A Case of Chronic Granulocytic Leukemia in
a
Dog
G. N. Joiner, C. J. Fraser, J. H. Jardine and J. M. Trujillo* ABSTRACT A case of chronic granulocytic leukemia was diagnosed in a ten year old miniature poodle and was observed for four and one half years. Methods of diagnosis and characteristic features are described. A persistent granulocytosis with a preponderance of mature forms and the absence of a detectable underlying pyogenic process were key diagnostic features which enabled distinction of this neoplastic process from acute granylocytic leukemia and a leukemoid reaction. Other features included dysplastic granulocytes in various developmental stages, marginal anemia and hyperplastic bone marrow (myeloid elements). No blast crisis occurred. This dog was euthanatized in August 1975.
processus neoplasique d'une leucemie granulocytaire aigue ou d'une reaction leucemoide. Au nombre des autres criteres figuraient des granulocytes anormaux a divers stades de developpement, une anemie marginale et une hyperplasie des elements myeloides de la moelle osseuse. Le chien ne manifesta jamais d'augmentation alarmante de cellules granulocytaires jeunes; on proc&da a son euthanasie, au cours du mois d'aoiut 1975.
INTRODUCTION
Reports of granulocytic (myelogenous) leukemia in dogs are relatively rare (3, 5, 6, 7, 8, 9). Of the reported cases the majority have been diagnosed without specifying the precise form (acute or chronic). Critical examination of the available material RESUMA suggests that only two of these reports are of the chronic variety (5, 6) in which inLes auteurs ont diagnostique un cas de stance both dogs died following an apleucemie granulocytaire chronique chez un parent blast crisis. Support for the diagcaniche miniature, age de dix ans, et ils l'ont nosis of chronic granulocytic leukemia observe de pres pendant quatre ans et demi. (CGL) in these two cases was based upon Ils decrivent leurs methodes de diagnostic et the relative maturity of the malignant les traits caracteristiques de cette affection. granulocytes and the protracted duration Une granulocytose persistante, comprenant of the disease. surtout des formes matures, et l'absence d'une In February 1971, a ten year old male condition pyogene sous-jacente decelable, miniature poodle was referred by a vetericonstituaient les principaux critieres de diag- nary practitioner to The University of nostic qui leur permirent de distinguer ce Texas System Cancer Center M. D. Anderson Hospital and Tumor Institute. This dog had been treated previously by several veterinarians because of an intractable *Section of Experimental Animals and the Department and intermittent diarrhea. The referring of Clinical Chemistry and Laboratory Medicine, The University of Texas System Cancer Centre, M. D. practitioner suspected a leukemic condition Anderson Hospital and Tumor Institute, Houston, Texas and the Department of Veterinary Public Health, of undetermined nature based upon an eleCollege of Veterinary Medicine, Texas A&M University, vated total white blood cell count (163,000 College Station, Texas 77843. The was WBC/cmm). dog moderately The cytogenetic studies were supported by PHS Grant ROI-CA-12687. anemic (PVC 30%, Hg 9.0 gm%, total RBC count 4,090,000/cmm). Submitted August 8, 1975.
Volume 40
-
April, 1976
153
MATERIALS AND METHODS CLINICAL FINDINGS
Although somewhat underweight (13 lbs), the dog was alert and displayed no regional lymphadenopathy. However, the spleen was readily palpable. The prostate gland was within size limits as determined by rectal palpation. Ventrodorsal and lateral radiographs of the thorax and abdomen confirmed the presence of an enlarged spleen. No other abnormalities were noted.
average monthly counts ranged from a high of 170,000/cmm to a low of 53,00{)/cmm (Fig. 1). Peripheral blood examinations throughout the 54-month period showed a persistent granulocytosis ranging from blast forms to the preponderantly mature segmented forms (Table I). Many of the neutrophils possessed segmented nuclei and bluish, immature-appearing cytoplasm suggestive of a dysplastic development. Occasionally, mitotic cells or immature granulocytes were observed (Fig. 2). Anemia as characterized by target cells, nucleated erythrocytes, hypochromia, anisocytosis and poikilocytosis also was observed con-
sistently. Material for bone marrow analysis was obtained with an Illinois pediatric biopsy LABORATORY FINDINGS needle. Aspirated marrow particles were removed from a paraffin coated watch glass A blood sample was cultured for the detection of Histoplasma capsulatum and with a microcapillary pipette and transa skin test was performed'. In addition, ferred to alcohol cleaned glass slides. Comblood samples were cultured on three suc- pressed material was stained subsequently cessive days at 24-hour intervals for the by the Wright-Giemsa method. Smears from detection of aerobic and anaerobic bac- these impressions were characterized by a teria. None of these attempts to isolate or granulocytic hyperplasia with many dysidentify an infectious agent was successful. plastic features. In most instances the A urinalysis and urine culture were un- nuclei of various developmental stages apdertaken in an additional attempt to clari- peared more