A spatial cell atlas of neuroblastoma reveals developmental, epigenetic and spatial axis of tumor heterogeneity.

Neuroblastoma is a pediatric cancer arising from the developing sympathoadrenal lineage with complex inter- and intra-tumoral heterogeneity. To chart this complexity, we generated a comprehensive cell atlas of 55 neuroblastoma patient tumors, collected from two pediatric cancer institutions, spanning a range of clinical, genetic, and histologic features. Our atlas combines single-cell/nucleus RNA-seq (sc/scRNA-seq), bulk RNA-seq, whole exome sequencing, DNA methylation profiling, spatial transcriptomics, and two spatial proteomic methods. Sc/snRNA-seq revealed three malignant cell states with features of sympathoadrenal lineage development. All of the neuroblastomas had malignant cells that resembled sympathoblasts and the more differentiated adrenergic cells. A subset of tumors had malignant cells in a mesenchymal cell state with molecular features of Schwann cell precursors. DNA methylation profiles defined four groupings of patients, which differ in the degree of malignant cell heterogeneity and clinical outcomes. Using spatial proteomics, we found that neuroblastomas are spatially compartmentalized, with malignant tumor cells sequestered away from immune cells. Finally, we identify spatially restricted signaling patterns in immune cells from spatial transcriptomics. To facilitate the visualization and analysis of our atlas as a resource for further research in neuroblastoma, single cell, and spatial-omics, all data are shared through the Human Tumor Atlas Network Data Commons at www.humantumoratlas.org.

Genomic and Immunophenotypic Landscape of Acquired Resistance to PD-(L)1 Blockade in Non-Small-Cell Lung Cancer.

PURPOSE: Although immune checkpoint inhibitors (ICI) have extended survival in patients with non-small-cell lung cancer (NSCLC), acquired resistance (AR) to ICI frequently develops after an initial benefit. However, the mechanisms of AR to ICI in NSCLC are largely unknown. METHODS: Comprehensive tumor genomic profiling, machine learning-based assessment of tumor-infiltrating lymphocytes, multiplexed immunofluorescence, and/or HLA-I immunohistochemistry (IHC) were performed on matched pre- and post-ICI tumor biopsies from patients with NSCLC treated with ICI at the Dana-Farber Cancer Institute who developed AR to ICI. Two additional cohorts of patients with intervening chemotherapy or targeted therapies between biopsies were included as controls. RESULTS: We performed comprehensive genomic profiling and immunophenotypic characterization on samples from 82 patients with NSCLC and matched pre- and post-ICI biopsies and compared findings with a control cohort of patients with non-ICI intervening therapies between biopsies (chemotherapy, N = 32; targeted therapies, N = 89; both, N = 17). Putative resistance mutations were identified in 27.8% of immunotherapy-treated cases and included acquired loss-of-function mutations in STK11, B2M, APC, MTOR, KEAP1, and JAK1/2; these acquired alterations were not observed in the control groups. Immunophenotyping of matched pre- and post-ICI samples demonstrated significant decreases in intratumoral lymphocytes, CD3e+ and CD8a+ T cells, and PD-L1-PD1 engagement, as well as increased distance between tumor cells and CD8+PD-1+ T cells. There was a significant decrease in HLA class I expression in the immunotherapy cohort at the time of AR compared with the chemotherapy (P = .005) and the targeted therapy (P = .01) cohorts. CONCLUSION: These findings highlight the genomic and immunophenotypic heterogeneity of ICI resistance in NSCLC, which will need to be considered when developing novel therapeutic strategies aimed at overcoming resistance.

Biomarkers of pembrolizumab efficacy in advanced anal squamous cell carcinoma: analysis of a phase II clinical trial and a cohort of long-term responders.

