Scorpion Amplification Refractory Mutation System Research Articles

Investigation of EGFR mutations in non-small cell lung cancer usually undetectable by PCR methods.

Epidermal growth factor receptor (EGFR) mutations are the most significant genomic drivers of non-small cell lung cancer (NSCLC) and determine the efficacy of EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy. PCR methods are used clinically for the detection of EGFR mutations. The Scorpion Amplification Refractory Mutation System (Scorpion-ARMS) and the cobas® EGFR Mutation Test v2 (cobas v2) are widely used PCR methods. However, those PCR methods only selectively detect the common EGFR mutations. The aim of the present study was to reveal the true frequency of EGFR mutations in NSCLC by investigating EGFR mutations usually undetectable by PCR methods by using direct sequencing. A total of 70 Japanese patients who underwent lung resection for NSCLC between September 2016 and March 2019 were included in the present study. Subsequently, PCR methods and direct sequencing were performed. In total, 29 mutations were detected by cobas v2. In total, 41 patients were identified as EGFR wild-type by cobas v2, among whom direct sequencing detected mutations in 3 patients. Subsequent Scorpion-ARMS was performed in the 3 patients in whom direct sequencing detected mutations. In total, one exon 21 L858R + G863D compound mutation was identified as a L858R single mutation, and two other mutations were undetectable. Moreover, 1 patient who was ‘wild-type’ on cobas v2 but ‘EGFR mutation’ on direct sequencing developed recurrence after surgery and responded to EGFR-TKI treatment. In present study, the percentage of undetectable EGFR mutations by cobas v2 was 9.4% in 32 mutations. It was inferred that the cause of the discrepancy in the mutation type (L858R + G863D in exon 21, and L858R in exon 21) between cobas v2 and Scorpion ARMS was due to the different limit of detection between these two PCR methods. In conclusion, the findings of the present study suggested that a selective mutation detection method may decrease the opportunity of patients with NSCLC to receive EGFR-TKI therapy. Thus, the development of a screening test to determine the EGFR status as wild-type or mutant is required for EGFR-TKI therapy.

Initial low dose of dacomitinib for first-line treatment of patients with EGFR exon 21 mutated non-small cell lung cancer.

e21083 Background: Dacomitinib showed superior efficacy for patients with epidermal growth factor receptor (EGFR) sensitive mutations as compared to that of gefitinib, especially for those harboring exon 21 L858R mutation. However, the grade 3-4 treatment related adverse events were more common in patients receiving 45mg of dacomitinib as initial treatment. Hence, we did this study to explore efficacy and safety of low dose dacomitinib for first-line treatment of patients with EGFR exon 21 mutated non-small cell lung cancer (NSCLC). Methods: We retrospectively analyzed data of patients with EGFR exon 21 mutated advanced NSCLC treated with 15 mg dacomitinib as initial therapy between August 2019 and December 2020 at Affiliated Dongguan Hospital, Southern Medical University, China. EGFR mutation was identified using the scorpion amplification refractory mutation system method (ARMS) or next-generation sequencing technology (NGS). A total of 9 NSCLC patients with exon 21 mutation were included. All of these patients received dacomitinib (15 mg, once a day) until disease progression or unacceptable toxicity. Response was assessed according to Response Evaluation Criteria In Solid Tumors version 1.1 (RECIST v1.1). Toxicities were recorded and classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 3.0. The last follow-up date is February 10, 2021. The primary end point was objective responsive rate (ORR). The secondary end points were disease control rate (DCR), median progression-free survival (PFS) and treatment related toxicity. Results: Among the 9 patients with EGFR exon 21 mutated NSCLC in this study, 8 patients harboring L858R mutation while one patients harboring L861Q mutation. The median age of treatment patients was 70 years (range, 39-87 years). There were 3 patients with brain brain metastases (33.3%) and had never received whole-brain radiation therapy (WBRT), surgery, radiosurgery or systemic treatment.They all received low dose of dacomitinib as initial treatment. The ORR was 77.8% and DCR was 100%. The median PFS data was not mature. None of these patients had a progression disease until the last follow-up date. Treatment related adverse events were generally mild. The most frequent toxicity was rash (44.4%), the remaining adverse events included rash, pruritus, diarrhea, abnormal hepatic function, anaemia and elevated creatinine, all of which occurred only once during the treatment period. None of the patients experienced grade 3 or higher toxicities. Conclusions: Our study showed that dacomitinib (15 mg, once a day) may be also effective for patients harboring EGFR exon 21 mutated NSCLC, with significantly reduced toxicities as compared to that of standard dose. Prospective or real-world studies are needed to further illuminate the effect of low dose of dacomitinib on EGFR exon 21 mutated patients.

Detection of EGFR Mutations in Plasma Cell-Free Tumor DNA of TKI-Treated Advanced-NSCLC Patients by Three Methodologies: Scorpion-ARMS, PNAClamp, and Digital PCR.

