DNA ploidy in early gastric carcinoma (T1): a flow cytometric study of 100 European cases

DNA ploidy of 100 early gastric carcinomas (T1) was analysed by flow cytometry on archival material from five European centres and was correlated to morphological features and clinical behaviour. Tumours were classified according to the macroscopic appearance, histological type, and growth pattern. Aneuploidy was observed in 39% of tumours. Aneuploidy was more frequent in submucosal than in mucosal tumours (p = 0.04), in raised than in flat or ulcerated lesions (p = 0.001), and in the intestinal histological than in the diffuse types (p = 0.016). The presence of lymph node metastasis in 10 cases had no obvious relation to DNA ploidy. Five related deaths occurred during the follow up (6 months--16 years) of 84 patients. These results are similar to those reported in a large Japanese series suggesting no major differences between the two populations. Although follow up data were insufficient to relate DNA ploidy to tumour behaviour in this study, the Japanese experience shows that particular attention should be paid to early direction and complete surgical excision of raised intestinal type T1 carcinomas that have a Pen A growth pattern and are aneuploid.     

Intratumoral heterogeneity in primary breast carcinoma: study of concurrent parameters

BACKGROUND AND OBJECTIVE: Intratumoral heterogeneity for prognostic factors (ploidy, proliferation, hormone receptor positivity) has been demonstrated in primary breast carcinoma by both flow cytometric and image analysis methods. Previously, heterogeneity in tumors had been demonstrated for only singular parameters. Our objective, using maps of tumors in which discrete regions can be analyzed simultaneously for DNA index (DI) and proliferative activity, was to demonstrate heterogeneity with respect to two parameters and to determine whether any interparametric relationships existed. METHODS: We analyzed 25 cases of archived, paraffin-embedded breast carcinoma (ductal) for Feulgen stain DNA analysis and MIB-1 immunohistochemistry using the CAS 200 Image Cytometer. For each tumor, four discrete regions were analyzed including tumor-host tissue interface sectors. RESULTS: Of 25 cases, 19 (76%) were homogeneously diploid or near-diploid aneuploid, and 6 (24%) were heterogeneous. Within the heterogeneous group, all cases had at least one diploid and one or more aneuploid populations from separate discrete regions. Five of six DI heterogeneous tumors displayed diploid values for the overall measurements of the respective tumors, based on analysis of 200 or more nuclei. Eight of 25 cases (32%) showed significant measurable variation for MIB-1 proliferative activity in various sectors of tumor. All the MIB-1 heterogeneous tumors, with one exception, were homogeneously diploid. CONCLUSIONS: These findings demonstrate that (1) heterogeneity is present with respect to DI and proliferative activity in breast carcinoma and is relatively common, (2) tumors homogeneous for one parameter may be heterogeneous for another, and (3) heterogeneity for proliferative activity is more common in homogeneously diploid tumors than in heterogeneous/aneuploid tumors.     

Heterogeneity in early and advanced gastric carcinomas by flow cytometric DNA analysis

We investigated 230 systematically sampled fresh specimens from 12 early and 26 advanced gastric cancer patients by DNA flow cytometry for heterogeneity in DNA content. Fifty-eight percent of the 12 early gastric cancers were uniformly diploid and 42% were uniformly aneuploid. Fifty-four percent of advanced cancers were uniformly diploid in superficial layers and 42% were uniformly diploid in deep layers, whereas 46% were uniformly aneuploid in superficial layers, and 50% were uniformly aneuploid and 8% were heterogeneously aneuploid and diploid in deep layers. Both diploid and aneuploid samples were obtained from 15% for advanced cancers, but ploidy heterogeneity did not occur in early cancers. Heterogeneity for DNA index (more than one aneuploid DNA index) occurred in 46% of whole thickness of advanced cancers, in 19% of superficial layers of advanced cancers, and in 8% of early cancers. We concluded that DNA ploidy determination using superficial layer specimens may be reliable in early gastric cancer but must be interpreted with care in advanced cancer.     

