The value of oestrogen receptor, progesterone receptor and keratins 5 and 14 immunohistochemistry in the evaluation of epithelial proliferations at cauterised margins in breast-conserving surgery specimens.

In breast conservative surgery, it is sometimes difficult to decide whether the cauterised tissue at the inked margin represents normal / hyperplastic or neoplastic tissue. We retrospectively assessed the value of ER, PR, CK5 and CK14 IHC in clarifying the nature of cauterised tissues at the margins concerning 34 lesions of 23 patients. 27 cases belonged to lesions that could not be adequately classified on the basis of the HE stains. Two thirds of them could be classified as non-neoplastic or neoplastic and two thirds of the remaining could be favourised as neoplastic or non-neoplastic, with 3/27 cases remaining uncertain. All 4 IHC reactions were helpful in classifying the lesions in almost half of the cases. However, 3 or 4 immunostains were supportive of the classification in 19/27. The most useful stains were the keratins, generally demonstrating a matching pattern of cell labelling with CK5 and CK14. ER and PR were somewhat less useful in classifying uncertain lesions. Considering all the 27 questionable lesions, IHC with ER, PR, CK5 and CK14 clarified the lesions at the cauterised margins in 23 cases. Taken all these considerations into account, CK5, CK14, PR and ER IHC may help in distinguishing between cautery damaged neoplastic and non-neoplastic tissues. All four IHC may yield the best support for decision making, but CK5 and/or CK14 may be sufficient in their own. The essential approach is that the results must be interpreted with caution, in the context of the given patient's disease, to avoid misinterpretations.

Clinical utility of keratin 14 expression measurement in reflecting the tumor properties and prognosis in patients with renal cell carcinoma: a study with long-term follow-up.

PURPOSE: Keratin 14 (KRT14) is hypothesized to be involved in the pathogenesis of renal cell carcinoma (RCC) based on its tumorigenic role in various cancers and its relationship with the prognosis of other urinary system malignancies. This study aimed to evaluate the correlation of KRT14 with tumor properties and prognosis in RCC patients. METHODS: Data from 180 RCC patients who received tumor resection were retrospectively reviewed. The KRT14 was assessed by immunohistochemistry (IHC) staining in tumor tissues and non-tumor tissues. RESULTS: KRT14 was insufficiently expressed in both tumor and non-tumor tissues, with median (interquartile range) IHC score of 2.0 (0.0-3.4) and 1.0 (0.0-2.0), respectively. While it was relatively higher in tumor versus non-tumor tissues (P < 0.001). Besides, tumor KRT14 was positively correlated with the pathological grade (P = 0.038), tumor size (P = 0.012), T stage (P = 0.006), and TNM stage (P = 0.018). Interestingly, tumor KRT14 high predicted shorter accumulating recurrence-free survival (RFS) (P = 0.003) and accumulating overall survival (OS) (P = 0.001), which was further verified by the multivariate Cox's regression analysis (both P < 0.05). Furthermore, tumor KRT14 high estimated shorter RFS and OS from the Gene Expression Profiling Interactive Analysis and Human Protein ATLAS databases (all P < 0.05). Subgroup analyses indicated that the correlation of tumor KRT14 with accumulating RFS and accumulating OS was more pronounced in RCC patients with better physical status (such as age < 65 years and better eastern cooperative oncology group performance status) and higher tumor stages (such as higher pathological grade). CONCLUSION: High KRT14 in tumor tissue could reflect an advanced tumor features and unsatisfying survival in RCC patients.

Immunohistochemical evaluation of keratins and involucrin in differentiating between palmoplantar pustulosis and pompholyx.

