Leukemia Research 23 (1999) 357–364
`
`Biologic characteristics of patients with hypocellular
`myelodysplastic syndromes
`
`Rajat Goyal a, Huma Qawi a, Irfan Ali a, Saleem Dar a, Suneel Mundle a,
`Vilasini Shetty a, Yifwayimare Mativi a, Krishnan Allampallam a, Laurie Lisak a,
`Jerome Loew b, Parameswaren Venugopal a, Sefer Gezer a, Erwin Robin c,
`Shelby Rifkin d, Azra Raza a,*
`a Rush Cancer Institute, Rush-Presbyterian-St. Luke’s Medical Center, Chicago, IL, USA
`b Department of Pathology, Rush-Presbyterian-St. Luke’s Medical Center, Chicago, IL, USA
`c Ingall’s Memorial Hospital, Har6ey, IL, USA
`d Northwest Community Hospital, Arlington Heights, IL, USA
`
`Received 17 August 1998; accepted 14 November 1998
`
`Abstract
`
`Rates of proliferation and apoptosis as well as expression of tumor necrosis factor alpha (TNF-a), transforming growth factor
`beta (TGF-b) and the number of macrophages were measured in bone marrow (BM) biopsies of 33 patients who presented with
`hypocellular (cellularityB30%) myelodysplastic syndromes (MDS). Results showed that 2:3 of the patients had high apoptosis,
`high cytokine levels and large number of macrophages in their biopsies while 1:3 did not. Apoptosis and TNF-a levels were
`directly related (r(cid:30)0.583, P(cid:30)0.003, n(cid:30)24) as was apoptosis and the degree of anemia (P(cid:30)0.033, n(cid:30)18). A subgroup of
`patients with abnormalities of chromosomes 5 or 7 had higher platelets (P(cid:30)0.026) and higher apoptosis (P(cid:30)0.038) when
`compared with the rest of the group. Eight patients had no evidence of apoptosis and almost no detectable TNF-a in their
`biopsies. We conclude that within the hypocellular variant of MDS, there may be two distinct sub-groups of patients, one who
`present with high cytokine-mediated intramedullary apoptosis and the other who may be better characterized as having a stem-cell
`failure defect since they showed no evidence of apoptosis. © 1999 Elsevier Science Ltd. All rights reserved.
`
`Keywords: Hypocellular myelodysplastic syndromes; Apoptosis; Proliferation; TNF-a; TGF-b; Chromosomes 5 and:or 7
`
`1. Introduction
`
`The myelodysplastic syndromes (MDS) are a group
`of hematopoietic disorders that primarily originate in a
`pluripotential bone marrow (BM) stem cell and tend to
`predominate in the elderly [1]. The vast majority of
`patients present with a refractory anemia that may or
`may not be associated with additional cytopenias and
`supportive care continues to be the mainstay of therapy
`
`Abbre6iations: MDS, myelodysplastic syndromes; ISEL, in situ end
`labeling of fragmented DNA; IudR, lododeoxyuridine; BrdU, bro-
`modeoxyuridine; TNF-a, tumor necrosis factor alpha; TGF-b, trans-
`forming growth factor beta; LI, labeling index; BM, bone marrow.
`* Corresponding author. Rush Cancer Institute, 2242 West Har-
`rison Street, Tech 2000 Building-Suite 108, Chicago, IL 60612-3515,
`USA. Tel.: (cid:27)1-312-4558474; fax: (cid:27)1-312-4558479.
`
`for these variable cytopenias [2]. Several recent ad-
`vances have been achieved in understanding the biolog-
`ical basis for the paucity of circulating blood cells, the
`most significant being the recognition of excessive in-
`tramedullary apoptotic death of hematopoietic cells
`belonging to all three hematopoietic lineages [3–6].
`This peculiar mode of suicidal cellular destruction ap-
`pears to be cytokine mediated in a substantive majority
`of patients with tumor necrosis factor alpha (TNF-a)
`being the prominent pro-apoptotic cytokine involved
`[7–9]. In summary then, the biological hall-marks of
`MDS are rapid intramedullary proliferation of hemato-
`poietic cells followed by an equally rapid and prema-
`ture apoptotic death of the vast majority of these cells
`in the presence of increased levels of pro-inflammatory
`cytokines such as TNF-a. It is important to note that
`
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`358
`
`R. Goyal et al. :Leukemia Research 23 (1999) 357–364
`
`these biological characteristics may not be present uni-
`versally in all MDS patients mainly because there are
`five different syndromes grouped under one umbrella.
