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.

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.

Combination of multicolor flow cytometry for circulating lymphoma cells and tests for the RHOAG17V and IDH2R172 hot-spot mutations in plasma cell-free DNA as liquid biopsy for the diagnosis of angioimmunoblastic T-cell lymphoma.

We applied two-step multicolor flow cytometry (FCM) for circulating lymphoma cells in the blood of 20 patients with angioimmunoblastic T-cell lymphoma (AITL) and confirmed neoplastic T-cells in all. Eleven exhibited dim expression of CD3 and 7 lost its expression. The proportion of CD10+ lymphoma cells ranged widely from 0 to 100%, with a median of 15.7%. Ten patients demonstrated expansion of a single T-cell receptor ß-chain repertoire. Lymphoma cells comprised 0.01 to 18.22% (median, 0.26%) of white cells and the absolute numbers ranged from 0.5 to 1491.6 cells (median, 29.3 cells) per microliter of blood. We next found that 14 (70%) and 3 (15%) patients carried RHOAG17V and IDH2R172 mutations, respectively, in cell-free DNA (cfDNA) in the plasma. The combination of multicolor FCM of the blood, and tests for RHOAG17V and IDH2R172 hot-spot mutations in plasma cfDNA provides a blood-based 'liquid biopsy' for the diagnosis of AITL.

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.

Pulmonary extranodal marginal zone lymphoma that presented with macroglobulinemia and marked plasmacytic cell proliferation carrying the t(14;18)(q32;q21)/MALT1-immunoglobulin heavy-chain fusion gene in pleural fluid.

An 80-year-old man presented with the accumulation of pleural fluid in the right thoracic cavity. Serum electrophoresis revealed an M-component and immunofixation confirmed IgM/λ. The level of IgM was 1,526 mg/dL. Imaging studies showed an infiltrative condition of the ipsilateral lung parenchyma. The fluid contained abundant neoplastic cells with the morphological and immunophenotypic features of plasma cells, which expressed IgM/λ monoclonal immunoglobulins on the cell surface and in the cytoplasm. The karyotype was 48,XY,+3,add(9)(p13),+12,add(14)(q32),del(16)(q22),-18,+mar, and a series of fluorescence in situ hybridization studies demonstrated that the add(14) chromosome represented der(14)t(14;18)(q32;q21), at which the MALT1-immunoglobulin heavy-chain (IGH) fusion gene was localized. A long-distance polymerase chain reaction amplified the fragment encompassing the two genes, showing that the junction occurred at the J6 segment of IGH and 3.7-kb upstream of the MALT1 breakpoint cluster. We propose that this case represents an extreme form of the plasmacytic differentiation of extranodal marginal zone lymphoma that developed in the lung.

Acute basophilic leukemia associated with the t(16;21)(p11;q22)/FUS-ERG fusion gene.

We herein report a rare case of acute basophilic leukemia with t(16;21)(p11;q22) generating the FUS-ERG fusion gene. The basophilic nature of leukemia blasts was demonstrated by cytomorphology, toluidine blue metachromasia, mature basophil-associated antigen expression, and characteristic granules under electron microscopy. The molecular link between t(16;21)/FUS-ERG and basophilic differentiation remains unclear.

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.

Establishment and characterization of a novel Hodgkin lymphoma cell line, AM-HLH, carrying the Epstein-Barr virus genome integrated into the host chromosome.

We describe the establishment and characterization of a cell line, AM-HLH, obtained from a patient with Epstein-Barr virus-positive (EBV+ ) nodular sclerosis-type Hodgkin lymphoma (HL). The cells were positive for CD2 and CD30 and negative for CD15. The immunoglobulin heavy- and κ light-chain genes were rearranged. The karyotype was of the triploid range. Southern blotting using the EBV terminal repeat probe detected 3 hybridizing bands that were identical to those of the parental HL material. The cells expressed EBV-encoded RNAs as well as latent genes (EBNA1, EBNA2, LMP1, and LMP2A) and lytic genes (BZLF1 and BALF2). Fluorescence in situ hybridization (FISH) with the cosmid pJB8 clone containing a fragment of EBV DNA as a probe revealed multiple hybridization signals at a marker chromosome. Additional FISH using whole chromosome painting and centromere probes in combination with multicolor FISH determined that multiple EBV copies were clustered within the chromosome 20 materials of the marker chromosome. Culture supernatants of AM-HLH contained IL-10 as measured by the bead-based immunoassay. It is possible that an integrated EBV genome and cellular genes on chromosome 20 were coamplified, leading to the enhanced expression of genes involved in cell growth control. The AM-HLH cell line will be useful to clarify the role of cytokines in the development of EBV+ HL.

Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia Is Separated into Two Subgroups Associated with Survival by BCR-ABL Fluorescence in situ Hybridization of Segmented Cell Nuclei: Report from a Single Institution.

Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) may include the lymphoid blast crisis of chronic myeloid leukemia (CML-BC). We applied fluorescence in situ hybridization (FISH) of the BCR-ABL fusion gene to peripheral blood and/or bone marrow smears to determine whether the fusion was restricted to mononuclear cell nuclei or if segmented cell nuclei representing mature neutrophils also carried the fusion (Seg-FISH). Among 20 patients with Ph+ ALL without a prior diagnosis of CML, 9 were Seg-FISH+ and 11 were Seg-FISH-. Seg-FISH+ cases were characterized by a higher rate of p210-type BCR-ABL transcripts, higher white cell and blast counts, and a higher rate of myeloid and T-lymphoid antigen expression than Seg-FISH- cases, in addition to 'major route' cytogenetic abnormalities associated with CML-BC. Eighteen patients were treated with tyrosine kinase inhibitors (TKIs) either alone or in combination with multiagent chemotherapy, and 7 underwent allogeneic hematopoietic stem cell transplantation. Progression-free and overall survivals were greater in the Seg-FISH+ group than in the Seg-FISH- group. These results suggest that the Seg-FISH+ group represents lymphoid CML-BC that occurs de novo, while the Seg-FISH- represents Ph+ ALL in the strict sense, and the two groups are associated with survival when treated with TKIs or TKI-combined therapy.

