RB2/p130 gene-enhanced expression down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in vivo.

Angiogenesis is an essential step in the progression of tumor formation and development. The switch to an angiogenetic phenotype can occur as a distinct step before progression to a neoplastic phenotype and is linked to genetic changes such as mutations in key cell cycle regulatory genes. The pathogenesis of the angiogenetic phenotype may involve the inactivation of tumor suppressor genes such as the "guardian of the genome," p53, and the cyclin-dependent kinase inhibitor p16. Retinoblastoma family member RB2/p130 encodes a cell cycle regulatory protein and has been found mutated in different tumor types. Overexpression of RB2/p130 not only suppresses tumor formation in nude mice but also causes regression of established tumor grafts, suggesting that RB2/p130 may modulate the angiogenetic balance. We found that induction of RB2/p130 expression using a tetracycline-regulated gene expression system as well as retroviral and adenoviral-mediated gene delivery inhibited angiogenesis in vivo. This correlated with pRb2/p130-mediated down-regulation of vascular endothelial growth factor protein expression both in vitro and in vivo.

Mutations in the retinoblastoma-related gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by retrovirus-mediated gene transfer.

The retinoblastoma (Rb) family consists of the tumor suppressor pRb/p105 and related proteins p107 and pRb2/p130. Recent immunohistochemical studies of the retinoblastoma family of proteins in 235 specimens of lung cancer show the tightest inverse association between the histological grading in the most aggressive tumor types and pRb2/p130. This led us to study a panel of human lung cancers for mutations in the RB2/p130 gene. Mutations in the Rb-related gene RB2/p130 were detected in 11 of 14 (78.5%) primary lung tumors by single-strand conformation polymorphism and sequence analysis. A Moloney leukemia virus-based retroviral system was set up, and a comparable viral concentration of 1 x 10(7) infectious units/ml was obtained. Retrovirus-mediated delivery of wild-type RB2/p130 to the lung tumor cell line H23 potently inhibited tumorigenesis in vitro and in vivo, as shown by the dramatic growth arrest observed in a colony assay and the suppression of anchorage-independent growth potential and tumor formation in nude mice. The tumors transduced with the RB2/p130 retrovirus diminished in size after a single injection, and a 12-fold reduction in tumor growth after RB2/p130 transduction compared with the Pac-transduced tumors (92% reduction, P = 0.003) and lacZ-transduced tumors (93% reduction, P < 0.001) was found to be statistically significant. These findings provide the missing confirmation that RB2/p130 is a "bona fide" tumor suppressor gene and strengthen the hypothesis that it may be a candidate for cancer gene therapy for lung cancer.

Expression of cell-cycle-associated proteins pRB2/p130 and p27kip in vulvar squamous cell carcinomas.

Squamous cell vulvar carcinoma accounts for 4% of all gynecologic malignancies. The cause of vulvar cancer is still unclear. Identification of new biologic factors involved in vulvar carcinogenesis may be useful in clarifying the natural history of this malignancy. We investigated the immunohistochemical expression of the retinoblastoma-related proteins pRB2/p130 and CKI p27kip1 in a series of 51 invasive squamous cell carcinomas of the vulva (ISCCs) and in synchronous normal vulvar skin, non-neoplastic epithelial disorders (NNED) and vulvar intraepithelial neoplasia (VIN). Normal vulvar skin staining showed positivity for both pRB2/p130 and p27kip1. Loss of pRB2/p130 occurred in 29 (57%) of 51 specimens of ISCCs, and in 1 of 7 specimens with VIN (14%; P = .04). We also observed a significant decrease of pRB2/p130 expression from NNED to neoplastic tissues (VIN and ISCCs) (P = .004). Loss of p27kip1 expression was found in 16 of 51 specimens (31%) of invasive carcinomas, in 1 (14%) of 7 specimens of VIN, and in 2 of 18 specimens of NNED (11%). pRB2/p130 and p27(kip1) did not correlate significantly with any of the clinicopathologic parameters examined. Our data indicate that loss of pRB2/p130 and p27kip1 are frequent events in invasive vulvar carcinomas compared with synchronous premalignant lesions, non-neoplastic epithelial disorders, and normal vulvar skin. The significant progressive decrease of pRB2/p130 expression from non-neoplastic epithelial alterations through intraepithelial neoplasia to invasive vulvar carcinomas suggests a role for this tumor suppressor gene in vulvar carcinogenesis.

Villous adenoma arising in Meckel's diverticulum.

