A new model using deep learning to predict recurrence after surgical resection of lung adenocarcinoma.

This study aimed to develop a deep learning (DL) model for predicting the recurrence risk of lung adenocarcinoma (LUAD) based on its histopathological features. Clinicopathological data and whole slide images from 164 LUAD cases were collected and used to train DL models with an ImageNet pre-trained efficientnet-b2 architecture, densenet201, and resnet152. The models were trained to classify each image patch into high-risk or low-risk groups, and the case-level result was determined by multiple instance learning with final FC layer's features from a model from all patches. Analysis of the clinicopathological and genetic characteristics of the model-based risk group was performed. For predicting recurrence, the model had an area under the curve score of 0.763 with 0.750, 0.633 and 0.680 of sensitivity, specificity, and accuracy in the test set, respectively. High-risk cases for recurrence predicted by the model (HR group) were significantly associated with shorter recurrence-free survival and a higher stage (both, p < 0.001). The HR group was associated with specific histopathological features such as poorly differentiated components, complex glandular pattern components, tumor spread through air spaces, and a higher grade. In the HR group, pleural invasion, necrosis, and lymphatic invasion were more frequent, and the size of the invasion was larger (all, p < 0.001). Several genetic mutations, including TP53 (p = 0.007) mutations, were more frequently found in the HR group. The results of stages I-II were similar to those of the general cohort. DL-based model can predict the recurrence risk of LUAD and identify the presence of the TP53 gene mutation by analyzing histopathologic features.

Revolutionizing Non-Small Cell Lung Cancer Diagnosis: Ultra-High-Sensitive ctDNA Analysis for Detecting Hotspot Mutations with Long-term Stored Plasma.

PURPOSE: Circulating cell-free DNA (cfDNA) has great potential in clinical oncology. The prognostic and predictive values of cfDNA in non-small cell lung cancer (NSCLC) have been reported, with epidermal growth factor receptor (EGFR), KRAS, and BRAF mutations in tumor-derived cfDNAs acting as biomarkers during the early stages of tumor progression and recurrence. However, extremely low tumor-derived DNA rates hinder cfDNA application. We developed an ultra-high-sensitivity lung version 1 (ULV1) panel targeting BRAF, KRAS, and EGFR hotspot mutations using small amounts of cfDNA, allowing for semi-quantitative analysis with excellent limit-of-detection (0.05%). MATERIALS AND METHODS: Mutation analysis was performed on cfDNAs extracted from the plasma of 104 patients with NSCLC by using the ULV1 panel and targeted next-generation sequencing (CT-ULTRA), followed by comparison analysis of mutation patterns previously screened using matched tumor tissue DNA. RESULTS: The ULV1 panel demonstrated robust selective amplification of mutant alleles, enabling the detection of mutations with a high degree of analytical sensitivity (limit-of-detection, 0.025%-0.1%) and specificity (87.9%-100%). Applying ULV1 to NSCLC cfDNA revealed 51.1% (23/45) samples with EGFR mutations, increasing with tumor stage: 8.33% (stage I) to 78.26% (stage IV). Semi-quantitative analysis proved effective for low-mutation-fraction clinical samples. Comparative analysis with PANAMutyper EGFR exhibited substantial concordance (κ=0.84). CONCLUSION: Good detection sensitivity (~80%) was observed despite the limited volume (1 mL) and long-term storage (12-50 months) of plasma used and is expected to increase with high cfDNA inputs. Thus, the ULV1 panel is a fast and cost-effective method for early diagnosis, treatment selection, and clinical follow-up of patients with NSCLC.

BRCA-mutated gastric adenocarcinomas are associated with chromosomal instability and responsiveness to platinum-based chemotherapy.

