Cell fusion: an approach to generating constitutively proliferating human tumor antigen-presenting cells.

Somatic cell hybrids of HLA-A2(+) EBV-transformed B- or dendritic cells (DC) and allogeneic HLA-A2(-) melanoma cell line Me15 were obtained by in vitro electrofusion using an electroporator. Before fusion, melanoma cells were stably transfected with green fluorescent marker protein (GFP) and neomycin resistance gene (neo(+)). Stably growing hybrid antigen-presenting cells (HAPC) expressing HLA-DR and HLA-A2 (or HLA-A30/31), and melanoma-associated antigens (MART-1, gp100) were selected by a double strategy of immunomagnetic MACS and neomycin selection. Fusion efficiency ranged between 3% and 18% (mean: 8.0+/-4.7%) as defined by simultaneous GFP and HLA-A2 detection. Expression of melanoma-associated antigens (MART-1, gp100) in hybrid cells was determined by reverse transcription-polymerase chain reaction (RT-PCR). HLA-restricted antigen-specific presentation of melanoma antigens was demonstrated by killing of semi-allogenic HAPC by HLA-A2-restricted MART-1 or gp100-specific cytotoxic T lymphocyte (CTL) clones. HLA restriction and antigen specificity were confirmed by inhibition of specific cytotoxicity by anti-HLA antibodies and cold target inhibition. During long-term (42-70 days) neomycin selection of HAPC, a drastic loss of antigen-presenting cell (APC)-derived determinants (e.g. HLA-DR, HLA-A2) was observed which, however, could be "reversed" by repeated MACSorting (days 10, 21 and 49). Our method allows the generation of semi-allogenic HAPC that constitutively proliferate in vitro. This opens the possibility of establishing a number of tumor-APC hybrids expressing defined HLA haplotypes and tumor antigens, of investigating their specific properties (e.g. antigen processing), and testing their diagnostic or therapeutic potential.

Gene therapy of cancer.

Gene therapy was initially thought of as a means to correct single gene defects in hereditary disease. In the meantime, cancer has become by far the most important indication for gene therapy in clinical trials. In the foreseeable future, the best way to achieve reasonable intratumoral concentrations of a transgene with available vectors is direct intratumoral injection with or without the aid of various techniques such as endoscopy or CT-guidance. At present, viral and non-viral methods of gene transfer are used either in vivo or ex vivo/in vitro. The most important viral vectors currently in use in clinical trials comprise retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses. None of the available vectors satisfies all the criteria of an ideal gene therapeutic system, and vectors with only minimal residues of their parent viruses ("gutless vectors") as well as completely "synthetic viral vectors" will gain more and more importance in the future. Non-viral gene therapy methods include liposomes, injection of vector-free DNA ("naked DNA"), protein-DNA complexes, delivery by "gene gun," calcium-phosphate precipitation, electroporation, and intracellular microinjection of DNA. The first clinical trial of gene therapy for cancer was performed in 1991 in patients with melanoma, and since then more than 5000 patients have been treated worldwide in more than 400 clinical protocols. With the exception of a case of fatal toxicity in a young man with hereditary liver disease treated intrahepatically with high doses of adenovirus, side effects have been rare and usually mild in all these studies and expression of the transgene could be demonstrated in patients in vivo. However, despite anecdotal reports of therapeutic responses in some patients, unequivocal proof of clinical efficacy is still lacking for most of the varied approaches to gene therapy in humans. As well as our only fragmentary understanding of the molecular pathophysiology of many diseases, the principal reason for the present lack of clinical success of gene therapy is the very low transduction and expression efficiency in vivo of available vectors. Despite the complexities of gene therapy for cancer, the numerous different approaches can be subdivided into three basic concepts: (1) strengthening of the immune response against a tumour, (2) repair of cell cycle defects caused by losses of tumour suppressor genes or inappropriate activation of oncogenes, and (3) suicide gene strategies. In addition, the importance of gene marker studies and gene therapeutic protection of normal tissue are briefly covered in this review.

Gene therapy of cancer.

