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PubMed1994

Optimal primer selection for clonality assessment by polymerase chain reaction analysis: II. Follicular lymphomas.

Prior studies have shown variable success in amplification of monoclonal B-cell populations from follicular lymphomas (FLs) of germinal center cell origin using the polymerase chain reaction (PCR). We examined 60 FLs by the PCR to determine optimal primer selection for detection of clonal immunoglobulin heavy chain gene (IgH) rearrangements in this common group of B-cell lymphomas. Each primer set included a 3' IgH joining region consensus primer; the 5' primer was different in each reaction. The first-line panel included the following: Set 1, bcl-2 major breakpoint region (mbr) primer; Set 2, bcl-2 minor cluster region (mcr) primer; Set 3, IgH variable region framework III consensus primer; and Set 4, seven separate IgH variable region framework I family-specific primers. A reserve panel also was used. The efficiency of monoclonal B-cell population amplification differed among primer sets. The bcl-2-targeted primer pairs (Sets 1 and 2) were most efficient and amplified 42 (70%) of 60 cases. Of these, the mbr and mcr primer sets detected monoclonality in 38 and four cases, respectively. Set 3 amplified monoclonal B-cell in 38 and four cases, respectively. Set 3 amplified monoclonal B-cell populations in 31 (52%) cases and Set 4 detected similar populations in only 27 (45%) samples. When results from primer Sets 1, 2, and 3 were combined, 49 of 60 (82%) FLs showed evidence of B-cell monoclonality by the PCR. Three of the 11 negative cases were documented as monoclonal with primer Set 4, and three additional samples were amplified only with our reserve panel.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1986

Temporal invariance and clonal uniformity of brain and cerebrospinal IgG, IgA, and IgM in multiple sclerosis.

Elevated cerebrospinal fluid (CSF) IgG and oligoclonal IgG bands on electrophoresis are valuable clinical markers for B cell proliferation in the brains of patients with multiple sclerosis (MS). Using two-dimensional electrophoresis, (2DE) we have established that the humoral immune response in MS brain is characterized by finite clonal complexity for the major Ig classes. An important question is whether this immune response is clonally stable or varies with time, related to the development of new lesions and random entry of B cells into the MS brain. To investigate this, we performed serial electrophoretic studies on CSF obtained from 19 patients with MS; the intervals ranged from 7 to 12 yr, with a mean of 8 yr. These analyses included studies of IgG, IgA, and IgM, and revealed that the humoral immune response in MS is clonally stable over long periods. Spontaneous fluctuations or reduction in CSF IgG levels by drugs did not qualitatively affect B cell clonal proliferation in MS brain, in that dominant bands and spots were not obliterated. It has been asserted that IgG synthesis in MS is nonsense antibody because the spectotypes of IgG isolated from different regions of MS brains differ. Factors other than clonal heterogeneity could account for differences found using one-dimensional analysis. B cell clonal products resolve into unique and well-resolved spots by 2DE; the method is uniquely suitable for analysis of restricted immune responses. Therefore, Ig were isolated from 11 regions of three MS brains and the 2DE patterns were compared. The similarity of the 2DE patterns indicate unequivocally that major clones are distributed uniformly although some clones are more prominent in some brain areas. IgA and IgM isolated from the same areas also showed similar patterns. Furthermore, the patterns of light and heavy chains in brain regions differed from serum but were similar to the autologous CSF, providing new evidence that CSF IgG in MS derives from synthesis in situ. Our results indicate that, once initiated, B cell clonal proliferation persists indefinitely and is little altered qualitatively at a clonal level over time, even when CSF IgG levels change or are altered by drugs. Our results are consistent with allotype and idiotype analysis of Ig production in MS and conflict with nonsense antibody proposals of the origin and nature of in situ synthesized Ig in MS.

Blood-Brain Barrier↗
PubMed1995

Analysis of meningiomas by methylation- and transcription-based clonality assays.

