T cells bet on sugars to help

● ● ● IMMUNOBIOLOGY

insideblood

1 DECEMBER 2005 I VOLUME 106, NUMBER 12









Comment on Underhill et al, page 3867



T cells bet on sugars to help

-------------------------------------------------------------------------------------

Linda G. Baum UCLA SCHOOL OF MEDICINE



Leukocytes emigrate into inflamed tissues by binding to selec-

tins on endothelial cells; while different leukocyte subsets traf-

fic to different tissue sites, the mechanisms regulating selective

expression of selectin ligands on specific cell subsets are not

known. Underhill and colleagues demonstrate that the signals

that drive Th1 cell differentiation also drive expression of the

glycosyltransferase enzymes that create selectin ligands on

these cells.

wo important scientific advances occurred Selectins mediate

T about 20 years ago. First, T-helper 1

(Th1) and Th2 subsets of CD4 T-helper cells

leukocyte rolling, al-

lowing the leukocyte to

were described.1 Second, lectin-carbohy- attach to endothelial

drate–mediated cell adhesion was shown to be cells and infiltrate into

the initial step in extravasation of leukocytes at the underlying tissue.

sites of inflammation.2 In subsequent years, Selectins can be ex-

several groups have noted homing of Th1 and pressed on leukocytes,

Th2 cells to different tissue sites, mediated by such as L-selectin on Regulated expression of specific glycosyltransferases during Th1 differentia-

specific selectin-ligand interactions between T lymphocytes, or on tion results in coordinated synthesis of selectin ligands on O-linked oligosac-

cells and endothelial cells. In this issue of endothelial cells, such charides. Modified from Underhill et al, in the article beginning on page 3867;

Blood, Kansas’s group has identified compo- as E-selectin and P- modified illustration by Paulette Dennis.

nents of the cellular signaling program that selectin; conversely,

instructs Th1 cells to specifically display the oligosaccharide ligands recognized by selectins coordinated expression of the glycosyltrans-

cell surface saccharide ligands recognized by are displayed by endothelial cells for L-selec- ferases in vivo to create the tetrasaccharide

endothelial cell selectins. tin or leukocytes for E- and P-selectins. The ligands remained unknown.

Th1 and Th2 cells are derived from naive basic selectin ligand is a tetrasaccharide, Because the roles of T-bet and Stat4 in

CD4 T cells, and differentiation of a naive Sia 2,3Gal 1,4(Fuc 1,3)GalNAc (ie, sialyl Th1 polarization are established, and because

CD4 T cell into a Th1 or Th2 cell is regulated Lex),3 that can be presented on different leuko- candidate glycosyltransferases that can create

by specific transcription factors, such as signal cyte and endothelial cell glycoprotein back- selectin ligands have been identified, Under-

transducer and activator of transcription 4 bones (see bottom of figure). Identification of hill and colleagues examined the roles of T-bet

(Stat4) and T-bet in Th1 cells and GATA-3 in the tetrasaccharide ligand suggested candidate and Stat4 in upregulating expression of these

Th2 cells. In addition to differences between glycosyltransferase enzymes that could make candidate glycosyltransferases during Th1

Th1 and Th2 subsets in cytokine production the structure. Analysis of mice deficient in polarization. In addition, to confirm that the

and responses to antigens, Th1 cells preferen- specific glycosyltransferases confirmed that glycosyltransferases are responsible for creat-

tially home to inflammatory sites in peripheral these enzymes could create the tetrasaccharide ing selectin ligands, Th1 cells generated from

tissues. Preferential trafficking of Th1 cells to ligands required for selectin-mediated leuko- mice deficient in specific glycosyltransferases

inflammatory sites results from expression on cyte trafficking and identified subtle differ- were analyzed. As structural analysis of oligo-

Th1 cells, but not Th2 cells, of high levels of ences in modifications of the tetrasaccharide to saccharide structures is technically challeng-

saccharide ligands recognized by selectins on promote recognition by different selectins.4,5 ing and requires amounts of material difficult

endothelial cells. However, the cellular mechanisms that control to obtain from primary T cells, creation of





blood 1 D E C E M B E R 2 0 0 5 I V O L U M E 1 0 6 , N U M B E R 1 2 3677

oligosaccharide ligands on Th1 cells was surface of mammalian cells and regulate cell strated the benefits of R-CHOP in indolent

shown by functional assays (ie, T-cell rolling adhesion, migration, proliferation, and sur- lymphomas, but the broad applicability of

on monolayers of cells expressing E- or P- vival, future studies will no doubt demonstrate their findings was uncertain due to limited

selectin). This functional approach allowed that signals regulating cellular glycosylation sample size and absence of a control group. A

identification of ST3Gal-VI ( 2,3-sialyl- are critical for the differentiation and function recent 9-year follow-up of their study and the

transferase VI) as a novel glycosyltransferase of all hematopoietic cells. ■ results of the present report, however, provide

involved in synthesis of selectin ligands on new evidence for the merits of R-CHOP and

Th1 cells. More important, these investiga- reshape treatment recommendations.2

tors’ past and current work are now synthe- REFERENCES

In this issue of Blood, Hiddemann and col-

1. Mossman TR, Cherwinski H, Bond MW, Giedlin MA,

sized in a model integrating signaling from leagues report the results of a randomized

Coffman RL. Two types of murine helper T cell clone.

T-cell and interleukin 12 (IL-12) receptors J Immunol. 1986;136:2348-2357. study of CHOP versus R-CHOP in 428 pa-

with transcriptional regulation resulting in 2. Stoolman LD, Rosen SD. Possible role for cell-surface tients with advanced-stage follicular lym-

coordinated expression of 4 different glycosyl- carbohydrate binding molecules in lymphocyte recircula-

tion. J Cell Biol. 1983;96:722-729.

phoma of grade I or II histology and requiring

transferases that create functional selectin treatment. Responding patients younger than

3. Phillips ML, Nudelman E, Gaeta FC, et al. ELAM-1

ligands on cell surface glycoproteins (see mediates cell adhesion by recognition of a carbohydrate 60 years of age were offered a second random-

figure). ligand, Sialyl-Lex. Science. 1990;250:1130-1132.

ization following CHOP or R-CHOP of either

In the emerging field of glycobiology, 4. Maly P, Thall AD, Petryniak B, et al. The (1,3) fuco-

syltransferase FucT-VII controls leukocyte trafficking

intensification with stem cell support or long-

mechanistic connection of cell signaling to the through an essential role in L-, E, and P-selectin ligand bio- term interferon- maintenance, whereas older

synthesis of specific oligosaccharide ligands synthesis. Cell. 1996;86:643-653. patients received interferon- maintenance.

during differentiation is an exciting and im- 5. Ellies LG, Tsuboi S, Petryniak B, Lowe JB, Fukuda M,

At the median follow-up time of 18 months

Marth JD. Core 2 oligosaccharide biosynthesis distin-

portant advance. As oligosaccharides on glyco- guishes between selectin ligands essential for leukocyte (range, 1-38 months), the risk of treatment

proteins and glycolipids cover much of the homing and inflammation. Immunity. 1998;9:881-890. failure was reduced by 60% in the R-CHOP

arm with benefit observed in patients younger

● ● ● CLINICAL OBSERVATIONS or older than 60 years and with low or high

international prognostic index scores. The

Comment on Hiddemann et al, page 3725 time to next therapy also demonstrated an

advantage for R-CHOP. Of great interest was

R-CHOP strikes again with survival the significant improvement in overall survival

with R-CHOP, albeit only modest at the rela-

benefit in follicular lymphoma

----------------------------------------------------------------------------------------------------------------

tively short follow-up time. A justifiable caveat

to a broad embrace of these findings is found in

the secondary treatment requirement. It is

Wyndham H. Wilson NATIONAL CANCER INSTITUTE

reassuring that the secondary randomizations

Hiddemann and colleagues demonstrate improved response duration and overall were stratified for risk and that the R-CHOP

survival with the addition of rituximab to CHOP, compared with CHOP alone, in benefit was irrespective of interferon- main-

symptomatic patients with untreated advanced-stage follicular lymphoma. tenance. It is notable though that the R-CHOP

benefit was not observed among patients ran-

he arrival of rituximab (R) onto the thera- rational basis for its combination with CHOP

T peutic landscape heralded a new era in the

treatment of B-cell lymphoma and provided a

(cyclophosphamide, doxorubicin, vincristine,

and prednisone). Czuczman et al1 first demon-

domized to stem cell consolidation, suggesting

that enhanced treatment efficacy and not the

unique biologic attributes of rituximab may

underlie the benefits of R-CHOP. It should

also not escape notice that differential access to

rituximab containing salvage treatment for

patients on the 2 arms could account for the

survival advantage of R-CHOP. Nevertheless,

these results as well as those of Czuczman et

al,2 where the observation time is quite ma-

ture, raise the specter of survival advantage, if

not cure, with R-CHOP in follicular lym-

phoma. While many opinions might favor the

notion that rituximab combined with CHOP

delays relapse in follicular lymphoma, the

prospect of cure should be considered with

appropriate circumspection.

