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Molecular determinants of clinical outcomes for anaplastic lymphoma kinase–positive non-small cell lung cancer in Chinese patients: A retrospective study

  • Maojing Guan
    Affiliations
    Department of Oncology, Anhui Chest Hospital, 397 Jixi Road, Hefei 230022, China

    Anhui Medical University Clinical College of Chest, 397 Jixi Road, Hefei 230022, China
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  • Jianping Xu
    Affiliations
    Department of Pathology, Anhui Chest Hospital, 397 Jixi Road, Hefei 230022, China

    Anhui Medical University Clinical College of Chest, 397 Jixi Road, Hefei 230022, China
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  • Qingming Shi
    Correspondence
    Corresponding author at: Department of Oncology, Anhui Chest Hospital, Anhui Medical University Clinical College of Chest, 397 Jixi Road, Hefei 230022, China,
    Affiliations
    Department of Oncology, Anhui Chest Hospital, 397 Jixi Road, Hefei 230022, China

    Anhui Medical University Clinical College of Chest, 397 Jixi Road, Hefei 230022, China
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Open AccessPublished:November 29, 2022DOI:https://doi.org/10.1016/j.cancergen.2022.11.005

      Highlights

      • Concomitant oncogene mutations and V3 variant indicate shorter PFS.
      • Baseline brain metastasis is an independent factor for overall survival.
      • TP53 mutations are the most common concomitant mutations.
      • The presence of TP53 mutations was associated with shorter PFS among patients who received ALK-TKI.

      Abstract

      Gene complexity affects the clinical outcomes of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). Here, we reviewed the medical records of patients with NSCLC between September 2015 and December 2020 in a single institution. We examined the clinical and genomic predictors of these outcomes using multivariate Cox proportional hazards analysis. Overall, 105 patients with ALK-rearranged NSCLC were included. Echinoderm microtubule-associated protein-like 4 (EML4) was the predominant fusion partner (96.2%). Five patients (4.8%) had non-EML4 fusion partners; three had novel partners. EML4::ALK variant 3 (36.5%) was predominant. One patient had the following three subtypes: E13::A20, E6ins33::A20, and E20::A20. Median progression-free survival (PFS), but not overall survival (OS), was significantly different between patients with variants 3 and 1. TP53 was the most common concomitant mutation (21.4%). The presence of TP53 mutations was associated with shorter PFS among patients who received ALK-TKI. Patients with concomitant oncogene mutations presented significantly shorter OS and PFS than those with only ALK rearrangement. In a multivariate Cox regression model, concomitant oncogene mutations and variant 3 carrier status were prognostic factors for PFS, whereas baseline brain metastasis was a prognostic factor for OS.

