Dr. Javier Hernández Losa of Vall d’Hebron University Hospital joins Absolute Gene-ius to discuss how biomarkers are shaping precision oncology and how researchers choose among qPCR, digital PCR, and NGS for different molecular questions. The conversation spans solid tumors, liquid biopsy, molecular residual disease, assay design, and the growing challenge of extracting more information from increasingly limited samples.
Precision oncology research increasingly depends on finding the right biomarker and choosing the right tool to measure it.
In this episode of Absolute Gene-ius, Dr. Javier Hernández Losa, leader of the Molecular Biology Laboratory at Vall d’Hebron University Hospital, joins Lisa Crawford and Jordan Ruggieri to discuss the evolving world of biomarker research. Javier explores the growing range of protein, DNA, RNA, and other molecular biomarkers, along with challenges posed by FFPE tissue, limited sample material, and liquid biopsy. He also explains how technology selection depends on the biological question: qPCR and digital PCR can be valuable for highly specific, sensitive analysis of a small number of known targets, while NGS can support broader profiling and discovery. The conversation also covers molecular residual disease, orthogonal validation, assay design, methylation, and the potential of combining multiple layers of molecular information.
In Career Corner, Javier reflects on 25 years in oncology research, the mentor who helped launch his career, and why young scientists should stay open to new technologies, questions, and directions. He also shares the familiar graduate-school promise that results will always be ready “on Monday.”
Jordan Ruggieri 00:00
Just double checking. You can't hear Grayson sword fighting in the background, can you?
Lisa Crawford 00:04
Yes.
Jordan Ruggieri 00:04
You can?
Lisa Crawford 00:05
Is that what that was?
Jordan Ruggieri 00:07
Just wait until he starts. Until he says what he wants to play, and he says, "I want to be an arrrgh matey!"
Jordan Ruggieri 00:25
Welcome to the Applied Biosystems Absolute Gene-ius Podcast, a series from Thermo Fisher Scientific. I'm Jordan Ruggieri.
Lisa Crawford 00:32
And I'm Lisa Crawford. Today, we're delighted to welcome Dr. Javier Hernández Losa to the show. Javier leads the Molecular Biology Laboratory at the Val d'Hebron University Hospital in Barcelona. His work spans everything from solid tumor biomarker testing to liquid biopsies.
Jordan Ruggieri 00:48
In our conversation, Javier shares incredible insights into biomarker classifications, assay designs, and how technologies like dPCR, qPCR, and NGS all have their moments to shine in the lab.
Lisa Crawford 01:00
It's a thoughtful and engaging discussion, so let's get started.
Jordan Ruggieri 01:07
Javier, thank you so much for joining us on today's episode of Absolute Gene-ius. We are thrilled to have you, and a topic that I know is definitely interesting for me, and something that I think our listeners are going to be really interested in. So thank you so much for joining us today. Can you give a little bit of background on who you are and what your research is about?
Javier Hernández Losa, PhD 01:30
Yes, certainly. So I think that I'm working as a molecular biology lab director here in the Vall d’Hebron Hospital. We are working within the Department of Pathology, but also within the Institute of Research from the Translational Molecular Pathology Unit, and so I’m here. I came here in 2003, and then there since this year. So we are performing many, many translational research based on biomarker testing, mainly.
Jordan Ruggieri 01:58
Are there different types or classifications of biomarkers? For instance, are some proteins versus some nucleic acid?
Javier Hernández Losa, PhD 02:07
Yes, obviously. I think that the source of the material that you are focused on naturally are completely different. So I think that protein expression, DNA or RNA alterations, but also probably why not another nucleic acid, probably microRNA, long noncoding RNAs, or whatever other RNAs also could be so important. So I think that, but indeed, it's important that whatever thing that you can distinguish between normality and activated process, I think that this is marked as a biomarker. For example, alterations in the hematological issues probably is important. If you are focused on the PET tags, also as a biomarkers, whatever protein expression measured by the different ligand or different ELISAs or different techniques that are capable to measure different amino acids, proteins, or whatever. I think that this is all of them are biomarkers.
