- Email: [email protected]
RS cells
Annals of Oncology 7 (SuppL 4): S41-S43, 1996. O 1996 Kluwer Academic Publishers. Printed in the Netherlands.
Review Single cell analysis of H/RS cells W. C. Chan1 & J. Delabie2 1
University of Nebraska Medical Center, Omaha, NE, U.S-A.; 2 University Hospitals ofLeuven,
Summary
Introduction
subpopulation amid a polyclonal background. Clonality was assessed independently in one NSHD case by determining the pattern of X-chromosome inactivation. This technique also failed to show that the H/RS cells were monoclonal. Our studies suggest that in both LPHD and NSHD, the H/RS cells may be a polyclonal proliferation at presentation and clonal populations may emerge from this polyclonal background. There is also some evidence of a correlation between the immunophenotype and the genotype of the H/RS cells. Single cell assays show great potential in elucidating the cell lineage and clonality of H/RS cells, as well as their in vivo evolution during the disease process. Key words: Hodgkin's disease, HUMARA gene assay, immunoglobulin gene rearrangement, Reed-Sternberg cells
In order to make further progress in delineating the lineage and clonality of H/RS cells, we and others have studied single isolated H/RS cells by highly sensitive polymerase chain reaction (PCR)-based assays [7-12]. Our data suggest that HD may begin as a polyclonal proliferation from which a monoclonal lesion may develop. There is also some correlation between immunophenotype and genotype.
It is the general consensus that Hodgkin's disease (HD) is a neoplastic condition and the large pleomorphic cells, Hodgkin/Reed-Sternberg (H/RS) cells, in the tumor are the neoplastic component [1]. However, the nature of the H/RS cells is still not entirely resolved. The difficulty arises in part from the fact that the H/RS cells usually constitute a minor component of the cellular population in the tumor. In addition, there seems to be considerable heterogeneity within the H/RS cell Results population, not only among different subtypes of HD, but even within the same tumor. Immunohistochemical We isolated H/RS cells from four cases of N-LPHD studies in the last decade have provided strong evi- based on their morphology and reactivity with antidence that the H/RS cells in nodular lymphocytic pre- EMA. The immunoglobulin heavy chain (IgH) gene dominance (N-LP) HD belong to the B-lineage, but lin- complementarity determining region (CDR) HI of eage derivation of the H/RS cells in the classic HD these cells was amplified by the PCR Single small Bremains unclear [2]. There is both cytogenetic and mo- and T-lymphocytes from the same tissue section acted lecular evidence for the existence of a clonal popula- as positive and negative controls, respectively. In this tion in some Hodgkin's tumors [3]. Studies utilizing study, similar proportions of H/RS cells (48%) and Epstein-Barr virus (EBV) as a clonal marker have pro- small B-cells (44%) showed amplified CDRffl provided evidence indicating that the H/RS cells constitute ducts suggesting that all the H/RS cells had rearranged the clonal population [4, 5]. However, it has been their IgH genes. This was in agreement with their B-cell observed that in some cases of HD showing immuno- phenotype. Interesting, the IgH CDRin amplicons of globulin heavy chain (IgH) gene rearrangement, the H/RS cells in each case were not identical as expected intensity of the rearranged band was lower than one of a monoclonal population. In fact, they were all difwould expect from the proportion of H/RS cell pre- ferent as seen in polyclonal B-cells [8]. sent, raising the possibility that the rearrangement is We later studied six cases of HD of the nodular sclepresent in only a subpopulation of the H/RS cells [6]. rosis (NS) type. Three of these cases had 5%, 10% and
Downloaded from http://annonc.oxfordjournals.org/ at University of Queensland on June 23, 2015
The nature of the Hodgkin/Reed-Sternberg (H/RS) cells in Hodgkin's disease (HD) is still unclear. We have studied isolated H/RS cells from four cases of lymphocytic predominance (LP) HD and six cases of nodular sclerosis (NS) HD and assayed the cells for immunoglobulin heavy chain (IgH) gene rearrangement by the polymerase chain reaction (PCR). The H/RS cells in all four cases of LPHD had rearranged their IgH gene in a polyclonal pattern. The H/RS cells of three cases of NSHD where at least a fraction of the cells expressed the B-cell marker CD20 had also rearranged their IgH gene. In two of the cases, the rearrangements were totally unrelated while in one case, two out of six rearrangements were identical in sequence indicating the presence of a clonal
Leuven, Belgium
