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Identification of hyperdiploidy in fixed cells from pediatric acute lymphoblastic leukemia cases using flow cytometry and cytogenetic analysis
Identification of Hyperdiploidy in Fixed Cells from Pediatric Acute Lymphoblastic Leukemia Cases Using Flow Cytometry and Cytogenetic Analysis M. C. Lowery, R. M. Bull, and C. G. Sciotto
ABSTRACT: Cytogenetic analysis is of value in predicting clinical outcome of pediatric cases of acute lymphoblastic leukemia (ALL). Hyperdiploidy in these patients has significant impact on therapeutic outcome. Because of the technologic limitations of cytogenetic analysis in determining hyperdiploidy in some of these cases, we devised a method to analyze cytogenetically fixed material for cellular DNA content by standard flow cytometric methods. This technique greatly enhances our ability to interpret the observance of single cell anomalies and assess whether these cells are predictive of clonal stemlines or are a result of random events.
INTRODUCTION Hyperdiploidy, classified as more than 50 chromosomes, is a fairly c o m m o n finding in acute lymphoblastic leukemia (ALL), accounting for approximately 24-30% of pediatric cases of ALL [1]. It is particularly frequent in ALL of early pre-B-cell precursor type [CD19 + ,CD10 + ,CD5 - ,CDT- ] and L1 or L2 morphology according to the French-AmericanBritish (FAB) classification [2, 3]. Hyperdiploidy is associated with improved therapeutic response and subsequent survival in pediatric patients with ALL [1, 3-6]. These patients with modal chromosome numbers greater than 55 achieve complete remission more readily and have increased survival. Cytogenetic analysis of hyperdiploid cells is often difficult owing to the suboptimal quality of leukemic chromosomes. Technical problems in cytogenetic analysis of ALL have often prevented identification of hyperdiploid stelnlines. Often these cells are not mitotically active in culture or are present in such a low percentage that they are not detectable by routine cytogenetic analysis. A more particular problem exists in patients in w h o m possibly only one or two hyperdiploid cells are observed cytogenetically. Whether these are true representations of a hyperdiploid clone or are merely random events is not clear. Because of the potential clinical ramifications of a hyperdiploid stemline in pediatric patients, we have combined techniques used in both flow cytometry and cytogenetics to evaluate these patients for the presence of a hyperdiploid
clone. We report a series of pediatric patients in w h o m this technique was useful in ascertaining the validity of a hyperdiploid stemline. CASE HISTORIES The patients ranged in age from 21 months to 19 years; age 21 years was defined as the cutoff age for the definition of pediatric patients. All patients were originally diagnosed with ALL. Table I summarizes the clinical histories of these patients. The pre-pre-B form of ALL designates cases that expressed CD19 but not cytoplasmic tt ilmnunoglobulin. The pre-T ALL cases were positive for T-call antigens such as CD3 or CD 7, but lacked CD2 antigen expression.
MATERIALS AND METHODS
Cytogenetics Cytogenetic studies were performed according to standard technique. Cells from the bone marrow (BM) aspirate were cultured for 24 hours before harvest in supplemented RPMI 1640 medium. Cells were harvested according to protocol and fixed in 3:1 methanol:glacial acetic acid fixative. Slides were prepared for cytogenetic analysis and Giemsa-trypsin banded [7]. The remaining pellet was stored at 4°C. Karyotypes were prepared and designated according to the International System for Human Cytogenetic Nomenclature [8].
Flow Cytometry From the Department of Pathology, Penrose-St. Francis Healthcare System, Colorado Springs, Colorado. Address reprint requests to: Mary C. Lowery, Ph.D., Penrose-St. Francis Healthcare System, 2215 N. Cascade Ave., Department of Pathology, Colorado Springs, Colorado 80907. Received March 2, 1992; accepted January 12, 1993. 136 Cancer Genet Cytogenet 67:136-140 (1993) 0165-4608/93/$06.00
Flow cytometric analysis of the fixed cell pellets followed standard protocol used in our laboratory for solid tumor analysis, with some modifications. These procedures are described briefly. The method is adapted from previously published data [9].
