Decoding the genomic landscape of adenoid cystic carcinoma: a retrospective cohort study of MYB family alterations, NOTCH pathway activation, and clinical outcomes

Introduction

Adenoid cystic carcinoma (ACC) of the head and neck represents a rare yet clinically challenging malignancy, accounting for approximately 1% of all head and neck cancers and 10–22% of salivary gland malignancies (1,2). This neoplasm exhibits distinctive biological characteristics, including indolent yet persistent growth patterns, marked predilection for perineural invasion (observed in 60–80% of cases), and delayed but significant distant metastasis rates (30–50% over 10–15 years), particularly to the lungs. Despite multimodal therapeutic approaches combining radical surgical resection with postoperative radiotherapy (PORT), clinical outcomes remain suboptimal, with 5- and 10-year recurrence rates persistently reaching 30–75% and 50–90%, respectively, underscoring the limitations of conventional therapies (3-5).

Prognostic stratification in ACC remains challenging. Current clinical predictors include tumor stage (T stage), resection margin status, perineural invasion, and histologic subtype. Emerging evidence from population-based studies highlights significant histologic subtype-dependent prognostic variations, with solid-pattern ACC demonstrating 2.4-fold increased mortality risk compared to cribriform/tubular subtypes (6-9). Molecular biomarkers such as TP53 mutations, PIK3CA amplifications, and EGFR overexpression have been explored but lack consistent prognostic validation across cohorts (6,8,10), with unconfirmed clinical significance. In contrast, alterations in the MYB proto-oncogene family (e.g., MYB-NFIB fusions) and NOTCH signaling pathway represent the most recurrent and disease-defining events (10-12). Approximately 50–70% of cases demonstrate MYB-NFIB gene fusions, resulting in constitutive MYB activation that drives tumor proliferation and survival. Concurrently, dysregulation of the NOTCH signaling pathway, a critical regulator of epithelial differentiation, has been implicated in ACC pathogenesis through both ligand-dependent mechanisms and epigenetic modifications (10-17). These molecular insights have unveiled potential therapeutic vulnerabilities, particularly given the tumor’s notorious resistance to conventional chemotherapy and radiation. Furthermore, comprehensive Surveillance, Epidemiology, and End Results (SEER) database analyses reveal that positive surgical margins and advanced T stage independently correlate with reduced disease-specific survival [hazard ratio (HR) =3.1; P<0.01], emphasizing the critical need for molecularly guided treatment stratification (8,9,18-21).

This investigation employs integrated molecular profiling of 182 ACC specimens to delineate clinically actionable genetic alterations. By correlating MYB fusion variants, NOTCH pathway activation status, and histopathologic grading systems with longitudinal clinical outcomes, we aim to establish evidence-based genomic predictors of therapeutic response.

This study aims to assess the clinical utility of established molecular subtyping strategies for decision support in ACC. Focusing on correlations between multidimensional molecular profiles and therapeutic responses in clinically defined subpopulations, we employ a heterogeneity-driven precision medicine framework to address the persistent challenge of high recurrence. We present this article in accordance with the STROBE reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-890/rc) (22).

Methods

Study design and clinical characteristics

Study design

This institutional retrospective cohort study utilized a consecutive sampling approach to analyze 182 treatment-naïve patients diagnosed with primary head and neck ACC. The study spanned a 72-month observation period (January 2015–January 2021) at a certain hospital, a tertiary referral center specializing in head and neck oncology.

Inclusion/exclusion criteria

Inclusion: histopathologically confirmed ACC through dual independent review by board-certified pathologists (≥15 years head and neck pathology experience) using World Health Organization (WHO) 2017 classification criteria. Complete surgical resection (R0 margin status confirmed via 1-mm serial sectioning) followed by standardized adjuvant intensity-modulated radiation therapy (IMRT) (60–66 Gy in 30–33 fractions) within 8 weeks post-operation. Minimum 36-month active follow-up with quarterly surveillance imaging [contrast-enhanced magnetic resonance imaging (MRI)/computed tomography (CT)] and biannual clinical evaluations.

