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Purpose:Neoadjuvant anti–PD-1 therapy has shown promise for resectable non–small cell lung cancer (NSCLC). We reported the first phase I/II trial of neoadjuvant nivolumab in resectable NSCLC, finding it to be safe and feasible with encouraging major pathological responses (MPR). We now present 5-year clinical outcomes from this trial, representing to our knowledge, the longest follow-up data for neoadjuvant anti–PD-1 in any cancer type.Patients and Methods:Two doses of nivolumab (3 mg/kg) were administered for 4 weeks before surgery to 21 patients with Stage I–IIIA NSCLC. 5-year recurrence-free survival (RFS), overall survival (OS), and associations with MPR and PD-L1, were evaluated.Results:With a median follow-up of 63 months, 5-year RFS and OS rates were 60% and 80%, respectively. The presence of MPR and pre-treatment tumor PD-L1 positivity (TPS ≥1%) each trended toward favorable RFS; HR, 0.61 [95% confidence interval (CI), 0.15–2.44] and HR, 0.36 (95% CI, 0.07–1.85), respectively. At 5-year follow-up, 8 of 9 (89%) patients with MPR were alive and disease-free. There were no cancer-related deaths among patients with MPR. In contrast, 6/11 patients without MPR experienced tumor relapse, and 3 died.Conclusions:Five-year clinical outcomes for neoadjuvant nivolumab in resectable NSCLC compare favorably with historical outcomes. MPR and PD-L1 positivity trended toward improved RFS, though definitive conclusions are limited by cohort size.

Breakdown of RET and ROS1 fusion positive patients

Figure S6

Retreatment Cohort - Initial irAEs: Clinical Characteristics and Features

Table S3

Co-occurring Level 1-3 alterations by MSK-IMPACT in 52 patients

The utility of circulating tumor DNA (ctDNA) as a biomarker in patients with advanced cancers receiving immunotherapy is uncertain. We therefore analyzed pretreatment (n = 978) and on-treatment (n = 171) ctDNA samples across 16 advanced-stage tumor types from three phase I/II trials of durvalumab (± the anti-CTLA4 therapy tremelimumab). Higher pretreatment variant allele frequencies (VAF) were associated with poorer overall survival (OS) and other known prognostic factors, but not objective response, suggesting a prognostic role for patient outcomes. On-treatment reductions in VAF and lower on-treatment VAF were independently associated with longer progression-free survival and OS and increased objective response rate, but not prognostic variables, suggesting that on-treatment ctDNA dynamics are predictive of benefit from immune checkpoint blockade. Accordingly, we propose a concept of “molecular response” using ctDNA, incorporating both pretreatment and on-treatment VAF, that predicted long-term survival similarly to initial radiologic response while also permitting early differentiation of responders among patients with initially radiologically stable disease.Significance:In a pan-cancer analysis of immune checkpoint blockade, pretreatment ctDNA levels appeared prognostic and on-treatment dynamics predictive. A “molecular response” metric identified long-term responders and adjudicated benefit among patients with initially radiologically stable disease. Changes in ctDNA may be more dynamic than radiographic changes and could complement existing trial endpoints.This article is highlighted in the In This Issue feature, p. 1775

Types of Clinical trials patients enrolled to according to level 1-4 and UMD assigned samples

Supplementary Table S1: List of actionable genes and potential matched therapy Supplementary Table S2: List of co-occurring Level 1-4 mutations Supplementary Table S3: EGFR mutations identified and associated therapy Supplementary Table S4: List of 410 genes in MSK-IMPACT assay

PDF file - 1601K, Representative case harboring a 9pb insertion mutation V769_D770insASV

PDF file - 76K, Comprehensive list of mutations tested by mass spectrometry (Sequenom)

Patients with Advanced NSCLC Treated at MSKCC with Immunotherapy From April 2011 to May 2016

Breakdown of MAP2K1, RAF1, ARAF, FGFR3-TACC3 fusion and CDKN2A loss patients

Retreatment Cohort - Recurrent irAEs: Characteristics of The Recurrent Immune-Related Toxicity

Time From First Dose of Immunotherapy to Date of The Initial irAE

PDF file - 61K, Summary of all cases tested for EGFR exon 20 insertions

Patient 1 Concurrent EGFR and KRAS mutations in a patient identified by MSK-IMPACT

Median number of mutations in samples sequenced by MSK-IMPACT in patients with a Level 1-4 alteration or assigned to the unknown mitogenic driver cohort.

