Genomic evolutional analysis of surgical resected specimen to assess osimertinib as a first-line therapy in two patients with lung cancer harboring EGFR mutation: Case series.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is crucial for patients with lung cancer harboring EGFR mutations. However, almost all patients experience disease progression, regardless of their response to the targeted therapy, necessitating the development of additional treatment options. Two patients with lung cancer harboring EGFR-L858R mutations in exon 21 were treated by surgical resection during successful osimertinib treatment. Because the pathological diagnosis was suspected to be pleural metastasis, osimertinib treatment was continued until disease progression. We analyzed the evolution of genomic alterations and the levels of AXL using tumor specimens obtained by repeated biopsies during the course of treatment: initial diagnosis, operation, and disease progression. Genetic alterations detected at the three time points were dramatically changed and showed reductions in numbers, while EGFR-L858R mutations were detected in all samples tested in both patients. Immunohistochemical expression of AXL remained positive from the beginning of analysis to disease progression. Clonal evolution under oncogenesis is related to gradual accumulation of genomic alterations during tumor growth. However, our case series revealed that volume reduction procedures may cause this phenomenon. Therefore, identification of intrinsic drug-resistant cells in tumors may be as important as detection of acquired genetic alterations.

Identification of uromodulin deposition in the stroma of perinephric fibromyxoid nephrogenic adenoma by mass spectrometry.

Nephrogenic adenoma (NA) is an epithelial lesion that usually occurs in the mucosa of the urinary tract. Rare cases of deep infiltrative or perinephric lesions have also been reported. Recently, NA with characteristic fibromyxoid stroma (fibromyxoid NA) has been proposed as a distinct variant. Although shedding of distal renal tubular cells due to urinary tract rupture has been postulated as the cause of NA in general, the mechanism underlying extraurinary presentation of NA and fibromyxoid stromal change in fibromyxoid NA remains unknown. In this study, we performed mass spectrometry (MS) analysis in a case of perinephric fibromyxoid NA of an 82-year-old man who underwent right nephroureterectomy for distal ureteral cancer. The patient had no prior history of urinary tract injury or radiation. Periodic acid-Schiff staining-positive eosinophilic structureless deposits in the stroma of fibromyxoid NA were microdissected and subjected to liquid chromatography/MS. The analysis revealed the presence of a substantial amount of uromodulin (Tamm-Horsfall protein). The presence of urinary content in the stroma of perinephric fibromyxoid NA suggests that urinary tract rupture and engraftment of renal tubular epithelial cells directly cause the lesion.

Distinct microRNA Signature and Suppression of ZFP36L1 Define ASCL1-Positive Lung Adenocarcinoma.

Achaete-scute family bHLH transcription factor 1 (ASCL1) is a master transcription factor involved in neuroendocrine differentiation. ASCL1 is expressed in approximately 10% of lung adenocarcinomas (LUAD) and exerts tumor-promoting effects. Here, we explored miRNA profiles in ASCL1-positive LUADs and identified several miRNAs closely associated with ASCL1 expression, including miR-375, miR-95-3p/miR-95-5p, miR-124-3p, and members of the miR-17∼92 family. Similar to small cell lung cancer, Yes1 associated transcriptional regulator (YAP1), a representative miR-375 target gene, was suppressed in ASCL1-positive LUADs. ASCL1 knockdown followed by miRNA profiling in a cell culture model further revealed that ASCL1 positively regulates miR-124-3p and members of the miR-17∼92 family. Integrative transcriptomic analyses identified ZFP36 ring finger protein like 1 (ZFP36L1) as a target gene of miR-124-3p, and IHC studies demonstrated that ASCL1-positive LUADs are associated with low ZFP36L1 protein levels. Cell culture studies showed that ectopic ZFP36L1 expression inhibits cell proliferation, survival, and cell-cycle progression. Moreover, ZFP36L1 negatively regulated several genes including E2F transcription factor 1 (E2F1) and snail family transcriptional repressor 1 (SNAI1). In conclusion, our study revealed that suppression of ZFP36L1 via ASCL1-regulated miR-124-3p could modulate gene expression, providing evidence that ASCL1-mediated regulation of miRNAs shapes molecular features of ASCL1-positive LUADs. IMPLICATIONS: Our study revealed unique miRNA profiles of ASCL1-positive LUADs and identified ASCL1-regulated miRNAs with functional relevance.

