Beyond the Slide is dedicated to exploring diagnostics in oncology.

This is the first episode in the Ovarian series, focused on Tumour Testing in Ovarian Cancer.

Today we’re talking about obtaining optimal specimens for tumour testing.

Hosted by Rachael Babin, from The Oncology Podcast, and joined by Professor Beena Kumar, the Program Director of Pathology at Monash Health, Victoria, and Professor Andreas Obermair, who specializes in surgery for gynaecological cancer at the Royal Brisbane and Women’s hospital in Brisbane, Australia.

We hope you enjoy listening.

 This podcast has been commissioned and funded by AstraZeneca. This podcast is intended for Health Care Professionals only and contains discussion of AstraZeneca Products. The thoughts and opinions expressed are the speaker's own and may not represent those of AstraZeneca.

TRANSCRIPT

[EPISODE]

Welcome to Beyond the Slide. An AstraZeneca podcast series, dedicated to exploring diagnostics in oncology.

Today we’re talking about obtaining optimal specimens for tumour testing in ovarian cancer.

I’m chatting to Beena Kumar and Andreas Obermair.

Professor Beena Kumar is the Program Director of Pathology at Monash Health, Victoria. Professor Andreas Obermair specializes in surgery for gynaecological cancer at the Royal Brisbane and Women’s Hospital in Brisbane.

Today’s episode is a fascinating dive into the nuances of ovarian cancer tissue specimens, covering neoadjuvant chemotherapy, optimal timing and tissue stewardship. There are many practical insights that will resonate with the broader ovarian cancer MDT.

You’ll find links to the resources, bios and Twitter handles in the notes on our website oncologynetwork.com.au.

This podcast has been commissioned and funded by AstraZeneca. This podcast is intended for Health Care Professionals only and may contain discussion of AstraZeneca Products. The thoughts and opinions expressed are the speaker's own and may not represent those of AstraZeneca.

This is Rachael Babin and this is Beyond The Slide.

As you understand, this has a major impact on the work volume in the laboratory. From the registrars who are cutting the specimens, to the scientists who are making those blocks and cutting glass lines, and the pathologist who instead of looking at five slides is probably looking at 10 slides to make a diagnosis. The morphology of the tumour is always better preserved in a specimen, which is sent to us for a primary diagnosis. This is primary, before debulking specimens, before chemotherapy.

Now, neoadjuvant chemotherapy causes fibrosis and adhesions in the peritoneal cavity. It not only interferes with the preoperative evaluation of the tumour spread, it also impacts the morphology and the microenvironment of the tumour. The amount of tumour DNA which can be extracted in a pre-chemo sample is always of better quantity and quality as the tissue has less background noise due to the inflammatory cells and the fibroblasts which cause the fibrosis.

When we come to talk about the tumour quantity, the tumour quantity is definitely less in post-chemo specimens when compared to a lesion which has had no chemo intervention. Obviously, after chemotherapy, there are increased number of macrophages and tumour lymphocytes. Also, there are a lot of necrotic tissue, which impacts on the quality of DNA, which is extracted for any molecular testing.

We are now able to achieve much better patient outcomes without compromising prognosis, and that is fantastic. I can remember how stressful it was in the old days to treat patients with upfront surgery where you couldn't really do a good job. But with that came some challenges. If I remember, when we started neoadjuvant chemotherapy, the only challenge that we had is, we needed to get a tissue diagnosis, right? So you just stick a needle in, and got some cells, and that was that. And we did some immunohistochemistry, and we had what we wanted.

But at the same time, this field of molecular genetics came out and emerged. Now, the requirements are, of course, a lot more advanced, and a lot more sophisticated than in the old days where we just needed a tissue diagnosis or a cytology diagnosis. So we are getting tissue or cells, and almost always, they will be good enough to establish a tissue diagnosis and establish the diagnosis of ovarian cancer. But now, the demands are a lot higher, so we need to cater for the demands of the molecular needs because they're so important, and they have so far-reaching patient impact. That has been a little bit of a challenge.

But it's probably not so relevant in the setting of post-chemo, because by the time the tumour has disappeared, the patient probably doesn't have any ascites. But in a primary tumour setting, it's actually a good tissue sample. Just to summarize, well-preserved, adequate quantity is what we're looking at.

