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

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

We have prospectively examined 66 consecutive initial diagnostic lymph node biopsies from unselected patients suspected of having malignant lymphoma for clonal immunophenotypic and immunogenotypic markers. By morphological and cell surface phenotypic criteria 52 had non-Hodgkin's lymphoma derived from the B-cell lineage and in these we compared surface immunoglobulin criteria for clonality with immunoglobulin gene rearrangement as detected by J_H, C_g, and C gene probes. We found that the addition of BglII and HindIII double digests to the standard BamHI and EcoRI restriction enzymes made it possible to detect rearrangement in the vast majority of lymphomas and that rearrangement of both J_H alleles is the rule. A rearranged heavy and/or light chain gene was detected in 47 of 52 (91%) tumors and the J_H probe alone detected rearrangements in 87% of tumors when multiple restriction enzymes were used. In contrast, surface immunoglobulin, the standard clonal marker for monoclonal B-cell malignancy, was either undetectable or did not exhibit light chain restriction in 29 of 52 tumors as detected by flow cytometric analysis. Further, in 24 of these 29 tumor DNAs we could detect an Ig gene rearrangement. In follicular (nodular) lymphoma which often gives ambiguous immunophenotypic results by cell suspension techniques, monoclonal gene rearrangements were detected in 16 of 18 tumor DNAs. Monoclonal surface immunoglobulin was detected in only 8 of 18 of this subset of cases.

The 52 tumors were also analyzed for potential oligoclonality. We found that the use of BglII, a restriction enzyme that closely spanned the J_H region, increased the sensitivity of detecting rearrangements and facilitated the identification of isotype switch variants. In only a single (1 of 52) tumor DNA were more than two rearranged bands seen with J_H, C_g and C probes, suggesting a multiclonal origin. Additional cases thought to potentially represent oligoclonality by immunophenotypic criteria proved to be isotype switch variants.

We conclude that Ig gene rearrangement is an extremely sensitive method for defining monoclonality in lymphoma cell populations, particularly if multiple restriction enzymes are used, and that the vast majority of the clonal diversity seen in initial diagnostic biopsies is the result of isotypic switch within a given clone rather than true oligoclonality.

¹ Presented in part at the Annual Meetings of the American Society of Hematology, New Orleans, Louisiana, December 1985 and December 1986 in San Francisco, California. Supported in part by Grants CA40725 and CA09429 from NIH.

² To whom requests for reprints should be addressed, at Division of Oncology/Hematology, New England Medical Center Hospitals, 750 Washington Street, Boston, MA 02111.

Received 4/11/88. Revised 8/11/88. Accepted 8/16/88.