Origin and prognostic value of circulating KRAS mutations in lung cancer patients.

Because of the current controversy on the origin and clinical value of circulating KRAS codon 12 mutations in lung cancer, we screened 180 patients using a combined restriction fragment-length polymorphism and polymerase chain reaction (RFLP-PCR) assay. We detected KRAS mutations in 9% plasma samples and 0% matched lymphocytes. Plasma KRAS mutations correlated significantly with poor prognosis. We validated the positive results in a second laboratory by DNA sequencing and found matching codon 12 sequences in blood and tumor in 78% evaluable cases. These results support the notion that circulating KRAS mutations originate from tumors and are prognostically relevant in lung cancer.

Combination of EGFR gene copy number and protein expression predicts outcome for advanced non-small-cell lung cancer patients treated with gefitinib.

BACKGROUND: Biological markers for optimal selection of patient to epidermal growth factor receptor (EGFR)-targeted therapies are not established in advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: EGFR/HER2 gene copy number by FISH, EGFR protein and pAKT expression by immunohistochemistry (IHC) and EGFR and KRAS mutations were tested in 204 gefitinib-treated NSCLC patients. RESULTS: Increased EGFR and HER2 gene copy number (FISH+), EGFR protein overexpression (IHC+), EGFR mutations and pAKT overexpression were all associated with significantly higher response rates (33%, 29%, 22%, 39% and 20% respectively). EGFR FISH+ (32%) and IHC+ (61%) correlated with improved survival, while EGFR mutations (27%), KRAS mutations (26%) and pAKT expression (69%) did not. In multivariate survival analysis EGFR FISH and IHC were independent predictive markers. EGFR FISH+/IHC+ patients (23%) had a median survival of 21 months versus 6 months for double-negative patients (30%). CONCLUSION: Combination of EGFR FISH and IHC is effective predictor for benefit from gefitinib. Patients with double-negative results are unlikely to benefit in western NSCLC populations.

Abnormalities of apoptotic and cell cycle regulatory proteins in distinct histopathologic components of benign prostatic hyperplasia.

INTRODUCTION: Benign prostatic hyperplasia (BPH) is a slowly progressive abnormal glandular enlargement with heterogeneous morphology. Disruption of apoptotic pathways has been suggested as an important regulatory mechanism in this common and significantly morbid disease. METHODS: Prostatic tissue from 20 patients with BPH and no prior or subsequent prostatic carcinoma was obtained by transurethral prostatectomy (TURP) at the University of California Davis. Apoptotic regulatory proteins: BCL2, BAX and p27 were analyzed by immunohistochemistry and evaluated for expression in four distinct histologic patterns: hyperplastic epithelium, nodules, dilated glands and atrophic/inflammatory glands. RESULTS: Particularly striking was the decreased expression of BAX and an abnormal BCL2 : BAX ratio within all nodules relative to expression in other epithelial patterns. p27 expression was decreased in 35% of the hyperplastic epithelial areas and 10% of the nodules. DISCUSSION: Overall, abnormal expression of BCL2, BAX and/or p27 was identified in the hyperplastic epithelium of 19 (90%) of specimens and all 12 (100%) of the hyperplastic nodules. The high frequency of abnormalities in apoptosis regulatory genes, suggests that alteration of apoptotic pathways is important for the development of this condition.

Activity of high-dose toremifene plus cisplatin in platinum-treated non-small-cell lung cancer: a phase II California Cancer Consortium Trial.

