Genomic landscape of glioblastoma without IDH somatic mutation in 42 cases: a comprehensive analysis using RNA sequencing data.

PURPOSE: Glioblastoma is a malignant brain tumor with a poor prognosis. Genetic mutations associated with this disease are complex are not fully understood and require further elucidation for the development of new treatments. The purpose of this study was to comprehensively analyze genetic mutations in glioblastomas and evaluate the usefulness of RNA sequencing. PATIENTS AND METHODS: We analyzed 42 glioblastoma specimens that were resected in routine clinical practice and found wild-type variants of the IDH1 and IDH2 genes. RNA was extracted from frozen specimens and sequenced, and genetic analyses were performed using the CLC Genomics Workbench. RESULTS: The most common genetic alterations in the 42 glioblastoma specimens were TP53 mutation (28.6%), EGFR splicing variant (16.7%), EGFR mutation (9.5%), and FGFR3 fusion (9.5%). Novel genetic mutations were detected in 8 patients (19%). In 12 cases (28.6%), driver gene mutations were not detected, suggesting an association with PPP1R14A overexpression. Our findings suggest the transcription factors SOX10 and NKX6-2 are potential markers in glioblastoma. CONCLUSION: RNA sequencing is a promising approach for genotyping glioblastomas because it provides comprehensive information on gene expression and is relatively cost-effective.

Current diagnosis and treatment of salivary gland-type tumors of the lung.

Salivary gland-type tumors of the lung are thought to originate from the submucosal exocrine glands of the large airways. Due to their rare occurrence, reports of their study are limited to small-scale or case reports. Therefore, daily clinical practices often require a search for previous reports. In the last 20 years, several genetic rearrangements have been identified, such as MYB::NF1B rearrangements in adenoid cystic carcinoma, CRTC1::MAML2 rearrangements in mucoepidermoid carcinoma, EWSR1::ATF1 rearrangements in hyalinizing clear cell carcinoma and rearrangements of the EWSR1 locus or FUS (TLS) locus in myoepithelioma and myoepithelial carcinoma. These molecular alterations have been useful in diagnosing these tumors, although they have not yet been linked to molecularly targeted therapies. The morphologic, immunophenotypic, and molecular characteristics of these tumors are similar to those of their counterparts of extrapulmonary origin, so clinical and radiologic differential diagnosis is required to distinguish between primary and metastatic disease of other primary sites. However, these molecular alterations can be useful in differentiating them from other primary lung cancer histologic types. The management of these tumors requires broad knowledge of the latest diagnostics, surgery, radiotherapy, bronchoscopic interventions, chemotherapy, immunotherapy as well as therapeutic agents in development, including molecularly targeted agents. This review provides a comprehensive overview of the current diagnosis and treatment of pulmonary salivary gland tumors, with a focus on adenoid cystic carcinoma and mucoepidermoid carcinoma, which are the two most common subtypes.

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.

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.

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.

Permissible Outcomes of Lobe-Specific Lymph Node Dissection for Elevated Carcinoembryonic Antigen in Non-Small Cell Lung Cancer.

Background and Objectives: Lobe-specific nodal dissection (L-SND) is currently acceptable for the dissection of early-stage non-small cell lung cancer (NSCLC) but not for cancers of more advanced clinical stages. We aimed to assess the efficacy of L-SND, compared to systemic nodal dissection (SND). Materials and Methods: We retrospectively collected the clinical data of patients with carcinoembryonic antigen (CEA) abnormality who underwent complete resection of NSCLC via lobectomy or more in addition to either SND or L-SND at two cancer-specific institutions from January 2006 to December 2017. Results: A total of 799 patients, including 265 patients who underwent SND and 534 patients who underwent L-SND, were included. On multivariate analysis, thoracotomy, more than lobectomy, cN1-2, advanced pathological stage, adjuvant treatment, and EGFR or ALK were strongly associated with SND. No significant differences were found in overall survival, disease-free survival, and overtime survival after propensity adjustment (p = 0.09, p = 0.11, and p = 0.50, respectively). There were no significant differences in local (p = 0.16), regional (p = 0.72), or distant (p = 0.39) tumor recurrence between the two groups. Conclusions: SND did not improve the prognosis of NSCLC patients with CEA abnormality. Complete pulmonary resection via L-SND seems useful for NSCLC patients with CEA abnormality.

