Outcomes Stratification of Head and Neck Cancer Using Pre- and Post-treatment DNA Methylation From Peripheral Blood.

PURPOSE: Established prognostic factors for head and neck squamous cell carcinoma (HNSCC) mostly consist of clinical and tumor features assessed before treatment. We report a novel application of DNA methylation in peripheral blood before and after radiation therapy to further improve outcomes stratification. METHODS AND MATERIALS: Peripheral blood samples from patients with nonmetastatic HNSCC were obtained for methylation analysis 1 week before and 1 month after radiation therapy. Patients were randomized 1:1 to a Discovery Cohort or a Validation Cohort. In the Discovery Cohort, associations between genome-wide methylation change (posttreatment minus pretreatment) and recurrence-free survival (RFS) as well as overall survival (OS) were evaluated using Cox regression. A methylation risk score (MRS) was then constructed from methylation levels at the top associated sites, filtered for residing within the regulatory regions of genes expressed in cells of hematopoietic lineage. The prognostic value of MRS was separately assessed in the Discovery and Validation Cohorts. RESULTS: Between December 2013 and September 2018, 115 patients participated in this study. Human papilloma virus negative status, oral cavity cancer, gastrostomy tube insertion, and higher neutrophil count before radiation therapy were associated with shorter RFS and OS (P < .05). Genes downstream of the methylation sites comprising MRS are HIF1A, SF1, LGALS9, and FUT5, involved in hypoxia response, blood cell maturation, and immune modulation. High MRS (in the top third) was significantly associated with worse RFS (hazard ratio [HR], 7.1; 95% confidence interval [CI], 1.4-35.5; P = .016) and OS (HR, 15.9; 95% CI, 1.6-153.6; P = .017) in the Discovery Cohort, independent of the aforementioned risk factors. These findings were replicated in the Validation Cohort, for which high MRS also independently predicted worse RFS (HR, 10.2; 95%, CI 2.4-43.4; P = .002) and OS (HR, 3.7; 95% CI, 1.3-10.4; P = .015). CONCLUSIONS: We successfully trained and validated a signature of DNA methylation in peripheral blood before and after radiation therapy that stratified outcomes among patients with HNSCC, implicating the potential for genomics-tailored surveillance and consolidation treatment.

Circulating Tumor DNA in Adults With Glioma: A Systematic Review and Meta-Analysis of Biomarker Performance.

BACKGROUND: Circulating tumor DNA (ctDNA) has emerged as a promising noninvasive biomarker to capture tumor genetics in patients with brain tumors. Research into its clinical utility, however, has not been standardized because the sensitivity and specificity of ctDNA remain undefined. OBJECTIVE: To (1) review the primary literature about ctDNA in adults with glioma to compare the sensitivity and specificity of ctDNA in the cerebrospinal fluid vs the plasma and (2) to evaluate the effect of tumor grade on detection of ctDNA. METHODS: PRISMA-guided systematic review and meta-analysis was performed using published studies that assessed ctDNA in either plasma or cerebrospinal fluid among adult patients with confirmed glioma. Summary receiver operating characteristic curves were generated using the Rücker-Schumacher method, and area under the curve (AUC) was calculated. RESULTS: Meta-analysis revealed improved biomarker performance for CSF (AUC = 0.947) vs plasma (AUC = 0.741) ctDNA, although this did not reach statistical significance ( P = .141). Qualitative analysis revealed greater sensitivities among single-allele PCR and small, targeted next-generation sequencing panels compared with broader panels. It additionally demonstrated higher sensitivity of ctDNA detection in high-grade vs low-grade gliomas, although these analyses were limited by a lack of specificity reporting in many studies. CONCLUSION: ctDNA seems to be a highly sensitive and specific noninvasive biomarker among adults with gliomas. To maximize its performance, CSF should be studied with targeted genetic analysis platforms, particularly in high-grade gliomas. Further studies on ctDNA are needed to define its clinical utility in diagnosis, prognostication, glioblastoma pseudoprogression, and other scenarios wherein neoadjuvant therapies may be considered.

Detection of Tumor Recurrence via Circulating Tumor DNA Profiling in Patients with Localized Lung Cancer: Clinical Considerations and Challenges.

