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Introduction: The current standard of care for patients with inoperable stage III non-small cell lung cancer includes chemoradiotherapy (CRT) followed by 1 year of checkpoint inhibitor (CPI) therapy. Nevertheless, the optimal duration of consolidation CPI remains unknown. Here, we characterized the relationship between circulating tumor DNA (ctDNA) minimal residual disease (MRD) and clinical outcomes of patients with unresectable locally advanced non-small cell lung cancer treated on a phase 2 trial of short-course consolidation immunotherapy after CRT, with the goal of testing whether ctDNA may be able to identify patients who do not require a full year of treatment., Methods: Plasma samples for ctDNA analysis were collected from patients on the Big Ten Cancer Research Consortium LUN 16-081 trial after completion of CRT, before day 1 of cycle 2 (C2D1) of CPI (i.e., 1 mo after treatment start), and at the end of up to 6 months of treatment. Tumor-informed ctDNA MRD analysis was performed using cancer personalized profiling by deep sequencing. Levels of ctDNA at each time point were correlated with clinical outcomes., Results: Detection of ctDNA predicted significantly inferior progression-free survival after completion of CRT (24-mo 29% versus 65%, p = 0.0048), before C2D1 of CPI (24-mo 0% versus 72%, p < 0.0001) and at the end of CPI (24-mo 15% versus 67%, p = 0.0011). In addition, patients with decreasing or undetectable ctDNA levels after 1 cycle of CPI had improved outcomes compared with patients with increasing ctDNA levels (24-mo progression-free survival 72% versus 0%, p < 0.0001). Progression of disease occurred within less than 12 months of starting CPI in all patients with increasing ctDNA levels at C2D1., Conclusions: Detection of ctDNA before, during, or after 6 months of consolidation CPI is strongly associated with inferior outcomes. Our findings suggest that analysis of ctDNA MRD may enable personalizing the duration of consolidation immunotherapy treatment., Competing Interests: Disclosure Dr. Durm receives support from Bristol-Myers Squibb; grants or contracts from AstraZeneca, Merck, and Mirati; consulting fees from Cook Biotech; payments or honoraria from Curio Science, DAVA Oncology, and AstraZeneca; and participates on an advisory board for Curio Science, DAVA oncology, and AstraZeneca. Dr. Ganti receives research grants or contracts from VA Office of Research and Development, Merck, TAB Biosciences, NEKTAR Therapeutics, Mirati Therapeutics, IOVANCE Therapeutics, and Apexigen; royalties or licenses from Oxford University Press; consulting fees from AstraZeneca, Flagship Biosciences, G1 Therapeutics, Jazz Pharmaceuticals, Cardinal Health, Mirati Therapeutics, Beigene Ltd., Sanofi Genzyme, Blueprint Medicines, and Regeneron Pharmaceuticals; payments or honoraria from MedLearning Group and Plexus Communications; participates on a data safety monitoring board for YmAbs Therapeutics; holds leadership or fiduciary roles at Academic and Community Cancer Research United and A Breath of Hope for Lung Cancer; and has received equipment or materials from Takeda Pharmaceuticals and Chimerx. Dr. Alizadeh receives research grants or contracts from the National Cancer Institute, National Heart, Lung and Blood Institute, National Institutes of Health, Celgene, Bristol-Myers Squibb, and Pfizer; consulting fees from ADCT, Celgene, Chugai, Genentech, Gilead, Janssen, Pharmacyclics, and Roche; has patents planned, issued, or pending with FortySeven Inc., Foresight Diagnostics, CiberMed Inc., and Roche; participates on a data safety monitoring board for Lymphoma Research Foundation; holds leadership or fiduciary roles at the American Society of Hematology, American Society of Clinical Oncology, American Society of Clinical Investigation, and Leukemia & Lymphoma Society; and has equity ownership interests in CiberMed Inc., Foresight Diagnostics, FortySeven Inc., and CARGO Therapeutics. Dr. Hanna receives grants or contracts from Merck, Genentech, Bristol-Myers Squibb, and Natera. Dr. Diehn receives grants or contracts from AstraZeneca and Varian Medical Systems; royalties or licenses from Roche and Foresight Diagnostics; consulting fees from AstraZeneca, Boehringer Ingelheim, Genentech, Illumina, Gritstone Bio, and Regeneron; payments or honoraria from Bristol-Myers Squibb, Japan, and Novartis; support for attending meetings or travel from Foresight Diagnostics and Regeneron; has patents planned, issued, or pending with Stanford University; holds leadership or fiduciary roles at Foresight Diagnostics; and holds stock or stock options in CiberMed Inc., Foresight Diagnostics, and Gritstone Bio. The remaining authors declare no conflict of interest., (Copyright © 2024 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Purpose: Small cell lung cancer (SCLC) is characterized by rapid progression after platinum resistance. Circulating tumor (ctDNA) dynamics early in treatment may help determine platinum sensitivity., Materials and Methods: Serial plasma samples were collected from patients receiving platinum-based chemotherapy for SCLC on the first 3 days of cycle one and on the first days of subsequent cycles with paired samples collected both before and again after infusions. Tumor-informed plasma analysis was carried out using CAncer Personalized Profiling by deep Sequencing (CAPP-Seq). The mean variant allele frequency (VAF) of all pretreatment mutations was tracked in subsequent blood draws and correlated with radiologic response., Results: ctDNA kinetics were assessed in 122 samples from 21 patients. Pretreatment VAF did not differ significantly between patients who did and did not respond to chemotherapy (mean 22.5% v 4.6%, P = .17). A slight increase in ctDNA on cycle 1, day 1 immediately post-treatment was seen in six of the seven patients with available draws (fold change from baseline: 1.01-1.44), half of whom achieved a response. All patients who responded had a >2-fold decrease in mean VAF on cycle 2 day 1 (C2D1). Progression-free survival (PFS) and overall survival (OS) were significantly longer in patients with a >2-fold decrease in mean VAF after one treatment cycle (6.8 v 2.6 months, log-rank P = .0004 and 21.7 v 6.4 months, log rank P = .04, respectively)., Conclusion: A >2-fold decrease in ctDNA concentration was observed by C2D1 in all patients who were sensitive to platinum-based therapy and was associated with longer PFS and OS.

Cerebrospinal fluid tumor-derived DNA (CSF-tDNA) analysis is a promising approach for monitoring the neoplastic processes of the central nervous system. We applied a lung cancer-specific sequencing panel (CAPP-Seq) to 81 CSF, blood, and tissue samples from 24 lung cancer patients who underwent lumbar puncture (LP) for suspected leptomeningeal disease (LMD). A subset of the cohort (N = 12) participated in a prospective trial of osimertinib for refractory LMD in which serial LPs were performed before and during treatment. CSF-tDNA variant allele fractions (VAFs) were significantly higher than plasma circulating tumor DNA (ctDNA) VAFs (median CSF-tDNA, 32.7%; median plasma ctDNA, 1.8%; P < 0.0001). Concentrations of tumor DNA in CSF and plasma were positively correlated (Spearman's ρ, 0.45; P = 0.03). For LMD diagnosis, cytology was 81.8% sensitive and CSF-tDNA was 91.7% sensitive. CSF-tDNA was also strongly prognostic for overall survival (HR = 7.1; P = 0.02). Among patients with progression on targeted therapy, resistance mutations, such as EGFR T790M and MET amplification, were common in peripheral blood but were rare in time-matched CSF, indicating differences in resistance mechanisms based on the anatomic compartment. In the osimertinib cohort, patients with CNS progression had increased CSF-tDNA VAFs at follow-up LP. Post-osimertinib CSF-tDNA VAF was strongly prognostic for CNS progression (HR = 6.2, P = 0.009). Detection of CSF-tDNA in lung cancer patients with suspected LMD is feasible and may have clinical utility. CSF-tDNA improves the sensitivity of LMD diagnosis, enables improved prognostication, and drives therapeutic strategies that account for spatial heterogeneity in resistance mechanisms., (© 2024. The Author(s).)

Radiotherapy (RT) is one of the primary treatments of head and neck squamous cell carcinoma (HNSCC), which has a high-risk of locoregional failure (LRF). Presently, there is no reliable predictive biomarker of radioresistance in HNSCC. Here, we found that mutations in NFE2L2, which encodes Nrf2, are associated with a significantly higher rate of LRF in patients with oral cavity cancer treated with surgery and adjuvant (chemo)radiotherapy but not in those treated with surgery alone. Somatic mutation of NFE2L2 led to Nrf2 activation and radioresistance in HNSCC cells. Tumors harboring mutant Nrf2E79Q were substantially more radioresistant than tumors with wild-type Nrf2 in immunocompetent mice, whereas the difference was diminished in immunocompromised mice. Nrf2E79Q enhanced radioresistance through increased recruitment of intratumoral polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and reduction of M1-polarized macrophages. Treatment with the glutaminase inhibitor CB-839 overcame the radioresistance induced by Nrf2E79Q or Nrf2E79K. RT increased expression of PMN-MDSC-attracting chemokines, including CXCL1, CXLC3, and CSF3, in Nrf2E79Q-expressing tumors via the TLR4, which could be reversed by CB-839. This study provides insights into the impact of NFE2L2 mutations on radioresistance and suggests that CB-839 can increase radiosensitivity by switching intratumoral myeloid cells to an antitumor phenotype, supporting clinical testing of CB-839 with RT in HNSCC with NFE2L2 mutations., Significance: NFE2L2 mutations are predictive biomarkers of radioresistance in head and neck cancer and confer sensitivity to glutaminase inhibitors to overcome radioresistance., (©2023 American Association for Cancer Research.)

