High cytokine production and effective antitumor activity of a recombinant vaccinia virus encoding murine interleukin 12

We have constructed a recombinant vaccinia virus (recVV), vKT0334 mIL-12, containing the genes encoding the p35 and p40 subunits of murine interleukin-12 (mIL-12). In vitro experiments demonstrated that vKT0334 mIL-12 efficiently infected a variety of murine and human tumor cell lines and produced very high amounts (1.5 micrograms/10(6) cells/24 h) of biologically active mIL-12. Mice injected s.c. with 10(6) MCA 105 sarcoma cells, followed by injection at the same site with saline or a control recVV, vKT033, containing no mIL-12 genes, all developed progressively growing tumor, whereas 60% of animals injected with vKT0334 mIL-12 remained tumor free (P < 0.0005). Furthermore, tumor growth was significantly reduced in the remaining mice treated with vKT0334 mIL-12 that did develop tumor compared with mice treated with vKT033 (P < 0.03) or saline (P < 0.0001). We conclude that recVV expressing high levels of mIL-12 offers an effective in vivo method of cytokine gene delivery and expression in tumors with subsequent antitumor effect.     

In vivo tumor transfection with superantigen plus cytokine genes induces tumor regression and prolongs survival in dogs with malignant melanoma

In vivo transfection of established tumors with immunostimulatory genes can elicit antitumor immunity. Therefore, we evaluated the safety and efficacy of intratumoral injections of a bacterial superantigen with a cytokine gene in dogs with malignant melanoma, a spontaneous and highly malignant canine tumor. 26 dogs with melanoma were treated with lipid-complexed plasmid DNA encoding staphylococcal enterotoxin B and either GM-CSF or IL-2. Dogs were evaluated for treatment-associated toxicity, tumor responses, immunologic responses, and survival times. The overall response rate (complete or partial remissions) for all 26 dogs was 46% (12 of 26), and was highest in patients with smaller tumors. Toxicity was minimal or absent in all dogs. Injected tumors developed marked infiltrates of CD4+ and CD8+ T cells and macrophages, and tumor regression was associated with development of high levels of antitumor cytotoxic T lymphocyte activity in peripheral blood lymphocytes. Survival times for animals with stage III melanomas treated by intratumoral gene therapy were prolonged significantly compared with animals treated with surgical tumor excision only. Thus, local tumor transfection with superantigen and cytokine genes was capable of inducing both local and systemic antitumor immunity in an outbred animal with a spontaneously developing malignant tumor.     

Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression

Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host-tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of direct in vivo gene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12-B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 x 10(7) pfu/mouse) of AdIL12-B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12-B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach for in vivo cancer therapy.     

Myocardial and forearm blood flow reserve in mild-moderate essential hypertensive patients

Interleukin-2 (IL-2) gene therapy alone and in combination with the herpes thymidine kinase gene (tk) was used to evaluate immunological responses and antitumor effects in head and neck cancer. Established floor of mouth squamous cell carcinomas in C3H/HeJ mice were directly injected with recombinant adenoviral vectors carrying both therapeutic and control genes. One week after adenoviral gene transfer, only the animals treated with combination IL-2+tk or tk alone demonstrated significant tumor regression. Residual tumors were harvested for microscopic evaluation and immunohistochemistry staining, which revealed a predominance of CD8+ lymphocytes in the tumor beds of the animals treated with IL-2. To evaluate the systemic immune effects of IL-2, animals treated with single or combination gene therapy received a second site challenge with parental tumor cells or a heterologous but syngeneic sarcoma cell line. Mice treated with combination IL-2 and tk demonstrated a protective systemic immunity specific to the parental tumor cell line, whereas no systemic immune response was evident in mice receiving IL-2 alone. In a separate experiment, a range of concentrations of the adenovirus IL-2 vector were used to treat established tumors. Even with the maximal single-dose adenovirus concentration, IL-2 alone was ineffective as a single therapy. These results support the use of adenovirus-mediated gene transfer of IL-2 as an effective immunotherapy when used adjuvantly with the tk "suicide gene".     

