Good afternoon everyone. First of all many thanks to Dr. Bonaventura and Dr. Arn for inviting

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 1 Good afternoon everyone. First of all many thanks to Dr. Bonaventura and Dr. Arn for inviting me here, it s great to be here and I m going to talk about the pathology of in-situ carcinoma. So what is in-situ carcinoma? I think Dr. Kelley already mentioned in her talk but pathologically that s how we define it. Proliferating cells with cancerous characteristics cytologically but limited to duct or lobules, that means they haven t crossed the basement membrane to invade the stroma, that is what ductal carcinoma in-situ or lobular carcinoma in-situ is. So just to understand pathologically how we diagnose I just put a picture of how a normal breast looks like. This is one of the ducts and this is a lobule and the junction here is called terminal duct-lobular unit. So any pathology that arises it actually starts here, then it gets into the duct that s where the ductal carcinoma in-situ tend to grow. Or it predominantly involves the lobule which is where lobular carcinoma in-situ tend to grow. But all lesions arise at this junction. So this is a high power view of the breast duct, here are those luminal cells, here is some secretions that s what normally happens in a duct, breast duct. And the cells lining the lumina are called luminal cells and the cells on the other side of the lumen are abluminal or stroma facing. These tiny little cells these are the so called basal cells or myoepithelial cells because they have characteristics of both epithelium and somewhat muscle like. And this is what s preventing anything that grows into the duct and here is the basement membrane which is so hard to see on this H&E section. And this is the breast stroma. And this is what happens when someone has ductal carcinoma in-situ all these luminal cells have

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 2 proliferated and dilated this duct so these are all cancerous cells. If the same cell is present here in the fat it s invasive cancer so that s why we call this entity cancer although it s not going to metastasize because it s limited by the basement membrane. Here is the normal adipose tissue of the breast. So the spectrum of in-situ carcinoma you can see presented on the slide, it s a wide spectrum and obviously we can t go through each lesion today. I m going to focus on ductal carcinoma in-situ the 3 grades, and the classical LCIS. Pleomorphic LCIS as Dr. Kelly mentioned it s considered more like DCIS clinically yes. Pathologically it s a different lesion but clinical behavior wise it s very similar to DCIS and it s treated like DCIS at the current time. Clinical presentation most of these lesions are diagnosed on screening mammography because they calcify and these are the calcifications which radiologists are biopsying and we see DCIS in the core biopsy. So that s how these are diagnosed most of the time. Very rarely they form a mass. This is one of the presentative pictures of these abnormal looking calcifications and the corresponding tissue specimen where you see these ducts. This is the area of necrosis, the same here. The pink thing is necrosis because there are no nuclei left which stain blue on hematoxylin and eosin stain and this is where the medius of calcification. The same thing, this is a more longitudinal section of the duct and you have necrosis and then calcification which correspond to the calcification seen on mammogram.

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 3 So there are a number of features pathologists report on when they see DCIs and these are the things that we tend to report especially on a core biopsy. There are additional things we do on resection which I'll come to in a minute. And I'll go very briefly into each one of these. So architectural morphologic patterns they are important for pathologists but prognostically it's one of the least important features that we talk about. It's just giving a name to a thing that we see on the microscope, that's why it's important for pathologists. It helps us also when something recurs how did it look like, that's what it means. And there are a number of different patterns. Apocrine patterns sometimes you see but it's not really a pattern but it's a change in a particular cell and this particular change can be seen in any of the above patterns, so we just say the apocrine differentiation is just a change. The cell becomes more pink looking because it accumulates more cytoplasm and there are some nuclear features associated with it. Cribriform DCIS you may have seen if you have seen the breast reports on DCIS. You will see cribriform DCIS very frequently reported. What it means is these are regular fenestrations within the duct and the cells, monotonous updating cells are linking around it and it resembles the save, like that's the picture of the save. So you can see how closely it resembles. So that's the picture matching you can do to diagnose DCIS, the cells are obviously clonal in origin and looks monotonous. But these are the fenestrations, that's the pattern we call cribriform. Under the high power view very round, sometimes we say cookie cut they are so round and well developed. That's how we diagnose this as DCIS. If you are considering ADH these things are

