DNA-ploidy analysis within selected regions of colorectal adenomas containing carcinoma

In order to better understand the relationship of DNA ploidy, dysplasia, early cancer, and colorectal tumor progression, 11 colorectal adenomas containing carcinoma invading the submucosa were investigated using DNA flow cytometry. Multiple frozen samples were taken from the selected sectors corresponding to adenoma tissue with low-grade dysplasia, high grade dysplasia and early cancer. Sampling accuracy was performed under histologic examination by multiple cryostatic sections. Data were compared with previously reported results in non-cancerous adenomas and advanced carcinomas. Incidence of DNA aneuploidy among the dysplastic regions of the adenomas containing carcinomas resulted higher than that observed in non-cancerous adenomas (p=0.02). Furthermore, among the DNA aneuploid populations, the frequency of clones with high DNA Index (DI>1.3) was slightly higher in adenomas with cancer than in adenomas without cancer (p=0.07). We suggest that differences may exist in DNA aneuploidy evolution between these two types of lesions. In early cancer, the near-diploid clones were 57% with respect to 18% (p=0.01) in advanced cancer since in this latter case the majority of the DNA abnormal clones were in the near-hypertriploid region (82%). Thus, the acquisition of the invasive phenotype appears to be linked with the expansion and stabilization of high DNA aneuploid clones. Further analysis on a larger number of cases of adenomas containing carcinoma are necessary to validate these interpretations.     

Frequency and degree of aneuploidy in benign and malignant thyroid neoplasms

The frequency and degree of aneuploidy in 44 benign and 124 malignant thyroid neoplasms were analyzed by DNA flow cytometry. Single aneuploid cell populations were found in 72% of the undifferentiated carcinomas, 64% of the follicular carcinomas, 24% of the papillary carcinomas and in 24% of the follicular adenomas. Multiple aneuploid cell populations were detected in 4% of the papillary and in 36% of the follicular carcinomas but not in undifferentiated carcinomas. A low degree of aneuploidy was found in well differentiated papillary carcinomas (mean DNA index of aneuploid populations: DI = 1.17; SD +/- 0.09). Significantly higher values were found for aneuploid moderately differentiated papillary carcinomas (DI = 1.46; SD +/- 0.29), well and moderately differentiated follicular carcinomas (DI = 1.61; SD +/- 0.33 and DI = 1.60; SD +/- 0.30, respectively) and undifferentiated carcinomas (DI = 1.72; SD +/- 0.19). High DNA indices were also found in several follicular adenomas (DI = 1.49; SD +/- 0.22). Comparison of the 10-year survival rates of patients with moderately versus well differentiated papillary carcinoma (79 vs. 98 months, respectively) indicates that loss of differentiation and progression of aneuploidy in this tumour type is associated with more aggressive clinical behaviour. Similarly, the high frequency and degree of aneuploidy in undifferentiated carcinomas is in agreement with the very poor survival rate (0% at 10 years) in this group of patients. However, the occurrence of highly aneuploid adenomas and (near)-diploid undifferentiated carcinomas does not point to a direct causal relationship between DNA-ploidy changes and clinical behaviour of these thyroid tumours.     

Cyclooxygenase-2 (COX-2) expression in high-risk premalignant oral lesions

Emerging data indicate a link between genetic instability and up-regulation of cyclooxygenase-2 (COX-2). To see if individuals at high risk of oral cancer are candidates for treatment with selective COX-2 inhibitors (coxibs), levels of COX-2 expression in healthy, premalignant and cancerous oral mucosa were compared with the occurrence of DNA ploidy status as a genetic risk marker of oral cancer. COX-2 gene product was evaluated immunohistochemically in 30 healthy persons, in 22 patients with dysplastic lesions without previous or concomitant carcinomas, and in 29 patients with oral carcinomas. The immunohistochemical findings were verified by western blotting. COX-2 expression was correlated to DNA content as a genetic risk marker of oral cancer. COX-2 was up-regulated from healthy to premalignant to cancerous oral mucosa. Thus, COX-2 expression was found in 1 case of healthy oral mucosa (3%). All specimens from healthy mucosa had a normal DNA content. In patients with premalignancies. In 29 patients with oral carcinomas, cyclooxygenase-2 expression was observed in 26 (88%), and aneuploidy was observed in 25 cases (94%, P=0.04). Notably, of 22 patients with dysplastic lesions, COX-2 was exclusively expressed in a subgroup of nine patients (41%) identified to be at high risk of cancer by the aberrant DNA content of their lesions. Seven of these patients were followed for 5 years or more. An oral carcinoma developed in six of them (85%; P=0.02). These findings emphasize the need to determine whether coxibs can reduce the risk of oral cancer in patients with high-risk precancerous lesions.     

