Staining Of Basal Cells Research Articles

CIN grades possessing different HPV RNA location patterns and RNAscope is helpful tool for distinguishing squamous intraepithelial lesions in difficult cervical cases

Background and objectivesThe precise grading and characterization of cervical intraepithelial neoplasia (CIN) has been the focus of pathologists for a long time. This study aimed to explore known strategies for the grading of CINs.MethodsAfter routine H&E review, 85 lesions graded CIN 1, 2, or 3 were investigated primarily by HPV RNAscope to detect HR-HPV and LR-HPV, in combination with an HPV-DNA test and P16/Ki67 immunohistochemistry (IHC). Then, the 85 cases were divided into a control group (49 cases) and a test group (36 cases). The former consisted of cases with consistency between morphology, HPV DNA detection and P16/Ki67 IHC. We used them to evaluate HPV RNA distribution patterns in CINs of different grades. The latter were ambiguous cases in which pathologists could not confirm the diagnosis because of inconsistencies between morphology, HPV DNA detection and P16/Ki67 IHC. We reassessed them by comparison to the pattern in the control group.ResultsThe expression patterns of HPV mRNA signals were different in different CIN lesions. LSIL/CIN1 lesions were mostly expressed in superficial epithelium with diffuse clustered nuclear or cytoplasmic staining; HSIL/CIN2 were characterised by nuclear/cytoplasmic punctate or diffuse cluster nuclear staining in the mid-surface layer, and scattered nuclear/cytoplasmic punctate staining in basal and parabasal cells; whereas HSIL/CIN3 showed full-thickness nucleus/cytoplasmic scattered staining with a punctate pattern. According to the staining pattern, we corrected the diagnosis of 22 cases (22/36, 61.1%).ConclusionBecause of its distinct location pattern, HPV RNAscope has obvious advantages over the HPV-DNA test, and combined with P16/Ki67 IHC, it can help pathologists correctly grade CIN. In addition, it can effectively discriminate true CIN from normal or CIN mimic lesions, such as immature squamous metaplasia, atrophy, and inflammatory/reactive changes. Therefore, HPV RNAscope is a valuable auxiliary diagnostic test to avoid the overtreatment and undertreatment of CIN lesions.

Cytokeratin 5 and cytokeratin 6 expressions are unconnected in normal and cancerous tissues and have separate diagnostic implications

Cytokeratins (CKs) 5 and 6 are functionally unrelated but often analyzed together using bispecific antibodies in diagnostic immunohistochemistry. To better understand the diagnostic utility of CK5 or CK6 alone, tissue microarrays with > 15,000 samples from 120 different tumor types as well as 608 samples of 76 different normal tissues were analyzed by immunohistochemistry. In normal tissues, both CKs occurred in the squamous epithelium; CK5 dominated in basal and CK6 in suprabasal layers. CK5 (not CK6) stained basal cells in various other organs. Within tumors, both CK5 and CK6 were seen in > 95% of squamous cell carcinomas, but other tumor entities showed different results: CK5 predominated in urothelial carcinoma and mesothelioma, but CK6 in adenocarcinomas. Joint analysis of both CK5 and CK6 obscured the discrimination of epithelioid mesothelioma (100% positive for CK5 alone and for CK5/6) from adenocarcinoma of the lung (12.8% positive for CK5 alone; 23.7% positive for CK5/6). CK5 and CK6 expressions were both linked to high grade, estrogen receptor, and progesterone receptor negativity in breast cancer (p < 0.0001 each), grade/stage progression in urothelial cancer (p < 0.0001), and RAS mutations in colorectal cancer (p < 0.01). Useful diagnostic properties which are commonly attributed to CK5/6 antibodies such as basal cell staining in the prostate, distinction of adenocarcinoma of the lung from squamous cell carcinoma and epithelioid mesothelioma, and identification of basal-type features in urothelial cancer are solely driven by CK5. At least for the purpose of distinguishing thoracic tumors, monospecific CK5 antibodies may be better suited than bispecific CK5/6 antibodies.

PTEN expression and morphological patterns in prostatic adenocarcinoma.

