Comparison of DNA ploidy in routine fine needle aspiration biopsy samples and paraffin-embedded tissue samples

The nuclear DNA content was determined by flow cytometry (FCM) from unfixed fine needle aspiration (FNA) biopsy samples of 31 human tumors, and from the same tumors after their excision, fixation with formalin and embedding in paraffin. The ploidy of the histograms was the same in 29 (94%) of the 31 cases. The disagreement in two cases may be explained by clonal heterogeneity of the tumors. The DNA index of the aneuploid cases was identical in fresh and fixed samples. The coefficient of variation of the diploid peaks (P less than .001) and the mean percentage of S-phase cells (P = .06) were larger in the fixed samples. It is concluded that routine FNA biopsy is a practical and reliable method for collecting cells for FCM DNA ploidy determination.     

Flow cytometric DNA analysis combined with fine needle aspiration biopsy in the diagnosis of palpable metastases

To determine the diagnostic value of flow cytometric (FCM) DNA analysis in combination with fine needle aspiration (FNA) biopsy, the nuclear DNA content was analyzed in FNA specimens from 155 superficial metastases and 60 benign lymph nodes. The results of FCM DNA analysis were considered to be positive for malignancy if DNA aneuploidy was found or if more than 12% S+G2M cells were present in the aspirate in diploid cases. The diagnostic accuracy of FCM DNA analysis alone in detecting malignancy was 92%, with a sensitivity of 91% and a specificity of 95%. FCM suggested the correct diagnosis in 5 of the 7 cytologically false-negative cases and in 9 of the 12 cytologically indeterminate or suspicious cases.     

Flow cytometric DNA ploidy analysis of soft tissue sarcomas. A comparative study of preoperative fine needle aspirates and postoperative fresh tissues and archival material

Flow cytometric (FCM) DNA ploidy measurements on frozen fresh samples of soft tissue sarcomas were compared with the corresponding analyses on preoperative fine needle aspirates and postoperative formalin-fixed archival tissues from the same tumors. A concordance in ploidy status (diploid versus non-diploid) was obtained for 63% of the fresh tissue-fine needle aspiration (FNA) sample comparisons and for 85% of the fresh tissue-archival material comparisons. The majority of discordances in the fresh tissue-FNA sample comparisons could be explained by FNA sampling errors. In the remaining discordant cases (3 of 27 FNA sample comparisons and 6 of 40 archival material comparisons), sampling errors could not explain the differences in ploidy status. The discordant cases were evenly distributed among the different sampling methods. Method reproducibility was not responsible for the differences in ploidy determinations; tumor heterogeneity may be an explanation for the discrepancies. This study showed that archival soft tissue sarcoma samples are as well suited for DNA ploidy analysis as are fresh frozen tissues.     

Diagnostic value of flow cytometric DNA determination combined with fine needle aspiration biopsy in thyroid tumors

The nuclear DNA content and the numbers of cells in the S and G2M phases of the cell cycle were determined by flow cytometry (FCM) in fine needle aspirates of 187 thyroid tumors to evaluate the diagnostic value of nuclear DNA content determination in combination with aspiration cytology. DNA aneuploidy was present in 4 of 5 follicular carcinomas, 2 of 3 anaplastic carcinomas, 5 of 15 excised follicular adenomas and 2 of 20 excised adenomatous goiters; all 7 papillary carcinomas and 4 lymphomas were diploid in the aspirate. Aneuploid carcinomas had easily distinguishable S and/or G2M phases, unlike the benign aneuploid tumors. None of the histologically benign tumors or the nonexcised tumors had greater than 6% S-phase cells, and only one benign tumor had greater than 9% G2M-phase cells. In contrast, all lymphomas had greater than 10% S-phase cells and four of seven papillary carcinomas had greater than 9% G2M-phase cells. The use of FCM determination in combination with fine needle aspiration biopsy cytology improved the diagnostic potential of the latter technique.     

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

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

Heterogeneity of DNA distribution pattern in colorectal carcinoma. A microspectrophotometric study of fine needle aspirates

For 50 consecutive patients with colorectal carcinoma, the nuclear DNA content in four separate fine needle aspiration samples taken from the resected tumors was analyzed using single-cell Feulgen cytometry. The DNA distribution patterns were divided into four classes according to their degree of euploidy/aneuploidy. Twenty tumors (40%) displayed a similar DNA pattern in all four samples while 30 tumors (60%) were heterogeneous, with a different DNA pattern within one or more of the four samples. None of the tumors were homogeneously diploid. These results illustrate the importance of multiple biopsy samples in the evaluation of the DNA pattern in colorectal tumors.     

