Novel gene fusions in human oropharyngeal carcinoma.

Few reports have analyzed the fusion genes involved in carcinogenesis in the oropharynx, where the incidence of human papillomavirus-associated tumors is relatively low. The aim of this study was to identify novel driver fusion genes in patients with oropharyngeal cancer. The study enrolled fifty-seven patients who were diagnosed with oropharyngeal carcinoma. RNA sequencing data from fresh-frozen specimens were used to identify candidate fusion genes via the JAFFA, arriba, and STAR-Fusion pipelines. Candidate fusion genes were confirmed by direct sequencing. The expression level of a candidate fusion gene was compared to that of tumors without fusion genes. Finally, filtering was performed for driver genes using the annoFuse pipeline. In addition, the VIRTUS pipeline was used to analyze the presence of human papillomavirus in the tumors. We identified 5 (8.8 %) novel potential driver in-frame fusion genes, MKNK2::MOB3A, ICMT::RPS6KA3, ATP1B3::GRK7, CSNK2A1::KIF16B, and FGFR3::MAEA, and 1 (1.8 %) known in-frame fusion gene, FGFR3::TACC3, in 57 patients with pharyngeal carcinoma. Our results suggest that sporadic fusion genes may contribute to tumorigenesis in oropharyngeal carcinomas.

Three-dimensional Liver Model Application for Liver Transplantation.

BACKGROUND: Children are removed from the liver transplant waitlist because of death or progressive illness. Size mismatch accounts for 30% of organ refusal. This study aimed to demonstrate that 3-dimensional (3D) technology is a feasible and accurate adjunct to organ allocation and living donor selection process. METHODS: This prospective multicenter study included pediatric liver transplant candidates and living donors from January 2020 to February 2023. Patient-specific, 3D-printed liver models were used for anatomic planning, real-time evaluation during organ procurement, and surgical navigation. The primary outcome was to determine model accuracy. The secondary outcome was to determine the impact of outcomes in living donor hepatectomy. Study groups were analyzed using propensity score matching with a retrospective cohort. RESULTS: Twenty-eight recipients were included. The median percentage error was -0.6% for 3D models and had the highest correlation to the actual liver explant (Pearson's R = 0.96, P < 0.001) compared with other volume calculation methods. Patient and graft survival were comparable. From 41 living donors, the median percentage error of the allograft was 12.4%. The donor-matched study group had lower central line utilization (21.4% versus 75%, P = 0.045), shorter length of stay (4 versus 7 d, P = 0.003), and lower mean comprehensive complication index (3 versus 21, P = 0.014). CONCLUSIONS: Three-dimensional volume is highly correlated with actual liver explant volume and may vary across different allografts for living donation. The addition of 3D-printed liver models during the transplant evaluation and organ procurement process is a feasible and safe adjunct to the perioperative decision-making process.

Utilization of Segmental Grafts Is Associated With Higher Transplant Rates in Pediatric Patients.

INTRODUCTION: In July 2017, a policy to increase the use of segmental grafts (SGs) was implemented at our institution. The aim was to compare changes in waitlist activity after implementation of this policy. METHODS: A single-center, retrospective study. Pediatric patients on the liver waiting list between January 2015 and December 2019 were screened. Patients were classified as receiving a liver transplant (LT) before (Period 1) or after (Period 2) policy changes. Primary end points were transplant rates and time to transplant. RESULTS: Sixty five first LT performed on 65 patients were included. Twenty nine LT were performed during Period 1 and 36 during Period 2. More than half (55%) of LT in Period 2 were SG, compared to 10.3% in Period 1 (P < 0.001). Forty nine and 56 pediatric candidates on the waiting list accounted for 38.78 and 24.48 person-years during Period 1 and Period 2, respectively. Transplant rates per 100 person-years on the waiting list increased from 85.09 during Period 1 to 187.87 in Period 2 (Rate ratio: 2.20; P < 0.001). Median time to receive a LT decreased from 229 d in Period 1 to 75 d during Period 2 (P = 0.013). One-year patient survival rates were 96.6% in Period 1 and 95.7% in Period 2. One-year graft survival rates were 89.7% and 88% in Period 1 and Period 2, respectively. CONCLUSIONS: A policy to increase the use of SG was associated with significantly higher transplant rates and lower waiting times. Implementation of this policy can be done successfully with no observed negative impact on patient and graft survival.

