Application of circulating tumor DNA analysis in detecting Minimal residual disease (MRD) after surgery for locally advanced head and neck squamous cell carcinoma: a preliminary study

The functional GRHL3-filaggrin axis maintains a tumor differentiation potential and influences drug sensitivity

Role of Hyaluronan-Mediated CD44 Signaling in Head and Neck Squamous Cell Carcinoma Progression and Chemoresistance

CHK1 plays a critical role in DNA damage repair (DDR) pathways as well as in coordinating DNA replication. Selective CHK1/CHK2 compounds are being tested in clinical trials but predictive biomarkers of patient response are lacking. A phase 1b expansion cohort study (I4D-MC-JTJA, NCT01115790) with the CHK 1 inhibitor, prexasertib, included patients with advanced, metastatic head and neck squamous cell carcinoma (HNSCC) or squamous cell carcinoma of the anus (SCCA). To identify genomic biomarkers associated with single-agent drug response, pre-treatment tissues (archived or biopsy) from 71 consented patients (HNSCC=47, SCCA=24) were subjected to next-generation sequencing (NGS) using the FoundationOne gene panel. In this subset of patients, the disease control rate (DCR) (Complete Response (CR) + Partial Response (PR) + Stable Disease (SD) based on RECIST Criteria (v 1.1)) was 60% (28/47) and 75% (18/24), respectively. We present here the observed genetic alterations corresponding to three pathways, Cell Cycle, DNA Damage Repair (DDR) and PI3K. In addition, patients’ human papillomavirus (HPV) carrier status was inferred from DNA sequencing using HPV-specific capture probes. HPV+ was 47% for HNSCC and 87% for SCCA. HPV+ and TP53 mutations were mutually exclusive across the two patient cohorts. In HNSCC patients with evaluable progression-free survival (PFS) data, greater clinical efficacy was observed in the HPV+ cohort (median PFS: 4.5 vs 1.4 months, log-rank p = 0.0008). Known or likely loss-of-function (LOF) mutations in FBXW7 and PARK2, two genes implicated in Cyclin E1 proteolysis, were noted in patients with favorable response in both tumor types. Across both HNSCC and SCCA cohorts, mutations and/or germline variants in the DDR genes BRCA1, BRCA2, MRE11A and ATR but not in Fanconi (FANC) pathway genes, were found in patients with treatment benefit. Whereas PIK3CA mutations were infrequent in the HNSCC cohort, in SCCA, mutations occurred in 5/8 (63%) patients with disease control vs 1/6 (17%) with PD. All 7 PI3KCA mutations observed in HPV+ HNSCC and SCCA patients mapped to the helical domain suggestive of Apobec-induced mutagenesis as their source of origin. The enhanced clinical benefit to prexasertib associated with HPV+ in HNSCC may reflect a prognostic effect. Alternatively, the clinical biomarker findings may support the hypothesis that oncogene-induced replication stress (RS) (i.e. arising from HPV E6/E7 and/or FBXW7 loss-dependent Cyclin E1 dysregulation) in the context of attenuated DDR (i.e. BRCA1/2, MRE11A mutations) may sensitize patients to prexasertib monotherapy. Citation Format: Ricardo Martinez, Sameera R. Wijayawardana, David Hong, Johanna Bendell, Anna Maria Russell, Richard P. Beckmann, Aimee Bence Lin. A clinical genomic biomarker study of the CHK1 inhibitor prexasertib in advanced head and neck squamous cancer and squamous cell carcinoma of the anus [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 1778. doi:10.1158/1538-7445.AM2017-1778