BACKGROUND: Recent trials suggest that programmed cell death 1 (PD-1)-directed immunotherapy may be beneficial for some patients with anal squamous cell carcinoma and biomarkers predictive of response are greatly needed. METHODS: This multicenter phase II clinical trial (NCT02919969) enrolled patients with metastatic or locally advanced incurable anal squamous cell carcinoma (n=32). Patients received pembrolizumab 200 mg every 3 weeks. The primary endpoint of the trial was objective response rate (ORR). Exploratory objectives included analysis of potential predictive biomarkers including assessment of tumor-associated immune cell populations with multichannel immunofluorescence and analysis of circulating tumor tissue modified viral-human papillomavirus DNA (TTMV-HPV DNA) using serially collected blood samples. To characterize the clinical features of long-term responders, we combined data from our prospective trial with a retrospective cohort of patients with anal cancer treated with anti-PD-1 immunotherapy (n=18). RESULTS: In the phase II study, the ORR to pembrolizumab monotherapy was 9.4% and the median progression-free survival was 2.2 months. Despite the high level of HPV positivity observed with circulating TTMV-HPV DNA testing, the majority of patients had low levels of tumor-associated CD8+PD-1+ T cells on pretreatment biopsy. Patients who benefited from pembrolizumab had decreasing TTMV-HPV DNA scores and a complete responder's TTMV-HPV DNA became undetectable. Long-term pembrolizumab responses were observed in one patient from the trial (5.3 years) and three patients (2.5, 6, and 8 years) from the retrospective cohort. Long-term responders had HPV-positive tumors, lacked liver metastases, and achieved a radiological complete response. CONCLUSIONS: Pembrolizumab has durable efficacy in a rare subset of anal cancers. However, despite persistence of HPV infection, indicated by circulating HPV DNA, most advanced anal cancers have low numbers of tumor-associated CD8+PD-1+ T cells and are resistant to pembrolizumab.

A cellular and spatial atlas of TP53 -associated tissue remodeling in lung adenocarcinoma.

TP53 is the most frequently mutated gene across many cancers and is associated with shorter survival in lung adenocarcinoma (LUAD). To define how TP53 mutations affect the LUAD tumor microenvironment (TME), we constructed a multi-omic cellular and spatial tumor atlas of 23 treatment-naïve human lung tumors. We found that TP53 -mutant ( TP53 mut ) malignant cells lose alveolar identity and upregulate highly proliferative and entropic gene expression programs consistently across resectable LUAD patient tumors, genetically engineered mouse models, and cell lines harboring a wide spectrum of TP53 mutations. We further identified a multicellular tumor niche composed of SPP1 + macrophages and collagen-expressing fibroblasts that coincides with hypoxic, pro-metastatic expression programs in TP53 mut tumors. Spatially correlated angiostatic and immune checkpoint interactions, including CD274 - PDCD1 and PVR - TIGIT , are also enriched in TP53 mut LUAD tumors, which may influence response to checkpoint blockade therapy. Our methodology can be further applied to investigate mutation-specific TME changes in other cancers.

Multicenter randomized controlled trial of neoadjuvant chemoradiotherapy alone or in combination with pembrolizumab in patients with resectable or borderline resectable pancreatic adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a challenging target for immunotherapy because it has an immunosuppressive tumor microenvironment. Neoadjuvant chemoradiotherapy can increase tumor-infiltrating lymphocyte (TIL) density, which may predict overall survival (OS). We hypothesized that adding programmed cell death protein 1 (PD-1) blockade to chemoradiotherapy would be well tolerated and increase TILs among patients with localized PDAC. METHODS: Patients were randomized 2:1 to Arm A (receiving pembrolizumab plus chemoradiotherapy (capecitabine and external beam radiation)) or Arm B (receiving chemoradiotherapy alone) before anticipated pancreatectomy. Primary endpoints were (1) incidence and severity of adverse events during neoadjuvant therapy and (2) density of TILs in resected tumor specimens. TIL density was assessed using multiplexed immunofluorescence histologic examination. RESULTS: Thirty-seven patients were randomized to Arms A (n=24) and B (n=13). Grade ≥3 adverse events related to neoadjuvant treatment were experienced by 9 (38%) and 4 (31%) patients in Arms A and B, respectively, with one patient experiencing dose-limiting toxicity in Arm A. Seventeen (71%) and 7 (54%) patients in Arms A and B, respectively, underwent pancreatectomy. Median CD8+ T-cell densities in Arms A and B were 67.4 (IQR: 39.2-141.8) and 37.9 (IQR: 22.9-173.4) cells/mm2, respectively. Arms showed no noticeable differences in density of CD8+Ki67+, CD4+, or CD4+FOXP3+ regulatory T cells; M1-like and M2-like macrophages; or granulocytes. Median OS durations were 27.8 (95% CI: 17.1 to NR) and 24.3 (95% CI: 12.6 to NR) months for Arms A and B, respectively. CONCLUSIONS: Adding pembrolizumab to neoadjuvant chemoradiotherapy was safe. However, no convincing effect on CD8+ TILs was observed.

