Dynamic change of cancer genome profiling in metachronous bilateral breast cancer with BRCA pathogenic variant.

A 61-year-old woman with BRCA2 pathogenic variant had been treated for 20 years and showed dynamic changes in the genomic profile of her metachronous bilateral breast cancer and metastases. She underwent right breast conservation surgery at age 42-Genome 1, lung metastasis and left axillary lymph node metastasis at age 51, partial excision under local anesthesia for left breast cancer at age 53-Genome 2, left axillary lymph node dissection was added 6 month later-Genome 3. Then, olaparib was administered, and subsequently, left mastectomy was performed for the recurrence of left breast cancer at age 59-Genome 4. Genomic profile of the tumor was analyzed at four points (Genome 1-3 were analyzed by in house breast cancer panel, and Genome 4 was analyzed by Foundation One CDx). Two interesting findings emerged from these analyses. First, the genomic profile revealed that the left axillary lymph node metastasis, considered histologically from right breast cancer, was a metastasis from the left breast cancer. The second finding is that as the disease progressed, mutation profile became more diverse. The profile of the left breast cancer removed after olaparib and other treatments showed reversion mutation of BRCA2 and was diagnosed as tumor mutation burden high. Subsequent response to pembrolizumab was favorable.

Brief Report: Tepotinib as a Treatment Option in MET Exon 14 Skipping-Positive Lung Cancers-Investigating Discordance Between ArcherMET and the Oncomine Dx Target Test.

Introduction: NSCLC is a leading cause of cancer-related mortality worldwide. Specific genetic alterations, such as MET exon 14 (METex14) skipping, have been identified in NSCLC, allowing targeted therapy. Tepotinib, a highly selective MET inhibitor, has displayed promise in patients with advanced NSCLC. Nevertheless, challenges arise when identifying treatment strategies for patients with discordant results regarding METex14 skipping detection between diagnostic tests. Methods: We investigated patients with NSCLC and discordant results for METex14 skipping between the Oncomine Dx Target Test (ODxTT) and ArcherMET. Clinical response, adverse events, and the duration of tepotinib treatment were assessed, and statistical analysis was performed. Results: Among the 19 patients deemed METex14 skipping positive by ODxTT, only 10 had concordant results with ArcherMET. The number of METex14 skipping reads detected by ODxTT was significantly lower in discordant cases. Of the 19 patients, 14 received tepotinib, and comparable response and disease control rates were observed in both concordant and discordant cases. The duration of treatment did not significantly differ between the two groups. Conclusions: Our findings suggest that tepotinib has comparable therapeutic effects in patients with METex14 skipping-positive NSCLC irrespective of the concordance of results between ODxTT and ArcherMET. Tepotinib is a possible treatment option for patients with METex14 skipping, even in patients with discordant test results.

Liquid biopsy of wash samples obtained via endoscopic ultrasound-guided fine-needle biopsy: Comparison with liquid biopsy of plasma in pancreatic cancer.

OBJECTIVES: Pancreatic cancer (PC) has a poor prognosis and limited treatment options. Liquid biopsy, which analyzes circulating tumor DNA (ctDNA) in blood, holds promise for precision medicine; however, low ctDNA detection rates pose challenges. This study aimed to investigate the utility of wash samples obtained via endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) as a liquid biopsy for PC. METHODS: A total of 166 samples (42 formalin-fixed paraffin-embedded [FFPE] tissues, 80 wash samples, and 44 plasma samples) were collected from 48 patients with PC for genomic analysis. DNA was extracted and quantified, and 60 significantly mutated genes were sequenced. The genomic profiles of FFPE tissues, wash samples, and plasma samples were compared. Finally, the ability to detect druggable mutations in 80 wash samples and 44 plasma samples was investigated. RESULTS: The amount of DNA was significantly lower in plasma samples than in wash samples. Genomic analysis revealed a higher detection rate of oncogenic mutations in FFPE tissues (98%) and wash samples (96%) than in plasma samples (18%) and a comparable detection rate in FFPE tissues and wash samples. Tumor-derived oncogenic mutations were detected more frequently in wash samples than in plasma samples. Furthermore, the oncogenic mutations detection rate remained high in wash samples at all PC stages but low in plasma samples even at advanced PC stages. Using wash samples was more sensitive than plasma samples for identifying oncogenic and druggable mutations. CONCLUSIONS: The wash sample obtained via EUS-FNB is an ideal specimen for use as a liquid biopsy for PC.

