Prediction of recurrence in giant cell bone tumors by DNA cytometry.

The most interesting therapeutic aspect of giant cell bone tumors is which patients can be cured without a risk of recurrence by intralesional surgery (curettage). To find out the suitability of some DNA cytometric and morphometric parameters for showing differences between this group of patients (n = 9) and those with recurrence (n = 12), the parameters mean ploidy, 2cDI (mean square deviation of the tumor cell DNA content from the normal 2c value), mean nuclear area and its variability were calculated from cytologic specimens prepared by a cell separation technique from formalin-fixed, paraffin-embedded tissues, measuring the values of 100 stromal cells per case by a TV image analysis system. Further measurements were performed on 19 cases of different diseases of the bone and on an additional 17 cases of giant cell tumors without follow-up. The 2cDI allowed us to distinguish the two groups of patients, with and without recurrence, without overlap; even the lowest value for patients with recurrence was higher than the highest value for cured ones. Mean ploidy analysis resulted in a less convincing discrimination of the patients. Mean nuclear area and its variability failed to predict recurrence. Single-cell DNA cytometry provided a parameter, 2cDI, that was able to predict recurrence in patients with giant cell bone tumors with high sensitivity.     

Bone metastases: prognostic applications of cytometric DNA analysis

OBJECTIVE: To examine DNA parameters as prognostic factors for developing metastases. STUDY DESIGN: Image cytometry was used to determine DNA content of 21 tumors and 28 metastases. DNA ploidy status, 2c deviation index (2cDI) and DNA malignancy grade (DNA-MG) (based on the variation of nuclear DNA content of tumor cells around the normal DNA [2c] peak) were examined for their prognostic value. RESULTS: Twenty of 21 tumors showed aneuploid content, and 1 tumor showed diploid DNA content. Twenty-one bone metastases showed aneuploid cells. In 6 cases both euploid and aneuploid cells were detected. In 1 metastasis only euploid cells were present. DNA-MG was increased in bone metastases (mean, 2.4) as compared to the corresponding primary tumor (mean, 2.2) in most of the cases. The mean value of the 2cDI was 30.07 in primary tumors and 42.5 in metastases. Twelve bone metastases had a higher 5cEE than did the primary tumor. CONCLUSION: Diploid and aneuploid cells were able to leave a tumor and establish metastases. DNA-MG and 2cDI were increased in metastases in comparison with the primary tumor, but even tumors with lower DNA-MG had metastatic potential.     

DNA ploidy and vimentin expression in primary breast cancer

DNA ploidy and vimentin expression in primary breast cancer
The DNA content of 50 breast cancers of varying tumour type, grade and stage was measured using static image cytometry, and correlated with vimentin expression in the tumour cells. A tendency to increased vimentin expression and aneuploidy was observed in high grade and late stage tumours¹. A statistically significant difference was observed in DNA index and ploidy balance between grade 1 and grades 2 and 3 carcinomas ( P <0.05) and between stage I and stage II carcinomas ( P <0.05). There was a significant difference in the expression of vimentin between grades 1, 2, 3 ( P <0,001), and stages I, II and III ductal carcinomas ( P <0.05). No significant difference was observed in the proliferation index and the degree of hyperploidy ( P >0.05). Clonal heterogeneity was observed in 25% of breast carcinomas, and was associated with increased vimentin expression. These changes may be indicative of genomic alteration and tumour aggressiveness.     

DNA content and p53 protein expression in ductal breast cancer

DNA content and p53 protein expression in ductal breast cancer
The DNA content of 85 ductal breast cancers of different histological grades was evaluated using static cytometry and correlated with immunocytochemical expression of p53 protein in tumour cells in cytological material. A statistically significant difference was observed between p53 protein expression and grade of malignancy ( P <0.001). The percentage of euploid tumours significantly decreased from grade I through grade II to grade III tumours ( P <0.001). Clonal DNA heterogeneity was observed in 26.6% of cases analysed and was correlated with p53 protein expression ( P <0.001). These changes probably reflect genomic alterations which may affect potential malignancy of breast cancer.     