Cribriform morphology, which includes intraductal carcinoma (IDCP) and invasive cribriform carcinoma, is an indicator of poor prognosis in prostate cancer. Phosphatase and tensin homologue (PTEN) loss is a predictor of adverse clinical outcomes. The association between PTEN expression and morphological patterns of prostate cancer is unclear. We explored the association between PTEN expression by immunohistochemistry, Gleason pattern 4 morphologies, IDCP and biochemical recurrence (BCR) in 163 radical prostatectomy specimens. IDCP was delineated from invasive cribriform carcinoma by p63 positive immunohistochemical staining in basal cells. Combined invasive cribriform carcinoma and IDCP were associated with a higher cumulative incidence of BCR [hazard ratio (HR) = 5.06; 2.21, 11.6, P < 0.001]. When including PTEN loss in the analysis, invasive cribriform carcinoma remained predictive of BCR (HR = 3.72; 1.75, 7.94, P = 0.001), while PTEN loss within invasive cribriform carcinoma did not. Glomeruloid morphology was associated with lower odds of cancer stage pT3 and lower cumulative incidence of BCR (HR = 0.27; 0.088, 0.796, P = 0.018), while PTEN loss within glomeruloid morphology was associated with a higher cumulative incidence of BCR (HR = 4.07; 1.04, 15.9, P = 0.043). PTEN loss within glomeruloid pattern was associated with BCR. The presence of any cribriform pattern was associated with BCR, despite PTEN loss not significantly associated with invasive cribriform carcinoma. We speculate that other drivers independent from PTEN loss may contribute to poor prognostic features in cribriform carcinoma.

Comparison of p53 immunohistochemical staining in differentiated vulvar intraepithelial neoplasia (dVIN) with that in inflammatory dermatoses and benign squamous lesions in the vulva.

Differentiated vulvar intraepithelial neoplasia (dVIN), the precursor lesion to human papillomavirus-independent vulvar squamous cell carcinoma (VSCC), can be difficult to distinguish from vulvar inflammatory dermatoses. Our goal was to determine if p53 could be a useful biomarker for dVIN, by characterizing p53 percentage, intensity and patterns of staining in dVIN and its histological mimics. We studied p53 immunohistochemical staining patterns in 16 dVIN cases and 46 vulvar non-neoplastic squamous lesions [12 lichen sclerosus (LS); seven lichen simplex chronicus; three lichen planus (LP); six psoriasis; 13 spongiotic dermatitis (SPO); and five candidiasis]. dVIN cases were adjacent to a p16-negative invasive VSCC in resection specimens. All dVIN cases showed null-type or moderate to strong uniform p53 staining in >70% of basal cells, with moderate to strong continuous parabasal staining extending to two-thirds of the epidermis. This was in contrast to weak or weak to moderate patchy p53 staining in the majority of other lesions. Moderate to strong and increased basal p53 staining (≥70%) was also observed in a subset of LS cases (5/12, 42%), LP cases (1/3, 33%), and SPO cases (36%, 4/11); however, in all categories, this was limited to the basal layer, and any staining in the parabasal layers was patchy. Strong and uniform p53 staining of basal cells, extending into the parabasal layers, and a complete absence of staining (null type) is useful in distinguishing dVIN from other mimics in the vulva. p53 staining of lesser intensity or quantity, particularly basal overexpression only, overlaps with that in vulvar inflammatory lesions.

P53 Immunohistochemical patterns in HPV-related neoplasms of the female lower genital tract can be mistaken for TP53 null or missense mutational patterns.

We have recently encountered p53 immunohistochemical (IHC) patterns in human papillomavirus (HPV)-associated carcinomas of the gynecologic tract, which were confused with absent (null) or overexpression TP53 mutational staining. We therefore evaluated p53 and p16 IHC in 25 squamous cell carcinomas (SCC) (16 vulva, 4 Bartholin's gland, and 5 cervix), 20 endocervical adenocarcinomas (EDAC), 14 high-grade squamous intraepithelial lesions (HSIL), 2 adenocarcinoma in situ (AIS), all of which exhibited morphologic features of HPV. Only cases showing diffuse/strong block-like p16 staining were included for further study. All EDACs underwent TP53 sequencing and HPV in situ hybridization (ISH) was performed in selected cases. p53 IHC staining fell into two main patterns. The most common was designated as "markedly reduced (null-like)" (absence or significantly attenuated staining in >70% of cells), which could be confused with true null mutational pattern. This was present in 14/25 (56%) SCCs, 7/14 (50%) HSILs, and 18/20 (90%) EDACs. The second notable pattern was "mid-epithelial (basal sparing)" (distinct absence of staining in basal cells juxtaposed with strong staining in parabasal cells), seen in 10/25 (40%) SCC, 7/14 (50%) HSIL, and none of the EDACs. There was scattered weak to moderate p53 staining (conventional wild type) in 1/25 (4%) SCC and 2/20 (10%) EDAC. No cases showed strong/diffuse overexpression. One EDAC had a TP53 missense mutation and exhibited "markedly reduced (null-like)" staining. HPV ISH revealed an inverse relationship with p53, cells positive for HPV mRNA were negative for p53. Knowledge of these patterns can help pathologists avoid misinterpreting p53 status in the setting of HPVA cancers.

