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.     

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.     

DNA distribution in non-small-cell lung carcinomas and its relationship to clinical behavior

A study of 187 surgical specimens of tumors of patients with non-small-cell lung carcinomas was carried out by means of flow cytometry. Eighty-four percent of the tumors were classified as tumors with abnormal DNA stemlines (DNA aneuploidy). Patients with tumors demonstrating DNA aneuploidy had significantly shorter survival times than those with tumors demonstrating DNA diploidy (p = .009). Cell cycle analysis was possible in 122 tumors. Patients whose tumors had 0-8% S-phase cells died later than patients whose tumors had 9-16% S-phase cells (p = .018). In addition, patients with tumors with a low fraction of labeled S-phase cells (autoradiography) had a better prognosis than patients with tumors with a high proportion of labeled S-phase cells (p = .041).     

DNA ploidy in colorectal cancer, heterogeneity within and between tumors and relation to survival

Flow cytometry was used to study the incidence of aneuploidy and to determine the significance of multiple sampling from colorectal tumors. DNA ploidy pattern has been proposed as a supplementary prognostic marker, but discrepancies in findings are major. DNA clonal heterogeneity, defined as two or more DNA aneuploid stemlines in the same tumor, is well established. However, most studies have been based on only one biopsy from each tumor. In our study multiple biopsies were taken from 163 patients (88 males and 75 females) electively operated for colorectal cancer. Tumor cells were harvested by fine needle aspiration from fresh frozen biopsies sampled at different sites of each tumor. DNA aneuploidy was detected in tumors from 145 patients (89%), and 18 patients (11%) had a solitary DNA diploid cell population. In a 79 month follow-up period 105 patients had died. Statistical analysis showed that distinction between diploidy and aneuploidy did not predict survival. However, grouping subpopulations into DNA diploid plus near diploid (DNA index (DI) 0. 97-1.15), DNA aneuploid with all aneuploid subpopulations in the interval 1.15-2.06, and DNA aneuploid with subpopulations with DI < 0.97 and/or DI > 2.06, showed a significant difference in survival in a Cox multivariate analysis including Dukes' stage P = 0.049 comparing the second group to the first and P = 0.01 comparing the third group to the first. In 21 (13%) patients only one subpopulation was found, 57 (35%) had two, 44 (27%) had three, and 41 (25%) had four or more different subpopulations. The association of DNA ploidy to survival is shown to be dependent on the number of biopsies analysed.     

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.     

Colorectal carcinoma. DNA ploidy pattern and prognosis with reference to tumor DNA heterogeneity.

Nuclear DNA content was measured in 72 colorectal carcinomas using single-cell microspectrophotometry on Feulgen-stained smears. Four samples were analyzed from each tumor. Patients were followed for 41-65 months (average, 53). DNA heterogeneity (both aneuploid and nonaneuploid patterns) was present in 44% of the cases. Sixty-eight percent of the tumors showed an aneuploid DNA pattern in at least one of the samples. Patients with nonaneuploid tumors tended to have a survival advantage over patients with homogeneously aneuploid tumors and demonstrated a significantly longer disease-free survival. The DNA ploidy pattern is of potential value in conjunction with histopathologic prognostic parameters in colorectal carcinoma. Since colorectal tumors exhibit pronounced DNA heterogeneity, multiple samples are required from each tumor to permit a proper evaluation of its DNA pattern. The DNA heterogeneity may represent tumor progression and can partly explain the conflicting results reported concerning DNA pattern and prognosis in colorectal carcinoma.     

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.     

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.     

Dilution effect of nontumor cells on flow cytometric DNA S-phase fraction in breast cancer fine needle aspirates

