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Viruses can be engineered or adapted for selective propagation in neoplastic tissues and further modified for therapeutic transgene expression to enhance their antitumor potency and druggability. Oncolytic viruses (OVs) can be administered locally or intravenously and spread to a variable degree at sites of tumor growth. OV-infected tumor cells die in situ, releasing viral and tumor antigens that are phagocytosed by macrophages, transported to regional lymph nodes, and presented to antigen-reactive T cells, which proliferate before dispersing to kill uninfected tumor cells at distant sites. Several OVs are showing clinical promise, and one of them, talimogene laherparepvec (T-VEC), was recently granted marketing approval for intratumoral therapy of nonresectable metastatic melanoma. T-VEC also appears to substantially enhance clinical responsiveness to checkpoint inhibitor antibody therapy. Here, we examine the T-VEC paradigm and review some of the approaches currently being pursued to develop the next generation of OVs for both local and systemic administration, as well as for use in combination with other immunomodulatory agents.

// Kevin Van der Jeught 1 , Lukasz Bialkowski 1 , Lidia Daszkiewicz 1 , Katrijn Broos 1 , Cleo Goyvaerts 1 , Dries Renmans 1 , Sandra Van Lint 1 , Carlo Heirman 1 , Kris Thielemans 1 and Karine Breckpot 1 1 Laboratory of Molecular and Cellular Therapy, Department of Immunology-Physiology, Vrije Universiteit Brussel, Laarbeeklaan, Jette, Belgium Correspondence: Karine Breckpot, email: // Keywords : Intratumoral, Immunotherapy, Tumor microenvironment, Immunomodulation, Vaccination Received : November 18, 2014 Accepted : December 24, 2014 Published : December 26, 2014 Abstract The identification of tumor-specific antigens and the immune responses directed against them has instigated the development of therapies to enhance antitumor immune responses. Most of these cancer immunotherapies are administered systemically rather than directly to tumors. Nonetheless, numerous studies have demonstrated that intratumoral therapy is an attractive approach, both for immunization and immunomodulation purposes. Injection, recruitment and/or activation of antigen-presenting cells in the tumor nest have been extensively studied as strategies to cross-prime immune responses. Moreover, delivery of stimulatory cytokines, blockade of inhibitory cytokines and immune checkpoint blockade have been explored to restore immunological fitness at the tumor site. These tumor-targeted therapies have the potential to induce systemic immunity without the toxicity that is often associated with systemic treatments. We review the most promising intratumoral immunotherapies, how these affect systemic antitumor immunity such that disseminated tumor cells are eliminated, and which approaches have been proven successful in animal models and patients.

Management of high-grade gliomas remains a complex challenge. Standard of care consists of microsurgical resection, chemotherapy and radiation, but despite these aggressive multimodality therapies the overall prognosis remains poor. A major focus of ongoing translational research studies is to develop novel therapeutic strategies that can maximize tumor cell eradication while minimizing collateral side effects. Particularly, biological intratumoral therapies have been the focus of new translational research efforts due to their inherent potential to be both dynamically adaptive and target specific. This two-part review will provide an overview of biological intratumoral therapies and summarize key advances and remaining challenges in intratumoral biological therapies for high-grade glioma. Part I focuses on discussion of the concepts of intratumoral delivery and immunotoxin therapies.

Management of high-grade gliomas (HGGs) remains a complex challenge with an overall poor prognosis despite aggressive multimodal treatment. New translational research has focused on maximizing tumor cell eradication through improved tumor cell targeting while minimizing collateral systemic side effects. In particular, biological intratumoral therapies have been the focus of novel translational research efforts due to their inherent potential to be both dynamically adaptive and target specific. This two part review will provide an overview of biological intratumoral therapies that have been evaluated in human clinical trials in HGGs, and summarize key advances and remaining challenges in the development of these therapies as a potential new paradigm in the management of HGGs. Part II discusses vector-based therapies, cell-based therapies and radioimmunotherapy.

Immune mechanisms have evolved to cope with local entry of microbes acting in a confined fashion but eventually inducing systemic immune memory. Indeed, in situ delivery of a number of agents into tumors can mimic in the malignant tissue the phenomena that control intracellular infection leading to the killing of infected cells. Vascular endothelium activation and lymphocyte attraction, together with dendritic cell–mediated cross-priming, are the key elements. Intratumoral therapy with pathogen-associated molecular patterns or recombinant viruses is being tested in the clinic. Cell therapies can be also delivered intratumorally, including infusion of autologous dendritic cells and even tumor-reactive T lymphocytes. Intralesional virotherapy with an HSV vector expressing GM-CSF has been recently approved by the Food and Drug Administration for the treatment of unresectable melanoma. Immunomodulatory monoclonal Abs have also been successfully applied intratumorally in animal models. Local delivery means less systemic toxicity while focusing the immune response on the malignancy and the affected draining lymph nodes.

Background: Resistance of pancreatic ductal adenocarcinoma (PDAC) to checkpoint blockade therapy is commonly attributed to its status as an immunologically cold tumor. However, it remains to be determined whether the lack of endogenous immunity to PDAC is predominantly due to a paucity of targetable neoantigens or to the unique tolerogenic environment of malignant pancreatic tissue. Using an implantable model derived from the Kras+/LSL-G12DTrp53+/LSL-R172HPdx1-Cre (KPC) mouse, we investigated how tissue implantation site and exogenous antigen presentation influenced immune surveillance of KPC tumors and response to combination immunotherapy. Methods: The KPC organoid-derived cell line mT4-2D was stably transduced with various expression constructs including luciferase (mT4-Luc), ovalbumin (mT4-OVA), or mCherry (mT4-Cherry). Each line was implanted subcutaneously (2.5x105 cells) or orthotopically at indicated doses to assess effects of exogenous antigen expression on engraftment efficiency. To evaluate how tissue environment governs response to therapy, mice were co-implanted with mT4-Luc at both orthotopic and subcutaneous sites and were exposed to combination therapy consisting of intratumoral STING agonist ML-RR-CDA (10ug/mouse) and checkpoint antagonists αCTLA-4 and αPD-1 beginning on day 14 post-implantation. Tumor growth at both sites was monitored weekly by IVIS bioluminescent imaging and mice were followed for survival, or tumors were harvested on day 25 for analysis of immune infiltrates by flow cytometry. Results: mT4 tumors grew aggressively at both subcutaneous and orthotopic sites, with doses as low as 3.5x104 causing mortality within 3 weeks post-implantation. However, expression of either OVA or mCherry resulted in spontaneous clearance in 100% of mice, regardless of implantation site. Luciferase expression yielded progressing tumors, and ex vivo peptide restimulation failed to identify Luciferase-specific T cells within mT4-Luc challenged mice; suggesting that Luciferase is a minimally immunogenic protein in C57BL/6 mice. Despite the poor immunogenicity of the mT4 model, we found that intratumoral injections of ML-RR-CDA delayed growth of subcutaneously-implanted mT4, and that concomitant administration of αCTLA-4/αPD-1 induced complete clearance in ~40% of mice. In contrast, intratumoral delivery of ML-RR-CDA was ineffective against orthotopic mT4 tumors, and combination therapy with αCTLA-4/αPD-1 achieved only modest delays in tumor progression. Flow cytometric analysis of tumor infiltrating immune populations in mice with co-implanted orthotopic and subcutaneous lesions revealed dichotomies in the density of CD8 T cells, CD4 effector T cells, and dendritic cell populations between tissue sites in response to local therapy. Conclusions: Although implantable cell lines isolated from KPC tumors are highly aggressive and resistant models of PDAC, we found that engraftment and growth of mT4-2D was completely negated upon forced expression of a single foreign protein antigen. This suggests that the lack of targetable mutanome products may explain the resistance of KPC and potentially human PDAC tumors to immunotherapy. However, when implanted subcutaneously, mT4-2D was sensitive to in situ STING activation in combination with checkpoint blockade, which indicates that immune responses against tissue-specific antigens are potentially sufficient to control growth of KPC tumors when removed from the suppressive pancreatic environment. Flow analysis revealed a greater capacity of CD8 T cells, CD4 T cells, and dendritic cells to infiltrate subcutaneous vs orthotopic tumors following intratumoral therapy, reinforcing the theory that therapies which enhance infiltration of select immune populations are needed in order to sensitize PDAC tumors to therapy. Citation Format: Casey R. Ager, Michael A. Curran. Effects of tissue site and antigenicity on KPC-derived pancreatic tumor growth and response to combination immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B52.

