TERT copy gain predicts the outcome of high-dose interferon α-2b therapy in acral melanoma

Introduction

Acral melanoma (AM) is the most predominant melanoma subtype among non-Caucasians,^1–3 particularly among Chinese, accounting for almost 50% of all cases of melanomas.⁴ Compared with common cutaneous melanoma, AM has a poorer prognosis^2,4,5 and shows a markedly different genomic landscape, with a far lower mutation burden dominated by larger-scale genomic rearrangements.^5–7 Vemurafenib and imatinib are more effective against advanced AM harboring BRAF and C-KIT mutations than traditional chemotherapies.^8–11 However, the mutation frequencies of BRAF and C-KIT in AM are only approximately 16%^12,13 and 12%,^13–15 respectively; therefore, a validated targeted therapy is unavailable for the majority of patients. Meanwhile, adjuvant HD-IFN is a year-long treatment modality associated with improved relapse-free survival (RFS)¹⁶ and is currently the primary treatment regimen for stage II/III AM after surgery.¹⁷ Despite the optimization of this adjuvant treatment, most patients will still develop distant metastases and eventually die. Moreover, biomarkers for identifying patients who would derive significant prognostic benefit from HD-IFN treatment have yet to be determined. To sum up, developing more effective therapeutic strategies and validating new candidate biomarkers for predicting treatment response are urgently needed.

Human telomerase contains two essential components, a functional human telomerase RNA (hTR, also named TERC) and a human telomerase reverse transcriptase (hTERT).¹⁸ TERT is located at 5p15.33, which regulates telomeric length. TERT upregulation plays a critical role in oncogenesis.¹⁹ TERT promoter mutations have been reported in up to 50% of all cases of cutaneous melanoma,^20,21 but only in 0%–7% of AM.^22–24 In addition, gene amplification is the most frequent mechanism for TERT activation and significantly reduced overall survival (OS) in AM.²⁵ TERT gene amplification has been evaluated via fluorescence in situ hybridization in a series of AM patients, and such amplification was detected in 6 of 34 cases (17.6%).²⁶ Another previous study detected TERT gene amplification in 5 of 16 (31.2%) patients with AM.²⁷ However, a small sample size was one of the limitations of these studies.

In the present study, we examined TERT copy gain in 573 melanoma samples. To the best of our knowledge, this is by far the largest study of patients with AM focusing on the copy gain of TERT, and we determined the association between TERT copy gain and the clinicopathological features of AM. Our study demonstrated prognostic value of TERT copy gain for different patient subgroups. These findings may also help to discover new potential molecular indicators for patients who may benefit from HD-IFN treatment.

Patients and methods

Study population

A total of 573 patients with AM who were treated at Peking University Cancer Hospital & Institute between 2008 and 2017 were included in this study (Table 1). These samples were evaluated to confirm the diagnosis of melanoma via H&E staining and immunohistochemistry for melanoma markers (S-100, HMB-45, or MART-1). Clinical data, including age, sex, tumor-node-metastases (TNM) stage, tumor thickness (Breslow), ulceration, and survival period (follow-up persisted until patient was lost to follow-up or death), were collected. Patients who received radiation treatment were excluded from our study. This investigation was performed after approval by the Ethics Committee of Peking University and was conducted according to the Declaration of Helsinki Principles. Written informed consent was obtained from each patient.

Table 1 Correlation of TERT copy number to clinical features of AM Note: aThe P-value of 573 patients was analyzed by the χ2 tests. Abbreviations: AM, acral melanoma; CNV, copy number variation.

