Comparison of the biomarkers for targeted therapies in primary extra-mammary and mammary Paget's disease.

BACKGROUND: Primary Extra-mammary Paget's disease (EMPD) is a very rare cutaneous adenocarcinoma affecting anogenital or axillary regions. It is characterized by a prolonged course with recurrences and eventually distant metastatic spread for which no specific therapy is known. METHODS: Eighteen EMPD (13 vulvar and five scrotal) and ten mammary Paget's disease (MPD) cases were comprehensively profiled for gene mutations, fusions and copy number alterations, and for therapy-relevant protein biomarkers). RESULTS: Mutations in TP53 and PIK3CA were the most frequent in both cohorts: 7/15 and 5/15 in EMPD; 1/6 and 4/7 in MPD HER2 gene amplification was detected in 4/18 EMPD (3 vulvar and 1 scrotal case) in contrast to MPD where it was detected in the majority (7/8) of cases. TOP2A gene amplification was seen in 2/12 EMPD and 1/6 MPD, respectively. Similarly, no difference in estrogen receptor expression was seen between the EMPD (4/15) and MPD (3/10). Androgen receptor was also expressed in the majority of both cohorts (12/16 EMPD) and (7/8 MPD).Here ARv7 splice variant was detected in 1/7 EMPD and 1/4 MPD cases, respectively. PD-L1 expression on immune cells was exclusively observed in three vulvar EMPD. In contrast to MPD, six EMPDs harbored a "high" tumor mutation burden (≥10 mutations/Mb). All tested cases from both cohorts were MSI stable. CONCLUSIONS: EMPD shares some targetable biomarkers with its mammary counterpart (steroid receptors, PIK3CA signaling pathways, TOP2A amplification). HER2 positivity is notably lower in EMPD while biomarkers to immune checkpoint inhibitors (high TMB and PD-L1) were observed in some EMPD. Given that no consistent molecular alteration characterizes EMPD, comprehensive theranostic profiling is required to identify individual patients with targetable molecular alterations.

Molecular subtyping of mammary Paget's disease using immunohistochemistry.

OBJECTIVE: To evaluate the molecular subtypes of Mammary Paget's disease (MPD) and the associated breast carcinomas. METHODS: This retrospective study was carried out at King Khalid University Hospital and King Faisal Specialist Hospital, Riyadh, Saudi Arabia. Data from MPD patient cases from January 2010 to June 2016 were reviewed. The molecular subtypes were determined based on estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expression with immunohistochemical staining. The relative frequencies of the luminal A and B, HER2-enriched and basal-like molecular subtypes were calculated and compared for MPD and the associated breast carcinomas. Results: Among 22 patients with MPD, HER2-enriched was the most frequently occurring molecular subtype and was observed in 11 (50%) patients. Mammary Paget's disease was classified as basal-like in 5 (22.7%) patients, and luminal A and B were each detected in 3 (13.6%) patients. The molecular subtype of MPD corresponded with the subtype of the associated breast carcinoma in 18 out of 20 patients (90%). CONCLUSIONS: The HER2-enriched subtype is the most frequently occurring molecular subtype in MPD. The molecular subtype of the associated breast carcinoma is usually similar to that of MPD. The molecular subtypes vary between MPD associated breast carcinoma and overall breast carcinoma. The HER2-enriched subtype is the most frequently occurring subtype of MPD associated breast carcinoma, while luminal subtypes are more common in overall breast carcinoma.

Whole-Exome Sequencing Reveals Frequent Mutations in Chromatin Remodeling Genes in Mammary and Extramammary Paget's Diseases.

Paget's disease (PD) is an intraepidermal adenocarcinoma of the skin at the breast (mammary PD) or urogenital locations (extramammary PD [EMPD]). At present, there is lack of clarity on PD's pathogenesis, the relationship between its subtypes, and its lineage link with the underlying invasive carcinomas. Here we describe that mammary PD and EMPD have similar mutational profiles, with the most frequent recurrent mutations occurring in the chromatin remodeling genes, such as KMT2C (MLL3, 39%) and ARID2 (22%), with additional recurrent somatic mutations detected in genes previously not known to be mutated in cancers, such as CDCC168 (34%), FSIP2 (29%), CASP8AP2 (29%), and BIRC6 (24%). In paired mammary PD and underlying breast carcinoma samples, distinct gene mutations were detected, indicating that they represent independent oncogenic events. Finally, multistage EMPD tissue sequencing revealed KMT2C gene occurring early in EMPD oncogenesis, and that multifocal EMPD samples share the same early gene mutations, suggesting clonal origin of multifocal EMPD. Our results reveal similar genomic landscapes between mammary PD and EMPD, including early aberrations in chromatin remodeling genes. In addition, mammary PD and underlying breast ductal carcinomas represent independent oncogenic events. These findings provide approaches for developing diagnostic tools and therapeutic interventions for PD.

