Nuclear and cytoplasmic c-myc staining in endometrial carcinoma and their relationship to survival.

OBJECTIVE: The role of the c-myc proto-oncogene in genomic instability is just becoming more fully understood. However, its role in endometrial cancer is essentially unknown. The objective of this study was to determine the relationship between cytoplasmic and nuclear c-myc staining, DNA index, and survival in patients with endometrial carcinoma. METHODS: One hundred and twenty-one patients with endometrial carcinoma were studied. Image analysis was used to determine DNA index. In addition to cytoplasmic and nuclear c-myc staining and DNA index, histologic type, stage, grade, depth of invasion, lymphvascular space invasion, and peritoneal cytology were evaluated as prognostic indicators. Univariate and multivariate analyses were performed. RESULTS: One hundred and twenty-one patients were followed for over 5 years. c-myc cytoplasmic staining was present in 75.2% of the patients' tumors, and nuclear staining was present in 66.9% (P = 0.99). DNA index was significantly higher in patients with nuclear c-myc staining and no cytoplasmic staining (DNA index 1.38) as compared to those patients whose tumors displayed cytoplasmic c-myc staining but no nuclear c-myc staining (1.18) (P = 0.016). Patients whose tumors stained positively for nuclear c-myc and negatively for cytoplasmic c-myc had significantly worse survival by Kaplan-Meier analysis (P < 0.0001). Seventeen patients died during the follow-up period of this study. By multivariate analysis, positive cytoplasmic c-myc staining with negative nuclear staining (P = 0.0076), negative cytoplasmic c-myc staining with positive nuclear staining (P = 0.011) and FIGO stage (P < 0.0001) were shown to be independent prognostic indicators predictive of survival. CONCLUSION: Nuclear and cytoplasmic c-myc staining, as well as FIGO stage, when assessed by multivariate analysis, were demonstrated to be important factors in predicting survival in the 121 patients in this study. While increasing FIGO stage was prognostic of decreased survival, the specific location of c-myc staining was also associated with prognosis. The expression of the c-myc protein is related to survival in patients with adenocarcinoma of the endometrium.

Nuclear size, shape, and density in endometrial carcinoma: relationship to survival at over 5 years of follow-up. Does analyzing only cells occupying the G0-G1 peak add useful information?

OBJECTIVE: The authors, using image analysis, previously demonstrated nuclear size and summed optical density to be independent prognostic indicators of recurrence in patients with endometrial carcinoma. The same tumors were analyzed by studying the optical features in the G0-G1 peak to see if this changed the values found as well as their importance as prognostic features at greater than 5 years of follow-up. METHODS: Tumors from 74 consecutive patients, surgically treated, with endometrial cancer, were evaluated. Survival, depth of invasion, lymphvascular space invasion, FIGO stage, grade, histology were analyzed. DNA index, progesterone receptor status, as well as nuclear size (NUSZ), shape (NUSH), and summed optical density (NUSD) were evaluated. NUSZ, NUSH, and NUSD were quantified using image analysis. RESULTS: Fifteen patients died from disease during the observation period of the study. Mean follow-up was 82 months with a median of 84 months. Forty-nine patients had stage I cancers, five stage II, 17 stage III, and three stage IV. NUSZ and NUSD were all significantly different between the original (entire cell cycle) and the re-measured (G0G1 only) values (both P < 0.001). Multivariate analysis showed both the original (P = 0.0001) and G0G1-only (P = 0.046) NUSZ and the original (P = 0.0002) and G0G1-only (P = 0.018) NUSD to be independent prognosticators of survival. CONCLUSION: Image analysis is able to quantify cellular and nuclear parameters not otherwise quantifiable. NUSD and NUSZ correlated with traditional prognostic indicators, were demonstrated independent predictors of survival at over 5 years of follow-up. Although the re-measured NUSZ and NUSD from only the G0-G1 peak were significantly different from the original NUSZ and NUSD, they were not as valuable as prognostic factors. Nuclear size and summed optical density measured from the entire cell cycle are independent prognostic indicators of survival at greater than 5 years of follow-up. Measuring nuclear morphometric features in the G0-G1 peak only does not add any new prognostic information.

p21 and p53 in ovarian carcinoma: their combined staining is more valuable than either alone.

