Myeloid and lymphoid neoplasms associated with
FGFR1
abnormalities (MLN-
FGFR1
abnormalities) are rare hematologic malignancies associated with chromosome 8p11.2 abnormalities. Translocations of ...8p11.2 were detected in 10 of 17,039 (0.06%) unique patient cytogenetic studies performed at nine institutions in Japan. No inversions or insertions of 8p11.2 were detected. Among the 10 patients with 8p11.2 translocations, three patients were diagnosed with MLN-
FGFR1
abnormalities, which were confirmed by FISH analysis. Peripheral blood eosinophilia was observed in all three patients, and all progressed to AML or T-lymphoblastic lymphoma/leukemia. The prevalence of 8p11.2 translocations in clinical practice and the proportion of MLN-
FGFR1
abnormalities in patients with 8p11.2 translocations in Japan were consistent with those in previous reports from Western countries.
Mutations in the Calreticulin (CALR) gene were identified in cases of myeloproriferative neoplasms (MPNs), and various functions of the CALR mutant protein are being elucidated. On the other hand, ...few data are available on the role of CALR in the hematopoietic system. The knockout (KO) mice of Calr are impaired in expression of transcription factors necessary for cardiac development and are embryonic lethal. To clarify the role of CALR in normal hematopoiesis and MPN pathogenesis, we generated hematopoietic cell-specific Calr KO mice.
Mice carrying floxed allele targeted exons 4-7 of Calr (Calrf/+ mice) and Calr heterozygous KO mice (Calr+/− mice) (Tokuhiro et al., Sci Rep 2015) were crossed with Mx1-Cre transgenic mice and obtained mice with three genotypes; Mx1-cre;Calr+/+, Mx1-cre;Calr+/−, and Mx1-cre;Calrf/−. Floxed alleles were then deleted by intraperitoneal injection with polyinosinic: polycytidilic acid.
No differences were found in the peripheral blood (PB) leukocyte count, hemoglobin levels, or platelet count among the three genotypes of mice. The proportions of Mac1+ or Gr1+ myeloid lineage cells, B220+ B cells, and CD3+ T cells among the three groups were comparable. In the bone marrow (BM), cell pellet from Mx1-cre;Calrf/− mice appeared anemic and the proportion of CD71+/Ter119+ erythroid cells and the number of CFU-E were significantly lower in Mx1-cre;Calrf/− BM cells compared to the other two genotypes of BM cells. On the other hand, no difference was found in other mature cells such as myeloid, T, B, or CD41+ megakaryocytes (Mks) in BM. As for HSCs and progenitors, Mx1-cre;Calrf/− mice exhibited a higher proportion of MPP and GMP compared to the other two genotypes of mice. We found no difference in other progenitor compartments including long- and short-term HSCs among the three groups. In contrast to the minor effect on BM and PB cells, The spleen weight in Mx1-cre;Calrf/− mice was about 2-fold heavier than that in Mx1-cre;Calr+/+ mice. In spleens from Mx1-cre;Calrf/− mice, the border between the white and red pulp was obscured. Mks and maturing myeloid cells had markedly infiltrated into the red pulp. In FACS analysis, mature myeloid cells, erythroid cells, and Mks were significantly increased in spleens from Mx1-cre;Calrf/− mice compared to those from the other two genotypes of mice. HSCs and most types of myeloid progenitors were also increased in the spleen. Hematopoiesis in the spleen may compensate for the reduced erythropoiesis in the BM, as no anemia was seen in Mx1-cre;Calrf/− mice. No onset of leukemia or myelofibrosis was observed, and no difference in survival was seen among the three groups following observation for 2 years.
As CALR plays a role as a chaperone in the endoplasmic reticulum (ER) during protein synthesis, we searched for proteins with expression that was reduced by Calr deficiency. The quantitative differences of proteins in Mac1+/Gr1+ BM cells between Mx1-cre;Calrf/− and Mx1-cre;Calr+/+ mice were compared using proteomics analysis by 2DICAL, a shotgun proteomics analysis system (Ono et al. Cellular Proteomics 2006). We found that only a few proteins, including myeloperoxidase and CALR itself, were reduced, and conversely, many proteins were increased by the absence of CALR. List of differentially upregulated proteins higher than those in WT samples were analyzed using Metascape (http://metascape.org) to determine enriched pathways. The most enriched cluster was “protein processing in the ER”, and 19 out of 76 upregulated (>1.8-fold) proteins were included in this cluster. In real-time PCR analysis, we confirmed that ER chaperon genes (BiP, Grp94, Hsp40, Pdia3, Pdia4, Pdia6) and genes involved in ER-related degradation (Trap, Bap31, Derl1, p97) were significantly upregulated in Mx1-cre;Calrf/− myeloid cells. These observations suggested that chaperone dysfunction due to Calr deficiency is compensated by upregulation of unfolded protein response (UPR) pathway.
