Staining Of Basal Cells Research Articles

Partial Atrophy on Prostate Needle Biopsy Cores: A Morphologic and Immunohistochemical Study

Partial atrophy is the most common benign mimicker of prostate cancer on needle biopsy. Of 3916 prostate needle core biopsy cases received in our consultation service over a period of 3 months (March 1, 2007 to May 31, 2007), 170 cases (4.3%) with partial atrophy were diagnosed as atypical glands by outside pathologists and prospectively identified. We supplemented our material with 108 cases of partial atrophy sent to our consultation service in 2006 from a single institution, which frequently uses a triple cocktail stain [p63, high molecular weight cytokeratin (HMWCK), alpha-methyl acyl-Coa racemase (AMACR)]. The morphologic features of the 278 cases and immunohistochemistry of 236 cases (198 with prostate cocktail and 38 with only basal cell makers) were analyzed. Forty-eight of 278 (17.3%) partial atrophy cases were mixed with postatrophic hyperplasia. Enlarged nuclei were visible in 43/278 (15.5%) cases, with prominent nucleoli seen in 58/278 (20.9%) cases (30 cases associated with nuclear enlargement). Of 198 cases with a prostatic cocktail stain, 48 (24.2%) had a cancer pattern for both basal cells and AMACR (p63-, HMWCK-, and AMACR+), 14 (7.1%) had a cancer pattern for basal cells (p63-, HMWCK-, and AMACR-), 89 (44.9%) had a cancer pattern for AMACR (p63+, HMWCK+, and AMACR+), and 47 (23.7%) had a totally benign pattern (p63+, HMWCK+, and AMACR-). Of the 198 cases using the cocktail stain, 136 (68.7%) had positive basal cell staining. The percentage of basal cells labeled with the combination of p63/HMWCK was: <5% in 42 (21.2%) cases, 5% to 75% in 58 (29.3%) cases, and >75% in 36 (18.2%) cases. An additional 38 cases immunostained only for p63 and/or HMWCK was negative in 2 (5.2%) cases, <5% (13.1%) in 5 cases, 5% to 75% in 19 (50%) cases, and >75% in 12 (31.6%) cases. In conclusion, partial atrophy is a benign mimicker of adenocarcinoma both as a result of its routine morphologic features and its immunohistochemical profile. Recognition of the classic morphology of partial atrophy on routine hematoxylin and eosin-stained sections is critical to avoid misdiagnosing partial atrophy as adenocarcinoma.

Partial Atrophy in Prostate Needle Biopsies: A Detailed Analysis of Its Morphology, Immunophenotype, and Cellular Kinetics

We systematically analyzed 73 prospectively collected partial atrophy (PA) foci from over 185 prostate needle biopsy cases to characterize them along 3 fronts: morphologic, as it can be a mimic of prostate cancer (PCa), immunohistochemical (basal cell markers and alpha-methyl acyl-CoA racemase), as it often shares the staining characteristics of PCa, and cellular kinetics (MIB-1 proliferation marker), as it belongs to the larger group of focal atrophy, some of which have been shown to be proliferative and associated with chronic inflammation. The following morphologic features were prominent at low magnification: small to mid-sized glands with circumscribed (70%) or disorganized growth pattern (30%), presence of stellate/undulated gland lumina (92%), associated few completely atrophic glands within the PA focus (97%), and scant apical but abundant lateral pale/clear cytoplasm similar to adjacent benign glands (100%). On higher magnification, 33% of foci contained micronucleoli, but all lacked nuclear enlargement (100%) or macronucleoli (100%), characteristic of PCa. No adjunctive features of PCa were seen. Patchy basal cell staining was observed in 52/71 (73%), whereas 4/71 (6%) were completely negative. alpha-methyl acyl-CoA racemase demonstrated variable expression, stronger than the benign glands in 7/72 (10%) foci. Associated pathology included PCa (42%), and complete atrophy (91%), distinct from PA foci. There was no difference between the mean proliferative index of the PA foci compared with the benign glands [5.5 (range 0 to 30) and 5.6 (range 0 to 31), respectively, P=0.97 by paired t test], as measured quantitatively by ChromaVision system. PA foci were rarely associated with inflammation (1%). Familiarity with these morphologic features and staining characteristics will allow its confident separation from cancer, especially in limited biopsy material. PA foci do not represent a spectrum of proliferative inflammatory atrophy, justifying its term.

