Refining Approaches to Overcome Challenges, Enhance Consistency, and Standardize Scoring and Reporting in HER2-Expressing Solid Tumors

[00:04:16]   

          Brief Overview of HER2 Signaling

So we'll start with a brief introduction of HER2 signaling in general. And HER2 signaling pathway is a key signaling pathway in cancer biology that we are all very familiar with. So I'm going to focus only on the information that's relevant to the rest of our discussion.

So HER2, also called ErbB2, belongs to the ErbB family of transmembrane tyrosine kinases. And the other family members include ErbB1 or HER1 and also called EGFR, ErbB3 or HER3, and ErbB4 or HER4.

Unlike other family members, the activation of HER2 does not rely on small molecule ligand. And the activation of HER2 is induced by dimerization of HER2 with itself, homodimer, or with other family members in heterodimerization form. And it’s the heterodimerization provides the most potent signaling of the entire pathway. And the downstream pathways include the MAP kinase pathway and PI3K/mTOR pathway, among others. And this leads to cell proliferation, survival, and angiogenesis.

And HER2 overexpression has been identified in multiple cancer types, and overexpression of HER2 provides overstimulation of all these downstream signaling pathways. And that as a result, there is uncontrolled cell proliferation, increased cell migration, and resistance to apoptosis. So HER2 is a really the key regulator, the master regulator of the entire pathway, and it's a prime therapeutic target.

There are multiple forms of HER2 therapy, such as monoclonal antibody, small molecule tyrosine kinase inhibitor, and antibody drug conjugate.

Another useful information I wanted to highlight on this slide is the mutation status of the RAS and RAF genes can be relevant in - when you're dealing with certain type of cancers, such as colorectal cancer. The RAS or RAF gene mutation lead to persistent activation of MAP kinase pathway that bypass HER2 signaling. And as a result, this renders resistant to HER2 therapy.

[00:06:47]

Tumor Agnostic Prevalence of HER2 Alterations (Mutations and Amplifications) in GI Cancers

So the prevalence of HER2 alterations varies significantly across cancer types with breast cancer and esophagogastric cancer has the highest rate of HER2 amplification. And lung - HER2 mutations play an important role in lung cancers, which we'll discuss later in the talk.

But within the GI system, about 10% to 30% of esophagogastric cancer harbor HER2 overexpression. And HER2 alterations also occurs to other GI cancers such as colorectal cancer, biliary cancer and pancreatic cancer, but with a much, much lower rate. And also in these cancer types, the percentage of mutation is relatively higher when you compare to that of esophagogastric cancer.

[00:07:40]

Guideline Recommendations for HER2 Testing in Advanced/Metastatic GI Cancers

So HER2 testing is critical in identifying patients who would benefit for HER2-targeted therapy. And for esophagogastric cancer, all patients with advanced and metastatic disease is recommended to have HER2 tested. And for colorectal cancer, HER2 testing is recommended for patients with wild-type RAS and RAF gene status with advanced disease who failed or progressed on standard therapy.

The testing indication for biliary cancer and pancreatic cancer is not as well defined, but we should consider HER2 testing for patients who have advanced or metastatic disease with limited therapeutic options.

And then the next question is, how do we test for HER2? Immunohistochemistry and in situ hybridization is the most common and the most well established testing method for HER2 overexpression. But next-generation sequencing is also a very useful tool if it is available to you, to the patients. And the next-generation sequencing not only provide HER2 amplification status but also provide information regarding HER2 mutations and along with alteration status of a long list of other therapeutic targets.

[00:09:08]

HER2 Immunohistochemistry Scoring: ToGA Study

Then how do we score HER2? So ToGA study is a landmark study that really established the HER2 scoring system for esophagogastric cancer. And the ToGA study also recognized the heterogeneity of HER2 expression in these cancer types. So 2 separate scoring systems are established. One is for surgical resection specimens, which usually come with larger amount of tissue. One is for biopsy specimens that usually come with smaller, very limited tissue.

So a 3+ HER2 score means strong complete basolateral or lateral membranous reactivity in more than 10% of tumor cells, in resection specimens. And for biopsy specimens, we only need strong basolateral or lateral membrane staining in any number of tumor cells. This—recognizing the heterogeneity of HER2 expression in these tumors.

And the 3+ staining is considered positive. And 2+ refers to weak to moderate complete basolateral or lateral membranous reactivity in more than 10% of tumor cells in resection specimens, or in any number of tumor cells in biopsy specimens, 2+ is considered equivocal, and this should be followed by in situ hybridization testing to further evaluate the amplification status of HER2.

And then 1+ refers to faint or barely perceptible membranous thinning in more than 10% of tumor cells in resection specimens, or in any number of tumor cells in biopsy specimens. And zero means no reactivity in less than 10% of tumor cells in biopsy - in resection specimens, or in any number of tumor cells for biopsy specimens. Both zero and 1+ are considered negative results.

[00:11:13]

HER2 Testing: Colon and Other Sites

For colorectal cancer, the HER2 storing system is largely similar as what we described for esophagogastric cancer, with a different cutoff. Here, the cutoff of positive staining cells is 50%. So 3+ positive result means intense membranous staining either circumferential, basolateral or lateral in more than 50% of tumor cells. So this increased cutoff for colorectal cancer increases the specificity of HER2 testing for colorectal cancers.

The HER2 testing, the scoring system is less well established for pancreatic and biliary cancers, and different cutoff points, including what being used for breast cancer, for GEJ cancers have been used in previous and ongoing trials, so direct comparison among these studies can be difficult.

Another challenge is the heterogeneity we mentioned earlier that has been observed in all GI tumors. And especially when you're dealing with biliary cancer and pancreatic cancer, you often have very limited sample. So this heterogeneity really affect the positivity rates when we're evaluating them with IHC.

And then although most alterations involving overexpression and amplification, active missense mutation of HER2 also represent a significant subset of HER2 alteration in GI system, especially colorectal, pancreatic, biliary. And the HER2 mutation would be missed by standard IHC and ISH study.

[00:12:58]

          Intratumoral Heterogeneity and Incomplete Membrane Staining of HER2

So just a few more words on the heterogeneity of HER2 expression in GI tumors. And it's well recognized that in contrast, breast cancer tend to have very homogeneous uniform staining, and the staining pattern tend to be complete membranous staining. But for GI, most of the GI tumors, we observe a more patchy staining pattern. And the positive cells often show basolateral or lateral staining only, or in other words, incomplete membranous staining.

[00:13:35]

What Are Key Differences in HER2 Testing inBreast and Gastric Cancers?