`Each syndrome presents with its own unique natural
`history and prognosis and each has its own spectrum
`of clinical heterogeneity. Thus it is important to iden-
`tify various subgroups within MDS and determine
`whether these patients have archetypal biologic char-
`acteristics which would distinguish them from other
`MDS patients, thereby allowing hematologists to de-
`sign therapeutic strategies tailored to suit their individ-
`ual needs. The present study is one such attempt.
`While the general rule in MDS is that the bone
`marrow is hypercellular despite the presence of vari-
`able cytopenias, 10–30% cases present with a hypocel-
`lular BM [1,2]. In the absence of a recognizable
`karyotypic abnormality, such cases are often difficult
`to distinguish from classic aplastic anemias, although
`preponderance of hypocellularity in the therapy-related
`myelodysplasias and its subsequent association with
`pathognomic cytogenetic abnormalities are often help-
`ful
`in negotiating such semantic matters in a small
`subsets of patients. In the present study, we have
`attempted to identify biologic characteristics which
`would be uniquely associated with hypocellular MDS
`cases. The most striking features of this study were
`related to findings of high intramedullary apoptosis of
`hematopoietic cells in association with high pro-apop-
`totic cytokines and presence of macrophages in ap-
`proximately 2:3 of the cases studied, thereby suggest-
`ing a possible explanation for the hypocellularity at
`least in a sub-set of these patients. Hemoglobin was
`inversely related to the level of apoptosis in this group
`with patients showing high apoptosis being more ane-
`mic. Finally, the subset of patients with hypocellular
`MDS who presented with abnormalities of chromo-
`somes 5 and:or 7 tended to have a higher platelet
`count than the rest of the group as well as a higher
`level of apoptosis.
`
`2. Materials and methods
`
`Thirty-three MDS patients are the subject of this
`report. These MDS patients were studied prior to
`starting a treatment protocol, but had been off all
`therapies including growth factors and vitamins for at
`least 4 weeks. Every individual received a 1 h infusion
`of IUdR (or in case of an iodine allergy, BrdU) at 100
`mg:M2 intravenously prior to undergoing a bone mar-
`row (BM) examination. Informed consent for the infu-
`sion protocol (MDS 90-02) was obtained from every
`individual. The infusion protocol was reviewed and
`approved by the Institutional Review Board (IRB) of
`the Rush-Presbyterian-St. Luke’s Medical Center, the
`National Cancer Institute (NCI) and the Food and
`
`Drug Administration (FDA). The IUdR and BrdU for
`these studies were supplied by the NCI. Immediately
`upon completion of the infusion, peripheral blood,
`BM aspirate and BM biopsies were obtained from the
`patient and transported on ice to Dr Raza’s labora-
`tory. The following studies were performed on the
`tissues.
`
`2.1. Detection of cytokines in the microen6ironment
`
`Levels of two cytokines TNF-a, and transforming
`growth factor beta (TGF-b) were determined semi-
`quantitatively in the bone marrow biopsies immuno-
`histochemically. All
`tissues were fixed in Bouin’s
`solution and embedded in glycol methacrylate. Two to
`three micron thick sections were obtained and placed
`on alcian blue coated coverslips. The sections were
`then individually labelled for each cytokine using the
`respective monoclonal antibodies as follows. After the
`tissues were dehydrated in distilled water for 10 min
`they were incubated with freshly diluted 3% H2O2 for
`30 min and then with pronase 1 mg:ml (Calbiochem,
`LaJolla, CA) for 45 min. Specimens were rinsed care-
`fully with 0.15 M phosphate buffered saline (PBS)
`[0.15 M sodium chloride in 0.1 M phosphate buffer,
`pH 7.5] after each incubation. Following the last 0.15
`M PBS rinse they were placed in 0.5 M PBS [0.5 M
`sodium chloride in 0.1 M phosphate buffer, pH 7.5]
`for 15 min. The sections were treated with 0.5 M PBS
`containing 1.5% horse serum for 60 min to block
`non-specificity. Subsequently the sections were incu-
`bated with the respective monoclonal anti TGF-b2b3
`(1:50) antibody (Oncogene Science, Manhasset, NY)
`or anti TNF-a (1:180) (Promega, Madison, WI) anti-
`body diluted in 0.5M PBS containing 1.5% horse
`serum for 60 min. This was followed by incubating the
`sections with biotinylated anti-mouse IgG [diluted
`(1:200) in 0.5M PBS with 1.5% horse serum] for 30
`min and with the avidin-biotin complex or ABC
`reagent. The horse serum, biotinylated anti-IgG and
`ABC complex were reagents in the Vectastain Elite
`ABC kit (Vector, Burlingham, CA). After each of the
`above incubations, specimens were rinsed in 0.5 M
`PBS. The color reaction was then developed using
`0.025%, 3,3% diamino benzidine tetrachloride (DAB) di-
`luted in 100 ml of 0.5 M Tris buffer, pH 7.5, with 0.01
`ml 30% H2O2 for 10 min and rinsed with distilled
`water. After processing, the coverslips were mounted
`on glass slides and examined by light microscopy.
