Lung cancer is one of the most common cancers and the leading cause of cancer death in the world (1). In the past decades, advances in molecular analysis and the development of targeted therapies have changed the identification of oncogenic drivers, the treatment and the survival for lung cancer. Anaplastic lymphoma kinase (ALK) rearrangement is one of the driver genes which detected in 3–7% of patients with non-small cell lung cancer (NSCLC) (2). For these patients, ALK tyrosine kinase inhibitors (TKIs) have been developed. Crizotinib is a first generation ALK-TKI that shows efficacy in the treatment of ALK rearrangement NSCLC (3). According to clinical studies of crizotinib (PROFILE 1005, PROFILE 1007, PROFILE1014), the major populations were lung adenocarcinoma (ADC) and an objective response rate (ORR) to crizotinib is approximately 60% and its median progression-free survival (PFS) is nearly 10 months (4). However, it is known that many cases ultimately acquired resistance to crizotinib (5). In addition, about 30% ALK-positive patients to the treatment of crizotinib demonstrate primary resistance which early progressive disease (PD) after the first month of treatment (6,7).
The mechanisms of acquired resistance included secondary mutations or copy number gain (CNG) in the ALK kinase domain and up regulation of bypass signaling pathways (8). However, to our knowledge, the mechanisms of primary resistance to ALK inhibitor for these patients remain elusive. And only some primary resistance mechanisms have been hypothesized, such as mutations of the EGFR pathway and BIM polymorphisms (9-11).
According to reports, the tumor suppressor of TP53 gene mutations occur in approximately 25–50% NSCLC patients (12,13). Ma et al. (14) has demonstrated regardless of EGFR and KRAS mutation status, non-disruptive TP53 mutations are independent markers of shorter overall survival (OS) in patients with advanced NSCLC. There is preclinical evidence in the human NSCLC cell lines NCI-H1299 and A549 showed a relationship between TP53 gene mutations and responsiveness to TKIs (15). Canale et al. (16) had reported that TP53 mutations reduce responsiveness to EGFR-TKIs and poor prognosis in EGFR-mutated NSCLC patients.
However, the relationship of TP53 gene status and the efficacy of crizotinib in ALK positive NSCLC patients was unclear. In this study we proposed to evaluate the role of TP53 mutation in ALK rearrangement advanced NSCLC patients that received crizotinib treatment. We analyzed the status of TP53 gene and its association with the effect of crizotinib in Chinese patients with ALK-positive NSCLC.
Eligible patients were required to have pathologically confirmed NSCLC and sufficient tissue for analysis. Clinical and pathologic data prospectively collected for analyses included age at diagnosis, gender, smoking status, and stage according to the new International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society multidisciplinary classification. This study was approved by the ethics committee of Fujian Provincial Cancer Hospital, Fujian Medical University Cancer Hospital, Fuzhou Fujian, China, and a written informed consent was obtained from each participant before the initiation of any study-related procedure.
Targeted next-generation sequencing
A total of 66 ALK positive patients treated with crizotinib. However, 28 specimens were not enough and 38 specimens were evaluated by NGS. For 38 patients, including parts of patients with ALK positive pts treatment before crizotinib, targeted region capture combined NGS was successfully performed in 21 patients. Genomic DNA sequencing libraries were prepared using the protocols recommended by the Illumina TruSeq DNA Library Preparation Kit. For samples close to the minimum input requirement, additional pre-capture PCR cycles were performed to generate sufficient PCR product for hybridization. The libraries were hybridized to custom-designed probes (Integrated DNA Technology) including all exons of 170 genes and selected intron of ALK, RET and ROS1 for the detection of Genomic rearrangements. DNA sequencing was performed on a HiSeq3000 sequencing system (Illumina, San Diego, CA) with 2×75 bp paired-end reads. The reads were aligned to the human genome build GRCh37 using BWA (a Burrows-Wheeler aligner). Somatic single nucleotide variant (sSNV) and indel calls were generated using MuTect and GATK, respectively. Somatic copy number alterations were identified with CONTRA. Genomic rearrangements were identified by the software developed in house analyzing chimeric read pairs.
Patients received crizotinib treatment (250 mg, twice daily) and had clinical data available including general characteristics, treatment efficacy and adverse reactions to treatment. Imaging data were independently reviewed by authors to evaluate their treatment responses according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 PFS was calculated from the date of initiating targeted drugs treatment to radiologic or clinical observation of disease progression.
