Methodologic aspects of nuclear DNA assessment of gliomas with astrocytic and/or oligodendrocytic differentiation. Correlation of image and flow cytometric studies on paraffin-embedded specimens

A total of 239 samples from paraffin-embedded, formalin-fixed astrocytic and/or oligodendrocytic gliomas from 111 patients were deparaffinized and disaggregated for image cytometric (ICM) and flow cytometric (FCM) DNA assessments. Each measurement technique produced evaluable histograms in about 85% of the samples analyzed. In the 10% that could not be analyzed by FCM, the background counts were too high and the coefficients of variation were too broad for precise evaluation. The failures with ICM were due to a shortage of Feulgen-stained tumor cell nuclei after the deparaffinization and disaggregation procedures. The results obtained were identical in 77% of the samples evaluable by both methods and practically identical (i.e., euploid versus aneuploid) in an additional 18%. The reasons for completely divergent DNA ploidy patterns in 5% of the samples could not be clarified. About 80% of the histopathologically highly malignant gliomas were found to consist of neoplastic cells with an aneuploid or tetraploid nuclear DNA distribution pattern. The results show that cytometric DNA assessments can be reliably performed on paraffin-embedded specimens of gliomas with astrocytic and/or oligodendrocytic differentiation by means of FCM and ICM on deparaffinized and disaggregated specimens.     

Dual-parameter flow cytometric analysis coupling the measurements of forward-angle light scatter and DNA content of archival ovarian carcinomas of low malignant potential

Paraffin-embedded archival specimens from 45 cases of ovarian carcinoma of low malignant potential (OCLMP) were analyzed by flow cytometry (FCM) using propidium iodide (PI) staining. Since single-parameter FCM analysis is often deficient in the resolution of subtle near-diploid DNA-aneuploid populations, forward-angle light scatter (FALS) was measured as a second parameter. DNA aneuploidy was identified in 15 cases (33%). In 7 of those 15 cases, aneuploidy was resolved with single-parameter FCM; in the remaining 8 cases, DNA aneuploidy was resolved only following dual-parameter analysis coupling DNA content and FALS. In all 15 cases, a single near-diploid aneuploid population was observed (mean DNA index = 1.2); there were no tetraploid aneuploid cases. The proliferative activity for all 45 cases studied ranged from 1.0% to 8.9%, with a mean of 3.5%. No difference in mean proliferative activity was observed between the aneuploid and diploid tumors (P greater than .05). To exclude the possibility that PI staining artifacts caused the observed aneuploidy, five of the eight cases shown to be aneuploid by dual-parameter analysis were further studied using an alternate DNA-binding dye, DAPI, yielding similar results. To exclude the possibility that contaminating stromal and/or inflammatory cells caused the observed aneuploidy, samples from a subset of the dual-parameter cases were sorted, revealing the aneuploid populations to be composed primarily of tumor nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow-fluorometric diagnosis of euploid and aneuploid human lymphocytes.

In this paper the potential of flow-fluorometric DNA determination as a convenient and economical alternative to conventional cytogenetics for the diagnosis of aneuploidy in human lymphocytes is explored. By comparing euploid and aneuploid samples, we found that the fluorescence signals emitted from propidium iodide (PI) stained cells are linearly proportional to DNA content. Variation in DNA content between euploid individuals of a given sex was sufficiently low to permit diagnosis of aneuploidy involving chromosomes with greater than 1.8% of the total diploid DNA (e.g., X, 8, 9, and 13). Interindividual DNA content variation with flow fluorometry was too substantial, however, to confidently diagnose trisomy 21. Fluorescent stains which exclude (variant) simple sequence DNA might overcome this limitation.     

Different opinions on classification of DNA histograms produced from paraffin-embedded tissue

Flow cytometric DNA ploidy determination has been regarded as an objective prognostic parameter in several types of human cancer. To test whether DNA histograms are similarly interpreted, a series of flow cytometric DNA histograms was posted to six investigators working in the field for independent classification. The histograms were produced from paraffin-embedded adrenal adenomas or non-neoplastic tissue and had several different patterns. Only 44% of the histograms were similarly classified by all investigators, and 85% by five of the six participants, when DNA ploidy was evaluated. Different criteria for tetraploidy existed, and also some uncertainty in classifying peridiploid and small aneuploid peaks. It is concluded that lack of consensus on histogram classification may result in widely varying percentages of DNA aneuploid tumors found even if the data are similar. Until general agreement is reached on the definition of DNA aneuploidy and its subclasses, classification of DNA histograms is variable and subjective.     