Analysis of circulating cell-free tumor DNA (cftDNA) has emerged as a specific and sensitive blood-based approach to detect epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. Still, there is some debate on what should be the preferential clinical method for plasma-derived cftDNA analysis. We tested 31 NSCLC patients treated with anti-EGFR tyrosine kinase inhibitors (TKIs), at baseline and serially during therapy, by comparing three methodologies in detecting EGFR mutations (L858R, exon 19 deletion, and T790M) from plasma: scorpions-amplification refractory mutation system (ARMS) methodology by using EGFR Plasma RGQ PCR Kit-QIAGEN, peptide nucleic acid (PNA) clamp and PANA RealTyper integration by using PNAClamp EGFR-PANAGENE, and digital real time PCR by using QuantStudio 3D Digital PCR System-Thermo Fisher Scientific. Specificity was 100% for all three mutations, independently from the platform used. The sensitivity for L858R (42.86%) and T790M (100%) did not change based on the method, while the sensitivity for Del 19 differed markedly (Scorpion-ARMS 45%, PNAClamp 75%, and Digital PCR 85%). The detection rate was also higher (94.23%) as measured by Digital PCR, and when we monitored the evolution of EGFR mutations over time, it evidenced the extreme inter-patient heterogeneity in terms of levels of circulating mutated copies. In our study, Digital PCR showed the best correlation with tissue biopsy and the highest sensitivity to attain the potential clinical utility of monitoring plasma levels of EGFR mutations.

Primary intraosseous Rosai-Dorfman disease: a clinicopathological analysis of fourteen cases

Objective: To investigate the clinicopathological characteristics, histogenesis, immunophenotypes and molecular genetic features of primary intraosseous Rosai-Dorfman disease (RDD) for improving diagnostic accuracy and differential diagnosis. Methods: This retrospective study included 14 RDD cases diagnosed from January 2009 to January 2019 at Beijing Jishuitan Hospital, China. The immunohistochemical staining for S-100, cyclin D1, CD1a and CD207 expression was analyzed. The BRAF V600E and KRAS mutation analyses were performed using the Scorpions amplification refractory mutation system (ARMS) fluorescence quantitative PCR. Results: There were 6 female and 8 male patients, aged from 2 to 64 years (mean 31.4 years). All of the 14 cases occurred in the bone without lymph node disease, while one patient developed additional lesions within vertebra and nasal cavity. Radiographically, the lesions were lytic with sclerotic margins. Histologically, the lesions percolated through the medullary cavity in an infiltrative fashion and alternating hyper- and hypo-cellular regions of histiocytic clusters (seen as alternating dark and light zones at low magnification). Large histiocytes also showed emperipolesis. Some cases had areas of fibrosis and dense lymphoplasmacytic infiltrates. There were vasculitis and an increased number of plasma cells in the cases involving multiple sites. One case showed concurrence of RDD and Langerhans cell histiocytosis(LCH) with inconspicuous increase of Langerhans histiocytes. Immunohistochemical staining showed that the large histiocytes were positive for S-100, CD68 and CD163 in all cases. The nuclear immunoreactivity for cyclin D1 was observed in 13 of the 14 cases. S-100, CD1a and CD207 were positive in the case with concurrence of RDD and LCH. ARMS-PCR results showed that BRAF V600E mutation was observed in the cases with concurrence of RDD and LCH, while there were no KRAS mutations (7/7). Follow-up information was available for 12 patients and ranged from 9 to 49 months. Three of the 12 patients experienced recurrences after the first surgery. Conclusions: Primary intraosseous RDD is rare, and its concurrence with LCH is a very rare phenomenon. Its clinical symptoms, imaging, and pathological manifestations need to be distinguished from other bone lesions. The molecular detection of BRAF V600E and the nuclear expression of cyclin D1 mutations can be used for the diagnosis and differential diagnosis of RDD.

Association Of Initial Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors Treatment And EGFR Exon 19 Deletion With Frequency Of The T790M Mutation In Non-Small Cell Lung Cancer Patients After Resistance To First-Line Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors.

BackgroundThe present study analyzed the relationship between clinical features and the T790M mutation in non-small cell lung cancer (NSCLC) patients resistant to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) treatment.MethodsNSCLC patients with resistance to first-generation EGFR-TKIs in which the disease control time was more than 6 months after initial TKI treatment were enrolled. T790M mutation analysis was performed using one of the following methods according to each manufacturer’s protocols: Cobas EGFR mutation test (41/105, 39.0%), digital PCR (42/105, 40.0%) or Scorpion amplification refractory mutation system (ARMS) (22/105, 21.0%). Sample type of T790M was from tissue only (53/105, 50.5%), plasma only (46/105, 43.8%), tissue and plasma (6/105, 5.7%).ResultsOf 105 patients, 57 were T790M-positive and 48 were T790M-negative. T790M-positive patients had longer progression-free survival (PFS) after initial EGFR-TKI treatment (p = 0.019). T790M positivity was more frequent in patients treated with gefitinib than in those treated with icotinib (65% vs 40.54%, p = 0.018). The rate of T790M positivity was lower in patients with EGFR L858R (44.44%, 12/27) before TKI treatment than in those with EGFR 19del (72.0%, 36/50, p = 0.036). Patients who achieved PR after initial EGFR-TKI treatment had a higher rate of T790M positivity than those with SD (75.76% vs 50%, p = 0.023). There was no relationship between T790M status and age, gender, primary site, metastasis site, or treatment before TKI.ConclusionProgression-free survival (PFS), drug type, response to initial EGFR-TKI treatment, and EGFR status before initial EGFR treatment were associated with the frequency of T790M mutation.