Jordan Ruggieri 03:17
What are common challenges that you have working with different biomarkers?
Javier Hernández Losa, PhD 03:23
Well, I think that probably one of the issues here also usually is the combination. So, for example, here if we are focused on alterations of the DNA or RNA, there are many different alterations. Namely, for example, mutations. We are focused on mutation. We are talking about the SNPs or in those changes, but they're completely different from different rearrangements. So as a result, we need to combine these SNP detection plus rearrangements, and if our research samples or our samples are fixed in formalin also embedded in paraffin. Also, this is a new challenge. Or, for example, the pre-analytic process from the liquid biopsy analysis, for example, in different pre-choice proposes. Also, this is a new challenge. So, I think that there are technical challenges in order to adequate or in order to capture all the biomarkers and the same chute.
Jordan Ruggieri 04:29
No, it makes a lot of sense if you have, especially if you have something that's formalin fixed. That tends to cause a lot of degradation, right? So it can be challenging there.
Javier Hernández Losa, PhD 04:39
Right. Also, if you add in of these challenges in terms of the fixation and the terms of the artifacts that you can find, and also you need to distinguish artifact with real alterations. As in addition, probably there are small pieces of samples, so I think that this is low amount, highly degraded. So yeah, your challenge to have success results are in this is very low sometimes.
Jordan Ruggieri 05:08
Yeah, that's a good point too. I didn't think about is if you have a very small sample, a biopsy sample or a tissue punch or something like that, where you have a very small amount. So just the amount of testing you can do on that and research you can do that can be challenging as well. Do, you mentioned that is that something that you run into frequently?
Javier Hernández Losa, PhD 05:32
Yes, I think that as much frequently recently that the place, the previous year. So I think in this field also the majority of the community want to collect as much information as possible in order to improve and go forward in terms of this biomarker validation. But also so at the end of the day, so the samples are currently less and less material, and the number of biomarkers have been increasing every year, so this formula is incredible because I think that in the next five of years, probably we have a small amount of pieces punch, and then the demanding is 20 or 30 biomarkers in this minor piece of samples. So I think the technology also need to help to us in order to do this properly.
Jordan Ruggieri 06:31
Have you seen a shift in the way that we look for biomarkers? Maybe away from solid samples and into liquid samples, like liquid biopsies with blood, urine, saliva, where it's a little bit less invasive? And what's your experience working with those types of samples?
Javier Hernández Losa, PhD 06:53
Yeah, I think that this is a new wave for acquiring the samples or the source of material in order to collect those biomarkers. So I think that the classical approach had been done basically by solid tumor, and initially I think when we started to do this biomarker testing, we need at that time using frozen material. I think that was a big challenge at that time because obviously it's not highly extended in different pathology labs. So I think that we move from the frozen to the FFPE material with the technology transformations in order to adapt this technology to acquire this and collect this analysis, and also the next wave of samples are liquid biopsy. But I fully trust, or I'm a good believer from the liquid biopsy implementation in the hospitals very soon.
Jordan Ruggieri 07:56
I have a bunch of questions around liquid biopsies. The first one being is you know you go from having the, you know, maybe the solid, you know, tissue sample to having something that is that is liquid. You know what we see and what we hear a lot is that that can also cause some challenges in terms of detecting the actual biomarker. Do you run into issues in terms of trying to find a rare target in a, you know, larger liquid sample instead of maybe testing or researching directly on the solid tumor?
