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26.  Regulation of innate immunity by extracellular nucleotides 
Extracellular ATP (eATP) is the most abundant among extracellular nucleotides and is commonly considered as a classical danger signal, which stimulates immune responses in the presence of tissue injury. In fact, increased nucleotide concentration in the extracellular space is generally closely associated with tissue stress or damage. However non-lytic nucleotide release may also occur in many cell types under a variety of conditions. Extracellular nucleotides are sensed by a class of plasma membrane receptors called P2 purinergic receptors (P2Rs). P2 receptors are expressed by all immunological cells and their activation elicits different responses. Extracellular ATP can act as an initiator or terminator of immune responses being able to induce different effects on immune cells depending on the pattern of P2 receptors engaged, the duration of the stimulus and its concentration in the extracellular milieu. Millimolar (high) concentrations of extracellular ATP, induce predominantly proinflammatory effects, while micromolar (low) doses exert mainly tolerogenic/immunosuppressive action. Moreover small, but significant differences in the pattern of P2 receptor expression in mice and humans confer diverse capacities of ATP in regulating the immune response.
PMCID: PMC3555188  PMID: 23358447
Extracellular nucleotides; P2 purinergic receptors; extracellular ATP; innate immunity
27.  Regulatory T-cells in chronic lymphocytic leukemia: actor or innocent bystander? 
Regulatory T (Treg) cells are now under extensive investigation in chronic lymphocytic leukemia (CLL). This small subset of T-cells has been, in fact, considered to be involved in the pathogenesis and progression of CLL. However, whether Treg dysregulation in CLL plays a key role or it rather represents a simple epiphenomenon is still matter of debate. In the former case, Treg cells could be appealing for targeting therapies. Finally, Treg cells have also been proposed as a prognostic indicator of the disease clinical course.
PMCID: PMC3555189  PMID: 23358515
Tregs; chronic lymphocytic leukemia; prognosis
28.  Gene mutations and molecularly targeted therapies in acute myeloid leukemia 
Acute myelogenous leukemia (AML) can progress quickly and without treatment can become fatal in a short period of time. However, over the last 30 years fine-tuning of therapeutics have increased the rates of remission and cure. Cytogenetics and mutational gene profiling, combined with the option of allogeneic hematopoietic stem cell transplantation offered in selected patients have further optimized AML treatment on a risk stratification basis in younger adults. However there is still an unmet medical need for effective therapies in AML since disease relapses in almost half of adult patients becoming refractory to salvage therapy. Improvements in the understanding of molecular biology of cancer and identification of recurrent mutations in AML provide opportunities to develop targeted therapies and improve the clinical outcome. In the spectrum of identified gene mutations, primarily targetable lesions are gain of function mutations of tyrosine kinases FLT3, JAK2 and cKIT for which specific, dual and multi-targeted small molecule inhibitors have been developed. A number of targeted compounds such as sorafenib, quizartinib, lestaurtinib, midostaurin, pacritinib, PLX3397 and CCT137690 are in clinical development. For loss-of-function gene mutations, which are mostly biomarkers of favorable prognosis, combined therapeutic approaches can maximize the therapeutic efficacy of conventional therapy. Apart from mutated gene products, proteins aberrantly overexpressed in AML appear to be clinically significant therapeutic targets. Such a molecule for which targeted inhibitors are currently in clinical development is PLK1. We review characteristic gene mutations, discuss their biological functions and clinical significance and present small molecule compounds in clinical development, which are expected to have a role in treating AML subtypes with characteristic molecular alterations.
PMCID: PMC3555190  PMID: 23358589
Acute myeloid leukemia; targeted therapy; mutation; FLT3; NPM1; CEBPA; JAK2
29.  Repeated treatment with high dose cyclophosphamide for severe autoimmune diseases 
High dose cyclophosphamide (HiCY) without stem cell rescue has been shown to induce remissions in patients with severe autoimmune disorders (SADS). However, up to 80% of these patients ultimately relapse. Here we review the outcomes of seven patients treated with two cycles and one patient treated with three cycles of HiCY. The diseases re-treated were scleroderma, multiple sclerosis, three patients with severe aplastic anemia (SAA), and three patients with myasthenia gravis (MG). All but two patients with SAA had received standard immunomodulatory therapy for their disease up front and had been refractory. All patients had complete hematologic recovery. Overall survival in this cohort was 100%. All patients relapsed after the initial cycle but event free survival thereafter was 93.3%. All are still in remission except two MG patients, one of whom relapsed after a severe GI infection requiring hospitalization, and the other relapsed 3 years after the second treatment and she did not respond to the third treatment. This shows that HiCY can be safely re-administered in patients with SAA and refractory SADS. The quality and duration of second remissions appears to be equal or superior to the first remission.
