VolitionRx Limited (VNRX) Earnings Call Transcript & Summary

Great. Good morning, everyone. I'm Alexandra Byrne. I'm a reporter at GenomeWeb, and I will be your moderator for today's webinar. The title of today's webinar is beyond the genome: Measuring Epigenetic Modifications Across Matrices for Biomarker and Drug Discovery and it is sponsored by Volition. Our speakers today are Eric Ariazi, Chief Development Officer of Helio Genomics; and Marielle Herzog, Research and Development Director at Volition. You may type in a question at any time during the webinar, you can do this through the Q&A panel, which appears on the right side of the webinar presentation. If you look to the bottom tray of your window, there is a series of widgets to enhance your webinar experience. And with that, I'll turn it over to Marielle.

Thank you very much. Hello, everyone. Good morning, good afternoon, good evening, wherever you might be. I'm delighted to be here with you today for this women focusing on Nu.Q Discover and how Nu.Q technology could help to accelerate epidrug development. About volition. So Volition is a multinational company. We have colleagues all around the world with 2 lab, 1 R&D lab and service in Belgium and 1 laboratory in the U.S. in San Diego. Our mission is really to stabilize and improve outcome of millions of people worldwide. But before diving into the core of this presentation, let's take a moment to have a look on the background behind the Nu.Q technology. So nucleosome, the fundamental structural unit of chromatin, they play a crucial role in compacting DNA into chromosome and allowing the genome to fit into the cell nucleus. Nucleosome in case of cell death are high cell turnover. Chromatin is fragmenting. And the nucleosome can be released into the environment or the blood -- like the blood flow. Nucleosomes are composed of approximately 140 base pairs of DNA wraparound in histone core proteins, histone and especially the histone cell are subject to Histone post-translational modification, also referred to as epigenetic modification such as methylation, acetylation, phosphorylation, ubiquitination and many others. Those modification can be added by enzyme also known as writer, could be removed by eraser or could serve as a docking site for other proteins. Writer, eraser and reader could be target for epidrugs. Those modifications are a key regulator of several processes like regulation of gene expression, DNA damage or cell differentiation. Alteration of nucleosome or histone PTM levels can lead to disease like cancer, but also inflammatory disease, autoimmune or neurodegenerative disease. Nu.Q assay consists of sandwich immunoassay with a capture antibody targeting specific epigenetic feature present on the nucleosome. And with an anti-nucleosome antibody in detection. Those assay are quantitative, results are expressed in nanogram per mL. And they allow to determine the level of circulating in nucleosome but also to profile the nucleosome. The assay analytically validated in K2 EDTA plasma, but we will see in a couple of slides that then can be used in other type of matrices. Only few microliters are required to perform the assay. Today, the Nu.Q Discover portfolio consists of 14 different assays, each assay targeting a specific epigenetic modification like methylation, acetylation or other PTM. We have also a test targeting a mutation. Those tests are highly specific with no or very limited cross-reactivity, are highly reproducible. It can detect up to few nanogram per mL of nucleosomes in the blood. This portfolio can be and will be expanded, and our experts are available to support you for any of your histone PTM needs. All those assays are available on an automated chemiluminescence immunoassay platform using magnetic beads. 2 of them are also available as a manual ELISA assay and a point-of-care test is currently in our development pipeline. As I mentioned previously, all those Nu.Q assay can be used with multiple different type of matrices from cell culture using supernatant or cell chromatin extract to tissue and blood with not only plasma but also some blood cells in animal model or in human, allowing to cover all the different steps of the drug development from discovery, preclinical to clinical. Just 3 examples, the first one on the cell line, where cell lines were treated either with an HDAC inhibitor here on the left or with an EZH2 inhibitor. Cells treated appears in the red and untreated in gray. Results are expressed as a ratio of the PTM over the Nu.Q H2.1. As you can see for the HDAC inhibitor treatment, we observed a clear decrease of the level of -- or clear increase, sorry, of level of acetylated mark in the treated cells without affecting the methylation mark, K9 or K27 methylation. Whereas for EZH2 inhibitor, which target H3K27 methylated mark, we observed a decrease of this mark in the treated cells, but specifically of the H3K27me3, but no effect on H3K9me3 or any other acetylated marker. A second example, after chromatin extraction, Nu.Q assay were able to profile the epigenetic modification present on different type of tissue, for example, here, lung, pancreas, prostate and also to compare normal tissue versus a tumor. Results are represented here as a heat map, where the red shows the highest level of histone PTM or specific histone PTM represented as a ratio and the blue at the lowest level. Then a third example where Nu.Q assay can track epigenetic modification on blood cell, for example, PBMC monocyte. And you can see that you can really have the different profile according to the different cells. So I gave you a global overview of this technology. Now I will hand over to Eric, who will show you some studies and results, illustrating how the Nu.Q Discover technology can be used, especially in plasma to really investigate key parameters like tumor burden, dose optimization for drug discovery, pharmacodynamic toxicity, but also to evaluate predictive biomarker or discover new biomarker. Eric?

