New and Potential Clinical Applications of Microbubble Sonography - HD

Introduction

Greetings and salutations.

I'm Dr. Art Fleischer from Vanderbilt University Medical Center, departments of Radiology and O-B-G-Y-N.

It is my honor to present a discussion of new applications of contrast ultrasound, particularly microbubble ultrasound.

And I hope this stimulates some thought and discussion of new applications of this very exciting modality.

Acknowledgments and Disclosures

At the beginning, I would like to acknowledge the very significant impact that one of my mentors, great mentors, Dr. Barry Goldberg and also Larry Waltrip, has made, using this Sona world as a vehicle, but have the Sona world has made a difference in the health of individuals really all around the world.

And I wanted to mention this. Thank you.

I have no relevant financial relationships to disclose, but I might have three others, that are not financial.

If you look at this picture, I'm really not an astronaut, though.

It's hard to believe, I think.

But some people call me spacey at times, but my first name really is not Kevin either.

Dedication

And this presentation I want to dedicate in memory of my mother, because she always, encouraged me to think big.

And here's John Ald Kennedy, describing his wish that the country be dedicated.

As he said, we choose to go to the moon in this decade and do the other things, not because they are easy, but because they were, they're hard.

And I think I put the challenge to everyone to think big.

My mother would've done that too.

Objectives

Objectives, both the diagnostic and therapeutic applications of microbubbles.

First, diagnostic part to look at tumor response, to use ultrasound as molecular imaging to label microbubbles to go to particular areas, to decrease collateral damage, for example, for oncologic treatment therapy, the microbubble can be used, for therapy and delivery of drugs through sono porion.

And, I will talk about this and enhanced treatment of, for example, Alzheimer's or brain tumors that normally there's a blood-brain barrier, which works very effectively.

The microbubbles can be used to improve drug delivery, and this is very exciting.

I wanna also familiarize you with a new term, theranostics, which is a combination of diagnostics and therapy because the border between diagnosis and therapy now is being blurred.

So this is a new term. As I said.

I'll talk about tumor response assessment with microbubbles labeled microbubbles and therapeutic applications.

Research Support

This is my research, support.

Thus far I'm very appreciative of several NIH grants, several grants from the A IUM, discovery grants.

I'm appreciative of Phillips's Healthcare, helping us out, and Broco and latus.

Tumor Angiogenesis Concepts

Okay, firstly, let's get some concepts down concerning tumor angiogenesis.

The picture on the left is an scanning electron micrograph of a few millimeter breast tumor.

And as you can see, this breast tumor has multiple tiny vessels surrounding it.

And it's the theory of Dr. Judah Folkman that in order for tumors to grow from maybe a few millimeters to a centimeter or so, they have to incite a new blood supply neo angiogenesis.

And they do this through what's called Vasogenic Endothelial Growth Factor vegf.

And what it produces is a tumor that has actually very irregular central blood flow.

There's areas of necrosis that occur very early on, and we know that tumors have increased interstitial pressure because they don't have the typical ordered lymphatics that normal tissue has.

Now, if we look at the vessel, process in tumor angiogenesis, again, I've referenced a beautiful article with these, diagrams.

What happens first is that there's fenestration of the basement membrane surrounding the, the small capillary, endothelial cell.

And then the fibroblasts come through this endothelial cell gap, and they set up a scaffold for neo vessels that eventually are, are made patent in the center of the vessel.

And thus, there is new blood supply to the tumor.

This is a comparative scanning electron micrograph, from Peter Choi and his colleagues at the NIH.

On the left, we see an artery going to an arterial, going to a capillary, going to a, a small venous structure, going to a venial.

And this is very organized hierarchy of vessels.

On the right is a comparable scanning electron micrograph of a tumor.

And as you can see, hopefully the new blood vessels are very irregular in their caliber, very clustered, very abnormal.

And if you're a red blood cell, just like going down a highway, you have an interstate going to a smaller road, going to a back road, et cetera, very organized here.

If you're a red blood cell, you just have a whole bunch of blind ending crazy network of vessels.

And of course, at least I think this indicates why we see, increased washout phase, at least in some of the tumors that we see and have studied.

