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Regular Article
LYMPHOID NEOPLASIA
P110a-mediated constitutive PI3K signaling limits the efficacy of
p110d-selective inhibition in mantle cell lymphoma, particularly with
multiple relapse
Sunil Iyengar, Andrew Clear, Csaba B¨od¨or, Lenushka Maharaj, Abigail Lee, Maria Calaminici, Janet Matthews,
Sameena Iqbal, Rebecca Auer, John Gribben, and Simon Joel
Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London, United Kingdom
Key Points
• The increased expression of
PI3K p110a in mantle cell
lymphoma, particularly at
relapse, suggests a role for
p110a in disease progression.
• A high PIK3CA/PIK3CD ratio
identifies patients unlikely to
respond to p110d inhibitors
and supports use of dual
p110a/p110d inhibitors in MCL.
Phosphoinositide-3 kinase (PI3K) pathway activation contributes to mantle cell lymphoma
(MCL) pathogenesis, but early-phase studies of the PI3K p110d inhibitor GS-1101 have
reported inferior responses in MCL compared with other non-Hodgkin lymphomas.
Because the relative importance of the class IA PI3K isoforms p110a, p110b, and p110d in
MCL is not clear, we studied expression of these isoforms and assessed their
contribution to PI3K signaling in this disease. We found that although p110d was highly
expressed in MCL, p110a showed wide variation and expression increased significantly
with relapse. Loss of pho sphatase and tensin homolog expression was found in 16%
(22/138) of cases, whereas PIK3CA and PIK3R1 mutations were absent. Although p110d
inhibition was sufficient to block B-cell receptor–mediated PI3K activation, combined
p110a and p110d inhibition was necessary to abolish constitutive PI3K activation. In
addition, GDC-0941, a predominantly p110a/d inhibitor, was significantly more active
compared with GS-1101 against MCL cell lines and primary samples. We found that
a high PIK3CA/PIK3CD ratio identified a subset of primary MCLs resistant to GS-1101 and
this ratio increased significantly with relapse. These findings support the use of dual p110a/p110d inhibitors in MCL and suggest
a role for p110a in disease progression. (Blood. 2013;121(12):2274-2284)
Introduction
Mantle cell lymphoma (MCL) is an aggressive disease in the vast
majority of patients and is incurable with conventional therapy.
Although there has been an improvement in median overall survival
(OS), from the 2- to 4-year range cited in earlier series to between 5
and 7 years more recently,
1
outcome is still one of the poorest among
B-cell lymphomas. MCL is characterized by t(11;14), which results
in juxtaposition of the IgH enhancer on chromosome 14 to the cyclin
D1 locus on chromosome 11, leading to the characteristic over-
expression of cyclin D1. Secondary hits primarily leading t o
defective DNA damage repair and cell -cycle dysregulation occur
in MCL,
2
and a number of studies have implicated activation
of the phosphoinositide-3 kinase (PI3K) pathway, one of the
most commonly dysregulated pathways in human cancer, in the
pathogenesis of this disease.
3-5
The serine-threonine kinase AKT,
which is the major downstream target of PI3K, is thought to be
important in MCL survival through its role in stabilizing cy clin D1
messenger RNA (mRNA), preventing nuclear export of cyclin D1
by phosphorylation of GSK-3b and increasing cyclin D1 translation
through mammalian target of the rapamycin (mTOR) activation.
6-8
PI3Ks are heterodimeric lipid kinases that have a regulatory and
a catalytic subunit. Class IA PI3Ks primarily signal downstream of
the B-cell receptor (BCR) and tyrosine kinase receptors to mediate
downstream effects that lead to increased cell metabolism, pro-
liferation, and survival. They have 3 catalytic subunit isoforms—
p110a,p110b, and p110d (encoded by PIK3CA, PIK3CB,and
PIK3CD, respectively)—that dimerize with a p85 regulatory subunit.
The p85 subunits (p85a,p85b,p55g,p55a, and p50a)recognize
phosphorylated tyrosine motifs.
9
These motifs are found in the
intracellular domains of CD19 and BCAP (the B-cell adaptor for
PI3K) and are phosphorylated upon BCR stimulation via phosphor-
ylation of Syk and Lyn.
10
On binding to these sites, the p110 catalytic
subunits are activated and convert cell membrane–bound PIP2
(phosphatidylinositol 4,5 biphosphate) to the important second
messenger PIP3 (phosphatidylinositol 3,4,5 triphosphate). PIP3
binds and activates proteins that have a pleckstrin homology domain,
such as AKT and PDK1. Full activation of Akt requires PI3K-
induced phosphorylation at threonine 308 and mTOR complex 2–
induced phosphorylation at serine 473. PTEN (phosphatase and
tensin homolog) is a lipid phosphatase that opposes activation of
this pathway by converting PIP3 back to PIP2.
