Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-08
2001-03-27
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C536S023100, C536S024300
Reexamination Certificate
active
06207376
ABSTRACT:
FIELD OF THE INVENTION
Allelic deletions on chromosome 5 were analyzed in microdissected human non-small cell lung cancers. Thirty-four primary squamous cell carcinomas, 15 primary adenocarcinomas, and 5 regional lymph node metastases were investigated for loss of heterozygosity (LOH) in chromosomal region 5p15-q21. The sites analyzed included the APC tumor suppressor gene at 5q21, five polymorphic microsatellite markers and the putative tumor suppressor locus del-27, that was assigned to chromosomal region 5p13-12 by FISH analysis. Allelic deletions encompassed larger genomic regions more often in squamous cell carcinomas than in adenocarcinomas. The del-27 and APC regions were identified as two distinct regions with the highest LOH frequencies within 5p15-q21. In squamous cell carcinomas LOH frequencies were 73% at the del-27 and 70% at the APC locus. In adenocarcinomas LOH at the del-27 and APC loci occurred in 38% of the informative cases. Allelic deletion of the APC gene and at the del-27 locus was also detected in the metastases. These results suggest involvement of at least two tumor suppressor genes from chromosome 5 in lung tumorigenesis.
BACKGROUND AND PRIOR ART
Inactivation of tumor suppressor genes appears to be the predominant genetic alteration in many solid tumors. In carcinomas inactivation of known tumor suppressor genes includes deletion of one allele and mutation of the remaining allele or homozygous deletion of both alleles (Rodriguez et al. 1994). To date, tumor suppressor genes p53, Rb1, MTS1 and MTS2 were found to be inactivated in a significant proportion of lung carcinomas (Takahashi et al. 1989; Harbour et al. 1988; Shapiro et al. 1995; Washimi et al. 1995). The MTS1 and MTS2 genes seem to be preferentially lost by homozygous deletion on chromosome 9p (Kamb et al. 1994). The p53 tumor suppressor gene on chromosome 17p and the Rb1 tumor suppressor gene on chromosome 13q are frequently inactivated by allelic deletion combined with mutations in the remaining allele (Kishimoto et al. 1992; Hensel et al. 1990). In non-small cell lung cancer (NSCLC), which is the most common type of lung cancer, allelic deletions detected as loss of heterozygosity (LOH) were reported for multiple chromosomal arms (Shiseki et al. 1994; Sato et al. 1994). This indicates inactivation of several tumor suppressor genes in lung tumorigenesis and progression. Generally, overall LOH incidences were higher in squamous cell carcinomas than in adenocarcinomas (Sato et al. 1994). More extensive analyses to determine homozygous and allelic deletions in NSCLC have been performed on chromosomes 3, 5 and 9. This led to the delineation of a minimal region of loss at 9p21, where the MTS1 and MTS2 genes are located (Merlo et al 1994). At least two regions have been identified on chromosomes 3 and 5 to harbor putative tumor suppressor genes involved in lung tumorigenesis (Yokoyama et al. 1992; Ashion-Rickardt et al. 1991; Wieland and Böhm, 1994).
On chromosome 5q allelic deletion of the known tumor suppressor gene APC at 5q21 was frequently observed in advanced NSCLC and it correlated with a poor prognosis (Hosoe et al. 1994; Fong et al. 1995). In squamous cell lung carcinomas LOH at 5q21 also correlates with tumor involvement of regional lymph nodes (Fong et al. 1995). Chromosomal region 5q33-35 is considered another target of frequent allelic deletion in advanced NSCLC (Hosoe et al. 1994). There was recently identified a region on chromosome 5 proximal to the APC gene that harbors the putative tumor suppressor locus del-27 (Wieland and Böhm, 1994). The del-27 sequence was isolated by genomic difference cloning, and it is homozygously deleted in a lung carcinoma cell line (Wieland et al. 1992 GeneBank M99171). Here it is shown by fluorescence in situ hybridization (FISH) that the del-27 locus is on chromosomal region 5p13-12. LOH at the del-27 locus occurred frequently in both, squamous cell and adenocarcinomas of the lung. LOH analysis using five polymorphic microsatellite markers in addition to polymorphic sites at the del-27 and APC loci, clearly identified the del-27 and APC region as two distinct regions with the highest LOH frequencies on chromosome 5.
SUMMARY OF THE INVENTION
The invention comprises a method of detection of polymorphism and/or loss of heterzygosity in the lung tumor suppressor gene located in chromosomal region 5p15-q21, its locus being characterized by GeneBank sequence M99171, said method being characterized by mapping human cell specimens using as probes the oligonucleotides 5′GATTTGAGGGTCATATTCA3′ (SEQ ID NO: 1) and/or 5′CAGTAAATGGTGCTTGGA3′ (SEQ ID NO:2).
Preferably, the probes are used as primers for mapping in a polymerase chain reaction.
REFERENCES:
Wieland et al, Cancer Research, vol. 54, pp 1775-1774, Mar. 1, 1994.*
Fong et al., Cancer Research, vol. 55, pp 220-223, Jan. 15, 1995.
Fulbright & Jaworski,LLP
Jones W. Gary
Roche Diagnostics GmbH
Souaya Jehanne
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