Philadelphia Chromosome inEosinophilic Leukemia*

HANS GRUENWALD M .D . . KOSMAS A . KIOSSOGLOU, M.D., `'V . J. MITUS, M .D . andWILLIAM DAMESHEK, M .D .

Boston, Massachusetts

E OSINOPIIILIC leukemia is a rare disorder .Dittrich [1] in 1952 found over 100 cases

reported by various investigators, but the ma-jority of these were discovered to have beenincompletely studied or were unacceptable whenrigid criteria were used [2a,3,4] . In a recentreview [5] only some twenty cases could beaccepted as well documented examples ofeosinophilic leukemia . The difficulty lies in thedifferential diagnosis from such disorders asparasitic infestation, hypersensitivity angiitis,Hodgkin's disease, tropical and familial eosino-philia, and various allergic disorders, all ofwhich may exhibit an eosinophilic leukemoidreaction [21 . In the past the existence of a trueeosinophilic leukemia was often denied [6,7],but it is now generally accepted as a clinicalentity [5,8] .

Whether eosinophilic leukemia should beconsidered a variant of chronic granulocyticleukemia or a completely separate entity is atthe moment controversial [2] . In favor of theformer interpretation are certain findings in theperipheral blood and bone marrow similar tothose of the usual "myelogenous" leukemia, i .e .,basophilia and eosinophilia, the difference beingmainly quantitative . That eosinophilic leukemiais a separate entity is indicated perhaps by itsusually much shorter course and its own peculiarmanifestations such as transient pulmonaryinfiltrates and myocardial involvement fre-quently leading to the development of muralthrombi [5] .

Recently we studied a patient with eosinophilicleukemia in whom the clinical and more par-ticularly the cytogenetic findings seemed toindicate a close relationship of the eosinophilic

form of chronic granulocytic leukemia to thetype more usually seen .

CASE REPORT

R. Y. (NECH 160-019), a forty-seven year oldwhite bakery supervisor, noted the sudden appear-ance of pain and swelling in his right leg on October24, 1963 ; a deep thrombophlebitis was diagnosed,and the condition cleared after two weeks of bed rest,applications of local heat, and heparin therapy. Ablood count at that time revealed a leukocyte count17,600 per cu. mm. with 36 per cent eosinophils ;subsequent blood counts showed persistent leuko-cytosis with marked eosinophilia, a shift to the leftof the eosinophilic series and an inconstant increasein basophils . In November 1963 the splenic tip be-came palpable. At this time night sweats and lowgrade fever were occasionally recorded ; the patientwas referred to us for further evaluation on Novem-ber 16, 1963 . He had lost 20 pounds in a period ofeight months .

The past history was essentially noncontributory .The patient denied having had allergies, respiratorysymptoms, gastrointestinal disturbances or musclepain . One brother had died from Hodgkin's disease atthe age of thirty-five ; otherwise the family historywas not significant .

Physical examination on admission revealed a welldeveloped man whose nutritional status was good .The temperature, pulse and blood pressure werenormal. The skin and mucosas were slightly pale .The abdomen was soft and nontender ; the liver edgewas palpable at the right costal margin ; a soft,smooth and nontender spleen was palpable 5 cm .under the left costal margin . No other abdominalmasses were felt. The extremities were symmetric .

On admission the hemoglobin value was 9.8 gm .per cent with a hematocrit of 35 per cent ; the redblood cell count was 3,130,000 per cu . mm ., reticulo-cytes 5.2 per cent . The platelet count was 513,420

* From the Blood Research Laboratory, Pratt Clinic-New England Center Hospital and the Department ofMedicine, Tufts University School of Medicine, Boston, Massachusetts . This investigation was supported with fundsfrom U. S. Public Health Service Grant CA-04168-06 from the National Cancer Institute . Manuscript receivedApril 9, 1965 .

VOL . 39, DECEMBER 1965

1003

1004 Eosinophilic Leukemia-Gruenwald el al .

Fm. 1 . Peripheral blood smear showing a basophil and an eosinophil with prominent basophilic granules .

Fie . 2 . Bone marrow aspirate with marked eosinophilia .