Microphotometric DNA analysis in moderate dysplasia of the uterine cervix. Correlation to the progression and regression of the lesion

Nuclear DNA content was determined by microphotometry in 27 cases of moderate dysplasia of the uterine cervix. All these cases were followed from one to ten years to study the biological behavior of moderate dysplasia in relation to cervical carcinogenesis. Ten of them showed progression to carcinoma in situ during a period of one to six years (median 34.8 months) and 17 cases showed regression to inflammation and normalcy during a period of one to ten years (median 44.2 months). DNA analysis was performed both on initial cervical smear and on representative smears collected during follow-up. Amongst ten cases which progressed to malignancy, nine had aneuploid DNA pattern and one had polyploid DNA pattern throughout the follow-up period. Amongst 17 cases which regressed to inflammation, 11 had euploid and six polyploid DNA pattern. The observation that moderate dysplasia with aneuploid DNA value often develops into carcinoma in situ in contrast to dysplasia with euploid or polyploid DNA value strengthens the conception that DNA aneuploidy may be considered as 'high risk indicator' for cervical precancerous lesions.     

Feulgen cytometry in simultaneous endometrial and ovarian carcinoma

The modal DNA value was measured in six patients with simultaneous endometrial and ovarian carcinoma by Feulgen static cytometry. One patient with low-grade endometrial and ovarian carcinoma manifested diploid indices at both sites. Another patient demonstrated aneuploid ovarian carcinoma and diploid endometrial carcinoma, indicating that these were separate neoplasms. The remaining four patients with Stage III disease had aneuploid endometrial and ovarian carcinomas with identical DNA indices. These data support a single neoplastic process with metastasis in the latter four patients. There was good correlation with the clinicopathologic impression on the likelihood of synchronous primaries versus metastatic neoplasms. It was concluded that DNA analysis is a useful adjunct in assessing the probability that spatially separate neoplasms represent metastasis.     

Comparison of flow cytometric DNA content in the primary tumor and in the corresponding lymph-node metastasis of patients with squamous cells carcinomas of the head and neck

The clonal DNA content of the cell populations of primary tumors and corresponding lymph node metastases was analysed by DNA flow cytometry in 48 patients with squamous cell carcinoma of the oral cavity. Three primary tumors were diploid, two associated with diploid metastases, one showing an aneuploid cell line in the secondary lesion. Fourty-five cases were aneuploid in both the primary tumors and in the synchronous metastases, all characterized by nearly identical DNA indices of the corresponding primary and secondary lesions. With few exceptions, aneuploidy formation appears to take place prior to dissemination of metastatic cells.     

Frequent loss of a p53 allele in carcinomas and their precursors in ulcerative colitis

Allelic deletions of the p53 gene previously were demonstrated by Southern hybridization to occur in high frequency in sporadic colon carcinomas and in a variety of other human tumors. We have examined the frequency of allelic loss of the p53 gene in carcinoma and dysplasia arising in patients with chronic ulcerative colitis who are heterozygous for the codon 72 polymorphism in exon 4 of the p53 gene. Cells derived from carcinoma and dysplasia specimens from 10 patients who were heterozygous at this locus were sorted by flow cytometry on the basis of DNA content. The p53 exon 4 region was amplified from diploid and aneuploid populations, via a polymerase chain reaction (PCR), and digested with BstUI. Three of three carcinomas, four of six dysplasias, and one patient who was indefinite for dysplasia demonstrated evidence of allelic loss of the p53 gene. Seven of ten cases of sporadic colon carcinoma, analyzed for comparative purposes, exhibited loss of a p53 allele. These results demonstrate that PCR analysis, followed by restriction endonuclease digestion of a polymorphic locus, can provide a rapid, definitive method for analyzing loss of heterozygosity in small numbers of cells from colonic mucosa. Such loss precedes cancer in ulcerative colitis and can be present in its earliest histologically identifiable precursor.     

