Reliability and significance of DNA measurements in interphase nuclei and division figures in histological sections.

DNA contents from single cells at interphase and division were analysed in histological sections and in imprints from 73 breast cancer specimens. Fetal livers from 18 terminations of normal pregnancies provided the standard for truly mitotic prophases, metaphases and telophases. The reliability of DNA quantities from image microphotometry was improved using paraffin-embedded tissue samples from which 4, 8 and 15 microns slices were Feulgen stained. Imprinted replicas from the mirror surface of each freshly cut specimen provided matching domains and represent the crucial approach in this project. A close positive relationship was observed between interphase nuclei in 8 microns sections and their imprinted counterparts (r = 0.992; n = 73). Interphase nuclei in 4 microns sections yielded insufficient DNA contents when compared with the imprints (r = 0.815; n = 21) and with endogenous lymphocyte nuclei. This 2 cDNA standard also calibrated 232 mitotic figures to 3.91 +/- 0.01 c in 15 microns sections from fetal liver. Prophases, metaphases and telophases were slightly scattered (coefficient of variation = 0.04 each). The 0.09 c deficiency to plain 4.0 c was read as an artifact from sectioning. However, the methodical bias did not challenge the most irregular DNA distribution profiles recorded from chromosome division figures (CDFs) in 15 microns sections of breast cancers. Poorly differentiated and aggressive breast cancer (Auer type IV, Zetterberg type A) exhibited a 4.5 c exceeding rate of 82.24% from a total of 752 CDFs in 10 randomly selected cases. Well differentiated, slowly growing cancer with diploid interphase nuclei (Auer I, Zetterberg D) surprisingly showed a 4.5 c exceeding rate of 29.26% from a total of 173 mitoses and CDFs in 10 randomly selected cases. The bulk of data beyond the mitotic 4.0 c level discriminates biological bias from methodical impairment. We concluded that 8 microns sections are sufficient for human interphase nuclei, whereas a depth of 15 microns preserves intact mitoses and CDFs.

The impact of tumour angiogenesis, p53 overexpression and proliferative activity (MIB-1) on survival in squamous cervical carcinoma.

Tumour angiogenesis (antifactor VIII-related antigen antibody), p53 overexpression (DO-1) and proliferative activity (MIB-1) were immunohistochemically analysed for the prediction of long-term survival in 113 patients with squamous cervical carcinoma. The median follow-up time was 82 months (range 72-99). In early stages (IB-IIA), neovascularisation was significantly related to tumour size. Significantly more patients in stage IIA had high tumour vascularity compared to stage IB (P < 0.01) but no significant difference was found between early and advanced stages (IIB-IVB) of cervical carcinoma. p53 overexpression was correlated to the stage of disease (P < 0.01). No relationship was found between tumour angiogenesis, p53 overexpression or MIB-1 and pelvic lymph node metastases, histological subtype or differentiation. Tumours with more than 50% p53 overexpression was significantly correlated with survival in the univariate analysis, but no independent predictive value was found. It is concluded that immunohistochemically detectable p53 overexpression as measured by DO-1 and proliferative activity as measured by MIB-1 seems of no clinical value for the prediction of long-term survival in squamous cervical carcinoma. The predictive value of tumour angiogenesis for survival outcome has still to be determined in squamous cervical carcinoma.

Immunohistochemical expression of the mutant p53 protein and nuclear DNA content during the transition from benign to malignant breast disease.