BACKGROUND: Palmoplantar pustulosis (PPP) and pompholyx are chronic diseases characterized by pustules and vesicles on the palms and soles. These disorders often have similar clinicopathological features, which lead to diagnostic difficulties. We aimed to investigate the expression patterns of keratins and involucrin in PPP and pompholyx using immunohistochemical staining. METHODS: Skin biopsies from patients with PPP (n = 40) and pompholyx (n = 22) were immunohistochemically analyzed for Keratin 5, 9, 14, and involucrin expression. RESULTS: K5 expression was higher in PPP than in pompholyx, with diffusely positive expression in the basal, spinous, and granular layers. K14 expression did not differ between groups. K9 expression was observed near the pompholyx vesicle (P = 0.014) and stratum spinosum (P < 0.001) but was almost absent around PPP pustules. Involucrin expression was diffused around the PPP pustules and partially around the pompholyx vesicles, but without statistical significance (P = 0.123). Involucrin expression was elevated in the basal layer of the PPP compared with that in the pompholyx (P = 0.023). CONCLUSION: PPP and pompholyx exhibited distinctive differentiation in the expression of K5, K9, and involucrin.

Association of current molecular subtypes in urothelial carcinoma with patterns of muscularis propria invasion.

Urothelial carcinoma is subdivided into luminal (L), basal (B), and p53-wild-type (WT) molecular subtypes, with basal and p53-WT groups showing more aggressive course and poor treatment response, respectively. The literature on molecular subtypes of UC includes a mixture of different stages. We investigated the molecular profile and outcome of pure cohort of muscle invasive bladder carcinoma (MIBC) considering two distinct patterns of muscularis propria (MP) invasion. Forty-three cystectomies harboring stage pT2 were retrospectively identified in 18 years. MP invasion was subclassified into patterns 1 (tumor encasing intact detrusor muscle bundles) and 2 (tumor dissecting/replacing detrusor muscle). Using IHC, B/L phenotypes, p53, and Ki67 were assessed, and survival data was collected. Pattern 1 invasion was noted in 16 (37%) and pattern 2 in 27 (63%), with mean age of pattern 1 being 10 years younger. B/L phenotypes were successfully determined in 83.7%; 48.8% and 34.8% revealed L and B phenotypes, respectively (indeterminate phenotype in 16.4%). Pattern 1 was associated with L phenotype (GATA3 and HER-2 expressions: p = 0.02 & p = 0.04, respectively). Ki67 ≥ 5/10HPF was noted in pattern 2 and B phenotype (p = 0.03). B phenotype showed association with p53-WT (p = 0.007). In median follow-up of 60.7 months, 63.6% of pattern 1 cases were alive without disease compared to 32% of pattern 2 (not significant). A panel of CK20 and GATA3 for luminal and CK5/6 and CK14 for basal subtypes can provide reliable molecular classification in UC. Also, morphology of MIBC can predict the molecular phenotype and the behavior of the UC.

Triple-marker immunohistochemical assessment of muscle-invasive bladder cancer: Is there prognostic significance?

BACKGROUND: Bladder cancer is the ninth most common cancer worldwide, and the third most common cancer in Lebanon. Immunohistochemistry (IHC) has been used to stratify muscle-invasive bladder cancer (MIBC) into different subtypes. However, to our knowledge, there exists no study that investigates the use of this low-cost technique to predict prognosis in bladder cancer patients in our region. AIM: To examine the feasibility of low-cost triple-marker IHC assessment for MIBC subtyping in order to predict patients' survival and cisplatin sensitivity. METHODS AND RESULTS: We collected the specimens of deceased patients diagnosed with MIBC on pathology at our institution. For each case, tumor tissue blocks were retrieved and stained for hematoxylin and eosin in addition to three molecular markers by IHC: cytokeratin 5/6, cytokeratin 14 staining basal BC, and GATA3 staining luminal BC. A cut-off of ≥20% was set as positive. Kaplan-Meier curves were built, factored by BC subtype, to predict overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFS). Hazard ratios in Cox regression were also created accounting for oncological factors and BC subtype. We categorized specimens as either luminal (GATA3 positive only) (n = 21; 56.7%) or as double-positive (GATA3 and basal cytokeratin 5/6 or cytokeratin 14 positive) (n = 16; 43.3%). The overall median survival was similar between the two categories (27.0 ± 4.82 months). Numbers favored luminal disease for PFS (Breslow P = .032). After adjusting for covariates, luminal molecular expression predicted PFS (0.28; [0.09-0.94]). Yet, the Cox model was not able to identify any predictors of OS or DSS. CONCLUSION: Specimens enriched with only a luminal molecular profile were more likely to exhibit cisplatin sensitivity. Despite the absence of guidelines recommending the utilization of molecular profiling in clinic practice, triple-marker IHC could serve as a potential low-cost prognostic indicator to identify patients at high risk of progression.

