Association between the presence of EGFR or ALK mutations and the location of primary non‑small cell lung cancer, as well as metabolic activity observed on ¹⁸F‑FDG PET/CT scans

Introduction

Lung cancer is the leading cause of cancer-related mortality worldwide, and non-small cell lung cancer (NSCLC) accounts for ~80% of all lung cancers, with adenocarcinoma being the most frequent type (1). The identification of specific gene mutations related to the development and progression of lung cancer has led to the emergence of new treatment targets. Mutations in the epidermal growth factor receptor (EGFR) gene are found in 15–50% of patients with lung cancer from various ethnic backgrounds (2). Alterations in the anaplastic lymphoma kinase (ALK) gene occur in 3–5% of patients with NSCLC (3). The emergence of EGFR and ALK inhibitor therapies in patients with lung cancer and EGFR and ALK genomic alterations has ushered in an era of targeted therapy for advanced NSCLC, thus changing therapeutic strategies from general chemotherapy to more targeted therapies (4). The accurate identification of these driver gene mutations from small biopsy specimens for molecular analysis is of notable clinical relevance. 18F-fluoro-2-deoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) is widely used for the diagnosis and initial staging of lung cancers, and the assessment of treatment response (5). Prior studies have analyzed the association between PET/CT-derived metabolic parameters and mutation status; however, results regarding the association between EGFR mutations and tumor glucose metabolism are conflicting, and to the best of our knowledge data comparing alterations in EGFR and ALK are limited. In addition to metabolic activity, prior studies have largely examined molecular subtypes; however, the possible role of anatomical constraints on these subtypes remains incompletely understood. Because EGFR mutations and ALK rearrangements are more common in specific patient subgroups, such as non-smokers and younger patients (6–8), these alterations may also be associated with different patterns of tumor anatomical localization. However, the association among tumor location, laterality and mutation status remains ambiguous. As such, the present study aimed to evaluate the association between anatomical tumor localization (lobar and central/peripheral distribution), glucose metabolism [based on maximum standardized uptake value (SUVmax)] and EGFR/ALK mutation status in patients with NSCLC. In contrast to previous studies that compared mutation-positive and wild-type tumors, the present study directly compared the anatomical and metabolic characteristics of tumors exhibiting EGFR mutations and ALK rearrangements.

Materials and methods

Study population

The present retrospective study included data from 112 patients with histopathologically confirmed NSCLC who underwent 18F-FDG PET/CT-based staging between January 2013 and December 2020. The inclusion criteria were documented EGFR mutation and/or ALK translocation, PET/CT for initial staging before any systemic or local therapy and diagnostically adequate images. The exclusion criteria were absence of baseline PET/CT data, long duration between imaging and molecular analysis, previous history of malignancy and poor image quality. Most patients (97%) had adenocarcinoma histology, and those with inadequate tissue for further classification were classified as NSCLC not otherwise specified (NOS). The present study was approved by the Ankara Atatürk Sanatorium Training and Research Hospital Clinical Research Ethics Committee (approval no. 2715). Given the retrospective nature of the study, the requirement for written informed consent was waived by the ethics committee. All procedures were conducted in accordance with The Declaration of Helsinki.

PET/CT imaging process

An integrated PET/CT scanner (Biograph 6 HI-REZ; Siemens Healthineers) was used. Patients were instructed to fast ≥6 h before the examination. The procedure was performed only in patients with blood glucose levels <180 mg/dl. Each patient rested for ~45 min after intravenous administration of 0.15 mCi/kg 18F-FDG. After the PET and low-dose CT scans, imaging was performed between the cranial region and the upper thigh without the use of intravenous contrast.