Thanks, Marielle. That was a nice introduction of the Volition technology, and now I can take it from here. I'm a translational cancer biologist and biotechnology executive with over 25 years of experience spanning academia and industry, advancing precision oncology and epigenetic assays from discovery through clinical development. Currently, I am the Chief Development Officer at Helio Genomics. I lead cross-functional teams to develop blood-based multi-omic cancer diagnostic assays. Some quick disclosures. I'm a former employee of ORIC Pharmaceuticals, and I have stock in ORIC Pharmaceuticals. I will be showing some data about ORIC's EED inhibitor, ORIC-944, and the case study data were originally presented from posters presented at prior AACR meetings. I'm also a former independent consultant for Volition. But first, I'd like to talk to you about an overview of histone modification. As Marielle mentioned, histones can be modified by writers and erasers. Writers deposit either acetylation or methylation marks. Acetylation marks open up the chromatin so it's more accessible to transcription factors and are associated with increased transcription. Methylation marks can either increase chromatin accessibility or decrease it to regulate transcription. For example, while H3K4 dimethylation and trimethylation marks usually occur at active genes, H3K9 trimethyl and H3K27 trimethyl marks are typically associated with gene repression. The Nu.Q assays for monitoring a variety of epigenetic marks are shown on the sides of the slide. At the bottom of the slide is a schematic showing the main methylation sites on histones H3 and H4. The methyl transferases are shown on top of the histone map and the demethylases at the bottom. As a case study, we will focus on the PRC2 complex with its EZH2 catalytic subunit that Trimethylated H3K27. Let's start by talking about detecting cancer using these Nu.Q assays for circulating cell-free nucleosomes. Volition collaborators took a panel of Nu.Q cell-free nucleosome assays for trimethylation of H3K27, H3K36, H3K9 and dimethylation of H3K4. They looked at the levels of these marks in non-small cell lung cancer sample versus healthy controls. They found that trimethylated H3K27 showed the highest increase of the markers in the panels with increases of approximately 3.7 or 2.9-fold in training and validation data sets when comparing the medians between non-small cell lung cancer group and the healthy group. Further, H3K27 trimethyl levels showed the best area under the Receiver Operator Characteristic Curve or ROC curve with values of approximately 0.9 in the data set and with a sensitivity of about 70% at 90% specificity. Based on these results with H3K27 trimethylation showing diagnostic potential, the volition collaborators performed an IHC staining study in non-small cell lung cancer tissues compared to healthy tissues. And what they saw was variable H3K27 trimethyl staining, but with increased staining associated with grade. Specifically, there was a lack of strong H3K27 trimethyl staining at Grade 1 but increased staining -- strong staining at Grade 2 and at grade 3. Further, Grade 3 cancers no longer showed weak staining compared to Grade 1 and 2 cancers. Thus, there was a significant association between H3K27 trimethyl staining and increasing grade. To investigate whether circulating H3K27 trimethyl nucleosomes could provide additional information for monitoring the presence of non-small cell lung cancer during treatment in combination with circulating tumor DNA, that is cell-free DNA fragments with cancer gene somatic alterations, H3K27 trimethyl nucleosome levels were measured in healthy subjects and then compared to that in the non-small cell lung cancer patients along with ctDNA status. In a healthy cohort, the upper limit of H3K27 trimethyl nucleosomes was established at 22.5 nanograms per mL based on one standard deviation above the 95 percentile. Using this cutoff, 38.8% of non-small cell lung cancer patients were categorized as H3K27 trimethyl positive. A next-generation sequencing analysis using a panel containing 78 cancer genes covering the majority of drivers of non-small cell lung cancer was used to detect ctDNA. The presence of at least one somatic alteration or copy number variation was considered a ctDNA positive sample. In this non-small cell lung cancer cohort, 43.1% of patients were categorized as ctDNA positive. When considering the additive value of combining H3K27 trimethyl status to ctDNA status, an extra 15.1% of patients could be identified as having circulating tumor material detected. This is significant because now the number of patients can be increased that can be tracked with the blood-based assay. Moreover, as demonstrated in our ROC curve analysis, using H3K27 trimethyl positive status alone achieved an AUC of 0.79, but adding ctDNA positive status improved the AUC to 0.87, demonstrating that combining the 2 analytes further improves tracking tumor burden as defined by liquid biopsy assays. So why would we find H3K27 trimethyl levels to be diagnostic in non-small cell lung cancer? Well, EZH2 is the histone methyltransferase that catalyzes trimethylation of H3K27. And its high expression is prognostic in non-small cell lung cancer and various other cancer types. Here is a meta-analysis of non-small cell lung cancer, including 10 different studies with almost 1,700 patients. And what we found was a combined hazard ratio of about 1.7, indicating that EZH2 is indeed prognostic for overall survival. Likewise, in myeloid neoplasms, we have another meta-analysis in 8 different studies. And again, a very significant overall hazard ratio. This time, it was 2.4. Furthermore, EZH2 is a