PMCID: PMC3555191  PMID: 23358715
Autoimmunity; cyclophosphamide; severe autoimmune diseases
30.  Suppression of STAT5A and STAT5B chronic myeloid leukemia cells via siRNA and antisense-oligonucleotide applications with the induction of apoptosis 
Signal transducers and activators of transcription (STAT) proteins function in the JAK/STAT signaling pathway and are activated by phosphorylation. As a result of this signaling event, they affect many cellular processes including cell growth, proliferation, differentiation, and survival. Increases in the expressions of STAT5A and STAT5B play a remarkable role in the development of leukemia in which leukemic cells gain uncontrolled proliferation and angiogenesis ability. At the same time, these cells acquire ability to escape from apoptosis and host immune system. In this study, we aimed to suppress STAT-5A and -5B genes in K562 CML cells by siRNA transfection and antisense oligonucleotides (ODN) targeting and then to evaluate apoptosis rate. Finally, we compared the transfection efficiencies of these approaches. Quantitative RT-PCR and Western blot results indicated that STAT expressions were downregulated at both mRNA and protein levels following siRNA transfection. However, electroporation mediated ODN transfection could only provide limited suppression rates at mRNA and protein levels. Moreover, it was displayed that apoptosis were significantly induced in siRNA treated leukemic cells as compared to ODN treated cells. As a conclusion, siRNA applications were found to be more effective in terms of gene silencing when compared to ODN treatment based on the higher apoptosis and mRNA suppression rates. siRNA application could be a new and alternative curative method as a supporting therapy in CML patients.
PMCID: PMC3555192  PMID: 23358828
Chronic myeloid leukemia; K562; STAT5; siRNA knockdown; antisense oligonucleotides; apoptosis
31.  Function of Ikaros as a tumor suppressor in B cell acute lymphoblastic leukemia 
The Ikaros transcription factor is crucial for many aspects of hematopoiesis. Loss of function mutations in IKZF1, the gene encoding Ikaros, have been implicated in adult and pediatric B cell acute lymphoblastic leukemia (B-ALL). These mutations result in haploinsufficiency of the Ikaros gene in approximately half of the cases. The remaining cases contain more severe or compound mutations that lead to the generation of dominant-negative proteins or complete loss of function. All IKZF1 mutations are associated with a poor prognosis. Here we review the current genetic, clinical and mechanistic evidence for the role of Ikaros as a tumor suppressor in B-ALL.
PMCID: PMC3555193  PMID: 23358883
B cell leukemia; Ikaros; tumor suppressor
32.  Bruton’s tyrosine kinase mediated signaling enhances leukemogenesis in a mouse model for chronic lymphocytic leukemia 
In chronic lymphocytic leukemia (CLL) signals from the B cell receptor (BCR) play a major role in disease development and progression. In this light, new therapies that specifically target signaling molecules downstream of the BCR continue to be developed. While first studies on the selective small molecule inhibitor of Bruton’s tyrosine kinase (Btk), Ibrutinib (PCI-32765), demonstrated that Btk inhibition sensitizes CLL cells to apoptosis and alters their migratory behavior, these studies however did not address whether Btk-mediated signaling is involved in the process of CLL leukemogenesis. To investigate the requirement of Btk signaling for CLL development, we modulated Btk expression in the IgH.ETμ CLL mouse model, which is based on sporadic expression of the simian oncovirus SV40 T-antigen in mature B cells. To this end, we crossed IgH.ETμ mice on a Btk-deficient background or introduced a human Btk transgene (CD19-hBtk). Here we show that Btk deficiency fully abrogates CLL formation in IgH.ETμ mice, and that leukemias formed in Btk haplo-insufficient mice selectively expressed the wild-type Btk allele on their active X chromosome. Conversely, Btk overexpression accelerated CLL onset, increased mortality, and was associated with selection of non-stereotypical BCRs into CLL clones. Taken together, these data show that Btk expression represents an absolute prerequisite for CLL development and that Btk mediated signaling enhances leukemogenesis in mice. We therefore conclude that in CLL Btk expression levels set the threshold for malignant transformation.