Clinical outcomes of muscle invasive bladder Cancer according to the BASQ classification.

BACKGROUND: We evaluated the clinical efficacy and prognosis of muscle-invasive bladder cancer according to the basal/squamous-like (BASQ) classification system based on immunohistochemical staining [CK5/6(+), CK14(+), GATA3(-), and FOXA1(-)]. METHODS: One hundred patients diagnosed with muscle-invasive bladder cancer (cT2-4 N0-3 M0) were included in the study. All patients underwent radical cystectomy after transurethral removal of bladder tumor. Immunostaining was performed for CK5/6, CK14, FOXA1, and GATA3 antibodies on tissue microarray slides, and expression patterns were quantitatively analyzed using a scanning program. RESULTS: The median follow-up time was 77.4 (interquartile range: 39-120.9) months. The mean age of the patients was 65.1 ± 11.2 years. FOXA1 or CK14 expression greater than 1% was respectively positively and negatively correlated with overall survival (OS; p = 0.011 and p = 0.042, respectively), cancer-specific survival (CSS; p = 0.050 for both), and recurrence-free survival (RFS; p = 0.018 and p = 0.040, respectively). For CK5/6+ and GATA3- or FOXA1- expression, 10% CK5/6+ cells were negatively correlated with OS (p = 0.032 and p = 0.039, respectively) and with RFS in combination with FOXA1- only (p = 0.050). CONCLUSIONS: In this study, CK14 expression was associated with a poor prognosis. The new classification system of bladder cancer based on molecular characteristics is expected to helpful tool for the establishment of personalized treatment strategies and associated prediction of therapeutic responses.

DNA Damage Response After Ionizing Radiation Exposure in Skin Keratinocytes Derived from Human-Induced Pluripotent Stem Cells.

PURPOSE: Epidermal cells are positioned on the body surface and thus risk being exposed to genotoxic stress, including ionizing radiation (IR), ultraviolet rays, and chemical compounds. The biological effect of IR on the skin tissue is a significant problem for medical applications such as radiation therapy. Keratinocyte stem cells and progenitors are at risk for IR-dependent tumorigenesis during radiation therapy for cancer treatment. To elucidate the molecular mechanism of genome stability in epidermal cells, we derived skin keratinocytes from human-induced pluripotent stem cells (iPSCs) and analyzed their DNA damage response (DDR). METHODS AND MATERIALS: Skin keratinocytes were derived from iPSCs and designated as first- (P1), second- (P2), and third- (P3) passage cells to compare the differentiation states of DDR. After 2 Gy gamma-ray exposure, cells were immunostained with DNA double-strand break markers γ-H2AX/53BP1 and cell senescence markers p16/p21 for DDR analysis. DDR protein expression level, cell survival, and apoptosis were analyzed by western blotting, WST-8 assay and TUNEL assay, respectively. DDR of constructed 3D organoid modeling was also analyzed. RESULTS: P1, P2, and P3 keratinocytes were characterized with keratinocyte markers keratin 14 and p63 using immunofluorescence, and all cells were positive to both markers. Derived keratinocytes showed high expression of integrin α6 and CD71 (real-time (qRT)-PCR ratio: iPSCs: integrin α6: 1.12, CD71: 1.25, P1: integrin α6: 7.80, CD71: 0.43, P2: integrin α6: 5.53, CD71: 0.48), suggesting that P1 and P2 keratinocytes have potential as keratinocyte progenitors. Meanwhile, P3 keratinocytes showed low expression of integrin α6 and CD71 (qRT-PCR ratio: P3: integrin α6: 0.55, CD71: 0.10), suggesting differentiated keratinocytes. After IR exposure, the P1 and P2 keratinocytes showed an increase in DNA repair activity by a γ-H2AX/53BP1 focus assay (P1: γ-H2AX: 28.0%, 53BP1: 17.0%, P2: γ-H2AX: 37.7%, 53BP1: 28.3%) but not in P3 keratinocytes (P3: γ-H2AX: 74.7%, 53BP1: 63.7%) compared with iPSCs (γ-H2AX: 57.0%, 53BP1: 55.0%). Furthermore, in derived keratinocytes, expression of the cellular senescence markers p16 and p21 were increased compared with iPSCs (P16: non irradiated, iPSCs: 0%, P1: 12.5%, P2: 14.5%, P3: 29.7%, IR, iPSCs: 0%, P1: 19.5%, P2: 34.8%, P3: 64.5%). DDR protein expression, cellular sensitivity, and apoptosis activity decreased in derived keratinocytes compared with iPSCs. CONCLUSIONS: We have demonstrated the derivation of keratinocytes from iPSCs and their characterization of differentiated states and DDR. Derived keratinocytes showed progenitors like character as a result of DDR. These results suggest that derived keratinocytes are useful tools for analyzing the effects of IR, such as DDR on the skin tissue from radiation therapy for cancer.

