At this year’s meeting we’ll be presenting a clinical trial of azacitidine in combination with nivolumab for patients with relapsed AML. The preclinical basis for this study was work that we had done in looking at expression of different T-cells, T-cell profiling, as well as expression of different immune checkpoints including PD-1 in patients with new AML and relapsed AML compared to healthy donors. We saw that the PD-1 was overexpressed on CD8 as well as CD4 factors in AML patients, especially in newly diagnosed, but even more so in relapsed AML and even further more so in multiply relapsed AML patients post-transplant. So based on this and also the fact that azacitidine significantly upregulates PD-1 on T-cells and PD-L1 on AML cells which we had actually seen in some of our other preclinical studies that we published. And also given the fact that azacitidine is the standard treatment used in high risk MDS as well as in AML and approved in Europe and on the NCCN guidelines in the United States we decided to do this combination.

What we saw with this combination, the initial study was done in the salvage setting and we started with the first six patients getting a standard dose of azacitidine, 75/m2 for seven days, and standard dose of nivolumab, 3mg/kg, on day one and fourteen repeated every two weeks. It was safe and so the study was then expanded to the phase II portion and a total of 70 patients have been treated.

So far what we are seeing is that the overall response rate, looking at it by the IWG criteria, is about 35% and this included 22% CR CRI with additional haematological improvement. The eight week mortality was very good at 6% which for a salvage AML population is lower than you would get with most regimens. But what was most interesting is that there were a group of patients who continued to have stable disease or haematological improvements that were durable up to a year or beyond without achieving a traditional response. So when we looked at the survival of the patients, either by survival or by response, we saw that those who achieved a response, either CR, CRI or haematological improvement, had a significantly improved survival. In fact, among the CR, CRI group almost all the CRs lasted more than a year without a stem cell transplant which again is beyond or higher than the norm we would expect with aza alone or with other chemotherapies in salvage AML. Furthermore, when we looked at the total salvage group the median survival was seven months but when we looked at subsets we saw then salvage 1, salvage 2 patients the median survival was almost ten months which in the salvage setting is actually equivalent to what has been shown in the front-line setting with azacitidine or decitabine in large phase III trials. So that was quite an encouraging finding.

We also did some molecular cytogenetic analysis on all the patients and found that the diploid population had a very high response rate of 50% or more and they also had a significantly higher median overall survival. So these are some interesting findings, we don’t know at this time why the diploid population is more sensitive but this is something we are looking at in our immunological correlative studies, both by flow cytometry, immunohistochemistry.

The last part of the study was a lot of immune profiling that was done on all the patients on the study at baseline, end of cycle 1, end of cycle 2, end of cycle 4 and so forth. This was done in collaboration with Jim Allison and Pam Sharma at our immunotherapy platform at MD Anderson. What we found was the key features were, number one, patients who had more T-cells, total T-cells, in the bone marrow aspirate or the tumour environment in an AML situation had a better chance of response, as would be expected. Furthermore, if you looked at the T-cell subsets, those who had more CD8 positive T-cells had a better chance of response and as the people responded among responders you saw that there was a development of further increased CD8 and CD3 populations indicative of immunological infiltrate which was not seen in the people who did not respond with the same treatment. The response was associated with the development of increased activation markers. So not only were there quantitatively more CD3 CD8 but they expressed activation markers like ICOS, indicating that this was truly an immunologically active population infiltrating to the bone marrow.

The last thing we saw which I think is clinically very important is that not among responders but among non-responders who had very low levels of CTLA-4 before treatment, they started having significant upregulation, two to three times higher, after cycle 1, cycle 2, indicating that if you block PD-1, one of the major T-cell checkpoints, the tumour escapes by CTLA-4, the other immune checkpoint leading to the concept that maybe if we do a combination blockade, just like has been done in solid tumours, we may be able to further double or even higher get these response rates. So that’s our next step is to do double checkpoints, both in salvage AML and then moving front line with the aza and nivo initially and then if we have a safe and effective regimen there to see if we can also move double checkpoints in the front line older AML. These studies are actually ongoing right now.