Perioperative chemoimmunotherapy induces strong immune responses and long-term survival in patients with HLA class I-deficient non-small cell lung cancer.

BACKGROUND: Loss of human leukocyte antigen (HLA) class I expression and loss of heterozygosity (LOH) are common events implicated in the primary resistance of non-small cell lung cancer (NSCLC) to immunotherapy. However, there is no data on perioperative chemoimmunotherapy (ChIO) efficacy or response mechanisms in the context of HLA class I defects. METHODS: Baseline HLA class I tumor status (HLA-deficient (HLA-DEF) or HLA-proficient (HLA-PRO)) was determined by DNA LOH combined with immunohistochemistry for protein levels in tissue of 24 patients with NSCLC treated with perioperative nivolumab plus chemotherapy from NADIM trial (NCT03081689). We integrated HLA tumor status with molecular data (programmed death-ligand 1 (PD-L1), TMB, TCR repertoire, TILs populations, bulk RNA-seq, and spatial transcriptomics (ST)) and clinical outcomes (pathological response and survival data) to study the activity of perioperative ChIO considering HLA class I defects. RESULTS: HLA-DEF tumors comprised 41.7% of analyzed tumors and showed a desert-like microenvironment at baseline, with lower PD-L1 levels and reduced immune infiltrate. However, perioperative ChIO induced similar complete pathological response (CPR) rates in both HLA-DEF and PRO tumors (50% and 60% respectively, p=0.670), as well as 3-year survival rates: Progression-free survival (PFS) and overall survival (OS) of 70% (95% CI 32.9% to 89.2%) for HLA-DEF, and PFS 71.4% (95% CI 40.6% to 88.2%) and OS 92.9% (95% CI 59.1% to 99.0%) for HLA-PRO (log-rank PFS p=0.909, OS p=0.137). Proof-of-concept ST analysis of a CPR HLA-DEF tumor after ChIO showed a strong immune response with tertiary lymphoid structures (TLS), CD4+T cells with HLA class II colocalization, and activated CD8+T cells. CONCLUSIONS: Our findings highlight the activity of perioperative ChIO, and the potential role of TLS and T-cell immune response, in NSCLC HLA-DEF tumors.

Pembrolizumab plus chemotherapy in frontline treatment of advanced ovarian cancer: Clinical and translational results from a phase 2 trial.

BACKGROUND: The efficacy and feasibility of pembrolizumab combined with chemotherapy in frontline management of advanced high-grade epithelial ovarian cancer (EOC) is unknown. Additionally, modification of the tumor microenvironment following neoadjuvant therapy is not well understood. METHODS: In this single-arm phase 2 trial (this study was registered at ClinicalTrials.gov: NCT02520154), eligible patients received up to 4 cycles of neoadjuvant chemotherapy followed by interval cytoreduction, 3 cycles of adjuvant intravenous carboplatin/weekly paclitaxel/pembrolizumab, and finally maintenance pembrolizumab until progression or toxicity (maximum 20 cycles). The primary endpoint was progression-free survival (PFS). Secondary endpoints included feasibility, toxicity, and overall survival (OS). PD-L1 staining, multiplex immunofluorescence staining, RNA sequencing, reverse-phase protein array analyses were performed on pre- and post-chemotherapy samples. FINDINGS: Thirty-one eligible patients were enrolled. Median PFS and OS was 14.88 (95% CI 12.39-23.00) and 57.43 months (95% CI 30.88-not reached), respectively. Among those with PD-L1 combined positive score (CPS) ≥10, the median PFS and OS were not reached compared to those with CPS <10 (10.50 and 30.90 months, respectively). Feasibility was met, with all patients completing their planned adjuvant cycles. Treatment discontinuation due to immune-related toxicity occurred in 6 patients (20%). Chemotherapy resulted in an infiltration of anti-tumor immune cells in the tumor microenvironment. Samples of patients with the best PFS demonstrated increased expression of NF-κB, TGF-ß, and ß-catenin signaling. CONCLUSIONS: Pembrolizumab with chemotherapy was feasible and resulted in PFS within the historical range for this EOC population. Patients with CPS ≥10 may benefit more from this regimen, and future studies should investigate this potential biomarker. FUNDING: This investigator-initiated trial was funded by Merck.

