Background/Aims: As targeted therapies are promising in the treatment of lung cancer (LC), it is important to identify the genetic variations in tumors. The present research aimed to determine the regional prevalence of alterations in ALK, ROS1, and EGFR genes. Materials and Methods: ALK rearrangement in 1152, ROS1 rearrangement in 390, and EGFR mutations in 1054 cases with LC were evaluated. Results: Alteration rates of ALK, ROS1, and epidermal growth factor receptor (EGFR) genes were 3.5%, 0.4%, and 11.2% in the samples, respectively. ALK rearrangements were mainly detected in young patients (P < 0.01) and in females (P < 0.01). Females were also more often inflicted by EGFR variations, especially from the exon 19 deletion. Exon 21 L858R mutations were more frequently found in men. However, any statistical significance between EGFR alterations and gender or age was not discovered. Conclusion: In this study, molecular changes were less frequent than expected. We thought that this low rate confirmed the aphorism of “smokes like a Turk, ” which could be because almost all patients were active or passive smokers.
Keywords: ALK rearrangements, EGFR mutations, lung cancer, ROS1 rearrangements
|How to cite this article:
Diniz G, Komurcuoglu B, Ozyilmaz B, Ozguzer A, Yucel N, Kirbiyik O. ALK, ROS1 and EGFR status of lung cancers in the Aegean Region of Turkey. Indian J Pathol Microbiol 2022;65:305-10
|How to cite this URL:
Diniz G, Komurcuoglu B, Ozyilmaz B, Ozguzer A, Yucel N, Kirbiyik O. ALK, ROS1 and EGFR status of lung cancers in the Aegean Region of Turkey. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 May 4];65:305-10. Available from: https://www.ijpmonline.org/text.asp?2022/65/2/305/343165
Lung cancer (LC) has become a leading cause of cancer-related mortality. Most cases of LC are diagnosed at an advanced stage when only a limited number of treatment alternatives can be offered to these patients., The development of LC involves multiple phases of tumorigenesis, including interactions among genetic, epigenetic, and environmental factors with resultant dysregulation of key oncogenes and tumor suppressor genes ending in the activation of cancer-related signaling pathways.,, The most common histological subgroup of LC is non-small cell LC (NSCLC), including adenocarcinoma, which accounts for 80% of all LCs., Molecular characterization of NSCLC has revealed multiple mutations for targeted therapies.,, These mutations, which induce LC growth, are more commonly detected in adenocarcinomas. Therefore, in histopathological examinations, emphasis is placed on separating adenocarcinomas from the other LCs to effectively select tumors for targeted molecular testing.,,,,, Some gene fusions involving tyrosine kinase receptor genes such as ALK and ROS1 have the capacity of undergoing oncogenic transformation.,, It was previously reported that activating rearrangements of ALK and ROS1 genes had been observed in 3%–7% and 1%–2% of the cases with NSCLC, respectively., Both mutations are clinically associated with a history of never smoking, younger age, female gender, and adenocarcinoma histologic type. In addition, they create treatment alternatives for tumors that have these alterations.,,,,,
Human epidermal growth factor receptor (EGFR) or HER-1 is one of the four tyrosine kinase receptors and belongs to the EGFR family After ligand-binding, EGFR receptors homo- and hetero-dimerize and induce autophosphorylation of the intracellular tyrosine kinase domain. Thereafter, the molecular cascade of events involving growth, cell proliferation, differentiation, and survival is triggered. Activation of driver EGFR oncogenes also increases the sensitivity of tumors to tyrosine kinase inhibitors (TKIs). Currently, many therapeutic approaches have been developed against the mutant EGFR.,,,,,, Although most EGFR mutations are associated with enhanced sensitivity to TKIs, some exon 20 insertions and T790M mutations are resistant to these drugs.,, EGFR mutation kits are generally designed to recognize some gene regions between exons 18–21, which are most severely mutated. In all kits, about 50 molecular changes, which can be grouped into mutations of exon 18 (G719A/S/C), exon 20 (T790M, S768I), and exon 21 (L858R, L861Q) with exon 19 deletion and exon 20 insertion, are usually investigated.,,,
Since the discovery of some driver mutations that have allowed new treatment options for LC, many drugs have been developed, most of which are TKIs. As a result, more prolonged survival has been obtained in patients diagnosed in advanced stages and whose survival is normally limited to months. Satisfactory data have been obtained on many issues, such as the relationship of these mutations with the clinical characteristics of patients, which drugs are more effective, and which changes cause drug resistance., Another feature revealed by studies about LC is that these mutations show the geographic difference. For example, there is a considerable difference between the mutation type and rate in LCs diagnosed in America and Northern European countries and the rates detected in Asian cases. However, data on the mutation rate in all parts of the world are limited. To shed light on the prevalence values in all other countries, it will contribute to the development of new treatment strategies for LC and measures to cope with the socio-economic problems caused by LCs. In the current research, we aimed to evaluate the ALK, ROS1, and EGFR status of LCs in the Aegean region of Turkey. In this way, we want to enlighten on the regional prevalence of these alterations.
