BACKGROUND Antibodies that block programmed death 1 (PD-1) protein improve survival in patients with advanced non–small-cell lung cancer (NSCLC) but have not been tested in resectable NSCLC, a condition in which little progress has been made during the past decade. METHODS In this pilot study, we administered two preoperative doses of PD-1 inhibitor nivolumab in adults with untreated, surgically resectable early (stage I, II, or IIIA) NSCLC. Nivolumab (at a dose of 3 mg per kilogram of body weight) was administered intravenously every 2 weeks, with surgery planned approximately 4 weeks after the first dose. The primary end points of the study were safety and feasibility. We also evaluated the tumor pathological response, expression of programmed death ligand 1 (PD-L1), mutational burden, and mutation-associated, neoantigen-specific T-cell responses. RESULTS Neoadjuvant nivolumab had an acceptable side-effect profile and was not associated with delays in surgery. Of the 21 tumors that were removed, 20 were completely resected. A major pathological response occurred in 9 of 20 resected tumors (45%). Responses occurred in both PD-L1-positive and PD-L1-negative tumors. There was a significant correlation between the pathological response and the pretreatment tumor mutational burden. The number of T-cell clones that were found in both the tumor and peripheral blood increased systemically after PD-1 blockade in eight of nine patients who were evaluated. Mutation-associated, neoantigen-specific T-cell clones from a primary tumor with a complete response on pathological assessment rapidly expanded in peripheral blood at 2 to 4 weeks after treatment; some of these clones were not detected before the administration of nivolumab. CONCLUSIONS Neoadjuvant nivolumab was associated with few side effects, did not delay surgery, and induced a major pathological response in 45% of resected tumors. The tumor mutational burden was predictive of the pathological response to PD-1 blockade. Treatment induced expansion of mutation-associated, neoantigen-specific T-cell clones in peripheral blood. (Funded by Cancer Research Institute–Stand Up 2 Cancer and others; ClinicalTrials.gov number, NCT02259621.)
Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.
Resistance to cancer therapies presents a significant clinical challenge. Recent studies have revealed intratumoral heterogeneity as a source of therapeutic resistance. However, it is unclear whether resistance is driven predominantly by pre-existing or de novo alterations, in part because of the resolution limits of next-generation sequencing. To address this, we developed a high-complexity barcode library, ClonTracer, which enables the high-resolution tracking of more than 1 million cancer cells under drug treatment. In two clinically relevant models, ClonTracer studies showed that the majority of resistant clones were part of small, pre-existing subpopulations that selectively escaped under therapeutic challenge. Moreover, the ClonTracer approach enabled quantitative assessment of the ability of combination treatments to suppress resistant clones. These findings suggest that resistant clones are present before treatment, which would make up-front therapeutic combinations that target non-overlapping resistance a preferred approach. Thus, ClonTracer barcoding may be a valuable tool for optimizing therapeutic regimens with the goal of curative combination therapies for cancer.
PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.
Mutation-associated neoantigens (MANA) are a target of antitumor T-cell immunity. Sensitive, simple, and standardized assays are needed to assess the repertoire of functional MANA-specific T cells in oncology. Assays analyzing in vitro cytokine production such as ELISpot and intracellular cytokine staining have been useful but have limited sensitivity in assessing tumor-specific T-cell responses and do not analyze antigen-specific T-cell repertoires. The FEST (Functional Expansion of Specific T cells) assay described herein integrates T-cell receptor sequencing of short-term, peptide-stimulated cultures with a bioinformatic platform to identify antigenspecific clonotypic amplifications. This assay can be adapted for all types of antigens, including MANAs via tumor exomeguided prediction of MANAs. Following in vitro identification by the MANAFEST assay, the MANA-specific CDR3 sequence can be used as a molecular barcode to detect and monitor the dynamics of these clonotypes in blood, tumor, and normal tissue of patients receiving immunotherapy. MANAFEST is compatible with high-throughput routine clinical and lab practices.
