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PubMed1997

Marginal zone B-cell lymphoma with monocytoid B-cell lymphocytes in pediatric patients without immunodeficiency. A report of two cases.

We report two cases of marginal zone B-cell lymphoma in two patients 6 and 18 years of age, respectively (cases 1 and 2) who had no clinical evidence of immunodeficiency or risk factors for human immunodeficiency virus (HIV) infection. Histologic analysis in both cases revealed diffuse nodal effacement by a monotonous population of atypical lymphoid cells with abundant pale cytoplasm and round to oval nuclei, with very infrequent mitotic activity. The neoplastic cells in both cases were of B-cell lineage (CD20 and CD79a positive), with CD43 coexpression. One case showed monoclonal light chain expression, and polymerase chain reaction analysis demonstrated clonal rearrangements of the immunoglobulin heavy chain gene in both cases. Abnormal cytogenetic findings were detected in case 2, in which metaphase spreads revealed trisomy 13 (karyotype 47, XY, +13). Although trisomy 13 has been described in association with acute nonlymphocytic leukemias and myelodysplastic syndromes, this case represents the first documented association of trisomy 13 with marginal zone B-cell lymphoma. Interphase cytogenetics analysis for trisomy 3, reported to be associated with mucosa-associated lymphoid tissue (MALT) lymphomas, was negative in both cases. Although low-grade lymphomas of the MALT type have been reported in HIV-positive patients, the two cases reported here are unique in that they occurred in young patients without HIV infection or any other evidence of immunodeficiency.

Adolescent↗
PubMed2004

Pancoast's syndrome in a patient with B-cell lymphoma diagnosed and confirmed with immunoglobulin gene rearrangement.

Pancoast's syndrome due to malignant lymphoma is extremely rare. A case of diffuse large B-cell lymphoma presenting as Pancoast's syndrome is described. A 66-year-old man complained of pain and weakness of the right arm, and CXR revealed a right apical lung tumour. Histological findings were consistent with it being a diffuse large cell type lymphoma and Southern blot analysis revealed clonal rearrangement of the immunoglobulin heavy-chain JH. Thus, the tumour in this patient was diagnosed to be diffuse large B-cell lymphoma. Malignant lymphoma is an extremely rare cause of Pancoast's syndrome and only five cases have been described. This is the first reported case of Pancoast's syndrome caused by B-cell lymphoma, which was accurately diagnosed by analysis of gene rearrangement.

Aged↗
PubMed1992

Clonal hybrids of the common laboratory fish Fundulus heteroclitus.

All-female hybrids of the killifishes Fundulus heteroclitus and Fundulus diaphanus, known from two sites in Nova Scotia, Canada, are shown to reproduce clonally. Isozyme analysis of crosses between female hybrids and male F. heteroclitus reveals that their progeny are genetically identical and show no evidence of recombination or paternal inheritance. Flow cytometric measurement of DNA content shows the hybrids to be diploid, with DNA values intermediate to those of the parental species. Because they are related to F. heteroclitus, a fish used widely as a model organism in experimental biology, the clonal hybrids are potentially valuable for experimental studies requiring subjects with a constant genetic background. In addition, the discovery of unisexuality and cloning in a fish whose reproductive physiology and development are so well characterized provides a unique opportunity to examine the underlying causes of clonal reproduction in vertebrates.

Alleles↗
PubMed2001

Heteroduplex analysis of T-cell receptor gamma gene rearrangement as an adjuvant diagnostic tool in skin biopsies for erythroderma.

