Robert J. C. Slebos

K-ras Oncogene Activation as a Prognostic Marker in Adenocarcinoma of the Lung

BACKGROUNDnThe capability of activated oncogenes to induce malignant transformation of immortalized cells in vitro has suggested that they have a similar role in the pathogenesis of human tumors. We previously found that activation of the K-ras oncogene by a point mutation in codon 12 occurs in about one third of human lung adenocarcinomas.nnnMETHODSnWe studied the clinical importance of this oncogene-activation in 69 patients with lung adenocarcinoma in whom complete resection of the tumor was possible. The polymerase chain reaction was used to amplify ras-specific sequences of DNA isolated from frozen or paraffin-embedded tumor samples. Ras point mutations were subsequently detected and classified with the use of mutation-specific oligonucleotide probes.nnnRESULTSnNineteen of the tumors harbored a point mutation in codon 12 of the K-ras oncogene. There was no association between the K-ras point mutation and the age at diagnosis, sex, or presence of previous or concurrent neoplasms. Tumors positive for K-ras point mutations tended to be smaller and less differentiated than those without mutations. The K-ras codon-12 point mutation was a strong (and unfavorable) prognostic factor: 12 of the 19 patients with K-ras point-mutation-positive tumors died during the follow-up period, as compared with 16 of the 50 patients with no mutation in the K-ras oncogene (P = 0.002). This difference in prognosis was also reflected in the duration of disease-free survival (P = 0.038) and in the number of deaths due to cancer (P less than 0.001).nnnCONCLUSIONSnThe presence of K-ras point mutations defines a subgroup of patients with lung adenocarcinoma in whom the prognosis is very poor and disease-free survival is not usually long despite radical resection and a small tumor load.

Mutational activation of the K-ras oncogene and the effect of chemotherapy in advanced adenocarcinoma of the lung: a prospective study.

PURPOSEnTo determine whether the clinical course and the response to chemotherapy of patients with advanced adenocarcinoma of the lung depends on the presence or absence of a ras mutation in the tumor. Mutational activation of K-ras is a strong adverse prognostic factor in stage I or II lung cancer and laboratory studies have suggested that ras mutations lead to resistance against ionizing radiation and chemotherapy.nnnPATIENTS AND METHODSnPatients with advanced adenocarcinoma of the lung with measurable or assessable disease received chemotherapy with mesna, ifosfamide, carboplatin, and etoposide (MICE). Archival biopsies, fresh biopsies, or fine-needle aspirations were tested for the presence of ras gene mutations. Associations of ras mutations with clinical characteristics, response to chemotherapy, and survival were studied.nnnRESULTSnThe presence or absence of ras gene mutations could be established in 69 of 83 patients (83%). A total of 261 courses of MICE were administered to 62 informative patients, 16 of whom were shown to have a K-ras mutation-positive tumor. The frequency of mutations (26%) and the type of mutations closely matched the pattern we have previously reported in operable disease. Patients with a ras mutation in their tumor were more likely to have a close relative with lung cancer, but other clinical characteristics, such as pattern of metastases, response, and survival, were similar between the ras mutation-positive and ras mutation-negative groups.nnnCONCLUSIONnPatients with advanced lung adenocarcinoma who harbor a ras mutation may have major responses to chemotherapy and have similar progression-free and overall survival as patients with ras mutation-negative tumors. K-ras mutations may represent one of several ways in which early tumors are enabled to metastasize to distant sites.

Resistance ot cisplatin and analogues: mechanisms and potential clinical implications

SummaryIn view of the important role of cisplatin (CDDP) in cancer chemotherapy, the frequent occurrence of resistance to the drug is a major clinical problem. The main cause for unresponsiveness of a tumor to CDDP is thought to be cellular drug resistance, which may be caused by (1) a decreased uptake of CDDP, (2) an increase in metallothioneins, (3) an increase in glutathione and/or glutathione-S-transferase, (4) increased DNA repair, or (5) increased tolerance to unrepaired lesions in DNA. Several mechanisms may be concomitantly operative. However, almost all data on CDDP resistance are derived from cell lines or experimental animal systems, and it is uncertain whether they are relevant for human tumors. Possible methods for overcoming CDDP resistance in cancer patients include the use of high-dose CDDP or carboplatin or of different formulations of platinum derivatives, the regional administration of CDDP, the inducement of hyperthermia, the depletion of glutathione by buthionine-S-R-sulfoximine (BSO), or the use of platinum analogues. The development of methods to detect and classify CDDP resistance in human tumor samples is urgently required for the development of modalities to overcome resistance.

