The Predictive Value of Cystatin C in Monitoring of B Non ...downloads.hindawi.com/journals/isrn.oncology/2013/752792.pdf · ISRNOncology 5 Table4:CorrelationofcystatinCwithclinicalstageofthediseaseandIPI

Hindawi Publishing CorporationISRN OncologyVolume 2013 Article ID 752792 6 pageshttpdxdoiorg1011552013752792

Research ArticleThe Predictive Value of Cystatin C in Monitoring of BNon-Hodgkin Lymphomas Relation to Biochemical andClinical Parameters

A SoftiT1 L BegiT1 A HalilbašiT2 T ViDin3 and J Kos34

1 Department of Biochemistry Faculty of Pharmacy University of Tuzla 75000 Tuzla Bosnia and Herzegovina2 Clinic for Oncology Haematology and Radiotherapy University Clinical Centre Tuzla 75000 Tuzla Bosnia and Herzegovina3 Chair of Pharmaceutical Biology Faculty of Pharmacy University of Ljubljana 1000 Ljubljana Slovenia4Department of Biotechnology Jozef Stefan Institute 1000 Ljubljana Slovenia

Correspondence should be addressed to A Softic adaleta myahoocom

Received 11 July 2013 Accepted 13 August 2013

Academic Editors G Gatti and G E Kim

Copyright copy 2013 A Softic et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The predictive value of cystatin C as a marker of course of the disease has been evaluated Fifty-two pairs of serum samples ofpatients with B non-Hodgkin lymphoma have been collected at the time of diagnosis and before fourth cycle of chemotherapyThelevels of cystatin C CRP 120573

2M LDH and IL-6 in samples have been measured and clinical parameters of course of the disease (B

symptoms clinical stage patientsrsquo age and IPI) have been noted In total patientrsquos group cystatin C levels correlated with 1205732M and

IPI In aggressive lymphomas the inhibitor levels correlated with clinical stage of disease and were significantly higher in patientswith elevated LDH activity In aggressive nodal lymphomas its levels correlated with 120573

2M IPI and clinical stage of disease The

cystatin C level was significantly increased in total group of patients over 60 years old while in particular types of lymphomano statistical significance has been obtained Our results indicate that cystatin C should be taken into consideration in diseasemonitoring However we expect that the disease-free and overall survival analysis will give the definitive answer about the reliabilityof cystatin C as an indicator of course of aggressive lymphomas

1 Introduction

Cysteine proteinase inhibitors cystatins are involved inmechanisms controlling intracellular and extracellular pro-tein degradation Under normal physiological conditionssmall amounts of catalytically active proteases released fromlysosomes or secreted from infected or dying cells are effec-tively blocked by cystatins A disbalance between proteinasesand their natural inhibitors leads to the development ofvarious diseases

Cystatin C amember of family II of cystatins is a nongly-cosylated 13 kDa protein inhibitor of cysteine proteases withwidespread distribution in almost all extracellular fluids thehighest levels having been determined in cerebrospinal fluidseminal plasma and synovial fluid [1]

A broad spectrum of biological roles has been suggestedfor cystatin C including control of protein catabolism

regulation of hormone processing and bone resorptioninflammation antigen presentation and T-cell dependentimmune response [2 3]

Cystatin C was suggested as a marker of glomerularfiltration rate since it is produced at a constant rate by allnucleated body cells freely filtered in the renal glomeruliand almost completely reabsorbed and catabolized in theproximal tubules [4]

Cystatin C has also been suggested as playing a roleassociated with alteration of the proteolytic system in cancerIn patients with colorectal lung and melanoma carcinomashigh serum levels of cystatin C are associated with pooroutcome of disease [5ndash7]

Thus cystatin C level was proposed for followup ofdiseases efficacy of chemotherapy and prediction of diseaseoutcome Our recent study showed that patients with Bnon-Hodgkin lymphomas had significantly higher cystatin C

2 ISRN Oncology

levels compared to healthy controls In addition patients withrelapse of the disease had significantly higher cystatin C levelcompared to patients without relapse [8]

For sake of evaluation of the predictive value of cystatinC as a marker of course of the disease the cystatin C levelswere compared with established biochemical parameters (Creactive protein (CRP) beta-2 microglobulin (120573

