Abstract
Introduction
Next-generation sequencing (NGS) is essential to the care of patients with NSCLC. Nevertheless, NGS is dependent on adequate material from biopsy. We evaluated the impact of biopsy method and needle gauge necessary for optimizing success in tissue NGS.
Methods
A total of 1660 formalin-fixed, paraffin-embedded samples were submitted to Caris Life Sciences from 2007 to 2022 for tumor profiling. The results of NGS assays were linked with retrospective biopsy data for patients with lung cancer treated at USC/Norris Cancer Center to create a database with the following parameters: demographics, biopsy method, tumor location (lung mass versus lymph node versus metastasis), needle gauge, number of needle passes, complications, tumor volume, DNA content, and status of NGS. Fisher’s exact test and analysis of variance were performed to determine the impact of biopsy method and needle gauge (G).
Results
In total, 77 computed tomography (CT)-guided transthoracic core needle (CT-TTCN) biopsies, 74 endobronchial ultrasound (EBUS)-guided transbronchial needle aspirations (TBNAs), 27 bronchial forceps biopsies, and 107 surgical resections were included. Furthermore, 41 of 77 CT-TTCN biopsies (53.2%), 43 of 74 EBUS-TBNAs (58.1%), 22 of 27 bronchial forceps biopsies (81.5%), and 105 of 107 surgical resections (98.1%) underwent successful NGS assays. The probability of successful NGS completion for lung cancers was highest in surgical resections and bronchial forceps biopsies. Needle-based biopsies were more successful when a needle larger than 20G was used. Complication rates were higher for CT-TTCN biopsies compared with EBUS-TBNA (p < 0.0001). Overall, the DNA yield was significantly higher in EBUS-TBNA compared with CT-TTCN biopsies in primary lung sites (p = 0.0002). EBUS-TBNA was found to have higher success rates in NGS compared with CT-TTCN for both primary lung lesions (p = 0.023) and lymph node targets (p = 0.035).
Conclusions
The less invasive EBUS-TBNAs had higher success rates in NGS than CT-TTCN biopsies and resulted in higher DNA concentrations. In CT-TTCN biopsies, use of 20G or smaller needles is associated with a higher risk of obtaining an inadequate specimen regardless of the number of passes taken. Surgical and bronchial forceps biopsies had highest success in achieving NGS.
Keywords: Next-generation sequencing (NGS), Non–small cell lung cancer (NSCLC), Biopsy method, Needle gauge, Transthoracic core needle biopsy, Fine-needle aspiration
Introduction
Guidelines for lung cancer diagnosis and treatment by the National Comprehensive Cancer Network strongly recommend genomic testing for patients with lung cancer at the time of diagnosis.1 Nevertheless, approximately only one in three patients with lung cancer in the United States undergoes successful next-generation sequencing (NGS) testing before initial therapy, and nearly half of patients still enter treatment with inadequate biomarker testing.2,3
A variety of methods are used for lung cancer biopsy and subsequent NGS analysis and include but are not limited to computed tomography (CT)-guided transthoracic core needle (CT-TTCN) biopsies, endobronchial ultrasound (EBUS)-guided transbronchial needle aspiration (TBNA), surgical biopsy, and bronchial forceps biopsy. The selection of biopsy method is multifactorial and includes tumor location, tumor size, and local availability of the specialist to perform the techniques. The selection of biopsy method weighs risks and benefits, including cost, convenience, and complication rate. One factor that may affect selection of method is the probability that biomarker testing will produce the quantity and quality of the tumor sample size necessary to inform treatment decisions. The aim of our study is to determine how selection of biopsy method affects the availability for biomarker testing using NGS.
Materials and Methods
A total of 1660 formalin-fixed, paraffin-embedded samples were submitted to Caris Life Sciences between 2007 and 2022 for NGS analysis. No pathologic review of specimen adequacy was performed at the local site before the decision to request NGS testing. Some requests for testing were sent long after initial specimen collection and run on archival tissue. Non-lung cancer cases were excluded from the study. There were 295 patients with a diagnosis of NSCLC and five patients with a diagnosis of SCLC. Three patients were found to having missing medical records and were excluded from the study. Furthermore, 12 patients with other biopsy techniques including bronchial brushings, bronchial alveolar lavage, thoracentesis, and multiple sample methods were excluded owing to small sample size. A retrospective chart review was conducted to abstract data from electronic medical records of eligible patients at University of Southern California/Norris Comprehensive Cancer Center and affiliated sites. A database was constructed and included the following parameters: demographics, biopsy method, tumor location (lung mass versus lymph node versus metastasis), needle gauge, number of passes, complications, tumor volume, DNA content, and success or failure of NGS. This study was approved by the University of Southern California Institutional Review Board. Informed consent was waived given the retrospective nature of this study.