Systematic benchmarking of imaging spatial transcriptomics platforms in FFPE tissues.

Emerging imaging spatial transcriptomics (iST) platforms and coupled analytical methods can recover cell-to-cell interactions, groups of spatially covarying genes, and gene signatures associated with pathological features, and are thus particularly well-suited for applications in formalin fixed paraffin embedded (FFPE) tissues. Here, we benchmarked the performance of three commercial iST platforms on serial sections from tissue microarrays (TMAs) containing 23 tumor and normal tissue types for both relative technical and biological performance. On matched genes, we found that 10x Xenium shows higher transcript counts per gene without sacrificing specificity, but that all three platforms concord to orthogonal RNA-seq datasets and can perform spatially resolved cell typing, albeit with different false discovery rates, cell segmentation error frequencies, and with varying degrees of sub-clustering for downstream biological analyses. Taken together, our analyses provide a comprehensive benchmark to guide the choice of iST method as researchers design studies with precious samples in this rapidly evolving field.

A multidimensional analysis reveals distinct immune phenotypes and tertiary lymphoid structure-like aggregates in the bone marrow of pediatric acute myeloid leukemia.

Because of the low mutational burden, children with acute myeloid leukemia (AML) are thought to have a 'cold' tumor microenvironment and consequently, a low likelihood of response to T cell-directed immunotherapies. Here, we provide a multidimensional overview of the tumor immune microenvironment in newly diagnosed pediatric AML. On a cohort level, we demonstrate wide variation in T cell infiltration with nearly one-third of cases harboring an immune-infiltrated bone marrow. These immune-infiltrated cases are characterized by a decreased abundance of M2-like macrophages, which we find to be associated with response to T cell-directed immunotherapy in adult AML. On an organizational level, we reveal the composition of spatially organized immune aggregates in pediatric AML, and show that in the adult setting such aggregates in post-treatment bone marrow and extramedullary sites associate with response to ipilimumab-based therapy. Altogether, our study provides immune correlates of response to T cell-directed immunotherapies and indicates starting points for further investigations into immunomodulatory mechanisms in AML.

Intertumoral lineage diversity and immunosuppressive transcriptional programs in well-differentiated gastroenteropancreatic neuroendocrine tumors.

Neuroendocrine tumors (NETs) are rare cancers that most often arise in the gastrointestinal tract and pancreas. The fundamental mechanisms driving gastroenteropancreatic (GEP)-NET growth remain incompletely elucidated; however, the heterogeneous clinical behavior of GEP-NETs suggests that both cellular lineage dynamics and tumor microenvironment influence tumor pathophysiology. Here, we investigated the single-cell transcriptomes of tumor and immune cells from patients with gastroenteropancreatic NETs. Malignant GEP-NET cells expressed genes and regulons associated with normal, gastrointestinal endocrine cell differentiation, and fate determination stages. Tumor and lymphoid compartments sparsely expressed immunosuppressive targets commonly investigated in clinical trials, such as the programmed cell death protein-1/programmed death ligand-1 axis. However, infiltrating myeloid cell types within both primary and metastatic GEP-NETs were enriched for genes encoding other immune checkpoints, including VSIR (VISTA), HAVCR2 (TIM3), LGALS9 (Gal-9), and SIGLEC10. Our findings highlight the transcriptomic heterogeneity that distinguishes the cellular landscapes of GEP-NET anatomic subtypes and reveal potential avenues for future precision medicine therapeutics.

