Both stem cell chemokine stromal cell-derived factor-1<i>&#x3b1;</i> (SDF-1<i>&#x3b1;</i>) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed<i> in vitro</i>. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs<i> in vitro</i>, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes.

Michael Laupheimer

Anna Skorska

Jana Gro&#xdf;e

Gudrun Tiedemann

Gustav Steinhoff

Robert David

Cornelia A. Lux

Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1<i>&#x3B1;</i> and ATP Gradient

Molecular studies have shown that multiple myeloma is a highly genetically heterogonous disease which may manifest itself as any number of diverse subtypes each with variable clinicopathological features and outcomes. Given this genetic heterogeneity, a universal approach to treatment of myeloma is unlikely to be successful for all patients and instead we should strive for the goal of personalised therapy using rationally informed targeted strategies. Current DNA sequencing technologies allow for whole genome and exome analysis of patient myeloma samples that yield vast amounts of genetic data and provide a mutational overview of the disease. However, the clinical utility of this information currently lags far behind the sequencing technology which is increasingly being incorporated into clinical practice. This paper attempts to address this shortcoming by proposing a novel genetically based &#x201c;traffic-light&#x201d; risk stratification system for myeloma, termed the RAG (Red, Amber, Green) model, which represents a simplified concept of how complex genetic data may be compressed into an aggregate risk score. The model aims to incorporate all known clinically important trisomies, translocations, and mutations in myeloma and utilise these to produce a score between 1.0 and 3.0 that can be incorporated into diagnostic, prognostic, and treatment algorithms for the patient.

Steven M. Prideaux

Emma Conway O&#x2019;Brien

Timothy J. Chevassut

The RAG Model: A New Paradigm for Genetic Risk Stratification in Multiple Myeloma

TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5&#x2032;-methylcytosine to 5&#x2032;-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5&#x2032;-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5&#x2032;-methylcytosines at key promoter sequences within genomic DNA.  This leads to relative hypomethylation of gene promoters as 5&#x2032;-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented.

Louis Norman

Paul Tarrant

Timothy Chevassut

TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing

Metastasis is a multistep process, which refers to the ability to leave a primary tumor through circulation toward the distant tissue and form a secondary tumor. Bone is a common site of metastasis, in which osteolytic and osteoblastic metastasis are observed. Signaling pathways, chemokines, growth factors, adhesion molecules, and cellular interactions as well as miRNAs have been known to play an important role in the development of bone metastasis. These factors provide an appropriate environment (soil) for growth and survival of metastatic tumor cells (seed) in bone marrow microenvironment. Recognition of these factors and determination of their individual roles in the development of metastasis and disruption of cellular interactions can provide important therapeutic targets for treatment of these patients, which can also be used as prognostic and diagnostic biomarkers. Thus, in this paper, we have attempted to highlight the molecular regulation of bone marrow metastasis in prostate and breast cancers.