PMCID: PMC3555194  PMID: 23359016
Chronic lymphocytic leukemia (CLL); bruton’s tyrosine kinase (Btk); B cell receptor signaling
33.  Kinetics of iron removal by phlebotomy in patients with iron overload after allogeneic hematopoietic cell transplantation 
Excess body iron could persist for years after allogeneic hematopoietic cell transplantation (HCT) with possible deleterious sequels. An iron depletive therapy with phlebotomy seems rational. Kinetics of iron removal by phlebotomy without erythropoietin support in non-thalassemic adult patients with iron overload after HCT and the impact of pre- and post-HCT hemochromatosis (HFE) genotype on iron mobilization were investigated. Patients and methods: Phlebotomy was initiated in 61 recipients of allografts due to hematologic malignancies (median age 48 years) after a median of 18 months. The prephlebotomy median serum ferritin (SF) was 1697ng/ml and the median number of blood transfusions 28 units. Alanine aminotransferase (ALT)/aspartate aminotransferase (AST), alkaline phosphates (AP), and bilirubin were elevated in 55.7%, 64% and 11.5% patients respectively. HFE-genotype was elucidated by polymerase chain reaction using hybridization probes and melting curve analysis. Results: Phlebotomy was well-tolerated irrespective of age or conditioning. A negative iron balance in 80% of patients (median SF 1086 ng/ml) and a rise in hemoglobin were observed (p<0.0001). Higher transfusional burden and SF were associated with a greater iron mobilization per session (p=0.02). In 58% of patients, a plateau after an initial steady decline in SF was followed by a second decline under further phlebotomy. The improvement in ALT (p=0.002), AST (p=0.03), AP (p=0.01), and bilirubin (p<0.0001) did not correlate with the decline in SF. Mutant HFE-gene variants were detected in 14/55 (25%) pre-HCT and 22/55 (40%) patients post-HCT. Overall, dissimilar pre- and posttransplantational HFE-genotypes were detected in 20/55 (40%) patients. Posttransplantational mutant HFE variants correlated with a slower decline in SF (p=0.007). Conclusions: Phlebotomy is a convenient therapy of iron overload in survivors of HCT. A negative iron balance and a rise in hemoglobin were observed in the majority of patients. Liver dysfunction improved irrespective of SF reduction suggesting a probable rapid decline of the deleterious labile plasma iron. In recipients of grafts with mutant HFE variants a “mixed chimerism” of HFE in body tissues might be created with a change in the set point for iron regulation. The transient plateau in SF after an initial decline might reflect iron mobilization from various tissues.
PMCID: PMC3512175  PMID: 23226624
Iron overload; ferritin; phlebotomy; allogeneic HCT
34.  Management of respiratory viral infections in hematopoietic cell transplant recipients 
Advances in stem cell transplantation procedures and the overall improvement in the clinical management of hematopoietic cell transplant (HCT) recipients over the past 2 decades have led to an increase in survival duration, in part owing to better strategies for prevention and treatment of post-transplant complications, including opportunistic infections. However, post-HCT infections remain a concern for HCT recipients, particularly infections caused by community respiratory viruses (CRVs), which can lead to significant morbidity and mortality. These viruses can potentially cause lower respiratory tract illness, which is associated with a higher mortality rate among HCT recipients. Clinical management of CRV infections in HCT recipients includes supportive care and antiviral therapy, especially in high-risk individuals, when available. Directed antiviral therapy is only available for influenza infections, where successful use of neuraminidase inhibitors (oseltamivir or zanamivir) and/or M2 inhibitors (amantadine or rimantadine) has been reported. Data on the successful use of ribavirin, with or without immunomodulators, for respiratory syncytial virus infections in HCT recipients has emerged over the past 2 decades but is still controversial at best because of a lack of randomized controlled trials. Because of the lack of directed antiviral therapy for most of these viruses, prevention should be emphasized for healthcare workers, patients, family, and friends and should include the promotion of the licensed inactivated influenza vaccine for HCT recipients, when indicated. In this review, we discuss the clinical management of respiratory viruses in this special patient population, focusing on commercially available antivirals, adjuvant therapy, and novel drugs under investigation, as well as on available means for prevention.
