RAPID COMMUNICATION |
Correspondence to: Gerard J. Nuovo, Ohio State Medical Center, Dept. Pathology, Div. Anatomic Pathology, E-411 Doan Hall, 410 W. 10th Ave., Columbus, OH 43210-1228.
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Summary |
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A definitive diagnosis of T-cell lymphoma may be contingent on the rearrangement profile of the T-cell receptor. This is most accurately done by molecular analysis of the beta-chain of the T-cell receptor (TCRß) by Southern blotting hybridization that requires unfixed tissue. We describe a reverse transcriptase in situ PCR (RT in situ PCR) method that permits the target-specific direct incorporation of the reporter nucleotide into the different transcripts that comprise the TCRß, using paraffin-embedded, formalin- fixed tissue. Each of the 25 possible Vß segment rearrangments was documented in three lymph nodes with nonspecific lymphadenitis, with clonal expansion evident in a case of metastatic melanoma. Monoclonal expression was documented in seven tissues diagnostic of a T-cell lymphoma. We analyzed five additional tissues for which a definitive diagnosis of T-cell vs B-cell lymphoma could not be rendered on the basis of histological, immunohistological, and flow cytometric analysis. RT in situ PCR for TCRß expression with CD3 co-labeling demonstrated which of these lesions was a B-cell-rich T-cell lymphoma. We conclude that the RT in situ PCR methodology will allow the routine determination of monoclonal vs multiclonal expression patterns of the TCRß using archival paraffin-embedded tissues.
(J Histochem Cytochem 49:139145, 2001)
Key Words: gene rearrangement, T-cell receptor, lymphoma, in situ PCR
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Introduction |
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THE DIAGNOSIS of a T-cell lymphoma may be difficult on the basis of cytological, histological, and immunophenotypic criteria (-chain, due to the relatively few variable region-defined families (
In situ hybridization (ISH) analysis of TCRß rearrangement is problematic for several reasons. A major difficulty is that standard ISH for either TCRß RNA or DNA would not be likely to detect the low copy number of the respective nucleic acids (
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Materials and Methods |
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Tissue Preparation
Fifteen formalin-fixed, paraffin-embedded tissues were retrieved from the surgical pathology archives of the Department of Pathology at Ohio State University Medical Center. The tissues included three lymph nodes obtained from resections of melanoma, breast cancer, and colon cancer. Metastastic melanoma was evident in the lymph node from the first case, whereas there was no evidence of metastastic disease in the latter two cases. We also analyzed seven tissues from five patients diagnosed with T-cell lymphoma on the basis of the clinical, histological, immunohistochemical and, when available, flow cytometric data. These seven tissues consisted of an HTLV-1-positive T-cell leukemia/lymphoma that had infiltrated the bone marrow, an angioimmunoblastic T-cell lymphoma in a lymph node and in the bone marrow from the same patient, one case of peripheral T-cell lymphoma of the lung, two biopsies of mycosis fungoides from the same patient (although the second biopsy after therapy was deemed suggestive but not diagnostic of mycoses fungoides), and one lymphomatoid papulosis of the skin. In addition, we evaluated five cases of lymphoma in which the distinction of T-cell-rich B-cell lymphoma vs B-cell-rich T-cell lymphoma could not be made on the basis of the clinical and pathological data.
RT In Situ PCR
Our RT in situ PCR protocol has been previously described (
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Immunohistochemistry
Our immunohistochemical protocol has been previously published (
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Results |
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To determine whether the histological distribution of TCRß expression was equivalent to the distribution of reactive T-cells, as defined by CD3 positivity, three lymph nodes were examined that were obtained from patients as part of the surgical treatment of their malignancy (breast and colon carcinoma and melanoma). The total polyclonal T-cell population in the lymph nodes would be expected to demonstrate each of the different Vß rearrangments of the TCRß (
For each of the three lymph nodes, a signal was evident with each of the five oligoprimer groups that in total comprised the entire TCRß sequence. More detailed analysis showed that TCRß mRNA could be demonstrated with each of the 25 individual Vß family-specific primers. The histological distribution of the T-cell receptor mRNAs was the same as that of the T-cell marker CD3. The percentage of T-cells that expressed a given TCRß rearrangement was equivalent (37% of the total T-cells) for each of the 25 Vß families for the lymph nodes from the patients with non-metastatic breast and colon cancer. However, in the lymph node with metastatic melanoma, certain transcription segments, specifically Vß5.1 and Vß16, were present in disproportionately greater numbers relative to the other Vß families (four to five times as many positive cells), although each of the 25 was present (data not shown). The cells expressing the TCRß Vß16 and Vß5.1 transcripts were in close proximity to the metastatic melanoma, and therefore their relative increase may represent oligoclonal expansion induced by tumor-related antigens.
We next tested seven tissues for which in each case an unequivocal diagnosis of T-cell lymphoma was made on the basis of the clinical, histological, and immunophenotypic analysis. This was done to determine whether the RT in situ PCR assay could demonstrate the expected monoclonality in these samples. The analyses were done blindly with respect to the histological diagnosis and which cases corresponded to the same patient (i.e., Cases 1 and 4 each had two biopsies taken at different time points; Table 2). In Cases 1 and 3, independent confirmation of T-cell clonality was demonstrated at outside laboratories using TCR analysis and HTLV-1 clonal insertion, respectively. These data are compiled in Table 2. In each case, a signal was evident in over 95% of the CD3-positive cells with only one of the 25 TCRß Vß family-specific primers. In these seven cases of unequivocal T-cell lymphoma, no other Vß family primer produced a signal in more than 1% of the T-cells, presumably reflecting reactive non-neoplastic T-cells among the malignant monoclonal T-cells. A representative case of angioimmunoblastic T-cell lymphoma is presented in Fig 1. Note that a signal was evident in the neoplastic cells in the lymph node and bone marrow specimen, taken several months apart from the same patient, using the TCRß Vß2-specific primers. In addition, the monoclonal rearrangement was localized in the bone marrow to the neoplastic T-cells and not to the adjoining normal hematopoetic elements. This serves as an internal control for the specificity of the RT in situ PCR assay. Furthermore, the signal was lost if HPV specific primers were employed or if RT in situ PCR was done with the Vß2 primer set after a 30-min pretreatment with RNase (Fig 1), demonstrating that the signal was RNA-based. Also note that in Case 4 the same T-cell receptor rearrangement (Vß22) was noted in the skin biopsy of mycoses fungoides and the subsequent biopsy done after therapy, in which the atypical lymphoid infiltrate was deemed suggestive but not diagnostic of mycoses fungoides due to the lack of epidermal invasion, presumably as a consequence of therapy.
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The final part of the study was use of the RT in situ PCR assay in five cases for which the clinical and pathological data were unequivocal for lymphoma but for which a definitive distinction of B-cell-rich T-cell lymphoma vs T-cell-rich B-cell lymphoma could not be made because of the presence of atypical CD20- and CD3-positive cells. In three of the cases, a monoclonal T-cell proliferation consistent with a T-cell lymphoma (Vß1, Vß12, Vß16, respectively, Fig 2) was seen after RT in situ PCR, whereas in the other two cases a polyclonal T-cell population was identified, consistent with a T-cell-rich B-cell lymphoma; these latter two cases showed monoclonal expression of Ig light chain kappa by RT in situ PCR (unpublished data). Co-labeling experiments in the former three cases confirmed that the cells that showed a monoclonal expression pattern of TCRß did co-label with CD3 (data not shown).
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Discussion |
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This study showed that the RT in situ PCR assay for TCRß rearrangment could detect transcripts from each of the 25 Vß families in cells from lymph nodes removed as part of the surgical management of carcinomas or melanoma. Furthermore, the assay could demonstrate the specific monoclonal TCR Vß rearrangement present in seven archival paraffin-embedded, formalin-fixed tissues diagnostic of a T-cell neoplasm. In two of these cases (Case 1 and Case 4; Table 2), the two separate biopsies from the same patient done blindly each showed exactly the same TCRß rearrangement. Importantly, the assay could differentiate those cases of lymphoma for which the histological and immunohistochemical data could not distinguish between a B-cell and a T-cell lymphoma.
It is well documented that there may be limited gene usage of both the variable and ß TCR sequences in T-lymphocytes in reactive inflammatory processes, such as multiple sclerosis (
The surgical pathologist must often deal with biopsies in which a T-cell lymphoma is considered in the differential diagnosis. The standard practice in such instances is to perform immunohistochemistry for a variety of T- and B-cell markers. However, even the demonstration of a predominant T-cell phenotype does not rule out a reactive non-neoplastic process nor the presence of a T-cell-rich B-cell lymphoma. Indeed, one of the cases in this study that was shown by RT in situ PCR to be a T-cell lymphoma was initially diagnosed as T-cell-rich B-cell lymphoma by an expert hematopathologist from an outside laboratory, underscoring the difficulty in such cases when clonality data are not available because of insufficient tissue. The RT in situ gene rearrangment analysis showed a monoclonal T-cell population and a polyclonal B-cell infiltrate in three of the five cases, demonstrating that these lesions were B-cell-rich T-cell lymphomas; the distinction has important clinical implications (
Received for publication September 5, 2000; accepted October 4, 2000.
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