Claudin-1 upregulation is associated with favorable tumor features and a reduced risk for biochemical recurrence in ERG-positive prostate cancer.

Claudin-1 is a membrane-tight junction protein and important for the sealing of the paracellular cleft in epithelial and endothelial cells. Differential expression of Claudin-1 is linked to disease outcome in various cancers. To evaluate the potential relevance of Claudin-1 expression in prostate cancer, a tissue microarray containing samples of 17,747 tumors with annotated clinico-pathological and molecular data was immunohistochemically analyzed for Claudin-1 expression. In normal prostate, glandular cells were always Claudin-1-negative while there was a strong staining of gland-surrounding basal cells. In contrast to normal prostatic glands, a positive Claudin-1 immunostaining, was found, however, in 38.7% of 12,441 interpretable cancers and was considered weak in 12.7%, moderate in 13.2%, and strong in 12.8% of cases. Positive Claudin-1 immunostaining was associated with favorable tumor features like low pT (p = 0.0032), low Gleason grade (p< 0.0001), and a reduced risk of PSA recurrence (p = 0.0005). A positive Claudin-1 staining was markedly more frequent in ERG-positive (63%) than in ERG-negative cancers (23%; p < 0.0001). Subset analyses revealed that all associations of Claudin-1 expression and favorable phenotype and prognosis were driven by ERG-positive cancers. Multivariate analyses revealed, however, that even in ERG-positive cancers, the prognostic impact of high Claudin-1 expression was not independent of established clinico-pathological parameters. Comparison with 12 previously analyzed chromosomal deletions identified conspicuous associations with PTEN and 12p13 deletions potentially indicating functional interactions. These data identify a peculiar role for Claudin-1 in prostate cancer. The protein is overexpressed in a fraction of prostate cancers and increased Claudin-1 expression levels predict a favorable prognosis in ERG-positive cancer.

Loss of Polycomb Repressive Complex 2 impairs lung bronchiolar cell growth and underlies chronic obstructive pulmonary disease

EZH2, the functional enzymatic component of the Polycomb Repressive Complex 2 (PRC2), is a histone methyl transferase that tri-methylates histone H3 at lysine 27 (H3K27me3). This protein plays a significant role in maintaining tissue specificity by silencing gene programs that control lineage fate. We hypothesized that PRC2 controls cell fate of the lung stem cells. In order to test this hypothesis, we generated mice in which we can conditionally delete Ezh2 upon ingestion of doxycycline. To examine growth, differentiation and self-renewal potentials of different lung epithelial cells, we FACS-isolated distal lung bronchioalveolar stem cells (BASCs) and cultured them in air-liquid-interface Matrigel cultures. We observed a 2fold decrease in organoid number of EZH2 null vs wild type bronchiolar cells and a significant difference in organoid diameter between Ezh2 wild type, heterozygous and null bronchiolar cells. To test in vivo potential of EZH2 depleted epithelium to repair, we performed naphthalene injury, which specifically injures the bronchiolar club cells. We observed that 7 days post naphthalene injury, Ezh2 knock-out lungs had significantly less repair, as measured by repopulation of bronchiolar lung cells. Given that we observed that lung stem cells were impaired when PRC2-mediated gene repression was perturbed, we sought to learn if PRC2 is also dysregulated in Chronic Obstructive Pulmonary Disease (COPD). We first performed Gene Set Enrichment Analysis and found that genes up-regulated in COPD patients relative to healthy smokers were also up-regulated in Ezh2 knock-out adenocarcinoma cells. We next obtained lung tissue from healthy patients and patients with COPD, and performed immunohistochemistry for the PRC2 mark H3K27me3 and immunofluorescence for basal and club cells. There was a significant decrease in H3K27me3, coupled with an increase in basal cells and goblet cells that contained high expressions of club cell markers in COPD lung compared to healthy controls. Finally, to determine if loss of PRC2 function drives basal cell fate and goblet cell differentiation, EZH2 transcripts were knocked down with short hairpins in human bronchiolar epithelial cells, and basal cell differentiation capacity was analyzed. We observed an increased expression of a goblet cell marker and decreased expression of a club cell marker in EZH2 knockdown cultures. Together our results indicate that PRC2 plays an important role in lung stem cell function as well as club cell differentiation fate. These mechanisms may also be perturbed in COPD patients. ChIP-seq and RNA-seq studies to understand the transcriptional and epigenetic changes leading to these phenotypes are underway Support or Funding Information NIH grant K22 CA 20103, Molecular Mechanisms of Toxicity Training Grant T32ES07266 There was a 2-fold decrease in organoid number of EZH2 null vs wild type bronchiolar cells. There was a significant difference in organoid diameter between Ezh2 wild type, heterozygous and null bronchiolar cells. Immunohistochemistry staining of EZH2 shows distinct differences in EZH2 expression between wild type, heterozygous and null organoids. Naphthalene exposure to mouse lung has been shown to kill club cells in the trachea and bronchioles. This injury typically results in club cell regeneration and subsequent lung cell repopulation. Club cells without EZH2 were unable to regenerate and repopulate the airway after naphthalene injury. Club cells that did regenerate in the Ezh2-null lungs lacked the GFP tag, indicating that these cells were likely not responding to doxycycline to delete Ezh2. Gene Set Enrichment Analysis was used to compare gene expression between murine Ezh2-deleted adenocarcinoma cells and COPD patients. It revealed that a significant number of the same genes are highly expressed in both states when compared to healthy lungs or the Ezh2 WT setting. Immunohistochemistry shows significantly lower abundance of H3K27me3 in the nuclei of COPD bronchiolar cells compared to normal. Immunofluorescence staining of club (CCSP and SOX2) and basal cells (KRT5 and SOX2) in normal vs COPD lung airway shows a mixture of metaplasia and hyperplasia along with collapsed airways in COPD airway compared to normal. There is an increase in basal cells and goblet cells that also contain high expressions of CCSP (a club cell marker). EZH2 transcripts were knocked down in immortalized, Human Bronchiolar Epithelial Cells (HBEC) which were, then, prompted to differentiate in air-liquid-interface cultures. Acetylated-Mucin5 was highly expressed while club cell secretory protein (CCSP) was lowly expressed in EZH2 knockdown cells compared to wild type. This suggest that EZH2 plays a role in club and goblet cell differentiation. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The identification of a novel antibody for CD133 using human antibody phage display.