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 4 falling short, they are not as round and not as monotonous so that's the difference between ADH and DCIS. Here is a picture where you see still cribriform DCIS but then also a duct which is completely filled with these cells and that's the pattern we call solid. This is also solid pattern. Then this is a pattern we call micropapillary because these are tiny little papilla coming out from the duct without a supporting fibrovascular core. And that's the difference between micropapillary and papillary. Papillary have a fibrovascular core within them but micropapillary don't. And as I said, prognostically these really do not matter, it's how to recognize these and diagnose it correctly. This is in contract papillary where you have 10 fibrovascular cores and not to misinterpret this thing as just a benign lesion but to recognize this is a papillary DCIS. This is a high power view of papillary DCIS, you have 10 fibrovascular core lined by these neoplastic appearing cells. The reason I say neoplastic because they look very similar, telling you it of clonal origin. This is another pattern sometimes can be misdiagnosed. This is called flat type because it's just flat. This is an area of necrosis and calcification and the reason it's DCIS there is not much proliferation but it's DCIS because the cytology is bad looking, that means the cells are really big and large, prominent nucleoli, that's why it's DCIS and it behaves like DCIS and it's associated with other recognizable forms of DCIS. That's why this thing is also DCIS. Another example of flat type.

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 5 So now we are coming to the more important part of what we report, grading of DCIS. This is one of the very important features prognostically of DCIS. Pathologists grade them as grade I, II and III, and what we are looking at is how big are the nuclei, what is the chromatin pattern, coarse chromatin, large nuclei, prominent nucleoli, these are all bad features. And what it likely correlates genomically is there is a large number of genomic changes as you start seeing the large cell and ugly looking nuclei. So that's how we divide them. If the cells are only slightly larger or similar to normal ductal epithelium then it's a grade I. And the reason this is called DCIS is because there is a monotonous proliferation of cells. There is clonal evolution, that's why it's a low grade DCIS. If the cells are very large or more than 2 times you call them as grade III and somewhere in between is the grade II. And these are prognostically important. This is an example of grade I, very small nuclei. That's grade II, yes of course it's cribriform pattern but it's a grade II. And here the cells are very large, prominent nucleoli, coarse chromatin, so it becomes grade III. Then necrosis and calcifications are two other things that we report. When present necrosis could be a large big area, central area of necrosis which we call comedonecrosis or it could be just focal and non-comedo type necrosis. And most of the time when you have comedonecrosis calcifications are associated with it. Calcifications are not prognostically important, unlike necrosis which is a prognostically important feature. Calcifications are important to report though because that's how radiologists correlate their findings with the pathologic findings.

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 6 So here are some examples. So there is almost no necrosis here, just tiny little some apoptotic debris. Some necrosis start appearing but not comedo type. But definitely here, this is no question comedonecrosis with associated calcifications. This thing I'm sure there will be a lot of interrupts over variability when pathologists look at it whether to call it comedo or not. But when you have central zone with this much necrosis most pathologists will still call it comedo. So the top two are not comedonecrosis but these two are comedonecrosis. Obviously this is very clear here and there is no necrosis there. This is an example where it's all calcified so a large number of calcifications are present in the background of necrosis. And this is a DCIS where there is no necrosis but still you can see calcification. That's calcification, this one, this one here, this one. So those are the findings we report on core biopsy but when we get resection specimens then these are the other two things we report which are important prognostically: size and margins. And almost all specimen that come to us after someone with diagnosed with DCIS on core biopsy they are oriented by the surgeons, so they are putting a marker clip. This is the deep, that means the posterior margin, and lateral. So once you know the laterality, right or left, you can figure out other margins. So in this case this area with be superior, this is lateral, medial, inferior, posterior and anterior is behind. So and then we ink them with 6 colors because that's how - that's the only way we can recognize which margin is positive or negative when we are examining it microscopically and we have standard inking key.