prognostic indicator in multiple other cancers as shown here, including breast, glioblastoma, gastric and gynecologic cancers. Next, how can these Nu.Q assays be used in epigenetic drug development? Well, this is where my case study with ORIC-944 comes into play. For this case study, I would like to tell you a little bit about PRC2. PRC2 is a multi-subunit complex known to play a role in regulating stem cell identity and cell differentiation through transcriptional silencing of target genes. EZH2 resides in the PRC2 complex. The PRC2 complex also contains SAS12 and EED subunits. SAS12 is a scaffolding subunit, while EED is the reader subunit that allosterically regulates the EZH2 subunit. So while EZH2 writes or catalyzes trimethylation of H3K27, it's the EED subunit that regulates whether EZH2 is active or not. Elevated PRC2 activity and trimethylation of H3K27 is associated with poor prognosis in patients with metastatic lesions. And first-generation EZH2 inhibitors such as tazemetostat have been approved in epithelioid sarcoma and follicular lymphoma. Since EED regulates EZH2, EED inhibitors offer another chance to target PRC2. At ORIC, we were interested in developing the EED inhibitor, ORIC-944. This EED inhibitor is orally available with picomolar activity in biochemical assays and nanomolar activity in cellular assays. At first, it was verified using the diffuse large B-cell lymphoma model, KARPAS-422, grown as xenografts in immunocompromised mice. This model has been widely used to demonstrate efficacy of tazemetostat. In this model, we looked at tazemetostat and compared it with ORIC-944. We observed that ORIC-944 at 100 and 200 milligrams per kilogram dosed once daily was more efficacious than 200 milligrams per kilogram tazemetostat that was dosed twice daily. Next, we were interested in metastatic castration-resistant prostate cancer. Since EZH2 has been implicated in this advanced form of prostate cancer and tested ORIC-944 in enzalutamide resistant 22RV prostate cancers. Here, we saw a strong single-agent activity ranging from 25 to 200 milligrams per kilogram of ORIC-944 dosed once daily. For comparison, 22RV1 tumors were resistant to the androgen receptor antagonist enzalutamide, consistent with prior reports. Now if we look at H3K27 trimethylation levels by IHC staining in the tumors itself, we can see that at baseline, there were very high levels of H3K27 trimethyl -- but with increasing doses of ORIC-944, there were increasing reductions in trimethylated H3K27 levels, demonstrating target engagement. Based on these results, ORIC-944 has gone into a Phase I clinical trial sponsored by ORIC. To implement this clinical trial, we needed to have a biomarker plan. So we developed 3 different biomarker assays in preclinical models. The first one is a skin punch biopsy. Mouse skin was stained for H3K27 trimethyl by IHC. The H3K27 trimethyl staining was evident in the epidermal layer of the skin and in the sebaceous gland. But in mice treated with ORIC-944 for 7 days, the staining disappeared. The staining was quantitated, and indeed, we saw a dose-dependent reduction in H3K27 trimethyl labeled cells. The advantages of this assay are, there was no tumor biopsy, and we did see a dose-dependent response. But the assay is only semi-quantitative and the dynamic ranges are limiting as the range of the values is large. Additionally, scoring all these positive cells takes a long time. Yet the skin punch biopsy assay has been employed in the clinic as demonstrated by this Phase I dose escalation study with tazemetostat. Skin punch biopsies were collected from 32 patients at baseline and on treatment. H3K27 trimethyl IHC staining was performed, and it was observed in the stratum spinosum layer of skin. Significant reductions were observed and taking this data and putting it into a maximum inhibitory effect model show that target inhibition of the H3K27 trimethylation mark was near maximum or near about 80% in the 800-milligram twice daily cohort. What this model says is if you were to double the dosage to 1,600 milligrams twice daily, you wouldn't have much more increase in the reduction of H3K27 trimethyl. Therefore, the skin punch biopsy assay is a viable assay to assess EZH2 inhibitor target engagement in the clinic and can be used to inform the dosage required for maximum target reductions. But the skin punch biopsy assay can be challenging if you want multiple time points, and this is not comfortable for the patient. Next, we went on to an AlphaLISA assay to measure H3K27 in mouse monocytes. The cell input for this assay was optimized to avoid a hook effect where excess analyte overwhelms the finite amounts of antibodies and beads in the assay. With this assay, we saw a very large reduction almost down to baseline. Likewise, when the EZH2, EZH1 dual inhibitor, CPI-0209, now known as tulmimetostat was being evaluated in a first-in-human trial, a similar monocyte assay was employed. Again, a dramatic reduction in H3K27 trimethyl levels were observed in human monocytes. However, if you look at the dosages, you can see that even the lowest dose achieved a near maximal reduction and not much more reduction was seen with increasing dose. In summary, with this monocyte assay, the advantages are, again, there's no tumor biopsy and you can perform longitudinal sampling, but it requires monocyte isolation from whole blood. Hence, you first need to collect whole blood from the patient and then send it to a central laboratory for monocyte isolation. But monocyte preps after shipping whole blood overnight, even on ice are poor because those monocytes start dying during shipping. Then you need to optimize the cell input into the assay to avoid a hook