Sparing of previously irradiated skin from erlotinib-induced acneiform rash

To the Editor: Erlotinib is a human epidermal growth factor receptor type 1 (Her1/EGFR) thyrosine kinase inhibitor used in the treatment of non–small cell lung cancer (NSCLC) and pancreatic cancer.1Cohen M.H. Johnson J.R. Chen Y.F. Sridhara R. Pazdur R. FDA drug approval summary: erlotinib (Tarceva) tablets.Oncologist. 2005; 10: 461-466Crossref PubMed Scopus (340) Google Scholar, 2Moore M.J. Goldstein D. Hamm J. Figer A. Hecht J. Gallinger S. et al.Erlotinib plus gemcitabine compared to gemcitabine alone in patients with advanced pancreatic cancer. A phase III trial of the National Cancer Institute of Canada Clinical Trials Group [NCIC-CTG]. American Society of Clinical Oncology 2005 Annual Meeting Proceedings (abstract 1).J Clin Oncol. 2005; 23: 1PubMed Google Scholar Typically appearing within the first 2 weeks, the major physical manifestation of erlotinib toxicity is a cutaneous rash, seen in almost 70% of patients; the face, neck, and upper torso are commonly involed.3Perez-Soler R. Saltz L. Cutaneous adverse effects with HER1/EGFR-targeted agents: is there a silver lining?.J Clin Oncol. 2005; 23: 5235-5246Crossref PubMed Scopus (449) Google Scholar We report a patient with NSCLC developing a typical acneiform rash sparing the area of previous radiotherapy. A 43-year-old male patient was admitted to the hospital with fatigue and a cough. A computed tomographic scan of his thorax showed an 86 mm × 75 mm mass in the left upper lobe, with multiple mediastinal and hilar lymphadenopathies. Biopsy was consistent with NSCLC. No evidence of distant metastasis was present. Treatment with gemcitabin plus cisplatin was commenced, and radiotherapy to the mass and mediastinum was applied between the third and fourth cycles. After the sixth cycle, the mass and the lymphadenopathies had largely regressed. With the patient's approval, treatment with erlotinib was started. After 2 weeks of erlotinib treatment, a typical acneiform rash developed on the face, trunk, and upper extremities. The skin overlying the areas of previous radiotherapy, however, was spared, on both the anterior and posterior sides of the trunk (Fig 1). We performed skin biopsies from the acne-spared, previously irradiated skin and from the neighboring affected nonirradiated skin. Histologic examination revealed suppurative folliculitis destroying follicular epithelium and adnexial structures in the nonirradiated skin specimen. Although eccrine glands and piloerector muscles were normal, there was no follicular structure in the previosly irradiated skin specimen (Fig 2). We performed EGFR staining to see if EGFR expression was altered in the radiated area. No difference was seen between irradiated and nonirradiated skin. In both specimens, moderate intracytoplasmic and membranous EGFR staining of basal epidermal cells and follicular epithelium were observed. Four months after treatment began, the irradiated area remained acne-free and showed no signs of disease progression. Rash is a common adverse affect of Her1/EGFR-targeted agents. Histology of the lesions associated with erlotinib, cetuximab, and gefitinib are similar, indicating a class effect of Her1/EGFR inhibitors. The pathogenesis of the rash is not completely understood. Her1/EGFR is expressed by normal keratinocytes, skin fibroblasts, and in the outer root sheath of the hair follicle. A neutrophilic infiltrate has been reported in dermal tissue, particularly the infundibular part of the hair follicle, but the sebaceous glands are not affected.3Perez-Soler R. Saltz L. Cutaneous adverse effects with HER1/EGFR-targeted agents: is there a silver lining?.J Clin Oncol. 2005; 23: 5235-5246Crossref PubMed Scopus (449) Google Scholar Histologic analysis of rash samples from patients receiving cetuximab showed lymphocytic perifolliculitis or suppurative superficial folliculitis without any infectious etiology.4Busam K.J. Capodieci P. Motzer R. Kiehn T. Phelan D. Halpern A.C. Cutaneous side-effects in cancer patients treated with the antiepidermal growth factor receptor antibody C225.Br J Dermatol. 2001; 144: 1169-1176Crossref PubMed Scopus (391) Google Scholar The skin changes may start within the first hours of radiotherapy and might last for months, or they may be permanent. The estimated D50 follicle dose is around 40 Gy.5Lawenda B.D. Gagne H.M. Gierga D.P. Niemierko A. Wong W.M. Tarbell N.J. et al.Permanent alopecia after cranial irradiation: dose-response relationship.Int J Radiat Oncol Biol Phys. 2004; 60: 879-887Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar The patient received 45 Gy total to the tumor bed, while the skin received 18.5 Gy. In the present case, we have shown that sparing of irradiated skin from erlotinib-related acneifom rash seems to be caused by radiation-induced follicular loss, and EGFR expresion is not altered.