mature than their correspondfy the cause of the granulocytosis. No sup- ing cytoplasmic elements. In addition, the port could be obtained for the diagnosis of cytoplasm of some of the granulocytes contained vacuoles as well as faintly disan infectious condition. cernible azurophilic granules. The nuclei A variety of tests were performed from peripheral blood samples for the evaluation of a few granulocytes were hypersegof renal, hepatic and pancreatic function. mented. There was a paucity of megakaryoTests performed included uric acid, total cytic and erythroid elements. Quantitative protein, A/G ratio, SGPT, SGOT, alkaline bone marrow analysis of a representative phosphatase, bilirubin, serum lipase and sample indicated an increased myeloid to serum amylase. A Coomb's test was per- erythroid ratio with a preponderance of formed at Texas A&M University. Seven mature elements (Table II). blood plasma samples were submitted for electrophoretic analysis during the first two years of observation. None of these CYTOGENETIC ANALYSIS clinical chemistry or hematological methMaterial collected for cytogenetic analods provided any insight into the nature of ysis was placed in tissue culture medium' the granulocytosis or the anemia. Total and differential white blood cell supplemented with 20% fetal calf serum counts coupled with morphological exami- and incubated for 24 hours at 37°C. These nation of stained smears from peripheral cells were then placed in tissue culture meblood and bone marrow provided strong dium and incubated 24 hours. After exevidence for the diagnosis of chronic granu- posure to colcemide for two hours, the cells locytic leukemia. During the initial 27- were treated with a hypotonic saline solumonth period of observation, weekly blood tion and fixed for "squash" preparations. cell counts were performed. Subsequent Cells from these preparations were stained samples were obtained once monthly. The with aceto-orcein and subsequently examined with a Zeiss phase microscope. 'Histoplasmin, Parke, Michigan.
154
Davis
and Company,
Detroit,
2Ham's F-10 Medium, Microbiological Associates, Bethesda, Maryland.
Can. J. comp. Med.
TABLE I. Representative Hematological Findings in a Dog with CGL
10/25/71a
3/9/71
Date i HB (gm ) .................. . ......... PCV (%) Nucleated RBC () Platelets cmm .. ......... . .......... WBC cmm
11.6 33 5
10.7 31 1
160,000 84,500
Myeloblasts ................... ... Promyelocytes. Myelocytes. .................. Metamyelocytes ........ Band Neutrophils ........ Segmented Neutrophils ...... . ......... Eosinophils . ........ Lymphocytes ........... Monocytes
0
1,267 2,535 4,647 10,985 54,925 1,690 5,070 3,380
...... 0 Unidentified Cells *Splenectomy performed prior to this sampling
6
398,000 115,000 .0
0 1.5 3 5.5 13 65 2 6 4 0
0 0 0
Yo
1,150 10,350 85,100 1,150 5,750 10,350 1,150
5/1/72 11.3 32
0 0 0 1 9 74 1 5 9 1
474,000 88,700 1,774
0 0 887
10,644 67,412 3,548 4,435 0 0
,'o.o
2 0 0 1 12 76 4 5 0 0
2/26/73 9.3 33 8 474,000 126,000 0 0 0
3,780 6,930 97,020 1,260 6,930 0 10,080
0 0 0 3 5.5 77 1 5.5 0 8
Average Monthly WBC Counts In A Dog With CGL
160 140
120
-
100
80
60 40
20 0
6 8
1971
1972
1973
10 12
6
i974
Time (Months) *Splenectomized
on May 12,1971
Fig. 1. Average monthly WBC counts in
Volume 40 - April, 1976
a
dog with CGL.
155
TABLE 11. Bone Marrow Analysis of a Dog with CGL
Myeloblasts ................. Promyelocytes .............. Myelocytes ................. Metamyelocytes ............. Neutrophils ................. Eosinophils ................. Basophils ................... Lymphocytes ............... Plasma cells ................. Monocytes .................. Reticulum cells .............. Prorubricytes ............... Metarubricytes ..............
No. 4
14 9 235 190 4 0 6 0 31 0 1 6
%o 0.8 2.8 1.8 47.0 38.0 0.8 0.0 1.2 0.0 6.2 0.0 0.2 1.2
M:E ratio = 456:7 = 65:1
SURGICAL BiopsY Portions of two ribs and wedge-shaped
portions of the spleen and liver
Fig. 2. Peripheral blood, CGL. A metamyelocyte, several neutrophilr and two primitive cells [blasts] are depicted. Tho WBC count was 126,OOO/cmm. Wright-Giemsa stain. X600.
Only eight metaphases were recovered from the bone marrow culture and two from the spleen culture. The range in chromosome number was 67 to 77 (all below the diploid number of 78 for the normal dog). The quality of these metaphases was such that no proper idiogram could be made. Material for this analysis was collected in May 1971. In December 1971, a heparinized peripheral blood sample was submitted for chromosome analysis. In this sampling the corrected total leukocyte count was 71,429/ cmm with four percent myeloblasts. Four cultures were initiated from the leukocyterich plasma obtained from the blood sample, three of which were not stimulated and one of which was stimulated with phytohemagglutinin3 (PHA). Each of the unstimulated cultures was harvested after 24, 48 and 72-hour incubations, respectively. The PHA-stimulated culture was harvested after 72 hours incubation. Only three mitotic cells were found on the preparations made from the 24-hour unstimulated cultures. The chromosome numbers were 69, 71 and 78. The other cultures yielded no mitotic cells for analysis. 3Phytohemagglutinin M, Difco Labs, Detroit, Michigan.