First-in-human dose escalation trial to evaluate the clinical safety and efficacy of an anti-MAGEA1 autologous TCR-transgenic T cell therapy in relapsed and refractory solid tumors.

RATIONALE OF THE TRIAL: Although the use of engineered T cells in cancer immunotherapy has greatly advanced the treatment of hematological malignancies, reaching meaningful clinical responses in the treatment of solid tumors is still challenging. We investigated the safety and tolerability of IMA202 in a first-in-human, dose escalation basket trial in human leucocyte antigen A*02:01 positive patients with melanoma-associated antigen A1 (MAGEA1)-positive advanced solid tumors. TRIAL DESIGN: The 2+2 trial design was an algorithmic design based on a maximally acceptable dose-limiting toxicity (DLT) rate of 25% and the sample size was driven by the algorithmic design with a maximum of 16 patients. IMA202 consists of autologous genetically modified cytotoxic CD8+ T cells expressing a T cell receptor (TCR), which is specific for a nine amino acid peptide derived from MAGEA1. Eligible patients underwent leukapheresis, T cells were isolated, transduced with lentiviral vector carrying MAGEA1-specific TCR and following lymphodepletion (fludarabine/cyclophosphamide), infused with a median of 1.4×109 specific T cells (range, 0.086×109-2.57×109) followed by interleukin 2. SAFETY OF IMA202: No DLT was observed. The most common grade 3-4 adverse events were cytopenias, that is, neutropenia (81.3%), lymphopenia (75.0%), anemia (50.0%), thrombocytopenia (50.0%) and leukopenia (25.0%). 13 patients experienced cytokine release syndrome, including one grade 3 event. Immune effector cell-associated neurotoxicity syndrome was observed in two patients and was grade 1 in both. EFFICACY OF IMA202: Of the 16 patients dosed, 11 (68.8%) patients had stable disease (SD) as their best overall response (Response Evaluation Criteria in Solid Tumors V.1.1). Five patients had initial tumor shrinkage in target lesions and one patient with SD experienced continued shrinkage in target lesions for 3 months in total but had to be classified as progressive disease due to progressive non-target lesions. IMA202 T cells were persistent in peripheral blood for several weeks to months and were also detectable in tumor tissue. Peak persistence was higher in patients who received higher doses. CONCLUSION: In conclusion, IMA202 had a manageable safety profile, and it was associated with biological and potential clinical activity of MAGEA1-targeting genetically engineered TCR-T cells in a poor prognosis, multi-indication solid tumor cohort. TRIAL REGISTRATION NUMBERS: NCT04639245, NCT05430555.

An atlas of epithelial cell states and plasticity in lung adenocarcinoma.

Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8+ alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8+ cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.

A randomized, non-comparative phase 2 study of neoadjuvant immune-checkpoint blockade in retroperitoneal dedifferentiated liposarcoma and extremity/truncal undifferentiated pleomorphic sarcoma.

Based on the demonstrated clinical activity of immune-checkpoint blockade (ICB) in advanced dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS), we conducted a randomized, non-comparative phase 2 trial ( NCT03307616 ) of neoadjuvant nivolumab or nivolumab/ipilimumab in patients with resectable retroperitoneal DDLPS (n = 17) and extremity/truncal UPS (+ concurrent nivolumab/radiation therapy; n = 10). The primary end point of pathologic response (percent hyalinization) was a median of 8.8% in DDLPS and 89% in UPS. Secondary end points were the changes in immune infiltrate, radiographic response, 12- and 24-month relapse-free survival and overall survival. Lower densities of regulatory T cells before treatment were associated with a major pathologic response (hyalinization > 30%). Tumor infiltration by B cells was increased following neoadjuvant treatment and was associated with overall survival in DDLPS. B cell infiltration was associated with higher densities of regulatory T cells before treatment, which was lost upon ICB treatment. Our data demonstrate that neoadjuvant ICB is associated with complex immune changes within the tumor microenvironment in DDLPS and UPS and that neoadjuvant ICB with concurrent radiotherapy has significant efficacy in UPS.

