Epidermal Growth Factor Receptor Mutation Testing Research Articles

Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer.

BackgroundBronchoscopy is a minimally invasive procedure for establishing the diagnosis of lung cancer. It sometimes fails to obtain tissue samples but readily collects cytological samples.MethodsWe developed PNA‐LNA dual‐PCR (PLDP), which amplified mutant sequences by a high‐fidelity DNA polymerase in the presence of a peptide nucleic acid (PNA) oligomer having a wild‐type sequence. Mutations are detected either by locked nucleic acid (LNA) probes for quick detection of a limited number of mutations, which are EGFR, KRAS, and BRAF mutations in the current study, or by direct sequencing for a comprehensive screening. In a total of 233 lung cancer samples, the results for cytological samples by PLDP were compared with those for tissue samples by cobas® EGFR mutation test (cobas) or by the PNA‐LNA PCR clamp method (P‐LPC). Moreover, the performance of PLDP using cell‐free DNA (cfDNA) was investigated.ResultsPeptide nucleic acid‐LNA dual‐PCR was able to detect each synthesized mutant sequence with high sensitivity. PLDP detected EGFR mutations in 80 out of 149 clinical samples, while the cobas or the P‐LPC detected in 66 matched. The correctness of PLDP was confirmed both by clinical response and by the results of sequencing using a next‐generation sequencer. PLDP detected mutations from cfDNA in approximately 70% of patients who harbors mutations in the tumor.ConclusionsPeptide nucleic acid‐LNA dual‐PCR exhibited an excellent performance, even using cytological samples. PLDP is applicable for the investigation of cfDNA. The combination of bronchoscopy and PLDP is attractive and will expand the utility of bronchoscopy in clinical practice.

P59.27 Complementary Utility of Combined ALK/ROS1 FISH with Immunohistochemistry for ALK/ROS1 Rearrangement Testing in Lung Cancer

Testing for EGFR mutations, ALK and ROS1 rearrangements and BRAF mutations are now recommended as the essential minimum predictive biomarkers to be tested in primary non-squamous NSCLC patients presenting with advanced disease. While the ALK D5F3 Ventana immunohistochemistry (IHC) Assay is approved for detection of ALK rearrangements and for targeted therapy, ROS1 IHC only serves as a screening tool for ROS1 rearrangements with demonstration of gene fusions by molecular methods required for confirmation and targeted treatment. Nevertheless, equivocal IHC results, although uncommon, still occur with the D5F3 ALK assay necessitating complementary methods for confirmation. The recent availability of combined ALK/ROS1 break-apart fluorescence in-situ hybridisation (FISH) probes offer the advantage of tissue conservation with ability to analyse both gene alterations in a single tissue section. The aim of the present study was to study the utility of the combined ALK/ROS1 FISH assay as a complement to ALK and ROS1 IHC in routine predictive biomarker testing algorithms. Over a 6-year study period, all EGFR wild type NSCLC samples were subject to automated ALK D5F3 Ventana IHC assay and ROS1 IHC using the D4D6 (Cell signalling technology) clone as a manual lab developed test. Diffuse strong granular staining in tumor cells for ALK and ROS1 protein was interpreted as positive. All cases of equivocal ALK staining and positive ROS1 staining was subject to combined ALK/ROS1 FISH using the FlexISH ALK/ROS1 DistinguISH Probe (Vysis) performed as per manufacturers’ instructions. Presence of ALK/ROS1 break apart signals and/or isolated ALK/ROS1 3’ signals in >15% of interpretable tumour nuclei was considered as positive for ALK or ROS1 rearrangement. The ALK Ventana Assay was performed in 711 formalin fixed paraffin embedded EGFR wild type NSCLC samples including biopsies, lymph node excisions and cell blocks. Of these, 12% (85/711) were positive, 2% (13/711) were equivocal, 75% (539/711) were negative, while in the remaining 11% (76/711), result could not be evaluated due to exhaustion of tumor tissue. FISH analysis in four of the ALK IHC equivocal cases show absence of ALK translocations while no tissue remained for FISH analysis in the remaining equivocal cases. ROS1 IHC was performed in 434 cases of the above cohort of which 7% (30/434) were positive. ROS1 immunopositivity was enriched among ALK IHC positive cases with 43% (19/44) of ALK IHC positive cases also showing protein expression of ROS1. FISH analysis revealed ROS1 translocation in two cases (both showing diffuse strong ROS1 protein expression and negative ALK IHC) while all dual ALK/ROS IHC positive cases tested by FISH showed presence of only ALK rearrangements with non-rearranged ROS1 signals. Tissue adequacy remains the major limiting factor for predictive biomarker testing in lung cancer. The use of combined ALK-ROS FISH allows for excellent demonstration of ALK and ROS1 rearrangements in the same section using the same criteria validated in single gene break apart FISH probes. ROS1 protein is commonly overexpressed in ALK rearranged NSCLC and may represent cross reactivity with ALK or a related protein.

