Flow cytometric analysis of DNA and nuclear protein in paraffin-embedded tissue

Human insulin-like growth factor I (IGF1) was labeled with 125I and the resulting mixture of iodination isomers was separated by reverse-phase HPLC. Three major radioactive peaks were isolated and identified by sequencing as the expected three monoiodinated species. The ranking of the affinities of the three isomers for the human IGF1 receptor was found to be Tyr24(125I) > Tyr31(125I) > Tyr60(125I). The Tyr31(125I) isomer was shown to have an affinity similar to that of unlabeled IGF1 and is thus the tracer of choice for IGF1. The tracers were stable upon storage at -20 degrees C for at least 3 months.     

Prognostic value of genomic damage in non-small-cell lung cancer

Genomic alterations have been analysed in 65 non-small-cell lung cancer (NSCLC) tissue samples by using the arbitrarily primed polymerase chain reaction (AP-PCR), which is a PCR-based genomic fingerprinting. We have shown that AP-PCR may be applied as a useful and feasible practical method for detection of the genomic alterations that accompany malignancy in NSCLC. Genomic changes detected by us consisted of: allelic losses or gains in anonymous DNA sequences, homozygously deleted DNA sequences and polymorphic DNA sequences. According to these genomic changes, lung tumours evaluated in the present study have been scored into three groups: low, moderate and high genomic damage tumours. The aim of this study was to investigate the effect of genomic damage on patient survival. Survival analysis was carried out in 51 NSCLC patients. Our results revealed that high genomic damage patients showed a poorer prognosis than those with low or moderate genomic damage (P = 0.038). Multivariate Cox regression analysis showed that patients with higher genomic alterations displayed an adjusted-by-stage risk ratio 4.26 times higher than the remaining patients (95% CI = 1.03-17.54). We can conclude that genomic damage has an independent prognostic value of poor clinical evolution in NSCLC.     

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.     

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.     

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)     

Image cytometric nuclear DNA quantitation of paragangliomas in tissue sections. Prognostic significance

BACKGROUND: It has been reported that dual therapy with high doses of omeprazole and amoxycillin proves efficient for Helicobacter pylori eradication. AIM: To compare the efficacy, safety and tolerability of eradicating regimens with omeprazole/amoxycillin. METHODS: In this randomized multicentre study, 267 duodenal ulcer patients were treated for 2 weeks with omeprazole 40 bid (Group A) or 20 mg bid (Group B), respectively, and with amoxycillin 0.5 g. qid followed by 4 weeks of 20 mg omeprazole om. Helicobacter pylori status was assessed by both histology and urease test in the antrum and the corpus. The patients were then followed-up for 9 months. RESULTS: Helicobacter pylori infection was cured in 62.9% of group A (95% CI: 53.8-71.4) and in 44.8% of group B (95% CI: 35.6-54.3; p = 0.007). Healing was achieved in 91.9% of patients in group A (95% CI:85.7-96.1), and in 87.9% of patients in group B (95% CI:80.6-93.2). The estimated probability of being in ulcer remission for cured patients was 0.95 (95% CI: 0.90-0.99) and for the not cured was 0.41 (95% CI: 0.24-0.59; p = 0.0001). However, between the two treatment groups no significant differences in symptom relief or ulcer recurrence were observed. Both regimens were well tolerated with minor side-effects occurring likewise within the two groups. At two months in cured patients antral histology revealed a total (group A + B) prevalence of 13.7% of active chronic gastritis. CONCLUSIONS: This long-term, large-size study clearly indicates that dual therapy does not represent a truly effective eradication therapy and this regime cannot be recommended.     

Diagnostic value of p53 protein and flow cytometric DNA analysis in the study of serous effusions

OBJECTIVE: To investigate the diagnostic value of p53 protein and DNA analysis in the study of serous effusions. STUDY DESIGN: A total of 76 samples of serous effusions were studied by immunohistochemistry for p53 protein and flow cytometric (FCM) DNA analysis. The results were correlated with final cytologic diagnoses, which were confirmed by immunohistochemistry using antibodies against cytokeratin, carcinoembryonic antigen, epithelial membrane antigen and fibronectin. RESULTS: Final cytologic diagnoses included 28 malignant effusions and 48 benign effusions. No expression of p53 protein was seen in benign effusions. In contrast, p53 protein expression was seen in 19/28 (sensitivity 68%) malignant effusions. FCM detected aneuploid cells in 12/28 (43% sensitivity) of malignant and 0/46 of benign effusions. Immunohistochemical determination of p53 protein combined with FCM DNA analysis increased sensitivity to 79%. CONCLUSION: Immunohistochemical determination of p53 protein and FCM DNA analysis can aid in making an accurate and specific diagnosis of serous effusions, but the principal limitation of these tests is their relatively low sensitivity.     

