Polyclonal Pattern Research Articles

Clonality of Isolated Eosinophils in the Hypereosinophilic Syndrome

The idiopathic hypereosinophilic syndrome (IHES) is a rare disorder characterized by unexplained, persistent eosinophilia associated with multiple organ dysfunction due to eosinophilic tissue infiltration. In the absence of karyotypic abnormalities, there is no specific test to detect clonal eosinophilia in IHES. Analysis of X-chromosome inactivation patterns can be used to determine whether proliferative disorders are clonal in origin. Methylation of HpaII andHha I sites near the polymorphic trinucleotide repeat of the human androgen receptor gene (HUMARA) has been shown to correlate with X-inactivation. In this study, we have used the polymerase chain reaction (PCR) with nested primers to analyze X-inactivation patterns of the HUMARA loci in purified eosinophils from female patients with eosinophilia. Peripheral blood eosinophils were isolated by their autofluoresence using flow cytometric sorting. Eosinophils purified from a female patient presenting with IHES were found to show a clonal pattern of X-inactivation. Eosinophil-depleted leukocytes from this patient were polyclonal by HUMARA analysis, thus excluding skewedness of random X-inactivation. After corticosteroid suppression of her blood eosinophilia, a clonal population of eosinophils could no longer be detected in purified eosinophils. In contrast, eosinophils purified from a patient with Churg-Strauss syndrome and from six patients with reactive eosinophilias attributed to allergy, parasitic infection, or drug reaction showed a polyclonal pattern of X-inactivation by HUMARA analysis. The finding of clonal eosinophilia in a patient presenting with IHES indicates that such patients may have, in reality, a low-grade clonal disorder that can be distinguished from reactive eosinophilias by HUMARA analysis. Further, the method described can be used to monitor disease progression.

Clonality of Isolated Eosinophils in the Hypereosinophilic Syndrome

AbstractThe idiopathic hypereosinophilic syndrome (IHES) is a rare disorder characterized by unexplained, persistent eosinophilia associated with multiple organ dysfunction due to eosinophilic tissue infiltration. In the absence of karyotypic abnormalities, there is no specific test to detect clonal eosinophilia in IHES. Analysis of X-chromosome inactivation patterns can be used to determine whether proliferative disorders are clonal in origin. Methylation of HpaII andHha I sites near the polymorphic trinucleotide repeat of the human androgen receptor gene (HUMARA) has been shown to correlate with X-inactivation. In this study, we have used the polymerase chain reaction (PCR) with nested primers to analyze X-inactivation patterns of the HUMARA loci in purified eosinophils from female patients with eosinophilia. Peripheral blood eosinophils were isolated by their autofluoresence using flow cytometric sorting. Eosinophils purified from a female patient presenting with IHES were found to show a clonal pattern of X-inactivation. Eosinophil-depleted leukocytes from this patient were polyclonal by HUMARA analysis, thus excluding skewedness of random X-inactivation. After corticosteroid suppression of her blood eosinophilia, a clonal population of eosinophils could no longer be detected in purified eosinophils. In contrast, eosinophils purified from a patient with Churg-Strauss syndrome and from six patients with reactive eosinophilias attributed to allergy, parasitic infection, or drug reaction showed a polyclonal pattern of X-inactivation by HUMARA analysis. The finding of clonal eosinophilia in a patient presenting with IHES indicates that such patients may have, in reality, a low-grade clonal disorder that can be distinguished from reactive eosinophilias by HUMARA analysis. Further, the method described can be used to monitor disease progression.

Heterogeneity of clonal development in chronic myeloproliferative disorders

Recent reports have suggested a previously unexpected variability in the expression of the dominant neoplastic clone in myeloproliferative disorders (MPD). We evaluated 49 female patients with MPD and informative at the X-linked androgen receptor (AR) locus to establish the X chromosome inactivation pattern of hemopoietic cells. Whereas in chronic myelogenous leukemia (CML) the granulocytes (PMN) were uniformly of monoclonal origin, a striking heterogeneity of clonal development was found in PMN from patients with other MPD, with up to 50% of them expressing a polyclonal pattern of X inactivation. Am. J. Hematol. 60:158–160, 1999. © 1999 Wiley-Liss, Inc.

