Preleukemia Cytogenetic Clues in Some Confusing Disorders Peter C. Nowell, MD
A number of disease states are considered "preleukemic" because they carry a significantly increased risk for the subsequent development of frank leukemia. These include a variety of cytopenias, myeloproliferative disorders, and childhood syndromes. Cytogenetic data suggest that these preleukemic disorders may not be qualitatively different from leukemia but simply represent quantitative differences in the degree of selective growth advantage enjoyed by a proliferating abnormal hemic population. Recent chromosome studies have indicated that a) this proliferation is characteristically clonal in both preleukemia and leukemia, apparently resulting from a heritable change in a marrow stem cel that allows it to escape to some degree from normal growth regulation; b) genetic instability in the clone, with additional genetic change, may often underlie clinical progression from the relative indolence of preleukemia or chronic leukemia to an aggressive stage comparable to acute leukemia; and c) certain specific chromosome segments carry genes important in the acquisition of growth advantage by hematopoietic stem cells, and many of these are common to both preleukemia and leukemia. Expansion of hemic clones may also be influenced significantly by alterations in the growth control mechanisms themselves. For instance, in various preleukemic states, preexisting marrow hypoplasia may permit clones with only minimal selective advantage to reach demonstrable size. Chromosome findings may help to establish the diagnosis and prognosis in preleukemic disorders, but additional long-term data are needed. (Am J Pathol 89:459-476, 1977)
ATTEM1PTS TO DISCUSS HUMAN PRELEUKENMIA are made difficult bv lack of agreement on a definition. In this paper, I will use the term rather broadlv and encompass three groups of disorders which carrv an increased risk of ultimately developing frank leukemia. It is recognized that these groups may overlap and that the risk of leukemia mav range from near 100% (perhaps better termed preclinical leukemia) to less than 10%. These three groups include: 1. A spectrum of marrow stem-cell dyscrasias characterized by reduction of one or more formed elements of the blood (cytopenia). The fully developed form is the pancytopenic state with near-normal marrow cellularitv and increased myeloblasts described by Linman and his colleagues From the Department of Patholog-, Univ ersity of Pennsv lvania School of MIedicine. Philadelphia. Pennsv l ania. Presented at the Seventy-fourth Annual Meeting of the American Association of Pathologists, Toronto, Ontario, Canada. March 15, 1977. Supported by Grants CA-1279 and CA-13822 from the N'ational Cancer Institute. N'ational Institutes of Health. Address reprint requests to Dr. Peter C. Now-ell. Department of Pathologp. School of Medicine, Universitv of Pennsvlsania, Philadelphia, PA 19104. 459
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as the preleukemic syndrome.1 These authors have suggested that various unexplained anemias (e.g., refractory anemia, sideroblastic anemia) may represent the earliest stage of this process and that depression in platelets, granulocytes, and monocytes appears subsequently. A high proportion of these cytopenias ultimately progress to nonlymphocytic acute leukemia. 2. Myeloproliferative disorders, as defined by the Polycythemia Vera Study Group.2 These are also considered marrow stem-cell dyscrasias and include polycythemia vera, myelofibrosis, undifferentiated myeloproliferative disorders, and primary thrombocytosis. A small proportion eventually progress to leukemia. 3. Certain congenital and childhood disorders carrying a somewhat increased risk of leukemia. These include the so-called chromosome breakage syndromes (Bloom syndrome, Fanconi anemia, ataxia telangiectasia, etc.),3 as well as some cases of unexplained marrow aplasia and hypoplasia. It is the major purpose of this paper to suggest that these various preleukemic disorders, as well as frank leukemia and lymphoma, are typically characterized by a clone' of cells with selective growth advantage replacing a proportion of the hematopoietic system. The difference between preleukemia and leukemia is considered as only quantitative, relating to the degree of selective advantage which the abnormal clone enjoys and the rate at which it is expanding at the expense of normal cells. The spectrum from preleukemia to chronic leukemia to acute leukemia is viewed as a continuum, with the distinction between "preneoplasia" and " true" neoplasia being essentially an arbitrary one determined by clinical necessity (Text-figure 1). Recent chromosome studies support this concept in three general areas: a) Proliferating clones of hemic cells have been identified by chromosome abnormalities in all of these disorders, preleukemic and leukemic. b) These clones appear to be genetically and cytogenetically unstable to some degree. Additional mutations, sometimes visible at the chromosome level, seem to occur with increased frequency, leading to selection of more deviant subpopulations and clinical progression from preleukemia or chronic leukemia to an acute leukemic phase. c) The cytogenetic abnormalities in these hemic clones are nonrandom; certain chromosome segments are involved with increased frequency, often with characteristic types of rearrangements. Many of these nonrandom changes are identical in preleukemia and leukemia. ° Clone is used here to indicate a population of cells descendant from a single cell of origin. It does not imply complete homogeneity, as subpopulations may coexist within the clone.
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The following sections will include a brief review of the cytogenetic evidence that both preleukemic and leukemic disorders do indeed involve clonal proliferation and that clinical progression to a more aggressive stage may be associated with additional genetic alterations in the expanding clone demonstrable at the chromosome level. Possible mechanisms underlying the development and progression of such clones will be considered, as well as the specific chromosome abnormalities involved. Some theoretical and practical implications will be suggested. It must be remembered throughout, however, that the evidence presented is cvtogenetic, and such observations always represent a relatively crude means of assessing genetically determined alterations in cells. Clones in Preleutemia and L*ukenia Chromosomal evidence for the clonal nature of these disorders and for clonal evolution in their development can be considered in reverse order of malignancy: acute leukemia, chronic leukemia, preleukemia. In the acute leukemias, with the greatest selective growth advantage, chromosome abnormalities are present in the neoplastic cells in approximately half of the cases. The same cytogenetic change is typically present in all leukemic cells in a given individual, indicating that the neoplastic population is a clone, the progeny of a single abnormal precursor. The altered karyotype may show only minor changes or may be highly aneuploid. Although the chromosomal pattem is often quite stable in the acute leukemias, clinical progression, especially resistance to therapy, has been associated with additional cytogenetic alterations in some instances. Recently, Rowley and Potter have described such cytogenetic evolution in 8 of 25 chromosomally abnormal acute leukemias studied by banding methods.4 Chronic granulocytic leukemia (CGL) has been more extensively studied cytogenetically than any other disorder of hematopoiesis. Typical CGL apparently results from a slight growth advantage afforded to a marrow stem cell by the Philadelphia chromosome. In a few instances, the expanding clone in the marrow has been identified by the Ph chromosome before the disease was clinically or hematologically demonstrable.5 When the inevitable alteration to the terminal "blast crisis" occurs, this seems to represent emergence of a new subpopulation, with the growth characteristics of acute leukemia, resulting from additional genetic alteration in the original Ph-positive clone (Text-figure 1 ).*7 This further genetic change in the neoplastic population can be recognized by additional chromosome abnormalities in about 75% of cases. With respect to the preleukemic disorders which were defined above,
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TEXT-FIGUtE 1-Simplified diaillustrating the hypothesis
gram w
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that the significant difference between acute leukemia, chronic . >N/ and preleukemia is the leukemia, .- ,)4,/rate at which an abnormal hemic clone is expahding. Progression oL / .- /from chronic to acute leukemia or from preleukemia to clinical leukemia (dotted arrows) reflects inCLINICAL DIAIS OF LEUKEIIIA creased rate of clonal expansion -due to additional genetic change in the clone (e.g., further chromosome alteration in CGL) or decrease in external growth regulation.