BACKGROUND: Homologous recombination defect is an important biomarker of chemotherapy in certain tumor types, and the presence of pathogenic or likely pathogenic mutations involving BRCA1 or BRCA2 (p-BRCA) mutations is the most well-established marker for the homologous recombination defect. Gastric cancer, one of the most prevalent tumor types in Asia, also harbors p-BRCA mutations. METHODS: To investigate the clinical significance of p-BRCA mutations, we analyzed 366 gastric cancer cases through next-generation sequencing. We determined the zygosity of p-BRCA mutations based on the calculated tumor purity through variant allelic fraction patterns and investigated whether the presence of p-BRCA mutations is associated with platinum-based chemotherapy and a certain molecular subtype. RESULTS: Biallelic p-BRCA mutation was associated with better response to platinum-based chemotherapy than heterozygous p-BRCA mutation or wild type BRCA genes. The biallelic p-BRCA mutations was observed only in the chromosomal instability subtype, while all p-BRCA mutations were heterozygous in microsatellite instability subtype. CONCLUSIONS: In conclusion, patients with gastric cancer harboring biallelic p-BRCA mutations were associated with a good initial response to platinum-based chemotherapy and those tumors were exclusively chromosomal instability subtype. Further investigation for potential association with homologous recombination defect is warranted.

An oncogenic CTNNB1 mutation is predictive of post-operative recurrence-free survival in an EGFR-mutant lung adenocarcinoma.

The Wnt/ß-catenin pathway is known to be frequently dysregulated in various human malignancies. Alterations in the genes encoding the components of Wnt/ß-catenin pathway have also been described in lung adenocarcinoma. Notably however, the clinical impacts of Wnt/ß-catenin pathway alterations in lung adenocarcinoma have not been fully evaluated to date. We here investigated the prognostic implications of single gene variations in 174 cases of surgically resected lung adenocarcinoma tested using targeted next-generation sequencing. Screening of the prognostic impact of single gene alterations identified an association between CTNNB1 mutation and poor recurrence-free survival in EGFR-mutant LUADs. Based on these results, the entire cohort was stratified into three groups in accordance with the mutational status of Wnt/ß-catenin pathway genes (i.e. oncogenic CTNNB1 mutation [CTNNB1-ONC], other Wnt/ß-catenin pathway gene mutations [Wnt/ß-catenin-OTHER], and wild type for Wnt/ß-catenin pathway genes [Wnt/ß-catenin-WT]). The clinicopathologic characteristics and survival outcomes of these groups were then compared. Oncogenic CTNNB1 and other Wnt/ß-catenin pathway gene mutations were identified in 10 (5.7%) and 14 cases (8.0%), respectively. The CTNNB1-ONC group cases displayed histopathologic features of conventional non-mucinous adenocarcinoma with no significant differences from those of the other groups. Using ß-catenin immunohistochemistry, we found that the CTNNB1-ONC group displayed aberrant nuclear staining more frequently, but only in 60% of the samples. The LUADs harboring a CTNNB1-ONC exhibited significantly poorer RFS outcomes than the other groups, regardless of the ß-catenin IHC status. This was a pronounced finding in the EGFR-mutant LUADs only in subgroup analysis, which was then confirmed by multivariate analysis. Nevertheless, no significant OS differences between these Wnt/ß-catenin groups were evident. Hence, oncogenic CTNNB1 mutations may be found in about 6% of lung adenocarcinomas and may predict post-operative recurrence in EGFR-mutant LUADs. Aberrant nuclear ß-catenin staining on IHC appears to be insufficient as a surrogate marker of an oncogenic CTNNB1 mutation.

Evaluation of Blood Tumor Mutation Burden for the Efficacy of Second-Line Atezolizumab Treatment in Non-Small Cell Lung Cancer: BUDDY Trial.