AMERICAN JOURNAL OP MEDICINE

per cu . mm . (by indirect method) . The white bloodcell count was 38,100 per cu . mm. with neutrophilicpolymorphonuclcars 12 per cent, neutrophilic bandforms 2 per cent, eosinophils 50 per cent (some withbasophilic granulations), basophils 23 per cent,lymphocytes 13 per cent. (Fig . 1 .) The erythrocytesedimentation rate was 104 mm . per hour (Wester-gren). The fasting blood sugar was 100 mg. per cent,the 24 hour postprandial blood sugar was 210 mg .per cent. Glucose tolerance test revealed a 1 hourlevel of 195 mg. per cent . The blood urea nitrogenwas 12 mg. per cent, creatinine 0 .9 mg . per cent anduric acid 6.5 mg. per cent . Serum electrolytes werenormal. The serum alkaline phosphatase was 2 .3Bodansky units, the serum total proteins were 5 .9 gm .per cent with albumin 3 .4 gm . per cent and globulin2.5 gm . per cent . Urinalysis was essentially negative .Thymol turbidity was 3 .0 units, the cephalin floccula-tion test result 4+ ; there was no bromsulfalein re-tention in 45 minutes, the serum glutamic oxalacetictransaminasc, serum glutamic pyruvic transaminaseand lactic dchydrogenase determinations revealed noabnormalities . Serum iron was 73 pg. per cent ; theunbound iron-binding capacity was 132 pg. per cent .Three stool examinations were negativee for ova andparasites . Urine cultures yielded no growth . The re-sults of the 'Irichina precipitin test and skin test werenegative .

The bone marrow was extremely hypercellular("packed"), with increases in both myeloid and ery-throid elements ; there was a marked increase ineosinophils and basophils with a shift to the left ofthese elements . Many erythroid elements which pre-sented megaloblastic features also were present to-gether with an increased number of crythrogones .(Fig. 2.) The myeloid :erythroid ratio was 2 .4 :1 .A differential count of 500 cells revealed thefollowing : myeloblasts 2 .4 per cent, promyelocytes 2per cent, eosinophilic promyelocytes 1 .6 per cent,eosinopllic myelocytes 18 .4 per cent, eosinophilicsegmented band forms 8 per cent, basophils 4 percent, neutrophilic myelocytes 10 per cent, ncutro .philic metatnyelocytes 6 .8 per cent, neutrophilicband forms 6 per cent, neutrophilic segmented bandforms 5 per cent, lymphocytes 5 .8 per cent, reticulumcells 1 .6 per cent, plasma cells 0.8 per cent, mega-karyocytes 0 .8 per cent, erythrogones (pronormo-blast) 4 .4 per cent, normoblast A (basophilic) 2 .4 percent, normoblast B (polychromatic) 8 .4 per cent,normoblast C (orthochromatic) 11 .6 per cent . Thealkaline phosphatase score of the mature neutrophilicgranulocytes of the blood was 25 (normal, 16 to 55) .

Needle biopsy of the liver showed a moderateeosinophilic leukocytic infiltration of the portal areas,without significant alteration of architecture, and arare parenchymal focus of extramedullary myelo-poiesis with many immature eosinophilic elements .The over-all pattern was considered to be consistentwith a leukemic process . Splenic aspiration showed

VoL . 39, DFOF.MnER 1965

Eosinophilic Leukemia-Gruenrx ald et al . 1005

marked infiltration by eosinophilic granulocytes,many of them immature .

The results of a Coombs' test were negative. Theradioactive chromium (Cru) autosurvival of the redcells showed a half-life of 18 .4 days, a mean cell lifeof forty-four days . The plasma radioactive iron (Fens)clearance had a half-life of 43 minutes ; the iron turn-over was 48.5 mg . per day. The incorporation of Fe59into red cells was decreased, with a maximum of 41per cent at seven days .

The patient remained essentially asymptomaticwhile in the hospital, apart from one temperatureelevation to 100 .6° F ., which subsided spontaneously .He was discharged on November 23, 1963 . No specifictherapy was recommended but, because of persistentfever and the recurrence of the thrombophlebitis,administration of busulfan (Myleran 0. ), 8 mg. per day,was started on December 4 ; on December 16 theuse of anticoagulants was resumed, with improvementin the thrombophlebitis of the leg . (Fig. 3 .)

On January 10, 1964, the patient was admittedagain to the local hospital because of increasing weak-ness, malaise, fever, weight loss, spontaneous bruisingand bleeding gums . Examination revealed a debili-tated and seriously ill man with widespread ecchy-moses. The pharynx presented patches of thick whiteexudate . A few small axillary and inguinal nodes werepalpable. 'Fhe spleen was massively enlarged, reach-ing to the iliac crest, and was moderately tender ; theliver was nontender and palpable 4 cm . below theright costal margin . The extremities appearednormal .

