T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy with diverse oncogenic drivers, the relative contributions of which remain obscure. Enforced expression of select oncogenes is ...sufficient to generate T-ALL in mice, however, mouse models are limited in their value for understanding human disease. We recently developed the first “synthetic” model of human T-ALL by lentiviral transduction of normal human CD34+ cord blood progenitors with a combination of four known T-ALL oncogenes: NOTCH1, LMO2, TAL1, and BMI1 (NLTB). These cells expand robustly in culture and produce aggressive, serially transplantable T-ALL in immunodeficient mice. Additionally, we found that LTB alone fails to perform in vitro or in vivo, highlighting an essential role of NOTCH1. To determine the minimal complement of oncogenes required to generate de novo human T-ALL, we executed a “leave-one-out” and “leave-two-out” strategy. We scored the following oncogene combinations in vitro and in vivo: NLB, NLT, NTB, NL, NT, and NB. We found that the various oncogene combinations yielded a spectrum of aberrant phenotypes affecting cell differentiation and proliferation, and a subset produced aggressive leukemias in mice. We also performed RNA-seq on non-leukemogenic and preleukemic cell populations, and fully transformed leukemic cells to define gene expression programs necessary for cellular transformation. Our approach allows us to attribute specific gene programs and cellular phenotypes to each oncogene individually and in combination. Our synthetic approach is flexible, reproducible, and experimentally tractable, allowing functional testing of individual genetic variants and can also serve as a customizable platform for testing of targeted therapeutics.
Background Patients treated with immunosuppressive drugs for autoimmune diseases may suffer “other iatrogenic immunodeficiency-associated lymphoproliferative disorders” (OIIA-LPD). Some OIIA-LPDs ...regress after drug withdrawal but others need cytotoxic chemotherapy. However, the factors associated with a response to drug cessation remain unknown.
Methods We collected clinical data on OIIA-LPD diagnosed between 2009 and 2018 at the University of Tsukuba Hospital and Toranomon Hospital in Japan. Clinicopathological features were retrospectively analyzed. The probability of overall survival (OS) and progression-free survival (PFS) was calculated using the Kaplan-Meier method. OS was defined as the time between the date of diagnosis of OIIA-LPD and the date of death or last follow-up. PFS was defined as the time from the date of diagnosis of OIIA-LPD to the date of commencing chemotherapy or the date of death or last follow up.
Results Fifty-four cases of OIIA-LPD were identified, of which 25 were diagnosed at the University of Tsukuba Hospital and 29 at Toranomon Hospital. The male to female ratio was 1:3.5 and the median age at diagnosis was 70 years (range, 35-85). Thirty-four patients (63%) had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Most patients (n=50 93%) had rheumatoid arthritis (RA). The remaining 4 patients had systemic lupus erythematosus (SLE), myasthenia gravis (MG), polymyalgia rheumatica (PMR), and polymyositis (PM), respectively. Methotrexate (MTX), tacrolimus, and biological agents were used in 49, 17, and 16 cases, respectively. Histological diagnoses of OIIA-LPD type were diffuse large B-cell lymphoma (DLBCL) (n=24), composite lymphoma of DLBCL and follicular lymphoma (FL) (n=1), Hodgkin lymphoma (HL) (n=18), MALT lymphoma (n=2), peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) (n=2), angioimmunoblastic T-cell lymphoma (AITL) (n=1), B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and classic HL (n=1), extranodal NK/T-cell lymphoma (ENKTL) (n=1), Burkitt lymphoma (n=1), and not specified (n=3). Epstein-Barr virus (EBV) was detected in 61% of cases tested (31/51) by in situ hybridization for EBER. At diagnosis of OIIA-LPD, 44 cases (81%) were under MTX treatment. MTX was discontinued in all of these, after which spontaneous regression was observed in 27 (61%). There was no clinical response in 5 patients (11%) within 2 months of MTX cessation, and 12 patients (27%) received chemotherapy without confirming whether stopping MTX would have been sufficient intervention. In 27 cases with spontaneous regression after cessation of MTX, 18 achieved complete remission without chemotherapy, but 9 needed chemotherapy due to re-initiation of LPD. Regarding the histological diagnosis, spontaneous regression was observed in OIIA-LPD patients with DLBCL in 12 of 24 cases (50%) and those with HL in 8 of 18 (44%). After achieving spontaneous regression, patients with DLBCL (10/12 cases, 83%) did not require any form of chemotherapy more often than those with HL (4/8 cases, 50%). Neither EBV positivity nor histology were associated with spontaneous regression. With a median follow-up of 26.1 months (range, 1-120.7), 2-year OS and PFS was 91.9% and 30.1%, respectively. The 2-year PFS of DLBCL and HL was 33.1% and 17.5%, respectively (p=0.533). Only four patients died due to the progression of OIIA-LPD.
