Miho Nakagawa

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.

A rare MYC/BCL3 double-translocation and protein-expression in a diffuse large B-cell lymphoma

The WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, published in 2017, proposed the category high-grade B-cell lymphoma (HGBL) with MYC and BCL2 and/or BCL6 rearrangements [1]...

Relapse of acute promyelocytic leukemia in the external auditory canal confirmed by PML/RARA dual-fusion and RARA break-apart fluorescence in situ hybridization

Dear Editor, A man in his fifties, who was receiving maintenance treatment with all-trans retinoic acid (ATRA) according to the AIDA-2000 protocol for intermediate-r isk acute promyelocytic leukemia (APL) [1], presented with hearing loss of the right ear associated with discharge and earache. On examination, the right external auditory canal (EAC) was narrowed due to marked swelling of the luminal lining and filled with exudative materials (Fig. 1a), and similar, albeit milder findings were observed in the left EAC. Computed tomography (CT) of the right temporal bone demonstrated that the middle ear cavity and mastoid air space were opacified and the EAC was narrowed by soft tissue density materials (Fig. 1b). F– fluorodeoxyglucose-positron emission tomography revealed the accumulation of the tracer along the right EAC. Since this condition did not resolve following the oral administration of cephalosporin and levofloxacin ear drops, biopsy was performed on the right EAC. The results obtained revealed that mononuclear cells were distributed or clustered within the thickened squamous epithelial layers (Fig. 1c). One year and 9 months before, the patient developed A P L w i t h t y p i c a l c y t o m o r p h o l o g i c a l a n d immunophenotypic features. A cytogenetic analysis revealed t(15;17)(q24;q21) and additional materials at the l o n g a r m o f c h r o m o s o m e 1 1 [ k a r y o t y p e : 46,XY,add(11)(q25),t(15;17)(q24;q21)]. A reverse transcriptase polymerase chain reaction (RT-PCR) amplified DNA corresponding to short-form PML/RARA fusion mRNA. The mRNA level of Wilms’ tumor-1 gene (WT1) was 1.6 × 10 copies/μgRNA. Paraffin-embedded tissue sections of EAC biopsy were subjected to fluorescence in situ hybridization (FISH) using a PML/RARA dual-color, dual-fusion probe and RARA dualcolor, break-apart probe. The results obtained showed that cell nuclei were labeled with yellow signals, indicative of the fusion of PML and RARA after the former probe, and a pair of split signals, indicative of the rearrangement of RARA after the latter (Fig. 1d). Although his blood cell count was normal and bone marrow (BM) showed normocellularity with no evidence of leukemic relapse, RT-PCR detected PML/RARA fusion mRNA in the material that was identical to that of the initial presentation. The WT-1 mRNA level was 1.2 × 10 copies/μg RNA. The patient was treated intravenously with arsenic trioxide (ATO), which led to the slow, but steady resolution of EAC infiltration as well as the amelioration of ear symptoms (Fig. 1a). BM became negative for PML/RARA mRNA. The patient then underwent allogeneic hematopoietic stem cell transplantation from an HLA-identical unrelated donor. EAC has been reported as a rare site for the relapse of APL [2–7]. We herein demonstrated that infiltrated cell nuclei carried the rearrangement of RARA with PML by FISH, con f i rm ing the r e l ap se o f APL a t th i s extramedullary site. EAC has been suggested to represent a specific sanctuary for disease involvement in APL [3] when patients are treated with ATRA-based regimens [2, 6]. This condition may occur at variable time intervals from first remission and is associated with or without molecular/hematological relapse [3]. Since this condition * Takashi Akasaka akasaka@tenriyorozu.jp

Cytogenetic evidence for the clonal hematopoietic cell origin of alveolar macrophages in myelodysplastic syndrome-associated pulmonary alveolar proteinosis

Dear Editor, Three major categories of pulmonary alveolar proteinosis (PAP) have been recognized: congenital form due to mutations in the genes for surfactant proteins or granulocyte macrophagecolony stimulating factor (GM-CSF) receptor, autoimmune PAP due to GM-CSF antibodies, and secondary PAP associated with heterogeneous conditions [1]. In a Japanese cohort of 404 PAP cases, 35 were associated with hematological disorders, including myelodysplastic syndrome (MDS) in 26 and other myeloid/lymphoid neoplasms in eight [2]. We previously described a 58-year-old female patient with the refractory cytopenia with multilineage dysplasia subtype of MDS [3]. Cytogenetic analysis of bone marrow cells revealed tandem triplication of the long arm of chromosome 1 [trp(1q)] as the sole cytogenetic abnormality [karyotype: 46,XX,trp(1)(q21q32)]. Fluorescence in situ hybridization (FISH) using a probe for the PBX1 (pre-B-cell leukemia homeobox 1) gene localized at 1q23 confirmed trp(1q) in both metaphase and interphase nuclei (Fig. 1a). The patient initially presented with prolonged productive coughing. Chest X-rays showed progressive infiltrates in both lungs, and computed tomography (CT) revealed patchy ground-glass opacification with thickened interlobular septa, predominantly in the lower lungs. The level of Kerbs von Lungren 6 antigen (KL-6), which is a biomarker of PAP and correlates with disease severity [4], was 3313U/mL (reference range, < 500 U/mL). Anti–GM-CSF autoantibody was negative. To confirm the association of PAP, we obtained bronchoalveolar lavage (BAL) fluids, showing a milky appearance and containing 660 cells per microliter in addition to globular materials with an amorphous structure (Fig. 1b) [1, 5, 6]. The cell differential was 0.5% neutrophils, 2.5% eosinophils, 53.5% lymphocytes, and 43.5% macrophages, and the macrophages had abundant foamy cytoplasm (Fig. 1b) [1]. FISH with the Vysis LSI TCF3/PBX1 dual color, dual fusion probe revealed that macrophage nuclei were labeled with four red (PBX1) signals, three of which were localized, and two green (TCF3, transcription factor 3 at 19p13.3) signals, while those of lymphocytes showed a two-red and two-green signal pattern (Fig. 1c–f). The patient underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) from an HLA-identical unrelated donor. The course was uneventful and donor-type hematopoiesis was promptly confirmed. CT 8 months after allo-HSCT revealed the improvement of pulmonary infiltrates, and the level of KL-6 had normalized. Unfortunately, she died of gastric cancer 18 months after transplant. We found trp(1q) in bone marrow cells and alveolar macrophages in common, indicating that PAP and MDS did not coincidently occur but were closely related disorders of clonal hematopoietic cell origin. The potential linkage between MDS and PAP is that alveolar macrophages derived from the MDS clone differentiate and proliferate locally and replace the normal macrophages, and, as MDS-clone– * Hitoshi Ohno hohno@tenriyorozu.jp