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Abstract

Background

Epidermal growth factor receptor (EGFR) mutation status is of a major clinical significance in non-small cell lung cancer (NSCLC) management, as it guides therapeutic decision making to target patients for a better response to therapy. This implicates the introduction of EGFR mutation analysis as the standard of care for Moroccan NSCLC patients, which in itself entails the implementation of targeted methods for routine EGFR mutation analysis in our laboratories. In this study, we aimed to present 2 targeted methods for EGFR mutation identification and to determine the prevalence and spectrum of EGFR mutations in NSCLC Moroccan patients.

Methods

A retrospective investigation of a cohort of 340 patients was undertaken to analyze somatic EGFR mutations in exons 18 to 21 using pyrosequencing and the Idylla^TM system.

Results

Of the enrolled patients, 70.9% were males and 29.1% were females. Predominately, 92% of cases had adenocarcinoma, and 53.7% of patients self-reported a history of smoking. Overall, 73 patients (21.7%) harbored an EGFR mutation, the most prevalent of which were the exon 19 deletions (53.4%) followed by exon 21 substitutions (31%). Exon 18 mutations and exon 20 alterations occurred in 8.1% and 6.7% of the positive EGFR mutation cases, respectively. Of the analyzed cases, all of the EGFR-mutated patients had adenocarcinoma. EGFR mutation prevalence was significantly higher in females (females vs males: 38.4% vs 14.5%, P < .001) and non-smokers (non-smokers vs non-smokers: 36% vs 10.3%, P < .001). The featured pyrosequencing and the Idylla^TM system are targeted methods endowed with high sensitivity and specificity as well as other compelling characteristics which make them great options for routine EGFR mutation testing for advanced NSCLC patients.

Conclusion

These findings underline the imperious need for implementing quick and efficient targeted methods for routine EGFR mutation testing among NSCLC patients, which is particularly useful in determining patients who are more likely to benefit from targeted therapy.

Keywords

non-small cell lung cancer epidermal growth factor receptor mutations pyrosequencing

Introduction

Lung cancer remains the leading cause of cancer-related mortality worldwide, accounting for 1.8 million deaths. In 2020, lung cancer was the second most prevalent malignancy with 2.2 million new diagnosed cases.¹ In Morocco, lung cancer incidence rate reached 17.2 per 100.000, a high figure if put in a regional context.² NSCLC accounts for 80% of lung cancers and is commonly diagnosed at advanced stages, not amenable to surgery. For this subset of cases, the prognosis remains poor.³

Epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) has been regarded as a distinct biological subset, characterized by the distinctive feature of high response rate to tyrosine kinase inhibitors (TKIs).⁴ EGFR mutations reported in NSCLC play a role in sensitizing the receptor to TKIs, as EGFR-mutated NSCLC patients show a 70% to 80% response rate to this class of anticancer drugs.⁵ EGFR exon 18 to 21 mutations are prevalent in patients with NSCLC, particularly in the adenocarcinoma subtype.⁶ Exon 19 deletions and exon 21 L858R substitutions make up for nearly 90% of all EGFR mutations in NSCLC.⁷ The frequency of EGFR mutations in NSCLC varies depending on sex, tobacco exposure, and ethnicity. The frequency of EGFR mutations among the Asian population is 40 to 60%, which is higher than the 10 to 30% reported in Caucasian populations.⁶

Despite the imperious necessity for an accurate depiction of the molecular epidemiology of EGFR, reports lack from certain regions of the world, namely, North Africa. This calls for a surge in EGFR mutation testing from these regions, in order to reflect accurate data about EGFR mutation frequencies and spectrum at population level. This entails the implementation of efficient and rapid targeted methods focused on the detection of well-characterized EGFR hotspots with clinical significance rather than the more complicated, lengthy, or and sometimes costly next generation sequencing (NGS). These targeted methods could change the landscape of lung cancer management in the country and facilitate the access of NSCLC patients to available targeted therapy drugs.

