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Identification of a rare MET variant in a familial case of extramammary Paget’s disease

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Introduction

Extramammary Paget’s disease (EMPD) is an intraepithelial adenocarcinoma that primarily affects the genital and axillary areas of elderly patients with a mean age of 60– 70 years1. EMPD is a rare disease, and its prevalence is 0.4 /1,000,000 persons annually1. The pathogenesis and cellular origin of EMPD remains unclear.

In sporadic EMPD cases, recurrent somatic variants in PIK3CA, ERBB2, TP53, RAS, and RAF have been identified in tumour tissues2. Pathogenic germline variants of mismatch repair (MMR) genes are associated with the development of EMPD3. Currently, a limited number of paired familial EMPD cases (i.e., parent-offspring, siblings) have been reported4; however, genomic analysis of these familial cases has not been adequately performed. We examined the first familial case of EMPD involving three siblings. To understand the common genetic background and pathogenesis of these cases, we performed multi-gene panel testing using tumour and peripheral blood DNA from the three affected siblings and their two offspring. Here, we report the results of genomic analyses of the familial case of EMPD.

Materials and Methods

Ethics statements

This study was approved by the Ethics Committee of Keio University (approval numbers: 20070081 and 20110159) and conducted in accordance with the Declaration of Helsinki. Written informed consent for the comprehensive genomic testing, approved by the Institutional Review Board of Keio University, was obtained from all the patients and their family members. Study protocols for the genetic analysis of germline variants were approved by the institutional review board (IRB) of the National Cancer Center (No.2013-303).

Multi-gene panel testing

Genomic DNA was extracted from formalin-fixed, paraffin-embedded tumour tissues derived from the surgical specimens from the three patients with EMPD. Microdissection (10 μm) was performed on in situ lesions to provide > 20 % tumour cells in the specimens and minimise the presence of necrosis. Genomic testing was performed using the Rapid-Neo testing platform, with slight modifications to the established clinical sequencing apparatus, as previously described5. The 145 genes examined in this study are listed in Table S1.

Blood samples were collected from the three affected siblings and their two offspring. Genomic DNA was extracted from peripheral blood lymphocytes. Nextgeneration sequencing was performed using custom-made gene panels using SureSelect Target Enrichment System6, NOP_FC ver3.0. The 147 genes examined are listed in Table S2.

Results

Clinical findings of the patients

Case 1 (Figure 1, III-9) was a 70-year-old Japanese man, the youngest of five siblings, who presented with erythema of the scrotum that appeared 5 years ago. Erythematous plaques with scales and erosion were predominantly distributed on the left side of the scrotum (Figure 2a), and hypopigmented areas were observed around the penis. No lesions were observed in the axillae. His two older sisters, Cases 2 and 3 (Figure 1 III-4 and III-8, 80 and 73 years old, respectively) had similar skin symptoms (Figures 2b and c).  

A skin biopsy from each patient revealed that Paget’s cells had proliferated in the epidermis (Figure 3a). Tumour cells were positive for CK7 (Figure 3b) and GATA3 (Figure 3c), and negative for CK20 and CDX2 (Figure 3d). Collectively, anogenital EMPD was diagnosed in all three cases, and they underwent surgical excision. No dermal invasion occurred in cases 1 and 2, whereas a slight invasion was detected in Case 3. None of the patients had metastatic carcinomas or received prior targeted therapies.  A close examination of Case 3 revealed non-syndromic bilateral clear cell renal cell carcinoma, and the patient underwent partial nephrectomy.

Multi-gene panel testing

Tumour-only multi-gene panel testing identified a heterozygous c.2997A>C (p.Glu999Asp) variant in the proto-oncogene MET (NM_000245.4) that occurred in all three affected siblings (Table 1; Figure 1, III-4, III-8 and III-9). Recurrent pathogenic variants, c.853G>A (p.Glu285Lys) in TP53 (NM_000546.6) and c.1633G>A (p.Glu545Lys) in PIK3CA (NM_006218.4), in only the youngest (III-9) of the three affected siblings were observed. Germline multi-gene panel testing revealed the same MET variant in all the examined family members, including the asymptomatic non-elderly offspring (Figures1, IV-2 and IV-8); no pathogenic variants in the MMR genes, such as MLH1, EPCAM, MSH2, MSH6, or PMS2, were identified.

