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PubMed2002

Global molecular epidemiology of the O15:K52:H1 extraintestinal pathogenic Escherichia coli clonal group: evidence of distribution beyond Europe.

Escherichia coli O15:K52:H1 is a significant extraintestinal pathogen in Europe (G. Prats et al., J. Clin. Microbiol. 38:201-209, 2000). To search for evidence of this clonal group outside of Europe, 75 non-European E. coli isolates of serogroup O15 were compared with five members of the O15:K52:H1 clonal group from Barcelona, Spain, according to genomic background, virulence genotypes, and antimicrobial resistance profiles. Amplification phylotyping showed that 16 (21%) of the 75 non-European O15 isolates corresponded with the O15:K52:H1 clonal group. The 16 non-European O15:K52:H1 clonal group members represented diverse geographic locales. They were isolated almost exclusively from humans with extraintestinal infections and accounted for 50% of all O15 isolates from five human clinical collections studied. Most non-European clonal group members exhibited a consensus virulence factor profile that included the F16 or F7-2 papA alleles (P fimbrial structural subunit), papG allele II (P fimbrial adhesin), iha (putative adhesin siderophore), and iutA (aerobactin receptor). This resembles the virulence profiles of (i) European representatives of the O15:K52:H1 clonal group and (ii) phylogenetically related "clonal group A," a recently recognized significant contributor to trimethoprim-sulfamethoxazole resistance in the United States (A. R. Manges et al., N. Engl. J. Med. 345:1007-1013, 2001). Antimicrobial resistance profiles were variable, and resistance was inconsistently transferred by conjugation. These findings indicate that the O15:K52:H1 clonal group is broadly distributed beyond Europe, exhibits previously unrecognized phenotypic and genotypic diversity, and contributes significantly to extraintestinal infections in humans.

Animals↗

PubMed2001

Frequency of clonal intraepithelial T lymphocyte proliferations in enteropathy-type intestinal T cell lymphoma, coeliac disease, and refractory sprue.

BACKGROUND: Clonal T cell receptor (TCR) gene rearrangements and loss of T cell antigens such as CD8 and TCR-beta in intraepithelial lymphocytes (IELs) may indicate the development of an enteropathy-type intestinal T cell lymphoma (EITCL) in patients with refractory sprue. AIMS: To define the diagnostic value of these markers in duodenal biopsies from patients with villous atrophy as a result of various underlying disorders. PATIENTS AND METHODS: Duodenal biopsies from eight patients with coeliac disease and five patients with villous atrophy caused by defined disorders were compared with three patients with refractory sprue evolving into overt EITCL, two patients with ulcerative jejunitis, and with eight patients with overt EITCL, for expression of CD3, CD4, CD8, and TCR-beta in IELs using immunohistochemistry and for clonal TCR-gamma gene rearrangements using polymerase chain reaction. In addition, biopsies from six consecutive patients with refractory sprue of uncertain cause were examined. RESULTS: Clonal TCR-gamma gene rearrangements were found in all resected tumours of patients with EITCL, in 3/8 duodenal biopsies of patients with EITCL, in 2/2 patients with ulcerative jejunitis, in 2/3 patients with refractory sprue evolving into overt EITCL, and in 1/6 patients with refractory sprue. No rearrangements were found in biopsies from patients with refractory sprue caused by defined disorders or those with coeliac disease. Clonality in duodenal biopsies was associated with an abnormal phenotype of IELs in all cases and in all but one case in patients with evidence of underlying coeliac disease. Specificity for detection of an EITCL using immunohistology was 77% for CD8 and for TCR-beta staining, and 100% for detection of a clonal TCR-gamma gene rearrangement. Sensitivity was 62% for staining with CD8 and clonality investigation, while sensitivity reached 100% for TCR-beta staining in all investigated patients with EITCL. CONCLUSIONS: Clonal proliferations of phenotypically abnormal IELs in refractory sprue represent an early manifestation of EITCL, for which the term "sprue-like intestinal T cell lymphoma" is proposed. This constellation is also found in duodenal biopsies from patients with an overt EITCL and is not related to other sprue syndromes, resulting in a high specificity for detection of an EITCL or refractory sprue evolving into EITCL. Overt EITCL may develop directly from coeliac disease without a precursor lesion (refractory sprue with clonal IELs) being demonstrable in duodenal biopsies or via a "sprue-like intestinal T cell lymphoma". This latter entity is a complication of coeliac disease.