Mucous Gland Adenoma of the Lung: A Neoplastic Counterpart of Mucinous Bronchial Glands.

Mucous gland adenoma (MGA) is a rare benign tumor that usually arises in the proximal airway and consists of mucus-secreting cells resembling bronchial glands. Here, we report 2 cases of MGAs and describe their morphologic, immunohistochemical, and molecular profiles in comparison with 19 pulmonary tumors of 5 other histologic types with mucinous cells (invasive mucinous adenocarcinoma, mucoepidermoid carcinoma, mixed squamous cell and glandular papilloma, bronchiolar adenoma/ciliated muconodular papillary tumor, and sialadenoma papilliferum). Two MGAs were found in 1 male patient and 1 female patient, located in the bronchus and trachea, respectively. One MGA was examined by RNA sequencing, and no putative driver mutations (including BRAF, KRAS, and AKT1 mutations) or gene fusions were identified. In another case of MGA, V600E mutations of BRAF and E17K mutations of AKT1 were not detected by allele-specific real-time PCR or digital PCR, respectively. However, a gene expression analysis revealed that the MGA presented a specific RNA expression profile with multiple genes enriched in the salivary gland. The gene expression of NKX3.1 was significantly higher in the MGA case in comparison to normal control lungs (P < .001). We then examined NKX3.1 immunohistochemistry for 2 MGAs and 19 tumors of 5 other histologic types. NKX3.1 was positive in MGA (2/2, 100%), whereas all constituent cells, including mucinous cells, were negative for NKX3.1 in other histologic types (0%, 0/19). In normal lung tissue, NKX3.1 was positive for mucinous acinar cells of the bronchial glands. In conclusion, the gene expression profile, taken together with the histologic similarity between MGA and bronchial glands, and the preferred location of the tumors (proximal airways with submucosal glands) suggest that MGA is a neoplastic counterpart of mucinous bronchial glands. NKX3.1 immunohistochemistry can be a sensitive and specific ancillary marker that distinguishes MGA from other histologic mimics.

Performance of a Prospective Anticoagulation Stratification Algorithm After Liver Transplantation.

Venous thromboembolism (VTE) occurs in 0.4% to 15.5% and bleeding occurs in 20% to 35% of patients after liver transplantation (LT). Balancing the risk of bleeding from therapeutic anticoagulation and risk of thrombosis in the postoperative period is challenging. Little evidence exists regarding the best treatment strategy for these patients. We hypothesized that a subset of LT patients who develop postoperative deep vein thromboses (DVTs) could be managed without therapeutic anticoagulation. We implemented a quality improvement (QI) initiative using a standardized Doppler ultrasound-based VTE risk stratification algorithm to guide parsimonious implementation of therapeutic anticoagulation with heparin drip. Methods: In a prospective management QI initiative for DVT management, we compared 87 LT historical patients (control group; January 2016-December 2017) to 182 LT patients (study group; January 2018-March 2021). We analyzed the rates of immediate therapeutic anticoagulation after DVT diagnosis within 14 d of LT, clinically significant bleeding, return to the operating room, readmission, pulmonary embolism, and death within 30 d of LT before and after the QI initiative. Results: Ten patients (11.5%) in the control group and 23 patients (12.6%; P = 0.9) in the study group developed DVTs after LT. Immediate therapeutic anticoagulation was used in 7 of 10 and 5 of 23 patients in the control and study groups, respectively (P = 0.024). The study group had lower odds of receiving immediate therapeutic anticoagulation after VTE (21.7% versus 70%; odds ratio = 0.12; 95% confidence interval, 0.019-0.587; P = 0.013) and a lower rate of postoperative bleeding (8.7% versus 40%; odds ratio = 0.14, 95% confidence interval, 0.02-0.91; P = 0.048). All other outcomes were similar. Conclusions: Implementing a risk-stratified VTE treatment algorithm for immediate post-LT patients appears to be safe and feasible. We observed a decrease in the use of therapeutic anticoagulation and a lower rate of postoperative bleeding without adverse impacts on early outcomes.

Biological Difference between L858R and Exon 19 Deletion Contributes to Recurrence-Free Survival of Resected Non-Small Cell Lung Cancer.

