Gene therapy for head and neck cancer

Surg Oncol Clin N Am 11 (2002) 589–606

Gene therapy for head and neck cancer Douglas K. Frank, MDa,b,c a

Department of Otolaryngology—Head and Neck Surgery and The Institute for Head and Neck Cancer, Beth Israel Medical Center, Phillips Ambulatory Care Center, 10 Union Square East, Suite 4J, New York, NY 10003, USA b Albert Einstein College of Medicine, 1300 Morris Avenue, Bronx, NY 10461, USA c Strang Cancer Prevention Center, 428 East 72nd Street, New York, NY 10021, USA

Squamous cell carcinoma of the upper aerodigestive tract, a term used synonymously with squamous cell carcinoma of the head and neck (SCCHN), is by far the most common head and neck solid tumor histology, excluding cutaneous and thyroid malignancies. This fact, along with easy accessibility in upper aerodigestive tract and metastatic cervical lymphatic sites, has made SCCHN the dominant histology for head and neck gene therapy intervention studies. Advances in organ preservation treatment strategies and postablation reconstructive techniques have improved the quality of life of head and neck cancer (SCCHN) patients. Overall prognosis, however, has not been impacted for several decades—SCCHN continues to have a dismal 50% survival rate when considering all patients [1]. Locoregional recurrence, second primary tumor development, and distant metastatic disease all contribute to this poor prognosis. It has become obvious to many researchers that novel treatments, such as gene therapy, need to be developed to possibly improve the prognosis for head and neck cancer. Despite its relative rarity, SCCHN remains one of the most studied tumors for preclinical and clinical human gene therapy work. Gene therapy for SCCHN has developed into a very broad field. Many different genes have been placed into head and neck cancer cells using an array of viral and nonviral gene transfer vectors. Although some head and neck cancer gene therapies are still confined to preclinical laboratory and animal work, others are in various phases of human clinical trial. Strategies to make gene transfer more efficient and even more specific for the individual cancer cell (vector targeting) are active areas of research, along with programs combining gene therapies with standard treatment modalities.

E-mail address: [email protected] (D.K. Frank). 1055-3207/02/$ - see front matter Ó 2002, Elsevier Science (USA). All rights reserved. PII: S 1 0 5 5 - 3 2 0 7 ( 0 2 ) 0 0 0 2 1 - 2

590

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

This article begins by highlighting the most important gene therapy vectors used in head and neck cancer research. This is followed by a broad summary of gene transfer strategies and the specific genes that have been used in preclinical as well as clinical head and neck cancer research. I then explore the strategies being developed to make head and neck cancer gene therapy more efficient and specific. Finally, there is a discussion of the foreseeable applications of head and neck cancer gene therapy.

Vectors Vectors are the messengers used to transfer genetic material into cancer cells. The ideal vector for cancer gene therapy would be nontoxic to the host, highly specific to the tumor of interest and its metastases, readily available, and would have the ability to transfer its gene of interest into cancer cells with extremely high efficiency. Another desirable attribute would be an ability to avoid attack from the patient’s own immune system. Unfortunately, no vector in common use today has all of these desirable attributes, but addressing concepts such as vector gene transfer efficiency and vector specificity for tumor are active areas of gene therapy research. Although some vectors are toxic to cancer cells by themselves and are thus therapeutic (therapeutic vectors), most are engineered to carry specific genes of therapeutic interest. Viruses are currently the workhorses of human cancer gene therapy, and head and neck cancer gene therapy is no exception (Table 1). The bulk of head and neck cancer gene transfer has used viral vectors in both the laboratory and clinical setting. Recently, nonviral gene transfer tools have gained interest in head and neck cancer gene therapy, Table 1 Characteristics of the major vectors used for head and neck cancer gene therapy

Natural tropism for tissues of the upper aerodigestive tract Can be delivered to dividing and non-dividing cells Immunogenicity Can be delivered by local tumor injection Systemic delivery (experimental) Transduction efficiency Vector toxicity

Can be targeted to tumor tissue

Adenovirus

Cationic liposomes

Yes

No

Yes

Yes

Yes Yes

No (pegylated cationic type) Yes

No

Yes

Relatively high Mild (limited to flu-like illness at high vector doses) Yes

Relatively low None

Yes

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

591

and may offer some distinct advantages over the traditional viral approaches outlined below. Viral vectors used in head and neck cancer gene therapy Viruses, because of their tropism for human cells, were the natural choice of researchers looking for messengers to transfer therapeutic genetic material into cancer cells. It was hypothesized that viruses could be engineered, through recombinant DNA techniques, to deliver any gene of interest. This hypothesis has largely proved to be true. The most commonly used viral vectors for human cancer gene therapy in general are adenovirus, retrovirus, and herpes virus. Head and neck cancer gene therapy strategies have focused on the adenovirus in both the laboratory and clinical setting [2–16]. The potential for using adenoviruses for gene transfer was recognized in the 1980s and was put into practical use in the early 1990s. Some of the earliest human investigations focused on lung cancer cells [17]. Ultimately, the adenovirus emerged as the logical choice for the developing field of head and neck cancer gene therapy [3,8,9]. Adenovirus has a natural tropism for the tissues of the upper aerodigestive tract, the primary sites of which give rise to SCCHN. These double-stranded DNA viruses are easy to grow and manipulate, and they can be cultured and stored at very high titres [17]. Through recombinant DNA techniques, these viruses have been engineered to transport a therapeutic gene of interest by insertion of a double stranded copy of the DNA of that gene (cDNA) into the native viral genome. When the recombinant adenovirus ‘‘infects’’ the target cancer cell, it deposits its genome (including therapeutic gene) into this cell. This genome, with its therapeutic gene insert, gets expressed using the host cell machinery [18,19]. Adenoviruses used for cancer gene therapy have been engineered to be replication deficient. Not only does this serve to focus the therapeutic efficacy of the vector on the gene it is transferring, but it also serves to limit toxicity to surrounding normal cells. The latter point holds true only if overexpression of the therapeutic gene is not harmful to normal cells. It is also important to note that adenovirus DNA does not incorporate into the host cell genome. The viral DNA remains episomal in the nucleus—its genes (including cDNA therapeutic gene inserts) only get expressed during the life of the individual host cell [19]. Episomal DNA does not get passed on to daughter cells once cell division occurs. When carrying out gene transfer with adenovirus, it is necessary to infect the individual target cell with multiple recombinant viral particles to ensure significant therapeutic gene overexpression. Recombinant adenoviral vectors are also engineered with a very active gene transcription promoter (frequently a cytolomegalovirus promoter) directly upstream from the therapeutic gene insert, further ensuring overexpression once in the target cell [2,3,8,9,17]. In vivo as well as clinical trial work using adenoviruses in head and neck cancer has relied on direct intratumoral injection [2–4,7,14]. The obvious