Adolescent↗

PubMed1996

Detection of clonal immunoglobulin gene rearrangements in the peripheral blood progenitor cells of patients with multiple myeloma: the potential role of purging with CD34 positive selection.

Aims-To determine the extent of clonal cell contamination of peripheral blood progenitor cell (PBPC) collections in patients with multiple myeloma (MM) and to assess the purging efficacy of CD34 positive selection.Methods-PBPC collections from 29 patients with MM were analysed for the presence of clonal immunoglobulin heavy chain (IgH) gene rearrangements with a fluorescence based PCR technique. In addition, the PBPC from eight of the 29 patients were "purged" by selection of CD34 positive haematopoietic progenitors with an avidin-biotin immunoabsorption column (Ceprate). In each case the unmanipulated PBPC, CD34 positive and waste fractions were all assessed for the presence of clonal IgH rearrangements.Results-Clonal IgH rearrangements (identical with those demonstrated in diagnostic bone marrow samples) were demonstrated in 10 (35%) of 29 cases and seemed to be confined to those with significant residual bone marrow disease. Clonal rearrangements were evident in the PBPC of two of the eight patients who underwent CD34 selection; in both instances a "clonal purge" was seen as it was not possible to demonstrate the clonal rearrangement in the CD34 positive fraction. In four of the six remaining cases the normal polyclonal fingerprint could not be demonstrated in the CD34 positive fraction, which is consistent with a significant reduction in contaminating B cells.Conclusions-Clonal cells contaminate PBPC collections in a significant proportion of patients with MM and may be eliminated by CD34 positive selection.

Journal Article↗

PubMed1986

Human fetal and adult chondrocytes. Effect of insulinlike growth factors I and II, insulin, and growth hormone on clonal growth.

Clonal proliferation of freshly isolated human fetal chondrocytes and adult chondrocytes in response to human insulinlike growth factors I and II (IGF I, IGF II), human biosynthetic insulin, and human growth hormone (GH) was assessed. IGF I (25 ng/ml) stimulated clonal growth of fetal chondrocytes (54 +/- 12 colonies/1,000 inserted cells, mean +/- 1 SD), but IGF II (25 ng/ml) was significantly more effective (106 +/- 12 colonies/1,000 inserted cells, P less than 0.05, unstimulated control: 14 +/- 4 colonies/1,000 inserted cells). In contrast, IGF I (25 ng/ml) was more effective in adult chondrocytes (42 +/- 6 colonies/1,000 inserted cells) than IGF II (25 ng/ml) (21 +/- 6 colonies/1,000 inserted cells; P less than 0.05, unstimulated control: 6 +/- 3 colonies/1,000 inserted cells). GH and human biosynthetic insulin did not affect clonal growth of fetal or adult chondrocytes. The clonal growth pattern of IGF-stimulated fetal and adult chondrocytes was not significantly changed when chondrocytes were first grown in monolayer culture, harvested, and then inserted in the clonal culture system. However, the adult chondrocytes showed a time-dependent decrease of stimulation of clonal growth by IGF I and II. This was not true for fetal chondrocytes. The results are compatible with the concept that IGF II is a more potent stimulant of clonal growth of chondrocytes during fetal life, whereas IGF I is more effective in stimulating clonal growth of chondrocytes during postnatal life.

Adult↗

PubMed2004

Early TCR-beta and TCR-gamma PCR detection of T-cell clonality indicates minimal tumor disease in lymph nodes of cutaneous T-cell lymphoma: diagnostic and prognostic implications.

