DNA and nuclear protein measurement in isolated nuclei of human endometrium

Feulgen-DNA and nuclear light green-protein measurements have been performed in isolated nuclei of normal (nonmalignant) and malignant human endometrial homogenates. The DNA content of the G0/G1 fraction of malignant endometrium showed much overlap with that of normal endometrium, or was slightly increased. Two of the 18 carcinomas were clearly aneuploid. No correlation was found between the histological grade and the DNA content. The tumors of clinical stage II and higher all had a higher DNA content than that of normal endometrium. The percentage of cells present in the proliferative fraction was higher in proliferative endometrium than in secretory and post-menopausal atrophic endometrium. For malignant endometrium, percentages were found comparable to that of normal endometrium or higher. No correlation was found with the histological grade. Tumors of stage II and higher had intermediate values compared to those of carcinomas below stage II. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. However, for post-menopausal women, most values of the carcinomas exceeded that of normal, atrophic, endometrium. Within the tumor population, no correlation was found with the histological grade. Higher values were found with tumors of clinical stage II and higher.     

Evaluation of DNA and nuclear protein features for their use in describing normal and malignant endometrium

Different feature sets (geometric, densitometric, and textural) derived from DNA and nuclear protein staining were evaluated for their use in describing atrophic, secretory, and proliferative endometrium, and well-differentiated stage I and moderately differentiated stage I adenocarcinomas of the endometrium. It was found that the pattern of significant differences among these groups varied between feature sets, while remaining consistent within a set of features. The DNA density and run-length features were not very effective in describing group mean differences, whereas the co-occurrence features revealed significant differences among most groups. The protein run-length features were the only ones that consistently showed a difference between proliferative endometrium and well-differentiated adenocarcinomas. Analyses repeated on only cells in the G0/G1 DNA region improved the differentiation between moderately differentiated adenocarcinomas and the other groups. It was concluded that the use of DNA and nuclear protein texture features are effective in describing group differences that cannot be described by DNA content only.     

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non-epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.     

DNA and nuclear protein characteristics in non-neoplastic and neoplastic endometrial tissue

Feulgen-DNA and nuclear protein (NP) measurements were performed on non-neoplastic and neoplastic endometrium. Non-neoplastic endometrial cells were exclusively characterized by euploid nuclear DNA content. The NP content may vary significantly in relation to the proliferative stage as reflected by a 2-3-fold increase NP/DNA ratio in growing as compared to growth arrested cells. Endometrial adenocarcinomas could be subdivided into euploid and aneuploid types. The euploid tumors were found to exhibit DNA and NP characteristics comparable with those of normal tissue. In contrast, aneuploid tumors showed DNA and NP characteristics indicating increased proliferative activity as well as a pronounced disorder between the DNA and protein cycle.     

DNA/nuclear protein content in the evaluation of cell cycle modifications during colon carcinogenesis

OBJECTIVE: To investigate the colorectal adenomacarcinoma sequence by biparametric DNA/nuclear protein flow cytometry with the aim of evaluating cell cycle modifications during carcinogenesis. STUDY DESIGN: Paraffin-embedded specimens of 27 adenomas with mild/moderate dysplasia, 20 adenomas with severe dysplasia/intramucosal adenocarcinomas, 28 adenocarcinomas and 14 normal colon mucosa specimens were analyzed by biparametric DNA/nuclear protein content flow cytometric analysis in order to evaluate cell cycle modifications during colorectal carcinogenesis. RESULTS: The mean G0-G1A fraction of the cell cycle was 50.6% (SD +/- 17.2), 25.7% (SD +/- 15.1), 27.8% (SD +/- 11.7) and 29% (SD +/- 13.8) for normal mucosa, adenomas with mild/moderate dysplasia, adenomas with severe dysplasia and adenocarcinomas, respectively. The difference between normal mucosa and the other groups was statistically significant (P < .05), while no significant differences were detectable between adenomas with different degrees of dysplasia and adenocarcinomas. CONCLUSION: Our results show a decrease in G0-G1A in adenomas with mild/moderate dysplasia, suggesting that modification of the cell cycle may represent an early step in colon carcinogenesis, and they support the hypothesis that disregulation of cell cycle-controlling genes is an early event in the adenoma-carcinoma sequence.     

