Apoptosis in Bone Marrow Biopsy Samples Involving Stromal and
`Hematopoietic Cells in 50 Patients With Myelodysplastic Syndromes
`
`By Azra Raza, Sefer Gezer, Suneel Mundle, Xue-Zhi Gao, Sairah Alvi, Raphael Borok, Shelby Rifkin, Amna Iftikhar,
`Vilasini Shetty, Agapi Parcharidou, Jerome Loew, Bridget Marcus, Zaineb Khan, Colette Chaney, John Showel,
`Stephanie Gregory, and Harvey Preisler
`
`Cell-cycle kinetics were measured in situ after infusions of
`iododeoxyuridine and/or bromodeoxyuridine in 50 patients
`with myelodysplastic syndromes (MDS) and the medianla-
`beling index in bone marrow (BM) biopsy samples was
`28.6%. Unfortunately, 26 of 50 patients showed that =75%
`of hematopoietic calls of all three lineages were undergoing
`programmedcell death (PCD) in their biopsy samples as
`shownby thein situ end labeling (ISEL) technique. Ten pa-
`tients had 1/3 and eight had 2/3 ISEL* cells. Stromal cells
`were frequently ISEL* and often S-phase cells were also
`found to be simultaneously ISEL*. Nucleosomal DNA frag-
`ments as a ladder in agarose gel were present in BM aspi-
`
`HE MYELODYSPLASTIC syndromes (MDS) are a
`group of acquired hematopoietic disorders with evi-
`dence oftrilineage dysplasia and an ~30% incidence of
`eventual
`transformation
`into
`acute myeloid leukemia
`(AML),.'? These are clonal disorders involving one or more
`clones,”* and normal hematopoiesis has been shownto si-
`multaneously coexist in the majority of cases.® Patients with
`evidence of ‘‘Abnormally Localized Immature Precursors’’
`(ALIP) in their bone marrow (BM) biopsy samples tend to
`die earlier than ALIP-negative patients because of a rapid
`transformation into acute leukemia.’ Cytogenetic abnormali-
`ties especially involving chromosomes 5 and 7 are com-
`monly detected,® whereas ras-mutationssignifying short sur-
`vival have been showed in ~10% to 40% patients.’ The
`disease is almost invariably fatal, and with the exception of
`BMtransplantation, there is no known cure."
`An apparent paradox in MDSis that patients with these
`disorders have peripheral cytopenias despite frequently hav-
`ing normo- or hypercellular BMs. One possible explanation
`for this contradictory finding may be that even though there
`are large numbers of cells in the BM, they are not exiting
`thal compartment because they are undergoing premature
`programmed cell death (PCD). To examinethis possibility,
`the present study was undertaken to define the incidence of
`PCD in the hematopoietic cells of MDS patients. Apoptosis
`is a gene-directed cellular self-destruction in which intracel-
`
`
`the
`From the Division of Hematology, Rush Cancer Institute,
`Department of Pathology, Rush-Presbyterian-St Luke's Medical
`Center, Chicago; and Northwest Community Hospital, Arlington
`Heights, IL.
`Submitted November 7, 1994; accepted February 9, 1995,
`Supported in part by a Grant No. (ROICA60085) from The Na-
`tional Cancer Institute.
`Address reprint requests to Azra Raza, MD, Professor of Medi-
`cine, Director, Leukemia Program, Rush Cancer Institute, Rush-
`Presbyterian-St Luke's Medical Center, 2242 W Harrison St, Suite
`108, Chicago, IL 60612.
`The publication costs of this article were defrayed in part by page
`charge payment. This article must
`therefore be hereby marked
`‘tadvertisement’’ in accordance with 18 U.S.C. section 1734 solely to
`indicate this fact.
`© 1995 by The American Society of Hematology.
`0006-4971/95/8601 -0026$3.00/0
`
`268
`
`rates of four patients who showed high ISEL and were ab-
`sent in two who had noISEL staining in biopsy samples,
`but only when DNA was extracted after a 4-hour in vitro
`incubation in complete medium. Therefore, laddering data
`confirmed the ISEL findings that the majority of hematopoi-
`etic cells in MDS are in early stages of PCD. We conclude
`that extensive intramedullary cell death may explain the par-
`adox of pancytopenia despite hypercellular marrows in MDS
`patients. Investigating approachesthat protect against PCD
`in some MDS subsets would beofinterest.
`© 1995 by The American Society of Hematology.
