SITC 2015: Intralesional Rose Bengal in Melanoma Elicits Tumor Immunity via HMGB1

Updated again below.

Abstract: Intralesional (IL) therapy is under investigation to treat dermal and subcutaneous metastatic cancer. Rose Bengal (RB) is a staining agent that was originally used by ophthalmologists and in liver function studies. Previously, IL injection of RB induced regression of injected and uninjected tumors in murine models. However, the relevant mechanism is yet unknown. In this study, we used an OVA-expressing B16 melanoma murine model and found that IL RB treatment led to increased tumor-specific T cells with memory characteristics. CD8+ T cell are crucial for tumor-specific response elicited by IL RB. IL RB therapy also increased antigen-specific T cell proliferation and enhanced tumor regression. In addition, IL RB facilitated dendritic cells (DCs) infiltrating lymph nodes draining from tumor. Incubation of melanoma cells with RB led to necrosis and the release of High Mobility Group Box 1 (HMGB1), which activated DCs via up-regulation of CD40 expression. The blockade of HMGB1 significantly reduced the antigen-presenting ability of DCs. To determine whether this mechanism was relevant in patients treated with IL RB, we performed a pilot clinical study in melanoma patients (NCT01760499). IL RB led to tumor regression in both RB-injected and uninjected lesions, associated with an increase in circulating T cells. Increased tumor-specific response was found from those circulating T cells of 5 out of 7 tested patients after IL RB treatment. HMGB1 levels in patient sera were also elevated. Together, these results reveal a clinically relevant immunoadjuvant pathway triggered by tumor cell death secondary to ablation with RB.

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Updated (11/3/15): Items in the abstract of note [to me]:

• "However, the relevant mechanism is yet unknown."

• Clinical (human) data: "Increased tumor-specific response was found from those circulating T cells of 5 out of 7 tested patients after IL RB treatment." {Underlined emphasisis is mine}

• "Together, these results reveal a clinically relevant immunoadjuvant pathway triggered by tumor cell death secondary to ablation with RB."

• "clinically relevant"
• "immunoadjuvant pathway"

Updated (11/3/15): "Clinically relevant" {Underlined emphasis is mine}

"In clinical research is not only important to assess the significance of the differences between the evaluated groups but also it is recommended, if possible, to measure how meaningful the outcome is (for instance, to evaluate the effectiveness and efficacy of an intervention). Statistical significance does not provide information about the effect size or the clinical relevance. Because of that, researchers often misinterpret statistically significance as clinical one. On one hand, a large sample size study may have a statistically significant result but a small effect size. Outcomes with small p-values are often misunderstood as having strong effect sizes. On the other hand, another misinterpretation is present when non statistical significant difference could lead to a large effect size but a small sample may not have enough power to reveal that effect." (Source)

"Ideally, a clinical trial should be able to demonstrate not only a statistically significant improvement in the primary efficacy endpoint, but also that the magnitude of the effect is clinically relevant...However, it is not only important to assess statistical significance, but also to assess the clinical relevance of the effect, and the assessment of clinical relevance has received much less attention in the statistical literature." (Source)

"Immunoadjuvant pathway triggered by tumor cell death:" {Underlined emphasis is mine}

"Conventional cancer treatments rely on radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Here we show that the success of some protocols for anticancer therapy depends on innate and adaptive antitumor immune responses. We describe in both mice and humans a previously unrecognized pathway for the activation of tumor antigen-specific T-cell immunity that involves secretion of the high-mobility-group box 1 (HMGB1) alarmin protein by dying tumor cells and the action of HMGB1 on Toll-like receptor 4 (TLR4) expressed by dendritic cells (DCs). During chemotherapy or radiotherapy, DCs require signaling through TLR4 and its adaptor MyD88 for efficient processing and cross-presentation of antigen from dying tumor cells. Patients with breast cancer who carry a TLR4 loss-of-function allele relapse more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele. These results delineate a clinically relevant immunoadjuvant pathway triggered by tumor cell death." (Source: Apetoh et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat. Med. 13, 1050–1059)

"Incubation of melanoma cells with RB led to necrosis and the release of High Mobility Group Box 1 (HMGB1), which activated DCs via up-regulation of CD40 expression."

"CD40 was initially characterized as a co-stimulatory molecule expressed on APCs that played a central role in B and T cell activation. However, this molecular pair functions in the regulation of both APCs and effector lymphocytes (Fig. 1). As we understand more about the number of different DC and T cell subsets, we are likely to find that CD40-CD40L interactions play important and distinct roles in regulating these novel subsets. In addition, as we continue to understand how innate immunity cells directly regulate B cells and antibody responses, the influence of CD40 and CD40L in these interactions should be further clarified. Further insights into the functions of CD40-CD40L interactions will advance our understanding of immune cell crosstalk and interdependent regulation of the immune system." (Source: Ma DY, Clark EA. The role of CD40 and CD40L in Dendritic Cells. Seminars in immunology. 2009;21(5):265-272.)

provectus IS a t-cell company

H/t InvestorVillage poster bradpalm1: A research team from the University of Illinois at Chicago (UIC) published a paper in July entitled The Potential of Intralesional Rose Bengal to Stimulate T-Cell Mediated Anti-Tumor Responses (Maker et al., J Clin Cell Immunol 2015, 6:4). This same team previously published results of their murine model work and research on PV-10 for colon cancer. See June 5, 2015 blog post Intralesional Injection of Rose Bengal Induces an Anti-tumor Immune Response and Potent Tumor Regressions in a Murine Model of Colon Cancer.