      Keywords

      Introduction

      Anaplastic lymphoma kinase (ALK) rearrangement is a unique molecular subtype of non-small cell lung cancer (NSCLC) that was first discovered in 2007 [
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      ]. ALK tyrosine kinase inhibitors (TKIs) are associated with a substantial improvement in patient survival and have been used as the first-line treatment [
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      ]. Some studies have emphasized the heterogenous molecular mechanisms underlying ALK, such as non-canonical fusion partners, different breakpoints in exons, and concomitant mutations.
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      EML4 and ALK are oppositely oriented genes located on the short arm of chromosome 2. A reciprocal inversion caused by paracentric inversion of these two genes leads to the formation of a transforming fusion gene, EML4::ALK. The fusion breakpoint in ALK is exon 20, which encodes the entire tyrosine kinase domain of the protein. Exon 2 of EML4 encodes the entire coiled-coil domain, which mediates the constitutive dimerization and activation of EML4::ALK. Different breakpoints within EML4 result in multiple variants of the EML4::ALK fusion gene, which have been named based on the chronological order of their discovery [
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      ]. The most common variants are EML4::ALK v1 (E13::A20), EML4::ALK v3a/b (E6::A20), EML4::ALK v2 (E20::A20), and EML4::ALK v5′ (E18::A20) [
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      Molecular and clinical analysis of Chinese patients with anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer.
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      Going beneath the tip of the iceberg. Identifying and understanding EML4-ALK variants and TP53 mutations to optimize treatment of ALK fusion positive (ALK+) NSCLC.
      ]. A schematic representation representing different EML4::ALK rearrangements is shown in Supplementary Figure 1. In vitro studies have suggested that variants differ in their protein stability and sensitivity to ALK-TKIs [
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      ], whereas other studies have not [
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      ].
      EML4::ALK was defined as being generally mutually exclusive to other oncogenic drivers, such as epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene (KRAS) [
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      ]. However, retrospective studies and case reports have shown that EML4::ALK can co-occur with oncogenes or tumor suppressor genes [
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      ,
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      • Jiang G.
      • Long H.
      • et al.
      Prevalence and Clinical Impact of Concomitant Mutations in Anaplastic Lymphoma Kinase Rearrangement Advanced Non-small-Cell Lung Cancer (Guangdong Association of Thoracic Oncology Study 1055).
      ,
      • Zhuang X.
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      • Chen X.
      • Ren S.
      • et al.
      Clinical features and therapeutic options in non-small cell lung cancer patients with concomitant mutations of EGFR, ALK, ROS1, KRAS or BRAF.
      ]. Yang et al. [
      • Yang J.J.
      • Zhang X.C.
      • Su J.
      • Xu C.R.
      • Zhou Q.
      • Tian H.X.
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      Lung cancers with concomitant EGFR mutations and ALK rearrangements: diverse responses to EGFR-TKI and crizotinib in relation to diverse receptors phosphorylation.
      ] reported that among treatment-naïve patients, the frequency of concomitant EGFR mutations and ALK rearrangements was 1.3% (13/ 977). Zhuang et al. [
      • Zhuang X.
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      Clinical features and therapeutic options in non-small cell lung cancer patients with concomitant mutations of EGFR, ALK, ROS1, KRAS or BRAF.
      ] found that among 227 treatment-naïve patients harboring an ALK rearrangement, 20 (8.8%) carried an additional alteration. The occurrence of concomitant mutations may cause resistance to TKIs and may be considered as negative prognostic factors [
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      ,
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      ].
      Several studies have focused on only one or two of these aspects. It is unclear which factor is the most important prognostic indicator and what types of gene rearrangements require more intense treatment. To address these questions comprehensively, in this study, we aimed to describe the characteristics and gene complexity of Chinese patients diagnosed with ALK-rearranged NSCLC, compare the effects of different gene variants on survival outcomes, and identify factors associated with real-world mortality risk.

      Materials and methods

      Study population

      In this retrospective analysis, we reviewed the medical records of 3194 patients diagnosed with NSCLC between September 2015 and December 2020. Briefly, the inclusion criteria were as follows: a) histologically confirmed NSCLC; b) age >18 years; c) positive results on immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), amplification refractory mutation system polymerase chain reaction, or NGS; and d) OS >3 months after diagnosis. All patients included in the study had a complete clinical characteristic record and tissue samples available for histologic review. This study was approved by the Institutional Review Board of Anhui Chest Hospital (approval number: K2021–009). Owing to the study's retrospective nature, the need for written informed consent from the patients was waived.

