The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line, LNCaP.

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10(-8) M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.

Fatty acid synthase: an early molecular marker of progression of prostatic adenocarcinoma to androgen independence.

PURPOSE: Changes in fatty acid synthase levels were investigated in the CWR22 xenograft model of prostatic adenocarcinoma after castration and during progression to androgen independence. MATERIALS AND METHODS: Male nude mice with CWR22 tumors were castrated and sacrificed 3, 7, 21, 28 or 42 days after castration. Some mice received testosterone replenishment 21 days after castration and were sacrificed 7 days later. In addition, other castrates were maintained for 7 to 10 months to allow tumors to relapse to androgen independence. Fatty acid synthase was measured by immunohistochemical study and Western blot analysis. RESULTS: Fatty acid synthase decreased rapidly after castration and after 28 to 42 days it was 5% to 10% of the level in tumors of intact controls. Temporal changes in fatty acid synthase after castration were associated with decreased proliferative potential and increased levels of the cyclin dependent kinase inhibitor p27Kip-1. Testosterone treatment of castrates at 21 to 28 days after castration increased fatty acid synthase expression to the level in tumors of intact controls. Tumors of long-term castrates with post-castration androgen independent growth had increased fatty acid synthase, as did small focal areas of androgen independent proliferation in tumors that had not increased in size by 7 to 10 months. In relapsed CWR22 tumors and in focal areas of androgen independent proliferation in nonrelapsed CWR22 tumors increased fatty acid synthase was associated spatially with increased proliferation and decreased p27Kip-1. CONCLUSIONS: Fatty acid synthase in CWR22 tumors depends initially on testosterone and it is associated with androgen mediated proliferation. Furthermore, increased fatty acid synthase levels associated with androgen independent proliferation may represent an early event in the development of the androgen independent phenotype.

Ep-Cam levels in prostatic adenocarcinoma and prostatic intraepithelial neoplasia.

PURPOSE: The monoclonal antibody (mAb) 323/A3, a second generation high affinity antibody of the 17-1A antibody family, recognizes a 40 kDa transmembrane glycoprotein that has been referred to as Ep-CAM, 17-1A recognized antigen, or EGP40. While Ep-CAM is expressed on the basolateral surface of a variety of epithelia, the strongest expression is frequently detected among several types of carcinoma. In this regard, Ep-CAM may be useful in therapy, in diagnosis, and/or in prognosis. We examined the distribution of Ep-CAM in normal, dysplastic, and malignant prostatic epithelium. MATERIALS AND METHODS: Paraffin sections of prostate tissue from 76 patients with clinically localized (pT2) prostatic adenocarcinoma were immunostained with mouse mAb 323/A3 using the avidin-biotin horseradish peroxidase method. RESULTS: Within benign prostatic epithelium, immunoreactivity typically was low and frequently was restricted to the luminal cells. In contrast, moderate to strong immunostaining was detected frequently in the luminal cells of high grade prostatic intraepithelial neoplasia (PIN). Furthermore, strong immunostaining usually was detected in the cells of adenocarcinomas. The immunostaining in PIN (p<0.0001) and in adenocarcinoma (p<0.0001) was significantly greater than that observed in the normal epithelium. Expression of Ep-CAM did not vary significantly with the Gleason score of tumors or the clinical outcome of patients. Expression of Ep-CAM was demonstrated also in the malignant prostatic cell lines LNCaP, DU145 and PC3 using immunohistochemistry and an immunoblot technique. CONCLUSIONS: These findings suggest that increased levels of Ep-CAM represent an early event in the development of prostatic adenocarcinoma.

Constrictive pericarditis: clinical and pathophysiologic characteristics.