Rapid on-site evaluation was performed across all EBUS, transthoracic, and bronchial forceps biopsies. The tumor samples underwent comprehensive genomic profiling with NGS. DNA analysis was done through either a 592 gene panel using Next Seq or whole exome sequencing using NovaSeq. RNA analysis was done by using either an Archer FusionPlex Solid Tumor Panel through ArcherDX or whole transcriptome sequencing using NovaSeq. All formalin-fixed, paraffin-embedded samples were microdissected to allow for tumor enrichment and yield a tumor DNA composition of more than or equal to 20%. NGS success was defined as a complete analysis of DNA, RNA, and protein.
Statistical analysis was performed using GraphPad Prism 9 (Dotmatics). A 95% confidence interval was used to calculate each parameter comparison. Fisher’s exact test and analysis of variance were used to assess the likelihood of success of NGS according to biopsy method and volume of tissue sample. Biopsy specimens collected using needles were categorized according to needle gauge. Complications were classified as minor or major. Minor complications required no intervention or just an overnight stay for observation. Major complications required intervention or longer hospital stay.
Results
Patient distribution and demographics including cancer subtype, age, sex, and race are found in Table 1. Of 285 patients with lung cancer, there were 77 CT-TTCN biopsies, 74 EBUS-TBNA, 27 bronchial forceps biopsies, and 107 surgical resections. Table 2 summarizes the NGS success and failure rates between different biopsy methods and biopsy locations. The probability of a successful NGS completion for lung cancers was statistically significant across CT-TTCN biopsy gauge sizes with 90% success rate (18 of 20) of NGS in 18G and 33.3% success rate (15 of 45) in 20G (p < 0.0001) (Fig. 1A).
Table 1.
Patient Characteristics
| Category | Total (N = 285), n (%) |
|---|---|
| NSCLC | 282 (98.9) |
| SCLC | 3 (1.1) |
| Male | 159 (55.8) |
| Female | 126 (44.2) |
| Race | |
| Asian | 90 (31.6) |
| Black | 19 (6.6) |
| Native Hawaiian or Pacific Islander | 2 (0.7) |
| White | 114 (40) |
| Other | 60 (21.1) |
| Ethnicity | |
| Hispanic | 29 (10.2) |
| Non-Hispanic | 248 (87) |
| Unknown | 8 (2.8) |
Table 2.
Comparison Between Biopsy Method/Location and NGS Success
| Category |
NGS Status |
|||
|---|---|---|---|---|
| Biopsy Type | Complete MI Profile | Limited Tissue | Partial QNS | QNS |
| Transthoracic (n = 77) | ||||
| Non-lung site (n = 25), n (%) | 19 (76.0) | 4 (16.0) | 1 (4.0) | 1 (4.0) |
| Lung (n = 45), n (%) | 15 (33.3) | 20 (44.4) | 2 (4.4) | 8 (17.8) |
| Lymph node (n = 7) | 7 | 0 | 0 | 0 |
| EBUS-TBNA (n = 74) | ||||
| Lung (n = 25), n (%) | 16 (64.0) | 8 (32.0) | 0 | 1 (4.0) |
| Lymph node (n = 49), n (%) | 27 (55.1) | 17 (34.7) | 3 (6.1) | 2 (4.1) |
| Surgical resections (n = 107), n (%) | 105 (98.1) | 1 (1.0) | 1 (1.0) | 0 |
| Bronch-forceps (n = 27), n (%) | 22 (81.5) | 2 (7.4) | 0 | 3 (11.1) |
Note. NGS success was defined as complete MI profile. NGS failure was defined as limited tissue, partial QNS, or QNS. Complete MI profile = all WES/592 gene panel + WTS/Archer panel + IHC were performed. Limited tissue = either WES/592 gene panel or WTS/Archer panel or IHC or combination of two. Partial QNS = either WES/592 gene panel or WTS/Archer panel or IHC or combination of two. QNS = quantity or quality not sufficient for any testing to be performed.