Discriminating the earliest stages of mammary carcinoma using myoepithelial and proliferative markers.

Mammographic screening has led to increased detection of breast cancer at a pre-invasive state, hence modelling the earliest stages of breast cancer invasion is important in defining candidate biomarkers to predict risk of relapse. Discrimination of pre-invasive from invasive lesions is critically important for such studies. Myoepithelial cells are the barrier between epithelial cells and the surrounding stroma in the breast ductal system. A number of myoepithelial immunohistochemistry markers have been identified and validated in human tissue for use by pathologists as diagnostic tools to distinguish in situ carcinoma from invasive breast cancer. However, robust myoepithelial markers for mouse mammary tissue have been largely under-utilised. Here, we investigated the utility of the myoepithelial markers smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC), cytokeratin-14 (CK14) and p63 to discriminate mammary intraepithelial neoplasia (MIN) from invasive disease in the C57BL/6J MMTV-PyMT transgenic model of mammary carcinoma. We identified that SMMHC and CK14 are retained in early in situ neoplasia and are appropriate markers for distinguishing MIN from invasive disease in this model. Additionally, the proliferation marker Ki67 is a superior marker for differentiating between normal and hyperplastic ducts, prior to the development of MIN. Based on this, we developed a scoring matrix for discriminating normal, hyperplasia, MIN and invasive lesions in this spontaneous mammary tumorigenesis model. This study demonstrates heterogeneous expression of myoepithelial proteins throughout tumour development, and highlights the need to characterise the most appropriate markers in other models of early breast cancer to allow accurate classification of disease state.

Differential expression of cytokeratin 14 and 18 in bladder cancer tumorigenesis.

It has been previously suggested that cytokeratins (CKs) are important diagnostic and prognostic biomarkers for urothelial lesions. Hence it is imperative to understand the expression pattern of cytokeratins during formation of papillary bladder cancer, which was the objective of the current study. Expression pattern of CK14 and CK18 were examined using immunohistochemical staining in a mice model of papillary bladder cancer. Twenty female mice were divided into two groups-group 1 (NT) and group 2, which received N-butyl- N-(4-hydroxybutyl) nitrosamine (BBN) for 20 weeks plus one week without treatment. Following histological classification of bladder lesions, CK14 and CK18 immunostaining was assessed according to its distribution and intensity. In NT animals, both basal cells and umbrella cells showed sporadic positive staining for CK14 and CK18, respectively. In BBN group, hyperplastic lesions showed significantly more CK14 and significantly less CK18 staining ( P < 0.05 in each case). Invasive carcinomas showed increased CK14 immunostaining in all epithelial layers. Cumulatively, our data indicate that altered CK14 (high) and CK18 (low) expression is perhaps an early event in bladder cancer tumorigenesis in females at least and is characteristic of both urothelial superficial pre-neoplastic and neoplastic lesions. Impact statement Studies have shown that expression of cytokeratins (CKs) or their altered distribution affects the bladder cancer pathogenesis and disease outcome, while the underlying mechanisms are not clear. The present study aims to explore the expression pattern of CK14 and CK18 during formation of papillary bladder cancer. The results showed that hyperplastic lesions showed significantly more CK14 and significantly less CK18 staining and invasive carcinomas showed increased CK14 immunostaining in all epithelial layers in N-butyl- N-(4-hydroxybutyl)nitrosamine (BBN)-induced mouse model. The results indicate that altered CK14 (high) and CK18 (low) expression is perhaps an early event in bladder cancer tumorigenesis and is characteristic of both urothelial superficial pre-neoplastic and neoplastic lesions, which may provide the early diagnosis index.