In this study, we aim to present 2 targeted methods for EGFR mutation identification that could be used as routine molecular characterization methods in our laboratories. Also, we seek to determine the frequency and spectrum of sensitizing EGFR mutations (exons 18 to 21) in NSCLC Moroccan patients, to position the findings in the international context, and to highlight the correlation between these alterations and patients’ clinicopathological characteristics. The present study is a collaborative work involving different interveners implicated in lung cancer management in Morocco: academic, hospital, and private sector molecular pathology center collaborators, in order to gain better insight into the burden and distinctive characteristics of lung cancer in Morocco.

Material and Methods

Study Design

This investigation is a bi-centric retrospective study on a total of 340 formalin-fixed paraffin-embedded (FFPE) blocks of Moroccan patients with NSCLC that were collected consecutively from the pathology department of Mohamed V Military Teaching Hospital of Rabat (N = 50) and from the department of pathology of the nations-unites pathology center (N = 290). Well-codified administrative procedures have been put in place, and patients’ clinicopathological characteristics including age, gender, histological subtype, and smoking history were determined.

This retrospective study was conducted using archived FFPE blocks and thus did not require ethics committee approval, according to national regulations. The data provided to the researchers in this study were anonymized. No identifiable data were available to the researchers. The study was performed in an ethical manner, according to the international guidelines of the Helsinki Declaration and its later amendments as well as our national legislation for medical research ethics.

The Idylla^TM EGFR Mutation Assay

FFPE blocks of 290 NSCLC patients were tested for EGFR exon 18 to 21 mutations. EGFR screening was performed using the Idylla^TM system, employing CE-IVD Idylla^TM EGFR Mutation Assay (Biocartis, Mechelen, Belgium). Detection of EGFR-specific targets is performed using fluorescently labelled probes. The Idylla^TM EGFR Mutation Assay is a fully automated process, where the entire run from FFPE sample deparaffinization to the results, including fully integrated sample preparation, DNA isolation, liberation of nucleic acids, and real-time PCR amplification, is carried out inside the cartridge.

Sample Preparation and Cartridge Setup

From each tested FFPE block, 10 μm tissue sections were sampled after hematoxylin-eosin staining was completed, to determine tumor content and area of the specimen. The FFPE tissue sections containing ≥10% neoplastic cells were placed between 2 10 mm disc of filter paper (Whatman grade 1), dampened with 10 μL of molecular grade water (to ensure adhesion), and placed directly into the disposable cartridge and then loaded into the Idylla^TM system, allowing integrated and automated DNA extraction and mutational hotspot analysis. For each sample tested, after placing the FFPE tissue section into the cartridge, DNA isolation takes place. The process starts with a deparaffinization step followed by the disruption of the tissue and cell lysis using a combination of chemical reagents, enzymes, heat, and high-intensity focused ultrasound. Subsequently, the extracted nucleic acids are sorted out using 5 parallel multiplex PCRs where highly selective target amplification primers are used. Target sequences are identified using fluorescent probes. For each of the 5 PCRs, the conserved fragment of the EGFR gene serves as a positive control to which the amount of amplifiable DNA and the quality of the run are referenced.

Analysis of Fluorescent Signals

The emanating fluorescent signals are analyzed by the software for capturing fluorescence signals and converted into curves, and for each valid curve, a cycle of quantification value (Cq) is calculated. The Idylla EGFR control Cq value is the average cycle quantification value for the EGFR sample processing controls in each of the 5 multiplex PCR reactions. The software interprets the presence of a mutation by calculating a ΔCq value obtained by the difference between the EGFR Total Cq and the Cq obtained for the mutant signal. Only samples of which the ΔCq value is within a predefined range of assay validity are considered valid, otherwise they are reported as wild type. The run is considered invalid in case no EGFR total reference signal was detected. Invalid calls may be due to insufficient DNA input, DNA fragmentation, presence of PCR reaction inhibitors, or cartridge-related problems.