Ploidy and morphology in osteosarcoma

In a cytometric DNA study of high-grade osteosarcoma, the relationship between DNA content and morphology was analyzed. The investigation, based on microspectrophotometry of tissue sections and flow cytometry (FCM), included both primary lesions and recurrences. FCM analysis, applied to a consecutive series of 47 primary osteosarcomas, disclosed that 2 were diploid and 45 were nondiploid, 8 of which were tetraploid. Multiple aneuploid peaks were detected in 13 tumors. Among the nondiploid tumors, there was no clear relationship between the peak DNA value(s) and the histologic subtype (osteoblastic, chondroblastic, fibroblastic) or grade (III-IV). The proliferative activity, as reflected by the percentage of S-phase cells, could be determined in 38 of the 47 tumors analyzed by FCM. The percentage was higher for aneuploid than for tetraploid lesions; however, the distribution of S-phase cells was not related to the histologic subtype or the grade of the tumors. To assess the reliability of a single sample for FCM, the DNA content of biopsy and surgical specimens was compared in 20 tumors; there was complete agreement in all cases with respect to the classification of the lesion as diploid, tetraploid or aneuploid. Analysis by FCM or microspectrophotometry of 12 local recurrences and 16 metastases and the corresponding 19 primary tumors showed that an aneuploid characteristic of the primary lesion was retained during progression of the disease. In 12 tumors analyzed by microspectrophotometry in tissue sections, comparison of chondroblastic and osteoblastic/fibroblastic areas within the same lesion consistently disclosed hyperploidy in both areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA ploidy assessment method applied to primary breast carcinoma FNA biopsies

The goal of our study was to assess suitability of FNA biopsy material as a source of samples (cell suspension) for DNA ploidy assessment in neoplastic tumors using flow cytometry. DNA ploidy is an established prognostic factor in many types of cancers. Aneuploid breast tumors are characterized by increased aggressiveness which manifests itself through rapid local progression and metastatic spread. Investigated specimens were breast cancer FNA biopsy cell suspensions. Measurements were performed using flow cytometry. Material studied comprised 143 cases analyzed in 1999-2000. We found in this group 101 carcinoma cases with aneuploid type and 42 cases of primary breast carcinoma with diploid type of cell cycle. Immunocytochemical assesssment of estrogen receptor and progesterone receptor status was performed in group of 105 cases. DNA ploidy was compared to receptor status of the investigated cells. DNA aneuploidy correlated with weak or no reaction for the presence of estrogen and progesterone receptors. Our study demonstrates the suitability of DNA ploidy assessment method applied to cytological material from FNA biopsies.     

Method of extracting DNA from fine needle aspirates of human solid tumors for Southern blot analysis

A rapid, simple, convenient method for extracting DNA from fine needle aspiration (FNA) samples of human solid tumors for Southern blot hybridization studies is described. After the preparation of an air-dried cytologic smear, the remaining sample in the needle was rinsed directly into a test tube for DNA extraction. The extraction procedure, in which manipulation of the sample is minimized, produced sufficient DNA for Southern blot analysis within 24 hours of the FNA biopsy in the ten consecutive cases studied. The DNA bound to the nylon membranes can be washed and reexamined with a variety of probes, allowing studies of lymphoid cell lineage, oncogene amplification or tumor progression. The assessment of cellularity on the cytologic specimen at the time of FNA provided a reliable guide to the need for further passes to obtain sufficient cells for DNA hybridization; the cytologic diagnosis could also be made on the smears.     

Dependence of DNA-histograms on the sampling techniques in fine needle aspirates of the breast.

