Levels of Osteopontin (SPP1), Osteonectin (SPARC) and Biglycan (BGN) in Acute Myeloid Leukemia Bone Marrow Biopsies Post-Induction Therapy Define the Status of Osteogenic Niche and Show Inverse Correlation with Therapeutic Response

Background: Acute Myeloid Leukemia (AML) has a five-year survival rate of 25% and its high mortality is linked to poor response to treatment and relapse. Our understanding of the molecular mechanisms controlling relapse and AML progression is limited. Animal models indicate that AML cells significantly modulate their bone marrow microenvironment inducing gradual loss of endosteal and vascular niches, both playing critical roles in support and maintenance of normal hematopoiesis. The goal of this study was to determine microenvironmental factors driving the gradual retraction of endosteal and vascular niches directly in the AML core bone marrow biopsies, and assess the treatment effect on hematopoietic and non-hematopoietic cells. Methods: Transcriptomics and histopathologic evaluations of matched human AML core bone marrow biopsies obtained at diagnosis (n=12) and day 14 post-induction therapy (n=12) with daunorubicin and cytarabine (7+3) were performed. Based on post-treatment frequency of blasts in the AML bone marrow aspirate, patients were classified as responders (<5% blasts) or non-responders (> 5% blasts). Three of 6 responders (3 men, 3 women, average age 59 yrs) had normal karyotype, and three of 6 non-responders (1 man, 5 women, average age 52.6 yrs), had normal karyotype. RNA was isolated from the core bone marrow biopsies and subjected to Clariom D Human Affymetrix arrays. Transcriptomics data were analyzed using Affymetrix Transcriptome Analysis Console with LIMMA R package and Gene Set Enrichment Analysis (GSEA). H&E stained bone marrow biopsy slides were subjected to blinded histopathological assessment. Results: Transcriptomic data analysis of responder vs. non-responder samples at diagnosis indicated significant loss of transcripts associated with heme metabolism (HBB, HBD, GYPE, CA1) suggesting decrease in frequency of erythroid progenitors (Fig.A). Trends of decreased frequency of erythroid progenitors were noted in both bone marrow biopsies and aspirates of diagnostic non-responder samples (Fig.B). Decreased frequency of lymphoid cells was also noted (Fig.B). Interestingly, while post-treatment we noted a relative increase in frequencies of lymphoid cells in both responder and non-responder samples, the increase was more prominent in responders (Fig.B). Trilineage hematopoiesis appeared affected more in diagnostic and post-treatment responder samples. Transcriptome analyses of diagnostic vs. post-treatment responder samples indicated significant increase in transcripts associated with activity within endosteal niche (SPARC, SPP1, DCN, VCAN, BGN) and significant loss of transcripts associated with DNA replication (TOP2, HELLS, E2F8) (Fig.C), the latter was consistent with treatment-related loss of cellularity. Only modest increase in SPARC, SPP1 or BGN levels and no significant decrease in DNA-replication associated transcripts were noted in non-responder post-treatment samples (Fig.1D). These data indicate greater loss of AML cells and greater activity within the endosteal niche in responder in comparison to non-responder samples. Finally, analyses performed on post-treatment responder vs. non-responder samples showed significant decrease in SPARC, SPP1, DCN, VCAN, BGN in non-responder post-treatment samples (Fig.E, F). Endosteal niche in histopathologic evaluation at diagnosis was generally unremarkable in both responder and non-responder samples with only rare osteoblasts present. In contrast, post-treatment, we found an elevated number of osteoblasts in responders in comparison to non-responder samples (Fig.G, H). Conclusions: Transcriptomic and histopathologic analyses of AML bone marrow biopsies procured at diagnosis and post-treatment from responder or non-responders indicate inverse correlation between the activity of endosteal niche and levels of transcripts involved in osteoblast maturation and homeostasis. Significant suppression of mesenchymal/osteoblast component of the niche is observed in non-responder samples. To our knowledge this is a first report showing the correlation between levels of osteopontin (SPP1), osteonectin (SPARC) and biglycan (BGN) and response to chemotherapy directly in the AML core bone marrow biopsies. Our data suggest that osteo-stimulatory factors could be used to achieve better therapeutic outcomes in AML. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Observations On Eosinophil Ifiltrates in The Bone Marrow Of Patients With Multiple Myeloma. Clinicopathological Correlates

Blood ◽

10.1182/blood.v122.21.5338.5338 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5338-5338

Author(s):

Finella MC Brito-Babapulle ◽

Tanya Cranfield ◽

Robert B Corser ◽

Helen Dignum ◽

Christopher James ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cell ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Osteolytic Lesion ◽

