- Email: [email protected]
The preleukemic syndrome
The Preleukemic Syndrome Correlation of In Vitro Parameters of Granulopoiesis Clinical Features
PETER L. GREENBERG,M.D BETH MARA, B.S. Stanford, Cufifornia
From the Department of Medicine, Veterans Administration Hospital, Palo Alto, California and Stanford Universitv School of Medicine, Stanford, California. Th’ls study was supported 1)~IJS. Public Health Service Research Grants CA13141 from the National Cancer Institute RR70 General Clinical Research Centers, Division of Research Resources, and Veterans Administration Research Funds. Requests for reprints should be addressed to Dr. Peter L. Grccnbcrg. Veterans Administration Hospital, 3801 Miranda Avenue. Palo Alto, California !)4304. Manuscript accepted fanuary 17,1979.
With
In vitro parameters of granulopoiesis were correlated with the clinical courses of 45 prospectively studied patients with the preleukemic syndrome. Clinical features of these cytopenic patients with marrow morphology showing hemopofetic dysplasia included the following: median age, 61 years; combined cytopenias, 60 per cent; low leukocyte alkaline phosphatase, 67 per cent; splenomegaly, 32 per cent; median survival, 16.9 months, and a 55 per cent two year actuarial probability of survival. Transformation into acute myeloid leukemia occurred in 19 patients (44 per cent) withfn a median perfod of 19.1 months. presenting pancytopenia was the only clinical feature indicative of subsequent acute transformation, with 69 per cent of these patients undergoing such evolution within a medfan period of 15 months. Lethal infections were frequent during the preleukemic period. Abnormalities of in vitro marrow myeloid clonal growth were initially present in 72 per cent of the patients showing significantly low granulocyte-monocyte colony formtng cell (CFU-GM) values and a high proportion of light density CFU-GM. Persisting or progressive decrements in CFU-GM sequentially occurred prior to or concomitant with acute transformation in six patients. Markedly diminished initial CFU-GM values (12 colonies/105 marrow cells) were predictive for a significantly decreased (19 per cent) two year probability of survival (p < 0.004),whereas no clinical feature showed this association. Marrow cell and urinary colony-stimulating activity output were normal during the chronic phase of the disorder. These in vitro myeloid culture studies are useful adjuncts to marrow morphology and clinical features for diagnostic and prognostic characterization of patients with this syndrome. Since the initial retrospective descriptions of a preleukemic syndrome preceding the development of a disorder resembling overt acute myeloid leukemia (AML), controversy has persisted regarding the ability to classify such patients prospectively [l-5]. These patients have had refractory cytopenias and subtle but characteristic qualitative bone marrow morphologic abnormalities, recently termed hemopoietic dysplasia [6,7]. Factors related.to patient survival or to the potential of the disorder to either remain indolent or undergo acute transformation have not been determined. The development of in vitro semisolid culture technics for myeloid marrow cells has permitted the assessment of the clonal growth of granulocytic-monocytic progenitor cells (CFU-GM) under the necessary influence of the humoral stimulatory substance colony-stimulating activity (CSA) in patients with a variety of hematologic abnormalities [8,9]. Alterations of these growth parameters have been described in
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patients with chronic myeloproliferative disorders, including some with preleukemia, and progressive changes were noted as these diseases evolved [lo-131. These studies have suggested diagnostic and prognostic implications of abnormalities of in vitro incidence of CFU-GM in marrow, the proportion of light density CFU-GM and urinary CSA output in these disorders. Cellular sources of CSA are necessary to stimulate human clonogenic granulocytic precursors in vitro [14,X4. Endogenous production of CSA by bone marrow cells occurs and may reflect microenvironmental factors critical for influencing granulopoiesis. Abnormalities of intramedullary cellular CSA production have been described in patients with AML and poor prognoses [14]. In an attempt to determine the clinical utility of in vitro marrow culture technics for evaluating the preleukemic syndrome and assessing their prognostic value, these in vitro parameters were prospectively correlated with clinical courses in 43 patients with this disorder. METHODS The in vitro technics employed for evaluating CFU-GM in marrow, the proportion of light density CFU-GM, and marrow cell and urinary CSA levels have been extensively described previously [10,11,14]. Briefly, the incidence of CFLJ-GM was determined by utilizing the double layer semisolid agar culture system with human leukocyte feeder layers. Colonies of greater than 50 granulocytic-monocytic cells were scored at seven and 10 days of culture. Light density CFU-GM were those less tlcnse than 1.062 g/cm3 as determined by the bovine serum albumin neutral density centrifugation technic. Marrow cell CSA production was determined by testing the ability of seven-day conditioned mediums from buoyant adherent marrow cells to promote colony formation by nonadherent buoyant human marrow target cells. Colonies formed with sequential dilutions of the test-conditioned mediums were compared to a stable leukocyte-conditioned medium standard CSA source. The effective concentrations were quantitatively analyzed by curve-fitting computer programs. Daily urinary CSA output was determined by testing processed (dialyzed and calcium phosphate gel adsorbed/eluted) urine specimens against C57Bl mouse marrow cells. Granulocyte-monocyte colony formation produced by dilutions of the urine specimen for CSA concentrations was quantitated by methods similar to those described for marrow cell CSA levels. Normal values for CFU-GM are 26 f 7.5 colonies/105 marrow cells (mean f standard deviation, SD); for the proportion of light density CFU-GM, 2.6 f 2.6 per cent: for adherent marrow cell CSA-GM, 34.4 f 26.1 U/ml; and for urinary CSA, 68,000 f 21,000 U/24 hours for adults less than 66 years old and 135,000f 83,000U/24 hours for adults more than 66 years old. Abnormal values were considered those beyond f 2 SD of control values. Patients. Between January 1970 and january 1978,43 patients with preleukemia were evaluated clinically and underwent in vitro marrow culture studies (see “Appendix”.) All patients or their records were available for follow-up evaluation, which was completed in June 1978. The median follow-up period of living patients was 20.7 months.
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Criteria for the diagnosis of preleukemia were similar to those previously described [1.5,7,11]and consisted of relatively prolonged courses of refractory anemia, neutropenia or thrombocytopenia, singly or in combination, associated with characteristically abnormal marrow morphology. We have maintained the designation preleukemia with the understanding that we are refetring to the group of patients having myelodysplastic disorders with the clinical characteristics described in whom the disease has a propensity to terminate in a syndrome morphologically resembling AML. As recently suggested. the descriptive term hemopoietic dysplasia may be more appropriate [6]. Marrow examination indicated dysplastic maturation of at least two, and generally all, of the granulocytic, erythroid and megakaryocytic cell lines, including qualitative changes such as myeloid nucleocytoplasmic asynchrony, megaloblastoid erythropoiesis, occasionally with ringed sideroblasts, and often bilobed megakaryocytes [16]. In addition, the presence of abnormal mononuclear cells was noted in the marrows of 39 of these 43 patients. The cells comprised approximately 5 to 10 per cent of the myeloid cells, had granular bluish grey cytoplasm with a prominent nuclear hof and a reticulated nuclear chromatin pattern with palestaining nucleoli. ne d&s have previously been prominently noted in other patients with preleukemia [3,11,17,18], and, although the precise nature is unknown, they have been identified as abnormal promyelocytes. The cellularity of the marrow sections was either normal, decreased or increased; when increased, this was due to erythroid but not granulocytic hyperplasia. A quantitative increase in marrow myeloblasts was not present. Particular attention was paid to exclude patients who were already leukemic (i.e., had subacute or smoldering myeloid leukemia, with a quantitative increase in marrow blasts) and those with only single hemopoietic line defects (e.g., idiopathic sideroblastic ineffective erythropoiesis, idiopathic ngutropenia an’d idiopathic thrombocytopenia). The 43 patients with preleukemia include 24 patients whose in vitro growth studies were reported previously [ll]. These patients are included in our present study since the longer follow-up period has permitted better definition of their clinical courses. No clinical differences were noted in the two groups of patients. Marrow aspirates for in vitro studies were obtained after informed consent was obtained according to guidelines established by the Stanford University Human Experimentation Committee. Analysis of the patients’ probability of survival and duration of disease prior to acute transformation was computed by Kaplan-Meier Actuarial survival curves. Statistical analysis of differences between these curves was performed by the method of Gehaland Mantel [19,20).The Wilcoxon two-tailed two-sample rank test and chi square analysis were also employed for certain computations. Results were considered to be statistically significant if p values were cO.05.