OBJECTIVE: To explore the efficacy and feasibility of interstitial chemotherapy using thermosensitive gel-coated ricin in hepatoma H22-bearing mice. METHODS: Ricin was purified by chromatography method. The purified ricin was identified by Western blot assay and the purity was determined by high-performance liquid chromatography. BALB/c mice were inoculated subcutaneously in right flank with hepatoma H22 cells. When the tumor size reached about 1.0 cm in diameter, 40 mice were randomly divided into untreated group, thermosensitive gel group, ricin group and thermosensitive gel-coated ricin group. Mice in each group were administered different agents by percutaneous intratumoral injection, including normal saline, thermosensitive hydrogel, ricin and thermosensitive gel-coated ricin. Fifteen days after treatment, the tumors were removed to calculate inhibition rate of tumor growth. The tumor tissues were made into pathological sections to perform histopathological examination. The ultrastructure of tumor tissue was examined by electron microscope examination as well. Blood was collected to detect the hepatic and renal functions. The caspase-3 activity of tumor tissue was determined by using zymologic method with a spectrophotometer. RESULTS: After intratumoral therapy, tumor weight in the thermosensitive gel-coated ricin group was lower than that in the untreated group, with a tumor growth inhibition rate of 71.31%. No obvious hepatic or renal toxicities were detected after thermosensitive gel-coated ricin treatment. Histopathologic observation of the tumor tissue showed massive necrosis and typical apoptosis phenomena, including chromatin margination and apoptotic body. Meanwhile, thermosensitive gel-coated ricin resulted in a significant increase in the caspase-3 activity as compared with the untreated group and the ricin group (P

In the treatment of adenocarcinoma of pancreas adjuvante and palliative treatment is of great importance. However the sucess of evidence based standard therapies in pancreatic carcinoma with Gemcitabine-monotherapy or other polychemotherapies are very limited, thus alternatives and innovative treatment options are required. This paper provides an overview of new strategies in surgical treatment and palliative local cytostatic and immunologic therapy options. The results of own studies using integrative approaches with mistletoe concomitant to chemotherapy are promising. Mistletoe therapy exerts positive effects on quality of life, chemotherapy related adverse effects and symptoms due to illness are reduced, time of hospitalization is shortened. Besides subcutaneous application of mistletoe intratumoral application is very effective. An own pilot study in hospital Havelhoehe in Berlin, Germany with 14 patients showed a local response in 92 % of patients concerning tumor reduction, prolongation of survival time and improvement of quality of life. Further complementary therapy options concomitant to chemotherapy like administration of antioxidants, vitamin C and E, selenium and glutathione are presented.

Purpose: IL13-PE38, a targeted cytotoxin comprised of interleukin 13 (IL-13) and a mutated form of Pseudomonas exotoxin, induces specific killing of tumor cells expressing abundant levels of the IL-13Rα2 chain. We hypothesized that tumor cells killed by the cytotoxin may release antigens and/or apoptotic bodies when cells are dying, which then induce adoptive immunity, and that the PE38 portion of IL13-PE38 may act as a stimulant for the induction of a CTL response.Experimental Design: To test this hypothesis, we established D5 melanoma tumors with or without expression of the IL-13Rα2 chain in both flanks of C57BL/6 mice, and then IL13-PE38 was injected in the right flank tumors only.Results and Conclusions: When animals with IL-13Rα2-expressing D5 tumor (right) were injected with IL13-PE38, right flank tumors expressing the IL-13Rα2 chain not only showed dramatic regression but contralateral tumors (left flank) also showed tumor regression. Cell depletion experiments in tumor-bearing animals indicated that both CD8+ and CD4+ T cells contribute to the regression of contralateral tumors through CTL activation in the periphery and cellular infiltration into tumors. In addition, intratumoral treatment into s.c. tumors of mice bearing metastatic lung tumors with IL13-PE38 showed not only the reduction of treated s.c. tumor but also the reduction of lung metastasis. Thus, IL13-PE38 mediates an antitumor effect not only directly but also indirectly by inducing a host CD8+ T cell immune response. Accordingly, targeted cytotoxins may be used to treat local disease even if they cannot be administered systemically, and yet may still induce a reasonable systemic antitumor response.

Purpose of review Despite advances in surgery, radiation therapy, and chemotherapy, malignant gliomas continue to be associated with a poor prognosis. Even the most intensive combinations of radiotherapy and chemotherapy are not curative. In recent years our understanding of how tumor cells overcome cell cycle control, evade programmed cell death, induce blood vessel formation, and escape immune regulation has increased substantially. Significant efforts are directed towards the development of novel experimental therapies to target these molecular and biological mechanisms that lead to the development and growth of brain tumors. This review summarizes the most recent developments in non-cytotoxic therapy for malignant gliomas, such as targeted molecular drugs, inhibitors of angiogenesis and intratumoral therapy. Recent findings The first generation of studies using these novel therapies is nearing completion. In general, most of these treatments are well tolerated, but single-agent activity is modest. There is significant interest in combining these therapies with each other and with conventional cytotoxic therapies such as radiation therapy and chemotherapy. Summary These new therapeutic approaches for malignant gliomas are showing modest activity. As we learn to use these agents more effectively, and as an increasing number of new and potentially promising agents are developed, it is likely that therapies for malignant gliomas will improve over the next few years.

