Significance of fetal hemoglobin-containing erythroblasts (F blasts) and the F blast/f cell ratio in myelodysplastic syndromes

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1 (2002) 16, Nture Publishing Group All rights reserved /02 $ Significnce of fetl hemoglobin-contining erythroblsts (F blsts) nd the F blst/f cell rtio in myelodysplstic syndromes JW Choi 1,2, Y Kim 1,3, M Fujino 4 nd M Ito 1 1 Deprtment of Pthology, Ngoy University Hospitl, Ngoy, Jpn; 2 Deprtment of Clinicl Pthology, College of Medicine, Inh University, Inchon, Kore; 3 Deprtment of Clinicl Pthology, College of Medicine, Ctholic University, Seoul, Kore; nd 4 First Deprtment of Pthology, Ngoy University School of Medicine, Ngoy, Jpn To investigte the reltionship between the fetl hemoglobincontining erythroblsts (F blsts) nd poptosis in myelodysplstic syndromes (MDS), we immunohistochemiclly ssessed F blsts, F cells, nd poptosis in 137 ptients with MDS. A mrked increse in the number of F blsts in the bone mrrow ws identified in 116 of 137 ptients (84.7%), nd the number of F cells ws elevted in 54 ptients (39.4%). Among the erythroblsts stined by nti-glycophorin C ntibody, the men percentge of F blsts ws % in MDS, which ws significntly higher thn tht in non-mds ptients with stress erythropoiesis ( %, P 0.01), lthough there were no significnt differences in the number of F cells between these groups. In prticulr, 62 of the 137 MDS ptients (45.3%) hd n pprent increse in F blsts but no elevtion of F cells. The poptotic rte ws significntly higher in the ptients with F blst/f cell (Fb/Fc) rtio 5.0 thn in those with Fb/Fc rtio 1.0 (P 0.01). The results indicte tht F cell precursors re incpble of mturing into functioning endstge F cells, presumbly owing to poptotic cell deth. The mesurement of F blsts in the bone mrrow is needed for the precise evlution of fetl-type erythropoiesis in MDS. (2002) 16, doi: /sj.leu Keywords: poptosis; myelodysplstic syndrome; F cells; F cell precursors Introduction Fetl hemoglobin (HbF) is produced minly in fetl life nd rpidly declines to extremely low levels by the ge of 1 yer. 1 In norml dults, HbF production is miniml nd HbF is restricted to specific popultion referred to s F cells. 2 However, the reemergence of HbF production my occur in certin inherited nd cquired conditions in dulthood, such s myelodysplstic syndromes (MDS) nd hemoglobinopthies. 3 Erythropoietic stress s seen in cute blood loss, chemotherpeutic mrrow bltion, mlignncies ffecting the erythropoietic system, nd cute hemolysis is lso ssocited with incresed F cell production. 4 6 Incresed F cells in MDS hve been reported to be of prognostic significnce. 7 The HbF concentrtion hs been ssessed by lkli denturtion, electrophoresis, or high performnce liquid chromtogrphy, 8 10 wheres F cells hve been mesured by n cid elution technique, n indirect immunofluorescence method on slides, or flow cytometry However, such methods do not provide ny informtion bout HbF expression or the distribution of F cell precursors in the bone mrrow. Recently, we reported method to detect HbF-contining erythroblsts in the bone mrrow by immunohistochemistry using new nti-hbf ntibody ginst synthetic peptide ntigen, nd defined HbF-contining erythroblsts or F cell precursors s F blsts. 14 F cells re produced from F cell precursors in hemto- Correspondence: M Ito, Deprtment of Pthology, Ngoy University Hospitl, 65 Tsurumi-cho, Show-ku, Ngoy, , Jpn; Fx: Received 7 December 2001; ccepted 11 Februry 2002 poietic orgns, nd the HbF concentrtion is lso bsed on the concentrtions of F cell hemolystes. Consequently, the mesurement of F cell precursors in the bone mrrow might provide more ccurte informtion on fetl-type erythropoiesis in dults. In contrst to the flow cytometric F cell ssy, immunohistochemistry for F blsts preserves the morphologic chrcteristics of the HbF-contining erythroid precursors in reltion to the histologic fetures nd llows detiled study of HbF production not only in dult bone mrrows, but lso in fetl erythropoietic orgns. An pprent prdox in MDS is tht ptients with this disese show peripherl cytopeni despite norml or high cellulrity in their bone mrrows. 