Flow and image cytometric DNA ploidy,including 5c exceeding cells,of serous borderline malignant ovarian tumors. Correlation with clinicopathologic characteristics

OBJECTIVE: To analyze DNA ploidy of serous borderline ovarian tumors by flow cytometry (FCM) and image cytometry (ICM), with 5c exceeding cells also analyzed, and to evaluate their correlation with clinicopathologic characteristics of patients and tumors. STUDY DESIGN: Cell suspensions were prepared according to a modified Hedley method from formalin-fixed, paraffin-embedded tissue blocks of 43 tumors. One part of the suspension was used for flow cytometric measurement; from the other part, filter slides were prepared for ICM. RESULTS: FCM and ICM found 2 aneuploid (peridiploid) serous borderline ovarian tumors, and FCM found 1. ICM found 3 tumors with 5c exceeding cells and 2 tumors with octaploid cells. There was no correlation between DNA aneuploidy and presence of 5c exceeding cells with tumor size, International Federation of Gynecology and Obstetrics stage or survival. CONCLUSION: The results confirm a good correlation between FCM and ICM DNA ploidy and the ability of ICM to detect 5c exceeding cells. The prognostic value of DNA ploidy and 5c exceeding cells in serous borderline malignant ovarian tumors warrant further evaluation.     

Diagnostic value of flow cytometric DNA determination combined with fine needle aspiration biopsy in thyroid tumors

The nuclear DNA content and the numbers of cells in the S and G2M phases of the cell cycle were determined by flow cytometry (FCM) in fine needle aspirates of 187 thyroid tumors to evaluate the diagnostic value of nuclear DNA content determination in combination with aspiration cytology. DNA aneuploidy was present in 4 of 5 follicular carcinomas, 2 of 3 anaplastic carcinomas, 5 of 15 excised follicular adenomas and 2 of 20 excised adenomatous goiters; all 7 papillary carcinomas and 4 lymphomas were diploid in the aspirate. Aneuploid carcinomas had easily distinguishable S and/or G2M phases, unlike the benign aneuploid tumors. None of the histologically benign tumors or the nonexcised tumors had greater than 6% S-phase cells, and only one benign tumor had greater than 9% G2M-phase cells. In contrast, all lymphomas had greater than 10% S-phase cells and four of seven papillary carcinomas had greater than 9% G2M-phase cells. The use of FCM determination in combination with fine needle aspiration biopsy cytology improved the diagnostic potential of the latter technique.     

Cytologic and DNA cytometric diagnosis and therapy monitoring of squamous cell carcinoma in situ and malignant melanoma of the cornea and conjunctiva

OBJECTIVE: To investigate the diagnostic accuracy of exfoliative cytology of the cornea and conjunctiva and DNA image cytometry for quality control and monitoring of therapy for malignant neoplasms. STUDY DESIGN: Conjunctival or corneal smears from six cases clinically suspicious for malignant melanomas and eight suspicious for carcinomas in situ were investigated. Smears from 18 cases clinically nonsuspicious for neoplastic diseases served as negative controls. Repeated smears were obtained during and after local mitomycin C (MMC) therapy. RESULTS: In none of 18 nonsuspicious cases, cytology revealed abnormal cells. DNA cytometry showed nonaneuploidy in all of these. All smears from patients with histologically proven malignant melanomas (MM) and squamous cell carcinomas in situ revealed abnormal cells. Image cytometry demonstrated DNA aneuploidy in 66.6% of patients with MM and 80% with carcinoma. Sensitivity of cytology thus was 100% for both MM and carcinoma; specificity also was 100%. DNA measurements after MMC therapy revealed euploid polyploidization of nonneoplastic squamous cells. DNA cytometry provided an objective identification of tumor cell regression. CONCLUSION: Cytologic examination of corneal and conjunctival smears is a noninvasive tool with high diagnostic accuracy for detection of epithelial neoplasms. DNA image cytometry can serve for quality control and for objective monitoring of the effect of local chemotherapy.     

