Deoxyribonucleic Acid Flow Cytometry Research Articles

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 40Gy. 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 64.80Gy. 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.

Synchronous lung cancers defined by deoxyribonucleic acid flow cytometry : Ichinose Y, Hara N, Ohta M. Department of Chest Surgery, National Kyushu Cancer Center, 1-1, 3-Chome, Notame, Minami-ku, Fukuoka 815. J Thorac Cardiovasc Surg 1991;102:418–24

Synchronous lung cancers defined by deoxyribonucleic acid flow cytometry : Ichinose Y, Hara N, Ohta M. Department of Chest Surgery, National Kyushu Cancer Center, 1-1, 3-Chome, Notame, Minami-ku, Fukuoka 815. J Thorac Cardiovasc Surg 1991;102:418–24

Prognostic significance of tumor deoxyribonucleic acid content in surgically resected small-cell carcinoma of lung

The prognostic role of deoxyribonucleic acid flow cytometry was investigated in 53 cases of surgically resected small-cell lung cancer. Deoxyribonucleic acid aneuploidy was detected in 26 patients (49.1%), the remaining tumors being either diploid or tetraploid. Patients with aneuploid tumors had a significantly reduced 2-year survival (38.5%) when compared with patients with diploid or tetraploid tumors (70.3%; p less than 0.05). This finding was independent of tumor stage on multiple logistic regression analysis. Diploid or tetraploid deoxyribonucleic acid content was associated with a particularly good 2-year survival (85%) in N0 or N1 disease. Tumor deoxyribonucleic acid ploidy should be taken into account in planning of management and assessment of prognosis in small-cell lung cancer.

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.

Cell Kinetics by Bromodeoxyuridine Labeling and Deoxyribonucleic Acid Ploidy in Prostatic Carcinoma Needle Biopsies

We studied 63 prostate carcinomas in needle biopsies after labeling in vitro by incubating the entire biopsy specimen with tritiated thymidine or bromodeoxyuridine before fixation. Biopsies from 176 patients were labeled, of which 98 were benign and 78 were carcinomas (15 carcinomas were excluded because of scant tissue). The procedure did not interfere with histological diagnosis. Median labeling index was 0.87% (range 0.1 to 29.3%). A labeling index exceeding 3% was unusual but it may indicate the potential for rapid clinical progression. Deoxyribonucleic acid (DNA) flow cytometry was performed on the last 44 carcinomas using cells shed by the prostatic needle biopsy tissue samples during transportation to the pathology laboratory. A sufficient number of cells were obtained for analysis in 37 cases, of which 6 were DNA aneuploid. Labeling index correlated with Gleason histological grade and score, and data from the 6 DNA aneuploid carcinomas suggest association between DNA aneuploidy and a high labeling index. Estimates of percentage of S-phase cells by flow cytometry did not correlate with variables other than DNA index, and appear to have been affected by a high noise-to-signal ratio (few proliferative cells relative to cellular debris) and inability to discriminate between benign and carcinomatous cells.

Synchronous lung cancers defined by deoxyribonucleic acid flow cytometry

When two pulmonary tumors are seen simultaneously in patients with lung cancer, it always raises a question as to whether the lesions are synchronous lung cancers or lung cancer with intrapulmonary metastasis. To settle this issue, we used deoxyribonucleic acid flow cytometry. Deoxyribonucleic acid ploidy patterns of tumors were able to be analyzed in 14 patients with two simultaneous pulmonary tumors resected by operation. These two tumors, with completely different patterns of deoxyribonucleic acid ploidy, were defined as synchronous lung cancers. Tumors were defined as lung cancer with intrapulmonary metastasis when both tumors showed diploidy or when at least one deoxyribonucleic acid index of abnormal clones between two aneuploid tumors was the same or almost identical. Tumors of the five patients with stage I disease were classified as synchronous lung cancers according to the criteria involving deoxyribonucleic acid flow cytometry. Both tumors were adenocarcinomas in four patients and large-cell and squamous cell carcinomas in one. Both tumors in four patients were located in the same lobe but different segments. All but one patient with different histologic types are alive without recurrence from 24 to 100 months after operation. Tumors of the nine patients with stage III disease in whom intrapulmonary metastasis was clinically suspected were classified as lung cancer with intrapulmonary metastasis according to the criteria. These data suggest that deoxyribonucleic acid flow cytometry of tumors may have diagnostic value in determining synchronous lung cancers.