11
The p110d isoform is a key messenger in BCR signaling and is
highly enriched in leukocytes,
12,13
making it an attractive target in
Submitted October 7, 2012; accepted December 18, 2012. Prepublished
online as Blood First Edition paper, January 22, 2013; DOI 10.1182/blood-
2012-10-460832.
J.G. and S.J. contributed equally to this study.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
© 2013 by The American Society of Hematology
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B-cell malignancies. However, in addition to quantitative isoform
expression, the mechanism of PI3K activation may predict sensitivity
to isoform selective inhibition. P110a (PIK3CA) is the only PI3K
catalytic unit isoform that harbors cancer-associated somatic muta-
tions and these occur at a high frequency in solid tumors.
14
PIK3CA
mutations have not been found in 2 separate studies of MCL
primary samples, but interestingly, gene amplification of PIK3CA
related to increased copy number has been described in this
disease.
3,15
Loss of PTEN expression is another mechanism leading
to constitutive activation of the PI3K pathway, and studies in solid
tumors have demonstrated a key role for p110b in PTEN-deficient
tumors.
16-18
Loss of PTEN expression has been described in
approximately 15% of MCLs.
15
Other mechanisms of PTEN in-
activation that have been suggested in MCL include phosphoryla-
tion of PTEN and negative regulation by the microRNA-17–92
cluster.
4,19
More recently, activation of all 3 class IA isoforms has
been described in association with somatic mutations in the gene
encoding the regulatory p85a subunit (PIK3R1) in solid tumors.
20
These mutations have not been studied in MCL.
Preclinical studies have demonstrated the potential of inhibiting
the PI3K pathway in MCL,
3,4,15,21
but early-phase studies of the first
p110d selective inhibitor, GS-1101, demonstrated more modest re-
sponses in patients with MCL compared with the impressive results
seen in indolent non-Hodgkin lymphoma (NHL) and chronic lym-
phocytic leukemia.
22
Because loss of PTEN expression (;15%) and
gene amplification of p110a (;68%) have been described in MCL,
15
understanding the relative contribution of the class IA isoforms in this
disease may help in targeting this important oncogenic pathway more
effectively.
We therefore studied the expression of class IA isoforms in MCL
and evaluated their contribution toPI3KsignalinginrelationtoBCR
activation, loss of PTEN expression, and increased PIK3C A expression.
We demonstrate that although p110d remains highly expressed in MCL,
tumor cells with increased PIK3CA expression can sustain constitutive
PI3K signaling despite p110d inhibition. Further, a PIK3CA/PIK3CD
ratio greater than twice that in healthy B-cell controls identified prima ry
MCL cases that were resistant to p110d inhibition but significantly
more sensitive to GDC-0941, a p110a/d inhibitor in vitro. We also
demonstrate a significant increase in both p110a expression and
the P IK3CA/PIK3CD ratio with MCL progression.
Materials and methods
Cell lines
Granta519 and Jeko-1 MCL cell lines were used after confirmation of their
identity by short tandem repeat profiling (LGC standards, Teddington, UK).
Jeko-1 was cultured in RPMI (Sigma, St. Louis, MO) and Granta519 in
Dulbecco’s modi fied Eagle medium (Sigma). Both were supplemented with
10% heat-inactivated FCS (Sigma) and 1% gentamicin (GIBCO, Life
Technologies, Paisley, UK).
Patient samples
In accordance with the updated Declaration of Helsinki, all samples were
obtained following ethical approval, and after informed consent from patients
treated at St Bartholomew’s hospital. Solid tissue used in tissue microarray
construction was fixed in formalin-fixed paraffin-embedded (FFPE) tissue.
Snap-frozen tissue was evaluated for tumor content using CD20 staining of
sections and homogenized using the Qiagen TissueLyserII (Qiagen, Hilden,
Germany) for DNA and RNA extraction. Mononuclear cells from peripheral
blood (PBMCs), bone marrow, and spleen-derived cell suspensions were
isolated using Ficoll-paque density gradient centrifugation, and 22 primary
MCL cell suspensions confirmed to have greater than 85% CD20-positive cells
by flow cytometry were used in experiments. Clinical details of these primary
samples are listed in supplemental Table 1. Cell suspensions were cultured in
Iscove modified Dulbecco medium (Sigma) supplemented with 10% human
serum, 1% gentamicin, 5 mg/mL bovine insulin, 50 mg/mL human transferrin,
and 1 mM sodium pyruvate. PBMCs for control B cells were obtained from
healthy volunteers and tonsil controls were obtained from tonsillectomies
performed for nonmalignant pathology.
