Genomic DNA and messenger RNA expression alterations of the CDKN2B and CDKN2 genes in esophageal squamous carcinoma cell lines.

The genes CDKN2B (MTS2) and CDKN2 (MTS1) encoding the proteins p15 and p16 are both located on chromosomal band 9p21, a locus at which frequent homozygous and heterozygous deletions occur in many primary human tumors, including esophageal carcinoma. CDKN2 and CDKN2B belong to a family of cyclin-dependent kinase 4 inhibitors (INK41) and control cell proliferation during the G1 phase of the cell cycle. Their inactivation may contribute to uncontrolled growth in human cancers. To investigate whether CDKN2B and CDKN2 are involved in esophageal tumorigenesis, we studied homozygous deletion, intragenic mutation, and messenger RNA (mRNA) expression of CDKN2 and CDKN2B in nine esophageal squamous cancer cell lines. Polymerase chain reaction (PCR) amplification revealed that five of the nine cell lines (55%) manifested homozygous deletions of CDKN2B, CDKN2, and/or flanking loci on chromosomal band 9p21. Reverse transcriptase-PCR (RT-PCR) was used to examine CDKN2 and CDKN2B mRNA in the nine cell lines. Lack of CDKN2 and CDKN2B mRNA correlated perfectly with homozygous deletion involving these genes. No subtle intragenic mutations of CDKN2B or CDKN2 were detected by DNA sequencing of their entire coding sequences in any cell lines lacking homozygous deletion. Two of the cell lines manifested homozygous deletions excluding CDKN2; one of these two deletions also excluded CDKN2B. These results suggest that inactivation of CDKN2B and CDKN2 may contribute to the malignant phenotype in esophageal cells and that homozygous deletion may be the predominant mechanism for inactivation of CDKN2B and CDKN2. Alternatively, a gene or genes adjacent to CDKN2B/CDKN2 may constitute the target(s) of deletion at this locus.

Adenomatous polyposis coli gene mutations in ulcerative colitis-associated dysplasias and cancers versus sporadic colon neoplasms.

Adenomatous polyposis coli (APC) gene mutations occur in most sporadic colonic adenomas and carcinomas. Precursor lesions of ulcerative colitis (UC)-associated colon carcinomas, although morphologically similar to sporadic adenomas, may be biologically distinct from them and are, in fact, managed differently. Since sporadic adenomas may also occur in UC, a method of discriminating between these forms of neoplasia could have clinical utility. We examined 33 patients with UC-associated dysplasias and cancers and 23 sporadic colon neoplasms in a side-by-side comparison for APC mutations. Codons 686-1693, containing 64% of all reported APC mutations (the mutation cluster region), were screened for truncating mutations using an in vitro synthesized protein assay. Two of thirty-three patients (6%) with UC-associated dysplasias and cancers had a total of three truncating APC mutations, all in frank carcinomas, while 17 of 23 (74%) with sporadic colonic neoplasms had mutations. DNA sequencing confirmed two mutations in codon 1460, replacing arginine with a stop codon, as well as one 2-base pair deletion, resulting in a frameshift and a stop at codon 1477. One specimen contained one each of these APC mutations. This apparent contrast in mutation rates at the mutation cluster region of APC is consistent with other biological characteristics separating sporadic colon neoplasms from UC-associated dysplasias and cancers. These data raise the possibility that nonadenomatous UC dysplasias may arise by a molecular pathway distinct from that prevailing in sporadic colon carcinogenesis, and they suggest a molecular assay to discriminate between sporadic adenomas and dysplasias occurring in UC.

The MTS1 gene is frequently mutated in primary human esophageal tumors.

Homozygous and heterozygous deletions involving chromosome 9p21 have been reported in a variety of primary human tumors in vivo, and point mutations have been reported in melanoma cell lines in vitro within a probable tumor suppressor gene, MTS1, located at chromosome 9p21. We describe six sequence alterations occurring among twenty-four primary esophageal squamous carcinomas and nineteen primary esophageal adenocarcinomas analyzed by DNA sequencing of MTS1 exon 2. Nucleotide substitutions were observed in five squamous cell carcinomas and in one adenocarcinoma. Two occurred in the germline, while four were somatic alterations. All six nucleotide changes resulted in marked alterations in amino acid sequence. Four were nonsense mutations leading to premature termination codons; nucleotide substitutions identical to two of these stop codons were previously reported in other tumor types. Loss of heterozygosity occurred in all five informative (constitutionally heterozygous) cases in which a sequence alteration was present. Esophageal cancer is one primary human tumor in which MTS1 constitutes an apparent target of heterozygous or homozygous deletions occurring at chromosome 9p21.

Frequent loss of heterozygosity on chromosome 9 in adenocarcinoma and squamous cell carcinoma of the esophagus.

