Development and clinical applications of an enclosed automated targeted NGS library preparation system.

The rapid development of next-generation sequencing (NGS) technology has promoted its wide clinical application in precision medicine for oncology. However, laborious and time-consuming manual operations, highly skilled personnel requirements, and cross-contamination are major challenges for the clinical implementation of NGS technology-based tests. The Automated NGS Diagnostic Solutions (ANDiS) 500 system is a fully enclosed cassette-dependent automated NGS library preparation system. This platform could produce qualified targeted amplicon library in three steps with only 15 min of hands-on time. Rigorous cross-contamination test using simulated contaminant plasmids confirmed that the design of disposable cassette guarantees zero sample cross-contamination. The BRCA1 and BRCA2 mutation detection panel and gastrointestinal cancer-related gene analysis panel for the ANDiS 500 platform showed 100% accuracy and precision in detecting germ-line mutations and somatic mutations respectively. Furthermore, those panels showed 100% concordance with verified methods in a prospective cohort study enrolling 363 patients and a cohort of 45 pan-cancer samples. In conclusion, the ANDiS 500 automated platform could overcome major challenges for implementing NGS assays clinically and is eligible for routine clinical tests.

Case report: TP53 and RB1 loss may facilitate the transformation from lung adenocarcinoma to small cell lung cancer by expressing neuroendocrine markers.

Introduction: Transformation from lung adenocarcinoma (LUAD) to small cell lung cancer (SCLC) is one of the mechanisms responsible for acquired EGFR-TKIs resistance. Although it rarely happens this event determines a rapid disease deterioration and needs specific treatment. Patient and method: We report a case of 75-year-old LUAD female with a p.L858R mutation in Epidermal Growth Factor Receptor (EGFR) who presented with SCLC transformation after responding to first line osimertinib treatment for only 6 months. To understand the underlying molecular mechanism, we retrospectively sequenced the first (LUAD) and the second (SCLC) biopsy using a 56 multi-gene panel. Immunohistochemistry (IHC) staining and Fluorescence In Situ Hybridization (FISH) was applied to confirm the genetic aberrations identified. Results: EGFR p.E709A and p.L858R, Tumor Protein p53 (TP53) p.A159D and Retinoblastoma 1 (RB1) c.365-1G>A were detected in both the diagnostic LUAD and transformed SCLC samples. A high copy number gain for Proto-Oncogene C-Myc (MYC) and a Phosphoinositide 3-Kinase Alpha (PIK3CA) p.E545K mutation were found in the transformed sample specifically. Strong TP53 staining and negative RB1 staining were observed in both LUAD and SCLC samples, but FISH only identified MYC amplification in SCLC tissue. Conclusion: We consider the combined presence of MYC amplification with mutations in TP53 and RB1 as drivers of SCLC transformation. Our results highlight the need to systematically evaluate TP53 and RB1 status in LUAD patients to offer a different therapeutic strategy.

Comprehensive analysis of aneuploidy status and its effect on the efficacy of EGFR-TKIs in lung cancer.

Background: Lung cancer has the highest mortality rate among cancers worldwide, and most patients are diagnosed with non-small-cell lung cancer (NSCLC), and evaluating the clinical efficacy of molecularly targeted cancer therapy remains a major challenge. Methods: This paper retrospectively investigated the outcome information of 291 lung cancer patients detected by next-generation sequencing (NGS) analysis and fluorescence in situ hybridization (FISH), including 63 patients with lung cancer who were followed up. We analyzed epidermal growth factor receptor (EGFR) mutation abundance and aneuploidy status to evaluate clinical efficacy. Results: The progress free survival (PFS) of patients diagnosed as euploidy was actually higher than that of patients diagnosed with aneuploidy, and was related to both the objective response rate (ORR) and disease control rate (DCR). Patients with an epidermal growth factor receptor (EGFR) mutation abundance ≥28.86% had slightly higher ORR and similar DCR. Two-way analysis of variance was used to assess the effects of EGFR mutation abundance and tumor aneuploidy status on patients' PFS. The results indicated a strong correlation between aneuploidy status and clinical efficacy, with euploid patients having a higher ORR and DCR. Conclusions: Aneuploidy status could effectively evaluate the clinical efficacy of patients with lung cancer. However, EGFR mutations abundance could not predict the extent of benefit from tyrosine kinase inhibitors (EGFR-TKI) treatment.

