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Clinical and prognostic implications of RET rearrangements in metastatic lung adenocarcinoma patients with malignant pleural effusion
Lung Cancer 88 (2015) 208–214
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Clinical and prognostic implications of RET rearrangements in metastatic lung adenocarcinoma patients with malignant pleural effusion Tzu-Hsiu Tsai a , Shang-Gin Wu b , Min-Shu Hsieh c , Chong-Jen Yu a , James Chih-Hsin Yang d,e , Jin-Yuan Shih a,∗ a
Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan Department of Internal Medicine, National Taiwan University Hospital Yun-Lin Branch, Yun-Lin, Taiwan c Department of Pathology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan d Department of Oncology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan e Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan b
a r t i c l e
i n f o
Article history: Received 11 December 2014 Received in revised form 20 February 2015 Accepted 24 February 2015 Keywords: RET rearrangements KIF5B–RET fusions CCDC6–RET fusions Lung adenocarcinoma EGFR NSCLC
a b s t r a c t Objectives: RET rearrangements represent one of the newest molecular targets in non-small cell lung cancer (NSCLC). However, the prevalence, clinical characteristics, and outcome of patients with RETrearranged lung adenocarcinoma in metastatic disease remain uncertain. Materials and methods: Multiplex reverse transcription-polymerase chain reaction (RT-PCR) was used to detect KIF5B–RET and CCDC6–RET fusions from specimens of malignant pleural effusion (MPE) in patients with metastatic lung adenocarcinoma. The demographic data and outcome of patients with RETrearranged tumors were compared with those with EGFR-mutant, KRAS-mutant, EML4–ALK-rearranged, and quadri-negative tumors. Results: Of the 722 patients with MPE of lung adenocarcinoma screened, 17 (2.4%) had RET-rearranged tumors. The detected RET rearrangements comprised 11 (65%) KIF5B–RET and 6 (35%) CCDC6–RET fusions, including 2 novel fusion variants identified. The presence of RET rearrangements was not associated with age at diagnosis, gender or smoking history, but predominantly seen in solid histological subtype. None of patients with RET-rearranged tumors had received kinase inhibitors with activity against RET kinase. The median overall survival was 22.4 months (95% CI, 8.8–36.0) for the 17 patients with RET-rearranged tumors, compared with 21.3 months (95% CI, 18.7–23.9; P = 0.57) for the 451 patients with EGFR-mutant tumors, 5.4 months (95% CI, 2.7–8.1; P = 0.002) for the 13 patients with KRAS-mutant tumors, 18.9 months (95% CI, 10.7–27.1; P = 0.82) for the 51 patients with EML4–ALK-rearranged tumors, and 12.0 months (95% CI, 9.0–15.0; P = 0.07) for the 190 patients with quadri-negative tumors. Conclusion: Multiplex RT-PCR from specimens of MPE is feasible for the screening of RET rearrangements in NSCLC. Metastatic RET-rearranged lung adenocarcinoma patients with MPE might have favorable survival comparable to those with metastatic EGFR-mutant tumors. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The landmark discovery that activating mutations in EGFR confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs) has spurred efforts to identify novel oncogenic drivers in non-small cell lung cancer (NSCLC) [1,2]. A number of mutations in key oncogenes,
∗ Corresponding author at: Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, #7, Zhongshan South Road, Taipei City 10002, Taiwan. Tel.: +886 2 2312 3456x62905; fax: +886 2 2358 2867. E-mail address: [email protected] (J.-Y. Shih). http://dx.doi.org/10.1016/j.lungcan.2015.02.018 0169-5002/© 2015 Elsevier Ireland Ltd. All rights reserved.
including EGFR, HER2, KRAS, NRAS, PIK3CA, BRAF, AKT1, and MAP2K1, have been identified as important genetic alterations in NSCLC [3,4]. In addition, chromosomal translocations involving kinase proto-oncogenes, such as ALK and ROS1, have been discovered in a small subset of patients with lung adenocarconoma [5,6]. These rearrangements lead to the formation of chimeric fusion kinases capable of oncogenic transformation [7]. Like EGFR mutations, ALK and ROS1 rearrangements are reported to be associated with unique clinical and pathological features in NSCLC, and confer sensitivity to specific TKIs such as crizotinib [8–11]. In 2012, four independent groups discovered RET rearrangements involving the KIF5B as a novel genetic alteration in NSCLC
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[12–15]. RET encodes a receptor tyrosine kinase that normally play a crucial part in the development of neural crest [16]. Although RET point mutations and rearrangements have long been recognized in medullary and papillary thyroid cancers, the rearrangements of RET oncogene represent one of the newest molecular targets in NSCLC [16–18]. To date, RET rearrangements have been identified as involving four fusion partners (KIF5B, CCDC6, NCO4, and TRIM33) that comprise 10 subtypes of fusion variants [12–15,19–26]. Although no selective RET inhibitors have yet been developed for clinical use, a few commercially available multi-kinase inhibitors, such as vandetanib, cabozantinib, sorafenib, and sunitinib, have activity against the RET kinase [16,27]. Although RET rearrangements are predicted to be a potential therapeutic target against NSCLC, the clinical characteristics of patients with RET-rearranged tumors in metastatic disease remains uncertain. This is because previous studies largely identified RET rearrangements in NSCLC by samples of surgical tissue, and accordingly addressed patients mainly with early and resectable disease. Also for this reason, the impact of RET rearrangements on the prognosis of metastatic NSCLC has not been explored. Here, using multiplex reverse transcription-polymerase chain reactions (RTPCR) and sequencing for samples of malignant pleural effusion (MPE), we identified patients with RET-rearranged tumors in a large cohort of patients with MPE of lung adenocarcinoma. The prevalence, clinical characteristics and outcome of these patients were examined. 2. Materials and methods
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RT-PCR of extracted RNA was carried out using a Qiagen One-Step RT-PCR Kit (Qiagen). The conditions and primers of RT-PCR for the amplification of EGFR and KRAS have been described previously [30]. The cDNA amplicons were purified and sequenced using a BigDye Terminator Sequencing Kit (Applied Biosystems, Foster City, CA). Sequencing products underwent electrophoresis on an automated ABI PRISM 3700 genetic analyzer (Applied Biosystems). Both the forward and reverse sequences obtained were analyzed. 2.4. Identification of KIF5B–RET and CCDC6–RET fusion genes Multiplex RT-PCR of extracted RNA for the screening of KIF5B–RET and CCDC6–RET fusions was performed using a Qiagen One-Step RT-PCR Kit (Qiagen). Briefly, 50 ng of total RNA was used as the template. The primer sets adopted from prior reports were as the following: 5 -TAAGGAAATGACCAACCACCAG-3 (forward primer, on exon 15 of KIF5B), 5 -AGCCACAGATCAGGAAAAGA-3 (forward primer, on exon 20 of KIF5B), 5 TGCAGCAAGAGAACAAGGTG-3 (forward primer, on exon 1 of CCDC6), and 5 -TCCAAATTCGCCTTCTCCTA-3 (reverse primer, on exon 12 of RET) [12,13,15]. In addition, we designed another primer: 5 -ATCGCAAACGCTATCAGCAAGA-3 (forward primer, on exon 24 of KIF5B) (Table 1). The RT-PCR reaction was initiated at 50 ◦ C for 30 min, heated to 95 ◦ C for 15 min, followed by 40 cycles of denaturation at 94 ◦ C for 50 s, annealing at 60 ◦ C for 50 s, extension at 72 ◦ C for 1 min, and a final extension at 72 ◦ C for 10 min. The RTPCR product was electrophoresed to screen for RET rearrangements, with different fusion variants subsequently confirmed by direct sequencing.
