LEUKEMIA IN THE F STRAIN OF MICE: OBSERVATIONS ON
CYTOLOGY, GENERAL MORPHOLOGY, AND
TRANSMISSION
ARTHUR KIRSCHBAUMZ
AND
LEONELL
C. STRONG
(From the Department of Anatomy, Yale University School of Medicine, New Haven, Conn.)
While many strains of mice with a high incidence of mammary tumors have
been studied, only one strain with a comparable incidence of leukemia has been
described (1). Stocks ’ with a frequent appearance of leukemia have, however, been reported ( 2 ) . The present communication concerns the incidence
morphology and transplantation of leukemia in a second highly inbred pedigreed strain, the F strain, in which this disease occurs frequently.
MATERIALS
AND METHODS
The F strain of mice has been inbred (3), brother to sister, for a period of twelve years,
for thirty generations. In the F, and subsequent generations enlarged spleens, lymph nodes,
and thymus glands have been observed in over 200 mice. Of the last 2 2 animals which
were sacrificed or died at over six months of age, 10 had either leukemia or mediastinal
lymphosarcoma. The present study is based on the last 17 spontaneous cases of leukemia
and lymphosarcoma which appeared in this strain, plus 295 cases of leukemia developing
in F mice following leukemic cell inoculations (Table I ) .
Old, non-breeding mice of the F strain were examined twice a week to determine
whether spleens and lymph nodes were enlarged. If these organs were found upon digital
palpation to be larger than normal, total white blood cell and differential counts were made,
the blood being obtained by cutting off the end of the tail and “milking.” The cover-slip
method of making blood smears was used throughout. Animals were killed by severing the
brain from the spinal cord. Dry imprints of spleen, lymph nodes, liver, bone marrow, and
thymus were made. The freshly cut surface of the tissue was touched lightly to a clean
slide, the imprinted material being waved dry immediately. On this type of preparation
Pappenheim’s May-Griinwald Giemsa staining combination was used. The May-Grunwald stain was allowed to remain on the slide for two minutes. An equal number of
drops of distilled water buffered to a pH of 6.4 were then added to the undiluted stain
and the diluted stain was allowed to remain on the slide until four minutes had elapsed
from the beginning of the staining process. The stain was then poured off (no rinsing)
and Giemsa solution (2 drops of stock solution to 1 C.C. of buffered distilled water) was
poured on the slide. After fifteen minutes the slide was de-stained by pipetting several
washings of buffered water over it. Blood smears were stained by the Giemsa solution for
only ten minutes. For the peroxidase reaction Richter’s method (4) was used. Following
the staining process smears and imprints were air-dried, dipped in xylol, and mounted in
damar.
Pieces of spleen, kidney, lung, lymph nodes, liver, thymus, and reproductive organs were
fixed in Helly’s fluid or, preferably, a saturated solution of mercuric chloride in 10 per cent
formalin, washed in running water overnight following eight to twelve hours’ fixation, stored
for a few days in iodized 70 per cent alcohol, and then passed through 2 changes of dioxan
1 This investigation was aided by grants from the Fluid Research Fund of Yale University,
the International Cancer Research Foundation, the Anna Fuller Fund, and the Jane Coffin Childs
Memorial Fund for Medical Research.
2 Alexander Brown Coxe Memorial Fellow in Anatomy, 1937-1938.
8
Stock ” indicates a less homogeneous group than “strain” since matings in the former are
not exclusively brother-sister.
400
401
LEUKEMIA I N F STRAIN OF MICE
TABLE
I : Spontaneous and Transplanted Leukemias and Lym$hosarcomas in the F Strain of Mice
Spontaneous case
mouse no.
125035
128011
120540
124309
121567
124184
121995
125016
130188
125745
129769
Non-pedigreed
128973
134006
130258
131668
Non-pedigreed
(splenectomized)
Average
Age of
NO.
of
in days transfers
398
322
540
43 1
503
379
476
525
374
505
349
365 (?)
445
230
415
299
52 7
9
29
-
416
80
* (I) Intraperitoneal.
NO.
of
Average
longevity
after
Type of leukemia
animals transplantation (days)
Myelogenous
Lymphatic
Lymphatic
Myelogenous
Myelogenous
Lymphatic
Lymphatic
Mixed (?)
Lymphatic
Lymphatic
17
9
3
12
1
?
Myelogenous
Lymphatic
Mediastinal lymphosarcoma
Lymphatic
Mediastinal lymphosarcoma
Lymphatic
295
t (S) Subcutaneous.
and 6 changes of paraffin and embedded in paraffin. Sections were cut at 5 or 6 micra and
stained primarily with Dominici’s eosin-orange G, toluidin-blue combination.