The first well documented case of villous adenoma arising in a Meckel's diverticulum not associated with a carcinoma is reported. A 21 year old man with long history of medically treated ulcerative colitis was admitted to hospital with severe pain and bleeding. Total abdominal colectomy and ileo-anal anastomosis was performed and during this procedure Meckel's diverticulum containing a villous adenoma showing minimal dysplasia was found and resected. The diverticulum was lined partly by ileal and partly by gastric epithelium, and the villous adenoma originated from the gastric mucosa.

Grading of upper urinary tract transitional cell carcinoma by computed DNA content and p53 expression.

OBJECTIVES: Transitional cell carcinomas of upper urinary tract (uttTCC) constitute 5% to 6% of all urothelial tumors. Ureteropyeloscopy has become the standard for clinical evaluation of uutTCC. Moreover, endoscopic treatments have been advocated as a conservative approach for low grade tumors or patients with intermediate grade tumors whose renal function is compromised. Therefore, grading has become the most predictive variable in defining therapeutic approach. In addition to morphologic evaluation, a series of biologic markers may be used to increase the accuracy of grading such as DNA analysis and p53 protein expression. In this study, we have evaluated these markers by means of cell image analysis with the SAMBA 400 system. METHODS: Thirteen cases of uttTCC were studied with cytologic smear, cell block, and histologic confirmation. DNA analysis was performed on cytologic smear. Immunostaining was performed on cell blocks. A grade was assigned on the basis of DNA evaluation and p53 expression quantitation. These grades were combined for each case and compared with the initial cytologic grading and the final histologic grading. RESULTS: Cytology alone diagnosed TCC in all but 1 case that was diagnosed atypical. Discrepancies were found in primary grading: cytologic grading concurred with histologic grading in 6 of the 13 cases. CONCLUSIONS: These results, although in a limited but selected number of cases, show the potential of computerized evaluation of biologic markers as parameters for a more objective grading of tumors.

The role of pRb2/p130 protein in diagnosing lung carcinoma on fine needle aspiration biopsies.

The retinoblastoma gene family is composed of three members: the retinoblastoma gene, one of the most studied tumor suppressor genes, and two related genes: p107 and pRb2/p130. These proteins are also known as the pocket proteins due to a unique structural and functional domain composed of subdomains A and B separated by a spacer region that is highly conserved among each of the proteins. These proteins exhibit unique growth suppressive properties that are cell type specific, suggesting that although the pocket proteins may complement each other, they are not fully functionally redundant. With the development of antibodies recognizing these three proteins it is now possible to detect expression in formalin-embedded specimens. Recent studies on 235 lung cancers, using immunohistochemical techniques, suggested an independent role for Rb2/p130 in the development and/or progression of human lung carcinoma. We found a statistically significant inverse relationship between the histological grading (degree of malignant potential) and the expression of pRb/p105, p107 and pRb2/p130 in squamous cell carcinomas, meaning that an increase in grading resulted in a significant decrease in protein expression. This phenomenon was particularly evident for pRb2/p130 (p < .0001) which had the highest percentage of undetectable levels in all the specimens examined and the tightest inverse correlation (p value) with both the histological grading and PCNA expression in the most aggressive tumor types, suggesting an important role for pRb2/p130 in the pathogenesis and progression of certain lung cancers. We further explored the expression of pRb2/p130 protein in routine archival FNAB cytological material from 30 Patients with lung cancer using immunocytochemical techniques, comparing protein expression with tumor type. Two pathologists evaluated the staining pattern and scored the percentage of positive cells. Of the 30 neoplasms, 27 displayed a positive staining for pRb2/p130. In particular, we detected pRb2/p130 in 9 (100%) squamous carcinomas, 11 (84%) adenocarcinomas, 5 (100%) BAC, and 2 (66%) SCC. The percentage of positive nuclei varied in different tumors with the highest expression level in adenocarcinomas. Immunocytochemistry represents a sensitive method for detection of pRb2/p130 expression in cytological or archival specimens, and the level of detection seems to be comparable to paraffin sections. Therefore, this methodology could be used in the preoperative evaluation of routine cytological specimens in order to improve the diagnostic and prognostic evaluation of lung cancer patients.

[The value of the Pap test in the diagnostic approach to HPV infection of the cervix uteri]. / Valore del Pap-test nell'approccio diagnostico dell'infezione da HPV della cervice uterina.