FP12.05 The Intrinsic Limitation and Clinical Impact of Mutant Allele-Specific Real-Time PCR-Based EGFR Mutation Assay in NSCLC

The detection of driver gene mutations, such as epidermal growth factor receptor (EGFR) mutations, plays a decisive role in the treatment of non-small cell lung cancer (NSCLC). Cobas EGFR mutation test is a mutant allele-specific real-time PCR assay that is commonly used in daily practice. It has a notable intrinsic detection limitation caused by its primer designs. Next-generation sequencing (NGS) is a comprehensive mutation assay that does not have the constraint. It will have a higher mutation detection rate but the clinical impact remains uncertain.

A large-scale, multicentered trial evaluating the sensitivity and specificity of digital PCR versus ARMS-PCR for detecting ctDNA-based EGFR p.T790M in non-small-cell lung cancer patients.

BackgroundDeveloping liquid biopsy technology with higher sensitivity and specificity especially for low-frequency mutations remains crucial. This study demonstrated superior performance of the newly developed digital PCR (dPCR) kit for ctDNA-based EGFR p.T790M detection in metastatic non-small-cell lung cancer (NSCLC) against ARMS-PCR.MethodsThis large-scale, multi-centered diagnostic study recruited 1,045 patients including 1,029 patients diagnosed with advanced NSCLC and 16 patients with specific samples between April 1st 2018 and November 30th 2019. EGFR p.T790M in plasma samples from mNSCLC patients were tested using dPCR with ADx-ARMS PCR and Cobas® EGFR Mutation Test V2 as comparator assays to confirm cut-off value for dPCR and evaluate its performance against ARMS-PCR-based assays. Efficacy was evaluated for patients with EGFR p.T790M detected by dPCR or ARMS-PCR, who underwent Osimertinib treatment.ResultsThe sensitivity, specificity, and concordance of dPCR against ADx-ARMS PCR was 98.15%, 88.66% and 90.16%, respectively for 1,026 plasma samples. Additional 9.26% patients were detected positive by dPCR. The majority of those samples had a mutation allele frequency between 0.1% and 1%. In 45 paired tissue and plasma samples, the sensitivity improved from 30.77% to 53.85% by dPCR with the specificity over 90%. The response of Osimertinib in 74 EGFR p.T790M-positive patients detected by dPCR, including 26 determined as negative by ARMS-PCR, were evaluated to have an ORR of 44.59% and a DCR of 90.54%.ConclusionsdPCR is a sensitive and accurate tool for ctDNA-based EGFR p.T790M detection due to its significantly improved sensitivity without compromising specificity, and dPCR is equivalent to ARMS-PCR as a companion diagnostic tool while benefiting more patients under Osimertinib treatment.Trial RegistrationChinese Clinical Trial Registry identifier: ChiCTR2100043147.

Surgical outcomes of one-stage resection for synchronous multiple primary lung adenocarcinomas with no less than three lesions

BackgroundMore and more synchronous multiple primary lung adenocarcinomas (SMPLA) have been diagnosed and surgical treatment has become the mainstay of treatment for them, but there are few reports on the surgical outcome of patients with ≥ 3 lesions who underwent surgical resection. Therefore, we summarized and analyzed the clinical characteristics and surgical outcomes of these patients, hoping to provide some experience in the diagnosis and treatment.MethodsClinical characteristics and treatment outcomes of patients with ≥ 3 lesions who have been diagnosed as SMPLA and underwent surgical resection in our hospital from March 2015 to July 2019 were retrospectively reviewed.ResultsTwenty-eight patients, 20 females and 8 males, with a mean age of 57.7 ± 5.69 (45–76) years, were finally included. A total of 95 lesions, 86.4% were ground-glass opacity (GGO) lesions (pure-GGO,45.3%; mixed-GGO,41.1%); 51 lesions had EGFR mutations and the mutation rate of invasive adenocarcinoma was significantly higher than that of other pathological subtypes (P < 0.001); the mutation rate of mGGO was also significantly higher than that of pGGO and solid nodule (SN) (P < 0.05). Four and 24 patients respectively underwent bilateral and unilateral surgical resection. The surgical procedure was mainly sublobar resection, and no severe postoperative complications or deaths occurred. After a median follow-up time of 32.2 months, the rates of overall survival and disease-free survival at 3 years were 94.7% and 88.9%, respectively.ConclusionsFor SMPLA with ≥ 3 lesions, one-stage resection may be safe and feasible, and surgical procedure was mainly sublobar resection as far as possible, which can yield satisfactory prognosis. EGFR mutation testing should be used routinely in the diagnosis and treatment of patients with SMPLA, especially in the presence of mGGO and invasive adenocarcinoma.