© 1993 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
H y p e r d i p l o i d y in ALL by F l o w Cytometry
Table I
Clinical history of patients
Patient/age la/5 yr 2a/4 yr 3b/2 yr 4/14 yr 5/21 mo 6/19 yr 7/9 yr 8/12 yr 9/12 yr
137
Diagnosis ALL, L1, ALL, L1, ALL ALL, L1, ALL, L1, ALL, L1, ALL, L1, ALL, L1, ALL, L2,
pre-pre-B, TdT + CD10 + pre-pre-B, TdT+ CD10 + pre-pre-B, TdT+ CD10 + pre-pre-B, TdT +, CD10 + pre-T, TdT +, CD7 + pre-pre-B, TdT +, CD1O + T cell, TdT +, CD2 +/CD7 + T cell, TdT +, CD2 +/CD7 +/CDla +
Abbreviations: CD, cluster designation; ALL, acute lymphoblastic leuke-
mia; TdT, terminal deoxynucleotidyl transferase. a Patients currently in remission. b Patient referred; limited clinical information available.
Data were acquired with a linear amplifier for the integrated and peak signal from the p r o p i d i u m i o d i d e (red) fluorescence. Estimates of cell cycle phases were determined with a commercial software program (DNAlysis, University of Utah). The DNA index was determined by dividing the mean channel n u m b e r of the a n e u p l o i d peak by the mean channel of the d i p l o i d peak. RESULTS
Cytogenetics/Flow Cytometry Cytogenetic and flow cytometric analyses of the patient samples are s u m m a r i z e d in Table 2. Representative examples of cellular morphology, karyotype, and flow cytometry results are shown in Figs. 1-3 (all data obtained from patient 4).
DISCUSSION Fixed cell pellets were centrifuged (1,200 r p m for 5 minutes) and the supernatant was discarded. The pellet was r e s u s p e n d e d in Hanks' buffered salt solution (HBSS) (3104170, GIBCO/BRL, Bethesda, MD) and centrifuged. The HBSS s u s p e n s i o n was repeated twice to remove all remaining fixative. After the last wash, the pellet was r e s u s p e n d e d in uns u p p l e m e n t e d RPMI 1640 m e d i u m (320-1875, GIBCO) and allowed to sit overnight at 4°C. The next day, the cells were centrifuged (1,200 r p m for 5 minutes) and r e s u s p e n d e d in a m o d i f i e d nuclear isolation m e d i u m [NIM: 1.714 g NaC1, 29.4 mg CaC12, 24.6 mg MgSO4.H20, 1.2 g nonidet po40 (N6507, Sigma, St. Louis, MO), 400 mg bovine serum albumin (BSA, A7511, Sigma), 10 mg p r o p i d i u m iodide (P4170, Sigma), 200 mg RNase, type I-AS (R5503, Sigma), 276 mg NaHzPO4.H20; q.s. solution to 200 ml with deionized water w i t h pH adjusted to 7.0]. The cell s u s p e n s i o n was then centrifuged (2,300 r p m for 10 minutes), the supernatant was discarded, and the cells were r e s u s p e n d e d in I ml NIM. A cell count was performed using a fluorescent microscope with cell counts optimized at 1-2 × 106 cells/ml. Normal d i p l o i d fixed cells, processed identically, were used as a control. Specimens were analyzed with a Coulter Profile II flow cytometer (Coulter Cytometry, Hialeah, FL) w i t h 488-nm argon laser (15 mW) excitation.