156
were re-
moved surgically. Several months later a splenectomy was performed. In addition, an intact splenic lymph node was removed for histological examination. Examination of tissues from the resected ribs revealed a hyperplastic bone marrow. Granulocytes in all stages of development comprised approximately 90% of the cellular population (Fig. 3). Primitive cells in the bone marrow stained positively with naphthol-ASD chloracetate esterase (NASD). Such a staining reaction is characteristic of myeloid cells (4). The spleen was enlarged by a factor of two. The histological appearance was more typical of a neoplastic process than a reactive one. Although Malpighian bodies were preserved the red pulp was altered significantly. Scattered among primitive mononuclear cells were recognizable granulocytic elements, megakaryocytes and cells which resembled megakaryocytic precursors. Hemosiderosis was pronounced. The overall appearance was characteristic of malignant myeloid metaplasia (Figs. 4 and 5). While the liver did not appear abnormal grossly, it contained scattered microscopic foci of hematopoietic elements. The wide range of maturation among these cells was suggestive of extramedullary hematopoiesis. The splenic lymph node appeared moderately enlarged. Microscopically, the node was characterized by a preponderance of granulocytes of varying maturity within the medullary cords, marked hemosiderosis
Can. J. comp. Med.
~~~~~~~~~~~~~~~~~~
eF
Figvple, CG
Not
%--. of
~~~~~~~~~~~~~~~~~~~~~e
the#heeooscl oua
*~~~~~~f
V
tion of mature and immature granulocytes, megakaryocyte. (M) ad mononuclear coils with vsidcular nuclei. A mitotic cebll is asbo observable (arrow). H & E stain, X380.
Fig. 5. Granlocytlc hprladsi, lbone mrrow. Nulmerous band and segmented neutrophils are scattered among primitive mononuclear cells. There is a paucity of erythrocytic elements. H & E stain. X600.
and a prominent nodular hyperplasia of the germinal centers. Asynchronous development was noted within some of the granulocytes. - 48.% . * '-
^>t.
ELECTRON MICROSCOPY
The vacuoles seen at the light microscopic on ultrastructural examevel *s l^inationwereto found j . be dilated and degenerative > a. !n~itochondria. There was asynchronous ] S ~~~! t % t -t 2§Fs development between nuclear and cytor e , -||* es plasmic elements. In some instances, a :-:^ "* >.~~~~~'W^ develmature nucleus coexisted withInlesser )-* IS 45j\* v~other inoped cytoplasmic granules. , %, ^ tr+,*_Nijx C 4 t stances, numerous well developed lysomal =i were associated with an immature 5granules 2 ¢*z_sv. 9 i4ffi-'*xt nucleus (Figs. 6 and 7) . The,e findings ^ . &> * p t
e-
8 as t~~4E~k
Fig 4. * ~
cytes (2, 12).
NECROPSY FINDINGS
t t w - ;w " ~tobarbital pen1975 with an intravenlous injection ofgross sodium. The most striking
Large mononuclear cells with disCGL.vacuolated Fg4Spleen, nucleoli' and cytoplasm are depicted. tinct Distinct granules are present in some of these cells (arrow). Band (B) and segmented (5) neutrophis are also present. Toluldine blue stain, one micron section.
This
dog was
euthanatized
in
August
feature was the extensive amount of reddish brown] bone marrow observed within
ADUU.
Volume 40
-
April, 1976
157
$'4.
-/
.
w9; -t
Fig. 6. Neutrophil, spleen, CGL. Note the condensation of chromatin along the nuclear membrane in a cell with a paucity of specific granules (asynchrony). Mitochondria are dilated and contain few cristae. Uranyl acetate and lead citrate stain. X24,000.
the cavity of ribs, midshaft femur, sternum and vertebral bodies. Mesenteric lymph nodes were enlarged. The liver and kidneys were not remarkably altered. Microscopically, the bone marrow was characterized by a prominent granulocytic hyperplasia with a preponderance of mature elements. Megakaryocytes were also numerous. The principle difference between the earlier biopsy material and the necropsy tissue was an increased amount of myelofibrosis in the latter instance. In the liver were scattered foci of hematopoiesis similar to that observed originally. Erythrophagocytosis was prominent within Kupffer cells. The medullary cords of the lymph nodes were comprised predominantly of granulocytes in various stages of maturation much like that observed in the biopsy specimen. The kidneys contained several large foci of neoplastic granulocytes in the cortex. Membranous glomerulonephritis was also observed.