INTRODUCTION: The differences in biological characteristics among different genotypes of classical EGFR mutations have not been clarified. This study aimed to clarify the clinical and biological differences between L858R and 19 deletion in NSCLC. METHODS: We analyzed a cohort of 191 consecutive cases of surgically resected NSCLC harboring EGFR driver mutations (L858R or 19 deletion) in which curative resection was performed in Aichi Cancer Center Hospital, Nagoya, Japan, from January 2006 to September 2021 and in which recurrence subsequently developed. We also subjected 61 surgically resected NSCLC specimens harboring EGFR driver mutations (L858R or 19 deletion) to an RNA sequencing analysis. RESULTS: In patients with stage I disease, the median time to recurrence did not differ to a statistically significant extent between the types of EGFR mutations; however, among those with stage II and III disease, the median time to recurrence in patients with the L858R genotype tended to be shorter in comparison to those with 19 deletion (log-rank test, p = 0.47 and 0.46, respectively). In comparison to 19 deletion tumors, L858R tumors had higher cytological malignancy (e.g., mitotic ability) and showed stronger immunogenicity. CONCLUSION: L858R and 19 deletion tumors are likely to have a slight difference in the time to recurrence. They suggest that even in EGFR driver tumors, which are treated as the same disease category, the biological characteristics of the tumors are different, which may leave room for innovations in postoperative treatment and treatment at recurrence.

Association of the KRAS genotype and clinicopathologic findings of resected non-small-cell lung cancer: A pooled analysis of 179 patients.

BACKGROUND: This study assessed the clinicopathological background of early-stage KRAS-mutated non-small-cell lung cancer and analyzed the biological process of KRAS-mutated tumor using an RNA sequencing procedure. PATIENTS AND METHODS: We used a cohort of consecutive series of 179 surgically resected early-stage non-small-cell lung cancers harboring KRAS mutations and analyzed the clinicopathological features, including the KRAS genotypes, affecting the recurrence-free survival and prognosis. Consequently, we performed RNA sequencing to determine the gene expression profiles of nineteen KRAS-mutated non-small-cell cancers. RESULTS: The most common KRAS genotype was p.G12C (57; 31.8%). A high p-stage (hazard ratio [HR], 4.181; P < 0.0001) and solid predominant adenocarcinoma histology (HR, 2.343; P = 0.0076) were significant independent prognostic factors for the recurrence-free survival. A high p-stage (HR, 3.793; P < 0.0001), solid predominant adenocarcinoma histology (HR, 2.373; P = 0.0147), and KRAS p.G12V genotype (HR, 1.975; P = 0.0407) were significant independent prognostic factors for the overall survival. A gene expression analysis of the two factors revealed the p.G12V genotype to be closer to those of stem cells, and the traits of e an enhanced fatty acid and amino acid metabolism. as well as And a solid predominant phenotype were shown to an acquired a trait that can withstand hypoxia and the effect of prostaglandin-endoperoxide synthase. CONCLUSION: The KRAS p.G12V genotype and solid predominant adenocarcinoma phenotype may be independent predictive factors of a poor clinical course in resected early-stage non-small-cell lung cancers, possibly due to the differentiation tendency observed in stem cells, the trait of an enhanced fatty acid and amino acid metabolism, and the effect of prostaglandin-endoperoxide synthase.

Sensitivity to dabrafenib and trametinib treatments in patients with non-small-cell cancer harboring BRAF compound mutations: A pooled analysis of BRAF p.V600E-positive advanced non-small-cell lung cancer.

PURPOSE: The present study clarified the sensitivity of the BRAF tyrosine kinase inhibitor mechanism in patients with BRAF compound mutation and predicted the sensitivity using molecular dynamics simulation. METHODS: We examined 16 BRAF tumors with p.V600E-positive non-small-cell lung cancer. RESULTS: One patient (6.2%) had a BRAF p.V600E and p.K601_W604 compound mutation with a good clinical response to dabrafenib and trametinib. Molecular dynamics simulation also complemented the effect. CONCLUSIONS: The combination of a genetic analysis and computational simulation model may help predict the sensitivity for dabrafenib in cases with a rare BRAF compound mutation. The construction of a genomic and simulation fused database is important for the development of personalized medicine in this field.

