Klinisk institutt 2

Midtveisevaluering - Benedicte Sjo Tislevoll




Mass cytometry reveal early signaling responses to induction therapy in immunophenotypically defined leukemic cell subsets that predict survival in AML

Benedicte Sjo Tislevoll 1, Oda Helen Eck Fagerholt 1, Monica Hellesøy 2, Stein-Erik Gullaksen1, Maria del Pilar Ayuda Duran 3, Laure Isabelle Piechaczyk 3, Jørn Skavland 1, Sonia Gavasso1, Randi Hovland 4, Tobias Gedde-Dahl 5, Øystein Bruserud 1,2, Jorrit Enserink 3,

Yngvar Fløisand 3,5, Nello Blaser 6 and Bjørn Tore Gjertsen 1,2

1. Centre for Cancer Biomarkers (CCBIO) Department of Clinical Science, University of Bergen, Bergen, Norway.

2. Department of Internal Medicine, Hematology Section, Haukeland University Hospital, Bergen, Norway

3. Department of Molecular Cell Biology, the Norwegian Radium Hospital, Oslo, Norway

4. Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway

5. Department of Hematology Oslo University Hospital, Rikshospitalet, Oslo, Norway.

6. Department of Mathematics, University of Bergen, Bergen, Norway

Current clinical practice in intensive induction treatment of AML recommends morphological response evaluation on day 14-17 after start of induction therapy. (Lowenberg B. et al NEJM 1999) However, alterations in genes and proteins related to chemotherapy resistance and cell survival can also be detected in vivo within hours after start of therapy. (Anensen N. et al Clin Cancer Res 2006, Gausdal G et al. Blood 2008, Øyan M. et al BMC Cancer 2009). The rapid kinetics in cell response to perturbation is underscored by in vitro studies that have shown an immediate response in intracellular signalling networks which can be detected within minutes after cells are exposed to perturbators. (Irish J. et al 2004).

Cytometry by time-of-flight (CyTOF) combines the platforms of conventional flow cytometry with mass spectrometry. This dramatically increases the numbers of antibodies that can be analyzed per single cell (>40) and permits a simultaneous analysis of both intracellular signaling networks and phenotypic complexity of AML cells.

Immediate evaluation of intracellular signaling after start of treatment in malignant and non-malignant cellular subsets could reveal the functional properties of resistant AML cancer clones, and how they may be therapeutically targeted.

We performed a 36- dimensional CyTOF analysis of 21 surface markers (CD66b, CD16, CD45, CD11b, CD25, CD3, CD4, CD8, CD56, CD20, CD38, CD34, CD117, CD33, CD64, CD14, HLA-DR, Axl-1H12, CD90, CD7, CD123) and 15 intracellular markers (cCaspase-3, p-4EB-P1, p-Stat 5, p-Stat 3, p-Stat 1, p-p38, p-ERK ½, p-Akt, p-NFkB, p-CREB, p-S6, p-Axl, p-Rb, Cyclin B1, p-Histone H3) of 32 de novo AML patients. Peripheral blood was sampled at 4 hours and at 18- 24 hours after start of standard “7+3” induction therapy (Lowenberg B. et al Blood 2017). Bone marrow and peripheral blood from healthy donors were also analyzed. Samples were barcoded, and a reference sample was included in each barcode for normalization of potential batch effects. FlowSOM, which applies self-organizing maps (SOMs), were used to perform unsupervised identification of leukemic and non-leukemic cell populations across the patient cohort (Van Gassen S. et al Cytometry A 2015).

FLOWSOM identified 10 different metaclusters in the 32 AML patients and 7 healthy donors based on well- characterized surface markers, as shown in the t-SNE (Fig 1A) and heatmap plots (Fig 1B).
To identify markers that could predict patient outcome, a lasso regression analysis correlating intracellular signaling markers to patient survival was performed.

Basal pre-treatment signaling levels were not found to be significantly correlated to survival in any of the signaling markers investigated. However, we found that the ratio of pERK 1/2 at 24 hours relative to pre-treatment in metacluster 9, significantly correlated to overall survival (p=5,93e-4) (Figure 1D). Thus, an increase in pERK1/2 at 24 hours compared to the pre-treatment sample, seems unfavorable. At the four-hour time point, we did not find any signaling alterations that correlated to patient survival in any of the markers investigated.

Unsupervised clustering analysis of over 30 de novo AML patients identified leukemic cell subpopulations that had unique patterns of intracellular signaling in response to induction therapy. We found that alterations in pERK1/2 during the first 24 hours after start of induction therapy significantly correlated with patient survival. The MAPK/ERK pathway is dysregulated in nearly 50% of all human malignancies and has an important role in cell proliferation, differentiation and survival. Cells with an increase in pERK as response to chemotherapy might represent cancer cell subsets that are resistant to conventional chemotherapy,  indicating that ERK could potentially be an  interesting therapeutic target in AML.  Monitoring of AML patients by CyTOF during therapy seems feasible in a clinical setting, using the therapy as a perturbator for unraveling functional defects in intracellular signaling that could identify suboptimal therapy responders.