Effect of sonication on paraffin-embedded tissue preparation for DNA flow cytometry

Since the publication of paraffin block extraction procedures, flow cytometric analysis of DNA ploidy and S-phase of tumor specimens has been widely applied. DNA aneuploidy, DNA tetraploid (elevated G2/M), and elevated S-phase are clinically significant in some tumor systems. True DNA tetraploid cell lines will contain a large 4c population and perhaps an 8c population; samples with cell aggregates will also contain a 6c population. Microscopic examination of samples having a 6c peak revealed nuclei with adhering debris and doublets, triplets, and larger nuclear aggregates. After sonication, a uniform suspension of single nuclei without adherent debris was seen. In addition to reducing the percent of G2/M cells, sonication also reduced S-phase percent such that it was closer to the bromodeoxyuridine labeling index. The DNA ploidy classification of specimens was also compared pre- and post-sonication. Four of 96 breast cancer samples changed classification; all were specimens in which the histogram became cleaner and a small DNA aneuploid peak became apparent after sonication.     

A comparison of maternal age, sex ratio and associated anomalies among numerically aneuploid, structurally aneuploid and euploid holoprosencephaly

During the period January 1987-July 2003, 59 cases of perinatally detected holoprosencephaly (HPE) with cytogenetic results were identified among 97,306 deliveries at Mackay Memorial Hospital. Among these 59 cases with HPE, 25 had euploidy, 27 had numerical aneuploidy, and 7 had structural aneuploidy. In the euploid cases, the male:female sex ratio was 0.39:1, whereas in the aneuploid cases, the ratio was 1:1. The mean (+/-SD) maternal ages for numerical aneuploidy, structural aneuploidy, and euploidy were 33.0 +/- 5.1 years, 27.9 +/- 2.1 years, and 27.8 +/- 5.0 years, respectively. The frequencies of associated major structural anomalies other than craniofacial defects in the cases with numerical aneuploidy, structural aneuploidy, and euploidy were 85.7%, 0%, and 16%, respectively. The present study of HPE suggests that a female excess appears only in the euploid cases, and advanced maternal age and structural anomalies are more commonly associated with the numerically aneuploid cases than the structurally aneuploid and euploid cases.     

Flow cytometric detection of multifocal DNA aneuploid cell populations in mastectomy specimens containing a primary breast carcinoma

DNA flow cytometry was used to study the presence of DNA aneuploid cell populations in macroscopically normal glandular tissue in mastectomy specimens from 30 patients with breast cancer. In the 13 patients with a DNA diploid primary tumor, no DNA aneuploidy could be found in any of the 39 distant specimens assessed. However, DNA aneuploid cell populations were demonstrated in four of the 17 (23%) patients with a primary DNA aneuploid carcinoma and in seven out of 54 (13%) distant tissue samples (P = 0.02). In all cases the DNA index of the DNA aneuploid cells found in the distant samples was identical to that of the primary tumor. The replicate aneuploid DNA indices and histologic controls taken in parallel very strongly suggest that these distant DNA aneuploid cell populations are metastases.     

A practical graphical method for estimating the fraction of cells in S in DNA histograms from clinical tumor samples containing aneuploid cell populations

A graphical method for the analysis of unperturbed DNA histograms is presented in which the area of the normalized histogram subtended by the fraction of cells in S is represented by a trapezoid whose dimensions are dependent on features common to all such histograms. The technique takes measurement variability into account. This method was applied to a variety of synthetic DNA histograms. Overall, calculated values for the fraction of cells in S correlated well with actual values. This method was applied to 36 diploid cases of non-Hodgkin lymphoma; results correlated well with those obtained by a computer-based method. The results of the graphical-method were also highly reproducible between different observers. The graphical method can be used in the presence of aneuploid cell populations. Techniques for calculating S fractions in the presence of aneuploidy in clinical samples are described. These techniques were applied to synthetic histograms of mixed diploid and aneuploid populations. Calculated values correlated well with actual values.     

Flow cytometric analysis of DNA aneuploidy in primary and metastatic human solid tumors