Real-World Analysis of the EGFR Mutation Test in Tissue and Plasma Samples from Non-Small Cell Lung Cancer.

Molecular evaluation of EGFR mutation is indispensable in treating non-small cell lung cancer (NSCLC). We compared the results of EGFR analysis using tissue DNA (tDNA) and circulating tumor (ctDNA) to evaluate the feasibility of plasma as an effective material for detecting EGFR mutation and the reliability of ctDNA analysis in real-world practice settings. We enrolled 554 NSCLC cases who had undergone ctDNA EGFR analysis between January 2019 and March 2020. EGFR mutations were detected in 240 (57.3%) of the 421 cases with EGFR mutations confirmed by tDNA analysis. In multivariate analysis, the size of the largest tumor deposits, disease progression, M stage, the detectable amount of tumor tissue with EGFR mutation in distant metastasis, liver metastasis, pleural seeding, and bone metastasis (p < 0.05) were identified as independent factors affecting the detection rate of EGFR mutations in ctDNA. Survival analysis revealed ctDNA status and M stage (p < 0.001) to be independent predictors of overall survival in the multivariate analysis. Our study demonstrates that EGFR analysis using ctDNA is a useful clinical tool and can aid in therapeutic decisions in real-world practical settings. However, clinicians should be aware of the possibility of false negatives and confirm EGFR analysis using tDNA in certain situations.

Testing for EGFR Mutations and ALK Rearrangements in Advanced Non-Small-Cell Lung Cancer: Considerations for Countries in Emerging Markets.

The treatment of patients with advanced non-small-cell lung cancer (NSCLC) in recent years has been increasingly guided by biomarker testing. Testing has centered on driver genetic alterations involving the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) rearrangements. The presence of these mutations is predictive of response to targeted therapies such as EGFR tyrosine kinase inhibitors (TKIs) and ALK TKIs. However, there are substantial challenges for the implementation of biomarker testing, particularly in emerging countries. Understanding the barriers to testing in NSCLC will be key to improving molecular testing rates worldwide and patient outcomes as a result. In this article, we review EGFR mutations and ALK rearrangements as predictive biomarkers for NSCLC, discuss a selection of appropriate tests and review the literature with respect to the global uptake of EGFR and ALK testing. To help improve testing rates and unify procedures, we review our experiences with biomarker testing in China, South Korea, Russia, Turkey, Brazil, Argentina and Mexico, and propose a set of recommendations that pathologists from emerging countries can apply to assist with the diagnosis of NSCLC.

Differences in Immunological Landscape between EGFR-Mutated and Wild-Type Lung Adenocarcinoma.

Recent clinical trials of lung adenocarcinoma with immune checkpoint inhibitors revealed that lung adenocarcinoma patients with EGFR mutations have a poor response to immunotherapy. However, the mechanisms have not been addressed. We performed immunohistochemistry analyses of resected lung adenocarcinoma tissues with and without EGFR mutations to investigate and compare the characteristics of the tumor microenvironment (TME). We retrospectively enrolled a total of 323 lung adenocarcinoma patients (164 had EGFR mutations), and their corresponding tissue samples were analyzed by the EGFR mutation test and immunohistochemistry. We selected the markers of the immune checkpoint molecule (PD1, PD-L1, and LAG-3) and immune cell (CD3, CD4, CD8, and Foxp3) as markers of the tumor microenvironment. Our results revealed that patients had a distinct tumor microenvironment between EGFR-mutant and wild-type lung adenocarcinomas; the expression of CD3, CD4, PD-L1, and Foxp3 in EGFR-mutant tumors was significantly higher than that in wild-type tumors, while the expression of LAG3 and PD-1 showed a positive correlation with EGFR-wild-type tumors. In survival analysis, EGFR-wild-type patients had longer disease-free survival (DFS) than EGFR-mutant patients (P = 0.0065). Our research demonstrates significant differences in tumor microenvironment composition between EGFR-mutant and wild-type patients. Our findings provide novel evidence that contributes to understanding the mechanism underlying the poor efficacy of immune checkpoint inhibitors.