TIME
clonal proliferation in the marrow, similar in many respects to the frank leukemias, has been demonstrable cytogenetically in a proportion of cases in each group. In some instances, additional chromosome changes have been observed in association with clinical progression to frank leukemia. Pierre 8 has studied 205 patients with various of the cytopenic dyscrasias previously described. He has found marrow chromosome abnormalities in 36%. In 17 cases followed sequentially, cytogenetic evolution was associated with the appearance of acute leukemia in eight instances. Similar results have been reported in the myeloproliferative disorders (polycythemia vera, myelofibrosis, etc.), although data relating to transition to leukemia are sparse because of its relative infrequency. It appears that perhaps 25% of untreated patients with these disorders have cytogenetically abnormal clones recognizable in the bone marrow and occasionally in the blood if immature forms are circulating.9-'3 Typically, these clones do not show extensive aneuploidy but have alterations involving only one or two chromosomes. The myeloproliferative disorders often have a relatively stable course without progression for a number of years, but an increased risk of ultimately developing leukemia, even without therapy, is recognized. The leukemic phase is characteristically associated with cytogenetic alterations in the neoplastic clone, and in several instances, it has been possible to correlate additional chromosome changes with progression from polycythemia vera to acute leukemia.12"3 More data will be forthcoming from long-term studies currently in progress under the Polycythemia Vera Study Group. In the childhood disorders considered preleukemic because of increased risk of the development of reticular neoplasms, data are insufficient to
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provide estimates on the frequencv of cytogeneticallv abnormal clones. Some information is available, however, particularlv on the chromosome breakage syndromes (Fanconi anemia, ataxia telangiectasia, Bloom svndrome).3 These patients show disordered hematopoiesis and immune deficiency to varving degrees, and in the first two syndromes, cytogeneticallv identifiable clones have been demonstrated in the hemic tissues of a number of individuals. In some cases, these have shown little tendency to expand and obviously represent a minimal growth advantage.14'15 In 1 patient, however, progression of such a disorder to frank leukemia has been associated with additional cytogenetic alterations within a proliferating clone.14 It is possible that preexisting marrow hypoplasia, whether or not it is associated with a chromosome breakage syndrome, may contribute to the emergence of clones with minimal growth advantage. We have recently observed 2 children with unexplained marrow hypoplasia and relatively stable, chromosomally abnormal clones in the hemic tissues (Figures 1 and 2). In 1 patient, a second clone, franklv leukemic, emerged in the bone marrow after several vears. Perhaps these patients had an unrecognized form of congenital instability of the genetic apparatus, but chromosome fragility was not demonstrably increased in lymphocyte cultures. Additional cases of childhood marrow aplasia and hy,poplasia, unrelated to recognized causes or syndromes, should be investigated cytogenetically; these dvscrasias carrv an increased risk of leukemia, and the present limited data suggest that further study might clarify their relationship to other clonal hemic disorders. It should be noted that the frequency of cvtogeneticallv abnormal clones in various preleukemic states, which has just been described, stands in clear contrast to the findings in normal individuals. Except for subpopulations of cells without a Y chromosome in elderly males (which seem to function normally),1' hemic clones are essentially never seen in the general population. In general, then, presently available cvtogenetic data support the view that clonal proliferation underlies a spectrum of disorders ranging from acute leukemia, with rapid expansion of the clone, to preleukemic states, in which the clone may have only a minimal growth advantage. The chromosome data also support the concept of genetic instability in these clones, with additional genetic changes, sometimes visible at the chromosome level, providing the basis for progression from preleukemia or chronic leukemia to a more rapidlv expanding and more clinicallv aggressive process (Text-figure 1). It should be pointed out that although this discussion has been restricted to cytogenetic findings, studies of enzvme
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patterns, immunoglobulins, and blood groups also indicate the clonal nature of both preleukemia and leukemia in man.17"18 Possible factors underlying the initial acquisition of growth advantage in such clones, as well as their subsequent progression, may now be considered further. Regulation of Hematopoiesis and Selective Growth Advantage in Hemic Cell Clones For a hematopoietic cell population to function as a malignancy, it need
only acquire the capacity to expand rapidly, at the expense of normal cells. The usual distinction between benign and malignant does not apply, as hemic cells invade and metastasize physiologically. Thus, unlike other organs, stage of malignancy and tumor progression in hematopoietic tissues need only reflect quantitative differences in the degree to which neoplastic cells have escaped from normal regulators of proliferation. Considerable evidence indicates that acquisition of such a selective growth advantage by hemic cells does not result from an increase in mitotic rate. Rather, it appears to reflect a change in the normal balance between proliferation and differentiation in the stem cell pool. The first step may well involve alteration in a single proliferating marrow cell, in one or another of the various stem cell compartments, such that the normal balance between the progeny of the aberrant cell which continue to proliferate and the progeny which differentiate is shifted in the direction of proliferation. Depending on the extent of the shift, the stem cell pool may expand gradually, producing the clinical picture of chronic leukemia with increased numbers of mature elements, or it may expand very rapidly, with concomitant marked reduction in differentiated forms, leading to clinical acute leukemia. The preleukemic disorders can be thought of as circumstances in which the shift in stem cell kinetics is minimal, so that clonal expansion proceeds at such a gradual rate and/or only to such a point that the clinical designation of leukemia is not warranted (Text-figure 1). In considering the growth regulatory mechanisms from which the altered stem cell may have escaped to some degree, there are at least four possibilities: a) local feedback controls from differentiated cells of the same lineage (chalones), perhaps best documented in the granulocytic series; 19 b) circulating hormones, such as erythropoietin; c) regulatory factors produced by nonhemic cells (particularly, reticular or fibroblastic elements) in the local microenvironment of the marrow; 20,21 and d) the immune system, with both stimulatory and suppressor effects of lym-
phocytes.22'23
None of these control mechanisms, except for erythropoietin, has been
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adequately characterized, but there is at least some evidence that each may play a role in the normal regulation of hematopoiesis and, further, that both leukemic and preleukemic human cells may show an altered response to such controls. Studies in tissue culture, particularly with the soft agar technique, are beginning to delineate more precisely some of these factors regulating hematopoiesis.2' The means by which a marrow stem cell might acquire this postulated altered response to growth regulators is perhaps most easily envisioned as a carcinogen-induced heritable change, analogous to the neoplastic transformation of fibroblasts induced in tissue culture by viruses, chemicals, or radiation. As with transformation in vitro, this heritable alteration in a stem cell could result from structural mutation, with or without visual chromosome change, or could initially be a reversible alteration in gene expression not involving structural damage to the genetic apparatus.25 The specific gene product(s) involved in escape from growth control are not known, but again an analogy to transformed cells in tissue culture might suggest a structural alteration in the glycoproteins or other elements of the external cell membrane, changing the stem cell's response to intercellular signals which normally regulate its proliferation. It should be recognized that, even in the acute leukemias, this autonomy is by no means absolute; there is both in vivo and in vitro evidence that in all of the various hematopoietic disorders under discussion, the cells still retain some degree of response to normal controls, and the postulated membrane change could well be quantitative not qualitative.2e In this regard, it is worth considering not only alterations in the proliferating stem cell and its progeny, but also the possibility of abnormalities in the external regulatory mechanisms as well. The relative importance of intracellular versus extracellular defects in the natural history of the various hemic disorders under consideration is by no means clear. One can consider that alterations in external regulatory factors might not only affect the initial establishment of hemic clones but also significantly influence the direction of differentiation within a particular clone at various times, determining whether granulocytes, monocytes, or erythrocytes are most prominent, and even account for the occasional disappearance of chromosomally demonstrable marrow clones.27 Farber " has pointed out that damage to the local parenchyma by liver carcinogens may play a very important role in the emergence of preneoplastic nodules, and similar considerations may obtain in the bone marrow. I have already noted that marrow hypoplasia in childhood may precede the appearance of both stable and aggressive clones (Figures 1 and 2). Similarly, marrow depression in adults, produced by benzene
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toxicity or by chemotherapy for multiple myeloma,2' may be followed by a sequence of events very similar to the preleukemic cytopenias described by Linman and his colleagues, eventuating in acute nonlymphocytic leukemia.' It is possible that some degree of unrecognized marrow suppression may be a primary event in many cytopenic preleukemic disorders, allowing a clone, initially only slightly altered, to begin its expansion. Such postulated hypoplasia might, in some cases, have an autoimmune basis; aplastic anemia mediated by suppressor T cells has recently been described.23 Clearly, more information is needed, not only on hematopoietic chalones and hormones, but also on the importance of the local microenvironment and the immune system in controlling both normal and abnormal hematopoietic proliferation and differentiation. This section can be concluded by simply listing several questions which may warrant further consideration: What growth regulatory influence do reticular cells have in the marrow? What is the significance of the fibrous tissue proliferation in myelofibrosis (it appears not to be part of the preleukemic hematopoietic clone 30)? Under what circumstances do lymphocytes and the immune system play a role in inhibition or stimulation of hematopoiesis? Chromosomal Sites Associated With Selective Growth Advantage in Hemic Cells Although specific genetic alterations responsible for the initiation of hemic neoplasia remain unknown, studies with the new chromosome banding techniques are beginning to identify cytogenetic alterations which occur nonrandomly in these disorders and which may suggest the location of genes important in the acquisition of selective growth advantage by a marrow stem cell. The neoplastic cells in chronic granulocytic leukemia are typically characterized by translocation of a portion of the long arm of Chromosome 22 to another chromosome (usually No. 9). It is not known whether the critical phenomenon is a structural alteration in the translocated segment or the separation of structural genes from regulatory genes by the translocation event. The Philadelphia chromosome is the only cytogenetic change consistently associated with nearly every case of a specific clinical entity, but in a number of other preleukemic and leukemic disorders, as well as some nonhemic tumors, a variety of nonrandom alterations in other chromosomes are now being reported. Most commonly, these involve Chromosomes 1, 5, 7, 8, 9, 14, 17, 20, and 21.6 7 When one of these chromosomes is aberrant in a neoplastic clone, it frequently shows a characteristic change.