Approximately 30% of patients with non-small-cell lung cancer (NSCLC) present with localized/non-metastatic disease and are eligible for surgical resection or other "treatment with curative intent". Due to the high prevalence of recurrence after treatment, adjuvant therapy is standard care for most patients. The effect of adjuvant chemotherapy is, however, modest, and new tools are needed to identify candidates for adjuvant treatments (chemotherapy, immunotherapy, or targeted therapies), especially since expanded lung cancer screening programs will increase the rate of patients detected with localized NSCLC. Circulating tumor DNA (ctDNA) has shown strong potential to detect minimal residual disease (MRD) and to guide adjuvant therapies. In this manuscript, we review the technical aspects and performances of the main ctDNA sequencing platforms (TRACERx, CAPP-seq) investigated in this purpose, and discuss the potential of this approach to guide or spare adjuvant therapies after definitive treatment of NSCLC.

Plasma Metabolic Phenotypes of HPV-Associated versus Smoking-Associated Head and Neck Cancer and Patient Survival.

BACKGROUND: Metabolic differences between human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) and smoking-associated HNSCC may partially explain differences in prognosis. The former relies on mitochondrial oxidative phosphorylation (OXPHOS) while the latter relies on glycolysis. These differences have not been studied in blood. METHODS: We extracted metabolites using untargeted liquid chromatography high-resolution mass spectrometry from pretreatment plasma in a cohort of 55 HPV-associated and 82 smoking-associated HNSCC subjects. Metabolic pathway enrichment analysis of differentially expressed metabolites produced pathway-based signatures. Significant pathways (P < 0.05) were reduced via principal component analysis and assessed with overall survival via Cox models. We classified each subject as glycolytic or OXPHOS phenotype and assessed it with survival. RESULTS: Of 2,410 analyzed metabolites, 191 were differentially expressed. Relative to smoking-associated HNSCC, bile acid biosynthesis (P < 0.0001) and octadecatrienoic acid beta-oxidation (P = 0.01), were upregulated in HPV-associated HNSCC, while galactose metabolism (P = 0.001) and vitamin B6 metabolism (P = 0.01) were downregulated; the first two suggest an OXPHOS phenotype while the latter two suggest glycolytic. First principal components of bile acid biosynthesis [HR = 0.52 per SD; 95% confidence interval (CI), 0.38-0.72; P < 0.001] and octadecatrienoic acid beta-oxidation (HR = 0.54 per SD; 95% CI, 0.38-0.78; P < 0.001) were significantly associated with overall survival independent of HPV and smoking. The glycolytic versus OXPHOS phenotype was also independently associated with survival (HR = 3.17; 95% CI, 1.07-9.35; P = 0.04). CONCLUSIONS: Plasma metabolites related to glycolysis and mitochondrial OXPHOS may be biomarkers of HNSCC patient prognosis independent of HPV or smoking. Future investigations should determine whether they predict treatment efficacy. IMPACT: Blood metabolomics may be a useful marker to aid HNSCC patient prognosis.

Association of Epigenetic Age Acceleration With Risk Factors, Survival, and Quality of Life in Patients With Head and Neck Cancer.

PURPOSE: Epigenetic age acceleration (EAA) is robustly linked with mortality and morbidity. This study examined risk factors of EAA and its association with overall survival (OS), progression-free survival (PFS), and quality of life (QOL) in patients with head and neck cancer (HNC) receiving radiation therapy. METHODS AND MATERIALS: Patients without distant metastasis were enrolled and followed before and at the end of radiation therapy and at 6 and 12 months after radiation therapy. EAA was calculated with DNAmPhenoAge at all 4 time points. Risk factors included demographic characteristics, lifestyle, clinical characteristics, treatment-related symptoms, and blood biomarkers. Survival data were collected until August 2020, and QOL was measured using Functional Assessment of Cancer Therapy-HNC. RESULTS: Increased comorbidity, symptoms unrelated to human papilloma virus, and more severe treatment-related symptoms were associated with higher EAA (P = .03 to P < .001). A nonlinear association (quadratic) between body mass index (BMI) and EAA was observed: decreased BMI (<35 kg/m2; P = .04) and increased BMI (≥35 kg/m2; P = .01) were linked to higher EAA. Increased EAA (per year) was associated with worse OS (hazard ratio [HR], 1.11 [95% confidence interval {CI}, 1.03-1.18; P = .004]; HR, 1.10 [95% CI, 1.01-1.19; P = .02] for EAA at 6 and 12 months after treatment, respectively) and PFS (HR, 1.10 [95% CI, 1.02-1.19; P = .02]; HR, 1.14 [95% CI, 1.06-1.23; P < .001]; and HR, 1.08 [95% CI, 1.02-1.14; P = .01]) for EAA before, immediately after, and 6 months after radiation therapy, respectively) and QOL over time (ß = -0.61; P = .001). An average of 3.25 to 3.33 years of age acceleration across time, which was responsible for 33% to 44% higher HRs of OS and PFS, was observed in those who died or developed recurrence compared with those who did not (all P < .001). CONCLUSIONS: Compared with demographic and lifestyle factors, clinical characteristics were more likely to contribute to faster biological aging in patients with HNC. Acceleration in epigenetic age resulted in more aggressive adverse events, including OS and PFS. EAA could be considered as a marker for cancer outcomes, and decelerating aging could improve survival and QOL.