Not enough can be enough: feasibility of the Idylla EGFR mutation test when reuse of stained tissue slides is the only option available

AimsThe minimally invasive procedures used in the diagnostic workup of patients with advanced non-small cell lung cancer (NSCLC) often provide poor yields of pathological material suitable for molecular analyses. Not...

Value of (18)F-FDG PET-CT imaging to predict epidermal growth factor receptor mutations in patients with lung squamous cell carcinoma

Objective: To investigate the value of (18)F-fluorodeoxy glucose ((18)F-FDG) positron emission tomography/computed tomography (PET-CT) in predicting the epidermal growth factor receptor (EGFR) mutations in patients with lung squamous cell carcinoma. Methods: We retrospectively analyzed the clinical data and (18)F-FDG PET-CT imaging data of 206 patients with lung squamous cell carcinoma confirmed by pathology and underwent EGFR mutation test in the First Affiliated Hospital of Nanjing Medical University from June 2013 to October 2018. Receiver operating characteristic (ROC) curve analysis was performed to quantify the predictive value of maximum standard uptake value (SUV(max)), metabolic tumor volume (MTV), total lesion glycolysis (TLG). The Chi-squared test was used to assess the difference in PET parameters. A multivariate Logistic regression analysis was performed to yield the parameters with statistic difference. Results: All of 206 patients with lung squamous cell carcinoma showed a high (18)F-FDG uptake. The median of SUV(max), MTV and TLG were 19.14, 37.69 cm(3) and 291.73, respectively. Among the 206 patients, EGFR mutations were identified in 14 cases, including 7 with exon 21 (L858R) mutation, 6 with exon 19 mutation and 1 with exon 20 mutation. ROC curve showed that the AUC of SUV(max), MTV and TLG were 0.624 (95% CI=0.454-0.794, P=0.122), 0.892 (95% CI=0.811-0.973, P<0.001) and 0.860 (95% CI=0.768-0.952, P<0.001), respectively. The median SUV(max) (19.14) was used as the cutoff points due to the small value of AUC. The cutoff point of MTV was 20.09 cm(3), the cutoff point of TLG was 211.07. Univariate analysis showed that the sex, smoking history, M stage, MTV and TLG were associated with EGFR mutations (all P<0.05). Logistic multivariate analysis showed that the sex, smoking history and TLG were the independent predictors of EGFR mutation (all P<0.05). Conclusion: TLG detected by (18)F-FDG PET/CT is an independent factor for predicting EGFR mutation in patients with lung squamous cell carcinoma, and has certain reference value for predicting EGFR mutation.

A Nationwide Study on the Impact of Routine Testing for EGFR Mutations in Advanced NSCLC Reveals Distinct Survival Patterns Based on EGFR Mutation Subclasses.

Simple SummaryThe presence of an EGFR activating mutation in tumors of non-small-cell lung cancer patients enables effective targeted therapy towards EGFR. Studies that describe a nationwide uptake of EGFR testing, the impact of the switch from single-gene EGFR to multi-gene testing, and the clinical response towards EGFR inhibitors in first-line treatment are limited. From 2013 to 2017 the percentage of patients routinely tested for EGFR mutations increased from 73% to 81% in the Netherlands. A strong shift towards EGFR testing as part of a multi-gene next generation sequencing analysis was observed. However, this did not change the percentage of EGFR mutations that were reported for this patient population, which remained stable at 12%. When treated with EGFR inhibitors that were available in a routine clinical setting prior to 2018, clear differences were observed between the type of EGFR mutation and survival. EGFR mutation analysis in non-small-cell lung cancer (NSCLC) patients is currently standard-of-care. We determined the uptake of EGFR testing, test results and survival of EGFR-mutant NSCLC patients in the Netherlands, with the overall objective to characterize the landscape of clinically actionable EGFR mutations and determine the role and clinical relevance of uncommon and composite EGFR mutations. Non-squamous NSCLC patients diagnosed in 2013, 2015 and 2017 were identified in the Netherlands Cancer Registry (NCR) and matched to the Dutch Pathology Registry (PALGA). Overall, 10,254 patients were included. Between 2013–2017, the uptake of EGFR testing gradually increased from 72.7% to 80.9% (p < 0.001). Multi-gene testing via next-generation sequencing (increased from 7.8% to 78.7% (p < 0.001), but did not affect the number of detected EGFR mutations (n = 925; 11.7%; 95% confidence interval (CI), 11.0–12.4) nor the distribution of variants. For patients treated with first-line EGFR inhibitors (n = 651), exon 19 deletions were associated with longer OS than L858R (HR 1.58; 95% CI, 1.30–1.92; p < 0.001) or uncommon, actionable variants (HR 2.13; 95% CI, 1.60–2.84; p < 0.001). Interestingly, OS for patients with L858R was similar to those with uncommon, actionable variants (HR 1.31; 95% CI, 0.98–1.75; p = 0.069). Our analysis indicates that grouping exon 19 deletions and L858R into one class of ‘common’ EGFR mutations in a clinical trial may mask the true activity of an EGFR inhibitor towards specific mutations.