Nucleolar organizer region-associated proteins in cancer cells. Quantitative investigations on gliomas, meningiomas, urinary bladder carcinomas and pleural lesions.

Using a modified silver staining technique, we investigated nucleolar organizer region-associated proteins (AgNORs) in paraffin sections of 156 neoplastic tissues and other lesions, including gliomas (n = 41), meningiomas (n = 20), urinary bladder carcinomas (n = 58), and neoplastic and reactive lesions of the mesothelium of the pleural cavity (n = 37). We found significant differences in the mean number and area of AgNORs per nucleus between nonanaplastic and anaplastic astrocytomas. In meningiomas AgNOR analysis may be useful to distinguish between mostly benign tumors (grade 1 tumors) and atypical ones. Urinary bladder carcinomas exhibited a statistically significant increase in both AgNOR number and area as the grade of malignancy increased. Diagnostically useful differences in the AgNOR configuration between inflammatory and neoplastic processes were found for mesothelial lesions. In general, a higher grade of malignancy correlated with an increase in the AgNOR number. This was accompanied by an increase in the total AgNOR area per nucleus, irrespective of whether the size of the individual AgNORs had changed.     

Prognostic significance of DNA image cytophotometry for osteosarcoma

OBJECTIVE: To investigate the prognostic significance of DNA image cytophotometric data. STUDY DESIGN: Twenty-six osteosarcomas in patients without lung metastases were investigated for several cytophotometric data. In 24 cases, these data were correlated with the clinical course of the patients to assess the prognostic value of nuclear DNA content in osteosarcomas. RESULTS: Of all osteosarcomas, 96% showed aneuploid DNA content. Patients with tumors having a 2c deviation index (2cDI) of 12.00, DNA malignancy grade (DNA-MG) of 2.0, a mean DNA content (MDC) of 4.95 c, DNA index (DI) of 1.75 or mean nuclear area (MNA) of 130 microns 2 had a significantly lower overall survival rate as compared to those with lower values (P < .05). CONCLUSION: Image cytophotometric features, such as 2cDI, DNA-MG, MDC, DI and MNA, are of prognostic value in patients with osteosarcoma and free of lung metastases.     

AgNOR count in resting cells (resting NOR) is a new prognostic marker in invasive bladder tumor.

PURPOSE: We have previously demonstrated that the AgNOR count in proliferating cells is a predictor of tumor recurrence in superficial bladder tumor (J. Urol. 162 (1999), 63-68). In the present study, we evaluate the type of AgNOR associated with cell cycles as a prognostic factor in invasive bladder tumor using a double staining technique employing both AgNOR and MIB-1 labelling. MATERIALS AND METHODS: Forty-four paraffin sections of invasive bladder tumors were stained simultaneously with AgNOR and MIB-1. The number of AgNORs in proliferating (MIB-1 positive) or resting (MIB-1 negative) cells were counted from a total of 100 nuclei. Correlations between MIB-1 associated AgNOR count and clinicopathological parameters were statistically analyzed. RESULTS: The AgNOR count in proliferating cells (proliferating NOR) was significantly higher than that in resting cells (resting NOR) (p<0.01). The resting NOR in tumors with distant metastases was significantly higher than that in tumors without metastases (p<0.05). Patients with a low resting NOR tumor had a better prognosis than those with a high resting NOR tumor, whereas the proliferating NOR was not associated with survival. Survival analysis revealed that the resting NOR was the most powerful prognostic marker in patients with invasive bladder tumor (p<0.05). CONCLUSIONS: Resting NOR had a predictive value in the prognosis of patients with invasive bladder tumor.     

Silver-stained nucleolar organizer proteins in chondrosarcoma.

Silver-stained nucleolar proteins (AgNORs) were counted in primary chondrosarcomas of three histologic grades and in metastatic chondrosarcomatous lesions in the lung. The AgNOR numbers of neoplastic cells in primary tumors increased stepwise from grade 1 (4.42 +/- 1.11) through grade 2 (4.94 +/- 1.31) to grade 3 (6.97 +/- 1.10). There was a significant difference in AgNOR numbers between grade 3 and both grades 1 and 2 (p less than 0.001). Furthermore, the mean number of AgNORs in metastatic lesions (9.75 +/- 0.83) was significantly higher than that in primary sites (p less than 0.001). The number of AgNORs therefore reflects the grade of the chondrosarcoma. The results in the present study indicate that silver colloid staining is a useful technique for determining the histologic grade and evaluating the proliferative activity of chondrosarcomas.     

