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Clonality analysis of B-cell lymphomas
HUMAN PATHOLOGY
Volume 26, No. 9 (September 1995)
Dr Groisman's previous publication in Human Pathology, and we encourage future investigators to employ CEA and other immunohistochemical techniques to better define these two related but distinct entities.
TIM C. D~ss, MSc MARGARETASHTON-KEY,DmRCPATH LANG-XINGPAN, PHD PETER G. ISAACSON,DM Department of Histopathology University College London Medical School University Street London, UK
F. RAAFAT
N.J. GREEN K. A. NATHAVlTHARANA I. W. BOOTH Department of Pathology The Institute of Child Health The Children's Hospital Birmingham, UK
1. Segal GH, Jorgensen T, Masih AS, et al: Optimal primer selection for clonality assessment by polymerase chain reaction analysis: L Low grade B-cell lyrnphoproliferative disorders of nonfollicular center cell type. HUM PATHOL 25:1269-1275, 1994 2. Segal GH, Jorgensen T, Scott M, et al: Optimal primer selection for clonality assessment by polymerase chain reaction analysis: II. Follicular lymphomas. HUM PATHOL 25:1276-1282, 1994 3. Diss TC, Peng HZ, Wotherspoon AC, et al: Detection of monoclonality in low-grade B-cell lymphomas using the polymerase chain reaction is dependent on primer selection and lymphoma type. J Pathol 169:291-295, 1993 4. Ling FC, Clarke CE, Corbett WEN, et al: Sensitivity of PCR in detecting monoclonal B cell proliferations. J Clin Pathol 46:624-627, 1993 5. Liu J, Johnson RM, Traweek ST: Rearrangement of the BCL-2 gene in follicular lymphomas. Diagn Mol Pathol 2:241-247, 1993
I. Raafat F, Green NJ, Nathavitharana KA, et al: Intestinal microvillous dystrophy: A variant of microvillous inclusion disease or a new entity? HUM PATHOL 25:1243-1248, 1994 2. Groisman GM, Ben-Izhak O, Schwersenz A, et al: The value of polydonal carcinoemb~yonic antigen inlmunostaining in the diagnosis of microvillous inclusion disease. HUM PATHOL 24:1232-1237, 1993
Clonality Analysis of B-Cell Lymphomas To the E d i t o r : l W e agree with Segal et al~.2 that the best polymerase chain reaction (PCR) strategy for confirmation of monoclonality in B-cell lymphoproliferative disorders is use of framework three and joining region (FRIII/JH) primers and that bcl-2 major breakpoint (MBR/JH) primers should be added to the analysis of putative follicular lymphomas. Our own studies using high molecular weight DNA from lymphomas that were preselected for monoclonality, have shown almost identical detection rates (96% for nonfollicle center, non-MALT type lymphomas and 70% for follicular lymphomas using FRIII/JH alone ~ and 83% for follicular lymphomas using FRIII/JH and MBR/JH combined [unpublished] ). However, other studies, especially those using paraffin processed samples, have shown lower sensitivities. 4'5 Our current studies using consecutive cases of follicular lymphoma taken from the histopathology archives, show a similarly reduced sensitivity of around 60% (using both FRIII/JH and MBR/JH primers), despite the fact that all samples were able to support amplification of FRIII/JH products. We believe that a significant factor in the reduction in sensitivity seen by us and others is the selection of cases rather than optimization of technique. Furthermore, the alternative primers employed by Segal et al, to increase the detection rate, are of limited benefit as the target sequences are too long to be reliably amplified from routine formalin-fixed, paraffin-embedded biopsy specimens. We consider it important that researchers embarking on clonality analysis of follicular and other lymphomas using routine histological samples are aware that the sensitivities achieved by Segal and coauthors will probably not be attainable and that PCR will be of benefit to diagnosis only when positive.
The above letter was referred to the authors of the article in question, who offer the following reply: To the Editor:--We appreciate the comments of Diss et al and agree that formalin-fixed, paraffin-embedded tissue is generally less optimal for polymerase chain reaction (PCR) analysis. Our studies, however, were focused on determining rational primer selection for the detection of clonality in lowgrade B-cell lymphoproliferative disorders in an optimal setting (ie, high molecular weight DNA extracted from nonformalin fixed tissue). The series was specifically designed in this fashion to prevent the introduction of formalin-related bias. We disagree with Diss and colleagues on the issue of the potential use of the reserve primer sets on fixed tissue. A study from 1993, by Kuppers et all has clearly documented that the immunoglobulin heavy chain gene variable region FRI primer set reactions are very capable of amplifying clonal B-cell populations from formalin-fixed, paraffin-embedded samples. Again, as with the first-line primers, one should expect a subset of cases to be falsely negative as a consequence of formalin-induced DNA damage.'
GLENN H. SEGAL,DO Department of Pathology University of Utah Health Sciences Center Salt Lake City, UT RAVE C. BRAYLAN,MD Department of Pathology and Laboratory Medicine University of Florida College of Medicine Gainesville, FL 1. Kuppers R, Zhao M, Rajewsky K, et al: Detection of clonal B-cell populations in paraffimembedded tissues by polymerase chain reaction. Am J Pathol 143:230-239, 1993
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Volume 26, No. 9 (September 1995)
Dr Groisman's previous publication in Human Pathology, and we encourage future investigators to employ CEA and other immunohistochemical techniques to better define these two related but distinct entities.
TIM C. D~ss, MSc MARGARETASHTON-KEY,DmRCPATH LANG-XINGPAN, PHD PETER G. ISAACSON,DM Department of Histopathology University College London Medical School University Street London, UK
F. RAAFAT
N.J. GREEN K. A. NATHAVlTHARANA I. W. BOOTH Department of Pathology The Institute of Child Health The Children's Hospital Birmingham, UK
1. Segal GH, Jorgensen T, Masih AS, et al: Optimal primer selection for clonality assessment by polymerase chain reaction analysis: L Low grade B-cell lyrnphoproliferative disorders of nonfollicular center cell type. HUM PATHOL 25:1269-1275, 1994 2. Segal GH, Jorgensen T, Scott M, et al: Optimal primer selection for clonality assessment by polymerase chain reaction analysis: II. Follicular lymphomas. HUM PATHOL 25:1276-1282, 1994 3. Diss TC, Peng HZ, Wotherspoon AC, et al: Detection of monoclonality in low-grade B-cell lymphomas using the polymerase chain reaction is dependent on primer selection and lymphoma type. J Pathol 169:291-295, 1993 4. Ling FC, Clarke CE, Corbett WEN, et al: Sensitivity of PCR in detecting monoclonal B cell proliferations. J Clin Pathol 46:624-627, 1993 5. Liu J, Johnson RM, Traweek ST: Rearrangement of the BCL-2 gene in follicular lymphomas. Diagn Mol Pathol 2:241-247, 1993
I. Raafat F, Green NJ, Nathavitharana KA, et al: Intestinal microvillous dystrophy: A variant of microvillous inclusion disease or a new entity? HUM PATHOL 25:1243-1248, 1994 2. Groisman GM, Ben-Izhak O, Schwersenz A, et al: The value of polydonal carcinoemb~yonic antigen inlmunostaining in the diagnosis of microvillous inclusion disease. HUM PATHOL 24:1232-1237, 1993
Clonality Analysis of B-Cell Lymphomas To the E d i t o r : l W e agree with Segal et al~.2 that the best polymerase chain reaction (PCR) strategy for confirmation of monoclonality in B-cell lymphoproliferative disorders is use of framework three and joining region (FRIII/JH) primers and that bcl-2 major breakpoint (MBR/JH) primers should be added to the analysis of putative follicular lymphomas. Our own studies using high molecular weight DNA from lymphomas that were preselected for monoclonality, have shown almost identical detection rates (96% for nonfollicle center, non-MALT type lymphomas and 70% for follicular lymphomas using FRIII/JH alone ~ and 83% for follicular lymphomas using FRIII/JH and MBR/JH combined [unpublished] ). However, other studies, especially those using paraffin processed samples, have shown lower sensitivities. 4'5 Our current studies using consecutive cases of follicular lymphoma taken from the histopathology archives, show a similarly reduced sensitivity of around 60% (using both FRIII/JH and MBR/JH primers), despite the fact that all samples were able to support amplification of FRIII/JH products. We believe that a significant factor in the reduction in sensitivity seen by us and others is the selection of cases rather than optimization of technique. Furthermore, the alternative primers employed by Segal et al, to increase the detection rate, are of limited benefit as the target sequences are too long to be reliably amplified from routine formalin-fixed, paraffin-embedded biopsy specimens. We consider it important that researchers embarking on clonality analysis of follicular and other lymphomas using routine histological samples are aware that the sensitivities achieved by Segal and coauthors will probably not be attainable and that PCR will be of benefit to diagnosis only when positive.
The above letter was referred to the authors of the article in question, who offer the following reply: To the Editor:--We appreciate the comments of Diss et al and agree that formalin-fixed, paraffin-embedded tissue is generally less optimal for polymerase chain reaction (PCR) analysis. Our studies, however, were focused on determining rational primer selection for the detection of clonality in lowgrade B-cell lymphoproliferative disorders in an optimal setting (ie, high molecular weight DNA extracted from nonformalin fixed tissue). The series was specifically designed in this fashion to prevent the introduction of formalin-related bias. We disagree with Diss and colleagues on the issue of the potential use of the reserve primer sets on fixed tissue. A study from 1993, by Kuppers et all has clearly documented that the immunoglobulin heavy chain gene variable region FRI primer set reactions are very capable of amplifying clonal B-cell populations from formalin-fixed, paraffin-embedded samples. Again, as with the first-line primers, one should expect a subset of cases to be falsely negative as a consequence of formalin-induced DNA damage.'
GLENN H. SEGAL,DO Department of Pathology University of Utah Health Sciences Center Salt Lake City, UT RAVE C. BRAYLAN,MD Department of Pathology and Laboratory Medicine University of Florida College of Medicine Gainesville, FL 1. Kuppers R, Zhao M, Rajewsky K, et al: Detection of clonal B-cell populations in paraffimembedded tissues by polymerase chain reaction. Am J Pathol 143:230-239, 1993
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