Clinicopathologic characteristics and diagnostic methods of RET rearrangement in Chinese non-small cell lung cancer patients.

Background: Rearranged during transfection (RET) rearrangement has been identified as one of the crucial oncogenic drivers in non-small cell lung cancer (NSCLC). Recently, two highly selective RET inhibitors have been approved by the US Food and Drug Administration and demonstrated remarkable responses. However, the clinical characteristics, outcomes and optimal diagnostic method of RET-rearrangements are not well understood. This study sought to evaluate the prevalence and characteristics of RET rearrangement, identify an effective diagnostic method for it, and correlate its presence with outcomes.
Methods: A total of 9,431 Chinese NSCLCs from two cancer centers who have undertaken targeted DNA-NGS were enrolled and 167 RET-positive cases were screened. Non-canonical RET rearrangements were confirmed by targeted RNA-NGS. If material was sufficient, positive cases were analyzed by fluorescence in situ hybridization (FISH) (n=30) and immunohistochemistry (IHC) (n=57). Clinicopathologic characteristics, molecular profiling and treatment outcomes of RET rearrangement were evaluated.
Results: The prevalence of RET rearrangement was 1.52% (138/9,101) in unfiltered cases and 8.79% (29/330) in EGFR/KRAS/BRAF/ALK-negative cases. RET rearrangement was common in females, never smokers, and lung adenocarcinoma patients. Additionally, 40.3% of stage IV RET-rearranged NSCLC patients developed brain metastases. TP53 was the most common concurrent mutation, and 8 patients harbored concurrent driver oncogenic alterations, including EGFR (N=5), KRAS (N=2), and ALK (N=1). Non-canonical fusion partners were identified in 13.8% (23/167) of cases by DNA-based NGS, and RNA-based NGS identified 3 new partners (EPS8, GOLGA5, and TNIP1). The concordance of FISH and NGS was 83.3% (25/30), while the concordance of IHC and NGS was only 28.1% (16/57). Both IHC and FISH demonstrated lower sensitivity for NCOA4-/other-RET fusions. The CCDC6-RET subgroup had significantly longer progression-free survival than the KIF5B-RET subgroup, both after chemotherapy (23 vs. 9.7 months; P=0.014). Conclusions: RET rearrangement occurs in 1.52% of Chinese NSCLCs and has identifiable clinicopathologic characteristics. RET IHC has a low sensitivity, disavowing its use in routine practice. While NGS and FISH has good performance in identifying RET rearrangement. Both IHC and FISH demonstrated lower sensitivity for NCOA4-/others-RET fusions. Clinical benefit with chemotherapy is different between CCDC6-RET and KIF5B-RET fusion patients, optimal treatment should be considered when selecting therapies for patients with RET-rearranged lung cancers. 2022 Translational Lung Cancer Research. All rights reserved.

Introduction

The discovery of targetable oncogenic drivers has led to significant improvements in the treatment of non-small cell lung cancer (NSCLC). Among these, a number of fusion drivers, such as anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1), and rearranged during transfection (RET), have been described as rare oncogenic events and recommended for routine testing at diagnosis (1).

The RET proto-oncogene was first identified in 1985 by Takahashi et al. (2). It encodes a transmembrane receptor tyrosine kinase that plays a key role in the differentiation of the kidneys and nervous system (3,4). The rearrangement of RET can lead to the constitutive activation of the RET tyrosine kinase domain and the recruitment of downstream signaling cascades, such as MAP kinase (MAPK), phosphatidylinositol-3-kinase (PI3K)/Akt, and Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways, which promote tumorigenesis (5).

Several multi-kinase inhibitors (MKIs), such as vandetanib and cabozantinib, have been used for targeted therapy to treat advanced RET-rearranged NSCLC patients. However, MKIs sometimes leads to significant “off-target” side effects, such as nausea, diarrhea, and hypertension (6,7). Recently, novel RET tyrosine kinase inhibitors (TKIs) with high selectivity, including selpercatinib (LOXO-292) and pralsetinib (BLU-667), have been approved by the Food and Drug Administration (FDA) for the treatment of advanced RET-rearranged lung and thyroid cancer (8-10). Therefore, it is important to rapidly and accurately identify RET-positive cases.