CD68 on rat macrophages binds tightly to S100A8 and S100A9 and helps to regulate the cells' immune functions.

S100A8 and S100A9 (S100 proteins) are regulators of immune cells of myeloid origin. Whereas S100 proteins are found at high concentrations in such cells, their immunologic roles remain unclear. We focused on cluster of differentiation 68 (CD68). The aim of this study is to investigate whether CD68 binds to extracellular S100A8 and/or S100A9 and subsequently participates in the regulation of the cells' immune functions. ELISA and affinity chromatography showed that both recombinant rat S100A8 (r-S100A8) and r-S100A9 bound to r-CD68, but not to r-CD14. Flow cytometry clearly showed evidences supporting above the 2 results. As analyzed by flow cytometry, a less amount of r-S100A8 or r-S100A9 bound to the macrophages treated with some deglycosylation enzymes. In an in vitro assay, the expression levels of S100A8 and S100A9 were significantly suppressed after the macrophages had been treated with an anti-CD68 antibody (ED1). As stimulated macrophages with r-S100A9, the expression of IL-1ß mRNA in macrophages, which were treated with anti-TLR4 or -RAGE antibodies, was significantly suppressed. r-S100A8 up-regulated IL-6 and IL-10 mRNAs, while r-S100A9 did TNF-α and IL-6 mRNAs, although these regulations were not statistically significant. Small interfering CD68 also significantly suppressed activation of macrophages through an autocrine pathway by r-S100A8 or r-S100A9. In macrophages stimulated with LPS, fluorescent immunologic staining showed that most CD68 colocalized with S100A8 or S100A9 and that the levels of all 3 molecules were markedly increased. In conclusion, CD68 on macrophages binds to S100A8 and S100A9 and thereby, plays a role in the regulation of the cells' immune functions.

[Application of Flow Cytometry in Clinical Laboratories and Its Future Direction, Focusing Upon Multicolor Flow Cytometry].

Flow cytometry (FCM) has been introduced into clinical laboratories to determine the lineage and functional stage of differentiation of hematopoietic tumor cells using multiple immunophenotypic markers. Multi-parametric analysis, which simultaneously measures multiple cellular characteristics, not only provides a large amount of novel information for each sample studied, but also requires a reduced volume of the specimen and avoids redundancies of reagents within antibody panels. Through a series of novel developments in FCM hardware, software, and dye-chemistry, recently developed multicolor flow cytometers are capable of detecting 10 or more fluorochromes simultaneously and expeditiously, taking advantage of not only cluster analysis but also detecting rare cellular populations within complex materials. In this study, we applied the multicolor FCM technology for: i) T-cell lineage assessment in early T-cell precursor acute lymphoblastic leukemia, ii) detection of a rare neoplastic population in angioimmunoblastic T-cell lymphoma composed of a complex mixture of cells, iii) assessment of bone marrow infiltration of B-cell lymphoma, and iv) sensitive detection of multiple myeloma cells with the use of a combination of antibodies, which is applicable to monitoring the response to treatment.

Relationship between plasma dabigatran concentration and activated partial thromboplastin time in Japanese patients with non-valvular atrial fibrillation.

BACKGROUND: Activated partial thromboplastin time (aPTT) is recommended for monitoring anticoagulant activity in dabigatran-treated patients; however, there are limited data in Japanese patients. To clarify the relationship between plasma dabigatran concentration and aPTT, we analyzed plasma dabigatran concentration and aPTT at various time points following administration of oral dabigatran in a Japanese hospital. METHODS: We enrolled 149 patients (316 blood samples) with non-valvular atrial fibrillation (NVAF) who were taking dabigatran. Patients had a mean age of 66.6±10.0 years (range: 35-84) and 66% were men. Plasma dabigatran concentrations and aPTT were measured using the Hemoclot(®) direct thrombin inhibitor assay and Thrombocheck aPTT-SLA(®), respectively. Samples were classified into eight groups according to elapsed times in hours since oral administration of dabigatran. RESULTS: Significantly higher dabigatran concentrations were observed in samples obtained from patients with low creatinine clearance (CLCr) (CLCr<50 mL/min). Dabigatran concentrations and aPTT were highest in the 4-h post-administration range. Additionally, there was a significant correlation between plasma dabigatran concentrations and aPTT (y=0.063x+32.596, r (2)=0.648, p<0.001). However, when plasma dabigatran concentrations were 200 ng/mL or higher, the correlation was lower (y=0.040x+38.034 and r (2)=0.180); these results were evaluated by a quadratic curve, resulting in an increased correlation (r (2)=0.668). CONCLUSIONS: There was a significant correlation between plasma dabigatran concentrations and aPTT. Additionally, in daily clinical practice in Japan, plasma dabigatran concentrations and aPTT reached a peak in the 4-h post administration range. Considering the pharmacokinetics of dabigatran, aPTT can be used as an index for risk screening for excess dabigatran concentrations in Japanese patients with NVAF.