Overall Survival for Patients in the Retreatment Cohort

PDF file - 66K, Summary of all mutations identified through clinical and extended mass spectrometry genotyping (n=1500)

Patient 2 Concurrent EGFR and KRAS mutations in a patient identified by MSK-IMPACT

Considering retreatment following recovery from an immune-related adverse event (irAE) is a common clinical scenario, but the safety and benefit of retreatment is unknown. We identified patients with advanced non–small cell lung cancer (NSCLC) treated with anti-PD-(L)1 who had treatment held due to irAEs and divided them into two groups: those retreated with anti-PD-(L)1 (retreatment cohort) or those who had treatment stopped (discontinuation cohort). Out of 482 NSCLC patients treated with anti-PD-(L)1, 68 (14%) developed a serious irAE requiring treatment interruption. Of these, 38 (56%) were retreated and 30 (44%) had treatment discontinued. In the retreatment cohort, 18 (48%) patients had no subsequent irAEs, 10 (26%) had recurrence of the initial irAE, and 10 (26%) had a new irAE. Most recurrent/new irAEs were mild (58% grade 1–2) and manageable (84% resolved or improved to grade 1). Two treatment-related deaths occurred. Recurrent/new irAEs were more likely if the initial irAE required hospitalization, but the initial grade and time to retreatment did not influence risk. Among those with no observed partial responses prior to the irAE, progression-free survival (PFS) and overall survival (OS) were longer in the retreatment cohort. Conversely, for those with objective responses prior to the irAE, PFS and OS were similar in the retreatment and discontinuation cohorts. Among patients with early objective responses prior to a serious irAE, outcomes were similar, whether or not they were retreated. Together, data suggest that benefit may occur with retreatment in patients with irAEs who had no treatment response prior to irAE onset. Cancer Immunol Res; 6(9); 1093–9. ©2018 AACR.

PDF file - 54K, Morphologic assessment of tumors harboring EGFR exon 20 insertions and comparison to a control group with classical EGFR sensitizing mutations

PDF file - 106k, Outline of sequential genotyping algorithm followed in this study.

Purpose:Neoadjuvant anti–PD-1 therapy has shown promise for resectable non–small cell lung cancer (NSCLC). We reported the first phase I/II trial of neoadjuvant nivolumab in resectable NSCLC, finding it to be safe and feasible with encouraging major pathological responses (MPR). We now present 5-year clinical outcomes from this trial, representing to our knowledge, the longest follow-up data for neoadjuvant anti–PD-1 in any cancer type.Patients and Methods:Two doses of nivolumab (3 mg/kg) were administered for 4 weeks before surgery to 21 patients with Stage I–IIIA NSCLC. 5-year recurrence-free survival (RFS), overall survival (OS), and associations with MPR and PD-L1, were evaluated.Results:With a median follow-up of 63 months, 5-year RFS and OS rates were 60% and 80%, respectively. The presence of MPR and pre-treatment tumor PD-L1 positivity (TPS ≥1%) each trended toward favorable RFS; HR, 0.61 [95% confidence interval (CI), 0.15–2.44] and HR, 0.36 (95% CI, 0.07–1.85), respectively. At 5-year follow-up, 8 of 9 (89%) patients with MPR were alive and disease-free. There were no cancer-related deaths among patients with MPR. In contrast, 6/11 patients without MPR experienced tumor relapse, and 3 died.Conclusions:Five-year clinical outcomes for neoadjuvant nivolumab in resectable NSCLC compare favorably with historical outcomes. MPR and PD-L1 positivity trended toward improved RFS, though definitive conclusions are limited by cohort size.

