CASE REPORT

Philadelphia Chromosome-Positive Chronic MyeloidLeukemia Expressing pi90BC*-AM'

Akimichi Ohsaka, Shigeo Shiina*, Masaru Kobayashi**, Hideyuki Kudo** and Ryuji Kawaguchi**

Abstract

We describe a case of Philadelphia chromosome-posi-tive chronic myeloid leukemia (Ph-positive CML)express-ing pl90BCTAfiL. Reverse transcription-polymerase chain re-action (RT-PCR) analysis of bone marrow cells showed a472-bp band using primers specific for the pl90*c* ABLbutnot \)210PCRmABL transcript. Sequencing analysis revealed thatthe PCR product was derived from the fusion between BCRexon el and ABLexon a2 (ela2). CMLexpressing pl90*c*"ABLis relatively rare. In a review of the literature, it may begrouped into 2 categories; approximately half of the pa-tients exhibited prominent monocytosis and intermediatehematological phenotype between CMLand chronicmyelomonocytic leukemia, and the remaining patientsshowed no monocytosis.(Internal Medicine 41: 1183-1187, 2002)

Key words: minor breakpoint cluster region, el a2 transcript,acute lymphoblastic leukemia, chronic myelomo-

nocytic leukemia

Introduction

Chronic myeloid leukemia (CML) is characterized by theconsistent involvement of the Philadelphia chromosome (Ph),which is derived from a reciprocal translocation between chro-mosome 9 and 22, t(9;22)(q34;ql1). It is found in up to 95% ofCMLpatients and in 20 to 30% of adults with acute lympho-blastic leukemia (ALL) (1). This translocation results in thefusion of the 3' part of the ABLgene on chromosome 9 withthe 5' part of the BCRgene on chromosome 22. The breakpointsin the ABLgene occur usually at 5' ofABLexon a2, whereasthose in the BCRgene are clustered in three regions. In mostpatients with CMLand in approximately one-third of patientswith Ph-positive ALL, the break occurs within the majorbreakpoint cluster region (M-bcr), spanning BCRexons el2 to

el6 (originally referred to as exons bl to b5), either betweenexons el3 and el4 or el4 and el5. These breakpoints produceBCR-ABLfusion genes that transcribe chimeric messenger RNA(mRNA) with either b2a2 or b3a2 junction encoding a p2 105C7?~ABL.In the remaining patients with Ph-positive ALLand in rarecases of CML(2), the breakpoint falls upstream of the M-bcrregion, within the first intron of the BCRgene, known as theminor breakpoint cluster region (m-bcr). In these cases, BCRexon el is fused to ABLexon a2 and a chimeric mRNAwithela2 junction encoding a smaller pigQBCR-ABLjs formed. A thirdbreakpoint location in the BCR gene has recently been identi-fied downstream from the M-bcr region, defined as the microbreakpoint cluster region (|Li-bcr) (2, 3). The resulting el9a2chimeric mRNAis translated into a larger p2305C/? AfiL. It hasbeen reported to be associated with a clinical picture of neutro-philic-CML characterized by a mild form of myeloprolifera-tive disorder (3). CML expressing p i905C7M5L is relatively rare,and to our knowledge only 17 cases were previously reportedin the English literature (4-16).In this article we describe a rare CMLpatient whoshowedprominent monocytosiswith a lowneutrophil/monocyte ratioin the peripheral blood, el a2 BCR-ABLtranscript and lung can-cer.

For editorial comment, see p 1092.