These results are of sufficient measure

Molecular prognostic model of follicular lymphoma based on signatures of infiltrating “immune” cells. to reshape the debate from “if ” to “when”



3678 1 DECEMBER 2005 I VOLUME 106, NUMBER 12 blood

patients should receive R-CHOP for the initial with rituximab responsiveness suggested the tion, and subcellular compartmentalization

treatment of advanced-stage disease, not to relevance of immune cells albeit as negative have been extensively studied in cultured en-

mention its role in early-stage disease. Ques- predictors.5 The prospective application of dothelial cells using simple flow protocols.2

tions of who will benefit from treatment with these technologies to future trials is vital for Other in vitro flow studies have attempted to

R-CHOP, versus less “hair-razing” regimens understanding outcome biology and to guide simulate the complex flow characteristics asso-

like R-CVP (rituximab– cyclophosphamide, the rational clinical development and use of ciated with sites of lesion susceptibility in the

vincristine, and prednisone), will occupy the new treatments. ■ arterial circulation.3 Such regions correlate

minds of oncologists and patients.3 Gene ex- with decreased eNOS transcript expression

pression profiling, while providing insight into REFERENCES even in lesion-free animals4 but the relation-

tumor biology, is also a powerful prognostic 1. Czuczman MS, Grillo-Lopez AJ, White CA, et al. ship between cause (complex flow) and effect

Treatment of patients with low-grade B-cell lymphoma

tool that may help identify beneficiaries of with the combination of chimeric anti-CD20 monoclonal (eNOS expression and localization) had not

such treatment. This technology has revealed antibody and CHOP chemotherapy. J Clin Oncol. 1999;17: been established in vivo.

268-276.

a central biologic role for infiltrating “reac- To address these issues, the Rotterdam

2. Czuczman MS, Weaver R, Alkuzweny B, Berlfein J,

tive” cells, termed the immune reaction (IR), Grillo-Lopez AJ. Prolonged clinical and molecular remis-

group generated transgenic mice that express

within the malignant lymph node on the sur- sion in patients with low-grade or follicular non-Hodgkin’s human eNOS in fusion with green fluorescent

lymphoma treated with rituximab plus CHOP chemo- protein (GFP) as a reporter of eNOS protein

vival of follicular lymphoma.4 The relative therapy: 9-year follow-up. J Clin Oncol. 2004;22:4711-

expression of 2 signatures, termed IR-1 and 4716. expression.5 To demonstrate hemodynamic

IR-2, that differentially express genes related 3. Marcus R, Imrie K, Belch A, et al. CVP chemotherapy cause-effect, gradations of and separations of

plus rituximab compared with CVP as first-line treatment flow were created by placement of a tapered

to T-cell markers, macrophages, and/or den- for advanced follicular lymphoma. Blood. 2005;105:1417-

dritic cells, can be used to predict survival (see 1423. cast around the midportion of the left common

figure). It is not unreasonable, given the early 4. Dave SS, Wright G, Tan B, et al. Prediction of survival carotid artery, resulting in a corresponding

in follicular lymphoma based on molecular features of tu- gradual narrowing of the artery lumen over a

benefits of rituximab on survival, to hypoth- mor-infiltrating immune cells. N Engl J Med. 2004;351:

esize that rituximab may impact tumors that 2159-2169. length of several millimeters. This is normally

predominantly express IR-2, as this signature 5. Bohen SP, Troyanskaya OG, Alter O, et al. Variation in a region of pulsatile laminar flow without flow

is associated with short survival. Indeed, a

gene expression patterns in follicular lymphoma and the separation and where forces are unidirectional.

response to rituximab. Proc Natl Acad Sci U S A. 2003;100:

recent study on gene expression associated 1926-1930.

It is also a site resistant to atherosclerotic lesion

development. Since is proportional to 1/(di-

ameter)3, shear stress increases rapidly through-

● ● ● HEMOSTASIS out the length of the cast. Downstream of the

cast, the lumen widens to create a short region

Comment on Cheng et al, page 3691 of oscillating separated flow similar to that

recorded at atherosclerosis-susceptible loca-

Hemodynamic manipulation of eNOS tions elsewhere. Within 24 hours following

placement, the hemodynamics were spatially

in vivo

----------------------------------------------------------------------------------------------------------------

mapped to face cell responses. Cheng and col-

leagues report that eNOS gene and protein

Peter F. Davies UNIVERSITY OF PENNSYLVANIA

expression was elevated as increased within

the tapered cast consistent with shear stress

In this issue of Blood, Cheng and colleagues at Erasmus University in Rotterdam experiments in vitro and that the intracellular

demonstrate that arterial eNOS expression in eNOS-GFP transgenic mice is re- redistribution of eNOS, including its activated

sponsive both to hemodynamic flow characteristics and to the magnitude of shear form (serine 1177 phosphorylation), was sig-

stress forces ( ). These experiments provide important in vivo validation of numer- nificantly increased both by elevated and

ous in vitro studies of the flow-eNOS relationships and reveal the spatial sensitivity oscillatory flow, although the fraction of total

of hemodynamic characteristics in the regulation of eNOS in intact arteries. eNOS that was phosphorylated remained

unaltered.

emodynamic regulation of vascular mediated generation of nitric oxide (NO) by

H physiology occurs through convective

mass transport and through forces imparted to

activation of endothelial nitric oxide synthase

(eNOS). Regulation of this enzyme is consid-

The studies are encouraging for investiga-

tions both in vivo and in vitro. They suggest

that flow-related mechanisms of eNOS regula-

the vessel wall. Flow is also a pathologic deter- ered to be of major physiologic and pathologic tion identified in reductionist experiments in

minant of the localization of atherosclerotic importance. Its activities play a dominant role tissue culture also occur in vivo, albeit with

lesions that originate at sites of geometric (and in vasodilatation by relaxing the smooth subtleties imparted by a more complex environ-

hemodynamic) complexity. A particular target muscle cells of arteries to reduce blood pres- ment. Furthermore, studies of flow disturbance

of flow forces is the arterial endothelium, an sure. NO also inhibits platelet aggregation, in relation to localized endothelial phenotype in

interface that is sensitive to the local (fric- leukocyte adhesion to the endothelium, and vivo can now be cautiously interpreted as cause-

tional) shear stress.1 An important physiologic cell migration. The effects of on eNOS tran- and-effect mechanisms rather than simply cor-

example of -endothelial interactions is flow- scription, translation, posttranslational activa- relative. ■



blood 1 D E C E M B E R 2 0 0 5 I V O L U M E 1 0 6 , N U M B E R 1 2 3679

REFERENCES oxide synthase are selectively up-regulated by steady with decreased porosity, the distinctly fine

laminar shear stress. Proc Natl Acad Sci U S A.