At this time the hemoglobin was 7 .6 gm . per cent,the white blood cell count 10,500 per cu . mm., andonly 26,000 platelets per cu . mm. Were present.Throat cultures grew Candida albicans .

The administration of anticoagulants and Myleranwas discontinued, and the patient was given vitaminK and repeated transfusions of fresh blood for controlof bleeding ; in addition, the administration of dexa-methasone, 12 mg . per day, was started . This regimenwas followed by a decrease in the fever and malaise ;the thrush was treated with nystatin (Mycostatin ® )gargles. On January 22 a course of radiotherapy tothe spleen was begun (eight applications of 150 reach), but the patient complained of left-sided ab-dominal discomfort, and on January 30 a splenicfriction rub was observed. He continued to bruisespontaneously and required numerous transfusionsof fresh blood . On February 17 the blood countshowed 21 per cent blasts ; a bone marrow aspirationrevealed practically complete replacement by myelo-blasts . He continued on a rapidly downhill course anddied on February 27, 1964 . Postmortem examinationwas not performed .

Of special interest were the results of bone marrowchromosomal studies, performed according to themethod described previously [9j . The combined Den-ver [101 and Patau [171 system of nomenclature was

1006 Eosinophilic Leukemia-Gruenwald et al .

TRANSFUSIONS

15-

10-

5-

450,000-

300,000 -

150,000 -

60,000

40,000 -

20,000-

BUSULFAN

4Mg.

DEXAMETHASONE 1 6Mg .

x-RAY

150

SPLEEN (A)

HEMOGLOBIN (Gm.%)

PLATELETS/ mm3

• = wec (TOTAL) /mm3

4 • EOSINOPHILS /mm3

• • BASOPHILS /mm3

O • MYELOBLASTS / mm3

NOV, I

DEC.

I

JAN .

I

FEB .

FIG . 3 . Graphic representation of clinical course .

used. In all, seventeen and forty-seven metaphase than forty-four to forty-seven on the second . Theplates were counted on two respective occasions and predominant cell line on the first occasion had forty-photomicrographed . (Table I.) six chromosomes. The Philadelphia (Ph 1) chromo-

The number of chromosomes ranged from forty- some could be demonstrated in fifteen of seventeenfive to forty-six on the first occasion and from less

cells counted (88 .2 per cent) . On the second specimen

TABLE ICHROMOSOMAL DISTRIBUTION

R .Y

22

12

2

1

1

11111

111

AMERICAN JOURNAL OF MEDICINE

Data MetaphasePlates

Chromosomes (no.)Ph 1

Chromosome<44 44 45 46 47

11/16/63Noo counted 17 0 0 2 15 0 15Per cent 100 0 0 11 .8 88 .2 2 .0 88 .211/21/63No. counted 47 1 1 2 19

24 43

Per cent 100 2 .1 2.1 4.3 40 .4

51 .1 91 .5

Eosinophilic Leukemia-Gneenttald et al .

obtained five days later, t,co stern lines sere present,one ~rith fore--six chromosomes and the other withlorry-seven . 13oth of these lines were positive for thePh 1 chromosome . The new line (forty-seven (hromo-somes), in addition to the Ph 1 chromosome, had anextra chromosome in the C6-X-12 series. The Ph 1chromosome could be identified in fort•- three offorty-seven (cIts counted (91 . 3 per cent) .

Karyotypes wire obtained (6 and 9 cells, re-spectively, on the two occasions) . (Table rt and Fig. 4and 5 .) A structural aberration in the form of a chro-matid break 'vas present . In addition, chromosomeswith blurred and fuzzy appearance were seen occa-sionally .

COMMENTS

The Ph 1 chromosome is known to he presentin almost every case of chronic granulocyticleukemia 112] when direct bone marrowpreparations are used for determina'.ion. Previ-ous cytogenetic studies in six cases described aseosinophilic leukemia 112--141 failed to revealthe presence of the Ph 1 chromosome, but thediagnosis in many of them was eduivocal .

The finding of the Ph 1 chromosome in thebone morrow cells of the patient here describedis of interest because it lends strong support tothe view that this disorder is a variant of chronicgranulocytic leukemia . The increase in basophilstends to support the contention that eosinophilicleukemia represents one of the manifestations ofa chronic neoplastic proliferation of the granulo-cyticc series ; in the classic cases of chronic granu-locytic leukemia some degree of basophilia andeosinophilic is commonly found [2,8] .

