KIAA1096, a gene on chromosome 1q,is amplified and overexpressed in bladder cancer

Chromosomal gain on 1q23-24 is a cytogenetic finding found in approximately 30% of bladder tumors. Currently, no defined or candidate tumor-associated genes from this region have been identified. The objective of this study was to identify and quantitate the expression of putative cancer genes located at this chromosome locus in normal urothelium, superficial, and muscle invasive bladder tumors. We examined both normal and bladder cancer tissue specimens (N = 40-80 RNA, DNA, and protein) by semiquantitative RT/PCR, genomic PCR, and by Western blotting. The KIAA1096 gene is located at 1q23-24 with no overexpression or amplification in normal urothelium, but was significantly upregulated in 30% of tumors (P = 0.0001). There was a trend towards increased expression in invasive compared to superficial lesions (P = 0.06). A significant increase in gene copy was also found in a 38% of TCC of the bladder compared to normal bladder mucosa or peripheral blood lymphocytes. Immunohistochemistry (IHC) demonstrated KIAA1096 expression in nonmalignant bladder mucosa tissue but apparent upregulation in invasive transitional cell carcinoma. Two other genes, CH1 and RGS5, which are situated in the same region of chromosome 1q, demonstrated disparate patterns of expression. In summary, KIAA1096 is a gene situated at 1q23-24, which demonstrated a pattern of RNA and DNA expression consistent with the 38% expression of cytogenetic amplification noted on previous studies. This gene may, therefore, be a putative marker for this cytogenetic phenomenon and provide an opportunity to evaluate the clinical significance of previous cytogenetic findings.     

Accuracy and reliability of flow cytometric DNA analysis using a simple, one-step ethidium bromide staining protocol

Sources of variation and error were investigated for a simple flow cytometric analysis of DNA content of detergent-isolated nuclei stained with ethidium bromide. Using the ploidy classes of mouse liver nuclei, deviations from linearity were assessed for three different instruments. In more extreme settings, the maximum deviations for a FACS instrument were up to 6 to 9%, but in general deviations were around 1% or lower for all instruments. As biological DNA standards, human peripheral lymphocytes and trout erythrocytes appeared to be suitable and easy to store frozen. The erythrocytes had dye-binding characteristics similar to those of human lymphocytes and a 20% lower fluorescence, thus being well suited as an internal standard, as was demonstrated in tumor ploidy analyses performed with varied tissue concentration. Staining homogeneity was improved when staining time was extended to 24 h, at which time male and female lymphocytes were completely separated with an average difference in DNA content of 1.9%. A small difference in fluorescence between mitogen-stimulated and unstimulated lymphocytes was reduced to less than 1% after 24 h of staining. In general, the manipulations of the conditions for the analysis resulted in maximum variations of around 1%, indicating the robustness and reliability of the technique.     

Measurement of O6-methylguanine-type adducts in DNA and O6-alkylguanine-DNA-alkyltransferase repair activity in normal and neoplastic human tissues.

1. Novel assays based on the use of the suicide repair enzyme O6-alkylguanine-DNA-alkyltransferase (AGT) to repair O6-alkylguanine-type adducts in DNA have been used for the analysis of extracts of human biopsy specimens of gastric mucosa, urinary bladder mucosa, colon and circulating lymphocytes. 2. Examination of these extracts revealed no detectable amounts of the precarcinogenic adduct O6-meG. 3. AGT measurements were the same among the normal and the autologous samples of all patients examined, which limits its prognostic value as a tumour marker. 4. AGT measurements from the cancer samples were much higher compared with the measurements of the other two groups which proves that AGT is just a marker of tumour burden. 5. Finally, AGT measurements from lymphocytes show that AGT from normal individuals have about the same value as that from patients suffering from cancer in urinary bladder mucosa and colon, but is much lower than that of patients with cancer in gastric mucosa.     

Trout and salmon erythrocytes and human leukocytes as internal standards for ploidy control in flow cytometry

Control of technique and use of biological standards in flow cytometry have become increasingly important due to the wider use of the method for ploidy determination of malignant tumors in clinical research. Trout (TRBC) and salmon erythrocytes and human buffy coat leukocytes were selected for a study of factors influencing the DNA stainability. Whether standard and test cells were mixed before or after enzymatic treatment and staining was found to be critical for the ploidy comparisons. Otherwise, artifactual differences of at least 20% may be noted, leading to an overestimation of DNA aneuploidy. The time from staining to analysis had minimal effect, with some exceptions. The proportions of different cells in the sample had no influence, and nonlinearity of measurements was negligible. Diploid cells in normal endometrium and benign ovarian tumors, as well as the diploid fraction of aneuploid tumor cells, were systematically measured to have a DNA staining 5-7% above human leukocytes.     