This study aimed to investigate the feasibility of blood-based biomarkers, including blood tumor mutation burden (bTMB), to predict atezolizumab efficacy in relapsed and advanced non-small cell lung cancer (NSCLC). Stage IV NSCLC patients who had previously received platinum-doublet chemotherapy were recruited and received 1200 mg of atezolizumab every three weeks. Blood was collected to obtain plasma cell-free DNA (cfDNA) before the first cycle (C0) and at the fourth cycle (C4). bTMB was measured by CT-ULTRA in patients with cfDNA over 10 ng. The objective response rate (ORR) of the enrolled 100 patients was 10%, and there was no difference in ORR according to bTMB (cutoff: 11.5 muts/Mb) at C0 (high bTMB: 8.1% vs. low bTMB: 11.1%). However, the C4/C0 bTMB ratio was significantly lower in the durable clinical benefit (DCB) patients. The cfDNA concentration at C0, the C4/C0 ratio of the cfDNA concentration, the highest variant allele frequency (hVAF), and the VAF standard deviation (VAFSD) were significantly lower in the DCB patients. In the multivariate analysis, a high cfDNA concentration at C0 (cutoff: 8.6 ng/mL) and a C4/C0 bTMB ratio greater than 1 were significantly associated with progression-free survival. These results suggest that baseline levels and dynamic changes of blood-based biomarkers (bTMB, cfDNA concentration, and VAFSD) could predict atezolizumab efficacy in previously treated NSCLC patients.

Whole-genome sequencing reveals an association between small genomic deletions and an increased risk of developing Parkinson's disease.

Single-nucleotide variants (SNVs) associated with Parkinson's disease (PD) have been investigated mainly through genome-wide association studies. However, other genomic alterations, including copy number variations, remain less explored. In this study, we conducted whole-genome sequencing of primary (310 PD patients and 100 healthy individuals) and independent (100 PD patients and 100 healthy individuals) cohorts from the Korean population to identify high-resolution small genomic deletions, gains, and SNVs. Global small genomic deletions and gains were found to be associated with an increased and decreased risk of PD development, respectively. Thirty significant locus deletions were identified in PD, with most being associated with an increased PD risk in both cohorts. Small genomic deletions in clustered loci located in the GPR27 region had high enhancer signals and showed the closest association with PD. GPR27 was found to be expressed specifically in brain tissue, and GPR27 copy number loss was associated with upregulated SNCA expression and downregulated dopamine neurotransmitter pathways. Clustering of small genomic deletions on chr20 in exon 1 of the GNAS isoform was detected. In addition, we found several PD-associated SNVs, including one in the enhancer region of the TCF7L2 intron, which exhibited a cis-acting regulatory mode and an association with the beta-catenin signaling pathway. These findings provide a global, whole-genome view of PD and suggest that small genomic deletions in regulatory domains contribute to the risk of PD development.

Establishing Patient-Derived Cancer Cell Cultures and Xenografts in Biliary Tract Cancer.

PURPOSE: Biliary tract cancers (BTCs) are rare and show a dismal prognosis with limited treatment options. To improve our understanding of these heterogeneous tumors and develop effective therapeutic agents, suitable preclinical models reflecting diverse tumor characteristics are needed. We established and characterized new patient-derived cancer cell cultures and patient-derived xenograft (PDX) models using malignant ascites from five patients with BTC. MATERIALS AND METHODS: Five patient-derived cancer cell cultures and three PDX models derived from malignant ascites of five patients with BTC, AMCBTC-01, -02, -03, -04, and -05, were established. To characterize the models histogenetically and confirm whether characteristics of the primary tumor were maintained, targeted sequencing and histopathological comparison between primary tissue and xenograft tumors were performed. RESULTS: From malignant ascites of five BTC patients, five patient-derived cancer cell cultures (100% success rate), and three PDXs (60% success rate) were established. The morphological characteristics of three primary xenograft tumors were compared with those of matched primary tumors, and they displayed a similar morphology. The mutated genes in samples (models, primary tumor tissue, or both) from more than one patient were TP53 (n=2), KRAS (n=2), and STK11 (n=2). Overall, the pattern of commonly mutated genes in BTC cell cultures was different from that in commercially available BTC cell lines. CONCLUSION: We successfully established the patient-derived cancer cell cultures and xenograft models derived from malignant ascites in BTC patients. These models accompanied by different genetic characteristics from commercially available models will help better understand BTC biology.

Clinicopathologic and genomic features of high-grade pattern and their subclasses in lung adenocarcinoma.