Case ReportA 77-year-old man was referred to the hospital in May1997for evaluation of leukocytosis. Whenthe patient had been ad-mitted to the hospital for the treatment of diabetes mellitus inFebruary 1989, his white blood cell (WBC) count was 4.8xlO9// with a normal differential count. On admission, the patientwas afebrile and he had a palpable liver but no splenomegaly.Hematological findings were hemoglobin (Hb) 15.3 g/dl, plate-lets 353x107/ and WBC33.8x107/ with 5% myelocytes, 2%metamyelocytes, 3%band-form neutrophils, 49%segmentedneutrophils, 2% eosinophils, 3% basophils, 22% monocytes

From the Department of Transfusion Medicine, Juntendo University School of Medicine, Tokyo, *the Department of Internal Medicine, Hitachi GeneralHospital, Ibaraki and **the Genomics Research Institute, SRL, Inc., Tokyo

Received for publication February 1 , 2002; Accepted for publication May 8, 2002Reprint requests should be addressed to Dr. Akimichi Ohsaka, the Department of Transfusion Medicine, Juntendo University School of Medicine, 2- 1- 1 Hongo,

Bunkyo-ku, Tokyo 113-8421

Internal Medicine Vol. 41, No. 12 (December 2002) 1183

Ohsaka et al

and 14%lymphocytes. Cytochemical study showed that circu-lating monocytes were positive for a-naphthyl butyrate esteraseactivity. The serumconcentrations of C-reactive protein andbacterial cultures were all negative. The neutrophil alkalinephosphatase score was decreased to 111 (normal range in ourhospital is 170-285). A bone marrow aspirate showed markedhypercellularity, myeloid hyperplasia (myeloid/erythroid ratiowas 10.7) and megakaryocytic hyperplasia without an increasein blasts. Cytogenetic analysis of bone marrow cells by con-ventional G-banding showed 45,X,-Y,t(9;22)(q34;ql l) in 19of 20 metaphase cells and the remaining one cell was normal(data not shown). He was diagnosed with CMLin the chronicphase and was treated with hydroxyurea, resulting in normalleukocyte counts but persistent monocytosis (20-30%).In March 2000, the patient was admitted to the orthopedic

department of the hospital because of lumbago and was diag-nosed with lumbar spondylosis. Hematological findings wereHb 12.8 g/dl, platelets 108xl09// and WBC6.0x107/ with 2%band-form neutrophils, 45% segmented neutrophils, 8% eosi-nophils, 22% monocytes and 23% lymphocytes. Subsequentphysical examination showed a reddish skin nodule of 3 cmin

diameter in his right chest wall, which was diagnosed patho-logically as skin metastasis of adenocarcinoma. Computedto-mography of the thorax revealed multiple nodular lesions inboth lungs and bony destruction of ribs, suggesting a diagnosisof lung cancer. He died of disseminated intravascular coagula-tion and respiratory failure in April 2000. There was no evi-dence suggestive of blast crisis (BC) of CMLin the course ofthe disease.

Molecular StudiesReverse transcription-polymerase chain reaction (RT-PCR)A highly sensitive nested PCRprocedure was performed as

described previously with some modification (13). RT-PCRanalysis of bone marrowcells at diagnosis using BCRexonel3 and ABLexon a3 primers showed no amplification frag-ment (Fig. 1A). On the other hand, RT-PCRanalysis using BCRexon el and ABLexon a3 primers revealed a 472-bp band (Fig.IB), suggesting that the breakpoint in the BCRgene fell withinthe m-bcr region.

M 1 2 3 M 1 2 3

(A) |HHH (A)å HHHH

(B) HHHBHi (B> MBMB^B

A B

Figure 1. A. RT-PCRanalysis for BCR-ABLchimeric mRNA(A) using BCRexon el3 and ABLexon a3 primers, and (3-actin (B) as the internal control. Lane 1, present case; Lane 2, HL-60 leukemic cell line as the negative control; Lane 3,K562 leukemic cell line as the positive control for b3a2 fusion transcript; and M, DNAsize marker. No specific band wasamplified in Lane 1 of the patient. B. RT-PCRanalysis for BCR-ABLchimeric mRNA(A) using BCRexon el and ABLexon a3 primers, and p-actin (B) as the internal control. Lane 1, present case; Lane 2, HL-60 leukemic cell line as thenegative control; Lane 3, another patient with Ph-positive ALLcarrying ela2 fusion transcript; and M, DNAsize marker.A 472-bp band was amplified in Lane 1 of the patient.