The clonal derivation of tumors can be determined by X chromosome inactivation analysis based on differential expression of genes or differential methylation of cytosine residues in CpG islands near polymorphic loci. In this report, we compared a transcription-based RNA analysis with a methylation-based DNA assay to determine clonality of meningiomas. Both clonality assays use PCR-based analysis at the hunan androgen-receptor gene (HUMARA) on the X chromosome. Among 23 meningiomas from female patients, 19 were informative heterozygotes at this locus (83%). The patterns of X chromosome inactivation in four patients were extremely skewed towards one allele in blood (unequal Lyonization), which precluded clonality determination in the tumor samples. Concordant clonality results with methylation- and transcription-based clonality assays were demonstrated in 9 of 13 informative tumors expressing the androgen receptor. Seven meningiomas were monoclonal, but surprisingly, two pathologically documented cases of meningiomas were polyclonal. There was disparity in 4 of 13 tumor specimens that were polyclonal by the methylation-based assay but monoclonal by the transcription assay. Clonality examination of these tumors by the methylation-based phosphoglycerate kinase assay provided identical results to the methylation-based analysis at the HUMARA locus. In addition, loss of heterozygosity (LOH) studies of chromosome 22, which is frequently deleted in meningiomas, showed that four of four informative samples of the six polyclonal tumors had partial LOH in tumor tissues. However, complete LOH was observed in primary cultured cells, which were also monoclonal by the methylation assay. Taken together, these data suggest that the disparity of the two assays in these four cases may be due to differences in the level of expression of the androgen receptor gene in tumors. Therefore, we conclude that: (a) clonal derivation of meningiomas determined by both transcription- and methylation-based clonality assays are in full agreement in many (9 of 13) but not all cases (4 of 13); and (b) most meningiomas (9 of 15) are monoclonal in origin, whereas some meningioma samples (6 of 15) are polyclonal or may contain heterogeneous components.

Adult↗
PubMed2005

Clonal relationship of tumor nodules in hepatocellular carcinoma: a hierarchical clustering analysis of comparative genomic hybridization data.

Information on the clonal relationship between tumor nodules in patients diagnosed with hepatocellular carcinoma (HCC) holds prognostic significance in the prediction of recurrence and postoperative treatment. Here, we investigated the clonal relationships in 11 focal nodules from 5 patients with HCC by comparative genomic hybridization (CGH) and spectral karyotyping. CGH analysis indicated tumor nodules in 5 (100%) of 5 cases to share similar patterns of genomic imbalances, suggesting nodules to be clonally related. This clonal relationship was further substantiated in 2 cases, where spectral karyotyping analysis indicated identical structural rearrangements between focal tumors. Few studies have attempted to differentiate multicentric growth from intrahepatic dissemination in HCC using CGH analysis; however, data interpretation remained subjective. In this study, we have extended our data to include published CGH findings on 57 nodules derived from 19 HCC cases. Simulation of CGH findings by unsupervised 2-way hierarchical clustering indicated tumor nodules in 20 (83.3%) of 24 cases to cluster concordantly, signifying a high incidence of clonal similarity. In sum, bioinformatic analysis of genomic profiles may represent a reliable research tool in analyzing clonal relationships among tumor nodules.

Adult↗
PubMedDate not supplied

Clonal composition of malignant fibrous histiocytoma: analysis by PCR-based assay of the human androgen receptor gene (HUMARA).

Malignant fibrous histiocytoma (MFH), the most common soft-tissue sarcoma, consists mainly of two different cell populations: histiocytelike and fibroblastlike cells. It has been suggested to contain a large amount of reactive histiocytes and fibroblasts hard to distinguish from the tumor cells. In this study, the clonality of MFH was determined by analyzing the patterns of X chromosome inactivation at the human androgen receptor gene (HUMARA) using DNA samples from archival snap-frozen and paraffin-embedded tissues. All the eleven informative female heterozygotes without severe inflammation showed the monoclonal pattern; 8 storiform-pleomorphic (6 distinct, 2 relative monoclonal pattern) and 3 myxoid (3 distinct monoclonal pattern) subtype. Although normal tissue DNA, amplifiable by the polymerase chain reaction, valid for the assay was not available in these cases, statistically at least 5 cases are monoclonal (p = 0.037 <0.05), even when markedly skewed lyonization were to primarily exist in the normal tissue at the highest rate as ever reported (33%). Experiments using the mixture of monoclonal and polyclonal DNA at varying ratios have suggested that a distinct monoclonal pattern is obtained only when the monoclonal component exceeds 80%. Our study demonstrates that most cells that are present in MFH are monoclonal in origin which may be the population of tumor cells.