PURPOSE: Although cisplatin is an important agent in non-small-cell lung cancer (NSCLC), de novo resistance is common and acquired resistance emerges rapidly during therapy. Proposed mediators of platinum resistance include the protein kinase C (PKC) signal transduction pathway and associated c-FOS overexpression. While estrogen administration has been reported to upregulate PKC and c-FOS expression, the triphenylethylenes tamoxifen and toremifene potentiate platinum cytotoxicity by inhibition of PKC. Downregulation of c-FOS expression has been reported to result from PKC inhibition. In view of these findings, we hypothesized that toremifene would reverse platinum resistance and that this interaction would be influenced by tumor estrogen receptor (ER) status. MATERIALS AND METHODS: A phase II trial of high-dose toremifene (600 mg orally daily on days 1-7) plus cisplatin (50 mg/m2 intravenously on days 4 and 11) every 28 days in NSCLC patients was conducted. A group of 30 patients with metastatic NSCLC who had been previously treated with platinum-based therapy were enrolled. RESULTS: All of the 30 patients were assessable for toxicity and 28 for tumor response. Therapy was well tolerated with minimal hematologic and non-hematologic toxicity. Common toxicity criteria grade 3 hematologic toxicity was seen in only three patients. Five patients achieved a partial response for an overall response rate of 18% (95% CI 6-37). Median overall survival was 8.1 months (95% CI 5.4-17). To assess PKC, ER, and c-Fos expression by immunohistochemistry, 12 informative pretreatment patient tumor specimens were obtained. Four patient tumor specimens were positive for one or both PKC isoforms (alpha and epsilon) while c-Fos was overexpressed in three. None of the responding patient tumors exhibited c-FOS or PKC-epsilon overexpression. ER expression was found to be infrequent (8%), contrasting with previous reports in this tumor type. CONCLUSION: While this phase II study indicates that high-dose toremifene plus cisplatin is feasible, active, and well tolerated in NSCLC patients previously treated with platinum compounds, the mechanism of action remains unclear. Further study of this regimen is warranted.

Apoptosis-related gene and protein expression in human lymphoma xenografts (Raji) after low dose rate radiation using 67Cu-2IT-BAT-Lym-1 radioimmunotherapy.

Despite low radiation dose rates, radioimmunotherapy (RIT) has proven particularly effective in the treatment of malignancies, such as lymphoma. Apoptosis has been suggested to be a major mechanism for cell death from continuous low-dose rate radiation from radioimmunotherapy. The goal of this study was to examine Raji lymphoma xenografts for induction of apoptosis and modulation of apoptosis-related gene and protein expression in response to 67Cu-2IT-BAT-Lym-1 RIT. In preclinical and clinical trials, 67Cu-2IT-BAT-Lym-1 has shown an exceptionally long tumor residence time associated with substantial cumulated radiation doses. The Raji model mirrors human lymphomas that have mutant p53 and increased BCL2 expression. Untreated athymic BALB/c nu/nu mice and mice treated with 400 micrograms Lym-1, or 335-500 microCi 67Cu on less than 400 micrograms Lym-1 antibody, were observed for toxicity and response over 84 days. Subgroups of 4-5 mice were sacrificed at 3, 6 and 24 h after therapy so that tumors could be examined for poly(ADP-ribose) polymerase (PARP) and DNA ladder evidence for apoptosis and for BCL2, p53, p21, GADD45, TGF-beta 1 and c-MYC gene and protein expression. Untreated tumors had little evidence of apoptosis and Lym-1 had no effect on apoptosis or gene expression. 67Cu-2IT-BAT-Lym-1 RIT induced an overall response rate of 50% with tolerable toxicity, and 29% of the tumors were cured at cumulated tumor radiation doses of about 1800 cGy. Apoptosis was greatly increased in the RIT treated Raji xenografts as evidenced by cleavage of PARP to the characteristic 85 kD fragment at 3 and 6 h and by the DNA cleavage pattern. BCL2 gene and protein expression were substantially decreased at 3 and 24 h, respectively, after 67Cu-2IT-BAT-Lym-1 RIT despite only modest cumulated radiation doses (56 cGy at 3 h). Evidence for apoptosis preceded tumor regression by 4-6 days. In these therapy-resistant, human lymphoma tumors treated with 67Cu-2IT-BAT-Lym-1, apoptosis was convincingly demonstrated to be a major mechanism for the effectiveness of RIT and occurred by p53-independent mechanisms.

