Cell cycle-dependent AgNOR analysis in invasive breast cancer

OBJECTIVE: To investigate to what extent analysis of silver-stained nucleolar organizer regions (AgNORs) is cell cycle dependent in breast cancer and to assess the prognostic value of an AgNOR analysis that takes into consideration the cell cycle status of tumor cells. STUDY DESIGN: In 97 cases of invasive breast carcinoma, morphometric AgNOR analysis was performed in tumor cells with immunohistochemical MIB-1 reactivity (NORcyc analysis) and in MIB-1-negative tumor cells (NORnon analysis). Additionally, conventional (NORconv) analysis without preceding MIB-1 staining was done. Findings were compared with the Nottingham prognostic index (NPI). RESULTS: In comparison to noncycling tumor cells, cycling ones exhibited significantly higher AgNOR numbers (mean values, 3.84 +/- 1.09 vs. 2.40 +/- 0.78 per nucleus), higher total AgNOR areas (5.95 +/- 3.17 vs. 5.62 +/- 3.05 micron 2, NS) and significantly lower mean AgNOR areas (2.08 +/- 1.14 vs. 2.93 +/- 1.69 micron 2). When related to NPI, correlation coefficients of NORnon analysis were higher than those of NORcyc analysis but lower than those of NORconv analysis. Among the different AgNOR parameters, total AgNOR area correlated best with NPI. CONCLUSION: Cell cycle status has a high impact on AgNOR analysis. However, the best prognostic information in breast cancer is derived from an AgNOR analysis that considers both cycling and noncycling tumor cells.     

Early diagnosis of mesothelioma in serous effusions using AgNOR analysis

OBJECTIVE: To compare the results of conventional cytology, DNA image cytometry, immunocytochemistry and argyrophilic nucleolar organizer region (AgNOR) analysis for the diagnosis of malignant cells in serous effusions. STUDY DESIGN: One hundred twenty effusions, 40 with carcinoses, 40 with malignant mesotheliomas and 40 without tumor cells on follow-up were studied by conventional cytology and three adjunctive methods. RESULTS: Unequivocal tumor cells were detected in 92.5% of effusions due to carcinoses and in 45% due to mesotheliomas. Applying immunocytochemistry with BerEP-4 positivity and DNA image cytometry with aneuploidy as a marker revealed 100% of carcinoses and 71.7% of mesotheliomas. Applying the experimentally found thresholds of 2.5 AgNORs as "satellites" and 4.5 AgNORs as "satellites and clusters" together as mean values per nucleus resulted in a 95% correct rate of mesothelioma and 100% rate of carcinoma cell identification without false positive diagnoses. CONCLUSION: AgNOR analysis may be a useful adjunct to other methods in the routine diagnosis of malignant serous effusions. It seems to be the most sensitive method in early cytologic diagnosis of mesotheliomas in effusions. Seventy-three percent of malignant mesotheliomas were diagnosed cytologically at first on effusions. Forty-seven percent of patients with malignant mesotheliomas were identified at the early tumor stage T1 N0 M0.     

Nuclear morphometry and AgNOR quantification: computerized image analysis on ovarian mucinous tumor imprints

OBJECTIVE: To determine the morphometric characteristics of nuclei and silver-stained nucleolar organizer regions (AgNORs) on cytologic imprints and their value in differential cytodiagnosis of benign, atypical proliferative (borderline) and malignant ovarian mucinous tumors. STUDY DESIGN: Forty-six mucinous ovarian tumor imprints (16 benign, 15 borderline, 15 malignant), were analyzed. Nuclear area, outline, "shape factor" and "form factor" were measured on Papanicolaou-stained smears. AgNOR quantification included 7 variables related to the number and area of single, cluster, total and relative AgNOR content per nucleus and the size distribution of AgNORs. RESULTS: Nuclear area and shape factor allowed distinguishing borderline and malignant tumors. The nuclear area in benign tumors was larger than that in borderline tumors; malignant tumors had the highest values. Single and cluster AgNORs were statistically significantly different in borderline tumors compared with malignant tumors, except for the cluster AgNOR area. The total AgNOR area, number and relative area increased from benign through malignant tumors, with statistically significant differences among all groups. By AgNOR size distribution, small AgNORs discriminate malignant from borderline and benign tumors. CONCLUSION: Combining nuclear morphometry and AgNOR analysis on cytologic imprints could be a diagnostically useful method in the assessment of mucinous ovarian tumors.     

