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

PURPOSE: To 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. PATIENTS AND METHODS: Patients 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. RESULTS: The 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. CONCLUSION: Patients 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.

Phase I clinical and pharmacologic study of chronic oral administration of the farnesyl protein transferase inhibitor R115777 in advanced cancer.

PURPOSE: To determine the maximum-tolerated dose, toxicities, and pharmacokinetics of R115777, a farnesyl transferase inhibitor, when administered continuously via the oral route. PATIENTS AND METHODS: Patients with advanced solid malignancies were treated with R115777 using an interpatient dose escalation scheme starting at 50 mg bid. Pharmacokinetics were assessed on days 1, 28, and 56. RESULTS: Twenty-eight patients were entered onto the study and the median duration of treatment was 55 days. The dose-limiting toxicities were myelosuppression and neurotoxicity. At a dose of 400 mg bid, grade 4 leukocytopenia and neutropenia were seen in two of four patients. Neurotoxicity grade 3 developed in one of five patients at 500 mg bid and in one of 13 at 300 mg bid after 8 weeks of treatment. Common nonhematologic toxicities were nausea, vomiting, and fatigue. The recommended dose for phase II/III testing in this scheme is 300 mg bid. The pharmacokinetic studies indicated dose proportionality. Little accumulation occurred and steady-state levels were reached within 2 to 3 days. Analyses of historic tumor material showed that five of 15 of patients had a K-ras mutation in codon 12. Three patients with pancreatic, colon, and cervix carcinomas had stable disease and one patient with a colon carcinoma had a minor response accompanied by a more than 50% decrease in carcinoembryonic antigen tumor marker. A fifth patient, with platinum-refractory non-small-cell lung cancer, showed a partial response that lasted for 5 months. CONCLUSION: Continuous dosing of R115777 is feasible with an acceptable toxicity profile at a dose of 300 mg bid.

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 microns 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.

Oligoclonal peripheral T-cell lymphocytosis as a result of aberrant T-cell development in a cortical thymoma.

A 42-year-old man presented with a locally invasive cortical thymoma. Before chemotherapy was commenced 36 months after presentation, an unusual peripheral lymphocytosis of 19 x 10(9)/l had slowly developed over time. After the first course of chemotherapy, the lymphocytosis showed a sharp decline to normal absolute cell numbers and subsequently remained at normal levels. Currently, the patient is in stable partial remission and doing well. Immunophenotypic analysis showed a mature T-cell phenotype with 78% TcR-a beta and 16% TcR-gamma delta in the absence of an immature component. Pretreatment Southern blot analysis of peripheral blood mononuclear cells showed an oligoclonal pattern with 13-20 rearranged fragments of different intensity for the TcR beta-gene. TcR gamma also showed a pattern compatible with an oligoclonal proliferation. After treatment, after normalization of absolute blood counts, the distribution of T-cell subsets still showed a slightly aberrant pattern. Immunophenotypic analysis of a blood sample taken 6 months later, also at normal absolute cell counts, showed an increase of thymocytes as well as of mature T cells with a polyclonal pattern on Southern blot analysis. These findings may be interpreted as the result of aberrant positive and negative selection and development of thymocytes in the microenvironment of neoplastic thymic epithelial cells and clonal selection through continuous peripheral stimulation. Moreover, this case stresses the importance of integrated interpretation of clinical, morphological, immunophenotypical, and molecular data to gain insight in unusual clinical problems.

Psychosocial effects of implant-retained overdentures.

The aim of this study was to compare implant-retained mandibular overdentures and two conventional treatments for their effects on functional ability (chewing, speaking, etc), patient satisfaction, and quality of life (psychosocial functioning). Assignment of 90 patients was executed by means of a balancing allocation computer program to ensure the pretreatment comparability of the three groups. Twelve months after treatment, the average scores for almost all specific quality-of-life measures had improved significantly in all three groups. On average, all patients experienced fewer restrictions in their social activities and had fewer psychological problems because of their full dentures. No impact on the general quality of life was established. One year after treatment, all three dental treatment modalities had a comparably positive effect on dental health-related quality of life.