DNA histograms were measured by flow cytometry for 656 human solid tumors (365 primary and 291 metastatic). The proportion of aneuploid cells in cell suspensions obtained by mechanical disaggregation was significantly higher than those obtained after enzymatic disaggregation (collagenase + DNAse) of the same tumor. A strong correlation was observed between the values of DNA-indices measured after staining with propidium iodide and with 4',-6-diamidino-2-phenylindole (r = 0.97). Aneuploid cells were observed in 430 tumors (66%); 30 of these had two aneuploid stemlines, and two had three aneuploid stemlines. The overall frequency of aneuploidy was 61% among primary and 71% among metastatic tumors. The median value of the DNA index was 1.67 for 224 primary aneuploid tumors and 1.68 for 206 metastatic aneuploid tumors. For most diseases, the largest proportion of aneuploid primary and metastatic tumors had DNA-indices in the hypertriploid region. No major differences in frequency and degree of aneuploidy was observed between primary and metastatic tumors. For carcinomas of the bladder and prostate, frequency of aneuploidy was higher among poorly differentiated, than among moderately and well-differentiated tumors. For carcinomas of the breast and for sarcomas, tumors with DNA-indices of greater than 2.0 were observed mostly in the poorly differentiated group. For patients with carcinomas of the bladder and prostate most tumors at earlier stages of disease were diploid; whereas most tumors at later stages of disease were aneuploid. For patients with carcinomas of the ovary, colon, and kidney, no relationship between stage of disease and aneuploidy was evident.     

Prognostic significance of DNA ploidy determined by high-resolution flow cytometry in breast carcinoma

OBJECTIVE: To assess the prognostic value of DNA ploidy in breast carcinoma and its relation to other established prognostic factors. STUDY DESIGN: We evaluated DNA ploidy in 303 breast carcinoma patients with a median follow-up of 63 months. Flow cytometry was performed on frozen tumor material, yielding histograms with narrow peaks (median coefficient of variation of 2.08). DNA ploidy pattern was classified as either diploid versus nondiploid, euploid (diploid and tetraploid) versus aneuploid or diploid/near-diploid (DNA index < 1.2) versus other, and correlated with relapse-free (RFS) and cancer-specific survival (CSS) along with tumor size, histologic grade and type, axillary lymph node involvement, menopausal and steroid receptor status, age and type of treatment. RESULTS: Seventy-one percent of tumors were DNA nondiploid (14% tetraploid and 57% aneuploid). There was a strong association between DNA ploidy and histologic grade. Histologic grade, lymph node status, tumor size and DNA ploidy (regardless of the classification used) were all significantly associated with RFS and CSS in multivariate analysis. CONCLUSION: These results suggest that DNA ploidy, at least when determined from frozen tumor tissue, is an independent prognostic factor in breast carcinoma; however, its prognostic power seems to be inferior to that of histologic grade, with which it strongly correlates.     

Comparison between slide and flow cytophotometric DNA measurements in breast tumors

Nuclear DNA analysis was performed in 37 human mammary adenocarcinomas in order to elucidate the difficulties and pitfalls connected with the interpretation of DNA histograms obtained using different methodologic approaches. For each tumor, DNA profiles were obtained by means of slide microspectrophotometry on a fine needle aspirate, slide cytophotometry on a 4-micron histologic section and flow cytometry on a suspension prepared from a cube of fresh tissue. When the DNA histograms were interpreted according to criteria usually applied to discriminate low-grade malignant tumors from high-grade malignant tumors, some tumors classified as euploid by one method were classified as aneuploid by another method. The main reasons for this weak correlation seem to be in specimen preparation and in tumor cell representation within the specimen between the methods. Another reason is that slide and flow techniques exhibit different sensitivities for malignancy-associated nuclear DNA changes: minor alterations of the DNA content of the tumor stemlines seem to be more exactly reported by means of the flow technique whereas structural alterations of the nuclear chromatin seem to be more sensitively recorded by means of the slide technique. It is suggested that thorough control of each step of the various DNA analysis procedures and the use of information obtainable by slide and flow techniques taken together may significantly improve the prognostic value of DNA measurements.     

Identification of Chromosomal Errors in Human Preimplantation Embryos with Oligonucleotide DNA Microarray

A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo) microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH) to evaluate accuracy of the results. We found that most (58.1%) of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s), partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal embryos.     

Intratumoral variations in DNA distribution patterns in mammary adenocarcinomas

Correlated flow-cytometric (FCM) and microspectrophotometric (MSP) techniques were applied to investigating whether intratumoral variations in the DNA distribution patterns of 21 primary mammary adenocarcinomas can occur. Although neoplastic cell populations with both diploid and tetraploid (i.e., euploid) distribution patterns could be found in varying proportions in some of the tumors, there was no evidence in any tumor nodule for the presence of euploid populations in one part and aneuploid populations in another. This statement was based on the results of the MSP technique, where the assessments were made on cytodiagnostically identified neoplastic cells. Also, when applying the FCM technique the statement was found to be essentially valid; only one of the tumor nodules showed a DNA distribution pattern that, by means of the criteria used in this procedure, was defined as being both euploid and aneuploid. Here, however, the technique consists of assessments made on a great number of microscopically non-identified cells. It was concluded that when conflicting reports are given from different laboratories on the prognostic value of the cytochemically assessed DNA distribution patterns in breast carcinomas, they are not likely to be attributed to intratumoral DNA heterogeneity but, rather, to differences in the methods used and in the criteria applied for the so-called ploidy assessments.     

