Prognostic significance of DNA index, multiploidy, and S-phase fraction in ovarian cancer

Paraffin-embedded tumor samples from 157 ovarian cancer patients were analyzed by DNA flow cytometry. Tumor ploidy had prognostic significance in both early and advanced ovarian cancer. After adjusting for stage, residual tumor mass, histopathologic type, treatment, and age of patient in a Cox regression analysis, the relative risk of death was two-fold higher in single DNA-aneuploid and sixfold higher in DNA-multiploid tumors as compared to DNA-diploid tumors (P less than 0.0001). A tetraploid DNA index was associated with a relatively low risk ratio, whereas hypertetraploid tumors were highly malignant. S-phase fraction (SPF) had prognostic value especially in DNA-diploid tumors. The simultaneous evaluation of DNA index, the number of aneuploid cell clones, and SPF gave more prognostic information than the determination of tumor ploidy alone. On the basis of these parameters we have developed a classification of tumor DNA histograms for better prognostic assessment of ovarian cancer.     

Prognostic significance of DNA image cytometry in libyan breast cancer

Background: We evaluated the relation of nuclear DNA content and clinicopathological features and prognosis in primary breast cancer of female Libyan patients with variable stage and grade and different treatment regimes. Patients and Methods: Histological samples from 104 patients of breast carcinoma were retrospectively studied by computerized nuclear DNA cytometry. Isolated nuclei from paraffin sections were stained with Feulgen stain and DNA was measured using a computer-assisted image analysis cytometry system. In each case, 200 nuclei were measured and the DNA histograms, S phase fraction (SPF) and number of cells above 5c and 9c were determined. We applied different approaches in the analysis of DNA to compare the DNA histograms with different clinicopathological features and survival. Results: The mean of DNA ploidy mode for all tumors was 3.43; 82.7% of tumors were aneuploid and 17.3% were diploid. The median SPF was 3.5% for DNA diploid and 13.5% for DNA aneuploid tumors. DNA aneuploid tumors and high SPF were associated with advanced stage, distant metastasis, high histological grade and lymph node involvement. The SPF was also associated with large tumor size and with younger patients (<50 years). In the overall population (median follow-up 51 months), patients with aneuploid DNA histograms and high SPF values had shorter survival times than those with diploid DNA histograms and low SPF values (p = 0.001, p < 0.0001, respectively). Also, short survival was associated with a multiploid DNA histogram and with DNA aneuploid cells ≥5 cells (p < 0.0001, p = 0.001, respectively). In a Cox multivariate analysis, DNA ploidy (p = 0.010), age (p = 0.038) and clinical stage (p = 0.001) were independent predictors of overall survival, and DNA ploidy (p = 0.018) and clinical stage (p = 0.001) also proved to be independent predictors of disease-specific survival. The SPF cutoff point of 11% might be applied to separate patients into good and poor prognosis groups. Conclusions: DNA image cytometry with careful analysis of the histograms may provide valuable prognostic information in Libyan breast cancer, with potential clinical implications in patient management, particularly in predicting the patients at high risk for metastasis and recurrence who should be considered as candidates for combined adjuvant therapy.     

Flow-cytometric analysis of colorectal cancer with hepatic metastases and its relationship to metastatic characteristics and prognosis

Studying the DNA ploidy patterns of 52 primary tumors, diploid tumors accounted for 48.1% and aneuploid tumors for 51.9%. Out of 31 patients with liver metastases, 35.5% had diploid tumors and 64.5%, aneuploid tumors. Heterogeneity (difference in DNA ploidy pattern between the primary lesion and liver metastases) was found in 20% of the patients examined. In 28 of the patients, the liver metastases were unresectable, and their prognoses were such that the 1- and 2-year survival rates from the diploid tumors were 42.9 and 14.3%, respectively, while 1-year survivors from aneuploid tumors died within 2 years. In resected cases of hepatic metastases, the DNA ploidy pattern of the metastatic lesions did not correlate with the metastasis period, extent of spread or number of lesions. The recurrence rate of aneuploid tumors in the residual livers was 50%, which was slightly higher than the rate of 36.4% for diploid tumors. The prognoses in patients with diploid tumors were significantly better than those in patients with aneuploid tumors: 5-year survival was 71.1% in diploid tumor patients, compared with 21% in aneuploid tumor patients.     

