Okay, I will try to talk briefly about the advances in the pathology of squamous cell carcinoma of the vulva and its precursors, okay? Now I have to tell you that some recent advances have been achieved in this vulvar pathology of squamous disease. The first is the recognition that we can see some lesions, intrapetile lesions, that simulate different intrapetile lesions correlated with different etiologies, okay? So I will start with lesions that are DVIN, differentiated vulvar intrapetile neoplasia-like, and also like sclerosis-like, but are actually related to HPV. So in this paper, the authors examined 326 DNA HPV-positive tumors, also with HPV typing and analysis of messenger RNA of HPV and immunohistochemistry for B16. And the conclusive association with HPV in these cases was based on B16 positivity and or RNA positivity, in addition to DNA HPV positivity. Well, less than 5% of the cases had unusual intrapetile lesions. Seven lesions looked like DVIN, five looked like sclerosis, and two lesions had mixed features which looked like DVIN and also like sclerosis. Well, HPV16, B16, and messenger RNA of B6 HPV were positive in three of the seven differentiated vulvar intrapetile neoplasia-like lesions, two of the five like sclerosis-like lesions, and one of the two lesions that had both areas simulating differentiated vulvar intrapetile neoplasia and like sclerosis. Well, B16 was positive in all those lesions that simulated differentiated vulvar intrapetile neoplasia and like sclerosis in tumors that were conclusively associated with HPV. So the pathologist has to be aware of this type of lesions, which can you see, as you can see here, recapitulate like in sclerosis, and also these lesions that I'm pointing here recapitulate differentiated vulvar intrapetile neoplasia. And below you can see the B16 immunohistochemistry in these lesions, all of that, all of them with block positivity, strong and diffuse, and nuclear and cytoplasmic positivity for B16, actually revealing that these are HPV-related lesions and not differentiated vulvar intrapetile neoplasia. Well, we have something about lesions which are actually HPV-related, which have superimposed like in simplest chronicles, and these lesions simulate differentiated vulvar intrapetile neoplasia. So as you can see here, a lot of hyperkeratosis, and also maturation of the lower layers of the epithelium in these lesions, okay? And when you have the B16 immunohistochemistry, you see these are B16 positive. And when you have B53 immunohistochemistry, you see that particular pattern of positivity, which spares the basal layer of the epithelium and stains the middle layers. As you can see here in higher magnification, this basal layer is spared and the middle layers of the atypical epithelium are stained with B53. In this paper, this lesion, which was recognized as a high-grade squamous intrapetile lesion, a classic or usual or undifferentiated vulvar intrapetile neoplasia, which superimposed like in simplest chronicles, had hyperchromasia in the basal layers, the basal most layers. Had a basal-to-full-thickness atypia, apoptosis, and the B16 positivity was reduced or lost in the upper layers. The B53 immunohistochemistry revealed parabasal and middle epithelium, which moderate positivity with sparing of the basal layer. So as you all know, we have HCU, which is the nomenclature of the last consensus, for classic or usual or undifferentiated vulvar intrapetile neoplasia, which is B16 positive, and also can have B53 positivity, but sparing the basal layer. We are, all of you are aware of differentiated vulvar intrapetile neoplasia, which is B16 negative, non-block positivity, and B53 has a pattern of positivity that is strong and continues in the basal layer, extending to the parabasal, to the upper layers. Well, so as I was talking about, we have something that you already know about a variation of differentiated vulvar intrapetile neoplasia, which has basaloid features, which is B16 negative and B53, a pattern that is correlated with mutation, as we will see later on. So this is a HCU-like differentiated vulvar intrapetile neoplasia. It is not HPV-related, and I was talking about HPV-related lesion, which is keratinizing, which is mature, which simulates differentiated vulvar intrapetile neoplasia, and as you can see, it's B16 positive, and the B53 pattern is not strong and continuous in the basal layer. Well, we have some proposals for B53 immunohistochemistry interpretation in the vulva. So when we have scattered positivity, heterogeneous, or when we have mid-epithelial positivity with sparing of the basal layer, possibly this is not related to B53 mutation. Well, when we have diffuse strong positivity, including the basal and parabasal layers, or something more restricted, but with a