Antibodies and reagents
Primary antibodies for wes tern blotting against p110a (#4249), glyceraldehyde-
3-phosphate dehydrogenase (GAPDH, #2118), total Syk (#2712), phospho-
Syk thr525/526 (#2710), total Akt (#9272), phospho-Akt ser473 and thr308
(#9271, #2965), phospho-GSK3b ser9 (#9323), total ribosomal S6 (#2217),
and phospho-S6 ser235/ser236 (#2211) were purchased from Cell Signaling
Technologies (Danvers, MA). Antibodies against p110b (sc-602) and p110d
(sc-55589) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA)
and anti-human PTEN (ABM-2052) from Cascade Bioscience (Winchester,
MA). Goat anti-human immunoglobulin (Ig)M F(ab9)
2
fragments were
purchased from Southern Biotech (Cambridge, UK). GDC-0941,
23
A66,
24
and TGX221
25
were purchased from Selleck Chem (Houston, TX) and CAL-
101/GS-1101 from Active Biochem (Maplewood, NJ). For cytotoxicity
studies, cells were treated in triplicate with increasing concentrations of
inhibitor (0.1-10 mM) for 72 hours while an inhibitor concentration of 1 mM
was used to assess downstream effects by western blotting.
Immunohistochemistry
Tissue microarrays (TMA) were constructed using triplicate 1-mm cores of
FFPE tissue. Clinical details of samples included in the tissue microarray are
listed in supplemental Table 2. Although antibodies against p110a, p110b,
and PTEN have previously been validated on FFPE tissue,
26
there was no
previous literature describing the use of a p110d-selective antibody for
immunohistochemistry (IHC) at the time of performing these experiments.
We therefore constructed a cell block microarray with high and low p110d–
expressing cell lines to optimize and validate this antibody (supplemental
Figure 1). Details of antibodies used, concentrations, and antigen retrieval
methods are listed in Table 1. All slides were scanned using an Olympus
scanning microscope to obtain high-resolution images that were analyzed on
Ariol SL-50 version 3.2 (Genetix, San Jose, CA) visual analysis software.
Briefly, images of cores were screened individually to exclude nontumor
tissue and the software was trained to calculate the percentage of positive
Table 1. Details of primary antibodies used for immunohistochemistry
Primary antibody Supplier/code Species (clone) Antigen retrieval Concentration Incubation
P110a Cell Signaling
#4249
Rabbit polyclonal
(C73F8)
Citrate pH6 1:250 40 seconds
P110b Abcam
ab55593
Mouse monoclonal Citrate pH6 1:250 40 seconds
P110d Santa Cruz
sc-55589
Mouse monoclonal (A-8) Citrate pH6 1:500 40 seconds
PTEN Cascade
ABM-2052
Mouse monoclonal (6H2.1) Citrate pH6 1:25 60 seconds
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cells and staining intensity of cores. Biopsies with only 1 evaluable core on
the TMA were excluded and IHC scores for duplicates and triplicates were
averaged. The IHC score was calculated as the product of percentage
positive cells and mean intensity. PTEN expression was scored indepen-
dently by 2 authors (S.I. and A.L.) as 21 (strong), 11 (moderate) or 0 (no
expression) based on expression in tumor cells compared with blood vessels
or nontumor cells in the TMA cores.
Western blotting
Briefly, cell lysates were prepared by resuspending cell pellets in lysis buffer
containing protease and phosphatase inhibitors (Roche, Basel, Switzerland).
Total protein was estimated using the Pierce BCA protein assay kit (Thermo
Scientific, Waltham, MA). Lysates were resolved on a NuPAGE 4% to 12%
gel (Invitrogen, Life Technologies, Paisley, UK) and transferred onto
a polyvinylidene difluoride membrane using the iBlot dry transfer method
(Invitrogen). Membranes were incubated with primary antibody either
overnight at 4°C or at room temperature for 2 hours at the recommended
dilutions. Membranes were then washed and incubated with horseradish
peroxidase-labeled secondary antibody (Dako, Denmark) for an hour at room
temperature. Electrochemiluminescence reagent (GE Health Care, Uppsala,
Sweden) was applied to visualize the blots using the Fuji LAS 2000 digital
imager (Fujifilm, Japan).
Quantitative real-time PCR
Total RNA was extracted from cell suspensions and frozen tissue using the
RNeasy mini kit (Qiagen, Hilden, Germany). A total of 2 mg RNA was rev erse
transcribed using the high-capacity reverse transcription kit (Applied Biosys-
tems, Life Technologies, Paisley, UK) as per manufacturer’s recommendations.
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed
on the ABI HT-7900 instrument (Applied Biosystems) using Taqman gene
expression assays (Applied Biosystems) for PIK3CA (Hs00192399_m1),
PIK3CB (Hs00927728_m1), and PIK3CD (Hs00180679_m1). Data were
analyzed using SDS2.1 software and relative expression values were calculated
using the DCt method with GAPDH (Hs99999905_m1) as endogenous
control. All reactions were run in triplicate.
PIK3CA and PIK3R1 mutation analysis
Genomic DNA was extracted from frozen lymph nodes (verified to have .80%
tumor content) and PBMCs of MCL patients using the DNeasy mini kit
(Qiagen). Primers used for PCR amplification of PIK3CA exons 9 and 20 and
PIK3R1 exons 9, 10, 11, 13, 15, and 16 are listed in supplemental Table 3. After
DNA purification, bidirectional Sanger sequencing was performed on the
purified PCR products.