Metastases and treatment-resistant lineages in patient-derived cancer cells of colorectal cancer.

Circulating tumor cells (CTCs) play an important role in metastasis and recurrence. However, which cells comprise the complex tumor lineages in recurrence and are key in metastasis are unknown in colorectal cancer (CRC). CRC with high expression of POU5F1 has a poor prognosis with a high incidence of liver metastatic recurrence. We aim to reveal the key cells promoting metastasis and identify treatment-resistant lineages with established EGFP-expressing organoids in two-dimensional culture (2DOs) under the POU5F1 promotor. POU5F1-expressing cells are highly present in relapsed clinical patients' blood as CTCs. Sorted POU5F1-expressing cells from 2DOs have cancer stem cell abilities and abundantly form liver metastases in vivo. Single-cell RNA sequencing of 2DOs identifies heterogeneous populations derived from POU5F1-expressing cells and the Wnt signaling pathway is enriched in POU5F1-expressing cells. Characteristic high expression of CTLA4 is observed in POU5F1-expressing cells and immunocytochemistry confirms the co-expression of POU5F1 and CTLA4. Demethylation in some CpG islands at the transcriptional start sites of POU5F1 and CTLA4 is observed. The Wnt/ß-catenin pathway inhibitor, XAV939, prevents the adhesion and survival of POU5F1-expressing cells in vitro. Early administration of XAV939 also completely inhibits liver metastasis induced by POU5F1-positive cells.

GRAMD2 + alveolar type I cell plasticity facilitates cell state transitions in organoid culture.

Alveolar epithelial regeneration is critical for normal lung function and becomes dysregulated in disease. While alveolar type 2 (AT2) and club cells are known distal lung epithelial progenitors, determining if alveolar epithelial type 1 (AT1) cells also contribute to alveolar regeneration has been hampered by lack of highly specific mouse models labeling AT1 cells. To address this, the Gramd2 CreERT2 transgenic strain was generated and crossed to Rosa mTmG mice. Extensive cellular characterization, including distal lung immunofluorescence and cytospin staining, confirmed that GRAMD2 + AT1 cells are highly enriched for green fluorescent protein (GFP). Interestingly, Gramd2 CreERT2 GFP + cells were able to form organoids in organoid co-culture with Mlg fibroblasts. Temporal scRNAseq revealed that Gramd2 + AT1 cells transition through numerous intermediate lung epithelial cell states including basal, secretory and AT2 cell in organoids while acquiring proliferative capacity. Our results indicate that Gramd2 + AT1 cells are highly plastic suggesting they may contribute to alveolar regeneration.

Whole Exome Sequencing of Thymoma Patients Exhibiting Exceptional Responses to Pemetrexed Monotherapy.

BACKGROUND: Pemetrexed is used for the chemotherapy of advanced thymoma. Exceptional responses of thymoma to pemetrexed treatment are not frequently observed. The underlying genetic mechanism of the exceptional responses remains unclear. We used whole-exome sequencing to explore the specific genomic aberrations that lead to an extreme and durable response. METHODS: Whole-exome sequencing using NovaSeq6000 (150 bp paired-end sequencing) was performed on nine formalin-fixed paraffin-embedded tissues from patients with advanced thymomas treated with pemetrexed (two exceptional responders and seven typical responders). RESULTS: We identified 284 somatic single-nucleotide variants (SNVs; 272 missense, 8 missense/splice-site, 3 stop-gain, and 1 stop-gain/splice-site), 34 insertions and deletions (Indels; 33 frameshift and one splice region), and 21 copy number variations (CNVs; 15 gains and six losses). No difference in the number of SNVs variants and distribution of deleterious Indels was observed between the exceptional and typical responders. Interestingly, arm-level chromosomal CNVs (15 gains and six losses) were detected in four patients, including an exceptional responder. The highest number of arm-level CNVs was observed in an exceptional responder. CONCLUSION: Exceptional responders to pemetrexed for metastatic thymomas may be characterized by arm-level CNVs. Further, whole-genome and RNA sequencing studies should be performed.