Localization using nonindividualized head-related transfer functions

The HPV proteins encoded by the early viral genes, including E6 and E7, are thought to subvert the normal regulatory pathways of infected cells to accommodate viral replication. Mechanistically some of this is accomplished by protein-protein interactions between viral proteins and a number of key cellular regulatory proteins that include tumor suppressor gene products. By undermining cellular regulatory pathways the HPV oncogenes cause hyperproliferation and the perturbation of normal cellular differentiation pathways. Although expression of the high-risk HPV-encoded E6 and E7 oncoproteins may be important prerequisites for cellular transformation, it is very likely that additional cellular changes are necessary for carcinogenic progression. The elucidation of the role of the early HPV genes in the initiation and/or maintenance of carcinogenic progression will continue to be a fascinating area of investigation and may reveal new opportunities for antiviral therapy and antitumor intervention.     

Stage-specific localization of basigin, a member of the immunoglobulin superfamily, during mouse spermatogenesis

We have previously established that a dimer repeat of the complete HPV 16 genome is sufficient to cause multiple organ malignancies, either carcinomas or T-cell lymphomas, in transgenic mice. Here, we report the expression of oncogenes supporting the notion that these tumors arose via multiple oncogenic pathways. In these mice, the transgenic HPV 16 genome cosegregated with the tumor phenotype. E6/E7 expression was observed in both carcinomas and T-cell lymphomas, while E2 expression was observed only in T-cell lymphomas. Some of the T-cell lymphomas revealed E2 expression alone, implying that oncogenic pathways of HPV other than the one involving E6/E7 existed in these transgenic mice. To establish that this is the case, expression of genes downstream from E6/E7 and oncogenes involved in T-cell lymphoma formation were analyzed. p53 mutations were observed in two of five tumors that lacked E6 expression. High levels of c-myc gene expression were observed in five of six tumors with E7 expression, suggesting that a pathway involving E7, inactivation of Rb, and activation of c-myc is important in tumorigenesis of HPV 16 in these transgenic animals. High levels of expression of the c-Pim gene were also noted in two of three c-myc-expressing T-cell lymphomas, suggesting cooperation between these two proto-oncogenes. Activation of Hox-11, Tal2/SCL-2, and Rbtn1/Ttg1 expression, which are highly associated with human T-cell acute lymphoblastic leukemia (T-ALL), was observed in three of three T-cell lymphomas with E2 expression but not E6/E7 expression, showing that pathways to tumor formation not involving E6/E7 exist in these transgenic animals. At least two oncogenic pathways to tumors in HPV 16 transgenic mice exist, one involving E6/E7 and c-myc and the other involving E2 and lymphomagenic oncogenes.     

Cellular proteins involved in papillomavirus-induced transformation

Human papillomaviruses (HPVs) are associated with at least 80% of cervical carcinomas and are classified as high-risk or low-risk based on whether or not they are commonly found in cervical cancers. The high-risk HPVs have early gene products (E6 and E7) that immortalize human keratinocytes and are at least partially responsible for causing cervical carcinoma. E6 and E7 from the high-risk viruses interact strongly with the tumor suppressors p53 and Rb; those from the low-risk HPVs do not. Transformation involves a multi-step process and requires additional factors besides high-risk HPV infection. High-risk HPVs are capable of immortalizing primary human keratinocytes in tissue culture, but such cells become transformed only after certain chromosomal changes take place, possibly having to do with oncogene activation. The DNA of high-risk HPVs is frequently (if not always) integrated into the genome of cancer cells; it is normally episomal in premalignant lesions. Integration disrupts the E2 and E5 genes and viral gene regulation. Cells containing integrated viral DNA show excessively high levels of E6 and E7. While there is some conflicting evidence, it appears that the p53 and Rb tumor-suppressor genes are more frequently mutated in HPV-negative tumors than they are in HPV-positive tumors, suggesting that for tumor formation to proceed the p53 and Rb proteins must be inactivated either by interaction with the viral proteins or by mutation. The presence of an activated oncogene in a cell lacking functional p53 or Rb may then be sufficient to cause tumor progression.     