Heterogeneity of clonal development in chronic myeloproliferative disorders.

Recent reports have suggested a previously unexpected variability in the expression of the dominant neoplastic clone in myeloproliferative disorders (MPD). We evaluated 49 female patients with MPD and informative at the X-linked androgen receptor (AR) locus to establish the X chromosome inactivation pattern of hemopoietic cells. Whereas in chronic myelogenous leukemia (CML) the granulocytes (PMN) were uniformly of monoclonal origin, a striking heterogeneity of clonal development was found in PMN from patients with other MPD, with up to 50% of them expressing a polyclonal pattern of X inactivation.

BCR/ABL− CD34+HLA-DR−Progenitor Cells in Early Chronic Phase, But Not in More Advanced Phases, of Chronic Myelogenous Leukemia Are Polyclonal

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) translocation and BCR/ABL gene rearrangement which occur in a pluripotent hematopoietic progenitor cell. Ph-negative (Ph-) hematopoiesis can be restored in vivo after treatment with -interferon or intensive chemotherapy, suggesting that normal stem and progenitor cells coexist with the Ph+ clone. We have previously shown that Ph- progenitors are highly enriched in the CD34(+)HLA-DR- fraction from early chronic phase (ECP) CML patients. Previous studies have suggested that the Ph-translocation represents a secondary clonal hit occurring in an already clonally mutated Ph- progenitor or stem cells, leaving the unanswered question whether Ph- CD34(+)HLA-DR- progenitors are normal. To show the clonal nature of Ph- CD34(+)HLA-DR- CML progenitors, we have compared the expression of BCR/ABL mRNA with X-chromosome inactivation patterns (HUMARA) in mononuclear cells and in CD34(+)HLA-DR+ and CD34(+)HLA-DR- progenitors in marrow and blood obtained from 11 female CML patients (8 in chronic phase and 3 in accelerated phase [AP] disease). Steady-state marrow-derived BCR/ABL mRNA-, CD34(+)HLA-DR- progenitors had polyclonal X-chromosome inactivation patterns in 2 of 2 patients. The same polyclonal pattern was found in the progeny of CD34(+)HLA-DR- derived long-term culture-initiating cells. Mobilization with intensive chemotherapy induced a Ph-, BCR/ABL mRNA- and polyclonal state in the CD34(+)HLA-DR- and CD34(+)HLA-DR+ progenitors from 2 ECP patients. In a third ECP patient, polyclonal CD34(+) cells could only be found in the first peripheral blood collection. In contrast to ECP CML, steady-state marrow progenitors in late chronic phase and AP disease were mostly Ph+, BCR/ABL mRNA+, and clonal. Further, in the majority of these patients, a Ph-, polyclonal state could not be restored despite mobilization with intensive chemotherapy. We conclude from these studies that CD34(+)HLA-DR- cells that are Ph- and BCR/ABL mRNA- are polyclonal and therefore benign. This population is suitable for autografting in CML.

BCR/ABL− CD34+HLA-DR−Progenitor Cells in Early Chronic Phase, But Not in More Advanced Phases, of Chronic Myelogenous Leukemia Are Polyclonal

AbstractChronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) translocation and BCR/ABL gene rearrangement which occur in a pluripotent hematopoietic progenitor cell. Ph-negative (Ph−) hematopoiesis can be restored in vivo after treatment with α-interferon or intensive chemotherapy, suggesting that normal stem and progenitor cells coexist with the Ph+ clone. We have previously shown that Ph− progenitors are highly enriched in the CD34+HLA-DR− fraction from early chronic phase (ECP) CML patients. Previous studies have suggested that the Ph-translocation represents a secondary clonal hit occurring in an already clonally mutated Ph− progenitor or stem cells, leaving the unanswered question whether Ph−CD34+HLA-DR- progenitors are normal. To show the clonal nature of Ph−CD34+HLA-DR− CML progenitors, we have compared the expression of BCR/ABL mRNA with X-chromosome inactivation patterns (HUMARA) in mononuclear cells and in CD34+HLA-DR+ and CD34+HLA-DR− progenitors in marrow and blood obtained from 11 female CML patients (8 in chronic phase and 3 in accelerated phase [AP] disease). Steady-state marrow-derived BCR/ABL mRNA−, CD34+HLA-DR−progenitors had polyclonal X-chromosome inactivation patterns in 2 of 2 patients. The same polyclonal pattern was found in the progeny of CD34+HLA-DR− derived long-term culture-initiating cells. Mobilization with intensive chemotherapy induced a Ph−, BCR/ABL mRNA−and polyclonal state in the CD34+HLA-DR−and CD34+HLA-DR+ progenitors from 2 ECP patients. In a third ECP patient, polyclonal CD34+ cells could only be found in the first peripheral blood collection. In contrast to ECP CML, steady-state marrow progenitors in late chronic phase and AP disease were mostly Ph+, BCR/ABL mRNA+, and clonal. Further, in the majority of these patients, a Ph−, polyclonal state could not be restored despite mobilization with intensive chemotherapy. We conclude from these studies that CD34+HLA-DR− cells that are Ph− and BCR/ABL mRNA− are polyclonal and therefore benign. This population is suitable for autografting in CML.

The clonality of nodules in recurrent goiters at second surgery.

Some studies showed that in multinodular goiters clonal and polyclonal nodules coexist. The clonality of nodules in recurrent goiters is, however, still unknown and may contribute to help explain the pathogenesis of this thyroid disease. The clonality of 14 nodules derived from recurrent goiters was assessed by means of an X-chromosome-inactivation method. Of 14 nodules, 10 showed a polyclonal pattern, 3 were clonal and, in 1 case, the result remained unclear. The mean age of the patients with recurrent goiter at the time of their first operation was significantly lower than the mean age of 50 patients who underwent thyroid surgery for the first time over the same period of time (34.6+/-10.9 years vs 53.7+/-13.5 years; P<0.05). The mean interval between first and second operation was 18 years. The finding that nodules in recurrent goiters are predominantly polyclonal suggests that these lesions have their origin in a de novo proliferation of different cohorts of thyrocytes due to unknown growth stimulating molecular events.

5 Clonality markers in polycythaemia and primary thrombocythaemia

Our current understanding of the pathogenesis of the myeloproliferative disorders is based on the assumption that they represent a clonal disorder resulting from transformation of a haematopoietic stem cell. Clonality assays exploit the fact that female cells have only one active X-chromosome. Methods for determining X chromosome inactivation have been devised at the protein (G6PD isoenzymes); DNA (HUMARA; phosphoglycerate kinase (PGK); hypoxanthine-phosphoribosyl transferase (HPRT) and RNA (G6PD; P55; IDS) levels. In this type of RNA assay the product of the active X chromosome is quantified by studying polymorphisms present in mRNA. The presence of skewed lyonization in normal females is a potential limitation to the method, although the use of T cells as a control makes it possible to distinguish clonal haematopoiesis from skewed lyonization. However, the phenomenon of acquired skewing in normal elderly women means that X-inactivation patterns in elderly patients must be interpreted with caution. Clonality studies have been conducted in polycythaemia vera (PV) and essential thrombocythaemia (ET) patients. They usually demonstrate a clonal X-inactivation pattern in granulocytes and/or platelets but a polyclonal pattern in T cells. However, in both ET and PV a significant minority of patients exhibit polyclonal haematopoiesis with polyclonal patterns in granulocytes/platelets. Female patients with polyclonal haematopoiesis differ from those with clonal haematopoiesis in terms of age and platelet count and possibly in their requirements for treatment. This new technology for the investigation of the MPD seems promising for understanding certain clinical aspects of these diseases and may be introduced for evaluation of new modalities of treatment.

Hepatitis C Virus and Immunoglobulin Gene Rearrangements: An Early Step in Lymphomagenesis?

