Naomi Kawashima

Immune-Mediated Hematopoietic Failure after Allogeneic Hematopoietic Stem Cell Transplantation: A Common Cause of Late Graft Failure in Patients with Complete Donor Chimerism

Late graft failure (LGF) without evidence of residual recipient cells is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-SCT) and often requires stem cell infusion from the same donor when the patient fails to respond to conventional therapies. We screened the peripheral blood (PB) of 14 patients who developed donor-type LGF at 2 to 132 months after allo-SCT for the presence of the markers for immune-mediated bone marrow (BM) failure. Increased glycosylphosphatidyl inositol-anchored protein-deficient (GPI-AP-) leukocytes, which accounted for .009% to 0.147% of the total granulocytes, were detected in 5 patients (severe aplastic anemia, n = 2; follicular lymphoma, n = 1; acute lymphoblastic leukemia, n = 1; myelodysplastic syndromes; n = 1) and 4.7% to 81.2% HLA-allele-lacking leukocytes (HLA-LLs) were detected in 2 patients (acute myelogenous leukemia, n = 1; and myelodysplastic syndromes, n = 1). Three of the 5 patients with increased GPI-AP- leukocytes were treated with antithymocyte globulin (ATG), and 2 patients achieved transfusion independence. These results suggest that immune mechanisms that are similar to acquired aplastic anemia underlie condition of approximately one-half of the patients with donor-type LGF, and that in patients with increased GPI-AP- cells, donor-derived hematopoiesis may be restored by ATG therapy alone without donor stem cell infusion.

Impact of T-cell chimerism on relapse after cord blood transplantation for hematological malignancies: Nagoya Blood and Marrow Transplantation Group study

Impact of T-cell chimerism on relapse after cord blood transplantation for hematological malignancies: Nagoya Blood and Marrow Transplantation Group study

Long-term outcomes of allogeneic hematopoietic cell transplantation with intensified myeloablative conditioning for refractory myeloid malignancy

Long-term outcomes of allogeneic hematopoietic cell transplantation with intensified myeloablative conditioning for refractory myeloid malignancy

Identification of the novel deletion-type PML-RARA mutation associated with the retinoic acid resistance in acute promyelocytic leukemia

All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are essential for acute promyelocytic leukemia (APL) treatment. It has been reported that mutations in PML-RARA confer resistance to ATRA and ATO, and are associated with poor prognosis. Although most PML-RARA mutations were point mutations, we identified a novel seven amino acid deletion mutation (p.K227_T233del) in the RARA region of PML-RARA in a refractory APL patient. Here, we analyzed the evolution of the mutated clone and demonstrated the resistance of the mutated clone to retinoic acid (RA). Mutation analysis of PML-RARA was performed using samples from a chemotherapy- and ATRA-resistant APL patient, and the frequencies of mutated PML-RARA transcript were analyzed by targeted deep sequencing. To clarify the biological significance of the identified PML-RARA mutations, we analyzed the ATRA-induced differentiation and PML nuclear body formation in mutant PML-RARA-transduced HL-60 cells. At molecular relapse, the p.K227_T233del deletion and the p.R217S point-mutation in the RARA region of PML-RARA were identified, and their frequencies increased after re-induction therapy with another type of retinoiec acid (RA), tamibarotene. In deletion PML-RARA-transduced cells, the CD11b expression levels and NBT reducing ability were significantly decreased compared with control cells and the formation of PML nuclear bodies was rarely observed after RA treatment. These results indicate that this deletion mutation was closely associated with the disease progression during RA treatment.

