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Alessandro Larchel: Thank you very much. My name is Alessandro Larchel, I am a urologist. I have the honor to present our program devoted to VHL disease, which basically means that I will take the credit for the work done by others, because this is a very multidisciplinary clinical research program basically involving the entire institute. Von Hippel-Lindau is a protein with a key role in renal cancer. If it doesn't work, HIF accumulates in the cells, thus promoting growth, androgenesis, cell proliferation, and ultimately cancer. The importance is critical, because of this apical position, very upstream relative to all the targets of all the systemic agents that are available for this disease. From a practical point of view, patients with the germline mutation in VHL disease have a very heterogeneous clinical phenotype. This is the most common cause of genetically defined renal cell carcinoma. According to those estimates, we expect an active population in Italy of 2000 living individuals, which is a lot of work for us as you can see, because this is the only center that is recognized by VHL Alliance in South Europe and in Italy.

This was critical for us, because since the very early development of the program, we wanted to work step by step with the patient association in the development of our clinical activity and also of our research program, which for us, basically it's one thing. When we designed our program, we wanted to assist our patients into three individual steps that correspond to three different moments in their management. Number one would be the indication for genetic referral and genetic testing, which basically is the patient that has fear to have the syndromic disease and we do not have a clear diagnosis yet. How to do that? First, we always want a pathologic confirmation of a primary, which is highly suspicious for VHL disease. Then if this is corroborated by, for example, a high-risk primary, by multiple low-risk primary, or by family history, which is suspicion, then we go for testing. There are recommendations which are endorsed by VHL Alliance as well, but the level of evidence that we have here, and this is a common problem that those of you who manage rare disease are very familiar with, are only expert-opinion based.

At the very beginning of our program, we have done this huge review of our repository of the institution and we were able to merge the clinical dataset of all those different clinician managing the primaries that are common in this disease. We merged the data together with the Central Pathology Unit and we merged also with the results of patients that were elected for genetic testing. This allowed us to create the kind of scale of the primary, that are most frequently associated with the syndromic disease. As you can see here in red, the proportion of patients with confirmed pathologic variants in VHL locus in case of central nervous system hemangioblastoma is 15%, in case of retinal hemangioblastoma, 35%, which are figures entirely different, for example, to patients that are treated for sporadic clear cell, renal cell carcinoma in which the risk of syndromic disease is less than 1%.

This is a balance between how often this disease appears in a sporadic setting relative to in a syndromic phase. Again, here the literature is very poor. What's next? Provided that the diagnosis is confirmed, what can we offer to our patients? This is a disease which is not the typical renal cancer that you take to the OR, you operate, and then you say goodbye. You will have a fine life, but it's a marathon that those patients have to run throughout their life, so surveillance is a key option to, let's say, plan the economy of multiple treatment that those patients will receive during their life. This is a common experience also of the overseas colleagues. We have a lot of patients traveling the country that want to be treated by us or managed by us, so we have to be as fast as possible. We manage the patient in an outpatient clinic and in three days we are able to offer all the instrumental and diagnostic examination, plus the clinical consultation with us as urologists, with the pancreatic surgeons, with the neurosurgeons, and of course with the medical oncologist.

This surveillance program is by definition an observational prospective study that allows us to basically gather key information that is dramatically lacking in the literature so far, because for example, it is unclear what is the burden of the disease, it is unclear what is the heterogeneity that we observe between patients. We have opened our program in January 2021 and now we are almost full, because we have a capacity of one patient per week. Why? Because those patients, as you can see in the table, are extremely problematic to manage and they stress the healthcare significantly, because of the burden of disease. Just consider that only 7% of the patients were free from any VHL primary and, basically if you see one of these patients, the chance that you will have to perform some kind of active treatment is more than 50%.

Another very interesting observation, since we have the option to have all these services in-house in the institution, we have a close collaboration with our geneticist and with the genetic counselor. One of the most intriguing line of research is to try to better decipher the relationship between the germline pathogenic variant and the clinical phenotype. Again, because you can have a 60-year-old gentleman without basically no significant disease and very young patients, which are instead badly affected. Surprisingly enough, we have stratified the clinical features of those patients according to the type of mutation distinguishing missense mutations relative to patients with a complete deletion. The patients with the missense mutation adds a higher burden of disease, which is strange, because it tells us that patients in which the protein is lacking at all fair better than those that can have misfolded or a still partially functioning VHL protein. That in a way it tells us that, even if the transmission pattern of this disease is autosomal dominant, the clinical burden from the patient is way, way more complex.

