Acquired Resistance Mechanism of EGFR Kinase Domain Duplication to EGFR TKIs in Non-Small Cell Lung Cancer.

PURPOSE: Epidermal growth factor receptor kinase domain duplication (EGFR-KDD) is a rare and poorly understood oncogenic mutation in non-small cell lung cancer (NSCLC). We aimed to investigate the acquired resistance mechanism of EGFR-KDD against EGFR-TKIs. MATERIALS AND METHODS: We identified EGFR-KDD in tumor tissue obtained from a patient with stage IV lung adenocarcinoma and established the patient-derived cell line SNU-4784. We also established several EGFR-KDD Ba/F3 cell lines: EGFR-KDD wild type (EGFR-KDDWT), EGFR-KDD domain 1 T790M (EGFR-KDDD1T), EGFR-KDD domain 2 T790M (EGFR-KDDD2T), and EGFR-KDD both domain T790M (EGFR-KDDBDT). We treated the cells with EGFR tyrosine kinase inhibitors (TKIs) and performed cell viability assays, immunoblot assays, and ENU (N-ethyl-N-nitrosourea) mutagenesis screening. RESULTS: In cell viability assays, SNU-4784 cells and EGFR-KDDWT Ba/F3 cells were sensitive to 2nd generation and 3rd generation EGFR TKIs. In contrast, the T790M-positive EGFR-KDD Ba/F3 cell lines (EGFR-KDDT790M) were only sensitive to 3rd generation EGFR TKIs. In ENU mutagenesis screening, we identified the C797S mutation in kinase domain 2 of EGFR-KDDBDT Ba/F3 cells. Based on this finding, we established an EGFR-KDD domain 1 T790M/domain 2 cis-T790M+C797S (EGFR-KDDT/T+C) Ba/F3 model, which was resistant to EGFR TKIs and anti-EGFR monoclonal antibody combined with EGFR TKIs. CONCLUSION: Our study reveals that the T790M mutation in EGFR-KDD confers resistance to 1st and 2nd generation EGFR TKIs, but is sensitive to 3rd generation EGFR TKIs. In addition, we identified that the C797S mutation in kinase domain 2 of EGFR-KDDT790M mediates a resistance mechanism against 3rd generation EGFR TKIs.

NTRK and RET fusion-directed therapy in pediatric thyroid cancer yields a tumor response and radioiodine uptake.

BACKGROUNDMolecular characterization in pediatric papillary thyroid cancer (PTC), distinct from adult PTC, is important for developing molecularly targeted therapies for progressive radioiodine-refractory (131I-refractory) PTC.METHODSPTC samples from 106 pediatric patients (age range: 4.3-19.8 years; n = 84 girls, n = 22 boys) who were admitted to SNUH (January 1983-March 2020) were available for genomic profiling. Previous transcriptomic data from 125 adult PTC samples were used for comparison.RESULTSWe identified genetic drivers in 80 tumors: 31 with fusion oncogenes (RET in 21 patients, ALK in 6 patients, and NTRK1/3 in 4 patients); 47 with point mutations (BRAFV600E in 41 patients, TERTC228T in 2 patients [1 of whom had a coexisting BRAFV600E], and DICER1 variants in 5 patients); and 2 with amplifications. Fusion oncogene PTCs, which are predominantly detected in younger patients, were at a more advanced stage and showed more recurrent or persistent disease compared with BRAFV600E PTCs, which are detected mostly in adolescents. Pediatric fusion PTCs (in patients <10 years of age) had lower expression of thyroid differentiation genes, including SLC5A5, than did adult fusion PTCs. Two girls with progressive 131I-refractory lung metastases harboring a TPR-NTRK1 or CCDC6-RET fusion oncogene received fusion-targeted therapy; larotrectinib and selpercatinib decreased the size of the tumor and restored 125I radioiodine uptake. The girl with the CCDC6-RET fusion oncogene received 131I therapy combined with selpercatinib, resulting in a tumor response. In vitro 125I uptake and 131I clonogenic assays showed that larotrectinib inhibited tumor growth and restored radioiodine avidity.CONCLUSIONSIn pediatric patients with fusion oncogene PTC who have 131I-refractory advanced disease, selective fusion-directed therapy may restore radioiodine avidity and lead to a dramatic tumor response, underscoring the importance of molecular testing in pediatric patients with PTC.FUNDINGThe Ministry of Science, ICT and Future Planning (NRF-2016R1A2B4012417 and 2019R1A2C2084332); the Korean Ministry of Health and Welfare (H14C1277); the Ministry of Education (2020R1A6A1A03047972); and the SNUH Research Fund (04-2015-0830).TRIAL REGISTRATIONTwo patients received fusion-targeted therapy with larotrectinib (NCT02576431; NAVIGATE) or selpercatinib (LOXO-RET-18018).