The lymph nodes are generally the first extracutaneous manifestation in patients with cutaneous T-cell lymphoma (CTCL); however, their early involvement is difficult to assess. The aim of our study was to define the diagnostic and prognostic value of T-cell clonality analysis for a more precise assessment of lymph node involvement in CTCL. T-cell clonality was determined by 2 independent polymerase chain reaction (PCR) assays, namely a recently developed T-cell receptor-beta (TCR-beta) PCR technique as well as an established TCR-gamma PCR. T-cell clonality was found in 22 of 22 lymph nodes with histologically detectable CTCL involvement as well as in 7 of 14 histologically noninvolved dermatopathic lymph nodes. The clonal T-cell populations in the lymph nodes were in all cases identical to those detected in the corresponding skin lesions, identifying them as the tumor cell population. T-cell clonality was not found in any of the 12 dermatopathic lymph nodes from 12 patients with inflammatory skin diseases. Clonal T-cell detection in 7 of 14 dermatopathic lymph nodes of patients with CTCL was associated with limited survival (74 months; confidence interval [CI], 66-82 months) as in patients with histologically confirmed lymph node involvement (41 months; CI, 35-47 months), whereas all patients without T-cell clonality in the lymph nodes (7 patients) were alive at the last follow-up. Thus, T-cell clonality analysis is an important adjunct in differentiating benign dermatopathic lymphadenitis from early CTCL involvement.

Adult↗

PubMed2002

Clonal heterogeneity in mycosis fungoides and its relationship to clinical course.

Mycosis fungoides (MF) is a cutaneous T-cell lymphoma characterized by multifocal disease and protracted clinical course. The few studies that have assessed T-cell receptor (TCR) gene rearrangements (GRs) present at different anatomic sites in MF have generally reported a common clone. We used a previously validated 4-color polymerase chain reaction (PCR) assay to assess the size and V-family usage of TCR-gamma GRs in 102 concurrent and/or sequential morphologically involved biopsy specimens (91 skin and 11 lymph nodes) from 39 MF patients. This assay detected TCR-gamma clonal GRs in 89 samples (87%) from 36 patients (92%). In 24 patients (77%), an identical clonal GR was present in at least 2 skin samples. However, in one third of these patients, additional different clonal GRs were also noted. Four patients (13%) had clonal GRs that were distinct in different skin samples. In 3 patients (10%), no GR was detected in any sample. In a comparison of lymph node and skin samples, 8 patients had the identical clonal GRs at both sites, 2 patients had different clonal GRs, and 1 patient had no GR identified at either site. Independent of clinical stage, patients who had the same GR detected in multiple concurrent biopsy specimens at the time of diagnosis were more likely to have progressive disease than those who had different GRs (P =.04). Four-color TCR-gamma PCR analysis can uncover multiple distinct clonal GRs in different samples consistent with multiclonal or oligoclonal disease in a significant proportion of MF patients. Demonstration of identical clonal GRs in multiple biopsy specimens at the time of diagnosis may provide prognostic information related to disease progression.

Adolescent↗

PubMed1993

Morphological and functional polymorphism within clonal thyroid nodules.

Thirty-nine thyroid nodules, removed because of recent growth, were analyzed morphologically by serial histological sections for the classical histomorphological hallmarks of follicular cell replication and for immunohistochemically demonstrable overexpression of the growth-associated ras-gene product p21ras. Clonal analysis was performed using the highly informative probe M27 beta that detects polymorphisms on the locus DXS255 of the X-chromosome. Twenty-four nodules were of clonal and 15 nodules were of poly-clonal origin. Only 3 out of the 24 clonal nodules were histomorphologically uniform. In all others, the structural hallmarks of active growth and the P21ras growth-marker expression were remarkably heterogeneous throughout the tumors. There were no histomorphological characteristics distinguishing these clonal tumors from polyclonal nodules. Even if a clonal thyroid tumor may be originally homogeneous in respect to the parameters studied here, mechanisms must exist that create wide heterogeneity of growth and of morphogenetic potential among the individual follicular cells during further expansion of the nodule. Thus, clonal nodules are much more common in nodular goiters than hitherto assumed on grounds of the classical morphological criteria. The diagnosis of a true monoclonal nodule can no longer rely on morphological and functional criteria alone but requires molecular or cytogenetic analysis of clonality.

Adult↗

PubMed1999

Cellular basis of B cell clonal populations in old mice.