Implementation of a pathological diagnosis and treatment pathway may improve the molecular detection of lung cancer.

Background: The 2018 Guideline from the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) established a benchmark turnaround time (TAT), according to which the results should be available to the treating oncologist within 10 working days. This article focused on the application process of a new protocol for pathological diagnosis and gene testing pathway and a sample collector. We want to solve the problem that there are not enough puncture samples for gene testing, and the benchmark turnaround time of gene detection was long in clinic. Methods: In this study, we established and validated a new protocol for a pathological diagnosis and treatment pathway that was tested in the Henan Cancer Hospital, China. The "Biology collector (BIOCO)" tool was designed by our team, was made of polyvinyl chloride (PVC) material (patent application number is 201820902335.6). It consisted of two round magnets on the collector that can be adsorbed on the microtome blade holder, thus making it to move arbitrarily. It collected specimens that were discarded when the wax block was trimmed. We analyzed the TAT, testing accuracy and anti-pollution of new protocol based on BIOCO, compared with the conventional process based on the Routine Collection (ROUCO). Results: The new pathway adopts a parallel approach to conventional pathology and molecular pathology, which significantly shortens the TAT to 4-6 days. The use of the BIOCO tool can effectively save pathological samples, avoid cross-contamination, and reduce the time delay caused by re-sampling. Most importantly, its accuracy and effectiveness are consistent with conventional collection methods. Conclusions: The new diagnosis and treatment pathway based on the BIOCO collector can be used as a practical approach for the molecular diagnostic platform of the hospital pathology department.

Perioperative chemotherapy with pemetrexed and cisplatin for pulmonary large-cell neuroendocrine carcinoma: a case report and literature review.

BACKGROUND: Pulmonary large-cell neuroendocrine carcinoma (LCNEC) is associated with poor prognosis, and its treatment strategy is still controversial, especially regarding chemotherapy regimens. CASE REPORT: We present the case of a 49-year-old Chinese male with primary pulmonary LCNEC treated with neoadjuvant and adjuvant chemotherapy with cisplatin plus pemetrexed. A suspected quasi-circular mass in the left lower pulmonary lobe and an enlarged mediastinal lymph node were found. The patient was diagnosed with adenocarcinoma with neuroendocrine differentiation based on computerized tomography-guided percutaneous lung biopsy. An EGFR gene mutation test showed negative results. Cisplatin and pemetrexed were administered as the neoadjuvant chemotherapy regimen. The primary lesion had reduced markedly, and the enlarged mediastinal lymph node had disappeared after two cycles of neoadjuvant chemotherapy. A left lower lobectomy and mediastinal lymph node dissection were performed. The lesion was confirmed as LCNEC based on postoperative histopathological analysis and immunohistochemical results. The patient underwent four cycles of adjuvant chemotherapy with cisplatin and pemetrexed for a month postoperatively, followed by postoperative adjuvant radiotherapy. The patient was still alive after a follow-up of 24 months, with no evidence of tumor recurrence. CONCLUSION: Cisplatin combined with pemetrexed is effective and safe for patients with pulmonary LCNEC.

Single nucleotide polymorphisms of casitas B-lineage lymphoma proto-oncogene-b predict outcomes of patients with advanced non-small cell lung cancer after first-line platinum based doublet chemotherapy.