New Insights into Macrophage Polarization and its Prognostic Role in Patients with Colorectal Cancer Liver Metastasis.

Background: As liver metastasis is the most common cause of mortality in patients with colorectal cancer, studying colorectal cancer liver metastasis (CLM) microenvironment is essential for improved understanding of tumor biology and to identify novel therapeutic targets. Methods: We used multiplex immunofluorescence platform to study tumor associated macrophage (TAM) polarization and adaptive T cell subtypes in tumor samples from 105 CLM patients (49 without and 56 with preoperative chemotherapy). Results: CLM exhibited M2 macrophage polarization, and helper T cells were the prevalent adaptive T cell subtype. The density of total, M2 and TGFß-expressing macrophages, and regulatory T cells was lower in CLM treated with preoperative chemotherapy. CLM with right-sided primary demonstrated enrichment of TGFß-expressing macrophages, and with left-sided primary had higher densities of helper and cytotoxic T cells. In multivariate analysis, high density of M2 macrophages correlated with longer recurrence-free survival (RFS) in the entire cohort [hazard ratio (HR) 0.425, 95% CI 0.219-0.825, p=0.011) and in patients without preoperative chemotherapy (HR 0.45, 95% CI 0.221-0.932, p=0.032). High pSMAD3-expressing macrophages were associated with shorter RFS in CLM after preoperative chemotherapy. Conclusions: Our results highlight the significance of a multi-marker approach to define the macrophage subtypes and identify M2 macrophages as a predictor of favorable prognosis in CLM.

Prolonged cytopenia following CD19 CAR T cell therapy is linked with bone marrow infiltration of clonally expanded IFNγ-expressing CD8 T cells.

Autologous anti-CD19 chimeric antigen receptor T cell (CAR T) therapy is highly effective in relapsed/refractory large B cell lymphoma (rrLBCL) but is associated with toxicities that delay recovery. While the biological mechanisms of cytokine release syndrome and neurotoxicity have been investigated, the pathophysiology is poorly understood for prolonged cytopenia, defined as grade ≥3 cytopenia lasting beyond 30 days after CAR T infusion. We performed single-cell RNA sequencing of bone marrow samples from healthy donors and rrLBCL patients with or without prolonged cytopenia and identified significantly increased frequencies of clonally expanded CX3CR1hi cytotoxic T cells, expressing high interferon (IFN)-γ and cytokine signaling gene sets, associated with prolonged cytopenia. In line with this, we found that hematopoietic stem cells from these patients expressed IFN-γ response signatures. IFN-γ deregulates hematopoietic stem cell self-renewal and differentiation and can be targeted with thrombopoietin agonists or IFN-γ-neutralizing antibodies, highlighting a potential mechanism-based approach for the treatment of CAR T-associated prolonged cytopenia.

Exploratory study of macrophage polarization and spatial distribution in colorectal cancer liver metastasis: a pilot study.

