Scand J Haematol (1975) 15, 201-206
Karyotypes in Infectious Mononucleosis P. PHILIP, M.D., P. ERNST, M.D.
&
G. LANGE WANTZIN, M.D.
Division of Haematology (Chief, Sven-Aage Killmann), Department of Medicine A , Rigshospitalet, University Hospital o f Copenhagen and University Clinic for Infectious Diseases (Chief, Viggo Faber), Blegdamshospitalet, Copenhagen, Denmark
Using a trypsin-Giemsa banding procedure, chromosome analysis was performed on blood from 21 consecutive patients hospitalized for infectious mononucleosis. Mitoses were harvested after 2 and 24 h in vitro incubation without PHA and after 48 h with PHA. No abnormalities were seen. Key words: infectious mononucleosis - karyotypes
Accepted for publication August 10, 1975 Correspondence to: Dr. Preben Philip, Department of Medicine A, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark
Neoplastic growth is often associated with chromosomal abnormalities but their possible pathogenetic role and significance for the course of neoplastic disease is still obscure. So far the Ph' in chronic myeloid leukaemia and a missing G group chromosome in most meningeomas are the only abnormalities consistently associated with well characterized pathologic pictures in man (Rowley 1974). The development of the banding preparation techniques made possible the recognition of each human chromosome pair (Caspersson et a1 1970). These methods are difficult to apply to human tumour cells without in vitro cultivation. While rather few reports on chromosome changes in solid tumours have been reported, the myeloproliferative dis-
eases have been quite extensively studied and several non-random chromosome abnormalities described (Philip 1975, Rowley 1975). Whether these can be correlated to the course of disease remains yet to be clarified. Mitoses are occasionally found in routine blood smears from patients with infectious mononucleosis (IM) (Sundberg 1960, Carter 1965, Cooper et a1 1967) but extremely rarely in peripheral blood from normal individuals. IM is a self limited lymphoprolliferative disorder with marked proliferation of lymphoid tissue which can closely resemble malignant lymphoma on pathological examination (Lukes et a1 1969, Salvador et a1 1971). Thus karyotype studies of the dividing cells in blood from IM pa-
202
P. PHILIP, P. ERNST & G. LANCE WANTZIN
tients would seem to be of considerable was added after 22 h incubation at 37O C and the mitoses were harvested as described 2 h later. interest. c) Blood cells were cultured for 48 h with P H A After incubation of IM leucocytes with and harvested as described. colchicine it is possible to collect mitoses Air dried spreads were prepared according to for chrolmosomal investigation. Only few the trypsin method introduced by Seabright studies of this kind have been reported. (1971). Karyograms were made from microphotoCarter (1965) found no abnormalities using graphs on Agfa-Ortho-25-Professional film, the a conventional cytogenetic technique. How- chromosomes being classified according to the convention given by the Paris Conference (1971). ever, the more recent banding methods have At least 12 spreads were examined from each provided a cytogenetic tool by which it is sample. sometimes possible to detect chromosomal rearrangements which would otherwise have RESULTS remained undetected (Milligan & Garson 1974, Mitelman & Brandt 1974). To the Mitoses were found in preparations from knowledge of the authors these methods 14 of the patients. These are listed in have not before been applied to the dividing Table I. Mitoses were found in only 4 cells of peripheral blood from IM patients. cases in the cultures which were not PHA We report here the chromosomal findings stimulated. Since this study is essentially in blood cells from a consecutive series of qualitative and designed to investigate IM patients using a trypsin-Giemsa banding whether or not chromosomal abnormalities technique. occur in the acute stage of IM, no attempts have been made to register mitotic indices. The results are given in Table I. No abnormalities which could not be attributed to MATERIALS AND METHODS chromosome loss during preparation were 21 consecutive patients hospitalized for IM were found. investigated. The patients, aged 7 t o 32 years, showed typical clinical signs of IM with positive Paul-Bunnell test and differential test. Chromosome analysis was performed on peripheral blood cells. Three different procedures were used. a) Direct method. In order to minimize the influence of the capability of in vitro survival, mitoses were harvested after 2 h in vitro incubation. 0.5 ml blood was added to 10 ml medium Fib 41 B (Fibiger Laboratory, Copenhagen) with 20 % foetal calf serum, 6 i.u./ml heparin and 0.05 p g h l colchicine. After 2 h at 37O C the cells were transferred to 0.015 M KCL for 20 min as a hypotonic treatment and subsequently fixed in methanol : acetic acid 3 : 1. b) 24 h in vitro incubation. In order t o harvest at the time of maximal mitotic rate (MacKinney 1965) blood cells were incubated for 24 h in vitro. 0.5 ml blood was added t o 10 ml medium as described above but without colchicine. Colchicine