42 arrangement was also demonstrable in single H/RS cells. Similar to N-LPHD, the rearrangements were not monoclonal. Hummel et al. [12], using a different technique of cell isolation, obtained similar results with their four cases of CD20 + NSHD. These results that the H/RS cells, in at least a subset of NSHD had undergone complete VDJ rearrangement and appeared to be polyclonal. Again, the number of cells analyzed was too small to exclude the presence of clonal subpopulations. Statistical analysis indicates that 34 cells with IgH gene IgH gene rearrangements need to be analyzed to detect a clonal population constituting 20% or more of the population with a 95% confidence. Indeed, in one of our cases, two clonally related cells out of six analyzed cells were detected indicating the presence of a subclone of considerable size. Similar observations were made in some cases of mixed cellularity HD with a B-cell phenotype in the report by Hummel and coworkers [12]. We considered the possibility that the H/RS cells in these cases might be a clonal population transformed prior to IgH gene rearrangement which Discussion occurred randomly later, giving rise to the appearance The immunophenotypic evidence for a B-lineage deri- of polyclonality when the IgH gene rearrangement was vation of the H/RS cells of N-LPHD is corroborated used as the criterion for clonality. The pattern of by genotypic evidence for B-lineage differentiation, i.e., X-chromosome inactivation was used, therefore, as an the complete rearrangement of the IgH gene. However, additional assay for clonality in three female cases. The in none of our cases did we detect a monoclonal popu- human androgen receptor gene on the X-chromosome lation by single cell analysis [8]. With the limited num- appears to be the best target for the single cell assay ber of cells studied in each case, we cannot exclude the [13]. In a case that was successfully studied, the H/RS presence of clonal subpopulation(s) in these cases. It cells did not inactivate consistently either the paternal also remains to be determined whether cases with or the maternal allele, again indicating the population is numerous H/RS cells or nodules of such cells would not monoclonal. We were able to demonstrate only rare more likely show evidence of a clonal population. Our IgH CDRUI rearrangements in the three cases of + data indicated a fundamental difference between NSHD with the classic HD phenotype (T", B", CD15 , + N-LPHD and B-non-Hodgkin's lymphoma (NHL) in CD30 ). This may mean that the H/RS cells had germthat the latter is a monoclonal proliferation at presenta- line IgH gene, IgH gene with only DJ rearrangement or tion while the former is often not Perhaps, we may even a clonal IgH gene rearrangement that was not consider N-LPHD as a pre-lymphomatous prolifera- amplifiable by the primers we used. Hence, we are not tion that has a propensity to evolve into a monoclonal able to make any conclusions regarding the lineage and B-NHL. Indeed, large cell lymphomas occur concur- clonality of H/RS cells in these cases. There have been some conflicting results reported rently or subsequently in a substantial proportion of N-LPHD [15] and histiocyte rich-B-cell lymphoma [16] from different research groups utilizing the single cell is also frequently associated with N-LPHD. Recently, a assay to study H/RS cells [9-12]. Part of the inconsisclonal relationship between the B-large cell NHL with tency may be related to the techniques employed and its associated N-LPHD has been demonstrated in some part of it may be due to different case selection criteria. cases supporting the concept of clonal evolution in If H/RS cells are picked from a tissue section by microN-LPHD [17]. N-LPHD generally has an indolent manipulation, it is likely that cells in a limited area of clinical behavior. Does this chronic course relate to a the tumor are being sampled. The possibility that H/RS generally prolonged polyclonal phase and should we cells in a geographically confined area, such as in a reconsider treatment options at this phase? Does the nodule in N-LP or NSHD, are likely to be clonal or emergence of a major clonal population signify a oligoclonal needs to be considered. To address this change or impending change in clinical behavior and question, cells from different nodules, especially those that are distant from each other, need to be sampled. hence require a different treatment strategy? Classic HD is immunophenotypically much more Another relevant consideration is the relationship bevariable than N-LPHD. We studied two groups of tween the abundance of H/RS cells in a tumor and the NSHD cases to assess whether there is a correlation likelihood of clonality in a case. If these two features between immunophenotype and genotype. In the three are related, the selection of cases with many H/RS cells cases where at least a fraction of the H/RS cells will yield a much higher incidence of monoclonal expressed the B-cell antigen CD20, IgH gene re- lesions. It has been reported [19] that there were HD
Downloaded from http://annonc.oxfordjournals.org/ at University of Queensland on June 23, 2015
100% of CD20 + H/RS cells, respectively, while the H/RS cells of the other three cases were CD20~. The H/RS cells were isolated based on their morphology and staining with either CD15 or CD30. The IgH CDRUI was amplified as described above with similar control populations. Only rare H/RS cells from the CD20" cases showed amplifiable products while the other three cases (CD20+) showed similar results as the previously studied N-LPHD, except that two cells with identical CDRm amplicons were detected in one of these cases consistent with the presence of a clonal population in a polyclonal background. We further studied one of the female CD20 + cases for evidence of monoclonality by the pattern of inactivation of the human androgen receptor (HUMARA) gene on the X-chromosome [13]. There was no exclusive inactivation of one of the alleles as expected of a monoclonal population [14].