Implication of BRAF V600E and CTNNB1 gene mutations in the pathological classification of craniopharyngioma

Objective: To investigate the clinicopathological significance of BRAF V600E and CTNNB1 gene mutations in adamantinomatous craniopharyngiomas (ACP) and papillary craniopharyngiomas (PCP). Methods: The retrospective study included a total of 67 craniopharyngiomas diagnosed from October 2009 to August 2018 at Xuanwu Hospital, Capital Medical University. The immunohistochemical staining for β-catenin and BRAF V600E expression, Sanger sequencing of exon 3 of CTNNB1, BRAF mutation analysis by scorpions amplification refractory mutation system (ARMS) fluorescence quantitative PCR were performed. Univariate survival analysis was used to correlate with tumor recurrence. Results: Of the 67 patients, 53 were ACPs and 14 were PCPs. Four patients underwent multiple operations and one of them presented with malignant transformation into squamous cell carcinoma. Histologically, ACPs were characterized by whorl-like cell clusters, peripheral palisaded layer, stellate reticulum, finger-shaped protrusions, ghost cells and wet keratinous substances. While PCPs usually consisted of mature squamous epithelium associated with fibrovascular stroma resulting in papillary appearance. The nuclear immunopositivity for β-catenin was observed in 73.6% (39/53) of ACPs, and it was absent in PCPs (0/14). The nuclear translocation of β-catenin usually presented at whorl-like structures or around ghost cells. Of all the cases, mutations analysis in exon 3 of β-catenin gene CTNNB1 were successful in 46 cases and 42.1% (16/38) of ACP showed CTNNB1 gene mutation, while none of the PCPs harbored CTNNB1 gene mutation (0/8). The cytoplasmic immunopositivity for BRAF V600E mutant protein was found in all PCPs (14/14) and negative in all ACPs (0/53). ARMS-PCR results showed that BRAF V600E mutations were observed in 13/14 of PCPs but not seen in ACPs (0/53). Follow-up data were available in 35 patients with duration of 2 to 120 months. Ten patients experienced recurrences after the first surgery. Upon univariate survival analysis, only subtotal excision was found to be associated with increased recurrence (P=0.032), while pathological type, postoperative radiotherapy and CTNNB1 gene mutation were not (P>0.05). Conclusions: There is significant difference in the expression of BRAF V600E and CTNNB1 genes between ACP and PCP, and their immunohistochemical and molecular detection therefore can be used in the diagnosis and differential diagnoses of craniopharyngiomas.

Frequency and significance of epidermal growth factor receptor mutations detected by PCR methods in patients with non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) is the most important driver gene of non-small cell lung cancer (NSCLC) as EGFR mutations determine the efficacy of EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy. In the present study, the comprehensive ability of widely used polymerase chain reaction (PCR) methods to detect EGFR mutations was determined. Among the 35 EGFR mutations detected via the direct sequencing of 73 patients with NSCLC, 11 types were identified in exons 18, 19 and 21. Among the 11 mutation types, all exon 18 and 21 mutations were identified by 2 widely used PCR methods, namely, Scorpion-Amplification Refractory Mutation System and cobas v2. However, among the 9 different exon 19 deletions, 3 types were not identified by the 2 methods. In addition, 25 samples with EGFR mutations were analyzed by the 2 methods, including a sample from a patient with an unidentified exon 19 deletion, the T751_I759 deletion and insertion S; this patient had long-term disease control as a result of EGFR-TKI therapy. The 2 methods could not detect this unidentified deletion, whereas sizing capillary electrophoresis for the comprehensive detection of exon 19 deletions detected this deletion. It is generally thought that patients with exon 19 mutations have higher response rates to EGFR-TKI therapy than patients with exon 21 mutations. The present study confirmed the EGFR mutation status by comparing the mutations with the Catalog Of Somatic Mutations In Cancer, which is the world's largest and most comprehensive resource for analyzing the effects of somatic mutations in human cancers. The predicted frequency of EGFR mutations identified by the 2 methods was 85%. The frequency of mutations detectable by the 2 methods was less for exon 19 than exon 21. Therefore, the results of the present study suggest that decreasing false-negative detection of exon 19 deletions is crucial for the clinical testing of EGFR mutations.