Adult↗
PubMed1993

Quantitation of murine hematopoietic stem cells in vitro by limiting dilution analysis of cobblestone area formation on a clonal stromal cell line.

Murine hematopoietic stem cells with varying proliferative capacity can be assayed by limiting dilution analysis of "cobblestone area" (CA) formation on stromal layers in microlong-term bone marrow cultures. Cobblestone area forming cell (CAFC) frequency determined at early time points (day 7) correlates with mature stem cells measured as day 8 CFU-S, whereas CAFC frequency determined at day 28 correlates more closely with long-term marrow repopulating ability. Here we report a modification of the CAFC assay in which a clonal bone marrow stromal cell line, GB1/6, is substituted for fresh marrow-derived stromal layers. This modification simplifies the initial culture setup, eliminates inhomogeneities in the stromal layer and reduces the need for mice. Normal bone marrow CAFC frequencies were the same for both types of stromal cell underlayer, demonstrating the ability of a clonal cell line to completely replace the heterogeneous microenvironment of fresh stroma for in vitro stem cell support.

Animals↗
PubMed2000

Multiplex polymerase chain reaction-based analysis of T-cell receptor gamma gene rearrangements for the determination of T-lymphocyte clonality.

Determination of the frequency of mutations at hprt or other loci in human lymphocytes provides a useful biomarker for human exposure to mutagens. One problem, however, is distinguishing between unique mutants and sibling mutants arising as progeny of an earlier mutant cell. We have developed a multiplex polymerase chain reaction (PCR)-based method to analyze T-cell receptor (TCR) gamma gene rearrangements for determination of T-cell clonality in mutational spectrum analysis. PCR primers for different subgroups of the V gene segment of the TCR gamma gene were selected at different sites in the TCR gamma gene so that the size of PCR products could define which V subgroup was involved in rearranged TCR gamma genes; gamma genes involving different V and J subgroups could be determined directly by PCR. Mutant T-lymphocytes with rearranged TCR gamma genes containing the same V and J subgroups were analyzed using PCR-based denaturing polyacrylamide gel electrophoresis. All of the 161 hprt mutant clones analyzed contained rearranged TCR gamma genes. Rearrangements among all subgroups of the V and J gene segments of the TCR gamma gene could be detected. VgammaI and Jgamma1/2 subgroups were involved in 69 and 71% of rearranged TCR gamma genes, respectively. This PCR-based analysis of TCR gamma gene rearrangements provides a simple and comprehensive method for identifying the clonality of mutant T-lymphocytes in human hprt mutant lymphocyte assay and mutational spectrum analysis.

Electrophoresis, Polyacrylamide Gel↗
PubMed2005

Application of BIOMED-2 primers in fixed and decalcified bone marrow biopsies: analysis of immunoglobulin H receptor rearrangements in B-cell non-Hodgkin's lymphomas.

The detection of clonality in lymphomas was recently improved by the BIOMED-2 approach, but analysis of fixed tissues is limited. Here, we adapted the BIOMED-2 protocol for examining immunoglobulin H (IgH) receptor rearrangements in fixed, decalcified bone marrow biopsies (BMBs) for clonality analysis in B-cell non-Hodgkin's lymphomas (B-NHL). The study included 111 decalcified BMBs (12 formalin fixed and 99 glutardialdehyde fixed), with B-NHL (n = 85), T-NHL (n = 8), or reactive infiltrates (n = 18). Initially, IgH FRIII polymerase chain reaction (PCR) analysis of crude DNA extracts from 75 glutardialdehyde-fixed BMBs (B-NHLs) using a standard seminested PCR resulted in clonal peaks in 46 of 75 (61.3%) BMBs compared with 19 of 70 (27.1%) for the original BIOMED-2 protocol. Modifications to both DNA extraction and PCR reaction improved the detection rate to 26 of 36 (72.2%) for BIOMED-2 primers, including 10 of 15 (66.7%) cases not detected by our standard IgH analysis. Moreover, introducing the same modifications for analysis of the FRII region by BIOMED-2 primers revealed clonal peaks in 19 of 36 (52.8%) B-NHLs compared with 5 of 70 (7.1%) for the original BIOMED-2 protocol. Together, analysis of FRII and FRIII regions by the modified BIOMED-2 protocol increased the detection rate to 31 of 36 (86.1%), particularly for BMBs with histological evidence of follicular lymphoma (FRIII, 70%; FRII, 90%). In summary, this study provides an improved protocol for detection of clonality by IgH-specific BIOMED-2 primers in fixed, decalcified bone marrow biopsies.

