Karyometric marker features in normal-appearing glands adjacent to human colonic adenocarcinoma.

The expression of nuclear marker features in normal-appearing tissue adjacent to colonic adenocarcinoma was investigated. Formalin-fixed, paraffin-embedded tissue sections of colon from 9 patients with adenocarcinoma and from 9 normal controls were cut 4 microns thick, Feulgen stained, and measured by a cell image analysis system using a Matrox MVP-AT/NP imaging board. Thirty nuclei in the tumor region, 30 nuclei 2 mm into the histologically normal-appearing distal margin, and the same number at 5, 10, 20, and 50 mm into the margin were measured for each patient. An additional 30 nuclei were recorded from 9 patients each free from colonic disease. Nuclear features were selected to discriminate between tumor nuclei and nuclei from normal control subjects and between nuclei measured in the histologically normal-appearing margin next to the tumor and control nuclei. Eight micromorphometric measures were found to be statistically significantly different in nuclei measured in the margin site, including features describing staining density (total absorbance, average absorbance 20% below mean, average absorbance 20% above mean) chromatin texture (cooccurrence matrix, run length, and peripheral tendency) and nuclear area. The category differences are statistically highly significant.

Evaluation of brush cytology as an independent technique for detection of esophageal carcinoma.

In this study the accuracy of indirect brush cytology for detection of esophageal carcinoma is evaluated against current standard methods of diagnosis and is compared with the known accuracy rate of endoscopically directed brush cytology. A standard endoscopic nylon brush placed inside a nasogastric tube was used in 203 patients with various esophageal problems. Correct diagnosis was made in 78% of cancers, 95% of potentially premalignant cases, and 100% of cases of normal esophageal mucosa with both indirect and directed brushing procedures. The technique meets most requirements of a new screening procedure as being simple, safe (no complications), relatively inexpensive, and acceptable to patients (98% compliance). Currently it is employed to monitor high-risk esophageal conditions and post-treatment courses of patients with pharyngoesophageal tumors for local recurrence or a new primary lesion in the esophagus.

Lectin binding in endometrial adenocarcinoma.

The relative increase in endometrial adenocarcinoma in women has increased the need for more objective criteria in the distinction of hyperplastic and neoplastic endometrium. The authors have used the ability of lectins to detect changes in surface glycoproteins to probe the differences among proliferative endometrium, endometrial adenomatous hyperplasia and adenocarcinomas. Paraffin-embedded sections of tissue obtained by Vakutage endometrial sampling technics were stained with each of 7 FITC lectin conjugates. Thirty-four specimens were examined (7 proliferative, 12 hyperplastic, and 15 adenocarcinomatous). Wheat germ agglutinin binding was detectable in all specimens with a distribution at the cell luminal border of glandular formations irrespective of diagnosis. However, adenocarcinoma cases showed distribution along the lumenal border and the cell periphery with loss of orientation of the lectin binding. Similar alterations and increased binding were noted for Concanavalin A. The WGA binding to sections was specifically inhibitable by oligosaccharides of N-acetyl-glucosamine. The results provide an objective criterion for detection of loss of cell orientation useful in the diagnosis of endometrial adenocarcinoma in tissue fragments.

Transcatheter bronchial brush and forceps biopsies. Histologic evaluation.

The histologic findings in transcatheter brush and forceps biopsies from 472 cases over a six-year period are evaluated. Diagnostic accuracy based on histologic findings was 38% and based on cytologic findings was 70%. When the two methods of examination were used, however, the overall accuracy was improved to 76%. In Hodgkin's disease and some inflammatory processes histologic examination is essential for the diagnosis. The transcatheter biopsies under fluoroscopic control are especially useful for securing tissues from the peripherally located lesions. When possible, examination of tissue obtained by transcatheter as well as forceps biopsies is encouraged.

DNA ploidy profiles as prognostic indicators in CIN lesions.