PMCID: PMC3512176  PMID: 23226621
RSV; influenza; parainfluenza; adenovirus; rhinovirus; metapneumovirus; HCT; transplant; cancer; immunocompromised host; antiviral therapy; infection prevention
35.  Growth factor independence 1 (Gfi1) regulates cell-fate decision of a bipotential granulocytic-monocytic precursor defined by expression of Gfi1 and CD48 
The transcriptional repressor Gfi1 regulates the expression of genes important for survival, proliferation and differentiation of hematopoietic cells. Gfi1 deficient mice are severely neutropenic and accumulate ill-defined CD11b+GR1int myeloid cells. Here we show that Gfi1 expression levels determine mono- or granulocytic lineage choice in precursor cells. In addition, we identify CD48 as a cell surface marker which enables a better definition of monocytes and granulocytes in mouse bone marrow. Using the CD48/Gr1/Gfi1 marker combination we can show that the CD11b+GR1int cells accumulating in Gfi1 deficient mice are monocytes and not granulocyte precursors. Expression of CD48, Gr1 and Gfi1 define different bone marrow subpopulations that are either committed to the granulocytic lineage, or bipotential precursors of granulocytes or monocytes. Finally, a comparison of genes differentially expressed between murine Gfi1 high granulocytic precursors and mature granulocytes with gene expression changes from human myeloblasts versus neutrophils show a strong resemblance of human and mouse differentiation pathways. This underlines the value of the markers CD48 and Gfi1 identified here to study human and murine granulo-monocytic differentiation.
PMCID: PMC3512177  PMID: 23226623
Gfi1; CD48; CD106; granulocyte; monocyte; myelopoiesis; neutropenia
36.  Acute myeloid leukemia with t(10;17)(p13;q12) chromosome translocation: a case report and literature review 
More than 50% of adult patients with acute myeloid leukemia (AML) carry chromosome abnormalities, like t(8;21)(q22;q22), t(15;17), t(8;21)inv(16) or t(16;16). t(10;17) translocation was very rare in AML. There are only 10 such cases reported in the literature. Here, we describe a case of acute myeloid leukemia with t(10;17)(p13;q12) chromosome translocation, who had complete remission after one course of chemotherapy.
PMCID: PMC3512178  PMID: 23226626
Acute myeloid leukemia; t(10;17)(p13;q12); chromosome translocation
37.  Prognostic significance of IDH1 mutations in acute myeloid leukemia: a meta-analysis 
Isocitrate dehydrogenase 1 (IDH1) gene aberrations have recently been reported in acute myeloid leukemia (AML). To evaluate the prognostic significance of IDH1 mutations in AML, we performed a meta-analysis. Fifteen studies covering a total of 8121 subjects were included in this analysis. The frequency of IDH1 R132 mutations were 4.4–9.3% for AML patients and 10.9–16.0% for cytogenetically normal (CN)-AML patients. The IDH1 mutations were associated with NPM1 mutations in 6 studies and normal cytogenetics in 5 studies. AML patients with IDH1 mutations had inferior overall survival compared to patients without the mutations (hazard ratio 1.17, 95% CI: 1.02–1.36). Additionally, in CN-AML patients, IDH1 mutations were associated with a lower complete remission rate (risk ratio 1.30, 95% CI: 1.04–1.63). Although the available literature is limited to observational studies, these results may justify the risk-adapted therapeutic strategies for AML according to the IDH1 status.
PMCID: PMC3512179  PMID: 23226625
Acute myeloid leukemia; IDH1; mutation; prognosis; meta-analysis
38.  Hematopoietic stem cells: interplay with immunity 
Ample evidence indicated that hematopoietic stem cells (HSCs) receive signaling from infection or other immune responses to adjust their differentiation and self-renewal. More recent reports also suggested that, while the bone marrow microenvironment or niche may provide the immune privilege for HSCs, HSCs can present surface immune inhibitors per se to suppress innate immunity and adaptive immunity to evade potential immune surveillance and attack. These findings support the hypothesis that HSCs are capable of interacting with the immune system as signal “receivers” and signal “providers”. On the one hand, HSCs are capable of directly sensing the signals from the immune system through their surface receptors to modulate their self-renewal and differentiation (“in” signaling); on the other hand, HSCs display surface immune inhibitory molecules to evade the attack from the innate and adaptive immune systems (“out” signaling). The continuing investigation of the interplay between HSCs and immunity may lead to the open-up of a new research filed – the immunology of stem cells.