Biopsy↗
PubMed2005

[Peripheral blood naive T cell level and its T cell receptor Vbeta repertoire usage profile in patients with chronic myelogenous leukemia].

OBJECTIVE: To analyze peripheral blood naive T cell level, its T cell receptor (TCR) Vbeta repertoire usage profile and clonality for evaluating the recent thymic output function and the expansion feature of TCR Vbeta subfamily T cells in patients with chronic myelogenous leukemia (CML). METHODS: Quantitative detection of T-cell receptor excision DNA circles (TRECs) in peripheral blood mononuclear cells (PBMNC) from 20 cases of CML was preformed by real-time PCR (TaqMan) analysis, and TRECs-number in T-cells was calculated from peripheral blood CD3-positive cell rate. The expression and clonality analysis were detected by RT-PCR and Genescan technique in PBMNC from 14 out of the 20 patients. Nine normal individuals served as controls. RESULTS: A dramatic reduction of TRECs value in patients with CML was detected as compared with that in normal controls. The mean value of TRECs was 0.06 +/- 0.16 copy/1000 CD3(+) cells in CML patients while 6.84 +/- 4.71 copies/1000 CD3(+) cells in normal controls (P < 0.01). The 1 - 12 Vbeta subfamilies were variably expressed in samples from 14 patients. Genescan analysis identified clonal expanded T cells of some Vbeta subfamily from 13 cases. Vbeta3, Vbeta10, Vbeta19, Vbeta21 and Vbeta22 subfamilies clonal T cells were more frequently seen. CONCLUSION: There is a prominent reduction of recent thymic output naive T cells function in CML. The predominant usage and clonal expansion of TCR Vbeta subfamily T cells could be identified, indicating that CML patients have specific immune response to leukemia associated antigen, in spite of their T cell immunodeficiency.

Adolescent↗
PubMed1993

Epstein-Barr virus (EBV) gene expression in EBV-positive peripheral T-cell lymphomas.

Epstein-Barr virus (EBV) DNA has been detected in peripheral T-cell lymphomas. In this study, analysis of the EBV termini indicated that the infection was clonal and nonpermissive. Analysis of viral expression detected the processed, spliced mRNAs representing EBNA1, LMP1, LMP2, and BamHI A transcripts in all EBV-positive peripheral T-cell lymphomas. The LMP1 protein was detected by immunofluorescence in a single specimen. In contrast, neither the EBNA2 protein nor the spliced EBNA2 mRNA were detected. These data indicate that EBV-infected T-cell lymphomas are clonal expansions of a single EBV-infected cell with a pattern of gene expression which is distinct from that detected in Burkitt's lymphomas or posttransplant lymphomas but similar to viral expression in nasopharyngeal carcinomas.

Antibodies, Monoclonal↗
PubMed2001

Assessments of clonal composition of colorectal adenomas by FISH analysis of chromosomes 1, 7, 13 and 20.

Chromosome banding analysis has shown that numerical aberrations, in particular gains of chromosomes 7, 13 and 20, are common in colorectal adenomas but cannot provide reliable information on the size of the abnormal clones in vivo. We examined interphase nuclei from 70 colorectal adenomas, of which 64 had been previously karyotyped, using fluorescence in situ hybridization (FISH) with probes for the pericentromeric regions of chromosomes 1, 7, 13 and 20. Gain of chromosome 7 was seen in 34% of the analyzed adenomas, +13 was seen in 44% and trisomy 20 was found in 32% of the adenomas, verifying that the trisomies are in vivo phenomena. The median proportion of cells with trisomy was larger than 50%. A comparison with the G-banding analysis showed a good correlation between the results yielded by the 2 methods. Based on the clonal size and karyotypic findings, a likely order of events during clonal evolution could be ascribed to each case. More than 1 numerical aberration was detected by FISH analysis in 16 adenomas. In 6 adenomas, a clone with only trisomy 7 was present alongside a clone with additional gain(s) of chromosomes 13 and/or 20. Seven cases had gain of chromosome 13 and/or gain of chromosome 20 in the largest clone, suggesting that a clone with either of these changes was present before the changes in chromosome 7 copy number took place. On the basis of the results of this combined meta- and interphase cytogenetic study, we conclude that gains of chromosomes 7, 13 and 20 are common in colorectal adenomas and that the trisomies usually are present in a large proportion of the cells. They seem to be primary chromosome aberrations in some adenomas, whereas in others they arise secondarily as part of the clonal evolution. Although the first gain usually is of chromosome 7, it is evident that it is the end result of the chromosomal aberrations, not the exact sequence in which they occur, that determines the pathogenetic consequences.