`
`lular endonucleases initially cleave the DNA into inter-
`nucleosomal fragments (180 to 200 bp ortheir integral multi-
`ples).'"' This process, which begins with fragmentation of
`DNA,endseventually with fragmentation of the nucleus and
`removal of the dead cell by macrophages. Although an end-
`stage karyorrhectic cell
`is easy to recognize under a light
`microscope,
`the earlier stages of PCD with limited DNA
`tragmentation and internucleosomal nicking cannot be mor-
`phologically identified. Thus, large numbers of cells may be
`undergoing apoptosis, yet there may be only a few cells in
`the morphologically identifiable karyorthectic stage. Conse-
`quently, an accurate estimation of the incidence of PCD
`must include a precise quantitation of cells in early stages
`of apoptosis in addition to the obviously karyorrhectic cells.
`The conventional method of detecting typical ‘‘DNA lad-
`dering’’ by gel electrophoresis may not be able to provide
`this information because very little low molecular-weight
`DNA fragments are formed in cells that are in the earlier
`stages of PCD.’* The techniqueofin situ end labeling (ISEL)
`of fragmented DNA appears to be a morereliable measure-
`ment of the early stages of apoptosis.'*'” Therefore, BM
`biopsy samples of 50 MDSpatients were processed for ISEL.
`The results obtained showed an exceptionally high rate of
`apoptosis in the hematopoietic cells ofall three lineages in
`these patients. These data confirmed our inilial hypothesis
`that high intramedullary death of hematopoietic cells may
`be the biologic basis for the paradox of pancytopenia despite
`hypercellular marrows in MDS patients.
`
`MATERIALS AND METHODS
`
`Rates of hematopoietic cellular proliferation and apoptosis were
`examined in fifty patients with a confirmed diagnosis of MDS. Each
`patient received a |-hour infusion of bromo- and/or iododeoxyuri-
`dine (BrdU and [UdR) at 100 mg/m’ as per protocols MDS 86-
`15 and/or MDS 90-02. Informed consent was obtained from every
`individual. The infusion protocols were approvedby thelocalInstitu-
`tional Review Board and the Food and Drug Administration and the
`drugs were provided by the National Cancer Institute. Some of these
`patients’ results have also been reported in earlier studies.””*?
`
`Studies of Cellular Proliferation
`
`Immediately at the end of the second thymidine analogue infusion,
`samples of BM aspirate (BM asp) and biopsy were obtained. The
`aspirates were used for the determination of durations of S-phase as
`
`Blood, Vol 86, No 1 (July 1), 1995: pp 268-276
`
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`APOPTOSIS IN BM BIOPSY SAMPLES
`
`described before.4 The BM biopsy samples were embedded in
`plastic using glycol methacrylate. Air-dried 2-3-ym-thick sections
`were placed on Alcian Blue—coated coverslips and processed for the
`estimation of labeling indices using an anti-IUdR/BrdU monoclonal
`antibody (MoAb) as described before.”*”*
`
`Studies to Measurethe Incidence of PCD or
`Apoptosis
`Two methods were used to measure the rale of apoptosis in these
`patients. The in situ end labeling (ISEL) of fragmented DNA as
`described by Wijsman et al'® was modified for use in plastic embed-
`ded biopsy samples for the detection of cells undergoing PCD. BM
`asps from six MDSpatients, three AML patients, and two normal
`controls were examined for the presence of the typical ladder pattern
`in agarose gels. Details of both methods are provided below.
`
`269
`
`mol/L TBSafter each of the above-mentioned incubations. Sections
`were then immersed in the following detection solution for 8 to 10
`minutes at 20°C. It contained Naphthol AS-MX phosphate (20 mg)
`freshly dissolved in 2 mL N,N-dimethylformamide. This was added
`to 100 mL 0.1 mol/L TRIS buffer, pH 8.2 followed by 0.1 mL 1
`mol/L Levamisole and 100 mg of Fast Blue BBsalt (Sigma Chemical
`Co, St Louis, MO). The solution was stirred for 2 minutes and
`filtered before use. Sections were washed in distilled water and
`mounted with Fluoromount.
`
`Control Experiments for ISEL Technique
`Control experiments for ISEL technique included two types of
`controls: negative and positive.
`
`Negative Controls
`ISEL solution devoid of DNA polymerase was used on control
`samples as negative controls. These slides were universally negative.
`
`Positive Controls
`
`Positive controls included DNAladdering versus ISEL of frag-
`mented DNA,detection of apoptosis in BM biopsy samples obtained
`from newly diagnosed high-risk patients with AML, and detection
`of apoptosis in normal BM biopsy samples obtained from patients
`with non-Hodgkin’s lymphoma (NHL).
`DNA laddering versus ISEL offragmented DNA. HL-60 cells
`were treated with a topoisomerase inhibitor etoposide (VP 16) at a
`concentration of 35 zmol/L/mL in RPMI 1640 medium containing
`20% fetal bovine serum (FBS). The controlcells were incubated in
`the same medium without etoposide. The incubation was continued
`for 4 hours at 37°C in 5% CO). At the end of the incubation, each
`sample was divided into two aliquots. Cells from one aliquot were
`embeddedin plastic, sectioned, and processed for ISEL to determine
`the percentage of apoptotic cells, whereas the other aliquot was used
`for DNA extraction. The DNA so obtained was mun on an agarose
`gel containing ethidium bromide.