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.     

Detection of p53 gene mutation in paraffin-embedded asbestos-related lung cancer tissue

The rearrangement of immunoglobulin heavy chain gene (IgH) and T cell receptor gamma gene (TcR gamma) was studied in 30 patients with acute lymphoblastic leukemia (ALL) by the polymerase chain reaction (PCR). 19 cases was found to have rearrangement of IgH gene, 12 of TcR gamma. Most of IgH rearrangement was characterized by one or two specific bands while some had more than two. Rearrangement of TcR gamma gene appeared as one specific band. A slight difference in number, size and lightness of bands was found among the patients. 4 different kinds of rearrangement were observed in the detection of IgH rearrangement in combination with TcR gamma gene. The rearranged patterns of IgH and TcR gamma gene as well as the clinical significance were discussed.     

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.     

Rapid preparation of tissue DNA from paraffin-embedded blocks and analysis by polymerase chain reaction

We have developed a new, easy, and more rapid method for DNA preparation, which avoids contamination. With this method, manual surgical blade scrapings from precisely targeted areas of paraffin block surfaces, without microtome cutting, were used to obtain tissues from 10 different neoplasms. Our results indicate the feasibility of DNA extraction from the scraped paraffin tissue for molecular genetic analysis. We applied this technique successfully to screen for the presence of human papillomavirus using the polymerase chain reaction (PCR) procedure in cases of endocervical, esophageal, and nasopharyngeal carcinomas, and to examine the expression of p53 gene from prostate and gastric adenocarcinomas. We conclude that this procedure is also suitable for purification of PCR products in analysis of the mutation or loss of allelic genes by Bstu I endonuclease digestion.     

骨转换标志物在非小细胞肺癌骨转移临床应用价值的研究

Objective:The purpose of this study was to assess the clinical application value of bone turnover markers in non-small-cell lung cancer (NSCLC) patients with bone metastases. Including diagnosing bone metastases, detecting bone metastatic spread. Methods: Alkaline phosphatase (AKP), p-C-terminal telopeptide of type I collagen (B-CTx), osteocalcin (OST) and bone alkaline phosphatase (BALP) were measured in 76 patients with bone metastases from NSCLC and 44 normal people. Results: The level of AKP, B-CTx and BALP in patients with bone metastasis was significantly higher than in the normal people. Significant correlation was observed among bone turnover markers. The levels of BALP and OST were significantly correlated with the extent of bone metastasis. The patients with high-level CTx and low-level BALP had higher risk of pathologic fracture. Conclusion: In NSCLC patients with bone metastases, bone turnover markers can help to make diagnosis and evaluate the severity. It will have a wide range of use in clinical practice.     

Prognostic parameters of renal cell carcinoma. Clinicopathologic and DNA image cytometric analysis in 133 pT3a cases

In 133 cases of patients with renal cell carcinoma which infiltrated locally into the renal fatty tissue (stage pT3a, TNM classification of 1987), the prognostic potential of the following parameters was investigated: symptoms, patient's age at the time of operation, tumor size and localization, grade of malignancy, cell type, growth pattern, and prognostic score according to St?rkel et al. [Eur Urol 1990;18(suppl 2):36]. Additionally, automated image analysis DNA cytometry was performed on 110/133 carcinomas. After an average observation period of 3.6 years (maximum 10.1 years), 59 (44.4%) of the patients died of their tumors. The cause-specific 5- and 10-year survival rates were 51.3 and 29.1%, respectively. Statistically significant differences (p < 0.05) within the individual parameters were found only for the grade of malignancy and the corresponding prognostic score. Using DNA cytometry, 13 types of renal cell carcinoma were differentiated; 90% of the tumors contained aneuploid cells. Significant differences between these relative to prognosis did not exist. In the case of locally advanced renal cell carcinoma, the DNA histogram does not seem to be superior to conventional prognostic criteria.     

