Extramammary Paget disease (EMPD) is a rare condition characterized by extreme pruritis and eczematoid-like lesions, most commonly of the skin of the external genitalia. EMPD of the vulva accounts for approximately 65% of EMPD. It occurs most often in White post- menopausal women and may be associated with un- derlying adenocarcinoma in 10%-30% of cases.
The mainstay of treatment of noninvasive EMPD is surgical resection, with some patients requiring mul- tiple resections over the course of the disease.2 Mi- croscopically positive margins after resection are frequent, and multiple studies have shown that dis- ease recurrence is common regardless of margin status. To spare patients repeated operations for recurrent disease, several nonsurgical modalities have been used to varying degrees of success. Among these, the topical immune-response modifier imiqui- mod, a toll-like receptor (TLR7) agonist, has shown therapeutic promise as an alternative to surgery or as a perioperative adjunct. Other treatment modalities, including radiation therapy and topical and systemic chemotherapy (eg, fluorouracil), have been used in inoperable disease, but their use is limited due to the lack of clinical data outside of case reports.5 Given the frequency of recurrence and morbidity of repeated surgical excision, alternative, conservative treatment options would benefit these patients greatly.
In the search for alternatives to surgery or systemic therapy, prior studies have demonstrated that a subset of intraepithelial and invasive EMPD shows an over- expression of HER2 protein and ERBB2 gene ampli- fication, as well as oncogenic mutations in PIK3CA and AKT1,6-11 which were associated with a more aggressive EMPD phenotype and poorer prognostic factors.
The goal of this study was to prospectively explore the molecular profile of primary noninvasive vulvar EMPD using massively parallel sequencing to identify potential therapeutic targets. We also report on a case of a patient with vulvar EMPD treated with targeted therapy on the basis of her tumor genomic mutations.
METHODS
Institutional review board approval for this retrospective analysis as well as for tumor molecular sequencing was obtained. Tumor and normal DNA were subjected to MSK- IMPACT—Memorial Sloan Kettering Cancer Center In- tegrated Mutation Profiling of Actionable Cancer Targets— sequencing, which targets 410-468 cancer-related genes (the number of genes analyzed was dependent upon the time when the tumor was subjected to analysis and the version of test available). Sequencing data analyses were performed, and mutations, copy number variations, and structural rearrangements were identified and anno- tated using validated bioinformatics approaches, as pre- viously described.14,15 These molecular alterations were further curated using OncoKB (Memorial Sloan Kettering Cancer Center, New York, NY), a precision oncology knowledge base, to identity clinically relevant cancer gene alterations. For the quantification of microsatellite in- stability (MSI), MSIsensor was used, as previously de- scribed, and samples with an MSIsensor score ≥ 10 were deemed MSI-high.
All pathology was confirmed by expert gynecologic pa- thologists. Electronic medical records were queried for all patients for demographics, clinical characteristics, treat- ment, and follow-up data. These features were integrated with the molecular findings. Research has shown that clinical outcomes are similar for patients with noninvasive and microscopically invasive (, 1 mm depth of invasion) disease, both groups were included in this report.
RESULTS
Clinical Features
We identified 26 patients with EMPD of the vulva whose tumors had undergone genomic sequencing (Table 1). Median age at diagnosis was 62 years (range, 29-77 years).
Seventeen patients (66%) were White, 4 (15%) were Black, and 5 (19%) were Asian. At the time of diagnosis, all patients had noninvasive or microinvasive (, 1 mm depth of invasion) tumors.
Primary treatment was surgery for 19 patients (73%) and imiquimod topical therapy for 7 (27%). Sixteen patients (62%) had recurrent disease after their initial treatment. Of these, 14 patients (87.5%) underwent a surgical resection at the time of diagnosis, whereas the other 2 (12.5%) were treated with imiquimod topical therapy. Seven patients (27%) had ≥ 2 surgeries as part of their clinical course, and 1 patient had undergone 5 vulvar resections.
Median time since initial diagnosis was 18.5 months (range, 3-226 months). Median follow-up time since tumor sequencing was 8 months (range, 0-26 months). Disease in 2 patients (8%) progressed to invasive adenocarcinoma, likely related to Paget disease, and both patients died of their disease.
Mutational Profiles of Vulvar EMPD
Molecular findings are summarized in Figure 1. The me dian coding mutation count was 2 (range, 0-29). Median allelic frequency of coding mutations in the samples ranged from 0%-32%. MSI was evaluated in 23 (88%) of the 26 samples. All 23 were microsatellite stable. Mutations were commonly seen in PIK3CA, TP53, and ERBB2.