The level of leukocyte alkaline phosphataseis usually very low in chronic granulocyticleukemia 115, Id 1 . The possibility was broachedat the time that the low leukocyte alkalinephosphatase level was related to the presence ofthe Ph 1 chromosome ; however, although thisassociation is very common, it is not invariable,there being occasio tat cases with the Ph 1chromosome and no-mal or high levels ofleukocyte alkaline phosphatase [17,18] . In thecase described herein, a normal level of leukocytealkaline phosphatase was found in the presenceof the Ph 1 chromosome . As the leukocytealkaline phosphatase is measured in matureneutrophilic granulocytes (and not in eosino-phils), it might be argued that the neutrophilicseries was not affected because eosinophilicleukemia is a separate entity unrelated to chronicgranulocvtic leukemia. However, it is unlikelythat the Ph 1 chromosome is limited to one type

voL. 39, nl[CEMBER 1965

TABLE zrCHROMOsOME ANAL%Sc,

1007

of granulocyte only, for it has been well docu-mented not only in the myeloid cells but also innucleated erythroid cells and probably in mega-karyocytes 1791 .

Chromosomes were studied in our patient ontwo occasions . The Ph 1 chromosome was foundeach time, but on the second occasion additionalchromosomal changes such as hyperdiploidywith an extra chromosome in the C series werefound. As hyperdiploidy was present in well over50 per cent of the cells analyzed on the secondoccasion, it is unlikely that such an abnormalitywas missed by chance on the first examination .A more likely possibility is that the chromosomalchanges encountered on the second examinationwere related to the clinical and morphologicpicture of acute leukemia ("blast crisis") thatdeveloped three months later . These may bethought of as the first objective manifestation inthis case of a higher degree of malignancy, sinceextra chromosomes in the C series have beenoccasionally associated with acute leukemiaand the blast crisis of chronic granulocyticleukemia [20] .

The development of the blast crisis in thispatient is a further indication of the close rela-tionship of eosinophilic leukemia to the muchmore common neutrophilic type of chronicgranulocytic leukemia . The course throughout,with increasing splenomegaly, asthenia andanemia ; the leukocytosis with the characteristicchromosomal abnormality ; the development ofa terminal blast crisis, is typical of the classicchronic (neutrophilic) granulocytic leukemia .

No. of

Chromo-C7Clls

somcs

FindingsKaryotyped

(no.)

11/16/634 . 46

All Ph 1 positive1 45

Ph I positive ; E 17 mono-somy

1 45

Pit 1 negative ; G-Y mono-soiny

I11/21/63

5 47 All Ph 1 positive ; an extrachromosome in the Go-X-12 series

3 46 All Ph 1 positive; onechrotnatid break

45 Ph 1 positive : G-Y mono-Sony

1008 Eosinophilic Leukemia-Gruenzeald et al .

Ftc . 4 . 13osinophilie leukemia . Top, metaphase plate containing forty-six chromosomes . Bottom, knryotypo of thesame cell with the Ph 1 chromosome .

AMERICAN JOURNAL O` MrDIC1NId

A1

Eosinophilic Leukemia -Gruenwald et al .

1009

A2 A3 B4 B5

Fm. 5 . Eosinophilic leukemia . Top, metaphase plate containing forty-seven chromosomes. Bottom, karyotype of thesame cell with the Ph 1 chromosome and an extra chromosome in the C6-X-12 series (C trisomy) .

VOL, 39, DECEMBER 1965

1010

On the other hand, the rather rapid course, themarked eosinophilia of the blood and honemarrow, the normal leukocytic alkaline phos-phatase, arc sufficiently different from the classiccase to warrant the designation of chronic eosin-ophilic granulocytic leukemia, i .e ., chroniceosinophiic leukemia .

SUMMARY

Clinical and cytogenetic findings in a patientwith eosinophilic leukemia are presented . ThePhiladelphia (Ph 1) chromosome was found in88 .2 per cent of bone marrow cells studied bythe direct method . The leukocyte alkaline phos-phatase score was 25 (normal, 16 to 55) . In thesecond bone marrow specimen, obtained fivedays later, in addition to the Ph 1 chromosome(91 .5 per cent), hyperdiploidy of forty-sevenchromosomes (51 .1 per cent) with an extrachromosome in the C6-X-12 series (C trisomy)was also observed . The patient died in "blastcrisis" three months later . The theoretical im-plications of the cytogenetic findings and theirsignificance are briefly discussed . It is concludedthat eosinophilic leukemia may in some cases bea variant of the much more common (neutro-philic) form of chronic granulocytic leukemia .