Immunohistochemical expression of the cellular phosphoprotein p53 was investigated in archival, formalin-fixed, and paraffin-embedded surgical breast tissue specimens from 543 patients using the polyclonal antibody CM-1. Cytometric DNA assessments were performed on histopathologically or cytopathologically identified cell nuclei using image analysis. The series included five samples of normal resting breast parenchyma, 35 benign lesions including benign tumors, 54 hyperplastic lesions with and without atypia, 109 carcinomas in situ, and 340 invasive adenocarcinomas. In 56 of the latter cases specimens from corresponding lymph node metastases also were investigated. Mutant p53 protein expression was absent in normal resting parenchyma and in benign lesions, including benign tumors and epithelial hyperplasias. However, 14 of the 54 hyperplasias (26%) were found to be of DNA aneuploid type. Thirteen of 109 (12%) carcinomas in situ and 79 of 340 (23%) invasive neoplasms expressed the mutant p53 protein. Eight of nine (89%) p53 immunoreactive carcinomas in situ and 62 of 78 (80%) invasive carcinomas with p53 expression were DNA aneuploid. In invasive carcinomas p53 expression was absent in well differentiate neoplasms. In contrast, 58 of 158 (37%) poorly differentiated invasive carcinomas immuoreacted. Intraductal carcinomas of comedo type and poorly differentiated invasive carcinomas of comedo type expressed the mutant p53 protein in seven of 18 cases (39%) and in 14 of 22 cases (64%), respectively. The staining behavior of lymph node metastases was the same as that of the corresponding primary tumors. The present findings suggest that chromosomal alterations as indicated by DNA aneuploidy occur in precancerous lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical c-erbB-2 protooncogene expression and nuclear DNA content in human mammary carcinoma in situ.

Immunohistochemical c-erbB-2 proto-oncogene expression and nuclear DNA distribution patterns were assessed in 119 formalin-fixed paraffin-embedded surgical specimens of human mammary carcinomas in situ (CIS). The series consisted of 107 ductal carcinomas in situ (DCIS), 9 lobular carcinomas in situ, and 3 cases of Paget's disease of the nipple. Nuclear DNA distribution patterns were assessed by image cytometric analysis of histopathologically identified cell nuclei. Fifty-one of 107 (48%) DCIS were immunoreactive for c-erbB-2, whereas specific cell membrane staining was absent in lobular carcinomas in situ. The neoplastic cell nuclei of 46 CIS (39%) were of DNA diploid type, and 73 CIS (61%) contained aneuploid nuclear DNA. Among various histopathologic subtypes of DCIS, significant differences in c-erbB-2 immunoreactivity and nuclear ploidy were observed. DCIS of the comedo type most often were c-erbB-2 positive and exhibited aneuploid nuclear DNA histograms. DCIS of micropapillary type was the second most frequently reactive c-erbB-2 expression, and aneuploidy were less common in solid, cribriform, and papillary DCIS. The results of the current study indicate that immunohistochemical expression of the c-erbB-2 proto-oncogene product is closely related to the histopathologic subtype and the nuclear DNA content of mammary CIS. Examples of CIS that are c-erbB-2 immunoreactive and DNA aneuploid seem to have a significantly higher risk for the subsequent development of infiltrating mammary carcinoma.

The relationship between aneuploidy and p53 overexpression during genesis of colorectal adenocarcinoma.

This paper describes the investigation of nuclear DNA content and p53 immunoreactivity in normal mucosa (n = 25), mildly (n = 15), moderately (n = 28) and severely atypical (n = 22) colorectal adenomas and in colorectal adenocarcinomas (n = 116). Twenty-seven per cent of the mildly atypical, 43% of the moderately, 77% of the severely atypical adenomas and 91% of the colorectal carcinomas were distinctly aneuploid. In the aneuploid lesions p53 immunoreactivity was not observed in mildly atypical adenomas, whereas 17% of the moderately atypical, 24% of the severely atypical adenomas and 66% of the adenocarcinomas were p53 positive. None of the diploid lesions were p53 immunoreactive. These data are interpreted to indicate that genomic instability as reflected by crude aneuploidy occurs early during genesis of colorectal carcinoma and represents a high risk factor for p53-gene mutation.

Prognostic significance of immunohistochemical c-erbB-2 proto-oncogene expression and nuclear DNA content in human breast cancer.