Development of systemic immunologic responses against hepatic metastases during gene therapy for peritoneal carcinomatosis with retroviral HS-tk and ganciclovir

Gene therapy with retroviral mediated gene transfer of the herpes simplex thymidine kinase (HS-tk) gene into a tumor mass confers sensitivity of the tumor cells to ganciclovir (GCV). Tumor-specific immunologic responses may develop following treatment of the primary tumor with retroviral HS-tk and GCV. In the present study we assessed whether GCV treatment of HS-tk transduced colon cancer (TK+) implanted in the peritoneal cavity induced a systemic antitumor response that would inhibit growth of a second wild-type (TK-) tumor implanted in the liver. DHDK12 rat colon cancer cells were transduced in vitro with the retroviral HS-tk vector and established as a permanent cell line (TK+ cells). TK+ or TK- DHDK12 cells (6x10(6) cells) were injected intraperitoneally on day 0 into BD-IX rats. On day 10, TK- cells (3x10(6) cells) were injected into the liver in all the groups. The animals were then treated with GCV (150 mg/kg) for 13 days. TK+ peritoneal tumors underwent significant regression during therapy with GCV (0.05+/-0.004 g; n=7) compared to wild-type (TK-) tumors (2.2+/-0.7g; n=6) (P<0.05). The volume of TK- tumors in the liver was significantly lower in GCV-treated rats with TK+ peritoneal tumors (12.5+/-8.3 mm3) compared to rats with TK- peritoneal tumors (96.7+/-18.1 mm3) (P<0.05). Histology of the liver tumors in the TK+ groups showed a dense monocytic infiltrate with fibrosis and only occasional viable tumor cells. Gene therapy with retroviral HS-tk vectors may provide a novel approach to treatment of gastrointestinal cancer by both direct cytotoxicity and an indirect mechanism that may include enhanced immuno logic responses against disseminated disease.     

Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis

The antitumor effect and mechanisms activated by murine IL-12 and IL-18, cytokines that induce IFN-gamma production, were studied using engineered SCK murine mammary carcinoma cells. In syngeneic A/J mice, SCK cells expressing mIL-12 or mIL-18 were less tumorigenic and formed tumors more slowly than control cells. Neither SCK.12 nor SCK.18 cells protected significantly against tumorigenesis by distant SCK cells. However, inoculation of the two cell types together synergistically protected 70% of mice from concurrently injected distant SCK cells and 30% of mice from SCK cells established 3 d earlier. Antibody neutralization studies revealed that the antitumor effects of secreted mIL-12 and mIL-18 required IFN-gamma. Interestingly, half the survivors of SCK.12 and/or SCK.18 cells developed protective immunity suggesting that anti-SCK immunity is unlikely to be responsible for protection. Instead, angiogenesis inhibition, assayed by Matrigel implants, appeared to be a property of both SCK.12 and SCK.18 cells and the two cell types together produced significantly greater systemic inhibition of angiogenesis. This suggests that inhibition of tumor angiogenesis is an important part of the systemic antitumor effect produced by mIL-12 and mIL-18.     

Immunotherapy for medullary thyroid carcinoma by a replication-defective adenovirus transducing murine interleukin-2

We have evaluated the feasibility of gene transduction using replication-defective adenovirus vector as a novel therapy for medullary thyroid carcinoma (MTC), a thyroid C cell neoplasm. Replication-defective adenoviruses were constructed to express murine interleukin-2 (mIL-2) gene and Escherichia coli beta-galactosidase (beta-gal; lacZ) gene under the control of the human cytomegalovirus (CMV) promoter (AdCMVmIL2, AdCMVbeta-gal) by homologous recombination. The efficiency of transduction was evaluated using AdCMVbeta-gal at different conditions. The gene transduction efficiency was dependent on multiplicity of infection, duration of exposure to the virus, and viral concentration. The expression of functional mIL-2 in transduced tumor cells was verified both in vitro and in vivo. Two cell lines (rat MTC and mMTC) secreted large amounts of functional mIL-2 after transduction, as tested in cytotoxic T lymphocyte (CTL) L-2 cells. When AdCMVmIL2-infected mMTC cells were injected s.c. into their host animals, tumors developed in 2 of 10 animals, in contrast to 9 of 10 animals injected with AdCMVbeta-gal-infected mMTC cells and all 10 animals injected with parental mMTC cells. Moreover protected animals developed a long lasting immunity against mMTC tumor cells and their splenocytes, showing cytotoxicity to parental tumor cells, and active natural killer (NK) cell activity. BALB/c-SCID (severe combined immune deficiency) mice were also used to evaluate the function of NK cells in antitumor activities. No tumor developed in SCID mice injected with AdCMVmIL2-infected cells, whereas all animals injected with either AdCMVbeta-gal-infected or parental mMTC cells developed tumors. Our data indicate that IL-2 production by MTC cells leads to rejection in syngeneic animals and suggest that both cytotoxic T cells and NK cells may play an important role. In addition, transduction of adenoviral vectors into tumor cells produces some nonspecific antitumor effects.     