Cancer Stem Cells Persist Despite Cellular Damage, Emergence of the Refractory Cell Population.

PURPOSE: Cancer stem cells (CSCs) are responsible for chemotherapy resistance and have unique properties that protect them from chemotherapy. Investigating CSCs may help to identify the population that is more resistant to treatments, leading to recurrence. We evaluated persisting CSCs, emerging after chemotherapy that cause tumor recurrence. METHODS: Using human colorectal cancer organoids prepared from surgical specimens, we looked at changes in CSCs, the emergence and changes in the original population, which single-cell analysis identified. RESULTS: With regards to changes in cancer stem cell markers, CD44 showed low levels after 5-fluorouracil administration. Once the CD44-ve population was sorted and cultured, the CD44+ve population gradually emerged, and the CD44-ve population decreased. Compared with the CD44-ve population of an organoid parent, the CD44-ve population proliferated after chemotherapeutic agent stimulation. The CD44-ve population was derived from the CD44+ve population before chemotherapeutic agents. In addition, when the CD44 variants were evaluated, the CD44v9 population remained. In single-cell analysis, we found that POU5F1 was highly expressed in the CD44low population. Velocity analysis showed that the CD44-ve population was induced after chemotherapy and expressed POU5F1. POU5F1-EGFP-Casp9 transfected organoids resulted in the appearance of a CD44-ve population after administration of a chemotherapeutic reagent. Both in vivo and in vitro, the dimerizer administration inhibited tumor growth significantly. CONCLUSIONS: POU5F1 is involved in chemotherapy resistance in relation to stemness. For the treatment against refractory tumors, such as the recurrence after chemotherapy, the treatment should target the emerging specific population such as CD44 (or CD44v9) and proliferative cancer cells.

An integrative epigenomic approach identifies ELF3 as an oncogenic regulator in ASCL1-positive neuroendocrine carcinoma.

Neuroendocrine carcinoma (NEC) is a highly aggressive subtype of the neuroendocrine tumor with an extremely poor prognosis. We have previously conducted a comprehensive genomic analysis of over 100 cases of NEC of the gastrointestinal system (GIS-NEC) and unraveled its unique and organ-specific genomic drivers. However, the epigenomic features of GIS-NEC remain unexplored. In this study, we have described the epigenomic landscape of GIS-NEC and small cell lung carcinoma (SCLC) by integrating motif enrichment analysis from the assay of transposase-accessible chromatin sequencing (ATAC-seq) and enhancer profiling from a novel cleavage under targets and tagmentation (CUT&Tag) assay for H3K27ac and identified ELF3 as one of the super-enhancer-related transcriptional factors in NEC. By combining CUT&Tag and knockdown RNA sequencing for ELF3, we uncovered the transcriptional network regulated by ELF3 and defined its distinctive gene signature, including AURKA, CDC25B, CLDN4, ITGB6, and YWAHB. Furthermore, a loss-of-function assay revealed that ELF3 depletion led to poor cell viability. Finally, using gene expression of clinical samples, we successfully divided GIS-NEC patients into two subgroups according to the ELF3 signature and demonstrated that tumor-promoting pathways were activated in the ELF3 signature-high group. Our findings highlight the transcriptional regulation of ELF3 as an oncogenic transcription factor and its tumor-promoting properties in NEC.

Clearance Profile of Circulating Tumor Human Papillomavirus DNA During Radiotherapy Predicts Clinical Outcomes in Human Papillomavirus-Related Oropharyngeal Cancer.

