Speaking of Science: Breast Cancer Awareness Month with Professor Belinda Parker

In recognition of Breast Cancer Awareness Month, an annual public health campaign that runs throughout October, we were joined by Co-Head of the Cancer Evolution and Metastasis Program and Head of the Cancer Metastasis and Precision Immunotherapy Laboratory at the Peter MacCallum Cancer Centre, Professor Belinda Parker.

Professor Parker discussed the research undertaken by her research lab, and how tailoring precision approaches for predicting and targeting aggressive breast cancers can lead to better patient outcomes.

Listen to Professor Parker ‘Speak of Science’ and delve into how we can improve early diagnosis and treatments for metastatic breast cancer and how we can effectively translate this from bench to bedside.

Recorded on Tuesday 29 October 2024 from 2:00PM – 3:00PM AEDT.

Video transcript

Professor Steve Wesselingh 0:00
I am Steve Wesselingh, I'm the chief executive officer of the NHMRC and actually by the time I finish my introduction that will allow the late people to join.

I think I might start and thank you all for joining us for the October instalment of Speaking of Science and today's instalment is in recognition of Breast Cancer Awareness Month.

But before we start, I'd like to acknowledge the Traditional Custodians of the lands on which we're all meeting today, and it's for people all over Australia. I would personally like to acknowledge the Ngunnawal people as the Traditional Custodians of the Australian Capital Territory where I am today. I acknowledge and respect their continuing culture and the contribution they make to the life of this city. I pay my respects to their Elders past, present and emerging and extend this respect to all Aboriginal and Torres Strait Islander people joining us today online.

A bit of housekeeping. There will be opportunities to ask questions at the end, so please use the Zoom chat function and then I'll turn your chat into a question and ask that at the end. We'll be recording this as we have recorded previous webinars and they're all available to stream on our website.

This afternoon we'll be speaking of breast cancer and the groundbreaking health and medical research that is happening to support the National Breast Cancer Foundation's vision of zero deaths from breast cancer by 2030. Which is a truly aspirational vision, but one I really think that we can achieve.

The breast cancer research that's happening around Australia is really pretty amazing. Breast cancer is the second most commonly diagnosed cancer in Australia, with 9 people dying every day from breast cancer. As a lot of you know, October is Breast Cancer Awareness Month, and it really does provide this opportunity to draw attention to the ongoing impact of breast cancer on those diagnosed and their loved ones.

Obviously by working together, we can ensure that thousands of Australians diagnosed with breast cancer have access to the very best treatment and hopefully the very best prevention as well because obviously if we can prevent breast cancer, that would be even better.

Belinda, who's joining us today, co-heads the Cancer Evolution and Metastasis Programme and leads the Microenvironmental Crosstalk and Therapeutics Laboratory at the Peter MacCallum Cancer Centre. I think Belinda you need to get a slightly shorter title to your lab, it’s quite a long title.
Belinda's team focuses on tumour cell crosstalk with the microenvironment, with particular interest in stromal biomarkers that predict the risk of early breast cancer recurrence. This research also focuses on tumour markers and targets for precision immunotherapeutic strategies in breast cancer and also in prostate cancer.

A specific area of interest for the Belinda Parker Lab at the Peter Mac is the tumour cell inherent immunoregulatory cytokines that dictate metastatic spread and therapeutic response.

Based on this expertise and all this work that Belinda has been doing, she's secured commercial collaborations to be the lead investigator on trials of specific families of cytokines in bone metastasis, a common sight of tumour spread in both prostate and breast cancer that is currently unfortunately incurable.

Belinda's also familiar to the NHMRC. She holds an NMHMRC Investigator Grant Fellowship, and has previously held fellowships with both the ARC and the Victoria Cancer Agency. I'd also like to note that Belinda was recommended to us by someone inside NHMRC, from a staff member at NHMRC, who'd previously been her PhD mentee. It's great to have someone who's very familiar to NHMRC and someone who's been doing such inspirational work.

Belinda, I'll hand over to you and really looking forward to your talk. Thanks, Belinda.

Professor Belinda Parker 4:28
Thanks very much, Steve, and thanks everyone for joining. Really appreciate it.

I'll just start the talk and Steve, are we good?

Professor Steve Wesselingh 4:36
Yeah, that looks good to me. Yeah.

Professor Belinda Parker 4:37
OK, fantastic.

OK, yes, very excited today to talk about our work.

I will focus on breast cancer as Steve mentioned. We've also been going into prostate cancer due to some similarities actually between breast and prostate cancer once they spread to the bone. But I will focus on our precision approaches for predicting and targeting aggressive types of breast cancer.

In our lab, we're really interested in how cancer cells interact with other cells because this can change when a cancer starts in very early stages in the breast, when it actually invades out of the breast and then into the circulation, but then also when these cells lodge into distant tissues and of course, there's also therapy.

When a patient is treated with therapy, changes in the cancer cells are very common and so when you're looking at cancer, you can't look at it at one stage. It definitely changes and evolves during the process and so we're really interested in 3 major themes in the lab.

One is in early breast cancer. Predicting the risk of a very early breast cancer recurring and becoming invasive cancer in another breast or the same breast or actually spreading to other tissues. We're really interested in this because we're actually thinking that we may be over-treating some patients diagnosed at these really early stages and I'll get into that a little bit in a minute.

We're also really interested in precision immunotherapy. Immunotherapeutics are being offered now particularly in triple negative breast cancer at the diagnostic stage in the neoadjuvant setting so before surgery. It's really important that we have biomarkers to predict who would respond and then precision ways to actually increase those responders. 
We've got a lot of activity in the lab, mainly in the metastatic setting. We look at the ability of cancer cells to spread to these tissues, how they change when they get there, and can we actually use fundamental biology to find targets to actually increase their response to therapy? Rather than giving these patients all these therapies and then doing reverse biology to look at why they may have responded, we're trying to utilise biology that we find in the lab to actually advise on better therapies for patients.