`
`2.2. Detection of macrophages
`
`The presence of macrophages was detected immuno-
`histochemically by the method described above. The
`monoclonal antibody used was EBM-11 (Dakopatts,
`Denmark).
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`

`R. Goyal et al. :Leukemia Research 23 (1999) 357–364
`
`359
`
`2.3. Detection of S-phase cells
`
`3. Results
`
`the two thymidine ana-
`The in-situ detection of
`logues IUdR and BrdU administered via intravenous
`infusions was carried out by using the protocols and
`immunohistochemical methods described before [5,6].
`After processing and mounting the coverslips with
`fluoromount, at least 2000 positively labeled S-phase
`myeloid cells were counted to determine the labeling
`index (LI). Erythroid and megakaryocytic cells were
`excluded.
`
`A total of 33 MDS patients are the subject of this
`report. According to the French–American–British
`(FAB) classification [10], 22 patients had refractory
`anemia (RA), one had RA with ringed sideroblasts
`(RARS), seven had RA with excess blasts (RAEB), two
`had RAEB in transformation (RAEB–t) and one had
`chronic myelomonocytic leukemia (CMMoL). There
`were 20 males and 13 females with a median age of 60
`years for the entire group. All patients had primary
`de-novo MDS.
`
`2.4. Measurement of apoptosis using in-situ end
`labeling (ISEL) of fragmented DNA
`
`3.1. Biologic characteristics
`
`ISEL was carried out on all the bone marrow biop-
`sies of the 33 patients as described in earlier studies
`[5,6]. Briefly, the sections following pre-treatment with
`sodium chloride sodium citrate (SSC)
`solution at
`80°C and with 1% Pronase (1 mg:ml in 0.15 M PBS;
`Calbiochem, LaJolla, CA) were incubated with a mix-
`ture of dATP, dCTP, dGTP (0.01M, Promega,
`Madison, WI), bio-dUTP (0.001M, Sigma) and DNA
`Polymerase I (20 U:ml, Promega) at 18°C. Incorpora-
`tion of bio-dUTP was finally visualized using avidin-
`biotin-peroxidase conjugate (Vectastain Elite ABC
`Kit, Vector, Burlingham, CA) and diamino benzidine
`tetrachloride. Thus cells labeled positively for ISEL
`showed brown staining in their nuclei under the light
`microscope.
`
`2.5. Interpretation of slides
`
`All the slides were observed on a televised screen
`by several
`investigators. A subjective quantitative
`scale was formulated to determine the degree of posi-
`(TGF-b, TNF-a),
`tivity of
`the different cytokines
`ISEL
`staining
`and
`the
`cellular
`component
`(macrophages) as follows: negative, low, intermediate
`and high. The intensity of
`staining:cell was not
`recorded. The percentage of cells were not calculated.
`Data on reproducibility of the results have been pro-
`vided in the past [5,8].
`
`Low
`Intermediate
`High
`
`1–3(cid:27)
`4–5(cid:27)
`6–8(cid:27)
`
`2.6. Statistical analysis
`
`The nonparametric Spearman test was used to de-
`termine correlations between groups.
