Russell M. Lebovitz

p53 protein accumulation in Barrett's metaplasia, dysplasia, and carcinoma: A follow-up study

BACKGROUND There is a significant interobserver and intraobserver variation in grading dysplasia in Barretts metaplasia. New markers are needed to optimize the assessment of potential risk of cancer development in these patients. The aim of this study is to explore the use of p53 as a marker of neoplastic progression in Barretts metaplasia. METHODS Immunohistochemistry was used to study p53 protein accumulation in 114 specimens from 54 patients with Barretts metaplasia. RESULTS Positive staining was found in 0% of the cases negative for dysplasia, 9% of those with low-grade dysplasia, 55% of those with high-grade dysplasia, and 87% of those with adenocarcinoma. Follow-up was available on 24 patients. Two patients who showed low-grade dysplasia and who were positive for p53 on biopsy showed high-grade dysplasia in follow-up biopsies. Of 21 patients who had biopsy specimens negative of p53, only one showed high-grade dysplasia on subsequent biopsy specimens. CONCLUSIONS Our data support the hypothesis that p53 plays an important role in the progression of Barretts metaplasia to adenocarcinoma. The follow-up study indicates that positive immunostaining for p53 may be an objective marker of neoplastic progression in Barretts metaplasia.

Prostate Specific Antigen and Gleason Grade: An Immunohistochemical Study of Prostate Cancer

Prostate cancer is histologically heterogeneous as reflected in the 5 patterns of the Gleason grading system. Gleason grade correlates with volume, extent and prognosis. Serum prostate specific antigen (PSA) levels also correlate with tumor volume but the degree to which grade correlates with PSA has not been precisely defined. To quantify this relationship further, we prepared maps of each grade of cancer in 86 radical prostatectomy specimens from patients with clinical stage T2 cancer. The median per cent of the volume of cancer per prostate composed of grade 1 was 0%, while it was 1% for grade 2, 84% for grade 3, 5% for grade 4 and 0% for grade 5. We stained 95 cancer foci (grades 1 to 5) in 40 of these specimens for PSA. The presence and intensity (0 to 3+) of staining in more than 33,000 acini (or cells) correlated inversely with grade (p < 0.0001). Nearly all acini in grade 1 and most in grade 2 stained positive (2 to 3+) for PSA; 87% were positive but with less intensity in grade 3. While many grade 4 (79%) and grade 5 (49%) cells were positive, the intensity of staining was weak. Serum PSA levels correlated with total tumor volume (r = 0.67) but serum PSA levels per cm.3 of cancer decreased with increasing grade (r = -0.24 and p < 0.02). These studies confirm the strong inverse correlation between Gleason grade and the PSA content of prostate cancer. Since more than 85% of grade 3 acini stained for PSA and grade 3 made up the largest portion (84%) of cancer, the predominant contributor to serum PSA levels from prostate cancer was Gleason grade 3. The other grades contribute relatively little to the serum PSA levels either because of the small volume (grades 1 and 2) or the diminished PSA content (grades 4 and 5).

Coinjection strategy for visual identification of transgenic mice

Transgenic mice were generated by coinjection of a dominant marker gene that induces fur and eye pigmentation (a tyrosinase minigene) plus an unrelated DNA construction that has a γ-glutamyl transferase (γGT) promoter linked to aras oncogene. Mice transgenic for γGT-ras could be identified in the first and all subsequent generations by simple visual inspection for pigmentation. Furthermore, the γ-glutamyl transferase promoter was active in kidney but not skin of the transgenic mice, indicating that the cointegrated DNA was active and independently expressed. These results confirm that the tyrosinase minigene can be used for coinjections to allow rapid visual identification of transgenic mice.

p53 immunostaining in the differentiation of reactive processes from malignancy in pleural biopsy specimens

