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Use of conditioned media in cell culture can mask cytogenetic abnormalities in acute leukemia
Use of Conditioned Media in Cell Culture Can Mask Cytogenetic Abnormalities in Acute Leukemia Guoxian Sun, H. Phillip Koeffler, Robert Peter Gale, Robert S. Sparkes, and Rhona R. Schreck
ABSTRACT: Conditioned media (CM)from a haman lung adenocarcinoma cell line expressing interleukins 1 and 6 (IL-1, IL-6), granulocyte (G), macrophage (M), and GM colony-stimulating factors (G, M, GM-CSF) and transforming growth factor/3 (TGF#) were used to stimulate growth of bone marrow (BM) cells from 18 persons with leukemia, myeladysplastic syndrome, or lymphoma. The objective was to increase numbers of analyzable metaphases and to enhance the likelihood of detecting cytogenetic abnormalities. Although more mitotic cells were observed with CM, the detection rate of cytogenetic abnormalities decreased in 12 of 18 cases. These data indicate that use of CMfor cytogenetic analyses may favor growth of normal versus leukemia cells and mask cytogenetic abnormalities.
INTRODUCTION Chromosome analyses of h u m a n leukemias provide potentially useful diagnostic and prognostic data. Accurate cytogenetic analysis of leukemia requires sufficient numbers of mitotic cells. This analysis is often compromised, however, by poor in vitro cell growth resulting in few metaphases and poor-quality chromosomes. Several laboratories have attempted to modify in vitro cell culture conditions to facilitate cytogenetic analyses. Recently, purified hematopoietic growth factors, or c o n d i t i o n e d m e d i a (CM) from cell lines w h i c h produce these factors, were used to facilitate in vitro cell growth and improve cytogenetic analyses of leukemia cells [1-3]. We used CM from the Lu-CSF-1 cell line to stimulate proliferation of bone marrow {BM) ceils from 18 persons with leukemia or other hematopoietic malignancies. A l t h o u g h we observed increased numbers and improved quality of metaphases, a decreased proportion of chromosomally abnormal cells was observed in most instances. From the Department of Medicine, School of Medicine, University of Californiaat Los Angeles (G. S., H. P. K., R. P. G., R. S. S.); and Medical Genetics-BirthDefectsCenter, Cedars Sinai Medical Center (R. R. S), Los Angeles, California. Address reprint requests to: Dr. Guoxian Sun, c/o Dr. Robert Peter Gale, Department of Medicine, Division of Hematology and Oncology, UCLA School of Medicine, Los Angeles, CA 90024-1678. Received July 17, 1989; accepted October 2, 1989.
107 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet46:107-113 (1990) 0165-4608/90/$03.50
108
G. Sun et al.
MATERIALS AND METHODS Cell Line The Lu-CSF-1 cell line was derived from a human lung adenocarcinoma established by H. P. Koeffler. It was maintained in a-minimal essential medium (a-MEM) (Flow Laboratories, McLean, VA) and 10% fetal calf serum (FCS) and subcultured weekly.
Conditioned Media Conditioned media were collected from confluent flasks of Lu-CSF-1 cultured for 7 days and centrifuged (300 g) to remove cells and debris. The supernatant was filtered through a 0.2-~m Nalgene filter, stored at 4°C, and used within I month.
Northern Blot Analysis Cytoplasmic RNA was prepared and analyzed by standard techniques [4-6]. The granulocyte-monocyte colony-stimulating factor (GM-CSF) cDNA probe [0.9 kilobase (kb), ECoR1-BamH1] was derived from plasmid pCSF-2. The M-CSF cDNA probe (1.6 kb) was derived from plasmid pcCSF-12. Human interleukin (IL)-6 cDNA (1.2 kb, Xbo-I) was derived from plasmid pXH, These plasmids were a gift of Dr. S. Clark (Genetics Institute, Inc., Cambridge, MA). A 30-base oligonucleotide was used as the G-CSF probe (gift of Dr. L. Souza, Aragon, Inc., Thousand Oaks, CA). This oligonucleotide was end-labeled with 7-32p-ATP [6]. DNA inserts were oligolabeled (random primed) as described [6]. 28S and 18S RNA were used as molecular weight markers.
