DNA ploidy in gastrointestinal B-cell lymphomas. An image analysis study of 43 cases

OBJECTIVE: To analyze the prognostic importance of DNA ploidy pattern on gastrointestinal (GI) B-cell lymphoma using image cytometry (ICM) and to compare the results with previously published flow cytometry (FCM) data. STUDY DESIGN: Forty-three cases of surgically resected primary GI B-cell lymphomas were examined. Thirty-eight tumors were located in the stomach, 2 in the small intestine, 1 in the large bowel and 2 in both the stomach and small intestine. Six cases were at stage E I 1, 15 at stage E I 2, 20 at stage E II 1 and 1 each at stages III and IV. Histologically, the lymphomas were classified as GI low grade marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) type (low grade, 12 cases), low grade MALT lymphoma with a high grade component (mixed type, 10 cases) and GI diffuse large B-cell lymphoma (DLBC) (high grade MALT lymphoma, 21 cases). After gross removal of nonneoplastic tissue, single cell suspensions were prepared from paraffin blocks and stained according to Feulgen. Ploidy analysis was done using a custom-made DNA cytometer and Optimas image analysis software (Optimas Corp., Seattle, Washington, U.S.A.). RESULTS: Aneuploidy was found in 42% (5/12 cases) of low grade MALT lymphoma, 90% (9/10 cases) of mixed type lymphoma and 100% (21/21 cases) of GI DLBCL. DNA ploidy had no significant impact on overall survival time (P = .73). CONCLUSION: ICM analysis showed a higher proportion of aneuploidy in GI lymphomas as compared to that in prior studies using FCM for ploidy determination. Whether DNA ploidy is an independent prognostic factor remains to be determined.     

Early detection of precancerous lesions in dysplasias of the lung by rapid DNA image cytometry

Hematoxylin-and-eosin-stained cytologic smears of sputum from 28 patients with dysplastic and suspicious cell findings were subjected to DNA image cytometry after Feulgen restaining. The nuclear DNA contents were measured with a TV-based image-analysis system, the Leitz TAS plus, combined with an automatic microscope. Computation of DNA data was performed according to an algorithm for the diagnosis and grading of malignancy. Of the 19 cases that were proven to be malignant in the follow-up, either by histologic examination, sputum cytology, fine needle aspiration biopsy or autopsy, the algorithm identified 17 as malignant in a stage (dysplasia) in which cytology was not yet able to present a definite diagnosis of malignancy. Only two cases of bronchial carcinoma were not detected in the state of dysplasia by this procedure. The periods between the DNA diagnosis of malignancy in dysplasia and the morphologic evidence of cancer varied from three days up to six months. Of the 11 cases that had been classified as benign by the algorithm, 9 were confirmed as benign during the clinical follow-up. Rapid DNA image cytometry appears able to separate squamous dysplasias of the lung into precancerous and nonprecancerous lesions.     

DNA cytometry in pleomorphic adenomas with cytologic atypia

OBJECTIVE: To investigate the nuclear DNA content of pleomorphic adenomas with cytologic atypia. STUDY DESIGN: Analysis was performed on new, fuchsin-stained samples of 10 selected cases of pleomorphic adenoma with cytologic atypia. Morphometric analysis was done by a computer-assisted image cytometry system and consisted of the determination of DNA indices, Auer DNA histogram types, and 3c and 5c exceeding rates. RESULTS: Eight cases were diploid and two cases aneuploid according to the DNA index. The Auer histogram was type I in five cases and type III in the others. In the two aneuploid cases the 3c exceeding rate was > 10% and the 5c exceeding rate > 1%. CONCLUSION: Atypical cells in pleomorphic adenomas with cytologic atypia carry abnormal amounts of DNA. Image cytometry can make detecting very low numbers of aneuploid cells easier due to its higher resolution as compared to that of flow cytometry.     

Predictive value of DNA cytometry in CIN 1 and 2. Image analysis of 193 cases

OBJECTIVE: To investigate DNA image cytometry for predicting the prognosis of cervical intraepithelial neoplasia (CIN). STUDY DESIGN: Smears from 151 women affected by CIN 1 or 2 on cytology with minimal follow-up of three years were included. Sixty-seven showed progression, with histologically confirmed carcinoma in situ or invasive cancer. Eighty-four cases showed regression of the disease, which was cytologically, histologically and colposcopically confirmed. Papanicolaou-stained smears were destained, and the Feulgen reaction was performed with consecutive image DNA cytometry of suspicious cells using an image analysis system (Cires, Zeiss, Germany). The DNA index of the greatest stemline and the number of single aneuploid cells, using 9c exceeding events, were computed. RESULTS: In the group with progression, an aneuploid DNA stemline was found in 25 smears (26.9%). In 64 cases (66.7%) more than one aneuploid event was detected. The total number of aneuploid cases in this group was 76 (81%). In the group without progression, the number of aneuploid stemlines was 2 (2%). Single aneuploid cells could be found in five cases (5%). The overall number of aneuploid cases in that group was five. The sensitivity was 74.3%, positive predictive value 85.2% and negative predictive value 77%. CONCLUSION: Aneuploidy is a marker for prospective malignancy in cervical Papanicolaou smears. DNA image cytometry, as an additional method, can be used to predict outcome in patients with CIN 1 and 2 of the cervix. DNA cytometry is not a screening method but can add further information for a treatment decision in doubtful cases.     