Genomic profiling of bronchoalveolar lavage (BAL) samples may be useful for tumor profiling and diagnosis in the clinic. Here, we compared tumor-derived mutations detected in BAL samples from subjects with non-small cell lung cancer (NSCLC) to those detected in matched plasma samples. Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) was used to genotype DNA purified from BAL, plasma, and tumor samples from patients with NSCLC. The characteristics of cell-free DNA (cfDNA) isolated from BAL fluid were first characterized to optimize the technical approach. Somatic mutations identified in tumor were then compared with those identified in BAL and plasma, and the potential of BAL cfDNA analysis to distinguish lung cancer patients from risk-matched controls was explored. In total, 200 biofluid and tumor samples from 38 cases and 21 controls undergoing BAL for lung cancer evaluation were profiled. More tumor variants were identified in BAL cfDNA than plasma cfDNA in all stages (P < 0.001) and in stage I to II disease only. Four of 21 controls harbored low levels of cancer-associated driver mutations in BAL cfDNA [mean variant allele frequency (VAF) = 0.5%], suggesting the presence of somatic mutations in nonmalignant airway cells. Finally, using a Random Forest model with leave-one-out cross-validation, an exploratory BAL genomic classifier identified lung cancer with 69% sensitivity and 100% specificity in this cohort and detected more cancers than BAL cytology. Detecting tumor-derived mutations by targeted sequencing of BAL cfDNA is technically feasible and appears to be more sensitive than plasma profiling. Further studies are required to define optimal diagnostic applications and clinical utility., Significance: Hybrid-capture, targeted deep sequencing of lung cancer mutational burden in cell-free BAL fluid identifies more tumor-derived mutations with increased allele frequencies compared with plasma cell-free DNA. See related commentary by Rolfo et al., p. 2826., (©2022 The Authors; Published by the American Association for Cancer Research.)

Profiling of circulating tumor DNA (ctDNA) in the bloodstream shows promise for noninvasive cancer detection. Chromatin fragmentation features have previously been explored to infer gene expression profiles from cell-free DNA (cfDNA), but current fragmentomic methods require high concentrations of tumor-derived DNA and provide limited resolution. Here we describe promoter fragmentation entropy as an epigenomic cfDNA feature that predicts RNA expression levels at individual genes. We developed 'epigenetic expression inference from cell-free DNA-sequencing' (EPIC-seq), a method that uses targeted sequencing of promoters of genes of interest. Profiling 329 blood samples from 201 patients with cancer and 87 healthy adults, we demonstrate classification of subtypes of lung carcinoma and diffuse large B cell lymphoma. Applying EPIC-seq to serial blood samples from patients treated with PD-(L)1 immune-checkpoint inhibitors, we show that gene expression profiles inferred by EPIC-seq are correlated with clinical response. Our results indicate that EPIC-seq could enable noninvasive, high-throughput tissue-of-origin characterization with diagnostic, prognostic and therapeutic potential., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Introduction: Increasing evidence suggests that consolidation durvalumab confers limited benefits for patients with stage III EGFR-mutated NSCLC. Induction or maintenance EGFR tyrosine kinase inhibitors (TKIs) added to concurrent chemoradiotherapy (CRT) may optimize definitive treatment, but there are limited data supporting an induction TKI strategy., Methods: We evaluated the efficacy and safety of induction EGFR TKIs administered before concurrent CRT in a retrospective series of patients with unresectable locally advanced EGFR-mutated NSCLC. Circulating tumor DNA (ctDNA) analysis was performed on a patient subset using CAPP-seq and correlated with outcomes., Results: Of six patients, three received erlotinib and three osimertinib as induction therapy before CRT. Induction TKIs were administered for a median of 2.5 months. The objective response rate after induction TKI was 83%. One patient had a complete response to induction erlotinib and continued erlotinib for 4 years until local progression, which was treated with CRT. Two patients completed maintenance erlotinib after CRT, and another received consolidation durvalumab. After a median follow-up of 20.5 months, only one patient developed disease recurrence, with rising ctDNA coinciding with recurrence. ctDNA remained undetectable in patients without recurrence, or low-level in a patient receiving maintenance erlotinib. Adverse events were mild and expected, and none developed pneumonitis., Conclusion: Induction EGFR TKI before CRT may achieve high disease control rates with promising signs of durability in patients with locally advanced EGFR-mutated NSCLC. ctDNA analysis after CRT can correlate well with clinical outcomes. Prospective studies are needed to define the role of induction EGFR TKIs in this setting., Competing Interests: Declaration of Competing Interest Dr. Aredo reports grants from the Conquer Cancer Foundation of ASCO. Dr. Wakelee reports grants to institution from ACEA Biosciences, Arrys Therapeutics, AztraZeneca/MedImmune, BMS, Celgene, Clovis Oncology, Exelixis, Genentech/Roche, Gilead, Merck, Novartis, Pharmacyclics, Seattle Genetics, Xcovery, Eli Lilly, Pfizer; honoraria from Novartis, AstraZeneca; and is on the advisory boards of AstraZeneca, Xcovery, Janssen, Daiichi Sankyo, Blueprint, Mirati, Helsinn, Merck (uncompensated), Takeda (uncompensated), Genentech/Roche (uncompensated), Cellworks (uncompensated), all outside the submitted work. Dr. Padda reports personal fees from Pfizer, G1 Therapeutics, Blueprint Medicines, AstraZeneca, Abbvie, Janssen Pharmaceuticals; grants to institution from Epicentrx, Bayer, Boehringer Ingelheim, Forty Seven Inc., all outside the submitted work. Dr. Guo reports grants to institution from Pliant Pharmaceuticals; consulting fees from Exact Sciences, BioMind.ai, Arterys, MORE Health; stock from Exact Sciences, all outside the submitted work. Dr. Diehn reports personal fees from Roche, AstraZeneca, Novartis, Genentech, BioNTech, RefleXion, Gritstone Oncology; grants from Varian Medical Systems and AstraZeneca; is on the advisory boards of AstraZeneca, Illumina, Gritstone Oncology, Genentech; other leadership roles of CiberMed, Foresight Diagnostics; non-financial support from Illumina, all outside the submitted work. In addition, Dr. Diehn has a patent related to cancer biomarkers with royalties paid to Roche and Foresight Diagnostics, outside the submitted work. Dr. Loo reports grants from Varian Medical Systems and is a board member of TibaRay, all outside the submitted work. Dr. Neal reports personal fees from Research to Practice, MLI Peerview, Medscape, Biomedical Learning Institute, Prime Oncology, Rockpointe, CME Matters, MJH Life Sciences, AstraZeneca, Jounce Therapeutics, Eli Lilly and Company, Calithera Biosciences, Amgen, Iovance Biotherapeutics, Genentech/Roche, Exelixis, Takeda Pharmaceuticals, UpToDate; grants from Genentech/Roche, Exelixis, Takeda Pharmaceuticals, Merck, Novartis, Boehringer Ingelheim, Nektar Therapeutics, Adaptimmune, GSK, AbbVie, all outside the submitted work. All remaining authors have declared no conflicts of interest., (Copyright © 2022. Published by Elsevier Ltd.)

