William Crabtree

Prognostic significance of Ki-67 immunostaining in nonmetastatic renal cell carcinoma.

PURPOSE Fresh tissue samples from nonmetastatic renal cell carcinoma (RCC) patients were analyzed by Ki-67 immunostaining to determine the prognostic significance of this tumor-biologic parameter. MATERIALS AND METHODS In a prospective study, Ki-67 immunostaining was performed on frozen sections of histologically proven node-negative RCC from 58 patients operated on between 1986 and 1988 to examine the methods prognostic value and its association with other clinicopathologic parameters such as tumor stage (pT) and grade (G). RESULTS The percentage of Ki-67-positive cells (ie, the proliferation rate [PR]) of all 58 RCC tumors ranged between 1% and 23%, while normal renal tissue exhibited PRs up to 2% only. In almost all cases, the highest PRs were observed in the peripheral zone of malignant tissue close to the normal renal tissue. PR did not correlate with pT, whereas a strongly significant correlation was observed between PR and G, as well as recurrence rate. Twenty-three of 58 patients (39.6%) developed tumor recurrence. Disease-free survival was strongly associated with PR. In a multivariate analysis, G and PR were independent prognostic markers. CONCLUSION The tumor-specific PR obtained by Ki-67 labeling seems to be an independent marker to describe the proliferative activity and aggressiveness of individual tumors. This new tumor-biologic marker detects RCC patients at high risk for recurrent disease, especially in those cases with identical pT and G.

Tumor Proliferative Activity is Predictive of Pathological Stage in Clinical Stage a Nonseminomatous Testicular Germ Cell Tumors

PURPOSE Traditional histopathological features have failed to predict accurately the pathological stage of clinical stage A nonseminomatous germ cell tumors of the testis. Based on pilot studies in nonconsecutive patients at our university, we evaluated nontraditional risk factors (cell cycle analysis by flow cytometry, deoxyribonucleic acid analysis by single cell cytophotometry [image analysis] and assessment of proliferative activity by immunohistochemistry) combined with histopathological features in consecutive patients with clinical stage A nonseminomatous testis cancer. MATERIALS AND METHODS Orchiectomy specimens from 105 consecutive patients with clinical stage A nonseminomatous germ cell tumors who underwent retroperitoneal lymph node dissection (76 with pathological stage A disease and 29 with proved metastasis) were recut, histopathologically reviewed, immunohistochemically stained with proliferation markers (for example Ki-67/MIB-1), and examined by flow cytometry and image analysis. RESULTS After multiple logistic regression analysis, the G2M+S cell cycle fraction of the aneuploid tumor stemline was the most predictive parameter of pathological stage (p = 0.0004). Using a cutoff of 41%, patients with metastasis were predicted with a sensitivity of 71%. Of 61 patients with a G2M+S value of less than 41%, 53 had pathological stage A cancer (negative predictive value 87%). A low volume of embryonal carcinoma was predominant in patients at low risk for metastasis and MIB-1 immunohistochemical staining identified 23% of patients with pathological stage A tumor who were at extremely low risk for metastatic disease. CONCLUSIONS Assessment of tumor cell proliferation cannot classify accurately high risk patients at a clinically applicable level. However, identification of patients at low risk for metastasis by flow cytometry, immunohistochemical proliferation markers and volume of embryonal carcinoma may be possible at the 90% level. MIB-1 staining is able to classify patients at extremely low risk for metastasis. These parameters deserve further study, since identification of patients at extremely low risk for metastasis could potentially decrease overall morbidity in the management of clinical stage A nonseminomatous testis cancer.

Predictive parameters of biologic behavior of early stage nonseminomatous testicular germ cell tumors

Background. Thirty percent of patients presenting with clinical stage A nonseminomatous testicular germ cell tumors in fact have pathologic stage B disease. This pilot study was performed to determine whether DNA content and cell cycle analysis by flow cytometry and single‐cell cytophotometry can improve clinical staging in these patients.

A reliable method for quantitating chromatin fragments by flow cytometry to predict the effect of total body irradiation and hyperthermia on mice

The frequencies of chromatin fragments, including micronuclei, in murine thymus cells, spleen cells and bone marrow cells have been used as a quantitative indicator of gamma-ray induced chromosome damage and could be used to screen potential radioprotective agents as well. The yield of chromatin fragments induced in mice receiving different dosage levels of total body irradiation alone and in mice also given whole body hyperthermia as a potent radioprotector were assessed by flow cytometric analysis. Our results demonstrated that chromatin fragments induced by irradiation in vivo was clearly dose-dependent and that chromatin fragments could potentially serve as a biological indicator of radiation damage. One hour of whole body hyperthermia at 40 degrees C (+/- 0.2 degree C) given 20 hours before a lethal dosage (900 cGy) of total body irradiation protects 100% of DBA/2 mice from an LD 100/16 irradiation dose (dose of irradiation that killed 100% of the mice in 16 days). This is in good agreement with the percent of chromatin fragments formed in the cells of the protected animals, which showed no significant difference from those observed in the normal mice. The results indicate that whole body hyperthermia protected the thymus and bone marrow from irradiation damage. This study provides further evidence which supports that whole body hyperthermia can act as a potent radioprotector in vivo. Measurement of the frequencies of chromatin fragments by flow cytometry is simple and reliable. The method can be applied to screen radioprotective agents.

