Chromosomal aberrations in UVB-induced tumors of immunosuppressed mice.

In immunocompromised individuals, such as organ transplant recipients, the risk of cutaneous squamous cell carcinoma (SCC) is increased 60-250 fold, and there is an increased likelihood to develop aggressive, metastatic SCC. An understanding of the genes involved in SCC tumorigenesis is critical to prevent SCC-associated morbidity and mortality. Mouse models show that different immunosuppressive drugs lead to SCCs varying in size, number, and malignant potential. In this study, we used mouse models that mimic adult transplant recipients to study the effect of immunosuppressive drugs and UV light on SCC development. UV-induced tumors from six treatment groups, control, tacrolimus (Tac), rapamycin (Rap), cyclosporin (CsA), mycophenolate mofetil (MMF), and Rap plus CsA, were evaluated by array comparative genomic hybridization. Mouse SCCs appear to show similar genomic aberrations as those reported in human SCCs and offer the ability to identify genomic changes associated with specific and combinatorial effects of drugs. Fewer aberrations were seen in tumors of mice treated with MMF or Rap. Tumors from Tac-treated animals showed the highest number of changes. Calcineurin inhibitors (Tac and CsA) did not cluster together by their genomic aberrations, indicating their contribution to UV mediated carcinogenesis may be through different pathways. The combination treatment (Rap plus CsA) did not cluster with either treatment individually, suggesting it may influence SCC tumorigenesis via a different mechanism. Future studies will identify specific genes mapping to regions of aberration that are different between treatment groups to identify target pathways that may be affected by these drugs.

Macrophage migration inhibitory factor (MIF) plays a critical role in pathogenesis of ultraviolet-B (UVB) -induced nonmelanoma skin cancer (NMSC).

Mounting evidence suggests that macrophage migration inhibitory factor (MIF) may serve as an important link between chronic inflammation and cancer development. The proinflammatory and proangiogenic activities of MIF position it as a potentially important player in the development and progression of nonmelanoma skin cancer (NMSC). To assess the role of MIF in the development and progression of NMSC, we exposed MIF(-/-) BALB/c mice to acute and chronic ultraviolet B (UVB) irradiation. Our studies demonstrate that MIF(-/-) BALB/c mice have a significantly diminished acute inflammatory response to UVB exposure compared to wild-type mice, as measured by myeloperoxidase activity, dermal neutrophil infiltration, and edematous response. Relative to wild-type mice, MIF(-/-) mice also show significantly lower vascular endothelial growth factor (VEGF) concentrations in whole skin and significantly lower 8-oxo-dG adduct concentrations in epidermal DNA following UVB exposure. Furthermore, MIF(-/-) mice showed significant increases in p53 activity, epidermal thickness, and epidermal cell proliferation following acute UVB insult. In response to chronic UVB exposure, MIF(-/-) mice showed a 45% reduction in tumor incidence, significantly less angiogenesis, and delayed tumor progression when compared to their wild-type counterparts. These data indicate that MIF plays an important role in UVB-induced NMSC development and progression.

The hairless mouse in skin research.

The hairless (Hr) gene encodes a transcriptional co-repressor highly expressed in the mammalian skin. In the mouse, several null and hypomorphic Hr alleles have been identified resulting in hairlessness in homozygous animals, characterized by alopecia developing after a single cycle of relatively normal hair growth. Mutations in the human ortholog have also been associated with congenital alopecia. Although a variety of hairless strains have been developed, outbred SKH1 mice are the most widely used in dermatologic research. These unpigmented and immunocompetent mice allow for ready manipulation of the skin, application of topical agents, and exposure to UVR, as well as easy visualization of the cutaneous response. Wound healing, acute photobiologic responses, and skin carcinogenesis have been extensively studied in SKH1 mice and are well characterized. In addition, tumors induced in these mice resemble, both at the morphologic and molecular levels, UVR-induced skin malignancies in man. Two limitations of the SKH1 mouse in dermatologic research are the relatively uncharacterized genetic background and its outbred status, which precludes inter-individual transplantation studies.

Sirolimus reduces the incidence and progression of UVB-induced skin cancer in SKH mice even with co-administration of cyclosporine A.

