Normal & Leukaemic haematopoiesis 2010 Dr. Liu Te Chih Dept - - PowerPoint PPT Presentation

Normal & Leukaemic haematopoiesis

2010

• Dr. Liu Te Chih

Dept of Haematology / Oncology National University Health Services Singapore

• Lineage assignment
• Differentiation of reactive from

leukaemic blasts

• Detection of Minimal Residual Disease

Use of Immunophenotyping today

• 1. Leukaemic cells are phenotypically aberrant

Normal (PBSCH) Leukaemic

CD34+ cells (red):

TJ - M4 AML : NG2/CD65/CD45/CD34

40% - 20%

Diagnostic

4% - 1%

PR: 5%

0.4% - 0.1%

CR Gated: CD34+ CD34+ blasts (yellow) CD34- blasts (red)

• 2. Detection Sensitivity: ~ 0.01%

5

Excitation Optics

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

Immunophenotyping today

• 8 Colour cytometers
• Improved hardware sensitivity

Normal haematopoiesis

Orkin, Cell 2008; 132:631

CD34+/90+/(Rho-lo)/lin-

• frequency: 0.02 - 0.1% of BM nucleated cells

Normal haematopoiesis

Orkin, Cell 2008; 132:631

CD34+/38-/90+/45RA-/ 10-/117+/FLT3R-/lin- CD34+/90-/38+/117-lo/10+ (or 7+)/FLT3R+/lin- CD123-lo/CD45RA+ CD123-lo/45RA- CD34+/90-/38+/117+/13+/33+/HLADR+/64- /41a-/9-/FLT3R+/lin- CD123-/CD45RA- CD34+/38-/90-/45RA-/ 10-/117+/FLT3R-/lin-

38- 38+

10+/IL7Ra+/ 117lo 90-

GATE OF CD34+ HPC

IMMUNOPHETOPYPIC CHARACTERIZATION OF IMMATURE / EARLY CD34+ HPC

Orfao, 2007

GATE OF CD34+ HPC

ERYTHROID & B-LYMPHOID DIFFERENTIATION IN BM

Orfao, 2007

Leukaemic situation

31yr Male Pancytopenia HLADR/117/45/34

Green - Leuk blasts Cyan - Myeloblasts Grey - Lymphocytes Yellow - Neutrophils Blue - Monocytes Pink - Eosinophils

SSC SSC SSC CD34 HLADR CD117 CD45 FSC

nTdt/cMPO/45/34

Green - Leuk blasts Cyan - Myeloblasts Grey - Lymphocytes Pink - B-lymphocytes

CD19/c79a/45/34 cCD3/7/45/34

nTdt cMPO SSC CD45 cCD79a CD7 CD19 cCD3

CD7/10/19/34

Green - Leuk blasts Cyan- Myeloblasts (presumed)

CD71/38/123/34

CD19 CD34 CD38 CD123 CD38 CD10

• Myeloblasts (10%)
• Lymphoblasts (90%) - ? T or ?B
• nuTdt+ / CD34+
• B-lineage: CD19+ / cyCD79a- / CD22- / 10+
• T-lineage: cyCD3- / CD7+

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

Orkin, Cell 2008; 132:631

CD34+/38-/90+/45RA-/ 10-/FLT3R-/lin- CD34+/90-/38+/117-lo/10+ (or 7+)/FLT3R+/lin- CD123-lo/CD45RA+ CD123-lo/45RA- CD34+/90-/38+/117+/13+/33+/HLADR+/64- /41a-/9-/FLT3R+/lin- CD123-/CD45RA- CD34+/38-/90-/45RA-/ 10-/FLT3R-/lin-

• Myeloblasts (10%)
• Lymphoblasts (90%) - T or B
• nuTdt+ / CD34+
• B-lineage: CD19+ / cyCD79a- / CD22- / 10+
• T-lineage: cyCD3- / CD7+

Ph+ (bcr-abl p190+) Acute Lymphoblastic Leukaemia

• Does AML originate from a HSC or does it

arise from a more mature (non-HSC) progenitor and aberrantly acquire self-renewal properties?

• majority are CD34+/90- blasts
• Holden. BLOOD 1995; 86:60
• variably repopulate irradiated NOD/SCID mouse
• most are not stem cells

Are AML aberrant HSC?

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

• 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

HSC numbers not sig higher as disease progresses

More MEP at Chronic Phase; More GMP at Blast Crisis

Increased b-catenin expression in progenitors, not HSC

• 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

• 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

PNAS 2000; 97:7521

• 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

Leukaemic cell of origin

Orkin, Cell 2008; 132:631

CML CML - BC

Rossi, Cell 2008; 132:681

• 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

Significance ? of LSC

Search for LSC specific markers important

• 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

• 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

• Results in diff types of AML:

• Results of transplantation into irradiated

mice:

• CD96+ cells engraft recipients

• 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

Cell 2009; 138: 271 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
• Majeti. Cell 2009: 138: 286

Rossi, Cell 2008; 132:681

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

LSC candidate markers

Marker Function

• CD123

unknown

• CD44

homes to HSC niche

• CLL-1

unknown

• CD96

unknown

• CD47

inhibits phagocytosis

Targetted anti-LSC Rx

• anti-CD47 depletes AML but spares normal

HSC

• (transplanted into NOD/SCID mice)
• Majeti. Cell 2009: 138: 286

Nature Medicine. 2006:12 1167

• (transplanted in NOD/SCID mice)

• (transplanted in NOD/SCID mice)

Cell Stem Cell. 2009; 5: 31

• 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

Remarks:

9% 6% 22% 62%

Case #2 total CD34+ 0.6%

CD34 subsets

Case #1 total CD34+ 0.7%

2% 2% 38% 57% CD34 subsets

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

Summary Display Live statistical input Reference Images

Data Visualisation Approaches