Flow cytometric analysis of DNA content in adult testicular germ cell tumors]

Flow cytometric DNA analysis was carried out in 54 patients with testicular germ cell tumors (GCTs) experienced at our hospital, to evaluate the clinical relevance of DNA index (DI) and provide some insight into the pathogenesis of testicular GCTs. Histological types with their incidences were seminomas in 31 patients and nonseminomatous germ cell tumors (NSGCTs) in 23 adults. DNA ploidy and DI were analyzed by flow cytometry in 158 paraffin embedded samples; 2.9 samples per case on the average. This study revealed that 52 cases (96%) of evaluable 54 adult GCTs were DNA aneuploid, while DNA diploid tumors were observed in only each one case of NSGCT and seminoma. There was a significant difference (p less than 0.01) between the distribution of DIs in adult NSGCTs (median DI = 1.50) and that in pure seminomas (median DI = 1.85). Although we found no significant correlation between DI and clinical staging of Japanese Urological Association, on the basis of Indiana University staging system, the median DI in NSGCT patients of the advanced extent was lower than those of the other extents. DNA heterogeneity was observed only in 4 of 23 NSGCT patients (17%) and 3 of those 4 patients were assigned to advanced extent. These data suggest that the lower DI and the presence of DNA heterogeneity may have prognostic relevance for NSGCTs.     

Tumor heterogeneity of renal cell carcinoma as analyzed by flow cytometry

DNA content in renal cell carcinoma was investigated to examine the tumor heterogeneity and was correlated with their morphologic grades. A total number of 147 paraffin-embedded samples (2-6 samples with a mean of 5) from 30 tumors were analyzed by flow cytometry. DNA aneuploid patterns were demonstrated in 5 of 9 grade 1 tumors (56%), 15 of 18 grade 2 tumors (83%) and 3 of all grade 3 tumors (100%), while aneuploid DNA histograms were exhibited in 20 of 57 grade 1 samples (35%), 45 of 81 grade 2 samples (56%) and 8 of 9 grade 3 samples (89%). DNA aneuploid patterns were demonstrated more significantly in grade 2 and 3 samples than in grade 1 samples (p less than 0.02 and p less than 0.01, respectively). Consequently, 73 samples (50%) showed DNA aneuploid patterns and 74 samples exhibited DNA diploid patterns. Eleven tumors (37%) showed homogeneous DNA ploidy patterns (7 tumors were diploid and 4 tumors were aneuploid only), while 19 tumors (63%) showed DNA heterogeneity, 17 of these 19 tumors demonstrating diploid as well as aneuploid samples. DNA heterogeneity was not found when the tumor samples examined were fewer than 3 samples. However, 19 of 25 tumors (76%) in which more than 4 samples were examined showed DNA heterogeneity, the incidence of which tended to be increased with upgrading of the tumor--3 of 9 (33%) in grade 1 tumors, 13 of 18 (72%) in grade 2 tumors and 3 of all (100%) in grade 3 tumors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric deoxyribonucleic acid analysis in stage I renal cell carcinoma

Tumor deoxyribonucleic acid (DNA) content was analyzed by flow cytometry in 60 consecutive patients with stage I renal cell carcinoma. Of 59 evaluable tumors 27 (46%) were homogeneously diploid, 1 (2%) was tetraploid and 31 (52%) were aneuploid. Of the 32 nondiploid tumors 25 were heterogeneous concerning ploidy. One of the 27 patients with diploid tumors had metastases compared to 5 of the 32 patients with nondiploid tumors (not significant). There was a significant difference in survival between patients with diploid and nondiploid tumors (p = 0.043). Neither nuclear grade, tumor cell type nor tumor size correlated with survival. Analysis of DNA content seems to predict survival significantly for patients with stage I renal cell carcinoma.     

Transitional cell carcinoma of the renal pelvis and ureter: prognostic relevance of nuclear deoxyribonucleic acid ploidy studied by slide cytometry: an 8-year survival time study.

