Basic Science Research

On-going Research within Dermatology Includes:

1. Clinical trial using Imiquimod (an immune modulator) to alter the course of atypical or dysplastic nevi.

Participants: Jane Grant-Kels, Carolyn Ray, Bijay Mukherji, Victor Moyo, Upendra Hegde, Michael Murphy, Nitya Chakraborty, Karren Slade.

Imiquimod cream, 5%, (FDA approved “Aldara” for topical treatment of external genital/perianal warts) is an immune response modifier that induces interferon production by monocytes/ macrophages. It has been shown to induce immunologic activity against human papillomavirus (HPV) by stimulating significant increases in numerous cytokines including interferon-alpha, interferon-gamma and IL-12. Additionally, there is a suggestion of activation of the helper T-cell type 1-mediated response by increases in CD4, CD8, and tumor necrosis factor-alpha (TNF-alpha) . In recent studies, Imiquimod has been shown to be an effective treatment of superficial basal cell carcinoma (sBCC) with response rates varying between 87.1% and 100% when used QD or BID respectively. There have been case reports of the use of Imiquimod in the treatment of Malignant Melanoma (MM); however, to our knowledge, there are no published studies on the use of imiquimod in the treatment of dysplatic nevi. The indication for interferon in the treatment of MM is well established. Therefore, we postulate that a topical agent capable of inducing interferon production might play a role in the treatment of dysplastic nevi. Given the unique mechanism of action of Imiquimod, the ease of its application to the skin, and its effect in the treatment of sBCC and MM we propose that it may also play a role in the treatment of dysplastic nevi. Our pilot study will examine whether topical administration of 5% Imiquimod cream will induce a localized inflammatory reaction capable of reversing dysplasia in dysplastic nevi. If such a response is observed, a case could be made for a potential role for Imiquimod in the treatment of dysplastic nevi and in the prevention of melanoma.

The immunologic effect of Imiquimod will be evaluated at the local and systemic levels. It has been shown that Imiquimod acts on the innate immune system by activating monocytes/macrophages and dendritic cells through specific toll-like receptors (TLRs). Although we do not anticipate major effect of topical application on systemic immunity, we will undertake a full phenotypic and functional characterization of the circulating immunocytes at baseline and several times within the treatment phase. This will include phenotypic analysis of the circulating monocytes, NK cells and B and T cells. Specific attention will be focused on their relative number, activation markers, and cytoplasmic cytokines (IFNgamma, IL-12, IL-2, TNF, etc.). This will be done using appropriate flow cytometric analysis. In addition, natural killer cell activities will be assessed in cytotoxicity assay against the NK sensitive target cells, K-562.

The local effect of Imiquimod will be evaluated by immunohistochemistry and by in situ hybridization techniques. Accordingly, a site treated with imiquimod (the IMTN) and an untreated site (the IMCN) will be excised one week after the initiation of topical Imiquimod application. These studies will examine the nature of the response, in situ, by histopathology and immunohistochemistry. This will allow us to determine the nature of the infiltrating cells: Are they T cells, B cells, or dendritic cells (DCs)? Do the infiltrating DCs express upregulated co-stimulatory molecules, MHC class II molecules? What type of cytokines [IL-2, IL-4, IL-10, IFNg , TNFa] can we detect in their cytoplasm?

In addition, considering that Imiquimod is likely to activate dermal antigen presenting cells (Langerhans cells and dermal DCs), a concerted effort will be made to determine if the Imiquimod treatment leads to the activation of DCs and antigen presentation so as to generate melanocyte-associated epitope-specific cytotoxic lymphocyte (CTL) response. For this purpose, T cells will be expanded in culture from the treated and untreated lesions. The expanded T cells will be phenotypically and functionally chracterized with specific attention focused on determining if the treatment activates and then recruits antigen-specific CTLs, in situ, as another potential mechanism.

2. Proposal for future research: To carry out a phase I/II clinical trial, in the melanoma model, with synthetic peptide-based vaccines administered intradermally in sites pretreated with imiquimod to create an inflammatory context as well as to recruit and activate DCs, in situ.

Participants: Bijay Mukherji, Upendra Hegde, Jane Grant-Kels, Philip Kerr.