Now, I have done some investigations on the adequacy or inadequacy of the tissue samples. I would like to give credit to my colleague, Dr. Margaret Cummings from Pathology Queensland who actually obtained these samples. At RBWH, we looked at adequacy or inadequacy in small versus large tissue samples. We pulled 49 samples, and from the 49 samples, 16 were small biopsies. From the 16, five were inadequate. That comes down to 45%. In other words, a small biopsy such as a core biopsy is inadequate in our experience, in 45% of samples. Whereas, in the large biopsies, so there are 33 large biopsies, there were only three inadequate. That is about 10%.

Then we also looked at – and you can imagine, that is a massive difference. We then looked at cytology specimen. We submitted for molecular testing 12 cytology specimen, and two were inadequate. That is 17%. I then thought, "Oh, this is really interesting. The difference of 45% inadequacy versus 10% inadequacy." That's massive, right? Because Rachael, imagine, these women line up for a core biopsy through radiology just for days and weeks, and then they'll wait for the results for days and weeks. Then we get a result of 45% inadequacy, which causes me a great deal of frustration, so I went back.

I credit Victoria Beshay from Peter Mac, because she pulled the most recent private data that I had. The first data that I've given you are from Margaret Cummings from RBWH. The second data are from private rooms that are submitted to Peter Mac. The most recent, 63 samples or 63 patients, there were only three inadequate. That is a 5% inadequacy, right?

Honestly, I can't remember when I had the last time an inadequate sample. My inadequacy, a percentage of 5% probably reflects that, because – I am a little bit biased by these results, because I'm saying like what I've felt is the right thing to do, according to the data that I have available today, suggest it is the right thing to do. To first of all, to take a large sample, and ideally take it before you start the chemotherapy.

I think more tissue is always better, but there's some reason why they can only do a core biopsy for whatever reason, then I think having a pathologist, or a scientist with the radiologist and assessing it on-site, and telling them that they are in the lesion, and they have taken out tumour. That has helped us in our practice. We do it for pancreatic cancers. We do it for lung cancers. Something like that could be done. But then if somebody can do laparoscopy, and give us more tissue, that's the best outcome for us.

That is a bit difficult to watch, and the amount of inadequate samples that I achieved in some places. I would encourage really, all department heads and interested gynaecologic oncologists to enquire with their pathology on it, what their inadequacy sample is because they would not even know very likely. That's what I would say.

Look, honestly, there may be even circumstances where we just can secure and confirm a diagnosis of ovarian cancer and we may not have molecular data. In some cases, we just need to move on. But we should remember that ideally, we want molecular data because they're so important. They just really help with decision-making, they help with expediting the patient's treatment, and personalise the treatment so that the patient has the greatest benefit from the treatment that she's receiving.

Just to add to what Andreas just said. In the public setting, what happens is, quite often, registrars end up doing any biopsies. That's because, there's no understanding that this is a very precious sample. You may or may not get tumour. In those circumstances, what happens is the registrar, or very young radiology consultant is trying and probably does not get a good yield. That's because they probably don't understand that this is so important for molecular testing. They don't have the knowledge base, or they are not tuned to the molecular tests, which have to happen after a primary diagnosis of cancer is made. I think that's the main problem.

I think when we have all these forums, or whenever there is an advance in the genomic medicine or personalised medicine, I think it's very important to involve the radiologists so that they understand where we are coming from. Separating the cores, yes, it is important, but I must reiterate that formalin-fixed tissue, and paraffin-embedded tissue is good enough for molecular test. Is it really important for the radiologist to put them into different containers? Yes and no. The advantage of being in different containers is, one can be primarily for histology and immunohistochemistry. The other container can be marked that this specimen is specifically for molecular testing.

That way, what happens is, the molecular sample is not touched for anything else, for immunohistochemistry. It's not cut again and again on the microtome and you lose tissue. So that is exclusively kept for the molecular tests. But I think, for this, the communication is very important from the surgical team, to the radiologist that this is what is required, and that has to percolate on to the pathology department. I think that multidisciplinary approach for these kinds of precious samples is very important, is what I think.