In summary, Calr deficiency induced erythroid hypoplasia in the BM, and induced splenomegaly and extramedullary hematopoiesis. This suggests that the amount of WT CALR expression remaining in CALR mutant cells may modify the phenotype of MPN patients. Absence of myeloperoxidase protein and upregulation of UPR pathway in myeloid cells from Calr KO mice indicates that CALR plays a significant role as a chaperone also in hematopoiesis and that CALR deficient cells are exposed to ER stress.
No relevant conflicts of interest to declare.
Myeloproliferative neoplasms are clonal hematopoietic stem cell disorders characterized by the proliferation of cells of one or more of the myeloid lineages. The three major subgroups of MPN, namely ...PV, ET, and PMF, have been shown to exhibit the same gene mutations such as JAK2, MPL, and CALR. As JAK2 mutant mice developed three type of MPNs as PV, ET, or PMF, and CALR mutant mice developed ET, both JAK2 and CALR mutants played a driver role in MPN. The frequencies of hematopoietic stem cells (HSCs) in bone marrow (BM) increased in mice with JAK2 or CALR mutation compared to WT mice, however, HSCs with JAK2 mutation or CALR mutation exhibited a reduced growth advantage relative to wild-type (WT) HSCs in competitive transplantation assay, suggesting that JAK2 mutation or CALR mutation is insuffi cient for MPN onset. In addition to the above driver mutations, mutations in epigenetic regulators have been reported in MPN patients. Among them, TET2 mutation was observed in 10-20% of MPN patients, and frequently associated with JAK2 mutation. Cooperation between mutant JAK2 and mutant TET2 compensated for impaired HSC functioning by JAK2 mutation, and developed MPNs in the serial transplantation assay.
The expected survival duration of polycythemia vera (PV) and essential thrombocythemia (ET) patients is not substantially lower than that of the general population. The current goal of therapy is to ...prevent thrombohemorrhagic events associated with PV and ET. The current first line therapy for PV is phlebotomy, hydroxyurea (HU), and aspirin, while that for ET was HU or anagrelide. The follow-up phase 3 randomized trial wherein the hematological response was evaluated in PV patients treated with ropeginterferon alfa-2b, a next-generation monopegylated IFN-α-2b, or HU, demonstrated a superior hematological effect and a lower incidence of adverse events in patients who were treated with ropeginterferon. The prognosis of primary myelofibrosis (PMF) is poorer than that of PV or ET. The only curative therapeutic option for PMF patients is allogeneic hematopoietic stem cell transplantation (HSCT). Other than HSCT options, ruxolitinib ameliorates splenomegaly and MF-associated symptoms and provides an overall survival benefit in PMF patients with intermediate-2 or high risk. Several different JAK inhibitors have been developed; however, many of them were discontinued because of toxicity concerns. Recently, promising results have been demonstrated for the effect of different JAK inhibitors as well as the drugs that directly target anemia and bone marrow fibrosis.
There are few prospective studies on patients with post-essential thrombocythemia myelofibrosis (PET-MF) and post-polycythemia vera myelofibrosis (PPV-MF). Therefore, we conducted a nationwide ...longitudinal prospective survey to clarify the clinical characteristics of these diseases. A total of 197 PET-MF and 117 PPV-MF patients diagnosed between 2012 and 2021 were analyzed. The median age at diagnosis was 70.0 years for both diseases. The time from diagnosis of ET or PV to that of MF was 9.6 and 10.4 years, respectively, with no significant difference. Patients with PPV-MF had higher hemoglobin levels and white blood cell counts than those with PET-MF, whereas those with PET-MF had higher platelet counts than those with PPV-MF. Although splenomegaly was more frequent in patients with PPV-MF at diagnosis, there was no difference in the frequency of constitutional symptoms. Ruxolitinib was the most common treatment administered to 74.6% and 83.8% of patients with PET-MF and PPV-MF, respectively. Patients with PET-MF and PPV-MF had similar prognoses, with 3-year overall survival (OS) of 0.742 in PET-MF and 0.768 in PPV-MF patients. In both diseases, leukemic transformation was the leading cause of death, followed by infection. The 3-year OS for patients with PET/PPV-MF and primary MF diagnosed during the same period was 0.754 and 0.626, respectively, with no significant difference. This survey provides real-world clinical features and prognostic data on secondary myelofibrosis in the ruxolitinib era.
