Leukemia Research Vol. 15, No. 1, pp. 59-63, 1991. Printed in Great Britain.

0145-2126/91 $3.00 + .00 Pergamon Press plc

KAPPA LIGHT CHAIN GENE R E A R R A N G E M E N T IN A T-CELL LYMPHOMA LUIGI MORETrI, MARIA TERESA MARIANO, AMEDEA DONELLI, GIULIANO MONTAGNANI, MARIO SARTI, MARCELLO GRANTINI, UBALDO DI PRISCO, UMBERTO TORELLI and FRANCO NARNI Servizio di Ematologia e Servizio di Immunoematologia Trasfusionale, Istituto di Clinica Medica II, Universith di Modena, Italy

(Received 31 May 1990. Accepted 30 July 1990) Abstract--Forty-three patients were studied to determine whether light chain gene rearrangements may occur in hematopoietic cells not pertaining to the B-lineage. In only one patient, affected by Tcell lymphoblastic lymphoma, one ~ light chain allele was rearranged. Neither at the protein level nor at the RNA level the rearranged gene was expressed. These data confirm that, although rarely, r light chain gene rearrangements may occur in neoplastic T-cells. Furthermore, as in our patient Ig heavy chain genes retained a germline configuration, the present data demonstrate that r light chain gene rearrangements may occur regardless of Ig heavy chain gene arrangement.

Key words: Immunoglobulin genes, light chain genes, gene rearrangements, lymphoma.

INTRODUCTION

nuclei, exhibiting focal acid phosphatase positivity (Fig. 1). Surface markers analysis showed that most cells were CD4/CD8 positive immature thymocytes, expressing Tio~fl antigen receptor (Table 1). A remission was obtained after six cycles of chemotherapy ( P R O M A C E / M O P P ) , and the patient received CNS irradiation (30 Gy) and methotrexate intratecally. Two months later, however, a systemic relapse occurred with massive bone marrow involvement and the patient died.

IMMUNOGLOBULIN (Ig) gene rearrangements have been extensively used as B-lineage markers [1-3]. Whereas lg heavy chain genes can frequently be rearranged in cell populations not pertaining to the B-lineage [4-7], only one case of ~ light chain gene rearrangement in a T-cell lymphoma has been reported so far [8]. Thus, r light chain gene rearrangement can be considered, at the molecular level, the most specific B-lineage marker. The aim of the present study was to determine at which frequency K light chain gene rearrangements can be observed in neoplastic T-cells or leukemic myeloid cells. Here we describe the only patient in our series exhibiting K light chain gene rearrangement.

M A T E R I A L S AND M E T H O D S

Patients Twenty-seven patients were affected by myeloid leukemias (eight by acute myeloid leukemia and 19 by chronic myeloid leukemia), and 16 by T-cell malignancies (eight by acute lymphoblastic leukemia or lymphoblastic lymphoma, eight by chronic lymphocytic leukemia). Diagnosis was made according to conventional morphological and cytochemical criteria and was confirmed by direct immunofluorescence and cytofluorograph readings (FACstar, Becton-Dickinson, Mountain View, CA, U.S.A.) using a series of monoclonal antibodies. All monoclonal antibodies listed in Table 1, except T6 (Coulter) and TCR-6-1 (T-cell Sciences) were from Becton-Dickinson. In all patient Ig heavy chain genes were in a germline configuration and in patients with T-cell malignancies the T-cell receptor flchain gene was rearranged.

CASE R E P O R T The patient we describe here (DS) was a 21-yearold male, admitted because of fever, chest pain and dyspnoea. A mediastinal mass and pleural effusion were demonstrated by X-ray. Pleural fluid obtained by thoracentesis contained a homogeneous population of small-sized blast cells with convoluted

Abbreviations: Ig, immunoglobulins; TCR, T-cell receptor; mRNA, messenger RNA. Correspondence to: Dr Franco Narni, Istituto di Clinica Medica II, Policlinico, via Del Pozzo 71, 41100 Modena, Italy.

Nucleic acid analysis High molecular weight DNA was extracted as previously 59

60

L. MORETrl et al.

TABLE 1. SURFACE MARKERS ANALYSIS OF BLAST CELLS FROM PATIENT D S . THE PERCENTAGE OF POSITIVE CELLS IS INDICATED

Leu 1 (CD5) Leu 4 (CD3) Leu 2 (CD8) Leu 3 (CD4) Leu2 + Leu3 Leu 9 (CD7) Leu 5 (CD2) Leu 17 (CD38)

98% 90% 98% 96% 93% 92% 94% 99%

T6 (CDla) TCR1 (Tio:fl) TCR-6-1 (yr) CALLA (CD10) Leu M3 (CD14) Leu 16 (CD20) HLA-DR I12-r (CD25)