Discussion

MET, a proto-oncogene, located on chromosome 7q31, encodes a member of the receptor tyrosine kinase family. The cytoplasmic domain of the MET protein is phosphorylated by the binding of Hepatocyte Growth Factor (HGF) to its receptor site and stimulates multiple downstream pathways, including the PI3K/AKT/mTOR, RAS/RAF/MAPK, and STAT signalling pathways (Figure S1)7. Mutationally activated MET promotes tumour cell proliferation, survival, adhesion, motility, and invasion. Since the initial discovery of missense MET mutations in hereditary papillary renal carcinoma, activation of MET mutations has been identified in various cancers8. Consequently, MET-targeted therapies have claimed the spotlight.

The c.2997A>C (p.Glu999Asp) variant of MET was not registered in the population databases, such as gnomAD (https://gnomad.broadinstitute.org/), ToGo Var (https://grch38.togovar.org), and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), and was classified as being of “uncertain significance” according to the ACMG classification guidelines. To evaluate the pathogenicity of the MET variant, we utilized prediction algorithms for the in silico analyses: CADD (https://cadd.gs.washington.edu/), PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (https://sift.bii.a-star.edu.sg/), and MutationTaster (http://www.mutationtaster.org/). Three algorithms [CADD: score 23.0, PolyPhen-2: probably damaging (0.991), and MutationTaster: disease causing] implied the pathogenicity of this variant, whereas SIFT (tolerated:0.31) did not (Table S3).

Missense variants can affect protein stability, cellular localisation, and molecular interactions9. Immunohistochemistry (IHC) of MET showed expression in tumour cells of our cases (Figure S2a, c and e). We confirmed that the expression level and cellular localisation of the protein were comparable to those in seven control specimens of EMPD with MET wild-type profiles (FigureS3). The PI3K/AKT pathway, for downstream MET signalling, is activated in EMPD tissues and could be a therapeutic target10. IHC of p-AKT displayed activation in tumour cells in our three affected cases (Figure S1b, d and f). We hypothesise that the MET variant identified in our case leads to overactivation of PI3K/AKT signalling. Future experimental studies are needed to assess the impact of this MET variant on downstream signalling. This study had limitations. We could not confirm whether the unaffected siblings also carried the MET variant because of the aging of relatives (III-1 or III-6), which made it difficult to obtain informed consent.

Furthermore, it was difficult to conclude whether two of the patients’ offspring with the MET variant (51 and 37 years old) were pre-symptomatic during our observational period, because EMPD typically develops in the elderly. Follow-up of these family members is required to determine the association of this MET variant with the onset of EMPD. Another limitation is the potential for genomic alteration beyond the scope of target sequencing to associate and play a role in the pathogenicity of EMPD. Nevertheless, these cases highlight the occurrence of rare familial EMPD in three siblings harbouring the same variant in proto-oncogene, MET. In conclusion, this is the first report of a familial EMPD with three affected siblings and the first reported genomic analysis. We identified a MET variant, but its contribution to the EMPD carcinogenesis remains to be elucidated. Further accumulation of genomic analyses in familial cases of EMPD will allow the establishment of pathogenic features. Family cases may have been overlooked in the past because only a limited number of cases have been reported. Dermatologists need to remember the possibility of familial occurrence of EMPD and check family history for early intervention.

References

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Authors: Yuki Kobayashi, Yoshio Nakamura, Umi Tahara, Kohei Nakamura, Kuniaki Nakanishi, Akihiro Miyagawa, Hiroto Horikawa, Kenta Kobayashi, Takeru Funakoshi, Kokichi Sugano, Mineko Ushiama, Teruhiko Yoshida and Toyoko Inazumi.

© The Author(s) 2024. Published by Oxford University Press on behalf of British Association of Dermatologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by -nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. 1

Funding sources: This research was supported in part by JSPS KAKENHI (Grant Number 20528244), Research Fund of Department of Dermatology, Tachikawa Hospital, Federation of National Public Service Personnel Mutual Aid Associations, The National Cancer Center Research and Development Fund (2023-A-18) and AMED (Grant Number JP21ck0106554).

Conflicts of interest: The authors declare no conflicts of interest.

Data availability: The data that support these findings are available from the corresponding author upon reasonable request.

Ethics statement: This study was approved by the Ethics Committee of Keio University (approval numbers: 20070081 and 20110159) and conducted in accordance with the Declaration of Helsinki. Written informed consent for the comprehensive genomic testing, approved by the Institutional Review Board of Keio University, was obtained from all the patients and their family members. Study protocols for the genetic analysis of germline variants were approved by the institutional review board (IRB) of the National Cancer Center (No.2013-303). The patients in this manuscript have given written informed consent to publication of their case details.

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