`
`The cellularity was assessed from BM biopsies and
`ranged between 5–30% (median(cid:30)20%). Table 1 shows
`the details of all the clinical:biological parameters mea-
`sured. The median white blood cell (WBC) count at
`presentation was 3.0(cid:29)109:l and the median hemo-
`globin was 9.4 Gm:dl (Table 1). The median labeling
`index (LI)
`for the entire goup was 22% (n(cid:30)16,
`range(cid:30)11–37%), median ISEL was 2 (n(cid:30)26, range(cid:30)
`0–8), median TNF-a level assessed in the BM biopsy
`was 3 (n(cid:30)29, range(cid:30)0–8), median TGF-b was 4
`(n(cid:30)28, range(cid:30)0–8) and the number of macrophages
`as measured by EBM-11 antibody were a median of 2
`(n(cid:30)30, range(cid:30)0–8).
`
`3.2. Apoptosis and cytokine studies
`
`Among the 33 hypocellular MDS cases, ISEL studies
`were available in 26 cases and 8:26 cases did not have
`any evidence of ISEL positivity in the entire biopsy.
`Ten of the 18 patients who had evidence of apoptosis
`showed greater than 4(cid:27) positivity. A highly significant
`relationship was found between ISEL positivity and
`TNF-a levels (r(cid:30)0.583, P(cid:30)0.003, n(cid:30)24). Futher-
`more, in patients who were ISEL-positive versus those
`that were ISEL-negative, there was a significant differ-
`ence in the level of TNF-a (P(cid:30)0.005). The median
`TNF-a level was 0 for the ISEL-negative patients (n(cid:30)
`7) and the median TNF-a was four for the ISEL
`positive cases (n(cid:30)17). Finally, level of apoptosis was
`also inversely related to the level of hemoglobin in these
`patients (r(cid:30) (cid:28)0.505, P(cid:30)0.033, n(cid:30)18).
`TNFa levels were available in a total of 29:33 pa-
`tients with a median value of 3(cid:27) (Table 1). Nine of
`these 29 patients showed no detectable TNF-a at the
`protein level in the BM biopsies. A significant differ-
`ence in the biological characteristics of patients with
`any detectable TNFa levels was noted in comparison
`with patients who were entirely negative. The most
`significant difference was related to the level of apopto-
`sis as already mentioned, TNF levels being high when
`ISEL was high (n(cid:30)18, P(cid:30)0.013) and low when ISEL
`
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`

`360
`
`R. Goyal et al. :Leukemia Research 23 (1999) 357–364
`
`Table 1
`Clinical:biological characteristics of patients with hypocellular myelodysplastic syndromes
`
`Sex
`
`Age
`
`FAB
`
`Cellularity
`
`BM-Bx-blasts*
`
`BM-Asp-blasts
`
`WBC
`
`HGB
`
`Platelet
`
`1.
`2.
`3.
`4.
`5.
`6.
`7.
`8.
`9.
`10.
`11.
`12.
`13.
`14.
`15.
`16
`17.
`18.
`19.
`20.
`21.
`22.
`23.
`24.
`25.
`26.
`27.
`28.
`29.
`30.
`31.
`32.
`33.
`
`M
`M
`M
`M
`M
`M
`M
`M
`M
`M
`F
`F
`F
`F
`F
`M
`F
`F
`M
`M
`M
`M
`F