INTRODUCTION: Recent lung adenocarcinoma (LUAD) grading system proposed by the International Association for the Study of Lung Cancer (IASLC) has emphasized the proportion of high-grade patterns (HGPs). We aimed to evaluate the clinicopathologic and genomic characteristics associated with HGP which has not yet been fully investigated. METHODS: Tissue samples from 174 patients who underwent surgical resection of LUAD from January to December 2015 were histologically evaluated. Proportions of HGPs, including solid, micropapillary, cribriform, and complex glandular patterns, were individually quantified. Prognostic implications of HGP proportion, both as a continuous variable and as subclasses divided by cutoffs of 20%, 50%, and 90% (low-intermediate grade [LIG], HGP <20%; high grade 1 [HG1], 20-<50%, HG2, 50-<90%; HG3, ≥90%) were evaluated. Different clinicopathologic factors and genomic alterations according to the HGP subclasses were assessed. RESULTS: Relative hazards of the HGP gradually elevated as its proportion increased over 20%, the cut-off value established by the IASLC grading system, and the cancer-specific overall survival (OS) of HG1 subclass was not significantly decreased compared to the LIG subclass on univariate analysis. However, further subgrouping showed significantly increased frequencies of male, advanced stage tumors, lymphovascular invasion, and spread through alveolar space in higher HGP subclasses. Also, common LUAD driver mutations, particularly EGFR mutations, were less frequent, whereas alterations in TP53 and cell cycle pathway-related genes were more frequent. Higher HGP subclasses and TP53 gene alteration were associated with shorter cancer-specific OS and RFS in multivariate survival analysis. CONCLUSIONS: HGP subclasses of LUAD displayed distinct clinicopathological characteristics and genomic alterations, including TP53 and cell cycle pathway, emphasizing the clinical value of these subclasses in LUAD. Higher HGP subclass and alteration in TP53 may be markers of poor post-operative survival.

Assessment of Anti-tumor Efficacy of Osimertinib in Non-Small Cell Lung Cancer Patients by Liquid Biopsy Using Bronchoalveolar Lavage Fluid, Plasma, or Pleural Effusion.

PURPOSE: This study was to evaluate anti-tumor efficacy of osimertinib in patients positive for acquired epidermal growth factor receptor (EGFR) T790M mutation in liquid biopsy using plasma, bronchoalveolar lavage fluid (BALF) or bronchial washing fluid (BWF), and pleural effusion. MATERIALS AND METHODS: Among patients benefited from previous EGFR‒tyrosine kinase inhibitor treatment followed by treatment failure, patients in whom T790M mutations are detected in at least one of the samples including tumor tissues, BALF/BWF, plasma, and pleural effusion were enrolled. T790M mutation was detected by extracting cell free DNA from liquid biopsy samples, using PANA Mutyper. Objective response rate (ORR) and progression-free survival (PFS) with osimertinib treatment were evaluated. RESULTS: Between January 2018 and December 2019, 63 patients were enrolled and received osimertinib. Mean age was 63 years, and 38 (60.3%) were female. Twenty-six patients had T790M mutation in both liquid and tissue samples (group A), 19 patients had only in tissue biopsy samples (group B), and 18 patients had T790M mutation only in liquid biopsy samples (group C). ORR in overall population was 63.5%, and was 61.5% in group A, 68.4% in group B, and 61.1% in group C, respectively. Median PFS in overall patients was 15.6 months (95% confidence interval, 10.7 to 24.2). There was no significant difference in ORR or PFS between groups. CONCLUSION: Osimertinib showed favorable efficacy in lung cancer patients with acquired resistance to prior EGFR-TKI therapies, who screened positive for harboring T790M mutation detected from cell free DNA extracted from plasma, BALF/BWF, and pleural effusion.

Immuno-genomic classification of colorectal cancer organoids reveals cancer cells with intrinsic immunogenic properties associated with patient survival.