Origin of multiple "primary" colon carcinomas. A retrospective flow cytometric study

Multiple, synchronous, apparently primary carcinomas of the colon are a relatively common occurrence. The DNA ploidy in 23 colon carcinomas from 10 patients was determined to see if this parameter supported common or independent origins for such synchronous lesions. Paraffin blocks of each tumor were prepared for flow cytometry, then analyzed for nuclear DNA content. In 3 of the 10 cases, the tumors within each colon differed with respect to DNA ploidy; in four cases all tumors were diploid; and in three cases both (all) tumors within each colon had identical aneuploid DNA indices. Tumors from the same colon with identical DNA histograms often had dissimilar histology. The replicate aneuploid DNA indices strongly suggest a common origin for the multiple tumors within these colons; tumors in the other groups are compatible with either single or multiple origins. These findings suggest that multiple "primary" colon carcinomas may, in some cases, arise as translumenal metastases from an initial single lesion.     

Flow cytometric analysis of nuclear DNA content in patients with recurrent epithelial ovarian cancer

Numerous reports have recently indicated that the DNA content of various malignant tumors can be of great value in predicting biological behavior and prognosis of the tumors. This study was undertaken to determine the nuclear DNA content in the primary and recurrent ovarian carcinoma of the same 20 consecutive patients by flow cytometry, and the results on clinical outcome was examined. The tissue samples of the recurrent tumors were obtained at second-look laparotomy. Of the primary tumors, 12 were diploid, 4 were pure aneuploid, and 4 were "mosaic", while of the recurrent tumors, 17 were diploid and 3 showed pure aneuploid. No significant difference of the DNA index at recurrence was observed. DNA ploidy was preserved in 12 out of 20 patients at recurrence. The time to recurrence after the initial treatment showed no significant difference versus DNA ploidy and change of ploidy. The sites and status of recurrence differed by DNA ploidy. At recurrence, the patients with DNA diploidy had a tendency to survive longer than those with DNA aneuploidy. The determination of DNA ploidy at recurrence may be useful as a variable parameter in predicting the survival of patients with ovarian carcinomas.     

Flow cytometric DNA analysis of poorly differentiated adenocarcinoma of the colorectum.

DNA ploidy patterns in 11 poorly differentiated adenocarcinomas of the colorectum were examined by flow cytometry using paraffin-embedded specimens. Measurements of DNA content were made of the superficial (Sup) half and deeper (Deep) half of the primary tumors in all cases, and of lymph node metastases in five cases. All the primary tumors showed invasion beyond the muscularis propria of the colorectum. Aneuploidy or polyploidy in either Sup or Deep of the primary tumor was found in six of the 11 (54.5%) tumors. Out of the six aneuploid tumors, five were in Dukes' stage C with distant metastases at the time of operation, and four died within one year of surgery. Conversely, out of five diploid tumors, none had distant metastases at the time of operation and two survived for longer than three years after surgery. The DNA ploidy pattern of Deep differed from that of Sup in four out of six aneuploid tumors, and two showed aneuploidy in Sup and diploidy in Deep. All the lymph node metastases in the five tumors had a diploid pattern, although three had aneuploid patterns in the primary tumors. The findings suggest the DNA ploidy pattern of a primary tumor to be correlated with the degree of metastasis at the time of operation or prognosis, but the population of tumor cells having different DNA contents may be apt to change between Sup and Deep in aneuploid tumor.     

The prognostic significance of nuclear DNA content in malignant epithelial tumors of the ovary