Fakher Rahim

Saeideh Hajizamani

Esmaeil Mortaz

Ahmad Ahmadzadeh

Mohammad Shahjahani

Saeid Shahrabi

Najmaldin Saki

Molecular Regulation of Bone Marrow Metastasis in Prostate and Breast Cancer

Autologous graft versus host disease (autoGVHD) is a rare transplant complication with significant morbidity and mortality. It has been hypothesized that patients with multiple myeloma might be predisposed to autoGVHD through dysregulation of the immune response resulting from either their disease, the immunomodulatory agents (IMiDs) used to treat it, or transplant conditioning regimen. Hematopoietic progenitor cell (HPC) products were available from 8 multiple myeloma patients with biopsy-proven autoGVHD, 16 matched multiple myeloma patients who did not develop autoGVHD, and 7 healthy research donors. The data on number of transplants prior to developing autoGVHD, mobilization regimens, exposure to proteasome inhibitors, use of IMiDs, and class I human leukocyte antigen types (HLA A and B) were collected. The HPC products were analyzed by flow cytometry for expression of CD3, CD4, CD8, CD25, CD56, and FoxP3. CD3<sup>+</sup> cell number was significantly lower in autoGVHD patients compared to unaffected controls (<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.2129908pt" id="M1" height="9.21094pt" version="1.1" viewBox="-0.0498162 -8.99795 53.2534 9.21094" width="53.2534pt"><g transform="matrix(.0135,0,0,-0.0135,0,0)"><path id="g113-81" d="M600 480C600 590 528 650 384 650H143L137 622C222 614 225 607 210 531L130 127C113 41 106 36 23 28L17 0H294L300 28C204 36 195 42 212 127L243 284L314 263C327 263 339 263 352 264C465 271 600 337 600 480ZM508 481C508 351 402 304 329 304C289 304 265 311 250 317L295 559C302 594 310 606 323 611C335 616 350 619 367 619C455 619 508 573 508 481Z"/></g><g transform="matrix(.0135,0,0,-0.0135,12.15,0)"><path id="g117-34" d="M535 323V373H52V323H535ZM535 138V188H52V138H535Z"/></g><g transform="matrix(.0135,0,0,-0.0135,23.887,0)"><path id="g113-49" d="M241 635C89 635 35 457 35 312C35 153 89 -12 240 -12C390 -12 443 166 443 312C443 466 390 635 241 635ZM238 602C329 602 354 454 354 312C354 172 330 22 240 22C152 22 124 173 124 313S148 602 238 602Z"/></g><g transform="matrix(.0135,0,0,-0.0135,30.39,0)"><path id="g113-47" d="M113 -12C146 -12 170 11 170 46C170 78 146 103 114 103S58 78 58 46C58 11 82 -12 113 -12Z"/></g><g transform="matrix(.0135,0,0,-0.0135,33.48,0)"><use xlink:href="#g113-49"/></g><g transform="matrix(.0135,0,0,-0.0135,39.983,0)"><path id="g113-53" d="M456 178V225H360V632H320C217 496 115 347 20 206V178H280V106C280 40 276 34 189 27V0H445V27C364 34 360 39 360 106V178H456ZM280 225H82C149 335 214 431 278 520H280V225Z"/></g><g transform="matrix(.0135,0,0,-0.0135,46.486,0)"><path id="g113-56" d="M447 623H65C61 580 56 530 47 475H76C100 541 106 550 172 550H388C308 376 196 170 91 -1L98 -12L172 -2C268 204 360 408 455 611L447 623Z"/></g></svg>). On subset analysis of CD3<sup>+</sup> cells, CD8<sup>+</sup> cells (but not CD4<sup>+</sup> cells) were found to be significantly lower in patients with autoGVHD (<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.2129908pt" id="M2" height="9.21094pt" version="1.1" viewBox="-0.0498162 -8.99795 53.2534 9.21094" width="53.2534pt"><g transform="matrix(.0135,0,0,-0.0135,0,0)"><use xlink:href="#g113-81"/></g><g transform="matrix(.0135,0,0,-0.0135,12.15,0)"><use xlink:href="#g117-34"/></g><g transform="matrix(.0135,0,0,-0.0135,23.887,0)"><use xlink:href="#g113-49"/></g><g transform="matrix(.0135,0,0,-0.0135,30.39,0)"><use xlink:href="#g113-47"/></g><g transform="matrix(.0135,0,0,-0.0135,33.48,0)"><use xlink:href="#g113-49"/></g><g transform="matrix(.0135,0,0,-0.0135,39.983,0)"><path id="g113-52" d="M285 378C315 398 338 416 353 432C373 451 384 474 384 503C384 579 325 635 236 635H235C182 635 136 610 108 579L65 516L85 496C110 533 150 575 205 575C258 575 300 543 300 481C300 407 232 369 141 339L147 310C163 315 188 321 211 321C268 321 338 284 338 192C338 94 288 40 217 40C160 40 119 68 93 91C85 98 77 97 69 91C60 84 47 71 46 58C44 46 48 35 62 22C75 10 116 -12 162 -12C234 -12 424 62 424 224C424 297 373 359 285 376V378Z"/></g><g transform="matrix(.0135,0,0,-0.0135,46.486,0)"><path id="g113-57" d="M249 635C141 635 70 555 70 471C70 401 114 353 179 316C143 294 106 267 90 252C68 231 45 202 45 157C45 50 130 -12 237 -12C322 -12 435 52 435 169C435 256 372 304 303 343C349 374 375 398 383 407C401 429 411 458 411 487C411 569 344 635 249 635ZM238 603C285 603 337 567 337 482C337 422 310 385 276 358C205 393 145 426 145 500C145 552 179 603 238 603ZM248 20C183 20 125 70 125 163C125 218 158 268 206 300C284 261 355 217 355 143C355 66 308 20 248 20Z"/></g></svg>). HLA-B55 expression was significantly associated with development of autoGVHD (<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.2129908pt" id="M3" height="9.21094pt" version="1.1" viewBox="-0.0498162 -8.99795 53.2534 9.21094" width="53.2534pt"><g transform="matrix(.0135,0,0,-0.0135,0,0)"><use xlink:href="#g113-81"/></g><g transform="matrix(.0135,0,0,-0.0135,12.15,0)"><use xlink:href="#g117-34"/></g><g transform="matrix(.0135,0,0,-0.0135,23.887,0)"><use xlink:href="#g113-49"/></g><g transform="matrix(.0135,0,0,-0.0135,30.39,0)"><use xlink:href="#g113-47"/></g><g transform="matrix(.0135,0,0,-0.0135,33.48,0)"><use xlink:href="#g113-49"/></g><g transform="matrix(.0135,0,0,-0.0135,39.983,0)"><use xlink:href="#g113-52"/></g><g transform="matrix(.0135,0,0,-0.0135,46.486,0)"><path id="g113-51" d="M412 140C382 77 369 73 315 73H129L270 222C362 320 402 379 402 466C402 571 322 635 234 635C177 635 130 609 99 576L42 495L64 475C90 514 133 568 201 568C274 568 318 519 318 435C318 349 255 267 193 193C144 135 87 78 32 23V0H405C417 45 427 89 440 131L412 140Z"/></g></svg>). Lower percentages of CD3<sup>+</sup> and CD8<sup>+</sup> T-cells and HLA-B55 expression may be predisposing factors for developing autoGVHD in myeloma.