BACKGROUND: Mutations in p53 are the most common genetic alterations in human malignancies. Expression of its protein product has been linked to decreased survival rate in ovarian carcinoma. Less is known about the importance of p21 expression. The purpose of this study was to determine the value of the combination of p21 and p53 expression in patients with epithelial ovarian malignancies. METHODS: One hundred three consecutive patients with epithelial ovarian carcinoma were studied using snap-frozen tissue specimens. Immunohistochemical staining utilizing the pAb1801 monoclonal antibody to p53 and NCL-WAF-1 monoclonal antibody to p21 was performed. Image analysis was used to determine whether nuclear staining for either antibody was present. In addition to p21 and p53, International Federation of Gynecology and Obstetrics stage, grade, histology, level of cytoreduction, and DNA index were analyzed as prognostic factors. Univariate and multivariate analyses was performed. RESULTS: One hundred three patients were observed for more than 5 years. Immunohistochemical staining for p21 and p53 were significantly inversely related (P = 0.041). Among the patients whose tumors showed p21 staining but no p53 staining, there were no recurrences and all patients were alive at 5-year follow-up. The 5-year survival rate for these patients was significantly better than for the patients with other combinations of p21/p53 staining (P < 0.0001). The DNA index between these 2 groups was not significantly different (P = 0.057). Multivariate analysis shows the combination of p21 and p53 (P = 0.013) staining to be more valuable as a prognostic indicator than either p53 (P = 0.015) or p21 (P = 0.5) alone. CONCLUSIONS: This study confirms the importance of the combination of p21 and p53 nuclear staining in patients with ovarian carcinoma. Cox regression analysis revealed combination of p21 positive and p53 negative to be a better independent indicator of prognosis and survival in patients with ovarian carcinoma than either p21 or p53 alone.

Immunohistochemical staining of the mismatch repair gene, hMSH2, and survival in patients with ovarian carcinoma.

OBJECTIVE: hMSH2 is a mismatch repair gene. The protein of this gene can be demonstrated by immunohistochemical methods. The authors wanted to analyze whether the percent of positive nuclear area staining of the hMSH2 protein correlated with survival in patients with ovarian carcinoma. METHODS: One hundred and two patients with epithelial ovarian carcinoma were studied. Slides were prepared from snap frozen tissue. Quantification of staining and DNA index were performed using image analysis. In addition to hMSH2, FIGO stage, grade, histology, and level of cytoreduction were analyzed as prognostic factors. RESULTS: Mean follow-up was 64 months and median was 59 months. Nineteen patients had stage I cancers, 4 stage II, 59 stage III, and 20 stage IV. Optimal cytoreduction was accomplished in 71% of patients. hMSH2 staining was significantly higher in better differentiated tumors (p=0.006), but there was no difference in staining among the FIGO stages (p=0.43). Cox regression analysis revealed FIGO stage (p=0.0005), level of cytoreduction (p=0.006) and hMSH2 staining (p=0.016) to be independent predictors of survival in patients with ovarian carcinoma. CONCLUSION: The hMSH2 protein can be demonstrated by immunohistochemical methods and quantified by image analysis. hMSH2 staining was shown to be an independent prognostic indicator of survival in patients with ovarian carcinoma.

p53 and bcl-2 in epithelial ovarian carcinoma: their value as prognostic indicators at a median follow-up of 60 months.