Conclusion Fifty-four cases of OIIA-LPD were retrospectively analyzed. Spontaneous regression after MTX discontinuation was observed in approximately 60% of these patients. No factors associated with response to drug cessation were associated with histological features or EBV positivity, but patients with DLBCL remained in complete remission more frequently than those with HL. Despite the fact that 2-year PFS is low, our retrospective analyses revealed favorable OS of OIIA-LPD because chemotherapy resulted in a good response regardless of whether MTX discontinuation was effective.
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No relevant conflicts of interest to declare.
Disseminated intravascular coagulation (DIC) is a lethal complication in patients with hematologic malignancies (HMs). DIC can be induced by the HM itself, but also by HM-associated secondary ...infection; however, whether difference of triggering factor impacts the outcome of DIC in HM patients remains unknown. The objective of this study is to clarify the difference between HM-induced DIC and infection-induced DIC in HM patients regarding treatment response and prognosis.
HM-induced DIC (158 episodes) and infection-induced DIC in HM patients (83 episodes) from a single center were retrospectively analyzed. Recombinant human thrombomodulin (rhTM) was administered in 149 episodes, while the remaining received conventional therapies.
In HM-induced DIC, improvement by day 7 was 46% (95% confidence interval CI, 38–54), and rhTM enhanced the improvement (hazard ratio HR, 1.7; 95% CI, 1.1–2.4). In contrast, improvement of infection-induced DIC was significantly worse (29%; 95% CI, 20–39 on day 7), and this was not influenced by rhTM (HR, 1.0; 95% CI, 0.50–2.2). Thirty-day survival in HM-induced DIC and infection-induced DIC was 87% (95% CI, 81–92) and 53% (95% CI, 42–63), respectively, and was not affected by treatment. A DIC score (Japanese Ministry of Health and Welfare criteria) of ≥5 was a predictor of worse survival in both types of DIC (HR, 2.5; 95% CI, 1.5–3.9).
This study showed the inadequacy of current therapeutic strategies for secondary infection-induced DIC, the prognosis of which was significantly worse than HM-induced DIC, and the limited efficacy of rhTM only in the improvement of HM-induced DIC.
•Triggering factors of DIC in hematological malignancy (HM) had impact on the outcome.•Survival rate of infection-induced DIC in HM was almost half that of HM-induced DIC.•Recombinant human thrombomodulin (rhTM) enhanced recovery only from HM-induced DIC.•Difference of the survival according to the treatment modalities was not observed.
Background:
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of blood cancer that can arise in both children and adults. Numerous studies have explored the effects of putative T-ALL ...oncogenes in mouse models and have contributed significantly to our understanding of disease pathogenesis. Nonetheless, it is clear there are important differences between mouse and human cells, particularly with respect to cellular transformation, and additional work is therefore needed to generate more accurate models of human disease. We sought here to create human T-ALL in the lab from normal CB progenitors by lentiviral transduction with a combination of known T-ALL oncogenes.
Methods:
Human CD34+ hematopoietic progenitor cells were isolated from pooled cord blood by magnetic bead/flow cytometric sorting (MACS/FACS). Sorted cells were then transduced by lentiviral vectors encoding a combination of four known T-ALL oncogenes including activated NOTCH1. NOTCH1 virus was marked with a GFP reporter (N1/GFP) while the other three accessory oncogenes were marked with a Cherry reporter (3xOnc/Cherry). Transduced cells were cultured on OP9-DL1 stromal feeders briefly prior to transplantation into NOD/SCID-IL2Rg-null (NSG) mice to assess leukemogenesis, or for longer periods to study their behavior in vitro.
