Vikram Mathews M'D

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Leukemic and stromal cell interactions -
relevance to therapy of acute leukemia

• Vikram Mathews M.D

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Survival in AML - ECOG protocols since 1973.
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Normal Hematopoietic microenvironment Stromal
cells hematopoietic Macrophages Endothelial
cells non hematopoietic Myofibroblasts
Fibroblastoid cells Osteogenic
precursors Adipocytes Extracellular matrix
(ECM) Proteoglycans and constituent
glycosaminoglycans Heparan sulfate
Chondroitin sulfate Dermatan sulfate
Hyaluronic acid Collagen types I, III,
IV, V, VI Fibronectin Thrombospondin
Sialoadhesin Laminin Tenascin Cytokin
es G-CSF, GM-CSF etc
Giles et al. Hematol 2002.
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Adhesion molecules on hematopoietic precursors
and stromal cells Cell adhesion molecules
(CAMS) classified into six distinct
superfamilies 1. Integrins heterodimeric
proteins ? and ? chains - ?1
integrins variety of VLA (very late
antigens) ?4?1 VLA-4 (VCAM-1, Fibronectin)
?5?1 VLA-5 (Fibronectin) - ?2
integrins LFA-1 (CD11a) (ICAM-1) Mac-1
(CD11b) 2. Selectins glycoproteins - L
selectin 3. Sialomucins CD34, CD45RA, CD164 4.
Immunoglobulin sequence homology to Ig
superfamily 3 CAMS of relevance to
hematopoiesis - VCAM-1 (VLA-4) -
ICAMS (LFA-1) - NCAMS (NCAM-1 - CD56) 5.
CD44 proteoglycan binds Hyaluronic acid widely
expressed on stromal cells, lower level on
HPC 6. Cadherins E, N and P cadherins.
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Table 2. Factors constitutively or inducibly
expressed by marrow stromal cells.
G-CSF IL-1 GM-CSF IL-1ß M-CSF IL-
6 Flt-3 ligand IL-7 SCF IL-8 LIF
IL-12 Thrombopoietin IL-14 TNF- IL-15
TNF-ß IL-16 TGF-ß IL-17 HGF IL-18
NGF BDNF SDF-1
Giles et al. Hematol 2002.
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Giles et al. Hematol 2002.
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Study illustrates 1. Expression pattern of
VLA-4 and VLA-5 in myeloid cell lines and in
patients with AML 2. Interaction of above with
appropriate ligands protects cells from
spontaneous and drug induced apoptosis 3.
Mechanism of this resistance to apoptosis 4.
Relevance of expression pattern to prognosis 5.
Intervention that can alter this resistance and
has potential for therapeutic application