Introduction: Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell lymphoma that is caused by HTLV-1. The prognosis of acute and lymphomatous variants of ATLL is poor, ranging from 2 weeks to ...>1 year. Compared to other types of malignant lymphomas, the organ infiltration is frequently observed in ATLL (Yamada et al. Leuk Lymphoma 1997). We previously reported the landscape of genetic mutations in ATLL, and showed that various mutations occurred in the TCR-NFκB pathway in more than 90% of ATLL cases (Kataoka et al. Nat Genet 2015). These somatic mutations are thought to develop ATLL in combination with viral genes such as HTLV-1 bZIP factor (HBZ). Among them, mutations in TET2, an epigenetic regulator, was observed in about 10% of ATLL cases. Higher frequencies inTET2 mutation was reported in other types of peripheral T-cell lymphoma (PTCL); it was observed in about 80% of angioimmunoblastic T-cell lymphoma (AITL) and in about 50% of PTCL, not otherwise specified. In PTCL, it has been reported that additional mutations in lymphoid progenitors derived from TET2 mutated hematopoietic stem cells cause increased cell proliferation and anti-apoptosis, leading to the disease progression. In ATLL, the role of TET2 mutation in disease progression is still unknown. In this study, we investigated the role of TET2 mutation in ATLL using mouse model and acute and lymphomatous variant ATLL cohort.
Materials and methods: As an animal model of HTLV-1 infection or ATLL, transgenic mice expressing HBZ under the control of the mouse CD4 promoter (HBZ-Tg) were generated with C57BL/6 background. Heterozygous TET2 knock-down mice (TET2KD) were generated with C57BL/6 background by gene trapping (Tang et al. Transgenic Res 2008; Shide et al. Leukemia 2012). HBZ-Tg/TET2KD compound mice (double mutant) were generated by crossing them. HBZ-Tg, TET2KD, and double mutant mice were investigated by cell counts, organ weight, FACS analysis, pathological analysis, and survival analysis. The relationship between the TET2 mutation status and the clinical feature was investigated using our acute and lymphomatous variant ATLL cohort (n=115).
Result: At 12 months, compared to wild type mouse (WT), sporadic splenomegaly and lymphadenopathy were observed in HBZ-Tg. No significant increase was observed in peripheral blood (PB) leukocyte and mononuclear cell (MNC) of BM and spleen, but an increase was observed in the estimated whole body MNC (Femur x 100/6 + spleen) (WT vs. HBZ-Tg; estimated whole body MNC (x106 cells/body), 416±162 vs. 621±147, p=0.01). In FACS analysis, the frequency of CD4+ T-cell was increased in PB, spleen, and BM (WT vs. HBZ-Tg; PB-CD4+ T-cell%, 4.9±0.9 vs. 28.2±22.8, p<0.05; spleen CD4+ T-cell%, 10.3±3.1 vs. 18.2±3.3, P<0.01; BM-CD4+ T-cell%, 1.2±0.6 vs. 2.5±1.1, p<0.05), and the estimated whole body CD4+ T-cell count was also increased (WT vs. HBZ-Tg; CD4+ T-cells (x106 cells/body) 13.6±7.6 vs. 41.9±24.4, p<0.01). In the survival analysis, compared to WT, the shortened overall survival (OS) was observed in HBZ-Tg (median survival time (MST, month), unreached vs. 11.1, p<0.01). In pathological analysis, HBZ-Tg showed increased leukocyte infiltration to various organs such as lung and liver, and the infiltrated cells were mainly composed of T-cells. In the lung, in addition to the cell infiltration, alveolar edema was observed, which was presumed to be the main cause of death.
Next, to elucidate the role of TET2 mutation in ATLL, the double mutant was analyzed. At six months, compared to HBZ-Tg, no increase was observed in the number of PB leukocyte, spleen-MNC, and BM-MNC, and also in the frequency and the number of CD4+ T-cells in PB, spleen and BM. However, in pathological and survival analysis, the double mutant showed severe cell infiltration in lung and liver and demonstrated inferior OS (median OS (month), 11.1 vs. 6.0, p<0.05). Further, the double mutant showed increased frequency of CD103 (integrin alpha E), an adhesion molecule, expressing cells (CD4+CD103+% in spleen; 6.7±1.0 vs. 10.9±1.9, p<0.05). In the acute and lymphomatous variant ATLL cohort analysis, genetic and clinical investigation revealed that organ infiltration detectable by imaging studies was frequently observed in TET2 mutated patients (WT-Pt (n=100) vs. TET2 mutated-Pt (n=15); extra nodular lesion, 78/100 vs. 14/15).