Comparative studies of nuclear DNA content in benign and malignant thyroid lesions

Currently available data suggest that DNA aneuploidy is associated with aggressive behavior of and unfavorable prognosis in several malignant human tumors as compared with diploid malignancies. However, the diagnostic and prognostic importance of flow cytometric DNA measurements in the case of thyroid neoplasms remains controversial. Therefore, the aim of our study was to evaluate utility of DNA index (DI) and proliferative index (PI) in distinguishing benign from malignant thyroid lesions taking into account the possible influence of intra-tumor heterogeneity and tissue preparation mode on DNA flow-cytometry measurements. A retrospective study was performed on 71 paraffin-embedded specimens from 57 patients with benign and malignant thyroid pathologies: 13 colloid goitres, 12 parenchymatous goitres, 19 adenomas and 13 carcinomas. In 14 of 57 cases two separate specimens taken from different areas of the same lesion were analysed and DNA parameters were compared. Additionally, flow cytometry DNA analysis was parallelly performed on 3 adjacent but differently processed tissue sections (fresh, formalin-fixed and paraffin-embedded) taken from each of 26 surgically excised thyroid lesions. DNA content was also analysed in both fresh and formalin-fixed twin specimens of normal pig thyroid glands (N = 6). We demonstrated that all tumors diagnosed as thyroid carcinomas were associated with abnormal nuclear DNA content although aneuploidy was not found specific to malignant thyroid tumors. Aneuploid samples of benign thyroid lesions exhibited higher proliferative activity, expressed as mean PI values, than diploid ones. In carcinomas the mean PI values were significantly higher than in benign lesions, independently whether they concerned aneuploid or diploid tissues. Considering intra-tumor heterogeneity, the flow cytometric DNA parameters can be assumed as reproducible despite differences in the mode of tissue fixation and preparation for analysis.     

Neutrophil gelatinase-associated lipocalin (NGAL) immunohistochemical expression in follicular cell-derived thyroid tumors: a novel diagnostic tool?

Discrimination of follicular cell-derived benign and malignant tumors of the thyroid is one of the major problems encountered in surgical pathology. In the present study, we evaluated the immunohistochemical expression of NGAL, an iron-binding protein involved in the infiltrative potential of cancer cells, in a cohort of tumors including 8 follicular adenomas (FA), 2 Hurthle cell adenomas (HA), 2 atypical adenomas (AA), 8 minimally invasive follicular carcinomas (MIFC), 9 widely invasive follicular carcinomas (WIFC), 3 Hurthle cell carcinomas (HC) and 8 papillary carcinomas (PC) with 5 follicular-variant PC (FVPC) and 3 not otherwise specified (PC-NOS). Our goal was to test whether evaluation of NGAL immunoexpression may be of use in the differential diagnosis of benign and malignant thyroid neoplasias. 92% of benign tumors (specificity) were negative for NGAL, whereby NGAL immuno-expression was found in 82% (sensitivity) of malignant tumors, and, specifically, in 100% of MIFC, in 87% of WIFC, in 100% of HC, in 80% of FVPC. None of the PC-NOS displayed NGAL staining. When only tumors with a follicular architecture were considered, NGAL specificity for malignant lesions was 92%; sensitivity, positive predictive value and negative predictive value were 92%, 96% and 85%. Diagnostic accuracy of NGAL expression in the differential diagnosis between benign and malignant follicular tumors was 92%. In conclusion, NGAL protein seems to represent a marker of malignant follicular cell-derived thyroid tumors, and especially of those with follicular architecture. Hence assessment of its expression might be of use with respect to differential diagnosis from follicular benign neoplasias.     

Comparison of morphonuclear features in normal, benign and neoplastic thyroid tissue by digital cell image analysis.