Flow cytometric analysis of DNA ploidy in canine mammary tumors

DNA ploidy has been determined using flow cytometry in 23 nonmalignant and 34 malignant (primary and metastatic) mammary tumors from 46 dogs. This parameter was compared with clinical stage, histology, and estrogen and progesterone receptor analysis. Twenty-one of 34 cancers (61.8%) from 32 dogs were DNA aneuploid. Aneuploidy was also found in 4 of 23 nonmalignant tumors (17.4%) from 20 dogs. Regional lymph nodes were involved in 6 of 10 diploid and 3 of 9 aneuploid cancers of dogs with operable disease. The aneuploidy incidence was higher in dogs that had distant metastasis at initial diagnosis (8 of 11) than in those presented with local or locoregional disease (9 of 19), although this difference was not statistically significant. DNA aneuploidy incidence was not found to be related to histological tumor type, histological malignancy grade, nuclear grade, or steroid receptor presence. Heterogeneity in DNA content was found in 4 of 32 cancers (30 dogs) in samples from primary or locally recurrent lesions. In 3 of 16 cancers that were analyzed both at the primary and at secondary sites of growth, a significant variation in DNA content was observed. The degree of aneuploidy in the dog cancers was much lower than seen for human breast carcinomas with a relatively high frequency of hypoploid stemlines (7 of 34 cancers, 20.6%). The frequency distribution of DNA indices in dog mammary cancers indicates that aneuploidy evolution probably differs from that of human breast cancer.     

DNA ploidy analysis by image cytometry helps to identify oral epithelial dysplasias with a high risk of malignant progression

Abnormal DNA content (aneuploidy) has been associated with malignant and premalignant epithelial lesions. The presence of aneuploidy in tumours at an early stage and in dysplastic lesions suggests that analysis of DNA content may be a useful marker for determination of prognosis in these lesions. The aim of this study was to use DNA image cytometry to evaluate aneuploidy in oral dysplastic lesions and to determine whether aneuploidy is associated with malignant progression. Forty-two lesions of oral epithelial dysplasias (OED) that had progressed to oral squamous cell carcinoma (OSCC) and 44 lesions that did not progress were analysed for DNA ploidy using image cytometry of nuclear monolayers prepared from paraffin-embedded tissue. Forty-two OSCC that had arisen from the OED cases and five samples of normal oral mucosa samples (NOM) were also examined. Aneuploidy was found in 14/42 (33.3%) of the OED that progressed, but in only 5/44 (11.3%) of OED that did not progress (p=0.01). A total of 19 OED were aneuploid of which 74% showed malignant progression compared to only 42% of the diploid lesions. The sensitivity and specificity of DNA image cytometry to detect cases with high risk of malignant progression was 0.33 and 0.88, respectively. The PPV and NPV were 0.74 and 0.58. We conclude that aneuploid oral dysplastic lesions have a high risk of malignant progression and that DNA image cytometry might help to identify those lesions most at risk.     

Flow cytometric DNA ploidy analysis of feline mammary tumors

Flow cytometric DNA analysis was performed on biopsies from 9 nonmalignant and 111 malignant (primary and metastatic) feline mammary lesions. In our series, 46.3% of the primary mammary carcinomas appeared to be aneuploid, whereas all but one benign breast lesion were diploid. The degree of aneuploidy in carcinomas was low, with a relatively high number of primary tumors (12 of 82) displaying hypodiploidy. Aneuploidy was not found to be correlated with any specific histological tumor type, vascular invasion, tumor size, or histological malignancy grade or with the separate components thereof. Comparison of the ploidy in primary and metastatic tumors from the same cases revealed a remarkable stability, both in time and location of appearance of the metastases. It is concluded that with respect to DNA ploidy feline mammary carcinoma has more in common with canine mammary carcinoma than with human mammary carcinoma. Further prospective studies are necessary to clarify the implications of aneuploidy in feline mammary carcinoma for tumor behavior and prognosis.     

