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Unexpected finding—Biologic truth or technical artifact?
Human PATHOLOGY VOLUME
22
October f991
NUMBER
10
Editorial Unexpected Finding-Biologic or Technical Artifact? Chancr cltL
Truth
support the diagnosis of myeloma (Fig 1 ;I, but some experts have demonstrated the limitations of‘ this method, which may not correlate with genetic ~clonality. Furthermore, the fixatives used and the methods of decalcification of bone marrow specimens mav cause high background or abolish immunoglohulin staining needed to demonstrate monoclonality of plas~na cells in JIlyeloma. For this reason, Humphries et al evaluated Southern blot detection of iInmunoglohulirl gene rearrangements, often useful in demonstrating clonality in lymphoid neoplasms. as a method to aid the diagnosis of plasma cell myeloma. Unexpectedly, the authors found no immunoglobulin gene rearrangements in seven of 36 (I 9%) cases of myeloma. Their studies were done on bone marrow aspirates separated by continuous gradient centrifugation. DNA extracted from the samples was digested with several restriction enzymes and analyzed by Southern blotting using probes for Ig heavy chain joining genes (JH) and kappa light chain joining genes. No correlation between the monoclonal serum Ig heavy or light chain isotype and the presence or absence of detectable gene rearrangements was found. The mean percentages of plasma cells in the bone marrow of patients without detectable rearrangements was 12.4%~. C)f the seven cases without detectable rearrangements, five had more than 10% plasma cells, well above the usual threshold of sensitivity for detection of clonalitv with I he Southern blot methoil. Humphries et al suggest that the failure of Southern blot analysis to detect clonality of plasma c.ells in some patients in their study brings into question the assumption that bone marrow plasma cells in myeloma are monoclonal. In support of their viewpoini, they cite studies of other investigators who failed to detect immunoglobulin gene rearrangements in my&ma bone marrow in which more than 1 O%, and usually more than 20%, plasma cells were present. In these studies, as wet1 as in the study of Humphries et al, Ig rearrangements were most frequently found in advanced disease with greater than 50% plasma cells. Some technical considerations that may explain the lack of Ig gene rearrangements found in myeloma cases
941
HUMAN PATHOLOGY
Volume 22, No. 10 (October
1991)
FIGURE 1. lmmunoperoxidase stain of bone marrow showing a large predominance of plasma cells stained for lambda light chain (left), but not stained for kappa light chain (right), in a newly diagnosed patient with plasma cell myeloma. The large number of plasma cells was not appreciated in the bone marrow biopsy stained with hematoxylin-eosin.
loss of plasma cells during the cell separation procedure, poor Southern blot transfer of rearranged bands (most commonly seen with high molecular weight bands), co-migration of rearranged bands with germline bands, and incomplete digestion of DNA. In studying 3 1 cases of myeloma with a JH probe, Zaccaria et al encountered five cases in which it was necessary to use five restriction enzymes before a rearranged band was detected, usually as a very faint band after digestion with BgI II enzyme.’ The DNA of two additional patients continued to reveal no gene rearrangements. In another series of 21 patients with B-cell malignancies other than myeloma, the same investigators found a discrete, intense rearranged band in all cases. Thus, the difficulty in obtaining rearranged bands appeared to be specific for myeloma. Zaccaria et al suggested that the unusual gene rearrangement results in myeloma patients are consistent with partial hybridization of the JH probe because of a more or less wide deletion of the area located 3’ to the J genes, which contains the enhancer.’ Other possible biologic explanations for the lack of Ig gene rearrangements include (1) a preponderance of nonmalignant plasma cells, presumably reactive to the neoplasm or induced to proliferate by a similar mechanism, such as interleukin-6 stimulation, which is increased in myeloma”; (2) deletion of Ig gene loci during gene rearrangement; (3) somatic Ig gene mutations during clonal evolution of the tumor cell population’; and (4) origin of the tumor cells from an early progenitor cell that has not yet undergone gene rearrangement. Of these, the latter is potentially the most interesting and may help to explain the frequent finding of myeloid antigens on myeloma cells,” megaloblastic or dysplastic changes in nonlymphoid cells in myeloma bone marrow,” and abnormalities of B lymphocytes in the blood of myeloma patients.’ The latter explanation seems unlikely since peripheral blood B lymphocytes of myeloma patients have been shown to be part of the malignant clone, having immunoglobulin gene rearrangements identical to those are the selective