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Chromosome 8 is usually trisomic, while Chromosome 7 is often monosomic. Alterations in Chromosome 1 commonly involve an extra copy of the long arm, while changes in Numbers 5 and 20 typically appear as deletions in the long arm. When Chromosome 17 is involved, an isochromosome for the long arm usually replaces a normal Number 17.6.7,31All of these abnormalities have been noted in a variety of both preleukemic and leukemic disorders, but in some instances a specific alteration is more frequently associated with a particular type of dyscrasia. A translocation from the long arm of Chromosome 8 to the long arm of Chromosome 14, for instance, appears largely restricted to lymphoproliferative disorders, especially the Burkitt tumor." Similarly, there are three rare syndromes (probably within the spectrum of the preleukemic cytopenias 1) in which the marrow clone in each instance is characterized by a specific, single chromosome alteration. These disorders all include refractory anemia, with variable patterns in the marrow and circulating granulocytes and platelets. The characteristic chromosome changes include: a) interstitial deletion of a portion of the long arm of number 5 (Figure 3), the so-called 5q- syndrome; 31 b) monosomy for Chromosome 7 (Figure 2), the 45 chromosome syndrome; 32 and c) an isochromosome for the long arm of Chromosome 17, replacing a normal Number 17 (Figure 4).n When these three disorders progress to acute nonlymphocytic leukemia, there may be additional chromosome changes. Whether they should be considered specific preleukemic syndromes or simply variants within the cytopenic group remains to be determined by more cases and longer follow-up." There are instances in the preleukemic childhood disorders when some of the nonrandom chromosome changes noted above have been present in relatively stable hemic clones which did not progress to frank leukemia. Several patients with ataxia telangiectasia have had circulating lymphocyte clones characterized by a translocation involving the long arm of Chromosome 14, and in only one instance has evolution to leukemia been reported.14 Harmden 15 has described a patient with Fanconi anemia in whom an expanding myeloid clone was characterized by an additional long arm of Number 1 attached to the short arm of Number 6. As illustrated in Figure 1, a strikingly similar rearrangement (with the extra long arm of Chromosome 1 on the long arm of Chromosome 6), has been observed in a patient with familial marrow hypoplasia in whom the clone was quite stable over many months."0 In another instance, illustrated in Figure 2, a child with unexplained marrow hypoplasia had a slowly expanding lymphocyte clone, trisomic for Number 8, in the peripheral
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blood for several years and ultimately developed an acute leukemia characterized by a separate marrow clone monosomic for a C group chromosome, probably Number 7. All of these various data indicate that a limited number of human chromosomal sites appear to carry genes which confer a selective growth advantage on hematopoietic stem cells. Perhaps only in the case of the Philadelphia chromosome does the acquired advantage consistently result in a specific clinical entity. In the other instances, the particular characteristics of the hemic disorder may be determined by other genetic alterations in the clone, not necessarily visible at the chromosome level, or by the environment in which the cells are proliferating. These cytogenetic observations obviously do not incriminate specific gene loci in hemic neoplasia, but cell hybridization techniques in tissue culture are rapidly expanding the map of the human genome, localizing more and more genes to specific chromosome segments and also defining incorporation sites for oncogenic viral DNA. Eventually it may be possible to locate certain genes and their products which are critical to neoplastic proliferation. Some progress in this direction has been made by Azumi and Sachs 35 in determining the chromosomal sites of genes controlling differentiation in mouse myeloid leukemia; many of the chromosomal segments noted above as nonrandomly involved in human neoplasia have already been shown to contain one or more genes involved in the control of nucleic acid biosynthesis or other metabolic pathways perhaps critical in growth regulation.6' 15 For example, imbalance of the long and short arms of Chromosome 17 (usually an isochromosome for the long arm) is a relatively common finding in hemic disorders (Figure 4); the long arm of Number 17 contains genes regulating thymidine kinase and also galactose kinase (perhaps important in membrane synthesis) and is also a preferential site for incorporation of both adenovirus 12 and SV40.6'36 In man, we cannot yet confidently relate neoplastic behavior in vivo to specific genetic loci, but the studies of human tumors with banding methods are indicating a limited number of chromosomal segments where such investigations might be focused. Progression of Preleukemic Clones to Clinical Leukemia The cytogenetic data presented thus far suggest that the initial establishment of proliferating clones in preleukemia, as well as leukemia, results from a heritable change in a marrow stem cell, often involving
specific chromosomal segments, and perhaps with significant contribution from alterations in the local environment. It has also been proposed that the presence of chromosome abnormalities in these clones indicates ge-
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netic instability and that further genetic change, sometimes visible at the chromosome level, may be the basis for clinical progression (Text-figure ).17 As already noted, progression from the chronic to the acute phase in CGL is accompanied, in most cases, by an additional chromosome change in the original Ph-positive clone. It seems a reasonable assumption that a similar phenomenon, although not as frequently visible at the chromosome level, could underly the progression of preleukemic disorders to frank leukemia. Some cytogenetic data which support this view of clonal evolution in preleukemia have already been mentioned. A variety of mechanisms, both intracellular and extracellular, could be responsible for these postulated additional mutational events. In the congenital chromosome breakage syndromes, where every cell has increased chromosome fragility (perhaps on the basis of a nucleic acid repair defect),'5 genetic lability resulting in either sequential mutations in an originally nonneoplastic clone or generation of a new (leukemic) clone can be readily understood. (It is recognized that the increased susceptibility of these cells to viral transformation, as well as the immune defects present in these patients, could also contribute to the process.3'15) In the other preleukemic disorders, it is possible that the original clone, in addition to its growth advantage, has also acquired, through somatic mutation, a defect in DNA repair comparable to that in the chromosome breakage syndromes. Other types of "mutagenic mutation" can also be postulated, resulting, for instance, in an error-prone DNA polymerase or in a defective mitotic apparatus throughout the clone, making additional genetic changes more likely than normal. There is evidence to support each of these mechanisms as operative in neoplastic cell populations.'7 37 One can speculate that different initial somatic mutations could determine subsequent differences in clinical course, as well as the likelihood of later genetic changes being visible at the chromosome level. For example, does the constant progression of CGL to an acute leukemic phase, usually accompanied by a visible additional chromosome change, reflect a specific type of mitotic instability induced by the Philadelphia chromosome? How is this genetic alteration different from that in polycythemia vera or myelofibrosis, where such a sequence of events is much less frequent? There is also the possibility, of course, that additional mutations in preleukemic clones could result, in some cases, not from acquired genetic instability in the proliferating cells but from repeated or continued exposure to the original carcinogen or the mutagenic effects of therapy.17 Also, prgression of a disorder from preleukemia to frank leukemia need not always involve an additional change in the genetic characteristics of
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the proliferating population; there could be a critical alteration in the external regulatory factors which have limited expansion of the proliferating clone for a period of time. This may be particularly true in those instances where progression of the disease is not associated with visible cytogenetic change, although it must again be stressed that visible chromosome aberrations undoubtedly represent only a minority of the genetic events occurring in the altered cells. In any event, if specific gene mutations are responsible for the further shift in stem cell kinetics which underlies the progression of preleukemia to leukemia, there is no evidence to indicate that the loci involved are different from those implicated in the earlier stages of these disorders. As already noted, the chromosomal rearrangements nonrandomly associated with both preleukemia and leukemia are not restricted to any particular phase of these dyscrasias. Those very commonly observed in the blast crisis of CGL, for instance, include a second Philadelphia chromosome, trisomy for Number 8, and/or an Isochromosome 17;6.7 the latter two alterations, as well as the other nonrandom changes described previously, have been observed both in stable preleukemic states and in acute leukemia. The findings to date seem simply to indicate that one or more of a limited number of specific cytogenetic rearrangements can confer a selective growth advantage when present in a human hemic cell clone. In some instances the acquisition of such a chromosome alteration may be the basis for clinical progression from preleukemia to a definitely leukemic phase. Clinical Implications in Human Preleukemia In the absence of greater knowledge concerning the significance of particular chromosome rearrangements in these disorders, diagnostic and prognostic conclusions concerning hemic cell clones in individual patients must presently be based primarily on clinical and hematologic findings, with only occasional assistance from the cytogenetic data. In the cytopenic states, particularly the fully developed pancytopenic preleukemic syndrome,' the finding of a chromosomally abnormal marrow clone is strong evidence in favor of rapid progression to clinically apparent leukemia.'0 This is particularly true if the clone is significantly aneuploid,