Comparison between Fluorimetry (Qubit) and Spectrophotometry (NanoDrop) in the Quantification of DNA and RNA Extracted from Frozen and FFPE Tissues from Lung Cancer Patients: A Real-World Use of Genomic Tests.

Background and Objectives: Panel-based next-generation sequencing (NGS) has been carried out in daily clinical settings for the diagnosis and treatment guidance of patients with non-small cell lung cancer (NSCLC). The success of genomic tests including NGS depends in large part on preparing better-quality DNA or RNA; however, there are no established operating methods for preparing genomic DNA and RNA samples. Materials and Methods: We compared the following two quantitative methods, the QubitTM and NanoDropTM, using 585 surgical specimens, 278 biopsy specimens, and 82 cell block specimens of lung cancer that were used for genetic tests, including NGS. We analyzed the success rate of the genomic tests, including NGS, which were performed with DNA and RNA with concentrations that were outliers for the Qubit Fluorometer. Results: The absolute value for DNA concentrations had a tendency to be higher when measured with NanoDropTM regardless of the type of specimen; however, this was not the case for RNA. The success rate of DNA-based genomic tests using specimens with a concentration below the lower limit of QubitTM detection was as high as approximately 96%. At less than 60%, the success rate of RNA-based genomic tests, including RT-PCR, was not as satisfactory. The success rates of the AmpliSeqTM DNA panel sequencing and RNA panel sequencing were 77.8% and 91.5%, respectively. If at least one PCR amplification product could be obtained, then all RNA-based sequencing was performed successfully. Conclusions: The concentration measurements with NanoDropTM are reliable. The success rate of NGS with samples at concentrations below the limit of detection of QubitTM was relatively higher than expected, and it is worth performing PCR-based panel sequencing, especially in cases where re-biopsy cannot be performed.

Positive Correlation Between the Number of Circulating Tumor Cells in the Pulmonary Vein and Tumor Spread Through Air Spaces in Resected Non-small Cell Lung Cancer.

BACKGROUND/AIM: Circulating tumor cells (CTCs) is one of the promising markers that predict dissemination and metastases. This study aimed to identify the relationship between CTCs in pulmonary vein (PuV) and spread through air space (STAS) in non-small cell lung cancers. MATERIALS AND METHODS: We applied a cytology-based microfluidic platform for rare cell isolation. Twenty-four patients were enrolled. RESULTS: The rate of CTC detection in PuV was 79.2%, and STAS was observed in 54.2% of the samples. When the definitive cut-off value was 1 CTC/1 ml, of the 14 CTC-PuV-high cases, 11 (78.6%) were STAS-positive, whereas 2 of the 10 (20.0%) CTC-PuV-low cases were STAS-positive, and the difference between the two groups was statistically significant (p=0.02). CTC-PuV-high exhibited a significantly poorer survival (p<0.01). CONCLUSION: The higher frequency of STAS is significantly associated with a higher number of CTCs in PuV, and the combination of STAS and CTC was significantly associated with poor prognosis.

Novel NR4A1 Arg293Ser Mutation in Patients With Familial Crohn's Disease.

BACKGROUND/AIM: The underlying etiology of Crohn's disease remains unknown. The aim of this study was to identify genomic alterations associated with the development of Crohn's disease in one Japanese family with a family history of Crohn's disease. MATERIALS AND METHODS: We performed whole-exome sequence and pedigree analysis of a Japanese family in which both sisters developed Crohn's disease. Whole-exome sequencing was performed using the Ion Torrent Proton™ system. Data from the Proton runs were initially processed using the Ion Torrent platform-specific pipeline software Ion Reporter. An autosomal dominant mode of inheritance was assumed, and stringent selection criteria were applied. RESULTS: A substitution in the NR4A1 gene at codon 293 resulting in an amino acid change from arginine to serine was identified only in the affected sisters. CONCLUSION: The impaired DNA-binding capacity of the NR4A1 protein due to an NR4A1 germline mutation may be a possible cause of Crohn's disease.

Alteration of DNA mismatch repair capacity underlying the co-occurrence of non-small-cell lung cancer and nonmedullary thyroid cancer.

