Analysis of nuclear DNA histograms in thymomas

The nuclear DNA content of normal thymuses and thymomas were measured by microphotometry. The DNA histograms were analyzed for 1) peak values, 2) cell with DNA contents of 4C or more (tetraploidy), and 3) average histograms. The DNA histograms of normal thymuses had peak values around 2C. However, the peak value on thymomal histograms moved to the 4C region (tetraploidy). Advances in stages were followed by an increase in the number of cells with high DNA contents. The number of cells with DNA contents of 8C or more (octaploidy) were significant only in stage III. Thymomal histograms were different from those of normal thymuses. The DNA histograms of thymomas changed with advances in stage, which may indicate that the epithelial cells of thymomas become neoplastic.     

DNA flow cytometric evaluation of spermatogenesis. Part 2: Studies on male infertility

Flow cytometric DNA analysis was performed on the testicular biopsy tissue obtained from 17 oligospermic men, 25 azoospermic men and 5 normal men. DNA histograms were made after viewing a small piece of biopsy tissue for a short time. The DNA histograms were classified by eye into four types (Type A: Aspermatogenesis without haploid cell, Type B: Maturation arrest at primary spermatocyte, Type C: Hypospermatogenesis, Type D: Normal spermatogenesis). Analysis of the DNA histograms accurately revealed the proportion of haploid, diploid and tetraploid cells. The DNA distributions for 5 normal men were 58.9 +/- 3.6% haploid cells, 24.3 +/- 3.8% diploid cells and 16.8 +/- 0.8% tetraploid cells. Significant correlation was found between the proportion of haploid cells (%haploid) and the testicular volume. The results of the investigation of the correlation between the DNA distributions and histological evaluations show that testicular degeneration increase proportionally to the decrease in haploid cells. Therefore, the %haploid appears to be an effective index for the quantitative evaluation of spermatogenesis.     

The accuracy of fine needle aspiration biopsy for flow cytometric determination of tumor DNA content

The use of fine needle aspiration (FNA) to obtain a diagnosis of malignancy is established in the practice of oncology, but there is little information on its accuracy in sampling tumor DNA content. We therefore compared flow cytometric DNA data obtained from FNA-derived samples with that obtained after digestion of the same murine tumor from which the aspirates had been taken. Fifteen female C3Hf/Kam mice were implanted with MCA-29 tumor cells from the same source tumor. MCA-29 is a multiploid mammary adenocarcinoma with two aneuploid populations (DNA Index of A = 1.67, B = 1.89). The tumors were grown to a mean size of 8.6 mm. After sacrifice, three FNAs were performed on each tumor, following which the whole tumor (WT) was excised and homogenized. All FNA and WT samples were digested with 0.04% pepsin and the nuclei stained with propidium iodide in preparation for flow cytometry. DNA histograms of the aspirates were compared with the corresponding WT histograms. Any single FNA detected population A in all (100%) cases and detected the less prominent population B in 94.3% of instances. Any single FNA was able to detect the same populations that were present in the whole tumor in 95.4% of cases, while the set of three aspirates matched the corresponding WT in 100% of cases. We conclude that FNA DNA histograms are accurate for the assessment of ploidy, but that in order to ensure detection of all tumor populations present, multiple aspirates are needed.     

Shape and position of the haploid peak in flow cytometric sperm histograms for the assessment of andrological diseases

Ejaculates from 45 patients with various andrological diseases and from healthy men were analyzed by flow cytometric DNA measurements. The sperm histograms obtained by this method were subjected to mathematical analysis. The height, width and position of the haploid (1 C) peak proved to be a useful criterion for the discrimination of spermatozoa from patients with heterogeneous testicular disorders (SHTD, "mixed atrophy") and homogeneous testicular disorders (SETD) with delivery of immature spermiogenetic cells as compared to healthy persons.     

Colorectal cancer. Dukes' stage, tumor site, preoperative plasma CEA level, and patient prognosis related to tumor DNA ploidy pattern

Flow cytometric DNA histograms of colorectal carcinomas from 264 patients were evaluated for the association of tumor site, Dukes' stage, tumor grade, and preoperative carcinoembryonic level with patient survival. The DNA nondiploid carcinomas were significantly more common from the left (descending and sigmoid) colon and the rectum. A poorer prognosis was found for patients with DNA nondiploid cancers than for patients with DNA diploid cancers. This was particularly true for patients with Dukes' stages B2 and C tumors with a small number (one to three) of lymph nodes with metastatic deposits. The DNA nondiploid cancers also had a relatively poorer prognosis in patients with unresectable disease. In a Cox multivariate analysis model, the DNA pattern was an independent prognostic variable for this group of 264 patients with resected colorectal carcinoma.     