Cytogenetic analysis continues to be an invaluable a d d i t i o n to current diagnostic strategies in h u m a n leukemias. Various chromosome abnormalities have been shown to be associated with either improved or worsened prognosis. Such information provides clinicians with information necessary to manage patients appropriately. Cytogenetic studies of pediatric ALLs have been especially fruitful in providing valuable clinical information. H y p e r d i p l o i d y is considered a favorable prognostic finding in pediatric ALLs. Several m e c h a n i s m s have been proposed to explain the successful remission in patients with h y p e r d i p l o i d y [5, 10], including a greater sensitivity to treatment with certain cell cycle phase-specific drugs [11] and their tendency toward terminal differentiation and resulting corticosteroid sensitivity [12]. H y p e r d i p l o i d cells generally show a lower frequency of translocations, w h i c h are u s u a l l y associated with poor prognoses [13-16]. Although h y p e r d i p l o i d y alone is considered favorable in ALL patients, there is controversy regarding the mixture of both cytogenetically normal and h y p e r d i p l o i d populations in such patients. Some data suggest that patients with both stemlines have a higher failure rate therapeutically than do those with a single h y p e r d i p l o i d p o p u l a t i o n [17]. The literature contains m u c h discussion and confusion
Table 2 Cytogenetic and flow cytometric analysis of patients with ALL Patient/age
Karyotype
DNA content
1/5 yr 2/4 yr 3/2 yr 4/14 yr
46,XX 46,XX 51-5516]/46,XY[12] 58,XY, ÷ X, ÷ Y, ÷ 4, ÷ 5, + 6,del(7)(p11), + 10,add(12)(p12), + 14, ÷ 14, + 21, ÷ 21,. 2mar[1]/46,XY [76]
Diploid Diploid Aneuploid Aneuploid, DNA index = 1.23
5/21 mo
57,XY,.X,+Y,÷4,÷6,.8,÷10,+14,+17,+18,.21,
Aneuploid, DNA index = 1.20
6/19 yr 7/9 yr 8/12 yr 9/12 yr
[15]/46,XY [7] 46,XY 46,XX 46,XY 53,XY, + Y, ÷ 8, + 10, + 11, + 11, + 13, + 19 [10]/46,XY [7]
Diploid Diploid Diploid Aneuploid, DNA index = 1.17
Abbreviation: ALL, acute lymphoblastic leukemia.
138
F i g u r e 1 Cytocentrifuge preparation of bone marrow aspirate showing convoluted lymphoblasts (Wright-Giemsa stain, original magnification x 600). F i g u r e 2 Giemsa-trypsin banded karyotype of single hyperdiploid cell found in cytogenetic analysis of the bone marrow: 58,XY, + X, +Y, + 4, + 5, + 6, del(7)(p11), + 10,add(12)(p12), + 14, + 14, + 21, + 21, + 2mar.
j
B
1
2
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3
~ t
C
12
18
11
m ty
20
G~pjm~
~ltt
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w
Hyperdiploidy in ALL by Flow Cytometry
139
Simple was run On 26 March 1991
Diploid stemline
- - D~,pk~d G2,-M - - Aneuplold
65~;:
C O U
N T
"l~r
Mean charmf~; CV
G2-M
li
Percenl area Total area = 39%
DNA index = 100 Aneuplold
~emline
i sl 0,-M
330-
70
134
Mean channe~
4.5
5.0
CV
45
10
6
Percent area
Total area = 61% DNA index = 123
0
f~
100
150
200
25O
DNA Fluorescence
Figure 3 DNA histogram of flow cytometry analysis on fixed cell pellet (CV, coefficient of variation). Only gross DNA content is evident; this technology does not indicate the presence of chromosome structural abnormalities. DNA index of aneuploid stemline = 1.23 (approximately equal to a chromosome count of 57).