We analyzed the morphonuclear features of thyroid cell nuclei from 10 normal tissues (10 multinodular goiters), 10 benign tissues (10 adenomas) and 10 neoplastic tissues (10 carcinomas--2 follicular, 2 medullary and 6 papillary). These morphonuclear features quantitatively described the nuclear size, DNA content and chromatin texture of 200-400 Feulgen-stained cell nuclei that were digitized by means of a cell image processor. Nuclear DNA estimations revealed that the benign thyroid tumors showed an aneuploidy level that was not different from that of the neoplastic tissues. Morphometric measurements showed a significant and positive correlation between an increase in nuclear area and the development of thyroid pathology--i.e., from normal to a neoplastic stage. We also observed a significant decrease in chromatin condensation and heterogeneity from normal to neoplastic thyroid tissue. In the specific case of thyroid tumors, such criteria were not found sufficient per se for a diagnosis of malignancy. More detailed data banks will be necessary for this purpose.     

Cytologic grading and DNA image cytometry of breast carcinoma on fine needle aspiration cytology smears

OBJECTIVE: To correlate the cytologic grade of breast carcinoma with DNA image cytometry (ICM) and nuclear area on fine needle aspiration cytology (FNAC) smears. STUDY DESIGN: In this prospective study, FNAC material from 28 breast carcinomas were studied for cytologic grade and DNA ICM. Breast carcinomas were classified as grade 1-3 (low to high). DNA histograms were classified by the modified Auer method. Degree of hyperploidy (DH), ploidy balance (PB) and nuclear area (NA) were measured on Feulgen-stained smears by a CAS 200 image cytometer. Cytologic grade was correlated with DNA ICM findings and NA. RESULTS: There were 3 cytologic grade 1, 13 grade 2 and 12 grade 3 breast carcinomas. Seven of eight cases of hypertetraploid aneuploidy were grade 3 tumors. All cytologic grade 1 tumors were diploid. There were significant differences in DH, PB and NA in different grades of breast carcinoma (one-way ANOVA). CONCLUSION: DNA image cytometry in combination with cytologic grading might offer additional information for the characterization of breast carcinomas diagnosed by FNAC. These observations are of particular interest with the introduction of preoperative chemotherapy.     

DNA content of Hurthle cell nodules in autoimmune thyroiditis

Hurthle cells are found in thyroid neoplasms and in reactive nodules in thyroiditis or goitrogenic processes. Cytometric studies have evaluated Hurthle cell neoplasms but not their reactive counterparts. DNA content of Hurthle cells in 22 cases of autoimmune thyroiditis was measured by flow cytometry and image content of Hurthle cells in 22 cases of autoimmune thyroiditis was measured by flow cytometry and image processing using nuclei extracted from paraffin-embedded tissue after microdissection of the Hurthle cell nodules. All 22 autoimmune thyroiditis Hurthle cell nodules were diploid, including 16 without associated neoplasms and six with associated malignant neoplasms (four papillary carcinomas, one follicular carcinoma and one follicular adenoma with papillary carcinoma). Concordance between flow cytometry and image processing was 100%. These findings indicate that the markedly atypical Hurthle cells in autoimmune thyroiditis are diploid by DNA quantitation. This suggests that atypia in Hurthle cells due to reactive or neoplastic processes may be differentiated by quantitative DNA analysis.     

DNA ploidy in soft tissue sarcoma: comparison of flow and image cytometry with clinical follow-up in 93 patients

In soft tissue sarcoma, the prognostic importance of DNA ploidy status is limited. One possible explanation may be technical; small non-diploid stemlines will be diluted in relation to the presence of normal diploid cells and may not be detected by flow cytometry (FCM). We assessed DNA ploidy status in 93 tumors with both FCM and image cytometry (ICM). ICM may permit the exclusion of non-relevant cells. The ability of the two methods to detect non-diploid stemlines was compared, as were the prognostic consequences. The patients (54 males) had a median age of 69 years. Surgical procedures were performed on all patients. None of the patients had received preoperative radiotherapy or chemotherapy. FCM and ICM were performed with standard methods. The prognostic value was assessed with univariate and multivariate analysis. In 82 of the 93 tumors, a concordant ploidy status by FCM and ICM was found. In 5 FCM type 1-2 tumors (diploid), the identification of non-diploid stemlines by ICM did not influence the metastatic rates. Increasing tumor size, histotype other than liposarcoma, increasing malignancy grade, tumor necrosis, and ICM non-diploidy were univariate prognostic factors for metastasis. In a multivariate analysis, only tumor size larger than 9 cm was a prognostic factor. In about 10% of the tumors, a discrepancy between FCM and ICM ploidy status was found, but we could not find a consistent prognostic consequence of this. Neither FCM nor ICM ploidy status was an independent prognostic factor.     