Blog post takeaways:

• Rose Bengal (PV-10)/PV-10 (Rose Bengal) induces/generates/creates/leads to anti-tumor immunity. To some the so-called Holy Grail of cancer therapy is achieving durable [system- or body-wide] anti-tumor immunity in a cancer patient.

• PV-10 can prime the immune system (through the direction injection of the compound into cancerous lesions), and help it defeat distant cancerous lesions around the body.

• The compound exposes antigens to the immune system that otherwise would not be exposed, or that otherwise would escape detection by (and thus presentation to) the immune system.

• PV-10 can help with T cell generation & circulation, even after treatment with immune checkpoint inhibitors fails.

• UIC's work reproduced Moffitt Cancer Center's (Moffitt's) work, which reproduced Provectus' founders' work.

• UIC's work that contributed to their paper was funded in part by a grant (non-specific to Provectus and PV-10) from the National Institutes of Health.

Recall that Provectus has a two-prong approach to fighting (killing) cancer: (i) local effect/tumor ablation (anti-tumor cytotoxicity) and (ii) systemic effect/tumor-specific immune response (induction of anti-tumor immunity)

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UIC and Moffitt independently reproduced the veracity of both prongs:

Click to enlarge. Image source

UIC's paper reviews PV-10's agnosticity in destroying tumors into which it is injected (i.e., anti-tumor cytotoxicity):

• Melanoma,
• Breast cancer,
• Ovarian cancer,
• Gastric cancer, and
• Sarcoma.

The paper discusses PV-10's agnosticity in generating anti-tumor immunity resulting from local tumor injection (i.e., from resultant anti-tumor cytotoxicity):

• Melanoma,
• Breast cancer, and
• Hepatocellular carcinoma (HCC).

The UIC team appears to have conducted murine model work on PV-10 in HCC, noting "Our current research is establishing the role of RB in generating anti-tumor immune responses in gastrointestinal cancer and liver metastases:"

"Furthermore, in syngeneic orthotopic models of murine hepatocellular carcinoma (HCC), RB similarly induced chemoablation in all treated tumors. Twenty-one to 81 days after RB treatment, when re-challenged with the same tumor, durable immunity was demonstrated in 14/14 animals without measurable tumor formation, whereas B16-F10 melanoma tumors, i.e., non-HCC cells, were able to be established in 13/13 animals [20]. Additionally, immunity to establishment of a new HCC tumor could be created through adoptive transfer of splenocytes from treated animals. This was an interesting finding given that analysis of splenic composition from animals with B16 melanoma that experienced distant lung tumor regression after RB treatment of flank tumors did not demonstrate any difference in the percent of T cells, Tregs, NK cells, B cells, myeloid derived suppressor cells, or macrophages [15]. The authors further demonstrated that bystander lesions disappeared or decreased in size in HCC models, whereas, no bystander tumors were ever observed to resolve in nude mice without a competent T-cell immune system. These experiments established that, similar to melanoma, in immunocompetent mice with orthotopic primary hepatocellular carcinoma flank tumors, an anti-tumor response can be induced by priming the animals with RB treatment of tumors. These experiments raise the possibility that RB induced cytotoxicity exposes antigens and mounts an immune response that may protects animals from additional tumor formation that can be adoptively transferred to other animals using splenocytes." {Underlined and bolded emphasis is mine}

Speaking of antigens, it's all about the antigens:

"These experiments established that in human and murine melanoma and breast cancer, there appeared to be an antigen-driven T-cell response that has the potential to activate T-cells and impart antitumor responses in bystander lesions and distant metastases."

Why are antigens important? Because they facilitate the priming of the immune system through their presentation to it:

"These findings may imply that tumor response in the primary lesion may be able to prime the immune system of patients for activity against distant lesions."

The UIC team's conclusions could be profound, among them:

• "RB-mediated tumor cell death may expose tumor antigens that may otherwise evade immune detection."

• "Though most profoundly described in melanoma cells, clearly an immunoreactive malignancy, the effect has been shown to be not limited to one specific type of malignancy. 

• "Decrease in tumor burden and stimulation of an immune response with PV-10 has been demonstrated in animal models of metastasis, and correlations of these responses in clinical studies is consistent with such results." Provectus' CTO Dr. Eric Wachter, PhD has said in a 2013 white paper: "PV-10 murine research demonstrated unambiguously, Dr. Wachter noted, that tumor burden is a critical variable in predicting response to a combination therapy." See Radiation (September 22, 2015) on the blog's Current News page.

• "That PV-10 treatment can potentially increase circulating cytotoxic T-cells, even in patients who were previously treated with immune-activating checkpoint blockade, supports the possibility that RB induced cytotoxicity may activate T-cells that are responsible for the bystander effect on untreated lesions."

• As such, intralesional therapy with RB may be a promising new mode of therapy to stimulate T-cell mediated anti-tumor immune responses."

UIC's work was conducted in part under an NIH grant.