|Materials and Methods|
In total, 1152 patients with LC diagnosed at …………. Hospital between April 2015 and March 2019 and referred to the pathology department of …………….. Hospital for molecular examination were included in this study. All fluorescence in situ hybridization (FISH) examinations were evaluated by two active pathologists and successful members of the Lung External Quality Assessment Scheme of the European Society of Pathology. Informed consent was obtained from all participants. This study was approved by the local ethics committee of …………… Hospital (2019/6-6; April 10, 2019).
ALK and ROS1 rearrangements: ALK and ROS1 rearrangements were detected using FISH without prior screening by immunohistochemical methods (IHC). FISH is the preferred method used to investigate ALK and ROS1 mutations and is a prerequisite for targeted therapy. Briefly, FISH was performed on unstained 4-mm-thick formalin-fixed paraffin-embedded (FFPE) tumor samples using the ZytoLight SPEC ALK Dual Color Break-Apart Probe and ZytoLight SPEC ROS1 Dual Color Break-Apart Probe (Zytovision; GmbH; Germany) following the manufacturer’s instructions. FISH images were captured using the manual digital system (Olympus) and scored by either one or two experienced pathologists. The proper tumor tissue sections were selected using a 10× objective lens to locate the nuclei. The nuclei were detected in those regions using a 100× oil immersion objective lens and a single band 40, 6-diamidino-2-phenylindole (DAPI)/Spectrum Green/Spectrum Orange dual and triple filters. Finally, the target nuclei were selected and scored by a pathologist using a specific algorithm for positive or negative signal patterns based upon the classifications described in the instruction manual of the kit. At least 50 tumor cell nuclei were counted according to the manufacturer’s instructions. ALK and ROS1 FISH-positive cases were defined as those with >25 nuclei (50%) with break-apart (BA) signals or isolated red signals (IRS) for ALK and isolated green signals (IGS) for ROS1. ALK and ROS1 FISH-negative samples were defined as those with <5 nuclei (<10%) with BA signals or IRS (for ALK) and IGS (for ROS1). ALK and ROS1 FISH cases were considered borderline if 5–10 cells (10%–20%) were positive. In the case of borderline results, a second reader evaluated the slides; 50 additional tumor cell nuclei were counted, and their percentages were calculated from a total of 100 cells. If <15% of the cells were positive, then the sample was considered negative. If ≥15% of the cells were positive, the sample was considered positive.
Detection of EGFR mutations: In our center, ALK and EGFR analyses were planned for each sample accepted by the pathology laboratory, and paraffin blocks or cytological smears were prepared for both tests. However, in some cases, EGFR tests could not be performed for different reasons, such as tissue depletion in the paraffin blocks, insufficient number of cytological slides, inability to deliver material to the laboratory, and economic problems. In 315 patients (27.3%), EGFR mutations were detected using Biocartis™ system (Idylla EGFR Mutation Assay (RUO) Detection Kit) at the pathology department of ……….. Hospital. This EGFR Mutation Assay allowed the detection of EGFR mutations directly from FFPE tissue sections or cytological smears within approximately 2 h. In addition, the tissue samples to be used in this assay were obtained by scraping from the paraffin blocks or stained cytology slides. Tissue sectioning, manual deparaffinization, or DNA extraction were not required. The tissue scraps were placed directly into the special cartridges, which contain all reagents.
In 739 patients (64.1%), tumor tissue specimens for the extraction of their DNAs were also prepared in our pathology department. Tissue sections were prepared from paraffin blocks or stained cytology smears were scraped away from glass slides, and all these samples were collected in microcentrifuge tubes. If required, for microdissection or macrodissection, tumor enrichment was performed on cytology smears. Most of the EGFR mutation analyses of these 739 patients were performed in the genetic laboratory of our hospital using real-time PCR.
