Hiroshi Akasaka

Molecular and Clinical Features of Non-Burkitt’s, Diffuse Large-Cell Lymphoma of B-Cell Type Associated With the c-MYC/Immunoglobulin Heavy-Chain Fusion Gene

PURPOSE t(8;14)(q24;q32) and/or c-MYC/immunoglobulin heavy-chain (IGH) fusion gene have been observed not only in Burkitts lymphoma (BL) but also in a proportion of non-BL, diffuse large-cell lymphoma of B-cell type (DLCL). We explored molecular features of DLCL with c-MYC/IGH fusion and the impact of this genetic abnormality on clinical outcome of DLCL. PATIENTS AND METHODS A total of 203 cases of non-BL DLCL were studied. Genomic DNA extracted from tumor tissues was subjected to long-distance polymerase chain reaction (LD-PCR) using oligonucleotide primers for exon 2 of c-MYC and for the four constant region genes of IGH. RESULTS Twelve cases (5.9%) showed positive amplification; one had a c-MYC/Cmicro, nine had a c-MYC/Cgamma, and two had a c-MYC/Calpha fusion sequence. Restriction and sequence analysis of the LD-PCR products, ranging from 2.3 to 9.4 kb in size, showed that breakage in the 12 cases occurred within a 1.5-kb region that included exon 1 of c-MYC in combination with breakpoints at the switch regions of IGH (10 of 12). In 10 cases, Myc protein encoded by the fusion genes demonstrated mutations and/or deletions. Six cases had additional molecular lesions in BCL-2 or BCL-6 and/or p53 genes. The age range of the 12 patients was 44 to 86 years, with a median age of 65.5 years. Five patients had stage I/II disease, and seven had stage III/IV disease. Lactate dehydrogenase was elevated in nine of 11 subjects. Seven showed involvement of the gastrointestinal tract. All patients were treated by surgery and/or chemoradiotherapy; six died of the disease within 1 year, resulting in the poorest 1- and 2-year survival rates among DLCL subgroups. CONCLUSION The c-MYC/IGH fusion gene of DLCL is identical to that of the sporadic type of BL (sBL). DLCL with c-MYC/IGH shares clinical features with sBL but is characterized further by an older age distribution.

Discontinuation of dasatinib in patients with chronic myeloid leukaemia who have maintained deep molecular response for longer than 1 year (DADI trial): a multicentre phase 2 trial

BACKGROUND First-line imatinib treatment can be successfully discontinued in patients with chronic myeloid leukaemia after deep molecular response has been sustained for at least 2 years. We investigated the safety and efficacy of discontinuing second-line or subsequent dasatinib after at least 1 year of deep molecular response. METHODS The Dasatinib Discontinuation trial was a prospective multicentre trial done in Japan. Eligible patients taking dasatinib and with confirmed stable deep molecular response were enrolled between April 1, 2011, and March 31, 2012. All patients received dasatinib consolidation therapy for at least 1 year. In those with sustained deep molecular response, dasatinib was discontinued. Patients were followed up every month in year 1 (clinical cutoff), every 3 months in year 2, and every 6 months in year 3 for deep molecular response and immunological profiles. The primary endpoint was the proportion of patients with treatment-free remission at 6 months after discontinuation. Molecular relapse was defined as loss of deep molecular response at any assessment. This study is registered, number UMIN000005130. FINDINGS 88 patients were enrolled in the consolidation phase, 24 were excluded from the discontinuation phase due to fluctuations in BCR-ABL1 transcript levels. One patient was excluded because of positive expression of major and minor BCR-ABL1 transcripts in chronic myeloid leukaemia cells and the detection of minor BCR-ABL1 transcripts during consolidation. Thus, 63 patients discontinued dasatinib treatment. The 25 patients who were excluded from discontinuation continued to receive dasatinib and none showed disease progression. Median follow-up was 20.0 months (IQR 16.5-24.0). Of the 63 patients who discontinued and were not excluded, 30 patients maintained deep molecular response while 33 patients had molecular relapses, all within the first 7 months after discontinuation. The estimated overall treatment-free remission was 49% (95% CI 36-61) at 6 months. No severe treatment-related toxic effects were seen. Treatment was restarted in the 33 patients with relapse; rapid molecular responses were seen in all 33 patients, of whom 29 (88%) regained deep molecular response within 3 months, as did the remaining four by 6 months. INTERPRETATION Dasatinib discontinuation after sustained deep molecular response for more than 1 year is feasible. FUNDING Epidemiological and Clinical Research Information Network (ECRIN).