DISCUSSION
It has recently been shown, that some carcinogenic agents such as 7,12-dimethylbenz(a)anthracene (DMBA) and certain viruses are associated with specific chromosomal abnormalities. Thus DMBA may give rise to either sarcoma or leukaemia in rats according to the mode of administration, but in both instances the same type of chromosomal deviation is found, which again is different from the abnormalities seen in Roussarcoma-virus induced sarcoma although this tumour is histologically indistinguishable from the DMBA induced sarcoma (Sugiyama et a1 1967, MacDougall 1971,
203
KARYOTYPES IN INFECTIOUS MONONUCLEOSIS TABLE I Banding analysed mitoses from peripheral blood cells o f 14 patients with infectious mononucleosis in whom mitotic figures could be found N o abnormalities were seen In vitro time
Patient FL
Ls JH MW HP ES PE
vu
KS HN SJ MR CM JO
Total no.
2h -PHA
I
Sex M ‘ M F F M F M F F F M F M M
I
24 h -PHA
48h
+PHA
No. of mitoses
No. of mitoses
11’ 0 0 5 0 0 0 0 0 0 0 0
5 9 0 0 0 0 1 0 0 0 0 0
0
0
2 12 7 10 3 4 7 2 10 4 5
0
0
7
16
15
80
No. of mitoses 5 0
1
Days from onset of symptoms to blood sample 12 13 27 17 13 7 14 10 5
7 7 13
11 15
* One mitosis missed a chromosome no. 20, probably lost during preparation. Mitelman & Levan 1972, Mitelman et a1 1972db). These studies have lead to the hypothesis, that each oncogenic agent induces specific karyotypic changes irrespective of the cell transformed (Mitelman & Levan 1972). At present, the general consensus is that Epstein-Barr virus (EBV) is the common etiologic agent to Burkitt’s lymphoma (BL) and IM (Henle & Henle 1974, Pattengale et a1 1974, Carter 1975). Several reports concerning the chromosome complement of BL-tumour cells (Jacobs et a1 1963, Stewart et a1 1965), and EBV-containing-cell lines derived from BL (Kohn et a1 1967, Huang et a1 1970, Zajac & Kohn 1970, Macek & Benyesh-Melnick 1972), IM lymphocytes (Diehl et al 1968, Huang et a1 1970, Steel et a1 1971, Macek & Benyesh-Melnick 1972), and from in vitro EBV-infected cells (Gerber et a1 1969, Whang-Peng et a1 1970,
Wahren et a1 1972, Amano et a1 1973) are available. The so called ‘C marker’ which is a C group chromosome, presumably a no. 10, with a pronounced secondary constriction subterminally located on the long arms is often mentioned but opinions differ as to the specificity of this abnormality (Kohn 1971, Steel et a1 1971, Whang-Peng 1971) in EBV infection. Using a banding technique, Manolov & Manolova (1972) described a 14q+ marker chromosome which they found present in 5 of 6 BL tumour biopsies and 7 of 9 tumour cell lines. The consistency of this marker has later been confirmed (Petit et a1 1972, Jarvis et a1 1974). It is possible, that the 14q+ marker will prove specific to EBV infection, but it should be noticed, that a similar marker has been reported in diseases not yet proven to be related to EBV such as multiple myeloma (Wurster-Hill et al 1973, Philip
204
P. PHILIP, P. ERNST
&,
1975) and Hodgkin’s disease (Reeves 1973) and that it was not found in any of the 31 established cell lines derived from patients with IM (Jarvis et a1 1974). However, the absence of chromosomal changes in IM cell lines hardly constitutes evidence against the hypothesis that the 14q+ marker is EBV specific. Even if the karyotyped cells carry the EBV genome this does not necessarily have to express itself by the marker chromosome since it appears that the EBV DNA is present in the peripheral cells of IM patients as a different type of infection from that shown by the virus in the malignantly transformed cells of BL (Rickinson et a1 1974). For the same reasons the negative results of the present study cannot be taken as evidence against the specificity of the 14q+ marker to EBV. Moreover, while BL cells and BL and IM cell lines bear B cell characteristics the atypical lymphoid cells in blood from IM patients comprise a mixed population of B and T cells (Gupta 1974, Carter 1975). Probably only B cells are infected with EBV (Jondal & Klein 1973, Yata et a1 1975) and it can not be ruled out that only T cells provided mitoses for analysis. PHA is a specific T cell stimulant. The PHA stimulated serie was included having in mind the interesting and somewhat surprising results of Dartnall et a1 (1973) who found in PHA stimulated samples from patients with multiple myeloma - a definite B cell disease - several mitoses with large acrocentric chromosomes about the size of the long arms of a normal chromosome 2. These may have originated from a no. 14 but banding analysis was not performed. The PHA response is markedly depressed in IM (Sheldon et a1 1973). Also in this study, rather few mitoses were seen in the PHA stimulated samples compared with
G. LANGE WANTZIN
cytogenetic routine preparations from normal individuals. No aberrations were found in any of the mitoses investigated.