Constrictive pericarditis is an uncommon disorder with various causes. Although most often idiopathic, it may also occur after cardiovascular surgery, radiation therapy, and tuberculosis, especially in developing countries. The encasement of the heart by a rigid, nonpliable pericardium results in characteristic pathophysiologic effects, including impaired diastolic filling of the ventricles, exaggerated ventricular interdependence, and dissociation of intracardiac and intrathoracic pressures during respiration. Constrictive pericarditis typically presents with chronic insidious signs and symptoms of predominantly systemic venous congestion. Notoriously difficult to diagnose and distinguish from restrictive cardiomyopathy (RCM), the use of cardiac catheterization, echocardiography (transthoracic and transesophageal), central venous (hepatic and pulmonary) and transvalvular Doppler measurements, and magnetic resonance imaging should secure the diagnosis in most cases, eliminating the need for diagnostic thoracotomy. Although medical treatment may temporarily alleviate symptoms of heart failure, patients do poorly without pericardiectomy.

Androgenic regulation of growth factor and growth factor receptor expression in the CWR22 model of prostatic adenocarcinoma.

The effects of androgen manipulation on epidermal growth factor (EGF) receptor, p185erbB-2 and transforming growth factor-alpha (TGF-alpha) levels were examined in prostatic adenocarcinoma. Male nude mice were inoculated with the CWR22 androgen-dependent human prostatic tumor or an androgen-independent (CWR22R) derivative. Mice with CWR22 tumors were castrated and subsequently killed at 3, 7, 21, 28 or 42 days post-castration. Other CWR22-bearing mice received s.c. testosterone pellets at 21 days post-castration and were killed 7 days later. EGF receptor, p185erbB-2 and TGF-alpha levels were examined by immuno-histochemistry. Strong EGF receptor and p185erbB-2 immunostaining was detected in CWR22 tumors from intact controls. EGF receptor immunostaining decreased by 65% to 70% at 21 to 42 days post-castration. Testosterone treatment at 21 to 28 days post-castration resulted in a 2-fold increase in EGF receptor immunostaining. p185erbB-2 immunostaining within CWR22 tumors did not decrease following castration and, in fact, was slightly increased at 7 days post-castration. The effects of castration on EGF receptor and p185erbB-2 levels were confirmed by Western blot analysis. Fewer than 10% of CWR22 tumor cells demonstrated strong TGF-alpha immunostaining, and androgen manipulation did not effect TGF-alpha immunostaining. In contrast, 30% of androgen-independent CWR22R tumor cells were strongly immunostained for TGF-alpha. Our findings indicate that EGF receptor levels, but not p185erbB-2 levels, are strongly dependent on testosterone in CWR22 tumors. The co-localization of TGF-alpha and the EGF receptor in CWR22R tumors suggests that these factors may constitute an autocrine pathway that regulates androgen-independent growth.

Fatty acid synthase expression is increased in neoplastic lesions of the oral tongue.

BACKGROUND: Fatty acid synthase (FASE) is required for fatty acid synthesis. Elevated levels of FASE have been observed in a variety of malignancies. METHODS: We examined the expression of FASE in 56 primary squamous cell carcinomas (SCC) of the tongue using immunohistochemistry (IHC) with a monoclonal antibody to FASE. RESULTS: Immunoreactivity was low in histologically normal epithelium (0.42 +/- .07, n = 43), moderate in mildly dysplastic epithelium (1.41 +/- 11, n = 40), and strong in SCC of the tongue (1.64 +/- 10, n = 50). Both mild dysplasia and SCC stained more strongly than histologically normal epithelium (p<0.00001). Well-differentiated tumors showed increased immunoreactivity when compared to less well-differentiated tumors (p=0.044). Decreased overall survival was observed among patients with tumors with low immunoreactivity (p = 0.04). CONCLUSIONS: Increased expression of FASE in dysplasia and squamous carcinomas of the oral tongue may be an indicator of both differentiation and early neoplastic change. FASE expression may be useful diagnostically, prognostically, and as a potential target for therapy.

Changes in cyclin dependent kinase inhibitors p21 and p27 during the castration induced regression of the CWR22 model of prostatic adenocarcinoma.