Interpretation of PET/CT results

FDG-PET/CT scans were reviewed by a nuclear medicine physician using an integrated workstation (Siemens Healthineers). The SUVmax was used as a quantitative measure of uptake levels on FDG-PET/CT and was defined as the maximum FDG concentration in the tumor normalized to the injected dose and patient body weight. The region of interest (ROI) was manually drawn around the pulmonary lesions to include the entire lesion within a single slice, and the maximum values within these ROIs were defined as the SUVmax. The location of the primary lung mass was then determined. Patients were divided into two groups (right and left) based on the lung side of the main mass. Tumors in the upper lobes of the right and left lungs and tumors in the middle lobe of the right lung were classified into the upper group, and lower lobe of the right and left lungs were classified into the lower group. The tumors were further classified according to their location as central or peripheral. Tumors in direct contact with the mediastinum or peribronchial location were considered to be central. PET/CT also revealed lymph nodes and distant metastases.

Testing for EGFR mutation(s) and ALK rearrangements

Pathologically confirmed samples were submitted to the Department of Pathology at Gazi University (Ankara, Turkiye) for molecular analysis.

ALK rearrangement analysis by fluorescence in situ hybridization (FISH)

ALK gene rearrangements were evaluated using formalin-fixed, paraffin-embedded (FFPE) tumor tissue sections. Tissue blocks were sectioned at a thickness of 4–5 µm and mounted on positively charged glass slides. For each case, a corresponding hematoxylin and eosin-stained slide (room temperature for 15 min) was reviewed by a board-certified pathologist to delineate the areas of highest tumor density and ensure a minimum of 50–100 viable tumor cells were present for analysis.

FISH was performed using a dual-color ‘break-apart’ probe set (Vysis ALK Break Apart FISH Probe Kit; Abbott Pharmaceutical Co. Ltd.). The probe set consists of a 3′ ALK spectrum orange/red fluorophore and a 5′ ALK spectrum green fluorophore, targeting the chromosome 2p23 region. In the absence of translocation, the close proximity of these probes results in a merged yellow signal or adjacent red/green signals. The FISH procedure followed standardized laboratory protocols. Initially, slides were incubated at 60°C 1 h to ensure tissue adherence, followed by deparaffinization in xylene and rehydration through a graded ethanol series.

Paraffin-embedded sections were incubated in an oven at 80°C for 1 h to achieve physical deparaffinization. Subsequently, chemical deparaffinization was performed by immersing the sections in xylene for 15 min. The sections were then rehydrated by incubation in 97 and 99% ethanol for 1 min each, respectively.

Antigen retrieval was carried out in a water bath at 96°C for 15 min using citrate buffer solution. The proteolytic treatment was performed using the Proteinase K solution supplied within the FISH kit (Abbott Pharmaceutical Co. Ltd.), which was applied to the marked area for 5 min according to the manufacturer's instructions to facilitate nuclear probe penetration

Target DNA and probes were co-denatured at 73°C for 5 min, followed by hybridization in a humidified chamber at 37°C for 16–24 h. Post-hybridization, stringency washes were conducted using a 0.4X SSC/0.3% NP-40 buffer at 72°C to remove non-specifically bound probes. Nuclei were counterstained with DAPI at room temperature for 10 min.

Slides were visualized using a fluorescence microscope equipped with appropriate excitation and emission filters (DAPI, green, orange and triple bandpass). Signal scoring was performed by two independent observers who counted a minimum of 50 non-overlapping tumor nuclei. A cell was defined as positive for ALK rearrangement if it exhibited either: Split signals, the red and green signals were separated by a distance greater than two signal diameters; or isolated single orange signals, representing the loss of the 5′ green signal associated with a rearrangement. A case was interpreted as ALK-positive if >15% of the counted cells displayed these rearrangement patterns. For cases showing borderline results (10–15% positive cells), an additional 50 nuclei were evaluated by a second reader to achieve a final consensus.