Effectiveness of computer-aided diagnosis (CADx) of breast pathology using immunohistochemistry results of core needle biopsy samples for synaptophysin, oestrogen receptor and CK14/p63 for classification of epithelial proliferative lesions of the breast.

AIMS: The aim of this study was to develop a computer-aided diagnosis (CADx) system for identifying breast pathology. METHODS: Two sets of 100 consecutive core needle biopsy (CNB) specimens were collected for test and validation studies. All 200 CNB specimens were stained with antibodies targeting oestrogen receptor (ER), synaptophysin and CK14/p63. All stained slides were scanned in a whole-slide imaging system and photographed. The photographs were analysed using software to identify the proportions of tumour cells that were positive and negative for each marker. In the test study, the cut-off values for synaptophysin (negative and positive) and CK14/p63 (negative and positive) were decided using receiver operating characteristic (ROC) analysis. For ER analysis, samples were divided into groups with <10% positive or >10% positive cells and decided using receiver operating characteristic (ROC) analysis. Finally, these two groups categorised as ER-low, ER-intermediate (non-low and non-high) and ER-high groups. In the validation study, the second set of immunohistochemical slides were analysed using these cut-off values. RESULTS: The cut-off values for synaptophysin, <10% ER positive, >10% ER positive and CK14/p63 were 0.14%, 2.17%, 77.93% and 18.66%, respectively. The positive predictive value for malignancy (PPV) was 100% for synaptophysin-positive/ER-high/(CK14/p63)-any or synaptophysin-positive/ER-low/(CK14/p63)-any. The PPV was 25% for synaptophysin-positive/ER-intermediate/(CK14/p63)-positive. For synaptophysin-negative/(CK14/p63)-negative, the PPVs for ER-low, ER-intermediate and ER-high were 100%, 80.0% and 95.8%, respectively. The PPV was 4.5% for synaptophysin-negative/ER-intermediate/(CK14/p63)-positive. CONCLUSION: The CADx system was able to analyse sufficient data for all types of epithelial proliferative lesions of the breast including invasive breast cancer. This system may be useful for pathological diagnosis of breast CNB in routine investigations.

Epithelial Fli1 deficiency drives systemic autoimmunity and fibrosis: Possible roles in scleroderma.

Systemic sclerosis (SSc), or scleroderma, is a multisystem autoimmune disorder characterized by vasculopathy and fibrosis in the skin and internal organs, most frequently in the esophagus and lungs. Hitherto, studies on SSc pathogenesis centered on immune cells, vascular cells, and fibroblasts. Although dysregulated keratinocytes in SSc have been recently reported, the contribution of epithelial cells to pathogenesis remains unexplored. In this study, we demonstrated the induction of SSc-like molecular phenotype in keratinocytes by gene silencing of transcription factor Friend leukemia virus integration 1 (Fli1), the deficiency of which is implicated in SSc pathogenesis. Keratin 14-expressing epithelial cell-specific Fli1 knockout mice spontaneously developed dermal and esophageal fibrosis with epithelial activation. Furthermore, they developed remarkable autoimmunity with interstitial lung disease derived from thymic defects with down-regulation of autoimmune regulator (Aire). Importantly, Fli1 directly regulated Aire expression in epithelial cells. Collectively, epithelial Fli1 deficiency might be involved in the systemic autoimmunity and selective organ fibrosis in SSc. This study uncovers unidentified roles of dysregulated epithelial cells in SSc pathogenesis.

Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells.

PURPOSE: Human and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs). METHODS: SGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis. RESULTS: SG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype. CONCLUSIONS: Epithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium.

A Comparative Study of Immunohistochemical Myoepithelial Cell Markers in Cutaneous Benign Cystic Apocrine Lesions.