Recent studies have indicated that the nuclear DNA content of certain malignant neoplasms can be used as an adjunct in predicting their biologic behavior. The DNA content of 99 ovarian carcinomas was determined by flow cytometric analysis of nuclei obtained from paraffin-embedded tissue. Of the 99 tumors, 51 were diploid and 48 showed one or more aneuploid peaks. The 5-year survival for patients with diploid tumors (50%) was significantly higher than for patients with aneuploid tumors (22%) (P less than 0.01). Other factors which significantly affected survival were clinical stage (P less than 0.001), tumor pattern grade (P less than 0.01), DNA index (P less than 0.01), the presence of ascites (P less than 0.001), peritoneal carcinomatosis (P less than 0.0001), and residual tumor at second-look laparotomy (P less than 0.05). Diameter of the primary ovarian tumor, diameter of the largest peritoneal implant before debulking, and the percent S-phase had no significant correlation with survival. Of 16 patients with aneuploid tumors who underwent second-look laparotomy, nine (56%) had residual tumor, compared to six of 22 of patients with diploid tumors (27%). Of seven patients with aneuploid tumors and a negative second-look laparotomy, four (57%) died from recurrent tumor. By comparison, of 16 patients with diploid tumors and a negative second-look laparotomy, only four (25%) died from recurrent tumor. The determination of DNA ploidy in ovarian carcinomas may be used as an adjunct in predicting tumor behavior, response to chemotherapy, and late recurrence of disease.     

Flow cytometric analysis of nuclear DNA content in ovarian tumors. Association of ploidy with tumor type, histologic grade, and clinical stage.

The authors undertook a prospective, flow cytometric study of nuclear DNA ploidy in 140 fresh ovarian tumors. There were 43 benign tumors, 27 borderline tumors, and 70 malignant tumors. Results of DNA ploidy analysis were compared to age at diagnosis, menopausal status, tumor size, histologic type, grade, and International Federation of Gynecology and Obstetrics (FIGO) stage. Although the majority of benign tumors were diploid, 19% were aneuploid. Among the benign tumors, DNA ploidy was significantly associated with tumor type and tumor size. All borderline tumors were diploid. Of the 70 malignant tumors, 64% were aneuploid. In the malignant tumors, DNA aneuploidy had significant univariate associations with histologic type, grade, and FIGO stage. By multivariate analysis, DNA aneuploidy remained significantly associated with stage and grade, both known predictors of survival in ovarian cancer. These results indicate that DNA ploidy varies with the aggressive potential of an ovarian cancer and may, at the time of initial diagnosis, provide additional information about tumor prognosis.     

The clinical significance of nuclear DNA ploidy pattern in 184 patients with pheochromocytoma.

Flow cytometric nuclear DNA analysis was performed on paraffin-embedded tissue samples taken from 184 patients with pheochromocytoma and paraganglioma treated between 1960 and 1987. The Hedley technique was used for measurement of nuclear DNA content. Thirty-five percent of the tumors were DNA diploid, 33% showed a DNA tetraploid pattern, and 32% had DNA aneuploid pattern. Familial pheochromocytoma and associated endocrine or neoplastic disorders were more common among patients with DNA nondiploid tumors. Eighty-four percent of the tumors that invaded blood vessels and all patients with regional or distant metastases had tumors classified as DNA tetraploid or DNA aneuploid. Of 22 patients who had disease progression, 21 (95%) had tumors with abnormal DNA ploidy pattern (P less than 0.001). All 12 patients who died of cancer-related disease had abnormal DNA ploidy; none of the patients with DNA diploid tumor (n = 64) have died of pheochromocytoma (P less than 0.01). These results suggest that nuclear DNA ploidy pattern is an important and independent prognostic variable for patients with pheochromocytoma and paraganglioma.     

Ploidy assessment of benign and malignant ovarian tumors by flow cytometry. A clinicopathologic study

In a prospective study, 112 fresh ovarian tumor samples were collected from 83 consecutive patients. Cellular DNA content was measured by flow cytometry. All the benign (n = 24) and semimalignant (n = 6) tumors were diploid. Of 50 malignant tumors, 24 (48%) were diploid and 26 (52%) were aneuploid. Aneuploidy was more frequent in the advanced stages of the disease, in tumors of low degree of differentiation, and in older patients. The patients with aneuploid tumors had smaller primary tumors and more often ascites. The fraction of cells with S-phase DNA content was higher in the aneuploid tumors. No association was seen to the tumor type. Ploidy determination is objective and reproducible. Aneuploidy associates to most negative prognostic factors in ovarian carcinoma and may reflect the aggressiveness of the tumor. The ploidy status may be taken into consideration in the stratification of patients of comparable risk for treatment studies.     