1184 Internal Medicine Vol. 41, No. 12 (December 2002)

CMLExpressing pl90BCR'ulL

Sequencing analysisThe RT-PCRproduct was purified from agarose gel and se-

quenced using a Taq Dye Deoxyá"terminator cycle sequenc-ing system (Perkin-Elmer, Foster City, CA). Sequencing analy-

Val Val Val Ser Glu Ala Thr lie Val Giy Val Arg Lys ThrGTT GTC GTG TCC GAG GCC ACC ATC GTG GGC GTC CGC AAG ACC

Gly Gin lie Trp Pro Asn Asp Gly Glu Gly Ala Phe His GlyGGG CAG ATC TGG CCC AAC GAT GGC GAG GGC GCC TTC CAT GGA

Asp Ala Glu Ala Leu Gin Arg Pro Val Ala Ser Asp Phe GluGAC GCA GAA GCC CTT CAG CGG CCA GTA GCA TCT GAC TTT GAG

A,BCR exonl -1- ABL exon2

Pro Gin Gly Leu Ser Glu Ala Ala Arg Trp Asn Ser Lys GluCCT CAG GGT CTG AGT GAA GCC GCT CGT TGG AAC TCC AAG GAA

Figure 2. Nucleotide sequences and amino acid residues of thechimeric BCR-ABLCDNAobtained from the patient. The PCRproduct was sequenced directly using a sequencing system. Thevertical arrow indicates the breakpoint between BCRexon el

and ABLexon a2.

sis revealed that the 472-bp PCR product was derived from thefusion between BCRexon el and ABLexon a2 (ela2, Fig. 2).

Discussion

We have described a rare CMLpatient carrying ela2 BCR-ABLtranscript who showed prominent monocytosis with a lowneutrophil/monocyte ratio in the peripheral blood. The patientwas associated with lung cancer during the course of the dis-ease.

Whenhigh WBCcounts are observed, monocytosis of higherthan lxlO9//, which is a simple defining feature of chronicmyelomonocytic leukemia (CMML)(17), is common in pa-tients with chronic myeloproliferative disorder. To avoidmisclassification of CMLas CMML,it has been proposed thatwhen leukocytosis is higher than 20x 109//, the absence of 'rela-tive' monocytosis (<8%) should be a criteria for the diagnosisof CML(18). However, when leukocytosis is below 20xl09//,the absence of 'absolute' monocytosis (<lxlO9//) is necessaryfor the diagnosis of CML(1 8). In the present study, we defineda monocytosis as an increase in both absolute number (>lxlO9//) and percentage (>8%) of circulating monocytes, because

Table 1. Hematological Data and Clinical Characteristics in CMLPatients Expressing p 19O*C*-A*Lwith Monocytosis*No. Author(year) (ref. Age/ WBCMonocytes Monocytes Basophils Immature Neutrophil/ Platelets NAPPhase of Splenomegaly Additional Survival

no.) Sex (xlO9//) (%) (xlO9//) (%) granulocytes Monocyte (xlO9//) score diagnosis karyotype (months)

( % ) change1 Seller! (1990) (4) 44/F 56.8 12 6.8 2 19 5.3 510 Low CP - ->+ No 60,Alive

2 Nakamura(1993) (6) 65/M 174.4 22 38.4 4 27 1.8 NA Low AP ++ No NA3 Melo(1994) (8) 81/F 32.5 19.9 6.5 0 16 3.0 335 Low CP - -X 21,Died4 Yamaguchi(1998) (12) 44/F 52 14.5 7.5 1.5 14 3.8 340 NA BO ++ No NA5 Ohno(1998) (13) 49/M 73.9 32 23.6 1 25 1.2 88 Low CP^BO - No 18,Alive6 Ravandi (1999) (15) 43/ 128.5 16 20.6 3 NA 4.6 298 NA AP - No 92,Alive