BACKGROUND: Casitas B-lineage lymphoma proto-oncogene-b (CBLB) influences the threshold of T cell activation and controlling peripheral T cell tolerance. In the present study, we hypothesize that potentially functional single nucleotide polymorphisms (SNPs) in CBLB are associated with clinical outcomes in patients advanced non-small cell lung cancer (NSCLC) treated with the first-line chemotherapy. METHODS: We genotyped three SNPs (rs2305035, rs3772534 and rs9657904) at CBLB in 116 advanced NSCLC patients with progression free survival (PFS) data and 133 advanced NSCLC patients with overall survival (OS) data, and we assessed their associations, 95% confidence interval (CI), with clinical outcomes by using Cox proportional hazards regression analyses. In silico functional analysis was also performed for the SNPs under investigation. RESULTS: We found that associations between the three SNPs and PFS/OS were not significant in the overall NSCLC patients. The rs2305035 AA genotype was associated with a worse PFS in female patients and those of non-smokers or light smokers (95% CI, 1.14-11.81, P=0.030; 95% CI, 1.42-10.24, P=0.008; and 95% CI, 1.39-9.93, P=0.009; respectively), compared with the GG+AA genotypes. We also found that the rs9657904 CC genotype was significantly associated with a worse OS than TT + TC genotypes in male advanced NSCLC patients. Further in silico functional analysis revealed that the rs965704 T allele was significantly associated with lower mRNA expression levels of the CBLB gene. CONCLUSIONS: Our findings identified two CBLB SNPs (rs2305035 and rs9657904) that were significantly associated with PFS and OS in several subgroups of Chinese advanced NSCLC patients after the first-line chemotherapy.

Pathological complete response to gefitinib in a 10-year-old boy with EGFR-negative pulmonary mucoepidermoid carcinoma: a case report and literature review.

BACKGROUND AND AIMS: Pulmonary Mucoepidermoid carcinoma (MEC) accounts for 0.1-0.2% of all lung cancer. It occurred in the 3-78 years old, and 50% patients younger than 30. MEC has no standard treatment, but recently reports indicated MEC without epidermal growth factor receptor (EGFR) mutations sensitive to gefitinib. OBJECTIVES: To explore a new standard treatment strategy for MEC, after reviewed literature related to MEC, we used Gefitinib to treatment a patient with EGFR-negative MEC, and observe its effects. METHODS: 10-year-old boy was diagnosed with low-grade MEC by bronchial lung biopsy, EGFR gene mutation test was negative. Gefitinib was administered as neoadjuvant therapy at 125 mg daily. RESULTS: The patient underwent right middle lobe, lower lobe resection and mediastinal lymph node dissection. After surgery, the patient had gained weight (5 kg) after 18 days of gefitinib therapy. A CT scan of the chest 1 month after surgical resection showed no recurrence, and followed for 22 months after treatment without tumour recurrence, suggesting that the patient was completely cured. CONCLUSION: Gefitinib has potential to become a standard treatment for pulmonary MEC patients, including pediatric patients. However, the mechanisms need further investigation.

Esophageal Adenocarcinoma and Its Rare Association with Barrett's Esophagus in Henan, China.

Incidence of esophageal adenocarcinoma (EAC) has increased sharply in Western Europe and United States over the past three decades. Nearly all cases of EAC in the west are thought to be associated with Barrett's esophagus (BE) at the time of diagnosis. Regions in the Henan province of China have one of world's highest incidences of esophageal cancer, yet recent temporal trends in the relative rates of EAC with respect to esophageal squamous-cell carcinoma (ESCC), as well as its association with Barrett's esophagus (BE), have not been reported. In this report, we present large-scale longitudinal clinical and histological data on 5401 esophageal cancers (EC) patients diagnosed during the recent 10-year period (2002-2011) at Henan Cancer Hospital, China. All 217 esophageal adenocarcinoma (EAC) patients from these 5401 EC patients were examined to better understand the relationship between Barrett's esophagus (BE) and EAC. We found that EAC was relatively rare and accounted for approximately 5% of all esophageal cancers each year during 2002-2011. There is no evidence of significant temporal trends in the rate of EAC relative to ESCC. Only 10 out of 217 (4.6%) EAC cases were detected to have any evidence of Barrett's esophagus. This result raises the possibility of a different etiological basis for EAC in China motivating more detailed epidemiological, clinical and molecular characterization of EAC in China in order to better understand the neoplastic development of EAC.

