RESUMEN
BACKGROUND: Genomic profiling in lung cancer is essential for precision medicine. Cytological specimens provide an alternative to formalin-fixed paraffin-embedded (FFPE) samples for comprehensive genomic analysis. However, this approach remains challenging when a limited number of tumor cells are available. We applied whole genome amplification (WGA) to cytology specimens to overcome this limitation. METHODS: Using a lung cancer panel targeting 58 genes, we performed next-generation sequencing of whole genome-amplified DNA extracted from cytological specimens containing 10-20 tumor cells (cyto-WGA) and DNA from corresponding FFPE tumor tissue. We compared sequencing data from cyto-WGA and FFPE samples to examine the detection accuracy of copy number variations and oncogenic and drug-matched variants. RESULTS: The DNA quality and quantity from cyto-WGA were higher than those from FFPE samples (p < .0005 and p < .05, respectively). Sequencing metrics of cyto-WGA and FFPE tissues showed no difference in the number of mapped reads and mean coverage depth, but there were significant differences in the on-target rate (p < .05) and uniformity (p < .0005). Copy number variations in cyto-WGA samples (n = 211) were higher than in FFPE samples (n = 9) (p < .0001). Fourty nine oncogenic variants were detected in cyto-WGA and 39 in FFPE. Of these variants, 34 (63%) were present in both samples. In addition, all 16 drug-matched variants were detected in FFPE and cyto-WGA samples with 100% concordance. CONCLUSION: Cyto-WGA can be a feasible and alternative method to detect oncogenic and drug-matched variants.
Asunto(s)
Variaciones en el Número de Copia de ADN , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , ADN , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Adhesión en Parafina , Formaldehído , Fijación del TejidoRESUMEN
BACKGROUND: Epidermal growth factor receptor (EGFR) is the gene with the highest mutation rate in non-small cell lung cancer (NSCLC) patients, and the accurate evaluation of its mutational status can facilitate patients receiving targeted drug therapy and thereby prolong patients' survival. The gene testing platform has adequacy requirements for the specimen quality in order to obtain accurate examination results. It has been reported that the number and proportion of tumor cells in samples will affect the detection rate of EGFR gene mutation. The present study aims to analyze the relationship between the quality of small biopsy specimens of NSCLC and the mutation rate of EGFR gene with amplification refractory mutation system (ARSM) test. METHODS: After collecting the clinical characteristics of 299 cases small biopsy of lung adenocarcinoma, DNA concentration of the specimens and the mutational status of EGFR gene, the number and proportion of tumor cells in HE stained sections evaluated using light microscopy, the relationship between specimen quality and the mutation rate of EGFR gene were analyzed. RESULTS: The mutation rates of EGFR for the groups with tumor cell number ≤500 and >500 were 40.7% (11/27) and 43.8% (119/272) respectively, without significant difference (P=0.764). The mutation rates for the groups with DNA concentration ≤20.4 ng/µL and >20.4 ng/µL were 42.7% (64/150) and 44.3% (66/149) respectively, without significant difference (P=0.776). The mutation rates for the groups with tumor cells proportion ≤30% and >30% were 29.4% (20/68) and 47.6% (110/231) respectively, demonstrating significant difference (P=0.008). Multivariate Logistic analysis showed that male, thyroid transcription factor-1 (TTF-1) negative, smoking history and tumor cell proportion less than 30% were main factors that contributes to the low detection rate of EGFR gene mutation. CONCLUSIONS: After meeting the minimum requirements for detection, the EGFR mutation rate is affected by the proportion of tumor cells in the sample. Therefore, it is necessary to re-evaluate the tumor cell proportion in the last section after the genetic test section. For samples with lower tumor cell proportion, enriching tumor cells through microdissection and other methods is recommended for a more accurate detection result. For specimens that cannot be enriched with tumor cells, circulating tumor DNA (ctDNA) test can be performed as a supplement. If the result is still negative, another biopsy should be considered to obtain enough tumor specimens for molecular testing.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Carcinoma de Pulmón de Células no Pequeñas/patología , Análisis Mutacional de ADN , Receptores ErbB/genética , Femenino , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación , Tasa de MutaciónRESUMEN
