IMMUNOLOGICAL CHARACTERIZATION OF PH+ ALL BONE MARROW BY MULTIPLEX IMMUNOHISTOCHEMISTRY
(Abstract release date: 05/18/17)
EHA Library. Hohtari H. 05/18/17; 182328; PB1614
Helena Hohtari
Contributions
Contributions
Abstract
Abstract: PB1614
Type: Publication Only
Background
The treatment results in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) have improved significantly in the era of tyrosine kinase inhibitors (TKIs). However, many patients relapse despite having received intense treatments with initially favorable responses. TKI therapy is known to modulate the immune system, and it may play a critical role in keeping the leukemia under control. However, little is known about the status of the immune system in patients with Ph+ ALL. Especially with the emerging immunotherapies in sight, it is vital to chart the immunological landmarks that could help us direct the treatment towards a more personalized course.
Aims
To characterize the immunological microenvironment in Ph+ ALL bone marrow (BM) by multiplex immunohistochemistry (IHC).
Methods
Ph+ ALL BM biopsies from the diagnosis stage were collected from Helsinki University Hospital and Tampere University Hospital (N=31). BM biopsies from non-leukemic (NL) controls (N=14) were used as a reference. Samples were hematopathologically evaluated and a tissue microarray (TMA) was constructed by selecting two BM cores with high leukemic cell infiltration per patient. The TMA sections were stained with both fluorescent and chromogenic dyes for six markers and nuclei simultaneously enabling cytometric analysis at cell-level resolution. Marker panels included T and B lymphoid cells, NK and dendritic cells, macrophages as well as myeloid derived suppressor cells. Furthermore, we analyzed immune checkpoint molecules (PD1, LAG3, OX40, TIM3, CTLA4) and their ligands (PD-L1, PD-L2, HLA-G, HLA-ABC) alongside with various activation markers (granzyme B, CD45RO, CD25, CD57, CD27). After the staining, the cells were segmented and quantified with the image analysis software CellProfiler and the cell analysis software FlowJo.
Results
The CD4+/CD8+ ratio was lower in Ph+ ALL BM versus NL BM (1.3 [Interquartile range (IQR) 1.0-1.9] vs. 2.0 [IQR 1.7-2.4], p=0.0134) indicating that there are relatively more CD8+ T cells in the leukemic than in the non-leukemic marrow. The ratio of memory CD4+CD45RO+ T cells in Ph+ ALL BM versus NL BM was elevated (21.0% [IQR 16.7-28.5] vs. 13.0 % [IQR 8.7-15.9] of CD4+ T cells, p=0.0044). The difference in memory CD8+CD45RO+ T cells was not significant (p=0.36).
Further analysis of the T cell phenotype showed increased proportion of both PD1-positive helper T cells and PD1-positive CD8+ T cells in Ph+ ALL BM vs. NL BM (29.7% [IQR 17.5-50.1] vs. 6.9% [IQR 5.7-8.9], of CD4+ cells, p<0.0001 and 28.8% [IQR 13.2-38.0] vs. 14.9% [IQR 9.6-18.7], of CD8+ cells, p=0.0107). The ratio of OX40-positive helper T cells was also higher in Ph+ ALL BM (27.1% [IQR 21.6-33.25] vs. 18.5% [IQR 14.8-21.9], of CD4+ cells, P=0.0001), but no difference was observed in the proportion of OX40-positive CD8+ T cells (P=0.49).
Conclusion
Multiplex IHC enables ample cytometric evaluation of different immune cell subtypes in their original microenvironmental context of the bone marrow. The TMA format not only allows analysis of tens of BM samples in parallel but also serves as a retrospective, easy-access archive for any follow-up studies. Ph+ ALL BM is characterized by a decrease in the CD4+/CD8+ ratio and an increase in the proportion of CD4+CD45RO+ T cells in comparison with the non-leukemic controls. The proportion of PD1-expressing T cells is also elevated. However, the heterogeneity between patients is marked. The analysis of other marker panels is presently ongoing, as well as correlation to clinical and treatment outcome parameters.