This is a beautiful diagram from Scientific American, an article written by Dr. Jane from Harvard, who is an expert on, tumor angiogenesis.

And I wanna make a few pertinent, comments.

So, diagrammatically, here's the tumor, and here are the blood vessels supplying this tumor.

But tumors produce these tiny, irregular blind ending, chaotic appearing vessels.

Well, why is this important?

Well, because in order to get the best chemotherapy and, trying to understand how to best treat lesions, we can understand that all these abnormal vessels, the concept of these abnormal vessels have to be taken care of.

And perhaps if we get rid of these vessels, we have a more efficient way of treating the tumor.

Again, microscopically tumor vessels are leaky, as shown in this diagram.

They're very irregular.

They're not hierarchical, in, in nature.

So the tumor microenvironment, we have dysfunctional vessels, which I've described, produce conditions of low oxygen, hypoxia, and high acidity.

Well, why is this important?

Because the ability to treat tumors, for example, in radiation, is related to their hypoxic or non hypoxic state.

Radiation in certain chemotherapies that require oxygen to kill are ineffective in tumors.

This is important. Immune cells that might attack cancer cells cannot function in an acidic environment in the tumor without oxygen.

Hypoxia causes changes to gene activity and promotes tumor cell migration in healthy tissues.

The fluid backup tumors, tissue swells, and this is, it, it, this is shown in the clinical world as, lymphedema, for example, causing painful symptoms.

Fluid pressure drives tumor generated proteins and cells toward healthy tissues into lymphatic vessels, increasing the chance of metastases.

So if we kind of understand what is shown in this diagram, I think we understand a little bit about tumors and how to not only diagnose them, but potentially, treat them.

Anecdote on Microbubbles

This is just a funny slide. Dr. Michelle Robin, who is chief of ultrasound in Birmingham, three hours south of Nashville.

And I were walking at the World Federation meeting in Seoul, Korea in 2006.

And we came across this, and I said to my wife, I said, this looks like macro bubbles.

We had just gotten outta session, concerning microbubbles.

Microbubbles and Perfusion Assessment

So, what we're interested in, in microbubbles, these are small structures, about a third, the size of a red blood cell.

Here's the animation showing a capillary, and the blue balls are, conceptually a microbubble.

Now, when we look at a slab of tissue, it would take a hundred vessels, with blood flow to get a adequate doppler, signal.

But really in cancer, the action is at the capillary level, not at the larger vessel.

So that if we could calculate the true perfusion defined as blood flow in ML per second over a particular volume, we could actually understand, tumor dynamics a lot better.

And when we look at the equation, which I'll show you in a minute, for profusion, there's an alpha and a beta.

Well, by understanding what we're seeing, with microbubble profusion, we can actually calculate these two parameters and thus come up with a estimation, a very close estimation, relative estimation of perfusion.

Okay, the microbubbles are small.

They're made up of a central gas surrounded by a lipid, shell.

And their diameter is one, is two to 15 microns.

Understanding a red blood cell is about seven microns.

And when exposed to ultrasound, they resonate, they oscillate.

They, as you can see here from this diagram, they can go from five microns to 50 microns, in their oscillation.

It's this oscillation that produces a harmonic that allows us to image them relative to surrounding echoes.

So our, our signal over, over noise is much better because these microbubbles, can be imaged with harmonics.

This is a picture of the definitive microbubbles.

And as you can see, they're pretty homogeneous in size.

And, and you can see them compared to the micro, to the scale at the bottom, which is five microns.

Now, this is a picture courtesy of Dr. Caskey and his colleagues at uc, Davis.

And this is a picture, kind of a animation, kind of a neat animation of the microbubble oscillating, and then breaking.

So to estimate profusion, as I mentioned, this is the, formula that we're, we're, looking at the alpha and the beta.

Now, to do accurate assessment of perfusion, one has to get the iv, ready and, and infusion rather than a bolus injection.

And, achieve a steady state, then increase your mechanical index, which basically breaks all the bubbles.

And then you watch the reperfusion, and you can calculate the beta from the slope of this line, and the, I'm sorry, the alpha from the slope of the line and the beta from its height.

