Search

Abstract Hodgkin Reed-Sternberg (HRS) cells of classic Hodgkin lymphoma (cHL), like many solid tumors, elicit ineffective immune responses. However, patients with cHL are highly responsive to PD-1 blockade, which largely depends on HRS cell-specific retention of MHC class II and implicates CD4+ T cells and additional MHC class I-independent immune effectors. Here, we utilize single-cell RNA sequencing and spatial analysis to define shared circulating and microenvironmental features of the immune response to PD-1 blockade in cHL. Compared with non-responders, responding patients have more circulating CD4+ naïve and central memory T cells and B cells, as well as more diverse CD4+ T cell and B cell receptor repertoires. Importantly, a population of circulating and tumor-infiltrating IL1β+ monocytes/macrophages is detectable in patients with cHL but not healthy donors, and a proinflammatory, tumor-promoting signature of these circulating IL1β+ monocytes is associated with resistance to PD-1 blockade in cHL. Altogether, our findings reveal extensive immune rewiring and complementary roles of CD4+ T cells, B cells and IL1β+ monocytes in the response to PD-1 blockade and suggest that these features can be captured with a peripheral blood test.

Abstract Background Brain metastasis (BM) is a rare but severe complication of head and neck squamous cell carcinoma (HNSCC), with limited knowledge of molecular characteristics and immunogenicity. Methods We analyzed 61 cases of HNSCC-BM from three academic institutions (n = 24) and Foundation Medicine Inc (FMI, n = 37). A subset of cases underwent next-generation sequencing, multiple immunofluorescence, and proximity ligation sequencing. Gene enrichment analysis compared alterations in FMI BM samples (n = 37) with local samples (n = 4082). Results Demographics included: median age of 59 years, 75% male, 55% current/former smokers, 75% oropharyngeal primary, and 67% human papillomavirus (HPV) +. ATM (54%), KMT2A (54%), PTEN (46%), RB1 (46%), and TP53 (46%) were frequently altered in BM samples from academic centers (62% HPV/p16+). Structural rearrangements ranged from 9 to 90 variants by proximity ligation sequencing. BMs had low densities of CD8+, PD-1+, PD-L1+, and FOXP3 + cells, and 92% had PD-L1 combined positive scores

Objectives Multiplex immunohistochemistry and immunofluorescence (mIHC/IF) are emerging technologies that can be used to help define complex immunophenotypes in tissue, quantify immune cell subsets, and assess the spatial arrangement of marker expression. mIHC/IF assays require concerted efforts to optimize and validate the multiplex staining protocols prior to their application on slides. The best practice guidelines for staining and validation of mIHC/IF assays across platforms were previously published by this task force. The current effort represents a complementary manuscript for mIHC/IF analysis focused on the associated image analysis and data management.Methods The Society for Immunotherapy of Cancer convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the quantitative image analysis of mIHC/IF output and data management considerations.Results Best-practice approaches for image acquisition, color deconvolution and spectral unmixing, tissue and cell segmentation, phenotyping, and algorithm verification are reviewed. Additional quality control (QC) measures such as batch-to-batch correction and QC for assembled images are also discussed. Recommendations for sharing raw outputs, processed results, key analysis programs and source code, and representative photomicrographs from mIHC/IF assays are included. Lastly, multi-institutional harmonization efforts are described.Conclusions mIHC/IF technologies are maturing and are routinely included in research studies and moving towards clinical use. Guidelines for how to perform and standardize image analysis on mIHC/IF-stained slides will likely contribute to more comparable results across laboratories and pave the way for clinical implementation. A checklist encompassing these two-part guidelines for the generation of robust data from quantitative mIHC/IF assays will be provided in a third publication from this task force. While the current effort is mainly focused on best practices for characterizing the tumor microenvironment, these principles are broadly applicable to any mIHC/IF assay and associated image analysis.