Some other interesting differences between HER2 testing in breast and gastric cancer that include correlation with morphology type. So HER2 overexpression seems to occur at a higher incidence. For gastric and GEJ cancer with intestinal morphology, where tumor with glandular differentiation. And for breast cancer, we observe more HER2 overexpression in higher grade and more aggressive type of breast cancer.

And HER2 overexpression seems to occur at a higher rate - slightly higher rate in GEJ cancers comparing to the other locations of the upper GI tract. And there is no correlation regarding location in HER2 expression for breast cancer.

[00:14:31]

HER2 Mutations vs HER2 Overexpression/Amplification in GI Cancers?

And going back to HER2 mutations vs overexpression or amplification, the current approvals in gastric and GEJ cancers and colorectal cancers are all based on trial criteria that requiring protein overexpression by IHC and the FISH. So only overexpression and amplification correlates with response in current data.

And also another interesting point - important point is bypassing IHC to test for FISH only is not recommended. And the rate of HER2 mutation varies by tumor locations and show varying prevalence of overlap with amplification. And this is important as the presence of HER2 mutations may affect response to therapy when they coexist with overexpression and amplification.

[00:15:31]

          AI, Deep Learning, and HER2 Scoring: Challenges and Limitations

So I think another hot topic for HER2 scoring is AI and the deep learning tools. This is definitely a very promising tool, but it's not without challenges and limitations such as like model training, algorithm development, validation, regulatory approval, and how to integrate it to our clinical workflow. And - and also very importantly, ethical and the legal considerations.

[00:15:58]

          Future Directions

And I think for the future, like AI tools and deep learning tools, it definitely will be used in our routine practice, especially multimodal AI that combine IHC and the ISH data with other biomarker evaluation for comprehensive cancer profiling. This has been studied extensively in breast cancer, and this will be available for cancer - other cancer types as well.

And AI model will be very useful for detect HER2-low expression because HER2-low has been very challenging topic for pathologists, and I think the AI tool will provide a more uniform and more accurate evaluation.

And hopefully in the near future, HER2 scoring, along with other AI tools, will integrate to our routine workflow that increase our efficiency and accuracy. And also it provides an opportunity for global standardization and harmonize all HER2 data across different laboratories and regions.

[00:17:13]

Let’s Return to an Earlier Question

So with that, let's return to our earlier question.

[00:17:16]

          Posttest 2

Which of the following staining pattern would constitute a HER2 IHC 2+ finding in a tumor cell cluster of a gastric cancer biopsy sample, suggesting the need for additional FISH testing?

So I think more people picked the second answer, which is the correct answer.

[00:18:01]   

          Posttest 2: Rationale

And the - the correct answer is weak to moderate complete basolateral or lateral membranous staining activity and overall heterogeneous staining pattern. And this is correct because 2+ is defined as weak to moderate basolateral and lateral membranous staining. And the samples from GI tumors tend to be very heterogeneous. So this - the second option fits best for the question.

And with that, I'm excited to move on to the next topic.

[00:18:38]

HER2 Alterations and Testing in NSCLC

I'll hand the floor to Dr Hung. Dr Hung is Associate Professor of Pathology at Harvard Medical School and an expert in thoracic pathology and biomarker testing. Dr Hung?

Dr Yin Hung (Harvard Medical School): Well, good morning, everyone, and nice meeting all of you. I'm not sure many - how many of you are path - thoracic pathologists here?

[00:19:01]   

          HER2 Mutations: Breast Cancer vs NSCLC

So HER2 is a new therapeutic biomarker and target in non-small-cell lung cancer. I will say a lot of our insights about HER2 biology was first derived in from breast cancer many, many, many years ago, in which one can interrogate HER2 status using either immunohistochemistry or using fluorescent in situ hybridization, FISH, to look for overexpression or amplification, respectively.

In lung cancer, it's a slightly different paradigm and concept. So the one particular way HER2 can drive certain subset of lung cancers basically by activating mutations. So DNA mutation, which I'll talk about later. It's a certain aspect that one should consider, as well as overexpression. And I will say the data on the gene amplification that is still ongoing.

[00:19:52]   

          Differences in HER2 Alterations in NSCLC

And so differences in terms of HER2 alteration lung cancer. So Dr Zhao already mentioned some HER2 biology. But HER2 is a transmembrane protein that's expressed on the cell surface. And - and it can be overexpressed in a subset of non-small-cell lung cancer.

In addition, HER2 activating mutation that involves exon 20, in particular can be seen in a subset of lung cancer. And then finally, as I mentioned before, HER2 amplification as well.

[00:20:18]

          Distribution of HER2 Mutations

And so specifically for the HER2 mutation that one can see in non-small-cell lung cancer, a lot of them—as you can see from the chart here, the majority of them over about 48%, involve an in-frame insertion, deletion or mutation that involved the exon 20. And so that comprises basically the majority of HER2 mutated non-small-cell lung cancer subtype.

But there can be other alterations that one can see, for instance, rarely involving the extracellular domain.

[00:20:55]

          What Is HER2-Positive NSCLC?

And so, in terms of HER2-positive non-small-cell lung cancer. So for mutation, one can see overall in about 2.6% of non-small cell lung cancer, non-squamous non-small-cell lung cancer, more commonly in never smoker and HER2 overexpression as defined by 2+ or 3+, HER2 IHC, that can be seen in H2 23% of non-small-cell lung cancer, in particular for the really high expresser 3+ that's seen in 4%.

For HER2 gene amplification, depending on cohort, one can see - depending on also how one defined the cutoff for the gene amplification, one can see in 7% to 22%. And so I think that the importance about HER2 recently is given that it's an actionable target given the new data on T-DXd that I will talk about later.

[00:21:46]

DESTINY-Lung02: Trastuzumab Deruxtecan in HER2-Mutated NSCLC

And so here I'll transition to talk about the T-DXd, trastuzumab deruxtecan. So recently there are a series of clinical trials, the DESTINY trials. So this - in this DESTINY-Lung02 trial, one can see the waterfall plot that, there's a good OR response for patients with HER2 mutated non-small-cell lung cancer regardless of whether the mutation is in the kinase domain or in the extracellular domain.

And you can also see at the bottom that some of these patients actually might have received other prior lines of therapy, for instance, even immunotherapy or systemic therapy. And still one can see a good waterfall plot.

[00:22:23]

DESTINY-Lung01: Trastuzumab Deruxtecan in HER2-Mutated or HER2-Overexpressing NSCLC

And so now this is again another waterfall plot. But here we're talking about HER2 overexpression of non-small-cell lung cancer. And as I mentioned before, it's defined by 2+ and 3+. And again, one can see from the waterfall plot that both 2+ and 3+ HER2-positive non-small-cell lung cancer show response to trastuzumab deruxtecan.

