Extramammary Paget disease (EMPD) is a rare, cutaneous malignancy that is most likely to occur in the genital area of the elderly population. Although there have been several reports on familial EMPD, its pathogenic germline mutations have not been analyzed. We herein report the results of the genetic analysis of an EMPD patient with a family history of the same disease, affecting her mother and sister.
A 47-year-old Japanese woman presented with a 5-month history of itchy erythema on the left side of the vulva. After a histopathological examination, we diagnosed this condition as EMPD and surgically removed the affected area. The patient was found to have a history of breast cancer at age 45, a family history of EMPD affecting her mother and sister, and a family history of breast cancer affecting the same sister (Fig. 1A). The affected area for the mother was both sides of the vulva; for the sister, it was the right side of the vulva. Their prognoses have been favorable, with the patient, her sister, and their mother all remaining recurrence-free for 6 years, 6 years, and 14 years after surgery, respectively. There was no history of malignancy affecting her father or brother. To identify the pathogenic germline mutations in this familial EMPD, we performed whole-exome sequencing (WES) on DNA from blood samples of the five family members, including the brother and father. This sequencing was done using the Ion Ampliseq Exsome RDY kit (A38264, Thermo Fisher Scientific) on the Ion S5 GeneStudio S5 system, and the variants were identified as previously reported [1]. WES revealed 64,304 candidate gene mutations. We narrowed down the candidate mutations according to the following exclusion criterion; 1) common only to the father, 2) other than those common among affected family members, 3) reported to be less pathogenic in the database (SnpEff and Clinvar database), 4) with a frequency of more than 1% (Tommo database, HVD database, and ExAC database), 5) other than heterogenetic or homogenetic mutations. Finally, 9 candidates were identified: UBAP2L V402M, ENKUR C5W, ERBB3 K318Q, GSE1 R389G, SYCP2 T798A, FAM118A M98V, ARHGEF35 H8R, DECR1 M220I and ELAVL2 R150H. We knocked down these candidates using breast cancer MCF-7 cells and immortalized keratinocyte HaCaT cells through siRNA transfection. After 24, 72 and 120 h, cell viability was assessed using Cell Titer Glo reagents (Promega). However, we found no difference in proliferation between the control cells and cells transfected with siRNAs targeting the candidate genes (data not shown). Also, in silico analysis from the Genome Unit at Keio University suggested that ERBB3 K318Q could be pathogenic (Fig. 1B). In silico analysis involved referencing the study by Liu et al. [2] and utilized databases such as fathmm, fitCons, and phyloP20way. Based on this result, we established MCF-7 and HaCaT cells expressing HER3 (wild-type ERBB3 or K318Q mutant ERBB3) constitutively, which displayed no significant differences in cell proliferation analysis (data not shown).
There have been four reports in the English literature on familial EMPD [3—6], and Zhang et al. reported identical immunostaining traits for HER2 in sibling-onset EMPD [3]. However, there have been no comprehensive reports on gene mutation analysis. In this study, due to the family history of EMPD, we excluded germline mutations of paternal origin and narrowed down the potential pathogenic mutation from germline mutations of maternal origin. As a result, ERBB3 K318Q was considered the most likely pathogenic mutation, but we could not prove its pathogenicity. ERBB3 K318 exists on HER3 protein domain II, and many of the pathogenic somatic mutations of ERBB3 are observed above HER3 protein domain II [7].
Only a few reports have included genetic analyses of EMPD. Zhang et al. and Kiniwa et al. performed WES on DNA from EMPD tumor tissue and reported its most frequent somatic mutations [8,9]. The only report on germline mutations in EMPD is an article by Kang et al., which re- ported that germline mutations in mismatch repair genes may be involved in the pathogenesis of EMPD, but no analysis of familial EMPD was conducted in their study [10]. Due to this patient s family history of breast cancer, we searched the database for genetic mutations that might be associated with familial breast cancer, such as BRCA1 and BRCA2, but we found no such mutations in the blood samples. Since no pathogenic germline mutations have been identified in about half of familial breast cancers, gene mutations of undetermined pathogenicity may be involved in the development of both EMPD and breast cancer in this family. On the other hand, Karam et al. have reported that cases of EMPD with concurrent breast cancer are very rare (only 10 of 1439 cases), suggesting that there is no obvious predisposition for the co-occurrence of EMPD and breast cancer [11].
To our best knowledge, there have been no studies on gene analysis (somatic or germline) in EMPD patients suspected of having hereditary cancer. In searches beyond EMPD, Rotunno M et al. conducted a sys- tematic review of whole-exome sequencing in patients potentially harboring hereditary tumors [12]. They reported that in studies on the function of the identified variants/genes, 60 tested for loss of hetero - zygosity in tumor samples (58% of which tested positive); 36 looked for somatic mutations in the same gene (69% of which were found); 22 looked for gene/methylation expression changes supporting a link with disease (86% with positive results); 35 checked for variant splicing (86% of which were verified); and 45 carried out in vitro experiments or other functional assays on the identified variants, 80% of which showed re- sults consistent with the function hypothesized for these variants. Regarding the inability to demonstrate the pathogenicity of the genetic mutations identified in this study, it is possible that the gene mutations may exhibit low functional activity.
In conclusion, we performed genetic analysis of blood samples from a family with a history of EMPD. Although we could not identify the pathogenic mutations in this case, we believe it may be possible to identify them by conducting gene mutation analysis of similar familial cases.
References
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Authors
Takuya Maeda, Teruki Yanagia, Shinya Kitamura, Hiroshi Nishihara, Yusuke Ono, Yusuke Mizukami, Shinya Tanaka, and Hideyuki Ujiiea.