OBJECTIVE: To analyze the proportion of nontumor cells in fine needle aspirates of breast carcinoma and its influence on flow cytometric S-phase fraction (SPF) estimation. STUDY DESIGN: We analyzed the proportion of nontumor cells in fine needle aspiration biopsy smears, performed flow cytometric analysis of DNA ploidy and SPF on freshly aspirated tumor material and analyzed histograms manually and automatically using Multi-Cycle AV software (Phoenix Flow Systems, San Diego, California, U.S.A.) for cell cycle analysis. We corrected SPF of diploid tumors for the dilution effect using an individually established percentage of nontumor cells (individual correction) and the mean proportion of nontumor cells in diploid tumors (factor correction). RESULTS: The proportion of nontumor cells ranged from 0.5% to 76.6% (mean, 12.6; SD, 15.7) in 55 diploid tumors and from 0.5% to 53% (mean, 8.6; SD, 8.9) in 84 aneuploid tumors (p=0.178). In 14 of 139 (10%) samples, the proportion of nontumor cells exceeded 20%. The mean SPF values of diploid tumors without correction were 4.9% (manually) and 6.5% (automatically) and of aneuploid tumors, 9.5% and 11.0%, respectively. In univariate Cox survival analysis, noncorrected SPF was a significant prognostic factor in overall survival (p < 0.001). Neither individual nor factor correction of SPF significantly changed its prognostic value. CONCLUSION: Fine needle aspirates contain low proportions of nontumor cells, having an insignificant dilution effect on SPF estimation. Most probably, SPF could be reliably estimated usingfreshly aspirated tumor material without any correction or adjustment.     

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 cytometric analysis of surgically treated squamous cell cancer of the uterine cervix, stage pT1b1-pT2b

OBJECTIVE: To determine the utility of DNA content and DNA-related variables of proliferative activity regarding prognosis in cervical cancer. STUDY DESIGN: DNA image (ICM) andflow cytometry (FCM) were performed to determine the DNA index (DI), 5c-exceeding rate (5c-ER), S-phase fraction (SPF) and proliferation index (PI) in 163 patients with surgically staged pT1b1-pT2b squamous cell cancer of the uterine cervix and treated with primary radical hysterectomy. ICM was performed on imprint cytology, obtained from fresh tumor tissue, which was also used for FCM. Results were analyzed using the chi2 test and Cox regression analysis for risk of pelvic lymph node involvement, tumor recurrence and recurrence-free survival (RFS). RESULTS: ICM was performed on all 163 and FCM on 133 samples. One-third of the tumors showed DNA aneuploidy. Analysis demonstrated prognostic significance of a DI > or = 1.70, with a (70:30) 2.3-fold risk of recurrence (P=.024) and reduced RFS of 10 months (P=.003) in cases of DI > or = 1.70. A high 5c-ER > 11% was associated with pelvic lymph node involvement and decreased RFS (P < or = .04). Significantly more relapses were found in tumors with SPF > 12% (70.8% vs. 29.2%, P=.007). RFS was markedly reduced in tumors with high SPF (52.3 vs. 61.1 months, P=.011). Low proliferative tumors (PI<25%) were associated with lower stage (P=.036) and increased RFS (61.2 vs. 47.1 months, P=.028). In multivariate analysis of clinicopathologic variables (pT category, nodal status, lymphovascular space involvement) and DNA related variables, pelvic lymph node involvement was the only significant predictor of RFS. In patients with nodal involvement, tumors with DI >1.70 were associated with lessfavorable outcomes. CONCLUSION: DNA-related variables of cell cycle analysis were valuablefor predicting prognosis in cervical cancer patients. Tumors with DI>1.70, 5c-ER >11% and high proliferative activity (SPF>12%, PI>25%) represent a subgroup with a poor prognosis.     

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.     

Flow and image cytometric DNA ploidy,including 5c exceeding cells,of serous borderline malignant ovarian tumors. Correlation with clinicopathologic characteristics

OBJECTIVE: To analyze DNA ploidy of serous borderline ovarian tumors by flow cytometry (FCM) and image cytometry (ICM), with 5c exceeding cells also analyzed, and to evaluate their correlation with clinicopathologic characteristics of patients and tumors. STUDY DESIGN: Cell suspensions were prepared according to a modified Hedley method from formalin-fixed, paraffin-embedded tissue blocks of 43 tumors. One part of the suspension was used for flow cytometric measurement; from the other part, filter slides were prepared for ICM. RESULTS: FCM and ICM found 2 aneuploid (peridiploid) serous borderline ovarian tumors, and FCM found 1. ICM found 3 tumors with 5c exceeding cells and 2 tumors with octaploid cells. There was no correlation between DNA aneuploidy and presence of 5c exceeding cells with tumor size, International Federation of Gynecology and Obstetrics stage or survival. CONCLUSION: The results confirm a good correlation between FCM and ICM DNA ploidy and the ability of ICM to detect 5c exceeding cells. The prognostic value of DNA ploidy and 5c exceeding cells in serous borderline malignant ovarian tumors warrant further evaluation.     

Flow cytometric bivariate analysis of DNA and cytokeratin in colorectal cancer.

