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Title: Identification of Driving ALK Fusion Genes and Genomic Landscape of Medullary Thyroid Cancer}
Article:
Abstract:
The genetic landscape of medullary thyroid cancer( MTC) is not yet fully understood, although some oncogenic mutations have been identified. To explore genetic profiles of MTCs, formalin-fixed, paraffin-embedded tumor tissues from MTC patients were assayed on the Ion AmpliSeq Cancer Panel v2. Eighty-four sporadic MTC samples and 36 paired normal thyroid tissues were successfully sequenced. We discovered 101 hotspot mutations in 18 genes in the 84 MTC tissue samples. The most common mutation was in the ret proto-oncogene, which occurred in 47 cases followed by mutations in genes encoding Harvey rat sarcoma viral oncogene homolog( N = 14), serine/threonine kinase 11( N = 11), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog( N = 6), mutL homolog 1( N = 4), Kiesten rat sarcoma viral oncogene homolog( N = 3) and MET proto-oncogene( N = 3). We also evaluated anaplastic lymphoma kinase( ALK) rearrangement by immunohistochemistry and break-apart fluorescence in situ hybridization( FISH). Two of 98 screened cases were positive for ALK FISH. To identify the genomic breakpoint and 5’ fusion partner of ALK, customized targeted cancer panel sequencing was performed using DNA from tumor samples of the two patients. Glutamine:fructose-6-phosphate transaminase 1( GFPT1)-ALK and echinoderm microtubule-associated protein-like 4( EML4)-ALK fusions were identified. Additional PCR analysis, followed by Sanger sequencing, confirmed the GFPT1-ALK fusion, indicating that the fusion is a result of intra-chromosomal translocation or deletion. Notably, a metastatic MTC case harboring the EML4-ALK fusion showed a dramatic response to an ALK inhibitor, crizotinib. In conclusion, we found several genetic mutations in MTC and are the first to identify ALK fusions in MTC. Our results suggest that the EML4-ALK fusion in MTC may be a potential driver mutation and a valid target of ALK inhibitors. Furthermore, the GFPT1-ALK fusion may be a potential candidate for molecular target therapy.
Introduction:
Many cancer gene profiling studies have recently been published, describing genetic trends that are not limited to specific cancers. Next-generation sequencing( NGS) is an important tool for detecting genetic alterations in many kinds of cancers, as it allows for millions of nucleic acid sequences to be simultaneously sequenced within a short period of time and is more cost-effective than older methods. Thus, many researchers and physicians anticipate that NGS will bring the concept of personalized cancer therapy to fruition. Medullary thyroid cancer( MTC) is a rare malignancy that accounts for up to 3–5% of thyroid cancers. It is derived from calcitonin-secreting para-follicular C cells and can arise in a familial( 25%) or sporadic( 75%) pattern. Genetic and epigenetic alterations play important roles in the progression and prognosis of MTC[1–3]. Genes encoding the ret proto-oncogene( RET) and Ras( RAS) are commonly mutated in MTC[4, 5]. The RET mutation is believed to be a causative event in both familial and sporadic MTC[6, 7]. In the Mitogen-activated protein kinase( MAPK) pathway, the RAS mutation is another genetic rearrangement that is prevalent in sporadic MTC and other types of thyroid cancer[2] but the prevalence and significance of other genetic mutations including BRAF in MTC remain unclear. MTC has a different response to treatment than that of well-differentiated thyroid cancers. Because radioactive iodine does not accumulate in MTC, few therapeutic options are available for advanced MTC. Inhibitors of RET, such as cabozantinib and vandetanib, have recently been shown to be effective in advanced MTC[8, 9]. However, whether the RET mutation is a predictive factor for the success of these drugs is unclear[9]. Recently, the rearrangement of anaplastic lymphoma kinase( ALK) was detected in a small but significant proportion of patients with non-small cell lung cancer( NSCLC)[10]. Several ALK inhibitors, including crizotinib, have achieved dramatic responses in cases of NSCLC harboring ALK rearrangements[11–13]. Although ALK rearrangement has also been episodically observed in a small set of other cancer types, little is known about ALK rearrangements in MTC[14, 15]. In this study, we used targeted NGS and various methods to examine the genetic profiles of MTC and detect ALK rearrangements.