[00:22:46]

HER2-Positive NSCLC: Prognosis and CNS Metastases

And so now here this is talking about - this slide I just showed some data on the prognosis and the idea about CNS metastases in non-small-cell lung cancer that's positive for HER2. It’s associated with high incidence of brain metastases during treatment, then KRAS or EGFR mutant diseases. And the HER2 exon 20 insertion mutation, that's associated with particularly aggressive disease with a poor outcome.

And separately, HER2 overexpression is also known to be associated with worse overall survival and disease-free survival.

[00:23:26]

Testing for HER2 Mutations in NSCLC: The Role of Molecular Profiling

And so now I'm going to transition to talk about how we can test for HER2, the role of molecular profiling and later talk about immunohistochemistry as well.

[00:23:33]

HER2 Testing Guidelines for Lung Cancer

And so for HER2 testing guidelines in lung cancer. So shown here, this is the blurb from the NCCN guideline. So for HER2 mutation, it is recommended as part of a broad molecular profiling using next-generation sequencing in patients with metastatic nonsquamous non-small-cell lung cancer. And it's also noted that test for HER2 mutation can be considered in patients with metastatic squamous cell carcinoma.

And then for HER2 amplification, that's particularly considered in the context of patients with acquired resistance to, for instance, EGFR mutant lung cancer with prior treatment to EGFR tyrosine kinase inhibitor, in a way to test for alternative mechanism of resistance.

[00:24:21]

          Guidance for HER2/ERBB2 Testing in Advanced NSCLC

And so here, some of you may already know this for non - advanced nonsquamous non-small-cell lung cancer. It can be driven by a variety of gene mutations, oncogenic drivers: EGFR, KRAS G12C, G12D and other alterations, ALK rearrangement, ROS1 rearrangement, BRAF V600E and non-V600E mutation, NTRK rearrangement, METex14 skipping, RET rearrangement, NRG1 rearrangement. And then HER2, the new kid on the block right - right at the bottom.

And so for the majority of these drivers, a lot of them actually mutually exclusive. So if you have a patient with a non-small-cell lung cancer with EGFR mutation, you're not going to find ALK or ROS1 and - and others. But HER2, I would say our data on it is still - is still ongoing. It is known that if a patient had a HER2-mutated lung cancer, you're not going to find the other driver mutations. Whereas for HER2 overexpression and for HER2 IHC positive, those parameters may—it's unlikely to be completely mutually exclusive with these other oncogenic drivers.

[00:25:21]

Biopsy: Establish Diagnosis, Determine Histologic Subtype, Biomarker Testing

And so in terms of what we think about day to day, right, for pathologists, if you get a case from a patient with non-small-cell lung cancer. And so what are you going to do with the tissue and how we think about it? Right.

So first histologic subtyping still is very important, right? The big decision tree is squamous vs nonsquamous because that can affect how we think about all the other biomarker testing, particularly for nonsquamous non-small-cell lung cancer. Then as I mentioned before for a patient with advanced disease, one would test for all these driver alterations ideally using a broad panel. And we also want to sort of test it in a way that thinking about tissue conscious, right. You want to make sure the biopsy is not exhaustive while we test for all these alterations.

For squamous non-small-cell lung cancer, for squamous cell carcinoma, one can certainly consider for testing, particularly in young patients, in those with minimal smoking history, because it could be from a cancer with mixed histology and squamous histology. And of course, in addition, some of you already know this, that for all non-small-cell lung cancer, PD-L1 is also a parameter that one should test for as well.

And also it is recommended to wait for the NGS result before acting on the PD-L1 result because, particularly for patients with known driver mutations with EGFR or ALK rearrangement, those patients might receive those TKI instead of immunotherapy, regardless of the PD-L1 results.

[00:26:45]

Select Principles of LDT Validation From the CAP Pathology/Laboratory Quality Center

And so now here, for some of you, you might send your HER2 out to a reference lab. Or maybe if you have a companion diagnostic, you perform your HER2 testing in-house. But if you want to develop a laboratory developed test for LDT for HER2, and that certainly can be done. Many academic centers and laboratories do that.

And so I'm just putting this slide from the archive talking about the general principle on validating laboratory developed tests that one want to make sure that achieve at least 90% overall concordance between your new test that you're trying to develop and the gold standard. And ideally, you want to use a variety of tissue that recapitulate how you test for the - how you practice it clinically. So you want to use whole sections and not simply rely on using tissue microarray.

[00:27:36]

Optimal Preanalytical Conditions for IHC

And so here this is the preanalytic condition. I think, you know, a lot of us know about it. And there's certainly a lot of preexisting literature about HER2 testing in breast cancer that we want to be very conscious on how the tissue is treated. Right.

And so, we right now test HER2 primarily on formalin-fixed paraffin-embedded tissue. And so we want to make sure the ischemic time is minimal. Use 10% neutral buffer formalin. Make sure that the tissue is fixed appropriately as listed here to maximize the - the tissue condition for proper HER2 immunohistochemistry.

And if you were to get a specimen, let's say from bony tissue metastatic site, avoid strong acid. so using EDTA decalcification reagent can certainly help preserve the HER2 immunohistochemistry status.

And of course, finally just want to make sure that, you know, if you're going to get a block of tissue, ideally cut many slices upfront to basically both. You know, you can use it for HER2 testing, PD-L1, next-generation sequencing rather than cut the block several times because that will lead to unnecessary wasting of tissue.

[00:28:43]

HER2 Expression by IHC in NSCLC

And so for HER2 over - for HER2 expression assessment in non-small-cell lung cancer, there are technically no established standard given how new this - this assessment is in non-small-cell lung cancer. And there are different ideas and different criteria used across trials. And there are multiple FDA approved tests for assessing HER2, but the HER2-positive here, at least in 1 of the trials defined as 3+, although I think 2+ and 3+ is also—can be used in some other instances.

And so you may ask me how are we going to score HER2, right? And so I'm glad I followed Dr Zhao on this. And so in terms of how we score HER2. One proposal is to basically use the GI, GEJ criteria.

[00:29:34]

ASCO/CAP Guidelines for HER2 Classification (Breast)

And so I'm just going to put here that some of you may know about breast cancer, how we score HER2 with what - and then how we use the HER2 status in a way think about the FISH status.

[00:29:44]

HER2 IHC Scoring Depends on Primary Tumor/Specimen Type

And so for - but here I'll just show a table of how there are differences between breast specimen, between gastric GEJ resection specimen and GEJ biopsy specimen. And so in the DESTINY trial, they actually used the GEJ resection biopsy criteria in assessing non-small-cell lung cancer HER2 expression.

And so that's the one that right now the majority of the community and the academic center in the field that does HER2 testing, that's the one that that's most popular.

