Netanel A. Horowitz

Colitis-Associated Cancer Is Dependent on the Interplay between the Hemostatic and Inflammatory Systems and Supported by Integrin αMβ2 Engagement of Fibrinogen

A link between colitis and colon cancer is well established, but the mechanisms regulating inflammation in this context are not fully defined. Given substantial evidence that hemostatic system components are powerful modulators of both inflammation and tumor progression, we used gene-targeted mice to directly test the hypothesis that the coagulation factor fibrinogen contributes to colitis-associated colon cancer in mice. This fundamental provisional matrix protein was found to be an important determinant of colon cancer. Fibrinogen deficiency resulted in a dramatic diminution in the number of colonic adenomas formed following azoxymethane/dextran sodium sulfate challenge. More detailed analyses in mice expressing a mutant form of fibrinogen that retains clotting function, but lacks the leukocyte integrin receptor alpha(M)beta(2) binding motif (Fibgamma(390-396A)), revealed that alpha(M)beta(2)-mediated engagement of fibrin(ogen) is mechanistically coupled to local inflammatory processes (e.g., interleukin-6 elaboration) and epithelial alterations that contribute to adenoma formation. Consistent with these findings, the majority of Fibgamma(390-396A) mice developed no discernable adenomas, whereas penetrance was 100% in controls. Furthermore, the adenomas harvested from Fibgamma(390-396A) mice were significantly smaller than those from control mice and less proliferative based on quantitative analyses of mitotic indices, suggesting an additional role for fibrin(ogen) in the growth of established adenomas. These studies show, for the first time, a unique link between fibrin(ogen) and the development of inflammation-driven malignancy. Given the importance of antecedent inflammation in the progression of numerous cancers, these studies suggest that therapies targeting fibrin(ogen)-alpha(M)beta(2) interactions may be useful in preventing and/or treating this important subset of malignancies.

Eradication of carbapenem-resistant Enterobacteriaceae gastrointestinal colonization with nonabsorbable oral antibiotic treatment: A prospective controlled trial

BACKGROUND Carbapenem-resistant Enterobacteriaceae (CRE) are emerging. In attempt to eradicate CRE colonization, we conducted a semirandomized, prospective, controlled trial using oral nonabsorbable antibiotics. METHODS Consecutive hospitalized CRE carriers were studied. Patients whose rectal isolates were gentamicin sensitive but colistin resistant were treated with gentamicin. Patients whose isolates were colistin sensitive but gentamicin resistant were treated with colistin. Patients whose isolates were sensitive to both drugs were randomized to 3 groups of oral antibiotic treatment: gentamicin, colistin, or both. Patients whose isolates were resistant to both drugs, and those who did not consent, were followed for spontaneous eradication. RESULTS One hundred fifty-two patients were included; 102 were followed for spontaneous eradication for a median duration of 140 days (controls), and 50 received 1 of the 3 drug regimens: gentamicin, 26; colistin, 16; both drugs, 8, followed for a median duration of 33 days. Eradication rates in the 3 treatment groups were 42%, 50%, and 37.5%, respectively, each significantly higher than the 7% spontaneous eradication rate in the control group (P < .001, P < .001, and P = .004, respectively) with no difference between the regimens. No significant adverse effects were observed. CONCLUSION Oral antibiotic treatment with nonabsorbable drugs to which CRE is susceptible appears to be an effective and safe for eradication of CRE colonization and, thereby, may reduce patient-to-patient transmission and incidence of clinical infection with this difficult-to-treat organism.

Thrombophilia and Cancer

Inherited and acquired thrombophilia are well-known risk factors for venous thromboembolism. The incidence of thrombotic events in cancer patients is increased compared to normal population. Data on inherited thrombophilia and cancer is limited. Most studies are small, and results are varied by geography, tumor type, stage of disease and therapy. Nevertheless, it seems that factor V Leiden and prothrombin mutation may increase VTE risk in cancer patients. Data also exist for hyperhomocysteinemia. Cancer patients with thrombophilia who have a central vein catheter are a special group with increased venous thrombotic event (VTE) risk. Among acquired risk factors, antiphospholipid antibodies are common in cancer patients, especially in lymphoproliferative diseases. Published data suggests that the risk of thrombotic events, venous and arterial, is increased in cancer patients with antiphospholipid antibodies. The questions of screening cancer patients for inherited thrombophilia or antiphospholipid antibodies are unclear. There is no data on primary VTE prophylaxis in cancer patients with thrombophilia and prophylaxis for cancer patients with central vein catheters is controversial.