Javier Hernández Losa, PhD 08:38
Yeah, I think that many differences between in terms of the sensitivity of the assay is that you are mandatory to be used depending on the source of material. So when we are talking about the solid tumor, I'm saying that the threshold in order to identify whatever attenuation usually 5%. This is very common, and also all the community has said the threshold of the 5%. So it means that in 100% you have five mutant alleles, and then also you are completely right. And over five, you are completely or almost sure that you are doing the right things. The other way around is if you are not detected this 5% you stay struggling in order to detect if it is real negative or not. But when we're shifting for the liquid biopsy, this formula is not the same, because if we are talking about, for example, plasma samples, and we are trying to identify CT alterations, so it means that the DNA alterations contained in the plasma samples are provided for the tumor. This is very a drop into the middle of the sea. So I think that proportion is very low. So as a result, if you are using the same methodologies, you never use because your threshold is not 5%, is below than 1%. So this is essential to know just previous to implement whatever technology in order to adequate to liquid biopsy.
Jordan Ruggieri 10:17
You know, you mentioned minimal residual disease. Can you talk a little bit about what that is and that concept itself?
Javier Hernández Losa, PhD 10:26
Yes, I think that we are talking about the minimal residual disease in order to, when any clinical activity are using for detecting tumor and that you are removing these tumors. So the way to monitor if this tumor had been removed completely and to predict the response or the relapse are used for minimal residual disease. Sometimes the new etiology, I said that the molecular residual disease. This is the other topic that also molecularly, if you identify any alteration on the tumor, that you can track and follow those alterations during the next months after the surgery, so you can detect this molecularly reactivation of the tumors, and as a result, anticipating the relapse. So this minimal residual disease usually is when you have established the molecular profile for the tumor, isolate or detect or select two or three or five alterations or whatever number of alterations that you can track during the follow-up. This means the molecular residual disease.
Jordan Ruggieri 11:45
No, again, makes a lot of sense, and MRD is definitely something that you know we've seen an increase in interest as well in terms of research. Is there any technology considerations looking at these types of studies? I mean, I can imagine you know you're looking like you said, you know, a drop in the sea sometimes for some of these biomarkers. How do you consider technologies to use like a qPCR or dPCR or NGS in your laboratory?
Javier Hernández Losa, PhD 12:24
Yeah, usually I think that depending of the intended of use or maybe what are you searching in terms of singular technology for these purposes? So, for example, if we are in in the last sample that I provide you, so in the MRD, if you detect the molecular profile, you analyze the molecular profile for any tumor, and you can monitor these alterations. If you are selecting only one of the single alterations, this is not necessary to do a huge screening every two months or every three months. Probably, is the best approach is go directly for these alterations, highly specifically. And high sensitivity technologies, so for example, digital PCR or qPCR, depending of the assays. But this is the thing, so basically, the theme is if you are focused only a breadth or maybe a minimal amount of samples or the minimal amount of markers, probably the multiplexing by qPCR or the digital PCR are very worthy. But if you need to do a more broadly approach in order to do a huge screening, molecular profiling, discovery, etc, etc, probably the NGS is the best solution today.
Lisa Crawford 13:50
Are there instances where you would use all three of those technologies in one workflow? For example, like would you start with NGS to narrow to figure out what you're looking for in a large sample, and then narrow it down from there? Does that happen?
Javier Hernández Losa, PhD 14:05
Yes, yes. I think that usually whatever technology that are in the discovery phase in your research are basically the omics approach. Let's say metabolomics, proteomics, genomics, etc, etc. So this is a huge stream in order to identify what are the candidates that you need to go in details and you do further validate it by not single or multiplex approaches. So I think this is in the classical scenario for approaches is discover whatever theme in order to do confirmatory tests. Usually, orthogonal tests are mandatory to be done.
Jordan Ruggieri 14:45
What are maybe some considerations for assay design? And we mentioned you talked about you know assays and looking at qPCR and dPCR. How do you approach assay design, and maybe which technology might work better?