Refinement of the BCL2/immunoglobulin heavy chain fusion gene in t(14;18)(q32;q21) by polymerase chain reaction amplification for long targets

The t(14;18)(q32;q21) translocation, involving the BCL2 gene and junctional segments (JH) of the immunoglobulin heavy chain gene (IGH), constitutes the most common chromosomal translocation in non‐Hodgkins lymphoma of B‐cell type. Although the breakpoints in BCL2 are largely clustered within the major breakpoint region (MBR) and minor cluster region (mcr), it is known that some breakpoints map away from these regions, resulting in negative amplification of the junctional sequence by polymerase chain reaction (PCR) for <1 kb targets. To circumvent this problem, we applied a novel PCR technology for long DNA targets, long‐distance (LD‐) PCR, to the detection of t(14;18) in clinical materials. Oligonucleotide primers were designed to be quite distant from the two known cluster regions in BCL2, and those for the corresponding IGH were complementary to the enhancer and constant regions. In all 52 cases identified as carrying BCL2/JH fusion by conventional Southern blot analysis, LD‐PCR successfully amplified fragments encompassing the junctions, which were readily identifiable on ethidium bromide‐stained gel. The size of the LD‐PCR products ranged from 3.9 kb to 10.7 kb in MBR/IGH fusion and 1.9 kb to 16 kb in mcr/IGH fusion. Furthermore, we established an LD‐PCR protocol for >20 kb targets, which covered the intervening region between the MBR and mcr. Restriction analysis of the LD‐PCR products revealed that breakpoints in 33 cases fell within the 150 bp‐MBR region, and in 3 cases were within the mcr determined previously by others. In contrast, the breakpoints of the remaining 16 cases were distributed over a large region from the MBR through mcr. Nucleotide sequence analysis of a potential cluster region revealed the presence of an Alu repeat sequence. Restriction analysis of LD‐PCR products with BstEII demonstrated a predominant usage of the JH6 segment (71%) at the BCL2/JH junctions. LD‐PCR using primers for the constant region genes showed that class switch recombination occurred in more than 80% of the IGH genes on the der(14) chromosome. Our study showed that LD‐PCR was capable of detecting virtually any t(14;18) that occurred within the approximately 30 kb region downstream of the MBR, and thus is suitable for initial diagnosis of lymphoma tissues. Furthermore, as amplified fragments obtained by the LD‐PCR contained distinctive regions of BCL2 and IGH, restriction analysis and nucleotide sequencing of the products refined the characteristics of t(14;18). Genes Chromosomes Cancer 21:17–29, 1998.

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.

Final 3-year Results of the Dasatinib Discontinuation Trial in Patients With Chronic Myeloid Leukemia Who Received Dasatinib as a Second-line Treatment

Micro‐Abstract We describe the results of a prospective trial of the discontinuation of second‐line dasatinib treatment in chronic myeloid leukemia patients who maintained a deep molecular response for > 1 year. The treatment‐free remission rate at 36 months was 44.4%. High natural killer cell counts before discontinuation correlated significantly with successful therapy discontinuation. Introduction We previously reported an interim analysis of the DADI (dasatinib discontinuation) trial. The results showed that 48% of patients with chronic myeloid leukemia in the chronic phase who maintained a deep molecular response (DMR) for ≥ 1 year could discontinue second‐ or subsequent‐line dasatinib treatment safely at a median follow‐up of 20 months. However, the results from longer follow‐up periods would be much more useful from a clinical perspective. Patients and Methods The DADI trial was a prospective, multicenter trial conducted in Japan. After confirming a stable DMR for ≥ 1 year, dasatinib treatment subsequent to imatinib or nilotinib was discontinued. After discontinuation, the loss of DMR (even of 1 point) was defined as stringent molecular relapse, thereby triggering therapy resumption. The predictive factors of treatment‐free remission (TFR) were analyzed. Results The median follow‐up period was 44.0 months (interquartile range, 40.5‐48.0 months). The estimated overall TFR rate at 36 months was 44.4% (95% confidence interval, 32.0%‐56.2%). Only 2 patients developed a molecular relapse after the 1‐year cutoff point. The presence of imatinib resistance was a significant risk factor for molecular relapse. Moreover, high natural killer cell and low &ggr;&dgr;+ T‐cell and CD4+ regulatory T‐cell (CD25+CD127low) counts before discontinuation correlated significantly with successful therapy discontinuation. Conclusion These findings suggest that discontinuation of second‐ or subsequent‐line dasatinib after a sustained DMR of ≥ 1 year is feasible, especially for patients with no history of imatinib resistance. In addition, the natural killer cell count was associated with the TFR.