Prognostic significance of DNA ploidy determined by high-resolution flow cytometry in breast carcinoma

OBJECTIVE: To assess the prognostic value of DNA ploidy in breast carcinoma and its relation to other established prognostic factors. STUDY DESIGN: We evaluated DNA ploidy in 303 breast carcinoma patients with a median follow-up of 63 months. Flow cytometry was performed on frozen tumor material, yielding histograms with narrow peaks (median coefficient of variation of 2.08). DNA ploidy pattern was classified as either diploid versus nondiploid, euploid (diploid and tetraploid) versus aneuploid or diploid/near-diploid (DNA index < 1.2) versus other, and correlated with relapse-free (RFS) and cancer-specific survival (CSS) along with tumor size, histologic grade and type, axillary lymph node involvement, menopausal and steroid receptor status, age and type of treatment. RESULTS: Seventy-one percent of tumors were DNA nondiploid (14% tetraploid and 57% aneuploid). There was a strong association between DNA ploidy and histologic grade. Histologic grade, lymph node status, tumor size and DNA ploidy (regardless of the classification used) were all significantly associated with RFS and CSS in multivariate analysis. CONCLUSION: These results suggest that DNA ploidy, at least when determined from frozen tumor tissue, is an independent prognostic factor in breast carcinoma; however, its prognostic power seems to be inferior to that of histologic grade, with which it strongly correlates.     

Independent prognostic value of hormone receptor expression and S-phase fraction in advanced breast cancer

OBJECTIVE: TO assess the prognostic value of immunocytochemically assessed hormone receptor expression and DNA flow cytometry data in advanced breast cancer. STUDY DESIGN: This prospective study with long-term follow-up evaluated the above parameters in relation to overall survival in 392 patients with advanced breast cancer (stages IIB, n = 106; IIIA, n = 66; IIIB, n = 174; and IV, n = 46) using fine needle aspiration cytology. RESULTS: Estrogen and progesterone receptor positivity was detected in 65.1% and 46.1% of the tumors, respectively. DNA aneuploidy was present in 70.9% of the cases, and the median S-phase fraction (SPF) was 9.4%. There was a significant correlation of aneuploidy and high SPF with lack of hormone receptors. The median SPF and SPF tertiles (cutoff values, 6.5% and 12%), applied in the whole series, showed a significant correlation with survival, whereas if SPF was used according to ploidy status, no prognostic significance was found. No differences in relation to survival among different DNA aneuploidy subclasses were verified. In univariate analysis, clinical staging, hormone receptors, DNA ploidy and SPF showed a statistically significant correlation with the overall survival. In multivariate analysis only DNA ploidy did not maintain independent prognostic significance. CONCLUSION: Hormone receptor expression and flow cytometric SPF are independent prognostic factors in advanced breast cancer.     

Prognostic value of carcinoma-associated antigen MK-1 in urinary bladder carcinoma

BACKGROUND: The present study has been performed to evaluate the expression of MK-1 in schistosomiasis-associated squamous cell carcinoma of the urinary bladder and to correlate this new marker with the conventional histopathological parameters. PATIENTS AND METHODS: Paraffin sections of 5-microm thickness from 81 cases were prepared for hematoxylin and eosin staining and immunohistochemical analysis of MK-1 expression was carried out. RESULTS: Forty-six cases (56.8%) were positive for MK-1 protein expression. Significant correlations between MK-1 expression and tumor grade (p=0.004), schistosoma (p=0.031), DNA ploidy (p=0.001), and tumor recurrence (p<0.001) were observed. MK-1, sex, tumor grade, stage, schistosoma, DNA ploidy, and recurrence were evaluated in relation to outcome. Univariate and multivariate analysis of survival were performed. The overall 5-year survival was 51.85%. In univariate analysis, MK-1 expression, tumor grade, DNA ploidy, and recurrence had a significant impact on the survival of these patients. In a Cox proportional hazards model, recurrence maintained its significant impact on survival. CONCLUSIONS: These findings suggest that MK-1 is a prognostic marker for recurrence: 34 (87.2%) of 39 recurrent cases were positive for MK-1 expression. However, only recurrence was an independent prognostic factor in patients with schistosomiasis- associated squamous cell carcinoma of the bladder.     