DNA Ploidy patterns of tumors diagnosed as metachronous or recurrent lung cancer

The relationship between the first tumor and the second tumor resected in 8 patients with non-small cell lung cancer was analyzed using deoxyribonucleic acid (DNA) flow cytometry. Of the 8 patients, 6 were clinically diagnosed as having metachronous lung cancers and 2, local recurrent tumors. The mean interval between operations in patients with metachronous lung cancers was 62 months (range, 15 to 128 months). Both tumors showed the same histology in 4 patients and a different histology in 2. In the 2 patients with local recurrent tumors, the interval between operations was 9 months and 39 months. In the analysis of DNA flow cytometry of the first and second tumors in the same patient, the tumors were defined as independent of each other when one tumor showed diploidy and the other, aneuploidy, or when each DNA index of abnormal clones between two aneuploid tumors was different. When both tumors showed diploidy or when at least one DNA index of abnormal clones between two aneuploid tumors was identical, the tumors were defined to be related to each other. According to these criteria, in 5 (83%) of the 6 patients clinically diagnosed as having metachronous lung cancers, the second tumor was classified as independent of the first tumor. On the other hand, in the 2 patients clinically diagnosed as having recurrent tumors, the second tumor was judged to be related to the first tumor. These data suggest that DNA flow cytometric analysis of tumors may be of value in the diagnosis of metachronous lung cancers.

Comparative Histopathology and Deoxyribonucleic Acid Flow Cytometry of Random Mucosal Biopsies in Untreated Bladder Carcinoma

In a study of 290 patients with untreated carcinoma of the bladder, histopathological studies of random mucosal biopsies were compared with the results of deoxyribonucleic acid (DNA) flow cytometry. By histopathology the findings were classified as severe atypia corresponding to primary grade 3 carcinoma in situ, atypia not fulfilling the criteria for carcinoma in situ and no atypia. The DNA histograms were classified as diploid or aneuploid. Aneuploid cell populations in mucosal biopsies were found mainly in cases with aneuploid tumors of grade 3.Of the biopsies classified as concomitant carcinoma in situ 76% were aneuploid. In biopsies exhibiting less severe or no atypia aneuploidy was found in 41 and 10%, respectively. For these 3 morphological categories the distributions of the aneuploid cell populations were similar irrespective of the histopathological findings and they were also the same as that found in primary carcinoma in situ. We concluded that gross chromosomal aberrations may appear at an early stage of the tumor development and before changes recognizable by morphology.The similarity of the DNA profiles of the aneuploid cell populations, regardless of morphological findings, indicates that apart from gross chromosomal aberrations changes of the phenotype are necessary for the expression of morphological changes.

Sensitivity of Urinary Quantitative Immunocytology with Monoclonal Antibody 486 P3/12 in 241 Unselected Patients with Bladder Carcinoma

We investigated the sensitivity of quantitative immunocytology with our monoclonal antibody 486 P3/12 in 241 unselected patients with transitional cell carcinoma. Immunocytology yielded a sensitivity of 91.8%, 89.4% and 92.9% for grade 1, 2 and 3 tumors, respectively. Standard cytology was positive in 59.2%, 63.8% and 84.7%, respectively. Deoxyribonucleic acid flow cytometry, used in the first 69 patients, was positive in only 27.7%, 48.6% and 57.1%, respectively.

