Significant progress has been made in recent years in the treatment of acute B-cell lymphoblastic leukemia. However, the disease in some cases persists giving rise to relapses, which are complex to treat. Understanding why resistance to treatment happens and how to effectively and rapidly deliver the most appropriate therapies for each patient was one of the goals set by physicians and researchers at IRCCS Ospedale San Raffaele, SR-Tiget and Università Vita-Salute San Raffaele with the study just published in Leukemia (part of the Nature Journals group).

 

The research, carried out with support from AIRC Foundation for Cancer Research, used innovative single cell RNA sequencing techniques and transcriptomic studies in collaboration with bioinformaticians. The study was conducted on both pediatric and adult samples in partnership with Tettamanti Center, IRCCS San Gerardo dei Tintori Foundation, Monza, and the Papa Giovanni XXIII Hospital, Bergamo.

 

Acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is a blood cancer that originates from particular white blood cells, the lymphocytes, and is characterized by an accumulation of these cells in the bone marrow, blood, and other organs. The disease is defined as "acute" because it progresses very quickly and, in ALL in particular, the B lymphocytes undergo a tumor transformation: the cell halts its maturation and begins to proliferate rapidly, invading first the bone marrow and then the blood, later also reaching lymph nodes, spleen, liver, and the central nervous system. Although ALL is a relatively rare disease (in Italy there are about 450 new cases in men and 320 in the women each year), it is the most frequent tumor in pediatric patients, accouning for 80 % of all leukemia and 25 % of all cancers diagnosed between 0 and 14 years of age * (* AIRC source).

 

Minimal residual disease: the prognostic factor

Thanks to the advancement of knowledge regarding the disease and of available treatments, particularly immunotherapies, the prognosis of patients with ALL today is much improved over the past. However, some patients experience relapse and the cells that persist after the initial treatment (induction), collectively referred to as Minimal Residual Disease or Minimal Residual Disease (MRD), are used as a prognostic factor. How? The lower the number of leukemic cells found at the end of the first treatment (low MRD), the better the patient's response to treatment and the lower the risk of relapse.  Conversely, the higher the MRD, the greater the likelihood of relapse. Quantifying MRD is therefore a key tool for stratifying patients and adjusting treatment ad hoc for each group of patients (low, medium and high risk of relapse).

 

The San Raffaele study

Another important aspect that remains to be clarified is how leukemic cells survive treatment, what underlies minimal residual disease, and how it then turns into relapse. In acute and chronic myeloid leukemia, the disease persistence is often associated with the existence of leukemic stem cells that can survive in a kind of hibernation state, even for a long time, and then "wake up" giving rise to relapse. 

"Just recently our laboratory carried out a study in which it was shown how a particular microRNA, miR-126, identifies precisely these leukemic stem cells, which are responsible for resistance to chemotherapy (Naldini et. al. 2023). In B-ALL we speak rather of intra-tumor heterogeneity: within a tumor multiple cell populations that differ in genetics, function and epigenetics coexist ," says Professor Bernhard Gentner, until recently group leader of the Laboratory of Stem Cells and Leukemia at the San Raffaele Telethon Institute for Gene Therapy and now a Professor at the University of Lausanne.

 

The researchers investigated whether miR-126 might also play a role in determining intra-tumor heterogeneity in human ALL and consequently also in disease persistence after treatment. To answer this question, they used genetic engineering techniques to mark leukemia cells with a fluorescent reporter able to highlight potential stem-like subpopulations. "We were surprised when we saw that most of the B-ALL samples examined contained a 'stem-like' subpopulation characterized by high miR-126 activity," explains Dr. Carolina Caserta, Postdoctoral fellow at the Translational Stem Cells and Leukemia Unit at IRCCS Ospedale San Raffaele. The study of the functional properties of this B-ALL subpopulation left few doubt about its importance: in animal models, it showed very aggressive and chemotherapy-resistant growth. But there is an easier way to identify this subpopulation in clinical samples.

Therefore, the researchers conducted molecular studies to look for changes at the DNA and RNA levels upstream and downstream of miR-126, defining, respectively, a DNA methylation pattern and a transcriptomic signature that characterize subpopulations with high miR-126 expression that can be easily identified by single cell RNA sequencing.

 

The second part of the study

"In the second part of our study, we focused understanding how all the experimental information obtained could be used in the clinic. We found that patients with B-ALL who more often showed these miR-126 high subpopulations, were more likely to be positive for MRD after chemotherapy," explains Dr. Silvia Nucera, a researcher and physician at the Tettamanti Foundation and former Postdoctoral fellow in Gentner's laboratory.

 

Perspectives for treating acute lymphoblastic leukemia

The idea of San Raffaele researcher group  is to introduce this prognostic test into the clinic in conjunction with current criteria: this would allow for a more accurate initial stratification of patients and for the identification of  candidates who would benefit more from a first-line immunotherapy approach. "Currently, to quantify MRDit is necessary to administer chemotherapy to the patients and to wait at least 1 month. Instead, the tests we developed could be performed directly on diagnostic samples before the start of treatment. This could, in the future, allow avoiding or reducing chemotherapy in poorly responsive patients by sparing them unnecessary treatment-related toxicity, a clear step forward in improving treatment," Professor Gentner concludes.