Results:
Initial transduction efficiencies were typically 3-5% for each virus with 1-2% doubly-transduced N1/GFP+, 3xOnc/Cherry+ cells (hereafter referred as 4xOnc cells). After 28 days culture in vitro, the 4xOnc population reproducibly expanded and outcompeted singly- and non-transduced populations, accounting for more than 70% of cells in mixed cultures. By absolute cell counts, non-transduced cells stopped expanding within the first few weeks; however, 4xOnc cells kept expanding even after 6 weeks of culture. To test leukemogenesis in vivo, CD45+ cells were FACS sorted after 10 days of culture on OP9-DL1 feeders (including doubly-, singly-, and non-transduced populations) and injected intrahepatically into NSG neonates. Engraftment of human cells was followed monthly by flow cytometry of peripheral blood. Engraftment of GFP+ Cherry+ 4xOnc cells was first detected 2 months after transplantation whereas no engraftment of singly- or non-transduced cells was detected. The level of engraftment was below 5% and did not increase substantially even after 6 months following transplantation. At day 203 post-transplant, the primary recipient was sacrificed and 4xOnc cells were recovered from bone marrow, spleen and thymus where the levels of engraftment were approximately 10%. 4xOnc cells from the primary recipient were then serially transplanted into secondary recipients. Engraftment of 4xOnc cells in secondary recipients was observed 5 weeks after transplant. Unlike the primary recipient, however, the percentage of 4xOnc cells in the peripheral blood of secondary recipients gradually increased and these animals developed clinically morbid disease by 20 weeks post-transplant. At the time of necropsy, splenomegaly, lymphadenopathy, and enlarged thymus were observed and the bone marrow contained 80-90% 4xOnc cells. By flow cytometric analyses, 4xOnc cells expressed CD2, CD3, CD7, CD38, and TdT supporting acute T-cell leukemia. Also, TCR gamma clonality assay was performed with genomic DNA from 4xOnc cells from secondary recipients and revealed of 5-7 distinct clonal populations. These in vitro and in vivo findings were observed with multiple experimental replicates and with different pools of cord blood.
Conclusion:
Our in vitro and in vivo results suggest that NOTCH1, in combination with 3 accessory oncogenes are sufficient to transform normal human blood cells into clonal T-ALL-like malignant cells. Although we cannot exclude the possibility of the spontaneous acquisition of additional co-operating genetic or epigenetic abnormalities, this model provides a significant step forward to reveal the mechanisms involved in human T-ALL pathogenesis.
No relevant conflicts of interest to declare.
Objective Graft failure (GF) is a life-threatening complication of hematopoietic stem cell transplantation (HSCT). A standardized conditioning regimen and an appropriate graft source of salvage HSCT ...for GF have not yet been established. Some case series have shown good hematopoietic recoveries after salvage HSCT using a short-term reduced-intensity preparative regimen consisting of fludarabine (30-90 mg/m2), cyclophosphamide (2 g/m2), and total-body irradiation (2 Gy). However, the dose of fludarabine has varied in these reports based on the clinical condition of the patients, resulting in very limited experiences with each dose of fludarabine. Methods We retrospectively analyzed 10 patients who developed GF after allogeneic HSCT and underwent salvage cord blood transplantation (CBT) using the above-mentioned conditioning regimen with a fixed dose (90 mg/m2) of fludarabine. Results Eight patients (80.0%) achieved neutrophil engraftment within 30 days from salvage HSCT with a median of 21 (range, 17-23) days. The 1-year overall survival (OS) rate after the salvage HSCT was 50.0%, and the median OS was 281 (range, 23-1,638) days. Cumulative incidences of non-relapse mortality and relapse at 1 year were 50.0% and 10.0%, respectively. Conclusion CBT using this short-term reduced-intensity conditioning regimen may be a promising salvage therapy for GF.
Follicular lymphoma (FL) is an indolent, but incurable malignancy as most patients eventually experience progressive disease. We hypothesized that clonal heterogeneity and patient-specific immune ...responses would contribute to variable clinical outcomes and that understanding the complexity of the entire tumor “ecosystem” would allow us to better match patients with specific types of tumor- and immune-targeted therapies.