942
seen in bone marrow plasma cells.x,!’ Peripheral
blood immunoglobulin gene rearrangements were significantly more common in patients with active disease compared with remission status and higher in untreated compared with treated patients. Patients with MGUS did not have gene rearrangements, although two myeloma patients with gene rearrangements had a preceding history of MGUS, suggesting that its detection may be an early sign of progression to multiple myeloma.” We are left without a biologic explanation for the production of a monoclonal paraprotein in the absence of Ig gene rearrangement, or the lack of correlation between the monoclonal serum Ig heavy or light chain isotype and the presence or absence of gene rearrangements detected by Southern blot method in the study of Humphries et al.’ Therefore, technical limitations of the Southern blot method remain the most likely explanation for these unexpected results. Failure to detect clonal gene rearrangements by the Southern blot method has been observed in other lymphoid malignancies, in which it has been unclear whether this was a limitation of the method used OI
provided new insight into the biology of the disease. Weiss et al detected no T-cell receptor (TCR) gene rearrangements in a group of seven peripheral T-cell lymphomas, five of which arose in the upper respiratory tract.“’ Ralfkaier et al noted TCR gene rearrangements in the tumor and plaque stages of cutaneous T-cell lymphoma (CTCL) but not in early patch-stage lesions.” These investigators suggested that early stage CTCL may be multiclonal. Whittaker et al did not detect TCR or Ig gene rearrangements in lymphomatoid papulosis type A lesions and suggested that these lesions have a nonT-, non-B-cell origin.” If correct, this finding would be most interesting since lymphomatoid papulosis type A lesions closely resemble Hodgkin’s disease, in which the nature of the malignant cell is controversial and TCR or Ig gene rearrangements often are not found.“’ It is often argued that negative gene rearrangement results, such as the examples cited above, are due to the
EDITORIAL
(Marshall E. Kadin)
limited sensitivity of the Southern blot method. This limitation can be overcome with the polymerase chain reaction (PCR) technique when suitable primers can be made. Indeed, PCR has been used successfully to detect clonal TCR gene rearrangements in some early patchstage lesions of CTCI, (unpublished observations) and lvmphomatoid papulosis type A.” A similar approach should be useful in studies of other lymphoproliferative disorders and can be used by Humphries et al and others to determine whether all cases of plasma cell myeloma are or are not monoclonal. Meanwhile, this interesting study emphasizes that the diagncjsis of a particular disease should not be determined by ;I single laboratory test, no matter how filshionable. Rather, each test, particularly those based on new u’nproven technologies, should be interpreted in context of other laboratory results, the patient’s history, and the c.linical findings. In the final analysis, if it waddles and quacks like a duck, it probably is a duck!
-&. Tonegawa S: Somatic :w2:575-581, l!W(
and Harvard
Medical
of antibodt
diver-sity. Narurr
5. Epstein ,J. Xiao H, He X-Y: Markers of multiple hematopoieticcell lineages in multiple myeloma. N Engl J Med .3??:664-668. 1900 6. Hobbs JR, Jacobs JR, Kremenchuzky S. ?t al: Inc idenre and pathogencsis of megaloblastic erythropoiesis in tmlltiple myelonra. J Clin Path01 20:699-705. I967 7. MeUstedt H. Hammarsrrom S. Helm G: Nlcmoclonal lymphocvte populations in human plasma cell rnveloma. Uin Exp Immunol 17:371-384. 1974 X. Berenson J, Wang R, Kim k, et al: F.Gdwce fox peripheral blood B lymphocytr but not T lynlphocvtr involwment in multiple mveloma. Blood 70:1.550-155.3. 1987 9. Chiu EKU’, Grneshaguru K. Hoffbr-and .4’1’. CI al: Cirwlating monoclonal B Ivmphocytes in multiple nrvrloma. Br J Hxmatol 72: 28-Y I. 1!I89 10. Weiss I.M. Picker LJ. Grogan I‘M, ct al: .\bsencr of clonal beta and gamma T-cell receptor- gene rearrangt‘meuts it] ‘, subset 01. peripheral T-cell lymphomas. Am J Pathol 1:10:436-q-12, I988
11. Ralfkaicr E. O’Connor 01 cutaneous T-cell lymphoma. Hospital
generation
School
NTJ, Crick 5.4. et al: (Genotkpic analysis J Invest Dermatol X8:762-765. I987
I?. Whittaker S. Smith N, Jones RR, ct aI, Analysis of beta. K&mma, and drlra T-cell receptor gem’s in I~rnphc)rnaroid papulosis: (:rllular basis of two distin1.t histolqqic wbsrts. J Invest Drrmatol 067X6-791. 1991 13. Raghavachar A. Binder T. Bartram CR: Inllnunoglob~~lin and TC ell ret rptol. gene I-earrangements in Hodgkin’\ disease. (:ancet Rr?, 4X:3591-Y5W, I!QCi 14. Kadin ME, Davis TH, Miller-Cassman R: Lymphomatoid papulosis. cutaneous T-cell lymphoma and Hodgkin’3 disease derivrd from a common T-cell clone. Blood WI 7X. I WI (wppl) (abstr) (in l”‘“5)