In 72 patients with urothelial carcinoma of the renal pelvis or ureter the ploidy, deoxyribonucleic acid (DNA) heterogeneity and counts of cell cycle phases in the tumor were analyzed by means of single cell DNA cytophotometry with the intention of finding new prognostic factors in addition to those already known (stage and grade). Followup ranged from 1 to 8 years. The results of the DNA analyses were related to the tumor categories, histopathological grading of the tumors and clinical course. Malignancy grade 1 tumors showed DNA frequency peaks in the diploid range, while tumors assessed as malignancy grade 2 showed heterogeneous DNA distribution patterns. Malignancy grade 3 tumors exhibited 71% aneuploid and 29% tetraploid DNA values. The proliferation rate of the tumor cells was statistically significantly higher in malignancy grades 2 and 3 than in malignancy grade 1. The prognosis for grade 1 tumors is good, whereas it is unfavorable in the case of grade 3 tumors. For these 2 groups (patients with grades 1 and 3 tumors) DNA ploidy affords no additional prognostic information. Grade 2 tumors, on the other hand, are heterogeneous in respect to DNA ploidy although they exhibit the same histomorphological degree of differentiation. These tumors can be subclassified as aneuploid (biologically aggressive) and diploid or tetraploid (biologically less aggressive) tumors. There was also a positive correlation between T category and DNA ploidy. The cell lines were aneuploid in 38% of the patients with stage T1 tumors, 56% with stage T2 tumors and almost 85% with stage T3, N+ tumors. A significant correlation was found between the results of DNA cytophotometry and the clinical course of the disease. Patients with diploid tumor cell nuclei had no metastases and no local tumor progression for up to 8 years, whereas patients with aneuploid tumor cell nuclei suffered metastasis and local tumor progression within 24 to 36 months. The patients died of the tumor 36 months after primary diagnosis on the average. The determination of DNA ploidy, tumor heterogeneity and tumor cell proliferation by means of DNA cytophotometry affords valuable clues as to prognosis.     

Renal cell carcinoma: DNA cytometry and its clinical significance. An 8 year survival study]

In 157 patients with renal cell carcinoma, the ploidy, DNA heterogeneity and the phases of the cell cycle occurring in the tumors were determined by means of single-cell DNA cytophotometry, in order to establish further prognostic factors in addition to the ones known so far (stage/grade). Patients with aneuploid tumors or tumors with more than one DNA frequency peak were found to have lymph node metastases intraoperatively and died earlier than patients with diploid tumors. Patients who had tumors with low proliferative activity survived longer than patients with highly proliferative tumor activity (p = 0.001).     

Flow cytometry analysis of urothelial cell DNA content according to pathological and clinical data on 100 bladder tumors

DNA content of 100 bladder tumors (34 grade I, 42 grade II and 24 grade III, WHO classification) were studied by flow cytometry. Ten normal bladder samples were used as control. The 100 bladder tumors could then be separated into two groups. A first group of 60 tumors (60%) had a unimodal distribution with a diploid peak and a DNA index close to 1.0, 32 grade I, 22 grade II and 6 grade III tumors displayed this pattern as did the 10 normal bladders. The second group (40%) had a bimodal distribution with two peaks, the first one (diploid peak) with a DNA index of 1.0, the second (aneuploid peak) with a DNA index greater than 1.0. Two grade I, 20 grade II and 18 grade III tumors belonged to this group. Frequency of the aneuploid peak increased with tumor grade and infiltration progression. Hence 6% of grade I, 48% of grade II and 75% of grade III tumors showed an aneuploid peak as well as 8% of Pa, 46% of P1, 73% of P2 and 87.5% of P3 stage tumors. This study showed that a good correlation exists between flow-cytometric, pathological and clinical data.     

Prognostic relevance of ploidy and proliferative activity of renal cell carcinoma

In 112 patients with renal cell carcinoma, the ploidy, DNA heterogeneity and the phases of the cell cycle occurring in the tumors were determined by means of single-cell DNA cytophotometry, in order to establish further prognostic factors in addition to the ones known so far (stage/grade). Patients with aneuploid tumors or tumors with more than one DNA frequency peak were found to have lymph node metastases intraoperatively and died earlier than patients with diploid tumors. Patients who had tumors with low proliferative activity survived longer than patients with highly proliferative tumor activity (p = 0.001).     