2.1. Patients and specimens 2.5. Identification of EML4–ALK fusion genes Between June 2005 and October 2012, we collected samples of cytology-proven MPE from patients with metastatic lung adenocarcinoma at the National Taiwan University Hospital. RNA extracted from the cell pellets obtained from these effusion specimens were used for genetic testing. The effusion samples might be obtained either at initial diagnosis or during the course of disease in these patients. All patients provided signed informed consent to allow for the use of samples in molecular analyses, and the study was approved by the Institutional Review Board of the hospital. Some of the materials have been examined previously, and reported in studies regarding EGFR mutations [28]. For all patients, medical records were reviewed to extract data on clinical characteristics, including age, gender, smoking history, histology, stage, and overall survival (OS). Patients who had smoked less than 100 cigarettes in their lifetime were categorized as never smokers. Adenocarcinoma histology was confirmed either by the pathology reports for biopsy of the primary or metastatic tumors, or by cell clocks of MPE with positive thyroid transcription factor1 staining by immunocytochemistry [29]. OS was measured from the date of initial diagnosis or relapse in metastatic disease until the date of death.
For those cases without identified EGFR mutations, KRAS mutations, or RET rearrangements, RT-PCR of the extracted RNA and electrophoresis were performed to screen EML4–ALK fusion genes, with the primers and conditions of RT-PCR as previously described [31]. The variants of identified EML4–ALK fusion genes were confirmed by subsequent sequencing. 2.6. Statistical analysis For clinical characteristics, categorical variables were analyzed with the chi-square test, except when a small size (less than five) necessitated the use of Fisher’s exact test. Wilcoxon rank sum test was applied to analyze continuous variables. Survival curves were plotted by the Kaplan–Meier method, and compared by the Wilcoxon (Breslow) test. Two-sided P values of less than 0.05 were considered to be statistically significant. The cutoff date for data collection was November 30, 2013. All analyses were performed using SPSS software (version 17.0 for Windows; SPSS Inc., Chicago, IL). 3. Results
2.2. Extraction of RNA from cell lysates of effusion samples 3.1. Genotypes of patients with MPE of lung adenocarcinoma The pleural fluid was collected and centrifuged at 250 × g for 10 min at 4 ◦ C, and the cell pellet was frozen in RNAlater (Qiagen, Valencia, CA) for storage. After the effusion samples were proved to contain tumors cells with cytology examination, cell-derived RNA was extracted using TRI reagent (Molecular Research Center, Cincinnati, OH) from the lysates of cell pellets. 2.3. Analysis of EGFR and KRAS mutations Analysis of EGFR (exons 18–21) and KRAS (exons 2 and 3) mutations with RT-PCR and direct sequencing was performed separately.
Between June 2005 and October 2012, there were 2228 patients diagnosed with metastatic lung adenocarcinoma at the National Taiwan University Hospital. Samples of cytology-proven malignant pleural effusion were collected from 722 patients for genotyping (Supplementary Fig. S1). The clinical characteristics of these patients enrolled in the study are presented in Table 2. All patients were of East Asian descent. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.lungcan.2015.02.018.
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Table 1 Primers used for multiplex reverse transcription-polymerase chain reactions (RT-PCR) to detect reported variants of RET rearrangement. RT-PCR primers
Ref
Detected variants
Amplicon size (bp)
Forward primer 5 -TAAGGAAATGACCAACCACCAG-3 (on exon 15 of KIF5B)
[15]
5 -AGCCACAGATCAGGAAAAGA-3 (on exon 20 of KIF5B)
[13]
5 -ATCGCAAACGCTATCAGCAAGA-3 (on exon 24 of KIF5B)
–
K15;R12 K16;R12 K15;R11 K22;R12 K23;R12 K24;R11 K24;R8 C1;R12
201 391 459 271 376 443 800 192
5 -TGCAGCAAGAGAACAAGGTG-3 (on exon 1 of CCDC6) Reverse primer 5 -TCCAAATTCGCCTTCTCCTA-3 (on exon 12 of RET)
[13] [12]
Table 2 Clinical characteristics of 722 genetically screened patients with malignant pleural effusion of lung adenocarcinoma. Characteristics
Patients
Total, n Age, years Median Range Age, n (%) >60 years ≤60 years Gender, n (%) Male Female Smoking history, n (%) Never smoker Current/former smoker Missing Stage, n (%) Relapsed Stage IV
722 66 28–96 460 (63.7%) 262 (36.3%) 345 (47.8%) 377 (52.2%) 489 (67.7%) 198 (27.4%) 35 (4.8%) 77 (10.7%) 645 (89.3%)
Among these 722 patients, 451 (62.5%) patients had tumors with identified EGFR mutations, 13 (1.8%) patients with KRAS mutations, 51 (7.1%) patients with EML4–ALK fusions, and 17 (2.4%) patients with RET rearrangements. The spectrum of identified EGFR mutations comprised exon 19 deletions in 191 (42.4%), exon 21 L858R point mutation in 224 (49.7%), and other types of mutations in 36 (8.0%). The subtypes of KRAS mutations included mutations in codon 12 (n = 10) and codon 13 (n = 1) of exon 2, as well as in codon 61 of exon 3 (n = 2). The major variants of EML4–ALK fusion gene were variant 1 with exon 13 of EML4 (n = 34), variant 2 with exon 20 of EML4 (n = 4), and variant 3a/b with exon 6a/b of EML4 (n = 12) fused to exon 20 of ALK, respectively. One hundred and ninety patients (26.3%) had tumors negative for EGFR mutations, KRAS mutations, EML4–ALK fusions, or RET rearrangements (hereafter referred to as quadri-negative). The presence of EGFR mutations, KRAS mutations, and RET rearrangements were mutually exclusive. The frequency of RET rearrangements was 6.3% among the 271 tumors harboring wide-type EGFR. Of the 159 tumors with wide-type EGFR and from never smokers, 12 (7.5%) had identified RET rearrangements. No patients with RET-rearranged tumors indicated a history of thyroid cancer or therapeutic radiation exposure. 3.2. Variants of RET fusion gene identified The RT-PCR primers used in this study enabled us to detect all eight KIF5B–RET and CCDC6–RET fusion variants identified to date (Table 1). Using RT-PCR and direct sequencing, 11 (65%) KIF5B–RET and 6 (35%) CCDC6–RET fusions were identified among the 17 tumors harboring RET rearrangements. The identified KIF5B–RET fusions comprised 8 KIF5B exon 15 fused to RET exon 12 (K15;R12), 2 KIF5B exon 22 fused to RET exon 12 (K22;R12), and 1 novel fusion variant involving a fusion of KIF5B exon 18 to RET exon 12 (K18;R12)
Fig. 1. Identification of two novel fusion variants of RET rearrangement. (A) A fusion of KIF5B exon 18 to RET exon 12. (B) A fusion of exon 5 and a proportion of intron 5 of CCDC6 to RET exon 11. Both of the fusion variants generate an in-frame RET fusion gene.