43
Acknowledgements This work was supported by LB506 awarded by the Department of Health, State of Nebraska and in part by USPHS CA36727 awarded by the National Cancer Institute, Department of Health and Human Services. References 1. Kaplan HS. Hodgkin's disease: Biology, treatment, prognosis. Blood 1981; 57: 813-22. 2. Hugh J, Poppema S. Immunophenotype of Reed-Sternberg cells. Int Rev Exp Pathol 1992; 33: 81-114. 3. Delabie J, Weisenburger DD, Chan WC. Hodgkin's disease: A monoclonal lymphoproliferative disorder? Histopathology 1995; 27: 93-6.
4. Weiss LM, Movahed LA, Warnke RA et al. Detection of Epstein-Barr viral genomes in Reed-Sternberg cells of Hodgkin's disease. N Engl J Med 1989; 320: 502-6. 5. Gulley ML, Eagan PA, Quintanilla-Martinez L et al. EpsteinBarr virus DNA is abundant and monoclonal in the ReedSternberg cells of Hodgkin's disease: Association with mixed cellularity subtype and Hispanic American ethnicity. Blood 1994; 83:1595-602. 6. Weiss LM. Gene analysis and Epstein-Barr viral genome studies of Hodgkin's disease. Int Rev Exp Pathol 1992; 33: 165-83. 7. Triimper LH, Brady G, Bagg A et al. Single-cell analysis of Hodgkin and Reed-Stemberg cells: Molecular heterogeneity of gene expression and p53 mutations. Blood 1993; 81: 3097115. 8. Delabie J, Tierens A, Wu G et al. Lymphocyte predominance Hodgkin's disease: Lineage and clonality determination using a single-cell assay. Blood 1994; 84:3291-8. 9. Kiippers R, Rajewsky K, Zhao M et al. Hodgkin disease: Hodgkin and Reed-Stemberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B-cells at various stages of development Proc Natl Acad Sci USA 1994; 91:10962-6. 10. Roth J, Daus H, Triimper L et al. Detection of immunoglobulin heavy-chain gene rearrangement at the single-cell level in malignant lymphomas: No rearrangment is found in Hodgkin and Reed-Stemberg cells. Int J Cancer 1994; 57: 799-804. 11. Daus H, Triimper L, Roth J et al. Hodgkin and Reed-Stemberg cells do not carry T-cell receptor y gene rearrangements: Evidence from single-cell polymerase chain reaction examination. Blood 1995; 85:1590-5. 12. Hummel M, Ziemann K, Lammert H et al. Hodgkin's disease with monoclonal and polyclonal populations of Reed-Sternberg cells. N Engl J Med 1995; 333: 901-6. 13. Willman CL, Busque L, Griffith BB et aL Langerhans'-cell histiocytosis (histiocytosis X) - a clonal proliferative disease. New Engl J Med 1994; 331:154-60. 14. Delabie J, Tierens A, Gavriil T et al. Phenotype, genotype and clonality of Reed-Stemberg cells in nodular sclerosis Hodgkin's disease: Results of a single-cell study (submitted). 15. Jaffe ES, Zarate-Osorno A, Kingma DW et al. The interrelationship between Hodgkin's disease and non-Hodgkin's lymphomas. Ann Oncol 1994; 5 (Suppl I}. S7. 16. Delabie J, Chan WC, Tierens A et al. Histiocyte rich B-cell lymphoma occurring in lymphocyte predominance Hodgkin's disease. Mod Pathol 1995; 8:108A. 17. Wickert RS, Weisenburger DD, Tierens A et al. Clonal relationship between lymphocytic predominance Hodgkin's disease and concurrent or subsequent large-cell lymphoma of B-lineage. Blood 1995; 86: 2312-20. 18. Kiippers R. Clonality of Hodgkin/Reed-Sternberg cells single cell analysis of HRS-cells picked from histologic sections. Third International Symposium on Hodgkin's Lymphoma, Cologne, Germany, September 1995. Correspondence to: Wing C. Chan, MD. Department of Pathology and Microbiology University of Nebraska Medical Center 600 South 42nd Street Omaha, NE 68198-3435 USA.
