The Department of Medicine, St. Elizabeth's Hospital
and Tufts Medical School, Boston, Massachusetts
*Supported in part by Grants HL 7542 and 5600 from the National
Heart and Lung Institute
Before considering the problem of leukemia it seems appropriate
to briefly review the normal regulation of hemopoiesis. I shall
confine my remarks to normal myelopoiesis and those leukemias associated
with disorders of myelopoiesis i. e. acute myelocytic, progranulocytic
and undifferentiated stem cellleukemias as well as chronic myelocytic
leukemias. A schematic outline of normal myelopoiesis (1) is shown
in figure 1. It is to be noted that the myeloid system shares a
common precursor cell with the erythroid aand megakaryocytic series.
This common precursor cell is referred to as the pluripotent hemopoietic
stem cell. Intermediate between the pluripotent stem cell and those
recognizable myeloid elements, which may be identified under the
light microscope, is a compartment which is committed to myelopoiesis.
Similar compartments exist for the erythroid and megakaryocytic
series. There is evidence) which has recently been reviewed ( 1),
that suggests that monocytes and myeloid elements derive from a
common committed precursor cell in which case the committed myeloid
stem cell compartment can not be considered truly unipotent. Further
the myeloblast and progranulocyte differentiates along one of three
lines the neutrophil, eosinophil or the basophil. We do not know
at what stage this commitment is made i. e. committed stem cell,
myeloblast or progranulocyte, nor what is the decision making mechnism.
In animals there is a technique to evaluate the pluripotent stem
cell compartment (2) but in man it cannot be assayed and inferences
must be drawn from animal experimentation. Briefly the assay for
the pluripotent stem cell or CFU consists of transplanting marrow
or spleen from the animals in question into heavily irradiated syngeneic
mice. The animals are sacrificed after 8-10 days by which time discrete
colonies are formed in the spleen. These colonies arise from a single
cell and morphologically may be erythroid, myeloid, megakaryocytic,
Fig. 1: Schematic representation of hemopoiesis: MK-Megakaryocytes;
MY -Myeloid; ER-Erythroid; EP-Erythropoietin; CSF-Colony Stimulating
Factor; NRA-Neutrophil Releasing Activity; solid line (EP) indicates
established in vivo activity; dashed line (CSF) indicates putative
activity. (From Progress in Hematology).
mixed. when a colony is dissected out and transplanted into a secondary
recipient, the colonies formed from this innoculum will have the
same spectrum of morphologic types as seen in the primary transplant,
indicating the pluripotent characteristics of the cells giving rise
to colonies as well as the capacity for self renewal. The committed
myeloid stem cell (CFC) may be evaluated in vitro by culturing bone
marrow in soft agar (3,4 ). The growth of cells from murine marrow
is dependent upon the presence of a glycoprotein-colony stimulating
factor. In man, however, autonomous growth is observed when the
cell concentration in the dish is sufficiently high. The latter
presumably reflects the presence in human marrow of a cell capable
of producing the colony stimulating factor. This cell is most likely
the monocyte ( 5, 6) . The hormone erythropoietin is responsible
for the differentiation of erythroid committed cells into pronormoblasts
and prolonged administration produces a sustained erythrocytosis.
It is tempting to suggest that colony stimulating factor serves
a similar function for myelopoiesis but as yet firm evidence in
the intact animal to establish a physiologic role of CSF has not
been forthcoming. There are, however, indirect data from which it
has been infered that CSF differentiates the myeloid committed stem
cells into myeloblasts in the intact animal (1). After differentiation,
the myeloid cells undergo a series of divisions while at the same
time maturing. The capacity for further DNA synthesis ceases at
the myelocytic stage but there is further maturation of the granulocytic
elements within the bone marrow prior to release into the peripheral
blood. Thus the bone marrow contains a large storage pool of nonproliferating
granulocytes, metamyelocytes, bands and mature polymorphonuclear
cells. It has been shown experimentally that certain experimental
manipulations (7, 8), e. g. the administration of endotoxin, effect
the release of a protein, which is capable of releasing granulocytes
from the storage compartment into the peripheral blood; this plasma
factor has been referred to as neutrophil releasing activity (1).
The mature granulocytes have a relatively short intravascular sojourn,
the half-time being of the order of 6.7 hours (9). During this time
the granulocytes are distributed between the central circulating
blood and the margins of capillaries ( 9) .Thus there is a marginal
and circulating pool of granulocytes, which are normally in equilibrium.