I saw a patient recently, and this is just gut-wrenching basically. But we should get a specimen at a the time of baseline before chemotherapy is started. Obviously, when you have a sample, a primary cytoreduction, then you have typically a very large specimen. When you do a laparoscopy before neoadjuvant chemotherapy, you also have a very large specimen. There are instances when you collect the specimen at interval surgical cytoreduction, where I'm thinking, well, I certainly had the experience that you get suboptimal tissues, and maybe no diagnosis. That is, for example, if a patient had a complete pathological response, sometimes you get that a patient basically had three cycles of neoadjuvant chemotherapy and all tumour just goes away. You have no sample anymore. If you waited until then, well, that is very unfortunate. It's fortunate for the patient, but very unfortunate, at the same time, because we don't have a good sample. Or if the sample is just inadequate, and there is a lot of fibrosis, then that is also very difficult then for molecular pathologist to get an appropriate sample.

Quite often, we see that it is a small bucket with a big ovarian tumour just squashed into the bucket with very little formalin, and that's going to just ruin the morphology, the immunohistochemical profile, and also the molecular analysis. It's good to fix the tissue at a lower temperature rather than room temperature. But this is not always possible because where you find a fridge to keep all your specimens in. It takes though – low temperature - takes more time. I think it's very good for molecule analysis, but I think we have to be practical there.

Then, looking at histology and cytology specimens, cellblocks are good. They are as good as formalin-fixed, paraffin-embedded blocks. But in our experience, we've had good DNA content out of smears, which are made from cytology specimens. The air-dried smears, you just scratch the cells out of the slide, glass slide and try to extract DNA out of that, it's fantastic results. We've had very good experience with that. In this journey, I think as we just discussed, there should be a good collaboration between the surgeons, the radiologist, the surgical, and the molecular pathologist, and the clinical oncologist as well.

That communication, and that ownership is very important. Everybody is responsible for that specimen, that specimen ownership has to start from the radiology department or the surgical theatres, right up to pathology. The other thing is, it's very important to have the tumour content. Once the block is made, slides are cut, you've made your primary diagnosis, how do you preserve tissue at that stage? For example, in our practice, any core biopsy, which comes to our department is really treated as a precious specimen. Few of the pathology departments cut into the blocks, and they get three levels to start off with. We don't do that. We just get one level of section, and we look at that morphology, and we say, "Yes, it's tumour, and these are the immunos we need so that we conserve and preserve our tissue."

Similarly, some departments also, once they cut sections on day one, before the diagnosis is made, they also cut some extra sections for immunohistochemistry. Whatever you can do to preserve, because each time you put the paraffin block on the microtome, which is the instrument we use to cut the block, you have to trim it, and you lose a lot of materials. If you can avoid mounting that paraffin block again and again on the microtome, that's a very important piece of practice or safe practice, which we all have to adhere to. Once we've done the diagnosis, we've come up to the molecular stage, I think it's very important that an anatomical pathologist who's trained in molecular pathology, who understands the importance of the tumour content, has a look at the slides, and gives you an accurate estimate of how much – what is the percentage of tumour on that slide. Anything above 50%, you can easily process it for molecular testing. There will be adequate DNA. But anything below 20% of tumour, it's not a good specimen at all. Actually, what you're doing is you're trying the test.

As Andreas said, after two weeks, you realise that there was no DNA at all. You have to make sure that you're choosing the right block, you're choosing the right – when I say block, if it is a tumour, if it's a core biopsy, you will have only one paraffin block. But if it is an excision specimen, you might have 10 paraffin blocks. But of the 10, you have to choose the one which has the maximum tumour content, so that you have adequate DNA, and your NGS test is not a failure, and you're getting a good answer for that.

All this is important in tissues to stewardship. A few years ago, four, or five years ago, it used to be a single gene assay. PCR-based assays where you only look for one mutation could be eGFR, it could be KRAS, whatever. But now, you have the next-gen sequencing, where you're looking at a whole panel of genes. I think that is a smart way of looking at mutations rather than doing the single PCR-based assays. Now, we're moving into a bit more complex field, which is called comprehensive genomic profiling. It is slightly expensive, but I think it gives you a whole lot of information about the tumour, the mutations, the MSI status, the tumour burden, everything. I think that's how we should be smart.