`Detection ofapoptosis in BM biopsy samples obtainedfrom newly
`diagnosed high-risk patients with acute myeloid leukemia (AML).
`Twenty AMLbiopsy samples were processed for ISEL beforestart-
`ing remission induction chemotherapy.
`Detection of apoptosis in normal BM biopsy samples obtained
`jrom patients with NHL. Two NHLpatients who had no BM
`involvement and whose BM biopsy samples were judged by hemato-
`pathologists to be indistinguishable from normal BM biopsy samples
`were also processed for ISEL technique to determine the incidence
`of apoptotic cell death.
`
`ISEL of Fragmented DNA
`Two-micrometersections from the previously mentioned BM bi-
`opsy samples were obtained. The sections were rehydrated in dis-
`tilled water for 10 minutes and then incubated with freshly diluted
`3% hydrogen peroxide (H,O,) for 30 minutes. Specimens were thor-
`oughly rinsed in 0.15 mol/L phosphate buffer solution (PBS) (0.15
`mol/L sodium chloride [NaCl] in 0.1 mol/L phosphate buffer, pH
`7.5). Subsequently they were incubated with sodium chloride, so-
`dium citrate solution (SSC) (0.3 mol/L NaCl and 30 mmol/L Na-
`citrate, pH 7.0) at 80°C for 20 minutesfollowed by thorough washing
`with 0.15 mol/L PBS. The sections were next treated with pronase
`(1.0 mg/mL in 0.15 mol/L PBS, Calbiochem, LaJolla, CA) at room
`temperature for 30 minutes. After this they were rinsedfirst in 0.15
`mol/L PBS and then in buffer A, pH 7.5 (50 mmoV/L TRIS HCl, 5
`mmol/L MgCh, 10 mmoV/L f-mercaptoethanol and 0.005% bovine
`serum albumin [BSA], fraction V; Sigma Chemical Co, St Louis,
`MO). Sections were then incubated with ISEL solution prepared in
`buffer A (0.01 mmol/L deoxyadenosine triphosphate, deoxyguano-
`sine triphosphate, deoxycytidine tiphosphate) (Promega, Madison,
`WI), 0.001 mmol/L biotin-11 deoxyuridine triphosphate (Sigma),
`and 20 U/mL Escherichia coli DNA polymerase I (Promega)at 18°C
`for 2 hours, followed by washing with buffer A and later with 0.5
`mol/L, PBS (0.5 mol/L NaC]in 0.1 mol/L phosphate buffer, pH 7.5).
`The specimens were then incubated with avidin-biotin-horseradish
`peroxidase conjugate (Vectastain Elite ABC Kit-Vector, Burl-
`ingame, CA; diluted 1:25 in 0.5 mol/L PBS containing 1% BSA
`and 0.5% Tween 20). Finally, samples were stained using 0.04%
`3,3’-diaminobenzidine tetrahydrochloride diluted in 0.05 mol/L
`TRIS buffer, pH 7.5, with 0.015% of 30% H,O, for 10 minutes and
`rinsed well with distilled water. The specimens were then mounted
`with Fluoromount G (Biotechnology Associate Inc, Birmingham,
`Interpretation of BM Biopsy Specimen Slide
`AL). Slides were left to dry overnight before estimation of PCD.
`All slides were examined underalight microscope attached to a
`television screen. Atleast three different investigators were involved
`in the interpretation of each slide (A.R. being one of them every
`time).
`
`Simultaneous Assessmentof Cell Birth
`(Proliferation) and Cell Death (Apoptosis) From the
`Same Biopsy Sample Section Using Double-Labeling
`by ISEL and Anti-IUdr/BrdU MoAb
`For the double-labeling technique designed to simultaneously de-
`tect S-phase cells and PCD, sections were first processed for ISEL
`as described above, and then rinsed thoroughly in 0.5 mol/L TBS
`(0.5 mol/L NaCl in 0.05 mol/L TRIS buffer, pH7.5). Subsequently
`the sections were treated with 4 N HCI for 15 minutes, and the anti-
`TUdR/BrdU MoAb 3D9 (20-22) diluted 1:200 in 0.5 mol/L TBS
`containing 0.25% Tween 20 for 60 minutes at room temperature,
`rabbit antimouse IgG (Dako, Carpenteria, CA) diluted 1:20 in 0.5
`mol/L TBS for 30 minutes and with mouse alkaline phosphatase—
`antialkaline phosphatase antibody (Dako, diluted 1:40 in 0.5 mol/L
`TBS) for 30 minutes. The sections were rinsed thoroughly with 0.5
`
`Proliferation Studies
`Immunohistochemical detection of IUdR- and/or BrdU-labeled S-
`phase cells was accomplished using either the anti-[UdR/BrdU
`MoAb 3D9orthe anti-BrdU MoAbBr-3. The nuclei of cells engaged
`in DNA synthesis were stained dark-brown in single-label slides.