Detection of EGFR mutations in liquid biopsy samples using allele-specific quantitative PCR: A comparative real-world evaluation of two popular diagnostic systems

PurposeThe detection of epidermal growth factor receptor (EGFR) mutations in plasma cell-free DNA (cfDNA) is an auxiliary tool for the molecular diagnosis of non-small cell lung cancer (NSCLC), especially when an adequate tumor tissue specimen cannot be obtained. We compared the diagnostic accuracy of two commonly used in vitro diagnostic-certified allele-specific quantitative PCR assays for detecting plasma cfDNA EGFR mutations. MethodsWe analyzed EGFR mutations in plasma cfDNA from 90 NSCLC patients (stages I–IV) before treatment (n ​= ​60) and after clinical progression on EGFR tyrosine kinase inhibitors (n ​= ​30) using the cobas EGFR mutation test v2 (Roche Molecular Systems, Inc.) and therascreen EGFR Plasma RGQ PCR kit (Qiagen GmbH). ResultsThere was higher concordance between plasma cfDNA and matched tumor tissue EGFR mutations with cobas (66.67%) compared with therascreen (55.93%). The concordance rate increased to 90.00% with cobas (Cohen's kappa coefficient, κ ​= ​0.80; p ​< ​0.0001) and 73.33% with therascreen (κ ​= ​0.49; p ​= ​0.0009) in advanced NSCLC patients. In treatment-naïve patients, cobas was superior to therascreen (sensitivity: 82.35% vs. 52.94%; specificity: 100% vs. 100%). In patients with clinical progression on EGFR tyrosine kinase inhibitors, EGFR exon 20 p.T790M was detected in 30% and 23% of cfDNA samples by cobas and therascreen, respectively. ConclusionsCobas was superior to therascreen for detection of plasma EGFR mutations in advanced NSCLC. Plasma cfDNA EGFR mutation analysis is complex; therefore, the diagnostic accuracy of commercially available assays should be validated.

The role of EGFR mutation testing in the choice for surgical tactics in NSCLC treatment

Lung cancer is the most commonly diagnosed cancer in the world, and currently the mortality rate from this disease is one of the highest. The detection of EGFR mutation plays an important role in the choice of treatment in clinical practice. As already known, over the past decade, EGFR mutations have been a predictive factor for the prescription of tyrosine kinase inhibitors (EGFR TKIs) in patients with advanced non-small cell lung cancer (NSCLC). The role of EGFR mutation as a prognostic factor for patients undergoing surgery and the possibility of using surgery to overcome the resistance mechanisms that develop in response to EGFR TKIs remain unexplored. This literature review examines key aspects of the EGFR mutation as a factor that can influence the treatment and using the surgical method.

Detection of epidermal growth factor receptor (EGFR) mutations from preoperative circulating tumor DNA (ctDNA) as a prognostic predictor for stage I-III non-small cell lung cancer (NSCLC) patients with baseline tissue EGFR mutations.

BackgroundPlasma circulating tumor DNA (ctDNA) may be a surrogate, minimally invasive approach to tissue-based epidermal growth factor receptor (EGFR) mutation detection in non-small cell lung cancer (NSCLC) patients. However, the predictive ability of preoperative ctDNA EGFR mutation test on long-term postoperative survival and tumor metastasis development has not been extensively investigated.MethodsStage I–III NSCLC patients with tissue EGFR mutations were enrolled in this study (n=174). The ctDNA EGFR mutations were identified in paired preoperative plasma samples. EGFR mutation testing was performed using Scorpion amplified refractory mutation system (ARMS) technology. The correlation between ctDNA EGFR mutation status and clinicopathologic parameters was analyzed. By combining at least 5 years of follow-up data, we assessed the relationship between ctDNA EGFR mutation status and disease-free survival (DFS) and overall survival (OS).ResultsPlasma-based ctDNA EGFR mutations were detected in 27 patients. The mutation types were exactly matched with those in paired tissue samples. Blood test sensitivity was closely associated with N stages, tumor-node-metastasis (TNM) stages and tumor differentiation (P<0.001). The overall 5-year survival rate was 18.5% versus 76.9% for ctDNA EGFR mutation-positive and ctDNA EGFR mutation-negative patients, respectively. For patients with ctDNA EGFR mutation positive, the median OS and DFS were 29.00±2.55 and 19.00±2.50 months, respectively, which were both significantly better than those in the ctDNA EGFR mutation-negative subgroup (P<0.001). ctDNA EGFR mutation was an independent risk factor of OS and DFS [hazard ratio (HR) 3.289, 95% confidence interval (CI), 1.816–5.956, P<0.001; HR, 4.860, 95% CI, 2.660–8.880, P<0.001]. For stage III patients with exon 19 deletion or L858R mutations in both tissue and plasma samples, tyrosine kinase inhibitor (TKI) therapy showed significantly better OS (P=0.025) and possible DFS benefit (P=0.060) than did chemotherapy.ConclusionsEGFR mutation testing using the Scorpion-ARMS method in preoperative plasma could be a strong predictor for postoperative survival and metastasis of NSCLC patients. Thus, the subset of this population may be benefit from targeted strategies and management.

