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Frequent FOXA1-Activating Mutations in Extramammary Paget's Disease

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Frequent FOXA1-Activating Mutations in Extramammary Paget's Disease cancers Article Frequent FOXA1-Activating Mutations in Extramammary Paget’s Disease 1, , 2, 1 3 Takuya Takeichi * y, Yusuke Okuno y, Takaaki Matsumoto , Nobuyuki Tsunoda , Kyogo Suzuki 4, Kana Tanahashi 1, Michihiro Kono 1 , Toyone Kikumori 3, Yoshinao Muro 1 and Masashi Akiyama 1 1 Department of Dermatology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; [email protected] (T.M.); [email protected] (K.T.); [email protected] (M.K.); [email protected] (Y.M.); [email protected] (M.A.) 2 Medical Genomics Center, Nagoya University Hospital, Nagoya 466-8550, Japan; [email protected] 3 Department of Breast and Endocrine Surgery, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; [email protected] (N.T.); [email protected] (T.K.) 4 Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; [email protected] * Correspondence: [email protected] These authors contributed equally to this work. y Received: 15 March 2020; Accepted: 27 March 2020; Published: 29 March 2020 Abstract: Extramammary Paget’s disease (EMPD) is a neoplastic skin disease of indeterminate origin with an unknown genetic cause. We performed a comprehensive genetic analysis or targeted gene sequencing in 48 patients with EMPD. We identified FOXA1 mutations, a GAS6–FOXA1 fusion gene, and somatic hotspot mutations in the FOXA1 promoter region in 11 of the 48 EMPD patients (11/48, 23%). Additional mutations were identified in PIK3CA (six patients) and in HIST1H2BB, HIST1H2BC, and SMARCB1 (one patient each), but none were found in other frequently mutated genes in cancer. A global gene expression analysis using EMPD clinical samples found the upregulation of PI3 kinase–AKT–mTOR signaling. ABCC11, which is specifically expressed in the apocrine secretory cells and is necessary for their sweat secretion, was upregulated in the EMPD samples. This upregulation suggests that Paget cells originate from apocrine secretory cells. Immunohistochemical staining revealed that FOXA1 expression was prevalent in all of the EMPD samples analyzed and was associated with estrogen receptor expression. Our genetic analysis indicates that EMPD frequently involves FOXA1 mutations. FOXA1 is a transcriptional pioneer factor for the estrogen receptor, and the present results suggest that certain treatments for hormone-dependent cancers could be effective for EMPD. Keywords: extramammary Paget’s disease; forkhead box A1; fusion gene; mammary Paget’s disease; somatic mutations; whole-genome sequencing 1. Introduction Paget’s disease (PD) is a neoplasm seen on the nipple/areola (mammary PD; MPD) or in extramammary body zones, such as in anogenital and perineal skin and the axilla (extramammary PD; EMPD) [1]. MPD is relatively rare, observed in 0.7–4.3% of all breast cancers. It is much more frequent in women, possibly because of the clear predominance of breast cancer in females (sex ratio of 1:50–200). MPD develops more often in people in their fifties (mean age: 57 years), i.e., in 70% of MPD cases in postmenopausal women, but it has been observed in adolescents and in elderly patients [1]. EMPD was first described by Crocker in 1889 [2]. It shares several clinicopathological similarities with Cancers 2020, 12, 820; doi:10.3390/cancers12040820 www.mdpi.com/journal/cancers Cancers 2020, 12, 820 2 of 13 its mammary homologue but shows some differences, namely, pathogenesis [1]. The precise incidence of EMPD is unknown, although it is possibly rarer than MPD, accounting for 6.5% of all reported cases of PD. It predominantly affects patients aged 65 to 70 years, and 90% of EMPD patients are older than 50 years [1]. In Asian populations, a male predominance for EMPD is observed [3], although there is a clear female preponderance of EMPD in non-Asian populations. Surgical resection is the first choice of treatment for resectable cases; once PD becomes unresectable, it is difficult to control by chemotherapy, and the prognosis is poor. For systemic metastases, including extensive lymph node metastases, no systemic chemotherapy improves the patient’s survival [4]. Moreover, as most patients with EMPD are elderly, the option of systemic chemotherapy is frequently limited [1,4]. Thus, invasion levels and the presence or absence of multiple lymph node metastases are important prognostic factors in EMPD [4]. Although cutaneous EMPD usually occurs in skin regions that are rich in apocrine glands, its exact cellular origin has yet to be determined. It has been proposed that EMPD arises from skin pluripotent stem cells [5]. Toker cells, which are a small population of benign pagetoid clear cells in normal nipples, have also been proposed as a candidate for the origin of Paget cells [6]. Several sequencing