A clonal expansion of peripheral blood mononuclear cells committed to IgM cryoprecipitating rheumatoid factor production has been demonstrated in hepatitis C virus (HCV)-associated mixed cryoglobulinemia (MC). To determine the role of HCV in B cell gene rearrangements we studied a series of 57 HCV-infected patients with and without MC. Clonal Ig gene rearrangements of both RNA and DNA were detected in 10 of the 13 patients with type II MC, 1 patient had gene rearrangement of the DNA only, and 2 had polyclonal patterns. 2 of the 17 patients with type III MC showed clonal rearrangement of both RNA and DNA, in 6 only the DNA was rearranged clonally and in 9 the patterns were completly normal. 14 of 27 patients with cryocrit <1% or without cryoglobulins had clonal DNA rearrangements without any in the RNA. These results suggest that clonal lesions in the DNA are related to HCV infection and that these changes antedate the appearance of mixed cryoglobulinemia.

Are Complicated Forms of Celiac Disease Cryptic T-Cell Lymphomas?

We assessed the clonality of duodenal mucosal T cells in patients with celiac disease and controls. Fifteen adult patients were studied. Four patients had a complicated celiac disease, 3 did not respond to a gluten-free diet, and 2 had an ulcerative jejunitis (including 1 patient with nonresponsive celiac disease). Seven patients had an untreated celiac disease responsive to a gluten-free diet. Histological examination of duodenal biopsies of these 11 patients showed benign-appearing celiac disease without evidence of lymphoma. Four patients with nonulcer dyspepsia and normal duodenal biopsies served as controls. TCRγ gene rearrangements were analyzed by multiplex polymerase chain reaction on DNA extracted from duodenal biopsies. Major clonal rearrangements of the T-cell receptor were found in 4 cases, all with complicated celiac disease. Monoclonality was confirmed by DNA sequencing of the junctional region in 3 cases and by hybridization with clone-specific oligoprobes. Patients with celiac disease responsive to gluten-free diet had mainly a polyclonal pattern, with 1 of them having an oligoclonal rearrangement. An oligoclonal pattern was also observed in 2 control patients. Three patients with complicated celiac disease evolved to T-cell lymphoma with liver (n = 2) or bone marrow (n = 1) invasion. Identical clones were found in the enteropathic duodenojejunum and peripheral blood in the patient with large-cell lymphoma with bone marrow invasion. This study suggests that complicated celiac disease is a cryptic T-cell lymphoma.

Are Complicated Forms of Celiac Disease Cryptic T-Cell Lymphomas?

We assessed the clonality of duodenal mucosal T cells in patients with celiac disease and controls. Fifteen adult patients were studied. Four patients had a complicated celiac disease, 3 did not respond to a gluten-free diet, and 2 had an ulcerative jejunitis (including 1 patient with nonresponsive celiac disease). Seven patients had an untreated celiac disease responsive to a gluten-free diet. Histological examination of duodenal biopsies of these 11 patients showed benign-appearing celiac disease without evidence of lymphoma. Four patients with nonulcer dyspepsia and normal duodenal biopsies served as controls. TCRgamma gene rearrangements were analyzed by multiplex polymerase chain reaction on DNA extracted from duodenal biopsies. Major clonal rearrangements of the T-cell receptor were found in 4 cases, all with complicated celiac disease. Monoclonality was confirmed by DNA sequencing of the junctional region in 3 cases and by hybridization with clone-specific oligoprobes. Patients with celiac disease responsive to gluten-free diet had mainly a polyclonal pattern, with 1 of them having an oligoclonal rearrangement. An oligoclonal pattern was also observed in 2 control patients. Three patients with complicated celiac disease evolved to T-cell lymphoma with liver (n = 2) or bone marrow (n = 1) invasion. Identical clones were found in the enteropathic duodenojejunum and peripheral blood in the patient with large-cell lymphoma with bone marrow invasion. This study suggests that complicated celiac disease is a cryptic T-cell lymphoma.