Advances in the treatment of non-small cell lung cancer: molecular markers take the stage.

Increasing evidence that non-small cell lung cancer is a systemic disease from the outset confirms the rationale for adjuvant chemotherapy. However, clinical trial evidence of benefit is still awaited. The position is clearer in the case of neoadjuvant therapy because long-term follow up of two trials now shows that patients randomized to chemotherapy before surgery were significantly more likely to survive to 5 years than patients treated with surgery alone. Early data suggest that neoadjuvant chemotherapy based on docetaxel (Taxotere; Aventis, Antony, France) (possibly used sequentially with other agents) may be as effective as older regimens and better tolerated. Because p53 status influences the expression of microtubule-associated proteins and hence the sensitivity of a tumor to taxanes, it is possible that molecular markers could be used to customize chemotherapy to individual patients. Generally, it is becoming clearer that molecular staging is a more sensitive means of demonstrating tumor dissemination than light microscopy. The Cancer and Leukemia Group B is undertaking a prospective study using reverse transcriptase-polymerase chain reaction to detect MUC-1 RNA in bone marrow and hilar and mediastinal lymph nodes removed at resection with the aim of distinguishing between stage I patients likely to remain disease-free for long periods and those at high risk of relapse. A study of small cell lung cancer is using automated fluorescence microscopy to detect keratin-positive cells in the marrow and blood of patients who have a complete response to initial therapy but are nevertheless at high overall risk of relapse. The identification of genetic lesions in a high proportion of patients with non-small cell lung cancer may guide the development of new therapies aimed at increasing rates of apoptosis among tumor cells.

BCL2 antisense transcripts decrease intracellular Bcl2 expression and sensitize LNCaP prostate cancer cells to apoptosis-inducing agents.

Prostate cancer (CaP) is the most commonly diagnosed cancer of aging men and the second leading cause of male cancer death in the United States. At present, no effective therapy is available for treating hormone independent CaP. Since Bcl2 is believed to play a role in protecting CaP cells from apoptosis, we investigated the effects of down-regulating Bcl2 expression on CaP cells. Genetically engineered LNCaP sublines were established by stably transfecting LNCaP cells with BCL2 antisense (BCL2-AS) transcript-expressing plasmids. Western blotting analysis showed that intracellular Bcl2 protein was decreased by 50-60% in BCL2-AS-transfected LNCaP cells. Expression of the antisense transcripts resulted in 50% growth inhibition of LNCaP cells in response to androgen withdrawal and markedly sensitized these cells to Adriamycin-induced apoptosis. These results suggest that down-regulation of Bcl2 protein using BCL2-AS transcripts could be exploited for improved treatment of advanced CaP.

3-Methylcholanthrene triggers the differentiation of alveolar tumor cells from canine bronchial basal cells and an altered p53 gene promotes their clonal expansion.

Alveolar type II cells arising in canine bronchial autografts following exposure to 3-methylcholanthrene (MCA) give rise to carcinomas of varying glandular and squamous growth patterns. To study the role of the tumor suppressor gene p53 in this process, sections from progressive lesions were immunostained for p53 protein; microdissected regions were screened for p53 mutations. Adjacent sections were examined for type II cell expression [cuboid cell shape, large roundish nucleus, cytoplasmic staining for surfactant protein A (SP-A)] and proliferating cell nuclear antigen expression. Evidence for an altered p53 function (nuclear staining, missense mutations) was found in most carcinomas of all histologic types and in all grades of bronchial dysplasia, but not in hyperplastic or normal bronchial epithelium. It was primarily associated with the hyperplastic type II cell populations present in the basal zone of the lesions. In addition, we found SP-A staining in hyperplastic (but not in normal) bronchial basal cells. These data suggest that MCA initiates type II cell differentiation through phenotypic selection (basal cells). Inactivation of the p53 gene promotes the clonal expansion of the type II cells into discernible populations of (squamous or glandular) alveolar tumor cells. This in vivo study is the first to show that p53 is involved in a specific pathway leading to bronchogenic carcinoma.