Prognostic significance of DNA cytometry in carcinoma of the uterine cervix FIGO stage IB and II.

OBJECTIVE: To assess the prognostic value of DNA-image cytometry in cervical carcinoma of the uterus and its relation to other established prognostic factors. STUDY DESIGN: The study included 116 cases of cervical carcinoma FIGO stages IB and II which were treated with radical abdominal hysterectomy. The median follow-up was 55 months (range 1-162 months). DNA image cytometry was performed on cytologic specimens prepared by enzymatic cell separation from formalin-fixed, paraffin-embedded tissues. DNA stemline ploidy, DNA stemline aneuploidy, 5c exceeding rate, 9c exceeding rate, 2c deviation index, and DNA malignancy grade were computed. DNA-variables as well as various clinical and histological variables were related to survival rates. RESULTS: In multivariate statistical analysis DNA stemline ploidy using 2.2c as a cut-off value and FIGO stage showed to be statistically significant available presurgery predictors of survival, whereas the postsurgical parameters lymphonodal status, tumor size and parametrial involvement were significantly correlated with survival. The synopsis of all parameters in a multivariate Cox model indicated that - with declining relevance - the number of positive pelvic lymph nodes, DNA stemline ploidy using a cut-off level at a modal value of 2.2c, largest pelvic lymph node, 5c exceeding rate, and ratio of carcinoma area to cervix area, were of predictive value for survival. CONCLUSIONS: Our results suggest that prognostic information deducted from classical staging parameters is successfully complemented by DNA image cytometry which can be applied pretherapeutically.     

Overall survival in advanced breast cancer: relevance of progesterone receptor expression and DNA ploidy in fine-needle aspirates of 392 patients

Fine-needle aspiration cytology (FNAC) is essential for making a diagnosis in advanced breast cancer. The determination of hormone receptors in the material obtained is useful for predicting patient response to endocrine therapy, but the prognostic value of hormone receptor expression as well as the clinical utility of DNA flow cytometry are controversial. The aim of this prospective study with long-term follow-up (median: 81 months) was to evaluate these biomarkers in relation to overall survival in a series of 392 patients with advanced breast cancer (stage IIB, n=106; IIIA, n=66; IIIB, n=174; and IV, n=46) using FNAC. Estrogen and progesterone receptor expression was found in 65.1% and 46.1% of the tumors, respectively. Hormone receptors were not found to be associated with clinical staging. DNA aneuploidy was present in 70.9% of the cases and the median S-phase fraction (SPF) was 9.4%. There was a significant correlation of aneuploidy and high SPF with lack of hormone receptors. In univariate analysis, advanced disease stage, absence of hormone receptors, DNA aneuploidy and high SPF showed a statistically significant correlation with poor clinical outcome. In multivariate analysis, disease stage, progesterone receptors and DNA ploidy retained independent prognostic significance in relation to overall survival. These data indicate that progesterone receptor expression and DNA ploidy are independent prognostic factors in advanced breast cancer.     

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

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

S-phase fraction determined on fine needle aspirates is an independent prognostic factor in breast cancer – a multivariate study of 770 patients

