Everybody, good morning, everybody. Apologies for the late start. I had some computer issues on my end. I am Adrian Suarez, the Director of Surgical Pathology at The Ohio State University. It is my distinct pleasure to introduce to you Dr. Stephanie Scala, who is the Gynecologic Pathology Fellowship Director and the Director of Surgical Pathology at the University of Michigan. She received her medical degree from Washington University in St. Louis, completed her APCP residency and Gynecology Pathology Fellowship at the University of Michigan before joining faculty in 2019. Her major research interest is HPV-independent vulvar neoplasia, and she has published multiple articles on this complex topic. Thank you so much for joining us, Dr. Scala. Before we get started, I should mention a couple of housekeeping items, a recording of this webinar will be available in the IGCS Education 360 Learning Portal within one business day. We encourage you to submit questions via the Q&A feature at the bottom of your screen, and we'll do our best to address as many as possible. Dr. Scala, I will now hand over the webinar to you. Welcome. Thank you. Thanks for inviting me. And let's see. Can you see my screen? I can. All right. All right, so we'll get started on this webinar about the evolving diagnostic criteria for HPV-independent vulvar neoplasia. The objectives for this talk are to understand the past terminology for vulvar neoplasia and the evolving current terminology for HPV-independent vulvar intrapithelial neoplasia. To understand some of the histologic features that pathologists look for for diagnosis of differentiated vulvar intrapithelial neoplasia, which more generically might be referred to as HPV-independent VIN with P53 abnormality. To recognize some of the challenges that we face in diagnosis of HPV-independent VIN with and without P53 abnormalities. To understand rationale for universal ordering of immunohistochemical stains for P16 and P53 when feasible. And to identify other entities in the differential diagnosis of HPV-independent VIN and how this might impact management decisions. So I thought it was interesting to read about some of the history of this field. And if we go back a hundred years, it was written that hardly any disease in gynecology presents so many interesting features for study as leukoplastic vulvitis. While considerable progress has been made in our knowledge of the histology of this condition and its relation to cancer, we are still far from a satisfactory answer as to why and wherefore. And as much as we have learned in the past 100 years, I think that a lot of the sentiment behind this statement still rings very true to this day. In 1971, the International Society for the Study of Vulvobaginal Diseases, or ISSVD, was organized. And in 1975, the ISSVD classification included vulvar dystrophies, such as hyperplastic dystrophy with or without atypia, with ekanthosis, elongation of epithelial folds or Reedy bridges, a variable granular layer, and hyperkeratosis. So this sounds similar to our descriptions of things like DVIN and HPV-independent P53 wild-type vulvar intrapithelial neoplasia, as well as arguably, possibly reactive conditions like lichen simplex chronicus. Vulvar dystrophies also included lichen sclerosis and then mixed dystrophy, which was lichen sclerosis with superimposed areas of epithelial hyperplasia with or without atypia. Vulvar atypia was defined as mild, moderate, or severe, with defining features similar to those that we use for HPV-associated vulvar intrapithelial neoplasia, one, two, and three. In 1976, it was written that we've begun to develop some insight into the natural history of these vulvar dystrophies. And at that time, it was thought that the relationship to vulvar carcinoma may not be as significant as was thought originally. At the time, there was little evidence of a causative relationship between lichen sclerosis and hyperplastic dystrophy. So there was some controversy over classifying both lichen sclerosis and hyperplastic dystrophy in the same category. I think today we can clearly say that lichen sclerosis is a common background for Deven to arise out of. So it sounds like the pendulum for this has been swinging back and forth for quite some time. Some other terminology that has previously been used includes squamous hyperplasia, where there's acanthosis, hyperkeratosis, and nocytologic atypia. And this was commonly seen adjacent to HPV-independent vulvar squamous cell carcinoma. Some will refer to Deven as vulvar intrapithelial neoplasia simplex type. And then among the non-Deven HPV-independent vulvar intrapithelial lesions or neoplasia, there have been terms used, including differentiated exophytic vulvar intrapithelial lesion or DVL, variciform acanthosis with altered differentiation or VAD, and variciform lichen simplex chronicus or VLSC. As you can see here, each of those had different subsets of morphologic features, which currently are being merged into some of the same categories. And we'll talk about some of those changes a little bit later. In