However, due to the low prevalence of RET fusions, there is only limited information about the clinical characteristics and outcomes of RET-rearranged NSCLC, especially in Chinese patients. Incidence of RET rearrangement ranges from 1% to 2% in NSCLC (11-13), and depends on the age, sex, smoking history and histological subtype. Different fusion partners have been identified in NSCLC, with the most major proportion being KIF5B (14). RET rearrangements tend to be mutually exclusive with other driver mutations in NSCLC such as EGFR, KRAS, ALK and ROS1, and associate with low PD-L1 expression (15) and low tumor mutation burden (16).

Furthermore, the screening methods for RET rearrangement have not yet been standardized. Several molecular diagnostic methods are used to identify gene fusions, including IHC, RT-PCR, FISH, and DNA/RNA-based NGS. Although IHC is an effective screening tool to detect ALK-positive patients, it showed low sensitivity (55–65%) and variable specificity (40–85%) in prior study (17) and may not be reliable to detect RET rearrangement. RT-PCR is specific, but it is limited to known fusion partners and thus may underestimate prevalence. RET FISH is highly sensitive (100%) but has suboptimal specificity (45–60%) (17,18). Moreover, break-apart displays low sensitivity in detecting non-canonical RET fusions (19). DNA-based NGS enables the detection of high-throughput genomic alterations and novel partners of gene rearrangement, but it fails to provide information on functional fusion transcripts (20). The main laboratory methods for RET rearrangements have not been systematically investigated and fully elucidated in NSCLC. Therefore, investigation of the prevalence, characteristics, and diagnostic methods in a large cohort of NSCLC patients may provide comprehensive genomic profiling and optimal strategy for the selection of RET-rearranged patients.

This study analyzed the molecular profile of 9,431 Chinese NSCLC patients who underwent targeted DNA-based NGS in daily clinical practice at two large cancer centers and identified 167 RET-rearranged NSCLC cases. Different techniques, including RNA-based NGS, FISH, and IHC, were performed to investigate their performance. The prevalence, clinicopathologic characteristics, molecular profiling, and therapeutic outcomes of the RET-rearranged cases were analyzed. We present the following article in accordance with the REMARK reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-202/rc).

Methods

Patients and study design

This retrospective analysis included 9,431 NSCLC patients in a multi-center study from January 2017 to August 2020 (including the Henan Cancer Hospital, Zhengzhou, China and National Cancer Center/Cancer Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China). All patients who met the following criteria were included in the analysis: confirmed NSCLC by pathology; detected the mutations by NGS (8/56 cancer-related genes). Altogether, the cohort included 9,101 NSCLC patients without mutation-based pre-selection and 330 epidermal growth factor receptor (EGFR)/KRAS proto-oncogene (KRAS)/B-Raf proto-oncogene (BRAF)/ALK-negative patients (EGFR/KRAS/BRAF mutation status was tested by PCR, and ALK fusion status was tested by IHC-Ventana) (). RET-positive cases were collected and analyzed. RNA-NGS were performed in non-canonical fusion subtypes. FISH (N=30) and IHC (N=57) assays were performed in RET-rearranged patients with sufficient tissue. Patients’ medical records were retrospectively reviewed to collect data on age, sex, smoking status, tumor stage at diagnosis, pathological diagnosis, and treatment histories. The stage of each patient was assessed following American Joint Committee on Cancer Staging Manual version 7. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the local ethics committee of The Affiliated Cancer Hospital of Zhengzhou University (No. 2021-KY-0092), and was also approved by the institutional review board of National Cancer Center/Cancer Hospital, Chinese Academy of Medical Science and Peking Union Medical College (No. 20/444-2640). Written informed consent was obtained from all individuals included in the study.

Figure 1

Study flow charts. A total of 9,431 patients from the Henan Cancer Hospital and the National Cancer Center/Cancer Hospital, Chinese Academy of Medical Science were enrolled in this study from January 2017 to August 2020. NSCLC, non-small cell lung cancer; RET, rearranged during transfection; NGS, next-generation sequencing; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization.

