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Abstract
There is a need to identify predictive biomarkers to guide treatment strategies in stage III non-small cell lung cancer (NSCLCs). In this multi-institutional cohort of 197 patients with stage III NSCLC treated with concurrent chemoradiation (cCRT) and durvalumab consolidation, we identify that low tumor aneuploidy is independently associated with prolonged progression-free survival (HR 0.63; p=0.03) and overall survival (HR 0.50; p=0.03). Tumors with high aneuploidy had a significantly greater incidence of distant metastasis and shorter median distant-metastasis free survival (p=0.04 and p=0.048, respectively), but aneuploidy level did not associate with local-regional outcomes. Multiplexed immunofluorescence analysis in a cohort of NSCLC found increased intratumoral CD8-positive, PD-1-positive cells, double-positive PD-1 CD8 cells, and FOXP3-positive T-cell in low aneuploid tumors. Additionally, in a cohort of 101 patients treated with cCRT alone, tumor aneuploidy did not associate with disease outcomes. These data support the need for upfront treatment intensification strategies in stage III NSCLC patients with high aneuploid tumors and suggest that tumor aneuploidy is a promising predictive biomarker.
- immune checkpoint inhibitors
- tumor biomarkers
- non-small cell lung cancer
- tumor microenvironment
Data availability statement
Data are available upon reasonable request.
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Introduction
Although durvalumab consolidation after concurrent chemoradiation (cCRT) in patients with unresectable stage III non-small cell lung cancer (NSCLC) has significantly prolonged survival, only a minority of patients achieve long-term cure.1 There remains a growing need to identify biomarkers of response to guide treatment intensification strategies. We have recently identified tumor mutational burden (TMB) and very-high PD-L1 (programmed death ligand 1) expression levels to independently predict for disease control.2 3 Aneuploidy may be a biomarker with special relevance in this patient population that may be informative beyond TMB and PD-L1.
Aneuploidy is defined as the presence of an abnormal number of chromosomes and is a hallmark of solid tumors.4 In advanced NSCLC treated with immune checkpoint inhibitors (ICIs), data find high aneuploidy5 6 to be associated with shorter survival outcomes, independent of TMB and PD-L1 expression.6 Recent studies in advanced NSCLC have also showed that there is a benefit to concurrent radiation with ICIs in high aneuploid tumors that is not observed in low-aneuploid disease,7 with the use of concurrent radiation leading to improved in-field and out-of-field disease outcomes. However, the impact of aneuploidy in patients with stage III NSCLC is yet to be assessed. Given that both radiotherapy and ICIs are employed to treat unresectable stage III NSCLCs, tumor aneuploidy may provide additional insights to guide management. In this multi-institutional analysis, we hypothesized that tumor aneuploidy would also be correlated with disease control and patterns of failure in patients with unresected stage III NSCLC treated with the standard of care regimen of cCRT followed by durvalumab consolidation.
Material and methods
Patients
This retrospective analysis included patients with American Joint Committee on Cancer (AJCC) 8th edition stage III NSCLC. Consecutive patients from Dana-Farber Cancer Institute (DFCI) and Memorial Sloan Kettering Cancer Center (MSKCC) were included who met the following criteria: (1) treated with platinum-based chemotherapy concurrently with definitive radiation therapy; (2) received at least one dose of durvalumab consolidation; and (3) had tumors that underwent targeted next-generation sequencing (NGS) such that aneuploidy could be assessed.
Aneuploidy assessment
To quantify aneuploidy levels, targeted sequencing data were analyzed using ASCETS (Arm-level Somatic Copy-number Events in Targeted Sequencing), as previously described8; this method uses segmentation files and log2 copy ratios for all interrogated genomic loci. A chromosomal arm was considered altered if at least 70% of its territory was either gained or deleted. The fraction of chromosomal arm alterations is defined as the number of altered chromosome arms divided by the number of chromosome arms assessed for each sample. Because of the relationship between fraction of chromosomal arm alterations and tumor content,8 we multiplied the fraction of chromosomal arm alterations by [1−(Tumor content/100)] to calculate the adjusted fraction of chromosomal arm alterations (FAA). Detailed methods are reported in the online supplemental methods.