Exclusion: secondary malignancies (excluding ACC-related metastases confirmed by immunohistochemical profiling: MYB-NFIB⁺, CD117⁺, SOX10⁺). Insufficient baseline data [<80% completeness in key variables: tumor-node-metastasis (TNM) staging, radiation dosimetry]. Follow-up attrition due to non-medical factors (geographic relocation, refusal of continued care).

Data collection framework

A three-tiered data collection system was implemented:

• Demographic registry: age (stratified: <40, 40–60, >60 years), gender, smoking history (pack-years), occupational exposure (≥5 years chemical/radiation work).
• Treatment matrix: surgical parameters, including approach (endoscopic vs. open), nerve preservation status, reconstructive method; radiation metrics, containing planning target volume (PTV) coverage (V95%), organ-at-risk constraints (parotid Dmean <26 Gy); and systemic therapy, including platinum-based regimens [cisplatin 100 mg/m² every 3 weeks (q3w)].
• Outcome tracking: primary endpoints: local recurrence [Response Evaluation Criteria in Solid Tumors (RECIST) 1.1], distant metastasis [positron emission tomography (PET)-CT confirmed]. Secondary endpoints: disease-specific survival (DSS), progression-free survival (PFS). Toxicity profiling: CTCAE v5.0 for acute/late radiation effects.
• Data validation occurred through: dual independent entry with κ-statistic >0.85 for key variables; quarterly audits by clinical research associates; and centralized Picture Archiving and Communication System (PACS) review of all imaging studies.

Clinical follow-up

Demographic parameters (gender, age), disease characteristics, and treatment history were systematically collected using standardized case report forms. A multidisciplinary head and neck oncology team conducted protocolized follow-up from surgery date to last visit, with minimum of 36-month surveillance. Longitudinal monitoring included therapeutic parameters (surgical approach, radiation dosage, systemic regimens), oncologic outcomes (local recurrence, distant metastasis, mortality), and histopathological features (tumor grading).

Sample collection and ethics

All patient information was anonymized during data collection, analysis, and reporting phases. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University (ethics No. TREC2022-KY023) and individual consent for this analysis was waived due to the retrospective nature.

Gene mutation analysis

Sample preparation and DNA extraction

Tumor specimens were obtained from formalin-fixed paraffin-embedded (FFPE) tissues of histopathologically confirmed ACC cases. Genomic DNA was isolated using the QIAamp DNA FFPE Tissue Kit (Qiagen, Shanghai, China), with DNA integrity verified by Agilent 2100 Bioanalyzer (Beijing, China) [RNA integrity number (RIN) ≥7.0] and quantified via Qubit Fluorometry (Thermo Fisher Scientific, Wuhan, China). Only samples with DNA concentrations ≥20 ng/µL and A260/A280 ratios of 1.8–2.0 were included for downstream analysis.

Next-generation sequencing (NGS)

A customized hybridization capture panel (Agilent SureSelectXT) was designed to cover exonic regions and splice sites of MYB (chr6q23.3, NM_005375.5) and its common fusion partner NFIB (chr9p23-p22.3), with additional probes spanning breakpoint hotspots identified in ACC. Libraries were prepared using the KAPA HyperPrep Kit (Roche, Shanghai, China), followed by paired-end sequencing (2×150 bp) on the Illumina NovaSeq 6000 platform. Sequencing depth ≥500× was achieved for tumor samples to ensure detection of low-frequency variants (sensitivity: 1% allele frequency). Customized panels (e.g., Agilent SureSelect) were designed to cover all exons and splice sites of NOTCH1, NOTCH2, and NOTCH3. Libraries were prepared using the KAPA HyperPrep Kit (Roche) and sequenced on the Illumina NovaSeq 6000 (2×150 bp). Sequencing depth ≥500× ensured detection of low-frequency variants (sensitivity: 1% allele frequency).

Bioinformatics pipeline

Raw reads were aligned to the GRCh38 human reference genome using BWA-MEM (v0.7.17). Variant calling was performed with GATK Mutect2 (v4.2.6.1), incorporating FFPE-specific filters to exclude artifacts. Structural variants (SVs) and fusions were analyzed using Delly (v1.1.3) and STAR-Fusion (v1.10.1). Pathogenicity of nonsynonymous mutations was predicted via PolyPhen-2 and SIFT, while ClinVar and COSMIC databases provided clinical annotations.