Near-diploid transitional cell carcinoma: A preliminary report

DNA ploidy analysis has been accepted as an important prognostic factor for patients with transitional cell carcinoma (TCC). However, there was few information dealing with the clinical relevance of slightly aberrant DNA content by flow cytometry (FCM). Here we present five cases of near-diploid (ND) tumours, with DNA index (DI) varying from 0.92 to 1.14, obtained from a prospective study of fifty-one cases (9.8%). The frequency of ND tumours showed a tendency to decrease with increasing tumour stage. Higher fraction of tumour proliferation, defined by Ki-67 index, showed a steady increment from 3.4 to 23.5% with occurrence of gross chromosomal changes. In contrast, the expression of epidermal growth factor receptor (EGFR) decreased from 48.3 to 35.3% for diploid (n=29) through aneuploid (n=17) tumours. All three ND bladder cancers had recurrence of one to three times with median follow-up of 36 months. The incidences of tumour recurrence (60%) and cancer death (20%) in ND tumours were intermediate between the aneuploid and diploid TCCs. But, flow DNA analysis of paraffin-embedded ND tumours revealed wide and symmetrical G0/G1 peak with DI varying from 5.6 to 13.0. Our limited experience suggests the necessity of special treatment for G0/G1 peaks having CV values greater than 5.5% from paraffin-embedded urothelial carcinomas.     

DNA content in correlation with postsurgical stage in non-small cell lung cancer.

The relationship between DNA content, TNM stage, tumor size, grade, histology, and disease-free survival was assessed in a retrospective study of patients with non-small cell lung cancer who had undergone resection and complete mediastinal lymph node dissection. Flow cytometric analysis was performed on paraffin-embedded tissue of 90 consecutive patients. The patients were analyzed both as a group and by individual stage. Median follow-up was 11 months (range, 1 to 35 months). Aneuploid tumors were not significantly different from diploid tumors with regard to pathologic TNM stage (p = 0.34), size (p = 0.5), grade (p = 0.5), or histology (p = 0.34). Disease-free survival of patients with aneuploid tumors was not significantly different than that of patients whose tumors had normal DNA content (p = 0.69). DNA content did not correlate with established prognostic factors in patients with non-small cell lung cancer who underwent resection and complete mediastinal lymph node dissection.     

Cellular DNA content and proliferative activity evaluated by flow cytometry versus histopathological and staging classifications in human bladder tumors

Flow cytometric analysis of cellular DNA content was performed on 78 biopsy bladder samples obtained from 61 patients with bladder tumors. All 6 normal tissue samples and 1 benign papilloma exhibited a cytometrically diploid DNA distribution, while 39 of 60 bladder carcinomas exhibited at least one aneuploid cell subpopulation. Furthermore, 13 of 39 aneuploid tumors were characterized by the presence of more than one aneuploid cell subpopulation. The results indicate a significant relationship between cytometric ploidy and morphological classification and stage: the occurrence of subpopulations with abnormal DNA content is associated with the increase in differentiation grade and stage. A significant statistical difference in the survival pattern between the diploid and aneuploid groups was observed. The percent of S cells extracted from DNA content distribution histograms indicates a statistically significant difference (p less than 0.01) between normal tissue (3.7 +/- 1.8), diploid tumor (8.4 +/- 3.9) and aneuploid tumor (14.9 +/- 6.0). Moreover the percent of S-phase cells increases with grade in only the aneuploid subgroup. Our results suggest that cytometric parameters in association with morphological and clinical criteria can contribute to a more accurate characterization of bladder tumors in prognostic terms.     

Transitional cell carcinoma of the upper urinary tract: evaluation of prognostic factors by histopathology and flow cytometric analysis

Prognostic factors in transitional cell carcinoma of the upper urinary tract were assessed with histopathological examination and flow cytometric analysis in a series of 127 patients operated upon between 1976 and 1988. In particular, we evaluated the usefulness of flow cytometry to identify patients who require adjuvant treatment among those with low grade and low stage disease (51% in this series). A multivariate analysis was done on 92 cases, considering patient age and sex, stage, grade and number of lesions (unifocal versus multifocal), site (renal pelvis versus ureter), presence of vesical tumors, recurrences along the urinary tract or in the bladder, type of operation and nuclear deoxyribonucleic acid (DNA) ploidy (diploid versus tetraploid/aneuploid tumors). Only the stage (p = 0.001), grade (p = 0.001) and, to a lesser extent, the DNA pattern (p = 0.031), as well as the number of lesions (p = 0.061) were determinant for prognosis. In regard to the subgroup of 41 patients with grade 2 or less, stage P1 or less tumors, no significant difference in survival was demonstrated between diploid and nondiploid tumor patients. However, 7 of 10 patients from the latter group are still under observation. Therefore, our conclusions may have to be modified in the future.     