Transcriptome analyses reveal FOXA1 dysregulation in mammary and extramammary Paget's disease.

Paget's disease (PD) is an uncommon intraepithelial adenocarcinoma with unknown pathogenesis. There are two anatomic subtypes: mammary (MPD) and extramammary (EMPD). Little is known about their molecular characteristics. Our objective was to discover novel molecular markers for PD and its subtypes. In the discovery phase, we used transcriptome analyses to uncover the most differentially expressed genes and pathways in EMPD biopsies compared with normal skin. In the validation phase, we performed immunohistochemistry analyses on the most promising marker (FOXA1) and other markers selected from a literature review (GATA3, estrogen receptor [ER], and androgen receptor [AR]) on independent biopsies of MPD (n = 86), EMPD (n = 59), and normal skin (n = 21). Transcriptome analyses revealed 210 genes differentially expressed more than 10-fold between EMPD and normal skin. These genes are involved in mammary and sweat gland development (FOXA1) and immune regulation, as well as epidermal differentiation. Immunohistochemistry staining revealed that FOXA1 was positive in 88% of both MPD and EMPD, whereas GATA3 was positive in 67% of MPD and 77% of EMPD, and ER was positive in 9% of MPD and 19% of EMPD. Finally, AR was positive in 33% of PD and 54% of EMPD. Mammary Paget's disease and EMPD share dysregulation of the glandular developmental regulator gene FOXA1, suggesting similarity in cell-specific transcriptional regulation. Further, FOXA1 may be a useful molecular target for developing PD therapies.

HER2 molecular subtype is a dominant subtype of mammary Paget's cells. An immunohistochemical study.

AIMS: To test the hypothesis that the similarity of the molecular subtypes of Paget's cells to the molecular subtypes of the underlying breast carcinomas favours the epidermotrophic theory of the origin of Paget's cells. METHODS AND RESULTS: The immunohistochemical expression of markers that define particular molecular subtypes of breast carcinomas were analysed. The whole analysis was performed by means of tissue microarrays in mammary Paget's disease and in the underlying breast carcinoma(s). Human epidermal growth factor receptor type 2 (HER2)-overexpression subtype [oestrogen receptor (ER(-) ); HER2(+) ] was a dominant molecular subtype of Paget's cells (37 of 43 analysed cases; 86%). Luminal B (ER(+) ; HER2(+) ) and luminal A (ER(+) ; HER(-) ) subtypes were identified in 12% and 2% of cases, respectively. None of the analysed tumours presented a basal-like phenotype. A similar distribution of molecular subtypes was identified in the underlying in situ breast carcinomas (HER2 subtype, 82%; luminal A, 6%; luminal B, 6%; basal-like, 6% of cases) and in the invasive component (HER2 subtype, 84%; luminal A, 8%; luminal B, 8%; basal-like, 0% of cases). CONCLUSIONS: HER2 molecular subtype is the dominant, but not the sole subtype seen in Paget's cells of the nipple. A similar distribution of molecular subtypes in both Paget's cells and in the underlying carcinomas strongly suggests their common origin.

Protocol for stage 2 of the GaP study (genetic testing acceptability for Paget's disease of bone): a questionnaire study to investigate whether relatives of people with Paget's disease would accept genetic testing and preventive treatment if they were available.

BACKGROUND: Paget's disease of bone (PDB) disrupts normal bone architecture and causes pain, deformity, deafness, osteoarthritis, and fractures. Genetic factors play a role in PDB and genetic tests are now conducted for research purposes. It is thus timely to investigate the potential for a clinical programme of genetic testing and preventative treatment for people who have a family history of PDB. This study examines the beliefs of relatives of people with PDB. It focuses particularly on illness and treatment representations as predictors of the acceptability and uptake of potential clinical programmes. Illness representations are examined using Leventhal's Common Sense Self-Regulation Model while cognitions about treatment behaviours (acceptance of testing and treatment uptake) are conceptualised within the Theory of Planned Behaviour. METHODS/DESIGN: A postal questionnaire of non-affected relatives of people with Paget's disease. The sample will include relatives of Paget's patients with a family history of Paget's disease and relatives of Paget's patients without a family history of Paget's disease. The questionnaire will explore whether a range of factors relate to acceptability of a programme of genetic testing and preventive treatment in relatives of Paget's disease sufferers. The questionnaire will include several measures: illness representations (as measured by the Brief Illness Perceptions Questionnaire); treatment representations (as measured by Theory of Planned Behaviour-based question items, informed by a prior interview elicitation study); descriptive and demographic details; and questions exploring family environment and beliefs of other important people. Data will also be collected from family members who have been diagnosed with Paget's disease to describe the disease presentation and its distribution within a family. DISCUSSION: The answers to these measures will inform the feasibility of a programme of genetic testing and preventive treatment for individuals who are at a high risk of developing Paget's disease because they carry an appropriate genetic mutation. They will also contribute to theoretical and empirical approaches to predicting diagnostic and treatment behaviours from the combined theoretical models.