`F
`M
`M
`M
`M
`F
`M
`F
`F
`F
`
`55
`55
`50
`62
`70
`59
`69
`75
`58
`59
`77
`53
`49
`76
`77
`46
`79
`47
`58
`66
`81
`83
`55
`53
`67
`50
`66
`57
`16
`60
`74
`72
`77
`
`RA
`RA
`RA
`RA
`RA
`RA
`RA
`RA
`RA
`RA
`RA
`RA
`RA
`RA
`RAEB-t
`RAEB
`RAEB
`CMMoL
`RA
`RA
`RA
`RA
`RA
`RA
`RAEB
`RAEB
`RAEB
`RAEB
`RAEB
`RAEB-t
`RA
`RA
`RARS
`
`20
`20
`20
`Hypo
`10-20
`Hypo
`20
`20
`Hypo
`10-20
`5
`30
`10
`Hypo
`Hypo
`10
`20
`10
`10
`5
`Hypo
`10
`20
`30
`10-20
`10-20
`20
`20
`Hypo
`20
`20
`5
`20
`
`3
`5
`5
`5
`5
`5
`5
`5
`5
`5
`5
`1
`5
`5
`25
`16
`8
`10
`5
`2
`5
`5
`5
`5
`35
`25
`9
`5
`5
`33
`0
`1
`5
`
`0
`0
`0
`5
`5
`5
`5
`5
`3
`1
`5
`2
`0
`0
`5
`31
`1
`22
`0
`2
`0
`5
`0
`2
`6
`5
`5
`10
`5
`30
`0
`1
`5
`
`2.1
`2.4
`3.9
`5.5
`4.5
`1.6
`3.0
`1.9
`4.0
`4.4
`3.9
`3.7
`5.4
`1.1
`2.4
`1.7
`5.0
`0.9
`4.2
`1.0
`3.3
`5.9
`3.1
`2.4
`1.6
`2.9
`8.6
`1.7
`0.8
`1.1
`4.4
`2.4
`4.2
`
`13.1
`9.6
`7.7
`9.0
`8.5
`9.2
`10.7
`9.5
`10.9
`11.2
`9.1
`8.9
`9.2
`10.1
`9.9
`13.3
`10.7
`7.4
`10.4
`7.9
`9.0
`9.7
`12.6
`7.9
`9.4
`12.8
`8.1
`7.8
`9.0
`10.0
`9.1
`9.2
`10.5
`
`143
`34
`31
`131
`18
`225
`397
`20
`54
`237
`131
`856
`292
`14
`224
`225
`309
`10
`10
`33
`197
`72
`225
`10
`32
`193
`628
`12
`33
`33
`77
`39
`280
`
`LI
`
`Ne
`11
`Ne
`Ne
`Ne
`26
`Ne
`Ne
`27
`Na
`Ne
`Na
`21
`Ne
`18
`28
`37
`24
`11
`19
`14
`23
`24
`Ne
`Na
`Ne
`29
`12
`11
`Na
`Na
`Na
`Na
`
`ISEL
`
`TNF-a
`
`TGF-b
`
`EBM-11
`
`4
`0
`8
`Na
`Na
`8
`Na
`2
`1
`4
`0
`4
`2
`0
`8
`0
`0
`8
`0
`Na
`8
`1
`1
`0
`2
`1
`0
`2
`5
`5
`Na
`Na
`Na
`
`8
`0
`8
`Ne
`3
`4
`5
`2
`2
`1
`0
`8
`3
`0
`4
`1
`0
`8
`0
`0
`8
`6
`3
`Na
`Ne
`5
`7
`3
`2
`0
`Na
`0
`0
`
`2
`3
`4
`Ne
`Na
`1
`7
`4
`5
`1
`Ne
`8
`4
`0
`2
`1
`5
`4
`3
`0
`2
`3
`4
`Ne
`0
`8
`0
`7
`3
`0
`Na
`Ne
`1
`
`6
`0
`0
`Ne
`4
`1
`6
`0
`3
`1
`0
`2
`4
`Ne
`0
`4
`5
`6
`0
`0
`7
`0
`3
`0
`4
`0
`4
`0
`3
`0
`Na
`0
`3
`
`* BM-Bx-blasts, % blasts in bone marrow biopsy; Ne, not evaluable; BM-Asp-blasts, % blasts in bone marrow aspirate; LI, labeling index (% S-phase cells); WBC, cells(cid:29)109:l; TNF-a: tumor
`necrosis factor alpha; Hgb, hemoglobin in gm:dl; TGF-b, transforming growth factor beta; Platelets, platelets(cid:29)109:l; EBM-11, surface antigen for macrophage; Na, not available for evaluation.
`
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`

`R. Goyal et al. :Leukemia Research 23 (1999) 357–364
`
`361
`
`Table 2
`Biologic correlates of TNF-a in patients with hypocellular myelodysplastic syndromes
`
`TNF-negative
`
`Median
`
`0
`1
`0
`
`n
`
`6
`7
`8
`
`TNF-positive
`
`Median
`
`3
`4
`3
`
`n
`
`18
`19
`20
`
`P
`
`0.013
`0.06
`0.06
`
`ISEL*
`TGF-b*
`EBM-11*
`
`* ISEL, in situ end labeling of fragmented DNA; TNF-a, tumor necrosis factor alpha; TGF-b, transforming growth factor beta; EBM-11,
`surface antigen for macrophage 11.