592

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

disadvantage of this technique is the fact that not all cancer cells will be exposed to vector. Nonetheless, direct intratumoral injection remains the standard delivery route for adenovirus-mediated head and neck cancer gene therapy. Now, systemic delivery would be complicated by the unknowns of massive delivery of virus to nontarget tissues. Nonviral vectors used in head and neck cancer gene therapy Viral vectors can theoretically be administered systemically to deliver their gene of interest. Concern remains, however, regarding the systemic delivery of the large viral titres that would be required to deliver therapeutic doses of gene to tumor tissue. Not only could this be potentially deleterious to the host, but it could also stimulate a massive immune response against the vector. Thus, it is clear that a significant limitation of head and neck cancer gene therapy using viral vectors is the requirement for direct tumor injection. Subsequent to the initiation of viral-mediated gene transfer work in head and neck oncology, interest in using nonviral gene transfer strategies began to develop. In the mid 1990s, a cationic liposome-mediated gene transfer system was developed for preclinical and clinical work for human cancer [20,21]. Liposomes are synthetic spheres consisting of a lipid membrane on their outer surface. They are capable of encapsulating a wide array of therapeutic molecules in their core for the purposes of delivery to cancer cells [22,23]. In fact, liposomes have been used for the purpose of chemotherapy drug delivery to human cancers for some time [24]. Recent modification of the outer lipid bilayer have made liposome particles more immune stable, thus significantly increasing their circulation half life in instances of systemic delivery [25,26]. Cationic liposomes are among the major nonviral vectors used for head and neck cancer gene therapy work. They appear to be virtually nontoxic to the host, can be delivered to dividing and nondividing cells, and are not significantly immunogenic, thus theoretically making them ideal for clinical use (see Table 1) [21,25,27]. Liposomal vectors are similar to viral vectors in that the therapeutic gene is placed inside the vector. Delivery of the genetic material to target cells is achieved through fusion of the liposome and target cell membranes, thus depositing the therapeutic DNA into the cell. Standard, nontargeted liposomal gene transfer is somewhat less efficient than adenoviral gene transfer, however [20].

Head and neck cancer gene therapy strategies There are several major categories of genes that have been used against head and neck cancer in the experimental and clinical setting. These include tumor suppressor genes, suicide genes, immunologic genes, and antisense to oncogenes (Table 2). We will discuss the relevant genes in each category, and their advantages for use where relevant.

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

593

Table 2 The major genes used in head and neck cancer gene therapy research and their characteristics Tumor SuppImmunoEnhances ressor Suicide logic AntiOncolytic Bystander Standard Gene Gene Gene Oncogene Adenovirus Effect Therapy p53 X p16 X p21 X HSV-tk IL-2/IL-12 Allovectin Cyclin-D1 Antisense ONYX-015

X

X

X X X

X X

X

SCCHN Human Clinical Trial X

X

X X

X

Tumor suppressor gene therapy Tumor suppressor genes are indigenous to the human genome. They act by inhibiting the growth of cells under various physiologic circumstances, including genetic damage induced by carcinogens. The mutation or deletion of tumor suppressor genes is thus a characteristic of cancer development. Cells with key tumor suppressor mutations can grow in an unregulated fashion, with the potential for local invasion and metastases depending on other acquired genetic characteristics. Because mutation or deletion of tumor suppressor genes is a characteristic of the malignant genotype, gene therapy using the wild-type form of these genes can be regarded as gene replacement therapy. The most common tumor suppressor genes that have been used in head and neck cancer gene therapy include p53, p16, and p21 (see Table 2) [2–5,8–10,14,27–30]. Only p53 gene transfer has progressed to human clinical trial [2,4]. p53 tumor suppressor gene therapy Mutation of the p53 tumor suppressor gene occurs in a majority of human malignancies, including SCCHN [8,9]. The discovery of p53 and its important role as a tumor suppressor in the 1980s led to a mountain of research to elucidate its functions. This research is still ongoing. p53 is an important inducer of cell-cycle arrest and apoptosis under circumstances of cellular stress [8,9]. These circumstances include carcinogenic insult with resultant DNA damage. After cellular stress, the exact mechanisms that govern the direction a cell will take upon wild-type p53 induction (cell-cycle arrest versus apoptosis) are not entirely clear. Given the emerging role of p53 as an important tumor suppressor and its mutation in a significant number of human cancers, it was logical that this gene became the target of researchers interested in the developing field of cancer gene replacement therapy. By the early and mid 1990s, a model was