943
22
October f991
NUMBER
10
Editorial Unexpected Finding-Biologic or Technical Artifact? Chancr cltL
Truth
support the diagnosis of myeloma (Fig 1 ;I, but some experts have demonstrated the limitations of‘ this method, which may not correlate with genetic ~clonality. Furthermore, the fixatives used and the methods of decalcification of bone marrow specimens mav cause high background or abolish immunoglohulin staining needed to demonstrate monoclonality of plas~na cells in JIlyeloma. For this reason, Humphries et al evaluated Southern blot detection of iInmunoglohulirl gene rearrangements, often useful in demonstrating clonality in lymphoid neoplasms. as a method to aid the diagnosis of plasma cell myeloma. Unexpectedly, the authors found no immunoglobulin gene rearrangements in seven of 36 (I 9%) cases of myeloma. Their studies were done on bone marrow aspirates separated by continuous gradient centrifugation. DNA extracted from the samples was digested with several restriction enzymes and analyzed by Southern blotting using probes for Ig heavy chain joining genes (JH) and kappa light chain joining genes. No correlation between the monoclonal serum Ig heavy or light chain isotype and the presence or absence of detectable gene rearrangements was found. The mean percentages of plasma cells in the bone marrow of patients without detectable rearrangements was 12.4%~. C)f the seven cases without detectable rearrangements, five had more than 10% plasma cells, well above the usual threshold of sensitivity for detection of clonalitv with I he Southern blot methoil. Humphries et al suggest that the failure of Southern blot analysis to detect clonality of plasma c.ells in some patients in their study brings into question the assumption that bone marrow plasma cells in myeloma are monoclonal. In support of their viewpoini, they cite studies of other investigators who failed to detect immunoglobulin gene rearrangements in my&ma bone marrow in which more than 1 O%, and usually more than 20%, plasma cells were present. In these studies, as wet1 as in the study of Humphries et al, Ig rearrangements were most frequently found in advanced disease with greater than 50% plasma cells. Some technical considerations that may explain the lack of Ig gene rearrangements found in myeloma cases
941
HUMAN PATHOLOGY
Volume 22, No. 10 (October
1991)
FIGURE 1. lmmunoperoxidase stain of bone marrow showing a large predominance of plasma cells stained for lambda light chain (left), but not stained for kappa light chain (right), in a newly diagnosed patient with plasma cell myeloma. The large number of plasma cells was not appreciated in the bone marrow biopsy stained with hematoxylin-eosin.
loss of plasma cells during the cell separation procedure, poor Southern blot transfer of rearranged bands (most commonly seen with high molecular weight bands), co-migration of rearranged bands with germline bands, and incomplete digestion of DNA. In studying 3 1 cases of myeloma with a JH probe, Zaccaria et al encountered five cases in which it was necessary to use five restriction enzymes before a rearranged band was detected, usually as a very faint band after digestion with BgI II enzyme.’ The DNA of two additional patients continued to reveal no gene rearrangements. In another series of 21 patients with B-cell malignancies other than myeloma, the same investigators found a discrete, intense rearranged band in all cases. Thus, the difficulty in obtaining rearranged bands appeared to be specific for myeloma. Zaccaria et al suggested that the unusual gene rearrangement results in myeloma patients are consistent with partial hybridization of the JH probe because of a more or less wide deletion of the area located 3’ to the J genes, which contains the enhancer.’ Other possible biologic explanations for the lack of Ig gene rearrangements include (1) a preponderance of nonmalignant plasma cells, presumably reactive to the neoplasm or induced to proliferate by a similar mechanism, such as interleukin-6 stimulation, which is increased in myeloma”; (2) deletion of Ig gene loci during gene rearrangement; (3) somatic Ig gene mutations during clonal evolution of the tumor cell population’; and (4) origin of the tumor cells from an early progenitor cell that has not yet undergone gene rearrangement. Of these, the latter is potentially the most interesting and may help to explain the frequent finding of myeloid antigens on myeloma cells,” megaloblastic or dysplastic changes in nonlymphoid cells in myeloma bone marrow,” and abnormalities of B lymphocytes in the blood of myeloma patients.’ The latter explanation seems unlikely since peripheral blood B lymphocytes of myeloma patients have been shown to be part of the malignant clone, having immunoglobulin gene rearrangements identical to those are the selective