Liquid Biopsy of Non-Plasma Body Fluids in Non-Small Cell Lung Cancer: Look Closer to the Tumor!

Liquid biopsy is a rapidly emerging field due to an increasing number of oncogenic drivers and a better understanding of resistance mechanisms to targeted therapies in non-small cell lung cancer (NSCLC). The sensitivity of the most widely used blood-based assays is, however, limited in particular in cases of low tumor volume where shed of tumor-derived material can be limited. A negative result thus requires biopsy confirmation using minimally invasive sampling procedures that can result in small specimens, which are often not suitable for genotyping. Liquid biopsy is not limited to plasma, and tumor DNA circulating in other body fluids such as urine, pleural fluid, cerebrospinal fluid, or cytology specimen-derived supernatant can be exploited. In comparison to cell blocks, these fluids in close contact to the tumor may contain a more abundant and less analytically demanding tumor DNA. In this review, we discuss the potential applications of circulating tumor DNA derived from cytology samples in NSCLC, from early stage (screening, nodule characterization) to metastatic disease.

miR-550-1 functions as a tumor suppressor in acute myeloid leukemia via the hippo signaling pathway.

MicroRNAs (miRNAs) and N6-methyladenosine (m6A) are known to serve as key regulators of acute myeloid leukemia (AML). Our previous microarray analysis indicated miR-550-1 was significantly downregulated in AML. The specific biological roles of miR-550-1 and its indirect interactions and regulation of m6A in AML, however, remain poorly understood. At the present study, we found that miR-550-1 was significantly down-regulated in primary AML samples from human patients, likely owing to hypermethylation of the associated CpG islands. When miR-550-1 expression was induced, it impaired AML cell proliferation both in vitro and in vivo, thus suppressing tumor development. When ectopically expressed, miR-550-1 drove the G0/1 cell cycle phase arrest, differentiation, and apoptotic death of affected cells. We confirmed mechanistically that WW-domain containing transcription regulator-1 (WWTR1) gene was a downstream target of miR-550-1. Moreover, we also identified Wilms tumor 1-associated protein (WTAP), a vital component of the m6A methyltransferase complex, as a target of miR-550-1. These data indicated that miR-550-1 might mediate a decrease in m6A levels via targeting WTAP, which led to a further reduction in WWTR1 stability. Using gain- and loss-of-function approaches, we were able to determine that miR-550-1 disrupted the proliferation and tumorigenesis of AML cells at least in part via the direct targeting of WWTR1. Taken together, our results provide direct evidence that miR-550-1 acts as a tumor suppressor in the context of AML pathogenesis, suggesting that efforts to bolster miR-550-1 expression in AML patients may thus be a viable clinical strategy to improve patient outcomes.

Amplification of Wild-type KRAS Imparts Resistance to Crizotinib in MET Exon 14 Mutant Non-Small Cell Lung Cancer.

PURPOSE: MET inhibitors can be effective therapies in patients with MET exon 14 (METex14) mutant non-small cell lung cancer (NSCLC). However, long-term efficacy is limited by the development of drug resistance. In this study, we characterize acquired amplification of wild-type (WT) KRAS as a molecular mechanism behind crizotinib resistance in three cases of METex14-mutant NSCLC and propose a combination therapy to target it. EXPERIMENTAL DESIGN: The patient-derived cell line and xenograft (PDX) DFCI358 were established from a crizotinib-resistant METex14-mutant patient tumor with massive focal amplification of WT KRAS. To characterize the mechanism of KRAS-mediated resistance, molecular signaling was analyzed in the parental cell line and its KRAS siRNA-transfected derivative. Sensitivity of the cell line to ligand stimulation was assessed and KRAS-dependent expression of EGFR ligands was quantified. Drug combinations were screened for efficacy in vivo and in vitro using viability and apoptotic assays. RESULTS: KRAS amplification is a recurrent genetic event in crizotinib-resistant METex14-mutant NSCLC. The key characteristics of this genetic signature include uncoupling MET from downstream effectors, relative insensitivity to dual MET/MEK inhibition due to compensatory induction of PI3K signaling, KRAS-induced expression of EGFR ligands and hypersensitivity to ligand-dependent and independent activation, and reliance on PI3K signaling upon MET inhibition. CONCLUSIONS: Using patient-derived cell line and xenografts, we characterize the mechanism of crizotinib resistance mediated by KRAS amplification in METex14-mutant NSCLC and demonstrate the superior efficacy of the dual MET/PI3K inhibition as a therapeutic strategy addressing this resistance mechanism.