The transmembrane glycoprotein CD133 is believed to be a marker of adult prostate stem cells and cancer stem/initiating cells. Investigating the role of CD133 in the normal biology of the prostate and in cancer is complicated by the lack of a sensitive and accurate antibody for its detection. Here, we describe the characterization of a unique antibody identified using human antibody phage display that can recognize CD133 in both formalin-fixed tissues and cell lines. A human single-chain variable fragment (scFv) antibody phage display library possessing a diversity of 8 × 109 was screened against fully glycosylated recombinant CD133. A counter screen was performed against deglycosylated CD133 to select for clones that preferentially recognized a glycosylation-independent epitope. The lead scFv was analyzed by flow cytometry and cloned into a rabbit immunoglobulin scaffold for immunohistochemistry (IHC). The antibody designated HA10 was found to bind a glycosylation-independent epitope on the peptide backbone of CD133 with high affinity. As a reagent for flow cytometry, HA10 detected CD133 more accurately than a commonly used commercially available antibody. IHC analysis with HA10 documented the staining of basal cells and luminal cells in healthy prostate sections. Weak staining of luminal cells was observed in adenocarcinoma sections at a very low frequency. Examination of a LuCaP patient-derived xenograft tissue microarray found that only three of the LuCaP models were positive for CD133. The three CD133pos LuCaP models all originated from non-AR driven metastatic prostate cancer with neuroendocrine differentiation. Subsequent interrogation of liver biopsies from a patient who failed second-generation anti-androgen therapy found high levels of CD133 staining. The original transurethral resection of the prostate from that patient was, however, absent of CD133. We have developed a novel antibody that was able to detect CD133 by both IHC and flow cytometry. Using HA10 as an IHC reagent, we found that CD133 is a marker for a very rare cell type in both healthy prostate and adenocarcinoma sections. Our preliminary investigation also suggests that there may be an association between CD133 and non-AR driven prostate cancer with neuroendocrine differentiation.

P40 as a Basal Cell Marker in the Diagnosis of Prostate Glandular Proliferations: A Comparative Immunohistochemical Study with 34betaE12.

Immunohistochemistry is important for the accurate diagnosis of basal cells in atypical glandular proliferations of the prostate. p40, an isoform of p63, may be an adjunct to a marker panel in this setting. Biopsies of 68 patients were analyzed by immunohistochemistry using antibodies to 34betaE12 and p40. Basal cell staining was classified as negative, partial (<60%), or diffuse (≥60%); irregular staining was defined as discordant staining patterns. In acinar proliferations (N = 41), partial staining for both markers was seen in 42%, and diffuse staining in 46% of reactive cases. An irregular reactivity was noted in one case only (2%). Finally, these lesions were signed out as benign. Acinar proliferations negative for both markers and limited amount of glands (≤4) were termed atypical small acinar proliferations (ASAP). Out of six PIN lesions two cases showed partial, three cases showed diffuse reactivity for both markers, and one case was stained irregular. All cases diagnosed as prostate carcinomas (N = 20) had no evidence of basal cell staining for neither of the markers. p40 expression is closely correlated to 34betaE12 with respect to demonstration of basal cells of prostate glands and may provide further information on the dignity of glandular proliferations of the prostate.