Transplants from the spontaneous cases of leukemia were made by either of two
methods. The spleen was removed under sterile conditions, placed in 2 c.c., or less, of
normal saline and cut into fine bits with a pair of sterile scissors. The emulsion was drawn
into a tuberculin syringe with a 25-gauge needle after the heavier pieces of spleen had settled
to the bottom of the dish. A hemocytometer was used to determine the number of cells
per unit volume. Doses estimated to be in the neighborhood of 70,000,000 cells, were
injected intraperitoneally for routine transfer. Successive transplants were made in this
manner from one generation of leukemic animals to the next. The alternative method of
transplantation was to place a few milligrams of fresh leukemic spleen or lymph node in a
sterile trocar and insert this tissue subcutaneously into the right axilla. With this method,
the incubation period was longer than following intraperitoneal inoculation. Recipient animals were in all cases normal pure-bred mice of the F strain from four to eight weeks of
age. Grafts were successful in practically 100 per cent of the cases. The technical procedures described above for cytological and histological study of spontaneous cases were
also used on animals developing leukemia as the result of transplantation of leukemic cells.
OBSERVATIONS
Most mice which developed leukemia spontaneously appeared emaciated,
with the abdomen distended by an enlarged spleen. Of the 1 7 leukemic (or
lymphosarcomatous) animals 14, representing both the myelogenous and the
lymphatic type of the disease, exhibited splenic enlargement. I n 2 of the
other 3 only the enlarged thymus, mediastinal nodes, and lungs (Fig. 30) were
infiltrated. The diagnosis in these animals has been given as “mediastinal
lymphosarcoma ” (Table I ) . The third mouse had been spIenectomized at
402
ARTHUR KIRSCHBAUM AND LEONELL
c.
STRONG
TABLE
I1 : Distribution of Leukemic Infiltrations in Seventeen Spontaneous Cases i n the F Strain
Mouse
125035
128011
125040
124309
121567
124184
121995
125016
130188
125745
129769
128973
Non-ped igreed
134006
130258
131668
Non-ped.
(splenectomized)
Blood*
Liver
Spleen
++
+++
++
++
+-+
++
++
+++
+++
++
++
++0
Lymph
Nodes
++
++
++
+++
+++
+++
Bone
Kidney Marrow
++
+++
++
++
7
++
-?
?
Lungs Thymust
++
++
++++
+++
++
++
+
++
-
-
++++
+++
+
+
*The blood was considered leukemic only if the leukocytosis was due to an increase in
leukemic cells.
t Gross thymic hypertrophy.
the age of seventy days. Enlargement of the lymph nodes varied, usually
being more pronounced in lymphatic than in myelogenous leukemia. Eight
out of 14 generally leukemic animals showed gross thymic hypertrophy
(Table 11).
Microscopically most of the lymph nodes in the mice with the spontaneous
disease were leukemic. Imprints of even small and only slightly enlarged
nodes showed considerable numbers of leukemic cells, many undergoing mitotic
division. Where enlargement of the node was pronounced, the normal architecture was destroyed. Open sinuses were visible in some cases (Fig. 34), but
in the majority of animals the sinuses were obliterated by leukemic cells.
Capsular infiltration and extension of leukemic cells into the surrounding connective tissue were common.
With lymphatic leukemia there was heavy periportal infiltration in the liver
(Fig. 33), plus a less pronounced sinusoidal infiltration. In myelogenous
leukemia a comparatively heavier sinusoidal involvement occurred and infiltrations in the red pulp of the spleen were marked. I n the enlarged spleens of
transplanted cases the leukemic cells of the pulp appeared to be encroaching
upon the non-leukemic malpighian corpuscles. In advanced cases of the
lymphatic type (Fig. 35) the spleen was a homogeneous mass of lymphocytes.
The hilus of the kidney and the perirenal fat were more heavily infiltrated than
the kidney itself in many cases. The kidney was infiltrated with leukemic
cells in only one of the myelogenous leukemias.
A true leukemic blood picture with an elevation of the leukocyte count due
to a predominance of leukemic cells was observed in 7 of the 14 spontaneous
cases (Table 11). Other leukemic mice had high white cell counts (20,00035,000) but the’elevation in these cases was due to a polymorphonuclear leuko-
LEUKEMIA IN F STRAIN OF MICE
403
cytosis. I n spontaneous case 121995 the blood count was normal, but animals
receiving leukemic cells from this mouse showed a pronounced leukemic blood
picture. Among the animals with spontaneous leukemia, the highest white
cell count, 665,000, was observed in mouse 125035 with a chronic myelogenous leukemia. The highest count in 8 spontaneous lymphatic leukemias
was 111,000, though some mice developing lymphatic leukemia as the result
of injection of leukemic cells had counts of over 300,000 per cu. mm., and in
the 5th transfer generation of the myelogenous leukemia originating in mouse
125035 a count of 920,000 was attained. Most animals with systemic leukemia were intensely anemic, as indicated by polychromatophilic red cells and
normoblasts in the blood. Replacement of normal hemopoietic bone marrow
by leukemic cells might explain the anemia.