Nucleolar organizer regions in the normal, hyperplastic and carcinomatous epithelium of endometrium.

A silver colloid technique to identify nucleolar organizer region associated protein (AGNORs) has been applied to paraffin sections in a total of 43 endometrial hyperplasias (24 adenomatous and 19 adenocystic) 26 endometrial carcinomas and 22 normal endometria (11 of proliferative and 11 of secretory phase). A morphometric analysis of highly magnified photographic images of AGNORs in light microscopic preparations was performed. Malignant tumor cells showed significantly higher AGNOR numbers, maximum diameter and mean area compared with normal and hyperplastic endometrium, with the exception of adenocystic hyperplasia whose Dmax and mean area were significantly larger. Regarding the distribution pattern of AGNOR dots in the cases studied, it was found that normal and hyperplastic endometrium had a mainly clustered distribution while endometrial adenocarcinomas revealed a scattered one. The significant differences observed in the number of AGNORs, their size and mean area between benign and malignant endometrial epithelia suggest that the AGNOR staining technique is of diagnostic importance in distinguishing between these two groups.     

Nucleolar organizer regions in the normal,hyperplastic and carcinomatous epithelium of endometrium

A silver colloid technique to identify nucleolar organizer region associated protein (AGNORs) has been applied to paraffin sections in a total of 43 endometrial hyperplasias (24 adenomatous and 19 adenocystic) 26 endometrial carcinomas and 22 normal endometria (11 of proliferative and 11 of secretory phase). A morphometric analysis of highly magnified photographic images of AGNORs in light microscopic preparations was performed. Malignant tumor cells showed significantly higher AGNOR numbers, maximum diameter and mean area compared with normal and hyperplastic endometrium, with the exception of adenocystic hyperplasia whose D_max and mean area were significantly larger. Regarding the distribution pattern of AGNOR dots in the cases studied, it was found that normal and hyperplastic endometrium had a mainly clustered distribution while endometrial adenocarcinomas revealed a scattered one. The significant differences observed in the number of AGNORs, their size and mean area between benign and malignant endometrial epithelia suggest that the AG-NOR staining technique is of diagnostic importance in distinguishing between these two groups.     

Flow cytometric analysis of human lung cancer. Correlation with histologic type and stage

DNA ploidy and cell-cycle characteristics of 65 operable lung cancers (41 adenocarcinomas, 19 epidermoid carcinomas, 3 large-cell carcinomas and 2 small-cell carcinomas) were analyzed using flow cytometry. Eighty percent of the tumors were aneuploid. The mean DNA index was lower in epidermoid than in adenocarcinoma. In adenocarcinoma, a low DNA index was correlated with early-stage disease; no correlation between DNA index and stage was observed in the other cell types. The %S-phase cells was highest in two cases of undifferentiated large-cell carcinoma and lowest in adenocarcinoma. The RNA index was increased approximately two-fold in all cell types. Longer follow-ups will be required to establish any correlation between the cell kinetic measurements reported here and survival times.     

Nuclear DNA in histologic sections from squamous-cell carcinoma and adenocarcinoma of the lung

The nuclear DNA content in morphologically identified tumor cells was analyzed in 4-micron histologic sections from 58 patients with lung carcinoma who survived for at least five years. Thirty-three of the carcinomas were invasive squamous bronchial carcinomas and 25 were pulmonary adenocarcinomas. In all squamous carcinomas, the majority of tumor cells were found to exhibit DNA values exceeding the normal tetraploid and/or diploid region. In contrast, some of the pulmonary adenocarcinomas were found to be composed of a majority of tumor cells with DNA values in the normal diploid region. The results indicate that invasive squamous bronchial carcinomas, in general, are tumors with aneuploid DNA patterns indicative of a high malignant potential and that malignancy grading based on DNA measurements does not add any significant prognostic information to that obtained by morphologic diagnosis.     