with gain or loss of more than two chromosomes, and if the chromosomes show "fuzziness" and overlapping."8 These criteria generally suggest a neoplastic process in any situation where the diagnosis is in doubt. In the earlier stages of the cytopenic disorders (refractory anemia, sideroblastic anemia, etc.),' the prognostic significance of a cytogenetically altered marrow clone is less certain. In those circumstances where a
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single chromosome change (5q-, Mlonosomv 7, or Iso-17) has been associated with a syndrome which probably belongs within this group of preleukemias, the cytogenetic abnormality appears to indicate a high probability of ultimate progression to frank leukemia, at least in the latter two instances. 31-33 However, these are rare disorders, and more data are needed. Among the other two groups of preleukemic dyscrasias, as defined earlier, present data would suggest that a karvotvpicallv altered clone in the myeloproliferative disorders (polvcvthemia vera, mvelofibrosis, etc. ) 2 may wTell provide evidence of genetic instability in the system, but as vet, there is no indication that the presence of such a clone has any value in predicting if or when a particular patient might progress to frank leukemia.9'10 The same is true of the relatively stable clones identified in the childhood disorders, wvhether or not they are associated with the chromosome breakage syndromes. Relatively few data are currently available, however, and here also longer follow-up and additional cases are required before one can indicate the degree of additional risk, if anv, imposed by minor rearrangements in general and specific cvtogenetic alterations in particular. Unfortunately, even when marked aneuploidy and poor chromosome morphology suggest that a frankly leukemic clone is beginnig to expand in the marrow of a preleukemic patient, therapy with presently available agents rarely improves the clinical course and may be deleterious. Cytogenetic data are helping to clarify the sequential relationship between preleukemia and leukemia, but more information is needed on the specific genes and gene products associated with selective growth advantage in hemic cells and on the environmental factors which influence their proliferation before a knowledgeable approach to modifxing the rate and frequency of progression from the preleukemic phase to frank leukemia can be undertaken. References 1. Linman JW. Bagby GC: The preleukemic syndrome: Clinical and laboratory features, natural course, and management. Blood Cells 2:11-31. 1976 2. Laszlo J: Nieloproliferative disorders (M PD): Myelofibrosis, mvelosclerosis. extramedullary hematopoiesis. undifferentiated MPD. and hemorrhagic thrombocythemia. Semin Hematol 12:409-432, 1973 3. German J: Genes which increase chromosomal instability in somatic cells and predispose to cancer. Prog Med Genet 8:61-101, 1972 4. Rowley JD, Potter D: Chromosomal banding patterns in acute nonlymphocytic leukemia. Blood 47:705-721. 1976
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Canellos GP, Whang-Peng J: Philadelphia chromosome positive preleukaemic state. Lancet 2:1227-1228, 1972 Mitelman F, Levan G: Clustering of abberations to specific chromosomes in human neoplasms. II. A survey of 287 neoplasms. Hereditas 82:167-174, 1976 Mark J: Chromosomal abnormalities and their specificity in human neoplasms: An assessment of recent observations by banding techniques. Adv Cancer Res 24:165-222, 1977 Pierre RV: Cytogenetic studies in preleukemia: Studies before and after transition to acute leukemia in 17 subjects. Blood Cells 1:163-170, 1975 Wurster-Hill D, Whang-Peng J, McIntyre OR, Hsu LYF, et al.: Cytogenetic studies in polycythemia vera. Semin Hematol 13:13-32, 1976 Nowell P, Jensen J, Gardner F, Murphy S, Chaganti RSK, German J: Chromosome studies in "preleukemia." III. Myelofibrosis. Cancer 38:1873-1881, 1976 Westin J, Wahlstrom J, Swolin B: Chromosome studies in untreated polycythemia vera. Scand J Haematol 17:183-196, 1976 Lawler SD, Millard RE, Kay HEM: Further cytogenetical investigations in polycythaemia vera. Eur J Cancer 6:223-235, 1970 Rowley JD: Do human tumors show a chromosome pattern specific for each etiologic agent? J Natl Cancer Inst 52:315-320, 1974 McCaw BK, Hecht F, Harnden DG, Teplitz RL: Somatic rearrangement of chromosome 14 in human lymphocytes. Proc Natl Acad Sci USA 72:2071-2075, 1975 Harnden DG: The relationship between induced chromosome aberrations and chromosome abnormality in tumor cells. Proceedings of the Fifth International Congress on Human Genetics, Mexico City, 1976 (In press) Pierre RV, Hoagland HC: Age-associated aneuploidy: Loss of Y chromosome from human bone marrow cells with aging. Cancer 30:889-894, 1972 Nowell PC: The clonal evolution of tumor cell populations: Acquired genetic lability permits stepwise selection of variant sublines and underlies tumor progression. Science 194:23-28, 1976 Salmon C: Blood group changes in preleukemic states. Blood Cells 2:211-220, 1976 Houck JC, Attallah AM: Chalones (specific and endogenous mitotic inhibitors) and cancer. Cancer, A Comprehensive Treatise, Vol 3. Edited by FF Becker. New York, Plenum Press, 1975, pp 287-326 Trentin JJ: Determination of bone marrow stem cell differentiation by stromal hemopoietic inductive microenvironments (HIM). Am J Pathol 65:621-628, 1971 Weiss L: The hematopoietic microenvironment of the bone marrow: An ultrastructural study of the stroma in rats. Anat Rec 186:161-184, 1976 Barr RD, Whang-Peng J, Perry S: Regulation of human hemopoietic stem cell proliferation by syngeneic T lymphocytes. Acta Haematol (In press) Good RA: Aplastic anemia: Suppressor lymphocytes and hematopoiesis. N Engl J Med 296:41-42, 1977 McCulloch EA, Buick R, Lan S, Till JE: Differentiation in human myeloblastic leukemia studies in cell culture. Am J Pathol 89:449-458, 1977 Pitot HC: Neoplasia: A somatic mutation or a heritable change in cytoplasmic membranes? J Natl Cancer Inst 53:905-911, 1974 Warren L, Buck CA, Tuszynski GP, Fuhrer JP: The membrane glycoproteins of normal and malignant cells. J Cell Biol (In press) Kamada N: Discussion of paper by Killmann. Blood Cells 2:104-105, 1976 Farber E, Solt DB, Cameron R, Laishes B, Ogawa K, Medline A: Newer insights into the pathogenesis of liver cancer. Am J Pathol 89:477-482, 1977 Dahlke MB, Nowell PC: Chromosomal abnormalities and dyserythropoiesis in the preleukaemic phase of multiple myeloma. Br J Haematol 31:111-116, 1975
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Van Slyck EJ, Weiss L, Dully NI: Chromosomal evidence for the secondary role of fibroblastic proliferation in acute myelofibrosis. Blood 36:729-735, 1970 31. Sokal G, Michaux JL, Van den Berghe H, Cordier A, Rodhain J, Ferrant A. Moriau M, DeBru%ere NI, Sonnet J: A new hematologic syndrome with a distinct kar3 otype: The 5q- chromosome. Blood 46:519-533, 19705 32. Whang-Peng J: Banding in leukemia: Techniques and applications. J Natl Cancer Inst 58:3-8, 1977 33. Nowell PC, Finan JB: Isochromosome 17 in atypical myeloproliferative and lymphoproliferative disorders. J Natl Cancer Inst 59:329-3,33. 1977 34. Zech L, Haglund U, Nilsson K, Klein G: Characteristic chromosomal abnormalities in biopsies and lmphoid-cell lines from patients with Burkitt and non-Burkitt lymphomas. Int J Cancer 17:47-6, 1976 35. Azumi JI, Sachs L: Chromosome mapping of the genes that control differentiation and malignancy in myeloid leukemic cells. Proc Natl Acad Sci USA 74:253-257, 1977 36. Croce CM: Assignment of the integration site for simian virus 40 to chromosome 17 in GM54VA, a human cell line transformed by simian virus 40. Proc Natl Acad Sci USA 74:315-318, 1977 37. Springgate CF, Loeb LA: Mutagenic DNA polyrmerase in human leukemic cells. Proc Natl Acad Sci USA 70:245-249, 1973 38. Jensen MK, Philip P: Cvtogenetic studies in haematological disorders which may terminate in acute leukaemia. Acta Med Scand 193:353-357. 1973
30.
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Legends for Figures Figure 1-Karyotype of an abnormal hemic clone in an 18-year-old boy with congenital marrow hypoplasia and severe thrombocytopenia. There is an extra copy of the long arm of Chromosome 1 attached to the long arm of Number 6 (arrow). This clone completely replaced the marrow but did not progress to frank leukemia during the year between the initial chromosome study and the patient's death from hemorrhage. A brother had a similar syndrome, which was not typical of Fanconi anemia, and did not show increased chromosome fragility.