Session topic: 1. Acute lymphoblastic leukemia - Biology
Keyword(s): Ph+ ALL, Immunohistochemistry
Abstract: PB1614
Type: Publication Only
Background
The treatment results in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) have improved significantly in the era of tyrosine kinase inhibitors (TKIs). However, many patients relapse despite having received intense treatments with initially favorable responses. TKI therapy is known to modulate the immune system, and it may play a critical role in keeping the leukemia under control. However, little is known about the status of the immune system in patients with Ph+ ALL. Especially with the emerging immunotherapies in sight, it is vital to chart the immunological landmarks that could help us direct the treatment towards a more personalized course.
Aims
To characterize the immunological microenvironment in Ph+ ALL bone marrow (BM) by multiplex immunohistochemistry (IHC).
Methods
Ph+ ALL BM biopsies from the diagnosis stage were collected from Helsinki University Hospital and Tampere University Hospital (N=31). BM biopsies from non-leukemic (NL) controls (N=14) were used as a reference. Samples were hematopathologically evaluated and a tissue microarray (TMA) was constructed by selecting two BM cores with high leukemic cell infiltration per patient. The TMA sections were stained with both fluorescent and chromogenic dyes for six markers and nuclei simultaneously enabling cytometric analysis at cell-level resolution. Marker panels included T and B lymphoid cells, NK and dendritic cells, macrophages as well as myeloid derived suppressor cells. Furthermore, we analyzed immune checkpoint molecules (PD1, LAG3, OX40, TIM3, CTLA4) and their ligands (PD-L1, PD-L2, HLA-G, HLA-ABC) alongside with various activation markers (granzyme B, CD45RO, CD25, CD57, CD27). After the staining, the cells were segmented and quantified with the image analysis software CellProfiler and the cell analysis software FlowJo.
Results
The CD4+/CD8+ ratio was lower in Ph+ ALL BM versus NL BM (1.3 [Interquartile range (IQR) 1.0-1.9] vs. 2.0 [IQR 1.7-2.4], p=0.0134) indicating that there are relatively more CD8+ T cells in the leukemic than in the non-leukemic marrow. The ratio of memory CD4+CD45RO+ T cells in Ph+ ALL BM versus NL BM was elevated (21.0% [IQR 16.7-28.5] vs. 13.0 % [IQR 8.7-15.9] of CD4+ T cells, p=0.0044). The difference in memory CD8+CD45RO+ T cells was not significant (p=0.36).
Further analysis of the T cell phenotype showed increased proportion of both PD1-positive helper T cells and PD1-positive CD8+ T cells in Ph+ ALL BM vs. NL BM (29.7% [IQR 17.5-50.1] vs. 6.9% [IQR 5.7-8.9], of CD4+ cells, p<0.0001 and 28.8% [IQR 13.2-38.0] vs. 14.9% [IQR 9.6-18.7], of CD8+ cells, p=0.0107). The ratio of OX40-positive helper T cells was also higher in Ph+ ALL BM (27.1% [IQR 21.6-33.25] vs. 18.5% [IQR 14.8-21.9], of CD4+ cells, P=0.0001), but no difference was observed in the proportion of OX40-positive CD8+ T cells (P=0.49).
Conclusion
Multiplex IHC enables ample cytometric evaluation of different immune cell subtypes in their original microenvironmental context of the bone marrow. The TMA format not only allows analysis of tens of BM samples in parallel but also serves as a retrospective, easy-access archive for any follow-up studies. Ph+ ALL BM is characterized by a decrease in the CD4+/CD8+ ratio and an increase in the proportion of CD4+CD45RO+ T cells in comparison with the non-leukemic controls. The proportion of PD1-expressing T cells is also elevated. However, the heterogeneity between patients is marked. The analysis of other marker panels is presently ongoing, as well as correlation to clinical and treatment outcome parameters.
Session topic: 1. Acute lymphoblastic leukemia - Biology
Keyword(s): Ph+ ALL, Immunohistochemistry
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