Topology of chromosomes 18 and X in human blastomeres from 3- to 4-day-old embryos.

The positions of chromosomes 18 and X fluorescence in situ hybridization signals were analyzed in blastomeres generated from human in vitro fertilization 3- to 4-day-old embryos after preimplantation screening of aneuploidy of chromosomes 13, 16, 18, 21, 22, X, and Y. Fluorescent signal localization compared with a three-dimensional sphere model of random signal distribution revealed significant differences, providing evidence of peripheral localization of chromosome 18 in aneuploid (p=0.0013) and aneuploid/euploid blastomeres (p=0.0011). No differences were found in localization of chromosome 18 in euploid and in chromosome X in euploid and aneuploid blastomeres.     

Comparison of aneuploidy frequencies between in vitro matured and unstimulated cycles oocytes by metaphase comparative genomic hybridization (mCGH)

A common observation after in vitro matured oocyte is that they yield poorer embryo quality compared to their in vivo counterparts. This study was designed to assess chromosomal status with metaphase comparative genomic hybridization after in vitro maturation (IVM) in unstimulated cycles and compare the results with those obtained after in vivo maturation. Patients without any obstetrical or gynecological pathology were admitted into the study. IVM oocytes were collected 36 h post hCG and matured in vitro at 37°C in 5% O₂, 6% CO₂, and 89% air for 36 h. All matured (metaphase II) oocytes were subject to polar body 1 (PB-1) biopsy and vitrified individually. PB-1 samples were transferred into 0.25 cc PCR tubes containing 2.5 μl of PBS. PB-1 samples from 12 IVM patients were studied. Twenty-six out of 63 PB-1 samples (41%) were determined as euploid and 37 samples (59%) were aneuploid, whereas these values were 42% euploid and 58% aneuploid in the control group (in vivo matured oocytes). No statistical differences were found between the IVM and the control groups for euploid–aneuploid samples (P = 0.900). More aneuploidy was observed on chromosomes 11, 13, 15, 21, and 22 after IVM. Results show a non-significant rate of abnormal PB-1 formation after IVM compared to in vivo maturation. More aneuploidy was observed in chromosomes 11, 13, 15, 21, and 22 in the IVM group.     

DNA quantification of squamous cell carcinoma of the oesophagus by flow cytometry and cytophotometric image analysis using formalin fixed paraffin embedded tissue.

This is the first comparative study of DNA quantification of oesophageal squamous cell carcinoma by flow cytometry (FCM) and image cytometry (ICM) using formalin fixed paraffin embedded tissue. The potential advantages of ICM include the identification of a reliable control cell population; avoidance of non-tumour stromal and inflammatory cell nuclei, nuclear fragments, degenerate cell nuclei and doublets, triplets etc., which are not possible with FCM using archival tissue. Twenty-eight cases, all of the same stage (stage 2a) and similar grade (well or moderately differentiated) were analysed. The cases were separated into two groups, those that had succumbed to tumour in less than 18 months (group A) and those that were tumour free at least 18 months post-resection (group B). Using ICM all 28 tumours yielded interpretable histograms by comparison to 25 of 28 using FCM. Aneuploidy was identified in 100% of cases in group A using ICM (in comparison to 73% by FCM) and in 73% of group B using ICM (in comparison to 44% by FCM). Any tumour aneuploid by FCM was also aneuploid by ICM. Nine cases aneuploid by ICM were euploid by FCM. The mean 5C exceeding rate (% of cells whose nuclei contain a DNA mass equivalent to > 5 sets of 23 chromosomes) was 21% in group A and 14% in group B (P < 0.01). Euploidy was confined to tumours of those patients disease free for more than 18 months. The conclusions of this study are that: firstly, ICM is superior in its yield of interpretable histograms to FCM using formalin fixed paraffin embedded tissue; secondly, ICM is more sensitive in the identification of aneuploid stemlines than FCM; and thirdly, euploid tumours (as detected by ICM) appear to have a better prognosis than aneuploid tumours of similar stage and grade.     