the current edition of the WHO, there is a single entry for vulvar intrapithelial neoplasia, HPV-independent. This is defined as non-invasive precursor of HPV-independent squamous cell carcinoma, characterized by atypia of the basal and parabasal keratinocytes in an otherwise well-differentiated epithelium. Acceptable terminology includes differentiated vulvar intrapithelial neoplasia. And then one of the places where this entry gets a bit confusing is that the DEVIL and VAD are listed as subtypes in this category, where acceptable terminology is listed as DEVIN. And I'm not sure that the WHO really intended to say that these other lesions, DEVIL and VAD, are subtypes of DEVIN. I think that was really meant to say those are subtypes of HPV-independent vulvar intrapithelial neoplasia. But admittedly, this is a bit confusing, even for someone who does look at a lot of vulvar pathology. So I would imagine that this is even more challenging for pathologists in locations where they do not commonly see these specimens. There's frequent abnormal p53 staining, but there are also TP53 wild-type HPV-independent intrapithelial lesions, which may have somatic mutations in other genes, such as PIK3CA, NOTCH1, or H-RAS. Listed as an essential criterion for HPV-independent VIN is atypia of the basal layer. And if we really dig into the literature that is referenced in this entry in the WHO, I think that this is a little bit misleading as well. So for DVIN, or the HPV-independent p53 abnormal or mutant vulvar intrapithelial neoplasia, you will see atypia of the basal layer. However, for these other lesions, such as DVIL and FAD, their definition does not include basal cytologic atypia. And in the WHO, they describe the clinical appearance of these lesions as thick keratinized plaques or erosive erythematous macules or plaques. So a very variable appearance is possible. Distinguishing between TP53 mutant and TP53 wild-type lesions likely has prognostic importance, particularly when we look at literature about vulvar squamous cell carcinoma and the three prognostic groups that have arisen, including HPV-associated vulvar squamous cell carcinoma, HPV-independent p53 mutant vulvar squamous cell carcinoma, and HPV-independent p53 wild-type vulvar squamous cell carcinoma, where the HPV-associated carcinomas have the most favorable prognosis. The p53 mutant cancers have the worst prognosis, and the p53 wild-type HPV-independent cancers are in some intermediate category. And those likely represent a heterogeneous group of various mutation types, various morphologies. And I don't think that we really have a great understanding of everything that makes up that group at this time. Among p53 wild-type acanthotic vulvar lesions, there are a variety of proposed terminologies. Coming from ISSVD, we have a recommendation for use of the term vulvar aberrant maturation, or VAM. And that would include many of these other entities that have been described, such as D-vul-VAD, burisiform lichen simplex chronicus. And some of the implications of this terminology, as opposed to terminology that has been proposed by a group of gynecologic pathologists, HPV-independent p53 wild-type burisiform acanthotic vulvar intraepithelial neoplasia, is that the vulvar aberrant maturation does not commit to defining these lesions all as being neoplastic. It leaves some uncertainty, some flexibility for consideration that these might, in some cases, be a reactive process. This might be in transition to neoplasia, but we're not, in all cases, completely sure that this is a neoplasm or a precancer. Either way, both of these entities sort of lump all of these other HPV-independent p53 wild-type lesions into those groups. And it seems to be a pretty widespread consensus that there doesn't seem to be a need to break this group down into specific categories, like D-vul-VAD, when we're not sure that there's really any biological difference between these. Differentiated vulvar intraepithelial neoplasia arises in the background of lichen sclerosis or lichen simplex chronicus. And as you may know, the diagnostic criteria are somewhat subjective. Those criteria include abnormal maturation or keratinization, which at a very late stage when this is transitioning into a carcinoma may include keratin pearls, but more often will include a more pink or hyper-eosinophilic appearance to the keratinocytes, increased cytoplasm throughout the whole thickness of the epithelium. We can see acanthosis or a thickening of that epithelium, elongated and sometimes anastomosing reedy ridges, perikeratosis, so hyperkeratosis at the surface with retained nuclei of those keratinocytes, and atypia in the basal layer. We'll talk a little bit more about the variety of types of atypia that can be seen in D-VIN and how that can be a challenge for pathologists as well. More and more, most pathologists are starting to require aberrant staining of P53 in order to consider an HPV-independent vulbar intraepithelial neoplasia to be truly D-VIN, meaning that higher risk pre-cancer lesion. P16 is typically not block