Immunohistochemical Antibody Cocktail Staining (p63/HMWCK/AMACR) of Ductal Adenocarcinoma and Gleason Pattern 4 Cribriform and Noncribriform Acinar Adenocarcinomas of the Prostate

Overexpression of alpha-methylacyl coenzyme A racemase (AMACR) in combination with absence of basal cell markers [ie, p63 and high molecular weight cytokeratin (HMWCK)] is typical of classic acinar prostatic adenocarcinoma. We studied the expression and diagnostic utility of p63/HMWCK/AMACR immunohistochemical cocktail staining in ductal adenocarcinoma and cribriform Gleason pattern 4 acinar prostate cancer and compared it to noncribriform Gleason pattern 4 acinar prostate cancer. One to 4 representative formalin-fixed paraffin-embedded archival tissue blocks from 62 radical prostatectomy specimens harboring prostate cancer of ductal (n=51), cribriform Gleason pattern 4 acinar (n=27), and noncribriform Gleason pattern 4 acinar adenocarcinoma (n=48) were included in this study. Immunohistochemistry was performed using a triple stain of AMACR, p63, and HMWCK. Only staining that was moderate or strong was considered positive. The percentage of staining intensity and the presence of occasional basal cells positive with p63/HMWCK were recorded in each histologic type of prostatic adenocarcinoma. Seventy-seven percent of ductal prostatic adenocarcinoma, 67% of cribriform acinar prostatic carcinoma, and 81% of noncribriform acinar prostatic carcinoma showed positive staining for AMACR. There was no statistically significant difference between AMACR staining among the 3 histologic types, although there was a trend for noncribriform acinar prostatic carcinoma to have greater expression of AMACR than cribriform acinar prostatic carcinoma (P=0.07). Staining was often heterogeneous, varying in staining intensities within the same histologic type of carcinoma. Basal cells were detectable by p63 and HMWCK in a patchy fashion in 31.4% (16/51) of ductal and 29.6% (8/27) of cribriform acinar carcinomas compared with 2.1% (1/48) of noncribriform acinar carcinomas. In summary: (1) the majority of prostatic ductal and cribriform acinar carcinomas strongly expressed AMACR, however, subpopulations of these prostatic carcinoma were either completely negative or only weakly positive; (2) AMACR staining was often heterogeneous in intensity in the same histologic type of tumor, even within the same case; (3) patchy basal cell staining in noncribriform acinar prostatic carcinoma is rare. In contrast, remnants of basal cells identified by p63/HMWCK were seen in a patchy fashion in a significant minority of both ductal and cribriform acinar prostatic adenocarcinoma, which most likely represents intraductal spread of tumor.

Proliferation and Differentiation of Transplantable Rabbit Epithelial Sheets Engineered with or without an Amniotic Membrane Carrier

To report a novel method of engineering transplantable, carrier-free corneal epithelial sheets by using a biodegradable fibrin sealant and to compare its characteristics with epithelial sheets cultivated on denuded amniotic membrane carriers. Stratified corneal epithelial sheets were prepared in culture dishes coated with biodegradable fibrin glue. Amniotic membrane (AM) carriers served as the control. The quality of cultivated sheets was compared by immunohistochemistry for cytokeratin (K)3, K12, K14, p63, occludin, and integrin beta1; electron microscopy; and colony-forming assays. K3 protein expression was compared by Western blot analysis. In a limbal-deficient rabbit transplantation model, postoperative adaptation and proliferation of BrdU-labeled cell sheets were examined by histology and anti-Ki67 staining. Epithelial sheets were successfully engineered by using a biodegradable fibrin sealant. Cell sheets in both groups were multilayered, expressed K3, K12, and K14, and had functioning occludin(+) apical tight junctions as well as p63 and integrin beta1 staining in basal cells. The carrier-free sheets appeared to be more differentiated than the AM sheets, which was also demonstrated by the higher levels of K3 in the Western blots. The colony-forming efficiency of dissociated cells was similar in both groups, although larger colonies were observed on the AM sheets. AM sheets retained higher levels of BrdU-labeled cells and fewer Ki67(+) cells compared with carrier-free sheets after transplantation. Tissue engineering with a commercially available fibrin sealant was an effective means of creating a carrier-free, transplantable corneal epithelial sheet. Carrier-free sheets were more differentiated compared with AM sheets, while retaining similar levels of colony-forming progenitor cells.

