Stage B prostate adenocarcinoma. Flow cytometric nuclear DNA ploidy analysis

Over a 16-year period (1966 to 1981), 349 patients underwent radical retropubic prostatectomy for pathologic stage B adenocarcinoma of the prostate. Nuclear DNA content was measured by flow cytometry on available archival material of 283 patients. Two hundred sixty-one patients (92%) had high-quality histograms. The ploidy distribution was as follows: DNA diploid, 177 (68%); DNA tetraploid, 74 (28%); and DNA aneuploid, 10 (4%). The average follow-up was 9.4 years. At the time of follow-up, 53 patients (20%) within the study group had developed tumor progression: 22 local, 23 systemic, and 8 both. The ploidy distribution of the population that developed tumor progression was 27 DNA diploid (51%), 16 DNA tetraploid (30%), and 10 DNA aneuploid (19%). This ploidy distribution is significantly different from that found for the nonprogression group with stage B disease. Overall, 31% of patients with DNA nondiploid tumors had tumors that progressed compared with 15% of patients with DNA diploid tumors. All (100%) DNA aneuploid tumors progressed. The DNA ploidy distribution of all pathologic stage B prostate cancers differs significantly from that found in more advanced stages (C and D1) previously reported for the same time interval. However, the ploidy distribution of stage B tumors that progressed closely resembles that of the stage C and D1 tumors. These results further support the working hypothesis that nuclear DNA content has marked prognostic significance for patients with adenocarcinoma of the prostate. It seems to us that analysis of ploidy by flow or static cytometry will become an essential tool for treating patients with localized prostate cancer.     

Deoxyribonucleic acid ploidy in the irradiated prostate

Summary A retrospective study was performed to determine whether the ploidy of localized prostate cancer was correlated with the response to radiation therapy. Prostate tissue was obtained from 69 patients prior to and at least 22 months following definitive radiation therapy. A positive postradiation biopsy of a hypoechoic lesion obtained under transrectal ultrasound guidance was taken as evidence of a failure to respond to therapy. Only 20% of these patients had negative posttherapy biopsies. This poor response rate was similar, regardless of whether the pretreatment tumor was diploid or aneuploid. Treatment failure was correlated with an increased incidence of aneuploid histograms after therapy. Prior to therapy, 80% of the tumors were diploid; after therapy, only 56% were diploid, whereas nontetraploid-aneuploid tumors had increased 3-fold. Among 44 patients who had diploid tumors prior to therapy, the residual prostate cancer was tetraploid in five cases and nontetraploid-aneuploid in ten cases.Supported in part by the Robert J. Mathews Foundation     

Evaluation of DNA ploidy combined with a cytometric proliferation index of imprints from core needle biopsies in prostate cancer.

OBJECTIVE: To evaluate if DNA ploidy analysis with a proliferation index (PI) derived from DNA cytometry of imprints from core needle biopsies predicts disease progression in patients with prostate cancer. METHODS: Touch imprints were done on a consecutive series of core needle biopsies taken by the same urologist from 240 patients with suspected prostate cancer, 137 (46%) of whom were found to have prostate cancer and included in the study. Scattered cells to the right of the image cytometry (ICM) ploidy-establishing peak, the S-phase fraction, and those in the G2M area of the ICM DNA histograms, were counted in percent of the total number of tumor cells, this value being designated the ICM PI. Based on previous results in archival fine needle aspiration material, the following classification was used: DNA group I, diploid tumor with a low PI; DNA group II, diploid tumor with an intermediate PI and tetraploid tumor with a low or intermediate PI, and DNA group III, diploid or tetraploid tumor with high PI and all tumors with an aneuploid pattern. RESULTS: Correlation was found to exist between DNA groups I-III and Gleason score (GS) (p < 0.0001), T-stage (p = 0.006), M-stage (p = 0.009) and disease progression (p < 0.0001). Among the 39 patients who had curative treatment and GS 5-7, the progression-free survival rate was 100% in DNA group I, as compared with only 38% in DNA group II and 55% in DNA group III within the follow-up period (p = 0.008). CONCLUSION: DNA ploidy combined with a PI derived from image cytometry of imprints from core needle biopsies yields additional prognostic information in patients with GS 5-7. Diploid tumors with a low PI (DNA group I) are associated with a low risk of disease progression.     