positive since this is an HPV-independent neoplasm or intraepithelial neoplasm. However, in very rare cases, P16 may have a block positive appearance in combination with aberrant staining for P53. And in that case, the reported cases that have been sequenced did indeed have TP53 mutations. In this bottom picture, we can see an example of a P53 stain with strong uniform intensity in the basal layer, and then some extension of fairly uniform, moderate to strong staining in some of those parabasal layers. This is another example of abnormal maturation or keratinization, where again, there's a very pink appearance to this lesion. Basal atypia, in some cases, will be a similar type of atypia that we're used to looking for in the HPV-associated vulvar intraepithelial neoplasia. So the sort of high-grade squamous intraepithelial lesion type atypia. There is some hyperchromasia or a more blue appearance to some of these nuclei from low power. In this case, it's extending up at least a handful of cell layers past the base. There's variation in nuclear size. We have some apoptotic debris and mitotic figures here. And another feature that is often talked about for Deven is prominent intercellular bridges, which we can also see here. That does have overlap with what we call spongiosis, a feature of some dermatoses. However, in this particular case, there doesn't seem to be a lot of inflammation within the epithelium. So we might feel a bit reassured that these prominent intercellular bridges are more a feature of this vulvar intraepithelial neoplasia than of a spongiotic reaction. In other cases, the type of basal atypia is very different from what we're used to looking for with HPV-associated neoplasia. Another type of atypia that has been described in Deben is sort of a cleared out or vesicular chromatin with one to multiple prominent nucleoli and nuclear enlargement with nuclei three times the size of a lymphocyte. I think here we can see some variation in the size of these nuclei. Some of them are more in the range of two times the size of a lymphocyte. But in any case, when in many locations in the body, we tend to think about nucleoli in association with inflammation, particularly as more of a reactive feature. This can be one huge reason for disagreement among pathologists, where some will see reactive atypia, others will see subtle basal cytologic atypia. Without really correlating with either the TP53 mutation status of this epithelium or IHC, if we are able to interpret that well, this can be a real challenge. Elongated reedy ridges are another one of those common features. In this case, we can also see very prominent hyperkeratosis, as well as perikeratosis with routine nuclei. This example does have a fairly prominent granular layer here as well. Getting into TP53 immunohistochemistry, there has been some great work done in Vancouver correlating the actual TP53 mutation status with the IHC staining patterns for P53. While this figure in some ways makes it seem straightforward, I have to say in practice, it's not always as easy as this might lead us to believe. But we'll walk through some of these patterns. The two wild-type patterns that have been identified are scattered cells, variable intensity. Here, we see some scattered, mostly basal keratinocytes with staining for P53. Here, that intensity of staining is very variable. The main thing that pathologists should be looking for in interpretation of the P53 is going to be the uniformity of moderate to strong staining. Even though there is also guidance saying we want 80% of these basal keratinocytes to be staining positive, the more important thing to focus on is uniform moderate to strong intensity in that number of basal keratinocytes, rather than just seeing that number of basal keratinocytes staining, which is not uncommon. There is some literature that has reported that P53 staining can be increased with topical steroid use. You might imagine if we're not focusing a lot on that uniformity of staining and the stronger intensity, the vast majority of these patients are likely using topical steroids. It stands to reason that a lot of these patients might have a lot of P53 staining of the type, but isn't really what we're looking for to make a diagnosis of DEBEN, and that can be a huge challenge. The other wild-type pattern that's been described, at first glance, might look a lot like this parabasal diffuse overexpression that is one of the mutant patterns, and for many years, I think that most people did interpret this mid-epithelial staining with basal sparing as being a mutant pattern of P53. However, this black arrow points out that the basal keratinocytes are actually negative for P53, and this is the pattern that we will most classically see in HPV-associated lesions. In our H cell, this is the pattern that we would expect to see. Mutant patterns include basal overexpression, which doesn't look entirely dissimilar to the scattered wild-type pattern, depending on how many of these cells are staining and what kind of intensity we see. In fact, there has been literature