T cell receptor β-chain repertoire in inclusion body myositis

Inclusion body myositis (IBM) is the most common muscle disease affecting individuals over 50 years of age. The inflammatory reaction is characterized by cell infiltrates predominated by CD8 + cytotoxic T cells. To analyze clonality of muscle infiltrating lymphocytes, we studied the complementarity determining region 3 (CDR3) length distribution of the T cell receptor (TCR). Muscle infiltrating lymphocytes were studied in three IBM patients and compared with peripheral blood lymphocytes (PBL) in two of these patients. The study was performed by reverse transcription polymerase chain reaction (RT-PCR) of RNA extracted from muscle tissue and PBL followed by analysis of fragment length distribution of the CDR3 region in each of 24 different Vβ families. There was a restricted usage of TCR Vβ gene families in muscle infiltrating T cells in all three patients. Some of the TCR Vβ gene families showed oligoclonal expansions but polyclonal patterns were dominating. The CDR3 distribution of most Vβ families differed between muscle infiltrating lymphocytes and PBL indicating that T cells have expanded locally or selectively accumulated in muscle.

Monoclonal IgH gene rearrangement in microdissected nodules from nodular sclerosis Hodgkin disease.

Recently, single-cell PCR studies have demonstrated that Hodgkin and Reed-Sternberg (HRS) cells are clonally related in many cases of Hodgkin disease. To investigate the lineage and clonality of neoplastic cells in local environments in nodular sclerosis Hodgkin disease (NSHD), we microdissected multiple distinct nodules from patients with NSHD and analyzed them for IgH gene rearrangement by PCR. These results were correlated with immunophenotype, Epstein-Barr-encoded RNA (EBER) expression, and clinical outcome. Forty individual nodules from 10 patients with NSHD (11 specimens) were microdissected from formalin-fixed paraffin-embedded tissue. DNA extracts were analyzed for IgH gene rearrangement by using PCR with FRIIIa and JHa primers. Cases were immunophenotyped in paraffin sections with antibodies to CD20(L26), CD79a(HM57), CD45RO(A6), CD15 (Leu-M1), and CD30(Ber-H2). Infection of HRS cells by Epstein-Barr virus was evaluated by using EBER in situ hybridization (EBER-ISH). DNA extracts from 12 of 40 microdissected nodules from 8 of 10 patients demonstrated a monoclonal pattern by IgH-PCR. Three patients demonstrated 2 individual nodules with different monoclonal patterns. One patient demonstrated 2 nodules with bands that appeared similar in size but were found to be different from one another upon further testing. All 28 remaining nodules demonstrated a polyclonal pattern. Six of 10 patients were positive for the Epstein-Barr virus genome by EBER-ISH. No correlation was found between IgH monoclonality, immunophenotypic features, Epstein-Barr virus infection, or clinical outcome. It was concluded that a subset of NSHD cases contain detectable monoclonality within individual nodules by IgH-PCR, suggesting that HRS cells are clonally related within local microenvironments.

Characterization of the Lymphoid Infiltrate in Hashimoto Thyroiditis by Immunohistochemistry and Polymerase Chain Reaction for Immunoglobulin Heavy Chain Gene Rearrangement

A close relationship between Hashimoto thyroiditis (HT) and low-grade B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) has been shown. We used immunohistochemistry to study paraffin sections from 40 unselected cases of HT and scored cases according to the lymphoid infiltrate and presence of lymphoepithelial lesions (LELs). Clonality was assessed by kappa/lambda immunohistochemistry and polymerase chain reaction for immunoglobulin heavy chain gene rearrangement (IgH PCR). Histologic findings were compared with 2 cases of primary thyroid MALT-type lymphoma. In HT, the lymphoid infiltrate consisted predominantly of T cells in all cases; B cells, associated with germinal centers, did not have the appearance of marginal zone cells. All cases had identifiable T-cell LELs; immunohistochemistry confirmed inconspicuous, rare B-cell LELs in 13 of 40 cases. In all cases, plasma cells were polyclonal and IgH PCR showed a polyclonal pattern. Clinical follow-up was available for 34 patients. Lymphoma developed in none. In contrast, a B-cell predominant infiltrate of marginal zone cells was present in the MALT-type lymphomas that was not confined to germinal centers. Cytokeratin stains demonstrated severe loss of epithelial elements and destructive LELs. LELs are not, in isolation, a useful criterion for distinguishing low-grade MALT-type lymphoma of the thyroid from HT. Features associated with low-grade MALT-type lymphoma include a predominance of B cells, marked loss of epithelial elements, and destructive LELs composed of marginal zone B cells. Unselected cases of HT do not contain monoclones detectable by IgH PCR.