I'll start in the early diagnosis area and cover that and then we'll move our way through metastasis and what we're interested in and what the important questions are now in the breast cancer space.

Most of you in the audience will know that breast cancer starts, if we take a cross section of a duct, breast cancer often starts in the epithelial cells that are inside this duct as a single layer. There can be other changes to the breasts that are not cancer such as certain types of hyperplasia and other benign conditions. But when the cancer starts and is confined to this breast duct, it's called ductal carcinoma in situ. Ductal carcinoma being one of the most common types of breast cancer, then cancer can become microinvasive.

The very earliest stages of when cancer starts invading beyond that ductal carcinoma in situ state and then invasive breast cancer which then invades through the surrounding tissue and extracellular matrix in the breast.

We're really interested in 2 sort of stages here. We know that mammographic screening has increased detection of early-stage breast abnormalities, so more women are getting biopsies. It’s really important to distinguish actual cancer from a benign abnormality and here I'm showing hyperplasia and DCIS looking quite different, but sometimes DCIS doesn't feel the whole duct like this. Distinguishing those states is quite important.

The other thing we're really interested in is the fact that 20 to 25% of breast cancers are now detected at that stage of ductal carcinoma in situ, so very good prognosis for that stage.

We really need precision therapeutic strategies for patients with DCIS. There aren't really any precision strategies at the moment. The patients are often treated as 1 group when they have ductal carcinoma in situ, depending on their stage.

One of the multidisciplinary approaches that I've been looking at in collaboration with Brian Abbey and Eugeniu Balaur at Latrobe and also with pathologist Sandra O'Toole, is actually using a novel slide technology to allow accurate diagnosis of those really early lesions. Because often pathologists will get these tissues to look at and sometimes, we're just looking at very few cells under the microscope that are quite hard to distinguish.

In collaboration, we actually developed a slide technology that can be utilised in a normal light microscope where we don't stain breast cancer tissue. We just take sections of it or of the other abnormality that's been seen. We just put it on the slide and pass normal light microscopy light through that and it gives off a different colour depending on the density of the tissue. That's because the slide actually has within it a silver coating with these little pits in it and you actually get movement of electrons and a different colour depending on the density of the tissue. That's the simplest way to put it.

If you have a look here, you can actually see this is a usual ductal hyperplasia lesion. Now that is not cancer and if you just looked by H&E, so hematoxin and ESM, what pathologists usually look at is the nucleus and the cytoplasm and stroma, sometimes it's quite hard to distinguish that from early stages of ductal carcinoma in situ.

But what you can see on the nano slide is that the usual ductal hyperplasia stays yellow and the ductal carcinoma in situ, so the cancer cells, give off this green-blue colour in the nucleus. This is something that we worked up over time to find the best formulation of the slide. We found this quite remarkable.

We actually did this in a number of human tissues. You can see that this is a normal breast duct. This is usual ductal hyperplasia that is somewhat proliferative, ductal carcinoma in situ, an invasive cancer, we can easily distinguish with no overlap the difference between cancer and non-cancer.

Where do we think this fits? Well at the moment when a biopsy is taken in the clinic, because you've had an unusual area on the mammogram screen, this will be sent to a pathology lab, a hemotoxin and eosin stain will be done, the pathologist will look at that, but then about 30% of those unclear cases where it can't be definitively diagnosed, the pathologist will send that on to get further immune histochemical stains such as eastern receptor and cytokine 5-6.

Where we think the NanoMSlide like this could be quite useful is as an adjunct to the H&E to bring the eye to those areas of differential colour. That could improve diagnosis and the speed of diagnosis, not only the accuracy in breast cancer. We’re also looking at larger cohorts at the moment of breast cancer to really look at this over a large scale and also other cancers. We've seen some really exciting findings in skin cancer, lung cancer, and we're now looking at many others, including colon and prostate because we think that wherever we've got early screening and we've got early detection, it's really important to be able to pick up those early cancers.

The other area, as I said, 25% of mammogram-screened cancers are now picked up at this early ductal carcinoma in situ stage where the cancer cells are still in the ducts surrounded by this myoepithelial layer, and this is like a structural cell that sort of keeps everything intact. It's known that the break of this myoepithelial layer really determines if you have invasive cancer or ductal carcinoma in situ and it does change your risk of metastasis whether you have 1 of these 2.

Now, if you leave a ductal carcinoma in situ, it does suggest that it will eventually develop into invasive, so you do need to do surgery to remove it. But it actually has an excellent survival rate with treatment, 99%. A lot of people could just have surgery and not need anything else.

However, because we don't know who those people are, a lot of women receive radiotherapy for ductal carcinoma in situ. It does reduce the risk of another invasive cancer or non-invasive cancer in the breast, but it doesn't change overall survival in terms of your risk of getting distant metastasis.

Because of that, we really need to have markers that can determine if a patient is likely to get invasive disease down the track. Because at the moment 70% of patients receive radiotherapy and that's a lot of people receiving therapy for something they may not need.

We've been working on a project actually supported by the National Breast Cancer Foundation and previously by the NBCF, to look at changes to the microenvironment around these breast lesions and to see if we can find markers that actually predict whether a patient is likely to go on to have invasive disease or whether they could be spared the extra radiotherapy and just get surgery.

We're looking at three different types of markers. One of them is the standard pathological markers that we tend to look at in breast cancer diagnosis and that can be anything from the progesterone receptor, oestrogen receptor, the proliferative status, etcetera. We're also looking at the myoepithelial cells. These cells, as I said, surround the breast duct. It's very evident that proteins in those cells dictate more so whether a cancer is likely to spread more so than the actual tumour cells themselves. The microenvironment is actually giving you really essential information on the risk of invasion. We're also looking at immune markers, and we've put these 3 together and we've found some really exciting markers. But a project from Christina Kozul has really developed this model where we can culture cancer cells with these myoepithelial cells and usually, they're kept in check. But if we delete some of the key proteins from these myoepithelial cells, in this case they're green, we can actually see that some cancers can invade out. We can find those proteins that are really suppressing this process and then look in human cancers and see if that's predicting invasive relapse.