PURPOSE: The expression of the cyclin dependent kinase inhibitors p21 and p27 was examined in prostatic adenocarcinomas following castration. MATERIALS AND METHODS: Male nude mice inoculated with the androgen dependent human prostatic tumor CWR22 were castrated when the tumors reached a volume of 0.8 to 1.1 cm.3 and were sacrificed at 3, 7, 21, 28 and 42 days post-castration. An additional group of mice received a subcutaneous testosterone pellet at 21 days post-castration and was sacrificed at 28 days post-castration. The expression of the Ki-67 antigen, p21 and p27 was examined by immunohistochemistry. RESULTS: The mitotic rate as well as the number of Ki-67 antigen positive cells decreased to 3% of intact control values by 7 days post-castration and were less than 0.01% of intact control values at 21, 28 and 42 days post-castration. The percentage of p21 expressing cells decreased from 15+/-2% in intact controls to less than 1% by 42 days post-castration. In contrast, the percentage of cells that expressed p27 increased from 25+/-3% in intact controls to 51+/-8% at 3 days post-castration and to 80 to 95% at days 7, 21, 28 and 42 days post-castration. Testosterone treatment from 21 to 28 days post-castration resulted in an increase in Ki-67 antigen positive cells to 200% of intact controls and a concomitant reduction in p27 expressing cells to about 50% of intact controls. Castration-induced changes in p27 expression were not observed in the CWR22R tumor, a transplantable relapsed derivative of the CWR22 tumor. CONCLUSION: These findings suggest that p27 expression is regulated negatively by androgens and that increased expression of p27 in CWR22 xenografts may be involved in the suppression of proliferation following castration.

Prehospital management of acute myocardial infarction: Electrocardiogram acquisition and interpretation, and thrombolysis by prehospital care providers.

OBJECTIVE: To review prehospital management of patients with suspected ST elevation acute myocardial infarction (AMI) based on the acquisition and interpretation of electrocardiograms (ECGs), and the effects of thrombolytic therapy initiated by prehospital care providers. DESIGN: MEDLINE was searched by combining the search phrases 'thrombolysis,' 'paramedics' and 'myocardial infarction' to identify all pertinent articles. The bibliographies were reviewed to search for other relevant articles. RESULTS: The earlier that treatment is initiated in AMI, the better the prognosis. Multiple randomized and nonrandomized trials indicate that prehospital care providers (including paramedics, nurses and doctors) are able to acquire prehospital ECGs with negligible increases in on-scene time, ranging from 30 s to 7 mins. With minimal training, they are capable of accurately interpreting ECGs and diagnosing ST elevation AMI, with results comparable with control ECGs obtained by physicians. Numerous studies have investigated the role of specially trained prehospital personnel in initiating thrombolysis. Trials outside of North America have predominantly used physicians, whereas North American studies employed paramedics. Thrombolysis has been shown to be safe and effective when started outside the hospital by physicians or paramedics, with a reduction in time to treatment and no increase in complications. The further a patient with ST elevation AMI is from hospital, the greater the potential benefit of prehospital thrombolysis. The European Myocardial Infarction Project (EMIP), the largest randomized trial of prehospital thrombolysis, demonstrated a trend towards reduced mortality but was underpowered to detect significant mortality differences. The Grampian Region Early Anistreplase Trial (GREAT), a rural study, is the only randomized trial to demonstrate a statistically significant mortality difference in patients receiving prehospital thrombolysis. Despite trends in favour of prehospital diagnosis and treatment of AMI, no urban study has been sufficiently powered to demonstrate mortality benefits. CONCLUSION: Prehospital treatment of patients with chest pain using ECGs and thrombolysis is safe. Though rural patients have significant reductions in total mortality when treated with thrombolysis in a prehospital setting, this has not been documented with an urban population. Prehospital identification of thrombolysis-eligible patients with ST elevation AMI via acquisition and interpretation of ECGs followed by triage to a hospital 'lytic team' has the potential to improve patient outcome and requires further investigation. A prehospital paramedic program for identifying and treating thrombolysis-eligible patients requires intensive planning, retrospective feasibility work, implementation and monitoring to establish effectiveness.

Cardiac murmur associated with an abnormal chest x-ray film.