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 7 After we ink, dry it, we section it. And it's sectioned perpendicular to the longest axis. In this case it was sectioned from superior to inferior resulting in 13 slices. And once you know how big the specimen was, number of slices you did, you can have an average size of about 4 mm thick. And that's the thickness of slice we keep because when you submit the tissue we cannot submit anything more than 5 mm. So most of our slices have to be of this thickness. And if you sequentially submit because it's so hard to measure DCIS because it could be present here, then here, here and here you cannot measure on slide. You have to go by how many sections is present. If you have submitted things sequentially then you can estimate the size of DCIS because grossly it's not visible unlike invasive cancer it's easy to measure the size. But DCIS the only way you can measure the extent or size of DCIS is by sequential method. So margins when we look at things microscopically this is DCIS obviously there are a lot of quarterly artifacts but we can clearly say this is DCIS. There is area of necrosis here and this is blue colored ink and it's going all the way, that's positive margin. When tumor is touching the ink or DCIS is touching the ink that's a positive margin. And when it's negative we just estimate how far this is. Here is DCIS and this distance is 4 mm. And we don't use any fancy things, just a regular ruler you can measure this 4 mm away. So these are the things I talked about, actually they are important, size, margin, how far the DCIS is, side of DCIS, grade and necrosis and it has been shown before, this is so-called Van Nuy's Prognostic Index developed by Dr. Mel Silverstein published in the '90s and it was a great tool where you scored them to give them a score for each of these findings. The lower the score the

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 8 less likelihood of recurrence, the higher the score high likelihood of recurrence. Radiation benefit lowest when the score is low, highest benefit when the score is high but these still do poorly. So they devised this in '95 but then further improved on this they added age as well, so now the score ranges from 4 to 12 for VNPR or Van Nuy's Prognostic Index, and as you can see the recurrence rate is lowest when the score is low and highest when the score is high. Radiation benefit almost nothing when the score is low, when it's intermediate to high score the radiation benefit is there but the high score patients still recur. And as you can see no one, almost no one dies of DCIS because it's an in situ disease. I also wanted to mention that now there are some molecular tests which are being marketed to say we can estimate the prognosis of a particular DCIS whether it will recur or not. One such test comes from Genomic Health, that makes oncotype DX. And they have devised a DCIS score and the way they have done it is using a cohort mostly of invasive cancer, not DCIS. When they devised this or selected the genes they were selected mainly from cases that have invasive cancer and DCIS associated with it. And they chose many of the proliferation genes. As you can see here an estrogen receptor signaling gene like progesterone receptor and GSTM1 which is glutathione (inaudible) gene which is somewhat involved in detoxification. So these genes they have used and have their proprietary formula which calculates the score and gives you a risk of recurrence. They have validated this in this ECOG trial. And if you look at the makeup of the patients who were in this trial most patients were towards the oldest age group, postmenopausal, relatively

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 9 small size DCIS with widely negative margins and almost everyone has been treated with - sorry 30% have been treated with Tamoxifen and almost all are ER positive. So it's a very defined group of patients on which this has been validated. And if you look at their low scores when you look at any risk of DCIS or invasive recurrence it divides in almost more like two groups rather than three groups, but when you are looking at 10 year risk of recurrence it divides them into three groups. And in their publication they have said there is a moderate correlation between DCIS score and the grade of comedonecrosis and weak correlation with tumor size. And the question come sup what we have been doing providing the grade, the margin, the necrosis and all of those features which creates the VNPI is DCIS score better then VNPI? And if you look at 10 year risk of recurrence with respect to lowest category of DCIS or modified VNPI intermediate and high risk it looks like the VNPI divides it into three different categories, DCIS will divide then into more like two different categories. But if you look at the patient population of these two assays that have been developed they are very different. As I mentioned it's older patient, ER positive, DCIS of limited extent, Tamoxifen treated and no radiation but VNPI was validated on cases of all types of DCIS, heterogeneously treated and 40% got radiation. So it's really you cannot compare the two assay at this time. And it seems like the utility of DCIS score is somewhat limited at the current time. And I think you will hear a lot more from Dr. Johnson and Dr. Haley regarding those decisions.