We're able to actually see with these models that the myoepithelial cells in green here actually encapsulate the tumour cells. But when we do change those proteins, the cancers can escape. We're now working with King's College on a large cohort, part of the DCIS Precision Programme, using a large cohort to actually look and determine if our microenvironment markers can help predict the risk of relapse in patients. So that'll be done on thousands of samples from women with known follow up.

I think this is really exciting because as a programme DCS position has looked at lots of tumour markers with limited success and it and by adding these immune markers and the myoepithelial markers, we feel that this could really add something to the field and so we're pretty excited about that.

Now on to metastatic disease. As I said, you know, we're getting better at diagnosing cancer early. One in 7 women will be diagnosed with breast cancer by the age of 85, so the actual incidence of breast cancer is rising. Also, what contributes to that is the early detection.

But what we see is, is that we're still getting a lot of deaths despite that early detection. We’re getting a rise in incidence and the deaths are staying pretty steady and the risk of metastatic spread despite therapy is 20 to 30%. So you can see that metastatic spread to distant tissues is stage 4 and the survival rate drops incredibly.

This is why breast cancer remains the second leading cause of cancer related deaths in women. We often hear the stat about the 90% or the 70% of women surviving depending on the stage that they're diagnosed with. But it's because it's so common that it's still the second leading cause of cancer related deaths in women. We need to do a lot better with targeting metastatic disease and some of these most aggressive cancers occur in young women pre-screen age. They haven't had their mammograms; they've been diagnosed potentially a little bit later when they have invasive disease or even their lymph nodes involved, and some of these young women have very aggressive disease.

Targeting metastasis is important and in our lab, we're particularly interested in bone metastasis. Over 70% of metastatic breast cancers occur in the bone and it can be the first sight of spread. Our lab has invested quite a bit of time in bone metastases. It's, as I said, 1 of the most common sites and it's commonly a first sight of spread with multiple sites involved. It's extremely painful. Patients with bone metastasis often complain of extreme pain, spinal cord compression, pathological fractures, bone marrow failure. It's quite a terrible disease, very morbid and eventually soft tissue spread will then rise and then the death of these patients and it's quite incurable at the moment.

We believe this is a therapeutic window to intervene. Can we block or target bone metastases early to block that secondary spread to other tissues? Not only to reduce the pain, but actually to help curing patients.

A little bit about bone metastasis that some of you may not know is that when breast and prostate cancer patients, because both of them can be diagnosed and have bone tumour cells in their bones, over 40% (depending on the study) of patients, will already have cancer cells sitting in their bone marrow at the time of diagnosis.

At the time where they're getting their primary tumour treated and removed, there's already cells that can't be detected unless we do very specific scientific analysis in the bone. They can sit there for many years and do nothing in a sort of dormant or sleeping state.

But in some patients, these can be activated and start growing and once they start growing, it's a very vicious cycle where they utilise the bone cells and they destroy the normal bone remodelling and that's how we get the problems with fractures and particularly in breast cancer, these seem to be osteolytic and these osteoclasts, the bone sort of degrading cells, are often involved in those horrible, painful bone fractures and lesions.

Now the problem with treatment of bone metastasis, we can either directly target the tumour cells with radiotherapy, chemotherapy or hormone targeted therapies if they're eastern receptor positive breast cancers, or we can target the bone cells and try and prevent this degradation of the bone and the release of growth factors that help the cancer to grow.

The issue is that the responses to these therapies are usually not curative. They can help with reducing the morbidity, reducing the pathologies, but actually we are not doing very well at curing these patients.

Because of that, there has been interest in immune therapies. There are a variety of immune therapies that are being used in the clinic with various results in different patients.

These include the checkpoint inhibitors that many of you would have heard of that have been quite great actually in Melanoma for about 50 to 60% of patients with metastatic melanoma responding to these antibodies that basically block suppression of the immune system.

There's also approaches using engineered T cells to go and fight cancers. These are working better in blood cancers rather than solar malignancies at the moment, but there's a lot of activity behind that and then there are other ways that you can try and stimulate an immune response in a tumour to try and increase the immune activation against a tumour.

But what we see in metastatic breast cancer is that it just doesn't work that well. The checkpoint inhibitors, this is an example of 1 study in both hormone interceptor positive in black and triple negative cases in red, where they were treated with the anti-PD1 inhibitor (Pembrolizumab) commonly used in Melanoma. There were only 3 patients out of 30 that had ongoing responses.

There was absolutely no improvement in progression-free survival. There was again no impact on their overall survival and when you look into the data of these trials, none of the patients responded if they had bone metastasis.

These therapies are having a lot of promise in the early treatment settings, such as in triple negative breast cancer, and I'll get into that a little bit later. But how can we target metastasis by harnessing our immune system? Is the immune system important in bone? And can we use another strategy if these don't work?

This is a very overwhelming picture, but the reason I wanted to put it there is because it is so complex. Breast cancers, when they develop and then enter into the bloodstream, they can travel alone or in clusters of circulating cells.

That can allow them to hide from the immune system, including when they interact with other types of cells, number of different cells, including platelets, that can allow them to sort of block their visibility to the immune system.

They then lodge or extravasate into distant tissues where again, they can sit in this dormant state that I discussed, or they can grow into large metastases. Now each one of these steps can be can controlled by a different immune mechanism.

It can be tissue-specific so that what happens in the bone could be different to the lung and so really studying all of this as a site-specific level is really important. Looking at the primary tumour is unlikely to tell you what a patient will respond to in terms of immunotherapy once their cancer has spread to a distant tissue.