BACKGROUND: Erythroderma, defined as red skin covering most of the body surface often accompanied or followed by exfoliation, is the clinical manifestation of at least six different underlying etiologies with allergic or irritant contact dermatitis, atopic/asteotic dermatitis, pityriasis rubra pilaris (PRP), psoriasis, and seborrheic dermatitis accounting for the majority of cases. Approximately 10% of cases are due to adverse drug reactions with roughly another 10% due to cutaneous T-cell lymphoma (CTCL), predominantly mycosis fungoides, or leukemia. It is clear from multiple studies that the clinical diagnosis of the underlying entity is often difficult, as these diseases can present in a very similar fashion. A skin biopsy is usually employed in this setting as a diagnostic tool. However, the histopathologic diagnosis of the underlying cause is complicated by the subtlety of the distinguishing histologic features. In this situation, an ancillary technique demonstrating the presence of a monoclonal T-cell proliferation could help to rule in or out CTCL in cases that clinically and histopathologically do not allow a definitive diagnosis. METHODS: We retrospectively studied 25 biopsies from sixteen patients who presented to the Stanford Dermatology Clinic with erythroderma. We examined the specimens morphologically and analyzed the gamma chain of the T-cell receptor (TCR- gamma) by polymerase chain reaction (PCR) followed by heteroduplex analysis for clonality. We then correlated the results of our PCR and heteroduplex analyses with the patients' clinical outcomes. RESULTS: Four biopsies, from three patients, contained clonal TCR-gamma rearrangements; the four biopsies, all of which were equivocal histologically, correlated to diagnoses of mycosis fungoides (MF) or Sézary syndrome (SS). Twenty-one biopsies contained polyclonal T-cell populations. Eighteen of these biopsies represent patients with inflammatory dermatoses. Three of these biopsies, all of which were taken from a single patient, correlate to a diagnosis of MF. CONCLUSION: TCR-gamma PCR heteroduplex analysis seems to represent an important adjuvant diagnostic tool that, used in conjunction with histopathology and clinical history, could help to clarify the underlying etiology of erythroderma.

Aged↗
PubMed2003

Lymphoid tumours of the ocular adnexa: a morphologic and genotypic study of 15 cases.

PURPOSE: To examine all lymphoproliferative lesions of the ocular adnexa diagnosed in Iceland during 1983-2000 and to determine whether polymerase chain reaction (PCR) methods to determine clonality are helpful in characterizing these lesions. METHODS: All patients diagnosed with lymphoproliferative lesions in the ocular adnexa in the years 1983-2000 were included in the study. Polymerase chain reaction studies for clonality were performed on these lesions. RESULTS: Fifteen cases were identified. Seven were classified as inflammatory pseudotumour, one as lymphoid hyperplasia, four as atypical lymphoid hyperplasia and three as lymphoma. Of 12 cases examined by PCR, three were monoclonal for B-cells (one lymphoma, one inflammatory pseudotumour and one atypical lymphoid hyperplasia) while the remaining lesions (including two lymphomas) appeared polyclonal. CONCLUSION: The results of this study suggest that analysis of clonality by PCR methods may be of limited use in classifying lymphoproliferative lesions of the ocular adnexa as benign or malignant. These results underscore the importance of using several techniques when determining clonality.

Adult↗
PubMed2001

Effect of chemotherapy and stem cell transplantation on T lymphocyte clones in familial haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL) is a rare disorder in infancy, curative only by an allogeneic stem cell transplantation (SCT). We recently confirmed the clonal evidence of T cells in FHL. To confirm the effect of chemotherapy and SCT in FHL, the change of T-cell clones was analysed in two patients using inverse reverse transcription-polymerase chain reaction (RT-PCR) of the T-cell receptor variable region (TCR V) gene, followed by PCR for the junctional region (Jbeta-PCR), a single-strand conformation polymorphism (SSCP) and sequencing analysis at diagnosis, after chemotherapy and after SCT. A high frequency (> 15%) of alphabeta T-cell clones and a predominant bias (Jbeta1:Jbeta2, 85:15) for the Jbeta1 subgroup were observed in the two patients at diagnosis. In one patient, however, an inverted predominant bias (Jbeta1:Jbeta2, 9:91) for the Jbeta2 subgroup and oligoclonal expansion were observed at relapse after chemotherapy. In the other patient, correction of both restricted Jbeta cluster usage and variation of TCR were observed after chemotherapy and SCT. Using sequence analysis, the clonal T cells detected at diagnosis were found to be substituted at low frequency (< 0.75%) by several new clones after chemotherapy and SCT. These results indicate that any genetic defect could influence the regulation of the T-cell network, and normalization of both the variation in each Vbeta repertoire and the Jbeta1/Jbeta2 ratio is needed to achieve remission, and might support the rationale that the only acceptable curative therapy of FHL is allogeneic SCT.