Cytotoxicity assays
Growth inhibition was measured with the Guava ViaCount assay (Millipore,
Billerica, MA). The ViaCount reagent was added to cells treated in triplicate
in a 96-well plate, and cell count and viability was determined on a Guava
express plus instrument (Millipore). The adenosine triphosphate (ATP)
cytotoxicity assay kit (Lonza, Basel, Switzerland) was used to measure the
cytotoxic effects of PI3K inhibitors on primary MCL cell suspensions.
Briefly, cells were plated in triplicate in a 96-well format and treated with
PI3K inhibitors at increasing concentrations before measurement of ATP
luminescence in a plate reader at 72 hours.
Statistical analysis
Prism version 5.03 (GraphPad, La Jolla, CA) was used for statistical
analysis. Normally distributed data sets were tested with paired or unpaired
t tests, as appropriate, whereas for 3 data sets, 1-way analysis of variance
followed by a Bonferroni multiple comparison post-hoc analysis was used.
Data sets that were not normally distributed were analyzed using the
Mann-Whitney test for unpaired samples or the Wilcoxon matched-
pairs signed-rank test for paired samples. A P value below .05 was
considered significant.
Results
P110a expression in MCL is significantly higher beyond
first relapse
Expression of p110a, b,andd was evaluated by IHC analysis of 144
evaluable biopsies from 109 MCL patients. P110d was cons istently
expressed at high levels in MCL. P110b expression was the weakest,
whereas p110a showed a wide variation in expression across biopsies
(Figure 1A). Median expression of p 110a (median IHC score 5 44.5 vs
33.7, P 5 .15) and p110d (median IHC scores 5 77.4 vs 81.1, P 5 .2) in
MCL was not different from that seen in the germinal center area of
tonsil controls, whereas P110b expression was significa ntly lower in
MCL (P 5 .01). As shown in Figure 1B, p110a expression was
significantly higher in biopsies taken beyond first relapse compared to
those taken at diagnosis (P 5 .04). These differences were even more
striking in sequential biopsies (P 5 .008)with5of6lymphomasamples
showing increased p110a expression beyond first relapse (Figure 1B-
D). Expression of p110a beyond first relapse was also significantly
higher than expression in to nsil controls (P 5 .024). No significant
change in expression of p110b or p110d was seen with relapse. We also
did not find a significant difference in ex pression of the individual
isoforms between blastoid and classical MCL (data not shown).
Loss of PTEN expression is relatively common in MCL, whereas
PIK3CA and PIK3R1 mutations are rare or absent
We assessed PTEN protein expression by IHC (Figure 2). In keeping
with a previous report,
15
we found loss of PTEN expression in 17% of
diagnostic (6/35) and 16% of all (22/138) biopsies (Figure 2C). A
higher proportion of tumors exhibiting blastoid morphology had loss
of PTEN expression compared to cases with classical histology, but
this was not statistically significant (20% vs 15%, P 5 .55 by Fisher’s
exact test). No significant change was f ound in the pattern of
isoform expression, particularly for p110b, in biopsies that had loss
of PTEN expression (Figure 2D). Loss of PTEN expression was
seen by western blotting in 2 of 12 primary MCL samples
(Figure 2E), and the results seen on western blotting corresponded
to those seen by IHC. Unlike reports in solid tumors,
16
we did not
find significant additional cytotoxicity in these 2 samples using the
combination of GS-1101 and TGX221 (p110b selective inhibitor)
compared to GS-1101 alone (Figure 2F). Exons 9 and 20 of PIK3CA
and exons 9, 11, 12, 13, 15, and 16 of PIK3R1 were sequenced in
a total of 20 primary MCL samples and 2 cell lines (Granta519, Jeko-
1); no mutations were found, suggesting these are rare or do not occur
in MCL.
P110d inhibition prevents BCR-induced PI3K activation in MCL,
whereas additional p110a inhibition is required to abolish
constitutive PI3K activation
The Jeko-1 MCL cell line exhibits low or undetectable levels of p-Akt
and was therefore used to study the effect of isoform selective
inhibition on agonist-induced BCR signaling. Whereas the p110d
inhibitor GS-1101 prevented phosphorylation of Akt (thr308) in
response to BCR stimulation with IgM F(ab9)
2
fragments, the
p110a inhibitor A66
24
and the p110b inhibitor TGX221
25
had
minimal effects on p-Akt (thr308) (Figure 3A). We then studied the
effect of isoform-selective inhibition on the Granta519 MCL cell
line, which exhibits constitutive Akt phosphorylation in association
with increased p110a expression as a result of increased PIK3CA
copy number and gene expression
15
(Figure 3B). Interes tingly, GS-1101
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(1 mM) was unable to abolish Akt phosphorylation in Granta519,
whereas at equimolar concentrations GDC-0941, a predominantly
p110a/d inhibitor, blocked Akt phosphorylation in a sustained
manner over 24 hours. This was reflected in reduced downstream
phosphorylation of ribosomal S6 as a result of mTOR inhibition
(Figure 3C). To establish that this effect on constitutive PI3K
activation was related to activity of the p110a isoform, we
combined GS-1101 separately with the p110a inhibitor A66 and
a p110b inhibitor TGX221 and tested these combinations along
with GS-1101 and GDC-0941 alone in serum-starved Granta519
cells. Combining GS-1101 with A66 had a similar effect as GDC-
0941, whereas combining GS-1101 with TGX221 made no significant
difference, confirming that residual constitutive PI3K signaling in
GS-1101–treated cells is attributable to p110a (Figure 3D).