regarding the importance of nonclonal chromosome abnormalities in BM specimens [18]. Because technical artifact can account for nonclonal (random] loss or gain and/or structural aberrations, it is important to be able to distinguish between true abnormalities and random events. The term "clones to come" has been used to identify possible nonrandom abnormalities as a means of emphasizing the importance of nonclonal aberrations [18], and appears to be favored especially in the event of sequential cytogenetic studies in which the same nonclonal abnormality may be visible in a different sample at a different time. In our analysis, we chose to use adjuvant methods to confirm the presence of an abnormal stemline. In the pediatric patients we report, flow cytometry was used to confirm the true presence of a hyperdiploid stemline, visualized in only one cell by traditional cytogenetic analysis. Although the utility of DNA content measurements in flesh BM specimens was previously demonstrated [11, 12], we have adapted existing techniques to fixed cell pellets remaining from our routine cytogenetic specimens. Often, too little specimen exists to warrant performing DNA content measurement initially. Processing the specimens for traditional cytogenetic analysis and then performing flow cytometric analysis on remaining material appears to be equally efficient. Although the technique is useful to detect numerical aberrations, more than a three chromosome number difference is required for alterations in overall DNA content. Therefore, acquisition/loss of one or two single chromosomes would not be detected. Furthermore, flow cytometry alone will not detect structural aberrations that will not alter gross DNA content. Structural abnormalities are clinically important facets in the overall prognosis of ALL patients [13-16].
We currently use flow cytometry in addition to routine cytogenetic analysis in all our pediatric ALL patients to ascertain the presence/absence of a hyper/hypodiploid population. Patients in w h o m we have detected one hyperdiploid cell by cytogenetic analysis have been shown by flow cytometry and to have normal diploid DNA content; the hyperdiploid cell was thus considered artifactual and/or due to a random event. Combined use of DNA ploidy analysis and cytogenetics is applicable to various hematopoietic disorders other than pediatric ALL; e.g., hyperdiploid BM in myelodysplasia has been associated with poorer survival than has hyperdiploid or normal DNA content [19]. We are now expanding our study to include more ALL patients and are compiling our results from all patients analyzed. We believe that the combination of flow cytometric analysis of DNA content and traditional cytogenetic analysis will enhance the information currently provided to clinicians, aiding in overall clinical management of patients.
REFERENCES 1. Williams DL, Tsiatis A, Brodeur GM, Look AT, Melvin SL, Bowman WP, Kalwinsky DK, Rivera G, Dahl GV (1982): Prognostic importance of chromosome number in 136 untreated children with acute lymphoblastic leukemia. Blood 60:864-871. 2. Bennett JM, Catovsky D, Daniel M-T, Flandrin G, Galton DAG, Gralnick HR, Sultan C (1976): Proposals for the classification of the acute leukemias. Br J Haematol 33:451-458. 3. Pui C-H, Williams DL, Roberson PK, Raimondi SC, Behm FG, Lewis SH, Rivera GK, Kalwinsky DK, Abromowitch M, Crist WM, Murphy SB (1988): Correlation of karyotype and im-
140 munophenotype in childhood acute lymphoblastic leukemia. J Clin Oncol 6:56-61. 4. Wodzinski MA, Watmore AE, Lilleyman JS, Potter AM (1991): Chromosomes in childhood acute lymphoblastic leukemia: Karyotypic patterns in disease subtypes. J Clin Patho144:48-51. 5. Pui C-H, Crist WM, Look AT (1990): Biology and clinical significance of cytogenetic abnormalities in childhood acute lymphoblastic leukemia. Blood 76:1449-1453. 6. Third International Workshop on Chromosomes in Leukemia 1980 (1981): Cancer Genet Cytogenet 4:95-142.
M . C . Lowery et al.
13.
14.
7. Seabright M (1971):A rapid banding technique for human chromosomes [Letter]. Lancet 2:971. 8. ISCN (1985): An International System for Human Cytogenetic Nomenclature (1985), Harden DG, Klinger HP (eds.); published in collaboration with Cytogenet Cell Genet (Karger, Basel, 1985); also in Birth Defects: Original Article Series, Vol. 21, No. 1 (March of Dimes Birth Defects Foundation, New York, 1985).
15.
9. Thornthwaite JT, Sugarbaker EV, Temple WJ (1980): Preparation of tissues for DNA flow cytometric analysis. Cytometry 1:229-237.
16.
10. Tsuchiya H, Matsuda I, Kaneko Y (1990): Why does childhood acute lymphoblastic leukemia with hyperdiploidy show a favorable prognosis? Cancer Genet Cytogenet 50:273-275.