Measurement of bromodeoxyuridine labeling index, Ki-67 score and Ag-NOR count in breast carcinomas. Comparison with DNA ploidy.

Nuclear DNA content and three cell proliferation markers, the bromodeoxyuridine (BrdUrd) labeling index, Ki-67 score and Ag-nucleolar organizer region (Ag-NOR) counts, were measured in 21 cases of breast carcinoma, and then relationships among them were investigated. The BrdUrd labeling indices and Ki-67 scores were strongly correlated with one another (r = 0.90, p less than 0.001). They were also slightly correlated with the Ag-NOR counts (p less than 0.05). Although growth activity varied greatly from case to case, even within the same histologic grade in breast carcinoma, high histologic grade carcinomas were frequently associated with high values of both the BrdUrd labeling index and Ki-67 score, as compared with low histologic grade ones. The DNA ploidy level (DNA index) was independent of these cell proliferation markers. In the present series, 17 of 21 cases (81%) were aneuploid. All cases without DNA aneuploidy fell into a group of low histologic grade, while 7 of 11 low grade tumors were aneuploid. All three cell proliferation markers were low in 2 of the 4 cases without DNA aneuploidy. It may be possible to identify cases with favorable prognosis in a group of low grade carcinomas on the grounds of diploid DNA content and low values of these markers.     

Comparative analysis of histology,DNA content,p53 and Ki- ras mutations in colectomy specimens with long-standing ulcerative colitis

Neoplastic progression in patients with chronic ulcerative colitis is characterized by the development of epithelial dysplasia, which is accompanied by genetic alterations. This study determined the time of onset of p53 and Ki-ras mutations as well as DNA aneuploidy during histological progression towards carcinoma. In all, 278 samples of 7 colectomy specimens were analyzed by flow cytometry, histology and single-strand conformation polymorphism analysis. Of the samples, 22% (61/278) were dysplastic and 43% (122/278) aneuploid, while 25% (71/278) showed p53 and 4% (11/278) Ki-ras mutations. The correlation between aneuploid status and p53 mutations varied among the patients. A strong correlation was noticed between histological progression from low-grade dysplasia to carcinoma and p53 mutations as well as DNA aneuploidy. Ki-ras mutations were found in 40% (2/5) of the carcinomatous samples. The correlation between p53 mutations and the histological status of the samples suggest the involvement of this genetic event in the development of colon cancer in patients with ulcerative colitis. In contrast to Ki-ras mutations, the appearance of p53 mutations is an early event. Therefore p53 analysis might be helpful in the classification of indefinite dysplasia and in the identification of patients at risk for cancer development. Further studies are necessary to detect the additional genetic alterations preceding the development of DNA aneuploidy. Int. J. Cancer 76:1–6, 1998.© 1998 Wiley-Liss, Inc.     

Regional ploidy variations in signet ring cell carcinomas of the stomach

Regional ploidy variations within individual tumors were analyzed by in-situ cytofluorometry of metaphase cells in Feulgen-stained paraffin sections, using 45 resected stomachs with early and advanced signet ring cell carcinomas. Aneuploid cells were found in one of 30 early cancers and in eight of 15 advanced cancers, and were almost always accompanied by diploid cancer cells in the mucosal part of the cancers. The diploid and the aneuploid cells were generally found to be distributed in different territories in the mucosa, and aneuploid foci were often included in the diploid area. These findings suggest the diploid origin of signet ring cell carcinomas and the occurrence of aneuploidy during the tumor development. Moreover, the aneuploid cells appeared to infiltrate beyond the mucosa more readily than the diploid cells; most of the aneuploid populations already invaded the extramucosal tissue, and the cancer cells infiltrating in the extramucosal tissue were predominantly aneuploid in six of the nine cancers with aneuploidy. Thus, it appears that the occurrence of aneuploid clones may accelerate the progression of signet ring cell carcinomas from early to advanced stages.     

DNA ploidy of liver biopsies from patients with liver cirrhosis and hepatocellular carcinoma: a flow cytometric analysis.