[00:30:12]

HER2 IHC Score Correlates With HER2 Amplification in NSCLC

And in terms of finally, the underlying biology. It's known that the HER2 immunohistochemistry score, particularly 3+, is strongly associated with the presence of HER2 high copy number gain amplification in non-small-cell lung cancer.

And so here I show this study from modern pathology recently, that this group, scoring a series of non-small-cell lung cancer using the HER2 IHC, regardless of whether they did it on the GI criteria on the left or the breast criteria on the right. Either way, the HER2 3+ is known to be strongly associated with HER2 amplification in non-small-cell lung cancer.

And so with that, I think this is probably the last slide. So in short, think about HER2 in non-small-cell lung cancer, it can be mutation. So one should test for HER2 mutation but also overexpression. And one can use the GEJ criteria. So that was it.

And then now I'm going to transition back to Dr Cleary.

[00:31:09]

Applying HER2 Testing Results to Treatment Decisions for Patients With Solid Tumors

Dr Cleary: Thank you. So I'm going to follow those 2 excellent talks from a pathology perspective, talking about a medical oncologist perspective of HER2-directed therapy in GI cancers.

[00:31:27]

ToGA: Chemotherapy ± Trastuzumab as First-line Therapy in HER2-Positive mGC

And Dr Zhao talked about the ToGA trial. And the ToGA trial was published back in 2010. And this was a time when the GI oncology was hungry, like lung cancer, to bring in targeted therapies to our patients.

And what we did with the ToGA trial is we combined chemotherapy, first-line chemotherapy with trastuzumab. And fortunately, we saw that it was beneficial. It improved overall survival by 2.7 months. So in the GI community, even though 2.7 months is a modest benefit, this was a big deal.

The other thing I will say about the ToGA trial is as time went on, we learned that the more strongly HER2-positive you are, the more benefit you get. So when you look at NGS testing, if you have very, very strong HER2 amplification, like 50-fold, 100-fold, those are the patients who really, really do well.

I saw a patient yesterday who's been on HER2-directed therapy for gastric cancer, who's been on it for 5 years. So there are some outliers who have very, very strong expression of HER2 that can do better than this.

[00:32:33]

KEYNOTE-811: First-line Trastuzumab and Chemotherapy ± Pembrolizumab in HER2+ Gastric/GEJ

However, for most patients the benefit is modest. And this is why the KEYNOTE-811 trial was really important. And this is a more modern trial. So what this did is it built off the ToGA trial, and it took patients who were getting chemotherapy and trastuzumab and asked a question of whether adding pembrolizumab would even make it more effective.

[00:32:53]

KEYNOTE-811: Pembrolizumab/Trastuzumab/CTAntitumor Response at Interim Analysis 3

And what they saw is that the addition of pembrolizumab to chemotherapy and trastuzumab was effective, and the objective response rate increased from 60% to 73%.

[00:33:06]

          KEYNOTE-811: PFS and OS (3rd Interim Analysis)

And when we looked at overall survival, particularly in the PD-L1 population down here in the lower right, we saw a really nice benefit that in patients who - who got the previous standard of care chemotherapy and trastuzumab, the median overall survival is only 15.7 months, whereas when patients got pembrolizumab combined with chemotherapy and trastuzumab, the median overall survival was 20 months.

And because of this trial, this became the standard of care. So for all patients with HER2 amplified PD-L1 tumors greater than CPS score of 1, the standard of care now is chemotherapy, trastuzumab and pembrolizumab.

[00:33:50]

DESTINY-Gastric01: Trastuzumab Deruxtecan in Previously Treated, HER2+ Gastric/GEJ Adenocarcinoma

One trial though, and it's already been mentioned a couple of times. Trastuzumab deruxtecan is an antibody drug conjugate. And the reason this has been so useful in the medical oncology community is patients who get acquired resistance to trastuzumab still can respond to this. Many people think that one of the really important parts of the trial design that led to this approval was they asked the question that after you get treated with, say, something like FOLFOX and trastuzumab, before you go on trastuzumab deruxtecan, you really want to make sure that HER2 is still overexpressed, because one of the resistance mechanisms to a trastuzumab-directed therapy is just losing HER2 overexpression.

So before you put patients on this trial, you had to confirm that HER2, in fact, was still overexpressed. And we do that clinically to this day.

But what you see in this trial is this is second-line. So patients who already were treated with trastuzumab-directed therapy that when patients got trastuzumab deruxtecan, the HER2-directed antibody drug conjugate compared to standard chemotherapy that there was a real benefit here. So that when you look at the median progression-free survival, patients who just got chemotherapy was 3.5 months and patients who got trastuzumab deruxtecan, it was 5.6 months.

[00:35:09]

DESTINY-Gastric01 Biomarker AnalysisT-DXd in HER2+ Advanced Gastric/GEJ Cancer After ≥2 Prior Regimens

And again, this slide really from a medical oncologist perspective, just shows the importance of, yes, you might have diagnosed a patient with a HER2 amplified gastric cancer at diagnosis, and they might have gotten trastuzumab-directed therapy in first-line. But you really need to confirm when you're going to second-line therapy that HER2 still overexpressed because it can be lost in some cases.

So 1 way you could do it is you could do next-generation sequencing on a newly acquired biopsy. But since that's hard, many of my colleagues just do cell-free DNA testing just looking to see if HER2 is still overexpressed.

[00:35:45]

GI Focus: CRC

I want to shift gears to colorectal cancer.

[00:35:49]

          HER2: Overexpressed/Amplified in 2% to 4% of CRC

So HER2 is overexpressed in about 2% to 4% of colorectal cancers.

[00:35:54]

HER2+ mCRC: A Distinct Subset

Interestingly, it's really seen primarily in left-sided colorectal cancer. So mainly in rectal cancers and sigmoid cancers. Similar to what you've already heard in breast cancer and in lung cancer, we do see a higher incidence of brain metastases in these patients. 80% of colorectal cancers that have HER2 amplify - amplification are RAS and BRAF wild-type.

Interestingly, though, we're starting to pay attention to the 20% of HER2 amplified CRCs who have a co-occurring oncogenic KRAS mutation. Because data is emerging that trastuzumab deruxtecan actually can be effective in that population.

And then finally, HER2 amplified CRC is unfortunately microsatellite stable. And I say unfortunately because we can't give these patients immunotherapy.

[00:36:45]

MOUNTAINEER: Tucatinib + Trastuzumab in Previously Treated HER2+ Metastatic Colorectal Cancer

So there's been several trials looking at this. But the 1 regimen that's FDA approved is tucatinib and trastuzumab. So to talk about tucatinib and trastuzumab, trastuzumab is an antibody that binds to HER2 in the extracellular space. But tucatinib actually hits HER2 in the kinase domain. So this is a way of hitting HER2 in 2 different spots.