Clinical Significance of Reporting Benign-Appearing Endometrial Cells in Pap Tests in Women Aged 40 Years and Over

OBJECTIVE To determine the clinical significance of reporting benign-appearing endometrial cells on Pap tests from women > or = 40 years. STUDY DESIGN Pap tests from 149 women demonstrating cytologically benign endometrial cells with histologic follow-up within 12 months were included. Age, menopausal status, hormone replacement therapy (HRT) status, abnormal uterine bleeding (AUB), number of children and information from subsequent endometrial sampling were recorded when available. RESULTS Of the total number of cases, 60.84% had no endometrial pathology (group 3), 35.66% presented with benign pathologic changes (group 2) and 3.50% demonstrated endometrial carcinoma (group 1). The average age for groups 1-3 was 51.40, 46.72 and 46.17 years, respectively. Overall, 13.99% were postmenopausal (13.79% of group 3, 9.80% of group 2 and 60.00% of group 1); 25.87% were known to use HRT (28.74% of group 3, 23.53% of group 2 and 0.00% of group 1) and 80.42% had AUB (75.86% of group 3, 86.27% of group 2 and 100.00% of group 1). CONCLUSION Benign-appearing endometrial cells in Pap tests from women > or = 40 years were associated with endometrial pathology in 39.16% of the cases, with 3.50% being carcinoma, demonstrating the efficacy of reporting their occurrence.

Predictive parameters in biologic assessment of low-stage retroperitoneal lymph-node dissection

SummaryIn all, 30% of patients felt to have clinical stage A nonseminomatous testis cancer in fact have pathologic stage B disease. Although patients with clinical stage A nonseminoma currently enjoy a very high change for cure, a better assignment of therapy at diagnosis could lead to an overall decrease in the morbidity of treatment. This study analyzed orchiectomy specimens from 102 patients with clinical stage A nonseminomatous testis cancer, all of whom underwent pathologic staging via retroperitoneal lymph-node dissection (RPLND). Various parameters of the orchiectomy specimen were analyzed to determine wheter or not clinical staging could be improved on the basis of these factors. Statistical analysis resulted in the following model. If the orchiectomy specimen consisted of 100% embryonal carcinoma the patient was classified as being at high risk for retroperitoneal metastasis. In the absence of this finding the aneuploid cell line as determined by flow cytometry was considered. If the percentage of aneuploid cells in the S phase was less than 29% the patient was felt to be at low risk for retroperitoneal metastasis. If this percentage was greater than 29% the patient was classified as being at high risk. Using this paradigm, 77% of pathologic stage A patients and 91% of pathologic stage B patients were correctly classified. The test efficiency was 82%. This pilot study resulted in an interesting model that should be tested prospectively in consecutive patients to determine whether it is clinically useful.

Micronuclei Assay — A Predictive Variable for Tumor Response to Treatment

Our preliminary data indicate that the formation of micronuclei (MN) in treated tumor cells is a predictive variable for tumor response to treatment. In a pilot study involving four patients who received both radiation therapy and hyperthermia, fine needle aspirate (FNA) samples were taken and analyzed before therapy, and after each 1000 centigray (cGy) up to 3000 cGy. The results indicate a correlation between increasing formation of micronuclei and decreasing tumor volume. All of the patients in this Study have had their tumors under control for at least one year. Our preliminary data demonstrated that a high level of micronuclei in tumor cells correlates with favorable response of the tumor to treatment with radiation and heat. The assay is easy to perform and FNA biopsy could be done in the clinic with minimal discomfort to the patient.

Reprocessing unsatisfactory ThinPrep papanicolaou tests using a modified SurePath preparation technique.

The frequency of unsatisfactory gynecologic specimens has increased in the study laboratory over the last few years due to the advent of personal lubricants. Similarly, lysed blood, protein, and necrotic debris present a challenge in terms of negative cell transference caused by a clogged filter. In the current study, the authors evaluated the potential use of a modified SurePath reprocessing technique to decrease the frequency of unsatisfactory specimens.

Impact of the Preparation Technique on DNA Content Measured by Image Analysis in Early Stage Human Testis Cancer

Current clinical staging which includes serum tumour markers and imaging techniques fails to identify 30-40% of clinical stage I nonseminomatous germ cell testicular tumour (NSGCT) patients who have occult metastatic disease at time of orchiectomy and who will, therefore, develop clinically evident metastases, usually within two years of follow-up. Therefore, there is a real clinical need to evaluate new biological parameters of the primary tumour which might be useful as predictors for occult metastatic disease. Some investigators have described that DNA content measured by image cytometry is of prognostic value in early stage NSGCT to detect patients at risk for occult metastatic disease. However, optimal preparatory techniques are mandatory in establishing new tumour biological markers in order to obtain reliable and reproducible results. This study has analyzed the impact of the sedimentation technique in comparison to the cytocentrifugation technique on DNA measurement in early stage NSGCT obtained by image cytometry. Different tissue blocks of formalin fixed, paraffin embedded primary testicular tumours (NSGCT) of 50 clinical stage I patients, who underwent retroperitoneal lymph node dissection between 1985 and 1989, were analyzed. Thirty (60%) patients had histologically proven lymph node involvement (pathological stage B), whereas 20 (40%) patients (pathological stage A) had neither lymph node metastases nor tumour recurrence during follow-up. The samples were prepared according to a modified Hedley technique: Individual tissue digestion times were monitored closely to avoid overdigestion. The times varied from 30 to 60 min depending on the constituents of the tissue section. Prolonged digestion times did not correlate with poor quality of the preparations and brief digestion times did not always yield optimal specimens. The impact of two different techniques (cytocentrifugation and gravity sedimentation) on the Feulgen staining results were compared. Cytocentrifuged samples consistently provided larger and paler nuclei with less well defined borders compared to slides from the same cell suspension processed by the sedimentation technique. Nuclei from pathologic stage II samples were more vulnerable to cytocentrifuge alteration than those of stage I. According to the results obtained in this study, the sedimentation slide preparation technique should be preferred for DNA ICM in NSGCT, and possibly in other human malignancies as well.