Inverse Correlation ◽

Eosinophil Infiltration ◽

Bone Marrow Biopsies ◽

Lytic Lesions

Abstract Mouse eosinophils have been shown in 2011 to be required for the maintenance of long lasting plasma cells in the bone marrow and in maintaining the bone marrow plasma cell microenvironment. Human eosinophils have been shown by Wong et al to support multiple myeloma cell proliferation via a mechanism independent of IL6. We looked at bone marrow biopsies taken from patients who had a paraprotein and in whom a diagnosis of multiple myeloma was suspected. These samples were taken solely for the purposes of diagnosisng multiple myeloma and were retrospectively reviewed from the point of view of degree of eosinophil infiltration and its correlation with tumour load, bone lytic lesions, plasma cell morphology, whether blastic, crystalline inclusions, Mott cells, flame cells and or lymphoplasmacytoid. There were no cases of IGD or E myeloma or osteosclerotic myeloma.Nonsecretory myeloma and cases of light chain myeloma with or without amyloid were included in the series. Biopsies were not performed from osteolytic lesion unless biopsy was necessary to make a diagnosis of myeloma. Myeloma was diagnosed when plasma cell infiltrate was greater than 10% on bone marrow aspirate with a paraprotein and or lytic lesions. Eosinophil infiltration did not correlate with any of the tumour clinicopathological markers but showed an inverse correlation with degree of plasmacytosis. Eosinophils were hardly ever found in marrow aspirates that had over 70% plasma cells. They were usually found in trephine sections of bone marrow in areas where there was Grade I/II fibrosis and were often found in close proximity to focal areas of plasma cell infiltration. Whether eosinophils play a role in preventing or maintaining malignant plasma cell recurrence is currently being studied. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Bone Marrow Pathology Predicts Mortality in Chronic Hemodialysis Patients

BioMed Research International ◽

10.1155/2015/160382 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Cheng-Hao Weng ◽

Kuan-Ying Lu ◽

Ching-Chih Hu ◽

Wen-Hung Huang ◽

I-Kwan Wang ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Biopsy ◽

Cox Regression ◽

Reference Group ◽

Hemodialysis Patients ◽

Chronic Hemodialysis ◽

Lymphoid Cells ◽

Maintenance Hemodialysis ◽

Bone Marrow Biopsies ◽

Cox Regression Analysis

Introduction. A bone marrow biopsy is a useful procedure for the diagnosis and staging of various hematologic and systemic diseases. The objective of this study was to investigate whether the findings of bone marrow studies can predict mortality in chronic hemodialysis patients.Methods. Seventy-eight end-stage renal disease patients on maintenance hemodialysis underwent bone marrow biopsies between 2000 and 2011, with the most common indication being unexplained anemia followed by unexplained leukocytosis and leukopenia.Results. The survivors had a higher incidence of abnormal megakaryocyte distributionP=0.001, band and segmented cellsP=0.021, and lymphoid cellsP=0.029than the nonsurvivors. The overall mortality rate was 38.5% (30/78), and the most common cause of mortality was sepsis (83.3%) followed by respiratory failure (10%). In multivariate Cox regression analysis, both decreased (OR 3.714, 95% CI 1.671–8.253,P=0.001) and absent (OR 9.751, 95% CI 2.030–45.115,P=0.004) megakaryocyte distribution (normal megakaryocyte distribution as the reference group), as well as myeloid/erythroid ratio (OR 1.054, CI 1.012–1.098,P=0.011), were predictive of mortality.Conclusion. The results of a bone marrow biopsy can be used to assess the pathology, and, in addition, myeloid/erythroid ratio and abnormal megakaryocyte distribution can predict mortality in chronic hemodialysis patients.

Download Full-text

Decitabine-Induced Early Platelet Response, a Predictor of Favorable Outcome during Hypomethylating Treatment of MDS, Is Associated with In Vivo Megakaryocytic Differentiation

Blood ◽

10.1182/blood-2019-128141 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4265-4265

Author(s):

Julia Stomper ◽

Annette M. May ◽

Tina E. Joeckel ◽

Peter Bronsert ◽

Martin Werner ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Treatment Cycle ◽

