Nucleic Acid Quality Assessment is Critical to the Success of the Oncomine Dx Target Test for Lung Cancer.

BACKGROUND AND OBJECTIVE: The Oncomine Dx Target Test (ODxTT) has been used as a companion diagnostic test for lung cancer. Here, we evaluated whether the amount of nucleic acid and the degree of RNA degradation are related to the success of the ODxTT. METHODS: This study included 223 samples from 218 patients with lung cancer. For all samples, DNA and RNA concentrations were quantified using Qubit, and the degree of RNA degradation was evaluated using the Bioanalyzer. RESULTS: Of the 223 samples, 219 samples were successfully analyzed in the ODxTT and four were not. DNA analysis failed in two samples, which were attributed to low DNA concentrations and both were cytology specimens. Meanwhile, RNA analysis failed in the other two samples. These samples had sufficient amounts of RNA, but it was highly degraded with DV200 (the percentage of RNA fragments > 200 base pairs) less than 30. Compared with RNA samples with DV200 ≥ 30, analysis of RNA with DV200 < 30 yielded significantly fewer reads for the internal control genes. This test showed actionable mutations were identified in 38% (83/218) of all patients and in 46.6% (76/163) of patients with lung adenocarcinoma. CONCLUSIONS: DNA concentration and degree of RNA degradation are key factors determining the success of diagnostic testing by the ODxTT.

Deep targeted sequencing of cytological tumor cells using whole genome amplification.

BACKGROUND: Genomic profiling in lung cancer is essential for precision medicine. Cytological specimens provide an alternative to formalin-fixed paraffin-embedded (FFPE) samples for comprehensive genomic analysis. However, this approach remains challenging when a limited number of tumor cells are available. We applied whole genome amplification (WGA) to cytology specimens to overcome this limitation. METHODS: Using a lung cancer panel targeting 58 genes, we performed next-generation sequencing of whole genome-amplified DNA extracted from cytological specimens containing 10-20 tumor cells (cyto-WGA) and DNA from corresponding FFPE tumor tissue. We compared sequencing data from cyto-WGA and FFPE samples to examine the detection accuracy of copy number variations and oncogenic and drug-matched variants. RESULTS: The DNA quality and quantity from cyto-WGA were higher than those from FFPE samples (p < .0005 and p < .05, respectively). Sequencing metrics of cyto-WGA and FFPE tissues showed no difference in the number of mapped reads and mean coverage depth, but there were significant differences in the on-target rate (p < .05) and uniformity (p < .0005). Copy number variations in cyto-WGA samples (n = 211) were higher than in FFPE samples (n = 9) (p < .0001). Fourty nine oncogenic variants were detected in cyto-WGA and 39 in FFPE. Of these variants, 34 (63%) were present in both samples. In addition, all 16 drug-matched variants were detected in FFPE and cyto-WGA samples with 100% concordance. CONCLUSION: Cyto-WGA can be a feasible and alternative method to detect oncogenic and drug-matched variants.

The Diagnostic Utility of Cell-Free DNA from Ex Vivo Bronchoalveolar Lavage Fluid in Lung Cancer.

Although bronchoscopy is generally performed to diagnose lung cancer, its diagnostic yield remains unsatisfactory. Assuming that lung cancer cells release cell-free DNA into the epithelial lining fluid, we hypothesized that lung cancer could be diagnosed by analyzing gene mutations in cell-free DNA in this fluid. This study included 32 patients with lung cancer who underwent surgery at our hospital. Bronchoalveolar lavage (BAL) was performed on the resected lung samples (ex vivo BAL model) after lobectomy. Each DNA sample (i.e., BAL fluid, primary lesion, and plasma) underwent deep targeted sequencing. Gene mutation analyses in the BAL fluid samples identified mutations identical to those in the primary lesions in 30 (93.8%) of 32 patients. In contrast, the microscopic cytology of the same BAL fluid samples yielded a diagnosis of lung cancer in only one of 32 patients, and the analysis of plasma samples revealed gene mutations identical to those in the primary lesions in only one of 32 patients. In conclusion, cell-free DNA released from lung cancer cells exists more abundantly in the airway than in the blood. The collection and analysis of the BAL fluid containing cell-free DNA derived from lung cancer can thus allow lung cancer diagnosis and the screening of driver mutations.

Lung Cancer Surgery With Persistent COVID-19 Infection.