95% 95% 2% 67% 4% ----

described [9] and digested with Eco RI, Hind IIl, and Barn HI restriction endonucleases according to the instructions of the manufacturer (BRL, Gaithersburg, MD, U.S.A.). Restriction fragments were size fractionated on a 0.8% agarose gel and transferred to Hybond-N membrane (Amersham, Arlington Heights, IL, U.S.A.), essentially as described by Southern [10]. Total RNA, extracted in the presence of guanidine thyocianate [11], was denatured, size fractionated and transferred as described [12]. Filters were hybridized to probes labelled to a high specific activity with 32p-dCTP [13]. The following probes were used: (1) a 6 kb Bam HI/Hind III genomic fragment including JH 16 sequences for the immunoglobulin heavy chain gene cluster [14]; (2) a cDNA clone containing C~ region sequences of the TCRfl gene, and (3) a cDNA clone containing V, D, J and C sequences of the TCR0: gene [15]; (4) a 2.5 kb Eco RI genomic fragment, spanning the entire Ck gene locus plus flanking sequences [16]; (5) a 2.8 kb Eco RI/Hind III genomic fragment corresponding to the major breakpoint region (mbr) of bcl-2 gene [17]; and (6) a Cla I/Eco RI genomic fragment (pMC41-Y) including the third exon of c-myc oncogene [18]. Hybridization and washing conditions of the filters have been described elsewhere [12].

RESULTS Overall, immunogenetic analysis showed that blast cells obtained from pleural effusion of patient DS were clonal T-lymphocytes. In addition to a germline band of approximately 24 kb, in fact, a rearranged band of 11.5 kb was found in Barn HI digested DNA using the TCRfl probe (Fig. 2A). After digestion with Eco RI the germline band of 12 kb was not visible, and an additional band corresponding to a restriction fragment of undetermined size (larger than 24 kb) was found (Fig. 2B). Using the JH probe only germline bands of 17, 19 and 11 kb were present in D N A digested with Bam HI, Eco RI and Hind III (not shown) respectively. After hybridization with the Ck probe, two bands of similar intensity were found in D N A extracted from our patient's blast cells digested with Bam HI: a germline band of 12.5 kb plus a rearranged band of 7.5 kb. In D N A extracted

from normal peripheral blood leukocytes of the same patient only a 12.5 kb germline band was found (Fig. 3). Although surface Ig were not present on the patient's blast cells (Table 1), we asked whether the rearranged x allele was expressed at the R N A level, No r light chain m R N A was found in total R N A extracted from the patient's blast cells, whereas abundant mRNAs were found using probes corresponding to TCR0:- and fl-chain genes (Fig. 4). In some cases the structure of t¢ light chain genes can be altered by chromosomal translocations involving, on the partner chromosome, either c-myc or bcl-2 oncogenes [19, 20]. The effect of these translocations would be a deregulation of the involved oncogene. Although such translocations are typical of B-cell malignancies, we analyzed the structure and the expression level of c-myc and bcl-2 (cytogenetic analysis was not available). A single germ line band of 22 kb was found in D N A digested with Barn HI after hybridization with a probe corresponding to the third exon of c-myc oncogene, and a germline band of 23 kb was present in D N A digested with the same enzyme after hybridization with a probe corresponding to the putative oncogene bcl-2. Consistently, c-myc was expressed at very low levels and bcl-2 m R N A was not detectable (not shown).

DISCUSSION In patient DS the T-lineage origin of the tumor cells was demonstrated by surface markers and, as far as Ig heavy chain and TCRfl-chain genes are concerned, it was confirmed by means of immunogenetic analysis. Nevertheless, one K light chain allele was rearranged. A genetic polymorphism can be ruled out, as the same rearrangement was not present in normal peripheral blood leukocytes obtained from the same patient. On the other hand, neither c-myc nor bcl-2 were rearranged or expressed at high levels: this would argue against the possibility of chromosomal translocations involving either chromosome 8 or chromosome 18. Thus, the rearrangement we observed using the Ck probe was probably a non-productive V-J rearrangement in a truly Tcell lymphoma. Both heavy and light chain gene rearrangements were observed in the only case of aberrant ic gene rearrangement previously reported [8]. In our patient, an additional atypical feature was represented by the occurrence of K light chain gene rearrangement in the absence of Ig heavy chain gene rearrangements. Although aberrant rearrangements involving light chain genes seem to be rare, this possibility should be considered when gene re-

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FIG. 2. DNA digested with Bam HI (A) and Eco RI (B) hybridized to TCRfl probe. Lane 1, normal DNA extracted from placenta; lane 2, DNA extracted from blast cells of patient DS. Fifteen micrograms of DNA were loaded on each lane. Dashes indicate germline bands, arrows indicate rearranged bands. FIG. 3. DNA digested with Barn HI, hybridized to Ck probe. Lane 1, placenta DNA; lane 2, DNA from blast cells of patient DS; lane 3, DNA from peripheral blood leukocytes of patient DS. Twenty micrograms of DNA were loaded on each lane. The arrow indicates the rearranged band. FIG. 4. Northern blot analysis of RNA extracted from blast cells of patient DS (lane 2) hybridized to Ck probe (A), to TCRo~ probe (B), and to TCR/3 probe (C). Positive controls (lane 1) were represented by RNA extracted from an IgAk secreting myeloma cell line (A) and from a T-ALL patient (B and C). Fifteen micrograms of RNA were loaded on each lane. 62

Kappa light chain gene rearrangement in a T-cell lymphoma arrangements are exploited as lineage markers in lymphoid malignancies. 10. Acknowledgements--This research was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.). M. T. Mariano was recipient of a fellowship from the A.I.R.C. We are grateful to Dr P. Leder and to Dr T. Mak for providing molecular probes.

11. 12.

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