Flow cytometric DNA analysis was used to assess cellular kinetics of needle liver biopsies from patients with liver cirrhosis and hepatocellular carcinoma (HCC). An abnormal DNA content was shown in 44.5% of liver cirrhosis cases and in 78.6% of tumor sites. The number of proliferating cells (S + G2M%) was significantly increased in cirrhotic liver (P < 0.05). Dysplasia was found in 66% of cirrhotic specimens. All negative dysplasia specimens showed a diploid pattern while 69% of positive dysplastic specimens were aneuploid (P < 0.001). In conclusion, cell proliferation, aneuploidy and liver cell dysplasia are important indicators in liver cirrhosis for the development of HCC.     

Flow cytometric DNA analysis of parathyroid tumors. Implication of aneuploidy for pathologic and biologic classification

The previous cytometric studies on parathyroid tumors have provided conflicting data regarding the relationship between DNA content and histopathology, resulting from differences in technical methods and data analysis. This study measured nuclear DNA of parathyroid tumors by flow cytometry in fresh material and determined whether DNA aneuploidy really assists in making a pathologic diagnosis of carcinoma or not. From May 1987 through April 1989, 65 consecutive patients operated on for primary hyperparathyroidism had DNA analysis of the freshly excised parathyroid tumors. Three of the patients had metastatic lesions of parathyroid carcinoma in the lung, cervical lymph nodes, and lung and mediastinal lymph nodes, respectively. Pathologic classifications of the lesions from the other 62 patients were 54 adenomas, four carcinomas, and four hyperplasias. In all the latter patients, hyperplasia was associated with a multiple endocrine neoplasia syndrome. Unequivocal evidence of aneuploidy was found in all of the metastatic lesions and 60% of the primary lesions of the carcinomas, in 9% of the adenomas and in 50% of the hyperplasias. Therefore, parathyroid carcinomas were more apt to be aneuploid than were adenomas (P = 0.0015, both-sided testing). In each of the cases of aneuploid hyperplasia, a small aneuploid peak was found. The high incidence of aneuploidy in patients with multiple endocrine neoplasia type 1 may indicate the presence of clonal heterogeneity of hyperplastic glands and the presence of an abnormal subset of cells that have malignant potential. Cell distribution analysis did not provide any significant information beyond ploidy level. In conclusion, DNA flow cytometric analysis of DNA ploidy patterns is a valuable adjunct to the histopathologic diagnosis of parathyroid neoplasms.     

Progression of diploid tumor cells in aneuploid head and neck squamous cell carcinomas

The development of aneuploid clones from diploid progenitor cells is a regular characteristic of head and neck squamous cell carcinoma progression. While the significance of aneuploidy formation for the acquisition of invasive and metastatic behavior is well documented, little is known about the contribution of diploid tumor cells after aneuploid clones have emerged. To distinguish diploid cells of epithelial origin from benign cellular components, we applied multiparameter flow cytometry of DNA content and cytokeratin (CK) expression to 36 primary tumors. Twenty-seven carcinomas accommodated aneuploid cell lines that stained positive for CK. All diploid cell populations obtained from aneuploid carcinomas contained CK-positive subpopulations as did all of nine tumors that consisted exclusively of diploid cells. The proportions of CK-positive diploid cells ranged between 6% and 80%, independent of whether they were achieved from entirely diploid or from aneuploid carcinomas. CK-gated diploid and aneuploid cell populations showed largely identical S-phase fractions. These results emphasize that diploid tumor cells regularly persist after the development of aneuploid clones and significantly contribute to local tumor progression. Despite the presence of diploid epithelial cells in aneuploid primary tumors, exclusively the aneuploid clones of eight corresponding lymph node metastases were CK-positive. This provides further evidence of a largely reduced metastatic potential of diploid tumor cells.     