ACKNOWLEDGEMENT This investigation was supported by grants from Ferdn. Hindsgaul’s Foundation, P. Carl Petersen’s Foundation, Anders Hasselbalch’s Fund against Leukaemia, the Danish Medical Research Council, the Danish Cancer Society and the Danish Foundation for the Advancement of Medical Science. We thank Mrs. Tonni Rem for skilful technical assistance.
REFERENCES Amano K, Shigeta S, Awano I & Hinuma Y (1973) Chromosomal aberrations of a lymphoblastoid cell line by experimental infection with EpsteinBarr virus. Gann 64, 309-11. Carter R L (1965) The mitotic activity of circulating atypical mononuclear cells in infectious mononucleosis. Blood 26, 579-86. Carter R L (1975) Infectious mononucleosis: Model for self-limiting lymphoproliferation. Lancet i, 846-49. Caspersson T, Zech L, Johansson C & Modest E J (1970) Identification of human chromosomes by DNA-binding fluorescent agents. Chromosoma (Berlin) 30, 215-27. Cooper E H , Hale A J &, Milton J D (1967) The proliferation of infectious mononucleosis lymphocytes in vitro. Acta Haematol (Basel) 38, 19-33. Dartnall J A, Mundy G R & Baikie A G (1973) Cytogenetic studies in myeloma. Blood 42, 22939. Diehl V, Henle G, Henle W & Kohn G (1968) Demonstration of a herpes group virus in cultures of peripheral leukocytes from patients with infectious mononucleosis. J Vi‘irol 2, 66369. Gerber G, Whang-Peng J & Monroe J H (1969) Transformation and chromosome changes induced by Epstein-Barr virus in normal human leukocyte cultures. Proc Natl Acad Sci USA 63, 740-47. Gupta S (1974) Atypical lymphocytes in infectious
KARYO’IYPES IN INFECTIOUS MONONUCLEOSIS mononucleosis. Lancet i, 1012. Henle W & Henle G (1974) Epstein-Barr virus and human malignancies. Cancer 34, 1368-74. Huang C C, Monowada J, Smith R T & Osunkoya B 0 (1970) Reevaluation of relationship between C chromosome marker and Epstein-Barr virus: Chromosome and immunofluorescence analyses of 16 human hematopoietic cell lines. J Natl Cancer Znst 45, 815-29. Jacobs P A, Tough I M & Wright D H (1963) Cytogenetic studies in Burkitt’s lymphoma. Lancet ii, 1144-46. Jarvis J E, Ball G, Rickinson A B & Epstein M A (1974) Cytogenetic studies on human lymphoblastoid cell lines from Burkitt’s lymphomas and other sources. Znt J Cancer 14, 716-21. Jondal M & Klein G (1973) Surface markers on human B and T lymphocytes. J Exp M e d 138, 1365-78. Kohn G, Mellman W J, Moorhead P S, Loftus J & Henle G (1967) Involvement of C group chromosomes in five Burkitt lymphoma cell linm. J Natl Cancer Inst 38, 209-22. Kohn G (1971) Letter. J Nut1 Cancer Znst 46, 68586. Lukes R J, Tindle B H & Parker J W (1969) ReedSternberg-like cells in infectious mononucleosis. Lancet ii, 1003-04. Macek M & Benyesh-Melnick M (1972) Chromosomal analysis of lymphoblastoid cell lines from patients with leukaemia, infectious mononucleosis, or Burkitt lymphoma. Neoplasma 19, 51-56. MacDougall J K (1971) Adenovirus-induced chromosome aberrations in human cells. J Genet Virol 12, 43-51. MacKinney A A (1965) Tissue culture of cells already in DNA synthesis from patients with infectious mononucleosis. Blood 26, 36-48. Manolov G & Manolova Y (1972) Marker band in one chromosome 14 from Burkitt lymphomas. Nature 237, 33-34. Milligan W J & Garson 0 M (1974) Giemsa banding of ‘normal’ leukaemic chromosomes: a preliminary report. 6, 143-46. Mitelman F & Brandt L (1974) Chromosome banding pattern in acute myeloid leukaemia. Scand J Haematol 13, 321-30. Mitelman F & Levan G (1972) The chromosomes of primary 7,12-dimethylbenz(a)anthracene-induced rat sarcomas. Hereditas 71, 325-34.