Flow cytometric analysis of DNA ploidy in colorectal adenoma

Flow cytometric measurement of nuclear DNA content in 159 colorectal adenomas was carried out to investigate the relationship between DNA ploidy and the histological findings. DNA aneuploidy was detected in 18 lesions (12.8%). The incidence of DNA aneuploidy was significantly higher in tubulovillous adenomas than in tubular adenomas (30.4% vs. 8.1%; p < 0.01). DNA aneuploidy was not found in any adenoma with mild dysplasia, but was noted in 19.1% of those with moderate dysplasia and in 33.3% of those with severe dysplasia. The mean size of the lesions was significantly larger in adenomas with aneuploidy than in those without aneuploidy (14.0 mm vs. 7.7 mm; p < 0.01). The DNA index values of 18 adenomas with aneuploidy were divided into two groups: one ranged from 1.07 to 1.23 and the other from 1.66 to 1.85. DNA index values correlated with the size of the lesions (p < 0.05), but not with the histologic type and degree of dysplasia.     

Comparative image cytometric DNA ploidy of liver cell dysplasia and hepatocellular carcinoma

Large-cell liver cell dysplasia (LCD), suggested to be a preneoplastic change that progresses to hepatocellular carcinoma (HCC), has a reported frequency of DNA aneuploidy by flow cytometry intermediate between that of nonneoplastic liver (0%) and HCC (80%). We assessed DNA ploidy by image cytometry of Feulgen-stained 5-microns sections of 30 livers with LCD and of 60 HCCs (29 with LCD in adjacent nonmalignant liver). All 30 LCDs were aneuploid, 27 (90%) of which were multiploid--11 (41%) with hyperdiploid and hypertetraploid peaks. Forty-eight (80%) HCCs were aneuploid; in nine of 20 (42%) with a hyperdiploid peak, a hyperdiploid peak was also present in the LCDs, but in none was there less than 0.24 between DNA indices. Besides the 12 (20%) diploid HCCs, a diploid peak was present in four heterogenous, three multiploid, and six HCCs with two phenotypes and two genotypes, one of which was diploid. One aneuploid/hyperdiploid peak in each of 22 nonneoplastic and 24 cirrhotic livers did not have a corresponding LCD or HCC aneuploid peak. These data do not suggest that dysplastic hepatocytes form a single mutant clone that progresses to HCC.     

Flow cytometric and quantitative image cell analysis of DNA ploidy in renal chromophobe cell carcinoma

Flow cytometry and quantitative image cell analyses were performed on a series of 31 chromophobe cell carcinoma and the findings were compared with those from 14 clear cell carcinomas. Thirty of 31 chromophobe cell carcinomas had significant hypodiploid cell clones with both techniques. By contrast, none of the 14 clear cell carcinomas was hypodiploid. Using quantitative image cell analyses, four groups of nuclei with hypodiploid, diploid, hyperdiploid, and tetraploid/hypertetraploid DNA patterns were identified, and their relative proportions were compared. In most of the chromophobe cell carcinomas, the predominant nuclear pattern was hypodiploid, and in clear cell carcinoma, diploid nuclei were most frequent. The number of binucleated cells in chromophobe cell carcinomas varied from 1.40% to 23% (mean, 10.8%) whereas, in clear cell carcinoma, these varied from 0.4% to 9.2% (mean, 2.5%). Evaluation of DNA content of double hypodiploid nuclei in chromophobe cell carcinomas showed that their combined DNA content was essentially similar to that of single hyperdiploid nuclei, thus suggesting polyploidy resulting from the fusion of these nuclei. Polyploidy may indeed be the basis for nuclear heterogeneity in chromophobe cell carcinoma. Scatterplots generated by plotting nuclear DNA mass against nuclear area produced patterns that were distinctive for the two types of carcinoma. We believe that the comparative findings in this study provide a comprehensive understanding of the ploidy status of chromophobe carcinoma and that these findings may be used as supportive evidence for establishing the diagnosis of chromophobe cell carcinoma.     