The use of immunohistochemical markers for myoepithelial cells (MEC) is a useful tool in the distinction of benign from malignant epithelial neoplasms. Although their use in breast tumors is well recognized, little is known concerning its application in comparable cutaneous lesions. Using benign cutaneous cystic apocrine lesions as a study model, the aim of this study was to compare 5 immunohistochemical markers [calponin, p63, smooth muscle actin (SMA), cytokeratin 14, and CD10] in their effectiveness to highlight MEC. Cases of apocrine hidrocystoma and cystadenoma (n = 44) were reviewed with a particular emphasis on proliferative features and apocrine change. The MEC staining pattern and the intensity and distribution scores in proliferative (n = 29) and nonproliferative (n = 15) lesions were assessed, and the differences between the 2 groups were statistically analyzed using Fisher exact test. Calponin and SMA stained MEC in the most consistent manner. Being a nuclear stain, p63 was easy to interpret but typically showed discontinuous staining. Cytokeratin 14 not only effectively highlighted MEC but also stained some luminal epithelial cells in an unpredictable manner. Because of prominent background dermal fibroblast staining, CD10 was often difficult to interpret. Only SMA and p63 showed a statistically significant difference in MEC staining intensity scores between the proliferative and nonproliferative groups. Our results show that immunohistological staining for MEC in benign cystic apocrine lesions of the skin is variable. The authors recommend that a panel of markers that includes calponin and p63 be used and highlight the need for awareness of specific caveats associated with individual markers.

Altered expression of keratin 14 in lesional epidermis of autoimmune skin diseases.

BACKGROUND: Keratin 14 (K14) is an intermediate filament protein that is mainly expressed in the basal layer of healthy stratified epithelia. K14 has been identified as an autoantigen in the autoimmune-mediated skin disease of Scurfy mice and patients with the "immune dysregulation polyendocrinopathy, enteropathy, and X-linked" syndrome. OBJECTIVES: To examine whether K14 is a target protein in autoimmune skin diseases (ASD), we analyzed the expression pattern of K14 in lesional skin of patients with lichen ruber, cutaneous lupus erythematosus, dermatomyositis, graft-versus-host disease, psoriasis, and pemphigus vulgaris, and evaluated the reactivity of patient sera with recombinantly expressed and epidermis-derived K14. METHODS: K14 expression was analyzed by immunohistochemistry on paraffin-embedded tissue sections of 17 healthy individuals and 58 patients with ASD. Sera from 10 healthy individuals and 41 patients with ASD were analyzed by Western blot for the presence of anti-K14 autoantibodies. RESULTS: In skin of patients with ASD, K14 expression is retained in suprabasal layers. In ASD with interface dermatitis, we observed focal loss of K14 within the basal layer and in hair follicles as well. A scattered dot-like K14 staining is seen in papillary dermis, most prominently in cutaneous lupus erythematosus and lichen ruber. Using Western blot, we show that sera of different patients with ASD recognize recombinantly expressed K14. CONCLUSION: We show focal loss of K14 in the basal epidermis correlating with interface dermatitis and hair follicle involvement. Moreover, enhanced reactivity of sera of patients with atopic dermatitis with K14 suggests K14 may function as an autoantigen in ASD.

A Rare Case of Mucoepidermoid Carcinoma ex Pleomorphic Adenoma arising in Minor Salivary Gland: Histopathological and Immunohistochemical Analysis.

Mucoepidermoid carcinoma ex pleomorphic adenoma (MCxPA) is a rare salivary gland tumor predominantly found in major salivary glands. A case of MCxPA involving the soft tissue and bone of the retromolar region of a 26-year-old man is presented. The histopathological features revealed a neoplasm with predominance of pleomorphic adenoma (PA) elements, and presence of mucoepidermoid carcinoma malignant epithelial cells in several areas. Histochemical and immunohistochemical studies were positive for periodic acid Schiff, alcian blue, cytokeratins 7, 13, 14, and 19, Bcl-2, c-erbB-2, FGF-2 and maspin in the malignant areas. The patient underwent a partial resection of the left side of the mandible with neck dissection and MCxPA diagnosis was confirmed.

BRCA-mutated Invasive Breast Carcinomas: Immunohistochemical Analysis of Insulin-like Growth Factor II mRNA-binding Protein (IMP3), Cytokeratin 8/18, and Cytokeratin 14.