Development of a molecular barcode detection system for pancreaticobiliary malignancies and comparison with next-generation sequencing.

BACKGROUND: Obtaining sufficient tumor tissue for genomic profiling is challenging in pancreaticobiliary cancer (PBCA). We determined the utility of molecular barcoding (MB) of liquid biopsies (bile, duodenal fluid, and plasma) for highly sensitive genomic diagnosis and detection of druggable mutations for PBCA. METHODS: Two in-house panels of 60 genes (non-MB panel) and 21 genes using MB (MB panel) were used for the genomic analysis of 112 DNA samples from 20 PBCA patients. We measured the yield of DNA and compared the genomic profiles of liquid samples obtained using the non-MB panel and the MB panel. The utility of the panels in detecting druggable mutations was investigated. RESULTS: A significantly greater amount of DNA was obtained from bile supernatants and precipitates compared to tumor samples (P < 0.001 and P = 0.001, respectively). The number of mutations per patient was significantly higher using the MB panel than using the non-MB panel (2.8 vs. 1.3, P = 0.002). Tumor-derived mutations were detected more frequently using the MB panel than the non-MB panel (P = 0.023). Five drug-matched mutations were detected in liquid samples. CONCLUSIONS: Liquid biopsy with MB may have utility in providing genomic information for the prognosis of patients with PBCA.

Influence of formalin fixation duration on RNA quality and quantity from formalin-fixed paraffin-embedded hepatocellular carcinoma tissues.

Analyzing RNA samples from formalin-fixed paraffin-embedded (FFPE) tissues is essential for precision medicine. We investigated RNA quantity and quality from FFPE tumor tissues fixed in formalin for various times and compared sequencing metrics from next-generation sequencing (NGS). Hepatocellular carcinoma (HCC) tissues were fixed in 10% neutral buffered formalin (1-240 h) and FFPE blocks were prepared. Total RNA was extracted, and the quantity and quality were assessed using the NanoDrop, Qubit and Bioanalyzer. After preparing sequencing libraries, NGS was performed on the Oncomine Dx Multi-CDx system. Total RNA yields of all samples met the threshold required for NGS, but longer fixation times resulted in decreased total RNA and long RNA fragment (>200 nt) yields. NGS analysis showed fewer sequencing reads of internal control genes from RNA with longer fixation times. RNA extracted from FFPE blocks stored for 500 days had reduced RNA yield and quality compared with RNA obtained from FFPE blocks prepared immediately. In conclusion, short and over-fixation should be avoided because of their negative impact on sequencing quality. Fixation process should be finished promptly within recommended guidelines (6-72 h) for cancer patients.

p53 Immunohistochemical Staining and TP53 Gene Mutations in Endometrial Cancer: Does Null Pattern Correlate With Prognosis?

Whether immunohistochemistry (IHC) of p53 accurately reflects the TP53 mutational status of endometrial carcinoma (EC) has not yet been established. This study aimed to clarify the relationship between p53 IHC and TP53 mutations in EC and to examine whether p53 IHC can be a more convenient prognostic marker than TP53 mutation in EC. We performed p53 IHC staining of EC samples obtained via surgery and genetic analyses using next-generation sequencing. p53 IHC results showed that of the 101 cases, 71 (70%) were wild-type (WT), 12 (12%) were overexpression (OE), and 18 (18%) were in the null group. Missense mutations were found in 9 cases (47.4%) in OE, 2 (10.5%) in null, and 8 (42.1%) in the WT group. Truncating mutations were found in 1 case (8.3%) in OE, 6 (50%) in null, and 5 (41.7%) in the WT group. The 5-year progression-free survival was 0% in OE, 74.8% in null, and 79.0% in the WT group. In the prognosis for each type of TP53 mutation, the 5-year progression-free survival was missense (32.2%), truncating (65.6%), and WT (79.7%). These survival comparisons showed that the p53 IHC OE had the poorest prognosis. These results suggest that the p53 IHC OE is an independent poor prognostic factor for EC and can be used as a simple and rapid surrogate marker for TP53 mutations. Contrastingly, the complete absence of p53 IHC-the null staining pattern-may not accurately predict a TP53 mutation in EC, and it is necessary to be more careful in making the diagnosis of "abnormal."