The prognostic significance of DNA ploidy measurements in cervical tissues was examined. Microphotometric measurements of 302 Feulgen-stained tissue sections (91 normal squamous epithelia, 14 condylomata, 29 cervical intraepithelial neoplasia (CIN) I, 78 CIN II, and 90 CIN III) were performed with a personal computer (PC)-based video microphotometry system. Analysis of these data shows that the DNA profile provides significant prognostic information: CINs with a polyploid DNA profile are more likely to return to normal than are those exhibiting an aneuploid pattern. Of 211 abnormal cases, 38% had polyploid DNA profiles and 62% were aneuploid. Eight-six percent of the cases that regressed were polyploid and 14% were aneuploid. Of the 130 aneuploid DNA cases, 95% remained static or progressed and only 5% regressed. Of these nonregressing aneuploid lesions, 90 remained static and 34 progressed, whereas within the nonregressing polyploid group 37 remained static and only 6 progressed. This result holds across diagnostic categories. Several other ploidy-related descriptors also showed prognostic significance (including mean ploidy, the 5N exceeding rate and 2N deviation index, and discriminant functions derived from order statistic analysis of the cumulative DNA histograms), but not to the degree or with the consistency of expression as the DNA profile categorization. These results indicate that important information about the prognosis of CIN lesions may be obtained by the DNA profile on Feulgen-stained tissue specimens. The data were acquired by a cytophotometry system of relatively modest cost consisting of readily available hardware components.

Correlation between visual clues, objective architectural features, and interobserver agreement in prostate cancer.

Three pathologists evaluated a number of designated architectural features to assign grades to 41 cases of well- to moderately differentiated adenocarcinoma, and their opinions were compared. The consensus opinion was obtained and evaluated against objective measurements of glandular architecture that were obtained by morphometric techniques. The observers agreed on gland size, gland uniformity, and the number of glands per field in only 49%, 31%, and 39% of cases, respectively. There were significant differences in the Gleason grades assigned by observers. Paired matching of individual Gleason grades showed agreement among observers in 44% (18 of 41), 56% (23 of 41), and 75% (31 of 41) of cases, respectively. This level of interobserver disagreement occurred even though cases with predominant patterns were selected carefully and those with variable patterns were excluded. A direct relationship appears to exist between increasing Gleason grade and increasing glandular variability, and there is an inverse relationship between Gleason grade, gland lumen area, and the number of glandular nuclei, as assessed by a group of pathologists.

Differentiation by TICAS analysis of cell populations of tracheal aspirates from hamsters with squamous-cell carcinoma.

Individual cells from the tracheal aspirates of hamsters exposed to benzo-a-pyrene were scanned at .5 mum in three colors. Features relating to size, shape, and color were extracted and calculated by computer. The single cells were then classified by these features into separate populations with varying degrees of atypia, extending up to frank cancer cells. A high degree of accuracy was attained in classification by these methods.

Study of viral integration of HPV-16 in young patients with LSIL.

AIMS: To investigate the physical status of human papillomavirus 16 (HPV-16) in low grade squamous intraepithelial lesions (LSILs) as a means of determining the percentage of viral integration. METHODS: Ninety two LSIL/HPV positive Thin Prep(TM) samples were initially tested for the E6 gene by the polymerase chain reaction (PCR) to identify the HPV-16 virus. To avoid false positive results, the specificity of the bands obtained from PCR was confirmed by Southern blot hybridisation with internal oligonucleotide probes. Next, a PCR screen for the E2 gene was performed to identify those samples in which the virus was integrated. Viral integration was detected in just over half of them. RESULTS: Twenty of the 92 samples were HPV-16 positive, as shown by PCR for the E6 gene. Southern blot analysis confirmed that 13 of these samples were positive for the viral E6 gene. Thus, viral integration was detected in just over a half of the samples positive for HPV-16. CONCLUSIONS: These data show that HPV-16 integration occurs in a subset of LSILs. The measurement of HPV-16 integration would be a helpful complementary tool for cytological evaluation in primary cervical screening to identify those patients at risk of developing high grade squamous intraepithelial lesions and cervical cancer.

Nuclear DNA analysis of insulinomas and gastrinomas.

The potential for malignancy of an islet cell tumor of the pancreas is difficult to cytologically judge when one evaluates only the primary lesion, because a malignant condition is usually determined by the presence of regional or distant metastases. Nuclear DNA cytometric measurements have proved helpful both in the evaluation of the malignant potential of other endocrine and nonendocrine lesions and in the determination of the "aggressiveness" of these tumors. Thirty-six islet cell tumors or their metastases from 25 patients were studied. Eleven patients had insulinomas and typical insulinoma syndromes, and 14 others had gastrinomas with the Zollinger-Ellison syndrome. Tissue from each tumor was stained by the Feulgen technique, and nuclear DNA cytometry was performed by means of the microTICAS system designed by the Cytopathology Laboratory of the University of Chicago. Ploidy measurements of insulinomas, taken alone, did not discriminate well between benign and malignant states. However, the single malignant insulinoma could be clearly recognized, for it was one of only two lesions in that group with 5N-exceeding rate (5N-ER) values of 1% or greater. (5N-ER is defined as the percentage of aneuploid nuclei with nuclear DNA content greater than 5N.) On the other hand, seven of eight malignant gastrinomas had ploidy values of 2.5N or greater (our definition of an aneuploid state) and/or had 5N-ER values of 1% or greater, while five of six benign gastrinomas had ploidy values of less than 2.5N and had 5N-ER values of 0%. In addition, the two most aggressive tumors had the highest ploidy and 5N-ER values. Nuclear DNA cytometric studies appear to offer promise as an aid in the evaluation of pancreatic islet cell tumors, particularly gastrinomas.

