young adult guinea pigs of strain 2 inbred line. This expenimental model appeared to be suitable for this study, particularly in view of the fact that leukemia induced in theseanimals is consistently accompanied by a generalized bloodinvolvement with peripheral WBC frequently exceeding300,000/cu mm in the terminal phase of the disease.
Accordingly, a study was undertaken to determine theincidence, extent, and area of cardiac involvement by leukemic cel! infiltration and the corresponding electrographicchanges in strain 2 guinea pigs with generalized leukemiainduced by inoculation of leukemic L2C cell suspensions.
MATERIALS AND METHODS
Animals. Strain 2 guinea pigs originally obtained fromHorton's Laboratory Animals Inc. in Los Gatos, Calif. , in1968 have been bred by brother-to-sister mating in ourlaboratory since that time. Young healthy animals from ourcolony of this inbred line have been used in this study. Theguinea pigs used in our study were 8 to 10 weeks old at theonset of the experiments and weighed about 350 to 500 geach.
L2C Strain of Luekemia. The L2C leukemia strain had itsorigin in a case of spontaneous leukemia that developed inone of the guinea pigs bred at the National Cancer Institutein Bethesda, Md., over 20 years ago (5) and has beentransplanted by cell graft, since that time, in young guineapigs of the same line. We obtained this leukemic strain in1968 from Dr. C. W. Jungeblut, who was then at Lenox HillHospital in New York City, and we have carried this Ieukemic strain by cell transfer in strain 2 guinea pigs in ourlaboratory (13).
Preparation of Leukemic Cell Suspensions. A guinea pigwith advanced leukemia, induced with a L2C leukemic cellinoculation, was sacrificed by ether inhalation. After theskin was shaved, the abdominal cavity was exposed. Fragments of spleen, mesentemic tumor, and in some instancesfragments of a peripheral lymph node also were removedaseptically, weighed, cut with scissors, and ground in amortar with sufficient sterile 0.9% NaCI solution added toobtain a cell suspension of 10% concentration. The cellsuspension was then passed through a sterile voile clothand serial dilutions were prepared. In most instances 0.5 mlof a 102 dilution was inoculated s.c.; in afew instances 10@or 10@ dilutions were used. All inoculated animals developed leukemia.
1998 CANCERRESEARCHVOL. 36
Electrocardiographic and Pathological Studies of the Heartin Experimental Guinea Pig Leukemia
Dino A. Belletti, Ludwik Gross,' Yolande Dreyfuss, Theodore Ehrenreich, Dorothy Feldman, and Lorraine A.Moore
Cancer Research unit, Veterans Administration Hospital, Bronx, New York 10468 (D. A. B. , L. G. , V. D. , T. E., D. F., L. A. MI, and Hoffman-La Roche Inc.,Department of Experimental Pathology, Nutley, New Jersey 07110 ID. F.)
SUMMARY
Electrocardiographic tracings were performed on 26strain 2 guinea pigs in which leukemia was induced byinoculation of L2C Ieukemic cells. All inoculated animalsdeveloped leukemia. In terminal phases of this disease significant electrocardiographic changes were observed in 20of the 26 animals; in 5 animals the electrocardiograms werenormal and in 1 guinea pig the changes were borderline.The most significant changes consisted of the onset of a Qwave or a T-wave inversion or both. Pathological examination of the heart removed in the terminal phase of thedisease revealed infiltration of the endocardium and epicardium and in the capillaries of the myocardium. Such areasof infiltration, when viewed with the electron microscope,revealed the presence of leukemic cells within the lumen ofcapillaries and in areas immediately surrounding the capillanes. Infiltration of myocardial fibers was not observed.The electrocardiographic changes observed in guinea pigleukemia may be related to the leukemic infiltration of myocardial capillaries and the resulting anoxia.
INTRODUCTION
Abnormal cardiac function has been observed frequentlyin patients with leukemia. The cause of abnormal functionmay be due to several factors, such as leukemic infiltrationof the heart (8, 15, 17), hemorrhage in its wall (14, 17),hypoxia resulting from severe anemia (10, 16), leukemicpericarditis (2, 4, 16, 22), heart block (3, 6, 7, 16), etc. Thefrequency of clinical signs of cardiac involvement, including electrocardiographic changes in patients with leukemia,has been reported to vary from 14 to 37% (1, 6, 8, 11, 12, 14,17, 18, 21).