Downloaded from http://annonc.oxfordjournals.org/ at University of Queensland on June 23, 2015
tumors with rearranged Ig kappa chain only. If the kappa chain is not assayed, such cases will be considered lacking Ig rearrangements. Aside from the above consideration, the problem of contamination is very real in the setting of single cell assays. Contamination may be due to the presence of DNA molecules in the fluid phase rather than in the cellular phase of the sample or the inadvertent aspiration of other cells in addition to the target H/RS cells into the micropipette. With the sensitivity of the PCR assay increased to the level of single molecules, contamination during the assay procedure is an ever present menace and all necessary precautions and controls must be used to safeguard the reliability of the experimental results. Single cell assay is at this infancy. Several methods have been developed and employed by different groups [8-10]. The limitations and advantages of each method will become apparent with more experience. This is a powerful technique that has an enormous potential for studying minute specimens, dissecting the heterogeneity of a cellular population and microanalyzing a biological process. The initial discordance of the reported findings in the study of H/RS cells may be more apparent than real and should not detract from the usefulness of this approach. Obviously, to make further progress, more cases and more cells per case need to be studied with great care to avoid contamination and sampling bias. Cases with varying morphology and immunophenotype should be included and correlated with the genotypic study. Sequential biopsies are of great value in determining clonal progression and the potential of H/RS cells to differentiate in vivo. The single cell assay is going to play a major role in unraveling the lineage and clonality of the enigmatic H/RS cell.
Review Single cell analysis of H/RS cells W. C. Chan1 & J. Delabie2 1
University of Nebraska Medical Center, Omaha, NE, U.S-A.; 2 University Hospitals ofLeuven,
Summary
Introduction
subpopulation amid a polyclonal background. Clonality was assessed independently in one NSHD case by determining the pattern of X-chromosome inactivation. This technique also failed to show that the H/RS cells were monoclonal. Our studies suggest that in both LPHD and NSHD, the H/RS cells may be a polyclonal proliferation at presentation and clonal populations may emerge from this polyclonal background. There is also some evidence of a correlation between the immunophenotype and the genotype of the H/RS cells. Single cell assays show great potential in elucidating the cell lineage and clonality of H/RS cells, as well as their in vivo evolution during the disease process. Key words: Hodgkin's disease, HUMARA gene assay, immunoglobulin gene rearrangement, Reed-Sternberg cells
In order to make further progress in delineating the lineage and clonality of H/RS cells, we and others have studied single isolated H/RS cells by highly sensitive polymerase chain reaction (PCR)-based assays [7-12]. Our data suggest that HD may begin as a polyclonal proliferation from which a monoclonal lesion may develop. There is also some correlation between immunophenotype and genotype.