effect. So this assay requires quite a bit of technical optimization, and then there's this lack of the dose response. After working on the monocyte assay, we wanted to look at circulating H3K27 trimethylation at the cell-free nucleosome level in plasma. And for this, we used the Volition Nu.Q assay. We used immunocompromised mice implanted with 22Rv1 prostate cancer cells and then treated them over the course of a week, sacrificed the animals, collected the blood, processed it to plasma and measured H3K27 trimethylation. After treating the mice for 7 days with 10 to 100 milligrams per kilogram ORIC-944 once daily, we saw a dose-dependent reduction in circulating levels of normalized H3K27 trimethylation. We also looked at the frequency of dosing. Over a 7-day period, we dosed for 3 days, 5 days or 7 days, and we saw a frequency-dependent decrease in normalized H3K27 trimethylation levels. Then we wanted to check, is H3K27 trimethyl coming from the tumor or is it coming from the host cells? In essence, is it tumor selective? For this experiment, we had both tumor-bearing and non-tumor-bearing mice. They were treated again for 1 week. If we look at the nontumor-bearing mice on the right side of the plot, the H3K27 trimethylation levels are already fairly low in the vehicle-treated group, and we did not see a further significant reduction in normalized H3K27 trimethylation. But when it comes to the tumor-bearing mice, the baseline levels of H3K27 trimethyl were elevated, and now we could see that reduction with ORIC-944. Therefore, because H3K27 levels are low in nontumor-bearing mice but high in mice with tumors, we concluded that H3K27 trimethyl is indeed selectively coming from the tumor. Furthermore, we concluded this blood-based assay is capable of reading out histone post-translational modification target engagement in the tumor itself. The same exact circulating H3K27 trimethylation assay normalized to total H3.1 was used by another group of Volition collaborators that were working on the CARE clinical trial. The CARE trial is a Phase II study testing tazemetostat in combination with an anti-PD-L1 antibody. As shown on the right -- as shown in the left panel, an evaluation of 191 patients samples across multiple time points in aggregate shows that at cycle 1, day 1, the H3 H3K27 trimethyl over H3.1 ratio was high at 0.56. But at day 1 of cycle 2 and cycle 3, the H3K27 trimethyl over H3.1 ratio was significantly reduced to 0.31 and was still reduced at the end of treatment. As shown on the right panel, if we take a subset of patients where all time points were evaluable, we can examine the data longitudinally. This longitudinal analysis confirmed the decrease in normalized H3K27 trimethylation levels was already occurring at cycle 2 and was maintained throughout the end of treatment. Therefore, this assay can be used in the clinic to follow pharmacodynamic activity of EZH2 inhibitors. In conclusion, for this section, I'd like to restate that H3K27 trimethylation as a pharmacodynamic biomarker can be measured using Nu.Q assays that are both quantitative and robust. We've shown dose dependency, frequency-dependent responses and tumor selectivity. It's noninvasive. It's already optimized and the turnaround time is rapid, often within a week. I'd like to point out that in the CARE trial, the trial's aim is to evaluate a combination of tazemetostat with an anti-PD-L1 antibody. Combining EZH2 inhibitors with Immune Checkpoint Blockade or ICB therapy is a growing area of interest in the clinic because there are multiple mechanisms by which EZ2 inhibitors can augment or potentiate ICB therapy. This slide illustrates how EZH2 inhibitors can enhance cancer immunotherapy, particularly when combined with immune checkpoint blockade. Starting on the left, we see the cancer immunity cycle, the multistep process by which the immune system detects and eliminates tumor cells. This cycle involves the expression of tumor antigens, activation of T cells and infiltration of effector immune cells into the tumor microenvironment. EZH2 normally suppresses several steps in the cycle such as antigen presentation, cytokine signaling and T cell infiltration, helping tumors evade immune detection. The right panel addresses how inhibiting EZH2 lifts this suppression. When EZH2 is inhibited, there is an increased expression of endogenous retroviruses, activation of interferon pathways and restoration of tumor antigen visibility. This boosts the recruitment and function of CD8-positive T cells, NK cells and invariant natural killer T cells. Thus, these effects of EZH2 inhibition helps convert cold tumors, those lacking immune infiltration into hot ones that are more likely to respond to immunotherapy. By targeting EZH2, we can also disrupt immune evasion tactics by blocking regulatory T cell-mediated suppression and blocking infiltration of myeloid-derived suppressor cells. Thus, EZH2 inhibition represents a compelling approach to overcome ICB resistance and extend the reach of immunotherapy to more patients. Here, I'd like to dive in a bit more into how EZH2 inhibition can increase expression of endogenous retroviruses or transposable elements to trigger a viral mimicry response that promotes antitumor immunity. In Panel A, we see the general mechanism. Transposable elements normally silenced by DNA methylation become reactivated when methylation is reduced during cancer progression or treatment. This DNA hypomethylation leads to reactivation of transposable elements, which produce double-stranded RNA. This mimics a viral infection, activating sensors like IFI-H1, RIG-1 and toll-like receptor 3, which in turn induce interferon beta and T cell activation. Panel B provides