Purpose: This was an open-label, dose-escalation trial of intratumoral administration of IL-4Pseudomonas exotoxin (NBI-3001) in patients with recurrent malignant glioma. Patients and methods: A total of 31 patients with histologically verified supratentorial grades 3 and 4 astrocytoma were studied. Of these, 25 patients were diagnosed with glioblastoma multiforme (GBM) while six were diagnosed with anaplastic astrocytoma. Patients were over 18 years of age and had Karnofsky performance scores ≥60. Patients were assigned to one of four dose groups in a dose-escalation fashion: 6 µg/ml × 40 ml, 9 µg/ml × 40 ml, 15 µg/ml × 40 ml, or 9 µg/ml × 100 ml of NBI-3001 administered via convection-enhanced delivery intratumorally using stereotactically placed catheters. Patients were followed with serial MRI scans and clinical assessments every four weeks for the first 16 weeks and then every eight weeks until week 26. Results: No drug-related systemic toxicity, as evident by lack of hematological or serum chemical changes, was apparent in any patients; treatment-related adverse effects were limited to the central nervous system. No deaths were attributable to treatment. Drug-related grade 3 or 4 toxicity was seen in 39% of patients in all dose groups and 22% of patients at the maximum tolerated dose of 6 µg/ml × 40 ml. The overall median survival was 8.2 months with a median survival of 5.8 months for the GBM patients. Six-month survival was 52% and 48%, respectively. Gadolinium-enhanced magnetic resonance imaging of the brain showed areas of decreased signal intensity within the tumor consistent with tumor necrosis following treatment in many patients. Conclusions: NBI-3001 appears to have an acceptable safety and toxicity profile when administered intratumorally in patients with recurrent malignant glioma.

With emerging drug delivery technologies becoming accessible, more options are expected to become available to patients with glioblastoma (GBM) in the near future. It is important for clinicians to be familiar with the underlying mechanisms and limitations of intratumoral drug delivery, and direction of recent research efforts. Tumor-adjacent brain is an extremely complex living matrix that creates challenges with normal tissue intertwining with tumor cells. For convection-enhanced delivery (CED), the role of tissue anisotropy for better predicting the biodistribution of the infusate has recently been studied. Computational predictive methods are now available to better plan CED therapy. Catheter design and placement—in addition to the agent being used—are critical components of any protocol. This paper overviews intratumoral therapies for GBM, highlighting key anatomic and physiologic perspectives, selected agents (especially immunotoxins), and some new developments such as the description of the glymphatic system.

The recent literature documents the growing interest in local intratumoral chemotherapy as well as systemic preoperative chemotherapy with evidence for improved outcomes using these therapeutic modalities. Nevertheless, with few exceptions, the conventional wisdom and standard of care for clinical and surgical oncology remains surgery followed by radiation and/or systemic chemotherapy, as deemed appropriate based on clinical findings. This, in spite of the fact that the toxicity of conventional systemic chemotherapy and immunotherapy affords limited effectiveness and frequently compromises the quality of life for patients. Indeed, with systemic chemotherapy, the oncologist (and the patient) often walks a fine line between attempting tumour remission with prolonged survival and damaging the patient's vital functions to the point of death. In this context, it has probably been obvious for more than 100 years, due in part to the pioneering work of Ehrlich (1878), that targeted or localized drug delivery should be a major goal of chemotherapy. However, there is still only limited clinical use of nonsystemic intratumoral chemotherapy for even those high mortality cancers which are characterized by well defined primary lesions i.e. breast, colorectal, lung, prostate, and skin. There has been a proliferation of intratumoral chemotherapy and immunotherapy research during the past two to three years. It is therefore the objective of this review to focus much more attention upon intratumoral therapeutic concepts which could limit adverse systemic events and which might combine clinically feasible methods for localized preoperative chemotherapy and/or immunotherapy with surgery. Since our review of intratumoral chemo-immunotherapy almost 20 years ago (McLaughlin & Goldberg 1983), there have been few comprehensive reviews of this field; only one of broad scope (Brincker 1993), three devoted specifically to gliomas (Tomita 1991; Walter et al. 1995; Haroun & Brem 2000), one on hepatomas (Venook 2000), one concerning veterinary applications (Theon 1998), and one older review of dermatological applications (Goette 1981). However, none have shed light on practical opportunities for combining intratumoral therapy with subsequent surgical resection. Given the state-of-the-art in clinical and surgical oncology, and the advances that have been made in intratumoral drug delivery, minimally invasive tumour access i.e. fine needle biopsy, new drugs and drug delivery systems, and preoperative chemotherapy, it is timely to present a review of studies which may suggest future opportunities for safer, more effective, and clinically practical non-systemic therapy.

We recommended increased doses of 6 MIU per individual lesions larger than 2 cm [3, 4]. IL-2 has different known dose dependent, in part opposite effects, like expansion of regulatory T cells and induction of their suppressive characteristics or on the other hand activation of cellular antitumor immunity [5]. Therefore, we assume that a high concentration of IL-2 at a tumor tissue is crucial for efficacy. These high concentrations can only insufficiently be reached through systemic therapy, with its dose-limiting toxicity [6] or by low doses of IL-2 injected in large metastases. Therefore and in contrast to our initial recommendations, we currently restrict intratumoral therapy with free IL-2 to lesions up to 1 cm in longest diameter and increase the dose per lesion as tolerated up to a maximum of 18 MIU units IL-2 per day. Recurrences developing during therapy should be included in subsequent treatments as soon as these become evident. In contrast, the applied dose in the patient presented by Moreno-Ramirez et al. with 6–10 MIU IL-2 applied on two bulky lesions, each with ~5 cm diameter, seems low. Recently, the use of an antibody-based targeted version of IL-2 has exhibited promising anticancer effects in patients with stage IIIC melanoma. The use of the L19–IL2 fusion protein has allowed us to reduce the number of injections and is accompanied by favorable time-to-stage IV profiles [2]. Using this dataset, we now analyzed the rate of complete responses according to longest diameter of the lesion (Table 1). Promising complete responses were likewise observed in lesions larger than 1 cm, but due to the low number of large lesions in this trial, it is not clear whether the targeted form of IL-2 offers an advantage over free IL-2 for treatment of larger lesions. If patients present several metastases of different diameters at baseline, a combined strategy, including initial Dear Editors

Preexisting lymphocytic infiltration of tumors is associated with superior prognostic outcomes in a variety of cancers. Recent studies also suggest that lymphocytic responses may identify patients more likely to benefit from therapies targeting immune checkpoints, suggesting that therapeutic efficacy of immune checkpoint blockade can be enhanced through strategies that induce tumor inflammation. To achieve this effect, we explored the immunotherapeutic potential of oncolytic Newcastle disease virus (NDV). We find that localized intratumoral therapy of B16 melanoma with NDV induces inflammatory responses, leading to lymphocytic infiltrates and antitumor effect in distant (nonvirally injected) tumors without distant virus spread. The inflammatory effect coincided with distant tumor infiltration with tumor-specific CD4(+) and CD8(+) T cells, which was dependent on the identity of the virus-injected tumor. Combination therapy with localized NDV and systemic CTLA-4 blockade led to rejection of preestablished distant tumors and protection from tumor rechallenge in poorly immunogenic tumor models, irrespective of tumor cell line sensitivity to NDV-mediated lysis. Therapeutic effect was associated with marked distant tumor infiltration with activated CD8(+) and CD4(+) effector but not regulatory T cells, and was dependent on CD8(+) cells, natural killer cells, and type I interferon. Our findings demonstrate that localized therapy with oncolytic NDV induces inflammatory immune infiltrates in distant tumors, making them susceptible to systemic therapy with immunomodulatory antibodies, which provides a strong rationale for investigation of such combination therapies in the clinic.