Although treatment of non-small cell lung cancer (NSCLC) with immune checkpoint inhibitors (ICIs) can produce remarkably durable responses, most patients develop early disease progression. Furthermore, initial response assessment by conventional imaging is often unable to identify which patients will achieve durable clinical benefit (DCB). Here, we demonstrate that pre-treatment circulating tumor DNA (ctDNA) and peripheral CD8 T cell levels are independently associated with DCB. We further show that ctDNA dynamics after a single infusion can aid in identification of patients who will achieve DCB. Integrating these determinants, we developed and validated an entirely noninvasive multiparameter assay (DIREct-On, Durable Immunotherapy Response Estimation by immune profiling and ctDNA-On-treatment) that robustly predicts which patients will achieve DCB with higher accuracy than any individual feature. Taken together, these results demonstrate that integrated ctDNA and circulating immune cell profiling can provide accurate, noninvasive, and early forecasting of ultimate outcomes for NSCLC patients receiving ICIs., Competing Interests: Declaration of Interests J.J.C. reports paid consultancy from Lexent Bio Inc. A.A.C. reports speaker honoraria and travel support from Roche Sequencing Solutions (RSS), Varian, and Foundation Medicine; a research grant from RSS; and has served as a paid consultant for Oscar Health. T.M. is a co-founder of Imvaq. J.W.N. reports research support from Genentech (GNE)/Roche, Merck, Novartis, Boehringer Ingelheim, Exelixis, Takeda Pharmaceuticals, Nektar Therapeutics, Adaptimmune, and GSK, and has served in a consulting or advisory role for AstraZeneca (AZ), GNE/Roche, Exelixis Inc., Jounce Therapeutics, Takeda Pharmaceuticals, and Eli Lilly. H.A.W. has received honoraria from Novartis and AZ and has participated on the advisory boards of Xcovery, Janssen, and Mirati. S.K.P. reports grant support from EpicentRx, Forty Seven, Bayer, and Boehringer Ingelheim and serves in a consulting or advisory role for AZ, AbbVie, G1 Therapeutics, and Pfizer. M. Das reports grant support from AbbVie, United Therapeutics, Varian, and Celgene and serves in a consulting role for AZ and Bristol-Myers Squibb (BMS). A.M.N. has patent filings related to expression deconvolution and cancer biomarkers and has served as a consultant for Roche, Merck, and CiberMed. M.D.H. reports paid consultancy from BMS, Merck, GNE, AZ/MedImmune, Nektar, Syndax, Janssen, Mirati Therapeutics, Shattuck Labs, and Blueprint Medicines; travel/honoraria from BMS and AZ; research funding from BMS; and a patent has been filed by MSK related to the use of tumor mutation burden to predict response to immunotherapy (PCT/US2015/062208), which has received licensing fees from Personal Genome Diagnostics. A.A.A. reports ownership interest in CiberMed and FortySeven, patent filings related to cancer biomarkers, and paid consultancy from GNE, Roche, Chugai, Gilead, and Celgene. M. Diehn reports research funding from Varian, ownership interest in CiberMed, patent filings related to cancer biomarkers, paid consultancy from Roche, AZ, and BioNTech, and travel/honoraria from Reflexion. M.Diehn, A.A.A., B.Y.N., and M.S.E. are co-inventors on a provisional patent application filed by Stanford University relating to this manuscript., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: Treatment with PD-(L)1 blockade can produce remarkably durable responses in patients with non-small cell lung cancer (NSCLC). However, a significant fraction of long-term responders ultimately progress and predictors of late progression are unknown. We hypothesized that circulating tumor DNA (ctDNA) analysis of long-term responders to PD-(L)1 blockade may differentiate those who will achieve ongoing benefit from those at risk of eventual progression., Experimental Design: In patients with advanced NSCLC achieving long-term benefit from PD-(L)1 blockade (progression-free survival ≥ 12 months), plasma was collected at a surveillance timepoint late during/after treatment to interrogate ctDNA by Cancer Personalized Profiling by Deep Sequencing. Tumor tissue was available for 24 patients and was profiled by whole-exome sequencing ( n = 18) or by targeted sequencing ( n = 6)., Results: Thirty-one patients with NSCLC with long-term benefit to PD-(L)1 blockade were identified, and ctDNA was analyzed in surveillance blood samples collected at a median of 26.7 months after initiation of therapy. Nine patients also had baseline plasma samples available, and all had detectable ctDNA prior to therapy initiation. At the surveillance timepoint, 27 patients had undetectable ctDNA and 25 (93%) have remained progression-free; in contrast, all 4 patients with detectable ctDNA eventually progressed [Fisher P < 0.0001; positive predictive value = 1, 95% confidence interval (CI), 0.51-1; negative predictive value = 0.93 (95% CI, 0.80-0.99)]., Conclusions: ctDNA analysis can noninvasively identify minimal residual disease in patients with long-term responses to PD-(L)1 blockade and predict the risk of eventual progression. If validated, ctDNA surveillance may facilitate personalization of the duration of immune checkpoint blockade and enable early intervention in patients at high risk for progression., (©2020 American Association for Cancer Research.)

Background: Tumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway., Result: To develop a deeper understanding of the interactions between cells within human lung tumors, we perform RNA-seq profiling of flow-sorted malignant cells, endothelial cells, immune cells, fibroblasts, and bulk cells from freshly resected human primary non-small-cell lung tumors. We map the cell-specific differential expression of prognostically associated secreted factors and cell surface genes, and computationally reconstruct cross-talk between these cell types to generate a novel resource called the Lung Tumor Microenvironment Interactome (LTMI). Using this resource, we identify and validate a prognostically unfavorable influence of Gremlin-1 production by fibroblasts on proliferation of malignant lung adenocarcinoma cells. We also find a prognostically favorable association between infiltration of mast cells and less aggressive tumor cell behavior., Conclusion: These results illustrate the utility of the LTMI as a resource for generating hypotheses concerning tumor-microenvironment interactions that may have prognostic and therapeutic relevance.

Radiologic screening of high-risk adults reduces lung-cancer-related mortality 1,2 ; however, a small minority of eligible individuals undergo such screening in the United States 3,4 . The availability of blood-based tests could increase screening uptake. Here we introduce improvements to cancer personalized profiling by deep sequencing (CAPP-Seq) 5 , a method for the analysis of circulating tumour DNA (ctDNA), to better facilitate screening applications. We show that, although levels are very low in early-stage lung cancers, ctDNA is present prior to treatment in most patients and its presence is strongly prognostic. We also find that the majority of somatic mutations in the cell-free DNA (cfDNA) of patients with lung cancer and of risk-matched controls reflect clonal haematopoiesis and are non-recurrent. Compared with tumour-derived mutations, clonal haematopoiesis mutations occur on longer cfDNA fragments and lack mutational signatures that are associated with tobacco smoking. Integrating these findings with other molecular features, we develop and prospectively validate a machine-learning method termed 'lung cancer likelihood in plasma' (Lung-CLiP), which can robustly discriminate early-stage lung cancer patients from risk-matched controls. This approach achieves performance similar to that of tumour-informed ctDNA detection and enables tuning of assay specificity in order to facilitate distinct clinical applications. Our findings establish the potential of cfDNA for lung cancer screening and highlight the importance of risk-matching cases and controls in cfDNA-based screening studies.

Background & Aims: Biomarkers are needed to risk stratify after chemoradiotherapy for localized esophageal cancer. These could improve identification of patients at risk for cancer progression and selection of additional therapy., Methods: We performed deep sequencing (CAncer Personalized Profiling by deep Sequencing, [CAPP-Seq]) analyses of plasma cell-free DNA collected from 45 patients before and after chemoradiotherapy for esophageal cancer, as well as DNA from leukocytes and fixed esophageal tumor biopsy samples collected during esophagogastroduodenoscopy. Patients were treated from May 2010 through October 2015; 23 patients subsequently underwent esophagectomy, and 22 did not undergo surgery. We also sequenced DNA from blood samples from 40 healthy control individuals. We analyzed 802 regions of 607 genes for single-nucleotide variants previously associated with esophageal adenocarcinoma or squamous cell carcinoma. Patients underwent imaging analyses 6-8 weeks after chemoradiotherapy and were followed for 5 years. Our primary aim was to determine whether detection of circulating tumor DNA (ctDNA) after chemoradiotherapy is associated with risk of tumor progression (growth of local, regional, or distant tumors, detected by imaging or biopsy)., Results: The median proportion of tumor-derived DNA in total cell-free DNA before treatment was 0.07%, indicating that ultrasensitive assays are needed for quantification and analysis of ctDNA from localized esophageal tumors. Detection of ctDNA after chemoradiotherapy was associated with tumor progression (hazard ratio, 18.7; P < .0001), formation of distant metastases (hazard ratio, 32.1; P < .0001), and shorter disease-specific survival times (hazard ratio, 23.1; P < .0001). A higher proportion of patients with tumor progression had new mutations detected in plasma samples collected after chemoradiotherapy than patients without progression (P = .03). Detection of ctDNA after chemoradiotherapy preceded radiographic evidence of tumor progression by an average of 2.8 months. Among patients who received chemoradiotherapy without surgery, combined ctDNA and metabolic imaging analysis predicted progression in 100% of patients with tumor progression, compared with 71% for only ctDNA detection and 57% for only metabolic imaging analysis (P < .001 for comparison of either technique to combined analysis)., Conclusions: In an analysis of cell-free DNA in blood samples from patients who underwent chemoradiotherapy for esophageal cancer, detection of ctDNA was associated with tumor progression, metastasis, and disease-specific survival. Analysis of ctDNA might be used to identify patients at highest risk for tumor progression., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Circulating tumor DNA (ctDNA) molecular residual disease (MRD) following curative-intent treatment strongly predicts recurrence in multiple tumor types, but whether further treatment can improve outcomes in patients with MRD remains unclear. We applied CAPP-Seq ctDNA analysis to 218 samples from 65 patients receiving chemoradiation therapy (CRT) for locally advanced NSCLC, including 28 patients receiving consolidation immune checkpoint inhibition (CICI). Patients with undetectable ctDNA after CRT had excellent outcomes whether or not they received CICI. Among such patients, one died from CICI-related pneumonitis, highlighting the potential utility of only treating patients with MRD. In contrast, patients with MRD after CRT who received CICI had significantly better outcomes than patients who did not receive CICI. Furthermore, the ctDNA response pattern early during CICI identified patients responding to consolidation therapy. Our results suggest that CICI improves outcomes for NSCLC patients with MRD and that ctDNA analysis may facilitate personalization of consolidation therapy.