EGFR mutation analysis

Molecular analysis for EGFR mutations was conducted using two specimen types: FFPE tumor tissue and/or liquid biopsy (plasma). For tissue samples, a pathologist performed a histological review to ensure a minimum tumor content of 10%, with a preference for samples >30% to optimize analytical sensitivity. For liquid biopsies, circulating cell-free DNA (cfDNA) was obtained from peripheral blood collected in specialized cell-free DNA BCT or EDTA tubes.

Genomic DNA from FFPE sections (10 µm thickness) and cfDNA from 2 ml of plasma were isolated using the cobas® DNA Sample Preparation Kit (cat. no. 05985536190; Roche Molecular Diagnostics) and the cobas cfDNA Sample Preparation Kit (cat. no. 07247737190; Roche Molecular Diagnostics), respectively. The FFPE extraction workflow included deparaffinization followed by proteinase K digestion. The plasma extraction utilized a magnetic bead-based process. All extracted DNA was quantified using fluorometric (Qubit) or spectrophotometric (NanoDrop; Thermo Fisher Scientific, Inc.) methods to ensure a minimum concentration of 2 ng/µl for tissue samples.

The cobas z 480 analyzer was employed for real-time PCR amplification and detection. The assay utilizes mutant-specific primers and fluorescently labeled TaqMan® (cobas® EGFR Mutasyon Test v2; Roche Molecular Systems, Inc.) probes to identify 42 mutations across exons 18, 19, 20 and 21 of the EGFR gene. The master mix included Uracil-N-glycosylase (AmpErase) (cobas® EGFR Mutasyon Test v2) to mitigate carry-over contamination by degrading previously amplified DNA. Thermocycling conditions were as follows: Initial denaturation was performed at 95°C for 15 min for 1 cycle. This was followed by 42 cycles of 95°C for 20 sec (denaturation), 53°C for 30 sec and 72°C for 20 sec (extension) and final extension step was carried out at 72°C for 5 min. Targeted variants included G719X in exon 18; deletions and complex mutations in exon 19; S768I, T790M and insertions in exon 20; and L858R and L861Q in exon 21.

Automated data analysis was performed using the cobas 4800 System Software. (05200881001cobas z 480 Roche®). Each reaction included a synthetic DNA internal control to monitor for PCR inhibitors and verify extraction efficiency. The software calculated the cycle threshold for each target; results were categorized as ‘mutation detected’ if the fluorescent signal exceeded the predefined threshold within the validated cycle range. Run integrity was maintained through the inclusion of a positive control (containing EGFR mutant sequences) and a negative control (nuclease-free water) in every batch.

Statistical analysis

Statistical analyses were performed using SPSS software (version 21.0; IBM Corp.). The normality of data distribution was tested using the Shapiro-Wilk test. Continuous variables were expressed as mean ± standard deviation or median [interquartile range (IQR), Q1-Q3], while categorical variables were presented as frequencies and percentages. Intergroup comparisons were conducted using the independent samples t-test (for normally distributed variables) and the Mann-Whitney U test (for non-normally distributed variables). Categorical variables were compared using the χ2 test or Fisher's exact test, as appropriate. P<0.05 was considered to indicate a statistically significant difference. Post hoc power analysis was performed using G*Power software (version 3.1.9.7; Heinrich-Heine-Universität Düsseldorf) (9) to assess sample size adequacy with α=0.05, total sample size of 112, and allocation ratio of 0.35. Post-hoc power was calculated to be 0.66 for a medium-to-large effect size (Cohen's h=0.5).