Conclusion: In both mice model and human cohort, TET2 mutation exacerbated organ infiltration of ATLL cells.
No relevant conflicts of interest to declare.
CALR exon9 frameshift mutations function as driver mutations in essential thrombocythemia (ET) and primary myelofibrosis patients with non-mutated JAK2 or MPL. The mutations augment signal transducer ...and activator of transcription activity in the presence of MPL, induce increased cell proliferation and growth factor independence in cell lines, and cause ET-like myeloproliferative neoplasms (MPN) in mice.
In tumor cells bearing the CALR mutation, mutant CALR protein expression occurs while wild-type (WT) CALR expression is decreased by half. Although the biological activity of mutant CALR has been elucidated in detail, the significance of CALR haploinsufficiency is unclear. The purpose of this study was therefore to clarify the influence of CALR haploinsufficiency on hematopoiesis in normal and CALR-mutated MPN in mice.
First, we analyzed the effect of CALR haploinsufficiency on hematopoiesis using CALR heterozygous knockout (CALR-hKO) mice (Tokuhiro et al. Sci Rep. 2015). Blood cell counts, liver and spleen weight, histology, cell fraction in bone marrow (BM) and spleen, and survival of CALR-hKO mice were all equivalent to that observed in WT mice. In the analysis of progenitor cells by fluorescence-activated cell sorting and colony formation assays, no difference was observed in the amount of progenitor cells and in colony replating capacity between WT mice and CALR-hKO mice. Interestingly, in competitive serial transplantation experiments using whole BM cells in primary and secondary transplanted mice, CALR-hKO cells showed higher levels of chimerism than WT cells.
Next, the effect of CALR haploinsufficiency on hematopoiesis in mutant CALR-del52 overexpressing mice was analyzed. We compared three groups of mice, WT mice, CALR-del52 transgenic (TG) mice (Shide et al. Leukemia 2017) and CALR-del52 TG/CALR-hKO double-mutant (TG-hKO) mice. Both TG mice and TG-hKO mice developed ET-like MPN. Compared to WT mice, increases in megakaryocytes, platelets and hematopoietic progenitor cells (including HSCs) were observed in these mice. However, no differences were observed between TG mice and TG-hKO mice.
Finally, 4000 LSK cells sorted from WT mice, TG mice, and TG-hKO mice (B6-Ly5.2) and B6-Ly5.1 competitor cells (1 × 106 WT BM cells) were mixed and injected into lethally irradiated B6-Ly5.1 recipient mice, and the percent chimerism of donor cells was followed for 1 year after transplantation. From 12 weeks after transplantation, the chimerism of TG cells was significantly lower than that of control WT cells, suggesting that the CALR mutation has a negative influence on clonal expansion of HSCs. The recipient mice transplanted with TG LSK cells showed very mild thrombocythemia. On the other hand, chimerism in TG-hKO cells was significantly higher than that in control WT cells up to 12 weeks after transplantation. Chimerism at 20 weeks after transplantation was equivalent to that in control WT cells, and the recipient mice transplanted with TG-hKO LSK cells showed severe thrombocythemia. These findings show that CALR haploinsufficiency compensates for HSCs functioning, which has been impaired by the CALR mutation, enhancing the ability of HSCs to develop ET.
Mice experiments have showed that HSCs with the JAK2V617F mutation are fragile, and it is considered that mutations in DNMT3A or TET2 often occurred prior to mutations in JAK2 to compensate for the defect in self renewal capacity. Conversely, since the CALR mutation is not typically preceded by any mutations, it is considered that the CALR mutation may not require any preceding mutations. Our results showed that, like JAK2 V617F mutants, overexpression of the CALR-del52 mutant impairs HSC functioning. Furthermore, we found that CALR haploinsufficiency restores the functions of impaired HSCs to the same extent as that found in WT HSCs. When an HSC acquires the CALR mutation, “defect” and “recovery” states thus occur simultaneously in the cell. This finding may explain why mutant CALR clones expand without needing to undergo any preceding mutation.
No relevant conflicts of interest to declare.