strong positivity and continuous positivity in the basal and parabasal layers, or we have basal positivity, only basal layer, but strong and continuous, or complete absence of positivity, these are probably related to B53 mutation and related to differentiated vulvar intrapetile neoplasia, not HPV-related, okay? This type of wild-type positivity are most related to HPV. Well, this paper is interesting because three molecular subtypes of vulvar squamous cell carcinoma and precursors were recognized. You can have one that is HPV-positive with B53 wild-type, the other one, which is the opposite, which is HPV-negative, B53 abnormal, mutated, which is the differentiated vulvar intrapetile neoplasia and related squamous cell carcinomas. And also there is a third pathway, which is HPV-negative and B53 wild-type with frequent NOTCH1 mutations. Well, the local recurrence rates of these three molecular subtypes of vulvar cancers and precancers is different. HPV-positive subtype has a lower local recurrence rate, the B53 abnormal subtype has the highest recurrence rate, and the third subtype, which is not HPV nor B53-related, has an intermediate recurrence rate. Well, HPV positivity is an independent prognostic factor for favorable outcome in multivariate analysis in this paper. Well, let me tell you something more about this new precursors of vulvar squamous cell carcinoma. This paper recognized atypical varusiform lesions, which were called atypical varusiform hyperplasia, and also included the vulvar acanthosis with alternate differentiation and varusiform lichen simplicis chronicles. These were compared with 14 HPV-negative squamous cell carcinomas, and this type of lesions have PIK3C8 and ARLD2 mutations, no B53 mutations, and one case of this type of lesions progressed to a B53-mutated squamous cell carcinoma. So this type of lesion, which were nominated as differentiated exophytic vulvar intertelial lesion, had varusiform architecture, abnormal differentiation, no invasion, absence of HPV changes, no significant basal tibia, as would be expected for differentiated vulvar intertelial neoplasia, and the P53 immunohistochemistry is wild type. Also there's a third type of precursor, which is possibly non-HPV related and not related also to B53 mutation. You know about P16 immunostaining, which allows for the accurate classification of vulvar squamous cell HPV-associated and independent carcinoma cases. So in this paper, which had a lattice of 201 tumors, well, in this paper, the P16 sensitivity and specificity for classification of vulvar squamous cell carcinoma as HPV-associated or HPV-independent was respectively 100% and 98.5%. Most discrepant cases in this paper were well-differentiated keratinizing tumors with P16 positivity. And 94% of these tumors were HPV-positive for PCR. So that's important that immunohistochemistry for P16 recognizes very well the HPV-related lesions. We have another paper on this type of P16 immunohistochemistry, which also tells us about prognosis. This paper had 114 vulvar squamous cell carcinomas. They had a morphological multimodal HPV analysis with PCR, DNA in-site hybridization, RNA in-site hybridization, and also P16 immunohistochemistry. Well, in this paper, on multivariate analysis, HPV morphology, P16 positivity, and RNA in-site hybridization positivity were associated with better five-year progression-free survival. So we should routinely report the HPV status in squamous cell carcinoma of the vulva. That's very important. That's recommended in our practice. Well, we also know about HPV or P16 positivity, the association with better progression-free survival and fewer relapses after radiotherapy in squamous cell carcinoma, HPV squamous cell carcinoma treated with radiotherapy. So HPV status is prognostic for this women. When we talk about P16 and P53, and this paper is very interesting because in this paper with 92 cases, stage one, well, these authors had the following conclusions. P16 positive, HPV positive patients were less likely to recur, no tumor-related deaths. Conversely, P53 positive patients in immunohistochemistry were three times more likely to recur and almost seven times more likely to die from vulvar cancer. And also in this paper, the tumor size above four centimeters was related to a four-fold increase in disease-specific mortality. So that's important. Both P16 and P53 immunohistochemistry related to prognosis. Another review article also tell us that P16 positive vulvar cancers had better survival compared to P16 negative, P53 positive vulvar cancers have a less favorable survival compared to P53 negative. And both of these markers may be clinically used for prognostic, of prognostic value in patients with vulvar cancer. Well there's a nice picture of perineural invasion by squamous