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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.

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)

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.

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.

Recurrence per PDL1 and MPR

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.

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.

Human gut microbial species found to associate with clinical responses to immune checkpoint inhibitors (ICIs) are often tested in mice using fecal microbiota transfer (FMT), wherein tumor responses in recipient mice may recapitulate human responses to ICI treatment. However, many FMT studies have reported only limited methodological description, details of murine cohorts, and statistical methods. To investigate the reproducibility and robustness of gut microbial species that impact ICI responses, we performed human to germ-free mouse FMT using fecal samples from patients with non-small cell lung cancer who had a pathological response or nonresponse after neoadjuvant ICI treatment. R-FMT mice yielded greater anti-tumor responses in combination with anti-PD-L1 treatment compared to NR-FMT, although the magnitude varied depending on mouse cell line, sex, and individual experiment. Detailed investigation of post-FMT mouse microbiota using 16S rRNA amplicon sequencing, with models to classify and correct for biological variables, revealed a shared presence of the most highly abundant taxa between the human inocula and mice, though low abundance human taxa colonized mice more variably after FMT. Multiple Clostridium species also correlated with tumor outcome in individual anti-PD-L1-treated R-FMT mice. RNAseq analysis revealed differential expression of T and NK cell-related pathways in responding tumors, irrespective of FMT source, with enrichment of these cell types confirmed by immunohistochemistry. This study identifies several human gut microbial species that may play a role in clinical responses to ICIs and suggests attention to biological variables is needed to improve reproducibility and limit variability across experimental murine cohorts.

Purpose: Activating mutations in the tyrosine kinase domain of HER2 (ERBB2) have been described in a subset of lung adenocarcinomas (ADCs) and are mutually exclusive with EGFR and KRAS mutations. The prevalence, clinicopathologic characteristics, prognostic implications, and molecular heterogeneity of HER2-mutated lung ADCs are not well established in U.S. patients.Experimental Design: Lung ADC samples (N = 1,478) were first screened for mutations in EGFR (exons 19 and 21) and KRAS (exon 2), and negative cases were then assessed for HER2 mutations (exons 19–20) using a sizing assay and mass spectrometry. Testing for additional recurrent point mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA, MEK1, and AKT was conducted by mass spectrometry. ALK rearrangements and HER2 amplification were assessed by FISH.Results: We identified 25 cases with HER2 mutations, representing 6% of EGFR/KRAS/ALK-negative specimens. Small insertions in exon 20 accounted for 96% (24/25) of the cases. Compared with insertions in EGFR exon 20, there was less variability, with 83% (20/24) being a 12 bp insertion causing duplication of amino acids YVMA at codon 775. Morphologically, 92% (23/25) were moderately or poorly differentiated ADC. HER2 mutation was not associated with concurrent HER2 amplification in 11 cases tested for both. HER2 mutations were more frequent among never-smokers (P < 0.0001) but there were no associations with sex, race, or stage.Conclusions:HER2 mutations identify a distinct subset of lung ADCs. Given the high prevalence of lung cancer worldwide and the availability of standard and investigational therapies targeting HER2, routine clinical genotyping of lung ADC should include HER2. Clin Cancer Res; 18(18); 4910–8. ©2012 AACR.

PDF file, 56K.

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.