15 Cytopeni in MDS is considered to be the result of pronounced hemtopoietic insufficiency, nd this ineffective hemtopoiesis is contributed to the incresed poptosis of bone mrrow precursors. 16 Severl investigtors hve reported higher incidence of F cells in ptients with MDS compred with controls. 7,17 However, ll the studies on fetl-type erythropoiesis in MDS hve thus fr been bsed minly on the mesurement of HbF concentrtions or the percentge of F cells in the peripherl blood. Furthermore, there hve been no studies which hve closely exmined the incidence of F blsts in the bone mrrow of MDS ptients nd the significnce of the F blst/f cell (Fb/Fc) rtio. Hence, in this study we ssessed the F blsts in the bone mrrow from ptients with MDS, nd investigted the reltionship between the Fb/Fc rtio nd poptosis. Subjects nd methods Ptients selection A totl of 137 ptients with MDS were selected from the bone mrrow cse files of the Deprtment of Pthology, Ngoy University Hospitl. The MDS ptients included 36 with refrctory nemi (RA), 15 RA with ringed sideroblsts (RARS), 31 RA with excess of blsts (RAEB), 43 RA with excess of blsts in trnsformtion (RAEB-t), nd 12 with chronic myelomonocytic leukemi (CMMoL). The study groupcomprised 75 men nd 62 women with men ge of 67 yers (rnge 36 to 89 yers). We studied 14 helthy individuls s controls, who hd no bnormlities in the bone mrrow or peripherl blood. As dditionl controls, we lso ssessed the F blsts in the bone mrrow from non-mds ptients (n 25) with stress erythropoiesis, such s cute hemolytic nemi (n 7) nd crcinom (n 10) or lymphom (n 8) involving the bone mrrow. The men ges of the two control groups were 61 yers (31 to 78 yers) nd 56 yers (37 to 81 yers), respectively.

2 Immunohistochemicl stining Seril sections (3 m) were immunohistochemiclly stined for HbF, single-strnded DNA (ssdna), glycophorin C (GPC), nd p53. To detect the F blsts nd F cells in the bone mrrow clot sections, we used polyclonl nti-hbf ntibody tht we recently developed using synthetic peptide s n immunogen. 14 The ssy for poptosis ws n immunohistochemicl method bsed on the binding of polyclonl ntibody to ssdna, s described previously. 18 Antibodies ginst ssdna, GPC, nd p53 were purchsed from Dko (Glostrup, Denmrk). Prffin-embedded bone mrrow clot sections were processed using streptvidin biotin complex method or n immunoperoxidse technique. Before pplying the HbF ntibody, slides were treted with 0.1% trypsin in 0.05 M Tris buffer (ph 7.6) t 37 C for 30 min. Known positive controls ( 29-week-old fetl liver for HbF, Burkitt s lymphom for ssdna, nd colorectl crcinom for p53) s well s negtive controls (sections in which the primry ntibody ws replced by non-immune mouse or rbbit serum) were lso stined in ech run. The proportion of F blsts ws defined s the HbF synthetic rte, which represented the percentge of F blsts identified by the nti-hbf ntibody mong 1000 erythroblsts stined by the nti-gpc ntibody. HbF-contining erythrocytes (F cells) were counted with light microscope (mgnifiction 400) in well-dispersed res of the bone mrrow clot sections, nd were expressed s percentge of the totl number of 10 4 erythrocytes. The poptotic rte nd the number of cells positive for p53 were expressed s percentge of immunorective nuclei out of 1000 bone mrrow cells. All slides were reviewed by two different investigtors, who were blinded to clinicl dt. Sttisticl tests The nonprmetric Mnn Whitney U test ws used for the comprison of men vlues between two prmeters. Correltions between the number of F blsts nd p53 expression were ssessed by the Person correltion coefficient. Dt nlysis ws performed using n SAS sttisticl progrm (SAS Institute, Cry, NC, USA). P 0.01 ws considered sttisticlly significnt. Results F blsts nd F cells in bone mrrow from ptients with MDS The proportions of F blsts nd F cells in the bone mrrow from 137 ptients with MDS re summrized in Tble 1. Among the erythroblsts stined by nti-gpc ntibody, the percentge of F blsts ws % in MDS, which ws significntly higher thn tht in the non-mds ptients with stress erythropoiesis ( %, P 0.01). However, there ws no significnt