Efficacy, safety and prognostic factors analysis of first-line icotinib treatment in advanced non-small cell lung cancer patients with mutated EGFR

Background: This study aimed to evaluate the efficacy, safety and prognostic factors of icotinib as first-line treatment in advanced non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR)-sensitive mutation. Methods: A total of 152 advanced NSCLC patients with EGFR-sensitive mutation underwent first-line icotinib treatment were retrospectively reviewed in this cohort study. Icotinib was given orally three times daily at 125 mg dose. Scorpion amplification refractory mutation system kit was used for EGFR mutation detection. The median follow-up duration was 15.0 (range: 1.0–44.0) months, and the last follow-up date was 2016/12/31. Results: The complete response (CR), partial response (PR), objective response rate (ORR), stable disease (SD) and progressive disease (PD) rates were 4% (N=6), 67.8% (N=103), 71.8% (N=109), 23.7% (N=36) and 4.6% (N=7) respectively. Median progression-free survival (PFS) and overall survival (OS) were 14.0 months (95% CI: 12.7–15.3 months) and 33.0 months (95% CI: 24.9–41.1 months) respectively. Multivariate logistic analysis indicated smoking history (P=0.013) was statistically correlated with non-ORR achievement, while EGFR mutation 19del (P=0.001) was statistically associated with ORR achievement. No factor could predict PFS or OS. As to safety profiles, there were 66 (43.4%), 35 (23.0%), 10 (6.6%), 9 (5.9%), 9 (5.9%), 4 (2.6%) and 3 (2.0%) patients appeared rash, diarrhea, abnormal liver function, paronychia, oral ulcer, fatigue and poor appetite, and most adverse events (AEs) were in mild grade. Conclusions: First-line icotinib treatment was efficient and well tolerated in patients with advanced NSCLC harboring EGFR mutations, and smoking history was negatively while EGFR mutation 19del was positively correlated with ORR achievement.

Serum VEGF levels in the early diagnosis and severity assessment of non-small cell lung cancer.

Background: Effective biomarkers are essential to the differential diagnosis and severity assessment of non-small cell lung cancer (NSCLC). This study explored the use of the serum vascular endothelial growth factor (VEGF) levels as a biomarker with the aim of achieving better management of NSCLC.Methods: Serum VEGF levels were assayed via enzyme-linked immunosorbent assay in 180 patients with NSCLC, 136 patients with benign pulmonary nodules, and 119 healthy controls. We additionally detected the serum concentration of three traditional biomarkers—carcinoembryonic antigen (CEA), cancer antigen (CA)-125, and cytokeratin 19 fragments (Cyfra 21-1)—to comparatively evaluate the efficiency and diagnostic value of VEGF in patients with NSCLC. We further evaluated the relationship between serum VEGF levels and clinicopathologic parameters. VEGF levels were compared between pro- and post-surgical patients using the Wilcoxon matched-pairs signed-rank test. DNA was isolated from the primary tumors. EGFR mutations were detected by Scorpions amplification refractory mutation system (ARMS).Results: Patients with NSCLC had significantly higher serum concentration of VEGF, compared to those with benign pulmonary nodules and healthy controls (P <0.0001). As a diagnostic biomarker of NSCLC, VEGF had area under the curve values of 0.824 and 0.839, sensitivities of 75.0% and 75.0%, and specificities of 93.3% and 95.6% when compared with healthy people and patients with benign pulmonary nodules, respectively; notably, these values were greater than those of CA125, Cyfra 21-1 and CEA. Furthermore, a model in which VEGF was combined with CEA, CA125, and Cyfra 21-1 was more effective for NSCLC diagnosis than VEGF alone (sensitivity, 85.0% and 84.4; specificity, 90.0% and 91.9% vs. healthy controls and patients with benign pulmonary nodules, respectively). When use to identify early-stage NSCLC, VEGF showed a better diagnostic efficacy than other biomarkers. The pro-surgical VEGF levels were significantly higher than those measured 25-30 days after surgery. Moreover, VEGF concentration differed significantly among cases according to TNM stages and malignant grades (P <0.0001). EGFR mutations and the size of benign pulmonary nodules did not affect the level of serum VEGF significantly.Conclusion: The serum VEGF levels exhibited relatively high sensitivity and specificity for NSCLC, and may therefore be a useful diagnostic biomarker. Furthermore, the serum VEGF levels could be used to assess prognosis and curative effects.

454P Efficacy of crizotinib in NSCLC with concomitant ALK fusion and c-MET overexpression

