Australian researchers utilising valuable samples from an initial clinical trial funded with the help of Leukaemia Foundation supporters have discovered why half of patients with a type of high-risk blood cancer will not respond to a common treatment.

The discovery could form the basis for a world-first clinical test to identify early whether or not patients with high-risk myelodysplastic syndrome (MDS) will respond to the frontline treatment azacitidine (AZA; Vidaza®). The findings could have potentially important translational outcomes, enabling for the early identification of such patients and will be incorporated into a clinical trial beginning next year.

AZA is not a cure for MDS, but about half of people who are treated with the chemotherapy drug will respond after four to six months, and may not progress to leukaemia.

Research fellow, Dr Ashwin Unnikrishnan, said an integral part of this project was also to find an effective alternative treatment for MDS.

The Leukaemia Foundation provided vital early funding* for the biological work that set the groundwork for the test that could become the world’s first prognostic tool to predict azacitidine response or resistance early in the treatment of high-risk MDs.

Dr Unnikrishnan said half of all high-risk patients treated with azacitidine respond to this treatment, showing an improvement in their blood counts and having a reduced risk of transforming to acute myeloid leukaemia.

“Unfortunately azacitidine is not a cure and three big questions have framed this research work right from the beginning,” he said.

“These are based around why approximately half of those treated with azacitidine never get a response, and why a significant number of the people who do respond will relapse within a two-year period.

“And the sad part is that the prognosis for those who initially fail to respond at all to azacitidine therapy, and those who initially response and then relapse, is very poor, and there is no other alternative treatment for them. There also is no way of predicting early whether someone will respond to azacitidine, or not.

“When a patient is given the treatment, it’s wait and watch, because it takes up to six months for a response, and if they don’t respond, there’s basically nothing more we can do for them,” said Dr Unnikrishnan.

“We thought this situation wasn’t good enough and we should do something about it.

“So another question was – can we identify people upfront, early on, before the six months, and can we do something for them?”

A small-scale investigator-driven clinical trial was opened in Australia in 2008 that recruited people with high-risk MDS and CMML, from NSW and the ACT, who were given compassionate access to azacitidine.

Bone marrow samples were collected before treatment started, with additional biopsies taken regularly during the six months the trial participants received azacitidine. By the end of the trial, in 2011, bone marrow samples had been collected from 21 patients – half with MDS and half with CMML.

“Our discoveries wouldn’t have been possible without the support of these patients and their contributions will help future patients,” said Dr Unnikrishnan.

“From the samples, we isolated the haematopoietic stem and progenitor cells (HSPCs) as we believe MDS and CMML arises from problems with the HSPCs.

“Using a new technology, called high-throughput sequencing, we could look at the expression of all the genes in a cell at the same time and identify which were turned ‘on’ or ‘off’’,” Dr Unnikrishnan explained.

“We used this information and the hypothesis we were testing (about who did and didn’t respond) to see if there was something that explained the difference.”

The analysis was carried out retrospectively, using the sample collections, and knowing which samples were from responders and non-responders.

“There were fundamental molecular differences, so we went on to validate these differences, through collaborations in the UK and Sweden. We wanted to ensure our findings weren’t parochial and that they occurred more generally, in larger numbers of patients from across the world, which they did.

“We found the cells of patients who responded to azacitidine went through the cell cycle more efficiently; their cells were dividing and active. The non-responders’ cells were quiescent – they were quiet and not doing much, and fewer cells were going through the cell cycle.

“We identified the cell cycle quiescence as a predictive quality, which we then validated.

“We used flow cytometry – a technique that uses a machine commonly available in hospitals – to look at the proteins produced by the genes. Again we identified, with high accuracy, markers of cell cycle quiescence that identified the responders and non-responders.

“Our work has always had a very translational focus, and our final goal is to move this predictive test into the clinic, to determine whether there is a high chance you are likely to respond or a high chance you will be a non-responder. We need to validate this prognostic tool in large numbers of patients and to define the boundaries of the response.

“We hope this test will be a diagnostic assay, which doesn’t exist anywhere in the world at the moment,” Dr Unnikrishnan said.

“Then what we want to do is provide an alternative treatment, to improve the response rates, and to give clinicians more tools in their arsenal to treat patients. We need a better treatment that targets those cells and eradicates them at a molecular level, for a more durable cure.

“We don’t know what we’ll find but we know what we need to do.

“Funding* from the Leukaemia Foundation was essential in establishing the initial trial that resulted in the valuable samples being banked.

“Discoveries take time and later additional funding from the Foundation was important to keeping things going.”

* Dr John Pimanda received a Leukaemia Foundation Career Establishment Grant of $50,000 per year from 2009-2011 (Identifying genes that are abnormally switched off in myelodysplasia), and in 2013, Dr Ashwin Unnikrishnan and Dr John Pimanda were awarded a Grant in Aid of $100,000 in 2013: (Identifying the differences between cancer stem cells and normal stem cells). They were invited to present their findings at the American Society of Hematology (ASH) annual conferences in 2015 and 2016, and at the European Hematology Association Congress in 2016.