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.

Negative reactions of BRAF mutation-specific immunohistochemistry to non-V600E mutations of BRAF.

BRAF mutations are rare driver mutations in non-small cell lung cancer (NSCLC), accounting for 1%-2% of the driver mutations, and the mutation spectrum has a wide range in contrast to other tumors. While V600E is a dominant mutation in melanoma, more than half of the mutations in NSCLCs are non-V600E. However, treatment with dabrafenib plus trametinib targets the BRAF V600E mutation exclusively. Therefore, distinguishing between V600E and non-V600E mutations is crucial for biomarker testing in NSCLC in order to determine treatment of choice. Immunohistochemistry (IHC) using the BRAF V600E mutation-specific antibody is clinically used in melanoma patients, but little is known about its application in NSCLC, particularly with regard to the assay performance for non-V600E mutations. In the present study, we examined 117 tumors with BRAF mutations, including 30 with non-V600E mutations, using BRAF mutation-specific IHC. None of the tumors with non-V600E mutations, including two compound mutations, showed a positive reaction. Furthermore, all V600E mutations were positive except for one case with combined BRAF V600E and K601_W604 deletion. Our findings confirmed that the BRAF V600E mutation-specific IHC is specific without any cross-reactions to non-V600E mutations, suggesting that this assay can be a useful screening tool in clinical practice.

Relationship between Paronychia and Drug Concentrations of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors.

PURPOSE: The purpose of the study was to evaluate the site of paronychia in patients with non-small cell lung cancer harboring an epidermal growth factor receptor (EGFR) gene activating mutation who were treated with EGFR tyrosine kinase inhibitors (EGFR TKIs). PATIENTS AND METHODS: The study included 55 patients with non-small-cell lung cancer who were treated with an EGFR TKIs. Resulting all toxicities were graded using the Common Terminology Criteria for Adverse Events version 4.0 system. Drug concentrations were determined with use of a quantum triple-quadrupole mass spectrometer and dried blood spots testing. RESULTS: Paronychia most commonly occurred in the thumb and the big toe. There was no correlation between the severity of paronychia and the drug concentration of each EGFR TKI at the site of paronychia. The mean penetration rates of the drug from plasma to the tip of the finger and toe were 74.1% (erlotinib), 82.2% (gefitinib), and 99.9% (afatinib). CONCLUSIONS: High concentrations of an EGFR TKI at the affected site did not play a role in the onset mechanism of paronychia. Therefore, educating patients about ways to avoid compression may be a better approach to managing this adverse event than reducing the dose of the EGFR-TKI or stopping treatment.

Validation of the digital PCR system in tyrosine kinase inhibitor-resistant EGFR mutant non-small-cell lung cancer.

The aim of this study was to compare the accuracy of the QuantStudio 3D Digital polymerase chain reaction (dPCR) system and a PCR-based next generation sequencing (NGS) system for detecting a secondary mutation in the epidermal growth factor receptor (EGFR) gene T790M in non-small cell lung cancer (NSCLC) patients previously diagnosed with EGFR-activating mutations. Twenty-five patients with NSCLC previously treated with EGFR-TKIs were examined. The patients were treated daily with either erlotinib or gefitinib. New biopsies, followed by DNA sequencing on an Ion Torrent systems using the Ion Torrent AmpliSeq Cancer Hotspot Panel and dPCR were performed. A comparison of NGS, sensitive PCR, and dPCR revealed that the sensitivities of NGS and dPCR were similar in this study. As well, T790M was detected in as low as about 5% of mutant allelic frequencies, which represented 5% of the total reads on site mapped reads in NGS and greater than 5% of the dPCR reads, which represented mutant and wild type copies. The strategy in which NGS sequencing is followed by revealed acquired mutation with dPCR may be a reasonable one. We demonstrated the utility of combining NGS and dPCR as a tool for monitoring T790M. NGS and dPCR with formalin-fixed paraffin-embedded (FFPE) specimens might become a standard genomic test for exploring acquired resistance to targeted molecular medicines.