Statistical analysis: SPSS software (version 25.0, IBM, SPSS) was used for all statistical analyses in this study. The quantitative data were recorded as mean values ± standard deviation and analyzed by using the Student’s t test for analysis between two groups, and the one-way analysis of variance (ANOVA) test was used to compare the data of three or more groups. Moreover, Turkey’s post hoc test was used to validate ANOVA for comparing measurement data among groups. In cases where the necessary conditions for parametric analysis were not met, Mann Whitney U was used to compare the quantitative data of the two groups, and the Kruskal Wallis test was used to compare the data of three or more groups. The Chi-square test or the Fisher’s exact test was used to compare categorical variables, which were recorded as percentage values. All P values reported were two-sided, and differences were considered significant when the P value was less than 0.05.
Among 1152 patients with LC, 908 (78.8%) were male, and the mean age of the patients was 60.4 ± 9.7 (23–93) years. The predominant histologic type was adenocarcinoma (921 cases; 79.9%), and almost all patients were smokers or ex-smokers (median: 20 packs/year). Information about the tumor stage was not available for any patients. Most of the study specimens (95%) were not obtained by excisional biopsy methods (cell blocks prepared from pleural effusion or percutaneous needle aspiration in 22.8%, bronchoscopic biopsy specimens in 56.8%, and cytological smear of endobronchial ultrasound-guided transbronchial needle aspiration or percutaneous needle aspiration in 15.4% of the cases). Five percent of the samples were obtained by surgical resection (lobectomy or wedge resection). In this period, 1152 FISH tests for ALK, 390 FISH tests for ROS1, and 1054 PCR analyses for EGFR were performed. Molecular analyses could be optimally performed in 895 (77.7%) patients for ALK, in 240 (20.8%) patients for ROS1, and in 1029 (89.3%) patients for EGFR. These analyses were not successful in the remaining patients due to an insufficient amount of tumor tissue samples. In particular, ROS1 analysis could not be performed as sufficient tumor cells could not be observed on the slides in almost all patients who were diagnosed with LC only from cytological smears and did not have tumor tissue samples. In the cell block sections, the least “insufficient material ” problem was experienced when all three tests were requested simultaneously. In 98 patients (8.5%), EGFR mutations could not be revealed due to lack of tissue in the block, failure to reach the report, patients going to another center, or his/her death. In only three (<1%) of 315 patients in whom EGFR mutations were investigated with PCR-based Biocartis system, sufficient DNA material could not be extracted, while the rate of insufficient DNA extraction was 2.9% in conventional RT-PCR tests.
When 257 (22.3%) patients with insufficient tumor tissue samples or those in whom any signal was not observed in the FISH test were excluded from the survey, ALK gene rearrangement was detected in 3.46% of the cases. ROS1 gene rearrangement was found in 0.04% of 240 patients who had been examined by optimal conditions. Because of the presence of ROS-1 gene rearrangement in only a 37-year-old female patient, we could not make any statistical comments about the relationship between age or sex and ROS1 status. In 16 (51.6%) male and 15 (48.4%) female patients, ALK rearrangements were detected with a significant difference between the genders and ALK positivities (Chi-square test, P < 0.01). The data we obtained illustrated that ALK rearrangements were more frequently detected in female patients. Meanwhile, ALK rearrangements were more frequently noted in young patients compared to patients older than 60 years of age [Figure 1], and there were also statistically significant differences between the age and rates of ALK positivity (Chi-square test, P < 0.01). Similarly, ALK rearrangement was almost always observed at a younger age, and the P value was found to be statistically significant with the independent samples t test (P = 0.005).
|Figure 1: Comparison of the distributions of the ALK rearrangements according to the age of patients|
In the present study, 17 ALK-positive cases (54.8%) dominantly revealed isolated red signal patterns, whereas 14 cases (45.2%) demonstrated a classical split pattern [Figure 2]. In addition, we determined borderline rates of 10- 15% among rearrangement-positive cells in 26 cases (2.9%).