PURPOSE: This study aimed to examine whether circulating tumor human papillomavirus type 16 (HPV16) DNA (ctHPV16DNA) can help identify patients with locally advanced HPV16-related oropharyngeal squamous cell carcinoma who may benefit from deintensified treatment. MATERIALS AND METHODS: We serially collected blood samples before, during, and after treatment from 22 patients who received 70 Gy radiotherapy alone and longitudinally quantified ctHPV16DNA using droplet digital polymerase chain reaction. We correlated the clearance profile of ctHPV16DNA with clinical outcomes. RESULTS: The percentage of patients with detectable ctHPV16DNA decreased after every 10 Gy of radiotherapy. By contrast, the percentage of patients who later developed treatment failure among patients with detectable ctHPV16DNA gradually increased as radiotherapy proceeded, reaching 100% after 60 Gy of radiotherapy. We defined patients with and without detectable ctHPV16DNA after receiving 40 Gy as having slow and rapid clearance profiles, respectively. All 12 patients with a rapid clearance profile remained disease-free after radiotherapy. Of the 10 patients with a slow clearance profile, three had persistent or progressive disease at response evaluation after radiotherapy and one developed distant metastasis during follow-up (ie, four patients experienced treatment failure). The median follow-up for surviving patients was 38.6 months, and the 3-year failure-free survival rates of patients with rapid and slow clearance profiles were 100% and 58%, respectively (P = .02). Neither baseline ctHPV16DNA levels nor metabolic tumor volume was an independent predictor of the pattern of the clearance profile. CONCLUSION: In patients with HPV16-related oropharyngeal squamous cell carcinoma receiving radiotherapy, a slow ctHPV16DNA clearance profile could prelude unfavorable outcomes. Monitoring ctHPV16DNA is essential for determining the clearance profile, which might help optimize treatment intensity individually.

Mid-term clinical outcomes of a posterior stabilized total knee prosthesis for Japanese patients: A minimum follow-up of 5 years.

BACKGROUND: A high-flexion posterior-stabilized total knee prosthesis has been developed for the Asian population. The component design was based on computed tomography images of Japanese osteoarthritic knees. The femoral component is composed of zirconia ceramics, which exhibit low friction and high durability. The present study aimed to evaluate the mid-term clinical outcomes of this implant. METHODS: This study included 334 knees of 210 patients who underwent primary total knee arthroplasty with this implant at our hospital between October 2010 and December 2014. The patients comprised 28 men and 172 women with an average age of 73 years. The average follow-up period was 5.9 years, and the follow-up rate was 71.1%. Clinical outcomes were assessed using the Knee Society scoring system, 2011 Knee Society questionnaire, and Knee Injury and Osteoarthritis Outcome Score. Kaplan-Meier survivorship analysis was performed to determine the cumulative prosthesis survival rate. RESULTS: In terms of clinical outcomes at the final follow-up, the average ranges of motion were -2.0 in extension and 126.7 in flexion. The Knee Society knee and function scores were 94.2% and 72.6%, respectively. With revision surgery or radiographic failure for any reason as the endpoint, the survival rates at 5 and 9 years were 98.2% and 95.5%, respectively. The most common reason for revision surgery or radiological failure was aseptic loosening. CONCLUSIONS: Despite several revision cases mainly due to aseptic loosening, the present study found that this new high-flexion posterior-stabilized total knee arthroplasty prosthesis design showed comparable results for Asian populations with other PS prosthesis. LEVELS OF EVIDENCE: Level Ⅱ (Prospective cohort study).

Inhibition of Epithelial-Mesenchymal Transition Maintains Stemness in Human Amniotic Epithelial Cells.

Human amniotic epithelial cells (hAECs), which are a type of placental stem cell, express stem cell marker genes and are capable of differentiating into all three germ layers under appropriate culture conditions. hAECs are known to undergo TGF-ß-dependent epithelial-mesenchymal transition (EMT); however, the impact of EMT on the stemness or differentiation of hAECs has not yet been determined. Here, we first confirmed that hAECs undergo EMT immediately after starting primary culture. Comprehensive transcriptome analysis using RNA-seq revealed that inhibition of TGF-ß-dependent EMT maintained the expression of stemness-related genes, including NANOG and POU5F1, in hAECs. Moreover, the maintenance of stemness did not affect the nontumorigenic characteristics of hAECs. We showed for the first time that TGF-ß-dependent EMT negatively affected the stemness of hAECs, providing novel insight into cellular processes of placental stem cells.

ASCL1 regulates super-enhancer-associated miRNAs to define molecular subtypes of small cell lung cancer.