Don't waste tissue when it comes to the laboratory. Keep in mind that you need it for so many tests, you need it for immunohistochemistry, you need it for molecular testing, you need it for comprehensive genomic profiling. For example, assay, which has only 20 genes, you may not need that amount of DNA. But if it is a comprehensive genomic profile, you will need more DNA. I think you have to have that mindset as pathologists that this is very precious, and I would probably need more tests on this patient so that he or she can get the right treatment. I think we have to be very mindful of what we're dealing with right at the start.

I just wanted to let people know who curate these tissue banks, that there may be a very reasonable request from time to time, that family will request access to tissue samples. That is just normal and just good behaviour. I would do the same if I was a patient, absolutely.

In my opinion, what is more important is, if it's a big specimen, it could be ovary, it could be breast, whatever. I think what is most important, and I think what preserves the morphology, and the DNA content, is slicing through those specimens, and making sure that you just don't have a big blob of ovary sitting in formalin just like that without being cut into slices. Because what will happen is, the outer surface will have formalin, but it will never go into the specimen. The inner part of the ovary has actually gone rotten, so you don't get to see the cells properly, you don't know what tumour it is. Is it high-grade serous? Is it clear cell? You wouldn't have a clue. I think if it's a Friday, we have to make sure that the specimens are sliced, and then so that formalin can seep into the entire specimen rather than just being on the surface. I think that's critical.

As I said, larger hospitals, tertiary health services, they all probably work six days instead of five days. The Friday issue is sort of becoming lesser and lesser these days. It's not as common as it used to be, even five or six years ago.

What I'm saying is that, I think I would predict that by taking small samples, the inadequacy rates will be jumping from 45% maybe to 65%. I would really encourage colleagues in different healthcare institutions to just really inquire about what are their institutional, or what are their inadequacy/adequacy rates in their institution, and maybe review that, and reflect on that, and see what they can do to optimise that.

But I think we should change our mindset and just look at the patient. Because behind every paraffin block, or every glass slide, there is a patient. If there is something which we can do for the patient to get better treatment, I think we should be excited about it like HRD. This is one more option, which a patient with ovarian cancer has, and that patient could be just 25 years old of age. It's quite a long life ahead. I think we have to look at it that way, and we have to look at all these advances as opportunities and not feel bogged down. I think this is more for the pathology world rather than the surgeons or the radiologists.

I'm not aware of Australian guidelines, UK guidelines, US guidelines. I'm only aware of very little primary data on that. We discussed them quite extensively in the last 45 minutes.

Pathology is a very, very protocol-driven department. I think we follow all that. So those are guidelines which are textbook kind of guidelines and very structured. But apart from that, with my own practical experience, I would say, as I mentioned, tissues is the issue. So be very conservative when you use them. Don't cut into the blocks by doing levels upfront, don't use a broad immunohistochemical panel. You just need to know whether it is serous carcinoma or clear cell carcinoma. You don't need 10 immunos to come to that conclusion. Especially if the patient has had a primary specimen, then you don't really need to repeat the immunos, on say, post-chemo specimen. So you have to be a bit smart in the way you do things.

Then, to be familiar with the molecular advances and nuances in each tumour stream, and that will make a pathologist more mindful of the tissue in hand and what needs to be done. Then, my last piece of advice would be, talk to your surgeon, talk to your oncologist, learn about the advances, see what they need to treat the patient. You may have the world's best pathology report, good, fantastic English, grammar and spelling, and everything is great, but then it lacks details which the surgeons want to treat the patient. Unless you talk to them and understand as to what they need, you won't have that connection.

I think for all this, the multidisciplinary meetings which we have, especially in a public hospital is really, really valuable. Because you go to the meetings, you present your case, you have the radiologist, then you have the surgeons, you have the oncologist, you understand as to what is required for the treatment. I think all these are very important. You have one set of guidelines, which are governed by WHO, and the College, and you have the other set of guidelines, which are more practical, which you need to understand and practice on a regular working day.

In the next episode of Beyond the Slide, I speak with Andrew Fellowes and Jeff Goh about HRD testing in Australia. Here’s a little preview.

Just a reminder that this podcast has been commissioned and funded by AstraZeneca. This podcast is intended for Health Care Professionals only and contains discussion of AstraZeneca Products. The thoughts and opinions expressed are the speaker's own and may not represent those of AstraZeneca.

If you enjoyed today’s podcast please share it with your colleagues. Thanks for listening.

This is Rachael Babin and this is Beyond The Slide.

[END]