Prognostic significance of DNA ploidy and proliferation rate in human astrocytic gliomas

DNA ploidy and the proliferative potential in 75 gliomas were investigated using bromodeoxyuridine labelling index (BrdUrd LI), S-phase fraction (SPF) and argyrophilic nucleolar organizer regions (AgNOR) technique. There were 53 highly malignant (AIII-AIV), and 22 low-grade (AI-AII) gliomas. One fragment of the tumour was fixed in Carnoy's solution for AgNOR test, while the other fragments were used for flow cytometric determination of the labelling index, SPF and DNA ploidy. For the BrdUrdLI, tumour samples from each patient were incubated in vitro for one hour at 37 degrees C with BrdUrd using the high pressure oxygen method. The tumours showed variability in the BrdUrdLI values, SPF and AgNOR counts/cell nucleus. The same percentage of DNA aneuploidy (55%) was found in high-grade as well as in low-grade gliomas. Univariate analysis showed that patients with grade I & II gliomas had significantly higher 3-year survival rate (p = 0.0193) than those with grade III and grade IV gliomas. Also patients with lower proliferation rate of tumours (BrdUrdLI < or =2.3% and AgNOR counts < or =2.6%/cell) had higher 3-year survival rate (p<0.03), which can be helpful in prognosis. Tumour ploidy or SPF had no influence on patients' survival (p = 0.7908). Cox multivariate analysis showed that only patients' age > 45 years and high tumour grade (III and IV) were significant unfavourable prognostic factors in terms of patients' survival.     

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.     

A new method for DNA extraction from feces and hair shafts of the South China tiger (Panthera tigris amoyensis)

It is commonly known that tigers (Panthera tigris) groom themselves by licking their coats, which leads to an abundance of hairs in their feces. These hairs are designated specially as “fecal hairs”. In our study, in order to explore fecal hairs potential as a DNA source for genetic analysis, 55 fecal hair samples were collected from 23 captive South China tigers (P. t. amoyensis). According to the amplification of mitochondrial primers loop F and loop R, DNA quality of noninvasive samples were grouped into three grades: grade I—the highest-quality DNA, grade II—high-quality DNA, and grade III—poor-quality DNA. No failed amplifications on microsatellite primers and only 0.27% genotyping errors occurred with grade I fecal hair DNA, as compared with 9.4% failed amplifications on microsatellite primers and 9.5% genotyping errors with grade II fecal hair DNA. It was found that 25.45% of fecal hair DNA was grade I and 65.45 and 10.00% of fecal hair DNA were grades II and III, respectively, as compared with 4.35% grade I fecal DNA and 34.78 and 60.87% grades II and III fecal DNA, respectively. Thus, higher-quality DNA can be extracted from fecal hairs than feces. In addition, DNA could be extracted from hair shafts of tigers and a minimum of 2000 hair shafts were required for visible DNA bands on a 1% agarose gel. These findings demonstrate that fecal hairs may serve as a convenient and reliable genomic DNA source for genotype analysis. Zoo Biol 28:49–58, 2009. © 2008 Wiley-Liss, Inc.     

Cell kinetics analysis in stage I endometrial carcinoma as determined by image cytometry and immunohistochemistry. Correlation with clinicopathologic characteristics