205
Mitelman F, Mark J & Levan G (1972a) Chromosomes of six primary sarcomas induced in the Chinese hamster by 7,12-dimethylbenz(a)anthracene. Hereditas 72, 311-18. Mitelman F,Mark J, Levan G & Levan A (1972b) Tumor etiology and chromosome pattern. Science 176, 1340-41. Paris Conference 1971. Standardization in Human Cytogenetics. Birth Defects: Original Article Series VIII: 7, 1972. The National Foundation, New York. Pattengale P K, Smith R W & Perlin E (1974) Atypical lymphocytes in acute infectious mononucleosis. N Engl J M e d 291, 1145-48. Petit P, Verhest A, van der Bilt F L & Jongsma A (1972) The chromosomes of the EB virus-positive Burkitt cell line P3J.HRlK studied by the fluorescent staining technique. Pathol Eur 7 , 17-21. Philip P (1975) Trisomy 8 in acute myeloid leukaemia. Scand J Haematol 14, 140-47. Philip P (1975) Marker chromosome 14q+ in multiple myeloma. Hereditas 80, 155-56. Reeves B R (1973) Cytogenetics of malignant lymphomas. Humangenetik 20, 231-50. Rickinson A B, Jervis J E, Crawford D H & Epstein M A (1974) Observations on the type of infection by Epstein-Barr virus in peripheral lymphoid cells of patients with infectious mononucleosis. Znt J Cancer 14, 704-15. Rowley J D (1974) D o human tumors show a chromosome pattern specific for each etiologic agent? J Natl Cancer Inst 52, 315-19. Rowley J D (1975) Nonrandom chromosomal abnormalities in hematologic disorders of man. Proc Natl Acad Sci USA 12, 152-56. Salvador A H, Harrison E G & Kyle R A (1971) Lymphadenopathy due to infectious mononucleosis: Its confusion with malignant lymphoma. Cancer 27, 1029-40. Seabright M (1971) A rapid banding technique for human chromosomes. Lancet ii, 971-72. Sheldon P J, Papamichail M, Hemsted E H & Holborrow E J (1973) Thymic origin of atypical lymphoid cells in i nfectious mononucleosis. Lancet i, 1153-55. Steel C M, McBeath S & O’Riordan M L (1971) Human lymphoblastoid cell lines. 11. Cytogenetic studies. J Natl Cancer Znst 47, 1203-14. Stewart S E, Lovelace E, Whang J J & Ngu V A (1965) Burkitt tumor: Tissue culture, cyto-
206
P. PHILIP, P. ERNST & G. LANGE WANTZIN
genetic and virus studies. J Nut1 Cancer Inst 34, 319-27. Sugiyama T, Kurita Y & Nishizuka Y (1967) Chromosome abnormality in rat leukemia induced by 7,12-dimetylbenz(a)anthracene. Science 158, 1058-59. Sundberg R D (1960) Lymphocytes: Origin, structure, and interrelationships. In J W Rebuck (ed) The lymphocyte and lymphocytic tissue, pp 121. Hoeber, New York. Wahren B, Lampert F & Goetz 0 (1972) Herpes viruses and chromosomal alterations seen with light and whole mount electron microscopy. Exp Cell Res 75, 271-74. Whang-Peng J (1971) Letter. J Nut1 Cancer Inst 46, 686-87. Whang-Peng J, Gerber P & Knutsen T (1970) So-
called C marker chromosome and EpsteinBarr virus. J Natl Cancer Inst 45, 831-39. Wurster-Hill D H, McIntyre 0 R, Cornwell 111 G G & Maurer L H (1973) Marker-chromosome 14 in multiple myeloma and plasma-cell leukaemia. Lancet ii, 1031. Yata J, Desgranges C, Nakagawa T, Favre M C & de-Th6 G (1975) Lymphoblastoid transformation and kinetics of appearance of viral nuclear antigen (EBNA) in cord-blood lymphocytes infected by Epstein-Barr virus. Int J Cancer 15, 377-84. Zajac B A & Kohn G (1970) Epstein-Barr virus antigens, marker chromosome, and interferon production in clones derived from cultured Burkitt tumor cells. J Nut1 Cancer Inst 45, 399406.