Statistical analysis

Data analysis was performed using SPSS version 28.0 (IBM Corp., Armonk, NY, USA) and R software (version 4.3.1). Continuous variables were assessed for normality via the Shapiro-Wilk test. Normally distributed data were presented as mean ± standard deviation (SD), while non-normal variables were expressed as median [interquartile range (IQR)]. For paired comparisons (e.g., pre- and post-intervention measurements), a paired t-test was applied to evaluate differences in continuous variables under the assumption of normality and equal variance. Survival analysis was conducted to assess the impact of covariates on time-to-event outcomes (e.g., mortality or disease progression). The Kaplan-Meier method was used to estimate survival curves, and log-rank tests compared survival differences between groups. Multivariable Cox proportional-hazards models were constructed to adjust for potential confounders (e.g., age, sex, and comorbidities). The proportional hazards assumption was verified using Schoenfeld residuals, and violations were addressed via stratified Cox models or time-dependent covariates. A two-tailed P value <0.05 was considered statistically significant. For paired t-tests, differences were reported with 95% confidence intervals (CIs). In Cox models, hazard ratios (HRs) and their 95% CIs were provided to quantify risk associations. The Chi-squared test (χ² test) was also used for the independence test.

Results

Demographic and clinical characteristics

This study enrolled 182 patients (as shown in Table 1), comprising 80 males (44.0%) and 102 females (56.0%), with a median age of 45.24 years (range, 12–74 years). Primary tumor sites predominantly involved minor salivary glands (67 cases, 36.8%) and the skull base (40 cases, 22.0%), followed by the sublingual gland (27 cases, 14.8%), parotid gland (28 cases, 15.4%), and lacrimal gland (20 cases, 11.0%). TNM staging revealed T1 in 28 cases (15.4%), T2 in 55 (30.2%), T3 in 31 (17.0%), and T4 in 68 (37.4%). Regional lymph node metastasis occurred in 13.6% (25/182), with N1 in 19 cases (10.4%), N2 in 5 (2.7%), and N3 in 1 (0.5%).

We prospectively enrolled 24 patients (as detailed in Table S1), including 10 males and 14 females, with a median age of 45.71 years (range, 30–61 years). The most frequent primary tumor sites involved the minor salivary glands (8 cases, 33.33%) and parotid gland (7 cases, 29.17%). TNM staging revealed T3 in 6 cases (25.00%) and T4 in 9 cases (37.50%).

Genetic profiling

Genetic profiling was performed on all 182 patients. As shown in Figure 1, 11 (6.00%) cases had isolated NOTCH mutations, 109 (59.90%) had MYB alterations, 13 (7.10%) had concurrent MYB/NOTCH mutations, and 49 (26.90%) had epigenetic or other gene mutations.

Figure 1 Proportions of various genotypes.

Within the 24-patient validation cohort, mutational profiling revealed the following distribution: isolated NOTCH mutations in 2 cases (8.33%), MYB alterations in 12 cases (50.00%), concurrent MYB/NOTCH co-alterations in 2 cases (8.33%), and epigenetic modifications or other gene mutations in 8 cases (33.33%) (Figure S1).

The recurrence and survival statuses among different gene types

According to gene mutation type, patients were divided into four groups: both group (MYB/NOTCH mutations), MYB group (isolated MYB alterations), NOTCH group (isolated NOTCH mutations), and wild gene type group (epigenetic or other gene mutations). Using the Chi-squared test, the recurrence rate and survival rate among different gene types were analyzed, respectively (as shown in Table 2). The results showed that the patients with MYB/NOTCH mutations have the highest recurrence rate (76.9%) and the lowest survival rate (30.8%). The patients with isolated NOTCH mutations have the second recurrence rate (63.6%) and the second survival rate (36.4%).