      Genetic analysis

      Most genomic profiling cases were analyzed at the Pathology Department of Anhui Chest Hospital. Fifty-six patients were diagnosed based on NGS findings. DNA was extracted using a TIANamp FFPE DNA Kit (TIANGEN, Beijing, China). A total of 400 ng of DNA per sample was used for the preparation of a DNA library. The concentration of the DNA library was measured using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). RNA was extracted using the RecoverAllTM Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific). The quality of the RNA obtained was assessed using Qubit RNA HS Assay Kits (Thermo Fisher Scientific). Total RNA (100 ng) was reverse transcribed using a random primer mix. Synthesized cDNA was end-repaired and then used to generate sequencing libraries according to the manufacturer's instructions. The sequencing library was generated using the Novogene library construction kit (Tianjin Novogene Bioinformatics Technology Co., Ltd., Tianjin, China) according to the manufacturer's instructions. This kit uses multiplex PCR capture and semiconductor sequencing technologies to purify DNA and RNA from formalin-fixed paraffin-embedded (FFPE) sections. The DNA and RNA were purified, then the DNA and cDNA fragments of the target region were then enriched using the multiplex PCR technique, and the enriched libraries were quantified and quality controlled. The quantified libraries were then sequenced using a gene sequencer (DA8600; Zhongshan University Daan Gene Co., Ltd., Registration No.: 20,143,401,961) to obtain the DNA and RNA sequences of the target regions, which were compared against the human genome database using the supporting software to determine whether mutations or fusions have occurred. Samples of plasma and pleural effusions were sequenced on the Illumina NovaSeq platform (Illumina Inc., San Diego, CA, USA). The 23 genes panel covered hotspot mutations in 19 genes (EGFR, PIK3CA, HER2, KRAS, NRAS, HRAS, BRAF, AKT1, FGFR1, FGFR2, GNA11, GNAQ, KIT, DDR2, PDGFRA, MAP2K1, PTEN, SMO, and TSC1) and fusions in four genes (ALK, MET, RET, and ROS1). The average sequencing depth of the DNA library was >2000X, and the number of RNA library sequences was >60,000. The mutation frequency thresholds for point mutations (single nucleotide variants [SNVs]) and insertion/deletion (InDel) mutations were 0.7% and 1%, respectively. If the number of sequences supported by a fusion type was ≥200, the fusion was considered positive; otherwise, it was considered negative. NGS-detected samples included FFPE tumor tissues (n = 44), malignant pleural effusions (n = 2), or plasma (n = 10).
      Forty-four patients were diagnosed based on findings from the amplification refractory mutation system (ARMS)-PCR. The total RNA was extracted according to a standard protocol (AmoyDx RNA Kit; Amoy Diagnostics Co., Xiamen, China). The quality and quantity of the extracted RNA were measured using an FLx800 spectrophotometer (BioTek Instruments, Inc.). Ten lung cancer-related genes, namely, ALK fusion gene, EGFR, PIK3CA, HER2, RET, MET, ROS1, KRAS, NRAS, and BRAF, were detected using ARMS-PCR with the ADx Gene Detection Kit (Amoy Diagnostics Co., Xiamen, China). Thirty-eight patients were diagnosed using the ten-gene panel, whereas six patients were tested only for ALK fusion gene, EGFR, and ROS1 using ARMS-PCR. PCR-detected samples included FFPE tumor tissues (n = 41), malignant pleural effusions (n = 1), or plasma (n = 2).
      Eighty-three patients were confirmed ALK-positive shortly after the diagnosis of NSCLC. Twenty-two were tested for ALK after failure on operation, chemo/radiotherapy, or immunotherapy. The median length of time from NSCLC diagnosis and identification of ALK fusion was 0.3 months (range, 0.1–80 months). Patients diagnosed using IHC or FISH have not been included in the survival analysis; therefore, the details of these methods are omitted. The study population is shown in Fig. 1.
      Fig. 1
      Fig. 1Flowchart showing the selection of the study participants. ALK, anaplastic lymphoma kinase; ALK+, ALK-positive; NSCLC, non-small cell lung cancer; PFS, progression-free survival; OS, overall survival; NGS, next-generation sequencing.

      Statistical analysis

      Progression-free survival (PFS) was calculated from the date of treatment initiation to disease progression or death. Patients who were alive without disease progression at the time of analysis were censored at their last follow-up. Overall survival (OS) was defined as the time from the first diagnosis to death due to any cause. Patients who were alive at the cut-off date were censored. Kaplan–Meier analysis was used to estimate survival outcomes, and the log-rank test was used to determine differences in survival curves between groups. Multivariate Cox proportional hazards models were used to evaluate predictors of PFS and OS. The models were adjusted for the following covariates: age, sex, smoking status, baseline brain metastasis, disease stage, V3 subtype, TP53 mutation status, and concomitant oncogene status. Data were analyzed using GraphPad Prism software (version 9.0.0) and R software (version 4.1.2). Statistical significance was assumed at a two-sided P-value of <0.05.