Investigation of a cardiac murmur and thrill in an asymptomatic 35-year-old man revealed a foreign body lying within the left ventricular cavity. Upon questioning, the man revealed that he had been shot in the chest years earlier. This is the first reported adult case of a retained intracardiac bullet causing a ventricular septal defect.

Nuclear accumulation of p53 in colorectal adenocarcinoma: prognostic importance differs with race and location of the tumor.

BACKGROUND: Although several studies have been conducted to examine the role of p53 genetic abnormalities and their prognostic value in colorectal carcinoma, the incidence of nuclear accumulation of p53 and the prognostic importance of nuclear accumulation of p53 in African-American and white patients have not been investigated separately. Therefore, the authors evaluated the prognostic significance of p53 nuclear accumulation in these two racial groups. METHODS: Nuclear accumulation of p53 was evaluated immunohistochemically in archival tissue specimens from 204 African-American and 300 white patients with primary colorectal adenocarcinomas who had undergone surgery. Survival times from colorectal adenocarcinoma were analyzed using Kaplan-Meier survival estimates and the Cox proportional hazards model for nuclear accumulation of p53 with adjustments for other confounding demographic and clinical variables. RESULTS: Approximately equivalent proportions of distal (54%) and proximal adenocarcinomas (47%) were positive for nuclear accumulation of p53 in African-American patients. In contrast, distal colorectal adenocarcinomas from white patients more frequently were positive for nuclear accumulation of p53 than adenocarcinomas of the proximal colon (63% vs. 38%, respectively). Nuclear accumulation of p53 was found to be a strong predictor of poor survival in white patients (hazard ratio = 6.77; P = 0.0001) but not in African-American patients with primary adenocarcinomas of the proximal colon. Nuclear accumulation of p53 was not of prognostic value in patients of either race with primary adenocarcinomas of the distal colorectum. CONCLUSIONS: Nuclear accumulation of p53 is a valuable indicator of poor prognosis only for white patients with adenocarcinomas of the proximal colon. The current study also suggests that the role of p53 dysregulation in colorectal adenocarcinomas may vary with the anatomic location of the tumor and the race of the patient. These findings suggest that the demographic characteristics of patients should be considered in the evaluation of prognostic markers of colorectal neoplasia.

Prognostic significance of Bcl-2 expression and p53 nuclear accumulation in colorectal adenocarcinoma.

The products of bcl-2 and p53 genes are involved in the regulation of apoptosis and proliferation and have been associated with prognosis in several malignancies, including colorectal adenocarcinoma. Although 2 European studies have reported a prognostic significance of Bcl-2 expression in colorectal adenocarcinomas, a study from the United States did not observe such an association. Therefore, we used immunohistochemistry to evaluate the prognostic significance of Bcl-2 expression, p53 nuclear accumulation and their concomitant expression in 134 US patients with colorectal adenocarcinoma. Antigen retrieval was required for adequate detection of Bcl-2 expression. Fifty percent of the colorectal tumors were classified as expressing Bcl-2, and Bcl-2 expression was associated with longer patient survival. Antigen retrieval was not necessary for detecting nuclear accumulation of p53 by immunohistochemistry. Nuclear accumulation of p53 was detected in 44% of colorectal adenocarcinomas and was associated with decreased patient survival. Tumors that did not express detectable levels of Bcl-2 but exhibited nuclear accumulation of p53 were associated with the shortest patient survival (log rank, p = 0.001). Multivariate Cox regression analysis demonstrated that Bcl-2 expression (p = 0.018), p53 nuclear accumulation (p = 0.024) and regional lymph-node metastasis (p = 0.005) were independent prognostic factors. Although a trend toward an inverse correlation between Bcl-2 and p53 expression was observed, the prognostic value of Bcl-2 expression was independent of p53 status. Thus, assessment of both Bcl-2 and p53 status may be valuable in predicting the prognosis of patients with colorectal adenocarcinomas.

Changes in biomarker expression in the development of prostatic adenocarcinoma.