Colony Formation in Soft Agar Informed consent was obtained from all subjects. Heparinized BM was obtained from healthy volunteers and patients by aspiration from the posterior iliac crest. Mononuclear cells were isolated by centrifugation on a Ficoll-Hypaque gradient, washed twice in phosphate-buffered saline and suspended in a-MEM containing 10% FCS and 1% penicillin and streptomycin (Irvine Scientific, Irvine, CA). Cells were plated in 35-ram Lux culture dishes (Miles Laboratories, Naperville, IL) in a two-layer soft agar system as described [7, 8]. The underlayer contained 0.5% agar and the upper layer contained 0.3% agar (Difco, Detroit, MI) dissolved in a-MEM. Lu-CSF-1 CM, 5% (vol/vol), was added to the agar underlayer. Cultures were incubated in a humidified atmosphere (5% CO2, 37°C), and colonies (-40 cells) were scored after 10 to 12 days with an inverted microscope. Experiments were performed in triplicate. Control plates without Lu-CSF-1 CM were included.
Bone Marrow Culture and Cytogenetic Analysis Bone marrow cells were obtained as described, pelleted, and resuspended in RPMI 1640 with 20% FCS with or without 10% Lu-CSF-1 CM. Cultures containing 1 × 107 nucleated cells/lO ml were incubated at 37°C for 24 or 48 hours. Colcemid (0.02 mg/ ml, GIBCO, Grand Island, NY) was added 30 minutes before the cells were harvested. The cells were centrifuged, resuspended in 0.4% KC1 for 30 minutes at 37°C, and exposed to three changes of a 3 : 1 methanol-acetic acid fixative. Microscope slides were prepared and trypsin-Giemsa banding performed by standard techniques |gJ. Culture conditions were identical for each pair of cultures (either 24 or 48 hours).
109
Masking of Cytogenetic Abnormalities by CM
Chromosome analyses were made of consecutive metaphases. Cytogenetic abnormalities were described according to the ISCN (1985) [10].
RESULTS Northern blot analysis showed that mRNA isolated from Lu-CSF-1 hybridized with probes for IL-1B, IL-6, G-CSF, GM-CSF, M-CSF, and transforming growth factor/~ (TGFB] (Fig. 1). Conditioned media from Lu-CSF-1 stimulated growth of myeloid colonies in mononuclear BM cells from normal subjects and persons with acute myelogenous leukemia. Control cultures showed more than five myeloid colonies per 2 x l0 s mononuclear cells plated. These rates increased to 87 -+ 18 with normal BM and to 56 -+ 24 with leukemia cells when 5% CM was added to cultures. Addition of 10% Lu-CSF-1 CM improved cell growth in vitro. The mitotic indices
Figure 1
Northernblot analysis of mRNA from Lu-CSF-1 cell line, hybridized with the 32p_
labeled probes indicated (described in Materials and Methods). Arrows indicate the expected signal for each respective probe, Molecular weight of each mRNA was determined by comparison to the 28 and 18S bands apparent on the ethidium bromide stained gels before RNA transfer. The size in kilobases of the transcripts are: granulocyte colony-stimulating factor (G-CSF), 1.6; granulocyte-macrophage CSF (GM-CSF), 0.9; M-CSF, 4.0; interleukin 1~ (IL-I~), 1.6; IL-6, 1.3; transforming growth factor ~(TGF)B, 2.2. Each lane represents a separate electrophoresis and
hybridization with the specific cytokine probe.
L U - CSF - I
:
CYTOKINE E X P R E S S I O N
! ~!~i~i!!~!!ii!ii~ ¸
~'i~i~ ii :~i'~~!i~'!:~i:!ii!'~!~i%~',',~'~J~,~,:~
~ i/~
2
G-CSF
M ~
CSF
M-CSF
IL-Ip IL-6
110
G. Sun et al.
• WithoutCM [ ] With CM
O
E rJ~
v
1
2
3
4
5
6
7
8
9
10
t6
17
18
Patient Number
Figure 2 Percent of abnormal cells in bone marrow samples from patients with acute leukemia, myelodysplastic syndrome, or lymphoma. Control cultures (solid bars}; cultures with 10% LuCSF-1 conditioned media (hatched bars}.