Next-generation sequencing of tyrosine kinase inhibitor-resistant non-small-cell lung cancers in patients harboring epidermal growth factor-activating mutations.

BACKGROUND: The aim of this study was to detect the epidermal growth factor receptor (EGFR)-activating mutations and other oncogene alterations in patients with non-small-cell lung cancers (NSCLC) who experienced a treatment failure in response to EGFR-tyrosine kinase inhibitors (TKIs) with a next generation sequencer. METHODS: Fifteen patients with advanced NSCLC previously treated with EGFR-TKIs were examined between August 2005 and October 2014. For each case, new biopsies were performed, followed by DNA sequencing on an Ion Torrent Personal Genome Machine (PGM) system using the Ion AmpliSeq Cancer Hotspot Panel version 2. RESULTS: All 15 patients were diagnosed with NSCLC harboring EGFR-activating mutations (seven cases of exon 19 deletion, seven cases of L858R in exon 21, and one case of L861Q in exon 21). Of the 15 cases, acquired T790M resistance mutations were detected in 9 (60.0%) patients. In addition, other mutations were identified outside of EGFR, including 13 cases (86.7%) exhibiting TP53 P72R mutations, 5 cases (33.3%) of KDR Q472H, and 2 cases (13.3%) of KIT M541L. CONCLUSIONS: Here, we showed that next-generation sequencing (NGS) is able to detect EGFR T790M mutations in cases not readily diagnosed by other conventional methods. Significant differences in the degree of EGFR T790M and other EGFR-activating mutations may be indicative of the heterogeneity of disease phenotype evident within these patients. The co-existence of known oncogenic mutations within each of these patients may play a role in acquired EGFR-TKIs resistance, suggesting the need for alternative treatment strategies, with PCR-based NGS playing an important role in disease diagnosis.

CT-guided fine-needle aspiration and core needle biopsies of pulmonary lesions: a single-center experience with 750 biopsies in Japan.

OBJECTIVE: CT-guided lung biopsy is a well-established diagnostic method for pulmonary lesions. The aim of our study was to evaluate the diagnostic outcomes and safety profile of conventional CT-guided lung biopsies. MATERIALS AND METHODS: We retrospectively analyzed the results of CT-guided lung biopsies for 750 patients to determine the diagnostic accuracy, complication rates, and independent risk factors for diagnostic failure and severe pneumothorax. RESULTS: Diagnostic accuracy was 92.9%. Independent risk factors for diagnostic failure were malignant lesions (odds ratio [OR], 4.20; 95% CI, 1.66-14.1; p = 0.001), lesions in the lower lobe (OR, 2.01; 95% CI, 1.17-3.47; p = 0.011), lesions 2.0 cm or smaller (OR, 2.87; 95% CI, 1.59-5.48; p < 0.001), and the presence of pneumothorax during the procedure (OR, 2.18; 95% CI, 1.27-3.78; p = 0.004). Pneumothorax requiring drainage occurred in 7% of patients. Independent risk factors for pneumothorax requiring drainage were age of 73 years or older (OR, 2.19; 95% CI, 1.21-4.05; p = 0.009), the presence of emphysema (OR, 4.29; 95% CI, 2.05-8.82; p < 0.001), benign lesions (OR, 2.33; 95% CI, 1.20-4.40; p = 0.012), supine positioning of the patient (OR, 2.61; 95% CI, 1.44-4.84; p = 0.001), and length from the pleura to the lesion of 1.5 cm or greater (OR, 3.08; 95% CI, 1.63-6.17; p < 0.001). CONCLUSION: CT-guided lung biopsy has a high diagnostic accuracy. Complication rates were acceptable and comparable to those of previous studies.