Both non-small-cell lung cancer cases in never-smokers and nonmedullary thyroid cancer cases have been increasing in developed countries. Some studies have shown an excess of co-occurrence of non-small-cell lung cancer and nonmedullary thyroid cancer. We aimed to clarify the underlying genetic factors that contribute to the occurrence of these two malignancies. We performed germline exome sequencing in a cohort of 9 patients with the two malignancies. In terms of candidate genes, we performed target resequencing, immunohistochemistry, and microsatellite instability testing on another cohort. Two rare missense heterozygous variants in MSH6 were identified and verified by Sanger sequencing. One available tumour specimen showed heterogeneous MSH6 status in immunohistochemistry. Further exploration with different cohorts (a total of 8 patients with the two malignancies) demonstrated that 2 out of 8 patients had a germline missense or promotor variant of MLH1 and four out of 10 tumour specimens revealed heterogeneous immunohistochemistry staining in any of the four mismatch repair proteins: MLH1, PMS2, MSH2 and MSH6. Although our cohort showed a different disease profile than Lynch syndrome, this study suggests causal roles of impaired DNA mismatch repair capacity in non-small-cell lung cancer and nonmedullary thyroid cancer.

Weak-evidence Fusion Candidates Detected by a FusionPlex Assay Using the Ion Torrent System.

BACKGROUND/AIM: The Archer FusionPlex platform is widely used for comprehensive fusion-gene detection in cancer tissues. This platform separately displays results for strong-evidence and weak-evidence fusion candidates (WEFCs). Distinctive fusion patterns are frequently found in the weak-evidence category and information about the patterns is clinically essential. PATIENTS AND METHODS: We describe the type and frequency of WEFCs observed using the FusionPlex Sarcoma Panel (S Panel) and the FusionPlex ALK, RET, and ROS1 ver2 Panel (ARR Panel). RESULTS: A total of 97 specimens were examined and 620 candidates were detected and categorized as WEFCs. A median of five WEFCs were detected per sample. In the S Panel group, there were 13 WEFCs with a frequency of more than 1%. In the ARR Panel group, a total of 16 WEFCs were detected with a frequency of more than 1%. CONCLUSION: Specific WEFCs were detected according to the panel selected.

AKT1 Mutations in Peripheral Bronchiolar Papilloma: Glandular Papilloma and Mixed Squamous Cell and Glandular Papilloma Is Distinct From Bronchiolar Adenoma.

Glandular papilloma (GP) and mixed squamous cell and glandular papilloma (MP) are rare benign pulmonary tumors occurring in the bronchi. Bronchiolar adenoma (BA) was recently characterized as a pulmonary tumor exhibiting alveolar spread. Both GP/MP and BA are composed of a mixture of glandular, ciliated, squamous, and basal cells. We aimed to clarify whether GP/MP and BA represent the same tumor. We evaluated the detailed histologic characteristics of 11 cases involving pulmonary peripheral tumors that exhibited histologic features of GP/MP or BA, and performed genetic analyses using targeted panel sequencing, allele-specific polymerase chain reaction, and digital polymerase chain reaction. Histologically, 4 and 7 tumors were classified as GP/MP and BA, respectively. GP/MP showed endobronchiolar papillary growth with a pseudostratified or stratified epithelium. In contrast, 5 BAs showed a predominant flat structure with a bilayered or pseudostratified epithelium, whereas 2 BAs showed a GP/MP-like papillary architecture. The mean epithelial thickness in each tumor was significantly larger in GP/MPs and BAs with a GP/MP-like morphology (103 to 242 µm) than in flat-predominant BA (23 to 47 µm, P=0.0010). AKT1 E17K mutations were detected in all GP/MPs and BAs with GP/MP-like morphology but were absent in the 5 flat-predominant BAs. AKT1 mutations were always concurrent with BRAF or HRAS mutations, and the variant allele frequency or percentage of mutant copies of AKT1 mutations was equal to those of BRAF or HRAS mutations. GP/MPs are characterized by AKT1 mutations concurrent with BRAF or HRAS mutations. Peribronchiolar papillary tumors with AKT1 mutations may also be classified as GP/MP.

Novel Resistance Mechanisms Including L1196Q, P1094H, and R1248_D1249 Insertion in Three Patients With NSCLC After ALK Tyrosine Kinase Inhibitor Treatment.

