Takamasa Katagiri

Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia.

BACKGROUND In patients with acquired aplastic anemia, destruction of hematopoietic cells by the immune system leads to pancytopenia. Patients have a response to immunosuppressive therapy, but myelodysplastic syndromes and acute myeloid leukemia develop in about 15% of the patients, usually many months to years after the diagnosis of aplastic anemia. METHODS We performed next-generation sequencing and array-based karyotyping using 668 blood samples obtained from 439 patients with aplastic anemia. We analyzed serial samples obtained from 82 patients. RESULTS Somatic mutations in myeloid cancer candidate genes were present in one third of the patients, in a limited number of genes and at low initial variant allele frequency. Clonal hematopoiesis was detected in 47% of the patients, most frequently as acquired mutations. The prevalence of the mutations increased with age, and mutations had an age-related signature. DNMT3A-mutated and ASXL1-mutated clones tended to increase in size over time; the size of BCOR- and BCORL1-mutated and PIGA-mutated clones decreased or remained stable. Mutations in PIGA and BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and longer and a higher rate of overall and progression-free survival; mutations in a subgroup of genes that included DNMT3A and ASXL1 were associated with worse outcomes. However, clonal dynamics were highly variable and might not necessarily have predicted the response to therapy and long-term survival among individual patients. CONCLUSIONS Clonal hematopoiesis was prevalent in aplastic anemia. Some mutations were related to clinical outcomes. A highly biased set of mutations is evidence of Darwinian selection in the failed bone marrow environment. The pattern of somatic clones in individual patients over time was variable and frequently unpredictable. (Funded by Grant-in-Aid for Scientific Research and others.).

Preemptive therapy of human herpesvirus-6 encephalitis with foscarnet sodium for high-risk patients after hematopoietic SCT.

Human herpesvirus-6 (HHV-6) is a major cause of limbic encephalitis with a dismal prognosis after allogeneic hematopoietic SCT (HSCT). A prospective, multicenter study was conducted to assess the safety and efficacy of preemptive therapy with foscarnet sodium (PFA) for the prevention of HHV-6 encephalitis. Plasma HHV-6 DNA was measured thrice weekly from day 7 until day 36 after umbilical cord blood transplantation (UCBT) or HSCT from HLA-haploidentical relatives. PFA, 90 mg/kg/day, was started when HHV-6 DNA exceeded 5 × 102 copies/mL. Mild and transient adverse events were associated with PFA in 7 of 8 patients. Twelve of 15 UCBT recipients became positive for HHV-6 DNAemia, defined by greater than 1 × 102 copies/mL of HHV-6 DNA in plasma. The virus exceeded 5 × 102 copies/mL in seven patients, whereas none of the five HLA-haploidentical HSCT recipients became positive. One patient developed mild limbic encephalitis just after initial PFA administration. Preemptive PFA therapy is safe, but as HHV-6 DNAemia can abruptly develop before neutrophil engraftment in UCBT recipients, prophylactic PFA administration from day 7 or earlier after UCBT may be needed.

Favorable outcome of patients who have 13q deletion: a suggestion for revision of the WHO 'MDS-U' designation.

To characterize bone marrow failure with del(13q), we reviewed clinical records of 22 bone marrow failure patients possessing del(13q) alone or del(13q) plus other abnormalities. All del(13q) patients were diagnosed with myelodysplastic syndrome-unclassified due to the absence of apparent dysplasia. Elevated glycosylphosphatidylinositol-anchored protein-deficient blood cell percentages were detected in all 16 with del(13q) alone and 3 of 6 (50%) patients with del(13q) plus other abnormalities. All 14 patients with del(13q) alone and 2 of 5 (40%) patients with del(13q) plus other abnormalities responded to immunosuppressive therapy with 10-year overall survival rates of 83% and 67%, respectively. Only 2 patients who had abnormalities in addition to the del(13q) abnormality developed acute myeloid leukemia. Given that myelodysplastic syndrome-unclassified with del(13q) is a benign bone marrow failure subset characterized by good response to immunosuppressive therapy and a high prevalence of increased glycosylphosphatidylinositol-anchored protein-deficient cells, del(13q) should not be considered an intermediate-risk chromosomal abnormality.