Background ALK fusion is a common driver gene in non-small-cell lung cancer (NSCLC), accounting for 3%-13%. Crizotinb is considered as first-line therapy for advanced ALK-positive patients. Patients with c-MET overexpression obtained clinical benefit from crizotinib, with the frequency of 31.9%. We investigated the efficacy of crizotinb in patients with concomitant ALK fusion and c-MET overexpression. Methods We identified 203 ALK-positive advanced NSCLC patients from January 2010 to November 2015 with any of the 3 methods:FISH, RACE-PCR, or Ventana IHC. A total of 113 patients were under the analysis of c-MET overexpression by IHC, in which 50% tumor cells with moderate to high intensity staining were defined as c-MET positive. Patients with ALK and c-MET positive are considered as Group A, while others as Group B. Besides, we defined patients with c-MET of 50% tumor cells with high intensity staining and ALK fusion as Group C, while others as Group D. All patients were identified without EGFR mutation by DNA direct sequencing or Scorpion amplification refractory mutation system. Results Among ALK-positive NSCLC patients, the frequency of c-MET positive was 29.2% (33/113). A total of 22 patients with concomitant ALK fusion and c-MET overexpression and 54 patients with only ALK fusion received crizotinib in their first- or further-line settings. There was no significant difference in age (P = 0.629), gender (P = 0.659), smoking history (P = 0.745), and histology (P = 0.498) between these two arms.Objective response rate (ORR) of crizotinib was 72.7% (16/22) in Group A and 63.0% (34/54) in Group B (P = 0.416). Median PFS of crizotinib was 12.8 vs 9.5 months in these two groups (P = 0.514).Median OS was 33.0 vs 29.5 months, with Hazard ratio (HR) of 1.038(P = 0.919). Eight patients were identified in group C with ORR of 100.0% (8/8), higher than those in group D (P = 0.046). Median PFS was 16.8 vs 9.3 months in these two groups, with HR of 0.548 (P = 0.079). Median OS was 34.0 vs 29.5 months, with HR of 0.633 (P = 0.347). Conclusions Similar efficacy with crizotinib was obtained in ALK-positive patients with or without c-MET overexpression. However, there was a trend of better efficacy with crizotinib in ALK-rearranged patients with more than 50% tumor cells of strong c- MET overexpression. Legal entity responsible for the study: Guangdong Lung Cancer Institute Funding Guangdong Lung Cancer Institute, Guangdong General Hospital & Guangdong Academy of Medical Sciences, Guangzhou, China Disclosure All authors have declared no conflicts of interest.

Clinical outcomes of advanced non-small-cell lung cancer patients with EGFR mutation, ALK rearrangement and EGFR/ALK co-alterations.

The co-occurrence of epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements constitutes a rare molecular subtype of non-small-cell lung cancer (NSCLC). Herein, we assessed the clinical outcomes and incidence of acquired resistance to tyrosine kinase inhibitors (TKIs) in this subtype. So we enrolled 118 advanced NSCLC treated with TKIs. EGFR mutations and ALK rearrangements were detected by DNA sequencing or Scorpion amplification refractory mutation system and fluorescence in situ hybridization respectively. Immunohistochemistry was used to evaluate the activation of associated proteins. We found that nine in ten patients with EGFR/ALK co-alterations had good response with first-line EGFR TKI, and the objective response rate (ORR) of EGFR TKIs was 80% (8/10) for EGFR/ALK co-altered and 65.5% (55/84) for EGFR-mutant (P = 0.57), with a median progression-free survival (PFS) of 11.2 and 13.2 months, (hazard ratio [HR]=0.95, 95% [CI], 0.49-1.84, P= 0.87). ORR of crizotinib was 40% (2/5) for EGFR/ALK co-altered and 73.9% (17/23) for ALK-rearranged (P= 0.29), with a median PFS of 1.9 and 6.9 months (hazard ratio [HR], 0.40; 95% [CI] 0.15-1.10, P = 0.08). The median overall survival (OS) was 21.3, 23.7, and 18.5 months in EGFR-mutant, ALK-rearranged, and EGFR/ALK co-altered (P= 0.06), and there existed a statistically significant difference in OS between ALK-rearranged and EGFR/ALK co-altered (P=0.03). Taken together, the first-line EGFR-TKI might be the reasonable care for advanced NSCLC harbouring EGFR/ALK co-alterations, whether or nor to use sequential crizotinib should be guided by the status of ALK rearrangement and the relative level of phospho-EGFR and phospho-ALK.

A sensitive and practical method to detect the T790M mutation in the epidermal growth factor receptor.

The current study aimed to develop a method to rapidly, sensitively and practically screen for the epidermal growth factor receptor (EGFR) T790M mutation. This method combines an allele-specific competitive blocker (ACB) with a TaqMan quantitative polymerase chain reaction (PCR) amplification refractory mutation system (ARMS) in a one-step reaction. Using a mimic of a human genomic DNA panel containing serially diluted mutant alleles, the performance efficacy of this method was assessed. Using this method, the EGFR T790M mutation was detected in tyrosine kinase inhibitor (TKI)-naïve samples obtained from 27 non-small cell lung cancer (NSCLC) patients with EGFR-activating mutations. The association between de novo T790M mutations and the clinical benefit of EGFR-TKI treatment was also analysed. The sensitivity of this method was as low as 0.01%. In the samples from the 27 NSCLC patients, this method identified 6 mutant patients (22.2%), which was higher than the detection rate with scorpion ARMS (0.0%). No clinical variables were associated with the occurrence of a de novo T790M mutation. The median progression-free survival time in the TKI-naïve patients with a T790M mutation was shorter that that of patients without the mutation, but the difference was not significant (3.2 vs. 19.5 months, respectively; P=0.256). The median overall survival time in the groups with or without T790M mutation also did not significantly differ (10 vs. 20 months, respectively; P=0.689). Overall, the ACB-ARMS PCR method could be useful for detecting the EGFR T790M mutation in clinical samples that contain only a small number of mutant alleles. The clinical significance of a de novo T790M mutation should be further investigated.

