Our study, at the end of the day, was to try to understand or get novel insights into the origin of CLL and how CLL evolves by studying CLL patients in which we do not identify a single CLL clone but multiple independent clones. So it's patients that don’t have a single basal neoplasm but have multiple CLL at the same time but of independent origin. This was already suggested, that a fraction of patients had this phenotype or this manifestation of their disease, we wanted to understand what was the magnitude of this phenomenon and which were the molecular features of these different clones.

What was the study design?

In our study we first started analysing more than 350 patients with CLL or what is called monoclonal B-cell lymphocytosis, which is the preceding phase to CLL. Also we compared what we found in the patients against a cohort of more than 120 healthy individuals, age-matched healthy individuals. We first studied samples from these patients and healthy individuals using next generation sequencing of the  immunoglobulin gene, which basically allows us to understand or characterise the architecture of the CLL, so to identify which patients have one CLL and which have multiple clones with different immunoglobulin genes.

When we characterised this we found that in CLL, for instance, there is around 12% of patients that have multiple CLL clones. Then the next question was to try to understand what were the molecular features of these different clones. For that we performed different single cell technologies. We performed single cell RNA-Seq, single cell DNA sequencing , single cell whole genome sequencing to understand which was the transcriptome and [??] alterations of these different clones.

What were the results?

The results that we found were that beyond this 12% of CLL patients that present this oligoclonal pattern, so multiple independent clones in their peripheral blood, when we went deep below, clones that were very, very minor, we also identified that in patients with CLL there is a bias in the B-cell repertoire in the patients compared to healthy individuals. So it’s not only that some patients might have more than one expanded clone but that, let’s say, to make it general and easy to understand, that most of the B-cells in the peripheral blood are biased towards CLL characteristics at an immunogenetic level.

Then when we performed all these single cell technologies we found that these clones, or these B-cell clones that are different from the main CLL population, also have that [??] profile of CLL made on the single cell RNA sequencing. Then we also observed that these different clones, even if they are very small in size, they can carry driver alterations of CLL that we typically observe in patients with CLL. Some of these driver alterations may be associated with aggressive disease, for instance TP53 mutations or the IGLV3-21R110 mutation can be found in very, very small clones.

Finally, what we found also using single cell whole genome sequencing is that these different clones  do not share any single somatic mutation in their genomes, suggesting that they originate from very independent, or completely independent, cells of origin. Using this technology also allows us to understand when these different clones start to emerge during the lifespan of the patient and we find, for instance, that in some cases the initial driver alterations in some of these clones were acquired before the age of 30 in patients who were diagnosed at the age of 60.

We could observe also how some of these clones start to grow and expand decades before the diagnosis of the disease. Just to give you some numbers, for instance we found that in some patients with CLL we identified up to nine different CLL clones, so different clones with different immunoglobulin genes, all of them with the phenotype of CLL.

What is the significance of these results and what can be done moving forward?

We started to analyse this phenomenon because we think, well there is some evidence, that in the early phases of CLL there is much higher oligoclonality. That is what we also found, that in the MBL there is 40% of patients presenting in this oligoclonal pattern. So by understanding this multiple oligoclonality founding CLL it’s a way to understand the initial steps of CLL development, so going back, let’s say, in time to understand the origin of this disease. Doing so, we can try to understand which are the initial driver alterations that are found in these clones, what makes some of these clones expand faster than others, so, let’s say, understand the initial steps of CLL origin and how these clones evolve.

This could also allow us in the future if we have good drugs that could target this CLL early and this is something that maybe in the future is shown. All this technology that we are developing here, the single cell technologies that allow us to identify these minor clones and so on, would allow us, for instance, to think in the future for the eventual early detection of CLL or early detection of more aggressive clones and this could lead to the development of potential early intervention schemes.