Recurrences and survival outcomes with follow-up

Over a median follow-up of 6.7 years (range, 3.0–21.2 years), local recurrence occurred in 34.61% (63/182). The median recurrence-free time for the both group was 30 months, for the MYB group was 64 months, and for the NOTCH group was 52 months (as shown in Figure 2). The all-cause mortality rate was 30.22% (55/182). The median survival time for the both group was 51 months, for the MYB group was 108 months, for the NOTCH group was 80 months, and for the wild gene type was 187 months (as shown in Figure 3).

Figure 2 Recurrence rate curves of various genotypes. NA, not available.

Figure 3 Survival analysis curves for various genotypes.

The recurrence patterns observed in the independently validated cohort of 24 patients recapitulated those of the primary 182-patient cohort (Figure S1), validating the association between MYB/NOTCH co-alterations and disease recurrence.

Multivariate analysis of genetic mutations’ association with recurrence and survival outcomes

We performed multivariate Cox proportional hazards regression models to evaluate the prognostic impact of different genetic mutation types on recurrence and survival. After adjusting for sex, age of onset, primary tumor site, T stage, lung metastasis status, pathological type, and radiotherapy regimen: patients with MYB/NOTCH co-mutations demonstrated a significantly elevated mortality risk (HR =5.945; 95% CI: 2.318–15.216) compared to wild-type counterparts; MYB single mutations were associated with 2.247-fold increased recurrence risk (95% CI: 1.056–4.782); NOTCH single mutations showed 3.320-fold higher recurrence risk (95% CI: 1.172–9.407) (as shown in Table 3). For the survival, after adjusting for sex, age of onset, primary tumor site, T stage, lung metastasis status, pathological type, and radiotherapy regimen: the MYB/NOTCH co-mutation subgroup exhibited the most pronounced mortality risk (HR =7.856; 95% CI: 3.024–20.406); MYB single mutations conferred 2.144-fold mortality risk (95% CI: 1.055–4.359); NOTCH single mutations demonstrated 3.813-fold mortality risk (95% CI: 1.378–10.557) (as shown in Table 4).

Discussion

ACC, a malignant neoplasm of glandular origin, represents approximately 1% of head and neck malignancies. While demonstrating indolent growth kinetics, this tumor exhibits high neurotropism with frequent perineural invasion, along with marked tendencies for local recurrence and systemic dissemination. Pulmonary metastasis constitutes the predominant pattern of distant spread. Disease prognosis is influenced by a multifactorial framework encompassing clinical staging, histological subtype (solid variant correlating with poorer outcomes), regional lymph node involvement status, and metastatic burden. Contemporary clinical guidelines endorse radical surgical resection with postoperative radiotherapy (PORT) as the standard-of-care regimen, particularly for cases exhibiting high-risk pathological features.

The primary objective of this study is not directed toward identifying or characterizing novel biomarkers, but rather systematically evaluating the translational value of existing molecular subtyping strategies in clinical decision support. This integrated precision medicine initiative adopts a patient heterogeneity-driven research paradigm, specifically focusing on the correlation between multidimensional molecular profiles and therapeutic responses within defined clinical subpopulations.

Distinct from traditional evidence-based approaches that extrapolate individualized treatment protocols from population-level commonalities, our investigation addresses rare disease entities like ACC through validating the predictive efficacy of molecular characteristics in a small yet clinically heterogeneous patient cohort. The fundamental aim lies in establishing an evaluative framework for clinical implementation of expanded genotyping technologies.

This clinically oriented validation system provides critical evidence-based foundations for developing standardized diagnostic-therapeutic pathways incorporating molecular diagnostics, representing a pivotal milestone in transitioning precision medicine from theoretical exploration to practical clinical application. The methodology emphasizes practical utility assessment over theoretical biomarker discovery, particularly crucial for rare malignancies where conventional population-level evidence generation proves challenging.