Adenoma↗
PubMed1988

Clonal dominance of primary tumours by metastatic cells: genetic analysis and biological implications.

A new method is described for analysing the clonal evolutionary dynamics of tumour growth and the lineage relationship of primary tumours to their metastases. It exploits random integrations of transfected plasmid or retroviral infected (proviral) DNA as a means of generating very large numbers of uniquely marked cell clones in a single-step selection whose fates can then be tracked during progressive tumour growth. Using a mouse breast adenocarcinoma we undertook experiments in which syngeneic mice were injected with a mixture of very large numbers of uniquely marked cell clones, only one or a few of which were metastatic, or with reconstituted mixtures containing a genetically tagged metastatic clone with an excess of non-marked non-metastatic tumour cells. Among the results we summarize is the finding that spontaneous metastases developed in a non-random fashion from genotypically distinct cell clones. They were clonal or biclonal at the time of analysis. We also found that the progeny of a single metastatic clone could eventually overgrow the primary tumour. Thus malignant (metastatic) cells may manifest a striking growth advantage within the primary tumour site as well as for dissemination and growth at distant, secondary sites. As a result, late-stage advanced primary tumours, if left intact, may evolve to become biologically similar or equivalent to distant metastases. This 'clonal dominance' phenomenon can reconcile many of the discrepant experimental findings with respect to the putative selective nature of metastatic phenotype. Furthermore, it has important consequences for understanding one source of biological variability in experiments in which different primary tumours are compared to each other or to metastases; it also has implications for theories regarding the clonal origin of neoplasms, and for the physiological and biochemical changes that cause malignant disease.

Animals↗
PubMed2003

Peripheral blood T-cell clonality in mycosis fungoides and nonlymphoma controls.

In mycosis fungoides (MF), T-cell clonality is reported in about 90% of skin and 40% of blood samples. However, identity of blood and cutaneous T-cell clone and prognostic relevance of blood T-cell clonality remain controversial. By PCR/fluorescence fragment analysis with estimation of clonal fragment lengths and relative peak heights, we objectively identified T-cell clonality unrelated to malignant lymphoproliferation in healthy donors (5/38), autoimmune dermatoses (3/8), and nonlymphoma skin cancer (9/39). This T-cell expansion of undetermined significance (TEXUS) was also found in 8/64 MF patients. Dissemination of neoplastic cells into blood, as identified by identical clonal fragment lengths in blood and skin, was detected in 23/64 MF patients. When monitoring for progression at TNM stage for a mean of 45.7 months, univariate analysis identified age of >60 years and detection of a related blood T-cell clone to be of prognostic relevance, whereas detection of TEXUS, sex, TNM stage at initial diagnosis, and detection of a cutaneous T-cell clone were irrelevant. Although multivariate analysis was not possible, further stratification clearly indicated an age of >60 years to be the predominating prognostic factor. In conclusion, investigation of T-cell clonality in skin and blood samples at the initial diagnosis cannot predict the clinical course of MF and the occurrence of TEXUS should be considered when assessing blood T-cell clonality.

Adult↗
PubMed1994

Cytogenetic analysis of multifocal breast carcinomas: detection of karyotypically unrelated clones as well as clonal similarities between tumour foci.