Immunoreactivity of the c-erbB-2 proto-oncogene product and nuclear DNA content were assessed in specimens from 211 breast cancer patients with a mean follow-up of 16 years (range 13-19 years). A routine immunoperoxidase technique was used and cytometrical DNA assessments were performed on cytodiagnostically identified tumour nuclei, using image analysis. C-erbB-2 cell membrane staining was observed in 29% of the cases and was found to be related to tumour size (P = 0.02), histopathological grade (P = 0.02) and nuclear DNA content (P < 0.01). In univariate analysis immunohistochemical c-erbB-2 expression was of prognostic significance among node-positive patients (P = 0.02), but not among women with node-negative disease. This prognostic ability was reduced by multivariate analysis and was no longer significant. In contrast, nuclear DNA content was significantly related to distant recurrence-free survival even in multivariate analysis after adjustment for nodal status and tumour size (P < 0.01). In conclusion, the findings of the present study indicate that c-erbB-2 expression is of limited prognostic value in a subgroup of patients, whereas nuclear DNA content seems to provide significant prognostic information even in node-negative patients.

Diagnostic impact of nuclear DNA content and proliferative activity in benign and malignant melanocytic lesions.

Nuclear DNA content was assessed, using image cytometry, in adult melanocytes in normal skin and in 20 intradermal naevi, 60 junction naevi, 107 compound naevi, 61 dysplastic naevi, 17 melanomas in situ and 101 primary malignant melanomas. Proliferation was estimated using mitotic counting and immunohistochemical staining by anti-Ki-67 (clone MIB1) monoclonal antibodies. All normal adult melanocytes; and intradermal naevi (97% junction naevi, 98% compound naevi, 66% dysplastic naevi) were diploid. Thirty-four percent of the dysplastic naevi, 88% of the melanomas in situ and 96% of the malignant melanomas were clearly aneuploid. Proliferation, as assessed by mitotic counting and MI81 index, was significantly higher in aneuploid invasive malignant melanomas than in aneuploid dysplastic naevi (P<0.0001). The results indicate that histomorphologically defined entities of melanocytic lesions are characterized by typical DNA distribution patterns. Distinct aneuploidy combined with high proliferation rates generally seem to be well correlated to an increased malignancy potential of the lesion. In dysplastic naevi, the clonic expansion of aneuploid naevus cells may be limited by host defence mechanisms. Thus, DNA aneuploidy seems to indicate increased risk of malignant transformation, but has to be combined with other data, such as proliferation, in order to differentiate more precisely between different melanocytic lesions.

Methodological aspects of DNA image cytometry in formalin-fixed paraffin-embedded material from pancreatic adenocarcinoma.

Deparaffinized and disintegrated material from conventionally formalin-fixed and paraffin-embedded surgical specimens of 100 cases of ductal adenocarcinoma of the pancreas was Feulgen-stained, and the cytochemical DNA distribution patterns of at least 100 single tumour cells and 50 "control" cells (fibrocytes) were assessed by means of image cytometry (ICM). In 77 cases a sufficient number of neoplastic cells could be obtained for these DNA assessments. The fairly high number (23) of cases that had to be excluded due to too small amounts of disintegrated cells or cell nuclei may be explained by the high content of connective tissue stroma in these pancreatic adenocarcinomas. The tumour cell nuclei in 76 of these 77 cases showed cytochemically a clear-cut "non-diploid" DNA distribution pattern. This observation reflects the well-known highly malignant growth potential of this carcinoma. Despite the fact that about 1/4 of the tumours had to be excluded, the main result of our methodological study is, after all that conventionally formalin-fixed paraffin-embedded specimens of most pancreatic adenocarcinomas can be successfully used for the deparaffinization-disintegration procedure preceding the nuclear DNA assessments by means of ICM. Additional studies are, however, required to obtain the diagnostic and prognostic impact of the results of such cytochemical analyses of the DNA distribution pattern in adenocarcinomas of the pancreas.

Genome instability in human tumorigenesis: microphotometry of interphase nuclei and pathologic mitoses reveals dysplasia.

