Peripheral blood CD26 leukaemia stem cell monitoring in CML during TKI treatment

Prof Monica Bocchia - University of Siena, Siena, Italy

We all know that the results in chronic myeloid leukaemia patients are extraordinary to date and to the TKIs which can let patients live, in a great majority, for their lives, for a normal lifespan. But even so we have some patients that lose response and this is because we know we have a reservoir in the body of leukaemia stem cells. This is not a new concept, of course, but it’s relatively new that we can now in chronic myeloid leukaemia detect leukaemia stem cells directly in the blood by flow cytometry. Indeed, a few years ago it was discovered that the CD26 marker is a specific marker for chronic myeloid leukaemia stem cells which is found in a subset population which is CD34 positive, CD38 negative and, of course, CD26 positive.

So we started to look at these chronic myeloid leukaemia stem cells in the blood, starting with a retrospective study in which we measured these cells in more than 200 patients in any stage of their disease. What we found was that even in those patients that were nearly cured, I would say, because they had a very, very low level of disease, meaning they had a deep and stable molecular response, still we could measure a small number, but still detectable, of chronic myeloid leukaemia stem cells in their blood. Even in those patients who are in so-called treatment for remission stage in which practically as they had such a profound and prolonged molecular response they can even stop their TKI treatment, even in those patients we found the persistence of chronic myeloid leukaemia stem cells.

In this particular study we wanted to see what is the destiny, actually, of these cells, what is the meaning of these cells. So we started a prospective study including many centres in Italy and at diagnosis started receiving the blood of these patients and we measured at a ten day line and then at different time points after treatment – six months, twelve months and so on – the amount of chronic myeloid leukaemia stem cells in their blood.

In this particular study we did a prospective study including many centres in Italy, haematological centres, in which we looked at the destiny of these chronic myeloid leukaemia stem cells in the blood starting from diagnosis. Practically we measured the number of CD26 positive leukaemia stem cells in the blood at time zero, at the baseline, and then at several time points during treatment.

100% of patients at diagnosis showed a consistent number of CD26 positive leukaemia stem cells in the range of 7 average per microliter, because we can measure them exactly in the number. But just after treatment this number dropped significantly and we had about 0.2, 0.1 cells per microliter at that time. Continuing the TKI treatment, regardless of the type of TKI treatment, these leukaemia stem cells remained detectable in the blood at a very, very low level but still detectable. This was practically seen in 182 patients that are included so far in this study and regardless of the type of treatment they received which was, of course, free for the physician to decide. Imatinib for the majority of patients but also several patients had nilotinib and other patients had dasatinib. So the behaviour of the chronic myeloid leukaemia stem cells is practically a fluctuating number along the treatment.

What was interesting in this study was that when we looked at characteristics of the patients included in the three treatments, for example we saw, and this is pretty understandable, that patients treated with dasatinib and nilotinib were much younger than the patients treated with imatinib. Today in Italy older patients usually receive imatinib while younger patients receive second generation TKIs. But when we looked at the number of leukaemia stem cells at diagnosis in these subtypes of patient, for example we saw that the level was higher, meaning that in imatinib patients we had about 4 cells per microliter while in the nilotinib and dasatinib group of patients we had 12 and 17 cells per microliter. It looks like in younger patients we may have more stem cells at diagnosis of the chronic myeloid leukaemia.

Another interesting part of the study was that we had 33 patients that switched TKI, 13 of them because of intolerance but 20 because of failure. When we looked at these 20 patients that switched because of failure and we looked at the amount of leukaemia stem cells at the beginning, at diagnosis, they had definitely the highest amount of CD26 positive leukaemia stem cells. So it looks like probably the number of leukaemia stem cells that you have at the beginning of your disease could somehow have a role in the future response to TKI treatment. But, of course, this data needs to be confirmed in a larger setting and also by following these patients longer than what we did so far.

Of course now we still have to answer several questions because these leukaemia stem cells exist but they persist also. They persist even in patients, as I said, that stop TKI treatment. So what do they do? Where do they go? What is their destiny? Practically we think that probably it’s the new system which can control them after a certain point and that’s why we are looking also at certain markers on the surface of these leukaemia stem cells which can better favour the control by the immune system.
One of these markers is PD-L1 which, of course, is a marker, it’s an antigen that can turn off the response of T-cells against the tumours. We looked in 61 patients at the expression of PD-L1 in these particular myeloid stem cells, chronic myeloid stem cell population. They are not all the same, the stem cells, and about half of them have, indeed, a strong positivity for PD-L1 but half of them, meaning half of the patients, don’t have this positivity. When we looked at the patients in which we found the high positivity for PD-L1, these are the patients in which the number of leukaemia stem cells at diagnosis was higher. So maybe the number has a meaning at diagnosis, maybe the type of leukaemia stem cells has a meaning. Indeed, when it’s PD-L1 positive it may be a type of stem cell that is more difficult to eradicate in the body.

So our study is going on and we are trying to answer these questions which I think are very important because they will tell us exactly, for example, if we can find a way to detect which patients can really stop TKIs because they have maybe a certain number of leukaemia stem cells, they have a certain type of leukaemia stem cells persisting, and so they can easily interact with the drug even, for example, if they didn’t have such a deep molecular response. Or instead find out which patients unfortunately cannot stop treatment but even in these patients we know now that continuing TKIs their life is anyway very good and their lifespan is practically normal.