      Results

      Clinicopathological characteristics

      Among the 3194 patients diagnosed with NSCLC, 105 patients were ALK-positive. Only one patient was tested using two methods (IHC and PCR), both yielding positive results. The demographic and clinico-pathological characteristics of the 105 patients (median age, 56 years; range, 30–84 years) are shown in Table 1. More than half of the population were males (51%) and had never smoked (80%). Almost all patients were diagnosed with adenocarcinoma (99%), except one patient with squamous carcinoma. The predominant fusion partner of ALK was EML4 (96.2%). Non-EML4 fusion partners, including kinesin family member 5B (KIF5B), thyroid peroxidase (TPO), AAA+ domain-containing protein 2B (ATAD2B), latent transforming growth factor beta-binding protein 1 (LTBP1), and microtubule-associated protein RP/EB family member 3 (MAPRE3), were found in five patients (4.8%). Among the 52 NGS-tested patients with clear variant information, two patients harbored double-fusion variants (EML4::ALK and LTBP1::ALK; MAPRE3::ALK and ALK::IR). The relative frequencies of EML4::ALK (E13::A20) and LTBP1::ALK (L32::A20) were similar (0.38% vs. 0.33%); the same was observed for those of MAPRE3::ALK and ALK::IR (1.86% vs. 1.81%).
      Table 1Clinico-pathological characteristics of patients.
      CharacteristicsALK-positive patients (N = 105)
      Sex, n (%)
      M54
      F51
      Age, mean (range)
      M55 (31–84)
      F57 (30–75)
      Smoking status, n (%)
      Smokers (former and current)21 (20)
      Non-smokers84 (80)
      Histology, n (%)
      Adenocarcinoma104 (99)
      Squamous carcinoma1 (1)
      Stage at diagnosis, n (%)
      IA10 (9)
      IIB4 (4)
      IIIA10 (9)
      IIIB10 (9)
      IIIC2 (2)
      IVA35 (33)
      IVB33 (31)
      Missing data1 (1)
      Testing method, n (%)
      IHC5(4)
      FISH1 (1)
      ARMS-PCR44 (42)
      NGS56 (53)
      Baseline brain metastasis, n (%)
      Yes15 (14)
      No87 (83)
      Unknown3 (3)
      Treatment, n (%)
      Surgery28 (27)
      ALK-TKI96(91)
      Chemo/radiotherapy58 (55)
      Immunotherapy4 (4)
      ALK, anaplastic lymphoma kinase; TKI, tyrosine kinase inhibitor; NGS, next-generation sequencing; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; ARMS-PCR, amplification refractory mutation system-polymerase chain reaction; M, male; F, female.