Recurrence per PDL1 and MPR

Postoperative radiation therapy (PORT) in resected non-small cell lung cancer (NSCLC) improves locoregional outcomes, but recent randomized data do not support its unselected use. We assessed if tumor mutational burden (TMB) and mutations in genes associated with radiation sensitivity can select patients for PORT.Patients with resected NSCLC treated with and without PORT who underwent tumor genomic profiling were examined. The incidence of locoregional failures (LRFs) in patients with deleterious mutations in DNA damage response and repair (DDR) genes and genes associated with radiation resistance (KEAP1/NFE2L2/STK11/PIK3CA) were investigated. Cox modeling and receiver operating characteristic curve (ROC) analysis assessed the relationship between TMB and locoregional control (LRC).Eighty-nine patients with NSCLC treated with PORT were analyzed, with a 2-year LRF rate of 19% (95% confidence interval, 10%-27%). Among patients treated with PORT, those with mutations in radiation resistance genes (n = 16 [18%]) had significantly more LRFs than patients without mutations (2-year LRF rate: 60% vs 11%; P.001). On multivariate analysis, radiation-resistance mutations were associated with LRF after PORT (hazard ratio, 7.42; P.001). Patients with mutations identified in DDR genes (n = 15 [17%]) had significantly improved LRC (P = .048) and no LRF events after PORT. On multivariate analysis, a higher TMB was associated with improved LRC after PORT (hazard ratio, 0.86; P = .01), and TMB was associated with PORT outcomes (area under ROC curve, 0.67-0.77). These genomic markers were not similarly associated with LRF in patients not treated with PORT.The data suggest that patients with radiation-resistance gene alterations may derive minimal benefit from PORT, whereas patients with high TMB and/or alterations in DDR genes may benefit from PORT and be suited for future precision-RT strategies. Prospective studies are necessary to validate these findings.

Despite the initial successes of immunotherapy, there is an urgent clinical need for molecular assays that identify patients more likely to respond. Here, we report that ultrasensitive measures of circulating tumor DNA (ctDNA) and T-cell expansion can be used to assess responses to immune checkpoint blockade in metastatic lung cancer patients (N = 24). Patients with clinical response to therapy had a complete reduction in ctDNA levels after initiation of therapy, whereas nonresponders had no significant changes or an increase in ctDNA levels. Patients with initial response followed by acquired resistance to therapy had an initial drop followed by recrudescence in ctDNA levels. Patients without a molecular response had shorter progression-free and overall survival compared with molecular responders [5.2 vs. 14.5 and 8.4 vs. 18.7 months; HR 5.36; 95% confidence interval (CI), 1.57–18.35; P = 0.007 and HR 6.91; 95% CI, 1.37–34.97; P = 0.02, respectively], which was detected on average 8.7 weeks earlier and was more predictive of clinical benefit than CT imaging. Expansion of T cells, measured through increases of T-cell receptor productive frequencies, mirrored ctDNA reduction in response to therapy. We validated this approach in an independent cohort of patients with early-stage non–small cell lung cancer (N = 14), where the therapeutic effect was measured by pathologic assessment of residual tumor after anti-PD1 therapy. Consistent with our initial findings, early ctDNA dynamics predicted pathologic response to immune checkpoint blockade. These analyses provide an approach for rapid determination of therapeutic outcomes for patients treated with immune checkpoint inhibitors and have important implications for the development of personalized immune targeted strategies.Significance: Rapid and sensitive detection of circulating tumor DNA dynamic changes and T-cell expansion can be used to guide immune targeted therapy for patients with lung cancer.See related commentary by Zou and Meyerson, p. 1038

PDF file, 54K, Summary of mutations detected by clinical mutation screening and extended mass spectrometry genotyping (n=1478 tumors).