RESULTS Composite presenting clinical and in vitro marrow culture features are indicated in Table I. Individual patient data are shown in the Appendix. As shown in Figure 1 and the Appendix, the initial incidence of CFU-GM in the marrow in preleukemic patients was significantly lower than normal (p < O.OOl],whether or not the patients subsequently underwent acute trans-
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formation, with a median value of 3 colonies per lo5 marrow cells plated. Seventy-two per cent of the initial CFLJ-GM values were less than normal. The CFU-GM values were not significantly different in the group of patients who eventually underwent transformation as opposed to those who did not. The proportion of light density CFU-GM was increased in seven of nine subjects, with a median of 15 per cent CFU-GM less dense than 1.062 g/cm3. These values were significantly above control levels (p < 0.61). The initial values of marrow adherent cell production of CSA and the urinary output of CSA were normal in 13 and nine patients, respectively. A linear relationship was noted between marrow cell CSA production (0to 74.6 U/ml) and adherent cell numbers (which ranged between 0.5 and 50 X lo5adherent cells/ml, with a correlation coefficient = 0.89,p < 0.001). The proportion of monocytes comprising the adherent cell population was 94.6 f 4.6 per cent [mean f SD] in these patients. In six patients, sequential evaluation indicated a persistent or progressive decrease in the incidence of CFU-GM prior to or concomitant with acute transformation (Figure 2). Clinical and in vitro parameters were evaluated to assess their predictive value for survival [Table II]. The median survival of the 43 patients was 13 months from the time of the first marrow culture and 18.9months from the time of diagnosis (Figure 3). The one, two and three year probability of survival from the time of diagnosis was 77 per cent, 55 per cent and 45 per cent, and from initial marrow culture 67 per cent, 47 per cent and 38 per cent, respectively (Figure 3). None of the clinical features analyzed and stated. in Table I predicted decreased survival when compared to their complementary parameters. However, correlation of in vitro studies with clinical courses indicated that survival of those with
TABLE I
Preleukemic Syndrome-Clinical Characteristics Per Cent
Characteristic 43 patients with Refractory cytopenia and Marrow morphology abnormal Hemopoietic dysplasia L 2 lines Age, median 61 years old >50 years old Cytopenia Single cytopenia Bicytopenia Pancytopenia 1 Leukocyte alkaline phosphatase Splenomegaly In vitro marrow culture 4 Initial incidence of CFU-GM t Initial % light density CFU-GM Acute transformation
77 40 30 30 67 33 72
78 44
NOTE: CFU-GM = granulocyte-monocyte
colony-forming
cells.
an incidence of CFU-GM in the marrow of <5 colonies/105 marrow cells (and particularly the 42 per cent with markedly decreased values of 12 colonies/105 cells) was significantly less than that of patients with higher colony-forming values (Table II, Figure 4). The one, two and three year probability of survival from the time of the initial marrow culture was 50 per cent, 19 per cent and 10 per cent, respectively, for patients with an incidence of CFU-GM of <2 colonies/W marrow cells, This contrasted with and was significantly less than the survival probabilities of 81 per cent, 76 per cent and 64 per cent, respectively, for patients with >2 colonies/l05 marrow cells (Figure 4) (p < 0.004). None of the clinical features analyzed correlated with differing CFU-GM
1
J In vitro parameters of granulopoiesis in preleukemic patients. Open boxes enclose the mean f 2 SD of normal values. CFU-GM = granulocyte-monocyte colony forming cells. CSAsM = adherent bone marrow cell colony stimulating activity production. CSA, = urinary colony stimulating activity excretion.
Frgure 1.
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PARAMETERS
IN PRELEUKEMIA-GREENBERG.
comparison with those who did not. Of the 12 patients who died without having undergone acute transformation, eight (60 per cent) died of infections, two (20 per cent) died of bleeding and the other two (20 per cent) died of causes unrelated to their hematologic disease. Among these patients, those who were neutropenic had a significantly shorter survival than those who were not neutropenic (8.9 months versus 21.1 months, p < 0.04). The potential for acute transformation was analyzed with respect to clinical and in vitro parameters (Table III). Forty-four per cent of the patients (19 of 43 patients) have so far undergone transformation within a median period of 19 months. Pancytopenia at the time of presentation was the only parameter which correlated with subsequent acute transformation. Despite a shorter period of survival, 69 per cent of these subjects underwent acute transformation as compared to 33 per cent of those without pancytopenia (p < 0.01). Transformation occurred in pancytopenic patients in a median time of 15 months from diagnosis in comparison with 45 months for those who were not pancytopenic, (p < 0.007) (Figure 5). The two year probability (from time of diagnosis) of patients undergoing transformation was 33 per cent for all patients, for those who were initially pancytopenic it was 66 per cent and for those who were not it was 18 per cent. The difference in transformation probability between the latter two groups of patients was statistically significant (p < 0.007)(Figure 5). The patients in the group that underwent transformation were of similar age’ compared to those who did not undergo transformation (63 years old, median], with a nonsignificant increased proportion of patients having pan-
Months prior to acute transformation I
MARA
I
F&lWs 2.
Sequential marrow granulocyte-monocyte colony forming cell (CFU-GM) values in preleukemic patients who underwent acute transformation.
levels (i.e., normal, low or very low]. Upon contrasting relatively short survivors (36 months), no clinical features were predictive. However, a significantly higher proportion of short-term survivors, 69 per cent (13 patients), had CFU-GM values 12/105 marrow cells compared to 13 per cent (eight patients) of the long-term survivors (p < 0.005). No difference was noted in the duration of survival of those who underwent acute transformation in
SURVIVAL
I OO
I
30
I
60
,I
I
90
I
120
I
150
I 160
I
210
MONTHS
:igure 3. Probability of survival of preleukemic patients from the time of diagnosis-and from the time of initial marrow culture study- - - -. The short vertical lines denote still surviving patients.