The prognostic significance of deoxyribonucleic acid flow cytometry in muscle invasive bladder carcinoma treated with preoperative irradiation and cystectomy.

Deoxyribonucleic acid (DNA) flow cytometry measurements were performed in nuclear suspensions obtained from paraffin-embedded biopsies from 83 patients with stages T2, T3 and T4a bladder carcinoma. All patients were treated with preoperative radiotherapy and cystectomy from 1976 through 1985. Of the tumors 13 (16%) were diploid, 18 (22%) tetraploid and 52 (63%) aneuploid. A total of 19 tumors (23%) had 2 or 3 stemlines in addition to the diploid cells. Post-radiotherapy stage reduction (absence of muscle infiltration in the cystectomy specimen) occurred more often in tumors with only 1 nondiploid stemline than in diploid tumors or nondiploid tumors with multiple stemlines. The 5-year survival rate was significantly poorer for patients with a diploid (33%) than for those with a nondiploid (66%) tumor (p = 0.05), although this was only marginally retained in a multivariate analysis (p = 0.11). The clinical significance of DNA ploidy in muscle infiltrating bladder cancer seems not to be as evident as has been shown for superficial bladder tumors but it may be of value in selecting patients for preoperative radiotherapy.     

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.     

Value of nuclear DNA ploidy patterns in patients with prostate cancer after radical prostatectomy.

Flow cytometric analysis of DNA ploidy was performed on prostatic adenocarcinoma specimens from 80 patients. In all these patients a radical retropubic prostatectomy had been performed. The nuclei for DNA ploidy determination were extracted from paraffin-embedded material of whole sections of the prostate from patients treated by radical prostatectomy between 1980 and 1985. DNA ploidy was a strong prognostic indicator independent of tumor grade and tumor stage. DNA ploidy offered additional information on both tumor stage and tumor grade. In stage C disease the likelihood of progression-free survival was 89.5% in diploid tumors and 27.8% in aneuploid tumors after 9 years. In tetraploid tumors all patients progressed after 9 years. The computed survival rates in stage C disease showed that patients with diploid tumors did significantly better than those with aneuploid or tetraploid tumor patterns. These data indicate therefore that DNA ploidy patterns determined by flow cytometric analysis provide important additional prognostic information on prostatic adenocarcinoma treated by radical prostatectomy.     

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

Nuclear DNA ploidy studies were performed by flow cytometry on extracted nuclei from 65 heptic metastases from colorectal cancer. In 25 patients, both primary and metastatic lesions were available for analysis. Primary carcinomas were DNA diploid pattern in 48.1%, DNA aneuploid in 51.9%. Of 31 hepatic metastases, 11 (35.5%) metastases showed a DNA diploid pattern, and 25 (64.5%) showed a DNA aneuploid pattern. Ploidy pattern was constant between primary and metastases in 80% of tumors. No significant relationship between metastatic characteristics and DNA ploidy pattern was found. The DNA aneuploid cancers had a relatively poorer prognosis in patients with unresectable hepatic metastasis. In resected hepatic metastases from colorectal cancer, rate of hepatic recurrence with DNA diploid pattern was lower than that with DNA aneuploid pattern. Survival of patients with DNA diploid metastases (71% alive at 5 years) was significantly better than that of patients with DNA aneuploid metastases (21% alive at 5 years) (p less than 0.05). These results demonstrated that flow cytometric DNA ploidy measurements may have prognostic value for patients with hepatic metastases from colorectal cancer.     