Standardized AgNOR analysis as a prognostic parameter in endometrial carcinoma, endometrioid type

OBJECTIVE: To evaluate interobserver reproducibility of histologic grade in endometrial adenocarcinomas of endometrioid type (EC), to assess the relationships between nuclear grade and the amount of argyrophilic nucleolar organizer region (AgNOR) proteins and to determine the prognostic value of AgNOR proteins and the main clinicopathologic parameters. STUDY DESIGN: Architectural and nuclear grading were independently assessed by two pathologists in 64 formalin-fixed, paraffin-embedded surgical samples of EC obtained from an equal number of patients (age range, 38-84 years; mean, 63.5). Interobserver agreement was determined using the kappa statistic; discrepant cases were reviewed, and a consensus was reached. Standardized AgNOR analysis was performed according to the guidelines of the Committee on AgNOR Quantification, measuring the mean area of AgNORs per nucleus (NORA) by an image analysis system. RESULTS: The kappa values for interobserver agreement were substantial for architectural grading and moderate for nuclear grading. When NORA values were compared to the nuclear grade assessed by different observers, the most significant linear correlation (r = .713, P < .001) was found for the nuclear assessment obtained by consensus of the two pathologists. Moreover, statistical analysis allowed discrimination of architectural grade 1 from grade 2 and 3 EC. By the Kaplan-Meier method, the prognosis was worse for patients with higher NORA values (> 4.212 micron 2), while, by Cox multivariate analysis, AgNOR quantity emerged as an independent prognostic variable. CONCLUSION: Use of standardized AgNOR analysis may be an additional and objective tool in the assessment of histologic grade as well as a reliable method of determining prognosis in EC.     

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.     

The AgNOR quantity as prognostic parameter in choroidal melanomas: a standardised analysis.

In order to assess the prognostic significance of silver-stained nucleolar organizer region (AgNOR) proteins, a standardised analysis has been performed on 34 ocular globes with choroidal melanomas. On formalin-fixed paraffin-embedded sections, the visualisation and quantification of AgNORs were achieved according to the guidelines of the Committee on AgNOR Quantification (1995); statistical analysis was performed on the mean AgNOR area values (NORA). We have encountered significantly higher NORA values in nonspindle shaped elements, in tumours of larger dimensions as well as in those with worse clinical course; no correlations were achieved when the AgNOR quantity was compared with age, sex and amount of pigment. The comparison of Kaplan-Meier survival curves revealed that patients affected by melanomas with higher NORA values (>3.327 microm2), non-spindle cell histotype and increased size of tumour had a worse prognosis; finally, by Cox multivariate analysis, the AgNOR quantity appeared the only independent prognostic variable to predict the final outcome of patients.     

AgNOR analysis of atypical ductal hyperplasia and intraductal carcinoma of the breast

OBJECTIVE: To determine the diagnostic and prognostic value of argyrophilic nucleolar organizer regions (AgNORs) in atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and microinvasive ductal carcinoma (MDCA) of the breast. STUDY DESIGN: Image analysis of histologic sections from biopsies of 46 breast ADH and DCIS and 18 cases of MDCA. Determination of morphometric features of cell nuclei and nucleolar organizer regions by using AMBA software system. Data were compared with the estrogen receptor/progesterone receptor (ER/PR) content as well as with the growth fraction, determined immunohistochemically. RESULTS: AgNOR number and total AgNOR area increased from ADH to DCIS. The highest values were recorded in cases of DCIS with microinvasion. Differences between ADH and intraductal or microinvasive ductal carcinoma were statistically significant. Within the group of intraductal carcinomas, the lowest values were measured in the solid type and highest values in the comedo type. A correlation was found between AgNOR features and growth fraction but not between these features and ER/PR status. CONCLUSION: Selected AgNOR features are relevant for differentiation between ADH and DCIS as well as between low and high grade DCIS and microinvasive ductal carcinoma. Therefore, objective and reproducible data obtained by AgNOR analysis may allow better evaluation of the prognostic significance of these lesions.     