594

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

developed whereby wild-type p53 was delivered to lung cancer cells through an adenovirus vector [17]. It was not long before head and neck cancer researchers became interested in adenovirus-mediated wild-type p53 (Adp53) gene therapy [3,8,9]. Ad-p53 gene therapy represented one of the earliest gene transfer strategies against SCCHN. In preclinical studies conducted at the University of Texas M.D. Anderson Cancer Center, Ad-p53 induced apoptosis in head and neck cancer cell lines, regardless of their p53 genotype (mutant versus wild-type) [8]. In vivo studies demonstrated that Ad-p53 reduced the growth of established tumors in xenograft models of SCCHN [9]. Furthermore, in a nude mouse xenograft model of microscopic residual disease, Ad-p53 prevented the establishment of tumors from subcutaneously deposited SCCHN cell lines [3]. These early laboratory studies paved the way for the first human clinical gene therapy trial for SCCHN. In 1998, Clayman et al reported Phase I dose escalation results of Ad-p53 gene transfer in patients with locoregionally recurrent SCCHN who were unsuccessfully treated with conventional therapy, including radiotherapy [2]. Tumors were directly injected at scheduled intervals until disease progression for the unresectable group. Resectable patients were directly injected preoperatively, and had their tumor beds ‘‘washed’’ with Ad-p53 intraoperatively after tumor extirpation. Expression of vector wild-type p53 as well as the downstream wild-type p53 transactivated cyclin dependent kinase inhibitor p21 was demonstrated immunohistochemically in treated tumor biopsy specimens. Antibodies were detected against the vector after early dosing, but this did not seem to affect the expression of downstream wild-type p53 activated genes in treated tumor biopsies. This suggested that the immune response against Ad-p53 in this early clinical work was not neutralizing. This study demonstrated that Ad-p53 gene therapy was feasible and very well tolerated. The most significant side effects were a brief flu-like illness at the higher vector dose titres. Phase II and III work with Ad-p53 against SCCHN is being conducted. Transfer of wild-type p53 into SCCHN has not been limited to the adenovirus vector. Cationic liposomes have also been used for this purpose in preclinical in vitro as well as in vivo work. Much of the work with p53 gene transfer in SCCHN using liposomal vectors has focused on its radiosensitizing and chemosensitizing properties [27,30]. That is to say that wild-type p53 gene transfer synergistically augments the anticancer effects of ionizing radiation on SCCHN. The reasons for and relevance of this latter important finding, also demonstrated with adenovirus-mediated p53 gene transfer [29], are discussed later in this article. Nonetheless, the radiosensitizing properties of wild-type p53 serve as an advantage of this type of tumor suppressor gene transfer in SCCHN. To date, clinical p53 gene therapy has employed direct intratumoral injection [2,4]. Although transduction efficiencies are relatively high when adenoviruses are used for the gene delivery, intratumoral injection is nonetheless

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

595

an inefficient way to deliver gene. Only a portion of the cells in a tumor are actually receiving vector with therapeutic gene. Currently, mainly liposomes are being modified to more specifically target tumor tissues (see below) to circumvent this problem [26,27,30]. An interesting property of p53 gene therapy is its ability to induce a bystander effect. A bystander effect is the process whereby cancer cells that have received a specific therapy are able to bring about growth inhibition in untreated cells in the same tumor population. Any anticancer therapy that can induce a bystander effect, particularly a therapy that relies on direct intratumoral delivery, would have profound therapeutic advantages. Adenovirus-mediated wild-type p53 gene transfer has demonstrated a modest bystander effect, both in vitro and in vivo [31,32]. The in vivo bystander effect, demonstrated in a murine lung cancer model, suggests that the antiangiogenic properties of wild-type p53 overexpression in the tumors upon gene tranduction are responsible [32]. Interestingly, a modest bystander effect has been demonstrated in vitro for SCCHN. The etiology of the bystander effect in the in vitro work is unclear, although mediation by gap junctional intercellular communication was suggested [31]. p16 and p21 tumor suppressor gene therapy Recent years have seen the cyclin-dependent kinase inhibitor, p16INK4A (p16) emerge as an important tumor suppressor [33]. Alteration resulting in inactivation of the p16 locus is now being recognized as a common event in SCCHN [14]. This point has made gene replacement with the p16 tumor suppressor an area of interest for investigators. Adenovirus-mediated p16 gene therapy (Ad-p16) has demonstrated a profound growth inhibitory response in preclinical studies of SCCHN, both in vitro and in vivo [14]. Ad-p16 was able to reduce the size of established SCCHN tumors in a murine model, paving the way for potential human clinical trial. The downstream wild-type p53 transactivated gene, p21, like p16, is also a cyclin-dependent kinase inhibitor and a tumor suppressor [5,10]. Activation of p21 is theorized to be one of the mechanisms whereby p53 overexpression induces cell-cycle arrest. With this in mind, it was proposed by investigators that p21 tumor suppressor gene therapy may be effective against SCCHN. In vitro work with p21 delivered to SCCHN using an adenovirus vector, however, has not shown profound antitumor effect [5,10]. Suicide gene therapy Suicide gene therapy delivers a gene to a cancer cell that expresses a protein with a specific and desired enzymatic activity. Thus, the delivered gene is not the normal wild-type counterpart of a mutated gene (such as a tumor suppressor gene) involved in the cancer development/progression process. The enzymatic activity of expressed suicide genes will convert

596

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

a subsequently delivered prodrug to an active form (in cells expressing the suicide gene) that is toxic and lethal to the transduced cell. Suicide gene therapy in the treatment of human cancer has mostly been conducted with the herpes simplex virus-thymidine kinase (HSV-tk) gene therapy system (see Table 2). Interest in this type of gene therapy for use against SCCHN began in the mid 1990s [6,16]. As in tumor suppressor gene replacement therapy, adenoviruses have been used to transduce SCCHN with the HSV-tk gene in preclinical in vitro and in vivo work [6,7,15, 16,34]. Once the cancer cells have been transduced with HSV-tk, they are treated with the prodrug, ganciclovir. In animal preclinical studies, the prodrug was typically given systemically (intraperitoneal delivery) [7,15,34]. Cells expressing the HSV-tk gene will convert ganciclovir to a phosphorylated form, which is toxic to the expressing cell upon further phosphorylation with native cellular enzymes [35]. HSV-tk suicide gene therapy for SCCHN, as in other human cancers, has resulted in impressive tumor cell kill in SCCHN preclinical in vitro and in vivo (murine) work [6,7,15,16,34]. An interesting advantage of HSV-tk gene therapy appears to be the presence of a profound bystander effect in preclinical investigations. In SCCHN and other tumor model systems, it appears that only 10% of a cell population needs to actually be transduced with the HSV-tk gene in order to kill the entire population [6,7,15,36–38]. Elegant work from several researchers has demonstrated that this bystander effect requires the nontransduced cells to be in contact with HSV-tk transduced cells. Furthermore, the contacting cells must express functional gap junctional intercellular channels [36,37,39]. It has been demonstrated that the toxic phosphorylated ganciclovir produced in HSV-tk transduced cells travels through these intercellular gap junctions to affect nontransduced cells. The profound bystander effect characteristic of HSV-tk gene therapy appears to make it an ideal treatment strategy, because the vectors currently in use are not able to deliver therapeutic gene to every cell in a tumor population. The loss of the gap junctional intercellular communication (GJIC) phenotype, however, is a common event in tumorigenesis, thus making HSV-tk gene therapy most effective in only a subpopulation of tumors [40,41]. There have not been any human clinical SCCHN trials using HSV-tk gene therapy, although limited and early human work has been seen in other organ systems [42,43]. Immunologic gene therapy Patients with head and neck cancer, like cancer patients in general, are somewhat immunosuppressed. Deficits in lymphocyte function and cytokine production are not unusual. Also, natural killer cell function may also be affected [44–47]. It is unclear whether the altered immune state in cancer patients is a product of carcinogenesis, or is primarily causative. Nonetheless,