17.
11. Look AT, Roberson PK, Williams DL, Rivera G, Bowman WP, Pui C-H, Ochs J, Abromowitch M, Kalwinsky DK, Dahl LGV, George S, Murphy SB (1985): Prognostic importance of blast cell DNA content in childhood acute lymphoblastic leukemia. Blood 65:1079-1086. 12. Smets LA, Homan-Blok J, Hart A, deVaanG, Behrendt H, Hah-
18.
19.
len K, deWaalFJ (1987):Prognostic implication of hyperdiploidy as based on DNA flow cytometric measurement in childhood acute lymphocytic leukemia-a multicenter study. Leukemia 1:163-166. Williams DL, Harber J, Murphy SB, Look AT, Kalwinsky DK, Rivera G, Melvin SL, Stass S, Dahl GV (1986): Chromosomal translocations play a unique role in influencing prognosis in childhood acute lymphoblastic leukemia. Blood 68:205-212. Bloomfield CD, Sacker-WalkerLM, Goldman AI, Van Den Berghe H, de la Chapelle A, Ruutu T, Alimena G, Garson OM, Golomb HM, Rowley JD, Kaneko Y, Whang-Peng J, Prigogina E, Philip P, Sandberg AA, Lawler SD, Mitelman F (1989): Six-yearfollowup of the clinical significance of karyotype in acute lymphoblastic leukemia. Cancer Genet Cytogenet 40:171-185. Raimondi SC, Privitera E, Williams DL, Look AT, Behm F, Rivem GK, Crist WM, Pui C-H (1991): New recurring chromosomal translocations in childhood acute lymphoblastic leukemia. Blood 77:2016-2022. Heerema NA, Palmer CG, Bachner RL (1985): Karyotypic and clinical findings in a consecutive series of children with acute lymphocytic leukemia. Cancer Genet Cytogenet 17:165-179. Yunis JJ (1986): Should refined chromosomal analyses be used routinely in acute leukemias and myelodysplastic syndrome? N Engl J Med 315:322-323. McConnell TS, Duncan MH, Foucar K, Southwest Oncology Group Leukemia Cytogenetics Subcommittee (1991):Do random (non-clonal) chromosome abnormalities in bone marrow predict a clone to come? Cancer Genet Cytogenet 53:257-263. Clark R, Peters S, Hoy T, Smith S, Whittaker K, Jacobs A (1986): Prognostic importance of hyperdiploid hematopoietic precursors in myelodysplastic syndromes. N Engl J Med 314:1472-1475.
ABSTRACT: Cytogenetic analysis is of value in predicting clinical outcome of pediatric cases of acute lymphoblastic leukemia (ALL). Hyperdiploidy in these patients has significant impact on therapeutic outcome. Because of the technologic limitations of cytogenetic analysis in determining hyperdiploidy in some of these cases, we devised a method to analyze cytogenetically fixed material for cellular DNA content by standard flow cytometric methods. This technique greatly enhances our ability to interpret the observance of single cell anomalies and assess whether these cells are predictive of clonal stemlines or are a result of random events.