Determination of DNA ploidy in archival tissue from non-Hodgkin's lymphoma using flow and image cytometry.

Paraffin-embedded tissue from a series of 40 cases of diffuse, large cell lymphoma was analyzed by both flow and image cytometry to compare the ability of these techniques to detect DNA aneuploid populations. Image cytometry (ICM) was performed both on nuclear suspensions and tissue sections. Twenty cases (50%) were non-diploid by at least one method of analysis. Twenty-five percent of the cases were aneuploid by flow cytometry (FCM) alone. The majority of these cases were near-diploid tumors which could not be resolved by ICM. Peri-tetraploid peaks were identified by ICM of tissue sections alone in 15% of the cases. There was an apparent loss of these peri-tetraploid cells during the preparation of the nuclear suspensions. The remaining cases showed a good correlation between all three methods in the determination of DNA ploidy. Flow and image cytometry are complimentary techniques when applied to archival tissue, however aneuploid populations may be missed if ICM is not performed on tissue sections.     

DNA and nuclear protein measurement in columnar epithelial cells of human endometrium

Propidium iodide DNA flow cytometry, Feulgen-DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content. For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage. Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false-positive cases.     

Image cytometry in nonproliferative fibrocystic breast changes. Ploidy evaluation and epidemiologic study

OBJECTIVE: To determine the usefulness of image cytometry on fine needle aspirates from patients with fibrocystic changes (FCCs) to assess the subsequent risk of breast cancer. STUDY DESIGN: Thirty-five hundred archival cases with fine needle aspiration (FNA) biopsy were assessed to select nonproliferative FCCs of the breast (500 cases), also classified as type 1 (FCC I). DNA evaluation was analyzed by means of image cytometry on ductal epithelial cells and on apocrine metaplastic cells; 97 quantifications were performed. Cytometric variables investigated were: DNA ploidy, G0/G1 peak of diploid nuclei, S-phase fraction, 5cER, 2cDI and coefficient of variation. Two groups each with 15 years of follow-up were formed: Simple pathology (SP), fibrocystic changes alone; associated pathology (AP), FCCs plus breast cancer. Each was studied separately and compared. RESULTS: In SP cases the average ploidy was 2.2 for epithelial cells and 4.2 for apocrine cells. The proportion of G0G1 diploid nuclei was 100%. In the study of AP, the average ploidy was 2.2 for epithelial ductal cells and 4.1 for apocrine ones, for a slight increase in SPF. Ductal cells were diploid, while apocrine cells were tetraploid, with statistical significance of P < .05. For the epidemiologic study we calculated the proportion of patients with FCC I who developed cancer by referring to a historical cohort study, obtaining a relative risk of 0.7. CONCLUSION: Our results prove that DNA image cytometry applied on FNA cytology is a very useful, minimally invasive and reliable tool to determine higher activity and risk for development of breast cancer in FCC I and thus to establish the need for closer follow-up of these patients. In addition, apocrine metaplastic cells of the breast display a tetraploid DNA histogram, while the other karyometric variables remain in the range of normality.     

DNA ploidy and vimentin expression in primary breast cancer

DNA ploidy and vimentin expression in primary breast cancer
The DNA content of 50 breast cancers of varying tumour type, grade and stage was measured using static image cytometry, and correlated with vimentin expression in the tumour cells. A tendency to increased vimentin expression and aneuploidy was observed in high grade and late stage tumours¹. A statistically significant difference was observed in DNA index and ploidy balance between grade 1 and grades 2 and 3 carcinomas ( P <0.05) and between stage I and stage II carcinomas ( P <0.05). There was a significant difference in the expression of vimentin between grades 1, 2, 3 ( P <0,001), and stages I, II and III ductal carcinomas ( P <0.05). No significant difference was observed in the proliferation index and the degree of hyperploidy ( P >0.05). Clonal heterogeneity was observed in 25% of breast carcinomas, and was associated with increased vimentin expression. These changes may be indicative of genomic alteration and tumour aggressiveness.     

Imaging cytometry by multiparameter fluorescence.

A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub-populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections.     