OBJECTIVE: p53 is the most common tumor suppressor gene involved with human malignancies. Mutations in p53 are present in approximately 50% of human malignancies. bcl-2 is a protooncogene. Expression of its protein product is related to better prognosis in several malignancies. METHODS: One hundred and three patients with epithelial ovarian carcinoma were studied. Immunohistochemical staining using the pAb1801 monoclonal antibody to p53 and the anti-bcl-2 124 monoclonal antibody to bcl-2 was performed. Image analysis was used to measure percentage positive nuclear area staining of mutant p53. In addition to bcl-2 and p53, FIGO stage, grade, histology, and level of cytoreduction were analyzed as prognostic factors. Univariate as well as Cox regression analysis was performed. RESULTS: One hundred and three patients were followed for a mean of 60 months. Twenty patients had FIGO stage I disease, 4 stage II, 59 stage III, and 20 stage IV. Immunohistochemical staining for mutant p53 was not significantly related to DNA index (P = 0.99) but was related to increasing FIGO stage (P < 0.001) and increasing histologic grade (P = 0.039). Using Cox regression analysis, increased mutant p53 staining was an independent predictor of survival in these patients (P = 0.0032), along with stage (P < 0. 0001) and level of cytoreduction (P < 0.0001). Although by itself bcl-2 was not an independent prognostic indicator (P = 0.18), the combination of p53 and bcl-2 was independently predictive of survival (P = 0.038). CONCLUSION: This study confirms the authors' earlier report on the importance of p53 as a prognostic indicator of survival in ovarian carcinoma. Cox regression analysis reveals mutant p53 staining to be a better independent indicator of prognosis and survival in patients with ovarian carcinoma than the combination of bcl-2 and p53.

MIB-1 in endometrial carcinoma: prognostic significance with 5-year follow-up.

OBJECTIVE: MIB-1, a monoclonal antibody to the Ki-67 antigen, has presumptively been shown to be predictive of recurrent disease in patients with endometrial cancer. In order to more conclusively establish whether MIB-1 staining can be used as a prognostic indicator of recurrent disease or survival, a larger group of patients with a minimum follow-up of 5 years was analyzed. METHODS: The tumors from 147 consecutive patients receiving primary surgical therapy for endometrial carcinoma were evaluated with the MIB-1 monoclonal antibody. Proliferation index was quantified by image analysis. Patients were followed for a minimum of 60 months. In addition to MIB-1 staining, histologic type, stage, grade, depth of invasion, lymphovascular space invasion, and peritoneal cytology were evaluated as prognostic indicators. RESULTS: Twenty-five of 147 patients died during the study period. MIB-1 staining was not significantly elevated in advanced (stage II, III, and IV) as opposed to early (stage I) carcinomas (P = 0.38). In patients whose tumor MIB-1 staining was less than 33.0%, no deaths occurred. By multivariate analysis, only MIB-1 staining (P < 0.001), FIGO stage (P = 0.005), and LVI (P = 0.005) were shown to be independent prognostic indicators predictive of survival. CONCLUSION: In this series of 147 consecutive patients with endometrial carcinoma, the monoclonal antibody MIB-1 was shown to be an independent prognostic indicator of 5-year survival. This follow-up further validates the previous work regarding the significance and potential usefulness of MIB-1 as a prognostic indicator.

p53 expression as a prognostic indicator of 5-year survival in endometrial cancer.

BACKGROUND: One of the most common genetic alterations to occur in human cancers is an alteration of the p53 tumor suppressor gene. The purpose of this article was to build upon the authors' previous work with p53 and determine whether p53 was a prognostic indicator of 5-year survival. METHODS: One hundred thirty-seven consecutively surgically treated patients with endometrial cancer had their p53 expression studied by immunoperoxidase staining and quantified by image analysis. All patients were evaluable for 5-year survival. RESULTS: One hundred three patients had endometrioid adenocarcinoma; 6, adenosquamous carcinoma; 14, papillary serous carcinoma; 10, clear cell carcinoma; and 4, undifferentiated carcinoma. p53 expression ranged from 0.0 to 58.2% positive nuclear area with a mean of 11.5% (median 2.6%) for the cohort. For the patients with endometrioid carcinoma, the mean p53 expression was 7.1% while for the nonendometrioid tumors it was 24.6% (P<0.001). Fifty-nine of the 103 endometrioid tumors (57.3%) stained positive for p53 while 32 of the 34 nonendometrioid (94.1%) tumors stained positive (P<0.001). Increasing histologic grade correlated with an increasing p53 expression (P = 0.003). The percentage of tumors expressing p53 was found to be higher in FIGO stage II, III, and IV than in FIGO stage I cancer (P = 0.003). However, mean p53 expression did not differ between early (stage I) and advanced (stage II, III, and IV) cancers (P = 0.088). Utilizing 5-year survival as the endpoint for multivariate analysis, FIGO stage (P = 0.0028) and p53 expression (P<0.001) were the only independent prognostic indicators found. CONCLUSION: p53 expression is more commonly found in nonendometrioid than in endometrioid adenocarcinoma of the endometrium. It, along with FIGO stage, is an independent prognostic indicator of 5-year survival.