7 Ravandi(1999) (15) 70/ 50 14 7.0 2 NA NA 305 Low CP -^BCt - No 33,Died8 Ravandi(1999) (15) 49/ 163 14 22.8 13 NA 4.8 33 Low AP ++ No 25,Died9 Ravandi(1999) (15> 76/ 251 10 2-5 8 NA 50 1124 Low CP^BC - No 104,Died

10 Presentcase 77/M 33-8 22 1A 3 7 2A 353 Low CP - "Y 35,Died

*Monocytosis was defined as an increase in both absolute number (>lxlO9//) and percentage (>8%) of peripheral blood monocytes in this study. fBiphenotypic blast crisis. WBC:white blood cells, NA: not available, NAP: neutrophil alkaline phosphatase, CP: chronic phase, AP: accelerated phase, BC: blast crisis.

Table 2. Hematological Data and Clinical Characteristics in CMLPatients Expressing pl90 BCR-ABLwithout Monocytosis*No. Author (year) (ref. Age/ WBCMonocytes Monocytes Basophils Immature Neutrophil/ Platelets NAPPhase of Splenomegaly Additional Survival

no.) Sex (xlO9//) (%) (xlO9//) (%) granulocytes Monocyte (xlO9//) score diagnosis karyotype (months)

(% ) change

1 Zaccaria(1990) (5) 77/F 63.3 4 2.5 1 38 12.5 261 Low CP - ->+ No 27,Died2 Guo(1993) (7) 52/M 180 6 10.8 13 24 8.3 187 NA CP + " No 20,Alive3 Kunieda(1994) (9) 62/M 79.6 NA NA NA NA NA 843 Low CP->BC + -Y 36,Died4 Costello(1995) (10) 32/F 4.5 6 0.3 1 27 2.5 77 NA BC* - -3,del(6)(ql6),-7 16,Alive

add(8)(pll),others5 Kirk(1996) (ll) 47/M 225 2 4.5 0 15 37.5 320 NA CP + No Died6 Solves(1999) (14) 31/F 268 0 0 1 36 NA 484 Low CP + No 108,Alive7 Ravandi(1999) (15) 55/ ll.7 1 0.1 2 NA 61 171 Low BC* - No 14,Died8 Roumier(1999) (16) 49/M 224 1+ 2.2 0 35 35 542 NA AP + inv(3)(q22q26),-7, 3,Died

-17,-22,others

*Monocytosis was defined as an increase in both absolute number (>lxlO9/Z) and percentage (>8%) of peripheral blood monocytes in this study. +The number of monocytes wasincreased to 30%in the course of the disease. *Lymphoidblast crisis. WBC:white blood cells, NA: not available, NAP:neutrophil alkaline phosphatase, CP: chronic phase, AP:accelerated phase, BC: blast crisis.

Internal Medicine Vol. 41, No. 12 (December 2002) 1185

Ohsaka et al

monocytosis is dependent on total WBCcounts.CMLwith ela2 BCR-ABLtranscript is relatively rare, andto our knowledge it has been reported previously in only 17

cases whose clinical data were available (4-16). Ten of the 18

reported cases (including the present case) showed prominentmonocytosiswith a low neutrophil/monocyte ratio in the pe-ripheral blood (Table 1), and the remaining 8 cases had nomonocytosis (Table 2). The hematological and clinical param-eters in the CMLpatients with monocytosis (n=10, group A)were compared with those in the CMLpatients without mono-cytosis (n=8, group B). Although the progression to BCin thegroup A patients was not different from that in the group Bpatients, the type of BCwas characteristic. Three of the 4 groupA patients with BC showed biphenotype, whereas 2 of the 3group B patients with BC exhibited lymphoid phenotype. There-fore, the involvement of lymphoid lineage was observed in 5of the 7 patients with BC. In addition, 2 of the 8 group B pa-tients showed additional chromosomal abnormalities except forthe loss of the sex chromosomes. No differences between the 2groups were observed in relation to other hematological andclinical characteristics. Melo et al (8) have suggested thatpigQBCR-ABL CMLmay be a specific form of CML? in which thecytomorphological characteristics are intermediate betweenCMLand CMML.However, 8 of the 18 reported cases showedthe characteristic features more commonlyfound in the classi-cal CMLwithout prominent monocytosis, indicating that CMLexpressing pi905C/? AfiL is a heterogeneous disease. It is prema-ture to claim any conclusion regarding the relationship betweengenotype and leukemia phenotype in the pl905O? ABLCML.Further studies of more cases are needed to clarify the issue.