The response rate among subgroups and survival was described with Kaplan-Meier methodology and the log-rank test was used to compare survival among subgroups. Statistical analysis was performed using SPSS version 19.0 software (IBM, Armonk, NY, USA). All P values were 2-sided, and a P value of <0.05 was considered statistically significant.
From July 2013 to May 2016, a total of 1,720 patients were enrolled in this study. Among them, 187 (10.87%) were identified as ALK-positive and 66 of them received oral crizotinib. Because of the tissue sample insufficient and specimen quality testing, 21 of them was successfully performed by targeted region capture combined NGS. The flow chart of the study design is shown in Figure 1. Clinic-pathological characteristics of patients were reported in Table 1. The majority of patients were male (52.38%, 11/21), less than 65 years old (61.90%, 13/21) and never smokers (80.95%, 17/21). All patients had a diagnosis of ADC (100%, 21/21). All patients received crizotinib (100%, 21/21). NGS detection found many accompanied genes, including mTOR pathway (PIK3CA p.E545K, FBXW7 p.R505L, STK11 p.D194E, PTEN p.R130G, MTOR p.E1799K, MTOR p.P2273L), RAS pathway (NRAS p.G12D), TP53 exon 5 (p.R158L, p.H179R), exon 6 (p.R213Q), exon 7 (p.G245S), exon 8 (p.Y271*, p.V272M, p.R273H, p.C277F) (Figure 2), SMARCA4 (p.R451L, p.E882K, p.R469W), AR (p.E355K), APC (p.P2559L, p.T1910S), CCND1 (p.M82V), CTNNB1 (p.S45P), BRAF (p.D594G), RB1 (p.G449E) and NF1 (p.A2437S).
Out of the 21 patients with successfully targeted region capture combined NGS, 8 (38.10%) patients showed a TP53 mutation: 25.00% (2/8) were on exon 5, 12.50% (1/8) on exon 6, 12.50% (1/8) on exon 7, and 50.00% (4/8) on exon 8 (Table 1). The majority of patients were male (75.00%, 6/8), less than 65 years old (62.50%, 5/8) and never smokers (75.00%, 6/8). There were no significant differences noted with regard to age, sex, smoking history, ECOG PS, histology, number of previous treatments, between patients with and without the TP53 gene (Table 2).
TP53 mutation and response to crizotinib
ORR and disease control rate (DCR) for crizotinib in the entire case series were 61.90% and 71.43%, respectively. TP53 gene wild group was found to be significantly associated with a higher ORR and DCR to crizotinib with respect to TP53 gene mutation group. An ORR of 76.92% and a DCR of 84.62% were observed in patients with TP53 gene wild group, and an ORR of 37.50% and a DCR of 50.00% were observed in patients with TP53 gene mutation group (Table 3).
TP53 mutation and survival
Statistically significant difference was observed in terms of PFS and OS between TP53 gene wild group and mutation group patients (P=0.038, P=0.021, respectively). The PFS in TP53 gene mutation group patients was shorter than that in TP53 gene wild group (3.3 vs. 10.4 months). The OS of TP53 gene mutation group was poorer than TP53 gene wild group (21.4 vs. 34.2 months). PFS and OS of TP53 gene on exon 5 were 9.3, 36.2 months and 1.0, 17.2 months), respectively; PFS and OS of TP53 gene on exon 6 were 7.3 and 34.6 months; PFS and OS of TP53 gene on exon 7 were 13.9 months, more than 21.4 months; PFS and OS of TP53 gene on exon 8 were 5.1 and 25.6 months, 1.0 and 15.3 months, 1.5 and 12.6 months, and 1.0 and 8.4 months, respectively (Table 1, Figure 3).
To our knowledge, our study is the first to explore the relationship between TP53 gene mutation status and the effect of crizotinib in Chinese patients with ALK-positive advanced NSCLC. NSCLC patients with ALK rearrangement are highly sensitive to crizotinib. Nowadays, a series of trial data (phase I–III) involving in ALK positive advanced NSCLC patients demonstrated the efficacy of crizotinib was good and adverse reactions could be tolerated (4,17,18). The data from East Asian patients also showed the ORR to crizotinib was 88% and its median PFS was 11.1 months (19). Unfortunately, some ALK-positive patients have been with shorter PFS after treatment with crizotinib which might be presence primary resistance to crizotinib. Up to now, the mechanisms of acquired resistance to ALK inhibitors can be divided into 2 types: ALK dominant or ALK non-dominant. ALK dominant includes secondary mutations and CNG in the ALK gene; ALK non-dominant includes the activation of bypass tracks, such as EGFR, MET, KIT, KRAS and IGF-1R (8). However, it is unclear that the causes of short PFS in ALK positive patients after crizotinib treatment. Some studies have explored the mechanisms of crizotinib primary resistance. Zhang et al. (9) reported patients with the BIM deletion polymorphism had a significantly shorter PFS (182 vs. 377 days, P=0.008) and lower ORR (44.4% vs. 81.7%, P=0.041) compared with those without in 69 ALK/ROS1 positive patients with crizotinib treatment. Doebele et al. (10) reported that a patient received re-biopsy after 61 days on crizotinib which showed stable disease. This biopsy demonstrated a lack of an ALK gene rearrangement by FISH, but the presence of an EGFR exon 21 mutation by direct sequencing. There is still a lack in the mechanisms reports of primary resistance to crizotinib with ALK-positive NSCLC.