Nucleic Acid Quality Assessment is Critical to the Success of the Oncomine Dx Target Test for Lung Cancer.

BACKGROUND AND OBJECTIVE: The Oncomine Dx Target Test (ODxTT) has been used as a companion diagnostic test for lung cancer. Here, we evaluated whether the amount of nucleic acid and the degree of RNA degradation are related to the success of the ODxTT. METHODS: This study included 223 samples from 218 patients with lung cancer. For all samples, DNA and RNA concentrations were quantified using Qubit, and the degree of RNA degradation was evaluated using the Bioanalyzer. RESULTS: Of the 223 samples, 219 samples were successfully analyzed in the ODxTT and four were not. DNA analysis failed in two samples, which were attributed to low DNA concentrations and both were cytology specimens. Meanwhile, RNA analysis failed in the other two samples. These samples had sufficient amounts of RNA, but it was highly degraded with DV200 (the percentage of RNA fragments > 200 base pairs) less than 30. Compared with RNA samples with DV200 ≥ 30, analysis of RNA with DV200 < 30 yielded significantly fewer reads for the internal control genes. This test showed actionable mutations were identified in 38% (83/218) of all patients and in 46.6% (76/163) of patients with lung adenocarcinoma. CONCLUSIONS: DNA concentration and degree of RNA degradation are key factors determining the success of diagnostic testing by the ODxTT.

Comparison of genomic profiling of circulating tumor DNA in pancreaticobiliary malignancies in plasma and bile.

BACKGROUND: Obtaining sufficient pancreaticobiliary tumor tissue for genomic profiling has limitations. Liquid biopsies using plasma do not provide sufficient sensitivity. Thus, this study aimed to determine the effectiveness of liquid biopsy between bile and plasma for identifying oncogenic and drug-matched mutations. METHODS: This study created a panel of 60 significantly mutated genes specific to pancreaticobiliary cancer (PBCA) and used it for genomic analysis of 212 deoxyribonucleic acid (DNA) samples (87 bile supernatant, 87 bile precipitate, and 38 plasma) from 87 patients with PBCA. The quantity of extracted DNA from bile and plasma was compared, as were genomic profiles of 38 pairs of bile and plasma from 38 patients with PBCA. Finally, we investigated 87 bile and 38 plasma for the ability to detect druggable mutations. RESULTS: The amount of DNA was significantly lower in plasma than in bile (p < .001). Oncogenic mutations were identified in 21 of 38 (55%) patients in bile and nine (24%) in plasma samples (p = .005). Bile was significantly more sensitive than plasma in identifying druggable mutations (p = .032). The authors detected 23 drug-matched mutations in combined bile and plasma, including five ERBB2, four ATM, three BRAF, three BRCA2, three NF1, two PIK3CA, one BRCA1, one IDH1, and one PALB2. CONCLUSIONS: Liquid biopsy using bile may be useful in searching for therapeutic agents, and using the obtained genomic information may improve the prognoses of patients with PBCA. PLAIN LANGUAGE SUMMARY: Genomic profiling of formalin-fixed paraffin-embedded tissues may provide actionable targets for molecular and immuno-oncological treatment. However, most pancreaticobiliary malignancies are unresectable and formalin-fixed paraffin-embedded tissues cannot be obtained. Although comprehensive genomic profiling tests using plasma have been used in recent years, the utility of those using bile is not clear. Our study revealed that bile identified more drug-matched mutations than plasma in advanced pancreaticobiliary cancer patients. Bile may help widen the patient population benefiting from targeted drugs.

