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Clonal Analysis of Untreated Non-Hodgkin's Lymphoma Utilizing Immunoglobulin Gene Rearrangement and Immunophenotype1

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Clonal Analysis of Untreated Non-Hodgkin's Lymphoma Utilizing Immunoglobulin Gene Rearrangement and Immunophenotype1 [CANCER RESEARCH 48, 6272-6277, November 15, 1988] Clonal Analysis of Untreated Non-Hodgkin's Lymphoma Utilizing Immunoglobulin Gene Rearrangement and Immunophenotype1 Richard A. Rudders,2 Satinder Dhillon, and Theodore G. Krontiris Hematology/Oncology Division, Department of Medicine, and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, and the New England Medical Center Hospitals, Boston, Massachusetts 02111 ABSTRACT is entirely absent. The recent development of molecular probes for genetic events associated with B-cell differentiation provides We have prospectively examined 66 consecutive initial diagnostic a potentially powerful approach for the study of lymphoma- lymph node biopsies from unselected patients suspected of having malig genesis. From the work of many investigators, it has been nant lymphoma for clonal immunophenotypic and immunogenotypic markers. By morphological and cell surface phenotypic criteria 52 had possible to determine the structure of the germ line Ig genes non-Hodgkin's lymphoma derived from the B-cell lineage and in these and construct a model of Ig gene rearrangement that is associ we compared surface immunoglobulin criteria for clonality with ¡iniiiu- ated with the maturation of B cells (1-3). Proceeding from this noglobulin gene rearrangement as detected by .In, ( ., and ( ...gene probes. scheme, several groups have shown that neoplasms of human We found that the addition of Bgl\l and ///mill I double digests to the B-cells such as leukemia and lymphoma regularly exhibit re standard /(umili and EcoRl restriction enzymes made it possible to arrangements of Ig genes that can serve as a diagnostic criterion detect rearrangement in the vast majority of lymphomas and that re for malignancy (4-8). arrangement of both JH alíelesis the rule. A rearranged heavy and/or In addition to their diagnostic utility, immunoglobulin gene light chain gene was detected in 47 of 52 (91%) tumors and the JH probe probes can be used to study lymphoma clonal evolution. Each alone detected rearrangements in 87% of tumors when multiple restriction individual tumor clone displays a characteristic Ig gene re enzymes were used. In contrast, surface immunoglobulin, the standard clonal marker for monoclonal B-cell malignancy, was either undetectable arrangement. The finding of distinct Ig gene rearrangements in or did not exhibit light chain restriction in 29 of 52 tumors as detected different populations of tumor cells (purified from the same by flow cytometric analysis. Further, in 24 of these 29 tumor DNAs we lymphoma on the basis of idiotype expression) has provided could detect an Ig gene rearrangement. In follicular (nodular) lymphoma additional evidence that lymphomas may have oligoclonal, which often gives ambiguous immunophenotypic results by cell suspen rather than monoclonal, origins (9-12). It should be noted, sion techniques, monoclonal gene rearrangements were detected in 16 of however, that this observation has been tempered by the finding 18 tumor DNAs. Monoclonal surface immunoglobulin was detected in that somatic mutation may alter idiotype expression within a only 8 of 18 of this subset of cases. monoclonal tumor and thus produce the artifactual appearance The 52 tumors were also analyzed for potential oligoclonality. We of oligoclonality (13). found that the use of Bgfl\, a restriction enzyme that closely spanned the The nature of Ig gene rearrangement studies in human lym JH region, increased the sensitivity of detecting rearrangements and facilitated the identification of isotype switch variants. In only a single phomas has, however, left several important issues unresolved (1 of 52) tumor DNA were more than two rearranged bands seen with since many have been carried out in selected and often previ JH, {',, and ( ', probes, suggesting a multiclonal origin. Additional cases ously treated patients. In this study we have examined an thought to potentially represent oligoclonality by immunophenotypic unselected series of initial lymphoma biopsies. We report herein criteria proved to be isotype switch variants. an analysis of the clonal composition of these tumors based on We conclude that Ig gene rearrangement is an extremely sensitive Ig gene rearrangement as well as on cell surface criteria. method for defining monoclonality in lymphoma cell populations, partic ularly if multiple restriction enzymes are used, and that the vast majority of the clonal diversity seen in initial diagnostic biopsies is the result of MATERIALS AND METHODS isotypic switch within a given clone rather than true oligoclonality. Tumor Samples. The initial tissue biopsies from 66 consecutive, previously untreated patients suspected of having lymphoma were pro INTRODUCTION spectively entered in the study. Biopsies of lymph