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TABLE Ii
Preieukemic Syndrome-Potential Survival
PARAMETERS
TABLE iii
For
IN PRELEUKEMIA-GREENBERG,
Preieukemic Syndrome-Potential Acute Transformation
Median Survival (mo) From First From Dlaanosis Marrow Culture Patients (43) Age >50 yr >60 yr Cytopenia SingWbicytopenia Pancytopenia 1 Leukocyte alkaline phosphatase Splenomegaly In vitro marrow culture 1 initial incidence of CFU-Gf+i <5 coionies/105 marrow ceils 12 colonies/105 marrow cells < 1 colonies/ 1O5 marrow ceils Acute transformation
18.9
13
17.9 21
11.3 15.8
21 11 21.1 33
14 8.9 15.9 7.8
26.6
12.1 11.1’ 10.2’ 11.1’ 18.9
l
for
Medianlime to Transformation lncldence of (mo) Transformation Fram From Initial Diagnosis Marrow Culture (q/o)
ParametersAssessed
44
19.1
15.9
69 }* 33
45.3 l5 }**
;;:;r*
57 56
32.5 19.5
9.2 16.5
52 45
31.0 20.0
16.9 13.3
1 initial incidence of CFU-GM 12 coionies/105 marrow cells
42
...
10.6
50
10.6
0 colonies/ 1O5 marrow ceils
75
13.3
Ail patients Cytopenia SingieIbicytopenia Pancytopenia Splenomegaiy 1 Leukocyte alkaline phosphatase Age (yr) <60 <50 In vitro marrow culture
l
NOTE: CFU-GM = granulocyte-monocyte colony forming ceils. Gehan analysis: p < 0.03, p < 0.004 relative to complementary parameter, other comparisons were not significantly different. l
MARA
l
NOTE: CFU-GM = granulocyte-monocyte colony forming ceils. Significantly different value relative to c&nplementary parameter, Gehan analysis: p < 0.01, ++ p < 0.007. Othercomparisons were not significantly different. l
SURVIVAL
All patients
-
CFU-GM 5 2 ---CFU-GM > 2---
0
0
I 10
I
20
I
30
I
I
40
I
50
I
60
I
70
I
60
90
MONTHS FROM INITIAL MARROW CULTURE
Figure 4. Probability of survival of preieukemic patients from the time of initial marrow culture relative to marrow granuiocyte-monocyte colony forming capacity (CFU-GM). A significantly decreased probability of survival was noted in the 16 patients with initially 12 colonies/105 marrow ceils compared to the 25 patients with >2 colonies/105 marrow ceils (p < 0.004). The short vertical lines denote still surviving patients.
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IN PRELEUKEMIA-GREENBERG,
MARA
TIME TO TRANSFORMATION I
I
I
All patients Pancytopenic
I
patients ----
Non pancytopenic
patients - - -
i loo0
I
30
I
I
60
90 MONTHS
120 FROM
1 150
I
180
I
210
DIAGNOSIS
Figure 5. Probability of acute transformation in preleukemic patients. A significantly increased probability of transformation was noted in the 13 patients who initially were pancytopenic compared to the 30 nonpancytopenic patients (p < 0.007). The short vertical lines denote patients who have not undergone transformation.
cytopenia (47 per cent versus 21 per cent], splenomegaly per cent versus 29 per cent) or low leukocyte alkaline phosphatase values (71 per cent versus 62 per cent) (Appendix). Decreased CFU-GM values in the marrow alone were not predictive of subsequent acute transformation. Median initial CFU-GM levels were the same and low in both groups (3 colonies/105 marrow cells plated). Comparisons of other initial in vitro parameters between the two groups were not possible as most of those data had been obtained only in patients who did not undergo transformation. Sequential analysis of these parameters is continuing. The median follow-up period of the 12 living patients who have not undergone transformation during the study is 20.7 months. Multivariate analysis of several parameters for predicting survival or the potential for transformation (e.g., pancytopenia plus low CFU-GM incidence) added little discriminatory power to that obtained with the single parameter alone. No particular type of cytopenia or the presence of pancytopenia was significantly correlated with low (or very low) CFU-GM values. (42
COMMENTS
The clinical features which characterized these 43 prospectively evaluated patients with preleukemia are similar to those previously described for retrospectively analyzed patients in whom AML had developed after they had had a preleukemic phase [l-4]. In our patients with refractory cytopenia and marrows showing hemopoietic dysplasia the incidence of combined cyto956
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penias was 60 per cent, of low leukocyte alkaline phosphatase levels 67 per cent and of splenomegaly 33 per cent. The median survival of these patients was 18.9 months with a 55 per cent two-year probability of survival. Transformation into AML occurred in 44 per cent of the patients within a median period of 19.1 months. The only clinical or in vitro parameter which was discriminating for the prognosis of survival was an initial markedly decreased incidence of CFU-GM (52 colonies/105 marrow cells). Sixty-nine per cent relatively short-term survivors had this finding in contrast to only 13 per cent of the long-term survivors. An abnormally low initial incidence of CFU-GM and a high proportion of light density CFU-GM were found in 72 per cent of the preleukemic patients, and 42 per cent had markedly decreased values. These patients with markedly decreased marrow myeloid colony formation had a significantly decreased duration of survival (10 month median, with a 19 per cent two year probability of survival from the time of initial marrow culture, p < 0.004). This shortened survival was due to the combination of deaths resulting from the consequence of acute transformation and from complications of the patients’ cytopenias, generally infectious, even in the absence of transformation. The in vitro culture studies thus suggest that markedly defective colony formation was associated with an inadequate marrow response to demand for new granulocytic cells. In the patients who did not undergo transformation, but who were neutropenic, decreased survival was related to infection. Qualitative
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APPENDIX
Patients-Clinical
and Sex
cytopenia
Splenomegsly
IN PRELEUKEMIA-GREENBERG,
MARA
and In Vitro Data
Age(yr) Case No.
