Characterizing kinase intergenic-breakpoint rearrangements in a large-scale lung cancer population and real-world clinical outcomes

Patients

A large cohort of lung cancer cases (N = 30 450) was subjected to comprehensive genomic profiling using targeted NGS of 425 cancer-related genes in a Clinical Laboratory Improvement Amendments-certified, College of American Pathologists-accredited laboratory (Nanjing Geneseeq Technology, Jiangsu, China), as previously described.¹⁹ Patients who had kinase IGRs were identified in our Laboratory Information Management System using a natural language search program. Patients’ demographic and clinical data, including age, sex, histology type, pathological stage, and treatment history, were retrospectively evaluated. This study was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University Hospital, Henan, China. All participants provided written informed consent before sample collection.

DNA extraction and targeted enrichment

Circulating tumor DNA from plasma was purified using the Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. Genomic DNA from white blood cells was extracted using the DNeasy Blood & Tissue Kit (Qiagen), while formalin-fixed paraffin-embedded (FFPE) genomic DNA was purified using the QIAamp DNA FFPE Tissue Kit (Qiagen). All DNA was quantified using the dsDNA HS Assay Kit on a Qubit Fluorometer (Life Technologies, Bleiswijk, The Netherlands). Sequencing libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, MA), as described previously.¹⁹ Indexed DNA libraries for all solid tumors were pooled for probe-based hybridization capture of the targeted 425 cancer-related genes.

Sequence data processing

Sequencing was carried out using the HiSeq 4000 platform (Illumina, Essex, UK), followed by data analysis, as previously described.²⁰ In brief, sequencing data were analyzed by Trimmomatic²¹ to remove low-quality (quality <15) or N bases, and were then mapped to the human reference genome, hg19, using the Burrows-Wheeler Aligner (https://github.com/lh3/bwa/tree/master/bwakit). PCR duplicates were removed by Picard (available at https://broadinstitute.github.io/picard/). The Genome Analysis Toolkit (GATK) (https://software.broadinstitute.org/gatk/) was used to perform local realignments around indels and base quality reassurance. Gene fusions were identified by Factera.²² Somatic single-nucleotide polymorphisms (SNPs) and indels were analyzed by VarScan2²³ and Mutect2, with the mutant allele frequency cut-off at 2% for tissue samples, 0.2% for cell-free DNA samples, and a minimum of three unique mutant reads. Common SNPs were excluded if they were present in >1% population frequency in the 1000 Genomes Project or the Exome Aggregation Consortium 65 000 exomes database. The resulting mutation list was further filtered by an in-house list of recurrent artifacts based on a normal pool of whole blood samples. The gene rearrangements, with one breakpoint within a coding region of kinase genes and the other in an intergenic region, were identified as kinase IGRs, the allele frequency cut-off of which was 0.3%.

RNA sequencing

Total RNA from FFPE samples was extracted using the RNeasy FFPE kit (Qiagen). The total RNA was quantified using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Ribosomal RNA and residual genomic DNA were depleted using the KAPA Stranded RNA-Seq Kit (KAPA Biosystems) with RiboErase (HMR) and DNase digestion, followed by purification using the Agencourt RNA Clean XP Beads (Beckman Coulter, Beverly, MA) according to the manufacturer's protocol. The KAPA Stranded RNA-Seq Library Preparation Kit was used to construct Illumina-compatible sequencing libraries, including RNA fragmentation and priming, double-stranded cDNA synthesis, adaptor ligation, and PCR amplification.

Sequencing was carried out on Illumina HiSeq NGS platforms (Illumina). To generate sequence reads in the FASTQ format, base calling was carried out on bcl2fastq v2.16.0.10 (Illumina, Inc.) and quality control was carried out with Trimmomatic (version 0.33).²¹ RNA-Seq reads were mapped to the human genome (hg19, Genome Reference Consortium GRCh37) using STAR (version 2.5.3a)²⁴ to identify individual exon, intron, and intergenic features. The average coverage of mapped reads across the base positions of the feature coordinates was calculated. Gene fusions were detected and visualized on the Integrative Genomics Viewer.²⁵