1. Davies PF. Flow-mediated endothelial mechanotrans- 251 networks are consistent with long-held

1996;93:10417-10422.

duction. Physiol Rev. 1995;75:519-560. views on the network of fibrin lack C, while

2. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, 4. Passerini AG, Polacek DC, Shi C, et al. Coexisting pro-

inflammatory and anti-oxidative endothelial transcription emphasizing the C role in lateral polymeriza-

Busse R, Zeiher AM. Activation of nitric oxide synthase in

endothelial cells by Akt-dependent phosphorylation. Na- profiles in a disturbed flow region of the adult porcine aorta. tion. The latter process is poorly understood,

ture. 1999;399:601-605. Proc Natl Acad Sci U S A. 2004;101:2482-2487.

in contrast to protofibril formation and

3. Topper JN, Cai J, Falb D, Gimbrone MA Jr. Identifi- 5. van Haperen R, Cheng C, Mees BM, et al. Functional

branching. The C pair, self-associated at

cation of vascular endothelial genes differentially respon- expression of endothelial nitric oxide synthase fused to

sive to fluid mechanical stimuli: cyclooxygenase-2, man- green fluorescent protein in transgenic mice. Am J Pathol. their ends and tethered to the central region,

ganese superoxide dismutase, and endothelial cell nitric 2003;163:1677-1686. become untethered following fibrinopeptide B

release. Whether they participate in lateral

polymerization in tethered, untethered, or

● ● ● HEMOSTASIS both forms remains unclear. Reptilase clots

display ample lateral polymerization, suggest-

Comment on Collet et al, page 3824

ing that either tethered C participates by an



Another fibrin C scene unmasked

----------------------------------------------------------------------------------------------------------------

unknown mechanism or other parts of the

molecule are involved. Be that as it may, par-

ticipation in lateral polymerization, while sig-

Dennis K. Galanakis STATE UNIVERSITY OF NEW YORK AT STONY BROOK nificant, may not be a major function of C

domains.

In this issue, Collet and colleagues identified C as the major determinant of vis- Decreased plasmin resistance by fibrin 251

coelastic clot properties and unambiguously highlighted its polymerization and clots appears inconsistent with reports of in-

fibrinolysis roles. creased plasmin resistance by normal fine net-

work clots. However, fibrin 251 results imply

ibrinogen1 consists of 3 pairs of disulfide- actions among juxtaposed molecules via their

F linked polypeptide chains, A , B , and ,

and possesses globular regions at each end and

C domains largely determine the normal

level of clot stiffness. Additionally, C do-

that interactions among C domains are more

important for plasmin resistance than other

clot network structure(s). Additionally, C

in its N terminal center. The carboxyl terminal mains play a significant role in minimizing the

regulates fibrinolysis, evident from its binding

A -chain region, commencing with residue irreversible deformation (plasticity) of clots.

of an impressive array of plasminogen activa-

220, is termed C and is required for certain Also, - bonds confer appreciable stiffness

tion and inhibitory proteins.5 These new re-

fibrin functions (see figure). Following release even before clots are cross-linked. Thus,

sults, moreover, support the view that the C

by thrombin of amino terminal fibrinopep- C- C compared with - bonds constitute

domains contribute directly to fibrinolysis

tides, fibrin monomers formed polymerize in a the major determinant of stiffness.

resistance. The impressive C repertoire

half-staggered manner, the central domain of Other likely minor determinants are -

tempts speculation that more scenes remain

one binding the outer domain of another. When and A knob–a hole bonds.4 Not surprisingly,

undiscovered. ■

600 to 800 nm long, the 2-stranded, twisting stiffness from the first 2 sets of determinants is

protofibrils assemble laterally forming fibrils. maximized by factor XIIIa– catalyzed

REFERENCES

Lateral contact pairs include B knobs– b holes; cross-links.

1. Weisel JW. Fibrinogen and fibrin. Adv Protein Chem.

C– C; and possibly2 (350-360)–(370-380). Decreased porosity in 251 clots, relative 2004;70:247-299.

Viscoelastic properties of thrombus are to that of normal clots, underscores the role of 2. Yang Z, Mochalkin I, Doolittle RF. A model of fibrin

regarded essential to its physiologic func- C. Under certain in vivo conditions (eg, vari- formation based on crystal structures of fibrinogen and fi-

brin fragments complexed with synthetic peptides. Proc

tions.3 The markedly decreased storage modu- able thrombin concentrations), a thrombus is Natl Acad Sci U S A. 2000;97:14156-14161.

lus (stiffness) by 251 clots implies that inter- likely to contain both increased and decreased 3. Weisel JW. The mechanical properties of fibrin for basic

porosity domains. The scientists and clinicians. Biophys Chem. 2004;112:267-276.

latter may be enhanced 4. Litvinov RI, Gorkun OV, Owen SF, Shuman H, Weisel

JW. Polymerization of fibrin: specificity, strength, and sta-

by the high-fibrinogen

bility of knob:hole interactions studied at the single mole-

excess at blood-throm- cule level. Blood. Prepublished on July 5, 2005, as DOI

bus interface(s). De- 10.1182/blood-2005-05-2039.



creased porosity do- 5. Mossesson MW. Fibrinogen and fibrin structure and

function. J Thromb Haemost. 2005;3:1894-1904.

mains, by limiting

intrathrombus circula-

tory flow, may shield

intrathrombus micro-

environments and by

extension enhance he-

mostatic/tissue repair

effectiveness and limit

Fibrin functions mediated in part or entirely by C. fibrinolysis. Along



3680 1 DECEMBER 2005 I VOLUME 106, NUMBER 12 blood

● ● ● CLINICAL OBSERVATIONS



¨

Comment on Dohner et al, page 3740; Schnittger et al, page 3733; and Verhaak et 116 nk-AML cases among 285 total AML

al, page 3747 cases with further embedding of nk-AML

cases among other cases with intermediate risk

Dueling mutations in normal on the basis of cytogenetic characteristics.

Thus, restricting direct comparison of essen-

karyotype AML

----------------------------------------------------------------------------------------------------------------

tial clinical trial features to reports D and S,

significant differences included median time

of follow-up (46 vs 16 months), median age

Robert E. Gallagher ALBERT EINSTEIN COLLEGE OF MEDICINE

( 48 vs 60 years), male-female ratio (0.78:1 vs

Nucleophosmin 1 (NPM1) mutations occur in about 50% of normal karyotype 1.02:1), treatment regimens, and possibly out-

AML patients, associated with increased probability of complete remission and, come. However, the essential conclusions re-

paradoxically, of FLT3 (Fms-like tyrosine kinase 3) mutations. Three European lated to NPM1 mutations were concordant in

leukemia groups now report that NPM1 mutations are also associated with in- all 3 reports. Of most importance, NPM1 mu-

creased survival but that this association is largely nullified by FLT3 internal tan- tations were found to predict for improved

dom duplication mutations. overall survival (see figure), relapse-free sur-

y cytogenetic analysis, currently the pri- al, but it was also associated with 2 adverse risk vival (reports D & S; P .001 and P .001), and

B mary method for prognostic subclassifi-

cation of acute myeloid leukemia (AML), 45%

factors, increased white blood cell (WBC)

count and internal tandem duplication (ITD)

event-free survival (reports S and V; P .001

and P .05) in either nk-AML or intermediate-

of cases have a normal karyotype.1 Although mutations of FLT3.3 Remarkably, only a few risk AML by Kaplan-Meier analysis only if

normal karyotype AMLs (nk-AMLs) are clas- months later, 3 European leukemia groups FLT3/ITD mutations were not coincidentally

sified as having intermediate prognosis, there have provided an extensive analysis of NPM1 present (ie, in the NPM1 FLT3 subgroup).

is marked heterogeneity in outcome. Recently, and FLT3 mutations, as well as other muta- There were no survival differences between

2 subgroups of nk-AML were identified by tions with prognostic significance in AML,1 in NPM1 FLT3 , NPM1 FLT3 , and NPM1

gene-expression analysis based on treatment cohorts of several hundred patients. These FLT3 subgroups. Other concordant conclu-

outcome, but no defining genetic abnormality expeditious reports are a tribute to the extraor- sions in the directly comparable D and S reports

was discovered.2 Thus, the recent finding by dinary leukemia group resources and effort were that NPM1 mutations are not related to