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

597

the generally poor immune status of cancer patients can impact upon tumor progression, development of systemic disease, and response to treatment. The boosting of the immune system with cytokines or biologic response modifiers has been an active area of research in head and neck cancer. Cytokines such as interleukin-2 (IL-2) boost immunity by increasing T cell and natural killer cell counts [48–51]. Unfortunately, systemic administration of cytokines can be quite toxic [52]. A human SCCHN trial of local administration of IL-2 in the early 1990s yielded only limited responses [53]. The promise of immunologic gene therapy lies in the theory that local tumor expression of immune stimulating genes (such as that encoding IL-2) could trigger an immune response against the cancer as a whole. Thus, every cell in the tumor need not be transduced with therapeutic gene. As indicated earlier, head and neck cancer gene therapy is limited in this regard by vector design and the direct tumor injection route of administration. Furthermore, immunologic gene therapy may be able to overcome some of the toxicities associated with systemic immunomodulation by keeping the immunomodulating agent, and thus the immune response, localized. In vivo murine gene transfer work with IL-2 and IL-12 has been conducted recently in SCCHN (see Table 2) [11,13]. The genes encoding these cytokines were introduced into tumor cells through adenoviral vectors. In the IL-2 work, cells were pretreated with Ad-IL-2 before animal implantation. This impacted significantly upon tumor development and progression after transduced cells were implanted. Partial responses were also seen using Ad-IL-2 against already established tumors. Normal major histocompatibility complex (MHC) proteins are cell surface molecules. They present antigen to the host immune system on the surfaces of cells, thus allowing for destruction of cells that have been altered in some fashion (that produce ‘‘foreign’’ antigens). Normal MHC proteins are absolutely required for foreign antigen presentation and the subsequent immune response process. Because cancer cells are phenotypically and genotypically different from normal cells, they should have a host of foreign antigens on their surfaces that are capable of triggering an antitumor response. Interestingly however, many human cancers, including head and neck cancer, no longer express (or express less) class I MHC, thus allowing them to avoid immune destruction [54–59]. With this in mind, a type of immunologic gene therapy has been developed that allows transduced cells to express class I MHC protein on their surfaces. This gene therapy, called alloantigen gene therapy (see Table 2), transduces cancer cells with the DNA that encodes for a class I MHC protein [60]. In theory, not only would this foreign MHC molecule initiate an antitumor response in the host, but it should also be able to trigger a response by complexing with tumor associated antigens. Alloantigen gene therapy is in early clinical trials against head and neck cancer. In phase I work, alloantigen gene therapy was administered to twenty patients with unresectable recurrent or persistent SCCHN [60]. The

598

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

gene was delivered by a liposomal vector through direct tumor injections. The gene therapy was regarded as quite safe because there were no adverse reactions. Some modest tumor responses were seen, and the transduced gene product (class I MHC HLA-B7) was expressed in treated tumor biopsy specimens. Other types of gene therapy Antioncogene gene therapy The genetic hallmarks of cancer cells are the mutation or deletion of tumor suppressor genes as well as the upregulation and overexpression of oncogenes. Tumor suppressor gene therapy, a type of gene replacement therapy, is an active area of head and neck cancer research as outlined above. Although it is not nearly as developed a research area, gene therapy directed against oncogenes has received the attention of some head and neck cancer investigators. Cyclins and their associated kinases act at different stages of the cell cycle to promote cell cycle progression, DNA synthesis, and ultimately cell division. Abnormal production of cyclins should in theory contribute to carcinogenesis by bringing about uncontrolled cell cycle progression and mitosis, not allowing for crucial cell cycle arrest and DNA repair when warranted. Indeed, cyclin D1, which is involved in progression from the G1 through the S (DNA synthesis) phases of the cell cycle, is amplified (at the DNA level) or overexpressed in human cancers, including SCCHN [61–63]. Cyclin D1 has thus been the target of some interesting laboratory research (see Table 2). Head and neck cancer cell lines transfected with an antisense gene to cyclin D1 have demonstrated decreased growth rates [64,65]. Furthermore, antisense to cyclin D1 transfected cell lines demonstrated decreased tumorigenicity in a nude mouse model [64]. Antisense genes are made by recombinant DNA techniques. Their transcribed messenger RNAs (mRNA) are designed to be complementary to the mRNAs of the native (sense) gene that is the target. The antisense mRNA will bind to its native sense homologue, thus rendering it inactive for translation into protein gene product. It should be possible, based on the results with cyclin D1, to transduce head and neck cancers with antisense genes directed against any number of oncogenes identified with this disease. Adenoviral or liposomal vectors, as in tumor suppressor gene therapy, could be used for this purpose. Oncolytic adenovirus ‘‘gene therapy’’ Treatment of head and neck cancer with the oncoloytic virus ONYX015 (see Table 2) deserves special mention. Although not a gene therapy per se because no therapeutic gene is actually transduced into cancer cells upon virus delivery, ONYX-015 takes advantage of some of the properties of the adenoviruses commonly used in cancer gene therapy. ONYX015 is an adenovirus that carries a deletion in its genome at the E1B