IHC and assessment

IHC staining was carried out as previously described.²⁶ In brief, paraffin embedded specimen (4 mm sections) slides were baked at 65°C for 30 min, then deparaffinized with xylene and rehydrated with ethanol. For antigen retrieval, slides were steamed in 1× EDTA (pH 9.0) at 99-100°C for 10 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. The slides were incubated with an anti-ALK antibody (1/250; D5F3; Cell Signaling Technology, Danvers, MA) overnight at 4°C in a humidified chamber. Using the OptView DAB IHC Detection Kit (Model: 6396500001; Roche, Basel, Switzerland), the tissue sections were treated with biotinylated anti-rabbit secondary antibody, followed by further incubation with streptavidin-horseradish peroxidase. The antigen-antibody complexes were visualized using 3,3′-diaminobenzidine (DAB) and counterstained with 10% Mayer’s hematoxylin, dehydrated, and mounted in Crystal Mount. The degree of immunostaining of the FFPE sections was reviewed and scored independently by two pathologists, based on both the proportion of positively stained tumor cells and the intensity of staining. Tumors with positive staining in >1% of tumor cells were defined as positive.

The landscape of kinase IGRs in patients with lung cancer

We retrospectively reviewed the mutational profiles of a large cohort of Chinese patients with lung cancer (N = 30 450) whose tumor tissue samples and/or liquid biopsies were subject to targeted NGS between June 2016 and July 2019. Approximately 11% of the cohort (3411/30 450) contained kinase gene rearrangements. In particular, a subset of 538 unique patients (1.8%) were identified to have kinase IGR events (Figure 1 and Table 1). Kinase IGRs were detected in diverse sample types, including tumor tissue (70%) and liquid biopsy specimens (30%). ALK was the most frequently rearranged kinase gene in the cohort, accounting for 117 out of the 624 kinase IGRs, followed by EGFR (54/624, 9%), RET (40/624, 6%), ERBB2 (37/624, 6%), and ROS1 (26/624, 4%) (Figure 2A). For each kinase IGR, the detection frequencies in tumor tissues and liquid biopsy samples were not significantly different (Supplementary Figure S1, available at https://doi.org/10.1016/j.esmoop.2022.100405). The other druggable kinase genes, including FGFRs and NTRK1, were also recurrent in the cohort with relatively low frequencies (3% and 1%, respectively).

We next examined the genomic position of intergenic breakpoints. Kinase gene rearrangements often occurred with intergenic breakpoints on the same chromosome (67.3%, 420/624), and the majority (71.5%, 446/624) of the kinase IGRs were 3′-kinases (Figure 2B). Notably, clinically actionable kinase genes, including ALK, EGFR, RET, ERBB2/3, ROS1, FGFR1/2/3, and NTRK1 were detected in a total of 316 kinase IGRs in 269 patients (Supplementary Table S1, available at https://doi.org/10.1016/j.esmoop.2022.100405). Fusions with an intact kinase domain were observed in 219 kinase IGRs (69.3%, 219/316). In most cases, the breakpoints spanned the entire coding regions of kinase genes, without significant hotspots. In contrast, ALK- and RET-involved IGR events tended to converge at the kinase domains. Specifically, 90.6% (106/117) of ALK breakpoints were located at exon 19/intron 19 and 94.9% (37/39) of that occurred between intron 10 and intron 11 of the RET gene. Intergenic breakpoints were rarely recurrent, however, except for one fusion that fused the intergenic region between NFYC and KCNQ4 to exon 48 of MTOR.

In addition, we identified 19 kinase IGR events in which a kinase gene was fused to a promoter region (Figure 3), including ALK (n = 1), RET (n = 3), ROS1 (n = 2), EGFR (n = 1), ERBB2 (n = 1), and FGFR3 (n = 1) (Supplementary Table S2, available at https://doi.org/10.1016/j.esmoop.2022.100405). ALK, RET, and ROS1 were observed to have an intact kinase domain in six cases, which may thereby result in the up-regulation of the oncogenic kinase caused by the promoter of the upstream fusion partner.