Inhibition of human squamous cell carcinoma growth in vivo by epidermal growth factor receptor antisense RNA transcribed from the U6 promoter

BACKGROUND: Squamous cell carcinomas of the head and neck (SCCHN), unlike normal mucosal squamous epithelial cells, overexpress epidermal growth factor receptor (EGFR) messenger RNA and protein. EGFR protein is required to sustain the proliferation of SCCHN cells in vitro. To determine whether EGFR expression contributes to tumor growth, we investigated the effect of suppressing EGFR expression in tumor xenografts through in situ expression of antisense oligonucleotides. METHODS: Intratumoral cationic liposome-mediated gene transfer was used to deliver plasmids capable of expressing sense or antisense EGFR sequences into human head and neck tumors, which were grown as subcutaneous xenografts in nude mice. The oligonucleotides were expressed under the control of the U6 RNA promoter. RESULTS: Direct inoculation of the EGFR antisense (but not the corresponding sense) plasmid construct into established SCCHN xenografts resulted in inhibition of tumor growth, suppression of EGFR protein expression, and an increased rate of apoptosis (programmed cell death). Sustained antitumor effects were observed for up to 2 weeks after the treatments were discontinued. CONCLUSION: These results suggest that interference with EGFR expression, using an antisense-based gene therapy approach, may be an effective means of treating EGFR-overexpressing tumors, including SCCHN.     

Intratumoral coinjection of adenoviral vectors expressing IL-2 and IL-12 results in enhanced frequency of regression of injected and untreated distal tumors

We have studied the ability of adenoviral (Ad) vectors expressing the cytokines IL-2 or IL-12 to mediate regression of established tumors in a mouse model of mammary adenocarcinoma. Previous results indicated that intratumoral injection of vectors expressing IL-2 (AdCAIL-2), or IL-12 (AdmIL-12.1) induced complete tumor regression in approximately 30-40% of treated animals. In the current studies, we investigated the mechanism of tumor killing in responding animals and the efficacy of AdIL-2 and AdIL-12 vector administration in combination compared with the use of either vector alone. Animals bearing subcutaneous mammary tumors were injected intratumorally with Ad vectors expressing IL-2 or IL-12 or were coinjected with both vectors. Animals receiving the combination treatment responded substantially better than animals which had received either vector alone, with 65% of animals treated with both vectors undergoing complete tumor regression. In all three treatment regimens, tumor regression was associated with the presence of specific antitumor antigen cytotoxic T-lymphocytes (CTLs), which secreted elevated levels of IFN-gamma. Consistent with circulating CTLs being involved in regression, when animals bearing bilateral tumors were inoculated in a single tumor with IL-2 or IL-12 expressing vectors, both tumors regressed in many cases. Again, treatment with both AdCAIL-2 and AdmIL-12.1 was most effective, with 63% of animals undergoing complete regression of both treated and untreated tumors, compared to 18 or 22% of animals injected with either AdCAIL-2 or AdmIL-12.1 alone. These data indicate that the combination of IL-2 and IL-12 is a more effective inducer of antitumor immune responses than either one alone, and that the resulting antitumor responses are effective in mediating the regression of distal untreated tumors, a property which may aid in the treatment of metastatic disease.     