were one to two times higher than cultures without CM (0.05% to 0.2% versus 0.02% to 0.1%, p < 0.01}. However, as shown in Table 1 and Fig. 2, patients 2, 3, 5, 8, 9 and 16-18 had karyotypically normal and abnormal cells when cells were cultured without CM. Addition of CM resulted in a much lower proportion of abnormal karyotypes. Patients 5 and 18, who had a minor clone of malignant cells in control cultures, showed only constitutionally abnormal and normal metaphases, respectively, with CM. The normal clone also predominated when CM was added to cultures from patients 2, 3, 8, 9, and 17, in whom the abnormal clone was dominant in control cultures. Patient 16 also showed a substantial increase in the proportion of normal metaphases with CM. Patients 1, 4, 6, and 7 had only abnormal metaphases in control cultures. In all four, some normal metaphases were observed in cultures with CM. Patient 6 is notable in that the routine culture showed only abnormal metaphases, whereas these constituted the minor clone in cultures with CM. In 12 of 13 patients with leukemias (acute myelogenous leukemia or acute lymphocytic leukemia) and in myelodysplastic syndrome, the CM culture had always an increased proportion of normal metaphases. No difference was noted in five patients with chronic myelogenous leukemia (CML) (patients 11-15), where only abnormal metaphases were observed with or without CM. DISCUSSION Cytogenetic analyses are important in hematologic malignancies, particularly leukemias. Recent studies used CM or molecularly cloned hematopoietic growth factors to increase the number and quality of chromosome preparations from persons with leukemia I1-3]. We used CM from the Lu-CSF-1 cell line because it expressed factors that stimulated growth of normal and leukemia cells in semisolid culture. We observed more and better quality metaphases when cells from persons with leukemia were cultured with (AM. Cytogenetic analyses from 12 of 13 persons with acute leukemia
66/M 46/F 36/M 59/M 39/M 6/F
85/M 57/M
11 12 13 14 15 16
17 18
MDS Lymphoma
CML CML CML CML CML MDS
AML AML
AML AML AML AML
ALL ALL ALL ALL
Diagnosis
Without CM
47,XX, + 21(17}/47,XX, + 21,dup(7)(q32-->q36)(3) 46,XY, - 7, + mar(24) 47,XY, + 8(20) 46,XX(5)/ 46,XY,dup(4}(q21-*q34),t(21;?}(q22;?}(14) 46,XX( 7 )/4 5,XX, - 17 ,t(12 ;17 )(p11;q11] (13 } 46,XX(1}/48,XX, + 8,t(3;9)(q13;p21), + t(3;9)(q13;p21)(13) 47,XY, + 8,t(9;22)(q34;q11)(10) 46,XX,t(9;22)(q34;q11)(20) 46,XY,t(9;22)(q34;ql 1)(20} 46,XY,t(9;22](q34;q11),i(17q)(20) 48,XY,t(9;22)(q34;ql 1)(15) 46,XX(11)/46,XX,del(8)(p12),t(12;?)(p 13;?), del(13)(q21q32)(7) 46,XY(4]/47,XY, + 15(16) 46,XY(17)/48,XY, + 2mar(3)
49,XY, + 7, + 8, + 14(20) 46,XY(2)/47,XY, + mar(10) 46,XX(6)/46,XX,i(9q),del(10)(q22q24}(10) 46,X¥,del{5)(q32),t(1;15)(q34;q21)(20} 46,XY(2)/49,XY, + 7, + 8, + 14(18} 46,XY(21)/47,XY, + mar(l) 46,XX(20)/46,XX,i(9q),del(10)(q22q24)(10) 46,XY(2)/ 46,XY,del(5)(q32),t(1;15)(q34;q21}(18) 47,XX, + 21(20) 46,XY(20}/46,XY, - 7, + mar(4) 46,XY(1)/47,XY, + 8(19) 46,XX(16)/ 46,XY,dup(4)(q21--~q34),t(21;?) (q22;?}(4) 4 6 , X X ( 1 6 ) / 4 5 , X X , - 17,t(12;17}(pll;qll}(4) 46,XX(1)/48,XX, + 8,t(3;9}(q13;p21}, + t(3;9)(q13;p21}(19) 47,XY, + 8,t(9;22)(q34;q11)(20) 46,XX,t(9;22)(q34;q11)(20) 46,XY,t(9;22)(q34;q 11)(20) 46,XY,t(9;22)(q34;ql 1),i(17q)(20) 46,XY,t(9;22)(q34;ql 1}(15} 46,XX(19)/46,XX,del(8)(p12) ,t(12;?)(p13;?), del(13)(q21q32)(1) 46,XY(28)/47 ,X¥, + 15(2) 46,XY(30)
With CM
24 24
24 48 24 24 24 48
24 24
24 48 24 48
24 24 48 24
Culture term (h)
Abbreviations: ALL, acute lymphocytic leukemia; AML, acute myelogenous leukemia; CML, chronic myelogenous leukemia; MDS, myelodysplasfic syndrome,
in relapse after bone marrow transplant.