BACKGROUND: The intrinsic immuno-ge7nomic characteristics of colorectal cancer cells that affect tumor biology and shape the tumor immune microenvironment (TIM) are unclear. METHODS: We developed a patient-derived colorectal cancer organoid (CCO) model and performed pairwise analysis of 87 CCOs and their matched primary tumors. The TIM type of the primary tumor was classified as immuno-active, immuno-exhausted, or immuno-desert. RESULTS: The gene expression profiles, signaling pathways, major oncogenic mutations, and histology of the CCOs recapitulated those of the primary tumors, but not the TIM of primary tumors. Two distinct intrinsic molecular subgroups of highly proliferative and mesenchymal phenotypes with clinical significance were identified in CCOs with various cancer signaling pathways. CCOs showed variable expression of cancer-specific immune-related genes such as those encoding HLA-I and HLA-II, and molecules involved in immune checkpoint activation/inhibition. Among these genes, the expression of HLA-II in CCOs was associated with favorable patient survival. K-means clustering analysis based on HLA-II expression in CCOs revealed a subgroup of patients, in whom cancer cells exhibited Intrinsically Immunogenic Properties (Ca-IIP), and were characterized by high expression of signatures associated with HLA-I, HLA-II, antigen presentation, and immune stimulation. Patients with the Ca-IIP phenotype had an excellent prognosis, irrespective of age, disease stage, intrinsic molecular type, or TIM status. Ca-IIP was negatively correlated with intrinsic E2F/MYC signaling. Analysis of the correlation between CCO immuno-genotype and TIM phenotype revealed that the TIM phenotype was associated with microsatellite instability, Wnt/ß-catenin signaling, APC/KRAS mutations, and the unfolded protein response pathway linked to the FBXW7 mutation in cancer cells. However, Ca-IIP was not associated with the TIM phenotype. CONCLUSIONS: We identified a Ca-IIP phenotype from a large set of CCOs. Our findings may provide an unprecedented opportunity to develop new strategies for optimal patient stratification in this era of immunotherapy.

Longitudinal change of genetic variations in cetuximab-treated metastatic colorectal cancer.

Recurrent gene mutations and copy number alterations in cancer patients are presumably associated with resistance to targeted therapy. In the present study, we assessed the gene mutations and copy number alterations that recurrently occurred in cetuximab-treated patients with metastatic colorectal cancer (mCRC). Targeted next-generation sequencing was performed in the tumor samples obtained pre- and postcetuximab treatment to assess the variations that occurred during cetuximab treatment. Moreover, we identified the emergent gene mutations (CDK6, EPHA3, ERCC2, MYC, PCMTD1, PIK3CA, PRIM2, RICTOR, and ZNRF3) and copy number alterations (ARAF, BCL2, BRCA2, EGFR, MYC, and SMAD4) that were recurrently observed only in postprogression samples and not in pretreatment or posttreatment samples from patients revealing clinical response. Furthermore, to identify the feasible candidate variations implicated in treatment resistance, we examined the variants with clonal expansion during treatment and discovered PCBP1 as a variant associated with posttreatment progression. Various recurrent mutations were enriched in the TGF-beta signaling pathway. Collectively, we identified recurrent variations in mCRC samples exhibiting post-cetuximab progression. Additionally, future studies are required to evaluate the therapeutic potential of these variations.

Comprehensive analysis of blood-based biomarkers for predicting immunotherapy benefits in patients with advanced non-small cell lung cancer.

BACKGROUND: This study aimed to investigate the feasibility of using circulating tumor cells (CTCs), peripheral blood cells (PBCs), and circulating cell-free DNA (cfDNA) as biomarkers of immune checkpoint inhibitor treatment response in patients with advanced non-small cell lung cancer (NSCLC). METHODS: We recruited patients diagnosed with advanced NSCLC who received pembrolizumab or atezolizumab between July 2019 and June 2020. Blood was collected before each treatment cycle (C1-C4) to calculate absolute neutrophil count (ANC), neutrophil-to-lymphocyte ratio (NLR), derived NLR (dNLR), and platelet-to-lymphocyte ratio (PLR). CTCs, isolated using the CD-PRIMETM system, exhibited EpCAM/CK+/CD45- phenotype in BioViewCCBSTM. The cfDNA was extracted from plasma at the beginning of C1 and C4. RESULTS: The durable clinical benefit (DCB) rate among 83 response-evaluable patients was 34%. CTC, PBC, and cfDNA levels at baseline (C1) were not significantly correlated with treatment response, although patients with DCB had lower CTC counts from C2 to C4. However, patients with low NLR, dNLR, PLR, and cfDNA levels at C1 had improved progression-free survival (PFS) and overall survival (OS). Patients with decreased CTC counts from C1 to C2 had higher median PFS (6.2 vs. 2.3 months; P=0.078) and OS (not reached vs. 6.8 months, P=0.021) than those with increased CTC counts. Low dNLR (≤2.0) at C1 and decreased CTC counts were independent factors for predicting survival. CONCLUSIONS: Comprehensive analysis of CTC, PBC, and cfDNA levels at baseline and during treatment demonstrated they might be biomarkers for predicting survival benefit. This finding could aid in risk stratification of patients with advanced NSCLC who are undergoing immune checkpoint inhibitor treatment.