Detection of false DNA aneuploidy and false DNA multiploidy in flow cytometric DNA analysis

Although false DNA aneuploid peaks have previously been described in normal tissue, criteria for distinguishing them from 'true' near-diploid peaks have not been established. Normal thyroid (n = 4) and kidney (n = 1) tissue were allowed to autolyze over a fixed period of time and DNA content was analyzed by flow cytometry (FCM). Autolysis was associated with the development of distinct separate G0/G1 peaks which had low DNA indices (1.09-1.18) and showed decreased forward light scatter (FSC) when compared to fresh tissue. Using DNA content and FSC measurements similar false DNA aneuploid peaks were identified in 29/94 surgical specimens. These cases included both benign and malignant lesions from thyroid (n = 63) with the remaining 31 neoplastic cases being from breast (16), lymphoma (8), sarcoma (4), lung (2) and uterine (1) tissue. In addition, false DNA multiploidy was identified. None of these cases showed histological evidence of necrosis. In a parallel comparison study using image cytometry (ICM) on the thyroid nodules, the presence of false DNA aneuploidy was supported. Investigators should routinely employ quality control criteria to identify possible cases of false DNA aneuploidy when measuring DNA content using FCM.     

Clonal heterogeneity in human esophageal squamous cell carcinomas on DNA analysis

Cancers are thought to arise through multistep accumulation of somatic mutations in the progeny of a single cell. Multiple mutations may induce molecular intratumor heterogeneity. Therefore, we examined molecular clonal heterogeneity in esophageal squamous cell carcinomas. Twenty-four esophageal squamous cell carcinomas and associated lymph node metastases were examined for microsatellite alterations, and abnormalities of the p53 and transforming growth factor-beta type II receptor (TGF-beta RII) genes. There were eight cases (33%) showing different patterns of loss of heterozygosity in primary tumors and metastatic lymph nodes with microsatellite markers. On the other hand, the abnormalities of p53 were identical in all these cases. No mutation was detected in the simple repeated sequences of the TGF-beta RII gene. These results indicate that molecular clonal heterogeneity exists in esophageal squamous cell carcinomas. Therefore, care is necessary in preoperative genetic diagnosis using biopsy samples.     

DNA ploidy analysis in breast carcinoma. Comparison of unfixed and fixed tissue analyzed by image and flow cytometry

We present here the isolation and characterization of four antimicrobial peptides produced by a European bumblebee Bombus pascuorum. A 51-residue insect defensin was characterized which, like the Apis mellifera defensins, had a highly conserved 12-residue extension to its C-terminal compared to defensins from other insects. Monoisotopic mass analysis of the C-terminal of B. pascuorum defensin confirmed that this molecule was C-terminally amidated. This defensin showed strong anti-Gram-positive activity and some anti-fungal activity; also, in contrast to other insect defensins, it showed anti-Gram-negative activity. A 17-residue apidaecin was characterized, showing anti-Gram-negative activity, and differing by a single amino acid substitution from the A. mellifera apidaecin. A 39-residue abaecin was isolated, the largest proline-rich antimicrobial peptide characterized to date, which showed activity against both Gram-negative and Gram-positive bacteria. Finally, we isolated an N-terminally blocked molecule, with a molecular mass of 10,122 Da, which showed activity against Gram-negative bacteria only. These characteristics are reminiscent of hymenoptaecin from the honeybee A. mellifera, but a definitive characterization of this molecule awaits further work. No evidence of lysozyme activity was found in the haemolymph of challenged or naive B. pascuorum.     

Autoradiographic assessment of blood flow heterogeneity in the hamster heart

OBJECTIVE: Provide regional flow measurement in the hearts of small mammals using a new, higher-resolution technique based on the deposition of a molecular marker. METHODS: We determined the instantaneous extraction and retention of the "molecular microsphere" radiolabeled desmethylimipramine in retrogradely perfused hamster hearts. In a separate series of experiments, autoradiography was used to measure regional myocardial deposition densities in hamster hearts of about 0.5 g with spatial area resolution of 16 x 16 microns. RESULTS: Radiolabeled desmethylimipramine is almost 100% extracted during a single transcapillary passage and is retained in the tissue for many minutes. Autoradiographic images demonstrated a spatial flow heterogeneity with standard deviations of 31 +/- 4% of the mean flow (N = 5) in 16 x 16 x 20-micronm3 voxels. This is equivalent to the projections made using fractal relationships from cruder observations obtained with microspheres in the hearts of baboons, sheep, and rabbits. CONCLUSIONS: Autoradiography using a molecular deposition marker provides quantitative information on myocardial flow heterogeneities with resolution at the size of cardiac myocytes. Because the regions resolved are smaller than the volume of regions supplied by single arterioles, the results must slightly exaggerate the true heterogeneity of regional flows.     

