Flow cytometric analysis of DNA abnormalities in colorectal carcinomas

We report a flow cytometric study on ploidy in 117 colorectal cancers. An aneuploid cell population was found in more than 70% of adenocarcinomas. Ploidy was found to be stage-related; aneuploid tumors with DNA index greater than or equal to 1.4 were found mainly in Dukes' C and Dukes' D stages (P less than 0.02). Tumors of the caecum and of the ascending colon were more often found to be diploid than those of the other sites. There was a progressive increase in the proportion of cells in S-phase depending on whether they were from normal tissue, inflammatory mucosa or adenocarcinomas. The proportion of cells in S-phase was significantly larger in aneuploid tumors (P less than 0.001). The data presented above suggest that aneuploidy and the proportion of non-resting cells could be important prognostic factors for colorectal cancers. The latter are independent of the stage of the disease and histological differentiation.     

Flow cytometric DNA index in the prognosis of colorectal cancer

The authors investigated the relationship between flow cytometric DNA index (DI, defined as the ratio of the DNA content of malignant cells to that of normal cells) and other prognostic factors (grade and stage, anatomical site, age and sex) with the survival of 115 patients with colorectal cancer. Multiple biopsy specimens from 62 patients were taken during colonoscopy before surgery. Additional samples from 53 patients were obtained from paraffin-embedded material. All patients were treated with surgery only. Fresh-frozen material gave higher incidence of DNA aneuploidy than paraffin-embedded material (79% versus 41%). The patients with DNA diploid tumors (DI = 1) had a better overall survival than those with DNA aneuploid tumors (DI = 1). Among DNA aneuploid tumors, those with DI greater than 1.2 (excluding DI = 2) were worse than those with DI = 1.2 (excluding DI = 1) and DI = 2. Cox's regression analysis showed that pathologic stage was more important for prognosis than DNA index, whereas age, sex, histologic grade, and anatomic site were removed from the analysis as not relevant for prognosis. Relative risk of death (RR), in reference to patients with DI = 1 and Stages A + B (RR = 1), were RR = 1.8 for patients with carcinomas with Stage C. RR = 2.7 for patients with carcinomas with DNA near-diploid and DNA tetraploid tumors. RR = 3.5 for those with DI greater than 1.2 (excluding DI = 2), and RR = 8.0 for those with Stage D. These data indicate that flow cytometrically evaluated DI values have a relevant independent power for predicting the clinical outcome of colorectal cancer patients.     

Flow cytometric DNA ploidy in colorectal adenomas and family history of colorectal cancer

Flow cytometric DNA ploidy of colorectal adenomas resected from 34 patients and the corresponding patient family history in first-degree relatives were evaluated. The samples with at least two separate G0-G1 peaks were defined as DNA aneuploid. The correlation between DNA ploidy and family history was evaluated using two-by-two contingency tables. This correlation was highly statistically significant: seven of nine patients (78%) with positive family histories, and five of 25 (20%) with negative family history had adenomas with DNA aneuploid stemlines (P = 0.0068). The overall DNA aneuploidy incidence was 12 in 34 cases (35.2%). The combined information of DNA aneuploidy and positive family history of colorectal cancer in patients with colorectal adenomas may help to better understand the process of colon carcinogenesis and to identify patients who have a higher risk for developing a malignancy.     

Flow cytometric analysis of ploidy in colorectal cancer: a multicentric experience.