It is the general consensus that Hodgkin's disease (HD) is a neoplastic condition and the large pleomorphic cells, Hodgkin/Reed-Sternberg (H/RS) cells, in the tumor are the neoplastic component [1]. However, the nature of the H/RS cells is still not entirely resolved. The difficulty arises in part from the fact that the H/RS cells usually constitute a minor component of the cellular population in the tumor. In addition, there seems to be considerable heterogeneity within the H/RS cell Results population, not only among different subtypes of HD, but even within the same tumor. Immunohistochemical We isolated H/RS cells from four cases of N-LPHD studies in the last decade have provided strong evi- based on their morphology and reactivity with antidence that the H/RS cells in nodular lymphocytic pre- EMA. The immunoglobulin heavy chain (IgH) gene dominance (N-LP) HD belong to the B-lineage, but lin- complementarity determining region (CDR) HI of eage derivation of the H/RS cells in the classic HD these cells was amplified by the PCR Single small Bremains unclear [2]. There is both cytogenetic and mo- and T-lymphocytes from the same tissue section acted lecular evidence for the existence of a clonal popula- as positive and negative controls, respectively. In this tion in some Hodgkin's tumors [3]. Studies utilizing study, similar proportions of H/RS cells (48%) and Epstein-Barr virus (EBV) as a clonal marker have pro- small B-cells (44%) showed amplified CDRffl provided evidence indicating that the H/RS cells constitute ducts suggesting that all the H/RS cells had rearranged the clonal population [4, 5]. However, it has been their IgH genes. This was in agreement with their B-cell observed that in some cases of HD showing immuno- phenotype. Interesting, the IgH CDRin amplicons of globulin heavy chain (IgH) gene rearrangement, the H/RS cells in each case were not identical as expected intensity of the rearranged band was lower than one of a monoclonal population. In fact, they were all difwould expect from the proportion of H/RS cell pre- ferent as seen in polyclonal B-cells [8]. sent, raising the possibility that the rearrangement is We later studied six cases of HD of the nodular sclepresent in only a subpopulation of the H/RS cells [6]. rosis (NS) type. Three of these cases had 5%, 10% and
Downloaded from http://annonc.oxfordjournals.org/ at University of Queensland on June 23, 2015
The nature of the Hodgkin/Reed-Sternberg (H/RS) cells in Hodgkin's disease (HD) is still unclear. We have studied isolated H/RS cells from four cases of lymphocytic predominance (LP) HD and six cases of nodular sclerosis (NS) HD and assayed the cells for immunoglobulin heavy chain (IgH) gene rearrangement by the polymerase chain reaction (PCR). The H/RS cells in all four cases of LPHD had rearranged their IgH gene in a polyclonal pattern. The H/RS cells of three cases of NSHD where at least a fraction of the cells expressed the B-cell marker CD20 had also rearranged their IgH gene. In two of the cases, the rearrangements were totally unrelated while in one case, two out of six rearrangements were identical in sequence indicating the presence of a clonal
Leuven, Belgium
42 arrangement was also demonstrable in single H/RS cells. Similar to N-LPHD, the rearrangements were not monoclonal. Hummel et al. [12], using a different technique of cell isolation, obtained similar results with their four cases of CD20 + NSHD. These results that the H/RS cells, in at least a subset of NSHD had undergone complete VDJ rearrangement and appeared to be polyclonal. Again, the number of cells analyzed was too small to exclude the presence of clonal subpopulations. Statistical analysis indicates that 34 cells with IgH gene IgH gene rearrangements need to be analyzed to detect a clonal population constituting 20% or more of the population with a 95% confidence. Indeed, in one of our cases, two clonally related cells out of six analyzed cells were detected indicating the presence of a subclone of considerable size. Similar observations were made in some cases of mixed cellularity HD with a B-cell phenotype in the report by Hummel and coworkers [12]. We considered the possibility that the H/RS cells in these cases might be a clonal population transformed prior to IgH gene rearrangement which Discussion occurred randomly later, giving rise to the appearance The immunophenotypic evidence for a B-lineage deri- of polyclonality when the IgH gene rearrangement was vation of the H/RS cells of N-LPHD is corroborated used as the criterion for clonality. The pattern of by genotypic evidence for B-lineage differentiation, i.e., X-chromosome inactivation was used, therefore, as an the complete rearrangement of the IgH gene. However, additional assay for clonality in three female cases. The in none of our cases did we detect a monoclonal popu- human androgen receptor gene on the