Granulocytosis may result from a shift of cells from the marginal
to the circulating pool, as is seen after epinephrine. There are
several lines of evidence to suggest that myelopoiesis normally
is governed by a feedback loop from the peripheral blood which is
regulated by a function of the circulating neutrophils. A feedback
loop requires a time delay between measurement of a particular function
in the peripheral blood and correction by changing the production
rate. This time delay would include an interval for the production
of a regulatory hormone, its effect on the committed stem cell and
the time required for maturation of the differentiated myeloblast
and its progeny. From this consideration one would expect oscillations
of the peripheral neutrophil count and further that the period of
these oscillations would be twice the time delay for differentiation,
maturation and release. Normally oscillations in the peripheral
granulocyte values are not seen. This is thought to reflect the
dampening effect of the intramedullary storage pool ( 10). If so,
then removal of the storage compartment should lead to detectable
oscillations in the peripheral granulocyte count. Morley and I (11)
observed that when animals were given cyclophosphamide, thus reducing
the stem cell compartment and in consequence the storage compartment,
a cyclical neutropenia developed and the period of the oscillations
was twice the projected time delay for bone marrow transit. Further
evidence for feedback control of granulopoiesis was provided by
studies in which one hind leg of an animal was shielded while the
remainder of the body was irradiated (12,13). The shielded limb
permitted us to measure the myeloid response to neutropenia. Different
levels of neutropenia were produced by varying the dose of irradiation.
As may be seen in figures 2 and 3 the concentration of both the
committed myeloid compartment, as assessed by the CFC technique,
and the differentiated proliferating myeloid cells (myeloblasts
and promyelocytes) varied as a log-linear function of the peripheral
neutrophil concentration. These data therefore provide firm evidence
that myelopoiesis is regulated by a feedback loop from the peripheral
blood. It seems most likely that the mechanism regulating myelopoiesis
is not the neutrophil concentration but rather a function thereof.
We have observed, as neutropenia developed, an increase in colony
stimulating factor ( 14) and a decrease in lipoprotein inhibitor
of in vitro colony formation in irradiated animals ( 15) .The time
course suggested that the increased generation of CSF might be due
to bacteremia and endotoxemia. This notion was supported by the
observation that germfree animals did not develop colony stimulating
factor after irradiation as did conventional animals (16 ). when
treated with endotoxin, however, germfree animals did produce CSF
(15 ). This led to the concept that endotoxemia was important in
CSF generation and in further experiments in normal animals a dose
response relationship was observed between the dose of endotoxin
and the amount of colony stimulating factor generated ( 17). It
is attractive to suggest therefore that the regulation of myelopoiesis
in a significant measure is related to intermittent bacteremia or
endotoxemia and perhaps also the breakdown products ofnormal tissues
which result in the release ofpyrogens or endotoxin. Thus in the
face of local infection or tissue necrosis one would
Fig. 2: Effect of polycythaemia on the size of the myeloblast-promyelocyte
compartment on day 4 after various doses of irradiation. Each point
shows the myeloblast-promyelocyte compartment compared with the neutrophil
count from a polycythaemic (White square) or normal (black square)
mouse. The regression lines were calculated by the method of least
squares. In each case the size of the myeloblast-promyelocyte compartment
is significantly (P > 0.01) related to the logarithm of the neutrophil
count but the regression line for the polycythaemic animals lies significantly
(P > 0.01 ) above that for the normal animals. (From Morley et al.
1970, reprinted from Br. J. Haematol. (24)).
Fig. 3: The relationship between the size of the CFC compartment
and the blood neutrophil count in ILS mice for days 2 -8 after irradiation.
CFC are the average of mean values for composite results of at least
three experiments per point. Correlation coefficient r and the regression
line are calculated from these values and are significant when p
> 0.005. (From Rickard et al. 1971. reprinted from Blood (13).)
anticipate increased CSF production which would produce the granulocytosis,
which is seen under such circumstances. Neutropenia of course is
associated wjth infection and increased CSF generation has been
reported ( 18) . This hypothesis is appealing but there are still
a number of questions to be resolved, principally the problem of
tolerance. It is well known that an animal repeatedly injected with
endotoxin will become tolerant to its pyrogenic effects and this
has been observed in respect to colony stimulating factor (15).