60/F 85/F
9 10
was
1 day/F 29/M 37/M 16/M
5 6 7 8"
a Patient
2/M 20/M 78/F 46/M
Age/sex
Karyotype (no. of cells)
C y t o g e n e t i c S t u d i e s i n 18 P a t i e n t s w i t h H e m a t o l o g i c M a l i g n a n c i e s
1 2 3 4
Patient no.
Table 1
112
G. Sun et al, showed different results w h e n cells were cultured with or without CM. Although the qualities of metaphases from both normal and abnormal cells were i m p r o v e d in these patients, the percentage of karyotypically normal cells increased w h e n CM was used. These data i m p l y that analysis of metaphases only from cell cultured w i t h CM, may miss cytogenetic abnormalities if the leukemia clone constitutes a small proportion of the d i v i d i n g cells. For example, patient 5 was a newborn with Down s y n d r o m e w i t h suspected acute leukemia, a situation difficult to distinguish from a transient l e u k e m o i d reaction. Control cultures showed a small proportion of cells w i t h an a d d i t i o n a l abnormality, a change suggestive of leukemia. Cytogenetic analysis w i t h CM s h o w e d only trisomy 21. The patient eventually died of leukemia. In patient 18, analysis of cultures of cells only with CM w o u l d have missed the abnormal clone. The decreased frequency of abnormal metaphases with CM containing hematopoietic growth factor(s) was not unanticipated. Bone marrow from persons with acute leukemia is often a mixture of normal and leukemia cells. Both p o p u l a t i o n s have low mitotic indexes. Normal cells consistently proliferate w h e n exposed to h e m a t o p o i e t i c growth factors. The response of leukemia cells is variable; often, mitotic indexes do not increase. Consequently, mixtures of normal and leukemia cells cultured w i t h hematopoietic growth factors often result in an increased proportion of normal metaphases. Neither can we exclude the possibility that the CM contains a factor(s) that inhibits proliferation of leukemia cells; this is unlikely in view of increased leukemia colony formation w h e n CM was added. This study underscores the c o m p l e x i t y of using h e m a t o p o i e t i c growth factors to improve results of cytogenetic analyses in leukemia. A l t h o u g h the n u m b e r and quality of metaphases may be increased, the proportion of abnormal cells m a y either be decreased or become undetectable. Thus, careful qualitative evaluation of the effects of any cell growth stimulating factor(s) should be undertaken before they are used clinically.
This study was supported in part by US Public Health Service Grants No. CA26038, CA32737, CA33938, and CA23175. G.S. is a fellow of the World Health Organization. R.P.G. is the Wald Foundation Scholar in Biomedical Communications.