Phase II study of durvalumab and tremelimumab in pulmonary sarcomatoid carcinoma: KCSG-LU16-07.

BACKGROUND: Pulmonary sarcomatoid carcinoma (PSC) is rare with a poor outcome and is resistant to conventional cytotoxic chemotherapy. The efficacy and safety of durvalumab and tremelimumab for treating recurrent or metastatic PSCs were assessed by a nonrandomized, open-label, phase II study. METHODS: A total of 18 patients with recurrent or metastatic PSC received 1500 mg of durvalumab and 75 mg of tremelimumab every four weeks, followed by 750 mg of durvalumab every two weeks until the disease progressed, or an unacceptable toxicity level was reached. The primary endpoint was the objective response rate (ORR). The secondary endpoints were progression-free survival (PFS), overall survival (OS), and toxicity. Genomic profiling of PSC by next-generation sequencing (NGS) and determination of peripheral blood lymphocyte subsets using flow cytometry were performed for exploratory analysis. RESULTS: A total of 15 out of 18 patients were evaluated for the analysis of the primary endpoint. At the data cutoff point, the ORR of 26.7% (95% confidence interval [CI]: 7.8-55.1) was achieved with the median follow-up duration of 12.0 months (range, 8.4-16.1). Median PFS and OS were 5.9 months (95% CI: 1.1-11.9) and 15.4 months (95% CI: 11.1-not reached), respectively. Treatment-related adverse events (AEs) of any grade were reported in 16 patients; the most common AEs were pruritus (n = 5), pneumonitis (n = 4), and rash (n = 4). Treatment was discontinued in two patients due to AEs of grade ≥ 3. CONCLUSIONS: Durvalumab and tremelimumab demonstrated clinical benefit with a prolonged survival and manageable toxicity profile in patients with recurrent or metastatic PSC.

A newly developed capture-based sequencing panel for genomic assay of lung cancer.

BACKGROUND: The increase in genetic alterations targeted by specific chemotherapy in lung cancer has led to the need for universal use of more comprehensive genetic testing, which has highlighted the development of a lung cancer diagnostic panel using next-generation sequencing. OBJECTIVE: We developed a hybridization capture-based massively parallel sequencing assay named Friendly, Integrated, Research-based, Smart and Trustworthy (FIRST)-lung cancer panel (LCP), and evaluated its performance. METHODS: FIRST-LCP comprises 64 lung cancer-related genes to test for various kinds of genetic alterations including single nucleotide variations (SNVs), insertions and deletions (indels), copy number variations (CNVs), and structural variations. To assess the performance of FIRST-LCP, we compiled test sets using HapMap samples or tumor cell lines with disclosed genetic information, and also tested our clinical lung cancer samples whose genetic alterations were known by conventional methods. RESULTS: FIRST-LCP accomplished high sensitivity (99.4%) and specificity (100%) of the detection of SNVs. High precision was also achieved, with intra- or inter-run concordance rate of 0.99, respectively. FIRST-LCP detected indels and CNVs close to the expected allele frequency and magnitude, respectively. Tests with samples from lung cancer patients also identified all SNVs, indels and fusions. CONCLUSION: Based on the current state of the art, continuous application of the panel design and analysis pipeline following up-to-date knowledge could ensure precision medicine for lung cancer patients.

A population-specific low-frequency variant of SLC22A12 (p.W258*) explains nearby genome-wide association signals for serum uric acid concentrations among Koreans.