Preexisting tumor-resident T cells with cytotoxic potential associate with response to neoadjuvant anti-PD-1 in head and neck cancer.

About 50% of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) experience recurrences after definitive therapy. The presurgical administration of anti-programmed cell death protein 1 (PD-1) immunotherapy results in substantial pathologic tumor responses (pTR) within the tumor microenvironment (TME). However, the mechanisms underlying the dynamics of antitumor T cells upon neoadjuvant PD-1 blockade remain unresolved, and approaches to increase pathologic responses are lacking. In a phase 2 trial (NCT02296684), we observed that 45% of patients treated with two doses of neoadjuvant pembrolizumab experienced marked pTRs (≥50%). Single-cell analysis of 17,158 CD8+ T cells from 14 tumor biopsies, including 6 matched pre-post neoadjuvant treatment, revealed that responding tumors had clonally expanded putative tumor-specific exhausted CD8+ tumor-infiltrating lymphocytes (TILs) with a tissue-resident memory program, characterized by high cytotoxic potential (CTX+) and ZNF683 expression, within the baseline TME. Pathologic responses after 5 weeks of PD-1 blockade were consistent with activation of preexisting CTX+ZNF683+CD8+ TILs, paralleling loss of viable tumor and associated tumor antigens. Response was associated with high numbers of CD103+PD-1+CD8+ T cells infiltrating pretreatment lesions, whereas revival of nonexhausted persisting clones and clonal replacement were modest. By contrast, nonresponder baseline TME exhibited a relative absence of ZNF683+CTX+ TILs and subsequent accumulation of highly exhausted clones. In HNSCC, revival of preexisting ZNF683+CTX+ TILs is a major mechanism of response in the immediate postneoadjuvant setting.

Targeting KDM2A Enhances T-cell Infiltration in NSD1-Deficient Head and Neck Squamous Cell Carcinoma.

In head and neck squamous cell carcinoma (HNSCC), a significant proportion of tumors have inactivating mutations in the histone methyltransferase NSD1. In these tumors, NSD1 inactivation is a driver of T-cell exclusion from the tumor microenvironment (TME). A better understanding of the NSD1-mediated mechanism regulating infiltration of T cells into the TME could help identify approaches to overcome immunosuppression. Here, we demonstrated that NSD1 inactivation results in lower levels of H3K36 dimethylation and higher levels of H3K27 trimethylation, the latter being a known repressive histone mark enriched on the promoters of key T-cell chemokines CXCL9 and CXCL10. HNSCC with NSD1 mutations had lower levels of these chemokines and lacked responses to PD-1 immune checkpoint blockade. Inhibition of KDM2A, the primary lysine demethylase that is selective for H3K36, reversed the altered histone marks induced by NSD1 loss and restored T-cell infiltration into the TME. Importantly, KDM2A suppression decreased growth of NSD1-deficient tumors in immunocompetent, but not in immunodeficient, mice. Together, these data indicate that KDM2A is an immunotherapeutic target for overcoming immune exclusion in HNSCC. SIGNIFICANCE: The altered epigenetic landscape of NSD1-deficient tumors confers sensitivity to inhibition of the histone-modifying enzyme KDM2A as an immunotherapeutic strategy to stimulate T-cell infiltration and suppress tumor growth.

Impact of Aneuploidy and Chromosome 9p Loss on Tumor Immune Microenvironment and Immune Checkpoint Inhibitor Efficacy in NSCLC.