48 fine needle aspiration biopsy (FNAB) samples from 25 breast cancer cases, originally used for cytodiagnosis were subjected to DNA cytometry. There were air dried smears stained with the MGG method, and samples stained with HE or PAP stain after 50% ethanol fixation and cytocentrifugation. Different sampling strategies were applied. Four methods were tested: method 1: cell groups measured, method 2: all cells measured, method 3: free cells measured, and method 4: atypical free cells measured. Method 4 showed most often DNA aneuploid histogram patterns, sampling method 1 had the highest number of DNA diploid histogram patterns. Diagnostic approaches may benefit from a sampling method detecting the hiding aneuploid cell population. Grading of neoplasm could potentially benefit from other approaches.     

Cytomorphometry of uveal melanoma. Comparison of fine needle aspiration biopsy samples with histologic sections.

A number of approaches are being investigated to increase the prognostic accuracy for uveal melanoma patients; the standard deviation of nucleolar area measurements and the DNA content appear to correlate better with survival than do classic histologic parameters. The utility of performing cytomorphometric measurements on fine needle aspiration (FNA) biopsy samples was prospectively analyzed for 24 eyes containing uveal melanomas that were examined with both 25-gauge FNA biopsy and standard histologic techniques. "Masked" analysis of the cellular composition of the 24 cases showed the presence or absence of epithelioid cells to be accurately predicted on the FNA samples in all cases. Image analysis cytomorphometric measurements of nucleolar area showed marked variability (with r less than 0.4) when FNA and histologic samples from the same case were compared. The relationship between these measurements was affected by cell type, sampling, specimen processing and investigator experience.     

Lipomatous lesions of the parotid gland. Potential pitfalls in fine needle aspiration biopsy diagnosis

Nine cases of benign fatty tumors of the parotid studied initially by fine needle aspiration (FNA) biopsy and subsequently diagnosed by open biopsy were reviewed. All lesions presented as enlargements of the parotid gland. The cytologic diagnoses were correct in only five of the nine cases. While four of six FNA samples from lipomas were correctly diagnosed by cytology, the other two samples were classified as inadequate. Three cases of diffuse infiltration of the parotid by mature-appearing adipose tissue were cytologically characterized as "no diagnostic change" in two cases and sialosis in one case. The clinical, radiologic, cytologic and histologic features of these tumors are described to increase the familiarity of cytologists with these lesions, which may prevent unnecessary surgical intervention in some patients.     

Comparison between flow cytometry and image cytometry in ploidy distribution assessments in gynecologic cancer.

The DNA content in 37 tumors from 34 women with gynecological cancer was measured by flow cytometry (FCM) and interactive image cytometry (ICM). Agreement was obtained in 81% of cases as regards ploidy levels, but seven tumors (19%) showed different ploidies. Of these, five were classified as diploid by FCM but either aneuploid (three cases) or polyploid (two cases) by ICM. Two other tumors were aneuploid by ICM but polyploid (one case) and unclassifiable (one case) by FCM. All tumors classified as aneuploid by FCM were also aneuploid by ICM, and all tumors classified diploid by ICM were also diploid by FCM. Of six patients whose tumors were classified as euploid (five diploid and one polyploid) by FCM but classified as aneuploid by ICM, five relapsed, and three of these have died of disease. On the basis of these findings, it is concluded that ICM must be performed in cases classified as diploid by FCM to ensure that small subpopulations of aneuploid tumor cells are not overlooked.     

Flow cytometric DNA analysis on fine needle aspiration biopsies of liver lesions

Flow cytometric DNA analysis on fine needle aspiration biopsies of liver lesions The DNA cell content of 39 fine needle aspiration biopsies (FNAs) from five benign liver lesions, nine hepatocellular carcinomas (HCCs), and 25 metastatic tumours was analysed in a prospective fashion by flow cytometry (FCM). All benign lesions were diploid. Aneuploidy was found in five (55.6%) HCCs and in nine (36%) metastatic tumours. DNA index (DI) differences were not significant. The S-phase fraction (SPF) was higher in the malignant tumours, both combined (P < 0.02) and separated primary and metastatic (P < 0.05). We could not demonstrate an association between diploidy and percentage of benign hepatocytes in the smears of malignant tumours. The serum alpha-fetoprotein (AFP) level did not correlate with ploidy, DI, or SPF in the HCCs. In conclusion, ploidy and DI do not discriminate between benign and malignant liver lesions, but the SPF is higher in malignant tumours. DNA analysis does not help to distinguish primary from metastatic liver tumours. The presence of benign hepatocytes in samples from malignant tumours does not seem to influence the analysis of ploidy by FCM.     