Supplemental material
Statistical analysis
The FAA distributions were normalized within different platforms by applying a natural logarithmic transformation followed by standardization to Z scores, as previously described.9 Progression-free survival (PFS) and overall survival (OS) were defined as the time from durvalumab start to progression or death. Local-regional and distant failures were defined from the start of durvalumab to disease progression, with distant failure defined as metastatic disease progression per AJCC 8th edition staging. Clinical outcomes were compared in patients based on tumor aneuploidy level. All p values are two-sided with significance predefined at <0.05.
Results
A total of 197 patients with stage III NSCLC treated with concurrent CRT and durvalumab consolidation were assessed. The median follow-up was 28.8 months (IQR 18.7–31.0 months). The median patient age was 68 years (range 44–85), 94% (n=185) had a history of tobacco use, and 82% had non-squamous histology. Most patients (n=127, 64%) had stage IIIB or IIIC disease. Patients were treated with a median of 8.0 months of durvalumab (range 1–12 months). Durvalumab consolidation started at a median of 6 weeks from end of radiation therapy (range 1–33 weeks). Aneuploidy was defined as high and low based on the median FAA value. Patients with low and high aneuploid tumors were generally similar, but more patients with low aneuploid tumors had an Eastern Cooperative Oncology Group (ECOG) 0 (51% vs 36%) performance status and tumors with PD-L1 TPS ≥90% (25% vs 4%) compared with the high aneuploidy group. Patients with high aneuploid tumors had a significantly higher median TMB (p=0.001), as has been previously described4 6 (online supplemental table 1).
Supplemental material
On univariable analysis, compared with tumors with high aneuploidy, low aneuploid tumors were associated with a longer but not significantly different PFS (median 21.3 vs 14.3 months, HR 0.72 (95% CI 0.49 to 1.05); p=0.08) and significantly longer OS (not reached vs 47.3 months, HR 0.51 (95% CI 0.28 to 0.91); p=0.02) (figure 1A,B). Tumors with high aneuploidy had a significantly greater cumulative incidence of distant metastasis versus low aneuploidy tumors (50.5% vs 35.7%, p=0.04), but there was no difference in the cumulative incidence of local-regional failure based on aneuploidy level (18.4% vs 21.2%, p=0.72) (figure 1C). Additionally, there was a significantly longer median distant-metastasis free survival in tumors with low versus high aneuploidy (not reached vs 20.4 months, HR 0.65 (95% CI 0.42 to 0.99); p=0.048), but no difference in local-regional failure free survival was observed by aneuploidy level (online supplemental figure 1).
Supplemental material
(A) Progression-free survival (PFS), (B) overall survival (OS), and (C) local-regional and distant cumulative incidence of recurrence by aneuploidy level.
To elucidate the predictive versus prognostic role of aneuploidy, we next examined the effect on outcomes in the context of cCRT without durvalumab consolidation. Among the 101 consecutive patients treated with cCRT between 2014 and 2017 (prior to the approval of durvalumab), there was no difference in terms of PFS, local-regional failure or OS based on tumor aneuploidy (online supplemental figure 2 and online supplemental table 2).
Supplemental material
Supplemental material
The impact of tumor aneuploidy on disease outcomes was further interrogated by incorporating TMB. When categorized by TMB-high (>50th percentile) and low (≤50th percentile), the TMBhigh Aneuploidylow group had the longest PFS (not reached) and OS (not reached), whereas the TMBlow Aneuploidyhigh group had the lowest PFS (8.0 months) and OS (46.9 months) (figure 2A,B). Similarly, the beneficial effect of low aneuploidy on local-regional or distant failure was most pronounced in the high TMB group, with a significantly lower cumulative incidence of local-regional (5% vs 23%, p=0.02) and distant (21% vs 48% p=0.004) failure compared with other subgroups (figure 2C).
Patients were classified into high and low tumor mutational burden (TMB) groups if their cancer had a TMB >50th or ≤50th percentile, respectively. Similarly, patients were also classified into high and low aneuploidy groups using the median value. (A) Progression-free survival (PFS), (B) overall survival (OS), and (C) local-regional and distant cumulative incidence of recurrence into four groups of the combined TMB and aneuploidy levels.