PARAMETERS
LAP
Initial CFU-GM (colonies/ 10s marrow cells)
_
Time to Transformation(mo.) From 1st Marrow From Culture Diagnosis
Duralion of Disease(mo.) From 1st Marrow From Culture Diagnosis
Patients With Acute Transformation 1
3 3 4 59 6 7 8 9*t 10 ll§
12t 13 14 15 16§ 17 18 19 20” 21§ 22t§ 23’ 249 259 26t§ 27’ 289 29 30’9 319 32.t 33 34’ 35 36t 37” 38t 39’ 409 419 42 43
68,F 61,F 26,M 59,M 71,M 88,F 63,F 39.F 68,M 62,F 61,M 84,M 56,M 67,F 61,M 38,F 48.F 89,M 77,F 80.M 77,M 60,M 34,M 71,M 57,F 62,M 64,F 81,M 54,F 36.F 57,F 70,M 81,M 50,M 36,M 68,M 75,M 84,M 63,M 51,F 60,F 36,F 73-M
AT NAT
+ +
T A
0 0 + 0 + + 0 + 0 0 + 0 + 0 0
N,A,T N,A,T N,A A A,T N,A,T N,A,T N,A,T T A A N,A,T N,A N,A,T N,A,T N N,A A N,A,T N,A,T N A ATT N,A N,A A N,T A N,A A,T T
0 0 + + 0 + 0 + 0 0 0
38.9
:’ T
35 30 39 14 19
65.1 72.4-F 11.3 26.3 1.4
i 1
10 6
74.6 47.5 43.9+
T -
31.5 1 0.5 0
20.9 13.5+ 8.9 4.6 37.4
7 i
0 0
11.1 14.5+ 15.9 18.9
0
2.5 0 Patients Without Acute Transformation f 1
39 76 38 23
27.1+ 20.7+ 4.4+ 25.2+
f
19 10 11 9 8 7 6 3.3 3 3 2 2.3
6.7+ 3.4 3.2+ 6.7 13.1 42.5+ 22.5+ 4.0+ 27.1+ 18.7 0.7 9.3 1.0+
H c* H
0 0 0 0
i
+ +
N,A,T A
0 0 + 0 0
N,A,T A,T
92
0
N,A,T A
A,T N
t
0
0
c*
t
1 1 0.8 0.5 00.5
1
0
27.2 2.1 27+ 6.3 4.8 13 15.8
47.6 139.5+ 65.1 11.3 1.4 26.6 119.5 84 45.2-I 13.5+ 35 8.9 8.5 52.3 59.4 19+ 18.9 17.9 11
37.1 67.9 50.8 8.7 1.2 19.6 39.6 9.6 43.5 5.6 16.9 8.6 4.1 34.3 8.7 10.6 16 15.9 1.1
45.8 135 50.8 8.7 1.2 20 111.5 19.1 44.7 5.6 31 8.6 8.0 49.3 57 15 16 17.9 9.6
39.0+ 20.7+ 4.4+ 72.0+ 6.7+ 3.2+ 46 21 13.1 223+ 40.5+ 4.0+ 39+ 18.7 1.7-I 21.9 0.7 27.2 2.1 32.4+ 10.3 4.8 19.3 15.8
NOTE: CFU-GM = granulocyte-monocyte colony forming cells. N = neutropenia. A = anemia. T = thrombocytopenia. -i- = splenomegaly. 0 = no splenomegaly. 1 = decreased. t = increased. - = normal. LAP = leukocyte alkaline phosphatase. per cent light density CFU-GM determined (see Figure 1). t Urinary colony stimulating activity output determined (see Figure 1). 5 Marrow cell colony stimulating activity production determined (see Figure 1). l
abnormalities in granulocyte chemotactic, phagocytic and bactericidal activities, and an increased incidence of lethal infections have also been previously reported in preleukemic patients [18,21,22]. The presence of pancytopenia at the time of diagnosis was predictive of subsequent acute transformation. This finding was also present in several early retrospective series of patients with this disorder, although those pa-
tients were not so analyzed [2-41. Recent studies have shown a higher incidence of chromosomal abnormalities and evolutionary potential in pancytopenic patients [23]. Thus, it is possible that the presence of pancytopenia reflects a qualitatively more severe degree of in vivo progression of clonal abnormalities than that in patients with other, less severe, types of cytopenias. The in vitro studies of marrow colony formation at the time
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CRANULOPOIETIC PARAMETERS IN PRELEUKEMIA-GREENBERG,
of diagnosis did not predict subsequent transformation. However, sequential studies in six preleukemic patients indicated a persisting or progressive decrease in the incidence of CFU-GM concomitant with or preceding progression of the disease. Enzymatic or cytogenetic analysis will be needed to determine whether this finding is a functional correlate of clonal evolution or whether it reflects progressive repopulation of the marrow by more abnormal granulopoietic cells. The finding that low colony formation per se was not predictive for subsequent transformation indicates that other factor(s) are necessary for this progression. Prior studies in patients with other chronic myeloproliferative disorders reported from our laboratory indicated that, in addition to a low or decreasing incidence of CFUGM, progressive abnormalities of urinary CSA levels developed as their disorders evolved [ll]. The patients with preleukemia described herein had normal levels of urinary CSA output and adherent marrow cell CSA production in the chronic stable phase of their diseases. Studies are ongoing to determine whether abnormalities of humoral regulatory substances develop in preleukemia with evolution of the disease. Low marrow colony formation, previously described
MARA
in preleukemia [lo-121, is not specific for this syndrome since it has also been found in certain neutropenic disorders and in acute, subacute and progressing chronic myeloid leukemia [9-11,24-271. However, the high proportion of light density CFU-GM found in preleukemia and other myeloproliferative disorders [11,13,24,26,28] is not present in the benign neutropenias [29], thus providing a useful adjunct to marrow morphology for distinguishing these entities. Although present in only a few patients in our series of preleukemit patients, other studies have shown an increased incidence of abortive cluster formation in patients with acute and chronic myeloproliferative disorders [11,24-261. Patients with AML, subacute myeloid leukemia and preleukemia may have similar in vitro clonal growth patterns despite differing pace and incidence of disease progression. Therefore, marrow morphology is necessary (i.e., showing quantitative rather than qualitative myeloid abnormalities) for the differential diagnosis of these conditions. As discussed herein for preleukemia, and in prior studies of other myeloproliferative disorders [11,14,24,25], the in vitro parameters of marrow granulopoiesis present in these diseases appear to provide information of prognostic value.
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Smith W. Ablin A. Goodman 1.et al.: Atvnical meaakarvocvtes in thepreleukemic phase’of AML. 9ood 42:535, i973. Breton-Gorius 1. Houssav D. Drevfus B: Partial mvelookroxidase deficiency in a’case of preleukemia. I. &dies of the fine structure and peroxidase synthesis of promyelocvtes. Br 1Haematol 30: 273, 1975. Dreyfus B: Preleukemic states. Blood Cell 2: 33, 1976. Gehan EA: A generalized 2-sample Wilcoxon test for doubly censored data. Biometrika 52: 650,1965. Mantel N: Ranking procedures for arbitrarily restricted observation. Biometrics 23: 65. 1967. Breton-Gorius J, Houssay D, Vilde J. et al.: Partial myeloperoxidase deficiency in a case of preleukemia. II. Defects of degranulation and abnormal bactericidal activity of blood neutrophils. Br J Haematol30: 279,1975. Ruutu P, Ruutu T. Vuopio P. et al.: Function of neutrophils in preleukemia. Stand J Haematoll8: 317,1977. Panani A. Paoavannis A. Kvrkou K. et al.: Cvtorenetic studies in preleukkmia using the G-banding staini& technique. Stand J Haematol18: 301,1977. Moore M, Spitzer G, Williams N, et al.: Agar culture studies in 127 cases of untreated acute leukemia. Blood 44: .I, 1974. Spitzer G, Dicke KA, Gchan EA, et al.: A simplified in vitro classification for prognosis in adult leukemia. The application of in vitro results in remission-predictive models. Blood 48: 795.1976. Moore MAS: In vitro studies in myeloid leukemia. Advances in Acute Leukemia [C&ton F, Crowther D, Malnas 1, edsl. New York, North Holland Publishing Company, i974, p 161.
27.
Mimer G, Testa N, Geary C, et al.: Bone marrow culture studies in refractory cytopenia and “smouldering leukacmia.” Br ] Haematoj 35: 251,1977. 28. Berthier R, Douady R, Hollard D: Cellular factors regulating granulopoiesis in myeloid leukemia. Blood Cells 3: 481, 1977. 29. Greenberg PL. Mara B, Mosny S: In vitro granulopoietic parameters in idiopathic neutropenia (abstract]. Proceedings of the XVII Congress, International Society of Hematology. Paris 1978, p 919.