Background: The liver is the most typical site of metastatic disease for patients with colorectal cancer (CRC), and up to half the patients with CRC will develop colorectal liver metastasis (CLM). Studying the tumor microenvironment, particularly macrophages and their spatial distribution, can give us critical insight into treatment. Methods: Ten CLMs (five treatment-naïve and five post-neoadjuvant chemotherapy) were stained with multiplex immunofluorescence panels against cytokeratins, CD68, Arg1, CD206, CD86, CD163, PD-L1, and MRP8-14. Densities of cell phenotypes and their spatial distribution in the tumor center and the normal liver-tumor interface were correlated with clinicopathological variables. Results: M2 macrophages were the predominant subtype in both the tumor center and the periphery, with a relatively higher density at the periphery. The larger tumors, more than 3.9 cm, were associated with higher densities of total CD68+ macrophages and CD68+CD163+ CD206neg and CD68+CD206+ CD163neg M2 macrophage subtypes. Total macrophages in the tumor periphery demonstrated significantly greater proximity to malignant cells than did those in the tumor center (p=0.0371). The presence of higher than median CD68+MRP8-14+CD86neg M1 macrophages in the tumor center was associated with poor overall survival (median 2.34 years) compared to cases with lower than median M1 macrophages at the tumor center (median 6.41 years) in univariate analysis. Conclusion: The dominant polarization of the M2 macrophage subtype could drive new therapeutic approaches in CLM patients.

Cellular and Molecular Mechanisms of Liver Fibrosis in Patients with NAFLD.

The expression of immune- and cancer-related genes was measured in liver biopsies from 107 NAFLD patients. The strongest difference in overall gene expression was between liver fibrosis stages F3 and F4, with 162 cirrhosis-associated genes identified. Strong correlations with fibrosis progression from F1 to F4 were observed for 91 genes, including CCL21, CCL2, CXCL6, and CCL19. In addition, the expression of 21 genes was associated with fast progression to F3/F4 in an independent group of eight NAFLD patients. These included the four chemokines, SPP1, HAMP, CXCL2, and IL-8. A six-gene signature including SOX9, THY-1, and CD3D had the highest performance detecting the progressors among F1/F2 NAFLD patients. We also characterized immune cell changes using multiplex immunofluorescence platforms. Fibrotic areas were strongly enriched in CD3+ T cells compared to CD68+ macrophages. While the number of CD68+ macrophages increased with fibrosis severity, the increase in CD3+ T-cell density was more substantial and progressive from F1 to F4. The strongest correlation with fibrosis progression was observed for CD3+CD45R0+ memory T cells, while the most significant increase in density between F1/F2 and F3/F4 was for CD3+CD45RO+FOXP3+CD8- and CD3+CD45RO-FOXP3+CD8- regulatory T cells. A specific increase in the density of CD68+CD11b+ Kupffer cells with liver fibrosis progression was also observed.

Gene expression profiling and multiplex immunofluorescence analysis of bullous pemphigoid immune-related adverse event reveal upregulation of toll-like receptor 4/complement-induced innate immune response and increased density of TH 1 T-cells.

BACKGROUND: Immune checkpoint inhibitor (ICI)-based cancer therapies cause a variety of cutaneous immune-related adverse events (irAEs) including immunobullous skin eruptions like bullous pemphigoid (BP). However, little is known about the underlying immunopathogenic drivers of these reactions, and understanding the unique gene expression profile and immune composition of BP-irAE remains a critical knowledge gap in the field of oncodermatology/oncodermatopathology. METHODS: BP-irAE (n = 8) and de novo BP control (n = 8) biopsy samples were subjected to gene expression profiling using the NanoString® Technologies nCounter PanCancer Immune Profiling Panel. Multiplex immunofluorescence (mIF) studies using markers for T-cells (CD3 and CD8), T helper 1 (TH 1) cells (Tbet), TH 2 cells (Gata3), TH 17 cells (RORγT), and regulatory T-cells (Tregs; FoxP3) were further evaluated using InForm® image analysis. RESULTS: Compared with de novo BP controls, BP-irAE samples exhibited upregulation of 30 mRNA transcripts (p < 0.025), including toll-like receptor 4 (TLR4) and genes associated with complement activation, and downregulation of 89 mRNA transcripts (p < 0.025), including genes associated with TH 2, TH 17, and B-cell immune response. BP-irAE demonstrated a greater density of Tbet+ (TH 1) cells in the dermis (p = 0.004) and fewer Tregs in the blister floor (p = 0.028) when compared with that of de novo control BP samples. CONCLUSIONS: BP-irAE exhibited activation of the TLR4/complement-driven classical innate immune response pathway, with dermal TH 1 immune cell polarization and decreased Tregs in the blister floor. TLR/complement signaling may underlie the immunopathogenesis of BP-irAE.