saying that we can see what can be interpreted, at least as basal overexpression, in a fair number of spongiotic dermatoses in lichen sclerosis and lichen simplex chronicus, and therefore, this is the pattern that's a bit more difficult to be sure about based on the IHC. If you're lucky, you'll have cases that have this really great, uniform, very strong parabasal diffuse overexpression, and in those cases, we can feel good that that really is indicative of a TP53 mutation. There is a mutant null pattern as well, so all of these nuclei are negative for P53. There can be some challenge here as well in terms of a very low level scattered wild-type pattern might raise concern for an absent mutant pattern. Sometimes you can have very weak staining in some cells, and as long as that's weaker than the adjacent normal epithelium, that's still compatible with an absent mutant expression pattern, and then sometimes you can also see the inflammatory cells extending into that epithelium, which will still show staining for P53, and that might make you back off from considering something to be a mutant pattern when, in fact, the actual squamous cells are negative. Cytoplasmic staining is a much less common mutant pattern that can be seen. So, all of the time spent on this, mostly just to say that even though P53 staining can be very helpful, it's still very challenging to interpret, especially in comparison to other situations like with endometrium and ovary where we can have a much higher level of confidence in interpreting a pattern as mutant, but given the maturation-dependent degradation of P53 in the squamous epithelium, this is really sort of a different game, and I have done some collaboration with Ling Hong in Vancouver, looked at her training modules of P53-IHC and known mutation status of TP53, and through looking at a lot of these cases of hers, I've found that my threshold for interpreting a pattern as being mutant, which I thought was already high, it seems that my threshold was actually too low, so it really seems even more complicated than I had appreciated, say, a year ago. So, it emphasizes the importance that if anything seems clinically off about a pathologic diagnosis or a situation, it might be helpful to get the opinion of a pathologist who is more familiar with the spectrum of changes we can see in the vulva. As sort of alluded to by the P53 staining patterns, we can see examples of H cell with morphologic features mimicking deep-in, and I think for most pathologists, if you showed this image of a thickened epithelium in the vulva with hyperkeratosis, a very pink appearance, some cytologic atypia near the base, and some of this washed-out stromal homogenization that looks like what we would see in lichen sclerosis, most of us would probably say this looks like it's most likely going to be a deep-in. However, here we can see a block-positive P16, a P53 that is mid-epithelial with basal sparing, and then high-risk HPV in situ hybridization, which is very positive in those basal keratinocytes. Some of the initial publications really emphasized that when you have more severe atypia or full-thickness cytologic atypia in this kind of context, you're more likely to be dealing with H cell, and I did think that for a while at first, but of course, if you give it enough time and enough biopsies that you look at, you'll see that there can be more severe and more full-thickness cytologic atypia in deep-in that truly does have P53 mutations as well, which just really highlights the importance of using immunohistochemical studies when available to help ensure accurate classification. I think especially in academics, there can be a bit of a tendency to try to prove that we are so good at interpreting the morphology of a lesion that we don't need these immunohistochemical studies, and that may be the case, and sure, in many cases, can we predict what the stains will do? Yes, but there are cases where we would be surprised by the staining, and it has a big implication on clinical management, and I think that the more we can admit that we might not always be right when we don't use these ancillary studies, the more that we do them, the more we can learn which cases we need to be applying those to, so this is sort of an involving area as well. In these H cell lesions with morphologic features mimicking deep-in, about a third of the cases will also have coexisting lichen sclerosis, and the average age is in the 60s, so clinically, given the age and the lichen sclerosis and also the morphologic appearance in many of these cases, it does seem that it would be more compatible with deep-in. However, this is H cell, which has a more favorable prognosis, doesn't necessarily need to be completely excised, etc. The European Society of Gynecological Oncology, ISSVD, European College for the Study of Vulval Disease, and European Federation for Colposcopy came out with some consensus statements on pre-invasive vulvar lesions where they talked about deep-in as something where conservative excision with negative margins and continuous