Surgical removal of cystic craniopharyngiomas in children is associated with significant operative morbidity and recurrence rates. The purpose of this study was to review our experience with a less invasive therapy, namely, intratumoral bleomycin, in the treatment of predominantly cystic craniopharyngiomas. All children with craniopharyngiomas treated at a tertiary care pediatric neurosurgical center since 1994, when bleomycin was first used, were reviewed retrospectively. Seven patients received intratumoral bleomycin therapy. Patients received 2–5 mg bleomycin per dose, 3 times per week, for 3–5 weeks as an initial course. Mean follow-up of these patients was 3 years. In 4 patients, treatment resulted in a significant decrease (>50%) in tumor size, which has remained stable. Two patients’ tumors progressed and underwent resection, and 1 patient had surgical removal because of persistent headaches, although no growth of residual tumor had been noted. One patient developed peritumoral edema as a result of bleomycin therapy. Intratumoral bleomycin is a useful alternative therapy for cystic craniopharyngiomas, and may control tumor growth and delay potentially harmful resection and/or radiotherapy in young children.

Intratumoral drug delivery is an inherently appealing approach for concentrating toxic chemotherapies at the site of action. This mode of administration is currently used in a number of clinical treatments such as neoadjuvant, adjuvant, and even standalone therapies when radiation and surgery are not possible. However, even when injected locally, it is difficult to achieve efficient distribution of chemotherapeutics throughout the tumor. This is primarily attributed to the high interstitial pressure which results in gradients that drive fluid away from the tumor center. The stiff extracellular matrix also limits drug penetration throughout the tumor. We have previously shown that neural stem cells can penetrate tumor interstitium, actively migrating even to hypoxic tumor cores. When used to deliver therapeutics, these migratory neural stem cells result in dramatically enhanced tumor coverage relative to conventional delivery approaches. We recently showed that neural stem cells maintain their tumor tropic properties when surface-conjugated to nanoparticles. Here we demonstrate that this hybrid delivery system can be used to improve the efficacy of docetaxel-loaded nanoparticles when administered intratumorally. This was achieved by conjugating drug-loaded nanoparticles to the surface of neural stem cells using a bond that allows the stem cells to efficiently distribute nanoparticles throughout the tumor before releasing the drug for uptake by tumor cells. The modular nature of this system suggests that it could be used to improve the efficacy of many chemotherapy drugs after intratumoral administration.

Endoscopic ultrasound (EUS) is continuously stepping into the therapeutic arena, simultaneously evolving in different directions, such as the management of pancreatic and biliary diseases, celiac neurolysis, delivering local intratumoral therapy, and EUS-guided endosurgery. EUS-guided vascular procedures are also challenging, considering the variety of vascular pathology, proximity of the vascular structures to the GI tract wall, high resolution, and real-time guidance offering an attractive access route and precise delivery of the intervention. The literature on vascular therapeutic EUS demonstrates techniques for the management of upper GI variceal and nonvariceal bleeding, pseudoaneurysms, and coiling and embolization procedures, as well as the creation of intrahepatic portosystemic shunts. The paucity of studies, diversity of study designs, and the number of animal model studies hamper a systematic approach to the conclusion and decision making important to clinicians and healthcare policy makers. Nevertheless, theoretical benefits and findings up to date concerning technical feasibility, efficacy, and safety of the procedures drive further research and development in this rather young therapeutic arena.

In Europe, commercially available extracts from the white-berry mistletoe (Viscum albumL.) are widely used as a complementary cancer therapy. Mistletoe lectins have been identified as main active components and exhibit cytotoxic effects as well as immunomodulatory activity. Since it is still not elucidated in detail how mistle toe extracts such as ISCADOR communicate their effects, we analyzed the mechanisms that might be responsible for their antitumoral function on a molecular and functional level. ISCADOR-treated glioblastoma (GBM) cells down-regulate central genes involved in glioblastoma progression and malignancy such as the cytokine TGF-βand matrix-metalloproteinases. Usingin vitroglioblastoma/immune cell co-cultivation assays as well as measurement of cell migration and invasion, we could demonstrate that in glioblastoma cells, lectin-rich ISCADOR M and ISCADOR Q significantly enforce NK-cell-mediated GBM cell lysis. Beside its immune stimulatory effect, ISCADOR reduces the migratory and invasive potential of glioblastoma cells. In a syngeneic as well as in a xenograft glioblastoma mouse model, both pretreatment of tumor cells and intratumoral therapy of subcutaneously growing glioblastoma cells with ISCADOR Q showed delayed tumor growth. In conclusion, ISCADOR Q, showing multiple positive effects in the treatment of glioblastoma, may be a candidate for concomitant treatment of this cancer.

✓ Targeted protein toxins are a new class of reagents with the potential for great tumor selectivity and cytotoxic potency. Two such compounds were studied: 1) Tf-CRM107, a conjugate of human transferrin (Tf) and diphtheria toxin with a point mutation (CRM107); and 2) 454A12-rRA, a conjugate of a monoclonal antibody (454A12) to the human Tf receptor and recombinant ricin A chain (rRA). Both compounds are potent and specific in killing human glioblastoma cell lines in vitro. The authors investigated the activity of these reagents administered intratumorally against solid U251 MG human gliomas in vivo. Nude mice with established U251 MG flank tumors (0.5 to 1.0 cm in diameter) were randomly assigned to be treated with 100-µl intratumoral injections of Tf-CRM107 (10 µg) or 454A12-rRA (10 µg), equimolar doses of CRM107 (4.3 µg), 454A12 antibody (7.5 µg), or rRA (1.5 µg), or phosphate-buffered saline (PBS) every 2 days for a total of four doses. Tumor volume and animal weight were assessed by a blinded observer before each treatment and biweekly for 30 days after initiating therapy. With Tf-CRM107 administration, tumor regression of greater than 95% occurred by Day 14 (p < 0.01) and tumors did not recur by Day 30. Treatment with 454A12-rRA caused a 30% decrease in tumor volume by Day 14 (p < 0.01). Treatment with equimolar doses of the unconjugated targeted protein toxin components CRM107, 454A12, or rRA caused significant U251 MG tumor growth inhibition, but the effects were less potent than the antitumor effects of the conjugates. This study also characterized the dose-response effect of Tf-CRM107 on tumor growth and tumor weight on Day 30. Nude mice with established U251 MG flank tumors (0.5 to 1.0 cm in diameter) were treated with 100-µl intratumoral injections of 10, 1.0, or 0.1 µg of Tf-CRM107 or PBS every 2 days for a total of four doses. All three doses of Tf-CRM107 significantly inhibited tumor growth by Day 14 (p < 0.01) and at Day 30 (p < 0.05), with a significant dose-response relationship. This study demonstrated in vivo efficacy of the targeted toxins Tf-CRM107 and 454A12-rRA against a human glioma. With intratumoral administration, the effect of Tf-CRM107 was tumor-specific and in some animals curative. Regional therapy with these potent tumor-specific agents using direct intratumoral infusion should limit systemic toxicity and may be efficacious against brain tumors.