Targeted DNA-NGS

Genomic DNA from formalin-fixed paraffin embedded (FFPE) tissue was isolated using the QIAamp DNA FFPE Tissue Kit (Qiagen, Duesseldorf, Germany). Libraries were prepared using commercial panels (Burning rock Technology, Guangzhou, China). Briefly, a panel covering 8 driver genes of NSCLC was used on 4,320 patients from the Henan Cancer Hospital. A panel covering 56 cancer-related genes was used on the other patients enrolled in the study, as previously reported (21). Samples were sequenced on the Nextseq 550 (Illumina, San Diego, CA, USA) with an average depth of 1,000×. All the reads were mapped to human genome 19, and alignments were visualized using the Integrative Genomics Viewer.

Targeted RNA-NGS

Total RNA from the FFPE tissue was extracted using the Magen FFPE DNA/RNA kit (Magen, Guangzhou, China). The quantity and quality of RNA were detected using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and the RNA Pico Sensitivity Reagent Kit (PerkinElmer), respectively. Libraries were prepared using the commercial panel (Burning rock Technology) containing 115 fusions and splice-region variants, which covered the entire coding region of RET. NGS was performed on the Novaseq-6000 platform (Illumina) with at least 25M reads per sample.

FISH and IHC

FISH was performed using the RET (10q11) dual-color break-apart rearrangement probe (LBP Medicine Science and Technology, Guangzhou) and following the manufacturer’s instructions. For each sample, >100 tumor cells were evaluated. Samples were considered RET-rearrangement when ≥15% of the tumor cells showed split signals or isolated 3' signals. Isolated 5' signals were thought to result from the deletion of the kinase domain and were considered negative. IHC was performed using a rabbit monoclonal anti-RET (EPR2871) antibody (ab134100, Abcam, Cambridge, MA). RET expression was evaluated according to the following intensity scores: 0: negative; 1+: weak; 2+: moderate, and 3+: strong in >10% of tumor cells. The FISH and IHC results were evaluated by 2 pathologists independently, blinded to the NGS results. We evaluated the RET-fusion using NGS as the reference standard in this study. FISH and IHC assays were performed in RET-rearranged patients with sufficient tissue. Consistency was defined as the percent of positive events to NGS results.

Statistical analysis

The Kaplan-Meier method was used to determine progression-free survival (PFS), and differences between groups were calculated using the log-rank test. The treatment response was assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (22). All the statistical analyses were conducted using GraphPad Prism 5 software. The clinical characteristics of the different groups, including gender, age, smoking history, histology, and brain metastasis, were compared by the χ2 test or Fisher exact test. Cox’s proportional-hazards model was used to estimate the hazards ratio (HR) and the corresponding 95% confidence interval (95% CI) for the covariates of interests. Variables included sex, smoking, age, histology, stage, RET-rearranged subtype, breakpoint, distant metastasis, brain metastasis were selected for univariate analysis. Covariates with P value <0.10 from univariate analysis were considered for multivariable model. Statistical significance was defined as a 2-sided P value <0.05.

Results

Prevalence and characteristics of RET-rearranged NSCLC

In total, 167 RET-rearranged NSCLC patients were detected in our study. Among the 9,101 NSCLC patients without mutation-based pre-selection, 1.52% (138/9,101) were detected to have RET rearrangement, while in EGFR/KRAS/BRAF/ALK-negative NSCLC patients, the prevalence of RET rearrangement was 8.79% (29/330) ().

The clinicopathological characteristics were accessible for 129 RET-rearranged NSCLC patients (). The median age at diagnosis was 57 years (range, 30–83 years). RET rearrangements were more frequent in females (65.1%) and never smokers (82.2%). The most common histological subtype of RET-rearranged NSCLC patients was lung adenocarcinoma (92.3%), but other subtypes were also detected, including squamous cell carcinoma (1.5%), large cell carcinoma (0.8%), and neuroendocrine carcinoma (1.5%). Among the 62 stage IV RET-rearranged patients, 74.2% (46/62) had distant metastasis, such as bone, brain, or liver metastasis. Notably, 40.3% (25/62) of the stage IV RET-rearranged patients had brain metastasis.