Immunization with human papillomavirus type 16 (HPV16) oncoprotein-loaded dendritic cells as well as protein in adjuvant induces MHC class I-restricted protection to HPV16-induced tumor cells

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical cancer. In this study, we demonstrate that dendritic cells (DCs) pulsed with HPV16 E7 protein are not only recognized in vitro by E7-specific CTLs but also elicit E7-specific CTL responses in vivo, associated with protection against a challenge with syngeneic HPV16-induced tumor cells. Vaccination with soluble E7 protein in incomplete Freund's adjuvant likewise induces E7-specific CTL responses associated with tumor protection. The presence of HPV16 E7-specific CTLs in vivo and the observation that depletion of CD8+ cells completely abolishes tumor protection demonstrate that CTLs are the major effector cells in mediating antitumor activity. The in vivo involvement of DCs in the activation of protective CTLs is suggested by the surface display of E7 peptide-loaded MHC class I molecules on these cells after E7 protein immunization. These data show that HPV16 E7 protein-pulsed DCs, as well as the administration of E7 protein antigen in adjuvant, can effectively stimulate tumor-specific MHC class I-restricted CD8+ T-cell-mediated protective immunity to HPV16-induced cancers.     

Intravenous RMP-7 increases delivery of ganciclovir into rat brain tumors and enhances the effects of herpes simplex virus thymidine kinase gene therapy

Herpes simplex virus thymidine kinase (HSV-tk) gene therapy for brain tumors depends on ganciclovir (GCV) and its transport across the blood-brain tumor barrier (BBTB). We examined whether RMP-7, the bradykinin analog and potent BBTB permeabilizer, could enhance the efficacy of GCV treatment of brain tumors by increasing the BBTB delivery of GCV. In vitro, a significant bystander cytocidal effect of GCV was shown in mixed HSV-tk-transduced (HSV-tk+) and control vector-transduced (HSV-tk-) C6 glioma cultures. A dose-dependent cytotoxic effect of GCV on untransformed C6 cells was also shown. In vivo, rats with 100% HSV-tk+ or 100% HSV-tk- intracerebral C6 gliomas were treated for 7 days with intravenous infusions of GCV alone or with GCV and RMP-7 (2.5 microg/kg/day). The growth of HSV-tk+ and HSV-tk- gliomas decreased with increasing doses of GCV. A high dosage (100 mg of GCV/kg/day) eradicated all HSV-tk- and HSV-tk+ tumors. An intermediate dosage (5 mg of GCV/kg/day) reduced the growth of HSV-tk- gliomas by 42% if given alone, and by 88% in combination with RMP-7. A low dosage (0.5 mg of GCV/kg/day) in combination with RMP-7 enhanced the regression of HSV-tk+ gliomas by 87% compared with GCV alone. Low-dose GCV was ineffective in HSV-tk- tumors. RMP-7 increased [3H] GCV tumoral uptake by 2.6- and 1.7-fold in the tumor center and periphery, respectively. We conclude that RMP-7 could be an important adjunctive treatment for suicide gene therapy of brain tumors, while an RMP-7/GCV combination may also have a significant antitumor effect in untransfected gliomas.     

Characterization of the antitumor immune response generated by treatment of murine tumors with recombinant adenoviruses expressing HSVtk, IL-2, IL-6 or B7-1

In a cancer gene therapy model recombinant adenoviruses expressing the herpes simplex virus thymidine kinase (HSVtk) gene were injected into tumors in situ, either alone or in combination with adenoviruses (Avs) engineered to express IL-2, IL-6 or the costimulatory molecule B7-1. HSVtk phosphorylates the prodrug ganciclovir, thus converting it into an antimetabolite which kills not only HSVtk expressing cells, but also by the 'bystander effect', neighboring untransduced tumor cells. The tumors regressed in 80% of mice upon AvTK/ganciclovir treatment: combinations with AvIL-2, AvIL-6, or AvB7-1 did not improve these results. Cured mice were protected from further challenge with wild-type tumor but not from challenges with an unrelated syngeneic tumor cell line. Since cytotoxic T lymphocyte responses in this tumor model were weak, we analyzed cytokine secretion from spleen cells of treated animals. The best correlate of antitumor immunity in this model was enhanced secretion of GM-CSF, while secretion of IL-2, IL-6 and IFN gamma was also frequently increased but not as consistently. The enhanced IFN gamma secretion associated with unchanged IL-4 secretion suggests that AvTK treatment results in a predominantly Th1-mediated antitumor immune response.     