      Prevalence and clinical effect of concomitant mutations

      Among 94 patients diagnosed using the ten-gene panel ARMS-PCR or NGS, the co-existence of EGFR and ALK variants was found in three patients (3.2%). KRAS was detected in two patients (2.1%). Patient No. 19 harbored ATAD2B::ALK and EGFR exon 21 L858R. Patient No. 35 harbored EML4::ALK(E6a::A20) and EGFR exon 19 delinsP. Patient No. 65 harbored EML4::ALK(E18::A19), EGFR exon 19 p.L747fs, KRAS exon 2 G12C, and TP53 exon 8 p.R283fs. Patient No. 80 harbored EML4::ALK(E6a::A20), KRAS exon 2 G12C, KRAS exon 2 G13C, and TP53 exon 5 p.H178fs. Other oncogene mutations, such as MET, RET, BRAF, DDR2, and PIK3CA, were individually detected in one patient (1.1%). ALK point mutations were found in three patients (3.3%). Two ALK resistance point mutations (p.L1196Q, p.I1171S) were found after treatment with second-generation ALK-TKIs. One patient harbored the EML4::ALK variant 1 and ALK exon3 p.R291C, received crizotinib as first‐line treatment, and achieved 12 months PFS.
      The median follow-up duration for all patients was 23.3 months. We divided the patients with advanced NSCLC into two groups: those with ALK rearrangement only (n = 65) and those with ALK rearrangement and concomitant oncogene mutations regardless of the TP53 status (n = 8). The characteristics of the two groups were similar. Age (P = 0.183), baseline brain metastasis (P = 0.137), stage (P = 0.311), sex (P = 0.148), and smoking history (P = 0.641) parameters were not statistically different from one another. In patients with concomitant oncogene mutations, the median PFS was eight months (95% confidence interval [CI] 2.5–13.5), while in those with ALK rearrangement only, the PFS was not reached (P = 0.0000) (Fig. 2G). Patients with oncogene mutations presented poorer OS than those without oncogene mutations (P = 0.0219). However, the median OS was not reached in both groups (Fig. 2H).
      Fig. 2
      Fig. 2Kaplan–Meier curves of progression-free survival (PFS) and overall survival (OS). Comparison of PFS between variant 1 and variant 3 (A), anaplastic lymphoma kinase (ALK) with TP53 mutations and ALK rearrangement only (C), “long” variants and “short” variants (E), and ALK rearrangement only and ALK with concomitant oncogenes (G). Comparison of OS between variants 1 and 3 (B), ALK with TP53 mutations and ALK rearrangement only (D), “long” variants and “short” variants (F), and ALK rearrangement only and ALK with concomitant oncogenes (H).

      Frequency and distribution of ALK fusion variants

      Among 56 patients diagnosed using NGS, the most frequent concomitant mutation was TP53 (n = 12, 21.4%). Among patients who received ALK-TKI, those carried TP53 mutations (n = 12) presented poorer PFS than those with ALK rearrangement only (n = 36) (15 months, 95% CI 7.5–22.5 months vs. not reached, P = 0.0046) (Fig. 2C). Patients with TP53 mutations presented poorer OS than those with ALK rearrangement only, although not significantly (P = 0.2156) (Fig. 2D).
      In NGS-tested patients, the variant in 52 patients was definite. The most common variant was variant 3 (E6::A20, 36.5%), followed by variant 1 (E13::A20, 34.6%), variant 2 (E20::A20, 11.5%), and variant 5′ (E18::A20, 3.8%). Six patients had variant 3a without the co-existence of variant 3b. One patient co-harbored three subtypes (E13::A20, E6ins33::A20, and E20::A20). A non-canonical breakpoint at exon 19 was detected in two patients (E13::A19 and E18::A19). The frequency of each ALK variant is listed in Table 2.
      Table 2Frequency and proportion of ALK variants in 52 rearrangements.
      ALK fusion typeNumberPercentage
      Variant 1 (E13∷A20)1834.6%
      Variant 2 (E20∷A20)611.5%
      Variant 3 (E6a/b∷A20)1325.0%
      Variant 3a (E6a∷A20)611.5%
      Variant 5′ (E18∷A20)23.8%
      E18∷A1911.9%
      E13∷A1911.9%
      E13∷A20,E6INS33∷A20,E20∷A2011.9%
      Non EML4 partner59.6%
      ALK, anaplastic lymphoma kinase.
      In the variant 1 group, the median PFS was significantly longer than that in the variant 3 group (34 months, 95% CI 17.7–50.3 months vs. 16 months, 95% CI 10.9–21 months) (P = 0.0252) (Fig. 2A). However, the median OS did not differ significantly between the variant 1 group and variant 3 group (P = 0.2914) (Fig. 2B).
      In the “long” variant group, which includes V1, V2, V5′, E18::A19, and E13::A19, the median PFS was significantly longer than that of the “short” variant group, including only V3 (34 months, 95% CI 10.7–57.3 months vs. 16 months, 95% CI 10.9–21 months) (P = 0.0266) (Fig. 2E). The median OS did not differ significantly between the groups (P = 0.1437) (Fig. 2F).