Pyrosequencing EGFR Mutation Assay

Genomic DNA Isolation

Tumor tissues, from 50 FFPE blocks, corresponding to the H&E-stained areas were manually micro-dissected using a microtome. Four sections of 6 μm were transferred into a microcentrifuge tube. Genomic DNA isolation was performed using the QIAamp DNA FFPE Tissue Kit (QIAamp DNA FFPE tissue, Qiagen, GmbH, Hilden, Germany), according to the manufacturer’s instructions. The sections were deparaffinized using 1 mL of xylene at 56°C for 10 min and resuspended in tissue lysis buffer and proteinase K, and then incubated at 56°C for 2 h. 1 mL of 100% ethanol is then added to the solution during the washing step. The lysate was transferred to the QIAamp MinElute column. During centrifugation, DNA binds to the membrane and contaminants flow through. After elution buffer addition, full-speed centrifugation was performed to collect pure and concentrated DNA.

DNA quantity and quality were measured using a Nanodrop-2000 spectrophotometer. DNA was then stored overnight at 4°C for further testing.

Polymerase Chain Reaction and Agarose Gel Electrophoresis

Polymerase chain reaction (PCR) was performed using the PyroMark PCR Kit (Manuel du kit Therascreen® EGFR Pyro®), according to the Kit’s manufacturer instructions. PCR was performed in 25 μL final volume by mixing 12.5 μL of PyroMark PCR Master Mix, 2.5 μL of CoralLoad Concentrate, 1 μL of primer mix, 4 μL of molecular-grade water, and 5 μL of the isolated DNA. An unmethylated wild-type genomic DNA, supplied with the kit, was used as a positive control for the PCR and sequencing reactions. Molecular- grade water was used as a negative control.

PCR reaction was performed as follows: an initial denaturation step at 95°C for 15 min, followed by 42 thermal cycles starting with denaturation at 95°C for 20 s, primer annealing at 53°C for 30 s, and primer extension at 72°C for 20 s. At the end of the last cycle, the PCR mixes were incubated at 72°C for 5 min. After amplification, PCR products were analyzed by electrophoresis on 2% agarose gels stained with ethidium bromide.

DNA Template Preparation and Pyrosequencing

DNA template preparation and sequencing reactions were performed using the Therascreen EGFR Pyro kit (Qiagen, GmbH, Hilden, Germany), according to the manufacturer’s instructions. Biotinylated PCR products were immobilized onto streptavidin-coated beads by mixing 10 μL of PCR product with 2 μL Streptavidin Sepharose suspension, 40 μL of the binding buffer, and 28 μL of water. To eliminate non-biotinylated DNA strands, samples were sequentially denatured using PyroMark Q24 Vacuum Prep Workstation Tool (Qiagen). Immobilized pure single-stranded DNA was then transferred to a microtiter plate containing target-specific sequencing primer in a buffer. Required volumes of substrates, enzymes, and nucleotides (Gold Reagent Kit, Qiagen) listed in the pre-run report were dispensed in a clean PyroMark Q24 Cartridge (Qiagen, GmbH, Hilden, Germany). Real-time sequencing was performed using PyroMark Q24 pyrosequencing instrument and software according to the manufacturer’s instructions. The Therascreen EGFR Pyro-plugin report incorporated the thresholds for mutation calls (detection limit for the mutation (LOD) + 3%). Pyrosequencing results were analyzed using the PyroMark Q24 software version 2.0.

Statistical Analysis

The potential correlations between EGFR mutations and patients’ clinicopathological characteristics were analyzed using χ2 statistics. A P-value less than .05 was considered statistically significant. All analyses were performed using SPSS (version 28.0.1.1; SPSS Inc., Chicago, IL).