158
DISCUSSION This poodle presented a diagnostic challenge during the initial stages of observation. The diarrhea which was reported in the history subsided within a brief period and therefore was attributed to dietary factors. The prominent granulocytosis with a preponderance of mature forms initially suggested a leukemoid reaction resulting from an underlying suppurative process such as prostatitis or pancreatitis. However, the magnitude of the neutrophilia appeared to exceed the upper limits of an infectious process. In addition, the occasional presence of granulocytic precursors and mitotic cells in the peripheral blood was more indicative of a leukemia than a leukemoid process (1). The dysplastic maturation process evidenced from bone marrow and peripheral blood smears also indicated a neoplastic
Can. J. comp. Med.
Fig. 7. Myeloblast, spleen, CGL. The nucleus is immature as indicated by even dispersion of chromatin. Granule formation, however, is well developed. Uranyl acetate and lead citrate stain. X24,000.
rather than leukemoid reaction. In the splenic red pulp the coexistence of mature and immature megakaryocytes and granulocytes provided additional evidence for a dysplasia. The overwhelming preponderance of similar appearing cells in the bone marrow of resected ribs coupled with the clinical pattern and persistently elevated WBC count confirmed the diagnosis of chronic granulocytic leukemia. Although few metaphases were recovered from the bone marrow culture the aneuploidy (hypodiploidy) observed in these cultures is usually associated with neoplasia (10). No firm conclusions could be drawn, however, because of the inadequate sampling. Chronic granulocytic leukemia, as contrasted with acute granulocytic leukemia and the acute lymphomas, is characterized by a relatively slow progression and a subtle clinical illness. Clinical signs are usually associated with a blast crisis which may occur on several occasions before the pa-
Volume 40
-
April, 1976
tient's eventual demise. Treatment is often unwarranted since it may be unduly damaging to bone marrow stem cells. The dog in this report failed to sustain a single blast crisis and lived a comfortable existence during the four and one half years under observation. It is therefore important to distinguish between acute and chronic granulocytic leukemia because of the significant difference in the clinical progression of each. This distinction can often be ascertained by the preponderance of a more mature population of granulocytic elements in CGL. This, in turn, must be differentiated from a leukemoid (infectious) reaction. Uinfortunately the use of the alkaline phosphatase stain for peripheral smears in distinguishing between CGL and leukemoid reactions is of no value in dogs, cats and certain other animals (11). Therefore, a differential diagnosis must rely heavily upon morphological features, a thorough search for a smouldering infection an(d clinical progression.
159
ACKNOWLEDGMENTS We wish to thank Drs. James J. Butler, M. J. Ahearn, R. A. Squire and K. R. Pierce for reviewing the case material. We would also like to thank Mary Ann Payne, Helen Jackson, Pam Perry and Kent Bennett for their excellent technical assistance.
REFERENCES 1. BESSIS, M. Cytology of the Blood and Blood-forming Organs. pp. 418-422. New York, New York: Grune and Stratton. 1956. 2. BESSIS, M. Ultrastructure of normal and leukemic granulocytes. In Proc. of International Conference on Leukemia - Lymphoma. C. J. D. Zarafonetis, Editor. pp. 281-303. Philadelphia: Lea and Febiger. 1968. 3. CAMERON, T. P., R. KINARD and R. JOHNSON. Irradiation of a dog with myelogenous leukemia. J. Am. vet. med. Ass. 154: 279-282. 1969.
160
4. LEDER, L. D. The selective enzymocytochemical demonstration of neutrophil myeloid cells and tissue mast cells in paraffin sections. Klin. Wchnschr. 42: 553-556. 1964. 5. LUCKE, V. M. and G. SUMNER-SMITH. J. Small Anim. Pract. Spec. Int. Issue 4: 23-28. 1963. 6. MEDWAY, W. and J. P. RAPP. A case of chronic granulocytic leukemia with thrombocytopenia purpura in a dog. Cornell Vet. 52: 247-260. 1962. 7. MEIER, H. Neoplastic diseases of the hematopoietic system in the dog. Zentbl. VetMed. 4: 633-688. 1957. 8. ROSCHER, A. A., R. S. BOATWRIGHT, H. G. KUPFER and R. H. EGHDAHL. Acute myelogenous leukemia with histopathologic studies, following total body irradiation of a dog. J. Am. vet. med. Ass. 136: 491-500. 1960. '. SCHALM, 0. W. Veterinary Hematology. 2nd Ed. pp. 564-565. Philadeluhia: Lea and Febiger. 1965. 10. TRUJILLO, J. M.. M. N. FERNANDEZ. C. C. SHULLENBERGER, L. H. RODRIGUEZ and A. CORK. Cytogenetic contributions to the study of human leukemias. In Proc. of Fourteenth Annual Clinical Conference on Cancer. pp. 105-122. Chicago: Yearbook Medical Publishers, Inc. 1970. 11. WILLSON, J. E. and D. E. BROWN. Leukemoid -eation resembling myelogenous leukemia in a dog. Failure of the leukocyte alkaline phosphatase test to aid in the differential diagnosis. Cornell Vet. 55: 55-63. 1965. 12. WINTROBE, M. M. Chronic myelogenous leukemia. In Clinical Hematology. pp. 1500-1519. Philadelphia: Lea and Febiger. 1974.