Evidence for false aneuploid peaks in flow cytometric analysis of paraffin-embedded tissue

It has recently been shown that bimodal histograms with false aneuploid peaks may be obtained by DNA flow cytometry from histologically normal tissue allowed to autolyze. To investigate if such peaks can be generated from surgically excised archival tissue, 198 paraffin blocks from 179 patients containing histologically normal spleen (n = 65), liver (n = 26), thyroid (n = 32), pancreas (n = 19), salivary gland (n = 49), or lymph node tissue (n = 7), obtained from the archives of two university pathology departments, were analyzed for nuclear DNA content. The great majority (n = 160, 83.8%) of the 191 interpretable histograms had a single symmetrical G1 peak; and 8 histograms, all produced from liver tissue had a tetraploid pattern. A slight or a prominent repeatable deviation in the G1 peak outline was present in 14 (7.3%) cases. A peak resembling an aneuploid G1 peak with a DNA index (DI) ranging from 1.14 to 1.38 was repeatedly produced from 9 (4.7%) blocks containing histologically normal or inflamed splenic (n = 3), pancreatic (n = 3), liver (n = 1), thyroid (n = 1), or lymph node (n = 1) tissue. The three abnormal peaks produced from pancreatic tissue were rounded in shape and resembled closely the ones that can be obtained from autolytic pancreatic tissue, and the six remaining extra peaks were all fused with the "diploid" peak. In conclusion, false peaks, probably caused by degradation of the nuclear contents during formalin fixation or before it, may rarely be obtained from surgical paraffin-embedded samples.     

Flow cytometric bivariate analysis of DNA and cytokeratin in colorectal cancer.

Different opinions about flow cytometric estimates of DNA aneuploidy and/or S-phase fraction (SPF) as supplementary prognostic markers in colorectal cancer are to some degree associated with methodology. Using univariate DNA analysis, we have previously investigated the DNA ploidy in colorectal cancer, its heterogeneity within and between tumors and its relation to survival. To improve detection of DNA aneuploid subpopulations and particularly estimation of their SPF's we investigated a method for bivariate DNA/cytokeratin analysis on fine-needle aspirates of 728 frozen biopsies from 157 colorectal tumors. Unfixed aspirates were stained with propidium iodide and FITC-conjugated anti-cytokeratin antibody in a saponin-buffer. A significant association between SPF and debris was observed. There were no substantial difference in DNA ploidy patterns between univariate and bivariate measurements (concordance was 92-95%). No new DNA aneuploid subpopulations were detected in cytokeratin-gated compared to ungated or univariate histograms. Debris-adjusted SPF's of cytokeratin-gated histograms were significantly higher than of ungated histograms, also for subpopulations with DI>1.4 (p<0.0001). There was no significant association between SPF and survival.     

Bone metastases: prognostic applications of cytometric DNA analysis

OBJECTIVE: To examine DNA parameters as prognostic factors for developing metastases. STUDY DESIGN: Image cytometry was used to determine DNA content of 21 tumors and 28 metastases. DNA ploidy status, 2c deviation index (2cDI) and DNA malignancy grade (DNA-MG) (based on the variation of nuclear DNA content of tumor cells around the normal DNA [2c] peak) were examined for their prognostic value. RESULTS: Twenty of 21 tumors showed aneuploid content, and 1 tumor showed diploid DNA content. Twenty-one bone metastases showed aneuploid cells. In 6 cases both euploid and aneuploid cells were detected. In 1 metastasis only euploid cells were present. DNA-MG was increased in bone metastases (mean, 2.4) as compared to the corresponding primary tumor (mean, 2.2) in most of the cases. The mean value of the 2cDI was 30.07 in primary tumors and 42.5 in metastases. Twelve bone metastases had a higher 5cEE than did the primary tumor. CONCLUSION: Diploid and aneuploid cells were able to leave a tumor and establish metastases. DNA-MG and 2cDI were increased in metastases in comparison with the primary tumor, but even tumors with lower DNA-MG had metastatic potential.     

DNA ploidy pattern and amplification of ERBB and ERBB2 genes in human gastric carcinomas

The DNA ploidy pattern and amplification of ERBB and ERBB2 genes were examined in paraffin-embedded tissue from gastric carcinomas using flow cytometry and a slot-blot hybridization technique. The incidence of aneuploidy in well differentiated adenocarcinomas (56%) was significantly higher (p less than 0.05) than that in poorly differentiated adenocarcinomas (21%). The DNA ploidy pattern was not remarkably different between the primary tumors and metastatic deposits in lymph nodes. Of the nine specimens having an aneuploid stem cell line in the primary tumor and/or in metastases, three showed ERBB2 gene amplification and one showed ERBB gene amplification. The incidence of epidermal growth factor (EGF) immunoreactivity in tumor cells showed no difference between diploid and aneuploid tumors. These findings indicate that aneuploidy is frequently associated with amplification of ERBB and ERBB2 genes.