To the Editor: Gene amplification is a common genetic alteration in cancer, contributing to tumor progression and treatment resistance. Never in mitosis gene A-related kinase 2 (NEK2) is a chromosomal instability gene that is aberrantly overexpressed in various cancers and is associated with poor prognosis.1 Previous results showed that NEK2 amplification correlates strongly with increased NEK2 expression in small cohorts of multiple myeloma (MM).2 Whether gene amplification contributes to abnormal NEK2 overexpression across different cancer types, particularly solid tumors, and its impact on patient prognosis remain underexplored. Here, methods to detect clinically significant NEK2 amplification in paraffin-embedded tissue samples were developed and validated Supplementary information, https://links.lww.com/CM9/C869. A total of 293 paraffin-embedded specimens were provided by Hunan Tumor Hospital, including 43 non-small cell lung cancer (NSCLC), 48 colorectal cancer (CRC), 157 breast cancer (BRC), and 45 multiple myeloma (MM) cases, with all patients giving written informed consent and with approval from the Medical Ethics Committee of the Xiangya School of Public Health, Central South University (No. XYGW-2022-109). Based on an initial survey of 15 major tumor types in the cBioPortal database (http://www.cbioportal.org/) Supplementary Figure 1A, https://links.lww.com/CM9/C869 and our previous study, three solid tumors with differing NEK2 amplification frequencies (NSCLC, 21.1%; CRC, 0.2%; BRC, 6.7%), and MM were selected for further analysis.2 High NEK2 expression was detected in 88.4% (38/43) of NSCLC patients, 60.4% (29/48) of CRC patients, 72.6% (114/157) of BRC patients, and 57.8% (26/45) of MM patients. NEK2 amplification was observed in 53.5% (23/43) of NSCLC cases, 54.2% (26/48) of CRC cases, 45.9% (72/157) of BRC cases, and 46.7% (21/45) of MM cases Figure 1A and Supplementary Figure 1B–D, https://links.lww.com/CM9/C869. Across all tumor types, NEK2 expression differed significantly between the NEK2-amplified (NEK2Amp) and NEK2-diploid (NEK2Diploid) tumors Figure 1A and B. Furthermore, a strong positive correlation between NEK2 amplification and NEK2 overexpression in NSCLC, CRC, BRC, and MM also were observed Supplementary Figure 2, https://links.lww.com/CM9/C869.Figure 1: NEK2 is amplified in multiple cancers, resulting in protein overexpression and association with poor prognosis. (A) Representative FISH images using probes specific for NEK2 (red) and corresponding IHC images showing NEK2 protein expression in paraffin-embedded samples from patients with NSCLC, CRC, BRC, and MM. (B) NEK2 protein expression levels in NSCLC, CRC, BRC, and MM patients with or without NEK2 amplification. Data are presented as means ± SD; P values were calculated using unpaired t tests. * P <0.001. (C) Kaplan–Meier analysis of OS in BRC patients with NEK2 Diploid or NEK2 Amp. (D) Kaplan–Meier analysis of OS in BRC patients stratified by NEK2 Diploid, NEK2 lowAmp, or NEK2 highAmp. (E) Kaplan–Meier analysis of OS in BRC patients with high NEK2 expression, with (NEK2 H-Amp) or without (NEK2 H-Diploid) NEK2 amplification. Amp: Amplification; BRC: Breast cancer; CI: confidence interval; CRC: Colorectal cancer; DAPI: 4’,6-Diamidino-2-phenylindole; ER: estrogen receptor; FISH: Fluorescence in situ hybridization; HER2: Human epidermal growth factor receptor 2; HR: Hazard ratio; IDC: Invasive ductal carcinoma; IHC: Immunohistochemistry; ILC: Invasive lobular carcinoma; M: Metastasis; MDLC: Mixed ductal–lobular carcinoma; MM: Multiple myeloma; N: Node; NEK2: Never in mitosis gene A-related kinase 2; NEK2 Amp: NEK2 amplification; NEK2 Diploid: NEK2 diploid; NEK2 highAmp: NEK2 High-level amplification; NEK2 H-Amp: High NEK2 expression with amplification; NEK2 H-Diploid: High NEK2 expression without amplification; NEK2 lowAmp: NEK2 Low-level amplification; NSCLC: Non-small cell lung cancer; OS: Overall survival; PR: Progesterone receptor; SD: Standard deviation; T: Tumor.To clarify the prognostic impact of NEK2 amplification, Kaplan–Meier survival analyses were conducted first on 116 paraffin-embedded BRC samples with