DNA ploidy analysis and cytologic examination of sorted cell populations from human breast tumors

Two different flow cytometric procedures were applied on cell samples from human breast tumors. One procedure involved DNA ploidy analysis on suspensions of isolated nuclei. The mean ploidy ratios of 27 benign breast lesions to chicken erythrocytes and rainbow trout erythrocytes were found to be 2.66 +/- 0.03 and 1.25 +/- 0.02, respectively. From the 45 stemlines found in a series of 43 carcinomas, 12 were diploid, 13 hyperdiploid and 20 near-tetraploid. No association was found between the lymph node status and the DNA ploidy level. The second procedure involved sorting fixed cells from DNA "windows" for the preparation of permanent cytologic specimens. The sorted cells appeared to be shrunken, but the morphologic quality was similar to that of imprint specimens from the same tumors, permitting discrimination between various types of normal cells and tumor cells. The combined use of both flow cytometric procedures may lead to greater insight into the relationship between the cytologic and cytogenetic heterogeneity of breast carcinomas.     

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

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

Use of leukocytes infiltrating bladder tumors as an internal reference for determining the DNA index by flow cytometry.

The use of leukocytes infiltrating bladder tumors as an internal standard for flow cytometric measurements of DNA and SG2M indices was investigated in comparison with the conventional use of chicken erythrocytes (chicken red blood cells; CRBC). The leukocytes were identified by indirect fluorescence, using a mouse anti-CD45 monoclonal antibody and a fluorescein isothiocyanate-conjugated goat anti-mouse Ig F(ab')2 fragment. DNA was stained with propidium iodide. The percentage of CD45-positive cells averaged 37% in 23 unimodal bladder tumors and 30% in 27 bimodal ones. The diploid peak of CD45-positive cells was always easily identified and permitted a more accurate calculation of the DNA index and the percentage of cells in SG2M phases than did the use of CRBC. Seven tumors that were classified as unimodal using CRBC as the standard did, in fact, contain a minor clone (with a DNA index of 1.80) that could be detected only when CD45-positive leukocytes were used as the internal standard.     

Flow cytometric analysis of paraffin-embedded material in human gastric cancer

Flow cytometric DNa analysis was performed on formalin-fixed, paraffin-embedded samples obtained by gastroscopic biopsy from 9 patients with histologically normal gastric mucosa (36 specimens) and by radical gastrectomy from 42 cases of human gastric cancer (120 specimens). Ploidy patterns and the distribution of cells in the different cell cycle phases were estimated, and the results were correlated with the histologic and clinical features. All samples of normal mucosa showed a diploid modal DNA content whereas DNA aneuploidy was encountered in 71.4% of the gastric tumors. The correlation between aneuploidy and histologic malignancy grading was statistically significant: aneuploidy was found in 36.4% of highly differentiated (grade 1 and grade 2) tumors and in 75.0% of poorly differentiated (grade 3) tumors (P less than .05). The percentage of cells in S-phase in normal gastric mucosa (median: 5.0%) was lower than that in the tumors (median: 11.3%) (P less than .05). There was a trend for grade 3 tumors to have higher median values (median: 13.4%) than grade 1 and 2 tumors (median: 9.3%); however, this was not statistically significant. An aneuploid DNA pattern was associated with a poorer prognosis, both in early and in advanced stages of gastric tumors, while proliferative activity did not correlate with postoperative survival.     

Selection of Microsatellite Markers for Bladder Cancer Diagnosis without the Need for Corresponding Blood

