Two recurrent types of IGH::5' BCL2 breakpoints representing cytogenetic ins(14;18)(q32;q21q21) and t(14;18)(q32;q21), mediated by the VDJ and class switch recombination processes, respectively.

We describe two types of IGH::BCL2 breakpoints involving the 5' region of BCL2 (5' BCL2). One was ins(14;18)(q32;q21q21) observed in 2 follicular lymphoma (FL) cases, in which IGH was cleaved at 3' of IGHD and 5' of IGHJ and BCL2 was cleaved at 5' BCL2 and downstream regions, and a 281- or 201-kilobase pair fragment containing the BCL2 protein-coding sequences was invertedly inserted into IGH. In another type observed in 2 FL and 2 chronic lymphocytic leukemia (CLL) cases, breakage and reunion occurred within the switch region associated with IGHM (Sµ) and 5' BCL2, creating IGH Sµ::5' BCL2 fusion sequences on der(18)t(14;18)(q32;q21). The former is considered to be mediated by VDJ-recombination, while the latter by the class switch recombination process. There were no particular features in FL or CLL cases with IGH::5' BCL2 breakpoints compared with those with t(14;18)(q32;q21)/IGH::BCL2 involving the 3' breakpoint cluster regions.

Diverse B-cell tumors associated with t(14;19)(q32;q13)/IGH::BCL3 identified by G-banding and fluorescence in situ hybridization.

We characterized 5 B-cell tumors carrying t(14;19)(q32;q13) that creates the IGH::BCL3 fusion gene. The patients' ages ranged between 55 and 88 years. Two patients presented with progression or recurrence of B-cell chronic lymphocytic leukemia (B-CLL)/small lymphocytic lymphoma (SLL), two with diffuse large B-cell lymphoma (DLBCL) of non-germinal center B-like phenotype, and the remaining one with composite angioimmunoblastic T-cell lymphoma and Epstein-Barr virus-positive DLBCL. The presence of t(14;19)(q32;q13) was confirmed by fluorescence in situ hybridization (FISH), showing colocalization of 3' IGH and 3' BCL3 probes on der(14)t(14;19) and 5' BCL3 and 5' IGH probes on der(19)t(14;19). One B-CLL case had t(2;14)(p13;q32)/IGH::BCL11A, and 2 DLBCL cases had t(8;14)(q24;q32) or t(8;11;14)(q24;q11;q32), both of which generated IGH::MYC by FISH, and showed nuclear expression of MYC and BCL3 by immunohistochemistry. The IGH::BCL3 fusion gene was amplified by long-distance polymerase chain reaction in 2 B-CLL/SLL cases and the breakpoints occurred immediately 5' of BCL3 exon 1 and within the switch region associated with IGHA1. The 5 cases shared IGHV preferentially used in B-CLL cells, but the genes were unmutated in 2 B-CLL/SLL cases and significantly mutated in the remaining 3. B-cell tumors with t(14;19)(q32;q13) can be divided into B-CLL/SLL and DLBCL groups, and the anatomy of IGH::BCL3 in the latter may be different from that of the former.

MYC/BCL2 double- and MYC/BCL2/BCL6 triple-hit follicular lymphomas associated with t(8;14;18)(q24;q32;q21).

We describe two follicular lymphoma (FL) patients with MYC/BCL2 double- and MYC/BCL2/BCL6 triple-hit translocations. The first patient (case 1) was a man in his 30s who presented with stage IV disease with leukemic manifestation. The second patient (case 2) was a man in his 60s who presented with relapsed FL, but his disease was in a limited stage. Histopathology of the lymph node biopsies revealed grade 3A FL in both cases. MYC positivity and the Ki-67-labeling index were 60-70 and 20% in case 1 and 30 and 50% in case 2, respectively. G-banding revealed t(8;14;18)(q24;q32;q21) in both cases and fluorescence in situ hybridization using MYC, IGH, and BCL2 break-apart probes confirmed t(8;14;18)(+5'BCL2,-3'MYC;+3'MYC,-5'IGH;+5'IGH,-5'BCL2). In case 2, additional materials of der(8)t(8;14;18) were duplicated and translocated to chromosome Y, and t(3;16)(q27;p13)/BCL6::CIITA was identified. We obtained BCL2-major breakpoint region::IGHJ5::IGHG1 and MYC exon 2::IGHA2 fusion sequences by long-distance polymerase chain reaction in case 1, and proposed that t(8;14;18) was generated by two-step translocations and that BCL2::IGH and MYC::IGH involved the same IGH allele. Both patients responded to the standard chemotherapy for FL. We suggest that the presence of t(8;14;18) in FL does not immediately indicate high-grade transformation and aggressive clinical behavior requiring intensive chemotherapy.