Results

Data from 112 patients, diagnosed with NSCLC between January 2013 and December 2020, were included (Table I). The mean age was 59.41±12.31 years, and most patients were older than 50 years (81.3%). The median tumor size was 40 mm (IQR, 25–55). Adenocarcinoma was the most frequent histological type among the patients diagnosed in 109/112 (97%) of patients, with total of three patients diagnosed with unclassified NSCLC NOS (2.7%). A larger percentage of tumors were located in the right lung [62/112 (55.4%)]; in addition, the primary tumors were located mostly in the upper lobes (right and left) [27 (24.1%)], followed by the left lower lobe [24 (21.4%)]. The least common site was the middle lobe of the right lung [13 (11.6%)] (Fig. 1). Among all patients, ALK rearrangements were detected in 29 (26%) and EGFR mutations in 83 (74.1%). None of the patients exhibited concurrent ALK and EGFR alterations; therefore, only the ALK-positive (+) cohort was compared with the EGFR+ cohort. Representative images of ALK status evaluated by break-apart FISH are presented in Fig. 2. In ALK-negative specimens, intact loci were identified by the presence of closely linked or fused orange/green signals (Fig. 2A). Conversely, ALK rearrangement was confirmed by two distinct positive signaling patterns: The classic split pattern showing marked physical separation of the 3′ (red) and 5′ (green) signals (Fig. 2B), and the atypical positive pattern characterized by isolated single 3′ (red) signals resulting from the deletion of the 5′ probe sequence (Fig. 2C). Clinical characteristics and imaging features of the patients are summarized in Table I.

Figure 1. EGFR and ALK positivity rate in central and peripheral tumors. EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma receptor tyrosine kinase. RUL, right upper lobe), RML, right middle lobe), RLL (Right lower lobe), LUL (Left upper lobe) and LLL (Left lower lobe.

Figure 2. Representative images of ALK status evaluated by break-apart FISH (A) ALK FISH-negative specimen showing shows a signal pattern consisting of two orange/green fusion signal. (B) ALK FISH-positive specimen showing split red and green signals. (C) ALK FISH-positive specimen showing isolated single red signals. ALK, anaplastic lymphoma receptor tyrosine kinase; FISH, fluorescence in situ hybridization.

Table I. Characteristics of patients and tumors (n=112).

Table I.

Characteristics of patients and tumors (n=112).

Characteristic	Value
Age, years	59.41 (12.31)
Sex, n (%)
Male	55 (49.1)
Female	57 (50.9)
Mutation status (%)
ALK (+)	29 (26)
EGFR (+)	83 (74)
Primary tumor
Tumor size, mm; median (IQR)	40 (25–55)
SUVmax; median (IQR)	12.42 (8.91–17.72)
Side of tumor, n (%)
Right	62 (55)
Left	50 (45)
Lobar localisation, n (%)
RUL	27 (24.1)
RML	13 (11.6)
RLL	21 (18.7)
LUL	27 (24.1)
LLL	24 (21.4)
Location, n (%)
Central	64 (57)
Peripheral	48 (43)
Lymphadenopathy, n (%)
Positive	102 (91)
ALK (+)	28 (97)
EGFR (+)	74 (89)
Negative	10 (9)
ALK (+)	1 (3)
EGFR (+)	9 (11)
Distant metastasis, n (%)
Positive	64 (57)
ALK (+)	20 (69)
EGFR (+)	44 (53)
Negative	48 (43)
ALK (+)	9 (31)
EGFR (+)	39 (47)
Histology, n (%)
Adenocarcinoma	109 (97)
NOS	3 (3)
Pleural effusion (%)	43 (38.4)

[i] EGFR, Epidermal growth factor receptor; ALK, Anaplastic lymphoma kinase; NOS, not otherwise specified; RUL, Right upper lobe; RML, Right middle lobe; RLL, Right lower lobe; LUL, Left upper lobe; LLL, Left lower lobe; SUVmax, The maximum standardized uptake value.

Among the patients with ALK+, there were 13 (44.8%) and 16 (55.2%) peripheral and central tumors, respectively, whereas for the patients with EGFR+, 48 (57.8%) had centrally located tumors and 35 (42.2%) had peripherally located tumors (Fig. 3). Of the patients with ALK+, 11 (37.9%) tumors were on the left and 18 (62.1%) on the right, whereas in those who were EGFR+, 39 (47.0%) were on the left and 44 (53.0%) were on the right. EGFR and ALK positivity were also more common in upper-lobe cancers (60.2 and 58.6%, respectively). There were no statistically significant associations between the side of the main tumor, lobar position, location type and occurrence of ALK positivity or EGFR mutations (P=0.530, P=0.147 and P=0.975, respectively).