Next step. In case of patients that we have to drop from the surveillance program because of an active indication for treatment, what to do. This is the first patient that we had to treat that is, as you can see, extremely problematic. It's a 52-year-old man that didn't want to follow surveillance program very, very precisely, so that was the scenario that we were facing. In transparent you have the adrenal parenchyma, which is basically zero on the left side. This kidney is silent on the renal scan. In purple, multiple renal cyst that were benign, and the orange one instead are primaries that we know that are clear renal cell carcinoma. Both kidneys were previously heavily treated owing to renal cell carcinoma and this is the 3D construction system that we use for sporadic renal cancer. To plan our surgery, in this case, the plan was way more complex, but we decided to perform a radical nephrectomy on the left kidney, because again, it was not functioning and to try to be as conservative as possible on the right one.

This is the left radical nephrectomy, as you can see. We were working in close contact with the engineers who developed the models with us, navigating the model together with us in the operating theater. This is the section of the left renal hilum and then the completion of the left radical nephrectomy. It is important that this approach is very useful for the multidisciplinary management of this disease, because also for the anatomy pathologist was key to this kind of map to understand basically what they were observing and grading. On the right side, this is the preparation of the right kidney. Again, here the plan was to mark and count each specific lesion. There were 10 in total. After that, we had to clear the kidney from all the disease to get the most accurate access to the kidney. I'm happy that our interventional radiologists are here with us, because they did an incredible job.

As you can see, we were basically targeting each lesion one by one with a local tumor ablation that in this peculiar setting is not a percutaneous maneuver, but is performed directly on the kidney in the surgical field together with us in the OR. Our strategy was, again, to save as much nephrons as possible. We treated all the lesions that were the most complex with the local tumor ablation, whereas those that were the most endophytic and let's say easier to nucleate, as you can see, were treated by means of partial nephrectomy. Again, the ultimate goal was to try to avoid dialysis for this man, so this kidney is not clamped and those resections were performed by keeping the perfusion, let's say, of the kidney and with close resection margins to the tumor as, let's say, as close and as aggressive as possible. The compression of the surgery, again, is the same that we performed for sporadic cancer with the reconstruction of the vessels and the closure of the colleges on the transaction bed.

Clearly, this patient was prepared for dialysis, but luckily enough, despite the expected spike in postoperative creatinine up to six, the patient always had a urinary output that was decent more than 100 CC per hours. Then the advice from our nephrology was to wait and not perform upfront dialysis. Luckily enough, on postoperative day 15, serum creatinine was 3.4, starting from a baseline of 2.4. Clearly, we gathered the clinical outcome of those data, but I would say the most intriguing part and the most intriguing opportunity that we have in dealing with this disease is asking ourselves, "What is the way best way to treat those patients?" Because for ourselves as surgeons, we do not want to perform this case, and we are convinced that the way forward is to work together and to identify systemic agents that can effectively treat those patients. The landscape and the offer for those patients has been dramatically changed one year ago by this pivotal phase-two trial testing an inhibitor of HIF2alpha.

The major issue that we have is, of course, that as you know, this drug is not approved in Europe and the evidence that we have is very, let's say, initial in this regard, because there are very strict inclusion criteria for these studies. Let's see this issue from the perspective of our clinic. If we try to test how many of our patient would fit into the inclusion criteria of this trial, it was only 23%. The reasons for the specific exclusion are listed in this table, that is one of our clinical study that is submitted to the upcoming conferences of next year's American and European Urological Association. What's the next step of our approach then? We save every single cell stemming from those patients and we have developed this protocol of a very thorough translational analysis in which we examine the clinical features of each individual primary. Plus, we have performed in this specific patient a single-cell RNA sequencing of five individual primaries with a specific approach that we saw in the previous presentation with the special attention, let's say, for the inter-tumoral and intratumoral heterogeneity.

I'm happy to share the first results of this investigation in which we, of course, have investigated the expression. Each color here corresponds to a different primary, the purple one instead is the normal tissue in which we have investigated the expression of HIF1alpha and HIF2alpha cluster genes. Why? The idea, of course, is what happens if I treat these patients with such a drug? Would I expect a consistent response among different primaries or not? Which of course is critical. The beauty of these approaches you're familiar with is that you can basically investigate whatever, you can query whatever marker of systemic agents that you can. For example, this is also the expression of markers of response to immune checkpoint inhibitors, which again, was consistent among the five primaries that we have investigated.

I want to close my presentation with just a glimpse of what we will be working on in the next month. On the left, this is the picture of the first patient derived organoid that we have cultured for one clear cell renal cell carcinoma. Plus, we want to replicate the same approach that we have so far established for renal cancer, only two extra renal primaries, which is going to be basically the focus of our investigation the next month. This is the specimen resulting of one of our patients treated with dronedarone necrosectomy owing to this narrow endocrine tumor that is currently under sequencing now. Thank you very much.