Post Treatment ◽

Platelet Response ◽

Absolute Increase ◽

Bone Marrow Biopsies ◽

Pre Treatment

Introduction Despite broad use of hypomethylating agents (HMAs) in MDS/AML treatment, the number of established outcome predictors is still very limited (Stomper and Lübbert, Sem Hematol 2019). One of them, the early, isolated and sometimes massive increase of platelets, is recurrently observed in patients with MDS treated with HMAs. It is not observed with non-HMA cytidine analogs such as low-dose cytarabine. In HMA-treated MDS patients, an early platelet increase is a predictor of overall and leukemia-free survival (van den Bosch et al., Leuk Res 2004; van der Helm et al., Br J Haematol 2011). HMAs induce cellular differentiation in vitro, by gene reactivation in the malignant cells. However, evidence for HMA-induced in vivo differentiation is still very limited. We hypothesized that the megakaryocytic cell lineage in MDS is a target for HMA-induced cellular maturation in vivo. Methods We systematically analyzed the bone marrow morphology of 34 higher-risk MDS patients (median age: 71.5 years, range 51-79) before and after 1 cycle of treatment with the HMA decitabine (DAC). All patients had been treated at a single center within 3 prospective clinical trials (Wijermans et al., Ann Hematol 2005; Lübbert et al., J Clin Oncol 2011). One treatment cycle consisted of 45 mg/m2 DAC per day (15 mg/m2 intravenously over 4 hours every 8 hours) for 3 consecutive days, repeated 6 weeks later. The early platelet response was evaluated after 1 cycle of DAC treatment. Based on the criteria of the International Working Group, an absolute increase in platelet count of 30x109/l or more compared to the pre-treatment count was defined as a platelet response. The histological analysis of the bone marrow specimens was performed by an experienced hematopathologist blinded to the treatment timepoints. Up to 200 megakaryocytes (MK) per sample were quantified at a magnification of 400 x using chloroacetate esterase staining. Results Thirteen of 34 patients (38%) showed a platelet response already after 1 cycle of DAC treatment, 21 (62%) did not. The median pre-treatment platelet count did not differ in patients with or without an early platelet response (median of 34x109/l in both groups, range 7-169 and 8-265, respectively). After 1 cycle of DAC treatment, patients with a platelet response had a median platelet count of 117x109/l (range 78-281), patients without this response had a median platelet count of 32 x109/l (range 4-155). Overall survival (OS) was measured from the time of early platelet response assessment after completion of 1 treatment cycle, i.e. after 6 weeks. The presence of a platelet response after 1 DAC cycle was associated with a longer OS compared to the absence of this early platelet response: median of 26.6 versus 14.0 months (p=0.04). Both pre- and post-treatment bone marrow biopsies of patients with an early platelet response showed higher numbers of MK, as well as significant differences in MK morphology compared to biopsies from patients without an early platelet response. Regarding MK numbers, increased MK density in specimens of patients with an early platelet response was observed both before (mean MK number per high power field 6.2 vs. 2.6, p=0.02) and after the application of DAC (mean MK number 10.4 vs. 3.1, p=0.01). Regarding MK maturation stage, more pre-treatment juvenile MK (on average 32.4% vs. 20.5% of all MK, p=0.03) and MK with typical myeloproliferative stigmata (present in 5/13 vs. 2/21 biopsies) were observed in patients with an early platelet response, compared to patients without this response. Regarding the induction of megakaryocytic maturation during this early treatment phase, more post-treatment "naked", mature MK nuclei indicative of active platelet shedding (on average 9.5% vs. 3.8% of all MK, p=0.01), were noted in patients with an early platelet response than in patients without an early platelet response. Conclusions This is, to the best of our knowledge, the first systematic hematopathological analysis of changes in quantitative and morphological MK features in bone marrow specimens of MDS patients during HMA treatment. DAC, which has in vitro differentiation-inducing effects on megakaryoblastic cells, induced maturation also of dysplastic MK in vivo in higher-risk MDS patients with an early platelet response to this HMA. The predictive value of an early platelet increase, a very easy-to-determine parameter, during this type of treatment is confirmed. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Cyclooxygenase-2 (COX-2) in Multiple Myeloma: Prognostic Marker or Therapeutic Target?.