Abstract 1196: BALF liquid biopsy provides a novel platform for speedy and accurate EGFR mutation testing in advanced NSCLC patients

Abstract Background EGFR mutation testing is an essential step for the therapeutic decision in newly diagnosed advanced NSCLC patients and usually performed by using DNA extracted from biopsied tumor tissue with a turnaround time(TAT) of 2-3 weeks. Plasma liquid biopsy using ctDNA is minimally invasive and less time-consuming, but shows limited usefulness due to low sensitivity. We conducted a prospective study to demonstrate clinical usefulness of bronchoalveolar lavage fluid (BALF) liquid biopsy using extracellular vesicle(EV)-derived DNA in advanced NSCLC patients by comparing the sensitivity and TAT with conventional tissue biopsy and plasma liquid biopsy Methods The analysis set for liquid EGFR genotyping is 224 BALF samples and 110 plasma samples obtained from newly diagnosed 224 advanced non-squamous NSCLC patients. EGFR genotyping was done through peptide nucleic acid(PNA)-mediated real-time PCR after extracting BALF EV DNA and plasma cfDNA separately. The sensitivity, specificity, and concordance rate of BALF liquid biopsy were calculated in comparison to matched tissue genotyping and plasma liquid biopsy. In addition, TAT was compared between BALF liquid biopsy and tissue genotyping. Results Tissue genotyping revealed 93 EGFR mutant cases (41.5%) and 131 wild-type cases (58.5%). The sensitivity, specificity, positive predictive value, negative predictive value, and concordance rate of BALF liquid biopsy to tissue genotyping were 97.8%, 96.9%, 96.8%, 98.4%, and 97.7%, respectively. BALF liquid biopsy showed almost identical performance with standard tissue genotyping, while only 5 cases were mismatched each other. On the other hand, plasma liquid biopsy using cfDNA (n=110) revealed 48.5% sensitivity, 86.3% specificity, and 63.6% concordance rate in comparison to tissue genotyping. The mean turnaround times of BALF liquid biopsy was significantly shortened to 2.6±2.0 days in contrast to 13.9±12.4 days of tissue genotyping. (p&amp;lt;0.001). Conclusion BALF liquid biopsy can be developed as a novel platform for EGFR mutation testing in advanced NSCLC with almost identical performance with conventional tissue genotyping and significantly shorten TAT to 1-2 days. Citation Format: In Ae Kim, Jae Young Hur, Hee Joung Kim, Wan Seop Kim, Kye Young Lee. BALF liquid biopsy provides a novel platform for speedy and accurate EGFR mutation testing in advanced NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1196.

Abstract 488: Computer-assisted three-dimensional quantitation of programmed death-ligand 1 expression in non-small cell lung cancer using tissue clearing technology on formalin-fixed, paraffin-embedded specimen