A 71-year-old man with a history of drug-induced interstitial pneumonia was diagnosed with COVID-19 infection and simultaneously found to have a pulmonary mass, suggesting a coexisting lung cancer. Approximately 1 month after COVID-19 pneumonia resolved, the patient electively underwent right upper lobectomy. Postoperatively, acute exacerbation of interstitial pneumonia occurred and the patient died on the fifteenth postoperative day. By quantitative reverse transcription polymerase chain reaction, high levels of COVID-19-derived RNA were detected in the specimen of lung parenchyma. Despite resolved COVID-19 infection, it may persist locally in the lungs, with the risk of acute exacerbation of interstitial pneumonia due to secondary stressors including surgery.

Discrimination Between Primary Lung Cancer and Lung Metastases by Genomic Profiling.

INTRODUCTION: In cases of lung tumors that occur after treatment for malignancies in other organs, the tumor may represent either a primary lung cancer or a solitary pulmonary metastasis from the other tumor. Because some lung tumors are difficult to differentiate on the basis of imaging and pathologic findings, treatment selection is often difficult. In this study, we attempted to make a genomic diagnosis of primary and metastatic lung tumors by analyzing tumor samples using next-generation sequencing and evaluated the efficacy and validity of the genomic diagnosis. METHODS: A total of 24 patients with a solitary lung nodule and a history of other malignancies were enrolled in this study. Tumor cells were selected from tissue samples using laser capture microdissection. DNA was extracted from those cells and subjected to targeted deep sequencing of 53 genes. RESULTS: The driver mutation profiles of the primary lung tumors were discordant from those of the primary tumors in other sites, whereas the mutation profiles of pulmonary metastases and previous malignancies were concordant. In all 24 patients, we could diagnose either primary lung cancer (six patients) or lung metastases (18 patients) on the basis of whether gene mutation profiles were concordant or discordant. In 12 patients (50.0%), discrepancies were observed between the genomic and clinical or histopathologic diagnoses. CONCLUSIONS: In patients with a solitary lung lesion and a history of cancer, tumor-specific mutations can serve as clonal markers, affording a more accurate understanding of the pathological condition and thus possibly improving both treatment selection and patient outcome.

Sphingomonas and Phenylobacterium as Major Microbiota in Thymic Epithelial Tumors.

The microbiota has been reported to be closely associated with carcinogenesis and cancer progression. However, its involvement in the pathology of thymoma remains unknown. In this study, we aimed to identify thymoma-specific microbiota using resected thymoma samples. Nineteen thymoma tissue samples were analyzed through polymerase chain reaction amplification and 16S rRNA gene sequencing. The subjects were grouped according to histology, driver mutation status in the GTF2I gene, PD-L1 status, and smoking habits. To identify the taxa composition of each sample, the operational taxonomic units (OTUs) were classified on the effective tags with 97% identity. The Shannon Index of the 97% identity OTUs was calculated to evaluate the alpha diversity. The linear discriminant analysis effect size (LEfSe) method was used to compare the relative abundances of all the bacterial taxa. We identified 107 OTUs in the tumor tissues, which were classified into 26 genera. Sphingomonas and Phenylobacterium were identified as abundant genera in almost all the samples. No significant difference was determined in the alpha diversity within these groups; however, type A thymoma tended to exhibit a higher bacterial diversity than type B thymoma. Through the LEfSe analysis, we identified the following differentially abundant taxa: Bacilli, Firmicutes, and Lactobacillales in type A thymoma; Proteobacteria in type B thymoma; Gammaproteobacteria in tumors harboring the GTF2I mutation; and Alphaproteobacteria in tumors without the GTF2I mutation. In conclusion, Sphingomonas and Phenylobacterium were identified as dominant genera in thymic epithelial tumors. These genera appear to comprise the thymoma-specific microbiota.

Actionable driver DNA variants and fusion genes can be detected in archived cytological specimens with the Oncomine Dx Target Test Multi-CDx system in lung cancer.

BACKGROUND: Molecular testing is critical for identifying actionable variants in lung cancer for precision medicine. When tumor tissue samples are unavailable, archived cytological specimens (ACSs) can be used. The authors examined whether oncogenic variants could be accurately detected in ACSs versus paired formalin-fixed, paraffin-embedded (FFPE) tumor tissues with in vitro diagnostic tests. METHODS: The authors collected 18 ACSs and 15 FFPE tissues from 15 patients with lung cancer and investigated genomic profiles with the Oncomine Dx Target Test Multi-CDx system, which is an integrated next-generation sequencing platform that comprehensively examines 4 companion diagnostic target genes (epidermal growth factor receptor [EGFR]; B-Raf proto-oncogene, serine/threonine kinase [BRAF]; anaplastic lymphoma kinase [ALK]; and ROS proto-oncogene 1, receptor tyrosine kinase [ROS1]). They compared the quantity and quality of extracted nucleic acids, the sequencing quality control (QC), and the detected variants between ACSs and FFPE tissues. RESULTS: The total amount of DNA and RNA obtained from 1 slide was higher in FFPE tissues than ACSs. The RNA integrity number was higher in ACSs. There were no differences in sequencing QC between ACSs and FFPE tissues. A total of 21 variants, including EGFR mutations and ALK and ROS1 fusion genes, were detected in both ACSs and FFPE tissues with 100% concordance. CONCLUSIONS: ACSs can be a feasible alternative with which to identify actionable mutations and fusion genes via the Oncomine Dx Target Test Multi-CDx system.