Falini et al3 that more than 50% of nk-AML required to consummate these integrated age, occur more often in females, are associated

cases harbor mutations in the NPM1 gene, the clinical-laboratory studies. There are signifi- with increased WBC count, are more frequently

highest incidence of any mutation in AML, cant differences among the reports. Dohner

¨ associated with the myelomonocytic phenotype

was a landmark discovery. and colleagues (report D) and Schnittger and and low CD34 expression, and occur less fre-

NPM1 mutation in nk-AML was an inde- colleagues (report S) report exclusively nk- quently with coincident partial tandem duplica-

pendent prognostic marker for achieving com- AML cases (n 300 and 401, respectively), tion of MLL. Apparent discrepancies between

plete remission (CR) in the study by Falini et while Verhaak and colleagues (report V) report reports included the findings of a decreased









Kaplan-Meier analysis of overall survival in normal karyotype or intermediate-risk AML in subgroups defined by the presence or absence of frameshift mutations in

¨

NPM1 and/or internal tandem duplication mutations of FLT3. The panels were modified from Figure 3B of Dohner et al (left), Figure 5A of Schnittger et al (middle),

and Figures 4A-B of Verhaak et al (right). The vertical hashmarks in the left and middle panels indicate censored patients. Illustration by Frank Forney.





blood 1 D E C E M B E R 2 0 0 5 I V O L U M E 1 0 6 , N U M B E R 1 2 3681

coincidence of NPM1 and CEBPA mutations related to the primary posttranslational target Lawrence and colleagues used elegant in vitro

and an independent survival advantage for level of aberrantly increased FLT3 tyrosine ki- and in vivo analyses to demonstrate normal

NPM1 mutation alone by multivariate analysis nase activity, where it may affect cellular path- numbers of immunophenotype-defined hema-

in reports S and V (in overall AMLs) but not ways regulating cell growth and survival.6 topoietic stem cells but significantly decreased

in report D, as well as a decreased incidence of Schnittger et al discuss the possibility that the hematopoietic stem cell proliferation in HoxA9

NPM1 mutations below age 35 in report V. cytoplasmic displacement of nucleophosmin 1 knockout mice. They made this interpretation

These founding NPM1 mutation reports (NPM1) protein by a multiplicity of frameshift acknowledging a limitation: HoxA9 deficiency

seem certain to have a major impact on clinical mutations that introduce a common nuclear down-modulates proliferation of hematopoi-

and molecular investigative approaches in export signal may increase susceptibility to che- etic progenitor cells, and functional stem cell

¨

nk-AML. Dohner et al found that allogeneic motherapy-induced apoptosis. This suggests assay readouts all depend on progenitor cell

stem cell transplantation did not improve the that dynamic molecular analyses at both tran- function in addition to stem cell function. Of

long-term 60% relapse-free survival rate in scriptional and posttranscriptional levels early interest, when Lawrence et al evaluated the

their NPM1 FLT3 cases but did so in the after exposure to chemotherapeutic agents with role of the nearest 5 neighbor of HoxA9, they

other subgroups, suggesting that this high-risk or without modulators, such as FLT3 inhibitors, found no hematopoietic stem cell defect in

procedure may not be indicated in first CR for might show differences between NPM1 FLT3 HoxA10-deficient mice.

this newly defined, favorable-prognosis sub- and NPM1 FLT3 nk-AML cells that could The 39 Hox family genes1 are clustered at 4

group. More speculatively, treatment with implicate important target molecules. It is hope- chromosomal loci (HoxA1-7, A9-11, and A13

recently developed Fms-like tyrosine kinase 3 ful that these example ideas are harbingers of at chromosome 7p15; HoxB1-9 and B13 at

(FLT3) inhibitors4 might be efficacious in advances that will lead to improvement in long- 17p21; HoxC4-6 and C8-13 at 12q13; and

NPM1 FLT3 patients by producing phar- static treatment outcome in AML. ■ HoxD1, D3, D4, and D8-13 at 2q31) in hu-

macologic conversion to a more chemotherapy- mans. All the mammalian Hox genes are

sensitive NPM1 FLT3 status. The interaction REFERENCES highly homologous to the HOM-C genes of

between NPM1 and FLT3 mutations could also 1. Marcucci G, Mrozek K, Bloomfield CD. Molecular het- Drosophila, discovered by Bridges and Mor-

provide important clues to key therapeutic tar- erogeneity and prognostic biomarkers in adults with acute gan,2 who in 1915 described the “bithorax”

gets in nk-AML. Verhaak et al, in agreement myeloid leukemia and normal cytogenetics. Curr Opin He-

matol. 2004;12:68-75. mutation, and later Lewis, who noted a “ho-

with a recently published gene expression meotic” mutant where an additional pair of

2. Bullinger L, Dohner K, Bair E. Use of gene-expression

study,5 found that NPM1 AML has a defined profiling to identify prognostic subclasses in adult acute legs was formed in place of the antennae. In

profile, not restricted to nk-AML cases, that is myeloid leukemia. N Engl J Med. 2004;350:1605-1616.

mammals as in flies, Hox genes exhibit striking

distinguished by up-regulation of several HOX 3. Falini B, Mecucci C, Tiacci E, et al. Cytoplasmic nucleo-

phosmin in acute myelogenous leukemia with a normal “collinearity” of expression and effect: during

genes and their TALE partner genes and by karyotype. N Engl J Med. 2005;352:254-266. embryonic body development Hox gene ex-

down-regulation of the CD34 gene, suggesting 4. Tallman MS, Gilliland DG, Rowe JM. Drug therapy for pression profiles correlate with body segments

that NPM1 mutation–associated leukemogen- acute myeloid leukemia. Blood. 2005;106:1154-1163.

and with developmental stage in a direct man-

esis involves transformation of a primitive 5. Alcalay M, Tiacci E, Bergomas R, et al. Acute myeloid

leukemia bearing cytoplasmic nucleophosmin (NPMc ner (ie, 3 genes such as HoxA1 and B1 are

CD34-negative hematopoietic stem cell. How- AML) shows a distinct gene expression profile character- expressed more anteriorly and earlier in devel-

ever, in neither report did the therapeutically ized by up-regulation of genes involved in stem-cell mainte-

opment than their 5 paralogs, which are ex-

nance. Blood. 2005;106:899-902.

influential FLT3 mutation status have a defining pressed later and in more posterior regions).

6. Takahashi S, Harigae H, Kumura Ishii K, et al. Over-

role in the gene expression profile. Perhaps, then, expression of Flt3 induces NF-kB pathway and increases However, there is no “one Hox, one segment”

the interactive effect of FLT3/ITD mutations is the expression of IL-6. Leuk Res. 2005;29:893-899.

rule; instead, chromosomally adjacent clusters

of Hox genes (eg, HoxA9-11) are expressed in

● ● ● STEM CELLS IN HEMATOLOGY clusters at a given developmental stage of a

given body segment. This redundancy is likely

Comment on Lawrence et al, page 3988 part of the reason that so few of the Hox genes

are individually necessary for any given func-

The wizard of Hox

----------------------------------------------------------------------------------------------------------------

tional role in hematopoiesis. Moreover, hema-

topoiesis seems to break the collinearity rule.

