In 2004, Aldara™ (imiquimod 5% cream, 3M Pharmaceutical, St Paul, MN, USA) labeling was extended by the Food and Drug Administration to include treatment of superficial basal cell carcinoma (BCC) based upon randomized controlled trials demonstrating complete histological clearance in 78% to 87% of superficial BCC treated topically 5 days per week for 6 weeks 12. Pilot-scale and investigator initiated trials had shown 90% to 100% clearance with q12 hours (twice per day) dosing 3.

Imiquimod belongs to a family of synthetic small nucleotide-like molecules with potent immuno-modulatory activity mediated through Toll-like receptor (TLR)-7 (and 8) signaling. When applied topically, these compounds display immune-mediated anti-tumoral activity without damaging normal tissues 134567 Imiquimod targets predominantly TLR-7 expressing plasmacytoid dendritic cells (pDCs) with secondary recruitment and activation of other DC and macrophage subtypes and induction of T helper₁ responses within three to five days of treatment 4. Stimulation of pDCs through TLR-7/myeloid differentiation response gene 88 (My-D88)/IRF-7 signaling induces expression of interferon (IFN)-α, which appears to act upon natural killer (NK) cells and conventional dendritic cells (DCs) to stimulate IFN-γ, tumor necrosis factor (TNF)-α, monocyte chemoattractant proteins (MCPs) and other cytokines 589 This immunological cascade leads within two weeks to apoptotic death of cancer cells and their substitution by a mononuclear cell infiltrate 3458

Although imiquimod function seems particularly associated with IFN-α-stimulated genes (ISGs) 10, it remains unclear whether this pathway is solely responsible for all the downstream effects ultimately resulting in tumor clearance. Indeed, a comprehensive and conclusive characterization of the events leading to tumor rejection based on a prospectively controlled study has never been reported. We previously characterized ISGs in vitro 11 and in vivo (Belardelli F and Arico' E, manuscript in preparation), compiling a road map for the interpretation of transcriptional surveys of biological conditions affecting the tumor microenvironment (Additional data file 1).

Here, we report a paired analysis of adjacent punch biopsies obtained pre- and post-treatment from 36 patients with BCC subjected to local application of imiquimod or a control cream in a blinded, randomized protocol.

A total of 65 subjects were screened, but 27 were ineligible due to their pre-enrollment biopsy excluding BCC and 2 were ineligible for other reasons. A total of 36 subjects were eligible for the study and started treatment with either imiquimod (n = 22) or vehicle cream (n = 14) (Table 1). After unblinding, treatment groups were color-coded to facilitate the discussion. Out of the subjects, 61% had nodular BCC, 17% superficial BCC, and 22% unspecified BCC. Of note is that all 4 subjects randomized to the imiquimod q12 hours × 4 days group had nodular BCC. Post-treatment biopsies were taken <12 hours after last dose for 17% of subjects, >36 hours after the last dose date for another 17%, and between 18 and 30 hours after last dose for 33%. This variability was uncontrollable and due to patient compliance. The locations of the tumors were: 41% on the face; 25% on the extremities; 22% on the trunk; and 11% on either the neck or scalp. Furthermore, patient (P) 23 and P28 did not complete treatment, missing two placebo and one imiquimod dose, respectively. The imbalance in the distribution of the elapsed time between last treatment dose and post-treatment biopsy did not significantly affect the results except, possibly, for the q24 × 8 (pink) cohort. Interestingly, at this early time point, already 9 of 22 imiquimod-treated BCCs were found to be clear of tumor cells, particularly among patients treated with the most intense schedule.

This is the first prospectively controlled study conducted to identify the early biological events associated with the eradication of BCC through an immune-mediated mechanism. By protocol design, tumor regression did not represent an endpoint and tumors were removed at the end of the study. Thus, the association between the molecular/genetic findings and tumor clearance is presumptive, based on the historical 80% to 90% clearance rates recognized by the Food and Drug Administration for the release of imiquimod for clinical use 2. However, it is interesting to note that 9 of 22 (41%) imiquimod-treated BCCs were devoid of cancer cells by EOT (2 to 8 days from beginning of treatment) while only 1 of 14 (7%) control-treated BCCs had no identifiable tumor cells (Fisher test p value = 0.05), suggesting that artifacts due to vehicle administration or surgical trauma were not responsible for the early tumor clearance.