Tumor necrosis factor alpha-mediated tumor regression by the in vivo transfer of genes into the artery that leads to tumors

We report that tumor necrosis factor (TNF) alpha induced a strong antitumor immune reaction when it was produced in arteries leading to tumors by gene transfer in vivo. We used a mouse model carrying a sarcoma-180 tumor in the right footpad and injected the fusogenic liposomes encapsulating the human TNF-alpha gene into the right femoral artery. Under this condition, human TNF-alpha was detected only in the artery leading to the tumor and in the tumor. There was a significant regression in tumor growth when the TNF-alpha gene was delivered into the right femoral artery, with 4 of 11 mice completely cured. No regression was observed when the TNF-alpha gene was delivered into the left femoral artery or into the tumor or when the luciferase gene was administered. Tumor regression was inhibited by the injection of anti-TNF-alpha, anti-CD4, or anti-CD8 monoclonal antibody, and CD8+ T cells accumulated in the tumors of TNF-alpha-treated mice. These results suggest that TNF-alpha expressed locally in the arteries leading to tumors efficiently suppresses tumor growth through reinforcement of an antitumor immune reaction. The significance of this phenomenon for cancer gene therapy was discussed.     

Transforming growth factor-beta receptors on human endometrial cells: identification of the type I, II, and III receptors and glycosyl-phosphatidylinositol anchored TGF-beta binding proteins

BACKGROUND: Mutation of the p53 tumor suppressor gene is the most commonly found genetic alteration in human cancer. The E6 gene product of human papillomavirus (HPV) 16 and 18 can inactivate the p53 protein by promoting its degradation. Because most HPV-positive cervical carcinoma cell lines contain wild-type p53 whereas HPV-negative cell lines have point mutations in the p53 gene, a major role in the development of HPV-negative cervical cancer has been attributed to p53. Recent studies, however, have observed no consistent presence of p53 mutation in HPV-negative primary cervical carcinomas. The MDM2 oncogene, which forms an autoregulatory loop with the wild-type p53 protein, has been found amplified in a high percentage of human sarcomas, thus abolishing the antiproliferative function of p53. METHODS: Forty-three primary cervical carcinomas and 10 autopsy-derived distant metastases from one patient were examined for p53 mutation and MDM2 amplification. These tumors had been selected from 238 cervical cancers that had been HPV-typed by Southern blot hybridization and polymerase chain reaction as a representative sample for their HPV status and their clinicopathologic characteristics. Seventeen of the cases had a remarkably good or poor clinical outcome. Human papillomavirus DNA sequences had been detected in 30 of these 43 primary tumors and 13 were negative for HPV by both methods. p53 mutation in the highly conserved exons 5-8 was studied by single-strand conformation polymorphism analysis and direct sequencing. MDM2 amplification was analyzed by Southern blot hybridization. RESULTS: Only two missense point mutations and one nucleotide sequence polymorphism were detected: a TAC-->TGC transition in codon 234 in exon 7, resulting in a Tyr-->Lys substitution, a CGT-->TGT transition in codon 273 in exon 8, resulting in an Arg-->Cys substitution and a polymorphism (CGA-->CGG) in codon 213 in exon 6. Both tumors revealing the point mutations were HPV-negative carcinomas. Amplification of the MDM2 gene was observed in 1 of the 53 specimens tested. CONCLUSIONS: In contrast to data derived from cultured cervical carcinoma cell lines and primary sarcomas, these results indicate that p53 mutation and amplification of the MDM2 oncogene are rare even in HPV-negative primary cervical carcinomas. However, to the authors; knowledge, this is the first observation of MDM2 amplification in humans outside sarcomas and neuroepithelial tumors.