The combination of viral vaccination with intratumoral (IT) administration of CpG ODNs is yet to be investigated as an immunotherapeutic treatment for solid tumors. Here, we show that such a treatment regime can benefit survival of tumor-challenged mice. C57BL/6 mice bearing ovalbumin (OVA)-expressing EG.7 thymoma tumors were therapeutically vaccinated with adenovirus type 5 encoding OVA (Ad5-OVA), and the tumors subsequently injected with the immunostimulatory TLR9 agonist, CpG-B ODN 1826 (CpG), 4, 7, 10, and 13 days later. This therapeutic combination resulted in enhanced mean survival times that were more than 3.5× longer than naïve mice, and greater than 40% of mice were cured and capable of resisting subsequent tumor challenge. This suggests that an adaptive immune response was generated. Both Ad5-OVA and Ad5-OVA + CpG IT treatments led to significantly increased levels of H-2 K(b)-OVA-specific CD8+ lymphocytes in the peripheral blood and intratumorally. Lymphocyte depletion studies performed in vivo implicated both NK cells and CD8+ lymphocytes as co-contributors to the therapeutic effect. Analysis of tumor infiltrating lymphocytes (TILs) on day 12 post-tumor challenge revealed that mice treated with Ad5-OVA + CpG IT possessed a significantly reduced percentage of regulatory T lymphocytes (Tregs) within the CD4+ lymphocyte population, compared with TILs isolated from mice treated with Ad5-OVA only. In addition, the proportion of CD8+ TILs that were OVA-specific was reproducibly higher in the mice treated with Ad5-OVA + CpG IT compared with other treatment groups. These findings highlight the therapeutic potential of combining intratumoral CpG and vaccination with virus encoding tumor antigen.

Malignant blue nevi are a rare variant of cutaneous melanoma often associated with metastatic disease and poor prognosis. Information on the treatment of this condition is limited. This case study reports the use of intralesional interferon -alpha2b (IFN-alpha2b) as a salvage option in a male patient with a congenital blue nevus on the right buttock. After initial diagnosis, the primary lesion (lesion 1) was partially resected and the patient received aggressive treatment with radiotherapy and concomitant polychemotherapy (cisplatin, vinblastine, and dacarbazine). However, the lesion progressed and the patient was treated second-line with carboplatin, paclitaxel, and sorafenib. Although this treatment stabilized the primary lesion, at cycle 6, the patient developed a measurable locoregional metastasis (lesion 2). At this stage, the patient was treated with intralesional IFN-alpha2b twice weekly at lesion 2, followed by weekly intralesional IFN-alpha2b therapy to both lesions, which resulted in partial disease regression. Biweekly maintenance therapy with IFN-alpha2b administered to both lesions maintained a partial response (ongoing at 23+ mo) and allowed complete occupational rehabilitation. Thus, intralesional therapy with IFN-alpha2b may be a viable treatment option in patients with locally advanced melanoma who have progressed on prior radiotherapy, polychemotherapy, and/or multikinase antiangiogenic-based therapy.

Glioblastoma is the most malignant, highly vascularized, infiltrative, and lethal brain tumor of astrocytic origin. Despite recent progress in diagonostic imaging, cytoreductive surgery, radiotherapy, and chemotherapy, the prognosis of patients with glioblastoma and their survival has not been substantially improved. Depriving glioblastoma tumor cells of oxygen and nutrients by preventing angiogenesis shows high promises for treatment of glioblastoma. So, new targeted therapeutics to deliver antiangiogenic molecules are effective weapons against this devastating brain cancer. A complex interplay between tumor cells, endothelial cells, and several angiogenic growth factors is a prerequisite for endothelial cell migration, proliferation, vascularization, and capillary formation. A better understanding of angiogenesis, angiogenic factors, and growth signaling in glioblastoma is thus necessary for therapeutic targeting of the multiple signal transduction pathways in this process. In this chapter, we discuss the molecular mechanisms of angiogenesis in glioblastoma and recently developed approaches inducing an antiangiogenic response such as protein tyrosine kinase inhibitors, nonreceptor tyrosine kinase inhibitors, intracellular effectors, gene delivery, vascular targeting, and encapsulated delivery of angiogenic inhibitors. Multimodality combination therapy of targeted antiangiogenic molecules along with radiation, chemotherapy, intratumoral therapy, immunotherapy, and vaccines may augment treatment efficacy and minimize toxicity. Novel clinical trials may evaluate efficacy of new drugs to overcome the current limitations in the treatment of glioblastoma patients and thus may provide new avenues for management of this malignancy.

Patients with high risk melanoma and neuroblastoma frequently experience recurrence despite surgical resection and appropriate adjuvant therapies. Immunotherapy with the immunocytokine hu14.18-IL2 (EMD-273063) was developed by means of fusion of two molecules of IL-2 to the monoclonal antibody, 14.18, that recognizes GD2, expressed on the earlier mentioned malignancies. This article will discuss the results of preclinical work using EMD-273063 therapy, including data suggesting that intratumoral therapy may have enhanced antitumor benefit compared with intravenous therapy. Initial clinical trials in adult melanoma and pediatric neuroblastoma have demonstrated acceptable toxicity profiles in dosing that induces immune activation. Preclinical and initial clinical data suggest greater efficacy in the setting of minimal residual disease; therefore, future clinical testing is planned to test the benefit of EMD-273063 in this setting.

Iodine-131 radiolabeled chimeric tumor necrosis therapy monoclonal antibody (131I-TNT) has been approved for the treatment of advanced lung cancer in China. In the present study, the immunogenicity of TNT was studied in advanced lung cancer patients using BIACORE and enzyme linked immunosorbent assay (ELISA) methods. Serum samples from 78 advanced lung cancer patients were analyzed for antibody development to TNT after systemic or intratumoral administration of two doses of 131I-TNT. Patients’ sera were obtained before, and 2 weeks and 2 months after 131I-TNT radioimmunotherapy. Four of 78 lung cancer patients (4/78 or 5.13%) developed antibodies to TNT as measured by ELISA method, and 7 of 78 patients (8.97%) development anti-TNT antibody as measured by BIACORE biosensor after 2 doses of 131I-TNT administration (P > 0.05). All the 4 ELISA-positive patients were also BIACORE-positive. Among the 7 BIACORE-positive patients, 5 (of 42, 11.9%) patients receiving intravenous TNT injection developed antibodies to TNT, and 2 (of 36, 5.56%) patients, receiving intratumoral therapy developed antibodies to TNT. The route of administration of the radiolabeled TNT antibody was not a statistically significant factor in the incidence of anti-TNT antibody. Detailed BIACORE serological analysis showed that the induced antibodies were mostly of the IgG1 subclass. 131I-TNT was immunogenic in only a small minority of advanced lung cancer patients (8.97%). The route of administration did not statistically influence the incidence of anti-TNT antibody after TNT radioimmunotherapy in lung cancer patients.

