Nested Variant of Urothelial Carcinoma Is a Luminal Bladder Tumor With Distinct Coexpression of the Basal Marker Cytokeratin 5/6.

OBJECTIVES: The nested variant of urothelial carcinoma (NVUC) is a rare bladder tumor that may possess a luminal molecular phenotype. We sought to determine whether a small immunohistochemical (IHC) panel using common surrogates for molecular phenotypes would reliably classify a cohort of pure NVUC cases. METHODS: IHC staining with a panel composed of markers for basal subtypes (CK5/6, CK14) and luminal subtypes (FOXA1, GATA3) was performed on pure small NVUC cases (n = 23) and 5 large NVUC cases (n = 5). Scoring of IHC stains was performed semiquantitatively. Individual cases were analyzed using previously reported IHC-based surrogates for molecular subtype. RESULTS: The phenotype of NVUC was classified as luminal from 60.1% (FOXA1+/CK5/6-) to 100% (GATA3+/CK14-) of cases using composite phenotypes. No cases possessed a basal or squamous cell carcinoma-like phenotype. The majority of small NVUC cases (69.5%) showed subset CK5/6 expression distinctly localized to the basal layers of tumor cell nests. Intratumoral heterogeneity was also noted in CK5/6 (21.7% of small NVUC cases) but no other markers. CONCLUSIONS: NVUC appears to express markers of both basal and luminal bladder tumors. Definitive gene expression profiling may be valuable to further characterize this unique histologic variant.

High frequency of p16 and SOX10 coexpression but not androgen receptor expression in triple-negative breast cancers.

Triple-negative breast cancers (TNBCs) represent approximately 12-17% of all breast cancers and have distinctively aggressive clinical courses. Because routine biomarkers for breast cancer do not apply for TNBCs, it is essential to find novel prognostic markers and potential targets for therapeutic agents. p16 and SOX10 are emerging biomarkers with relatively unexplored expressions in TNBCs. We present an analysis of the expression of p16 and SOX10 in combination with that of androgen receptor (AR) and cytokeratin (CK) 5/6 in TNBCs. In addition, we used tissue microarrays (TMAs) to compare frequencies of p16 and SOX10 between TNBCs and non-TNBCs. Fifty-six TNBC samples with clinical data were stained immunohistochemically with p16, SOX10, AR, and CK5/6. Fifty-four cases (96.4%) were invasive ductal carcinoma, not otherwise specified, and 46 cases (82.1%) were Nottingham histologic grade 3. The majority of TNBC cases were positive for p16 (n = 44; 78.6%) and SOX10 (n = 48; 85.7%). AR was positive in 15 cases (26.8%). CK5/6 was positive in 24 cases (42.9%), which were classified as basal-like breast cancer (BLBC) subtype. The frequencies of p16 and SOX10 expression in BLBC and non-BLBC subtypes did not reveal significant statistical difference in a separate analysis. Using archived TNBC and non-TNBC TMAs, we observed that 56% of TNBC cases were positive for p16 compared with 16% of non-TNBC cases (p-value <0.0001). SOX10 was positive in 80% of TNBC cases compared with 35% of non-TNBC cases (p-value <0.0001). A significant correlation was observed between p16 and SOX10 coexpression in TNBC cases (n = 56/80, p = 0.02) but not in non-TNBC cases (n = 23/348; p = 0.626). In conclusion, p16 and SOX10 are frequently expressed in TNBC, regardless of CK5/6 expression. Furthermore, p16 and SOX10 are often coexpressed in TNBCs compared with non-TNBCs.

The coexpression of fibroblast activation protein (FAP) and basal-type markers (CK 5/6 and CD44) predicts prognosis in high-grade invasive urothelial carcinoma of the bladder.