And you can come up with a number that roughly quantitate perfusion.

Tumor Response Assessment

So let's look at the first new application that is tumor response.

There's a group of physicians in France at their cancer hospitals, led by Dr. Nancy LaSalle.

This was first, very nicely detailed in an article in radiology in 2011 where they studied patients with hepatocellular carcinoma and their response to anti-angiogenic treatment.

And she found that the waveforms and analysis that I went over were quite predictive of tumor response.

And in cancer, what's called the progression-free interval.

And in fact, overall survival.

So the microbubble profusion assessment, does correlate to both short term and long term tumor response.

A similar study was reported by Williams, looking at the, tumor response of microbubbles, a tumor response to an, to anti angiogenic, agents in renal cell carcinomas.

And he found that the findings on microbubble profusion actually predicted tumor response much earlier than changes in tumor size, which is the resist category used for CT assessment of tumor, response.

There's, other papers that have used this, and I've, including them here.

There's a recent paper on perfusion changes in cervical carcinoma with, with, successful anti, with oncologic treatment.

And of course, there's also some very nice papers describing this technique for preclinical studies.

For example, if you're gonna study whether or not a oncologic drug is going to be effective, it's important to know that.

And these are preclinical IE experimental or animal studies that have reported the use of a particular labeled microbubble.

To assess this now,

I wanna just show you our very limited, admittedly so, clinical experience with a drug that was made as an anti-angiogenic treatment for HEPA cellular carcinoma.

These patients had failed standard hepatocellular carcinoma, chemotherapy and treatment.

So let's look at these, patients.

And I must acknowledge Dr. Andre Ock, my colleague, who did, the vast majority of this work.

What we have here is a 3D color Doppler image of a patient that has a paracellular carcinoma.

We see this in the long axis, for example, in the orthogonal plane, coronal plane, and with 3D color Doppler.

This is pre, pre-treatment.

The volume was calculated at 19 cubic centimeters.

Now, at we study these patients at 15 days post-treatment.

I think you can appreciate that this tumor, has really not changed in, in size.

Now I'm showing the video of at day zero, the tumor, and this is the fundamental image and the harmonic image.

And I'm gonna show this to you one more time.

So we start at the echogenic, appearance of breaking all the bubbles, and we put a region of interest here.

And our software is doing the, the work, calculating the alpha and the beta.

This is the same patient, same mass at 15 days post administration of drug.

And obviously it doesn't really look like much difference to the eye, but when we calculate the bolus perfusion, okay, this is intensity over time.

The blue line is the pre-treatment, diagram.

The red is the post-treatment at 15 days.

You can see there's a marked decrease in profusion if we do our destruction.

Reperfusion, sequence.

You again, you see that even though the the mass has not changed in size, this is indication of a responder.

And here's the side-by-side graphs and the side-by-side comparison of the volume, the contrast enhancement in DB decibels, microvascular density, decibels, and the blood velocity.

So this is a good responder.

This is another patient that has an hepatocellular carcinoma being treated by, anti-angiogenic treatment.

Here's the baseline 3D images.

This is at day 15.

The volume has not changed, but let's look at the video, the video again, if we had to do this just by eyeballing, it doesn't look like a big difference.

This is at the, baseline.

And now, at day 15, this, this tumor here.

So visually it doesn't look like much difference.

But let's look at the graphs here.

The yellow is pre-treatment, and the red is at day 15.

You can see a nice decrease in blood flow or profusion, and a similar decrease in profusion on the destruction profusion sequence.

And here's the side by side comparisons using, a different color scheme, but the information is very similar.

What does a poor responder look like?

And here's a, again, an HCC right here.

This is after the initial injection and the killing of the microbubbles with high mechanical index.

And here we see perhaps a difference in side-by-side profusion.

Obviously, this has to be quantitative using offline analysis.

And here we have our bolus, and this is pre-treatment, post-treatment, I'm sorry, this is pre-treatment and then post-treatment.

And you could see that the post-treatment is actually more flow than the pre-treatment, pre-treatment post-treatment, poor responder.

And if we quantitate in this, in this fashion, our three cases we see there's a significant difference in the non-responder.