Clone Cells↗
PubMed2003

Microbead display of proteins by cell-free expression of anchored DNA.

Gene expression technologies where nucleic acid sequences remain physically linked to their corresponding gene products are important tools for selection and identification of rare variants in large protein libraries. Here, we describe a gene expression system, which combines the potential of bead-based suspension array technology (SAT) with gene expression and clonal identification. Using streptavidin-coated polystyrene micrometer-sized beads as solid supports for anchored PCR products, we have investigated conditions for cell-free expression and bioaffinity technology to provide clonal co-anchoring of corresponding gene products. Experiments showed that coupled transcription and translation of PCR product expression cassettes resulted in display of affinity-anchored proteins whose binding characteristics could be analyzed via direct and selective interaction with a fluorescently labeled target protein. Interestingly, experiments performed with differently biotinylated PCR products showed that the efficiency of display was dependent on the directionality of the expression cassette relative to the bead surface. In spiked systems, using small immunoglobulin binding proteins as models, we demonstrate efficient flow cytometric sorting of beads corresponding to the target interacting clones, verified by post-sorting analysis and clonal identification at DNA level. The use of this technology, including alternative formats, for different proteomics applications is discussed.

Adsorption↗
PubMed1996

Characterization of Prevotella intermedia and Prevotella nigrescens by enzyme production, restriction endonuclease and ribosomal RNA gene restriction analyses.

Restriction endonuclease analysis, rRNA gene restriction analysis (ribotyping), multilocus enzyme electrophoresis and lipase production were investigated for their potential to differentiate isolates belonging to the closely-related species Prevotella intermedia and Prevotella nigrescens. Of 122 strains identified originally as P. intermedia, 52 were assigned to P. intermedia and 68 to P. nigrescens using multilocus enzyme electrophoresis. All 39 P. intermedia and 52 out of 53 P. nigrescens tested produced lipase. Restriction endonuclease analysis identified clonal variants, but did not facilitate the differentiation of strains into species. Taq I ribotyping of 99 strains revealed that all P. intermedia demonstrated a species-specific fragment of 0.40 kbp, which was always associated with a second fragment of 0.57 kbp, and all P. nigrescens tested shared a species-specific fragment of 2.21 kbp. Two strains atypical by multilocus enzyme electrophoresis had none of the above species-specific fragments. Thus, lipase production and restriction endonuclease analysis did not distinguish between P. intermedia and P. nigrescens, but Taq I ribotyping did and also allowed the characterization of individual strains.

Bacterial Typing Techniques↗
PubMed1991

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.

B-Lymphocytes↗
PubMed1999

Rapid screening of T-cell receptor (TCR) variable gene usage by multiplex PCR: application for assessment of clonal composition.

The selection of various T-cell receptor (TCR) gene families and complex rearrangements during intra-thymic differentiation provide the basis for the expression of antigen specificity by mature T cells. TCR beta variable (TCRBV) transcripts can be identified by RT-PCR, but multiple reactions are required to detect all genes of the TCRBV subfamilies. We describe here a multiplex PCR method that amplifies 46 functional genes comparing 23 TCRBV families in 5 reactions where each reaction contains 4 to 7 specific primers together with a single fluorescence-tagged TCR beta constant region primer. Between 8 and 10 distinct subtypes within each of the 23 TCRBV families can be identified by analysis of the CDR3 length. Multiplex PCR products isolated from agarose gels can be subjected to direct sequencing for confirmation and definitive clonotyping if necessary. The data illustrated here show that the multiplex PCR technique is useful for screening TCRBV usage and can be easily adapted for analysis of clonal composition in T-cell populations.

