e14526 Background: We developed a novel technology, Mutant-enriched liquidchip (MEL), which integrates the sensitive mutant enriched PCR and quantitative high throughput liquidchip (suspension array), to detect circulating EGFR mutations (Exon 19 deletion and exon 21 L858R mutation) in patients with advanced non-small cell lung cancer (NSCLC). Methods: To enrich mutant EGFR, a unique restriction site is introduced into the mutation alleles so that the wild type sequence can be selectively removed by restriction digestion, and the undigested mutated DNA is amplified by PCR. The product is then hybridized to complementary probes (including 15 types of exon 19 deletion and exon 21 L858R mutation) which had been conjugated to beads coding with different fluorescent dye, followed by measuring through Luminex 200 system. Plasmid DNA mixture with different EGFR genotypes was applied to determine the sensitivity and accuracy of MEL. Afterwards, the MEL was validated in 49 patients whose EGFR genotypes of tissue specimen had been tested with direct sequencing The circulating genomic DNA was obtained from serum sample of other 201 Chinese stage IIIB or IV NSCLC patients without EGFR-TKI administration, and the EGFR mutation status was analyzed by using of MEL. Results: The results shows that MEL is capable of detecting as few as 20 copies of mutant EGFR alleles with a sensitivity limit of at least mutant/wild-type ratio of 0.1%. It also shows that MEL can not only confirm EGFR mutations status in tissue specimens already known by direct sequencing (13/49), but also detect mutations in some of those showing wild type by sequencing (16/49). Overall, 54% of patients had circulating EGFR mutation. 34% of patients had an exon 19 deletion and 29.6% had L858R. 63.1% of mutations were found in females and 67.6% in never-smokers. Conclusions: This novel MEL method allows for highly sensitive and reproducible detection of human somatic mutations in heterogeneous specimens, and could be applicable to test EGFR mutations non-invasively in advanced NSCLC patients for predicting response to targeted therapy. No significant financial relationships to disclose.
ABSTRACT. Drug resistance in cells is a major impedance to successful treatment of lung cancer. Taxus chinensis var. inhibits the growth of tumor cells and promotes the synthesis of interleukins 1 and 2 and tumor necrosis factor, enhancing immune function. In this study, T. chinensis var.-induced cell death was analyzed in lung cancer cells (H460) enriched for stem cell growth in a defined serum-free medium. Taxus-treated stem cells were also analyzed for Rhodamine 123 (Rh-123) expression by flow cytometry, and used as a standard functional indicator of MDR. The molecular basis of T. chinensis var.-mediated drug resistance was established by real-time PCR analysis of ABCC1, ABCB1, and lung resistance-related protein (LRP) mRNA, and western blot analysis of MRP1, MDR1, and LRP. Our results revealed that stem cells treated with higher doses of T. chinensis var. showed significantly lower growth inhibition rates than did H460 cells (P < 0.05). The growth of stem and H460 cells treated with a combination of T. chinensis var. and cisplatin was also significantly inhibited (P < 0.05). Rh-123
Rationale:With the recent advancements in molecular biology research, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have emerged as excellent therapies for patients with EGFR-mutant cancers. However, these patients inevitably develop cross-acquired resistance to EGFR-TKIs. Transformation to small-cell lung cancer (SCLC) is considered a rare resistance mechanism against EGFR-TKI therapy. Here, we report a case of TKI resistance due to SCLC transformation and demonstrate its mechanisms and clinical features.Patient concerns:A 54-year-old Chinese man with a history of smoking for 40 years complained of an intermittent cough in March 2019.Diagnosis:Transbronchial lung biopsy was performed on the basal segment of the left lower lobe, which confirmed lung adenocarcinoma. In January 2020, repeat biopsy was performed, and the results of immunohistochemistry (IHC) staining showed TTF-1 (+), CK7 (+), napsin A (+), syn (+), and CD56 (+), with a Ki-67 (+) index 80% of small cell carcinomas. Infiltrating adenocarcinomas and small cell carcinomas were observed.Interventions:Icotinib (125 mg thrice daily) was administered as a first-line treatment from June 2019. We subsequently administered a chemotherapy regimen consisting of etoposide (180 mg, days 1–3) plus cisplatin (45 mg, days 1–3) every 3 weeks for 1 cycle after recurrence. As the patient could not tolerate further chemotherapy, he continued taking icotinib orally and received whole-brain radiotherapy 10 times to a total dose of 30 Gy after brain metastases.Outcomes:The patient relapsed after successful treatment with icotinib for 9 months. A partial response was achieved after 4 cycles of chemotherapy, and despite the brief success of chemotherapy, our patient exhibited brain metastasis and metastases of the eleventh thoracic spine and the second lumbar vertebra with pathological fracture. The patient eventually died of aggressive cancer progression.Lessons:Our case highlights the possibility of SCLC transformation from EGFR-mutant adenocarcinoma and the importance of repeat biopsy for drug resistance. Serum neuron-specific enolase levels may also be useful for detecting early SCLC transformation.
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