So our next speaker is, sorry, Dr. Anna Kemeny on anti-tumor efficacy of anti-PD-L1 in ACTH CPD-P2-3 adenovirus. Thank you. So my name is Hannah Kemeny, and I'm a rising fourth-year medical student at Duke. I wanted to start by first just saying thank you to AANS for the opportunity to present our work, as well as my mentor, Dr. Peter Fecci. And I will be talking to you about the expression of PD-L1 on pituitary adenomas and the role for immunotherapy. So pituitary adenomas are among the most common intracranial tumors, affecting about 20% of the general population. Additionally, there are about 10,000 diagnoses of pituitary adenomas a year. Typically, these tumors are benign, and their symptoms are a result of mass effect. There are a subset of tumors that are functional in that they secrete physiologic hormones, and therefore, even at small sizes, can result in systemic comorbidities. One such functional tumor is an ACTH-producing adenoma or Cushing's disease, which results in the secretion of cortisol by the adrenal glands. And this results in a variety of systemic comorbidities, including metabolic abnormalities such as diabetes, immune deficiencies, as well as cardiovascular abnormalities. Unfortunately, a true cure for Cushing's disease remains a challenge. By and large, surgical intervention remains the first-line intervention for these tumors. However, about 25% recur in about five years. As such, new treatment modalities are necessary in order to provide therapies for those that do not respond to the standard care. Within the oncology cancer field, immunotherapy remains a promising area that has garnered a lot of attention and is believed to have the opportunity to revolutionize cancer care. One such area is the use of immune checkpoints or immune modulation. An axis that has been highly studied is the PD-L1, PD-1 axis. So PD-L1 is a protein that is overexpressed by tumor cells. And upon binding to the PD-1 receptor on activated T-cells, these T-cells are turned off and unable to...and it inhibits their ability to have a cytotoxic antitumor function. Therefore, therapies have been focused on blocking these receptors, so either PD-1 or PD-L1, and by inhibiting this attachment, T-cells remain on and they're able to have an antitumor function. And a lot of therapies have used this and have been FDA-approved and have had overwhelming success in solid tumors such as lung cancer, melanoma, breast cancer, and renal cell carcinoma. Though not extensively studied, our collaborators in Boston found that PD-L1 expression does occur in human pituitary adenomas. So given this expression, this kind of had us believe...or question whether or not checkpoint blockade, so specifically against PD-L1, would be an opportunity to provide relief for patients that are refractory to standard of care for pituitary adenomas. So we hypothesized that it would, and in order to do this, we wanted to first identify whether or not PD-L1 was expressed in these pituitary adenomas. Next, we wanted to establish our mirroring model for Cushing's disease and then see whether or not anti-PD-L1 therapy would be beneficial in both the subcutaneous model as well as an intracranial model. So first, we looked at about 70 of the human pituitary adenomas from our institution that had been banked, and these ranged from both functional to non-functional adenomas. And here, when looking at the functional adenomas, we found that about 36% had PD-L1 expression compared to actually none of our non-functioning samples. And given the...so this is kind of just a representative slide of this expression. So here you can see the positive control with the placenta, and then we have expression of PD-L1 in our pituitary adenoma as well. So we then wanted to translate this into our mirroring model. So we identified a well-studied pituitary adenoma cell line that is in mice that secretes ACTH, and we first looked at this in vitro, given that our hypothesis was dependent on whether or not this had PD-L1 expression. And we found that the cell line itself did have PD-L1 expression by flow. We then wanted to transition this into our mirroring model. So just for initial ease, we started by implanting them subcutaneously. And here, we confirmed through both IHC and our HME staining that this was, in fact, a pituitary adenoma. Next, we wanted to identify whether or not this PD-L1 expression remains in vivo. So in order to do this, we allowed the tumors to grow, harvested them, and then saw that we had PD-L1 expression, as you can see in the bottom panel, compared to the unstained control. Additionally, using flow cytometry, we found that the subcutaneous tumor had higher levels of PD-L1 expression compared to the normal pituitary gland. Next, given that we wanted to create this mirroring model of Cushing's disease, we wanted to confirm that this tumor did, in fact, have the ACTH secreting properties. So again, we completed IHC, and we found that we did have positive ACTH staining. Additionally, we used an ELISA kit. And here, we took blood from our MICEP baseline. And then following tumor growth for about eight weeks, we then took an additional blood sample, and we saw that we had significantly elevated levels of ACTH following growth. Additionally, we saw that as these tumors grew, we had significant weight gain that accompanied the growth, which was an expected pathologic response to the higher levels of ACTH secretion. Additionally, we saw that our mice took on the Cushing-Goyt phenotype. So as I mentioned, they had the increase in weight. And additionally, their body contour changed as they had the deposits of the subcutaneous fat. And then next, we wanted to identify could we, the anti-tumor efficacy of anti-PD-L1. So to do this, we implanted, again, the tumors into the flank, or the tumor cell line into the flank. And then every three days, we administered an anti-PD-L1 antibody. And here, we, again, measured the tumor growth over the course of those three days. And here, we saw that the tumor growth was substantially smaller with the treatment group compared to the isotype. Additionally, we then, using that ELISA, again, compared the baseline ACTH levels in the blood to the blood following treatment. And we found that the treatment group had lower levels of ACTH compared to the treatment, or excuse me, compared to the group that did not receive any treatment. And then we wanted to recapitulate these findings and see if we found them in an intracranial model. So we took our cell line, and we implanted these into the right frontal lobe using a stereotactic frame. And we used a dose that was uniformly lethal at 24 days. And again, following our same model, we treated every three days with intraperitoneal injections. And we, again, here saw that we did have anti-tumor efficacy. And we had about a 40% survival rate in our treated group. So in conclusion, we found that we know that first-line treatment for Cushing's disease is surgical resection. But there are a subset of patients who do have recurrence and are in need for a different treatment modality. We confirmed that both human and mouse pituitary adenomas have expression of PD-L1, making this a viable target. And indeed, we did see efficacy after anti-PD-L1 therapy, both in reducing the tumor growth as well as increasing survival. So we believe that we have this model for Cushing's disease that we can use kind of in our preclinical work to evaluate the role of immunotherapies in treating Cushing's disease. And for future directions, we're looking at the mechanism by which this works as well as other therapeutic combinations. So specifically with PD-L1 and then other checkpoint inhibitors. Thank you.

anti-PD-L1 therapy

pituitary adenomas

immunotherapy

tumor growth

Cushing's disease