(Fig. 1). Of the tumors harboring CCDC6–RET fusions, the majority (5 of 6) involved CCDC6 exon 1 fused to RET exon 12 (C1;R12). In addition, we identified a novel fusion variant involving the fusion of exon 5 and a proportion of intron 5 of CCDC6 with RET exon 11, which was in an in-frame fashion (Fig. 1). 3.3. Clinical characteristics of patients with RET-rearranged tumors As shown in Table 3, the median age of patients with RETrearranged tumors did not significantly differ from those with EGFR-mutant, KRAS-mutant, EML4–ALK-rearranged, or quadrinegative tumors. The frequency of RET rearrangements among the 722 screened patients was also not associated with age (>60 versus ≤60 years, 2.2% versus 2.7%; P = 0.67), gender (males versus females, 2.6% versus 2.1%; P = 0.67), or smoking history (never smokers versus current or former smokers, 2.5% versus 2.5%; P = 0.96). By contrast, EGFR-mutant tumors were significantly more commonly present in female (55.4% versus 69.0%; P < 0.001) and never smokers (67.5% versus 46.0%; P < 0.001), while quadri-negative tumors occurred preferentially in male (32.2% versus 21.0%; P = 0.001) and current or former smokers (20.9% versus 42.4%; P < 0.001). Tumors with EML4–ALK fusions were more frequently seen in patients with age of 60 years or less (5.4% versus 9.9%; P = 0.02). The clinical details of the 17 patients with MPE of lung adenocarcinoma harboring RET rearrangements are provided in Table 4. The median age of these 17 patients was 63 years (range, 29–89 years), with equal distribution of gender (9 males versus 8 females). Twelve (71%) were never smokers, likely reflecting the increased frequency of never smoker in the entire study population (67.7%).
P < 0.001 P = 0.43 38 (7.8%) 12 (6.1%) P = 0.21
We observed that approximately half of patients (8 of 17, 47%) had a small primary lesion (≤3 cm). In addition to malignant effusion or pleural seeding, the most frequent sites of distant metastasis involved lung (5 of 17, 29%), brain (4 of 17, 24%) and bone (4 of 17, 24%), but liver (1 of 17, 6%) and adrenal (0 of 17, 0%) metastases were rare. Biopsy tissues for histological analysis were available from 7 of the 17 cases with RET-rearranged tumors (Table 4). Among these 7 cases, 6 (85.7%) were sub-classified as solid-predominant morphology. One case exhibited signet-ring cell appearance.
P < 0.001
7 (1.4%) 6 (3.0%)
P = 0.50 5 (1.4%) 8 (2.1%)
3.4. Outcome of patients with RET-rearranged tumors
There were 35 patients with missing data regarding smoking history. *
12 (2.5%) 5 (2.5%) 489 198
P = 0.96
330 (67.5%) 91 (46.0%)
P < 0.001 9 (2.6%) 8 (2.1%) 345 377
P = 0.67 10 (2.2%) 7 (2.7%) 460 262
P = 0.67
102 (20.9%) 84 (42.4%)
P = 0.001 111 (32.2%) 79 (21.0%) 29 (8.4%) 22 (5.8%)
P = 0.18
125 (27.2%) 65 (24.8%) P = 0.02 25 (5.4%) 26 (9.9%) P = 0.39 10 (2.2%) 3 (1.1%) 290 (63.0%) 161 (61.5%)
P = 0.67
67 53–77 66 31–96 63 29–89
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Fig. 2. Overall survival after the initial diagnosis or relapse in metastatic disease in patients with malignant pleural effusion of RET-rearranged, EGFR-mutant, KRASmutant, EML4–ALK-rearranged, and quadri-negative lung adenocarcinomas. OS, overall survival.
Age, years Median Range Age, n (%) >60 years ≤60 years Gender, n (%) Male Female Smoking history, n (%)* Never smoker Current/former smoker
66 28–96
191 (55.4%) 260 (69.0%)
67 29–91 60 28–85
P = 0.49
Quadri-negative (n = 190) EGFR mutation (n = 451) RET rearrangement (n = 17) Total (n = 722) Characteristics
Table 3 Clinical characteristics of genotype-specific subsets of patients with malignant pleural effusion of lung adenocarcinoma.
KRAS mutation (n = 13)
ALK rearrangement (n = 51)
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The median follow-up time was 14.5 months for the 722 screened patients, and 15.0 months for the 17 patients with tumors harboring RET rearrangements. On the cutoff date for data collection, 195 (27.0%) patients were still alive, including 2 patients with RET-rearranged tumors. The median overall survival for the entire study population was 18.1 months (95% CI, 16.3–19.9). None of the 17 patients with RET-rearranged tumors had received multi-kinase inhibitors with activity against RET kinase, while 412 (91.4%) of the 451 patients with EGFR-mutant tumors had received EGFR TKIs. Fifteen (29.4%) of the 51 patients with tumors harboring EML4–ALK fusions had been treated with crizotinib. As shown in Fig. 2, the median OS was 22.4 months (95% CI, 8.8–36.0) for patients with RET-rearranged tumors, compared with 21.3 months (95% CI, 18.7–23.9; P = 0.57) for patients with EGFR-mutant tumors, 5.4 months (95% CI, 2.7–8.1; P = 0.002) for patients with KRAS-mutant tumors, 18.9 months (95% CI, 10.7–27.1; P = 0.82) for patients with EML4–ALK-rearranged tumors, and 12.0 months (95% CI, 9.0–15.0; P = 0.07) for patients with quadri-negative tumors. Patients with EGFR-mutant and EML4–ALK-rearranged tumors had significant longer OS than patients with quadri-negative (P < 0.001 and P = 0.012, respectively) and KRAS-mutant tumors (P < 0.001 and P = 0.003, respectively). 4. Discussion Our study represents a large survey of metastatic RETrearranged lung adenocarcinoma assessed in the context of multiplexed genotyping from MPE. It allows for an unprecedented comparison of RET-rearranged lung adenocarcinoma with other genomic subsets in the setting of metastatic disease with
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Table 4 Clinical details of 17 patients with malignant pleural effusion of lung adenocarcinoma harboring RET rearrangements. Case No.
Age (yr)
KIF5B–RET fusions 1 57 2 64 75 3 29 4 76 5 41 6 57 7 66 8 9 63 10 55 11 73 CCDC6–RET fusions 55 12 61 13 89 14 53 15 67 16
17
79
Gender
Smoking (pack yr)
Histology
F M M F F M
0 0 40 0 0 40
M M M F F
0 60 0 0 0
– Solid Solid – – Acinar, micropapillary – Solid – – –
M F M M F
0 0 30 30 0
F
0
– – – Solid Solid, signet ring cells with mucin staining Solid, micropapillary
Nodal status
Metastasis site
OS (month)
RET fusion variants
2.5 2.6 2.6 1.7 5.4 1.7
N1 N0 N2 N2 N1 N0
MPE, lung Brain MPE, liver, bone MPE, MPCE, bone MPE Pleural, brain
10.5 30.5 28.4 10.5 8.1 52.7*
K15;R12 K15;R12 K15;R12 K15;R12 K15;R12 K15;R12
3.6 11.0 4.5 3.0 4.7
N2 N3 N0 N0 N2
MPE MPE MPE, lung MPE, lung MPE, brain, bone
13.9* 4.2 11.7 11.6 32.7
K15;R12 K15;R12 K22;R12 K22;R12 K18;R12
3.4 1.8 1.3 7.7 7.0
N2 N3 N0 N3 N3
MPE, bone Lung MPE MPE, MPCE MPE, lung
22.4 17.1 30.3 33.7 22.7
C1;R12 C1;R12 C1;R12 C1;R12 C1;R12
4.5
N3
Brain
Tumor size (cm)
3.8
C5 ins intron 5 (AGTCTTGCTGTGTTGC);R11
Abbreviations: OS, overall survival; F, female; M, male; MPE, malignant pleural effusion; MPCE, malignant pericardial effusion. * Still alive on the cutoff date for data collection.