Results

Clinical Characteristics of Study Population

A total of 340 NSCLC unselected patients were enrolled in this study. Clinicopathological features of the included patients are summarized in Table 1. Among the analyzed cases, male patients were predominant, accounting for 70.9% (241), while female patients accounted for 29.1% (99). The median age was 56 years, with a range of 31 to 87 years. The histological subtype was defined in 95.5% (325) of the cases. Predominately, 92% (313) of patient had adenocarcinoma, 3.2% (11) had squamous cell carcinoma, and 1 patient presented with sarcomatoid carcinoma (0.3%). 4.4% (15) of patients presented with NSCLC NOS. Data regarding smoking history were lacking, as only 54 patients reported their smoking habits. There were more smokers than non-smokers, as 53.7% (29) self-reported a history of smoking. 66.7% (97) of patients presented with a metastatic disease, 21.3% (31) with a localized disease, and 11.7% (17) with a locally advanced disease. 72.6% (213) of the samples were of pulmonary origin and 27.4% (80) from extra-pulmonary sites. The latter included the following sites: pleura (38), lymph nodes (16), bone (7), brain (6), liver (6), skin (3), (1) pericardium, (1) peritoneum, (1) vertebrae, and (1) adrenal. A total of 32.6% (111) of the tumors were poorly differentiated, 17% (58) moderately differentiated, and 3% (10) well differentiated.

Table 1.

Clinicopathological Characteristics of the Study Population.

Clinicopathological Characteristics	Variables Studied	Percentage (n)
Gender	Female	29.1 (99)
Gender	Male	70.9 (241)
Age	<60	27 (92)
Age	≥60	73 (248)
Smoking status	Smoker	53.7 (29)
Smoking status	Non-smoker	46.3 (25)
Histological subtype	ADK	92 (313)
	SCC	3.2 (11)
	SC	.3 (1)
	NSCLC NOS	4.4 (15)
Disease stage	Localized	21.3 (31)
	Locally advanced	11.7 (17)
	Metastatic	66.7 (97)
Tumor site	Pulmonary	72.6 (213)
Tumor site	Extra-pulmonary	27.4 (80)

Distribution of EGFR Mutations Among the EGFR-Mutated Patients

Overall, 73 (21.7%) patients harbored an EGFR mutation. The most prevalent were the exon 19 deletions (53.4% [39]) and exon 21 substitutions (31.5% [23]), of which the L858R substitution was the most frequent, accounting for 23.3% (17) of EGFR mutations. The L861Q substitution accounted for 6.8% (5) and a single L861R exon 21 substitution was reported (1.3%). The frequency of exon 18 mutations was 8.1% (6), all of which were G719X substitutions (G719A [2.7%], G719C [4.1%], and G719S [1.3%]). As for exon 20 alterations, the mutation rate was 6.7% (5), made up of 2.7% (2) of the S768I substitution and exon 20 insertions, each, and 1.3% (1) of the T790M substitution (Table 2).

Table 2.

Spectrum and Frequency of EGFR Mutations in the Study Population.

Mutation Type		Prevalence, % (N)
Exon 18	G719X substitution	8.2 (6)
Exon 19	Exon 19 deletions	53.4 (39)
Exon 20	Exon 20 insertion	2.7 (2)
	S768I substitution	2.7 (2)
	T790M substitution	1.3 (1)
Exon 21	L858R substitution	23.3 (17)
	L861Q substitution	6.8 (5)
	L861R substitution	1.3 (1)
Concomitant mutations	G719A/S768I	1.3 (1)
	G719A/L861Q	1.3 (1)
	T790M/S768I	1.3 (1)

Concurrent mutations were found in 4.1% (3) of the EGFR-mutated patients. One patient harbored an exon 18 G719A substitution and an exon 20 S768I mutation. Another patient carried an exon 18 G719A substitution and an exon 21 L861Q alteration. The third patient had an exon 20 T790M substitution and an exon 21 S768I mutation (Table 2).

Correlation of EGFR Mutational Status and Patients’ Clinicopathological Features

EGFR mutations were found to correlate with patients’ clinicopathological characteristics, as shown in Table 3. Overall, EGFR mutation prevalence was higher in females (females vs males: 38.4% vs 14.5%, P < .001). All of the EGFR-mutated patients had adenocarcinoma (adenocarcinoma vs non-adenocarcinoma: 100% vs .0%). Among the analyzed patients, we could determine the smoking history of 54 patients. EGFR mutations were more frequent in non-smoking patients (36%) than in smokers (10.3%), a statistically significant correlation was seen between the 2 groups (P < .001).