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

599

locus [66,67]. The E1B gene product normally binds to the key p53 tumor suppressor gene product in the host cell after infection, rendering it inactive [67,68]. The virus subsequently replicates and ultimately kills the infected cell. Because ONYX-015 is engineered to be E1B deficient, it should not be able to replicate in and destroy cells that express wild-type p53. These properties of ONYX-015 were of interest to cancer investigators because up to 60% of human malignancies are mutant at the p53 locus. Thus, it was hypothesized that ONYX-015 would selectively kill p53 mutant (cancer) cells [66–68]. It became clear early in preclinical development that some wild-type p53 cancer cells were sensitive to ONYX-015 [69,70]. This finding probably reinforces the fact that that there are post-transcriptional and post-translational factors that also govern p53 function in cells, even if the gene itself is not deleted or mutated. As noted, ONYX-015 appears to be most active in p53 mutant cells. At least 40% of human cancers, however, including head and neck cancer, are p53 wild-type. A healthy subset of these cancers will undoubtedly also have normal p53 expression, thus rendering them least sensitive to ONYX-015. This is a potential significant drawback to ONYX-015 cancer therapy. Nonetheless, ONYX-015 is in early clinical trial in the treatment of human malignancy, including SCCHN. A phase I clinical trial of direct intratumoral injection of ONYX-015 in patients with advanced SCCHN revealed that the treatment was well tolerated at high viral titres [71]. A phase II clinical trial in patients with unresectable, recurrent SCCHN was recently published [67]. About one third of patients in this trial had a partial response. Two patients had a complete response. No wild-type p53 tumors achieved a response, again highlighting a potential drawback of ONYX-015 in this genotypic subpopulation of tumors.

Enhancing head and neck cancer gene therapy efficiency and specificity Adenovirus and liposomal vectors are the workhorses of head and neck cancer gene therapy. In general, these vectors suffer from an inability to deliver therapeutic gene to every cell in a tumor population, thus limiting their efficiency as an anticancer tool. This is a significant obstacle that needs to be overcome in order to make cancer gene therapy in general more practical. Research is ongoing on this issue. The primary areas of interest of researchers in improving the efficiency of gene transfer include vector targeting and bystander effects. Certain types of gene therapy may also inherently be more efficient. Vector targeting Currently available vectors for gene transfer may have poor tropism for some tumors. The efficiency of gene transfer may also be hindered by the

600

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

inadvertent delivery of therapeutic gene into a nontarget cell. With this in mind, there has been progress made in targeting cationic liposomal vectors to tumor cells. Human tumor cells, including some SCCHNs, overexpress transferrin and folic acid receptors on their cell surfaces. Liposomal vectors have been engineered with the transferrin or folic acid ligand located on their surface to take advantage of this [26,27,30]. A transferrin ligandtargeted cationic liposomal vector markedly enhanced the transfection efficiency of head and neck cancer cells in vitro [27]. Because liposomal vectors are nontoxic and lack immunogenicity, systemic administration may be plausible when coupled with tumor associated ligands such as transferrin or folic acid. Systemic administration of such targeted liposomes would not only address the primary tumor, but possibly metastases as well. Preclinical in vivo murine work has demonstrated that transferrin ligand-liposome-p53 complex cationic liposomes can be delivered systemically and can sensitize various cancer cells types (including SCCHN) to either radiation or cisplatin based chemotherapy [27,30]. Tumor growth inhibition, tumor regression, and prevention of the development of metastases have been observed with this vector and similar (folic acid) ligand-targeted liposomes.

Bystander effects Clearly, any gene transfer treatment strategy that could also have an effect on tumor cells that have not been directly transduced would have tremendous therapeutic advantages. As indicated earlier, HSV-tk gene therapy is associated with a GJIC-mediated bystander effect in human tumor model systems [6,7,15,36–38]. Because the cancer phenotype is frequently associated with loss of GJIC [40,41], the HSV-tk-mediated bystander effect may have no practical value in the treatment of a large subset of malignancies. The protein building blocks of gap junctions are called connexins. The genes encoding connexins are usually not mutated during carcinogenesis—the decreased GJIC phenotype is a manifestation of decreased connexin production and poor localization into the cell membrane [36,40,41]. Thus, one way to get around the problem of decreased tumor GJIC and enhance an HSV-tk-mediated bystander effect would be to upregulate connexin production. There are multiple substances that do this, including retinoids. Retinoids have been shown to enhance the efficacy of HSV-tk gene therapy in human tumor model systems through the upregulation of GJIC [72–74]. The upregulation of GJIC seems to occur at the level of connexin production, possibly at the gene transcription level. The use of retinoids for this purpose in SCCHN would not only be valuable from a gene therapy viewpoint, but retinoids by themselves have therapeutic benefits against epithelial malignancies [75].