Can. J. comp. Med.
a
Dog
G. N. Joiner, C. J. Fraser, J. H. Jardine and J. M. Trujillo* ABSTRACT A case of chronic granulocytic leukemia was diagnosed in a ten year old miniature poodle and was observed for four and one half years. Methods of diagnosis and characteristic features are described. A persistent granulocytosis with a preponderance of mature forms and the absence of a detectable underlying pyogenic process were key diagnostic features which enabled distinction of this neoplastic process from acute granylocytic leukemia and a leukemoid reaction. Other features included dysplastic granulocytes in various developmental stages, marginal anemia and hyperplastic bone marrow (myeloid elements). No blast crisis occurred. This dog was euthanatized in August 1975.
processus neoplasique d'une leucemie granulocytaire aigue ou d'une reaction leucemoide. Au nombre des autres criteres figuraient des granulocytes anormaux a divers stades de developpement, une anemie marginale et une hyperplasie des elements myeloides de la moelle osseuse. Le chien ne manifesta jamais d'augmentation alarmante de cellules granulocytaires jeunes; on proc&da a son euthanasie, au cours du mois d'aoiut 1975.
INTRODUCTION
Reports of granulocytic (myelogenous) leukemia in dogs are relatively rare (3, 5, 6, 7, 8, 9). Of the reported cases the majority have been diagnosed without specifying the precise form (acute or chronic). Critical examination of the available material RESUMA suggests that only two of these reports are of the chronic variety (5, 6) in which inLes auteurs ont diagnostique un cas de stance both dogs died following an apleucemie granulocytaire chronique chez un parent blast crisis. Support for the diagcaniche miniature, age de dix ans, et ils l'ont nosis of chronic granulocytic leukemia observe de pres pendant quatre ans et demi. (CGL) in these two cases was based upon Ils decrivent leurs methodes de diagnostic et the relative maturity of the malignant les traits caracteristiques de cette affection. granulocytes and the protracted duration Une granulocytose persistante, comprenant of the disease. surtout des formes matures, et l'absence d'une In February 1971, a ten year old male condition pyogene sous-jacente decelable, miniature poodle was referred by a vetericonstituaient les principaux critieres de diag- nary practitioner to The University of nostic qui leur permirent de distinguer ce Texas System Cancer Center M. D. Anderson Hospital and Tumor Institute. This dog had been treated previously by several veterinarians because of an intractable *Section of Experimental Animals and the Department and intermittent diarrhea. The referring of Clinical Chemistry and Laboratory Medicine, The University of Texas System Cancer Centre, M. D. practitioner suspected a leukemic condition Anderson Hospital and Tumor Institute, Houston, Texas and the Department of Veterinary Public Health, of undetermined nature based upon an eleCollege of Veterinary Medicine, Texas A&M University, vated total white blood cell count (163,000 College Station, Texas 77843. The was WBC/cmm). dog moderately The cytogenetic studies were supported by PHS Grant ROI-CA-12687. anemic (PVC 30%, Hg 9.0 gm%, total RBC count 4,090,000/cmm). Submitted August 8, 1975.
Volume 40
-
April, 1976
153
MATERIALS AND METHODS CLINICAL FINDINGS
Although somewhat underweight (13 lbs), the dog was alert and displayed no regional lymphadenopathy. However, the spleen was readily palpable. The prostate gland was within size limits as determined by rectal palpation. Ventrodorsal and lateral radiographs of the thorax and abdomen confirmed the presence of an enlarged spleen. No other abnormalities were noted.