DNA content of Hurthle cell nodules in autoimmune thyroiditis

Hurthle cells are found in thyroid neoplasms and in reactive nodules in thyroiditis or goitrogenic processes. Cytometric studies have evaluated Hurthle cell neoplasms but not their reactive counterparts. DNA content of Hurthle cells in 22 cases of autoimmune thyroiditis was measured by flow cytometry and image content of Hurthle cells in 22 cases of autoimmune thyroiditis was measured by flow cytometry and image processing using nuclei extracted from paraffin-embedded tissue after microdissection of the Hurthle cell nodules. All 22 autoimmune thyroiditis Hurthle cell nodules were diploid, including 16 without associated neoplasms and six with associated malignant neoplasms (four papillary carcinomas, one follicular carcinoma and one follicular adenoma with papillary carcinoma). Concordance between flow cytometry and image processing was 100%. These findings indicate that the markedly atypical Hurthle cells in autoimmune thyroiditis are diploid by DNA quantitation. This suggests that atypia in Hurthle cells due to reactive or neoplastic processes may be differentiated by quantitative DNA analysis.     

Exfoliative-cytologic diagnosis of basal-cell carcinoma, with the use of DNA image cytometry as a diagnostic aid

Eighty-four skin lesions clinically suspected of being basal-cell carcinomas were investigated by exfoliative cytology. Specific criteria were found for the cytologic diagnosis of basal-cell carcinoma. All 37 cases of basal-cell carcinoma were correctly classified cytologically and could be differentiated from the 8 cases of squamous-cell carcinoma. There were no false-positive or false-negative diagnoses. Insufficient cellular material was obtained in 17% of the cases. The technique for collecting exfoliated epidermal cells with a new swab is described. DNA image cytometry was used as a diagnostic aid in doubtful cases. DNA image cytometry showed that 83% of the basal-cell carcinomas had an aneuploid nuclear DNA content.     

Quantitative DNA analysis of low grade cervical intraepithelial neoplasia and human papillomavirus infection by static and flow cytometry.

Quantitative deoxyribonucleic acid (DNA) analysis of cervical biopsy specimens from 26 women with cytological, colposcopic, and histological evidence of mild cervical atypia consistent with cervical intraepithelial neoplasia grade I, reactive atypia, or human papillomavirus infection alone or in combination was performed in a comparative evaluation of Feulgen microspectrophotometry, the fast interval processor image analysis system, and flow cytometry. The fast interval processor image analysis system showed a distinct advantage over the other methods, being faster and allowing the operator to see the cells that were selected for measurement. The three methods of measurement together showed that the DNA content of at least 2% of the cells measured exceeded 5C (C being the haploid amount of DNA in a normal cell and 2C representing the diploid complement of a normal cell) in all cases of cervical intraepithelial neoplasia grade I and reactive atypia and in 87% of those reported as showing human papillomavirus infection alone. In contrast, the DNA content of cervical biopsy specimens from the transformation zone of 11 normal controls did not exceed 4C. This study shows the value of using a DNA threshold--that is, the "5C exceeding rate"--to distinguish between normal and neoplastic appearances of the cervix. These results support the view that cervical infection by human papillomavirus is a true precursor of neoplasia.     

Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.

BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.     

Fine needle aspiration biopsy of cystic benign lymphoepithelial lesion of the parotid gland in patients at risk for the acquired immune deficiency syndrome

Cystic benign lymphoepithelial lesion (BLL), a previously rare lesion of the parotid gland consisting of marked lymphoid hyperplasia with accompanying squamous-lined cysts, has recently been described in patients with the acquired immune deficiency syndrome (AIDS) or AIDS risk factors. Thirteen fine needle aspiration (FNA) samples of parotid gland masses from patients with AIDS (one case), AIDS risk factors (five cases) or denial of AIDS risk factors (two cases) and a histopathologic diagnosis of BLL were examined. The FNA features that correlated best with the histopathologic findings were (1) a heterogeneous lymphoid population, (2) scattered single and/or clustered foamy macrophages and (3) superficial and/or anucleated squamous cells. Most aspirates showed some combination of these three components. The differential diagnostic considerations, the clinical and radiologic correlations and the relationship of this lesion to HIV infection are discussed. Patients with parotid masses whose aspirates consist of some combination of squamous cells, lymphocytes and foamy macrophages should be questioned for possible AIDS risk factors.     