Anaerobic bacteria and intrahepatic stones: detections of Clostridium sp. and Bacteroides fragilis.

OBJECTIVE: To detect anaerobic bacteria Clostridium sp. and Bacteroides fragilis in intrahepatic stones by molecular genetic method. METHODS: DNA was extracted from 59 stone samples and subjected to polymerase chain reaction (PCR) amplification targeting the 16S rRNA gene of Clostridium sp. and the glutamine synthetase gene of Bacteroides fragilis. Single-strand conformational polymorphism (SSCP) analysis was performed to identify the Clostridium sp. RESULTS: 16S rRNA gene sequences for Clostridium sp. were identified in 49 stones (83%, 49/59). The two most common groups were detected in 19 (41%) and 17 (37%) of the 46 samples using SSPC analysis, and 25/59 (42%) stones were tested positive for Bacteroides fragilis. CONCLUSIONS: Anaerobes such as Clostridium sp. and Bacteroides fragilis present in intrahepatic stones and may play a role in stone formation. PCR is a useful technique to detect fastidious pathogens, which are difficult to culture. SSCP of PCR products is a rapid method in differentiating bacterial species.

Effect of 67Cu-2IT-BAT-Lym-1 therapy on BCL-2 gene and protein expression in a lymphoma mouse model.

Radioimmunotherapy using monoclonal antibodies against tumor-associated antigens has been particularly promising in the treatment of radiosensitive malignancies such as lymphoma. 67Cu has excellent physical and biochemical properties for radioimmunotherapy. 67Cu-2IT-BAT-Lym-1 has been used in preclinical and clinical trials, where an exceptionally long residence time of 67Cu on tumor was observed. BCL-2, a proto-oncogene that promotes cell survival by blocking apoptotic cell death, is overexpressed in most B-cell lymphomas including Raji human Burkitt's lymphoma cells. In this study, therapeutic efficacy and BCL-2 gene and protein expression levels were examined in Raji xenografts in mice after 67Cu-2IT-BAT-Lym-1 radioimmunotherapy. 67Cu-2IT-BAT-Lym-1 therapy induced a response rate (complete and partial responses) of approximately 50%. BCL-2 gene expression was decreased 3 h after radioimmunotherapy, followed by a decrease in Bcl-2 protein by 24 h. Decreases in BCL-2 gene and protein expression preceding observations of 67Cu-2IT-BAT-Lym-1 therapeutic effect suggest that down-regulation of BCL-2 leaves cells more likely to be killed by low dose-rate radiation from radioimmunotherapy.

Use of a yeast assay to detect functional alterations in p53 in prostate cancer: review and future directions.

BACKGROUND: While many studies have suggested that p53 mutations are common in human cancers, the functional activity of these mutant alleles has not yet been fully addressed. We believe that information about the functional status of individual p53 mutants will prove to be important for a better understanding of the role of p53 in tumor development and progression. Ultimately, this information could also influence treatment decisions for individual cancer patients. METHODS: A recently developed yeast functional assay can be used to assess the transactivational activity of p53 mutants. Furthermore, this assay is more sensitive than single strand conformational polymorphism (SSCP) for detection of p53 mutations. In this review, we summarize the mechanism of this new technique and describe its applications in cancer research, with an emphasis on prostate cancer. RESULTS: The use of the yeast functional assay provides a simple, sensitive, and reproducible method for detecting p53 mutations and for determining the transactivational activity and dominant-negative role of individual p53 mutants. CONCLUSIONS: This method may be adapted to analyze other transcriptional factors, including the human androgen receptor.

RB status as a determinant of response to UCN-01 in non-small cell lung carcinoma.