Two cases of primary diffuse large B-cell lymphoma of the CNS associated with t(8;14)(q24;q32) or t(3;14)(q27;q32) identified by G-banding and fluorescence in situ hybridization applied to metaphase spreads.

We describe two patients with primary diffuse large B-cell lymphoma of the central nervous system (PCNS-DLBCL). The first patient (case 1) was a woman in her late 70s who presented with a tumor in the left frontal lobe, whereas the second patient (case 2) was a man in his early 70s who presented with a left frontal lobe tumor associated with intratumoral hemorrhage. The histopathology of the tumor specimen disclosed the proliferation of large cells with centroblastic (case 1) or immunoblastic/plasmablastic (case 2) cytomorphology and an accumulation of the tumor cells within the perivascular space. The cells in both cases were positive for CD20, CD79a, BCL6, IRF4/MUM1, MYC, and BCL2 and negative for CD5 and CD10. G-banding revealed t(8;14)(q24;q32) in case 1, and the tetraploid-range karyotype including two or three copies of der(3)t(3;14)(q27;q32) and der(14)t(3;14)(q27;q32) in case 2. Fluorescence in situ hybridization applied to metaphase spreads confirmed colocalization of MYC and IGH (case 1) and BCL6 and IGH (case 2) hybridization signals on the relevant derivative chromosomes. Case 1 carried the MYD88L265P mutation. This case report provides clear evidence for the occurrence of t(8;14)(q24;q32) and t(3;14)(q27;q32) in PCNS-DLBCL using metaphase-based cytogenetic analysis.

t(9;14)(p13;q32)/PAX5-IGH translocation as a secondary cytogenetic abnormality in diffuse large B-cell lymphoma.

A 75-year-old man presented with an ileocecal tumor composed of diffuse proliferation of large cells with immunoblastic morphology. Lymphoma cells were positive for CD20, CD79a, IRF4/MUM1, and BCL2, negative for CD5, CD10, and MYC, and partially positive for BCL6. PAX5 was positive with variable staining intensity among the cell nuclei. The V-D-J sequence of IGH showed the mutated configuration. The G-banding karyotype demonstrated two cytogenetic clones with or without t(9;14)(p13;q32), but the two shared other structural and numerical abnormalities. Fluorescence in situ hybridization using PAX5 and IGH probes confirmed the presence or absence of t(9;14)(p13;q32)/PAX5-IGH in each clone. The breakpoints of t(9;14)(p13;q32) were mapped 2,170 bp upstream of the coding region of PAX5 alternative exon 1B and within the IGHJ6-Eµ enhancer intron of IGH. It is suggested that t(9;14)(p13;q32) in this case was a secondary cytogenetic abnormality and the translocation is not necessarily involved in initial malignant transformation of B-cells but can occur later during the course of diffuse large B-cell lymphoma.

Identification of Functional Domains of CXCL14 Involved in High-Affinity Binding and Intracellular Transport of CpG DNA.

Some CXC chemokines, including CXCL14, transport CpG oligodeoxynucleotides (ODNs) into dendritic cells (DCs), thereby activating TLR9. The molecular basis of this noncanonical function of CXC chemokines is not well understood. In this study, we investigated the CpG ODN binding and intracellular transport activities of various CXC chemokines and partial peptides of CXCL14 in mouse bone marrow-derived dendritic cells. CXCL14, CXCL4, and CXCL12 specifically bound CpG ODN, but CXCL12 failed to transport it into cells at low dose. CXCL14 N-terminal peptides 1-47, but not 1-40, was capable of transporting CpG ODN into the cell, resulting in an increase in cytokine production. However, both the 1-47 and 1-40 peptides bound CpG ODN. By contrast, CXCL14 peptides 13-50 did not possess CpG ODN binding capacity or transport activity. The chimeric peptides CXCL12 (1-22)-CXCL14 (13-47) bound CpG ODN but failed to transport it. These results suggest that amino acids 1-12 and 41-47 of CXCL14 are required for binding and intracellular transport of CpG ODN, respectively. We found that an anti-CXCL14 Ab blocked cell-surface binding and internalization of the CpG ODN/CXCL14 complex. On the basis of these findings, we propose that CXCL14 has two functional domains, one involved in DNA recognition and the other in internalization of CXCL14-CpG DNA complex via an unidentified CXCL14 receptor, which together are responsible for eliciting the CXCL14/CpG ODN-mediated TLR9 activation. These domains could play roles in CXCL14-related diseases such as arthritis, obesity-induced diabetes, and various types of carcinoma.