As indicated by qPCR, IFN-γ transcription was more prevalent than IFN-α transcription. This is in line with the evidence of predominant NK, CD8 and CD4 T cell activity in this study. Sullivan et al. 30 had indeed previously observed similar cellular infiltrates (particularly CD4 and CD56 expressing cells) in a smaller, open-label, matched controlled, non-randomized study in which six patients with BCC treated with imiquimod at daily intervals for a total of ten administrations were compared with six patients receiving comparable vehicle cream treatment. The predominance of IFN-γ transcription suggests that pDCs trigger other immune functions through the production of IFN-α, which in turn activates resident T and NK cells, selective producers of IFN-γ 31. We hypothesize that these secondary immune effector mechanisms induce destruction of target cells, providing antigen to professional antigen presenting cells for priming of naive T-cells in draining lymph nodes 3132. Indeed, several of the transcripts associated with imiquimod treatment show activation of T and NK cells and induction of IFN-γ stimulated genes (Figure 3). The cytotoxic T and NK cell signatures identified here (granzymes, perforin and other NK cell-related genes) have recently been described in a mouse model of IFN-α and IFN-γ-producing killer DCs (IKDCs) 33, which simultaneously display cytotoxic and pro-inflammatory functions. Thus, IKDCs could summarize in a cellular unit our findings of ISG activation combined with broader cytotoxic and pro-inflammatory properties. At present, IKDCs have not been characterized in humans, nor it is known whether they express TLR-7; future studies should address their role as putative mediators of immune rejection.

Imiquimod treatment stands as a unique opportunity to study the mechanisms of immune-mediated rejection directly in human tissues. This TLR-7 agonist links multiple immune pathways. Of these, IFN-α plays a consistent but not exclusive role. Previous transcriptional surveys have provided a broad view of the biological processes associated with immune-mediated tissue destruction, identifying convergent characteristics. Neoplastic inflammation approaches the unresolving process of chronic hepatitis C virus (HCV) infection where the presence of antigen-specific immune responses do not lead to clearance of the pathogen in the majority of cases 3435. Both diseases are characterized by the expression of ISGs that do not seem sufficient to clear the pathogenic procress. Similar signatures can be identified in liver biopsies from patients with chronic HCV infection 36 and in chronic allograft rejection controlled with standard immune suppression 37. ISGs are also consistently expressed in melanoma metastases following the systemic administration of IL-2 independent of clinical outcome 38. Thus, it appears that ISGs are part of immunological processes associated with chronic inflammation insufficient to clear its cause. On the contrary, several non-ISGs identified by this study delineate potent inflammatory (CCL7/MCP-3, CCL4/MIP-β, and so on) and cytotoxic (granzymes, perforin, NKG-5, and so on) functions rarely observed in chronically inflamed tissues but described in the acute inflammation associated with destruction of a tumor 38 or allograft 37, liver damage in HCV-induced cirrhosis 36 or gut dysfunction during flares of Crohn's disease 39. This study corroborates the impression that immune-mediated tissue destruction comprises at least two components: a baseline cluster of ISGs that may be necessary but insufficient to induce tissue rejection and a less common activation of broad cytotoxic and other potent pro-inflammatory innate immune effector functions that are more tightly associated with rejection. Our findings are supported by the recent description of clearance of established cancers by the adoptive transfer of innate immune effector cells in the powerful model of spontaneous regression/complete resistance mice 40.

Sarwal M et al. 37 reported strikingly similar results evaluating the transcriptional behavior of renal cell allograft during acute rejection, basing the analysis on a similar array platform and utilizing the same RNA amplification method 41 (Figure 3, transcripts labeled in red). In spite of these similarities, they also reported a B cell signature characterized by enhanced expression of CD20 and several immunoglobulins that we did not identify in our study. This discrepancy could be explained by a specific role that B cell-mediated immunity may play in the context of allo-recognition. In the case of BCC, the strong pro-inflammatory stimulus induced by imiquimod through TRL-7 signaling might bypass the requirement for an endogenous, tissue specific insult responsible for the secondary triggering of the cellular immune effector mechanisms identified by both studies. The signatures identified by both studies also match the anecdotal identification of the same genes in a melanoma metastasis that underwent regression following systemic IL-2 therapy 38.