Table 3.

Distribution of EGFR Mutations According to the Patients’ Clinicopathological Characteristics.

Characteristics Parameters	EGFR Mutations (n = 73)	EGFR Wild Type (n = 267)	P-Value
Gender % (n)			<.001
Male	14.5 (35)	85.5 (206)
Female	38.4 (38)	61.6 (61)
Smoking status % (n)			<.001
Smoker	10.3 (3)	89.7 (26)
Non-smoker	36 (9)	64 (16)

EGFR Status Evaluation Using Pyrosequencing and Idylla^TM System

In this study, EGFR status analysis was performed using pyrosequencing and the IdyllaTM system. Both targeted techniques are endowed with high sensitivity and specificity. Although both techniques are able to detect hotspot mutations in exons 18 to 21, the EGFR mutation panel of the IdyllaTM system covers 30 more activating mutations than those covered by the Qiagen Therascreen Pyro kit, as The IdyllaTM EGFR Mutation Assay (Biocartis) detects 51 mutations and indels vs 21 mutations and indels detected by the Therascreen EGFR Pyro Kit (Qiagen). On the basis of turnaround time and hands-on time, the IdyllaTM approach is much faster (time to results in a few hours) and requires less than 5 minutes of hands on time, as the whole process is taking place inside of the cartridge. The IdyllaTM system being automated the total cost being mainly related to cartridge price as it includes all reagents needed to complete the analysis and the hands on time is quite reduced. As for pyrosequencing, the cost is divided on the much extended hands on time, the reagent kit, and consumables for the extraction. The Idylla^TM method requires a smaller amount of fixed tissue to be loaded in the cartridge (1 10 μm thick tissue section) than that needed for the pyrosequencing method (4 6 μm thick sections). Although The Idylla^TM system being fully automated is a great advantage, it is not possible to obtain the residual DNA extracted after the EGFR mutation analysis, while extracting DNA prior to pyrosequencing analysis allows to perform subsequent analysis on other genetic alterations in the event that no EGFR mutation is detected. One advantage that is common for both methods is the ease of results interpretation, as they are both endowed with simple data interpretation. Pyrosequencing is a quantitative sequencing method that is highly sensitive but takes longer, as prior to the PCR and sequencing, a DNA isolation step is needed. The Idylla^TM system, being fully automated, requires less hands-on time and takes a shorter time to deliver EGFR mutational status results, and with a minimum amount of fixed tissue (Table 4).

Table 4.

Comparative Analysis of Pyrosequencing and Idylla^TM in EGFR Mutation Testing.

Criteria	Idylla^TM	Pyrosequencing
Sensitivity and specificity⁸	High	High
Tissue saving	Optimal	Unideal
Extraction time and analysis	Less	More
Mutation coverage	More	Less
Interpretation of the results	Easy	Easy
Hands-on time	Less	More
DNA availability for additional testing	Unavailable	Available
Cost	180 $	220 $

Discussion

The clinical significance of EGFR mutations implicates the introduction of EGFR testing as the standard of care in NSCLC management, which in itself entails the implementation of targeted methods for routine molecular characterization of EGFR mutations in our laboratories in order to guide therapeutic decision making.

Overall, 73 patients (21.7%) harbored mutations in at least 1 of the 4 exons (exons 18 to 21). Among the detected mutations, exon 19 deletions were the most prevalent, accounting for 53.4%, followed by exon 21 substitutions (31.5%), of which the L858R mutation was the most prevalent accounting for 23.3%. The frequency of exon 18 mutations was 8.1%, all of which were G719X substitutions. The most rarely encountered EGFR mutations were exon 20 alterations accounting for 6.7%, made up of 2.7% of the S768I substitution and exon 20 insertions, each, and 1.3% of the T790M substitution. In concordance with well-established knowledge about the correlation of EGFR mutations and patient and tumor characteristics, the occurrence of EGFR mutations was significantly correlated with gender (females vs males: 38.4% vs 14.5%, P < .001) and smoking status (smokers vs non-smokers: 10.3% vs 36%, P < .001). Of the analyzed patients, all the EGFR-mutated cases had adenocarcinoma.