In this study, we performed 38-dimensional single-cell phenotyping by mass cytometry (CyTOF) to simultaneously characterize both the substructure of malignant B cell populations as well as the T cell microenvironment in a cohort of 77 diagnostic patient FL biopsies and 35 benign reactive LN (rLN) biopsies.
We first applied the t-distributed Stochastic Neighbour Embedding (t-SNE) algorithm to explore intra- and inter- tumoral heterogeneity among malignant B cell populations. t-SNE mapping of individual samples showed that more than a third of FL samples contain at least two phenotypically distinct tumor subpopulations, supporting the notion of multi-clonal tumor architectures presumably due to ongoing clonal evolution. Batched analysis combining all 77 FL cases together with 35 rLN samples revealed two distinct tumor subtypes comprising about 25% (type “A”) and 10% (type “B”) of total FL samples, respectively, with individual tumors within each subtype showing highly similar and partially overlapping phenotypes. Mapping the same data using Uniform Manifold Approximation and Projection (UMAP), a dimensional reduction algorithm similar to t-SNE but preserves global structure more accurately, revealed that type A tumors localized in close proximity to normal germinal center (GC) B cells, thus fulfilling conventional expectations as to the histogenesis of FL. In contrast, type B tumors localized more closely to pre-GC B cells, implying the existence of an alternate histogenic path in FL. Importantly, we also performed single-cell RNA-Seq on a subset of FL cases which independently confirmed the type A vs type B distinction in whole transcriptomic space.
We next analyzed matching T cell data using a modified Statistical Scaffold algorithm in order to place distinct subsets in context with conventionally defined normal T cell populations. Clustering analysis using multi-layer phenograph performed on T cells from all FL and rLN samples combined yielded hundreds of small, but phenotypically distinct populations that were then annotated according to the nearest conventionally defined T cell subset. These imputed designations were used as features to perform hierarchical clustering of samples which revealed 3 major clusters. Cluster1 was characterized by mostly naive T cell populations and contained the majority of rLN samples. Cluster2 was characterized by more differentiated effector T cell populations and was dominated by FL samples. Samples within Cluster2 could be further divided into Tfh, Treg and Th1-rich subgroups. Cluster3 was characterized by a diverse T cell environment including naive, memory and differentiated effector subsets and contained a mixture of rLN and FL samples. Integrative analysis correlating B- and T- cell features revealed type B FL tumors were associated with a Tfh-rich immune landscape.
Taken together, these data reveal pervasive phenotypic heterogeneity in both malignant and immune cell compartments of patient FL samples and suggest that defining tumoral subtypes as well as the status of the local immune response within individual samples will support more refined diagnostic classification and highlight functional interactions most amenable to therapeutic targeting.
Gascoyne:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Celgene: Consultancy, Honoraria; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Janssen: Research Funding. Steidl:Juno Therapeutics: Consultancy; Seattle Genetics: Consultancy; Nanostring: Patents & Royalties: patent holding; Bristol-Myers Squibb: Research Funding; Tioma: Research Funding; Roche: Consultancy.
Article Note: Ainsleigh J. Hill and Chaoran Zhang contributed equally as first authors. Andrew P. Weng and Jeffrey W. Craig contributed equally as senior authors. Funding information Terry Fox ...Research Institute Byline: Ainsleigh J. Hill, Chaoran Zhang, Manabu Kusakabe, Kevin Gowing, Xuehai Wang, Ryan R. Brinkman, Andrew P. Weng, Jeffrey W. Craig
In follicular lymphoma (FL), clinical progression/histologic transformation occurs as a result of emergence of biologically aggressive malignant clones that escape the immune response. Whether such ...clones are preexisting or arise as a consequence of spontaneous or therapy-induced DNA mutation/genomic instability remains an open question; however, it has become increasingly clear that it will be important to understand the multi-clonal structure of tumors in order to treat them more effectively. As well, tumor-infiltrating immune cells represent a complex and heterogeneous population and are hypothesized to either help or hinder tumor progression. Several studies have searched for prognostic value in various infiltrating immune cell subsets, but results have been inconsistent, likely in part because most studies focus only on a few, incompletely characterized subsets at a time. Furthermore, the potential for co-variance between malignant subclones and the complement of infiltrating immune cells has not yet been explored. We would propose that understanding the entire tumor "ecosystem" in FL is needed to make further strides in predicting biological behavior and ultimately in guiding selection of the most appropriate of available rational therapies.