Myelofibrosis (MF) associated with myeloproliferative neoplasms (MPN) has been considered to be a reactive phenomenon caused by mesenchymal stromal cells (MSCs) stimulated by cytokines such as TGFb-1 ...overproduced by neoplastic megakaryocytes (MKs) and platelets. TGFb-1 stimulates non-neoplastic mesenchymal cells to produce collagen and fibronectin and to induces bone marrow (BM) fibrosis. However, the involvement of neoplastic fibrocyte in MF has recently been reported (Verstovsek et al. JEM 2016), and among blood cells, monocytes in particular are considered to be the main source of neoplastic fibrocytes. In this study, we assesed the role of neoplastic fibrocytes using a mouse model of MPN induced by Jak2V617F (Shide et al. Leukemia 2008).
First, the distribution of neoplastic fibrocyte in the BM of Jak2V617F transgenic (TG) mice was examined. We transplanted wild-type (WT) or Jak2V617F TG cells (B6-CD45.2), together with WT BM cells (B6-CD45.1) into irradiated WT recipient mice (B6-CD45.1). Only recipient mice transplanted with a mixture of Jak2V617F cells and WT cells developed BM fibrosis. In immunofluorescent staining of fibrotic BM, cells expressing the fibrocyte marker CD45/Collagen-1(Col-1) were observed much more than cells expressing the fibroblast marker CD90(usually positive for MSCs)/Col-1. As for CD45/Col-1 positive cells, cells expressing CD45.2/Col-1 were much more than cells expressing CD45.1/Col-1, clearly indicating that these cells were derived from Jak2V617F mutant blood cells. On the other hand, in the BM of recipient mice transplanted with control WT cells, few cells expressing CD45/Col-1 or CD90/Col-1 were present. To examine the differentiation ability of Jak2V617F blood cells to fibrocytes directly, peripheral blood (PB) mononuclear cells (MNC) of Jak2V617F mice or WT mice were cultured in vitro. After 5 days of culture, PB MNCs from Jak2V617F mice differentiated into mature fibrocytes exhibiting a long spindle shape with Col-1 expression. On the other hand, there were very few fibrocytes differentiated from PB MNC from WT mice.
Next, we depleted monocytes, the main source of fibrocytes, and observed its effects on BM fibrosis in vivo. Jak2V617F TG mice were mated with CD11b-diphtheria toxin receptor (DTR) TG mice (Duffield et al. JCI 2005) to obtain Jak2V617F/CD11b-DTR double TG mice. Mice transplanted with BM cells from Jak2V617F/CD11b-DTR double TG mice (hereinafter called Jak2V617F/CD11b-DTR mice) exhibit leukocytosis, thrombocytosis, anemia, splenomegaly, and BM fibrosis with increased megakaryocytes. Jak2V617F/CD11b-DTR mice was administered diphtheria toxin (DT) intraperitoneally to deplete monocytes. One day after DT administration, the number of PB monocytes (CD11b+/F4/80+) drastically decreased in Jak2V617F/CD11b-DTR mice, and the reduction of monocyte was maintained by every-other-day DT administration. After 8 weeks DT treatment, mice were sacrificed and analyzed. As a control group, Jak2V617F/CD11b-DTR mice treated with PBS were examined. DT treatment drastically decreased the number of neoplastic fibrocytes expressing CD45.2/Col-1 in BM and spleen of Jak2V617F/CD11b-DTR mice compared with control mice treated with PBS. Consistently, reticulin fibers were eliminated almost completely and collagen fibers almost fully disappeared in BM, which led to a reversal of the decrease in BM cellularity, although the number of MKs was not affected. Similar findings were observed in the spleen, although not completely. Plasma TGF-b1 level were about 2-fold higher in Jak2V617F/CD11b-DTR mice than in WT mice. Neoplastic monocyte depletion significantly decreased TGF-b1 level. Since MK numbers did not change, this indicates that fibrocytes are one of the main sources of TGF-b1. In other features of MF in Jak2V617F/CD11b-DTR mice, splenomegaly was ameliorated by DT treatment. Microscopic analysis revealed an improvement in the damaged spleen architecture and the disappearance of splenic fibrosis.
In summary, most collagen-producing cells in BM were neoplastic fibrocytes in Jak2V617F-induced MPN, indicating that neoplastic fibrocytes played an essential role and mesenchymal fibroblasts had a minor contribution in fibrosis in MPN. Depletion of neoplastic monocytes also improved splenomegaly as well as BM fibrosis in mice, and this cell fraction could be a promising therapeutic target.
No relevant conflicts of interest to declare.
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