      Univariate and multivariate Cox proportional hazard models

      We performed univariate and multivariate Cox proportional hazard models on 52 patients diagnosed using NGS (Fig. 3). Eight patients had early stage (I-IIIA) NSCLC and received surgery with or without chemotherapy. Forty-three patients had advanced stage (IIIB-IV) NSCLC and received TKI as first-line treatment. Three patients who harbored both EGFR and EML4::ALK received EGFR-TKI as first-line treatment, which was switched with crizotinib treatment after disease progression. Patient No. 35 and No. 80 showed primary resistance to ALK-TKI. Of 26 advanced-stage patients with disease progression, 14 (53.8%) were re-tested. Twelve patients switched to treatment with another ALK-TKI, whereas fourteen patients switched to a combined treatment of chemo/radiation with ALK-TKI.
      Fig. 3
      Fig. 3Genomic landscape of patients with anaplastic lymphoma kinase (ALK) fusion (n = 52). The top part represents the number of mutations detected in each sample, whereas the bottom part represents clinical information, including age, sex, variants, and censor.
      Based on both the results of previous studies and our own findings on survival analysis, we selected particular variables for further analysis [
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      • et al.
      Impact of TP53 mutation status on systemic treatment outcome in ALK-rearranged non-small-cell lung cancer.
      ]. In a univariate analysis, which included age, sex, smoking status, baseline brain metastasis, disease stage, V3 variant status, oncogene mutations, and TP53 mutation status, we found that baseline brain metastasis (P = 0.045), V3 variant carrier status (P = 0.030), and oncogene mutations (P = 0.006) were significantly associated with PFS. The TP53 mutations (P = 0.174), age (P = 0.927), sex (P = 0.744), disease stage (P = 0.243), and smoking status (P = 0.158) were not statistically associated with PFS. In the multivariate Cox regression model, both concomitant oncogene mutations (hazard ratio [HR] 5.439 [95% CI 1.441–19.862]; P = 0.012) and V3 variant carrier status (HR 3.396 [95% CI 1.186–9.725]; P = 0.023) remained significant risk factors for poor PFS (Table 3). However, the effect of baseline brain metastasis (HR 2.269 [95% CI 0.573–8.977]; P = 0.243) was not significant in the multivariate analysis.
      Table 3Results of univariate and multivariate survival analyses of lung adenocarcinoma patients with ALK fusion.
      VariableCategoryPFS analysisOS analysis
      UnivariateMultivariateUnivariateMultivariate
      HR95% CIP valueHR95% CIP valueHR95% CIP valueHR95% CIP value
      Age≥65/<651.0530.348–3.1850.9270.4760.129–1.7520.2640.8910.195–4.0780.8820.5700.110–2.9660.504
      SexMale/female1.1630.469–2.8840.7442.2100.569–8.5760.2521.3500.428–4.2570.6092.2630.378–13.5580.371
      SmokingYes/no2.1760.739–6.4130.1585.1230.997–26.3280.0501.3640.365–5.0910.6441.6230.231–11.4240.626
      StageI II/III IV0.0380.000–9.2390.2430.0000.000–7.030E+2450.9650.0390.000–47.0990.3710.000NA0.979
      BBMYes/no3.1921.028–9.9110.0452.2690.573–8.9770.2437.0922.221–22.6450.0018.0971.830–35.8190.006
      V3 variantYes/no3.0251.114–8.2080.0303.3961.186–9.7250.0232.2780.642–8.0740.2023.6650.949–14.1490.059
      TP53 mutationYes/no2.0680.726–5.8940.1741.3550.336–5.4700.6701.6140.428–6.0940.4801.4430.329–6.3330.627
      OncogenesYes/no3.7861.479–9.6930.0065.4391.441–19.8620.0123.1550.947–10.5060.0612.9930.660–13.5740.155
      ALK, anaplastic lymphoma kinase; PFS, progression-free survival; OS, overall survival; BBM, baseline brain metastasis; CI, confidence interval; HR, hazard ratio.
      In the univariate analysis, we found that baseline brain metastasis (P = 0.001) was significantly associated with worse OS. The presence of oncogene mutations was also associated with OS, although not significantly (P = 0.061). In the multivariate Cox regression model, baseline brain metastasis was the only significant independent risk factor for poor OS (P = 0.006). The V3 subtype tended to be associated with OS, but not significantly (P = 0.059).

