So our last speaker is Dr. Akita Bhatnagar on Enhanced 5-ALA Induced Fluorescence in Hormone-Secreting Pituitary Adenomas. Take your time. That's okay. You don't have to run. Hi, everyone. My name's Akita, and I'm a third-year medical student at George Washington. Firstly, I'd like to thank AANS for giving me this opportunity to present my work. I have no conflicts of interest. Okay. So Cushing's disease is an endocrine disorder that is caused by pituitary adenomas. And these are microadenomas as they're less than one centimeter in size, making them the smallest surgical target in the entire field of medicine. They're hormone-secreting tumors. They secrete ACTH that in turn leads to high levels of cortisol in the body. And it is this state of hypercortisolemia that is found in a Cushing's disease patient that leads to significant morbidity and high rates of mortality. So the treatment of choice for Cushing's disease is transfedoidal adenectomy, as displayed in this video here, which leads to successful surgical outcomes. And that's why preoperative MR imaging becomes extremely important to make the surgeon's job easier to perform the surgery. However, the problem is that these tumors are oftentimes missed on MR imaging, like 40% of the time, because of their small size. And that's why our research efforts is towards improving the imaging paradigm in Cushing's disease, for which we looked at 5-ALA, also called 5-aminolevulinic acid. So what is this 5-ALA? It is an endogenous metabolite that is found in the heme biosynthesis pathway that is present in all of our cells. Aminolevulin, as shown here, is inherently fluorescent that lights up most of these cells. But tumor cells have 50 to 100 times greater uptake and conversion of ALA into protoporphyrin. That is the basis of targeted tumor imaging and cancer specificity, as seen with 5-ALA. So 5-ALA is a clinical standard, as of now, for gliomas in Europe. The way it works is that a patient would drink a solution of 5-ALA. It equilibrates in the body over a period of two to three hours. A surgeon would come and shine blue light over the area of interest. 5-ALA gets excited and photo-converted into protoporphyrin that has an emission in the red spectra, which lights up only the tumor cells. And as you can see in this image here, there's a clear demarcation of tumor from normal, as seen by the protoporphyrin fluorescence. The intraoperative use of 5-ALA for pituitary adenomas has also been studied in one clinical trial so far, again in Europe. And again, as we see here, there's a nice demarcation of pituitary tumor from normal. The sensitivity and specificity of MR-negative microadenomas has been documented to be around 75%, and it's close to around 80% for macroadenomas, which are MR-positive. So one of the reasons why tumor cells have a greater uptake in conversion of 5-ALA into protoporphyrin is because this heme synthesis pathway is tightly linked to the mitochondrial respiration chain, as seen over here. And we know that tumor cells, at least most of them, have greater mitochondrial respiration rates, as seen by the oxygen consumption, as compared to normal, which leads to a greater uptake of 5-ALA, which is, again, the basis of cancer specificity. So we first started off our studies with the aim to define this energy phenotype of pituitary tumor cell lines obtained from a mouse model called ATT20s and the normal counterpart. Now, using a seahorse experiment, what we see is that the overall OCR, which stands for oxygen consumption rate, is much higher as seen in pituitary tumors as compared to normal, which is what we expected. We then went ahead and did some flow cytometry. We tested the 5-ALA fluorescence emanated from human pituitary adenoma obtained from surgery and its normal adjacent gland. So first, what we see is the blue peaks, which is the untreated cells, both normal human pituitary tissue and human pituitary tissue. We then treated both these samples with ALA. So the first thing that we see is a two-fold increase from the normal sample, which is quite expected as we do have baseline levels of 5-ALA on all of our normal cells. However, the point to be noted here is that there is a tenfold increase in expression from the adenomatous part. And this is, again, the basis for clear tumor demarcation intraoperatively that is achieved with 5-ALA. We, again, continued our studies with some in vitro fluorescence diagnostics in murine pituitary cells. So we treated the ATT20 cells and the normal pituitary cells with 5-ALA, and we see a sevenfold increase from the tumor cell lines. We were then interested to see what happens to 5-ALA once we add dexamethasone and CRH which are, again, involved in the tight hormone regulatory loop as seen in a Cushing's disease patient. So after we add dexamethasone to 5-ALA treated ATT20 cells, we see a 31% increase in fluorescence levels as compared to the cell lines that don't have any dexamethasone. However, CRH, in fact, showed a slight depression in fluorescence levels. But the point that I want to bring out over here is this increase in expression levels after dexamethasone addition. So this finding perhaps suggests that Cushing's disease patients already have super physiological levels of glucocorticoids in their body that could, in fact, enhance the signal intensity achieved by the 5-ALA imaging system. Another use of 5-ALA is for photodynamic therapy or cell kill as being currently used for melanomas. So we wanted to see what happens to the ATT20s, which are the mouse pituitary cell lines after the addition of 5-ALA. So first, these are the untreated cells, the first panel, and the second panel is the treated cells with 5-ALA. The blue is the stain for nuclei, red is the stain for mitochondria, and yellow independently is the stain for protoporphyrin. So in the first panel of untreated cells, we see nice, robust, healthy cells. However, after the addition of 5-ALA, we incubate these samples for around four hours. We see a fragmentation of mitochondria as seen by the dispersion of the red signal over here, which, again, is one of the suggested bases of cell kill as achieved by 5-ALA. At a higher magnification, we see these green punctae in the ALA-treated ATT20 cell lines, which is basically the overlay of the yellow signal of protoporphyrin over the red mitochondria, which again supports the mitochondrial localization and effect of protoporphyrin. So in conclusion, 5-ALA is a promising agent for fluorescence-guided resection in pituitary microadenomas. The signal could be enhanced by superphysiological levels of glucocorticoids, as seen in a Cushing's disease patient. The mitochondrial localization and disruption of 5-ALA is probably the basis for photodynamic therapy. For future studies, we'd like to investigate the autofluorescence as emanated by 5-ALA from even the normal cells. We'd like to establish further the toxicity profile of 5-ALA, and we'd like to study the photobleaching effect of protoporphyrin. And I'd like to thank the principal investigator of this study, Dr. Prashant Chiribuena, and the rest of the lab at NIH. Thank you. Thank you very much, and thank you to all the speakers. Let's give a round of applause. And with this, we conclude the WNS meeting. Thank you.

5-ALA

pituitary adenomas

Cushing's disease

imaging

treatment