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The Diagnostic Significance of Myf-3 Hypermethylation in Malignant

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The Diagnostic Significance of Myf-3 Hypermethylation in Malignant Leukemia (2001) 15, 583–589 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu The diagnostic significance of Myf-3 hypermethylation in malignant lymphoproliferative disorders JME Taylor,1,2 PH Kay2 and DV Spagnolo1 1The Western Australian Centre for Pathology and Medical Research, Nedlands; and 2Department of Pathology, The University of Western Australia, Nedlands, Western Australia, Australia Deregulated methylation of cytosine in DNA is a frequent find- tiation and proliferation by controlling gene expression.4 Cyto- ing in malignancy that is reflected by general genomic hypome- sine bases usually within a 5′ CpG 3′ dinucleotide are methyl- thylation and regional hypermethylation that includes the myogenic gene Myf-3. In this study of 198 DNA samples from ated by one or more of a series of 5-methyltransferase 186 patients with a wide range of lymphoproliferative disorders enzymes called DNMT1, 2, 3a and 3b. The activities of (LPD), the methylation status of Myf-3 was assessed to evalu- DNMT1, 3a and 3b for example, are increased in many types ate its significance in the diagnosis of malignant LPD. DNA was of neoplastic cells.5,6 Increased activity of the cytosine methyl- digested with the restriction endonucleases HpaII and MspI, ating systems in neoplastic cells is associated with hyperme- and using the Southern blot (SB) technique, the size and den- thylation of specific regions of the genome including the short sity of fragments that hybridized with a Myf-3 probe were used arm of chromosome 117 which includes the myogenic gene to assign the methylation status. None of the samples from 45 8 9 patients from a wide age range with benign LPDs had evidence Myf-3 (Myo-D1 ). of altered Myf-3 methylation and there was no age-related Myf-3 is rich in 5′ CpG 3′ dinucleotides and it is hypome- methylation change. By contrast, 115/123 (93%) of samples thylated in non-malignant cells. By contrast, in some types of from patients with non-Hodgkin lymphoma (NHL) or lymphoid neoplastic cells it has been shown to be hypermethylated. In leukemia had increased Myf-3 methylation. There was no our early studies we showed that Myf-3 is hypermethylated in methylation alteration in 22/24 (92%) of samples from patients DNA from a small number of malignant LPDs but not from with Hodgkin lymphoma (HL), nor in five of six samples from 10 LPDs that had atypical histopathologic features which were not benign LPDs. Moreover, our investigations into the methyl- diagnostic of lymphoma, while the remaining sample of atypi- ation status of Myf-3 in breast and colon neoplasms showed cal LPD had hypermethylated Myf-3 fragments. There was an that it may be useful as a prognostic indicator.11–13 association between increasing Myf-3 methylation and higher This study of the methylation status of Myf-3 in DNA from histopathologic grade of malignancy within specific lymphoma benign and malignant lymphoid tissues from a large number categories. It is concluded that the detection of increased Myf- 3 methylation is a sensitive and specific test of malignancy of patients with a variety of LPDs was undertaken to determine which may complement other molecular methods that are cur- its diagnostic significance in lymphoproliferative disorders. rently used for the assessment of clonality. It may be of parti- cular diagnostic use in natural killer (NK) and null cell malig- nancies for which other indicators of clonality are lacking. Materials and methods Furthermore, methylation status may prove to be of potential prognostic value. Leukemia (2001) 15, 583–589. Keywords: diagnosis; DNA methylation; leukemia; lymphoma; DNA isolation Myf-3 Cases for study were selected retrospectively from archival freshly frozen lymphoid tissues or from EDTA anticoagulated Introduction peripheral blood leukocytes and bone marrow aspirates. All solid tissues were stored at −80°C prior to DNA extraction. DNA was isolated from each sample using a standard The application of molecular techniques has greatly advanced 14 the ability to distinguish between malignant and non-malig- phenol/chloroform, ethanol precipitation method. DNA was nant lymphoproliferative disorders (LPDs). For example, extracted from 198 samples from 186 patients with benign or Southern blotting (SB) and polymerase chain reaction (PCR)- malignant LPDs (one patient had metachronous NHL and HL). based techniques are routinely used to detect monoclonal DNA samples were obtained from 134 lymph nodes, 11 rearrangements of immunoglobulin (Ig) and T cell receptor splenic samples, eight peripheral blood leukocyte specimens, (TCR) genes which in an appropriate clinical setting are seven tonsils, six bone marrow aspirates, and 32 other extra- characteristic of malignant LPDs.1–3 Furthermore, some lym- nodal tissues. phoma categories have specific chromosomal translocations that occur at high frequency. However, these current markers of monoclonality have limited prognostic value and cannot Case classification be used to assess the clonality