Table 11 summarizes observations on the presence or absence of leukemic
infiltrations in the organs in spontaneous cases.
Lesions in mice receiving transplants were not always the same as in the
spontaneous cases from which the transplanted material was derived. Mouse
124184, for example, showed no infiltration of the liver, kidney, or bone marrow but in animals inoculated with cells from this case such infiltration was
present. Systemic lesions were the same whether the cells were introduced
subcutaneously or intraperitoneally. A local tumor formed at the site of subcutaneous implantation in most instances. As observed by Richter and MacDowell (5) , characteristic lesions were found for each transmission-line of
the disease. High white blood counts characterized certain lines, particularly
in the late stages of the disease. A leukemic blood picture was observed in
most of the transmission-lines, whereas only half of the spontaneous cases had
a leukocytosis resulting from an increase of leukemic cells. The interval between inoculation time and death was shortened considerably by frequent
tr msplantation.
Since very few cases of spontaneous myelogenous leukemia occurred in
MacDowell’s C58 strain, and none was transplanted, it is of interest to report
that 4 out of 17 cases in the F strain were myelogenous and injection of cells
frqm these mice into normal animals of the F strain resulted in the development of this type of leukemia. As in the case of lymphatic leukemia, systemic
leukemia was produced by either subcutaneous or intraperitoneal inoculation
of cells. As previously indicated, the F strain has a tendency to develop leukemia (45 per cent of 22 mice). Other strains studied (A, C,H, CBA, C,,)
are practically non-leukemic, although they may show occasional cases of leukemia or lymphosarcoma. Myelogenous leukemia has not been observed thus
far in these strains.
CYTOLOGICAL
OBSERVATIONS
Since the leukemic cells multiply rapidly in the actual lesions, dry imprints
of leukemic tissues (bone marrow, lymph nodes, spleen, liver) were employed.
This type of preparation offers several advantages. First, all types of leukemic
cells are present; not only those which gain entrance to the blood. Second,
cells are more easily classified than in sections (compare Plate I with Fig. 35).
4 “Line ” is used to indicate cell lineage in successive transfers, following Richter and MacDowell.
PLATE I
LEUKEMIA,8TH TRANSPLANT
GENFIG. I. STEM CELL FROM LYMPHNODEO F MYEUJGENOUS
SHOWINGTHREENUCLEOLI,DELICATESIEVE-LIKENUCLEAR
CHROMATIN,
AND LIGHTLY
BASOPHILIC
CYTOPLASM
EARLYDIFFERENTIATION
FIG. 2. CELL FROM SAMELYMPH NODEAS FIG. 1, ILLUSTRATING
TOWARDS A LEUKOCYTE
The chromatin pattern is still immature, but a central hole has appeared in the nucleus.
AS ABOVE
FIG. 3. ATYPICALMASTCELL FROM THE SAME MATERIAL
The nucleus is oval with immature chromatin pattern. Such cells were prominent in !ocal
tumors resulting from the subcutaneous transplantation of leukemic spleen or lymph node tissue
of this line.
FIG.4. SAMETYPEOF CELLAS FIG 3, SHOWING
WATER-SOLUBLE
GRANULES
FIG. 5. STEMCELLFROM LYMPHNODEOF SPONTANEOUS
I‘ MIXEDLEUKEMIA
” ( ?), SHOWING
6 OR 7 NUCLEOLI,
SIEVE-LIKE
NUCLEAR
CHROMATIN,
AND MYELOID
AZUROPHILIC
GRANULES
I N THE
LIGHTLYBASOPHILICCYTOPLASM
STEMCELLFROM BLOODOF SAMEMOUSEAS FIG. 5 , SHOWING 5 NUCLEOLI,
FIG. 6. SMALLER
AND AZUROPHILIC
CYTOPLASMIC
GRANULES
SLIGHTLY
COARSER
CHROMATIN
PATTERN,
FIG. 7 . STEM CELL FROM BLOODOF MYELOGENOUS
LEUKEMIA,6TH TRANSPLANT
GENERATION,
SHOWING5 NUCLEOLI,
DELICATE
CHROMATIN
PATTERN,
AND LIGHTLY
BASOPHILICCYTOPLASM
FIG. 8. YOUNGCELLSHOWING
LEUKOCYTE
DIFFERENTIATION
A central hole has appeared in the nucleus, but the chromatin pattern is that of the stem cell
and 3 sharp nucleoli are present.