Abstract Immune checkpoint blockade therapy has revolutionized cancer treatment, achieving sustained remission even in a proportion of patients with metastatic disease. However, not all patients respond to these therapies. Currently, histological detection of PD-L1 expression in tumor specimens is the standard method for guiding checkpoint blockade therapies involving anti-PD-1/PD-L1 antibodies. Nevertheless, accumulating evidence from clinical studies indicates that the pathological assessment of PD-L1 expression is neither a consistent nor a reliable predictor of anti-PD-1/PD-L1 treatment outcome. This inconsistency can be partly attributed to tumor heterogeneity in PD-L1 expression, as well as the common practice of assessing PD-L1 expression based on a single tissue section made from limited tumor biopsy specimen. In order to overcome this obstacle, we developed a novel method that can make formalin-fixed, paraffin-embedded (FFPE) tissue translucent to light, allowing three-dimensional imaging of the specimen. Our specialized protocol can process non-small cell lung cancer tissue up to 150 micrometer in thickness, allowing fluorescence-labeled anti-PD-L1 antibody staining throughout the entire tissue, and produce high-resolution 3D images. Compared to a traditional single 4-micrometer section of the same specimen, our 3D image provides 30 times more coverage of the specimen, assessing PD-L1 expression of approximately 15 times more cells. We also developed a computer-assisted PD-L1 signal quantitation algorithm, integrating artificial intelligence (AI) models of tumor region segmentation and PD-L1 positive cell classifier to generate PD-L1 tumor proportion score (TPS) along the specimen depth. Both AI models have greater than 90% accuracy compared to pathologist's assessment. Analysis of 3D images from seven lung adenocarcinoma specimens showed that TPS variation may reach up to 23.76% at different tissue depth levels. One of the seven cases has TPS below 1% at certain depth level but above 1% at other levels, a difference that can potentially influence clinical decision making. Importantly, our technology permits recovery of the processed tissue for subsequent analysis, including traditional histology examination, immunohistochemistry, and DNA analysis, such as EGFR mutation test. In conclusion, our novel method has the potential to increase the accuracy of tumor PD-L1 expression assessment and enable precision deployment of cancer immunotherapy. Citation Format: Yen-Yu Lin, Lei-Chi Wang, Yu-Han Hsieh, Sih-Kai Chuang, Yung-An Chen, Yu-Chieh Lin, Yen-Yin Lin, Teh-Ying Chou. Computer-assisted three-dimensional quantitation of programmed death-ligand 1 expression in non-small cell lung cancer using tissue clearing technology on formalin-fixed, paraffin-embedded specimen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 488.

A Real-World, Observational, Prospective Study to Assess the Molecular Epidemiology of Epidermal Growth Factor Receptor (EGFR) Mutations upon Progression on or after First-Line Therapy with a First- or Second-Generation EGFR Tyrosine Kinase Inhibitor in EGFR Mutation-Positive Locally Advanced or Metastatic Non-Small Cell Lung Cancer: The 'LUNGFUL' Study.

Simple SummaryNon-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases, with few patients carrying driver mutations in the gene encoding for epidermal growth factor receptor (EGFR). Advances in translational research have established EGFR tyrosine kinase inhibitors (TKIs) as the standard first-line therapy for NSCLC patients with activating EGFR mutations. The aim of our observational study was to assess the frequency of T790M acquired resistance and predictors of its presence, in patients with EGFR-mutated locally advanced or metastatic NSCLC who have progressed in the first-line EGFR-TKI treatment setting with first- or second-generation TKIs and have undergone molecular testing in tissue and/or plasma biopsy. The study highlights the challenges of performing tissue re-biopsy in routine care settings, which can lead to patients considered non-eligible for certain therapies from which they can benefit, and merits further actions from the healthcare community, in order to establish re-biopsy as a standard procedure.Background: Real-world data on the molecular epidemiology of EGFR resistance mutations at or after progression with first- or second-generation EGFR-TKIs in patients with advanced NSCLC are lacking. Methods: This ongoing observational study was carried out by 23 hospital-based physicians in Greece. The decision to perform cobas® EGFR Mutation Test v2 in tissue and/or plasma at disease progression was made before enrollment. For patients with negative/inconclusive T790M plasma-based results, tissue re-biopsy could be performed. Results: Ninety-six (96) eligible patients were consecutively enrolled (median age: 67.8 years) between July-2017 and September-2019. Of the patients, 98% were tested upon progression using plasma and 2% using tissue/cytology biopsy. The T790M mutation was detected in 16.0% of liquid biopsies. Tissue re-biopsy was performed in 22.8% of patients with a T790M-negative plasma result. In total, the T790M positivity rate was 21.9%, not differing between patients on first- or second-generation EGFR-TKI. Higher (≥2) ECOG performance status and longer (≥10 months) time to disease progression following EGFR-TKI treatment initiation were associated with T790M positivity. Conclusions: Results from plasma/tissue-cytology samples in a real-world setting, yielded a T790M positivity rate lower than previous reports. Fewer than one in four patients with negative plasma-based testing underwent tissue re-biopsy, indicating the challenges in routine care settings.

Analytical Validation of a Pan-Cancer Panel for Cell-Free Assay for the Detection of EGFR Mutations.

