Biomedical subjects

F M Waldman

Publications and source records attributed to F M Waldman.

At least 55 records · Page 3Linked to original sources

PubMed1997

Frequent homozygous deletion of cyclin-dependent kinase inhibitor 2 (MTS1, p16) in superficial bladder cancer detected by fluorescence in situ hybridization.

Deletion of all or part of chromosome 9 is a well-described genetic alteration in bladder tumors. It has been proposed that inactivation of a tumor-suppressor gene on chromosome 9 is an important event in tumor development. Recent reports have supported cyclin-dependent kinase inhibitor 2 (CDKN2, also known as MTS1, INK4, p16) at 9p21 as a candidate tumor-suppressor gene in solid tumors. However, the prevalence of CDKN2 mutations in primary bladder tumors has been controversial. Therefore, we applied gene-specific probes for CDKN2 and the interferon alpha gene (IFNA), also located at 9p21, to characterize further the genomic deletions at this locus in bladder cancer. Seventeen superficial (pTa or pT1) bladder tumor specimens were examined for gene deletion by fluorescence in situ hybridization. Dual-labeling hybridization with a repetitive pericentromeric probe for chromosome 9 and a gene-specific probe for CDKN2 was performed to characterize the gene copy number in relation to the chromosome 9 copy number on a cell-by-cell basis. Homozygous deletion for CDKN2 without homozygous IFNA deletion was found in 5 of 17 tumors tested. Both genes were deleted in one additional case, and one tumor showed deletion of IFNA without deletion of CDKN2. Homozygous deletion at the 9p21 locus was found only in tumors having monosomy for the chromosome 9 centromeric signal. These results indicate that the homozygous deletion of the CDKN2 gene is a frequent and early event in superficial bladder cancer.

Adult↗

PubMed1997

Chromosome 8p deletions are associated with invasive tumor growth in urinary bladder cancer.

Alterations of chromosome 8, including deletions of 8p, occur frequently in many tumors. In this study, fluorescence in situ hybridization was used to study the relationship between 8p deletions, 8q gains, and phenotype in bladder cancer. Cells from 87 tumors were examined by dual-labeling fluorescence in situ hybridization with a centromere 8 probe (pJM12) and P1 probes for 8p22, 8p12, 8q12, and 8q24. Both 8p22 deletions and 8q24 gains were strongly associated with tumor phenotype. There was a marked difference in 8p22 deletions between noninvasive (pTa) tumors (3/33) and minimally invasive (pT1) tumors (8/19; P = 0.005) whereas there was no significant difference between pT1 and muscle-invasive (pT2-4) tumors (19/35; P = 0.3926). Six tumors with 8p22 deletion were examined at 8p12. Three of these tumors showed no 8p12 deletion, narrowing down the site of a putative tumor suppressor gene distal to 8p12. In one other case, there was a marked increase in 8p12 copy number (> 40 per cell; amplification), suggesting the presence of an oncogene involved in bladder cancer at 8p12. The marked difference in 8p22 deletions between noninvasive (pTa) and minimally invasive (pT1) tumors is consistent with a role of a putative tumor suppressor gene on 8p for development of invasive tumor phenotype.

Chromosome Aberrations↗

PubMed1996

Interlaboratory variability in fluorescence in situ hybridization analysis. The NCI Bladder Tumor Marker Network.

Reliable interpretation of fluorescence in situ hybridization (FISH) data, especially data that have been generated in more than one laboratory, requires knowledge of the sources of variability inherent in FISH analysis. Possible sources of variation may derive from differences in sample preparation, probes used, intrasample heterogeneity, hybridization protocols, counting criteria within and between scorers, fluorescence microscopes, and filters. This study characterized the relative weight of some of these factors in order to determine the degree to which FISH results are comparable between laboratories. We used a hierarchical partitioned chi 2 analysis to measure sources of variation. We found that replicate counts varied no more than expected based on counting statistics (i.e., multinomial variation). However, with replicate hybridizations done in two separate laboratories, the variability increased significantly. Thus, care must be taken when interpreting FISH data that are derived from more than one institution. Previously agreed upon counting criteria as well as standardized FISH hybridization protocols may decrease this variability.

Chi-Square Distribution↗

PubMed1996

Genetic aberrations detected by comparative genomic hybridization are associated with clinical outcome in renal cell carcinoma.