Figure 2-Karyotype of a leukemic clone in a six-year-old girl with a 4-year history of marrow hypoplasia. There is monosomy for a C-group chromosome, probably Number 7 (arrow). This clone first appeared in the bone marrow concurrently with the development of acute monomyelocytic leukemia which led to death within 4 months. An apparently unrelated clone, trisomic for Number 8, was present among her peripheral blood lymphocytes throughout her 4-year course, gradually expanding from 15 to 65% of PHA-stimulated metaphases. Both clones are considered somatic alterations; skin fibroblasts had a normal karyotype. The patient's disorder was not familial, and there was no evidence of increased chromosome fragility. There are some similarities to the preleukemic monosomy-7 syndrome described in adults.32
Figure 3-Karyotype of an abnormal clone filling the marrow of a 53-year-old woman who has marked thrombocytosis with slight anemia and leukocytosis. There is a deletion in the long arm of Chromosome 5 (arrow), suggesting the 5q- syndrome,3' but the hematologic findings are not typical. Her condition has been unchanged for 18 months since the initial chromosome study. Figure 4-Metaphase from an abnormal clone filling the marrow of an 82-year-old woman who had a disorder characterized by severe anemia, slight neutrophilia with acquired Pelger-Huet anomally, and marrow changes including disordered erythropoiesis and increased myeloblasts. The only alteration is an isochromosome for the long arm of Number 17 (arrow) replacing a Normal 17. She died in 6 months, without leukemia, but the few similar cases previously reported with an iso-17 clone all progressed to frank leukemia.3
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A number of disease states are considered "preleukemic" because they carry a significantly increased risk for the subsequent development of frank leukemia. These include a variety of cytopenias, myeloproliferative disorders, and childhood syndromes. Cytogenetic data suggest that these preleukemic disorders may not be qualitatively different from leukemia but simply represent quantitative differences in the degree of selective growth advantage enjoyed by a proliferating abnormal hemic population. Recent chromosome studies have indicated that a) this proliferation is characteristically clonal in both preleukemia and leukemia, apparently resulting from a heritable change in a marrow stem cel that allows it to escape to some degree from normal growth regulation; b) genetic instability in the clone, with additional genetic change, may often underlie clinical progression from the relative indolence of preleukemia or chronic leukemia to an aggressive stage comparable to acute leukemia; and c) certain specific chromosome segments carry genes important in the acquisition of growth advantage by hematopoietic stem cells, and many of these are common to both preleukemia and leukemia. Expansion of hemic clones may also be influenced significantly by alterations in the growth control mechanisms themselves. For instance, in various preleukemic states, preexisting marrow hypoplasia may permit clones with only minimal selective advantage to reach demonstrable size. Chromosome findings may help to establish the diagnosis and prognosis in preleukemic disorders, but additional long-term data are needed. (Am J Pathol 89:459-476, 1977)
ATTEM1PTS TO DISCUSS HUMAN PRELEUKENMIA are made difficult bv lack of agreement on a definition. In this paper, I will use the term rather broadlv and encompass three groups of disorders which carrv an increased risk of ultimately developing frank leukemia. It is recognized that these groups may overlap and that the risk of leukemia mav range from near 100% (perhaps better termed preclinical leukemia) to less than 10%. These three groups include: 1. A spectrum of marrow stem-cell dyscrasias characterized by reduction of one or more formed elements of the blood (cytopenia). The fully developed form is the pancytopenic state with near-normal marrow cellularitv and increased myeloblasts described by Linman and his colleagues From the Department of Patholog-, Univ ersity of Pennsv lvania School of MIedicine. Philadelphia. Pennsv l ania. Presented at the Seventy-fourth Annual Meeting of the American Association of Pathologists, Toronto, Ontario, Canada. March 15, 1977. Supported by Grants CA-1279 and CA-13822 from the N'ational Cancer Institute. N'ational Institutes of Health. Address reprint requests to Dr. Peter C. Now-ell. Department of Pathologp. School of Medicine, Universitv of Pennsvlsania, Philadelphia, PA 19104. 459
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as the preleukemic syndrome.1 These authors have suggested that various unexplained anemias (e.g., refractory anemia, sideroblastic anemia) may represent the earliest stage of this process and that depression in platelets, granulocytes, and monocytes appears subsequently. A high proportion of these cytopenias ultimately progress to nonlymphocytic acute leukemia. 2. Myeloproliferative disorders, as defined by the Polycythemia Vera Study Group.2 These are also considered marrow stem-cell dyscrasias and include polycythemia vera, myelofibrosis, undifferentiated myeloproliferative disorders, and primary thrombocytosis. A small proportion eventually progress to leukemia. 3. Certain congenital and childhood disorders carrying a somewhat increased risk of leukemia. These include the so-called chromosome breakage syndromes (Bloom syndrome, Fanconi anemia, ataxia telangiectasia, etc.),3 as well as some cases of unexplained marrow aplasia and hypoplasia. It is the major purpose of this paper to suggest that these various preleukemic disorders, as well as frank leukemia and lymphoma, are typically characterized by a clone' of cells with selective growth advantage replacing a proportion of the hematopoietic system. The difference between preleukemia and leukemia is considered as only quantitative, relating to the degree of selective advantage which the abnormal clone enjoys and the rate at which it is expanding at the expense of normal cells. The spectrum from preleukemia to chronic leukemia to acute leukemia is viewed as a continuum, with the distinction between "preneoplasia" and " true" neoplasia being essentially an arbitrary one determined by clinical necessity (Text-figure 1). Recent chromosome studies support this concept in three general areas: a) Proliferating clones of hemic cells have been identified by chromosome abnormalities in all of these disorders, preleukemic and leukemic. b) These clones appear to be genetically and cytogenetically unstable to some degree. Additional mutations, sometimes visible at the chromosome level, seem to occur with increased frequency, leading to selection of more deviant subpopulations and clinical progression from preleukemia or chronic leukemia to an acute leukemic phase. c) The cytogenetic abnormalities in these hemic clones are nonrandom; certain chromosome segments are involved with increased frequency, often with characteristic types of rearrangements. Many of these nonrandom changes are identical in preleukemia and leukemia. ° Clone is used here to indicate a population of cells descendant from a single cell of origin. It does not imply complete homogeneity, as subpopulations may coexist within the clone.
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The following sections will include a brief review of the cytogenetic evidence that both preleukemic and leukemic disorders do indeed involve clonal proliferation and that clinical progression to a more aggressive stage may be associated with additional genetic alterations in the expanding clone demonstrable at the chromosome level. Possible mechanisms underlying the development and progression of such clones will be considered, as well as the specific chromosome abnormalities involved. Some theoretical and practical implications will be suggested. It must be remembered throughout, however, that the evidence presented is cvtogenetic, and such observations always represent a relatively crude means of assessing genetically determined alterations in cells. Clones in Preleutemia and L*ukenia Chromosomal evidence for the clonal nature of these disorders and for clonal evolution in their development can be considered in reverse order of malignancy: acute leukemia, chronic leukemia, preleukemia. In the acute leukemias, with the greatest selective growth advantage, chromosome abnormalities are present in the neoplastic cells in approximately half of the cases. The same cytogenetic change is typically present in all leukemic cells in a given individual, indicating that the neoplastic population is a clone, the progeny of a single abnormal precursor. The altered karyotype may show only minor changes or may be highly aneuploid. Although the chromosomal pattem is often quite stable in the acute leukemias, clinical progression, especially resistance to therapy, has been associated with additional cytogenetic alterations in some instances. Recently, Rowley and Potter have described such cytogenetic evolution in 8 of 25 chromosomally abnormal acute leukemias studied by banding methods.4 Chronic granulocytic leukemia (CGL) has been more extensively studied cytogenetically than any other disorder of hematopoiesis. Typical CGL apparently results from a slight growth advantage afforded to a marrow stem cell by the Philadelphia chromosome. In a few instances, the expanding clone in the marrow has been identified by the Ph chromosome before the disease was clinically or hematologically demonstrable.5 When the inevitable alteration to the terminal "blast crisis" occurs, this seems to represent emergence of a new subpopulation, with the growth characteristics of acute leukemia, resulting from additional genetic alteration in the original Ph-positive clone (Text-figure 1 ).*7 This further genetic change in the neoplastic population can be recognized by additional chromosome abnormalities in about 75% of cases. With respect to the preleukemic disorders which were defined above,
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TEXT-FIGUtE 1-Simplified diaillustrating the hypothesis
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that the significant difference between acute leukemia, chronic . >N/ and preleukemia is the leukemia, .- ,)4,/rate at which an abnormal hemic clone is expahding. Progression oL / .- /from chronic to acute leukemia or from preleukemia to clinical leukemia (dotted arrows) reflects inCLINICAL DIAIS OF LEUKEIIIA creased rate of clonal expansion -due to additional genetic change in the clone (e.g., further chromosome alteration in CGL) or decrease in external growth regulation.