A lot of work in our lab previously has actually looked at a particular pathway, that I will get into briefly, where we look at cancers that are growing in the bone and this is just the blue is bone. We've tracked cancer cells that are just sitting there in a sort of dormant state and they're the ones with the red and sort of looking yellow here. The green population here is a cancer that started to divide. When we compare these and see what's the biggest difference? What causes this outgrowth in the bone? It's all about suppression of the immune system.

It's all about the ability to block immune activation and to block the ability of your antigens in your body being processed and presented on the outside of the cell so the cell goes unrecognised.

The key pathway is called the type 1 interferon pathway. We've found that that's completely suppressed in bone. Every time we look at bone, and that can be from breast or prostate cancer, it can be from the models we use in our lab or from patients, we see that we don't see this pathway in bone metastasis, and it looks like they're completely cold and that's how they've survived in that environment once they grow into large metastasis.

This is the pathway. We've done a lot of work around that that I won't go through just to say that it's the cancer cells’ ability to produce these little cytokines that are important in immunity, that's really important in any anti-cancer response. We’ve found that the T cells, so cytotoxic T cell killing of tumour cells, is really critical that you have this pathway on. Same with the innate response NK cells that are recognising stress ligands, etcetera, stress cells.

But also critically, as I said, if the tumour doesn't have this active pathway, they don't have the ability to show themselves on the surface through presenting on MHC+1. They're pretty much invisible to multiple therapies actually that require immunity.

This is just an example of an interferon high cancer in the red is the tumour cells. These are all the immune cells in multiple colours in breast cancer and an interferon low cancer.

What we find is the interferon low cancer will have more myeloid cells or suppressive macrophages in the tumour microenvironment, whereas that with a high interferon will have more T cells and active T cells and those that seem to have a memory against the tumour cells. The loss of this pathway is obviously causing a complete loss of the ability of the immune system to respond through multiple mechanisms.

The reason this is so critical is because, and I know this is a busy slide, but it's important to say that the interferon pathway is actually critical in response to chemotherapy, immunotherapy, many therapies.

Because when you have damage to a cell by radiotherapy or chemotherapy, it breaks up the DNA into pieces. Usually that DNA is recognised by these damage recognition receptors that are also important in recognising viruses and bacterial infections. The problem is because that recognition system and all of the transcription factors are lost in bone metastases, you don't get that response. They can't see those damaged cells from chemotherapy, and they can't then stimulate the immune system to have a response against those cells.

We've actually published quite a few papers on the requirement of this pathway for response to chemotherapy, to the immune therapies that I highlighted before, the anti-PD1 and others have also reported that this pathway now comes up a lot when someone's looking at response to therapy.

We obviously need to do better. We're stimulating the pathway to get patient response.

This is an example in a mouse model. This model is a very aggressive triple negative breast cancer model. What we do is we inject the cells into the mammary gland, so it's an orthotopic site, and it mimics a breast cancer, I guess. Then in this case we treat it with Doxorubicin, a common chemotherapeutic using triple native breast cancer.

We removed the tumour and then we waited for evidence of metastasis that you can visualise in these mice, and you can see that despite some effects of the Doxorubicin, the chemotherapy on the primary tumour, chemotherapy did nothing to metastasis. Each 1 of these steps down is a mouse dying from metastasis. When we see evidence of metastasis, chemotherapy did nothing. This is an interferon low model, so the mice, the tumours didn't express a lot of interferon in their tumours.

When we restore the pathway molecularly by putting in a key transcription factor, they survive for a lot longer. But if we combine that with chemotherapy, hardly any of the mice get spread of their cancers. That was really exciting.

But of course, that's mice. What about patients? Well, if we go back to triple negative breast cancer patients and we look at their cancers before they get chemotherapy and then take another sample mid-chemotherapy and then after for whoever has tumour left, we can actually find that a key transcription factor is called interferon regulatory factor 9 (IRF9). The expression of it in brown here actually predicts those patients that are unlikely to have spread of their cancer and this is at the time of neoadjuvant therapy. This is very early on.

We're actually predicting if they're going to get spread of their cancer despite chemotherapy. Then you can see here, this is quite a small cohort, but you can see here loss of IRF9. These patients are succumbing very quickly to triple negative breast cancer in distant sites and they're succumbing to disease.

This 1 marker tells you all about the environment of that cancer, that it has the whole pathway suppressed if it's gone, that there's none of these what's called tissue-resident memory T cell signatures, that have been identified previously by Sherene Loi to actually tell you if you're going to have response in the triple negative neoadjuvant setting, and then also in the adjuvant setting when you take the cancer out first before therapy, we're also seeing that this IRF9 that comes up brown is either there or it's not. If it's there, there's very little risk of spread of cancer.

That's quite important at the moment because as I said, immunotherapy is now being used for neoadjuvant triple negative breast cancer. We're getting treatment before the cancer's removed.

This has been happening with chemotherapy and all of these dotted lines are chemotherapy alone, but the addition of the immunotherapy, in this case a PDL1 inhibitor, is increasing patient responses, their complete pathological response. The tumours completely dissolve and that's a really good marker of a good prognosis later on.

The different colours here are just whether they express PDL1, so the target of the immune therapy or not. Even those that don't express PDL1 are having a fantastic response because of course other cells may express them other than the tumour and many other reasons.

But we're getting these much bigger responses when we add the immunotherapy. But why? Why do some of these respondents, some don't? Who needs chemotherapy alone? Who needs chemotherapy plus immune therapy? We still don't know. So we give it to everybody.

Obviously, there's side effects associated with the immune therapy added to the chemo, so you do want to know who needs it and who doesn't. Plus, you want to be able to increase the patients that respond by giving them something else if they're not going to respond to this combination.

We're working with a company at the moment, Prelude DX. We have a number of markers that we've developed that are quite interesting, part of the interferon pathway, and we want to use those markers to determine what is the patient need in the triple negative breast cancer setting.