Background: The prognostic significance of DNA ploidy and the S-phase fraction (SPF) have been extensively studied in breast cancer, but their clinical utility remains controversial. The type of tumour material can substantially influence flow cytometric DNA measurements. Material obtained by fine needle aspiration (FNA) biopsy is very suitable for flow cytometric DNA analysis because it contains a low proportion of non-tumour cells and less debris than tissue samples. Methods: The prognostic significance of DNA ploidy and SPF, determined on FNA samples, was analysed in 770 breast cancer patients, diagnosed between 1992 and 1997. DNA ploidy and SPF were determined at the time of diagnosis as part of the diagnostic work-up. The median follow-up was 90 months. Survival analysis included overall cancer specific survival (OS), disease free survival (DFS) and survival after recurrence (SAR). Other variables included in survival analyses were age, histological grade, histological type, lymph node status and tumour size. Disease free interval and the site of recurrence were also included in SAR analysis. Results: DNA ploidy and SPF correlated with tumour type, size, lymph node involvement and, especially, tumour grade. In a univariate analysis, both aneuploidy and high SPF were associated with shorter OS, DFS and SAR, but only SPF retained its independent prognostic significance in multivariate analyses. Independent prognostic variables for OS were node status, histological grade, SPF and tumour size. Node status, histological grade and SPF were independent predictors of DFS, while the site of recurrence, SPF, histological grade, disease free interval and age were independent predictors of SAR. Conclusions: DNA ploidy and SPF can be efficiently and routinely determined on FNA samples. High SPF is independently associated with a worse clinical outcome of patients with breast cancer. Although SPF and histological grade share prognostic information to some degree, SPF provides additional, less subjective prognostic information. The prognostic value of SPF determined on FNA samples could be even more relevant in neoadjuvant settings and for patients not amenable for surgical treatment, when histological grade cannot be assessed.     

Flow cytometric DNA ploidy, p53, PCNA, and c-erbB-2 protein expressions as predictors of survival in surgically resected gastric cancer patients

In order to determine retrospectively the impact of some cytometric and immunohistochemical parameters on the overall survival of gastric cancer patients treated with surgery alone, paraffin-embedded tumor samples from 137 gastric carcinoma patients undergoing curative resection from 1987-1993 were analyzed by flow cytometry (FCM) and immunohistochemistry (p53, c-erbB-2, and PCNA expression). FCM-derived parameters were DNA ploidy and fraction of S-phase cells (SPF). Multiple regression analysis was applied to determine the prognostic significance of the conventional clinicopathologic findings together with the flow cytometric and immunohistochemical parameters on overall survival. When all parameters were entered simultaneously into the Cox regression model, stage and DNA ploidy (DNA index >1.35) clearly emerged as the only independent prognostic factors. When the stages were analysed separately, the independent prognostic factors resulted DNA ploidy in early stages (I-II) and grading in stage IIIA tumors. For stage IIIB tumors, no independent prognostic factor was found. These results indicate that the DNA ploidy pattern is a valuable predictor of survival in curatively resected gastric cancer patients, especially when less advanced tumors are taken into consideration.     

Is agNOR and DNA ploidy analysis useful for evaluating thyroid neoplasms?

OBJECTIVE: To correlate the subjective AgNOR counting method and DNA content with histologic diagnoses of thyroid cancer and invasion. STUDY DESIGN: Eighty-one consecutive cases of thyroid carcinoma were selected for DNA and AgNOR analysis. The diagnoses were: papillary carcinoma (n = 40), follicular carcinoma (n = 31), Hürthle cell adenocarcinoma (n = 4), and undifferentiated carcinoma (n = 6). Seven normal thyroids were used as controls. DNA quantitative measurement was performed with Vidas 2.0 software (Kontron Bildanalyse, Munich, Germany) connected to an MPM 210 photometer microscope (Carl Zeiss, Oberkochen, Germany). The DNA index was obtained using histograms. Counting the NORs was performed by subjectively counting the NORs in 200 malignant cells. RESULTS: DNA ploidy analysis showed all Hürthle cell adenocarcinomas, 21 (67%)follicular tumors, 23 (57%) papillary tumors and 4 (67%) undifferentiated carcinomas to be aneuploid. DNA analysis correlated with histologic type of the tumor (p = 0.032). There was no statistical significance to the AgNOR counting variables studied. Statistical analysis showed correlation between ploidy and histologic diagnosis, but not AgNOR counting, to have prognostic value. CONCLUSION: DNA ploidy is more useful than subjective counting of NORs as an adjunct method for thyroid lesion analysis.     

Nasopharyngeal carcinoma heterogeneity of DNA content identified on cytologic preparations.