an example in triple-negative breast cancer where treatment with paclitaxel alters S-adenosylmethionine's metabolism and causes DNA hypomethylation, but tumors can still escape immune detection. When EZH2 targets the transposable element promoters by tri-methylating H3K27 to maintain their silencing despite DNA hypomethylation. However, if we inhibit EZH2, this histo-mediated repression is lifted, allowing for the transposable element re-expression and double-stranded RNA accumulation, which in turn activates the viral mimicry pathway. This enhances innate immune signaling and can make tumors more visible to the immune system, especially when combining with immunotherapy. Based on the aforementioned effects of EZH2 inhibitors helping to overcome resistance to ICB therapy, there's a growing body of clinical trial activity designed to test this in patients. The CARE trial -- as shown previously and on the third trial shown here is one such example. As this slide shows, at least 6 clinical trials as of 2024 are currently investigating combining an EZH2 inhibitor with an anti-PD-1, PD-L1 or CTLA-4 antibody in solid tumors or in refractory diffuse large B-cell lymphoma. These trials span Phase I and II, indicating early but expanding interest in this therapeutic avenue. This represents a strategic shift, moving from monotherapy to rational immunoepigenetic combinations to overcome resistance and broaden the reach of immunotherapy. Now I'd like to switch gears and talk about how can the patient population be expanded to offer PRC2 inhibitors to the most individuals possible. In lymphomas, the H3K27 trimethylation is elevated because there's frequently a mutation in EZH2 or there's co-expression of both EZH1 and EZH2 at very high levels, they can lead to accumulation of H3K27 trimethylation. But there are also other epigenetic factors that oppose PRC2, such as the SWI/SNF complex, also known as BAF and PBAF complexes in mammals, which remodel nucleosomes in an ATP-dependent fashion to regulate chromatin accessibility and allowing Histone acetyltransferases or HATs to acetylate histones. So if there are inactivating mutations in SWI/SNF subunits such as ARID1 or SMARCA4 or in other factors like BAP1 and MLL2, then they no longer oppose PRC2 and H3K27 trimethylation accumulates. BAF and PBAF subunit members are mutated frequently in human cancers and in total are mutated in approximately 20% of all cancers. Specifically, SMARCB1 is mutated in 99% of rhabdoid tumors, a deadly pediatric cancer of the CNS as well as frequently mutated in kidney and soft tissues, while ARID1A is also mutated in about 40% of endometrial cancers and 26% of bladder cancers. With this epigenetic antagonism between SWI/SNF and PRC2 in mind, there is a Phase II clinical trial testing the EZH1, EZH2 dual inhibitor, tulmimetostat, where patients were recruited into 6 tumor cohorts. Of these 4 solid tumor cohorts were based on ARID1A mutation or BAP1 loss. Two other cohorts were lymphoma and prostate cancer because there has been activity in those indications before. Among the solid tumors, the ovarian, endometrial and mesothelioma cohorts with ARID1A and BAP1 alterations showed good efficacy as seen here in the waterfall plot. These cohorts achieved eligibility for Stage 2 expansion. Complete and partial responses were also observed in the lymphoma cohort. These findings support including patients with tumors containing ARID1A alterations or BAP1 loss and continuing investigations of EZH inhibitors. These clinical results bring up the question, can H3K27 trimethylation be used as a biomarker in SWI/SNF family drug development? This figure shows the BAF complex, which as previously mentioned, is a mammalian version of the SWI/SNF complex discovered in yeast. Considering the BAF complex, there exists a synthetic lethal relationship being exploited by pharma companies between the catalytic ATPase paralogs, BRM or SMARCA2 and BRG1 or SMARCA4 and also between the DNA targeting paralogs, ARID1A and ARID1B. In the case of synthetic lethality between BRG1 and BRM, BRG1 is mutated in 10% of non-small cell lung cancer. So if there is a non-small cell lung cancer with an inactivating mutation in BRG1, you can treat with an inhibitor against BRM and completely block BAF complex activity in the tumor but not infect normal cells in the body, which contain wild-type BRG1. Similarly, ARID1A is frequently mutated in up to 40% of uterine cancers. So if you treat ARID1A mutated uterine cancer with an ARID1B degrader or inhibitor, then you should again block the activity of the BAP complex in the tumor, but not in normal cells. Now this is important relative to H3K27 trimethylation because, as mentioned, if you completely inactivate the BAP complex in tumors, this should lead to accumulation of H3K27 trimethylation which can be measured in circulation with the Nu.Q assay. Additionally, since SWI/SNF-mediated nucleosome remodeling also allows for improvement of half the genomic regions, blocking SWI/SNF activity may also be monitored by measuring reductions in circulating acetylated marked H3K27 nucleosomes. In summary, we discussed a wide range of topics. We discussed how Nu.Q circulating nucleosomes can be used to detect non-small cell lung cancer. We examined how H3K27 trimethyl normalized to H3.1 can be used for pharmacodynamic monitoring of PRC2 inhibitors. We reviewed how EZH2 inhibitors could overcome ICB resistance, and we looked at how patient selection and pharma activity is expanding to SWI/SNF family member mutations, and this is another opportunity for H3K27 trimethyl levels to be used as a biomarker. We've taken a lot of your time. And with that, I'd like to switch over to questions.