Table 1

Clinicopathological characteristics of RET-rearranged NSCLC patients (N=129)

Patient characteristics	No. (%) of patients (N=129)
Median age, years [range]	57 [30–83]
Gender
Female	84 (65.1)
Male	45 (34.9)
Smoking
Never	106 (82.2)
Smoker	23 (17.8)
Histology
Adenocarcinoma	119 (92.3)
Squamous	2 (1.5)
Adenosquamous	5 (3.9)
Large cell carcinoma	1 (0.8)
Neuroendocrine carcinoma	2 (1.5)
Ki-67 (%)
<20%	9 (7.0)
20–39%	13 (10.1)
40–59%	9 (7.0)
≥60%	18 (13.9)
NA	80 (62.0)
Stage at diagnosis
I	37 (28.7)
II	7 (5.4)
III	23 (17.8)
IV	62 (48.1)
Distant metastasis (% of stage IV)
No	16 (25.8)
Yes	46 (74.2)
Brain metastasis (% of stage IV)
No	37 (59.7)
Yes	25 (40.3)

RET, rearranged during transfection; NSCLC, non-small cell lung cancer; NA, not available.

Partners of RET fusion

Among the 167 RET rearrangements, the most common fusion partner was KIF5B (68.2%, 114/167), followed by coiled-coil domain containing 6 (CCDC6) (16.8%, 28/167), and nuclear receptor coactivator 4 (NCOA4) (1.2%, 2/167) (). The breakpoint of RET was most frequently observed in intron 11, but other breakpoints included intron 10 and exon 11 (). In relation to the fusion partners, the most common breakpoints were KIF5B intron 15 (91% of KIF5B-RET patients) and CCDC6 intron 1 (93% of CCDC6-RET patients) ().

Figure 2

Overall landscape of RET-rearranged NSCLCs detected by NGS (N=167). (A) Proportions of different RET rearrangement partners; (B) percent of RET breakpoint positions according to the fusion subtypes; (C) distribution of RET-fusion partners’ breakpoints; (D) concurrent genetic alteration analysis demonstrated by oncoPrint. The top bar indicates the number of mutations in each patient. The right-side bar demonstrates the number of patients harboring a specific mutation. Different colors indicate different mutation type categories. RET, rearranged during transfection; NSCLC, non-small cell lung cancer; NGS, next-generation sequencing.

Different characteristics were compared between the 90 KIF5B-RET patients and 23 CCDC6-RET patients with accessible clinical records (Table S1). The incidence of brain metastasis and distant metastasis in KIF5B-RET patients was higher than that in CCDC6-RET patients; however, the differences were not statistically significant.

Additionally, non-canonical fusion partners were also identified in 23 patients, including ADAM metallopeptidase with thrombospondin type 1 motif 2 (ADAMTS2), Rho GTPase activating protein 12 (ARHGAP12), centrosomal protein 128 (CEP128), epidermal growth factor receptor pathway substrate 8 (EPS8), and intergenic fusions. The relevant clinical information is listed in . Of the fusion partners, 3 have been reported as individual cases in the literature (23-25), but others have never been reported. RNA-based NGS was then performed to verify non-canonical RET rearrangement. Among the 23 non-canonical fusion cases, 10 cases had samples available for RNA-NGS and were all proven to have functional RET fusions at the RNA level (). It is of great clinical significance to evaluate the RET-TKI efficacy in such cases.

Table 2

Non-canonical fusion cases and their relevant clinical information (N=23)