Microsatellite markers are used for loss-of-heterozygosity, allelic imbalance and clonality analyses in cancers. Usually, tumor DNA is compared to corresponding normal DNA. However, normal DNA is not always available and can display aberrant allele ratios due to copy number variations in the genome. Moreover, stutter peaks may complicate the analysis. To use microsatellite markers for diagnosis of recurrent bladder cancer, we aimed to select markers without stutter peaks and a constant ratio between alleles, thereby avoiding the need for a control DNA sample. We investigated 49 microsatellite markers with tri- and tetranucleotide repeats in regions commonly lost in bladder cancer. Based on analysis of 50 blood DNAs the 12 best performing markers were selected with few stutter peaks and a constant ratio between peaks heights. Per marker upper and lower cut off values for allele ratios were determined. LOH of the markers was observed in 59/104 tumor DNAs. We then determined the sensitivity of the marker panel for detection of recurrent bladder cancer by assaying 102 urine samples of these patients. Sensitivity was 63% when patients were stratified for LOH in their primary tumors. We demonstrate that up-front selection of microsatellite markers obliterates the need for a corresponding blood sample. For diagnosis of bladder cancer recurrences in urine this significantly reduces costs. Moreover, this approach facilitates retrospective analysis of archival tumor samples for allelic imbalance.     

Ploidy and proliferative activity of human gastric carcinoma: a cytofluorometric study on fresh and on paraffin embedded material

Flow cytometric DNA analysis was performed on both fresh and on paraffin embedded samples obtained by gastroscopic biopsies in 5 patients with histologically normal gastric mucosa (20 specimens) and by radical gastrectomies in 9 cases of human gastric cancer (36 specimens). Ploidy and the distribution of cells in the different cell cycle phases were estimated. Results from fresh tissue were compared with those from paraffin embedded material. All the samples of normal mucosa showed a diploid modal DNA content. Unimodal DNA distribution was found both in fresh and paraffin embedded material of 3 well differentiated gastric adenocarcinomas, whereas the remaining 6 tumors (1 moderately differentiated, 4 poorly differentiated and 1 undifferentiated adenocarcinomas) showed aneuploidy. The percentage of cells in S phase in normal tissue and in tumors were respectively 9.0% and 11.9% in the fresh material, and 10.0% and 15.0% in the paraffin embedded material. No statistically significant differences were found between fresh and paraffin embedded samples, whereas the proliferative activity was, in both cases, statistically higher in tumors than in normal mucosa (p less than .01 and p less than .001 respectively). The quality of DNA flow cytometry from paraffin embedded material was comparable with that from fresh samples.     

Karyometry in rectal mucosa of patients with previous colorectal adenomas

OBJECTIVE: To determine whether karyometric measurements taken in biopsies from histologically normal-appearing rectal mucosa could serve as a biomarker for the risk of recurrence of polyps. MATERIALS AND METHODS: Biopsies were taken from the rectal mucosa of cases with a prior history of colonic polyps at the baseline of the study. In 57 cases recurrent polyps occurred (R cases); in 72 cases no recurrent disease was found at the end of the study (NR cases). From each biopsy 100 nuclei were recorded at high resolution. After segmentation, feature extraction and selection of a discriminating subset of features, a number of discriminant functions were derived. Also, measures of nuclear abnormality were computed. RESULTS: The differences in karyometricfeature values for nuclei from biopsies of cases with recurrent or nonrecurrent disease were very small and not notably expressed in the majority of nuclei. It was possible by focusing on nuclei showing clear deviations from normal to derive a discriminant function that exhibited a shift for the NR and R data sets. The distributions of discriminant function scores were then subjected to a second-order discriminant analysis to separate cases according to recurrence status. This function showed a statistically highly significant correlation with recurrence. At one extreme of its score distribution were 11 of 57 cases that had a recurrence, and at the other extreme were 8-10 of 72 cases that had no recurrence. The distributions of nuclear abnormality values for these subsets of cases were drastically different, with an average value of 1.72 for the group that may be at high risk for another recurrence and 1.02 for the group possibly at low risk. All cases with a prior history of colonic polyps showed a nuclear abnormality deviating from normal. CONCLUSION: Measurement of a sample of 100 nuclei from the rectal mucosa will suggest, for approximately 10% of cases, that a high risk for recurrence of adenomatous polyps exists and, for a slightly lower proportion, confirm that the nuclei deviate only slightly from those from individuals with no history of colonic polyps. For the majority of cases with a prior history of adenoma, the nuclei in the biopsy show a notable deviation from normal, but the deviation is practically the same for cases that had a recurrence and those that did not. However, a tentative discriminant function (DF I,3) derived from the characteristics of the extreme cases correctly classified approximately 64% of nonrecurrent and 83% of recurrent cases using a Bayesian decision boundary.     

Reversal of cell surface abnormalities of T lymphocytes in hodgkin's disease after in vitro incubation in fetal sera.