X-chromosome lation by single cell analysis [8]. With the limited num- appears to be the best target for the single cell assay ber of cells studied in each case, we cannot exclude the [13]. In a case that was successfully studied, the H/RS presence of clonal subpopulation(s) in these cases. It cells did not inactivate consistently either the paternal also remains to be determined whether cases with or the maternal allele, again indicating the population is numerous H/RS cells or nodules of such cells would not monoclonal. We were able to demonstrate only rare more likely show evidence of a clonal population. Our IgH CDRUI rearrangements in the three cases of + data indicated a fundamental difference between NSHD with the classic HD phenotype (T", B", CD15 , + N-LPHD and B-non-Hodgkin's lymphoma (NHL) in CD30 ). This may mean that the H/RS cells had germthat the latter is a monoclonal proliferation at presenta- line IgH gene, IgH gene with only DJ rearrangement or tion while the former is often not Perhaps, we may even a clonal IgH gene rearrangement that was not consider N-LPHD as a pre-lymphomatous prolifera- amplifiable by the primers we used. Hence, we are not tion that has a propensity to evolve into a monoclonal able to make any conclusions regarding the lineage and B-NHL. Indeed, large cell lymphomas occur concur- clonality of H/RS cells in these cases. There have been some conflicting results reported rently or subsequently in a substantial proportion of N-LPHD [15] and histiocyte rich-B-cell lymphoma [16] from different research groups utilizing the single cell is also frequently associated with N-LPHD. Recently, a assay to study H/RS cells [9-12]. Part of the inconsisclonal relationship between the B-large cell NHL with tency may be related to the techniques employed and its associated N-LPHD has been demonstrated in some part of it may be due to different case selection criteria. cases supporting the concept of clonal evolution in If H/RS cells are picked from a tissue section by microN-LPHD [17]. N-LPHD generally has an indolent manipulation, it is likely that cells in a limited area of clinical behavior. Does this chronic course relate to a the tumor are being sampled. The possibility that H/RS generally prolonged polyclonal phase and should we cells in a geographically confined area, such as in a reconsider treatment options at this phase? Does the nodule in N-LP or NSHD, are likely to be clonal or emergence of a major clonal population signify a oligoclonal needs to be considered. To address this change or impending change in clinical behavior and question, cells from different nodules, especially those that are distant from each other, need to be sampled. hence require a different treatment strategy? Classic HD is immunophenotypically much more Another relevant consideration is the relationship bevariable than N-LPHD. We studied two groups of tween the abundance of H/RS cells in a tumor and the NSHD cases to assess whether there is a correlation likelihood of clonality in a case. If these two features between immunophenotype and genotype. In the three are related, the selection of cases with many H/RS cells cases where at least a fraction of the H/RS cells will yield a much higher incidence of monoclonal expressed the B-cell antigen CD20, IgH gene re- lesions. It has been reported [19] that there were HD
Downloaded from http://annonc.oxfordjournals.org/ at University of Queensland on June 23, 2015
100% of CD20 + H/RS cells, respectively, while the H/RS cells of the other three cases were CD20~. The H/RS cells were isolated based on their morphology and staining with either CD15 or CD30. The IgH CDRUI was amplified as described above with similar control populations. Only rare H/RS cells from the CD20" cases showed amplifiable products while the other three cases (CD20+) showed similar results as the previously studied N-LPHD, except that two cells with identical CDRm amplicons were detected in one of these cases consistent with the presence of a clonal population in a polyclonal background. We further studied one of the female CD20 + cases for evidence of monoclonality by the pattern of inactivation of the human androgen receptor (HUMARA) gene on the X-chromosome [13]. There was no exclusive inactivation of one of the alleles as expected of a monoclonal population [14].
43
Acknowledgements This work was supported by LB506 awarded by the Department of Health, State of Nebraska and in part by USPHS CA36727 awarded by the National Cancer Institute, Department of Health and Human Services. References 1. Kaplan HS. Hodgkin's disease: Biology, treatment, prognosis. Blood 1981; 57: 813-22. 2. Hugh J, Poppema S. Immunophenotype of Reed-Sternberg cells. Int Rev Exp Pathol 1992; 33: 81-114. 3. Delabie J, Weisenburger DD, Chan WC. Hodgkin's disease: A monoclonal lymphoproliferative disorder? Histopathology 1995; 27: 93-6.