Burkitt leukemia with precursor B-cell features that developed after ruxolitinib treatment in a patient with hydroxyurea-refractory JAK2V617F-myeloproliferative neoplasm.

A 62-year-old woman, who had a 16-year history of JAK2V617F-mutated myeloproliferative neoplasm (MPN), developed Burkitt leukemia (BL) 16 months after treatment with ruxolitinib to control hydroxyurea-refractory conditions. BL cells were CD10+, CD19+, CD20-, CD34-, cytoplasmic CD79a+, and TdT+, and lacked surface immunoglobulins but expressed the cytoplasmic µ heavy chain. In the bone marrow, nuclear MYC+ BL cells displaced the MPN tissues. t(8;14)(q24;q32) occurred at a CG dinucleotide within MYC exon 1 and at the IGHJ3 segment, and an N-like segment was inserted at the junction. The V-D-J sequence of the non-translocated IGH allele had the unmutated configuration. DNA from peripheral blood at a time of the course of MPN exhibited homozygous JAK2V617F mutation, while that at BL development included both JAK2V617F and wild-type DNAs. Although the association between JAK1/2 inhibitor therapy for MPN and secondary development of aggressive B-cell neoplasm remains controversial, this report suggests that, in selected patients, close monitoring of clonal B-cells in the BM is required before and during treatment with JAK1/2 inhibitors.

Diffuse large B-cell lymphoma carrying t(9;14)(p13;q32)/PAX5-immunoglobulin heavy chain gene is characterized by nuclear positivity of MUM1 and PAX5 by immunohistochemistry.

We described four patients with diffuse large B-cell lymphoma (DLBCL) carrying t(9;14)(p13;q32) that places the PAX5 adjacent to the immunoglobulin heavy chain (IGH) gene. Ages ranged between 63 and 80, and three were female. One developed a nodal disease, and the other three involved extranodal organs. The lymphoma cells were CD10- /BCL6- /MUM1+ in three and CD10+ /BCL6+ /MUM1+ in one. BCL2 was weak or negative. All had t(9;14)(p13;q32), and three had additional 14q32/IGH translocations or +der(14)t(9;14)(p13;q32). Fluorescence in situ hybridization using the PAX5 break-apart probe showed that the locus was disrupted between the 5' and 3' probes or within the 5' probe. Immunohistochemistry (IHC) using a monoclonal antibody against PAX5 showed strong nuclear positivity in all four patients. Cell block IHC of a CD30+ DLBCL cell line, KIS-1, which carried the t(9;14)(p13;q32) and PAX5-IGH fusion gene, reproduced the CD10- /BCL6- /MUM1+ immunophenotype, low-level BCL2, and strong nuclear PAX5. Uniform nuclear positivity of MUM1 in all four cases and KIS-1 cells suggest that these lymphomas arose at a late stage of B-cell differentiation, where expression of PAX5 physiologically becomes downregulated. It is therefore possible that high-level PAX5 resulting from t(9;14)(p13;q32) at this stage of differentiation perturbs the plasma cell differentiation program initiated by PAX5 repression, thereby contributing to the development of a fraction of DLBCL.

Mechanisms of aggregation and fibril formation of the amyloidogenic N-terminal fragment of apolipoprotein A-I.

