Pediatric Hematology and Oncology, 26:157–164, 2009Copyright C© Informa Healthcare USA, Inc.ISSN: 0888-0018 print / 1521-0669 onlineDOI: 10.1080/08880010902754917

PLASMA HEPARANASE AS A SIGNIFICANT MARKER

OF TREATMENT RESPONSE IN CHILDREN WITH HODGKIN

LYMPHOMA: Pilot Study

Myriam Weyl Ben Arush, MD � Department of Pediatric Hematology Oncology,Meyer Children’s Hospital, Rambam Health Care Campus, Haifa, Israel; and The BruceRappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

I. Shafat, PhD � Cancer and Vascular Biology Research Center, Haifa, Israel; and TheBruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

Ayelet Ben Barak, MD � Department of Pediatric Hematology Oncology, MeyerChildren’s Hospital, Rambam Health Care Campus, Haifa, Israel

Rachel Bar Shalom, MD � Department of Nuclear Medicine, Rambam Health CareCampus, Haifa, Israel

Eugene Vlodavsky, MD and Neta Ilan, PhD � Cancer and Vascular BiologyResearch Center, Haifa, Israel; and The Bruce Rappaport Faculty of Medicine,Technion-Israel Institute of Technology, Haifa, Israel

� Introduction: The aim of this pilot study was to determine heparanase plasma levels (HP)at diagnosis and at restaging in children diagnosed with Hodgkin lymphoma and to investigatewhether this parameter provides prognostic information for response to treatment after inductiontherapy. Patients and Methods: HP levels of 19 pediatric patients (mean age: 10.3 years (y)(range, 4–18 y), 9 girls, 10 boys) with Hodgkin lymphoma were assayed at diagnosis and at restag-ing. HP levels were determined using an ELISA anti-human heparanase immunoassay kit. Ac-cording to diagnosis, CAT scan and/or FDG/ PET-CT fusion were performed to assess response totreatment after 2–3 courses of chemotherapy. Two patients received VAMP protocol (1 stage IA, 1stage IIA), 1 received AV-PC (nonbulky stage IIA), 4 received COPP/ABV (3 stage IIA bulky, 1stage IIIA nonbulky), 4 received ABVE-PC (2 stage IIB, 1 stage IIA bulky, 1 stage IIIA bulky), 2received ABVD (1 stage IIA bulky, 1 stage IIIA), and 6 received escalated BEACOPP (1 stage IIIB,3 stage IVA, 2 stage IVB). Results: Changes in HP levels were found to correlate with responseto treatment for most of the children. At diagnosis, average HP level was 1019 pg/mL (range,141–5733 pg/mL), decreasing at restaging to 588 pg/mL (range, 62–3267 pg/mL) (p = .034).At diagnosis, the average HP of the 16 patients in CR or VGPR was 1104 pg/mL; it had decreased

Address correspondence to Dr. Myriam Weyl Ben Arush, Department of PediatricHematology–Oncology, Meyer Children’s Hospital, Rambam Health Care Campus, POB 9602, Haifa31096, Israel. E-mail: m benarush@rambam.health.gov.il

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at restaging to 586 pg/mL (p = .032). At diagnosis, the average HP level for the 3 patients withTP or PR was 1704 pg/mL; it had increased to 1938 pg/mL at restaging (p = .166). Due to thesmall number of patients, no correlation was observed between HP levels at diagnosis, staging, orany other clinical prognostic factor. Conclusions: Changes in plasma HP levels correlated withresponse to treatment for children diagnosed with Hodgkin lymphoma. This provides a rationale forexploring clinical interest in plasma heparanase measurements of a larger group, using the test forclinical trials of antiangiogenic therapies.

Keywords children, ELISA, heparanase, Hodgkin lymphoma, prognostic factor

Hodgkin lymphoma is a heterogeneous disease, which varies in biologi-cal expression and clinical course. Depending on risk factors, approximately80–90% of patients achieve complete response (CR) and the majority ofthese patients can expect to be cured. In contrast, patients not achievingCR after first-line treatment have a worse long-term outcome [1]. There-fore, it is of interest to identify prognostic factors in children who do notachieve an adequate response to standard first-line therapy but who may berescued.