Purpose: Neoadjuvant PD-1 blockade is a promising treatment for resectable non-small cell lung cancer (NSCLC), yet immunologic mechanisms contributing to tumor regression and biomarkers of response are unknown. Using paired tumor/blood samples from a phase II clinical trial (NCT02259621), we explored whether the peripheral T-cell clonotypic dynamics can serve as a biomarker for response to neoadjuvant PD-1 blockade. Experimental Design: T-cell receptor (TCR) sequencing was performed on serial peripheral blood, tumor, and normal lung samples from resectable NSCLC patients treated with neoadjuvant PD-1 blockade. We explored the temporal dynamics of the T-cell repertoire in the peripheral and tumoral compartments in response to neoadjuvant PD-1 blockade by using the TCR as a molecular barcode. Results: Higher intratumoral TCR clonality was associated with reduced percent residual tumor at the time of surgery, and the TCR repertoire of tumors with major pathologic response (MPR; <10% residual tumor after neoadjuvant therapy) had a higher clonality and greater sharing of tumor-infiltrating clonotypes with the peripheral blood relative to tumors without MPR. Additionally, the posttreatment tumor bed of patients with MPR was enriched with T-cell clones that had peripherally expanded between weeks 2 and 4 after anti-PD-1 initiation and the intratumoral space occupied by these clonotypes was inversely correlated with percent residual tumor. Conclusions: Our study suggests that exchange of T-cell clones between tumor and blood represents a key correlate of pathologic response to neoadjuvant immunotherapy and shows that the periphery may be a previously underappreciated originating compartment for effective antitumor immunity. See related commentary by XXX, p. XXX
CONFLICTS OF INTERESTDMP and KNS have filed for patent protection on a subset of the technologies described herein (US provisional patent application no. 62/407,820). AD, ALC, FD, DMP, JB, and KNS have filed for patent protection on a subset of the technologies described herein (US provisional patent application no. 63/135,534). SZ is a founder of, holds equity in, and serves as a consultant to Personal Genome Diagnostics. SZ holds equity in Thrive Earlier
8524 Background: Improved therapy is needed for patients (pts) with early-stage non-small cell lung cancer (NSCLC), as the majority relapse after curative resection. Our group reported the first trial of neoadjuvant PD-1 blockade in resectable NSCLC, finding therapy to be safe and feasible. Here we report extended clinical follow-up and long-term molecular response data from this trial. Methods: IV nivolumab 3 mg/kg was given every 2 weeks for 2 doses prior to surgery in 20 pts with resectable NSCLC at Johns Hopkins and MSKCC. Blood for correlative studies was taken prior to each dose of nivolumab, prior to surgery, 2-4 weeks post-surgery, and during long-term follow up. In a subgroup of pts, longitudinal molecular data was assessed in peripheral blood for circulating tumor DNA (ctDNA) and dynamics of tumor-infiltrating T-cell clonotypes. Results: At median follow up of 30 months (m), 15 of 20 pts are disease-free and alive. Two pts have died (one from relapsed disease). Median recurrence free survival (RFS) has not been reached. The 24m RFS rate is 69% (95% CI: 51-93). Thus far, presence of ctDNA at diagnosis and major pathologic response (MPR - ≤10% viable tumor in resected specimen) do not associate with RFS. One long-term immune-related adverse event has occurred (skin, G3). All pts who on pathologic review had ≥30% reduction in viable tumor in response to nivolumab demonstrated clearance of detectable ctDNA from blood prior to surgery. Pts with MPR experienced expansion of neoantigen-specific T-cells in peripheral blood. In one patient with ongoing disease free status, expansion of tumor-associated T-cells has persisted in peripheral blood beyond 15m from surgery. By contrast, in a patient who had detectable peri-operative ctDNA and 75% residual disease at surgery, minimal T-cell expansion was observed in peripheral blood, with a decreasing frequency of expanded T-cell clones over time that correlated with eventual cancer relapse. Conclusions: Long-term follow up reinforces the safety of neoadjuvant nivolumab in resectable NSCLC. Analysis of ctDNA and peripheral T-cell expansion in responders compared with non-responders suggests potential biomarkers for response and surveillance. While RFS data is encouraging, phase 3 trials are ongoing to evaluate efficacy of PD-(L)1 blockade in early-stage NSCLC. Clinical trial information: NCT02259621.
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