Heparanase-neutralizing antibodies attenuate lymphoma tumor growth and metastasis

Significance Heparanase is the predominant enzyme that cleaves heparan sulfate (HS) in mammals, a linear polysaccharide that is normally attached to a core protein, forming HS proteoglycans (HSPGs) that are abundant in the cell surface and extracellular matrix (ECM). Cleavage of HS by heparanase results in structural alterations of the ECM and release of ECM-bound factors that together stimulate cancer metastasis and angiogenesis. Here we provide evidence that heparanase is expressed by B-lymphomas, and heparanase inhibitors restrain tumor growth. Furthermore, we describe, for the first time to our knowledge, the development and characterization of heparanase-neutralizing monoclonal antibodies (mAbs) that inhibit cell invasion and tumor metastasis. Moreover, we show that these mAbs attenuate lymphoma growth by targeting heparanase in the tumor microenvironment. Heparanase is an endoglycosidase that cleaves heparan sulfate side chains of proteoglycans, resulting in disassembly of the extracellular matrix underlying endothelial and epithelial cells and associating with enhanced cell invasion and metastasis. Heparanase expression is induced in carcinomas and sarcomas, often associating with enhanced tumor metastasis and poor prognosis. In contrast, the function of heparanase in hematological malignancies (except myeloma) was not investigated in depth. Here, we provide evidence that heparanase is expressed by human follicular and diffused non-Hodgkins B-lymphomas, and that heparanase inhibitors restrain the growth of tumor xenografts produced by lymphoma cell lines. Furthermore, we describe, for the first time to our knowledge, the development and characterization of heparanase-neutralizing monoclonal antibodies that inhibit cell invasion and tumor metastasis, the hallmark of heparanase activity. Using luciferase-labeled Raji lymphoma cells, we show that the heparanase-neutralizing monoclonal antibodies profoundly inhibit tumor load in the mouse bones, associating with reduced cell proliferation and angiogenesis. Notably, we found that Raji cells lack intrinsic heparanase activity, but tumor xenografts produced by this cell line exhibit typical heparanase activity, likely contributed by host cells composing the tumor microenvironment. Thus, the neutralizing monoclonal antibodies attenuate lymphoma growth by targeting heparanase in the tumor microenvironment.

Reproductive organ involvement in non-Hodgkin lymphoma during pregnancy: a systematic review

Data for pregnancy-associated non-Hodgkin lymphoma are limited to case reports, making it difficult to define this disorder. We did a systematic search for articles published between 1967 and 2011 with the aim to determine the characteristics, management, and outcome of pregnancy-associated non-Hodgkin lymphoma. We identified 121 patients from 74 papers. Most patients with stage information available presented with stage IV disease (75%, 82 of 108 patients). Patients were classified into three clinical groups; those with indolent lymphomas accounted for 5% (five of 108), aggressive lymphomas (diffuse large B-cell lymphoma and T-cell lymphomas) made up 48% of patients (52 of 108), and highly aggressive lymphomas (Burkitts lymphoma, immunoblastic lymphoma, and unspecified highly aggressive lymphomas) accounted for 47% of patients (51 of 108). Reproductive organ involvement (breast, ovary, uterus, placenta) was reported in 49% of 110 patients with information available on extranodal involvement, and prevailed in endemic Burkitts lymphoma (100%, 19 of 19), followed by non-endemic Burkitt lymphoma (70%, 14 of 20), immunoblastic lymphoma (67%, two of three), peripheral T-cell lymphoma (46%, six of 13), and indolent (40%, two of five) and diffuse large cell lymphoma (23%, nine of 40). Most patients received antepartum (45%, 55 of 121) or postpartum therapy (45%, 54 of 121), resulting in 6-month survival of 53% for mothers and a livebirth rate of 83%. Pregnancy-associated non-Hodgkin lymphoma has unique clinical characteristics with frequent reproductive organ involvement. Collaborative prospective studies are needed to further characterise pathophysiological and clinical aspects of this complication.

Bortezomib-induced peripheral neuropathy is related to altered levels of brain-derived neurotrophic factor in the peripheral blood of patients with multiple myeloma