The purpose of this study was to determine whether electrocardiographic changes were present in animals with experimentally induced leukemia. We had a choice in thislaboratory to study leukemia induced in either mice, rats, orguinea pigs. The guinea pig was chosen because the relatively large size of this animal makes it easier to carry outthe electrocardiographic tracings. We have at our disposala leukemic strain (L2C) that induces stem-cell leukemiafollowing inoculation of leukemic cell suspensions into
I To whom requests for reprints should be sent, at Veterans Administra
tion Hospital, 130 West Kingsbridge Road, Bronx, N. Y. 10468.Received November 4, 1975; accepted February 16, 1976.
Electrocardiographic Tracings. Electrocardiograms weretaken of 7 young healthy guinea pigs as an initial preliminary study; these animals were not used for further expenimentation. Prior to inoculation, electrocardiograms weretaken of another group consisting of 12 animals. Followinga base-line electrocardiogram, each of the 12 guinea pigs inthe 2nd group received s.c. inoculations of leukemic cells.At 2- to 3-day intervals, a 7-lead electrocardiogram, WBC,and differential blood cell examination were made until theterminal phase of the induced disease was reached (Table1). Finally, in the 3rd group, consisting of 14 animals, noelectrocardiograms were taken prior to inoculation; these14 animals received s.c. inoculations of leukemic cells. Allinoculated animals developed leukemia within 2 to 3 weeksafter inoculation and were sacrificed at the terminal stage ofthe disease, after a time interval varying from 15 to 44 daysafter inoculation. Electrocardiograms were taken in animalsof this group only at the terminal phase of the disease (Table2).
The electrocardiograms2 were taken under light ether anesthesia. In each guinea pig 6 limb leads and 1 precordiallead were taken as a base line (Fig. 3). The 1 precordial leadcorresponds to a point in between the V1 and V2 humanchest lead. The site in the guinea pig was usually at thelower end of the sternum in the center. This point wasmarked with indelible ink, enabling us to apply the chestelectrode at the same site during subsequent electrocardiograms.
The electrocardiograms were considered abnormal if asignificant T-wave change or appearance of a significant Qwave appeared as compared with the original base-linestudy. Since the normal variations in the electrocamdiogmams of guinea pigs have not been established, onlysignificant changes in the electrocardiograms were considered abnormal. The electrocardiograms were analyzed forheart rate, PR interval duration, QRS axis, the presence orabsence of Q-wave, and whether the T-wave was upright orinverted.
Pathological Examination. At autopsy, the hearts wereremoved and fixed in Bouin's solution. Multiple serial homizontal sections were made, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The degreeand localization of the leukemic cell infiltration of the myocardium, epicardium, and endocardium were studied withlight microscopy.
Electron Microscopy. Specimens were fixed in 4% phosphate-buffered glutamaldehyde followed by 1% phosphatebuffered osmic acid. They were then processed as previously described (9). Tissue blocks were sectioned with aPortem-Blum microtome using a diamond knife. Sectionswere carbon coated , stained with umanylacetate and leadcitrate (20), and examined in a Philips 300 electron microscope.
RESULTS
Electrocardiographic Tracings in Normal Guinea Pigs
The heart matein normal young healthy guinea pigs variedfrom 260 to 320 beats/mm. PR interval varied from 0.05 to0.08 sec. The QRS duration varied from 0.02 to 0.03 sec.The QRS electrical axis varied from —10to 100°.No Qwaves were noted in Leads 1, 2, and 3 or in Lead AVF. In 1guinea pig a 0-wave was noted in Lead V,; this was considemedto be a normal variation. The T-wave was upright inLeads 1, 2, and 3 and either upright or inverted in precordialLead V,. No ST changes were noted in the electrocardiograms of normal healthy guinea pigs (Fig. 1).