The clinical behavior of renal cell carcinoma (RCC) cannot be predicted by histological and other markers. In this study, comparative genomic hybridization was used to evaluate whether the number of genomic aberrations has prognostic significance in 41 nonmetastatic clear cell RCC extending beyond the renal capsule. Losses were most prevalent at 3p (56%) and 9p and 13q (24% each). The number of DNA losses per tumor was associated with recurrence-free survival (P = 0.03). DNA gains most often involved chromosome 5q (17%) and chromosome 7 (15%). The number of DNA gains was not associated with clinical outcome. Loss of chromosome 9p was the only individual locus associated with recurrence (P = 0.04), suggesting that a tumor suppressor gene on chromosome 9p may play a role in RCC progression.

Carcinoma, Renal Cell↗

PubMed1996

Comparative genomic hybridization for genetic analysis of renal oncocytomas.

Renal oncocytomas are uncommon tumors of the kidney that are considered to be of low malignant potential. Neither conventional cytogenetic nor restriction fragment length polymorphism analyses have identified consistent genetic alterations in their genomic DNA. The purpose of the present study was to identify the genetic alterations associated with the development of renal oncocytomas. We studied 13 renal oncocytomas by using comparative genomic hybridization, and we identified loss of genetic material from chromosomes 1 and/or 14 in six of these tumors. These alterations may represent early genetic events in the development of these tumors.

Adenoma, Oxyphilic↗

PubMed1996

Amplifications of oncogene erbB-2 and chromosome 20q in breast cancer determined by differentially competitive polymerase chain reaction.

A new method of measuring gene copy number in small samples of DNA was used to measure amplification of the erbB-2 gene and of chromosome 20q in breast cancer. This method, termed 'differentially competitive polymerase chain reaction' (DC-PCR) combines the advantages of two other techniques for measuring amplification by PCR, namely differential PCR and competitive PCR. The DC-PCR methodology was evaluated for sensitivity and specificity by comparing amplification of erbB-2 measured by DC-PCR with that obtained by fluorescence in situ hybridization (FISH) for 42 cases or Southern blotting and/or slot blot analysis for 34 cases. There was over 90 percent concordance with both FISH and Southern blotting and/or slot blot analysis. DC-PCR was used to further characterize the newly described amplicon at chromosome 20q. By analyzing DNA from 10 breast cancer cell lines at 7 different loci, we identified a potential common region of amplification of approximately 5 centimorgans at chromosome 20q13 bordered by loci D20S52 and RMC20C100-S1. One hundred and seventeen cases of primary breast cancer were evaluated for amplification at these two loci. Amplification at one or more loci, defined as > 1.5 fold higher copy number than that of normal DNA, was found in 25 cases (21%). Sixteen cases were amplified at only one of the two probes (12 cases for RMC20C001-S1 and 4 cases for D20S52), suggesting that the target gene lies between the two markers or that there are two independent target genes within a small chromosome region.

Blotting, Southern↗

PubMed1996

Molecular cytometry of cancer.

The application of molecular probes to diagnosis and prognosis of malignancies has redefined our perceptions of disease, allowing diagnosis by genotypic rather than phenotypic criteria. DNA analysis is especially useful when applied to pathological material in situ, because this allows the pathologist to combine information from both morphological and molecular observations. DNA in situ hybridization is a useful approach for the molecular pathologist, especially when combined with cytometric analysis. Potential clinical applications for in situ hybridization and the recently described technique of comparative genomic hybridization in tumor diagnosis and prognosis are described.

Chromosome Aberrations↗

PubMed1996

Comparative genomic hybridization analysis of archival formalin-fixed paraffin-embedded uveal melanomas.

Comparative genomic hybridization (CGH) was used to analyze seven autologous uveal melanomas with both formalin-fixed, paraffin-embedded and fresh-frozen specimens. In addition, DNA from two archival formalin-fixed tumors more than 45 years old were also analyzed. The most frequent genetic changes were loss of chromosome 3; increase in copy number of 6p and loss of 6q; and increase in copy number of 8q. A comparison of CGH data from the fresh-frozen tumors and their autologous formalin-fixed tumors revealed a correlation coefficient of 0.83. Comparative genomic hybridization (CGH) analysis of 45-year-old specimens identified genetic changes similar to those found in more recent tumors including loss of chromosome 3 and increase in copy numbers of 6p and 8q. The results indicate that there is a good agreement between data obtained from formalin-fixed and fresh-frozen specimens using CGH. Furthermore, the results demonstrate the applicability of this technique in analyzing archival formalin-fixed tumors that were previously not accessible to cytogenetic analysis.