Safety of pre-engraftment prophylactic foscarnet administration after allogeneic stem cell transplantation

K. Ishiyama, T. Katagiri, K. Ohata, K. Hosokawa, Y. Kondo, H. Yamazaki, A. Takami, S. Nakao. Safety of pre‐engraftment prophylactic foscarnet administration after allogeneic stem cell transplantation. Transpl Infect Dis 2011. All rights reserved

Mismatch of minor histocompatibility antigen contributes to a graft-versus-leukemia effect rather than to acute GVHD, resulting in long-term survival after HLA-identical stem cell transplantation in Japan

We determined the alleles of five polymorphic molecules including HA-1 and four adhesion molecules for 106 patients transplanted with HLA-identical stem cell grafts and investigated the association of mismatches as correlates of relapse and graft-versus-host disease (GVHD). All 106 recipients underwent stem cell transplantation (SCT) after myeloablative conditioning between 1985 and 2002. Risk status of disease at SCT was standard (n=63) and high (n=42). After SCT, 36, 49 and 33 developed acute GVHD, chronic GVHD and relapsed, respectively. Our patients relapsed at rates of 16.7 and 38.6% with one or more and without incompatibilities (P=0.013). The relapse rates of patients with CD62L, CD31 codon 563, CD31 codon 125, HA-1 and CD49b incompatibilities were 5.9, 11.8, 15.4, 16.0 and 33.3%, respectively. The frequency of acute GVHD did not differ regardless of incompatibilities. In standard-risk group, the accumulated relapse rates of 19 and 44 patients with and without minor histocompatibility antigen incompatibility were 22% and unexpectedly 66%, respectively (P=0.02). The probability of 12-year survival was 88% in the former and 66% in the latter patients (P=0.03). Our data suggest that incompatibility of CD62L, CD31 codon 563 and CD31 codon 125 contributes to a graft-versus-leukemia effect rather than to GVHD, resulting in prolonged survival after HLA-identical SCT.

Identification of an HLA class I allele closely involved in the autoantigen presentation in acquired aplastic anemia.

To identify HLA alleles closely involved in the autoantigen presentation in acquired aplastic anemia (AA), we studied the HLA allelic loss frequencies of 312 AA patients, including 43 patients with loss of heterozygosity of 6p chromosome (6pLOH). An analysis of the HLA alleles contained in the lost haplotype revealed HLA-B*40:02 to be the most frequently lost allele. When we examined 28 AA (12 6pLOH[+] and 16 6pLOH[-]) patients with HLA-B*40:02 for the presence of leukocytes lacking HLA-B4002 (B4002-) using a new monoclonal antibody specific to this allele, B4002- granulocytes were detected not only in all 6pLOH(+) patients but also in 9 (56%) of the 16 6pLOH(-) patients. Furthermore, 10 (83%) of the 12 6pLOH(+) patients possessed 1.0% to 78% B4002- granulocytes that retained the HLA-A allele on the same haplotype (B4002-A+), suggesting the frequent coexistence of granulocytes that underwent mutations restricted to HLA-B*40:02 with 6pLOH(+) (B4002-A-) granulocytes. Deep sequencing of the HLA-B*40:02 of sorted B4002-A+ granulocytes revealed various somatic mutations, such as frameshift, nonsense, and splice site mutations, in all 15 patients studied. Surprisingly, missense mutations in the α-3 domain of HLA-B*40:02 that are not involved in the antigen presentation were detected exclusively in the B4002+ granulocytes of 3 patients possessing B4002- granulocytes. The markedly high prevalence of leukocytes lacking HLA-B4002 as a result of either 6pLOH or structural gene mutations, or both, suggests that antigen presentation by hematopoietic stem/progenitor cells to cytotoxic T cells via the HLA-B allele plays a critical role in the pathogenesis of AA.

Induction of HLA‐B*40:02‐restricted T cells possessing cytotoxic and suppressive functions against haematopoietic progenitor cells from a patient with severe aplastic anaemia