Prediction of Lymph Node Metastases in Bladder Carcinoma with Deoxyribonucleic Acid Flow Cytometry

The deoxyribonucleic acid patterns in lymph node metastases in relation to the degree of ploidy and proliferation in the primary bladder tumor were evaluated in 162 patients who underwent cystectomy for muscle invasive bladder carcinoma. The 34 diploid tumors had given rise to lymph node metastases in only 2 cases (6%), whereas 34% of the aneuploid tumors had metastasized. The frequency of lymph node metastases increased with the proportions of S-phase cells. In transitional cell carcinoma all tumors with a proportion of S-phase cells exceeding 20% had metastasized, while tumors with a proportion of S-phase cells between 10 and 20%, and less than 10% had metastases in 50 and 33%, respectively. The degree of ploidy of aneuploid tumors seems to be of importance for the potential to give rise to metastases, since the majority of metastatic tumors were in the triploid-tetraploid and hyperdiploid but not hypertetraploid regions. The degree of ploidy of lymph node metastases was in good agreement with that of the primary tumors. (J. Urol., 144: 884–887, 1990)

Deoxyribonucleic Acid Flow Cytometry in Predicting Response to Radical Radiotherapy of Bladder Cancer

Using flow cytometry tumors can be classified according to their deoxyribonucleic acid content as diploid or aneuploid with 1 or several cell lines and to the ploidy level of the aneuploid cell lines, as well as to the proportion of proliferating cells. We used this technique in 73 patients with invasive transitional cell carcinoma of the bladder who received radical radiation therapy with followup of surviving patients for at least 5 years. The effects of therapy were related to the deoxyribonucleic acid patterns before irradiation. All diploid tumors disappeared, while aneuploid tumors with 1 cell line disappeared in 55% and those with several cell lines in 30% of the patients. Aneuploid tumors with cell lines exceeding 4c appeared to be more radiosensitive than those in the triploid region. Thus, tumors unresponsive to therapy as well as those that recurred after irradiation were mostly triploid. Since most of the tumors had high proliferation rates the proportion of S-phase cells was of only limited value for further classification of the various tumors.In the choice between radical radiotherapy, in which bladder function is maintained, and cystectomy an analysis of tumor ploidy provides at least some basis on which radiation response can be predicted. In the choice of therapy we must also consider the fact that almost 20% of the patients who failed to respond to therapy revealed aneuploidy only in bladder washings after irradiation, indicating existence of concomitant carcinoma in situ.

Optimum testicular aspiration technique for deoxyribonucleic acid flow cytometric analysis of spermatogenesis in primates (Papio anubis)

Deoxyribonucleic acid flow cytometry on testicular tissue is an established method to evaluate spermatogenesis. Needle aspiration is less invasive and permits sequential sampling as compared with open testis biopsy. Using seven primates, we examined the needle aspiration technique. There was no significant difference among locations aspirated; the Westcott needle (Bard Urological, Covington, GA) provided the most rapid analysis, and both the Biopty gun (Bard Urological) and Westcott needle were easy to use. Baseline means +/- SD for ploidy distributions were 1N: 65.73% +/- 7.1; 2N: 18.95% +/- 5.5; 4N: 11.4% +/- 2, Experimental data generated from primate models using testis aspiration and flow cytometry may elucidate human infertile conditions.

Deoxyribonucleic Acid Ploidy and the Direct Assay of Prostatic Acid Phosphatase and Prostate Specific Antigen in Fine Needle Aspiration Biopsies as Diagnostic Methods in Prostatic Carcinoma

We used fine needle biopsies from prostatic tumors at routine examinations in 133 patients. Cytological grading was performed with a scoring system. Cellular prostatic acid phosphatase and cellular prostate specific antigen from the aspirates were quantitated. Deoxyribonucleic acid flow cytometry was performed and the tumors were subdivided into diploid, tetraploid and aneuploid groups. Tumor staging was assessed by digital examination. A decrease in the biochemical markers was significantly correlated with the increase in malignancy grade, tumor stage and a shift from diploid to aneuploid tumors. Cellular prostatic acid phosphatase and cellular prostate specific antigen as well as tumor ploidy may contribute to the objective determination of the malignancy potential of the prostatic carcinoma. (J. Urol, 144: 299-302, 1990)

