High-quality RNA and DNA from flow cytometrically sorted human epithelial cells and tissues.

Microarray technologies have made possible comprehensive analyses of nucleic acid sequence and expression. However, the technology to obtain efficiently high-quality RNA and DNA suitable for array analysis from purified populations of neoplastic cells from human tissues has not been well addressed. Microdissection can enrich for populations of cells present in various tumor tissues, but it is not easily automated or performed rapidly, and there are tissues in which cells of interest cannot be readily isolated based on morphologic criteria alone. Here we describe a protocol for efficient RNA and DNA isolation from flow cytometrically purified whole epithelial cells from primary tissue. The aqueous reagent, RNAlater, which preserves RNA, allows immunolabeling and purification of whole epithelial cells by flow sorting without special instrument preparation to reduce RNase activity. We used real-time PCR to determine RNA quality afterflow sorting. High-quality RNA and DNA suitable for expression and genotype analysis can be readily obtained from flow cytometrically purified populations of neoplastic cells from human tissues.

Loss of heterozygosity in childhood de novo acute myelogenous leukemia.

A genome-wide screening for loss of heterozygosity (LOH), a marker for possible involvement of tumor suppressor genes, was conducted in 53 children with de novo acute myelogenous leukemia (AML). A total of 177 highly polymorphic microsatellite repeat markers were used in locus-specific polymerase chain reactions. This comprehensive allelotyping employed flow-sorted cells from diagnostic samples and whole-genome amplification of DNA from small, highly purified samples. Nineteen regions of allelic loss in 17 patients (32%) were detected on chromosome arms 1q, 3q, 5q, 7q (n = 2), 9q (n = 4), 11p (n = 2), 12p (n = 3), 13q (n = 2), 16q, 19q, and Y. The study revealed a degree of allelic loss underestimated by routine cytogenetic analysis, which failed to detect 9 of these LOH events. There was no evidence of LOH by intragenic markers for p53, Nf1, or CBFA2/AML1. Most lymphocytes lacked the deletions, which were detected only in the leukemic myeloid blast population. Analysis of patients' clinical and biologic characteristics indicated that the presence of LOH was associated with a white blood cell count of 20 x 10(9)/L or higher but was not correlated with a shorter overall survival. The relatively low rate of LOH observed in this study compared with findings in solid tumors and in pediatric acute lymphoblastic leukemia and adult AML suggests that tumor suppressor genes are either infrequently involved in the development of pediatric de novo AML or are inactivated by such means as methylation and point mutations. Additional study is needed to determine whether these regions of LOH harbor tumor suppressor genes and whether specific regions of LOH correlate with clinical characteristics. (Blood. 2001;98:1188-1194)

Evolution of neoplastic cell lineages in Barrett oesophagus.

It has been hypothesized that neoplastic progression develops as a consequence of an acquired genetic instability and the subsequent evolution of clonal populations with accumulated genetic errors. Accordingly, human cancers and some premalignant lesions contain multiple genetic abnormalities not present in the normal tissues from which the neoplasms arose. Barrett oesophagus (BE) is a premalignant condition which predisposes to oesophageal adenocarcinoma (EA) that can be biopsied prospectively over time because endoscopic surveillance is recommended for early detection of cancer. In addition, oesophagectomy specimens frequently contain the premalignant epithelium from which the cancer arose. Neoplastic progression in BE is associated with alterations in TP53 (also known as p53) and CDKN2A (also known as p16) and non-random losses of heterozygosity (LOH). Aneuploid or increased 4N populations occur in more than 90-95% of EAs, arise in premalignant epithelium and predict progression. We have previously shown in small numbers of patients that disruption of TP53 and CDKN2A typically occurs before aneuploidy and cancer. Here, we determine the evolutionary relationships of non-random LOH, TP53 and CDKN2A mutations, CDKN2A CpG-island methylation and ploidy during neoplastic progression. Diploid cell progenitors with somatic genetic or epigenetic abnormalities in TP53 and CDKN2A were capable of clonal expansion, spreading to large regions of oesophageal mucosa. The subsequent evolution of neoplastic progeny frequently involved bifurcations and LOH at 5q, 13q and 18q that occurred in no obligate order relative to each other, DNA-content aneuploidy or cancer. Our results indicate that clonal evolution is more complex than predicted by linear models.

p16INK4a expression is frequently decreased and associated with 9p21 loss of heterozygosity in sporadic melanoma.