Review of negative and low‐grade cervical smears in women with invasive cervical cancer after the first 3 years of the national cervical screening programme in Slovenia

A. Repše‐Fokter, A. Pogačnik, V. Snoj, M. Primic‐Žakelj and M. S. Fležar
Review of negative and low‐grade cervical smears in women with invasive cervical cancer after the first 3 years of the national cervical screening programme in Slovenia Objective: The purpose of the study was to perform a national review of negative, low‐grade and inadequate smears reported during the latest screening period before cervical cancer diagnosis in 2006, after the first 3 years of the screening programme. Methods: Among 162 new cervical cancer cases there were 47 (29%) without previous cytology, 47 (29%) with one high‐grade smear prior to diagnosis and 68 (42.0%) with at least one previous negative, low‐grade, atypical or inadequate smear 1–40 months before diagnosis. Of the latter 68 cases, 37 patients with 59 smears (together with 118 control slides) were included in the review as 31 had smears reported at laboratories no longer operating. Findings were related to the last cytology report before diagnosis as well as to histological type and stage of the cancer. Results: In our study group, 19 (51%) of 37 patients had squamous cell carcinoma, 15 (41%) adenocarcinoma and 3 (8%) adenosquamous carcinoma, compared with 121 (75%), 26 (16%), 12 (7%), respectively, and 3 (2%) other types, for all carcinomas. Twenty‐one of 37 women also had high‐grade cytology prior to diagnosis of cancer. Women with previous cytology (with or without recent high‐grade smears) were more likely to have stage I cancers than those without cytology (P < 0.0001). The expert group upgraded 17/33 smears in the patients with squamous carcinomas, which was more than in those with adeno‐ and adenosquamous carcinomas (5/24, P < 0.05). Conclusion: As expected, a higher proportion of smears preceding adenocarcinomas were true negative. Under‐diagnosed smears were not related to cancer stage or last cytology report before diagnosis.     

Nuclear planimetry in renal-cell tumors

A morphometric study using conventional planimetry was carried out in 73 renal-cell tumors (64 adenocarcinomas and 9 adenomas) in order to correlate the objective nuclear measurements with some histologic and clinical data believed to have prognostic value. The use of a discriminant function to correctly separate adenomas and low-grade adenocarcinomas was also investigated. There was a strong association between the nuclear grade and the following parameters: nuclear area, nuclear major diameter and nuclear elongation. There was also a relationship between an adverse outcome and the major nuclear diameter and nuclear elongation. In a univariate survival analysis, only the clinicopathologic stage, the nuclear grade and the histologic presence of a pseudosarcomatous cell population had predictive value. Multivariate survival analysis using Cox models showed that the clinicopathologic stage, major nuclear diameter, presence of anaplastic cells and nuclear area had predictive value. Although the minor nuclear diameter reached a significant level, a discriminant function that would correctly separate adenomas and low-grade renal-cell carcinomas could not be formulated.     

Expression of p53, p21/waf, bcl-2, bax, Rb and Ki67 proteins in colorectal adenocarcinomas

This study investigated the combined immunoexpression of p53, p21, bcl-2, bax, Rb and Ki67 proteins in colorectal adenocarcinomas and correlated expression patterns with tumour stage and grade. Paraffin sections from 98 cases of colorectal adenocarcinomas were stained by immunohistochemistry for p53, p21, bcl-2, bax, Rb and MIB-1 (Ki67) proteins. In addition, 12 cases of colorectal adenomas and normal colorectal mucosa were studied in parallel. P53, p21, bcl-2, bax, Rb and Ki67 proteins were detected in at least 5% of tumour cells in 63/98, 72/98, 52/98, 96/98 and 98/98 adenocarcinomas, respectively. Comparative study of the normal-adenoma-carcinoma tissues revealed abrogation of the normal immunotopography in adenomas and adenocarcinomas, and considerable modifications, increase or reduction, of the expression of p53, p21, bcl-2, bax, Rb and Ki67 proteins in adenocarcinomas when compared with normal mucosa and adenomas. Statistically significant correlations were found between low bax expression and Dukes C stage of carcinomas, Ki67 expression and carcinoma grade, and Ki67 and Rb expression. P53, p21, bcl-2 and Rb immunoexpression did not correlate with tumour stage or grade. Our findings show that low bax immunoexpression is frequently related to colorectal adenocarcinomas with lymph node metastases suggesting that low levels of bax expression play a role in late stage colorectal cancer. The correlation between Ki67 and Rb expression, in view of previous data that the hyperphosphorylated inactive Rb protein is frequently increased in colorectal adenocarcinomas, suggests that Rb protein is somewhat ineffective in inhibiting the cell-cycle progression in these malignancies. Furthermore, our findings provide immunohistochemical evidence that the abrogation of the normal immunotopography and the modifications of the expression of p53, p21, bcl-2, bax, Rb and Ki67 proteins reflect important events in colorectal oncogenesis.     