Acquired aplastic anaemia (AA) is a syndrome characterized by bone marrow (BM) hypoplasia and pancytopenia. Autoimmunity to haematopoietic stem/progenitor cells (HSPCs) has been considered as a possible cause (Young et al, 2006). We recently described AA patients possessing clonal/oligoclonal haematopoiesis who had specifically lost either human leucocyte antigen (HLA) haplotype containing the HLA-A*02:06 or -B*40:02 (Katagiri et al, 2011). We surmized that selective pressure against HSPCs by specific cytotoxic T lymphocytes (CTLs) via these HLA molecules led to an escape of HSPCs without the causative HLA haplotype. We sought to determine whether HLA-B*40:02-restricted CTLs specific for HSPCs were present in patient peripheral blood or BM, and if so, how the CTLs induced BM failure as well as clonal haematopoiesis in AA patients who improved with immunosuppressive therapy (IST). Method details are provided in Data S1. This case report describes a 14-year-old male diagnosed with severe AA. Because he had no suitable donor, he received antithymocyte globulin and ciclosporin (CsA) and subsequently achieved remission (Fig 1A). His AA recurred twice but was successfully treated with CsA, indicating his haematopoiesis was CsA-dependent. He possessed a risk allele of HLA-B*40:02. His blood cells lacked one HLA haplotype containing HLA-B*40:02 (Figure S1) due to acquired copy number-neutral loss of heterozygosity of the 6p arms (6pLOH) (data not shown). To isolate HLA-B*40:02-restricted CTLs that could have triggered his BM failure (time point 4 in Fig 1A), we chose K562/B*40:02 as alternative antigen-presenting cells in place of the patient’s rare BM HSPCs because K562 has characteristics of HSPCs (Lozzio et al, 1981). The T cell line generated lysed K562/B*40:02 more intensely than K562/A*02:01, but did not lyse the non-haematopoietic cell line 293T/B*40:02 (Fig 1B). Of 84 putative clones after CTL cloning, clone A6 (CTL-A6) was selected for further analyses as a representative clone (Fig 1C). CTL-A6 did not recognize immortalized B and T lymphocytes expressing HLA-B*40:02 (Fig 1D), suggesting that mature lymphocytes might not express the target antigen. We also examined whether CTL-A6 recognized HLA-B*40:02-transduced cell lines derived from various haematological malignancies, but none of them was lysed by CTL-A6 (Fig 1E), indicating that CTL-A6 recognized antigen expressed only in K562 among the cell lines tested and might have been involved in the development of AA in the patient. Finally, we questioned whether reactivity of CTL-A6 to K562/B*40:02 would be altered with differentiation of K562. Phorbol 12-myristate 13-acetate (PMA) treatment (Witt et al, 2000) completely abrogated reactivity of CTL-A6 against K562/B*40:02 (Fig 1F) while reactivity of CTL-EH6 (Torikai et al, 2007) against simultaneously treated K562/A*02:01 expressing HLA-A*02:01-restricted minor histocompatibility antigen (mHag) HA-1H (den Haan et al, 1998) was retained (Fig 1G). Butyrate treatment produced a similar trend, but to a lesser extent (Colamonici et al, 1986). Morphological changes were apparent but their viability remained unchanged (Figure S2A). Flow cytometry revealed slight upregulation of HLA class I expression and diminished glycophorin A expression by PMA (Figure S2B). These data suggest that CTL-A6 recognized an antigen expressed in HSPC with limited maturation. We then examined the effects of CTL-A6 on HSPC growth using CD34 cells from normal BM. We identified BM from a HLA-A*02:01, B*40:02 healthy donor that was positive for HLA-B*40:02-restricted mHag encoded by SLC1A5 (Kamei et al, 2009), but negative for HA-1H. CTL-3B6 (Kamei et al, 2009) and CTL-EH6, corresponding to these mHags, were used as positive and negative controls, respectively (Fig 2A). While CD34 cells cultured with CTL-EH6 allowed expansion of immature and differentiated cells, those with CTL-3B6 showed no growth (Fig 2B). When CD34 cells were cocultured with CTL-A6, either partial growth inhibition (Experiment 1 with erythropoietin, left) or no inhibition (Experiment 2 without erythropoietin, right) was observed. After 14 d coculture in Experiment 2, cells cocultured with CTL-EH6 contained mature myeloid cells and some less mature myeloid cells (Fig 2C, upper left); the majority of those with CTL-A6 were mature myeloid cells, including some with degraded morphology (upper right); cells in the CTL-EH6 coculture contained CD13-, CD14or CD33-antigen high fractions (Fig 2C, lower left), whereas cells in the CTL-A6 coculture contained weakly positive and negative cells for CD13, CD14 or CD33; one-third of the latter contained CD8 cells most probably corresponding to residual CTL-A6 (lower right). All types of colony-forming units (CFU) were diminished by two to threefold, with the exception of mixed lineage CFU (CFU-GEMM), which showed greater than fourfold decrease, in the coculture with CTL-A6 compared with those with CTL-EH6 (Fig 2D). Altogether, correspondence

Increased glycosylphosphatidylinositol‐anchored protein‐deficient granulocytes define a benign subset of bone marrow failures in patients with trisomy 8