Curt I. Civin JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE Clearly, global transcriptomic and proteomic

methodologies should be of considerable util-

In this issue of Blood, Lawrence and colleagues provide strong evidence that ity in dissecting the physiologic functions of

HoxA9 plays a key physiologic role in the proliferation of early mouse hematopoi-

the Hox family and pathways in hematopoi-

etic stem progenitor cells.

esis.

embers of this group showed previously studies (especially since forced expression of As the normal physiology is being pains-

M that overexpression of HoxA9 drove

expansion of mouse hematopoietic stem pro-

each of several Hox genes resulting from chro-

mosomal translocations can drive leukemia

takingly illuminated, the ability of certain Hox

genes and TAT-Hox proteins to stimulate in

genitor cells. Indeed, many, perhaps all, Hox cell proliferation) but only the loss of HoxB4 vivo and ex vivo hematopoietic stem cell ex-

genes may be sufficient to “pharmacologically” (or HoxB3 and B4 together) had previously pansion is already nearing clinical application,

drive stem cell expansion in gain-of-function resulted in impaired stem cell functionality. especially with HoxB4. In this context we



3682 1 DECEMBER 2005 I VOLUME 106, NUMBER 12 blood

wonder the following. (1) Should we prefer Hox genes are regulated may permit efficient represents the rate-limiting step in the reac-

any one Hox for clinical stem cell expansion? stimulation of the physiologic expression pro- tion. This conclusion is consistent with an-

Specifically, should we test HoxA9 now that file to optimally drive hematopoietic stem cell other very recent report of Wajih et al.3

we know it is not only sufficient but also neces- expansions. ■ The finding of Sun et al has important

sary for optimal stem cell function in vivo (in practical implications. Some vitamin K– de-

mice)? Or will most or all of these Hox para- pendent coagulation factors are of commercial

logs function similarly? (2) Can we further REFERENCE interest as factor IX and rFVIIa for the treat-

enhance stem cell expansion by combined ap- 1. Grier DG, Thompson A, Kwasniewska A, McGonigle

GJ, Halliday HL, Lappin TR. The pathophysiology of

ment of hereditary or acquired bleeding disor-

plication of more than one Hox molecule? (3) HOX genes and their role in cancer. J Pathol. 2005;205: ders and as activated protein C for the treat-

As we learn more about stem cell functional 154-171. ment of sepsis. The yield of carboxylated and

wiring diagrams, can we productively improve 2. Bridges CB, Morgan TH. The third-chromosome group

accordingly functional active protein in cell

of mutant characters of Drosophila melanogaster. Publ Car-

the “context” for stem cell expansion by up- negie Inst. 1923;327:1-251. lines coexpressing VKORC1 will be much

regulating or down-regulating Hox or other 3. Lewis EB. A gene complex controlling segmentation in higher than in currently used cell lines.

pathways? (4) Finally, determining how the Drosophila. Nature. 1978;276:565-570. The role of VKORC1 as a main regulator

of the carboxylation reaction has gained par-

● ● ● HEMOSTASIS ticular attention in view of its function as the

molecular target of coumarin derivatives (eg,

Comment on Sun et al, page 3811 warfarin), the most prescribed drug for

therapy of thromboembolic events. Recently,

VKORC1: the little big protein

----------------------------------------------------------------------------------------------------------------

frequent VKORC1 gene variants have been

reported that significantly alter the level of

Johannes Oldenburg INSTITUTE OF EXPERIMENTAL HAEMATOLOGY AND TRANSFUSION MEDICINE VKORC1 activity and consequently affect the

therapeutic dose of the drug.4,5 In fact, these

VKORC1, the recently identified protein of the vitamin K cycle, catalyzes vitamin observations suggest VKORC1 as the principal

K epoxide to vitamin K and further to vitamin K hydroquinone, thus representing genetic modulator of the interindividual and

the rate-limiting enzyme for the carboxylation of vitamin K– dependent proteins. interethnic differences in warfarin response.

he key gene of the vitamin K cycle encod- from 52% to 92% by coexpressing VKORC1 Moreover, we are tempted to speculate that these

T ing the molecular target of coumarin-type

anticoagulants, vitamin K epoxide reductase

(see figure). The explanation for this observa-

tion is that most likely VKORC1 is responsible

naturally occurring VKORC1gene variants may

also have an important influence on downstream

(VKORC1; Online Mendelian Inheritance in for both the conversion of vitamin K epoxide function of vitamin K– dependent proteins, in-

Man [OMIM]*608547; 16p11.2), has recently to vitamin K and vitamin K to vitamin K hy- cluding matrix Gla protein and osteocalcin,

been identified by our group and Sun and col- droquinone. Thus, although -glutamyl car- which have been suggested to play a role in the

leagues.1,2 VKORC1 recycles vitamin K 2,3 boxylase (GGCX) is the enzyme that accom- pathogenesis of atherosclerosis, myocardial in-

epoxide to vitamin K hydroquinone, which plishes the carboxylation reaction, VKORC1 farction, and stroke.

functions as the essential cofactor for -car-

boxylation of Gla-domain proteins such as

coagulation factors II, VII, IX, and X; proteins

C, S, and Z; osteocalcin; matrix Gla protein

(MGP); and Gas6. This gene extended over

5126 base pairs and comprised 3 exons encod-

ing a small protein of 163 amino acids with a

calculated relative molecular mass of about 18

kDa.1,2 Mutations in VKORC1 cause 2 dis-

tinctive phenotypes: a homozygous missense

mutation in the VKORC1 gene leads to com-

bined deficiency of vitamin K– dependent

coagulation factors type 2 (VKCFD2),1 and

heterozygous missense mutations are respon-

sible for hereditary warfarin resistance in hu-

mans, rats, and mice.1

In this issue of Blood, Sun and colleagues

report further key information about

VKORC1. Using a HEK 293 cell line overpro-

ducing factor X, they found that the fraction of Separation of -carboxylated and uncarboxylated FX by hydroxylapatite chromatography. See the complete

carboxylated factor X increases dramatically figure in the article beginning on page 3811.





blood 1 D E C E M B E R 2 0 0 5 I V O L U M E 1 0 6 , N U M B E R 1 2 3683

The work of Sun et al demonstrates for vitamin K epoxide reductase. Nature. 2004;427:541- the information on only precursor thrombo-

544.

VKORC1 as a main regulator of the carboxyla- cytes that are marked by the expression of

3. Wajih N, Sane DC, Hutson SM, Wallin R. The inhibi-

tion reaction, which, in view of the multiple tory effect of calumenin on the vitamin K-dependent

CD41 promoter. Thus, whether these large

downstream pathways affected by this protein, gamma-carboxylation system: characterization of the sys- GFP cells are in fact thrombocyte precursors

suggests further exciting studies in the near tem in normal and warfarin-resistant rats. J Biol Chem. remains to be established. It is entirely pos-

2004;279:25276-25283.

future. ■ sible that there is expression of CD41 in the

4. Rieder MJ, Reiner AP, Gage BF, et al. Effect of

VKORC1 haplotypes on transcriptional regulation and war- nonthrombocytic lineage and the cells could

REFERENCES farin dose. N Engl J Med. 2005;352:2285-2293. be the multipotent hematopoietic precursors.

1. Rost S, Fregin A, Ivaskevicius V, et al. Mutations in 5. Geisen C, Watzka M, Sittinger K, et al. VKORC1 hap- Thus, caution must be exercised in extrapolat-

VKORC1 cause warfarin resistance and multiple coagula- lotypes and their impact on the inter-individual and inter-

tion factor deficiency type 2. Nature. 2004;427:537-541. ing the current findings. In fact, the authors

ethnical variability of oral anticoagulation. Thromb Hae-

2. Li T, Chang CY, Jin DY, et al. Identification of the gene most. 2005;94:773-779. are aware of the expression of CD41 in cell

types other than megakaryocytes in birds and

mammals. Fluctuations in gene expression

● ● ● HEMATOPOIESIS

during development are not unprecedented

Comment on Lin et al, page 3803 and indeed the authors themselves identified

expression of CD41 promoter in unfertilized



A green light for the thrombopoietic zebrafish eggs. Even though this is not relevant

to thrombopoiesis, this observation is novel



program

----------------------------------------------------------------------------------------------------------------

and raises several questions. Is there mater-

nally derived CD41 mRNA in unfertilized

eggs? If so, what is the role for CD41 in early

Pudur Jagadeeswaran UNIVERSITY OF NORTH TEXAS development? Is there a species-specific differ-

ence for the role of CD41 since lack of func-

The opportunity to study the dynamics of thrombopoeisis in real time has long

tional CD41 in mice and men seem to have no

been awaited.

apparent developmental abnormalities?

n their new paper, Lin and colleagues have tral region of the aorta that roughly corre- In conclusion, the current work will initiate

