21eha disclose to interest of conflicts no are There
162-P
Flaer
(days) Time Flaer
15 10 5 0 PIGH. of expression
0 BV 0nM azacytidine 0nM BV
% GPI positive cells lack also subcultures negative GPI
HP 0nM azacytidine 0nM HP ALL - B in variants escape negative CD52/GPI of
expression analysis showed that the the that showed analysis expression 5
MOCK BV 0.5nM azacytidine 0.5nM BV
might be a promising therapeutic strategy to prevent outgrowth outgrowth prevent to strategy therapeutic promising a be might cell cultures by FACS. mRNA mRNA FACS. by cultures cell
methylated
M
10
Highly or fully fully or Highly HP 0.5nM azacytidine 0.5nM HP ALL ALL - Leiden the from generated were
epigenetic modulatory drugs with alemtuzumab alemtuzumab with drugs modulatory epigenetic Combining
methylated Partially L
Inge JEDEMA Inge Acute- leukemia lymphoblastic Biology 2 subcultures negative and positive GPI
15
Unmethylated U
NGFR (marker gene) NGFR region promoter the
cells - B 20 ALL ALL
U U U U U U U U U U U U U U U U
by methylation of of methylation by regulation down epigenetic to due possibly
Healthy CD52
peptide T2A autocleaving the via - BV Leiden -
HP Leiden
PBGD
25
L L L L M M L L L M M M M L M L coupled to PIGH or PIGA PIGA or PIGH to coupled markergene a for - GPI BV but but mutations, DNA to due not was mRNA PIGH of Loss
PIGA +
assay Demethylation
L L L L L L L L L L L L L L L L
GPI+ BV
Construct used for transductions encodes encodes transductions for used Construct
L L L L L L L L L M L M M L L L PIGH
- GPI HP
transduction retroviral by expression
empty (marker)
L L L L L L L L L L L L L L L L
GPI+ HP
PIGH, PIGA, or or PIGA, PIGH,
T2A NGFR
was stable, but could be corrected by enforced PIGH PIGH enforced by corrected be could but stable, was
TSS
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
CpG
ALL cell subcultures subcultures cell ALL - B of phenotype negative CD52/GPI The
sequencing bisulfite by assay methylation Promoter
vector PIGH +
VBK synthesis anchor GPI in component key a , expression mRNA
MHX
or empty empty or PIGA not but construct,
•
reexpression anchor GPI in resulted azacytidine using Demethylation
Patient Patient
Patient Patient
PIGH PIGH of loss to due expression CD52/GPI lost cells These
PIGH PIGH the with transduction •
subcultures negative GPI in methylation promoter of level Higher
GPI GPI
subcultures upon retroviral retroviral upon subcultures neg neg •
aberrations genetic by caused not was mRNA PIGH of Loss
ALL - BV ALL - HP
ALL ALL - B negative GPI the in
diagnosis at present were populations sample patient original the to comparable highly phenotypically showed:
subcultures cell negative GPI
Restored GPI anchor expression expression anchor GPI Restored
were samples patient ALL - B two from generated cultures Cell patients CD52/GPI negative cell cell negative CD52/GPI patients ALL - B of majority the In Detailed analysis of the GPI negative and GPI positive ALL subcultures subcultures ALL positive GPI and negative GPI the of analysis Detailed Flow cytometric analysis of the transduced transduced the of analysis cytometric Flow
PIGH of downregulation Epigenetic expression GPI restores wtPIGH cultures ALL - B GPI and GPI Conclusions
pos neg
control loading a as GAPDH with samples, (n=3) genes GPI 26 the for expression mRNA
ALL ALL - B in analysis expression mRNA PIGH for screened sample ALL - B a of Example
regimen culturing day 14
sample ALL - B a in compartment cell B CD19+
ALL - B adult in expression blood Peripheral PB, marrow; Bone BM, **
SL15 PIGO
Azacitidine in a a in Azacitidine - 5 using assay demethylation a for used was subculture
anchor negative cells within the the within cells negative anchor - CD52/GPI
0.00 674.114 BM PMX8367 18
mechanism underlying the loss of CD52/GPI anchor anchor CD52/GPI of loss the underlying mechanism
6995 AOR
promoter CpG methylation by bisulfite sequencing. GPI negative negative GPI sequencing. bisulfite by methylation CpG promoter
Example of a flow cytometric analysis of of analysis cytometric flow a of Example PGAP3 PIGN
0.00 514,778 PB MRQ3306 17
The aim of the current study was to further unravel the the unravel further to was study current the of aim The
•
GPI negative and positive subcultures were used to compare the the compare to used were subcultures positive and negative GPI
0.00 21,305 PB MGR9751 16 8262 JHO
PGAP2
DOI:10.3252/pso.eu.21EHA.2016 PIGM
) anchor GPI ( Flaer 0.00 45,701 BM JWK9219 15
0747 PGZ
PGAP1 PIGL
vector empty an or PIGA 0.00 356,142 BM CPH6760 14
expression
, , PIGH encoding constructs with studies transduction retroviral perform 0.00 103,698 PB ASC5968 13 6179 MEP PIGZ PIGK