Different opinions about flow cytometric estimates of DNA aneuploidy and/or S-phase fraction (SPF) as supplementary prognostic markers in colorectal cancer are to some degree associated with methodology. Using univariate DNA analysis, we have previously investigated the DNA ploidy in colorectal cancer, its heterogeneity within and between tumors and its relation to survival. To improve detection of DNA aneuploid subpopulations and particularly estimation of their SPF's we investigated a method for bivariate DNA/cytokeratin analysis on fine-needle aspirates of 728 frozen biopsies from 157 colorectal tumors. Unfixed aspirates were stained with propidium iodide and FITC-conjugated anti-cytokeratin antibody in a saponin-buffer. A significant association between SPF and debris was observed. There were no substantial difference in DNA ploidy patterns between univariate and bivariate measurements (concordance was 92-95%). No new DNA aneuploid subpopulations were detected in cytokeratin-gated compared to ungated or univariate histograms. Debris-adjusted SPF's of cytokeratin-gated histograms were significantly higher than of ungated histograms, also for subpopulations with DI>1.4 (p<0.0001). There was no significant association between SPF and survival.     

Cell cycle flow cytometric analysis in the diagnosis and management of colorectal carcinoma

OBJECTIVE: To establish prognostic models and protocols for individualized management in colorectal carcinoma patients based on both clinical and DNA flow cytometric parameters. STUDY DESIGN: Prospective study of 88 colon carcinoma patients with a minimum follow-up of 12 months, operated on with the intent to cure and not treated with radiotherapy or chemotherapy. All the cases were subjected to a clinical evaluation: age, sex, tumor localization and size, histologic grade, tumor stage, disease-free interval, survival and flow cytometric study (ploidy, DNA index and S-phase fraction [SPF]). RESULTS: From the total of 88 neoplasms studied, 56 (63.6%) were from males and 32 (36.4%) from females; 30 (34%) were located in the right side of the colon, 7 (8%) in the transverse colon and 51 (58%) in the left side of the colon. Eleven (12.5%) were stage I, 52 (59.1%) stage II and 25 (28%) stage III. Forty-two (47.7%) were diploid and 46 (52.3%) aneuploid. The S-phase mean was 14.6% (12% for diploids and 16.9% for aneuploids). During the follow-up period, 26.1% of diploid tumors recurred, whereas aneuploid tumors recurred in 36.9% (P < .05). SPF from diploid and aneuploid tumors was analyzed separately. CONCLUSION: Regarding relapse-free interval, the behavior of diploid tumors with a high SPF was similar to that of aneuploid ones. Two kinetic profiles were established, favorable (diploid tumors with low S phase) and unfavorable (diploid with high S phase and all aneuploid tumors), that had significant prognostic value for progression and survival and that allowed identification of patients at high risk of recurrence. We formulated a prognostic index according to SPF and tumor stage that has discriminatory capacity for biologic behavior in colorectal tumors.     

Optimizing flow cytometric DNA ploidy and S-phase fraction as independent prognostic markers for node-negative breast cancer specimens.

Developing a reliable and quantitative assessment of the potential virulence of a malignancy has been a long-standing goal in clinical cytometry. DNA histogram analysis provides valuable information on the cycling activity of a tumor population through S-phase estimates; it also identifies nondiploid populations, a possible indicator of genetic instability and subsequent predisposition to metastasis. Because of conflicting studies in the literature, the clinical relevance of both of these potential prognostic markers has been questioned for the management of breast cancer patients. The purposes of this study are to present a set of 10 adjustments derived from a single large study that optimizes the prognostic strength of both DNA ploidy and S-phase and to test the validity of this approach on two other large multicenter studies. Ten adjustments to both DNA ploidy and S-phase were developed from a single node-negative breast cancer database from Baylor College (n = 961 cases). Seven of the adjustments were used to reclassify histograms into low-risk and high-risk ploidy patterns based on aneuploid fraction and DNA index optimum thresholds resulting in prognostic P values changing from little (P < 0.02) or no significance to P < 0.000005. Other databases from Sweden (n = 210 cases) and France (n = 220 cases) demonstrated similar improvement of DNA ploidy prognostic significance, P < 0.02 to P < 0.0009 and P < 0.12 to P < 0.002, respectively. Three other adjustments were applied to diploid and aneuploid S-phases. These adjustments eliminated a spurious correlation between DNA ploidy and S-phase and enabled them to combine independently into a powerful prognostic model capable of stratifying patients into low, intermediate, and high-risk groups (P < 0.000005). When the Baylor prognostic model was applied to the Sweden and French databases, similar significant patient stratifications were observed (P < 0.0003 and P < 0.00001, respectively). The successful transference of the Baylor prognostic model to other studies suggests that the proposed adjustments may play an important role in standardizing this test and provide valuable prognostic information to those involved in the management of breast cancer patients.     