Ploidy and cell proliferation determined by flow cytometry were assessed on colorectal cancers from patients admitted to two Italian cancer research centres. A total of 181 patients were followed prospectively for 4 years at the Istituto Regina Elena (IRE) of Rome and at the Istituto Nazionale Tumori (INT) of Milan. Fresh (at the IRE) or frozen (at the INT) tumour material and similar procedures were used for subsequent sample preparation. Similar frequencies of aneuploid tumours (63% vs 66%) and superimposable median DNA indices (1.6) were observed for the two case series. In both series, DNA ploidy was generally unrelated to clinico-pathological factors, except for a higher frequency of aneuploid tumours in Dukes'' D (88%) than in Dukes'' A stage (33%) in the IRE experience. DNA ploidy was a weak prognostic indicator at 3 years but not at 4 years in the IRE case series, and it never exhibited a clinical relevance in the INT experience. Conversely, multiploidy was an indicator of worse relapse-free and overall survival at 4 years in the IRE and INT case series.     

Flow cytometric analysis of nuclear DNA heterogeneity in gastric cancer

Flow cytometric analysis of nuclear DNA ploidy was performed to evaluate the clinical significance of DNA-ploidy heterogeneity and DNA-index heterogeneity between the superficial layer and the deep layer of the tumor obtained from 88 advanced gastric cancer patients. DNA-ploidy heterogeneity was observed in 28 patients (31.8%) and characterized mainly by diploidy in the superficial layer and aneuploidy in the deep layer. More than 10% difference in the DNA index among aneuploidy (DNA-index heterogeneity) was observed in 10 (26.3%) of 38 patients with aneuploidy. There was no tendency for the DNA index to increase with deep infiltration. DNA-ploidy and DNA-index heterogeneities were not correlated with the various clinicopathological characteristics. Patients with aneuploidy had significantly poorer prognosis than did those with diploidy. The survival rate for patients with DNA heterogeneity was not significantly different from that for patients without DNA heterogeneity. These results suggest that the DNA-ploidy pattern may be an important prognostic factor, but that DNA heterogeneity may not have an impact on the survival in advanced gastric cancer.      

Flow cytometric DNA analysis of hepatocellular carcinoma.

The prognostic value of nuclear DNA content was studied retrospectively using flow cytometry in 203 cases of resected hepatocellular carcinoma. The occurrence of DNA aneuploidy, which was detected in 50% of patients, correlated significantly with tumor size and the presence of vascular invasion or intrahepatic metastasis. Overall, patients with DNA aneuploid tumors had a significantly worse prognosis than those with DNA diploid tumors (P less than 0.001) and, also in subdivided groups by tumor size (P less than 0.01). Among DNA aneuploid patients, the survival times were significantly shorter for patients with a low DNA index (less than 1.5) than for those with a high DNA index (greater than or equal to 1.5) (P less than 0.05). In a Cox multivariate analysis, nuclear DNA content provided significant prognostic value (P = 0.008), as did vascular invasion (P = 0.001) and intrahepatic metastasis (P = 0.005). These results indicated that nuclear DNA content has an important prognostic value in hepatocellular carcinoma.     

Flow and image cytometric DNA analysis in rhabdomyosarcoma

Rhabdomyosarcoma is the most common malignant soft-tissue tumor in childhood, with an overall 3-year disease-free survival of 73%. DNA content is known to correlate with prognosis and therapy response in many cancers. To determine the role of DNA content in rhabdomyosarcoma, 23 tumor samples were studied retrospectively: 18 primary tumors and 5 post-chemotherapy recurrences or specimens obtained at second-look surgeries. The DNA analysis was performed on disaggregated paraffin-embedded tissue nuclei by flow and image cytometry and correlated with the histology and clinical history. Of the primary tumors 4 were diploid, 4 polyploid, and 10 aneuploid (9 with a single aneuploid G0G1 peak and 1 multiploid) by flow cytometry. The concordance rate between flow and image cytometry was 19 of 23 (83%); one case did not have flow cytometry available. Most embryonal rhabdomyosarcomas were aneuploid (10 of 12; 83%), and they had a high incidence of recurrence in Stages III and IV (4 of 12; 33%). Although aneuploidy in pediatric cancers may predict a therapeutic response and good prognosis, this was not supported by our findings in rhabdomyosarcoma. The tumor DNA content correlated with the clinical stage but not with the patient's clinical course or tumor histopathological type. DNA content did not appear to be as important a prognostic tool as tumor stage.     