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 10 Talking about differential diagnosis of DCIS there are a number of different lesions that we have to consider to make the correct diagnosis. One is definitely ADH and then DCIS which versus LCIS. So I'll try to show you as many I can. ADH ultimately is atypical ductal hyperplasia. This is basically a continuum of what is ADH versus a low grade DCIS because if you look at if you think clonality is equal to neoplasia then you have to think again because 8 clonality things have been shown in ADH. So that thing, that clonal evolution starts happening at this stage of atypical ductal hyperplasia. But the pathologic definition of atypical ductal hyperplasia is it has some but not all feature of DCIS. So it will start mimicking DCIS but it's no reaching to the point where pathologists will make the call. So there is some degree of subjectivity. The way at least we practice in our group is when almost all pathologists agree it's a DCIS then it's a DCIS. If some but not all pathologists agree it's not going to be DICIS, that's a simple rule. Everyone should agree once you are making that diagnosis of DCIS. So here you can see there is cribriforming but as I mentioned these are not so cookie cut. There is some hesitation in calling it DCIS. Again here there is not as much proliferation. There is some micropapillary but they are not well developed. So this is what we say so-called diagnostic of ADH although the definition is it has some, but not all features of DCIS. Another lesion which should not be confused, flat epithelial atypia, which is a kind of atypical ductal hyperplasia but without the architectural features these are just low grade nuclei compared

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 11 to a flat type of DCIS which have really high grade nuclei. So once you go at high power you can distinguish the two. These are really ugly, nasty looking nuclei which will behave like DCIS. Sometimes - most of the time it's quite easy to distinguish whether the cells have penetrated the basement membrane, that means is it invasive cancer or DCIS? But sometimes DCIS can involve preexisting sclerosing adenosis and it almost looks invasive. So in those cases pathologists use myoepithelial cell markers. I showed you those basal cells or myoepithelial cells, you can use immunohistochemical stains to mark those if you can't see them on H&E, like here it almost looks invasive. But once we did myoepithelial marker P63 which is a nuclear marker for it you can clearly see it's all around the lesion. And a cytoplasmic marker for myoepithelial cell called smooth muscle myosin heavy chain it also marks. So this is DCIS actually involving sclerosing adenosis and it's not - and it's not invasive cancer. So sometimes when we review slides from other institutions once in a while we disagree or there is a discordance between the diagnosis. Sometimes DCIS involves the lobules and if you are not aware of this particular phenomenon then you may not even recognize it. And the only way you can recognize are these highly atypical cells just tracking into the lobular. Do you remember that TDLU I showed you that globular unit but not extracting retrograde into the lobules. And obviously if this thing is close to the margin we will say DCIS is close to the margin or involving the margin because these cells are DCIS cells.

