The effects of frozen tissue storage conditions on the integrity of RNA and protein.

Unfixed tissue specimens most frequently are stored for long term research uses at either -80° C or in vapor phase liquid nitrogen (VPLN). There is little information concerning the effects such long term storage on tissue RNA or protein available for extraction. Aliquots of 49 specimens were stored for 5-12 years at -80° C or in VPLN. Twelve additional paired specimens were stored for 1 year under identical conditions. RNA was isolated from all tissues and assessed for RNA yield, total RNA integrity and mRNA integrity. Protein stability was analyzed by surface-enhanced or matrix-assisted laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS, MALDI-TOF-MS) and nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). RNA yield and total RNA integrity showed significantly better results for -80° C storage compared to VPLN storage; the transcripts that were preferentially degraded during VPLN storage were these involved in antigen presentation and processing. No consistent differences were found in the SELDI-TOF-MS, MALDI-TOF-MS or nLC-ESI-MS/MS analyses of specimens stored for more than 8 years at -80° C compared to those stored in VPLN. Long term storage of human research tissues at -80° C provides at least the same quality of RNA and protein as storage in VPLN.

Analysis of chromosome 9p21 deletion and p16 gene mutation in salivary gland carcinomas.

The chromosomal locus 9p21 contains the p16(INK4a/CDKN2/MTS1) tumor suppressor gene that has been implicated in a variety of tumor types, including carcinomas of the head and neck, esophagus, and pancreas. To determine whether the loss of this gene is involved in salivary gland cancers, 35 carcinomas and paired nonneoplastic specimens were analyzed for loss of heterozygosity (LOH) of polymorphic genetic markers located in the region of interest. Five types of salivary gland tumors were studied: mucoepidermoid carcinoma, salivary duct carcinoma, adenoid cystic carcinoma, acinic cell carcinoma, and polymorphous low-grade adenocarcinoma. Seven of 9 salivary duct carcinomas showed LOH of 1 or more polymorphic markers. In 1 case of salivary duct carcinoma with LOH, we confirmed a deletion of bp 240-254 within exon 2. In addition, another salivary duct carcinoma showed a homozygous deletion of p16 in differential polymerase chain reaction analysis. Loss of heterozygosity was found in 1 of 10 adenoid cystic carcinomas and 1 of 8 mucoepidermoid carcinomas and was absent in the remaining subtypes. No mutations in exon 1 or exon 2 or homozygous deletion of p16 were found in these 2 particular neoplasms with LOH. These results suggest that inactivation of p16 is important in the development or progression of at least some salivary duct carcinomas, but we found no evidence that its alteration plays a role in the other subtypes examined.

Mapping of genetic deletions on the long arm of chromosome 4 in human esophageal adenocarcinomas.

Loss of the long arm of chromosome 4 has been identified previously as a common occurrence in adenocarcinomas of the esophagus and gastroesophageal junction by relatively low resolution genetic surveys. To better define the extent of 4q deletion in these neoplasms we isolated DNA from 29 primary carcinomas using microdissection, and used DNA obtained from xenografts of 14 carcinomas grown in immunodeficient mice in an assay of loss of heterozygosity of 25 polymorphic microsatellite markers distributed along the chromosomal arm. Two carcinomas exhibited widespread microsatellite instability and were excluded from deletion mapping. In the remaining 41 carcinomas, loss of heterozygosity was detected in 33 (80%). Twenty-three cancers showed complete or extensive reduction to homozygosity along the length of the long arm. Ten cancers had smaller discrete areas of loss and were principally useful in discerning three non-overlapping areas of consensus genetic deletion. Area 1 centered on marker D4S1534 at 4q21.1-22, area 2 centered on marker D4S620 at 4q32-33, and area 3 centered on marker D4S426 at 4q35. No known tumor suppressor genes map to these loci, but the frequent deletion of these areas in gastroesophageal carcinomas and in other carcinomas suggests that undiscovered tumor suppressor genes may reside here.

Mutations of c-kit JM domain are found in a minority of human gastrointestinal stromal tumors.

The c-kit gene encodes a transmembrane receptor kinase (KIT) which is expressed in the majority of human gastrointestinal stromal tumors (GISTs), a subtype of gastrointestinal mesenchymal neoplasms. A previous study identified mutations in the juxtamembrane (JM) domain of c-kit in five of six GISTs (Science 279: 577, 1998). To better define the frequency and spectrum of c-kit gene mutations in mesenchymal neoplasms of the GI tract that had been characterized for KIT protein expression, we examined archived tissue samples for mutations in the JM domain by PCR amplification and DNA sequencing. c-kit JM domain mutations were found in nine of 56 mesenchymal tumors (46 GISTs, eight leiomyomas, two leiomyosarcomas) and occurred exclusively in GISTs (21%). Seven of the nine mutations consisted of intragenic deletions of one to 19 codons. There was one insertion mutation that added 12 codons and one missense mutation (Val560Asp). None of the mutations disrupted the downstream reading frame of the gene. The single missense mutation (Val560Asp) is very similar to the only other missense mutation reported in GISTs (Val599Asp). Of the 46 GISTs, 43 were strongly positive for KIT protein expression and negative for diffuse expression of desmin. Neither KIT expression nor gene mutations were found in gastrointestinal leiomyomas or leiomyosarcomas. We conclude that mutation of the c-kit JM domain does not occur in gastrointestinal mesenchymal neoplasms with well developed-smooth muscle differentiation, and is restricted to GISTs. However, since these mutations are only found in a minority of GISTs, further investigation into the mechanisms of c-kit gene activation in this group of neoplasms is warranted.

Allelic deletion in 11p15 is a common occurrence in esophageal and gastric adenocarcinoma.

BACKGROUND: Esophageal adenocarcinoma and gastric adenocarcinoma have distinct epidemiologic characteristics, but they are morphologically identical and sometimes clinically indistinguishable. Recent work in molecular oncology suggests that cancer types have distinct molecular genetic profiles that may explain their biologic differences. Gastric adenocarcinoma has previously been shown to have a relatively high rate of deletion in chromosome 11. To determine whether similar genetic loci are involved in esophageal adenocarcinoma, we assayed for genetic loss in chromosome 11 in samples of these cancers. METHODS: Dissection of neoplastic and nonneoplastic tissue was performed under direct microscopic visualization from histologic sections of 15 gastric adenocarcinomas and 15 esophageal adenocarcinomas. After DNA extraction, polymerase chain amplification products of a series of polymorphic microsatellite markers on chromosome 11 were analyzed by polyacrylamide gel electrophoresis. Tumor specific chromosomal deletion was signaled by the loss of microsatellite alleles. RESULTS: A panel of 3 polymorphic markers in 11p15 revealed overall incidences of loss of heterozygosity (LOH) of 53.3% in esophageal adenocarcinomas and 61.5% in gastric adenocarcinomas. A panel of 3 polymorphic markers in 11q22-23.3 revealed overall incidences of LOH of 14.3% in esophageal adenocarcinomas and 31% in gastric adenocarcinomas. Diffuse microsatellite instability, which was consistent with a replication error phenotype, was found in 2 of 15 (13.3%) gastric adenocarcinomas but was not deleted in esophageal adenocarcinomas. CONCLUSIONS: Esophageal and gastric adenocarcinoma have a similar significant incidence of genetic loss in 11p15. This is suggestive of the presence of a tumor suppressor gene that may be inactivated in both tumor types.