Electrocardiographic Tracings in Leukemic Guinea Pigs
In the 1st group consisting of 12 guinea pigs, electrocardiograms were taken at 2- to 3-day intervals following inoculation with Ieukemic cell suspensions. The 1st electrocamdiographic changes occurred 14 days after inoculation;they became more frequent as the disease progressed(Figs. 2 and 2a). In this group, 4 guinea pigs developedsignificant 0-wave and T-wave changes, 4 other guinea pigsdeveloped only T-wave changes, and 1 guinea pig developed a flat T-wave in Lead 1. The remaining guinea pigs hadnormal electrocardiograms (Table 1).
In the 2nd group, 14 guinea pigs were inoculated withleukemic cells suspensions; in this group electrocardiograms were taken only in the terminal phase of the disease. Twelve guinea pigs in this group had abnormal electrocardiograms, described in Table 2, and 2 guinea pigs hadnormal electrocardiograms.
All 12 animals in the 1st group and 14 animals in the 2ndgroup, including those that had normal electrocardiogmams,had extensive leukemic cell infiltration of the endocardium and epicardium, revealed on pathological examination after they were sacrificed in terminal phase of thedisease.
In summary, of 26 guinea pigs with generalized leukemiaexamined in terminal phase of leukemia, 20 (77%) had abnormal electrocardiograms, 1 had a borderline electrocardiogram, and 5 had normal electrocardiograms.
Pathological Findings
General Description. The hearts were not appreciablyenlarged or deformed. The principal site of leukemic infiltration was at the base of the heart. Small, dark brown, 1- to2-mm lesions were noted on the surfaces of the aunicles andthe great vessels at their junction with the heart. In manyinstances, the small lymph nodes adjacent to the greatvessels were enlarged. Small punctate lesions, appearing tobe leukemic infiltrations, were seen on the epicardium andendocardium of the right and left ventricles. There were noother anatomic sites of leukemic involvement. Pemicardialcavity effusion or leukemic infiltration of the penicardial sacwere not noted. Hemorrhage was not observed in any of theanatomic sites in the heart.
2Theelectrocardiographictermsusedare: L, Lead1; L2,Lead2; L:1,Lead 3; AVR, augmented chest lead, right upper limb; AVL, augmentedchest lead, left upper limb; AVF, augmented chest lead, both lower limbs;VI, unipolar precordial Lead 1; V2, unipolar precordial Lead 2; Q, R, 5, T,
standard wave designations in electrocardiographic tracings; Inv. T, invertedT-wave.
E!ectrocardiographic changes in advancedguinea pig leukemia
‘IElectrocardiograms taken in terminal phase of leukemia immediately prior to sacrifice of the animals.
b Blood taken from earlobe.
Light Microscopy. Leukemic cells in clusters and occasionally in large sheets were noted in the lesions of theepicardium at the base of the heart and around the greatvessels (Figs. 4 and 5). Lymph nodes often showed extensive leukemic invasion. In a few animals, the lymph nodes
were completely replaced by leukemic cells (Fig. 4) andcould represent the source of the leukemic infiltration of thebase of the heart. Leukemic infiltration in the other anatomic sites of the heart described above was relativelyslight. Both ventricles were involved; there were small clus
2000 CANCERRESEARCHVOL. 36
a Electrocardiogram taken in terminal phase of leukemia immediately prior to sacrifice of the animals. All guinea pigs in this group alsohadelectrocardiogramstaken when in good health, before leukemic cell inoculation and all had normal electrocardiogramsat that time.
b Blood taken from earlobe.
Table 2Electrocardiographic changes in advancedguinea pig leukemia
tens of leukemic cells infiltrating the epicardium and endocardium. These leukemic deposits were generally small butoccasionally they infringed on the underlying myocardium(Fig. 6). Infiltration of the myocardium appeared to resultfrom direct extension of the epicardial or endocardial infiltration. However, in appropriately thin histological sectionsand on high power, the leukemic cells were found to be indilated capillaries situated between myocardial fibers(Fig. 7). Thus, the leukemic cells appeared to be separatedfrom the myofibrils by capillary endothelium and only anoccasional cell was noted outside the capillary wall. Microscopic hemorrhage was not noted.