      Discussion

      In this study, 105 patients with ALK fusions were retrospectively evaluated. Among them, 56 (53%) patients had rearrangement of ALK as confirmed using NGS. Moreover, we discovered three novel ALK fusion partners and identified concurrent mutations that could affect survival.
      Consistent with the finding of a previous study [
      • Ou S.I.
      • Zhu V.W.
      • Nagasaka M.
      Catalog of 5′ Fusion Partners in ALK-positive NSCLC Circa 2020.
      ], EML4 was the most common fusion partner (96.2%), but we also reported some novel and dual fusion partners. One patient harboring TPO::ALK (T7::A20) received chemoradiotherapy because of the first-time negative gene analysis using AMRS-PCR and died within 22 months. Therefore, the sensitivity of this novel variant to ALK inhibitors was not assessed. Two patients with double-fusion variants (EML4::ALK and LTBP1::ALK; MAPRE3::ALK and ALK::IR) responded well to alectinib treatment (PFS of 24 and 13 months, respectively). Our observations are in line with those of a previous study, which reported that intergenic sequence-ALK or the co-existence of fusions did not have a significant effect on the therapeutic benefit of crizotinib treatment [
      • Cai C.
      • Tang Y.
      • Li Y.
      • Chen Y.
      • Tian P.
      • Wang Y.
      • et al.
      Distribution and therapeutic outcomes of intergenic sequence-ALK fusion and coexisting ALK fusions in lung adenocarcinoma patients.
      ].
      Similar to the case presented by Kunimasa et al. [
      • Kunimasa K.
      • Hirotsu Y.
      • Kukita Y.
      • Ueda Y.
      • Sato Y.
      • Kimura M.
      • et al.
      EML4-ALK fusion variant.3 and co-occurrent PIK3CA E542K mutation exhibiting primary resistance to three generations of ALK inhibitors.
      ], patient no. 50, who harbored variant 3a and had both PIK3CA p.E542K and TP53 mutations, exhibited primary resistance to crizotinib. Our study confirmed that the co-existence of ALK and other oncogenes is rare, and the concomitant presence of oncogenes affects both PFS and OS. However, because of the rather limited number of patients included in the Cox regression analysis, the difference in OS was close to but did not reach statistical significance (P =0.061).
      In vivo and in vitro studies [
      • Heuckmann J.M.
      • Balke-Want H.
      • Malchers F.
      • Peifer M.
      • Sos M.L.
      • Koker M.
      • et al.
      Differential protein stability and ALK inhibitor sensitivity of EML4-ALK fusion variants.
      ,
      • Woo C.G.
      • Seo S.
      • Kim S.W.
      • Jang S.J.
      • Park K.S.
      • Song J.Y.
      • et al.
      Differential protein stability and clinical responses of EML4-ALK fusion variants to various ALK inhibitors in advanced ALK-rearranged non-small cell lung cancer.
      ,
      • Su Y.
      • Long X.
      • Song Y.
      • Chen P.
      • Li S.
      • Yang H.
      • et al.
      Distribution of ALK Fusion Variants and Correlation with Clinical Outcomes in Chinese Patients with Non-Small Cell Lung Cancer Treated with Crizotinib.
      ] have reported that different EML4::ALK fusion variants exhibit different levels of protein stability and sensitivity to ALK inhibitors. EML4 protein contains a tandem atypical beta-propeller domain (TAPE) and an N-terminal coiled-coil trimerization domain. The V3 variant does not contain the TAPE domain, while other variants (V1, V2, V7, etc.) contain parts of the TAPE domain. The truncated TAPE structural domain of EML4 results in an unstable EML4::ALK fusion protein that is more sensitive to ALK-TKIs, making it evident that the lack of the TAPE domain in the V3 variant leads to resistance against ALK-TKIs [
      • Sabir S.R.
      • Yeoh S.
      • Jackson G.