Flow cytometric studies of human brain tumors. Part 4: Metastatic tumors

The distribution of DNA content in 12 cases of metastatic brain tumor, 6 cases of primary lung cancer and 6 cases of primary breast cancer were studied by flow cytometry. According to the pattern of DNA histogram, 12 specimens of metastatic brain tumors were classified into 3 types. Six cases of type I showed a sharp peak in 2C with large numbers of cells in 4C, 4 cases of type II showed the highest peak near 3C with a small peak in 2C, and 2 cases of type III showed a shift of the peak to the right near 4-5C. Type II and III seemed to have two different kinds of the cells which were mixed with normal brain cells or blood cells showing small peak in 2C and tumor cells showing the highest peak in 3C to 5C. Primary lung cancer and breast cancer were also divided into 2 types of DNA histogram, which were similar to those of metastatic brain tumors as type I and type II. Although there was no case of metastatic brain tumor having a DNA histogram of primary tumor in the same case, DNA patterns of metastatic brain tumors might be related to those of primary tumors because of the similarity of the histograms. It was difficult to disclose the primary sites or histological types of metastatic brain tumors from the type of DNA histograms but flow cytometric analysis revealed the characteristic patterns such as type II and III in metastatic brain tumors. We consider that these patterns might be of clinical diagnostic value in flow cytometric study.     

Improved application of impulse cytophotometry for the diagnosis of urinary bladder carcinoma

Summary Using pulse cytophotometry, almost quantitative separation of the leucocyte fraction from DNA histograms was possible by means of an anticoincidence discrimination device. This modified technique was employed for biparametric DNA/protein measurements of voided urine samples, bladder washings, and tumour tissues. The results show a high degree of correlation between these samples so that, for tumour diagnosis from DNA histograms, voided urine specimens can be used rather than bladder washings. The criteria for the bladder tumour diagnosis are derived from DNA measurements of 32 controls and 35 tumour patients. The diagnostic sensitivity of this method is 0.91 and the specifity 0.75.Supported by grant No. 01 ZO 029-ZA/NT/MT 299 of the DEVLR (ministry of research and technology)     

Comparison of histologic and flow cytometric evaluation of cyclophosphamide-induced testicular damage

This study evaluates the ability of DNA histograms obtained by flow cytometry to detect and quantify reversible alterations in spermatogenesis induced by cyclophosphamide, a known inhibitor of spermatogenesis. Evaluation of per cent of cells in each of the haploid (lc), diploid (2c), and tetraploid peaks (4c) as determined by flow cytometry in treated and control Balb/C mice over a six-week period, and comparison with routine histologic evaluation have led us to conclude that DNA histogram evaluation is a rapid and accurate means of identifying testicular damage and recovery. This technique may be useful in sequential monitoring of the effects of malignancy and/or treatments applied on spermatogenesis in young men.     

Flow cytometry in prostatic adenocarcinoma

Summary We have examined the reproducibility of DNA histograms produced by flow cytometry in two strains of the Dunning R3327 rat prostatic tumor model, determining the extent of variability between tumors in the same animal, from animal to animal within the same strain and from strain to strain. We have also investigated the ability of flow cytometry to monitor changes in DNA histograms produced by tumor age and hormonal manipulation. We have found that there is a considerable difference in aneuploidy in tumors from the two strains, and also much variability in its extent within each strain. While orchiectomy (ORCH) tended to decrease the extent of aneuploidy, this could only be reliably detected if each tumor acted as its own control, with sampling being done pre- and post-ORCH.     

Differences in the DNA-stainability of spermatozoa from fertile and suspected infertile men

The aim of this work was to determine whether it is possible to distinguish between fertile (control group, already fathers) and infertile men (suspected infertility), by comparing the fluorescence intensity of the sperm-DNA after incubation with appropriate dyes. First we examined two different DNA-specific dyes (DAPI and YOYO-1) using bull spermatozoa. Based on good results in immunohistochemical applications, YOYO-1 was chosen for further work. The fluorescence-intensity of 200 single, morphologically normal spermatozoa in each semen sample were measured in a cytophotometer, means + SD determined and histograms delineated. Of 20 samples from the control group, 17 had markedly higher fluorescence-intensity than did 7/15 of the suspected infertile men. It is concluded that the DNA of the latter seven samples was less accessible to the dye than was the DNA of the control group. There are cases of infertility known in which there is loss of one or more of the DNA-binding proteins, which in spermatozoa are mainly (85%) protamines. The relationship between the stainability of the sperm-DNA and the packaging with DNA-binding proteins is discussed. Two of the histograms showed abnormalities in the distribution of the fluorescence-intensities, one sample was extremely fragile and most of the sperm lysed during the staining-procedure. Five samples showed normal histograms in comparison with the control group.     