7-Hydroxystaurosporine (UCN-01), a protein kinase inhibitor in clinical development, demonstrates potent antineoplastic activity. To determine whether specific genetic abnormalities would modulate the response to UCN-01, a model of human non-small cell lung carcinoma (NSCLC) cell lines with differential abnormalities of p16CDKN2, RB, and p53 was used for these studies. Cell growth was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and cell cycling was studied using flow cytometric analysis of DNA content. Changes in protein levels and phosphorylation were assessed by Western blotting. In cell lines expressing wild-type RB (A549 and Calul), UCN-01 treatment resulted in dose-dependent growth inhibition, arrest of cells in G1, and a reduction of cells in S phase. p16CDKN2-null cells showed similar growth inhibition to normal fetal lung fibroblasts. UCN-01-induced growth arrest was accompanied by induction of p21CDKN1 and a shift of Rb to the hypophosphorylated state in both p53 wild-type and mutant cell lines. In contrast, UCN-01 treatment of the RB-null cell line H596 resulted in less growth inhibition. To test the role of RB in response to UCN-01, effects of treatment were examined in two human isogenic models of RB expression: the bladder cancer cell line 5637 (RB-null) and the prostate cancer cell line DU-145 (RB-mutant). In the Rb-expressing 5637 subline (RB5), UCN-01 treatment resulted in Rb hypophosphorylation and an accumulation in G1 in contrast to the parent line. Similarly, the wild-type Rb-expressing DU-145 sublines (DU1.1 and B5) showed increased G1 arrest compared with the parent cells. We conclude that UCN-01-induced G1 arrest can occur in cells null for p53 and p16CDKN2, and that RB status influences the ability of UCN-01 to induce a G1 arrest. These data suggest that the molecular profile of cell cycle regulating genes in individual tumors may predict responsiveness and provide insight into optimal therapeutic application of this new antineoplastic agent.

Overexpression of cyclin D1 is rare in human prostate carcinoma.

BACKGROUND: Overexpression of cyclin D1 has been documented in a number of human cancers. Increased expression of cyclin D1 can contribute to cellular transformation and abnormal proliferation. METHODS: Quantitative RT-PCR and/or Western blot analysis were used to determine the level of cyclin D1 expression in 96 human prostate tumors, 15 benign prostate hyperplasias, 4 prostate cancer cell lines, and 3 xenografts. RESULTS: Our results demonstrate that 4.2% of the prostate tumors examined overexpressed cyclin D1 transcripts. In the cell lines, expression was normal, with the exception that reduced levels of cyclin D1 transcript and protein were observed in the DU145 cell line, as expected from cells with mutant RB. Normal levels of cyclin D1 were found in all xenograft tumors and BPH specimens examined. CONCLUSIONS: These data show that overexpression of cyclin D1 occurs rarely in human prostate tumors. However, when overexpression of cyclin D1 does occur, it may identify a subset of tumors with a different molecular biology.

Efficient recombination of Lym-1 scFv gene using multiple doubly-restricted DNA fragments.

In order to improve radioimmunotherapy of lymphoma, a Lym-1 single-chain antigen-binding (scFv) protein molecule was produced. Because the commonly used polymerase chain reaction (PCR) method frequently causes unexpected mutations, we developed a non-PCR method for scFv gene assembly. The method involved a stepwise linkage of doubly-restricted DNA fragments and re-digestion of the resultant concatamers. Using this strategy, the Lym-1 scFv expression gene was readily constructed without mutations. The recombinant gene was cloned into an expression vector and scFv protein was expressed. The method can be used for other genes or DNA recombination.

Tumors of the pancreas with osteoclast-like and pleomorphic giant cells: an immunohistochemical and ploidy study.