The capacity of periphal blood lymphocytes from patients with untreated Hodgkin's disease to form E rosettes with sheep erythrocytes and to respond in vitro to PHA stimulation were found to be profoundly impaired. In 49% of the patients, the percentage of E rosette-forming cells (E-RFC) was more than two standard deviations below the mean for normal donors. Overnight incubation of the peripheral blood lymhocytes from these patients in culture media containing 20% fetal calf serum was followed by restoration of the percentage of E-RFC up to normal levels. Similar results have been observed after incubation in fetal human serum, but not in adult human AB serum or adult bovine serum. Incubation of peripheral blood lymphocytes from untreated patients in20% fetal calf serum also resulted in a remarkable restoration of their capacity to respond normally to PHA. Possible mechanisms involved in these reversible cell surface and in vitro lymphocyte function abnormalities in Hodgkin's disease are discussed.     

In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors

Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproducibility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.     

Relative quantification of glycated Cu-Zn superoxide dismutase in erythrocytes by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESIMS) was used for relative quantification of glycated Cu-Zn superoxide dismutase (SOD-1) in human erythrocytes. SOD-1 samples were prepared from erythrocytes by removing hemoglobin using hemoglobind gel followed by ethanol and chloroform extraction. The reproducibility in measurement of the relative percentage of glycated protein was good, and the standard deviation of each measurement was 4.0%. From the mass spectral analysis of a mixture of commercial SOD-1 and in vitro partially glycated SOD-1 in several ratios, it was found that free and glycated SOD-1 have the same ionization efficiencies. The percentage of glycation on SOD-1 was measured in 30 individuals, including patients with diabetes mellitus. The glycation levels ranged from 4.5% to below the detection limit. The SOD-1 sample extracted from erythrocytes was fractionated by Glyco-Gel B chromatography, and the separated fractions were analyzed by MS. The mass spectra of absorbed fraction showed significant amounts of non-specific binding of non-glycated proteins to Glyco-Gel B.     

In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors

Summary Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproductbility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.     

Quantitative Analysis of Torso FDG-PET Scans by Using Anatomical Standardization of Normal Cases from Thorough Physical Examinations

Understanding of standardized uptake value (SUV) of 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography (FDG-PET) depends on the background accumulations of glucose because the SUV often varies the status of patients. The purpose of this study was to develop a new method for quantitative analysis of SUV of FDG-PET scan images. The method included an anatomical standardization and a statistical comparison with normal cases by using Z-score that are often used in SPM or 3D-SSP approach for brain function analysis. Our scheme consisted of two approaches, which included the construction of a normal model and the determination of the SUV scores as Z-score index for measuring the abnormality of an FDG-PET scan image. To construct the normal torso model, all of the normal images were registered into one shape, which indicated the normal range of SUV at all voxels. The image deformation process consisted of a whole body rigid registration of shoulder to bladder region and liver registration and a non-linear registration of body surface by using the thin-plate spline technique. In order to validate usefulness of our method, we segment suspicious regions on FDG-PET images manually, and obtained the Z-scores of the regions based on the corresponding voxels that stores the mean and the standard deviations from the normal model. We collected 243 (143 males and 100 females) normal cases to construct the normal model. We also extracted 432 abnormal spots from 63 abnormal cases (73 cancer lesions) to validate the Z-scores. The Z-scores of 417 out of 432 abnormal spots were higher than 2.0, which statistically indicated the severity of the spots. In conclusions, the Z-scores obtained by our computerized scheme with anatomical standardization of torso region would be useful for visualization and detection of subtle lesions on FDG-PET scan images even when the SUV may not clearly show an abnormality.     

Tumor-infiltrating lymphocytes in colorectal tumors display a diversity of T cell receptor sequences that differ from the T cells in adjacent mucosal tissue

Tumors from colorectal cancer (CRC) are generally immunogenic and commonly infiltrated with T lymphocytes. However, the details of the adaptive immune reaction to these tumors are poorly understood. We have accrued both colon tumor samples and adjacent healthy mucosal samples from 15 CRC patients to study lymphocytes infiltrating these tissues. We apply a method for detailed sequencing of T-cell receptor (TCR) sequences from tumor-infiltrating lymphocytes (TILs) in CRC tumors at high throughput to probe T-cell clones in comparison with the TCRs from adjacent healthy mucosal tissue. In parallel, we captured TIL counts using standard immunohistochemistry. The variation in diversity of the TIL repertoire was far wider than the variation of T-cell clones in the healthy mucosa, and the oligoclonality was higher on average in the tumors. However, the diversity of the T-cell repertoire in both CRC tumors and healthy mucosa was on average 100-fold lower than in peripheral blood. Using the TCR sequences to identify and track clones between mucosal and tumor samples, we determined that the immune response in the tumor is different than in the adjacent mucosal tissue, and the number of shared clones is not dependent on distance between the samples. Together, these data imply that CRC tumors induce a specific adaptive immune response, but that this response differs widely in strength and breadth between patients.     