follow-up are recommended, and they very strongly discourage medical treatment or ablation for deep-in given the higher risk of progression to vulvar squamous cell carcinoma, which typically will be in a much shorter timeframe, say like one to two years, in comparison to the longer time that we have until cancer develops out of an HPV-associated lesion. Because deep-in does arise out of dermatoses, treatment-resistant dermatoses need to be carefully inspected, and punch or excisional biopsies should be performed of any suspicious lesion. Multiple biopsies should be done when there's variation in appearance or multiple lesions. And as a pathologist, I have to throw in there that it is great that they emphasized punch or excisional biopsies because if we don't get to see the full thickness of this lesion, we can't evaluate for basal cytologic atypia, we can't evaluate for the possibility of any unexpected stromal invasion, and it really just can't be overstated how helpful the punches are. I mean, even if we do see invasion, if we don't have a punch biopsy, it's more likely that we'll have tangential sectioning of that biopsy and be unable to really provide a depth of invasion if we can confidently say there's invasion at all. Another recommendation for deep-in is that increased cancer risk is reduced by treatment of lichen sclerosis with high-potency topical corticosteroids, and those high-potency topical corticosteroids should be given even after excision of deep-in to help reduce the risk of progression to carcinoma. Steroid use, they say, should be continued even when asymptomatic, at least weekly, and there should be lifelong, every 6 to 12 month checkups in order to ensure that more deep-in lesions or cancers are not arising. On the other hand, for each cell, careful examination of the whole anogenital area, which is at risk for HPV-associated dysplasia. These patients should be asked about possible symptoms of anal squamous cell carcinoma, and here they do say that excisional or ablative procedures can be used. For any ablative procedures, biopsies should be done to exclude unexpected invasion, and again, I'll make a plug here to say that we really do want to see punches or excisional biopsies if we're able to reliably tell you whether or not there is invasion. I imagine it can't be pleasant to receive a report where we say there's tangential sectioning, we're not sure if this is H cell or if it might be a cancer, doesn't feel good to write the reports, probably doesn't feel super helpful to receive them, and having those excisional or punch biopsies is a good way to avoid that. Patients with positive margins for each cell can be followed without immediate re-excision if there's no visible residual lesion, medical management can be considered, and HPV vaccines may reduce recurrence. So given these different sort of levels of concern and follow-up, it would make sense that we want to be as sure as we can when we're classifying something as HPV-independent or HPV-associated. So we'll quickly run through some of these other things. One of the biggest questions for the HPV-independent p53 wild-type acanthotic lesions is where do we draw the line between reactive or inflammatory and neoplastic? As I brought up earlier, the ISSPD, Difficult Pathologic Diagnosis Committee, argues that these are lesions rather than neoplasia, and we don't always know whether this is a response to inflammation or to scratching or whether this is a neoplastic transition. They argue that not calling these things VIN allows for individualized management based on assessment of the neoplastic versus surgical risks. They do agree with separating these from DVIN where we're defining that as being a p53 mutant or aberrant p53 IHC in an HPV independent VIN and close follow-up is warranted. Whatever you want to call these lesions, they may or may not show a background of lichen spherosis or lichen simplex chronicus. They too tend to be clinically discrete lesions in patients over 60 years old. Most of the recommendations I've seen are to consider excision with negative margins versus close surveillance with follow-up biopsies. Again, this is just kind of going through the spectrum of what these things have been called in the past. Of note, there should not be a p53 abnormality in these lesions. And the literature about these talks about absent to mild basal and parabasal atypia. I will say another level of complexity comes in to say the lesions that have cytologic atypia otherwise have similar features but do not have evidence of a p53 abnormality. There's not as much information about what happens in those cases. If there is really prominent cytologic atypia, do those behave more like D-VIN? Do they behave more like this VAM or HPV independent p53 wild-type VABIN? So, in an ideal world, we really have multidisciplinary care for these patients as much as possible. Clinical photos, multiple biopsies, good communication about previous diagnoses and history and treatment are really important. When possible, we really want to use p16 and p53 stains because