Previous studies from this laboratory have shown that >85% of old mice have stable B cell clonal populations detectable by Ig heavy chain complementary-determining region 3 mRNA size analysis and confirmed by sequence analysis. B cells from the same clone are frequently detected in several lymphoid compartments of the same mouse. We now report the phenotype of all ten stable B cell clonal populations detected in five 20-month-old C57BL/6 mice. These clonal B cells appear to develop in the periphery and nine of the ten B cell clonal populations expressed the CD5 cell surface marker. Stable B cell expansions may be dominated by cells at two stages of differentiation. Some B cell populations were detected with DNA as well as RNA and represent large clonal populations of B cells, detectable in several lymphoid compartments. These populations are found predominantly in B cell populations expressing CD45R/B220 and the mRNA coding for the membrane-bound form of the mu Ig heavy chain, which suggests a predominance of B lymphocytes in these populations. In other cases, smaller clonal populations were detected only in splenic RNA samples. These clonal populations were found predominantly among CD45R/B220- B cells and did not express the membrane-bound form of the micro Ig heavy chain. We offer the hypothesis that the B cell clonal populations present in old mice may be precursors of the two types of B cell neoplasms which are dominated by CD5+ B cells (B cell chronic lymphocytic leukemia) or plasma cells (multiple myeloma).

Aging↗

PubMed1999

Demonstration of frequent occurrence of clonal T cells in the peripheral blood but not in the skin of patients with small plaque parapsoriasis.

Clinical, immunohistological, and molecular biological data suggest the chronic dermatosis small plaque parapsoriasis (SPP) to be a precursor of mycosis fungoides (MF). However, most data are contradictory and confusing due to inexact definition of SPP. Recently, clonal T cells were detected in skin and blood samples of early MF. Because demonstration of identical T-cell clones in skin and blood of SPP patients would indicate a close relationship of SPP to MF, we investigated the clonality of skin and blood specimens from 14 well-defined SPP patients. By a polymerase chain reaction (PCR) amplifying T-cell receptor gamma rearrangements and subsequent high-resolution electrophoresis, clonal T cells were detected in 9 of 14 initial and 32 of 49 follow-up blood samples, but in 0 of 14 initial skin specimens. Even a clone-specific PCR showing the persistence of the initial blood T-cell clone in 20 of 20 follow-up samples, failed to detect the T-cell clone in the skin. In 2 patients, the clonal T cells were shown to be CD4(+). For the first time, the majority of SPP patients was shown to carry a T-cell clone in the peripheral blood. Although a relation between circulating clonal T cells and SPP cannot directly be proven by the applied techniques, our results indicate blood T-cell clonality to be a characteristic feature of SPP and CTCL because analysis of multiple controls and clinical workup of our SPP patients excluded other factors simulating or causing a clonal T-cell proliferation. A sufficient cutaneous antitumor response but also an extracutaneous origin of the T-cell clones might explain the failure to detect skin infiltrating clonal T cells.

Aged↗

PubMed2001

Denaturing HPLC for identification of clonal T-cell receptor gamma rearrangements in newly diagnosed acute lymphoblastic leukemia.

BACKGROUND: Denaturing HPLC (DHPLC) can be used to screen DNA for known and unknown mutations. We describe a novel, HPLC-based method for discrimination among polyclonal, oligoclonal, and/or clonal T-cell receptor gamma (TCR-gamma) rearrangements in samples from children with newly diagnosed acute lymphoblastic leukemia. METHODS: TCR rearrangements were PCR amplified from initial leukemic samples and, after heteroduplex-induction, the clonality status of each product was evaluated. To attain this, we used DHPLC on a high-resolution micropellicular matrix. Running conditions were established by melting-curve analysis of known clonal and polyclonal products and melting-point prediction software. Elution profiles were studied at 50 degrees C (native) and, to achieve optimal separation, at different column temperatures between 56 and 64 degrees C. RESULTS: For VgammaI-Jgamma1.3/2.3 rearrangements, an analysis temperature of 60 degrees C with a linear triethylammoniumacetate-acetonitrile gradient separated clonal bands from the polyclonal background amplification. Less than 15% clonal PCR product was detectable in mixtures of initial leukemic cell DNA and polyclonal DNA. Biallelic rearrangements produced two sharp peaks. Clonality of PCR products from 100 initial leukemic samples was completely identified in all investigated cases. CONCLUSIONS: Heteroduplex analysis with standardized DHPLC conditions simplifies the detection of unknown clonal or polyclonal TCR rearrangements in newly diagnosed leukemias. Clonal targets for detection of minimal residual disease are available after a short, automated analysis of PCR amplified rearrangements.

Base Sequence↗

PubMed1992

Clonal growth and differentiation of rabbit meibomian gland epithelium in serum-free culture: differential modulation by EGF and FGF.