      • Bayliss R.
      EML4-ALK Variants: biological and Molecular Properties, and the Implications for Patients.
      ,
      • Heuckmann J.M.
      • Balke-Want H.
      • Malchers F.
      • Peifer M.
      • Sos M.L.
      • Koker M.
      • et al.
      Differential protein stability and ALK inhibitor sensitivity of EML4-ALK fusion variants.
      ]. In this study, we included patients with advanced NSCLC taking ALK inhibitors and patients with early-stage NSCLC. The same conclusion that variant 3 is associated with a worse prognosis was reached.
      The TP53 mutations are a negative prognostic factor for PFS under most treatment conditions [
      • Kron A.
      • Alidousty C.
      • Scheffler M.
      • Merkelbach-Bruse S.
      • Seidel D.
      • Riedel R.
      • et al.
      Impact of TP53 mutation status on systemic treatment outcome in ALK-rearranged non-small-cell lung cancer.
      ]. However, in our study, poor PFS due to TP53 mutations was observed only in patients receiving ALK-TKIs. In the univariate analysis, when patients undergoing surgery were considered, the difference was no longer statistically significant. Tao et al. reported similar findings by analyzing resected NSCLC [
      • Tao H.
      • Shi L.
      • Zhou A.
      • Li H.
      • Gai F.
      • Huang Z.
      • et al.
      Distribution of EML4-ALK fusion variants and clinical outcomes in patients with resected non-small cell lung cancer.
      ]. Further studies are needed to determine the prognostic value of TP53 mutations in early-stage ALK-rearranged NSCLC.
      Our study had some limitations. First, it was a retrospective, single-institution study. Follow-up intervals and treatment protocols were not uniform, which might have impacted the results. The experimental group included a small number of cases due to the scarcity of the concomitant mutations, which might have led to false-positive results. Second, the difference in OS rates between the groups did not reach statistical significance, probably owing to a relatively short follow-up time. Lastly, owing to the limited number of patients diagnosed based on NGS findings and the rather small NGS panel, a definite conclusion on which aspect of comprehensive mutation profiling concerns the most cannot be drawn. As a result, a prospective study with a larger sample size is needed to clarify these effects.
      In conclusion, ALK patients with NSCLC who carried variant V3 and concomitant oncogenic mutations had a shorter PFS. The presence of TP53 mutations was associated with a shorter PFS among patients who received ALK-TKI. Baseline brain metastasis was the only significant prognostic factor for OS. The identification of novel partners of co-existing variants and other oncogenes revealed the complexity of ALK rearrangement. A better understanding of ALK gene complexity will help optimize therapeutic strategies. Larger scale studies are needed to generate more data on factors that affect prognosis.

      Author contributions

      Maojing Guan: Conceptualization, Methodology, Data collection, Software, Writing- Original draft preparation and revision. Jianping Xu: Specimen collection, Software, Suggestion. Qingming Shi: Methodology, Supervision, Revision.

      Funding

      This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

      Data Availability

      • The authors do not have permission to share data.

      Declaration of Competing Interest

      None

      Acknowledgments

      We thank Li Huifang, Dai Ke, and Chen Fei for their contributions to data collection for this research. We would like to thank Editage (www.editage.com) for their writing support on the Abstract.

      Appendix. Supplementary materials

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