Seven tumors (27%) harbored an oncogenic TP53 mutation. Four tumors (15%) had an ERBB2 mutation, and an additional 3 tumors (12%) had an oncogenic ERBB2 amplification. The point mutations were all classified as OncoKB level 3B, which includes mutations that are candidate predictive biomarkers for US Food and Drug Administration–approved drugs being used off-label or in investigational agents.16 Specific mutations that are characterized as OncoKB level 3B are summarized in Appendix Table A1.
Nine tumors (35%) harbored a PIK3CA mutation. The mutations were all classified as OncoKB level 3B. Of these, 3 tumors (33%) had the hotspot mutations p.E542K or p.E545K. All tumors harboring PIK3CA mutations were noninvasive, and in the time since diagnosis, only 1 patient (EMPD-04, whose tumor harbored a p.E542K mutation) had a recurrence requiring treatment.
Multiple copy number and structural variants were iden- tified in 11 samples (69%). Most of these alterations were variants of uncertain significance, with a few predicted to be oncogenic based on OncoKB but clinically not actionable.
Targeted Treatment of Vulvar EMPD
One patient in whom an oncogenic PIK3CA mutation was identified elected to enroll in a phase II clinical trial for treatment with a novel agent targeting the mutation. She was initially diagnosed with vulvar EMPD at the age of 49 years and was treated with a wide local excision of the tumor. Margins were positive and there was no evidence of invasion. She then had a total vulvectomy 3 years later. Over the next 15 years, she was followed closely with multiple biopsies performed but did not require additional treatment. A symptomatic recurrence in 2015 prompted her to seek treatment, and treatment with topical imiquimod was initiated. Her treatment was limited to 7 months before she self-discontinued for substantial adverse effects and worsening disease.
The patient’s tumor underwent sequencing, and a PIK3CA mutation, p.E542K, was identified. She enrolled in a trial of treatment with a molecule targeting the mutation and had a partial pathologic response with complete symptom resolution. Her disease was well controlled for 19 months before new symptomatic lesions led to drug discontinuation. The patient, once again, was treated with surgical resection. At 10 months since her surgery, she is experiencing minimal symptoms and has no new concerning lesions.
DISCUSSION
Noninvasive EMPD of the vulva is a chronic disease that causes substantial morbidity. The mainstay of treatment is surgical resection, with patients often requiring repeated excisions, leading to disfiguring cosmetic outcomes. Alternatives to surgery include imiquimod and, to a lesser, extent radiation therapy, photodynamic therapy, CO2 laser, topical 5-fluorouracil, and topical bleomycin.19 Topical 5-fluorouracil and bleomycin are highly toxic and are associated with poor response rates. Localized radiation therapy can lead to complete regression in up to 80% of patients, but it is also associated with potential adverse effects.21 Topical 5% imiquimod cream has been prospectively shown to be a feasible option for women with recurrent EMPD of the vulva, with a complete response rate of 55%-75%. Even imiquimod, however, is associated with intolerable adverse effects that can lead to treatment discontinuation. Alternative treatments are needed for this disease.
Over the past decade, many cancers have been successfully treated with targeted therapies on the basis of tumor molecular profiling. Recent publications have re- ported a subset of EMPD tumors with an overexpression of HER2 protein and ERBB2 gene amplification.6-8 In fact, Barth et al11 reported on a case of a patient with inva- sive EMPD who responded completely to single-agent trastuzumab. This patient’s tumor was determined to be HER2 positive by immunohistochemistry; however, next-generation sequencing did not identify an ERBB2 mutation or gene amplification. These findings led to an ongoing phase II trial of combining chemotherapy with trastuzumab in advanced EMPD (UMIN Clinical Trials Registry ID No. UMIN000021311). In our cohort, we identified patients with both ERBB2 gene amplifications and mutations. The point mutations identified are all classified as OncoKB level 3B: candidate predictive biomarkers for drug efficacy. Early evidence suggests that patients with EMPD with a somatic ERBB2 mutation would benefit from HER2-targeting agents, similar to those with HER2 gene amplification.
Another study of EMPD tumors from both male (79%) and female (21%) patients looked at mutations in 10 genes in the RAS/RAF, PI3K/AKT, and WNT pathways, and found mutant RAS and RAF genes in 19% of cases and oncogenic PIK3CA and AKT1 mutations in 35% of cases. Interestingly, the majority of EMPD tumors with a PIK3CA and AKT1 mutation had an invasive disease phenotype. The authors postulated that activation of the PI3K/AKT pathway may be a precursor in the development of EMPD. Of note, the upregulation of the PI3K/AKT pathway is an important pathway in a multitude of cancers and may be associated with worse clinical outcomes and resistance to therapies.