P.166 Salivary urea,creatinine,ascorbic and oxalic acid in CRF patients

To gain preliminary data on the location of a 195 kD cell peripheral protein of epidermal keratinocytes recognized by monoclonal antibody AE11, immunofluorescence staining was carried out on cultured human epidermal cells. B11 antidesmosomal polyclonal antibody and AE3 antikeratin monoclonal antibody staining were used as comparative reference controls. Desmosomal antigens span the cell membrane and keratins constitute a family of cytoplasmic proteins. The cultured cells were systematically used either unfixed, fixed with 4% paraformaldehyde, or fixed with methanol at 4°C. The former two preparations were designed to expose only the surface antigens, whereas the latter method permeated the membrane to allow cytoplasmic staining. Differences in staining patterns were observed in the basal layer as compared with the upper layers with all three antibodies. B11 antidesinosomal antibody staining was consistent in pattern after either paraformaldehyde or methanol fixation. In addition to the punctate staining along adjacent cell surfaces as shown in the basal layer, the surface planes of the upper cells exhibited parallel arrays of linear streaks, demonstrating the distribution of desmosomal proteins. Antikeratin staining by AE3 showed the typical filamentous staining in basal cells. However, a homogeneous patchy staining of suprabasal cells was observed. The presence of punctate surface staining using antikeratin antibody on paraformaldehyde fixed cells suggests leakage of keratin to the cell surface. AE11 showed stronger staining in the top cells on methanol treated cultures and a punctate surface staining of the cells fixed with paraformaldehyde. These observations provide useful preliminary information in localizing peripheral proteins in epidermal keratinocytes.

Effects of Phyto-Oestrogens on Veal Calf Prostate Histology

In veal calf production plant-based proteins are frequently included in milk replacer fed to the animals. Since soy products, which are mostly used, are known for their high levels of phyto-oestrogens, the effects of these feeds on the veal calf prostate were examined. Goal was to determine whether these compounds could interfere with histological screening for oestrogenic growth promoters. In a feeding experiment, four groups of veal calves fed plant-based protein-supplemented milk replacer (PBM), containing 5% soy concentrate, 5% soy isolate, 5% wheat gluten and 2% potato protein, for 4 weeks were compared to animals fed dairy-based control feed (DBM); animals treated with estradiol benzoate, diethylstilbestrol and ethinylestradiol served as positive controls. Daidzein and genistein levels measured in feed and urine showed high levels of genistein and daidzein in the soy isolate and soy concentrate supplemented feeds. Genistein and daidzein were also found in the urine of the animals that were fed these feeds. Haematoxylin-eosin-stained prostate sections of PBM-fed animals showed slight hyperplasia and some dilated tubules as compared to the DBM-fed group, but no metaplasia, which is used for screening for oestrogenic hormones. The positive controls showed extensive squamous metaplasia. Immunohistochemical staining for cytokeratin 5 (using RCK 103 monoclonal antibody) in basal cells showed a normal staining pattern of basal cells in the DBM-fed calves and extensive basal cell proliferation and squamous metaplasia in the oestrogen-treated positive control animals. PBM-fed calves showed no increase of basal cell staining but showed elongations of the basal cells in most animals, sometimes resulting in circular figures. It is concluded that the feeds examined in this study did not interfere with histological screening for oestrogens in male veal calves.

Differential expression and distribution of epithelial adhesion molecules in non-small cell lung cancer and normal bronchus

Background: Changes in epithelial cell interactions have been implicated in carcinogenesis, tumour invasion and metastasis. Aim: To screen for altered expression of epithelial adhesion genes in lung cancer development. Methods:...

Utilidad de Bcl-2 como nuevo marcador de expresión de células basales en patología prostática

Introduction and objectivesThe diagnosis of invasive adenocarcinoma of the prostate is often difficult in needle prostatic cores, where, additionally, the assessment of the presence of basal cells has demonstrated to be of paramount importance. currently, the immunohistochemical expression of 34betae12 antigen and p63 protein are the most utilized markers. in our study, we analyzed comparatively the expression of 34betae12, p63, bcl-2 and alpha-methylacyl-coa racemase in order to evaluate the usefulness of bcl-2 as a new marker of the basal cells in prostatic pathology. Methods and resultsThis study comprises radical prostatectomy specimens from 48 patients which were studied in order to determine the lack of staining of basal cells in invasive tumor areas together with the expression of racemase. Likewise, the presence of basal cells in areas of atrophy, hyperplasia, adenosis, and high-grade prostatic intraepithelial neoplasia (PIN) was also examined. Within the areas of adenosis and PIN a discontinuous pattern of basal cell expression was found in some cases. In 2 out of 48 cases (4,2%) of invasive carcinoma a weak bcl-2 expression without a basal cell distribution was found. Moreover, the expression of bcl-2 in the stromal lymphocytes appeared to be essential as an internal positive control of the technique. ConclusionsIn addition to classical markers, we demonstrated the diagnostic value of bcl-2 as a new basal cell marker.