I'll just quickly go through this. We believe that we'll be able to determine those people that just need chemotherapy, those that need the addition of immunotherapy because they'll have a presence of this interferon pathway either at baseline or induced.

But then there'll be these patients that don't will have a poor outcome with those anyway And to decrease their metastatic risk, we need to give some sort of sensitizer to increase their response to these immunotherapies.

I sort of like to just show this in a basic way. What we want to do is turn these cold tumours, which are the IRF9 or interferon low tumours into hot tumours and then we can see that we can actually restore their response to chemotherapy and immune therapy, which we've seen before in our mouse models, and it does mirror what we're seeing in clinical trials.

I'm not going to go through this pathway. All I want to say is that the drugs that you can use in the clinic to stimulate interferons are similar to mimicking, for example, a viral infection. For example, Poly (I:C) that we use as sort of an investigational agent, 3M-052 is a TLR 7-8 agonist. It just stimulates the DNA damage responses in this pathway and there are other agonists that you may have heard of just stimulating a viral response or a DNA damage response.

What I want to say is these therapies work really well in early cancer, so probably could be given in triple negative breast cancer. But once you have metastases that spread to the bone, they won't respond to these therapies. What we think is, is that when the cancers get to the bone, they've actually epigenetically reprogrammed those cells, not at the gene code level, but they've actually changed their methylation status so that they actually switch off these genes and they can't be restored by these drugs.

This is an example of where it does work in early breast cancer. When you treat with Poly (I:C) again, investigational agent mimics a viral infection and you add the immune therapy, anti-PD1, they work really well together. The immune therapy alone does nothing. Same as in patients, if you have low interferon, you can see you can't even detect cancers in the lungs in this case.

You can also see from these graphs, just believe me, because so I don't have to go through them completely, is that we're getting an increased immune response against the cancers even in the lungs. Even though we only treated in this case for a couple of weeks, we're still picking up these cells in the lung that are actually tumour-specific T cells. That's really exciting if treated early.

But look at this, this is a survival curve with anti-PD1 and Poly (I:C). This is a really aggressive model. Seeing an increase in survival, takes longer to see metastatic spreading these mice if you treat early in the neoadjuvant setting before you remove primary tumours. But if you wait until they've got metastatic disease, it does nothing. There's nothing that you can do at the moment about that.

What we think is there's a real need to change the way we use these therapies. We can't use interferons in a metastatic setting because we need something else to bolster their response. In the early treatment setting, however, we believe there are biomarker guided therapies that can be used in combination with chemotherapy, radiotherapy and anti-PD1. We’re really working on that area while we're trying to solve what to do about metastatic disease.

Let’s show you quickly what I'm doing with metastatic disease, bearing in mind the time. What we're doing is we've got a way that we can take bone metastases from patients. We can put a reporter system in them that tells us if this interferon pathway is switched back on and that's because one of the key promoters in DNA is called the ISRE (interferon stimulated response element). We have a fluorescent marker and luciferase so bioluminescent marker under the control of that promoter. When it's turned on, we can see bioluminescence and fluorescence.

Miriam started this project in the lab looking at different drugs that could actually turn on the pathway in these 4T1.2 bone metastatic cells that are quite aggressive and also in human cells and other mouse lines to see if we can find a drug that could switch the pathway on so we could use it in combination therapies.

Indeed, we did find that there were quite a few epigenetic drugs, so those that change the ability of genes to be expressed, HDAC inhibitors and DNA methyl transferase inhibitors that seem to work quite well at increasing the pathway.

This is an example of a drug that we took forward which was called Decitabine. It's a DNMT inhibitor, so DNA methyl transferase inhibitor. We could see that when we treated mice and this, in this case, again, it's a really aggressive model and we forced them straight to the bone actually through the heart. You can see that even that drug alone and you can just see by the colour, it's bioluminescence and these are spines from the mice, you can see that you could hardly detect any bone mets.

Then in combination with that checkpoint inhibitor that does nothing alone, we're actually removing metastasis in these mice. We're also seeing that if we look at the RNA expression that the biggest pathway that's turned back on is the interferon pathway.

We're doing what we want to do by treating these mice and also, we did methylation arrays, and it shows that that methylation that was turning off the genes is now there's a demethylation event. They’re no longer methylated to be turned on and we're getting those T cell responses back. Just focus on the increase in the MHC class 1, presenting antigen so that the cell can be seen by the immune system.

But also, we're recovering that T cell response to those cells when we take tumour specific T cells and incubate them with these cells, now they can see them, and they can kill them. In this case, if we're just looking at a release of interferon gamma, it just tells us that there's a functional response.

I won't, in the interest of time, go through this, but we're actually looking at more specific ways now to target interferons. Why is that important? Well, what we've found is in the bone, as I've said, there's a complete loss of the pathway. That means you're blocking any immune responses that tumours can grow. What we want is to turn this back on, targeting the mechanisms of loss in the first place so that we can restore these, the ability to show antigen and get a T cell response.

What we don't want to do is restore it too much. If you give something like an epigenetic drug, it's possible that lots of cells will then produce all this interferon. Not only will you target other cells, but you could cause this chronic response, and this chronic response is a problem because it will cause this immune sort of suppressive environment and inflammatory environment that is also detrimental and can cause metastasis. That balance is critical.

What we're doing instead is finding what are the genes that cause the loss of this pathway in the bone and we're doing a CRISPR in vivo screen. This is work done by Tom Chadwick and he's Co supervised by Marco Herald at the Olivia Newton John Cancer Centre, where he takes those really aggressive cells, he then takes bone metastases from these mice and then he puts a library in them and does the same reporter experiment that we did with the drugs to see what do you have to inhibit to increase this pathway.