Exploring the genomic profile of EMPD in greater depth, Kiniwa et al29 performed whole-exome sequencing on 3 tumors from patients with EMPD and identified recurrent somatic mutations in TP53, PIK3CA, and ERBB2.29 This work contributes to our understanding of the molecular and genetic underpinnings of vulvar EMPD. Similar to previous reports, we identified a subset of patients with PIK3CA mutations and ERBB2 mutations or amplifications.
Multiple PI3K inhibitors are in development, and here we have described a patient who received benefit from such a targeted agent. ERBB2 inhibitors, including tyrosine kinase inhibitors and monoclonal antibodies, have demonstrated particular benefit in patients with breast cancer. Although these medications are only approved for tumors with HER2 protein overexpression or ERBB2 gene amplification, evidence suggests that patients with ERBB2 mutation-positive cancers can benefit from ERBB2 targeting agents.
Similar to treatments aimed at tumor genetic mutations, immunotherapy drugs have become vital to the treatment of a variety of malignancies. Immunotherapy drugs are effective in DNA mismatch repair protein deficient or MSI-high tu mors, and pembrolizumab, an antibody targeting anti- programmed cell death protein 1 (PD-1), has been ap- proved by the Food and Drug Administration for use in these cancers. Previous studies have reported conflicting con- clusions about programmed death-ligand 1 (PD-L1) ex- pression in EMPD tumors or associated lymphocytes. Mauzo et al31 noted PD-L1 expression in 14% of EMPD tumors and in 83% of the tumor-infiltrating lymphocytes. Conversely, Karpathiou et al32 noted no expression of PD-L1 in any of the 22 EMPD tumors or associated immune infiltrates they ex- amined. Furthermore, Tse et al33 found that no EMPD case in their series was either MSI-high or stained positive for PD-L1.
Although there may be a subset of tumors that express PD- L1, all the tumors that underwent MSI testing in our study were microsatellite stable, and no tumor was MSI-high, likely limiting the utility of immunotherapy in these EMPD tumors. It is also notable that, to date, there is no published literature, to our knowledge, reporting on patients with EMPD responding to checkpoint inhibitors.
Our study is limited by the small number of cases included, making it difficult to draw definitive conclusions. However, vulvar EMPD is a rare disease and our data support findings of previous publications suggesting that some patients with poorly controlled disease may be able to benefit from novel, targeted agents. We believe that although noninvasive EMPD is not lethal, the severity of symptoms and the lack of effective non- surgical treatment options warrant including patients with EMPD in basket trials evaluating drugs targeting molecular aberrations.
REFERENCES
1. McDaniel B, Crane JS: Extramammary Paget disease, in StatPearls. Treasure Island, FL, StatPearls Publishing, 2019
2. Nitecki R, Davis M, Watkins JC, et al: Extramammary Paget disease of the vulva: A case series examining treatment, recurrence, and malignant transformation. Int J Gynecol Cancer 28:632-638, 2018
3. Black D, Tornos C, Soslow RA, et al: The outcomes of patients with positive margins after excision for intraepithelial Paget’s disease of the vulva. Gynecol Oncol 104:547-550, 2007
4. Onaiwu C.O., Salcedo MP, Pessini SA, et al: Paget’s disease of the vulva: A review of 89 cases. Gynecol Oncol Rep 19:46-49, 2016
5. Edey K.A., Allan E, Murdoch JB, et al: Interventions for the treatment of Paget’s disease of the vulva. Cochrane Database Syst Rev 6:CD009245, 2019
6. Masuguchi S, Jinnin M, Fukushima S, et al: The expression of HER-2 in extramammary Paget’s disease. Biosci Trends 5:151-155, 2011
7. Richter C.E., Hui P, Buza N, et al: HER-2/NEU overexpression in vulvar Paget disease: The Yale experience. J Clin Pathol 63:544-547, 2010
8. Tanaka R, Sasajima Y, Tsuda H, et al: Human epidermal growth factor receptor 2 protein overexpression and gene amplification in extramammary Paget disease. Br J Dermatol 168:1259-1266, 2013
9. Kang Z, Xu F, Zhang QA, et al: Oncogenic mutations in extramammary Paget’s disease and their clinical relevance. Int J Cancer 132:824-831, 2013
10. Kang Z, Xu F, Zhang QA, et al: Correlation of DLC1 gene methylation with oncogenic PIK3CA mutations in extramammary Paget’s disease. Mod Pathol