Validity of pathological diagnosis for early colorectal cancer in genetic background.

BACKGROUND: This study aimed to investigate the validity of pathological diagnosis of early CRC (E-CRC) from the genetic background by comparing data of E-CRC to colorectal adenoma (CRA) and The Cancer Genome Atlas (TCGA) on advanced CRC (AD-CRC). METHODS: TCGA data on AD-CRC were studied in silico, whereas by next-generation sequencer, DNA target sequences were performed for endoscopically obtained CRA and E-CRC samples. Immunohistochemical staining of mismatch repair genes and methylation of MLH1 was also performed. The presence of oncogenic mutation according to OncoKB for the genes of the Wnt, MAPK, and cell-cycle-signaling pathways was compared among CRA, E-CRC, and AD-CRC. RESULTS: The study included 22 CRA and 30 E-CRC lesions from the Chiba University Hospital and 212 AD-CRC lesions from TCGA data. Regarding the number of lesions with driver mutations in the Wnt and cell-cycle-signaling pathways, E-CRC was comparable to AD-CRC, but was significantly greater than CRA. CRA had significantly more lesions with a driver mutation for the Wnt signaling pathway only, versus E-CRC. CONCLUSIONS: In conclusion, the definition of E-CRC according to the Japanese criteria had a different genetic profile from CRA and was more similar to AD-CRC. Based on the main pathway, it seemed reasonable to classify E-CRC as adenocarcinoma. The pathological diagnosis of E-CRC according to Japanese definition seemed to be valid from a genetic point of view.

Paraganglioma with High Levels of Dopamine, Dopa Decarboxylase Suppression, Dopamine ß-hydroxylase Upregulation and Intra-tumoral Melanin Accumulation: A Case Report with a Literature Review.

Object Exclusively dopamine-producing pheochromocytoma/paraganglioma (PPGL) is an extremely rare subtype. In this condition, intratumoral dopamine ß-hydroxylase (DBH), which controls the conversion of norepinephrine from dopamine, is impaired, resulting in suppressed norepinephrine and epinephrine production. However, the rarity of this type of PPGL hampers the understanding of its pathophysiology. We therefore conducted genetic and immunohistological analyses of a patient with an exclusively dopamine-producing paraganglioma. Methods Paraganglioma samples from a 52-year-old woman who presented with a 29.6- and 41.5-fold increase in plasma and 24-h urinary dopamine, respectively, but only a minor elevation in the plasma norepinephrine level was subjected to immunohistological and gene expression analyses of catecholamine synthases. Three tumors carrying known somatic PPGL-related gene variants (HRAS, EPAS1) were used as controls. Whole-exome sequencing (WES) was also performed using the patient's blood and tumor tissue. Results Surprisingly, the protein expression of DBH was not suppressed, and its mRNA expression was clearly higher in the patient than in the controls. Furthermore, dopa decarboxylase (DDC), which governs the conversion of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) to dopamine, was downregulated at the protein and gene levels. In addition, melanin, which is synthesized by L-DOPA, accumulated in the tumor. WES revealed no PPGL-associated pathogenic germline variants, but a missense somatic variant (c.1798G>T) in CSDE1 was identified. Conclusion Although pre-operative plasma L-DOPA was not measured, our histological and gene expression analyses suggest that L-DOPA, rather than dopamine, might have been overproduced in the tumor. This raises the possibility of pathophysiological heterogeneity in exclusively dopamine-producing PPGL.

Deep targeted sequencing of cytological tumor cells using whole genome amplification.

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.

Giant pulmonary bulla causing respiratory compromise in a very low-birthweight infant.

Acquired cystic lung disease in premature infants is a serious respiratory complication, and pulmonary interstitial emphysema (PIE) has been widely reported. We report a rare case of giant pulmonary bulla in an infant treated with bullectomy where chest computed tomography was useful in directing treatment.

