Nasopharyngeal carcinoma and Burkitt's lymphoma in a Canadian family. I. HLA typing, EBV antibodies and serum immunoglobulins J.H. JONCAS,*t. MD, PH D E. Rioux,§ MD, PH D J.P. WASTIAUX,* MD; M. LEYRITZ,* M Sc; L. ROBILLARD,* B Sc; J. MENEZES,t. DVM, PH D
Two nasopharyngeal carcinomas of the lymphoepithelioma type and two Burkitt's lymphomas with the characteristic histopathologic features developed in three siblings and one first-degree cousin in a large French-Canadian family. Epstein-Barr virus antibody titres in the two lymphoepithelioma cases but not in the Burkitt's lymphoma cases were, as expected, greatly elevated- HIA typing of the family members failed to disclose HLA antigens A2 and B Sin-2, which have been associated with lymphoepithelioma in Asia. The occurrence, however, of a plasmacytoma in one other first-degree cousin and low serum IgA values in several siblings and cousins suggests the possibility of a genetically determined predisposing B-cell dysfunction in the development of these tumours. Deux carcinomes nasopharyngiens de type lymphoepitheliome et deux lymphomes de Burkitt ayant toutes les caracteristiques physiopathologiques sont apparus chez trois membres d'une fratrie et un cousin au premier degr6 au sein d'une famille canadiennefran9aise nombreuse. Tel qu'on s'y attendait, dans les deux cas de lympho6pitheliome les titres en anticorps antivirus Epstein-Barr etaient eleves, mais non dans les cas de lymphome de Burkitt. La determination du type HLA chez les membres de cette famille n'a pu mettre en evidence les antig.nes HLA-A2 et B Sin-2, qui ont ete associes au lymphoepitheliome en Asie. L'apparition, toutefois, dun plasmocytome chez un autre cousin du premier degre, et Ia pr6sence de faibles taux s6riques d'lgA chez plusleurs membres d'une fratrie et leurs cousins, suggerent Ia possibilite d'un d.reglement d'ordre g6netique des cellules B, predisposant au developpement de ces tumeurs.
An association has been reported between HLA-A2 and HLA-B Sin-2 histocompatibility antigens and nasopharyngeal carcinoma of the lymphoepithelioma type in Asia.14 In contrast, this relation was not observed in Tunisians.4 Burkitt's lymphoma has not, to date, been associated with any particular HLA histocompatibility antigen.5 The present study of a Canadian family with several cases of nasopharyngeal carcinoma (lymphoepithelioma) and Burkitt's lymphoma was undertaken to determine the importance of a genetic HLA-linked factor in the development of these tumours. Methods and patients
Methods Titres of antibodies to Epstein-Barr virus (EBV) nuclear antigen (EBNA) were measured by the anticomplement immunofluorescence method of Reedman and Klein6 as modified by Henle, Henle and Horwitz.7 Titres of antibodies to EBV early antigen (EBV-EA) were measured by an indirect immunofluorescence test on acetone-fixed Raji cells 6 days following 5-bromodeoxyuridine (BUDR) induction of the antiESNA EBV-EA EBV-VCA IlL-A
gens. Control tests were done with EBV viral capsid antigen (EBV-VCA)positive, EA-negative serum on BUDRactivated Raji cells and VCA-positive, EA-positive serum on normal antigennegative cells. Titres of antibodies to EBV-VCA in the patient's serum were measured by indirect immunofluorescence in two-fold dilutions with the use of the HRlK clone of P3J lymphoblastoid cells as antigen-positive cells and Hyland fluorescein-conjugated goat antiserum to human IgG (heavy and light chains). Raji lymphoblastoid cells were used as antigen-negative control cells. Known positive and negative sera were used as control antisera. HLA typing was done by the microcytotoxicity assay.8 Serum immunoglobulin concentrations were measured by nephelometry using the Technicon AutoAnalyzer 2. Patients The family under study was composed of a mother, 11 living children and two living maternal cousins (Fig. I). The father, three additional siblings and two additional maternal cousins died prior to the study. Patient 1: A 25-year-old woman (N.e.)
ANA 10 320
128 1721
IJt O
LTJ
o
3,32 7,14
6.6.6i.U.ooo
b. IL?