It is reported that TP53 gene mutations occur in approximately 50% of lung cancer patients and are more common in squamous cell lung carcinoma than in lung ADC (12). In the lung ADC cancer, TP53 mutation rates ranges from 25% to 40% (20,21). In addition, some reports showed the prevalence of ALK-rearranged with p53 mutation was about 9.1% (1/11) to 28.6% (2/7) (22,23). In our study, we observed a mutation percentage of 38.1% in 21 ALK positive ADC patients. Therefore, coexisting with TP53 mutations in ALK positive ADC patients is still further to explore in a larger research, especially for comparing ALK rearrangement patients with ALK negative patients. The p53 protein regulates cellular response to a variety of cellular stress signals by inducing cell cycle arrest (24). The normal function disruption of p53 disrupts this cellular response, leading to possible malignant cell transformation. Gene mutations lead to a loss of p53 functions, however non-disruptive mutations could remain some of the p53 protein functional properties (25). Therefore, we analyzed the relationship of efficacy and survival between ALK positive and TP53 gene status with crizotinib therapy.
It is the first time to put forward that patients with TP53 gene mutation can no response to crizotinib. In our study, TP53 gene mutation group was found to be significantly associated with a lower ORR (76.92% vs. 37.50%) and DCR (50.00% vs. 84.62%) to crizotinib with respect to TP53 gene wild group. In addition, significant shorter PFS (3.3 vs. 10.4 months, P=0.038) and OS (21.4 vs. 34.2 months, P=0.021) were observed in patients with TP53 gene mutation group patients than TP53 gene mutation group. In particular, 4 of 8 TP53 mutated patients showed PD within two months of crizotinib therapy. Therefore, we think ALK positive patients with TP53 gene mutation would influence the efficacy of crizotinib treatment. Likewise, in EGFR mutation populations, Canale et al. (16) also demonstrated TP53 mutations were associated with a significantly lower DCR respect to wild patients. However, no statistically significant difference was observed in terms of PFS and OS between two groups. Their most significant result was that TP53 exon 8 mutation was associated with the worst prognosis. Our study also found ALK positive patients with TP53 exon 8 mutation were poorer survival than other mutation types. The TP53 mutant type has been found to be an important factor for gefitinib-induced apoptosis in NSCLC cell lines and reduces gefitinib-induced apoptosis (26). We think that TP53 could have a role as prognostic factor to ALK inhibitors, rather than predictive factor. In the future, we should carry out well-designed prospective multicenter studies to demonstrate the correlation between TP53 gene status and ALK inhibitors including other generations in patients with ALK-positive disease.
We also recognize that there are several limitations to our study. First, due to a small sample, the conclusions should be bias a certain extent. Second, this was a retrospective study, and therefore selection bias was inevitable. Hence, future research will continue to increase the population in the hope of reducing the bias to some extent.
In conclusion, we described the relationship between TP53 gene and the efficacy of crizotinib in ALK rearrangement patients. And the TP53 mutant patients were associated with poor ORR and shorter PFS. However, more work and further studies should be performed to explore the significance of TP53 gene in patients with ALK-positive NSCLC who were treated with ALK inhibitors.
Funding: This study was supported in part by grants from the National Clinical Key Specialty Construction Program (2013); Leading Project Foundation of Science Department of Fujian Province (2016Y0019); Leading Project Foundation of Science Department of Fujian Province (2015Y0011).
Conflicts of Interest: The authors have no conflicts of interest to declare.
Ethical Statement: This study was approved by the ethics committee of Fujian Provincial Cancer Hospital, Fujian Medical University Cancer Hospital, Fuzhou Fujian, China, and a written informed consent was obtained from each participant before the initiation of any study-related procedure.
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