We have established a serum-free clonal culture system to study the growth and differentiation of individual progenitor epithelial cells of the meibomian gland independent of other cell types and undefined serum factors. Single meibomian gland epithelial cells were obtained by subjecting whole meibomian glands to a brief EDTA treatment, needle aspiration, and nylon mesh filtration. The cells had been isolated by a previously described method using enzymatic and microsurgical techniques. Four to five hundred cells obtained were seeded on a 35 mm or 60 mm dish with serum-free MCDB 151 medium containing insulin, transferrin, selenium, ethanolamine, o-phosphorylethanolamine, dimethyl sulfoxide, and calcium. Control cultures with this basic medium did not show continuous clonal growth. Addition of epidermal growth factor (EGF) from 1 to 5 and 10 ng/ml enhanced clonal growth with a decreasing effect as measured by colony forming efficacy and colony size. Clonal growth was associated with the occurrence of two types of colony morphology and an increase in intracellular lipid production in the BrdU-labelled cells, shown by Nile red fluorescent staining. In contrast, the clonal growth stimulated by addition of acidic fibroblast growth factor (aFGF) from 1 to 100 ng/ml exhibited a pattern of an initial increase followed by a decrease, with the maximum noted at 10 ng/ml. Moreover, the aFGF-stimulated clonal growth was associated with a uniform colony morphology and minimal lipid synthesis even in the non-BrdU-labelled cells. These results indicate that clonal growth of meibomian gland epithelium can be achieved in a serum-free culture by adding either of these two peptide growth factors. Furthermore, the clonal growth stimulated by EGF was associated with progressive cellular differentiation more so than that of aFGF. Further exploration of such a differential regulation of proliferation and differentiation by EGF and aFGF may provide a better understanding of normal meibomian gland function and pathogenesis of various meibomian gland disorders.

Animals↗

PubMed2005

Prognostic significance of clonal diversity of immunoglobulin gene rearrangements in patients with diffuse large B-cell lymphoma.

Clonal diversity of the immunoglobulin (Ig) gene rearrangement represents the oligoclonality of B-cell neoplasm, and has been shown to be a marker for poor prognosis in acute lymphoblastic leukemia. However, no previous report has addressed its prognostic impact in diffuse large B-cell lymphoma (DLBCL). We investigated the clinical significance of clonal diversity in DLBCL patients. Lymph node samples from 98 DLBCL patients were examined for Ig heavy and light chain gene rearrangements using Southern blot analysis. Clonal diversity was defined as oligoclonality detected on Southern blotting as previously described, and PCR analysis for IgH oligoclonality was performed on parts of DLBCL samples with clonal diversity for confirming the Southern blot analysis results. We found that clonal diversity could be detected in 36 (36.7%) of DLBCL patients, and PCR analysis showed concordant results. Regarding the clinical relevance, clonal diversity was significantly associated with relapse or refractory disease. Survival analysis showed that clonal diversity is an independent prognostic factor in DLBCL (p=0.05, Cox's proportional hazard method), and stratified analyses found the most significant subgroup is the high-intermediate risk category (p=0.01, log-rank test). We conclude that clonal diversity of Ig gene rearrangements is associated with a high risk of relapse or refractory disease in DLBCL patients. It is also a factor of poor prognosis in DLBCL, especially for high-intermediate risk category.

Adolescent↗

PubMed1994

Detection of clonal histiocytes in Langerhans cell histiocytosis: biology and clinical significance.

Although the first clinical description of Langerhans cell histiocytosis (LCH) was published over a century ago, the aetiology and pathogenesis of this enigmatic disorder are still remained unknown. Viral, immunological, neoplastic and other pathogenetic mechanisms have been considered, but none has been proven. The prevailing opinion is that LCH is a reactive disorder rather than a neoplastic process, but this presumption has never been definitively tested. A key feature of a neoplasm is its clonal derivation from a single cell. To determine if LCH is a polyclonal reactive or a clonal disorder, we and others have recently used molecular biological techniques to assess clonality in LCH. Using X chromosome-linked DNA probes that can detect clonal or polyclonal X chromosome inactivation patterns in female tissues, clonal CD1a+ histiocytes have now been detected in the lesional tissues in each of 16 females affected with LCH. Most of these patients were studied prior to the initiation of therapy. Lymphoid clonality was excluded in all cases in which it was studied, confirming that the clonal cells in LCH are the CD1a+ dendritic cells presumed to be pivotal in this disorder. Two distinct lesions (a pre-treatment bone biopsy and a lymph node biopsied 3 years later) have been studied in only one case to date; the same clonal pattern of X chromosome inactivation was observed, consistent with persistence of the same clone during this patient's disease course.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

Trypanosoma and Leishmania have clonal population structures of epidemiological significance.