To explore the mechanisms by which NSCLCs with low aneuploidy are more responsive to cCRT and durvalumab, we performed multiplexed immunofluorescence for CD8, PD-1, and FOXP3 in a separate cohort of 462 NSCLC samples that also underwent NGS at DFCI. We found a significant association between low-aneuploidy level and increased CD8-positive T-cell, PD-1-positive cells, double-positive PD-1 CD8 cells, and FOXP3-positive T-cell counts intratumorally (figure 3A). Additionally, TMBhigh Aneuploidylow tumors had significantly higher tumor-associated immune cells compared with other subgroups (figure 3B).
(A) CD8+, PD-1+, PD-1+CD8+, and FOXP3+ cells/mm2 by aneuploidy level. (B) CD8+, PD-1+, PD-1+CD8+, and FOXP3+ cells/mm2 by four groups of the combined tumor mutational burden (TMB) and aneuploidy levels. (C) Multiplexed immunofluorescence using the ImmunoProfile platform on three samples from non-small cell lung cancers.
Multivariable analysis
A multivariable model was created incorporating aneuploidy level and other variables that associated with PFS and OS on univariable analysis including ECOG PS, disease stage, PD-L1 and TMB (defined as p<0.1 for either PFS or OS) (online supplemental table 3). After adjusting for confounding factors, low tumor aneuploidy independently associated with improved PFS (HR 0.63 (95% CI 0.41 to 0.95); p=0.03) and OS (HR 0.50 (95% CI 0.27 to 0.92); p=0.03). Of note, even after adding an interaction term with ECOG PS and PD-L1 expression in the multivariable model, aneuploidy was significantly associated with shorter PFS (HR 0.65, p=0.042) and OS (HR 0.49, p=0.040).
Supplemental material
Discussion
These multi-institutional data identify that in patients with stage III NSCLC treated with cCRT and durvalumab consolidation, high tumor aneuploidy is independently associated with poor PFS and OS. Furthermore, the poor outcomes among these high aneuploid tumors are driven by a significantly higher incidence and earlier development of distant failures. These data suggest that upfront treatment intensification strategies may be of particular benefit in patients with high aneuploid tumors. Furthermore, we show that TMB and tumor aneuploidy can be used together to identify patients at greater and lower risk for progression. Additionally, our correlative data show that tumor aneuploidy and TMB are associated with distinct tumor immune microenvironments in patients with NSCLC.
In advanced NSCLC, high aneuploid tumors have been found to be resistant to ICI therapies.6 Our findings of poor PFS and OS among high aneuploid tumors are consistent with this literature. But interestingly, we did not find tumor aneuploidy to associate with local-regional outcomes. This finding is in line with recent work suggesting an interaction between radiotherapy and high aneuploid tumors.7 Recent work by Spurr et al suggest that tumor aneuploidy can be used to identify a subset of patients with advanced NSCLC who would benefit from concurrent radiation with ICI therapy.7 In this analysis, patients with high aneuploid tumors treated with radiation concurrent with ICI were less likely to have disease progression in radiated and unirradiated sites compared to patients who received sequential radiotherapy and ICI. Our study focused on patients with stage III NSCLC that received cCRT to local-regional disease immediately prior to ICI consolidation. We therefore hypothesize that our finding that high aneuploid tumors have poor distant outcomes, but not poor local-regional outcomes suggest that high aneuploid tumors are sensitive to radiation therapy and/or potentially the upfront radiation led to mechanisms that improved the responsiveness of the local-regional disease to ICI therapy. While our analysis is most suitable for hypothesis-generating data, our findings that high aneuploid stage III tumors have an earlier onset and greater incidence of distant failures further suggest that treatment intensification with combination ICI agents, or ICI concurrent with chemoradiation, should be explored in this patient population.
To gain further insight into the mechanism by which high aneuploidy produces a deleterious effect, we also evaluated immune cells infiltrates by multiplexed immunofluorescence in a cohort of NSCLC. We found that NSCLC with high aneuploidy display a distinct immunophenotype consistent with immune evasion with significantly lower levels of CD8+T cells. In addition, the TMBhigh Aneuploidylow subgroup, which had the most favorable disease control, had significantly higher levels of PD-1 expressing CD8+T cells within the tumor microenvironment, an observation associated with favorable clinical outcomes to PD-1 blockade.10 11 Further supporting these findings is that we did not find tumor aneuploidy to associate with disease outcomes in a separate exploratory cohort of patients treated with cCRT alone without durvalumab.