Among the genes mutually reported by the previous three studies, NK4/IL-32 was recently recognized as a central mediator of Crohn's disease 42 and associated with liver damage during HCV infection 36. NK4/IL-32 is a potent inducer of pro-inflammatory cytokines and it is selectively expressed by immune cells stimulated with IFN-γ IL-2 or the combination of IL-12 and IL-18 1439. Indeed, we found NK4/IL-32, together with other genes associated with cytotoxic function, to be constitutively expressed by NK cells but only by activated CD8+ T cells 43. Moreover, we recently observed NK4/IL-32 to be preferentially expressed in metastatic melanoma compared with other less immune responsive cancers 44. It is possible that NK4/IL-32 may play a central role during imiquimod treatment by amplifying inflammatory stimuli through the induction of a cytokine cascade. Thus, this novel cytokine emerges as a central player in immune rejection or autoimmunity.

Dermatologists have long used imiquimod to treat BCC 44546 Imiquimod mimics the action of single-stranded viral RNA 31, activating a pro-inflammatory cascade as a chemical prototype of the danger model of immune activation 47. Meanwhile, tumor immunologists have struggled to explain the paradoxical co-existence of tumor antigen-specific T cells induced by vaccination with growing tumor tissues. Indirect evidence suggests that vaccine-induced T cells reach the tumor site 48 and recognize tumor cells producing IFN-γ˜ MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2Caerbhv2BYDwAHbqedmvETj2BSbqee0evGueE0jxyaibaiKI8=vI8tuQ8FMI8Gi=hEeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciGacaGaaeqabaqadeqadaaakeaaiiaacuWFZoWzgaacaaaa@34E8@ However, this is not sufficient for tumor rejection since other effector mechanisms are not simultaneously activated 49 because cancers do not provide the danger signal necessary for full implementation of the immune responses 50. Thus, immunization successfully affects the afferent loop of the immune response by eliciting TA-specific T cells but cannot affect T cell activation at the receiving end 5152. The cancer specificity of TLR agonists consists of the preferential attraction of TLR-7 expressing pDCs to chronically inflamed tissues and their enhanced recruitment 53. Similar conclusions were recently reached by Torres et al. 54, who followed the biological events induced by imiquimod when administered to patients with actinic keratosis. Thus, TLR agonists exemplify how the gap between the induction of TA-specific T cells by immunization and their activation at the receiving end could be closed. It is thus conceivable that preparations of TLR agonists suitable for systemic administration may be used in the future as single agent therapy for other tumor types (trials are currently ongoing in Europe for melanoma) or as adjuvants to enhance the effectiveness of active-specific immunization approaches 555657.

This study stands as a proof of principle that, when tissues are easily accessible, mechanistic observation about the effects of a treatment can be easily performed in humans by combining minimally invasive techniques (fine needle aspirates, through cut or punch biopsies) with high-fidelity mRNA amplification; such approaches are fundamental to refresh scientific hypotheses through direct human observation. Second, it provides insights into the early events leading to tumor rejection in a most powerful human model. Finally, it suggests that immune-mediated tumor rejection is only one aspect of tissue-specific destruction, which follows a constant immunological pathway shared by other anti-cancer immunotherapies, acute allograft rejection, autoimmune disease and tissue damage during chronic pathogen infections.

Detailed methods are available as Additional data file 2.

The following additional data are available with the online version of this paper. Additional data file 1 provides a list of genes previously shown to be associated with the stimulation of various cell types with IFN-α. Additional data file 2 is an extended version of the Materials and methods, providing full disclosure of the methodology used. Additional data file 3 provides a complete list of the 637 genes specifically induced by imiquimod treatment based on the statistical approach presented in the text. Additional data file 4 is a diagram illustrating the mining strategy that was implemented for the preparation of Figure 1c,d.