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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

Given the success of immunotherapy (IO), multiple IO-based options exist for advanced non-small cell lung cancer (NSCLC). Currently used biomarkers do not fully predict clinical outcomes and response assessment remains limited to radiological evaluation. Dynamic biomarkers that evaluate both tumor and host immune responses to IO are needed. We studied patients with NSCLC that received IO and chemo-IO to identify predictive and longitudinal biomarkers of clinical response using circulating tumor DNA (ctDNA) and plasma proteomic dynamics. We conducted deep targeted error-correction sequencing (TEC-Seq) of plasma cell-free DNA (cfDNA) and matched white blood cell (WBC) genomic DNA (gDNA) to identify ctDNA variants. We performed multiplexed antibody-based proximity extension assays of serial plasma samples to detect immune-related proteins. From separate training (n=31) and validation (n=29) cohorts, a total of 288 plasma cfDNA and 52 WBC gDNA specimens underwent TEC-Seq. A total of 260 variants were detected in plasma cfDNA and 78 variants in WBC gDNA. Almost a third of the plasma cfDNA variants (32%, n=61 of 188) were also found in WBC gDNA and filtered out. ctDNA variants were identified in 82% of patients (n=50). In the training cohort, longitudinal decreases in ctDNA variant levels were observed in patients with durable clinical benefit (DCB). Molecular response, defined as the loss of detectable ctDNA, was associated with longer progression-free (PFS; p=0.0004, log-rank) and overall survival (OS; p=0.017, log-rank). We incorporated on-treatment ctDNA dynamics including molecular response, recrudescence, and emergence of new variants into a logistic regression model to predict clinical benefit. This integrative model predicted DCB at a sensitivity of 84%, specificity of 76%, and with an area under the curve (AUC) of 0.90, better than baseline tumor PD-L1 expression alone (AUC 0.70, p=0.044, bootstrap method). In the validation cohort, molecular response was associated with longer PFS (p=0.00098, log-rank) and OS (p=0.0037, log-rank) and the integrative model predicted DCB at a sensitivity of 100%, specificity of 78%, and AUC of 0.89. In a subset (n=28 of 31) of the training cohort, plasma proteomics analysis revealed that increased baseline levels of IL15 and DCN were independently associated with DCB (p Citation Format: Joseph C. Murray, Karlijn Hummelink, Lamia Rhymee, Alessandro Leal, Leonardo Ferreira, Mara Lanis, James R. White, Noushin Niknafs, Kristen Marrone, Jarushka Naidoo, Benjamin Levy, Samuel Rosner, Christine Hann, Josephine Feliciano, Vincent Lam, David Ettinger, Qing K. Li, Peter Illei, Kim Monkhorst, Hatim Husain, Julie R. Brahmer, Victor Velculescu, Patrick Forde, Robert B. Scharpf, Valsamo Anagnostou. Longitudinal dynamics of circulating tumor DNA and plasma proteomics predict clinical outcomes to immunotherapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1668.

Alterations in EGFR, KRAS, and ALK are oncogenic drivers in lung cancer, but how oncogenic signaling influences immunity in the tumor microenvironment is just beginning to be understood. Immunosuppression likely contributes to lung cancer, because drugs that inhibit immune checkpoints like PD-1 and PD-L1 have clinical benefit. Here, we show that activation of the AKT–mTOR pathway tightly regulates PD-L1 expression in vitro and in vivo. Both oncogenic and IFNγ-mediated induction of PD-L1 was dependent on mTOR. In human lung adenocarcinomas and squamous cell carcinomas, membranous expression of PD-L1 was significantly associated with mTOR activation. These data suggest that oncogenic activation of the AKT–mTOR pathway promotes immune escape by driving expression of PD-L1, which was confirmed in syngeneic and genetically engineered mouse models of lung cancer where an mTOR inhibitor combined with a PD-1 antibody decreased tumor growth, increased tumor-infiltrating T cells, and decreased regulatory T cells. Cancer Res; 76(2); 227–38. ©2015 AACR.