The functional role of U2AF1 mutations in lung adenocarcinomas (LUADs) remains incompletely understood. Here, we report a significant co-occurrence of U2AF1 S34F mutations with ROS1 translocations in LUADs. To characterize this interaction, we profiled effects of S34F on the transcriptome-wide distribution of RNA binding and alternative splicing in cells harboring the ROS1 translocation. Compared to its wild-type counterpart, U2AF1 S34F preferentially binds and modulates splicing of introns containing CAG trinucleotides at their 3' splice junctions. The presence of S34F caused a shift in cross-linking at 3' splice sites, which was significantly associated with alternative splicing of skipped exons. U2AF1 S34F induced expression of genes involved in the epithelial-mesenchymal transition (EMT) and increased tumor cell invasion. Finally, S34F increased splicing of the long over the short SLC34A2-ROS1 isoform, which was also associated with enhanced invasiveness. Taken together, our results suggest a mechanistic interaction between mutant U2AF1 and ROS1 in LUAD.

Increasing evidence suggests that ionizing radiation therapy (RT) in combination with checkpoint immunotherapy is highly effective in treating a subset of cancers. To better understand the limited responses to this combination we analysed the genetic, microenvironmental, and immune factors in tumours derived from a transgenic breast cancer model. We identified two tumours with similar growth characteristics but different RT responses primarily due to an antitumour immune response. The combination of RT and checkpoint immunotherapy resulted in cures in the responsive but not the unresponsive tumours. Profiling the tumours revealed that the Axl receptor tyrosine kinase is overexpressed in the unresponsive tumours, and Axl knockout resulted in slower growth and increased radiosensitivity. These changes were associated with a CD8 + T-cell response, which was improved in combination with checkpoint immunotherapy. These results suggest a novel role for Axl in suppressing antigen presentation through MHCI, and enhancing cytokine release, which promotes a suppressive myeloid microenvironment.

Cervical cancer remains the third most frequently diagnosed and fourth leading cause of cancer death in women worldwide. We sought to develop a micro-RNA signature that was prognostic for disease-free survival, which could potentially allow tailoring of treatment for cervical cancer patients. A candidate prognostic 9-micro-RNA signature set was identified in the training set of 79 frozen specimens. However, three different approaches to validate this signature in an independent cohort of 87 patients with formalin-fixed paraffin-embedded (FFPE) specimens, were unsuccessful. There are several challenges and considerations associated with developing a prognostic micro-RNA signature for cervical cancer, namely: tumour heterogeneity, lack of concordance between frozen and FFPE specimens, and platform selection for global micro-RNA expression profiling in this disease. Our observations provide an important cautionary tale for future miRNA signature studies for cervical cancer, which can also be potentially applicable to miRNA profiling studies involving other types of human malignancies.

Purpose: Despite significant improvement in locoregional control in the contemporary era of nasopharyngeal carcinoma (NPC) treatment, patients still suffer from a significant risk of distant metastasis (DM). Identifying those patients at risk of DM would aid in personalized treatment in the future. MicroRNAs (miRNAs) play many important roles in human cancers; hence, we proceeded to address the primary hypothesis that there is a miRNA expression signature capable of predicting DM for NPC patients., Methods and Results: The expression of 734 miRNAs was measured in 125 (Training) and 121 (Validation) clinically annotated NPC diagnostic biopsy samples. A 4-miRNA expression signature associated with risk of developing DM was identified by fitting a penalized Cox Proportion Hazard regression model to the Training data set (HR 8.25; p < 0.001), and subsequently validated in an independent Validation set (HR 3.2; p = 0.01). Pathway enrichment analysis indicated that the targets of miRNAs associated with DM appear to be converging on cell-cycle pathways., Conclusions: This 4-miRNA signature adds to the prognostic value of the current "gold standard" of TNM staging. In-depth interrogation of these 4-miRNAs will provide important biological insights that could facilitate the discovery and development of novel molecularly targeted therapies to improve outcome for future NPC patients.

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer which is prevalent in southern China, south-east Asia and northern Africa. The development and stepwise progression of NPC involves accumulation of multiple gross genetic changes during the clonal expansion of Epstein-Barr virus (EBV)-infected nasopharyngeal epithelial cell population. Here, using paired-end whole-transcriptome sequencing, we discovered a number of chimeric fusion transcripts in a panel of EBV-positive tumour lines. Among these transcripts, a novel fusion of ubiquitin protein ligase E3 component n-recognin 5 (UBR5) on 8q22.3 and zinc finger protein 423 (ZNF423) on 16q12.1, identified from the NPC cell line C666-1, was recurrently detected in 12/144 (8.3%) of primary tumours. The fusion gene contains exon 1 of UBR5 and exons 7-9 of ZNF423 and produces a 94 amino acid chimeric protein including the original C-terminal EBF binding domain (ZF29-30) of ZNF423. Notably, the growth of NPC cells with UBR5-ZNF423 rearrangement is dependent on expression of this fusion protein. Knock-down of UBR5-ZNF423 by fusion-specific siRNA significantly inhibited the cell proliferation and colony-forming ability of C666-1 cells. The transforming ability of UBR5-ZNF423 fusion was also confirmed in NIH3T3 fibroblasts. Constitutive expression of UBR5-ZNF423 in NIH3T3 fibroblasts significantly enhanced its anchorage-independent growth in soft agar and induced tumour formation in a nude mouse model. These findings suggest that expression of UBR5-ZNF423 protein might contribute to the transformation of a subset of NPCs, possibly by altering the activity of EBFs (early B cell factors). Identification of the oncogenic UBR5-ZNF423 provides new potential opportunities for therapeutic intervention in NPC., (© 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Introduction: Despite improvements in treatment strategies for head and neck squamous cell carcinoma (HNSCC), outcomes have not significantly improved; highlighting the importance of identifying novel therapeutic approaches to target this disease. To address this challenge, we proceeded to evaluate the role of iron in HNSCC., Experimental Design: Expression levels of iron-related genes were evaluated in HNSCC cell lines using quantitative RT-PCR. Cellular phenotypic effects were assessed using viability (MTS), clonogenic survival, BrdU, and tumor formation assays. The prognostic significance of iron-related proteins was determined using immunohistochemistry., Results: In a panel of HNSCC cell lines, hemochromatosis (HFE) was one of the most overexpressed genes involved in iron regulation. In vitro knockdown of HFE in HNSCC cell lines significantly decreased hepcidin (HAMP) expression and intracellular iron level. This in turn, resulted in a significant decrease in HNSCC cell viability, clonogenicity, DNA synthesis, and Wnt signalling. These cellular changes were reversed by re-introducing iron back into HNSCC cells after HFE knockdown, indicating that iron was mediating this phenotype. Concordantly, treating HNSCC cells with an iron chelator, ciclopirox olamine (CPX), significantly reduced viability and clonogenic survival. Finally, patients with high HFE expression experienced a reduced survival compared to patients with low HFE expression., Conclusions: Our data identify HFE as potentially novel prognostic marker in HNSCC that promotes tumour progression via HAMP and elevated intracellular iron levels, leading to increased cellular proliferation and tumour formation. Hence, these findings suggest that iron chelators might have a therapeutic role in HNSCC management.

The down-regulation of microRNA-196b (miR-196b) has been reported, but its contribution to cervical cancer progression remains to be investigated. In this study, we first demonstrated that miR-196b down-regulation was significantly associated with worse disease-free survival (DFS) for cervical cancer patients treated with combined chemo-radiation. Secondly, using a tri-modal approach for target identification, we determined that homeobox-B7 (HOXB7) was a bona fide target for miR-196b, and in turn, vascular endothelial growth factor (VEGF) was a downstream transcript regulated by HOXB7. Reconstitution of miR-196b expression by transient transfection resulted in reduced cell growth, clonogenicity, migration and invasion in vitro, as well as reduced tumor angiogenesis and tumor cell proliferation in vivo. Concordantly, siRNA knockdown of HOXB7 or VEGF phenocopied the biological effects of miR-196b over-expression. Our findings have demonstrated that the miR-196b/HOXB7/VEGF pathway plays an important role in cervical cancer progression; hence targeting this pathway could be a promising therapeutic strategy for the future management of this disease.