Compared to previous Moroccan studies, we found that a recent study by Sow et al reported a prevalence of 21.9% of EGFR mutations in a cohort of 334 Moroccan patients. These mutations were mainly detected in exon 19 (65.8%), followed by exon 21 (23.3%) and exon 18 (6.8%). Mutations in exon 20 were the least frequent, with 4.1%.⁹

EGFR mutations were found to be heavily influenced by ethnicity and geography. Putting our results in the regional context, we found that EGFR mutation prevalence among Moroccan NSCLC patients is higher than that seen in NSCLC patients of Caucasian ethnicity (≈15%) but is lower than that identified among Asian NSCLC patients (≈50%).¹⁰ Comparing our results with those reported in the Middle East and North Africa (MENA) region, EGFR mutation rate in the Moroccan population was higher than that reported in Tunisian,¹¹ Jordanian,¹² Lebanese,¹³ Egyptian,¹⁴ and Turkish⁶ NSCLC patients. Disparities in EGFR prevalence rates can be explained by the implication of different factors including ethnicity, study sample sizes, and each country’s population size. Conversely, higher EGFR mutation frequency was seen in Algerian,¹⁵ Iraqi,¹⁶ Iranian,¹⁷ and the Gulf region¹⁸ NSCLC patients, in comparison to their Moroccan counterparts. Discrepancies in EGFR mutation rates seen in these countries relative to Morocco are largely due to the difference in ethnicity, the MENA region being a broad and non-homogeneous area. However, despite the common ethnicity and demographic features of the Moroccan, Algerian, and Tunisian populations (North Africa), we found a difference in EGFR mutation rate which could be attributed to the population scope of the studies, and specimens and techniques used for EGFR testing.

In concordance with previous reports, Rondell et al reported a frequency of 16.1% of EGFR-mutated cases among African and Middle Eastern NSCLC patients, in a large-scale study involving 23757 patients from different parts of the world: northern Asia, southern Asia, Europe, Africa (including the Middle East), South America, and North America. Among the studied cases, Taiwan had the highest rate of EGFR-activating mutations (55% [2802/5103]_, followed by China (37% [1009/2702]_, then Japan (29% [9644/32935]), and lastly India with a rate of 29% (605/2077). While the highest rates were recorded in Asia, the lowest were in South America with 7.9% (114/1439). In Europe, the frequency of EGFR mutations was 13.4% (138/1030). In North America, where the largest studied population was 86654 patients, 9.2% carried EFGR mutations.¹⁹

These findings emphasize the imperious necessity to make EGFR testing the standard of care for NSCLC patients’ management in Morocco. The targeted methods featured in this study are simple and rapid methods that can be implemented as routine EGFR testing techniques in our molecular oncology laboratories. These methods are endowed with a high specificity, sensitivity, and simple data interpretation. As demonstrated in previous studies, NGS despite its numerous advantages is not without challenges; the most prominent of which is ensuring testing is clinically relevant, cost-effective, and can be integrated into clinical care.²⁰ Among the targeted methods widely used for EGFR testing, real-time PCR (qPCR) and digital droplet PCR (ddPCR) are the methods of choice in many pathology labs. ddPCR is endowed with an unmatched specificity and sensitivity, being able to quantify EGFR mutations at single-molecule-level and detect rare mutants in a high background of wild-type sequences, making it an ideal tool for the detection of the T790M TKI-resistant mutation. Furthermore, ddPCR may have good application prospects in patients’ follow-up, as it can detect EGFR mutations in various types of specimens, namely, tumor cells, blood plasma, and urine. While qPCR essays are very sensitive, they require high quality and concentration for DNA templates and depend on the threshold and quantification cycle (Cq) value for result interpretation. As for ddPCR, the samples are partitioned into reaction chambers, and subsequently the presence and absence of targeted molecules are determined in each part of the sample following end-point PCR.^21,22