Caspase-4 promotes metastasis and interferon-γ-induced pyroptosis in lung adenocarcinoma.

Caspase-4 (CASP4) is a member of the inflammatory caspase subfamily and promotes inflammation. Here, we report that CASP4 in lung adenocarcinoma cells contributes to both tumor progression via angiogenesis and tumor hyperkinesis and tumor cell killing in response to high interferon (IFN)-γ levels. We observe that elevated CASP4 expression in the primary tumor is associated with cancer progression in patients with lung adenocarcinoma. Further, CASP4 knockout attenuates tumor angiogenesis and metastasis in subcutaneous tumor mouse models. CASP4 enhances the expression of genes associated with angiogenesis and cell migration in lung adenocarcinoma cell lines through nuclear factor kappa-light chain-enhancer of activated B cell signaling without stimulation by lipopolysaccharide or tumor necrosis factor. CASP4 is induced by endoplasmic reticulum stress or IFN-γ via signal transducer and activator of transcription 1. Most notably, lung adenocarcinoma cells with high CASP4 expression are more prone to IFN-γ-induced pyroptosis than those with low CASP4 expression. Our findings indicate that the CASP4 level in primary lung adenocarcinoma can predict metastasis and responsiveness to high-dose IFN-γ therapy due to cancer cell pyroptosis.

Candidate tumor-specific CD8+ T cell subsets identified in the malignant pleural effusion of advanced lung cancer patients by single-cell analysis.

Isolation of tumor-specific T cells and their antigen receptors (TCRs) from malignant pleural effusions (MPE) may facilitate the development of TCR-transduced adoptive cellular immunotherapy products for advanced lung cancer patients. However, the characteristics and markers of tumor-specific T-cells in MPE are largely undefined. To this end, to establish the phenotypes and antigen specificities of CD8+ T cells, we performed single-cell RNA and TCR sequencing of samples from three advanced lung cancer patients. Dimensionality reduction on a total of 4,983 CD8+ T cells revealed 10 clusters including naïve, memory, and exhausted phenotypes. We focused particularly on exhausted T cell clusters and tested their TCR reactivity against neoantigens predicted from autologous cancer cell lines. Four different TCRs specific for the same neoantigen and one orphan TCR specific for the autologous cell line were identified from one of the patients. Differential gene expression analysis in tumor-specific T cells relative to the other T cells identified CXCL13, as a candidate gene expressed by tumor-specific T cells. In addition to expressing CXCL13, tumor-specific T cells were present in a higher proportion of T cells co-expressing PDCD1(PD-1)/TNFRSF9(4-1BB). Furthermore, flow cytometric analyses in advanced lung cancer patients with MPE documented that those with high PD-1/4-1BB expression have a better prognosis in the subset of 57 adenocarcinoma patients (p = .039). These data suggest that PD-1/4-1BB co-expression might identify tumor-specific CD8+ T cells in MPE, which are associated with patients' prognosis. (233 words).

Effect of the BCL2 gene polymorphism on survival in advanced-stage non-small cell lung cancer patients who received chemotherapy.

INTRODUCTION: This study aimed to evaluate the association between BCL2 single-nucleotide polymorphisms and survival outcome in advanced non-small cell lung cancer (NSCLC). METHODS: One hundred and sixty-eight patients with advanced NSCLC who were treated with anti-cancer drugs and could be evaluated for therapeutic response between April 2005 and March 2010 at Kyoto University Hospital were enrolled. DNA was extracted from peripheral blood samples. The BCL2 polymorphisms -938 C→A (rs2279115) and +21 A→G (rs1801018) were genotyped using the 5'-nuclease assay. The univariate relationship between each independent clinicopathologic variable and BCL2 genotype was examined using Fisher's exact test. To evaluate risk factors associated with prognosis, a Cox proportional hazards regression model with a step-down procedure was used. RESULTS: The median survival time of patients with the -938 AA and AC genotypes were significantly shorter than those with the -938 CC genotype (p = 0.027 by log-rank test). Based on multivariate analysis, poor performance status [hazard ratio (HR) 2.424, 95% confidence interval (CI) 1.727-3.262; p < 0.0001], non-adenocarcinoma histology (HR 1.512, 95% CI 1.167-1.938; p = 0.0048) and the BCL2 -938 AA + AC genotype (HR 1.219, 95% CI, 1.024-1.456; p = 0.0256) were significant independent prognostic factors for survival. CONCLUSIONS: Polymorphisms in BCL2 may be associated with survival in advanced-stage NSCLC patients who received chemotherapy.

