Episode Transcript
[00:00:00] Speaker A: Foreign.
Welcome to Base by Base, the papercast that brings genomics to you wherever you are. Today we are taking a deep dive into, well, frankly, a revolutionary diagnostic tool. It's designed for one of the most aggressive and rarest childhood cancer syndromes out there, Constitutional Mismatch Repair Deficiency, or cmmrd. Just imagine a condition where the risk of getting cancer is over 90% before a child even hits their 20s. I mean, for these kids and their families, a delayed diagnosis isn't just frustrating, it's. It can be catastrophic.
[00:00:45] Speaker B: Absolutely. And the huge challenge has always been the genetics. It's complex, creating this massive bottleneck and getting answers quickly when time is so critical.
[00:00:53] Speaker A: And that bottleneck often comes down to one specific gene, doesn't it?
[00:00:57] Speaker B: Uh, yes, it's often the PMS2 gene. It's actually the most common culprit in CMRD. But it's, well, a genetic nightmare to sequence accurately because there's just so much DNA noise around it.
[00:01:07] Speaker A: Noise, like interference?
[00:01:08] Speaker B: Exactly. From pseudogenes, bits of DNA that look almost identical but don't function. But this research we're discussing today presents a way to sort of bypass that complexity entirely.
[00:01:18] Speaker A: Okay, so here's the breakthrough, as I understand it. They've developed this highly sensitive functional assay. It doesn't just sequence the gene. It looks at a specific pattern of cellular mistakes, a unique kind of mutational signature.
And this signature apparently not only diagnoses CMMRD faster than ever, but it can actually tell us which specific gene is broken. So let's really unpack this because it sounds potentially game changing.
[00:01:44] Speaker B: It really is significant. So just to define it clearly, CMMRD is this rare, devastating syndrome. It's extraordinarily penetrant, meaning the risk is incredibly high if you have the genetic cause. And that causes it's caused by pathogenic variants in the mismatch repair genesis. Mmmmr genes for short. The main ones are MLH1, MSH6, MSH2, and that tricky one, PMS2.
[00:02:06] Speaker A: Pathogenic variants. Meaning broken copies.
[00:02:09] Speaker B: Yes. And critically, for CMMRD to manifest, these variants must be bialelic. That means both copies of the gene, the one you inherit from your mother and the one from your father, are affected. They both have pathogenic variants.
[00:02:20] Speaker A: Got it. Both copies knocked out.
[00:02:22] Speaker B: Precisely. And this functional defect means the cell's natural DNA proofreading system, the mismatch repair system, is essentially broken. And it can't fix errors made during DNA replication.
[00:02:34] Speaker A: And the consequence of that is instability.
[00:02:36] Speaker B: Massive instability. It leads to an extremely high incidence of really aggressive cancers, typically high grade brain tumors, certain blood cancers, hematological malignancies, and very early onset colorectal cancers, often in childhood or adolescence.
[00:02:52] Speaker A: And the average age of onset is something like seven and a half years.
[00:02:55] Speaker B: That's the mean age. Yes. It highlights the urgency.
[00:02:58] Speaker A: And it's not just about knowing the risk for surveillance, is it? Treatment decisions actually depend on this CMMRD status.
[00:03:04] Speaker B: Absolutely critical. For instance, CMMRD associated tumors, because of their underlying biology, are often resistant to some common chemotherapy agents like temozolomide, which is often used for brain tumors.
[00:03:15] Speaker A: So standard chemo might not work.
[00:03:17] Speaker B: Right. But on the flip side, these same patients, because their tumors have such high mutational burdens due to the MMR deficiency, might be excellent candidates for newer immunotherapies. Things like immune checkpoint inhibitors.
[00:03:31] Speaker A: Ah, so knowing the status quickly opens up potentially life saving treatment avenues.
[00:03:37] Speaker B: Exactly. It's truly life saving information.
[00:03:39] Speaker A: Okay, so the stakes are incredibly high. Early diagnosis, right? Treatment. And yet we keep hitting this snag, diagnosing those pathogenic variants, especially in PMS2. You said it accounts for a lot of cases.
[00:03:52] Speaker B: Around 60% of CMMRD cases involve PMS2.
But its genetic analysis is notoriously complicated.
[00:04:00] Speaker A: Because of those pseudogenes you mentioned?
[00:04:02] Speaker B: Correct. These pseudogenes are stretches of DNA that are non functional, basically genetic fossils. But they look virtually identical to the actual PMS2 gene sequence.
[00:04:11] Speaker A: So when you try to sequence the.