Heterogeneity of salivary gland tumors studied by flow cytometry

Intratumor DNA heterogeneity was investigated by flow cytometric analysis of multiple samples taken from different sites of 8 benign and 16 malignant primarily resected salivary gland tumors. All benign tumors had diploid DNA content. The overall incidence of DNA diploidy in 16 malignant cases examined was 50%. Intratumor differences in DNA ploidy were observed in four malignant tumors (25%); 2 of these 4 heterogenous tumors contained both aneuploid and diploid cell clones. The remaining 12 tumors showed a homogeneous DNA content in the different specimens; 8 were diploid, 3 aneuploid, and 1 was polypoid. The DNA nondiploid tumors were clinically more advanced than the DNA diploid ones (p < 0.01). The tumor proliferation rate (fraction of cells in S-phase) was higher in DNA nondiploid samples than in diploid ones (p < 0.01). The DNA nondiploid tumors seemed to recur more often than DNA diploid ones did. The data emphasize the usefulness of DNA measurements for the characterization of malignant salivary gland tumors but also the importance of adequate sampling in assessing their DNA ploidy.     

Prognostic factors in head and neck cancer: histologic grading, DNA ploidy, and nodal status.

Histopathologic malignancy score and DNA ploidy were investigated as prognostic factors for 72 cases of squamous cell carcinoma of the head and neck (HNSCC). The malignancy grading was based upon four different morphologic characteristics for the tumor cell population and four characteristics for the tumor-host relationship. DNA ploidy was determined through flow cytometry on fresh-frozen tumor samples. The median malignancy score was 20, with 71% of the tumors scoring less than 20 being diploid and 68% of the tumors scoring greater than or equal to 20 being nondiploid (p = 0.003). Univariate analysis revealed that tumors scoring less than 20 and diploid tumors had a significantly higher proportion of complete response and better survival as compared to tumors scoring greater than or equal to 20 and nondiploid tumors, respectively. There was a tendency toward better survival among patients without regional metastasis (N0) as compared with patients with regional spread (N+), whereas the other single factors, patient age, clinical stage, histologic grade, and tumor size did not correlate with prognosis. In N+ patients both malignancy score and DNA ploidy were predictive for survival, whereas in N0 patients only malignancy score was related to prognosis. A multivariate analysis showed that the combination of malignancy score and nodal status were the strongest predictors for survival. DNA ploidy did not contribute further information in this test, due to its close relation with the histopathologic malignancy score.     

Expression of MIB-1, mitotic index and S-phase fraction as indicators of cell proliferation in suerficial bladder cancer

Cell proliferation of transitional cell bladder cancer (TCC) was determined by MIB-1 immunolabeling, volume-corrected mitotic index (M/V index) and S-phase fraction measurement in 207 patients with superficial (Ta-T1) bladder cancer. The results were compared to T category, WHO grade and DNA-ploidy. The MIB-1 score was related to T category (P<0.001), WHO grade (P<0.001), DNA ploidy (P<0.0001), M/V index (P<0.0001) and fraction of cells in S phase (P<0.0001). The mean MIB-1 score was 6.37% for G1, 14.59% for G2 and 28.59% for G3 carcinomas (P<0.001). The MIB-1 score for Ta tumors was 9.24% and for T1 tumors 25.34% (P<0.001). The M/V index was 3.9 for G1, 11.5 for G2 and 25.9 for G3 tumors (P<0.0001). The M/V index for Ta tumors was 6.4 and 25.3 for T1 tumors (P<0.0001). WHO grade 1 tumors had 7.7%, grade 2 tumors 13.8% and grade 3 tumors 21.8% of cells in S phase (P<0.001). Of grade 1 tumors, 97% were diploid and 3% aneuploid, and 78% of grade 2 tumors were diploid and 22% aneuploid. Of grade 3 tumors, 30% were diploid and 70% aneuploid (P<0.001). Of Ta tumors, 92% were diploid and 8% aneuploid, respectively, whereas 40% of T1 tumors were diploid and 60% aneuploid (P<0.0001). The results show that quantitative cell proliferation indices are associated with T category and WHO grade in superficial bladder cancer. The prognostic value of the S-phase fraction and mitotic index has been demonstrated in several previous analyses of prognostic factors while the value of MIB-1 score on bladder cancer prognosis remains to be established in further follow-up studies.     