Streptococcus australis and Ralstonia pickettii as Major Microbiota in Mesotheliomas.

The microbiota has been reported to be correlated with carcinogenesis and cancer progression. However, its involvement in the pathology of mesothelioma remains unknown. In this study, we aimed to identify mesothelioma-specific microbiota using resected or biopsied mesothelioma samples. Eight mesothelioma tissue samples were analyzed via polymerase chain reaction (PCR) amplification and 16S rRNA gene sequencing. The operational taxonomic units (OTUs) of the effective tags were analyzed in order to determine the taxon composition of each sample. For the three patients who underwent extra pleural pneumonectomy, normal peripheral lung tissues adjacent to the tumor were also included, and the same analysis was performed. In total, 61 OTUs were identified in the tumor and lung tissues, which were classified into 36 species. Streptococcus australis and Ralstonia pickettii were identified as abundant species in almost all tumor and lung samples. Streptococcus australis and Ralstonia pickettii were found to comprise mesothelioma-specific microbiota involved in tumor progression; thus, they could serve as targets for the prevention of mesothelioma.

Association of Mutation Profiles with Postoperative Survival in Patients with Non-Small Cell Lung Cancer.

Findings on mutations, associated with lung cancer, have led to advancements in mutation-based precision medicine. This study aimed to comprehensively and synthetically analyze mutations in lung cancer, based on the next generation sequencing data of surgically removed lung tumors, and identify the mutation-related factors that can affect clinical outcomes. Targeted sequencing was performed on formalin-fixed paraffin-embedded surgical specimens obtained from 172 patients with lung cancer who underwent surgery in our hospital. The clinical and genomic databases of the hospital were combined to determine correlations between clinical factors and mutation profiles in lung cancer. Multivariate analyses of mutation-related factors that may affect the prognosis were also performed. Based on histology, TP53 was the driver gene in 70.0% of the cases of squamous cell carcinoma. In adenocarcinoma cases, driver mutations were detected in TP53 (26.0%), KRAS (25.0%), and epidermal growth factor receptor (EGFR) (23.1%). According to multivariate analysis, the number of pathogenic mutations (≥3), presence of a TP53 mutation, and TP53 allele fraction >60 were poor prognostic mutational factors. The TP53 allele fraction tended to be high in caudally and dorsally located tumors. Moreover, TP53-mutated lung cancers located in segments 9 and 10 were associated with significantly poorer prognosis than those located in segments 1-8. This study has identified mutation-related factors that affect the postoperative prognosis of lung cancer. To our knowledge, this is the first study to demonstrate that the TP53 mutation profile varies with the site of lung tumor, and that postoperative prognosis varies accordingly.

Primary Driver Mutations in GTF2I Specific to the Development of Thymomas.

Thymomas are rare mediastinal tumors that are difficult to treat and pose a major public health concern. Identifying mutations in target genes is vital for the development of novel therapeutic strategies. Type A thymomas possess a missense mutation in GTF2I (chromosome 7 c.74146970T>A) with high frequency. However, the molecular pathways underlying the tumorigenesis of other thymomas remain to be elucidated. We aimed to detect this missense mutation in GTF2I in other thymoma subtypes (types B). This study involved 22 patients who underwent surgery for thymomas between January 2014 and August 2019. We isolated tumor cells from formalin-fixed paraffin-embedded tissues from the primary lesions using laser-capture microdissection. Subsequently, we performed targeted sequencing to detect mutant GTF2I coupled with molecular barcoding. We used PyClone analysis to determine the fraction of tumor cells harboring mutant GTF2I. We detected the missense mutation (chromosome 7 c.74146970T>A) in GTF2I in 14 thymomas among the 22 samples (64%). This mutation was harbored in many type B thymomas as well as type A and AB thymomas. The allele fraction for the tumors containing the mutations was variable, primarily owing to the coexistence of normal lymphocytes in the tumors, especially in type B thymomas. PyClone analysis revealed a high cellular prevalence of mutant GTF2I in tumor cells. Mutant GTF2I was not detected in other carcinomas (lung, gastric, colorectal, or hepatocellular carcinoma) or lymphomas. In conclusion, the majority of thymomas harbor mutations in GTF2I that can be potentially used as a novel therapeutic target in patients with thymomas.