INTRODUCTION Hyperdiploidy, classified as more than 50 chromosomes, is a fairly c o m m o n finding in acute lymphoblastic leukemia (ALL), accounting for approximately 24-30% of pediatric cases of ALL [1]. It is particularly frequent in ALL of early pre-B-cell precursor type [CD19 + ,CD10 + ,CD5 - ,CDT- ] and L1 or L2 morphology according to the French-AmericanBritish (FAB) classification [2, 3]. Hyperdiploidy is associated with improved therapeutic response and subsequent survival in pediatric patients with ALL [1, 3-6]. These patients with modal chromosome numbers greater than 55 achieve complete remission more readily and have increased survival. Cytogenetic analysis of hyperdiploid cells is often difficult owing to the suboptimal quality of leukemic chromosomes. Technical problems in cytogenetic analysis of ALL have often prevented identification of hyperdiploid stelnlines. Often these cells are not mitotically active in culture or are present in such a low percentage that they are not detectable by routine cytogenetic analysis. A more particular problem exists in patients in w h o m possibly only one or two hyperdiploid cells are observed cytogenetically. Whether these are true representations of a hyperdiploid clone or are merely random events is not clear. Because of the potential clinical ramifications of a hyperdiploid stemline in pediatric patients, we have combined techniques used in both flow cytometry and cytogenetics to evaluate these patients for the presence of a hyperdiploid
clone. We report a series of pediatric patients in w h o m this technique was useful in ascertaining the validity of a hyperdiploid stemline. CASE HISTORIES The patients ranged in age from 21 months to 19 years; age 21 years was defined as the cutoff age for the definition of pediatric patients. All patients were originally diagnosed with ALL. Table I summarizes the clinical histories of these patients. The pre-pre-B form of ALL designates cases that expressed CD19 but not cytoplasmic tt ilmnunoglobulin. The pre-T ALL cases were positive for T-call antigens such as CD3 or CD 7, but lacked CD2 antigen expression.
MATERIALS AND METHODS
Cytogenetics Cytogenetic studies were performed according to standard technique. Cells from the bone marrow (BM) aspirate were cultured for 24 hours before harvest in supplemented RPMI 1640 medium. Cells were harvested according to protocol and fixed in 3:1 methanol:glacial acetic acid fixative. Slides were prepared for cytogenetic analysis and Giemsa-trypsin banded [7]. The remaining pellet was stored at 4°C. Karyotypes were prepared and designated according to the International System for Human Cytogenetic Nomenclature [8].
Flow Cytometry From the Department of Pathology, Penrose-St. Francis Healthcare System, Colorado Springs, Colorado. Address reprint requests to: Mary C. Lowery, Ph.D., Penrose-St. Francis Healthcare System, 2215 N. Cascade Ave., Department of Pathology, Colorado Springs, Colorado 80907. Received March 2, 1992; accepted January 12, 1993. 136 Cancer Genet Cytogenet 67:136-140 (1993) 0165-4608/93/$06.00
Flow cytometric analysis of the fixed cell pellets followed standard protocol used in our laboratory for solid tumor analysis, with some modifications. These procedures are described briefly. The method is adapted from previously published data [9].
© 1993 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
H y p e r d i p l o i d y in ALL by F l o w Cytometry
Table I
Clinical history of patients
Patient/age la/5 yr 2a/4 yr 3b/2 yr 4/14 yr 5/21 mo 6/19 yr 7/9 yr 8/12 yr 9/12 yr
137
Diagnosis ALL, L1, ALL, L1, ALL ALL, L1, ALL, L1, ALL, L1, ALL, L1, ALL, L1, ALL, L2,
pre-pre-B, TdT + CD10 + pre-pre-B, TdT+ CD10 + pre-pre-B, TdT+ CD10 + pre-pre-B, TdT +, CD10 + pre-T, TdT +, CD7 + pre-pre-B, TdT +, CD1O + T cell, TdT +, CD2 +/CD7 + T cell, TdT +, CD2 +/CD7 +/CDla +
Abbreviations: CD, cluster designation; ALL, acute lymphoblastic leuke-
mia; TdT, terminal deoxynucleotidyl transferase. a Patients currently in remission. b Patient referred; limited clinical information available.
Data were acquired with a linear amplifier for the integrated and peak signal from the p r o p i d i u m i o d i d e (red) fluorescence. Estimates of cell cycle phases were determined with a commercial software program (DNAlysis, University of Utah). The DNA index was determined by dividing the mean channel n u m b e r of the a n e u p l o i d peak by the mean channel of the d i p l o i d peak. RESULTS
Cytogenetics/Flow Cytometry Cytogenetic and flow cytometric analyses of the patient samples are s u m m a r i z e d in Table 2. Representative examples of cellular morphology, karyotype, and flow cytometry results are shown in Figs. 1-3 (all data obtained from patient 4).
