BRAF-MAD1L1 and BRAF-ZC3H7A: novel gene fusion in Rhabdomyosarcoma and Lung adenocarcinoma

Jiang Da Hebei Medical University Fourth A liated Hospital and Hebei Provincial Tumor Hospital Hui Jin Hebei Medical University Fourth A liated Hospital and Hebei Provincial Tumor Hospital Xinliang Zhou Hebei Medical University Fourth A liated Hospital and Hebei Provincial Tumor Hospital Shaoshuang Fan Hebei Medical University Fourth A liated Hospital and Hebei Provincial Tumor Hospital Mian Xu Origimed Xiaoyan Chen (  xum@origimed.com ) The Hospital od Shunyi District Beijing


Background
Rhabdomyosarcoma (RMS) is an aggressive malignancy of soft-tissue in children and adolescents (1). There are 4.5 cases per 1 million in children per year and it accounts for 4-5% of all childhood malignancies (2). Despite most cases occur in children younger than 10 years, adult RMS still accounts for 41% of all RMS patients in the Surveillance, Epidemiology and End Results database between 1973 and 2005 (3). Pediatric patients with RMS are generally treated by standard protocols or within clinical studies. In the United State, most pediatric patients with RMS can be signi cantly improved with multimodality therapy (surgery, chemotherapy and radiotherapy) and the 5-year overall survival (OS) is 55% and 71% for IRS-I and IRS-IV, respectively (4)(5)(6). However, the treatment for adult RMS older than 19 years old is still non-standardized and depends on different physicians' experience and personal choice.
The treatment results of adult RMS have been shown to be inferior to that of pediatric RMS (3,7). A recent study assessed the failure pattern and clinical outcome of adult patients with RMS who received multimodality treatment (surgery,chemotherapy and radiotherapy) in the rst-line treatment of metastatic disease, observing approximately 21.7 months of median OS and 6.1 months of median OS especially for genitourinary (8). Lower 3-year DFS (0% versus 26.7%; p = 0.616) and OS (0% versus 53.5%; p = 0.12) of high-risk patients demonstrates the aggressive behavior of this disease.
Lung adenocarcinoma (LADC) is the most common type of lung cancer and occurs in both smokers and never smokers (9). Treatment strategy for LADC in advanced stages have changed obviously from the traditional platinum-based chemotherapy to a gene-based targeted therapy as a rst-line treatment when the tumor carried targetable mutations (10). In primary LADC, screening for genomic alterations is becoming a clinical standard for guiding individual treatment options and identifying new targets (11). However, the majority of patients treated with targeted kinase inhibitors ultimately relapse, so new targets must be identi ed to improve the OS of patients with LADC.
Gene fusion is an important genomic alternation that has been observed in various cancers, including LADC (12) and RMS (13); such fusion is likely to contribute to cancer progression. Recent studies have highlighted the important role of gene fusion in cancers, which is expected to provide new ideas for treatment and improving prognosis. It is reported that KIAA1549-BRAF gene fusion is a prognostic factor in pilocytic astrocytomas and it associates with a better outcome (14). Recurrent BRAF gene fusions with features reminiscent of infantile brosarcomas expand the spectrum of fusion-positive spindle cell sarcomas (15). A novel gene fusion, LMO7-BRAF, was identi ed in papillary thyroid carcinoma and behaves as an oncogenic alteration (16). All the above studies indicate that BRAF gene fusion is involved in the development of cancer.
Herein, we describe the rst case of RMS harboring a BRAF-MAD1L1 fusion and one case of LADC harboring a BRAF-ZC3H7A fusion revealed by next-generation sequencing. These fusions result in BRAF kinase domain activation. However, both patients were not treated with BRAF inhibitors, resulting in a poor prognosis clinically.  2B). The patient was treated with ifosfamide and doxorubicin hydrochloride liposomes for 3 cycles and received a partial response (PR) (Fig. 2C). In August 2018, after 6 cycles of91; chemotherapy, CT scans showed multiple low-density nodules in thyroid and multiple masses around the spleen and retroperitoneum, with increased size and PD (Fig. 2D). After 3 cycles of second-line treatment with albumin paclitaxel, the size of the mass signi cantly increased with PD (Fig. 2E). In October 2018, the patient received anlotinib hydrochloride (12 mg 1/day, d1-d14 for 3 weeks). In December 2018, pelvic tumors increased slightly, and new liver metastases were observed with PD.