Prognostic significance of DNA flow cytometric analysis in patients with nasopharyngeal carcinoma

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a prevalent malignant tumor among Southern Chinese. Previously, the authors described the prognostic significance of a serum antibody assay to a recombinant Epstein-Barr virus Bam HI-Z replication activator protein (ZEBRA) in NPC patients with long term follow-up. In this study, the authors further reported the use of DNA flow cytometry (DNA-FCM) as an additional technique for determining the prognosis of NPC patients in the same series. METHODS: One hundred and forty-three archival biopsies from 110 NPC patients were deparaffinized and subjected to DNA-FCM analysis. DNA ploidy state and various proliferative indices (PI) of the tumors were correlated with patient survival and frequency of recurrence. RESULTS: Among the biopsies analyzed, 119 were histologically positive NPC and 24 were negative. Fifty-one tumor biopsies that fulfilled the guideline criteria of the DNA Cytometry Consensus Conference were correlated with the clinical manifestations of the patients. Among them, 43 tumors (84%) were DNA diploid and 8 (16%) were aneuploid. Two PI, S-phase fraction (SPF) and proliferation fraction (PF), appear to be potentially useful prognostic indicators. For example, PF in patients who developed locoregional recurrence (15.1%) and distant recurrence (16.4%) after radiation therapy both were significantly higher than PF in patients who were in complete remission (8.2%) (P = 0.0005 and P = 0.004, respectively). Significant differences in SPF between patients with distant recurrence (10.6%) and those in remission (5.7%) also was found (P = 0.005). Using Kaplan-Meier analysis, patients with high PF, high SPF, and aneuploid tumors had significantly poorer 12-year survival rates (35%, 26%, and 28%, respectively) than those patients with low PF, low SPF, and diploid tumors (77%, 67%, and 59%, respectively) (P < 0.0009, P < 0.004, and P < 0.01, respectively). CONCLUSIONS: Determination of tumor PI and DNA ploidy state by DNA-FCM at diagnosis of NPC can be potentially useful in selecting a poor prognostic subgroup of NPC patients. These parameters may enable oncologists to plan for more stringent treatment strategies such as hyperfractionated and accelerated radiation therapy or concomitant chemoradiotherapy for these patients.     

A comparison of cytogenetic studies and flow cytometry in breast carcinomas

The increased use of DNA-based typing techniques has improved the accuracy and reliability of HLA types assigned to patients requiring an allogeneic bone marrow transplant and their potential donors, facilitating better donor selection. The benefits of this technology, which will completely replace serologic typing within the next few years, have not yet been fully exploited since the typing information obtained in the form of DNA sequence polymorphisms is presently converted to an HLA allelic type for submission to a registry. Furthermore, within the registry, the allelic type may be further converted to a serologic antigen for use in the search and match process. Because the current nomenclature system makes selection of donors who are likely to provide optimal matches difficult, it is critical that DNA-based HLA typing data be recorded by registries as sequence polymorphisms tested as positive or negative rather than interpreted HLA types and that searching and matching utilize these polymorphisms. Until this transition is complete, we will not fully utilize this powerful HLA typing technology to best serve those patients seeking unrelated donors.     

