A Rare EGFR–SEPT14 Fusion in a Patient with Colorectal Adenocarcinoma Responding to Erlotinib

Abstract
Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. Growing evidence supports gene fusions as good candidates for molecularly targeted therapy in CRC. Here we describe a case of a 63-year-old man who had a radical right hemicolectomy procedure 24 months ago. Pathological diagno- sis indicated colorectal adenocarcinoma with stage pT4N2bMx. During re-examination in December 2016, positron emission tomography/computed tomography scans indicated relapse with multiple lymph nodes metastasis. Then the patient received a nine-cycle combination treatment of XELOX and bevacizumab and showed progressive disease (PD). Subse- quently, the patient was treated with bevacizumab plus FOLFIRI for 2 months before discontinuation because of adverse events. Paraffin sections of postoperative colorectal tissue were sub- jected to next-generation sequencing, and epidermal growth factor receptor (EGFR) amplification and rare EGFR–SEPT14 fusion were identified. The patient then received erlotinib, an EGFR tyrosine kinase inhibitor (TKI), and achieved a partial response. However, the patient subsequently showed PD, and a new variant, EGFRvIII, appeared in metastasis, which may be involved in erlotinib resistance. We suggest that there is value in treating patients harboring EGFR fusions with EGFR TKI ther- apy, and EGFR–SEPT14 fusion may be used as a therapeutic tar- get for CRC.

The patient was a 63-year-old man who had undergone radical right hemicolectomy in 2014. The postoperative pathological diagnosis indicated a moderately differentiated colorectal ade- nocarcinoma at stage pT4N2bMx. Immunohistochemical staining showed positive for mutS homolog 2 (MSH2) and mutS homolog 6 (MSH6). Family history revealed that his mother had colorectal cancer at the age of 83. When re- examined in December 2016, positron emission tomography/ computed tomography scans showed a thickened intestinal wall at the anastomosis and multiple pathologically enlarged lymph nodes in his abdominal aorta and root of the mesentery (Fig. 1A), which suggested relapse with multiple lymph nodes metastasis. Meanwhile, genetic testing did not detect the mutations of KRAS, NRAS, BRAF, or PIK3CA in colorectal tissue. The patient went through a nine-cycle combination treatment of XELOX (oxaliplatin 200 mg intravenously [IV],D1, and capecitabine, 1,500 mg p.o., b.i.d., days 1–14, every 21 days) and bevacizumab (400 mg IV, day 1, every 21 days) from December 2016 to October 2017. In December 2017, a computed tomography (CT) scan showed left adrenal nodules and enlarged lymph nodes in his abdominal aorta and root of the mesentery (Fig. 1B), suggesting progressive disease (PD). The patient was then treated with bevacizumab (300 mg IV, day 1, every 14 days) plus FOLFIRI (irinotecan 300 mg IV, day 1, leucovorin 300 mg IV, day 1, and 5-fluorouracil 500 mg IV bolus, day 1 plus 4,000 mg over 46 hours, every 14 days) from December 2017 to January 2018.

Therapy was discontinued as a result of intestinal perforation around the anastomotic stoma. In December 2017, paraffin sections of postoperative colorectal tissue were subjected to next-generation sequenc- ing (NGS), and epidermal growth factor receptor (EGFR) ampli- fication and EGFR–SEPT14 fusion were identified. The tumorwas microsatellite stable. From January 2018 to May 2018, the patient recuperated and did not receive any further drug treatment.Genotyping Results and Interpretation of the Molecular ResultsNGS-based ultra-deep panel sequencing was performed on tumor samples and matched blood in a Clinical LaboratoryImprovement Amendments–certified and College of American Pathologists–accredited laboratory (OrigiMed) [1]. Briefly, genomic DNA from a formalin-fixed paraffin-embedded tis- sue specimen containing more than 20% tumor content was fragmented to ~250 bp by sonication. A DNA library was constructed using KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, MA). Hybrid-capture-selected libraries were sequenced to a mean coverage of 1,000× on an Illumina NextSeq-500 Platform (Illumina Incorporated, San Diego, CA). Genomic alterations including single base substitution, copynumber variants, short and long insertions/deletions, and gene rearrangement and fusions were assessed. The tumor mutational burden was estimated by analyzing somatic mutations including coding base substitution and indels per megabase of the panel sequences examined. The results showed EGFR amplification and EGFR–SEPT14 fusion.As members of a highly conserved GTPase family, sep- tins were first described in Saccharomyces cerevisiae [2]. Septins have been involved in multiple cellular functions such as cytokinesis, cell cycle control, mitotic spindle forma- tion, and plasma membrane compartmentalization [3].