Abstract Spatial omics technologies decipher functional components of complex organs at cellular and subcellular resolutions. We introduce Spatial Graph Fourier Transform (SpaGFT) and apply graph signal processing to a wide range of spatial omics profiling platforms to generate their interpretable representations. This representation supports spatially variable gene identification and improves gene expression imputation, outperforming existing tools in analyzing human and mouse spatial transcriptomics data. SpaGFT can identify immunological regions for B cell maturation in human lymph nodes Visium data and characterize variations in secondary follicles using in-house human tonsil CODEX data. Furthermore, it can be integrated seamlessly into other machine learning frameworks, enhancing accuracy in spatial domain identification, cell type annotation, and subcellular feature inference by up to 40%. Notably, SpaGFT detects rare subcellular organelles, such as Cajal bodies and Set1/COMPASS complexes, in high-resolution spatial proteomics data. This approach provides an explainable graph representation method for exploring tissue biology and function.

Abstract Highly multiplexed protein imaging is emerging as a potent technique for analyzing protein distribution within cells and tissues in their native context. However, existing cell annotation methods utilizing high-plex spatial proteomics data are resource intensive and necessitate iterative expert input, thereby constraining their scalability and practicality for extensive datasets. We introduce MAPS (Machine learning for Analysis of Proteomics in Spatial biology), a machine learning approach facilitating rapid and precise cell type identification with human-level accuracy from spatial proteomics data. Validated on multiple in-house and publicly available MIBI and CODEX datasets, MAPS outperforms current annotation techniques in terms of speed and accuracy, achieving pathologist-level precision even for typically challenging cell types, including tumor cells of immune origin. By democratizing rapidly deployable and scalable machine learning annotation, MAPS holds significant potential to expedite advances in tissue biology and disease comprehension.

Abstract Cellular organization and functions encompass multiple scales in vivo. Emerging high-plex imaging technologies are limited in resolving subcellular biomolecular features. Expansion Microscopy (ExM) and related techniques physically expand samples for enhanced spatial resolution, but are challenging to be combined with high-plex imaging technologies to enable integrative multiscaled tissue biology insights. Here, we introduce Expand and comPRESS hydrOgels (ExPRESSO), an ExM framework that allows high-plex protein staining, physical expansion, and removal of water, while retaining the lateral tissue expansion. We demonstrate ExPRESSO imaging of archival clinical tissue samples on Multiplexed Ion Beam Imaging and Imaging Mass Cytometry platforms, with detection capabilities of > 40 markers. Application of ExPRESSO on archival human lymphoid and brain tissues resolved tissue architecture at the subcellular level, particularly that of the blood-brain barrier. ExPRESSO hence provides a platform for extending the analysis compatibility of hydrogel-expanded biospecimens to mass spectrometry, with minimal modifications to protocols and instrumentation.

Abstract Although concurrent chemoradiation (CRT) and durvalumab consolidation has become a standard treatment for stage III non-small cell lung cancer (NSCLC), clinicopathologic and genomic factors associated with its efficacy remain poorly characterized. Here, in a multi-institutional retrospective cohort study of 328 patients treated with CRT and durvalumab, we identify that very high PD-L1 tumor proportion score (TPS) expression ( ≥ 90%) and increased tumor mutational burden (TMB) are independently associated with prolonged disease control. Additionally, we identify the impact of pneumonitis and its timing on disease outcomes among patients who discontinue durvalumab: compared to patients who experienced early-onset pneumonitis (