MPE. In our series, 17 (2.4%) of 722 patients with MPE of lung adenocarcinoma had tumors harboring KIF5B–RET or CCDC6–RET fusions, which included 2 novel fusion variants. The presence of RET-rearranged tumors was not associated with age, gender, or smoking history, but predominantly seen in solid histological subtype. Of note, our results suggest that the survival of metastatic RET-rearranged lung adenocarcinoma patients with MPE compares favorably with that of patients with EGFR-mutant tumors who had been treated with EGFR TKIs. In the earlier screening studies, RET rearrangements were reported to be present in 1–2% of unselected NSCLC patients of both East Asian and Caucasian descent [12–14,19,21,24,26,32]. By comparison, the frequency of RET rearrangements (2.4%) in our patients was modestly higher. We could not exclude the possibility that the higher prevalence of RET rearrangements in our series come from the selection of patients with metastatic disease. Several studies have implied that RET-rearranged tumors might be more prevalent in patients at advanced stage. For example, of the cohort reported by Wang et al. [19], the prevalence of RET rearrangements in lung adenocarcinoma was 1.3% (5 of 388) among patients with stage 1 or 2 disease, and 2.4% (6 of 245) among patients with stage 3 and 4 disease. Similarly, in the large series reported by Tsuta et al. [26], RET rearrangements were present in 1.1% (16 of 1496) of patients with stage 1 or 2 disease, and in 1.6% (6 of 364) of patients with stage 3 or 4 disease. Furthermore, probably because we enrolled patients with MPE of lung adenocarcinoma, our cohort included many female and never-smoker patients. As previously reported, adenocarcinoma in Asian never-smoker female patients may have distinct spectrum of genetic alterations [33]. It has been reported that patients with RET-rearranged NSCLC were relatively younger and preferentially never- or light-smokers, reminiscent of the patients characteristics of ALK-rearranged NSCLC [12,13]. However, the age in our cohort was similar among patients with RET-rearranged, EGFR-mutant, KRAS-mutant, and quadri-negative tumors. We also observed that the frequency of RET rearrangements was not associated with gender or smoking history. Thus, the relationship among age, smoking status, sex, and RET rearrangements remains unclear owing to the very low percentage of such populations. However, it is worth noting that
there were many female and never-smoker patients, along with a few young cases, among those with RET-rearranged tumors in this cohort. Of note, solid histologic subtype accounted for the majority of RET-rearranged tumors (six of seven with available tissue for histological analysis, 85.7%) in the current study, which is similar to the previous report by Wang et al. [19]. Knowledge of this clinical profile may help physicians select patients for the identification of this uncommon genetic alteration. In the current study conducted in Taiwan, we identified 11 (65%) KIF5B–RET and 6 (35%) CCDC6–RET fusions among the 17 RET-rearranged tumors. To date, KIF5B–RET fusions are present in approximately 90% of the RET rearrangements reported, with CCDC6–RET fusions accounting for the remaining 10% [27]. In the report by Takeuchi et al. [13], there was only one case (8%) with CCDC6–RET fusion gene in 13 identified RET-rearranged tumors from Japanese patients. The rate of CCDC6–RET fusions in RETrearranged tumors (3 of 22, 14%) was also low in another Japanese cohort [26]. By contrast, 3 of the 13 (23%) RET-rearranged tumors from Chinese patients contain CCDC6–RET fusions, as reported by Wang et al. [19]. The higher rate of CCDC6–RET fusions in our cohort is in line with that reported in Chinese patients. It is currently not clear whether this discrepancy is a result of the difference in genetic and environmental backgrounds among the screened populations. In the report by Drilon et al., the median overall survival of the five patients with metastatic RET-rearranged NSCLC was as long as 27 months [20,34]. However, three patients among them had received treatment with carbozantinib, a multi-kinase inhibitors with activity against RET kinase. Therefore, it is difficult to determine the prognostic effect of RET rearrangements in metastatic NSCLC. Although not statistically significant (probably owing to the limited number of patients with RET-rearranged tumor), our study suggested that metastatic RET-rearranged lung adenocarcinoma patients with MPE might have longer survival than those with quadri-negative tumors. Indeed, the survival of patients with RETrearranged tumors in our series was comparable to that of patients with EGFR-mutant tumors. It should be noted that the vast majority of our patients with EGFR-mutant tumors had been treated with EGFR TKIs, which might have prolonged the survival in this genomic subset. Our data raises the question of whether RET rearrangements
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may confer a more indolent phenotype and better outcome. In papillary thyroid cancer with CCDC6–RET fusions, it has been reported the expression of RET protein from the upstream CCDC6 promoters was relatively low [35]. This means that the oncogenic RET activity may be modest in tumors with CCDC6–RET rearrangements, which may correlate with the relatively benign phenotype of papillary thyroid cancer [16]. Further studies are needed to explore the transcription profile of RET protein in lung cancer harboring RET rearrangements, as well as its influence on the prognosis. Since RET-rearranged NSCLC potentially represents a therapeutically subcategory, an issue is to define an optimal biomarker assay for screening in practice. A variety of methods can be used, including immunohistochemistry (IHC), fluorescent in situ hybridization (FISH) and RT-PCR. Currently, there are no reliable RET antibodies for the detection of RET rearrangements in paraffin sections [19,32,36]. FISH is traditionally the standard technology to detect chromosomal translocations in tumor tissue. However, because of the high cost and need for technical expertise, FISH may not be an effective way to screen RET rearrangements that are present at a very low frequency in NSCLC. By contrast, RT-PCR is a rapid and sensitive method, that is, less expensive in terms of cost, and can be practically applied to cytological specimens. The RT-PCR primers used in this study were able to detect all KIF5B–RET and CCDC6–RET fusion variants identified to date. We could not exclude the possibility that a few RET-rearranged tumors, such as those harboring NCO4–RET and TRIM33–RET fusions, may have been missed. However, with the screening of approximately 5000 cases in total in previous studies, each one of these two RET rearrangements only appeared in an individual case, respectively [27]. We believe that NCO4–RET and TRIM33–RET rearrangements should be extremely rare in NSCLC. There are a number of limitations regarding this study that should be highlighted. Firstly, as we screened for RET rearrangements using specimens of MPE, the enrolled patients with metastatic lung adenocarcinoma were limited to those with MPE. Those with metastatic lung adenocarcinoma but without MPE will not be included in this study. Secondarily, because most of our patients had tissue diagnosis by small biopsy or cytology, comprehensive histological analysis was not feasible for all cases with RET-rearranged tumors. Finally, RET-rearranged tumors identified in this study were not subjected to RET FISH analysis to confirm the diagnostic accuracy of RT-PCR. 5. Conclusions The current study provides insights into the understanding of RET rearrangements in metastatic lung adenocarcinoma, and suggests a potential prognostic role of RET rearrangements in metastatic disease. We demonstrate the feasibility of multiplex RT-PCR with cytological samples for the screening of RET rearrangements in NSCLC. This screening strategy could be applied to facilitate the identification of these molecularly defined patients. Conflict of interest statement All authors report that they have no conflicts of interest. Acknowledgments The authors would like to thank the third core facility at National Taiwan University Hospital for technical assistance and facility support. This study was supported from grants 101-2314-B002169-MY3 and 101-2314-B002-170-MY3 (Ministry of Science and Technology, Taiwan), grant MOHW103-TDU-PB-211-144002 (Ministry of Health and Welfare, Taiwan), grant 103R7601-3 (National
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Taiwan University, Taiwan), and grant 103-FTN17 (National Taiwan University Hospital, Taiwan) to J.-Y.S.