A Rapid and Simple Procedure for the Routine Detection of ras Point Mutations in Formalin-Fixed, Paraffin-Embedded Tissues

The use of the polymerase chain reaction (PCR) to detect specific DNA sequences in small amounts of tissues or cells has become a widespread tool in the field of molecular biology. With the better understanding of the clinical significance of oncogene activations in human tumors, the application of PCR in a routine setting is rapidly gaining importance. We have developed a rapid and simple procedure for the detection of mutated ras oncogenes in routinely fixed, paraffin-embedded tissue samples. DNA is isolated from three 10 μm tissue sections by incubation with a nonionic detergent and proteinase K, and can be directly used for amplification by PCR. The amplified DNA fragments are then dot-blotted onto nylon membranes and are hybridized to radioactively labeled oligodeoxynucleotides, specific for each of the mutated ras sequences. After a selective washing procedure, only fully matched oligodeoxynucleotides remain bound to the membrane, thus revealing the nature of the sequences that were present in the starting material. With this method, the detection of point mutations in ras genes can be performed in a routine setting, and the results of the analyses can be available in as few as 3–4 days.

Cellular protoonocogenes are infrequently amplified in untreated non-small cell lung cancer.

To examine a potential contribution of protooncogene abnormalities other than point-mutational activation of the K-ras protooncogene in the classification of non-small cell lung cancer, amplification of cellular protooncogenes was studied in 47 lung tumour specimens obtained at thoracotomy and in four lung tumour cell lines. The primary tumours included 21 adenocarcinomas, nine large-cell carcinomas, 13 epidermoid carcinomas, one carcinoid and three metastases of primaries outside the lung. The copy numbers per haploid genome of 11 protooncogenes in every tumour sample were determined: H-ras, K-ras, N-ras, c-myc, N-myc, L-myc, erbB, mos, myb, ncu (erbB-2) and ral amplifications. The c-myc gene was amplified 5-7-fold in two adenocarcinomas, the H-ras gene 3 5-fold in one adenocarcinoma, while the K-ras and the neu gene were amplified in lung metastases from a colorectal and a breast cancer primary respectively. None of the tumours with an amplified protooncogene simultaneously harboured a mutationally activated K-ras gene. We conclude that amplification of the investigated protooncogenes is a rare event in non-small cell lung cancer. In view of the two c-myc amplifications detected, a systematic study of c-myc expression levels in non-small cell lung cancers appears worthwhile.

ras oncogene activation and the expression of ras-related genes in human lung cancer

Abstract Mutational activation of a ras oncogene is detected in about 30% of human adenocarcinomas of the lung. Most mutations involve codon 12 of the K- ras gene. K- ras mutations are rare in non-adenocarcinomas and very rare or non-existent in small-cell lung cancer. K- ras mutations occur with significantly higher frequency in smokers than in non-smokers and indicate a poor prognosis, even in patients with (clinically) early disease after apparently radical operation. It is still uncertain whether the poor prognosis is in part caused by increased resistance to radiation and/or chemotherapy of ras mutation-positive tumors. A possible role of ras -related genes that code for small monomeric GTP binding proteins in human lung cancer is under investigation. The rasac and racA genes were expressed in most, and K rev in all 16 lung cancer samples and 5 lung cancer cell lines investigated. No point mutations were revealed by Single-Strand Conformation Polymorphism (SSCP) analysis in any of these genes, but three samples had unexpected smaller-sized bands on K rev analysis, both under denaturing and non-denaturing conditions. The significance of this finding is being investigated.