942
seen in bone marrow plasma cells.x,!’ Peripheral
blood immunoglobulin gene rearrangements were significantly more common in patients with active disease compared with remission status and higher in untreated compared with treated patients. Patients with MGUS did not have gene rearrangements, although two myeloma patients with gene rearrangements had a preceding history of MGUS, suggesting that its detection may be an early sign of progression to multiple myeloma.” We are left without a biologic explanation for the production of a monoclonal paraprotein in the absence of Ig gene rearrangement, or the lack of correlation between the monoclonal serum Ig heavy or light chain isotype and the presence or absence of gene rearrangements detected by Southern blot method in the study of Humphries et al.’ Therefore, technical limitations of the Southern blot method remain the most likely explanation for these unexpected results. Failure to detect clonal gene rearrangements by the Southern blot method has been observed in other lymphoid malignancies, in which it has been unclear whether this was a limitation of the method used OI
provided new insight into the biology of the disease. Weiss et al detected no T-cell receptor (TCR) gene rearrangements in a group of seven peripheral T-cell lymphomas, five of which arose in the upper respiratory tract.“’ Ralfkaier et al noted TCR gene rearrangements in the tumor and plaque stages of cutaneous T-cell lymphoma (CTCL) but not in early patch-stage lesions.” These investigators suggested that early stage CTCL may be multiclonal. Whittaker et al did not detect TCR or Ig gene rearrangements in lymphomatoid papulosis type A lesions and suggested that these lesions have a nonT-, non-B-cell origin.” If correct, this finding would be most interesting since lymphomatoid papulosis type A lesions closely resemble Hodgkin’s disease, in which the nature of the malignant cell is controversial and TCR or Ig gene rearrangements often are not found.“’ It is often argued that negative gene rearrangement results, such as the examples cited above, are due to the
EDITORIAL
(Marshall E. Kadin)
limited sensitivity of the Southern blot method. This limitation can be overcome with the polymerase chain reaction (PCR) technique when suitable primers can be made. Indeed, PCR has been used successfully to detect clonal TCR gene rearrangements in some early patchstage lesions of CTCI, (unpublished observations) and lvmphomatoid papulosis type A.” A similar approach should be useful in studies of other lymphoproliferative disorders and can be used by Humphries et al and others to determine whether all cases of plasma cell myeloma are or are not monoclonal. Meanwhile, this interesting study emphasizes that the diagncjsis of a particular disease should not be determined by ;I single laboratory test, no matter how filshionable. Rather, each test, particularly those based on new u’nproven technologies, should be interpreted in context of other laboratory results, the patient’s history, and the c.linical findings. In the final analysis, if it waddles and quacks like a duck, it probably is a duck!
-&. Tonegawa S: Somatic :w2:575-581, l!W(
and Harvard
Medical
of antibodt
diver-sity. Narurr
5. Epstein ,J. Xiao H, He X-Y: Markers of multiple hematopoieticcell lineages in multiple myeloma. N Engl J Med .3??:664-668. 1900 6. Hobbs JR, Jacobs JR, Kremenchuzky S. ?t al: Inc idenre and pathogencsis of megaloblastic erythropoiesis in tmlltiple myelonra. J Clin Path01 20:699-705. I967 7. MeUstedt H. Hammarsrrom S. Helm G: Nlcmoclonal lymphocvte populations in human plasma cell rnveloma. Uin Exp Immunol 17:371-384. 1974 X. Berenson J, Wang R, Kim k, et al: F.Gdwce fox peripheral blood B lymphocytr but not T lynlphocvtr involwment in multiple mveloma. Blood 70:1.550-155.3. 1987 9. Chiu EKU’, Grneshaguru K. Hoffbr-and .4’1’. CI al: Cirwlating monoclonal B Ivmphocytes in multiple nrvrloma. Br J Hxmatol 72: 28-Y I. 1!I89 10. Weiss I.M. Picker LJ. Grogan I‘M, ct al: .\bsencr of clonal beta and gamma T-cell receptor- gene rearrangt‘meuts it] ‘, subset 01. peripheral T-cell lymphomas. Am J Pathol 1:10:436-q-12, I988
11. Ralfkaicr E. O’Connor 01 cutaneous T-cell lymphoma. Hospital
generation
School
NTJ, Crick 5.4. et al: (Genotkpic analysis J Invest Dermatol X8:762-765. I987
I?. Whittaker S. Smith N, Jones RR, ct aI, Analysis of beta. K&mma, and drlra T-cell receptor gem’s in I~rnphc)rnaroid papulosis: (:rllular basis of two distin1.t histolqqic wbsrts. J Invest Drrmatol 067X6-791. 1991 13. Raghavachar A. Binder T. Bartram CR: Inllnunoglob~~lin and TC ell ret rptol. gene I-earrangements in Hodgkin’\ disease. (:ancet Rr?, 4X:3591-Y5W, I!QCi 14. Kadin ME, Davis TH, Miller-Cassman R: Lymphomatoid papulosis. cutaneous T-cell lymphoma and Hodgkin’3 disease derivrd from a common T-cell clone. Blood WI 7X. I WI (wppl) (abstr) (in l”‘“5)
943