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Adhesion molecule expression on leukemic
cells - Leukemic blasts express many of the
adhesion molecules seen on normal HPC - Early
studies attempted to show differential expression
pattern compared to normal HPC. - not
conclusive - wide heterogenity - Studies by the
Westmead group - Sydney - AML cells bind to
stromal cells via a combination of ?1 and ?2
integrin mechanisms - Similar role in pre B cell
ALL Other molecules such as CD31, CD44, non ?1
, non ?2 integrins etc also have a role -
Importance of SDF-1/CXCR4 interaction in BM
engraftment of pre B cell leukemic blasts
Reuss-Borst et al. Leukemia 1995. De Waele et al.
Eur J Hematol 1999
Gottlieb et al. Blood 1993. Bradstock et al. Leuk
Lymphoma 1995.
Bradstock et al. Leukemia 2000. Bradstock et al.
Exp Hematol 2001.
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Adhesion to stroma protects AML cells from
spontaneous and drug induced apoptosis - Exp
Hematol. 1994 Dec22(13)1252-60. Bone marrow
adherent layers inhibit apoptosis of acute
myeloid leukemia cells. Bendall LJ, Daniel A,
Kortlepel K, Gottlieb DJ. - Exp Hematol. 2001
Apr29(4)448-57. Acute myeloid leukemia cells
are protected from spontaneous and drug-induced
apoptosis by direct contact with a human bone
marrow stromal cell line (HS-5). Garrido SM,
Appelbaum FR, Willman CL, Banker DE. - Leukemia.
2002 Sep16(9)1713-24. Stromal cells prevent
apoptosis of AML cells by up-regulation of
anti-apoptotic proteins. Konopleva M, Konoplev
S, Hu W, Andreeff M.
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Studies with clinical correlation - Expression
of VLA molecules on acute leukemic cells
relationship with disease characteristics.
Archimbaud et al. Exp Hematol 1995. AML
n67 ALL n 40 Expression of VLA-4 associated
with high bone marrow blast involvement at
diagnosis and increased peripheral blood blasts
in AML. No hematological correlation with
ALL No correlation in either with disease
outcome.
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Vla-4 negative n 10
Two AML cell lines expression pattern ()
?4 ?5 ?1 U937 95.9 95.4 94.6 HL60 98.8 0.8 9
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Vla-4 positive n 20
On the basis of discriminate function cutoff
level for VLA-4 positivity was 34.55 No
difference between VLA-4 -/ patients for foll
risk factors FAB, PS, WBC, Karyotype,
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AML cell lines incubated on fibronectin(o), BSA
() or VCAM-1() coated with DNR or Ara-C.
Viable cells assayed after 24 hours by trypan
blue exclusion. Data shown as mean SD
(triplicate cultures). Representative data from
one of three independent experiments. U937 HL
60
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U937 cells
Is the interaction with fibronectin which confers
resistance to drug induced apoptosis mediated via
VLA-4 or VLA-5? Repeat cultures with blocking
antibodies to to VLA-4 and VLA-5 at 10
mcg/ml Viable cells assayed after 24 hours by
trypan blue exclusion. Data shown as mean SD
(triplicate cultures). Representative data from
one of three independent experiments.
BSA coated plates Fibronectin coated plates
Caspase -3 activity had a similar pattern as
Annexin V staining.
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When leukemic cell lines were cultured on stromal
cells. Similar protective effect as seen with
incubation on fibronectin coated plates. Which
could be negated by using VLA-4 blocking
antibodies. Suggests that this could be an
important drug resistance mechanism in
vivo. Similar results generated using patients
leukemic cells instead of leukemic cell lines.
Viability of cells post incubation of patients
leukemic cells with DNR on fibronectin coated
plates proportional to expression of VLA-4 level.
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Next series of experiments address the mechanism
of resistance to drug induced apoptosis
following VLA-4 - fibronectin interaction. - up
regulation of bcl-2 - mediated via PI-3K/AKT
signaling pathway.
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Anti VLA-4 and Ara-C prevents MRD in
mice SCID mice
Radiation 400 rads
U937 cells 5 x 106 cells
Each group n10 mice Vehicle - normal
saline Vehicle Ara C Control antibody and
vehicle Control antibody and Ara C VLA-4
vehicle VLA-4 Ara C Intra-peritoneal
administration Antibodies 1 mg AraC 20 mg
DAY 7
Observe x 62 days
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Vehicle Vehicle Ara C Control antibody and
vehicle Control antibody and Ara C VLA-4
vehicle VLA-4 Ara C also PCR negative both at
24 hrs and at 62 days
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Similar experiment with human leukemic
cells NOS-SCID recipients 400 rads
conditioning Donors Patient 1 VLA-4
-ve Patient 2, 3 VLA-4 ve Injected 1 x 107
leukemic cells Day 3 and 4 IP AraC 40 mg and
anti VLA-4 2 mg / isotypic antibody 2
mg Analyzed Day 5/6
AraC isotype AB
AraC anti VLA-4
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VLA-4 on leukemic cells defines patient
prognosis n25 (excluded 5 patients with
APL) Protocol JALSG AML 97 VLA-4 ve VLA-4
-ve n 15 10 Induction CR 60 100 (p0.02
9) Relapse rate 55.6 0 (p0.011) 5 year
DFS 44.4 100 (P0.0094) 5 year
OS 25 100 (p0.0011)
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The authors conclude 1. VLA-4 expression should
be used as a prognostic marker in the management
of AML. 2. Propose the use of VLA-4 specific
antibodies combined with chemotherapy to
eradicate MRD in AML.
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Abstract Stromal cell-derived factor-1 (SDF-1)
is a key regulator of the behavior of normal and
leukemic precursor-B (pre-B) cells. It is
possible that inhibiting SDF-1-driven processes
in pre-B acute lymphoblastic leukemia (ALL) may
have therapeutic implications.
----------------- ------ Similar results were
obtained with ALL samples. AMD3100 (1 M)
completely blocked SDF-1-induced chemotaxis and
attenuated the migration of pre-B ALL cells into
bone marrow stromal layers. ------- -------In
addition, AMD3100 enhanced the cytotoxic and
antiproliferative effects of the cytotoxic
agents vincristine and dexamethasone. The
ability of SDF-1 inhibitors to modulate these
biologically important functions of leukemic
cells warrants further investigation. Leukemia
(2003) 17, 1294-1300. doi10.1038/sj.leu.2402998
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Cell adhesion mediated - drug resistance in
human myeloma cell line. Damiano JS et al Blood
1999.