cell carcinoma of the vulva. And when we talk about perineural invasion, well, we have a nice paper from Mila Salcedo. We also have Kathleen here that tell us about perineural invasion that should be considered a poor prognostic factor and associated with higher stage and also with poor overall survival. Both in univariate and multivariate or multivariable model. Well what to do about poorly oriented and or fragmented specimens that is our real life when we deal with vulvar biopsies. How we measure the depth of invasion in this type of samples. Well I have to tell you an old paper, not so old, telling about the inter-observer agreement for assessing invasion stage 1a vulvar squamous cell carcinoma. Look at these black lines, how different pathologists used these black lines for measuring the depth of invasion. Look, different types of measures, different landmarks in order to measure the depth of invasion. In this paper with 45 cases with depth of invasion originally reported as equal to or below 5 millimeters, 11 experienced gynecological pathologists were involved in this paper. The agreement for diagnosing invasion was only fair, the agreement for measuring depth of invasion and tumor fixings was moderate, and interpretation of the location of adjacent most superficial dermal papillae vary among observers that I was trying to show you in this picture. Well an alternative method to assess the depth of invasion was proposed in another paper. This paper has 148 patients with no lymph node data with figure stage 1b or higher. The median depth of invasion in the traditional method that is depicted in this letter A was 5.5 millimeters versus 3.6 millimeters in the alternative method which is depicted in letter B here. Well 69 stage 1b patients in this paper, 13 were downstaged to stage 1a based on the traditional method to assess the depth of invasion, sorry the alternative method. Now the downstaged patients developed less recurrences and had higher disease-specific survival than patients who remained at stage 1b even after trying to measure the depth of invasion by the alternative method. In a more recent paper, 50 digitally scanned slides of over squamous cell carcinoma with a depth of vision of approximately 1 millimeter were distributed and analyzed by 10 specializes in foreign training pathologists. The depth of invasion was measured with both the conventional and alternative methods. Well the conventional method had a moderate agreement and also the alternative method compared to the conventional method, no notable difference, but there were some pitfalls. Which invasiveness is the deepest one? The presence of invasive growth and where it starts, curved surface in some samples, carcinoma located on the edge of its cell block in some samples, posteration, different measuring methods. So as you see again, oh I'm sorry, as you can see again there's different lines telling us that different observers were using different landmarks in order to measure the depth of invasion. So that's a difficult task. I'm just, I have a few more minutes, just have to tell you about PD-L1 suppression as a favorable prognostic factor in over squamous cell carcinoma. Also as you can see here, the p16 status is also related to better prognosis. And the PD-L1 staining in immune cells, not in cancer cells, was also related to better prognosis. In this graphic you can see in p16 positive tumors, the staining of immune cells with PD-L1 was related to better prognosis. Well in this paper, 84 tumors were analyzed with p16 DNA HPV and markers of immune cells. Well the positivity on cancer cells was correlated to a higher infiltration of CD4 positive, CD8 positive, FOXP3 positive and CD68 positive cells. Well the positivity on perituminal immune cells was an independent favorable prognostic factor for overall survival in this paper. The positivity of cancer cells but not immune cells was more frequent in p16 negative tumors. Well high risk HPV was not correlated with PD-L1 status, that's important. And I guess I will finish by now. This paper is very interesting and that shows that high numbers of activated helper T cells are associated with better outcome in early stage of cancer, irrespective of HPV or p53 status that we were talking about earlier. This is the first study demonstrating association between interrupted T cells and clinical outcome. Our data suggests that abnormal p53 expressed in vulvar splenocell carcinomas mostly are co-tumors, whereas HPV driven vulvar splenocell carcinomas, the p16 positive, are strongly T cell infiltrated and maybe this could explain the better prognosis of this type of tumors, the HPV related ones. Okay. Well, that's it. I'm sorry for the...

squamous cell carcinoma

vulva

HPV

subtypes

recurrence rates

prognostic factors