High-grade urothelial carcinoma (UC) of the bladder is a heterogeneous disease with dismal prognosis. Bladder tumors with basal phenotype are intrinsically aggressive, and morphological parameters that define disease staging remain main prognosticators. We intend to evaluate the role of cancer-associated fibroblasts (CAFs) in the prognosis of bladder cancer and its association with basal and luminal phenotypes. Clinical and pathological parameters, including the immunohistochemical expression of fibroblast activation protein (FAP) and markers of basal (CK5/6, CD44) and luminal (CK20, GATA3) phenotypes, have been investigated in a series of 121 patients with UC of the bladder treated by radical cystectomy with lymph node dissection, and their implication in long-term cancer-specific survival has been evaluated. A cytoplasmic immunostaining of FAP in CAFs implies worse disease-specific survival (hazard ratio [HR] = 1.68; P = .048). FAP expression is associated with tumor staging (P < .0001), with best discrimination at T2a/T2b level, and with negative expression of markers of luminal phenotype, such as CK20 (P < .0001) and GATA3 (P = .005). In the multivariate analysis, simultaneous expression of FAP, CK5/6, and CD44 is a strong prognosticator of disease-specific survival (HR = 2.3; P = .001), together with nodal invasion (HR = 3.47; P < .0001) and bladder infiltration up to deep muscle or beyond (HR = 2.47; P = .02). There is no association between positive FAP expression in primary tumor and nodal disease (P = .22). FAP expression in CAFs favors tumor invasion in high-grade invasive UC of the bladder with basal phenotype. This new immunohistochemical marker could be added to the routine immunohistochemical protocol to predict clinical behavior in these patients.

Predicting outcomes in non-muscle invasive (Ta/T1) bladder cancer: the role of molecular grade based on luminal/basal phenotype.

Bladder cancer tumors can be divided into two molecular subtypes referred to as luminal or basal. Each subtype may react differently to current chemotherapy or immunotherapy. Likewise, the technology required for comprehensive molecular analysis is expensive and not yet applicable for routine clinical diagnostics. Therefore, it has been suggested that the immunohistochemical expressions of only two markers, luminal (CK20+, CK5/6-) and basal (CK5/6+, CK20-), is sufficient to identify the molecular subtypes of bladder cancer. This would represent a molecular grade that could be used in daily practice. Molecular classification is done using immunohistochemistry to assess luminal-basal phenotype based on tissular expression of CK20 and CK5/6 as surrogate for luminal or basal subtypes, respectively. A series of 147 non-muscle-invasive bladder carcinoma cases was selected, and the tumors were divided into four subgroups based on the presence of CK20 and/or CK5/6, that is, null (CK20-, CK5/6-), mixed (CK20+, CK5/6+), basal (CK20-, CK5/6+), and luminal (CK20+, CK5/6-) categories. Survival analysis was estimated using the Kaplan-Meier method and the log-rank test. Hazard ratios were calculated by Cox multivariate analysis. The molecular grade included cases with null (n = 89), mixed (n = 6), basal (n = 20), and luminal (n = 32) phenotypes with differences in recurrence-free, progression-free and cancer-specific survival associated with molecular-grade categories in patients with low- or high-grade Ta, or high-grade T1 tumors. The multivariate analysis identified the luminal phenotype as a predictor of more aggressive neoplasms. Our findings provide a rationale to investigate luminal and basal subtypes of bladder cancer using two gene expression signatures as surrogate markers and show that non-muscle-invasive bladder carcinoma can be stratified into biologically and clinically different subgroups by using an immunohistochemical classifier.

Expression of PTEN, Androgen Receptor, HER2/neu, Cytokeratin 5/6, Estrogen Receptor-Beta, HMGA2, and PLAG1 in Salivary Duct Carcinoma.

Salivary duct carcinoma (SDC) is an aggressive neoplasm that resembles high-grade invasive ductal carcinoma of the breast. It can develop de novo or from the malignant transformation of pleomorphic adenoma (PA). We performed immunohistochemical stains for phosphatase and tensin homologue [PTEN androgen receptor (AR)], HER2/neu, cytokeratin 5/6, estrogen receptor-beta, high-mobility group AT-hook 2 (HMGA2), and pleomorphic adenoma gene 1 (PLAG1) on tissue microarray samples of 75 SDCs and 31 adenocarcinomas, not otherwise specified (NOS). Our data showed the following in SDC samples: loss of PTEN was found in 17 of 60 (28.3%); AR was expressed in 43 of 62 (69.4%); HER2/neu was overexpressed in 25 of 58 (43.1%); cytokeratin 5/6 was expressed in 14 of 54 (25.9%); estrogen receptor-beta was expressed in 37 of 56 (66.1%); HMGA2 was expressed in 29 of 63 (46.0%); and PLAG1 was expressed in 0 of 62 (0%). In addition, there was no statistically significant difference in the age at onset between patients with HMGA2-positive SDCs (range 32-85 years; mean: 64.3 years; median: 64.5 years) and those with HMGA2-negative SDCs (range 41-79 years; mean: 62.5 years; median: 64.5 years). There was also no statistically significant difference in overall survival between patients with HMGA2-positive and HMGA2-negative SDCs (follow-up period range 3-201 months; mean: 49.8 months; median: 30 months). Among 10 patients with a definite PA component (SDC ex-PA), 6 were positive and 4 were negative for HMGA2. Our data were consistent with previous findings that AR and estrogen receptor-beta are expressed in most SDCs, whereas HER2/neu overexpression and loss of PTEN are expressed in a subset of SDCs. In our cohort of patients, HMGA2 was expressed in approximately half of SDCs. HMGA2 and PTEN are promising therapeutic targets for salivary gland tumors.