`The labeling index (LI) was obtained by counting only the labeled
`myeloid cells as described before.?*?* Frequently erythroid islands
`as well as occasional megakaryocytes were also in S-phase and
`these were easily recognizable because of their unique morphologic
`appearance. These labeled erythroid and megakaryocytic cells and
`their unlabeled counterparts were excluded from the LI calculations.
`At least 2,000 cells from five different fields were counted.
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`

`270
`
`PCD Studies by ISEL
`Coverslips processed for ISEL were mounted onto glass slides
`and examined on the television screen by a group of observers (AR
`was always present). The slides werefirst scanned under low power.
`ISEL* cells showed clear, punctate brownstaining over their nuclei,
`often being most marked in the perinuclear area, Areas of apoptosis
`and PCD wereidentified if these were present in compartmentalized
`pockets. The following scoring system was used to approximately
`quantitate the incidence of PCD: absent. only an occasional ISEL*
`cell, definitely constituting +15% ofall cells; low, up to one third
`of the biopsy sample containing ISEL™ cells; intermediate, between
`one third and two thirds of the biopsy sample containing ISEL~
`cells; high, =75% ISEL*cells.
`
`Simultaneous Proliferation and PCD Studies
`In these double-labeled slides, the nuclei of S-phase cells were
`stained blue, the nuclei of ISEL* cells were stained brown and those
`S-phase cells that were also ISEL* showed the presence of both
`brown and blue staining.
`
`Detection of DNA Laddering
`Detection of DNA laddering was performed on BM aspcells of
`six MDS and three AMLpatients as well as two BM asp samples
`that were completely normal (obtained from BM transplant donors).
`After density separation on Ficoll-Hypaque,
`1
`[0° cells/mL were
`incubated for 4 hours at 37°C in 5% CO, atmosphere and used
`for DNA extraction. Cells without the 4-hour incubation were also
`similarly used for DNA extraction. The reason for incubating cells
`over virious time points before extracting DNA wasas follows: Our
`hypothesis based on the ISEL data in BM biopsy samples wasthat
`Most of the ceils appeared to be in earlier stages of PCD with only
`low amounts of DNA damagethat may not be sufficient to produce
`laddering in gels. However, leaving the cells in complete medium
`over specified time points may be enough time for the cells to
`complete the process of upoptosis with generation of low molecular-
`weight DNAthat would produce the typical DNA laddering. Thus,
`although initial experiments used a variety of time points (0, 1, 2,
`3, and 4 hours) for in vitro incubation of cells in complete medium
`before DNA extraction, later experiments have used only the 0- and
`4-hour time points because they uppear to be the two most informa-
`tive intervals.
`Briefly, the cells were washed in 0.1 mol/L PBS, pH 7.5 (0.1 mol/
`L NaClin 0.1 mol/L PBS) to removetraces of medium, lysed with
`guanidine isothiocyanate (GITC) at room temperature and layered
`on 5.7 mol/L cesiumchloride (CsCl) gradient. Afler overnight cen-
`trifugation at 32,000 rpm, the DNA fraction al the interface of CsCl
`and GITC was aspirated and pooled together with the RNAfraction
`trom the bottom of CsCl gradient tube. Subsequently, this prepara-
`tion wastreated with chilled 100% and 80% ethanol, incubated with
`Proteinase K (Boehringer Mannheim, Mannheim, Germany) and re-
`peatedly treated with phenol:chloroform:isoamy! alcohol mixture
`(25:24:1, Sigma) to remove proteins. DNA wasthen precipitated by
`adding Na-acetate (3 mol/L, pH 6.0) and double volume of chilled
`100%. ethanol for 1 hour at —20°C. This DNA fraction was then
`treated with RNase conceniration | zg/mL (final concentration 20
`pe/mL) and subjected to reextraction using phenol:chloroform:iso-
`umyl alcohol mixture followed by treatment with Na-acetae and
`chilled 100% ethano! as described earlier. Finally, the total DNA
`content in each sample was cstimated by optical density at 260 nm
`and 5 pg DNA was loaded in 1.5% agarose gel containing ethidium
`bromide. After electrophoresis, the gel was photographed underul-
`traviolet (UV)light,
`
`RAZA ET AL
`
`RESULTS
`
`Fifty patients wilh a confirmed diagnosis of MDSare the
`subject of this report. The French-American-British (FAB)
`classification was used to identify the various subtypes of
`MDSpatients.** There were 19 cases ofrefractory anemia
`(RA), 6 cases of refractory anemia with ring sideroblasts
`(RARS),
`[7 cases of refractory anemia with excess blasts
`(RAEB), 7 cases of RAEB in transformation (RAEB-t) and
`1 case of chronic myelomonocytic leukemia (CMMOL). BM
`cellularity was also available in all 50 cases. Table | de-
`scribes the details of FAB type, apoptosis, cell cycle kinetics,
`biopsy sample cellularity and complete blood counts in ull
`50 patients.