Figure 3. Distribution of EGFR-mutated and ALK-rearranged non-small cell lung cancer according to primary tumor location. EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma receptor tyrosine kinase.

All 112 primary lung cancers exhibited notably elevated FDG uptake. The median SUVmax of ALK+ lesions was 11.94 (IQR, 8.02–15.95), and 12.42 (IQR, 9.17–17.96) for those who were EGFR+; the difference between the groups was not statistically significant (P=0.451) (Fig. 4). Representative F-18 FDG PET/CT images are shown in Fig. 5.

Figure 4. SUVmax value of primary tumor in EGFR and ALK positive groups. EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma receptor tyrosine kinase; SUVmax, maximum standardized uptake value.

Figure 5. Representative F-18 FDG PET/CT images of patients with non-small cell lung cancer. (A) EGFR-positive case demonstrating primary tumor FDG uptake (B) ALK-positive case demonstrating primary tumor FDG uptake. FDG-PET/CT, 18F-fluoro-2-deoxyglucose positron emission tomography/computed tomography.

There was no significant difference in tumor size between the ALK+ and EGFR+ groups (P=0.472). The mean ages of ALK+ and EGFR+ patients were 61±13.74 and 58.86±11.80 years, respectively, with no significant association between patient age and mutation status (P=0.422). The prevalence of nodal involvement was not significantly different according to mutation status (P=0.229; Table II). The median SUVmax of lymph nodes in the cohort with ALK rearrangement was 6.38 (IQR, 3.42–9.71) and in the cohorts with EGFR mutation was 5.50 (IQR, 3.46–11.91) without a statistically significant difference (P=0.334). The clinical information, tumor localization and metabolic characteristics of the EGFR-positive and ALK-positive groups are presented in Table II. EGFR mutation positivity was higher among men [43/83 (55%)] and ALK positivity was slightly higher among women [17/29 (59%)] (Fig. 6). Distant metastasis was noted in the majority of patients with ALK+ [20/29 (69%)] compared with 44 (53%) with EGFR mutation positivity. Mutation positivity was not significantly associated with distant metastasis (P=0.135; Table II). PET/CT revealed lymph node involvement in 102 (91%) patients. FDG PET/CT identified 74 (89%) lymphadenopathies in patients with EGFR(+). In addition, 28/29 (97%) of patients with ALK+ exhibited lymph node involvement. Pleural effusions were observed in 43 patients. Specifically, pleural effusion at diagnosis was present in 13/29 (45%) patients with ALK+ and in 30/83 (36%) patients with EGFR+, with no statistically significant association between mutation status and development of effusion (P=0.408; Tables I and II).

Figure 6. Relationship between EGFR and ALK mutation status and sex. EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma receptor tyrosine kinase.

Table II. Comparison of baseline clinical, anatomical, and metabolic characteristics between EGFR-mutated and ALK-rearranged patients with non-small cell lung cancer.

Table II.

Comparison of baseline clinical, anatomical, and metabolic characteristics between EGFR-mutated and ALK-rearranged patients with non-small cell lung cancer.