Immunohistochemistry of cytokeratin (CK) 5/6, CD44 and CK20 as prognostic biomarkers of non-muscle-invasive papillary upper tract urothelial carcinoma.

AIMS: Immunohistochemical (IHC) staining for cytokeratin (CK) 5/6, CD44 and CK20 has been significantly associated with the prognosis of urinary bladder urothelial carcinoma, and probably reflects its molecular characteristics. We aimed to investigate the IHC-based subgroups and their prognostic effects on non-muscle-invasive papillary upper tract urothelial carcinoma (UTUC). METHODS AND RESULTS: IHC staining for CK5/6, CK20 and CD44 was analysed in 211 patients with non-muscle-invasive papillary UTUC. Staining was classified as showing a negative, positive or normal pattern. We found that CK5/6-negative, CD44-negative and CK20-positive tumours were distinctly high-risk subgroups that were associated with high grade (CK5/6-negative, P < 0.001; CD44-negative, P < 0.001; CK20-positive, P = 0.017) and frequent intravesical recurrence (CK5/6-negative, P = 0.002). Using survival analysis with Kaplan-Meier and log-rank tests, we found that these IHC subgroups were correlated with poor progression-free (CK5/6-negative, P = 0.001; CD44-negative, P = 0.009; CK20-positive, P = 0.031) and cancer-specific (CK5/6-negative, P = 0.009) survival. Furthermore, CK5/6 negativity was an independent prognostic factor for shorter progression-free (P = 0.009) and cancer-specific (P = 0.045) survival. CK5/6 improved Harrell's C-indices for progression-free (0.68-0.77, P = 0.029) and cancer-specific (0.59-0.77, P < 0.001) survival. When markers were combined, luminal-like subtypes showed poor prognoses. CONCLUSIONS: We demonstrated that IHC staining for CK5/6, CD44 and CK20 was significantly associated with the clinicopathological characteristics and prognoses of patients with non-muscle-invasive papillary UTUC. The IHC subgroups may be correlated with the molecular characteristics of non-muscle-invasive papillary UTUC.

Is prolactin a negative neuroendocrine regulator of human skin re-epithelisation after wounding?

Chronic wounds remain a major unmet healthcare challenge, associated with substantial morbidity and economic costs. Therefore, novel treatment strategies and therapeutic approaches need to be urgently developed. Yet, despite the increasingly recognized importance of neurohormonal signaling in skin physiology, the neuroendocrine regulation of cutaneous wound healing has received surprisingly little attention. Human skin, and its appendages, locally express the pleiotropic neurohormone prolactin (PRL), which not only regulates lactation but also hair follicle cycling, angiogenesis, keratinocyte proliferation, and epithelial stem cell functions. Therefore, we examined the effects of PRL in experimentally wounded female human skin organ culture. Overall, this revealed that PRL slightly, but significantly, inhibited epidermal regeneration (reepithelialisation), cytokeratin 6 protein expression and intraepidermal mitochondrial activity (MTCO1 expression), while it promoted keratinocyte terminal differentiation (i.e. involucrin expression) ex vivo. If the current pilot data are confirmed by further studies, PRL may serve as one of the-rarely studied-negative regulators of cutaneous wound healing that control excessive reepithelialisation. This raises the intriguing and clinically relevant question of whether PRL receptor antagonists could actually promote epidermal repair after human skin wounding.

Assessment of markers expressed in human hair follicles according to different skin regions.