`
`Results of Control Experiments
`
`A comparison of ISEL technique with DNA laddering
`detected by gel electrophoresis was performed on HL-60
`cells treated with etoposide, Whereas untreated HL-60 cells
`did not show any low molecular-weight DNA,
`the cells
`treated with etoposide showed a characteristic DNA ladder
`pattern (Fig 1A). Results obtained by ISEL correlated very
`well with these data. The percentage of apoptotic (ISEL*)
`cells in the control, untreated HL-60 cells was 1.59%,
`whereasthis percentage in etoposide-treated HL-60 cells in-
`creased to 13.9% (Fig 1, B and C). The DNA laddering and
`ISEL methods were similarly compared in at least three more
`sets of experiments and results obtained repeatedly showed
`that an accurate detection of apoptotic cells can be accom-
`plished using etther. Once satisfied that ISEL is a highly
`accurate method of detecting PCD, we proceeded to use
`plastic embedded BM biopsy samples obtained from MDS
`patients for precise quantitation of apoptotic cel] death.
`Incidence of spontaneous apoptosis was quantitatively
`scored in 20 BM biopsy samples obtained from AML pa-
`tients studied at the time of diagnosis and before receiving
`any remission induction chemotherapy. Noneof these biopsy
`samples showed greater than 10% to 15% of the total cells
`positive for ISEL staining. The leukemic blasts were univer-
`sally negative. The few ISEL* cells were usually present in
`small clusters and appeared morphologically to represent
`areas of residual normal hematopoiesis. Furthermore, dou-
`bie-labeling showed that none of the S-phase cells were
`ISEL".
`PCD wasalso studied in two normal BM biopsy samples
`obtained from NHL patients. Both the patients showed geo-
`graphically well-defined areas of apoptotic cell death scat-
`tered throughout the sumples. Within these ‘‘geographically
`restricted islands of death,’’ all three lineages of cells were
`ISEL*, as were occasional stromal cells. On the whole, such
`areas accounted for approximately one third of the BM bi-
`opsy sample area, but it was never as extensive as in MDS
`patients. Finally. S-phase cells were never double-Jabeled
`for ISEL in these samples.
`
`Proliferation Studies
`
`Statistical Analysis
`Mann Whitney tcsts were uscd for two sample comparisons of
`continuous vaniubles, Contingency tables, with chi squarestatistics
`or Fisher's exact test, were used for analyzing.
`
`Sequential labeling of S-phase cells was performed in vivo
`using infusions of IUdR and/or BrdU. Labeling indices (LI)
`trom BM biopsy samples were available in 46 patients. The
`median LI was 28.6% with a range of 13% to 49.1%. Dura-
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`APOPTOSIS IN BM BIOPSY SAMPLES
`
`271
`
`Table 1. Cell Cycle Kinetics, PCD, and Clinica! Characteristics of Patients With MOS
`Te
`Cellularity
`WBC Count
`Hb Count
`Platelets Count
`
`3 No.