Flow cytometric DNA analysis of frozen samples

Now that flow cytometric DNA analysis is becoming an established procedure, many samples that are being received from various hospitals cannot all be analyzed within a day. Therefore, samples must be stored for subsequent analysis in a flow system. To determine a proper temperature for storage by freezing, samples of the same material were stored for 2 weeks at 5 degrees C, -80 degrees C, and -195 degrees C. Measurement and analysis at the end of this period revealed no major differences among the subsequent histograms of these samples. Thus, it seems that a flow cytometric DNA analysis can be made of frozen samples for diagnostic and prognostic evaluations.     

Prognostic significance of flow cytometric analysis of DNA content in colorectal cancer: A prospective study

We prospectively analyzed the tumor DNA content by flow cytometry in 100 patients who underwent a curative resection for colorectal cancer between August 1989 and May 1992 in order to evaluate the prognostic significance of DNA ploidy and the DNA index (DI). Patients with aneuploid tumors were found to have a significantly shorter disease-free survival than those with diploid tumors (P = 0.014). In addition, patients who had tumors with a DI greater than 1.6 had a significantly shorter disease-free survival than those who had tumors with a DI of less than 1.6 (P = 0.0001). After stratification by stage, this association was only seen in Dukes' stage C disease (P = 0.0065). Cox's regression analysis demonstrated that the DI (below or above 1.6) rather than DNA ploidy was an important independent predictor of disease-free survival. These results suggest that the DI rather than DNA ploidy provides us with important prognostic information in patients undergoing curative surgery for colorectal cancer. © 1993 Wiley-Liss, Inc.     

Flow cytometric DNA analysis and chromosomal aberrations in malignant glioblastomas.

In this study we combined flow cytometry with fluorescence in situ hybridization to detect numerical aberrations in chromosomes. Fifty-nine human malignant gliomas were examined by flow cytometry for DNA-content and cell cycle analysis and for numerical aberrations of chromosome 1 by in situ hybridization using a chromosome specific centromere probe. Of the gliomas analysed, 42% were diploid and 58% showed aneuploid tumour cell populations. The DNA index was heterogeneous ranging from 1.0 to 2.3. The S-phase analysis showed proliferation activity from a very low range of 0.7% up to 17.0%. In general, diploid gliomas exhibited a lower S-phase activity than aneuploid gliomas. Of the aneuploid gliomas, 15% showed a peridiploid pattern with a DNA index mean of 1.1. In these peridiploid tumours a trisomy of chromosome 1 could be detected by fluorescence in situ hybridization (FISH). The frequency of trisomic chromosome 1 in malignant gliomas reflects a very slight increase in DNA index from diploid to peridiploid (DNA index 1.1). Comparison of chromosome numbers and DNA content gave good correlation. Also important, the results reflects the cell cycle, specifically the extent of S-phase activity. In general, cell proliferation of diploid and peridiploid gliomas is much less than in higher aneuploid gliomas. The analysis of DNA content may thus yield results with respect to the biological behaviour of tumours in general.     

Flow cytometric DNA analysis of gastric smooth muscle tumors.

BACKGROUND. To better understand the malignant grade of gastric smooth muscle tumors, the DNA content of these tumors was studied. METHODS. In 43 patients with gastric smooth muscle tumors, the cellular DNA content was determined by flow cytometry and compared with the histologic classification and the prognosis. RESULTS. Flow cytometry indicated that all 21 leiomyomas and 9 of the 10 low-grade leiomyosarcomas were diploid; 10 of the 12 high-grade leiomyosarcomas were aneuploid. All patients with leiomyomas and 8 of the 11 patients with diploid leiomyosarcomas had neither local recurrence nor metastasis. By contrast, 8 of the 10 patients with aneuploid leiomyosarcomas died of their disease (mean survival, 41 months; range, 18-78 months). CONCLUSIONS. These results indicate that the DNA ploidy pattern shown by flow cytometry is related closely to the histologic classification and prognosis of gastric smooth muscle tumors.     

