18th International Myeloma Workshop
New MM patients with high levels of circulating tumour cells distinguished by increased bone marrow plasma cell proliferation
Dr Cathelijne Fokkema - Erasmus MC Cancer Institute, Rotterdam, Netherlands
Despite the fact that the progression free survival in multiple myeloma patients is improving, some patients respond poorly to treatment highlighting the need to identify these patients at higher risk for a more unfavourable disease course. It has been recognised multiple times that high levels of circulating tumour cells, as for instance demonstrated by our group and the group, are related to an unfavourable disease course. However, the pathobiology behind this unfavourable disease course is unknown and therefore our group has been focussing on this for a few years.
What was the methodology used in this study?
For this study we analysed paired bone marrow plasma cell samples of newly diagnosed myeloma patients and their peripheral blood samples, the plasma cells from there. For the method we used we decided to perform single cell RNA sequencing. Through this method we can analyse the transcriptome per cell in every patient so this is a very specific method. Through this we can analyse specific subpopulations of the different bone marrow plasma cells and circulating tumour cells and we can easily analyse the differences between the two groups.
What were your findings?
We analysed the bone marrow and the peripheral blood samples of five paired patients with high levels of circulating tumour cells – between 0.5% and 8% of circulating tumour cells. Between the peripheral blood and bone marrow samples of these patients we did not see any differences, not in their clustering, so we did not identify a specific subpopulation in the peripheral blood, but also not in differently expressed genes between the two sources.
Then we decided to analyse the bone marrow samples of 20 patients because we now thought that the difference in biology seen in the disease may lie in the bone marrow and not in the peripheral blood. So for this we analysed the bone marrow samples of eight patients with high levels of circulating tumour cells. They had plasma cell levels in the peripheral blood between 3% and 22% and very low levels of circulating tumour cells. These patients had CTC levels between 0.006% and 0.02%.
Now when we looked into the specific bone marrow samples of these two groups we did see that there were genes upregulated in the patients with high levels of circulating tumour cells related to proliferation and cell cycle. Because we had the whole transcriptome of these patients available we could actually perform a very sensitive method to analyse in which cell cycle the patients were, namely the cell-cycle analysis. With the cell-cycle analysis we load in lists of known G2, M and S phase markers and each cell we performed single cell RNA sequencing on was labelled in which phase they were, so cycling or non-cycling. What we then saw is that patients with high levels of circulating tumour cells had a significantly higher percentage of cycling cells in their bone marrow samples. This indicates that patients with high levels of circulating tumour cells have a more proliferative phenotype in their bone marrow and this may be the mechanism behind this more unfavourable disease course in this group of patients.
How can these results impact the future treatment of multiple myeloma?
As I mentioned in the beginning, we tried to identify those patients with highly at risk multiple myeloma and patients with high levels of circulating tumour cells are identified as high risk multiple myeloma patients. We do, however, treat them as regular myeloma patients but based on their gene signature and based on previous studies we know that it could be that these patients respond worse to therapy and may need a more personal-based therapy or more close monitoring in the future. Hopefully with further analysing this mechanism we are now starting on, these are the very first results of this study, we hope to be a part of that.
What is next for this study?
What we saw is that patients with high levels of circulating tumour cells have a more proliferative phenotype. However, what is the reason for this proliferative phenotype is something we do not know. So we now have two hypotheses – one that this proliferative phenotype is based on a cell intrinsic mechanism. So, for instance, well-known mutations in myeloma make the myeloma cells proliferate more. However, because the common mutations in myeloma, such as del 17p and translocation (11;14) were evenly divided between the two groups, this is something we think is more unlikely. However, we will be performing DNA sequencing on a few of these patients to further analyse specific mutations in patients with high levels of circulating tumour cells versus low levels of circulating tumour cells.
A second option we are working on which is very exciting is that there may be an extrinsic source that makes these myeloma cells proliferate more and pushes them more into the cell cycle, namely the microenvironment. So for this we have started single-cell RNA sequencing on the microenvironment of these plasma cell samples we already analysed and we will be hoping to further have these results in the future.
Is there anything else important that you would like to mention?
I would like to thank all my teammates from the Rotterdam Cupedo and Sonneveld group and my supervisors, Pieter and Tom Webb have been of great help in this research.