Wilms' tumor. Role of fine needle aspiration and DNA ploidy by image analysis in prognostication

OBJECTIVE: To evaluate the utility of cytomorphologic features and DNA ploidy estimation in fine needle aspirates (FNAs), from Wilms' tumors for prognostication. STUDY DESIGN: Twenty-three cases of Wilms' tumor having FNA and follow-up data were selected. Cytomorphology was analyzed by two observers. DNA ploidy was determined in 19 cases by image cytometry by destaining Papanicolaou-stained slides and restaining with Feulgen stain. Various parameters and patient outcomes were compared, and statistical evaluation was done. RESULTS: Poor outcome (12/23 cases) was associated with age < 2 years (P = .01), severe pleomorphism of blastemal cells (4/23 cases, P < .05), very large nucleoli (5/23 cases, P = .075), atypical mitosis (6/23 cases, P = .032) and aneuploidy/tetraploidy of tumor cells (6/29 cases, P = .005). Term unfavorable cytology is proposed when a combination of severe pleomorphism, very large nucleoli and atypical mitosis is seen in FNA smears. Four Wilms' tumor FNAs were characterized as showing unfavorable cytology, and all had a poor outcome (P = .0351). Three of the six cases with aneuploid/tetraploid features also showed unfavorable cytology. CONCLUSION: Unfavorable cytology and aneuploidy/tetraploidy in FNA smears of Wilms' tumor are associated with a poor prognosis.     

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.     

K-ras2 activation and genome instability increase proliferation and size of FAP adenomas.

The possible role of K-ras2 mutations and aneuploidy toward increase of proliferation and adenoma size in Familial Adenomatous Polyposis (FAP) adenomas is not known. The present study addresses these issues by investigating 147 colorectal adenomas obtained from four FAP patients. The majority of adenomas had size lower than or equal to 10 mm (86%), low grade dysplasia (63%), and were preferentially located in the right colon (60%). Normal mucosa samples were obtained from 19 healthy donors. Three synchronous adenocarcinomas were also investigated. K-ras2 mutation spectrum was analysed by PCR and Sequence Specific Oligonucleotide (SSO) hybridization, while flow cytometry (FCM) was used for evaluating degree of DNA ploidy and S-phase fraction. Overall, incidences of K-ras2 mutations, DNA aneuploidy and high S-phase values (>7.2%) were 6.6%, 5.4% and 10.5%, respectively. In particular, among the adenomas with size lower than 5 mm, K-ras2 mutation and DNA aneuploidy frequencies were only slightly above 1%. Statistically significant correlations were found between K-ras2 and size, DNA ploidy and size and K-ras2 and S-phase (p < 0.001). In particular, among the wild type K-ras2 adenomas, high S-phase values were detected in 8% of the cases versus 57% among the K-ras2 mutated adenomas (p = 0.0005). The present series of FAP adenomas indicates that K-ras2 activation and gross genomic changes play a role toward a proliferative gain and tumour growth in size.     

DNA and S-phase distribution and incidence of metastasis in human primary lung carcinoma

The aim of the study was to investigate the relationship between DNA and S-phase distribution in primary non-small-cell lung carcinomas with the incidence of metastasis. Patients with non-small-cell lung carcinomas were divided into two groups depending on whether at time of surgery there were metastases or not, and these groups were correlated with the data obtained by flow cytometry or autoradiography. As expected from other studies, survival time was significantly longer for those patients without metastases at time of surgery (P = .0002) and the incidence of metastasis was significantly higher when the primary tumor was greater than or equal to 70 cm3 (P = .026). In this study, a total of 185 fresh specimens of lung carcinomas were investigated by flow cytometry. Patients with aneuploid tumors had a higher tendency to have metastases (P = .016). Patients with tumors with a higher proportion of S-phase cells measured by either flow cytometry or autoradiography demonstrated significant increase in the formation of metastases (P = .02 and P = .05). We feel that these results warrant further investigation with other primary tumors. A comparison of primary tumors that are known to rapidly metastasize vs. those that either slowly or rarely metastasize may prove to yield valuable insight into the important factors associated with metastatic potential.