Blood ◽

10.1182/blood.v108.11.5051.5051 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5051-5051

Author(s):

Roger G. Owen ◽

Im Fan ◽

Sheila J.M. O’Connor ◽

Faith E. Davies ◽

Rebecca A. Rollett ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Bone Marrow ◽

Therapeutic Target ◽

Plasma Cells ◽

Prognostic Significance ◽

Cyclooxygenase 2 ◽

Lymphoid Cells ◽

Bone Marrow Biopsies ◽

Cox 2

Abstract Cyclooxygenase-2 (COX-2) is the key enzyme involved in prostaglandin synthesis. It appears to support the growth of a number of solid tumours including colon, breast, ovary, lung and uterine cervix and may be an important therapeutic target in at least some of these tumours. COX-2 expression has recently been evaluated (by immunohistochemistry using polyclonal anti-COX-2 antibodies) in multiple myeloma (MM) where expression was documented in 33–57% of patients. COX-2 expression in these studies was strongly associated with an adverse outcome. In addition there is some emerging data to suggest that the use of aspirin in MM may improve survival rates. In order to further evaluate this we have used a monoclonal antibody (Clone SP21, Labvision, Fremont, Ca) to assess COX-2 expression in both normal and neoplastic plasma cells. 52 specimens were assessed using standard streptavidin-biotin immunoperoxidase techniques using a known COX-2+ colon cancer as a positive control. Strong uniform COX-2 expression was seen in 32/33 (97%) of myeloma patients assessed and was also documented in all patients with MGUS (n=10). COX-2 expression was also documented in reactive plasmacytic lesions (oral mucosa, skin and lymph node, n=6) as well as normal bone marrow plasma cells (n=6). Megakaryocytes stained positively in all bone marrow biopsies examined and provided a useful positive internal control while erythroid, myeloid and lymphoid cells were consistently negative. We would conclude that COX-2 is strongly expressed by both normal and neoplastic plasma cells suggesting that COX-2 is a potential therapeutic target in MM. The apparent increase in the proportion of myeloma patients expressing COX-2 in the present study reflects the use of a monoclonal antibody in our immunohistology studies. The fact that polyclonal antibodies identify a lower proportion of patients who appear to have an inferior outcome suggests that the level of expression is of prognostic significance rather than its presence or absence. This is worthy of further study using more appropriate techniques such as RQ-PCR.

Download Full-text

Gene Expression Profiling (GEP) in Multiple Myeloma (MM): Comparison of Purified MM Cells (PMM), Random Bone Marrow Biopsies (RBX) and Fine Needle Biopsies from Focal Lesions (FNBX).

Blood ◽

10.1182/blood.v106.11.1535.1535 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1535-1535

Author(s):

Yongsheng Huang ◽

Fenghuang Zhan ◽

Bart Barlogie ◽

John Shaughnessy

Keyword(s):

Bone Marrow ◽

Normal Karyotype ◽

The Other ◽

Standard Laboratory ◽

Focal Lesions ◽

Bone Marrow Biopsies ◽

Ct Guided ◽

Fine Needle ◽

Central Processing ◽

Laboratory Features

Abstract Background: GEP has become a useful tool for the distinction of genetic subgroups of patients with malignant disease of a common pathologic diagnosis, including MM. The aim of this study was to determine whether whole bone marrow biopsies could replace the laborious magnetic bead purification of CD 138-positive plasma cells, with the potential added advantages of: 1. less sample manipulation and thus facilitating shipping to a central processing and analysis facility; 2. encompassing the bone marrow micro-environment as an important contributor to MM survival and drug resistance. Patients and Methods: Samples included paired PMM and RBX from 190 newly diagnosed patients treated on Total Therapy protocols TT2 (n = 85) and TT3 (n = 105); 29 of these patients also had CT-guided fine needle biopsies (FNBX); 5 subjects with MGUS and 5 normal donors were used for comparison. Total RNA was applied to Affymetrix U133 Plus2 microarray chips. Unsupervised hierarchical clustering (UHC) was performed on Log2 transformed GEP intensity values of probe sets with >95% present detection calls and >0.5 standard deviation. Results: UHC analysis of RBX (MM, MGUS, normal) identified 2 major subgroups: one containing both normal and MGUS samples (“normal-like”, N-L) and the other comprising only MM samples (“myeloma-like”, M-L) (Figure 1). In the context of previously defined 7 GEP subgroups (Zhan et al, Blood 2004), the N-L group contained higher proportions of hyperdiploid and normal karyotype cases, whereas the M-L group comprised the majority of MAF/MAFB, FGFR3/MMSET and proliferation cases. Good-risk standard laboratory features (non-IgA isotype, lower B2M and CRP levels, higher Hb concentrations) were over-represented in the N-L group. When applied to TT2 patients with a median follow-up of 3 years, N-L patients had superior 3-yr EFS (85% vs 55% for M-L group; p=.02). A further UHC analysis addressed similarities/differences among PMM, RBX and FNBX samples available in 29 patients. The dendrogram had 2 major branches; one comprising all PMM samples and the other all BX with sub-branches separating RBX and FNBX subgroups. Finally, 90% of all MAF, MAFB, FGFR3, MMSET and CCND1 “spikes” identified in PMM (with well established prognostic implications) were also detectable in RBX and FNBX samples. Conclusion: GEP analysis of RBX distinguished 2 subgroups, M-L and N-L, in the context of comparison with normal subjects and MGUS. Compared to M-L, N-L patients (comprising more favorable genetic and standard laboratory features) had superior EFS. Both RBX and FNBX were adequate tissue sources for the detection of prognosis- and possibly therapy-relevant translocation-associated “spiked” genes. Genes differentiating PMM, RBX and FNBX will be reported at the meeting. BX as tissue source should make GEP studies of MM feasible in the context of multi-institutional trials.