25:1160-1168, 2012
11. Barth P, Dulaimi Al-Saleem E, Edwards KW, et al: Metastatic extramammary Paget’s disease of scrotum responds completely to single agent trastuzumab in
a hemodialysis patient: Case report, molecular profiling and brief review of the literature. Case Rep Oncol Med 2015:895151, 2015
12. Cheng D.T., Mitchell TN, Zehir A, et al: Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): A hybridization
capture-based next-generation sequencing clinical assay for solid tumor molecular oncology. J Mol Diagn 17:251-264, 2015
13. Zehir A, Benayed R, Shah RH, et al: Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat Med 23: 703-713, 2017 [Erratum: Nat Med 23(8):1004, 2017]
14. Smith ES, Da Cruz Paula A, Cadoo KA, et al: Endometrial cancers in BRCA1 or BRCA2 germline mutation carriers: Assessment of homologous recombination DNA repair defects. JCO Precis Oncol 3:1-11, 2019
15. Weigelt B, Bi R, Kumar R, et al: The landscape of somatic genetic alterations in breast cancers from ATM germline mutation carriers. J Natl Cancer Inst 110:1030-1034, 2018
16. Middha S, Zhang L, Nafa K, et al: Reliable pan-cancer microsatellite instability assessment by using targeted next-generation sequencing data. JCO Precis Oncol 2017:10.1200/PO.17.00084, 2017
17. Crawford D, Nimmo M, Clement PB, et al: Prognostic factors in Paget’s disease of the vulva: A study of 21 cases. Int J Gynecol Pathol 18:351-359, 1999
18. Chakravarty D, Gao J, Phillips S, et al: OncoKB: A precision oncology knowledge base. JCO Precis Oncol 1:1-6, 2017
19. Wollina U: Extensive invasive extramammary Paget’s disease: Surgical treatment. J Cutan Aesthet Surg 6:41-44, 2013
20. Lam C, Funaro D: Extramammary Paget’s disease: Summary of current knowledge. Dermatol Clin 28:807-826, 2010
21. Son S.H., Lee JS, Kim YS, et al: The role of radiation therapy for the extramammary Paget’s disease of the vulva; experience of 3 cases. Cancer Res Treat 37:365-369, 2005
22. Cowan R.A., Black DR, Hoang LN, et al: A pilot study of topical imiquimod therapy for the treatment of recurrent extramammary Paget’s disease. Gynecol Oncol 142:139-143, 2016
23. Sawada M, Kato J, Yamashita T, et al: Imiquimod 5% cream as a therapeutic option for extramammary Paget’s disease. J Dermatol 45:216-219, 2018
24. Bose R, Kavuri SM, Searleman AC, et al: Activating HER2 mutations in HER2 gene amplification negative breast cancer. Cancer Discov 3:224-237, 2013
25. Salvesen H.B., Carter SL, Mannelqvist M, et al: Integrated genomic profiling of endometrial carcinoma associates aggressive tumors with indicators of PI3 kinase activation. Proc Natl Acad Sci USA 106:4834-4839, 2009
26. Woenckhaus J, Steger K, Sturm K, et al: Prognostic value of PIK3CA and phosphorylated AKT expression in ovarian cancer. Virchows Arch 450:387-395, 2007
27. Ludovini V, Bianconi F, Pistola L, et al: Phosphoinositide-3-kinase catalytic alpha and KRAS mutations are important predictors of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in patients with advanced non-small cell lung cancer. J Thorac Oncol 6:707-715, 2011
28. Li SY, Rong M, Grieu F, et al: PIK3CA mutations in breast cancer are associated with poor outcome. Breast Cancer Res Treat 96:91-95, 2006
29. Kiniwa Y, Yasuda J, Saito S, et al: Identification of genetic alterations in extramammary Paget disease using whole exome analysis. J Dermatol Sci 94:229-235, 2019
30. Dean L: Trastuzumab (Herceptin) therapy and ERBB2 (HER2) genotype, in Pratt V, McLeod HL, Rubinstein WS, et al:(eds), Medical Genetics Summaries [Internet]. August 5, 2015. Bethesda, MD, National Center for Biotechnology Information, August 5, 2015
31. Mauzo SH, Tetzlaff MT, Milton DR, et al: Expression of PD-1 and PD-L1 in extramammary Paget Disease: Implications for immune-targeted therapy. Cancers (Basel) 11:E754, 2019
32. Karpathiou G, Chauleur C, Hathroubi S, et al: Expression of CD3, PD-L1 and CTLA-4 in mammary and extra-mammary Paget disease. Cancer Immunol Immunother 67:1297-1303, 2018
33. Tse, J, Elvin JA, Vergilio J, et al: Extra-mammary Paget’s disease (EMPD) of the skin: A comprehensive genomic profiling (CGP) study. J Clin Oncol 37:9591, 2019 (15 suppl)
Authors: Marina Stasenko, MD; Gowtham Jayakumaran, MS; Renee Cowan, MD; Vance Broach, MD; Dennis S. Chi, MD; Anthony Rossi, MD; Travis J. Hollman, MD, PhD; Ahmet Zehir, PhD; Nadeem R. Abu-Rustum, MD; and Mario M. Leitao Jr, MD