Dna heterogeneity determined by flow cytometry in prostatic adenocarcinoma—necessitating multiple site analysis

Although DNA ploidy analysis of prostate cancer is generally associated with grade, stage, clinical outcome, and responsiveness to androgen therapy, one possible reason cited for contrary reports may be tumor heterogeneity. A preliminary report using flow cytometric analysis of punch biopsies demonstrated DNA heterogeneity in five of nine patients. We evaluated 75 patients by cutting whole mounts of formalin fixed prostatectomy tissue every 0.6 cm. All malignant areas and a selected normal area were circumscribed, excised, remounted, and 1–3 50 μ thick sections removed. The nuclei were extracted by a Hedley technique and the DNA stained with propidium iodide. Each whole mount had an average of 1 distinct malignant area (range of 1–6 areas per whole mount block). Nuclei were analyzed on a Becton Dickinson (San Jose, CA) FACScan flow cytometer equipped with RFIT DNA software program. After excluding histograms with CVs > 8.0% and/or “suspicious” diploid histograms having a right “shoulder,” 75 or 87 patients still had ≥2 malignant sites available for analysis (average 4, range 2–9 malignant sites/patient). The 322 histograms had an average CV of 4.4%. Thirty of 75 patients (40%) showed DNA heterogeneity in multiple samples taken from the same prostate. There were 37 prostates with only diploid (D), 1 with only tetraploid (T), 7 with only aneuploid (A), 20 with D plus A, 7 with D plus T, 2 with D plus T plus A, and 1 with a D plus suspected hypodiploid DNA content. Exclusion of the tetraploid and “near diploid aneuploid” cases still resulted in 16% (12/75) of the patients having a diploid versus aneuploid DNA content heterogeneity. Because 40% of the prostates contained a different ploidy depending on which area was sampled, this report suggests multiple sites of malignancy must be analyzed to more accurately assess the ploidy status of prostatic adenocarcinoma. © 1995 Wiley-Liss, Inc.     

Relationship of DNA content to conventional prognostic factors in clinically localised carcinoma of the prostate

One hundred and nine patients treated by total prostatectomy for apparently localised carcinoma were analysed to investigate the prognostic significance of capsular invasion and penetration, seminal vesicle invasion, lymph node metastases, grade as assessed by the Gleason and MD Anderson Hospital (MDAH) systems and DNA content measured by flow cytometry of nuclear material extracted from paraffin embedded tumour. Comparison of DNA content was made with 36 benign prostates. Mean follow-up/survival was 60.7 months, at which time 21 patients had evidence of recurrence or had died. Only 5 patients had local recurrence. Tumour grade, as assessed by both the Gleason Sum Score and the MDAH system, correlated with anatomical extent and was the most important determinant of time to recurrence. Fifty-nine tumours were diploid, 44 tetraploid and 6 aneuploid. One of 36 benign prostates showed aneuploidy. Ploidy did not correlate with the anatomical extent of the tumour or with grade. Tetraploid tumours recurred earlier than diploid tumours. None of 6 aneuploid tumours have recurred, although only 3 have been followed beyond 5 years. Multivariate analysis showed that after accounting for grade, none of the other variables, including ploidy, contributed any additional significant prognostic information. Although the results must be regarded as preliminary, in view of the small number of patients with recurrence, they suggest that DNA content offers limited prognostic information in clinically localised prostate cancer.     

Adrenocortical carcinoma: nuclear deoxyribonucleic acid ploidy studied by flow cytometry

Nuclear deoxyribonucleic acid (DNA) ploidy studies with use of paraffin-embedded specimens were performed by flow cytometry on 52 adrenocortical carcinomas. Specimens were prepared by the combined techniques of Hedley and Vindel?v. Clinical course was obtained by chart review and follow-up examination. Nine (17%) tumors had a normal (diploid) DNA pattern, 13 (25%) were DNA tetraploid, and 30 (58%) were DNA aneuploid. The DNA aneuploid group was subdivided: 18 tumors with one stemline and 12 tumors with two stemlines of abnormal DNA cells. For tumors that were resected for cure, the 5-year Kaplan-Meier disease-free survival rates of the five patients with DNA diploid tumors and of the six patients with DNA tetraploid tumors were 80% and 33%, respectively. For 21 patients of whom 12 had one-stemline and nine had two-stemline DNA aneuploid tumors, the survival was 67% and 0%, respectively. Following palliative resection, the 4-year survival rates of the four patients with DNA diploid, seven with DNA tetraploid, five (omitting one with short follow-up) with one-stemline DNA aneuploid, and three with two-stemline DNA aneuploid tumors were 0%, 0%, 0%, and 33%, respectively. Although adrenocortical carcinoma is in general markedly aggressive, the addition of nuclear DNA ploidy studies may help to identify certain groups of patients who have a relatively favorable prognosis.     