In spite of the prevalence of prostatic adenocarcinoma, the development and natural history of this malignancy is poorly understood. This paper reviews the current knowledge of biomarker expression during the development and progression of prostatic adenocarcinoma. Emphasis is placed on the comparison of biomarker expression in benign prostatic epithelium, intraepithelial neoplasia (PIN), a putative preinvasive lesion, and prostatic adenocarcinoma. Within the benign epithelium, the proliferative potential is restricted to the basal cells as demonstrated by the expression of proliferating cellular nuclear antigen (PCNA). The strong expression of the bcl-2 protein, an inhibitor of a apoptosis, supports the concept that the basal cells or a subpopulation of the basal cells represent the stem cell of the epithelium. In addition, the strong expression of growth factor receptors such as the epidermal growth factor receptor (EGFr), p185erbB-2, p180erbB-3, and c-met suggests that the growth of the basal cells is regulated by autocrine or paracrine factors. The luminal cells express secretory products such as prostate specific antigen and prostatic acid phosphatase, but demonstrate little expression of PCNA as well as growth factor receptors and proto-oncogene products. These observations are consistent with the theory that the luminal cell population is derived from the differentiation of the basal cells. In contrast to the normal epithelium, PCNA expression is frequently detected in the dysplastic luminal cells of the PIN lesion. Likewise, strong expression of p185erbB-2, p180erbB-3 and the c-met proto-oncogene product is also detected in the luminal cells of PIN lesions. Other factors which are strongly expressed by the dysplastic luminal cells include the nm23-H1 gene product, tumor associated glycoprotein-72 (TAG-72), fatty acid synthetase (FASE) and proteolytic enzymes. These findings suggest that PIN lesions are derived from an impairment of the differentiation of basal cells. The majority of biomarkers such as PCNA, p185erbB-2, P180erbB-3, TAG-72, nm23-H1 and FASE which are strongly expressed in PIN lesions are also expressed in prostatic adenocarcinoma supporting the concept that PIN is a preinvasive lesion. Mutations of the p53 tumor suppressor gene, as well as strong expression of transforming growth factor alpha and bcl-2 typically occur in advanced stage prostatic adenocarcinomas and therefore likely represent late events in the development of prostatic adenocarcinoma.

The use of biomarker expression to characterize neoplastic processes.

Biomarkers have been used by pathologists to aid the diagnosis of tumors for almost three decades. Their use has resulted in the re-evaluation and reclassification of several types of tumors. Currently, biomarkers are required to differentiate certain specific tumors with similar histologic patterns. Additional uses of biomarkers in the characterization of neoplastic processes are discussed including their use is prognosis, detecting early neoplastic processes, identifying tumor recurrence, measuring the effectiveness of various therapies (surrogate end point biomarkers), and identifying targets for novel therapies including immunotherapy and gene therapy. We propose that these newer uses of biomarkers will be just as important to pathology in the future as the uses of biomarkers in diagnosis have been over the past two decades.

Hormone levels and mammary epithelial cell proliferation in rats treated with a regimen of estradiol and progesterone that mimics the preventive effect of pregnancy against mammary cancer.

Women who bear their first child by their late teens have about half the risk of developing breast cancer relative to nulliparous women. The rat is a good model for studying the role of hormones in breast cancer since, for example, young rats become nearly refractory to mammary carcinogenesis after delivering a litter of pups. Short term administration of estradiol and progesterone (E & P) provides virgin rats protection from mammary carcinogenesis as effectively as pregnancy. The purpose of these studies were twofold: first, to evaluate potential long-term toxicity of the E & P treatments and second, to compare hormone treated rats and pregnant rats with respect to circulating E & P levels as well as mammary epithelial cell proliferation and differentiation. To test for toxicity, rats were treated with E & P (20 micrograms and 4 mg, respectively) or vehicle by s.c. injections 5 times per week for 5 weeks beginning at 40 days of age. The animals were weighed biweekly and sacrificed at 500 days of age when detailed necropsies were performed. No significant difference in weight gain was observed between the two groups nor was any toxicity grossly observable in the hormone-treated rats. Furthermore, there was no increase in the number of spontaneous mammary or pituitary tumors in the E & P treated group relative to controls. To evaluate serum hormone titers and mammary proliferation, rats were treated with steroids or vehicle daily beginning at 65 days of age. At 6 and 24 hours after the 1st, 14th and 35th injection, serum E & P were measured by RIA and mammary epithelial cell proliferation by immunohistochemistry (PCNA). At 6 hours after each injection, E & P levels were 3 to 5 fold those observed late in pregnancy. By 24 hours, however, E & P levels subsided to late pregnancy levels or lower. The mammary epithelial cell proliferation index in either E & P treated or late pregnant rats was 6 to 14%. Histologic sections and wholemounts of mammary glands showed a similar degree of differentiation between rats treated with E & P for 14 days or longer and late pregnant rats. These data further suggest that E & P treatments are a non-toxic means of mimicking the protective effect of pregnancy against mammary cancer and that pregnancy or hormone treatments may achieve this prophylaxis through a differentiation mechanism.