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor with dismal prognosis. Recently, molecular subtypes of SCLC have been defined by the expression status of ASCL1, NEUROD1, YAP1, and POU2F3 transcription regulators. ASCL1 is essential for neuroendocrine differentiation and is expressed in the majority of SCLC. Although previous studies investigated ASCL1 target genes in SCLC cells, ASCL1-mediated regulation of miRNAs and its relationship to molecular subtypes remain poorly explored. Here, we performed genome-wide profiling of chromatin modifications (H3K27me3, H3K4me3, and H3K27ac) by CUT&Tag assay and ASCL1 knockdown followed by RNA sequencing and miRNA array analyses in SCLC cells. ASCL1 could preferentially regulate genes associated with super-enhancers (SEs) defined by enrichment of H3K27ac marking. Moreover, ASCL1 positively regulated several SE-associated miRNAs, such as miR-7, miR-375, miR-200b-3p, and miR-429, leading to repression of their targets, whereas ASCL1 suppressed miR-455-3p, an abundant miRNA in other molecular subtypes. We further elucidated unique patterns of SE-associated miRNAs in different SCLC molecular subtypes, highlighting subtype-specific miRNA networks with functional relevance. Notably, we found apparent de-repression of common target genes of different miRNAs following ASCL1 knockdown, suggesting combinatorial action of multiple miRNAs underlying molecular heterogeneity of SCLC (e.g., co-targeting of YAP1 by miR-9 and miR-375). Our comprehensive analyses provide novel insights into SCLC pathogenesis and a clue to understanding subtype-dependent phenotypic differences.

Performance of oral HPV DNA, oral HPV mRNA and circulating tumor HPV DNA in the detection of HPV-related oropharyngeal cancer and cancer of unknown primary.

A biomarker that is useful for the detection of human papillomavirus (HPV)-related oropharyngeal cancer (OPC) and cancer of unknown primary (CUP) is indispensable. We evaluated the diagnostic performance of HPV DNA and mRNA in oral gargle samples and circulating tumor HPV16 DNA (ctHPV16DNA) in blood samples. Oral HPV DNA and mRNA were analyzed using commercially available HPV assays of the GENOSEARCH HPV31 and Aptima, respectively. ctHPV16DNA was analyzed using in-house droplet digital polymerase chain reaction. Seventy-four patients with OPC and eight patients with CUP were included. The sensitivity and specificity of oral HPV DNA, oral HPV mRNA, and ctHPV16DNA were 82% (95% confidence interval [CI] = 66-92) and 100% (95% CI = 88-100), 85% (95% CI = 69-94) and 94% (95% CI = 73-100), and 93% (95% CI = 81-99) and 97% (95% CI = 84-100), respectively, for HPV16-related OPC, while those were 20% (95% CI = 1-72) and 100% (95% CI = 3-100), 0% (95% CI = 0-52) and 100% (95% CI = 3-100), and 100% (95% CI = 54-100) and 100% (95% CI = 16-100), respectively, for HPV16-related CUP. The sensitivity of ctHPV16DNA for HPV16-related OPC was higher than that of oral biomarkers, though the difference was not statistically significant. ctHPV16DNA remarkably correlated with the anatomic extent of disease, total metabolic tumor volume and HPV16 copy number per tumor genome in patients with HPV16-related OPC/CUP, whereas oral biomarkers did not. In conclusion, ctHPV16DNA is a potentially promising biomarker for HPV16-related OPC, while further studies are required for HPV16-related CUP.

Comprehensive Genomic Profiling of Neuroendocrine Carcinomas of the Gastrointestinal System.

The neuroendocrine carcinoma of the gastrointestinal system (GIS-NEC) is a rare but highly malignant neoplasm. We analyzed 115 cases using whole-genome/exome sequencing, transcriptome sequencing, DNA methylation assays, and/or ATAC-seq and found GIS-NECs to be genetically distinct from neuroendocrine tumors (GIS-NET) in the same location. Clear genomic differences were also evident between pancreatic NECs (Panc-NEC) and nonpancreatic GIS-NECs (Nonpanc-NEC). Panc-NECs could be classified into two subgroups (i.e., "ductal-type" and "acinar-type") based on genomic features. Alterations in TP53 and RB1 proved common in GIS-NECs, and most Nonpanc-NECs with intact RB1 demonstrated mutually exclusive amplification of CCNE1 or MYC. Alterations of the Notch gene family were characteristic of Nonpanc-NECs. Transcription factors for neuroendocrine differentiation, especially the SOX2 gene, appeared overexpressed in most GIS-NECs due to hypermethylation of the promoter region. This first comprehensive study of genomic alterations in GIS-NECs uncovered several key biological processes underlying genesis of this very lethal form of cancer. SIGNIFICANCE: GIS-NECs are genetically distinct from GIS-NETs. GIS-NECs arising in different organs show similar histopathologic features and share some genomic features, but considerable differences exist between Panc-NECs and Nonpanc-NECs. In addition, Panc-NECs could be classified into two subgroups (i.e., "ductal-type" and "acinar-type") based on genomic and epigenomic features. This article is highlighted in the In This Issue feature, p. 587.