4. Weiss LM, Movahed LA, Warnke RA et al. Detection of Epstein-Barr viral genomes in Reed-Sternberg cells of Hodgkin's disease. N Engl J Med 1989; 320: 502-6. 5. Gulley ML, Eagan PA, Quintanilla-Martinez L et al. EpsteinBarr virus DNA is abundant and monoclonal in the ReedSternberg cells of Hodgkin's disease: Association with mixed cellularity subtype and Hispanic American ethnicity. Blood 1994; 83:1595-602. 6. Weiss LM. Gene analysis and Epstein-Barr viral genome studies of Hodgkin's disease. Int Rev Exp Pathol 1992; 33: 165-83. 7. Triimper LH, Brady G, Bagg A et al. Single-cell analysis of Hodgkin and Reed-Stemberg cells: Molecular heterogeneity of gene expression and p53 mutations. Blood 1993; 81: 3097115. 8. Delabie J, Tierens A, Wu G et al. Lymphocyte predominance Hodgkin's disease: Lineage and clonality determination using a single-cell assay. Blood 1994; 84:3291-8. 9. Kiippers R, Rajewsky K, Zhao M et al. Hodgkin disease: Hodgkin and Reed-Stemberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B-cells at various stages of development Proc Natl Acad Sci USA 1994; 91:10962-6. 10. Roth J, Daus H, Triimper L et al. Detection of immunoglobulin heavy-chain gene rearrangement at the single-cell level in malignant lymphomas: No rearrangment is found in Hodgkin and Reed-Stemberg cells. Int J Cancer 1994; 57: 799-804. 11. Daus H, Triimper L, Roth J et al. Hodgkin and Reed-Stemberg cells do not carry T-cell receptor y gene rearrangements: Evidence from single-cell polymerase chain reaction examination. Blood 1995; 85:1590-5. 12. Hummel M, Ziemann K, Lammert H et al. Hodgkin's disease with monoclonal and polyclonal populations of Reed-Sternberg cells. N Engl J Med 1995; 333: 901-6. 13. Willman CL, Busque L, Griffith BB et aL Langerhans'-cell histiocytosis (histiocytosis X) - a clonal proliferative disease. New Engl J Med 1994; 331:154-60. 14. Delabie J, Tierens A, Gavriil T et al. Phenotype, genotype and clonality of Reed-Stemberg cells in nodular sclerosis Hodgkin's disease: Results of a single-cell study (submitted). 15. Jaffe ES, Zarate-Osorno A, Kingma DW et al. The interrelationship between Hodgkin's disease and non-Hodgkin's lymphomas. Ann Oncol 1994; 5 (Suppl I}. S7. 16. Delabie J, Chan WC, Tierens A et al. Histiocyte rich B-cell lymphoma occurring in lymphocyte predominance Hodgkin's disease. Mod Pathol 1995; 8:108A. 17. Wickert RS, Weisenburger DD, Tierens A et al. Clonal relationship between lymphocytic predominance Hodgkin's disease and concurrent or subsequent large-cell lymphoma of B-lineage. Blood 1995; 86: 2312-20. 18. Kiippers R. Clonality of Hodgkin/Reed-Sternberg cells single cell analysis of HRS-cells picked from histologic sections. Third International Symposium on Hodgkin's Lymphoma, Cologne, Germany, September 1995. Correspondence to: Wing C. Chan, MD. Department of Pathology and Microbiology University of Nebraska Medical Center 600 South 42nd Street Omaha, NE 68198-3435 USA.
Downloaded from http://annonc.oxfordjournals.org/ at University of Queensland on June 23, 2015
tumors with rearranged Ig kappa chain only. If the kappa chain is not assayed, such cases will be considered lacking Ig rearrangements. Aside from the above consideration, the problem of contamination is very real in the setting of single cell assays. Contamination may be due to the presence of DNA molecules in the fluid phase rather than in the cellular phase of the sample or the inadvertent aspiration of other cells in addition to the target H/RS cells into the micropipette. With the sensitivity of the PCR assay increased to the level of single molecules, contamination during the assay procedure is an ever present menace and all necessary precautions and controls must be used to safeguard the reliability of the experimental results. Single cell assay is at this infancy. Several methods have been developed and employed by different groups [8-10]. The limitations and advantages of each method will become apparent with more experience. This is a powerful technique that has an enormous potential for studying minute specimens, dissecting the heterogeneity of a cellular population and microanalyzing a biological process. The initial discordance of the reported findings in the study of H/RS cells may be more apparent than real and should not detract from the usefulness of this approach. Obviously, to make further progress, more cases and more cells per case need to be studied with great care to avoid contamination and sampling bias. Cases with varying morphology and immunophenotype should be included and correlated with the genotypic study. Sequential biopsies are of great value in determining clonal progression and the potential of H/RS cells to differentiate in vivo. The single cell assay is going to play a major role in unraveling the lineage and clonality of the enigmatic H/RS cell.