Mutation analysis of therapy-related myeloid neoplasms

We analyzed the genetic mutation status of 13 patients with therapy-related myeloid neoplasms (t-MN). Consistent with previous reports, t-MN cells preferentially acquired mutations in TP53 and epigenetic modifying genes, instead of mutations in tyrosine kinase and spliceosome genes. Furthermore, we compared the mutation status of three t-MN cells with each of the initial lymphoid malignant cells, and identified common mutations among t-MN and the initial malignant cells in two patients. In a patient who developed chronic myelomonocytic leukemia (CMML) after follicular lymphoma (FL), TET2 mutation was identified in both CMML and FL cells. Notably, the TET2 mutation was also identified in peripheral blood cells in the disease-free period with the same allelic frequency as CMML and FL cells, but not in a germ-line control, indicating that the TET2 mutation occurred somatically in the initiating clone for both malignant cells. On the other hand, a germ-line MYB mutation was identified in a patient who developed myelodysplastic syndromes (MDS) after FL. These results suggest that germ-line deposition and clonal hematopoiesis are closely associated with t-MN susceptibility; however, further analysis is necessary to clarify the mechanism required to provide the initiating clone with lineage commitment and clonal expansion.

Iodine staining and p16 immunohistochemistry as a novel screening for secondary esophageal neoplasm after hematopoietic stem cell transplantation

Regarding allogeneic hematopoietic stem cell transplantation (HSCT) survivors, several studies have reported a twofold to fourfold increased risk of secondary solid tumors, and incidence ratios of oral, pharyngeal and esophageal cancers were significantly higher than the general population [1–3]. Notably, increased risk of oral squamous cell carcinoma (SCC) has been reported in patients with chronic graft-versus-host disease (GVHD) [4–6]. Considering the dismal prognosis of esophageal cancer found in the advanced stage, detection in the asymptomatic early stages is highly desirable. Human papillomavirus (HPV) infection is suspected as an associated risk factor for oral and esophageal SCC in patients with immunodeficiency caused by the human immunodeficiency virus, solid organ transplantation, or allogeneic HSCT [5, 7, 8]. A case report has assessed the usefulness of p16 immunohistochemistry, a surrogate marker for HPV, for the early diagnosis of secondary oral and esophageal malignancy after HSCT [4]. Indeed, p16 immunohistochemistry is known as a pathologically good tool for the detection of oral and esophageal squamous cell neoplasm (SCN). The HPV-associated oncoprotein E6 and E7 interfere with cell cycle and inactivate tumor-suppressor protein p53 and pRb (retinoblastoma protein), which result in increased p16 expression levels caused by negative feedback control [9]. In patients with oral and pharyngolaryngeal SCC, the esophagus should be examined and screened for another SCN [4, 5]. In addition, esophagogastroduodenoscopy (EGD) with iodine staining and narrow band imaging (NBI) of the esophagus is a powerful tool for the detection of early esophageal SCN. In this study, we screened esophageal SCN using EGD examination with iodine staining, as well as pathological examination with p16 immunostaining, in patients after allogeneic HSCT. We performed EGD screening in patients who survived at least 1 year after the first allogeneic HSCT and who visited Japanese Red Cross Nagoya First Hospital from January 2009 to January 2017. Patients with oral mucosal lesions, primarily oral GVHD, but without gastrointestinal symptoms were selected. All patients provided informed consent in accordance with the Declaration of Helsinki. This study was designed as a prospective observational survey, and approved by the ethical committee of our institutional review board. EGD was performed by using an Olympus GIF-H290Z, GIF-H260Z, or GIF-H260 scope (Olympus Corporation, Tokyo, Japan). All patients were examined with normal white light, NBI, and iodine staining, respectively. Endoscopic biopsies were performed for abnormal endoscopic findings, even if it were minor changes. Biopsy specimens were stained with hematoxylin and eosin (HE) and p16 mouse monoclonal antibody (Ventana, Arizona, USA) immunostaining by using an autostainer BOND Max (Leica Biosystems, Newcastle, UK) according to the instruction manual, and then we compared p16 staining status with patient’s clinical characteristics. Each patient had a single-time EGD screening; however, annual EGD examinations were recommended for patients that were p16 positive. * Masafumi Ito itom@nagoya-1st.jrc.or.jp

Clinical significance of ASXL2 and ZBTB7A mutations and C-terminally truncated RUNX1-RUNX1T1 expression in AML patients with t(8;21) enrolled in the JALSG AML201 study