Is DNA ploidy of prognostic significance in stage I cutaneous melanoma?

Recent studies have suggested that the presence of DNA aneuploidy in stage I cutaneous melanoma carries a poor prognosis. To see if our experience correlated with these reports, we used DNA analysis by flow cytometry of propidium iodide-stained nuclei disaggregated from formalin-fixed paraffin-embedded tissue of biopsy specimens to retrospectively study 55 patients who had cutaneous stage I melanomas. The patients had been treated from 1977 to 1987 with a mean follow-up of 5.4 years. Thirty-nine (71%) of the 55 histograms were diploid, and 16 (29%) of the histograms were aneuploid. DNA content was significantly associated with other conventional prognostic factors, including growth pattern, ulceration, pathologic stage, tumor thickness, and Clark's level. DNA aneuploidy was significantly related to disease-free survival and predicted a poorer prognosis (p less than 0.05), but when stratified for tumor thickness it lost significance. A multivariate discriminant function analysis of 12 factors in melanoma showed six factors to be independently significant in determining prognosis. DNA content (p = 0.034) ranked fifth in importance behind growth pattern (p less than 0.001), ulceration (p less than 0.001), thickness (p = 0.001), and pathologic stage (p less than 0.005). DNA content, although significantly associated with conventional prognostic factors and disease-free survival, is not the best indicator of biologic behavior of melanomas in this study. Further investigation into its usefulness is necessary before DNA content can become a routine diagnostic modality in the work-up of stage I cutaneous melanomas.     

Correlations between nuclear DNA content and nuclear morphometry versus histological grading of prostatic carcinoma

The nuclear area, N/C ratio and anisonucleosis by morphometry and the DNA content by image photo-cytometry of paraffin-embedded tissues of 9 cases of prostatic hypertrophy and 48 cases of carcinoma obtained by open surgery, needle biopsy or by autopsy were correlated with histological grading. In histological grading, in addition to 9 benign hypertrophy cases, 11 were classified as well differentiated adenocarcinoma, 19 as poorly differentiated adenocarcinoma and 1 as small cell carcinoma. All 14 tumors taken from distant metastasis were classified as poorly differentiated adenocarcinoma. Correlation of the data by tumor cell morphometry with those of nuclear image photo-cytometry was carried out on surgical materials alone. In autopsy materials, DNA ploidy patterns were compared between primary lesions and metastases. The DNA histogram patterns of most cases were classified into 4 groups. Hypertrophy was differentiated from carcinoma by the histogram patterns. There were significant correlations between mean DNA value and morphometric factors. One of the 4 histogram patterns had a higher mortality than others. The DNA histograms did not distinguish between the primary and metastatic lesions of the same patients. However, the DNA histograms constructed from metastasis frequently showed a single stem-line of the primary lesion. This study disclosed several correlations between histological grading and morphometrical factors.     

Flow cytometric analysis of DNA ploidy using paraffin-embedded germ cell tumors

The DNA content of paraffin-embedded materials was determined retrospectively using flow cytometry (FCM) in 36 germ cell tumors, and related to histological type, clinical staging and tumor marker. These histograms were classified from the basis of mode and variance into a diploid and an aneuploid pattern. We could evaluate the DNA histograms in 20 of 36 specimens (56%). Aneuploid patterns were found in 11 of 20 evaluated cases, but there was no correlation between ploidy patterns and histological types. Aneuploid patterns were demonstrated in 2 of 6 stage I cases (33%), and 4 of 5 stage II cases (80%) in seminomas. The difference between stage I and II cases was not statistically significant. There was no correlation between clinical staging and DNA content in non-seminomas. Of the seminomas with elevated human chorionic gonadotropin (HCG) titers, 3 of 4 cases showed aneuploid patterns. These findings indicate that the determination of DNA ploidy in seminomas may prove to be of prognostic value.     

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.     

Application of flow cytometry to the diagnosis of bladder carcinoma

DNA histograms obtained by flow cytometry (FCM) in patients with bladder tumor were analyzed. DNA histogram pattern and calculated proliferation index (PI) can be used to evaluate the presence or the absence of tumor cells and this technique can be applied to the automated cytology in patients with bladder tumor. Furthermore flow cytometry is considered to provide us with useful information about the malignant potential of the tumor as well as the effect of the treatment. In the follow-up examination, our results suggest that flow cytometry may be a valuable addition to the routine urological examination in the conservatively treated patients with superficial bladder tumors.     