Abstract Atezolizumab with chemotherapy has shown improved progression-free and overall survival in patients with metastatic PD-L1 positive triple negative breast cancer (TNBC). Atezolizumab with anthracycline- and taxane-based neoadjuvant chemotherapy has also shown increased pathological complete response (pCR) rates in early TNBC. This trial evaluated neoadjuvant carboplatin and paclitaxel with or without atezolizumab in patients with clinical stages II-III TNBC. The co-primary objectives were to evaluate if chemotherapy and atezolizumab increase pCR rate and tumor infiltrating lymphocyte (TIL) percentage compared to chemotherapy alone in the mITT population. Sixty-seven patients (ages 25–78 years; median, 52 years) were randomly assigned – 22 patients to Arm A, and 45 to Arm B. Median follow up was 6.6 months. In the modified intent to treat population (all patients evaluable for the primary endpoints who received at least one dose of combination therapy), the pCR rate was 18.8% (95% CI 4.0–45.6%) in Arm A, and 55.6% (95% CI 40.0–70.4%) in Arm B (estimated treatment difference: 36.8%, 95% CI 8.5–56.6%; p = 0.018). Grade 3 or higher treatment-related adverse events occurred in 62.5% of patients in Arm A, and 57.8% of patients in Arm B. One patient in Arm B died from recurrent disease during the follow-up period. TIL percentage increased slightly from baseline to cycle 1 in both Arm A (mean ± SD: 0.6% ± 21.0%) and Arm B (5.7% ± 15.8%) (p = 0.36). Patients with pCR had higher median TIL percentages (24.8%) than those with non-pCR (14.2%) (p = 0.02). Although subgroup analyses were limited by the small sample size, PD-L1-positive patients treated with chemotherapy and atezolizumab had a pCR rate of 75% (12/16). The addition of atezolizumab to neoadjuvant carboplatin and paclitaxel resulted in a statistically significant and clinically relevant increased pCR rate in patients with clinical stages II and III TNBC. (Funded by National Cancer Institute).

Abstract A single dose of bevacizumab reduced the density of angiopoietin-2-positive vessels while improving the infiltration of CD4+ T and CD8+ T cells, and mature dendritic cells in patients with primary triple-negative breast cancer. Our findings provide a rationale for including bevacizumab during neoadjuvant treatment to enhance the efficacy of immune checkpoint blockers in this disease.

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I–low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

Background Responses to conventional donor lymphocyte infusion for postallogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell–based therapy is a promising modality to treat post-HCT relapse.Methods We initiated this ongoing phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5 to 10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High-resolution profiling with mass cytometry and single-cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion.Results In the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires.Conclusion Given their rapid expansion and long-term persistence in an immune-compatible environment, CIML NK cells serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies.Trial Registration ClinicalTrials.gov NCT04024761.Funding Dunkin’ Donuts, NIH/National Cancer Institute, and the Leukemia and Lymphoma Society.

Abstract: Disease relapse remains the leading cause of failure after autologous stem cell transplantation (ASCT) for patients with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL). We conducted a phase 2, multicenter, single-arm study of the anti–PD-1 monoclonal antibody pembrolizumab given after ASCT in patients with chemosensitive DLBCL, hypothesizing that it would improve the progression-free survival (PFS) at 18 months after ASCT (primary endpoint) from 60% to 80%. Pembrolizumab was administered at 200 mg IV every 3 weeks for up to 8 cycles, starting within 21 days of post-ASCT discharge. Twenty-nine patients were treated on this study; 62% completed all 8 cycles. Seventy-nine percent of patients experienced at least one grade 3 or higher adverse event, and 34% experienced at least one grade 2 or higher immune-related adverse event. Overall, 59% of patients were alive and progression free at 18 months, which did not meet the primary endpoint. The 18-month overall survival was 93%. In conclusion, pembrolizumab was successfully administered as post-ASCT consolidation in patients with R/R DLBCL, but the PFS did not meet the protocol-specific primary objective and therefore does not support a larger confirmatory study. This trial was registered at www.clinicaltrials.gov as #NCT02362997.

Abstract: 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.

B-cell non-Hodgkin lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL, such as diffuse large B-cell lymphoma, have been comprehensively interrogated at the genomic level, but rarer subtypes, such as mantle cell lymphoma, remain less extensively characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth, including diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, and Burkitt lymphoma. We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumors from each subtype, including frequent genetic deregulation of the ubiquitin proteasome system. In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.