Fusion	Breakpoints	Locus of the partner gene	Gender	Age (years)	Histology	Literature	RNA-NGS results	FISH	IHC
ADAMTS2-RET	Intron10_Exon3	5q35.3	M	54	ADC	NA	NA	NA	NA
ARHGAP12-RET	Intron4_Intron11	10p11.22	F	65	ADC	NA	KIF5B-RET (Exon15_Exon12)	+	1+
CEP128-RET	Intron18_Intron10	14q31.1	F	63	ADC	NA	NA	NA	NA
EPS8-RET	Intron12_Intron11	12p12.3	F	56	ADC	NA	EPS8-RET (Exon12_Exon12)	NA	1+
ERC1-RET	Intron8_Intron11	12p13.33	M	54	ADC	PMID: 32737449	NA	NA	−
KIAA1217-RET	Intron1_Intron11	10p12.2-p12.1	F	70	ADC	PMID: 31162284	KIF5B-RET (Exon15_Exon12)	+	2+
PLCXD3-RET/LINC01264-RET	Intron2_Intron11/intergenic_Intron11	5p13.1; 10q11.21	M	83	ADC	NA	KIF5B-RET (Exon15_Exon12)	NA	NA
SLC6A11-RET	Intron5_Intron11	3p25.3	M	71	ADC	NA	NA	NA	NA
SPECC1L-DORA2A-RET	Intron10_Intron11	22q11.23	F	60	ADC	PMID: 31917708	NA	NA	NA
STK33-RET	Intron1_Intron11	11p15.4	F	65	ADC	NA	NA	NA	NA
BET1-RET	Intergenic_Intron11	7q21.3	F	56	ADC	NA	KIF5B-RET (Exon15_Exon12)	+	1+
CENPK-RET	Intergenic_Exon12	5q12.3	M	58	SCC	NA	NA	NA	NA
FXYD4-RET	Intergenic_Intron11	10q11.21	F	54	ADC	NA	NA	NA	NA
LINC00680-RET	Intergenic_Intron10	6p11.2	F	63	ADC	NA	GOLGA5-RET (Exon7_Exon12)	+	1+
KIAA0146-RET	Intergenic_Intron11	8q11.21	M	65	SCC	NA	NA	NA	NA
LOC105378330-RET	Intergenic_Intron11	10q21.3	F	64	ADC	NA	NA	NA	NA
LOC105378470-RET	Intergenic_Intron11	10q25.1	F	64	ADC	NA	KIF5B-RET (Exon15_Exon12)	NA	NA
LOC441666-RET	Intergenic_Intron11	10q11.21	F	67	ADC	NA	NA	NA	NA
MARCH8-RET	Intergenic_Intron11	10q11.21-q11.22	F	56	ADC	NA	KIF5B-RET (Exon15_Exon12)	+	2+
NAMPTL-RET	Intergenic_Intron11	10p11.21	F	49	AdCa	NA	NA	NA	NA
OR13A1-RET	Intergenic_Intron11	10q11.21	M	58	ADC	NA	NA	NA	NA
TNIP1-RET/ RASGEF1A-RET	Intron8_Intron11/intergenic_Intron11	5q33.1; 10q11.21	M	65	ADC	NA	TNIP1-RET (Exon8_Exon12)	−	3+
TBC1D14-RET	Intergenic_Intron11	4p16.1	F	65	ADC	NA	KIF5B-RET (Exon15_Exon12)	NA	NA

F, female; M, male; ADC, adenocarcinoma; SCC, Squamous Cell Carcinoma; AdCa, Adenosquamous; NA, not available; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; −, negative; +, positive; 1+, weak; 2+, moderate; 3+, strong.

Mutation profile and concurrent driver gene alterations

As shows, 106 RET-arranged patients were detected using 56 cancer-related gene panel. Co-occurring genetic aberrations were found in 77 patients (77/106, 73%). We constructed a heatmap to demonstrate the alterations co-occurring with the RET rearrangements (). Tumor protein 53 (TP53) was the most commonly altered (34/77, 44%), followed by BRCA2 (8/77, 10%), PTCH1 (7/77, 9%), ATM (6/77, 8%), EGFR (6/77, 8%), and TSC2 (6/77, 8%). Other genomic alterations, including MYC, CDK4, MET, FGFR3, and PIK3CA, were also observed. Among the 106 RET-rearranged patients, 8 (7.55%) harbored concurrent driver gene alterations, including EGFR L858R (N=3), EGFR 19del (N=2), KRAS G12X (N=2), and EML4-ALK (N=1) (for further details, see Table S2).