TGF-ß-mediated epithelial-mesenchymal transition and tumor-promoting effects in CMT64 cells are reflected in the transcriptomic signature of human lung adenocarcinoma.

Epithelial-mesenchymal transition (EMT) is a cellular process during which epithelial cells acquire mesenchymal phenotypes. Cancer cells undergo EMT to acquire malignant features and TGF-ß is a key regulator of EMT. Here, we demonstrate for the first time that TGF-ß could elicit EMT in a mouse lung adenocarcinoma cell line. TGF-ß signaling activation led to cell morphological changes corresponding to EMT and enhanced the expression of mesenchymal markers and EMT-associated transcription factors in CMT64 lung cancer cells. RNA-sequencing analyses revealed that TGF-ß increases expression of Tead transcription factors and an array of Tead2 target genes. TGF-ß stimulation also resulted in alternative splicing of several genes including Cd44, tight junction protein 1 (Tjp1), and Cortactin (Cttn). In parallel with EMT, TGF-ß enhanced cell growth of CMT64 cells and promoted tumor formation in a syngeneic transplantation model. Of clinical importance, the expression of TGF-ß-induced genes identified in CMT64 cells correlated with EMT gene signatures in human lung adenocarcinoma tissue samples. Furthermore, TGF-ß-induced gene enrichment was related to poor prognosis, underscoring the tumor-promoting role of TGF-ß signaling in lung adenocarcinoma. Our cellular and syngeneic transplantation model would provide a simple and useful experimental tool to study the significance of TGF-ß signaling and EMT.

Targeting hepatocyte growth factor in epithelial-stromal interactions in an in vitro experimental model of human periodontitis.

Periodontitis is a chronic inflammatory disease leading to progressive connective tissue degradation and loss of the tooth-supporting bone. Clinical and experimental studies suggest that hepatocyte growth factor (HGF) is involved in the dysregulated fibroblast-epithelial cell interactions in periodontitis. The aim of this study was to explore effects of HGF to impact fibroblast-induced collagen degradation. A patient-derived experimental cell culture model of periodontitis was applied. Primary human epithelial cells and fibroblasts isolated from periodontitis-affected gingiva were co-cultured in a three-dimensional collagen gel. The effects of HGF neutralizing antibody on collagen gel degradation were tested and transcriptome analyses were performed. HGF neutralizing antibody attenuated collagen degradation and elicited expression changes of genes related to extracellular matrix (ECM) and cell adhesion, indicating that HGF signaling inhibition leads to extensive impact on cell-cell and cell-ECM interactions. Our study highlights a potential role of HGF in periodontitis. Antagonizing HGF signaling by a neutralizing antibody may represent a novel approach for periodontitis treatment.

Circulating tumor HPV DNA complements PET-CT in guiding management after radiotherapy in HPV-related squamous cell carcinoma of the head and neck.