B-cell receptor (BCR) signaling pathway components represent promising treatment targets in multiple B-cell malignancies including diffuse large B-cell lymphoma (DLBCL). In in vitro and in vivo model systems, a subset of DLBCLs depend upon BCR survival signals and respond to proximal BCR/phosphoinositide 3 kinase (PI3K) blockade. However, single-agent BCR pathway inhibitors have had more limited activity in patients with DLBCL, underscoring the need for indicators of sensitivity to BCR blockade and insights into potential resistance mechanisms. Here, we report highly significant transcriptional upregulation of C-X-C chemokine receptor 4 (CXCR4) in BCR-dependent DLBCL cell lines and primary tumors following chemical spleen tyrosine kinase (SYK) inhibition, molecular SYK depletion or chemical PI3K blockade. SYK or PI3K inhibition also selectively upregulated cell surface CXCR4 protein expression in BCR-dependent DLBCLs. CXCR4 expression was directly modulated by fork-head box O1 via the PI3K/protein kinase B/forkhead box O1 signaling axis. Following chemical SYK inhibition, all BCR-dependent DLBCLs exhibited significantly increased stromal cell-derived factor-1α (SDF-1α) induced chemotaxis, consistent with the role of CXCR4 signaling in B-cell migration. Select PI3K isoform inhibitors also augmented SDF-1α induced chemotaxis. These data define CXCR4 upregulation as an indicator of sensitivity to BCR/PI3K blockade and identify CXCR4 signaling as a potential resistance mechanism in BCR-dependent DLBCLs.

Objectives The interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) analyses are emerging technologies that can be used to help quantify immune cell subsets, their functional state, and their spatial arrangement within the tumor microenvironment.Methods The Society for Immunotherapy of Cancer (SITC) convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the optimization and validation of mIHC/mIF assays across platforms.Results Representative outputs and the advantages and disadvantages of mIHC/mIF approaches, such as multiplexed chromogenic IHC, multiplexed immunohistochemical consecutive staining on single slide, mIF (including multispectral approaches), tissue-based mass spectrometry, and digital spatial profiling are discussed.Conclusions mIHC/mIF technologies are becoming standard tools for biomarker studies and are likely to enter routine clinical practice in the near future. Careful assay optimization and validation will help ensure outputs are robust and comparable across laboratories as well as potentially across mIHC/mIF platforms. Quantitative image analysis of mIHC/mIF output and data management considerations will be addressed in a complementary manuscript from this task force.

Abstract: Classical Hodgkin lymphoma (cHL) is characterized by nearly universal genetic alterations in 9p24.1, resulting in constitutive expression of PD-1 ligands. This likely underlies the unique sensitivity of cHL to PD-1 blockade, with response rates of ∼70% in relapsed/refractory disease. There are now numerous clinical trials testing PD-1 inhibitors in earlier stages of treatment and in combination with many other therapies. In general, non-Hodgkin lymphomas (NHLs) do not display a high frequency of 9p24.1 alterations and do not share cHL's vulnerability to PD-1 blockade. However, a few entities have genetic or immunologic features that may predict sensitivity to immune checkpoint blockade. These include primary mediastinal B cell lymphoma, primary central nervous system lymphoma, and primary testicular lymphoma, which harbor frequent alterations in 9p24.1, as well as Epstein Barr virus (EBV)–infected lymphomas, where EBV infection leads to increased PD-L1 expression. Although these subtypes may be specifically vulnerable to PD-1 blockade, the majority of NHLs appear to be minimally sensitive to PD-1 blockade monotherapy. Current investigations in NHL are therefore focusing on targeting other checkpoints or studying PD-1–based combination therapy. Looking forward, additional insight into the most common mechanisms of resistance to immune checkpoint inhibitors will be important to guide rational clinical trial design. In this review, we describe the biological basis for checkpoint blockade in cHL and NHL and summarize the clinical data generated to date. Guided by our rapidly evolving understanding of the pathobiology of various lymphoma subtypes, we are hopeful that the role of checkpoint inhibitors in lymphoma treatment will continue to grow.