The clinical usefulness of flow cytometric DNA analysis in prostatic cancer]

Flow cytometry was used to measure the DNA content in archival paraffin-embedded prostatic cancer specimens from 54 patients with known outcomes. The specimens were obtained by transurethral resection of the prostate. DNA ploidy as a predictor of prognosis was compared with histological grade and clinical stage. Although no significant correlation between histological grade or clinical stage and ploidy pattern was demonstrated, an increased percentage of DNA aneuploid tumors was seen in higher histological grade and in advanced clinical stage. The survival rate calculated by Kaplan-Meier analysis showed that DNA ploidy pattern was a more reliable indicator to predict survival probability than histological grade or clinical stage. All patients with a near diploid pattern (11 patients) survived more than 5 years, whereas all those with an aneuploid pattern (21 patients) died within 3.5 years. Of 22 patients with a tetraploid pattern, 15 died of tumor progression within 5 years. The remaining 7 patients with favorable outcome had a relatively lower proliferation index (less than 65) in DNA histogram and none of them suffered from stage D disease. In conclusion, the results from this retrospective study suggest that flow cytometric DNA analysis in prostatic cancer would be useful as a means of providing prognostic information.     

DNA pattern and cytological findings in fine-needle aspirates of untreated prostatic tumors. A flow-cytofluorometric study

The cellular DNA content in fine-needle prostatic aspirates from 500 untreated patients was determined by flow cytofluorometry. According to the DNA patterns diploid, tetraploid, and non-tetraploid aneuploid cases were identified. In 301 cytologically benign cases more than 90% showed diploid DNA patterns. Among 166 carcinomas the incidence of aneuploid DNA values increased with the degree of anaplasia, ie, 44% in well-differentiated, 78% in moderately differentiated, and 97% in poorly differentiated tumors. In aneuploid cases of well-differentiated carcinomas almost exclusively tetraploid DNA patterns were observed, while in poorly differentiated carcinomas about 80% showed non-tetraploid aneuploid DNA distributions. Among aneuploid cases of moderately differentiated carcinomas 2/3 were tetraploid and 1/3 non-tetraploid aneuploid. Morphologically similar tumors may thus be separated by the DNA profiles. The biological significance of these results must be further evaluated by clinical follow-up of the patients.     

Flow cytometric determination of nuclear DNA content in benign adrenal pheochromocytomas

Nuclear DNA content of paraffin-embedded tissue from 19 clinically benign adrenal pheochromocytomas and 18 control adrenal glands was analyzed using flow cytometry. All control adrenal glands and 6 pheochromocytomas were diploid. Four tumors were tetraploid. Nine were aneuploid with relative DNA indices in the near diploid range in 2, in the peritriploid range in 5, and in the near tetraploid range in 2. These results indicate that aneuploid DNA content is a frequent occurrence in benign adrenal pheochromocytomas. Aneuploidy per se is not a specific marker of malignancy in these tumors as has been suggested by previous reports.     

Evaluation of DNA nuclear pattern as a prognostic determinant in resected pancreatic ductal adenocarcinoma.

From a cohort of 72 patients who underwent radical resection for ductal adenocarcinoma of the pancreas between 1951 and 1980, 62 paraffin-embedded specimens were analyzed by flow cytometry. Patients were divided into two groups according to the length of survival: long-term survivors (19 patients who survived 3 or more years after operation) and short-term survivors (43 patients who died within 12 months after resection). In 30 specimens (48%), the nuclear DNA pattern was diploid, whereas 32 were nondiploid (two tetraploid and 30 aneuploid). There were no significant differences in the number of diploid/nondiploid patterns, the fraction of cells in DNA synthetic (S) phase, or the DNA index between the two groups. These data suggest that there is no difference in the DNA content analysis of patients with pancreatic ductal adenocarcinoma when comparing long-term with short-term survivors following resection.     