CD52 membrane expression due to loss of GPI anchor anchor GPI of loss to due expression membrane CD52 0.00 226,151 PB LMJ5903 12 using FACS into pure GPI negative and GPI positive subcultures to to subcultures positive GPI and negative GPI pure into FACS using
PIGY PIGH 7504 HRG
• 0.00 40,682 BM LOW9484 11 variants expressed normal CD52 mRNA levels, but lacked lacked but levels, mRNA CD52 normal expressed variants ALL samples were cultured, and subsequently divided divided subsequently and cultured, were samples ALL - B different Two
0.03 795,546 PB HBP6230 10 PIGX PIGF
9115 KYE
ALL. These These ALL. - B human with engrafted model mouse a in shown
0.03 628,534 BM AOR6995 9
electrophoresis
negative escape variants after alemtuzumab treatment was was treatment alemtuzumab after variants escape negative PIGW
PIGC CD52
0.04 331,127 PB MHJ8932 8
conventional PCR using specific primers and visualized by gel gel by visualized and primers specific using PCR conventional
et al, 2010), outgrowth of CD52 CD52 of outgrowth 2010), al, et Nijmeijer ( study previous a In
PIGV PIGB 0.11 31,171 PB HBZ8516 7
that comprise the GPI anchor biosynthesis pathway was analyzed by by analyzed was pathway biosynthesis anchor GPI the comprise that
0.18 938,627 BM EBG6102 6
6102 EBG PIGU PIGA
mRNA was isolated, cDNA synthesized, and expression of the 26 genes genes 26 the of expression and synthesized, cDNA isolated, was mRNA
0.28 25,465 BM JHO8262 5
variants escape ALL - B negative CD52
fluorescence activated cell sorting (FACS) and from these populations populations these from and (FACS) sorting cell activated fluorescence
PIGT GPAA1 0.65 769,559 BM PGZ0747 4
clinical efficacy, which could be caused by the outgrowth of of outgrowth the by caused be could which efficacy, clinical
•
ALL populations were purified by by purified were populations ALL - B negative GPI and positive GPI
0.91 36,783 PB KYE9115 3
PIGS DPM3
-- + -- + GPI:
inositol (GPI) anchored CD52 protein has shown modest modest shown has protein CD52 anchored (GPI) inositol
2.14 274,959 PB MEP6179 2 KYE9115
at21 EHA Poster PIGQ DPM2 - glycophosphatidyl the targets which alemtuzumab contrast, In 4.98 385,018 PB HRG7504 1
%) 0.01 limit (detection counterstaining
10th June 2016 June 10th
type nr (%) screened
FLAER and CD52 and CD19 CD19 and CD52 and FLAER aerolysin specific - GPI labeled
**
PIGP DPM1 ID Patient
Sample Sample Sample Sample GPI negative negative GPI cells cells - B #
presented (HCL) patients and healthy donors by flow cytometry with a fluorescently fluorescently a with cytometry flow by donors healthy and patients (HCL)
) ALL - (B leukemia lymphoblastic acute cell
on B ) 5 (n= donors healthy in or ), 6 (n= HCL ), 5 (n= leukemia (CLL), mantle cell lymphoma (MCL), and hairy cell leukemia leukemia cell hairy and (MCL), lymphoma cell mantle (CLL), leukemia
: in outcomes treatment improved has antigens CD20 or CD19
bone marrow samples from treatment naïve B naïve treatment from samples marrow bone ALL, chronic lymphocytic lymphocytic chronic ALL, - MCL ), 5 (n= CLL as such malignancies cell B other in not but samples,
involved in GPI anchor biosynthesis, in purified GPI negative B negative GPI purified in biosynthesis, anchor GPI in involved cells ALL -
The development of novel antibody based therapies targeting targeting therapies based antibody novel of development The
• Frequency of CD52/GPI anchor negative cells was analyzed in blood and and blood in analyzed was cells negative anchor CD52/GPI of Frequency
ALL - B 18 of out 10 in present were populations cell negative /GPI 52 CD mRNA expression, but of none of the other genes genes other the of none of but expression, mRNA PIGH of loss Recurrent
Background ALL cells at diagnosis at cells ALL - B GPI methods and Material
cells GPI in expression PIGH No
neg neg
Department of Dermatology, Leiden University Medical Center, The Netherlands. The Center, Medical University Leiden Dermatology, of Department Department of Hematology, Hematology, of
2 1
Maarten H Maarten , Zoutman . H Wim , Egmond van Esther . H.M , Rijs Kevin , Loeff C Floris . Vermeer . Jedema Inge , J.M Constantijn , J.H.Frederik , . Halkes . Falkenburg
2 1 1 1 1 1 1 2
TO B CELL ACUTE LYMPHOBLASTIC LEUKEMIA LYMPHOBLASTIC ACUTE CELL B TO
LACKING CD52 MEMBRANE EXPRESSION, CONFERRING ALEMTUZUMAB RESISTANCE RESISTANCE ALEMTUZUMAB CONFERRING EXPRESSION, MEMBRANE CD52 LACKING
LOSS OF PIGH EXPRESSION FREQUENTLY RESULTS IN A GPI A IN RESULTS FREQUENTLY EXPRESSION PIGH OF LOSS NEGATIVE SUBCLONE SUBCLONE NEGATIVE -