To evaluate the expression of insulin-like growth factor II mRNA-binding protein (IMP3), CK8/18, and CK14 in BRCA mutated and sporadic invasive breast carcinoma. Immunohistochemistry for IMP3, CK8/18, and CK14 was performed on 39 cases of invasive breast carcinomas with BRCA mutation (24 BRCA1, 14 BRCA2, and 1 dual BRCA1/BRCA2) and 54 cases of sporadic invasive breast carcinomas. The relationship between the IMP3, CK8/18, and CK14 and the tumor grade and molecular phenotypes were analyzed. IMP3, CK8/18, and CK14 positivity were present in 20 (51%), 22 (56%), and 14 (36%) of 39 BRCA-mutated breast carcinomas, and 11 (20%), 53 (98%), and 24 (44%) of 54 sporadic breast carcinomas respectively. The rates of IMP3 expression and absence of CK8/18 (44% versus 2%) in BRCA-mutated breast carcinomas was significantly higher than the sporadic breast carcinomas (p = 0.002 and p < 0.001). No significant difference was observed for CK14 among the two groups (p = 0.408). No significant difference was observed among BRCA1-related and BRCA2-related breast carcinomas in the immunoprofile for IMP3, CK8/18, and CK14. No significant correlation was identified between the expression of IMP3 and CK8/18 and the tumor grade in both BRCA-mutated and sporadic breast carcinomas (p > 0.05). In cases with luminal A and B phenotypes, the rates of expression of IMP3 and loss of CK8/18 were significantly higher in BRCA-mutated as compared to sporadic breast carcinoma (p < 0.001). In cases with basal-like phenotype, the absence of CK8/18 expression was significantly higher in BRCA-mutated breast carcinomas (54% versus 0%, p = 0.001), while no difference was observed for IMP3 expression (p = 0.435). Regardless of mutation type, histologic grade, or molecular phenotype, the absence of CK8/18 expression and presence of IMP3 expression are seen at much higher rate in BRCA mutated breast carcinomas.

The clinical use of a P63/cytokeratin7/18/cytokeratin5/14 antibody cocktail in diagnostic breast pathology.

An antibody cocktail directed against p63, cytokeratin (CK)5/14, and CK7/18 is reported to be useful in distinguishing noninvasive from invasive breast lesions and for the characterization of intraductal epithelial proliferations. However, limited studies evaluate its use in clinical practice. A retrospective review of breast material at a university medical center identified cases that were immunostained with the above antibody cocktail. Additional p63 immunostaining alone was performed to further determine the utility of the antibody cocktail in the evaluation of invasion. Of 50 breast cases identified, the antibody cocktail was used to confirm or exclude invasion in 44 (88%). Twenty-two (50%) of these had easily identifiable p63/CK5/14-positive myoepithelial cells, whereas the remainder lacked such staining, confirming the diagnosis of invasive carcinoma. In 27 cases with available diagnostic material for additional p63 immunostaining, the cocktail better highlighted myoepithelial cells by staining nuclei and cytoplasm. Easier identification of invasion was also facilitated by CK7/18 expression in invasive foci, especially those composed of single cells. Ten cases were immunostained to help determine the nature of an intraductal proliferation. The cocktail demonstrated a mosaic staining pattern of both CK7/18- and CK5/14-positive epithelial cells in 3 (30%) cases consistent with usual hyperplasia; homogenous CK7/18 expression in the remaining cases supported the diagnosis of atypical ductal hyperplasia or carcinoma in situ. In summary, the p63/CK7/18/CK5/14 cocktail stain appears to be a useful tool in diagnostic breast pathology, in the evaluation of possible invasion, particularly in the setting of minute foci of invasion as well as in epithelial proliferations.

Determination of cytokeratins 1, 13 and 14 in oral lichen planus.