Moreover, there appears to be cross-tolerance between various endotoxins.
A major problem in assessing the granulopoietic response to avariety
ofstimuli has been the storage pool. This has made it difficult
to separate granulocytosis due to a true increase in proliferation
from release of granulocytes from the storage pool. Further, the
possibility exists that the depletion of the storage pool triggers
an intramedullary feedback loop which may affect myelopoiesis. The
use of the millipore diffusion chamber has been introduced to circumvent
this problem (19, 20). Normal bone marrow is implanted into these
chambers which are then placed intraperitoneally into host animals.
Manipulation of the hosts prior to implantation of the chambers
provides an opportunity for the study of growth regulation within
the in vivo culture system. Using this technique Tyler and his associate
(21) have demonstrated that the proliferation of pluripotent stem
cells as well as the growth of myeloid elements is enhanced in neutropenic
animals. The best explanation for these results is that a humoral
factor is produced in the neutropenic host which diffuses into the
chamnbers. The nature of this humoral factor and its relation to
the colon y stimulating factor have not been established. From the
above We may conclude that rn yelopoiesis is governed by a feedback
regulatory mechanism from the peripheral blood and tissues. There
is indirect evidence to suggest that this regulation may be accomplished
by the glycoprotein, colony stimulating factor, which is responsible
for the differentiation of myeloid elements in the soft agar culture
system. There is, however, no direct in vivo evidence that CSF plays
this central role. Finally the mechanisls governing the production
of CSF have not been completely resolved. It is clear that many
tissues have the capacity to produce this protein and that endotoxin
plays an important role in its generation. Further studies, however,
will be necessary before final definitive conclusions can be drawn
about its ph ysiologic role in the regulation of mnyelopoiesis.
The Leukemic Cell
The two principal types ofmyelocytic leukemia, are the acute leukemmias,
including acute myelocytic, progranulocytic and myelomonocytic leukemia,
and chronic nnyelocytic leukemia. Patients with chronic myrelocytic
leukemia after a variable period of time may enter what is termed
a blast cell crisis which in all respects appears to be similar
to acute myelocytic leukemia. This is most likely a reflection of
the natural history of the disease although the role of chemotherapeutic
agents and irradiation in effecting the transformation from CML.
to blast cell crisis has not been fully evaluated. It is clear from
cytogenetic studies that chronic myeloytic Ieukemia is basically
a disease of the pluripotent stem cell compartment. The vast majority
of patients have an abnormal chromosome, the Philadelphis chromosome
(PHI) which is seen in erythroid and megakaryocytjc cells as well
as in the myelocytic; cells. Differentiation in many respects is
normal and growth by and large is confined to the bone marrow and
spleen, which might be considered a normal hemopoietic organ~ The
lack of the enzyme alkaline phosphatase in the mature granulocytes
of CML indicates that differentiation is not entirely normal. Further,
the intravascular life of the granulocyte in CML is prolonged. Immature
cells, myelocytes, promyelocytes and myeloblasts are released into
the peripheral blood suggesting some change in the transit pattern
from bone marrow to peripheral blood. It has been suggested that
the release of granulocytes is normally regulated by membrane properties
(22). If so, then the premature release of immature cells suggest
a change in the membrane and is further evidence of abnormal differentiation.
Although there are these demonstrable abnormalities of the granulocytes,
it is clear that in some measure normal regulatory mechanism persists.
Oscillation of the peripheral white count and also the platelet
count has been documented in a number of patients with CML (23),
indicating the existence of a feedback mechanism. In most instances,
however, the period of oscillation is longer than normal. The prolonged
life span of the granulocytes together with the retention of a measure
of feedback control suggests that during its initial phase chronic
myelocytic leukemia in part is an accumulative disease. Undoubtedly
there is additional input from the stem cell compartment, because
arithmetically the prolonged intravascular life span could not account
for the level of granulocytosis seen in some patients nor for the
oscillation seen in those where oscillatory phenomenon have been
documented. The acute myelocytic leukemias may show varying patterns.
In some, examination of the bone marrow reveals only abnormal myeloid
elements; in others erythropoiesis and megakaryocytopoiesis may
be present. Even during the preleukemic phase there may be identifiable
morphologic abnormalities in megakaryocytes and erythroid elements.
when cytogenetic abnormalities exist, these may be present in the
megakaryocytes and erythroid elements as well as the myeloid. These
observations suggest that acute myeloblastic leukemia at least in
some instances originates from the stem cell. It seems likely to
me that this is true for all patients with acute myeloblastic leukemias.