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 12 Sometimes benign looking lesions can mimic cribriform type DCIS. One example is collagenous spherulosis where there is an accumulation of basement membrane-like material and it almost (inaudible) which I was telling you that is cribriform DCIS but I think these need to be distinguished from DCIS. Obviously experience helps. And then you have to go at high power to look at are these the monotonous cell of DCIS or just normal cells or sometimes even atypical lobular hyperplasia can involve collagenous spherulosis making it very difficult to distinguish from ductal carcinoma in situ. In that particular case it was actually atypical lobular hyperplasia involving collagenous spherulosis mimicking DCIS. But we did an e-cadherin stain which is membrane protein that is absent in all type of lobular lesions, atypical lobular hyperplasia, lobular carcinoma in situ which really helps to show that these cells are e-cadherin negative and it's actually ALH, atypical lobular hyperplasia involving collagenous spherulosis rather than DCIS. And sometimes the ducts are full, ti's differential diagnosis is solid type of DCIS or because the cells look a little discohesive, not attached together is it LCIS? In those cases just like in the last case we do e-cadherin staining and also combine with another protein called P120. So when e- cadherin is muted the P120 protein that normally resides in the membrane of the cell comes into the cytoplasm. And here the e-cadherin protein is present in the brown and P120 with red. And as you can see this particular solid lesion there is no e-cadherin which is lost in all lobular lesions as I said. And P120 red is cytoplasmic, so this is LCIS and this is the stain combination we use to diagnose DCIS, sorry LCIS in distinguishing it from DCIS.

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 13 This is another example of LCIS where there is pagetoid extension. These are the native luminal epithelium cells but the cells burrowing underneath are the LCIS cells. That's called pagetoid extension of LICIS into the duct. And using that dual color stain e-cadherin being brown, so normal ductal epithelium, luminal epithelium is membranous brown but then lobular carcinoma in situ showing P120 cytoplasmic expression confirming this is LCIS. So lobular carcinoma in situ is considered a risk lesion, there is a relative risk of 8. That means these patients over their lifetime have 8 times higher risk than someone who does not have LCIS. But there is also data to suggest that it has a precursor role and it's possibly related to the amount of LCIS. The current management of LCIS is it is excised when it is diagnosed on core needle biopsy because there is an upstate weight of about 15%. That means once you excise the area where LCIS was diagnosed and on core biopsy you will see DCIS or invasive cancer in 15% of cases. But when we see this LCIS at the margin or close to the margin on a resection specimen we do not report margins because it's still considered a risk lesion. So there is no point keep going back to remove something which is considered a risk lesion. So that's - this is for classical LCIS, I'm not discussion today pleomorphic LCIS which is treated like DCIS. That means if it's at the margin you try to clear the margin before patient is referred for radiation therapy.

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 14 So this is a slide showing you the precursor rule of LCIS. Here is LCIS and LCIS ILC, invasive lobular carcinoma, is just budding out of it. So that clearly shows this is a precursor lesion for ILC in this particular case. Just a couple of slides for prognostic predictive markers for DCIS. Estrogen receptor, most low and intermediate grade DCIS are estrogen receptor positive. Almost half of high grade DCIS are also ER positive, that we cannot really predict so you have to really do the stain. But high grade DCIS, especially the ones that have apocrine differentiation, that means large cells, eosinophilic, prominent nucleoli those are the ones that are ER negative. PR reactivity, it's somewhat difficult to predict based on the nuclear grade of DCIS. But DCIS with apocrine differentiation is often negative, so apocrine differentiation is inversely correlated with hormone receptors. HER 2 reactivity in DCIS is seen often with high grade nuclei of DCIS, apocrine differentiation and comedonecrosis. However currently HER 2 testing on DCIS is not recommended. No one is treating DCIS at the current time with Trastuzumab based therapy, so HER 2 testing is not performed clinically on DCIS cases. KI-67, it's a proliferation marker and generally correlates with mitotic activity. Again there is no clinical utility at the time for doing KI-67 in DCIS. There is not much data on its usefulness.

PATHOLOGY IN-SITU CARCINOMA, ROHIT BHARGAVA, MD 15 But it's like that it will correlate with DCIS oncotype score because DCIS oncotype score is actually looking at proliferation in high grade nuclei or the molecular grading of DCIS. So in summary, DCIS and LCIS are not difficult to diagnose pathologically but one should be aware of the pitfalls. The reporting of nuclear grade necrosis, size, margin status on resection specimen are important for clinical management of DCIS. Classical LCIS is a risk factor but it's also a precursor lesion. And thank you for your attention.