DNA cytophotometry and prognosis in ovarian tumors of borderline malignancy. A clinicomorphologic study of 80 cases.

Surgical specimens of 80 ovarian tumors of borderline malignancy (OTBM) were investigated by scanning DNA cytophotometry. Diploid or euploid DNA histograms were found for 21 tumors, whereas 59 OTBM showed noneuploid or aneuploid DNA patterns. All patients were followed-up after surgery for at least 3 years (mean observation period, 6.7 years). Follow-up showed 11 cases of recurrent disease and 6 deaths. DNA findings and several other morphologic and clinical details (including patient age, histologic type and stage of disease, and extent of therapy) were correlated to the postoperative course. Statistical analyses disclosed that, of these parameters, only DNA content significantly affected prognosis. Recurrences and deaths resulting from tumor exclusively were observed among patients with noneuploid or aneuploid OTBM, whereas none of the diploid or euploid tumors recurred (P less than 0.05). DNA cytophotometry thus might be regarded as an effective complementary means to assess the prognosis of individual OTBM cases.     

DNA ploidy in intraductal breast carcinomas

Cellular DNA-ploidy in 74 clinically detected intraductal breast carcinomas (IDCs) was analysed by flow cytometry. The histograms were classified as either diploid or aneuploid, and the DNA ploidy pattern compared with that of invasive breast carcinomas and normal breast tissue. All normal breast tissues were diploid while 28 (38%) of the IDCs were aneuploid, the DNA indices ranging from 1.32 to 2.00. The frequency of aneuploidy in invasive ductal carcinomas (73%) was significantly higher (P = 0.003), DNA index ranging from 1.34 to 2.92, compared with that in IDCs. Retrospectively, 14.5% of the patients had invasive breast cancer 16–166 months after the diagnosis of IDC. Neither DNA ploidy nor histopathological classification alone predicted clinical outcome, but patients with DNA diploid non-comedo IDC had a more favourable course.     

Large-scale genomic instability predicts long-term outcome for women with invasive stage I ovarian cancer.

BACKGROUND: The objective was to evaluate the value of DNA ploidy using high-resolution image cytometry in predicting long-term survival of patients with early ovarian cancer. PATIENTS AND METHODS: A retrospective analysis of 284 cases with FIGO stage I ovarian carcinoma treated during the period 1982-1989 was performed. Clinical follow-up information was available for all patients. RESULTS: Patients with diploid and tetraploid tumors had a 10-year relapse-free survival of 95% and 89%, respectively, compared with 70% and 29% for polyploid and aneuploid tumors, respectively. DNA ploidy analysis was the strongest predictor of survival in multivariate analysis (diploid/tetraploid versus polyploid/aneuploid; relative hazard 9.0) followed by histological grade, including clear cell tumors in the group of poorly differentiated tumors (grade 1-2 versus grade 3 or clear cell; relative hazard 2.7), and FIGO stage (Ib/Ic versus Ia; relative hazard 2.0). In a stratified Kaplan-Meier analysis, patients with grade 1-2, diploid or tetraploid tumors had a 10-year relapse-free survival of 95%, forming a low-risk group. Patients with grade 3 or clear cell, diploid or tetraploid tumors had 10-year relapse-free survival of 86%, forming an intermediate-risk group, while all patients with aneuploid/polyploid tumors formed a high-risk group, with 10-year relapse-free survival of 34%. CONCLUSIONS: This study points to the importance of including DNA ploidy analysis by image cytometry when selecting patients with early ovarian cancer for adjuvant treatment after surgery.     

Are DNA ploidy and epidermal growth factor receptor prognostic factors for untreated ovarian cancer? A prospective study