Of paramount importance in considering the pathophysiology of acute
myelocytic leukemia is the fact that complete remissions can be
achieved with chemotherapy and normal hemopoiesis re-established.
Thus, normal pluri-potent stem cells must be present in the bone
marrow together with the leukemic stem cells. This suggests the
existence of clones of pluripotent cells some of which are leukemic
the other normal. Thus, if leukemia is of viral origin the abnormal
information transmitted by the virus is present in some but not
all pluripotent stem cell lines. One might suggest as a corollary
that in some instances the viral infection of a stem cell will result
in a lethal transformation and death of what might otherwise have
become a leukemic clone. In others non-lethal information develops
and an abnormal line of differentiation results producing leukemia.
In still others the viral infection may not be manifest or affects
DNA in such away that normal myelopoiesis is unaffected. The alternative
explanation of course would be that some stem cells become infected
with virus but others do not. Since the introduction of the soft
agar technique for the culture of myeloid precursors there has been
considerable interest in a number of laboratories as to the role
of the myeloid committed stem cell and the differentiating colony
stimulating factor in leukemic patients. It has been reported that
normal differentiation may be achieved when bone marrow from aleukemic
patient is cultured in vitro. In a rather large survey Moore (24)
found that the marrow from about 60% of patients with acute leukemia
developed small clusters in culture rather than normal colonies,
20 % developed colonies and 20 % failed to grow. The central issue
is whether in cultures of bone marrow from leukemic patients normal
colonies develop from a normal pluripotent stem cell or under these
cultural conditions are normal myeloid elements differen tiated
from a leukemic stem cell line ? Moore and Metcalf ( 25) have reported
that cytogenetic markers present in leukemic cells were found in
all metaphases of cultured leukemic marrow. Unfortunately pooled
rather than single colonies were examined. These data indicate that
leukemic cells are proliferating in culture but do not provide the
final answer that normal granulocytes develop in culture from leukemic
progenitors. To be candid there is relatively little data published
on the morphologic aspects of the differentiated colonies from culture
of leukemic cells and in a recent conference the photomicrographs
which were shown of leukemic cultures did not persuade me that anyone
had succeeded in differentiating leukemic cells into normal myeloid
elements. It has also been suggested by Mintz and Sachs (26) that
an inhibitor may be involved in the failure of leukemic myeloblast
to differentiate. Clearly, further information is needed on the
characteristics of leukemic cells in the soft agar system. Are normal
stem cells differentiating? Are leukemic cells capable of normal
differentiation and the production of normal differentiated polymorphonuclear
cells? What, if any, is the role of colony stimulating factor in
the failure of differentiation? What, if any, is the role of inhibitors
in this failure of differentiation ? The concept that myeloid leukemia
represents merely a failure of differentiation from the myeloblast
stage due to an impaired interaction between colony stimulating
factor and the target cells seems to oversimplify the issue. This
would be somewhat analogous to pernicious anemia in which the morphologic
abnormalities may be as striking as those seen in acute leukemia.
This disease has been shown to be the result of abnormal differen
tiation due to a deficiency of B 12. Administration of the vitamin
immediately corrects the abnormal proliferation. I t seems to me
that those who consider acute leukemia in this light overlook one
major and important characteristic of the leukemic cell. In pernicious
anemia the abnormal cellular proliferation is confined to the normal
areas of hemopoiesis -the bone marrow. In acute myelocytic leukemia
as in other neoplastic diseases proliferation occurs not only in
the site of normal cell production but elsewhere in the body. Thus
in acute myelocytic leukemia leukemic proliferation may be seen
virtually anywhere, the skin, the heart, the liver, brain, etc.
Thus the normal mechanisms which restrict cell production to a specific
anatomic area are no longer operative. It seems most likely that
the normal mechanism controlling the site of cell production is
an interaction between the cells and their immediate environment
or microenvironment. Thus in acute myelocytic leukemia the growth
pattern reflects not only a failure of differentiation but other
cellular abnormalities, most likely in the membrane structure, which
permit metastatic growth. In this respect acute myeloblastic leukemia
appears to be more than a simple failure of differentiation of the
myeloblast. For this reason it seems unlikely that the resolution
of acute leukemia will result from efforts directed solely at differentiation
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