Gene therapy was initially thought of as a means to correct single gene defects in hereditary disease. Since then, cancer has become the most important indication for gene therapy in clinical trials. In the foreseeable future, the best way to achieve reasonable intratumoral concentrations of a transgene with available vectors will be direct intratumoral injection with or without the help of various techniques such as endoscopy or computed tomography guidance. At present, viral and nonviral methods of gene transfer are used either in vivo or ex vivo/in vitro. The most important viral vectors currently used in clinical trials are retroviruses, adenoviruses, adeno-associated viruses and herpes viruses. However, none of them satisfies all the criteria of an ideal gene therapeutic system, and vectors with only minimal residues of their parent viruses (gutless vectors) and completely synthetic viral vectors are gaining importance. Nonviral methods of gene therapy include liposomes, injection of vector-free (naked) DNA, protein-DNA complexes, delivery by gene gun, calcium-phosphate precipitation, electroporation and intracellular microinjection of DNA. The first clinical trial of human gene therapy was performed in 1990 and since then more than 5000 patients have been treated worldwide in over 400 clinical protocols. Side effects were rare and mostly mild in all of these studies and expression of the transgene was demonstrated in patients in vivo. Despite anecdotal reports of therapeutic responses in some patients, there is still no unequivocal proof of clinical efficacy of most approaches to gene therapy in cancer, primarily due to very low transduction and expression efficacy in vivo of available vectors. Strategies for gene therapy of cancer can be subdivided into four basic concepts: 1) strengthening of the immune response against a tumor, 2) repair of cell cycle defects caused by loss of tumor suppressor genes or inappropriate activation of oncogenes, 3) suicide gene strategies and 4) inhibition of tumor angiogenesis. Gene marker studies and gene protection of normal tissue are also discussed.

bcl-2 expression is reciprocal to p53 and c-myc expression in metastatic human colorectal cancer.

Apoptosis (programmed cell death) inhibition may be an important mechanism by which gastrointestinal mucosal cells containing damaged DNA evade normal clearance mechanisms and grow to become invasive tumours. Since bcl-2 is an apoptosis inhibitor, bcl-2 mRNA expression was measured in 21 metastases of colorectal cancer using reverse transcription-polymerase chain reaction analysis. The mean bcl-2 mRNA expression (0.45 U, P < 0.0001) was lower than that of normal mucosal controls (= 1 U). p53 expression was inversely correlated with bcl-2 expression (P = 0.021) in 19 evaluable samples, and in tumours where p53 expression was over twice that of normal colonic mucosal values, bcl-2 mRNA was significantly decreased (mean 0.30, P = 0.0052). c-myc was also inversely correlated with bcl-2 expression (P = 0.025). Decreased bcl-2 expression in metastatic colorectal cancer may be partly due to allelic loss, given the proximity of bcl-2 to the frequently deleted DCC gene on chromosome 18q. However, the inverse correlation to p53/c-myc suggests an active downregulation of bcl-2, possibly following delegation of its apoptosis inhibiting role to other genes.

Gene therapy with cytokine-transfected xenogeneic cells in metastatic tumors.

On the basis of compelling preclinical data in cats and dogs we initiated a clinical gene therapy study in nine patients with advanced solid tumors using xenogeneic fibroblasts secreting human IL-2 (Vero-IL-2 cells). Cohorts of three successive patients with tumors accessible to CT- or ultrasound-guided injection were treated repeatedly with 5 x 10(5), 5 x 10(6), or 5 x 10(7) Vero-IL-2 cells. Endpoints of the study were feasibility, toxicity, and clinical and biological effects of this novel approach to immunotherapy of cancer. Histopathological, immunological and molecular analyses were performed on biopsy specimens of tumors and blood samples from before, during and after treatment. Low levels of serum antibodies to Vero cells developed in 2/9 patients. Analysis of tumor biopsies showed increased expression of CD3 mRNA and enhanced tumor infiltration with varying lymphocyte subpopulations after treatment. In addition, monoclonal alterations of the TCR repertoire of blood and tumor lymphocytes were observed. Treatment was well tolerated and toxicity consisted of transient fever in one patient and short-lived, mild itching and erythema in two others. One patient with soft tissue sarcoma showed a more than 90% and more than 50% reduction of the volume of two distant, non-injected metastases, respectively, lasting for 22+ months. Four other patients showed stabilization of their disease for three to nine months, among whom was a patient with melanoma who developed marked vitiligo. We conclude that repeated injection of up to 5 x 10(7) Vero-IL-2 cells was safe and showed biological and clinical activity in heavily pretreated patients with advanced solid tumors. Further evaluation of intratumoral application of Vero-IL-2 seems warranted.