Javier Hernández Losa, PhD 15:04
Yeah, I think that this is a good point. So I think that this is a combination. Probably the best approach, usually and the first is to do a research through the PubMed or whatever bibliographic approach in order that any other colleagues in the community have been validate any assay. So I think that the best approach is copy and paste because they are previously validated. If not, you need to do at the beginning, so starting points or defined assays. Usually, in my experience, I think that there are the FAS people who are involved in the specialists from the different companies. Usually, very helpful because they know perfectly how to design or how to improve your design. So we, as a scientist, we know exactly which alteration do we map. But probably the best approach is that the engineers and the technology behind of that help to to design or provide you the best assays to covering your interrogated analysis.
Jordan Ruggieri 16:13
What are common pitfalls or problems that you run into with assay design and with these technologies?
Javier Hernández Losa, PhD 16:21
All of us always in the scientific community tell us the good things. Basically, you know, this is the good titer, the good results have been published in a good journals, but this is not the real life. The real life is sometimes is good, sometimes it's not so good. So I think that you need to figure out that there are different, how do you say there are different reviews, or there are different consortia or agreements in terms to follow the design and the validation of the assay. So there are good papers for the communities, for the different associations in terms of you need to do this step, this step, and this step basically. So you need to follow these guidelines in order to adequate your assays. The artifacts are on that. So this is the real things you need to study. You need to analyze very careful your results, and then also completely trustable your results, and then also in some specific cases you need to cross validate with different assays or different technologies in order to be sure just previous to go ahead or to do any statement that are standard.
Jordan Ruggieri 17:49
What are you most excited about, maybe over the next 10 years, in in this field?
Javier Hernández Losa, PhD 17:56
Well, for me, the most the most excited technologies also that are coming up probably in the next coming years, probably. We will bring for our hospitals is those technologies who combine different layers of information. It means that, for example, we try to do our best in order to incorporate the methylation approach. I see that the methylation provide in some tumors, some risk classification, some risk stratifications, and why not for potential future treatment, and also you can combine the DNA alteration based on the classical molecular DNA alterations or methylation in the same assay. I think that provide more information than the only one key or only one layer of information. This is one thing, and probably in the biomarkers, in terms of the research, also the combinations for different sorts of material plus artificial intelligence that also are boosting our research program. So I think that we will understand at least a one step forward the molecular alterations in different tumor types.
Jordan Ruggieri 19:23
Can you talk a little bit about methylation and what that is, and maybe a little bit about epigenetics, and how that impacts cancer biology and research?
Javier Hernández Losa, PhD 19:36
Yeah, I think that also when we are talking about methylation, I think that one of the key processes here are based on the methodology. So I think the technology at today also are based on the at the same for the biomarkers, and I, and I saying before, so I think initially we started with the frozen material, and then also it was very worthy in order to adequate and know the alterations. Today, also there are new technologies that are used in methylation and FFPE, at the same time with the whole genome analysis, and why not in the next coming years probably using only liquid biopsy at the same time, very rapidly and also cost effective. Because I think that 10 years ago none of us are using the whole genome sequencing because there are some expensive. You invest a lot of time in order to do the full results. But today, I think that technology overcome those barriers. So we are now in the barrier that we have the whole genome sequencing in 10 days, also basically. So I think that probably the next pushing for the technology is why not combining with methylation. Yeah.
Lisa Crawford 21:06
As you know, we love dPCR here at Absolute Gene-ius and dPCR technologies can be extremely helpful in MRD research, especially alongside qPCR and NGS technologies.
Jordan Ruggieri 21:16
That's right, Lisa. dPCR excels at quantifying low-abundance targets and can also aid in longitudinal surveillance of select markers with shorter turnaround time and lower cost after the biomarker of interest is identified via NGS.
Lisa Crawford 21:29
With the Absolute Q, reliable and precise digital PCR can be as simple as preparing your samples, loading onto the plate, and running the instrument. And the Absolute Q utilizes more than 95% of the sample input, so you won't have to worry about missing rare targets due to dead volume. There are also multiplex assays available for targets like BRAF and ESR1, able to detect down to 0.1% variant allele frequency.