Treatment and clinical outcomes of RET-rearranged NSCLC

Among the 129 RET-rearranged NSCLC patients with available treatment information, approximately 33.3% (43/129) underwent platinum-based doublet chemotherapy as the first-line treatment, 22.5% (29/129) received chemotherapy combined with antiangiogenic therapy as the first-line treatment, and 3.1% (4/129) received immune checkpoint inhibitors in clinical trials (). To evaluate the chemotherapy efficacy among different subtypes, the survival data of 36 late-stage patients were analyzed, including 28 KIF5B-RET patients and 8 CCDC6-RET patients. We found that patients with the CCDC6-RET subtype had significantly longer PFS than those with the KIF5B-RET subtype (23 vs. 9.7 months; P=0.014) (). Additionally, no significant difference was observed between the different breakpoints of RET (intron 11 vs. other locations) (). As KIF5B-RET patients appeared to suffer from brain and distant metastasis more than CCDC6-RET patients (Table S1; albeit the difference was not statistically significant), RET-rearranged subtypes were included in the Cox proportional-hazards model with other clinical characteristics. The results of the univariate and multivariate Cox proportional-hazards model based on the 36 RET-rearranged cases are listed in Table S3. Covariates with a P value <0.10 in the univariate analysis were included in the multivariable model. According to the multivariable analysis, CCDC6-RET cases had significantly better PFS than KIF5B-RET cases (HR =0.192, 95% CI: 0.044–0.831; P=0.027). Research with a sufficiently large cohort needs to verify these findings.

Figure 3

Outcomes of 129 RET-rearranged NSCLCs and representative FISH image and IHC staining pattern of RET-rearranged cases. (A) First-line treatment strategies of 129 RET-rearranged NSCLCs. (B) PFS analysis between KIF5B-RET and CCDC6-RET subtypes treated with chemotherapy (left). PFS analysis between different RET breakpoints in patients treated with chemotherapy (right). (C) RET FISH and IHC staining (i: 200×; ii-iii: 1,000×; iv-ix: 100×). HE-stained section of a lung adenocarcinoma with RET rearrangement (i). Representative image of RET-FISH using a break-apart probe (ii). Example of RET FISH testing showing equivocal signals (iii). RET-IHC negative NSCLC (iv). RET-IHC showing positive (3+) reaction in a KIF5B-RET case (v), 2+ positivity in a KIF5B-RET (vi), 2+ positivity in a CCDC6-RET case (vii), and a 1+ positivity in a NCOA4-RET case (viii). Expression of the RET protein was detected in nonneoplastic tracheal tissue (ix). RET, rearranged during transfection; NSCLC, non-small cell lung cancer; Chemo, platinum-based doublet chemotherapy; NA, not available; TKI, tyrosine kinase inhibitor; IHC, immunohistochemistry; HE, hematoxylin-eosin; FISH, fluorescence in situ hybridization; PFS, progression-free survival.

Only 4.7% (6/129) of the patients had access to RET-TKI, mainly due to the inaccessibility of RET-TKI at that time. A 54-year-old male with poorly differentiated lung adenocarcinoma had disease progression after receiving surgery, radiotherapy, and chemotherapy. The targeted NGS revealed an ERC1-RET fusion. Subsequently, after being started on Cabozantinib, SD was achieved. The patient continued to receive Cabozantinib treatment for 10 months before disease progression with new lung metastasis (Figure S1).

None of the 8 patients with concurrent driver gene alterations received RET-TKI treatment. One lung adenocarcinoma patient (Case No. 1) with EGFR L858R and KIF5B-RET has received Icotinib for almost 2 years and achieved stable disease (SD). When the patient’s disease progressed, KIF5B-RET continued to be detected, but EGFR L858R disappeared. Another lung adenocarcinoma patient with EGFR 19del (Case No. 5) has been receiving Gefitinib for 2 years, and CCDC6-RET was detected as a resistant mechanism in the EGFR-TKI relapsed tumor (Table S2).

Detection of RET rearrangement

Among the 167 RET-positive cases detected by DNA-NGS, 144 were canonical fusion subtypes, and 23 were non-canonical fusion subtypes. A total of 10 non-canonical fusion samples were available for RNA-NGS, and all were proven to have functional RET fusions at the RNA level (). Representative fusion patterns at DNA and RNA levels are shown in Figure S2 using IGV.