I described the generation of transgenic ze-

brafish with green fluorescent thrombocytes.

sponds to aorta-gonads-mesonephros (AGM),

which is the site of hematopoiesis in mamma-

further studies on thrombocyte differentiation

and will open new avenues to explore dynam-

This fish was created using a zebrafish CD41 lian development. However, these GFP cells ics of thrombopoiesis in real time. ■

gene promoter that drives the expression of appear after the dissolution of AGM and are

jellyfish green fluorescent protein (GFP). Us- too caudal. Thus, the authors may have un-

REFERENCES

ing this fish, the authors have identified that earthed yet another novel site for hematopoi-

1. Gregory M, Jagadeeswaran P. Selective labeling of ze-

there are 2 populations of thrombocytes that esis. All the above point out that there will be brafish thrombocytes: quantitation of thrombocyte function

are made in kidney marrow. The authors claim novel unidentified programs in thrombocyte and developmental detection. Blood Cells Mol Dis. 2002;

28:417-427.

that one population of cells that expresses low development and the transgenic tool will help

2. Thattaliyath B, Cykowski M, Jagadeeswaran P. Young

levels of GFP seems to be the precursor for the in exploring such programs. However, one thrombocytes initiate the formation of arterial thrombi in

other cells that have high levels of GFP and limitation of this study is that it will enhance zebrafish. Blood. 2005;106:118-124.



that this invention will be useful in studying

thrombopoiesis. Even though thrombocytes ● ● ● IMMUNOBIOLOGY

have been identified earlier in zebrafish devel-

opment and in circulation,1,2 the current re- Comment on Airoldi et al, page 3846

port is important in following thrombocytes in

real time in development and in circulation

and thus merits attention.

Cytokine receptor gene plays

The significance of the above work is the

fact that the zebrafish thrombocytes have par-

antioncogene

----------------------------------------------------------------------------------------------------------------

allels to megakaryopoiesis and platelet produc- Giorgio Trinchieri NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES

tion in mammals. Since almost all mammalian

Mice genetically deficient for the IL12RB2 gene develop systemic lymphocyte

genes exist in fish, it would not be surprising

activation, spontaneous autoimmunity, and malignancy, particularly plasmacy-

that knockdown of zebrafish genes using anti-

toma and lung carcinoma.

sense morpholinos and the above transgenic

nterleukin-12 (IL-12) is a heterodimeric is formed by 2 chains, IL-12R 1 and IL-

line could identify novel players in megakaryo-

poiesis. In this context it is interesting to note

that the suggested precursor GFP cells are

I cytokine formed by 2 chains, IL-12 p35 or

-chain and IL-12 p40 or -chain.1 The IL-12

12R 2. IL-12R 2 is specific for the IL-12

receptor, whereas IL-12R 1 also associates

large, similar to other known hematopoietic p40 chain can also associate with IL-23 p19 to with IL-23R to form the receptor for IL-23.

progenitor cells. GFP cells appear in the ven- form the IL-23 cytokine. The IL-12 receptor IL-12 is considered a typical proinflammatory



3684 1 DECEMBER 2005 I VOLUME 106, NUMBER 12 blood

cytokine produced mostly by myeloid cells and the inability of the animals to control aberrant the complex role of proinflammatory cytokines

dendritic cells, and its biologic effects, in par- B-cell activation or an effect of the chronic in either promoting or preventing tumor initia-

ticular the ability to induce production of in- inflammatory environment on B-cell neoplas- tion and progression. ■

terferon (IFN- ) and to support T-helper 1 tic transformation and tumor progression.

(Th1) type T-cell responses, have been stud- The occurrence in some animals of lung ad-

REFERENCES

ied particularly on natural killer (NK) and T enocarcinoma may have an opposite mecha-

1. Trinchieri G. Interleukin-12 and the regulation of innate

cells that constitutively or upon activation nism and be linked to defective innate antitu- resistance and adaptive immunity. Nat Rev Immunol.

express functional IL-12 receptors. Although mor surveillance in the animals lacking IL-12 2003;3:133-146.



an activity of IL-12 on B-cell functions and functions, possibly secondary to a reduced 2. Jelinek DF, Braaten JK. Role of IL-12 in human B lym-

phocyte proliferation and differentiation. J Immunol.

immunoglobulin production has been de- production of IFN- . Future studies analyz- 1995;154:1606-1613.

scribed in early studies,2 whether B cells ex- ing the specific role of IL-12 in regulation of 3. Airoldi I, Gri G, Marshall JD, et al. Expression and

press functional IL-12 receptors has long been B-cell activation and transformation, autoim- function of IL-12 and IL-18 receptors on human tonsillar B

cells. J Immunol. 2000;165:6880-6888.

a controversial issue. More recently, however, munity, and solid tumor immunosurveillance

4. Airoldi I, Di Carlo E, Banelli B, et al. The IL-12Rbeta2

it was clearly established that normal B cells will shed new light on the mechanisms of ho- gene functions as a tumor suppressor in human B cell malig-

express both chains of the IL-12 receptor and meostatic regulation of B-cell activation and on nancies. J Clin Invest. 2004;113:1651-1659.

that they respond to IL-12 with increased im-

munoglobulin secretion, expression of the ● ● ● TRANSPLANTATION

IL-18 receptors, and, particularly in the pres-

ence of IL-18, production of a high level of Comment on Burroughs et al, page 4002

IFN- .3 However, Airoldi et al4 have shown

that in malignant B cells the IL12RB2 gene

was silenced, probably by hypermethylation.

Antagonizing CXCR4 accelerates CD34

When the IL12RB2 gene expression was re-

established either by treatment of the cells

cell mobilization

----------------------------------------------------------------------------------------------------------------

with a DNA methyltransferase inhibitor or by

Steven M. Devine THE OHIO STATE UNIVERSITY COMPREHENSIVE CANCER CENTER

gene transfection, IL-12, both in vitro and in

vivo, induced apoptosis and growth inhibition A novel bicyclam CXCR4 antagonist, AMD3100, appears capable of mobilizing a

of the malignant B cells.4 fully functional hematopoietic allograft within just 6 hours following a single injec-

On the basis of the data mentioned above, tion.

Airoldi et al4 have postulated that IL-12R 2

ecent studies have demonstrated that the showed that a single dose of AMD3100 in-

functions as a tumor suppressor in human

B-cell malignancies. In a paper in the present

R interaction between the chemokine stro-

mal-derived factor 1 (SDF-1/CXCL12) and

duces HSC and HPC mobilization within a

few hours, allowing for apheresis to be per-

issue of Blood, Airoldi and colleagues tested

its only known receptor, CXCR4, serves as a formed on the same day of drug administra-

this hypothesis by analyzing the appearance of

key regulator of hematopoietic stem cell tion. Following both autologous and alloge-

malignancies in aging IL12rb2– deficient mice.