Cell Line↗
PubMed2000

Different risk factors and prognosis for early and late intrahepatic recurrence after resection of hepatocellular carcinoma.

BACKGROUND: Recent studies have shown that the prognosis of recurrent hepatocellular carcinoma (HCC) after resection was dependent on the time of recurrence. The current study investigated whether early and late intrahepatic recurrences were associated with different risk factors and prognostic factors. METHODS: After curative resection of HCC, 246 patients were followed prospectively for recurrence. Intrahepatic recurrences were classified into early (</= 1 year) and late (> 1 year) recurrences. Risk factors for recurrence and prognostic factors for survival after recurrence in each group were analyzed. RESULTS: Early and late intrahepatic recurrences developed in 80 patients and 46 patients, respectively. By multivariate analysis, preoperative tumor rupture (P = 0.022) and venous invasion (P < 0.001) were independent risk factors for early recurrence, whereas cirrhosis (P = 0.018) was the only significant risk factor for late recurrence. By comparing histologic features of resected recurrent and primary tumors, 8 of 9 resected early recurrent tumors (89%) were classified as intrahepatic metastases, whereas all 6 resected late recurrent tumors (100%) were multicentric occurrences. Despite similar treatments, the prognosis for patients with early recurrence was worse than that of patients with late recurrence (median survival of 15.8 months vs. 29.6 months; P = 0.005). Independent prognostic factors for early recurrence were serum albumin level and initial tumor pTNM classification, whereas only serum bilirubin level was found to be an independent prognostic factor for late recurrence. CONCLUSIONS: Early and late intrahepatic recurrences after resection of HCC were associated with different risk factors and prognostic factors. Early recurrences appear to arise mainly from intrahepatic metastases, whereas late recurrences are more likely to be multicentric in origin. The current study suggests that different strategies may be needed for the prevention and management of early and late recurrences. Further studies based on genetic analysis of clonal origins of tumors are required to clarify fully the mechanism of early and late recurrences after resection of HCC.

Adult↗
PubMed2001

Molecular cytogenetic definition of three distinct chromosome arm 14q deletion intervals in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms characterized by frequent chromosome arm 14q losses. In this study, the 14q changes in a series of 39 histologically and immunohistochemically confirmed GISTs were analyzed in detail by metaphase and/or interphase fluorescence in situ hybridization (FISH) studies using 21 genetically well-characterized, region-specific 14q11-24 YAC clones. By conventional cytogenetic analysis, acquired clonal chromosome aberrations were found in 17 out of 35 tumors. Chromosome 14 was involved in 13 cases; six specimens showed complete chromosome 14 loss, while the remaining seven had structural abnormalities with the breakpoints residing within the intervals 14q11-13 or 14q22-24. Other recurrent chromosome aberrations included frequent deletions of chromosome 1p (11/17), losses of chromosome 22 (7/17), losses or deletions of chromosome arm 13 (6/17) or 15 (4/17), and gains or translocations involving chromosome 17 (4/17). Combining cytogenetic data with double-color FISH analysis, total or partial losses of 14q material were detected in 29 out of 36 tumors (81%). The 14q losses were found in all stages and histological subtypes. Two most frequent common deletion regions flanked by YACs 931B1 and 761D4, and 802E7 and 892C11 at 14q23-24 (25/30 of each; 83%) could be identified. Furthermore, 21 tumors (70%) shared a region of deletion defined by YACs 957H10 and 931E5 at 14q11-12. Our results suggest the presence of at least three distinct critical deletion regions on chromosome 14 in GISTs.