Proangiogenic cytokines play important roles in the pathogenesis ofcancer [2]. Most angiogenic factors are soluble molecules released by tumorcells that mediate an angiogenic response. Measurement of such biologicalfactors reflecting underlying mechanisms of pathogenic importance mayadd to prognostic information. Probably the most important angiogenesisstimulators are vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) [3]. Quantification of angiogenesis stimulatorsin children with malignancies has already been reported by Pavlakovic etal. [3], who showed elevation of serum VEGF in children with solid tumorsand elevated pretherapeutic serum VEGF levels, which declined to normallevels when the children achieved complete response [3]. Tabone et al. [4]showed correlation between serum VEGF at diagnosis and survival. Recentlywe published a study on the significance of serum VEGF as a marker oftumor response in pediatric malignancies [5] and, in a further report, weperformed a subset analysis of children with Hodgkin lymphoma, measuringbaseline and post-treatment serum levels [6].

Heparanase is an endo-β-glucuronidase that cleaves heparan-sulfate(HS) side chains in a distinctive manner [7, 8]. Cleavage of HS, an im-portant constituent of the extracellular matrix (ECM) and basement mem-branes, is considered critical for cell invasion associated with inflammation,angiogenesis, and tumor metastasis. Furthermore, heparanase upregulationwas documented in an increasing number of primary human tumors, cor-relating with enhanced local and distant metastasis, increased microvesseldensity, and reduced postoperative survival of cancer patients. Heparanaseinduction in human malignancies, as well as in several other pathologies,such as cirrhosis, nephrosis, and diabetes [9], implies that the enzyme mayserve as a valuable diagnostic marker.

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Plasma Heparanase in Hodgkin Lymphoma 159

Recently, one author reported the development of an ELISA methodcapable of the detection and quantification of heparanase in urine samplesand demonstrated an elevation of heparanase levels in the urine of cancerand diabetes patients [10]. In this study, we examined the ability of theELISA method to detect and quantify heparanase levels in the blood samplesof children with Hodgkin lymphoma at diagnosis and at restaging.

PATIENTS AND METHODS

Antibodies and Reagents

Anti-heparanase 1E1 monoclonal antibody and 1453 and 733 polyclonalantibodies have been previously described (HRP-conjugated goat anti-rabbitantibody was purchased from Jackson ImmunoResearch (West Grove, PA).Microtiter 96-well plates (Maxisorp) came from Nunc (Roskilde, Denmark).HRP colorimetric substrate 3,30,5,50-tetramethylbenzidine (TMB) was pur-chased from Dako (Glostrup, Denmark). Bovine serum albumin (BSA) wasfrom MP Biomedicals (France). Single-chain active heparanase (GS3) geneconstruct was kindly provided by Christian Steinkuhler (IRMB/Merck Re-search Laboratories, Pomezia, Italy), and the protein was purified from theconditioned medium of baculovirus-infected insect cells [11].

Patients (Table 1)

Heparanase plasma levels of 19 consecutive pediatric patients withHodgkin lymphoma were assayed at diagnosis and at restaging. Mean age:10.3 years (y) (4–18 y), 9 girls, 10 boys. According to diagnosis, CAT scan andFDG/PET-CT fusion were performed to assess response to treatment aftertwo-three courses of chemotherapy. Two patients received VAMP protocol[12] (1 stage IA, 1 stage IIA); 1 patient received AV-PC (nonbulky stageIIA) Children’s Oncology group (COG) protocol low risk; 4 patients re-ceived COPP/ABV [13] (3 stage IIA bulky, 1 stage IIIA nonbulky); 4 patientsreceived ABVE-PC Children’s Oncology group (COG) protocol intermedi-ate risk (2 stage II, 1 stage IIA bulky, 1 stage IIIA bulky); 2 patients receivedABVD [13] (1 stage IIA bulky, 1 stage IIIA); and 6 patients received escalatedBEACOPP [14] (1 stage IIIB, 3 stage IVA, 2 stage IVB).

Sample Collection

Blood samples were collected at diagnosis and at restaging (2–3 coursesof chemotherapy). A total of 3 mL of peripheral blood was collected inEDTA-containing tubes. Samples were cooled and plasma was obtained assoon as possible by centrifugation (1500 × g , 15 min at 4◦C). All sampleswere frozen and thawed once.