Flow cytometric analysis of primary and metastatic bladder cancer

A total of 22 patients with high grade P2-4N+ transitional cell carcinoma of the bladder underwent flow cytometric analysis of nuclei obtained from paraffin embedded specimens from the primary (bladder) and metastatic (lymph node) sites. Tumor heterogeneity was defined as polyclonal aneuploidy of the primary tumor (not identified in the population studied) or as a difference in the deoxyribonucleic acid index of the primary and metastatic sites of 0.20 or more (8 patients). With these criteria 8 patients (36%) had heterogeneous tumors and 14 (64%) had homogeneous tumors. The median survival of 14 patients with aneuploid and 8 with diploid primary tumors was 17.5 and 8.0 months, respectively (p equals 0.08, Lee-Desu test). When patient survival was compared to the ploidy of the metastatic site, or in patients with diploid primary and metastatic lesions versus deoxyribonucleic acid aneuploidy at either the primary and/or metastatic site, the aneuploid tumors had a longer survival but this difference was not significant (p equals 0.13 and 0.23, respectively). Our study demonstrates the value of flow cytometry to identify primary metastatic tumor heterogeneity. It also suggests that the presence of metastasis may be a more important factor to define the biological potential of transitional cell carcinoma than is deoxyribonucleic acid ploidy.     

Needle biopsy DNA ploidy status predicts grade shifting in prostate cancer

DNA ploidy analysis of prostate needle biopsy specimens was performed to determine whether ploidy status could predict tumor grade shifting at radical prostatectomy. The paired needle biopsy and radical prostatectomy specimens from 111 randomly selected men with prostate cancer were obtained from the surgical pathology files of the Albany Medical Center Hospital. The original tumor grades were assigned by a staff of 12 surgical pathologists according to the Gleason system. Tumors with original Gleason scores < or = 6 were classified as low grade, and tumors with scores of > or = 7 were considered high grade. DNA ploidy analysis was performed on the needle biopsy specimens using the CAS 200 image analyzer (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA) on Feulgen stained 5-microm tissue sections. There were 88 diploid and 23 nondiploid cases. Thirty-eight of 111 (34%) of cases had grade shifting from needle biopsy to radical prostatectomy specimens. Of 89 low-grade needle biopsy cases, 28 (31%) were upgraded at radical prostatectomy. Of 22 high-grade needle biopsy cases, 10 (45%) were downgraded to low grade at radical prostatectomy. Of the 28 low-grade needle biopsy specimens that were upgraded at radical prostatectomy, 19 (68%) featured an aneuploid histogram and 9 (32%) were diploid. Nineteen of 28 (68%) of aneuploid low-grade tumors on needle biopsy became high-grade at radical prostatectomy. Nine of 10 (90%) diploid high-grade tumors at needle biopsy became low-grade at radical prostatectomy. Of the 38 cases in which ploidy and grade were incongruous, 28 (74%) had grade shifting. In a multivariate regression analysis, a high-grade Gleason score on radical prostatectomy specimens correlated significantly with needle biopsy ploidy (p = 0.0001) but not with needle biopsy grade (p = 0.15). The sensitivity of the needle biopsy grade in the detection of high-grade tumors on radical prostatectomy was 30%, and the specificity was 86%. The sensitivity of ploidy status in the prediction of high grade at radical prostatectomy was 78%, and the specificity was 96%. With a prostate-specific antigen (PSA) level of >0.4 ng/ml as the indicator of post-radical prostatectomy disease recurrence on a subset of 106 patients, on univariate analysis, disease recurrence was predicted by needle biopsy ploidy (p = 0.001) and radical prostatectomy grade (p = 0.04) but not by needle biopsy grade (p = 0.39). On multivariate analysis, needle biopsy DNA ploidy status independently predicted disease recurrence (p = 0.002), whereas needle biopsy and prostatectomy grade did not. These results indicate that DNA ploidy analysis of needle biopsy specimens of prostate cancer predicts grade shifting, that it is a more sensitive and specific indicator of final tumor grade at radical prostatectomy than is the original needle biopsy grade, and that ploidy status independently predicts postoperative disease recurrence.     