[00:04:12] Speaker B: Real gene, you get tremendous noise and signal interference. Standard sequencing methods often can't distinguish perfectly, leading to ambiguous results. Or what we call variants of unknown significance. Vus.
[00:04:23] Speaker A: Which leaves families and doctors in limbo.
[00:04:25] Speaker B: An impossible limbo. Yes, you suspect cmmrd, but the genetic test isn't definitive. That's why we desperately needed a reliable functional test. Something that just tells us, is the proofreading system actually working properly? Yes or no.
[00:04:40] Speaker A: Okay, so how did they tackle that? They looked for the consequences of the broken system.
[00:04:44] Speaker B: Precisely. They turned to looking for the downstream effects in cells. Specifically, they focused on using NGS next generation sequencing based methods and to detect something called microsatellite instability or msi.
[00:04:58] Speaker A: Microsatellites? Those are repetitive DNA sequences, right?
[00:05:01] Speaker B: Exactly. Short, repetitive sequences scattered throughout the genome. They are inherently prone to errors during replication, like little stutters. A working MMR system constantly sixes these stutters.
[00:05:12] Speaker A: But if the MMR system is broken.
[00:05:14] Speaker B: Like in cmmrd, then these microsatellites become wildly unstable. You see lots of insertions or deletions in their lengths. Finding this instability, this msi, especially in Non cancerous tissue like blood cells, LEUKOC acts as a functional indicator, a surrogate marker for the underlying constitutional MMR deficiency.
[00:05:32] Speaker A: So they developed a specific test for this?
[00:05:34] Speaker B: Yes. Their core solution is the highly sensitive assessment of MSI assay or HS msi. What's clever is they didn't try to look at millions of markers across the whole genome, which can be complex and expensive.
[00:05:46] Speaker A: What did they do instead?
[00:05:47] Speaker B: They developed a custom NGS DNA panel. It targets only 192 very carefully selected microsatellites.
These specific markers were chosen because they are known to be particularly sensitive. They are almost guaranteed to become unstable when the MMR system fails A targeted.
[00:06:03] Speaker A: Approach and the output is the assay.
[00:06:06] Speaker B: Calculates an HS MSI score. It's simply the percentage of unstable microsatellites. Out of those 192 that were analyzed, how many show changes in length?
[00:06:15] Speaker A: And they found a threshold, a cutoff point.
[00:06:17] Speaker B: Yes. Through extensive validation work, they established a crucial diagnostic threshold.
An HS MSI score in a blood sample must exceed 4.57% to be considered positive for CMRD.
[00:06:29] Speaker A: 4.57%. That sounds very precise.
[00:06:31] Speaker B: It reflects the sensitivity needed. Remember, in blood, the instability might be subtle compared to a tumor. So you need a highly sensitive test and a well defined cutoff.
[00:06:39] Speaker A: And validating something like this for a rare disease requires, well, a lot of collaboration.
[00:06:45] Speaker B: Absolutely. We really should acknowledge the huge international effort here. This involved the Hereditary Cancer Group at Idabel in Spain, the Arthur and Sarnia Labatt Brain Tumor research center at SickKids in Toronto. And crucially, they leveraged data and samples from large consortia. The International Replication Repair deficiency consortium, or IRRD, and the European consortium care for CMMRD C4CMMRD.
[00:07:11] Speaker A: That kind of global teamwork is essential for rare diseases, isn't it?
[00:07:15] Speaker B: Indispensable. You simply can't get enough diverse cases or robust data. Otherwise, by pooling resources, they could test this HS MSI assay on a really strong and varied group of samples.
[00:07:27] Speaker A: So tell us about that testing cohort. They didn't just use confirmed CMRD cases, did they? They needed to make sure it didn't misfire on similar conditions.
[00:07:33] Speaker B: Exactly right. They were very thorough. They tested 66 blood samples that were blinded. The lab didn't know the diagnosis beforehand. This included confirmed CMMRD individuals, but also crucial non CMMRD controls.
[00:07:45] Speaker A: Like healthy individuals?
[00:07:46] Speaker B: Healthy individuals, yes. But also patients with other hereditary cancer syndromes that can sometimes overlap clinically or genetically. Like lynch syndrome, which involves MMR genes, but usually only one Faulty copy or lie. Fraumeni syndrome.
[00:08:00] Speaker A: Okay, so really challenging the specificity. And they also looked at tumors?
[00:08:04] Speaker B: Yes. They also analyzed 24 tumor samples known to be associated with CMMRD, covering different types like brain tumors and lymphomas.