[Operator Instructions] We would like to ask attendees to take a moment after the webinar has ended to take our exit survey to give us your feedback. The first question will be for Marielle, which is just where and how do I access the assays?

Thank you. So you can contact directly Volition, but we are also happy to share that you can find our assays through [indiscernible] And you can scan this QR code to learn more how to get access through them.

Great. And on to the next question, which will be for Eric. You showed some data demonstrating that post translationally modified cell-free nucleosomes could aid in the detection and monitoring of non-small cell lung cancer. Do they also serve as prognostic markers?

Thank you. So I have a prepared answer for this. In the webinar, I presented several meta-analyses demonstrating that EZH2 is prognostic in non-small cell lung cancer, myeloid neoplasms and other malignancies. Given this, it is biologically possible that elevated levels of H3K27 trimethyl as a downstream product of EZH2 activity and measured on CF nucleosomes could also serve as a prognostic biomarker. This hypothesis is supported by a study presented at the 2025 European Lung Cancer Conference as a follow-up to the 2023 non-small cell lung cancer study shown in this webinar. An interim analysis of the follow-up study examined over 600 non-small cell lung cancer patients and demonstrated higher baseline levels of H3K27 CF nucleosomes were indeed associated with significantly worse outcomes. Specifically, patients with high H3K27 trimethyl nucleosome levels had a mean survival of 13.4 months, compared to more than double the survival time of 27.4 months in patients with lower levels of this marker. The hazard ratio was 3.56, indicating a strong and statistically significant prognostic impact. It's worth noting that this analysis used a higher cutoff of 53.7 nanograms per mL of H3K27 trimethyl CF nucleosomes as opposed to the 22.5 nanogram per mL threshold reported in the earlier 2023 study. This suggests that the threshold optimization may be important when applying this biomarker for prognostic purposes. Overall, these findings support the potential of H3K27 trimethyl cell-free nucleosomes as a noninvasive, epigenetically informed prognostic biomarker in non-small cell lung cancer. Thank you.

Yes. And then another question. Can you just talk about the ways in which the Nu.Q assays facilitate early toxicity detection during drug development?