MTS1 expression and prognosis in acute myeloid leukemia.

MTS1, a tumor suppressor gene located on chromosome 9p21, has been shown to be altered in a number of human tumor cell lines, primary solid tumors, and leukemias. In this study we found low expression of MTS1 in lymphocytes from seven of nine healthy donors, but in none of eight granulocyte samples from the same controls, suggesting a physiological role of MTS1 in peripheral blood cells capable of proliferation, but not in end-stage differentiated cells. We detected MTS1 mRNA expression in 38 of 57 patients (66%) with acute myelogenous leukemia (AML) treated in a standardized clinical protocol. No deletion of the MTS1 gene was found in any of the AML samples tested. There was no significant association between expression of MTS1 and response to therapy, progression-free, or overall survival. Except for a negative correlation between MTS1 level and leukocyte count at diagnosis (p = 0.03), there was also no association with any of the known prognostic parameters in AML. We conclude that MTS1 shows a significantly higher expression in leukemic than in normal peripheral blood cells, that deletion of MTS1 is not a frequent event in AML, and that its expression is not significantly correlated with outcome of the disease.

The status of p53 in the metastatic progression of colorectal cancer.

In order to investigate the role of TP53 in tumour progression and metastasis, we analysed 33 liver metastases of colorectal carcinomas and 19 primary colon carcinomas from the same hospital with respect to mutational changes, loss of heterozygosity and expression of the TP53 tumour suppressor gene. Direct sequencing of PCR products corresponding to the coding region of TP53 revealed that 13 of 19 primary tumours (68%) and 23 of 33 liver metastases (70%) had mutations in the TP53 gene. The distribution of mutations along the coding region of TP53 was similar in liver metastases compared to primary tumours. Thus, codon specificity did not seem to be a relevant factor and cells carrying specific TP53 mutations seem to have no selective advantage in the metastasising process. Comparing our data with the mutational spectra found in other countries did not reveal differences in the distribution of mutations along the coding region. Most of the metastases analysed showed loss of heterozygosity (LOH, 9 of 12 cases, 75%) and strong nuclear staining in immunohistochemistry (10 of 17 cases, 59%). Furthermore, with respect to mRNA expression levels, tumours carrying TP53 mutations showed significantly higher p53 mRNA levels compared to those without TP53 mutations. Thus, regulation of p53 mRNA levels seems to be subject to selection processes in tumourigenesis.

[Possibilities and prospects in gene therapy in cancer patients]. / Möglichkeiten und Aussichten der Gentherapie bei Krebspatienten.

The many different oncological concepts of gene therapy can be classified into three major groups. These include immunopotentiation, compensation of oncogene and tumor suppressor gene alterations, and suicide gene strategies. Gene marker studies and the transfer of drug resistance genes to normal cells have also shown to be of some importance in oncology. Although there is no clear demonstration of the efficacy of gene therapy so far, it will most likely become part of the therapeutic armamentarium of oncologists within the next two to three decades.

Overexpression and amplification of c-myc during progression of human colorectal cancer.

Overexpression and amplification of the c-myc oncogene occur in approximately 70 and 10% of human primary colorectal carcinomas, respectively, indicating the importance of this gene in colorectal tumorigenesis. Little, however, is known about the involvement of c-myc in the progression of colorectal cancer. We therefore determined c-myc gene expression and amplification in a group of primary tumors and metastases from patients with colorectal cancer using quantitative PCR-based tests. While the percentage of metastases overexpressing c-myc (13/26 = 50%) was in the same range as reported for primary tumors by others, gene amplification of c-myc was significantly (p = 0.001) more frequent in metastases (16/27 = 59%) compared to primary tumors (1/23 = 4%) in our series. Interestingly, in 23 metastases where both expression and amplification of c-myc could be determined, there was no correlation between gene copy number and expression level (p = 0.18; r = 0.19). We conclude that amplification but not overexpression of c-myc is related to metastatic progression of colorectal cancer and that overexpression of c-myc is driven by mechanisms other than the number of c-myc copies in the tumors studied.