PMCID: PMC3512180  PMID: 23226622
Hematopoietic stem cells; immunity; immune privilege; CD47; CD274; LILRB2; PIR-B; immune inhibitory receptors; infection; inflammation
39.  Unbalanced replication as a major source of genetic instability in cancer cells 
The origin of genetic instability in tumors is a matter of debate: while the prevailing model postulates a mutator phenotype resulting from an alteration in a caretaker gene as a prerequisite for genetic alterations leading to tumor formation, there is evidence against this model in the majority of cancers. A model for chromosomal instability should take into account the role of oncogenes in directly stimulating DNA and cellular component replication, creating aberrant structures when overexpressed. I will distinguish here two distinct mechanisms for the genetic instability of tumors: primary and secondary. Primary genetic instability is dependent on the inactivation of genes involved in maintaining genetic stability (caretaker genes), whereas secondary genetic instability is dependent on genes involved in tumor progression, i.e. oncogenes and tumor suppressor genes of the gatekeeper type. Secondary genetic instability, the most frequent condition, can be explained by the fact that some of the genes involved in tumor progression control replication of cell structures from within, leading to replication unbalance.
PMCID: PMC3484411  PMID: 23119227
Genetic instability; tumorigenesis; oncogenes; tumor suppressor genes; DNA replication; cell replication; replication unbalance; chromosomal instability
40.  Gastrointestinal malignant lymphoma: a pathologic study of 37 cases in a single Japanese institution 
The author reviewed pathologic features of 37 cases of malignant lymphoma in the gastrointestinal organs in the last 10 years in our pathology laboratory. The current WHO classification was adopted. The 37 cases consisted of 20 males and 17 female, and the age ranged from 46 to 89 years with a median of 69 years. Of the 37 cases, 25 cases (68%) were gastric lymphomas, 6 cases (16%) were small intestinal lymphomas, and 6 cases (16%) were colon lymphomas. Of the 37 cases, 35 cases (95%) were B-cell neoplasms and 2 cases (5%) were T-cell neoplasms. In the 25 gastric lymphomas (male:female=14:11, age range 46-84 years, median 70 years) 11 cases were diffuse large B-cell lymphomas, and 14 cases were extranodal marginal zone B-cell lymphomas (MALT lymphomas). The clinical endoscopic diagnosis was gastritis in 3, gastric ulcer in 3, gastric carcinoma in 7, carcinoid in 1, submucosal tumor in 1, malignant lymphoma in 2, and suspected MALT lymphoma in 8. In the 6 small intestinal lymphomas (male:female=2:4, age range 49-89 years, median 70 years), all cases were located in the ileum. Of the 6 cases, 4 were diffuse large B-cell lymphoma and 2 were peripheral T-cell lymphoma. One case showed multiple lymphomas, and one case was associated with rectal adenocarcinoma and one case with gastric MALT lymphoma. The clinical diagnosis was adenocarcinoma in 2, suspected lymphoma in 2, and ileal tumor in 2. In the 6 colon lymphomas (male:female=4:2, age range 69-86 years, median 74 years), 5 cases were diffuse large B-cell lymphomas and one case was follicular lymphoma. Clinical endoscopic diagnosis was GIST in 1, colon carcinoma in 4, and colon polyp in 1. Cases of Hodgkin’s disease, mantle cell lymphoma, Burkitt lymphoma were not recognized in the present series. In summary, the author reported pathologic features of 37 cases of gastrointestinal malignant lymphoma in our laboratory in the last 10 years.