PI3K inhibition with GDC-0941 results in greater cytotoxicity to
MCL cells compared with GS-1101
We compared the cytotoxicity of GS-1101 and GDC-0941 on
growth and survival of MCL cell lines and primary samples. GDC-
0941 induced significantly higher growth inhibition at 72 hours in
2 MCL cell lines with intact PTEN expression (Granta519 and
Jeko-1) compared with GS-1101, whereas the p110a-selective
inhibitor A66 had a minimal effect on both cell lines (Figure 3F-
G). Using an ATP cytotoxicity assay, a similar significant effect
was observed on treating 12 primary MCL samples (Figure 3H)
and GDC-0941 was significantly more cytotoxic than GS-1101
above concentrations of 0.1 mM(P 5 .046 at 0.1 mM, .008 at 1
mM, .005 at 5 mM, and .035 at 10 mM). Basal phospho-Akt levels
were not predictive of sensitivity to either inhibitor in both cell
lines and primary samples. We also treated B cells isolated from 3
healthy donors with GS-1101 and GDC-0941 and found no
statistically significant difference in cytotoxicity even at higher
concentrations.
A high PIK3CA/PIK3CD mRNA ratio can predict resistance to
p110d-selective inhibition
Our findings in the Granta519 MCL cell line led us to hypothesize
that gene expression of the PI3K isoforms in MCL cells can predict
sensitivity to isoform selective inhibition. We measured PIK3CA,
PIK3CB, PIK3CD, and GAPDH mRNA levels by qRT-PCR in the
same 12 MCL cell suspensions that we previously treated with GS-
1101 and GDC-0941 and compared them with healthy B-cell
controls. PIK3CD was highly expressed with gene expression of
both PIK3CA and PIK3CD significantly up-regulated compared with
healthy B-cell controls. PIK3CB showed the weakest expression
(Figure 4A). Neither PIK3CA nor PIK3CD levels predicted
sensitivity or resistance to the inhibitors independently. However,
by plotting the ratio of PIK3CA to PIK3CD (PIK3CA/PIK3CD),
Figure 1. IHC expression of class IA PI3K isoforms in mantle cell lymphoma. (A) Dot plot showing expression of class IA isoforms in MCL biopsies (144 biopsies
evaluable for all 3 isoforms from 109 patients) compared with tonsil controls (n 5 14). Each dot represents the average IHC score of triplicate cores and bars represent median
expression. P110d is highly expressed in MCL and p110a shows a wide range of expression, whereas p110b expression is the weakest. (B) Dot plots showing significant
increase in p110a expression, but not p110b or p110d, beyond first relapse. (C) This finding is more striking in 12 sequential cases, 6 of whom had biopsies beyond first
relapse (connected by gray lines). (D) Representative IHC images (original magnification 3200) of sequential biopsies from 2 patients showing a significant increase in p110a
expression with relapse *P , .05, **P , .01. ns, not significant.
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Figure 2. Loss of PTEN expression in MCL. (A) Core from a PTEN-null adenocarcinoma control showing PTEN-negative tumor islands surrounded by PTEN-positive
stroma by IHC (original magnifications 350 and 3200). (B) Representative images of MCL cores with and without loss of PTEN expression (original magnifications 350 and
3200). Macrophages and blood vessels were used as internal controls. (C) Pie chart showing the proportion of all biopsies with PTEN loss and blastoid morphology
accompanied by a bar graph of distribution of PTEN loss in blastoid and nonblastoid MCL. (D) Dot plot comparing p110b expression levels between cores with and without
loss of PTEN expression showing no difference among the 3 groups (PTEN2, PTEN 11, and PTEN21). There was also no significant difference in expression of p110a and d
between these groups (data not shown). (E) Western blot for PTEN expression in 12 MCL cell suspensions showing loss of expression in 2 samples (indicated by arrows). (F)
Results of ATP cytotoxicity assay after 72 hours’ treatment showing no benefit from addition of a p110b-selective inhibitor to GS-1101 in 2 MCL suspensions exhibiting loss of
PTEN expression.