BACKGROUND@#Epidermal growth factor receptor (EGFR) is the gene with the highest mutation rate in non-small cell lung cancer (NSCLC) patients, and the accurate evaluation of its mutational status can facilitate patients receiving targeted drug therapy and thereby prolong patients' survival. The gene testing platform has adequacy requirements for the specimen quality in order to obtain accurate examination results. It has been reported that the number and proportion of tumor cells in samples will affect the detection rate of EGFR gene mutation. The present study aims to analyze the relationship between the quality of small biopsy specimens of NSCLC and the mutation rate of EGFR gene with amplification refractory mutation system (ARSM) test.@*METHODS@#After collecting the clinical characteristics of 299 cases small biopsy of lung adenocarcinoma, DNA concentration of the specimens and the mutational status of EGFR gene, the number and proportion of tumor cells in HE stained sections evaluated using light microscopy, the relationship between specimen quality and the mutation rate of EGFR gene were analyzed.@*RESULTS@#The mutation rates of EGFR for the groups with tumor cell number ≤500 and >500 were 40.7% (11/27) and 43.8% (119/272) respectively, without significant difference (P=0.764). The mutation rates for the groups with DNA concentration ≤20.4 ng/μL and >20.4 ng/μL were 42.7% (64/150) and 44.3% (66/149) respectively, without significant difference (P=0.776). The mutation rates for the groups with tumor cells proportion ≤30% and >30% were 29.4% (20/68) and 47.6% (110/231) respectively, demonstrating significant difference (P=0.008). Multivariate Logistic analysis showed that male, thyroid transcription factor-1 (TTF-1) negative, smoking history and tumor cell proportion less than 30% were main factors that contributes to the low detection rate of EGFR gene mutation.@*CONCLUSIONS@#After meeting the minimum requirements for detection, the EGFR mutation rate is affected by the proportion of tumor cells in the sample. Therefore, it is necessary to re-evaluate the tumor cell proportion in the last section after the genetic test section. For samples with lower tumor cell proportion, enriching tumor cells through microdissection and other methods is recommended for a more accurate detection result. For specimens that cannot be enriched with tumor cells, circulating tumor DNA (ctDNA) test can be performed as a supplement. If the result is still negative, another biopsy should be considered to obtain enough tumor specimens for molecular testing.
RESUMEN
BACKGROUND: We previously reported cryobiopsy (Cryo) with endobronchial ultrasonography-guide sheath (EBUS-GS) for peripheral pulmonary lesions (PPLs) provides significantly larger tissues than transbronchial biopsy (TBB) and provides high quantity and quality DNA for gene analysis by next generation sequencing. However, the tumor cell yields and programmed death ligand 1 (PD-L1) expression between each approach have not been compared. Here, we assessed the tumor cell numbers and PD-L1 expression for Cryo with EBUS-GS for PPLs and TBB in patients with lung cancer. METHODS: Sixteen patients were enrolled in this prospective study from June to November 2017 at Tokyo Women's Medical University Hospital. The number of tumor cells from a single biopsy, total number of tumor cells, average number of tumor cells, and 22C3 PD-L1 expression (≥ 50% and ≥ 1%) were compared between Cryo and TBB. RESULTS: The numbers of tumor cells from a single biopsy, total numbers of tumor cells, and average numbers of tumor cells obtained by Cryo were significantly larger than those obtained by TBB (Cryo [means ± standard errors of the means]: 1321 ± 303.7, 1981 ± 411.7, and 1406 ± 310.3; TBB: 208.8 ± 38.24, 1044 ± 189.0, and 208.8 ± 37.81; P < 0.0001, P = 0.0474, P = 0.0006, respectively). PD-L1 ≥ 50% and ≥ 1% patients for Cryo were 18.8 and 56.3%, respectively, whereas those for TBB were 12.5 and 37.5%, respectively. The sensitivity, specificity, positive predictive value, negative predictive value, concordance, and κ coefficient based on Cryo for TBB were 66.7, 100, 100, 92.9, 93.8%, and 0.7647, respectively, for PD-L1 ≥ 50%; and 44.4, 71.4, 66.7, 50, 56.3%, and 0.1515, respectively, for PD-L1 ≥ 1%. CONCLUSION: Cryo with EBUS-GS may be a useful diagnostic approach for lung cancer, with advantages over TBB for gene analysis and whole exon sequencing. Particularly, it could contribute to patients taking pembrolizumab as first-line therapy when PD-L1 was negative by evaluating TBB specimens. It could also provide ample tissue for PD-L1 expression analysis in addition to accurate diagnosis and gene analysis.