Transplant immunosuppressants have been implicated in the increased incidence of non-melanoma skin cancer in transplant recipients, most of whom harbor considerable UVB-induced DNA damage in their skin prior to transplantation. This study was designed to evaluate the effects of two commonly used immunosuppressive drugs, cyclosporine A (CsA) and sirolimus (SRL), on the development and progression of UVB-induced non-melanoma skin cancer. SKH-1 hairless mice were exposed to UVB alone for 15 weeks, and then were treated with CsA, SRL, or CsA+SRL for 9 weeks following cessation of UVB treatment. Compared with vehicle, CsA treatment resulted in enhanced tumor size and progression. In contrast, mice treated with SRL or CsA+SRL had decreased tumor multiplicity, size, and progression compared with vehicle-treated mice. CsA, but not SRL or combined treatment, increased dermal mast cell numbers and TGF-beta1 levels in the skin. These findings demonstrate that specific immunosuppressive agents differentially alter the cutaneous tumor microenvironment, which in turn may contribute to enhanced development of UVB-induced skin cancer in transplant recipients. Furthermore, these results suggest that CsA alone causes enhanced growth and progression of skin cancer, whereas co-administration of SRL with CsA causes the opposite effect. JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article please go to http://network.nature.com/group/jidclub

Clinically relevant immunosuppressants influence UVB-induced tumor size through effects on inflammation and angiogenesis.

Immunosuppressive therapies allow long-term patient and transplant survival, but are associated with increased development of UV-induced skin cancers, particularly squamous cell carcinomas. The mechanisms by which CsA, MMF, tacrolimus (TAC) or sirolimus (SRL), alone or in dual combinations, influence tumor development and progression are not completely understood. In the current study, chronically UV-exposed mice treated with SRL alone or in combination with CsA or TAC developed more tumors than mice treated with vehicle or other immunosuppressants, but the tumors were significantly smaller and less advanced. Mice treated with CsA or TAC developed significantly larger tumors than vehicle-treated mice, and a larger percentage in the CsA group were malignant. The addition of MMF to CsA, but not to TAC, significantly reduced tumor size. Immunosuppressant effects on UVB-induced inflammation and tumor angiogenesis may explain these findings. CsA enhanced both UVB-induced inflammation and tumor blood vessel density, while MMF reduced inflammation. Addition of MMF to CsA reduced tumor size and vascularity. SRL did not affect inflammation, but significantly reduced tumor vascularity. Thus the choice of immunosuppressants has important implications for tumor number, size and progression, likely due to the influence of immunosuppressants on UVB-induced inflammation and angiogenesis.

Depletion of CD4+ cells exacerbates the cutaneous response to acute and chronic UVB exposure.

Solid organ transplant recipients have a 60-250-fold increased likelihood of developing sunlight-induced squamous cell carcinoma (SCC) compared with the general population. This increased risk is linked to the immunosuppressive drugs taken by these patients to modulate T cell function, thus preventing organ rejection. To determine the importance of T cells in the development of cutaneous SCC, we examined the effects of selectively depleting Skh-1 mice of systemic CD4+ or CD8+ T cells, using monoclonal antibodies, on ultraviolet B (UVB) radiation-induced inflammation and tumor development. Decreases in systemic CD4+ but not CD8+ T cells significantly increased and prolonged the acute UVB-induced cutaneous inflammatory response, as measured by neutrophil influx, myeloperoxidase activity, and prostaglandin E2 levels. Significantly more p53+ keratinocytes were observed in UVB-exposed CD4-depleted than in CD4-replete mice, and this difference was abrogated in mice depleted of neutrophils before UVB exposure. Increased acute inflammation was associated with significantly increased tumor numbers in CD4-depleted mice chronically exposed to UVB. Furthermore, topical treatment with the anti-inflammatory drug celecoxib significantly decreased tumor numbers in both CD4-replete and CD4-depleted mice. Our findings suggest that CD4+ T cells play an important role in modulating both the acute inflammatory and the chronic carcinogenic response of the skin to UVB.

Human CD4+CD25low adaptive T regulatory cells suppress delayed-type hypersensitivity during transplant tolerance.