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clonal proliferation in the marrow, similar in many respects to the frank leukemias, has been demonstrable cytogenetically in a proportion of cases in each group. In some instances, additional chromosome changes have been observed in association with clinical progression to frank leukemia. Pierre 8 has studied 205 patients with various of the cytopenic dyscrasias previously described. He has found marrow chromosome abnormalities in 36%. In 17 cases followed sequentially, cytogenetic evolution was associated with the appearance of acute leukemia in eight instances. Similar results have been reported in the myeloproliferative disorders (polycythemia vera, myelofibrosis, etc.), although data relating to transition to leukemia are sparse because of its relative infrequency. It appears that perhaps 25% of untreated patients with these disorders have cytogenetically abnormal clones recognizable in the bone marrow and occasionally in the blood if immature forms are circulating.9-'3 Typically, these clones do not show extensive aneuploidy but have alterations involving only one or two chromosomes. The myeloproliferative disorders often have a relatively stable course without progression for a number of years, but an increased risk of ultimately developing leukemia, even without therapy, is recognized. The leukemic phase is characteristically associated with cytogenetic alterations in the neoplastic clone, and in several instances, it has been possible to correlate additional chromosome changes with progression from polycythemia vera to acute leukemia.12"3 More data will be forthcoming from long-term studies currently in progress under the Polycythemia Vera Study Group. In the childhood disorders considered preleukemic because of increased risk of the development of reticular neoplasms, data are insufficient to
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provide estimates on the frequencv of cytogeneticallv abnormal clones. Some information is available, however, particularlv on the chromosome breakage syndromes (Fanconi anemia, ataxia telangiectasia, Bloom svndrome).3 These patients show disordered hematopoiesis and immune deficiency to varving degrees, and in the first two syndromes, cytogeneticallv identifiable clones have been demonstrated in the hemic tissues of a number of individuals. In some cases, these have shown little tendency to expand and obviously represent a minimal growth advantage.14'15 In 1 patient, however, progression of such a disorder to frank leukemia has been associated with additional cytogenetic alterations within a proliferating clone.14 It is possible that preexisting marrow hypoplasia, whether or not it is associated with a chromosome breakage syndrome, may contribute to the emergence of clones with minimal growth advantage. We have recently observed 2 children with unexplained marrow hypoplasia and relatively stable, chromosomally abnormal clones in the hemic tissues (Figures 1 and 2). In 1 patient, a second clone, franklv leukemic, emerged in the bone marrow after several vears. Perhaps these patients had an unrecognized form of congenital instability of the genetic apparatus, but chromosome fragility was not demonstrably increased in lymphocyte cultures. Additional cases of childhood marrow aplasia and hy,poplasia, unrelated to recognized causes or syndromes, should be investigated cytogenetically; these dvscrasias carrv an increased risk of leukemia, and the present limited data suggest that further study might clarify their relationship to other clonal hemic disorders. It should be noted that the frequency of cvtogeneticallv abnormal clones in various preleukemic states, which has just been described, stands in clear contrast to the findings in normal individuals. Except for subpopulations of cells without a Y chromosome in elderly males (which seem to function normally),1' hemic clones are essentially never seen in the general population. In general, then, presently available cvtogenetic data support the view that clonal proliferation underlies a spectrum of disorders ranging from acute leukemia, with rapid expansion of the clone, to preleukemic states, in which the clone may have only a minimal growth advantage. The chromosome data also support the concept of genetic instability in these clones, with additional genetic changes, sometimes visible at the chromosome level, providing the basis for progression from preleukemia or chronic leukemia to a more rapidlv expanding and more clinicallv aggressive process (Text-figure 1). It should be pointed out that although this discussion has been restricted to cytogenetic findings, studies of enzvme
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patterns, immunoglobulins, and blood groups also indicate the clonal nature of both preleukemia and leukemia in man.17"18 Possible factors underlying the initial acquisition of growth advantage in such clones, as well as their subsequent progression, may now be considered further. Regulation of Hematopoiesis and Selective Growth Advantage in Hemic Cell Clones For a hematopoietic cell population to function as a malignancy, it need
only acquire the capacity to expand rapidly, at the expense of normal cells. The usual distinction between benign and malignant does not apply, as hemic cells invade and metastasize physiologically. Thus, unlike other organs, stage of malignancy and tumor progression in hematopoietic tissues need only reflect quantitative differences in the degree to which neoplastic cells have escaped from normal regulators of proliferation. Considerable evidence indicates that acquisition of such a selective growth advantage by hemic cells does not result from an increase in mitotic rate. Rather, it appears to reflect a change in the normal balance between proliferation and differentiation in the stem cell pool. The first step may well involve alteration in a single proliferating marrow cell, in one or another of the various stem cell compartments, such that the normal balance between the progeny of the aberrant cell which continue to proliferate and the progeny which differentiate is shifted in the direction of proliferation. Depending on the extent of the shift, the stem cell pool may expand gradually, producing the clinical picture of chronic leukemia with increased numbers of mature elements, or it may expand very rapidly, with concomitant marked reduction in differentiated forms, leading to clinical acute leukemia. The preleukemic disorders can be thought of as circumstances in which the shift in stem cell kinetics is minimal, so that clonal expansion proceeds at such a gradual rate and/or only to such a point that the clinical designation of leukemia is not warranted (Text-figure 1). In considering the growth regulatory mechanisms from which the altered stem cell may have escaped to some degree, there are at least four possibilities: a) local feedback controls from differentiated cells of the same lineage (chalones), perhaps best documented in the granulocytic series; 19 b) circulating hormones, such as erythropoietin; c) regulatory factors produced by nonhemic cells (particularly, reticular or fibroblastic elements) in the local microenvironment of the marrow; 20,21 and d) the immune system, with both stimulatory and suppressor effects of lym-
phocytes.22'23
None of these control mechanisms, except for erythropoietin, has been
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adequately characterized, but there is at least some evidence that each may play a role in the normal regulation of hematopoiesis and, further, that both leukemic and preleukemic human cells may show an altered response to such controls. Studies in tissue culture, particularly with the soft agar technique, are beginning to delineate more precisely some of these factors regulating hematopoiesis.2' The means by which a marrow stem cell might acquire this postulated altered response to growth regulators is perhaps most easily envisioned as a carcinogen-induced heritable change, analogous to the neoplastic transformation of fibroblasts induced in tissue culture by viruses, chemicals, or radiation. As with transformation in vitro, this heritable alteration in a stem cell could result from structural mutation, with or without visual chromosome change, or could initially be a reversible alteration in gene expression not involving structural damage to the genetic apparatus.25 The specific gene product(s) involved in escape from growth control are not known, but again an analogy to transformed cells in tissue culture might suggest a structural alteration in the glycoproteins or other elements of the external cell membrane, changing the stem cell's response to intercellular signals which normally regulate its proliferation. It should be recognized that, even in the acute leukemias, this autonomy is by no means absolute; there is both in vivo and in vitro evidence that in all of the various hematopoietic disorders under discussion, the cells still retain some degree of response to normal controls, and the postulated membrane change could well be quantitative not qualitative.2e In this regard, it is worth considering not only alterations in the proliferating stem cell and its progeny, but also the possibility of abnormalities in the external regulatory mechanisms as well. The relative importance of intracellular versus extracellular defects in the natural history of the various hemic disorders under consideration is by no means clear. One can consider that alterations in external regulatory factors might not only affect the initial establishment of hemic clones but also significantly influence the direction of differentiation within a particular clone at various times, determining whether granulocytes, monocytes, or erythrocytes are most prominent, and even account for the occasional disappearance of chromosomally demonstrable marrow clones.27 Farber " has pointed out that damage to the local parenchyma by liver carcinogens may play a very important role in the emergence of preneoplastic nodules, and similar considerations may obtain in the bone marrow. I have already noted that marrow hypoplasia in childhood may precede the appearance of both stable and aggressive clones (Figures 1 and 2). Similarly, marrow depression in adults, produced by benzene
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toxicity or by chemotherapy for multiple myeloma,2' may be followed by a sequence of events very similar to the preleukemic cytopenias described by Linman and his colleagues, eventuating in acute nonlymphocytic leukemia.' It is possible that some degree of unrecognized marrow suppression may be a primary event in many cytopenic preleukemic disorders, allowing a clone, initially only slightly altered, to begin its expansion. Such postulated hypoplasia might, in some cases, have an autoimmune basis; aplastic anemia mediated by suppressor T cells has recently been described.23 Clearly, more information is needed, not only on hematopoietic chalones and hormones, but also on the importance of the local microenvironment and the immune system in controlling both normal and abnormal hematopoietic proliferation and differentiation. This section can be concluded by simply listing several questions which may warrant further consideration: What growth regulatory influence do reticular cells have in the marrow? What is the significance of the fibrous tissue proliferation in myelofibrosis (it appears not to be part of the preleukemic hematopoietic clone 30)? Under what circumstances do lymphocytes and the immune system play a role in inhibition or stimulation of hematopoiesis? Chromosomal Sites Associated With Selective Growth Advantage in Hemic Cells Although specific genetic alterations responsible for the initiation of hemic neoplasia remain unknown, studies with the new chromosome banding techniques are beginning to identify cytogenetic alterations which occur nonrandomly in these disorders and which may suggest the location of genes important in the acquisition of selective growth advantage by a marrow stem cell. The neoplastic cells in chronic granulocytic leukemia are typically characterized by translocation of a portion of the long arm of Chromosome 22 to another chromosome (usually No. 9). It is not known whether the critical phenomenon is a structural alteration in the translocated segment or the separation of structural genes from regulatory genes by the translocation event. The Philadelphia chromosome is the only cytogenetic change consistently associated with nearly every case of a specific clinical entity, but in a number of other preleukemic and leukemic disorders, as well as some nonhemic tumors, a variety of nonrandom alterations in other chromosomes are now being reported. Most commonly, these involve Chromosomes 1, 5, 7, 8, 9, 14, 17, 20, and 21.6 7 When one of these chromosomes is aberrant in a neoplastic clone, it frequently shows a characteristic change.