available follow-up data. Patients with NEK2 amplification had significantly shorter overall survival (OS) (median: 78 months, range: 17–90 months) than those with normal NEK2 diploid status (median: 84 months, range: 12–94 months; P = 0.0047) Figure 1C. The degree of amplification, however, did not affect OS, as both high- and low-amplification groups showed a median OS of 78 months (range 17–90 months in low-amplification group, and range 20–82 month in high-amplification group) Figure 1D. Among patients with high NEK2 expression, those with NEK2 amplification also had shorter OS (median: 79 months, range: 17–84 months) compared with NEK2 diploid patients (median, 84 months, range 16–85 months) Figure 1E These results indicate that NEK2 amplification is more closely associated with poor prognosis than the extent of amplification. To determine whether NEK2 amplification independently predicted OS in BRC patients, multivariable Cox regression analyses adjusting for age, clinical stage, tumor–node–metastatic (TNM) stage, histologic subtype, and progesterone receptor, estrogen receptor and human epidermal growth factor receptor 2 (HER2) status were performed. NEK2 amplification remained significantly associated with OS (HR = 2.432, 95% CI = 1.146–5.159, P = 0.021) Supplementary Figure 3, https://links.lww.com/CM9/C869, indicating its independent prognostic value in BRC. Comparable results regarding the prognostic impact of NEK2 amplification in NSCLC and CRC were obtained using the cBioPortal database Supplementary Figure 4, https://links.lww.com/CM9/C869, suggesting that NEK2 amplification may be associated with poorer outcomes across multiple cancer types. The clinical and pathological characteristics of patients with NSCLC, CRC, BRC, and MM who showed high NEK2 expression with amplification (NEK2H-Amp) were compared with those showing high NEK2 expression without amplification (NEK2H-Diploid), to identify features associated with NEK2 amplification Supplementary Tables 1–4, https://links.lww.com/CM9/C869. NEK2 amplification was observed in 57.9% (22/38) of NSCLC patients, particularly those with lung squamous cell carcinoma (26.3% low, 21.1% moderate, and 10.5% high amplification); 79.3% (23/29) of CRC patients with glandular epithelial tumors (44.8% low, 24.1% moderate, and 10.3% high); 53.5% (61/114) of BRC patients with mammary epithelial tumors (22.8% low, 20.2% moderate, and 10.5% high); and 65.4% (17/26) of MM patients (26.9% low, 26.9% moderate, and 11.5% high). The results revealed no association between tumor differentiation and NEK2 amplification status or extent in NSCLC, CRC, or BRC, as amplification frequencies were similar in well- and poorly differentiated tumors Supplementary Figure 5, https://links.lww.com/CM9/C869. These findings suggest that NEK2 amplification is independent of tumor cell origin and differentiation. In addition, patients in the NEK2H-Amp group presented with more advanced disease at diagnosis across tumor types, with a significantly greater proportion diagnosed at stages III or IV compared with the NEK2H-Diploid group in NSCLC, CRC, and BRC, and at more advanced international staging system (ISS) stages in MM Supplementary Figure 6 and Supplementary Tables 1–4, https://links.lww.com/CM9/C869. These findings suggest that higher levels of NEK2 amplification are associated with a more aggressive disease course. Because tumor metastasis is a major determinant of poor prognosis in solid tumors, lymph node and distant metastasis were evaluated in NEK2H-Amp and NEK2H-Diploid groups among NSCLC, CRC, and BRC patients with high NEK2 expression. The NEK2H-Amp group showed a higher frequency of distant metastasis than the NEK2H-Diploid group (18.2% vs. 12.5% in NSCLC, 30.4% vs. 0% in CRC, and 10.7% vs. 0% in BRC). Although significant group differences were not detected, the percentage of NEK2 amplification tended to be higher in patients with lymph node or distant metastasis in NEK2H-Amp cases Supplementary Figure 7A–H and Supplementary Table 1–3, https://links.lww.com/CM9/C869. In MM, bone disease and renal dysfunction represented the most common forms of target organ damage and indicated disease progression. Both conditions were more prevalent in the NEK2H-Amp group than in the NEK2H-Diploid group, with bone disease observed in 76.5% vs. 66.7% of patients and renal dysfunction in 41.2% vs. 0% Supplementary Figure 7I–L and Supplementary Table 4, https://links.lww.com/CM9/C869. Together, these findings suggest a modest association between NEK2 amplification and tumor progression. A previous study demonstrated an association between TP53 mutations or deletions and NEK2.2 Dysfunctional p53 protein resulting from TP53 alterations can accumulate in the nucleus and show aberrant expression in multiple cancers.3,4 As such, p53 protein expression in solid tumors was examined by IHC and its relationship with NEK2 amplification was assessed. TP53 alterations was significantly more frequent in the NEK2H-Amp group than in the NEK2H-Diploid group in NSCLC and BRC, and a similar trend was found for CRC though without statistical significance. In addition, the NEK2H-Amp group showed a higher prevalence of two molecular subtypes in BRC compared with the NEK2H-Diploid group, including increased proportions of HER2 overexpressed (29.5% vs. 15.1%) and triple-negative disease (8.2% vs. 5.7%) Supplementary Figure 8, https://links.lww.com/CM9/C869. These findings suggest that TP53 alterations are more closely associated with the presence of NEK2 amplification than with its extent in NSCLC and BRC, and that NEK2 amplification may be associated with less favorable molecular subtypes in BRC. Amplification of oncogenes and drug-resistance genes can drive protein overexpression in tumors, influence clinical outcomes, and may serve as molecular markers for early diagnosis and as targets for targeted therapy. NEK2 regulates mitotic processes, and its aberrant expression has been associated with poor prognosis. In this study, fluorescence in situ hybridization (FISH) methods to detect NEK2 amplification in clinical paraffin-embedded samples were developed. It was found that NEK2 amplification, accompanied by increased protein expression, is common across multiple cancers and is associated with tumor progression and poor prognosis, supporting its potential as a therapeutic target. Gene amplification in cancer can act both as a therapeutic target and as a mechanism of treatment resistance. Detecting gene amplification can therefore inform targeted treatment strategies, particularly in cancers driven by specific genomic gains like HER2 and mesenchymal–epithelial transition factor (MET) gene.5,6 Although challenges in inhibiting NEK2 in solid tumors, a strategy for directly targeting cancer-associated gene amplification using triplex-forming oligonucleotides may enable targeted therapy of NEK2. A framework connects TP53 loss and NEK2 amplification in tumor progression, aiding patient stratification for NEK2 inhibitors. The development of efficient NEK2 probes for detecting amplification in paraffin-embedded tumor samples was a key challenge in this study. Small sample size and single-center design limit result generalizability and introduce bias, while short follow-up affects NEK2’s reliability. In summary, our data highlight NEK2 amplification as a clinically relevant biomarker across multiple cancer types, with implications for prognosis and treatment strategies, and support its potential as a therapeutic target and necessitating. Further clinical and mechanistic studies are needed to clarify the role of NEK2 amplification in tumor biology and to support the development of targeted therapeutic approaches. Acknowledgments We would like to thank the members of the Biomedical Center of the Institute for Advanced Study of Central South University for providing the experimental platform. Funding This study was supported by grants from the National Natural Science Foundation of China (Nos. 82270219, 82430008, and 82130006), the Natural Science Youth Foundation of Xinjiang Medical University (No. 2024XYZR01), and the Independent Exploration and Innovation Project for Postgraduates of Central South University (No. 2022ZZTS0928). Conficts of interest None.
Abudumijiti et al. (Mon,) studied this question.