Phase II trial of neoadjuvant sitravatinib plus nivolumab in patients undergoing nephrectomy for locally advanced clear cell renal cell carcinoma.

Sitravatinib is an immunomodulatory tyrosine kinase inhibitor that can augment responses when combined with programmed death-1 inhibitors such as nivolumab. We report a single-arm, interventional, phase 2 study of neoadjuvant sitravatinib in combination with nivolumab in patients with locally advanced clear cell renal cell carcinoma (ccRCC) prior to curative nephrectomy (NCT03680521). The primary endpoint was objective response rate (ORR) prior to surgery with a null hypothesis ORR = 5% and the alternative hypothesis set at ORR = 30%. Secondary endpoints were safety; pharmacokinetics (PK) of sitravatinib; immune effects, including changes in programmed cell death-ligand 1 expression; time-to-surgery; and disease-free survival (DFS). Twenty patients were evaluable for safety and 17 for efficacy. The ORR was 11.8%, and 24-month DFS probability was 88·0% (95% CI 61.0 to 97.0). There were no grade 4/5 treatment-related adverse events. Sitravatinib PK did not change following the addition of nivolumab. Correlative blood and tissue analyses showed changes in the tumour microenvironment resulting in an immunologically active tumour by the time of surgery (median time-to-surgery: 50 days). The primary endpoint of this study was not met as short-term neoadjuvant sitravatinib and nivolumab did not substantially increase ORR.

PD-L1+ macrophages are associated with favorable features in primary mediastinal (thymic) large B-cell lymphoma.

Primary mediastinal (thymic) large B-cell lymphoma (PMBCL) is a rare, aggressive subtype of non-Hodgkin lymphoma and has a complex inflammatory microenvironment. Although most patients can be cured with standard-of-care immunochemotherapy, patients who have disease relapse have an unfavorable prognosis. Pre-treatment prognostic biomarkers in PMBCL are needed. In this retrospective study, we analyzed the clinical features and outcomes of PMBCL patients and their association with immune cell subpopulations identified by multiplex immunofluorescence at initial diagnosis. Two different antibody panels were used to assess macrophages in tissue biopsy specimens collected before the initiation of induction therapy. Twelve PMBCL patients, including five patients who had disease relapse, were included in the analysis. At a median follow-up time of 32.2 months, the median progression-free and overall survival durations were not reached. Our findings suggest that a high density of PD-L1+ macrophages is associated with favorable features, such as early disease stage and the absence of B-symptoms, and indicate that a high percentage of PD-L1+ macrophages and high densities of CD30+PD-L1+ cells and CD30+ cells might be associated with a lower risk of relapse within 12 months of therapy initiation. Further studies are needed to develop a biomarker signature predictive of treatment response with therapeutic consequences for patients with newly diagnosed PMBCL.

Neoadjuvant chemotherapy plus nivolumab with or without ipilimumab in operable non-small cell lung cancer: the phase 2 platform NEOSTAR trial.

