Russell B. Myers

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. Int. J. Cancer 74:346–358, 1997.

THE EFFECT OF ISOLATED SOY PROTEIN ON PLASMA BIOMARKERS IN ELDERLY MEN WITH ELEVATED SERUM PROSTATE SPECIFIC ANTIGEN

PURPOSE We performed a randomized double-blind crossover pilot study in elderly men with elevated prostate specific antigen (PSA) on the effects of the daily consumption of 2 soy beverages, each containing 20 gm. of isolated soy protein, on the isoflavone concentration in blood and urine, and on the 3 serum biomarkers cholesterol, PSA and the soluble p105 component of the p185erbB-2 proto-oncogene. MATERIALS AND METHODS A total of 34 men supplemented their diet by consuming 1 of 2 soy protein beverages assigned randomly twice daily for a 6-week period. In a second 6-week period they consumed the other soy protein beverage. The beverage ISP+ provided 42 mg. of genistein and 27 mg. of daidzein daily, whereas the other beverage, ISP-, provided only 2.1 and 1.3 mg. of these isoflavones daily, respectively. Blood and 24-hour urine samples were obtained before the study, at 2-week intervals during the study and 2 weeks after study completion. RESULTS ISP+ and to a lesser extent ISP- substantially increased the serum concentration and urinary output of the isoflavones and their metabolites. Serum cholesterol was significantly decreased by ISP+ irrespective of the order in which the 2 soy beverages were administered and in apparent correlation with the total isoflavone concentration. There was no significant effect of the soy beverages on serum PSA and p105erbB-2 values. CONCLUSIONS This study reveals that short-term exposure of elderly men with elevated serum PSA values to soy protein containing isoflavones decreases serum cholesterol but not the serum biomarkers PSA and p105erbB-2.

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.

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 Bouins 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.

Introduction to the Theory and Practice of Fixation of Tissues

Abstract Many approaches to fixation and types of fixatives have been developed and tested over the last-century. The mechanisms by which fixatives act to harden and preserve tissues fall into broad categories, including dehydrants, heat effects, cross-linkers, effects of acids, and combinations of these categories. Each fixative has advantages and disadvantages, including specific molecules retained within “fixed” tissues, swelling or shrinkage of fixed tissues, variations in the qualitv of histochemical and immunohistochemical staining, and varying capabilities to maintain the structures of cellular organelles. One of the major problems with formaldehyde type (cross-linking) fixatives has been the loss of antigen immunorecognition; correcting this usually requires some method of antigen recovery. Similarly, the extraction of mRNA and DNA from formalin fixed tissue in paraffin blocks is problematic. All widely used fixatives are selected by compromise—good aspects are balanced against less desirable features. This article discusses the basics of fixation and provides the formulas for the fixatives currently used in pathology, histology, and anatomy and discusses good and bad aspects of specific fixatives. (The J Histotechnol 24:173, 2001)

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

Fatty acid synthase (FASE) is required for fatty acid synthesis. Elevated levels of FASE have been observed in a variety of malignancies.

Biomarker expression in prostatic intraepithelial neoplasia.

OBJECTIVE This study was conducted to gain a better understanding of the underlying cellular events involved in the development of prostatic intraepithelial neoplasia (PIN) and to clarify the relationship of PIN to invasive prostatic adenocarcinoma (PCa). METHOD This article reviews previous studies from our laboratory and others of biomarker expression in PIN and PCa. RESULTS The development of PIN is characterized by increased expression of several biomarkers which may influence the proliferative potential of the dysplastic cells. Increased expression of the growth factor receptors P185erbB-2, p180erbB-3, as well as the product of the c-met proto-oncogene is frequently detected in the dysplastic luminal cells as well as malignant cells of the prostate. Likewise, the expression of the nm-23H1 gene product is strongly expressed in dysplastic and malignant cells. Increased proliferative potential of the dysplastic cells is directly reflected by increased expression of PCNA. In contrast to the enhanced expression of the biomarkers associated with proliferation, decreased expression of prostate-specific antigen (PSA), prostatic acid phosphatase (PAP) and Leu 7 by dysplastic luminal cells is indicative of an impairment of the process of cellular differentiation. Aberrant glycosylation as well as the inappropriate expression of glycosylated tumor antigens is demonstrated by enhanced binding of the lectin Ulex europaeus and increased expression of tumor-associated glycoprotein 72 (TAG-72) and the Lewis Y antigen in dysplastic and malignant cells. Finally, enhanced expression of proteolytic enzymes such as cathepsin D and the 72-kD form of collagenase IV by dysplastic cells may represent an integral event in the development of invasive PCa. CONCLUSION The studies described in this review clearly demonstrate phenotype similarities of PIN to invasive PCa and furthermore support the concept that PIN represents a preinvasive lesion.

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.

Evaluation of biomarker modulation by fenretinide in prostate cancer patients.

An NCI-sponsored, phase II trial of N-(4-hydroxyphenyl)- retinamide (4-HPR) in patients with organ-confined prostate cancer in the period prior to radical prostatectomy was carried out. Thirty-seven men with the histologic diagnosis of prostate cancer planning to have radical prostatectomy entered the study after informed consent and were given 4-HPR (or matching placebo) as a single daily dose (two 100-mg capsules of 4-HPR or two capsules of placebo daily) for 3 weeks prior to surgery. Four men dropped out for unrelated reasons. Thirty-three men completed the study. At the time of surgery, repeat biopsies of the prostate were performed to study the effects of the drug on potential surrogate endpoint biomarkers (SEBs) of malignancy within the tissue. The panel of potential SEBs of malignancy include p53, cytomorphometric indices, ploidy, PNCA, erbB-2, erbB-3, EGF receptor, TGF-α tumor-associated glycoprotein-72, fatty acid synthetase and Lewis Y antigen. Twenty-three patients had matching pre- and posttherapy lesions and were considered informative. Results from the patients indicate significant differential expression of biomarkers in pretreatment specimens of uninvolved prostatic tissue (normal-appearing epithelia) prostatic intraepithelial neoplasia (PIN) and prostate cancer. The mean erbB-2 expression was 0.58 in uninvolved vs. 1.04 in PIN (p = 0.002); while the mean erbB-2 expression was 1.35 in prostate cancer (p = 0.0007, uninvolved vs. prostate cancer). A similar pattern of increased biomarker expression between uninvolved and PIN or prostate cancer tissues can be observed for EGF receptor (mean = 1.21, 1.87 and 1.76 for uninvolved, PIN and prostate cancer, respectively) and erbB-3 (mean = 0.81, 1.59 and 1.30 for uninvolved, PIN and prostate cancer, respectively). There were no statistically significant differences in biomarkers observed in the 4-HPR-treated patients when compared with placebo-treated control patients. There was a posttreatment up-regulation of biomarkers observed in both groups of patients. This observation is most likely explained by an effect due to the diagnostic sextant biopsy equally affecting both groups of patients. Results from this study do not demonstrate a chemoprevention effect of 4-HPR on tissue-based SEBs at the dose given.

Elevated serum levels of p105erbB‐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.