The role of DNA flow and image cytometry in the evaluation of body cavity fluids

In addition to conventional cytomorphologic study, 50 body cavity fluid specimens (benign and malignant) were analyzed by both flow cytometry (FCM) and image cytometry (ICM) in order to evaluate the potential application of these techniques in the diagnosis of malignancy. While 88% of the fluids were similarly classified by FCM and ICM as being either diploid (66%) or nondiploid (22%), with similar DNA index values, nondiploid peaks were identified by ICM alone in 12% of the fluids. Aneuploid populations were present in 92% of the cytologically positive fluids and in 15% of the cytologically negative fluids. All of the latter fluids came from patients who clinically had tumors involving the respective body cavities. These results show that (1) both FCM and ICM are useful adjuvant techniques in the evaluation of body cavity fluid specimens and (2) ICM is superior to FCM in the identification of small aneuploid subpopulations. The identification of aneuploid populations by either method is highly suggestive of malignancy.     

Is agNOR and DNA ploidy analysis useful for evaluating thyroid neoplasms?

OBJECTIVE: To correlate the subjective AgNOR counting method and DNA content with histologic diagnoses of thyroid cancer and invasion. STUDY DESIGN: Eighty-one consecutive cases of thyroid carcinoma were selected for DNA and AgNOR analysis. The diagnoses were: papillary carcinoma (n = 40), follicular carcinoma (n = 31), Hürthle cell adenocarcinoma (n = 4), and undifferentiated carcinoma (n = 6). Seven normal thyroids were used as controls. DNA quantitative measurement was performed with Vidas 2.0 software (Kontron Bildanalyse, Munich, Germany) connected to an MPM 210 photometer microscope (Carl Zeiss, Oberkochen, Germany). The DNA index was obtained using histograms. Counting the NORs was performed by subjectively counting the NORs in 200 malignant cells. RESULTS: DNA ploidy analysis showed all Hürthle cell adenocarcinomas, 21 (67%)follicular tumors, 23 (57%) papillary tumors and 4 (67%) undifferentiated carcinomas to be aneuploid. DNA analysis correlated with histologic type of the tumor (p = 0.032). There was no statistical significance to the AgNOR counting variables studied. Statistical analysis showed correlation between ploidy and histologic diagnosis, but not AgNOR counting, to have prognostic value. CONCLUSION: DNA ploidy is more useful than subjective counting of NORs as an adjunct method for thyroid lesion analysis.     

Pulmonary fine needle aspiration biopsy. Assessing the negative diagnosis.

OBJECTIVE: To assess the significance of the "negative for malignancy" category when applied to pulmonary transthoracic fine needle aspiration biopsy (FNAB). STUDY DESIGN: Transthoracic lung FNABs diagnosed as "negative for malignancy" were identified from the files of Barnes-Jewish Hospital's South and North Campus over a period of five and nine years, respectively. Histologic correlation and clinical follow-up were obtained. RESULTS: Of the 1,181 lung FNABs performed during the study period, 108 cases (9%) had a negative cytologic diagnosis. Histologic correlation was available in 46 cases (43%), of which 23 cases had benign histologic findings, and 19 cases were malignant. Thirty-five of the 62 cases without histologic correlation had clinical follow-up consistent with a benign process. CONCLUSION: Based on the histologic correlation and clinical data available, the negative predictive value was 77%. Inadequate sampling was responsible for all false negative cytologic diagnoses in this series.     