Background and Objectives Twenty-seven patients with refractory liver metastases from colorectal cancer took part in a Phase II study of the light infusion technology™ (Litx™) light-activated drug/device system to assess safety and evaluate time to tumor progression (TTP). Methods Litx™ consists of the light-activated drug, talaporfin sodium (LS11), activated intratumorally by a catheter-like array of light-emitting diodes (LEDs). After placement of the array via ultrasound or computed tomography (CT) guidance, LS11 was administered intravenously, followed 15–60 min later by light infusion for 2.8 hr. Patients were assessed for adverse events and tumor response using physical examination, laboratory values, and CT scan evaluation over a period of 60 days. Results The observed occurrence of Litx™ treatment-related adverse events was minimal and cumulative toxicity did not occur when combined with chemotherapy. Assessment of TTP and tumor response rate, although statistically non-robust, suggest potential improvement. Conclusions The Litx™ system was shown to be safe for treating liver metastases from colorectal cancer and there was no cumulative toxicity when combined with standard systemic therapy. Preliminary assessments of TTP and tumor response rate justify further evaluation in a Phase III follow-up study. J. Surg. Oncol. 2007;96:518–524. © 2007 Wiley-Liss, Inc.

BACKGROUND: Using a special methodology for radiolabeling lipid nanoparticles with therapeutic radionuclides, we have developed 186Re-nanoliposomes (186RNL). The liposomes, not unlike the membranes of human cells, are composed of a bilayer of distearoylphosphatidylcholine and cholesterol, and confer drug delivery control on beta emitting 186Re. This then permits the delivery of 186Re radiation of high specific activity which is retained within the tumor. METHODS: A phase 1 dose escalation study of 186RNL in recurrent glioma utilizing a standard 3 + 3 design was undertaken to determine the maximum tolerated dose of 186RNL by convection enhanced delivery following stereotactic biopsy. 186RNL is administered by the use of the SmartFlow Flex catheter (BrainLab) by convection enhanced delivery intra-tumorally. Catheter placement is guided using iPlan Flow and the Varioguide systems (BrainLab). Following catheter placement, patients are taken to a nuclear medicine suite where direct visualization of 186RNL infusion is followed under whole body planar imaging and SPECT/CT. Repeat imaging is performed immediately following, and at 1, 5, and 8 days after administration to obtain radiation dosimetry and distribution. RESULTS: Three patients have been treated to date with 1mCi to achieve a treatment field volume of 5mL. Catheter placement for all 3 cases has closely matched the stereotactic plan based upon MRI fusion with SPECT/CT. No treatment related adverse effects have been observed to date despite absorbed doses up to 140Gy (37 MBq). For patient 01, loco-regional retention was 76.8% of administered radioactivity on average from time 0 to 72 hours. During the 120 hour monitoring period, there was a trend toward slow loco-regional dispersion of radioactivity, which is indicated by larger extended radioactivity distribution volumes increasing from 19mL at 40 minutes to 31mL at 120 hours. CONCLUSION: Initial results of 186RNL delivery by CED are promising and the study will continue until an MTD is reached.

The objective of this work was to characterize a polymeric paste formulation of the anticancer drug paclitaxel that was injectable through a narrow gauge needle at room temperature and set to a solid implant in vivo for the intratumoral treatment of localized cancer. Pastes were manufactured from a triblock copolymer composed of poly(D,L-lactide-co-caprolactone)-block-polyethylene glycol-block-poly(D,L-lactide-co-caprolactone) (PLC-PEG-PLC) or triblock blended with a low molecular weight polymer methoxypolyethylene glycol (MePEG). Characterization of pastes was performed using differential scanning calorimetry (DSC), gel permeation chromatography (GPC) and drug release studies. Paste integrity in water was measured by determining the degree of fragmentation under initial agitation. MePEG was found to be miscible with the triblock polymer and paclitaxel dissolved in various blends of these polymers up to 15% drug loading. Pastes composed of 40:60 triblock:MePEG blends and 10% paclitaxel were found to inject through a 23-gauge needle and set to a solid pellet in phosphate-buffered saline at 37 degrees C. Such pellets released paclitaxel in a controlled manner over 7 weeks. Pastes composed of 40:60 triblock:MePEG blends containing 10% paclitaxel are proposed as suitable injectable formulations of the drug for intratumoral therapy.

The level of N7-(2-hydroxyethyl)guanine (N7-HOEtG), one of the DNA alkylation products formed by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment, was measured in human brain tumor samples by high performance liquid chromatography with electrochemical detection. The tumors from 6 recurrent chemotherapy-naive patients with recurrent glioblastoma multiforme were analyzed as controls. The mean level of N7-HOEtG in DNA of these specimens was 0.42 pmol/mg DNA. Samples were also obtained from a patient with a recurrent glioblastoma multiforme after direct intratumoral therapy with BCNU in ethanol (DTI-015). The levels of N7-HOEtG in the samples distal, medial, and adjacent to the site of injection were 0.8, 2.6, and 369.5 pmol/mg DNA, respectively. Comparison of the level of N7-HOEtG detected in the distal sample after injection with BCNU in ethanol with the mean level of the untreated samples indicated that it was not sufficiently different to be ruled out as a chance occurrence. Comparison of the levels of N7-HOEtG in the medial and adjacent brain tumor samples with the mean level of the control samples showed values that were approximately 6- and 879-fold higher. These results demonstrate that intratumoral administration of BCNU in ethanol produces significant levels of DNA alkylation and suggest that DNA adduct measurements provide a unique molecular dosimeter to evaluate delivery of alkylating agents to brain tumors.

Obstructing esophageal cancer produces severe dysphagia with ensuing death within 90 days. Palliation is possible with modalities like stent placement, laser, and photodynamic therapy. However, these treatments have a high rate of complications, and the overall mortality is not altered. A new alternative treatment evaluated in this study is endoscopic intratumoral injection with cisplatin/epinephrine (CDDP/epi) gel. CDDP/epi gel injections were administered weekly for 3 to 8 weeks in nine patients, median age, 72 years; mean tumor volume (+/-SEM), 41.44 (+/-22.4) cm3. Eight patients had stage IV, and one had stage III esophageal carcinoma. The mean dysphagia score (+/-SEM) was 3.5 (+/-0.17). All patients were followed up until death. Dysphagia resolved in eight patients with reduction in mean dysphagia score (+/-SEM) from 3.5 (+/-0.17) to 0.75 (+/-0.28; p = 0.005). Tumor volume was reduced by 75% in one patient and by 50% in two patients. The median survival was 4 months. The longest follow-up has been 15 months (458 days). In this pilot study, intratumoral injection of CDDP/epi gel restored swallowing in eight of nine patients and was an effective and safe outpatient treatment in patients with obstructive esophageal cancer.