Electron Microscopy. Electron microscopic examinationof sections from tissue blocks containing leukemic infiltration revealed the presence of leukemic cells within thelumen of capillaries and immediately surrounding capillamies. Fig. 8 demonstrates a leukemic cell within a capillary,proximal to the myocardium. In Fig. 9, a leukemic cellappears outside a capillary. In higher magnification of theoutlined area (Fig. 9a), a guinea pig leukemic virus particleis shown (arrow). Fig. 10 illustrates a group of guinea pigleukemia virus particles (arrow) within a leukemic cell adjacent to heart muscle.
DISCUSSION
In this study, all guniea pigs inoculated with leukemiccells developed generalized leukemia. Cardiac involvementin the form of leukemic cell infiltration was present to somedegree in all animals, involving usually the endocardium,epicardium, and occasionally also the myocardium. A largernumber of leukemic cells was observed in the epicardiumthan in the endocandium.
The infiltration with leukemic cells was more pronouncedin the aunicles than in the ventricles. This observation was incontrast with the recent report of Urbaneck et a!. (19) whoobserved that in lambs in which leukemia was inducedfollowing inoculation of blood from leukemic cattle, theventricles had a heavier infiltration with leukemic cells ascompared with the aunicles. In the group of guinea pigsused in experiments here reported we did not observe pencardial effusion in the leukemic animals. However, amongother leukemic animals, not included in this study, we haveseen 2 or 3 guinea pigs with penicardial effusion.
The electrocard iograms showed a significant abnormalityin 20 of 26 (77%) of the leukemic guinea pigs. The earliestelectrocardiographic changes were noticed on the 14th dayafter inoculation with leukemic cells; however, in someanimals significant electrocardiographic changes were notnoted until the terminal phase of the disease, i.e. , up to 45days after inoculation. There was a correlation between theonset of the electrocardiographic changes and the penipheral WBC elevation; in those animals in which there wereelectrocardiographic changes, the WBC were markedly elevated. The significant electrocardiographic changes wererepresented by the onset of a 0-wave or a T-wave inversionor both. There was no significant change in the heart mate.No ST changes were noted. No evidence of atmioventnicular
block and interventniculam conduction disturbance wasnoted.
There was striking invasion of myocardial capillaries byleukemic cells. The pathological changes in these vesselsmay explain some of the electrocardiographic changesnoted during the terminal phase of the disease. Very fewleukemic cells actually infiltrated the muscle fibers of themyocardium. The great majority of these cells were withinthe capillaries of the endocardium, myocardium, and epicardium.
It is possible that the electrocardiographic changes notedduring the terminal phase of the disease were most probably related to anoxic changes secondary to either completeor partial occlusion of the capillaries of the heart or thatthey resulted from compression of the myocardial cells byoverdistended capillaries.
REFERENCES
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2. Bierman, H. R., Perkins, E. K., and Ortega, P. Pericarditis in Patientswith Leukemia. Am. Heart J., 43: 413-422, 1952.
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4. Bregano, P., and Perrotta, P. II Cuore Nelle Leucemie: Aspetti Clinici edElettrocardiografici. Folia Cardiol. 19: 193-207, 1960.
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7. Dresdale, D. T., Spain, D., and Perez-Pina, F. Heart Block and LeukemicCell Infiltration of Interventricular Septum of Heart. Am. J. Med., 6: 530—533, 1949.
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9. Feldman, D. G., and Gross, L. Electron Microscopic Study ofthe GuineaPig Leukemia Virus. Cancer Res., 30: 2702-271 1, 1970.
10. Forkner, C. E. Leukemia and Allied Disorders, 333 pp. New York: TheMacMillan Company, 1938.
11. Gigante, D. , and Serra, N. Sul Comportamento Dell' Elettrocardiogramma in 56 Casi di Leucemia, Folia Cardiol., 13: 131-158, 1954.
12. Giraud, G., Latour, H., Puech, P., Roujon, J., and Page, A. InfiltrationLymphoblastique du Myocarde; Troubles Rythmiques Dissociatifs Interessant es Trois Etages du Coeur. Arch. Maladies Coeur Vaisseaux, 50:720-734,1957.