MDR1 expression correlates with mutant p53 expression in colorectal cancer metastases.

Overexpression of the multidrug resistance MDR1 gene is thought to contribute to drug resistance in non-responsive cancers like colorectal carcinoma. Little is known about the mechanisms by which expression of MDR1 is regulated in human tumours. However, there is growing evidence that regulation primarily takes place at the transcriptional level and that the process of tumour progression is related to activation of the MDR1 gene. Mutations in the p53 tumour-suppression gene occur in approximately 70% of colorectal cancers. As a transcriptional regulator, p53 might be involved in regulation of MDR1 expression in these tumours. We therefore determined MDR1 expression using the differential polymerase chain reaction technique in 30 colorectal tumours (4 primaries and 26 metastates) and correlated our results with previously reported data on p53 in the same group of patients. We found a significant positive correlation between p53 and MDR1 expression in p53-mutated tumours (P = 0.005; r = 0.596), but not in tumours without a p53 mutation. In addition, we observed a tendency towards higher MDR1 expression levels in tumours carrying p53 mutations (P = 0.14) compound to wild-type p53 tumours. These data indicate that mutant p53 may play a role in the regulation of MDR1 expression in human cholorectal cancer.

Evidence for a mutual regulation of p53 and c-myc expression in human colorectal cancer metastases.

BACKGROUND: Alterations of the c-myc and the p53 genes occur in a majority of human colorectal cancers, and functional interaction between these two genes has recently been suggested. PATIENTS AND METHODS: We analyzed p53 sequence and c-myc and p53 mRNA expression in 26 metastases and 4 advanced primaries of human colorectal cancer. RESULTS: Twenty-one of 30 tumors (=70%) carried mutations of the p53 gene. In these samples, c-myc and p53 were overexpressed in 70% (15/21) and 71% (14/20) of evaluable cases, respectively, while in tumors carrying only wild-type p53, overexpression of c-myc and p53 was observed in only 33% (3/9; p < 0.05) and 22% (2/9; p < 0.01), respectively. Expression of p53 and c-myc were positively correlated (p = 0.014; r = 0.563) in tumors carrying a p53 mutation, but not in those with only wild-type p53. CONCLUSION: We conclude that c-myc might induce p53 expression in human colorectal cancer and that wild-type but not mutant p53 might be involved in a negative feedback regulation of c-myc expression. The abrogation of this normal control mechanism seems to be an essential step during colorectal tumorigenesis and metastatic progression.

[Adjuvant treatment of colonic carcinoma]. / Adjuvante Behandlung des Kolonkarzinoms.

After many years of negative trials of adjuvant chemotherapy in colon cancer, two studies in the years 1989 and 1990 of the NCCTG and the Intergroup Trial, respectively showed a significant reduction of relapse and improved survival in patients treated with 5-FU and levamisole in an adjuvant setting. The absolute and relative reductions in 5-year-relapse and death rates were approximately 35% and 17%, respectively. Intraportal perfusion of the liver in an adjuvant perioperative setting seems to be of similar benefit. Preliminary data of the adjuvant therapy with 5-FU and folinic acid also show that this combination seems to have at least the same efficacy as the current standard 5-FU/levamisole in the adjuvant therapy of colon cancer. At the current time, patients with colon cancer of Dukes stage C should be offered adjuvant chemotherapy with 5-FU/levamisole outside of clinical studies.

Gene expression analysis by a competitive and differential PCR with antisense competitors.