`Patient GMA No,
`FAB
`ISEL
`Li%
`Ts (hs}
`{hs}
`(%)
`(x 10°/L)
`(g/dL)
`(x10)
`1
`207/88
`RA
`High
`39.6
`15.6
`38
`30
`45
`11.6
`92
`2
`131/90
`RAEB-t
`High
`47
`19
`40
`15
`17
`12.3
`182
`3
`130/89
`RA
`High
`26
`NA
`NA
`90
`9.4
`10.6
`433
`4
`250/87
`RA
`High
`25.1
`9.7
`38.6
`50
`2.7
`10.1
`68
`5
`14/87
`RAEB
`High
`21.3
`15.2
`17.3
`95
`12.8
`7.4
`44
`6
`3/87
`RA
`High
`15
`7.4
`47
`10
`3.9
`10.1
`29
`7
`268/90
`RARS
`High
`29.3
`65.8
`224.6
`90
`38.1
`8.2
`360
`8
`215/90
`RARS
`High
`13
`8.06
`62
`90
`13
`9.6
`409
`9
`21/91
`RARS
`High
`37
`6.72
`18.16
`40
`4.5
`11.8
`71
`10
`43/93
`RAEB
`High
`24
`NA
`NA
`20
`0.7
`6.1
`24
`11
`11/93
`RAEB
`High
`26
`NA
`NA
`60
`37
`9.3
`127
`12
`5/93
`RAEB
`High
`18
`NA
`NA
`20
`1.6
`9.2
`225
`13
`63/93
`RA
`High
`25
`NA
`NA
`70
`2.9
`10.1
`256
`14
`169/90
`RAEB-t
`High
`32
`NA
`NA
`70
`2.5
`9.3
`61
`16
`616/87
`RAEB-t
`High
`31
`22
`71
`60
`NA
`NA
`NA
`16
`390/87
`RAEB
`High
`33.6
`14,7
`43.7
`80
`14.6
`12
`219
`17
`19/93
`RAEB-t
`High
`18
`NA
`NA
`70
`10.1
`8.4
`15
`18
`21/93
`RAEB
`High
`18
`NA
`NA
`40
`1.5
`9.1
`45
`19
`20/91
`RARS
`High
`30.6
`11.82
`38
`40
`75
`8.4
`417
`20
`86/94
`RA
`High
`24
`NA
`NA
`80
`3.1
`10.6
`63
`21
`98/94
`RAEB
`High
`28.7
`4.6
`16.1
`70
`47
`79
`224
`22
`150/94
`RA
`High
`31.5
`27.9
`88.6
`80
`2.5
`11.1
`162
`23
`64/94
`RAEB-t
`High
`28.5
`5.6
`19.8
`40
`1.3
`8
`25
`24
`130/94
`RAEB
`High
`24.5
`24.9
`101.8
`40
`25,7
`9.3
`43
`25
`281/94
`RAEB
`High
`18.7
`5
`27
`70
`9.8
`9.4
`99
`26
`88/94
`RAEB
`High
`23.9
`3.6
`15
`90
`1.6
`8.3
`56
`27
`382/88
`RA
`Intermediate
`46.6
`NA
`NA
`35
`NA
`NA
`NA
`28
`128/90
`RARS
`Intermediate
`49.1
`6.8
`13.8
`90
`1.4
`8.3
`43
`29
`34/89
`RA
`Intermediate
`39
`24
`61.5
`65
`15.2
`9.2
`729
`30
`100/89
`RAEB-t
`Intermediate
`39,3
`17.5
`44.5
`50
`49
`9.2
`32
`31
`182/80
`RARS
`Intermediate
`32.3
`16.2
`50.15
`70
`3.4
`8
`11
`32
`609/87
`RAEB-t
`Intermediate
`31
`22
`71
`50
`4
`8.8
`53
`33
`136/90
`RAEB
`Intermediate
`29
`32.3
`111.3
`60
`6.7
`8.2
`196
`34
`292/94
`RAEB
`Intermediate
`33.8
`6.9
`20.4
`80
`3.2
`9.1
`93
`36
`390/87
`RA
`Low
`33.1
`13.8
`41.7
`55
`1
`8.7
`212
`36
`74/87
`RA
`Low
`25.3
`14.6
`57.5
`50
`3.1
`10.5
`70
`37
`25/93
`RA
`Low
`41
`NA
`NA
`70
`8.7
`9.3
`52
`38
`46/93
`RA
`Low
`16
`NA
`NA
`30
`17
`74
`79
`a9
`103/93
`RAEB
`Low
`38
`NA
`NA
`70
`0.8
`8.8
`28
`40
`26/91
`RA
`Low
`35
`5.8
`16.5
`80
`3.4
`7.8
`104
`41
`112/94
`RA
`Low
`NA
`5.3
`NA
`60
`3.7
`13.2
`126
`42
`23/94
`CMMOL
`Low
`19.1
`NA
`NA
`60
`11.8
`6.5
`173
`43
`104/94
`RA
`Low
`NA
`NA
`NA
`30
`6.3
`9.9
`30
`44
`48/94
`RAEB
`Low
`NA
`NA
`NA
`60
`6.3
`12.8
`915
`45
`245/94
`RA
`Low
`23.9
`3
`12.6
`30
`2.5
`6.7
`52
`46
`51/93
`RAEB
`Negative
`14
`NA
`NA
`50
`3.1
`12,9
`250
`47
`68/87
`RAEB
`Negative
`24
`NA
`NA
`70
`2.5
`9.2
`1,551
`48
`282/94
`RAEB
`Negative
`24.9
`5.8
`23.1
`80
`7.4
`10
`90
`49
`275/94
`RA
`Negative
`44.5
`41
`9.1
`10
`1.1
`8.1
`29
`
`88/93 RA NA NA NA NA 1 4 10.950 35
`
`
`
`
`
`
`
`
`
`
`Abbreviations: GMA, glycol methacrylate biopsy; WBC, white blood cell; Hb, hernoglobin; NA, not available.
`
`tion of S-phase (Ts) was available in 30 patients with a
`median of 12.8 hours and a range of 3.0 to 65.8 hours. Total
`cell cycle time (Tc) was available in 29 with a median of
`40.0 hours (range, 9.1 to 224.6 hours). These data are also
`provided in Table 1 for individual patients.
