Patient eligibility
The study was authorized and approved by the IRB of, and patients were recruited from three centers: The University of California Davis Comprehensive Cancer Center, Cedars Sinai Medical Center, and VA David Grant Medical Center. The study design and conduct complied with all relevant regulations regarding the use of human study participants and was conducted in accordance with the criteria set by the Declaration of Helsinki. All patients provided written informed consent prior to any study procedures. The trial was registered on clinicaltrials.gov (NCT02599454). The first patient was enrolled on 5/3/2016, and the last patient was enrolled on 8/9/2019. Eligible patients were ≥18 years of age with histologically confirmed T1-3 NSCLC ≤ 7 cm diameter. Although patients with EGFR or ALK mutations would not be expected to respond to ICI based on data from advanced NSCLC, these patients were not excluded since there is limited tissue available for NGS testing in this population. Patients with chest wall invasion (T3) and 2 nodules within the same lobe of the lung were eligible. Patients were required to have one or more features predictive of increased recurrence risk: diameter ≥1 cm for the phase I component and ≥2 cm for the expansion cohort; SUV ≥ 6.2 on FDG PET; or moderately/poorly differentiated histology. Patients were required to be either medically inoperable as determined by multidisciplinary evaluation or to have refused surgery, had a forced expiratory volume over 1 s (FEV1) ≥ 700 cc, and a diffusing capacity for carbon monoxide (DLCO) ≥ 5.5 m/min/mm Hg on pulmonary function testing (PFT), and had a Zubrod performance status (PS) ≤ 2. Exclusion criteria included New York Heart Association class II or greater cardiovascular disease, severe infections within 4 weeks of enrollment, history of autoimmune disorders, idiopathic pulmonary fibrosis, and active human immunodeficiency virus, hepatitis B, or hepatitis C. Prior malignancies did not disqualify as long as they were not active at the time of enrollment. Required staging workup included PFTs within 3 months of registration and computed tomography (CT) of the chest within 28 days of registration. PET/CT staging was not mandated but was encouraged.
Study design
The trial was designed as a proof-of-concept phase I study with a standard 3 + 3 dose escalation design followed by a patient expansion cohort (Fig. 1A). Atezolizumab was delivered intravenously (IV) in 21-day cycles. Three atezolizumab dose levels were evaluated: 3 mg/kg IV, 10 mg/kg IV, and 1200 mg IV flat dosing (Fig. 1A). Patients received a planned 6 cycles of atezolizumab with SABR initiated with cycle 3, 24–48 h following the atezolizumab infusion. SABR was delivered to 50 Gy over 4–5 fractions. DLT was defined as ≥grade 3 immune-related adverse event or other ≥grade 3 treatment-related adverse events that did not resolve to ≤grade 2 within 14 days of onset, or grade 2 diarrhea, aspartate aminotransferase (AST)/alanine transaminase (ALT) > 3× the upper limit of normal with bilirubin > 2× upper limit of normal, or pneumonitis that required holding treatment >14 days. The DLT period was 9 weeks. Patients were assessed with labs, including a completed blood count, metabolic panel, c-reactive protein, and thyroid function tests prior to each cycle. Tumor assessment was performed with either PET/CT or CT at the discretion of the treating physician every 2 cycles during treatment. After completion of treatment, tumor assessment was performed every 3 months, year 1–2, and every 6 months, year 3–5. The full trial protocol is included in the Supplementary information file.
Stereotactic ablative radiotherapy
All patients underwent CT simulation with slice thickness ≤3.0 cm with reliable immobilization. Four-dimensional (4D) CT simulation was strongly encouraged but not required. For patients simulated with 4DCT, an internal target volume (ITV) was created using the 10 respiratory phases, and a 5 mm planning target volume (PTV) margin was added in all directions. For patients simulated without 4DCT, a 5 mm PTV margin was added in the axial plane, and a 1.0–1.5 cm margin craniocaudally was added depending on tumor motion as assessed by fluoroscopy. A motion management strategy (abdominal compression, respiratory gating, tumor tracking, or breath-hold) was required if respiratory motion, as assessed by fluoroscopy, exceeded 1 cm. SABR was delivered to 50 Gy over 4 fractions for peripherally located tumors (>2 cm from the proximal bronchial tree and not touching mediastinal pleural) and to 50 Gy over 5 fractions for centrally located tumors. Fractions were delivered 40–96 h apart. The prescription isodose surface was chosen such that 95% of the PTV was covered by the prescription isodose line, and 99% of the PTV received a minimum of 90% of the prescription dose. Dose constraints included combined lung-GTV volume receiving 20 Gy (V20) < 10%, with less than 1500 cc combined lung receiving 12.5 Gy and less than 1000 cc combined lung receiving 13.5 Gy. Full normal tissue dose constraints are provided in Supplemental Table 1.