|Figure 2: Normal signal appearance in a case with native ALK gene (a), Presence of pathological split signals (b), and Presence of pathological single red signals (c)|
EGFR gene mutations were detected in 115 (11.2%) of 1029 patients from whom adequate tumor tissue samples could be harvested. EGFR mutations seen in the order of frequency can be listed as follows: exon 19 deletion, exon 21 L858R mutation, exon 20 insertion, exon 20 S768I mutation, exon 21 L861Q mutation, and exon 18 G719X mutation. In addition, there were rare double alterations, such as L858R with S768I mutations, G719X with S768I mutations, and exon 19 deletion with T790M mutation [Table 1]. EGFR variations were detected in 59 male (51.4%) and 56 female (48.6%) patients. EGFR gene variations were more frequently detected in female NSCLC patients, and there were statistically significant differences between the gender and EGFR variations (Chi-square test, P < 0.01). Exon 19 deletions were more frequently found in women than men, and exon 21 L858R mutations were more often found in men than women. However, any statistically significant differences were not revealed between male and female patients according to the types of EGFR gene variations (Chi-square test, P = 0.155). Meanwhile, the coexistence of exon 19 del and L858R mutation, which was the most common EGFR gene variation, was less frequently detected in patients younger than 60 years of age without any significant difference between the age of the patients and EGFR variations (Chi-square test, P = 0.362) [Figure 3]. As EGFR mutations were observed at all age groups, the P value was not found to be statistically significant with the independent samples t test, either (P = 0.712).
|Table 1: Comparison of the distribution of EGFR mutations between males and females|
|Figure 3: Comparison of the distributions of the EGFR variations according to the age of the patients|
LC can be described as one of the most common cancers with a high mortality rate and the leading cause of cancer-related deaths worldwide., Most patients present at the advanced stage of the disease with a poor prognosis and one-year survival rate of less than 20%. At an advanced stage, surgical resection is not possible because diagnosis and identification of molecular alterations can only be made by examining small biopsy material or cytology specimens.,, As targeted therapies are promising in the treatment of LC, it is important to determine the genetic variations in tumors for improvement of patients’ survival. Prolonged median overall survival times have been reported for patients with NSCLC who received targeted therapy when a molecular target was identified. Previous reports on patients with EGFR-mutated and ALK-rearranged tumors have demonstrated median overall survival times that exceeded three years.,,,,, In this study, we could not obtain follow-up data of the patients; thus, the survival rates of patients are unknown. The most important limitation of our study is the lack of information about the survival rates of the patients. However, as our aim in this study is to shed light on the regional prevalence of three mutations that create treatment options in LC, we think that the lack of follow-up results may not be considered as an important limitation.
In the advanced stage of LC, the diagnosis is based on the analysis of cytology samples, cell blocks, or small biopsy materials.,, Therefore, in a significant proportion of patients, molecular alterations cannot be tested due to tissue depletion in paraffin blocks or due to an insufficient number of cytological slides. In the present study, we aimed to evaluate the status of ALK, ROS1, and EGFR for each material sent to the pathology laboratory of our center. However, in most cases, some of the diagnostic and molecular tests were performed in different laboratories. Both tests were performed in the same laboratory, and results were obtained within a shorter time with a lower likelihood of missing EGFR test results when compared to those realized in external centers.,,,,, Therefore, especially in tumors that are diagnosed in advanced stages, such as lung tumors with insufficient tumor samples, performing diagnostic and molecular tests in the same center ensures the realization of a greater number of analyses with lesser tissue samples.
An examination of genomic alterations is often required for many carcinomas before initiating systemic treatment.,, FISH, IHC, and real-time PCR can be used to detect ALK fusion genes in tumor samples. Although the FISH break-apart assay is the gold standard diagnostic tool, FISH is not a cost-effective technique to use in all cases of LC. Many authors reported that ALK IHC is a relatively cost-effective and easily interpreted test and shows high sensitivity and specificity for the detection of ALK rearrangements. ALK IHC is also approved for patient selection for treatment with TKIs.,, It was previously reported that ALK rearrangements appear to be homogenously distributed in the tumor., Therefore, it is not necessary to select a specific tumor area based on the immune profile. In this study, ALK and ROS1 rearrangements were detected using the FISH method without prior screening by IHC. Therefore, we cannot comment on the relationship between ALK rearrangements and ALK IHC tissue expression.