BACKGROUND: Tumors of the pancreas with osteoclast-like giant cells are of uncertain histogenesis and aggressiveness. Their relationship, if any, to undifferentiated (anaplastic) carcinomas of the pancreas with pleomorphic giant cells is also not clear. METHODS: Eleven tumors with osteoclast-like giant cells were studied by immunohistochemistry for epithelial and mesenchymal markers, as well as for a proliferation marker (Ki67) and p53 protein expression. Cytometric image analysis for nuclear DNA content was also performed. K-ras mutations were investigated by DNA sequence analysis. RESULTS: Neoplastic, predominantly spindle-shaped cells and osteoclast-like giant cells were positive for mesenchymal markers CD68, LCA, and A1ACT. These spindle-shaped cells were also positive for human muscle actin. Spindle-shaped cells of seven tumors were also positive for epithelial markers carcinoembryonic antigen, epithelial membrane antigen, or keratin. Nine tumors contained a variable number of pleomorphic giant cells in addition to osteoclast-like giant cells. Pleomorphic giant cells were much less positive for mesenchymal markers than were osteoclast-like giant cells, but they were positive for some epithelial markers. A high percentage of spindle-shaped and pleomorphic giant cells were positive for Ki67. Diploid and aneuploid populations were present in varying proportions in both spindle cells and pleomorphic giant cells. The nuclei of osteoclast-like giant cells, however, were diploid and not proliferating. Spindle-shaped and pleomorphic giant cells were positive for p53 protein in 5 of 10 cases. Five of six tumors studied were positive for K-ras mutations. CONCLUSION: The distinction between tumors with osteoclast-like giant cells and undifferentiated carcinomas with pleomorphic giant cells is often not clear-cut. Both types of tumors have mesenchymal and epithelial characteristics in varying proportions and may arise from an undifferentiated pancreatic stem cell. Long-term survival of two patients suggests that some tumors with osteoclast-like giant cells may have a better prognosis than the usual pancreatic ductal adenocarcinoma.

Yttrium-90-DOTA-peptide-chimeric L6 radioimmunoconjugate: efficacy and toxicity in mice bearing p53 mutant human breast cancer xenografts.

UNLABELLED: The novel radioimmunoconjugate, 90Y-DOTA-peptide-chimeric L6 (ChL6), was designed to reduce radiation to critical normal tissues with an exceptionally stable 90Y chelate moiety and a biodegradable linker. Human breast cancer tumors (HBT 3477) in mice were treated with 90Y-DOTA-peptide-ChL6 to examine the effects of increasing dose on the therapeutic efficacy and toxicity of this new agent. METHODS: Groups of athymic mice bearing HBT 3477 xenografts received 4.1- to 14.1-MBq doses of 90Y-DOTA-peptide-ChL6 intravenously. The lethal dose (LD)(50/30), general well-being (weight loss), hematotoxicity and therapeutic efficacy were studied. RESULTS: The LD(50/30) was 12.8 MBq, which corresponded to doses of 17.9 and 50.9 Gy to the total body and tumor (200 mm3), respectively. Deaths were associated with hematotoxicity; no deaths occurred at doses of 9.6 MBq or less. At sublethal doses, the rate of tumor response (cures +/- complete responses + partial responses) increased with increasing dose: 4.1 MBq, 27%; 5.9 MBq, 41%; 8.5 MBq, 69%; and 9.6 MBq, 79% (maximum tolerated dose, MTD). In mice receiving doses of 4.1-9.6 MBq, 6 of 74 (8%) of tumors were cured. Increasing the 90Y dose led to smaller tumor size at nadir and longer tumor regrowth delay but no increase in cure. Although the HBT 3477 p53 gene was found to be mutant resulting in p53 protein not binding DNA breaks, tumors at MTD demonstrated evidence of apoptosis. CONCLUSION: In the human breast cancer athymic mouse model, 90Y-DOTA-peptide-ChL6 had a high therapeutic index and LD(50/30) leading to a 79% response rate at the MTD. The evidence of apoptosis as a mechanism for this tumor response in p53 mutant breast cancer warrants further studies because these observations are relevant to the treatment of lethal breast cancer.