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TABLE 1 Patient Characteristics

Age CurrentGender (years) Stage Chemotherapy status

M 4 IIA Nonbulky COPP/ABV CRF 17 IVA Bulky es BEACOPP CRM 14 IIA Bulky COPP?ABV CRM 6 IA Nonbulky VAMP TPF 12 IVB Bulky es BEACOPP CRM 9 IVA Bulky es BEACOPP CRM 6 IIIB Bulky es BEACOPP CRM 15 IIA Bulky ABVE-PC CRF 18 IIA Bulky ABVD CRF 15 IIA Nonbulky VAMP CRF 4 IIIB Bulky COPP/ABV TPM 15 IIIA Bulky ABVD CRF 16 IVA Bulky es BEACOPP CRM 16 IIIA Bulky ABVE-PC CRM 16 IIA Bulky COPP/ABV VGPRM 15 IIB Bulky ABVE-PC PRF 15 IVB Bulky es BEACOPP CRF 15 IIB Nonbulky ABVE-PC CRF 7 IIA Nonbulky AV-PC CR

Note. M, male; F, female; COPP, cyclophosphamide, vincristine, prednisone, procarbazine; ABV,doxorubicin, bleomycin, vinblastine; es, escalated; BEACOPP, bleomycin, etoposide, doxorubicin, cy-clophosphamide, vincristine, prednisone, procarbazine; VAMP, vincristine, doxorubicin, methotrexate,prednisone; ABVE-PC, doxorubicin, bleomycin, vincristine, etoposide, procarbazine, cyclophosphamide;AV-PC, doxorubicin, vincristine, prednisone, cyclophosphamide.

ELISA Method

The ELISA method was carried out as described [10]. Briefly, wells ofmicrotiter plates were coated (18 h, 4◦C) with 1 µg/mL of 1E1 monoclonalanti-heparanase antibody in 50 µL of coating buffer (0.05 M Na2CO3, 0.05M NaHCO3, pH 9.6) and were then blocked with 1% BSA in PBS for1 h at 37◦C. Samples were diluted with 0.5% BSA (1:1) and a total of100 µL was loaded in duplicate and incubated for 2 h at room temperature,followed by the addition of 100 µL antibody 1453 (1 µg/mL) for additional2 h at room temperature. HRP-conjugated goat anti-rabbit IgG (1:20,000)in blocking buffer was added (1 h, room temperature) and the reactionwas visualized by the addition of 100 µL chromogenic substrate (TMB) for30 min. The reaction was stopped with 100 µL H2SO4, and absorbance at450 nm was measured with reduction at 630 nm using an ELISA plate reader.Plates were washed 5 times with washing buffer (PBS, pH 7.4, containing0.1% (v/v) Tween 20) after each step. As a reference for quantification,a standard curve was established by a serial dilution of recombinant 8 +50 GS3 active heparanase enzyme (390 pg/mL to 25 ng/mL).

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Plasma Heparanase in Hodgkin Lymphoma 161

TABLE 2 Heparanase Levels in the Plasma of 19 Patients with Hodgkin Lymphoma

Heparanase levels (pg/mL)

At diagnosis At restaging Normal

Overall average 1019 ± 334 588 ± 183 163 ± 18p value (diagnosis vs. restaging) .035 <.0001 (diagnosis)

.0085 (restaging)Median 433 259 155Average patient at CR (n = 16) 1104 ± 390 586 ± 195 0.013 (restaging)p value (diagnosis vs. restaging) .033Median patient at CR 455 342Correlation 13 of 16 patients at CR had

reduced heparanase levels(81.3%)

Statistical Analysis

Data were analyzed by the Prism software (GraphPad, San Diego, CA,USA). One-tailed paired t test and the nonparametric Mann-Whitney testwere employed. p < .05 was considered as statistically significant.

RESULTS (Table 2)

Fifteen children had complete response, 1 very good partial response,2 children had tumor progression, and 1 had stable disease according tothe PET-CT performed after 2–3 courses of chemotherapy. The averagelevel of plasma heparanase in these patients was determined to be 1019 ±334 pg/mL at diagnosis (6-fold increase from normal controls) and 588 ±173 pg/mL at restaging (Figure 1A), a statistically significant reduction(p = .035). At diagnosis, the average HP of the 16 patients in CR or VGPRwas 1104 pg/mL; at restaging, this had decreased to 586 pg/mL (p = .032)(Figure 1B). At diagnosis, the average HP level for the 3 patients with TPor PR was 1704 pg/mL, which had increased to 1938 pg/mL at restaging(p = .166).