Programmed death-ligand 1 (PD-L1) is a trans-membrane protein and co-inhibitory factor of the immune response. PD-L1 can combine with its receptor (PD-1) to reduce the proliferation of PD-1-positive cells and induce apoptosis. Therefore, PD-L1 was employed as a prognostic marker and a target for anti-cancer immunotherapy in blocking the PD-1 and PD-L1 checkpoints. PD-L1 is highly expressed in various malignancies, including head and neck squamous cell carcinoma (HNSCC). HNSCC is the sixth most common cancer worldwide, with high incidence and mortality rates in China. Although PD-L1 expression has been widely investigated, the PD-L1 expression status in Chinese HNSCC patients (pts) is largely unrevealed. The primary objective of this study was to determine the prevalence of PD-L1 expression with the Combined Positive Score (CPS) ≥20 in Chinese pts with recurrent or metastatic (R/M) HNSCC. The secondary objectives were to determine the prevalence of CPS≥1 in pts with R/M HNSCC and to study the difference in the demographic characteristics, clinicopathological parameters, treatment status, and other available biomarkers between the PD-L1 CPS≥20 group and the PD-L1 CPS<20 group. This study was a multi-center retrospective analysis of data from six centers in China from August 9, 2021, to February 28, 2022. Pts with histologically/cytologically confirmed diagnoses of R/M HNSCC were included. PD-L1 expression was assessed by IHC using the 22C3 PharmDx assay (Agilent, Santa Clara, CA, USA) and was determined using a CPS. The Chi-square test, Fisher’s exact test, or Wilcoxon rank sum test would be used to compare the prevalence of these variables among pts between the PD-L1 CPS≥20 group and the PD-L1 CPS<20 group. Out of 406 enrolled pts with R/M HNSCC, 402 testing pts were included in the final analysis. For all testing R/M HNSCC pts, 168 pts (41.8% [95% CI 36.92-46.78]) had PD-L1 expression with CPS ≥20. In addition, 337 pts (83.8% [95% CI 79.86-87.29]) had PD-L1 expression (CPS≥1). For the testing pts, there were statistically significant differences in variables of gender (P < 0.001), smoking habit (P = 0.0138 for non-smokers versus current smokers), and primary tumor site (P < 0.001 for hypopharynx versus oral cavity and P = 0.0304 for larynx versus oral cavity) between the PD-L1 CPS≥20 group and the PD-L1 CPS<20 group. In summary, in Chinese R/M HNSCC pts, most pts (83.8%) had PD-L1 expression, and two-fifths (41.8%) had PD-L1 expression with CPS ≥20. Prevalence of PD-L1 among Chinese pts with R/M HNSCC was consistent with that reported in the global KEYNOTE-048 study. PD-L1 expression was significantly associated with gender, smoking history and primary tumor site. Our findings of the variables related to the PD-L1 expression levels can supply adjuvant evidence for clinical practice and are a solid basis for future research on immunotherapy. Citation Format: Haizhen Lu, Dong Kuang, Lili Jiang, Jingping Yun, Qingxin Xia, Jian Wang, Pei Duan, Ping Zhou, Shengbing Zang, Yiping Jin, Xiangnan Jiang, Jielin Li, Wenmin Tang, Jiansong Zhou, Jihua Chen, Jianming Ying. The PD-L1 protein expression in Chinese patients with recurrent or metastatic head and neck squamous cell carcinoma: A multi-center retrospective study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5463.

Head and neck squamous cell carcinomas (HNSCCs) that develop from the mucosal epithelium in the oral cavity, pharynx, and larynx are a heterogeneous group of malignant tumors. A lack of appropriate screening and diagnostic methods leads to late diagnoses, with the majority of patients having locally advanced disease, which is associated with a high risk of local recurrence and a poor prognosis and is usually treated with combination therapies. Biomarkers for predicting the therapy response and risk of recurrence in HNSCC patients are urgently needed. Liquid biopsy, e.g., the profiling of circulating biomarkers in bodily fluids, is a promising approach with increasing utility in the early detection and diagnosis of cancer, monitoring cancer progression, patient stratification and treatment selection, detecting minimal residual disease (MRD), and predicting recurrence across different cancer types, including HNSCC. Among liquid biomarkers, circulating tumor DNA (ctDNA), which is based on detecting tumor-specific mutations, insertions/deletions, copy number alterations, and methylation, is the most promising transformative tool in cancer management and personalized cancer treatment. In this review, we provide an update of recent data on the role of non-viral ctDNA in the management of HNSCC patients. Accumulating data suggests the enormous potential of ctDNA profiling by serial sampling during and after definitive therapy in detecting MRD and predicting recurrence in HNSSC patients treated with a single treatment modality (surgery or radiotherapy) or with combination therapies, including immune-checkpoint-inhibitor-based immunotherapy. By incorporating the latest immunotherapy trials and organizing the data by the treatment modality, this review offers a novel perspective not found in previous surveys.

Phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases that regulate cellular activities such as proliferation, survival, motility and morphology. Recent studies reported that the p110alpha (PIK3CA), catalytic subunit of PI3-kinase is somatically mutated in human cancers. Hot- spot mutations (E542K, E545K and H1047R) are reported to have higher oncogenic potential. Although PIK3CA mutations were reported in head and neck squamous cell carcinomas (HNSCC) of limited ethnicity, the functional consequences of HNSCC-associated PIK3CA mutations have not been examined. Status of PI3K signaling related genes (PTEN-RAS-EGFR) in the presence of PIK3CA mutation have not been reported. In this study, we analyzed exons 9 and 20 of PIK3CA in 54 samples, including 17 HNSCC cell lines, 19 Indian and 18 Vietnamese primary tumors. We found mutations in 29.4% (5/17) of HNSCC cell lines, 10.5% (2/19) of Indian tumors and no mutation (0/18) in Vietnamese tumors. Two homozygous PIK3CA mutations were found in cell lines and a novel insertion mutation with oncogenicity in Indian tumor. Analysis of PI3K signaling related genes showed that PIK3CA and PTEN mutations were mutually exclusive, though PTEN mutation is uncommon in HNSCC. However, PIK3CA mutation coexisted with H-RAS mutation. Furthermore, PIK3CA mutations were mutually exclusive to EGFR amplification. All the 5 mutants that we found in HNSCC, showed increased PI3 kinase activities, followed by growth factor independent higher colony forming efficiency, changes in morphology, higher rates of migration and invasion compared with PIK3CA wild-type. Our study is the first to examine the oncogenic potential of PIK3CA mutants associated with HNSCC and report on PIK3CA mutations in Indian and Vietnamese ethnicity. These results suggest that PIK3CA mutations in HNSCC are likely to be oncogenic and may significantly contribute to HNSCC carcinogenesis and pave attractive target for therapeutic prevention.

Low-Level Expression of miR-375 Correlates with Poor Outcome and Metastasis While Altering the Invasive Properties of Head and Neck Squamous Cell Carcinomas

IA18: Tumor suppressor gene silencing by somatic mutations and promoter methylation leads to genomic instability in head and neck squamous cell carcinoma

Large and comprehensive genomic analyses of head and neck squamous cell carcinomas (HNSCC) are greatly increasing our understanding of the heterogeneity of this disease and identifying the key genomic changes that drive these tumors. HNSCC tumors associated with HPV are distinct from HNSCC disease linked to tobacco exposure (HPV-positive versus HPV-negative). HPV- tumors are characterized by: 1) amplification of receptor tyrosine kinases (RTK); 2) genomic loss of TP53 and loss of CKDN2A/RB1 by a variety of mechanisms including truncating mutation, deletion, and/or alternative splicing; 3) amplification of CCND1/CDK4/CDK6; 4) activation of NFE2L2/KEAP1/CUL3; and 5) loss of NOTCH1/FAT1/AJUBA. In contrast, HPV-positive HNSCCs harbor: 1) RTK amplification; 2) FGFR2/3 mutations and/or FGFR3-TACC3 fusions; 3) CDKN2A loss; and 4) E2F1 amplification. Interestingly, activating mutations in the PIK3CA oncogene are found in approximately one third of HPV-negative HNSCC and in over fifty percent of HPV-positive tumors. Mutually exclusive mutations in RAS family genes, specifically RHOA, KRAS, and HRAS, are present but infrequently found in HNSCC (6% of TCGA cases). Amplification of chromosome 3q, a region containing the TP63, SOX2, and PIK3CA genes, is seen in the majority of both HPV-negative and HPV-positive HNSCC. HPV-negative HNSCCs demonstrate clear evidence of molecular heterogeneity, as demonstrated by expression profiling studies indicating diverse biologic subclasses within HPV-negative disease, including a class of tumors without EGFR amplification and/or overexpression, previously termed “atypical” HNSCCs, which consist of approximately 20% of HPV-negative cases and the majority of HPV-positive HNSCCs. An intriguing mutational pattern identified by TCGA was a subset of HPV-negative HNSCC originating in the oral cavity with few to no copy-number alterations statistically enriched for HRAS, CASP8, and PIK3CA mutations and lack of TP53 mutation. With increasing genetic profiling of HNSCC cell lines and patient-derived xenografts, there is increasing opportunity to rationally select preclinical models to test specific precision medicine hypotheses. Citation Format: Jennifer R. Grandis. Comprehensive genomic characterization of head and neck squamous cell carcinomas [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr IA09.