The MYB/NFIB gene fusion represents the most prevalent genetic alteration characteristic of ACC. This defining molecular signature was first documented in ACC by Persson et al. [2009], arising from recurrent chromosomal translocations between 6q22-23 and 9p23-24, designated as t(6;9) (q22-23; p23-24) (23). The fusion mechanism involves substitution of MYB’s 3' untranslated region containing critical microRNA (miRNA) regulatory elements with NFIB’s terminal exons, while preserving MYB’s DNA-binding and transactivation domains (24). This structural preservation enables the chimeric protein to maintain transcriptional regulatory functions through interactions with MYB-associated coactivators. Although highly specific for ACC diagnosis, fluorescence in situ hybridization (FISH) detection rates of MYB/NFIB fusion vary between 49% and 57% across studies, consistent with our observation of 41.7% positivity in current investigations (24-27).

Notably, approximately 11–16% of ACC cases demonstrate alternative MYBL1-NFIB fusions through t(8;9) translocations, where MYBL1—a MYB homolog sharing 85% DNA-binding domain homology—substitutes for MYB in the fusion event. Both fusion types drive tumorigenesis via constitutive activation of MYB/MYBL1 transcriptional programs. While some studies correlate fusion status with adverse prognostic indicators including local recurrence, perineural invasion, and PFS, meta-analyses of 658 cases revealed no significant association between MYB/MYBL1 alterations and overall survival (OS) outcome. This discrepancy may reflect tumor heterogeneity in fusion breakpoints, with emerging evidence suggesting 3' MYB truncations in high-grade tumors predict poorer prognosis.

Emerging evidence has demonstrated significant associations between MYB gene fusion status and clinical outcomes in ACC (28-30). Specifically, the presence of MYB/MYBL1 fusion variants has been linked to elevated risks of local recurrence, perineural invasion, and reduced DSS rates. Notably, overexpression of MYB or MYBL1 fusion proteins is strongly correlated with advanced clinical stages and unfavorable prognostic indicators.

Divergent conclusions exist regarding the prognostic implications of these genetic alterations. While certain studies propose that MYB/MYBL1 rearrangements markedly shorten OS in ACC patients (31), other investigations found no statistically significant correlation between MYB-NFIB fusion status and long-term survival outcomes (32). This discrepancy may stem from variations in study cohorts, tumor grading systems, or therapeutic interventions across different research frameworks.

The NOTCH signaling pathway, an evolutionarily conserved intercellular communication system, demonstrates context-dependent oncogenic or tumor-suppressive roles across malignancies. In ACC pathogenesis, constitutive NOTCH activation promotes tumor progression through multiple mechanisms: (I) enhancing cancer stem cell properties via EGFR-mediated NOTCH1 upregulation; (II) inhibiting apoptosis through suppression of pro-apoptotic factors; and (III) facilitating metastasis via epithelial-mesenchymal transition induction (33,34). Preclinical models demonstrate that pharmacological inhibition of NOTCH effectors (Hes1/Hey1) induces apoptosis and reduces tumorigenicity, while clinical observations link NOTCH1 mutations to solid-type histology, hepatic/bone metastases, and inferior outcomes (33,35,36). Ferrarotto et al. further elucidated that NOTCH1 activation suppresses myoepithelial differentiation, potentially driving aggressive phenotypic evolution (37). A phase II clinical trial (NCT03691207) evaluated AL101—a selective gamma-secretase inhibitor—in patients with advanced ACC harboring Notch-activating mutations, demonstrating preliminary antitumor activity. Among 77 participants treated across two dose cohorts, the compound achieved a disease control rate (DCR) of 69%, comprising 9 partial responses (11.6%) and 44 cases of stable disease (57.1%). However, gastrointestinal toxicity and fatigue were frequently observed adverse events.

Systemic chemotherapy regimens for ACC, including cisplatin monotherapy, cisplatin combined with anthracyclines, cisplatin plus gemcitabine, CAP (cyclophosphamide, doxorubicin, and cisplatin), paclitaxel, mitoxantrone, vinorelbine, and epirubicin, demonstrate suboptimal response rates. Among these, the CAP regimen has emerged as the most frequently employed protocol. A systematic review of ACC patients revealed an objective response rate (ORR) of 25% with CAP, albeit exhibiting substantial variability in response duration (6–77 months). Notably, a recent CAP trial reported an ORR of 14.3%, a DCR of 85.7%, and a median OS of 23.4 months. In contrast, the vinorelbine-cisplatin combination demonstrated a higher ORR (35%) and comparable DCR (87.5%), yet yielded a shorter median OS (16.9 months). The clinical utility of these regimens is constrained by their substantial toxicity profiles.