FIG. 9. STEMCELLFROM BLOOD
OF HUMAN
MYELOGENOLS
LEUKEMIA
Nucleoli and chromatin pattern simulate the structure seen in young cells of mouse leukemia.
FIG. 10. YOUNGCELL (LYMPIIOBLAST)
FROM LYMPH NODE 01” SPONTANEOUS
LYMPHATIC
LEUKEMIA,
SHOWING
SAMENUCLEAR
FEATURES
AS THE MYELOID
STEMCELL
FIG.11. STEMCELLFROM NORMAL
MOUSEBONEMARROW
Prominent nucleoli and sieve-like chromatin pattern characterize normal as well as malignant
young cells.
FIGS,12-17. PROGRESSIVE
STAGESIN THE MATURATION
OF POLYMORPHONUCLEAR
(HETEROPHILE) LEUKOCYTE:
CELLS FROM BLOODOF MOUSEWITH MYELOGENOUS
LEUKEMIA,
3 D TRANSPLANT
GENERATION.
Fig. 12. Stem cell with nucleoli and fine chromatin pattern. Fig. 13. Leukoblast, with coarse
chromatin pattern and azurophilic granules in cytoplasm. Fig. 14. Promyelocyte, in which the
chromatin pattern is assuming the form of that seen in the adult leukocyte, basichromatin becoming
delimited from the parachromatin; central hole in the nucleus. Fig. 15. Myelocyte, showing
chromatin pattern of mature cell and larger central nuclear hole. Fig. 16. Metamyelocyte; beginning lobulation of nucleus. Fig. 17. Atypical mature heterophile leukocyte.
FIG. 18. YOUNGLYMPHOID
CELLFROM DRY IMPRINT
OF NORMAL
MOUSETHYMUS(THREE
MONTHS)
Delicate chromatin pattern and nucleoli make the nucleus quite similar to that of lymphoid
leukemic cells (Figs. 10 and 19).
FIG.19. LYMPHOBLAST
FROM BLOODO F THIRDTRANSPLANT
GENERATION
OF LYWPHATIC
LEUKEMIA
Nuclear chromatin pattern is quite lymphocytic, 3 nucleoli are evident, indicating immaturity.
FIG. 20. LARGELYMPHOBLAST
FROM DRY IMPRINT
OF LYMPH NODE FROM SPONTANEOUS
PATTERN
RATHERLYMPHOCYTIC
; 5 PROMINENT
NUCLEOLI
LYMPHATIC
LEUKEMIA:CHROMATIN
FIG. 21. LYMPHOCYTE
OF MATURE
APPEARANCE
FROM SAMEMOUSEAS FIGURE
19
A cell with this structure might be found in normal mouse blood.
FIG. 22. METAPHASE
ST&E IN DIVISIONFROM DRY IMPRINT
OF SPONTANEOUS
LYMPHATIC
LEUKEMIA
Atypical granules are present in the cytoplasm. The well-preserved chromosomes indicate
that the dry imprint technic does not damage some cells to any great degree.
FIG. 23. MITOSISI N YOUNG CELL, DRYIMPRINT O F LIVER,MYELOGENOUS
LEUKEMIA,
3D TRANSPLANT GENERATION
(SAMEMOUSEAS FOR F m 12-17)
Greenish-brown granules in cytoplasm indicate positive peroxidase reaction.
FIG. 24. “ MALIGNANT
LYMPHOCYTE
” OF FURTH:MATURE
CHROMATIN STRUCTURE, DEEPLY
FROM BLOOD
OF SPONTANEOUS
LYMPHATIC
LcUKEMIA
BASOPHILIC
CYTOPLASM,
Cells with this structure may be found occasionally in normal mouse blood.
FIG. 25. PROPRASE
IN MITOSISFROM DRY IMPRINT
OF LYMPIXNODE,SPONTANEOUS
LYMPHATIC LEUKEMIA
Note large size of cell and spireme.
ERATION,
a
404
1
I
I,
PLATE I
(All figures except 9, 11 and 18 are from dry tissue imprints or dry blood smears of mouse
leukemia. Fig. 9 is from the blood of human myelogenous leukemia, Fig. 11 is from a dry imprint
of normal mouse bone marrow, Fig. 18 from a dry imprint of normal mouse thymus. MayGrunwald-Giemsa stain was used in all cases. Original drawings in water color. X 1600.)
405
406
ARTHUR KIRSCHBAUM A N D LEONELL C. STRONG
Third, the same criteria for morphological identification of cells can be used
here as in blood smears (see Plate I).