Objective: Head and neck squamous cell carcinoma (HNSCC) affects approximately 500,000 people per year, with 50,000 affected in the United States. Depending on stage and pathologic features, treatment modalities consist of surgery with or without adjuvant therapy, or primary chemoradiation. However, monitoring with serial examination and imaging including CT or PET-CT has significant limitations with a delay between actual and detectable tumor response. Circulating cell-free tumor DNA (ctDNA) has shown promise as a biomarker for monitoring cancer treatment response, recurrence, and prognosis. We utilize a novel, barcoded next-generation sequencing approach called SimSen-Seq to facilitate ultrasensitive detection of ctDNA, and we are applying it to samples from patients with head and neck squamous cell carcinoma (HNSCC). The purpose of this study is to evaluate the feasibility of using tumor mutations that are detectable in ctDNA to monitor treatment response and recurrence in patients with HNSCC. Methods: Longitudinal blood samples were obtained from patients with Stage I-IV HNSCC, with the initial sample obtained immediately before surgery. Imaging studies and pathology reports were reviewed to determine disease stage. Tumor samples were obtained from surgical specimens, and tumor DNA was sequenced using a targeted panel to identify mutations. SimSen-Seq assays were developed for each patient and used to generate sequencing libraries from cell-free DNA isolated from plasma. Mutations in plasma were identified, quantified, and associations with disease stage and response to therapy were explored. Results: Preliminary results from 5 patients with HNSCC demonstrate detection of ctDNA, and correlation of ctDNA fraction with treatment response and recurrence. Four of these patients had stage IV disease, and ctDNA was detectable immediately preoperatively. One patient demonstrated positive ctDNA 3 weeks postoperatively, with no clinical evidence of residual disease, but subsequently recurred and died soon afterward. Two other patients demonstrated negative ctDNA at 3-week postoperative blood draws, but subsequently demonstrated metastatic recurrence over 6 months later. Conclusions: ctDNA can be detected in the plasma of HNSCC patients, and can be detected prior to clinical evidence of recurrence. Furthermore, positive detection early after surgery may suggest a poor prognosis. Additional samples have been collected, and more work to evaluate detection rates based on stage plus response to therapy and recurrence is ongoing. Citation Format: Stefanie S. Saunders, Matthew Egyud, Anand Devaiah, Scharukh Jalisi, Tony Godfrey. Detection of cell-free circulating tumor DNA in plasma as a biomarker for treatment response, prognosis, and recurrence in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 57.