PMCID: PMC3484413  PMID: 23119230
Malignant lymphoma; gastrointestinal organs
41.  Phosphorylation of p47phox is required for receptor-mediated NADPH oxidase/NOX2 activation in Epstein-Barr virus-transformed human B lymphocytes 
The phagocyte NADPH oxidase (NOX2) is known to be expressed in Epstein-Barr virus (EBV)-transformed human B lymphocytes. Phosphorylation of the NOX2 cytosolic subunit p47phox is required for phorbol myristate acetate (PMA)-induced NOX2 activation in EBV-transformed B lymphocytes, however the role of this process in receptor-mediated NOX2 activation is not known. Here, we used pansorbin which acts by cross linking cell surface IgG and transfected cells with mutated p47phox to address if the phosphorylation of this subunit is required for receptor-mediated NOX2 activation. We show that pansorbin induced NOX2 activation in a time and concentration-dependent manner, albeit at levels only of 20% of those induced by PMA. GF109203X, a PKC selective inhibitor, inhibited pansorbin as well as PMA-induced NOX2 activation. Using specific anti-phospho serine antibodies we showed that pansorbin induced p47phox phosphorylation on Ser304, 315, 320, 328, and 345 and kinetics of these phosphorylations preceed NOX2 activation. To determine whether the phosphorylation of p47phox is required for pansorbin-induced NOX2 activation, we transfected EBV-transformed lymphocytes deficent in p47phox with a plasmid expressing wild type p47phox or p47phox with all the phosphorylated serines mutated to alanines, p47phoxS(303-379)A. Results show that pansorbin-induced NOX2 activation was greatly decreased in lymphocytes expressing the mutant as compared to the wild-type p47phox. These results show that pansorbin induced p47phox phosphorylation on multiple sites in EBV-transformed B lymphocytes and this process is required for pansorbin-induced NADPH oxidase activation in these cells.
PMCID: PMC3484414  PMID: 23119229
NADPH oxidase; NOX2; p47phox; B lymphocytes; pansorbin; ROS; phosphorylation
42.  Benefits of hypoxic culture on bone marrow multipotent stromal cells 
Cultivation of cells is usually performed under atmospheric oxygen tension; however, such a condition does not replicate the hypoxic conditions of normal physiological or pathological status in the body. Recently, the effects of hypoxia on bone marrow multipotent stromal cells (MSCs) have been investigated. In a long-term culture, hypoxia can inhibit senescence, increase the proliferation rate and enhance differentiation potential along the different mesenchymal lineages. Hypoxia also modulates the paracrine effects of MSCs, causing upregulation of various secretable factors, including the vascular endothelial growth factor and interleukin-6, and thereby promoting wound healing and diabetic fracture healing. Finally, hypoxia plays an important role in mobilization and homing of MSCs, primarily by its ability to induce stromal cell-derived factor-1 expression along with its receptor, CXCR4. After transplantation, an ischemic environment, that is the combination of hypoxia and lack of nutrition, can lead to apoptosis or cell death, which can be overcome by the hypoxic preconditioning of MSCs and overexpression of prosurvival genes like Akt, HO-1 and Hsp70. This review emphasizes that hypoxia is an important factor in all major aspects of stem cell biology, and the mechanism involved in the hypoxic inducible factor-1signaling pathway behind these responses is also discussed.
PMCID: PMC3484415  PMID: 23119226
Mesenchymal stem cells; hypoxia; hypoxic preconditioning; proliferation; differentiation potential; apoptosis; migration; engraftment; HIF-1
44.  Emerging therapeutic options for myelofibrosis: a Canadian perspective 
Myelofibrosis (MF) is a clonal stem cell disorder characterized by cytopenias, splenomegaly, marrow fibrosis, and systemic symptoms due to elevated inflammatory cytokines. MF is associated with decreased survival. The quality of life of patients with MF is similar to other advanced malignancies. Allogeneic hematopoietic cell transplantation is a curative treatment, but is applicable to a minority of patients with MF. None of the conventional therapies are known to alter the natural history of the disease. Significant progress has been made in the last few years in the understanding of disease biology of MF. Discovery of the JAK2V617F mutation paved the way for drug discovery in MF, and the first JAK1/2 inhibitor, ruxolitinib, has been approved by FDA and Health Canada. Several other JAK1/2 inhibitors are at various stages of clinical development. As a consequence, the therapeutic landscape of MF is changing from a disease where no effective therapies existed to one with several novel treatment options on the horizon. In this report, we assess the changing therapeutic options for MF, and critically analyze the position of novel treatments in the current armamentarium.