Paget's disease of the nipple: a copy number of the genes ERBB2 and CCND1 versus expression of the proteins ERBB-2 and cyclin D1.

Paget's disease of the nipple (PDN) is characterized by Paget's cells infiltrating from an underlying breast carcinoma into the epidermis of the nipple. The tumor cells were reported to overexpress ERBB-2 protein. There is no evidence, whether the overexpression is caused by amplification of the ERBB2 gene (as is the case of invasive duct carcinomas) or by other causes. Because the Paget's cells proliferate vividly we were interested also in cyclin D1 expression and in its relation to the number of CCND1 gene copies. To address these issues, we performed a study using fluorescence in situ hybridization on interphasic nuclei (I-FISH) on formalin fixed / paraffin embedded tissue sections from twelve women with PDN. We compared immunohistochemical (IHC) expression of the ERBB-2 protein and cyclin D1 with the copy number of the gene ERBB2 and CCND1, respectively (I-FISH). In all cases of our series we found overexpression of the ERBB-2 protein (3+), and in all of them there was a strong amplification of the ERBB2 gene (more than 10 signals per a nucleus, usually about 20). Keratinocytes of the epidermis had two signals of the gene. IHC and I-FISH revealed comparable findings in successive biopsies in two patients. Cyclin D1 was positive in seven cases. We found a moderate amplification of the CCND1 gene (up to 10 signals per a nucleus) in three patients only. Any correlation between the cyclin D1 overexpression anda copy number of the CCND1 gene was observed. Combined numerical changes of chromosome 17 and of chromosome 11 were found in seven women. It is concluded that in PDN, the ERBB-2 protein overexpression is caused by amplification of the ERBB2 gene. A specific immuno-therapy with trastuzumab (Herceptin) used in patients with invasive duct carcinoma may be also effective in patients with PDN.

HCR, a candidate gene for psoriasis, is expressed differently in psoriasis and other hyperproliferative skin disorders and is downregulated by interferon-gamma in keratinocytes.

We have previously shown that HCR is a good candidate gene for psoriasis based on its location in the PSORS1 locus, predicted secondary structure change of the associated allele, and expression pattern. To understand better the function of HCR, we studied how HCR expression is altered in hyperproliferative skin diseases other than psoriasis and in cancers. We examined also its regulation by different cytokines, growth factors, and antipsoriatic agents using quantitative RT-PCR (TaqMan) analysis and its location by immunostaining of keratinocyte cultures. Compared to psoriasis, HCR protein had a different distribution in chronic dermatitis, pityriasis rubra pilaris, mycosis fungoides, and chronic skin ulcers. In three of six grade III squamous cell carcinomas of the skin, four of four adenocarcinomas of the lung, and two of two ductal breast adenocarcinomas, positive cytoplasmic staining in cancer cells was detected. As in psoriasis, Ki67 did not colocalize with HCR. In cell cultures, HCR staining was detected perinuclearly in the cytoplasm and in the nuclei, suggesting that the protein may have a role in both compartments. A 2-fold downregulation of HCR mRNA expression was observed on stimulation with interferon-gamma. Based on the observations that HCR is detected in cancers of epithelial origin in Ki67-negative areas and that interferon-gamma downregulates its expression, we suggest it to have an antiproliferative function.

Intraepidermal cells of Paget's carcinoma of the breast can be genetically different from those of the underlying carcinoma.