Introduction: Most head and neck squamous cell carcinoma (HNSCC) patients are diagnosed with locoregionally advanced disease and face high recurrence rates despite standard-of-care treatments. Using circulating tumor DNA (ctDNA) to detect minimal residual disease (MRD) has emerged as a promising biomarker for assessing recurrence risk and treatment efficacy across various cancers. However, the sensitivity of commercially available assays for detecting MRD remains limited, often failing to capture subtle dynamic changes. As a result, the dynamics of ctDNA immediately following treatment and its potential as a reliable biomarker for guiding early post-operative adjuvant treatment decision making remain poorly understood. Methods: Our group has developed MAESTRO (minor-allele-enriched sequencing through recognition oligonucleotides) that uses patient-specific probes to selectively enrich ctDNA carrying thousands of genome-wide tumor mutations, enabling highly sensitive MRD detection with minimal sequencing. We utilized MAESTRO to prospectively monitor MRD in HNSCC patients after surgery during the immediate post-operative window—serial blood samples taken while patients were in the hospital recovering from surgery—and the surveillance period, aiming to evaluate its potential to predict disease progression. Kaplan Meier survival analysis and Cox proportional hazards regression were applied to assess the association of MRD detection during the immediate post-operative window with event-free survival (EFS). Results: 24 HNSCC patients were enrolled. Patients were pre-dominantly HPV-negative [23/24 (95.8%)] and had advanced stage III/IV disease [22/24 (91.7%)]. Serial sampling in the days following surgery revealed rapid ctDNA clearance on the first post-operative blood draw (1-3 days postop) of patients without MRD. MAESTRO detected ctDNA in 13 of the 15 (86.7%) patients who experienced an event (recurrence or death). In 8 of the 13 (61.5%) patients with an event and detectable MRD, tumor fractions were below the limit of detection of leading commercial assays. MRD detection was associated with worse EFS (HR = 27.4; 95% CI 3.5-214.5; P < 0.0001) and remained an independent predictor of EFS when controlling for high-risk pathologic features (23.8; 3.0-192.1; P = 0.003). Conclusions: Tumor fractions of HNSCC patients treated effectively with surgery were undetectable as early as the first postoperative day. Most tumor fraction levels detected by MAESTRO were outside the sensitivity range of leading commercially available assays. MRD detection of patients in the immediate post-operative period was predictive of recurrence and worse survival. MAESTRO-based MRD detection may enable highly-sensitive identification of high-risk HNSCC patients and guide therapy including whether treatment escalation may be necessary. Citation Format: Edward S. Sim, 1 Justin Rhoades, 2 Kan Xiong, 2 Laurel Walsh, 2 Andjela Crnjac, 2 Timothy Blewett, 2 Yana Al-Inaya, 1 Julia Mendel, 1 Daniel Ruiz, 1 Vasileios Efthymiou, 1 Gjystina Lumaj, 1 William J. Benjamin, 1 G. Mike Makrigiorgos, 3 Shervin Tabrizi, 2 Viktor A. Adalsteinsson, 2 Daniel L. Faden1. Immediate postoperative minimal residual disease detection with MAESTRO predicts recurrence and survival in head and neck cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3251.