Follow-up study of male and female offspring of DES-exposed mothers.

This follow-up study presents the effects of DES on the genital tract of male and female offspring of mothers who were part of a double-blind, placebo-controlled investigation during 1951 and 1952 aimed at determining the effect of DES on pregnancy. Epididymal cysts, hypotrophic testes, and capsular induration were the more common genital lesions found in 25% of 163 DES-exposed males as compared to 6% in 168 control males. Semen analysis data on 39 subjects of the DES-exposed group and 25 subjects of the control group showed that 26% of the DES-exposed group produced an ejaculate volume under 1.5 ml; no such cases were observed in the control group. The average values for sperm density ant total motile spermatozoa per ejaculate, although in the normal range, were more than two times lower in the DES-exposed group as compared to the controls. A quality score of greater than 10 ("severely pathologic semen") was found in 28% of the DES-exposed group as compared to 0 in the control group. An association of pathologic semen quality with physical abnormalities was found only in the DES-exposed group. Two cases of azoospermia, one without genital abnormalities on physical examination and one with bilateral hypotrophic testes were observed so far in the DES-exposed group. Eighteen percent of 229 DES-exposed female patients had irregular menstrual cycles (oligomenorrhea) as compared to 10% of 136 controls. The history of pregnancy revealed a lower incidence of pregnancy in the DES-exposed group (18%) than in the control group (33%). Circumferential ridges of the vagina and cervix were seen in 40% of 229 DES-exposed females but in none of 136 controls. Colposcopic findings in the vagina revealed adenosis in 66.8% of the DES-exposed females and in 3.6% of the control group. Dysplastic lesions were more prevalent in the vagina and cervix of the DES-exposed subjects. No cases of cancer were observed in either the male or female offspring.

Cytologic diagnosis of occult breast lesions using stereotaxic needle aspiration. A preliminary report.

In mammographically detected breast lesions, only 10% to 25% of biopsy specimens are malignant. Furthermore, the current method of needle localization of these lesions is cumbersome and inefficient. Stereotaxic needle aspiration was used to examine 84 patients. Successful localization with the needle tip within 1 to 2 mm of the suspected lesion was possible in 80 cases (95.2%). Following aspirate cytology, the lesion was localized with indigo carmine and Kopans' wire and every patient underwent a standard open excisional biopsy. Twelve cases of breast cancer were diagnosed histologically. Eleven of these cases were correctly diagnosed cytologically, while one case yielded a false-negative result. In the remaining 72 histologically benign cases, four lesions were reported cytologically to be atypical. There were no complications. Stereotaxic needle aspiration localizes occult breast lesions precisely and in conjunction with mammography, and it is an acceptable preoperative method of diagnosing nonpalpable breast tumors.

Ureteroscopic biopsy: technique and specimen preparation.

Because tissue samples obtained ureteroscopically are small, the techniques for biopsy and for handling and processing the samples are crucial. Our aim is to describe the biopsy technique in great detail so other centers can reproduce it. Patients were evaluated by retrograde ureteropyelography and ureteroscopy for diagnosis. The entire collecting system was examined using a combination of small-diameter rigid and flexible ureteroscopes. Samples were retrieved by aspiration, saline lavage, or, when possible, biopsy of visible tumor by a basket or cup forceps. Multiple samples were obtained from all patients. Fresh specimens were hand delivered to the cytopathology laboratory, where they were evaluated with the cytospin technique. A cell block was prepared whenever there was any visible tissue in the sample. Since we have practiced this technique of handling specimens, our ability to diagnose and grade upper tract neoplasms ureteroscopically has improved markedly. Use of this technique can improve the diagnostic accuracy of ureteroscopic biopsy.

Use of p53 in the diagnosis of upper-tract transitional cell carcinoma.