Karyotypes in Infectious Mononucleosis P. PHILIP, M.D., P. ERNST, M.D.
&
G. LANGE WANTZIN, M.D.
Division of Haematology (Chief, Sven-Aage Killmann), Department of Medicine A , Rigshospitalet, University Hospital o f Copenhagen and University Clinic for Infectious Diseases (Chief, Viggo Faber), Blegdamshospitalet, Copenhagen, Denmark
Using a trypsin-Giemsa banding procedure, chromosome analysis was performed on blood from 21 consecutive patients hospitalized for infectious mononucleosis. Mitoses were harvested after 2 and 24 h in vitro incubation without PHA and after 48 h with PHA. No abnormalities were seen. Key words: infectious mononucleosis - karyotypes
Accepted for publication August 10, 1975 Correspondence to: Dr. Preben Philip, Department of Medicine A, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark
Neoplastic growth is often associated with chromosomal abnormalities but their possible pathogenetic role and significance for the course of neoplastic disease is still obscure. So far the Ph' in chronic myeloid leukaemia and a missing G group chromosome in most meningeomas are the only abnormalities consistently associated with well characterized pathologic pictures in man (Rowley 1974). The development of the banding preparation techniques made possible the recognition of each human chromosome pair (Caspersson et a1 1970). These methods are difficult to apply to human tumour cells without in vitro cultivation. While rather few reports on chromosome changes in solid tumours have been reported, the myeloproliferative dis-
eases have been quite extensively studied and several non-random chromosome abnormalities described (Philip 1975, Rowley 1975). Whether these can be correlated to the course of disease remains yet to be clarified. Mitoses are occasionally found in routine blood smears from patients with infectious mononucleosis (IM) (Sundberg 1960, Carter 1965, Cooper et a1 1967) but extremely rarely in peripheral blood from normal individuals. IM is a self limited lymphoprolliferative disorder with marked proliferation of lymphoid tissue which can closely resemble malignant lymphoma on pathological examination (Lukes et a1 1969, Salvador et a1 1971). Thus karyotype studies of the dividing cells in blood from IM pa-
202
P. PHILIP, P. ERNST & G. LANCE WANTZIN
tients would seem to be of considerable was added after 22 h incubation at 37O C and the mitoses were harvested as described 2 h later. interest. c) Blood cells were cultured for 48 h with P H A After incubation of IM leucocytes with and harvested as described. colchicine it is possible to collect mitoses Air dried spreads were prepared according to for chrolmosomal investigation. Only few the trypsin method introduced by Seabright studies of this kind have been reported. (1971). Karyograms were made from microphotoCarter (1965) found no abnormalities using graphs on Agfa-Ortho-25-Professional film, the a conventional cytogenetic technique. How- chromosomes being classified according to the convention given by the Paris Conference (1971). ever, the more recent banding methods have At least 12 spreads were examined from each provided a cytogenetic tool by which it is sample. sometimes possible to detect chromosomal rearrangements which would otherwise have RESULTS remained undetected (Milligan & Garson 1974, Mitelman & Brandt 1974). To the Mitoses were found in preparations from knowledge of the authors these methods 14 of the patients. These are listed in have not before been applied to the dividing Table I. Mitoses were found in only 4 cells of peripheral blood from IM patients. cases in the cultures which were not PHA We report here the chromosomal findings stimulated. Since this study is essentially in blood cells from a consecutive series of qualitative and designed to investigate IM patients using a trypsin-Giemsa banding whether or not chromosomal abnormalities