Prognostic significance of standardized AgNOR analysis and Ki-67 immunostaining in gastrointestinal stromal tumors

OBJECTIVE: To evaluate the prognostic utility of argyrophilic nucleolar organizer region (AgNOR) protein parameters and Ki-67-immunostained growth fraction (Ki-67 labelling index) and to correlate AgNORs with Ki-67 LI and the main clinicopathologic parameters in gastrointestinal stromal tumors (GISTs). STUDY DESIGN: On 55 patients with surgically excised GISTs, visualization and quantification of AgNORs were performed as specified in the guidelines of the Committee on AgNOR Quantification. RESULTS: AgNOR protein area (NORA) > or = 5.28 microns 2 was statistically associated with mitotic rate > or = 5 x 10 high-power fields (hpfs) (P < .001) and presence of necrosis (P < .001); Ki-67 LI > or = 9.69% was significantly associated with mitotic rate > or = 5 x 10 hpfs (P < .001), size > or = 5 cm (P = .033) and presence of necrosis (P < .001). Ki-67 LI and NORA strongly correlated. Preliminary Kaplan-Meier survival analysis showed that an increased value of NORA, Ki-67 LI, mitotic rate, tumor size and presence of necrosis had a negative influence on patient survival. Multivariate regression analysis showed that only NORA and Ki-67 LI were independent parameters in predicting the clinical outcome for patients with GISTs. Mitotic rate and necrosis remained as independent prognostic factors when NORA and Ki-67 LI were not allowed to enter in models. CONCLUSION: AgNOR protein quantity, as determined by image cytometry, and Ki-67 immunostaining seem to represent reliable predictive parameters in GISTs and are independent of mitotic rate, tumor dimension and necrosis.     

AgNOR proteins from morphologically intact isolated nucleoli.

AgNOR staining has been proposed as a useful tool for the diagnosis and prognosis of cancer. The AgNOR proteins, however, have not yet been clearly identified and characterized, possibly due to the partial character of the results obtained when studying the proteins extracted from altered nucleoli isolated by "standard" methods. In the present study, we analysed, on western blots, the AgNOR staining profiles obtained with protein extracts from Ehrlich tumor cell nucleoli isolated by a recent procedure that preserves the nucleolar ultrastructure. In addition to the well-known C23 and B23 protein bands, we readily detected an extra band at approximately 125 Kda. By immunoblotting, we showed that this polypeptide may be related to the nucleolar phosphoprotein pp135 evidenced in rat-cell nucleoli. By immunoelectron microscopy, we detected this protein in the dense fibrillar component and fibrillar center of the nucleoli as well as the coiled bodies. The distribution coincides with the cytochemical AgNOR staining pattern obtained at the ultrastructural level.     

Quantitative analysis of AgNOR proteins in exfoliative cytology specimens of oral mucosa from smokers and nonsmokers

OBJECTIVE: To determine the cell proliferation rate and possible effects of cigarette smoking on the oral mucosa lining through analysis of silver-stained nucleolar organizer regions (AgNORs) in exfoliative cytology specimens. STUDY DESIGN: Exfoliative cytology was performed on the left side of the border of the tongue and of the floor of the mouth in 25 smoking patients and 25 nonsmoking patients. The inclusion criterion for smokers was the consumption of more than 20 cigarettes per day for a minimum of 30 years. RESULTS: The slides were stained by histochemical AgNOR method. In the nonsmoking group the mean number of AgNORs per nucleus was 2.732 +/- 0.236 in the tongue border and 2.918 +/- 0.195 in the floor of the mouth. In smoking patients the mean number of AgNORs per nucleus was 3.372 +/- 0.375 in the tongue border and 3.245 +/- 0.237 in the floor of the mouth. CONCLUSION: The results suggest higher cell proliferation quantified by the histochemical AgNOR technique in exfoliative cytology specimens obtained from the oral mucosa lining of smokers presenting no clinical alterations.     