DISCUSSION Fixed cell pellets were centrifuged (1,200 r p m for 5 minutes) and the supernatant was discarded. The pellet was r e s u s p e n d e d in Hanks' buffered salt solution (HBSS) (3104170, GIBCO/BRL, Bethesda, MD) and centrifuged. The HBSS s u s p e n s i o n was repeated twice to remove all remaining fixative. After the last wash, the pellet was r e s u s p e n d e d in uns u p p l e m e n t e d RPMI 1640 m e d i u m (320-1875, GIBCO) and allowed to sit overnight at 4°C. The next day, the cells were centrifuged (1,200 r p m for 5 minutes) and r e s u s p e n d e d in a m o d i f i e d nuclear isolation m e d i u m [NIM: 1.714 g NaC1, 29.4 mg CaC12, 24.6 mg MgSO4.H20, 1.2 g nonidet po40 (N6507, Sigma, St. Louis, MO), 400 mg bovine serum albumin (BSA, A7511, Sigma), 10 mg p r o p i d i u m iodide (P4170, Sigma), 200 mg RNase, type I-AS (R5503, Sigma), 276 mg NaHzPO4.H20; q.s. solution to 200 ml with deionized water w i t h pH adjusted to 7.0]. The cell s u s p e n s i o n was then centrifuged (2,300 r p m for 10 minutes), the supernatant was discarded, and the cells were r e s u s p e n d e d in I ml NIM. A cell count was performed using a fluorescent microscope with cell counts optimized at 1-2 × 106 cells/ml. Normal d i p l o i d fixed cells, processed identically, were used as a control. Specimens were analyzed with a Coulter Profile II flow cytometer (Coulter Cytometry, Hialeah, FL) w i t h 488-nm argon laser (15 mW) excitation.
Cytogenetic analysis continues to be an invaluable a d d i t i o n to current diagnostic strategies in h u m a n leukemias. Various chromosome abnormalities have been shown to be associated with either improved or worsened prognosis. Such information provides clinicians with information necessary to manage patients appropriately. Cytogenetic studies of pediatric ALLs have been especially fruitful in providing valuable clinical information. H y p e r d i p l o i d y is considered a favorable prognostic finding in pediatric ALLs. Several m e c h a n i s m s have been proposed to explain the successful remission in patients with h y p e r d i p l o i d y [5, 10], including a greater sensitivity to treatment with certain cell cycle phase-specific drugs [11] and their tendency toward terminal differentiation and resulting corticosteroid sensitivity [12]. H y p e r d i p l o i d cells generally show a lower frequency of translocations, w h i c h are u s u a l l y associated with poor prognoses [13-16]. Although h y p e r d i p l o i d y alone is considered favorable in ALL patients, there is controversy regarding the mixture of both cytogenetically normal and h y p e r d i p l o i d populations in such patients. Some data suggest that patients with both stemlines have a higher failure rate therapeutically than do those with a single h y p e r d i p l o i d p o p u l a t i o n [17]. The literature contains m u c h discussion and confusion
Table 2 Cytogenetic and flow cytometric analysis of patients with ALL Patient/age
Karyotype
DNA content
1/5 yr 2/4 yr 3/2 yr 4/14 yr
46,XX 46,XX 51-5516]/46,XY[12] 58,XY, ÷ X, ÷ Y, ÷ 4, ÷ 5, + 6,del(7)(p11), + 10,add(12)(p12), + 14, ÷ 14, + 21, ÷ 21,. 2mar[1]/46,XY [76]
Diploid Diploid Aneuploid Aneuploid, DNA index = 1.23
5/21 mo
57,XY,.X,+Y,÷4,÷6,.8,÷10,+14,+17,+18,.21,
Aneuploid, DNA index = 1.20
6/19 yr 7/9 yr 8/12 yr 9/12 yr
[15]/46,XY [7] 46,XY 46,XX 46,XY 53,XY, + Y, ÷ 8, + 10, + 11, + 11, + 13, + 19 [10]/46,XY [7]
Diploid Diploid Diploid Aneuploid, DNA index = 1.17
Abbreviation: ALL, acute lymphoblastic leukemia.