Cystic degeneration during neo-adjuvant chemotherapy predicts squamous metaplasia of triple negative breast cancer: report of two cases.

Neo-adjuvant chemotherapy (NAC) has become a standard treatment for advanced breast cancer because of the advantage of monitoring drug sensitivity and enabling breast-conserving therapy. The changes during NAC are also important to know the biological characteristics of the tumor. We experienced two cases with cystic degeneration and enhancement of the cyst wall during NAC for triple negative breast cancer (TNBC). They were diagnosed to have breast cancer with squamous metaplasia. In case 1, a 37-year-old woman with right breast cancer diagnosed as TNBC, T3N3M0, Stage 3b was treated with NAC. MRI showed a cystic degeneration with a diameter of 3.5 cm and enhancement of the cyst wall, and the other nodules were extinguished. The histopathological finding of the surgical specimen revealed solid tubular carcinoma with squamous metaplasia. In case 2, a 58-year-old woman with right breast cancer diagnosed as HER2 enriched subtype, T2N0M0 stage 2 was treated with NAC containing trastuzumab. The post-NAC MRI showed extinguishment of the mass in the right breast, but showed a cystic lesion with 24 mm in diameter and enhancement of its wall in the left breast. She underwent breast conserving surgery for bilateral breast cancer, and histopathological finding of the surgical specimen indicated complete remission of right breast cancer and squamous cell carcinoma developed in the left breast. These changes are impressive and remind us that there are metaplastic changes (especially for squamous metaplasia) with resistance to chemotherapy.

Detection of actionable mutations in cytological specimens obtained by endoscopic ultrasound-guided fine needle aspiration with rapid onsite evaluation in pancreatic cancer.

BACKGROUND: It is not clear whether archived cytological specimens (ACSs) obtained with endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) with rapid onsite evaluation (ROSE) can be used for genomic profiling of tumors. We used ACSs to perform genomic analysis of specimens to identify oncogenic and druggable mutations. METHODS: A panel of 60 significantly mutated genes specific to pancreatobiliary cancer was created and used for genomic analysis of 113 specimens of 44 formalin-fixed paraffin-embedded (FFPE) tissues and 69 ACSs obtained by EUS-FNA with ROSE were included. The quantity and quality of DNA extracted from FFPE tissues and ACSs were compared. We also compared DNA from spray and touch ACSs. Next, genomic profiles were compared. We also evaluated detection of target gene mutations in each specimen. RESULTS: The amount of DNA in FFPE tissues was greater than in ACSs (P = 0.014), but the quality of DNA was comparable (P = 0.378). There was no quantitative or qualitative difference between spray and touch ACSs (P = 0.154 and P = 0.734, respectively). Oncogenic mutations were shared at 82 % in FFPE tissues and ACSs and 82 % in spray and touch ACSs. The sensitivity of genomic analysis in ACSs was 97 % (67 of 69), which was comparable to that of cytology (62 of 69, 90 %; P = 0.165), and was significantly higher than that of histology (32/44, 73 %; P < 0.001). Drug-matched mutations were identified in five of the 44 lesions (11 %). CONCLUSION: Genomic analysis of ACSs is useful in the prognosis of pancreatic cancer because detection of driver mutations is similar to detection in FFPE tissues.

Genomic profiling amplifies the utility of endoscopic ultrasound-guided fine needle biopsy by identifying clinically applicable druggable mutations in pancreatic cancer.