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

601

Foreseeable applications of head and neck cancer gene therapy Tremendous strides are being made in human cancer gene therapy technology, making it more efficient and specific. Nonetheless, it is unlikely that gene therapy for head and neck cancer will be used as a single curative treatment modality. It is most likely that gene therapy for head and neck cancer will find its way into the multidisciplinary treatment paradigm that has been taking shape for this disease. This multidisciplinary paradigm now includes surgery, external beam and interstitial radiation therapy, chemotherapy, and developing non-gene therapy molecular intervention strategies, such as inhibitors of the epidermal growth factor [76,77]. Some of the best examples of using head and neck cancer gene therapy in combination with other more standard treatment modalities can be seen with wild-type p53 gene transfer [27,29,30]. The DNA damaging effects of ionizing radiation and chemotherapy are known to upregulate wild-type p53 expression in cells with this capability, thus inducing cell-cycle arrest and or apoptosis [8,9]. It therefore seems logical that introducing exogenous wild-type p53 into head and neck cancer cells (the majority of which are p53 mutant) should sensitize these cells to the effects of radiation and chemotherapy. Indeed, wild type p53 gene transfer has been demonstrated to sensitize head and neck cancer cells to the effects of ionizing radiation and cytotoxic chemotherapy in vitro and in vivo [27,29,30]. This laboratory finding could be translated into a clinical situation where less radiotherapy and or chemotherapy could be delivered, along with wild-type p53 gene transfer into a given tumor. Improved clinical outcomes along with reduced radiation and or chemotherapy associated toxicities could result. Despite the removal of all gross and microscopic histologic disease during surgery for head and neck cancer, there remains a high rate of locoregional treatment failure. It has been demonstrated that treatment failure is associated with abnormalities at the molecular level in histologically normal appearing tumor resection margins, despite the delivery of standard postoperative radiation therapy [78]. Mutation at the p53 locus as well as overexpression of the eIF4E translation initiation factor are two examples of molecular derangements that are associated with treatment failure in histologically normal appearing SCCHN resection margins [79,80]. Thus, the use of tumor suppressor gene replacement therapy or antisense gene therapy directed against overexpressed tumor specific oncogenes just after surgical resection may prove to be a useful adjuvant to deal with the problem of microscopic residual disease. In the published phase I head and neck cancer gene therapy trial conducted at the University of Texas M.D. Anderson Cancer Center, Ad-p53 was delivered to a subset of patients in a surgical adjuvant setting (just after surgical extirpation). The clinical feasibility and safety of this approach was well demonstrated in these incurable patients [3]. Although gene therapy research has focused on the treatment of SCCHN, applications for the treatment of premalignacy are realistic. Also,

602

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

patients treated for head and neck cancer are notorious for the development of synchronous and metachronous second primary head and neck cancers. In fact, this is a common cause of mortality in the head and neck cancer patient. Thus, a potential future gene therapy application exists for these patients as well. Mutation at tumor suppressor and oncogene loci exists in patients with upper aerodigestive tract premalignancy, as well as in patients who have already been treated for a head and neck cancer. Such patients may ultimately be very good candidates for gene transfer directed at their specific abnormalities to prevent malignant progression or the development of recurrent or second primary tumors. References [1] American Cancer Society Facts and Figures. Publication # 93–400. Washington, DC: American Cancer Society; 1993. [2] Clayman GL, El-Naggar AK, Lippman SM, et al. Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma. J Clin Oncol 1998;16:2221–32. [3] Clayman GL, El-Naggar AK, Roth JA, et al. In vivo molecular therapy with p53 adenovirus for microscopic residual head and neck squamous carcinoma. Cancer Res 1995; 55:1–6. [4] Clayman GL, Frank DK, Bruso PA, et al. Adenovirus-mediated wild-type p53 gene transfer as a surgical adjuvant in advanced head and neck cancers. Clin Cancer Res 1999; 5:1715–22. [5] Clayman GL, Liu TJ, Overholt SM, et al. Gene therapy for head and neck cancer: comparing the tumor suppressor gene p53 and a cell cycle regulator WAF1/CIP1 (p21). Arch Otolaryngol Head Neck Surg 1996;122:489–93. [6] Goebel EA, Davidson BL, Graham SM, et al. Adenovirus-mediated gene therapy for head and neck squamous cell carcinomas. Ann Otol Rhinol Laryngol 1996;105:562–7. [7] Goebel EA, Davidson BL, Graham SM, et al. Tumor reduction in vivo after adenoviral mediated gene transfer of the herpes simplex thymidine kinase gene and ganciclovir treatment in human head and neck squamous cell carcinoma. Otolaryngol Head Neck Surg 1998;119:331–6. [8] Liu TJ, El-Naggar AK, McDonnell TJ, et al. Apoptosis induction mediated by wild-type p53 adenoviral gene transfer in squamous cell carcinoma of the head and neck. Cancer Res 1995;55:3117–22. [9] Liu TJ, Zhang WW, Taylor DL, et al. Growth suppression of human head and neck cancer cells by the introduction of wild-type p53 gene via a recombinant adenovirus. Cancer Res 1994;54:3662–7. [10] Mobley SR, Liu TJ, Hudson JM, et al. In vitro growth suppression by adenoviral transduction of p21 and p16 in squamous cell carcinoma of the head and neck: a research model for combination gene therapy. Arch Otolaryngol Head Neck Surg 1998;124:88–92. [11] Myers JN, Mank-Seymour A, Zitvogel L, et al. Interleukin-12 gene therapy prevents establishment of SCC VII squamous cell carcinomas, inhibits tumor growth, and elicits long-term antitumor immunity in syngeneic C3H mice. Laryngoscope 1998;108:261–8. [12] O’Malley BW, Chen SH, Schwartz MR, et al. Adenovirus-mediated gene therapy for human head and neck squamous cell cancer in a nude mouse model. Cancer Res 1995;55: 1080–5. [13] O’Malley Jr B, Li D, Buckner A, et al. Limitations of adenovirus-mediated interleukin-2 gene therapy for oral cancer. Laryngoscope 1999;109:389–95.