Deoxyribonucleic acid flow cytometry in invasive bladder carcinoma: a possible predictor for successful bladder preservation following transurethral surgery and chemotherapy-radiotherapy.

Tumor deoxyribonucleic acid (DNA) ploidy was evaluated as an objective parameter that may correlate better with the responsiveness of bladder cancer to chemotherapy plus radiotherapy than do clinical features or histopathological subtypes. A total of 40 patients with localized muscle-invading bladder cancer (clinical stages T2 to T4) underwent prospective treatment on a potential bladder preserving protocol. Tumors of 37 of the 40 patients were analyzed by DNA flow cytometry of multiple paraffin embedded specimens. Transurethral resection, neoadjuvant chemotherapy and 40 Gy. radiotherapy plus cisplatin were followed by urological reevaluation of the tumor (a complete response required a negative biopsy and a negative urine cytology study). A total of 7 noncomplete response patients underwent immediate radical cystectomy whereas the full bladder sparing treatment (radiotherapy to 64.80 Gy. plus cisplatin) was given to 23 complete response patients and 7 noncomplete response patients who were unsuited for surgery. Of the tumors 22 (59%) were purely aneuploid and 10 (27%) were purely diploid. Five tumors contained aneuploid and diploid patterns in different tumor specimens (partly diploid). Current status with a 30-month median followup in surviving patients includes an 82% overall survival rate in the aneuploid group versus 47% in the diploid/partly diploid group. Of all the patients 68% are free of invasive tumor: 82% in the aneuploid group versus 47% in the diploid/partly diploid group. By multivariate analysis pure aneuploidy was significantly (p = 0.05) correlated with freedom from invasive tumor in the bladder (either as persistence or as recurrence) and approached significance (p = 0.08) in correlation with overall patient survival. A longer observation time will be required to confirm this unexpectedly good outcome for patients with pure aneuploid tumors. We hypothesize that pretreatment DNA ploidy status may become a clinically useful prognostic factor in selecting patients for successful treatment with transurethral surgery and neoadjuvant chemotherapy plus radiotherapy.     

The significance of tumor heterogeneity for prediction of DNA ploidy of prostate cancer

OBJECTIVE: In a previous study, we mapped the ploidy heterogeneity of prostate cancer using flow cytometry in 676 tumor samples from 50 radical prostatectomy specimens. Ploidy heterogeneity was common (42% of tumors) and was found in all non-diploid tumors. The volume of non-diploid tumor was estimated and found to predict extra-prostatic extension and seminal vesicle invasion. The aim of this study was to evaluate the impact of tumor heterogeneity on preoperative ploidy assessment. MATERIAL AND METHODS: In 50 men at least six core biopsies were taken before prostatectomy. Sections from biopsies with cancer were Feulgen-stained for image cytometry. After exclusion of biopsies with insufficient material, 123 histograms from 48 men (mean 2.6; range 1-7) remained for analysis. RESULTS: In 32 men, biopsies were diploid. In 16 men, at least one biopsy was non-diploid (14 tetraploid, two aneuploid) and 10 of them also had diploid biopsies. In 34 men (71%), the prostatectomy specimens were correctly predicted as being either diploid (48%) or non-diploid (23%). The sensitivity and specificity of biopsies for predicting non-diploid cancer were 55% and 82%, respectively, and the positive and negative predictive values were 69% and 72%, respectively. The ploidy status of tumors with and without ploidy heterogeneity was correctly predicted in 55% and 82% of cases, respectively (p=0.04). Biopsies underestimated ploidy in 9/20 tumors (45%) with heterogeneous ploidy status. Underestimation mainly occurred when one or two cores were analyzed. CONCLUSIONS: Preoperative prediction of the ploidy status of prostate cancer is hampered by tumor heterogeneity. Analysis of multiple biopsies is important for correct preoperative ploidy estimation.     