INTRODUCTION: Although gene-level copy number alterations have been studied as a potential biomarker of immunotherapy efficacy in NSCLC, the impact of aneuploidy burden and chromosomal arm-level events on immune checkpoint inhibitor (ICI) efficacy in NSCLC is uncertain. METHODS: Patients who received programmed cell death protein 1 or programmed death-ligand 1 (PD-L1) inhibitor at two academic centers were included. Across all 22 chromosomes analyzed, an arm was considered altered if at least 70% of its territory was either gained or deleted. Among nonsquamous NSCLCs which underwent targeted next-generation sequencing, we retrospectively quantified aneuploidy using the adjusted fraction of chromosomal arm alterations (FAA), defined as the number of altered chromosome arms divided by the number of chromosome arms assessed, adjusted for tumor purity. RESULTS: Among 2293 nonsquamous NSCLCs identified, the median FAA increased with more advanced cancer stage and decreased with higher PD-L1 tumor proportion score (TPS) levels (median FAA in TPS < 1%: 0.09, TPS 1%-49%: 0.08, TPS ≥ 50%: 0.05, p < 0.0001). There was a very weak correlation between FAA and tumor mutational burden when taken as continuous variables (R: 0.07, p = 0.0005). A total of 765 advanced nonsquamous NSCLCs with available FAA values were treated with ICIs. With decreasing FAA tertiles, there was a progressive improvement in objective response rate (ORR 15.1% in upper tertile versus 23.2% in middle tertile versus 28.4% in lowest tertile, p = 0.001), median progression-free survival (mPFS 2.5 versus 3.3 versus 4.1 mo, p < 0.0001), and median overall survival (mOS 12.5 versus 13.9 versus 16.4 mo, p = 0.006), respectively. In the arm-level enrichment analysis, chromosome 9p loss (OR = 0.22, Q = 0.0002) and chromosome 1q gain (OR = 0.43, Q = 0.002) were significantly enriched in ICI nonresponders after false discovery rate adjustment. Compared with NSCLCs without chromosome 9p loss (n = 452), those with 9p loss (n = 154) had a lower ORR (28.1% versus 7.8%, p < 0.0001), a shorter mPFS (4.1 versus 2.3 mo, p < 0.0001), and a shorter mOS (18.0 versus 9.6 mo, p < 0.0001) to immunotherapy. In addition, among NSCLCs with high PD-L1 expression (TPS ≥ 50%), chromosome 9p loss was associated with lower ORR (43% versus 6%, p < 0.0001), shorter mPFS (6.4 versus 2.6 mo, p = 0.0006), and shorter mOS (30.2 versus 14.3 mo, p = 0.0008) to immunotherapy compared with NSCLCs without 9p loss. In multivariable analysis, adjusting for key variables including FAA, chromosome 9p loss, but not 1q gain, retained a significant impact on ORR (hazard ratio [HR] = 0.25, p < 0.001), mPFS (HR = 1.49, p = 0.001), and mOS (HR = 1.47, p = 0.003). Multiplexed immunofluorescence and computational deconvolution of RNA sequencing data revealed that tumors with either high FAA levels or chromosome 9p loss had significantly fewer tumor-associated cytotoxic immune cells. CONCLUSIONS: Nonsquamous NSCLCs with high aneuploidy and chromosome 9p loss have a distinct tumor immune microenvironment and less favorable outcomes to ICIs.

High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly.

Resolving fundamental molecular and functional processes underlying human synaptic development is crucial for understanding normal brain function as well as dysfunction in disease. Based upon increasing evidence of species-divergent features of brain cell types, coupled with emerging studies of complex human disease genetics, we developed the first automated and quantitative high-content synaptic phenotyping platform using human neurons and astrocytes. To establish the robustness of our platform, we screened the effects of 376 small molecules on presynaptic density, neurite outgrowth, and cell viability, validating six small molecules that specifically enhanced human presynaptic density in vitro. Astrocytes were essential for mediating the effects of all six small molecules, underscoring the relevance of non-cell-autonomous factors in synapse assembly and their importance in synaptic screening applications. Bromodomain and extraterminal (BET) inhibitors emerged as the most prominent hit class and global transcriptional analyses using multiple BET inhibitors confirmed upregulation of synaptic gene expression. Through these analyses, we demonstrate the robustness of our automated screening platform for identifying potent synaptic modulators, which can be further leveraged for scaled analyses of human synaptic mechanisms and drug discovery efforts.