REFERENCES
1. Minamihisamatsu M, Odaka T, linnai I, Ishihara T (1986): A culture technique for chromosome analysis in human myeloid leukemias. Cancer Genet Cytogenet 19:345-50 2. Michael l, Lever I, Rochmilewitz EA, Fibach E (1986): Myeloid leukemia cells in culture: Applications for cytogenetic analysis. Blood 68:790-3. 3. Morgan S, Hecht BK, Morgan R, Hecht F (1987): Qualitative and quantitative enhancement of bone marrow cytogenetics by addition of giant cell tumor conditioned medium. Karyogram 113:39-40. 4. Clemen MJ (1984): Purification of eukaryotic mRNA. In: Haines BD, Higgins SJM, eds. Transcription and Translation. IRL Press, Washington, D.C., 1984:211-20. 5. Goldberg DA (1980): Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene. Proc Natl Acad Sci USA 77:5794-8. 6. Maniatis T, Fritsch EF, Sambrook J (1982): Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 7, Douer D, Koeffler PIP (1982): Retinoic acid-inhibition of clonal growth of human myeloid leukemia cells. J Clin Invest 69:277-83. 8. Douer D, Koeffler HP (1982): Retinoic acid enhances colony stimulating factor induced clonal growth of normal human myeloid progenitor cells in vitro. Exp Cell Res 138:193-8.
Masking of Cytogenetic Abnormalities by CM
113
9. Seabright M (1971): A rapid banding technique for human chromosomes. Lancet 2:971-2. 10. ISCN (1985): An International System for Human Cygoenetic Nomenclature, Harnden DE, 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).
ABSTRACT: Conditioned media (CM)from a haman lung adenocarcinoma cell line expressing interleukins 1 and 6 (IL-1, IL-6), granulocyte (G), macrophage (M), and GM colony-stimulating factors (G, M, GM-CSF) and transforming growth factor/3 (TGF#) were used to stimulate growth of bone marrow (BM) cells from 18 persons with leukemia, myeladysplastic syndrome, or lymphoma. The objective was to increase numbers of analyzable metaphases and to enhance the likelihood of detecting cytogenetic abnormalities. Although more mitotic cells were observed with CM, the detection rate of cytogenetic abnormalities decreased in 12 of 18 cases. These data indicate that use of CMfor cytogenetic analyses may favor growth of normal versus leukemia cells and mask cytogenetic abnormalities.
INTRODUCTION Chromosome analyses of h u m a n leukemias provide potentially useful diagnostic and prognostic data. Accurate cytogenetic analysis of leukemia requires sufficient numbers of mitotic cells. This analysis is often compromised, however, by poor in vitro cell growth resulting in few metaphases and poor-quality chromosomes. Several laboratories have attempted to modify in vitro cell culture conditions to facilitate cytogenetic analyses. Recently, purified hematopoietic growth factors, or c o n d i t i o n e d m e d i a (CM) from cell lines w h i c h produce these factors, were used to facilitate in vitro cell growth and improve cytogenetic analyses of leukemia cells [1-3]. We used CM from the Lu-CSF-1 cell line to stimulate proliferation of bone marrow {BM) ceils from 18 persons with leukemia or other hematopoietic malignancies. A l t h o u g h we observed increased numbers and improved quality of metaphases, a decreased proportion of chromosomally abnormal cells was observed in most instances. From the Department of Medicine, School of Medicine, University of Californiaat Los Angeles (G. S., H. P. K., R. P. G., R. S. S.); and Medical Genetics-BirthDefectsCenter, Cedars Sinai Medical Center (R. R. S), Los Angeles, California. Address reprint requests to: Dr. Guoxian Sun, c/o Dr. Robert Peter Gale, Department of Medicine, Division of Hematology and Oncology, UCLA School of Medicine, Los Angeles, CA 90024-1678. Received July 17, 1989; accepted October 2, 1989.
107 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet46:107-113 (1990) 0165-4608/90/$03.50
108
G. Sun et al.
MATERIALS AND METHODS Cell Line The Lu-CSF-1 cell line was derived from a human lung adenocarcinoma established by H. P. Koeffler. It was maintained in a-minimal essential medium (a-MEM) (Flow Laboratories, McLean, VA) and 10% fetal calf serum (FCS) and subcultured weekly.
Conditioned Media Conditioned media were collected from confluent flasks of Lu-CSF-1 cultured for 7 days and centrifuged (300 g) to remove cells and debris. The supernatant was filtered through a 0.2-~m Nalgene filter, stored at 4°C, and used within I month.