Volume 66
PETER L. GREENBERG,M.D BETH MARA, B.S. Stanford, Cufifornia
From the Department of Medicine, Veterans Administration Hospital, Palo Alto, California and Stanford Universitv School of Medicine, Stanford, California. Th’ls study was supported 1)~IJS. Public Health Service Research Grants CA13141 from the National Cancer Institute RR70 General Clinical Research Centers, Division of Research Resources, and Veterans Administration Research Funds. Requests for reprints should be addressed to Dr. Peter L. Grccnbcrg. Veterans Administration Hospital, 3801 Miranda Avenue. Palo Alto, California !)4304. Manuscript accepted fanuary 17,1979.
With
In vitro parameters of granulopoiesis were correlated with the clinical courses of 45 prospectively studied patients with the preleukemic syndrome. Clinical features of these cytopenic patients with marrow morphology showing hemopofetic dysplasia included the following: median age, 61 years; combined cytopenias, 60 per cent; low leukocyte alkaline phosphatase, 67 per cent; splenomegaly, 32 per cent; median survival, 16.9 months, and a 55 per cent two year actuarial probability of survival. Transformation into acute myeloid leukemia occurred in 19 patients (44 per cent) withfn a median perfod of 19.1 months. presenting pancytopenia was the only clinical feature indicative of subsequent acute transformation, with 69 per cent of these patients undergoing such evolution within a medfan period of 15 months. Lethal infections were frequent during the preleukemic period. Abnormalities of in vitro marrow myeloid clonal growth were initially present in 72 per cent of the patients showing significantly low granulocyte-monocyte colony formtng cell (CFU-GM) values and a high proportion of light density CFU-GM. Persisting or progressive decrements in CFU-GM sequentially occurred prior to or concomitant with acute transformation in six patients. Markedly diminished initial CFU-GM values (12 colonies/105 marrow cells) were predictive for a significantly decreased (19 per cent) two year probability of survival (p < 0.004),whereas no clinical feature showed this association. Marrow cell and urinary colony-stimulating activity output were normal during the chronic phase of the disorder. These in vitro myeloid culture studies are useful adjuncts to marrow morphology and clinical features for diagnostic and prognostic characterization of patients with this syndrome. Since the initial retrospective descriptions of a preleukemic syndrome preceding the development of a disorder resembling overt acute myeloid leukemia (AML), controversy has persisted regarding the ability to classify such patients prospectively [l-5]. These patients have had refractory cytopenias and subtle but characteristic qualitative bone marrow morphologic abnormalities, recently termed hemopoietic dysplasia [6,7]. Factors related.to patient survival or to the potential of the disorder to either remain indolent or undergo acute transformation have not been determined. The development of in vitro semisolid culture technics for myeloid marrow cells has permitted the assessment of the clonal growth of granulocytic-monocytic progenitor cells (CFU-GM) under the necessary influence of the humoral stimulatory substance colony-stimulating activity (CSA) in patients with a variety of hematologic abnormalities [8,9]. Alterations of these growth parameters have been described in
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patients with chronic myeloproliferative disorders, including some with preleukemia, and progressive changes were noted as these diseases evolved [lo-131. These studies have suggested diagnostic and prognostic implications of abnormalities of in vitro incidence of CFU-GM in marrow, the proportion of light density CFU-GM and urinary CSA output in these disorders. Cellular sources of CSA are necessary to stimulate human clonogenic granulocytic precursors in vitro [14,X4. Endogenous production of CSA by bone marrow cells occurs and may reflect microenvironmental factors critical for influencing granulopoiesis. Abnormalities of intramedullary cellular CSA production have been described in patients with AML and poor prognoses [14]. In an attempt to determine the clinical utility of in vitro marrow culture technics for evaluating the preleukemic syndrome and assessing their prognostic value, these in vitro parameters were prospectively correlated with clinical courses in 43 patients with this disorder. METHODS The in vitro technics employed for evaluating CFU-GM in marrow, the proportion of light density CFU-GM, and marrow cell and urinary CSA levels have been extensively described previously [10,11,14]. Briefly, the incidence of CFLJ-GM was determined by utilizing the double layer semisolid agar culture system with human leukocyte feeder layers. Colonies of greater than 50 granulocytic-monocytic cells were scored at seven and 10 days of culture. Light density CFU-GM were those less tlcnse than 1.062 g/cm3 as determined by the bovine serum albumin neutral density centrifugation technic. Marrow cell CSA production was determined by testing the ability of seven-day conditioned mediums from buoyant adherent marrow cells to promote colony formation by nonadherent buoyant human marrow target cells. Colonies formed with sequential dilutions of the test-conditioned mediums were compared to a stable leukocyte-conditioned medium standard CSA source. The effective concentrations were quantitatively analyzed by curve-fitting computer programs. Daily urinary CSA output was determined by testing processed (dialyzed and calcium phosphate gel adsorbed/eluted) urine specimens against C57Bl mouse marrow cells. Granulocyte-monocyte colony formation produced by dilutions of the urine specimen for CSA concentrations was quantitated by methods similar to those described for marrow cell CSA levels. Normal values for CFU-GM are 26 f 7.5 colonies/105 marrow cells (mean f standard deviation, SD); for the proportion of light density CFU-GM, 2.6 f 2.6 per cent: for adherent marrow cell CSA-GM, 34.4 f 26.1 U/ml; and for urinary CSA, 68,000 f 21,000 U/24 hours for adults less than 66 years old and 135,000f 83,000U/24 hours for adults more than 66 years old. Abnormal values were considered those beyond f 2 SD of control values. Patients. Between January 1970 and january 1978,43 patients with preleukemia were evaluated clinically and underwent in vitro marrow culture studies (see “Appendix”.) All patients or their records were available for follow-up evaluation, which was completed in June 1978. The median follow-up period of living patients was 20.7 months.
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Criteria for the diagnosis of preleukemia were similar to those previously described [1.5,7,11]and consisted of relatively prolonged courses of refractory anemia, neutropenia or thrombocytopenia, singly or in combination, associated with characteristically abnormal marrow morphology. We have maintained the designation preleukemia with the understanding that we are refetring to the group of patients having myelodysplastic disorders with the clinical characteristics described in whom the disease has a propensity to terminate in a syndrome morphologically resembling AML. As recently suggested. the descriptive term hemopoietic dysplasia may be more appropriate [6]. Marrow examination indicated dysplastic maturation of at least two, and generally all, of the granulocytic, erythroid and megakaryocytic cell lines, including qualitative changes such as myeloid nucleocytoplasmic asynchrony, megaloblastoid erythropoiesis, occasionally with ringed sideroblasts, and often bilobed megakaryocytes [16]. In addition, the presence of abnormal mononuclear cells was noted in the marrows of 39 of these 43 patients. The cells comprised approximately 5 to 10 per cent of the myeloid cells, had granular bluish grey cytoplasm with a prominent nuclear hof and a reticulated nuclear chromatin pattern with palestaining nucleoli. ne d&s have previously been prominently noted in other patients with preleukemia [3,11,17,18], and, although the precise nature is unknown, they have been identified as abnormal promyelocytes. The cellularity of the marrow sections was either normal, decreased or increased; when increased, this was due to erythroid but not granulocytic hyperplasia. A quantitative increase in marrow myeloblasts was not present. Particular attention was paid to exclude patients who were already leukemic (i.e., had subacute or smoldering myeloid leukemia, with a quantitative increase in marrow blasts) and those with only single hemopoietic line defects (e.g., idiopathic sideroblastic ineffective erythropoiesis, idiopathic ngutropenia an’d idiopathic thrombocytopenia). The 43 patients with preleukemia include 24 patients whose in vitro growth studies were reported previously [ll]. These patients are included in our present study since the longer follow-up period has permitted better definition of their clinical courses. No clinical differences were noted in the two groups of patients. Marrow aspirates for in vitro studies were obtained after informed consent was obtained according to guidelines established by the Stanford University Human Experimentation Committee. Analysis of the patients’ probability of survival and duration of disease prior to acute transformation was computed by Kaplan-Meier Actuarial survival curves. Statistical analysis of differences between these curves was performed by the method of Gehaland Mantel [19,20).The Wilcoxon two-tailed two-sample rank test and chi square analysis were also employed for certain computations. Results were considered to be statistically significant if p values were cO.05.