Anu Batra

Michele Cottler-Fox

Terry Harville

Bobbie S. Rhodes-Clark

Issam Makhoul

Mayumi Nakagawa

Autologous Graft versus Host Disease: An Emerging Complication in Patients with Multiple Myeloma

Though discovered later than osteoblastic niche, vascular niche has been regarded as an alternative indispensable niche operating regulation on hematopoietic stem cells (HSCs). As significant progresses gained on this type niche, it is gradually clear that the main work of vascular niche is undertaking to support hematopoiesis. However, compared to what have been defined in the mechanisms through which the osteoblastic niche regulates hematopoiesis, we know less in vascular niche. In this review, based on research data hitherto we will focus on component foundation and various functions of vascular niche that guarantee the normal hematopoiesis process within bone marrow microenvironments. And the possible pathways raised by various research results through which this environment undergoes its function will be discussed as well.

Ningning He

Lu Zhang

Jian Cui

Zongjin Li

Bone Marrow Vascular Niche: Home for Hematopoietic Stem Cells

Hepatitis C is a significant public health problem in Egypt where the highest prevalence (14.7&#x25;) of hepatitis C virus (HCV) exists. HCV prevalence is even higher among clinical populations and groups at risk of exposure to infection. Chronic HCV infection is associated with several hematological complications that may necessitate bone marrow (BM) examination. The aim of this study is to estimate HCV prevalence among patients referred for BM examination and to explore hematological and BM findings among HCV positive patients. One hundred adult patients referred for BM examination were included in the study and screened for HCV antibodies. Patients&#x2019; clinical, hematological, and BM findings were recorded. The seroprevalence of HCV among patients referred for BM examination was 42&#x25;. The most common indication for BM examination among HCV positive patients was peripheral cytopenias (88.1&#x25;). The most common cytopenia detected was thrombocytopenia (85.7&#x25;). The most common diagnosis among HCV positive patients was hypersplenism (52.4&#x25;) followed by B-lymphoproliferative disorders (19&#x25;) and then immune thrombocytopenic purpura (11.9&#x25;). In conclusion, HCV prevalence among patients referred for BM examination is higher than that estimated in the general population. Patients with unexplained peripheral cytopenias should be tested for HCV.