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Clinical and prognostic implications of RET rearrangements in metastatic lung adenocarcinoma patients with malignant pleural effusion Tzu-Hsiu Tsai a , Shang-Gin Wu b , Min-Shu Hsieh c , Chong-Jen Yu a , James Chih-Hsin Yang d,e , Jin-Yuan Shih a,∗ a
Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan Department of Internal Medicine, National Taiwan University Hospital Yun-Lin Branch, Yun-Lin, Taiwan c Department of Pathology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan d Department of Oncology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan e Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan b
a r t i c l e
i n f o
Article history: Received 11 December 2014 Received in revised form 20 February 2015 Accepted 24 February 2015 Keywords: RET rearrangements KIF5B–RET fusions CCDC6–RET fusions Lung adenocarcinoma EGFR NSCLC
a b s t r a c t Objectives: RET rearrangements represent one of the newest molecular targets in non-small cell lung cancer (NSCLC). However, the prevalence, clinical characteristics, and outcome of patients with RETrearranged lung adenocarcinoma in metastatic disease remain uncertain. Materials and methods: Multiplex reverse transcription-polymerase chain reaction (RT-PCR) was used to detect KIF5B–RET and CCDC6–RET fusions from specimens of malignant pleural effusion (MPE) in patients with metastatic lung adenocarcinoma. The demographic data and outcome of patients with RETrearranged tumors were compared with those with EGFR-mutant, KRAS-mutant, EML4–ALK-rearranged, and quadri-negative tumors. Results: Of the 722 patients with MPE of lung adenocarcinoma screened, 17 (2.4%) had RET-rearranged tumors. The detected RET rearrangements comprised 11 (65%) KIF5B–RET and 6 (35%) CCDC6–RET fusions, including 2 novel fusion variants identified. The presence of RET rearrangements was not associated with age at diagnosis, gender or smoking history, but predominantly seen in solid histological subtype. None of patients with RET-rearranged tumors had received kinase inhibitors with activity against RET kinase. The median overall survival was 22.4 months (95% CI, 8.8–36.0) for the 17 patients with RET-rearranged tumors, compared with 21.3 months (95% CI, 18.7–23.9; P = 0.57) for the 451 patients with EGFR-mutant tumors, 5.4 months (95% CI, 2.7–8.1; P = 0.002) for the 13 patients with KRAS-mutant tumors, 18.9 months (95% CI, 10.7–27.1; P = 0.82) for the 51 patients with EML4–ALK-rearranged tumors, and 12.0 months (95% CI, 9.0–15.0; P = 0.07) for the 190 patients with quadri-negative tumors. Conclusion: Multiplex RT-PCR from specimens of MPE is feasible for the screening of RET rearrangements in NSCLC. Metastatic RET-rearranged lung adenocarcinoma patients with MPE might have favorable survival comparable to those with metastatic EGFR-mutant tumors. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The landmark discovery that activating mutations in EGFR confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs) has spurred efforts to identify novel oncogenic drivers in non-small cell lung cancer (NSCLC) [1,2]. A number of mutations in key oncogenes,
∗ Corresponding author at: Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, #7, Zhongshan South Road, Taipei City 10002, Taiwan. Tel.: +886 2 2312 3456x62905; fax: +886 2 2358 2867. E-mail address: [email protected] (J.-Y. Shih). http://dx.doi.org/10.1016/j.lungcan.2015.02.018 0169-5002/© 2015 Elsevier Ireland Ltd. All rights reserved.
including EGFR, HER2, KRAS, NRAS, PIK3CA, BRAF, AKT1, and MAP2K1, have been identified as important genetic alterations in NSCLC [3,4]. In addition, chromosomal translocations involving kinase proto-oncogenes, such as ALK and ROS1, have been discovered in a small subset of patients with lung adenocarconoma [5,6]. These rearrangements lead to the formation of chimeric fusion kinases capable of oncogenic transformation [7]. Like EGFR mutations, ALK and ROS1 rearrangements are reported to be associated with unique clinical and pathological features in NSCLC, and confer sensitivity to specific TKIs such as crizotinib [8–11]. In 2012, four independent groups discovered RET rearrangements involving the KIF5B as a novel genetic alteration in NSCLC
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[12–15]. RET encodes a receptor tyrosine kinase that normally play a crucial part in the development of neural crest [16]. Although RET point mutations and rearrangements have long been recognized in medullary and papillary thyroid cancers, the rearrangements of RET oncogene represent one of the newest molecular targets in NSCLC [16–18]. To date, RET rearrangements have been identified as involving four fusion partners (KIF5B, CCDC6, NCO4, and TRIM33) that comprise 10 subtypes of fusion variants [12–15,19–26]. Although no selective RET inhibitors have yet been developed for clinical use, a few commercially available multi-kinase inhibitors, such as vandetanib, cabozantinib, sorafenib, and sunitinib, have activity against the RET kinase [16,27]. Although RET rearrangements are predicted to be a potential therapeutic target against NSCLC, the clinical characteristics of patients with RET-rearranged tumors in metastatic disease remains uncertain. This is because previous studies largely identified RET rearrangements in NSCLC by samples of surgical tissue, and accordingly addressed patients mainly with early and resectable disease. Also for this reason, the impact of RET rearrangements on the prognosis of metastatic NSCLC has not been explored. Here, using multiplex reverse transcription-polymerase chain reactions (RTPCR) and sequencing for samples of malignant pleural effusion (MPE), we identified patients with RET-rearranged tumors in a large cohort of patients with MPE of lung adenocarcinoma. The prevalence, clinical characteristics and outcome of these patients were examined. 2. Materials and methods
209
RT-PCR of extracted RNA was carried out using a Qiagen One-Step RT-PCR Kit (Qiagen). The conditions and primers of RT-PCR for the amplification of EGFR and KRAS have been described previously [30]. The cDNA amplicons were purified and sequenced using a BigDye Terminator Sequencing Kit (Applied Biosystems, Foster City, CA). Sequencing products underwent electrophoresis on an automated ABI PRISM 3700 genetic analyzer (Applied Biosystems). Both the forward and reverse sequences obtained were analyzed. 2.4. Identification of KIF5B–RET and CCDC6–RET fusion genes Multiplex RT-PCR of extracted RNA for the screening of KIF5B–RET and CCDC6–RET fusions was performed using a Qiagen One-Step RT-PCR Kit (Qiagen). Briefly, 50 ng of total RNA was used as the template. The primer sets adopted from prior reports were as the following: 5 -TAAGGAAATGACCAACCACCAG-3 (forward primer, on exon 15 of KIF5B), 5 -AGCCACAGATCAGGAAAAGA-3 (forward primer, on exon 20 of KIF5B), 5 TGCAGCAAGAGAACAAGGTG-3 (forward primer, on exon 1 of CCDC6), and 5 -TCCAAATTCGCCTTCTCCTA-3 (reverse primer, on exon 12 of RET) [12,13,15]. In addition, we designed another primer: 5 -ATCGCAAACGCTATCAGCAAGA-3 (forward primer, on exon 24 of KIF5B) (Table 1). The RT-PCR reaction was initiated at 50 ◦ C for 30 min, heated to 95 ◦ C for 15 min, followed by 40 cycles of denaturation at 94 ◦ C for 50 s, annealing at 60 ◦ C for 50 s, extension at 72 ◦ C for 1 min, and a final extension at 72 ◦ C for 10 min. The RTPCR product was electrophoresed to screen for RET rearrangements, with different fusion variants subsequently confirmed by direct sequencing.