Positron emission tomography and computed tomography (PET-CT) is widely used to assess the response to radiotherapy. However, the ability of PET-CT to predict treatment failure in human papillomavirus (HPV)-related squamous cell carcinoma of the head and neck (HNSCC) is unsatisfactory. We quantified circulating tumor HPV type16 DNA (ctHPV16DNA) using optimized droplet digital PCR in 35 patients with HPV16-related HNSCC, who received radiotherapy with or without chemotherapy, and prospectively correlated ctHPV16DNA and metabolic response with treatment failure. After a median follow-up of 21 months, ctHPV16DNA and PET-CT had similar negative predictive values (89.7% vs 84.0%), whereas the positive predictive value was much higher in ctHPV16DNA than in PET-CT (100% vs 50.0%). Notably, six patients who had detectable posttreatment ctHPV16DNA all had treatment failure irrespective of metabolic response, whereas none of five patients who had partial metabolic response without detectable posttreatment ctHPV16DNA had treatment failure. The risk of treatment failure was high in patients who had incomplete metabolic response with detectable posttreatment ctHPV16DNA (hazard ratio [HR], 138.8; 95% confidence interval [CI], 15.5-3366.4; P < .0001) and intermediate in patients who had discordant results between metabolic response and posttreatment ctHPV16DNA (HR, 4.7; 95% CI, 0.8-36.2, P = .09) as compared with patients who had complete metabolic response without detectable posttreatment ctHPV16DNA. One-year event-free survival rates of each risk group were 0%, 88% (95% CI, 46-98) and 95% (95% CI, 72-99), respectively (P < .0001). In conclusion, posttreatment ctHPV16DNA complements PET-CT and helps guide decisions managing patients with HPV16-related HNSCC after radiotherapy.

E74-Like Factor 3 Is a Key Regulator of Epithelial Integrity and Immune Response Genes in Biliary Tract Cancer.

The transcription factor E74-like factor 3 (ELF3) is inactivated in a range of cancers, including biliary tract cancer (BTC). Here, we investigated the tumor-suppressive role of ELF3 in bile duct cells by identifying several previously unknown direct target genes of ELF3 that appear to be implicated in biliary duct carcinogenesis. ELF3 directly repressed ZEB2, a key regulator of epithelial-mesenchymal transition, and upregulated the expression of CGN, an integral element in lumen formation. Loss of ELF3 led to decreased cell-cell junctions and enhanced cell motility. ALOX5 and CXCL16 were also identified as additional direct targets of ELF3; their corresponding proteins 5-lipoxygenase and CXCL16 play a role in the immune response. Conditioned medium from cells overexpressing ELF3 significantly enhanced the migration of natural killer cells and CD8+ T cells toward the conditioned medium. Gene expression profiling for BTC expressing high levels of ELF3 revealed significant enrichment of the ELF3-related genes. In a BTC xenograft model, activation of ELF3 increased expression of ELF3-related genes, enhanced the tubular structure of the tumors, and led to a loss of vimentin. Overall, our results indicate that ELF3 is a key regulator of both epithelial integrity and immune responses in BTC. SIGNIFICANCE: Thease finding shows that ELF3 regulates epithelial integrity and host immune responses and functions as a tumor suppressor in biliary tract cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/2/489/F1.large.jpg.

Integrated Single-Cell RNA-Sequencing Analysis of Aquaporin 5-Expressing Mouse Lung Epithelial Cells Identifies GPRC5A as a Novel Validated Type I Cell Surface Marker.

Molecular and functional characterization of alveolar epithelial type I (AT1) cells has been challenging due to difficulty in isolating sufficient numbers of viable cells. Here we performed single-cell RNA-sequencing (scRNA-seq) of tdTomato+ cells from lungs of AT1 cell-specific Aqp5-Cre-IRES-DsRed (ACID);R26tdTomato reporter mice. Following enzymatic digestion, CD31-CD45-E-cadherin+tdTomato+ cells were subjected to fluorescence-activated cell sorting (FACS) followed by scRNA-seq. Cell identity was confirmed by immunofluorescence using cell type-specific antibodies. After quality control, 92 cells were analyzed. Most cells expressed 'conventional' AT1 cell markers (Aqp5, Pdpn, Hopx, Ager), with heterogeneous expression within this population. The remaining cells expressed AT2, club, basal or ciliated cell markers. Integration with public datasets identified three robust AT1 cell- and lung-enriched genes, Ager, Rtkn2 and Gprc5a, that were conserved across species. GPRC5A co-localized with HOPX and was not expressed in AT2 or airway cells in mouse, rat and human lung. GPRC5A co-localized with AQP5 but not pro-SPC or CC10 in mouse lung epithelial cell cytospins. We enriched mouse AT1 cells to perform molecular phenotyping using scRNA-seq. Further characterization of putative AT1 cell-enriched genes revealed GPRC5A as a conserved AT1 cell surface marker that may be useful for AT1 cell isolation.