Outcomes
The primary objective was to determine the maximum tolerated dose (MTD) of atezolizumab when delivered with SABR in early-stage, medically inoperable NSCLC. MTD was defined as the highest dose at which no more than one of six patients developed a DLT or dose Level 3 if the MTD was not reached. Secondary objectives included the safety profile of the experimental regimen using common toxicity criteria for adverse events (CTCAE) version 4 and preliminary efficacy data by objective response rate (ORR) and PFS by response evaluation in solid tumors (RECIST) version 1.140. This patient population is at very high risk for developing additional primary aero-digestive tract malignancies due to “field cancerization” effects. In order to distinguish the development of a new primary NSCLC from local or systemic disease progression, the development of disease in the contralateral lung without evidence of ipsilateral or systemic recurrence was reviewed and characterized by a multidisciplinary tumor board. In addition to PFS, OS, and FFP were analyzed. FFP (time from enrollment to documentation of disease progression) was a post-hoc analysis undertaken because of the significant rate of death from an intercurrent illness unrelated to cancer or study treatment. Survival and progression were measured from the day of trial enrollment. ORR was assessed following cycle 2, prior to initiation of SABR, as the lung changes post-SABR make response assessment inaccurate.
Correlative science tissue collection
Baseline tumor samples were required for study participation and were taken from tissue blocks or fresh tumor biopsies. Samples were formalin-fixed and paraffin-embedded (FFPE), and fresh sections of FFPE were used for immunohistochemistry (IHC) and multi-plex immune fluorescence (IF). Blood samples were collected at baseline, after every cycle, and end of treatment. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated from the whole blood. PBMCs were cryopreserved for batched analysis. Stool samples were collected at baseline, end of cycle 2, and end of treatment. Determining response by RECIST criteria after radiotherapy ablation in NSCLC is difficult to interpret and is generally not performed as radiotherapy scar tissue cannot be distinguished from tumors. This makes applying RECIST criteria and determining response rates after lung SBRT problematic in this population which only has a single lesion to evaluate for a response. As an indicator of ICI efficacy, early response to ICI alone (before SBRT) is reported. Post-ablation imaging was not used to determine or confirm responses.
Tissue analysis
Tumor samples were stained for PD-L1 by immuno-histochemistry on the Ventana Discovery Ultra autostainer (Roche) using the clone E1L3N (cell signaling) at a dilution of 1:100 as previously described41. Briefly, “4 µm FFPE sections were mounted on Superfrost Plus microscope slides (Thermo Fisher Scientific) and dried overnight. Sections were deparaffinized, followed by antigen retrieval in CC1 buffer (pH 9, 95 °C; Roche), endogenous peroxidase blocking, and then incubation with the primary antibodies. Chromogenic detection was performed with Chromomap DAB (Roche), followed by counterstaining with hematoxylin. Sections stained with and without primary antibody were used as positive and negative controls.” Membrane staining was verified and quantified by a board-certified pathologist blinded to outcomes using TPS as previously described42. Additional sections, when available, were also stained and analyzed by multi-plex IF for CD3, Ki-67, and granzyme b (GZB) as previously described26. Briefly, “FFPE histology sections were deparaffinized and subjected to antigen retrieval using EDTA buffer (Sigma-Aldrich) pH = 8.0 and boiled for 20 min at 97 °C in a pressure-boiling container (PT module, Lab Vision). Slides were then incubated with dual endogenous peroxidase block (DAKO #S2003) for 10 min at room temperature and subsequently with a blocking solution containing 0.3% bovine serum albumin in 0.05% Tween solution for 30 min. Slides were stained with 4′,6-Diamidino-2-Phenylindole (DAPI) for visualization of all cells, CK to detect tumor epithelial cells, CD3 for T-lymphocytes, GZB for T-cell cytolytic potential and Ki-67 as a cell proliferation marker. Primary antibodies included CK clone AE1/AE3 (catalog # M3515) from DAKO used with a concentration of 0.12 mg/ml, CD3 clone SP7 (catalog # NB600-1441) from Novus biologicals dilution 1:100 (culture supernatant), GZB clone 4E6 (catalog # ab139354) from Abcam with a concentration of 5 μg/ml and Ki-67 clone MIB1 (catalog # M724029-2) from DAKO with a concentration of 0.46 μg/ml. Secondary antibodies and fluorescent reagents used were goat anti-rabbit Alexa546 (Invitrogen; 1:100 dilution), anti-rabbit Envision (K4009, DAKO, 1:100 stock dilution) with biotinylated tyramide/Streptavidine-Alexa750 conjugate (Perkin-Elmer); anti-mouse IgG1 antibody (eBioscience) with fluorescein-tyramide (Perkin-Elmer), anti-mouse IgG2a antibody (Abcam) with Cy5-tyramide (Perkin-Elmer). Residual horseradish peroxidase activity between incubations with secondary antibodies was eliminated by exposing the slides twice for 7 min to a solution containing benzoic hydrazide (0.136 mg) and hydrogen peroxide (50 µl).” Fluorescence was quantified using the AQUA method, and the QIF score was calculated by dividing the target pixel intensities by the area of positivity in the sample. Slides were examined by a pathologist to exclude defective areas and staining artifacts.