Prolonged hyperuricemia is a cause of gout and an independent risk factor for chronic health conditions including diabetes and chronic kidney diseases. Genome-wide association studies (GWASs) for serum uric acid (SUA) concentrations have repeatedly confirmed genetic loci including those encoding uric acid transporters such as ABCG2 and SLC9A2. However, many single nucleotide polymorphisms (SNPs) found in GWASs have been common variants with small effects and unknown functions. In addition, there is still much heritability to be explained. To identify the causative genetic variants for SUA concentrations in Korean subjects, we conducted a GWAS (1902 males) and validation study (2912 males and females) and found four genetic loci reaching genome-wide significance on chromosomes 4 (ABCG2) and 11 (FRMD8, EIF1AD and SLC22A12-NRXN2). Three loci on chromosome 11 were distributed within a distance of 1.3 megabases and they were in weak or moderate linkage disequilibrium (LD) states (r2 = 0.02-0.68). In a subsequent association analysis on the GWAS loci of chromosome 11 using closely positioned markers derived from whole genome sequencing data, the most significant variant to be linked with the nearby GWAS signal was rs121907892 (c.774G>A, p.W258*) of the SLC22A12 gene. This variant, and each of the three GWAS SNPs, were in LD (r2 = 0.06-0.32). The strength of association of SNPs with SUA concentration (negative logarithm of P-values) and the genetic distance (r2 of LD) between rs121907892 and the surrounding SNPs showed a quantitative correlation. This variant has been found only in Korean and Japanese subjects and is known to lower the SUA concentration in the general population. Thus, this low-frequency variant, rs121907892, known to regulate SUA concentrations in previous studies, is responsible for the nearby GWAS signals.

Predominant DICER1 Pathogenic Variants in Pediatric Follicular Thyroid Carcinomas.

Background: Pediatric thyroid cancer has characteristics that are distinct from adulthood thyroid cancer. Due to its very low prevalence, little is known about the genetic characteristics of pediatric follicular thyroid cancer (FTC). Methods: We investigated genetic alterations in tumor tissues from 15 patients aged <20 years (median: 14.3 years; range: 2.4 - 19.0 years) using multifaceted approaches. Whole-exome sequencing, targeted next-generation sequencing using a cancer gene panel, and Sanger sequencing of the major exons of the H/K/N-RAS and DICER1 genes and the promoter region of the TERT gene were performed. Normal tissues or blood of patients with DICER1- or PTEN-positive tumors was also evaluated to determine whether the variant is germ line. Results: The median tumor size was 3.1 cm (range: 0.6 - 6.4 cm). Four patients exhibited angioinvasion and one extensive capsular invasion; none showed evidence of disease over a median of 8.1 years. Eight patients (53.3%) had DICER1 variants, including four with DICER1 syndrome (three patients were <10 years of age). One patient had a germ line PTEN frameshift variant with the diagnosis of PTEN hamartoma tumor syndrome. One patient had a PAX8/PPARγ rearrangement, and two patients had no genetic driver alteration other than multiple loss of heterozygosity with or without copy number alterations in their tumors. No RAS or TERT variants were found. Nodular hyperplasia and follicular adenoma (FA) coexisted in DICER1 variant-positive FTCs more frequently than variant-negative FTCs (p = 0.026). All DICER1 variant-positive FTCs had a somatic missense variant at metal binding sites (six at codon p.E1813 and two at codon p.D1709) within the RNase IIIb domain; seven had other missense, nonsense, or frameshift variants in the DICER1 gene. Six coexisting FAs of two patients with DICER1 syndrome (three of each) had additional somatic variants at metal binding sites within the RNase IIIb domain (codon p.E1705, p.D1709, p.D1810, or p.E1813), different from each other and from the indexed FTC tumor. Conclusions: Pediatric FTCs have distinct genomic alterations and pathogenesis compared with adults, particularly those characterized by DICER1 variants. The DICER1 variant should be considered in pediatric FTCs, especially in cases <10 years of age. In all DICER1 variant-positive FTCs and FAs, recurrent hotspot variants were found at metal binding sites within the RNase IIIb domain, suggesting they impact tumorigenesis.

Genomic Landscape of Young-Onset Bladder Cancer and Its Prognostic Implications on Adult Bladder Cancer.

Due to the rare occurrence of young-onset bladder cancer (YBC), its genomic characteristics remain largely unknown. Twenty-nine biopsy-proven YBC cases were collected using a nation-wide search for bladder cancer diagnosed at 20 years or younger. Whole exome sequencing and RNA sequencing were carried out in 21 and 11 cases, respectively, and compared with those of adult bladder cancer (ABC) cases obtained from public databases. Almost all YBCs were low grade, non-invasive papillary tumors. YBC had a low mutation burden and less complex copy number alterations. All cases harbored putative driver mutations. Mutations were most commonly found in HRAS (10 cases), with a preference for exon 5. FGFR3 gene fusions were noted with various partner genes (7 cases). The alterations on HRAS and FGFR3 occurred in a mutually exclusive manner. Others included KRAS mutations (2 cases), chromosomes 4p and 10q arm-level deletions (1 case), and ERCC2 mutation (1 case). There were no point mutations in TP53 and FGFR3. The gene expression profiles of YBC were similar to those of the ABC group with good prognosis. None of the YBCs and ABCs with YBC-like mutations showed progression to muscle-invasive tumors. Our results suggest that bladder cancer with YBC-like mutations represents an indolent bladder tumor, regardless of age.