The p l 905C/?~ABL transcript is more frequently associated withPh-positive ALLthan CML. The higher transforming abilityof pi90fia?-A5L (19) can cause an immediate transformation ofthe lymphoid progenitor cells originating from the abnormalstem cells. However, it has been shown that p\90BCRABLtran-script is frequently expressed at low levels using competitivePCRassays in p2105C7? ABLCML,although its pathogenetic sig-nificance is uncertain (20). In addition, there are sporadic casesof chronic phase CMLexpressing only pl90sc*-ABL (4-16). Al-though wehave no clear explanation whythese cases do notprogress rapidly towards ALL, the possibility is that a molecu-lar background, genetically determined, is able to block, at leastpartially, the transforming ability of pl905C/? A5L (21). Alterna-tively, three forms of BCR-ABLtranscript (p l 905C* A5L, p2 1 05C/?"ABL and p230*c/?-ABL) might have identical leukemogenic proper-ties, but their expression might be largely restricted to differ-ent hematopoietic lineages because a particular breakpoint inthe BCRgene is favored in a given lineage during the forma-tion of the Ph. The BCRintron 1 breakpoint might be frequentin B lymphoid progenitor cells but uncommonin hematopoi-etic stem cells, explaining the rarity of pWO**ABLin CML(22).Therefore, pl905C/? A5L per se can induce the myeloprolifera-tive process including granulocytic and monocytic lineages,resulting in promotion of chronic phase CML.This is supportedby experiments which used a murine bone marrow transduc-tion/transplantation model, where the three oncogenesinduced

an identical fatal CML-like disease in recipient mice, with noappreciable differences in histopathology, hematologic param-eters or disease latency (22).Another issue of this study is that the present case was asso-ciated with lung cancer during the course of the disease. Naguraet al (23) have reported multiple primary cancers associatedwith hematological malignancies in Japanese patients. Therewas no association between CMLand lung cancer in the 231cases collected (23). This suggests that the association of thetwo diseases found in our patient may be coincidental.

References

1) Thijsen SFT, Schuurhuis GJ, van Oostveen JW, Ossenkoppele GJ. Chronicmyeloid leukemia from basics to bedside. Leukemia 13: 1646-1674, 1999.

2) Melo JV. The diversity of BCR-ABLfusion proteins and their relationshipto leukemia phenotype. Blood 88: 2375-2384, 1996.

3) Pane F, Frigeri F, Sindona M, et al. Neutrophilic-chronic myeloid leuke-mia: a distinct disease with a specific molecular marker (BCR/ABLwithC3/A2 junction). Blood 88: 2410-2414, 1996.

4) Selleri L, von Lindern M, Hermans A, Meijer D, Torelli G, Grosveld G.Chronic myeloid leukemia may be associated with several bcr-abl tran-scripts including the acute lymphoid leukemia-type 7 kb transcript. Blood

75: 1146-1153, 1990.

5) Zaccaria A, Tassinari A, Testoni N, et al. Alternative BCR/ABLtranscriptsin chronic myeloid leukemia. Blood 76: 1663-1665, 1990.

6) Nakamura Y, Hirosawa S, Aoki N. Consistent involvement of the 3' halfpart of the first BCRintron in adult Philadelphia-positive leukaemia with-out M-bcr rearrangement. Br J Haematol 83: 53-57, 1993.

7) Guo JQ, Hirsch-Ginsberg CF, Xian YM, et al. Acute lymphoid leukemiamolecular phenotype in a patient with benign-phase chronic myelogenousleukemia. Hematol Pathol 7: 91-106, 1993.