Loss of heterozygosity (LOH) affecting chromosome 9p has been shown to occur frequently in head and neck cancer, glioma, mesothelioma, melanoma, lung cancer, and numerous other tumor types. Chromosome 9p is therefore presumed to contain a tumor suppressor gene or genes. Since esophageal cancer shares characteristics with some of the above tumor types, we performed a detailed examination of 60 patients with squamous cell carcinoma or adenocarcinoma of the esophagus for LOH at loci D9S162, IFNA, D9S171, D9S126, D9S104, D9S165, and D9S163. Multiplex polymerase chain reactions were performed with the inclusion of one radiolabeled nucleotide, and products were electrophoresed on denaturing polyacrylamide gels. Thirty-six of the 60 patients (60%) exhibited LOH at one or more loci on chromosome 9p. Eight of 17 patients (47%) with adenocarcinoma manifested LOH, while 28 of 43 (65%) with squamous cell carcinoma showed LOH. LOH was most frequent at loci D9S171 (19 of 23, or 83%) and D9S165 (24 of 32, or 75%). These data support the hypothesis that a tumor suppressor gene or genes located on this portion of chromosome 9p exert(s) an effect on esophageal cancer development.

Microsatellite instability in ulcerative colitis-associated colorectal dysplasias and cancers.

Microsatellites are short nucleotide repeat sequences present throughout the human genome. Alterations of microsatellites, comprising extra or missing copies of these sequences, have been termed microsatellite instability. This abnormality occurs in sporadic and hereditary adenocarcinomas of the proximal colon, as well as in many other tumor types. We determined whether microsatellite instability occurred in ulcerative colitis-associated cancers or precancerous dysplasias. Sixty-three patients were evaluated, consisting of 188 samples of genomic DNA (63 normal controls, 68 cancers, 52 dysplasias, and 5 adjacent tissues) at loci D2S119, D2S123, D2S147, D10S197, and D11S904. Multiplex polymerase chain reaction was performed using one radiolabeled nucleotide, and the products were electrophoresed on denaturing polyacrylamide gels. Seventeen of the 63 patients (27%) possessed lesions showing instability at 1 or more loci. Fourteen of 68 tumor samples (21%) and ten of 52 dysplasias (19%) displayed instability. There was no tendency for a greater number of loci to manifest instability in more advanced lesions. Neither anatomic location nor loss of heterozygosity at the p53 locus were associated with microsatellite instability by 2-way table analysis. These data support a role for defective DNA repair in the generation of a subset of both early and advanced ulcerative colitis-associated colorectal neoplastic lesions.

In vivo immunization against autologous glioblastoma-associated antigens.

A glioblastoma patient was immunized in vivo with a mixture of autologous and homologous glioblastoma cells coupled to adjuvant peptide and cord-factor analog. Immune activity of peripheral blood lymphocytes was measured in a short-term 51chromium-release assay against autologous tumor target cells. The patient developed direct cell-mediated cytotoxicity against tumor-associated antigens, which appeared to be T-cell mediated.

T cell nature of adjuvant peptide--induced antitumor cell-mediated cytotoxicity.

The direct cell-mediated antitumor cytotoxicity induced by coupling a murine methylcholanthrene-induced sarcoma with N-acetylmuramyl-L-alanyl-D-isoglutamine (adjuvant peptide) was eliminated by treatment of effector cells with anti-Thy 1.2 or anti-Lyt 2.2 monoclonal antibody and complement. Cytotoxicity was not H-2 restricted.

Use of covalently bound cord factor analog to increase tumor immunogenicity.

Mice were immunized in vitro and in vivo against the tumor-associated antigens of a methylcholanthrene-induced tumor, using immunogen cells coupled to a cord factor analog (trehalosedihemisuccinatemonohexadecylamide). Immune activity was measured with a short-term 51Cr-release assay. The cord factor analog potentiated immunity induced by adjuvant peptide.

In vitro immunization against autologous glioblastoma cells coupled to adjuvant peptide.

A glioblastoma patient was immunized in vitro against autologous tumor cells. Tumor cell immunogens coupled to adjuvant peptide induced direct cell-mediated cytotoxicity, which was measured with a short-term 51chromium release assay. Plain tumor cells were non-antigenic. Cytotoxicity was largely eliminated by erythrocyte-sedimentation of effectors. These results suggest the feasibility of immunizing glioblastoma patients against autologous tumor-associated antigens.

Hyperreactivity to myelin basic protein in multiple sclerosis.

Multiple sclerosis patients, control patients with other neurological diseases, and normal volunteers were assayed in a short-term 51chromium release assay for cell-mediated cytotoxicity against lymphocyte targets coated with myelin basic protein. Multiple sclerosis patients, compared to the other two groups, were hyperreactive to myelin basic protein, both before and after in vitro boost with additional myelin basic protein. The boost served to augment the difference between multiple sclerosis patients and controls.