OBJECTIVE: To evaluate tumor heterogeneity of DNA content in nasopharyngeal carcinoma (NPC) performed on cytologic specimens. STUDY DESIGN: Image cytometric analysis of DNA ploidy status of 40 NPCs was performed on nasopharyngeal brushing smears stained with the Feulgen method after hematoxylin eosin staining. If the DNA distribution pattern from the same tumor exhibited diploid, aneuploid or/and tetraploid peaks or some combination of these patterns, the presence of tumor heterogeneity of DNA content was identified. RESULTS: Thirty-four cases (85%) had a nondiploid DNA pattern among the 40 NPCs. Twenty-eight cases exhibited tumor heterogeneity of DNA content (70%). Of the 28 tumors, 13 (46%) had a combination of diploid and tetraploid patterns, 10 (37%) had a combination of diploid and aneuploid patterns, 3 cases (11%) had a combination of tetraploid and aneuploid patterns, and 2 cases had two aneuploid stem lines. The relationship between DNA ploidy pattern and tumor histologic and cytologic morphology was also examined. CONCLUSION: There is a high incidence of DNA content heterogeneity in NPC. The relevance of tumor heterogeneity to the biologic behavior of NPC awaits further study. DNA quantification with image cytometry on destained cytologic preparations is feasible and reliable.     

Evaluation of prognostic factors following flow-cytometric DNA analysis after cytokeratin labelling: II. Cervical and endometrial cancer.

In gynecologic oncology valid prognostic factors are necessary to define biologically similar subgroups for analysis of therapeutic efficacy. This study is the first published prospective study concerning prognostic significance of DNA ploidy and S-phase fraction in cervical and endometrial cancer following enrichment of tumor cells by cytokeratin labelling. Epithelial cells were labeled by FITC-conjugated cytokeratin antibody (CK 5, 6, 8, and CK 17) prior to flow cytometric cell cycle analysis in 91 specimens of cervical cancer and 73 samples of endometrial cancer. In cervical cancer neither DNA-ploidy nor S-phase fraction were relevant prognostic parameters. But CV of the G(0)G(1)-peak showed prognostic relevance in cervical cancer cells, even in multivariate analysis. This interesting observation, however, seems to have no therapeutic consequence due to the small discrimination capacity of CV. In endometrial carcinoma, gross DNA-aneuploidy (DNA-index > 1.3) and a high percentage of proliferating cells (>75th percentile) were univariate and multivariate highly significant prognostic factors for recurrence-free survival. Especially DNA-aneuploidy (DI>1.3) is one of the most important independent molecular biological prognostic factors. While diagnostic curettage we could identify risk patients even preoperatively by determination of the prognostic factors like histologic tumor type, grading, cervical involvement and DNA-ploidy. Thereby these patients could be treated primarily in an oncologic center. In conclusion, our investigations showed that the determination of DNA-ploidy should be done in endometrial carcinoma. In cervical cancer no clinical significance for determination of DNA-parameters was found.     

Prognostic significance of DNA ploidy and proliferation rate in human astrocytic gliomas

DNA ploidy and the proliferative potential in 75 gliomas were investigated using bromodeoxyuridine labelling index (BrdUrd LI), S-phase fraction (SPF) and argyrophilic nucleolar organizer regions (AgNOR) technique. There were 53 highly malignant (AIII-AIV), and 22 low-grade (AI-AII) gliomas. One fragment of the tumour was fixed in Carnoy's solution for AgNOR test, while the other fragments were used for flow cytometric determination of the labelling index, SPF and DNA ploidy. For the BrdUrdLI, tumour samples from each patient were incubated in vitro for one hour at 37 degrees C with BrdUrd using the high pressure oxygen method. The tumours showed variability in the BrdUrdLI values, SPF and AgNOR counts/cell nucleus. The same percentage of DNA aneuploidy (55%) was found in high-grade as well as in low-grade gliomas. Univariate analysis showed that patients with grade I & II gliomas had significantly higher 3-year survival rate (p = 0.0193) than those with grade III and grade IV gliomas. Also patients with lower proliferation rate of tumours (BrdUrdLI < or =2.3% and AgNOR counts < or =2.6%/cell) had higher 3-year survival rate (p<0.03), which can be helpful in prognosis. Tumour ploidy or SPF had no influence on patients' survival (p = 0.7908). Cox multivariate analysis showed that only patients' age > 45 years and high tumour grade (III and IV) were significant unfavourable prognostic factors in terms of patients' survival.     