8) Melo JV, Myint H, Galton DAG, Goldman JM. P19OBCRABLchronic my-eloid leukaemia: the missing link with chronic myelomonocytic leu-

kaemia? Leukemia 8: 208-21 1, 1994.9) Kunieda Y, Okabe M, Kurosawa M, Itaya T, Kakinuma M, Miyazaki T.Chronic myeloid leukemia presenting ALL-type BCR/ABLtranscript. Ann

Hematol 69: 189-193, 1994.10) Costello RT, Gabert J, Brunei V, et al. Minor breakpoint cluster region(m-BCR) positive chronic myeloid leukaemia with an acute lymphoblas-tic leukaemia onset: a case report. Br J Haematol 91: 428^30, 1995.

1 1) Kirk JA, Radich J, Edmands S, et al. Unusual expression ofmRNAtypi-cal of Philadelphia positive acute lymphoblastic leukemia detected inchronic myeloid leukemia. AmJ Hematol 52: 129-134, 1996.

12) Yamaguchi H, Inokuchi K, Shinohara T, Dan K. Extramedullary presen-tation of chronic myelogenous leukemia with p i 90 BCR/ABLtranscripts.Cancer Genet Cytogenet 102: 74-77, 1998.

1 3) Ohno T, Hada S, Sugiyama T, Mizumoto T, Furukawa H, Nagai K. Chronicmyeloid leukemia with minor-bcr breakpoint developed hybrid type ofblast crisis. AmJ Hematol 57: 320-325, 1998.

14) Solves P, Bolufer P, Lopez JA, et al. Chronic myeloid leukemia with ex-pression of ALL-typeBCR/ABLtranscript: a case-report and review ofthe literature. Leuk Res 23: 851-854, 1999.

15) Ravandi F, Cortes J, Albitar M, et al. Chronic myelogenous leukaemiawith pl85BC/?MfiL expression: characteristics and clinical significance. Br J

Haematol 107: 581-586, 1999.16) Roumier C, Daudignon A, Soenen V, et al. pl90 bcr-abl rearrangement: a

secondary cytogenetic event in somechronic myeloid disorders?Haematologica 84: 1075-1080, 1999.

17) Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the classifica-tion of the myelodysplastic syndromes. Br J Haematol 51: 189-199, 1982.

18) Kantarjian HM, Kurzrock R, Talpaz M. Philadelphia chromosome-nega-tive chronic myelogenous leukemia and chronic myelomonocyticleuke-

mia. Hematol Oncol Clin North Am 4: 389-404, 1990.

1186Internal Medicine Vol. 41, No. 12 (December 2002)

CMLExpressing v\ 9Qbcr-abl

19) McLaughlin J, Chianese E, Witte ON. Alternative forms of the BCR-ABLoncogene have quantitatively different potencies for stimulation of im-mature lymphoid cells. Mol Cell Biol 9: 1866-1874, 1989.

20) van Rhee F, Hochhaus A, Lin F, Melo JV, Goldman JM, Cross NCR pl90BCR-ABLmRNAis expressed at low levels in p210-positive chronicmyeloid and acute lymphoblastic leukemias. Blood 87: 5213-5217, 1996.

21) Saglio G, Pane F, Martinelli G, Guerrasio A. BCR/ABL transcripts andleukemia phenotype: an unsolved puzzle. Leuk Lymphoma26: 281-286,

1997.

22) Li S, Ilaria RLJr, Million RP, Daley GQ, Van Etten RA. The P190, P210,and P230 forms of the BCR/ABLoncogene induce a similar chronic my-eloid leukemia-like syndrome in mice but have different lymphoid leuke-mogenic activity. J Exp Med 189: 1399-1412, 1999.

23) Nagura E, Kawashima K, Yamada K. Multiple primary cancers associ-ated with hematological malignancies. Jpn J Clin Oncol 15 Suppl 1: 21 1-

222,1985.

Internal Medicine Vol. 41, No. 12 (December 2002) 1187