Role of NF‐κB in the pathogenesis of psoriasis elucidated by its staining in skin biopsy specimens

NF-κB is a transcription factor belonging to the Re1 family, and it regulates the activity of a large number of proinflammatory genes. Its role in psoriasis, which is a prototype chronic inflammatory disease, is therefore expected to be considerable. It has been found that many of the triggering factors for psoriasis initiate inflammation by activation of NF-κB. Skin biopsies of 74 psoriatic cases were studied for the grade of NF-κB staining. They were then correlated with the histopathological indices of severity, especially epidermal hyperplasia and inflammatory infiltrate. Epidermal nuclear positivity for NF-κB correlated with the grade of epidermal hyperplasia (P = 0.043). There was a significant correlation between grade of basal cell positivity of NF-κB and inflammatory infiltrate (P = 0.004). Lymphocyte staining showed a strong correlation with epidermal and basal cell staining pattern for NF-κB (P = 0.004 and 0.003, respectively). There was, however, no correlation of lymphocyte staining with any histopathological parameter. There was also a peculiar propensity for NF-κB to show a positive nuclear stain at the tips of dermal papillae. The study has demonstrated that translocation of NF-κB in the epidermis and basal cells is responsible for two of the key pathological features of psoriasis, epidermal hyperplasia, and inflammation. We have postulated that translocation of NF-κB in lymphocytes, in psoriatic skin leads to NF-κB translocation in the epidermis and basal cells. NF-κB is therefore likely to be one of the key molecules in the pathogenesis of psoriasis.

Glutamate, a metabolic biomarker of aggressiveness and a potential therapeutic target for prostate cancer

Glutamate, a metabolic biomarker of aggressiveness and a potential therapeutic target for prostate cancer

1922 FEATURES OF ATYPICAL GLANDS ON INITIAL PROSTATE BIOPSY AS POSITIVE OR NEGATIVE PREDICTORS OF MALIGNANCY ON SUBSEQUENT PROSTATE BIOPSY

You have accessJournal of UrologyProstate Cancer: Detection and Screening III1 Apr 20121922 FEATURES OF ATYPICAL GLANDS ON INITIAL PROSTATE BIOPSY AS POSITIVE OR NEGATIVE PREDICTORS OF MALIGNANCY ON SUBSEQUENT PROSTATE BIOPSY Sriram Venigalla, Chen Zhao, and Hiroshi Miyamoto Sriram VenigallaSriram Venigalla Rochester, NY More articles by this author , Chen ZhaoChen Zhao Rochester, NY More articles by this author , and Hiroshi MiyamotoHiroshi Miyamoto Rochester, NY More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.2079AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Patients whose prostate biopsy reveals the presence of atypical glands suspicious for prostatic carcinoma (AGSC) are subject to close monitoring and repeat biopsies, perhaps unnecessarily, as clinicians do not have a clear basis for understanding and stratifying the significance of these findings. The value of histologic atypia on an initial prostate biopsy in predicting the detection of prostate cancer on subsequent biopsies should thus be further studied. METHODS In a retrospective, blinded manner, we examined the initial biopsies of 82 patients (93 foci) reported as AGSC. Five factors assessed in these biopsies include the size of the nucleoli of cells composing AGSC (graded on a scale of 1-4 with “1” denoting small, inconspicuous nucleoli and “4” denoting macronucleoli), the presence of high-grade prostatic intraepithelial neoplasia (HGPIN) adjacent to or separate from AGSC, the number of AGSC, the presence of intraluminal crystalloid, mucin, or dense secretions within AGSC, and the presence of basal cells in AGSC highlighted by immunohistochemical staining. RESULTS On subsequent biopsies, 31 (38%) patients were found to have prostate cancer (PCA) while no carcinoma was identified in the remainder (62%) of cases (NOCA). Nucleolar grade was significantly higher (p=0.010) in PCA cases (mean ± SD: 2.43 ± 0.80) than in NOCA cases (mean ± SD: 1.98 ± 0.70). Adjacent (p=0.235) and separate (p=0.132) HGPINs were found in 8/37 (22%) foci and 8/31 (26%) cases in PCA, respectively, and in 6/56 (11%) foci and 6/51 (12%) cases in NOCA, respectively. The number of atypical glands was similar (p=0.950) between PCA (mean ± SD: 7.03 ± 5.35) and NOCA (mean ± SD: 7.09 ± 4.18). The presence of intraluminal crystalloid, mucin, or dense secretions in AGSC did not show a statistically significant correlation between PCA [5/37 (14%)] and NOCA [13/56 (23%)] (p=0.293). Immunohistochemical stains demonstrated patchy basal cells in 17/39 (44%) of NOCA whereas basal cells were absent in 24/27 (89%) PCA (p=0.006). CONCLUSIONS On an atypical initial prostate biopsy, the size of nucleoli in AGSC is a positive predictor of prostate cancer on subsequent biopsy. On the other hand, as expected, even patchy staining of basal cells in AGSC represents a negative predictor of prostate cancer. However, HGPIN adjacent to or separate from AGSC, the total number of AGSC, and intraluminal materials do not reliably predict the subsequent diagnosis of prostate cancer. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e775-e776 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sriram Venigalla Rochester, NY More articles by this author Chen Zhao Rochester, NY More articles by this author Hiroshi Miyamoto Rochester, NY More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The value of triple antibody (34βE12 + p63 + AMACR) cocktail stain in radical prostatectomy specimens with crushed surgical margins