Somatic loss-of-function mutations of the additional sex combs-like transcriptional regulator 1 (ASXL1) gene are common genetic abnormalities in human myeloid malignancies and induce clonal expansion of mutated hematopoietic stem cells (HSCs). To understand how ASXL1 disruption leads to myeloid cell transformation, we generated asxl1 haploinsufficient and null zebrafish lines using genome-editing technology. Here, we show that homozygous loss of asxl1 leads to apoptosis of newly formed HSCs. Apoptosis occurred via the mitochondrial apoptotic pathway mediated by upregulation of bim and bid. Half of the asxl1+/− zebrafish had myeloproliferative neoplasms (MPNs) by 5 months of age. Heterozygous loss of asxl1 combined with heterozygous loss of tet2 led to a more penetrant MPN phenotype, while heterozygous loss of asxl1 combined with complete loss of tet2 led to acute myeloid leukemia (AML). These findings support the use of asxl1+/− zebrafish as a strategy to identify small-molecule drugs to suppress the growth of asxl1 mutant but not wild-type HSCs in individuals with somatically acquired inactivating mutations of ASXL1.

Abstract Epigenetic alterations by histone/protein deacetylases (HDACs) are one of the many mechanisms that cancer cells use to alter gene expression and promote growth. HDAC inhibitors have proven to be effective in the treatment of specific malignancies, particularly in combination with other anticancer agents. We conducted a phase I trial of panobinostat in patients with unresectable stage III or IV melanoma. Patients were treated with oral panobinostat at a dose of 30 mg daily on Mondays, Wednesdays, and Fridays (Arm A). Three of the six patients on this dose experienced clinically significant thrombocytopenia requiring dose interruption. Due to this, a second treatment arm was opened and the dose was changed to 30 mg oral panobinostat three times a week every other week (Arm B). Six patients were treated on Arm A and 10 patients were enrolled to Arm B with nine patients treated. In nine patients treated on Arm B, the response rate was 0% (90% confidence interval [CI]: 0–28%) and the disease‐control rate was 22% (90% CI: 4–55%). Among all 15 patients treated, the overall response rate was 0% (90% CI: 0–17%) and the disease‐control rate was 27% (90% CI: 10–51%). There was a high rate of toxicity associated with treatment. Correlative studies suggest the presence of immune modifications after HDAC inhibition. Panobinostat is not active as a single agent in the treatment of melanoma. Further exploration of this agent in combination with other therapies may be warranted.

Abstract: Diagnostics and supportive care for patients with non-Hodgkin lymphoma (NHL) in lower- and middle-income countries (LMICs) are lacking. We hypothesized that high-throughput transcription-based diagnostics could classify NHL specimens from Malawi amenable to targeted therapeutics. We established tissue microarrays and classified 328 cases diagnosed by hematoxylin and eosin as NHL at University of Malawi College of Medicine using immunohistochemistry (IHC) for conventional markers and therapeutic targets. A subset was analyzed using NanoString-based expression profiling with parsimonious transcriptional classifiers. Overall, 72% of lymphomas were high-grade B-cell tumors, subsets of which were enriched for expression of MYC, BCL2, and/or PD-L1. A 21-gene transcriptional classifier, previously validated in Western cohorts, divided 96% of diffuse large B-cell lymphomas (DLBCLs) with 100% of B-cell lymphomas, unclassifiable, into 1 cluster and 88% of Burkitt lymphomas into a separate cluster. Cell-of-origin categorization of 36 DLBCLs by NanoString lymphoma subtyping test (LST) revealed 69% concordance with IHC. All discordant cases were classified as germinal center B cell–like (GCB) by LST but non-GCB by IHC. In summary, utilization of advanced diagnostics facilitates objective assessment and segregation of biologically defined subsets of NHL from an LMIC without expert review, thereby establishing a basis for the implementation of effective and less toxic targeted agents.