6056 Background: Highly sensitive minimal residual disease (MRD) assays using circulating tumor (ct)DNA have broad clinical potential and are impacting cancer care. Circulating human papillomavirus (HPV) DNA has emerged as a biomarker of occult MRD among patients (pts) with HPV+ oropharyngeal cancers, but most head and neck squamous cell carcinomas (HNSCC) are not virally driven. Despite multimodality therapy, nearly half of pts with locoregionally advanced HPV-negative HNSCC relapse. As ctDNA has the potential to identify MRD, predict outcomes, and guide treatment for these pts, we performed a retrospective cohort study to evaluate the performance of a custom-built, clinically and commercially available ctDNA assay for this population. Methods: Pts with newly diagnosed HPV-negative HNSCC (all mucosal sites) treated at a single academic institution with pre-treatment ctDNA testing (Signatera, Natera) performed during clinical care were identified. Signatera utilizes up to 16-plex PCR from matched tumor and blood to develop a personalized ctDNA assay. A subset of patients had additional ctDNA testing during follow-up. Study objectives were to understand baseline ctDNA detection in relation to disease characteristics, and to correlate ctDNA changes on- and post-treatment with disease status and survival. Results: Testing was performed in 92 of 115 (80%) pts with HPV-negative HNSCC (median age: 66, 69% male, 64% smokers); ctDNA testing could not be performed in 23 (20%) pts due to insufficient tumor tissue. Oral cavity (43%) and larynx (22%) were the most common subsites; with most having clinical T2-3 (54%) and N1-2 (51%) disease (AJCC 2017 8th edition) treated with definitive surgery (46, 40%) and/or chemoradiation (59, 51%). Pre-treatment, 69/92 (75%) pts had detectable ctDNA (range: 0.03-4049.69 mean tumor molecules/mL). No baseline independent clinical features predicted pre-treatment ctDNA detectability or levels (multiple logistic/linear regression modeling), but levels varied by T and N stage in univariate analysis (both p<0.05; Kruskal-Wallis test). At a median follow-up of 5.2 months (range: 0.2-15.1), 47 pts (51%) had >1 test result (range: 1-7; 170 total samples). Of 47 pts, 17 (36%) had ctDNA detected after treatment. Disease-free survival was significantly worse for pts who were ctDNA positive vs. negative after treatment (HR 4.35, 95%CI: 1.68-11.21, p<0.01); 1-year overall survival was 83.7% vs. 100% for pts who were ctDNA positive vs. negative. Conclusions: Tumor-informed, personalized ctDNA testing is feasible among pts with non-virally mediated HNSCC. ctDNA positivity as an early indicator of MRD positivity post-treatment was associated with inferior survival, identifying a high-risk subgroup. Further research is warranted to understand whether ctDNA may be leveraged to guide therapy and improve outcomes for HNSCC.

Clinical Medicine The Journal of Clinical Investigation Genetic landscape of metastatic and recurrent head and neck squamous cell carcinoma Matthew L. Hedberg, 1,2,3 Gerald Goh, 4,5 Simion I. Chiosea, 6 Julie E. Bauman, 7 Maria L. Freilino, 3 Yan Zeng, 3 Lin Wang, 6 Brenda B. Diergaarde, 8 William E. Gooding, 9 Vivian W.Y. Lui, 10 Roy S. Herbst, 4 Richard P. Lifton, 4,5 and Jennifer R. Grandis 11 Medical Scientist Training Program, University of Pittsburgh–Carnegie Mellon University, Pittsburgh, Pennsylvania, USA. 2 Department of Pharmacology and Chemical Biology and Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. 4 Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA. Howard Hughes Medical Institute, New Haven, Connecticut, USA. 6 Department of Pathology and 7 Department of Medicine, Division of Hematology/Oncology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. 8 Department of Epidemiology, Graduate School of Public Health, University of Pittsburgh, and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA. Biostatistics Facility, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA. 10 Department of Pharmacology and Pharmacy, Department of Biochemistry, Li-Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong. 11 Department of Otolaryngology–Head and Neck Surgery, UCSF, San Francisco, California, USA. BACKGROUND. Recurrence and/or metastasis occurs in more than half of patients with head and neck squamous cell carcinoma (HNSCC), and these events pose the greatest threats to long-term survival. We set out to identify genetic alterations that underlie recurrent/metastatic HNSCC. METHODS. Whole-exome sequencing (WES) was performed on genomic DNA extracted from fresh-frozen whole blood and patient-matched tumor pairs from 13 HNSCC patients with synchronous lymph node metastases and 10 patients with metachronous recurrent tumors. Mutational concordance within and between tumor pairs was used to analyze the spatiotemporal evolution of HNSCC in individual patients and to identify potential therapeutic targets for functional evaluation. RESULTS. Approximately 86% and 60% of single somatic nucleotide variants (SSNVs) identified in synchronous nodal metastases and metachronous recurrent tumors, respectively, were transmitted from the primary index tumor. Genes that were mutated in more than one metastatic or recurrent tumor, but not in the respective primary tumors, include C17orf104, inositol 1,4,5-trisphosphate receptor, type 3 (ITPR3), and discoidin domain receptor tyrosine kinase 2 (DDR2). Select DDR2 mutations have been shown to confer enhanced sensitivity to SRC-family kinase (SFK) inhibitors in other malignancies. Similarly, HNSCC cell lines harboring endogenous and engineered DDR2 mutations were more sensitive to the SFK inhibitor dasatinib than those with WT DDR2. CONCLUSION. In this WES study of patient-matched tumor pairs in HNSCC, we found synchronous lymph node metastases to be genetically more similar to their paired index primary tumors than metachronous recurrent tumors. This study outlines a compendium of somatic mutations in primary, metastatic, and/or recurrent HNSCC cancers, with potential implications for precision medicine approaches. FUNDING. National Cancer Institute, American Cancer Society, Agency for Science, Technology and Research of Singapore, and Gilead Sciences Inc. Introduction Head and neck squamous cell carcinoma (HNSCC) is the sev- enth most common incident cancer worldwide, with more than 600,000 new cases each year (1). The major risk factors for HNSCC are tobacco use, alcohol consumption, and/or infec- tion with oncogenic strains of HPV, primarily HPV 16 (2). Despite advances in multimodality treatment, including surgery, radiation, and chemotherapy, 5-year overall survival has improved modestly Authorship note: Matthew L. Hedberg and Gerald Goh contributed equally to this work. Conflict of interest: The authors have declared that no conflict of interest exists. Submitted: March 27, 2015; Accepted: October 22, 2015. Reference information: J Clin Invest. 2016;126(1):169–180. doi:10.1172/JCI82066. over 3 decades, and this persistent mortality is largely due to high rates of regional metastasis and locoregional recurrence (3). HNSCC metastases almost always arise first in the cervical lymph nodes (4). Most patients are diagnosed with locally advanced disease, and more than half have cervical lymph node metastases present at initial diagnosis (5). Clinically, this is classified as syn- chronous nodal metastasis and is an indicator of a poor prognosis. Five-year adjusted survival rates range from approximately 30% to 60% for patients with synchronous nodal metastasis compared with approximately 85% for patients whose cancer has not metas- tasized (6). Patients with synchronous nodal metastasis are also more likely to develop locoregional or distant metastatic recur- rence of HNSCC after completing curative-intent therapy (7). jci.org Volume 126 Number 1 January 2016