Tumorigenesis goes with genome instability comprising point mutations and gross chromosome aberrations due to defective DNA repair mechanisms and multiple overrun of cell cycle checkpoints. Pathologic mitoses (CDFs) occur in human precancers and cancers and were detected by nuclear DNAs deviating more or less than 0.5 c from 4.0 c values. Abundant CDFs were recorded above 4.5 c threshold in lesions of the uterine cervix and the stomach. Low-grade dysplasias and well differentiated carcinomas showed 3%-10% CDFs, high-grade dysplasias 30%-44% CDFs in total divisions. Poorly differentiated cancers comprised some 50% CDFs. Most telophase CDFs showed asymmetric morphology and unbalanced DNA content in their corresponding "halves". CDFs precede DNA aneuploidy of interphase nuclei not only in precancers, but also in cancer. Chromatin bridges and lagging chromosomes suggest that unbalanced telophases are caused by somatic nondisjunction. Tumour progression from low-grade to high-grade dysplasia and cancer is characterised by recurrent shifts from 2 c to 4 c interphase nuclei and a remarkable increase in the 5 c exceeding rate. Clonal selection is the gateway in tumorigenesis for aberrant karyotypes.

Mammary carcinoma. Comparison of nuclear DNA content from in situ and infiltrative components.

Twenty-eight mammary carcinomas were analyzed with respect to their nuclear DNA content. Ten of the carcinomas were entirely in situ (noninfiltrative) while 18 showed areas of both infiltrative and noninfiltrative growth. The DNA content of individual tumor cells was measured in sections from the original paraffin-embedded specimens. In the tumors that had noninfiltrative as well as infiltrative zones, DNA analyses were performed in both areas. Comparison between the DNA patterns obtained from these different areas of the same tumor showed very close agreement. Both groups of tumors (those with and those without areas of invasion) contained some cases that showed a euploid DNA pattern and some cases that showed an aneuploid pattern. Furthermore, analysis of the DNA content of regional lymph node metastases in seven of the invasive cases did not show an increased aneuploidy in the metastases. The results suggest that, in mammary carcinomas, invasive and noninvasive tumors cannot be distinguished by DNA analysis and that tumor progression does not seem to be associated with a significant alteration of the nuclear DNA content.

Nuclear DNA changes during pathogenesis of squamous carcinoma of the cervix in 3,4-benzopyrene-treated mice.

The pathogenesis of squamous carcinoma of the uterine cervix was studied by means of morphologic and cytochemical techniques in mice treated with 3,4-benzopyrene. The results indicate that carcinogen-induced transformation of normal cervical epithelium into squamous carcinoma occurs through a sequence of cellular alterations, demonstrated by progressively increasing cellular atypia and a progressively increasing and scattered DNA content.

Prognostic significance of DNA measurements in 409 consecutive breast cancer patients.

Four hundred nine consecutive breast cancer patients were studied retrospectively. Microspectrophotometric DNA measurements were performed using archival, fine-needle slide preparations upon which the primary diagnoses had been based 8 to 13 years earlier. The DNA distribution patterns of the tumor cell populations were analyzed according to various criteria and the cytochemical data were correlated to the clinical course, defined as distant recurrence-free survival. The results demonstrated a strong relationship between nuclear DNA content of the breast cancer cells and prognosis. Tumors exhibiting DNA values within the limits of normal tissues (DNA euploidy) were found to be correlated with a favorable prognosis. In contrast, tumors with increased and scattered DNA values (DNA aneuploidy) were found indicative of poor prognosis. This was found to be the case regardless whether the percentage of cells above 2.5c or 5c, DNA index/modal value, or the histogram typing according to Auer et al were utilized to discriminate low-grade from high-grade malignant cases. All of these DNA variables were also shown to be significantly correlated. With the aid of the Cox regression method, the additional prognostic value of any given variable was tested against the others. The statistical analyses showed that the histogram typing gives significant prognostic information in addition to that provided by any other variable. In conclusion, the current study demonstrates that tumor nuclear DNA content is a strong indicator of prognosis in patients suffering from invasive breast adenocarcinoma. However, the results also show that simple determination of the stemline position is not the optimal DNA measure of intrinsic tumor malignancy potential. The fraction of cells scattered outside the modal peaks of the histograms are of utmost importance for adequate cytochemical malignancy grading in breast carcinomas.

Predictive value of nuclear DNA content in breast cancer in relation to clinical and morphologic factors. A retrospective study of 227 consecutive cases.