This paper presents three results concerning the population structure of Trypanosoma cruzi, the agent of Chagas disease: (1) The mode of propagation of T. cruzi in nature is clonal; sexual reproduction is either totally absent or so rare that it leaves no traces in the population structure of the parasite. (2) The genetic diversity of the clonal lineages is large: extant T. cruzi represent lineages of descent that have evolved independently for long time spans (up to 40 million years). (3) Some genetically identical clonal lineages ("clonets") are geographically widespread ("ubiquitous"). However, most clonets are endemic, restricted in geographic distribution. These results have each in turn consequences of epidemiological significance: (1) In a sexually-reproducing organism the individual genotype is ephemeral; the entity that persists and evolves is the species ("gene pool"), and a few individuals contain most of the genetic variability of the species. In a clonally-propagating organism, the entity that persists and evolves is the clonal lineage; the genetic diversity of the species can only be captured by extensive sampling of distinct lineages. (2) The extensive genetic divergence among clonal lineages implies proportionally diverse biological characteristics, which are likely to include pathological effects, host propensity, vulnerability to drugs and vaccines, and other medically significant attributes. The extant T. cruzi lineages diverged much before human origins; hence, specific adaptation to human hosts, to whichever extent it exists, has evolved independently in separate lineages, and may not have evolved at all in some T. cruzi. (3) Epidemiological surveys and medical characterization, including search for specific vaccines and drugs, should not proceed randomly; rather, preliminary surveys must identify those clonets that are ubiquitous and target them for investigation. Review of published literature shows that Leishmania (and other parasitic protozoa) also has a clonal population structure. We advance a taxonomic and nomenclatural proposal that is appropriate for clonal organisms, yet simple.

Americas↗

PubMed1995

Clonal hematopoiesis as defined by polymorphic X-linked loci occurs infrequently in aplastic anemia.

We evaluated the methylation status of the X-linked gene phosphoglycerate kinase (PGK1) and the DXS 255 locus detected by probe M27 beta to study clonality in acquired aplastic anemia (AA). A total of 30 females were suitable for clonal analysis of peripheral blood polymorphonuclear cells (PMN) and mononuclear cells using a polymerase chain reaction-based procedure in 24 patients and Southern blotting in 9. Overall, 10 of 30 patients exhibited an imbalanced X-inactivation pattern. However, in 4 patients, analysis of constitutional DNA suggested a skewed methylation pattern and 2 further cases had to be excluded because of the lack of an appropriate control. A truly clonal pattern was thus established in 4 of 30 (13%) patients. In 7 patients who later developed clonal disorders of hematopoiesis, X-inactivation analysis did not predict this event in any case. In patients with a paroxysmal nocturnal hemoglobinuria phenotype, there was no correlation between the proportion of phosphatidylinositol glycan anchored protein (PIG-AP)-deficient blood cells and the corresponding X-inactivation pattern. X-inactivation analysis detected clonal hematopoiesis in only 3 of 10 patients with a deficiency in PIG-AP in the cell population under study, but sorting of nucleated cells on the basis of PIG-AP expression showed the clonal nature of PIG-AP-deficient cells. We conclude that the majority of patients with AA show polyclonal hematopoiesis using X-linked clonal analysis, but that minor clonal populations, such as PIG-AP-deficient cells, may not be detected unless sorted cell populations are separately analyzed.

Adolescent↗

PubMed1995

Detection of clonal T-cell receptor gamma gene rearrangements with the use of the polymerase chain reaction in cutaneous lesions of mycosis fungoides and Sézary syndrome.