Low tumor uptake and normal tissue toxicity limit the efficacy of RIT for the treatment of solid tumors. In this study, an intratumoral injectable gel drug delivery system for local administration of RIT was evaluated using the LS174T human colon cancer xenograft model in SCID mice. The injectable gel is a collagen-based drug delivery system designed for intratumoral (i.t.) administration, which has previously been shown to enhance drug retention at the injection site and reduce systemic drug exposure. We compared the local (tumor) retention and biodistribution of 111In-labeled NR-LU-10 monoclonal antibody given i.t. in the injectable gel versus simple aqueous solution. 111In gel given i.t. and 111In-NR-LU-10 given intraperitoneally (i.p.) were used as controls. The results showed that tumors treated with 111In-NR-LU-10 gel maintained the highest levels of radioactivity for up to 96 h. At 48 h after the administration of 111In-NR-LU-10 gel i.t., 111In-NR-LU-10 solution i.t., 111In gel i.t., or 111In-NR-LU-10 i.p., the level of radioactivity remaining in each gram of tumor was 98, 49, 45, and 16% of the injected dose, respectively. It was estimated that if 100 microCi of 90Y-NR-LU-10 were administered similarly, tumor treated with 90Y-NR-LU-10 gel i.t. would receive a dose of 90.0 Gy, whereas normal tissues in the same animal would receive a dose of approximately 2.43 Gy. In contrast, if 90Y-NR-LU-10 were delivered i.p., a comparable tumor would receive a dose of 16.8 Gy and corresponding normal tissues would receive 3.36 Gy. Consistent with these estimates, enhanced antitumor efficacy was observed when 90Y-NR-LU-10 gel was administered i.t. Tumor growth delay time was 6.9-fold (P < 0.01) longer in these animals (14.4 days) than in animals treated with 90Y-NR-LU-10 i.p. (2.1 days). Systemic toxicity was also significantly reduced in gel-treated animals as monitored by loss of body weight. This study demonstrated that intratumoral delivery of 90Y-NR-LU-10 gel markedly increased the retention of the radioisotope in tumors, enhanced the antitumor efficacy, and reduced systemic toxicity compared to systemic administration of the radiolabeled antibody. This injectable gel drug delivery system may allow for improvement in the therapeutic index for RIT.

Background: High risk neuroblastoma is incurable using current treatment regimens in the majority of patients. Oncolytic virotherapy offers a novel approach to treating cancer. In vitro and in vivo studies in adult tumor models have shown efficacy with the use of cancer selective, attenuated, replication-competent Herpes Simplex Virus (HSV)-derived vectors. Such viruses are currently in clinical trials for several adult cancers.

The formation of antibodies with interleukin-2 (IL-2)-neutralizing capacity and their possible impact on successful locoregional PEG-IL-2 therapy was studied in the guinea pig Line 10 (L10) tumor model. Previously it was shown in this animal model that intratumoral therapy with polyethylene glycol-modified human recombinant IL-2 (PEG-IL-2) induced tumor regression and eradication of lymph node metastases. Therefore, the putative negative effects of IL-2-neutralizing antibodies can be studied in this model. In two similar experiments, animals were immunized by subcutaneous injections (3x/week for 5 weeks) with PBS/BSA, rIL-2, or PEG-IL-2. One week after the last injection, L10 tumor cells were inoculated contralaterally on the flank. Seven days after tumor transplantation, intratumoral treatment with PBS/BSA or PEG-IL-2 was started. During immunization and subsequent intratumoral therapy, sera were collected from the guinea pigs and tested in the CTLL-16 bioassay for IL-2-inhibitory activity. Intratumoral injections with PBS/BSA after immunization only incidently resulted in cure of tumor and metastases, suggesting that by sensitization treatment alone, the immune system was not sufficiently activated to eradicate a tumor inoculum. PBS/BSA immunization followed by intratumoral PEG-IL-2 therapy resulted in complete cure of primary tumor and lymph node metastases in all treated animals. Immunization with rIL-2 evoked IL-2-inhibitory activity in the serum of all guinea pigs. Nonetheless, six of these 11 rIL-2-immunized animals were completely cured by intratumoral PEG-IL-2 therapy. PEG-IL-2 immunization resulted in IL-2-inhibitory serum activity in three of 12 guinea pigs. The guinea pigs with high IL-2-neutralizing activity in their serum showed progressive growth of tumor and metastases, whereas the other animals were completely cured. The level of IL-2-neutralizing capacity in the sera from rIL-2-pretreated animals was higher than that from PEG-IL-2-pretreated guinea pigs, confirming a reduced immunogenicity of PEG-IL-2 as reported after intravenous administration. Passage of the sera from the immunized guinea pigs over a protein G column drastically reduced IL-2-inhibitory activity, implying IgG-related IL-2 inactivation. In conclusion, IL-2-neutralizing activity, possibly mediated by IgG antibodies, is more readily induced by intradermal rIL-2 administration than by PEG-IL-2 injections. Our data suggest that local treatment of cancer patients with a second cycle of PEG-IL-2 after previous therapy with rIL-2 or PEG-IL-2 may reduce the therapeutic efficacy of PEG-IL-2.

Direct intratumoral injection of interleukin-2 (IL-2) was evaluated in a murine model. Balb/c mice received 5 x 10(4) Line 1 alveolar carcinoma cells (L1C2) by subcutaneous injection. On the third day following tumor implantation, mice received injections of IL-2 (5 x 10(3)-5 x 10(4) units) or diluent twice daily, either by i.p. or intratumoral injection, 5 days/week for 3 weeks. Intratumoral injection of 5 x 10(4) units IL-2 significantly reduced tumor volume (P0.05 versus control), increased median survival time (P = 0.0001), and resulted in a 23.5% cure rate (P = 0.008). There were no long-term survivors in the other treatment groups. Both tumor-infiltrating lymphocytes (TIL) and splenic lymphocytes isolated directly from IL-2-treated mice demonstrated enhanced cytolytic activity compared to diluent-treated controls. To determine whether non-T-cell-mediated antitumor responses were active in our model, intratumoral immunotherapy was evaluated in athymic Balb/c nu/nu mice. In order to decrease the recruitment of lymphocyte precursors, nude mice were splenectomized and received cyclophosphamide prior to tumor injection and IL-2 therapy. Intratumoral IL-2 immunotherapy also significantly decreased tumor volume in these immunodeficient mice (P0.02), but did not lead to long-term survival. We conclude that both TIL and splenic lymphocytes are activated in vivo in response to intratumoral IL-2 immunotherapy, suggesting that intratumoral therapy with IL-2 activates both local and systemic antitumor responses.

T-bet is a T-box transcription factor (Tbx21) previously identified as a master regulator of Type-1 polarization in T cells. We have previously demonstrated that ectopic expression of hT-bet converts dendritic cells (DC) into Type 1-biasing APCs. In the current study, we investigated whether mouse DC overexpressing mT-bet promote superior Type 1 anti-tumor T cell responses when applied as an intratumoral therapy. CD11c+ DCs were then purified and transduced with recombinant adenovirus to express high levels of mT-bet. DC. mT-bet stimulated stronger Tc1 and Th1 T cell responses when compared with control DC and retained the capacity to kill and then uptake tumor cells/debris, allowing for the cross-priming of specific T cell responses in vitro. Surprisingly, in contrast to human DC. T-bet, CD40L-activated DC. mT-bet produced elevated levels (vs. control DC) of IL-12p70/TNF-α. DC. mT-bet exerted potent anti-tumor activity in the (day 7 established) s.c. CMS4 sarcoma after intratumoral administration. These anti-tumor effects were mediated primarily by Tc1 cells, however, both CD4+ T cells and NK cells appeared to also be involved in therapeutic response. In situ imaging suggested that DC. mT-bet treatment inhibited the accumulation of CD11b+Gr1+ myeloid-derived suppressor cells and tumor-associated vascular structures. Collectively, these findings suggest that DC. mT-bet-based therapy has the potential to promote superior Type-1 anti-tumor immunity in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3824.