RESULTS Composite presenting clinical and in vitro marrow culture features are indicated in Table I. Individual patient data are shown in the Appendix. As shown in Figure 1 and the Appendix, the initial incidence of CFU-GM in the marrow in preleukemic patients was significantly lower than normal (p < O.OOl],whether or not the patients subsequently underwent acute trans-
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GRANLJLOPOIETICPARAMETERS IN PRELEUKEMIA-GREENBERG. MARA
formation, with a median value of 3 colonies per lo5 marrow cells plated. Seventy-two per cent of the initial CFLJ-GM values were less than normal. The CFU-GM values were not significantly different in the group of patients who eventually underwent transformation as opposed to those who did not. The proportion of light density CFU-GM was increased in seven of nine subjects, with a median of 15 per cent CFU-GM less dense than 1.062 g/cm3. These values were significantly above control levels (p < 0.61). The initial values of marrow adherent cell production of CSA and the urinary output of CSA were normal in 13 and nine patients, respectively. A linear relationship was noted between marrow cell CSA production (0to 74.6 U/ml) and adherent cell numbers (which ranged between 0.5 and 50 X lo5adherent cells/ml, with a correlation coefficient = 0.89,p < 0.001). The proportion of monocytes comprising the adherent cell population was 94.6 f 4.6 per cent [mean f SD] in these patients. In six patients, sequential evaluation indicated a persistent or progressive decrease in the incidence of CFU-GM prior to or concomitant with acute transformation (Figure 2). Clinical and in vitro parameters were evaluated to assess their predictive value for survival [Table II]. The median survival of the 43 patients was 13 months from the time of the first marrow culture and 18.9months from the time of diagnosis (Figure 3). The one, two and three year probability of survival from the time of diagnosis was 77 per cent, 55 per cent and 45 per cent, and from initial marrow culture 67 per cent, 47 per cent and 38 per cent, respectively (Figure 3). None of the clinical features analyzed and stated. in Table I predicted decreased survival when compared to their complementary parameters. However, correlation of in vitro studies with clinical courses indicated that survival of those with
TABLE I
Preleukemic Syndrome-Clinical Characteristics Per Cent
Characteristic 43 patients with Refractory cytopenia and Marrow morphology abnormal Hemopoietic dysplasia L 2 lines Age, median 61 years old >50 years old Cytopenia Single cytopenia Bicytopenia Pancytopenia 1 Leukocyte alkaline phosphatase Splenomegaly In vitro marrow culture 4 Initial incidence of CFU-GM t Initial % light density CFU-GM Acute transformation
77 40 30 30 67 33 72
78 44
NOTE: CFU-GM = granulocyte-monocyte
colony-forming
cells.
an incidence of CFU-GM in the marrow of <5 colonies/105 marrow cells (and particularly the 42 per cent with markedly decreased values of 12 colonies/105 cells) was significantly less than that of patients with higher colony-forming values (Table II, Figure 4). The one, two and three year probability of survival from the time of the initial marrow culture was 50 per cent, 19 per cent and 10 per cent, respectively, for patients with an incidence of CFU-GM of <2 colonies/W marrow cells, This contrasted with and was significantly less than the survival probabilities of 81 per cent, 76 per cent and 64 per cent, respectively, for patients with >2 colonies/l05 marrow cells (Figure 4) (p < 0.004). None of the clinical features analyzed correlated with differing CFU-GM
1
J In vitro parameters of granulopoiesis in preleukemic patients. Open boxes enclose the mean f 2 SD of normal values. CFU-GM = granulocyte-monocyte colony forming cells. CSAsM = adherent bone marrow cell colony stimulating activity production. CSA, = urinary colony stimulating activity excretion.
Frgure 1.
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GRANULOPOIETIC
PARAMETERS
IN PRELEUKEMIA-GREENBERG.
comparison with those who did not. Of the 12 patients who died without having undergone acute transformation, eight (60 per cent) died of infections, two (20 per cent) died of bleeding and the other two (20 per cent) died of causes unrelated to their hematologic disease. Among these patients, those who were neutropenic had a significantly shorter survival than those who were not neutropenic (8.9 months versus 21.1 months, p < 0.04). The potential for acute transformation was analyzed with respect to clinical and in vitro parameters (Table III). Forty-four per cent of the patients (19 of 43 patients) have so far undergone transformation within a median period of 19 months. Pancytopenia at the time of presentation was the only parameter which correlated with subsequent acute transformation. Despite a shorter period of survival, 69 per cent of these subjects underwent acute transformation as compared to 33 per cent of those without pancytopenia (p < 0.01). Transformation occurred in pancytopenic patients in a median time of 15 months from diagnosis in comparison with 45 months for those who were not pancytopenic, (p < 0.007) (Figure 5). The two year probability (from time of diagnosis) of patients undergoing transformation was 33 per cent for all patients, for those who were initially pancytopenic it was 66 per cent and for those who were not it was 18 per cent. The difference in transformation probability between the latter two groups of patients was statistically significant (p < 0.007)(Figure 5). The patients in the group that underwent transformation were of similar age’ compared to those who did not undergo transformation (63 years old, median], with a nonsignificant increased proportion of patients having pan-
Months prior to acute transformation I
MARA
I
F&lWs 2.
Sequential marrow granulocyte-monocyte colony forming cell (CFU-GM) values in preleukemic patients who underwent acute transformation.
levels (i.e., normal, low or very low]. Upon contrasting relatively short survivors (
SURVIVAL
I OO
I
30
I
60
,I
I
90
I
120
I
150
I 160
I
210
MONTHS
:igure 3. Probability of survival of preleukemic patients from the time of diagnosis-and from the time of initial marrow culture study- - - -. The short vertical lines denote still surviving patients.