The product of the p16/INK4a/CDKN2/MTS1 tumor-suppressor gene acts as a negative cell cycle regulator by inhibiting G1 cyclin-dependent kinases that phosphorylate the retinoblastoma protein. p16 is inactivated in a wide range of human malignancies, including familial melanoma. However, its expression and function in sporadic melanoma has not been extensively investigated. We studied p16 expression in 62 archival melanomas and 30 archival nevi and lentigines by immunohistochemistry. Eighteen of 26 (69%) superficial spreading melanomas, 17 of 28 (61%) nodular melanomas, all of three lentigo maligna melanomas, and all of five melanoma metastases were found to harbor less than 10% p16-positive tumor cells. In contrast, only six of 24 (25%) nevi had less than 10% positive cells. No correlation between tumor thickness and loss of p16 expression was found. Using DNA from micro-dissected tumor and matched normal tissues, five of seven (71%) p16-negative melanoma cases had 9p21 loss of heterozygosity (LOH), and one of these 9p21 LOH cases had promoter region hypermethylation of the remaining p16 allele. These data demonstrate that partial or complete loss of p16 expression is prevalent in sporadic melanoma and is frequently associated with 9p21 LOH.

Inactivation of p16 in human mammary epithelial cells by CpG island methylation.

Proliferation of human mammary epithelial cells (HMEC) is limited to a few passages in culture due to an arrest in G1 termed selection or mortality stage 0, M0. A small number of cells spontaneously escape M0, continue to proliferate in culture, and then enter a second mortality stage, M1, at which they senesce. Evidence that M0 involves the Rb pathway comes from the observation that expression of human papillomavirus type 16 E7 alleviates the M0 proliferation block, and we further show that the Rb-binding region of E7 is required to allow cells to bypass M0. In contrast, E6 does not prevent HMEC from entering M0 but, rather, is involved in M1 bypass. Here we show that inactivation of the D-type cyclin-dependent kinase inhibitor p16INK4A is associated with escape from the M0 proliferation block. Early-passage HMEC express readily detectable amounts of p16 protein, whereas normal or E6-expressing HMEC that escaped M0 expressed markedly reduced amounts of p16 mRNA and protein. This initial reduction of p16 expression was associated with limited methylation of the p16 promoter region CpG island. At later passages, a further reduction in p16 expression occurred, accompanied by increased CpG island methylation. In contrast, reduction of p16 expression did not occur in E7-expressing HMEC that bypassed M0, due to inactivation of Rb. These observations in the E6-expressing HMEC correlate well with the finding that CpG island methylation is a mechanism of p16 inactivation in the development of human tumors, including breast cancer.

Genetic analysis of long-term Barrett's esophagus epithelial cultures exhibiting cytogenetic and ploidy abnormalities.

BACKGROUND & AIMS: Progression to cancer in Barrett's esophagus occurs through an accumulation of cell cycle and genetic abnormalities that have been documented in vivo. To better study neoplastic evolution in Barrett's esophagus, the aim of this study was to establish in vitro cultures from preneoplastic tissues. METHODS: Mechanical and enzymatic dissociation methods were used to initiate Barrett's epithelial cultures from endoscopic biopsy specimens, and the cells were characterized using flow-cytometric, cytogenetic, and molecular genetic analyses. RESULTS: Four long-term cultures were established from 39 attempts. All cultures contain cytogenetic abnormalities and elevated flow-cytometric 4N DNA content fractions. Molecular genetic abnormalities detected include the following: 9p and/or CDKN2/p16 abnormalities in 4 of 4 cultures, 17p loss of heterozygosity and p53 mutation in 3 of 4 cultures, and 5q loss of heterozygosity in 1 of 4 cultures. Inactivation of p53 was statistically associated with successful long-term culture. CONCLUSIONS: These cultures contain cell cycle and molecular genetic abnormalities that closely parallel those previously documented to occur early in cancer development in Barrett's esophagus in vivo. These alterations also appear to be associated with successful growth in vitro. The cultures may provide a premalignant in vitro system in which to test potential therapies for Barrett's esophagus as well as to examine etiologic factors and genetic intermediates important in neoplastic progression.

p16INK4a promoter is hypermethylated at a high frequency in esophageal adenocarcinomas.