Cytologic and DNA-cytometric early diagnosis of oral cancer.

OBJECTIVE: The aim of this prospective study was to report on the diagnostic accuracy of conventional oral exfoliative cytology taken from white-spotted, ulcerated or other suspicious oral lesions in our clinic. In addition we checked DNA-image cytometry as an adjuvant diagnostic tool. Our hypothesis is that DNA-aneuploidy is a sensitive and specific marker for the early identification of tumor cells in oral brushings. STUDY DESIGN: 251 cytological diagnoses obtained from exfoliative smears of 181 patients from macroscopically suspicious lesions of the oral mucosa and from clinically seemingly benign oral lesions which were excised for establishing histological diagnoses were compared with histological and/or clinical follow-ups of the respective patients. Additionally nuclear DNA-contents were measured after Feulgen restaining using a TV image analysis system. RESULTS: Sensitivity of our cytological diagnosis on oral smears for the detection of cancer cells was 94.6%, specificity 99.5%, positive predictive value 98.1% and negative predictive value 98.5%. DNA-aneuploidy was assumed if abnormal DNA-stemlines or cells with DNA-content greater 9c were observed. On this basis the prevalence of DNA-aneuploidy in smears of oral squamous cell carcinomas in situ or invasive carcinomas was 96.4%. Sensitivity of DNA-aneuploidy in oral smears for the detection of cancer cells was 96.4%, specificity 100%, positive predictive value 100% and negative 99.0%. The combination of both techniques increased the sensitivity to 98.2%, specificity to 100%, positive predictive value to 100% and negative to 99.5%. CONCLUSIONS: Brush cytology of all visible oral lesions, if they are clinically considered as suspicious for cancer, are an easily practicable, cheap, non-invasive, painless, safe and accurate screening method for detection of oral precancerous lesions, carcinoma in situ or invasive squamous cell carcinoma in all stages. We conclude that DNA-image cytometry is a very sensitive, highly specific and objective adjuvant tool for the early identification of neoplastic epithelial cells in oral smears.     

Histological grading, DNA content, cell proliferation and survival of patients with astroglial tumors

Light microscopy, image cytometry (ICM), and flow cytometry (FCM) were used to study the degree of differentiation, DNA content, and S-phase of astrocytomas and glioblastoma multiforme in 102 patients. The postoperative real survival time (RST) was also studied. Using ICM, 62 astrocytomas were investigated. Grade I astrocytomas were composed of DNA-diploid cell lines, while grade III and glioblastoma multiforme consisted predominantly of DNA-aneuploid lines. Moderately differentiated astrocytomas were divided as follows: 14 DNA-diploid and 18 DNA-aneuploid. Forty astrocytomas were studied by FCM. Using the DNA index (DI) value, cases with abnormal DNA cell lines were established in all astrocytomas, with their number increasing in grades II and III astrocytomas. FCM indicated the same subdivision of moderately differentiated astrocytomas: 12 with DNA-diploid and 12 with DNA-aneuploid stem lines. Patients with DNA-diploid cell lines in the astrocytomas and low S-fraction survived longer than patients with abnormal DNA cell populations and higher S-fraction. The results from this study indicate that, together with the degree of differentiation of astroglial tumors, the appearance of cell lines with abnormal DNA value and higher S-fractions also have prognostic value.     

Roles of DNA cytometry and detection of EBERs in predicting a diagnosis of nasopharyngeal carcinoma.