[00:08:12] Speaker A: And I found this fascinating how they benchmarked it. They compared their 192 marker panel not just to older methods, but to a whole genome instability score.
[00:08:21] Speaker B: That's right. They compared their HS MSI scores against the LADOX score, sometimes called mmrdnis. This is derived from low pass whole genome sequencing and essentially measures instability across millions of microsatellites genome wide.
[00:08:34] Speaker A: So the question was, can our targeted panel of 192 markers perform as well as looking at, well, pretty much everything?
[00:08:41] Speaker B: That was the key question for validation. And the results. How did it perform exceptionally well in identifying CM MRD from blood samples? The HS MSI assay achieved 99% sensitivity, meaning it caught almost every true case, and 100% specificity, meaning it didn't wrongly flag any of the controls as having CMMRD across the unique cases. That's about as accurate as a functional diagnostic test can get.
[00:09:05] Speaker A: Wow. So the targeted 192 marker strategy was definitely sufficient. And it wasn't just accurate in blood. Right? You mentioned the tumors.
[00:09:12] Speaker B: Yes. The robustness in tumors was also a major finding. The HS MSI assay successfully detected MSI in all 24 CMMRD associated tumors.
[00:09:21] Speaker A: They tested all 24, regardless of type.
[00:09:24] Speaker B: Regardless of the organ of origin, high grade glioma, lymphoma, others. This is a massive improvement. Traditional MSI testing methods, the ones often used for colorectal cancer, have historically performed very poorly on these non colorectal CMMRD tumors.
Their sensitivity was estimated at only about 18%. 18%. So often completely missing the mark. This new assay hit 100% in their cohort.
[00:09:46] Speaker A: That's a huge leap. And the correlation with the whole genome logica scores, did that pan out?
[00:09:51] Speaker B: It did, and it strongly validated their targeted approach. They found a very strong positive correlation between the HS MSI score from 192 markers and the logic rescore from millions of markers.
The correlation coefficient R was 0.89 in blood samples and 0.898 in tumor samples. That's extraordinarily high.
[00:10:12] Speaker A: So it proves you don't need the whole genomic library, so to speak. You just needed to pick the right 192 index cards very carefully.
[00:10:18] Speaker B: Precisely. It showed their selected panel was capturing the essential instability signature effectively.
[00:10:24] Speaker A: Okay, but now we get to what seems like the real aha moment of this research. It wasn't just that the microsatellites were unstable, but how they were unstable. They found a specific pattern, a genotype specific signature.
[00:10:37] Speaker B: This is where it gets really fascinating and adds another layer of diagnostic power. Think of the MMR system like a spell checker for DNA, especially focusing on those repetitive sequences, the microsatellites. When the spell checker is broken, specific kinds of mistakes happen more often.
In this context, the mistakes are usually either inserting an extra base, a letter into the repeat, or deleting one. These are called insertion deletion variants or indels.
[00:11:03] Speaker A: Okay, insertions or deletions. Indels.
[00:11:05] Speaker B: What they crucially found was that the type of INDEL mistake that predominated was different depending on which MMR gene was broken by those biallelic variants.
[00:11:14] Speaker A: Different mistakes depending on the broken gene. How?
[00:11:16] Speaker B: Patients who had biallelic pathogenic variants, specifically in PMS2, that really common and hard to sequence gene, their unstable microsatellites were uniquely enriched with one base insertions. They kept making mistakes that added an extra letter to the repeats.
[00:11:31] Speaker A: Wait, hang on. So if PMS2 is the broken gene, the cell's characteristic mistake is adding extra bases, like a stutter that adds sound exactly like that.
[00:11:38] Speaker B: It's a specific functional fingerprint of PMS2 deficiency. And in striking contrast, what about the others?
[00:11:44] Speaker A: MLH1, MSH2, MSH6, carriers of pathogenic variants.
[00:11:49] Speaker B: In those other MMR genes, MLH1, MSH2 or MSH6 showed the opposite pattern. They had a significantly higher proportion of one base deletions. Their characteristic mistake was skipping a letter in the repeat.
[00:12:02] Speaker A: Wow. So insertions point towards PMS2, deletions point towards the others. That difference is consistent.
[00:12:08] Speaker B: Highly consistent. The difference in this INDEL pattern allowed the assay to specifically predict whether the underlying defect was in PMS2 or one of the other MMR genes. With an accuracy classification rate in AUC of 0.997.