Neoadjuvant ipilimumab + nivolumab (Ipi+Nivo) and nivolumab + chemotherapy (Nivo+CT) induce greater pathologic response rates than CT alone in patients with operable non-small cell lung cancer (NSCLC). The impact of adding ipilimumab to neoadjuvant Nivo+CT is unknown. Here we report the results and correlates of two arms of the phase 2 platform NEOSTAR trial testing neoadjuvant Nivo+CT and Ipi+Nivo+CT with major pathologic response (MPR) as the primary endpoint. MPR rates were 32.1% (7/22, 80% confidence interval (CI) 18.7-43.1%) in the Nivo+CT arm and 50% (11/22, 80% CI 34.6-61.1%) in the Ipi+Nivo+CT arm; the primary endpoint was met in both arms. In patients without known tumor EGFR/ALK alterations, MPR rates were 41.2% (7/17) and 62.5% (10/16) in the Nivo+CT and Ipi+Nivo+CT groups, respectively. No new safety signals were observed in either arm. Single-cell sequencing and multi-platform immune profiling (exploratory endpoints) underscored immune cell populations and phenotypes, including effector memory CD8+ T, B and myeloid cells and markers of tertiary lymphoid structures, that were preferentially increased in the Ipi+Nivo+CT cohort. Baseline fecal microbiota in patients with MPR were enriched with beneficial taxa, such as Akkermansia, and displayed reduced abundance of pro-inflammatory and pathogenic microbes. Neoadjuvant Ipi+Nivo+CT enhances pathologic responses and warrants further study in operable NSCLC. (ClinicalTrials.gov registration: NCT03158129 .).

CD30 expression is frequently decreased in relapsed classic Hodgkin lymphoma after anti-CD30 CAR T-cell therapy.

Chimeric antigen receptor (CAR) T-cells anti-CD30 is an innovative therapeutic option that has been used to treat cases of refractory/relapsed (R/R) classic Hodgkin lymphoma (CHL). Limited data are available regarding the CD30 expression status of patients who relapsed after this therapy. This is the first study to show decreased CD30 expression in R/R CHL in patients (n = 5) who underwent CAR T-cell therapy in our institution between 2018 and 2022. Although conventional immunohistochemical assays showed decreased CD30 expression in neoplastic cells in all cases (8/8) the tyramide amplification assay and RNAScope in situ hybridisation detected CD30 expression at different levels in 100% (n = 8/8) and 75% (n = 3/4), respectively. Hence, our findings document that certain levels of CD30 expression are retained by the neoplastic cells. This is not only of biological interest but also diagnostically important, as detection of CD30 is an essential factor in establishing a diagnosis of CHL.

Analysis of Immune Intratumor Heterogeneity Highlights Immunoregulatory and Coinhibitory Lymphocytes as Hallmarks of Recurrence in Stage I Non-Small Cell Lung Cancer.

Our understanding of the molecular mechanisms underlying postsurgical recurrence of non-small cell lung cancer (NSCLC) is rudimentary. Molecular and T cell repertoire intratumor heterogeneity (ITH) have been reported to be associated with postsurgical relapse; however, how ITH at the cellular level impacts survival is largely unknown. Here we report the analysis of 2880 multispectral images representing 14.2% to 27% of tumor areas from 33 patients with stage I NSCLC, including 17 cases (relapsed within 3 years after surgery) and 16 controls (without recurrence ≥5 years after surgery) using multiplex immunofluorescence. Spatial analysis was conducted to quantify the minimum distance between different cell types and immune cell infiltration around malignant cells. Immune ITH was defined as the variance of immune cells from 3 intratumor regions. We found that tumors from patients having relapsed display different immune biology compared with nonrecurrent tumors, with a higher percentage of tumor cells and macrophages expressing PD-L1 (P =.031 and P =.024, respectively), along with an increase in regulatory T cells (Treg) (P =.018), antigen-experienced T cells (P =.025), and effector-memory T cells (P =.041). Spatial analysis revealed that a higher level of infiltration of PD-L1+ macrophages (CD68+PD-L1+) or antigen-experienced cytotoxic T cells (CD3+CD8+PD-1+) in the tumor was associated with poor overall survival (P =.021 and P =.006, respectively). A higher degree of Treg ITH was associated with inferior recurrence-free survival regardless of tumor mutational burden (P =.022), neoantigen burden (P =.021), genomic ITH (P =.012) and T cell repertoire ITH (P =.001). Using multiregion multiplex immunofluorescence, we characterized ITH at the immune cell level along with whole exome and T cell repertoire sequencing from the same tumor regions. This approach highlights the role of immunoregulatory and coinhibitory signals as well as their spatial distribution and ITH that define the hallmarks of tumor relapse of stage I NSCLC.