BACKGROUND: Genomic profiling of tumors is available, but whether the small fragment obtained via endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) is sufficient for these examinations is unknown. Here we investigated whether EUS-FNB specimens are suitable for genomic profiling to identify oncogenic and drug-matched mutations. METHODS: We constructed a pancreatobiliary cancer panel for targeted panel sequencing that covered 60 significantly mutated genes and compared the results with those of whole-exome sequencing (WES). In total, 20 and 53 formalin-fixed paraffin-embedded tissues obtained via surgery and EUS-FNB were analyzed, respectively. First, we examined the DNA quality and genomic profiles of 20 paired samples from 20 malignant lesions obtained via surgery and EUS-FNB. We then tested 33 samples obtained via EUS-FNB from 24 malignant and 9 benign lesions for the discrimination of malignancy. Finally, we explored drug-matched mutations from EUS-FNB specimens. RESULTS: Although the DNA quantity obtained via surgery was higher than that obtained via EUS-FNB (P = 0.017), the DNA quality and mean depth were equivalent (P = 0.441 and P = 0.251). Panel sequencing of EUS-FNB specimens identified more oncogenic mutations than WES (90 % vs. 50 %). Furthermore, the number of oncogenic mutations did not differ between EUS-FNB and surgically resected specimens. Genomic profiling of EUS-FNB specimens enabled the discrimination of malignancy with 98 % accuracy. Of 44 malignant lesions, drug-matched alterations were identified in 14 % (6/44) of malignant lesions. CONCLUSION: EUS-FNB specimens can be widely utilized for diagnostic purposes, discrimination of malignancy, and detection of drug-matched mutations for the treatment of pancreatic cancer.

Simple IHC reveals complex MMR alternations than PCR assays: Validation by LCM and next-generation sequencing.

Evaluation of the status of mismatch repair (MMR) in tumors is crucial for determining the application of immune checkpoint inhibitors (ICIs). Conventional PCR (MSI-PCR) is the gold standard for confirming the MMR status. However, it requires visual confirmation and presents difficulties in determining MMR status. Immunohistochemistry (IHC) is a simple method and can confirming MMR protein expression in the whole tumor. We aim to investigate IHC is more suitable for evaluating MMR status in the tumor. We compared MSI-PCR and IHC by testing 319 samples from 284 patients across 14 cancer types. In discordant cases, we performed laser-capture microdissection and microsatellite instability assay by next-generation sequencing (MSI-NGS). The concordance rate between IHC and MSI-PCR testing was 98.1% (313/319). Two reasons for these discrepancies were ambiguous MSI-PCR results and heterogeneous MSI status within the tumor. Among six cases (1.9%), three were judged as MSI-H by MSI-PCR but with proficient MMR by IHC. The results of MSI-NGS revealed microsatellite stable in these three cases. The remaining three cases, two of three were MSI-H and one was MSS in whole tumor in MSI-PCR. IHC showed a "mosaic" pattern containing both proficient MMR and deficient MMR portions by IHC in all three cases. We performed microdissection and MSI-PCR and found intratumoral heterogeneity of MMR status. These results indicated the advantages of IHC and performed expanded samples (n = 1082) and two additional mosaic cases were identified. Our results clearly indicated that simple IHC is the best choice for determining MMR alterations in critical cases for ICIs treatment.

The Diagnostic Utility of Cell-Free DNA from Ex Vivo Bronchoalveolar Lavage Fluid in Lung Cancer.

Although bronchoscopy is generally performed to diagnose lung cancer, its diagnostic yield remains unsatisfactory. Assuming that lung cancer cells release cell-free DNA into the epithelial lining fluid, we hypothesized that lung cancer could be diagnosed by analyzing gene mutations in cell-free DNA in this fluid. This study included 32 patients with lung cancer who underwent surgery at our hospital. Bronchoalveolar lavage (BAL) was performed on the resected lung samples (ex vivo BAL model) after lobectomy. Each DNA sample (i.e., BAL fluid, primary lesion, and plasma) underwent deep targeted sequencing. Gene mutation analyses in the BAL fluid samples identified mutations identical to those in the primary lesions in 30 (93.8%) of 32 patients. In contrast, the microscopic cytology of the same BAL fluid samples yielded a diagnosis of lung cancer in only one of 32 patients, and the analysis of plasma samples revealed gene mutations identical to those in the primary lesions in only one of 32 patients. In conclusion, cell-free DNA released from lung cancer cells exists more abundantly in the airway than in the blood. The collection and analysis of the BAL fluid containing cell-free DNA derived from lung cancer can thus allow lung cancer diagnosis and the screening of driver mutations.