EBUS-TBNA, endobronchial ultrasound guided transbronchial needle aspiration; IHC, immunohistochemistry; MI, molecular intelligence; NGS, next-generation sequencing; QNS, quality not sufficient; WES, whole exome sequencing; WTS, whole transcriptome sequencing.
Figure 1.
(A) Number of successful NGS and failed NGS cases in different CT-TTCN biopsy gauge sizes (∗∗∗∗p < 0.0001). (B) DNA yield between biopsy method/location (∗∗∗p = 0.0002). CT-TTCN, computed tomography–guided transthoracic core needle; EBUS-TBNA, endobronchial ultrasound–guided transbronchial needle aspiration; NGS, next-generation sequencing.
DNA Yield Between Biopsy Methods
The DNA yield was subdivided between biopsy methods and biopsy location (Fig. 1B). For CT-TTCN biopsies, the DNA yield was 14.8 ± 11.5 μg/μL for primary lung lesions, 40.5 ± 13.8 μg/μL for accessible lymph nodes, and 39.7 ± 18 μg/μL for extrathoracic non-lymph node metastatic sites. For EBUS-TBNA, the DNA yield was 43.9 ± 19.4 μg/μL for mediastinal and hilar lymph nodes and 41.4 ± 16.4 μg/μL for accessible central lung lesions. For transbronchial forceps biopsies, the diagnostic yield was 44 ± 14.2 μg/μL for primary lung tumors, compared with 46.3 ± 13 μg/μL by surgical wedge resections. Overall, the DNA yield was significantly higher in EBUS-TBNA compared with CT-TTCN biopsies in primary lung sites (p = 0.0002).
Number of Passes Between CT-TTCN Biopsies and EBUS-TBNAs
There was no significant difference in the number of passes between needle gauge sizes in both CT-TTCN biopsies and EBUS-TBNA. For CT-TTCN biopsies, the mean number of needle passes was 4.3 ± 2.0 for primary lung lesions, 3.3 ± 0.8 for lymph nodes, and 3.7 ± 1.7 for extrathoracic non-lymph node metastatic sites. For EBUS-TBNA, the mean number of needle passes was 5.6 ± 2.7 for lymph nodes and 5.1 ± 3.6 for central lesions. Moreover, the number of passes was significantly higher in EBUS-TBNA compared with CT-TTCN biopsies in lymph nodes (p = 0.036).
Complications
In CT-TTCN biopsies, the most common complications were pneumothorax and hemorrhage. The incidence of pneumothorax was 18.2% (14 of 77), whereas the incidence of hemorrhage was 2.6% (two of 77). There was one major complication of minor postbiopsy hemorrhage with pneumothorax, which resolved after chest tube placement. Complication rate for CT-TTCN biopsies with a 18G needle was 10% (two of 20) compared with 35.6% (16 of 45) with a 20G needle (p = 0.04).
In EBUS-TBNA, there was one major complication of bleeding, respiratory failure, and hemoptysis, which resolved after intubation for several days. Overall complication rate for CT-TTCN biopsies and EBUS-TBNA was 26% (20 of 77) and 1.4% (one of 74), respectively (p < 0.0001).
Discussion
Our study concludes that EBUS-TBNA had higher success rates in NGS compared with CT-TTCN. Furthermore, CT-TTCN biopsies with a larger needle yielded strikingly higher success rate (90% versus 33%) in NGS compared with using smaller needles. Overall complication rate was higher for CT-TTCN biopsies compared with EBUS-TBNA, but this was not driven by the patients in whom larger biopsy needles were used.
Others have found similar results with regard to the impact of biopsy method on biomarker success but have not categorized success with regard to biopsy needle size.4, 5, 6, 7 Although surgical resections and forceps biopsy are clearly the best method for obtaining a tissue, not all patients have these specimens available or obtainable at diagnosis, and often, needle biopsy is the preferred method on the basis of anatomical and logistical considerations. We found that the failure rate with non-coring needles was much higher than a prior study by Zheng et al.,7 which determined that surgical biopsy specimens had lower failure rates (0.7%) compared with small biopsy (5.8%) and fine-needle aspiration (3.1%) specimens. The increased failure rate from our study may be due to the increased requirements of tumor content necessary to do whole exome and whole transcriptome analysis compared with the hotspot panel used in the prior study.