technique. occur in the acute stage of IM, no attempts have been made to register mitotic indices. The results are given in Table I. No abnormalities which could not be attributed to MATERIALS AND METHODS chromosome loss during preparation were 21 consecutive patients hospitalized for IM were found. investigated. The patients, aged 7 t o 32 years, showed typical clinical signs of IM with positive Paul-Bunnell test and differential test. Chromosome analysis was performed on peripheral blood cells. Three different procedures were used. a) Direct method. In order to minimize the influence of the capability of in vitro survival, mitoses were harvested after 2 h in vitro incubation. 0.5 ml blood was added to 10 ml medium Fib 41 B (Fibiger Laboratory, Copenhagen) with 20 % foetal calf serum, 6 i.u./ml heparin and 0.05 p g h l colchicine. After 2 h at 37O C the cells were transferred to 0.015 M KCL for 20 min as a hypotonic treatment and subsequently fixed in methanol : acetic acid 3 : 1. b) 24 h in vitro incubation. In order t o harvest at the time of maximal mitotic rate (MacKinney 1965) blood cells were incubated for 24 h in vitro. 0.5 ml blood was added t o 10 ml medium as described above but without colchicine. Colchicine
DISCUSSION
It has recently been shown, that some carcinogenic agents such as 7,12-dimethylbenz(a)anthracene (DMBA) and certain viruses are associated with specific chromosomal abnormalities. Thus DMBA may give rise to either sarcoma or leukaemia in rats according to the mode of administration, but in both instances the same type of chromosomal deviation is found, which again is different from the abnormalities seen in Roussarcoma-virus induced sarcoma although this tumour is histologically indistinguishable from the DMBA induced sarcoma (Sugiyama et a1 1967, MacDougall 1971,
203
KARYOTYPES IN INFECTIOUS MONONUCLEOSIS TABLE I Banding analysed mitoses from peripheral blood cells o f 14 patients with infectious mononucleosis in whom mitotic figures could be found N o abnormalities were seen In vitro time
Patient FL
Ls JH MW HP ES PE
vu
KS HN SJ MR CM JO
Total no.
2h -PHA
I
Sex M ‘ M F F M F M F F F M F M M
I
24 h -PHA
48h
+PHA
No. of mitoses
No. of mitoses
11’ 0 0 5 0 0 0 0 0 0 0 0
5 9 0 0 0 0 1 0 0 0 0 0
0
0
2 12 7 10 3 4 7 2 10 4 5
0
0
7
16
15
80
No. of mitoses 5 0
1
Days from onset of symptoms to blood sample 12 13 27 17 13 7 14 10 5
7 7 13
11 15
* One mitosis missed a chromosome no. 20, probably lost during preparation. Mitelman & Levan 1972, Mitelman et a1 1972db). These studies have lead to the hypothesis, that each oncogenic agent induces specific karyotypic changes irrespective of the cell transformed (Mitelman & Levan 1972). At present, the general consensus is that Epstein-Barr virus (EBV) is the common etiologic agent to Burkitt’s lymphoma (BL) and IM (Henle & Henle 1974, Pattengale et a1 1974, Carter 1975). Several reports concerning the chromosome complement of BL-tumour cells (Jacobs et a1 1963, Stewart et a1 1965), and EBV-containing-cell lines derived from BL (Kohn et a1 1967, Huang et a1 1970, Zajac & Kohn 1970, Macek & Benyesh-Melnick 1972), IM lymphocytes (Diehl et al 1968, Huang et a1 1970, Steel et a1 1971, Macek & Benyesh-Melnick 1972), and from in vitro EBV-infected cells (Gerber et a1 1969, Whang-Peng et a1 1970,
Wahren et a1 1972, Amano et a1 1973) are available. The so called ‘C marker’ which is a C group chromosome, presumably a no. 10, with a pronounced secondary constriction subterminally located on the long arms is often mentioned but opinions differ as to the specificity of this abnormality (Kohn 1971, Steel et a1 1971, Whang-Peng 1971) in EBV infection. Using a banding technique, Manolov & Manolova (1972) described a 14q+ marker chromosome which they found present in 5 of 6 BL tumour biopsies and 7 of 9 tumour cell lines. The consistency of this marker has later been confirmed (Petit et a1 1972, Jarvis et a1 1974). It is possible, that the 14q+ marker will prove specific to EBV infection, but it should be noticed, that a similar marker has been reported in diseases not yet proven to be related to EBV such as multiple myeloma (Wurster-Hill et al 1973, Philip