Quantification of DNA using QuantiGene Plex 2.0 assay in FFPE tissues

Genomic DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) specimens using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). The QuantiGene Plex 2.0 reagent system (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect the copy number of TERT according to the manufacturer’s recommended protocols. Briefly, each patient’s DNA sample was mixed with an oligonucleotide probe. The mixture was then added to a 96-well plate, and the reagent system captured the target DNA and control DNA. Afterward, working samples were incubated overnight at 54°C. After washing unbound material with 200 μL of wash buffer, sequential hybridization of DNA amplifier molecules, preamplifier hybridization, amplifier hybridization, and label probe hybridization were then performed. The plate was then prepared for analysis by a professional analyzer. After dividing by the sample value, the gene copy numbers of the patients were calculated as follows: a copy number of 2 or less was considered no gain, while a copy number >2 was considered a copy number gain of TERT. In these amplified populations (copy number >2), we further classified the copy numbers as median gain (>2 but <4) or high copy number gain (4 or greater).

Statistical analysis

All statistical analyses were performed using SPSS 21.0 software (IBM Corporation, Armonk, NY, USA). OS was calculated and plotted using the Kaplan–Meier method. Differences between groups were compared via log-rank test. A 2-sided χ² test was used to examine the correlations between gains of TERT gene and clinicopathological parameters. The Cox proportional hazard regression model was used for multivariate analysis.

Results

Copy gain of TERT in patients with acral melanoma

We used pretreatment biopsy tissue samples from 573 patients with AM. The tumor specimens were derived from primary lesions. All the tumor sections were collected at the time of diagnosis before initiating the treatments. The patients’ characteristics are shown in Table 1.

To study the copy number variation of TERT, we performed QuantiGene Plex 2.0 Assays (Thermo Fisher Scientific) for AM specimens. We found that 257 of the 573 patients (44.9%) displayed TERT copy gain (ie, a gain >2-fold change, Figure 1A). Of them, 81 patients (31.5%) had a high copy number (ie, a gain >4-fold change) (Figure 1B). Patients with ulceration showed a significantly higher copy gain rate of TERT than those without ulceration (P=0.028). Patients with tumors thicker than 4 mm also had a higher copy gain rate of TERT than those with tumors <4 mm thick (P=0.048). No relationship was noted between TERT copy gain and sex, age, clinical stage, or metastasis site (Table 1).

Figure 1 TERT copy gain in 573 AM specimens. Notes: (A) A DNA copy number of >2 copies was determined as gene copy gain, and TERT copy gain was observed in 44.9% (257/573) of the AM specimens (above the X-axis). (B) The scatter plot showed different degrees of copy gain of TERT as follows: no copy gain is defined as a copy number of 2 or less, while a copy gain is defined as a copy number >2. In these amplified populations, we further defined <2 copy number and <4 as median copy number gain. A copy number <4 is defined as high copy number gain. Abbreviation: AM, acral melanoma.

Relationship between TERT copy gain and representative gene of melanoma

The rates of BRAF wild-type and mutant genes among the 257 patients with TERT copy gain were 88.6% and 11.4%, respectively, while they were 84.4% and 15.6%, respectively, among patients with no TERT copy gain. No correlation between BRAF mutation and TERT copy gain was observed (P=0.143). In addition, we also analyzed the relationship between 3 other common mutations (KIT, PDGFRA, and NRAS) and TERT copy gain in 573 patients with AM. No correlation was found between TERT copy gain and common gene mutation (Table 2).

Table 2 Analysis of the relationship of TERT copy number status with other gene mutations in AM Note: aThe P-value was analyzed by the χ2 tests. Abbreviations: AM, acral melanoma; CNV, copy number variation; WT, wild type.

Influence of TERT copy gain on OS of patients

Among the 573 patients with AM, 278 (70.4%) received HD-IFN regimen. Meanwhile, 117 patients underwent different treatment modalities, namely, dacarbazine (PUDEPHARM, Shanxi Datong, China) (n=62 patients), temozolomide (Temodal, Orion Corporation Orion Pharma, Espoo, Finland) (n=14 patients), and other chemotherapeutic drugs or targeted therapy (n=41 patients). Out of the 41 patients, 11 and 1 were treated with imatinib (Glivec, Novartis, Beijing, China) and vemurafenib (Zelbraf, Roche, Basel, Switzerland), respectively.