Melanin bleaching in argyrophilic staining of AgNORs in pigmented lesions: a morphometric evaluation

OBJECTIVE: To establish a procedure that can effectively bleach melanin from pigmented lesions without affecting quantification of argyrophilic staining of nucleolar organizer regions (AgNORs). STUDY DESIGN: Twenty banal compound nevi, five from each of nonpigmented, slightly pigmented, moderately pigmented and heavily pigmented groups, were bleached by 10% H202 for periods of 0 (nonbleached controls) and 24 hours. AgNOR size and count parameters of nevomelanocytic nuclei were measured by video image analysis. Melanin bleaching using KMnO4 was also investigated. RESULTS: In all lesions treated with 10% H202 for 24 hours, the melanin was bleached effectively, with no qualitative change in AgNOR appearance. There were no significant differences in mean AgNOR number per nucleus (AgNOR number), mean individual AgNOR size (AgNOR size) or mean percentage of AgNOR area per nucleus (% nuclear area) between nonbleached and bleached sets in both the nonpigmented and slightly pigmented groups. However, disintegration of AgNOR dots was observed in those treated with 1% KMnO4 for 5, 10 and 15 minutes. There were significant decreases in AgNOR size (P = .002) and % nuclear area (P = .003) and increase in AgNOR number (P = .05) in the slightly pigmented group evaluated when treated with 1% KMnO4 for five minutes. CONCLUSION: Melanin in pigmented lesions can be bleached effectively with an H202 procedure without significantly affecting AgNOR staining properties in contrast to bleaching with KMnO4.     

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.     

Morphometry and digital AgNOR analysis in cytological imprints of benign, borderline and malignant serous ovarian tumours

AIM: The aim of the study was to determine values of a quantitative morphometry analysis of nuclear characteristics and argyrophilic nucleolar organizer regions (AgNORs) in differential cytodiagnosis of benign, atypically proliferating (borderline) and malignant serous ovarian tumours. METHODS: Cytological imprints of benign (n = 20), borderline (n = 19) and malignant (n = 20) ovarian serous tumours were analysed. A computerized, digital analysis was used to determine morphometric nuclear features, the number and characteristics of single AgNORs, cluster AgNORs, total AgNOR and AgNOR area/nucleus (relative area) ratio. According to their size AgNORs were classified in three categories. A one-way variance analysis and post hoc test (Scheffé) were used for statistical analysis. RESULTS: The morphometric nuclear analysis showed that benign, borderline and malignant serous ovarian tumours are statistically different (P < 0.001) according to the area and outline, the values being highest in malignant tumours and lowest in the borderline group. Digital analysis of AgNORs in benign, borderline and malignant groups showed that the total AgNOR number increases with progression of the lesion (meaning tumour malignancy) significantly (P < 0.001) between benign and malignant as well as between borderline and malignant serous ovarian tumours (P < 0.001). The progression of the lesion malignancy was accompanied by a significant (P < 0.001) progressive increase of the total and relative AgNOR area per nucleus. The AgNOR size increases from benign to malignant tumours and a statistically significant difference (P < 0.001) was observed in all three groups regarding small and large AgNORs. CONCLUSION: Combining different markers of morphometric nuclear characteristics and AgNOR values could improve differential cytodiagnosis of benign, borderline and malignant serous ovarian tumours.     

Prognostic value of nucleolar organizer regions in neuroendocrine tumours of the lung

The value of the number and size of the nucleolar organizer regions (NORs) as prognostic indicators in human neuroendocrine lung tumours was evaluated in a quantitative study of 57 cases, including 33 small cell carcinomas (SCLCs), 9 well-differentiated neuroendocrine carcinomas (WDNECs) and 15 classic carcinoids. NORs were visualized on paraffin sections by an argyrophilic technique (AgNOR) and measured by automatic image analysis. In each case, the mean number and area of AgNORs were evaluated; the results were compared with clinical follow-up and survival. AgNOR values for both number and area were significantly higher in SCLCs than in WDNECs and carcinoids. WDNECs had insignificantly higher AgNOR values than carcinoids. Among SCLCs, AgNOR values of the oat cell subtype and the intermediate cell subtype did not differ significantly. Regardless of the histological tumour type, AgNOR values strongly correlated with prognosis, with more and larger AgNORs indicating a more progressive clinical course. In the present study we demonstrate for the first time that the biological behaviour of neuroendocrine lung tumours is correlated with the number and size of AgNORs. Thus the measurement of AgNORs may serve as an additional prognostic indicator in these neoplasms, particularly in the separation of SCLCs from WDNECs with a more favourable prognosis.     