Cytologic and DNA cytometric diagnosis and therapy monitoring of squamous cell carcinoma in situ and malignant melanoma of the cornea and conjunctiva

OBJECTIVE: To investigate the diagnostic accuracy of exfoliative cytology of the cornea and conjunctiva and DNA image cytometry for quality control and monitoring of therapy for malignant neoplasms. STUDY DESIGN: Conjunctival or corneal smears from six cases clinically suspicious for malignant melanomas and eight suspicious for carcinomas in situ were investigated. Smears from 18 cases clinically nonsuspicious for neoplastic diseases served as negative controls. Repeated smears were obtained during and after local mitomycin C (MMC) therapy. RESULTS: In none of 18 nonsuspicious cases, cytology revealed abnormal cells. DNA cytometry showed nonaneuploidy in all of these. All smears from patients with histologically proven malignant melanomas (MM) and squamous cell carcinomas in situ revealed abnormal cells. Image cytometry demonstrated DNA aneuploidy in 66.6% of patients with MM and 80% with carcinoma. Sensitivity of cytology thus was 100% for both MM and carcinoma; specificity also was 100%. DNA measurements after MMC therapy revealed euploid polyploidization of nonneoplastic squamous cells. DNA cytometry provided an objective identification of tumor cell regression. CONCLUSION: Cytologic examination of corneal and conjunctival smears is a noninvasive tool with high diagnostic accuracy for detection of epithelial neoplasms. DNA image cytometry can serve for quality control and for objective monitoring of the effect of local chemotherapy.     

DNA ploidy in breast lesions. A comparative study using two commercial image analysis systems and flow cytometry

DNA ploidy determinations on a series of 24 breast specimens were performed independently utilizing flow cytometry (FCM) and two separate commercially available computerized image analysis systems for image cytometry (ICM). The tissues analyzed were obtained from 20 carcinomas, 2 benign neoplasms and 2 benign reductive procedures. The results showed a close correlation between the DNA indices (DIs) obtained by all methods in 14 of the 24 cases. In four cases, all methods showed aneuploid peaks, but with differing DIs. In six cases (two benign and four malignant) FCM showed diploidy while ICM showed peridiploid cell populations. The results obtained with the two image analysis systems were in agreement for 20 of the 24 cases. ICM is an acceptable alternative to FCM for reproducible ploidy analysis. ICM-based measurements have the advantage of the visual discrimination of abnormal cells and therefore may have a greater sensitivity in identifying small aneuploid populations. Populations with DIs in the range of 1.0 to 1.3 need to be assessed carefully in ICM-based determinations due to the potential that these "aneuploid" peaks may represent shifted diploid populations.     

Image cytometric DNA analysis in human breast cancer analysis may add prognostic information in diploid cases with low S-phase fraction by flow cytometry.

Measurements of DNA ploidy can be performed either with image cytometry (ICM) or flow cytometry (FCM); both methods provide independent prognostic information in primary breast cancer. The aim of the present investigation was to compare the two methods and to relate the findings to prognosis (median follow-up 42 months). Concordance in ploidy status (diploid, tetraploid, aneuploid) was obtained in 76% of the samples (168/222). When the fraction of S-phase cells (SPF) from FCM analysis was also taken into consideration, four different groups of samples were obtained (Flow I-IV), which were considered to correspond to the Auer classification (Auer I-IV) of DNA histograms obtained from image cytometry. Complete concordance between the two techniques now was 70% (155/222). Samples classified as Flow I (diploid or near-diploid with low SPF) and Auer I had a distant metastasis rate of 3/60 (5%), as compared to 62/154 (40%) for all other combinations of the Flow and Auer classifications taken together. Thus, the only findings of prognostic importance were that some samples were Flow I but not Auer I, or vice versa. These two groups represent 17 (7.7%) and 14 (6.3%), respectively, of the total number of samples, and had frequencies of distant metastasis similar to those of the other high-risk groups, namely, 7/17 and 5/14, respectively. In a multivariate analysis, flow cytometric S-phase value was a stronger prognostic factor than either the Flow and Auer classification. We conclude that when routine FCM DNA analysis is used, diploid or near-diploid samples with a low S-phase value should be reanalyzed with ICM.     

Cell cycle effects of the DNA topoisomerase inhibitors camptothecin and m-AMSA in lymphoblastoid cell lines from patients with Fanconi anemia

Patients with the autosomal recessive disorder Fanconi anemia (FA) present with progressive pancytopenia, skeletal abnormalities and a predisposition to leukemia. In addition to elevated rates of spontaneous chromosome aberrations occurring in cultured fibroblasts and lymphoblastoid cell lines, an increased susceptibility to DNA cross-linking agents and oxygen has been found. To explain this hypersensitivity to clastogenic agents a defective function of DNA topoisomerase I or II could be invoked, a suggestion which is supported by the co-localization of the DNA topoisomerase I gene and a putative FA gene to chromosome 20q. In order to investigate the function of DNA topoisomerases in FA, the sensitivity of lymphoid B-cell lines derived from FA patients and control cell lines to inhibitors of DNA topoisomerases I and II was compared using continuous bromodeoxyuridine labeling and bivariate Hoechst/ethidium bromide flow cytometry. Both agents inhibited cell proliferation mainly by arresting cells in the G2 phase of the cell cycle. However, no difference was found in sensitivity towards both DNA topoisomerase inhibitors between control and FA cell lines.     

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.