Correlation of Ki-67 and MCM-2 proliferative marker expression with grade of histological malignancy (G) in ductal breast cancers

The study aimed at examining a relationship between expression of Ki-67 antigen and minichromosome maintenance 2 protein (MCM-2) and a grade of histological malignancy G in ductal breast cancers. The function of widely used marker of proliferation Ki-67 is still not clear. In contrast, the MCM-2 protein is well known to play an important role in controlling the cell cycle. Both proteins represent small protein molecules, which manifest nuclear expression only during cell division of normal and neoplastic cells. Their expression is noted in several malignant tumours. These studies were conducted on 56 archival paraffin blocks of ductal breast cancers. Immunohistochemical reactions were performed using monoclonal Ki-67- and MCM-2-specific antibodies. Statistical analysis demonstrated a positive correlation between expressions of two proteins (r=0.6; p<0.05). The most intense expression of these two markers was demonstrated in G3 grade cancers. Statistical analysis showed more pronounced expression of Ki-67 antigen in G3 grade cancers as compared to cancers of G1 and G2 grades (p<0.001) and, in the case of MCM-2 protein, a more pronounced expression in G3 grade cancers, as compared to those of G1 (p<0.05) or G2 grade (p<0.01). The results obtained in our study suggest that MCM-2 could be used as a marker of proliferation in breast carcinomas.     

Pap smears in women with endometrial carcinoma

OBJECTIVE: To correlate Pap smear findings with the histology of endometrial carcinoma and stage of the disease. STUDY DESIGN: Between 1995 and 1998, 76 women with endometrial carcinoma, having had Pap smears done within two to three months of hysterectomy at Memorial Sloan-Kettering Cancer Center, formed the basis for this study. All Pap smears and histologic sections were reviewed. RESULTS: Thirty-four patients had normal Pap smears (45%), and 42 had abnormal ones (55%). The mean age of the two groups was 65.1 and 65.2 years, respectively. Histologic subtypes included 44 International Federation of Gynecology and Obstetrics (FIGO) grade 1 endometrioid adenocarcinoma (low grade) and 32 high grade carcinomas, including 19 FIGO grade 2 or 3 endometrioid adenocarcinomas, 5 papillary serous carcinomas (PSC), 2 clear cell carcinomas (CC), 1 adenosquamous carcinoma, 3 endometrioid adenocarcinomas mixed with PSC and 2 endometrioid adenocarcinomas mixed with CC. The proportions of patients with low and high grade tumors with abnormal Pap smears were 43% (19/44) and 72% (23/32), respectively (P=.01). The proportions of patients with abnormal Pap smears and no myometrial invasion, invasion of <50% and >50% myometrial thickness were 40% (8/20), 62% (26/42) and 57% (8/14), respectively (P =.27). Vascular invasion was identified in 56% (9/16) of patients with abnormal Pap smears and in 55% (33/60) of patients with normal ones (P = .93). The proportions of patients having abnormal Pap smears with stage I and stages II, III or IV disease were 48% (30/62) and 86% (12/14), respectively (P =.01). CONCLUSION: Although the Pap smear is not a sensitive screening test for endometrial cancer and a negative Pap smear does not rule it out, this study revealed that abnormal Pap smears are significantly associated with high grade of tumor and stage II-IV endometrial carcinoma. However, they are not associated with patient age, depth of myometrial invasion or vascular invasion.     