The beginning of a new era in induction treatment for operable non-small cell lung cancer: a narrative review.

Background and Objective: The survival benefit of induction therapy for non-small cell lung cancer (NSCLC) remains controversial. Recently, the outcomes of systemic therapy for NSCLC have dramatically changed with the advent of molecular target drugs and immune checkpoint inhibitors (ICIs). The present review was conducted to investigate the outcomes of induction therapy with reference to randomized control trials (RCTs). Methods: We reviewed RCTs and ongoing clinical trials between 1990 and 2022 using relevant databases: PubMed, Web of Science, and EMBASE database. We investigated the outcomes of induction therapy. Key Content and Findings: Induction therapy was associated with longer overall survival in comparison to surgery alone in several RCTs for stage III disease. However, its benefit in early-stage (I-II) disease was unclear. Regarding induction chemotherapy and chemoradiotherapy, the safety and survival outcomes did not differ between the two arms. Epidermoid growth factor receptor (EGFR) tyrosine kinase inhibitors as induction therapy in patients with proven EGFR mutations may be a sufficient choice for the improvement of overall survival. In ongoing single arm clinical trials and a randomized control study, the administration of ICIs as induction therapy was associated with a good pathological response and satisfactory safety, which will lead to a better survival outcome. Long-term observation is needed to evaluate the toxicity and survival impact of induction therapy with ICIs. Conclusions: Induction chemotherapy and EGFR-TKIs for stage IIIA NSCLC may contribute to the improvement of survival outcomes although the effect of systemic therapy on stage I-II remains controversial. ICIs may be considered as a valuable treatment option because of their feasibility and safety for induction therapy.

Changes in serum DAMPs and cytokines/chemokines during near-infrared photoimmunotherapy for patients with head and neck cancer.

BACKGROUND: Near-infrared photoimmunotherapy (NIR-PIT) for head and neck cancer is a recently developed therapy. However, there is limited data on patients receiving NIR-PIT in real clinical settings. METHODS: Seven NIR-PIT sessions were administered to five patients with head and neck squamous cell carcinoma (HNSCC). Serum damage-associated molecular patterns (DAMPs) (HMGB1 and Hsp70 levels), and cytokine and chemokine production, were compared before and after NIR-PIT. RESULTS: The serum concentration of HMGB1 increased after NIR-PIT (p = 0.031, Wilcoxon test) in all patients except one who did not achieve a clinical response. Chemokines MIP-1α (CCL3) and MIP-1ß (CCL4) increased significantly 1-3 days after treatment (CCL3, p = 0.0036; CCL4, p = 0.0016, Wilcoxon test). A low pre-treatment neutrophil-to-lymphocyte ratio (NLR) was associated with a better response to therapy and survival. CONCLUSIONS: The release of DAMPs, and cytokine/chemokine production, were detected in the patients' peripheral blood. The baseline NLR may predict patient outcomes in response to NIR-PIT.

Diagnostic utility of DNA integrity number as an indicator of sufficient DNA quality in next-generation sequencing-based genomic profiling.