Correlative assays performed on each sample

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; 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 Henick, p. 1205

PDF file, 76K, Comprehensive list of mutations tested by mass spectrometry (Sequenom).

Figures S1-S8

Supplementary Figures related to Figures 1, 2, 3, and 4.

Table S2: Surveillance plasma sample ctDNA metrics. Table S3: Baseline plasma sample ctDNA metrics. Table S4: Surveillance plasma sample variants in ctDNA detected samples. Table S5: Baseline plasma variants in available samples. Table S6: Tumor variants detected by whole exome and targeted sequencing. Table S7: Canonical genes implicated in clonal hematopoiesis.

Table S1 shows summary patient demographics, pathologic features, and treatment details

Supplementary Table S1. Summary of patient characteristics Supplementary Table S2. Summary of plasma samples analyzed Supplementary Table S3. Summary of peripheral blood lymphocytes and tumor infiltrating lymphocytes samples analyzed Supplementary Table S4. Genes analyzed by TEC-Seq Supplementary Table S5. Summary of TEC-Seq Characteristics Supplementary Table S6. Somatic sequence alterations detected in cfDNA Supplementary Table S7. Mutation Cellularity Analysis for tumor-specific cfDNA variants Supplementary Table S8. TCR-beta sequencing analysis Supplementary Table S9. Differentially abundant clones for CGLU111 Supplementary Table S10. Differentially abundant clones for CGLU117 Supplementary Table S11. Differentially abundant clones for CGLU127 Supplementary Table S12. Differentially abundant clones for CGLU212 Supplementary Table S13. Differentially abundant clones for CGLU135 Supplementary Table S14. Differentially abundant clones for CGLU161 Supplementary Table S15. Differentially abundant clones for CGLU159 Supplementary Table S16. Differentially abundant clones for CGLU162 Supplementary Table S17. Differentially abundant clones for CGLU203 Supplementary Table S18. Differentially abundant clones for CGLU243

PDF file, 56K, Flow chart of patients screened for mutations under the clinical and extended panels.

Purpose:Treatment with PD-(L)1 blockade can produce remarkably durable responses in patients with non–small cell lung cancer (NSCLC). However, a significant fraction of long-term responders ultimately progress and predictors of late progression are unknown. We hypothesized that circulating tumor DNA (ctDNA) analysis of long-term responders to PD-(L)1 blockade may differentiate those who will achieve ongoing benefit from those at risk of eventual progression.Experimental Design:In patients with advanced NSCLC achieving long-term benefit from PD-(L)1 blockade (progression-free survival ≥ 12 months), plasma was collected at a surveillance timepoint late during/after treatment to interrogate ctDNA by Cancer Personalized Profiling by Deep Sequencing. Tumor tissue was available for 24 patients and was profiled by whole-exome sequencing (n = 18) or by targeted sequencing (n = 6).Results:Thirty-one patients with NSCLC with long-term benefit to PD-(L)1 blockade were identified, and ctDNA was analyzed in surveillance blood samples collected at a median of 26.7 months after initiation of therapy. Nine patients also had baseline plasma samples available, and all had detectable ctDNA prior to therapy initiation. At the surveillance timepoint, 27 patients had undetectable ctDNA and 25 (93%) have remained progression-free; in contrast, all 4 patients with detectable ctDNA eventually progressed [Fisher P < 0.0001; positive predictive value = 1, 95% confidence interval (CI), 0.51–1; negative predictive value = 0.93 (95% CI, 0.80–0.99)].Conclusions:ctDNA analysis can noninvasively identify minimal residual disease in patients with long-term responses to PD-(L)1 blockade and predict the risk of eventual progression. If validated, ctDNA surveillance may facilitate personalization of the duration of immune checkpoint blockade and enable early intervention in patients at high risk for progression.