To evaluate the prognostic value of TERT for predicting the OS of patients, Kaplan–Meier survival analysis was performed for all the 573 patients. The median OS of patients with and without TERT gain was 73.7 months (95% CI: 69.9–78.5 months) and 62.7 months (95% CI: 59.5–67.9 months), respectively. The no TERT gain group did not show improved OS relative to the whole population (P=0.890, Figure 2A). Among the 278 patients treated with IFN α-2b, those with no TERT gain did not demonstrate better OS than those with TERT copy gain (P=0.389). Among the patients treated with IFN, we analyzed whether TERT copy gain affected survival in those with stage II and stage III AM. Our results showed that OS was not significantly different between patients with and without TERT gain (n=221, P=0.228, Figure 2B).

Figure 2 Influence of the TERT copy gain on patients’ OS. Notes: (A) Kaplan–Meier survival analysis was performed in all the 573 patients. The copy gain of TERT did not correlate with the OS of all patients (P=0.890). (B) Among the 573 patients with AM, 278 received a high-dose IFN regimen. A total of 221 patients had stage II/III disease. Kaplan–Meier survival analysis showed no significant difference in OS between the TERT copy gain group and no copy gain group (P=0.228). Abbreviations: AM, acral melanoma; IFN, interferon; OS, overall survival.

Influence of TERT copy gain on patient response to high-dose interferon treatment and RFS

IFN α-2b is the most commonly utilized adjuvant regimen for patients with AM in China. Thus, we analyzed whether TERT copy gain can affect the efficacy of IFN therapy. The median RFS of the patients with and without TERT copy gain was 18.2 months (95% CI: 10.6–25.3 months) and 29.1 months (95% CI: 21.7–36.2 months), respectively. We used Kaplan–Meier survival analysis to evaluate the predictive value of TERT and RFS for postoperative adjuvant therapy, and the results demonstrated significant correlation between TERT copy gain and RFS in cases treated with IFN α-2b. TERT copy gain significantly predicted worse RFS (P=0.008) (Figure 3A). Then, we further analyzed the efficacy of IFN therapy in patients with stage II and stage III disease. Among these patients, the median RFS was significantly different between patients with and without TERT gain (n=221, P=0.034, Figure 3B). Collectively, these results indicate that TERT copy gain in patients with AM can predict worse RFS among patients treated with HD-IFN, particularly for those with stage II and III disease.

Figure 3 Influence of TERT copy gain on RFS in patients with AM treated with HD-IFN. Notes: (A) TERT copy gain is correlated with short RFS in patients with AM treated with HD-IFN. The curves demonstrated significant correlation between TERT copy gain and RFS in cases treated with IFN α-2b (P=0.008). (B) Among the 221 patients with stage II/III, TERT copy gain is correlated with short RFS (P=0.034). Abbreviations: AM, acral melanoma; HD-IFN, high-dose interferon; RFS, relapse-free survival.

The results of multivariate Cox regression assays showed that TERT copy gain (hazard ratio =1.50; P=0.019) and TNM stage (hazard ratio =2.92; P<0.001) were independent prognostic factors of RFS for patients with AM undergoing HD-IFN therapy (Table 3).

Table 3 Cox regression analysis of the correlation between TERT copy number and clinicopathologic factors with RFS Note: aThe P-value was analyzed by the χ2 tests. Abbreviations: HR, hazard ratio; Mut, mutation; RFS, relapse-free survival; TNM, tumor-node-metastasis; WT, wild type.

Discussion

AM is distinguishable from other melanoma subtypes by its unique clinical, epidemiological, and genetic features.^1,2,7,28 AM is the predominant melanoma subtype in non-Caucasians and has a notably worse prognosis than common cutaneous melanoma.^4,29 In recent years, AM was found to have a markedly different genomic landscape from other subtypes of melanoma, with a far lower mutation burden dominated by large-scale structural variants⁷ and a higher frequency of focal amplifications.¹³ In this study, we focused on the gene copy gain of TERT on a large scale, with an aim to determine its prognostic significance in patients with AM and to analyze the relationship between TERT and the efficiency of the HD-IFN.