BACKGROUND: Body region-dependent hair follicle (HF) characteristics are concerned with follicular size and distribution, and have been demonstrated to have characteristics for each region of the body. OBJECTIVES: The aim of the present study was to investigate the expression patterns of the markers called cytokeratin 15 (K15), cytokeratin 6 (K6) and monoclonal antibody Ki-67, and also apoptosis in HFs, which can be observed in different parts of the human body. MATERIAL AND METHODS: In this study, healthy human HFs were taken by biopsy from 5 various donor sites of the human body: the scalp, the leg, the abdomen, the back and waist. HF-containing skin specimens taken using cryosection were stained with hematoxylin & eosin (H&E) and K15, K6, Ki-67 and terminal deoxynucleotidyl transferase-mediated digoxigenin-dNTP nick end-labelling (TUNEL) immunofluorescence staining protocol was performed. RESULTS: Different skin regions from the human body were examined histologically. While the HFs of scalp tissue showed anatomically obvious hair layers, some hair sections from other regions, like the leg, the abdomen, back and waist, were not as distinct as in the scalp region. According to our findings, K15 expression was highest in the scalp. In addition, the immunoreactivity (IR) intensity of K15 was significantly decreased in the HFs on the waist and abdominal regions, compared to the scalp and back regions (p < 0.001). However, the IR intensity of K6 in the scalp region was statistically significantly higher than the IR intensity of K6 in the abdomen region (p < 0.05). Moreover, we showed intraepithelial apoptosis and proliferation of keratinocytes in the bulge of HF. In the study, Ki-67-positive and TUNEL-positive cell numbers were not statistically significant (p > 0.05). CONCLUSIONS: Our findings are important for further investigation of molecular aspects of the human hair follicle stem cells compartments in health and disease, which might be a promising model for comparative studies with different human diseases.

Cytokeratin 5/6 expression in bladder cancer: association with clinicopathologic parameters and prognosis.

OBJECTIVES: Well differentiated keratinized squamous component as a part of urothelial carcinoma can be easily appreciated; however non-keratinizing squamous differentiation closely resembles urothelial differentiation. In addition prognostic significance of CK 5/6 expression in the absence of apparent squamous differentiation is still unclear. Therefore, in the present study we aimed to evaluate the frequency of CK 5/6 expression in 127 cases of urothelial carcinoma and its prognostic significance in loco-regional population. RESULTS: Positive CK5/6 expression was noted in 6.3% (8 cases) and 13.4% (17 cases) revealed focal positive CK 5/6 expression. On the other hand, 80.3% (102 cases) showed negative CK5/6 staining. Significant association of CK5/6 expression was noted with tumor grade and muscularis propria invasion, however no significant association was noted with overall and disease free survival. On the basis of the results of our study we can conclude that CK5/6 is an independent prognostic biomarker in urothelial carcinoma and therefore can be used in the prognostic stratification of the patients with bladder cancer.

Nrf2 Promotes Keratinocyte Proliferation in Psoriasis through Up-Regulation of Keratin 6, Keratin 16, and Keratin 17.

Psoriasis is a chronic inflammatory skin disease characterized by keratinocyte hyperproliferation of epidermis. Although hyperproliferation-associated keratins K6, K16, and K17 are considered to be the hallmarks of psoriasis, the molecular basis underlying the overexpression of these keratins remains unclear. Nrf2 regulates cell proliferation. Therefore, we investigated whether Nrf2 regulates keratinocyte proliferation via promoting expression of K6, K16, and K17 in psoriasis. We initially found that psoriatic epidermis exhibited elevated expression of Nrf2. Furthermore, Nrf2 promoted expression of K6, K16, and K17 in both HaCaT cells and primary human keratinocytes by binding to the ARE domains located in the promoter of these genes. Additionally, upon stimulation with IL-17 or IL-22, Nrf2 translocated to the nucleus and initiated expression of targeted keratins. In mice of imiquimod-induced psoriasis-like dermatitis, topical application of Nrf2 small interfering RNA alleviated the epidermal hyperplasia with reduced expression of these keratins. More importantly, Nrf2 promoted the proliferation of human keratinocytes through up-regulation of K6, K16, or K17. These data suggested that inflammatory cytokines promoted Nrf2 nuclear translocation in psoriatic epidermis, which led to elevated expression of K6, K16, and K17, thus promoting keratinocyte proliferation and contributing to the pathogenesis of psoriasis.

The expression of keratin 6 is regulated by the activation of the ERK1/2 pathway in arsenite transformed human urothelial cells.