Cytogenetic analysis was performed on short-term cell cultures of two foci (A and B) from each of three multifocal breast carcinomas. In case I, four clones (three related and one unrelated) were detected in sample A. In sample B, two of the three related clones and the unrelated clone seen in A were found, as was also a third subclone showing a pattern of clonal evolution slightly different from that detected in A. In cases II and III, multiple cytogenetically unrelated clones were found in A and B, with only one clone being shared by both foci in each case. Our finding of cytogenetic similarities between macroscopically distinct tumour lesions indicates that the multifocality reflects intramammary tumour spread rather than the synchronous emergence of pathogenetically independent carcinomas within the same breast. On the other hand, the detection of karyotypic heterogeneity in the form of cytogenetically unrelated clones in all foci suggests that human breast carcinoma may be polyclonal. This polyclonality may be part of the explanation for the cellular heterogeneity commonly seen at the phenotypic level in breast cancer.

Aged↗
PubMed1995

Southern blot detection of immunoglobulin lambda light chain gene rearrangements for clonality studies.

Southern blot analysis of immunoglobulin (Ig) genes has proven to be important for detection of clonal rearrangements in patients with lymphoproliferative diseases. To improve the detection of clonal Ig lambda (Ig lambda) gene rearrangements, we carefully determined the precise restriction map of the J-C lambda gene region, developed a suitable C lambda probe (IGLC3), and evaluated relevant restriction enzymes in combination with the IGLC3 probe. For the latter purpose, we selected 75 B cell malignancies with proven expression of Ig lambda protein chains in order to be sure that each malignancy contained at least one clonally rearranged IG lambda allele. Our extensive Southern blot analyses with the IGLC3 probe in EcoRI and/or HindIII digests revealed that combined EcoRI/HindIII digestion detected Ig lambda gene rearrangements in 95% of the 75 patients and 94% of the 98 rearranged alleles. In contrast, HindIII and EcoRI single digests allowed detection of rearrangements in only 78% and 83% of the patients, and 67% and 79% of rearranged alleles, respectively. We conclude that the use of the IGLC3 probe in combined EcoRI/HindIII digests is superior to EcoRI and HindIII single digests. This probe/enzyme combination is informative for clonality studies in approximately 95% of patients.

Alleles↗
PubMed1996

Optimal primer selection for clonality assessment by polymerase chain reaction analysis. III. Intermediate and high-grade B-cell neoplasms.

Previous studies have reported that low-grade B-cell lymphoproliferative disorders have variable B-cell monoclonality detection rates by polymerase chain reaction (PCR) analysis. For instance, monoclonal B-cell populations from chronic lymphocytic leukemia/small lymphocytic leukemia and mantle cell lymphoma are most often readily amplified with a single primer pair, whereas follicular lymphomas and plasma cell neoplasms require alternative strategies to approach these higher diagnostic sensitivity standards. Because most published reports have not focused on the variation in PCR B-cell monoclonality detection among subtypes of intermediate and high-grade B-cell neoplasms, additional information is necessary to determine primer selection strategies and identify problematic tumor subtypes within this group. The current investigation, the third part in a series, was aimed at documenting the efficiency of B-cell monoclonality detection by PCR in 71 aggressive B-cell neoplasms of various types using a comprehensive approach. A predetermined panel of primer sets was used in an algorithmic fashion. Specifically, all samples were analyzed with the standard VH-FRIII/JH assay previously shown to have the highest efficiency of monoclonality detection within low-grade B-cell lymphoproliferative disorders. Negative samples were further evaluated with primer sets in the following order until a positive result was observed, or all primer sets were used: (1) bcl-2/JH, (2) VH-FRI family specific/JH, and (3) VH-FRI consensus/JH. Forty-three (61%) of the 71 B-cell neoplasms evaluated with VH-FRIII/JH showed monoclonal B-cell populations. Sequential use of the three reserve primer sets in samples negative with this initial primer pair resulted in an overall improvement in PCR detection from 61% to 82% (58 of 71 specimens) (P < .001). The VH-FRI family specific assay identified B-cell monoclonality in 11 (73%) of these 15 specimens and was the most productive reserve primer set. Individual categories exhibited the following initial (I) and final (F) PCR detection rates: acute lymphoblastic leukemia/lymphoblastic lymphoma, 11 specimens (I = 91% to F = 91%); small noncleaved cell lymphoma, 14 specimens (I = 79% to F = 86% [P > .25]); diffuse large cell lymphoma, 33 specimens (I = 52% to F= 85% [P < .005]) and large cell, immunoblastic lymphoma, 13 specimens (I = 38% to F = 62% [P < .01]). The authors have shown that comprehensive PCR analysis is capable of detecting B-cell monoclonality in a significant proportion of samples from each subtype of intermediate and high-grade B-cell neoplasm. The VH-FRIII/JH assay was an adequate initial primer set, but required augmentation with the reserve PCR assays to attenuate the false negative rate and improve diagnostic sensitivity. The B-cell clonality PCR assay is optimally used as a screening tool and when used in this fashion, the more laborious and time-consuming restriction fragment-Southern blot hybridization (RF-SBH) method for IgH gene rearrangement detection may be limited to a relatively small proportion of PCR-negative aggressive B-cell neoplasms.