Annexin A8 Is Up-Regulated During Mouse Mammary Gland Involution and Predicts Poor Survival in Breast Cancer

Microarray studies have linked Annexin A8 RNA expression to a "basal cell-like" subset of breast cancers, including BRCA1-related cancers, that are characterized by cytokeratin 5 (CK5) and CK17 expression and show poor prognosis. We assessed Annexin A8's contribution to the overall prognosis and its expression in normal, benign, and cancerous tissue and addressed Annexin A8's physiologic role in the mammary gland. Using microarrays and reverse transcription-PCR, the Annexin A8 expression was studied during mouse mammary gland development and in isolated mammary structures. Reverse transcription-PCR on cultured human luminal and basal cells, along with immunocytochemistry on normal and benign breast tissues, was used for cellular localization. Annexin A8's prognostic relevance and its coexpression with CK5 were assessed on tissue arrays of 1,631 cases of invasive breast cancer. Coexpression was further evaluated on a small cohort of 14 BRCA1-related breast cancers. Annexin A8 was up-regulated during mouse mammary gland involution and in pubertal ductal epithelium. Annexin A8 showed preferred expression in cultured basal cells but predominant luminal expression in normal human breast tissue in vivo. Hyperplasias and in situ carcinomas showed a strong staining of basal cells. Annexin A8 expression was significantly associated with grade (P < 0.0001), CK5 (P < 0.0001), and estrogen receptor status (P < 0.0001); 85.7% BRCA1-related breast tumors coexpressed Annexin A8 and CK5. Annexin A8 is involved in mouse mammary gland involution. In humans, it is a luminally expressed protein with basal expression in cell culture and in hyperplasia/ductal carcinoma in situ. Expression in invasive breast carcinomas has a significant effect on survival (P = 0.03) but is not independent of grade or CK5.

Differential expression of p63 isotypes (Δ N and TA) in salivary gland neoplasms: biological and diagnostic implications

To determine the association between the expression of p63 gene isoforms (TA and DeltaN) and salivary gland tumorigenesis, we performed reverse transcription-polymerase chain reaction analysis of these markers in 71 benign and malignant salivary gland neoplasms. The results were correlated with the expression of Notch ligand JAG1 gene and the clinicopathologic features and the full-length p63 protein expression by immunohistochemistry. Both p63 isoforms were either negative or weakly expressed in normal salivary gland tissues. TAp63 was highly expressed in most benign tumors and was either negative or weakly positive in most carcinomas. Conversely, DeltaNp63 was negative or faintly positive in most benign neoplasms and was highly expressed in adenoid cystic, mucoepidermoid, and myoepithelial carcinomas. Immunohistochemical analysis using anti-full-length p63 protein showed ubiquitous nuclear staining in basal and myoepithelial cells in both benign and malignant neoplasms. JAG1 was expressed in most benign and malignant tumors and did not correlate with p63 isoforms expression. We conclude that (1) p63 isoforms are differentially expressed in most benign and malignant tumors and may play distinct biological roles in certain salivary gland neoplasms; (2) p63 immunostaining do not correlate with the isoforms expression; and (3) isoform-specific antibodies are required for better cellular localization and biological correlations.

Using an AMACR (P504S)/34βE12/p63 Cocktail for the Detection of Small Focal Prostate Carcinoma in Needle Biopsy Specimens

We assessed the usefulness of immunohistochemical analysis with a 3-antibody cocktail (alpha-methylacyl coenzyme A racemase [AMACR, or P504S], 34betaE12, p63) and a double-chromogen reaction for detection of limited prostate cancer in 138 needle biopsy specimens, including 82 with small foci of prostatic adenocarcinoma and 56 benign prostates. When carcinoma was present, red cytoplasmic granular staining (AMACR) in the malignant glands and cells and dark brown nuclear (p63) and cytoplasmic (34betaE12) staining in basal cells of adjacent nonmalignant glands were found. Of 82 cases of small foci of prostatic adenocarcinoma, 78 (95%) expressed AMACR; all malignant glands were negative for basal cell staining. All benign glands adjacent to malignant glands were recognized easily by basal cell marker positivity and little or no AMACR expression. No benign glands were simultaneously positive for AMACR and negative for basal cell markers (specificity, 100%). There were no differences in intensity and numbers of positive glands with double-chromogen staining compared with using 1-color staining. Our results indicate that immunohistochemistry with a 3-antibody cocktail and double chromogen is a simple and easy assay that can be used as a routine test, which overcomes the problems of studying small lesions in prostate needle biopsies with multiple immunohistochemical stains.

Using an AMACR (P504S)/34bE12/p63 Cocktail for the Detection of Small Focal Prostate Carcinoma in Needle Biopsy Specimens

We assessed the usefulness of immunohistochemical analysis with a 3-antibody cocktail (α-methylacyl coenzyme A racemase [AMACR, or P504S], 34βE12, p63) and a double-chromogen reaction for detection of limited prostate cancer in 138 needle biopsy specimens, including 82 with small foci of prostatic adenocarcinoma and 56 benign prostates. When carcinoma was present, red cytoplasmic granular staining (AMACR) in the malignant glands and cells and dark brown nuclear (p63) and cytoplasmic (34βE12) staining in basal cells of adjacent nonmalignant glands were found. Of 82 cases of small foci of prostatic adenocarcinoma, 78 (95%) expressed AMACR; all malignant glands were negative for basal cell staining. All benign glands adjacent to malignant glands were recognized easily by basal cell marker positivity and little or no AMACR expression. No benign glands were simultaneously positive for AMACR and negative for basal cell markers (specificity, 100%). There were no differences in intensity and numbers of positive glands with double-chromogen staining compared with using 1-color staining. Our results indicate that immunohistochemistry with a 3-antibody cocktail and double chromogen is a simple and easy assay that can be used as a routine test, which overcomes the problems of studying small lesions in prostate needle biopsies with multiple immunohistochemical stains.