204
P. PHILIP, P. ERNST
&,
1975) and Hodgkin’s disease (Reeves 1973) and that it was not found in any of the 31 established cell lines derived from patients with IM (Jarvis et a1 1974). However, the absence of chromosomal changes in IM cell lines hardly constitutes evidence against the hypothesis that the 14q+ marker is EBV specific. Even if the karyotyped cells carry the EBV genome this does not necessarily have to express itself by the marker chromosome since it appears that the EBV DNA is present in the peripheral cells of IM patients as a different type of infection from that shown by the virus in the malignantly transformed cells of BL (Rickinson et a1 1974). For the same reasons the negative results of the present study cannot be taken as evidence against the specificity of the 14q+ marker to EBV. Moreover, while BL cells and BL and IM cell lines bear B cell characteristics the atypical lymphoid cells in blood from IM patients comprise a mixed population of B and T cells (Gupta 1974, Carter 1975). Probably only B cells are infected with EBV (Jondal & Klein 1973, Yata et a1 1975) and it can not be ruled out that only T cells provided mitoses for analysis. PHA is a specific T cell stimulant. The PHA stimulated serie was included having in mind the interesting and somewhat surprising results of Dartnall et a1 (1973) who found in PHA stimulated samples from patients with multiple myeloma - a definite B cell disease - several mitoses with large acrocentric chromosomes about the size of the long arms of a normal chromosome 2. These may have originated from a no. 14 but banding analysis was not performed. The PHA response is markedly depressed in IM (Sheldon et a1 1973). Also in this study, rather few mitoses were seen in the PHA stimulated samples compared with
G. LANGE WANTZIN
cytogenetic routine preparations from normal individuals. No aberrations were found in any of the mitoses investigated.
ACKNOWLEDGEMENT This investigation was supported by grants from Ferdn. Hindsgaul’s Foundation, P. Carl Petersen’s Foundation, Anders Hasselbalch’s Fund against Leukaemia, the Danish Medical Research Council, the Danish Cancer Society and the Danish Foundation for the Advancement of Medical Science. We thank Mrs. Tonni Rem for skilful technical assistance.
REFERENCES Amano K, Shigeta S, Awano I & Hinuma Y (1973) Chromosomal aberrations of a lymphoblastoid cell line by experimental infection with EpsteinBarr virus. Gann 64, 309-11. Carter R L (1965) The mitotic activity of circulating atypical mononuclear cells in infectious mononucleosis. Blood 26, 579-86. Carter R L (1975) Infectious mononucleosis: Model for self-limiting lymphoproliferation. Lancet i, 846-49. Caspersson T, Zech L, Johansson C & Modest E J (1970) Identification of human chromosomes by DNA-binding fluorescent agents. Chromosoma (Berlin) 30, 215-27. Cooper E H , Hale A J &, Milton J D (1967) The proliferation of infectious mononucleosis lymphocytes in vitro. Acta Haematol (Basel) 38, 19-33. Dartnall J A, Mundy G R & Baikie A G (1973) Cytogenetic studies in myeloma. Blood 42, 22939. Diehl V, Henle G, Henle W & Kohn G (1968) Demonstration of a herpes group virus in cultures of peripheral leukocytes from patients with infectious mononucleosis. J Vi‘irol 2, 66369. Gerber G, Whang-Peng J & Monroe J H (1969) Transformation and chromosome changes induced by Epstein-Barr virus in normal human leukocyte cultures. Proc Natl Acad Sci USA 63, 740-47. Gupta S (1974) Atypical lymphocytes in infectious
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205
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