Endothelial Cells in B-Cell Lymphomas
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《新英格兰医药杂志》
To the Editor: Although Streubel et al. (July 15 issue)1 provide evidence that lymphoma-associated endothelial cells have genetic changes of lymphomas, their data do not fully support this conclusion. Fluorescence in situ hybridization (FISH) studies use fluorescent probes that produce pinpoint images according to the focal plane of analysis. Nonspecific signals, characteristic of such preparations, are a potential source of false positivity. Such aberrant signals, which could falsely label endothelial cells, are not accounted for statistically in this article. Specifically, the mean and range of false positive signals of the controls are not reported.
Another concern is whether endothelial cells are truly being identified. The putative CD31-positive endothelial cells shown in Figure 1 of the article lack a discernible fluorescent signal with membrane distribution that is expected of membrane antigen CD31. Of greatest concern is the fact that the two-dimensional images may not accurately depict the three-dimensional relationships of cells. Consequently, FISH signals may be presumed to be of endothelial origin when, in fact, overlying lymphocytes are being localized. Methods with higher three-dimensional spatial resolution, such as confocal microscopy, multifluor microscopy,2 or electron microscopy,3 could address this possibility.
Lawrence D. True, M.D.
Karen Swisshelm, Ph.D.
University of Washington
Seattle, WA 98195-6100
ltrue@u.washington.edu
References
Streubel B, Chott A, Huber D, et al. Lymphoma-specific genetic aberrations in microvascular endothelial cells in B-cell lymphomas. N Engl J Med 2004;351:250-259.
Rubin MA, Zerkowski MP, Camp RL, et al. Quantitative determination of expression of the prostate cancer protein alpha-methylacyl-CoA racemase using automated quantitative analysis (AQUA): a novel paradigm for automated and continuous biomarker measurements. Am J Pathol 2004;164:831-840.
Fidler IJ, Ellis LM. Neoplastic angiogenesis -- not all blood vessels are created equal. N Engl J Med 2004;351:215-216.
The authors reply: To exclude false positivity, we investigated endothelial cells in 20 reactive lymph nodes for each set of probes. The cutoff value ranged from 3 percent to 7 percent (median, 5 percent). In the endothelial cells of the tumor samples, translocations were found in 15 to 85 percent (median, 37 percent), which is clearly above the cutoff value. The results were confirmed by two independent sets of translocation probes. In addition, reactive T-cells in the B-cell tumors were negative for the translocations.
The images are not strictly two-dimensional, because they are the result of a stepwise capturing of a row of images at several levels within a section with a thickness of about 5 μm. This may also influence the membrane staining for CD31, which in addition is impaired by denaturation steps, because as in the fluorescence immunophenotyping and interphase cytogenetics (FICTION) procedure, FISH is performed after the immunostaining.
The finding that endothelial cells cultured from lymphomas also harbor the specific translocations strongly supports the in situ results.
Berthold Streubel, M.D.
Oswald Wagner, M.D.
Andreas Chott, M.D.
Medical University of Vienna
A-1090 Vienna, Austria
andreas.chott@meduniwien.ac.at
Another concern is whether endothelial cells are truly being identified. The putative CD31-positive endothelial cells shown in Figure 1 of the article lack a discernible fluorescent signal with membrane distribution that is expected of membrane antigen CD31. Of greatest concern is the fact that the two-dimensional images may not accurately depict the three-dimensional relationships of cells. Consequently, FISH signals may be presumed to be of endothelial origin when, in fact, overlying lymphocytes are being localized. Methods with higher three-dimensional spatial resolution, such as confocal microscopy, multifluor microscopy,2 or electron microscopy,3 could address this possibility.
Lawrence D. True, M.D.
Karen Swisshelm, Ph.D.
University of Washington
Seattle, WA 98195-6100
ltrue@u.washington.edu
References
Streubel B, Chott A, Huber D, et al. Lymphoma-specific genetic aberrations in microvascular endothelial cells in B-cell lymphomas. N Engl J Med 2004;351:250-259.
Rubin MA, Zerkowski MP, Camp RL, et al. Quantitative determination of expression of the prostate cancer protein alpha-methylacyl-CoA racemase using automated quantitative analysis (AQUA): a novel paradigm for automated and continuous biomarker measurements. Am J Pathol 2004;164:831-840.
Fidler IJ, Ellis LM. Neoplastic angiogenesis -- not all blood vessels are created equal. N Engl J Med 2004;351:215-216.
The authors reply: To exclude false positivity, we investigated endothelial cells in 20 reactive lymph nodes for each set of probes. The cutoff value ranged from 3 percent to 7 percent (median, 5 percent). In the endothelial cells of the tumor samples, translocations were found in 15 to 85 percent (median, 37 percent), which is clearly above the cutoff value. The results were confirmed by two independent sets of translocation probes. In addition, reactive T-cells in the B-cell tumors were negative for the translocations.
The images are not strictly two-dimensional, because they are the result of a stepwise capturing of a row of images at several levels within a section with a thickness of about 5 μm. This may also influence the membrane staining for CD31, which in addition is impaired by denaturation steps, because as in the fluorescence immunophenotyping and interphase cytogenetics (FICTION) procedure, FISH is performed after the immunostaining.
The finding that endothelial cells cultured from lymphomas also harbor the specific translocations strongly supports the in situ results.
Berthold Streubel, M.D.
Oswald Wagner, M.D.
Andreas Chott, M.D.
Medical University of Vienna
A-1090 Vienna, Austria
andreas.chott@meduniwien.ac.at