Comparison of 34βE12 and P63 in 100 Consecutive Prostate Carcinoma Diagnosed by Needle Biopsies

P63, a homologue of p53, was recently identified as a useful basal cell-specific marker. We compared the sensitivity and specificity of p63 with the widely used high-molecular-weight keratin 34betaE12 for the diagnosis of prostate carcinoma in needle biopsies. We selected 100 consecutive prostate carcinoma diagnosed by needle biopsies with an adequate number of cancerous glands on the slide. We chose 1 representative hematoxylin and eosin-stained slide from each case and gave it a Gleason score. The same paraffin block was retrieved for 34betaE12 and p63 stains. We compared staining patterns of 34betaE12 and p63 on both malignant glands and benign glands and recorded basal cell density (percentage of basal cells with positive staining in the benign glands). The cases were divided into 3 groups according to the Gleason score: 5 to 6 (31 cases), 7 (46 cases), and 8 to 10 (23 cases). In 20 cases, focal and patchy staining in a basal cell distribution in malignant glands (range, 1%-20%; mean, 6.6%) was demonstrated (19 by both stains and 1 by 34betaE12 only). In 1 case with a Gleason score of 9, the cancer cells, not the basal cells, were stained focally by p63 but not by 34betaE12. Higher-grade tumors demonstrated higher numbers of malignant glands with basal cell staining (1.65% for Gleason 7, 1.26% for Gleason 8-10, compared with 0.42% for Gleason 5-6). The overall specificity of the absence of basal cell staining in the malignant glands for 34betaE12 and p63 was 98.63% and 98.60%, respectively. In 17 cases, both stains revealed total absence of basal cell staining in some benign glands (range, 1%-10%; mean, 3.5%). The overall sensitivity in identifying basal cells in benign glands was 99.48% and 99.44% for 34beta12 and p63, respectively. Basal cell density was higher for 34betaE12 in comparison with p63 (92% vs. 87%). For diagnosing prostate carcinoma in the needle biopsies, p63 is as specific and sensitive Hospital as 34betaE12 and therefore can be used as a complementary basal cell-specific stain for 34betaE12 in difficult cases.

Usefulness of Basal Cell Cocktail (34βE12 + p63) in the Diagnosis of Atypical Prostate Glandular Proliferations

We evaluated the diagnostic usefulness of the 34βE12-p63 cocktail, compared with 34βE12 and p63 used alone, in 34 prostate needle biopsy (NBXs) and 3 transurethral resection specimens containing atypical glandular proliferations and in 18 NBXs containing unequivocal prostate carcinoma (PCa). Staining intensity; percentage of basal cells staining in benign, atypical, and malignant glands; number of benign glands lacking basal cell staining; and staining variance were analyzed. All NBXs with unequivocal PCa were negative with all 3 markers. Diagnoses were as follows for the atypical cases after staining for the 3 markers: PCa, 9; postatrophic hyperplasia, 12; high-grade prostatic intraepithelial neoplasia (HGPIN), 5; atypical adenomatous hyperplasia, 6; benign atypical proliferations, 4; and HGPIN with adjacent small atypical acinar proliferation suggestive of PCa, 1. The cocktail demonstrated consistently strong staining intensity and improved basal cell staining in morphologically benign and benign atypical glands compared with p63 and 34βE12 alone; it had the smallest staining variance compared with 34βE12 (F < 0.0001) and p63 (F = 0.31), although its advantage for resolving individual atypical cases was limited compared with 34βE12 and p63 alone. Of 37 atypical cases, 1 (3%) additionally was resolved as benign using the cocktail and p63. Because the diagnosis of PCa is supported by lack of basal cell staining, the immunohistochemical analysis with highest possible sensitivity and lowest possible variability is critical to ensure that a negative reaction is true. The cocktail provides a simple, cost-effective improvement in basal cell immunohistochemical analysis of difficult prostate lesions.