Case Presentation
Peripheral blood samples were collected for next-generation sequencing to identify additional therapeutic options. Genomic testing revealed a BRAF-MAD1L1 fusion (Fig. 3A), FGFR1 gene ampli cation, TP53 R248W mutation, and KMT2C/VWC2 rearrangement as well as high bTMB (14.05 Muts/Mb). Firstgeneration sequencing (Sanger) con rmed BRAF-MAD1L1 fusion in the RMS patient ( Fig. 3B-C). On December 28, 2019, the patient's blood was drawn for ctDNA test, during which the disease progressed rapidly, and the patient died in March 2019.

Case 2
A female patient at age 72 was diagnosed with LADC in November 2016 (Fig. 4). Pemetrix combined with cisplatin was applied as a rst-line chemotherapy regimen for ve cycles with SD in February 2017. In Although present data and the published literature demonstrate BRAF fusions are enrichment in many cancers, including pilocytic astrocytoma, spitzoid melanoma, pancreatic acinar carcinoma and papillary thyroid cancer (18)(19)(20)(21), it has not previously been described in RMS. In this report, we rst reported a novel BRAF-MAD1L1 fusion and BRAF-ZC3H7A fusion resulted from a genomic rearrangement in the RMS patient and LADC patient, respectively.
To our knowledge, MAD1L1, the mitosis checkpoint gene, and other gene fusion partner, contains a conserved an N-terminal coil domain and C-terminal domain (595-718 amino acids) that plays a role in the kinetochore targeting (22). Depletion of MAD1L1 genes in mammal cells severely affects the spindle checkpoint function, leading to aneuploidy and tumorigenesis (23), and abnormal expression of MAD1L1 is associated with poor prognosis (24). A patient with melanoma harboring a MAD1L1-BRAF fusion (intron 16 of MAD1L1 and intron 7 of BRAF), which is a similar translocation with case 1, demonstrated a partial response to treatment with certain types of RAF inhibitors, such as sorafenib (25). So, BRAF-MAD1L1 fusion keeps yet the intact in-frame BRAF kinase domain observed in case1 is likely to be effective in the treatment of certain RAF inhibitors. More evidence needs further research.
ZC3H7A (zinc nger CCCH domain-containing protein 7A) is a member of the CCCH zinc nger family and was highly enriched in macrophage-related organs, suggesting that they may play a role in the regulation of innate immunity and in ammatory response. A study had con rmed that ZC3H7A, together with TP53 and KRAS, were clustered into a single network that is strongly related to cancer development (26). Here, we also detected ZC3H7A and TP53 mutations in case 2. These preclinical results suggest the role of BRAF-MAD1L1 and BRAF-ZC3H7A fusion as oncogenic-drivers in a variety of tumors, deserving targeted inhibition by small molecule kinase inhibitors.
As the rst BRAF inhibitor, sorafenib is used in the rst-line treatment for advanced liver cancer and kidney cancer. Another class of BRAF inhibitors, vemurafenib, which have high inhibitory activity against BRAF, especially BRAF V600E, is mainly used to treat melanoma. However, results of ongoing preclinical studies could not provide a signal of the e cacy of BRAF inhibitors on these diseases. The ARMC10-BRAF fusion patient-derived xenograft PDX models showed the highest growth rate in melanomas and signi cant response to downstream MAPK inhibition with trametinib, a MEK inhibitor, but no response to vemurafenib; a metastatic spitzoid melanoma featured with a ZKSCAN1-BRAF fusion responded to treatment with the MEK inhibitor trametinib (27). Sorafenib monotherapy for current or progressive lowgrade astrocytomas harboring KIAA1549-BRAF fusion showed signi cant progression upon treatment (19). Here, both patients did not use trametinib or sorafenib as a therapeutic drug, and the cancers developed rapidly and led to the patients' death, suggesting that the combination of trimetidine or sorafenib may have a better therapeutic effect on patients with BRAF-fused cancers.
In this study, we reported two novel BRAF fusions, BRAF-MAD1L1 and BRAF-ZC3H7A fusion, which has resulted in a poor prognosis in RMS and LADC. With regard to the treatment strategy of these novel BRAF fusions, our cases suggest that the trametinib or sorafenib may bene t patients. A single drug or multidrug combination could be selected according to the patient's physical status. And With the rapid development of NGS techniques, newly identi ed gene fusions and concomitant molecular alterations in oncology practice may provide novel therapeutic targets and bring signi cant clinical improvement for RMS and LADC patients.

Declarations
Ethics approval and consent to participate This study was approved by the ethics committee of Fourth Hospital of Hebei Medical University. Written informed consent for the study was obtained from the patients.

Consent for publication
Written informed consent for publication has be obtained from the patients.

Availability of data and materials
All data generated or analysed during this study are included in this published article.