Urothelial cancers have an environmental etiological component, and previous studies from our laboratory have shown that arsenite (As+3) can cause the malignant transformation of the immortalized urothelial cells (UROtsa), leading to the expression of keratin 6 (KRT6). The expression of KRT6 in the parent UROtsa cells can be induced by the addition of epidermal growth factor (EGF). Tumors formed by these transformed cells have focal areas of squamous differentiation that express KRT6. The goal of this study was to investigate the mechanism involved in the upregulation of KRT6 in urothelial cancers and to validate that the As+3-transformed UROtsa cells are a model of urothelial cancer. The results obtained showed that the parent and the As+3-transformed UROtsa cells express EGFR which is phosphorylated with the addition of epidermal growth factor (EGF) resulting in an increased expression of KRT6. Inhibition of the extracellular-signal regulated kinases (ERK1/2) pathway by the addition of the mitogen-activated protein kinase kinase 1 (MEK1) and MEK2 kinase inhibitor U0126 resulted in a decrease in the phosphorylation of ERK1/2 and a reduced expression of KRT6. Immuno-histochemical analysis of the tumors generated by the As+3-transformed isolates expressed EGFR and tumors formed by two of the transformed isolates expressed the phosphorylated form of EGFR. These results show that the expression of KRT6 is regulated at least in part by the ERK1/2 pathway and that the As+3-transformed human urothelial cells have the potential to serve as a valid model to study urothelial carcinomas.

Basal cell carcinoma: CD56 and cytokeratin 5/6 staining patterns in the differential diagnosis with Merkel cell carcinoma.

BACKGROUND: Basal cell carcinoma (BCC) can resemble Merkel cell carcinoma (MCC) on histopathological examination and while CK20 is a useful marker in this differential, it is occasionally negative in MCC. CD56, a sensitive marker of neuroendocrine differentiation, is sometimes used to identify MCC, but has been reportedly variably positive in BCC as well. In contrast, CK5/6 consistently labels BCC but is not expressed in neuroendocrine tumors. METHODS: We evaluated 20 cases of BCC for the pattern of CD56 and cytokeratin 5/6 (CK5/6) staining, hypothesizing that these 2 stains could differentiate BCC from MCC in difficult cases. Seventeen cases of MCC previously stained with CD56 were also examined. RESULTS: All BCCs showed patchy expression of CD56 except for 2 cases, which showed staining of greater than 70% of tumor. CK5/6 was diffusely positive in all cases of BCC. Fifteen of 17 MCCs were diffusely positive for CD56. The difference in the pattern of CD56 expression between MCC and BCC (diffuse vs patchy, respectively) was statistically significant (P < .05). CONCLUSION: BCC typically shows patchy CD56 expression and diffuse CK5/6 positivity. These 2 markers can be used to distinguish between BCC and MCC in challenging cases.

[Metaplastic carcinoma of the breast and the impact of the p63 and cytokeratin 5/6: experience of 40 patients]. / Carcinoma metaplásico de mama y repercusión de la expresión de p63 y citoqueratina 5/6: experiencia de 40 pacientes.

BACKGROUND: Metaplasic carcinoma of the breast was initially described by Huvos in 1974. It is a rare and aggressive entity characterized by the presence of mesenchymal and epithelial components. OBJECTIVE: To know the incidence and biologic behaviour of the metaplasic carcinoma of the breast at the Instituto de Enfermedades de la Mama, FUCAM, AC. METHODS: Data on women diagnosed with metaplasic carcinoma of the breast between January 2005 and December 2014 was collected by retrospectively reviewing in FUCAM. Clinical, pathological and immunohistochemical characteristics were assessed. The five-year disease-free survival (DFS) and overall survival (OS) were evaluated. RESULTS: a total of 4198 patients have been diagnosed with breast cancer in our institution, 40 (0.95%) of them with metaplasic carcinoma. The median age of the patients was 46 years (27-73). 60% of the patients were diagnosed with an advanced clinical stage (III) and the triple-negative subtype was the most frequently found. A mean follow-up of 24 months showed rates of overall survival and disease-free survival of 80% and 69.9%, respectively. The presence of both, cytokeratins 5/6 and p63, seems to have a negative impact in local recurrence. CONCLUSION: this study demonstrates that metaplasic carcinoma is a rare and aggressive disease. Expression of both tumor cytokeratins was associated with a worse outcome.

Meibomian Gland Dysfunction Model in Hairless Mice Fed a Special Diet With Limited Lipid Content.