To determine the utility of positive p53 protein immunostaining as an adjunct in the diagnosis of malignancy in pleural biopsy specimens, we reviewed 73 recently obtained pleural biopsy specimens that represented the typical range of diagnoses encountered in the evaluation of a proliferative pleural process. Immunohistochemistry was performed on paraffin sections of each biopsy specimen using a monoclonal antibody to the p53 suppressor gene product clone BP53-12 (BioGenex, San Ramon, CA) and a standard capillary gap (Microprobe, Fischer Scientific, Pittsburgh, PA) avidin-biotin complex technique with a citrate buffer antigen retrieval solution. Of the pleural biopsy specimens with unequivocal malignancy, 19 of 40 mesotheliomas and nine of 18 metastatic adenocarcinomas were immunopositive for p53 protein. All 13 of the biopsy specimens with reactive mesothelial hyperplasia or organizing pleuritis were negative. Two pleural biopsy specimens, which were interpreted as suspicious but inconclusive for malignancy, were positive for p53 protein and subsequent pathology specimens confirmed the presence of metastatic carcinoma in both of these biopsy specimens. Our findings suggest that p53 protein immunostaining is relatively sensitive and highly specific in differentiating reactive processes from primary or metastatic malignancies in histopathologically equivocal pleural biopsy specimens.

Functional identification of LZTS1 as a candidate prostate tumor suppressor gene on human chromosome 8p22

Deletions in the 8p21-22 region of the human genome are among the most common genetic alterations in prostate carcinomas. Several studies in different tumor tissues, including prostate, indicate that there are probably multiple tumor suppressor genes (TSGs) present in this region. To identify candidate TSGs on 8p22 a YAC contig spanning this region was assembled and YAC clones retrofitted with a selectable marker (neo) were transferred into rat prostate AT6.2 cells. Two overlapping YAC clones showed greatly reduced colony-forming efficiency, indicating they may carry a TSG. Two BAC clones encompassing the overlapping region also appeared to exert suppressive effects on the growth of AT6.2 cells. Database searches for genes mapped to the critical region identified a gene known as FEZ1 (LZTS1) as a potential candidate suppressor gene. Subsequent experiments showed that over-expression of LZTS1 cDNA inhibited stable colony-forming efficiencies of AT6.2, HEK-293 and LNCaP cells. In contrast, LZTS1-transfected Rat-1 and RM1 cells were growth-stimulated. Database searches also identified additional isoforms of the LZTS1 mRNA, as well as LZTS1 protein domains reminiscent of those found in transcription factors. Together these data suggest that the LZTS1 gene is involved in the regulation of cell growth and its loss of function may contribute to the development of prostatic carcinomas, as well as other cancers.

p53 accumulation in benign breast biopsy specimens

Several studies of benign breast lesions using methacran-fixed, paraffin-embedded tissues and cytological preparations have suggested that p53 accumulation in these lesions as detected by immunohistochemical (IHC) staining is rare to absent. As a result, several different investigators have suggested that p53 immunoreactivity in breast specimens infers a diagnosis of malignancy or may identify premalignant lesions. We immunostained 271 breast biopsy specimens from 271 patients with the monoclonal anti-p53 antibody BP-53-12 and found positive nuclear staining in seven of 23 malignant lesions (30%) and 39 of 248 benign biopsy specimens (16%). Of the benign lesions, 30% of fibroadenomas, nonpremalignant breast lesions, were positive. Long-term follow-up information was available on 48 patients with benign biopsy specimens and showed that 12% of those positive and 7% of those negative for p53 developed breast carcinoma. This difference was not significant (P > .2). We conclude that (1) p53 immunoreactivity in breast lesions should not be used as exclusive evidence of malignancy and (2) p53 immunoreactivity in benign breast lesions may not identify a subset of patients at increased risk for breast carcinoma.

Cloning of cDNA and genomic structure of the mouse γ-glutamyl transpeptidase-encoding gene

Abstract We have isolated and characterized cDNA and genomic clones containing the coding region for the mouse γ-glutamyl transpeptidase (GGT). The sequences of the full-length cDNAs for three of the seven known mouse Ggt RNAs (types I, II and III) were determined and found to be identical in the coding region. Comparisons of the deduced amino-acid sequence of mouse GGT with that of rat and human reveal 95 and 79% overall identities, respectively. The mouse Ggt gene has 12 coding exons and spans approx. 12 kb. We have also re-analyzed rat genomic Ggt clones previously isolated by us and found that the rat and mouse genes share the same intron/exon boundaries. Our findings are of interest because they define the structure of the mouse and rat Ggt genes and will allow comparison with human GGT genes which, recent findings suggest, have diverged substantially from rodents.