difference in the percentge of F cells between the MDS ptients nd the non-mds ptients with stress erythropoiesis. Among the 137 ptients, 116 (84.7%) hd percentge of F blsts in the bone mrrow tht exceeded 0.3%, which ws tken s the provisionl cutoff vlue bsed on the men 2s.d. for the control group. The percentge of F cells ws greter thn 0.7% in 39.4% (54/137) of ptients, F cell precursors nd poptosis in MDS Tble 1 F blsts nd F cells in bone mrrow from ptients with MDS nd non-mds ptients with stress erythropoiesis Subjects Cses F blsts F cells b (n) (men (men s.d.,%) s.d.,%) Ptients with MDS Non-MDS ptients Control group MDS, myelodysplstic syndromes; F blsts, fetl hemoglobin (HbF)-contining erythroblsts; F cells, HbF-contining erythrocytes. Men vlues of F blsts nd F cells in ech group were compred by the Mnn Whitney U test. Percentges of F blsts in the bone mrrow from ptients with MDS were significntly higher thn for the controls nd for the non-mds ptients with stress erythropoiesis (P 0.01, respectively). b There were no significnt differences in the F cell levels between the MDS nd the non-mds ptients. the cutoff vlue lso bsed on the men 2 s.d. for the controls. To further investigte the ssocition between the incresed F blst proportions nd F cell levels, the 137 MDS ptients were subdivided into three groups ccording to the percentge of F blsts nd F cells in the bone mrrow (Tble 2). An increse in both F blsts ( 0.3%) nd F cells ( 0.7%) ws observed in 39.4% (54/137) of MDS ptients nd in 76.0% (19/25) of non-mds ptients with stress erythropoiesis (P 0.01). It is noteworthy tht ptients who hd n incresed F blst percentge ( 0.3%) but no increse in the percentge of F cells ( 0.7%) were identified in 45.3% (62/137) of the MDS ptients; however, there were no such cses in the non- MDS ptients with stress erythropoiesis. F blst/f cell (Fb/Fc) rtio nd poptotic rte The F blst percentge in ech subtype of MDS, long with the corresponding F cells nd Fb/Fc rtio re listed in Tble 3. There were no significnt differences in the men proportions of F blsts or F cells mong the subtypes of MDS, lthough dvnced MDS, especilly RAEB or RAEB-t, showed trend towrd n incresed percentge of F blsts in the bone mrrow. Among the subtypes of MDS, the Fb/Fc rtio ws the highest in RAEB nd ws significntly bove the vlues in RA or RARS (P 0.01, in ech group). The expression of ssdna nd p53 ws elevted in 42 ptients (30.6%) nd in 101 ptients (73.7%) by greter thn 1.0%, respectively, which ws the cutoff vlue tken from the men 2s.d. for controls. The poptotic rte verged 5.4% in the MDS ptients, which ws significntly bove the vlues in the control subjects (0.5%, P 0.01). RAEB nd RAEB-t showed reltively high poptotic rtes compred with RA, RARS, nd CMMoL. No significnt correltions between the F blst percentges nd p53 expression were noted. As shown in Tble 4, it is pprent tht ssdna expression ws significntly higher in the ptients with n Fb/Fc rtio 5.0 thn in those with n Fb/Fc rtio 1.0 (P 0.01); however, no significnt difference in p53 expression ws observed between the two groups. 1479

3 1480 Tble 2 F cell precursors nd poptosis in MDS Groupings bsed upon the percentge of F blsts nd F cells Clssifiction F blsts (%) F cells b (%) No. of cses (%) in MDS ptients No. of cses (%) in non-mds ptients P (n 137) with stress erythropoiesis (n 25) vlues Group (39.4) 19 (76.0) 0.01 Group (45.3) 0 (0.0) 0.01 Group (15.3) 6 (24.0) NS NS, not significnt. F blsts, 0.3%; b F cells, 0.7%: cutoff vlues bsed on the men 2 s.d. of the controls. Tble 3 HbF production, poptotic rte nd p53 expression in ech subtype of MDS Subtypes Cses HbF production Apoptotic rte p53 expression b (n) (%) F blsts F cells Fb/Fc rtio p53-positive p53-positive (%) (%) cells (%) cse (n) MDS RA (69.4) RARS (73.3) RAEB (77.4) RAEB-t (79.1) CMMoL (58.3) Fb/Fc rtio, F blst/f cell rtio. Dt re expressed s the men sd. Apoptotic rte in controls: %; b p53 expression in controls: %. There were no significnt differences in the men vlues of F blsts, F cells, or p53 mong the MDS subtypes; however, the Fb/Fc rtio ws significntly higher in RAEB thn in RA or RARS (P 0.01, respectively). Among