Analysis of Variance↗

PubMed1996

Patterns of epidermal growth factor receptor amplification in malignant gliomas.

Amplification of the gene for epidermal growth factor receptor (EGFR) is a common finding in malignant gliomas. We found that 18 of 29 grade 3 and grade 4 gliomas had EGFR amplification when assayed using fluorescence in situ hybridization. The amplification pattern suggests that the amplicon is contained within double minute chromosomes in most cases. EGFR copy number can differ by 20-fold in amplified cells within a single case. Polysomy 7 occurs frequently in both EGFR-amplified and -unamplified cells. More than one-third of the cases had < or = 10 percent of cells with amplified EGFR, and it is likely that these cases would not have been identified by methods that do not examine DNA on a cell by cell basis.

Brain Neoplasms↗

PubMed1996

Renal cell carcinoma genetic analysis by comparative genomic hybridization and restriction fragment length polymorphism analysis.

PURPOSE: To compare comparative genomic hybridization (CGH) with restriction fragment length polymorphism (RFLP) analysis in renal cell carcinoma (RCC). MATERIALS AND METHODS: Fifteen RCC specimens were analyzed by both CGH and RFLP analysis at 18 loci. RESULTS: Restriction fragment length polymorphism analysis was informative on 90 chromosomal arms. Allelic imbalance was identified on 27 chromosomal arms by RFLP and on 26 arms by CGH. Data from CGH and RFLP demonstrated a high degree of concordance (p < 0.001). Comparative genomic hybridization identified previously documented areas of interest in RCC as well as potential new areas of interest including loss of genetic material on chromosome 2 and gains of genetic material on chromosome 16p. CONCLUSIONS: Comparative genomic hybridization can successfully be performed in RCC specimens. As it surveys the entire genome simultaneously, it may be more efficient than conventional cytogenetics or RFLP analysis in analyzing RCC.

Carcinoma, Renal Cell↗

PubMed1995

Reutilization of previously hybridized slides for fluorescence in situ hybridization.

Application of fluorescence in situ hybridization (FISH) to clinical material is sometimes limited by sample size. In addition, heterogeneity among slides prepared from a single sample may lead to variation in FISH analyses. Reutilization of material for repeated FISH analyses would help to alleviate these problems. We have developed a simple procedure for repeated FISH analyses with directly conjugated probes. Previously hybridized probes are removed by incubation in denaturing solution, and slides can then be rehybridized without residual signals remaining. Several cycles of this procedure allow a full complement of chromosomal loci to be analyzed on the same population of cells. Advantages of this protocol include gaining more cytogenetic information from small samples and eliminating the problem of intratumor variability.

Centromere↗

PubMed1995

ERBB-2 (HER2/neu) gene copy number, p185HER-2 overexpression, and intratumor heterogeneity in human breast cancer.

Amplification of the ERBB-2 (HER-2/neu) gene is accompanied by overexpression of its cell surface receptor product, p185HER-2. Heterogeneity has been observed for both the gene copy number and the level of overexpression of its protein product. To better understand their relationship, correlation between the level of cellular expression of p185HER-2 and ERBB-2 gene amplification was studied in four human breast cancer cell lines (BT-474, SK-BR-3, MDA-453, and MCF-7) and in a primary human breast tumor sample. The relative expression of p185HER-2 was measured by immunofluorescence by using flow and/or image cytometry while correlated DNA analysis was performed on the same cells by fluorescence in situ hybridization to determine ERBB-2 gene and chromosome 17 copy numbers. Marked heterogeneity was observed in both protein expression and ERBB-2 copy number. Despite this heterogeneity, and in accordance with previous studies, the average levels of p185HER-2 expression correlated well with average ERBB-2 gene copy numbers in the four lines examined (r = 0.99). When the relationship between copy number and protein expression was studied on a cell-by-cell basis, p185HER-2 expression correlated with both the absolute number of ERBB-2 gene copies/cell (r = 0.59-0.63) and chromosome 17 copy number (r = 0.45-0.61). It is of interest that there was weak or no correlation between p185HER-2 protein expression and the ERBB-2 copy number:chromosome 17 copy number ratio (r = 0.0-0.25). In more than one-half of cells expressing a high level of p185HER-2, the chromosome 17 copy number was high (two or three times the average copy number), whereas < 2% of an unselected population had a high chromosome 17 copy number. Bromodeoxyuridine incorporation indicated that the S-phase-labeling index was homogeneous across various p185HER-2-expressing subpopulations in the SK-BR-3 cell line. Analysis of the primary breast tumor sample showed results similar to the cell lines, supporting the strong possibility of a mechanistic link among p185HER-2 overexpression, ERBB-2 amplification, and high chromosome 17 copy number.