In view of the growing interest in the more efficient targeted therapies, more efforts should be deployed to facilitate the access of Moroccan NSCLC patients harboring EGFR mutations to TKIs. Therefore, methods that deliver rapid and reliable results of EGFR mutation status, with an affordable cost, should be used for routine EGFR testing to target patients for better response to TKIs. For advanced or metastatic NSCLC, hotspot mutation analysis using targeted methods prove more useful, as patients need to be treated rapidly with molecules that have already been approved for treatment, given the rapid tumor progression and the availability of effective targeted therapy.⁸ In this context, targeted techniques like the Idylla^TM and pyrosequencing offer a lot of these advantages, as the fully automated Idylla^TM method combines the short turnaround time and the minimal fixed tissue usage, whereas pyrosequencing offers a high sensitive EGFR mutation analysis and allows further molecular testing using the extracted DNA. This will allow performing broader mutational analysis to identify other actionable genetic alterations, in the case no mutation is detected for EGFR.

Limitations of our study are the lack of information on smoking habits and survival data. That being said, our series remains the largest homogeneous study in North Africa on EGFR mutation status in NSCLC. Also, our study features 2 targeted methods (pyrosequencing and the Idylla^TM method) endowed with high sensitivity and specificity as well as other compelling characteristics that make them great options for routine EGFR mutation testing for advanced NSCLC patients. In conclusion, our findings confirm the global heterogeneity of the EGFR molecular epidemiology in NSCLC. It has been shown that EGFR mutation prevalence in Moroccan patients with NSCLC is higher than that seen in NSCLC patients of Caucasian ethnicity but is lower than that identified in Asian NSCLC patients. Several patients and tumor features were proven to significantly modify the frequency of EGFR mutations, namely, ethnicity, gender, smoking history, and the histologic subtype. Since EGFR mutation rates vary depending on, inter alia, ethnicity, EGFR molecular testing should be a standard of care in Morocco in order to reflect more accurate and realistic data on EGFR mutation frequencies, which entails the need for further research implementations in the country.

Footnotes

Author Contributions

YB has conceived the study, exploited data, and drafted the paper. SELZ contributed to data collection. AL and BB critically reviewed the manuscript. MG, AB, and FK were involved in data analysis. MO, KE, and YS contributed to data collection and review of the manuscript. All authors have read and agreed to the published version of the manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Ethical Approval

This is a retrospective study conducted on archived FFPE blocks and thus did not require ethics committee approval, according to national regulations. Ethical review and approval are waived for this study due to its retrospective nature. The data provided to the researchers in this study were anonymized. No identifiable data were available to the researchers. The study was performed in an ethical manner, according to the international guidelines of the Helsinki Declaration and its later amendments as well as our national legislation for medical research ethics.

ORCID iD

Youssra Boustany

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Advanced Non-small Cell Lung Cancer: EGFR Mutation Analysis Using Pyrosequencing and the Fully Automated qPCR-Based Idylla TM System

Abstract

Background

Methods

Results

Conclusion

Keywords

Introduction

Material and Methods

Study Design

The IdyllaTM EGFR Mutation Assay

Sample Preparation and Cartridge Setup

Analysis of Fluorescent Signals

Pyrosequencing EGFR Mutation Assay

Genomic DNA Isolation

Polymerase Chain Reaction and Agarose Gel Electrophoresis

DNA Template Preparation and Pyrosequencing

Statistical Analysis

Results

Clinical Characteristics of Study Population

Distribution of EGFR Mutations Among the EGFR-Mutated Patients

Correlation of EGFR Mutational Status and Patients’ Clinicopathological Features

EGFR Status Evaluation Using Pyrosequencing and IdyllaTM System

Discussion

Footnotes

Author Contributions

Declaration of Conflicting Interests

Funding

Ethical Approval

ORCID iD

References

The Idylla^TM EGFR Mutation Assay

EGFR Status Evaluation Using Pyrosequencing and Idylla^TM System