Acknowledgment : We wish to thank Dr . JohnGardner of New Bedford, Massachusetts, forreferring this patient and for his help and co-operation in the study by providing us withessential information .

REFERENCES

1 . DIETRICH, H. Zur Frage der eosinophilen Leukamie .Acta haemat ., 7 : 230, 1952 .

2. (a) DAMESHEK, W . and GCNZ, F . Leukemia . 2nd

Eosinophilic Leukemia-Gruen¢t'ald el al .

and KRIvrr, W . Eosinophilic leukemia . Am. J.Med ., 30 : 310, 1961 .

6. NAEGELI . O . Blutkrankheiten and Blut Diagnostik .Berlin, 1931 . Springer-Verlag . Cited by Belfiore,F. Contributo clinico alla conoscenza delta leuee-mia eosinofila . Halmatologica, 49 : 273, 1964 .

7. Di GCGLIELMO, G . Cited by Belfiorc, F . Contributoclinico alla conoscenza delta leucemia cosinofila.Haematologica, 49 : 273, 1964 .

8. PINEY, A . La leuc€mia a eosinophiles existe-t-elle'Rev. himat ., 4: 3, 1949 .

9 . Ksossocrou, K . A., Mires, W . J . and DAMESHEK,W. A direct method for chromosome studies ofhuman bone marrow . Am . J. Clin . Path ., 41 : 183,1964.

10. A proposed standard system of nomenclature ofhuman mitotic chromosomes . Lancet, 1 : 1063,1960 .

11. PATAU, K. Chromosome identification and theDenver report . Lancet, 1 : 933, 1961 .

12. NOWELL, P. C. and HUNCERFORD, D . A. Chromo-some changes in human leukemia and a tentativeassessment of their significance . Ann . New YorkAced. Sc ., 113 : 654, 1964 .

13 . KRAUSS, S ., SOKAL. J . E. and SANDBERG, A. A.Comparison of Philadelphia chromosome-positiveand negative patients with chronic myelocyticleukemia . Ann . Int . Med ., 61 : 625, 1964 .

14. GOH, K ., SWISHER, S. N. and ROSENBERG, C. A.Cytogenctic studies in eosinophilic leukemia . Therelationship of cosinophilic leukemia and chronicmyelocytic leukemia . Ann . Int . Med ., 62 : 80, 1965 .

15. VALENTINE, W . N., BECK, W . S ., FOLLETTE, J . H .,MILLS, 11. and LAWRENCE, J . S . Biochemicalstudies in chronic nryelocytic leukemia, poly-cythemia vera and other idiopathic myeloproli£-erative disorders . Blood. 7 : 959, 1952 .

16. MITeS, W. J., BERCNA, L . J ., MEDNICOFF, 1. B. andDAMESHEK, W. Alkaline phosphatase of matureneutrophils in chronic forms of the myeloprolifera-tive syndrome . Am . .1. Clin . Path ., 30 : 285, 1958 .

17. BOWEN, P. and LEE, C . S . N . Ph 1 chromosome inthe diagnosis of chronic myeloid leukemia : Re-port of a case with features stimulating myelofi-brosis . Bull . Johns Hopkins Hosp ., 113 : 1, 1963 .

18. HEATH, C. W., JR. and MOLONEY, W . C. ThePhiladelphia chromosome in an unusual case ofmyeloproli£erative disease . Blood, in press .

AMERICAN JOURNAL OF MEDICINE

ed., p . 226, New York, 1964 . Grune Stratton, Inc .(b) Ibid„ pp . 221-222 .

19. WHANO, J ., FREI, in, E ., Tpo, J. H., CARBONE,P. P. and BRECHER, G . The distribution of the

3. PAPACEORCtOU, A . Zoe Differentialdiagnose dereosinophilen Leukamie . Blut, 8 : 338, 1962 .

4. EvANS, T. S. and NESBIT, R . R. Eosinophilic leu- 20 .

Philadelphia chromosome in patients with chronicmyelogenous leukemia . Blood, 22 : 664, 1963 .

KsossocLOU, K . A ., MITUS, W . J. and DAMESHEK,keinia . Blood, 4 : 603, 1949 .

S . BENTLEY, H . P ., REARDON, A . E ., KNOEDLER, J . P .W. Chromosomal aberrations in acute leukemia .Blood, in press .