DNA/RNA content and proliferative fractions of colorectal carcinomas: a five-year prospective study relating flow cytometry to survival

In our prospective study, flow cytometric analysis of cellular DNA and RNA content was performed on unfixed fresh specimens of colorectal adenocarcinoma taken from 176 patients. Of the 176 tumors, 113 (64%) were aneuploid. There was no correlation between aneuploidy and tumor stage, grade, location, or size. After a median follow-up of 5.6 years, no correlation between DNA or RNA content and patient survival was found. DNA content alone was not an independent prognostic factor when the colorectal carcinomas were segregated by curable and incurable stages. However, normal mucosa, diploid tumors, and aneuploid tumors showed progressively higher proliferation and higher RNA and DNA indices. Proliferative fraction--defined as the percentage of cells in S + G2 and M phases of the cell cycle--was significantly related to ploidy and to Dukes' stage. Despite these correlations, we did not detect a significant influence of proliferative fraction on survival when patients were segregated above or below the mean proliferative fraction for all tumors. More accurate methods of identifying the proliferative fraction of tumor cells are currently being pursued. While the role of flow cytometry in the evaluation and management of patients with colorectal carcinoma is still undefined for a number of other cellular parameters, it seems unlikely that DNA index, RNA index, or the proliferative fractions calculated from the DNA histogram, will, of themselves, represent independent prognostic factors.     

Flow cytometric analysis of DNA content in human bladder tumors and irrigation fluids

Flow cytometry (FCM) was used to study the DNA distribution of 99 tumor biopsy specimens and 41 bladder irrigation samples from patients with transitional cell carcinoma of the bladder. For tumor biopsy and cystectomy specimens, the frequency of aneuploidy increased with advancing tumor stage and grade. All T0 tumors were diploid. Twenty-seven percent of T1, 71.4% of T2, and 75% of T3 and T4 tumors were aneuploid. All Grade I tumors were diploid. Thirty percent of Grade II and 76.9% of Grade III tumors were aneuploid. The frequency of aneuploidy of tumors in the early stages (Ta, T1) is similar to the incidence of subsequent progression by these tumors described in the literature. For irrigation fluids, the relationship between grade and stage and the frequency of aneuploidy was similar to the relationship seen with tumor specimens. All four patients with only carcinoma in situ had aneuploid cells in their irrigations. The comparison of FCM data of bladder biopsy and bladder irrigation from the same cystoscopic evaluation suggests adequate representation of tumor cells in the irrigation fluids for almost all cases. The authors conclude that DNA ploidy analysis by FCM appears useful in a clinically important group of patients with aneuploid superficial tumors of moderate or high grade. Bladder irrigation analysis appears useful in the follow-up of patients with a history of carcinoma in situ and those with aneuploid tumors.     

Flow cytometric DNA analysis of parathyroid tumors with special reference to its diagnostic and prognostic value in parathyroid carcinoma

The nuclear DNA content of paraffin-embedded parathyroid tumors from 49 patients with proven primary hyperparathyroidism was determined by flow cytometric analysis. The lesions included 14 primary and 11 locally recurrent or metastatic lesions from 16 carcinoma patients, 28 single adenomas from 28 patients, and 15 hyperplastic glands from five patients with familial multiple endocrine neoplasia type 1. No abnormal DNA stemline was found in any of the hyperplastic glands. One (3.6%) of the adenomas was aneuploid. There was no difference in ploidy patterns between the primary and recurrent lesions of the carcinomas and five (31%) of the carcinomas expressed aneuploidy. Four of the five patients with aneuploid carcinoma had recurrences including pulmonary metastases. One of them died of this disease 12 years after the initial operation, and all except one of the others are hypercalcemic even after removal of the successive recurrent or metastatic tumors. Of the 11 patients with diploid carcinoma, four had either local recurrence or pulmonary metastasis. Two of them are living with normocalcemia 3 and 6 years, respectively, after removal of the recurrent tumors and the others are alive with mild hypercalcemia. The remaining seven patients with diploid carcinoma, however, have no recurrence 2 to 5 years after the initial operation. Thus aneuploid parathyroid carcinomas are likely to show more malignant behavior than those with a diploid DNA pattern. All of the patients with adenoma and hyperplasia have been normocalcemic after a mean follow-up interval of 37 months. This study indicates that flow cytometric analysis of nuclear DNA content is a valuable adjunct to histologic examination in the diagnosis of parathyroid carcinoma and the prediction of the clinical outcome.