Discussion:
We identified two types of ALK fusion genes in MTC by sequencing via IHC, FISH, and NGS analyses. Of the two fusion types, the EML4-ALK fusion was the same as the most commonly detected variant in NSCLC,[19] where the EML4-ALK fusion is a strong predictive factor for the efficacy of ALK inhibitors[13, 21, 22]. In the current study, the patient with metastatic MTC harboring the EML4-ALK fusion showed a dramatic response to crizotinib. We are the first to report an MTC case with a targetable EML4-ALK fusion gene. Previously, Kelly et al. used the Illumina HiSeq sequencing system to identify one papillary thyroid cancer case with an EML4-ALK fusion[15]. However, they also tested 22 medullary carcinoma cases and did not find any cases with the EML4-ALK fusion, as evaluated by reverse transcription-PCR. Their failure to detect the ALK rearrangement in MTC is understandable, given that our prevalence rate of ALK fusions in the current study was only 2%( 2 out of 98 cases). This suggests that more efficient strategies are needed to detect the ALK rearrangement. Results from the current study suggest that IHC-based screening, along with FISH-based confirmation and targeted NGS, may be a cost-effective and reliable method to detect ALK rearrangements. Most importantly, we detected a novel GFPT1-ALK fusion that has not been reported in any type of cancer. GFPT1 is a key enzyme in the biosynthesis of N-acetylglucosamine and is required for critical events in neuromuscular transmission[23]. Until now, several fusion partners of ALK have been reported in various cancers[24–28]. Among them, huntingdon-interacting protein( HIP1)-ALK and RAN-binding protein 2( RANBP2)-ALK, which have been reported to exist in NSCLC and inflammatory myofibroblastic tumors, respectively, show clinical responses to crizotinib[25, 26]. In the current study, the MTC case harboring the GFPT1-ALK fusion showed strong ALK protein expression and did not exhibit co-existing genetic mutations; both of these factors may support an important role for this fusion gene in the pathogenesis of this MTC case. However, we were unable to validate whether GFPT1-ALK was a driving oncogene or a therapeutically targetable gene. Whether GFPT1-ALK is also a predictor for ALK inhibitors is unclear. Currently, vandetanib and cabozantinib are approved for the treatment of MTC by the U. S. Food and Drug Administration. However, the prognosis of patients with metastatic MTC is still poor, due to the inherent resistance to radioiodine therapy and aggressive nature of this disease. Furthermore, the rarity of MTC makes it hard to perform prospective studies to find new agents. Therefore, the comprehensive genetic analysis of MTC can help to identify effective ways to improve its prognosis. Despite the low frequency of ALK rearrangements in MTC, our techniques can be used to detect target genes in other rare diseases. In addition, our sequencing analysis of MTC is the largest to date. Previously, Agrawal et al. published the largest genomic analysis of MTC[5], where they performed whole-exome sequencing of 17 sporadic MTCs and 40 additional MTCs( hereditary or sporadic) for validation. RET was the dominant mutation( 43/57) in that study. We used a larger sample size and accurate verification by comparing 36 pairs of MTC with matched normal thyroid tissues that were acquired from the same person. In the comparison analyses, all five hereditary cases were observed to have germ-line RET mutations in both MTC and control tissues. However, M918T RET( N = 10), Q61K HRAS( N = 7), KRAS( N = 2), and MET( N = 1) mutations were harbored dominantly in MTCs. Simbolo et al. identified RET, HRAS, KRAS and STK11 mutations as significant somatic mutations in MTCs, whereas TP53, KDR, KIT, MET, PIK3CA and ATM mutations were classified as nonpathogenic germ-line variants[29]. Our current data are compatible with that report. Interestingly, however, the F354L STK11 mutation, regarded as significant somatic mutation by Simbolo et al., was observed in both MTCs and control tissues of our seven cases. Therefore, we presume that the F354L STK11 mutation is a germ-line mutation in MTC. In conclusion, we report that the EML4-ALK fusion, which was found for the first time in MTC, could be an effective molecular target of crizotinib. Furthermore, our results also suggest that the novel GFPT1-ALK fusion can be a potential candidate for molecular target therapy. This study included the largest set of molecular profile data in MTC to date, which was achieved by using high-depth NGS panel sequencing, and also presented the genetic landscape of MTC. Further translational research is needed to determine the oncogenic roles of these mutations in MTC.
Little is known about the molecular biology of medullary thyroid cancer ( MTC ) , which is a rare disease . Genomics are increasingly being used to improve our knowledge about disease biology and to identify therapeutic targets in many cancers . Here , we report the largest genomic results of MTC to date . MTC tissue frequently included several mutations . For the first time , anaplastic lymphoma kinase ( ALK ) rearrangements were detected in MTC: one case with a glutamine:fructose-6-phosphate transaminase 1 ( GFPT1 ) -ALK fusion , and another case with an echinoderm microtubule-associated protein-like 4 ( EML4 ) -ALK fusion . The fusion mechanism of the novel GFPT1-ALK fusion was successfully investigated using molecular biology techniques . In addition , an inhibitor of ALK ( crizotinib ) dramatically decreased the number of metastatic MTC lesions harboring the EML4-ALK fusion , thus verifying the fusion as a promising target in MTC . Our findings suggest that using rapidly improving sequencing techniques and accumulated genomic data to comprehensively perform genetic analyses on rare tumors , such as MTC , will help to improve the poor prognosis of orphan diseases .