To identify prognostic factors for untreated ovarian cancer, DNA ploidy, proliferative index (P.I.) and epidermal growth factor receptor (EGFR) expression were analyzed in a prospective series of 40 patients with ovarian cancer and 7 patients with borderline malignant ovarian tumor followed up for 5 years or more (median, 77 months). The frequency of aneuploid cells was 53.8% (21/39) in ovarian cancer and 14.3% (1/7) in borderline malignancy. There was no significant association between DNA ploidy and the clinicopathologic findings, in which aneuploid ovarian cancer was more common among advanced tumors. The S-phase fraction and P.I. value were higher in the patients with aneuploid tumors (p = 0.076). EGFR expression was detected in 76.9% (30/39) of ovarian cancers and 42.9% (3/7) of borderline malignant ovarian tumors, and the mean EGFR level was 5.8 +/- 12.1 (range: 0-49.5) and 28.3 +/- 71.1 (range: 0-189.4) fmol/mg protein, respectively. There was no correlation between EGFR expression and DNA ploidy, P.I., and clinicopathologic findings analyzed. The 5-year survival rate in patients with aneuploid tumors was significantly worse in patients with ovarian cancer (p = 0.0165, log-rank test). No significant relationship was shown between P.I., EGFR expression, and 5-year survival. Cox multivariate analysis showed that DNA ploidy, P.I., and EGFR expression are not associated with the risk of death (p = 0.5917, p = 0.9924, and p = 0.6840, respectively), although clinical stage shows a significant relationship (p = 0.0027). Our data showed that DNA ploidy is significantly related to the prognosis by univariate analysis, but DNA ploidy, P.I., and EGFR expression were not independent prognostic factors for the untreated ovarian cancer.     

DNA content flow cytometry as a prognostic factor for node-positive breast cancer. The role of multiparameter ploidy analysis and specimen sonication

The DNA content was analyzed in paraffin-embedded material from 167 patients with node-positive breast cancer to learn whether specimen sonication and multiparameter ploidy analysis (MPPA) (using DNA content and light scatter) could improve the strength of ploidy as a prognostic variable. Sonicated specimens were found to have fewer aggregates, a lower percentage of cells in S-phase (%S) and G2M phase than the corresponding nonsonicated specimens. The results using MPPA predicted the prognosis better because they allowed detection of small aneuploid peaks in histograms classified as diploid or tetraploid using DNA content alone. Ploidy was a significant univariate factor, and patients with tetraploid tumors had the best survival. In the multivariate analysis, if other routine factors were examined preferentially, ploidy and %S did not provide additional prognostic information for survival. This study of paraffin-embedded breast cancers suggested that sonication and MPPA may improve the ploidy analysis in certain cases and that tetraploidy may be a favorable ploidy pattern in this group.     

Regional DNA content heterogeneity in colonic adenocarcinoma: prognostic significance in patients with liver metastases.

We retrospectively examined by flow cytometry the DNA ploidy pattern in tissue blocks from 25 primary colon adenocarcinomas and their lymph node and liver metastases. Intratumoral heterogeneity was present in 22% of primary tumors and 21% of metastatic liver deposits. Intertumoral heterogeneity, measured between the primary tumor and its lymph node and liver metastases, was 0% and 20%, respectively. Of 24 patients who underwent successful resection of their liver metastases, 8 neoplasms had uniformly diploid DNA content, while 16 tumors had aneuploid DNA pattern in either the primary tumor, the metastases, or both. Five-year survival was better in the diploid group (38% vs. 7%, P = 0.10 by log rank analysis). Three of eight patients in the diploid group remain free of disease, while all 16 patients with aneuploid cell populations have died of recurrent disease.     

Correlation of DNA ploidy and proliferative activity in human gastric cancer

Analysis of DNA ploidy patterns was performed on 129 cases of primary gastric cancer and the results were correlated with histologic findings and in vivo bromodeoxyuridine (BrdU) labeling. Forty-nine cases were diploid (38%) and 80 cases were aneuploid (62%). There was no correlation between DNA ploidy and histologic type. In aneuploid tumors, incidence of lymphatic invasion, lymph node metastasis, and rate of advanced cases were significantly higher than those in diploid tumors. During the follow-up period of 5 to 10 years, 23 of 40 patients (55%) with aneuploid tumors died of disease within 3 to 120 months. Only 13 of 36 patients (36%) with diploid tumors died of disease. The BrdU labeling indices (BrdU LI) ranged from 2.8% to 26.7%, with a mean of 10.4%. There was no correlation between BrdU LI and histologic type or stage. The mean BrdU LI of early cancers was 8.1%. The mean BrdU LI of advanced cancers was 11.9%. The BrdU LI of cancers with lymphatic invasion or lymph node metastasis was higher than those without them. The mean BrdU LI of diploid cancers was 6.0%. The mean BrdU LI of aneuploid cancers was 11.9%. There was a good correlation between BrdU LI and DNA ploidy patterns. These results indicate that DNA ploidy patterns and BrdU LI may possibly be useful prognostic markers for gastric cancers.     