The predictive value of nuclear DNA content in breast cancer in relation to clinical and morphologic factors was studied in 227 consecutive cases of invasive breast adenocarcinomas with follow-up periods of 8 to 13 years. The results show that, with the use of Cox multivariate analysis nuclear DNA content provided significant prognostic information additional to that given by all other clinical and histomorphologic variables taken together. This fact indicates that the DNA content of breast cancer cells reflects biological properties, associated with the malignant behavior of the tumor, other than those determining the stage of the disease. Nuclear DNA content was strongly correlated to histopathologic grading of the ductal carcinomas, with poorly differentiated tumors more likely to be aneuploid. On the other hand, no clear correlation was found to exist between nuclear DNA content and axillary node status, indicating that these two factors are independent prognostic parameters. It is noteworthy that DNA content provided additional prognostic information within both the node-negative and node-positive patient groups. In summary, the results shown here indicate that nuclear DNA content, as an objective biological marker of tumor aggressiveness, can significantly improve our prognostic capabilities within the currently designated stages.

DNA content and survival in mammary carcinoma.

In a retrospective study of archival fine needle aspiration biopsy material from 112 patients with primary mammary carcinoma, the DNA distribution patterns of the cancer cell populations were determined. A distinct correlation was found between the occurrence of certain types of DNA distribution histograms and the survival time of the patients. Thus, the data indicate that DNA determinations can give prognostic information, in the individual case, over and above that furnished by clinical staging and morphologic criteria.

DNA ploidy in human colorectal adenomas.

The DNA content of 101 colorectal adenomas of various histologic types resected from 83 patients was determined by image cytometric measurements in order to investigate if a correlation between DNA ploidy and particular histomorphologic features exists and if DNA measurements can be of additional diagnostic value. Overall, 67 of 101 (66%) adenomas showed an aneuploid DNA distribution pattern, including 8 of 19 (42.1%) mildly atypical, 32 of 48 (66.7%) moderately atypical and 27 of 34 (79.4%) severely atypical adenomas. Correlating DNA content with the histologic type, 17 of 42 (40.5%) tubular, 28 of 37 (75.7%) tubulovillous and all 22 villous adenomas exhibited aneuploid DNA histograms. Aneuploidy was also observed more frequently in larger adenomas. The results show a good correlation between tumor size, histomorphologic features and DNA content. The most remarkable observation is that as many as 42% of the adenomas histomorphologically considered mildly atypical exhibited aneuploidy. Since aneuploidy has been demonstrated to indicate premalignant or malignant cellular alterations, DNA image cytometry is suggested for providing valuable additional information on the diagnosis of colorectal adenomas.

Dissociation of cellular protein and DNA content in mild and moderate dysplasia as a reflection of the degree of aneuploidy in cancer.

Tumor progression was analyzed in vivo. The bronchial epithelium in five beagle dogs was weekly treated by 20-methylcholanthrene (20-MC). Bronchial cells were harvested before each application of the drug. The cytologic criteria used in the diagnostic procedure were based on a grading developed for Papanicolaou-stained preparations of human squamous bronchial epithelium. The cells were then destained and restained by Feulgen-naphthol yellow S technique. An increased variation in the protein/DNA ratio was an early event in tumorigenesis; it occurred even before aneuploid cells appeared in mild dysplasia, as compared with the control cells. A large increase in the coefficient of variation (CV) in the protein/DNA ratio in mild dysplasia vis-a-vis the control cells was positively correlated to the degree of aneuploidy occurring later in tumorigenesis. These results were compared with the findings in breast cancer cells from patients with near-diploid, aneuploid and near-tetraploid tumors. The CV in the protein/DNA ratio was significantly higher in the aneuploid tumors, indicating an increased dissociation between cell growth and DNA synthesis.

Immunohistochemical expression of the mucin-type glycoprotein A-80 and prognosis in human breast cancer.