And conceptually, I think this is a nice therapy because it's harder to develop resistance when you're putting pressure on HER2 in 2 different ways. And this trial was extremely effective. The overall response rate was 38%. The median overall survival was 24 months.

And similar to gastric cancer, we're finding that the patients who do really well, the outliers are patients who have really strong HER2 overexpression. So I'm taking care of a patient who has next-generation sequencing 120-fold amplification of HER2. And he's been on this treatment for four years.

[00:37:43]

          Recent Data on HER2-Targeted Regimens for mCRC

There are multiple HER2-directed therapies that are approved in the NCCN guidelines.

[00:37:50]

          DESTINY-CRC01: Trastuzumab Deruxtecan in HER2-Amplified mCRC

But the one I wanted to focus on again was trastuzumab deruxtecan. And this concept that trastuzumab deruxtecan can overcome acquired resistance is not only true in gastric cancer but also is true in colorectal cancer.

And when you look at the lower part of this graph over here, it talks about patients who got previous HER2-directed therapy. And here you'll see that even patients who got previous HER2-directed therapy, the response rate to trastuzumab deruxtecan is 43%, which is incredibly impressive. And the reason I get excited about this as an oncologist is this way I can use that HER2 target much longer in my patients.

[00:38:31]

Trastuzumab Deruxtecan Can Overcome Resistance to Trastuzumab-Based Combinations

And this is a patient that I took care of, and I published with my colleagues Harsh Singh and Jason Hornick. And it shows a patient with colorectal cancer, like most patients with HER2 amplified colorectal cancer, she had a rectosigmoid cancer. She had 141 copy amplification, and she was on trastuzumab and lapatinib, another HER2 kinase inhibitor, for 4 years.

And then we saw using cell-free DNA that she developed several resistance mutations that made this stop working. She had a HER2 resistance mutation and 3 – PIK3CA mutations. But despite this, and you see this at the edge of this curve over here. When we put her on trastuzumab deruxtecan, she still was able to respond.

[00:39:18]

GI Focus: BTC

Zanidatamab (ZW25): A Humanized IgG1-like Biparatopic Antibody

Finally, the new kid on the block is biliary tract cancers. And zanidatamab was just FDA approved for biliary tract cancers in December. And zanidatamab is a very interesting antibody to HER2 because it's a biparatopic antibody. And the idea here is zanidatamab has been designed to bind to 2 different areas of the HER2 protein. And that's thought to cause cross-linking and more efficacy in terms of targeting HER2.

[00:39:50]

HERIZON-BTC-01: Zanidatamab for Previously Treated HER2‑Amplified Biliary Tract Cancer

And the results were this were impressive. So in the HERIZON-BTC-01 trial, they tested zanidatamab in previously treated HER2 amplified biliary tract cancers. And they saw a response rate of 40%. One of the reasons as someone who takes care of biliary tract cancer patients that this is impressive to me is typically when we think about cytotoxic chemotherapy in second-line biliary tract cancers, the response rate is like 5%. Second-line chemotherapy really is very poor.

And seeing a therapy that we can give in second-line that has an overall response rate of 40%, it's just wonderful. A really good option for our patients.

[00:40:30]

Zanidatamab BTC Approval

So as I said, this has now an accelerated FDA approved - FDA approval for biliary tract cancers.

[00:40:36]

          Zanidatamab: Key Select Trial Data Across Solid Tumors and Ongoing Phase III Trials

And there are multiple trials going on with zanidatamab in other GI malignancies, including combining with chemotherapy and gastroesophageal cancer and also combining with first-line chemotherapy in biliary tract cancers.

[00:40:49]

Trastuzumab Deruxtecan Has Activity in HER2-Positive Biliary Tract Cancers

And then finally, again going along with this theme that trastuzumab deruxtecan can target HER2 therapies and overcome acquired resistance, trastuzumab deruxtecan also has activity in biliary tract cancers.

[00:41:04]   

          Additional Select Ongoing/Planned Phase III Trials With HER2-Targeted Therapies

Here are HER2—I actually—in thinking about clinical trials for GI cancers, HER2 has really shown that this is a therapy we could - this is a target that we can really get a lot of leverage out of and really can help our patients. So the pharma community is really developing many, many agents targeting HER2. So this is a really crowded space. But it's wonderful. But I think we'll have more and more therapies for our patients in the future.

[00:41:29]

Let’s Return to an Earlier Question

So I want to return to an earlier question.

[00:41:34]

          Posttest 3

For patients with HER2 IHC 3 - 3+ metastatic biliary tract cancer that has progressed after first-line gemcitabine and cisplatin with immunotherapy, which of the following would be an FDA approved option for targeted therapy? And the answers are listed below.

Great. And so the answer to that several of you got is zanidatamab.

[00:42:14]

          Posttest 3: Rationale

And that's based on the trial that actually showed a 40% response rate of this biparatopic antibody. And this is brand new. This just got FDA approved in December. And actually it only became available last month. So this is something you're just going to be starting hearing about in the biliary tract cancer space.

[00:42:33]

HER2-Targeted Therapy in NSCLC

So for the next 2 presentations, they're going to be video speakers talking. And the first one is going to be about HER2-targeted therapy in lung cancer.

Dr Todd Bauer (Tennessee Oncology): Hi. I'm Todd Bauer with Tennessee Oncology and the Greco Haynesworth Centers for Research in Nashville, Tennessee. And I appreciate you spending your time with me to hear more about HER2-targeted therapy in non-small-cell lung cancer.

[00:42:55]

          Biomarker Testing and Targeted Treatment in Advanced NSCLC

Well, if we go back a little bit into the history of targeted therapy in lung cancer, we go all the way back to EGFR and its classical L858R mutation and the deletion 19. And then fast forward over the next 20 years or so, and we've got an EGFR and ALK and ROS and BRAF and NTRK and RET and MET and KRAS, and now ultimately HER2. So that's a lot of things to remember.

And many of these have first-line treatment options but some of them are second-line. So a couple of points on this. First, sending broad-based next-generation sequencing is the most appropriate way to test our lung cancer patients based on these number of alterations. Two, it's really important to know the difference between the mutations and the fusions and the overexpression, because those - those what seem to be minor detail, actually have massive impact on the predictive nature of those tests and the response to the approved therapies.

And third, again, recognizing which treatment options offer first-line vs second-line therapies is very important.

[00:43:55]

          Trastuzumab Deruxtecan NSCLC Approval

So trastuzumab deruxtecan has an accelerated FDA approval for patients who's unresectable or metastatic non-small-cell lung cancers, who harbor an activating HER2 mutation and who have received a prior systemic therapy. So HER2 mutation and second-line treatment, patients are eligible for trastuzumab deruxtecan.