Flow cytometric DNA ploidy analysis of oral cancer comparison with histologic grading

It has been reported that DNA content analysis provides prognostic information eliminating the subjective component involved in routine microscopic decision making. In an attempt to establish a relationship between pathological findings and flow cytometric analysis, 36 formalin-fixed, paraffin-embedded tumour tissue samples were prepared according to Hedley's method and analysed by means of an Epics Profile II flow cytometer. DNA aneuploidy was observed in 15 tumours (41%). A statistically significant correlation was identified between DNA index and mitoses, cellular response and degree of differentiation, but not the ploidy status. It was not possible to identify a significant association of sex, age, and site of the tumour to DNA index. We found a strong correlation between histologic malignancy and DNA index; an increase in DNA index as malignancy score increases was noted. It was concluded that DNA index shows a good correlation with the histologic features of oral cancer, being a complement of differentiation and histologic grading analysis. The use of DNA analysis as a complement to pathological studies would help to diminish the subjective component of assessment of head and neck cancers. Ploidy status was not statistically associated with the differentiation of tumours.     

Flow cytometric DNA analysis does not predict the radiochemoresponsiveness of esophageal cancer

The relationship between the DNA pattern and the responsiveness to chemotherapy or chemoradiotherapy has been evaluated in 30 patients with squamous cell carcinoma of the esophagus. In 24 patients polychemotherapy with cisplatin (100 mg/m² on day 1) and 5-fluorouracil (1,000 mg/m²/ 24 h, continuous infusion of 120 h) every 3 weeks, was performed. Six other patients received chemoradiotherapy with cisplatin 80 mg/m² on day 1 and 18.5 Gy (split course). Before treatment, at least three endoscopic biopsies were taken from each tumor and frozen at ?85°C. Five patients were excluded from the evaluation, three because of interrupted treatment and two due to unsuitable biopsy material obtained endoscopically. The response rate to the cytoreductive treatment was 40% (10/25). DNA content was analyzed with flow cytometry. Out of 25 evaluable patients, a diploid and aneuploid tumor was present in 8 (32.0%) and 17 (68.0%) patients, respectively. According to the DNA pattern, a major response was observed in 4 of 8 patients with a diploid tumor and in 6 of 17 patients with an aneuploid tumor (P = 0.5). No relationship between the percentage of cells in the S-phase and the response to the cytoreductive treatment was evident. Although a slightly higher percentage of major responses was found in euploid tumors, there is no evidence that flow-cytometric DNA analysis can be helpful in the selection of patients for chemotherapy or chemoradiotherapy.© 1993 Wiley-Liss, Inc.     

Flow cytometric DNA analysis of adrenocortical tumors in children

Flow cytometric DNA analysis of isolated nuclei was performed on 14 lesions occurring in ten children with adrenocortical tumors. Unimodal DNA content distributions were obtained from seven tumors occurring in patients without metastases 2 to 18 years after diagnosis. Abnormal DNA contents were detected in all four primary lesions, which subsequently metastasized, and in the tumor of one patient who was followed for less than 2 years. Paraffin and frozen preparations were virtually identical, as were the analyses of the primary, recurrent, and metastatic disease occurring in one patient. These observations suggest that DNA content abnormalities detected by flow cytometry correlate with metastases, and may provide an objective measure of the biologic potential of these tumors.     

Flow cytometric analysis of colorectal mucosa from patients with Crohn's disease, ulcerative colitis and cancer.