OBJECTIVE: To analyze the suitability of DNA cytometry and detection of Epstein-Barr virus (EBV)-encoded RNAs (EBERs) on nasopharyngeal brushings for predicting a diagnosis of nasopharyngeal carcinoma (NPC). STUDY DESIGN: Cytologic preparations in 66 cases suspicious for NPC were evaluated for NPC diagnosis in comparison with the histologic diagnosis. Based on cytologic examination, 38 cases containing cytologically proven cancer and 8 cases interpreted as cytologically negative for cancer with adequate cellularity in the smear specimens were analyzed for DNA ploidy with an image analyzer and for EBER expression by in situ hybridization (ISH). RESULTS: Based on the cytologic diagnosis, DNA aneuploidy analysis, DNA nondiploidy analysis and EBER detection demonstrated a sensitivity of 50%, 84% and 92%, respectively, with the same specificity, 100%, for predicting a diagnosis of cancer. Their negative predictive values were 30%, 57% and 73%, respectively. There was a significant difference between DNA aneuploidy analysis and EBER analysis in sensitivity (P < .001) and in negative predictive value (P < .05) but not between DNA nondiploidy analysis and EBER analysis even though EBER analysis showed a slightly higher value in both parameters (P > .1 and P > .5, respectively). CONCLUSION: ISH for EBERs in cytologic smears showed a role superior to that of DNA aneuploidy analysis in the diagnosis of NPC. Considering its advantages of simple experimental conditions and lower cost as compared with DNA measurement, EBER detection can play a practical and important diagnostic role in patients suspected of having primary NPC.     

DNA ploidy in breast lesions. A comparative study using two commercial image analysis systems and flow cytometry

DNA ploidy determinations on a series of 24 breast specimens were performed independently utilizing flow cytometry (FCM) and two separate commercially available computerized image analysis systems for image cytometry (ICM). The tissues analyzed were obtained from 20 carcinomas, 2 benign neoplasms and 2 benign reductive procedures. The results showed a close correlation between the DNA indices (DIs) obtained by all methods in 14 of the 24 cases. In four cases, all methods showed aneuploid peaks, but with differing DIs. In six cases (two benign and four malignant) FCM showed diploidy while ICM showed peridiploid cell populations. The results obtained with the two image analysis systems were in agreement for 20 of the 24 cases. ICM is an acceptable alternative to FCM for reproducible ploidy analysis. ICM-based measurements have the advantage of the visual discrimination of abnormal cells and therefore may have a greater sensitivity in identifying small aneuploid populations. Populations with DIs in the range of 1.0 to 1.3 need to be assessed carefully in ICM-based determinations due to the potential that these "aneuploid" peaks may represent shifted diploid populations.     

Predictive value of DNA cytometry in CIN 1 and 2. Image analysis of 193 cases

OBJECTIVE: To investigate DNA image cytometry for predicting the prognosis of cervical intraepithelial neoplasia (CIN). STUDY DESIGN: Smears from 151 women affected by CIN 1 or 2 on cytology with minimal follow-up of three years were included. Sixty-seven showed progression, with histologically confirmed carcinoma in situ or invasive cancer. Eighty-four cases showed regression of the disease, which was cytologically, histologically and colposcopically confirmed. Papanicolaou-stained smears were destained, and the Feulgen reaction was performed with consecutive image DNA cytometry of suspicious cells using an image analysis system (Cires, Zeiss, Germany). The DNA index of the greatest stemline and the number of single aneuploid cells, using 9c exceeding events, were computed. RESULTS: In the group with progression, an aneuploid DNA stemline was found in 25 smears (26.9%). In 64 cases (66.7%) more than one aneuploid event was detected. The total number of aneuploid cases in this group was 76 (81%). In the group without progression, the number of aneuploid stemlines was 2 (2%). Single aneuploid cells could be found in five cases (5%). The overall number of aneuploid cases in that group was five. The sensitivity was 74.3%, positive predictive value 85.2% and negative predictive value 77%. CONCLUSION: Aneuploidy is a marker for prospective malignancy in cervical Papanicolaou smears. DNA image cytometry, as an additional method, can be used to predict outcome in patients with CIN 1 and 2 of the cervix. DNA cytometry is not a screening method but can add further information for a treatment decision in doubtful cases.     