Characteristic	Category	ALK(+) (n=29)	EGFR (+) (n=83)	Total (n=112)	P-value
Tumor laterality	Left	11 (37.9%)	39 (47.0%)	50 (44.6%)	0.530
	Right	18 (62.1%)	44 (53.0%)	62 (55.4%)
Lobar distribution	Upper lobe	17 (58.6%)	50 (60.2%)	67 (59.8%)	0.147
	Middle lobe	5 (17.2%)	7 (8.4%)	12 (10.7%)
	Lower lobe	7 (24.2%)	26 (31.4%)	33 (29.5%)
Tumor location	Peripheral	13 (44.8%)	35 (42.2%)	48 (42.9%)	0.975
	Central	16 (55.2%)	48 (57.8%)	64 (57.1%)
Age, years)	-	61.00±13.74	58.86±11.80	59.41±12.31	0.422
Tumor size (mm)	-	40 (27.5–55)	35 (25–55)	40 (25–55)	0.472
Distant metastasis	Positive	20 (69.0%)	44 (53.0%)	64 (57.1%)	0.135
	Negative	9 (31.0%)	39 (47.0%)	48 (42.9%)
Lymph node involvement	Positive	28 (96.6%)	74 (89.2%)	102 (91.1%)	0.229
	Negative	1 (3.4%)	9 (10.8%)	10 (8.9%)
Pleural effusion	Present	13 (44.8%)	30 (36.1%)	43 (38.4%)	0.408
	Absent	16 (55.2%)	53 (63.9%)	69 (61.6%)
Primary tumor SUVmax	-	11.94 (8.02–15.95)	12.42 (9.17–17.96)	12.42 (8.91–17.72)	0.451
Lymph node SUVmax	-	6.38 (3.42–9.71)	5.50 (3.46–11.91)	5.62 (3.45–11.52)	0.334

[i] Data are presented as n (%), mean ± standard deviation or median (Q1-Q3). Pearson χ² or Fisher's exact test was used for categorical variables, while independent samples t-test (bootstrap method) or Mann-Whitney U test (Monte Carlo simulation) was used for continuous variables, as appropriate. No patients with concurrent ALK rearrangement and EGFR mutation were identified. ALK, anaplastic lymphoma kinase; EGFR, epidermal growth factor receptor; SUVmax, maximum standardized uptake value.

Discussion

The present investigation aimed to compare the anatomical distribution and metabolic characteristics derived from 18F-FDG PET/CT between EGFR-mutated and ALK-rearranged NSCLC. The present findings revealed that tumor side, lobar distribution, central versus peripheral localization and SUVmax were not significantly associated with mutation status, suggesting the low discriminatory power of conventional PET/CT parameters in this setting.

EGFR mutations and ALK rearrangements are notable therapeutic targets, and non-invasive imaging biomarkers have been widely studied as adjuncts to tissue-based molecular testing. Although 18F-FDG PET/CT has been proposed for mutation assessment (10), the current evidence remains controversial. Potential associations between tumor location and driver mutations have been suggested in previous studies (6,11–13); however, the present study did not observe a significant association between tumor location and mutation status.

Conflicting results have been reported in previous studies regarding the localization of tumors and molecular alterations in NSCLC. Some studies have proposed a higher incidence of central tumors in EGFR(+) cases (14), whereas others have not shown any significant association with ALK status (15). Other reports have described a higher incidence of central tumors in ALK+ NSCLC (16,17). These discrepancies may be explained by different patient populations, cohort sizes and the definition of tumor centrality.

Lung cancer is more often found in the upper lobes, and the present study found a greater frequency of both EGFR-mutant and ALK-rearranged tumors in this location, which is consistent with previous studies (7,18–21). While the literature indicates a relative predominance of EGFR mutations in the left upper lobe (22) and a more heterogeneous distribution for ALK-positive tumors (10,23), this upper lobe predominance did not reliably distinguish between the two molecular subtypes in the present cohort. However, differences in anatomical classification can influence the comparability between studies (24).

SUVmax, which reflects glucose metabolism in tumors, has been extensively explored as a potential imaging biomarker of genetic alterations in NSCLC; however, results have been inconclusive. Some studies have reported lower SUVmax values in EGFR-mutant tumors (8,25,26), whereas others have not found any significant association (27–30). Studies have identified an association between PET parameters and ALK rearrangement (4,15,31) The SUVmax did not vary significantly between the EGFR-mutant and ALK-rearranged groups in the present cohort, supporting its limited, utility for differentiating these groups. This is consistent with meta-analytic data demonstrating modest diagnostic performance of 18F-FDG PET/CT for predicting EGFR mutation (32).