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Figure 3. Role of class IA isoforms in BCR-induced and constitutive PI3K signaling. (A) Jeko-1 cells were pretreated for an hour with isoform-selective inhibitors (1 mM)
as indicated, followed by IgM stimulation with 10 mg/mL anti-human IgM F(ab’) fragments for 10 minutes. Phospho-Akt (thr308) levels were compared by western blotting with
nonstimulated and IgM-stimulated Jeko-1 controls. P-Syk (tyr525/526) was used as a marker of BCR activation. GS-1101 (p110d-selective) blocked p-Akt production,
whereas A66 (p110a-selective) and TGX-221 (p110b-selective) did not. (B) Real-time PCR and western blotting confirming increased expression of PIK3CA and p110a in
Granta519 MCL cell lines compared with the lymphoblastoid cell line NcNc. (C) Western blot comparing downstream effects of GS-1101 (1 mM) and GDC-0941 (1 mM), at 2
time points demonstrating incomplete and nonsustained effect of GS-1101 on p-Akt, p-GSK3b, and p-S6. (D) Western blots were performed with serum-starved (4 hours)
Granta519 cells treated with GS-1101, GDC-0941, and combinations of GS-1101 with A66 or TGX-221 (all 1 mM), for 2 hours, confirming the effect of p110a on constitutive
PI3K activation. (E) Comparison of 50% inhibition/inhibitory concentration of GS-1101 and GDC-0941 for the 4 class I isoforms. GS-1101 is highly p110d-selective, whereas
p110a is predominantly p110a/d-selective. The activity of both inhibitors against p110g is comparable. (F) Western blot showing expression of p-Akt (t308) and the class IA
catalytic unit isoforms in Jeko-1 and Granta519. (G) Greater growth inhibition is seen in Granta519 and Jeko-1 with GDC-0941 compared with GS-1101, whereas A66 has
a minimal effect. (H) Dot plots showing greater cytotoxicity (ATP assay) with GDC-0941 compared with GS-1101 in 12 primary MCL samples with significant toxicity at and
above 0.1 mM. (I) Western blot showing p-Akt (thr308) expression in the same 12 MCL cell suspensions. (J) Comparative cytotoxicity (ATP assay) of GDC-0941 and GS-1101
in 3 healthy B cells showing somewhat greater, but not statistically significant, cytotoxicity at all concentrations with GDC-0941. *P , .05, **P , .01.
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we were able to separate the samples into 2 groups—1 with ratios
similar to healthy B cells that had similar responses to GS-1101
andGDC-0941and1withsignificantly higher expression. PIK3CA/
PIK3CD greater than twice that seen in healthy B-cell controls
identified a group that was resistant to GS-1101 and showed a
significantly greater response to GDC-0941 (P 5 .03 at 0.1 mM,
P 5 .008 at 1 mM) (Figure 4B). The 2 samples that were most
sensitive to GS-1101 reassuringly fell in the group with the lower
ratios. These differences were seen at 0.1 and 1 mM, concentrations
at which the drugs are likely to be most selective for their targets.
We validated our findings in a second independent cohort of 10
primary MCL samples using 1 mM GS-1101 and GDC-0941
(Figure 4C). We also treated these 10 samples with the p110a
inhibitor A66 (1 mM) alone but found no significant cytotoxicity in
any of the samples treated (supplemental Figure 2). As may be
expected, we found some primary samples, mainly in the low-
ratio group, that were relatively resistant to both GS-1101 and
GDC-0941 at these lower concentrations, even when phosphor-
ylation of Akt was blocked, suggesting alternative survival pathways
were active in these tumors (data not shown).
A high PIK3CA/PIK3CD mRNA ratio is frequently associated
with MCL progression
To investigate whether disease progression was associated with
achangeinPIK3CA/PIK3CD, qRT-PCR was performed on RNA
extracted from frozen serial biopsies from 4 MCL patients and
2 tonsil controls. Protein was extracted simultaneously for western
blotting from these biopsies. Whereas PIK3CD was highly expressed
and up-regulated compared with tonsil controls (Figure 5A),
PIK3CA/PIK3CD was higher in all 4 cases at relapse compared with
diagnosis (Figure 5C). Compared with PBMCs, PIK3CA expression,
and as a result PIK3CA/PIK3CD, was relatively lower in lymph
nodes. However, protein expression by western blotting showed
upregulation of p110a compared with tonsil controls and also showed
a marked increase with relapse in 2 of 4 serial biopsies (Figure 5B).
To investigate whether differences existed in isoform expression
between MCL and other NHL, we extracted PI3K class IA isoform
expression data from publicly available gene expression profiling
(www.ebi.ac.uk) from a study comparing diagnostic samples of MCL
to indolent NHL.
27
Even in these diagnostic samples, median
expression of only the PIK3CA isoform, and therefore PIK3CA/
PIK3CD, was significantly higher in MCL compared with chronic
lymphocytic leukemia and indolent NHL (Figure 6A). There was no
publicly available data comparing gene expression in relapsed
lymphomas that we could analyze at the time of this study, but we
performed immunohistochemistry for p110a in sequential biopsies
from 10 follicular lymphoma patients, 4 of whom had paired
biopsies beyond first relapse, and did not find evidence of increase
in expression with relapse (Figure 6B-C).