Abstract 329: Establishment of three-dimensional primary tumor cell culture method and novel drug sensitivity test

Abstract Background Predicting sensitivity to anticancer drug and selecting the most effective drug may help to enhance the therapeutic value, leading to improve patients’ QOL. For these purposes, primary cancer cell from patient specimen is being studied on anticancer drug sensitivity test. In general cell culture method, primary cancer cells are difficult to culture because of fibroblast cells are increasing predominantly than cancer cells. For resolving the problem, collagen-gel-embedding culture method has been developed. However this culture method remain having some problems, for example, complexity of the cell culture preparation from small cancer tissue like a biopsy sample, low efficiency of primary cancer cells and difficulty in multi-drug sensitivity test. Therefore we developed a three dimensional (3D) cancer cell culture kit that could conduct a primary tumor cell culture and an evaluation of the anticancer drug sensitivity in one-step process after dissociated small cancer tissue. For 3D cell culture we have used NanoCulture Plate (NCP) which is scaffold based 3D culture system and easily form spheroids than other culture systems. Spheroids microenvironment are closely resemble to actual tumor biology, and are therefore more relevant for drug sensitivity. In this report, we carried out a sensitivity test in a lung adenocarcinoma specimen harboring EGFR gene exon19 deletion mutation indicating susceptibility to gefitinib to confirm the efficacy of the system. Method Dissociation of cancer tissue and primary cancer cell culture were performed by using 3D cancer cell culture kit and NCP. Partially digested cancer fragments from surgical specimens were seeded and cultured for 3 days. The spheroids were composed of both CK-18 expressing cancer cells and α-SMA expressing fibroblast cells, then spheroids were treated with gefitinib. After 4 days of drug treatment, the susceptibility of the cells to gefitinib was investigated an index of viability by ATP assay. Detection of EGFR mutations was performed by SRL Inc., using real-time PCR that combines scorpion technology and Amplification Refractory Mutation System (ARMS). Result and conclusion We found that primary cells with EGFR exon19 deletion were highly susceptible to gefitinib, whereas the cells with intact EGFR were not. These finding indicated the feasibility of 3D cancer cell culture kit and NanoCulture Plate for primary cell culture and screening of effective anticancer drugs. The cell lines generated through this procedure may help to advance our knowledge of certain forms of lung cancer and may also be useful for developing patient-specific anti-cancer drug screening procedures. To clarify the causal relationship between the mutation type and susceptibility, additional experiment and optimization of this kit are currently underway. Citation Format: Hiroshi Goji, Manami Shimomura, Yasushi Uemura, Tetsuya Nakatsura, M.Mamunur Rahman, Manabu Itoh. Establishment of three-dimensional primary tumor cell culture method and novel drug sensitivity test. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 329. doi:10.1158/1538-7445.AM2015-329

Efficacy of bronchoscopic biopsy for the detection of epidermal growth factor receptor mutations and anaplastic lymphoma kinase gene rearrangement in lung adenocarcinoma.

BackgroundTo explore the efficacy of bronchoscopic biopsy for the detection of epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) gene rearrangement in lung adenocarcinoma.MethodsAll patients with bronchoscopic biopsy-proven lung adenocarcinoma at the Peking Union Medical College Hospital from January 2009 to November 2011 were enrolled. Scorpion amplification refractory mutation system (ARMS) was used to detect EGFR gene mutations and fluorescence in situ hybridization (FISH) to detect ALK rearrangement. The correlation of immunohistochemistry (IHC) results with standard methods for EGFR mutation status and ALK rearrangement were checked.ResultsBronchoscopic specimens were successfully used to detect EGFR mutation and ALK rearrangement with success rates of 85.2% and 71.3%, respectively, in non-small cell lung cancer patients. EGFR analysis by ARMS yielded a positive result in 35.8% (33/92) and positive ALK rearrangement was detected by FISH in 7.8% (6/77) of cases. It was more likely to be unsuccessful in patients with tumor cells less than 100/high power field and the ratio tumor numbers in 0–10%. In EGFR-IHC, the sensitivity and specificity of E746-A750 deletions were 73.3% (11/15) and 93.3% (70/75), respectively, and those of L858R were 93.3% (14/15) and 93.2% (69/74), respectively. In ALK-IHC, the sensitivity and specificity were 50% (3/6) and 100% (71/71), respectively.ConclusionsSmall bronchoscopic specimens could achieve higher successful detection rates via EGFR mutation and ALK gene rearrangement.