DNA ploidy in soft tissue sarcoma: comparison of flow and image cytometry with clinical follow-up in 93 patients

In soft tissue sarcoma, the prognostic importance of DNA ploidy status is limited. One possible explanation may be technical; small non-diploid stemlines will be diluted in relation to the presence of normal diploid cells and may not be detected by flow cytometry (FCM). We assessed DNA ploidy status in 93 tumors with both FCM and image cytometry (ICM). ICM may permit the exclusion of non-relevant cells. The ability of the two methods to detect non-diploid stemlines was compared, as were the prognostic consequences. The patients (54 males) had a median age of 69 years. Surgical procedures were performed on all patients. None of the patients had received preoperative radiotherapy or chemotherapy. FCM and ICM were performed with standard methods. The prognostic value was assessed with univariate and multivariate analysis. In 82 of the 93 tumors, a concordant ploidy status by FCM and ICM was found. In 5 FCM type 1-2 tumors (diploid), the identification of non-diploid stemlines by ICM did not influence the metastatic rates. Increasing tumor size, histotype other than liposarcoma, increasing malignancy grade, tumor necrosis, and ICM non-diploidy were univariate prognostic factors for metastasis. In a multivariate analysis, only tumor size larger than 9 cm was a prognostic factor. In about 10% of the tumors, a discrepancy between FCM and ICM ploidy status was found, but we could not find a consistent prognostic consequence of this. Neither FCM nor ICM ploidy status was an independent prognostic factor.     

Prognostic significance of DNA aneuploidy and p21 ras oncoprotein expression in colorectal cancer and their role in the determination of treatment modalities

The purpose of the present study was to investigate the prognostic significance of DNA ploidy, S-phase fraction and p21 ras oncoprotein expression in patients with colorectal cancer and to correlate these factors with the clinical behavior of the tumors and their response to therapy. Of 79 patients with colorectal cancer 57% (45/79) had early stage disease. Forty-one percent (32/79) had aneuploid tumors while 30% (24/79) of the tumors had a high (>10%) S-phase fraction. p21ras oncoprotein expression was detected in 38% (30/79) of tumors. Patients with aneuploid tumors had a worse prognosis than patients with diploid tumors (p=0.0002). Similarly, patients with high S-phase fraction tumors had a shorter survival than those with low S-phase fraction tumors (p=0.005). No such difference was found between p21 raspositive and p21 ras-negative tumor subgroups. In early stage colorectal cancer, aneuploidy was closely correlated with disease outcome (p=0.029). Early stage patients with diploid tumors who received radiotherapy and chemotherapy had a better prognosis than patients with aneuploid tumors. In conclusion, DNA ploidy is a significant and independent prognostic factor in colorectal cancer. Aneuploidy and genetic alteration of the p21 ras oncoprotein are important in determining the biological aggressiveness of colorectal cancer. Furthermore, DNA ploidy may identify those subgroups of patients with early stage disease who may benefit from more aggressive treatment.     

Comparison of quantitative histomorphometry and DNA ploidy in tissue sections of prostate carcinoma

OBJECTIVE: To examine the ability of quantitative histomorphometry to predict DNA ploidy of prostate carcinoma in biopsy tissue sections assigned after quantitation by nuclear digital image analysis. STUDY DESIGN: Thirty-five diploid, 35 tetraploid and 35 aneuploid prostatic carcinomas in biopsies, assessed by the CAS 200 image analyzer (Bacus Laboratories, Lombard, Illinois, U.S.A.), were reevaluated by the Bacus Laboratories Incorporated Slide Scanner, a microscope that quantifies histologic images. Thirty-one histomorphometric features from cancer cells were captured at 40 x magnification, averaged across tilesfor each case and incorporated into a multivariate discriminant model to determine which features predicted ploidy interpretation by nuclear image analysis using the CAS 200. RESULTS: On average, 60 and 15 minutes were required to perform nuclear image analysis and histomorphometry, respectively. The multivariate discriminant model identified configurable run length, difference variance, contrast, inverse difference moment, sum entropy and diagonal variance as histomorphometry features capable of distinguishing diploid from nondiploid tumors (P < .05). Cross-validation studies showed the model correctly classified 74.3% of the diploid and 57.1% of the nondiploid cases. CONCLUSION: Quantitative histomorphometry can predict the ploidy of prostate carcinoma in biopsy tissue sections. Quantitative histomorphometry has potential as a method of rapidly assessing DNA ploidy otherwise earmarked for nuclear image analysis, resulting in savings of time and expense.     