This work is limited by its retrospective nature but provides important insights to guide clinical trial design and future treatment strategies. Additionally, further analyses evaluating the impact of aneuploidy across larger panels of patients with mutations in genes associated with disease outcomes are needed. With several phase III trials in progress evaluating unselected approaches at treatment intensification including combinational immunotherapy agents, induction immunotherapy before CRT, and immunotherapy concurrent with CRT,12 there remains an unmet need to identify factors for patient selection. Our data suggest that tumor aneuploidy is a promising predictive biomarker to guide treatment intensification.
Data availability statement
Data are available upon reasonable request.
Ethics statements
Patient consent for publication
Acknowledgments
The Molecular Diagnostics Service in the Department of Pathology, and the Marie-Josee and Henry R. Kravis Center for Molecular Oncology at Memorial Sloan Kettering Cancer Center.
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
Twitter @alessi_joao, @XinAnnWang, @ArielleElkrief, @emilylebow, @TrishaSantosMD, @PhDMariaThor, @AdamJSchoenfeld, @DrMarkAwad
Correction notice This article has been corrected since it was first published online. In the original article, Daniel R Gomez had been omitted from the joint senior authorship. Daniel R Gomez, Mark M Awad and Narek Shaverdian are now listed as joint senior authors.
Contributors JVA: Conceptualization; data curation; formal analysis; investigation; methodology; visualization; roles/writing—original draft; writing—review and editing. AP: Data curation; methodology; writing—review and editing. ALR: Data curation; methodology; writing—review and editing. BR: Methodology; writing—review and editing. XW: Methodology; validation; writing—review and editing. AE: Writing—review and editing. FP: Data curation; methodology; validation; writing—review and editing. ADF: Data curation; methodology; validation; writing—review and editing. MMG: Writing—review and editing. ESL: Data curation; methodology; writing—review and editing. MT: Data curation; methodology; validation; writing - review and editing. PMGS: Data curation; writing—review and editing. AR: Data curation; methodology; validation; writing—review and editing. AS: Validation; writing—review and editing. JEC: Validation; writing—review and editing. BJ: Methodology; validation; writing—review and editing. DRG: Conceptualization; methodology; project administration; resources; supervision; validation; writing—review and editing, MA: Conceptualization; methodology; project administration; resources; supervision; validation; writing—review and editing. NS: Conceptualization; methodology; project administration; resources; supervision; validation; writing—review and editing.
Funding This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748 and by Elva J. and Clayton L. McLaughlin Fund for Lung Cancer Research and V Foundation.
Competing interests NS reports research funding from Novartis. MA serves as a consultant to Merck, Bristol-Myers Squibb, Genentech, AstraZeneca, Nektar, Maverick, Blueprint Medicine, Syndax, AbbVie, Gritstone, ArcherDX, Mirati, NextCure and EMD Serono. Research funding: Bristol-Myers Squibb, Lilly, Genentech and AstraZeneca. BJ receives post marketing royalties for EGFR mutation testing from Dana-Farber Cancer Institute, is a paid consultant to Novartis, Checkpoint Therapeutics, Hummingbird Diagnostics, Daichi Sankyo, AstraZeneca, G1 Therapeutics, BlueDotBio, GSK, Hengrui Therapeutics, Simcere Pharmaceutical, and unpaid member of a steering committee for Pfizer, and receives research support from Novartis and Cannon Medical Imaging. DRG has received consulting fees from Johnson and Johnson, Medtronic, AstraZeneca and GRAIL. He has received honoraria from MedLearning Group and Varian. He has received research funding from Varian and AstraZeneca. ESL has an equity interest and fiduciary role in Oncia Technologies. ALR reports grants from Varian Medical Systems, Boehringer Ingelheim, Pfizer, Astra Zeneca and Merck in addition to personal fees from Astra Zeneca, Merck, Cybrexa, Research to Practice, and MoreHealth, and reports non-financial support from Philips/Elekta. AS reports grants from GSK, PACT pharma, Iovance Biotherapeutics, Achilles Therapeutics, Merck and Harpoon Therapeutics, and consulting fees from J&J, KSQ Therapeutics, BMS, Enara Bio, Perceptive Advisors and Heat Biologics. JEC reports grants from Merck, Brystol Myers Squibb, Genentech and AstraZeneca.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.