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

603

[14] Rocco JW, Liggett Jr WH, Duan L, et al. p16INK4A adenovirus-mediated gene therapy for human head and neck squamous cell cancer. Clin Cancer Res 1998;4:1697–704. [15] Sewell DA, Li D, Duan L, et al. Safety of in vivo adenovirus-mediated thymidine kinase treatment of oral cancer. Arch Otolaryngol Head Neck Surg 1997;123:1298–302. [16] Wilson KM, Stambrook PJ, Bi WL, et al. HSV-tk gene therapy in head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 1996;122:746–9. [17] Zhang WW, Fang X, Mazur W, et al. High-efficiency gene transfer and high-level expression of wild-type p53 in human lung cancer cells mediated by recombinant adenovirus. Cancer Gene Ther 1994;1:5–13. [18] Berkner KL. Development of adenovirus vectors for the expression of heterologous genes. Biotechniques 1988;6:616–29. [19] Hitt MA, Addison CL, Graham FL. Human adenovirus vectors for gene transfer into mammalian cells. Adv Pharmacol 1997;40:137–206. [20] Bebok Z, Abai AM, Dong JY, et al. Efficiency of plasmid delivery and expression after lipidmediated gene transfer to human cells in vivo. J Pharmacol Exp Ther 1996;279:1462–9. [21] Felgner PL, Tsai YJ, Sukhu L, et al. Improved cationic lipid formulations for in vivo gene therapy. Ann N Y Acad Sci 1995;771:126–38. [22] Felgner PL, Ringold GM. Cationic liposome-mediated transfection. Nature 1989;337: 387–9. [23] Felgner PL, Zaugg RH, Norman JA. Synthetic recombinant DNA delivery for cancer therapeutics. Cancer Gene Ther 1995;2:61–5. [24] Gregoriadis G, Wills EJ, Swain CP, et al. Drug-carrier potential of liposomes in cancer chemotherapy. Lancet 1974;1:1313–6. [25] Harrington KJ, Mohammadtaghi S, Uster PS, et al. Effective targeting of solid tumors in patients with locally advanced cancers by radiolabeled pegylated liposomes. Clin Cancer Res 2001;7:243–54. [26] Xu L, Chang EH. Tumor-targeted gene delivery by ligand-PEG ‘‘post-coated’’ cationic liposomes [abstract 502]. In: Proceedings of the 1999 AACR, NCI, EORTC International Conference. Washington, DC; 1999. p. 102. [27] Xu L, Pirollo KF, Chang EH. Transferrin-liposome-mediated p53 sensitization of squamous cell carcinoma of the head and neck to radiation in vitro. Hum Gene Ther 1997;8: 467–75. [28] Chang EH, Jang YJ, Hao Z, et al. Restoration of the G1 checkpoint and the apoptotic pathway mediated by wild-type p53 sensitizes squamous cell carcinoma of the head and neck to radiotherapy. Arch Otolaryngol Head Neck Surg 1997;123:507–12. [29] Pirollo KF, Hao Z, Rait A, et al. p53-mediated sensitization of squamous cell carcinoma of the head and neck to radiotherapy. Oncogene 1997;14:1735–46. [30] Xu L, Tang WW, Pirollo KF, et al. Chemosensitization of tumor cells by systemically delivered, ligand-directed, cationic liposome mediated wtp53 gene therapy [abstract 535]. In: Proceedings of the 1999 AACR, NCI, EORTC International Conference. Washington, DC; 1999. p. 109. [31] Frank DK, Frederick MJ, Liu TJ, et al. Bystander effect in the adenovirus-mediated wildtype p53 gene therapy model of human squamous cell carcinoma of the head and neck. Clin Cancer Res 1998;4:2521–7. [32] Nishizaki M, Fujiwara T, Tanida T, et al. Recombinant adenovirus expressing wild-type p53 is antiangiogenic: a proposed mechanism for bystander effect. Clin Cancer Res 1999;5: 1015–23. [33] Okamoto A, Demetrick DJ, Spillare EA, et al. Mutations and altered expression of p16INK4 in human cancer. Proc Natl Acad Sci USA 1994;91:11045–9. [34] Sewell DA, Li D, Duan L, et al. Optimizing suicide gene therapy for head and neck cancer. Laryngoscope 1997;107:1490–5. [35] Reid R, Mar EC, Huang ES, et al. Insertion and extension of acyclic, dideoxy, and ara nucleotides by herpes-viridae, human alpha and human beta polymerases: a unique

604

[36] [37]

[38] [39] [40] [41] [42]

[43]

[44] [45]

[46] [47] [48]

[49]

[50]

[51]

[52] [53] [54] [55]

[56]

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606 inhibition mechanism for 9-(1,2-dihydroxy-2-propoxymethyl)guanine triphosphate. J Biol Chem 1988;262:3898–904. Mesnil M, Piccoli C, Tiraby G, et al. Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins. Proc Natl Acad Sci USA 1996;93:1831–5. Mesnil M, Yamasaki H. Bystander effect in herpes simplex virus-thymidine kinase/ganciclovir cancer gene therapy: role of gap-junctional intercellular communication. Cancer Res 2000;60:3989–99. Wu JK, Cano WG, Meylaerts SAG, et al. Bystander tumoricidal effect in the treatment of experimental brain tumors. Neurosurgery 1994;35:1094–103. Pitts JD. Cancer gene therapy: a bystander effect using the gap junction pathway. Mol Carcinog 1994;11:127–30. Holder JW, Elmore E, Barrett JC. Gap junction function and cancer. Cancer Res 1993;53: 3475–85. Yamasaki H. Gap junctional intercellular communication and carcinogenesis. Carcinogenesis 1990;11:1051–8. Alvarez RD, Curiel DT. A phase I study of recombinant adenovirus vector-mediated intraperitoneal delivery of herpes simplex thymidine kinase gene and intravenous ganciclovir for previously treated ovarian and extraovarian cancer patients. Hum Gene Ther 1997;8:597–613. Sterman DH, Treat J, Litzky LA, et al. Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a phase I clinical trial in malignant mesothelioma. Hum Gene Ther 1998;9:1083–92. Cortesina G, Sacchi M, Galeazzi E, et al. Immunology of head and neck cancer: perspectives. Head Neck 1993;15:74–7. Mickel RA, Kessler DJ, Taylor JMG, et al. Natural killer cell cytotoxicity in the peripheral blood, cervical lymph nodes and tumor of head and neck cancer patients. Cancer Res 1988; 48:5017–22. Schantz SP, Shillitoe EJ, Brown B, et al. Natural killer cell activity and head and neck cancer: a clinical assessment. J Natl Cancer Inst 1986;77:869–75. Vitolo D, Letessier EM, Johnson JT, et al. Immunologic effector cells in head and neck cancer. J Natl Cancer Inst 1992;13:203–8. Melioli G, Margarino G, Scaka M, et al. Perilymphatic injections of recombinant interleukin-2 (rIL-2) partially correct the immunologic defects in patients with advanced head and neck squamous cell carcinoma. Laryngoscope 1992;102:572–8. Rabinowich H, Vitolo D, Altarac S, et al. Role of cytokines in the adoptive model of human head and neck cancer by human IL-2 activated natural killer cells. J Immunol 1992; 149:340–9. Salter J, Maclennan KA, Moore J, et al. The phenotypic changes in tumour infiltrating lymphocytes and tumour cells following intra-arterial infusion of interleukin-2 in patients with squamous cell carcinoma. J Pathol 1995;176:167–73. Whiteside TL, Letessier E, Hirabayashi H, et al. Evidence for local and systemic activation of immune cells by peritumoral injections of interleukin 2 in patients with advanced squamous cell carcinoma of the head and neck. Cancer Res 1993;53:5654–62. Siegel JP, Puri RK. Interleukin-2 toxicity. J Clin Oncol 1991;9:694–704. Cortesina G, De Stefani A, Sacchi M, et al. Immunomodulation therapy for squamous cell carcinoma of the head and neck. Head Neck 1993;15:266–70. Esteban F, Concha A, Delgado M, et al. Lack of MHC class I antigens and tumour aggressiveness of squamous cell carcinoma of the larynx. Br J Cancer 1990;62:1047–51. Honma S, Tsukada S, Honda S, et al. Biological-clinical significance of selective loss of HLA-class-I allelic product expression in squamous cell carcinoma of the uterine cervix. Int J Cancer 1994;57:650–5. Houck JR, Sexton FM, Zajdel G. HLA class I and class II antigen expression on squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg 1990;116:1181–5.