OBJECTIVES: To assess the usefulness of p53 staining of cytology specimens obtained ureteroscopically in the diagnosis of upper-tract transitional cell carcinoma (TCC). METHODS: We collected specimens from 43 patients undergoing a total of 50 ureteroscopic procedures for a variety of indications, including the diagnosis of TCC. Specimens were obtained by direct biopsy with forceps or basket whenever possible. We examined specimens for evidence of TCC by cytospin as well as cell block for any visible fragments. Each specimen was then stained for overexpression of p53 by immunohistochemical staining, and the degree of staining was graded. Eight patients subsequently underwent nephroureterectomy; the pathologic specimens were stained for p53 and compared with the cytology results. RESULTS: Staining for p53 was positive in specimens from 36 of 50 procedures, including all 28 with ureteroscopic or cytologic evidence of TCC (P < 0.0001). By contrast, cytology accurately diagnosed only 23 of the 28. Specimens from all 14 procedures that were negative also stained negative for p53. Specimens from all 8 procedures with no tumor seen and atypical cytology stained positive for p53; 4 of 5 patients with adequate follow-up have had a tissue diagnosis of TCC at that site. Overall, 35 of the 36 specimens that stained positive for p53 were obtained from patients with some history of TCC (P < 0.0001). No significant association could be found between degree of staining and grade (P = 0.3034). The degree of staining of ureteroscopic biopsy specimens was identical to that of the nephroureterectomy specimen in 6 of 8 cases. CONCLUSIONS: p53 nuclear protein staining of cytology specimens obtained ureteroscopically appears to correlate well with the presence of upper-tract urinary TCC. Further study is needed to determine if it can provide a definitive diagnosis in cases with indeterminate cytologic findings.

Utility of the Bard BTA test in detecting upper urinary tract transitional cell carcinoma.

OBJECTIVES: The Bard BTA test has been shown in early studies to be useful in diagnosing transitional cell carcinoma (TCC) of the bladder. However, the utility of this test has not been evaluated for TCC of the upper urinary tract. We therefore evaluated the clinical utility of the BTA test for upper urinary TCC. METHODS: We tested 71 specimens from the ureter and/or renal pelvis in 22 patients with a history or clinical suspicion for TCC and 9 patients with benign disorders. RESULTS: When compared to cytologic diagnoses, BTA had a sensitivity of 65%, a specificity of 40% (when correlated with clinical history), a false-positive rate of 33%, and a false-negative rate of 62%. The test had a positive predictive value of 83% and a negative predictive value of 32%. CONCLUSIONS: The BTA does not have any clinical value in detecting upper urinary tract TCC.

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.

Retrograde brush biopsy of the ureter and renal pelvis.

In resonse to the limitations of urinary cytology which have resulted in a high incidence of false negative reports, a technique of retrograde brush biopsy has been developed. This technique aids in the diagnosis of carcinomas of the ureteral and renal collecting systems, thereby permitting earlier and more intensive treatment of malignant lesions of the urinary tract.

Clinical utility of chromosome 17 alpha satellite probe in distinguishing benign mesothelium from malignant cells: a pilot study using routinely fixed specimens.

Fluorescent in situ hybridization has shown promise in detecting malignant cells in body cavity effusions. However, this requires special preparatory techniques not used in many laboratories. We developed an in situ hybridization (ISH) procedure specifically for ethanol-fixed specimens, and using it tested the clinical utility of the chromosome 17alpha satellite probe (C17alpha). ISH with C17alpha was used in 12 malignant and 10 benign ethanol-fixed body cavity effusions. Cells were pretreated with protease K prior to ISH. The probe was detected by an anti-digoxigenin-horseradish peroxidase method. Signals were counted in 100 nuclei and the chromosome index (CI) and percent diploid cells calculated in each case. ISH was successfully performed in all cases. Malignant cells had an average CI of 2.23 with less than 44% diploid nuclei and 50% of specimens exhibited bizarre signals. Benign effusions had an average CI of 1.98 with over 84.6% diploid nuclei. Questionably bizarre signals were seen in two (20%) benign specimens. ISH can be performed on ethanol-fixed specimens. The C17alpha probe may prove valuable in detecting malignant cells in body cavity effusions.

Evaluation of renal masses including retrograde renal brushing.