In most cases, the ALK fusion gene is formed by a rearrangement occurring on the short arm of chromosome 2 and involves the genes encoding for ALK (2p23.2) and EML4 or rarely genes on other chromosomes. As the kinase domain of ALK tyrosine kinase is encoded by the 3´ part of the gene, the unpaired 3´ signal indicates the fusion gene other than ALK- EML4. In summary, the total number of rearranged cells can be expressed as the sum of the number of cells with split and isolated 3´ patterns.,,,, In the present study, more than half of the ALK-positive cases revealed predominantly or solely the isolated red signal pattern. It was previously reported that the prevalence of genomic alteration in LCs changes according to the geographic region, country, and race of patients. We thought that fusion genes other than ALK-EML4 may be more common in our geographic region as well as in Turkey. A larger population study is required for describing genomic alterations and outcomes of LC.
In our study, changes in the EGFR, ALK, and ROS-1 genes that could be treated with targeted therapy were less frequently seen than previously reported rates. However, as is known, these changes are often at their highest levels among young people, women, and non-smokers. These low rates may be because almost all of our patients were active or passive smokers at some point in their lives. For this reason, the aphorism “smoking like a Turk ” is used worldwide. In addition, in our study, the rate of ALK rearrangements was found to be 10%–15% in 26 patients who underwent optimal FISH testing. Two separate experienced pathologists re-evaluated a greater number of cells on the same slides as recommended in such cases. FISH tests were also repeated in cases with ALK rearrangement rates just below 15% if sufficient material was available. In contrast to other tumors in which mutation status drives treatment modalities, the prognostic significance of mutations in LC has been much more recently discovered. Therefore, we do not yet have sufficient data on the results of the treatment of LC demonstrating molecular changes.,,,,,, For example, since the development of tailored treatment for EGFR2 positive breast cancers in the early 2000s, the cut-off value of 30% for membranous EGFR2 overexpression rate in tumor cells has been used for years in treatment protocols. However, because of the studies and meta-analyses conducted over 10 years, the cut-off value has been lowered to 10% from 30% since 2013. We think that the cut-off value for the rates of ALK rearrangement may be also reduced in future treatment protocols according to the results of prognostic studies.
Unlike the ALK tests, ROS1 IHC has been recommended only as a screening tool and should be followed by confirmatory FISH testing.,,, There has also been debate over the prevalence of ROS1 abnormality and its mutual exclusiveness to other mutations. Previous studies have reported that the rearrangements in ROS1 are generally distinct from EGFR and ALK mutational status. However, recently, it was reported that ROS1 translocations can occur in conjunction with other driver mutations such as EGFR, KRAS, and BRAF. It was also found that ROS1 expression is a stage-independent predictor of favorable survival. We determined only one ROS1-positive tumor with a solid pattern in a 37-year-old woman. Therefore, we could not arrive at conclusions about the relationship between ROS1 rearrangement and other features of LC.
In summary, several important conclusions can be drawn from our study. First, we found that the frequency of molecular changes was less than reported, which may be related to the smoking status of the patients in our country because it was previously reported that EGFR, EGFR2, ALK, ROS1, and BRAF alterations have been much more frequently observed in never/light smokers, whereas KRAS mutations are more frequently found in heavy smokers.,,,,,, For example, comprehensive genomic profiling appears to be particularly beneficial as targetable genomic alterations were identified in up to 65% of tumors of the patients with less than 15 packs/year of smoking. In addition, we determined that preparing cell blocks from cytological materials and all molecular tests performed in these blocks in the same pathology laboratory where these cases were diagnosed reduced the insufficient material problem. Similarly, we concluded that the presence of a single red signal in patients with predominantly ALK rearrangements may be due to the frequency of fusions other than the ALK-EML4 gene fusion. In our series, the number of patients with ALK rearrangement was just under 15%, almost equal to the number of ALK-positive cases. We thought that the prognostic studies in larger patient series may change the cut-off value of ALK positivity to a lower rate in the future.
The study was approved by the Local Ethics Committee of the Hospital (2019/6-6; April 10, 2019).
General informed consent was obtained from patients before the surgical operation for using their specimens for scientific studies.
This article was edited in English by Gurkan Kazanci who professional translator.
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Conflicts of interest
There are no conflicts of interest.
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Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) clinical practice guideline update. J Clin Oncol 2013;31:3997-4013.
Source of Support: None, Conflict of Interest: None