Objective: To determine the safety and efficacy of targeted antitumor therapy with cisplatin/epinephrine injectable gel in patients with advanced squamous cell carcinoma of the head and neck. Design: Two prospective, double-blind, placebo-controlled phase III trials of identical design. Crossover from blinded to open-label phase was permitted for patients with disease progression. Setting: Tertiary referral centers in North America and Europe. Patients: One hundred seventy-nine intensively pretreated patients with recurrent or refractory squamous cell carcinoma of the head and neck. Intervention: Cisplatin/epinephrine injectable or placebo gel was administered by direct intratumoral injection; up to 6 weekly treatments. Dose was 0.25 mL of active or placebo gel per cubic centimeter of tumor up to 10 mL total. Patient benefit after local tumor control of the most symptomatic tumor was assessed by patients and physicians using the Treatment Goals Questionnaire. Main Outcome Measures: Local tumor response and patient benefit attributable to improvements in tumor-related symptoms. Results: Combined results for the 178 patients with evaluable data in the 2 trials confirmed objective tumor responses in 35 (29%) of 119 patients, including 23 (19%) complete responses achieved with cisplatin/epinephrine gel, vs 1 (2%) of 59 for placebo (P

A s. c. experimental model of Wilms' tumor in rats was used to compare the effects of intratumoral treatment with vincristine plus actinomycin D to i.v. treatment with these chemotherapeutic drugs. The Wilms' tumor model is a fast-growing solid tumor that has been shown to be resistant to traditional clinical treatment procedures used for Wilms' tumor in man. Injection of the chemotherapeutic drugs directly into the tumor mass was found to be more effective than i.v. therapy in causing long-term remission of the tumor. Intratumoral therapy was also less toxic to the animals than i.v. therapy when measured by post-treatment survival rates and weight loss during the 1st week following treatment. However, intratumoral treatment caused an initial fibrosis of the tumor tissue, which resulted in a slower rate of absorption of the resultant fibrotic tumor mass than was seen in tumors treated i.v. Also, intratumoral injection resulted in necrosis of the overlying skin, which healed as the fibrotic tumor tissue was absorbed. Intratumoral treatment of a cervical tumor was found to cause the remission of a second major tumor mass located at some distance from the initial injection (i.e., in the lumbar region). No significant benefits were noted when dimethylsulfoxide (DMSO) was used in place of aqueous mannitol as a vehicle to deliver the chemotherapeutic agents. There was a significant correlation between the drug dose-to-tumor-size ratio (D/T ratio) and the effectiveness of the chemotherapy. When this ratio was high enough, a single treatment with a combination of vincristine and actinomycin D usually resulted in total remission of the experimental Wilms' tumor in response to either intratumoral or i.v. therapy.

Object. To overcome the problems associated with using stereotactic techniques to establish intracranial xenografts in nude mice and to treat engrafted tumors with intratumoral therapies (such as gene or viral therapies), the authors developed an implantable guide-screw system. In this study, they describe the guide-screw system, its method of implantation, and their experience with establishing xenografts and delivering intratumoral therapy.Methods. The system consists of a 2.6-mm guide screw with a central 0.5-mm diameter hole that accepts the 26-gauge needle of a Hamilton syringe. The screw is implanted into a small drill hole made 2.5 mm lateral and 1 mm anterior to the bregma. A stylet is used to cap the screw between treatments. Tumor cells or therapeutic agents are injected in a freehand fashion by using a Hamilton syringe and a 26-gauge needle fitted with a cuff to determine the depth of injection. To test this system, guide screws were successfully implanted in 44 (98%) of 45 nude mice. After 1 to 2 weeks of recovery, 38 mice were inoculated with U87MG cells and killed 5 days later. On histological studies in 37 (97%) of these animals, xenografts were evident within the caudate nucleus (mean diameter 2.5 mm). To determine whether injections into the center of an established xenograft could be reproducibly achieved with the guide-screw system, an adenovirus vector containing the β-galactosidase gene was injected 3 days after cell implantation in 15 of the mice. All of these animals demonstrated transduced cells within the tumor. To demonstrate that engrafted animals have a uniform survival time that is indicative of reproducible tumor growth, the survival of six mice was assessed after engraftment with U87MG cells. All six animals died within 28 to 35 days.Conclusions. The guide-screw system allows a large number of animals to be rapidly and reproducibly engrafted and for intratumoral treatments to be accurately delivered into established xenografts.

Recombinant vaccinia viruses derived from strain Praha, clone P13, and strain MVA were used for intratumoral delivery and expression of IL12 genes in tumors induced by HPV16 E6+E7 oncogenes in mice. Intratumoral injection of 10(3) PFU of P13-IL12 virus resulted in an increase of intra-tumoral IL12 on days 6-13, while only low levels of IL12 were found in sera. After the inoculation of 10(6) PFU of MVA-IL12, the same levels of IL12 were found as in animals injected with control virus. The intratumoral inoculation of 10(3) PFU P13-IL12 resulted in only approximately 30% of the tumors being virus positive, which was a consequence of reduced multiplication of the recombinant virus in vivo. The number of virus-positive tumors was not increased by repeated inoculations on three consecutive days. Intratumoral therapy with a dose of 10(3) PFU of P13-IL12 slowed down the growth of TC1 tumors, but never caused their regression. When local P13-IL12 treatment was combined with antigen-specific, DNA-vaccination therapy, no synergy between the two treatments was observed. The treatment with IL12-expressing virus retarded tumor growth to some degree, but did not change the number of regressing tumors. The highest efficacy of intra-tumoral P13-IL12 therapy was observed when the TC-1/A9 cell subline, with downregulated MHC class I expression, was used. TC-1/A9 tumors are less refractory to treatment with 10(3) P13-IL12/EL than are parental TC-1 cells.

To the Editor:— It is my considered belief and experience that the local infiltration of fluorouracil ( 208 :536, 1969) in extensive cancers is rarely, if ever, applicable. We have used this technique in small primary and secondary skin cancers to determine if the tumor is sensitive to the agent in a given patient. 1 We saw no objective tumor regressions in this test series to warrant intratumoral therapy. Certainly, large fungating lesions, in my opinion, would be difficult to inject uniformly and are not suitable for this type of therapy. In their case presentation, the dose of fluorouracil was such that the tumor regression might well be due to a systemic effect of the drug. I think the above letter upon which I am commenting could mislead many physicians into using this drug by local injection and result in undue trauma.