The Multivariate Prognostic Index and nuclear DNA content are independent prognostic factors in primary breast cancer patients

The predictive value of a previously described Multivariate Prognostic Index (which incorporates weighted values of the mitotic activity index, tumor size, and the axillary lymph node status), and the nuclear DNA content (DNA) was evaluated in 156 patients with primary invasive ductal breast cancer, diagnosed between 1980 and 1983. The results were analysed with respect to the occurrence of distant recurrence and survival of the patients after at least 3 yr of follow-up (range 36-73 months; median 44 months). Known prognostic factors such as lymph node status, tumor size, and the mitotic activity index correlated independently with distant recurrence. Furthermore, in respect to survival, the investigated prognostic factors (except DNA content) were significantly correlated. The results indicate that the predictive value of the Multivariate Prognostic Index (MPI) is stronger (P less than 0.001) than of the nuclear DNA content (P less than 0.005) with respect to distant recurrence. In a Cox multivariate regression analysis DNA ploidy turned out to be an independent prognostic factor once the MPI was selected. Furthermore, in Cox's analysis, DNA ploidy was the fourth selected variable after lymph node status, mitotic activity index, and tumor size in individual parameter analysis. The results of this study indicate that, with respect to breast cancer screening programs, it seems worthwhile to integrate morphometric features, the MPI, and DNA ploidy in a new prognostic model.     

DNA ploidy of bladder cancer using bladder biopsy supernate specimens

OBJECTIVE: To perform DNA image cytometry on 119 bladder biopsy supernate (BBS) specimens of transitional cell carcinoma (TCC) bladder to: (1) test the suitability of this cytologic specimen for use in DNA ploidy analysis, and (2) assess the value of DNA ploidy measured on this specimen as to the risk of tumor recurrence and survival. STUDY DESIGN: The histologic grade and cytologic grade were correlated, and the DNA ploidy produced was determined by image analysis of Feulgen-stained nuclei. Kaplan-Meier curves related age, sex, grade and DNA ploidy to recurrence of tumor and survival. Log rank analyses were used to ascertain the difference between the curves for each categorical variable. RESULTS: Urothelial cells derived from the BBS specimen were demonstrated to be representative of the tumor. The tumor recurrence rate was significantly higher (P = .0001) and the survival rate significantly lower (P = .0002) for patients with aneuploid tumors compared to those with diploid tumors. Patients with TCC 2 tumors had a significantly shorter time to recurrence (P = .003), although the relationship between ploidy and survival in this group was of marginal significance. CONCLUSION: The specimen was free of many of the problems associate with the other specimen types used for measuring DNA ploidy. The results show that the BBS specimen is diagnostically useful and suitable for DNA analysis, providing prognostically relevant information.     

Cytometric and histologic predictors of prognosis in ampullary carcinoma treated with pancreatico-duodenectomy

The association between several clinical, histologic and karyometric variables and the survival rates of patients with ampullary carcinoma was examined. Cases were limited to those treated exclusively by pancreaticoduodenectomy for which follow-up data was available, eliminating those cases with deaths due to other causes. The histologic type was classified as papillary, intestinal or mixed while the differentiation was recorded as well, moderate or poor. The stroma was categorized as scanty, moderate or abundant, and the tumor stage was evaluated according to Martin's classification. High-resolution morphometric and microphotometric (DNA content) evaluation of Feulgen-stained nuclei was performed using the microTICAS system. Statistical analyses were performed to examine the relationship between these variables and survival. Neither the tumor stage nor the presence of positive lymph nodes was a significant prognostic indicator, nor was the degree of differentiation or the amount of stroma. However, the survival showed a significant association with several karyometric variables and with the histologic type. Specifically, aneuploid DNA ploidy profiles, higher mean ploidy values and larger nuclei were associated with a lower survival rate. Short-term survivors (less than five years) had a mean ploidy of 2.8N, a mean 5N exceeding rate of 8.3% and a mean nuclear area of 41 sq microns, while long-term survivors (greater than or equal to five years) had corresponding means of 1.9N, 0.6% and 26 sq microns. These differences are all significant at a two-tailed significance level of less than .05 using a separate variance estimate t-test. In addition, papillary tumors showed a better prognosis than did intestinal or mixed tumors (Breslow P less than .04 and Mantel-Cox P less than .009).     