Obviously, this has to be tested in larger series, but if you look at the data from AL in France, where it's used clinically every day, multiple patients, this looks very promising.

Unanswered Questions

So there's still a lot of unanswered questions.

What defines a good, poor, or fair response?

And according to Dr. Lasso, the area under the curve, the a UC, if there's a 40% reduction, that is a good responder.

How does this correlate with clinical response and survival?

Yes, in Dr. Lassos very large series, it does.

How about multiple lesions, which one to select?

We don't really know how, how about the effect of areas of necrosis?

Another question to be examined.

Does the treatment decrease interstitial pressures and improve hypoxic areas?

At least theoretically it's possible. Okay.

Other Applications of Microbubbles

Some other applications of microbubbles that have been recently reported sono pation, that is use of microbubbles to get drug across that gap.

Junction has been utilized in theory in patients with pancreatic cancer, which we know is a very deadly disease.

The typical diagnosis to, demise, time is three to five months.

So anything that could improve would be great.

And in a very small series of 10 patients in Bergen, Norway, they report that their patients that had microbubble administered, actually survived up to a year.

Very exciting. How about in pediatric, patients that unfortunately have been refractive to typical chemotherapy.

This work being done by Beth McCarville from St. Jude's Hospital in Memphis, it seems that Memphis always gets confused with Nashville.

Memphis is where Elvis lived.

It's in the west part of the state, about two hours west of Nashville.

Anyway, she reported that the contrast enhanced ultrasound predicted which patients would be responsive to chemotherapy in her series of 13 unfortunate, children with advanced cancer.

There is has been discussion of the use of contrast enhanced ultrasound and in fact, sheer wavy elastography in finding, cancer foci in the prostate.

This was described in urology today in September.

Future Opportunities and Molecular Imaging

So the future opportunities for microbubbles, and this of course, goes into the area of treatment.

We have a diagram of a microbubble with the lipid and a targeting ligand on the end.

It could theoretically be told to go to areas where there's a complementary, ligand.

So we can quantitate profusion.

We can monitor angiogenesis as we know the tumors grow, where there are blood vessels that are rapidly growing.

We could target drug therapy theoretically, and we can enhance, gene therapy.

This is a diagram courtesy of Evan Unger from Arizona.

And the I'm a, rx um, company.

So what are we doing? This is a diagram showing the, typical vessel endothelial cell and an antibody to VEGF that is, that is hanging off the luminal side of these cells.

Now, when we inject a contrast that has these, a ligand, a microbubble, that has a ligand attached to it, we let it flow.

And where there is a match, the microbubble stays, otherwise it there, there's flow in the, the, the microbubbles just pass through the blood supply at seven minutes only the blood bound protein, the VEGF, for example, microbubbles remains.

This has shown electron microscopy and with optical image from this group.

And we can see the red dots, in fact are microbubbles in vessels that are neoplastic diagrammatically.

This is what goes on.

Here's a picture of an endothelial, cell in a tumor, with the, mi the antibody, the, VEGF receptor.

And when there is, the, the, when there is VEGF finds this receptor, angiogenesis can continue.

However, if there's blockage, this theoretically would block tumor growth.

This is a diagram that Andre made now several years ago of the VGF receptor, the second VEGF receptor.

Here's the microbubble that is so-called glue.

Part of it is the bison strep adin.

And this is the anti-VEGF two to show kind of the concepts of molecular imaging.

And this is a diagram, maybe a fancier diagram.

Of course, it cost us much more money than the one that Andre, did himself.

So how do we look for this labeled, imaging using microbubbles?

We inject, this is in a mouse model, and we, have a total a signal here.

And at a point in time, further out, we use high mechanical index and we destroy the bubbles.

While the bubbles that are still attached are the bubbles that, we're interested in.

Here is a picture from a vivaan vionic image.

These are millimeters, not centimeters.

This is a, a tumor that's implanted in the thigh that Andre did.

And you can see, some echoes, but the green actually, shows where the vessels inside of this tiny tumor, are.

And this is, again, with a very high frequency, scanner.

So, this is another picture of, the bubbles coming in, producing the, the image in green of the areas of tumor vessels.