Analysis of EML4-ALK gene fusion mutation in patients with non-small cell lung cancer

背景与目的非小细胞肺癌（non-small cell lung cancer, NSCLC）是肺癌的主要类型，相关位点突变检测研究已经成为肺癌分子靶向治疗的热门方向，研究NSCLC肿瘤组织中动物微管相关蛋白4与间变性淋巴瘤激酶融合基因（echinodem microtubule associated protein like 4-Anaplastic lymphoma kinase, EML4-ALK）与表皮生长因子受体（epidermal growth factor receptor, EGFR）的基因突变状态，比较免疫组织化学（immunohistochemistry, IHC）与蝎形探针扩增阻滞突变系统（Scorpions amplification refractory mutation system, Scorpions ARMS）荧光定量PCR与荧光原位杂交（fluorescence in situ hybridization, FISH）检测EML4-ALK融合基因与EGFR基因突变的敏感性。方法应用IHC、ARMS荧光定量PCR及FISH技术检测85例NSCLC石蜡包埋肿瘤组织以及癌旁正常肺组织中EML4-ALK融合基因状态，并应用ARMS方法检测EGFR基因第18、19、20和21外显子突变状态。结果115例NSCLC中IHC显示32例有ALK（D5F3）表达，表达率为27.8%;ARMS检测27例存在EML4-ALK融合基因突变，突变检出率为23.5%;53例检出EGFR突变，突变率为46%。而FISH检测23例存在EML4-ALK融合基因突变，检出率为20%，稍低于ARMS检测结果，提示ARMS的敏感度更高。结论联合运用IHC/ARMS荧光定量PCR/FISH技术能够对EML4-ALK融合基因状态做出快速、准确评价。

Pemetrexed versus gefitinib as a second-line treatment in advanced nonsquamous nonsmall-cell lung cancer patients harboring wild-type EGFR (CTONG0806): a multicenter randomized trial

CTONG0806 assessed the efficacy of pemetrexed versus gefitinib as second-line treatment in advanced nonsquamous nonsmall-cell lung cancer (NSCLC) harboring wild-type epidermal growth factor receptor (EGFR). Patients with locally advanced or metastatic nonsquamous NSCLC harboring wild-type EGFR, detected by direct sequencing, and previously treated with platinum-based chemotherapy were randomized to receive gefitinib (250 mg/day) orally or pemetrexed (500 mg/m(2)) i.v. on day 1 of a 21-day cycle until disease progression or unacceptable toxicity. The primary end point was progression-free survival (PFS). The Independent Review Committee (IRC) evaluated all pictorial data. From February 2009 to August 2012, 161 patients were enrolled, and 157 were assessable (81 in the gefitinib arm, 76 in the pemetrexed arm). Baseline characteristics were balanced between the two arms. The median PFSs were 4.8 versus 1.6 months in the pemetrexed and gefitinib arms, respectively [hazard ratio (HR) 0.54, 95% confidence interval (CI) 0.40-0.75, P < 0.001] as confirmed by IRC evaluation (5.6versus 1.7 months, HR 0.53, 95% CI 0.38-0.75, P < 0.001). The median overall survival (OS) showed a trend of superiority in the pemetrexed arm (12.4 versus 9.6 months, HR 0.72, 95% CI 0.49-1.04, P = 0.077). Quality-of-life assessment showed no marked difference between the arms. No unexpected adverse events were found. Of 108 patients with sufficient DNA samples, EGFR mutation status was re-tested by Scorpion amplification refractory mutation system (ARMS); 32 (29.6%) tested positive (19 in the pemetrexed arm, 13 in the gefitinib arm; median PFS: 8.1 versus 7.0 months, HR 0.94, 95% CI 0.43-2.08, P = 0.877). CTONG0806 is the first trial to show significant improvement in PFS and an improved OS trend with pemetrexed compared with gefitinib as second-line setting treatment of EGFR wild-type advanced nonsquamous NSCLC. ARMS is superior to direct sequencing in excluding false-negative patients. NCT00891579.

Suitability of Small Bronchoscopic Tumour Specimens for Lung Cancer Genotyping

Background: Biomarker-driven clinical trials in advanced non-small cell lung cancer (NSCLC) usually accept biopsy specimens only, as cytology specimens are supposed to be more challenging due to low neoplastic cell content and suboptimal DNA quantity. Objectives: We aimed to evaluate 2 aspects of bronchoscopic biopsy and cytology specimens: (1) the proportion of neoplastic cells and quantity of DNA extracted, and (2) the detection limit of the Scorpion amplification refractory mutation system on endoscopic samples obtained in daily clinical practice. Methods: We screened 679 patients with advanced-stage NSCLC for the presence of an activating EGFR mutation according to the guidelines of the European Society of Medical Oncology. Their diagnostic tumour tissue samples were characterized. A dilution experiment was performed to determine the minimal proportion of neoplastic cells for a reliable test result. Results: Surgical biopsies, bronchoscopic forceps biopsy samples and needle aspiration cytology specimens exhibited a median tumour cell proportion of 70 versus 30 versus 20% and a DNA quantity of 2,500 versus 1,610 versus 1,440 ng, respectively. The overall EGFR mutation rate was 11%, with no differences between different sample types. Dilution experiments showed that the detection limit depends on the type of mutation. A neoplastic cell content of at least 10 and 25% for exon 19 deletions and exon 21 L858R point mutation, respectively, was required for a true negative result. Conclusions: Bronchoscopic forceps biopsy and needle aspiration cytology specimens are suitable for accurate EGFR mutation analysis using single-gene quantitative real-time polymerase chain reaction. Technologies with a better analytical sensitivity are evolving and should consider these endoscopic tumour specimens.