In this study, we sought to use mass cytometry (CyTOF) to characterize both the clonal substructure of malignant populations and the infiltrating immune repertoire with unparalleled breadth, yet at single-cell resolution. We designed and optimized a 2-tube, 40-parameter CyTOF panel in which one tube is dedicated to characterizing population substructure among malignant B-cells and the other to profiling the composition of the infiltrating T-cell repertoire. We accessed viably frozen single cell suspensions from excess lymph node biopsy material remaining after diagnostic flow cytometry have been prospectively banked for the past 2 decades.
We stained 12 FL, 23 diffuse large B-cell lymphoma (DLBCL), and 5 reactive lymph node (rLN) samples using the B-cell marker tube, acquired CyTOF data, and performed analyses using tSNE and Complicity mapping algorithms, which led us to make the following observations: First, over half of FL samples contain at least two phenotypically distinct tumor subclones. In contrast, very few of the DLBCL samples that we have examined thus far exhibit definitive phenotypic subclones. Lower intra-tumoral heterogeneity in DLBCL may imply that these tumors represent outgrowth of a highly evolved, dominant clone as compared to a less evolved collection of "untested" clones in FL. Second, tSNE mapping of FL tumors revealed two distinct subtypes, one in which the individual tumors showed highly similar and partially overlapping phenotypes that localized in proximity to normal germinal center B cells ("GC" subtype), and the other which was composed of more phenotypically heterogeneous tumors that were localized more distantly from normal germinal center B cells ("non-GC" subtype).
We also stained 6 FL and 5 rLN samples using the T-cell marker tube, acquired CyTOF data, and performed analysis using tSNE and Scaffold mapping algorithms. We found the relative abundance of T-cells in FL samples was comparable to rLN (36.1% vs. 37.4% of total cells, p=0.90); however, most of the FL samples showed elevated CTL fractions (20.1% vs. 13.0% of total T-cells, p=0.02). Using Scaffold mapping, we were able to resolve a dramatic diversity of T and T/NK cell subsets, each of whose abundance varied substantially from one sample to the next. For example, the majority of CTL cells in rLN samples exhibited a relatively homogeneous, naïve-like phenotype, while CTL cells in FL typically contained a heterogeneous mixture of activated and exhausted subsets. The T-helper compartment exhibited a similar picture with relative population homogeneity within rLN samples, contrasting with heterogeneity within FL samples. Composite analysis correlating B- and T-cell features within the same FL specimen revealed that loss of HLA-DR and CD124 on malignant cells was correlated with CTL exhaustion, suggesting a potential immune escape mechanism.
Our findings illustrate that novel information is revealed by 40-dimensional CyTOF analysis and provide insight into important, but understudied aspects of intra-tumoral heterogeneity and variable host immune response in lymphoma.
Scott:Janssen: Consultancy; Celgene: Consultancy; BC Cancer Agency: Patents & Royalties: Inventor on a patent licensed to NanoString Technologies; Roche: Honoraria.
Backgrounds Intravascular large B cell lymphoma (IVLBCL) is a rare subtype of extranodal diffuse large B-cell lymphoma (DLBCL) that features lymphoma cells lodged in lumina of small vessels. Because ...of the lack of mass formation, diagnosis by tumor biopsy is often difficult, and thus, cell-free DNA (cfDNA) analysis might be useful for assisting diagnosis of IVLBCL. In this study, we first aimed at investigating the prevalence of mutations in genes involved in the B-cell receptor, Toll-like receptor/interleukin-1 receptor, and nuclear factor kappa B pathways. We then evaluated the liquid biopsies for detection of hot-spot mutations by droplet digital polymerase chain reaction (ddPCR).