OBJECTIVES: The purposes of this study are to clarify the details of the ALK tyrosine kinase inhibitor (TKI) resistance mechanism in rebiopsy cases and to predict novel resistance gene alterations using molecular dynamics simulation. METHODS: A total of 21 patients with ALK-positive NSCLC who underwent a rebiopsy after ALK TKI failure were included in this analysis. ALK fluorescence in situ hybridization and reverse transcription polymerase chain reaction were performed with paired initial and rebiopsy tumor specimens. RESULTS: Nine patients had no known ALK resistance mechanisms. Four had ALK amplification. L1196M, I1171N, and G1269A, mutations that are known to indicate resistance to ALK TKIs, were detected in one patient each. Small cell carcinoma and sarcomatoid transition were found in one case each. L1196Q, P1094H, and exon 24 76-base pair insertion were detected after the second-generation ALK TKIs. CONCLUSIONS: The combination of a genetic analysis and a computational simulation model may make a prediction of resistance mechanisms for overcoming ALK TKI resistance, and the construction of a genomic and simulation fused database is important for the development of personalized medicine in this field.

Efficacy of local therapy for oligoprogressive disease after programmed cell death 1 blockade in advanced non-small cell lung cancer.

Immune checkpoint inhibitors (ICIs) have dramatically changed the strategy used to treat patients with non-small-cell lung cancer (NSCLC); however, the vast majority of patients eventually develop progressive disease (PD) and acquire resistance to ICIs. Some patients experience oligoprogressive disease. Few retrospective studies have evaluated clinical efficacy in patients with oligometastatic progression who received local therapy after ICI treatment. We conducted a retrospective analysis of advanced NSCLC patients who received PD-1 inhibitor monotherapy with nivolumab or pembrolizumab to evaluate the effects of ICIs on the patterns of progression and the efficacy of local therapy for oligoprogressive disease. Of the 307 patients treated with ICIs, 148 were evaluated in our study; 42 were treated with pembrolizumab, and 106 were treated with nivolumab. Thirty-eight patients showed oligoprogression. Male sex, a lack of driver mutations, and smoking history were significantly correlated with the risk of oligoprogression. Primary lesions were most frequently detected at oligoprogression sites (15 patients), and 6 patients experienced abdominal lymph node (LN) oligoprogression. Four patients showed evidence of new abdominal LN oligometastases. There was no significant difference in overall survival (OS) between the local therapy group and the switch therapy group (reached vs. not reached, P = .456). We summarized clinical data on the response of oligoprogressive NSCLC to ICI therapy. The results may help to elucidate the causes of ICI resistance and indicate that the use of local therapy as the initial treatment in this setting is feasible treatment option.

Targeted RNA sequencing with touch imprint cytology samples for non-small cell lung cancer patients.

BACKGROUND: RNA-based sequencing is considered ideal for detecting pathogenic fusion-genes compared to DNA-based assays and provides valuable information about the relative expression of driver genes. However, RNA from formalin-fixed paraffin-embedded tissue has issues with both quantity and quality, making RNA-based sequencing difficult in clinical practice. Analyzing stamp-derived RNA with next-generation sequencing (NGS) can address the above-mentioned obstacles. In this study, we validated the analytical specifications and clinical performance of our custom panel for RNA-based assays on the Ion Torrent platform. METHODS: To evaluate our custom RNA lung panel, we first examined the gene sequences of RNA derived from 35 NSCLC tissues with diverse backgrounds by conventional methods and NGS. Next, we moved to the clinical phase, where clinical samples (all stamp-derived RNA) were used to examine variants. In the clinical phase we conducted an NGS analysis while simultaneously applying conventional approaches to assess the feasibility and validity of the panel in clinical practice. RESULTS: In the prerun phase, all of the variants confirmed with conventional methods were detected by NGS. In the clinical phase, a total of 80 patients were enrolled and 80 tumor specimens were sequenced from February 2018 to December 2018. There were 66 cases in which the RNA concentration was too low to be measured, but sequencing was successful in the vast majority of cases. The concordance between NGS and conventional methods was 95.0%. CONCLUSIONS: RNA extraction using stamp specimens and panel sequencing by NGS were considered applicable in clinical settings. KEY POINTS: Significant findings of the study Next-generation sequencing using RNA from stamp specimens was able to detect driver gene changes in non-small cell lung cancer including fusion genes with the same accuracy as conventional methods. What this study adds Using RNA from stamp specimens obtained from biopsy increases the number of candidate cases for RNA sequencing in clinical settings.