Download Full-text

SFRP1 Is Estrogen Inducible in Bone Marrow Stromal Cells and Suppresses the Earliest Events in Lymphopoiesis.

Blood ◽

10.1182/blood.v110.11.611.611 ◽

2007 ◽

Vol 110 (11) ◽

pp. 611-611 ◽

Cited By ~ 2

Author(s):

Takafumi Yokota ◽

Kenji Oritani ◽

Karla P. Garrett ◽

Taku Kouro ◽

Makoto Nishida ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Lymphoid Cells ◽

Mouse Bone Marrow ◽

B Lymphopoiesis ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Lymphoid Progenitors ◽

Frizzled Receptors ◽

Rag 1

Abstract It has long been known that lymphopoiesis is transiently suppressed during pregnancy, and that this can be experimentally simulated by treatment with estrogen. Indeed, sensitivity to estrogen made it possible to identify very primitive lymphoid cells in the Lin- c-kitHi Sca-1+ fraction that is enriched with hematopoietic stem cells (HSC) and multipotent progenitors of mouse bone marrow. However, analyses showing how the earliest events in lymphopoiesis is suppressed in those circumstances have not been performed. Hematopoietic and environmental cells are both potential hormone targets and, because of this complexity, very little has been known regarding mechanisms. In the present study, we performed high resolution analysis using RAG-1/GFP reporter mice and confirmed that early lymphoid progenitors (ELP) in the Lin-c-kitHi Sca-1+ fraction are particularly depressed in pregnancy or after estrogen injection. The Lin- c-kitLo fraction containing common lymphoid progenitors (CLP)/pro-lymphocytes was very sensitive to estrogen in stroma-free serum-free culture, but earlier stages of lymphopoiesis seemed to be blocked in vivo. Thus, we focused on identifying mechanisms involving bone marrow environment. Two independent strategies with macroarrays or differential display-PCR were used to isolate stromal cell genes that were both estrogen regulated and associated with inability to support B lymphopoiesis. We have identified secreted frizzled related protein 1 (sFRP1) as an estrogen inducible gene, and its expression corresponded to inability to support lymphopoiesis. This member of the complex Wnt family has been previously described as both an agonist and an antagonist of canonical, β-catenin dependent signaling. We found that sFRP1, like recombinant Wnt3a, stimulated the canonical pathway in the Lin- c-kitHi Sca-1+ fraction, and blocked progression in the B lymphocyte lineage even when exogenous Wnt ligands were not provided. The suppressive effects of sFRP1 and Wnt3a on B lymphopoiesis were canceled out when both were present simultaneously. To better understand the vulnerability of early progenitors to the Wnt signaling, we examined the expression pattern of Frizzled receptors. Interestingly, the HSC-enriched fraction (Lin- c-kitHi Sca-1+ IL-7Rα-RAG-1/GFP-) highly expressed 7 out of 9 Frizzled receptors, which markedly declined as they differentiated to ELP (Lin- c-kitHi Sca-1+ IL-7Rα- RAG-1/GFP+) and CLP (Lin- c-kitLo Sca-1Lo/- IL-7Rα+). Myelo-erythroid progenitors were less affected by sFRP1 in culture, suggesting that it is similar to estrogen with respect to lineage specificity. Bone-lining stromal cells express sFRP1 and the transcripts were elevated in bone marrow by pregnancy or estrogen injection. In summary, these observations implicate sFRP1 as a possible mediator in hormone regulation of the earliest events in lymphopoiesis.

Download Full-text

P-ERK1/2 Is a Predictive Factor of Response to ESA Treatment in Myelodysplastic Syndromes.