[00:44:15]   

          DESTINY-Lung01: T-DXd in HER2-Mutated or HER2-Overexpressing NSCLC

So we got that approval based on the DESTINY-Lung01 trial, which was T-DXd in HER2-mutated or HER2 overexpressing non-small-cell lung cancer patients. They had to be refractory to standard therapy and no prior HER2-targeted therapy. They had to have good performance status, of course.

These patients were broken down into 2 cohorts: those that were HER2 expressing by IHC 2+ or 3+. And then within that cohort, dosages of 6.4 mg/kg every 3 weeks, or 5.4 mg/kg every 3 weeks.

Second cohort were those patients who had HER2 mutations, again subdivided into those 2 dosing schemas.

The primary endpoint was confirmed objective response by an independent review committee.

[00:44:58]

          HER2-Mutated NSCLC Cohort in DESTINY-Lung01: Best Percent Change in Tumor Size

When we look at the HER2-mutated patients, you see an objective response rate of 55%. But overall, many patients who did not make that metric of 30% reduction tumor still had stable disease or better. Indeed, only one patient had clear trend towards progression at the - at the study cutoff.

The median progression-free survival in this arm was 8.2 months, and the median overall survival was 17.8 months. So really strong results in patients who had HER2 mutations.

[00:45:29]   

          DESTINY-Lung02: Trastuzumab Deruxtecan in HER2-Mutated NSCLC

This led to DESTINY-Lung02, which was T-DXd in HER2-mutated non-small-cell lung cancer. Again, similar patient population, but just in mutated patients. No more overexpressed patients in this. They are randomized 2:1 to a dose of 5.4 mg/kg every 3 weeks, or 6.4 mg/kg every 3 weeks.

These arms were not powered to test against each other, but rather to see if the lower limit of the 95% confidence interval in both arms was better than the benchmark objective response rate of 26%, basing that on the REVEL trial, which was ramucirumab plus docetaxel.

[00:46:10]   

          DESTINY-Lung02: Response by BICR

Looking at the results of that, we see that the confirmed objective response rate in the 5.4-mg arm was 49% and the 6.4-mg arm was 56%. Again, these were not designed to be compared but rather see if they were better than the objective response rate benchmark. And both doses were better than that benchmark.

Guys, I hope you found this information useful. I appreciate your time and have a great afternoon.

[00:46:35]

Tumor-Agnostic Indications for HER2-Targeted Agents and Emerging Therapeutic Approaches

Dr Funda Meric-Bernstam (MD Anderson Cancer Center): Hi everyone. I'm Funda Meric-Bernstam. I'm the Chair of the Department of Investigational Cancer Therapeutics, and the Medical Director of the Institute of Personalized Cancer Therapy at MD Anderson Cancer Center.

It is my pleasure today to talk about Tumor-Agnostic Indications for HER2-Targeted Agents and Other Emerging Therapeutic Approaches.

[00:46:54]

          DESTINY-PanTumor02: T-DXd for HER2+ Solid Tumors

You know, there's been a lot of excitement about HER2 as a target across tumor types, and this really intensified with the success of DESTINY-PanTumor02. In this study, we did a multicenter phase II trial of trastuzumab deruxtecan, or T-DXd, where we gave patients that had either 2+ or 3+ HER2 expression on immunochemistry across tumor types.

Trastuzumab deruxtecan of 5.4 mg/kg every 3 weeks. You can see really the really clinically meaningful objective responses shown with the overall cohort having objective response rate of 37.1% with a duration of response of 11.3 months.

If we looked at patients that were HER2 IHC 3+ on central testing, the objective response rate was 61.3% with the 21 - 22.1 month duration of response. The data presented here had a median follow up of 12.75 months. You can see the activity was really robust with responses seen across a variety of different tumor types, especially striking in endometrial cancer, cervical cancer, ovarian cancer. But really meaningful activity also in several other tumor types, including the other tumor cohort, which was a basket cohort.

[00:48:15]

          Trastuzumab Deruxtecan Tumor-Agnostic Approval

The data was then subsequently further reviewed looking at the patients who had IHC 3+ on their enrollment test. Three quarters of the patients were enrolled based on local testing. A quarter of the patients were enrolled in DESTINY-PanTumor02 were enrolled with central testing.

Evaluated in this way, the objective response rate in DESTINY-PanTumor02 was 51.4% for patients that had a 3+ IHC. Maybe see that this was again with a median - median duration of response that was quite meaningful at 19.4 months.

This data was supplemented by the data seen in DESTINY-Lung01, where patients were enrolled with 3+ IHC, again, with objective response rate of 52.9 months. And the colorectal cancer study, where objective response rate of 46.9 months.

Based on the data from these 3 studies, cumulatively from 192 people, you can see that T-DXd for patients that have high - high expression defined as 3+ is really clinically active, leading to a very first PanTumor HER2 agent approval on April 5th for trastuzumab deruxtecan.

[00:49:26]

          DESTINY-PanTumor01: T-DXd for Solid Tumors With HER2-Activating Mutations

Now, there are other, you know, interesting data that's emerging regarding activity, and other therapeutic opportunities. For example, T-DXd was also explored for patients with HER2 activating mutations. T-DXd is already FDA approved for patients that have HER2 activating mutations in the context of lung cancer.

If you see that when we looked at a variety of tumor types, there was also activity seen in HER2 activating mutations, across a variety of different tumor types, with a cumulative objective response rate of 29.4%, suggesting that, you know, genomic testing and looking at HER2 mutations may be something we also need to keep an eye on.

There are also several drugs in development in this space, including multiple small molecule inhibitors.

[00:50:10]

          SGNTUC-019: Tucatinib + Trastuzumab for Previously Treated HER2+ or Mutant Advanced Solid Tumors

Speaking of small molecule inhibitors, an interesting combination that's emerged is the combination of a small molecule inhibitor of HER2, tucatinib, with HER2 monoclonal antibody, trastuzumab. You can see that there's data emerging from a multi-cohort basket trial.

Here the waterfall plot we are looking at is from biliary cancer showing very meaningful clinical activity with the objective response rate of 46.7%.

[00:50:38]

          MyPathway: Pertuzumab + Trastuzumab for HER2-Altered Advanced Solid Tumors

Another interesting combination that has been explored across a variety of tumor types is 2 different monoclonal antibodies: pertuzumab and trastuzumab. This was studied in the context of a basket study through MyPathway study.