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.     

Nucleostemin基因在卵巢上皮性肿瘤的表达及与病理分型的关系

目的：研究核干细胞因子Nucleostemin（NS）基因在卵巢上皮性肿瘤中的表达,探讨其与肿瘤病理分型的关系。方法：采用RT-PCR及Western blot检测36例卵巢癌组织手术标本,32例卵巢良性上皮肿瘤组织手术标本,12例正常卵巢组织标本中Nu-cleostemin基因及相应蛋白的表达,采用分组对照的方法对比3组样本中NS基因及蛋白的表达情况,并进行相对定量研究。采用统计学方法检测NS基因的表达是否与临床病理分级及血清CA125存在关联。结果：①卵巢癌组织中NS的阳性表达率显著高于良性肿瘤组织及正常卵巢组织;②卵巢癌组织中,淋巴结转移组NS的表达水平高于未转移组;③临床分期Ⅲ期组的表达水平高于ⅠB期组;④中、低分化组的表达水平高于高分化组。结论：卵巢癌组织中存在NS基因的高表达,其表达量与组织类型无关,而与临床TNM分期及组织分级正相关。     

Image cytometry of aneuploidy, growth fraction (MoAb Ki-67) and hormone receptors (ER, PR) immunocytochemical assays in breast carcinomas

DNA nuclear content was assessed in human breast carcinomas (n = 132) using image cytometry. Optical density histograms of Feulgen stained cell imprints from fresh tissue samples, subsequently frozen for immunocytochemical assays, were determined by the SAMBA system and used for the DNA index, the ploidy balance (PB) and the proliferation index (PI) computation. The three parameters were correlated to (i) histological data (tumour grade, vascular and/or lymph node invasion) and to (ii) growth fraction (Ki67), hormone receptor antigenic sites (ER, PR) and intramedullar (bone marrow) biopsies and anti-KL1-positive epithelial cells. It was shown that 57% of breast carcinomas were aneuploid. Aneuploidy PI significantly correlated to the criteria of poor prognosis such as high tumour grade, vascular and lymphatic invasion and to increased Ki67-positive cells, and the absence of or low ER and PR. Since image cytometry is easy to handle and perfectly suitable for current diagnostic practice in pathology departments, particularly for tumour cell ploidy assessment and standardized analysis of immunostaining procedures with morphological control of the preparation, we conclude that image cytometry, as performed with the SAMBA, must be regarded as a relevant tool for prognosis evaluation and therapy guidance in individual patients.     

DNA ploidy and S-phase fraction in carcinoma of the gallbladder related to histopathology, number of gallstones and survival.

Gallstones are a risk factor for the development of gallbladder cancer. We studied DNA ploidy and cell cycle composition by flow cytometry in archival specimens from 52 gall bladder carcinomas in relation to histopathological grade, tumour stage, gallstone number and survival. 69% of the gallbladder carcinomas showed aneuploidy. All tumours with single stones (N=11) were aneuploid while only 61% of tumours with multiple stones (N=41) were aneuploid (p=0.002). DNA aneuploidy was related to increase in T-category (p=0.01), grade (p=0.02), and nuclear pleomorphism (p=0.0005). The distribution of DNA ploidy shifted from tetraploid in low stage towards triploid positions in high stage tumours (p=0.02) combined with higher S-phase values in triploid tumours (p=0.05). S-phase fraction increased during development from normal tissue to dysplasia, cancer in situ and cancer in diploid cases (p=0.0002), and further at the change from diploid to aneuploid (p=0.004). At a median cancer specific survival time of four months patients with diploid tumours had a better survival than those with aneuploid tumours (p=0.02). In multivariate analysis of the tumour characteristic, only T-category and tumour grade were independent prognostic factors.The shift from diploid to aneuploid and the further shift of ploidy within aneuploid tumours are in agreement with the concept of a clonal development of gallbladder cancer. These changes are combined with a stepwise increase in the fraction of S-phase cells. Low frequency of symptoms in single stone patients may be the reason for detection of malignancy at a late stage of tumour development.     

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.