Mutation Profile of Aggressive Pheochromocytoma and Paraganglioma with Comparison of TCGA Data.

In pheochromocytoma and paraganglioma (PPGL), germline or somatic mutations in one of the known susceptibility genes are identified in up to 60% patients. However, the peculiar genetic events that drive the aggressive behavior including metastasis in PPGL are poorly understood. We performed targeted next-generation sequencing analysis to characterize the mutation profile in fifteen aggressive PPGL patients and compared accessible data of aggressive PPGLs from The Cancer Genome Atlas (TCGA) with findings of our cohort. A total of 115 germline and 34 somatic variants were identified with a median 0.58 per megabase tumor mutation burden in our cohort. The most frequent mutation was SDHB germline mutation (27%) and the second frequent mutations were somatic mutations for SETD2, NF1, and HRAS (13%, respectively). Patients were subtyped into three categories based on the kind of mutated genes: pseudohypoxia (n = 5), kinase (n = 5), and unknown (n = 5) group. In copy number variation analysis, deletion of chromosome arm 1p harboring SDHB gene was the most frequently observed. In our cohort, SDHB mutation and pseudohypoxia subtype were significantly associated with poor overall survival. In conclusion, subtyping of mutation profile can be helpful in aggressive PPGL patients with heterogeneous prognosis to make relevant follow-up plan and achieve proper treatment.

Real-world utility of next-generation sequencing for targeted gene analysis and its application to treatment in lung adenocarcinoma.

PURPOSE: This study investigated the clinical utility of next-generation sequencing (NGS) for detection of genetic alterations and its implications on treatment of lung adenocarcinoma in real-world practice. PATIENTS AND METHODS: Data were reviewed for 391 patients with lung adenocarcinoma who underwent NGS between March 2017 and October 2018. Formalin-fixed, paraffin-embedded archival samples were used for performing NGS targeting 382 genes, including all exons of 199 genes, 184 hotspots, and the partial introns of 8 genes often rearranged in cancer. Survival analysis was performed for stage IV disease. RESULTS: Among the 391 patients, at least one actionable mutation was identified in 294 patients (75.2%). The most commonly mutated gene was EGFR (n = 130, 33.2%), involving EGFR exon 19 deletion (n = 48, 12.3%), L858R (n = 47, 12%), and others (n = 35, 9%), followed by KRAS (n = 48, 12.3%), ALK (n = 40, 10.2%), RET (6%), MET (3%), ROS-1 (3%), and BRAF (2%) mutations. TP53 (46.9%) and CDKN2A (12.6%) mutations were common co-mutations in patients with AMs. With a median follow-up duration of 16.8 months, median overall survival was 36.8 months in patients with stage IV disease. Patients treated with the corresponding targeted therapy for AMs based on NGS reports lived significantly longer than those not treated with such therapy (p < 0.001). After multivariate analysis, targeted therapy for AM was a significantly favorable factor for survival (AM without targeted therapy vs. AM with targeted therapy, hazard ratio 2.58, 95% confidence interval 1.57-4.25; p < 0.001). CONCLUSION: This study revealed that AMs could be comparably detected using NGS. Based on these NGS results, a suitable targeted therapy can be selected, which may improve survival in patients with lung adenocarcinoma. This NGS-based approach is useful in real-world practice to provide guidance when selecting targeted therapy.