2.1. Patients and specimens 2.5. Identification of EML4–ALK fusion genes Between June 2005 and October 2012, we collected samples of cytology-proven MPE from patients with metastatic lung adenocarcinoma at the National Taiwan University Hospital. RNA extracted from the cell pellets obtained from these effusion specimens were used for genetic testing. The effusion samples might be obtained either at initial diagnosis or during the course of disease in these patients. All patients provided signed informed consent to allow for the use of samples in molecular analyses, and the study was approved by the Institutional Review Board of the hospital. Some of the materials have been examined previously, and reported in studies regarding EGFR mutations [28]. For all patients, medical records were reviewed to extract data on clinical characteristics, including age, gender, smoking history, histology, stage, and overall survival (OS). Patients who had smoked less than 100 cigarettes in their lifetime were categorized as never smokers. Adenocarcinoma histology was confirmed either by the pathology reports for biopsy of the primary or metastatic tumors, or by cell clocks of MPE with positive thyroid transcription factor1 staining by immunocytochemistry [29]. OS was measured from the date of initial diagnosis or relapse in metastatic disease until the date of death.
For those cases without identified EGFR mutations, KRAS mutations, or RET rearrangements, RT-PCR of the extracted RNA and electrophoresis were performed to screen EML4–ALK fusion genes, with the primers and conditions of RT-PCR as previously described [31]. The variants of identified EML4–ALK fusion genes were confirmed by subsequent sequencing. 2.6. Statistical analysis For clinical characteristics, categorical variables were analyzed with the chi-square test, except when a small size (less than five) necessitated the use of Fisher’s exact test. Wilcoxon rank sum test was applied to analyze continuous variables. Survival curves were plotted by the Kaplan–Meier method, and compared by the Wilcoxon (Breslow) test. Two-sided P values of less than 0.05 were considered to be statistically significant. The cutoff date for data collection was November 30, 2013. All analyses were performed using SPSS software (version 17.0 for Windows; SPSS Inc., Chicago, IL). 3. Results
2.2. Extraction of RNA from cell lysates of effusion samples 3.1. Genotypes of patients with MPE of lung adenocarcinoma The pleural fluid was collected and centrifuged at 250 × g for 10 min at 4 ◦ C, and the cell pellet was frozen in RNAlater (Qiagen, Valencia, CA) for storage. After the effusion samples were proved to contain tumors cells with cytology examination, cell-derived RNA was extracted using TRI reagent (Molecular Research Center, Cincinnati, OH) from the lysates of cell pellets. 2.3. Analysis of EGFR and KRAS mutations Analysis of EGFR (exons 18–21) and KRAS (exons 2 and 3) mutations with RT-PCR and direct sequencing was performed separately.
Between June 2005 and October 2012, there were 2228 patients diagnosed with metastatic lung adenocarcinoma at the National Taiwan University Hospital. Samples of cytology-proven malignant pleural effusion were collected from 722 patients for genotyping (Supplementary Fig. S1). The clinical characteristics of these patients enrolled in the study are presented in Table 2. All patients were of East Asian descent. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.lungcan.2015.02.018.
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Table 1 Primers used for multiplex reverse transcription-polymerase chain reactions (RT-PCR) to detect reported variants of RET rearrangement. RT-PCR primers
Ref
Detected variants
Amplicon size (bp)
Forward primer 5 -TAAGGAAATGACCAACCACCAG-3 (on exon 15 of KIF5B)
[15]
5 -AGCCACAGATCAGGAAAAGA-3 (on exon 20 of KIF5B)
[13]
5 -ATCGCAAACGCTATCAGCAAGA-3 (on exon 24 of KIF5B)
–
K15;R12 K16;R12 K15;R11 K22;R12 K23;R12 K24;R11 K24;R8 C1;R12
201 391 459 271 376 443 800 192
5 -TGCAGCAAGAGAACAAGGTG-3 (on exon 1 of CCDC6) Reverse primer 5 -TCCAAATTCGCCTTCTCCTA-3 (on exon 12 of RET)
[13] [12]
Table 2 Clinical characteristics of 722 genetically screened patients with malignant pleural effusion of lung adenocarcinoma. Characteristics
Patients
Total, n Age, years Median Range Age, n (%) >60 years ≤60 years Gender, n (%) Male Female Smoking history, n (%) Never smoker Current/former smoker Missing Stage, n (%) Relapsed Stage IV
722 66 28–96 460 (63.7%) 262 (36.3%) 345 (47.8%) 377 (52.2%) 489 (67.7%) 198 (27.4%) 35 (4.8%) 77 (10.7%) 645 (89.3%)
Among these 722 patients, 451 (62.5%) patients had tumors with identified EGFR mutations, 13 (1.8%) patients with KRAS mutations, 51 (7.1%) patients with EML4–ALK fusions, and 17 (2.4%) patients with RET rearrangements. The spectrum of identified EGFR mutations comprised exon 19 deletions in 191 (42.4%), exon 21 L858R point mutation in 224 (49.7%), and other types of mutations in 36 (8.0%). The subtypes of KRAS mutations included mutations in codon 12 (n = 10) and codon 13 (n = 1) of exon 2, as well as in codon 61 of exon 3 (n = 2). The major variants of EML4–ALK fusion gene were variant 1 with exon 13 of EML4 (n = 34), variant 2 with exon 20 of EML4 (n = 4), and variant 3a/b with exon 6a/b of EML4 (n = 12) fused to exon 20 of ALK, respectively. One hundred and ninety patients (26.3%) had tumors negative for EGFR mutations, KRAS mutations, EML4–ALK fusions, or RET rearrangements (hereafter referred to as quadri-negative). The presence of EGFR mutations, KRAS mutations, and RET rearrangements were mutually exclusive. The frequency of RET rearrangements was 6.3% among the 271 tumors harboring wide-type EGFR. Of the 159 tumors with wide-type EGFR and from never smokers, 12 (7.5%) had identified RET rearrangements. No patients with RET-rearranged tumors indicated a history of thyroid cancer or therapeutic radiation exposure. 3.2. Variants of RET fusion gene identified The RT-PCR primers used in this study enabled us to detect all eight KIF5B–RET and CCDC6–RET fusion variants identified to date (Table 1). Using RT-PCR and direct sequencing, 11 (65%) KIF5B–RET and 6 (35%) CCDC6–RET fusions were identified among the 17 tumors harboring RET rearrangements. The identified KIF5B–RET fusions comprised 8 KIF5B exon 15 fused to RET exon 12 (K15;R12), 2 KIF5B exon 22 fused to RET exon 12 (K22;R12), and 1 novel fusion variant involving a fusion of KIF5B exon 18 to RET exon 12 (K18;R12)
Fig. 1. Identification of two novel fusion variants of RET rearrangement. (A) A fusion of KIF5B exon 18 to RET exon 12. (B) A fusion of exon 5 and a proportion of intron 5 of CCDC6 to RET exon 11. Both of the fusion variants generate an in-frame RET fusion gene.