Brief Communication on Pathologic Assessment of Persistent Stable Metastatic Lesions in Patients Treated With Anti-CTLA-4 or Anti-CTLA-4 + Anti-PD-1 Therapy.

Despite the wide use of immune checkpoint inhibition for the treatment of melanoma, the mechanisms leading to long-term stable disease are incompletely understood. Patients with metastatic melanoma who had received ipilimumab alone or ipilimumab plus nivolumab 2+years prior and attained at least 6 months of stable disease were identified. Positron emission tomography/computed tomography (PET/CT) was performed. Pretreatment and posttreatment biopsies of areas of stable disease were assessed for tumor, fibrosis, and inflammation. Seven patients underwent PET/CT and tissue biopsy. Fluorodeoxyglucose avid lesions on PET/CT ranged from no activity to an SUV of 22. In 6 patients, the residual stable lesions were composed of necrosis and fibrosis with a prominent pigment containing macrophages and no residual melanoma. In 1 patient, a nodal lesion demonstrated melanoma with active inflammation. In most patients with durable stable disease after treatment with ipilimumab or ipilimumab/nivolumab, residual lesions demonstrated predominantly necrosis and fibrosis consistent with resolving lesions. The presence of melanophages in these samples may suggest ongoing immune surveillance. One patient did demonstrate residual melanoma, indicating the need for ongoing monitoring of this patient population.

Inference of single cell profiles from histology stains with the Single-Cell omics from Histology Analysis Framework (SCHAF).

Tissue biology involves an intricate balance between cell-intrinsic processes and interactions between cells organized in specific spatial patterns, which can be respectively captured by single-cell profiling methods, such as single-cell RNA-seq (scRNA-seq), and histology imaging data, such as Hematoxylin-and-Eosin (H&E) stains. While single-cell profiles provide rich molecular information, they can be challenging to collect routinely and do not have spatial resolution. Conversely, histological H&E assays have been a cornerstone of tissue pathology for decades, but do not directly report on molecular details, although the observed structure they capture arises from molecules and cells. Here, we leverage adversarial machine learning to develop SCHAF (Single-Cell omics from Histology Analysis Framework), to generate a tissue sample's spatially-resolved single-cell omics dataset from its H&E histology image. We demonstrate SCHAF on two types of human tumors-from lung and metastatic breast cancer-training with matched samples analyzed by both sc/snRNA-seq and by H&E staining. SCHAF generated appropriate single-cell profiles from histology images in test data, related them spatially, and compared well to ground-truth scRNA-Seq, expert pathologist annotations, or direct MERFISH measurements. SCHAF opens the way to next-generation H&E2.0 analyses and an integrated understanding of cell and tissue biology in health and disease.

Mechanisms of response and resistance to combined decitabine and ipilimumab for advanced myeloid disease.

The challenge of eradicating leukemia in patients with acute myelogenous leukemia (AML) after initial cytoreduction has motivated modern efforts to combine synergistic active modalities including immunotherapy. Recently, the ETCTN/CTEP 10026 study tested the combination of the DNA methyltransferase inhibitor decitabine together with the immune checkpoint inhibitor ipilimumab for AML/myelodysplastic syndrome (MDS) either after allogeneic hematopoietic stem cell transplantation (HSCT) or in the HSCT-naïve setting. Integrative transcriptome-based analysis of 304 961 individual marrow-infiltrating cells for 18 of 48 subjects treated on study revealed the strong association of response with a high baseline ratio of T to AML cells. Clinical responses were predominantly driven by decitabine-induced cytoreduction. Evidence of immune activation was only apparent after ipilimumab exposure, which altered CD4+ T-cell gene expression, in line with ongoing T-cell differentiation and increased frequency of marrow-infiltrating regulatory T cells. For post-HSCT samples, relapse could be attributed to insufficient clearing of malignant clones in progenitor cell populations. In contrast to AML/MDS bone marrow, the transcriptomes of leukemia cutis samples from patients with durable remission after ipilimumab monotherapy showed evidence of increased infiltration with antigen-experienced resident memory T cells and higher expression of CTLA-4 and FOXP3. Altogether, activity of combined decitabine and ipilimumab is impacted by cellular expression states within the microenvironmental niche of leukemic cells. The inadequate elimination of leukemic progenitors mandates urgent development of novel approaches for targeting these cell populations to generate long-lasting responses. This trial was registered at www.clinicaltrials.gov as #NCT02890329.