Discussion
Several studies have described the importance of the PI3K pathway
in the pathogenesis of MCL.
3-5
The relatively inferior effects seen
in MCL in early-phase trials of the PI3K p110d selective inhibitor
GS-1101 emphasize the need to study the contribution of the other
class IA PI3K isoforms to MCL survival.
In keeping with previous observations,
3,15
we did not find PIK3CA
mutations in 20 MCL primaries, and PTEN loss occurred at a
frequency of about 16%, with an apparently, but not significantly,
higher incidence in blastoid MCL. In addition, activating PIK3R1
mutations were shown to be rare or absent in MCL. Although gene
amplification of PIK3CA related to increased copy number has been
previously described in MCL, we demonstrate for the first time
that a high PIK3CA/PIK3CD mRNA ratio can predict resistance
to p110d-selective inhibition and is frequent with relapse. The
mechanism for this increase of expression with relapse is unclear.
Our observation that the increase in p110a expression is particularly
significant beyond first relapse may imply a chemotherapy-induced
phenomenon or selection and expansion of a chemotherapy-resistant
clone with high PIK3CA expression, but this needs further study.
We demonstrate that although p110d continues to be the pre-
dominant isoform in terms of expression and agonist-induced BCR
signaling in MCL, an increase in p110a expression can maintain
constitutive PI3K signaling in MCL cells despite p110d inhibition.
We explored the option of an small interfering RNA study to
demonstrate the reliance of Granta519 on p110a but it has been
found that knocking down the expression of any 1 of the class I
PI3K isoforms can result in a compensatory increase or decrease in
activity of other isoforms and lead to artifacts.
28-31
We therefore
elected to use a highly selective p110a inhibitor, A66, for our
studies.
24
Studies in healthy mouse lymphocytes have demonstrated
that although PI3K p110d plays a predominant role in both agonist-
dependent and agonist-independent BCR signaling, p110a contrib-
utes to agonist-independent signaling.
32
The increased expression
of p110a in MCL, particularly with relapse, may therefore allow
tumor cells to survive independent of agonist-induced activation
of the PI3K pathway. Of note, a similar phenomenon has been
observed in immortalized macrophages, where p110a takes on
a more prominent role in PI3K signaling, in contrast to primary
macrophages where p110d is the predominantly active isoform.
33
It
is possible that PI3K inhibitors may also have an effect on the tumor
microenvironment; this issue is not explored here. However, the
clear effect in MCL cell lines and primary MCL cells in the absence
of the microenvironment, together with the relative increase of
PIK3CA in MCL cells themselves, strongly suggest that this is
likely to result in a differential effect with greater toxicity to the
tumor cells compared with an effect on the microenvironment.
P110b shows the weakest expression at both the RNA and protein
level in MCL and we found no additional effect on PI3K-induced Akt
phosphorylation on combining a highly selective p110b inhibitor
(TGX-221) with GS-1101. We also demonstrate that, unlike obser-
vations in solid tumors,
16
there is a lack of additional cytotoxicity
from combining TGX-221 and GS-1101 in MCL samples that exhibit
loss of PTEN expression, but this requires confirmation on a larger
number of samples. GDC-0941, an inhibitor with high selectivity for
p110a and p110d, w as significantly more cytotoxic in both MCL
cell lines and primary samples. Although inhibiting additional
PI3K isoforms is likely to be associated with greater toxicity, this
inhibitor has shown favorable toxicity profiles in early-phase clinical
trials in solid tumors.
34
Our findings are unlikely to be influenced
by p110g, the only class IB catalytic unit isoform, because both
GS-1101 and GDC-0941 have similar efficacy against p110g
(Figure 3E). The greater contribution of p110a to PI3K signaling with
MCL relapse can e xplain the relativel y modest effects seen in relapsed/
refractory MCL patients recruited into early-phase trials of GS-1101.
We propose that PIK3CA/PIK3CD is a potential biomarker that
can identify patients who are resistant to p110d inhibition and may
benefit most from combined p110a/d inhibitor therapy. This ratio
may identify tumors that can use p110a-mediated constitutive PI3K
signaling for microenvironment-independent survival. Our data also
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Figure 4. The PIK3CA/PIK3CD mRNA ratio can predict resistance to GS-1101. (A) Gene expression of class IA PI3K isoforms, relative to GAPDH, in 12 diagnostic MCL
PBMCs and 3 healthy B-cell controls, showing significant increase in PIK3CA and PIK3CD compared with controls (gray) and very low levels of PIK3CB.(B)APIK3CA/
PIK3CD greater than twice the mean ratio in healthy B cells identifies a subset of primary samples (5/12) that are resistant to 0.1 mM and 1 mM GS-1101 (GS), but show
significantly higher toxicity with equimolar concentrations of GDC-0941 (GDC). (C) This cutoff, when applied to an independent validation cohort (n 5 10), was able to identify
GS-1101–resistant samples (primary samples treated with 1 mM of GS-1101 and GDC-0941).