[00:12:23] Speaker A: Almost perfect accuracy. Just based on the type of mistake the unstable microsatellites show.
[00:12:27] Speaker B: Yes.
[00:12:27] Speaker A: So this functional assay doesn't just say yes, CMMRD is present, it effectively says yes, CMMRD is present and it's highly likely due to PMS2 or yes, CMMRD is present and is likely due to one of the other genes. All without directly sequencing PMS2 and battling those pseudogenes.
[00:12:42] Speaker B: That's the power of it. It functionally pinpoints the likely culprit gene group.
[00:12:46] Speaker A: The clinical value there seems immense if PMS2 is the most common and the hardest to sequence traditionally, this functional test basically tells you if you need to focus your efforts there or elsewhere.
[00:12:56] Speaker B: Precisely. It bypasses that major diagnostic hurdle for the most frequent cause of cmmrd. It provides strong evidence to guide further, perhaps more targeted genetic confirmation if needed, or helps interpret those ambiguous VUS results.
[00:13:11] Speaker A: And practically speaking, is this HS MSI assay something labs can actually run?
[00:13:16] Speaker B: That's another key advantage because it uses a targeted NGS panel, just 192 markers, rather than whole genome sequencing. It offers high robustness and accuracy, but with a relatively fast turnaround time and comparatively lower computational requirements.
[00:13:32] Speaker A: So more feasible for standard clinical labs to adopt.
[00:13:35] Speaker B: Much more feasible. It's a pathway to making this kind of accurate functional diagnosis more accessible globally, not just in specialized research centers. It can provide that definitive support needed to confirm or even discard a CMMRD diagnosis, especially in those tricky cases with uncertain genetic findings.
[00:13:51] Speaker A: And there was one more finding regarding the score itself, wasn't there? The amount of instability seemed to correlate with something important.
[00:13:58] Speaker B: Yes, the quantitative score, the actual percentage of unstable markers, seemed to carry prognostic information.
They observed a significant negative correlation between the HS MSI score in the blood and the age at diagnosis of the patient's first tumor.
[00:14:13] Speaker A: Negative correlation meaning higher instability score correlated.
[00:14:17] Speaker B: With a younger age at first cancer diagnosis. The correlation coefficient was R A chelation 0.4, which is statistically significant.
[00:14:26] Speaker A: So the more unstable the DNA proofreading system is, the earlier cancer seems to strike.
[00:14:31] Speaker B: That's what the data suggests. It implies that the degree of the mismatch repair deficiency, how broken the system is functionally measured by this score, might actually influence how quickly the disease manifests, affecting the syndrome's penetrance or timeline.
[00:14:44] Speaker A: That's extremely thought provoking. It's not just a yes no diagnosis, but potentially a functional metric for risk level or disease aggressiveness.
[00:14:52] Speaker B: It hints at that possibility. Yes. However, it's important to note, and the researchers acknowledge this, that while this correlation is very promising, we really need larger prospective studies. Following patients over time with these scores is necessary to fully figure out how useful they are for actual clinical cancer risk. Stratification and tailoring patient follow up plans.
[00:15:12] Speaker A: Right. Validation in a real world clinical follow up setting is the next step.
[00:15:16] Speaker B: Exactly. But the foundation is incredibly strong.
So if we boil it down, the central insight here is that this HS MSI assay is now validated as a highly accurate ancillary diagnostic tool for cmmrd. It's uniquely capable of reliably detecting that low level MSI signature even in non cancerous blood tissue, which is key for.
[00:15:36] Speaker A: Early diagnosis before a tumor even develops.
[00:15:39] Speaker B: Correct, and it massively improves upon older methods for detecting MSI in the CMMRD associated tumors themselves.
But perhaps most critically, it uses those specific mutational signatures, the predominance of one base insertions versus one base deletions, to functionally identify the likely affected gene group, particularly flagging the diagnostically challenging PMS2 cases.
[00:15:59] Speaker A: So, looking ahead, what does this all really mean for treating childhood cancer? How might combining this kind of sensitive functional signature, measuring both the degree and the type of instability with traditional genetic testing lead us towards, I don't know, truly personalized cancer risk assessment? Could it mean earlier, more targeted therapies or surveillance for kids with cmrd? That really seems to be the frontier. This research has just opened up. This episode was based on an Open Access article under the CC BY 4.0 license. You can find a direct link to the paper and the license in our episode description. If you enjoyed this, follow or subscribe on your podcast app and leave a five star rating. If you'd like to support our work, use the donation link in the description.
Thanks for listening and join us next time as we explore more science base by base.