Paget's carcinoma (PC) of the breast is characterized by neoplastic cells of "glandular" type located within the epidermis of the nipple-areolar complex, often associated with an underlying ductal carcinoma, either in situ or invasive. At present the origin of PC cells is controversial, although there is a widespread opinion that PC cells are "foreign" elements to the epidermis resulting from an epidermotropic migration of neoplastic elements from an underlying ductal carcinoma. An alternative view is that some cases result from neoplastic transformation of preexisting, innocent intraepidermal clear cells of the nipple-areolar complex (Toker cells) that migrate from nonneoplastic ducts. Consequently, 10 cases were studied using methods for clonality (ie, loss of heterozygosity and mitochondrial DNA displacement loop sequence analysis). Microdissection of intraepidermal neoplastic cells and of cells from underlying duct carcinomas and metastases was performed. In no fewer than 2 cases, PC cells were genetically different from underlying lesions, which showed consistent homology among themselves. Therefore, it is suggested that the rule of epidermotropism by neoplastic cells from an underlying carcinoma is not applicable to all cases, and that in some cases PC cells might be the result of neoplastic transformation of preexisting intraepidermal nonneoplastic cells. Consequently, the underlying tumors are coincidental neoplastic lesions (collision tumors).

The role of HER-2/neu oncogene and vimentin filaments in the production of the Paget's phenotype.

The histogenesis as well as the biological and molecular differences in mammary Paget's disease (MPD) and extramammary Paget's disease (EPD) are not well understood. HER-2/neu oncogene overexpression is associated with poor prognosis in breast cancer patients. It is also believed that the spread of Paget's cells through the epidermis is induced by a motility factor that acts via the HER-2/neu receptor. However, previous studies on HER-2/neu expression in MPD and EPD have given conflicting results. Recent studies have suggested that vimentin expression in breast cancer confers a more aggressive phenotype with a possible role in tumor invasion and metastasis. We examined 58 cases of MPD and EPD for HER-2/neu overexpression and vimentin status to study the role of these markers in the production of the Paget's phenotype. Thirty-five of the 38 cases (92.1%) of MPD were associated with an underlying carcinoma, while none of the cases of EPD were associated with an underlying malignancy. Thirty-six of the 38 cases of MPD (94.7%) overexpressed the HER-2/neu oncoprotein and 17 cases (44.7%) showed vimentin expression. In contrast, only 1 of the 20 cases of EPD (5%) showed positivity for HER-2/neu oncoprotein and all were negative for vimentin. Our results indicate that the cell motility enhancing effect of HER-2/neu oncoprotein and possibly vimentin plays a significant role in the pathogenesis of MPD which appears to be a pagetoid spread of an underlying ductal malignancy (secondary), while EPD is an in situ malignant transformation of a totipotential epidermal cell or glandular epithelium.

HER2 oncogene amplification in extramammary Paget's disease.

AIMS: To study HER2 oncogene amplification and over-expression in skin samples of 23 patients with extramammary Paget's disease (EMP). EMP is a rare intra-epidermal adenocarcinoma, which has been reported to over-express the HER2 oncoprotein. METHODS AND RESULTS: HER2 gene amplification, detected by chromogenic in-situ hybridization, was found in 43% (10/23) of the lesions. HER2 protein over-expression (3+ immunostaining intensity) was found in 12 tumours (52%), including all 10 tumours with gene amplification. Two tumours showed low-level (2+) HER2 immunostaining. Mammary Paget's lesions, which were used as controls, showed HER2 amplification and over-expression in all 10 cases studied. CONCLUSIONS: These results indicate that HER-2 protein over-expression in EMP is common and due exclusively to gene amplification. They open up the possibility of HER2-targetted immunotherapy for patients with HER2+ disease.

Morphological types of breast cancer in family members and multiple primary tumours: is morphology genetically determined?

BACKGROUND: We conducted the present study to determine whether breast cancer morphology is genetically determined. METHODS: Using the nationwide Swedish Family Cancer Database, which includes data on 10.2 million individuals and over 25,000 morphology-specific breast cancers, we followed morphological types in familial cancers between mothers and daughters and between sisters. Additionally, we recorded morphological data in women who presented with two primary breast cancers and in those who presented with an invasive and in situ breast cancer. We used kappa statistics to examine the association between genetics and morphology. A kappa value of 0 indicates that the process is random and a value of 1 indicates that it is completely determined (i.e. genetic); values between 0.40 and 0.60 are considered to indicate a moderately determined process. RESULTS: The study sample included a total of 25,730 first and 3394 second invasive breast cancers, and 2990 in situ breast cancers. Ductal, lobular, tubuloductal and comedo were the most common invasive types. We identified 164 mother-daughter pairs with breast cancer of a defined morphology, yielding a low kappa value of 0.08. Among 100 sister pairs the kappa value was 0.002. In individuals with two primary breast cancers the kappa values were 0.22 and 0.01 for two invasive and in situ-invasive pairs, respectively. However, for a tumour with a subsequent tumour detected in the contralateral breast less than 1 year later the kappa value was 0.47. CONCLUSION: The data suggest that breast cancer morphology is not genetically determined. However, because of mixed morphologies and the overwhelming prevalence of ductal morphology, the results for rare morphologies should be interpreted with caution.