He's found some really exciting candidates already, including, I'm sorry, I can't tell you what it is, but it's a methyl transferase complex. So again, inhibiting that similar to inhibiting a DNA methyl transferase with a drug looks like it's restoring the pathway, but this is specific. These are specific DNA methyl transferase and it's an interesting pair. We're really interested in following that up in other models.

Just a last thing I sort of wanted to go through is these are mouse models. How do I know if that's going to work in patients? I know that the pathway is important in patients, but how do I know if any of the genes or the drugs we find are going to work?

What we're doing now is we're taking samples from patients at the time of death rapid autopsy programme, and we've developed a way to grow these cancers on a bone scaffold because it's actually really hard to grow samples from the bone, it's almost impossible. But we're growing them on the bone scaffold to try and keep them alive with the normal stress conditions that they would be under when they're on bone.

We're collaborating with engineers, with bone imagers, clinicians, and we're putting it into a microfluidic bio chamber so we can image at all times the cells going onto the scaffold and then delivering drugs, but also delivering matched immune cells from those patients to see if we can turn back on the immune system using what we've found in the mice but take it into a human system.

I just want to show you this is what the at the moment we're using a femur from a cow bone cutting it into very specific sizes. This is an example of breast metastasis growing on one of these scaffolds. The red cells are the cells that are actually viably growing on them. Now that was a patient-derived cell line. But what we've now done is taken cancer from a patient at the time of their autopsy, taken it straight back to the lab and we've shown for the first time that we can grow these cancers on these scaffolds. We're really excited about that because this is the first time we've seen bone metastasis growing on such a scaffold.

What next? We want to monitor with the drugs how we turning back on this interferon signalling pathway. We test the therapies that we've identified and again, we really want to add those matched immune cells to see if in real time, those interactions, and have more confidence about what we're taking forward to the clinic.

This is just an example of what we've been looking at. I'm hoping you can see the red cells or the viable cells. The blue is HLABC, so that's MHC class 1, so what presents peptide, and we can actually pick up the increase in that on the bone and we recently got some funding to get a better multi photon microscope. We're really excited about looking at more of these markers in parallel.

We are also working towards bone biopsy assessment in patients. At the moment, we are doing biomarker studies in patients that get bone biopsies, and we've talked to consumers and if we could personalise therapy, they'd be willing to get bone biopsies. We're really interested in personalising therapy going forward based on this technique. But we are quite far off.

I just wanted to sort of finish there. This is quite a busy slide, but what I just wanted to say was we do work with samples we get from clinical trials at Peter Mac. We then look at our biomarkers to see if we're predicting response and then we go back into our models, whether that be the mouse models or now the scaffold model, to test ways that we can increase the response for patients or we can find new ways to treat patients that aren't responding.

We are involved in a number of trials around that. We will be looking at samples directly taken from the neoadjuvant trials that Sherene Loi is running at Peter Mac. I already discussed the biomarkers and the bone scaffold, but we're also actually looking at the interaction between different bone cells and cancer cells that leads to this. We've identified some really cool markers that we can target with CAR-T cells, so we're doing some work in that.

We're also looking at hormone therapies because really a lot of hormone receptor positive patients get bone metastasis. What's the best hormone therapy to give early and what is the impact on the bones? We've got a project funded by the NBCF for that.

It just leaves me to thank my team. I've really sort of talked about a lot of these people, but someone that's really important in this, 2 people actually, are Lisa and Heather that actually go into the autopsies and help us get the tissues and of course my clinical colleagues, and I'll leave it there.

Thank you.

Professor Steve Wesselingh 41:16
Thanks very much, Belinda. Fantastic. What amazing work.

Maybe if we bring your slides down there, we can see your face. Just fantastic.

I just encourage everyone to think of some questions and put them on the chat and then I can take those questions and ask Belinda.

While people are doing that, I guess one of my questions going back right at the beginning, if we got everything right, is breast cancer actually a preventable illness?

Professor Belinda Parker 41:48
Prevention.

I knew that that the question would come up.

Look, I think at the moment, we'd have to get everything right, because prevention at the moment is tamoxifen, for example.

So, do you mean before?

Professor Steve Wesselingh 42:01
I guess I'm thinking if everyone went and had a mammogram, we got the mammogram, right, could we prevent breast cancer?

Professor Belinda Parker 42:13
If everyone was getting screened?

Not every cancer is picked up by mammogram, so you'd also have to get the best screening at the very early stages. There's a lot of things around that.

But yeah, early detection, what's happening with early detection at the moment is for every person, I think you save, you overtreat about 4 so this ratio of overtreating.

But if we can get the markers better and really personalise that treatment and we're actually better at picking them up and we're screening people that are younger because at the moment there's a lot of women that aren't at that screening age that are still coming up with aggressive cancer. There's this bit of a balance there.

But yes, if we could get all of those things right and have the markers to actually predict whether they need the addition of radiotherapy or not, yes, I think that's a better place to prevent subsequent cancers. At the moment where we're not there yet with preventing that in the first place, like Tamoxifen is given to patients and it's got a lot of side effects. It’s really important to predict who needs that and get better preventatives. But I think at the moment the best place is to look at the early screening and doing the right thing for those patients.

Professor Steve Wesselingh 43:22
Fantastic, fantastic.

We've got a question from Zoe. Can you please outline where HERD2 and hormone receptors come in?

Are you finding success in predicting invasive metastatic carcinoma by looking at HERD2 in the microenvironment?

Professor Belinda Parker 43:37
That's a really good question.

At the moment the prognostic work works best in triple negative breast cancer because the loss of the pathway happens early and that's why you can treat early, and you can target all the metastases.

We have looked at signatures, so interferon signatures in patients that have hormone receptor positive disease or in prostate cancer patients that have obviously ER+, we can actually predict the risk of bone metastasis in them. But the single marker RF9 is not good enough.