average monthly counts ranged from a high of 170,000/cmm to a low of 53,00{)/cmm (Fig. 1). Peripheral blood examinations throughout the 54-month period showed a persistent granulocytosis ranging from blast forms to the preponderantly mature segmented forms (Table I). Many of the neutrophils possessed segmented nuclei and bluish, immature-appearing cytoplasm suggestive of a dysplastic development. Occasionally, mitotic cells or immature granulocytes were observed (Fig. 2). Anemia as characterized by target cells, nucleated erythrocytes, hypochromia, anisocytosis and poikilocytosis also was observed con-
sistently. Material for bone marrow analysis was obtained with an Illinois pediatric biopsy LABORATORY FINDINGS needle. Aspirated marrow particles were removed from a paraffin coated watch glass A blood sample was cultured for the detection of Histoplasma capsulatum and with a microcapillary pipette and transa skin test was performed'. In addition, ferred to alcohol cleaned glass slides. Comblood samples were cultured on three suc- pressed material was stained subsequently cessive days at 24-hour intervals for the by the Wright-Giemsa method. Smears from detection of aerobic and anaerobic bac- these impressions were characterized by a teria. None of these attempts to isolate or granulocytic hyperplasia with many dysidentify an infectious agent was successful. plastic features. In most instances the A urinalysis and urine culture were un- nuclei of various developmental stages apdertaken in an additional attempt to clari- peared more mature than their correspondfy the cause of the granulocytosis. No sup- ing cytoplasmic elements. In addition, the port could be obtained for the diagnosis of cytoplasm of some of the granulocytes contained vacuoles as well as faintly disan infectious condition. cernible azurophilic granules. The nuclei A variety of tests were performed from peripheral blood samples for the evaluation of a few granulocytes were hypersegof renal, hepatic and pancreatic function. mented. There was a paucity of megakaryoTests performed included uric acid, total cytic and erythroid elements. Quantitative protein, A/G ratio, SGPT, SGOT, alkaline bone marrow analysis of a representative phosphatase, bilirubin, serum lipase and sample indicated an increased myeloid to serum amylase. A Coomb's test was per- erythroid ratio with a preponderance of formed at Texas A&M University. Seven mature elements (Table II). blood plasma samples were submitted for electrophoretic analysis during the first two years of observation. None of these CYTOGENETIC ANALYSIS clinical chemistry or hematological methMaterial collected for cytogenetic analods provided any insight into the nature of ysis was placed in tissue culture medium' the granulocytosis or the anemia. Total and differential white blood cell supplemented with 20% fetal calf serum counts coupled with morphological exami- and incubated for 24 hours at 37°C. These nation of stained smears from peripheral cells were then placed in tissue culture meblood and bone marrow provided strong dium and incubated 24 hours. After exevidence for the diagnosis of chronic granu- posure to colcemide for two hours, the cells locytic leukemia. During the initial 27- were treated with a hypotonic saline solumonth period of observation, weekly blood tion and fixed for "squash" preparations. cell counts were performed. Subsequent Cells from these preparations were stained samples were obtained once monthly. The with aceto-orcein and subsequently examined with a Zeiss phase microscope. 'Histoplasmin, Parke, Michigan.
154
Davis
and Company,
Detroit,
2Ham's F-10 Medium, Microbiological Associates, Bethesda, Maryland.
Can. J. comp. Med.
TABLE I. Representative Hematological Findings in a Dog with CGL
10/25/71a
3/9/71
Date i HB (gm ) .................. . ......... PCV (%) Nucleated RBC () Platelets cmm .. ......... . .......... WBC cmm
11.6 33 5
10.7 31 1
160,000 84,500
Myeloblasts ................... ... Promyelocytes. Myelocytes. .................. Metamyelocytes ........ Band Neutrophils ........ Segmented Neutrophils ...... . ......... Eosinophils . ........ Lymphocytes ........... Monocytes
0
1,267 2,535 4,647 10,985 54,925 1,690 5,070 3,380
...... 0 Unidentified Cells *Splenectomy performed prior to this sampling
6
398,000 115,000 .0
0 1.5 3 5.5 13 65 2 6 4 0
0 0 0
Yo
1,150 10,350 85,100 1,150 5,750 10,350 1,150
5/1/72 11.3 32
0 0 0 1 9 74 1 5 9 1
474,000 88,700 1,774
0 0 887
10,644 67,412 3,548 4,435 0 0
,'o.o
2 0 0 1 12 76 4 5 0 0
2/26/73 9.3 33 8 474,000 126,000 0 0 0
3,780 6,930 97,020 1,260 6,930 0 10,080
0 0 0 3 5.5 77 1 5.5 0 8
Average Monthly WBC Counts In A Dog With CGL
160 140
120
-
100
80
60 40
20 0
6 8
1971
1972
1973
10 12
6
i974
Time (Months) *Splenectomized
on May 12,1971
Fig. 1. Average monthly WBC counts in
Volume 40 - April, 1976
a
dog with CGL.
155
TABLE 11. Bone Marrow Analysis of a Dog with CGL
Myeloblasts ................. Promyelocytes .............. Myelocytes ................. Metamyelocytes ............. Neutrophils ................. Eosinophils ................. Basophils ................... Lymphocytes ............... Plasma cells ................. Monocytes .................. Reticulum cells .............. Prorubricytes ............... Metarubricytes ..............
No. 4
14 9 235 190 4 0 6 0 31 0 1 6
%o 0.8 2.8 1.8 47.0 38.0 0.8 0.0 1.2 0.0 6.2 0.0 0.2 1.2
M:E ratio = 456:7 = 65:1
SURGICAL BiopsY Portions of two ribs and wedge-shaped
portions of the spleen and liver
Fig. 2. Peripheral blood, CGL. A metamyelocyte, several neutrophilr and two primitive cells [blasts] are depicted. Tho WBC count was 126,OOO/cmm. Wright-Giemsa stain. X600.
Only eight metaphases were recovered from the bone marrow culture and two from the spleen culture. The range in chromosome number was 67 to 77 (all below the diploid number of 78 for the normal dog). The quality of these metaphases was such that no proper idiogram could be made. Material for this analysis was collected in May 1971. In December 1971, a heparinized peripheral blood sample was submitted for chromosome analysis. In this sampling the corrected total leukocyte count was 71,429/ cmm with four percent myeloblasts. Four cultures were initiated from the leukocyterich plasma obtained from the blood sample, three of which were not stimulated and one of which was stimulated with phytohemagglutinin3 (PHA). Each of the unstimulated cultures was harvested after 24, 48 and 72-hour incubations, respectively. The PHA-stimulated culture was harvested after 72 hours incubation. Only three mitotic cells were found on the preparations made from the 24-hour unstimulated cultures. The chromosome numbers were 69, 71 and 78. The other cultures yielded no mitotic cells for analysis. 3Phytohemagglutinin M, Difco Labs, Detroit, Michigan.