Prognostic significance of flow cytometric DNA analysis in colorectal cancer

Significance of flow cytometric DNA analysis for assessing malignant potential and survival of colorectal cancer was investigated using paraffin-embedded materials from 144 patients with primary colorectal cancer who had been treated from 1971 to 1985. Forty-four percent of colorectal cancer were composed of diploid and 56 percent were aneuploid. DNA indices (DI) of aneuploid tumors showed a bimodal distribution. There was no significant correlation between ploidy pattern and clinicopathological factors. While, DI level showed significantly higher in poorly differentiated adenocarcinomas and in clinicopathological stage III and V tumors. Overall survival in the patients with aneuploid tumor was significantly worse than that in those with diploid tumor (p less than 0.001). Survival rate was poorer in the patients with aneuploid tumor than in those with diploid tumor, who were stratified according to categories of curable resection, stage, histological type, negative peritoneal or hepatic involvement and negative node metastases. However, there was no significant relation between DI and survival among the patients with aneuploid tumor. From these results, it was concluded that the nuclear DNA content of colorectal cancer may represent biological malignant potential of the disease, and that the DNA ploidy pattern may be an important prognostic indicator, being independent of clinicopathological factors.     

DNA ploidy and S phase fraction in human bladder cancer. Relation to survival and histological grade (WHO)

The nuclear DNA content of archival paraffin-embedded bladder cancer samples (70 patients) of WHO grades I-III has been measured by flow cytometry. The female/male ratio was 15/55. The mean follow-up time was 13 years (range 9.6-22.0 years). 37 of 70 (53%) patients had DNA index 1.0 (diploid DNA content), and the remaining 33 (47%) patients had an aneuploid tumor. There was no significant difference in the age (mean +/- SD) of the patients having a diploid (66 +/- 9 years) or an aneuploid tumor (68 +/- 11 years) at the time of diagnosis. 47 deaths occurred during the follow-up period; 24 (51%) of these were due to bladder cancer (12 diploid, 12 aneuploid tumors). No significant difference was found after radical treatment during the disease-free interval (mean +/- SD) between diploid (48 +/- 45 months) and aneuploid (35.5 +/- 35 months) groups of patients. Recurrences during the follow-up period were equally common among aneuploid and diploid tumors. A statistically significant relation between histological grade and survival could be demonstrated, but DNA ploidy and S phase fraction had little prognostic value in this respect. There was no statistically significant difference in survival between aneuploid (30%) and diploid (35%) groups of tumors during the follow-up period. The study suggests that flow cytometric determination of nuclear DNA ploidy from paraffin-embedded samples in bladder tumors does not add to the prognostic power of subjective histological grading.     

Prognostic relevance of DNA ploidy and proliferative activity in urothelial carcinoma of the renal pelvis and ureter. A study on a follow-up period of 6 years

In 55 patients with urothelial carcinoma of the renal pelvis or ureter, the ploidy, the DNA heterogeneity and the counts of cell cycle phases in the tumor were examined by means of single-cell DNA cytophotometry in order to find more prognostic factors than those already known (stage and grade). Follow-up periods ranged from 1 to 6 years. At the time of first diagnosis, 42 (76%) of the patients had tumors of the renal pelvis, 13 (24%) of them had ureteral tumors. 23 (42%) patients were in stage pT 1 N 0, 15 (27%) in stage pT 2 N 0, 12 (22%) in stage pT 3 N 0, and 5 (9%) were in stage pT 3 N+. The histological malignancy grade most frequently seen in the patients examined--i.e. in 51% of cases--was malignancy grade II. 25% of the patients had grade III tumors whereas only 24% had grade I tumors. With malignancy grade I, DNA cytophotometry showed DNA frequency peaks to be in the diploid range while tumors with malignancy grade II showed heterogenous DNA patterns. 71% of the patients with malignancy grade III showed aneuploid DNA values; 29% of them had polyploid DNA values. For malignancy grades II and III, the proliferation rate of the tumor cells was statistically significantly higher than for malignancy grade I. The determination of tumor heterogeneity and tumor cell proliferation by means of DNA cytophotometry gives valuable clues regarding prognosis.