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CRANULOPOIETIC
TABLE Ii
Preieukemic Syndrome-Potential Survival
PARAMETERS
TABLE iii
For
IN PRELEUKEMIA-GREENBERG,
Preieukemic Syndrome-Potential Acute Transformation
Median Survival (mo) From First From Dlaanosis Marrow Culture Patients (43) Age >50 yr >60 yr Cytopenia SingWbicytopenia Pancytopenia 1 Leukocyte alkaline phosphatase Splenomegaly In vitro marrow culture 1 initial incidence of CFU-Gf+i <5 coionies/105 marrow ceils 12 colonies/105 marrow cells < 1 colonies/ 1O5 marrow ceils Acute transformation
18.9
13
17.9 21
11.3 15.8
21 11 21.1 33
14 8.9 15.9 7.8
26.6
12.1 11.1’ 10.2’ 11.1’ 18.9
l
for
Medianlime to Transformation lncldence of (mo) Transformation Fram From Initial Diagnosis Marrow Culture (q/o)
ParametersAssessed
44
19.1
15.9
69 }* 33
45.3 l5 }**
;;:;r*
57 56
32.5 19.5
9.2 16.5
52 45
31.0 20.0
16.9 13.3
1 initial incidence of CFU-GM 12 coionies/105 marrow cells
42
...
10.6
50
10.6
0 colonies/ 1O5 marrow ceils
75
13.3
Ail patients Cytopenia SingieIbicytopenia Pancytopenia Splenomegaiy 1 Leukocyte alkaline phosphatase Age (yr) <60 <50 In vitro marrow culture
l
NOTE: CFU-GM = granulocyte-monocyte colony forming ceils. Gehan analysis: p < 0.03, p < 0.004 relative to complementary parameter, other comparisons were not significantly different. l
MARA
l
NOTE: CFU-GM = granulocyte-monocyte colony forming ceils. Significantly different value relative to c&nplementary parameter, Gehan analysis: p < 0.01, ++ p < 0.007. Othercomparisons were not significantly different. l
SURVIVAL
All patients
-
CFU-GM 5 2 ---CFU-GM > 2---
0
0
I 10
I
20
I
30
I
I
40
I
50
I
60
I
70
I
60
90
MONTHS FROM INITIAL MARROW CULTURE
Figure 4. Probability of survival of preieukemic patients from the time of initial marrow culture relative to marrow granuiocyte-monocyte colony forming capacity (CFU-GM). A significantly decreased probability of survival was noted in the 16 patients with initially 12 colonies/105 marrow ceils compared to the 25 patients with >2 colonies/105 marrow ceils (p < 0.004). The short vertical lines denote still surviving patients.
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GRANULOPOIETIC
PARAMETERS
IN PRELEUKEMIA-GREENBERG,
MARA
TIME TO TRANSFORMATION I
I
I
All patients Pancytopenic
I
patients ----
Non pancytopenic
patients - - -
i loo0
I
30
I
I
60
90 MONTHS
120 FROM
1 150
I
180
I
210
DIAGNOSIS
Figure 5. Probability of acute transformation in preleukemic patients. A significantly increased probability of transformation was noted in the 13 patients who initially were pancytopenic compared to the 30 nonpancytopenic patients (p < 0.007). The short vertical lines denote patients who have not undergone transformation.
cytopenia (47 per cent versus 21 per cent], splenomegaly per cent versus 29 per cent) or low leukocyte alkaline phosphatase values (71 per cent versus 62 per cent) (Appendix). Decreased CFU-GM values in the marrow alone were not predictive of subsequent acute transformation. Median initial CFU-GM levels were the same and low in both groups (3 colonies/105 marrow cells plated). Comparisons of other initial in vitro parameters between the two groups were not possible as most of those data had been obtained only in patients who did not undergo transformation. Sequential analysis of these parameters is continuing. The median follow-up period of the 12 living patients who have not undergone transformation during the study is 20.7 months. Multivariate analysis of several parameters for predicting survival or the potential for transformation (e.g., pancytopenia plus low CFU-GM incidence) added little discriminatory power to that obtained with the single parameter alone. No particular type of cytopenia or the presence of pancytopenia was significantly correlated with low (or very low) CFU-GM values. (42
COMMENTS
The clinical features which characterized these 43 prospectively evaluated patients with preleukemia are similar to those previously described for retrospectively analyzed patients in whom AML had developed after they had had a preleukemic phase [l-4]. In our patients with refractory cytopenia and marrows showing hemopoietic dysplasia the incidence of combined cyto956
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penias was 60 per cent, of low leukocyte alkaline phosphatase levels 67 per cent and of splenomegaly 33 per cent. The median survival of these patients was 18.9 months with a 55 per cent two-year probability of survival. Transformation into AML occurred in 44 per cent of the patients within a median period of 19.1 months. The only clinical or in vitro parameter which was discriminating for the prognosis of survival was an initial markedly decreased incidence of CFU-GM (52 colonies/105 marrow cells). Sixty-nine per cent relatively short-term survivors had this finding in contrast to only 13 per cent of the long-term survivors. An abnormally low initial incidence of CFU-GM and a high proportion of light density CFU-GM were found in 72 per cent of the preleukemic patients, and 42 per cent had markedly decreased values. These patients with markedly decreased marrow myeloid colony formation had a significantly decreased duration of survival (10 month median, with a 19 per cent two year probability of survival from the time of initial marrow culture, p < 0.004). This shortened survival was due to the combination of deaths resulting from the consequence of acute transformation and from complications of the patients’ cytopenias, generally infectious, even in the absence of transformation. The in vitro culture studies thus suggest that markedly defective colony formation was associated with an inadequate marrow response to demand for new granulocytic cells. In the patients who did not undergo transformation, but who were neutropenic, decreased survival was related to infection. Qualitative
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Prekukemic
APPENDIX
Patients-Clinical
and Sex
cytopenia
Splenomegsly
IN PRELEUKEMIA-GREENBERG,
MARA
and In Vitro Data
Age(yr) Case No.