Germline p53 mutation in a patient with multiple primary cancers.

We report a case of multiple primary cancers having a germline missense mutation of the p53 gene. The patient was a Japanese female and had a history of five different types of cancers. PCR/direct sequencing analysis revealed the presence of a nucleotide substitution, AGC (Ser) to AGG (Arg), at codon 106 of the p53 gene in DNA from non-cancerous breast tissue. This is the first case of germline p53 mutation at codon 106, and could contribute to establishing correlations between the types and locations of germline p53 mutations and their phenotypical consequences.

Identification of women at high genetic risk of breast cancer through the National Health Service Breast Screening Programme (NHSBSP).

Breast cancer is a multifactorial disease with an inherited predisposition being implicated in around 5% of all cases. Using previous epidemiological data assessing risks for the relatives of women with breast cancer, we have identified 154 women (from a screened population of 35,505) and 289 of their relatives between 50 and 64 years who have more than twice the age related risk of developing breast cancer. This constitutes 1.24% of the breast screening population attending the North East Scotland NHSBSP. For each woman identified to be at high risk, we have found 1.87 female relatives between 50 and 64 years and 1.85 relatives under 50 years also to be at high risk. Around 78% of the women identified with a significant family history of breast or other cancer have attended for counselling about their risks. The breast screening programme can be used to identify women at high risk of breast cancer in order to offer them (and their relatives) access to genetic counselling and appropriate screening.

A comparative study of cytokeratin expression in Paget cells located at various sites.

BACKGROUND: Extramammary Paget disease appears in anogenital, axillary, or other areas. In this study, the authors addressed the question of whether the histogenesis of 35 cases of Paget disease arising at different sites was the same. METHODS: Specimens of 35 cases of extramammary Paget disease (16 genital; 9 invasive carcinomas of genital; 6 axillary; 1 periumbilical; and 3 perianal), 4 cases of mammary Paget disease, 4 cases of breast carcinomas, and 6 cases of anal carcinomas of perianal spread from primary rectal adenocarcinomas were retrieved and stained by the avidin-biotin-complex method, using various kinds of monoclonal antikeratin antibodies. RESULTS: There was no significant difference in cytokeratin expression among these cases of extramammary Paget disease. Simple epithelial keratins were expressed in Paget cells in extramammary Paget disease, but no expression of differentiation-specific or noncornifying stratified squamous epithelial keratins was observed, regardless of the degree of invasion. Paget cells in extramammary Paget disease revealed a similar cytokeratin expression to that in secretory cells of normal apocrine or eccrine glands. In addition, there was no significant difference in cytokeratin expression in tumor cells among extramammary and mammary Paget disease, breast carcinomas, and anal carcinomas. CONCLUSIONS: Cases of Paget disease arising at different locations could not be distinguished from each other based on cytokeratin expression. In addition, antikeratin antibodies against simple epithelial keratins were demonstrated to be more useful for the identification of Paget cells in the paraffin sections than were conventional antibodies, such as an antibody against carcinoembryonic antigen (CEA).

c-erbB-2 oncoprotein expression in mammary and extramammary Paget's disease: an immunohistochemical study.

Over-expression of the c-erbB-2 oncogene occurs in a proportion of human adenocarcinomas and in breast carcinoma is associated with poorer prognosis. Sections of formalin-fixed, paraffin-embedded tumour tissue from 22 patients with mammary and extramammary Paget's disease have been stained immunohistochemically using a monoclonal antibody (NCL-CB11) raised against a synthetic peptide from the C-terminal end of the predicted sequence of the c-erbB-2 protein product. All 12 cases of mammary Paget's disease showed membrane staining of intra-epidermal cells, indicating c-erbB-2 over-expression. Sections of underlying ductal breast carcinoma were available in nine cases; all nine tumours were c-erbB-2 positive and in eight the in situ component was of comedo or solid type. There was membrane staining of tumour cells in four of the 10 cases of extramammary Paget's disease; staining intensity was generally weaker than that observed in the cases of mammary disease. The possible implications of these findings for the histogenesis of both mammary and extramammary Paget's disease are discussed.