( a ) Lymphatic Leukemia: When a leukemic blood picture was present, 65
to 95 per cent of the white blood cells were lymphoid (Fig. 28). This was
true for both spontaneous and transplanted leukemia. Many of the lymphoid
cells were mature in appearance (Figs. 2 1 and 24), but in variable proportions
the nucleus showed the presence of nucleoli, indicating immaturity (Fig. 19)It has been our experience that some lymphatic leukemias of the F strain, perhaps the more chronic ones, have mature cell types in the blood, most of the
nuclei possessing dense chromatin patterns without nucleoli. On the other
hand, cytologically and symptomatically ( ? ) acute forms of leukemia were
found, with cells of the lymphoblastic (Fig. 10) or myeloblastic (Fig. 6) type
in the blood.
I n all the lymphatic leukemias, even those with mature cells in the blood,
dry imprints of the lymph nodes and spleen showed a great increase in lymphoblastic cell types with a fine nuclear chromatin pattern and nucleoli (Fig. 28).
Imprints of normal mouse lymph nodes revealed very few cells which might be
called ‘‘ lymphoblastic,” showing mostly medium-sized lymphocytes with a
mature chromatin pattern. I t might be deduced, by inference, that since the
cells which have increased in number in the leukemic lymph nodes are lymphoblastic, these are the so-called “ malignant cells.” But the lymphoblastic cells
(Figs. 10, 19 and 20), although presumably malignant, are not indubitably so,
for some lymphoid cells of the normal mouse thymus (Fig. 18) and lymph
nodes possess the same distinctive morphological characteristics of immaturity
(sieve-like chromatin pattern and nucleoli) as leukedc lymphocytes (Fig. 10).
Anaphase stages in mitosis appeared in what seemed to be smaller lymphocytes, but the fact that most prophases were in the largest cells (Fig. 2 5 ) again
indicates that the large lymphoblastic type is malignant.
In some cases of lymphatic leukemia the blood was characterized-by large
intensely basophilic lymphocytes (Fig. 24) ; some of these had a more delicate
(immature) chromatin pattern than normal blood lymphocytes, while others
had a coarse nucleus with heavy blocks of chromatin, differing from normal
lymphocytes primarily in their cytoplasmic basophilia (Fig. 24). Many of
the large lymphocytes with coarse nuclei were similar to large lymphocytes
found in normal mouse blood. Intensely basophilic cells, such as those described above, had morphologic homologues in normal lymph nodes (dry imprints).
Although cells of the tissues in “lymphatic leukemia resembled immature
(lymphoblastic) cells of normal lymphoid tissues, certain cytological differences between normal and leukehic lymphoblasts $ere observed. Nucleoli
were more distinct in leukemic lymphoblasts. The chromatin of immature
leukemic cells stained more intensely, and the pattern was sharper (Fig. 29).
This “ hyperchromatic ” appearance has been said to be characteristic of malignant cells in general. The sieve-like chromatin arrangement probably indicates ‘‘ prekinesis.” The average size of leukemic lymphocytes is greater than
that of normal lymphocytes.
As in human leukemia, the cells of the less differentiated leukemias (cytologically) resembled the myeloblasts of normal bone marrow and the myeloid
LEUKEMIA IN F STRAIN OF MICE
407
leukemic stem cells more closely than normal lymphoid stem cells. In one
spontaneous case it was difficult to determine whether there had been a " shift
to the left " in the bone marrow, many myeloblasts being present, or whether
the bone marrow had been invaded by leukemic lymphocytes which were
dispersed among the maturing granulocytes. In leukemic lymphoblasts, as
pointed out above, the nucleoli were more sharply defined and the chromatin
pattern more distinct than in the immature cells of normal lymph nodes.
( b ) Myelogenous Leukemia: Myelogenous leukemia has been considered
to be uncommon in mice (6, 7, 8). Because of this, passage through successive generations of mice is necessary to prove the myelogenous nature of the
disease. Lymphatic leukemia with a secondary inflammatory reaction might
confuse the picture. The previous reports on transplantable myelogenous leukemia in mice are those of Furth and Barnes (6, 7, 8), Kaalund;J#rgensen
(9),Hall and Knocke ( 2 ) and the present authors (10). The spontaneous
disease was well described by Simonds ( 11).
Of the 4 spontaneous myelogenous leukemias appearing in our material, 2
were rather chronic symptomatically, a third was acute, and the fourth was
not transplanted, but appeared cytologically to be chronic. Two out of the 4
mice with spontaneous myelogenous leukemia had greenish lymph nodes. I n
one transmission-line green lymph nodes appeared in each of the first 8 transfer
generations. This corresponds to the chloroleukemia described by Hall and
Knocke ( 2 ) . In mouse 130258, however, though the lymph nodes and thymus
were green, the leukemia was of the lymphoid type.