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Chromosome 8 is usually trisomic, while Chromosome 7 is often monosomic. Alterations in Chromosome 1 commonly involve an extra copy of the long arm, while changes in Numbers 5 and 20 typically appear as deletions in the long arm. When Chromosome 17 is involved, an isochromosome for the long arm usually replaces a normal Number 17.6.7,31All of these abnormalities have been noted in a variety of both preleukemic and leukemic disorders, but in some instances a specific alteration is more frequently associated with a particular type of dyscrasia. A translocation from the long arm of Chromosome 8 to the long arm of Chromosome 14, for instance, appears largely restricted to lymphoproliferative disorders, especially the Burkitt tumor." Similarly, there are three rare syndromes (probably within the spectrum of the preleukemic cytopenias 1) in which the marrow clone in each instance is characterized by a specific, single chromosome alteration. These disorders all include refractory anemia, with variable patterns in the marrow and circulating granulocytes and platelets. The characteristic chromosome changes include: a) interstitial deletion of a portion of the long arm of number 5 (Figure 3), the so-called 5q- syndrome; 31 b) monosomy for Chromosome 7 (Figure 2), the 45 chromosome syndrome; 32 and c) an isochromosome for the long arm of Chromosome 17, replacing a normal Number 17 (Figure 4).n When these three disorders progress to acute nonlymphocytic leukemia, there may be additional chromosome changes. Whether they should be considered specific preleukemic syndromes or simply variants within the cytopenic group remains to be determined by more cases and longer follow-up." There are instances in the preleukemic childhood disorders when some of the nonrandom chromosome changes noted above have been present in relatively stable hemic clones which did not progress to frank leukemia. Several patients with ataxia telangiectasia have had circulating lymphocyte clones characterized by a translocation involving the long arm of Chromosome 14, and in only one instance has evolution to leukemia been reported.14 Harmden 15 has described a patient with Fanconi anemia in whom an expanding myeloid clone was characterized by an additional long arm of Number 1 attached to the short arm of Number 6. As illustrated in Figure 1, a strikingly similar rearrangement (with the extra long arm of Chromosome 1 on the long arm of Chromosome 6), has been observed in a patient with familial marrow hypoplasia in whom the clone was quite stable over many months."0 In another instance, illustrated in Figure 2, a child with unexplained marrow hypoplasia had a slowly expanding lymphocyte clone, trisomic for Number 8, in the peripheral
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blood for several years and ultimately developed an acute leukemia characterized by a separate marrow clone monosomic for a C group chromosome, probably Number 7. All of these various data indicate that a limited number of human chromosomal sites appear to carry genes which confer a selective growth advantage on hematopoietic stem cells. Perhaps only in the case of the Philadelphia chromosome does the acquired advantage consistently result in a specific clinical entity. In the other instances, the particular characteristics of the hemic disorder may be determined by other genetic alterations in the clone, not necessarily visible at the chromosome level, or by the environment in which the cells are proliferating. These cytogenetic observations obviously do not incriminate specific gene loci in hemic neoplasia, but cell hybridization techniques in tissue culture are rapidly expanding the map of the human genome, localizing more and more genes to specific chromosome segments and also defining incorporation sites for oncogenic viral DNA. Eventually it may be possible to locate certain genes and their products which are critical to neoplastic proliferation. Some progress in this direction has been made by Azumi and Sachs 35 in determining the chromosomal sites of genes controlling differentiation in mouse myeloid leukemia; many of the chromosomal segments noted above as nonrandomly involved in human neoplasia have already been shown to contain one or more genes involved in the control of nucleic acid biosynthesis or other metabolic pathways perhaps critical in growth regulation.6' 15 For example, imbalance of the long and short arms of Chromosome 17 (usually an isochromosome for the long arm) is a relatively common finding in hemic disorders (Figure 4); the long arm of Number 17 contains genes regulating thymidine kinase and also galactose kinase (perhaps important in membrane synthesis) and is also a preferential site for incorporation of both adenovirus 12 and SV40.6'36 In man, we cannot yet confidently relate neoplastic behavior in vivo to specific genetic loci, but the studies of human tumors with banding methods are indicating a limited number of chromosomal segments where such investigations might be focused. Progression of Preleukemic Clones to Clinical Leukemia The cytogenetic data presented thus far suggest that the initial establishment of proliferating clones in preleukemia, as well as leukemia, results from a heritable change in a marrow stem cell, often involving
specific chromosomal segments, and perhaps with significant contribution from alterations in the local environment. It has also been proposed that the presence of chromosome abnormalities in these clones indicates ge-
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netic instability and that further genetic change, sometimes visible at the chromosome level, may be the basis for clinical progression (Text-figure ).17 As already noted, progression from the chronic to the acute phase in CGL is accompanied, in most cases, by an additional chromosome change in the original Ph-positive clone. It seems a reasonable assumption that a similar phenomenon, although not as frequently visible at the chromosome level, could underly the progression of preleukemic disorders to frank leukemia. Some cytogenetic data which support this view of clonal evolution in preleukemia have already been mentioned. A variety of mechanisms, both intracellular and extracellular, could be responsible for these postulated additional mutational events. In the congenital chromosome breakage syndromes, where every cell has increased chromosome fragility (perhaps on the basis of a nucleic acid repair defect),'5 genetic lability resulting in either sequential mutations in an originally nonneoplastic clone or generation of a new (leukemic) clone can be readily understood. (It is recognized that the increased susceptibility of these cells to viral transformation, as well as the immune defects present in these patients, could also contribute to the process.3'15) In the other preleukemic disorders, it is possible that the original clone, in addition to its growth advantage, has also acquired, through somatic mutation, a defect in DNA repair comparable to that in the chromosome breakage syndromes. Other types of "mutagenic mutation" can also be postulated, resulting, for instance, in an error-prone DNA polymerase or in a defective mitotic apparatus throughout the clone, making additional genetic changes more likely than normal. There is evidence to support each of these mechanisms as operative in neoplastic cell populations.'7 37 One can speculate that different initial somatic mutations could determine subsequent differences in clinical course, as well as the likelihood of later genetic changes being visible at the chromosome level. For example, does the constant progression of CGL to an acute leukemic phase, usually accompanied by a visible additional chromosome change, reflect a specific type of mitotic instability induced by the Philadelphia chromosome? How is this genetic alteration different from that in polycythemia vera or myelofibrosis, where such a sequence of events is much less frequent? There is also the possibility, of course, that additional mutations in preleukemic clones could result, in some cases, not from acquired genetic instability in the proliferating cells but from repeated or continued exposure to the original carcinogen or the mutagenic effects of therapy.17 Also, prgression of a disorder from preleukemia to frank leukemia need not always involve an additional change in the genetic characteristics of
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the proliferating population; there could be a critical alteration in the external regulatory factors which have limited expansion of the proliferating clone for a period of time. This may be particularly true in those instances where progression of the disease is not associated with visible cytogenetic change, although it must again be stressed that visible chromosome aberrations undoubtedly represent only a minority of the genetic events occurring in the altered cells. In any event, if specific gene mutations are responsible for the further shift in stem cell kinetics which underlies the progression of preleukemia to leukemia, there is no evidence to indicate that the loci involved are different from those implicated in the earlier stages of these disorders. As already noted, the chromosomal rearrangements nonrandomly associated with both preleukemia and leukemia are not restricted to any particular phase of these dyscrasias. Those very commonly observed in the blast crisis of CGL, for instance, include a second Philadelphia chromosome, trisomy for Number 8, and/or an Isochromosome 17;6.7 the latter two alterations, as well as the other nonrandom changes described previously, have been observed both in stable preleukemic states and in acute leukemia. The findings to date seem simply to indicate that one or more of a limited number of specific cytogenetic rearrangements can confer a selective growth advantage when present in a human hemic cell clone. In some instances the acquisition of such a chromosome alteration may be the basis for clinical progression from preleukemia to a definitely leukemic phase. Clinical Implications in Human Preleukemia In the absence of greater knowledge concerning the significance of particular chromosome rearrangements in these disorders, diagnostic and prognostic conclusions concerning hemic cell clones in individual patients must presently be based primarily on clinical and hematologic findings, with only occasional assistance from the cytogenetic data. In the cytopenic states, particularly the fully developed pancytopenic preleukemic syndrome,' the finding of a chromosomally abnormal marrow clone is strong evidence in favor of rapid progression to clinically apparent leukemia.'0 This is particularly true if the clone is significantly aneuploid,