Clinicopathological and molecular analysis of multinodular and vacuolating neuronal tumors of the cerebrum.

Multinodular and vacuolating neuronal tumor (MVNT) of the cerebrum is a recently recognized rare neuronal tumor, and its pathogenesis is unclear. We analyzed 7 cases of histologically typical MVNT: 6 were adults (mean age, 43.0 years [range, 23-56 years]) and 1 was a child (age, 10 years). The most common symptoms were seizures (n = 4) and headache (n = 2). The tumors were supratentorial (temporal, 5; frontal lobes, 2) in origin as reported. Vacuolated tumor cells were robustly positive for α-INA and Olig2 and at least partly positive for synaptophysin and MAP2, but negative for Neu-N, nestin and CD34. GFAP and vimentin were expressed in reactive astrocytes but not in tumor cells. Negative results were obtained for p53, IDH-1, BRAFV600E, H3 K27M, EGFR, Lin28A, and L1CAM. ATRX, BRG1, INI-1, and TMHH were retained. The Ki-67 labeling index was very low (<1%), and pHH3 revealed no mitotic figure. Ultrastructural features of tumor cells were comparable with those of immature neuronal cells, with several intracytoplasmic myelin-like autophagosomes and pericellular vacuolization. No IDH1/IDH2 and BRAFV600E mutations were found upon direct sequencing. Whole-exome sequencing revealed FGFR2-ZMYND11 gene fusion in 1 case. After gross total resection, all patients were alive without seizures. There was no tumor recurrence during an average period of 68 months (range, 23-101 months). The analysis of 7 typical cases of MVNT suggested that these lesions may be clonal tumors because FGFR2-ZMYND11 fusion was found (1 case).

Genome-Wide Association Study Reveals Distinct Genetic Susceptibility of Thyroid Nodules From Thyroid Cancer.

Context: Thyroid nodules are very common, and 7% to 15% of them are diagnosed as thyroid cancer. However, the inherited genetic risk factors for thyroid nodules and their associations with thyroid cancer remain unknown. Objective: To identify the genetic variants associated with susceptibility to thyroid nodules in comparison with thyroid cancer. Design and Setting: We performed a three-stage genome-wide association study for thyroid nodules. The discovery stage involved a genome-wide scan of 811 subjects with thyroid nodules and 691 subjects with a normal thyroid from a population-based cohort. Replication studies were conducted in an additional 1981 cases and 3100 controls from the participants of a health checkup. We also performed expression quantitative trait loci analysis of public data. Results: The most robust association was observed in TRPM3 (rs4745021) in the joint analysis (OR, 1.26; P = 6.12 × 10-8) and meta-analysis (OR, 1.28; P = 2.11 × 10-8). Signals at MBIP/NKX2-1 were replicated but did not reach genome-wide significance in the joint analysis (rs2415317, P = 4.62 × 10-5; rs944289, P = 8.68 × 10-5). The expression quantitative trait loci analysis showed that TRPM3 expression was associated with the rs4745021 genotype in thyroid tissues. Conclusions: To the best of our knowledge, we have performed the first genome-wide association study of thyroid nodules and identified a susceptibility locus associated with thyroid nodules, suggesting that thyroid nodules have a genetic predisposition distinct from that of thyroid cancer.

Genome-wide association and expression quantitative trait loci studies identify multiple susceptibility loci for thyroid cancer.

Thyroid cancer is the most common cancer in Korea. Several susceptibility loci of differentiated thyroid cancer (DTC) were identified by previous genome-wide association studies (GWASs) in Europeans only. Here we conducted a GWAS and a replication study in Koreans using a total of 1,085 DTC cases and 8,884 controls, and validated these results using expression quantitative trait loci (eQTL) analysis and clinical phenotypes. The most robust associations were observed in the NRG1 gene (rs6996585, P=1.08 × 10-10) and this SNP was also associated with NRG1 expression in thyroid tissues. In addition, we confirmed three previously reported loci (FOXE1, NKX2-1 and DIRC3) and identified seven novel susceptibility loci (VAV3, PCNXL2, INSR, MRSB3, FHIT, SEPT11 and SLC24A6) associated with DTC. Furthermore, we identified specific variants of DTC that have different effects according to cancer type or ethnicity. Our findings provide deeper insight into the genetic contribution to thyroid cancer in different populations.