Somatic mutations and amplification are significant sources of genetic diversity. In our study, the copy gain rate of TERT in AM was 44.9% (257 of the 573) (gain >2-fold change). This result was significantly higher than those of previous studies (17.6%–31.2%).^26,30 This is probably due to the small sample size of these studies.

The tumor thickness, ulceration, and stage are known prognostic factors for predicting the outcomes of melanoma.³¹ Our results showed that ulceration (P=0.028) and thickness (P=0.048) were associated with TERT copy gain. However, TERT copy gain is not correlated with the stage of primary melanomas. In addition, unlike a previous study with a cohort of 43 AM patients,²⁵ we found no association between TERT copy gain and OS in our study. We obtained the same result after stratified analysis according to TNM stage. Our data suggest that the TERT copy gain may not play a significant role in the OS of patients with AM.

IFN-α has been the only drug observed to improve RFS in high-risk postoperative patients with melanoma, and the US Food and Drug Administration approved HD-IFN for melanoma in 1996.³² A randomized phase II trial conducted in Chinese high-risk patients with AM showed that 1-year adjuvant HD-IFN (15×10⁶ U/m² for days 1–5/week for 4 weeks +9×10⁶ U twice a week for 48 weeks) may yield clinical benefits in patients with stage IIIb–IIIc AM or those with ≥3 nodal metastases.¹⁶ The median RFS of patients with AM treated with 1-year adjuvant HD-IFN was 17.9 months; however, no specific factor can predict the treatment efficacy.

In our study, the RFS of patients with TERT copy gain was significantly shorter than those with normal TERT copy number and stage II/III patients treated with HD-IFN. The Cox proportional hazards model also revealed that TERT copy gain was an independent adverse prognostic factor of RFS for all patients with AM treated with HD-IFN. This result can be explained as follows. The molecular mechanism of IFN α-2b includes direct and indirect activities. Direct activity occurs through the inhibition of cancer cell growth via cell cycle arrest, apoptosis, or differentiation. Indirect activity occurs through the activation of immune cells, inhibition of vascularization, and induction of cytokines.³³ Many studies have reported that IFN α-2b can regulate more than 300 genes in cellular signal transduction pathways.³⁴ IFN α-2b can activate JAK-1 and TYK-2 and induce antiproliferative and antiviral activity.^35,36 IFN α-2b can also inhibit the interaction of ERK with MEK or the interaction of MEK with other kinases.³⁷

Given the mechanism by which IFN α-2b influences the growth of various cancer cells via the JAK/STAT and MAPK signal transduction pathways, some researchers have also found a relationship between TERT and these pathways. They found that the expression levels and enzymatic activity of TERT are regulated by multiple signaling molecules and pathways, including the JAK/STAT, RAS/RAF/MEK/MAPK, PI3K/Akt/mTOR, IKK/NFκB, TGFβ/Smads, and PKC pathways.³⁸ These studies may help to explain why the HD-IFN group with no copy gain of TERT exhibited longer RFS than the TERT copy gain group. However, the exact mechanism remains unknown; thus, further research is warranted. In addition, to achieve more satisfactory effects, we also suggest that the TERT copy number of patients be detected before administering HD-IFN treatment.

Conclusion

In this study, we have shown the status and the clinical significance of TERT copy number in a large cohort of patients with AM. We found that the TERT copy gain might not be a prognostic factor of OS in patients with AM. However, TERT copy gain may predict the outcome of HD-IFN treatment for AM.

Acknowledgments

This work was supported by grants from the Major State Basic Research Development Program of China (2013CB911004), National Natural Science Foundation of China (81672696, 81772912), Beijing Municipal Natural Science Foundation (7152033), Beijing Baiqianwan Talents Project, Beijing Municipal Administration of Hospitals Clinical medicine Development of special funding support (ZYLX201603), and Beijing Municipal Science & Technology Commission (Z151100003915074).

Author contributions

All authors contributed toward data analysis, drafting, and critically revising the paper and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.

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