PURPOSE: A novel meibomian gland dysfunction (MGD) model was developed to facilitate understanding of the pathophysiology of MGD and to evaluate treatment with azithromycin ophthalmic solution (azithromycin). MGD was induced in HR-1 hairless mice by feeding them a special diet with limited lipid content (HR-AD). METHODS: Male HR-1 hairless mice were fed an HR-AD diet for 16 weeks. Development of MGD was assessed by histopathology at 4-week intervals. The lid margin was observed by slit-lamp examination. After cessation of the HR-AD diet, the mice were fed a normal diet to restore normal eye conditions. Expression of cytokeratin 6 was determined by immunostaining. We evaluated the effects of topically applied azithromycin on the plugged orifice in this model. RESULTS: After mice were fed the HR-AD diet, histopathology analysis showed hyperkeratinization of the ductal epithelium in the meibomian gland. Ductal hyperkeratinization resulted in the loss of acini, followed by atrophy of the gland. Slit-lamp examination revealed a markedly plugged orifice, telangiectasia, and a toothpaste-like meibum compared with that of a normal eyelid. Cessation of feeding with HR-AD ameliorated both the MGD signs and the expression of cytokeratin 6, restoring the tissue to a histologically normal state. Azithromycin treatment significantly decreased the number of plugged orifices and ameliorated atrophy, as revealed by histopathologic analysis. CONCLUSIONS: We developed a novel model that mimics human MGD signs in HR-1 hairless mice fed an HR-AD diet. Azithromycin treatment led to therapeutic improvement in this model. This MGD model could be useful for the evaluation of drug candidates for MGD.

A conditional transgenic mouse line for targeted expression of the stem cell marker LGR5.

LGR5 is a known marker of embryonic and adult stem cells in several tissues. In a mouse model, Lgr5+ cells have shown tumour-initiating properties, while in human cancers, such as basal cell carcinoma and colon cancer, LGR5 expression levels are increased: however, the effect of increased LGR5 expression is not fully understood. To study the effects of elevated LGR5 expression levels we generated a novel tetracycline-responsive, conditional transgenic mouse line expressing human LGR5, designated TRELGR5. In this transgenic line, LGR5 expression can be induced in any tissue depending on the expression pattern of the chosen transcriptional regulator. For the current study, we used transgenic mice with a tetracycline-regulated transcriptional transactivator linked to the bovine keratin 5 promoter (K5tTA) to drive expression of LGR5 in the epidermis. As expected, expression of human LGR5 was induced in the skin of double transgenic mice (K5tTA;TRELGR5). Inducing LGR5 expression during embryogenesis and early development resulted in macroscopically and microscopically detectable phenotypic changes, including kink tail, sparse fur coat and enlarged sebaceous glands. The fur and sebaceous gland phenotypes were reversible upon discontinued expression of transgenic LGR5, but this was not observed for the kink tail phenotype. There were no apparent phenotypic changes if LGR5 expression was induced at three weeks of age. The results demonstrate that increased expression of LGR5 during embryogenesis and the neonatal period alter skin development and homeostasis.

The significance of combined CK5/6 and p63 immunohistochemistry in predicting the risks of subsequent carcinoma development in intraductal papilloma of the breast.

Prediction of subsequent risks of breast carcinoma (BC) development in intraductal papilloma (IDP) has remained controversial with the exception of atypical papilloma (AP). The potential value of immunohistochemistry (IHC) of cytokeratin 5/6 [CK5/6] and p63 have been proposed but its standardization has also remained controversial. We studied 17 patients initially diagnosed as IDP or AP who subsequently developed BC with 34 age-matched controls. We compared histological features, results of IHC (estrogen receptor [ER], progesterone receptor [PR], human epidermal growth factor receptor 2 [HER2], p63, CK5/6, Ki67), and ultrasound findings. Univariate conditional logistic regression analysis revealed that the status of both CK5/6 and p63/CK5/6 were significantly associated with subsequent BC development (P < 0.05). BC development in CK5/6 positive patients was 17.9% and p63/CK5/6 double positive patients 8.6%, respectively. Ultrasound evaluation was not significantly associated with any of the parameters examined and subsequent carcinoma development. Despite CK5/6 positivity, the subsequent incidence of BC development was nearly 20%. However p63/CK5/6 double positive status could predict a significantly lower subsequent carcinoma incidence, indicating a more accurate prognostic utility. Combining p63/CK5/6 with histological findings could be easily applied and could predict the subsequent BC development of the patients diagnosed as IDP at biopsy.