FISH (N=30) and IHC (N=57) assays were performed in RET-rearranged patients with sufficient tissue. The FISH results revealed the RET rearrangement in 25/30 patients, resulting in the FISH/NGS concordance of 83.3% (). IHC intensity scores were 0 in 3.5% (2/57), 1+ in 35.1% (20/57), 2+ in 33.3% (19/57), and 3+ in 28.1% (16/57) of the RET-rearranged patients. IHC 3+ was considered positive, and the concordance of IHC and NGS was 28.1%. The staining pattern of RET-IHC varied in different fusion subtypes. Notably, KIF5B-RET showed diffuse, 2+/3+ cytoplasmic staining, while CCDC6-RET and others showed granular and patchy staining with weak intensity (). The normal tracheal epithelium also showed RET-IHC staining, which might lead to a staining pitfall in the interpretation of IHC results. We noted that IHC had extremely low sensitivity in non-KIF5B-RET patients (CCDC6-RET, 8%, NCOA4-RET, 0%, and other-RET, 25%), while FISH also showed unsatisfying sensitivity in non-KIF5B-RET patients. FISH or IHC were not able to detect NCOA4-RET cases.

Table 3

Concordance of different RET-fusion testing techniques

Test result	NGS status
	KIF5B-RET	CCDC6-RET	NCOA4-RET	Others-RET	All patients
RET-IHC
N	39	12	2	4	57
3+	14	1	0	1	16
2+	14	5	0	0	19
1+	11	6	1	2	20
0	0	0	1	1	2
Concordance	35.9%	8.3%	0.0%	25.0%	28.1%
RET-FISH
N	19	7	2	2	30
Positive	18	6	0	1	25
Negative	1	1	2	1	5
Concordance	94.7%	85.7%	0.0%	50.0%	83.3%

RET, rearranged during transfection; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; 0, negative; 1+, weak; 2+, moderate; 3+, strong; NGS, next-generation sequencing.

Discussion

To the best of our knowledge, this study has one of the largest RET-rearranged NSCLC cohorts for which a comprehensive analysis of molecular profiling, clinical outcomes, and detection methods has been performed. This study enrolled 9,431 Chinese NSCLC patients, among whom NGS identified 167 RET-rearranged patients. In 9,101 Chinese NSCLC patients without molecular-based pre-selection, the prevalence of RET rearrangement was 1.52%, reflecting the findings of previous reports (26,27). In 330 EGFR/KRAS/BRAF/ALK-negative NSCLC patients, the prevalence of RET rearrangement was up to 8.8%, indicating the necessity of RET detection in NSCLC when other driver genes are negative. Similar to ALK- and ROS1-rearranged NSCLC (28), RET rearrangement was more common in female, never smokers, and lung adenocarcinoma patients. Importantly, 40.3% (25/62) of the stage IV RET-rearranged NSCLC patients had brain metastasis. This is much higher than the average brain metastasis rate reported for advanced NSCLC (10–20%) (29), and is especially high in KIF5B-RET patients (43%). Previous studies have also reported that RET fusion is an independent risk factor of brain metastasis (30,31). This observation may reinforce the importance of evaluating the intracranial therapeutic response of RET-TKIs based on the molecular subclass of tumors. Selective RET inhibitors, including pralsetinib and selpercatinib, have been approved by the FDA, and both of them have shown a significant ability to cross the blood-brain barrier (31-33).

To date, at least 15 RET-rearranged subtypes have been reported in NSCLC, including KIF5B-RET, CCDC6-RET (34), NCOA4-RET (35), TRIM33-RET (36), KIAA1217-RET (37), ERC1-RET (38), and MYO5C-RET (39). In this study, diverse RET fusion partners were identified, including canonical partners, such as KIF5B (68.2%, 114/167), CCDC6 (16.8%, 28/167), and NCOA4 (1.2%, 2/167). Rare partners, such as KIAA1217 and TBC1D32, were also identified, which have been reported in a previous study (40). Among the 23 non-canonical fusion subtypes identified in our study by DNA-NGS, 10 with sufficient tumor tissue were verified to harbor functional fusion transcripts by RNA-NGS, and 3 novel partners (EPS8, GOLGA5, and TNIP1) were found. Numerous breakpoints of ALK rearrangement have been reported to be associated with clinical benefits (17,41). However, the breakpoints of RET and its partners were relatively concentrated in our study, mainly in RET intron 11, KIF5B intron 15, and CCDC6 intron 1 and no significant survival difference for chemotherapy was observed between the different RET breakpoints.