In one line of myelogenous leukemia the degree of cellular differentiation
did not extend beyond the myelocyte (Figs. 1, 2 and 2 7 ) after several transfers had been made. Fig. 26 shows the blood of a mouse of the third transfer
generation. Although in the first few transfer generations the stem cells
(Fig. 1) matured to myelocytes (Fig. 1 5 ) , in the later transplants cellular
differentiation in the tissues was merely sufficient to indicate that the leukemia
was myeloid (Fig. 2 7 ) , most of the cells being of the undifferentiated type
(Fig. 1). Many of the latter possessed myeloid azure granules and were
peroxidase positive. Although there was a very high white blood cell count
in the first 4 transfer generations (400,000 per cu. mm.), later generations
showed a much lower count (20,000-40,000 per cu. mm.), with relatively fewer
leukemic cells. The presence of atypical cells wjth basophile granules (Figs.
3 and 4) was a constant feature. I n tumors resulting from subcutaneous
transplantation of lymph node or spleen they were especially abundant. The
immature chromatin pattern of these cells suggested malignancy, but division
stages were seldom observed. The basophiles may represent differentiated
products of the " malignant " cells. They were somewhat similar in appearance to the tissue mast cells of the normal mouse. Since they were found in
the leukemic blood they must be considered hematogenous elements in this
case. The granules were water soluble (Fig. 4). Barnes and Furth (6, 7, 8)
observed these cells in one myelosis studied by them. Unless they were present in the tissues of their material, grafts were unsuccessful.
A second myelogenous leukemia presented the degree of leukocytic differentiation shown in Figs. 7 and 8. Fig. 7 represents a myeloblast and Fig. 8 a
promyelocyte with myeloblastic nucleoli still prominent. I n contrast to the
J
PLATE I1
(All preparations stained with May-Griinwald-Giemsa combination)
IN
LEUKEMIA,3D TRANSFER
GENERATION:
VARIOUS STAGES
FIG. 26. BLOODSMEAR, MYELOGENOUS
LEUKOCYTE
DEVELOPMENT
(SEE FIGS. 1, 2 , 7, 8, 12-17]
The cell in the upper right-hand corner is a stem cell (see Figs. 1, 7 and 1 2 ) . X 1100.
FIG. 27. DRY IMPRINT
OF LYMPH NODE,MYELWENOUS
LEUKEMIA,1 1 TRANSFJLR
~ ~ GENERA-
TION
The 4 cells with ( I doughnut ” nuclei are early stages in leukocyte development. Leukocytic
differentiation was incomplete (see Figs. 1 and 2 ) . X 1300.
[Legend cont. on next page]
408
LEUKEMIA I N F STRAIN OF MICE
409
limited degree of maturation in this leukemia and in the one described above,
the line originating in mouse 1 2 5035 showed many differentiated leukocytes
(Figs. 12-17). The developmental stages were similar to those observed in
normal bone marrow leukopoiesis. Immature cells of dry imprints gave a
positive peroxidase reaction (Fig. 2 3 ) . Terminally, when the white blood cell
count approached a million, a tremendous number of quite mature heterophile
leukocytes were seen in the blood. Although there is little doubt that the
leukemic myeloblast (Fig. 1 2 ) differentiated to more mature stages of leukocyte development, the great number of fully differentiated leukocytes suggests
the possibility that many cells were not of leukemic cell parentage.
The stem cells of lymphatic and myelogenous leukemia may be quite similar (Figs. 1 0 and 1 2 ) . So-called myeloid azure granules were seldom seen,
however, in lymphoblasts, though they were not infrequent in leukemic myeloblasts. The atypical granulation in Fig. 2 2 , a dividing leukemic lymphoblast,
was only rarely found in this type of cell. Fig. 9 represents a myeloblast
from human myelogenous leukemia. The morphologic similarity between leukemic cells of the two species is well illustrated when this cell is compared with
Fig. 12, a myeloblast of mouse myelogenous leukemia.
DISCUSSION
The leukemic blood cells are not normal immature cells since they have
the capacity to multiply in a fashion apparently not under the control of the
rest of the body. Morphologically, however, with the technics used here,
there is nothing of primary significance to distinguish these cells from ordinary
immature cells, from many intensely basophilic lymphocytes of the lymph
nodes or from myeloblasts or myelocytes of the bone marrow. It has been
concluded by other authors ( 1 2 ) that leukemic lymphocytes of the mouse are
morphologically similar to normally developing, incompletely differentiated
lymphocytes.