The effects of dihydrotestosterone on the expression of p185(erbB-2) and c-erbB-2 mRNA in the prostatic cell line LNCaP.

The c-erbB-2 proto-oncogene encodes a 185000 molecular weight protein (p185(erbB-2)) which shares structural homology with the epidermal growth factor (EGF) receptor. We examined the effects of dihydrotestosterone (DHT) on the expression of p185(erbB2) and c-erbB-2 mRNA in the human malignant prostatic cell line LNCaP. LNCaP cells grown in steroid-depleted media were treated with DHT (10(-11)-10(-6) M) for 48 h and p185(erbB-2) expression was determined by Western blotting and immunoprecipitation of 35S-methionine labelled p185(erbB-2). c-erbB-2 mRNA levels were determined using a competitive quantitative reverse transcription-polymerase chain reaction (RT-PCR) based technique. DHT at concentrations of 10(-9) M or greater resulted in decreased expression of p185(erbB-2). In contrast, DHT at these levels stimulated EGF receptor protein expression and cellular proliferation. c-erbB-2 mRNA levels declined to 30-50% of control levels following treatment with DHT of 10(-10) M or greater. Furthermore, the inhibitory effects on c-erbB-2 mRNA were rapid, occurring within 6-12 h of treatment. In summary, these results demonstrate that DHT, at concentrations that stimulate cell growth, inhibits the expression of p185(erbB-2) and c-erbB-2 mRNA.

Elevated serum levels of p105(erbB-2) in patients with advanced-stage prostatic adenocarcinoma.

Expression of a truncated or extracellular form (p105erbB-2) of p185erbB-2 has been demonstrated in the sera of breast cancer patients. We examined the levels of p105erbB-2 in the sera of patients with various stages of prostatic adenocarcinoma, in patients with benign prostatic hyperplasia (BPH) and in a series of control male patients hospitalized for illnesses unrelated to the prostate. p105erbB-2 levels did not differ between the controls and BPH patients or between these groups and patients with stage A, B or C adenocarcinomas. In contrast, serum p105erbB-2 levels of patients with stage D adenocarcinomas were significantly elevated when compared with either control or BPH patients. There was no correlation between PSA and p105erbB-2 levels among controls, patients with BPH or patients with prostate cancer. Patients with poorly differentiated tumors (combined Gleason score >7) or moderately differentiated tumors (combined Gleason score 5-7) had higher p105erbB-2 levels as compared to patients with well-differentiated tumors (combined Gleason score <5), though this difference was not statistically significant. There was no correlation between serum p105erbB-2 levels and p185erbB-2 expression in malignant tissue, as determined by immunohistochemistry. However, patients with moderate to strong expression of p185erbB-2 within the adenocarcinomas were approximately 4 times more likely to demonstrate elevated serum p105erbB-2 levels as compared with patients with low expression of p185erbB-2.

Expression of nm23-H1 in prostatic intraepithelial neoplasia and adenocarcinoma.

