Normal & Leukaemic haematopoiesis. Dr. Liu Te Chih Dept of Haematology / Oncology National University Health Services Singapore

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1 Normal & Leukaemic haematopoiesis 2010 Dr. Liu Te Chih Dept of Haematology / Oncology National University Health Services Singapore

2 Use of Immunophenotyping today Lineage assignment Differentiation of reactive from leukaemic blasts Detection of Minimal Residual Disease

3 1. Leukaemic cells are phenotypically aberrant Normal (PBSCH) CD34+ cells (red): Leukaemic

4 2. Detection Sensitivity: ~ 0.01% Diagnostic 40% - 20% PR: 5% 4% - 1% CR 0.4% - 0.1% TJ - M4 AML : NG2/CD65/CD45/CD34 Gated: CD34+ CD34+ blasts (yellow) CD34- blasts (red)

5 Excitation Optics fiber optic cables prisms focusing lens steering plate red laser flow cell blue laser violet laser 5

6 Immunophenotyping today 8 Colour cytometers Improved hardware sensitivity

7 Normal haematopoiesis Orkin, Cell 2008; 132:631

8 CD34+/90+/(Rho-lo)/lin- - frequency: % of BM nucleated cells

9 Normal haematopoiesis CD34+/38-/90-/45RA-/ 10-/117+/FLT3R-/lin CD34+/38-/90+/45RA-/ 10-/117+/FLT3R-/lin- CD34+/90-/38+/117+/13+/33+/HLADR+/ /41a-/9-/FLT3R+/lin- CD34+/90-/38+/117-lo/10+ (or 7+)/FLT3R+/lin- 10+/IL7Ra+/ CD123-lo/45RA- CD123-/CD45RA- CD123-lo/CD45RA+ 117lo Orkin, Cell 2008; 132:631

10 IMMUNOPHETOPYPIC CHARACTERIZATION OF IMMATURE / EARLY CD34 + HPC GATE OF CD34 + HPC Orfao, 2007

11 ERYTHROID & B-LYMPHOID DIFFERENTIATION IN BM GATE OF CD34 + HPC Orfao, 2007

12 Leukaemic situation

13 31yr Male Pancytopenia SSC SSC HLADR/117/45/34 Green - Leuk blasts Cyan - Myeloblasts Grey - Lymphocytes Yellow - Neutrophils Blue - Monocytes Pink - Eosinophils SSC FSC CD117 CD45 CD34 HLADR

14 SSC cmpo ntdt/cmpo/45/34 CD45 ntdt CD19/c79a/45/34 ccd3/7/45/34 ccd79a CD7 Green - Leuk blasts Cyan - Myeloblasts Grey - Lymphocytes Pink - B-lymphocytes CD19 ccd3

15 CD10 CD7/10/19/34 CD19 CD71/38/123/34 CD38 CD38 Green - Leuk blasts Cyan- Myeloblasts (presumed) CD123 CD34

16 Myeloblasts (10%) Lymphoblasts (90%) -? T or?b nutdt+ / CD34+ B-lineage: CD19+ / cycd79a- / CD22- / 10+ T-lineage: cycd3- / CD7+

17 Phenotype: CD45-/34+/38-/123+/10+/19+/7dim/117-/HLADR+/nuTdt+/71- Lineage Negative: cy79a/cy22/cympo/cy3/4/8/56/light chain/41/42a CD34+/38-/90+/45RA-/ 10-/FLT3R-/lin- CD34+/90-/38+/117-lo/10+ (or 7+)/FLT3R+/lin- CD34+/90-/38+/117+/13+/33+/HLADR+/64- /41a-/9-/FLT3R+/lin- CD123-lo/45RA- CD34+/38-/90-/45RA-/ 10-/FLT3R-/lin- CD123-/CD45RA- CD123-lo/CD45RA+ Orkin, Cell 2008; 132:631

18 Ph+ (bcr-abl p190+) Acute Lymphoblastic Leukaemia Myeloblasts (10%) Lymphoblasts (90%) - T or B nutdt+ / CD34+ B-lineage: CD19+ / cycd79a- / CD22- / 10+ T-lineage: cycd3- / CD7+

19 Does AML originate from a HSC or does it arise from a more mature (non-hsc) progenitor and aberrantly acquire self-renewal properties?