Combined Fine Needle Aspiration Biopsy, and Immunophenotypic and Genotypic Approach to Posttransplantation Lymphoproliferative Disorders

To report our experience with a combined approach to posttransplant lymphoproliferative disorders (PT-LPDs) that utilizes fine needle aspiration biopsy. A review of the files in the Department of Pathology, Saint Louis University Health Sciences Center; from 1988 to 1996 identified six patients with a diagnosis of PT-LPD who underwent either percutaneous or radiologically guided fine needle aspiration biopsy (FNAB). In all cases, material was collected for cytomorphology, flow cytometric analysis and, in selected cases, DNA polymerase chain reaction (PCR). Subsequent evaluations and clinical outcomes were obtained from the medical record. The six transplant recipients (4 men and 2 women; 3 cardiac, 2 renal and 1 hepatic transplant) had an age range of 16-65 years. The aspirate material on these six patients had a polymorphic pattern of lymphoid cells with varying sizes. By flow cytometry, two were monoclonal, while four had a polyclonal pattern. DNA PCR analysis on two FNABs demonstrated a monoclonal rearrangement of the immunoglobulin heavy chain gene. FNAB provides cytomorphologic characterization of PT-LPDs in transplantation patients and sufficient material for successful use of flow cytometry immunophenotyping and DNA PCR analysis. FNAB, therefore, has an important role in the evaluation of organ transplantation patients and is a valuable tool for assessing and diagnosing PT-LPD.

Low prevalence of monoclonal b cells in Helicobacter pylori gastritis patients with duodenal ulcer

We have studied the prevalence of B-cell clonality among a large group of 320 patients with Helicobacter pylori gastritis and duodenal ulcer. These patients underwent endoscopic examination with multiple gastric biopsies at diagnosis and were followed 2 and 12 months after therapy. Histopathologic examination of 809 sets of biopsy specimens showed lymphoid gastritis with lymphoid aggregates or follicles, but without lymphoepithelial lesion, in 302 samples corresponding to initial biopsy specimens ( n = 130) or to posttreatment biopsy specimens ( n = 172). DNA extracted from fresh antral specimens allowed the amplification of Helicobacter pylori DNA in all cases before therapy. The arrangement of the immunoglobulin heavy chain gene was studied by polymerase chain reaction (PCR) in the 302 selected lymphoid gastritis samples. Single or dominant bands were seen only in four specimens from three patients (1.3%), whereas a polyclonal pattern was seen in the other 298 samples. The detection threshold of our PCR technique was approximately 3% of clonal B cells diluted in a polyclonal population. This threshold appeared to be a reliable cutoff between polyclonal gastritis and clonal MALT lymphoma. In our experience, Helicobacter pylori lymphoid gastritis appeared mainly as a benign polyclonal condition.

Evidence for multiclonality in multicentric Kaposi's sarcoma.