Trisomy 8 (+8), one of the most common chromosomal abnormalities found in patients with myelodysplastic syndromes (MDS), is occasionally seen in patients with otherwise typical aplastic anemia (AA). Although some studies have indicated that the presence of +8 is associated with the immune pathophysiology of bone marrow (BM) failure, its pathophysiology may be heterogeneous. We studied 53 patients (22 with AA and 31 with low‐risk MDS) with +8 for the presence of increased glycosylphosphatidylinositol‐anchored protein‐deficient (GPI‐AP−) cells, their response to immunosuppressive therapy (IST), and their prognosis. A significant increase in the percentage of GPI‐AP− cells was found in 14 (26%) of the 53 patients. Of the 26 patients who received IST, including nine with increased GPI‐AP− cells and 17 without increased GPI‐AP− cells, 14 (88% with increased GPI‐AP− cells and 41% without increased GPI‐AP− cells) improved. The overall and event‐free survival rates of the +8 patients with and without increased GPI‐AP− cells at 5 yr were 100% and 100% and 59% and 57%, respectively. Examining the peripheral blood for the presence of increased GPI‐AP− cells may thus be helpful for choosing the optimal treatment for +8 patients with AA or low‐risk MDS.

Individual Hematopoietic Stem Cells in Human Bone Marrow of Patients with Aplastic Anemia or Myelodysplastic Syndrome Stably Give Rise to Limited Cell Lineages

Mutation of the phosphatidylinositol N‐acetylglucosaminyltransferase subunit A (PIG‐A) gene in hematopoietic stem cells (HSCs) results in the loss of glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) on HSCs, but minimally affects their development, and thus can be used as a clonal maker of HSCs. We analyzed GPI‐APs expression on six major lineage cells in a total of 574 patients with bone marrow (BM) failure in which microenvironment itself is thought to be unaffected, including aplastic anemia (AA) or myelodysplastic syndrome (MDS). GPI‐APs‐deficient (GPI‐APs−) cells were detected in 250 patients. Whereas the GPI‐APs− cells were seen in all six lineages in a majority of patients who had higher proportion ([dbmtequ]3%) of GPI‐APs− cells, they were detected in only limited lineages in 92.9% of cases in the lower proportion (<3%) group. In all 250 cases, the same lineages of GPI‐APs− cells were detected even after 6–18‐month intervals, indicating that the GPI‐APs− cells reflect hematopoiesis maintained by a self‐renewing HSC in most of cases. The frequency of clones with limited lineages seen in mild cases of AA was similar to that in severe cases, and clones with limited lineages were seen even in two health volunteer cases. These results strongly suggest most individual HSCs produce only restricted lineages even in a steady state. While this restriction could reflect heterogeneity in the developmental potential of HSCs, we propose an alternative model in which the BM microenvironment is mosaic in supporting commitment of progenitors toward distinct lineages. Our computer simulation based on this model successfully recapitulated the observed clinical data. STEM CELLS2013;31:536–546

GPI‐anchored protein‐deficient T cells in patients with aplastic anemia and low‐risk myelodysplastic syndrome: implications for the immunopathophysiology of bone marrow failure

Glycosylphosphatidylinositol‐anchored protein‐deficient (GPI‐AP−) T cells can be detected in some patients with bone marrow failure (BMF), but the link between these cells and BMF pathophysiology remains to be elucidated. To clarify the significance of GPI‐AP− T cells in BMF, peripheral blood from 562 patients was examined for the presence of CD48−CD59−CD3+ cells using high‐resolution flow cytometry (FCM), and the GPI‐AP− T cells were characterized with regard to their phenotype and sensitivity to inhibitory molecules, including herpesvirus entry mediator (HVEM) and a myelosuppressive cytokine, TGF‐β. A multi‐lineage FCM analysis detected CD48−CD59−CD3+ T cells in 72 (12.8%) of the patients, together with GPI‐AP− myeloid cells. Unexpectedly, 12 patients (10 with aplastic anemia and 2 with myelodysplastic syndrome‐refractory anemia, 2.1%), who showed clinical features similar to those of other BMF patients with GPI‐AP− myeloid cells, such as a good response to immunosuppressive therapy, displayed 0.01–0.3% GPI‐AP− cells exclusively in T cells. The CD48−CD59− T cells consisted of predominantly effector memory (EM) and terminal effector cells, while CD48−CD59− T cells from non‐BMF patients who had received anti‐CD52 antibody only showed EM and central memory phenotypes. TGF‐β and HVEM capable of inhibiting T‐cell proliferation via its GPI‐AP CD160 ligation suppressed the in vitro proliferation of GPI‐AP+ T cells more potently than that of GPI‐AP− T cells from the same patients. The presence of GPI‐AP− T cells, as well as GPI‐AP− myeloid cells, may therefore reflect the immunopathophysiology of BMF in which cytokine‐mediated suppression of hematopoietic stem cells via GPI‐AP‐type receptors takes place.