Characterization of early postzygotic somatic mutations through multi-organ analysis.

Mosaicisms caused by postzygotic mutational events are of increasing interest because of their potential association with various human diseases. Postzygotic somatic mutations have not been well characterized however in terms of their developmental lineage in humans. We conducted whole-genome sequencing (WGS) and targeted deep sequencing in 15 organs across three developmental lineages from a single male fetus with polycystic kidney disease (PKD) of 21 weeks gestational age. This fetus had no detectable neurological abnormalities at autopsy but germline mutations in the PKHD1 gene were identified that may have been associated with the PKD. Eight early embryonic mosaic variants with no alteration of protein function were detected. These variants were thought to have occurred at the two or four cell stages after fertilization with a mutational pattern involving frequent C>T and T>C transitions. In our current analyses, no tendency toward organ-specific mutation occurrences was found as the eight variants were detected in all 15 organs. However different allele fractions of these variants were found in different organs, suggesting a tissue-specific asymmetric growth of cells that reflected the developmental germ layer of each organ. This indicated that somatic mutation occurrences, even in early embryogenesis, can affect specific organ development or disease. Our current analyses demonstrate that multi-organ analysis is helpful for understanding genomic mosaicism. Our results also provide insights into the biological role of mosaicism in embryonic development and disease.

Genetic Alterations Detected by Targeted Next-generation Sequencing and Their Clinical Implications in Neuroblastoma.

BACKGROUND/AIM: This study was conducted to evaluate the clinical usefulness of panel next-generation sequencing (NGS) and to investigate the spectrum of genetic alterations and their clinical implications in neuroblastoma. PATIENTS AND METHODS: Formalin-fixed, paraffin-embedded archival samples from 41 cases of neuroblastoma were used for targeted sequencing. RESULTS: A total of 145 somatic mutations were identified, including 51 synonymous, 86 missense, 3 nonsense, 2 frameshift deletion, 2 splice-site, and 1 in-frame deletion mutations. The most frequently mutated gene was ALK (9 missense mutations). The common copy number variations (CNVs) were amplification at 2p24.2 and deletion at 11q22.3 and 1p36.21. ALK mutations were more frequent in patients with stage 4 or 4S (0% vs. 33.3%, p=0.017). Among 27 patients with high-risk disease, the 5-year overall survival was inferior in patients with ALK mutations to those without (25.0% vs. 67.0%, p=0.009). CONCLUSION: Genetic analysis using targeted NGS was feasible and helpful in detecting point mutations and CNVs in neuroblastoma. Targeted NGS could predict prognosis and be used to find molecular target-based therapies for neuroblastoma.

Spontaneous mutations in the single TTN gene represent high tumor mutation burden.

Tumor mutation burden (TMB) is an emerging biomarker, whose calculation requires targeted sequencing of many genes. We investigated if the measurement of mutation counts within a single gene is representative of TMB. Whole-exome sequencing (WES) data from the pan-cancer cohort (n = 10,224) of TCGA, and targeted sequencing (tNGS) and TTN gene sequencing from 24 colorectal cancer samples (AMC cohort) were analyzed. TTN was identified as the most frequently mutated gene within the pan-cancer cohort, and its mutation number best correlated with TMB assessed by WES (rho = 0.917, p < 2.2e-16). Colorectal cancer was one of good candidates for the application of this diagnostic model of TTN-TMB, and the correlation coefficients were 0.936 and 0.92 for TMB by WES and TMB by tNGS, respectively. Higher than expected TTN mutation frequencies observed in other FLAGS (FrequentLy mutAted GeneS) are associated with late replication time. Diagnostic accuracy for high TMB group did not differ between TTN-TMB and TMB assessed by tNGS. Classification modeling by machine learning using TTN-TMB for MSI-H diagnosis was constructed, and the diagnostic accuracy was 0.873 by area under the curve in external validation. TTN mutation was enriched in samples possessing high immunostimulatory signatures. We suggest that the mutation load within TTN represents high TMB status.

Whole transcriptome analysis of gestational trophoblastic neoplasms reveals altered PI3K signaling pathway in epithelioid trophoblastic tumor.