HPV infection of the low genital tract is the most diffuse STD world wide. For this reason it's necessary to follow a simple flow chart to reach a correct diagnosis and to practice an adequate therapy. The authors relate on their experience in the management of a group of patients they have observed in a STD clinic. In this group it was executed a diagnostic protocol including Pap test, in situ hybridization, dot blot and histologic examination on tissue biopsies. From the analysis of the obtained data, the authors state that the Pap test is very useful to start in the diagnostic protocol, for it is precise, inexpensive and allows to identify the patients to follow with further diagnostic procedures such as colposcopy and histologic examination, in order to reach an exhaustive diagnosis and to assess the proper therapy.

Belief network for grading prostate lesions.

A Bayesian belief network for grading prostatic lesions into eight primary Gleason grades was developed and tested. The network employs 13 diagnostic clues, 8 based on tissue architectural features and 5 based on nuclear features. For every diagnostic clue, three to five different outcomes are specified by membership functions. The network works in a robust fashion and attained agreement with consensus visual grading in 241 of 256 microscopic fields.

[Immunohistochemistry in cytologic diagnosis using fine needle aspiration]. / L'immunoistochimica nella diagnostica citologica per aspirazione con ago sottile.

The role of immunocytochemistry in fine-needle aspiration cytology. The Authors review the literature about fine-needle aspiration biopsy cytology in different organs and show the feasibility of the application to cytological specimen of immunocytochemical methods. Thus, they analyze 15 cases of tumors of various localization diagnosed by F.N.A.C. where immunocytochemistry succeeded in their correct typization. They also show the encouraging results of the immunocytochemical search of specific hormones in pituitary adenomas. Therefore the Authors emphasize the great usefulness of immunocytochemical techniques for the improvement of the diagnostic possibilities in the fine-needle aspiration cytology.

An algorithm for automatic tracking of nuclear boundaries.

An image segmentation algorithm, based on boundary tracking, was introduced to achieve automatic segmentation of nuclei. This will improve reliability and reproducibility for the computer-assisted grading of routinely stained material, especially from biopsies, which often offer only scanty clinical material. Nuclear grading systems using karyometric features were developed earlier. However, hematoxylin and eosin-stained tissues have proven difficult for automatic segmentation, which is a crucial part of an objective grading system. In this paper we describe an automatic tracking method that traces nuclear boundaries on the basis of edge information and local boundary features. There were two phases to the procedure. First, approximate boundaries were extracted by automatic thresholding; then, boundaries were refined through interactive tracking. The results are encouraging.

[Preoperative cytologic diagnosis of medullary carcinoma of the thyroid]. / La diagnosi citologica preoperatoria nel carcinoma midollare della tiroide.

The Authors emphasize the importance of preoperative aspiration cytology in the diagnosis of 5 medullary types out of 58 thyroid carcinomas observed in the Institute of Pathology of the Università Cattolica del S. Cuore of Rome in the period 1983-88. All the cases were diagnosed preoperatively by mean of a fine-needle aspiration cytological specimen; the case where morphological diagnosis was doubtful were studied with immunohistochemical techniques for calcitonin and CEA. Out of all cases, they report one case of multiple endocrine adenomatosis type II with medullary thyroid carcinoma and pheochromocytoma and another one with cervical node metastases without a clinically detectable thyroid swelling. Therefore, the authors propose the fine-needle aspiration cytology as a simple and important mean in the preoperative diagnostic procedure in case of medullary thyroid carcinoma.

Role of computed quantitation of immunohistochemical staining of Ki-67 antigen in diagnosing ampullary lesions.

OBJECTIVE: To determine the possible role of Ki-67 antigen expression by visual and computed quantitation in diagnosing ampullary lesions. STUDY DESIGN: Twenty-two cases of ampullary lesions treated at Thomas Jefferson University Hospital between 1989 and 1994 were analyzed. Four cases of adenoma, 4 of epithelial dysplasia in adenoma, 7 of well-differentiated adenocarcinoma and 7 of high grade adenocarcinoma were included. For each case three consecutive sections were obtained from the paraffin-embedded blocks. The first slide was stained with hematoxylin & eosin for visual diagnosis; the other two were immunoprocessed to evaluate the expression of Ki-67 antigen. Visual quantitation of Ki-67 was evaluated by light microscopy, and computed quantitation was performed utilizing the SAMBA 4000 cell image analysis system. RESULTS: Immunohistochemical analysis of the ampullary lesions showed a positive correlation of Ki-67 expression, both by visual and computed quantitation, with biologic grade. The cell proliferation sequence was carcinoma, adenoma with dysplasia and adenomia. CONCLUSION: Ki-67 antigen expression correlated highly with the progression of malignancy in ampullary lesions. Computed quantitation of Ki-67 was more sensitive than visual quantitation, especially in differentiating between low and high grade adenocarcinomas.