the subtypes of MDS, RAEB nd RAEB-t showed reltively high poptotic rte compred to RA, RARS nd CMMoL. Tble 4 rtio Apoptotic rte nd p53 expression bsed on the Fb/Fc Tble 5 Distribution pttern of F blsts in the bone mrrow from ptients with elevted F blsts Apoptosis Fb/Fc rtio Fb/Fc rtio P vlues nd p Cses (n) NT Apoptotic rte (%) 10.9 ± ± p53 expression (%) 18.7 ± ± Distribution of F blsts in bone mrrow Severl distinct ptterns of HbF expression in erythroid precursors were observed in ptients with MDS. In ll the controls who hd F blsts in their bone mrrow or in 63.2% of the non-mds ptients, F blsts were mostly dispersed in widespred re without forming ny clusters of cells. Clusters of F blsts (smll cell colonies with more thn three F blsts) were seen in ptients with MDS (56.0%) nd in non-mds ptients with stress erythropoiesis (36.8%). However, sheets of F blsts were observed only in ptients with MDS (31.9%), which were chrcterized by confluence of cells with more thn 50 F blsts (Tble 5 nd Figure 1). HbF expression ws lrgely restricted to immture erythroid precursors or dysplstic erythroblsts. Discussion This study evluted the percentge of F blsts nd HbF production in the bone mrrow from ptients with MDS nd n Distribution MDS Non-MDS Controls pttern (n 116) (n 19) (n 6) Dispersed 14 (12.1) 12 (63.2) 6 (100.0) Smll cluster 65 (56.0) 7 (36.8) 0 (0.0) Sheet of cells 37 (31.9) 0 (0.0) 0 (0.0) Dispersed: scttered without ggregtes or smll colonies; Cluster: ggregtes of F blsts with 3 cells per cluster; Sheet: confluent cell collections of F blsts with 50 cells. Controls: only few F blsts were observed in six cses. ssocition between the Fb/Fc rtio nd poptosis. We found significnt increse in F blsts in the bone mrrow from ptients with MDS compred to non-mds ptients with stress erythropoiesis or helthy controls. In prticulr, nerly hlf of the MDS ptients, who hd mrkedly incresed proportions of F blsts, hd norml levels of F cells. We lso discovered n increse of poptosis predominntly in MDS ptients with n elevted Fb/Fc rtio. Similr dt on HbF expression in erythrocytes were reported previously by other investigtors, who found elevted HbF concentrtions in upto 50% of MDS ptients tested. 9,19,20 Crig et l 21 found tht the men F cell percentge in MDS ptients ws not sttisticlly different from tht in norml blood donors. In contrst, in the present study, HbF-contining erythrocytes in the bone mrrow were seven times s bundnt in ptients with MDS s in helthy controls. Mesurement of the HbF concentrtion or the F cell ssy my

4 F cell precursors nd poptosis in MDS 1481 Figure 1 Immunohistochemicl stining of bone mrrow clot sections from ptients with myelodysplstic syndromes ( d, f, nd g, originl mgnifiction 200; e nd h, originl mgnifiction 400). A number of F blsts re observed, forming cluster ( nd b) or sheet of cells (c nd d). (e) F blsts re seen predominntly in dysplstic erythroid precursors from ptient with MDS. (f) Erythroblsts in bone mrrow clot section re identified by nti-glycophorin C ntibody for the estimtion of the percentge of F blsts mong 1000 erythroblsts. (g) Prffinembedded bone mrrow clot section shows F cells stined by nti-hbf ntibody. (h) A lrge percentge (more thn 50%) of the nucleted cells is positive for nti-ssdna ntibody in representtive ptient with MDS, subtype RAEB. hve differed between the two studies. In this study, we used polyclonl nti-hbf ntibody, which we developed to confirm its chrcteriztion with high specificity nd sensitivity to fetl hemoglobin. 14 Rectivtion of HbF production in ptients with MDS is common phenomenon nd n elevted F cell count hs been reported to be possible prognostic fctor for MDS. 7,17,21 The present study is the first documenttion tht F blsts re found frequently in most ptients with MDS. We investigted the F blst levels in MDS ptients nd non-mds ptients with stress erythropoiesis. The men percentge of F blsts ws three times higher in the MDS ptients thn in the non-mds ptients; however, there were no significnt differences in the F cell levels between the two groups. Furthermore, ptients who hd elevted F blsts but no increse in F cell levels were identified in pproximtely hlf of the MDS ptients, wheres there were no such subjects in the non-mds ptients with stress erythropoiesis. These results suggest tht F cell precursors re incpble of mturing effectively to the level of circulting F cells in MDS. MDS re bone mrrow stem cell disorders chrcterized by ineffective hemtopoiesis leding to blood cytopenis despite the presence of cellulr mrrow, nd propensity towrds leukemic trnsformtion. 