`
`Apoptosis Studies
`
`In situ end labeling of fragmented DNA was performed
`on the plastic embedded biopsy samples of all 50 patients.
`
`A distinct brown staining in a variety of patterns over the
`nucleus identifies a cell as being engaged in DNAcleavage.
`Amongthe 50 cases being reported, one biopsy sample was
`excluded because of poor quality of the sample whereas four
`cases showed ‘‘absent’’ apoptosis or only occasional ISEL*
`cells and were considered as ISEL~ (Table 1). Of the re-
`maining 45 cases, 26 had high, 8 had intermediate and 10
`had low ISEL-positivity.
`Interestingly, all three lineages of hematopoietic cells in-
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`aeSaeed
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`(A) Agarose gel electrophoresis of DNA ex-
`Fig 1.
`tracted from HL-60 calls incubated with etoposide at
`varying concentrations. Lane 1, no etoposide; lane
`Z, treated with etoposide at 17.5 ;mol/L for 4 hours,
`lane 3, treated with etoposide at 36 yzmol/L for 4
`hours; lane 4, DNA molecular-weight marker, 100-bp
`DNAladder(GIBCO Life Technologies, GrandIsland,
`NY). Note the appearance of laddering in lane 3. {B)
`In situ end labeling on contro! HL-60 cells without
`etoposide treatment show no positive staining. (C)
`ISEL of etoposide-treaated HL-60 calls show distinct
`
`llml RAZA ET AL
`ISEL staining over clearly karyorrhectic cells.
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`APOPTOSIS IN BM BIOPSY SAMPLES
`
`cluding the myeloid. erythroid and megakaryocytic cells
`were found to be undergoing apoptosis. Figure 2 shows some
`of these ISEL* cells. Furthermore, almost
`in every case,
`stromal cells were also found to be apoptotic including endo-
`thelial cells, fat cells, and fibroblasts. In Figure 2, ISEL” as
`well as negative fibroblasts and endothelial cells can be seen.
`Figure 3 shows the typical crescent-shaped chromatin con-
`densation in an AML patient's leukemic blast as stained by
`the ISEL technique.
`
`Simultaneous Labeling for Proliferation and
`Apoptosis
`
`Double labeling using ISEL and anti-[UdR/BrdU antibody
`techniques wis performed on 15 MDS patients. In every case,
`S-phasecells were frequently found to be undergoing apoptosis
`as well. Figure 4 clearly shows a megakaryocyte thatis synthe-
`sizing DNA (blue staining) and is also undergoing apoptosis
`(brownstaining) or is in a state of ‘‘Antonymy.’” Because all
`of the first 15 consecutive patients examined by this double-
`libeling technique showed the same concurrent cell-birth/cell-
`death pattern, the remaining biopsy samples were not double-
`labeled. In a numberofpatients, macrophagesfilled with ISEL*
`debris of apoptotic cells were recognizable (Fig 5).
`
`Results of DNA Laddering in MDS, AMLs, and
`Normal Controls
`
`DNA wasextracted from BM asps of six MDS patients,
`(Table L, patients no. 22, 24, 25, 34, 48, and 49), three AML
`patients and two normal individuals who were BM transplant
`donors. Figure 6 shows the resulis in these 11 individuals.
`Lane | shows the DNA molecular marker, (100-bp DNA
`ladder). The next two Janes show the results in 2 normal
`donors whose BM asps were incubated for 4 hours in com-
`plete medium before extraction of DNA. No evidence of
`DNAladdering can be seenin cither ofthe norma] samples.
`Similarly, the next four lanes show three AMLpaticnts and
`none of them show any DNA laddering cither. On the other
`hand, lanes 8 through 18 represent 0- and 4-hour samples
`ofstx MDSpatients. The first four of these (Table |, patients
`no, 22, 24, 25, and 34) show laddering in the 4-hour sumples,
`but not in the 0-hourlanes. All 4 four ofthese individuals
`had evidence of ISELpositivily in their biopsy samples. The
`last two MDS patients (Table 1, patients no. 48 and 49) had
`no ISELstaining in their biopsy samples and show no DNA
`laddering even after 4 hours of incubation in complete me-
`dium (lanes I$ through 18). Thus, the data obtained by gel
`clectrophoresis confirmed the results obtained by ISEL stain-
`ing in all cases examined,
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`273
`
`
`
`Flg 6. Agarose gel electrophorasis of DNA extracted from Ficoll-
`separated BM asp mononuclearcells (BMAMNC)}before(t,) and after
`(t,) incubation in RPMI 1640 medium containing 10% FBS,for 4 hours
`at 37°C, Ethidium bromide staining photographedin ultravioletlight.