And on the - on the figure shown here, we're looking at the objective response rate by tumor type in patients that were HER2 amplified or overexpressed and KRAS wild-type. You can see that there were, you know, clinically important activity seen across a variety of tumor types, including salivary cancer, colon cancer, and biliary cancer.

And for these 3 tumor types, there's now an NCCN guideline inclusion for this treatment combination.

[00:51:18]

Zanidatamab (ZW25): A humanized IgG1-like Biparatopic Antibody  

Another interesting agent that's involved is zanidatamab. This is a biparatopic antibody that binds HER2 to 2 separate locations, the same binding sites in fact, for trastuzumab and pertuzumab.

[00:51:29]

          Zanidatamab for HER2+ Solid Tumors

The phase I clinical trial had a large expansion enrolling a variety of tumor types. It showed an objective response rate of 37% with responses across a variety of tumor types. Ultimately, based on the activity seen during the phase I for biliary cancer, a dedicated biliary tract cancer trial, HERIZON-BTC-01 was conducted.

And zanidatamab has received accelerated FDA approval in this setting. Zani is being further explored across a variety of tumor types in monotherapy as well as combination therapy now.

[00:52:04]

          Disitamab Vedotin for HER2+ Cancers

Going back to ADCs, there's a lot of development in ADCs still with several agents in development for HER2. A really interesting agent that's evolved is disitamab vedotin. This is an antibody drug conjugate that's a novel HER2-targeted antibody that's coupled to MMAE with a cleavable linker.

This drug is already has regulatory approval for gastric cancer in China. And you can see has really interesting activity demonstrated in urothelial cancer in this waterfall plot. You can see objective responses not only in patients that are HER2-positive with an overexpression or a 2+ expression and amplification, but also in patients that are HER2 IHC 2+ with FISH negative.

[00:52:47]

          Select Other HER2-Directed Therapies in Development

You can see this is just the beginning. There are several other agents targeting HER2 and there are small molecule inhibitors that are looking really very interesting, especially being developed in HER2-mutant lung cancer, but being explored in a variety of other tumor types.

There are several other antibody drug conjugates that have either different payloads or different antibodies or, you know, novel strategies altogether.

Further, there are many other novel takes on HER2 targeting, including bispecifics, CARs. So this is definitely a very important area of develop - drug development and important to think about HER2 testing across the board for solid tumors.

Thank you for letting me share this information.

[00:53:35]

Let's Return to an Earlier Question

          Posttest 1

Dr Cleary: So we're going to return to an earlier question. Which of the following pathology findings related to HER2 alterations would you flag as actionable with an approved targeted therapy for patients with previously treated advanced non-small cell lung cancer?

[00:54:30]

          Posttest 1: Rationale

Great. And so most people put HER2 expression or activating mutations, which is the correct answer that trastuzumab deruxtecan is approved in patients in lung cancer who have activating HER2 mutations, but also it's approved in patients who have overexpression of HER2 as well.

[00:54:39]

          Poll 4

Poll question number four. Do you plan to make changes in your clinical practice based on what you learned in today's program?

1. Yes;
2. No; or
3. Uncertain.

[00:54:59]

          Poll 5

Poll question number 5. Please take a moment to text in one key change that you plan to make in your clinical practice based on this education?

[00:55:26]

Q&A

Super. So we have a series of questions. The first question that was submitted was, can you get HER2 mutations in a next-generation sequencing with a negative HER2 IHC stain? What's the significance of this? So again, the question is, can you get HER2 mutations in next-generation sequencing with a negative HER2 IHC stain? What's the significance of this?

I was thinking especially in lung cancer, that's - that's where this is relevant.

Dr Hung: Yeah. Okay, great. Yeah. So that's a great question actually. So I think the complexity of it is it depends on the 2 variables. So number one is where is the epitope of your HER2 antibody, right?

So you can imagine a scenario in which there can be a HER2 activating mutation, but it's somehow destroy the epitope of the antibody hitting it. And therefore you get a negative result from HER2 IHC but it's still an oncogenic HER2 mutation. So that scenario can certainly happen. So I think that's the option one.

And the other thing too is as I mentioned before, the way to drive a lung cancer, there are 2 different ways one can activate HER2. So either by HER2 gene amplification overexpression or by HER2 mutation. And so those 2, they don't necessarily go together actually. And so you can certainly get cases in which there's no mutation is amplified or overexpressed, or you can get cases in which there is a mutation, but when you stain it, and the staining is not impressive. So the analogy is similar to, let's say, EGFR mutant lung cancer, in which there's actually a very poor correlation between the EGFR protein expression and the presence of EGFR mutation.

Dr Cleary: Thank you. The second question is someone online wrote in, do you perform HER2 IHC for both adenocarcinoma and squamous cell carcinoma of the lung routinely or only if requested?

Dr Hung: That's a great question. Only as requested I would say right now because I - the - so our typical workflow for - thinking about patients with advanced non-small-cell lung cancer. So we will - right now we test for both using a next-generation sequencing panel. Look for those targeted mutations and then a PD-L1 immunostaining. And then we have a few blanks left to do certain things.

And so HER2 alteration can certainly be seen in a subset of patients. But I would say that in terms of epidemiology, most of the time one might pick up other alterations EGFR, KRAS mutation, KRAS most commonly. And PD-L1 expression, if those are very high, I think that leads to treatment to a certain paradigm and pathway first. And so for HER2 immunohistochemistry, we usually only perform as requested by clinician. But we usually make sure there's enough tissue left so that when they ask for it, we will not say the case is exhausted so that they have to think about rebiopsy.

Dr Cleary: Thank you. The next question is a GI question. Do you suggest HER2 tests for all colon cancers same as MSI high testing?

I'll say from my perspective, yes. I think even though HER2 amplification is mainly seen in left-sided colorectal cancers. Occasionally, we see them in right-sided colorectal cancers. But - but also more to the point is we really want to see if - in colon cancer we're starved for targeted therapy. So I want to look at every single patient to see if there is HER2 amplification. As an oncologist, I typically use next-generation sequencing for this.

Dr Zhao: Yeah, that's a great answer. And it's again a good question. We don't perform HER2 routinely for all colon cancer biopsies. The rationale from pathologists is we - we - we think HER2 testing is reserved for late-stage metastatic or advanced disease that have limited options. And most of the biopsy we receive do have other options. And they could be early stage, or they could have a suitable candidate for surgery.

So we - it's hard for us to determine how to stratify like which test is best for colorectal cancer patients. So I think it's great to hear your thoughts on like how useful HER2 testing would be. And like I think we should, as a pathology community, maybe think more about how to integrate this biomarker testing in our routine practice.