Exosomes and microvesicles (MVs) are nanometer-sized, membranous vesicles secreted from many cell types into their surrounding extracellular space and into body fluids. These two classes of extracellular vesicles are regarded as a novel mechanism through which cancer cells, including virally infected cancer cells, regulate their micro-environment via the horizontal transfer of bioactive molecules: proteins, lipids, and nucleic acids (DNA, mRNA, micro-RNAs; oncogenic cargo hence often referred to as oncosomes). In head and neck cancer (HNC), exosomes and MVs have been described in Epstein Barr Virus (EBV)-associated nasopharyngeal cancer (NPC), as well as being positively correlated with oral squamous cell carcinoma (OSCC) progression. It has therefore been suggested that HNC-derived vesicles could represent a useful source for biomarker discovery, enriched in tumor antigens and cargo; hence fundamentally important for cancer progression. This current review offers an overall perspective on the roles of exosomes and MVs in HNC biology, focusing on EBV-associated NPC and OSCC. We also highlight the importance of saliva as a proximal and easily accessible bio-fluid for HNC detection, and propose that salivary vesicles might serve as an alternative model in the discovery of novel HNC biomarkers., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Deregulation of miRNAs is associated with almost all human malignancies. Human papillomavirus (HPV)-associated oropharyngeal carcinoma (OPC) has a significantly more favorable outcome compared with HPV-negative OPCs; however, the underlying mechanisms are not well understood. Hence, the objectives of this study were to determine whether miRNA expression differed as a function of HPV status and to assess whether such miRNAs provide prognostic value beyond HPV status., Methods: Global miRNA profilings were conducted on 88 formalin-fixed and paraffin-embedded (FFPE) OPC biopsies (p16-positive: 56; p16-negative: 32), wherein the expression levels of 365 miRNAs plus 3 endogenous controls were simultaneously measured using quantitative real-time (qRT)-PCR. Seven FFPE specimens of histologically normal tonsils were used as controls., Results: Overall, 224 miRNAs were expressed in more than 80% of the investigated samples, with 128 (57%) being significantly differentially expressed between tumor versus normal tissues (P < 0.05). Upregulated miR-20b, miR-9, and miR-9* were significantly associated with HPV/p16-status. Three miRNA sets were significantly associated with overall survival (miR-107, miR-151, miR-492; P = 0.0002), disease-free survival (miR-20b, miR-107, miR-151, miR-182, miR-361; P = 0.0001), and distant metastasis (miR-151, miR-152, miR-324-5p, miR-361, miR492; P = 0.0087), which retained significance even after adjusting for p16 status. The associated biologic functions of these miRNAs include immune surveillance, treatment resistance, invasion, and metastasis., Conclusion: We have identified several miRNAs, which associate with HPV status in OPC; furthermore, three candidate prognostic sets of miRNAs seem to correlate with clinical outcome, independent of p16 status. Furthermore, evaluations will offer biologic insights into the mechanisms underlying the differences between HPV-positive versus HPV-negative OPC.

Despite improvements in therapeutic approaches for head and neck squamous cell carcinomas (HNSCC), clinical outcome has remained disappointing, with 5-year overall survival rates hovering around 40-50%, underscoring an urgent need to better understand the biological bases of this disease. We chose to address this challenge by studying the role of micro-RNAs (miRNAs) in HNSCC. MiR-193b was identified as an over-expressed miRNA from global miRNA profiling studies previously conducted in our lab, and confirmed in HNSCC cell lines. In vitro knockdown of miR-193b in FaDu cancer cells substantially reduced cell proliferation, migration and invasion, along with suppressed tumour formation in vivo. By integrating in silico prediction algorithms with in vitro experimental mRNA profilings, plus mRNA expression data of clinical specimens, neurofibromin 1 (NF1) was identified to be a target of miR-193b. Concordantly, miR-193b knockdown decreased NF1 transcript and protein levels significantly. Luciferase reporter assays confirmed the direct interaction of miR-193b with NF1. Moreover, p-ERK, a downstream target of NF1 was also suppressed after miR-193b knockdown. FaDu cells treated with a p-ERK inhibitor (U0126) phenocopied the reduced cell proliferation, migration and invasion observed with miR-193b knockdown. Finally, HNSCC patients whose tumours expressed high levels of miR-193b experienced a lower disease-free survival compared to patients with low miR-193b expression. Our findings identified miR-193b as a potentially novel prognostic marker in HNSCC that drives tumour progression via down-regulating NF1, in turn leading to activation of ERK, resulting in proliferation, migration, invasion, and tumour formation.

Purpose: Despite recent improvements in local control of head and neck cancers (HNC), distant metastasis remains a major cause of death. Hence, further understanding of HNC biology, and in particular, the genes/pathways driving metastasis is essential to improve outcome., Experimental Design: Quantitative reverse transcriptase PCR (qRT-PCR) was used to measure the expression of miR-375 and metadherin (MTDH) in HNC patient samples. Targets of miR-375 were confirmed using qRT-PCR, Western blot analysis, and luciferase assays. Phenotypic effects of miR-375 reexpression and MTDH knockdown were assessed using viability (MTS), clonogenic survival, cell migration/invasion, as well as in vivo tumor formation assays. The prognostic significance of miR-375 or MTDH in nasopharyngeal carcinoma (NPC) was determined by comparing low versus high expression groups., Results: MiR-375 expression was significantly reduced (P = 0.01), and conversely, MTDH was significantly increased (P = 0.0001) in NPC samples. qRT-PCR, Western blots, and luciferase assays corroborated MTDH as a target of miR-375. Reexpression of miR-375 and siRNA knockdown of MTDH both decreased cell viability and clonogenic survival, cell migration/invasion, as well as in vivo tumor formation. NPC patients whose tumors expressed high levels of MTDH experienced significantly lower survival and, in particular, higher distant relapse rates (5-year distant relapse rates: 26% vs. 5%; P = 0.005)., Conclusions: Dysregulation of miR-375 and MTDH may represent an important oncogenic pathway driving human HNC progression, particularly distant metastases, which is now emerging as a major cause of death for HNC patients. Hence, targeting this pathway could potentially be a novel therapeutic strategy by which HNC patient outcome could be improved., (©2011 AACR.)

Several microRNAs have been implicated in human breast cancer but none to date have been validated or utilized consistently in clinical management. MicroRNA-301 (miR-301) overexpression has been implicated as a negative prognostic indicator in lymph node negative (LNN) invasive ductal breast cancer, but its potential functional impact has not been determined. Here we report that in breast cancer cells, miR-301 attenuation decreased cell proliferation, clonogenicity, migration, invasion, tamoxifen resistance, tumor growth, and microvessel density, establishing an important oncogenic role for this gene. Algorithm-based and experimental strategies identified FOXF2, BBC3, PTEN, and COL2A1 as candidate miR-301 targets, all of which were verified as direct targets through luciferase reporter assays. We noted that miR-301 is located in an intron of the SKA2 gene which is responsible for kinetochore assembly, and both genes were found to be coexpressed in primary breast cancer samples. In summary, our findings define miR-301 as a crucial oncogene in human breast cancer that acts through multiple pathways and mechanisms to promote nodal or distant relapses., (©2011 AACR.)

Nasopharayngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignancy most common in East Asia and Africa. Here we report frequent downregulation of the microRNA miR-218 in primary NPC tissues and cell lines where it plays a critical role in NPC progression. Suppression of miR-218 was associated with epigenetic silencing of SLIT2 and SLIT3, ligands of ROBO receptors that have been previously implicated in tumor angiogenesis. Exogenous expression of miR-218 caused significant toxicity in NPC cells in vitro and delayed tumor growth in vivo. We used an integrated trimodality approach to identify targets of miR-218 in NPC, cervical, and breast cell lines. Direct interaction between miR-218 and the 3'-untranslated regions (UTR) of mRNAs encoding ROBO1, survivin (BIRC5), and connexin43 (GJA1) was validated in a luciferase-based transcription reporter assay. Mechanistic investigations revealed a negative feedback loop wherein miR-218 regulates NPC cell migration via the SLIT-ROBO pathway. Pleotropic effects of miR-218 on NPC survival and migration were rescued by enforced expression of miR-218-resistant, engineered isoforms of survivin and ROBO1, respectively. In clinical specimens of NPC (n=71), ROBO1 overexpression was significantly associated with worse overall (P=0.04, HR=2.4) and nodal relapse-free survival (P=0.008, HR=6.0). Our findings define an integrative tumor suppressor function for miR-218 in NPC and further suggest that restoring miR-218 expression in NPC might be useful for its clinical management., (© 2011 AACR.)

Head and neck cancer (HNC) is the eighth most common malignancy worldwide, comprising a diverse group of cancers affecting the head and neck region. Despite advances in therapeutic options over the last few decades, treatment toxicities and overall clinical outcomes have remained disappointing, thereby underscoring a need to develop novel therapeutic approaches in HNC treatment. Uroporphyrinogen decarboxylase (UROD), a key regulator of heme biosynthesis, was identified from an RNA interference-based high-throughput screen as a tumor-selective radiosensitizing target for HNC. UROD knockdown plus radiation induced caspase-mediated apoptosis and cell cycle arrest in HNC cells in vitro and suppressed the in vivo tumor-forming capacity of HNC cells, as well as delayed the growth of established tumor xenografts in mice. This radiosensitization appeared to be mediated by alterations in iron homeostasis and increased production of reactive oxygen species, resulting in enhanced tumor oxidative stress. Moreover, UROD was significantly overexpressed in HNC patient biopsies. Lower preradiation UROD mRNA expression correlated with improved disease-free survival, suggesting that UROD could potentially be used to predict radiation response. UROD down-regulation also radiosensitized several different models of human cancer, as well as sensitized tumors to chemotherapeutic agents, including 5-fluorouracil, cisplatin, and paclitaxel. Thus, our study has revealed UROD as a potent tumor-selective sensitizer for both radiation and chemotherapy, with potential relevance to many human malignancies.

There is increasing evidence supporting the role of members of the polycomb group (PcG) gene family in tumor development and progression. However, their precise role in tumorigenesis and mechanisms of their regulation remain to be elucidated. Using nasopharyngeal carcinoma (NPC) as a disease model, a comprehensive analysis was undertaken on the clinical significance of EZH2 expression, identification of the cellular processes regulated by EZH2, and the mechanisms of its deregulated expression. Herein, we report EZH2 as being associated with a higher risk of relapse in NPC patients (P = 0.002). Genome-wide microarray and bioinformatics identified several vital cellular processes (such as differentiation, development, and apoptosis) to be regulated by EZH2, corroborated by in vitro lethality, and delayed tumor formation in vivo upon EZH2 depletion. The combination of global microRNA (miR) profiling in primary NPC specimens, and in silico analyses provided several candidate miRs that could regulate EZH2. Using a luciferase-based assay, miR-26a, miR-101, and miR-98 were validated as bona fide regulators of EZH2 expression. In particular, miR-98 was underexpressed in relapsed patient samples, strongly suggesting an important role for the miR-98 and EZH2 axis in NPC biology.