DNA quantification in colorectal carcinoma using flow and image analysis cytometry

Numerous studies using flow cytometry (FCM) have shown that DNA quantification and ploidy classification can provide information of prognostic significance for patients with colorectal carcinoma; recent advances in image analysis cytometry (image cytometry, ICM) provide a new, alternative technique for DNA quantification. This study investigated whether (1) patients with colorectal carcinomas that exhibit a diploid pattern of DNA distribution have improved five-year survival statistics as compared to their non-diploid counterparts and (2) ICM provides quantitative data comparable to that obtained by FCM. DNA quantification and ploidy classification of 27 cases of primary colorectal carcinoma was performed on archival paraffin-embedded tissue by both FCM and ICM; 70% (19) of the tumors were classified as nondiploid by ICM while 56% (15) were similarly classified by FCM. Diploid tumors were associated with Dukes' stage A while nondiploid tumors were associated with Dukes' stage D. The overall five-year survival rate was 75% for patients with ICM diploid tumors and 67% for patients with FCM diploid tumors. The five-year survival was only 53% for patients with nondiploid tumors identified by both techniques. This study confirmed that DNA quantification is an important prognostic indicator for patients with colorectal carcinoma. It also showed that ICM provides data comparable to that of FCM and may be more sensitive.     

Influence of section thickness, mean nuclear diameter and nuclear crowding on DNA ploidy in histologic sections of melanocytic skin lesions

OBJECTIVE: To determine the influence of section thickness, nuclear diameter (MND) and area percentage of nuclei (a measure of nuclear crowding) on histologic DNA ploidy assessed by image cytometry (ICM) of primary melanocytic skin neoplasms (MSNs). STUDY DESIGN: Initially a feasibility study was performed to determine if comparable DNA ploidy histograms could be obtained from cell disaggregates and tissue sections. Following this, DNA ICM was performed on Feulgen-stained tissue sections (4, 6, 8 and 10 microns thick) from 30 primary MSNs (20 benign, 10 malignant) with nuclear diameters from 5.6 to 8.6 microns. Area percentage of nuclei was assessed in all cases at all section thicknesses. RESULTS: The feasibility study produced comparable results for cytocentrifuge and tissue section preparations. For sectioned MSNs, DNA ploidy histograms from 4-micron sections had a higher coefficient of variation of the 2c peak than those from 6-, 8- and 10-micron sections. Ten-micrometer sections had marked overlapping of nuclei, and only small numbers of cells could be measured, giving inadequate results. MND and area percentage of nuclei did not have an important influence on the results. CONCLUSION: Adequate DNA ploidy profiles can be obtained by DNA ICM on 6- and 8-micron-thick histologic sections of MSNs, provided that a strict measurement protocol is followed.     

DNA ploidy and immunomarking of bladder urothelial tumors before and after intravesical bacillus Calmette-Guérin treatment

OBJECTIVE: To investigate DNA ploidy and immunoexpression of Ki-67 and p53 as predictivefactors in cases of superficial urothelial cell carcinoma (UCC) treated with bacillus Calmette-Guérin (BCG). STUDY DESIGN: Samples were obtained from 66 patients with UCC (pTa grade 3 or high grade and pT1 independent of grade or with concomitant carcinoma in situ) before and after intravesical BCG treatment. DNA ploidy analysis (ploidy balance, degree of hyperploidy and aneuploidy, proliferation index) was done by static cytometry. Ki-67 and p53 were analyzed immunohistochemically in paraffin-embedded tissue, and their quantification was carried out using an image analysis system. RESULTS: During a mean follow-up of 63.8 months, 31 of the 66 patients developed recurrent tumors (46.9%). DNA ploidy analysis showed that ploidy balance as well as degree of hyperploidy and aneuploidy were not statistically different between recurrent and nonrecurrent tumors. Only proliferation index was statistically significant between recurrent and nonrecurrent tumors. No statistically significant difference was observed in the percentage of Ki-67- and p53-positive cells between primary tumors that recurred and those that did not. CONCLUSION: These findings suggest that only proliferation index has predictive value for recurrence and progression in UCC treated with BCG.