studies using targeted or whole-exome sequencing approaches have revealed that EMPD and MPD carry driver mutations in PIK3CA, KRAS, BRAF, AKT1, and other genes [7–10]. Although the aforementioned genes are frequently mutated in various cancers [11] and are not particularly characteristic of PD, it is important to gather information on these candidate driver mutations towards understanding the underlying molecular genetic basis of EMPD. In this study, we performed a comprehensive genetic analysis, including whole-genome sequencing, of our series of EMPD patients and a targeted sequencing analysis of MPD patients to elucidate the molecular pathogenesis of EMPD. 2. Results 2.1. Whole-Genome Sequencing of Two Patients with EMPD We performed whole-genome sequencing using frozen specimens containing Paget cells and peripheral blood mononuclear cells derived from two patients with EMPD (Figure1, Table S3). In one patient (UPN1), we identified a novel in-frame fusion involving GAS6 (encoding growth arrest-specific protein 6) and FOXA1 (encoding forkhead box A1 or hepatocyte nuclear factor 3α), along with 43 nonsynonymous somatic point mutations (Figure1A). The GAS6–FOXA1 fusion gene identified in UPN1 was generated by a balanced translocation between chromosomes 13 and 14 (Figure1C and Figure S1). This translocation connected the initial two exons and the second intron of GAS6 to a point 10 kb upstream of FOXA1, which was validated by both Sanger sequencing and RNA sequencing (Figure1D). The resulting transcript contained exons 1 and 2 of GAS6 and exon 2 of FOXA1, whose combination resulted in an in-frame fusion of the two proteins. While all functional domains of GAS6, except a truncated Gla domain, were lost in the fusion, the FOXA1 forkhead domain, the essential domain for its transcriptional activity, was conserved (Figure1E). Based on the protein structure of GAS6–FOXA1, we considered that the function of this gene fusion is to upregulate the transcriptional activity of FOXA1 using GAS6 promoter activity. Cancers 2020, 12, x FOR PEER REVIEW 3 of 14 (https://cancer.sanger.ac.uk/cosmic/accessed at 04/10/2019), although the affected amino acid residue is a known target of histone acetylation [14]. CancersCombined,2020, 12, 820 the whole-genome analysis found FOXA1 to be affected in both patients. Somatic3 of 13 copy number abnormalities were not detected in these patients. A UPN1 B UPN2 PIK3CA (p.E81K) GAS6-FOXA1 (fusion gene) FOXA1 (promoter) HIST1H2BC (p.K24N) C UPN1 Chromosome 13 (complement) Chromosome 14 (complement) 114,566 114,564 114,562 38,074 38,07238,070 38,068 38,066 38,064 38,062 38,060 (kb) 114,561,638 38,074,584 12 GAS6 TTC6121 FOXA1 D UPN1 cDNA 8283 84 85 25 26 27 28 AA Asp Pro Glu Thr Ala Tyr Ser Ser CDS 244 255 73 84 G A C CCCG A G A CCCG G T A C T C C T CC GAS6 exon 2 FOXA1 exon 2 E UPN1 1 30 53 85 94 116 278 298 678 Signal EGF- EGF- EGF- EGF- Laminin Laminin Gla GAS6 peptide like 1 like 2 like 3 like 4 G-like 1 G-like 2 1 25 169 260 472 FOXA1 Forkhead Signal Forkhead GAS6-FOXA1 peptide 533 aa FUPN2 Chromosome 14 (complement) 38,080 38,078 38,076 38,074 38,07238,070 38,068 38,066 38,064 38,062 38,060 (kb) g.38064406G>A 2 TTC6121 FOXA1 FigureFigure 1. 1. FOXA1-activatingFOXA1-activating mutations mutations in extramammary in extramammary Paget’s Paget’s disease disease (EMPD) (EMPD) identified identified by whole- by genomewhole-genome sequencing. sequencing. (A,B) Summary (A,B) Summary of somatic of somaticmutations mutations identified identified in patients in patients UPN1 (A UPN1) and( UPN2A) and UPN2 (B). Dots indicate nonsynonymous mutations, and the blue arch indicates gene fusion. We identified Cancers 2020, 12, 820 4 of 13 43 somatic point mutations and a gene fusion of GAS6 and FOXA1 in UPN1. A total of 190 somatic point mutations were identified in UPN2, 3 of which were possible driver mutations. (C) Chromosomal structure of the GAS6–FOXA1 fusion gene. Genome coordinates, transcripts, and the breakpoint (dashed line) are indicated. (D) Complementary DNA sequence of the GAS6–FOXA1 fusion gene. Exon 2 of GAS6 is joined to exon 2 of FOXA1, resulting in an in-frame fusion. (E) Predicted protein structure of GAS6–FOXA1. (F) Position of the FOXA1 promoter mutation (g.38064406G>A), which is 81 bp upstream of the transcription start site of FOXA1. In another patient (UPN2), we identified several possible driver mutations, including a mutation affecting the promoter region of FOXA1 (g.chr14:38064406G>A in the hg19 genome coordinate), a PIK3CA (encoding phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) p.E81K mutation, and a HIST1H2BC (encoding histone cluster 1 H2B family member c) p.K24N mutation (Figure1B). The FOXA1 promoter mutation is located 81 bp upstream of the gene’s transcription start site (Figure1F and Figure S2) and has been reported to upregulate FOXA1 expression [12].
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