Atsuko Okumura

Molecular features of a new human lymphoma cell line carrying both BCL2 and BCL6 gene rearrangements

Chromosomal translocations and/or their molecular equivalents involving the BCL6 gene on 3q27 band have been suggested to be involved in the development of non-Hodgkins lymphoma of B-cell type (B-NHL). The rearrangement of BCL6 sometimes coexists with other translocations specific to B-NHL. Here, we report a novel B-cell lymphoma cell line, YM, established from a patient with diffuse large cell lymphoma. The YM cells expressed B-cell-associated antigens in addition to μδ/κ monoclonal immunoglobulin. Southern blot analysis of DNA from YM cells demonstrated rearrangement of the BCL2 gene within the 5′ flanking region (5′-BCL2). Polymerase chain reaction (PCR) using primer pairs for the BCL2 exons 1 and 2, and for the constant region of the immunoglobulin κ light chain gene (IGκ) revealed PCR products encompassing the 5′-BCL2/IGκ fusion, indicating that the YM cells had a t(2;18)(p11;q21) translocation. The BCL6 gene was rearranged at a point within the first intron, and cloning of the rearranged BCL6 revealed unidentified sequences juxtaposed to the 5′ side of the gene. The isolated clones were mapped to 16p11.2 by high resolution fluorescence in situ chromosomal hybridization. Thus, the YM cells carried a 3q27 translocation involving 16p11.2 as a partner. Chromosome painting of metaphase spreads confirmed that the YM cells had both t(2;18) and t(3;16). Northern blot analysis using a fragment immediately adjacent to the breakpoint on 16p11.2 revealed transcriptional activity within this locus. The YM cells expressed abundant transcripts with aberrant sizes from BCL2 and BCL6, indicating deregulated overexpression of the two genes resulting from the t(2;18) and t(3;16). The YM cell line will therefore be useful to study whether BCL2 and BCL6 genes collaborate in the pathogenesis of B-NHL.

Crizotinib resistance in acute myeloid leukemia with inv(2)(p23q13)/RAN binding protein 2 (RANBP2) anaplastic lymphoma kinase (ALK) fusion and monosomy 7.

This is the first report on the development of a p.G1269A mutation within the kinase domain (KD) of ALK after crizotinib treatment in RANBP2-ALK acute myeloid leukemia (AML). An elderly woman with AML with an inv(2)(p23q13)/RANBP2-ALK and monosomy 7 was treated with crizotinib. After a short-term hematological response and the restoration of normal hematopoiesis, she experienced a relapse of AML. Fluorescence in situ hybridization using the ALK break-apart probe confirmed the inv(2)(p23q13), while G-banded karyotyping revealed the deletion of a segment of the short arm of chromosome 1 [del(1)(p13p22)] after crizotinib therapy. The ALK gene carried a heterozygous mutation at the nucleotide position g.716751G>C within exon 25, causing the p.G1269A amino acid substitution within the ALK-KD. Reverse transcriptase PCR revealed that the mutated ALK allele was selectively transcribed and the mutation occurred in the ALK allele rearranged with RANBP2. As both the del(1)(p13p22) at the cytogenetic level and p.G1269A at the nucleotide level newly appeared after crizotinib treatment, it is likely that they were secondarily acquired alterations involved in crizotinib resistance. Although secondary genetic abnormalities in ALK are most frequently described in non-small cell lung cancers harboring an ALK alteration, this report suggests that an ALK-KD mutation can occur independently of the tumor cell type or fusion partner after crizotinib treatment.

Genetic analysis of 8;21 chromosomal translocation without AML1 gene involvement in MDS‐AML

We have investigated a case of acute myelocytic leukaemia derived from myelodysplastic syndrome (MDS‐AML) with an 8;21 translocation. In this case the AML1/MTG8 (ETO) fusion transcript was not detected by reverse transcriptase–polymerase chain reaction (RT‐PCR), and the rearrangement of the AML1 gene locus was not detected by Southern blot nor pulse field gel electrophoresis (PFGE) analyses using specific probes for the AML1 gene. Fluorescence in‐situ hybridization (FISH) study using cosmid probes for 21q22 revealed that the breakpoint of 21q22 was telomeric to the AML1 gene locus and centromeric from D21S259, 351, 3421 loci. This is the first report concerning the t(8;21)(q22;q22) carrying AMLs (de novo AML, MDS‐AML and therapy‐related AML) to show that the breakpoint at 21q22 is located outside the AML1 gene locus. It is also noteworthy that the cell‐surface antigen expression pattern of the bone marrow (BM) blasts was changed from CD7+CD2+CD13+CD33+CD19−CD11b+CD14+CD36+ to CD7− CD2− CD13+ CD19+ CD11b− CD14− CD33+ CD34+ CD36−CD56+ during leukaemic progression, and the pattern in leukaemic phase was similar to the characteristic phenotype of de novo AML cases with t(8;21), when the AML1/MTG8 fusion transcripts are always detected by RT‐PCR.

A unique subtype of diffuse large B-cell lymphoma primarily involving the bone marrow, spleen, and liver, defined by fluorodeoxyglucose-positron emission tomography combined with computed tomography

Abstract We describe 10 cases of diffuse large B-cell lymphoma (DLBCL) confined to the bone marrow (BM), spleen, and liver, as evidenced by the uniformly increased uptake of fluorodeoxyglucose (FDG) on positron emission tomography combined with computed tomography (PET/CT). Ages ranged from 56 to 87. All, but one patient presented with ‘B’ symptoms, a poor performance status, and hepatosplenomegaly. All patients showed cytopenia and elevated lactate dehydrogenase levels and were classified into the high-risk category of the International Prognostic Index scoring. BM infiltration was diffuse, interstitial/intrasinusoidal, or mixed, and all showed the nongerminal center B immunophenotype. Five patients had a rearrangement involving 3q27/BCL6, while six had increased copies of MYC, BCL2, or BCL6. All patients were initially treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone, leading to complete responses in six out of eight evaluable patients. We propose BM, spleen, and liver-type DLBCL, which is defined by the findings of FDG-PET/CT.