Quantification of EGFR mutations in primary and metastatic tumors in non-small cell lung cancer.

BACKGROUND: EGFR mutation detection has been widely applied in the prediction of TKIs therapy in Non-Small Cell Lung Cancer (NSCLC). Metastatic tumors rather than primary tumors were usually assayed for those patients in advanced stages. Although the difference of EGFR mutation status in primary and metastatic tumors has been reported, the quantitative difference (ratio of mutated EGFR among total EGFR) in primary and metastatic tumors as well as in different sites of primary tumors was not clear. METHODS: Genomic DNA in Formalin Fixed-Paraffin Embedded samples of primary and metastatic tumors of 50 NSCLC patients was extracted. Real-time fluorescent PCR was performed to quantify the EGFR mutation ratios. RESULTS: The EGFR mutation ratios detected in different sites of primary tumors were highly concordant, whereas the EGFR mutation ratios in metastatic tumors were lower than those in primary tumors. CONCLUSIONS: Randomly chosen sample may reliably represent the type and ratio of mutations of EGFR in primary tumors. EGFR mutation ratios in primary tumors and metastatic tumors are different. If metastatic tumors are used for the detection of EGFR mutation, the sensitivity of the detection assay must be considered.

Prognostic impact of p16 and p21 on gastroenteropancreatic neuroendocrine tumors.

Aberrant expression of the cell cycle kinase inhibitors, p16 and p21, has been associated with poor prognosis in a number of human malignancies. These proteins may also be involved in the development and progression of gastroenteropancreatic neuroendocrine tumors (GEP-NETs). The present study aimed to investigate protein levels of p16 and p21 in GEP-NETs and to evaluate their clinical significance. p16 and p21 protein expression was tested immunohistochemically in the tissue samples of 68 GEP-NETs. The association between expression and clinicopathological characteristics and overall survival was assessed. Low expression of p16 (no positive nuclear staining) was found in 37 (54%) cases and high p21 expression (≥5% positive nuclear staining) was detected in 23 (34%) cases. Low p16 protein levels indicated a poorer prognosis for patients graded as G2 subgroup in the univariate analysis (relative risk, 4.4; 95% CI, 1.8-10.6). No significant correlation was found between the expression of p21 and any of the clinicopathological variables. The present study indicates a prognostic relevance for p16 immunoreactivity. Low levels of p16 protein were associated with a shorter survival in the G2 subgroup of GEP-NETs. p21 protein expression was not identified to be useful as a predictive indicator in GEP-NETs.

Prognostic impact of cyclin D1, cyclin E and P53 on gastroenteropancreatic neuroendocrine tumours.

Conventional classifications of gastroenteropancreatic neuroendocrine tumours (GEP- NETs) are rather unsatisfactory because of the variation in survival within each subgroup. Molecular markers are being found able to predict patient outcome in more and more tumours. The aim of this study was to characterize the expression of the proteins cyclin D1, cyclin E and P53 in GEP- NETs and assess any prognostic impact. Tumor specimens from 68 patients with a complete follow-up were studied immunohistochemically for cyclin D1, cyclin E and P53 expression. High cyclin D1 and cyclin E immunostaining (≥ 5% positive nuclei) was found in 48 (71%) and 24 (35%) cases, and high P53 staining (≥ 10% positive nuclei) in 33 (49%) . High expression of P53 was more common in gastric neuroendocrine tumors and related to malignant behavior, being associate with a worse prognosis on univariate analysis (RR=1.9, 95%CI=1.1-3.2). High expression of cyclin E was significantly associated with shorter survival in the univariate analysis (RR=2.0, 95%CI=1.2-3.6) and multivariate analysis (RR=2.1, 95%CI=1.1-4.0). We found no significant correlation between the expression of cyclin D1 and any clinicopathological variables. Our study indicated a prognostic relevance for cyclin E and P53 immunoreactivity. Cyclin E may be an independent prognostic factor from the 2010 WHO Classification which should be evaluated in further studies.