Spatial PD-L1, immune-cell microenvironment, and genomic copy-number alteration patterns and drivers of invasive-disease transition in prospective oral precancer cohort.

BACKGROUND: Studies of the immune landscape led to breakthrough trials of programmed death-1 (PD-1) inhibitors for recurrent/metastatic head and neck squamous cell carcinoma therapy. This study investigated the timing, influence of somatic copy-number alterations (SCNAs), and clinical implications of PD-L1 and immune-cell patterns in oral precancer (OPC). METHODS: The authors evaluated spatial CD3, CD3/8, and CD68 density (cells/mm2 ) and PD-L1 (membranous expression in cytokeratin-positive intraepithelial neoplastic cells and CD68) patterns by multiplex immunofluorescence in a 188-patient prospective OPC cohort, characterized by clinical, histologic, and SCNA risk factors and protocol-specified primary end point of invasive cancer. The authors used Wilcoxon rank-sum and Fisher exact tests, linear mixed effect models, mediation, and Cox regression and recursive-partitioning analyses. RESULTS: Epithelial, but not CD68 immune-cell, PD-L1 expression was detected in 28% of OPCs, correlated with immune-cell infiltration, 9p21.3 loss of heterozygosity (LOH), and inferior oral cancer-free survival (OCFS), notably in OPCs with low CD3/8 cell density, dysplasia, and/or 9p21.3 LOH. High CD3/8 cell density in dysplastic lesions predicted better OCFS and eliminated the excess risk associated with prior oral cancer and dysplasia. PD-L1 and CD3/8 patterns revealed inferior OCFS in PD-L1 high intrinsic induction and dysplastic immune-cold subgroups. CONCLUSION: This report provides spatial insight into the immune landscape and drivers of OPCs, and a publicly available immunogenomic data set for future precancer interrogation. The data suggest that 9p21.3 LOH triggers an immune-hot inflammatory phenotype; whereas increased 9p deletion size encompassing CD274 at 9p24.1 may contribute to CD3/8 and PD-L1 depletion during invasive transition. The inferior OCFS in PD-L1-high, immune-cold OPCs support the development of T-cell recruitment strategies.

Urinary T cells are detected in patients with immune checkpoint inhibitor-associated immune nephritis that are clonotypically identical to kidney T cell infiltrates.

Acute kidney injury (AKI) occurs in ~20% of patients receiving immune checkpoint inhibitor (ICI) therapy; however, only 2-5% will develop ICI-mediated immune nephritis. Conventional tests are nonspecific in diagnosing disease pathology and invasive procedures (i.e. kidney biopsy) may not be feasible. In other autoimmune renal diseases, urinary immune cells correlated with the pathology or were predictive of disease activity. Corresponding evidence and analysis are absent for ICI-mediated immune nephritis. We report the first investigation analyzing immune cell profiles of matched kidney biopsies and urine of patients with ICI-AKI. We demonstrated the presence of urinary T cells in patients with immune nephritis by flow cytometry analysis. Clonotype analysis of T cell receptor (TCR) sequences confirmed enrichment of kidney TCRs in urine. As ICI therapies become standard of care for more cancers, noninvasively assessing urinary immune cells of ICI therapy recipients can facilitate clinical management and an opportunity to tailor ICI-nephritis treatment.

The immune landscape of undifferentiated pleomorphic sarcoma.