Noticeably, all 13 (68.4%) patients with lower heparanase levels at restag-ing were in complete remission. Of the 6 patients with higher heparanaselevel, 3 exhibited stable disease or disease progression and a modest elevationof heparanase levels (Figure 1C).

DISCUSSION

Heparanase activity has been traditionally correlated with cell invasionassociated with cancer metastasis, a consequence of structural modificationthat loosens the ECM barrier [15]. More recently, heparanase upregulation

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FIGURE 1 Plasma samples of pediatric patients with Hodgkin lymphoma were obtained at diagnosisand at restaging, following 2–3 courses of chemotherapy, and heparanase levels were quantified for theentire group (A, n = 19), patients diagnosed to undergo complete remission (CR) (B, n = 16), andpatients who exhibited disease progression (C, n = 3).

has been documented in an increasing number of human carcinomas andhematological malignancies [16]. In many cases, heparanase inductioncorrelated with increased tumor metastasis, vascular density, and shorterpostoperative survival rate, thus providing strong clinical support for thepro-metastatic and pro-angiogenic function of the enzyme [17]. In additionto the well-studied catalytic feature of the enzyme, heparanase was noted toexert biological functions apparently independent of its enzymatic activity.This function requires heparanase secretion and is thought to be mediatedby an as yet unidentified heparanase receptor. Thus, active and inactiveheparanase secreted by tumor cells or the tumor microenvironment mayexert local and systemic effects and is the subject of anti-cancer drug de-velopment programs [18]. One author recently reported the developmentof an ELISA method capable of quantifying heparanase levels in urinesamples [10]. In this study, we extended the utility of the ELISA method anddemonstrated that it can successfully be applied also to quantify heparanasein plasma. As expected, heparanase levels were significantly elevated inplasma obtained from pediatric cancer patients. Interestingly, heparanaselevels in the plasma closely reflected the patient’s condition followinganti-cancer treatment. Thus, heparanase levels were markedly reducedin most patients under remission but remained stable or slightly elevatedin patients who responded poorly to treatment and exhibited tumorprogression.

Increased pretreatment serum levels of angiogenic molecules have beenimplicated in the prognosis of patients with Hodgkin lymphoma. Mainou-Fowler et al. reported increased microvessel density and expression ofVEGF in patients with Hodgkin lymphoma in progression [19]. Doussis-Anagnostopoulou et al. [20] demonstrated by immuno-histochemistry thatthe neoplastic population clearly expressed VEGF in 70% of cases of classicHodgkin lymphoma, contrary to Foss et al. [21], who analyzed 20 cases of

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Plasma Heparanase in Hodgkin Lymphoma 163

classic Hodgkin lymphoma and found a large number of VEGF-positive cellsthat were reactive and nonlymphoid, with only one case of positive neoplas-tic cells. Doussis-Anagnostopoulou et al. believed that the neoplastic ReedSternberg and Hodgkin cells were responsible for the upregulation of VEGF.VEGF mRNA was demonstrated in both cells, using RT-PCR. Later, Agarwaland Naresh reported the co-expression of VEGF and vascular endothelialgrowth factor receptor 2 (VEGFR-2) in Reed Sternberg cells [22].

The results of this study show that the plasma concentration values ofheparanase are a significant marker as a predictor to response to treatmentin children with Hodgkin lymphoma. Post-therapy plasma heparanase levelswere significantly reduced in children who were in good partial or completeremission as proved by PET-CT. We observed the same results in our laststudy [6], showing changes in serum levels of VEGF in a smaller group ofchildren with Hodgkin lymphoma.

Our findings confirm that heparanase participation in neoangiogenesisin Hodgkin lymphoma may be as important as the participation of VEGF.Additional studies need to be performed to better define the angiogenicprofile in Hodgkin patients and to develop anti-angiogenic therapies forpatients with refractory Hodgkin lymphoma.

Declaration of Interest: The authors report no conflicts of interest. Theauthors alone are responsible for the content and writing of the paper.

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