BackgroundTriple antibody cocktail immunohistochemical staining is routinely used as an ancillary method to establish a diagnosis of prostate cancer in biopsies with small foci of atypical glands. Crush artefact can...

P53 expression and mutation analysis of odontogenic cysts with and without dysplasia

Overexpression of p53 protein is well described in odontogenic cystic lesions (OCLs), including those with epithelial dysplasia; however, most p53 antibodies stain both wild-type and mutated p53 protein and may not reflect genotype. Direct sequencing of the p53 gene has not identified mutations in OCLs with dysplasia. The purpose of this study was to determine the molecular basis of p53 expression in several types of OCLs with and without dysplasia. The study material comprised 13 OCLs: odontogenic keratocyst (n = 5), orthokeratinized odontogenic cyst (n = 5), dentigerous cyst (n = 2), lateral periodontal cyst (n = 1), and unspecified developmental odontogenic cyst (UDOC) (n = 1). Five of these had features of mild or moderate epithelial dysplasia. One intraosseous squamous cell carcinoma (SCC) that was believed to have arisen from an antecedent dysplastic orthokeratinized OC was also included. Immunohistochemistry was performed using the DO7 monoclonal antibody that recognizes wild-type and mutated p53. DNA was extracted from microdissected tissue for all samples and exons 4 to 8 of the p53 gene direct sequenced. In 4 of 5 OCLs with dysplasia there was strong nuclear staining of basal and suprabasal cells. In all cases without dysplasia, nuclear expression in basal cells was either negative or weak and was absent in suprabasal cell nuclei. A mutation in exon 6 of the p53 gene (E224D) was identified in both the dysplastic orthokeratinized OC and the subsequent intraosseous SCC. OCLs with features of dysplasia show increased expression of p53 protein that does not reflect p53 mutational status. One dysplastic OC shared the same p53 mutation with a subsequent intraosseous SCC, indicating that p53 mutation may be associated with malignant transformation in this case.

Role of bcl-2 oncoprotein in oral potentially malignant disorders and squamous cell carcinoma: An immunohistochemical study

The product of bcl-2 gene, bcl-2 protein, an anti-apoptotic protein, is known to be over-expressed in potentially malignant disorders and squamous cell carcinoma (SCC) of the oral cavity. The aim of this study is to compare the topographical aspect and degree of bcl-2 over-expression in potentially malignant disorders including leukoplakia, oral submucous fibrosis (OSMF), and oral lichen planus (OLP), with that of the oral squamous cell carcinoma (OSCC), and to determine whether bcl-2 protein can be considered as a tumor marker. A group of 60 histo-pathologically diagnosed, formalin-fixed, paraffin embedded tissue samples was included in the study. The study group was further subdivided into four groups: Group I, consisting of oral leukoplakia; Group II, OSMF; Group III, OLP and Group IV, OSCC. These samples were collected from Government Dental College, Bangalore, and then subjected to immunohistochemical (IHC) staining using indirect immunoenzyme labeled streptavidin biotin (LSAB) method. Out of 30 cases of OSCC: 11 (36.7%) cases showed greater supra-basal keratinocyte staining; 15 (50%) cases showed greater number of positive cells in the basal cell layer, with relatively less number of supra-basal cells showing positive staining; and, rest of the 4 (13.3%) cases did not show convincing staining. Among the total 30 cases of potentially malignant disorders: 10 each of leukoplakia, OSMF and OLP, 2 (20%), 2 (20%), 4 (40%) of the cases showed greater supra-basal cell layer positive staining and 8 (80%), 6 (60%), 6 (60%) of them showed greater basal cell staining, respectively. Two cases of OSMF did not show convincing staining. In the cases that were bcl-2 positive: 2 (6.67%) of the OSCC, 3 (30%) of leukoplakia, 2 (20%) of OSMF and 1 (10%) of OLP, showed more than 50% of the cells positive. 25-50% cells were positive in 21 (70%) of OSCC, 6 (60%) of leukoplakia, 4 (40%) of OSMF and 6 (60%) of OLP cases. 10-25% of cells were positive in 4 (13.3%) of OSCC, 1(10%) of leukoplakia, 2 (20%) of OSMF and 3 (30%) of OLP cases. Less than 10% of cells were positive in 3 (10%) of OSCC and 2 (20%) of OSMF cases. CLINICAL SIGNIFICANCE AND CONCLUSION: As definite number of cases showed bcl-2 over expression in our study, the role of bcl-2 in the development and progression of oral neoplasia needs further investigation along with other oncogenes.