Extreme cytoplasmic basophilia of lymphocytes with a coarse nuclear
chromatin pattern may in some mouse leukemias represent a distinctive pathological alteration. Such cells have been designated as “ malignant lymphocytes ” ( 13). Although a significant increase of extremely basophilic cells
with mature chromatin pattern has been observed in transmission-lines of other
strains, these cells have not been observed in the F strain in sufficient numbers
to warrant their designation as specifically ‘ I malignant.” Individual cells
corresponding morphologically to these lymphocytes might be selected from
normal lymph node imprints. No uniform “ malignant cell type ” has been
found for either lymphatic or myelogenous leukemia. Although specific
morphological criteria for malignancy have been sought, none has been observed. The only strikingly consistent cytological observation made in the
present study was the characteristic shift to immature cells in the hemopoietic
BLOOD SMEAR, LYMPHATIC
LEUKEMIA,
3D TRANSFER
GENERATION
Over 90 per cent of the cells in the circulating blood were lymphoid. Note nucleolus in nucleus
of cell in upper center portion of the field. For cytological detail see Figs 19 and 21. X 1100.
FIG.29. DRYIMPRINT
OF LYMPHNODE,
SPONTANEOUS
LYMPHATIC
LEUKEMIA
Mitoses are well shown (see Figs. 22, 23 and 25). The leukemic lymphocytes have a sieve-like
nuclear chromatin pattern and nucleoli (cell in lower right-hand corner; see Fig. 10). X 1300.
FIG.28.
PLATE I11
MEDIASTINAL
LYMPHOSARCOMA
FIG.30. LUNG,SPONTANEOUS
Lungs and thymus (with adjacent lymph nodes) were the only organs involved. Collars of
lymphosarcomatous tissue surround vessels and bronchioles. Helly-hematoxylin-eosin. X 70.
FIG. 31. KIDNEY,SPONTANEOUS
LYMPHATIC
LEUKEMIA:
HEAVYLEUKEMIC
INFILTRATION
BETUBULES
AND SURROUNDING
GLOMERULI.
HELLY-DOMINICI.
X 70
[Legend cont. on next pugel
TWEEN
410
LEUKEMIA IN F STRAIN OF MICE
411
organs of leukemic animals and the presence of similar cells in the liver, kidney, and other non-hemopoietic organs (Figs. 27 and 29). The increased frequency of mitoses in leukemic tissues (Fig. 29) was characteristic.
Strumia (14) concluded that the young cells in mouse leukemia have less
significance than in human leukemia. Our observations, especially on tissue
imprints, do not support this conception, Lewis (15) cultured lymphocytes
of lymphosarcomas which appeared in mice injected with dibenzanthracene.
She says: “ In appearance and locomotion the malignant lymphoid cells resembled greatly enlarged lymphocytes rather than lymphoblasts.” She agreed
with Furth, Seibold and Rathbone (13) that the malignant cell is a pathological lymphocyte and not a lymphoblast. In a note at the end of the paper,
however, she reported that in one instance “ the malignant cells appeared to be
lymphoblasts.”
Cells of certain structure may for a particular line of leukemia be called
“malignant.” I t is difficult, however, to set up general criteria for malignancy, since cells of distinctive morphology characterize each separate transmission-line and morphological features found in many leukemic cells, as
nucleoli, sieve-like chromatin pattern, cytoplasmic basophilia, etc., are found
also in normal cells of the hemopoietic organs. Prominence of nucleoli and
very distinct nuclear chromatin pattern (Plate I ) , due probably to greater
stainability of the chromatin, characterized many leukemic cells observed in
this study.
Physiologically a vast difference exists between the morphologically similar
immature cells of normal mouse hemopoietic organs and those of the leukemic
infiltration. The former do not have the capacity of multiplying wildly and
infiltrating tissues when introduced into a normal mouse of the same inbred
strain. Whatever the origin of the first leukemic cells of the spontaneous case,
transplantation experiments have proved that, once they are produced, they can
divide and metastasize like cancer cells. There is no conclusive evidence that
the introduction of leukemic cells into a normal susceptible mouse provokes
hyperplasia or metaplasia of host tissues. The fact that each line of leukemia
has cells of a rather specific morphology would argue against reaction of host
tissue. It is unlikely that the action of each line is so specific that the new
host’s mesenchymal tissues react to produce cells morphologically identical
with those introduced.
Leukemic cells of man have been said to arise from the reticular cells of
FIG. 32. LUNG,MYELOGENOUS
LEUKEMIA,
STH TRANSFER
GENERATION:
HEAVYINFILTRATION
BETWEEN
ALVEOLI
One vessel is filled with leukemic cells of the circulating blood; a portion of the other vessel
shown has numerous cells in one portion of the lumen. Helly-Dominici. X 70.