27,,
EBNA EBY-EA
640 160 320 40
EBv-VCA 1230 320
10 10 0 20 2 10 20 0000000 10 80 10 80 40 80 20 1,32 14,17
HL-A
2.32 14,21
26,32 14,21
168 37
MUG) II
80 0 30
20 0 20
28,32 14,21
1,3 7,17
12,14
1,32 14.17
59
QEQO
160 80 40 000 160 10 40
1,3 7,17 IgA 1gM
CL GL) 16y
160 0 160
ALL 67
2,32
1,3 7,17
2,32 12,14 24
30
53
From 8the department of virology, Institut Armand-Frappier, Ville de Laval; tcentre de recherche p.diatrique, H6pital Sainte-Justine, Montr6al; tthe departments of microbiology and immunology and pediatrics, University of Montreal; and §the department of hematology, H6pital de l'Enfant-J.sus, QuEbec
EBNA EBv-EA EBV.VCA ANA NPC BL ALL MM 1
Reprint requests to: Dr J.H. Joncas, Institut Armand-Frappier, 531, boul. des Prairies, CP 100, Laval-des-Rapides, PQ H7N 4Z3
FIG. 1-Results of HLA typing and Epstein-Ban virus (EBV) serology of family under study.
858
Epstein Barr nuclear antigen antihedy titer 0. tit@r< ./2 Epstein Barr virus early antigen antibedy titer 0: titer < l/. Epstein Barr virus viral capsid antigen antibody titer 0. titer< ./S Antinuclear antibady an EBV negative Melt central cells Nasepharyngeal carcinema tlympheepltlseliama Burkitt. lymptmema. IL? Lymplseblastic sarcema 119571 Acute lympheblastic leukemia Multiple myaloma Iplasmocytama IgGI Deceased
CMA JOURNAL/NOVEMBER 6, 1976/VOL. 115
Normal values tinge/el 190 570 - 1920 1gM: 47-147 IgA: 61 - 330
g
10-
Table I-Results of Epstein-Barr virus (EBV) serologic study of patient with nasopharyngeal carcinoma (lymphoepithelioma) Titre of serum antibodies to Time EBV.VCA* EBV-EAI EBNA. 1972 1:80 < 1:5 1:80 1974 1:160 1:20 1:160 January1975 1:320 1:40 1:10 April 1975 1:160 1:10 1:80 *VCA = viral capsid antigen. fEA = early antigen. .EBNA = Epstein-Barr virus nuclear antigen.
those of the two patients with nasopharyngeal carcinoma, but more striking was the absence of EBV-EA antibodies in all relatives of these two patients except the mother (titre, I / 10). Several siblings and cousins were found to have low serum IgA values, although patient I had a normal serum IgA value and a somewhat low serum 1gM value 1 month before she died. Discussion The failure to find HLA antigens A2 and B Sin-2 in the family under study suggests that these histocompatibility antigens are not, at least outside Asia, associated with an unusual susceptibility to nasopharyngeal carcinoma.
The finding of high titres of antibodies to EBNA and EBV-VCA and, in particular, to EBV-EA in the serum of the two patients with nasopharyngeal carcinoma (lymphoepithelioma) agrees with the findings in other reports. Nasopharyngeal carcinoma anywhere in the world is associated with high titres of EBV antibodies, whereas in Burkitt's lymphoma this association appears to be limited to the African tumour. The occurrence of a plasmacytoma in a first-degree cousin and the low serum IgA values in several siblings and cousins suggest the possibility of B-cell dysfunction in the development of these tumours in this family. Provisor and colleagues9 have reported progressive hypo- and agammaglobulinemia in three siblings following infec-
S
I 9
*
tious mononucleosis; the EBV infection most likely uncovered an underlying B-cell dysfunction, which led to againmaglobulinemia.'0 Purtilo and colleagues11 have described a lymphoproliferative disease associated with combined variable immunodeficiency in 6 of 18 boys in the Duncan kindred. Infectious mononucleosis occurred during or preceding terminal events in three cousins, and lymphomas of the ileum and central nervous system were observed in two half-brothers. In certain genetically predisposed hosts, therefore, such as members of the family we have studied, B-cell dysfunction could possibly lead to hypogammaglobulinemia or B-cell lymphomas or both as late sequelae to EBV infection, since the main if not the only target cell of this virus is the B-lymphocyte. Experiments are in progress to document further the B-cell dysfunction, particularly in relation to the EBV infection in the family under study. The postulated genetic defect in this family would probably be autosomal and not X-linked as in Duncan's disease. The authors are grateful to Dr. Malcolm Simons for supplying us with the Sin-2 antiserum. This work was supported in part by a Medical Research Council of Canada grant (no. 5518) and a grant from the Department of Education of the Province of Quebec (FCAC). References
FIG 3A-Section of ovarian mass in padent 2 characteristic "starry sky" appearauce due to scattered histlocytes in mass of lymphoid tumour cells (HPS; x300)
FIG. 3B-Higher magnification of section in Fig. 3A (x500).