1. The evaluation of renal masses has become an increasingly important topic because of the increasing incidence of kidney cancer, the improved cure rate of renal carcinoma with the proper preoperative diagnosis, and the proliferation in renal mass diagnostic methodology. 2. A variety of benign entities can produce an abnormal renal mass with attendant difficulties in being distinguished from malignant neoplasms. Among these benign lesions are: simple renal cysts, polycystic kidneys, congenital variations in renal size and shape, segmental renal hypertrophy, renal infarcts, intrarenal hematomas, renal hamartomas, renal leiomyomas, renal adenomas, renal angiomas, renal fibrolipomatosis, hydronephrosis of a duplicated collecting system, renal abscesses, and xanthogranulomatous pyelonephritis. 3. Nephrotomography, nephrosonography (ultrasound), adrenalin renal arteriography, selective magnification renal arteriography, renal venography and cavography, lymphangiography, renal scintillation scanning, abnormal levels of enzymes in blood and urine, immunologic studies (circulating antibodies and tumor-associated antigens), percutaneous needle aspirations, and retrograde renal brushing have all increased the diagnostic accuracy of determining the etiology of renal masses. None of these diagnostic procedures is infallible. A judicious combination of procedures gives the most reliable diagnostic results. 4. A search continues for (a) chemical agent(s) or a chemical profile in the blood or urine which is (are) specific for renal carcinoma, but as yet this is an investigational area and not a practical clinical reality.

Influence of polyethylene glycol and acetone on the in vitro biotransformation of tamoxifen and alprazolam by human liver microsomes.

The use of in vitro hepatic microsomal models as a method of studying the biotransformation of xenobiotics can be complicated by the insolubility of a lipophilic substrate. Addition of solvent to an in vitro system is an approach used to increase solubility of such substrates, but solvents may interact with the microsomal enzymes and cause changes in the kinetic parameters. This study focused on the solvents polyethylene glycol-400 (PEG) and acetone and their influence on the human microsomal in vitro biotransformation of the antineoplastic agent tamoxifen (TAM). The antianxiety agent alprazolam (ALP), a verified P450 3A substrate, was also studied. TAM is a nonpolar drug that is metabolized to two oxidative metabolites, N-desmethyltamoxifen (DMT) and 4-hydroxytamoxifen (4-OH-TAM), by cytochrome P450 isoforms. DMT is formed primarily by 3A isoforms, and the pathway(s) responsible for 4-OH-TAM formation are unknown. Biotransformation of ALP is mediated by P450 3A isoforms, which form alpha-hydroxyalprazolam (alpha-OH-ALP) and 4-hydroxyalprazolam (4-OH-ALP). Both PEG and acetone at 5% of the total microsomal incubation volume were found to solubilize TAM in the in vitro system. However, both solvents had an effect on the P450 mediated metabolism of ALP and TAM. For ALP, PEG was a noncompetitive inhibitor of alpha-OH-ALP (mean Ki = 2.06%) and 4-OH-ALP (mean Ki = 2.37%) formation. Acetone stimulated the production of alpha-OH-ALP, but had no apparent influence on 4-OH-ALP formation. The solvent's influence on TAM metabolism varied. PEG decreased the amount of DMT formed by the microsomes as compared to the system containing no solvent (control); however, 4-OH-TAM formation in the presence of PEG was 146-226% of controls. Samples containing acetone produced smaller quantities of both DMT (39-63%) and 4-OH-TAM (45-69%) as compared to controls. Since PEG and acetone increased the solubility of TAM in the incubation buffer but inhibited or accelerated enzymatic reactions compared to buffer alone, actual solubility in buffer was not a determinant of the rate of TAM metabolism. TAM appeared to be taken up into the microsomal fraction, making it available for biotransformation by the P450 isoforms. Although solvents may increase the solubility of nonpolar agents in aqueous systems, careful evaluation of the effects of solvent on metabolite formation, as compared to buffer controls, is needed in order to properly evaluate an in vitro metabolic model.

Brush cytology for the early detection of esophageal carcinoma among patients with upper aerodigestive malignancies.

The incidence of associated esophageal carcinoma (EC) among patients with upper aerodigestive tract malignancies is high. Esophageal brush cytology, as developed and evaluated as a screening device for early detection of EC among villagers of northeastern Iran, was employed to examine 56 clinic patients with known tumors of the upper aerodigestive tract on 106 occasions. Two asymptomatic EC were detected and are presented. The procedure was also used as an adjunct to endoscopy in order to monitor the response of tumors under treatment. Sensitivity of 40% and specificity of 90% were found and could be improved with more judicious application of the procedure. The use of this simple test for early detection of EC among this high risk subset of clinic patients is recommended.