Kaposi's sarcoma (KS) develops in a variety of clinical states and is the most common tumor seen in patients with HIV-1 infection. KS develops as a multifocal mucocutaneous disease with subsequent spread to visceral organs, and it has been argued to be a benign proliferation caused by its multifocality at initial presentation, lack of aneuploidy, and spontaneous regression upon withdrawal of immunosuppressive agents in iatrogenically induced disease. We wished to determine whether KS lesions are clonal, indicative of a true neoplasm. Also, we tested whether multifocal KS lesions are clonally related, derived from a common progenitor cell or of independent cellular origin. We studied the X-chromosome inactivation pattern of the human androgen receptor gene in tumor biopsies of women with KS. This procedure tests for the clonality of a tissue specimen, a hallmark of neoplasia. Each specimen was microdissected to minimize normal cell contamination. Of 12 evaluable cases, 10 were HIV-seropositive and 2 were HIV-seronegative. Twenty-four biopsies from the 12 patients were examined. Five cases were consistent with individual KS lesions being clonal. In two cases, multiple KS specimens derived from the individual patients had different androgen receptor alleles inactivated, proving unequivocally that these KS lesions arose independently from distinct transformed cells. In seven cases, only a polyclonal pattern of inactivation was observed, whereas two others had tumor areas of both clonal and polyclonal inactivation patterns. These findings suggest that KS can be a clonal neoplasm, and in some of the cases multiple KS lesions in a given patient can arise from independent cellular origins and acquire clonal characteristics. The polyclonal inactivation pattern observed in other KS lesions may represent a premalignant stage or false negative results.

Thyroid nodules in recurrent multinodular goiters are predominantly polyclonal.

Not only thyroid adenomas and carcinomas, but also the majority of single and well delimited goiter nodules, even if morphologically heterogeneous, are of clonal origin. However, it is still unknown whether the nodules of rapidly growing, recurrent goiters are clonal or polyclonal. We investigated by PCR-based analysis of exon 1 of the human androgen receptor gene clonality of nodules grown in recurrent multinodular goiters (MNG) of 14 female patients. The total goiter volume varied widely between 15 ml and 170 ml. The mean age of patients undergoing surgery for recurrent goiter at the time of their first operation was significantly lower with 34.6 +/- 10.9 yr in comparison to 50 consecutive patients who were operated for MNG for the first time (53.7 +/- 13.5 yr). The interval between first and recurrent operation was 18 +/- 8.5 yr. The mean volume of well circumscribed nodules selected for the present investigation was 3.8 +/- 1.4 ml. Assessment of clonality in at least 2 samples of each lesion revealed a polyclonal pattern in 10 out of 14 nodules, whereas only 3 nodules were clonal and in one case the result remained unclear. The unexpected finding that most nodules within MNG, that had re-grown after a first subtotal thyroidectomy, were of polyclonal rather than clonal composition, suggests that these lesions are generated by de novo-proliferation of cohorts of differing thyrocytes sharing the common trait of an exceedingly high intrinsic growth rate or alternatively, by unknown growth stimulating molecular events acting focally on clusters of cells derived from different ancestors. In addition, the relatively young age of patients with recurrent MNG at the time of their first surgery and the comparatively short interval between first and second operation point to a genetic element in the occurrence of growth-prone thyrocytes.

Comparison of in situ hybridisation and polymerase chain reaction in the diagnosis of B cell lymphoma.

AIM: To compare the sensitivity of the detection of immunoglobulin light chain messenger RNA (mRNA) restriction by in situ hybridisation (ISH) and clonal immunoglobulin heavy chain gene rearrangements by polymerase...

Polyclonal expansion of cells with trisomy 7 in synovia from patients with osteoarthritis

Trisomy 7 as the single chromosome aberration has been found in a variety of neoplasms and in normal tissue in the proximity of tumors, as well as in non-neoplastic lesions. Recently, we described a nonrandom pattern of chromosome aberrations, in particular, a gain of chromosome 7, in synovia, cartilage, and osteophytes from patients with osteoarthritis. To study the clonal origin of trisomy 7 in osteoarthritis, multiple synovial samples were collected from five women, all of whom were informative heterozygotes with regard to the X-linked human androgen receptor gene (AR). From each case, three to four independent cell cultures were initiated. Trisomic cell populations were subcloned from the individual cultures, and it was established whether or not the same allele of AR was inactivated in trisomic cells from different parts of the same joint. The finding of a polyclonal X-inactivation pattern in two of the cases provides strong evidence that gain of an extra copy of chromosome 7 occurs independently in multiple cells.