Reticular basement membrane fragmentation and potential epithelial mesenchymal transition is exaggerated in the airways of smokers with chronic obstructive pulmonary disease

In COPD, the airways are chronically inflamed, and we have now observed fragmentation of the reticular basement membrane (Rbm). This appears to be a hallmark of the process known as epithelial mesenchymal transition (EMT), in which epithelial cells migrate through the Rbm and differentiate into fibroblasts. The aim of this study was to confirm the extent and relevance of Rbm fragmentation in smokers and patients with COPD, and to undertake a preliminary analysis of some classical markers of EMT. Endobronchial biopsies from current smokers (CS; n = 17) and ex-smokers with COPD (ES; n = 15), smokers with normal lung function (NS; n = 16) and never-smoking control subjects (NC; n = 15) were stained for the EMT markers, S100A4, vimentin, epidermal growth factor receptor and matrix metalloproteinase-9. Compared with NC, there was significant Rbm fragmentation in the CS, ES and NS groups, which was positively associated with smoking history in subjects with COPD. Staining for basal epithelial S100A4, epithelial epidermal growth factor receptor and matrix metalloproteinase-9 in cells within Rbm clefts, and for S100A4 in Rbm cells, was increased in the CS, NS and ES groups compared with the NC group. There was also increased Rbm cell S100A4 staining in the CS group compared with the ES and NS groups. Basal epithelial cell staining for S100A4 was inversely correlated with airflow limitation. Double staining for both S100A4 and vimentin further strengthened the likelihood that these changes represented active EMT. This is the first detailed description of fragmentation and cellularity of the Rbm in smokers, which were most marked in subjects with COPD. The data are consistent with active EMT in these subjects.

Distribution of Basal/Myoepithelial Markers in Benign and Malignant Bronchioloalveolar Proliferations of the Lung

We investigated the staining pattern of commonly used basal cell/myoepithelial markers, such as p63 (a p53-homologous nuclear protein), basal cell-specific cytokeratin antibody (34betaE12, K903), and smooth muscle myosin heavy chain (SMMHC) in benign and malignant bronchioloalveolar proliferations of the lung. We studied 85 lung lesions consisting of 35 bronchioloalveolar carcinoma, 30 well-differentiated adenocarcinoma, and 20 cases of benign lung lesions. In normal lung, p63, K903, and SMMHC decorated the basal cells of large and small airways and occasional cells of terminal bronchioles. In reactive processes, a distinctive staining pattern was present in 19/20 (95%) of the cases characterized by staining of basal cells of the airways and bronchiolar epithelium and squamous metaplastic epithelium for p63 and K903, whereas 12/20 (60%) stained with SMMHC. Respiratory ciliated cells, alveolar epithelial cells, and nonepithelial cells were negative. In bronchioloalveolar carcinoma, a discontinuous peripheral rim of p63-immunoreactive cells was retained surrounding and intermingled with the malignant bronchioloalveolar proliferation in 31/35 (88.5%) cases, SMMHC in 28/35 (80%) cases, and K903 in 20/35 (57%) cases. For adenocarcinoma, a majority of the cases (28/30, 93%) were negative for p63 and K903; however, SMMHC showed artifactual staining in the desmoplastic stroma in 6/30 (20%) cases. Our results highlighted the differential expression of basal cell markers across various bronchioloalveolar lesions. The staining pattern of basal cells in bronchioloalveolar carcinoma supports that these neoplasms may actually be carcinoma in-situ.