Loss of heterozygosity (LOH) of 9p21, which contains the p16INK4a tumor suppressor gene locus, is one of the most frequent genetic abnormalities in human neoplasia, including esophageal adenocarcinomas. Only a minority of Barrett's adenocarcinomas with 9p21 LOH have a somatic mutation in the remaining p16 allele, and none have been found to have homozygous deletions. To determine whether p16 promoter hypermethylation may be an alternative mechanism for p16 inactivation in esophageal adenocarcinomas, we examined the methylation status of the p16 promoter in flow-sorted aneuploid cell populations from 21 patients with premalignant Barrett's epithelium or esophageal adenocarcinoma. Using bisulfite modification, primer-extension preamplification, and methylation-specific PCR, we demonstrate that the methylation assay can be performed on 2 ng of DNA (approximately 275 cells). Eight of 21 patients (38%) had p16 promoter hypermethylation and 9p21 LOH, including 3 patients who had only premalignant Barrett's epithelium. Our data suggest that promoter hypermethylation with LOH is a common mechanism for inactivation of p16 in the pathogenesis of esophageal adenocarcinomas.

Allelic loss of 9p21 and mutation of the CDKN2/p16 gene develop as early lesions during neoplastic progression in Barrett's esophagus.

High frequency allelic loss of chromosome 9p21 has been reported in a number of human cancers, including those of the esophagus. The CDKN2 gene on chromosome 9p21 that encodes the p16 inhibitor of cyclinD/Cdk4 complexes is a target of allelic loss and inactivation in a variety of human cancers and cell lines. However, the roles of 9p21 allelic losses and CDKN2 mutations in human neoplastic progression in vivo remain controversial. We determined the prevalence of allelic loss at 9p21 and mutations in CDKN2 in esophageal adenocarcinomas and investigated the order in which they occurred relative to the development of aneuploidy and cancer during neoplastic progression. Aneuploid cell populations from 32 patients with Barrett's esophagus who had premalignant epithelium, cancer, or both, were purified by DNA content flow cytometric cell sorting and evaluated by polymerase chain reaction. Twenty-four of 32 informative patients (75%) had allelic loss at 9p21 in aneuploid cell populations. Premalignant epithelium was available for seven of the patients who had 9p21 allelic losses in cancer; allelic loss of 9p21 was detected before cancer in all seven (100%). Allelic loss of 9p21 preceded the development of aneuploidy in 13 of 15 patients (87%) who had aneuploid cell populations detected in premalignant epithelium, and the two events were detected simultaneously in the remaining two patients. Five of 22 aneuploid populations (23%) with 9p21 loss had somatic mutations in the remaining CDKN2 allele. The same mutations and 9p21 allelic losses were also found in the corresponding diploid cells from premalignant epithelium in all three cases that were evaluable. However, there was no evidence for mutation or homozygous deletion of p16 in the other 17 patients with 9p21 allelic loss. Our results indicate that 9p21 allelic losses and CDKN2 mutations develop as early lesions in diploid cells before aneuploidy and cancer during neoplastic progression in Barrett's esophagus.

Allelic loss and mutational analysis of the DPC4 gene in esophageal adenocarcinoma.

DPC4, a recently cloned gene located on 18q2l.l, is inactivated in almost one half of pancreatic adenocarcinomas. To determine whether DPC4 inactivation is involved in esophageal adenocarcinoma, we have analyzed aneuploid populations from biopsies of 35 patients with Barrett's esophagus who had premalignant epithelium, adenocarcinoma, or both. Sixteen of 35 patients (46%) had allelic loss at l8q21.1, including 7 patients who had only premalignant tissue present in their Barrett segment. In addition, three of four patients (75%) with l8q21.1 loss in their aneuploid populations had the allelic loss present in diploid cells. Mutational analysis of DPC4 did not reveal any inactivating alterations in the gene. These data indicate that allelic losses at l8q are selected during neoplastic progression in Barrett's esophagus, but the targeted gene remains to be identified.