Immunohistochemical expression of the tumour associated mucin-type glycoprotein A-80 was investigated in a series of 173 breast cancer patients with a clinical follow-up between 13 and 19 years. A routine immunoperoxidase technique was used in formalin-fixed, paraffin-embedded surgical tumour specimens. One hundred and fifty of 173 tumours (87%) immunostained with MAb A-80. The degree of A-80 immunoreactivity was related to the tumour grade but not to lymph node status, tumour size, or nuclear DNA distribution pattern. In univariate analysis the degree of A-80 expression was found to be of significant prognostic value both in node negative and in node positive breast cancer patients (P = 0.03). Patients with non-A-80 immunoreactive tumours had significant longer distant metastases-free survival times and fewer relapses than women with carcinomas composed of A-80 immunoreactive tumour cells. This prognostic value was reduced in a multivariate analysis, including lymph node status, tumour size, and nuclear DNA distribution pattern, but retained borderline significance (P = 0.08). In conclusion, the findings of this study indicate that expression of the mucin-type glycoprotein A-80 as determined by immunohistochemistry seems to be related to clinical outcome in breast cancer patients.

Association of immunohistochemical p53 tumor suppressor gene protein overexpression with prognosis in highly proliferative human mammary adenocarcinomas.

An increasing body of evidence suggests that in addition to conventional histopathologic tumor characteristics, DNA content measurements, cell kinetic data, and investigations of tumor suppressor gene expressions might be of valuable information in breast cancer patients. Against this background we investigated immunohistochemically overexpression of the interphase associated protein proliferating cell nuclear antigen (PCNA) and the mutant p53 protein in routinely paraffin-embedded surgical specimens from 180 breast cancer patients with known nuclear DNA profiles. The mean clinical follow-up was 16 years (range 13-20 years). The percentage of PCNA immunoreactive tumor cell nuclei ranged between < 5% and 60% (mean 13.59 +/- 10.85%). There was a direct association between high levels of PCNA expression (> 20%) and p53 protein overexpression (p = 0.001), high histologic tumor grade (p = 0.009), and DNA aneuploidy (p = 0.019). Mutant p53 protein overexpression was found in 44 of 180 (24%) cases and was significantly related to high histologic tumor grade (p = 0.004), DNA aneuploidy (p = 0.001), and high levels of PCNA expression (p = 0.001). Patients with highly proliferative carcinomas (> 20% PCNA expression) had a shortened distant metastases-free survival when their neoplasms overexpressed p53. In contrast, the distant metastases-free survival of patients with highly proliferative, p53-negative tumors was significantly longer (p = 0.03). Immunohistochemical p53 protein overexpression thus appears to be indicative of an increased malignant potential in breast cancer patients. Highly proliferative tumors composed of p53 immunoreactive neoplastic cells clinically seem to behave more aggressively than the highly proliferative p53-negative tumors.

Prognostic value of the combined assessment of proliferating cell nuclear antigen immunostaining and nuclear DNA content in invasive human mammary carcinomas.

The expression of the S-phase associated, nuclear protein proliferating cell nuclear antigen (PCNA) was investigated in routinely paraffin-embedded surgical specimens from 209 breast cancer patients. Cytometric DNA assessments were performed on fine-needle aspirates, upon which the primary diagnosis of breast cancer had been based. The mean clinical follow-up was 16 years (range 13-20 years). The percentage of PCNA immunoreactive tumour cells ranged between less than 5 to 60% (mean value 13.34%). There was a direct association between PCNA expression, high histological tumour grade (p < 0.01), and DNA aneuploidy (p = 0.009). In a subgroup of 22 patients with near-diploid DNA distribution patterns the PCNA expression yielded additional prognostic information. Patients with tumours of near-diploid DNA histograms and more than 20% of PCNA immunoreactive neoplastic cells had a significantly worse clinical course, than patients with near-diploid tumours containing less than 20% PCNA immunoreactive cells (p = 0.0001). In contrast, the PCNA immunoreactivity did not yield additional prognostic information for patients with distinctly diploid or highly aneuploid tumour variants. In a multivariate analysis comprising all 209 patients, nodal status (p < 0.01), tumour size (p < 0.01), and DNA ploidy (p < 0.01) were found to have significant prognostic effect. The findings indicate that carcinomas characterised by high proliferative activity and near-diploid DNA distribution patterns can show rapid tumour progression. The combined assessment of the PCNA immunoreactivity and of the nuclear DNA content in routinely processed surgical specimens of breast cancer patients appears to be of prognostic value.