Adult↗
PubMed2004

T-cell receptor gammadelta-large granular lymphocytic leukemia associated with an aberrant phenotype and TCR-Vbeta20 clonality.

Large granular lymphocytic (LGL) leukemia is a rare heterogenous disorder of mature lymphocytes with a characteristic morphology, multiple autoimmune disorders and indolent clinical course. Most cases exhibit a T-cell phenotype of CD3, CD8 and CD57 positivity, while the minority exhibit a CD2, CD56, and CD16 positive NK-cell phenotype. We report a case of a 71-year-old female suffering from a TCRgammadelta positive T-cell leukemia with a morphology compatible to LGL leukemia. She referred to the hospital for investigation of mild anemia, lymphocytosis, neutropenia and hyperglobulinemia. Peripheral blood and bone marrow were occupied by mature large granular lymphocytes with abundant azurophilic granules. The immunophenotype was CD3+, CD2+, CD5+, CD7+, CD4-, CD8-, CD16-, CD56-, CD57- and the Vbeta repertoire analysis showed clonal reactivity with Vbeta20 mAb. The patient was diagnosed as having T-LGL and was treated with G-CSF. So far, she experiences an indolent clinical course. To our knowledge, this is a rare case of TCRgammadelta positive T-LGL leukemia with the aberrant immunophenotype of CD3+, CD4-, CD8-, CD16-, CD56-, CD57-.

Aged↗
PubMed1997

Chromosome 2 hypersensitivity and clonal development in murine radiation acute myeloid leukaemia.

Acute myeloid leukaemias induced by ionizing radiation in mouse are characterized by chromosome (chr) 2 aberrations. While it is known that chr 2 aberrations form early and in abundance post-irradiation, unequivocal evidence for hypersensitivity of chr 2 in the first post-irradiation mitoses is lacking. Here it is established that chromosomal aberrations detected in bone marrow cells by chromosome painting are induced in all mice at an approximately 2-fold greater frequency in chr 2 by comparison with chrs 1 and 3 at 24 and 48 h following in vivo whole-body X-irradiation. Long-term follow up studies (to 15 months post-irradiation) indicated that chromosomal hypersensitivity is accounted for largely by the existence of hot-spots for aberration formation on sensitive chromosomes. Analysis of clonal developments suggested that chr 2 aberrant clones are selected for entry into the proliferating bone marrow cell compartment in preference to cells with other aberrations and that these clones in general have a higher proliferative potential. However, neither the induction of chr 2 aberrations nor the presence of a chr 2 aberrant clone specifically predict the development of AML in an individual irradiated mouse. Nonetheless these events or sub-groups of these events are necessary for AML development.

Acute Disease↗
PubMed1989

Specificity of HLA restricting elements for human nickel reactive T cell clones.

In order to study the fine specificity of HLA class II restriction, we have established nickel specific T cell clones from a nickel allergic patient. Cells were cloned by limiting dilution after primary stimulation and selection of nickel specific blasts. Several clones were established which were all shown to carry the CD4 marker. All clones were shown to be completely blocked by monoclonal antibodies directed against DR antigens, but unaffected by antibodies against DQ or DP, thus demonstrating their DR specificity. For the study of HLA class II restriction, a panel of cell donors was carefully HLA typed by including the use of DRB and DQB cDNA probes. Specificity analysis, using allogeneic antigen presenting cells, revealed that the clones were either restricted to DR3- or DR4-like molecules, which is consistent with the fact that the donor was DR3, DR4 positive. However, the studies also revealed that the fine specificity of the DR3 and DR4 restriction could not be completely assessed by serological and genomic typing of panel cells. This indicates that cellularly defined HLA restriction elements recognized by T cells cannot be defined properly with available class II typing methods, and the results of these experiments documented the additional polymorphism of class II restriction elements. The clonal specificity analysis has shed further light on the biologically relevant level of DR polymorphism.