Samples of colorectal mucosa from patients with Crohn's disease, ulcerative colitis and cancer were analyzed by means of flow cytometry. S- and G2-phase fractions were determined and mean values were calculated for different groups of patients. Almost identical results were obtained for inflamed and normal appearing mucosa from patients with Crohn's disease as well as inflamed mucosa from patients with ulcerative colitis. The mean S- and G2-phase fractions in normal appearing mucosa from cancer patients, however, were significantly higher. This seems to be due to the fact that patients with Crohn's disease are between 15 and 45 years old, while cancer patients are mostly over 45. A detailed analysis of the S- and G2-phase fractions in different age groups revealed a slight, but significant increase in colorectal proliferation between 25 and 75 years.     

Flow cytometric analysis of the nuclear DNA content of hepatoblastoma.

The nuclear DNA content of 15 hepatoblastoma cases was determined in paraffin-embedded tissues by flow cytometry. The DNA index (DI) was calculated, and the ploidy pattern of nuclear DNA was estimated. The correlation between the ploidy pattern and clinicopathologic findings was studied, and the prognostic significance of the ploidy pattern was investigated. An aneuploid pattern was seen in 50% of the lesions with histologic embryonal and anaplastic types. It was not seen in the fetal type. In the tumors with combined epithelial components, the fetal-type component had a diploid pattern in all five cases. The embryonal-type component was associated with aneuploidy in two of five cases. In aneuploid tumors, vascular invasion (tumor emboli in the vessels) was observed more frequently. The prognosis of the patients with an aneuploid tumor was significantly poorer. These results indicate that nuclear DNA ploidy pattern analysis might be useful in investigating the prognosis of hepatoblastoma.     

Flow cytometric DNA analysis of medulloblastoma. Prognostic implication of aneuploidy

Paraffin-embedded surgical specimens from 26 infants and children with medulloblastomas treated between 1972 and 1981 were examined for DNA ploidy by flow cytometry (FCM). All patients received a standard treatment (a combination of maximum debulking of medulloblastoma and postoperative craniospinal irradiation with a posterior fossa boost of 5000 rad or more). They were studied to correlate the results of the findings of FCM DNA analysis with their final outcome, DNA ploidy, and extent of tumor resection. All seven patients with totally resected aneuploid medulloblastoma are alive, whereas only one of six patients with subtotally resected diploid medulloblastoma is alive (P = 0.0047). The current study suggests both DNA ploidy and extent of surgical resection are the most important determinant of patients' prognosis. Patients in selected group, particularly those with subtotally resected diploid tumor, are advised to undergo aggressive adjuvant chemotherapy.     

Flow cytometric DNA analysis as a diagnostic aid for cervical condyloma and cancer

Flow cytometric DNA analysis data (FCDA) were obtained from 324 samples provided through the Gynecology-Oncology Clinic. These samples consisted of 294 combined endoectocervical and vaginal smears and 30 peritoneal washings. Using a conventional scheme for G0/G1, S + G2/M and the coefficient of variation with computer correction for the cell-cycle kinetics, it was possible to assign a diagnostic Class I, II, III or V similar to that used by the Cytology Laboratory. These data were then compared with the histopathologic and colposcopic diagnoses. The correlation between FCDA and cytologic results were essentially similar to the previous data obtained from only endocervical sampling. The most interesting finding in this study was the recognition of an FCDA pattern showing a higher DNA content in the G0/G1 and the early S regions in 70 of 94 (74.5%) of samples from patients with condyloma acuminata. All condyloma samples were diagnosed either by cytologic, histopathologic, or colposcopic examination, or a combination of two or three. All biopsy specimens were then reviewed by one pathologist to verify any discrepancies. The relationship of this pattern to the viral etiology of this disease is discussed with the three methods of diagnosis and electron microscopic observations. It is suggested that, based on this study. FCDA analysis of pap smears may also be useful in determining the presence of condyloma in a gynecology clinic. The potential value of FCDA analysis from peritoneal washings for the diagnosis of gynecologic cancer can not be ascertained in this preliminary investigation because of insufficient samples.