PMCID: PMC3484412  PMID: 23119228
Myelofibrosis; JAK1/2; ruxolitinib; splenomegaly; treatment options
45.  The involvement of Galectins in the modulation of the JAK/STAT pathway in myeloproliferative neoplasia 
In patients with myeloproliferative neoplasia (MPN) the development of fibrosis and increased vessel density correlate with poor prognosis. The JAK2V617F mutation constitutively activates JAK2, which phosphorylates signal transducer activator of transcription (STAT), up-regulating vascular endothelial growth factor (VEGF), which might be responsible for angiogenesis in MPN. Galectins are involved in the development of fibrosis and angiogenesis and might also be involved in activation of the JAK/STAT pathway in MPN.
106 MPN patients, 36 essential thrombocythemia (ET), 25 polycythemia vera (PV) and 45 primary myelofibrosis (PMF), were analyzed for the expression pattern of galectin-1, galectin-3, pSTAT3, pSTAT5 and MVD by immunostaining of bone marrow biopsy sections followed by automated image analysis. The JAK2 mutational status was analysed through real time PCR in blood samples.
The expression of galectin-1 was significantly higher in all MPN patients compared to normal controls. Galectin-3 was expressed more in PV patients. MVD was significantly higher in all MPN patients and correlated with galectin-1 and pSTAT5 expression. pSTAT5 expression showed a trend of higher expression in patients carrying the JAK2V617F mutation as well as in PV patients. PMF patients and all JAK2V617F positive patients showed a significantly higher pSTAT3 expression compared to control and ET patients.
The findings suggest the involvement of galectin-1 in MPN development, regardless of the subtype. Furthermore involvement of galectin-3 in PV development, pSTAT5 in that of PV and JAK2V617F positive patients and angiogenesis, as well as pSTAT3 is involved in the pathogenesis of PMF.
PMCID: PMC3384397  PMID: 22762031
MPN; myeloproliferative neoplasia; galectin; JAK; STAT; angiogenesis; MVD
46.  Quality of life and physicians' perception in myelodysplastic syndromes 
To detect factors associated with quality of life (QOL) of patients with myelodysplastic syndrome (MDS) and to compare the MDS patients’ self-assessed QOL with that perceived by their physicians. In an observational, non-interventional, prospective, multicentre study, QOL was evaluated in 148 patients with newly diagnosed low- and intermediate-risk IPSS MDS. QOL measures (QOL-E v.2, LASA and EQ-5D) and patient-related candidate determinants of QOL were assessed for up to 18 months. Patients' QOL scores were compared with those obtained by appointed hematologists’ assessment and with ECOG performance status (PS). Fatigue was not prevalent at diagnosis, though physical QOL and energy levels were low. Transfusion-dependent patients had worse QOL scores. In multivariate analysis, Hb levels and comorbidities were a major determinant of QOL. Physicians’ perception of patients’ well-being correlated with patients’ QOL. Physicians underestimated the impact of disturbances on patients’ QOL, mainly in the MDS-specific components. ECOG PS did not discriminate patients according to QOL status. In conclusion, the association of anemia with QOL is confirmed, while co-morbidities emerge as an independent predictor of QOL in MDS. Fatigue is not a major concern. ECOG PS is not a valuable surrogate of patient’s QOL, thus highlighting that physician’s judgment of patient’s well-being must not substitute patient-reported outcomes. Appropriate questionnaires should be used to assess MDS patients’ QOL in order to improve communication and therapeutic choice.
PMCID: PMC3384400  PMID: 22762033
Myelodysplastic syndromes; quality of life; comorbidities; anemia; transfusion-dependence; patient-reported outcomes
47.  Time and temperature stability of T-cell subsets evaluated by a dual-platform method 
T-cell subset enumeration in HIV patients is routinely performed for monitoring infection stage and response to antiretroviral therapy. Studies have examined the effect of specimen refrigeration and age for single-platform (SP) methods, but there is limited data for time and temperature requirements of dual-platform (DP) methods.
Using a DP method, we analyzed peripheral blood (PB) from 52 HIV patients at room temperature (RT) at 24, 72, and 96 hours. PBs from 34 HIV patients had baseline RT analysis within 24 hours, and then were refrigerated and analyzed at 24, 48, and 72 hours. The coefficient of variation (CV) and residuals (changes in lymphocyte subsets) were recorded at each time point and compared to assess the precision and bias under the various conditions. Testing performance under different conditions was compared by linear regression.