We report a sensitive method for the reproducible and accurate measurement of gene expression from small samples of RNA. This method is based on a combination of two PCR techniques: First, an endogenous reporter gene and the gene of interest are simultaneously amplified in one tube after random-primed reverse transcription (RT) of RNA (differential RT-PCR). Second, exogenous homologous fragments of both genes with artificially introduced mutations are added and coamplified in the same reaction (competitive PCR). The first-strand cDNA, and the mutated antisense homologues of the reporter as well as the target gene compete for their respective primers and are therefore amplified with equal efficiencies. After PCR, restriction enzyme digestion allows visualization of the quantitative differences between the four resulting reaction products. The ratios of products that competed during PCR provide the quantitative information. The initial amount of a specific cDNA can be calculated from any competitor/cDNA ratio of reliably measurable PCR product amounts. Extensive competitor titration to experimentally approach the equilibrium is therefore unnecessary. The differential counterpart of competitive and differential RT-PCR (CD-RT-PCR) allows expression of the levels in reference to a reporter gene. MDR1 expression was determined in tumor cells by CD-RT-PCR.

Detection of allelic loss within the beta 1-interferon gene in childhood acute lymphoblastic leukemia using differential PCR.

Deletion of the short arm of chromosome 9p involving the beta 1-interferon (IFN) gene has been implicated in the process of malignant transformation in lymphomas and acute lymphoblastic leukemias. Since cytogenetic analysis is frequently unsuccessful in clinical samples, we used a recently described differential PCR technique to detect losses within the beta 1-IFN gene in 86 acute leukemias. Using differential PCR, no beta 1-IFN deletion was detected in 44 acute myeloid leukemia (AML) and eight control samples. However, five of 42 acute lymphoblastic leukemia (ALL) probes (12%) exhibited loss of the beta 1-IFN gene (three common ALL, two T-ALL). Cytogenetic analysis was performed independently in three of these five cases and revealed abnormalities of chromosome 9p in two samples. Two of five T-ALL cases exhibited a loss within the beta 1-IFN gene, compared with 3/29 c-ALLs, suggesting a predominance of IFN gene loss in T-ALLs. These data indicate that PCR can be used for rapid detection of gene dosage phenomena in clinical leukemia samples.

[Hepatocellular carcinoma: systemic chemotherapy, perspectives of gene or immunotherapy]. / Das Hepatozelluläre Karzinom: systemische Chemotherapie, Perspektiven der Gen- oder Immuntherapie.

The systemic chemotherapy of hepatocellular carcinoma with single agent or combination chemotherapy has not resulted in reproducible response rates above 20%. Correspondingly, an effect on the survival of the treated patient population has not been shown so far. The most effective single agent is doxorubicin, which should, however, not be used outside of clinical studies in the systemic treatment of hepatocellular carcinoma. The results of hormonal treatment of hepatocellular carcinoma are also disappointing: the response rates of agents like tamoxifen, ketoconazol, nilutamid, LHRH-agonists or megestrol are usually below 10% and the survival of patients can not be improved. New approaches that are currently tested in vitro and in mouse models are pharmacological or immunological interactions with tumor cells that make use of molecular biology techniques and are summarised under the term "gene therapy". Some of these projects are in clinical phase I studies and first results can be expected in the next three to five years.

Molecular alterations in a patient with Turcot's syndrome.

Cells of a patient with Turcot's syndrome and of her parents were evaluated for the presence of molecular alterations in the p53 and the Ki-ras gene. Deletions on chromosome 17p, overexpression and point mutations of the p53 gene as well as mutations of the Ki-ras gene were detected in primary and metastatic tumour but not in the germline of the patient nor in her parents.

Position specificity of Ki-ras oncogene mutations during the progression of colorectal carcinoma.