Markov optical texture parameters as prognostic indicators in ovarian carcinoma.

Texture is a descriptive property of a surface describing the morphometric heterogeneity of complex structures. Computer aided image analysis allows optical texture measurement and analysis of gray-scale images. The authors, utilizing image analysis, prospectively studied Markov nuclear texture features to determine their relevance as prognostic indicators of survival in patients with epithelial ovarian carcinoma. Ninety-nine consecutive patients with ovarian cancer, treated initially with surgery were evaluated for their length of survival, level of cytoreduction, FIGO stage, grade, histology, and DNA index, as well as 20 Markov texture features. Markov nuclear texture features were quantified using image analysis. Mean follow-up for the study population was 64 months (median 59) with a range from 51 to 89 months. Five optical texture features showed significant correlation with length of survival. Difference entropy (P = 0.033) and information measure A (P = 0.041) were both indirectly correlated with survival while information measure B (P = 0.030), correlation coefficient (P = 0.045), and the maximum correlation coefficient (P = 0.041) were directly correlated. Only sum entropy (P = 0.035), FIGO stage (P = 0.0031), and level of cytoreduction (P < 0.0001) were independent predictors of survival in this population. Optical texture can be quantified by image analysis. Utilizing multivariate analysis, the Markov texture feature, sum entropy, was demonstrated to be an independent prognostic indicator of survival in patients with epithelial ovarian cancer. FIGO stage and optimal cytoreduction also were independent prognostic indicators of survival.

Lack of bcl-2 persistence: an independent prognostic indicator of poor prognosis in endometrial carcinoma.

OBJECTIVE: bcl-2 is a protein which prohibits programmed cell death. The purpose of this study was to determine whether bcl-2 staining was related to traditional prognostic factors and/or recurrence in patients with endometrial carcinoma. METHODS: One hundred twenty consecutively surgically treated patients with endometrial carcinoma had their tumors studied immunohistochemically for bcl-2 staining. RESULTS: The mean follow-up of the patients was 53 months with a median of 56 months (range 30 to 68 months). bcl-2 staining was positive in 44.0% of patients with endometrioid carcinomas and in 23. 1% of patients with nonendometrioid carcinomas (P < 0.001). Increasing depth of invasion (P = 0.014), grade (P = 0.011), and FIGO stage (P = 0.018) were each correlated with decreasing bcl-2 staining. bcl-2 staining was positive in 44.1% of patients whose tumors showed no lymphovascular space invasion and in 11.1% of patients with lymphovascular space invasion (P < 0.001). Only 1 of 26 patients with recurrent disease had persistence of bcl-2 staining. Multivariate analysis revealed FIGO stage (P = 0.0051), histologic grade (P = 0.050), and lack of staining for bcl-2 (P = 0.012) to be independent predictors of recurrence. CONCLUSION: bcl-2 persistence is more common in endometrioid than in nonendometrioid adenocarcinomas of the endometrium. It appears to be inversely correlated with the universally recognized prognostic factors of depth of invasion, histologic grade, and FIGO stage. Lack of bcl-2 persistence was an independent predictor of recurrence of disease. This group of patients continues to be followed to determine the role of bcl-2 persistence or lack of persistence as a predictor of 5-year survival of patients with endometrial carcinoma.

Heat shock protein 27: an independent prognostic indicator of survival in patients with epithelial ovarian carcinoma.

OBJECTIVE: Heat shock protein 27 (HSP27) is produced in response to pathophysiologic stress in animal cells. The purpose of this study was to determine prospectively whether HSP27 was associated with known prognostic factors as well as survival in patients with epithelial ovarian carcinoma. METHODS: Ninety-nine patients with epithelial ovarian carcinoma were studied. Slides were prepared from fresh tissue. Patient records were examined for FIGO stage, grade, histology, level of cytoreduction, and survival. RESULTS: Immunohistochemical staining for HSP27 was not related to histologic grade, level of cytoreduction or histologic subtype. A statistically significant difference in HSP27 staining was found in relation to FIGO stage (P = 0.013). HSP27 staining was found to be an independent predictor of 2-year survival in these patients (P = 0.041). CONCLUSION: The level of HSP27 significantly decreases as the FIGO stage increases and is an independent prognostic indicator of survival in patients with epithelial ovarian carcinoma.