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Figure 5. PIK3CA/PIK3CD increases with relapse in MCL. (A) Gene expression of the PI3K class IA isoforms compared with 2 tonsil controls (gray). Serial biopsies from
the same patient are in the same color (B) western blot showing very weak expression of p110b and a clear increase in MCL p110a compared with tonsil while p110d protein
expression in MCL is similar to tonsil controls. Increase in p110a with relapse is apparent in 2 out of 4 serial biopsies (C) Dot plot showing a significantly higher PIK3CA/
PIK3CD ratio in all relapsed samples compared with matched diagnostic samples and tonsil controls.
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suggest that Akt phosphorylation, when present, is a useful marker to
study responses to PI3K inhibition but absence of phosphorylation
does not preclude sensitivity to PI3K inhibition. Also, we did not find
higher Akt phosphorylation in MCL exhibiting increased PIK3CA/
PIK3CD. This is not entirely surprising because, for instance, Akt-
independent PI3K signaling has been found in cancers harboring
PIK3CA mutations
35,36
and a similar mechanism could operate in
MCLs that have a high PIK3CA/PIK3CD ratio.
In summary, our findings suggest that MCL requires blockade of
both p110a and p110d, particularly at relapse, for effective PI3K
inhibition. This will require testing in a clinical trial, but the as-
sessment of the relative gene expression of these isoforms may help
identify those patients that are most likely to respond to these exciting
agents.
Acknowledgments
The authors thank Bart Vanhaesebroeck, Ezra Aksoy, the European
Hematology Association (EHA)-ASH Translational Res earch Training
in Hematology faculty, particularly Donna Neuberg, David Williams,
and Linda Burns, for their valuable advice and suggestions. The
authors also thank Jacek Marzec for bioinformatics, John Riches and
Eleni Kiotsiu for providing healthy B cells, and all the patients at
Barts Can cer Centre who kindly consented to provide samples for
this research.
This work was supported by grants from the David Pitblado Foun-
dation, National Cancer Institute (P01 CA81538) (J.G.), Cancer
Research UK (C1574/A6806) , and an EHA Partner Fellowship
awarded by the European Hematology Association (2009/01) (C.B.).
Authorship
Contribution: S.I., J.G., and S.J. designed experiments; S.I., A.C.,
and C.B. performed the experiments; R.A., L.M., S.I., and J.M.
provided essential reagents, clinical samples, and related clinical
information; M.C. and A.L. assisted with tissue microarray
constructi on and imm unohis tochemistry scoring. S.I. prepared
the manuscript with input from all authors.
Conflict-of- interest disclosure: J.G. and R.A. have received
honoraria from Gilead for attendance at advisory boards. J.G. has
received ho norar ia fr om R oche for attendance at a dvisory boards. The
remaining authors declare no competing financial interests.
Correspondence: John Gribben, Centre for Haemato-Oncology,
Barts Cancer Institute, London EC1M 6BQ, UK; e-mail: j.gribben@
qmul.ac.uk.
Figure 6. P110a expression does not increase with relapse in follicular lymphoma and gene expression of PIK3CA is significantly higher in MCL compared with
indolent NHL and CLL. (A) Gene expression of class IA PI3K isoforms at diagnosis in MCL was compared with indolent NHL using publicly available Affymetrix data
(European Bioinformatics Institute, www.ebi.ac.uk ref: E-GEOD-16455) with the web-based software O-miner. cMCL, conventional MCL, CLL, chronic lymphocytic leukemia,
FL, follicular lymphoma; HCL, hairy cell leukemia, SMZL, splenic marginal zone lymphoma. PIK3CA expression is significantly higher in MCL compared with indolent NHLs
(Benjamini-Hochberg multiple testing adjusted P values: cMCL vs HCL 5 .005, cMCL vs SMZL 5 .0001 cMCL vs CLL 5 .003, cMCl vs FL 5 .05) in this study, whereas
PIK3CB and PIK3CD expression is not. (*P , .05, **P , .01, ***P , .001). (B) IHC images of 4 follicular lymphoma diagnosis: second-relapse pairs showing no increase in
p110a expression with relapse (original magnification 3200). (C) Dot plot comparing expression of p110a in sequential biopsies from follicular lymphoma patients (n 5 10). No
evidence of increased p110a expression is seen with relapse. Pairs are connected by straight lines.
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2284 IYENGAR et al BLOOD, 21 MARCH 2013
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online January 22, 2013
originally publisheddoi:10.1182/blood-2012-10-460832
2013 121: 2274-2284
Matthews, Sameena Iqbal, Rebecca Auer, John Gribben and Simon Joel
Sunil Iyengar, Andrew Clear, Csaba Bödör, Lenushka Maharaj, Abigail Lee, Maria Calaminici, Janet
relapse
-selective inhibition in mantle cell lymphoma, particularly with multiple
δ-mediated constitutive PI3K signaling limits the efficacy of p110αP110
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