OBJECTIVES: DNA integrity number (DIN) is a metric for assessing DNA degradation, calculated based on electrophoresis using the Agilent TapeStation System. The utility of DIN as a diagnostic indicator of sufficient DNA quality in clinical next-generation sequencing (NGS) has not been well described. METHODS: We evaluated the DINs of 166 tumor formalin-fixed, paraffin-embedded (FFPE) tissue samples submitted for 124-gene panel sequencing. We also investigated a new metric on the electropherogram that could improve the predictive accuracy of the DIN. RESULTS: A DIN cutoff of 2.5 discriminated samples with successful analysis (n = 143) from samples with failed analysis (n = 23), with a sensitivity of 0.84 and a specificity of 0.78 (area under the curve [AUC] = 0.88). The DIN was positively correlated with the mean coverage (r = 0.72, P < .0001) but could not discriminate success from failure when the DIN was below 2.5 (negative predictive value, 0.44). We introduced a new metric, the peak/base ratio, that distinguished success from failure with higher accuracy than the DIN (cutoff = 1.6; sensitivity = 0.98, specificity = 0.83, and AUC =0.96). CONCLUSIONS: To predict successful NGS, the DNA quality of FFPE tissue can be easily and reliably assessed using the DIN and peak/base ratio.

Expression of CD5 in salivary gland tumors: an ancillary marker for carcinoma showing thymus-like differentiation (CASTLE) of the major salivary gland.

Recently, cases of carcinoma showing thymus-like differentiation (CASTLE) occurring in major salivary glands have been identified. To assess the diagnostic value of CD5 immunohistochemistry in distinguishing salivary CASTLE from other types of salivary gland tumors, we evaluated CD5 expression in 109 salivary gland tumors, encompassing 23 different histological types, including salivary CASTLE. In addition, we reviewed 10 previously reported cases of salivary CASTLE. Most salivary CASTLE cases (10/11, 91%) showed strong CD5 expression. In contrast, 104 of 108 (96%) non-salivary CASTLE tumors were negative for CD5, while the remaining four tumors (3.7%), all of which were histologically Warthin tumors, showed focal positivity for CD5 with weak to moderate intensity. In conclusion, the findings in this study support the potential use of CD5 immunohistochemistry for distinguishing salivary CASTLE from other histological types of salivary gland tumors. Aberrant CD5 expression in this tumor may be linked to the tumor microenvironment.

Single-cell sequencing on CD8+ TILs revealed the nature of exhausted T cells recognizing neoantigen and cancer/testis antigen in non-small cell lung cancer.

BACKGROUND: CD8+tumor infiltrating lymphocytes (TILs) are often observed in non-small cell lung cancers (NSCLC). However, the characteristics of CD8+ TILs, especially T-cell populations specific for tumor antigens, remain poorly understood. METHODS: High throughput single-cell RNA sequencing and single-cell T-cell receptor (TCR) sequencing were performed on CD8+ TILs from three surgically-resected lung cancer specimens. Dimensional reduction for clustering was performed using Uniform Manifold Approximation and Projection. CD8+ TIL TCR specific for the cancer/testis antigen KK-LC-1 and for predicted neoantigens were investigated. Differentially-expressed gene analysis, Gene Set Enrichment Analysis (GSEA) and single sample GSEA was performed to characterize antigen-specific T cells. RESULTS: A total of 6998 CD8+ T cells was analyzed, divided into 10 clusters according to their gene expression profile. An exhausted T-cell (exhausted T (Tex)) cluster characterized by the expression of ENTPD1 (CD39), TOX, PDCD1 (PD1), HAVCR2 (TIM3) and other genes, and by T-cell oligoclonality, was identified. The Tex TCR repertoire (Tex-TCRs) contained nine different TCR clonotypes recognizing five tumor antigens including a KK-LC-1 antigen and four neoantigens. By re-clustering the tumor antigen-specific T cells (n=140), it could be seen that the individual T-cell clonotypes were present on cells at different stages of differentiation and functional states even within the same Tex cluster. Stimulating these T cells with predicted cognate peptide indicated that TCR signal strength and subsequent T-cell proliferation and cytokine production was variable but always higher for neoantigens than KK-LC-1. CONCLUSIONS: Our approach focusing on T cells with an exhausted phenotype among CD8+ TILs may facilitate the identification of tumor antigens and clarify the nature of the antigen-specific T cells to specify the promising immunotherapeutic targets in patients with NSCLC.