20 Are AML aberrant HSC? majority are CD34+/90- blasts Holden. BLOOD 1995; 86:60 variably repopulate irradiated NOD/SCID mouse most are not stem cells

21 Bonnet. Nat Med 1997: 3: 730 LSC within the CD34+/38- fraction Re-populating cells within fractions: CD34++/38-; CD34++/71-; CD34++/HLADR- Blair. BLOOD 1998; 92: 4325

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23 At diff disease stages, isolated proportions of: Haem Stem Cells (HSC) Common Myeloid Progenitors (CMP) Granulocyte/Macrophage Precursors (GMP) Megakaryocyte/Erythrocyte Precursors (MEP) compared bcr-abl transcript levels in diff populations

24 HSC numbers not sig higher as disease progresses

25 More MEP at Chronic Phase; More GMP at Blast Crisis

26 Increased b-catenin expression in progenitors, not HSC

27 Conclusion: CML blasts are GMP cells, not HSC or MPP cells Blasts acquire stem cell (self-renewal) properties via activation of wnt/b-catenin pathway

28 PNAS 2000; 97:7521 paradox: some pt have persistent low level t(8;21)+ yet w long-term remission? t(8;21) PCR+ MRD isolated diff populations from pt in CR & checked for t(8;21) by PCR

29 Results: at presentation: leuk cells are CD34+/38+/90-; PCR+ also in some B-cells but not T-cells; also in some CFU- E, CFU-mega, CFU-GM at CR: PCR+ in some stem cells (CD34+/38-/90+), mono & B-cells; also in some CFU-E, CFU-mega, CFU-GM Conclusion: t(8;21) mutation occur at totipotent cell but in itself, is insuff to result in leukaemia additional downstream mutations required for persistent proliferation & self-renewal (Leukaemia) LSC is downstream of totipotent HSC

30 Leukaemic cell of origin CML CML - BC Orkin, Cell 2008; 132:631

31 Rossi, Cell 2008; 132:681

32 Significance? of LSC More aggressive disease Good risk AML have lower LSC numbers Ailles. BLOOD 1999; 94:1761 MRD indicator if LSC phenotype is different from normal progenitors Potential therapeutic target

33 Search for LSC specific markers important

34 search for LSC-specific surface markers in t(8;21) made cdna from stem-cell enriched fraction screened for genes with surface expression using modified Signal Sequence Trap - Retroviral Expression (SST-REX) PCR underlying aim: for development of LSC-targetted Rx PNAS 2007; 104:11008

35 Results: 35 candidate genes isolated Expression levels of each candidate gene were compared in t(8;21) vs Normal BM CD34+/38- cells CD96 only gene w high expression in leuk over normal HSC

36 Results in diff types of AML:

37 Results of transplantation into irradiated mice: CD96+ cells engraft recipients

38 Conclusion: Leuk SC phenotype CD34+/38-/90-/96+ this phenotype in normal person is not self-renewing phenotype similar in t(8;21)+ & t(8;21)- patients differ from norm HSC CD34+/38-/90+/96- CD96 possibly not a good therapeutic target CD96+ in T, NK-cells, kidney tubules, gut mucosa, vascular endothelium

39 Cell 2009; 138: 271 Cell 2009; 138: 286

40 Majeti. Cell 2009: 138: 286 CD47+ in all mouse AML cell lines CD47+ in human AML cells Differential expression of CD47 on Stem cells CD47hi in AML LSC but CD47lo in norm HSC CD47 expression inhibits phagocytosis

41 Rossi, Cell 2008; 132:681

42 CD47 a/w poor outcome Majeti. Cell 2009: 138: 286 CD47 a/w FLT3 ITD; t(8;21) mostly CD47-

43 LSC candidate markers Marker CD123 CD44 CLL-1 CD96 CD47 Function unknown homes to HSC niche unknown unknown inhibits phagocytosis

44 Targetted anti-lsc Rx

45 Majeti. Cell 2009: 138: 286 anti-cd47 depletes AML but spares normal HSC (transplanted into NOD/SCID mice)

46 (transplanted in NOD/SCID mice) Nature Medicine. 2006:

47 (transplanted in NOD/SCID mice) Cell Stem Cell. 2009; 5: 31

48 Remarks: Clinical impetus for detection and characterisation of early CD34+ subsets LSC detection earliest upstream progenitor w aberrant phenotype more reflective of MRD status than the downstream blast count targetting Rx at LSC promising in-vivo chemo / ex-vivo purging pre-auto SCT

49 CD34 subsets 2% 2% 38% 57% CD34 subsets 6% 9% 22% 62% Case #1 total CD % Case #2 total CD %

50 Immunophenotyping today 8 Colour cytometers interactions and artefacts are compounded analysis more complex Newer approaches to analysis / visualisation of results Remove some of the subjectivity from the analysis

51 Data Visualisation Approaches Reference Images Summary Display Live statistical input