Blood ◽

10.1182/blood.v114.22.294.294 ◽

2009 ◽

Vol 114 (22) ◽

pp. 294-294

Author(s):

Emilie Frisan ◽

Patrycja Pawlikowska ◽

Cécile Pierre-Eugène ◽

Valérie Bardet ◽

Laure Gibault ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Myelodysplastic Syndromes ◽

Conflicts Of Interest ◽

Marrow Cell ◽

Wilcoxon Test ◽

Response To Treatment ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

Bone Marrow Biopsies

Abstract Abstract 294 Endogenous serum erythropoietin (sEPO) less than 500UI/L and a transfusion requirement lower than 2 units per month are the best predictive factors for response to treatment by erythropoiesis-stimulating agents (ESA) in low/int-1 myelodysplastic syndromes (MDS). However, the highest response rate hardly reaches 60% suggesting that other factors may influence the response. To investigate the biological signature of response to ESA, we enrolled 100 low/int-1 MDS patients in a prospective study of erythropoiesis at diagnosis before they were treated with ESA. According to the IWG 2006 criteria, 43 patients were non-responders. These patients had significantly higher serum EPO level, higher number of transfusion per month, and lower number of bone marrow-deriving BFU-E and CFU-E than responders. Analysis of CD34+-deriving erythroid progenitors by in vitro liquid culture, demonstrated that all MDS patients (n=54) had an increased apoptosis and a delayed expression of erythroid marker, glycophorin A (GPA). A collapse of EPO-induced DNA synthesis was observed in non-responders, while EPO-dependent erythroid cell differentiation and survival to Fas-induced apoptosis was equivalent in the two groups. Thus, non-responders exhibited an early and isolated default in EPO-induced cell proliferation, suggesting a defect in EPO-R signaling. Immunofluorescence to p-ERK1/2 before and after EPO-R stimulation in immature erythroblasts was negative in 6/8 non-responders, and positive in all 11 responders. Immunohistochemistry to p-ERK1/2 on bone marrow biopsies in 5 non-responders was negative and positive in immature cells in 4 responders. By flow cytometry, p-ERK1/2 expression in the CD71+/GPA− bone marrow cell fraction corresponding to immature erythroblasts (n=30) was significantly lower in non-responders (n=16) than in responders (n=14; Wilcoxon-test: p<0.0001). Receiver operator curve (ROC) analysis of the flow cytometry test demonstrated a good predictive value for the response to ESA with a 0.96 area under the curve (AUC) [95%CI: 0.89 – 1.00]. ROC were also constructed for BFU-E number, serum EPO level, and number of transfusion per month and the AUC were computed. p-ERK1/2 was equivalent to BFU-E and superior to serum EPO level or number of transfusion in predicting the response to ESA. Although requiring validation in a larger cohort, these results suggest that p-ERK1/2 is a ready tool available for the prediction of response to ESA in MDS patients. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Evaluation of Angiogenesis and the CD57+ Lymphocytic Population on Bone Marrow Biopsies (BM) in Multiple Myeloma Patients Treated with Thalidomide (Thal)

Blood ◽

10.1182/blood.v118.21.5095.5095 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5095-5095

Author(s):

Edvan Crusoe ◽

Adriana Quero ◽

Eliana C M Miranda ◽

Manuella Sampaio ◽

Ana Lucia Peres ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Day Treatment ◽