The absence of an association between tumor location and mutation status indicates that the distribution of tumor location is primarily determined by structural and environmental factors (such as bronchial anatomy and carcinogen exposure) (26), rather than by oncogenic changes. Similarly, the absence of metabolic differences indicates that FDG uptake is associated with complex tumor biology rather than mutation-specific effects. The EGFR and ALK signaling pathways activate downstream cascades, such as PI3K/AKT and MAPK, and tumor metabolism is modulated by intratumoral heterogeneity, hypoxia and the tumor microenvironment (33–36), thus limiting the utility of SUVmax as a surrogate biomarker.

The clinical importance of these findings lies in the complementary role of 18F-FDG PET/CT in the initial diagnostic workup. SUVmax and tumor location were not independent predictors of EGFR or ALK status; however, the metabolic profile may be an additional aspect of the overall assessment of tumor behavior. Specifically, ALK-rearranged tumors are more often associated with advanced metastatic disease, consistent with their known aggressive phenotype (8,31). PET/CT can be helpful in assessing the extent of disease when tissue sampling is limited; however, it should not be used in place of molecular testing.

Recent studies have demonstrated the potential of combining PET/CT parameters, clinical parameters, and radiomic or deep learning-based features to improve the molecular characterization of NSCLC (37–41). Imaging must be considered within the framework of multimodal diagnosis, in which treatment selection must be based on molecular testing as the gold standard. In previous studies, mutation status has been associated with clinicopathological factors such as age, sex and tumor size (42–45). In the present cohort, these associations were not significant, which may be due to the limited sample size and heterogeneity of the population.

The present study has several limitations. First, its retrospective, single-center design, relatively small sample size and imbalance between the EGFR and ALK subgroups may limit the generalizability of the findings. In addition, metabolic assessment was limited to SUVmax, and no advanced PET-derived parameters such as metabolic tumor volume or total lesion glycolysis were included. Differences in anatomical classification, such as the right middle lobe being grouped within the upper lobes, may also have influenced the comparisons. A post hoc power analysis demonstrated 66% statistical power, indicating that smaller associations may not have been detected. Internal validation strategies such as resampling or subgroup analyses were not performed because of the limited subgroup size. Another notable limitation is the lack of direct mechanistic validation. Although potential biological pathways linking EGFR and ALK signaling to tumor metabolism were discussed, these interpretations remain speculative because integrated molecular or functional analyses were not available. Therefore, the findings should be interpreted as exploratory and hypothesis-generating.

In conclusion, tumor localization and SUVmax derived from 18F-FDG PET/CT data yielded limited utility in differentiating EGFR-mutant and ALK-rearranged NSCLC. These findings should be interpreted in the context of the study limitations and validated in larger multicenter cohorts. Future studies including advanced metabolic and radiomic approaches may improve non-invasive molecular characterization and support more precise personalized management of patients diagnosed with NSCLC.

Acknowledgements

The authors would like to thank Professor Cengiz Bal (Department of Biostatistics, Medicine Faculty, Osmangazi University, Eskişehir, Turkiye) for his support to the statistical analysis. The present manuscript was presented as a poster presentation at the 36th National Nuclear Medicine Congress, Cyprus (May 08–12, 2024).

Funding

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

IUB, UY, TIC and NA contributed to the conception and design of the study. SK, PAK, DK and EGA were responsible for data collection and literature search. IUB analyzed the images, interpreted the results and wrote the initial draft of the manuscript. UY, TIC and NA supervised the study and revised the manuscript. IUB and TIC confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

The Ethics Committee of Atatürk Sanatorium Training and Research Hospital approved the study with approval no. 2715, and the study was conducted in accordance with the principles set forth in The Declaration of Helsinki.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

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References