Allelic loss at the GPx-1 locus in cancer of the head and neck

Glutathione peroxidase is a selenium-containing, antioxidant enzyme previously implicated in the risk and development of lung and breast cancer, in part the result of allelic loss at the GPx-1 locus. This study examined allelic loss at the same locus in squamous cell carcinomas of the head and neck. The frequency of a polymorphism at codon 198 resulting in either a leucine or a proline at that position was surveyed by comparing 133 DNA samples obtained from head and neck tumors and 517 samples obtained from cancer-free individuals. Tumor DNAs exhibited fewer pro/leu heterozygotes as compared to DNA obtained from the cancer-free population. Fewer GPx-1 heterozygotes were verified by determining the frequency of highly polymorphic alanine repeat sequences in the same gene. The analysis revealed an approximately 42% reduction in heterozygosity in the DNA from the tumor samples. In order to assess loss of heterozygosity (LOH) at the GPx-1 locus, DNA was genotyped from peripheral lymphocytes, tumor tissue, and microscopically normal tissues adjacent to the tumor, derived from the same patients. These studies indicated LOH at the GPx-1 locus in each of the three tumor/normal tissues sample sets examined. Furthermore, LOH in the microscopically normal tissues at the tumor margin occurred in two of the three sample sets examined. These data implicate GPx-1 in the development of squamous cell carcinoma the head and neck and suggest that allelic loss of this gene, or one tightly linked to it, is an early event in the development of this type of malignancy. Author to whom all correspondence and reprint requests should be addressed. These authors contributed equally to this work.     

DNA ploidy and proliferative activity of human pulmonary epithelium

DNA ploidy and distribution has been determined in normal and abnormal bronchial, bronchiolar and alveolar epithelium from 22 patients, aged between 0 and 85 years, 9 of whom had received chemotherapy for malignant disease. The DNA ploidy was diploid in all the specimens examined. The S + G2/M fraction was significantly greater in diseased than normal bronchial trees. In the bronchial epithelium, mean values +/- the standard deviation (SD) were 5.5 +/- 2.2% vs 1.1 +/- 0.6%, in bronchiolar epithelium 4.6 +/- 1.6% vs 1.0 +/- 0.9% and in alveolar epithelium 4.6 +/- 1.6% vs 0.8 +/- 0.5%. The highest S + G2/M value of 8.9% was obtained from inflamed bronchial epithelium. Polyploid cells up to the octaploid range occurred infrequently but their incidence was slightly increased to between 0.16% and 0.9% in diseased lungs and in patients who had received chemotherapeutic drugs. It was concluded that (1) non-cancerous drugs. It was concluded that (1) non-cancerous pulmonary epithelium is diploid, that (2) pulmonary epithelium shows steady-state renewal at all ages and polyploid cells are rare under normal conditions and that (3) the S + G2/M fraction increases up to approximately 10% in reactive proliferative states.     

Hydrogen exchange kinetics of human hemoglobins. The pH dependence of solvent accessibility in cyanomet-, oxy-, and deoxyhemoglobin.

The hydrogen exchange kinetics of human oxy-, deoxy-, and cyanomethemoglobin have been measured as a function of pH by the tritium tracer method. At 5 degrees C and in phosphate buffer both liganded and unliganded forms of ferrohemoglobin exhibit deviations from the regular pH dependence of exchange that is characteristic of cyanomethemoglobin. In oxyhemoglobin, the deviation from the normal exchange pattern is centered at pH 7.4 and is in the direction of increased exchange or solvent accessibility. The effect in deoxyhemogloin, while occurring at the same pH and being of the same order of magnitude, is in the opposite direction, thus suggesting a pH-induced conformational transition leading to a less accessible structure. The width of these pH-induced deviations in solvent accessibility is approximately 1 pH unit in both cases. We propose a model in which specific interactions between charged groups in both froms of ferrohemoglobin account for these deviations.