The thing is, is that a lot of patients are getting that loss once they get to the bone in the hormone receptor positive. Some of them already have a loss and it might be a clonal selection of some that are already aggressive and have lost the pathway.

But it's also happening in the bone microenvironment. So yes, I think in HERD2+ and ER+ disease that it does hold a place.

What we're trying to do now is also develop some circulating methylation markers because that would be really fantastic too to monitor that over time. I hope that answers the question. But yeah, it's just different stages, different proportion of sort of hazard ratio that you would get based on the fact that we do get a loss in the bone environment as well.

Professor Steve Wesselingh 44:53
OK.

The next one also telling you that it was a great talk, lovely talk.

Did you use a whole genome library for in vivo CRISPR screening? If so, would you find more if you use the targeted library?

Professor Belinda Parker 45:06
I love that. I do like that question because I just had that meeting with my student yesterday.

We did use a whole genome.

But what happens is because you have less coverage of each gene, your statistics may limit the number of targets because you want it to be at least in 3 of the 4 guides that are there for each gene to be present. But you're sort of working against yourself a little bit.

We’ve done the whole genome CRISPR screen, but we're actually going to do an epigenetic screen specifically. We're also doing a screen with the top 100 genes that were found. But again, that could miss out some really important ones. We're doing that, it's important.

Professor Steve Wesselingh 45:46
The next person's, questions coming from everywhere here, has this progressed to a phase 1 trial? If so, which and if not, what would be the first area that you would choose?

Professor Belinda Parker 46:00
Yeah. So there have been trials using the TLR agonist in triple negative breast cancer, but we haven't progressed anything in bone metastasis. I'd really like to see it work on the bone scaffold.

I probably could have progressed some of the bone metastasis work further, but I just want to see what works the best. Because when we use things like Decitabine, it's also similar to using a chemotherapeutic. It works really well, but you will get other effects.

We're seeing that we wipe out myeloid cells, for example. There are literally no myeloid cells after using it.

Even though that could be good because that model is very myeloid driven, myeloid suppressor cell driven, I think we've got to be careful using that in a clinical trial in that setting. I mean there are some clinical trials using Decidabine that have looked really good in early treatment settings. I do think using it in bone metastasis could be useful with the right combination. But at the moment we haven't progressed anything to trials. We sort of want to get the bone scaffold to work and then work with our clinical partners that are very close to us

I am involved in with, with Shanine Sandhu in prostate cancer, in bone metastasis. She's doing a trial with PARP inhibitors. PARP inhibitors are important in that DNA damage response and allowing that to happen and then combining that with the immune checkpoint inhibitor.

Again, we're looking at our biomarkers because we think that those sort of therapies would work better if we could switch back on the interferon pathway properly. Only some patients will have a response. There'll be some exceptional responders, but most won't. We're very active in that space and then we're going to be using the clinicians to help us progress them when we're very confident.

Professor Steve Wesselingh 47:44
The next question, I was recently diagnosed with invasive lobular carcinoma via mammogram as it formed a lump, but it normally spreads by fingers and not picked up by mammograms and ultrasounds.

Is this the second largest cause of cancer?

Should we not screen with MRIS?

Would invasive lobular carcinoma be seen in bone?

Professor Belinda Parker 48:07
Oh, good question. I don't know.

Everything I've worked on is ductal carcinoma. I'm sorry.

It doesn't mean lobular carcinoma is not critical.

It’s just because of the bone metastatic prevalence and the samples I had available to me and the models were ductal carcinoma.

I'm not a clinician and I don't do screening, but I'm always for, picking up cancers earlier.

It's a problem because the biggest risk factor for breast cancer is just being a woman.

One in 7 women get breast cancer. Do we screen everybody and from what age? That's a constant discussion that I think breast, Breast Screen Victoria and other screening entities are having.

I won't give my specific opinion on that just because I'm really not in the right place, but I do believe in getting better at risk assessment and early detection and bringing those 2 together may be the case.

In the prevention space, they've found a lot about, you know, in terms of preventing cancer, they've also discovered a lot of risk factors. Combining the risk factors to determine if you should be screened earlier than the general population, that's where we could be in a really good spot.

Professor Steve Wesselingh 49:28
Thanks for that question and that great question.

I agree. I think looking at prevention obviously is an important area.

The next question is about the nano slide results and whether these were interpreted by software or were they interpreted just by eye?

Professor Belinda Parker 49:43
The first stage was independent pathology assessment.

That was also by myself, but I'm not a pathologist. It was also Sandra O'Toole in Sydney where we did independent scoring and saw that they were very consistent even with a scientist and a pathologist.

It was then analysed digitally, and we have a huge amount of interest in digital pathology. That’s actually probably where it will go because it's actually able to really distinguish sort of the different spectrum of colour much better than the human eye. If for example, someone was colour blind, you could actually distinguish more range of colours using digital pathology.

We're definitely interested in digital pathology. We've done a little bit of it and it looks really fantastic and I think we're going to go into that space a bit more.

Professor Steve Wesselingh 50:35
Doesn't appear to be any more coming up, but they'll come up.

Going right back to the beginning where you were talking about the decision of whether to have radiotherapy or not, and you talked about some markers and protein expression that you were looking at on cells.

I am aware of decision RT, which is a programme that looks at any of some of those things. Are you aware of that and how does that combine with your work?

Professor Belinda Parker 51:00
That's interesting. Decision RT, I work with them that they're Prelude. They're testing my epithelial markers at the moment.

Decision RT, as you would know, is specifically about predicting whether a patient should receive radiotherapy, and it actually looks quite good.

It's not just about are you at risk of recurrence, it's about whether you would receive a benefit from radiotherapy. Some of those markers including the progesterone receptor also come up in our screen.

What we've done is we've asked them to add some amount of myoepithelial markers for their next generation screen. They are testing that at the moment.