`
`was negative (P(cid:30)0.013, n(cid:30)6). TGF-b was similarly
`high (median(cid:30)4, n(cid:30)19) in the TNF-positive group
`in the TNF-negative
`and low (median(cid:30)1, n(cid:30)7)
`group (P(cid:30)0.06). Macrophages were quite prominent
`in the high TNF-positive:TGF-b-positive group (me-
`dian(cid:30)3, n(cid:30)20) and low in the TNF-negative:TGF-b
`negative group (median(cid:30)0, n(cid:30)8). These relation-
`ships were not statistically significant (P(cid:30)0.06). The
`summary of the data is shown in Table 2.
`
`3.3. Cytogenetic studies
`
`Karyotypes were determined in all 33 patients.
`Thirteen patients had normal chromosomes and 20
`had an abnormal karyotype. Among these 20 pa-
`tients, 13 had abnormalities of chromosomes 5 (n(cid:30)9)
`and 7 (n(cid:30)4), one had del (20)(q11 q13) and three
`had trisomy 8. The remaining three patients had vari-
`able anomalies listed in Table 3 (del 16,(cid:27)15, del 9,
`del 15,(cid:27)21 and marker chromosome). There were no
`statistically significant differences between individuals
`who had normal cytogenetics versus those who had
`any abnormal metaphases. However, when the subset
`of hypocellular MDS cases who presented with ab-
`normalities of chromosomes 5 or 7 were compared to
`all other patients (normal as well as any other kary-
`otypic abnormality), two statistically significant differ-
`ences were noted.
`Firstly, the platelet count was higher for patients with
`5 or 7 abnormalities (median 247(cid:29)109:l, n(cid:30)13 vs.
`101(cid:29)109:l, n(cid:30)20, P(cid:30)0.026) as shown in Table 4.
`Secondly, this subgroup of patients also had a statis-
`tically higher level of apoptosis in their bone marrow
`when compared to normal plus any chromosomal ab-
`normality except
`that of 5 or 7 group (median
`ISEL(cid:30)4.0, n(cid:30)10 vs. median 1.0, n(cid:30)16, respec-
`tively, P(cid:30)0.038). These data are shown in Table 4.
`
`4. Discussion
`
`Hypocellular myelodysplastic syndromes have been
`recognized as a distinct variant of MDS however, no
`
`differences in the natural history, prognosis or re-
`sponse to treatment have been appreciated between
`hypocellular versus normo:hypercellular MDS [11–
`15]. The main source of confusion continues to be the
`difficulty of discriminating hypocellular MDS from
`aplastic anemia patients, in the absence of chromoso-
`mal abnormalities, however recent suggestions to treat
`these hypocellular MDS patients with immunosup-
`pressive aplastic anemia-like therapies have resulted in
`modest successes thereby obviating the need to cate-
`gorically make this distinction in every case [16]. The
`presentation of an identical clinical syndrome in the
`setting of such starkly contrasting bone marrow cellu-
`larity as seen in hypo versus hypercellular MDS how-
`ever, poses an interesting biological challenge. The
`present study was therefore undertaken to determine
`whether the recently recognized biological characteris-
`tics commonly associated with hypercellular MDS are
`also a consistent feature in patients who present with
`the hypocellular variant. What we found in this study
`of 33 hypocellular MDS cases was in fact quite simi-
`lar to what has already been described for the more
`frequently encountered hypercellular variety. In sum-
`mary, 2:3 of these patients demonstrated the presence
`of
`intramedullary apoptosis accompanied by higher
`levels of TNF-a, TGF-b and macrophages when com-
`pared with 1:3 of the group that did not have apop-
`tosis. Profundity of anemia was
`related to high
`apoptosis and patients with abnormalities of chromo-
`somes 5 or 7 appeared to have more apoptotic cells
`in their marrows compared to patients who had a
`normal karyotype and cytogenetic abnormalities af-
`fecting chromosomes other than 5 and:or 7.