The Ki-ras proto-oncogene is converted into an active oncogene by mutations in codon 12, 13, or 61. The incidence of mutations in the Ki-ras oncogene in colorectal adenomas and primary colorectal carcinomas has been shown to be 50-75 and 40-65%, respectively. To determine the role activation of the Ki-ras oncogene plays in the progression of colorectal carcinoma, we analyzed DNA from 11 nude-mouse xenografts and from 24 metastases of 22 patients with colorectal carcinoma, using the polymerase chain reaction technique and hybridization with labeled mutation-specific oligomers. Eleven of the 24 metastases (46%) carried mutations, 7 in codon 12 and 4 in codon 13, whereas only 1 nude-mouse tumor (9%) harbored a Ki-ras codon-12 mutation. Eleven of these 12 mutations in advanced stages of colorectal cancer were localized to the second position of either codon 12 or codon 13, whereas a majority of published ras mutations in earlier stages are in the first position of codon 12 of the Ki-ras oncogene. We conclude that there is a position specificity of Ki-ras oncogene mutations in advanced stages of colorectal carcinoma. In general, however, these mutations do not seem to play an important role in the progression of this cancer.

PCR-determined expression of the MDR1 gene in chronic lymphocytic leukemia.

To determine the role the multiple drug-resistance (MDR 1) gene plays in chronic lymphocytic leukemia (CLL), we measured the expression of the MDR 1 gene in 30 patients with this disease. A rapid, highly sensitive, and nonradioactive technique based on the polymerase chain reaction (PCR) was used for that purpose. In this technique, called differential PCR, the target (MDR 1) and a reference gene (beta 2-microglobulin) are co-amplified by PCR from random hexamer-primed cDNA in the same reaction vessel. The level of target gene expression is reflected in the ratio between the intensities of the two resulting PCR product bands, as measured by high-performance liquid chromatography (HPLC). MDR 1 gene expression was detectable in 29/30 (97%) patients with CLL, with a median expression level of 0.36 U (human placenta = 1 U). There was no correlation between expression of the MDR 1 gene and clinical stage, time from diagnosis, absolute lymphocyte count, several lymphocyte surface markers, or prior treatment in the patients analyzed. Immunocytochemical studies of the same material using the monoclonal antibody C219 showed a very low or undetectable expression of the P-glycoprotein in the lymphocytes of all patients studied, whereas granulocytes were significantly more immunoreactive. We conclude that the level of expression of the MDR 1 gene in CLL is generally low, that the removal of granulocytes is important in studies of expression of MDR 1 mRNA in CLL, and that differential PCR provides a rapid and reliable method for quantifying the amount of a specific mRNA, even in very small samples of total RNA.

Multiple drug resistance gene expression in human renal cell cancer is associated with the histologic subtype.

Overexpression of p170 glycoprotein, the product of the multiple drug resistance (mdr) gene, has been associated with resistance to various cytotoxic drugs used in the treatment of human neoplasms. Normal renal epithelial cells express p170 as a function of their secretory capacity. Because renal cell carcinomas (RCC) respond poorly to chemotherapeutic regimens, p170 expression was studied in primary RCC. Such expression was measured in 40 human RCC and normal kidney tissues using immunohistochemical staining with the monoclonal antibody C-219. Staining intensities of the whole tumor and of different areas of the cryostat sections were transformed into digital numbers using an algorithm designed for this purpose. In most tumors, an inhomogeneous staining pattern and a correlation between grade of differentiation and C-219 immunoreactivity was observed. A comparison of the tumors according to their histopathologic subtypes showed clear differences. The means (range) of the staining intensities of the different types of RCC: clear cell carcinoma Grade 1 (n = 3), 2.0 (2.0 to 2.0); clear cell carcinoma Grade 2 (n = 19), 0.8 (0.0 to 2.9); clear cell carcinoma Grade 3 (n = 5), 0.1 (0.0 to 0.2); tubular carcinoma (n = 4), 2.0 (2.0 to 3.0); anaplastic carcinoma (n = 8), 0.05 (0.0 to 0.2); oncocytoma (n = 1), 0.0 (0.0 to 0.0); and normal kidney (n = 40), 0.5 (0.0 to 2.0). The differences between anaplastic, clear cell, and tubular carcinoma were significant (P less than 0.001 by Kruskal-Wallis test). In addition, the difference between the three subgroups of clear cell carcinoma was significant (P less than 0.01). It was concluded that the histopathologic subtypes of RCC correlate with the degree of mdr gene expression, as determined by staining with the C-219 monoclonal antibody.