Loss of nerve growth factor-mediated neuronal survival has recently been proposed as a candidate mechanism underlying bortezomib-induced peripheral neuropathy (BIPN) (Broyl et al, 2010). However the literature does not reveal any data from patients that can support this hypothesis. Brain-derived neurotrophic factor (BDNF) is a neuronal growth factor that is crucial for neuronal survival and repair (Hofer & Barde, 1988). We report on alterations in BDNF peripheral blood levels and the development of BIPN in patients with multiple myeloma (MM). Following approval of the Ethics Committee and obtaining patient’s informed consent, peripheral neuropathy was assessed and graded in 25 MM patients using the abbreviated version of the Total Neuropathy Score (TNS) tool (Argyriou et al, 2008). These patients were examined at diagnosis and after receiving four courses of a bortezomib-based regimen given intravenously either weekly or biweekly (Table I). Patients that developed TNS ≥ 2 during treatment were determined to have BIPN and three patients with TNS ≥ 2 at diagnosis were excluded from the study. At the time of neurological examination, a venous blood sample was obtained and soluble BDNF (sBDNF) level was determined by a commercial enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA) in platelet-poor-plasma. One of our patients passed away after two courses of bortezomib. Eight of the remaining patients (36%) developed BIPN. We found lower levels of sBDNF in the plasma of patients that developed BIPN as compared to patients that did not develop BIPN [BDNF level standard deviation (SD) in BIPN 2 164 0 721 vs. 4 62 0 61 ng/ml in non-BIPN; P = 0 007; Fig 1A]. By analysing the difference between BDNF levels at diagnosis and after four courses of treatment of each patient, we demonstrated that sBDNF level was reduced in patients developing BIPN while it remained stable or even minimally elevated in patients that did not develop BIPN (difference in BDNF SD in BIPN 1 668 0 670 vs. 0 405 0 708 ng/ml in non-BIPN P = 0 02) (Fig 1B). Platelets are thought to be responsible for the homeostasis of BDNF in the blood as they take up, store and secrete BDNF (Fujimura et al, 2002). As we did not observe differences in the platelet counts of patients that developed BIPN compared to patients that did not develop BIPN (data not shown), we reasoned that decreased sBDNF levels in the plasma of patients with BIPN may result from lack of secretion of BDNF from the platelets. To test this hypothesis, we determined the content of BDNF in the platelets of patients that developed BIPN relative to those who did not develop BIPN by two different methods. First, platelets that were collected and stored in 20°C at the time of the patients’ examination were lysed and the BDNF immunoreactive band was determined by gel electrophoresis using specific rabbit polyclonal anti-human BDNF mAb (Abcam, Cambridge, MA, USA). Second, BDNF and the platelet activation marker CD62p were determined in freshly isolated platelets of patients with BIPN and patients without BIPN using flow cytometry. Briefly, 20 ll of platelet-rich-plasma from each patient was stained with phycoerythrin-cyanin 5 (PE-CY5) Anti human CD41a monclonal antobody (mAb) (Beckmann Coulter, Brea, CA, USA) and PE anti human CD62p mAb (Biolegends, San Diego, CA, USA) or fixed permeabilized (Invitrogen, Frederick, MD, USA) and stained with ATTO-488 Anti human proBDNF pAb (Alomone Labs Ltd., Jerusalem, Israel). The intensity of surface CD62p or intracellular BDNF signal was determined in 10 000 platelets. Analysis of BDNF in platelets by gel electrophoresis suggested that BDNF content was increased in platelets of patients who developed BIPN as compared to platelets of patients that did not (mean BDNF expression level post-treatment relative to pre-treatment SD in BIPN 4 33 1 15-fold vs. 1 67 0 57-fold in non-BIPN P = 0 02) (Fig 1C). Flow cytometric analysis confirmed the increase of BDNF content in platelets of patients with BIPN, as detected by higher intensity of proBDNF compared to platelets of patients without BIPN [mean fluorescence intensity (MFI) of BDNF SD in BIPN 43 12 6 04 vs. 18 267 10 162 ng/ml in non-BIPN P = 0 009]. As expected, the increased BDNF signal in platelets of patients with BIPN coincided with lower

Haematological malignancies in pregnancy: An overview with an emphasis on thrombotic risks

With increase of maternal age, the incidence of haematological malignancies during pregnancy is rising and posing diagnostic and treatment challenges. Lymphoma is the fourth most common malignancy diagnosed in pregnancy; Hodgkin lymphoma is more frequent in pregnant women than non-Hodgkin lymphoma (NHL). The proportion of highly aggressive lymphomas in pregnant women is significantly higher than in non-pregnant women of reproductive age. Reproductive organ involvement is observed in almost half of pregnant women with NHL. The association of acute leukaemia and pregnancy is infrequent and it is assumed that pregnancy does not accelerate the disease course. Both cancer and pregnancy induce a procoagulant state which can lead to maternal venous thromboembolism (VTE) and placental occlusion. Pregnancy in woman with myeloproliferative neoplasms (MPN) promotes thrombotic environment, associating with an augmented risk of placental thrombosis, intrauterine growth retardation or loss and maternal thrombotic events.Haematological malignancies during pregnancy often require urgent diagnosis and management and are associated with potential adverse fetal outcomes. Most chemotherapeutic agents are teratogenic and should be avoided during the first trimester. Their use during the second and third trimesters may cause intrauterine growth restriction, premature birth and intrauterine fetal death. All chemotherapeutic drugs should be administered only after a detailed discussion with the patient and with close fetal monitoring. Chemotherapy and biological agents might also augment thrombotic risk. Guidelines for VTE prophylaxis in pregnant women with hematologic malignancies, apart from MPN, are currently unavailable, and therefore, clinical judgment should be made in each case.