Breast Neoplasms↗

PubMed1995

Heterogeneity of chromosome 17 and erbB-2 gene copy number in primary and metastatic bladder cancer.

To study the relationship of tumor genomic heterogeneity with bladder cancer phenotype and p53 gene alterations, 138 primary bladder tumors were examined by dual labeling fluorescence in situ hybridization (FISH) using probes for chromosome 17 centromere (p17H8) and p53 (17p13.1). The number of different aneusomic populations > 5% (and monosomic populations > 20%) of cells served as a marker for heterogeneity. Nuclear p53 overexpression and Ki67 labeling index (Ki67 LI) were determined by immunohistochemistry. The number of aneusomic populations was 0 in 53 tumors, 1 in 18, 2 in 47, 3 in 9, and > 3 in 11 tumors. Presence of aneusomy was associated with tumor grade and stage (P < 0.0001 each). Ki67 LI was low in disomic tumors (11.0 +/- 7.7), higher in tumors with 1-3 aneusomic populations (17.4 +/- 11.3), and highest in tumors with > 3 aneusomic populations (25.8 +/- 10.9; P = 0.02 for > 3 vs. 1-3 populations). Aneusomy and heterogeneity were associated with p53 alterations. Aneusomy was seen in 35% of tumors with neither p53 expression nor p53 deletion but in 97% of tumors with both p53 deletion and expression. Nine of 11 tumors with > 3 aneusomic populations exhibited both p53 deletion and overexpression. To study genomic heterogeneity in tumor progression, two recurrences and three metastases of a tumor with known erbB-2 amplification were examined for centromere 17 and erbB-2 copy number. A considerable heterogeneity in centromere 17 and erbB-2 gene copy number was found in both recurrences and metastases, indicating a marked genomic instability in these metastatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Transitional Cell↗

PubMed1995

Autofluorescence correction for fluorescence in situ hybridization.

Optimal sensitivity of fluorescence in situ hybridization (FISH) requires bright signals and low background fluorescence. Use of locus-specific probes is especially dependent on high sensitivity. Some tissue preparations show high autofluorescence, masking small or dim signals. We have developed a new method for subtracting autofluorescence from digital images on a pixel-by-pixel basis. It is based on the observation that fluorescent labels for FISH have narrower excitation and emission spectra than the chemical components responsible for autofluorescence. Our new approach uses calculation of the ratio of autofluorescence between multiple color images for correction of autofluorescence in each individual image. By subtracting autofluorescence components, we were able to enhance centromeric signals and make previously indistinguishable cosmid signals clearly visible. This image-processing approach to autofluorescence correction may widen the applicability of gene-specific probes in FISH analysis of tumor material.

Artifacts↗

PubMed1995

Y chromosome loss detected by FISH in bladder cancer.

To examine the significance of Y chromosome losses in bladder cancer, fluorescence in situ hybridization (FISH) was used to determine its prevalence and associations with known parameters of malignancy. Cells were dissociated from formalin-fixed paraffin-embedded bladder tumors from 68 male patients and from 11 post-mortem bladder washes of male patients with a negative bladder cancer history, and were examined by FISH using centromeric probes for chromosomes X, Y, 7, 9, and 17. Nullisomy for chromosome Y was seen in 23 of 68 tumors (34%), monosomy in 28 of 68 tumors (41%), and polysomy in 17 of 68 tumors (25%). There was no association between chromosome Y loss and tumor grade, stage, tumor growth fraction (Ki67 LI), p53 immunostaining, and presence of p53 deletions. Patient age was higher for tumors with a Y loss (73.5 +/- 12.0 years) than for tumors without Y loss (66.6 +/- 10.8 years; p = 0.0207). In one normal bladder wash, a distinct subpopulation (38% of cells) with Y nullisomy was seen. These data suggest that Y loss is a frequent event that can occur early in bladder cancer, although there is no evidence for a role of Y loss in tumor progression.