Liquid biopsies have increasingly shown clinical utility. Although next-generation sequencing has been widely used for the detection of somatic mutations from plasma, performance characteristics vary by platform. Therefore, thorough validation is mandatory for clinical use. This study aimed to evaluate the analytical validity of the Oncomine Pan-Cancer Cell-Free Assay. A massively parallel sequencing for the assay was performed using the Ion S5 XL System with Ion 540 kit. The analytical sensitivity and precision were evaluated using pre-characterized reference materials. The specificity was evaluated using plasma from healthy subjects. A comparison with the Cobas EGFR Mutation Test v2 was performed using reference materials and plasma from lung cancer patients. For SNVs and short indels, the analytical sensitivities at variant allele frequencies (VAFs) of 0.1%, 0.5%, and 1% were 50%, 93.4%, and 100% with 20 ng of input, respectively. The overall precision of the true positive variants was 98% at a VAF of 1% with 20 ng input. The assay showed a similar sensitivity to that of the Cobas EGFR Mutation Test v2 at a VAF of 0.5% with 20 ng of input and 100% concordance on clinical samples. The Pan-Cancer Cell-Free Assay can be applied to detect EGFR mutations in advanced lung cancer patients, although follow-up studies will be needed to evaluate the analytical validity for other types of genes and aberrations using clinical samples.

Detection of EGFR Mutations in Plasma cfDNA and Paired CTCs of NSCLC Patients before and after Osimertinib Therapy Using Crystal Digital PCR.

Simple SummaryLiquid biopsy is a useful tool during longitudinal monitoring of NSCLC patients and requires highly sensitive and reliable technologies for accurate detection of genomic alterations. We evaluated and used crystal digital PCR to detect and quantify EGFR mutations in plasma cfDNA and paired CTCs of NSCLC patients before treatment with osimertinib and at progression of disease.Circulating tumor DNA (ctDNA) analysis has clinical utility in EGFR mutant NSCLC. Circulating tumor cells (CTCs) consist a unique source of information at the cellular level. Digital PCR (dPCR) is a valuable tool for accurate and valid analysis of gene mutations in liquid biopsy analysis. In the present study we detected EGFR mutations in ctDNA and paired CTCs under osimertinib therapy at two time points using crystal dPCR and the naica® system (Stilla Technologies). We quantified mutation allele frequencies (MAF) of EGFR mutations in 91 plasma cfDNA samples of 48 EGFR mutant NSCLC patients and in 64 matched CTC-derived genomic DNA samples, and the FDA-cleared cobas® EGFR mutation test in 80 identical plasma samples. Direct comparison between crystal dPCR and the cobas EGFR assay revealed a high concordance for all EGFR mutations. Our comparison of crystal dPCR results in ctDNA with the corresponding primary tissue has shown a strong correlation. EGFR mutations analysis in paired CTC-derived gDNA revealed a high heterogeneity. Crystal dPCR offers the unique advantages of high analytical sensitivity, precision, and accuracy for detecting and quantifying multiple EGFR mutations in plasma cfDNA and CTCs of NSCLC patients.

The diagnostic and predictive efficacy of 18F-FDG PET/CT metabolic parameters for EGFR mutation status in non-small-cell lung cancer: A meta-analysis

PurposeTo investigate the predictive performance of the maximum standardized uptake value (SUVmax) and mean standardized uptake value (SUVmean) of primary lesions based on 18 F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) for EGFR mutation status in patients with non-small cell lung cancer (NSCLC). MethodsThe PubMed/Medline, Embase, Cochrane Library and Web of Science databases were searched as of January 1, 2021. Studies whose reported data could be used to construct contingency tables were included. Study characteristics were extracted, and methodological quality assessment was conducted by two separate reviewers using the Quality Assessment of Diagnostic Accuracy Studies. The pooled sensitivity, specificity and area under the summary receiver operating characteristic curve (AUROC) were calculated. The possible causes of heterogeneity were analysed by meta-regression. ResultsThe 18 included studies had a total of 4024 patients. The majority of the studies showed a low to unclear risk of bias and concerns of applicability. For differentiating EGFR-mutant NSCLC from wild-type NSCLC, the pooled sensitivity and specificity were 71 % and 60 % for SUVmax and 64 % and 63 % for SUVmean, respectively. The summary AUROCs of SUVmax and SUVmean were 0.69 (95 % CI, 0.65–0.73) and 0.68 (95 % CI, 0.64–0.72), respectively. The meta-regression analysis indicated that blindness to EGFR mutation test results, the number of readers and the number of PET/CT scanners were possible causes of heterogeneity. ConclusionsOur meta-analysis implied that SUVmax and SUVmean of primary lesions from 18F-FDG PET/CT harboured moderate predictive efficacy for the EGFR mutation status of NSCLC.