David S. Reinhold

Molecular staging of colorectal cancer: K-ras mutation analysis of lymph nodes upstages Dukes B patients.

PURPOSE. Multiple attempts have been made to improve the clinical/pathologic staging system of Dukes to focus adjuvant therapy decisions. The purpose of this study was to determine whetherK-ras mutational status of regional nodes in patients with Dukes B2 colorectal cancer could be used to stage their disease more accurately. METHODS: Using formalin-fixed, paraffin-embedded archival material, tumor samples were screened forK-ras mutations using a mutation-specific polymerase chain reaction method, followed by gel electrophoresis in a 96-well array. Patients with Dukes B2 tumors that have mutations in codon 12 or 13 of theK-ras gene were identified. RESULTS: Mutational analysis of the lymph nodes from these patients revealed an 80 percent (16/20) incidence of the same mutations in regional lymph nodes. None of the four patients with mutation-free nodes developed recurrence compared with 37.5 percent (6/16) withK-ras positive lymph nodes. CONCLUSIONS: The data suggest that patients with Dukes B2 colorectal cancers that have mutations in codon 12 or 13 of theK-ras gene are at high risk for the development of nodal metastases. Mutational analysis of the lymph nodes identifies high-risk patients who should be considered for adjuvant chemotherapy. Therefore,K-ras mutational analysis should be considered for molecular staging of colorectal cancer.

Nickel subsulfide is similar to potassium dichromate in protecting normal human fibroblasts from the mutagenic effects of benzo[a]pyrene diolepoxide

The cellular response to multiple carcinogen treatment has not been extensively studied, even though the effect of individual carcinogens is, in many cases, well known. We have previously shown that potassium dichromate can protect normal human fibroblasts from the mutagenic effects of benzo[a]pyrene diolepoxide (BPDE), and that this effect may be via an oxidative stress mechanism [Tesfai et al. (1998) Mutat Res 416:159–168]. Here, we extend our previous work by showing that nickel subsulfide can produce the same effect. Normal human fibroblasts, preincubated with nickel subsulfide for 46 hr followed by a coincubation of nickel subsulfide and BPDE for 2 hr, showed a dramatic reduction in the mutant frequency of the hypoxanthine (guanine)phosphoribosyl‐transferase (HPRT) gene when compared to cells treated only with BPDE. The preincubation period with nickel subsulfide was necessary to see the antagonistic effect, since it was not observed if the cells were simply incubated with both carcinogens for 2 hr. The extent of the antagonistic effect was nickel subsulfide dose‐dependent and also appeared to be species‐specific, since the effect was not observed when Chinese hamster fibroblasts were tested. Finally, the antagonistic effect of the nickel subsulfide was eliminated by vitamin E, suggesting that production of reactive oxygen species by the nickel may be required. This data, along with our previous work, suggest that the antagonistic effect we observe is not chromium‐specific, and that it could be species‐specific. Environ. Mol. Mutagen. 33:211–218, 1999

Chromium can reduce the mutagenic effects of benzo[a]pyrene diolepoxide in normal human fibroblasts via an oxidative stress mechanism

The interaction of multiple carcinogens on human cells has not been extensively examined. This study reports the results of experiments in which normal human fibroblasts were exposed to both benzo[a]pyrene diolepoxide (BPDE) and potassium dichromate. The effect of four different treatment protocols on the cloning ability of the cells and the mutant frequency of the HPRT gene was determined. The combined treatment of both carcinogens caused a slightly greater than additive decrease in the cloning ability of the cells when compared to cells treated with the individual carcinogens. The result was the same regardless of the treatment protocol used in the experiment. The results of the mutant frequency experiments, however, varied dramatically with the protocol employed. The mutant frequency in cells which were simultaneously treated with both carcinogens was dramatically reduced from the mutant frequency found when cells were treated with BPDE alone. This antagonistic effect was not present when cells were either pretreated with potassium dichromate prior to BPDE or incubated with potassium dichromate following BPDE treatment. The observed antagonistic effect was the result of oxidative stress produced by chromium since it was completely or nearly completely reversed by the addition of either vitamin E or catalase to the cultures.