Adaptive T regulatory (T(R)) cells mediate the suppression of donor-specific, delayed-type hypersensitivity (DTH) in tolerant organ transplant recipients. We hypothesized that cells belonging to the CD4(+)CD25(+) T cell subset but distinct from natural T(R) cells may fulfill this role. To test this hypothesis, PBMC and biopsy samples from two tolerant kidney transplant recipients (K1 and K2) were analyzed. When transferred with recipient APC into a SCID mouse footpad, CD4(+) T cells were hyporesponsive in DTH to donor type HLA-B Ags and derivative allopeptides. However, anti-human TGF-beta1 Ab revealed a response to immunodominant allopeptides in both patients, suggesting that CD4(+) T effector (T(E)) cells coexisted with suppressive, TGF-beta1-producing CD4(+) T(R) cells. During in vitro culture, allopeptide stimulation induced both IFN-gamma-producing and surface TGF-beta1(+) T cells. The relative strength of the latter response in patient K1 was inversely correlated with the level of systemic anti-donor DTH, which varied over a 6-year interval. Allopeptide-induced surface TGF-beta1 expression was found primarily in Forkhead box P3 (FoxP3)-negative CD4(+)CD25(low) T cells, which could adoptively transfer suppression of donor-specific DTH. Biopsy samples contained numerous surface TGF-beta1(+) mononuclear cells that costained for CD4 and, less frequently CD25, but were negative for FoxP3. The CD4(+)TGF-beta1(+) T cells were localized primarily to the tubulointerstitium, whereas TGF-beta1(-)FoxP3(+)CD25(+) cells were found mainly in lymphoid aggregates. Thus, adaptive T(R) cells suppressing T(E) cell responses to donor allopeptides in two tolerant patients appear to be functionally and phenotypically distinct from CD4(+)CD25(high)FoxP3(+) T cells.

Dietary (n-3) polyunsaturated fatty acids inhibit HER-2/neu-induced breast cancer in mice independently of the PPARgamma ligand rosiglitazone.

Overexpression of human epidermal growth factor receptor 2 (HER-2/neu) characterizes a molecular subtype of breast cancer associated with poor clinical outcome. Preventive strategies for HER-2/neu-positive breast cancer, which is often estrogen and progesterone receptor negative, remain undefined. Activators of peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear hormone receptor also expressed in breast cancer, hold potential as cancer prevention agents. PPARgamma ligands include specific fatty acids and synthetic compounds, such as the thiazolidinediones, which appear to inhibit cell proliferation and tumorigenesis. We hypothesized that a thiazolidinedione, rosiglitazone, may serve as a chemopreventive agent for HER-2/neu-associated mammary carcinogenesis, but that efficacy may be influenced by dietary fat content. We studied the effects of diets enriched with corn or fish oil (25% of energy) with and without rosiglitazone (12 g/kg) in a 2 x 2 factorial design on mammary tumorigenesis in murine mammary tumor virus (MMTV)-HER-2/neu transgenic mice. Despite in vitro evidence of antiproliferative effects in an MMTV-HER-2/neu tumor cell line, rosiglitazone did not affect mammary carcinogenesis in vivo. Interestingly, fish oil-based diets markedly suppressed breast tumor incidence (57% of mice vs. 87% of corn oil-fed mice, P = 0.0001) as well as tumor multiplicity (P = 0.001) and mammary gland dysplasia (P = 0.001). These findings demonstrate a potent preventive effect of (n-3) PUFA on HER-2/neu-mediated mammary carcinogenesis, without interaction with a synthetic PPARgamma activator. Further studies focusing on the mechanisms by which (n-3) fatty acids suppress HER-2/neu signaling pathways involved in the pathogenesis of breast cancer are warranted.

IFN-gamma gene polymorphisms associate with development of EBV+ lymphoproliferative disease in hu PBL-SCID mice.

Posttransplantation lymphoproliferative disorder (PTLD) is a devastating post-transplantation complication often associated with Epstein-Barr virus (EBV). Although the type and length of immunosuppression are risk factors, a patient's inherent immune capacity also likely contributes to this disorder. This report uses severe-combined immunodeficient mice given injections of human peripheral blood leukocytes (hu PBL-SCID [Severe Combined Immunodeficient] mice) to test the hypothesis that cytokine genotype associates with the development of EBV-associated lymphoproliferative disease (LPD). We observed that the A/A (adenosine/adenosine) genotype for base + 874 of the interferon gamma (IFN-gamma) gene was significantly more prevalent in PBLs producing rapid, high-penetrance LPD in hu PBL-SCID mice, compared to PBLs producing late, low-penetrance LPD or no LPD. In examining the relationship between genotype and cytolytic T-lymphocyte (CTL) function, transforming growth factor beta (TGF-beta) inhibited restimulation of CTLs in PBLs with adenosine at IFNG base + 874, but not in PBLs homozygous for thymidine. Importantly, neutralization of TGF-beta in hu PBL-SCID mice injected with A/A genotype PBLs resulted in reduced LPD development and expanded human CD8(+) cells. Thus, our data show that TGF-beta may promote tumor development by inhibiting CTL restimulation and expansion. Further, our data indicate that IFNG genotype may provide valuable information for both identifying transplant recipients at greater risk for PTLD and developing preventive and curative strategies.