The N-terminal (1-83) fragment of the major constituent of plasma high-density lipoprotein, apolipoprotein A-I (apoA-I), strongly tends to form amyloid fibrils, leading to systemic amyloidosis. Here, using a series of deletion variants, we examined the roles of two major amyloidogenic segments (residues 14-22 and 50-58) in the aggregation and fibril formation of an amyloidogenic G26R variant of the apoA-I 1-83 fragment (apoA-I 1-83/G26R). Thioflavin T fluorescence assays and atomic force microscopy revealed that elimination of residues 14-22 completely inhibits fibril formation of apoA-I 1-83/G26R, whereas Δ32-40 and Δ50-58 variants formed fibrils with markedly reduced nucleation and fibril growth rates. CD measurements revealed structural transitions from random coil to ß-sheet structures in all deletion variants except for the Δ14-22 variant, indicating that residues 14-22 are critical for the ß-transition and fibril formation. Thermodynamic analysis of the kinetics of fibril formation by apoA-I 1-83/G26R indicated that both nucleation and fibril growth are enthalpically unfavorable, whereas entropically, nucleation is favorable, but fibril growth is unfavorable. Interestingly, the nucleation of the Δ50-58 variant was entropically unfavorable, indicating that residues 50-58 entropically promote the nucleation step in fibril formation of apoA-I 1-83/G26R. Moreover, a residue-level structural investigation of apoA-I 1-83/G26R fibrils with site-specific pyrene labeling indicated that the two amyloidogenic segments are in close proximity to form an amyloid core structure, whereas the N- and C-terminal tail regions are excluded from the amyloid core. These results provide critical insights into the aggregation mechanism and fibril structure of the amyloidogenic N-terminal fragment of apoA-I.

The novel double-hit, t(8;22)(q24;q11)/MYC-IGL and t(14;15)(q32;q24)/IGH-BCL2A1, in diffuse large B-cell lymphoma.

An 82-year-old woman presented with generalized lymphadenopathy and skin involvement. Lymph node biopsy revealed diffuse large B-cell lymphoma with a high proliferation index. G-banding and fluorescence in situ hybridization showed a hypertetraploid karyotype with two copies of t(8;22)(q24;q11), generating the fusion of MYC and the immunoglobulin λ chain gene (IGL), and two copies of the novel immunoglobulin heavy chain gene (IGH) translocation, t(14;15)(q32;q24). A long-distance inverse polymerase chain reaction (PCR) using nested primer combinations designed for each constant gene of IGH showed that Cγ4 was juxtaposed to the downstream sequence of the BCL2A1 (BCL2-related protein A1) gene through the Sγ4 switch region. As a result of t(14;15)(q32;q24), BCL2A1 and IGH Sγ4-Cγ4 were aligned in the same transcriptional orientation at a distance of 64 kb. Reverse transcriptase-mediated PCR showed high BCL2A1 mRNA levels in a lymphoma specimen. Since BCL2A1, mapped at 15q24.3 or 15q25.1, encodes a protein that is an anti-apoptotic member of the BCL2 protein family, we herein described the novel double-hit, t(8;22)(q24;q11)/MYC-IGL and t(14;15)(q32;q24)/IGH-BCL2A1, in which BCL2A1 is considered to play a role equivalent to that of BCL2 in the most frequent double-hit, MYC/BCL2.

Neuropeptide Y-immunoreactive neuronal system and colocalization with FMRFamide in the optic lobe and peduncle complex of the octopus (Octopus vulgaris).

The distribution of neuropeptide Y (NPY)-like immunoreactivity and its colocalization with FMRFamide were investigated in the optic lobe and peduncle complex of the octopus ( Octopus vulgaris) by using immunohistochemical techniques. In the optic lobe cortex, NPY-immunoreactive (NPY-IR) fibers were observed in the plexiform layer, although no NPY-IR somata were observed in the outer or inner granular cell layers. In the optic lobe medulla, NPY-IR somata were seen in the cell islands, and abundant NPY-IR varicose fibers were observed in the neuropil. Most of the NPY-IR structures in the medulla showed FMRFamide-like immunoreactivity. In the peduncle lobe, abundant NPY-IR and FMRFamide-IR (NPY/FMRF-IR) varicose fibers were seen in the basal zone neuropil of the peduncle lobe. In the olfactory lobe, NPY/FMRF-IR varicose fibers were also abundant in the neuropil of the three lobules. NPY/FMRF-IR somata, with processes running to various neuropils, were scattered in the median and posterior lobules. In the optic gland, many NPY/FMRF-IR varicose fibers formed a honeycomb pattern. These observations suggest that NPY/FMRF-IR neurons in the optic lobes participate in the modulation of visual information and that those in the optic gland are involved in the regulation of endocrine function.