A randomized phase 2 study of neoadjuvant carboplatin and paclitaxel with or without atezolizumab in triple negative breast cancer (TNBC) - NCI 10013.

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).

Expansion, persistence, and efficacy of donor memory-like NK cells infused for posttransplant relapse.

BackgroundResponses 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.MethodsWe 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.ResultsIn 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.ConclusionGiven 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 RegistrationClinicalTrials.gov NCT04024761.FundingDunkin' Donuts, NIH/National Cancer Institute, and the Leukemia and Lymphoma Society.

Correction: Comprehensive Immunoprofiling of High-risk Oral Proliferative and Localized Leukoplakia.

[This corrects the article DOI: 10.1158/2767-9764.CRC-21-0060.][This corrects the article DOI: 10.1158/2767-9764.CRC-21-0060.].

Low peripheral blood derived neutrophil-to-lymphocyte ratio (dNLR) is associated with increased tumor T-cell infiltration and favorable outcomes to first-line pembrolizumab in non-small cell lung cancer.

BACKGROUND: An elevated peripheral blood derived neutrophil-to-lymphocyte ratio (dNLR) is a negative prognostic marker for patients with non-small cell lung cancer (NSCLC) receiving chemotherapy and immune checkpoint inhibitors. Whether dNLR is also associated with clinical outcomes to first-line pembrolizumab among patients with NSCLC and a programmed cell death ligand 1 (PD-L1) Tumor Proportion Score (TPS) of ≥50% is uncertain. How dNLR relates to the tumor immune microenvironment is also unclear. METHODS: In two participating academic centers, we retrospectively analyzed the dNLR (defined as the absolute neutrophil count/white cell count - absolute neutrophil count) prior to initiation of first-line pembrolizumab in patients with metastatic NSCLC and a PD-L1 TPS ≥50% and lacking genomic alterations in EGFR and ALK. An unbiased recursive partitioning algorithm was used to investigate an optimal dNLR cut-off with respect to objective response rate (ORR). Multiplexed immunofluorescence for CD8+, FOXP3+, PD-1+, and PD-L1 was performed on a separate cohort of NSCLCs to determine the immunophenotype associated with dNLR. RESULTS: A total of 221 patients treated with first-line pembrolizumab were included in this study. The optimal dNLR cut-off to differentiate treatment responders from non-responders was 2.6. Compared with patients with a dNLR ≥2.6 (n=97), patients with dNLR <2.6 (n=124) had a significantly higher ORR (52.4% vs 24.7%, p<0.001), a significantly longer median progression-free survival (mPFS 10.4 vs 3.4 months, HR 0.48, 95% CI 0.35 to 0.66, p<0.001), and a significantly longer median overall survival (mOS 36.6 vs 9.8 months, HR 0.34, 95% CI 0.23 to 0.49, p<0.001). After adjusting for age, sex, tobacco use, performance status, histology, serum albumin level, oncogenic driver status, and PD-L1 distribution (50%-89% vs ≥90%), a dNLR <2.6 was confirmed to be an independent predictor of longer mPFS (HR 0.47, 95% CI 0.33 to 0.67, p<0.001) and mOS (HR 0.32, 95% CI 0.21 to 0.49, p<0.001). Among advanced NSCLC samples with a PD-L1 TPS of ≥50%, those with a dNLR <2.6 had significantly higher numbers of tumor-associated CD8+, FOXP3+, PD-1 +immune cells, and PD-1 +CD8+T cells than those with a dNLR ≥2.6. CONCLUSIONS: Among patients with NSCLC and a PD-L1 TPS ≥50%, a low dNLR has a distinct immune tumor microenvironment and more favorable outcomes to first-line pembrolizumab.