Use of computer graphic filters for the nuclear grading of hematoxylin and eosin-stained specimens from prostatic lesions.

An inexpensive workstation is being developed to assist pathologists in diagnosing routine hematoxylin and eosin-stained slides. A linear discriminant model was applied to karyometric features of prostate lesions, and a grade according to Mostofi was determined from the discriminant values. Twenty cases, five of hyperplasia and five each of carcinoma Mostofi grades I, II and III, for a total of 600 nuclei, were selected to train the model. Computer graphic filters were constructed from the discriminant values. Each segmented nucleus has a colored frame (the graphic filter) displayed around it. The color, determined from discriminant values and correlated with the grades, ranges from green for hyperplasia, yellow for low grade, orange for medium grade and red for high grade. An additional 20 cases, 5 of hyperplasia and 5 each of the Mostofi grades, for a total of 538 nuclei, were selected to test the graphic filter. Ninety-six percent of the hyperplasia nuclei were framed in green, 84% of low grade nuclei were framed in yellow, 90% of medium grade nuclei were framed in orange, and 89% of high grade nuclei were framed in red. These results indicate the potential of the color graphic filter to show the pathologist immediate and accurate visual information about the grade of a nucleus. This method may help with the difficult diagnosis of borderline lesions and may help in making the diagnosis from scanty biopsy material.

Importance of different nuclear morphologic patterns in grading prostatic adenocarcinoma. An expanded model for computer graphic filters.

The aim of this work was to continue the development of an interactive workstation for the nuclear grading of prostatic lesions by including a large range of nuclear patterns. A previous model was based on four groups: hyperplasia, Mostofi grade 1, Mostofi grade 2 and Mostofi grade 3. Each group included the most common nuclear patterns of the lesions. One set used to test the model included cases showing patterns different from the typical ones of the model. Poor results were obtained for low and medium grades. A review of all the cases in our database led to the conclusion that different nuclear patterns can belong to the same "nuclear grade." Thus, in this work the model was expanded to include six groups: hyperplasia, two subgroups for Mostofi grade 1, two subgroups for Mostofi grade 2 and Mostofi grade 3. A set of 900 nuclei, 150 in each group, was selected to test the model. An additional 300 nuclei, 50 in each group, were used for a test set. The overall success rate for classifying the nuclei in the test set using the new model was 93% as compared to a rate of 71% obtained for the similar test set, described above, using the previous model. Moreover, correlating karyometric features with nuclear morphology indicated a role for nucleoli in nuclear grading. The good results obtained with large and heterogeneous sets of cases indicate that the procedures used to develop this model may be adapted for the development of models for the nuclear grading of other tumors.

Value of the cribriformity factor in grading prostatic carcinoma.

Architectural and histometric features for the objective grading of prostate adenocarcinoma in histologic specimens were analyzed in five cases each of Gleason primary grades 2, 3A, 3B, 3C, 4A and 4B, selected as "typical" for the histopathologic images. Tissue sections from the selected cases were stained by the Feulgen method. Fifteen fields for each grade, for a total of 4,430 glands, were digitized by a video-based microphotometer at low resolution (pixel spacing of 2 microns). Outer and inner outlines of the glandular epithelium were traced manually using a mouse. For each field the number of glands, the gland area, the lumen area, the area of the glandular epithelium and the cribriformity factor were computed. The gland area and its variance proved to be useful indicators for lower-grade lesions, whereas the variance of cribriformity resulted in an excellent grading indicator in the Gleason 3-4 range when cribriform glands were present.

Morphologic and planimetric diagnosis of follicular thyroid lesions on fine needle aspiration cytology.

During 1989-1992, 2,729 fine needle aspiration biopsies were performed; 585 with histologic controls were reviewed. The aim of the study was to demonstrate the incidence of carcinoma in follicular-structured smears without nuclear enlargement, evaluated with planimetric techniques, and to suggest a new cytodiagnostic classification. Of 398 follicular-structured smears, 188 were colloid nodules, 38 were thyreocytic hyperplasias without nuclear atypia, 146 were predominantly follicular lesions (PFL) and 26 were follicular lesions with nuclear pleomorphism. The last showed a high incidence of neoplasia (69.2%) and carcinoma (46.1%) and the largest planimetric values for nuclear area, perimeter and maximum diameter. The second and third categories showed only a difference in the incidence of benign neoplasms (32.9 vs. 15.8%). These results suggest that six months of expectant management might be useful in simple follicular lesions, whereas a follicular pattern with nuclear enlargement requires surgical treatment for the strong possibility of carcinoma.