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.

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.

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. METHODS: The orchiectomy specimens of 102 patients with clinical stage A disease were analyzed retrospectively using histopathologic classification, flow cytometry, and single-cell cytophotometry. All patients had undergone retroperitoneal lymph node dissection. RESULTS: The multivariate analysis in this group of patients resulted in the following model: If the primary tumor consisted of 100% embryonal carcinoma, the patient was classified as high risk for retroperitoneal metastasis. If the patient was found to have less than 100% embryonal carcinoma in the primary tumor, the percent of aneuploid tumor cells in S-phase as identified by flow cytometry was most predictive for pathologic stage. Using this approach, 91% of all patients with pathologic stage B, and 77% of the patients with pathologic stage A were correctly classified; test efficiency was 82%. CONCLUSIONS: These results demonstrate an improvement in clinical staging in this group of patients. This paradigm, developed from retrospective analysis, will be tested prospectively in consecutive patients to determine if it is clinically useful.

Quantitative DNA measurement by flow cytometry and image analysis of human nonseminomatous germ cell testicular tumors.

Current clinical staging, which includes the use of serum tumor markers and imaging techniques, fails to identify the 30-40% of clinical stage I (CS I) nonseminomatous germ cell testicular tumor (NSGCT) patients who have occult metastatic disease. Therefore, there is a real clinical need to evaluate new biological parameters of the primary tumor that might be useful as predictors of occult metastatic disease. This study was undertaken to compare quantitative DNA measurements by flow cytometry and image analysis in CS I NSGCT, and to analyze the relevance of these parameters for predicting occult lymph node involvement. Different blocks of formalin-fixed, paraffin-embedded NSGCTs of 62 CS I patients who underwent retroperitoneal lymph node dissection between 1985 and 1989 were prepared according to the Hedley technique, and analyzed by quantitative cytometry. Thirty-six (58.1%) patients had histologically proven lymph node involvement (pathological stage II), whereas 26 (41.9%) patients (pathological stage I) had neither lymph node metastases according to retroperitoneal lymph node dissection (RPLND) specimens nor tumor recurrence during follow-up. Concordant results were found in 76.5% of the samples by both cytometric techniques. For flow cytometry, the percentages of aneuploid cells in the S- and the G2M + S-phase were the most robust predictive parameters for lymph node involvement, whereas for image analysis the 5c exceeding rate (5cER) had the most predictive significance. Based on the experience obtained in this study, both cytometric techniques provide additional information on tumor aggressiveness that might be useful in therapeutic selection of early stage NSGCT patients for either RPLND or surveillance only.

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

In 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 whether 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.

Comparison of image analysis and flow cytometric measurements of DNA content of canine transitional cell carcinomas.

The DNA content of neoplasms is generally determined by flow cytometry, but quantitative microscopic image analysis is an alternative technique for DNA quantification. To compare these two methods, the DNA content of 31 paraffin-embedded canine transitional cell carcinomas was measured by flow cytometry and image analysis. Interpretable data were available for 26 samples by flow cytometry and 27 samples by image analysis. For 23 neoplasms with interpretable data by both methods, a good correlation (r = .80, P < .001) was obtained between the DNA indices determined by flow cytometry and image analysis. Concordance of DNA ploidy classification was reached in 21 samples; however, for 4 samples an aneuploid cell population, usually tetraploid, was identified by image analysis and not by flow cytometry. Two samples also had aneuploid peaks by flow cytometry and not by image analysis. Although a good correlation was seen between the measurement of DNA content of paraffin-embedded canine tissue by image analysis and flow cytometry, this study did demonstrate that aneuploid cell populations can be missed by both methods and that image analysis was better than flow cytometry in the detection of aneuploid cell populations, particularly tetraploid ones, in samples with excessive cellular debris.

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 method's 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.

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.

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.