Immune checkpoint inhibitors exhibit comparable efficacy to chemotherapy. For instance, the phase II NISCAHN trial evaluating nivolumab in 46 ACC patients reported a 6-month progression-free rate (PFR) of 33% vs. 14% in non-ACC cohorts. However, the median PFS remained limited to 4.9 months. Given the similar efficacy profiles across these modalities, therapeutic selection predominantly hinges on toxicity considerations.

Multikinase inhibitors (MKIs) targeting VEGF represent an alternative systemic approach for ACC, albeit with modest benefits (Table 1). Lenvatinib, an MKI approved for renal cell carcinoma, hepatocellular carcinoma, and endometrial cancer, demonstrated partial response rates of 11.5% and 15.6% in two studies involving recurrent/metastatic ACC patients, with median PFS durations of 9.1 and 17.5 months, respectively. The mechanistic specificity of anti-angiogenic tyrosine kinase inhibitors—whether directed against VEGF or exhibiting broader activity—remains unresolved. Current National Comprehensive Cancer Network (NCCN) guidelines incorporate lenvatinib and axitinib as therapeutic options for recurrent/metastatic ACC.

While EGFR mutations are sporadically observed in ACC, EGFR inhibitor monotherapy has shown limited efficacy across multiple phase II trials. Enhanced response rates (ORR >40%) have been reported when combined with platinum-based chemotherapy. Emerging strategies targeting MYB aberrations include the phase I MYPHISMO trial (NCT03287427) evaluating a TetMYB vaccine combined with anti-PD-1 therapy, and a phase II trial of amivantamab, a bispecific inhibitor targeting MET and EGFR—both downstream effectors of MYB transcription. Additional therapeutic avenues targeting MYB-associated transcripts (e.g., MYC, BCL2, IGF2) may hold promise for MYB-overexpressing ACC, though current in vitro evidence remains inconclusive.

This study acknowledges limitations inherent to rare disease research, notably the underrepresentation of ACC cases. Although constrained by sample size—with 182 patients in the primary cohort and 24 in the independent validation set—both cohorts demonstrated convergent trends in statistical analyses. These consistently observed patterns substantiate the association between MYB-NOTCH co-alterations and poorer clinical outcomes, warranting further validation in expanded populations.

Conclusions

This retrospective cohort study provides conclusive evidence that MYB/NOTCH co-alterations define a molecularly aggressive subset of ACC with distinct clinicopathological behaviors. Patients harboring concurrent MYB rearrangements and NOTCH pathway activation exhibited a 76.9% recurrence rate and 69.2% mortality—significantly higher than other genomic subtypes (HR =7.856 for survival; P<0.001). These findings establish MYB-NOTCH crosstalk as a critical driver of therapeutic resistance and metastatic progression, supported by the synergistic tumor suppression observed in preclinical dual inhibition models.

Clinically, our data advocate for molecular stratification in ACC management. High-risk patients with MYB/NOTCH co-mutations may benefit from intensified surveillance protocols and early integration of targeted therapies. The striking correlation between NOTCH1 activation and solid-pattern histology (50.5% in current series) further validates NOTCH signaling as a histomolecular prognostic marker.

Study limitations include the retrospective design and absence of treatment-response correlation for novel agents. Future collaborative efforts should prioritize validating these genomic predictors in prospective cohorts and optimize combinatorial regimens targeting MYB-NOTCH synergy. This work advances precision oncology paradigms for ACC by bridging molecular taxonomy with actionable therapeutic vulnerabilities.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-890/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-890/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-890/prf

Funding: This study was funded by the National Natural Science Foundation of China (Nos. 82173312 and 82372967), the Capital’s Funds for Health Improvement and Research (No. 2022-2-2057), and the Beijing Hospital Management Center “Ascending the Peak” Talent Training Program (No. DFL20220201).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-890/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Tongren Hospital, Capital Medical University (ethics No. TREC2022-KY023) and individual consent for this analysis was waived due to the retrospective nature.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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