of NK and null cell prolifer- ations where neither Ig or TCR genes are rearranged. Three groups of well-characterised cases of benign, malignant and atypical LPDs were used. Classifications were confirmed Cytosine methylation is a part of the heritable epigenetic 1,2 3 machinery which is thought to influence cellular differen- immunophenotypically and genotypically by PCR and SB based methods as previously described. The diagnosis of malignant B and T cell LPDs was supported by the detection of clonal antigen receptor gene rearrangements. Malignant Correspondence: JME Taylor, Tissue Pathology Division, The Western LPDs were categorized according to the proposed WHO Australian Centre for Pathology and Medical Research (PathCentre), 15 Locked Bag 2009, Nedlands, Western Australia, Australia 6909; classification (Table 1). Fax: 08 9346 4122 Follicular lymphoma (FL) and diffuse large B cell lymphoma Received 20 October 2000; accepted 19 December 2000 (DLBCL) samples were sub-grouped according to cytologic Myf-3 hypermethylation in malignant LPD JME Taylor et al 584 Table 1 Myf-3 methylation status and classification of lymphoproliferative disorders in the study15 Diagnostic category n Hypermethylation Minor change No change Benign LPD: 45 samples from 45 patients 0 (0) 0 (0) 45 (100) Non-specific reactive hyperplasia 37 Granulomatous lymphadenitis 7 Lymphoid hamartoma 1 Atypical lymphoid hyperplasia: 6 samples from 6 patients 6 1 (17) 0 (0) 5 (83) B cell neoplasms: 92 samples from 84 patients 71b (79) 16 (18) 3 (3) Precursor B lymphoblastic leukemia/lymphoma 6 B cell small lymphocytic lymphoma 10 B cell prolymphocytic leukemia 1 Splenic marginal zone B cell lymphoma 1 Plasma cell myeloma 2 Extra-nodal marginal zone B cell lymphoma of MALT type 7 Nodal marginal zone B cell lymphoma 1 Follicular lymphoma 34 [31] Mantle cell lymphoma 10 [9] Diffuse large B cell lymphoma 17 Atypical Burkitt lymphoma 3 T and NK cell neoplasms: 31 samples from 29 patients 19 (63) 6a (20) 5 (17) Precursor T lymphoblastic leukemia/lymphoma 7 T cell prolymphocytic leukemia 1 T cell granular lymphocytic leukemia 4 [3] Extra-nodal NK/T cell lymphoma, nasal type 2 Hepatosplenic γδ T cell lymphoma 2 [1] Sezary syndrome 1 Peripheral T cell lymphoma, not otherwise characterized 9 Anaplastic large cell lymphoma T/null cell, primary systemic type 4 Unclassified high grade cutaneous CD4 and CD56 positive NK 1 cell lymphoma Hodgkin lymphoma: 24 samples from 23 patients 2 (8) 0 (0) 22 (92) Nodular lymphocyte predominance Hodgkin lymphoma 3 Nodular sclerosis Hodgkin lymphoma 11 Mixed cellularity Hodgkin lymphoma 10 [9] n, sample number, where multiple samples were examined from the same patient, patient numbers are given in square brackets. Two non-specific reactive hyperplasia samples had evidence of Epstein–Barr virus (EBV) infection using an in situ hybridization method. A further patient whose biopsy showed borderline histopathologic features of malignancy without evidence of clonality, later developed a NHL that had hypermethylation of Myf-3 (later sample not included). Four patients each had two different metachronous B-NHL and another had B cell lymphoma and Hodgkin lymphoma in two separate biopsies. One FL sample with hypermethylated Myf-3 was originally diagnosed as atypical follicular hyperplasia but was later shown to have a clonal Ig heavy chain gene rearrangement. Percentage is shown in parenthesis (%). Symbols indicate 1 (a) or 2 (b) excluded repeat samples where the initial and repeat samples had identical maximum Myf-3 fragment sizes. criteria that reflected the histopathologic grade of malignancy. from a patient with lymphomatoid papulosis who had a Lower grade FL had follicular growth and comprised predomi- lymphoid infiltrate with malignant cytological features that nantly small cleaved cells (grade 1) or mixed small cleaved lacked immuno- or genotypic evidence of clonality. None of and large cells (grade 2), whereas higher grade 3 FL(l/d) had the atypical LPD samples had SB or PCR-based evidence of predominantly large cells with a follicular or follicular and clonal antigen receptor gene rearrangements and none had diffuse growth pattern. Lower grade DLBCL had unspecified clinical evidence of lymphoma subsequent to biopsy. cytologic features, whereas higher grade DLBCL(p/i) had a pleomorphic or immunoblastic morphology. Peripheral T cell lymphomas not otherwise characterized (PTL), were sub-grouped according to histopathologic grade Influence of age of malignancy based on cell size. Lower grade PTL samples were composed of medium or mixed medium and large size cells, whereas higher grade PTL(l) samples were comprised Several reports have shown that the methylation status of predominantly of large cells. some regions of the genome changes with age.16–19 Conse- The atypical LPD samples comprised five lymph nodes from quently, studies were undertaken to determine whether the five patients who had follicular hyperplasia with atypical his- increased Myf-3 methylation associated with different types of topathologic features that neither confirmed nor excluded a LPD was attributable to malignancy or to the age of the diagnosis of malignancy.
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