Prognostic significance of nuclear deoxyribonucleic acid ploidy patterns in resected hepatic metastases from colorectal carcinoma

Nuclear deoxyribonucleic acid (DNA) ploidy studies of paraffin-embedded archival tumor specimen blocks were performed by flow cytometry on extracted nuclei from 101 surgically resected hepatic metastases from colorectal cancer. In 28 patients, the corresponding primary carcinoma of the metastases was also studied. Tumor clinicopathology and clinical course of the patients were reviewed. Preparation of paraffin-embedded tissue specimens was performed by the technique of Hedley et al. and stained with propidium iodide according to the method of Vindelov et al. Eighty-eight of 101 metastatic tumors and 26 of 28 primary tumors yielded evaluable DNA histograms. Twenty-six metastases showed a DNA diploid pattern, 25 showed a significantly increased 4C peak (DNA tetraploid/polyploid), and 37 had a DNA aneuploid peak. Ploidy pattern was constant between primary and metastases in 84.6% of tumors. No significant relationship between host and tumor characteristics and ploidy pattern was found except for a correlation between grade 3 metastases and DNA aneuploid. Survival of patients with DNA aneuploid metastases was significantly less than that of patients with DNA diploid metastases (p = 0.03). However, among DNA nondiploid metastases, survival was significantly less for low DNA index metastases (less than or equal to 1.5) than for high DNA index (greater than 1.5) metastases (p less than 0.05). Flow cytometric DNA ploidy measurements may have prognostic value for patients with resected hepatic metastases from colorectal carcinoma.     

Is voided urine suitable for flow cytometric DNA analysis?

Samples of bladder washings are frequently used to provide cellular material for flow cytometric DNA analysis. Since voided urine is a potential source of similar material, the aim of this study was to determine whether voided urine yields satisfactory specimens. We compared the qualitative results of flow cytometric DNA analysis of the cells in 53 specimens of voided urine and 109 samples of bladder washings; 45% (24/53) of urine specimens gave satisfactory DNA histograms compared with 93% (101/109) of bladder washings. This difference was apparent regardless of whether the bladder contained a tumour. It was concluded that bladder washings provide superior material for flow cytometric DNA analysis and that flow cytometric DNA analysis of freshly voided urine may have deficiencies which preclude its use in routine clinical practice.     

Deoxyribonucleic Acid Flow Cytometry in the Assessment of Spermatogenesis

Purpose

We compared deoxyribonucleic acid (DNA) flow cytometric analysis of testicular tissue to quantitative assessment of spermatogenesis.

Materials and Methods

We studied 35 infertile men with azoospermia or oligospermia. All patients underwent incisional testicular biopsies. DNA flow cytometric analysis was performed on each specimen to evaluate the ability of the method to quantify alterations in spermatogenesis. The results were compared to quantitative histological examination. At least 100 spermatic tubules were examined on each specimen and the number of spermatids per tubule was counted. All histological specimens were examined by the same pathologist.

Results

Of the 35 specimens analyzed with DNA flow cytometry 5 were normal, while the percentage of haploid cells (spermatids and spermatozoa) was decreased (hypospermatogenesis) in 14, complete maturation arrest was noted in 2 and almost complete absence of haploid cells was found in 14. Comparing the findings on histological examination with histograms, excellent correlation was noted in cases of the Sertoli-cell-only syndrome and complete maturation arrest, while 3 of 14 histograms with hypospermatogenesis demonstrated normal spermatogenesis on histological examination. Additionally 1 of 5 histograms with normal spermatogenesis demonstrated hypospermatogenesis on histological examination.

Conclusions

DNA flow cytometry of the testicular tissue seems to be an objective and quantified method that can be used to investigate spermatogenesis in infertile men. It is also less time-consuming than any histological examination, permits management decisions within 1.5 hours after biopsy and may replace testicular histopathological study. Flow cytometric diagnoses correlated well with histopathological findings.     

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.     

DNA flow-cytometric,histological and hormonal analysis of sertoli cell only syndrome (SECOS)

Sertoli cell only syndrome (SECOS) was identified on histology in 21 cases(16,28%) among 129 testicular biopsies performed in our department forazoospermia over the last 5 years. In these patients history, clinicalfeatures, hormonal levels, and histological findings were analyzed. Inaddition DNA flow-cytometric analysis was performed and showed an almostcomplete absence of haploid cells. All patients presented with elevatedserum FSH levels suggesting a Sertoli cell damage or reduced productionof inhibin due to the absence of sermatogenic cells. An good correlation wasfound between histological findings and DNA histograms.In conclusion SECOS is a syndrome of unknown aetiology presenting in menwith azoospermia. DNA flow-cytometric analysis is a reliable, rapid and easymethod in the diagnosis of SECOS, and can replace histological examination.