PARAMETERS
LAP
Initial CFU-GM (colonies/ 10s marrow cells)
_
Time to Transformation(mo.) From 1st Marrow From Culture Diagnosis
Duralion of Disease(mo.) From 1st Marrow From Culture Diagnosis
Patients With Acute Transformation 1
3 3 4 59 6 7 8 9*t 10 ll§
12t 13 14 15 16§ 17 18 19 20” 21§ 22t§ 23’ 249 259 26t§ 27’ 289 29 30’9 319 32.t 33 34’ 35 36t 37” 38t 39’ 409 419 42 43
68,F 61,F 26,M 59,M 71,M 88,F 63,F 39.F 68,M 62,F 61,M 84,M 56,M 67,F 61,M 38,F 48.F 89,M 77,F 80.M 77,M 60,M 34,M 71,M 57,F 62,M 64,F 81,M 54,F 36.F 57,F 70,M 81,M 50,M 36,M 68,M 75,M 84,M 63,M 51,F 60,F 36,F 73-M
AT NAT
+ +
T A
0 0 + 0 + + 0 + 0 0 + 0 + 0 0
N,A,T N,A,T N,A A A,T N,A,T N,A,T N,A,T T A A N,A,T N,A N,A,T N,A,T N N,A A N,A,T N,A,T N A ATT N,A N,A A N,T A N,A A,T T
0 0 + + 0 + 0 + 0 0 0
38.9
:’ T
35 30 39 14 19
65.1 72.4-F 11.3 26.3 1.4
i 1
10 6
74.6 47.5 43.9+
T -
31.5 1 0.5 0
20.9 13.5+ 8.9 4.6 37.4
7 i
0 0
11.1 14.5+ 15.9 18.9
0
2.5 0 Patients Without Acute Transformation f 1
39 76 38 23
27.1+ 20.7+ 4.4+ 25.2+
f
19 10 11 9 8 7 6 3.3 3 3 2 2.3
6.7+ 3.4 3.2+ 6.7 13.1 42.5+ 22.5+ 4.0+ 27.1+ 18.7 0.7 9.3 1.0+
H c* H
0 0 0 0
i
+ +
N,A,T A
0 0 + 0 0
N,A,T A,T
92
0
N,A,T A
A,T N
t
0
0
c*
t
1 1 0.8 0.5 00.5
1
0
27.2 2.1 27+ 6.3 4.8 13 15.8
47.6 139.5+ 65.1 11.3 1.4 26.6 119.5 84 45.2-I 13.5+ 35 8.9 8.5 52.3 59.4 19+ 18.9 17.9 11
37.1 67.9 50.8 8.7 1.2 19.6 39.6 9.6 43.5 5.6 16.9 8.6 4.1 34.3 8.7 10.6 16 15.9 1.1
45.8 135 50.8 8.7 1.2 20 111.5 19.1 44.7 5.6 31 8.6 8.0 49.3 57 15 16 17.9 9.6
39.0+ 20.7+ 4.4+ 72.0+ 6.7+ 3.2+ 46 21 13.1 223+ 40.5+ 4.0+ 39+ 18.7 1.7-I 21.9 0.7 27.2 2.1 32.4+ 10.3 4.8 19.3 15.8
NOTE: CFU-GM = granulocyte-monocyte colony forming cells. N = neutropenia. A = anemia. T = thrombocytopenia. -i- = splenomegaly. 0 = no splenomegaly. 1 = decreased. t = increased. - = normal. LAP = leukocyte alkaline phosphatase. per cent light density CFU-GM determined (see Figure 1). t Urinary colony stimulating activity output determined (see Figure 1). 5 Marrow cell colony stimulating activity production determined (see Figure 1). l
abnormalities in granulocyte chemotactic, phagocytic and bactericidal activities, and an increased incidence of lethal infections have also been previously reported in preleukemic patients [18,21,22]. The presence of pancytopenia at the time of diagnosis was predictive of subsequent acute transformation. This finding was also present in several early retrospective series of patients with this disorder, although those pa-
tients were not so analyzed [2-41. Recent studies have shown a higher incidence of chromosomal abnormalities and evolutionary potential in pancytopenic patients [23]. Thus, it is possible that the presence of pancytopenia reflects a qualitatively more severe degree of in vivo progression of clonal abnormalities than that in patients with other, less severe, types of cytopenias. The in vitro studies of marrow colony formation at the time
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957
CRANULOPOIETIC PARAMETERS IN PRELEUKEMIA-GREENBERG,
of diagnosis did not predict subsequent transformation. However, sequential studies in six preleukemic patients indicated a persisting or progressive decrease in the incidence of CFU-GM concomitant with or preceding progression of the disease. Enzymatic or cytogenetic analysis will be needed to determine whether this finding is a functional correlate of clonal evolution or whether it reflects progressive repopulation of the marrow by more abnormal granulopoietic cells. The finding that low colony formation per se was not predictive for subsequent transformation indicates that other factor(s) are necessary for this progression. Prior studies in patients with other chronic myeloproliferative disorders reported from our laboratory indicated that, in addition to a low or decreasing incidence of CFUGM, progressive abnormalities of urinary CSA levels developed as their disorders evolved [ll]. The patients with preleukemia described herein had normal levels of urinary CSA output and adherent marrow cell CSA production in the chronic stable phase of their diseases. Studies are ongoing to determine whether abnormalities of humoral regulatory substances develop in preleukemia with evolution of the disease. Low marrow colony formation, previously described
MARA
in preleukemia [lo-121, is not specific for this syndrome since it has also been found in certain neutropenic disorders and in acute, subacute and progressing chronic myeloid leukemia [9-11,24-271. However, the high proportion of light density CFU-GM found in preleukemia and other myeloproliferative disorders [11,13,24,26,28] is not present in the benign neutropenias [29], thus providing a useful adjunct to marrow morphology for distinguishing these entities. Although present in only a few patients in our series of preleukemit patients, other studies have shown an increased incidence of abortive cluster formation in patients with acute and chronic myeloproliferative disorders [11,24-261. Patients with AML, subacute myeloid leukemia and preleukemia may have similar in vitro clonal growth patterns despite differing pace and incidence of disease progression. Therefore, marrow morphology is necessary (i.e., showing quantitative rather than qualitative myeloid abnormalities) for the differential diagnosis of these conditions. As discussed herein for preleukemia, and in prior studies of other myeloproliferative disorders [11,14,24,25], the in vitro parameters of marrow granulopoiesis present in these diseases appear to provide information of prognostic value.
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Block CA, Jacobson LO, Bethard WF: Preleukemic acute leukemia. JAMA 152: 1018,1953. Meacham GC, Weisberger AS: Earlv atvoical manifestations of leukemia. Ann Intern Med 411786,*1954. Fisher W. Armentrout S. Weisman R. et al.: “Preleukemia.” Arch Intern Med 132:.226,1973. Blair TR. Bayrd ED, Pease GL: Atypical leukemia. JAMA 198: 21, 1966. Saarni M. Linman 1:Preleukemia. The hematoloaic svndrome preceding acuteleukemia. Am J Med 55: 38,-1973. General Discussion: I. Is preleukemic states an adequate designation? Blood Cells 2: 347.1976. Linman JW, Bagby GC: The preleukemic syndrome (hemopoietic dysplasia). Cancer 42: 854, 1978. Metcalf D: In vitro cloning techniaues for hemoooietic cells: clinical applications. Ann Intern Med 87: 483, 1977. Greenberg PL: Imolications of in vitro marrow clonal growth studies for human disorders of granulopoietic regulation. Hemopoietic Cell Differentiation, ICN-UCLA Symposia on Molecular and Cellular Biology, vol X, [Golde DW, Cline MJ, Metcalf D, Fox CF, eds), New York, Academic Press, 1978, p 295. Greenberg PL. Nichols WC. Schrier SL: Granuloooiesis in acute myeloid leukemia and preleukemia. N Engl J Med 284: 1225,197l. Greenberg PL. Mara B, Bax I, et al.: The myeloproliferative disorders. Correlation between clinical evolution and altcrations of granulopoiesis. Am 1Med 61: 878, 1976. Scnn IS. Pinkerton PH: Defective in vitro colonv formation by human bone marrow preceding overt leukemia. Br J Haemato123: 277,1972. Moore M, Williams N, Metcalf D: In vitro colony formation by normal and leukemic human hemopoietic cells. Characterization of the colony forming cells. J Nat1 Cancer Inst 50: 603, 1973. Greenberg PL, Mara B, Heller P: Marrow adherent cell colony-stimulating activity production in acute myeloid leukemia. Blood 52: 362,1978. Messner H., Till JE, McCulloch EA: Interacting cell populations affecting granulopoietic colony formation by normal and leukemic human marrow cells. Blood 42: 701,1973.
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