DNA flow cytometric analysis in patients with operable non-small cell lung carcinoma

Fresh surgical specimens of tumors from 60 patients with previously untreated non-small cell lung carcinoma (NSCLC) who underwent radical surgery between January 1991 and October 1992 were investigated by means of flow-cytometry. The nuclear DNA measurement was carried out using a Facscan (Becton, Dickinson, USA). Analysis of the DNA content was performed in all 60 patients whilst cell cycle analysis was possible in 41 cases (68.3%). Forty-two of the 60 cases (70%) were aneuploid and 18 (30%) were diploid. The overall mean value of DNA index was 1.5. Diploid NSCLC were compared with aneuploid tumors: no significant differences in age distribution, sex ratio, histology and staging were found between the two groups (P > 0.05). An S-phase proportion of more than 10% was found in 30 out of 41 patients (73.2%). Early cancer deaths were reported in four patients (6.6%): the aneuploidy rate was very close in these patients (75%) and in the remaining surviving patients (69.6%). An S-phase proportion of more than 10% was found in 100% of early cancer deaths and in 70.2% of the remaining cases; such a difference seems of some importance although it was not statistically significant (P = 0.071). In conclusion, flow-cytometry studies seem to be a useful tool in the understanding of the biological behavior of patients with NSCLC. In the present prospective report there were no significant correlations between DNA measurements and clinical outcome, however, these results suggest that a high S-phase proportion should be seen as a possible prognostic indicator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric DNA measurements in aspiration biopsies and surgical specimens of breast cancer

OBJECTIVE: One of the prognostic factors in breast cancer is the proliferation activity of the tumor. This study sought knowledge of this activity before surgery to benefit the design and timing of therapy. STUDY DESIGN: Flow cytometric DNA analysis data from 52 diagnostic fine needle aspirates were compared with data from subsequent surgical specimens. RESULTS: The data showed that the coefficient of variation of the G1 peak was lower in the aspirates. Small, near-diploid peaks were detected more frequently in aspirate histograms than in surgical specimens. DNA analyses by flow cytometry from aspirates, which can be obtained prior to surgical treatment, were as reliable as those obtained from surgical specimens, provided that the cellular material was diagnostic of cancer. CONCLUSION: Our results suggest that flow cytometry DNA analysis from the first preoperative cytologic specimen from a breast tumor will permit faster planning and coordination of breast cancer care.     

Flow cytometric DNA analysis of invasive carcinomas detected by screening mammography: use of specimen mammography-guided fine-needle aspirates

Clinical studies of flow cytometric DNA analysis of breast carcinoma are often limited by the lack of fresh tissue samples from smaller, nonpalpable carcinomas. In addition, most studies measuring DNA in the current literature focus on larger palpable masses that may have less relevance to the smaller, nonpalpable lesions. A prospective study of flow cytometric DNA analysis of in vitro specimen mammography-guided fine-needle aspirates (FNAs) of 103 consecutive nonpalpable invasive carcinomas detected by screening mammography was performed to determine efficacy and explore associations with mammographic and pathological features. For 62 (60%) lesions for which DNA analysis on both FNA and standard tissue incision samples was performed, there was excellent (89%) agreement for ploidy determinations (kappa=0.77) and poor agreement for S-phase percentage determinations (kappa=0.23). Specimen mammography-guided FNA analysis detected aneuploidy in 36% of lesions overall, including 34% of 41 lesions for which standard tissue procurement was not possible. Mammographic microcalcifications had a higher aneuploid rate (14 of 28 lesions, 50%) as compared with soft tissue masses (22 of 75 lesions, 29%), P < 0.01. Lobulated masses with indistinct margins had a higher aneuploid rate (5 of 6 lesions, 83%) as compared with more irregular, spiculated masses (7 of 27 lesions, 26%), P < 0.01. The aneuploidy rate was independent of specific histological diagnosis, lesion size, nuclear grade, or nodal or estrogen receptor status. Flow cytometric DNA analysis of mammographic lesion-specific, fresh, cellular FNA samples obtained under specimen mammographic guidance can assess early invasive carcinomas when gross fresh tissue procurement is not possible. This technique could be incorporated into larger clinical follow-up studies to determine the prognostic significance of flow cytometric DNA analysis for these very early breast carcinomas.     