P63 expression in assessment of bronchioloalveolar proliferations of the lung

Discrimination of well-differentiated pulmonary adenocarcinoma from reactive bronchioloalveolar epithelium can be difficult on routine histology, especially with small biopsies. Ancillary studies to help in this distinction are desirable. p63, a p53-homologous nuclear protein, is a marker of reserve cells of the bronchus and terminal lobular unit. In this study, 33 cases of adenocarcinoma (20 open lung and 13 transbronchial/percutaneous biopsies) and 43 cases of benign lungs with fibrosis and metaplasia (22 open lung and 21 transbronchial/percutaneous biopsies) were studied for nuclear p63 expression by immunohistochemistry (Dako, Carpinteria, CA, USA). Five additional cases each of atypical adenomatous hyperplasia and adenosquamous carcinoma and three cases of squamous carcinoma (all open lung biopsies) were also stained. The diagnostic categories of benign lung conditions were usual interstitial pneumonia, parenchymal scar, cryptogenic organizing pneumonia and diffuse alveolar damage. In neoplastic cases, p63 positivity was calculated as percentage of all tumor cells examined. In areas of normal lung, p63 decorated the reserve cells of large and small airways and occasional cells of the distal lobular unit. In fibrotic reactive processes, an interrupted but distinct pattern of nuclear staining was present in all cases, with staining of basal cells of the airways as well as bronchiolar- and squamous-metaplastic epithelium (43/43, 100%). p63 immunoreactivity was less uniform in areas of acute lung injury within these cases. One adenocarcinoma and two cases of atypical adenomatous hyperplasia showed strong immunoreactivity (>80%), while three adenocarcinomas highlighted only rare tumor nuclei (<5% of tumor cells). Morphologic areas where p63 immunostaining was not helpful included the junction of normal lung and lepidic growth of adenocarcinoma, and retrograde spread of adenocarcinoma into small airways. Our results highlight the differential expression of p63 across various bronchioloalveolar lesions. Moreover, p63 may be helpful in distinguishing reactive from neoplastic glandular proliferations in the lung.

K16 expression in uninvolved psoriatic skin: a possible marker of pre-clinical psoriasis.

K16, a type I keratin, is upregulated in hyperproliferative states including psoriasis. It has been used as a marker of psoriasis and its expression is upregulated in relapsing psoriasis and downregulating in resolving. We evaluated non-lesional psoriatic skin for K16 expression. Sixty-seven non-lesional and lesional skin samples from patients with psoriasis and normal skin from 19 non-psoriatic patients were studied by immunohistochemistry on frozen sections with K16. Seventeen of 19 normal skin samples showed staining of basal cells in the deeper part of the rete ridges. Sixty-two non-lesional psoriatic skin samples showed intense basal staining of K16. Of the remaining five non-lesional samples, diffuse intense suprabasal staining in one, pan-epidermal staining in two, and no staining was seen in two samples. Suprabasal (37), diffuse (14), sandwich (12), and basal (3) pattern staining were seen in psoriatic skin. One psoriatic skin sample did not show any expression. Our results demonstrate that K16 expression is also observed in non-lesional psoriatic skin and may serve as a marker of preclinical psoriasis.

110. Helper-Dependent Adenovirus with the Cytokeratin-18 Promoter Produces Greater Transepithelial Chloride Current and More Persistent Transgene Expression Than the CMV Promoter in Differentiated Human Airway Epithelia

Gene transfer of the wild-type CFTR cDNA to airway epithelia could rescue the electrolyte transport defect and represent an attractive strategy for treating the genetic disease cystic fibrosis (CF). Numerous gene transfer vectors have expressed CFTR under control of the cytomegalovirus (CMV) enhancer/promoter because it generates robust expression in many cell types. However, this promoter could drive expression in cell types that do not normally express CFTR and may yield limited persistence. An alternative enhancer/promoter is from the cytoskeletal protein cytokeratin-18 (K18); cytokeratin-18 is constitutively expressed in differentiated epithelia and has a tissue distribution similar to CFTR. To test the K18 promoter, we used helper-dependent adenoviral vectors and compared transgene expression driven by the K18 and CMV promoters. We delivered them to well-differentiated primary cultures of CF airway epithelia grown at the air-liquid interface. We applied vector to the basolateral surface to facilitate infection through binding of the adenovirus receptor CAR. With a β-galactosidase transgene, the CMV promoter generated much greater β-galactosidase activity and total protein levels than the K18 promoter. However, the opposite pattern emerged when the two promoters drove CFTR expression; the K18 promoter generated greater transepithelial chloride current than the CMV promoter. These results suggested that the two promoters might drive expression in different cell types. We found that the CMV promoter generated the most intense X-Gal staining in basal cells. In contrast, the K18 promoter generated diffuse staining in columnar epithelial cells. These results suggest that the K18 promoter produced relatively greater CFTR expression in columnar epithelial cells that touch the apical surface and are responsible for transepithelial chloride transport, whereas the CMV promoter generated greater expression in basal cells. We also compared the persistence of transgene expression. Over the course of 12 weeks, transgene expression driven by the K18 promoter remained relatively constant, while transgene expression from the CMV promoter fell rapidly. These data emphasize the importance of appropriate regulatory elements in developing gene transfer vectors for CF airway epithelia and suggest that the K18 promoter may have advantages over the CMV promoter.