Patients and Methods Twenty-seven IVLBCL patients whose archived samples were available were included in this study. The patients were diagnosed as IVLBCL from January 2005 to May 2017 in University of Tsukuba Hospital, JA Toride Medical Center, and Kameda Medical Center. The study was approved by the review board in each institution, and conducted according to the Declaration of Helsinki. Tissue-derived DNA (tdDNA) was extracted from formalin-fixed paraffin-embedded specimens and/or cryo-preserved bone marrow samples, in which the lymphoma cell infiltration had been pathologically confirmed. cfDNA was extracted from 1 ml of serum or plasma obtained prior to chemotherapy. We performed targeted sequence for 8 genes (B2M, BTG2, CARD11, CD79B, MYD88, PIM1, PRDM1, and TNFAIP3) in paired tdDNA and cfDNA from 9 IVLBCL subjects by Ion Torrent Personal Genome Machine (Thermo Fisher Scientific). MYD88 L265P (c.794T>C) mutational analysis was performed by ddPCR assays (BioRad) for 46 samples in total collected from 27 subjects. Statistical analysis was performed with EZR version 1.3.2. Paired t-test was used to compare variant allele frequencies (VAFs) in paired samples.
Results All cases were categorized as the non-GCB by Hans classification. Random skin biopsies (RSB) and bone marrow biopsies (BMB) were performed in 21 and 27 patients. The diagnostic yield of RSB was 81.0% (17 of 21). Initial BMB detected lymphoma cells in 48.1% (13/27) patients, but repeated BMB increased successful detection up to 66.7% (18/27). tdDNA was available in 26 out of 27 subjects, and in 9 of which paired cfDNA was also available (Figure 1). In one subject, only cfDNA but not tdDNA was available. Targeted sequencing for 8 genes demonstrated at least one mutation in CD79B, MYD88, PIM1, or PRDM1 in both tdDNA and cfDNA, or only in cfDNA, in 8 of 9 subjects (Figure 2). We identified 3 missense CD79B mutations in 6 (66.7%; Y196C in 2, Y196H in 3, and L199P in 1). All MYD88 mutations were L265P, which were found in 5 of 9 (55.6%). Together, either Y196 CD79B or L265P MYD88 mutation was found in 7 of 9 (77.8%). PIM1 mutations and PRDM1 mutations were seen each in 3 (33.3%). VAFs were higher in cfDNA than in tdDNA (mean, 45.6% vs. 3.7%; P=0.0006, Figure 3). Four mutations in 3 patients were detected only in cfDNA. On the other hand, all variants detected in tdDNA were confirmed in cfDNA. ddPCR assay for the L265P MYD88 mutation with all 46 samples including tdDNA and cfDNA revealed the L265P MYD88 mutation in 24/46 (52.2%) samples from 16/27 (59.3%) IVLBCL patients. Furthermore, sequential analysis of cfDNA was performed in one of the MYD88 -mutated patients. The L265P MYD88 mutation was detected (VAF=30%) in cfDNA collected 17 weeks before the diagnosis of IVLBCL, whereas the mutation was not detected in DNA derived from the bone marrow sample that was collected at the same timing and resulted in the negative pathological finding.
Conclusion Mutations in MYD88 and CD79B are frequent in IVLBCL . Targeted sequencing suggested that tumor cell-derived DNA is abundant in serum/plasma. Liquid biopsy to detect L265P MYD88 and Y196 CD79B may provide a powerful tool for approaching diagnosis of IVLBCL.
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Chiba:Nippon Shinyaku: Honoraria, Research Funding.
c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the activated protein-1 super family of basic leucine zipper proteins. Despite its overexpression ...in hematologic malignancies, the physiologic roles c-Maf plays in normal hematopoiesis have been largely unexplored. On a C57BL/6J background, c-Maf−/− embryos succumbed from severe erythropenia between embryonic day (E) 15 and E18. Flow cytometric analysis of fetal liver cells showed that the mature erythroid compartments were significantly reduced in c-Maf−/− embryos compared with c-Maf+/+ littermates. Interestingly, the CFU assay indicated there was no significant difference between c-Maf+/+ and c-Maf−/− fetal liver cells in erythroid colony counts. This result indicated that impaired definitive erythropoiesis in c-Maf−/− embryos is because of a non–cell-autonomous effect, suggesting a defective erythropoietic microenvironment in the fetal liver. As expected, the number of erythroblasts surrounding the macrophages in erythroblastic islands was significantly reduced in c-Maf−/− embryos. Moreover, decreased expression of VCAM-1 was observed in c-Maf−/− fetal liver macrophages. In conclusion, these results strongly suggest that c-Maf is crucial for definitive erythropoiesis in fetal liver, playing an important role in macrophages that constitute erythroblastic islands.
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