1. Su.ior..s Mi, DAY NE, WEE GB, et al: Nasopharyngeal carcinoma. V. Immunogenetic studies of Southeast Asian ethnic groups with high and low risk for the tumour. Cancer Res 34: 1192, 1974 2. SIMoNs Mi, WEE GB, CHAN SH, et a!: Probable identification of an HL-A second-locus antigen associated with a high risk of nasopharyngeal carcinoma. Lancet 1: 142, 1975 3. Sn.sor..s MJ, WEE GB, DAY NE, et al: Immunogenetic aspects of nasopharyngeal carcinoma. I. Differences in HL-A antigen profiles between patients and control groups. mt / Cancer 13: 122, 1974 4. BETUEL H, CAMOUN M, COLOMBANI J, et al:
The relationship between nasopharyngeal carcinoma and the HL-A system among Tunisians. mt i Cancer 16: 249, 1975 5. DAussvr J, Hoiis J: Some contributions of the HL-A complex to the genetics of human
diseases. Transplant Rev 22: 44, 1975 6. REEDMAN M, KLEIN G: Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. mt i Cancer 11: 499, 1973 7. HENLE G, HENLE W, HoRwITz CA: Antibodies to Epstein-Barr virus-associated nuclear antigen in infectious mononucleosis. J infect Dis 130: 231, 1974 8. TERASAKI P1, MCCLELLAND iD: Micro droplet
assay of human serum cytotoxins. Nature 204: 998, 1964 9. PIioviso. AJ, IAcouNE JJ, CHILCOTE RR, et al: Acquired agammaglobulinemia after a life-threatening illness with clinical and laboratory features of infectious mononucleosis in three related male children. N Engli Med 293: 62, 1975 10. NISSENBLATT Mi: Immunologic sequelse of infectious mononucleosis. Ibid, p 668 11. PURTILO DT, CAssEL CK, YANG iPS, Ct a!:
FIG. 4-Section of retropentoneal tumour in patient 3: appearance compatible with diagnosis of Burkiff s lyinphoma, American type (lIPS; x500).
860 CMA JOURNAL/NOVEMBER 6, 1976/VOL. 115
X-linked recessive progressive combined variable immunodeficiency (Duncan's disease). Lancet 1: 935, 1975
Two nasopharyngeal carcinomas of the lymphoepithelioma type and two Burkitt's lymphomas with the characteristic histopathologic features developed in three siblings and one first-degree cousin in a large French-Canadian family. Epstein-Barr virus antibody titres in the two lymphoepithelioma cases but not in the Burkitt's lymphoma cases were, as expected, greatly elevated- HIA typing of the family members failed to disclose HLA antigens A2 and B Sin-2, which have been associated with lymphoepithelioma in Asia. The occurrence, however, of a plasmacytoma in one other first-degree cousin and low serum IgA values in several siblings and cousins suggests the possibility of a genetically determined predisposing B-cell dysfunction in the development of these tumours. Deux carcinomes nasopharyngiens de type lymphoepitheliome et deux lymphomes de Burkitt ayant toutes les caracteristiques physiopathologiques sont apparus chez trois membres d'une fratrie et un cousin au premier degr6 au sein d'une famille canadiennefran9aise nombreuse. Tel qu'on s'y attendait, dans les deux cas de lympho6pitheliome les titres en anticorps antivirus Epstein-Barr etaient eleves, mais non dans les cas de lymphome de Burkitt. La determination du type HLA chez les membres de cette famille n'a pu mettre en evidence les antig.nes HLA-A2 et B Sin-2, qui ont ete associes au lymphoepitheliome en Asie. L'apparition, toutefois, dun plasmocytome chez un autre cousin du premier degre, et Ia pr6sence de faibles taux s6riques d'lgA chez plusleurs membres d'une fratrie et leurs cousins, suggerent Ia possibilite d'un d.reglement d'ordre g6netique des cellules B, predisposant au developpement de ces tumeurs.
An association has been reported between HLA-A2 and HLA-B Sin-2 histocompatibility antigens and nasopharyngeal carcinoma of the lymphoepithelioma type in Asia.14 In contrast, this relation was not observed in Tunisians.4 Burkitt's lymphoma has not, to date, been associated with any particular HLA histocompatibility antigen.5 The present study of a Canadian family with several cases of nasopharyngeal carcinoma (lymphoepithelioma) and Burkitt's lymphoma was undertaken to determine the importance of a genetic HLA-linked factor in the development of these tumours. Methods and patients
Methods Titres of antibodies to Epstein-Barr virus (EBV) nuclear antigen (EBNA) were measured by the anticomplement immunofluorescence method of Reedman and Klein6 as modified by Henle, Henle and Horwitz.7 Titres of antibodies to EBV early antigen (EBV-EA) were measured by an indirect immunofluorescence test on acetone-fixed Raji cells 6 days following 5-bromodeoxyuridine (BUDR) induction of the antiESNA EBV-EA EBV-VCA IlL-A
gens. Control tests were done with EBV viral capsid antigen (EBV-VCA)positive, EA-negative serum on BUDRactivated Raji cells and VCA-positive, EA-positive serum on normal antigennegative cells. Titres of antibodies to EBV-VCA in the patient's serum were measured by indirect immunofluorescence in two-fold dilutions with the use of the HRlK clone of P3J lymphoblastoid cells as antigen-positive cells and Hyland fluorescein-conjugated goat antiserum to human IgG (heavy and light chains). Raji lymphoblastoid cells were used as antigen-negative control cells. Known positive and negative sera were used as control antisera. HLA typing was done by the microcytotoxicity assay.8 Serum immunoglobulin concentrations were measured by nephelometry using the Technicon AutoAnalyzer 2. Patients The family under study was composed of a mother, 11 living children and two living maternal cousins (Fig. I). The father, three additional siblings and two additional maternal cousins died prior to the study. Patient 1: A 25-year-old woman (N.e.)
ANA 10 320
128 1721
IJt O
LTJ
o
3,32 7,14
6.6.6i.U.ooo
b. IL?
27,,
EBNA EBY-EA
640 160 320 40
EBv-VCA 1230 320
10 10 0 20 2 10 20 0000000 10 80 10 80 40 80 20 1,32 14,17
HL-A
2.32 14,21
26,32 14,21
168 37
MUG) II
80 0 30
20 0 20
28,32 14,21
1,3 7,17
12,14
1,32 14.17
59
QEQO
160 80 40 000 160 10 40
1,3 7,17 IgA 1gM
CL GL) 16y
160 0 160
ALL 67
2,32
1,3 7,17
2,32 12,14 24
30
53
From 8the department of virology, Institut Armand-Frappier, Ville de Laval; tcentre de recherche p.diatrique, H6pital Sainte-Justine, Montr6al; tthe departments of microbiology and immunology and pediatrics, University of Montreal; and §the department of hematology, H6pital de l'Enfant-J.sus, QuEbec
EBNA EBv-EA EBV.VCA ANA NPC BL ALL MM 1
Reprint requests to: Dr J.H. Joncas, Institut Armand-Frappier, 531, boul. des Prairies, CP 100, Laval-des-Rapides, PQ H7N 4Z3
FIG. 1-Results of HLA typing and Epstein-Ban virus (EBV) serology of family under study.
858
Epstein Barr nuclear antigen antihedy titer 0. tit@r< ./2 Epstein Barr virus early antigen antibedy titer 0: titer < l/. Epstein Barr virus viral capsid antigen antibody titer 0. titer< ./S Antinuclear antibady an EBV negative Melt central cells Nasepharyngeal carcinema tlympheepltlseliama Burkitt. lymptmema. IL? Lymplseblastic sarcema 119571 Acute lympheblastic leukemia Multiple myaloma Iplasmocytama IgGI Deceased
CMA JOURNAL/NOVEMBER 6, 1976/VOL. 115
Normal values tinge/el 190 570 - 1920 1gM: 47-147 IgA: 61 - 330
g
10-
Table I-Results of Epstein-Barr virus (EBV) serologic study of patient with nasopharyngeal carcinoma (lymphoepithelioma) Titre of serum antibodies to Time EBV.VCA* EBV-EAI EBNA. 1972 1:80 < 1:5 1:80 1974 1:160 1:20 1:160 January1975 1:320 1:40 1:10 April 1975 1:160 1:10 1:80 *VCA = viral capsid antigen. fEA = early antigen. .EBNA = Epstein-Barr virus nuclear antigen.
those of the two patients with nasopharyngeal carcinoma, but more striking was the absence of EBV-EA antibodies in all relatives of these two patients except the mother (titre, I / 10). Several siblings and cousins were found to have low serum IgA values, although patient I had a normal serum IgA value and a somewhat low serum 1gM value 1 month before she died. Discussion The failure to find HLA antigens A2 and B Sin-2 in the family under study suggests that these histocompatibility antigens are not, at least outside Asia, associated with an unusual susceptibility to nasopharyngeal carcinoma.