FIG. 33. LIVER,SPONTANEOUS
LYMPHATIC
LEUKEMIA
Periportal connective tissue is heavily infiltrated with leukemic lymphocytes. Infiltration in
sinusoids is more diffuse. Helly-Dominici. X 70.
FIG. 34. LYMPHNODE,SPONTANEOUS
LYMPHATIC
LEUKEMIA
Sinuses are open; otherwise architecture is lost. Sinuses are usually obliterated by leukemic
cells, the node then being a mass of lymphocytes with no special arrangement. Helly-Dominici.
X 70.
FIG. 35. SPLEEN,SPONTANEOUS
LYMPHATIC
LEUKEMIA
This picture at high magnification shows the large leukemic lymphocytes which have crowded
out all normal lymphocytes. Red and white pulp could not be distinguished in this spleen, all
areas having the structure shown. Helly-Dominici. X 700.
412
ARTHUR KIRSCHBAUM AND LEONELL C. STRONG
the lymph nodes ( 16) and the mesenchymal tissues of other organs (17).
Reticular hyperplasia was evident (dry imprints) in small leukemic nodes of
spontaneous cases in the F strain. Whether this was merely a reactive process or the first stage in leukemic cell formation cannot be proved by observation alone. Transplantation experiments indicated that leukemic cells form
tumors when injected subcutaneously and that cells metastasize from such tumors to produce systemic leukemia. In transplanted leukemia the reticuloendothelial system was not observed to participate in the formation of leukemic
cells. Potter, Victor and Ward (18) have observed, however, that in spontaneous cases in the C58 strain of mice the preleukemic change in lymph node
or liver is probably one of reticular cell proliferation. Removal of a node
showing such proliferative activity in the medullary portion could in some
instances prevent the appearance of spontaneous leukemia for many months,
whereas transplantation of the node into normal mice would produce systemic
leukemia.
SUMMARY
(1) Observations were made on 17 cases of spontaneous leukemia and
lymphosarcoma in the F strain of mice. Two hundred and ninety-five F mice
developed leukemia as the result of inoculation of leukemic cells. F strain
mice were 100 per cent susceptible to inoculation of leukemic cells.
( 2 ) Lymphosarcoma, myelogenous leukemia, and lymphatic leukemia may
be grouped together as “ leukemic conditions ” appearing frequently in the F
strain. Myelogenous leukemia was transplanted with the same technic as
lymphatic leukemia, that is, by either subcutaneous or intraperitoneal inoculation of cells.
( 3 ) The rather specific morphology of each line of leukemic cells during
frequent transfer suggested that mice receiving transplants developed leukemia
as the result of proliferation of introduced cells. There was no positive evidence that reticulo-endothelial or other mesenchymal cells played a r6le in the
histogenesis of transplanted leukemia.
( 4 ) The leukemic cells appeared to be neoplastic. Local tumors were
formed by these cells when they were introduced subcutaneously into normal
mice; subsequently systemic leukemia usually appeared. When the cells were
introduced intraperitoneally systemic disease resulted, with or without tumor
formation in the peritoneal cavity.
( 5 ) Leukemic cell types showed a wide degree of cytological variation,
there being no specific cytomorphological criteria for malignancy. The “ immature” cells of human leukemia are morphologically similar in many instances to the “ leukemic ” cells of the tissues and blood in certain lines of
mouse leukemia.
( 6 ) Young cells of normal hemopoietic organs of the mouse have certain cytological characteristics in common with leukemic cells (dry imprint
technic). These are ( a ) nucleoli in the nucleus, ( b ) sieve-like nuclear
chromatin pattern, (c) cytoplasmic basophilia of young lymphocytes, ( d )
myeloid azure granules in the cytoplasm of myeloblasts. Lymphoblast nuclei
of mouse leukemia have more prominent nucleoli and are more “hyper-
LEUKEMIA IN F STRAIN OF MICE
41 3
chromatic ” (intense chromatin stain) than the nuclei of lymphoblasts coming
from normal lymph nodes.
NOTE:The authors wish to acknowledge their gratitude to Mrs. Charmian Johnstone
Manning for her painstaking work in drawing the blood cells of Plate I.
ADDENDUM:
Nine lymphatic and 4 myelogenous leukemias have been observed since completion of this manuscript. Twenty-one leukemias and 2
lymphosarcomas have appeared in the last 62 animals permitted to live beyond
six months of age. Thirty spontaneous cases have been studied microscopically; 8 of these were myelogenous leukemia, 2 were mediastinal lymphosarcoma. The microscopic studies were made on mice of the 29th to 31st
inbred generations.
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5
The stocks of mice referred to by Hall and Knocke were developed by J. Furth.