Original articles
Interphase Detection of t(4;14)(p16.3;q32.3) by In Situ Hybridization and FGFR3 Overexpression in Plasma Cell Malignancies

https://doi.org/10.1016/S0165-4608(99)00155-7Get rights and content

Abstract

The immunoglobulin (Ig) genes are frequently involved in chromosomal rearrangements with a wide variety of partner loci in multiple myeloma (MM). However, several partner chromosomes have not been detected by conventional cytogenetic methods; for example, 4p16.3 (FGFR3), 6p25.3 (IRF4), and 16q23 (c-maf). To clarify the incidence of t(4;14)(p16.3;q32.3) in primary tumors of MM and to evaluate possible correlations with specific manifestations of the disease, G-banding, double-color fluorescence in situ hybridization (DC-FISH), and/or reverse-transcriptase polymerase chain reaction (RT-PCR) were performed on 40 patients with MM—two with plasmacytoma (PCM) and three with plasma cell leukemia (PCL). All patients were studied by DC-FISH; 40 were studied by G-banding and 36 were studied by RT-PCR. The FISH probes consisted of a cosmid pC385.12 containing the FGFR3 gene, a YAC Y6 containing VH, and a phage Igγ1-10 containing the γ1 constant region (Cγ). We identified eight patients with either FGFR3/Cγ fusion or FGFR3 overexpression: six patients with both FGFR3/Cγ fusion and FGFR3 overexpression, one patient with FGFR3/Cγ, and one with FGFR3 overexpression. FGFR3/Cγ fusion was demonstrated at a frequency of 19% to 38% on interphase nuclei in seven of the 45 patients. Lytic bone lesions were found to be associated with FGFR3 overexpression. Interphase FISH with FGFR3 and Cγ probes combined with RT-PCR proved to be an effective tool for detection of this fully cryptic translocation, thus facilitating the characterization of clinical features of MM patients with t(4;14).

Introduction

Multiple myeloma (MM), plasma cell leukemia (PCL), and plasmacytoma (PCM) are neoplasms of the long-lived plasma cell [1]. In these plasma cell malignancies, chromosomal translocations most frequently involve the immunoglobulin heavy chain (IgH) gene locus at 14q32.33, which implicates it in the pathogenesis of MM via dysregulation of proto-oncogenes located at partner chromosomal loci 2, 3, 4, 5. We have previously detected this IgH translocation by means of fluorescence in situ hybridization (FISH) in approximately 70% patients with MM [6]. A large number of chromosome partners have been identified recurrently in this characteristic translocation (e.g., 11q13.3, 8q24.1, 4p16.3, 18q21.3, 16q23, 6p21.1, 7q32.1, 1q21, and 9p13) 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, three of which, 4p16.3, 11q13.3, and 16q23, are most commonly involved 1, 5. On the other hand, several partner chromosomes cannot be detected by means of conventional cytogenetic methods, for example 4p16.3 (FGFR3), 6p25.3 (IRF4), and 16q23 (c-maf). These genes are located at the terminal bands of their respective chromosomes. In addition, many housekeeping genes have been assigned to the telomeric regions which are particularly recombination-prone [16].

The t(4;14)(p16.3;q32.3) translocation has recently been identified in MM cell lines, and is associated with overexpression of the fibroblast growth factor receptor 3 gene 3, 17. Because this translocation is a subtle chromosomal abnormality involving two of the most telomeric regions detectable by molecular cytogenetic techniques, no cytogenetic data have been available for assessing the incidence of t(4;14) in patients with plasma cell malignancies. In order to clarify the incidence of t(4;14) in primary tumor of plasma cell malignancies and to evaluate possible correlations with specific manifestations of the disease, we performed G-banding, interphase FISH, and/or reverse-transcriptase polymerase chain reaction (RT-PCR) analyses on 45 patients with these malignancies. As a result, six patients were identified with both fusion between the FGFR3 and IgH genes (FGFR3/IgH fusion) and FGFR3 overexpression, one patient with FGFR3/IgH fusion, and one with FGFR3 overexpression.

Section snippets

Patients and Cell Lines

The bone marrow (BM) or peripheral blood (PB) cells of 45 patients with plasma cell malignancies was studied. Forty patients had MM, two had plasmacytoma (PCM), and three had PCL. The PCM tumors were detected in the orbita and the skin in each patient. Diagnosis of PCL was based on plasmacytosis exceeding 2 × 109/L of plasma cells in PB [18]. Thirty patients were male and 15 female, and their median age was 66 years (range, 39 to 89) at diagnosis. The types of M-protein identified were IgGκ in

Cytogenetic Findings

Metaphase spreads were available for karyotypic analysis of 34 patients with MM, three with PCL, and two with PCM. Karyotypes of 14 of the patients (nos. 22–26, 28, 30, 31, 35, 39, and 42–45) were described previously 6, 9. Twenty-three patients showed normal karyotype, and 15 abnormal karyotypes, including three with 14q+ chromosome: add(14)(q32) in patient no. 26, three-way translocation of t(8;14;11)(q24;q32;q13) in no. 34, and t(11;14) in no. 43 (Table 1).

Controls

PCR products of Y6 and a phage

Discussion

Interphase DC-FISH with FGFR3 and Cγ probes detected t(4;14)(q16.3;q32.3) in seven of 45 patients with plasma cell malignancies. On the other hand, mRNA of FGFR3 gene overexpressed in seven of the 36 patients studied by RT-PCR. FISH analysis of six of these seven patients with FGFR3 overexpression showed FGFR3/IgH fusion. These results indicate that t(4;14) significantly correlates with overexpression of the FGFR3 gene. In previous molecular studies on primary tumors, t(4;14) was detected at

Acknowledgements

This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture (10670962 and 08266109), Japan.

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