Liquid biopsy of fine-needle aspiration supernatant for lung cancer genotyping.

BACKGROUND: Tumor genotyping is transforming lung cancer care but requires adequate tumor tissue. Advances in minimally invasive biopsy techniques have increased access to difficult-to-access lesions, but often result in smaller samples. With the advent of highly sensitive DNA genotyping methods used for plasma analysis, we hypothesized that these same methods might allow genotyping of free DNA derived from fine needle aspiration supernatant (FNA-S). METHODS: We studied patients with known or suspected lung cancer undergoing fine needle aspirate (FNA). After spinning the sample for cellblock, the FNA-S (usually discarded) was saved for genotyping. Supernatant cell-free DNA (SN-cfDNA) was extracted and tested by both droplet digital PCR (EGFR, BRAF, KRAS mutations) and highly sensitive amplicon-based next-generation sequencing (NGS). RESULTS: 17 samples were studied, including 11 FNAs from patients with suspected lung cancer and 6 FNAs from patients with lung cancer and acquired drug resistance. Of 6 newly diagnosed adenocarcinomas, 4 had a driver mutations (1 EGFR, 2 KRAS, 1 HER2) found on tissue; all of these could be detected in SN-cfDNA. The EGFR driver mutation was detected in all 5 adenocarcinomas with acquired EGFR resistance and the EGFR T790 M in three cases, in agreement with cellblock. CONCLUSIONS: FNA-S is a rich source of fresh tumor DNA, potentially increasing the diagnostic yield from small FNAs. Through use of emerging techniques for highly sensitive genotyping, this widely available biospecimen has potential for facilitating rapid cancer genotyping at diagnosis and after drug resistance.

Validation of a Plasma-Based Comprehensive Cancer Genotyping Assay Utilizing Orthogonal Tissue- and Plasma-Based Methodologies.

Purpose: To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility.Experimental Design: Analytic validation was conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison with clinical droplet digital PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of digital sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples.Results: Digital sequencing technology enabled variant detection down to 0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24-2.76 copies 95% limits of detection while maintaining high specificity [prevalence-adjusted positive predictive values (PPV) >98%]. Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across >750 patients demonstrated high accuracy and specificity [positive percent agreement (PPAs) and negative percent agreement (NPAs) >99% and PPVs 92%-100%]. Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (>99.6% technical success rate) and clinical sensitivity (85.9%), with high potential actionability (16.7% with FDA-approved on-label treatment options; 72.0% with treatment or trial recommendations), particularly in non-small cell lung cancer, where 34.5% of patient samples comprised a directly targetable standard-of-care biomarker.Conclusions: High concordance with orthogonal clinical plasma- and tissue-based genotyping methods supports the clinical accuracy of digital sequencing across all four types of targetable genomic alterations. Digital sequencing's clinical applicability is further supported by high rates of technical success and biomarker target discovery. Clin Cancer Res; 24(15); 3539-49. ©2018 AACR.

False-Positive Plasma Genotyping Due to Clonal Hematopoiesis.

Purpose: Plasma cell-free DNA (cfDNA) genotyping is increasingly used in cancer care, but assay accuracy has been debated. Because most cfDNA is derived from peripheral blood cells (PBC), we hypothesized that nonmalignant mutations harbored by hematopoietic cells (clonal hematopoiesis, CH) could be a cause of false-positive plasma genotyping.Experimental Design: We identified patients with advanced non-small cell lung cancer (NSCLC) with KRAS, JAK2, or TP53 mutations identified in cfDNA. With consent, PBC DNA was tested using droplet digital PCR (ddPCR) or next-generation sequencing (NGS) to test for CH-derived mutations.Results: We first studied plasma ddPCR results from 58 patients with EGFR-mutant NSCLC. Two had KRAS G12X detected in cfDNA, and both were present in PBC, including one where the KRAS mutation was detected serially for 20 months. We then studied 143 plasma NGS results from 122 patients with NSCLC and identified 5 JAK2 V617F mutations derived from PBC. In addition, 108 TP53 mutations were detected in cfDNA; for 33 of the TP53 mutations, PBC and tumor NGS were available for comparison, and 5 were present in PBC but absent in tumor, consistent with CH.Conclusions: We find that most JAK2 mutations, some TP53 mutations, and rare KRAS mutations detected in cfDNA are derived from CH not tumor. Clinicians ordering plasma genotyping must be prepared for the possibility that mutations detected in plasma, particularly in genes mutated in CH, may not represent true tumor genotype. Efforts to use plasma genotyping for cancer detection may need paired PBC genotyping so that CH-derived mutations are not misdiagnosed as occult malignancy. Clin Cancer Res; 24(18); 4437-43. ©2018 AACRSee related commentary by Bauml and Levy, p. 4352.