Polo-like kinase 1 (Plk1) is a critical regulator of many stages of mitosis; increasing evidence indicates that Plk1 overexpression correlates with poor clinical outcome, yet its mechanism of regulation remains unknown. Hence, a detailed evaluation was undertaken of Plk1 expression in human nasopharyngeal cancer (NPC), the cellular effects of targeting Plk1 using siRNA in combination with ionizing radiation (RT) and potential upstream microRNAs (miRs) that might regulate Plk1 expression. Using immunohistochemistry, Plk1 was observed to be overexpressed in 28 of 40 (70%) primary NPC biopsies, which in turn was associated with a higher likelihood of recurrence (p = 0.018). SiPlk1 significantly inhibited Plk1 mRNA and protein expression, and decreased Cdc25c levels in NPC cell lines. This depletion resulted in cytotoxicity of C666-1 cells, enhanced by the addition of RT, mediated by G2/M arrest, increased DNA double-strand breaks, apoptosis, and caspase activation. Immunofluorescence demonstrated that the G2/M arrest was associated with aberrant spindle formation, leading to mitotic arrest. In vivo, transfection of C666-1 cells and systemic delivery of siPlk1 decreased tumour growth. MicroRNA-100 (miR-100) was predicted to target Plk1 mRNA, which was indeed underexpressed in C666-1 cells, inversely correlating with Plk1 expression. Using luciferase constructs containing the 3'-UTR of Plk1 sequence, we document that miR-100 can directly target Plk1. Hence, our data demonstrate for the first time that underexpressed miR-100 leads to Plk1 overexpression, which in turn contributes to NPC progression. Targeting Plk1 will cause mitotic catastrophe, with significant cytotoxicity both in vitro and in vivo, underscoring the important therapeutic opportunity of Plk1 in NPC.

Purpose: The objective of this study is to investigate the significance of microRNAs (miRNA) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC)., Experimental Design: A global miRNA profiling was done on 51 formalin-fixed archival HNSCC samples using quantitative reverse transcription-PCR approach, correlated with patients' clinical parameters. Functional characterization of HNSCC-associated miRNAs was conducted on three HNSCC cell lines. Cell viability and proliferation were investigated using MTS and clonogenic assays, respectively; cell cycle analyses were assessed using flow cytometry., Results: Thirty-eight of the 117 (33%) consistently detected miRNAs were significantly differentially expressed between malignant versus normal tissues. Concordant with previous reports, overexpression of miR-21, miR-155, let-7i, and miR-142-3p and underexpression of miR-125b and miR-375 were detected. Upregulation of miR-423, miR-106b, miR-20a, and miR-16 as well as downregulation of miR-10a were newly observed. Exogenous overexpression of miR-375 in HNSCC cell lines reduced proliferation and clonogenicity and increased cells in sub-G(1). Similar cellular effects were observed in knockdown studies of the miR-106b-25 cluster but with accumulation of cells in G(1) arrest. No major difference was detected in miRNA profiles among laryngeal, oropharyngeal, or hypopharyngeal cancers. miR-451 was found to be the only significantly overexpressed miRNA by 4.7-fold between nonrelapsed and relapsed patients., Conclusion: We have identified a group of aberrantly expressed miRNAs in HNSCC and showed that underexpression of miR-375 and overexpression of miR-106b-25 cluster might play oncogenic roles in this disease. Further detailed examinations of miRNAs will provide opportunities to dissect the complex molecular abnormalities driving HNSCC progression.

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer commonly occurring in southern China. To decipher the molecular basis of this cancer, we performed high-resolution array CGH analysis on eight tumour lines and 10 primary tumours to identify the genes involved in NPC tumorigenesis. In this study, multiple regions of gain were consistently found at 1q21-q24, 7q11-12, 7q21-22., 11q13, 12p13, 12q13, 19p13 and 19q13. Importantly, a 2.1 Mb region at 12p13.31 was highly amplified in a NPC xenograft, xeno-2117. By FISH mapping, we have further delineated the amplicon to a 1.24 region flanked by RP11-319E16 and RP11-433J6. Copy number gains of this amplicon were confirmed in 21/41 (51%) primary tumours, while three cases (7.3%) showed high copy number amplification. Among the 13 genes within this amplicon, three candidate genes, lymphotoxin beta receptor (LTbetaR), tumour necrosis factor receptor superfamily memeber 1A (TNFRSF1R) and FLJ10665, were specifically over-expressed in the NPC xenograft with 12p13.3 amplification. However, only LTbetaR was frequently over-expressed in primary tumours. LTbetaR is a member of the TNF family of receptors, which can modulate NF-kappaB signalling pathways. Over-expression of LTbetaR in nasopharyngeal epithelial cells resulted in an increase of NF-kappaB activity and cell proliferation. In vivo study showed that suppression of LTbetaR by siRNA led to growth inhibition in the NPC tumour with 12p13.3 amplification. These findings implied that LTbetaR is a potential NPC-associated oncogene within the 12p13.3 amplicon and that its alteration is important in NPC tumorigenesis.

Overexpression of BMI1 correlates with cancer development, progression, and therapy failure; however, the underlying molecular mechanisms remain to be fully elucidated. Using the C666-1 nasopharyngeal cancer (NPC) model, the role of BMI1 in mediating response of NPC cells to radiation therapy (RT) was investigated. The results showed a novel radioresistance function for BMI1 in NPC, wherein BMI1 depletion sensitized NPC cells to RT. Cell cycle analysis and transmission electron microscopy (TEM) showed apoptosis as the major mode of cell death, and the mitochondria as a primary targeted cellular organelle. Genome-wide microarray and pathway analyses revealed that the P53 pathway is a critical mediator of this process. Cotransfection with siP53 rescued C666-1 cells from cytotoxicity upon BMI1 depletion and RT, thereby corroborating the role for P53. Pretreatment with the antioxidant, Trolox, inhibited apoptosis, indicating that production of reactive oxygen species (ROS) is also mediating cytotoxicity. In vivo, BMI1 depletion combined with RT abrogated tumor-forming capacity in SCID mice, showing the relevance of this process in a more complex tumor environment. Hence, we show a novel role for BMI1 in conferring radioresistance in cancer cells through the downregulation of p53-mediated apoptosis. These results suggest a potential strategy of BMI1 depletion combined with RT for tumors wherein BMI1 appears to be driving disease progression.

Purpose: Seliciclib is a small-molecule cyclin-dependent kinase inhibitor, which has been reported to induce apoptosis and cell cycle arrest in EBV-negative nasopharyngeal carcinoma cell lines. Because most nasopharyngeal carcinoma patients harbor EBV, we proceeded to evaluate the cytotoxic effects of seliciclib in EBV-positive nasopharyngeal carcinoma models., Experimental Design: Cytotoxicity of seliciclib was investigated in the EBV-positive cell line C666-1 and the C666-1 and C15 xenograft models. Caspase activities and cell cycle analyses were measured by flow cytometry. Efficacy of combined treatment of seliciclib with radiation therapy was also evaluated., Results: Seliciclib caused significant cytotoxicity in the C666-1 cells in a time- and dose-dependent manner, with accumulation of cells in both sub-G(1) and G(2)-M phases, indicative of apoptosis and cell cycle arrest, respectively. Caspase-2, -3, -8, and -9 activities were all increased, with caspase-3 being the most significantly activated at 48 h after treatment. These cells also showed a reduction of Mcl-1 mRNA and protein levels. Combined treatment of seliciclib with radiation therapy showed a synergistic interaction with enhanced cytotoxicity in C666-1 cells and delayed repair of double-strand DNA breaks. For in vivo models, significant delays in tumor growth were observed for both C666-1 and C15 tumors, which were associated with enhanced apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunohistochemistry analyses., Conclusions: Seliciclib enhanced the antitumor efficacy of radiation therapy in EBV-positive nasopharyngeal carcinoma, characterized by G(2)-M arrest, and apoptosis, associated with an induction in caspase activity. This process is mediated by reduction in Mcl-1 expression and by attenuation of double-strand DNA break repair.