13. Gross, L., Dreyfuss, V., Ehrenreich, T., and Moore, L. A. ExperimentalStudies on Leukemia in Guinea Pigs. Acta Haematol., 43: 193-209, 1970.
14. Horeau, J., and Robin, C. Cardiac Manifestations of Leukemia. Actual.Cardiol. Angiol. Intern., 10: 193-201 , 1961.
15. Kirshbaum, J. D.. and Preuss, F. S. Leukemia. A Clinical and PathologicStudy of One Hundred and Twenty-Three Fatal Cases in a Series of14,400 Necropsies. Arch. Internal Med., 71: 777-792, 1943.
16. Nogrette, P. Le Coeurdans les Leucemies, les Lymphoreticulosarcomeset Ia Maladie de Hodgkin. Coeur Med. Interne, 5: 27-36, 1966.
17. Ponomareva, E. D. On Cardiovascular Disorders in Acute Leukemia.Soy. Med., 28: 3-10, 1964.
18. Roberts, W. C., Bodey, G. P., and Wertlake, P. T. The Heart in AcuteLeukemia. Am. J. Cardiol., 21: 388—412,1968.
19. Urbaneck, D., Beyer, J., Wittmann, W., and Seils, H. Pathomorphologische Befunde bei Schafleukose aus Ubertragungsversuchen mit Material hamatologisch leukosepositiver Rinder. Arch . Exptl. Veterinaermed.,27: 75-87, 1973.
20. Venable, J. H., and Coggeshall, R. A Simplified Lead Citrate Stain forUse in Electron Microscopy. J. Cell BioI., 25: 407-408, 1965.
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Fig. 1. A typical normal tracing in a young healthy guinea pig.Fig. 2. An electrocardiogram taken from a guinea pig in terminal phase of leukemia, revealing a 0-wave and a T-wave inversion in Lead 1. Such changes
suggest leukemic involvement of the anterolateral region of the left ventricle.Fig. 2a. Higher magnification of Fig. 2, representing the originalelectrocardiogram taken from a guinea pig in the terminal phase of leukemia, revealing a
0-wave in Lead 1 and a T-wave inversion in Lead 1.Fig. 3. An anesthetized guinea pig with the electrodes in place.Fig. 4. Transverse coronal section of heart with a large, thick-walled, slightly dilated left ventricle and a much smaller right ventricle. Above the left auricle
there is an enlarged, leukemic lymph node. Small leukemic deposits are present in epicardium and endocardium (arrows). H & E, x 5.Fig. 5. Multiple, horizontal, transverse heart sections. Top left, base of heart and large vessels, bottom right, apex. Large leukemic infiltration present at
base of heart and small leukemic deposits present elsewhere in epicardium and endocardium (arrows). H & E, x 3.Fig. 6. Low-power view of left ventricular wall showing extensive leukemic infiltration of epicardium with moderate involvement of the underlying
myocardium. H & E. x 150.Fig. 7. Detail of Fig. 6 illustrating the leukemic cell infiltration in the myocardium. Leukemic cells are present within capillaries and are separated from
myocardial fibers by the capillary wall and endothelium (arrow). Myocardial fibers are compressed where the leukemic cells distend the capillaries (upper). Afew cells are present outside the capillary wall (thick arrow). H & E, x 35o.
Fig. 8. Electron micrograph of heart muscle from a leukemic guinea pig. Also shown is a leukemic cell within the lumen of a capillary. x 21.200.Figs. 9 and 9a. A leukemic cell in an intercellular space proximal to cardiac muscle. In a higher magnification (Fig. 9a) of the outlined area a leukemic virus
particle (arrow) is shown. Fig. 9, x 22,640; Fig. 9a, x 58,000.Fig. 10. A leukemic cell, containing a group of leukemic virus particles (arrow), is situated adjacent to heart muscle. x 71.000.
1976;36:1998-2006. Cancer Res Dino A. Belletti, Ludwik Gross, Yolande Dreyfuss, et al. Experimental Guinea Pig LeukemiaElectrocardiographic and Pathological Studies of the Heart in
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