BACKGROUND AND DESIGN: We used the amplification of junctional V (variable)-J joining sequences of the rearranged T-cell receptor gamma (TCR gamma) genes by polymerase chain reaction for rapid and sensitive detection of a clonal T-cell population in a total of 51 skin specimens obtained from 45 patients with mycosis fungoides, five patients with Sézary syndrome, and 29 patients with chronic inflammatory dermatoses. RESULTS: A clonal TCR gamma gene rearrangement was present in all tumors (3/3, 100%) and in most infiltrated plaques (16/22, 73%) and erythrodermas (10/12, 83%). In the patch stage, a clonal subset was found in more than half of the cases (8/14, 57%), whereas no clonality was observed in the controls. We also amplified the V-J sequences of the Igh locus coding for the heavy chain of immunoglobulins, without evidence of clonal rearrangement. These data were compared with those from in situ immunophenotypic analysis. Moreover, by using the same assay with successive dilutions of standard clonal T-cell DNA, a semiquantitative study of the T-cell clone was carried out in some cases. The highest ratios of clonal DNA were observed in advanced stages. CONCLUSIONS: These data validate polymerase chain reaction V gamma-J gamma as a rapid, sensitive tool that can be used in the routine analysis of clonality in cutaneous lesions of mycosis fungoides and in the early diagnosis of mycosis fungoides and Sézary syndrome. Semiquantitative studies suggest that the malignant T-cell clone follows a selective process during the course of the progressive form of mycosis fungoides.

Clone Cells↗

PubMed1995

Identification of a clonal form of HIV in early Kaposi's sarcoma: evidence for a novel model of oncogenesis, "sequential neoplasia".

We recently reported clonal human immunodeficiency virus (HIV involvement in four acquired immune deficiency syndrome (AIDS)-associated non-B-cell lymphoproliferations. In three of these cases HIV expression was localized to tumor-associated macrophages. Because one of the cases had a major component involved in angioproliferation, we speculated that some form of Kaposi's sarcoma (KS), which also has a major component of angioproliferation, might be involved clonally with HIV. The current study is an evaluation of four cases of KS and control tissues taken from four patients who died with complications of HIV disease. With use of the inverse polymerase chain reaction technique to identify clonal forms of HIV, a clonal form of HIV was found in one of four KS cases. The HIV-positive tumor was an early KS lesion of the bowel, and uninvolved bowel from the same patient showed no clonal HIV. Immunohistochemical analysis demonstrated the presence of prominent HIV-expressing macrophages that also coexpressed high levels of HIV tat, basic fibroblast growth factor, and interleukin-6. These data provide evidence for a pathogenic process termed "sequential neoplasia," wherein a clonal macrophage provides a growth factor milieu stimulating the proliferation of a responder cell population that ultimately becomes autonomous. In the current case, the macrophages expressing HIV were located adjacent to the KS tumor tissue and were found to be producing known KS growth factors. The absence of finding clonal HIV in three more advanced KS lesions suggests that the clonal macrophage may be required only for early pathogenesis and that sequential neoplastic changes occurring in the endothelial cells gave rise to autonomous KS.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1994

Polymerase chain reaction amplification analyses of clonality in T-cell malignancy including peripheral T-cell lymphoma.

Clonality in T-cell malignancy was investigated using T-cell receptor (TcR) V beta 1-20 family primers and polymerase chain reaction amplification (PCR) of cDNA prepared from tissue biopsies and blood involved with tumour. Secondary PCR amplification of the VDJ joints of primary PCR products was performed to distinguish clonal from polyclonal products, and clonal V beta gene products were confirmed by direct PCR sequencing in the majority of cases. In eight T-cell malignancies including T-cell acute lymphoblastic leukaemia (T-ALL) and T-cell chronic lymphocytic leukaemia (T-CLL) shown to be clonal by Southern blot analysis, one or two primary PCR products were identified and shown to be clonal. In five cases of peripheral T-cell lymphoma (PTCL) all V beta 1-20 families were identified after primary PCR amplification, and clonal products were identified in two cases after secondary amplification; TcR V beta clonal families could not be demonstrated in the remaining three cases. These data were in agreement with previous Southern blot analysis of these cases, and confirmed the presence of reactive T cells in PTCL as well as providing further evidence for the genotypic heterogeneity of this entity. In the remaining case, a blood lymphocytosis, primary PCR amplification produced predominant TcR V beta 6 and V beta 12 family products, of which the V beta 6 family proved clonal after secondary PCR amplification. There was no evidence for overrepresentation of TCR V beta families by the tumour populations in this study, furthermore the data confirm the involvement of reactive cells in T-cell malignancy and the genetic heterogeneity of PTCL.

Base Sequence↗