22 Incresed poptosis in MDS hs been proposed s possible explntion for the ineffective hemtopoiesis To investigte why enhnced F blst production is not ccompnied by n elevtion of F cells in some ptients with MDS, we exmined the reltionship between the Fb/Fc rtio nd poptosis. There were no significnt differences in the poptotic rte between the subjects with n Fb/Fc rtio 1.0 nd those with Fb/Fc rtio 1.0. However, the men vlues of the poptotic rte showed significnt differences when we compred the subjects with n Fb/Fc rtio 5.0 nd those with n Fb/Fc rtio 1.0: the poptotic rte ws three-fold higher in the subjects with n Fb/Fc rtio 5.0 thn in the subjects with n Fb/Fc 1.0. Interestingly, n increse in the Fb/Fc rtio ws prominent in the RAEB nd RAEB-t subtypes, which showed reltively high poptotic rte. These results indicte tht only the ptients with modertely incresed Fb/Fc rtio re relevnt to poptosis. Our dt

5 1482 F cell precursors nd poptosis in MDS lso suggest tht the norml percentge of F cells in the ptients with enhnced F blst production is t lest prtly due to poptotic cell deth in MDS. In this respect, the F cell ssy does not seem to reflect sufficiently the extent of fetltype erythropoiesis in MDS. We therefore believe tht F blsts should be mesured to investigte HbF production in MDS. Our results for the poptotic rte in MDS were in generl greement with the previous report of Shimzki et l 27 who found tht the poptotic rte ws higher in the MDS ptients thn in the control subjects nd ws predominnt in the erythroblsts. Severl studies hve confirmed higher poptotic rte in the bone mrrow of MDS ptients compred with controls, but these incidences vried widely between uthors. Rz et l 28 using n in situ end-lbelling (ISEL) technique to identify frgmented DNA ends generted during poptosis, demonstrted tht more thn 50% of MDS ptients hd excessive trilinege intrmedullry cell deth. In contrst, Lepelley et l 29 reported tht moderte increse in the percentge of poptotic cells (2.5 to 5%) ws observed in 15% of cses, nd ws predominnt in erythroblsts. In this study, using n immunohistochemicl ssdna ssy, which is sensitive nd specific to erly stges of poptosis, we observed significntly incresed poptosis in ptients with MDS compred with controls. It is not known whether HbF is synthesized by the bnorml clonogenic cells of the MDS or whether it represents the bility of the bone mrrow to rectivte fetl erythrocyte progenitors. 30 The higher proportion of F blsts in MDS thn in stress erythropoiesis mens tht HbF production in MDS is beyond the level imposed by fetl erythropoietic drive during stress erythropoiesis. Furthermore, the HbF production ws lrgely restricted to dysplstic erythroblsts or immture erythroid precursors, forming clusters or sheets of F blsts. Crig et l 21 demonstrted tht HbF production ws significntly higher in MDS ptients with n bnorml kryotype thn in those with norml kryotype. Koc et l 31 found tht the incresed trnscription of the lph nd gmm genes were prtly responsible for the elevtion of HbF in MDS. These results suggest tht fetl-type erythropoiesis is ssocited with the bnorml clonogenic cells in MDS. However, Reinhrdt et l 7 reported tht there is no ssocition between incresed HbF production nd kryotype bnormlities in ptients with MDS. Anlysis t the single-cell level in ptients with positive cytogenetic mrkers of the clonogenic cells will be needed to resolve this issue. In conclusion, high poptosis rte occurs in MDS ptients with n elevted Fb/Fc rtio, suggesting tht considerble number of F blsts fil to mture into functioning end-stge F cells, despite vigorous F blst production in the bone mrrow. 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