Flow cytometry and ex-vivo stimulation assays
In this report, we focus on samples obtained pre-treatment and after cycles 1–3 to discover potential early biomarkers of clinical outcomes. Analysis focusing on additional cycles and the effects of SABR on the immune response will be reported elsewhere. PBMCs were immunophenotyped using flow cytometry. Briefly, PBMCs were thawed and incubated with Fc-block (BD Bioscience, Franklin Lakes, NJ) on ice for 15 min. Then, cells were stained with specific antibody cocktails (supplemental Table 2) for 1 h on ice and then stained with Aqua-LIVE/DEAD (Invitrogen, Carlsbad, CA) for 30 min at room temperature. Cells were washed after each step using PBS containing 0.5% BSA and before being processed on a BD Fortessa flow cytometer (BD Bioscience, Franklin Lakes, NJ). Data were analyzed using FlowJo software version 10.6.2 (Tree Star Inc. Ashland, OR). Selected samples were stimulated ex-vivo (see supplemental figure 8A) using eBioscience stimulation cocktail (Invitrogen, Carlsbad, CA). Cytokine secretion was prevented using eBioscience Brefeldin A (Invitrogen, Carlsbad, CA). Cells were then stained with a specific antibody cocktail (Supplemental Table 2). Intracellular cytokine staining used the eBioscience FOXP3/Transcription Factor Staining Buffer Set (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol.
Transcriptomic analysis
RNA was extracted from cryopreserved PBMCs and sequenced for RNA expression and TCR analysis43. Total RNA was extracted using a Quick-RNA MagBead kit (Zymo Research) from cryopreserved PBMC. RNA concentrations were then quantified using a Qubit Fluorometer (Invitrogen), and RNA integrity was assessed using the Agilent TapeStation (Agilent).
Indexed libraries were constructed using the SMARTer® Stranded Total RNA-Seq Kit v3 (Takara Bio) following the manufacturer’s instructions. The quantity and quality of the libraries were assessed by Qubit and Agilent 2100 Bioanalyzer, respectively. Libraries’ molar concentrations were validated by qPCR for library pooling. Sequencing was performed on the Illumina HiSeq 4000 platform using PE150 chemistry (Illumina).
The raw data were aligned to the hg38 reference genome, and the number of reads per gene was counted using STAR v.2.5.144. The DESeq2 R package45 was applied for differential expression analysis and gene expression normalization. All p values were corrected using the false discovery rate (FDR) method. Genes with corrected p-values less than 0.05-, and two or more-fold change differences are considered differentially expressed. Gene expression data were log-transformed for principal components and clustering analysis. The clustered heatmap was created with the R package “pheatmap”46.
Statistics and reproducibility
The clinical trial was a non-randomized phase I trial. For the dose escalation component, a traditional 3 + 3 design was used. By study design, 3–6 patients were expected to enroll. An additional 15-patient expansion cohort was planned. With 6 patients treated at the MTD in the dose escalation phase and an additional 15-patient expansion cohort, a minimum of 21 patients were expected to be available for assessing safety and estimating efficacy at the MTD. The trial closed early following the accrual of 5 patients to the expansion cohort due to activation of the phase 3 SWOG/NRG S1914, which enrolled this population. A total of 20 patients were enrolled. Descriptive statistics (including frequency, proportion, median, and range) were used to summarize patient demographics, tumor characteristics, toxicity, and response status. A waterfall plot was generated to describe the relative changes in tumor size using R version 4.0.4. OS, PFS, and FFP were analyzed by the Kaplan–Meier method47 and compared between subgroups by log-rank tests using GraphPad Prism version 8.3.
The sample size for correlative studies was based on the presence of sufficient material to perform the studies. No statistical method was used to predetermine the sample size for correlative studies. No data were excluded from the analyses. This trial did not include randomization, and investigators were not blinded during experiments and outcome assessment. Statistical analysis of IHC/IC data was performed using descriptive statistics. Comparisons across two groups (e.g., responders vs. non-responders or non-progressor vs. progressor) were performed using a two-sided t-test. Data were analyzed using GraphPad Prism version 8.3. For the evaluation of PBMCs and transcriptomic, a two-sided t-test was used to compare two groups, and ANOVA was used to evaluate multiple groups or time points. Flow cytometry data were analyzed using Flowjo software (v. 6.10.2). Percent cells were examined for normality and log transformed when necessary. Statistical analyses were performed using the R statistical package (v.4.1.1)48. A two-sided t-test was used to compare two groups of patients, and p-value less than 0.05 were considered statistically significant. Since these analyses were hypothesis-generating in nature to discover potential biomarkers that will be further analyzed in an ongoing phase III trial, we did not control for multiple comparisons (with the exception of transcriptomic analyses). Potential biomarkers were analyzed using logistic regression implemented in the R package logistf49. The logistic regression ROC analysis was performed with the “ROCR” package50.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.