Neoadjuvant PD-1 Blockade in Resectable Lung Cancer

Abstract

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 .).

Figures

Figure 1.. Patterns of Radiologic and Pathological Response to Neoadjuvant Therapy with Nivolumab.

Panel A shows computed tomographic (CT) imaging of the chest of a 62-year-old male smoker (Patient 1) with stage IIB squamous lung cancer before and after the administration of nivolumab. In the upper row, the pretreatment scan before the infusion of nivolumab shows a primary tumor mass measuring 8 cm in diameter in the lower right lobe of the lung (arrow).A scan performed before surgery shows 35% shrinkage with associated tumor cavitation (arrow). In the lower row, shown are representative sections of tumor specimens obtained from Patient 1 before the administration of nivolumab (left) and after the administration (right) (hematoxylin and eosin staining). This patient had 100% pathological regression of the large primary lung tumor but had residual lymph-node metastases in the resection specimen. Panel B shows CT of the chest of a 78-year-old female former smoker (Patient 5) with stage IIIA lung adenocarcinoma, who received two doses of nivolumab preoperatively. In the upper row, the tumor is larger on imaging performed after the administration of nivolumab than before administration (arrows), possibly because of infiltration of immune cells into the tumor. In the lower row, shown are representative sections of tumor specimens obtained from Patient 5 before the administration of nivolumab (left) and after administration (right) (hematoxylin and eosin staining). Neoplastic cells are present throughout the pretreatment specimen, whereas in the post-treatment specimen, there was 90% tumor regression.

Figure 2.. Pathological Assessment of Response to Neoadjuvant Blockade of Programmed Death 1 (PD-1).

Panel A shows pathological regression in the resected primary lung tumor after neoadjuvant administration of nivolumab, according to the percentage of remaining viable tumor cells, for each of the 20 patients who underwent surgical resection. The gray horizontal line indicates the threshold for a major pathological response (90% regression). Clinical and pathological features that include the presence or absence of lymph-node (LN) metastases in the surgical specimen and preoperative radiologic response (according to Response Evaluation Criteria in Solid Tumors [RECIST]) are annotated for each patient. AC denotes adenocarcinoma, SCC squamous-cell carcinoma, PR partial response, SD stable disease, and PD-L1 programmed death ligand 1. Also shown are biopsy specimens obtained before (Panel B) and after (Panel C) neoadjuvant administration of nivolumab in a patient (MD043-008) who had a major pathological response (multiplex immunofluorescence staining). With this staining technique, visible structures include cytokeratin-positive tumor cells (orange), CD68+ macrophages (magenta), FoxP3+ regulatory T cells (yellow), CD8+ T cells (green), PD-1+ cells (red), and PD-L1+ cells (white). In the pretreatment specimen, only a few intratumoral macrophages are seen expressing PD-L1. However, there are multiple foci where PD-L1 and PD-1 are expressed in close proximity to each other (inset with white circle) in the pretreatment specimen. Focal, geographic tumor-cell PD-L1 expression was observed in an adaptive pattern (not shown in this image). After two doses of nivolumab, the tumor is infiltrated by liquid containing CD8+ and PD-1+ immune cells. Some of the infiltrating immune cells express PD-L1, which is consistent with an adaptive immune resistance mechanism.,

Figure 3.. Association between Mutational Burden and Pathological Response to PD-1 Blockade.

Panel A shows the number of sequence alterations in pretreatment tumor samples obtained from 11 patients who underwent surgical resection and had sufficient pretreatment tissue available for sequencing, with differential responses to PD-1 blockade. Patients who had a major pathological response were found to carry a significantly higher number of somatic sequence alterations than those without a major pathological response, with a mean (±SE) number of 311±55 and 74±60, respectively (P=0.01 by exact Wilcoxon test). Panel B shows the number of sequence alterations per tumor, which was found to be inversely associated with the percentage of residual viable tumor cells after nivolumab treatment (Spearman’s rho, −0.75; P=0.008). The dashed black line indicates the linear regression line, and the dashed gray lines indicate the upper and lower boundaries of the 95% confidence interval. AC denotes adenocarcinoma, AS adenosquamous carcinoma, and SCC squamous-cell carcinoma.

Figure 4.. Identification of Mutation-Associated, Neoantigen-Specific T Cells after Neoadjuvant Treatment with Nivolumab.

An antigen-recognition assay that evaluates in vitro expansion of T-cell clones after peptide stimulation was performed with the use of 47 algorithm-predicted, candidate mutation-associated neoantigens on peripheral-blood T cells obtained from Patient 1 before the administration of nivolumab (on day −28) and after administration (on day 44 after surgical resection) to determine the repertoire of functional mutation-associated neoantigen recognition. Seven mutation-associated neoantigens were recognized at both time points. The 47 peptides that were chosen for analysis represented the top 10 ImmunoSelect-R pipeline (Personal Genome Diagnostics) peptides that were predicted to bind specifically to each of the patient’s five HLA class I alleles. Three peptides were excluded because they were predicted to bind multiple HLA alleles. Three T-cell clones from a peripheral-blood sample obtained on day 44 that specifically expanded in culture to mutation-associated neoantigen (MANA) number 7 were found in the pretreatment tumor-biopsy specimen and the resection specimen (Panel A). Also shown are the frequencies of the three T-cell clones in longitudinal analysis of peripheral-blood T cells before and after treatment (Panel B) and in a tumor-biopsy sample before treatment, in resected tumor, in normal lung, and in tumor-involved and tumor-uninvolved lymph nodes at resection (Panel C). TCR Vβ CDR3 AA denotes T-cell receptor Vγ complementarity-determining region 3 amino acid.