Northern Blot Analysis Cytoplasmic RNA was prepared and analyzed by standard techniques [4-6]. The granulocyte-monocyte colony-stimulating factor (GM-CSF) cDNA probe [0.9 kilobase (kb), ECoR1-BamH1] was derived from plasmid pCSF-2. The M-CSF cDNA probe (1.6 kb) was derived from plasmid pcCSF-12. Human interleukin (IL)-6 cDNA (1.2 kb, Xbo-I) was derived from plasmid pXH, These plasmids were a gift of Dr. S. Clark (Genetics Institute, Inc., Cambridge, MA). A 30-base oligonucleotide was used as the G-CSF probe (gift of Dr. L. Souza, Aragon, Inc., Thousand Oaks, CA). This oligonucleotide was end-labeled with 7-32p-ATP [6]. DNA inserts were oligolabeled (random primed) as described [6]. 28S and 18S RNA were used as molecular weight markers.
Colony Formation in Soft Agar Informed consent was obtained from all subjects. Heparinized BM was obtained from healthy volunteers and patients by aspiration from the posterior iliac crest. Mononuclear cells were isolated by centrifugation on a Ficoll-Hypaque gradient, washed twice in phosphate-buffered saline and suspended in a-MEM containing 10% FCS and 1% penicillin and streptomycin (Irvine Scientific, Irvine, CA). Cells were plated in 35-ram Lux culture dishes (Miles Laboratories, Naperville, IL) in a two-layer soft agar system as described [7, 8]. The underlayer contained 0.5% agar and the upper layer contained 0.3% agar (Difco, Detroit, MI) dissolved in a-MEM. Lu-CSF-1 CM, 5% (vol/vol), was added to the agar underlayer. Cultures were incubated in a humidified atmosphere (5% CO2, 37°C), and colonies (-40 cells) were scored after 10 to 12 days with an inverted microscope. Experiments were performed in triplicate. Control plates without Lu-CSF-1 CM were included.
Bone Marrow Culture and Cytogenetic Analysis Bone marrow cells were obtained as described, pelleted, and resuspended in RPMI 1640 with 20% FCS with or without 10% Lu-CSF-1 CM. Cultures containing 1 × 107 nucleated cells/lO ml were incubated at 37°C for 24 or 48 hours. Colcemid (0.02 mg/ ml, GIBCO, Grand Island, NY) was added 30 minutes before the cells were harvested. The cells were centrifuged, resuspended in 0.4% KC1 for 30 minutes at 37°C, and exposed to three changes of a 3 : 1 methanol-acetic acid fixative. Microscope slides were prepared and trypsin-Giemsa banding performed by standard techniques |gJ. Culture conditions were identical for each pair of cultures (either 24 or 48 hours).
109
Masking of Cytogenetic Abnormalities by CM
Chromosome analyses were made of consecutive metaphases. Cytogenetic abnormalities were described according to the ISCN (1985) [10].
RESULTS Northern blot analysis showed that mRNA isolated from Lu-CSF-1 hybridized with probes for IL-1B, IL-6, G-CSF, GM-CSF, M-CSF, and transforming growth factor/~ (TGFB] (Fig. 1). Conditioned media from Lu-CSF-1 stimulated growth of myeloid colonies in mononuclear BM cells from normal subjects and persons with acute myelogenous leukemia. Control cultures showed more than five myeloid colonies per 2 x l0 s mononuclear cells plated. These rates increased to 87 -+ 18 with normal BM and to 56 -+ 24 with leukemia cells when 5% CM was added to cultures. Addition of 10% Lu-CSF-1 CM improved cell growth in vitro. The mitotic indices
Figure 1
Northernblot analysis of mRNA from Lu-CSF-1 cell line, hybridized with the 32p_
labeled probes indicated (described in Materials and Methods). Arrows indicate the expected signal for each respective probe, Molecular weight of each mRNA was determined by comparison to the 28 and 18S bands apparent on the ethidium bromide stained gels before RNA transfer. The size in kilobases of the transcripts are: granulocyte colony-stimulating factor (G-CSF), 1.6; granulocyte-macrophage CSF (GM-CSF), 0.9; M-CSF, 4.0; interleukin 1~ (IL-I~), 1.6; IL-6, 1.3; transforming growth factor ~(TGF)B, 2.2. Each lane represents a separate electrophoresis and
hybridization with the specific cytokine probe.