Somaia Mohammed Mousa

Hepatitis C among Egyptian Patients Referred for
 Bone Marrow Examination: Seroprevalence and Analysis of Hematological Findings

Loss of heterozygosity (LOH) is a common event in malignant cells. In this work we introduce a new approach to identify patients with loss of heterozygosity in the HLA region either at first diagnosis or after HLA mismatched allogeneic HSCT. Diagnosis of LOH requires a high purity of recipient target cells. FACS is time consuming and also frequently prevented by rather nonspecific or unknown immune phenotype. The approach for recipient cell enrichment is based on HLA targeted complement-dependent cytotoxicity (CDC). Relative fluorescent quantification (RFQ) analysis of HLA intron length polymorphisms then allows analysis of HLA heterozygosity. The approach is exemplified in recent clinical cases illustrating the detection of an acquired allele loss. As illustrated in one case with DPB1, distinct HLA loci in donor and patient were sufficient for both proof of donor cell removal and evaluation of allele loss in the patient's leukemic cells. Results were confirmed using HLA-B RFQ analysis and leukemia-associated aberrant immunophenotype (LAIP) based cell sort. Both results confirmed suspected loss of HLA heterozygosity. Our approach complements or substitutes for FACS-based cell enrichment; hence it may be further developed as novel routine diagnostic tool. This allows rapid recipient cell purification and testing for loss of HLA heterozygosity before and after allogeneic HSCT in easily accessible peripheral blood samples.

Klaus Witter

Roland Reibke

Marion Subklewe

Robert Zahn

Teresa Kauke

Karsten Spiekermann

Michael Spannagl

Johanna Tischer

Wolfgang Hiddemann

Andrea Dick

Combination of Complement-Dependent Cytotoxicity and Relative Fluorescent Quantification of HLA Length Polymorphisms Facilitates the Detection of a Loss of Heterozygosity

Angiogenesis and proliferation as measured by microvessel density (MVD) and proliferation index (PI) are essential correlates of malignancy. The aim of our study was to evaluate difference between these values in AML and ALL and also to study the modulation in these parameters following achievement of remission in acute lymphoblastic leukemia. Differences between adult and adolescent cases of acute leukemia in relation to these values were also studied. We also tried to assess the relationship between angiogenesis and proliferation. Fifty-five patients with acute leukemia were included in the study. Trephine biopsies were immunostained with CD34 and factor VIIIrAg to demonstrate angiogenesis measured as MVD. Immunostaining with PCNA and Ki-67 was done to study proliferation. We found a significant increase in MVD and PI in cases when compared with controls (<svg     style="vertical-align:-0.30096pt;width:75.025002px;" id="M1" height="11.375" version="1.1" viewBox="0 0 75.025002 11.375" width="75.025002"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,11.112)"><path id="x1D443" d="M619 482q0 -69 -40.5 -119t-96 -72.5t-118.5 -26.5h-44l-70 20l-31 -151q-14 -67 0.5 -83t89.5 -22l-5 -28h-287l8 28q65 7 81.5 22t29.5 83l79 398q12 56 0.5 70.5t-78.5 20.5l7 28h255q108 0 164 -43t56 -125zM524 478q0 141 -146 141q-25 0 -47 -8q-16 -6 -20.5 -13.5&#xA;t-10.5 -39.5l-43 -241q37 -13 83 -13q67 0 125.5 45t58.5 129z" /></g><g transform="matrix(.017,-0,0,-.017,15.582,11.112)"><path id="x3C" d="M512 -3l-437 233v51l437 233v-58l-378 -200v-2l378 -199v-58z" /></g><g transform="matrix(.017,-0,0,-.017,30.286,11.112)"><path id="x30" d="M241 635q53 0 94 -28.5t63.5 -76t33.5 -102.5t11 -116q0 -58 -11 -112.5t-34 -103.5t-63.5 -78.5t-94.5 -29.5t-95 28t-64.5 75t-34.5 102.5t-11 118.5q0 58 11.5 112.5t34.5 103t64.5 78t95.5 29.5zM238 602q-32 0 -55.5 -25t-35.5 -68t-17.5 -91t-5.5 -105&#xA;q0 -76 10 -138.5t37 -107.5t69 -45q32 0 55.5 25t35.5 68.5t17.5 91.5t5.5 105t-5.5 105.5t-18 92t-36 68t-56.5 24.5z" /></g><g transform="matrix(.017,-0,0,-.017,38.446,11.112)"><path id="x2E" d="M113 -12q-24 0 -39.5 16t-15.5 42q0 24 16 40.5t40 16.5t40 -16.5t16 -40.5q0 -26 -16 -42t-41 -16z" /></g><g transform="matrix(.017,-0,0,-.017,42.321,11.112)"><use xlink:href="#x30"/></g><g transform="matrix(.017,-0,0,-.017,50.481,11.112)"><use xlink:href="#x30"/></g><g transform="matrix(.017,-0,0,-.017,58.64,11.112)"><use xlink:href="#x30"/></g><g transform="matrix(.017,-0,0,-.017,66.8,11.112)"><path id="x31" d="M384 0h-275v27q67 5 81.5 18.5t14.5 68.5v385q0 38 -7.5 47.5t-40.5 10.5l-48 2v24q85 15 178 52v-521q0 -55 14.5 -68.5t82.5 -18.5v-27z" /></g>