nodes, tumor masses, Current concepts are consistent with the view that human or both were received fresh in the laboratory in sterile, cold saline after malignant lymphomas are expansions of single clones ot lymph- a portion was removed for routine histopathological examination, and were processed immediately. A portion was snap frozen in liquid N2 oid cells that have undergone neoplastic transformation. The for DNA studies and the remainder was converted to a single cell major immunophenotypic criterion for defining malignancy in suspension for immunophenotypic analysis. When multiple biopsy these tumors has been the expression by B-cell clones of either specimens were submitted (as was the case when exploratory laparot surface or secretory Ig with a restricted light chain type. In omies were performed), portions of each were processed and analyzed practical usage this definition has its limitations. For instance, separately. normal cell populations frequently dilute out tumor cells, ob Immunophenotypic Analysis. Cells were examined for surface anti scuring the malignant clone(s). Also, Ig is often either not gens with a panel of monoclonal and polyclonal antibodies by using clearly expressed on the cell surface of B-cell-derived tumors or standard immunofluorescent techniques as previously described (14). Briefly, cell suspensions were incubated with monoclonal or polyclonal Received 4/11/88; revised 8/11/88; accepted 8/16/88 antibodies directed against a, /.¡.-,.& heavy and light Ig chains, Bl The costs of publication of this article were defrayed in part by the payment (CD20), B4 (CD19), la, T3 (CD3), T4 (CD4), T8 (CDS), TÕO(CD38), of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Til, Leu 1 (CDS), IL2R (CD25), and CALLA (CD10). Polyclonal 1Presented in part at the Annual Meetings of the American Society of antibodies used were affinity purified F(Ab')2 fragments. All antibodies Hematology, New Orleans. Louisiana, December 1985 and December 1986 in were regularly titrated against known positive controls. Stained cell San Francisco, California. Supported in part by Grants CA40725 and CA09429 populations were analyzed by flow cytometry on a Becton Dickinson from NIH. 2To whom requests for reprints should be addressed, at Division of Oncology/ (Sunnyvale, ÇA)FACS analyzer. Hematology, New England Medical Center Hospitals, 750 Washington Street, DNA Probes. DNA clones representing the human JH (15), C. (16) Boston, MA 02111. and CA(17) genes were generously provided by Dr. Philip Leder, Boston, 6272 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1988 American Association for Cancer Research. CLONAL ANALYSIS OF UNTREATED NON-HODGKIN'S LYMPHOMA MA. The JH probe that was used in our study consisted of a 2.5-kilobase were detected with BamHl and EcoRl that were not detected Sau3A fragment of JH. with Bgl\\. In contrast, in 11 of 22 DNAs the Bglll digest DNA Purification and Analysis. Tissue that had been snap-frozen in revealed an increased number of rearranged bands (Fig. 1). This liquid nitrogen was pulverized over dry ice. Cells in the resulting powder represents an almost 4-fold difference in sensitivity. Nine of were lysed in a solution containing 0.5% SDS, 0.15 M NaCI, 10 mM these 11 cases also were digested with BamH'md and/or Tris-HCI, pH 7.5, and 100 HIMEDTA. Proteinase K was added to 200 /jg/ml and the mixture was incubated at 65"C for 30 min. Following £coHind. An increase in the number of rearranged bands seen two extractions with phenol. phenol:chloroform:isoamyl alcohol in 1 of the 9 cases. We were also able to compare directly the results of Bglll, (25:24:1), and finally. CHCl^isoamyl alcohol, the DNA was precipi BamH'md, and/or EcoH'md digests with our JM probe in 26 tated in two volumes of 95% ethanol. The DNA was redissolved, incubated sequentially with RNase A (100 ¿ig/ml)and proteinase K, cases where BamH\ and EcoRl digests displayed a single rear extracted again with phenol and chloroform/isoamyl alcohol, and re- ranged band or none at all. In 5 of 26 cases Bgl\\ digests precipitated in two volumes of ethanol. The pellet was washed in 70% revealed more bands than double digests and in 7 of 26 cases ethanol and dissolved in 10 mM Tris-HCI, pH 7.5. and 10 mM EDTA. double digests revealed additional bands not seen with Bglll Five ¿jgof DNA were digested with BamHl, EcoRl, Bglll, and (Fig. 2). In the majority of cases (14 of 26) no new information /tomHI///fndIII, and £foRl////ndIII restriction endonuclease accord ing to the manufacturer's (New England Biolabs, Beverly, MA) speci was obtained. This suggests that the larger increment in sensi fications and electrophoresed through 0.7% agarose gels. Southern tivity is obtained by adding Bgl\\ or double digests to standard transfer (18) was to GeneScreen Plus filters (New England Nuclear, BamHl and EcoRl but that doing both will still yield an Boston. MA). Probe was nick translated (19) to a specific activity of additional increment of undetected bands. greater than 10' cpm/Mg DNA and hybridized to filter-bound DNA at Rearranged Ig Genes.
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