Mean CV was ≤7.3% and median residuals were <30/μl for absolute CD4 and CD8 determinations. There was good correlation between baseline analysis data at RT and at various time points, both at RT and 4°C.
Our results are similar to those published for SP methods for aging or refrigerated specimens. The high level of agreement between measurements supports the robustness of this DP methodology.
PMCID: PMC3384401  PMID: 22762032
HIV; Absolute CD4 counts; flow cytometry; dual platform; specimen stability
48.  A long road of T-cells to cure cancer: from adoptive immunotherapy with unspecific cellular products to donor lymphocyte infusions and transfer of engineered tumor-specific T-cells 
The notion that immunocompetent cells, contained within adult bone marrow or peripheral blood, are capable of mediating an antitumor effect was first validated experimentally in 1957. T-cell immunotherapy for malignant disease is now routinely used in the context of allogeneic bone marrow transplantation. After 50 years of investigations into the use of T-cells for cancer therapy, adoptive cellular immunotherapy for cancer has progressed from the delivery of unspecific cellular products to the transfer of engineered tumor-specific T-cells. Adoptive cellular immunotherapy for cancer has now reached a stage of increasing feasibility and efficacy.
PMCID: PMC3384398  PMID: 22762028
Immunocompetent cells; antitumor effect; T-cell immunotherapy; allogeneic bone marrow transplantation; tumor-specific T-cells
49.  Advances and application of radioimmunotherapy in non-Hodgkin lymphoma 
The activity of radio-immuno conjugate in Non-Hodgkin Lymphoma (NHL) has resulted in FDA approval of two antibodies, Y90 Ibritumomab tiuxetan and I131 tositumomab. Both these agents target CD20, a receptor widely expressed in B-Cell NHL. These immunoconjugates deliver their radioactive payload to the malignant clone in the bone marrow and lymph node. Their use has been associated with modest improvement in survival end points among several lymphoma histologies. The promising effect on disease control as well as their efficacy in disease relapse is encouraging in low grade lymphoma. Radioimmunotherapy (RIT) is increasingly being explored in the setting of consolidation as well as conditioning regimens prior to stem cell transplantation. Here, we summarize the clinical use, complications and future applications of RIT in NHL.
PMCID: PMC3384399  PMID: 22762027
Radioimmunotherapy; non-Hodgkin lymphoma; stem cell transplantation; CD20 target
50.  Subtle bone marrow involvement by large B-cell lymphoma with pronormoblast-like morphology and prominent but not exclusive sinusoidal distribution 
Primary bone marrow presentation of diffuse large B-cell lymphoma (DLBCL) is unusual, and appreciation of the diffuse growth pattern may be difficult in cases with low-level involvement. In particular, subtle sinusoidal and interstitial bone marrow involvement and morphologic overlap of the tumor cells with pronormoblasts may result initially in a missed diagnosis. We describe the clinicopathologic features of 13 cases of morphologically subtle DLBCL involving the bone marrow, which were only identified with the aid of immunohistochemistry. The overwhelming majority of cases (12/13, 92%) presented with cytopenias, and 5 of 7 cases, with available information, had splenomegaly. The morphology of the tumor cells in the aspirate smears overlapped with pronormoblasts (immature erythroid precursors) in 12 of 13 cases. Similarly, in histologic sections, the tumor cells in virtually all cases (12/13) demonstrated round nuclear contours and oblong nucleoli, mimicking pronormoblasts. A CD20 immunohistochemical stain was essential in identifying the neoplastic infiltrate in all cases. The majority of cases (73%, 10/13) showed low-level bone marrow involvement by lymphoma, 10% or less. CD20 immunohistochemistry highlighted individually dispersed and small clusters of large lymphoid cells in a sinusoidal and/or interstitial growth pattern. Most of the cases that were assessed showed a non-germinal center phenotype (CD10-, BCL6-/+, IRF4/MUM1+). There was an aggressive disease course with a median overall survival of 6 months. We would recommend performing a CD20 immunostain in patients who present with unexplained cytopenias and/or splenomegaly. Further investigation is warranted to better describe the features of this unique and aggressive variant of DLBCL.
PMCID: PMC3384402  PMID: 22762030
Diffuse large B cell lymphoma; bone marrow; immunohistochemistry; CD20; pronormoblast-like

Results 26-50 (85)