Post Treatment ◽

Unexpected Finding ◽

Bone Marrow Biopsies ◽

Overall Response ◽

Significant Difference ◽

Angiogenic Effect

Abstract Abstract 5095 Introduction – Multiple myeloma (MM) is a plasma cell dyscrasias characterized by the bone destruction, renal insufficiency, anemia and hypercalcemia. Studies suggest that the disease activity and the degree of angiogenesis in the bone marrow (BM) are related. It has been shown that increased microvessel density (MVD) in MM patients`BM specimens is associated with poor prognosis, and BM MVD at diagnosis is an important prognostic factor for survival of patients. Due to the anti-angiogenic effect, Thal is becoming consolidated as a therapeutic option for the treatment of MM. The presence of cytotoxic CD57+ T lymphocytes in the BM in MM patients who have never been treated correlates with the clinical progression of the patients. The present study has the objective of presenting findings of the anti-angiogenic effect of thalidomide, correlating with the reduction of MVD in the obtained response, as well as verifying the presence of CD57+ lymphocytes before and after the treatment, correlating this with the rate response. Materials and methods– BM were collected from the posterior iliac crest in MM patients who had never been treated at least 12 weeks after having initiated treatment with Thal or up to the fourth week after discontinuing its use. Patients who had previously received Thal were excluded. The bone marrow biopsies were done by two pathologists who were blind as to the disease treatment phase. Angiogenesis was estimated based on the MVD, utilizing the anti-CD34 antibody as an immunohistochemical marker. The slides were photographed at 03 sites of densely concentrated vessels selected by counting under 400X magnification. The final density of the microvessel site median was determined. The CD57+ lymphocyte analysis was made for plasmocyte concentration zones, determining the final count using the median of three sites. Statistical analysis was performed with the SPSS 15.0 for Windows, a t-parametric test for equal averages and Spearman correlation. Results– There was a total of 20 patients (pre- and post-treatment). The median age was 64 (40–82 years), 65% of the cases being male. The Durie-Salmon Staging distribution: IIA/B= 10 % and IIIA/B=90%, and ISS: 2=45% and 3=35%. The IgG isotype was present in 70% of the cases. The therapeutic schedule made use of target doses of thalidomide at 200mg/d. Fourteen patients utilized the Thal and dexamethasone (TD) schedule, four patients, cyclophosphamide+TD (CTD), one patient, Melphalan+prednisone+Thal (MPT) and one patient combination TD+CTD. Eleven patients received a 90-day treatment between collections, seven, a 120-day treatment, one, a 150-day treatment and one, a 270-day treatment. The median MVD count of pre-thal CD34 was 11.42, and post-thal, 7.17 (p=0.01). The pre-thal CD57 median was 26.42 and the post-treatment, 21.58 (not significant). There was a negative post-CD34 versus overall response correlation (p=0.04) and a positive CD57+ versus overall response correlation (p=0.05). Pre-CD34 versus International Staging System correlations (p=0.01) were observed. Another unexpected observation was the analysis of the gender as an independent variable, it was observed that the post-treatment CD57+ was significantly different between the sexes (p=0.008). Conclusion– This study confirms the anti-angiogenic effect of Thal in MM patients, with reduced MVD by CD34 analysis, in addition to its correlation with the overall response. It also demonstrates the significant correlation between the post-treatment CD57+ lymphocytic population and the overall response. The unexpected finding was the significant difference in the quantity of lymphocytes present in the post-treatment BM between the genders (increased in men and reduced in women). Furthermore, it was observed that a greater quantity of MVD correlates with the worst ISS staging. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C

Science Signaling ◽

10.1126/scisignal.aap7336 ◽

2019 ◽

Vol 12 (598) ◽

pp. eaap7336 ◽

Cited By ~ 12

Author(s):

Laura F. Bennett ◽

Chang Liao ◽

Michael D. Quickel ◽

Beng San Yeoh ◽

Matam Vijay-Kumar ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Proinflammatory Cytokines ◽

Lymphoid Cells ◽

Sterile Inflammation ◽

Erythroid Progenitors ◽

Gene Encoding ◽

Immune Effector ◽

Effector Cells ◽

Stress Erythropoiesis

Inflammation alters bone marrow hematopoiesis to favor the production of innate immune effector cells at the expense of lymphoid cells and erythrocytes. Furthermore, proinflammatory cytokines inhibit steady-state erythropoiesis, which leads to the development of anemia in diseases with chronic inflammation. Acute anemia or hypoxic stress induces stress erythropoiesis, which generates a wave of new erythrocytes to maintain erythroid homeostasis until steady-state erythropoiesis can resume. Although hypoxia-dependent signaling is a key component of stress erythropoiesis, we found that inflammation also induced stress erythropoiesis in the absence of hypoxia. Using a mouse model of sterile inflammation, we demonstrated that signaling through Toll-like receptors (TLRs) paradoxically increased the phagocytosis of erythrocytes (erythrophagocytosis) by macrophages in the spleen, which enabled expression of the heme-responsive gene encoding the transcription factor SPI-C. Increased amounts of SPI-C coupled with TLR signaling promoted the expression ofGdf15andBmp4, both of which encode ligands that initiate the expansion of stress erythroid progenitors (SEPs) in the spleen. Furthermore, despite their inhibition of steady-state erythropoiesis in the bone marrow, the proinflammatory cytokines TNF-α and IL-1β promoted the expansion and differentiation of SEPs in the spleen. These data suggest that inflammatory signals induce stress erythropoiesis to maintain erythroid homeostasis when inflammation inhibits steady-state erythropoiesis.

Download Full-text

Graft Versus Host Disease Leads to Elimination of Tissue-Resident Innate Lymphoid Cells Following Experimental and Clinical Allogeneic Transplantation

Blood ◽

10.1182/blood-2019-128966 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3204-3204

Author(s):

Paola Vinci ◽

Elena Garcia-Martinez ◽

Margaret H. O'Connor ◽

Anastasiya Egorova ◽

Jason Kuttiyara ◽

...