EGFR and KRAS mutations as criteria for treatment with tyrosine kinase inhibitors: retro- and prospective observations in non-small-cell lung cancer.

Results of individualized therapy guided by mutational tumor profile of patients with non-small-cell lung cancer are presented. After confirming the importance of epidermal growth factor receptor (EGFR) and KRAS mutations for (non)response on gefitinib in a retrospective series of patients, EGFR mutations were looked for before--and were a condition for--treatment with gefitinib or erlotinib. To increase the chance to find such a mutation, we selected patients on the basis of smoking status, gender and histopathology. Out of 41 patients selected, 13 (32%) were found to harbor an EGFR mutation. In nine of them it concerned deletions in exon 19 and in none of them KRAS mutations were detected. All nine patients with an exon 19 deletion had a favorable and continuing response to tyrosine kinase inhibitors (TKIs), while four other patients with point mutations responded less favorably: stable disease or a response of short duration. These observations confirm the potential role of EGFR and KRAS mutations in predicting (non)response to TKIs. Exon 19 deletions that are associated with the best responses might be used for first-line treatment selection, while KRAS mutations could play a role in excluding patients from treatment with TKIs.

Goblet cell carcinoid of the appendix: a specific type of carcinoma.

AIMS: Goblet cell carcinoid is a poorly understood tumour of the appendix. The aim of this study was to determine whether it should be regarded as a separate entity or as a variant of classical carcinoid. METHODS AND RESULTS: The immunohistochemical expression pattern of 21 markers and the mutation status of KRas codon 12 were determined in 16 goblet cell carcinoids and compared with 14 classical carcinoids, 19 colonic adenocarcinomas and 10 appendiceal mucinous cystadeno (carcino)mas. The results were subjected to a stepwise linear discriminant analysis. Goblet cell carcinoids were significantly different from the control groups. The most important markers for discriminating between the groups were CEA (classical carcinoid versus all others), KRas mutation (present in all mucinous cystadeno (carcino)mas), beta-catenin (goblet cell carcinoid versus left sided colonic adenocarcinoma) and chromogranin (goblet cell carcinoid versus right sided colonic adenocarcinoma). Expression of Math1 and HD5 was similar in goblet cell carcinoid and colonic adenocarcinoma but absent in classical carcinoid. CONCLUSION: The results suggest that goblet cell carcinoids should be regarded as a separate entity. The formerly used term 'crypt cell carcinoma' may be more appropriate because it reflects the more aggressive clinical behaviour of these tumours as well as their greater similarity to adenocarcinomas rather than to carcinoids.

Monitoring gastric lymphoma in peripheral blood by quantitative IgH allele-specific oligonucleotide real-time PCR and API2-MALT1 PCR.

Gastric extranodal marginal zone lymphoma (EMZL) often shows prolonged localised disease, but the present study demonstrated the presence of tumour cells in peripheral blood (PB) of low stage patients. We studied the presence of tumour cells in PB in gastric lymphoma patients harbouring or lacking t(11;18)(q21;q21), by real-time immunoglobulin (Ig)H allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) and API2-MALT1 PCR. Tumour cells were exclusively detected in PB of t(11;18)(q21;q21)+-EMZL patients. The presence of tumour cells in PB and gastric biopsy follow-up samples showed a good correlation in these patients, suggesting clinical relevance for monitoring of tumour cells in PB of gastric t(11;18)(q21;q21)+-EMZL patients.

Absence of ras gene mutations in early gastric carcinomas.