Author Correction: Targeted inhibition of STAT/TET1 axis as a therapeutic strategy for acute myeloid leukemia.

The original version of this Article contained an error in the spelling of the author James C. Mulloy, which was incorrectly given as James Mulloy. This has now been corrected in both the PDF and HTML versions of the Article.

Cell-Free DNA in Oncology: Gearing up for Clinic.

In the past several years, interest in the clinical utility of cell-free DNA as a noninvasive cancer biomarker has grown rapidly. Success in the development of plasma genotyping assays and other liquid biopsy assays has widened the scope of cell-free DNA use in research and the clinic. Already approved by the US Food and Drug Administration in the narrow context of epidermal growth factor receptor-mutated non-small cell lung cancer, plasma genotyping assays are currently being investigated in a wide array of clinical settings and modalities. These include plasma genotyping as a tool for early diagnosis, the detection of minimal residual disease, and the evaluation of treatment response/progression. In this review, we assess the clinical landscape of plasma genotyping assays and propose strategies for their further expansion into routine clinical care.

Targeted inhibition of STAT/TET1 axis as a therapeutic strategy for acute myeloid leukemia.

Effective therapy of acute myeloid leukemia (AML) remains an unmet need. DNA methylcytosine dioxygenase Ten-eleven translocation 1 (TET1) is a critical oncoprotein in AML. Through a series of data analysis and drug screening, we identified two compounds (i.e., NSC-311068 and NSC-370284) that selectively suppress TET1 transcription and 5-hydroxymethylcytosine (5hmC) modification, and effectively inhibit cell viability in AML with high expression of TET1 (i.e., TET1-high AML), including AML carrying t(11q23)/MLL-rearrangements and t(8;21) AML. NSC-311068 and especially NSC-370284 significantly repressed TET1-high AML progression in vivo. UC-514321, a structural analog of NSC-370284, exhibited a more potent therapeutic effect and prolonged the median survival of TET1-high AML mice over three fold. NSC-370284 and UC-514321 both directly target STAT3/5, transcriptional activators of TET1, and thus repress TET1 expression. They also exhibit strong synergistic effects with standard chemotherapy. Our results highlight the therapeutic potential of targeting the STAT/TET1 axis by selective inhibitors in AML treatment.

Discrimination of Germline EGFR T790M Mutations in Plasma Cell-Free DNA Allows Study of Prevalence Across 31,414 Cancer Patients.

Purpose: Plasma cell-free DNA (cfDNA) analysis is increasingly used clinically for cancer genotyping, but may lead to incidental identification of germline-risk alleles. We studied EGFR T790M mutations in non-small cell lung cancer (NSCLC) toward the aim of discriminating germline and cancer-derived variants within cfDNA.Experimental Design: Patients with EGFR-mutant NSCLC, some with known germline EGFR T790M, underwent plasma genotyping. Separately, deidentified genomic data and buffy coat specimens from a clinical plasma next-generation sequencing (NGS) laboratory were reviewed and tested.Results: In patients with germline T790M mutations, the T790M allelic fraction (AF) in cfDNA approximates 50%, higher than that of EGFR driver mutations. Review of plasma NGS results reveals three groups of variants: a low-AF tumor group, a heterozygous group (∼50% AF), and a homozygous group (∼100% AF). As the EGFR driver mutation AF increases, the distribution of the heterozygous group changes, suggesting increased copy number variation from increased tumor content. Excluding cases with high copy number variation, mutations can be differentiated into somatic variants and incidentally identified germline variants. We then developed a bioinformatic algorithm to distinguish germline and somatic mutations; blinded validation in 21 cases confirmed a 100% positive predictive value for predicting germline T790M. Querying a database of 31,414 patients with plasma NGS, we identified 48 with germline T790M, 43 with nonsquamous NSCLC (P < 0.0001).Conclusions: With appropriate bioinformatics, plasma genotyping can accurately predict the presence of incidentally detected germline risk alleles. This finding in patients indicates a need for genetic counseling and confirmatory germline testing. Clin Cancer Res; 23(23); 7351-9. ©2017 AACR.