(Fig. 1). Of the tumors harboring CCDC6–RET fusions, the majority (5 of 6) involved CCDC6 exon 1 fused to RET exon 12 (C1;R12). In addition, we identified a novel fusion variant involving the fusion of exon 5 and a proportion of intron 5 of CCDC6 with RET exon 11, which was in an in-frame fashion (Fig. 1). 3.3. Clinical characteristics of patients with RET-rearranged tumors As shown in Table 3, the median age of patients with RETrearranged tumors did not significantly differ from those with EGFR-mutant, KRAS-mutant, EML4–ALK-rearranged, or quadrinegative tumors. The frequency of RET rearrangements among the 722 screened patients was also not associated with age (>60 versus ≤60 years, 2.2% versus 2.7%; P = 0.67), gender (males versus females, 2.6% versus 2.1%; P = 0.67), or smoking history (never smokers versus current or former smokers, 2.5% versus 2.5%; P = 0.96). By contrast, EGFR-mutant tumors were significantly more commonly present in female (55.4% versus 69.0%; P < 0.001) and never smokers (67.5% versus 46.0%; P < 0.001), while quadri-negative tumors occurred preferentially in male (32.2% versus 21.0%; P = 0.001) and current or former smokers (20.9% versus 42.4%; P < 0.001). Tumors with EML4–ALK fusions were more frequently seen in patients with age of 60 years or less (5.4% versus 9.9%; P = 0.02). The clinical details of the 17 patients with MPE of lung adenocarcinoma harboring RET rearrangements are provided in Table 4. The median age of these 17 patients was 63 years (range, 29–89 years), with equal distribution of gender (9 males versus 8 females). Twelve (71%) were never smokers, likely reflecting the increased frequency of never smoker in the entire study population (67.7%).
P < 0.001 P = 0.43 38 (7.8%) 12 (6.1%) P = 0.21
We observed that approximately half of patients (8 of 17, 47%) had a small primary lesion (≤3 cm). In addition to malignant effusion or pleural seeding, the most frequent sites of distant metastasis involved lung (5 of 17, 29%), brain (4 of 17, 24%) and bone (4 of 17, 24%), but liver (1 of 17, 6%) and adrenal (0 of 17, 0%) metastases were rare. Biopsy tissues for histological analysis were available from 7 of the 17 cases with RET-rearranged tumors (Table 4). Among these 7 cases, 6 (85.7%) were sub-classified as solid-predominant morphology. One case exhibited signet-ring cell appearance.
P < 0.001
7 (1.4%) 6 (3.0%)
P = 0.50 5 (1.4%) 8 (2.1%)
3.4. Outcome of patients with RET-rearranged tumors
There were 35 patients with missing data regarding smoking history. *
12 (2.5%) 5 (2.5%) 489 198
P = 0.96
330 (67.5%) 91 (46.0%)
P < 0.001 9 (2.6%) 8 (2.1%) 345 377
P = 0.67 10 (2.2%) 7 (2.7%) 460 262
P = 0.67
102 (20.9%) 84 (42.4%)
P = 0.001 111 (32.2%) 79 (21.0%) 29 (8.4%) 22 (5.8%)
P = 0.18
125 (27.2%) 65 (24.8%) P = 0.02 25 (5.4%) 26 (9.9%) P = 0.39 10 (2.2%) 3 (1.1%) 290 (63.0%) 161 (61.5%)
P = 0.67
67 53–77 66 31–96 63 29–89
211
Fig. 2. Overall survival after the initial diagnosis or relapse in metastatic disease in patients with malignant pleural effusion of RET-rearranged, EGFR-mutant, KRASmutant, EML4–ALK-rearranged, and quadri-negative lung adenocarcinomas. OS, overall survival.
Age, years Median Range Age, n (%) >60 years ≤60 years Gender, n (%) Male Female Smoking history, n (%)* Never smoker Current/former smoker
66 28–96
191 (55.4%) 260 (69.0%)
67 29–91 60 28–85
P = 0.49
Quadri-negative (n = 190) EGFR mutation (n = 451) RET rearrangement (n = 17) Total (n = 722) Characteristics
Table 3 Clinical characteristics of genotype-specific subsets of patients with malignant pleural effusion of lung adenocarcinoma.
KRAS mutation (n = 13)
ALK rearrangement (n = 51)
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The median follow-up time was 14.5 months for the 722 screened patients, and 15.0 months for the 17 patients with tumors harboring RET rearrangements. On the cutoff date for data collection, 195 (27.0%) patients were still alive, including 2 patients with RET-rearranged tumors. The median overall survival for the entire study population was 18.1 months (95% CI, 16.3–19.9). None of the 17 patients with RET-rearranged tumors had received multi-kinase inhibitors with activity against RET kinase, while 412 (91.4%) of the 451 patients with EGFR-mutant tumors had received EGFR TKIs. Fifteen (29.4%) of the 51 patients with tumors harboring EML4–ALK fusions had been treated with crizotinib. As shown in Fig. 2, the median OS was 22.4 months (95% CI, 8.8–36.0) for patients with RET-rearranged tumors, compared with 21.3 months (95% CI, 18.7–23.9; P = 0.57) for patients with EGFR-mutant tumors, 5.4 months (95% CI, 2.7–8.1; P = 0.002) for patients with KRAS-mutant tumors, 18.9 months (95% CI, 10.7–27.1; P = 0.82) for patients with EML4–ALK-rearranged tumors, and 12.0 months (95% CI, 9.0–15.0; P = 0.07) for patients with quadri-negative tumors. Patients with EGFR-mutant and EML4–ALK-rearranged tumors had significant longer OS than patients with quadri-negative (P < 0.001 and P = 0.012, respectively) and KRAS-mutant tumors (P < 0.001 and P = 0.003, respectively). 4. Discussion Our study represents a large survey of metastatic RETrearranged lung adenocarcinoma assessed in the context of multiplexed genotyping from MPE. It allows for an unprecedented comparison of RET-rearranged lung adenocarcinoma with other genomic subsets in the setting of metastatic disease with
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Table 4 Clinical details of 17 patients with malignant pleural effusion of lung adenocarcinoma harboring RET rearrangements. Case No.
Age (yr)
KIF5B–RET fusions 1 57 2 64 75 3 29 4 76 5 41 6 57 7 66 8 9 63 10 55 11 73 CCDC6–RET fusions 55 12 61 13 89 14 53 15 67 16
17
79
Gender
Smoking (pack yr)
Histology
F M M F F M
0 0 40 0 0 40
M M M F F
0 60 0 0 0
– Solid Solid – – Acinar, micropapillary – Solid – – –
M F M M F
0 0 30 30 0
F
0
– – – Solid Solid, signet ring cells with mucin staining Solid, micropapillary
Nodal status
Metastasis site
OS (month)
RET fusion variants
2.5 2.6 2.6 1.7 5.4 1.7
N1 N0 N2 N2 N1 N0
MPE, lung Brain MPE, liver, bone MPE, MPCE, bone MPE Pleural, brain
10.5 30.5 28.4 10.5 8.1 52.7*
K15;R12 K15;R12 K15;R12 K15;R12 K15;R12 K15;R12
3.6 11.0 4.5 3.0 4.7
N2 N3 N0 N0 N2
MPE MPE MPE, lung MPE, lung MPE, brain, bone
13.9* 4.2 11.7 11.6 32.7
K15;R12 K15;R12 K22;R12 K22;R12 K18;R12
3.4 1.8 1.3 7.7 7.0
N2 N3 N0 N3 N3
MPE, bone Lung MPE MPE, MPCE MPE, lung
22.4 17.1 30.3 33.7 22.7
C1;R12 C1;R12 C1;R12 C1;R12 C1;R12
4.5
N3
Brain
Tumor size (cm)
3.8
C5 ins intron 5 (AGTCTTGCTGTGTTGC);R11
Abbreviations: OS, overall survival; F, female; M, male; MPE, malignant pleural effusion; MPCE, malignant pericardial effusion. * Still alive on the cutoff date for data collection.