156
were re-
moved surgically. Several months later a splenectomy was performed. In addition, an intact splenic lymph node was removed for histological examination. Examination of tissues from the resected ribs revealed a hyperplastic bone marrow. Granulocytes in all stages of development comprised approximately 90% of the cellular population (Fig. 3). Primitive cells in the bone marrow stained positively with naphthol-ASD chloracetate esterase (NASD). Such a staining reaction is characteristic of myeloid cells (4). The spleen was enlarged by a factor of two. The histological appearance was more typical of a neoplastic process than a reactive one. Although Malpighian bodies were preserved the red pulp was altered significantly. Scattered among primitive mononuclear cells were recognizable granulocytic elements, megakaryocytes and cells which resembled megakaryocytic precursors. Hemosiderosis was pronounced. The overall appearance was characteristic of malignant myeloid metaplasia (Figs. 4 and 5). While the liver did not appear abnormal grossly, it contained scattered microscopic foci of hematopoietic elements. The wide range of maturation among these cells was suggestive of extramedullary hematopoiesis. The splenic lymph node appeared moderately enlarged. Microscopically, the node was characterized by a preponderance of granulocytes of varying maturity within the medullary cords, marked hemosiderosis
Can. J. comp. Med.
~~~~~~~~~~~~~~~~~~
eF
Figvple, CG
Not
%--. of
~~~~~~~~~~~~~~~~~~~~~e
the#heeooscl oua
*~~~~~~f
V
tion of mature and immature granulocytes, megakaryocyte. (M) ad mononuclear coils with vsidcular nuclei. A mitotic cebll is asbo observable (arrow). H & E stain, X380.
Fig. 5. Granlocytlc hprladsi, lbone mrrow. Nulmerous band and segmented neutrophils are scattered among primitive mononuclear cells. There is a paucity of erythrocytic elements. H & E stain. X600.
and a prominent nodular hyperplasia of the germinal centers. Asynchronous development was noted within some of the granulocytes. - 48.% . * '-
^>t.
ELECTRON MICROSCOPY
The vacuoles seen at the light microscopic on ultrastructural examevel *s l^inationwereto found j . be dilated and degenerative > a. !n~itochondria. There was asynchronous ] S ~~~! t % t -t 2§Fs development between nuclear and cytor e , -||* es plasmic elements. In some instances, a :-:^ "* >.~~~~~'W^ develmature nucleus coexisted withInlesser )-* IS 45j\* v~other inoped cytoplasmic granules. , %, ^ tr+,*_Nijx C 4 t stances, numerous well developed lysomal =i were associated with an immature 5granules 2 ¢*z_sv. 9 i4ffi-'*xt nucleus (Figs. 6 and 7) . The,e findings ^ . &> * p t
e-
8 as t~~4E~k
Fig 4. * ~
cytes (2, 12).
NECROPSY FINDINGS
t t w - ;w " ~tobarbital pen1975 with an intravenlous injection ofgross sodium. The most striking
Large mononuclear cells with disCGL.vacuolated Fg4Spleen, nucleoli' and cytoplasm are depicted. tinct Distinct granules are present in some of these cells (arrow). Band (B) and segmented (5) neutrophis are also present. Toluldine blue stain, one micron section.
This
dog was
euthanatized
in
August
feature was the extensive amount of reddish brown] bone marrow observed within
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Fig. 6. Neutrophil, spleen, CGL. Note the condensation of chromatin along the nuclear membrane in a cell with a paucity of specific granules (asynchrony). Mitochondria are dilated and contain few cristae. Uranyl acetate and lead citrate stain. X24,000.
the cavity of ribs, midshaft femur, sternum and vertebral bodies. Mesenteric lymph nodes were enlarged. The liver and kidneys were not remarkably altered. Microscopically, the bone marrow was characterized by a prominent granulocytic hyperplasia with a preponderance of mature elements. Megakaryocytes were also numerous. The principle difference between the earlier biopsy material and the necropsy tissue was an increased amount of myelofibrosis in the latter instance. In the liver were scattered foci of hematopoiesis similar to that observed originally. Erythrophagocytosis was prominent within Kupffer cells. The medullary cords of the lymph nodes were comprised predominantly of granulocytes in various stages of maturation much like that observed in the biopsy specimen. The kidneys contained several large foci of neoplastic granulocytes in the cortex. Membranous glomerulonephritis was also observed.