The product of the nm23-H1 gene has been reported to be related to the metastatic potential of several tumors. Although several studies have characterized the expression of the nm23-H1 gene product in prostatic adenocarcinoma, little is known of the expression of this protein in prostatic intraepithelial neoplasia (PIN), a putative precancerous lesion. The authors used immunohistochemistry to examine the expression of the nm23-H1 protein in PIN as well as benign and malignant prostatic tissue. A monoclonal antibody to nm23-H1 (Novocastra, Newcastle upon Tyne, UK, clone 37.6) and a biotin/strepavidin detection system were used for antigen localization. Weak to moderate immunostaining was consistently detected in the benign glandular epithelium of 28 radical prostatectomy specimens. In contrast, strong immunostaining was detected in the glandular epithelium in PIN lesions of 19 radical prostatectomy specimens examined. Strong immunostaining was also frequently detected in the malignant cells of 39 localized prostatic adenocarcinomas, as well as 7 metastatic lesions. These findings show a phenotypic similarity of PIN to prostatic adenocarcinoma with respect to nm23-H1 expression. Furthermore, strong expression of nm23-H1 likely represents an early event in the development of prostatic adenocarcinoma.

Effects of fixation and tissue processing on immunohistochemical demonstration of specific antigens.

Identification of biomarkers in archival tissues using immunochemistry is becoming increasingly important for determining the diagnosis and prognosis of tumors, for characterizing preinvasive neoplastic changes in glandular tissues such as prostate, for evaluating the response of tumors and preinvasive neoplastic changes to certain therapies (i.e., as a surrogate intermediate end point), for selecting patients who are candidates for specific therapies (e.g., immunotherapy) and for retrospective studies. For detecting specific biomarkers it is important to understand the limitations imposed by the fixation methods and processing of the tissues. This study was designed to determine the effects of fixation on the detection in archival paraffin blocks of selected antigens postulated to be important in tumor biology. We evaluated the antigens TGF alpha, p185erbB-2, broad spectrum keratins, p53, and TAG-72 (B72.3). Fixatives evaluated included standard preparations of neutral buffered formalin, acid formalin, zinc formalin, alcoholic formalin, ethanol, methanol, and Bouin's fixative. We found that in general neutral buffered formalin is the poorest fixative for maintaining antigen recognition by immunohistochemistry and that no single fixative was best for all antigens. The dehydrating (coagulant) fixatives (e.g., ethanol and methanol) preserved immunorecognition of p53 and broad spectrum keratins best while the slow cross-linking fixatives (e.g., unbuffered zinc formalin) were best for demonstrating TGF alpha and p185erbB-2. Fixatives other than neutral buffered formalin produced equivalent recognition of the epitope of TAG-72 by B72.3. In formalin fixed archival tissues, only a portion of the antigen signal can be detected by routine immunohistologic methods.

CD44 expression in dysplastic epithelium and squamous-cell carcinoma of the esophagus.

The molecular events involved in the development of esophageal dysplasia and carcinoma are poorly understood. We examined the expression of CD44, a cell-adhesion molecule, in normal and dysplastic epithelia and in squamous-cell carcinoma (SCC) of the esophagus. A monoclonal antibody (MAb) which recognized all CD44 isoforms and 2 MAbs specific to the CD44v3 and CD44v6 splice variants were used to detect CD44 isoforms in 50 archival specimens. A semi-quantitative scoring system based on the extent and intensity of the immunostaining was used to quantify CD44 expression. In normal epithelium, expression of CD44 was strongest in the basal-cell layer and weak or absent in surface cells. Expression of CD44 was increased in dysplastic epithelium as compared with normal epithelium. The extent of this increase correlated directly with the severity of dysplasia. CD44 was expressed in all SCCs, but the extent and intensity of immunostaining varied with areas of tumor differentiation. The well-differentiated components showed greater CD44 expression than the moderately and poorly differentiated components. The patterns of expression of CD44v3 and CD44v6 were strikingly similar to that of total CD44 in normal, dysplastic and malignant esophageal epithelia. Thus, changes in expression of these splice variants likely account to some extent for the changes in total CD44 expression observed in the dysplastic and malignant transformation of the esophagus. Our results suggest that changes in the expression of CD44 may be involved in the development of esophageal dysplasia as well as SCC.