Sure. The H3.1 assay as a readout of total cell-free nucleosomes in circulation can serve as an indicator of toxicity as it provides a sensitive method for detecting systemic cell death in a patient. For instance, Volition has developed the H3.1 Nu.Q assay to detect sepsis, a condition characterized by acute cell death. But the same principle applies to detecting lower levels of cell death induced by therapeutic agents. Importantly, the detection of H3.1 CF nucleosomes as a readout of cell death does not require the compound to be epigenetically active. Therefore, if a therapeutic agent induces toxicity in any tissue, cancerous or noncancerous, it should result in increased H3.1 levels in circulation. In preclinical oncology studies, animal weight is commonly used as a surrogate indicator for toxicity. However, weight loss can also reflect other nonspecific effects such as reduced food intake, altered metabolism or CNS effects. Incorporating the H3.1 Nu.Q assay alongside weight measurements can help discern true cytotoxic effects from nonspecific effects. Moreover, formal preclinical toxicity assessments can leverage this assay by measuring H3.1 CF nucleosome levels in nontumor-bearing animals treated with investigational compound and comparing them to vehicle-treated controls. Tissue-specific toxicity, such as hepatotoxicity could also be evaluated using liver explant models with H3.1 levels measured in conditioned media as a proxy for cell death. Overall, the H3.1 Nu.Q assay offers a robust and highly sensitive tool for early toxicity detection across multiple contacts and drug development. Thank you.

Yes. And then one more for you, Eric. How do the Nu.Q assays support the discovery and validation of predictive biomarkers that can identify responders and nonresponders in clinical trials?

For therapeuters that target the epigenome, that is epidrugs, it is reasonable to hypothesize that clinical responders may be enriched in the subset of patients with elevated levels of specific histone post-translational modifications targeted by the epidrug. Nu.Q assays detect specific histone PTMs, making them well suited for patient preselection and predictive biomarker development. For instance, in the non-small cell lung cancer study presented in this webinar, multiple histone PTMs, including H3K27 trimethyl were elevated in subsets of patients. In this context, Nu.Q assays could serve as minimally invasive liquid biopsy tools to preselect those patients who are more likely to benefit from specific epidrugs.

Great. And then we have some questions for Marielle. So I wanted to first ask, is this an immunoassay or a sequencing-based assay?

It's an immunoassay. It's a sandwich immunoassay using a capture antibody that is specific to some epigenetic feature and an anti-nucleosome antibody in detection. So it is sandwich immunoassay.

And another one for you. Someone asked, how specific is the antibody to detect H3K27e3 versus monoenzymal dilation?

Thank you. It's a really good question. In fact, you should know that one of the first experiments we did when we developed an assay is to check the specificity and we rejected a lot of antibody there. And for the H3K27Methyl3 Nu.Q immunoassay, we have checked the cross-reactivity with non-methylated mono or di, and there is no cross-reactivity with those histone modification. So H3K27Methyl3 is really specific to this premethylated mark.

And then one more for you, Marielle. What are the logistical considerations for sample collection, shipping and processing, especially when using the Nu.Q in global clinical trials?

Yes. So indeed, there are some [indiscernible] condition or sample processing. Obviously, it will also depend on the Matrices. As we mentioned at the beginning, we can work on Nu.Q assay can be used in different type of matrices, tissue, blood and so on. But just as a first, our experts are available to discuss with you and to guide you. But as example, for K2EDTA plasma sample, we recommend to centrifuge blood within the first 4 hours and then to store either at 4 degrees for immediate testing or if you want to ship us, it has to be stored and shipped on ice because they have to be stored on minus 80 degrees. The other recommendation is also to avoid multiple freezestore cycles. So it's much better to have 1 or maximum 2 freezestore cycles before analyzing your blood samples, for example.

And another for you, Marielle, somebody asked, is the [indiscernible] test FDA approved?

Not yet. This test is a research use only test from now.

Great. And we'll pop back to Eric, a question for you. How do Nu.Q assays integrate with complementary biomarker modalities such as ctDNA and circulating tumor proteins to track responses to therapeutics?

Sure. Nu.Q assays can be integrated with other biomarker platforms that use blood such as genomic and proteomic liquid biopsy assays. And this provides a more comprehensive view of tumor biology and treatment response. A compelling example comes from the non-small cell lung cancer story presented here, where ctDNA sequencing identified 43.1% of patients undergoing treatment. In parallel, Nu.Q assays targeting histone PTM such as H3K27 trimethyl detected additional patients, capturing 15.1% of cases not identified by ctDNA alone. This demonstrates Nu.Q assays can serve as complementary tools to ctDNA sequencing, improving sensitivity in detecting circulating tumor material and enhancing the overall ability to monitor tumor responses or recurrence of minimal residual disease. In addition, Nu.Q assays can complement existing circulating protein tumor markers. For example, tumor protein markers like CEA in colorectal, breast, lung and GI cancers or CA125 in ovarian cancer or PSA in prostate cancer are commonly used to monitor treatment response. Given that EZH2 is a prognostic factor across these tumor types, the Nu.Q H3K27 trimethyl assay could potentially identify patients not captured by these traditional tumor markers. This combination can broaden the range of patients effectively monitored by liquid biopsy, particularly in cancers with heterogeneous expression profiles.