OBJECTIVE: Genomic characteristics of gestational trophoblastic neoplasm (GTN) are mostly unknown. This study reveals the molecular features of malignant GTN, including choriocarcinoma (CC), epithelioid trophoblastic tumor (ETT), and placental site trophoblastic tumor (PSTT), by whole transcriptome sequencing analysis. METHODS: Data obtained from the total RNA sequencing of 2 CC, 4 ETT, and 4 PSTT were evaluated for differential gene expression, pathway alteration, fusion gene, infiltrating immune cell type, PD-L1 and PTEN expression level, and mutation analysis was performed. RESULTS: The transcriptome data were correlated with known biomarkers, including HDS3B1, p63, hCG, and hPL for all tumor types. ETT and PSTT were more closely clustered compared with CC in clustering analysis using gene expression; however, ETT showed various altered signaling pathways, including PI3K-Akt-mTOR, with frequent loss of PTEN protein expression. This finding was both well correlated with PIK3CA c.3140A > G pathogenic mutation, detected in 1 ETT, and further confirmed using the MassARRAY method. PSTT showed an overexpressed gene cluster associated with muscle contraction and G protein-coupled receptor activity. No significant fusion gene was seen in all 10 cases. In tumor-infiltrating immune cell profiles, CD4 memory T cell and macrophage signature were relatively high in ETT and PSTT. PD-L1 mRNA expression level was high in all cases, which was significantly correlated with the PD-L1 level by immunohistochemistry (p = 0.03) with positivity in all 10 cases. CONCLUSIONS: ETT and PSTT were similar at the transcriptome level, with a high level of PD-L1 expression in all tumor types; however, specific pathways, such as PI3K signaling, were altered in ETT.

Prediction of Core Signaling Pathway by Using Diffusion- and Perfusion-based MRI Radiomics and Next-generation Sequencing in Isocitrate Dehydrogenase Wild-type Glioblastoma.

Background Next-generation sequencing (NGS) enables highly sensitive cancer genomics analysis, but its clinical implications for therapeutic options from imaging-based prediction have been limited. Purpose To predict core signaling pathways in isocitrate dehydrogenase (IDH) wild-type glioblastoma by using diffusion and perfusion MRI radiomics and NGS. Materials and Methods The radiogenomics model was developed by using retrospective patients with glioma who underwent NGS and anatomic, diffusion-, and perfusion-weighted imaging between March 2017 and February 2019. For testing model performance in predicting core signaling pathway, patients with IDH wild-type glioblastoma from a retrospective analysis from a registry (ClinicalTrials.gov NCT02619890) were evaluated. Radiogenomic feature selection was performed by using t tests, least absolute shrinkage and selection operator penalization, and random forest. Combining radiogenomic features, age, and location, the performance of predicting receptor tyrosine kinase (RTK), tumor protein p53 (P53), and retinoblastoma 1 pathways was evaluated by using the area under the receiver operating characteristic curve (AUC). Results There were 120 patients (52 years ± 13 [standard deviation]; 61 women) who were evaluated. Eighty-five patients (51 years ± 13; 43 men) were in the training set and 35 patients with IDH wild-type glioblastoma (56 years ± 12; 19 women) were in the validation set. Radiogenomics model identified 71 features in the RTK, 17 features in P53, and 35 features in the retinoblastoma pathway. The combined model showed better performance than anatomic imaging-based prediction in the RTK (P = .03) and retinoblastoma (P = .03) and perfusion imaging-based prediction in the P53 pathway (P = .04) in the training set. AUC values of the combined model for the prediction of core signaling pathways were 0.88 (95% confidence interval [CI]: 0.74, 1) for RTK, 0.76 (95% CI: 0.59, 0.92) for P53, and 0.81 (95% CI: 0.64, 0.97) for retinoblastoma in the validation set. Conclusion A diffusion- and perfusion-weighted MRI radiomics model can help characterize core signaling pathways and potentially guide targeted therapy for isocitrate dehydrogenase wild-type glioblastoma. © RSNA, 2019 Online supplemental material is available for this article.