L U - CSF - I
:
CYTOKINE E X P R E S S I O N
! ~!~i~i!!~!!ii!ii~ ¸
~'i~i~ ii :~i'~~!i~'!:~i:!ii!'~!~i%~',',~'~J~,~,:~
~ i/~
2
G-CSF
M ~
CSF
M-CSF
IL-Ip IL-6
110
G. Sun et al.
• WithoutCM [ ] With CM
O
E rJ~
v
1
2
3
4
5
6
7
8
9
10
t6
17
18
Patient Number
Figure 2 Percent of abnormal cells in bone marrow samples from patients with acute leukemia, myelodysplastic syndrome, or lymphoma. Control cultures (solid bars}; cultures with 10% LuCSF-1 conditioned media (hatched bars}.
were one to two times higher than cultures without CM (0.05% to 0.2% versus 0.02% to 0.1%, p < 0.01}. However, as shown in Table 1 and Fig. 2, patients 2, 3, 5, 8, 9 and 16-18 had karyotypically normal and abnormal cells when cells were cultured without CM. Addition of CM resulted in a much lower proportion of abnormal karyotypes. Patients 5 and 18, who had a minor clone of malignant cells in control cultures, showed only constitutionally abnormal and normal metaphases, respectively, with CM. The normal clone also predominated when CM was added to cultures from patients 2, 3, 8, 9, and 17, in whom the abnormal clone was dominant in control cultures. Patient 16 also showed a substantial increase in the proportion of normal metaphases with CM. Patients 1, 4, 6, and 7 had only abnormal metaphases in control cultures. In all four, some normal metaphases were observed in cultures with CM. Patient 6 is notable in that the routine culture showed only abnormal metaphases, whereas these constituted the minor clone in cultures with CM. In 12 of 13 patients with leukemias (acute myelogenous leukemia or acute lymphocytic leukemia) and in myelodysplastic syndrome, the CM culture had always an increased proportion of normal metaphases. No difference was noted in five patients with chronic myelogenous leukemia (CML) (patients 11-15), where only abnormal metaphases were observed with or without CM. DISCUSSION Cytogenetic analyses are important in hematologic malignancies, particularly leukemias. Recent studies used CM or molecularly cloned hematopoietic growth factors to increase the number and quality of chromosome preparations from persons with leukemia I1-3]. We used CM from the Lu-CSF-1 cell line because it expressed factors that stimulated growth of normal and leukemia cells in semisolid culture. We observed more and better quality metaphases when cells from persons with leukemia were cultured with (AM. Cytogenetic analyses from 12 of 13 persons with acute leukemia
66/M 46/F 36/M 59/M 39/M 6/F
85/M 57/M
11 12 13 14 15 16
17 18
MDS Lymphoma
CML CML CML CML CML MDS
AML AML
AML AML AML AML
ALL ALL ALL ALL
Diagnosis
Without CM
47,XX, + 21(17}/47,XX, + 21,dup(7)(q32-->q36)(3) 46,XY, - 7, + mar(24) 47,XY, + 8(20) 46,XX(5)/ 46,XY,dup(4}(q21-*q34),t(21;?}(q22;?}(14) 46,XX( 7 )/4 5,XX, - 17 ,t(12 ;17 )(p11;q11] (13 } 46,XX(1}/48,XX, + 8,t(3;9)(q13;p21), + t(3;9)(q13;p21)(13) 47,XY, + 8,t(9;22)(q34;q11)(10) 46,XX,t(9;22)(q34;q11)(20) 46,XY,t(9;22)(q34;ql 1)(20} 46,XY,t(9;22](q34;q11),i(17q)(20) 48,XY,t(9;22)(q34;ql 1)(15) 46,XX(11)/46,XX,del(8)(p12),t(12;?)(p 13;?), del(13)(q21q32)(7) 46,XY(4]/47,XY, + 15(16) 46,XY(17)/48,XY, + 2mar(3)