Global micro-RNA (miR) profiling of human malignancies is increasingly performed, but to date, the majority of such analyses have used frozen tissues. However, formalin fixation is the standard and routine histological practice for optimal preservation of cellular morphology. To determine whether miR analysis of formalin-fixed tissues is feasible, quantitative real-time PCR (qRT-PCR) profiling of miR expression in 40 archival formalin-fixed paraffin-embedded (FFPE) breast lumpectomy specimens were performed. Taqman Low Density Arrays (TLDAs) were used to assess the expression level of 365 miRs in 34 invasive ductal carcinomas and in 6 normal comparators derived from reduction mammoplasties. Its technical reproducibility was high, with intra-sample correlations above 0.9 and with 92.8% accuracy in differential expression comparisons, indicating such global profiling studies to be technically and biologically robust. The TLDA data were confirmed using conventional single-well qRT-PCR analysis, showing a strong and statistically significant concordance between these two methods. Paired frozen and FFPE breast cancer samples from the same patients showed a similar level of robust correlation of at least 0.94. Compared with normal breast samples, a panel of miRs was consistently dysregulated in breast cancer, including earlier-reported breast cancer-related miRs, such as upregulated miR-21, miR-155, miR-191, and miR-196a, and downregulated miR-125b and miR-221. Additional novel miR sequences of potential biological relevance were also uncovered. These results show the validity and utility of conducting global miR profiling using FFPE samples, thereby offering enormous opportunities to evaluate archival banks of such materials, linked to clinical databases, to rapidly acquire greater insight into the clinically relevant role for miRs in human malignancies.

Bladder cancer is the ninth most common cancer in the world. Urothelial carcinoma (formerly known as transitional cell carcinoma) comprises the majority of bladder cancers. In order to decipher the genetic alteration leading to the carcinogenesis of urothelial cancer, we performed genome-wide allelotyping analysis using 384 microsatellite markers spanning 22 autosomes together with comparative genomic hybridization (CGH) in 21 urothelial cancer. High frequency of allelic imbalance was observed in chromosome arm 1q (61.9%), 3p (61.9%), 4q (66.67%), 8p (57.14%), 9p (76.2%) and 9q (66.67%). Allelic imbalance with frequency above average was also observed in chromosome arm 2q, 10p, 10q, 11p, 11q, 12q, 13q, 15q, 17p and 19q. The allelic imbalance of each case and fractional allelic loss for each chromosome was associated with higher tumor grade and stage (P<0.05). We have also delineated several minimal deletion regions on chromosome 3p, 4q, 8p, 9p, 9q, 11p, 13q, 16q and 17p. By CGH analysis, common chromosomal alterations included gain of 1p, 1q, 12q, 16p, 17q and 19p as well as loss of 4q and 9p in most of the cases. Our findings may provide valuable information to locate putative oncogenes and tumor suppressor genes in the carcinogenesis of bladder cancer in this locality.

Purpose: Nasopharyngeal carcinoma (NPC) is a malignancy of the head and neck region that is associated with EBV latency. Curative treatments for NPC achieve modest survival rates, underscoring a need to develop novel therapies. We evaluated the therapeutic potential of a mutant vesicular stomatitis virus (VSVDelta51) as single treatment modality or in combination with ionizing radiation (RT) in NPC., Experimental Design: MTS assay was used to assess cell viability in vitro; apoptosis was measured using propidium iodide staining and caspase activation. In vivo experiments were conducted using tumor-bearing nude mice with or without local RT (4 Gy). Apoptosis was assessed in excised tumor sections with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining., Results: Our data showed that NPC cells are exquisitely sensitive to VSVDelta51 oncolysis, which correlated with the presence of EBV. Efficacy of VSVDelta51 against NPC cells was further augmented when combined with RT. A single systemic injection of VSVDelta51 achieved 50% survival in treated mice, which increased to 83% when combined with local tumor RT. In addition to induction of apoptosis, an antiangiogenic effect of VSVDelta51 was observed in vivo, suggesting a novel tumoricidal mechanism for VSVDelta51. This virus also prevented growth of NPC sphere-forming cells in vitro, showing potential utility in targeting NPC-initiating cells., Conclusions: Our data represent the first report showing that EBV-positive NPC cells are exquisitely sensitive to VSVDelta51 oncolysis and documenting the successful utilization of this combinatorial regimen as a novel curative therapeutic strategy for NPC.

In this study, we have elucidated the chromosomal imbalances in the multistep pathogenesis and delineated several critical tumor suppressor gene (TSG) loci in multiple myeloma (MM). By using comparative genomic hybridization, allelotyping, and multicolor interphase fluorescence in situ hybridization, 5 MM cell lines and bone marrow CD138+ plasma cells from 88 Chinese patients with monoclonal gammopathy of undetermined significance (MGUS) and early and advanced stages of MM were investigated. In all MGUS and MM samples, chromosome copy number abnormalities were detected. A higher number of chromosomal imbalances and specific genetic alterations are involved in MGUS to MM transition (-6q, +3p, and +1p) and MM progression (+2p and +9q). In addition to -13q, we first found high frequencies (42% to 46%) of -4q involving high percentages (70% to 74%) of clonal plasma cells in both MGUS and MM, suggesting that inactivation of TSG in this region is also a potentially critical genetic event in MM tumorigenesis. By high-resolution allelotyping, we defined a common deletion region on 4q13.3 and found that a candidate TSG, platelet factor 4, was frequently silenced by promoter hypermethylation in MM (15 of 28) and MM cell lines (5 of 5). These data have opened up a new approach in the molecular targeting therapy and provide novel insights into MM tumorigenesis.

Nasopharyngeal carcinoma is highly prevalent in Southern China and Southeast Asia. To unveil the molecular basis of this endemic disease, high-resolution comparative genomic hybridization arrays were used for systematic investigation of genomic abnormalities in 26 nasopharyngeal carcinoma samples. A comprehensive picture of genetic lesions associated with tumorigenesis of nasopharyngeal carcinoma was generated. Consistent chromosomal gains were frequently found on 1q, 3q, 8q, 11q, 12p, and 12q. High incidences of nonrandom losses were identified on chromosomes 3p, 9p, 11q, 14q, and 16q. In addition to previously characterized regions, we have identified several novel minimal regions of gains, including 3q27.3-28, 8q21-24, 11q13.1-13.3, and 12q13, which may harbor candidate nasopharyngeal carcinoma-associated oncogenes. In this study, gain of 11q13.1-13.3 was the most frequently detected chromosomal aberration and a 5.3-Mb amplicon was delineated at this region. Within this 11q13 amplicon, concordant amplification and overexpression of cyclin D1 (CCND1) oncogene was found in nasopharyngeal carcinoma cell lines, xenografts, and primary tumors. Knockdown of cyclin D1 by small interfering RNA in nasopharyngeal carcinoma cell lines led to significant decrease of cell proliferation. The findings suggest that cyclin D1 is a target oncogene at 11q13 in nasopharyngeal carcinoma and its activation plays a significant role in nasopharyngeal carcinoma tumorigenesis.

Purpose: The aim of this study is to comprehensively characterize genome copy number aberrations in medulloblastomas using high-resolution array comparative genomic hybridization., Experimental Design: High-density genomic arrays containing 1,803 BAC clones were used to define recurrent chromosomal regions of gains or losses throughout the whole genome of medulloblastoma. A series of 3 medulloblastoma cell lines and 16 primary tumors were investigated., Results: The detected consistent chromosomal aberrations included gains of 1q21.3-q23.1 (36.8%), 1q32.1 (47.4%), 2p23.1-p25.3 (52.6%), 7 (57.9%), 9q34.13-q34.3 (47.4%), 17p11.2-q25.3 (89.5%), and 20q13.31-q13.33 (42.1%), as well as losses of 3q26.1 (57.9%), 4q31.23-q32.3 (42.1%), 6q23.1-25.3 (57.9%), 8p22-23.3 (79%), 10q24.32-26.2 (57.9%), and 16q23.2-q24.3 (63.2%). One of the most notable aberrations was a homozygous deletion on chromosome 6q23 in the cell line DAOY, and single copy loss on 30.3% primary tumors. Further analyses defined a 0.887 Mbp minimal region of homozygous deletion at 6q23.1 flanked by markers SHGC-14149 (6q22.33) and SHGC-110551 (6q23.1). Quantitative reverse transcription-PCR analysis showed complete loss of expression of two genes located at 6q23.1, AK091351 (hypothetical protein FLJ34032) and KIAA1913, in the cell line DAOY. mRNA levels of these genes was reduced in cell lines D283 and D384, and in 50% and 70% of primary tumors, respectively., Conclusion: Current array comparative genomic hybridization analysis generates a comprehensive pattern of chromosomal aberrations in medulloblastomas. This information will lead to a better understanding of medulloblastoma tumorigenesis. The delineated regions of gains or losses will indicate locations of medulloblastoma-associated genes. A 0.887 Mbp homozygous deletion region was newly identified at 6q23.1. Frequent detection of reduced expression of AK091351 and KIAA1913 genes implicates them as suppressors of medulloblastoma tumorigenesis.

Nasopharyngeal carcinoma (NPC) is a distinct geographical disease with high incidence in Southeast Asia. Previous CGH studies have located multiple regions of chromosomal gains and losses in NPC. To elucidate the regions of gain and amplification, a high-resolution array comparative genomic hybridization (array CGH) was applied to characterize the common amplicons in NPC cell lines and xenografts. Consistent with our previous CGH findings, frequent gains at chromosomes 1q, 3q, 7, 8, 9q, 12q and 20q were detected. High incidence of gains were identified on chromosome 3q and 12q. The findings were further confirmed by FISH analysis. Using 16 BAC clones on 3q26-28 and 6 BAC clones on 12q13, the smallest regions of gain at 3q and 12q were defined on five NPC cell lines. Chromosome 3q26.33 (RP11-510K16) and 12q13.2-q13.3 (RP11-183H16) showed the highest amplification frequency in FISH analysis with 100 and 66.7%, respectively. PIK3CA, a candidate oncogene located at 3q26.32 adjacent to this 3q amplicon was investigated by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Overexpression of this gene was found in 4 NPC cell lines and 3 xenografts. The findings implied that PIK3CA may be a putative oncogene involved in the tumorigenesis of NPC.