Age Factors↗

PubMed1995

Selective cell culture of primary breast carcinoma.

We have used culture conditions which simulate the microenvironment of breast tumors for the isolation and propagation of primary breast tumor cells in vitro. In this monolayer setup, the mixture of cells dissociated from primary breast tumors is subjected to self-created gradients of oxygen and nutrients as well as metabolic waste and extracellular pH. The tumor populations isolated under these novel conditions have displayed phenotypic properties characteristic of breast carcinomas, including homogeneous expression of cytokeratin 19, and increased mitochondrial retention of the cationic dye rhodamine 123. Nonmalignant cultures from reduction mammoplasty were unable to survive these conditions. One tumor population which reached passage 10 was aneuploid for chromosomes 15 and 17, and displayed a p53 mutation in exon 8. These studies strongly suggest that the culture conditions described here can suppress the growth of normal breast cells, thereby allowing selective isolation of some populations of slow-growing primary tumor cells in vitro.

Breast↗

PubMed1995

Karyotypic heterogeneity and its relation to labeling index in interphase breast tumor cells.

We have used fluorescence in situ hybridization (FISH) with chromosome-specific probes and immunofluorescent detection of in vivo bromodeoxyuridine (BrdUrd) incorporation to evaluate simultaneously numerical chromosome aberrations and proliferative activity of breast cancers. The number of distinct hybridization domains specific for repetitive pericentromeric sequences on chromosomes 1, 7, 11, 15, 17, and X was used as an indicator of copy number of these chromosomes in interphase tumor cells from 23 human breast cancers. Every tumor analyzed showed a heterogeneous distribution of copy number for at least one chromosome type. The copy number distribution for different chromosomes within a tumor frequently showed differing patterns. Major cell populations showing monosomy were relatively rare, occurring only in five cases for chromosome 17, once for chromosome 1, and once for chromosome 15. Flow cytometric analysis of DNA ploidy correlated well with FISH analysis, although flow cytometry failed to detect aneuploidy when only a few chromosomes were affected. To determine whether cell populations with different chromosomal copy numbers have identical proliferation characteristics in vivo, BrdUrd incorporation and centromeric copy number were detected simultaneously. Comparison of the chromosome copy number distribution in BrdUrd-positive cells vs. the distribution of the entire cell population showed different distributions in seven of the 20 cases analyzed. This study demonstrates the common occurrence of chromosome copy number heterogeneity and suggests that a cell phenotype (proliferation) may be associated with genotypic subpopulations.

Adult↗

PubMed1995

Chromosome-9 loss detected by fluorescence in situ hybridization in bladder cancer.

A loss of chromosome-9 material is one of the most frequent genomic aberrations known in bladder cancer. In order to better understand the role of chromosome-9 losses in bladder cancer, 125 formalin-fixed and 37 unfixed bladder tumors were examined using fluorescence in situ hybridization (FISH). A repetitive probe for a pericentromeric region on 9q12 (pHUR98) was applied for chromosome-9 copy-number enumeration. Under-representation of chromosome 9 was found in 74 of 162 cases. There was no association between loss of chromosome 9 and increased grade or stage, papillary growth pattern, p53 protein expression, or tumor-cell proliferation (Ki-67). These data show that chromosome-9 loss is an early event in bladder-cancer development, occurring independently of p53 alterations. In order to determine the prevalence of large sub-regional chromosome-9 deletions, dual hybridizations with pHUR98 and cosmid probes for 9q34, 9q22, and 9p21 were performed. Partial deletion was detected in only 1 of 36 cases for 9q34 and in 1 of 24 cases for 9p21. Surprisingly, amplification of the interferon alpha locus on 9p21 was seen in 1 of 24 tumors. The finding of 9p amplification may indicate the site of an oncogene relevant for bladder cancer.

Chromosome Deletion↗