A phase I study of interleukin 12 with trastuzumab in patients with human epidermal growth factor receptor-2-overexpressing malignancies: analysis of sustained interferon gamma production in a subset of patients.

PURPOSE: On the basis of preclinical studies, we hypothesized that interleukin (IL)12 would potentiate the antitumor actions of an antihuman epidermal growth factor receptor-2 (HER2) monoclonal antibody (trastuzumab). We conducted a Phase I trial to determine the safety and optimal biological dose of IL-12 when given in combination with trastuzumab. PATIENTS AND METHODS: Patients with metastatic HER2-positive malignancies received trastuzumab on day 1 of each weekly cycle. Beginning in week 3, patients also received intravenous injections of IL-12 on days 2 and 5. The IL-12 component was dose-escalated within cohorts of 3 patients (30, 100, 300, or 500 ng/kg). Correlative assays were conducted using serum samples and peripheral blood cells obtained during the course of therapy. RESULTS: Fifteen patients were treated, including 12 with HER2 2+ or 3+ breast cancer. The regimen was well tolerated with IL-12-induced grade 1 nausea and grade 2 fatigue predominating. Evaluation of dose-limiting toxicity and biological end points suggested that the 300 ng/kg dose was both the maximally tolerated dose and the optimal biological dose of IL-12 for use in combination with trastuzumab. Two patients with HER2 3+ breast cancer within the 500 ng/kg dose level experienced grade 1 asymptomatic decreases in left ventricular ejection fraction of 12% and 19% after 3 and 10 months of therapy, respectively. There was one complete response in a patient with HER2 3+ breast cancer metastatic to the axillary, mediastinal, and supraclavicular nodes, and 2 patients with stabilization of bone disease lasting 10 months and >12 months, respectively. Correlative assays showed sustained production of interferon (IFN)gamma by natural killer cells only in those patients experiencing a clinical response or stabilization of disease. Elevated serum levels of macrophage inflammatory protein-1alpha, tumor necrosis factor-alpha, and the antiangiogenic factors IFN-gamma inducible protein-10 and monokine induced by gamma were also observed in these patients. Patient genotyping suggested that a specific IFN-gamma gene polymorphism might have been associated with increased IFN-gamma production. The ability of patient peripheral blood cells to conduct antibody-dependent cellular cytotoxicity against tumor targets in vitro did not correlate with clinical response or dose of IL-12. CONCLUSIONS: The addition of IL-12 to trastuzumab therapy did not appear to enhance the efficacy of this antibody treatment. Sustained production of IFN-gamma and other cytokines were observed in three patients: One who exhibited a complete response and two others who had stabilization of disease lasting over 6 months. Given the small sample size and heterogeneity of the patient population, the effects of IL-12 on the innate immune response to trastuzumab therapy should be further explored in the context of a larger clinical trial.

Clinical significance of MHC-reactive alloantibodies that develop after kidney or kidney-pancreas transplantation.

The purpose of this study was to determine the relationships between acute rejection, anti-major histocompatibility complex (MHC) class I and/or class II-reactive alloantibody production, and chronic rejection of renal allografts following kidney or simultaneous kidney-pancreas transplantation. Sera from 277 recipients were obtained pretransplant and between 1 month and 9.5 years post-transplant (mean 2.6years). The presence of anti-MHC class I and class II alloantibodies was determined by flow cytometry using beads coated with purified MHC molecules. Eighteen percent of recipients had MHC-reactive alloantibodies detected only after transplantation by this method. The majority of these patients produced alloantibodies directed at MHC class II only (68%). The incidence of anti-MHC class II, but not anti-MHC class I, alloantibodies detected post-transplant increased as the number of previous acute rejection episodes increased (p = 0.03). Multivariate analysis demonstrated that detection of MHC class II-reactive, but not MHC class I-reactive, alloantibodies post-transplant was a significant risk factor for chronic allograft rejection, independent of acute allograft rejection. We conclude that post-transplant detectable MHC class II-reactive alloantibodies and previous acute rejection episodes are independent risk factors for chronic allograft rejection. Implementing new therapeutic strategies to curtail post-transplant alloantibody production, and avoidance of acute rejection episodes, may improve long-term graft survival by reducing the incidence of chronic allograft rejection.