While circulating tumor DNA (ctDNA) is a promising biomarker for minimal residual disease (MRD) detection in head and neck squamous cell carcinoma (HNSCC), more sensitive assays are needed for accurate MRD detection at clinically-relevant timepoints. Ultrasensitive MRD detection immediately after surgery could guide adjuvant therapy decisions, but early ctDNA dynamics are poorly understood. We applied MAESTRO, a whole-genome, tumor-informed, mutation-enrichment sequencing assay, in a pooled testing format called MAESTRO-Pool, to plasma samples from HNSCC patients collected immediately after surgery and during surveillance. We evaluated whether early MRD detection could predict outcomes. Among 24 predominantly HPV-independent (95.8%) HNSCC patients, rapid ctDNA clearance occurred by the first postoperative sample (1-3 days postoperatively) in 9 patients without an event (recurrence or death). 13/15 patients with an event were MRD+ (PPV = 92.9%; NPV = 80%) with a median tumor fraction (TFx) of 54 ppm (range 6-1,177 ppm). In the first and last sample of the immediate postoperative window, 8/13 and 10/13 patients had TFx below 100 ppm, respectively, the detection limit of leading commercial assays. Early MRD detection correlated with worse overall survival (HR = 8.3; 95% CI: 1.1-66.1; P = 0.02) and event-free survival (HR = 27.4; 95% CI: 3.5-214.5; P < 0.0001) independent of high-risk pathology. Immediate postoperative MRD detection by MAESTRO was predictive of recurrence and death. Given the ultralow TFxs observed, ultrasensitive assays will be essential for reliable MRD detection during early postoperative timepoints to enable personalized adjuvant therapy decision-making in HNSCC.

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