Flow cytometric analysis of variations in the DNA content of superficial and deep layers in colorectal cancer

Experiments on s.c. rat tumours (DS sarcoma) were performed to determine whether chronic or acute changes in tumour perfusion necessarily lead to changes in tissue oxygenation and bioenergetic status since, as a rule, blood flow is thought to be the ultimate determinant of the tumour bioenergetic status. Based on this study, there is clear experimental evidence that growth-related or acute (following i.v. administration of tumour necrosis factor alpha) decreases in tumour blood flow are accompanied by parallel alterations in tissue oxygenation. In contrast, tumour energy status remains stable as long as flow values do not fall below 0.4-0.5 ml g-1 min-1, and provided that glucose as the main substrate can be recruited from the enlarged interstitial compartment. Perfusion rate seems to play a paramount role in determining energy status only in low-flow tumours or low-flow tissue areas.     

Carcinoembryonic antigen, tissue polypeptide antigen and neuron-specific enolase pleural levels used to classify small-cell and non-small-cell lung cancer patients by discriminant analysis

The classification of lung cancer into small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) is essential for disease prognosis and treatment. For this purpose, we have tried to optimize the use of three tumour markers determined on pleural effusions, to differentiate SCLC from NSCLC by means of a canonic variable, generated by discriminant analysis, including subjects with histologically proven lung cancer. Discriminant analysis was performed by using carcinoembryonic antigen, neuron-specific enolase and tissue polypeptide antigen pleural levels, determined in 65 consecutive and unselected patients, histologically classified as 49 NSCLC and 16 SCLC. To validate the formula generated, a control group of 37 lung cancer patients (10 SCLC and 27 NSCLC), enrolled subsequently, was employed. Applying the discriminant analysis to SCLC and NSCLC patients a good classification was obtained (92% rate of correct classification). The aforementioned formula, applied to the validation group, showed a 92% rate of correct classification. This method, which is rapid, inexpensive and routinely applicable to malignant pleural effusions, may be reliably used to classify lung cancer patients.     

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.     

Multiparameter flow cytometric analysis of a pH sensitive formyl peptide with application to receptor structure and processing kinetics

Environmentally sensitive molecules have many potential cellular applications. We have investigated the utility of a pH sensitive ligand for the formyl peptide receptor, CHO-Met-Leu-Phe-Phe-Lys (SNAFL)-OH (SNAFL-seminaphtho-fluorescein), because in previous studies (Fay et al.: Biochemistry 30:5066-5075, 1991) protonation has been used to explain the quenching when the fluoresceinated formyl pentapeptide ligand binds to this receptor. Moreover, acidification in intracellular compartments is a general mechanism occurring in cells during processing of ligand-receptor complexes. Because the protonated form of SNAFL is excited at 488 nm with emission at 530 nm and the unprotonated form is excited at 568 nm with emission at 650 nm, the ratio of protonated and unprotonated forms can be examined by multiparameter flow cytometry. We found that the receptor-bound ligand is sensitive to both the extracellular and intracellular pH. There is a small increase in the pKa of the ligand upon binding to the receptor consistent with protonation in the binding pocket. Once internalized, spectral changes in the probe consistent with acidification and ligand dissociation from the receptor are observed.     

Prognostic significance of postoperative empyema in lung cancer

OBJECTIVE: To evaluate the safety and feasibility of laparoscopic choledocholithotomy via choledochotomy for the treatment of choledocholithiasis. DESIGN: A prospective series of 1332 consecutive patients who underwent laparoscopic cholecystectomies, with a mean follow-up of 21.2 months. SETTING: University-affiliated referral center. Patients: Forty-three patients (3%) with documented common bile duct stones from January 1991 to February 1995. INTERVENTIONS: Laparoscopic choledocholithotomy with choledochotomy and T tube drainage were performed in 40 patients. Postoperative endoscopic sphincterotomy after laparoscopic cholecystectomy was performed in three patients. MAIN OUTCOME MEASURES: Documented removal of common bile duct stones and procedure-related complications. RESULTS: Laparoscopic choledocholithotomy via choledochotomy was successful in 35 (88%) of 40 patients in whom this procedure was attempted. The mean (+/- SD) operation time was 191.3 +/- 75.4 minutes, and the mean (+/- SD) length of postoperative stay was 10.4 +/- 2.7 days. Seven complications (18%) were recorded, including three major complications (8%) and two retained stones (5%). CONCLUSIONS: Laparoscopic choledocholithotomy via choledochotomy can be performed safely, without increasing the morbidity rate as compared with that of open choledocholithotomy. Thus, some of the advantages of minimally invasive surgery are preserved.