Introduction: Undifferentiated pleomorphic sarcoma (UPS) can be associated with a relatively dense immune infiltration. Immune checkpoint inhibitors (anti-PD1, anti-PDL1, and anti-CTLA4) are effective in 20% of UPS patients. We characterize the immune microenvironment of UPS and its association with oncologic outcomes. Material and methods: Surgically resected UPS samples were stained by immunohistochemistry (IHC) for the following: tumor-associated immune cells (CD3, CD8, CD163, CD20), immune checkpoints (stimulatory: OX40, ICOS; inhibitory: PD-L1, LAG3, IDO1, PD1), and the adenosine pathway (CD73, CD39). Sections were reviewed for the presence of lymphoid aggregates (LA). Clinical data were retrospectively obtained for all samples. The Wilcoxon rank-sum and Kruskal-Wallis tests were used to compare distributions. Correlations between biomarkers were measured by Spearman correlation. Univariate and multivariate Cox models were used to identify biomarkers associated with overall survival (OS) and disease-free survival (DFS). Unsupervised clustering was performed, and Kaplan-Meier curves and log-rank tests used for comparison of OS and DFS between immune clusters. Results: Samples analyzed (n=105) included 46 primary tumors, 34 local recurrences, and 25 metastases. LA were found in 23% (n=10/43), 17% (n=4/24), and 30% (n=7/23) of primary, recurrent, and metastatic samples, respectively. In primary UPS, CD73 expression was significantly higher after preoperative radiation therapy (p=0.009). CD39 expression was significantly correlated with PD1 expression (primary: p=0.002, recurrent: p=0.004, metastatic: p=0.001), PD-L1 expression (primary: p=0.009), and CD3+ cell densities (primary: p=0.016, recurrent: p=0.043, metastatic: p=0.028). In recurrent tumors, there was a strong correlation between CD39 and CD73 (p=0.015), and both were also correlated with CD163+ cell densities (CD39 p=0.013; CD73 p<0.001). In multivariate analyses, higher densities of CD3+ and CD8+ cells (Cox Hazard Ratio [HR]=0.33; p=0.010) were independently associated with OS (CD3+, HR=0.19, p<0.001; CD8+, HR= 0.33, p=0.010) and DFS (CD3+, HR=0.34, p=0.018; CD8+, HR=0.34, p= 0.014). Unsupervised clustering of IHC values revealed three immunologically distinct clusters: immune high, intermediate, and low. In primary tumors, these clusters were significantly associated with OS (log-rank p<0.0001) and DFS (p<0.001). Conclusion: We identified three immunologically distinct clusters of UPS Associated with OS and DFS. Our data support further investigations of combination anti-PD-1/PD-L1 and adenosine pathway inhibitors in UPS.

The Single-Cell Immunogenomic Landscape of B and Plasma Cells in Early-Stage Lung Adenocarcinoma.

Tumor-infiltrating B and plasma cells (TIB) are prevalent in lung adenocarcinoma (LUAD); however, they are poorly characterized. We performed paired single-cell RNA and B-cell receptor (BCR) sequencing of 16 early-stage LUADs and 47 matching multiregion normal tissues. By integrative analysis of ∼50,000 TIBs, we define 12 TIB subsets in the LUAD and adjacent normal ecosystems and demonstrate extensive remodeling of TIBs in LUADs. Memory B cells and plasma cells (PC) were highly enriched in tumor tissues with more differentiated states and increased frequencies of somatic hypermutation. Smokers exhibited markedly elevated PCs and PCs with distinct differentiation trajectories. BCR clonotype diversity increased but clonality decreased in LUADs, smokers, and with increasing pathologic stage. TIBs were mostly localized within CXCL13+ lymphoid aggregates, and immune cell sources of CXCL13 production evolved with LUAD progression and included elevated fractions of CD4 regulatory T cells. This study provides a spatial landscape of TIBs in early-stage LUAD. SIGNIFICANCE: While TIBs are highly enriched in LUADs, they are poorly characterized. This study provides a much-needed understanding of the transcriptional, clonotypic states and phenotypes of TIBs, unraveling their potential roles in the immunopathology of early-stage LUADs and constituting a road map for the development of TIB-targeted immunotherapies for the treatment of this morbid malignancy. This article is highlighted in the In This Issue feature, p. 2483.