(HSC) trafficking.1 In clinical practice, the neic transplantation, the cells collected

They observed not only a very significant inci-

hematopoietic cytokine granulocyte colony- following mobilization with AMD3100 alone

dence of plasmacytoma and lung carcinoma

stimulating factor (G-CSF) is widely used to were capable of reconstituting hematopoiesis

but also immune complex mesengial glomeru-

lonephritis with serum antinuclear antibodies induce the mobilization of HSCs and hemato- with neutrophil and platelet recovery kinetics

and multiorgan lymphoid infiltrates with sys- poietic progenitor cells (HPCs) for reconstitu- similar to those observed following transplan-

temic B- and T-cell activation in all aging ani- tion of hematopoiesis following myelosuppres- tation of G-CSF–mobilized cells. One canine

mals. The observed autoimmune pathology sive therapy. Several groups have demonstrated allogeneic recipient appeared to develop acute

may in part be secondary to an up-regulation that G-CSF causes mobilization primarily graft-versus-host disease (GVHD), but the

of IL-6 in the IL12rb2– deficient animals, and through its indirect disruption of the SDF-1/ overall incidence of this serious transplanta-

the data presented suggest that there is a recip- CXCR4 interaction, inducing its cleavage by tion complication did not seem to be different

rocal down-regulation between IL-6 and IL- serine proteases or via down-regulation of from what would be expected following trans-

12. These results strongly support the conclu- SDF-1 mRNA.2-4 Whatever the precise plantation of G-CSF–mobilized cells. All dogs

sions that IL-12 may be important in mechanism(s), this implies that agents that that received a transplant achieved full donor

controlling aberrant or excessive B-cell activa- directly inhibit this interaction may be effec- hematopoietic chimerism following myeloab-

tion and that the absence of signaling of this tive mobilizers. In this issue of Blood, Bur- lative radiation.

proinflammatory cytokine paradoxically re- roughs and colleagues have demonstrated that These intriguing preliminary data suggest

sults in a state of systemic B- and T-cell activa- AMD3100, a direct antagonist of CXCR4, in- that by directly antagonizing CXCR4, a more

tion. These findings open a new perspective on duces the rapid mobilization of hematopoietic rapid mobilization of clinically relevant

the physiologic role of IL-12. The high fre- cells with both short- and long-term repopu- CD34 cells can be induced. This stands in

quency of plasmacytoma observed in the aging lating capacity. Using a clinically relevant my- stark contrast to the 4 to 5 days of G-CSF

IL12rb2– deficient animals may reflect either eloablative canine transplantation model, they treatment normally required to mobilize



blood 1 D E C E M B E R 2 0 0 5 I V O L U M E 1 0 6 , N U M B E R 1 2 3685

sufficient HSCs and HPCs. A recent clinical affect the quality and quantity of other impor- nized that a subset of seropositive patients will

trial in patients with non-Hodgkin lymphoma tant cell subsets cells mobilized in an allograft develop clinical disease.

and multiple myeloma demonstrated that such as T and natural killer (NK) cells, and The limited interactions of fondaparinux

when AMD3100 is combined with G-CSF, how might this influence the risk of GVHD with PF43 and lack of serologic cross-reactivity

the yield of CD34 cells mobilized and avail- and relapse? Ongoing studies should provide of drug with preformed HIT antibodies4 have

able for collection is considerably greater than some clues over the next year. Stay tuned as led to the general perception that fondapa-

following G-CSF alone.5 These canine studies the story unfolds. ■ rinux is likely nonimmunogenic. In this issue

now suggest that AMD3100 could theoreti- of Blood, Warkentin and colleagues dispel this

cally be used alone to mobilize HSCs and REFERENCES notion through serologic investigations of pa-

HPCs from healthy donors in a 1-day proce- 1. Lapidot T, Dar A, Kollet O. How do stem cells find their tients enrolled in 2 large orthopedic trials

way home? Blood. 2005;106:1901-1910.

dure. An ongoing trial is currently testing this (Pentathlon5 and Pentamaks6) comparing

2. Papayannopoulou T. Current mechanistic scenarios in

hypothesis. hematopoietic stem/progenitor cell mobilization. Blood.

enoxaparin to fondaparinux for thrombopro-

Despite early enthusiasm for this agent, a 2004;103:1580-1585. phylaxis after surgery. For their analysis,

number of questions arise. Will blocking 3. Petit I, Szyper-Kravitz M, Nagler A, et al. G-CSF in- samples from over 2700 patients (or 80% of

duces stem cell mobilization by decreasing bone marrow the overall study population from the 2 trials)

SDF-1/CXCR4 inhibit the normal homing SDF-1 and up-regulating CXCR4. Nat Immunol. 2002;

and migration of stem and progenitor cells 3:687-694. were assayed for PF4/heparin antibodies at

following transplantation or, alternatively, 4. Semerad CL, Christopher MJ, Liu F, et al. G-CSF po- baseline and at days 5 to 9 of anticoagulant

tently inhibits osteoblast activity and CXCL12 mRNA ex- therapy and correlated with clinical outcomes.

might homing of these cells be more effective? pression in the bone marrow. Blood. Prepublished on July

Are the qualities of the HSCs mobilized fol- 21, 2005, as DOI 110.1182/blood-2004-01-0272. There were several surprising but clinically

lowing AMD3100 similar to those mobilized 5. Flomenberg N, Devine SM, DiPersio JF, et al. The use noteworthy findings in this study. The authors

by G-CSF or are there important differences?

of AMD3100 plus G-CSF for autologous hematopoietic document similar seroconversion rates in both

progenitor cell mobilization is superior to G-CSF alone.

Finally, how will treatment with AMD3100 Blood. 2005;106:1867-1874.

treatment groups. PF4/heparin antibodies

were detected in 1.5% and 2.8% of fondapa-

rinux-treated patients compared with 1.1%

● ● ● CLINICAL OBSERVATIONS and 5.2% of the enoxaparin-treated patients

(Pentathlon and Pentamaks trials, respec-

Comment on Warkentin et al, page 3791

tively; P value not significant for fondaparinux

vs enoxaparin). Antibodies induced by fondapa-

The immune paradox of fondaparinux

----------------------------------------------------------------------------------------------------------------

rinux were comparable to those induced by

enoxaparin with respect to immunoglobulin G

Gowthami M. Arepally DUKE UNIVERSITY MEDICAL CENTER (IgG) isotype and capacity to trigger UFH-

dependent platelet activation. There were no

In this issue of Blood, Warkentin and colleagues demonstrate that fondaparinux cases of clinical HIT among the seropositive

elicits an immune response to PF4/heparin. Antibodies induced by the drug do not patients in either treatment group. The most

recognize complexes of PF4/fondaparinux but, paradoxically, bind complexes of intriguing findings of this study were related

PF4/heparin and PF4/LMWH. to the serologic specificities of fondaparinux-

induced antibodies. As shown in the figure,

n 1979, Rosenberg and Lam1 noted that the life-threatening com-

I anticoagulant activity of heparin resides in

only a subpopulation of heparin molecules that

plication caused by

platelet factor 4 (PF4)/

bind antithrombin. They subsequently identi- heparin antibodies.

fied an invariant tetrasaccharide sequence re- PF4/heparin antibod-

sponsible for heparin’s biologic activity.1 A ies are frequently in-

modified version of this sequence has since duced by heparin

been developed as a synthetic pentasaccharide therapy and, depend-

compound (fondaparinux; molecular weight ing on the clinical set-

[MW] 1728) with potent factor Xa inhibi- ting, occur in approxi-

tory activity. In several large clinical trials, mately 8% to 21% of

fondaparinux has proven to be comparable to medical patients ex-

low-molecular-weight heparin (LMWH) in posed to unfraction-

safety and efficacy for the prevention and ated heparin (UFH)

treatment of venous thromboembolism. and in 2% to 8% of

The clinical trials of fondaparinux, how- patients treated with

ever, did not directly address one important LMWH.2 Although

biologic aspect of this heparin-like drug, its the immune pathogen- Ratio of antibody binding to PF4/polysaccharide complexes compared to PF4

immunogenicity and potential for initiating esis of HIT is not well alone by fluid-phase EIA. See the complete figure in the article beginning on

heparin-induced thrombocytopenia (HIT), a understood, it is recog- page 3791.