49,XY, + 7, + 8, + 14(20) 46,XY(2)/47,XY, + mar(10) 46,XX(6)/46,XX,i(9q),del(10)(q22q24}(10) 46,X¥,del{5)(q32),t(1;15)(q34;q21)(20} 46,XY(2)/49,XY, + 7, + 8, + 14(18} 46,XY(21)/47,XY, + mar(l) 46,XX(20)/46,XX,i(9q),del(10)(q22q24)(10) 46,XY(2)/ 46,XY,del(5)(q32),t(1;15)(q34;q21}(18) 47,XX, + 21(20) 46,XY(20}/46,XY, - 7, + mar(4) 46,XY(1)/47,XY, + 8(19) 46,XX(16)/ 46,XY,dup(4)(q21--~q34),t(21;?) (q22;?}(4) 4 6 , X X ( 1 6 ) / 4 5 , X X , - 17,t(12;17}(pll;qll}(4) 46,XX(1)/48,XX, + 8,t(3;9}(q13;p21}, + t(3;9)(q13;p21}(19) 47,XY, + 8,t(9;22)(q34;q11)(20) 46,XX,t(9;22)(q34;q11)(20) 46,XY,t(9;22)(q34;q 11)(20) 46,XY,t(9;22)(q34;ql 1),i(17q)(20) 46,XY,t(9;22)(q34;ql 1}(15} 46,XX(19)/46,XX,del(8)(p12) ,t(12;?)(p13;?), del(13)(q21q32)(1) 46,XY(28)/47 ,X¥, + 15(2) 46,XY(30)
With CM
24 24
24 48 24 24 24 48
24 24
24 48 24 48
24 24 48 24
Culture term (h)
Abbreviations: ALL, acute lymphocytic leukemia; AML, acute myelogenous leukemia; CML, chronic myelogenous leukemia; MDS, myelodysplasfic syndrome,
in relapse after bone marrow transplant.
60/F 85/F
9 10
was
1 day/F 29/M 37/M 16/M
5 6 7 8"
a Patient
2/M 20/M 78/F 46/M
Age/sex
Karyotype (no. of cells)
C y t o g e n e t i c S t u d i e s i n 18 P a t i e n t s w i t h H e m a t o l o g i c M a l i g n a n c i e s
1 2 3 4
Patient no.
Table 1
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G. Sun et al, showed different results w h e n cells were cultured with or without CM. Although the qualities of metaphases from both normal and abnormal cells were i m p r o v e d in these patients, the percentage of karyotypically normal cells increased w h e n CM was used. These data i m p l y that analysis of metaphases only from cell cultured w i t h CM, may miss cytogenetic abnormalities if the leukemia clone constitutes a small proportion of the d i v i d i n g cells. For example, patient 5 was a newborn with Down s y n d r o m e w i t h suspected acute leukemia, a situation difficult to distinguish from a transient l e u k e m o i d reaction. Control cultures showed a small proportion of cells w i t h an a d d i t i o n a l abnormality, a change suggestive of leukemia. Cytogenetic analysis w i t h CM s h o w e d only trisomy 21. The patient eventually died of leukemia. In patient 18, analysis of cultures of cells only with CM w o u l d have missed the abnormal clone. The decreased frequency of abnormal metaphases with CM containing hematopoietic growth factor(s) was not unanticipated. Bone marrow from persons with acute leukemia is often a mixture of normal and leukemia cells. Both p o p u l a t i o n s have low mitotic indexes. Normal cells consistently proliferate w h e n exposed to h e m a t o p o i e t i c growth factors. The response of leukemia cells is variable; often, mitotic indexes do not increase. Consequently, mixtures of normal and leukemia cells cultured w i t h hematopoietic growth factors often result in an increased proportion of normal metaphases. Neither can we exclude the possibility that the CM contains a factor(s) that inhibits proliferation of leukemia cells; this is unlikely in view of increased leukemia colony formation w h e n CM was added. This study underscores the c o m p l e x i t y of using h e m a t o p o i e t i c growth factors to improve results of cytogenetic analyses in leukemia. A l t h o u g h the n u m b e r and quality of metaphases may be increased, the proportion of abnormal cells m a y either be decreased or become undetectable. Thus, careful qualitative evaluation of the effects of any cell growth stimulating factor(s) should be undertaken before they are used clinically.
This study was supported in part by US Public Health Service Grants No. CA26038, CA32737, CA33938, and CA23175. G.S. is a fellow of the World Health Organization. R.P.G. is the Wald Foundation Scholar in Biomedical Communications.
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