Phase II study of erlotinib for previously treated patients with EGFR wild-type non-small-cell lung cancer, following EGFR mutation status reevaluation with the Scorpion Amplified Refractory Mutation System.

While assessing the efficacy of erlotinib in patients with epidermal growth factor receptor (EGFR) wild-type (WT) non-small-cell lung cancer (NSCLC), the sensitivity of the method used for the EGFR mutation analysis may affect the evaluation of the efficacy. We conducted a phase II study of erlotinib for previously treated patients with EGFR WT NSCLC screened by the peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) clamp method, which is known to be highly sensitive. The primary endpoint was the objective response rate (ORR). Preplanned reevaluation of the EGFR genotype as an exploratory endpoint was performed using the Scorpion Amplification Refractory Mutation System (S-ARMS) assay. Erlotinib was administered daily until disease progression or development of unacceptable toxicity. A total of 53 evaluable patients were enrolled. The histological subtypes were adenocarcinoma in 40 patients, squamous cell carcinoma in 9 patients and not otherwise specified NSCLC in 4 patients. Partial response (PR) was achieved in 6 patients (4 with adenocarcinoma and 2 with squamous cell carcinoma). The ORR was 11.3% [95% confidence interval (CI): 4.3-23.0]. The median progression-free survival (PFS) was 1.8 months (95% CI: 1.2-2.3). Samples from 26 of the 53 patients (49.0%) were available for EGFR mutation reanalysis with the S-ARMS assay. Of these 26 samples, only 1 sample of adenocarcinoma was found to be EGFR mutation-positive (exon 19 deletion) and the patient achieved a PR. The EGFR WT genotype was reconfirmed by the S-ARMS assay in the remaining 25 patients and 2 of these patients exhibited a PR. This study did not meet the primary endpoint, although erlotinib was found to be moderately effective in pretreated patients with EGFR WT NSCLC, even when the EGFR mutational status was confirmed by the highly sensitive PNA-LNA clamp PCR method.

A Prospective, Molecular Epidemiology Study of EGFR Mutations in Asian Patients with Advanced Non–Small-Cell Lung Cancer of Adenocarcinoma Histology (PIONEER)

Introduction:PIONEER (NCT01185314) was a prospective, multinational, epidemiological study of epidermal growth factor receptor (EGFR) mutations in patients from Asia with newly diagnosed advanced lung adenocarcinoma.Methods:Eligible patients (aged ≥20 years) had untreated stage IIIB/IV adenocarcinoma. The EGFR mutation status (primary end point: positive, negative, or undetermined) of tumor samples (biopsy, surgical specimen, or cytology) was determined (Scorpion amplification refractory mutation system). EGFR mutation frequency was calculated and compared between demographic and clinical subgroups.Results:Of 1482 patients from seven Asian regions, 43.4% of patients were female, median age was 60 years (range, 17–94), and 52.6% of patients were never-smokers. EGFR mutation status was evaluable in tumors from 1450 patients (97.8%) (746 [51.4%] positive; 704 [48.6%] negative). Country, sex, ethnicity, smoking status, pack-years (all p < 0.001), disease stage (p = 0.009), and histology type (p = 0.016) correlated significantly with EGFR mutation frequency. Mutation frequency was 61.1% in females, 44.0% in males; lower in patients from India (22.2%) compared with other areas (47.2%–64.2%); highest among never-smokers (60.7%); and decreased as pack-year number increased (>0–10 pack-years, 57.9%; >50 pack-years, 31.4%) (similar trend by sex). Ethnic group (p < 0.001) and pack-years (p < 0.001) had statistically significant associations with mutation frequency (multivariate analysis); sex was not significant when adjusted for smoking status.Conclusion:PIONEER is the first prospective study to confirm high EGFR mutation frequency (51.4% overall) in tumors from Asian patients with adenocarcinoma. The observed high mutation frequency in demographic/clinical subgroups compared with white populations suggests that mutation testing should be considered for all patients with stage IIIB/IV adenocarcinoma, even males and regular smokers, among Asian populations.

Adrenal cortical neoplasms: a study of clinicopathological features related to epidermal growth factor receptor gene status

BackgroundAdrenocortical carcinoma (ACC) is a rare but highly malignant neoplasm with limited treatment options.MethodsIn this study, the clinicopathological features of 22 ACCs and 22 adrenocortical adenomas (ACA) were analyzed, and the EGFR protein expression, EGFR gene mutation status and EGFR gene copy number alteration of all tumors were examined using immunohistochemistry, fluorescence in situ hybridization (FISH), and the Scorpion Amplification Refractory Mutation System (ARMS), respectively.ResultsEGFR protein expression was detected in 63.6% of the ACC samples, and EGFR FISH was positive in 50% of the ACC samples (all were high polysomy on chromosome 7). In contrast, 27.3% of the ACA samples demonstrated EGFR expression, and none of the ACA samples tested positive by FISH. There were significant differences between the ACC and ACA in terms of protein expression (P = 0.035) and gene copy number alterations (P < 0.001).ConclusionsEGFR protein expression and high polysomy on chromosome 7 are frequent abnormalities in ACC than in ACA.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2068470757103500.