Evaluation of routine application of P504S, 34βE12 and p63 immunostaining on 250 prostate needle biopsy specimens

We aimed to determine whether a standard P504S, 34betaE12 and p63 immunostaining of prostate core needle biopsy specimens can optimize diagnostic accuracy of conventional staining methods. The staining properties of all three antibodies were evaluated on 250 prostate biopsies formerly diagnosed as benign. Lack of basal cell staining in more than three glands for 34betaE12 and p63 occurred in 41 (27.5%) and 9 (6%) respective cases from the General Hospital pool. Respective figures from the Uropathology department specimens were 18 (18%) for 34betaE12 and 8 (8%) for p63. With the aid of P504S positivity, a case of prostate cancer as well as another of atypical small acinar proliferation was discovered. Despite its potential for important aid in accurate diagnosis, standard application of immunohistochemistry in prostate biopsy is not justified and should be reserved for equivocal cases where conventional pathology fails to be conclusive.

Cytoplasmic and Nuclear Maspin Expression in Lung Carcinomas

Maspin, a serine protease inhibitor related to the serpin family, can inhibit invasion and metastasis of malignancies although direct evidence of the clinicopathologic significance of cytoplasmic relative to nuclear expression is limited. Here, maspin expression was examined on tissue microarrays containing lung carcinoma (n=155) and adjacent noncancerous tissue (n=20) and also 4 lung carcinoma cell lines (LC-1/Sq, LC-IF, PC-14, and AoI) by immunohistochemistry. Maspin expression was compared with clinicopathologic parameters of the tumors. Maspin expression showed positive nuclear staining in basal cells, LC-IF, and PC-14 cell lines, and also cytoplasmic immunoreactivity in secretory and ciliated cells, LC-1/Sq cell line. Cytoplasmic staining was the lowest in adenocarcinoma (AD) and the highest in squamous cell carcinoma as compared with other types of lung carcinoma (P<0.05), and positively correlated with expression of p53 and caspase-3 (P<0.05). The cytoplasmic one showed stronger immunoreactivity in male carcinoma patients than female ones (P<0.05). The nuclear maspin expression gradually increased through squamous cell carcinoma, AD, large cell carcinoma to small cell carcinoma (P<0.05) and was also positively associated with the levels of vascular epithelial growth factor and extracellular matrix metalloproteinase inducer expression (P<0.05). Kaplan-Meier analysis indicated that the cytoplasmic or nuclear maspin expression was not a good prognostic marker for lung carcinomas overall (P>0.05), but the cytoplasmic pattern pointed to good survival for AD cases (P<0.05). It was concluded that the cytoplasmic and nuclear expression patterns of maspin are involved in the cellular differentiation of normal lung tissue and the histogenesis of different lung carcinomas. The cytoplasmic maspin may play an important role in lung carcinomas by regulating apoptosis and thus is a favorable prognostic marker for AD patients, whereas the nuclear location may be linked to promotion of angiogenesis.

Expression of integrin-beta1 in oral leukoplakia and early invasive carcinoma and its relationship with cell proliferation

To investigate the expression and significance of integrin-beta1 in oral leukoplakia and early invasive carcinoma. The expression of integrin-beta1 and Ki-67 was examined in 12 normal oral mucosa, 10 simple hyperplasia, 24 epithelial dysplasia and 14 early invasive carcinoma by immunohistochemistry. In normal and simple hyperplasia epithelium, integrin-beta1 was mostly expressed in the basal cell membrane, and Ki-67 in the nuclei of basal and the parabasal layers. In dysplasia epithelium and early invasive carcinoma, integrin-beta1 showed membrane staining and Ki-67 showed nuclear staining in dysplastic basal cells, sprinkle cells and SCC cells. Integrin-beta1 and Ki-67 were overexpressed in 7 and 10 of 24 dysplasia cases and in 8 of 14 early invasive carcinoma cases respectively. There was a significant difference in integrin-beta1 and Ki-67 expression among the four groups (chi2 = 10.651, P = 0.014; chi2 = 14.831, P = 0.002), in integrin-beta1 expression among normal oral mucosa, simple hyperplasia and early invasive carcinoma (P = 0.008, P = 0.013) and in Ki-67 expression among normal oral mucosa and dysplasia, early invasive carcinoma (P = 0.026, P = 0.001), and among simple hyperplasia and early invasive carcinoma (P = 0.005). A significant correlation between integrin-beta1 and Ki-67 (R = 0.442, P < 0.01) was found. Integrin-beta1 showed increased staining in dysplasia epithelium cells and SCC cells, and may correlate to the proliferation of the dysplasia cells.