Jordan Ruggieri 21:54
You can learn more at thermofisher.com/oncologypanels or visit the Absolute Gene-ius webpage. Now let's get back to our guest.
Jordan Ruggieri 22:04
All right, over to Lisa for Career Corner.
Lisa Crawford 22:07
Yeah, so you've talked a lot about your research. You've given a great explanation of that, but we do want to learn a little bit more about you as well. So what I like to ask is, thinking back to when you were younger, what was it about science specifically that interested you? Not necessarily, you know, going into oncology, but just the beginnings of your career in science or your interest in science.
Javier Hernández Losa, PhD 22:32
I think for myself, I think that I'm, when I was young, so I think that I'm very clear to do research because I'm focused on an oncologist. To be honest, I think that probably one of the things is probably in first of I study biology, and in the first of the career, I think that I did a summer school, and the summer school I attend a course for the molecular oncology, and at that time, so I think that key professional, key speaker from Spanish scientists such as Mariano Barbasid or Joan Massague are very well international world recognition of this investigator. I think that provides some speakers and some speech regarding the molecular oncology. And at that time, probably I very was impressive. After this, and during the next four years, during my career, also I attended from the Molecular Oncology Forum, and as much attended was much interested for me. And at the end of my career, my mentor, Dr. Ramón y Cajal, I entered in his lab more than 20 years ago, to be honest, and also starting this crazy idea for starting for doing research in oncology, and well, that's it. So I think that 25 years later, I'm continuous doing this, so I'm very proud for this.
Lisa Crawford 24:11
You mentioned a mentor whose lab you went into. Tell us a little bit more about him, how you met him, and you know what kind of inspiration and support that he gave you in your career.
Javier Hernández Losa, PhD 24:25
Well, I think that for me, my mentor, I knew him in a in another summer's course. He's one of the key speaker, and then he provided me and talked regarding the molecular pathology. That this is my field right now. And then also at the end of the meeting, I think that I met him, and I provide my interest in order to know his work and so on, and then also for me, I think that I have luck at that time because at this moment I think that there are only one space in the lab in order to say, “Okay, on September please come to my lab and yeah, I do join you,” and then well, what's going on. If you have to more research or not, or which type of project that you are liking, well, this type of things, and then also after arrival to the lab, this lab published in a Nature journal. So I think that this was a Nature Medicine publication at that time, and was impressive because many students want to be at that time in this laboratory, but there are no space, there are no more staffs, and also I was the last one in order to enter in this train. So I think that we track the train. My mentor is for me during the last, during the whole my career, also is the person who guide me in terms of the research and my position. So I think that this is incredible. In fact, this is not retired. This is my boss here from the research, so it's the same boss in the last 20 years. So I think that is my mentor is continuous today, teaching me and well guiding me in the direction.
Jordan Ruggieri 26:19
Wow, that's awesome!
Lisa Crawford 26:20
Yeah, I love that he's still your boss.
Javier Hernández Losa, PhD 26:23
Yes, yes, I think that there are only the last two years. I think, so in two years he will retire, and then also I think that some of us are substituting and there are doing more activities and more research projects.
Lisa Crawford 26:40
Yeah, I mean that's a thread we see a lot. Is just you know in science in particular, mentorship is so important to everybody's career, and along those lines, is there anything that you would tell a young scientist who's just now getting into this field or interested in getting in this field? Something that you would tell them that maybe you wish you knew, or something that's important for them to keep in mind as they start their career?