How often does alpha-methylacyl-CoA-racemase contribute to resolving an atypical diagnosis on prostate needle biopsy beyond that provided by basal cell markers?

Alpha-methylacyl-CoA-racemase (AMACR), a recently discovered tumor marker for prostate cancer, is being used increasingly in conjunction with hematoxylin and eosin (H&E) histology and basal cell markers in the workup of difficult prostate needle biopsies. However, it is not known how often a positive AMACR staining is used merely to support a malignant diagnosis that could otherwise be established based on routine H&E histology and negative basal cell staining. This study included 307 prostate needle biopsies that were sent to us for consultation diagnosed as "atypical" by contributing pathologists. Immunohistochemistry for AMACR, high molecular weight cytokeratin, and p63 was performed. AMACR staining intensity was graded as negative, weak, moderate, and strong. Only staining that was significantly stronger than that of background benign glands was considered positive. An "expert review" diagnosis was first rendered as benign, atypical, or cancer based on the H&E-stained section and basal cell marker stains without the knowledge of AMACR expression. The AMACR stains were then reviewed and a final diagnosis was rendered. A total of 215 cases had a final diagnosis of cancer following evaluation of the H&E-stained section, basal cell markers, and AMACR. Of these 215 cases, 176 (81.9%) were positive and 39 (18.1%) were negative for AMACR staining. Of 81 cases with a final diagnosis of atypical following review of all material, 42 (51.9%) were positive and 39 (48.1%) were negative for AMACR staining. When AMACR staining was negative, in no case was the initial cancer, atypical, or benign diagnosis (based on routine histology and negative basal cell markers) changed based on AMACR stain results. Of 115 cases called atypical after expert review, 76 were positive for AMACR; of these 76 cases, 34 (44.7%) were changed to a final diagnosis of cancer. AMACR expression was positive, with moderate and strong staining in 30 of the 34 cases (88.2%), for which the immunohistochemical result converted the expert review atypical diagnosis to a final cancer diagnosis. Of these 34 cases, 11 underwent radical prostatectomy, and cancer was found in all cases. Three additional patients underwent repeat biopsy, and cancer was present in the repeat biopsy in 2 patients. The cases whose diagnosis was changed from "atypical" on expert review to cancer were all highly suspicious for cancer based on H&E histology and negative basal cell markers, yet a definitive cancer diagnosis could not be established because of small size, insufficient cytologic atypia, or biopsy artifact. Interpretation and use of AMACR staining should be executed with caution. A negative AMACR stain can be seen in approximately 18% of cases considered to be cancer based on H&E stain combined with negative basal cell markers. A positive AMACR staining converted an atypical diagnosis, based on suspicious histology and negative basal cell marker stains, to cancer in approximately 10% (34 of 307) of cases thought to be atypical by contributing pathologists and in approximately 50% (34 of 76) of cases thought be atypical on expert review by a specialist in genitourinary pathology.

Up-regulation of cathepsin X in prostate cancer and prostatic intraepithelial neoplasia.

Evidence is accumulating that several proteases are involved in prostate cancer progression. A locus which is often amplified in prostate cancer is the chromosomal region 20q13. Interestingly, one of the genes encoding the cysteine protease cathepsin X maps to this region. The aim of this study was to assess the expression pattern of cathepsin X in malignant and non-malignant prostatic tissue samples. Matched malignant and non-malignant tissue specimens were obtained from 56 men after radical prostatectomy. Cathepsin X was quantified at both protein and mRNA levels using several detection methods: Western blotting, immunohistochemistry, quantitative RT-PCR, and in situ hybridization. Furthermore, genomic DNA was analyzed by PCR for possible gene amplification. Immunohistochemical analysis of formalin-fixed, paraffin-embedded sections of radical prostatectomy specimens was performed utilizing a polyclonal antibody against human procathepsin X and revealed staining of acinar basal cells in normal prostate glands. Prostatic intraepithelial neoplasias (PINs) and prostate carcinomas stained highly positive for cathepsin X, showing a significant difference to the staining of normal prostate glands. In contrast, relatively weak and heterogeneous staining was observed for cathepsins F, B, and L. Up-regulation of cathepsin X at the protein level was confirmed by Western blotting. No statistically significant difference was observed at the mRNA level. PCR of genomic DNA revealed that cathepsin X up-regulation most likely occurs in the absence of genomic amplification. The high expression levels of cathepsin X both in PIN and invasive adenocarcinomas of the prostate suggest that cathepsin X may play a role in the early tumorigenesis of prostate cancer. Further studies are needed to define the utility of this cysteine protease as a diagnostic marker for the early detection of prostate cancer.