Keyword(s):

Bone Marrow ◽

Lamina Propria ◽

Peripheral Blood ◽

Significant Loss ◽

Duodenal Biopsy ◽

Lymphoid Cells ◽

Post Transplant ◽

Tissue Protection ◽

Target Organs ◽

Duodenal Biopsies

A potential role for endogenous innate lymphoid cells (ILCs) in prevention of gastrointestinal (GI) GVHD has recently been described in murine and clinical models, and experimental approaches for adoptive cellular therapy with ILCs have recently been described as well. However, the specific in vivo roles of tissue-resident ILCs and their capacity for reconstitution post-transplant remain unclear. We thus sought to characterize the significance and tissue distribution of ILCs after allogeneic bone marrow transplant (allo-BMT) in mouse models and in patients. In order to specifically assess the functional role of ILCs after allo-BMT, we established a clinically relevant acute GVHD model based on sex mismatched H-Y antigens. Lethally irradiated Rag2-/-male mice (Thy1.2, B6) were transplanted with T cell-depleted (TCD) BM and purified T cells obtained from wild-type female mice (Thy1.1, B6). To specifically deplete host-derived ILCs (and not donor cells), recipient mice were treated with an anti-Thy1.2 antibody or an anti-isotype antibody prior to BMT. Pre-transplant depletion of ILCs induced significantly worse systemic signs of GVHD, as well as increased thymic injury and intestinal GVHD pathology, indicating an important role for endogenous ILCs in tissue protection after allo-BMT in this context. To better understand the roles of tissue-resident ILCs in reducing pathology post-BMT, we evaluated ILC subsets in different GVHD target organs after allo-BMT. Two weeks after MHC-mismatched (C57BL6 into BALB/c) TCD-BMT, both donor- and host-derived ILCs could be detected in the liver, lungs, and intestines. Liver was characterized by a high number of NK cells and ILC1s, while lungs contained a high proportion of ILC2s. The small intestine contained a relatively greater proportion of ILC3s. For all tissues analyzed, the majority of ILCs were host-derived. During T cell replete BMT resulting in GVHD, both donor and host-derived ILCs were significantly reduced in all three tissues compared to TCD-BMT mice, and this was true for all ILC lineages. To better understand the loss of donor-derived ILCs in GVHD, we investigated ILC precursors in recipient marrow post-BMT, hypothesizing that an impairment of ILC precursors could explain the loss of all ILC lineages in the various tissues analyzed. Indeed, we found a significant loss of CLPs, αLPs, CHILPs, and ILC2Ps in mice with GVHD, suggesting that bone marrow involvement with GVHD induced a loss of ILC precursors impairing their reconstitution. We next sought to evaluate if the loss of tissue-resident ILCs in GVHD target organs after experimental BMT was relevant for clinical transplantation. To evaluate ILC frequencies in patient tissues post-transplant, duodenal biopsies were collected from patients undergoing clinically indicated endoscopy to work up symptoms of acute upper GI (UGI) GVHD. In total, duodenal samples were collected from ten patients post-transplant and 6/10 had histologic evidence of GVHD on biopsy (Table 1). While frequencies were low, ILCs could be identified by FACS in digested lamina propria of duodenal biopsy specimens. Notably, ILC frequencies were significantly lower in patients with biopsy-proven UGI GVHD, and duodenal ILCs could be identified in only 1/6 patients with histologic evidence of GVHD (Fig 1). Among the ILCs that could be identified, ILC1s represented the greatest proportion of ILC populations within the duodenal lamina propria. Accordingly, the frequency of ILC1s was significantly reduced in transplant patients with biopsy-proven UGI GVHD. Additionally, we were able to evaluate paired peripheral blood and duodenal biopsy samples in a small subset of patients. Although the numbers were limiting, this cohort suggested that ILC2s may represent a greater proportion of ILCs in peripheral blood than in the duodenum post-transplant. Overall, clinical findings were consistent with experimental models demonstrating a reduction of ILCs in GVHD. In conclusion, tissue-resident ILCs contribute to tissue protection after allo-BMT, but GVHD leads to elimination of pre-existing ILCs and loss of ILC precursors in the marrow, impairing their reconstitution. Most duodenal ILCs in clinical biopsy samples post-transplant were ILC1s in this patient cohort, and patients with biopsy-proven UGI GVHD demonstrated significant loss of the few lamina propria ILCs that could be identified in clinical duodenal biopsies. Disclosures Hanash: Nexus Global Group LLC: Consultancy.

Download Full-text