Javier Hernández Losa, PhD 27:08
Yeah, as in the my advice for the young researchers is that yes, try to learn as much as possible because sometimes the people are focused on different topics or very focused on a specific situation in the oncology world. But the oncology are say are shifting and also are so changing and rapidly, more rapidly than that we expect to be honest. And then, for example, in my case, I started to analyze a signature from the PRKACA MAP kinase in response for Cisplatin, mainly signal interaction and the consequence for the chemotherapy treatment, and try to identify why the this treatment is involved in the in the cell cultures and how to. Prevent the mechanism of resistance for this treatment. This was the origin, and then during the last 10 years and 15 years, I personally moved from this from the adenoviral therapies, so using for the molecular genetic alterations, for protein expression identification, so there are many fields within the oncology. So I think that the learning curve here is okay, choosing the best that you want, but don't go back if there are no feel comfortable. So maintain because there are some different branches that you can identify the questions that you are looking at the beginning. So I think that this is one year in one position. This is okay. If you're comfortable, go ahead. If not, moving for another because I think that the research is research. There are open questions that you need to follow, and there are not the right response. There are different responses to the questions, and then also you need to follow up on tracking your new career.
Lisa Crawford 29:24
So leave yourself open because things are always changing, and you never know where you might go next?
Javier Hernández Losa, PhD 29:29
Yes. Yes. Yes. I I know I know for example many other colleagues that are working now in the oncology field that starting with genetic approach in Drosophila melanogaster, and then they starting they learn the technologies they learn the techniques they know the procedures, but there are some other questions in other fields that are very broad to be understandable and to very brilliant scientists that are moving from the basic to the to the bench to the clinic to the clinic to the bench. So I think that not fixed in any laboratory. This is my advice. So I think that as much open as better as possible.
Jordan Ruggieri 30:15
Good advice.
Lisa Crawford 30:16
So I think we just have one more question, and this is a fun question we like to ask everybody. But in all of your career, can you think of any moments in the lab that were maybe your most embarrassing moment or time something went wrong and a little bit crazy that you then had to deal with. On the other side, also, what would be a proud moment that you've had? So we talk about the silly, and then we talk about the proud.
Javier Hernández Losa, PhD 30:43
Yeah, I think that the first, the former question saying that at the beginning when was an PhD student, we did lab meeting every Friday afternoon, and then all of us says, "When do you have the results? " And they say, "On Monday. On Monday.” Always we say, “On Monday", because on Friday we didn't have the results, or we don't have the expected results that this is completely different, yeah. And then also, I think that this is the feeling that you are likely stressful, not just previous to the boss, and then there are all of your colleagues are in front of the boss trying to define and to present the data, and this was at the beginning. Obviously, this is an leveling course. Today, I think that is very proud to have an overview regarding the latest years in the in the research field. So when I move my phrase to the last five years, I think that the growing evidence and the growing team that we created during the research, I think that I'm very proud to do this team. So initially, you have an you stay as a PhD student, you stay alone with the group of the laboratory, but you didn't know anything. You didn't know anyone, so I think that you stay along. Yeah. Or 25 years later, I think that they have a very excellent team, more than nine people working, and so happy to have it, and hoping to increase obviously in the next years, increase our team to do more research, if it's possible. Obviously.
Lisa Crawford 32:36
Very cool.
Jordan Ruggieri 32:37
It's awesome.
Lisa Crawford 32:38
We're definitely excited to see what comes out of your lab.
Jordan Ruggieri 32:41
Javier. I just want to say thank you so much for joining us, and we really enjoyed the conversation. Very insightful, and thank you for taking the time to talk about your research with us.
Javier Hernández Losa, PhD 32:52
This is completely my pleasure, and so thanks for the questions, especially for the personal question. That, to be honest, is very special for me. So thank you.
Lisa Crawford 33:04
That was Dr. Javier Hernández Losa, leader of the Molecular Biology Laboratory at Val d'Hebron University Hospital in Barcelona. We've got more great conversations around the corner in upcoming episodes, so stay curious, and we'll see you next time.
Jordan Ruggieri 33:18
This episode of the Applied Biosystems Absolute Gene-ius podcast was produced by Sarah Briganti, Matt Ferris, and Matthew Stock. All products mentioned in this episode are for research use only. Not for use in diagnostic procedures.