Clone Cells↗
PubMed1987

Surface markers of human lymphokine-activated killer cells and their precursors. Analysis at the population and clonal level.

Lymphokine-activated killer (LAK) activity was first analyzed on PBL populations fractionated on the basis of the expression of T11 or T3 antigen. LAK cell precursors were found to be present in both T11+ and T11- populations, but only in the T3- cell fraction. The generation of LAK activity in highly purified T3- populations of PBL was not accompanied by expression of T3 antigen during a 5-day culture period. LAK activity was next analyzed at the level of limiting dilution clonal microcultures. T11+T3- and T11+T3+ cells, cloned under optimal culture conditions, gave a frequency of proliferating cells of approximately 1 cell in 1.25 for T11+T3+ and 1 cell in 10 for T11+T3- cells. Clones were screened for their ability to lyse fresh ovarian carcinoma cells and K562 target cells. The majority of LAK clones were derived from the T11+T3- cells; moreover, most of the clones derived from these cells displayed LAK activity. Clones displaying LAK activity lysed a panel of fresh or cultured tumor target cells, but failed to lyse PHA-activated lymphoblasts. Surface marker analysis indicated that all the clones had maintained the original T11/T3 phenotype. Whereas 2 T3+ selected LAK clones expressed the T8+T4- phenotype, only 1 out of 9 T3- clones was T8+T4-, all the others lacking both T4 and T8 antigens.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1994

Heterogeneity in DNA content & proliferative status of human brain tumours.

Intra-tumour and inter-tumour heterogeneity in the cytokinetic organization was studied in 235 primary human brain tumours. DNA index (DI; relative tumour cell DNA content) and proliferating fraction (%PF; a measure of proliferative status) were analyzed in tumour biopsy by flow cytometry using a DNA specific fluorochrome (DAPI) and internal standards (chicken erythrocytes, CE). Incidence of micronuclei was studied in tumour biopsy tissue as well as in explants maintained in organ culture. Clonal diversity (implied by the presence of multiple peaks in the DNA histograms) was highest among medulloblastomas (44%) followed by gliomas (19%) and meningiomas (14%). Nearly 85 per cent of the malignant gliomas analyzed (histological grade III/IV) exhibited a great deal of regional variation in the proliferative status as well as micronuclei frequency as compared to meningiomas. Inter-tumoural variations in the DNA content was highest among gliomas (0.9 < DI < 3.6) and lowest among schwannomas (1.7 < DI < 2.2). Similarly, the distribution of %PF values was also broader (10-49%) in gliomas as compared to the other primary brain tumours (5-36%). Analysis of tumours taking both DI and %PF values improved the ability to discern histologically graded low and high tumours. Analysis of clonal diversity and spatial heterogeneity in the cytokinetic parameters could complement the clinicopathological findings in assessing the biological behaviour of human brain tumours, facilitating the prognostification and design of otpimal treatment regimen.

Brain Neoplasms↗
PubMed2000

[Clonal rearrangement of intratumoral T-cell receptor beta-chain gene in two patients with thymoma accompanied by pure red cell aplasia].

We encountered two cases of thymoma accompanied by pure red cell aplasia and demonstrating clonal rearrangement of the T-cell receptor beta-chain gene (TCR-beta) in lymphocytes. Patient 1 was a 55-year-old man and Patient 2 was a 43-year-old woman. Both had severe anemia and mediastinal tumors. Bone marrow aspiration was performed and pure red cell aplasia diagnosed. Thymoma was the presumptive diagnosis for the mediastinal tumors, and extended thymectomy was performed. The post-operative diagnosis was invasive thymoma (spindle-cell type) in Patient 1 and non-invasive thymoma (mixed lympho-epithelial type) in Patient 2. The cell compositions (%) obtained with T-cell surface marker analysis were as follows: [table: see text] Southern blot analysis disclosed clonal rearrangement of TCR-beta genes in thymoma thymocytes from both patients.

Adult↗