MPE. In our series, 17 (2.4%) of 722 patients with MPE of lung adenocarcinoma had tumors harboring KIF5B–RET or CCDC6–RET fusions, which included 2 novel fusion variants. The presence of RET-rearranged tumors was not associated with age, gender, or smoking history, but predominantly seen in solid histological subtype. Of note, our results suggest that the survival of metastatic RET-rearranged lung adenocarcinoma patients with MPE compares favorably with that of patients with EGFR-mutant tumors who had been treated with EGFR TKIs. In the earlier screening studies, RET rearrangements were reported to be present in 1–2% of unselected NSCLC patients of both East Asian and Caucasian descent [12–14,19,21,24,26,32]. By comparison, the frequency of RET rearrangements (2.4%) in our patients was modestly higher. We could not exclude the possibility that the higher prevalence of RET rearrangements in our series come from the selection of patients with metastatic disease. Several studies have implied that RET-rearranged tumors might be more prevalent in patients at advanced stage. For example, of the cohort reported by Wang et al. [19], the prevalence of RET rearrangements in lung adenocarcinoma was 1.3% (5 of 388) among patients with stage 1 or 2 disease, and 2.4% (6 of 245) among patients with stage 3 and 4 disease. Similarly, in the large series reported by Tsuta et al. [26], RET rearrangements were present in 1.1% (16 of 1496) of patients with stage 1 or 2 disease, and in 1.6% (6 of 364) of patients with stage 3 or 4 disease. Furthermore, probably because we enrolled patients with MPE of lung adenocarcinoma, our cohort included many female and never-smoker patients. As previously reported, adenocarcinoma in Asian never-smoker female patients may have distinct spectrum of genetic alterations [33]. It has been reported that patients with RET-rearranged NSCLC were relatively younger and preferentially never- or light-smokers, reminiscent of the patients characteristics of ALK-rearranged NSCLC [12,13]. However, the age in our cohort was similar among patients with RET-rearranged, EGFR-mutant, KRAS-mutant, and quadri-negative tumors. We also observed that the frequency of RET rearrangements was not associated with gender or smoking history. Thus, the relationship among age, smoking status, sex, and RET rearrangements remains unclear owing to the very low percentage of such populations. However, it is worth noting that
there were many female and never-smoker patients, along with a few young cases, among those with RET-rearranged tumors in this cohort. Of note, solid histologic subtype accounted for the majority of RET-rearranged tumors (six of seven with available tissue for histological analysis, 85.7%) in the current study, which is similar to the previous report by Wang et al. [19]. Knowledge of this clinical profile may help physicians select patients for the identification of this uncommon genetic alteration. In the current study conducted in Taiwan, we identified 11 (65%) KIF5B–RET and 6 (35%) CCDC6–RET fusions among the 17 RET-rearranged tumors. To date, KIF5B–RET fusions are present in approximately 90% of the RET rearrangements reported, with CCDC6–RET fusions accounting for the remaining 10% [27]. In the report by Takeuchi et al. [13], there was only one case (8%) with CCDC6–RET fusion gene in 13 identified RET-rearranged tumors from Japanese patients. The rate of CCDC6–RET fusions in RETrearranged tumors (3 of 22, 14%) was also low in another Japanese cohort [26]. By contrast, 3 of the 13 (23%) RET-rearranged tumors from Chinese patients contain CCDC6–RET fusions, as reported by Wang et al. [19]. The higher rate of CCDC6–RET fusions in our cohort is in line with that reported in Chinese patients. It is currently not clear whether this discrepancy is a result of the difference in genetic and environmental backgrounds among the screened populations. In the report by Drilon et al., the median overall survival of the five patients with metastatic RET-rearranged NSCLC was as long as 27 months [20,34]. However, three patients among them had received treatment with carbozantinib, a multi-kinase inhibitors with activity against RET kinase. Therefore, it is difficult to determine the prognostic effect of RET rearrangements in metastatic NSCLC. Although not statistically significant (probably owing to the limited number of patients with RET-rearranged tumor), our study suggested that metastatic RET-rearranged lung adenocarcinoma patients with MPE might have longer survival than those with quadri-negative tumors. Indeed, the survival of patients with RETrearranged tumors in our series was comparable to that of patients with EGFR-mutant tumors. It should be noted that the vast majority of our patients with EGFR-mutant tumors had been treated with EGFR TKIs, which might have prolonged the survival in this genomic subset. Our data raises the question of whether RET rearrangements
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may confer a more indolent phenotype and better outcome. In papillary thyroid cancer with CCDC6–RET fusions, it has been reported the expression of RET protein from the upstream CCDC6 promoters was relatively low [35]. This means that the oncogenic RET activity may be modest in tumors with CCDC6–RET rearrangements, which may correlate with the relatively benign phenotype of papillary thyroid cancer [16]. Further studies are needed to explore the transcription profile of RET protein in lung cancer harboring RET rearrangements, as well as its influence on the prognosis. Since RET-rearranged NSCLC potentially represents a therapeutically subcategory, an issue is to define an optimal biomarker assay for screening in practice. A variety of methods can be used, including immunohistochemistry (IHC), fluorescent in situ hybridization (FISH) and RT-PCR. Currently, there are no reliable RET antibodies for the detection of RET rearrangements in paraffin sections [19,32,36]. FISH is traditionally the standard technology to detect chromosomal translocations in tumor tissue. However, because of the high cost and need for technical expertise, FISH may not be an effective way to screen RET rearrangements that are present at a very low frequency in NSCLC. By contrast, RT-PCR is a rapid and sensitive method, that is, less expensive in terms of cost, and can be practically applied to cytological specimens. The RT-PCR primers used in this study were able to detect all KIF5B–RET and CCDC6–RET fusion variants identified to date. We could not exclude the possibility that a few RET-rearranged tumors, such as those harboring NCO4–RET and TRIM33–RET fusions, may have been missed. However, with the screening of approximately 5000 cases in total in previous studies, each one of these two RET rearrangements only appeared in an individual case, respectively [27]. We believe that NCO4–RET and TRIM33–RET rearrangements should be extremely rare in NSCLC. There are a number of limitations regarding this study that should be highlighted. Firstly, as we screened for RET rearrangements using specimens of MPE, the enrolled patients with metastatic lung adenocarcinoma were limited to those with MPE. Those with metastatic lung adenocarcinoma but without MPE will not be included in this study. Secondarily, because most of our patients had tissue diagnosis by small biopsy or cytology, comprehensive histological analysis was not feasible for all cases with RET-rearranged tumors. Finally, RET-rearranged tumors identified in this study were not subjected to RET FISH analysis to confirm the diagnostic accuracy of RT-PCR. 5. Conclusions The current study provides insights into the understanding of RET rearrangements in metastatic lung adenocarcinoma, and suggests a potential prognostic role of RET rearrangements in metastatic disease. We demonstrate the feasibility of multiplex RT-PCR with cytological samples for the screening of RET rearrangements in NSCLC. This screening strategy could be applied to facilitate the identification of these molecularly defined patients. Conflict of interest statement All authors report that they have no conflicts of interest. Acknowledgments The authors would like to thank the third core facility at National Taiwan University Hospital for technical assistance and facility support. This study was supported from grants 101-2314-B002169-MY3 and 101-2314-B002-170-MY3 (Ministry of Science and Technology, Taiwan), grant MOHW103-TDU-PB-211-144002 (Ministry of Health and Welfare, Taiwan), grant 103R7601-3 (National
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Taiwan University, Taiwan), and grant 103-FTN17 (National Taiwan University Hospital, Taiwan) to J.-Y.S.
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