Prognostic Value of DNA Flow Cytometry in Thymomas and Thymic Carcinomas

Thymomas are the most common anterior mediastinal masses. Malignant potential and prognosis are unrelated to histologic appearance. Deoxyribonucleic acid (DNA) flow cytometry is of prognostic significance in a variety of tumors. We reviewed the records of 35 patients who on pathologic examination had a thymoma or thymic carcinoma. Flow cytometric studies, including DNA indices (ploidy) and S phase fraction, were done on paraffin block specimens from 31 patients. We believe this is the first report of DNA flow cytometric studies in thymic pathology. Mean survival was 63.5 +/- 13.3 months for patients with benign thymomas, 10.5 +/- 4.6 months for patients with malignant thymomas, and 19.3 +/- 4.1 months for patients with thymic carcinomas. Patients with benign thymomas lived significantly longer than those with malignant thymomas (P = .001) and thymic carcinomas (P = .03). DNA flow cytometry demonstrated four aneuploid tumors (two benign thymomas and two malignant thymomas). All thymic carcinomas were diploid. There was no statistically significant difference among the groups. The mean S phase fraction was 15.22% for benign thymomas, 11.15% for malignant thymomas, and 14.31% for thymic carcinomas. No statistically significant difference was found among the groups. We conclude that flow cytometry is not a useful guide to malignant potential or prognosis in thymomas and thymic carcinomas.

A Clinically Applicable Method to Preserve Urine and Bladder Washing Cells for Flow Cytometric Monitoring of Bladder Cancer Patients

We describe a method to fix exfoliated bladder cells that is suitable for followup of bladder cancer patients by deoxyribonucleic acid flow cytometry. After fixation with room temperature methanol plus acetic acid (20:1, volume:volume) urine and bladder washing samples from these patients can be stored at room temperature for 3 to 7 days and then assessed reliably for the presence of aneuploidy and the percentage of hyperdiploid cells. For those with active transitional cell carcinoma diagnostic accuracy comparing fresh to fixed specimens was improved from 58 to 92% with urine and from 50 to 100% with washing samples. For patients with a history of transitional cell carcinoma who currently are free of disease the false positive rate remains unchanged after fixation. The procedure described is suitable for use in the outpatient clinic and should permit shipping of samples without refrigeration to a central flow cytometry facility for analysis. (J. Urol, 143: 700–705, 1990)

Deoxyribonucleic acid flow cytometric analysis of cervical adenocarcinoma: Prognostic significance of deoxyribonucleic acid ploidy and S-phase fraction

Paraffin-embedded tumor samples from 125 patients with cervical adenocarcinoma were analyzed by deoxyribonucleic acid flow cytometry. Thirty-one percent of the tumors were aneuploid. Triploid deoxyribonucleic acid content predominated (51.3%) and one third of the deoxyribonucleic acid aneuploid tumors were tetraploid, whereas near-diploid deoxyribonucleic acid aneuploidy was seen infrequently. Deoxyribonucleic acid aneuploidy was associated with tumor size, histologic grade, clinical stage, and high S-phase fraction. Deoxyribonucleic acid ploidy and S-phase fraction value were independent prognostic parameters, together with the presence of lymph node metastases and tumor size. In conclusion, our results indicate that flow cytometric deoxyribonucleic acid analysis helps to predict the prognosis and may thus influence the choice of treatment.

Improved survival after pulmonary resection of metastatic colorectal carcinoma

While hepatic resection of metastatic colorectal carcinoma is accepted as effective in selected patients, resection of metastases to other solid organs has not gained wide acceptance. We retrospectively reviewed the records of 49 patients who had resection of isolated pulmonary (18 patients) and hepatic (31 patients) metastases from the large bowel, comparing disease-free survival and overall survival. Tumor parameters analyzed included Dukes' stage, deoxyribonucleic acid (DNA) flow cytometry, and number of metastases. Dukes' B and diploid tumors had longer disease-free survival and overall survival than did Dukes' C and aneuploid tumors, though the difference was not significant. Patients with a single lung metastasis had a significantly longer disease-free survival (P = .02) than did patients with multiple metastases. Mean and median survival were longer in patients with lung metastases. Five-year actuarial survival was 19% for patients with liver metastases and 47% for patients with lung metastases. Resection of isolated pulmonary metastases from the large intestine results in survival comparable to or better than resection of hepatic metastases. An aggressive surgical approach is warranted in patients with isolated resectable pulmonary metastases of colorectal carcinoma.