We analyzed the clinical significance and genetic features of ASXL2 and ZBTB7A mutations, and the alternatively spliced isoform of the RUNX1-RUNX1T1 transcript, which is also called AML1-ETO9a (AE9a), in Japanese CBF-AML patients enrolled in the JALSG AML201 study. ASXL2 and ZBTB7A genes were sequenced using bone marrow samples of 41 AML patients with t(8;21) and 14 with inv(16). The relative expression levels of AE9a were quantified using the real-time PCR assay in 23 AML patients with t(8;21). We identified ASXL2 (34.1%) and ZBTB7A (9.8%) mutations in only AML patients with t(8;21). ASXL2-mutated patients had a significantly higher WBC count at diagnosis (P = 0.04) and a lower frequency of sex chromosome loss than wild-type patients (33 vs. 76%, respectively, P = 0.01). KIT mutations were the most frequently accompanied with both ASXL2 (36%) and ZBTB7A (75%) mutations. Neither ASXL2 nor ZBTB7A mutations had an impact on overall or event-free survival. Patients harboring cohesin complex gene mutations expressed significantly higher levels of AE9a than unmutated patients (P = 0.03). In conclusion, ASXL2 and ZBTB7A mutations were frequently identified in Japanese AML patients with t(8;21), but not in those with inv(16). Further analysis is required to clarify the detailed biological mechanism of AE9a regulation of the cohesin complex.

Successful treatment with allogeneic stem cell transplantation followed by DLI and TKIs for e6a2 BCR-ABL-positive acute myeloid leukaemia: A case report and literature review

Rationale: Patients with the e6a2 BCR-ABL transcript, 1 of the atypical transcripts, have been reported to have a poor prognosis, and allogeneic stem cell transplantation (ASCT) can be considered as additional therapy. However, long-term survival after ASCT for this disease is rare. Patient concerns: This report concerns a 55-year-old female patient with e6a2 BCR-ABL-positive acute myeloid leukemia including the outcome of ASCT followed by donor lymphocyte infusion (DLI). Diagnoses: The breakpoint was confirmed by direct sequencing. Her minimal residual disease could be detected by nested reverse-transcription polymerase chain reaction using primers for the minor BCR-ABL (e1a2) transcript. Interventions: Treatment with tyrosine kinase inhibitors (TKIs) and ASCT followed by DLI. Outcomes: Despite multiple cytogenetic and molecular relapses after ASCT, she remains in molecular remission at 46 months after ASCT. Lessons: This case indicates the efficacy of the combination of the graft-versus-leukemia effect and TKIs for e6a2 BCR-ABL-positive acute leukemia. When the Philadelphia chromosome with an unusual chromosomal breakpoint is suggested, we should clarify the breakpoint because that information can aid molecular assessments and decisions to provide an additional or alternative therapy.

Increase of bone marrow macrophages and CD8 + T lymphocytes predict graft failure after allogeneic bone marrow or cord blood transplantation

Graft failure (GF) remains an obstacle to survival after allogeneic hematopoietic stem cell transplantation. However, differentiating GF from delayed engraftment (DE) can be difficult. Host CD8+ lymphocytes have been reported to mediate graft rejection, but the impact of macrophages on DE or GF is yet to be clarified. Peri-engraftment bone marrow (BM) specimens of 32 adult patients with normal engraftment, DE or GF were retrospectively evaluated to identify the potential associations of CD163+ macrophage and CD8+ lymphocyte infiltration into BM. The macrophage or CD8+ lymphocyte number/total nucleated cell number was defined as the Mac ratio and CD8 ratio, respectively. Both DE and GF groups had significantly higher Mac ratios at day 14 than the normal group (P<0.0001), but no significant difference was observed between the DE and GF groups (P=1.000). The CD8 ratio at day 14 was significantly higher in the GF than in the normal group (P=0.005), whereas the CD8 ratios of the DE and normal groups were similar (P=0.07). A high Mac ratio at day 14 was associated with a risk of DE or subsequent GF. Patients with increased CD8 ratio at day 14 had a further risk of GF. The Mac ratio and the CD8 ratio appear to be well suited for predicting engraftment status.