OBJECTIVE: To test the significance of various proliferative indices in endometrial carcinoma (EC) since previous investigations failed to yield consistent results that would establish them as factors of clinical importance. STUDY DESIGN: Seventy patients with EC were evaluated for various proliferative indices (mitotic index, Ki-67 index, argyrophilic nucleolar organizer (AgNOR) number and area per nucleus, and p53 protein expression) in relation to image cytometry (nuclear area, diameter and roundness) and standard clinicopathologic features (age, histologic type and grade, and depth of invasion). We also tested the proliferation index (PI), which combines the Ki-67 index and AgNOR area in Ki-67-positive nuclei. Slides from each case were double stained for Ki-67 antigen and AgNOR proteins for this purpose. RESULTS: Mitotic counts were significantly higher in papillary-serous (vs. endometrioid) tumors (P = .0001), high grade (vs. low grade) tumors (P = .0001), deeply invasive (P = .017) and p53-positive tumors (P = .017). AgNOR counts correlated only with age (higher in older women, P = .002), while the PI correlated with mitotic counts (P = 0.28) and marginally with depth of invasion (P = .06). Morphometric variables were associated just with histologic type and grade. p53 Protein was expressed exclusively in invasive tumors and was related strongly to histologic type (P = .0029) and grade (P = .0001). CONCLUSION: Our data reestablish the value of classic histopathologic features (mitotic index, histologic type and grade) as the most important tools for EC evaluation. In addition, we suggest that p53 immunostaining may be used for predicting aggressive behavior in EC.     

DNA ploidy in gastrointestinal B-cell lymphomas. An image analysis study of 43 cases

OBJECTIVE: To analyze the prognostic importance of DNA ploidy pattern on gastrointestinal (GI) B-cell lymphoma using image cytometry (ICM) and to compare the results with previously published flow cytometry (FCM) data. STUDY DESIGN: Forty-three cases of surgically resected primary GI B-cell lymphomas were examined. Thirty-eight tumors were located in the stomach, 2 in the small intestine, 1 in the large bowel and 2 in both the stomach and small intestine. Six cases were at stage E I 1, 15 at stage E I 2, 20 at stage E II 1 and 1 each at stages III and IV. Histologically, the lymphomas were classified as GI low grade marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) type (low grade, 12 cases), low grade MALT lymphoma with a high grade component (mixed type, 10 cases) and GI diffuse large B-cell lymphoma (DLBC) (high grade MALT lymphoma, 21 cases). After gross removal of nonneoplastic tissue, single cell suspensions were prepared from paraffin blocks and stained according to Feulgen. Ploidy analysis was done using a custom-made DNA cytometer and Optimas image analysis software (Optimas Corp., Seattle, Washington, U.S.A.). RESULTS: Aneuploidy was found in 42% (5/12 cases) of low grade MALT lymphoma, 90% (9/10 cases) of mixed type lymphoma and 100% (21/21 cases) of GI DLBCL. DNA ploidy had no significant impact on overall survival time (P = .73). CONCLUSION: ICM analysis showed a higher proportion of aneuploidy in GI lymphomas as compared to that in prior studies using FCM for ploidy determination. Whether DNA ploidy is an independent prognostic factor remains to be determined.     

Grading follicular lymphoma on fine needle aspiration specimens. Comparison with proliferative index by DNA image analysis and Ki-67 labeling index

OBJECTIVE: To determine whether follicular lymphoma (FL) can be graded on fine needle aspiration (FNA) biopsies by determining the percentage of centroblasts in the neoplastic follicles on the smears. STUDY DESIGN: Eighty-nine cases of histologically confirmed cases of FL, including 31 grade 1, 46 grade 2 and 12 grade 3, were evaluated. Proliferative index (PI) by DNA image analysis (DIA) and Ki-67 labeling index (LI) were obtained on all cases. A minimum of 200 cells were counted per case (range, 200-800 cells) at 40x magnification, and the number of large cells (centroblasts) was expressed as a percentage of the total number of cells counted within the follicles. RESULTS: The percentage of centroblasts in the follicular aggregates was 9.7 +/- 2.9% in grade 1 FLs, 24.7 +/- 5.6% in grade 2 and 48.4 +/- 7.5% in grade 3. These differences were significant (P < .05). DNA image analysis of PI and Ki-67 LI differed significantly between grade 1 FLs and grade 2 and 3 FLs (P < .05), but there were no significant differences between grade 2 and 3 FLs. CONCLUSION: Determining the percentage of centroblasts in the follicular aggregates on FNA specimens is a good method of grading FLs. Using the percentage of centroblasts per follicular structure, FL grades 1, 2 and 3 were adequately distinguished. PI by DIA and Ki-67 LI clearly distinguished FL grade 1 from FL grades 2 and 3; however, it did not clearly distinguish between grades 2 and 3.     