`The paradox of variable cytopenias despite hypercel-
`lular BMs in MDS in general has been explained on the
`basis of excessive intramedullarly cytokine-induced
`death of hematopoietic cells [5–7,17]. Obviously, this
`cannot apply universally to all MDS patients since at
`least 1:3–1:2 of MDS cases do not show evidence of
`excessive apoptosis [17]. In those patients who have a
`hypercellular BM but no evidence of apoptosis, it is
`conceivable that the rapidly proliferating hematopoietic
`cells are being retained in the marrow for abnormally
`
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`

`

`362
`
`R. Goyal et al. :Leukemia Research 23 (1999) 357–364
`
`Table 3
`Cytogenetic characteristics of patients with hypocellular myelodysplastic syndromes
`
`GMA
`
`Cytogenetics
`
`ISEL*
`
`TNF-alpha*
`
`0
`0
`
`08
`
`6
`3
`na
`ne
`5
`7
`3
`
`20
`
`1
`0
`4
`0
`8
`3
`0
`na
`5
`8
`8
`4
`8
`0
`1
`
`0
`3
`ne
`2
`2
`
`472:95
`408:96
`419:97
`191:93
`236:97
`176:96
`337:96
`506:97
`390:97
`23:95
`238:96
`502:95
`163:95
`191:97
`194:97
`234:93
`198:98
`55:98
`161:97
`329:94
`208:98
`504:97
`516:97
`340:94
`5:93
`1113:94
`211:96
`107:98
`
`199:98
`477:97
`98:95
`278:97
`384:96
`
`0
`46, XY
`0
`46, XY
`na
`46, XY
`8
`46, XY
`1
`46, XY
`1
`46, XX
`0
`46, XX
`2
`46, XY
`1
`46, XY
`0
`46, XY
`2
`46, XY
`5
`46, XX
`5
`46, XY
`4
`46, XY, del(5)(q13q33)[19]:46, XY[1]
`0
`46, XX, del(5)(q22)[4]:46, XX [16]
`8
`46, XX[3]:47-48,X,-X,del(5)(q?12q21)
`na
`46, XX[19]:NCA:46, XX,del (5) (?q15?q35) [1]
`4
`46,XX, del(5)(q15q33)[18]:47, XX,(cid:27)8[2]
`2
`46,XX,del(5)(q31q33)[26]:46,XX[2]: NCA 47,XX,(cid:27)8
`0
`46,XX[2]:46,XX,del(5)(q22q35)[6]:47,XX,(cid:27)8[2]
`na
`46, XX, del(5)(q15q33)[18]:47, XX,(cid:27)8[2]
`46,XY,del(5)(q23q33),del(9)(q21q32)[10]: 46,idem,?del(20)(q13.1)[4]: 46,XY[5] NCA:46,XY,del(9) (q21q32) na
`4
`46,XY,der(7)t(1;7)(q10;p10)[18]:46,XY[2]
`46,XY,[9]:46,XY,der,(7)t(1;7)(q21;q11)[7]
`8
`46,XY[6] :46,XY,del(7)(q22q32)[19]
`8
`45xy,-7[7]:46xy[9]
`8
`0
`45,X,-Y[5]:47,XY,(cid:27)8[10]:47, idem,del(16)(q22)[2]:46,XY[3]
`0
`60,XXY,(cid:27)1,add(2)(q33?),(cid:27)8,(cid:27)9x2,(cid:27)10,(cid:27)11,(p11.2) (cid:27)13,(cid:27)14,(cid:27)15,(cid:27)20,add(21)(p11.2),(cid:27)22x2,(cid:27)
`mar[21]: 46,XY,[3]
`46, XX,del(13) (q12q22)[8]:47, XX,(cid:27)8[2]:46, XX[10]
`46, XY, del(20)(11.2q13.1)[20]
`46,X,-Y,(cid:27)15
`46, XY, del(15)(?q21q24)[2]:46, XY[18]
`47,XY,(cid:27)21[3]:46,XY[17]
`
`na
`na
`na
`2
`1
`
`* na, not available for evaluation; ne, not evaluable; ISEL, in situ end labeling of fragmented DNA; TNF-alpha, tumor necrosis factor alpha.
`
`time. Overexpression of adhesion
`long periods of
`molecules could result in such a probability. The situa-
`tion of hypocellularity in MDS on the other hand,
`could be explained by excessive apoptosis which is not
`matched by an equal rapidity of cell-birth thereby
`tipping the balance in favor of cellular destruction.
`Hypocellularity in the absence of apoptosis is a more
`intriguing challenge and this small group of patients
`
`Table 4
`Relationship between cytogenetics and apoptosis
`
`ISEL* (median)
`
`Platelet (mean9
`SEM)
`
`Abnormalities of Chromo-
`some 5:7
`Normal:others
`
`4 (n(cid:30)10)
`
`1 (n(cid:30)16)
`
`P value
`
`0.038
`
`247.1959.8
`(n(cid:30)130)
`100.7932.5
`(n(cid:30)20)
`0.026
`
`* ISEL, in situ end labeling for fragmented DNA.