		
	
</svg>). In addition cases with ALL had a significantly higher MVD compared to those with AML (<svg     style="vertical-align:-0.30096pt;width:58.712502px;" id="M2" height="11.375" version="1.1" viewBox="0 0 58.712502 11.375" width="58.712502"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,11.112)"><use xlink:href="#x1D443"/></g><g transform="matrix(.017,-0,0,-.017,15.582,11.112)"><use xlink:href="#x3C"/></g><g transform="matrix(.017,-0,0,-.017,30.286,11.112)"><use xlink:href="#x30"/></g><g transform="matrix(.017,-0,0,-.017,38.446,11.112)"><use xlink:href="#x2E"/></g><g transform="matrix(.017,-0,0,-.017,42.321,11.112)"><use xlink:href="#x30"/></g><g transform="matrix(.017,-0,0,-.017,50.481,11.112)"><use xlink:href="#x31"/></g>

		
	
</svg>). The patients with ALL who went into remission showed a significant reduction in MVD; PI remained high. The cases which did not achieve remission showed no significant reduction in either MVD or PI. All adolescent cases of ALL were similar to adults with respect to MVD and PI.

Prabhavati Jothilingam

Debdatta Basu

Tarun K. Dutta

Angiogenesis and Proliferation Index in Patients with Acute Leukemia: A Prospective Study

<i>Aims</i>. To study the etiology, diagnostic features, and clinical significance of myelonecrosis. <i>Methods</i>. A retrospective review of all trephine biopsies done over 12 years (January 2000 to December 2012) in Department of pathology was done and all trephine biopsies showing MN were identified and studied. <i>Results</i>. Twenty-five cases accounting for 0.4&#x25; were identified. Fever and generalized weakness were the common presenting symptoms. Anemia was seen in all cases followed by thrombocytopaenia. Malignancy was the underlying cause in 64&#x25; of cases; hematolymphoid malignancy was seen in two-thirds and solid malignancies in one-third of the cases. Tuberculosis accounted for 16&#x25; of the cases and the etiology was unknown in 12&#x25;. <i>Conclusions</i>. The causes of MN are varied and hematological malignancy and solid malignancies are the most common causes. Presence of myelonecrosis is associated with a poor prognosis. Myelonecrosis may obscure the underlying disorder and hence a thorough search in the bone marrow biopsy itself with the help of immunohistochemistry may prove worthwhile in identifying the underlying disease.

Jinkala Sree Rekha

Rakhee Kar

Myelonecrosis: A Clinicopathological Study from a Tertiary Care Center in South India over a Twelve-Year Period

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Bone Marrow Research