AgNOR quantification in cells of normal oral mucosa exposed to smoking and alcohol. A cytopathologic study

OBJECTIVE: To evaluate cell proliferative activity by counting and measuring argyrophilic nucleolar organizer regions (AgNORs) per nucleus in cell smears from mucosa clinically exposed to smoking and alcohol. STUDY DESIGN: Group 1 (control) consisted 17 patients, group 2 (smoking) of 25 and group 3 (smoking and alcohol) of 18. Cell smears collected from the mucosa of the lower lip, border of the tongue and floor of the mouth underwent AgNOR staining. Mean number and mean area of AgNORs per nucleus were calculated for the first 50 cells in each smear. ANOVA and the Tukey test were used for statistical analyses at a 5% significance level. RESULTS: Statistical analyses revealed a greater mean number and larger mean area of AgNORs per nucleus in groups 2 (smoking) and 3 (smoking and alcohol). Samples from the border of the tongue had the lowest mean values for number and area of AgNORs per nucleus in comparison with samples from the lower lip and floor of the mouth in the 3 groups. CONCLUSION: Anatomic sites exposed to smoking or to smoking and alcohol had increased cellular proliferative activity.     

Proliferative activity of gastric epithelial cells in Helicobacter pylori infected children

Helicobacter pylori infection has been associated with gastric carcinogenesis. Gastric epithelial cells proliferative rate is accelerated in H. pylori infected adult patients. Our study was performed to evaluate proliferative cell activity in gastric epithelium in the course of H. pylori infection in the early stage of its natural history. Gastric antral biopsy specimens were obtained from thirteen H. pylori positive and seven negative children. To assess replication rates we used nucleolar organiser regions staining with colloidal silver nitrate technique (AgNOR). The number of AgNORs per nucleus, area of single AgNOR, and the quotient of these two parameters (AgNOR content) were analysed. The mean area of AgNOR was lower in H. pylori positive than in negative children. Conversely, both the mean number of AgNOR per nucleus and AgNOR content were higher in infected than non infected subjects. These results show accelerated proliferation of gastric antral epithelial cells in the course of H. pylori infection in children. Such alteration of cell replication occurring in an initial phase of natural history of long lasting infection provides an explanation for the association between acquisition of H. pylori infection in the first years of life and the development of gastric cancer.     

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.     

Different proliferation patterns in breast cancer: AgNOR measurements in ER-negative and ER-positive tumor cells.

The relation between estrogen receptors (ER) and argyrophilic nucleolar organizer regions (AgNORs) in situ within human breast cancer cells was analyzed. For AgNOR measurements in 49 invasive breast carcinomas, a new reproducible staining method for dual demonstration of ER and AgNORs was applied. Quantitative AgNOR variables were determined in ER-positive and ER-negative tumor cells by digital image analysis. The relationships between AgNOR parameters of ER-positive and ER-negative cells and other prognostic factors of breast cancer [Bloom-Richardson-Grading and growth fraction (Ki-67 index)] were investigated. A higher AgNOR content in ER-negative cells and a special clustering phenomenon in ER-positive tumor cells were found. Correlation with other criteria of malignant potential could be exclusively demonstrated for ER-negative cells. ER-negative cells of breast cancer can be characterized as the more malignant and possibly prognosis-dictating cell fraction. Thus, ER-negative cells probably contribute more to the progression of the tumor disease and furthermore to the prognosis than ER-positive cells. We recommend measurement AgNORs exclusively in ER-negative cells of breast cancer.     

Argyrophilic nucleolar organizer regions (AgNORs) in interphases and metaphases of normal and neoplastic gill cells of Macoma balthica (Bivalvia: Tellinidae) from the Gulf of Gdansk, Baltic Sea

Chromosome analysis of gill cells of different populations of Macoma balthica (L.) from the Bay of Gdansk (Baltic Sea) revealed 2 clam categories, 1 with neoplastic features and 1 without. Silver-staining was performed on interphase and metaphase cells of both categories. The mean argyrophilic nucleolar organizer region (AgNOR) count per abnormal interphase cell was significantly higher than in normal interphase cells. Normal silver-stained metaphases had 3 nucleolar organizer region (NOR) chromosome phenotypes. The location of the NORs in the most frequent phenotype (55.6% in 54 metaphases scored) was interstitial on the largest metacentric chromosome pair, Pair No. 1. Abnormal silver-stained metaphases had a higher number of active NOR sites. Different phenotypes were observed (frequency greater than 10% for 67 metaphases scored); 2 were similar to those in normal metaphases and 5 were ectopic. The higher activity of AgNORs observed in abnormal cells confirmed the diagnosis of malignant neoplasia.