`
`may be more like the aplastic anemia patients in that
`they suffer more from a stem-cell failure disorder rather
`than apoptotic death of their progeny following rapid
`proliferation. The description of MDS progenitor cell
`as having a proliferation defect is compatible with such
`hypocellular cases and no evidence of apoptosis. In the
`present study, of the eight patients who showed no
`apoptosis by ISEL, the LI was low (11%, 11%) for two
`and high (28, 37 and 29%) for three patients while it
`could not be reliably measured in three patients due to
`technical problems posed by the extremely hypocellular
`marrows. In other words, there are no unique prolifera-
`tive properties associated with this group of MDS
`patients who have a hypocelluar BM with no detectable
`apoptosis. It must be noted here that only one tech-
`nique of in situ end labeling was utilized in the present
`study to document apoptosis. It is equally probable
`that these patients also have excessive apoptosis which
`is not detectable by the tests used. If only large molecu-
`lar weight DNA segments were produced for example,
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`

`R. Goyal et al. :Leukemia Research 23 (1999) 357–364
`
`363
`
`then ISEL as well as gel electrophoretic techniques
`would fail to detect these and a pulsed field elec-
`trophoretic assay would be required. In fact, we have
`previously demonstrated in a group of eight patients
`who showed no evidence of apoptosis either by ISEL
`or gel electrophoresis, six out of eight showed the
`presence of high molecular weight DNA fragments
`when studied by pulsed field electrophoresis [18]. In
`summary therefore, all we can say is that eight pa-
`tients with hypocellular MDS did not show ISEL pos-
`itivity as a sign of apoptotic death of hematopoietic
`cells in their bone marrows. On the other hand, the
`majority of hypocellular MDS cases have high cell-
`birth, high cell-death and high pro-apoptotic cytoki-
`nes very similar to the majority of hypercellular MDS
`cases. Thus, the fact that cases of hypocellular MDS
`have a similar clinical course and responsiveness to
`therapy as hypercellular MDS is matched by the bio-
`logic characteristics measured here.
`In the present study, two other associations are of
`interest. First, patients who had evidence of apoptosis
`in their marrows showed a statistically significant in-
`verse relationship with hemoglobin levels. We have
`not shown such a correlation in the hypercellular vari-
`ety of MDS patients. Second, patients with abnormal-
`ities of chromosomes 5 and:or 7 appeared to have a
`higher incidence of apoptosis in their bone marrows
`than those patients who showed either a normal kary-
`otype or some other cytogenetic abnormality. Once
`again, we have not found this association between
`abnormalities of 5 and:or 7 chromosomes and apop-
`tosis in the more commonly encountered hypercellular
`variety of MDS. Both of the above observations are
`statistically significant only in the hypocellular MDS
`indicating that hypocellular MDS cases are more ho-
`mogeneous in their characteristics than are hypercellu-
`lar patients. This should come as no surprise. Within
`each category of MDS, there exists remarkable het-
`erogeneity, an obvious example being the cellularity of
`the marrow. By separating the hypocellular MDS pa-
`tients with specific karyotypic abnormalities, the pop-
`ulation being analyzed is clearly far more ‘pure’ or
`less heterogeneous and therefore the relationships be-
`tween biological parameters are far more significant.
`In the case of hypercellular MDS, even if we separate
`patients by karyotype, it is possible that in some indi-
`viduals it is the erythroid precursors that are con-
`tributing to the cellularity in a major way as opposed
`to others in whom myeloid or megakaryocytic precur-
`sors may be more prominent. Thus, we would have a
`far more heterogeneous marrow in a hypercellular set-
`ting as compared to a hypocellular situation. The re-
`sulting weak or no relationships between biological
`characteristics are only to be expected in such cases.
`In conclusion, a group of 33 hypocellular MDS
`cases were studied for a variety of biological and clin-
`
`ical characteristics. The resulting insights should help
`us understand the genesis of cytopenias in MDS bet-
`ter. The ultimate hope of course is that such an im-
`proved understanding will be translated into better
`therapies for our patients.
`
`Acknowledgements
`
`The authors wish to thank the nurses of the Hema-
`tology Division, Rush Cancer Institute for their caring
`and thoughtful attitude towards protocol patients and
`Sandra Howery and Lakshmi Venugopal for their ex-
`cellent administrative:secretarial assistance.
`
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`