The aims of this study were to assess the prevalence and type of activating point mutations at codons 12, 13, and 61 of the Ki-, Ha-, and N-ras genes in a series of early gastric carcinomas in white patients and to correlate these ras gene mutations, if any, with the histological type (Lauren classification), the type of growth pattern, and with the Helicobacter pylori status. Haematoxylin and eosin and Giemsa stained sections from 45 formalin fixed, paraffin wax embedded early gastric carcinomas were used to assess the Lauren type, the type of growth pattern, and the antral H pylori status. DNA was extracted according to standard procedures. Mutations at codon 12 of the Ki-ras gene were examined with a polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) method and dot blot hybridisation with allele-specific 32P-labelled oligodeoxynucleotide (ASO) probes. All other ras genes were analysed with specific PCR amplification and dot blot hybridisation with ASO probes. Mutations were detected by overnight autoradiography at -70 degrees C. Some 20 intestinal-type and 25 diffuse-type early gastric carcinomas were seen. According to growth pattern, there were 24 small mucosal type early gastric carcinomas, five superficial spreading type early gastric carcinomas, and 16 penetrating type early gastric carcinomas (four penetrating A type, 12 penetrating B type). H pylori was found in the antral mucosa of 28 early gastric carcinomas (62%). Activating ras gene mutations were not found. It was discovered that activating point mutations at codons 12, 13, and 61 of the Ki-, Ha-, and N-ras genes do not play a part in the development of early gastric carcinomas in white subjects, irrespective of Lauren type. Moreover, differences in biological behaviour between early carcinomas with different types of growth pattern are not related to these ras gene mutations. Finally, H pylori positive and H pylori negative gastric carcinomas cannot be discriminated on the basis of ras gene mutational analysis.

Comparative analysis of p53 gene mutations and protein accumulation in human non-small-cell lung cancer.

A series of 54 resected primary non-small-cell lung carcinomas was analyzed for p53 gene mutations and for p53 protein accumulation and the findings were correlated with clinical parameters. Mutations in exons 5 through 8 of the p53 gene were identified by a denaturing gradient gel electrophoresis (DGGE) assay and cycle sequencing, whereas p53 protein accumulation was detected in paraffin-embedded tissue by immunostaining using 2 different murine monoclonal antibodies (MAbs) (BP53-12 and DO7). A p53 gene mutation and/or p53 protein accumulation was found in 37 of 54 tumors. Mis-sense mutations were closely associated with positive immunostaining, which was intense in 15 out of 17 cases with a mutation. In 10 tumors, obvious p53 accumulation was detected in the absence of mutations in exons 5 through 8. Conversely, only one of 8 p53 non-sense mutations led to detectable p53 accumulation. The most frequent single base changes were G --> T transversions and C --> T transitions. The presence of a p53 alteration was not related to age, tumor size, stage or histology. However, we found a significant inverse correlation between p53 alterations and the presence of a K-ras mutation. This was reflected in the overall postoperative survival data: patients with p53 alterations in their tumors tended to have a better prognosis than those without a p53 alteration; however, this difference was lost when cases with a K-ras mutation were omitted from the analysis.

N- and KRAS mutations in primary testicular germ cell tumors: incidence and possible biological implications.

Recently, conflicting results have been reported on the incidence of RAS mutations in primary testicular germ cell tumors of adults (TGCTs). In four studies a low incidence of mutations (less than 15%) in a variety of TGCTs or derived cell lines was found, whereas in two other studies a high incidence of N- or KRAS mutations (over 40%) was shown. A total of 62 testicular seminomas (SE) and 34 nonseminomatous TGCTs (NS) were studied thus far. The largest series consisted of 42 TGCTs, studied on paraffin embedded tissue. We present the results of analysis for the presence of N- and KRAS mutations, in codons 12, 13, and 61, in snap frozen samples of 100 primary TGCTs, comprising 40 SE and 60 NS. Using the polymerase chain reaction (PCR) and allele specific oligonucleotide hybridization (ASO), mutations were found in five SE (three in NRAS and two in KRAS, all codon 12), and in one NS (KRAS, codon 12). To exclude underestimation of the incidence of RAS mutations in TGCTs due to the presence of an excess of wild type alleles in the analyzed sample, a PCR technique preferentially amplifying KRAS alleles with a mutation in codon 12 was applied to all SE. This approach, allowing a 250 times more sensitive assay, resulted in the detection of only one additional SE with a mutation. Based on a critical analysis of published data and on our results from the largest series of frozen samples investigated thus far, we conclude that N- or KRAS mutations are rare and apparently not essential for initiation or progression of TGCTs.