17p (p53) allelic losses, 4N (G2/tetraploid) populations, and progression to aneuploidy in Barrett's esophagus.

Increased 4N (G2/tetraploid) cell populations have been postulated to be genetically unstable intermediates in the progression to many cancers, but the mechanism by which they develop and their relationship to instability have been difficult to investigate in humans in vivo. Barrett's esophagus is an excellent model system in which to investigate the order in which genetic and cell cycle abnormalities develop relative to each other during human neoplastic progression. Neoplastic progression in Barrett's esophagus is characterized by inactivation of the p53 gene, the development of increased 4N (G2/tetraploid) cell fractions, and the appearance of aneuploid cell populations. We investigated the hypothesis that patients whose biopsies have increased 4N (G2/tetraploid) cell fractions are predisposed to progression to aneuploidy and determined the relationship between inactivation of p53 and the development of 4N abnormalities in Barrett's epithelium. Our results indicate that increased 4N (G2/tetraploid) populations predict progression to aneuploidy and that the development of 4N abnormalities is interdependent with inactivation of the p53 gene in Barrett's esophagus in vivo.

Determination of the frequency of loss of heterozygosity in esophageal adenocarcinoma by cell sorting, whole genome amplification and microsatellite polymorphisms.

It is well established that the progression to human cancer is characterized by the evolution of clones of cells with accumulated genetic abnormalities. However, technical difficulties limit the ability to study this process in some premalignant and malignant conditions. For example, the progression to esophageal adenocarcinoma in the premalignant condition Barrett's esophagus is characterized by the evolution of genetic and cell cycle abnormalities, but it has been difficult to characterize this process completely because of the small size of biopsies and the relative abundance of genetically normal stromal cells in some esophageal adenocarcinomas and premalignant mucosa. We have combined flow cytometric cell sorting to obtain purified populations of neoplastic cells with whole genome amplification and analysis of microsatellite polymorphisms to determine the frequency of allelic loss on every nonacrocentric autosomal arm in 20 esophageal adenocarcinomas and two high-grade dysplasias. DNA samples of purified flow-sorted aneuploid and corresponding normal tissue were amplified with a degenerate 15mer primer. Aliquots of these reactions were then screened with forty-three highly polymorphic simple sequence repeat markers in PCR-based assays. Allelic losses were observed at polymorphic loci in 38 of the 40 chromosome arms that were analysed and the median fractional allelic loss (FAL) observed in the samples was 0.28. The background allelic loss frequency was estimated at 0.23 with the highest rates of loss observed at 17p (100%), 5q (80%), 9p (64%), 13q (43%), 18q (43%) and 1p (41%). These data represent the first comprehensive allelotype of esophageal adenocarcinoma and show the feasibility of multiloci analyses with small highly purified human biopsy material.

Genotypic analysis of multiple loci in somatic cells by whole genome amplification.

To screen multiple loci in small purified samples of diploid and aneuploid cells a PCR-based technique of whole genome amplification was adapted to the study of somatic lesions. DNA samples from different numbers of flow-sorted diploid and aneuploid cells from biopsies were amplified with a degenerate 15mer primer. Aliquots of these reactions were then used in locus-specific reactions using a single round of PCR cycles with individual sets of primers representing polymorphic markers for different regions. As a result, polymorphic markers for different chromosomal regions, including VNTRs and dinucleotide repeats, can be used to perform up to 30 locus-specific PCR assays with a single sample obtained from fewer than 1000 cells.

Mother-child HIV disease: a model for collaborative care.

Women Connected and the Maternal Child Clinic are two unique dyad-centered programs for providing quality care to HIV-infected women and their children. Each is a collaborative effort to maintain standard of care for these patients within a large county facility. Women Connected facilitates their inpatient treatment and care, while the Maternal Child HIV Clinic provides follow-up outpatient health and social services.

DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells.

In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild-type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziin-resistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in beta-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.