Sensitivity and optimal clinicopathological features for mutation-targeted liquid biopsy in pN0M0 EGFR-mutant lung adenocarcinoma.

PURPOSE: Liquid biopsy for early-stage lung cancer diagnosis is challenging, and optimal candidates' clinicopathological features are unknown. We investigated utility and clinicopathological features of optimal candidates in somatic mutation-targeted liquid biopsy using droplet digital polymerase chain reaction (ddPCR) in pN0M0 EGFR mutation-positive lung adenocarcinoma patients. METHODS: We performed EGFR mutation-targeted ddPCR liquid biopsy in 100 patients with resected pN0M0 invasive lung adenocarcinoma, whose tumor diameter in high-resolution computed tomography (HRCT) was ≤ 5 cm. Peripheral blood-derived serum was collected preoperatively. Two representative EGFR somatic variants (exon 19 [E746-A750 del (2235_2249 del)]; exon 21 (L858R)) were utilized as liquid biopsy targets. Clinicopathological features including radiological appearance, subhistology, and invasive status were compared between ddPCR-positive and ddPCR-negative patients. RESULTS: Among the 100 patients, 98 showed part-solid or pure-solid appearance in HRCT and 2 showed non-solid appearance; 98 were pathological stage IA1-IB. Of the 66 patients with EGFR mutation detection in ddPCR, 12 were significantly positive and 10 (83.3%, 10/12) exhibited pure-solid appearance in HRCT. Clinical invasive tumor ratio was significantly higher in ddPCR-positive than in ddPCR-negative patients (median: 100% vs. 85.4%, P = 0.0212), whereas other clinicopathological features were not significantly different. CONCLUSION: Mutation-targeted liquid biopsy using ddPCR detected lung cancer in 12.0% (12/100) of pN0M0 EGFR-mutant lung adenocarcinoma patients. In 83.3% of the ddPCR-positive patients, tumors showed pure-solid appearance in HRCT. The detection ratio increased to 21.3% (10/47) among patients with pure-solid appearance tumors. Tumor appearance might be useful for better selection of liquid biopsy candidates.

Synchronicity of genetic variants between primary sites and metastatic lymph nodes, and prognostic impact in nodal metastatic lung adenocarcinoma.

PURPOSE: Nodal positive lung adenocarcinoma includes wide range of survival. Several methods for the classification of nodal-positive lung cancer have been proposed. However, classification considering the impact of targetable genetic variants are lacking. The possibility of genetic variants for the better stratification of nodal positive lung adenocarcinoma was estimated. METHODS: Mutations of 36 genes between primary sites and metastatic lymph nodes (LNs) were compared using next-generation sequencing. Subsequently, mutations in EGFR and BRAF, rearrangements in ALK and ROS1 were evaluated in 69 resected pN1-2M0 adenocarcinoma cases. Recurrence-free survival (RFS), post-recurrence survival (PRS), and overall survival (OS) were evaluated with respect to targetable variants and tyrosine kinase inhibitor (TKI) therapy after recurrence. RESULTS: About 90% of variants were shared and allele frequencies were similar between primary and metastatic sites. In 69 pN1-2M0 cases, EGFR/ALK were positive in primary sites of 39 cases and same EGFR/ALK variants were confirmed in metastatic LNs of 96.7% tissue-available cases. Multivariate analyses indicated positive EGFR/ALK status was associated with worse RFS (HR 2.366; 95% CI 1.244-4.500; P = 0.009), and PRS was prolonged in cases receiving TKI therapy (no post-recurrence TKI therapies, HR 3.740; 95% CI 1.449-9.650; P = 0.006). OS did not differ with respect to targetable variants or TKI therapy. CONCLUSIONS: Cases harbouring targetable genetic variants had a higher risk of recurrence, but PRS was prolonged by TKI therapy. Classification according to the targetable genetic status provides a basis for predicting recurrence and determining treatment strategies after recurrence.

Therapeutic strategies and genetic profile comparisons in small cell carcinoma and large cell neuroendocrine carcinoma of the lung using next-generation sequencing.

Small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC) of the lung are classified as variants of endocrine carcinoma and subdivided into pure or combined type. Clinical benefit of target therapy has not been established in these tumors. This study aimed to compare genetic and clinicopathological features between SCLC and LCNEC or pure and combined types, and explore the possibility of target therapy using next-generation sequencing. In 13 SCLC and 22 LCNEC cases, 72 point mutations, 19 deletions, and 3 insertions were detected. As therapeutically targetable variants, mutations in EGFR (L858R), KRAS (G12D, G12A, G12V), and PIK3CA (E545K) were detected in 5 cases. The case harboring EGFR mutation showed response to EGFR-tyrosine kinase inhibitor. However, there are no clinicopathological features associated with therapeutically targetable cases. And there was no significant genetic feature between SCLC and LCNEC or pure and combined types. In conclusion, although patients with SCLC and LCNEC may benefit from target therapy, they were not identifiable by clinicopathologic background. And there was not significant genetic difference between SCLC and LCNEC, including between pure and combined types. Classifying SCLC and LCNEC in same category is reasonable. However, distinguishing the pure type from combined type was not validated. Comprehensive genetic analysis should be performed to detect targetable variants in any type of SCLC and LCNEC.

Circulating free DNA as non-invasive diagnostic biomarker for childhood solid tumors.

PURPOSE: Our aims are to determine circulating free DNA (cfDNA) in childhood solid tumor patients who underwent surgical intervention and to analyze any relationships with clinical parameters. METHODS: Fourty-four consenting children admitted with solid tumors between 2010 and 2014 were recruited. CfDNAs isolated from 0.5mL plasma obtained before and 1-30days after surgery were analyzed by next-generation sequencing (NGS: IonTorrent Cancer Hotspot panel) and by gene amplification analysis using a digital PCR (dPCR) platform. RESULTS: Total amounts of cfDNA were 54-825ng and were significantly associated with stage of disease. In cfDNA, 15 mutations or deletions (2 ALK, 2 TP53, 1 WT1, 3 CTNNB1, 1 APC, 1 KIT, 1 RET, 1 CDNK2AT, and 3 SMARCB1) were identified. In 10 neuroblastoma suspected cases, 2 showed high copy numbers of MYCN using dPCR. The positive rate in our cohort was 36%, and all of these aberrations were detected in the original tumors. None of the aberrations were detectable in cfDNA after surgery except for three cases whose tumors remained after surgery. CONCLUSIONS: These data demonstrate the feasibility and potential utility of mutation/deletion/amplification screening in cfDNA using NGS and dPCR for the detection of tumor biomarkers in children with solid tumors. These markers also have the potential utility to evaluate complete resection after surgery.