I have to say, for upper GI tumors, especially esophagogastric cancer, we have a much larger panel that even for initial biopsies, even we have - don't have a clear understanding of the stage of the - the patients. We still do a relatively large panel just to make sure we provide as much biomarker status for these patients. But somehow I think our like panel designing is relatively less thoughtful for colorectal cancer.

Dr Cleary: Yeah, I really like Dr Zhao’s answer. You know, as she said, we don't do it for all tumors. And actually, I think that's an important point. So for patients with stage I to stage III, it's not going to affect their management. So we don't have to do it there.

For - for metastatic patients, as a medical oncologist, we just order next-generation sequencing. So she said we typically don't do HER2 IHC because we can get all the information we need from next-generation sequencing. So I think that's right on the money.

The next question is, how do you assess HER2 status in biliary tract cancer? How is this done in zanidatamab pivotal trials?

Really good question. I have to say, similar to colon cancer, as an oncologist, I actually just use next-generation sequencing. So I'm really looking - and this is for patients with advanced biliary tract cancers. I'm using - I'm looking for using next-generation sequencing. Although in the zanidatamab trial, they also did IHC as well.

The next - the next question is, are there efforts to provide HER2 testing guidelines for lung or colon or biliary tract cancers? Or are the guidelines for breast and gastric cancer sufficient?

Dr Zhao: Again, a wonderful question. Yeah, it's various cutoff points and also like a scoring system being used or in like previous and ongoing trials, I find this is a really good question. And I'm not so sure if there's any efforts in unifying the scoring system, because it's just difficult to corroborate, like all the prior data into this with the newer system too. What do you think?

Dr Hung: So my - my take on it is we can think about how long it takes them for the community to harmonize the whole PD-L1 immunostaining. Right? It took about a decade to harmonize all the scoring systems, TPS, CPS scoring, immune cells and different antibodies. So I think in the sense of HER2, I think we are still in our early days, right? So I think that was 1 challenge.

Maybe we will harmonize it 10 years from now. But recently, I mean, I particularly like our last presentation mentioning that right now HER2 3+ is in fact the indication for HER2-targeted therapy is actually pan cancer.

So I have even gotten requests to score HER2 in thymic cancer. I mean, that - or other cancer type, right? Or sarcomas and other things, right? And so how do we score them? And so right now I would say the approval is 3+ but which criteria do we use? Breast, GEJ biopsy or resection?

I would say the GEJ biopsy resection is what we, you know in a way what we used. And so by - by default but - but in terms of is it the best system we score in the future? I mean I don't think - I think we're still in the data free zone. And so perhaps in the future we'll finally sort it out to harmonize this whole scoring system.

Dr Cleary: Super. The next one is a very good question is, is HER2 amplification always associated with HER2 expression?

Dr Zhao: Oh, very interesting question.

Dr Hung: So the - I would say based on the - at least in non-small-cell lung cancer, the association is very high. So if you have a case in which you know there's a HER2 gene amplification, high copy number gain amplification, most cases do show 3+ IHC staining and - as I've shown in my last slide.

But you can - I mean they were like very, you know, minority of cases that can be HER2 1+ or negative. And so I think the problem for those study is you wonder is it negative due to some sort of pre-analytic variable, right?

Is it just suboptimal tissue staining and condition? Or is it due to a secondary mutation that somehow kills the epitope in which the antibody binds? And so I think we're still trying to sort it out.

Dr Cleary: Yeah, I think we have similar data in colon cancer. Jason Hornick published a paper looking at this next-generation sequencing always have correspondence with IHC. And interestingly, we found that for colon cancer, it does that when you see HER2-positive 3+, it always is going to mean, at least in our institution, that HER2 is going to be amplified on next-generation sequencing.

The next question is for large-sized solid tumor biopsies, would the pathologist on the panel recommend still using the gastric biopsy scoring system? So for large-sized solid tumor biopsies, do you still recommend using the gastric biopsy scoring system?

Dr Zhao: Yeah, I haven't really thought about this.

Dr Hung: Large biopsy. It was a small resection.

Dr Zhao: Right. Yeah, I guess it really depends on how big the tumor it is like, because biopsy is really just a sample from that tumor mass. And if - and we assume it's not going to represent the entire tumor, that's why like in recognizing heterogeneity of the HER2 overexpression, I think we continue to use the biopsy criteria for scoring. But yeah, it is an interesting question. I don't think any - we have any data that analyzes this kind of type of sample.

Dr Hung: Completely agree. Actually, I would say the - the biopsy criteria I feel like is - perhaps it's the one that one should consider. Even though we saw in the data free zone trying to compare the two. But - but in a way, I think the biopsy criteria allow us to make sure we don't under call HER2 in a way to think about patients eligible for HER2-targeted therapy.

Dr Cleary: This next question is really good because it's something we struggle with in clinic. Will you prioritize testing metastatic site biopsy rather than localized biopsy or resection specimens?

I will say this. As a medical oncologist, it's one of the problems we have is there is some heterogeneity with HER2. And - and so my personal preference is to use next-generation - is to use cell-free DNA. Because the one advantage of cell-free DNA is that with cell-free DNA, you're really getting a sense of what all the tumors are doing. So I like doing that.

But in terms of what's more important, the metastatic biopsy or the - the initial resection specimen, we really don't know. So I think either are fine. I don't know if either of you have a thought on that.

Dr Hung: So another – it’s a great question. So again I use PD-L1 as an analogy. It took us about 10 years, 15 years to sort things out. So in non-small-cell lung cancer, it's known that if you were to test the metastatic site as compared to the primary, you see high PD-L1 in the metastatic site statistically actually. So there's certainly an intra-tumoral heterogeneity, tumor evolution over time.

But I would say from a practical standpoint, and I mentioned in my slides before, if you were to test a metastatic site and I've seen it many times, particularly, you know, patients might have bone metastases. Just make sure don't just put the acid in strong - don't put the tissue in strong acid because I think that will give you a negative HER2 - false negative HER2.

Dr Cleary: The next question is, in the era of tumor agnostic tumors, do you perform ISH testing on patients with - with a 2+ positive HER2 staining?

Dr Zhao: I guess I haven't gotten any results like a request on that for that question to treat other solid like the types of solid tumors.

Dr Hung: I would say haven't actually. I was - I would say from the few, you know, cases of rare other rare cancers, stomach carcinoma, sarcomas with HER2, IHC staining, we haven't gotten request to - to FISH them. But 3+ I would say sometimes the oncologist might want to FISH them. But - but I - but I would say in my limited experience, limited - I haven't seen requests for FISH and HER2 2+.

Dr Cleary: Thank you. So first we again want to thank all of you for coming so early and listening to this talk on HER2. We'll be around here afterwards if people have additional questions, but we're going to wrap it up here. Thank you.

[END OF TRANSCRIPT]