Genome analysis of peeling archival cytology samples detects driver mutations in lung cancer.

INTRODUCTIONS: When tumor tissue samples are unavailable to search for actionable driver mutations, archival cytology samples can be useful. We investigate whether archival cytology samples can yield reliable genomic information compared to corresponding formalin-fixed paraffin-embedded (FFPE) tumor samples. PATIENTS AND METHODS: Pretreatment class V archival cytology samples with adequate tumor cells were selected from 172 lung cancer patients. The genomic profiles of the primary lung tumors have been analyzed through whole-exome regions of 53 genes. We compared the genomic profiles based on the oncogenicity and variant allele frequency (VAF) between the archival cytology and the corresponding primary tumors. We also analyzed the genomic profiles of serial cytological samples during the treatment of EGFR-TKI. RESULTS: A total of 43 patients were analyzed with the paired samples for DNA mutations and other three patients were analyzed for their fusion genes. A total of 672 mutations were detected. Of those, 106 mutations (15.8%) were shared with both samples. Sixty of seventy-seven (77.9%) shared mutations were oncogenic or likely oncogenic mutations with VAF ≧10%. As high as 90% (9/10) actionable driver mutations and ALK and ROS1 fusion genes were successfully detected from archival cytology samples. Sequential analysis revealed the dynamic changes in EGFR-TKI-resistant mutation (EGFR p.T790M) during the course of treatment. CONCLUSION: Archival cytology sample with adequate tumor cells can yield genetic information compared to the primary tumors. If tumor tissue samples are unavailable, we can use archival cytology samples to search for actionable driver mutations.

Identification of Clonality through Genomic Profile Analysis in Multiple Lung Cancers.

In cases of multiple lung cancers, individual tumors may represent either a primary lung cancer or both primary and metastatic lung cancers. In this study, we investigated the differences between clinical/histopathological and genomic diagnoses to determine whether they are primary or metastatic. 37 patients with multiple lung cancers were enrolled in this study. Tumor cells were selected from tissue samples using laser capture microdissection. DNA was extracted from those cells and subjected to targeted deep sequencing. In multicentric primary lung cancers, the driver mutation profile was mutually exclusive among the individual tumors, while it was consistent between metastasized tumors and the primary lesion. In 11 patients (29.7%), discrepancies were observed between genomic and clinical/histopathological diagnoses. For the lymph node metastatic lesions, the mutation profile was consistent with only one of the two primary lesions. In three of five cases with lymph node metastases, the lymph node metastatic route detected by genomic diagnosis differed from the clinical and/or pathological diagnoses. In conclusion, in patients with multiple primary lung cancers, cancer-specific mutations can serve as clonal markers, affording a more accurate understanding of the pathology of multiple lung cancers and their lymphatic metastases and thus improving both the treatment selection and outcome.

Dual-molecular barcode sequencing detects rare variants in tumor and cell free DNA in plasma.

Conventional next generation sequencing analysis has provided important insights into cancer genetics. However, the detection of rare (low allele fraction) variants remains difficult because of the error-prone nucleotide changes derived from sequencing/PCR errors. To eliminate the false-positive variants and detect genuine rare variants, sequencing technology combined with molecular barcodes will be useful. Here, we used the newly developed dual-molecular barcode technology (Ion AmpliSeq HD) to analyze somatic mutations in 24 samples (12 tumor tissues and 12 plasma) from 12 patients with biliary-pancreatic and non-small cell lung cancers. We compared the results between next generation sequencing analysis with or without molecular barcode technologies. The variant allele fraction (VAF) between non-molecular barcode and molecular barcode sequencing was correlated in plasma DNA (R2 = 0.956) and tumor (R2 = 0.935). Both methods successfully detected high VAF mutations, however, rare variants were only identified by molecular barcode sequencing and not by non-molecular barcode sequencing. Some of these rare variants in tumors were annotated as pathogenic, and therefore subclonal driver mutations could be observed. Furthermore, the very low VAF down to 0.17% were identified in cell free DNA in plasma. These results demonstrate that the dual molecular barcode sequencing technologies can sensitively detect rare somatic mutations, and will be important in the investigation of the clonal and subclonal architectures of tumor heterogeneity.

PD-L1 Expression and Tumor-Infiltrating Lymphocytes in Thymic Epithelial Neoplasms.