Prospective study on prognostic significance of DNA ploidy and Ki-67 expression in colorectal cancer

The aim of the study was to correlate tumoral DNA ploidy and Ki-67 expression with therapy response, Overall Survival (OS), Disease Specific Survival (DSS) and Disease Free Survival (DFS). Three samples of colorectal cancer were collected from each patient. One sample of normal tissue was our internal control. DNA ploidy was evaluated by FACSCalibur cytometer and Ki-67 by immunohistochemistry. We studied 67 patients and we found aneuploidy in 65,7 percent of carcinoma with a Ki-67 median expression of 55 percent. After surgery and chemotherapy in 35 percent of the patients with aneuploid carcinoma and high proliferative activity (Ki-67 greater than 55 percent) there were no evidence of disease versus 100 percent of patients with DNA diploidy and low proliferative activity (Ki-67 less than 55 percent). Tumoral aneuploidy significantly correlated with lower OS, DSS and DFS (18 percent vs 86 percent at 30 months). Univariated analysis demonstrated a significant correlation between aneuploidy and develop disease progression (p=0,033, odd ratio=5.7), while the cut-off of 55 percent for Ki-67 expression did not correlate with OS, DSS and DFS. Preliminary results (the study is still in progress) seemed to suggest that DNA ploidy has a prognostic and predictive significance in colorectal carcinoma.     

Prognostic biomarkers in renal cell carcinoma: relevance of DNA ploidy in predicting disease-related survival

OBJECTIVE: To investigate the prognostic value of DNA ploidy, Ki-67 index and p53 expression in relation to disease-related survival in a consecutive series of patients with renal cell carcinoma (RCC). MATERIAL AND METHODS: The study group consisted of 64 RCC patients treated by radical nephrectomy. Histological type, pathological staging and nuclear anaplasia were assessed according to the WHO classification, TNM system and Fuhrman grading criteria, respectively. Ploidy was determined by DNA flow cytometry using two sampling methods (frozen vs paraffin-embedded tissue). Ki-67 and p53 were evaluated by immunohistochemistry techniques using two cutoff points (10% vs mean value) for staining interpretation. Kaplan-Meier and Cox regression analyses were used for prognostic evaluation. RESULTS: Thirty-one tumors (48.4%) showed DNA diploidy and 33 (51.6%) were DNA aneuploid. Concordance between both ploidy measurement methods was found in 85.5% of cases (p=0.0455). The mean values for Ki-67 and p53 immunostaining were 3.65% (0-23.5%) and 5.90% (0-55.9%), respectively. DNA ploidy significantly correlated with staging, tumor size (pT), nuclear grading, and Ki-67 (mean value cutoff). Ki-67 (10% cutoff) correlated with staging and pT, while p53 (mean value cutoff) was associated with Ki-67 (mean value cutoff). There were significant differences between survival curves for pathological stage, pT, nuclear grade, ploidy, Ki-67 (both cutoffs), and p53 (10% cutoff). By univariate regression analysis, stage III and stage IV, pT3, aneuploidy, high Ki-67 (both cutoffs), and p53 overexpression (10% cutoff) showed significant correlations with worse disease-related survival. In addition, DNA aneuploidy significantly correlated with poor prognosis within stages I/II (p=0.0355) and stages III/IV (p=0.0138) of the disease. CONCLUSION: The results indicate that DNA ploidy has relevant prognostic value in RCC, adding useful information to the classic histopathological indicators of clinical outcome.