Deoxyribonucleic Acid Flow Cytometry on Primary Adenocarcinoma of the Bladder: An Analysis of 36 Cases

Deoxyribonucleic acid flow cytometry was applied retrospectively to 36 primary pure adenocarcinomas of the bladder, stages A through D. Six tumors were enteric, 3 mucinous, 11 signet ring, 3 papillary, 5 unspecified and 8 mixed. Eight tumors were urachal in origin and 28 were nonurachal. The deoxyribonucleic acid pattern was diploid in 12 cases, aneuploid in 19, tetraploid in 3 and uncertain in 2. Nineteen patients died of disease after a mean of 27.4 months, 7 were well at a mean of 73.9 months, 8 had died of an unrelated cause and 1 was alive with metastatic disease. Ploidy pattern did not correlate with tumor stage, histological pattern or type of outcome: 6 of 12 patients with diploid and 12 of 22 with nondiploid tumors died of disease. However, if the tumor was urachal 1 of 4 patients with a diploid pattern died of disease, while 3 of 4 with an aneuploid pattern either died or were alive with disease. Our data suggest that deoxyribonucleic acid ploidy pattern apparently is not a significant predictor of outcome for primary adenocarcinoma of the bladder except possibly when the origin is urachal. (J. Urol., 142:1206-1210, 1989)

Deoxyribonucleic Acid Flow Cytometry of Benign Prostatic Disease

Deoxyribonucleic acid flow cytometry was performed on aspirated prostatic cells from 198 patients who had benign cytological or histological findings. Unsatisfactory acellular histograms were obtained from 10.6 per cent of the cases. Three-fourths of the satisfactory samples (more than 5,000 cells after subtracting debris) showed the expected single peak deoxyribonucleic acid diploid to near diploid histograms. Unexpectedly, the remaining samples were deoxyribonucleic acid aneuploid, most having 2 peridiploid peaks (deoxyribonucleic acid index 0.82 to 1.31). Usually, proliferation was low with less than 20 per cent hyperdiploid cells and with 2.5 ± 1.5 per cent G2 cells. In 10 per cent of the single peak histograms there was evidence of inflammation, identified as an increase in hyperdiploid cells without an increased percentage of G2 cells but with a tail of high channel values. The aforementioned histogram features were considered to be benign findings. Seven per cent of the samples had deoxyribonucleic acid histograms suggestive of prostate cancer. Of these samples 7 had diploid or peridiploid aneuploid histograms with high proliferation (more than 20 per cent hyperdiploid cells with 8.5 ± 3.8 per cent G2 cells), while 5 had histograms with deoxyribonucleic acid aneuploidy other than peridiploidy.

The Clinical Application of Aspiration Deoxyribonucleic Acid Flow Cytometry to Neurologically Impaired Men Entering an Electroejaculation Program

For the neurologically impaired patient electroejaculation with a rectal probe can furnish sperm for artificial insemination. However, documentation of pregnancies in this patient group has been limited. Deoxyribonucleic acid flow cytometry has been shown to be a reproducible quantitative method with which to evaluate spermatogenesis. We assessed the ability of deoxyribonucleic acid flow cytometry on testicular aspirates to predict the quality of sperm production in 13 men undergoing electroejaculation.Semen analysis was performed on antegrade and retrograde specimens. Deoxyribonucleic acid histograms from testicular aspirates were evaluated for the relative proportions of haploid, diploid and tetraploid cells. These ratios were compared to the means reported for our own series of 25 patients who at vasovasostomy had sperm intraoperatively and to the similar figures from a group of 10 accident victims.Five patients had normal testicular ploidy compartments defined as within 2 standard deviations from control means. Of the 5 patients 4 had normal sperm counts and motilities (30 per cent or greater). Two patients have contributed sperm for artificial insemination; 1 has resulted in pregnancy. Three patients had moderately abnormal (2 to 4 standard deviations) testicular ploidy classes. These patients had adequate sperm counts but low motilities (5 per cent or less). Of the 5 patients with markedly abnormal (greater than 4 standard deviations) histograms 4 remained azoospermic despite repeated attempts at electroejaculation.Our data support the use of deoxyribonucleic acid flow cytometry on testicular aspirates as a predictor of which patients might succeed and those who are unlikely to succeed in an electroejaculation program.