we cannot always distinguish these things based on routine microscopy. And this becomes particularly important when we're biopsying treatment-resistant lesions within lichen sclerosis, suspected D-VIN or HPV-independent lesions, or presumed H cell in women older than 45 years or women who also have LS or LP, or when there's not response to laser or imiquimod. And because there can be some differences, more aggressive resection for D-VIN or ablative procedures for HPV-associated things, we don't necessarily want to aggressively excise H cell, but we also don't want to misdiagnose D-VIN as H cell and think that it is not as high risk as it is. So, communication is very key. Some other pitfalls include just lichen simplex chronicus, a response to this chronic itch-scratch cycle. You see thickening of the epithelium, elongated reedy ridges, hyperkeratosis. Hypergranulosis is an important finding that in some cases might help us to, if there's a D-VIN that has a similar appearance but lacks a granular layer, we know that we shouldn't be blaming that on just the mechanical rubbing for lichen simplex chronicus. But admittedly, it can be very hard to tell the difference, and sometimes you'll see lichen simplex chronicus superimposed on other things, like these dermatoses can be itchy and they get scratched, and then you have a combination of these features. The other thing that can be commonly missed, if it's not thought about, is infectious causes. This case was sent in as a consultation total vulvectomy for a reported biopsy result of vulvar squamous cell carcinoma. By these green dots, you can see something that appears that it could be invasion. We've got thickening, we've got elongated reedy ridges here, a little bit of a more keratinizing sort of paradoxical maturation appearance, but tons of chronic inflammation in the dermis with numerous plasma cells, a lot of intraepithelial neutrophils, so we decided to do a little bit more of an infectious workup on this case. There had been a GMS stain done to look for fungus, which was negative. However, we did a spirochete stain to assess for syphilis, and this was blazingly positive. So again, a lot of these features may look similar to cancer or to devin, but in the setting of intense dermal chronic inflammation and intraepithelial acute inflammation, or a variety of different dermatitis patterns, it's really important to consider syphilis. If we don't consider it, we'll never be able to confirm that that's what is happening. And another similar example with tons of inflammation, but in this case we see some cells that are kind of sloughing off the surface, multinucleation, margination of chromatin, so it's just sort of clearing out the center and going to the edges of cells, and these are features that we see with herpes infection. In some cases, we may have something, a hypertrophic lesion of herpes in the vulva rather than the typical ulcer, and in these cases, pseudoepitheliomatous hyperplasia may mimic invasion, particularly in immunocompromised patients. There can be cytologic atypia, there can be mitotic activity, but we're really looking for those viral cytopathic features, and of course, confirmatory HSV staining can show us that this is indeed herpes. These are my references, and I'll take questions. Thank you very much, Dr. Scala. So, we have received a few questions via the chat, so I'm gonna read them to you. First one is, do you need p53 immunohistochemistry at the margin of HPV-independent squamous cell carcinoma? How would you explain that when reported to the clinician? That is an ongoing question. So, there is a clinical trial in Australia, New Zealand, and Canada, the STRIVE trial, where part of that is performing p53 IHC on the margins of these resections and trying to excise those p53 abnormal margins, whether or not there's morphologic evidence of necessarily something that most people would consider a devin. And there is a decent amount of literature saying that these devins and cancers come out of these p53 abnormal fields of dysplasia, and when those extend to the margin, there is a higher risk of recurrence. And at least based on some of the literature from the group in Vancouver, there is a faster recurrence with actual morphologic devin along with that IHC abnormality at the margin. But p53 abnormalities at the margin do have about the same recurrence risk. It just takes a little bit longer than if you can morphologically see that lesion. I think that's something that deserves further study. And on top of that, I think there's still a question of, even if we are reducing the risk of recurrence, we need to balance that with, say, the area that we're excising. We don't necessarily want to be all the time super aggressive about excising everything. So how much of a clear margin do you need, given this sensitive area? I think that there's a lot of interesting discussion that will likely come out of that in the near future. Right. And also what happens if your re-excision itself has a p53 abnormal margin? Are you going to continue? You know, right? So, yeah. Yeah. It's