The principle on which we and others had based the approach to cancer vaccine with peptide loaded APCs or with specific peptides alone has been validated to a certain extent. These approaches have shown biological and even some clinical activities, however modest they may be. The modest clinical gains have understandably led to a pessimistic view on peptide and DC-based vaccines on one hand and raised the yearning for better and more ideal cancer antigens as well as for alternate forms of vaccination on the other. The major rationale for our proposal is that, while the field waits for ideal antigens and alternative forms vaccines, time has come for innovative steps that could improve upon the result and move the field forward. Considering that dealing with ex vivo cultured DCs has many drawbacks yet recognizing the powerful and indispensable role of DCs in antigen presentation, we believe that developing a strategy(s), alternative to but still centering around the pivotal role of recruiting DCs in the peptide vaccination process, will be a step in the right direction. Since most vaccines work through resident DCs and DCs recruited at the sites, a strong rationale exists to develop an in vivo strategy that could recruit as well as activate DCs in the process. Adjuvants usually serve the purpose. Unfortunately, a good adjuvant suitable for human use in peptide or protein-based vaccines is not yet available although a number of agents are being tested. We believe that imiquimod has the properties that are essential – if not ideal – for the purpose of activating resident DCs plus LCs, recruiting plus activating DCs from the DC pool, and creating an inflammatory milieu through the induction of critical pro-inflammatory cytokines, in situ.

Imiquimod has undergone extensive testing prior to receiving FDA approval and additional information has since emerged to clearly establish its biological and immunological properties. Even its chronic topical use is well tolerated yet it is locally inflammatory after two- three applications. Most importantly, recent works have provided compelling proofs of its properties as a biologic response modifier, in general, and its effect on dermal DCs and LCs, in particular. As such, we believe that all the essential ingredients are in place to move it to human clinical trial as a potentially useful vaccine adjuvant without additional in vitro studies or in vivo testing in animal models. Finally, unprecedented opportunity now exists to carry out more precise quantitative as well as qualitative assessments of the outcome (i.e., generation of peptide specific and tumor reactive CTL response). For example, some of the latest state of the art assays – such as tetramer staining assay, ELIspot assay, and Fastimmune assays – have changed the very nature of patient monitoring by making it exquisitely precise and sensitive. Thus, any improvement of outcome can be reliably monitored.

Imiquimod, an imidazoquinoline, is now used in the treatment of anogenital warts and a number viral or non-viral cutaneous neoplasms with considerable success. Imiquimod does not possess direct anti-viral activities in in vitro assays. The mechanism of its anti-tumor effect is also not fully understood. It is believed that the anti-viral and anti-tumor activities, in vivo, might be mediated through the induction of cytokines and enhancement of cell mediated immunity. Indeed, a considerable body of data has emerged to suggest that imiquimod stimulates the innate as well as the acquired arms of the immune system. Further, when applied topically, it activates resident DCs and LCs in the skin, and importantly, imiquimod stimulates innate immunocytes – such as macrophages, DCs, etc. – via the toll receptor TLR7 and induces a number of inflammatory cytokines (IFNa, IL-12, TNFa,, etc.) in DC precursors and DCs. These properties, therefore, suggest that imiquimod might be an ideal agent to create an inflammatory context, in situ, for specific peptide-based immunization. In short, imiquimod is a potentially useful topical adjuvant for vaccine therapy.

The field of peptide-based cancer vaccine approach has been too good to be abandoned. It needs additional innovative strategies to harness the full potential of the basic approach. We have proposed to test a novel approach based on a sound hypothesis. We, therefore, believe that the proposed work could move the filed forward and if our hypothesis is proven to be correct, the approach will have application beyond melanoma. This, then, is the major significance of our proposal.

Past Investigations

• 2003 – 2004: Jane Grant-Kels, M.D., in collaboration with Richard Nuccitelli, Ph.D.
  Worked with Mouse model for melanoma in which you inject mouse melanoma cells just beneath the skin in a mouse and a tumor developed within five days. The melanomas were then investigated with the bioelectric field imager.
  The specific aim of this animal research project was to determine if a new instrument designed for BioElectric Field Imaging (BFI) could be used for the diagnosis of melanomas by measuring local perturbations in the electric fields in skin near the melanoma. Our hypothesis was that melanomas exhibit increased rates of cell division and this could lead to a reduction in the local transepidermal resistance that could be manifested as a local electric field perturbation melanomas.
• 2003 – 2004: In collaboration with Tim Hla, Ph.D., Michael Murphy, M.D., Jane Grant-Kels, M.D., and Michael Kruk, Ph.D., and M.D., student . Study using archival tissue of the expression of S1P1/Edg-1 in melanoma and psoriasis.
• 2002 – 2004: Differentiation of histologic response to collagen injection Vs particulate fascia lata injection for treatment of dermal defects. Jane Grant-Kels, M.D., in collaboration with Neil Sadick, M.D., 772 Park Avenue, NY, NY 10021 and Cornell University/New York Hospital Department of Dermatology.