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

605

[57] Isakov N, Katza VS, Feldman M, et al. Loss of expression of transplantation antigens encoded by the H-2K locus on Lewis lung carcinoma cells and its relevance to the tumor’s metastatic properties. J Natl Cancer Inst 1983;71:139–45. [58] Lampson LA, Fisher CA, Whelan JP. Striking paucity of HLA-A, B, C and beta 2-microglobulin on human neuroblastoma cell lines. J Immunol 1983;130:2471–8. [59] Mattijssen V, de Mulder PHM, Schalkwijk L, et al. HLA antigen expression in routinely processed head and neck squamous cell carcinoma primary lesions of different sites. Int J Cancer Suppl 1991;6:95–100. [60] Gleich LL. Gene Therapy for Head and Neck Cancer. Laryngoscope 2000;110:708–26. [61] Jiang W, Kahn S, Tomita N, et al. Amplification and expression of the human cyclin D gene in esophageal cancer. Cancer Res 1992;52:2980–3. [62] Lammie G, Fantl V, Smith R, et al. D11S287, a putative oncogene on chromosome 11q13 is amplified and expressed in squamous cell and mammary carcinomas and linked to BCL-1. Oncogene 1991;6:439–44. [63] Schuuring E, Verhoeven E, Mooi W, et al. Identification and cloning of two overexpressed genes U21B31/PRAD1 and EMS1, within the amplified chromosome 11q13 region in human carcinomas. Oncogene 1992;7:355–61. [64] Nakashima T, Clayman GL. Antisense inhibition of cyclin D1 in human head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 2000;126:957–61. [65] Wang MB, Billings KR, Venkatesan N, et al. Inhibition of cell proliferation in head and neck squamous cell carcinoma cell lines with antisence cyclin D1. Otolaryngol Head Neck Surg 1998;119:593–9. [66] Bischoff JR, Kirn DH, Williams A, et al. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science 1996;274:373–6. [67] Nemunaitis J, Ganly I, Khuri, et al. Selective replication and oncolysis in p53 mutant tumors with ONYX-015, an E1B–55kD gene-deleted adenovirus, in patients with advanced head and neck cancer: a phase II trial. Cancer Res 2000;60:6359–66. [68] Debbas M, White E. Wildtype p53 mediates apoptosis by E1A, which is inhibited by E1B. Genes Dev 1993;7:546–54. [69] Goodrum FD, Ornelles DA. p53 status does not determine outcome of E1B 55-kilodalton mutant adenovirus lytic infection. J Virol 1998;72:9479–90. [70] Turnell AS, Grand RJ, Gallimore PH. The replicative capacities of large E1B-null group A and group C adenoviruses are independent of host cell p53 status. J Virol 1999;73:2074–83. [71] Ganly I, Kirn D, Eckhardt S. A phase I study of ONYX-015, an E1B attenuated adenovirus, administered intratumorally to patients with recurrent head and neck cancer. Clin Cancer Res 2000;6:798–806. [72] Mehta PP, Bertram JS, Lowenstein WR. The actions of retinoids on cellular growth correlate with their actions on gap junctional communication. J Cell Biol 1989;108: 1053–65. [73] Park JY, Elshami AA, Amin K, et al. Retinoids augment the bystander effect in vitro and in vivo in herpes simplex virus thymidine kinase/ganciclovir-mediated gene therapy. Gene Ther 1997;4:909–17. [74] Rogers M, Berestecky JM, Hossain MZ, et al. Retinoid-enhanced gap junctional communication is achieved by increased levels of connexin 43 mRNA and protein. Mol Carcinog 1990;3:335–43. [75] Lotan R. Suppression of squamous cell carcinoma growth and differentiation by retinoids. Cancer Res 1994;54(suppl):1987–90. [76] Robert F, Ezekiel MP, Spencer SA. Phase I study of anti-epidermal growth factor receptor antibody cetuximab in combination with radiation therapy in patients with advanced head and neck cancer. J Clin Oncol 2001;19:3234–43. [77] Shin DM, Donato NJ, Perez-Soler R, et al. Epidermal growth factor receptor-targeted therapy with C225 and cisplatin in patients with head and neck cancer. Clin Cancer Res 2001;7:1204–13.

606

D.K. Frank / Surg Oncol Clin N Am 11 (2002) 589–606

[78] Brennan JA, Mao L, Hruban RH, et al. Molecular assessment of histopathologic staging in squamous cell carcinoma of the head and neck. N Engl J Med 1995;332:429–35. [79] Koch WM, Brennan JA, Zahurak M, et al. p53 mutations and locoregional treatment failure in head and neck squamous cell carcinoma. J Natl Cancer Inst 1996;88:1580–6. [80] Nathan CO, Liu L, Li B, et al. Detection of the proto-oncogene eIF4E in surgical margins may predict recurrence in head and neck cancer. Oncogene 1997;15:579–84.