Co-alterations

Analyses of concurrent alterations revealed that 31% of samples (167/546) lacked any oncogenic alterations (Figure 4A). EGFR mutations (38.6%) and ALK fusions (6.4%) were the most frequently detected mutations and structural variants in the entire cohort, followed by other oncogenic driver alterations, including TP53 (21.2%), PIK3CA (6.2%), KRAS (3.3%), ROS1 fusion (1.5%), and RET fusion (1.3%). EGFR L858R was the most abundant variant, accounting for 36.1% of all EGFR mutations (Figure 4B). To gain an understanding of the therapeutic implications of the druggable kinase IGRs, we focused on a total of 275 samples collected from 269 patients (Supplementary Table S1, available at https://doi.org/10.1016/j.esmoop.2022.100405), which were classified into three categories: treatment-naïve (Figure 4C, N = 25), formally-treated (Figure 4D, N = 132), and treatment history unknown (Figure 4E, N = 118). In the treatment-naïve and treatment history unknown subgroups, the majority of kinase IGRs were detected in tissue samples (treatment-naïve: 22/25, 88%; unknown: 94/118, 80%). Relatively equal tissue and liquid samples were analyzed in the formally-treated subgroup (tissue: 65 versus liquid: 67); however, no significant differences in kinase IGR frequencies were found between tissue and liquid biopsies in all three categories. Consistent throughout the entire cohort, EGFR and TP53 were the most frequently mutated genes in all three categories.

Notably, kinase IGRs were detected in 13 treatment-naïve samples (52%) that had no accompanying oncogenic driver mutations or canonical structural variants (IGR alone). Among them, one patient (patient 6: ALK IGR) received crizotinib treatment based on the NGS results, which led to a 14-month clinical benefit.

Clinical outcomes of targeted therapies in patients with ALK and ROS1 IGRs

Among the 167 patients containing kinase IGRs without oncogenic alterations, 28 patients received targeted therapies in real-world practice. In particular, targeted treatments were administrated in six patients after kinase IGR detection, and three of which exhibited a favorable response from crizotinib (Figure 5A, D, and E). Therefore, the clinical significance of kinase intergenic fusions remained to be determined. Alternative detection methods such as RNA sequencing, IHC, or FISH are typically recommended to validate the presence of functional fusion products. As shown in Table 2, one patient, patient 6 (Figure 5A), who was diagnosed with NSCLC in March 2017, underwent surgical removal and received six cycles of adjuvant chemotherapy (pemetrexed and cisplatin). An ALK IGR, of which the 5′ intergenic breakpoint was located downstream of LPIN1 on chromosome 2, was identified in the patient’s tumor tissue, without any other concomitant oncogenic alterations. RNA sequencing further revealed messenger RNA transcripts of EML4-ALK in the same tumor sample, and the patient demonstrated clinical benefits from crizotinib (14 months) and alectinib. A similar observation was also made in a second patient (as shown in Table 2), patient 145 (Figure 5B), who harbored an ALK IGR, without other known oncogenic mutations. Patient 145 experienced a 5-month stable disease following crizotinib treatment, before the discovery and validation of an EML4-ALK fusion by RNA sequencing and IHC in the patient’s primary tumor tissue (Figure 5C).

Furthermore, we also observed favorable clinical outcomes in two other patients who harbored ROS1 IGRs. As shown in Figure 5D and E, after the failure of multiple lines of chemotherapy, patients 32 and 11 underwent targeted NGS which uncovered a ROS1 IGR, but without any other canonical oncogenic mutations. ROS1 was fused to the promoter region of P2RX4, which may have up-regulated the expression of the ROS1 kinase domain. Both patient 32 and patient 11 achieved favorable clinical benefits from crizotinib treatment for 13 and 19 months, respectively. Neither case, however, was validated by alternative approaches due to sample shortages.

Conclusion

IGR is rarely detected and reported in patients with lung cancer, with very limited clinical information available. Our study substantially expanded the dataset of NGS-detected IGRs in a real-world setting. Using comprehensive molecular analyses and clinical information, we reported on the potential of IGRs to serve as the druggable targets, however, further validation at the RNA and/or protein levels is highly recommended.