And so Andre and his, colleagues, several years ago, showed that there was a difference in tumors with very high VEGF and tumors that have relatively low VEGF.

And this is written up in Journal of Ultrasound in Medicine.

So again, the concept that the targeted microbubble can perhaps even contain medication in its center, go to the area of the tumor, so-called magic bullet.

Increase mechanical index, break the bubble, and deliver the bubble into the tumor in interstitium.

This is a, very nice, picture of such a micro bubble where the center is gas.

And of course, there's thought about replacing what's in the center, what we use as a gas, per fluorocarbon is used in most microbubbles, a sulfur compound as well.

Maybe if we could use oxygen, deliver more oxygen to the tumor, we could decrease, well increase its radiation, sensitivity, for example.

And this is a study that is ongoing, a discovery study at our place.

And we're testing locally delivering the chemotherapy agent to the xenograft and applying radiation and looking at differences in, in, animals where the tumor had, in fact, the microbubbles.

And those were, there was no microbubbles.

So the concept is to locally enhance, oxygen and make it more radio sensitive.

Therapeutic Applications

This is also work done by Dr. Charles Caskey at our Vanderbilt Imaging Institute.

Diagrammatically. What should, what goes on?

This is a endothelial cell, with the vascular gaps.

Here's a microbubble.

When the ultrasound is turned on, the microbubble swells, it oscillates asymmetrically, and then when it breaks, it forces a field, a, a jet of fluid to go across the gap junctions.

And this is kind of pictures, neat pictures of what occurs microscopically when the bubble enlarges and when it breaks, it produces these little eddy currents.

Well, think about it.

If you have drug in the lumen of the vessel and you're trying to get it to the interstitium, the microbubbles can enhance that.

This was a study that he did in his group at, university of of California at Davis, where they had tumors in, in mice, and they let them grow.

And at day, at week three, they, treated them with the microbubbles.

And normally these tumors would just continue to grow.

But he observed and others that in some of the tumors, there was a rapid decrease in the profusion.

And so theoretically, what the microbubbles are doing is they're helping to deliver drug across the blood-brain barrier.

Very exciting work.

Well, another concept that is very exciting is, the use of microbubbles to get antibodies to these amyloid plaques in Alzheimer's disease to enhance the transport of the antibodies from the vessel lumen out into the interstitial across the blood brainin barrier to attack these deposits of amyloid characteristic of the abnormalities seen in in Alzheimer's.

Admittedly, this is in a mouse, model, but it looks extremely exciting that the micro bubbles can enhance, drug delivery across these gap junctions.

This is a group from Wrist vein Australia.

Conclusion

So in conclusion, I would like to just, have you understand this and give it, give much thought to it, that contrast enhanced ultrasound, we know is excellent for the differential diagnosis of liver lesions, renal lesions, and ovarian masses.

But the microbubble ultrasound combination has potential to assess tumor response, and this in initial phases seems to be better than looking for changes in size and tumor on CT or MRR or possibly pet.

We don't know. There's a potential to enhance specifically targeted therapy.

And this term is called, theranostics combination of diagnosis and therapy.

And there's a potential for using micro bubbles.

I didn't describe this, but for breaking up a blood clot, and I did basically mention the use of micro bubbles to attack, to let, antibodies go across the blood brain barrier to attack the amyloid in Alzheimer's disease.

Acknowledgments

I wanna thank my colleagues and acknowledge them.

Charles Caskey, Vanderbilt Imaging Institute, Dr. Andre Ach, who is now at Jefferson, but did some pioneering work when he was at Vanderbilt, and I'm fortunate in continuing to collaborate with him and the group at Jefferson.

The various companies that have supplied the microbubbles and software and drug.

In one case. I want to acknowledge our media specialist, John Bobbit, and I wanna thank the directors of these areas.

Dr. John Gore, who's the director of the Vanderbilt Imaging Science, Dr. Reed Oy, who is chairman of Department of Radiology.

They set up an environment where someone who is primarily a clinical person can be involved in clinical research.

And thank you for listening, and I hope this presentation is both informative and thought provoking.

Thank you.