Does Gender Matter in Non-Hodgkin Lymphoma? Differences in Epidemiology, Clinical Behavior, and Therapy

Non-Hodgkin lymphoma (NHL) is one of the most common hematologic malignancies worldwide. The incidence of NHL has been rising for several decades; however, in the last 20 years, it reached a plateau. NHL incidence among males is significantly higher than in females. In addition to gender itself, gravidity has a protective role against NHL occurrence. Gender also matters in terms of NHL clinical characteristics. For example, female predominance was found in three extra-nodal sites (the breast, thyroid, and the respiratory system) occasionally involved in NHL. The diagnosis of NHL during pregnancy is associated with a unique clinical behavior. It is usually diagnosed in the second or third trimester and in advanced stage. Furthermore, the histological subtype is highly aggressive, and reproductive organ involvement is common. The reduced rate of NHL among females may be explained by direct effects of estrogens on lymphoma cell proliferation or by its effect on anti-tumor immune response. Gender has an important role in responsiveness to standard B cell NHL treatment. Among older adults, women benefited more from the addition of the anti-CD20 antibody rituximab to standard chemotherapy regimens. This phenomenon can be explained by the difference in clearance rate of rituximab that was found to be significantly lower among older females than older males. In mantle cell lymphoma, women receiving lenalidomide have higher rates of response. An understanding of the mechanisms responsible for gender-associated NHL differences will ultimately improve the clinical approach, allowing for a more accurate assessment of prognosis and patient-tailored treatment.

JC polyomavirus reactivation is common following allogeneic stem cell transplantation and its preemptive detection may prevent lethal complications

Extended application of allogeneic stem cell transplantation (alloSCT) is expected to increase the frequency of JC polyomavirus (JCPyV)-related progressive multifocal leukoencephalopathy (PML). The aim of this study was to assess frequency, risk factors and course of JCPyV reactivation in allografted hematology patients. This retrospective study included consecutive adult patients, treated with alloSCT between January 2008 and December 2011. Quantitative JCPyV-PCR analysis was performed on whole blood DNA samples, originally drawn for cytomegalovirus detection since transplant date. The study included 164 patients diagnosed with hematological malignancies. Patients received reduced-intensity conditioning (n=74) or myeloablative conditioning (n=90), followed by alloSCT. Twenty patients developed transient and 20 had persistent JCPyV reactivation. Two of the patients with persistent reactivation showed a gradual increase in JCPyV levels, preceding PML development by 96 and 127 days. Cessation of immunosuppression resulted in complete resolution of neurological symptoms in one patient, while the other died of PML. Seventy percent of the ‘persistently reactivating’ patients died. Multivariate analysis confirmed age to be the only significant predictive factor for JCPyV reactivation. In conclusion, JCPyV reactivation occurs in a quarter of allografted patients. Preemptive detection of JCPyV reactivation in high-risk subjects and early discontinuation of immunosuppressive therapy may prevent development of lethal PML.

Development of multifocal leukoencephalopathy in patients undergoing allogeneic stem cell transplantation—can preemptive detection of John Cunningham virus be useful?

Therapeutic options for progressive multifocal leukoencephalopathy (PML) caused by reactivation of John Cunningham virus (JCV) are limited and inefficient in preventing neurological progression and death. The current study investigated the course of JCV reactivation resulting in PML in patients undergoing allogeneic stem cell transplantation (allo-SCT) and assessed the feasibility and potential significance of preemptive JCV detection in peripheral blood, enabling early cessation of immunosuppressive therapy and immune restoration. Two allografted patients were diagnosed with PML at 188 and 808 days post-allo-SCT. Stored DNA samples of both patients, originally obtained for quantitative cytomegalovirus PCR analysis since transplantation, were evaluated for JCV. JCV reactivation in peripheral blood was found to precede the appearance of neurological symptoms by 126 and 105 days. JCV blood levels were found to be highly correlated with the steroid dosage administered for treating graft-versus-host disease (GVHD). In one patient, the cessation of immunosuppression, including steroids, led to the disappearance of JCV in peripheral blood, with a remarkable improvement in neurological symptoms. In conclusion, the current study suggests the feasibility of early detection of JCV reactivation in blood. Immune restoration at that point may prevent PML development; however prospective studies are warranted to elucidate these issues.