A comparison of static cytofluorometry and flow cytometry for the estimation of ploidy and DNA replication in human breast cancer

Summary 332 primary invasive breast carcinomas were analysed by static cytofluorometry and flow cytometry. The ploidy distributions were similar, and 54% of the tumors were judged DNA aneuploid by both methods.The coefficient of variation of the G₀–G₁ peaks ranged from 2.0 to 8% with both techniques, but the mean was somewhat lower with flow cytometry — 4.1%, compared to 4.9% for the static measurements.The proportion of S-phase cells was possible to estimate from 80% of the flow histograms and 70% of the static histograms. S-phase was not estimated from the static histograms if less than 150 tumor cells were measured. With 160 tumors S-phase was measured by both methods. The range was 0 to 27% with the static measurements and 0.7 to 25% with flow cytometry. Corresponding mean values were 7.6% and 8.2%, which are similar to thymidine labeling index results with breast cancers reported in some studies. A close correlation was obtained (r = 0.927) comparing S-phase fractions estimated from aneuploid tumors with flow cytometry and static cytofluorometry if more than 200 cells were measured with the latter. The proportion of S-phase cells was significantly lower for the diploid tumors.We conclude that both techniques can be useful for the estimation of DNA ploidy and replication in human breast cancer.     

Invasive and noninvasive implants in ovarian serous tumors of low malignant potential

Ovarian serous tumors of low malignant potential ("borderline" serous tumors) are classified according to the histologic features of the primary ovarian tumor, without regard to any coexisting extraovarian disease. The peritoneal implants display a range of histologic appearances, ranging from benign glands (endosalpingiosis), to noninvasive papillary glandular proliferations resembling the ovarian neoplasms, to irregular glands associated with a desmoplastic stroma and having features of invasive disease. This review of 16 patients with histologically documented extraovarian tumor implants seen at Indiana University Medical Center, Indianapolis, and 13 patients whose tumor implants have been previously described in the literature indicates that the clinical stage of disease has much greater prognostic significance than does the implant histologic features. There is a tendency for patients with more advanced disease to have invasive implants. However, within a given clinical stage, disease progression or recurrence was not influenced by the presence or absence of invasive histologic characteristics in the tumor implants.     

Peritoneal implants of ovarian serous borderline tumors. Histologic features and prognosis

The clinicopathologic features of 56 cases of ovarian serous borderline tumors (SBT) associated with peritoneal implants were reviewed. Data from 368 person-years of follow-up (median follow-up, 6.0 years) were analyzed to investigate the possibility that the histologic features of implants of this type of tumor may correlate with the prognosis. Eighty-five percent of the 56 patients were clinically free of tumor at the time of death or at last contact. Thirteen percent of the patients died of tumor, and one patient (2%) was alive with widespread progressive tumor. The product-limit estimate of the probability of death from tumor (+/- standard error) was 4% (+/- 3%) at 5 years and 23% (+/- 9%) at 10 years. The following three histologic features of the implants correlated with an adverse prognosis: (1) invasion (P = 0.0004), (2) severe cytologic atypia in both invasive and noninvasive implants (P = 0.0008) and in noninvasive implants alone (P = 0.02), and (3) the presence of mitotic activity in both types of implants (P = 0.02) and in noninvasive implants alone (P = 0.02). The only other feature that correlated with the prognosis was the presence of residual tumor postoperatively as assessed by the surgeon (P = 0.01). The product-limit estimate of death of tumor in patients with at least one of these four adverse prognostic factors was 56% (+/- 20%) at 10 years. Whether or not the patients received radiation therapy, chemotherapy, or both had no statistically significant effect on the outcome. These data and the results of a stratified analysis suggest that patients may benefit from additional therapy if adverse prognostic factors are present, especially invasiveness or severe cytologic atypia. It is unlikely that additional therapy is necessary in patients without adverse prognostic features, because no deaths occurred in this group.