Object: Few studies have been conducted to investigate the genomic survey of oncogene amplification in medulloblastoma. Low frequency of N-myc, C-myc, and epidermal grow factor receptor (EGFR) gene amplification (< 10%) has been reported in medulloblastoma. Previous comparative genomic hybridization (CGH) study of primary medulloblastomas has revealed chromosomal amplification on 2p21, 3p, 5p15.3, 7q, 8q24, 11q22.3, and 17q. The aim of this study was to detect common oncogenes involved in medulloblastoma tumorigenesis., Methods: The authors studied a series of 14 samples by performing CGH and array-based CGH. The CGH analysis detected nonrandom losses on 8p, 17p, 16q, 8q, and 1p, whereas gains were found on 17q, 12q, 7q, and 1p. Array-based CGH was conducted to investigate amplification of 58 oncogenes throughout the genome of these samples. Gene amplifications identified for the first time included PGY1 at 7q21.1, MDM2 at 12q14.3-q15, and ERBB2 at 17q21.2. The highest frequencies of oncogene gain were detected in D17S1670 (61.5%), PIK3CA (46.2%), PGY1 (38.5%), MET (38.5%), ERBB2 (38.5%), and CSE1L (38.5%). The gain in gene copy numbers was confirmed in 34 additional archival medulloblastoma cases by using fluorescence in situ hybridization analysis., Conclusions: This is the first genome-wide survey of multiple oncogene amplifications involved in the development of medulloblastoma. Gains of several candidate oncogenes such as D17S1670, ERBB2, PIK3CA, PGY1, MET, and CSE1L were frequently detected. These genes may be used as molecular markers and therapeutic targets of medulloblastomas.

Background: The discovery of fetal DNA in maternal plasma has opened up an approach for noninvasive prenatal diagnosis. Despite the rapid expansion in clinical applications, the molecular characteristics of plasma DNA in pregnant women remain unclear., Methods: We investigated the size distribution of plasma DNA in 34 nonpregnant women and 31 pregnant women, using a panel of quantitative PCR assays with different amplicon sizes targeting the leptin gene. We also determined the size distribution of fetal DNA in maternal plasma by targeting the SRY gene., Results: The median percentages of plasma DNA with size >201 bp were 57% and 14% for pregnant and nonpregnant women, respectively (P <0.001, Mann-Whitney test). The median percentages of fetal-derived DNA with sizes >193 bp and >313 bp were 20% and 0%, respectively, in maternal plasma., Conclusion: Plasma DNA molecules are mainly short DNA fragments. The DNA fragments in the plasma of pregnant women are significantly longer than those in the plasma of nonpregnant women, and the maternal-derived DNA molecules are longer than the fetal-derived ones.

Deletion of 11q23 is a common genetic aberration in nasopharyngeal carcinoma (NPC). Multiple candidate tumor suppressor genes (TSG) were mapped to this region but few of them were investigated in NPC. TSLC1 (tumor suppressor in lung cancer) is recently reported to be a putative TSG on 11q23. This gene was found to be inactivated by promoter hypermethylation in non-small cell lung carcinoma (NSCLC), liver cancer, and breast cancer. To study the role of TSLC1 gene in NPC tumorigenesis, we screened for mutations and aberrant methylation of TSLC1 gene in 5 NPC cell lines, 3 NPC xenografts, and 38 primary NPC cases. No somatic mutations of TSLC1 were detected in the NPC samples, but a 9-bp (CCACCACCA) deletion in exon 8 was found in a primary NPC and its corresponding blood sample. Bisulfite sequencing revealed aberrant methylation of TSLC1 promoter in four NPC cell lines. Loss of TSLC1 gene expression was found in two cell lines (HK-1 and CNE-2) with dense methylation. Expression of this gene was restored in these cell lines after treatment with demethylating agent 5-aza-2'-deoxycytidine. Our results showed that silencing of TSLC1 gene expression in NPC was associated with promoter hypermethylation. Promoter hypermethylation of TSLC1 gene was further illustrated in 34.2% (13/38) of primary NPCs. No aberrant promoter methylation was found in any of the four investigated normal nasopharyngeal epithelia. Frequent epigenetic inactivation of TSLC1 gene in NPC suggested that this gene is one of the target tumor suppressor genes of this endemic cancer., (Copyright 2003 Wiley-Liss, Inc.)

Nasopharyngeal carcinoma (NPC) cell lines and xenografts represent valuable models for functional and therapeutic studies on this common malignancy in Southeast Asia. The karyotypic information in most NPC cell lines and xenografts, however, remains largely unclear to date. We have characterized the chromosomal aberrations in six commonly used human NPC cell lines and xenografts using the molecular cytogenetic technique of comparative genomic hybridization (CGH). Genomic imbalances identified in cell lines were further correlated with structural abnormalities indicated from spectral karyotyping (SKY) analysis. CGH revealed consistent overrepresentations of 8q (six out of six cases) with a smallest overlapping region identified on 8q21.1 approximately q22. Other common gains included 7p (4/6 cases), 7q (4/6 cases), 12q (4/6), and 20q (4/6 cases), where minimal overlapping regions were suggested on 7p15 approximately p14, 7q11.2 approximately q21, and 12q22 approximately q24.1. Common losses were detected on 3p12 approximately p21 (4/6 cases) and 11q14 approximately qter (4/6 cases). Although SKY analysis on cell lines revealed predominantly unbalanced rearrangements, reciprocal translocations that involved chromosome 2 [i.e., t(1;2), t(2;3), and t(2;4)] were suggested. Furthermore, SKY examination illustrated additional breakpoints on a number of apparently balanced chromosomes. These breakpoints included 3p21, 3q26, 5q31, 6p21.1 approximately p25, 7p14 approximately p22, and 8q22. Our finding of regional gains and losses and breakpoints represents information that may contribute to NPC studies in vitro.

Ganglioglioma is a mixed neuronal and glial tumor first described by Perkin in 1926. Because of its rare occurrence in the central nervous system, the pathogenesis of this neoplasm is still largely unknown. Previous studies of ganglioglioma mainly focused on histologic features, immunohistochemical analysis, clinical treatment, and patient outcome. Very few cytogenetic and molecular genetic studies have been reported on this neoplasm. To better understand the mechanism underlying the development of ganglioglioma, we performed comparative genomic hybridization analysis to investigate chromosomal imbalances across the entire genome in five cases of gangliogliomas. Loss of genetic material on the short arm of chromosome 9 was a common genetic alteration found in three of five cases. Overrepresentation of partial or the whole chromosome 7 was another recurrent chromosomal imbalance, confirmed by fluorescence in situ hybridization. Immunohistochemical analysis was performed; all five cases revealed no reaction or low expression for epidermal growth factor receptor antibody. Our study highlights chromosomal regions for further fine mapping and investigation of candidate tumor suppressor genes involved in the pathogenesis of ganglioglioma.

We have applied the method of genomic microarray to investigate amplification of oncogenes throughout the genome of nasopharyngeal carcinoma (NPC). Array based comparative genomic hybridization (array CGH) allows simultaneous examination of 58 oncogenes commonly amplified in various human cancers. In the present study, we have examined 15 NPC samples including five cell lines, two xenografts and eight primary tumours with array CGH to reveal the particular oncogenes associated with this cancer. This is the first genome wide survey of multiple oncogene amplifications involved in the development of NPC. Non-random gene amplifications were identified for the first time in NPC on MYCL1 in 1p34.3 and on TERC and PIK3CA at 3q26.3. Other high level amplified oncogenes included NRAS, RAF1, MYB, EGFR, FGF4, EMS1, and D17S167. Highest frequencies of gain of novel oncogenes were detected on MYCL1 (66.7%), TERC (46.7%), ESR (46.7%), PIK3CA (40%), LAMC2 (33.3%), and CSE1L (33.3%).

Pleomorphic xanthoastrocytoma (PXA) is a rare, low-grade astrocytic tumor found in the central nervous system. Histologically, the tumor is characterized by markedly pleomorphic and lipidized cells. Although most of the patients have a favorable prognosis, a small number of cases undergoing recurrence or progression to anaplastic astrocytoma were reported. Very few genetic studies have been performed on PXA because of its rarity and the pathogenesis of this neoplasm is largely unknown. In order to provide an overview of genetic alterations in PXA, we performed comparative genomic hybridization to identify chromosomal imbalances (DNA gains and losses) in three cases of PXA. Genetic imbalance was detected on at least one chromosome for each case. One case, which revealed multiple genetic alterations, showed a poor prognosis. DNA gain on chromosome 7 and loss on 8p were demonstrated in two of three cases, suggesting that the candidate gene(s) located on these regions may play a role in the development of PXA. Further studies are needed to identify the residing candidate genes that are involved in the tumorigenesis of PXA. In addition, the histopathological features and previous genetic studies on PXA are reviewed.