The finding of high titres of antibodies to EBNA and EBV-VCA and, in particular, to EBV-EA in the serum of the two patients with nasopharyngeal carcinoma (lymphoepithelioma) agrees with the findings in other reports. Nasopharyngeal carcinoma anywhere in the world is associated with high titres of EBV antibodies, whereas in Burkitt's lymphoma this association appears to be limited to the African tumour. The occurrence of a plasmacytoma in a first-degree cousin and the low serum IgA values in several siblings and cousins suggest the possibility of B-cell dysfunction in the development of these tumours in this family. Provisor and colleagues9 have reported progressive hypo- and agammaglobulinemia in three siblings following infec-
S
I 9
*
tious mononucleosis; the EBV infection most likely uncovered an underlying B-cell dysfunction, which led to againmaglobulinemia.'0 Purtilo and colleagues11 have described a lymphoproliferative disease associated with combined variable immunodeficiency in 6 of 18 boys in the Duncan kindred. Infectious mononucleosis occurred during or preceding terminal events in three cousins, and lymphomas of the ileum and central nervous system were observed in two half-brothers. In certain genetically predisposed hosts, therefore, such as members of the family we have studied, B-cell dysfunction could possibly lead to hypogammaglobulinemia or B-cell lymphomas or both as late sequelae to EBV infection, since the main if not the only target cell of this virus is the B-lymphocyte. Experiments are in progress to document further the B-cell dysfunction, particularly in relation to the EBV infection in the family under study. The postulated genetic defect in this family would probably be autosomal and not X-linked as in Duncan's disease. The authors are grateful to Dr. Malcolm Simons for supplying us with the Sin-2 antiserum. This work was supported in part by a Medical Research Council of Canada grant (no. 5518) and a grant from the Department of Education of the Province of Quebec (FCAC). References
FIG 3A-Section of ovarian mass in padent 2 characteristic "starry sky" appearauce due to scattered histlocytes in mass of lymphoid tumour cells (HPS; x300)
FIG. 3B-Higher magnification of section in Fig. 3A (x500).
1. Su.ior..s Mi, DAY NE, WEE GB, et al: Nasopharyngeal carcinoma. V. Immunogenetic studies of Southeast Asian ethnic groups with high and low risk for the tumour. Cancer Res 34: 1192, 1974 2. SIMoNs Mi, WEE GB, CHAN SH, et a!: Probable identification of an HL-A second-locus antigen associated with a high risk of nasopharyngeal carcinoma. Lancet 1: 142, 1975 3. Sn.sor..s MJ, WEE GB, DAY NE, et al: Immunogenetic aspects of nasopharyngeal carcinoma. I. Differences in HL-A antigen profiles between patients and control groups. mt / Cancer 13: 122, 1974 4. BETUEL H, CAMOUN M, COLOMBANI J, et al:
The relationship between nasopharyngeal carcinoma and the HL-A system among Tunisians. mt i Cancer 16: 249, 1975 5. DAussvr J, Hoiis J: Some contributions of the HL-A complex to the genetics of human
diseases. Transplant Rev 22: 44, 1975 6. REEDMAN M, KLEIN G: Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. mt i Cancer 11: 499, 1973 7. HENLE G, HENLE W, HoRwITz CA: Antibodies to Epstein-Barr virus-associated nuclear antigen in infectious mononucleosis. J infect Dis 130: 231, 1974 8. TERASAKI P1, MCCLELLAND iD: Micro droplet
assay of human serum cytotoxins. Nature 204: 998, 1964 9. PIioviso. AJ, IAcouNE JJ, CHILCOTE RR, et al: Acquired agammaglobulinemia after a life-threatening illness with clinical and laboratory features of infectious mononucleosis in three related male children. N Engli Med 293: 62, 1975 10. NISSENBLATT Mi: Immunologic sequelse of infectious mononucleosis. Ibid, p 668 11. PURTILO DT, CAssEL CK, YANG iPS, Ct a!:
FIG. 4-Section of retropentoneal tumour in patient 3: appearance compatible with diagnosis of Burkiff s lyinphoma, American type (lIPS; x500).
860 CMA JOURNAL/NOVEMBER 6, 1976/VOL. 115
X-linked recessive progressive combined variable immunodeficiency (Duncan's disease). Lancet 1: 935, 1975