Thymic epithelial tumors (TETs) are rare malignant mediastinal tumors that are difficult to diagnose and treat. The programmed death 1 (PD-1) receptor and its ligand (PD-L1) are expressed in various malignant tumors and have emerged as potential immunotherapeutic targets. However, the immunobiology of TETs is poorly understood. We evaluated PD-L1 expression and the presence of tumor-infiltrating lymphocytes (CD8 and CD3 expression) in surgical TET specimens from 39 patients via immunohistochemistry and determined their relation to clinicopathological parameters. Cases with membranous reactivity of the PD-L1 antibody in ≥1% of tumor cells were considered positive. Positive PD-L1 expression was observed in 53.9% of cases. Histologically, PD-L1 expression was positive in 2/6 type A, 2/6 type AB, 3/9 type B1, 4/4 type B2, 5/6 type B3, and 5/8 type C TET cases. Thus, the number of cases with PD-L1 expression and the percent expression of PD-L1 were significantly higher in more aggressive thymomas (type B2 or B3). CD3+ and CD8+ tumor-infiltrating lymphocytes were diffusely and abundantly distributed in all cases. These data suggest that a PD-1/PD-L1 blockade is a promising treatment for TETs, with more beneficial treatment effects for aggressive thymomas such as type B2 or B3.

Clinical Implications of Noncoding Indels in the Surfactant-Encoding Genes in Lung Cancer.

Lung cancer arises from the accumulation of genetic mutations, usually in exons. A recent study identified indel mutations in the noncoding region of surfactant-encoding genes in lung adenocarcinoma cases. In this study, we recruited 94 patients with 113 lung cancers (88 adenocarcinomas, 16 squamous cell carcinomas, and nine other histologies) who had undergone surgery in our department. A cancer panel was designed in-house for analyzing the noncoding regions, and targeted sequencing was performed. Indels in the noncoding region of surfactant-encoding genes were identified in 29/113 (25.7%) cases and represent the precise cell of origin for the lung cancer, irrespective of histological type and/or disease stage. In clinical practice, these indels may be used as clonal markers in patients with multiple cancers and to determine the origin of cancer of unknown primary site.

Genomic Characteristics of Invasive Mucinous Adenocarcinomas of the Lung and Potential Therapeutic Targets of B7-H3.

Pulmonary invasive mucinous adenocarcinoma (IMA) is considered a variant of lung adenocarcinomas based on the current World Health Organization classification of lung tumors. However, the molecular mechanism driving IMA development and progression is not well understood. Thus, we surveyed the genomic characteristics of IMA in association with immune-checkpoint expression to investigate new potential therapeutic strategies. Tumor cells were collected from surgical specimens of primary IMA, and sequenced to survey 53 genes associated with lung cancer. The mutational profiles thus obtained were compared in silico to conventional adenocarcinomas and other histologic carcinomas, thereby establishing the genomic clustering of lung cancers. Immunostaining was also performed to compare expression of programmed death ligand 1 (PD-L1) and B7-H3 in IMA and conventional adenocarcinomas. Mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS) were detected in 75% of IMAs, but in only 11.6% of conventional adenocarcinomas. On the other hand, the frequency of mutations in epidermal growth factor receptor (EGFR) and tumor protein p53 (TP53) genes was 5% and 10%, respectively, in the former, but 48.8% and 34.9%, respectively, in the latter. Clustering of all 78 lung cancers indicated that IMA is distinct from conventional adenocarcinoma or squamous cell carcinoma. Strikingly, expression of PD-L1 in ≥1% of cells was observed in only 6.1% of IMAs, but in 59.7% of conventional adenocarcinomas. Finally, 42.4% and 19.4% of IMAs and conventional adenocarcinomas, respectively, tested positive for B7-H3. Although currently classified as a variant of lung adenocarcinoma, it is also reasonable to consider IMA as fundamentally distinct, based on mutation profiles and genetic clustering as well as immune-checkpoint status. The immunohistochemistry data suggest that B7-H3 may be a new and promising therapeutic target for immune checkpoint therapy.

Surgery for congenital bronchial atresia.

A 22-year-old woman presented with a feeling of chest tightness. Chest computed tomography showed a dendritic shadow in the left segment 1 + 2c, surrounded by hyperlucent emphysematous changes. Bronchoscopy revealed loss of the orifice of bronchus 1 + 2c. Thus a diagnosis of bronchial atresia was made, and a left upper division segmentectomy was performed. Bronchial atresia should be considered in the differential diagnosis of young patients with an abnormal chest shadow. It can be diagnosed based on the characteristic imaging and bronchoscopic findings. Surgery is indicated for symptomatic bronchial atresia and can provide complete amelioration of the condition.