrelated to HRS cell expression of MHC class I but closely associated with HRS cell expression of MHC class II, highlighting the potential role of CD4+ T-cell effectors (J Clin Oncol 2018;36:942-50). To define genetic bases of response and resistance to PD-1 blockade and identify complementary treatment targets, we performed whole exome sequencing (WES) of HRS cells. We first used a previously described multi-color flow cytometric sorting protocol (Methods 2012; 57:368-75) to obtain highly purified CD30+ HRS cells and normal B cells from the excisional biopsies of 25 newly diagnosed cHLs. The isolated HRS cells and paired normal B cells were then subjected to WES using an optimized workflow for low input samples and an expanded bait set to capture structural variants (SVs). We used established analytical pipelines to identify significantly mutated genes (candidate cancer genes [CCGs], MutSig2CV), SCNAs (GISTIC2.0) and SVs (4 algorithms). With improved methodology and purity (median of 80%) of the isolated HRS cells, we defined 15 significantly mutated CCGs, 21 recurrent SCNAs, including 6 CN gains (4 focal and 2 arm level) and 15 CN losses (14 focal and 1 arm level), and low frequency SVs. We identified 2 cHLs as hypermutators with MSI signatures due to splice site mutations in MSH2 or missense mutations in POLE. Excluding the 2 hypermutators, the analyzed cHLs had a median mutational density of 6.4 mutations/Mb, that falls within the top quartile of reported cancer mutational frequencies (Nature 2013 499:214). We also identified a previously unappreciated high incidence of ARID1A mutations (24%) in cHL. This is noteworthy because ARID1A deficiency increases mutational load and augments the efficacy of PD-1 blockade in murine models (Nature Med 2018;24:556). Together, the observed MSI signatures, relatively high mutational burden and newly identified ARID1A mutations in cHL represent additional potential genetic bases for the efficacy of PD-1 blockade. Notably, these cHLs also exhibited recurrent 9p24.1 copy gain (80%) and multiple genetic bases of enhanced JAK/STAT signaling including JAK2 copy gain (80%), STAT6 mutations (32%) involving known hotspots (D419 and N421) in the DNA-binding domain and frequent inactivating SOCS1 mutations (68%). We also identified multiple genetic bases for immune evasion, including B2M inactivating mutations (36%), HLA-B mutations (16%) and 6p21.32/HLA-B copy loss (28%), copy loss of the larger 6p21.32 region and inactivating CIITA SVs (8%). Additional signaling pathways were perturbed by multiple genetic mechanisms in these cHLs. For example, NF-κB pathway alterations included: TNFAIP3 mutations (24%) and 6q23.2/TNFAIP3 copy loss (56%), 12% biallelic; NFKBIE mutations (24%) and 6q21.32/NFKBIE copy loss (12%); and NFKBIA mutations (16%). The gene encoding the nuclear export protein, XPO1, was perturbed by E571K mutations (24%) and frequent 2p15/XPO1 copy gain (72%). Additionally, GNA13, an activator of RHOA and modifier of PI3K signaling, was mutated in 24% of cases. Of interest, cHL recurrent alterations including B2M, TNFAIP3, STAT6, and GNA13 mutations and 6q23.2 and 9p24.1 SCNAs were also identified in > 20% of examined primary mediastinal B-cell lymphomas, highlighting shared pathogenetic mechanisms in these diseases. In summary, comprehensive genomic analyses of purified HRS cells reveal new genetic bases of immune evasion, potential mechanisms of response and resistance to PD-1 blockade and additional targetable alterations. KW, BC, CS, AD and DW contributed equally. JF, GG and MS contributed equally. Disclosures Rodig: Affimed: Research Funding; KITE: Research Funding; Merck: Research Funding; Bristol Myers Squibb: Research Funding. Shipp:Merck: Research Funding; Bayer: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria.