The rapid detection of clonal T-cell proliferations in patients with lymphoid disorders.

A series of T-cell proliferations in peripheral blood, bone marrow, or tissue samples were analyzed for clonality. The technique used employs the polymerase chain reaction to amplify portions of the rearranged T-cell receptor beta chain genes, using primers recognizing conserved sequences of the variable, diversity, and joining region segments. We examined 17 cases of T-cell lymphoma or leukemia; a clone was identified in 13 cases (76%) overall and in 7 of 8 cases (87.5%) in which both beta-chain alleles were known to be rearranged, as shown by restriction enzyme analysis. No clonal rearrangements were detected in samples from 13 non-T-cell disorders, including B-cell lymphomas, reactive lymphoid proliferations, and nonlymphoid tumors. This method is useful for detecting clones in thymic and post-thymic T-cell malignancies and has the advantages of being extremely rapid (a result is obtained within hours of the biopsy procedure), requiring no radiolabeling, using only a small amount of tissue, and being applicable to formalin-fixed, paraffin-embedded tissue.     

A strategy for demonstrating the clonal origin of small numbers of T lymphocytes in histopathological specimens

A strategy is described for detecting large T-cell clones among small numbers of lymphoid cells in fresh or formalin-fixed tissue samples using the polymerase chain reaction (PCR) to amplify and identify rearrangements of the V and J genes of the T-cell gamma receptor. Following hybridization of primers with gene-specific sequences at judiciously selected locations on each of the eight potentially active V and five potentially active J genes, the PCR can theoretically amplify DNA segments which span the join between rearranged V and J genes and are of approximately 384 different sizes, each segment size reflecting different gamma gene clonal rearrangements. Large monoclonal populations of T lymphocytes, indicated by excessive amounts of particular PCR segment sizes, can be further characterized by direct nucleotide sequencing of the hypervariable N regions of these segments, and the presence of such clones can be confirmed directly in tissue sections by in situ hybridization with N region-specific oligonucleotide probes.     

Characterization of the breakpoint of a t(14;14)(q11.2;q32) from the leukemic cells of a patient with T-cell acute lymphoblastic leukemia

The leukemic cells and derivative cell line from a 74-year-old male with T-cell acute lymphoblastic leukemia showed chromosomal abnormalities including a t(14;14)(q11.2;q32). This translocation is characteristic of a variety of T-cell malignancies, particularly T-cell prolymphocytic leukemia and the clonal proliferations of peripheral T cells in patients with ataxia-telangiectasia. Using DNA probes that spanned the T-cell receptor alpha chain (TCRA) joining (J) locus, the DNA rearrangement caused by the translocation was identified, cloned, and sequenced. The breakpoint shows site-specific juxtaposition of a TCRA joining segment and DNA from a region of 14q32 centromeric to the immunoglobulin heavy chain locus. Comparison of restriction map and nucleotide sequence from this translocation with other related chromosomal breakpoints suggests a dispersion of breakpoints throughout the 14q32 region.     

Positive immunoglobulin gene rearrangement study by the polymerase chain reaction in a colonic adenocarcinoma.

The detection of clonal rearrangements of the immunoglobulin heavy chain gene by the polymerase chain reaction provides a rapid method to differentiate monoclonal from polyclonal B-lymphocyte proliferations. It has been shown to be highly specific and so far, no false-positive results have been described. A case of a poorly differentiated colonic adenocarcinoma that showed a "false positive" clonal immunoglobulin heavy chain gene rearrangement by the polymerase chain reaction technique is reported. DNA contamination was unlikely because of the strict adherence to the laboratory polymerase chain reaction protocol and also the repeated demonstration of the same amplified band in a separate experiment using DNA extracted from another piece of tumor tissue. The apparent monoclonal immunoglobulin heavy chain gene rearrangement in the first polymerase chain reaction may be related to a combination of the paucity of lymphoid cells in the tissue sample and the presence within this small number of lymphocytes of a clonal reactive cell population. It is, therefore, important to correlate the routine microscopic and immunohistochemical findings in the interpretation of polymerase chain reaction results, especially when working with nonlymphoid tumors and lymphocyte-poor lesions.     

Polymerase chain reaction (PCR) amplification demonstrates the absence of human T-cell lymphotrophic virus (HTLV)-I specific pol sequences in peripheral T-cell lymphomas

HTLV-I seronegative patients in nonendemic areas have been described with T-cell proliferations the DNA of which contains specific HTLV-I viral sequences. We have looked for the presence of HTLV-I DNA sequences in 27 HTLV-I seronegative patients with peripheral T-cell lymphomas, distinct from adult T-cell leukemia (ATL), and four HTLV-I seropositive patients, three with an ATL and one with a tropical spastic paraparesis. Using HTLV-I pol specific primers, the genomic DNA from peripheral blood mononuclear cells and lymph nodes massively infiltrated by tumor cells was analyzed by the enzymatic gene amplification procedure. In contrast to the peripheral blood lymphocytes from the four HTLV-I seropositive patients, the peripheral T-cell lymphoma samples did not harbor HTLV-I pol sequences. The data show that the detection of HTLV-I nucleotide sequences by the polymerase chain reaction correlates with serologic analysis in this series.     

Evaluation of biotinylated DNA probes for the detection of gene rearrangements in clinical specimens

To determine whether a nonisotopic procedure is suitable for analyzing clinical specimens for gene rearrangements, the authors hybridized DNA from 15 specimens of lymphoid tissue with biotinylated DNA probes directed to J beta I + J beta II (T-cell receptor beta chain gene), JH (immunoglobulin gene heavy chain J region), and J kappa (immunoglobulin gene kappa light chain J region). Five cases of benign lymphoid hyperplasia, one case of dermatopathic lymphadenopathy, and one case of small noncleaved follicular center cell lymphoma had germline hybridization patterns when digested with Bam HI, Eco RI, and Hind III restriction endonucleases. Four cases of B-cell lymphoma and three cases of T-cell lymphoma had clearly detectable rearrangements of the genes for immunoglobulin or the T-cell receptor or both. One case of dermatopathic lymphadenopathy had a faint, clonal rearrangement of the T-cell receptor after digestion with Eco RI and Bam III. The authors conclude that biotinylated DNA probes can be useful for analyzing gene rearrangements in clinical specimens.     

Absence of clonal beta and gamma T-cell receptor gene rearrangements in a subset of peripheral T-cell lymphomas.

The authors describe a set of seven peripheral T-cell lymphomas that lack detectable rearrangements of T-cell receptor (TCR) genes. All cases showed antigenic profiles consistent with T-cell lymphoma, including expression of Leu-5 (CD2) antigen. However, few other T-lineage markers were found, and none of the cases tested (6 of 7) bound antibody recognizing the constant region of the beta TCR protein. Each case showed exclusively germline configurations of DNA for the beta TCR genes in Southern blot analyses with the use of several different combinations of restriction enzymes and DNA hybridization probes. One case contained clonal rearrangements of the gamma TCR gene and of the immunoglobulin heavy chain gene. Our results suggest that certain cases of peripheral T-cell lymphoma may lack rearrangements of TCR genes--particularly those cases expressing restricted numbers of T-lineage antigens. In view of these findings, failure to detect rearrangements of TCR genes by Southern blot analyses is not necessarily inconsistent with malignant lymphocytic proliferations in T-lineage neoplasia.     

Detection of T-cell receptor-gamma gene rearrangement in lymphoproliferative disorders by temperature gradient gel electrophoresis

OBJECTIVE: Polymerase chain reaction amplification of DNA for T-cell receptor (TCR) gene rearrangement analysis is helpful in the evaluation of T-cell lymphoproliferative disorders. Detection of polymerase chain reaction products is limited by the poor resolution of bands analyzed by agarose or polyacrylamide gel electrophoresis. To improve the detection of a clonal T-cell population, we used temperature gradient gel electrophoresis (TGGE) as an alternative method for analysis of TCR gene rearrangement. DESIGN: One hundred eighteen archival DNA samples were randomly selected based on previous Southern blot analysis results. Samples included 58 T-cell neoplasms with positivity for TCR beta gene rearrangement, 22 cases of reactive hyperplasia with germline pattern for both TCR beta and J(H), and 38 patients with B-cell lymphoma. MOLT-16, a T-cell lymphoblastic cell line, was used for the sensitivity assay. Polymerase chain reaction was performed using GC-clamped multiplex primers to amplify the TCR gamma locus and was analyzed by TGGE. The range of temperature gradients was empirically determined for optimal resolution of bands. RESULTS: The sensitivity of TGGE was 0.1% when DNA from the MOLT-16 cell line was serially diluted with DNA from reactive lymphoid tissue. Fifty-four (93%) of 58 T-cell neoplasms with TCR beta gene rearrangements showed rearrangement patterns by TCR gamma TGGE, and only 1 of 60 samples (reactive or B-cell lymphomas) showed evidence of gene rearrangement by TGGE. Patients with T-cell neoplasm and involvement of multiple sites showed an identical migration pattern by TGGE analysis. CONCLUSION: We demonstrate that TGGE is an effective method for analysis of TCR gene rearrangement in the evaluation of nodal and extranodal lymphoid lesions.     

Advances in the assessment of T-cell clonality

The diagnosis of T-cell lymphomas is often more challenging than that of B-cell lymphomas and can be difficult even for monospecialised haematopathologists. Their identification involves histomorphological assessment and the recognition of aberrant immunophenotypes but may additionally require polymerase chain reaction (PCR)-based analysis of T-cell receptor (TR) gene rearrangements (clonality analysis). TR gene rearrangements occur naturally during T-cell development, acting as a unique barcode for each cell: in neoplastic proliferations subsets of these barcodes become expanded and can therefore be detected as clonal populations. Here we examine the current role of clonality analysis, discussing limitations and possible future directions which may improve diagnostic efficacy and efficiency. This review will provide helpful insight to histopathology trainees as well as non-specialist consultants who may encounter T-cell lymphomas in their routine diagnostic practice. The article is also suitable for practising haematopathologists as a refresher on theory and an insight into possible future developments.     

A PCR membrane spot assay for the detection of plum pox virus RNA in bark of infected trees

A procedure for sensitive detection of plum pox virus RNA in infected bark of trees is described. The method is based on the extraction of bark material with buffer containing proteinase K followed by partial purification of RNA using QUIAGEN anion exchange resin. The RNA is then reverse transcribed, the single stranded cDNA is amplified by the polymerase chain reaction using biotinylated deoxynucleotides as label. The amplified cDNA can subsequently be detected by spotting the reaction mixture onto a nitrocellulose membrane. After fixation and washing the incorporated label is detected enzymatically using streptavidin-alkaline phosphatase. It was shown that this non-radioactive detection system is more sensitive than ELISA and a DNA/RNA hybridization test using 32P-labelled probes. It is also possible to detect plum pox virus infection with this assay in trees in the non-vegetative period.     

The polymerase chain reaction for detection of T-cell antigen receptor expression.

Applications of the polymerase chain reaction have revolutionized the field of immunogenetics, particularly in studies of human leukocyte antigen class II polymorphism, and more recently in the analysis of T-cell receptor usage. However, the enormous diversity and variability of the T-cell receptor complex have made the amplification of the complete repertoire difficult. Several methods have been devised to address this problem. Each system is described with recent examples of its use and an assessment of its advantages and disadvantages. The use of quantitative polymerase chain reaction in T-cell receptor analysis is also discussed. The elucidation of the T-cell repertoire involved in a pathogenic process can have therapeutic implications, given the success of reversing experimental autoimmune disorders by directing specific forms of immunotherapy against V region gene products.     

A T-cell receptor gamma polymerase chain reaction assay using capillary electrophoresis for the diagnosis of cutaneous T-cell lymphomas.

Detection of clonal T-cell receptor gamma rearrangements by polymerase chain reaction (TCRgamma PCR) followed by high-resolution electrophoresis has now become a valuable tool in the diagnosis of cutaneous T-cell lymphoma (CTCL). The identification of clonal TCRgamma PCR products by fluorescent fragment analysis (FFA) on a capillary DNA sequencer is described here and compared with an established hetero-duplex temperature gradient gel electrophoresis (HD-TGGE). Of 55 CTCL derived lesional skin samples, clonality was obtained in 46 samples by FFA (83.6%) and in 45 samples by HD-TGGE (81.8%). Of 35 control skin specimens from various nonmalignant dermatoses, two samples (pityriasis lichenoides chronica) showed clonality by both methods, one sample (chronic dermatitis) only by FFA. The sensitivity of FFA was established using three clonal T-cell lines and peripheral blood mononuclear cells. The detection limit for clonal material was approximately 1% to 2.5% in mixtures of DNA and 1% to 3% in cell dilutions. For cell dilution series, we confirmed a linear correlation between the clonal/polyclonal peak-size ratios and the portion of clonal cells up to about 10%. Thus, the initial ratio between mono-and polyclonal template is correctly displayed by FFA within that concentration range. In conclusion, FFA on capillary DNA sequencer is a well-suited separation technique in TCRgamma PCR-based clonality analysis also exhibiting quantitative properties.     

A simplified method of detection of clonal rearrangements of the T-cell receptor-gamma chain gene.

A series of T-cell proliferations in peripheral blood, bone marrow, or tissue samples, together with seven T-cell lines, were analysed for clonality. The technique used employs the polymerase chain reaction (PCR) to amplify rearranged T-cell receptor gamma genes, using primers recognising conserved sequences in the variable and joining gene segments. Of the 20 cases of T-cell leukaemia or lymphoma analysed, a clone was detected in 14 (70%): Of seven T-cell lines, a clone was detected in 6 (84%). No positive results were recorded in eight non-T-cell disorders (including nonlymphoid malignancies and reactive disorders). When the results of this technique were combined with the results of our previously published method for the detection of clonally rearranged T-cell receptor-beta (TCR-beta) genes using PCR, 9 of 10 (90%) T-cell tumours were detected. This method uses only four primer combinations in two tubes, and is therefore simple and rapid: it requires no radiolabelling, uses only a small amount of tissue, and can be performed on formalin-fixed, paraffin-embedded tissue.     

Amplification of human papillomavirus DNA sequences by using conserved primers.

The polymerase chain reaction has potential for use in the detection of small amounts of human papillomavirus (HPV) viral nucleic acids present in clinical specimens. However, new HPV types for which no probes exist would remain undetected by using type-specific primers for the polymerase chain reaction before hybridization. Primers corresponding to highly conserved HPV sequences may be useful for detecting low amounts of known HPV DNA as well as new HPV types. Here we analyze a pair of primers derived from conserved sequences within the E1 open reading frame for HPV sequence amplification by using the polymerase chain reaction. The longest perfect homology among HPV sequences is a 12-mer within the first exon of E1M. A region of conserved amino acids coded by the E1 open reading frame allowed the detection of another highly conserved region about 850 base pairs downstream. Two 21-mers derived from these conserved regions were used to amplify sequences from all HPV DNAs used as templates. The amplified DNA was shown to be specific for HPV sequences within the E1 open reading frame. DNA from HPVs whose sequences were not available were amplified by using these two primers. HPV DNA sequences in clinical specimens could also be amplified with the primers.     

Angiocentric immunoproliferative lesions: a molecular analysis of eight cases.

Angiocentric immunoproliferative lesions (AILs) are believed to represent a unique type of extranodal malignant lymphoma on the basis of clinicopathologic and immunophenotypic evidence. However, molecular studies to assess clonality have been performed on a small number of cases. In this study we assessed the clonality of eight AILs using restriction fragment analysis, the Southern blot technique, and probes to assess the configuration of the T-cell receptor beta, gamma, and delta chain genes and the immunoglobulin heavy and K light chain genes. In addition, the presence of the Epstein-Barr (EB) viral genome was assessed by using both Southern blot analysis (seven cases) and polymerase chain reaction amplification (five cases). Our results demonstrate that gene rearrangements are rare in AILs. A clonal gene rearrangement was identified in only one case, a grade III AIL with a rearrangement of the T-cell receptor delta chain gene. In two additional AILs (both grade III), the EB viral genome was detected as a single band by Southern blot analysis with a probe derived from the terminal repeat region of the virus, suggesting that a single episomal configuration of the EB viral genome was present in each case, as would occur in a clonal population of infected cells. In the remaining cases there was no evidence of clonality, although EB sequences were detected in one of four cases using the polymerase chain reaction. The rarity or absence of gene rearrangements in AILs is difficult to explain if AILs are malignant, presumably monoclonal lymphomas. However, their frequent association with the EB virus may suggest an analogy between AILs and lymphoproliferative disorders that occur in immunosuppressed patients. These findings further emphasize the unique clinicopathologic aspects of AILs and may also be useful diagnostically in the differential diagnosis of lymphoproliferative disorders.     

Improved method for the routine identification of toxigenic Escherichia coli by DNA amplification of a conserved region of the heat-labile toxin A subunit.

This report describes a DNA amplification procedure for routine identification of heat-labile-toxin-producing Escherichia coli. Two oligonucleotide primers were used in a polymerase chain reaction procedure to amplify a highly conserved region of the A subunit of the heat-labile enterotoxin gene. Amplifications were done directly on E. coli colonies from plates when Salmonella, Shigella, or parasite infections were excluded as agents of the severe diarrhea in the patients. The conditions for the polymerase chain reaction method were empirically determined, and the procedure is inexpensive, sensitive, and specific. Positive results can be obtained over a wide variation in bacterial numbers, with no inhibition of Thermus aquaticus DNA polymerase. Detection of the amplified product can be done by agarose gel electrophoresis, which is specific and sensitive enough for routine diagnosis of this pathogen in clinical isolates. If greater sensitivity and specificity are required, hybridization with 32P- or alkaline phosphatase-labeled oligonucleotide probes can be used. Our results suggest that heat-labile-toxin-producing E. coli is responsible for about 9% of nondiagnosed diarrhea cases in Tygerberg Hospital, Tygerberg, Republic of South Africa.     

Detailed analysis of the T-cell lymphocytic infiltrate in penile lichen sclerosus: an immunohistochemical and molecular investigation

AIM: To determine the extent of clonal outgrowth in the lymphocytic tissue infiltrate of lichen sclerosus (LS). The presence of T cells with a monoclonally rearranged T-cell receptor gamma-gene (TCRgamma) has been described in up to 50% of biopsies of vulvar and penile LS. MATERIAL AND RESULTS: We analysed 33 foreskin specimens with LS for the presence of clonal T cells by conventional polymerase chain reaction (PCR) analysis and with TCRgamma-PCR-based fluorescent fragment analysis. Eighteen of 33 patients revealed a band indicating a monoclonally rearranged TCRgamma on conventional PCR analysis. Subsequent TCRgamma-PCR-based fluorescent fragment analysis identified 8/18 patients with monoclonal T-cell DNA ranging from 1.4% to 23.1% of total T-cell DNA analysed and a size range from 56 to 72 base pairs. Four of 18 patients had an oligoclonal and 6/18 patients revealed a polyclonal banding pattern. The lymphocytic infiltrate contained low numbers of gammadelta T cells and cytotoxic T cells in comparable numbers to the low percentage of clonal TCRgamma DNA. CONCLUSIONS: The low percentage of clonal TCRgamma DNA argues against a systemic neoplastic disease, but rather for a local immune disorder. The target antigen of the clonal outgrowth is unknown, but an exaggerated antigen-dependent proliferation of T cells due to chronic local antigen exposure, probably an infectious antigen, is the most likely explanation.     

VJ REARRANGEMENTS OF THE TCRγ LOCUS IN PERIPHERAL T-CELL LYMPHOMAS: ANALYSIS BY POLYMERASE CHAIN REACTION AND DENATURING GRADIENT GEL ELECTROPHORESIS

Using Southern blotting for the diagnosis of clonality in peripheral T-cell lymphomas (PTCLs), analysis of the T-cell receptor (TCR) γ gene rearrangement was shown to be more informative than that of the TCR β gene rearrangement. In order to amplify every VJγ rearrangement, a polymerase chain reaction (PCR) procedure using newly designed GC-clamp primers has been developed. All primers can be mixed in a single multiplex PCR. PCR products are analysed by denaturing gradient gel electrophoresis (DGGE), providing tumour-specific imprints inasmuch as the procedure characterizes N sequence polymorphism at the VJ junctions. In a series of 30 PTCL cases, the PCR procedure demonstrated 27 cases to be clonally rearranged and failed in three cases. PCR was more accurate than Southern blotting, showing 47 rearranged γ alleles, four of which were undetectable on the Southern blot. When lymphomas were studied at different sites and at relapse, the DGGE pattern remained unchanged. In PTCL, the proposed PCR is helpful for the diagnosis and staging of the disease and should improve the follow-up monitoring. The undetectability of clonal rearrangements in a few cases is discussed in the light of concepts of lymphomagenesis and T-cell differentiation.     

Sensitive detection of clonal immunoglobulin rearrangements in frozen and paraffin embedded tissues by polymerase chain reaction heteroduplex analysis.

Molecular detection of a clonal population of B or T cells through analysis of rearranged antigen receptor genes is an essential adjunct to the morphologic, flow cytometric, and immunohistochemical evaluation of tissue specimens for the presence of leukemia or lymphoma. Combining polymerase chain reaction (PCR) with heteroduplex annealing and polyacrylamide gel electrophoresis (PAGE) has been used to detect clonal T-cell receptor rearrangements, particularly in skin biopsy specimens. The authors have developed a similar PCR heteroduplex assay for detection of clonal VDJ immunoglobulin gene rearrangements using two sets of primers based on relatively conserved consensus regions in the J(H) and framework I and 2 regions of the immunoglobulin heavy chain V region gene. This method is able to detect a clonal rearrangement when the clone comprises as little as 1% of the population in a polyclonal B-cell background. It may be used on fresh, frozen, or paraffin-embedded tissue and detects a clonal population in a majority of lymphoma subtypes. Compared with conventional PCR analysis, this method requires only a short additional cycle of denaturation and slow renaturation before PAGE. Interpretation is simplified as the clonal PCR product migrates away from the polyclonal background products.     

Detection of clonal lambda light chain gene rearrangements in frozen and paraffin-embedded tissues by polymerase chain reaction.

A method was established to detect clonal lambda light chain gene rearrangements in peripheral blood lymphocytes and frozen or paraffin-embedded tissues. V lambda-gene-family-specific primers were used together with a J lambda primer mix in separate reactions to amplify V lambda gene rearrangements by the polymerase chain reaction. Clonal lambda gene rearrangements were detected in seven of seven lambda-expressing B cell leukemias, in four of five lambda-expressing non-Hodgkin's lymphomas with frozen tissues, and in seven of nine cases of lambda-expressing non-Hodgkin's lymphomas for which formalin-fixed, paraffin-embedded specimens were available. Clonality of amplified polymerase chain reaction products was confirmed by sequence analysis for several cases. The present study shows that it is possible to amplify clonal lambda gene rearrangements in the majority of lambda-expressing B cell leukemias and lymphomas. The method described here, therefore, is a useful supplement to the previously described approach of VH and VK gene amplification to detect clonal B cell populations and allows the study of V lambda gene usage and somatic mutation in lambda-expressing normal and malignant B cells.