Prognostic value of S-phase fraction in lymph-node-negative breast cancer by image and flow cytometric analysis

A two-year multicentre prospective study was performed from 1992 to 1995 in order to evaluate the real value of various kinds of coral blocks as bone substitute in maxillofacial surgery. This study was supported by the French National Agency for Research Valorization (GBM/TEP procedure). Ten Maxillofacial Surgery Units were included. During this time, 28 coral blocks (23 patients) of two different shapes were used as malar implants for correction of congenital or acquired zygomatic hypoplasia. The mean follow-up was 1.8 year (min: 1.5; max: 2). The tolerance was perfect for 89% of cases. The radiologic opacity never decreased more than 30% and the volume augmentation was always stable at the end of the follow-up period. Three implants were removed because of septic complications. Rigid fixation between the implant and the zygomatic bone appears to be the most important factor of success. On the other hand, the surgical approach (endo- or exo-buccal) does not seem to influence the success rate. The aesthetic improvement was always evaluated as satisfactory and stable by the patients and the surgeons. The authors discuss the real value of the various kinds of biomaterials and especially coral, comparing their personal data with those of the literature. Coral blocks clearly constitute a safe and reliable bone substitute, but further investigations are required to determine its long-term behavior.     

Flow cytometric DNA analysis of breast cancers with predominance of carcinoma in situ: a comparison of the premalignant and malignant components

Flow cytometric DNA analysis was performed on unfixed frozen tissue samples from 48 cases of invasive breast cancer (IC) with a predominance of ductal carcinoma in situ (DCIS). In 15 cases the samples contained only the DCIS component, in 17 cases only the IC component, whereas in 16 cases separate samples from the DCIS as well as the IC part within the individual lesion were available. In the latter 16 cases, complete or partial accordance in DNA ploidy between DCIS and IC was found in 12 cases, whereas no correspondence could be demonstrated in the remaining 4 cases, possibly due to intratumoral DNA heterogeneity. Comparison of the DNA index distribution in samples of DCIS and IC from the 48 cases showed concordant results except for the DNA hyperdiploid subclass, in which 6 clones were found in the DCIS portion compared to 18 clones in the IC portion. S-phase fractions were also comparable in the two groups. A comparison of the DCIS component from the present series of breast cancers to our previous series of pure DCIS also showed similar results with respect to the DNA index distribution, DNA heterogeneity, and S-phase fraction. No differences could be demonstrated between DCIS with and without invasion. The results indicate that the DNA ploidy pattern of breast cancer, as detected by flow cytometric DNA analysis, is established at the preinvasive stage of carcinogenesis.     

Multiparameter flow cytometric DNA analysis of effusions: a prospective study of 36 cases compared with routine cytology and immunohistochemistry

Single-parameter flow cytometry (SFCM) is limited in its ability to detect aneuploid and diploid malignant cells or accurately estimate S-phase fractions (SPF) in effusions because of the high degree of contamination by benign mesothelial cells and inflammatory cells. We examined 36 pleural and peritoneal fluids by conventional cytology and multiparameter FCM (MFCM) to analyze the DNA content of cells expressing epithelial markers cytokeratin, epithelial membrane antigen, carcinoembryonic antigen, BRST-1, or BRST-3 (B72.3) and compared the results to those found with SCFM. The cases were also studied by immunohistochemistry using the same antibody panel. By routine cytology, 14 of the 36 cases were classified as carcinomas, 11 as reactive, 1 as mesothelioma, and 10 as suspicious. MFCM allowed reclassification of 5 of the 10 suspicious cases as carcinomas and the remaining 5 as reactive cases based on ploidy and marker expression. Whereas SFCM detected only 13 nondiploid carcinomas, MFCM detected 4 diploid and 15 nondiploid carcinomas. All reactive cases were diploid by SFCM or MFCM. The mesothelioma case showed were distinct peaks by MFCM, a diploid peak with SPF of 13.4% and a tetraploid peak with SPF of 36.1%. The SPF of the nondiploid carcinomas ranged from 5.9 to 50.4% and diploid carcinomas, from 3.5 to 14.5% when gated on epithelial cells. The reactive cases had SPF ranging from 0.4 to 4.4%.(ABSTRACT TRUNCATED AT 250 WORDS)