UNLABELLED: INTRODUCCION: Cytokeratins (CK) are molecules of the cytoskeleton that contribute to the cellular differenciation. We studied the expression of CK1, CK13 and CK14 in thirty-three patients with OLP. The biopsied lesions were located in the dorsal surface of the tongue, the palatal keratinized mucosa and the nonkeratinized buccal mucosa. OBJECTIVES: This study aimed to determine the expression of CK1, CK13 and CK14 in oral lichen planus (OLP) and its relations with: clinical patterns, prognosis, drugs and tobacco intake and histopathological features. STUDY DESIGN: Immunohistochemical analysis, retrospective, descriptive, observational and no randomized study. RESULTS: No significant difference was observed in the expression of CK1 in patients with or without drug treatment. No association was found with the amount of drugs intake or smoking nor with the histopathological features examined. Samples immunostained with CK13 were all positive in the suprabasal layers, and 13 of them in the basal layer. In these last ones, statistical analysis showed significance in the grade of vacuolization of the basal layer (p=0.023) and in the degree of exocytosis (p=0.0025), this, making the degree of affection higher for both parameters. Thirty-two tissue sections were immunostained with CK14. CK14 was expressed in the basal layer in 97% of samples and in the suprabasal layer in 94% of samples. CONCLUSIONS: The three CK were altered in OLP. CK1 does not have a direct connection with the presence of orthokeratosis. The finding of the CK13 in the basal layer is related to the agression of the lymphocytic infiltration in the epithelium, due to the basal stratum vacuolization and the increase in lymphocytic exocitosis. The presence of CK14 in the suprabasal stratums is not a parameter to predict malignancy. The CK in OLP do not follow the normal pattern of keratinized or non-keratinized mucosa.

A novel method for isolation of epithelial cells from ovine esophagus for tissue engineering.

PURPOSE: The yield of a critical number of basal epithelial cells with high mitotic rates from native tissue is a challenge in the field of tissue engineering. There are many protocols that use enzymatic methods for isolation of epithelial cells with unsatisfactory results for tissue engineering. This study aimed to develop a protocol for isolating a sufficient number of epithelial cells with a high Proliferating Index from ovine esophagus for tissue engineering applications. METHODS: Esophageal mucosa was pretreated with dispase-collagenase solution and plated on collagen-coated culture dishes. Distinction of the various types of epithelial cells and developmental stages was done with specific primary antibodies to Cytokeratins and to Proliferating Cell Nuclear Antigen (PCNA). RESULTS: Up to approximately 8100 epithelial cells/mm2 of mucosa tissue were found after one week of migration. Cytokeratin 14 (CK 14) was positive identified in cells even after 83 days. At the same time the Proliferating Index was 71%. CONCLUSION: Our protocol for isolation of basal epithelial cells was successful to yield sufficient numbers of cells predominantly with proliferative character and without noteworthy negative enzymatic affection. The results at this study offer the possibility of generation critical cell numbers for tissue engineering applications.

Effect of the antineoplastic agent busulfan on rat molar root development.

OBJECTIVE: The antineoplastic bifunctional-alkylating agent busulfan (Bu) induces developmental anomalies. We examined histopathological changes in the molar roots of rats that received Bu at different stages of root formation. DESIGN: At different developmental stages, i.e., on postnatal days (P) 13, 15, and 19, rats were administered 7.5 mg/kg of Bu dissolved in dimethyl sulfoxide (DMSO) and then killed on P 30. After micro-computed tomography analysis, the maxillary first molars underwent immunohistochemical analysis for cytokeratin 14 (CK14), nestin, and dentin sialoprotein (Dsp). This was followed by histomorphometric analysis. RESULTS: The rats receiving Bu at an early stage (i.e., P 13 and P 15) showed osteodentin formation and complete destruction of the Hertwig's epithelial root sheath (HERS). Cells around osteodentin showed nestin and Dsp immunoreactivity. The root lengths in rats treated with Bu at P 13 (1228.44 ± 62.17 µm) and P 15 (1536.08 ± 109.71 µm) were lower than that in the control rats (1674.10 ± 40 µm). A narrowed apical foramen and an increased amount of osteodentin were also present, depending on the rat's age at the time of treatment (P < 0.05). CONCLUSION: Busulfan treatment in juvenile rats resulted in abnormal root development, depending on the stage at which Bu was administered. This abnormal development may result from the destruction of the HERS. The administration of Bu caused a shortage of HERS cells, which are required for normal root development. This disturbs root formation, resulting in osteodentin formation and a narrowed apex foramen.