138
F i g u r e 1 Cytocentrifuge preparation of bone marrow aspirate showing convoluted lymphoblasts (Wright-Giemsa stain, original magnification x 600). F i g u r e 2 Giemsa-trypsin banded karyotype of single hyperdiploid cell found in cytogenetic analysis of the bone marrow: 58,XY, + X, +Y, + 4, + 5, + 6, del(7)(p11), + 10,add(12)(p12), + 14, + 14, + 21, + 21, + 2mar.
j
B
1
2
t
3
~ t
C
12
18
11
m ty
20
G~pjm~
~ltt
p
w
Hyperdiploidy in ALL by Flow Cytometry
139
Simple was run On 26 March 1991
Diploid stemline
- - D~,pk~d G2,-M - - Aneuplold
65~;:
C O U
N T
"l~r
Mean charmf~; CV
G2-M
li
Percenl area Total area = 39%
DNA index = 100 Aneuplold
~emline
i sl 0,-M
330-
70
134
Mean channe~
4.5
5.0
CV
45
10
6
Percent area
Total area = 61% DNA index = 123
0
f~
100
150
200
25O
DNA Fluorescence
Figure 3 DNA histogram of flow cytometry analysis on fixed cell pellet (CV, coefficient of variation). Only gross DNA content is evident; this technology does not indicate the presence of chromosome structural abnormalities. DNA index of aneuploid stemline = 1.23 (approximately equal to a chromosome count of 57).
regarding the importance of nonclonal chromosome abnormalities in BM specimens [18]. Because technical artifact can account for nonclonal (random] loss or gain and/or structural aberrations, it is important to be able to distinguish between true abnormalities and random events. The term "clones to come" has been used to identify possible nonrandom abnormalities as a means of emphasizing the importance of nonclonal aberrations [18], and appears to be favored especially in the event of sequential cytogenetic studies in which the same nonclonal abnormality may be visible in a different sample at a different time. In our analysis, we chose to use adjuvant methods to confirm the presence of an abnormal stemline. In the pediatric patients we report, flow cytometry was used to confirm the true presence of a hyperdiploid stemline, visualized in only one cell by traditional cytogenetic analysis. Although the utility of DNA content measurements in flesh BM specimens was previously demonstrated [11, 12], we have adapted existing techniques to fixed cell pellets remaining from our routine cytogenetic specimens. Often, too little specimen exists to warrant performing DNA content measurement initially. Processing the specimens for traditional cytogenetic analysis and then performing flow cytometric analysis on remaining material appears to be equally efficient. Although the technique is useful to detect numerical aberrations, more than a three chromosome number difference is required for alterations in overall DNA content. Therefore, acquisition/loss of one or two single chromosomes would not be detected. Furthermore, flow cytometry alone will not detect structural aberrations that will not alter gross DNA content. Structural abnormalities are clinically important facets in the overall prognosis of ALL patients [13-16].
We currently use flow cytometry in addition to routine cytogenetic analysis in all our pediatric ALL patients to ascertain the presence/absence of a hyper/hypodiploid population. Patients in w h o m we have detected one hyperdiploid cell by cytogenetic analysis have been shown by flow cytometry and to have normal diploid DNA content; the hyperdiploid cell was thus considered artifactual and/or due to a random event. Combined use of DNA ploidy analysis and cytogenetics is applicable to various hematopoietic disorders other than pediatric ALL; e.g., hyperdiploid BM in myelodysplasia has been associated with poorer survival than has hyperdiploid or normal DNA content [19]. We are now expanding our study to include more ALL patients and are compiling our results from all patients analyzed. We believe that the combination of flow cytometric analysis of DNA content and traditional cytogenetic analysis will enhance the information currently provided to clinicians, aiding in overall clinical management of patients.
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