Early detection is a major goal for reducing mortality of human cancer. However, non-invasive detection of early stage tumors has remained a challenge. We have developed an approach called Targeted Error Correction Sequencing (TEC-Seq) for ultra-sensitive analyses of circulating cell-free tumor DNA (ctDNA). This methodology involves in-solution targeted capture of multiple regions of the genome and deep sequencing (~30,000x) of cell-free DNA fragments. Laboratory and bioinformatic methods were optimized to enrich for rare ctDNA molecules and to reduce potential amplification, sequencing, and contamination errors. We have used this approach to examine 58 cancer related genes, and demonstrated a limit of detection of mutant to wild-type DNA of 0.05% and a specificity >99.999% across targeted regions of interest. We applied this method to analyze plasma from healthy individuals as well as over 200 individuals with breast, lung, colorectal and ovarian cancers. Analysis of plasma from 44 healthy individuals revealed no tumor-related somatic mutations and identified alterations in genes related to myelodysplasia in a subset of cases. Among patients with cancer, we detected measurable ctDNA in 56%, 71%, 57%, and 56% of patients with early stage (I and II) breast, colorectal, lung and ovarian cancer. Over three quarters of patients with late stage (III and IV) disease had detectable ctDNA among all cases analyzed. Analyses of mutations in the circulation revealed a high concordance with alterations in the independently analyzed tumors of these patients. These analyses provide a widely applicable, ultra-sensitive, and non-invasive method for detection of ctDNA, and have important implications for detection of early stage disease and management of patients with cancer. Citation Format: Jillian A. Phallen, Mark Sausen, Vilmos Adleff, Alessandro Leal, Jacob Fiksel, Carolyn Hruban, Savannah Speir, Eniko Papp, Valsamo Anagnostou, Mai-Britt W. Orntoft, Thomas Reinert, Brian D. Woodward, Derek Murphy, Sonya Parpart-Li, David Riley, Monica Nesselbush, Naomi Sengamalay, Andrew Georgiadis, Rob Scharpf, Qing K. Li, Sian Jones, Samuel Angiuoli, Joost Huiskens, Cornelis Punt, Nicole van Grieken, Remond Fijneman, Gerrit Meijer, Hatim Husain, Luis Diaz, Torben Ørntoft, Hans J. Nielsen, Claus L. Anderson, Victor E. Velculescu. Detection of circulating tumor DNA in early stage cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-246. doi:10.1158/1538-7445.AM2017-LB-246

Oligodendroglial tumors (OTs) are a major subtype of glioma. They are characterized by IDH mutation and a combined deletion of chromosomes 1p and 19q. OT is an interesting entity in neuro-oncology due to the strong association of 1p/19q codeletion with favorable prognosis and chemosensitivity. Although the mechanism underlying the predictive power of 1p/19q codeletion in prognosis and treatment response is not known, chromosomes 1p and 19q likely contain OT-related genes. Recent exomic sequencing study in OTs revealed two novel mutated genes, CIC and FUBP1, that map to 19q13 and 1p31, respectively. The aims of this study were to (1) determine the incidences of 1p/19q codeletion and mutations of IDH, CIC and FUBP1, and (2) evaluate the clinical relevance of these molecular aberrations in 55 OTs diagnosed at our hospital. Our results showed that somatic mutations of CIC and FUBP1 occurred in 47% and 15% of OTs examined. CIC mutations included 57% indels, 39% missense mutations and 4% nonsense mutation. Missense mutations in CIC were localized to the high-mobility group domain and the C1 motif with repressor activity. More than 75% of missense mutations were predicted to confer damaging effects on cic function. FUBP1 mutations included 3 indels, 3 splicing alterations and 1 nonsense mutation. Six of 7 FUBP1-mutated tumors were concurrently mutated in CIC. 1p/19q codeletion and IDH mutation were observed respectively in 44% and 91% of OTs examined. About 60% of CIC-mutated OTs also showed 1p/19q codeletion, suggesting that, at least in this tumor subset, CIC might act as a candidate tumor suppressor. Follow-up data were available for 44 patients. Survival analysis revealed correlation of tumor grade and patient age of Citation Format: Jesse C. Pang, Aden K. Chan, Nellie Y. Chung, Kay Li, Wai Sang Poon, Ho-Keung Ng. Mutation analysis and prognostic relevance of CIC and FUBP1 in oligodendroglial tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1919. doi:10.1158/1538-7445.AM2013-1919