Algorithms↗
PubMed1997

Genotypic analysis of cutaneous T-cell lymphoma: a comparative study of Southern blot analysis with polymerase chain reaction amplification of the T-cell receptor-gamma gene.

The diagnosis of early cutaneous T-cell lymphoma (CTCL) is a difficult point in dermatology. Recently, Southern blot analysis (SBA) and polymerase chain reaction (PCR) have been used to detect clonality in initial lesions in which clinical and histological findings are unspecific. Forty-one samples from 25 patients with CTCL were investigated for the presence of T-cell receptor-gamma gene rearrangement using a nested PCR technique and analysed by polyacrylamide gel electrophoresis (PAGE). Conventional SBA was also performed on 28 samples from 20 of these patients. In addition, 20 samples corresponding to patients with large plaque parapsoriasis (LPP), cutaneous B-cell lymphoma (CBCL) and eczema were analysed by PCR in the same way as were the CTCL specimens. Most of the CTCL specimens (81%) showed clonality on PCR analysis. Among patients with mycosis fungoides, 71% of initial patch lesions and 100% of plaques and tumours showed clonal disease. Clonality could be detected in three of four histologically negative post-treatment lesions. Clonal rearrangement was detected in one of three patients with LPP and in three of 10 patients with CBCL. None of the samples corresponding to patients with eczema showed positive results. SBA was significantly less sensitive than PCR in detecting clonality in CTCL patients (42% among early disease and 60% among advanced cases). The results indicate that this PCR/PAGE technique is a reliable and useful method for the detection of clonality in early skin lesions of CTCL patients and probably in the identification of silent extracutaneous involvement.

Adolescent↗
PubMed2000

T-Cell clonality in pityriasis lichenoides et varioliformis acuta: a heteroduplex analysis of 20 cases.

BACKGROUND: Cutaneous lesions of pityriasis lichenoides et varioliformis acuta (PLEVA), a T cell-mediated cutaneous inflammatory condition, are clinically similar to lymphomatoid papulosis (LyP), leading some authors to hypothesize that they are part of the same spectrum of lymphoproliferative disorders, although reports of the development of cutaneous lymphoma in patients with PLEVA are not as frequent as they are for patients with LyP. Furthermore, unlike in cases of LyP, no systematic search for a dominant T-cell clone has been carried out in cases of PLEVA, whereas clones have been detected in a few cases of PLEVA using mainly Southern blot analysis. OBJECTIVE: To investigate T-cell clonality in a series of archival PLEVA lesions. TISSUES: Archival paraffin-embedded biopsy specimens from 20 clinically and pathologically typical cases of PLEVA were selected. MAIN OUTCOME MEASURE: Identification of a dominant T-cell clone by polymerase chain reaction and heteroduplex analysis targeted on the TCRgamma gene. Peripheral blood mononuclear cells (PBMCs) and Jurkat cells were used as negative and positive controls. Serial dilutions of Jurkat T-cell lymphoma DNA in PBMC DNA were used to assess the sensitivity of the method. RESULTS: Analysis of 13 (65%) of 20 PLEVA biopsy specimens revealed the presence of a dominant T-cell clone. Positive and negative controls confirmed the specificity of the procedure. The sensitivity was determined to be between 1% and 5% of the total T-cell infiltrate. CONCLUSIONS: This study provides further evidence for the presence of a dominant T-cell clone in skin lesions of some patients with PLEVA and supports the hypothesis that PLEVA is part of the spectrum of clonal-T-cell cutaneous lymphoproliferative disorders.

Adolescent↗