Assessment of histogenesis and proliferative potential in cytologic specimens of human brain tumors. Value of immunocytochemistry and nucleolar organizer regions.

The value of immunocytochemistry and nucleolar organizer regions (NORs) for the histogenetic identification and the estimation of the proliferative potential of brain tumors was assessed by the investigation of imprint smears of 51 neurosurgical tumor specimens. A panel of five monoclonal antibodies was used to cover a broad range of immunohistochemical markers. For the assessment of NORs, a silver staining technique (AgNOR) was used. NORs were enumerated and measured by means of an interactive image analysis system. The immunocytochemical results were similar for the smears and paraffin-embedded sections for 95.6% of the investigations performed and for 76.2% of the cases. Glial fibrillary acidic protein (GFAP) was positive in 9 of 17 tumors of glial origin, but was negative in 9 metastatic tumors. Vimentin was positive in 10 of 10 and fibronectin in 9 of 10 meningiomas investigated. The number of NORs increased steadily with the increasing grade of malignancy. Especially in glioblastomas, the number of NORs per cell exhibited a wide range, which might reflect the heterogeneity of these neoplasms. Metastases revealed a higher number of NORs per cell than did glioblastomas. In the cytologic differential diagnosis of these tumors, an absence of GFAP expression combined with a high NOR count is suggestive of a metastatic tumor.     

Hepatocellular carcinoma and preneoplastic lesions of the liver: evaluation of argyrophilic nucleolar organizer regions (AgNORs).

The authors applied a silver colloid technique to identify Argyrophilic Organiser Region (AgNOR) to 8 groups of hepatic lesions: alcoholic hepatitis with dysplasia (3 cases); chronic active hepatitis with dysplasia (4 cases); cirrhosis with dysplasia (5 cases); focal nodular hyperplasia (4 cases) and hepatocellular carcinomas (3 cases of grade I, 3 cases of grade II and 5 cases of grade III of Edmondson). Four cases of non-specific reactive hepatitis were used as control. This work suggests the simplicity and utility of simultaneous application of clumps per cell, AgNORs per clump and total AgNORs counts in the evaluation of neoplastic and preneoplastic lesions of the liver. The results show, in hepatocellular carcinomas, a relationship between the number of clumps, the AgNORs per clump, the total number of AgNORs and the grading of Edmondson. The nodular lesions that can be considered in the differential diagnosis with carcinoma are sufficiently well discriminated using the two parameters AgNORs per clump and total number of AgNORs.     

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 ploidy and pS2 protein expression in breast cancer

The DNA content of ductal breast carcinomas of varying histological grade was measured using static image cytometry and correlated with pS₂ expression in the tumour cells. Our study was performed on imprint of surgical biopsies of 60 women with ductal breast cancer. A statistically significant difference was observed between pS₂⁺ expression and grade of malignancy ( P <0.001). The percentage of euploid tumours significantly decreased from grade I to grade II to grade III ( P =0.01). The percentage of aneuploid tumours increased from pS₂⁺ to pS₂⁻ breast tumours ( P <0.001). These findings may be indicative of pS₂ and DNA ploidy alterations and tumour aggressiveness.     

Nucleolar organizing regions in renal cell carcinoma, renal oncocytoma and renal adenoma

Nucleolar organizing regions (NORs), as demonstrated by the silver-colloid staining technique, have been counted in 75 renal cell carcinomas (20 grade 1, 22 grade 2, 17 grade 3 and 16 sarcomatoid), eight renal oncocytomas and nine renal adenomas. Mean NOR counts were 3.27, 6.28, 9.24 and 8.12, respectively, for grades 1, 2, 3 and sarcomatoid tumours, 3.09 for renal oncocytomas and 2.63 for renal adenomas. Analysis of data using the unpaired Student's t-test showed significant difference between NOR counts of grade 1, 2 and 3/sarcomatoid renal cell carcinoma, and grades 2, 3 and sarcomatoid renal cell carcinomas when compared to renal oncocytomas and adenomas. The association between type and grade of tumour, NOR value and tumour proliferation is discussed.