I do think it's a really exciting test because it's sort of identifying a couple of groups of patients. What we need to do though is identify the group of patients that despite radiotherapy will still go on to get relapse. But what's really important about that is the patients that will have a good prognosis without the radiotherapy. Making sure we're confident in that space that the patient just receives surgery.

I know those guys quite well and hoping we can contribute to that area.

Professor Steve Wesselingh 52:11
Because the second point you made is probably quite important because you can't, as I understand, have radiation therapy more often or at perhaps not even twice, so you don't really want to treat someone who's not going to respond to the radiotherapy in case they need it in the future.

Professor Belinda Parker 52:27
But also all of the side effects, lymphedema. Like there's a lot of side effects and risk of other cancers. I mean, if you knew you were getting a response to it, you would 100% do it. I think that's the problem speaking to the consumers is we just need to give these patients more information about their own cancer, so they feel like it's personalised treatment.

I'm sure that's what most of the scientists are trying to do and the clinicians is trying to actually be able to give someone confidence and that they're making the right decision in in what therapies that they're getting. Especially there's a lot of women out there not knowing if they should be receiving chemotherapy and radiotherapy when they've had an early diagnosis. I think it's really interesting area and a really important one.

Professor Steve Wesselingh 53:12
Yeah, thanks for that.

This question comes from my infectious diseases background because I was really interested in your type 1 interferon stuff.

A long time ago before science knew a lot, people did actually treat other things by giving people things like biostim, which was just smashed up bacteria, which obviously just stimulated cytokine pathways, including interferon.

Have you thought about whether, I mean, you're having trouble getting the type 1 interferon pathway activated, what about giving people a viral infection?

Professor Belinda Parker 53:48
In triple negative breast cancer, actually 1 of our trials is using a virus. Dexede virus

Professor Steve Wesselingh 53:52
All right, well, there we go.

Professor Belinda Parker 53:56
But in the early treatment setting that that could be an option, but my preference is tailor agonist because you don't have all the other issues with giving a virus.

But the that would maybe work in an early treatment setting. But the responses that are required to those viruses are still those pattern recognition response pathways and they're lost in in in the bone metastasis.

What we did do was in triple in early breast cancer, we asked Sherene Loi to go back to her patients and see of her patients who had an infection post mastectomy or who had some sort of documented infection and did that actually result in a decrease in their risk of metastasis? Just thinking of a natural sort of way to look at it.

But in that particular study, again, we're only looking at a site, it depends if it was localised or became systemic of course. We only, the only thing that could predict metastasis was the interferon signature. That study didn't tell us what we wanted to look at. But we haven't been able to do that properly.

But I guess my opinion is that the TLR agonists work really well. We once did a single intra-tumoral injection into a tumour with a TLR 7-8 agonist and it completely wiped out metastasis, even though we only gave 1 injection.

We’re really interested in that area and then we have to use something else for distant metastasis.

Professor Steve Wesselingh 55:30
Perhaps maybe this will be the last question from Justin.

Can you comment on how you go about establishing overseas collaborations and the importance of these to your research and any advice you could provide to EMCRs about this side of your work?

Professor Belinda Parker 55:41
I've been quite proactive at reaching out to people and speaking to clinicians. You just have to try and make that contact and show them your work and show them how exciting and how excited you are and passionate about it.

For example, with an AstraZeneca collaboration I had, I flew over there and presented to them. I'm quite proactive at talking to people and being associated with clinicians, don't underestimate how that helps you as well, because a lot of them have links with either international pharma, international scientists.

For example, with the DCIS, precision cohort, I was originally, I guess given those contacts through Bruce Mann, who I worked with and how I met Bruce Mann, I went and waited in his rooms to speak to him between patients.

I think making those initial contacts is just having the ability to go and speak to them and say, can I show you my data? Can I tell you my ideas?

Going to conferences is obviously critical. Talking to people at conferences, presenting your work, asking them in advance when you know they're going to be there. Could you please come and look at my poster? I'd really love to show you my work.

Those sort of things as an early career person are really important.

I would make lots of meetings, especially with clinicians, because that's where my work was going translationally. That's actually led to a lot of collaborations with others.

But again, meeting people and telling them as a postdoc, I would write to someone that even had conflicting data to me and say, could you come and look at my poster? It was great and we would actually talk about why you're getting something different. Then I collaborated with that person for years, Bonnie Sloan at Wayne State.

I think that just backing yourself and showing them your data and so that they believe in it.

Professor Steve Wesselingh 57:30
Great answer. Actually, I said it was going to be the last, but this will be the last. What's the best piece of advice you've ever received?

Professor Belinda Parker 57:36
Oh gosh, you couldn't have sent me one that one in advance?

I guess it is just believing that, I mean, this is going to sound really corny, but just believe in yourself. Everyone thinks that someone else knows more than them, that they can do an experiment that you can't do that. They've got, they do more work, they've got more data. It's not true.

We all have something to give. I guess you know, when someone once said to me “you can't do everything”, collaborate with as many people as you can, be open to that.

I think for me, that's probably the best advice I received. I didn't feel like I had to do everything myself. It's all about getting the right people around you and you don't need to know everything. You don't need to be an expert at everything. We all think we don't know much about each other's area. That was probably the best advice.

Professor Steve Wesselingh 58:30
Alright, we might end on that. That's been terrific, the science has been terrific and then the last couple of answers I think really would be great for the early and mid-career researchers that have been listening as well. Thank you, Belinda.

That's fantastic work on a really, really important area and you know, wouldn't it be fantastic by 2030 if no one dies from breast cancer? Wouldn't that be extraordinary?

Then following on from that, maybe we can get to a point where no one gets breast cancer, or we prevent or identify it so early that we can make out it's not breast cancer. Just fantastic.

Thank you very much and thanks everyone else for joining us. It was a great audience and great questions and looking forward to next month's webinar. Thank you.

Professor Belinda Parker 59:17
Thanks everyone.

End of transcript.