Aspiration biopsy findings in amyloid tumor of the cervical vertebra. A case report

BACKGROUND: The differential diagnosis of destructive lytic lesions of the spine includes amyloid tumors. The diagnosis of amyloid tumor with fine needle aspiration biopsy (FNA) is challenging. Previous reports of FNA of osseous amyloid tumors have detailed the cytologic appearance of amyloid along with lymphocytes, plasma cells and histiocytes, occasionally multinucleate or forming granulomatous lesions. CASE: An 84-year-old man presented with neck pain. Radiologic studies showed a destructive, lytic lesion of C-6, with a large, soft tissue mass. FNA yielded many acellular smears containing abundant amyloid that was confirmed with special stains of corresponding tissue cores and subsequent surgical biopsies. CONCLUSION: Osseous amyloid tumors are destructive, lytic lesions that mimic other processes. Amyloid can be distinguished from other substances in FNA samples and amyloid tumor identified, even when amyloid is present without typical cellular components.     

Spermatogenesis in azoospermic,formerly cryptorchid men. Use of needle aspiration techniques

OBJECTIVE: To assess the use of testicular needle aspiration techniques to evaluate fertility potential in azoospermic, formerly cryptorchid men. STUDY DESIGN: Fifteen consecutive adult azoospermic, formerly cryptorchid patients (eight unilateral and seven bilateral) were examined by needle aspiration techniques, fine (FNA) and large needle (LNAB) testicular aspiration biopsy, for cytologic and histologic analysis. Five of the 15 subsequently underwent surgical biopsy for attempted assisted fertilization. RESULTS: Spermatozoa or spermatids were detected by FNA cytology or LNAB histology in one or both testicles in 87.5% of the unilateral and 28.6% of the bilaterally affected, formerly cryptorchid patients (P = .041, Fisher's exact test). The addition of LNAB to FNA identified spermatids in one patient with unilateral cryptorchidism and only Sertoli cells on FNA cytology. Furthermore, LNAB differentiated testicles with the cytologic finding of only Sertoli cells into those with or without diffuse fibrosis. In the five patients in whom assisted fertilization was attempted, the needle aspiration techniques predicted the presence or absence of spermatozoa in the subsequent surgical biopsy. CONCLUSION: The two needle aspiration techniques can be used to assess the fertility potential of azoospermic, formerly cryptorchid men and to select patients for assisted fertilization.     

Autolysis is a potential source of false aneuploid peaks in flow cytometric DNA histograms

Serial flow cytometric nuclear DNA content analyses were performed from normal human spleen, thyroid, liver, and pancreas removed from ten patients at autopsy and stored for up to 8 d without any preservative to study the effect of autolysis on DNA histograms. Fine needle aspiration biopsy (FNAB) samples were taken in diluted ethanol and tissue biopsies from the same area in formalin for embedding into paraffin at the time of autopsy and serially thereafter. Histograms obtained from samples taken within 10 h after death had a symmetrical G1 peak with a small coefficient of variance (CV) except histograms produced from paraffin-embedded pancreatic tissue, but bimodal distributions similar to those seen in aneuploid tumors were obtained from many samples stored longer than for 20 h. The DNA indices of the bimodal histograms were usually less than 1.3. The false peaks were more prominent in FNAB samples than in paraffin-embedded samples. The time of appearance of the false aneuploid peaks varied individually, and they were usually first seen in samples taken from the pancreas, followed by the liver, the thyroid and the spleen. Because neoplasms may become necrotic in vivo and fixation of fresh surgical samples may be slow and incomplete, increased DNA staining caused by autolysis may be a source of false aneuploid peaks in DNA content analysis.     

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

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

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

Flow cytometric DNA analysis in the diagnosis of lung tumors. A comparison with conventional methods

The efficiency of flow cytometric (FCM) DNA analysis in the diagnosis of lung carcinoma was compared with that of conventional cytologic techniques on bronchial brushing and fine needle aspiration samples from 461 patients. The main advantage of FCM was the rapid delivery of results. Unfortunately, this was offset by a poor sensitivity in the detection of bronchial tumors. Nevertheless, DNA analysis may still prove useful in determining the prognosis and in evaluating the effects of chemotherapy on known tumor stem lines.