158
DISCUSSION This poodle presented a diagnostic challenge during the initial stages of observation. The diarrhea which was reported in the history subsided within a brief period and therefore was attributed to dietary factors. The prominent granulocytosis with a preponderance of mature forms initially suggested a leukemoid reaction resulting from an underlying suppurative process such as prostatitis or pancreatitis. However, the magnitude of the neutrophilia appeared to exceed the upper limits of an infectious process. In addition, the occasional presence of granulocytic precursors and mitotic cells in the peripheral blood was more indicative of a leukemia than a leukemoid process (1). The dysplastic maturation process evidenced from bone marrow and peripheral blood smears also indicated a neoplastic
Can. J. comp. Med.
Fig. 7. Myeloblast, spleen, CGL. The nucleus is immature as indicated by even dispersion of chromatin. Granule formation, however, is well developed. Uranyl acetate and lead citrate stain. X24,000.
rather than leukemoid reaction. In the splenic red pulp the coexistence of mature and immature megakaryocytes and granulocytes provided additional evidence for a dysplasia. The overwhelming preponderance of similar appearing cells in the bone marrow of resected ribs coupled with the clinical pattern and persistently elevated WBC count confirmed the diagnosis of chronic granulocytic leukemia. Although few metaphases were recovered from the bone marrow culture the aneuploidy (hypodiploidy) observed in these cultures is usually associated with neoplasia (10). No firm conclusions could be drawn, however, because of the inadequate sampling. Chronic granulocytic leukemia, as contrasted with acute granulocytic leukemia and the acute lymphomas, is characterized by a relatively slow progression and a subtle clinical illness. Clinical signs are usually associated with a blast crisis which may occur on several occasions before the pa-
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tient's eventual demise. Treatment is often unwarranted since it may be unduly damaging to bone marrow stem cells. The dog in this report failed to sustain a single blast crisis and lived a comfortable existence during the four and one half years under observation. It is therefore important to distinguish between acute and chronic granulocytic leukemia because of the significant difference in the clinical progression of each. This distinction can often be ascertained by the preponderance of a more mature population of granulocytic elements in CGL. This, in turn, must be differentiated from a leukemoid (infectious) reaction. Uinfortunately the use of the alkaline phosphatase stain for peripheral smears in distinguishing between CGL and leukemoid reactions is of no value in dogs, cats and certain other animals (11). Therefore, a differential diagnosis must rely heavily upon morphological features, a thorough search for a smouldering infection an(d clinical progression.
159
ACKNOWLEDGMENTS We wish to thank Drs. James J. Butler, M. J. Ahearn, R. A. Squire and K. R. Pierce for reviewing the case material. We would also like to thank Mary Ann Payne, Helen Jackson, Pam Perry and Kent Bennett for their excellent technical assistance.
REFERENCES 1. BESSIS, M. Cytology of the Blood and Blood-forming Organs. pp. 418-422. New York, New York: Grune and Stratton. 1956. 2. BESSIS, M. Ultrastructure of normal and leukemic granulocytes. In Proc. of International Conference on Leukemia - Lymphoma. C. J. D. Zarafonetis, Editor. pp. 281-303. Philadelphia: Lea and Febiger. 1968. 3. CAMERON, T. P., R. KINARD and R. JOHNSON. Irradiation of a dog with myelogenous leukemia. J. Am. vet. med. Ass. 154: 279-282. 1969.
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4. LEDER, L. D. The selective enzymocytochemical demonstration of neutrophil myeloid cells and tissue mast cells in paraffin sections. Klin. Wchnschr. 42: 553-556. 1964. 5. LUCKE, V. M. and G. SUMNER-SMITH. J. Small Anim. Pract. Spec. Int. Issue 4: 23-28. 1963. 6. MEDWAY, W. and J. P. RAPP. A case of chronic granulocytic leukemia with thrombocytopenia purpura in a dog. Cornell Vet. 52: 247-260. 1962. 7. MEIER, H. Neoplastic diseases of the hematopoietic system in the dog. Zentbl. VetMed. 4: 633-688. 1957. 8. ROSCHER, A. A., R. S. BOATWRIGHT, H. G. KUPFER and R. H. EGHDAHL. Acute myelogenous leukemia with histopathologic studies, following total body irradiation of a dog. J. Am. vet. med. Ass. 136: 491-500. 1960. '. SCHALM, 0. W. Veterinary Hematology. 2nd Ed. pp. 564-565. Philadeluhia: Lea and Febiger. 1965. 10. TRUJILLO, J. M.. M. N. FERNANDEZ. C. C. SHULLENBERGER, L. H. RODRIGUEZ and A. CORK. Cytogenetic contributions to the study of human leukemias. In Proc. of Fourteenth Annual Clinical Conference on Cancer. pp. 105-122. Chicago: Yearbook Medical Publishers, Inc. 1970. 11. WILLSON, J. E. and D. E. BROWN. Leukemoid -eation resembling myelogenous leukemia in a dog. Failure of the leukocyte alkaline phosphatase test to aid in the differential diagnosis. Cornell Vet. 55: 55-63. 1965. 12. WINTROBE, M. M. Chronic myelogenous leukemia. In Clinical Hematology. pp. 1500-1519. Philadelphia: Lea and Febiger. 1974.
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