Role of planimetric analysis in diagnosing thyroid follicular lesions on fine needle aspiration biopsies: a study with histologic correlation.

BACKGROUND: Follicular lesions represent a gray area of interpretation in fine needle aspiration biopsy (FNAB) of the thyroid, with as much as 25% inconclusive reports. We identified "predominantly follicular lesions" (PFLs) as the cytologic category most apt to take advantage of planimetric analysis to reach a more definitive diagnosis. STUDY DESIGN: Sixty-eight cases of FNAB were diagnosed as PFL among the 1,296 FNABs submitted to our institution between January 1994 and June 1995. These cases underwent planimetric analysis with a Leica semiautomatic image analyzer. A smear from a colloid nodule was used as a reference slide. Nuclear areas, perimeters, form factors and maximum diameters were evaluated. Cases in which nuclear areas and maximum nuclear diameters values were found to be > or = 30% higher than the corresponding values found in the reference slide were reported to the clinician as suspicious for malignancy ("flagged" by the computer). These cases required closer follow-up with repeat FNAB within a month, ultrasound and nuclear imaging studies. Nineteen of these cases underwent surgical resection. RESULTS: Histologic reports diagnosed 9 cases of follicular carcinomas, 4 cases of follicular adenomas and 6 cases of nodular hyperplasia. When nuclear areas, perimeters and maximum diameters were all utilized, all the malignant lesions were reported correctly by the computer analysis as flagged, and all the benign lesions were reported as "not flagged." The sensitivity and specificity were 100%, and statistically significant correlations were proven. CONCLUSION: Although the above data provide strong evidence for the value of planimetric analysis in differentiating between follicular lesions, we cannot reach definitive conclusions on the basis of such a limited number of cases. However, the results stimulated our current efforts in applying planimetry along with the evaluation of other biologic markers to a larger set of cases.

Whole liver intraoperative irradiation after partial hepatectomy produces minimal functional and pathologic lesions.

Intraoperative radiation therapy (IORT) was delivered to remnant rat liver after partial hepatectomy to determine the chronic effects of treatment on survival, blood chemistry, liver weight, and histology. Survival at one year was 100%. Remnant liver weight was markedly increased in all animals. Liver function appeared to be unaltered in all groups and at all observation times. Inflammatory cell infiltration occurred immediately after treatment in all animals, showing a slight progression until day 45; by day 180 the values had returned to baseline. Vascular changes were seen early in all groups, then progressively decreased; the vascular score was back to baseline at days 180 and 365. Nuclear alterations were observed in both irradiated and nonirradiated hepatic cells; in all cases these were limited to isolated or focal areas of hepatocytes. There was little fibrosis formation and by day 180 all scores were back to baseline. We conclude that the chronic effects of whole liver IORT after one-third hepatectomy are minimal in the rat and are similar to those observed after surgery alone.

Chromatin texture features in hematoxylin and eosin-stained prostate tissue.

A pilot study was undertaken to determine the expression of certain nuclear features in prostatic lesions. Twenty cases, 5 of hyperplasia and 5 each of carcinoma, Mostofi grades I-III, were selected as a training set, and an additional 20 cases were used as a test set, including 5 cases of hyperplasia and 5 cases each in Mostofi grades I-III. Images of hematoxylin and eosin-stained, 4-microns paraffin sections were obtained with a JVC BY-110 three-color camera and digitized by an IBM personal computer with a Matrox MVP-AT/NP imaging board. Thirty nuclei for each case from the training set, for a total of 600 nuclei, and 10 nuclei for each case from the test set, for a total of 200 nuclei, were analyzed by quantitative cytometric software on a SUN 3/60 workstation. A linear discriminant model was used for statistical analysis. One hundred percent of the hyperplasia group, 98% of the low grade group, 92% of the medium grade group and 82% of the high grade group were classified correctly in the test set with an overall success rate of 93%. Statistically significant chromatin texture features included heterogeneity, condensation, margination, run length nonuniformity, long run emphasis, gray level nonuniformity and inertia. Area, roundness and staining intensity (total extinction) were also significant. The results with standard hematoxylin and eosin-stained tissue sections were similar to those previously obtained with Feulgen-stained material. These results indicate that routine hematoxylin and eosin-stained material offers consistent diagnostic clues.