`Lane 1, DNA molecular-weight markers, 100-bp DNA ladder (GIBCO
`BRL}; lanes 2 and 3, t, DNA from BMAMNCof normal healthy donors;
`lane 4, t,; and lanes 5 through7, t, DNA from BMAMNC ofthree AML
`patients; lanes 8 through 11, t, and t DNA from BMAMNC of MDS
`patients, The normal donors as well as AML patients did not show
`any evidence of low molecular-weight DNAat 0 or 4 hours. Interest-
`ingly, 4 of 6 MDS patients although showed intact DNA at 0 hours
`claarly demonstrated a characteristic laddering at 4 hours, However,
`the remaining 2 MDS patients did not show laddering even at 4
`hours. The prepensity of BMAMNC te underge apoptosis in vitro
`detected by agarose gel electrophoresis and the degree of spontane-
`ous apoptosis measured in BM biopsy samples by ISEL carrelated
`very well,
`
`Other Clinical and Biologic Characteristics Versus
`Proliferation and Apoptosis
`The rates of cell birth and cell death were compared in
`the various FAB categories. Apoptosis was maximum in
`RAEB-t patients, all 7 having greater than 50% ISEL* cells,
`whereas 9 RA patients had high and 9 had low ISEL-posi-
`tivity (P = .057) with two patients having intermediate posi-
`tivity. Most importantly, even though there was so much
`cel] death in RAEB-t patients, clusters of ISEL~ myeloblasts
`(ALIPs) were clearly recognizable. On the other hand, L.1
`was below median for 5 of 7 RAEB-t patients, whereas half
`the RA patients had above and half had below median LI.
`ISEL results were also corelated with other parameters such
`as LI (P = .314), Ts (P = .234), Te (P = .275), WBC (P
`= .160), Hb (P = .801), platelets (P = .273), and biopsy
`sample cellularity (P = .648). No statistically significant
`relationships were identified.
`DISCUSSION
`
`A recently described technique for detecting PCD that
`uses ISEL of fragmented DNA**” has been used in the
`
` +
`
`Fig 2. Plastic embedded BM biopsy from an MDSpatient showing large numbers of apoptoticcells Jabeled brown by JSEL. Note the many
`negative cells in the field. Stromal cells such as fibroblasts and endothelial cells are also ISEL”.
`Fig 3. Chromatin condensationin the form of a typical crescent usually seen in earlier stages of programmedcell death stained by ISELin
`the BM biopsy sample of a patient with acute myeloid leukemia.
`Fig 4. Double labeling to simultaneously detect calls engaged in DNA synthesis (anti-1\UdR/BrdU antibody) and apoptosis (ISEL), Blue marks
`S-phase cells whereas brown marks apoptosis. Note the large megakaryocyte that is simultaneously engaged in both processes (blue and
`brown staining). This state has been termed ’’Antonymy.”
`Fig 5. A macrophagethat contains ISEL* debris of apoptotic cells in the BM biopsy sample of an RAEB-t patient.
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`RAZA ET AL
`
`present study to determine the incidence of intramedullary
`cell death in patients with myelodysplastic syndromes. Four
`unique observations have been made in the present study.
`First, that myelodysplastic syndromes are highly prolifera-
`tive disorders with almost one third of the marrow cells
`engaged in DNA synthesis (median LI for myeloid cells,
`29%; median cell cycle time, 44 hours). Second, that unfortu-
`nately this high rate of cell birth is cancelled by an cqually
`high rate of cell-death resulting in a functionally aplastic
`BM,an observation that constitutes the most likely basis for
`the apparent paradox of pancytopenia despite hypercellular
`marrows encountered in the majority of these patients. This
`high rate of apoptosis affected not only all three lineages of
`the hematopoietic cells, but also involved cells of stromal
`origin (fat cells, endothelial cells, fibroblasts). Third,
`that
`many cells are simultaneously engaged in DNA synthesis
`and apoptosis. Fourth, that the DNAextracted immediately
`upon density separation of BM asps showed noladdering in
`agarose gels, whereas DNAextracted after a 4-hour incuba-
`tion of density-separated cells in complete medium showed
`the characteristic laddering in 4 of 4 MDS patients with
`250% ISEL* cells in the matched biopsy samples, but none
`in the 2 of 2 MDSpatients with no ISEL staining in their
`biopsy samples.
`While a high rate of apoptosis has previously been hypoth-
`esized to be a possible explanation for the ineffective hema-
`topoiesis observed in these disorders on the basis of finding
`increased apoptotic nuclei in trephine sections,” the present
`report is the first documented proof that large numbers of
`marrow cells are engaged inearlier stages of PCD. Several
`features of this study are unexpected. To begin with,
`the
`extent of apoptosis encountered was previously unsuspected.
`In 26 of 50 patients examined, there was evidence that =75%
`of all hematopoietic cells were apoptotic.