Septin 14 (SEPT14) is a member of septin family molecules and is abundantly expressed in developing cerebral cortex in neu- ronal development [3]. SEPT14 maps to 7p11.2 in humans and includes a conserved GTPase domain and a carboxy-terminus coil-coiled domain, which is characteristic of other septins [4].EGFR (also known as ErbB1) is a 170-kDa transmembrane tyrosine kinase whose main ligands are epidermal growth fac- tor and transforming growth factor-α. As one of the most studied receptor tyrosine kinases, EGFR plays an essential role during embryonic development and adult homeostasis and is often aberrantly activated in cancer [5]. EGFR contributes to tumor development and progression. In the patient’s tumor, EGFR–SEPT14 fusion was detected. The exon 24 on EGFR wasfused to the exon 10 on SEPT14, while retaining the receptor tyrosine kinase domain of EGFR (Fig. 2).Functional and Clinical Significance of the Specific Mutation in the Particular CancerEGFR fusions have been previously reported in gliomas [6], and four fusions have been identified in lung cancer, including EGFR–TNS3, EGFR–PURB, EGFR–RAD51, and EGFR–ZCCHC6[7, 8]. EGFR–SEPT14 fusion was first reported in glioblastoma, the structure of which involved EGFR at the N terminus, pro- viding a receptor tyrosine kinase domain that was fused to a coiled-coil domain from SEPT14 [9]. EGFR–SEPT14 fusion was also identified in a 62-year-old never-smoking female with lung adenocarcinoma [10]. This patient responded to icotinib treatment and had no treatment-related adverse events.

One study showed that EGFR–SEPT14 fusion could activate signal transducer and activator of transcription 3 signaling, confer mitogen independence, and impart sensitivity to EGFR kinase inhibition [9]. Here, by using a comprehensive NGS assay, we identified a rare EGFR–SEPT14 fusion in advanced colorectal adenocarcinoma. To our knowledge, this is the first report of EGFR–SEPT14 fusion identified in colorectal cancer.Potential Strategies to Target the Pathway and Implications for Clinical PracticeCancers with EGFR mutations usually depend on EGFR signaling for growth and survival and are often sensitive to EGFR-targeted inhibitors [11]. Given the function of EGFR in diverse cellular pro- cesses, two therapeutic approaches, including tyrosine kinaseinhibitors (TKIs) and monoclonal antibodies, are currently being developed and employed for targeting EGFR in various human cancers [12]. It has been confirmed that patients with cancer have shown benefit from EGFR-targeted agents, including non- small cell lung cancer, colorectal cancer, squamous cell carci- noma of the head and neck, and breast cancer [13–17].

As a first-generation EGFR-mutant-selective TKI, erlotinibincrease in PI3Kp110δ [27]. A study reported that patients with glioblastomas with EGFRvIII mutant had worse survival after treatment with erlotinib [21], suggesting that EGFRvIII mutation may be involved in erlotinib resistance. Taken together, the new gene variant, EGFRvIII, might be responsi-ble for erlotinib resistance of this patient.was approved by the Food and Drug Administration for patients with metastatic non-small cell lung cancer whose tumors have EGFR exon 19 deletions or exon 21 (L858R) substitution muta- tions [18]. Preclinical studies have shown that EGFR–SEPT14 fusion was sensitive to the EGFR inhibitor erlotinib [9]. Four cases with lung cancer harboring EGFR–RAD51 fusion showed significant clinical benefit from treatment with erlotinib [7].

Conclusion
The current report described a Chinese patient with a rare EGFR–SEPT14 fusion and EGFR amplification who showed an antitumor response from treatment with the EGFR TKI erlotinib. EGFRvIII mutation might contribute to erlotinib resistance. On May 4, 2018, a CT scan showed enlarged lymph nodes in the retroperitoneum and mesentery (Fig. 1C), and given that the patient was carrying EGFR–SEPT14 fusion, he started erlotinib (150 mg, once daily) therapy. A CT scan showed a reduction of para-aortic lymph nodes on May 24, 2018 (Fig. 1D), indicating a partial response. Of note, his rising carcino-embryonic antigen reduced from 13.02 ng/mL to 7.98 ng/mL during erlotinib treatment. However, during a re-examination in July 2018, the result of the CT scan suggested PD (Fig. 1E–G). NGS was performed for mediasti- nal lymph nodes, and a new gene variant, EGFR variant III (EGFRvIII), was detected. As a common EGFR genomic alter- ation, EGFRvIII results from an in-frame deletion of exons 2–7 (801 bp) of EGFR [19]. It has been shown that EGFRvIII can activate antiapoptotic signals through the PI3K–Akt sig- naling pathway, which plays a critical role for cell survival, proliferation, and motility [20]. EGFRvIII is found in many human cancers, including glioblastomas, lung cancer, and head and neck cancer [21–23], but is rare in colorectal can- cer [24–26]. EGFRvIII is highly oncogenic, and its expression confers resistance to EGFR TKIs. EGFRvIII can regulate resis- tance to erlotinib in EGFR-amplified glioblastoma via an Fusion/Rearrangement: Recombination of two unlinked segments of human DNA, exhibited as sequencing reads uniquely aligned to two different genes or tow apart DNA segments.