with gain or loss of more than two chromosomes, and if the chromosomes show "fuzziness" and overlapping."8 These criteria generally suggest a neoplastic process in any situation where the diagnosis is in doubt. In the earlier stages of the cytopenic disorders (refractory anemia, sideroblastic anemia, etc.),' the prognostic significance of a cytogenetically altered marrow clone is less certain. In those circumstances where a
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single chromosome change (5q-, Mlonosomv 7, or Iso-17) has been associated with a syndrome which probably belongs within this group of preleukemias, the cytogenetic abnormality appears to indicate a high probability of ultimate progression to frank leukemia, at least in the latter two instances. 31-33 However, these are rare disorders, and more data are needed. Among the other two groups of preleukemic dyscrasias, as defined earlier, present data would suggest that a karvotvpicallv altered clone in the myeloproliferative disorders (polvcvthemia vera, mvelofibrosis, etc. ) 2 may wTell provide evidence of genetic instability in the system, but as vet, there is no indication that the presence of such a clone has any value in predicting if or when a particular patient might progress to frank leukemia.9'10 The same is true of the relatively stable clones identified in the childhood disorders, wvhether or not they are associated with the chromosome breakage syndromes. Relatively few data are currently available, however, and here also longer follow-up and additional cases are required before one can indicate the degree of additional risk, if anv, imposed by minor rearrangements in general and specific cvtogenetic alterations in particular. Unfortunately, even when marked aneuploidy and poor chromosome morphology suggest that a frankly leukemic clone is beginnig to expand in the marrow of a preleukemic patient, therapy with presently available agents rarely improves the clinical course and may be deleterious. Cytogenetic data are helping to clarify the sequential relationship between preleukemia and leukemia, but more information is needed on the specific genes and gene products associated with selective growth advantage in hemic cells and on the environmental factors which influence their proliferation before a knowledgeable approach to modifxing the rate and frequency of progression from the preleukemic phase to frank leukemia can be undertaken. References 1. Linman JW. Bagby GC: The preleukemic syndrome: Clinical and laboratory features, natural course, and management. Blood Cells 2:11-31. 1976 2. Laszlo J: Nieloproliferative disorders (M PD): Myelofibrosis, mvelosclerosis. extramedullary hematopoiesis. undifferentiated MPD. and hemorrhagic thrombocythemia. Semin Hematol 12:409-432, 1973 3. German J: Genes which increase chromosomal instability in somatic cells and predispose to cancer. Prog Med Genet 8:61-101, 1972 4. Rowley JD, Potter D: Chromosomal banding patterns in acute nonlymphocytic leukemia. Blood 47:705-721. 1976
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Canellos GP, Whang-Peng J: Philadelphia chromosome positive preleukaemic state. Lancet 2:1227-1228, 1972 Mitelman F, Levan G: Clustering of abberations to specific chromosomes in human neoplasms. II. A survey of 287 neoplasms. Hereditas 82:167-174, 1976 Mark J: Chromosomal abnormalities and their specificity in human neoplasms: An assessment of recent observations by banding techniques. Adv Cancer Res 24:165-222, 1977 Pierre RV: Cytogenetic studies in preleukemia: Studies before and after transition to acute leukemia in 17 subjects. Blood Cells 1:163-170, 1975 Wurster-Hill D, Whang-Peng J, McIntyre OR, Hsu LYF, et al.: Cytogenetic studies in polycythemia vera. Semin Hematol 13:13-32, 1976 Nowell P, Jensen J, Gardner F, Murphy S, Chaganti RSK, German J: Chromosome studies in "preleukemia." III. Myelofibrosis. Cancer 38:1873-1881, 1976 Westin J, Wahlstrom J, Swolin B: Chromosome studies in untreated polycythemia vera. Scand J Haematol 17:183-196, 1976 Lawler SD, Millard RE, Kay HEM: Further cytogenetical investigations in polycythaemia vera. Eur J Cancer 6:223-235, 1970 Rowley JD: Do human tumors show a chromosome pattern specific for each etiologic agent? J Natl Cancer Inst 52:315-320, 1974 McCaw BK, Hecht F, Harnden DG, Teplitz RL: Somatic rearrangement of chromosome 14 in human lymphocytes. Proc Natl Acad Sci USA 72:2071-2075, 1975 Harnden DG: The relationship between induced chromosome aberrations and chromosome abnormality in tumor cells. Proceedings of the Fifth International Congress on Human Genetics, Mexico City, 1976 (In press) Pierre RV, Hoagland HC: Age-associated aneuploidy: Loss of Y chromosome from human bone marrow cells with aging. Cancer 30:889-894, 1972 Nowell PC: The clonal evolution of tumor cell populations: Acquired genetic lability permits stepwise selection of variant sublines and underlies tumor progression. Science 194:23-28, 1976 Salmon C: Blood group changes in preleukemic states. Blood Cells 2:211-220, 1976 Houck JC, Attallah AM: Chalones (specific and endogenous mitotic inhibitors) and cancer. Cancer, A Comprehensive Treatise, Vol 3. Edited by FF Becker. New York, Plenum Press, 1975, pp 287-326 Trentin JJ: Determination of bone marrow stem cell differentiation by stromal hemopoietic inductive microenvironments (HIM). Am J Pathol 65:621-628, 1971 Weiss L: The hematopoietic microenvironment of the bone marrow: An ultrastructural study of the stroma in rats. Anat Rec 186:161-184, 1976 Barr RD, Whang-Peng J, Perry S: Regulation of human hemopoietic stem cell proliferation by syngeneic T lymphocytes. Acta Haematol (In press) Good RA: Aplastic anemia: Suppressor lymphocytes and hematopoiesis. N Engl J Med 296:41-42, 1977 McCulloch EA, Buick R, Lan S, Till JE: Differentiation in human myeloblastic leukemia studies in cell culture. Am J Pathol 89:449-458, 1977 Pitot HC: Neoplasia: A somatic mutation or a heritable change in cytoplasmic membranes? J Natl Cancer Inst 53:905-911, 1974 Warren L, Buck CA, Tuszynski GP, Fuhrer JP: The membrane glycoproteins of normal and malignant cells. J Cell Biol (In press) Kamada N: Discussion of paper by Killmann. Blood Cells 2:104-105, 1976 Farber E, Solt DB, Cameron R, Laishes B, Ogawa K, Medline A: Newer insights into the pathogenesis of liver cancer. Am J Pathol 89:477-482, 1977 Dahlke MB, Nowell PC: Chromosomal abnormalities and dyserythropoiesis in the preleukaemic phase of multiple myeloma. Br J Haematol 31:111-116, 1975
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Legends for Figures Figure 1-Karyotype of an abnormal hemic clone in an 18-year-old boy with congenital marrow hypoplasia and severe thrombocytopenia. There is an extra copy of the long arm of Chromosome 1 attached to the long arm of Number 6 (arrow). This clone completely replaced the marrow but did not progress to frank leukemia during the year between the initial chromosome study and the patient's death from hemorrhage. A brother had a similar syndrome, which was not typical of Fanconi anemia, and did not show increased chromosome fragility.
Figure 2-Karyotype of a leukemic clone in a six-year-old girl with a 4-year history of marrow hypoplasia. There is monosomy for a C-group chromosome, probably Number 7 (arrow). This clone first appeared in the bone marrow concurrently with the development of acute monomyelocytic leukemia which led to death within 4 months. An apparently unrelated clone, trisomic for Number 8, was present among her peripheral blood lymphocytes throughout her 4-year course, gradually expanding from 15 to 65% of PHA-stimulated metaphases. Both clones are considered somatic alterations; skin fibroblasts had a normal karyotype. The patient's disorder was not familial, and there was no evidence of increased chromosome fragility. There are some similarities to the preleukemic monosomy-7 syndrome described in adults.32
Figure 3-Karyotype of an abnormal clone filling the marrow of a 53-year-old woman who has marked thrombocytosis with slight anemia and leukocytosis. There is a deletion in the long arm of Chromosome 5 (arrow), suggesting the 5q- syndrome,3' but the hematologic findings are not typical. Her condition has been unchanged for 18 months since the initial chromosome study. Figure 4-Metaphase from an abnormal clone filling the marrow of an 82-year-old woman who had a disorder characterized by severe anemia, slight neutrophilia with acquired Pelger-Huet anomally, and marrow changes including disordered erythropoiesis and increased myeloblasts. The only alteration is an isochromosome for the long arm of Number 17 (arrow) replacing a Normal 17. She died in 6 months, without leukemia, but the few similar cases previously reported with an iso-17 clone all progressed to frank leukemia.3
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