like, to some extent, is this similar to excising cancers on sun-exposed skin of an older patient, where if you keep trying to excise to clear margins for any of these lesions, you might just get into another field of dysplasia and then try to clear that, and then there's no more epithelium left. Right, right. Another question, is there any evidence that p53 mutations that result in overexpression cause different disease when compared to no p53 mutations? I'm not entirely sure whether that has been heavily investigated, but that could be an interesting question. Okay. And a question from me, what do you see as the most significant challenges for pathologists in low resource environments when it comes to like, you know, implementation of new systems for vulvar pathology? Yeah, I think even in very, like, academically-based, resource-rich conditions, this is incredibly challenging. So, definitely in most places, there will be, in most places, there will be, you know, kind of, I don't know. On one hand, it seems like it could be much more challenging to not necessarily have all of the same tools to rely on, but I guess at the same time, if you don't always have these tools to rely on, might that help you to sort of see more commonly which morphologic changes might lead to different kind of clinical courses? I don't know, but that is a great question in terms of using p53 to decide, is something deep in or is it not? And I think one thing that would be really interesting is to see, is it always the p53 mutations that make the difference, or could we go partly based off of the degree of cytologic atypia? Like, are cytologically atypical HPV-independent VIN without p53 abnormalities similarly high-risk to DVIN? And if so, might that be a clinically meaningful way to stratify these groups as well? Actually, a related question that came in the chat is, should we grade DVIN as high-grade and low-grade? Yeah, so by convention, at least at this time, all DVIN should be considered to be high-grade, regardless of how subtle the cytologic atypia may be. But with how much things kind of swing back and forth over time, and we'll say that this is how it should be one day, and then 10 years later, we're like, no, actually, the classification before that was more correct, so let's go back to that. I would say at this time, no, but especially as we understand more about these HPV-independent VIN without p53 abnormalities, and whether or not the degree of cytologic atypia is meaningful, or to bring in a whole other caveat, like, is there clinical significance to HPV-independent VIN that has a morphologic appearance more like H cell? You know, might there be a role in the future for some grading of the cytologic atypia in these cases? I can't confidently say that there wouldn't be, but at the present time, they should all be considered high-grade. All right, here's another one. So, what is the significance of p53 abnormalities in Likens sclerosis? That's another great question. So, I think that most of the literature looking at p53 abnormalities in Likens sclerosis doesn't necessarily have a lot of clinical follow-up to be able to say for sure. The other caveat to that being that interpreting p53 in vulvar skin or vulvar mucosa is very challenging, and how much can we rely on just, like, stated interpretations of p53 if we're not able to look at it ourselves and see, like, by current criteria, would we be considering this abnormal or would we not? It's possible that there may be some differences if we got very rigid about our interpretation of that. I have a project that I've been working on with one of our residents interested in GYN pathology where, at least based on our original interpretation of the p53 stains, we thought that it was pretty common to see equivocal or what we thought was mutant pattern p53 in these, but for everything that we thought was equivocal or aberrant by IHC, we did next-generation sequencing and looked for mutations including p53, and the number of those that truly had p53 mutations was much, much lower than what we were guessing based on the IHC, and at least based on that, we were looking at both a group with benign follow-up for two to three years and a group that had HPV-independent vanin follow-up, and the only cases we saw true tp53 mutations in were cases that did end up progressing to devin, so I think there's a possibility that there is something there, but I think it's very much complicated by the fact that interpretation of this stain is very difficult in this context. Mm-hmm. All right. Well, that is all the time we have for today. Thank you very much, everybody, for attending, and thank you, Dr. Escala, for sharing this magnificent, fantastic talk. A reminder, a recording of today's session will be available on the IGCS 360 Education Learning Portal within one day. We wish you all health and safety. Thank you so much. Thank you.

HPV-independent vulvar neoplasia

terminology

VIN

histologic features

immunohistochemical stains

P16

P53

accurate classification

multidisciplinary care