3686 1 DECEMBER 2005 I VOLUME 106, NUMBER 12 blood

fondaparinux-induced antibodies do not bind spective studies and experience with fondapa- riority of HDT for either achievement of CR

complexes of PF4/fondaparinux but, rather, rinux are needed to understand the true clini- (17% vs 15%) or prolongation of OS (58 vs 53

show reactivity toward complexes of PF4/ cal significance of the immune response months).4

UFH and PF4/enoxaparin. invoked by this new agent. Until then, the ´

The study by Blade and colleagues pub-

Based on these findings, the authors con- sensitizing effects of fondaparinux and its po- lished in this issue of Blood, the fifth study

clude that fondaparinux-sensitized patients tential for inducing clinical HIT must be taken comparing HDT versus SDT, shows signifi-

are at low risk for developing HIT, as antibod- seriously. ■ cantly higher CR rates after HDT (30% ver-

ies do not bind PF4/fondaparinux complexes sus 11%), without statistically significant im-

REFERENCES

and therefore cannot induce drug-dependent provement in progression-free survival (PFS;

1. Rosenberg RD, Lam L. Correlation between structure

platelet activation. This conclusion is likely and function of heparin. Proc Natl Acad Sci U S A. 1979;76: 42 versus 33 months) and OS (61 versus 66

premature for several reasons. In the original 1218-1222. months). Since only 12% patients in the SDT

reports of the 2 clinical trials,5,6 thrombocyto- 2. Lee DH, Warkentin TE. Frequency of heparin-induced cohort received HDT as salvage and survival

thrombocytopenia. In: Warkentin TE, Greinacher A, eds.

penia ( 100 109/L) was reported in 2% to Heparin-Induced Thrombocytopenia. 3rd ed. New York, after relapse in both arms was equivalent (15.9

4% of patients receiving either drug. Whether NY: Marcel Dekker AG; 2004:107-148. versus 16.4 months), the lack of OS benefit of

thrombocytopenia in these 2 clinical trials was 3. Rauova L, Poncz M, McKenzie SE, et al. Ultralarge HDT cannot be due to salvage transplants in

complexes of PF4 and heparin are central to the pathogene-

drug induced or associated with development sis of heparin-induced thrombocytopenia. Blood. 2005;105: the conventional dose therapy arm. This re-

of PF4/heparin antibodies was not addressed 131-138. port differs from prior randomized trials,

by Warkentin et al. This lack of clinical and 4. Savi P, Chong BH, Greinacher A, et al. Effect of since only patients responding to initial

fondaparinux on platelet activation in the presence of hepa-

serologic information on thrombocytopenic therapy were eligible for randomization. A

rin-dependent antibodies: a blinded comparative multi-

patients is an important limitation of this study center study with unfractionated heparin. Blood. 2005;105: prior retrospective study of patients who

and makes it difficult to estimate the true risk 139-144.

were candidates for, but did not receive,

of clinical HIT associated with either fondapa- 5. Turpie AG, Bauer KA, Eriksson BI, Lassen MR, Com-

mittee PSS. Postoperative fondaparinux versus postopera- HDT also supports equivalent patient out-

rinux or enoxaparin. Because of the cross- tive enoxaparin for prevention of venous thromboembolism comes with SDT.5

reactivity profile of fondaparinux-induced after elective hip-replacement surgery: a randomised

double-blind trial [erratum appears in Lancet. 2002;360: Who benefits from HDT? In this study,

PF4/heparin antibodies, it is clear that sensi- 1102]. Lancet. 2002;359:1721-1726. increased CR rate does not translate into a

tized patients are at risk for developing HIT if 6. Bauer KA, Eriksson BI, Lassen MR, Turpie AG, Steer- survival benefit; to date, a clear benefit of

they are subsequently exposed to UFH or ing Committee of the Pentasaccharide in Major Knee Sur-

gery S. Fondaparinux compared with enoxaparin for the HDT is observed only in those randomized

LMWH. Fondaparinux-sensitized patients prevention of venous thromboembolism after elective major studies with significantly lower CR rates after

are also theoretically at risk for developing knee surgery. N Engl J Med. 2001;345:1305-1310.

SDT, supporting the view that achieving

subacute or delayed-onset HIT, as PF4/hepa- 7. Wallis DE, Workman DL, Lewis BE, Steen L, Pifarre R,

higher CR rates is associated with prolonged

Moran JF. Failure of early heparin cessation as treatment

rin antibodies can often elicit disease in the for heparin-induced thrombocytopenia. Am J Med. 1999; survival. Novel therapies like thalidomide,

absence of circulating drug.7 Additional pro- 106:629-635.

bortezomib, and lenalidomide (Revlimid) with

activity in relapsed refractory MM are now

being used as initial therapy to achieve higher

frequency of CR and may thereby improve

● ● ● CLINICAL OBSERVATIONS outcome. Nonetheless, 6 of 9 patients in this

study who were unresponsive to initial therapy

´

Comment on Blade et al, page 3755 underwent HDT and achieved partial re-

sponse (PR), suggesting that HDT can achieve



To transplant or not to transplant?

----------------------------------------------------------------------------------------------------------------

responses even in patients with primary re-

fractory disease.

Although 3 of 5 randomized studies show

Nikhil C. Munshi and Kenneth C. Anderson DANA FARBER CANCER INSTITUTE; HARVARD MEDICAL SCHOOL that HDT achieves prolongation of EFS and

OS ranging from 4 to 12 months and from 1 to

´

In this issue of Blood, Blade and colleagues report that HDT does not improve less than 23 months, respectively, few, if any,

overall or event-free survival in patients responding to initial chemotherapy. patients are cured. In the current study, no

se of high-dose melphalan with stem cell significant increased complete response (CR) benefit in either EFS or OS is observed after

U transplantation is a major advance in the

therapy of multiple myeloma (MM). To date,

rates, with prolonged event-free survival

(EFS) and overall survival (OS), in the patient

HDT. Given these modest benefits, it is criti-

cal to assess quality of life achieved after HDT;

5 randomized studies have compared the out- cohorts receiving HDT. In contrast, the My- indeed, a single study has shown that quality

come of patients treated with high-dose elome-Autogreffe Group (MAG) study does of life was inferior at 6 months after HDT than

therapy (HDT) versus standard-dose therapy not show superiority of HDT for EFS and SDT.6

(SDT). The Intergroupe Francais du My- OS3; and the US Intergroup trial, which ran- Two major strategies are under evaluation

elome 90 (IFM90)1 and the Medical Research domized patients to HDT versus SDT with to improve outcome after HDT. First, re-

Council (MRC) VII2 trials show statistically delayed HDT at relapse, does not show supe- peated or tandem HDT has improved PFS



blood 1 D E C E M B E R 2 0 0 5 I V O L U M E 1 0 6 , N U M B E R 1 2 3687

and OS in some studies, with benefit of second REFERENCES survival in multiple myeloma (MM) with high dose therapy

(HDT) employing mel 140 mg/m2 TBI 12 Gy auto-

HDT especially for those who do not achieve 1. Attal M, Harousseau JL, Stoppa AM, et al. A prospec-

transplants versus standard dose therapy VBMCP and no

tive, randomized trial of autologous bone marrow transplan-

CR or near CR after single HDT. Second, benefit from interferon (IFN) maintenance: results of inter-

tation and chemotherapy in multiple myeloma: Intergroupe

attempts are under way integrating novel Francais du Myelome. N Engl J Med. 1996;335:91-97. group trial S9321 [abstract]. Blood. 2003;102:42a.



agents such as thalidomide, bortezomib, and 2. Child JA, Morgan GJ, Davies FE, et al. High-dose che- 5. Blade J, San Miguel JF, Fontanillas M, et al. Survival of

motherapy with hematopoietic stem-cell rescue for multiple multiple myeloma patients who are potential candidates for

lenalidomide into the transplantation para-

myeloma. N Engl J Med. 2003;348:1875-1883. early high-dose therapy intensification/autotransplantation

digm not only to enhance response before 3. Fermand JP, Ravaud P, Chevret S, et al. High-dose and who were conventionally treated. J Clin Oncol. 1996;14:

HDT, but also as maintenance therapies to therapy and autologous peripheral blood stem cell trans- 2167-2173.

prolong PFS and OS after transplantation. plantation in multiple myeloma: up-front or rescue treat- 6. Gulbrandsen N, Wisloff F, Brinch L, et al. Nordic Myeloma

ment? Results of a multicenter sequential randomized clini- Study Group: health-related quality of life in multiple my-

Novel agents therefore may improve outcome cal trial. Blood. 1998;92:3131-3136. eloma patients receiving high-dose chemotherapy with autolo-

and ultimately obviate the need for HDT. ■ 4. Barlogie B, Kyle RA, Anderson KC, et al. Comparable gous blood stem-cell support. Med Oncol. 2001;18:65-77.









3688 1 DECEMBER 2005 I VOLUME 106, NUMBER 12 blood