DNA yields were similar for all biopsy types in large part because the DNA yield is dependent on the volume of tissue that undergoes microdissection and is then submitted for sequencing. Pathologists will perform this task to obtain the tissue required. In this case of our study, this was in the range of 40 to 50 μg/μL for all biopsy methods except for CT-TTCN biopsies of the lungs, which typically produced under 20 μg/μL of material. Most methodologies recommend a total input of 50 ng DNA.8 In contrast, the expanded panel in this study requires an input of 60 ng. EBUS-TBNA yielded significantly higher DNA concentration from lung sites as compared with CT-TTCN biopsies along with fewer complications in our study, which may suggest that EBUS-TBNA biopsy method is preferable to CT-TTCN biopsy. This is in contrast to prior work by Yao et al.9 which found no significant difference in complication rates between EBUS-TBNA and CT-TTCN biopsies.
Our paradoxical finding of lower complication rates with larger needles is explained by differences in the biopsy target in both groups. All 20 CT-TTCN biopsies in our series using a 18G needle were performed on a non-lung site, such as an adrenal gland or lymph node, which may explain the lower complication rate in the 18G needle group. Nevertheless, others have found the larger needles safe in the lungs. Elshafee et al.10 found that CT-guided lung biopsies with a 18G needle is a safe diagnostic technique with a minor complication rate of 45.6% and a major complication rate of 8%. More recently, new advanced techniques, such as cryobiopsy with radial-endobronchial ultrasound (Cryo-Radial), were found to have similar yield as CT-TTCN biopsies in peripheral lung lesions at a lower major complication rate of 4.2%.11
This study was retrospective and our data are limited to information available in the medical records. A prospective study of biopsy using different needle sizes and types would be required to definitively reveal the relative benefit to larger needles. As a single-institution study, the number of physicians carrying out these procedures was small and the findings may not be applicable along the full range of skills and experience levels that exist in all centers caring for patients with lung cancer. Similar analysis by other centers is needed to confirm or refute our findings.
CRediT Authorship Contribution Statement
Raymond Diep: Conceptualization, Methodology, Formal analysis, Investigation, Data Curation, Writing—Original Draft, Writing—Review and Editing.
Madeline MacDonald: Methodology, Investigation, Data Curation, Writing—Original Draft.
Ryan Cooper: Conceptualization.
Anna Grzegorczyk: Conceptualization.
Rastko Rakocevic: Conceptualization.
Ching-Fei Chang: Methodology, Writing—Review and Editing.
Angeline Uy: Writing—Review and Editing.
Nicholas Cowgill: Writing—Review and Editing.
Jorge Nieva: Conceptualization, Methodology, Writing—Review and Editing, Supervision.
Acknowledgments
This work was supported by the National Cancer Institute, United States (R25 CA225513). This research was conducted through the University of Southern California/Children's Hospital Los Angeles Summer Oncology Research Fellowship Program and was supported in part by the National Cancer Institute, United States R25 grant CA225513 at the Norris Comprehensive Cancer Center (Los Angeles, CA). This work was also supported by the Children’s Hospital Los Angeles, United States, Concern Foundation for Cancer Research, United States, and Tri Delta, United States.
Footnotes
Disclosure: Dr. Nieva reports providing consulting for Aadi Biosciences, AstraZeneca, Bristol-Myers Squibb, Fujirebio, G1 Therapeutics, Genentech, Mindmed, Naveris, Takeda, and Western Oncolytics; receiving research support from Genentech and Merck; having intellectual property in Cansera; and having ownership interests in Cansera, Epic Sciences, Indee Bio, and Quantgene. Dr. Uy and Mr. Cowgill are employees of Caris Life Sciences. The remaining authors declare no conflict of interest.
Cite this article as: Diep R, MacDonald M, Cooper R, et al. Biopsy method and needle size on success of next-generation sequencing in NSCLC: a brief report. JTO Clin Res Rep. 2023;4:100497.
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