Flow cytometric analysis of cellular DNA content in human astrocytomas and oligodendrogliomas

This report presents a flow-cytometric analysis of cellular DNA in biopsies and primary cell cultures of 21 human astrocytomas and 19 oligodendrogliomas. A distinct correlation between histological dedifferentiation and pathological DNA distribution was found. Classification was made according to increasing histological anaplasia, corresponding to a four-grade scale and proliferation index (PI). Four types of gliomas were defined according to characteristic DNA patterns and proliferative activities in comparison to their histological grading: 1. purely diploid DNA patterns with low 4C (premitotic) peaks and PI values up to 10 in well-differentiated gliomas; 2. increase of tetraploid cells and PI of 10-16 in tumors with histological grades II or II–III; 3. diploid-tetraploid DNA distribution with PI values up to 30–31 and malignancy grade III; 4. polyploid and aneuploid karyograms with excessive 4C increase, emerging in grade III and especially grade III–IV of these gliomas. Varying DNA distribution during tumor development could be observed in a malignant transformation of an oligodendroglioma I to a glioblastoma after a course of 3 1/2 years. A more detailed subdivision of these tumors according to their DNA content and proliferative activity was achieved. With the exception of occasional variation in karyograms, DNA distribution usually remained stable in primary tissue cultures (PTC).     

Significance of ploidy in laryngeal cancer

We studied 48 patients with laryngeal or hypopharyngeal primary tumors with DNA flow cytometry. Twenty-four of the tumors were glottic, 18 were supraglottic, and 6 were from the pyriform sinus. Patients were followed for a minimum of 12 months, with a mean follow-up of 23 months. Twenty-three of the 48 primary tumors (48 percent) were clearly aneuploid, 22 percent were tetraploid, and 30 percent were diploid. We concluded that patients with aneuploid primary tumors, high DNA levels, or both have a significantly better prognosis than those with diploid tumors, and this remained statistically significant when clinical outcome was adjusted for tumor status, stage, and nodal status.     

DNA ploidy and the prognosis of stage pT1 bladder cancer

The histopathological grade, proportion of "S"-phase nuclei and DNA ploidy values were linked and of prognostic significance in a retrospective series of stage pT1 bladder cancers. Nuclei were extracted from paraffin sections of 75 biopsies (56 patients). DNA ploidy and the proportion of "S"-phase nuclei were measured using flow cytometry. Progressive disease (pT2 or greater) developed within 3 years in 35% (6/17) of patients with poorly differentiated tumours, 35% (8/23) with aneuploid tumours and 35% (7/20) of those with a high proportion of "S"-phase nuclei. Of 8 tumours with all 3 features, progressive disease developed in 6 cases (75%). Of 9 patients who developed progressive disease, 8 (89%) had aneuploid tumours. Progressive disease did not develop in 11 patients with well differentiated tumours, compared with 4% (1/24) in diploid/tetraploid tumours and 7% (2/27) in those with a low/medium percentage of "S"-phase nuclei. In contrast to muscle-invasive disease, recurrent superficial tumours developed with a high incidence in all groups. Only 6/56 patients (11%) remained alive and disease-free for 3 years. It is concluded that these 3 features are of similar prognostic significance and accuracy in identifying patients requiring more aggressive therapy.     

Nuclear DNA analysis of prostate tissues: correlation with stage and grade of tumour

Flow cytometry was used to analyse the DNA content of 20 unfixed prostatic specimens obtained from patients with cancer of the prostate. The tumours were of different stages and grades of differentiation and the numbers of cells in each phase of the cell cycle were compared to those obtained from 26 patients with benign prostatic hyperplasia. It was noted that, as the cancer progressed from a well to a poorly differentiated state, there was a distinct shift in the distribution of cells in the G0/G1 phase towards the S and G2+M phases. This was particularly marked in cases with a summed Gleason score of 7-10, and was independent of the stage and bulk of the tumour. Furthermore, all tumours with a Gleason score of 7 or more exhibited a tetraploid histogram, whereas only 2 of 14 patients with a well or moderately well differentiated cancer had tetraploid histograms. Two patients were found to have tumours with aneuploid DNA content, and both died within 2 years from the time of diagnosis. Although it is too early to speculate on the place flow cytometry in the clinical management of patients with cancer of the prostate, the results generated from this present study suggest that flow cytometry may be capable of providing prognostic information not supplied from histopathological analysis.