Okay. Great. And I have another question for Marielle. Someone asks, is there any contamination from this based on ethylation protein other than from nucleosome? Do you need an enrichment step to enrich nucleosomes from plasma samples?

Thank you for this question. So our sandwich immunoassay are designed to detect intact circulating nucleosome. So we are not detecting any free histone. We are really detecting circulating nucleosome with a specific histone PTMs. That's for the first part of the question. And for the second part, no, we do not enrich our sample. You can use directly 50 microliter of plasma sample in the immunoassays. So there is no enrichment, you use directly plasmas.

And Marielle, while I have you, can you talk a little bit about how Volition supports pharma partners in customizing these assays or developing new biomarkers that are tailored to specific drug programs?

Yes, for sure. So another very good question. So definitely we can support and customize assay, for example, different matrices that we have not explored yet. And we are there to help to maybe optimize assays to design protocols that would work for these new matrices, for example. But we are also really open to work for and to develop new assay for our new Histone PTM that we have not targeted yet in our portfolio. For this, we have really a team that is dedicated to assay development. They have already developed a 14 assay present in our current portfolio. So they have -- they are highly -- have a huge expertise to assay development. So we are really there and to be open to discuss with you to support your histone PTM research and really to customize any assays.

And then a question for Eric. How do you -- how do EZH inhibitors improve immunogenicity of tumor cells for immunotherapy?

Thank you for that question. For this, I did prepare a slide. So EZH inhibitors reverse epigenetic mediated gene repression. This leads to increased tumor visibility to the immune system and supports T cell infiltration and activation. Exciting emerging data now show that EZH inhibition also significantly enhances the efficacy of adoptive cell therapies, including CAR-T and TCR-engineered T cells across both hematologic malignancies and solid tumors. EZH inhibition does this by increasing tumor immunogenicity, which in turn supports T cell activation, persistence and function. As shown in 2 recent 2025 preclinical studies published in cancer cell and summarized here schematically, EZH inhibition reprograms tumor cells through reduction of H3K27 trimethyl marks resulting in upregulation of MHC proteins, co-stimulatory ligands and adhesion molecules. EZH inhibition also increases inflammatory cytokine production. These reprogramming changes make tumor cells more visible, leading to improved recruitment and engagement of CAR-T and TCR-engineered T cells, which makes the tumor cells more susceptible to T cell-mediated killing. In lymphoma models, EZH inhibition not only enhance infiltration of CAR T cells, but also prolong interaction between T cells and tumor cells, improving cytotoxicity. Additionally, pretreating CAR T cells with EZH2 inhibitors improve their stemness, expansion and resistance to exhaustion, enabling better in vivo persistence and sustained antitumor activity. Importantly, dual EZH1/2 inhibition, for instance, with valemetostat showed even greater enhancement of adoptive cell therapy across a range of tumor antigens and cancer types, including solid tumors. Overall, EZH2, EZH inhibition acts on both tumor cells and T cells, reprogramming the tumor microenvironment to be more permissive to immune attack and supporting durable responses to adoptive cell therapies. Ongoing clinical trials are now evaluating these combinations in relapsed/refractory lymphomas and other malignancies. Thank you.

Thanks, Eric. I'm going to hop back to Marielle. We have someone in the audience just wondering, do we need to do the centrifugation twice for plasma separation or is one time enough?

No. For the plasma preparation, we do not need -- you do not need to do twice centrifugation. You can do 1 or 2 centrifugation as you are used to. Anyway, the Nu.Q assay step include another centrifugation before running the assay, so basically, you can do 1 or 2 centrifugation depending on your current protocols for plasma.

Great. So thank you both. That's all the time we have for today. So I'd like to thank Eric and Marielle and our sponsor Volition. If we didn't get to them -- if we didn't get to your question, we'll try to follow up with our experts, and they'll be able to get back to you. But thank you, everyone, for your questions. And as a reminder, please look out for the survey after you log out to provide your feedback. If you missed any part of this webinar or would like to listen to it again, an archived version will be e-mailed to all attendees. Thank you for joining us for this Genome webinar.