We also characterized the mutational profile of the RET-rearranged patients and found that TP53 was the most common concurrent alteration. Previous studies have suggested that TP53 concomitant mutations have a strong negative effect on the outcomes of patients with EGFR-mutant (42-45) and ALK-rearranged NSCLC (43,46,47). The poor prognostic effect of the TP53 mutation on tumors may be due to the loss of tumor inhibitory function and the elevated level of genomic instability (48). It is generally believed that RET rearrangement occurs exclusively with other oncogenic drivers in treatment-naive lung cancers (49). However, 8 RET-rearranged NSCLC patients in our study harbored concurrent oncogenic driver gene alterations. CCDC6-RET was found to be a resistant mechanism in an EGFR L858R patient, and the 7 other RET-rearranged NSCLC patients with concurrent driver gene alterations were all treatment-naïve patients. Among these treatment-naïve patients, only one KIF5B-RET patient with EGFR L858R received EGFR-TKI Icotinib treatment, and that patient had a PFS time of 23 months. Intratumor heterogeneity may explain multi-driver gene alterations. Sun (50) and Kim (51) reported that RET fusion could occur as an acquired resistance mechanism to Osimertinib. Additionally, McCoach (52) reported that RET rearrangement could also act as an acquired resistance mechanism of ALK-TKI. Thus, we believe that screening for RET fusion in post-treatment settings is clinically significant.

Due to the inaccessibility of the RET-TKI at the time of diagnosis in our study cohort, most RET-rearranged patients received chemotherapy. Pemetrexed-based chemotherapy for NSCLC patients with RET fusion-positive metastatic NSCLC has been shown to provide a durable benefit (53). In our study, the CCDC6-RET subgroup had a significantly longer PFS than the KIF5B-RET subgroup. Tan et al. reported that overall survival was more prolonged in CCDC6-RET fusion than KIF5B-RET fusion-positive patients (54). However, the reasons for a better prognosis in CCDC6-RET than KIF5B-RET patients remain unclear.

Precision medicine for tumors depends on an effective and reliable detection method. This is even more important for mutations which exist only in a small proportion of patients. Ideally, molecular detection should be highly sensitive, specific, and feasible in most diagnostic laboratories. At present, there is no gold-standard for RET rearrangement detection. FISH has been the gold-standard assay for diagnosing ALK- and ROS1-fusions (55). It is available in most pathology laboratories, and has a low tumor tissue requirement. However, our study revealed that RET FISH might lead to false-negative results, especially in CCDC6-RET and non-canonical RET-fusion subtypes. Technically, the RET FISH test may be more challenging than most other break-apart assays, as RET and its most common fusion partners are situated very near to each other on chromosome 10 (approximately 7.9–17.9 Mb apart) and are thus difficult to interpret. Radonic indicated that FISH is a sensitive but unspecific technique for RET screening (56). RET-IHC has not generally been recommended in previous studies, as IHC is more likely to yield false-negative results (17,57,58), which was also observed in our study. The current impediments of RET-IHC include the low-level expression of the RET-fusion protein and the lack of specific antibodies. We also observed RET expression in the normal tracheal epithelium, which might lead to a false-positive result.

Targeted DNA-NGS in RET detection is accurate and comprehensive and thus provides a unique advantage in exploring novel partners and the simultaneous testing of multiple genes. However, DNA-NGS has limitations in identifying complex fusions, and RNA-NGS adds value to accurate detection (Figure S2). The discordance of RET fusion at the DNA and RNA level may be due to alternative splicing and the flexible break-induced repair mechanisms (59,60). Taking all these factors into consideration, we recommend DNA-NGS as a preliminary screening strategy for patients who have been newly diagnosed. A FISH analysis may be an appropriate method when the specimens have too low tumor cell content. For unusual results of DNA-NGS or FISH, RNA-NGS can be used as a validation technique.

There are several limitations of our study. It is a retrospective study, in only two major centers. The NGS methods were not completely the same in the whole study cohort. Another limitation of our study is that due to the unavailability of targeting agents, the number of patients receiving RET-TKIs was small. Therefore, no correlation could be done between the clinical efficacy of RET-TKIs with fusion. We intend to conduct further studies that include more clinical features of RET-rearranged patients and prognostic evaluations of different RET-fusion subtypes.

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