2M) lactate

dehydrogenase (LDH) levelsactivities) with clinical parame-ters of disease progression (the presence of B symptoms clin-ical stage age and international prognostic index (IPI)) andwith suggested prognostic factor in patients with lymphoma[9] (the levels of cytokine interleukin 6 (IL-6))

2 Materials and Methods

21 Subjects Serum samples of patients with diagnosis of Bnon-Hodgkin lymphoma have been collected from February2008 until May 2011 at the Clinic for Oncology Hematologyand Radiotherapy of the University Clinical Centre of Tuzlawith the approval of the local ethical committee and thewritten consent of the participants Two samples of bloodfrom each patient were taken first at the time of diagnosisbefore therapy and the second immediately before fourthcycle of chemotherapy Thus it was possible to exclude thepossibility of increasing the cystatin C sera level due toinfluence of corticosteroids which are the common part ofchemotherapeutic protocols [10ndash12]

Fromfifty-two pairs of collected samples three pairs werefrom the patients with the relapse of the disease Elevenpatients had the indolent type of lymphoma and 41 hadthe aggressive form of those 18 with nodal and 23 withextranodal disease presentation

Thirty-six patients have been receiving CHOP regimen ofchemotherapy (cyclophosphamide doxorubicin hydrochlo-ride oncovin and prednisone) Among them thirty patientshave been receiving Rituximab and one patient bleomycinEight patients have been receiving CHOEP protocol (CHOP+ etoposide) and 6 of them with addition of immunotherapyThree patients have been receiving COP protocol (cyclophos-phamide oncovin and prednisone) 2 patients FCDprotocol(fludarabine cyclophosphamide and dexamethasone) onepatient high doses of methotrexate while 2 patients withhighly aggressive lymphomas have been treated by modifiedYugoslav protocol for acute lymphoblastic leukemia Theselast two patients have been included in group of aggressivelymphomas

For each patient his age type of lymphoma and IPI havebeen noted and his biochemical parameters like CRP 120573

2M

LDH and creatinine in serum have been measuredThe serum creatinine level has been used as a criterion for

selection of patients those with the creatinine level above theupper physiologic limit have not been included in the studybecause of possible renal dysfunction Also patients withautoimmune diseases asthma and other malignant diseaseshave been excluded from the study

Seven milliliters of blood per patient has been collectedThe blood was clotted at room temperature and subsequentlycentrifuged at 3000 rpm Serumwas separated aliquoted and

stored at minus20∘C For analysis delayed for a longer period oftime serum was stored at ndash80∘C

Reevaluation was carried out after three cycles ofchemotherapy by repeating the tests which had results out ofnormal range A complete response was defined as the disap-pearance of all clinical laboratory and radiographic evidenceof lymphoma Responding patients with minimal residualradiographic abnormalities were classified as showing partialresponse Lesser objective responses were classified as poorpartial response

22 Methods The levels of cystatin C in sera of lymphomapatients were measured by specific sandwich-type ELISAdeveloped at the Department of Biochemistry andMolecularBiology Jozef Stefan Institute and Krka dd as described[7] A microtiter plate was coated with rabbit affinitypurified anti-cystatin C polyclonal antibody Recombinanthuman cystatin C was used for preparation of calibrationcurves Sera from patients were diluted 1 100 prior tobeing applied to wells of microtiter plate As a detectionantibody 1A2 monoclonal antibody was used conjugatedto horseradish peroxidase A substrate for peroxidase was331015840551015840-tetramethylbenzidine liquid substrate system (TMBSigma) The reaction was stopped by adding of 2M H

2SO4

and the absorbance was measured at 450 nm on minireader(TECAN Sunrise) Measured values of diluted samples andcontrols were compared with the calibration curve andexpressed in ngmL of serum

The levels of IL-6 in sera of lymphoma patients weremeasured by commercial sandwich-type ELISA (Quan-tikine RampD Systems Inc Minneapolis USA) on minireader(TECAN Sunrise) ELISA assays were performed in dupli-cate and the mean value determined

Biochemical parameters were measured at the Polyclinicfor Laboratory Diagnostics University Clinical Centre TuzlaCRP was measured by immunoturbidimetric method onArchitect c8000 analyzer 120573

2M was measured by Micropar-

ticle Enzyme Immunoassay (MEIA) (Abbott) on AxSYMsystem LDH was measured by enzyme method (Abbott) onArchitect c8000 analyzer and enzyme method (Siemens) onDimension system Creatinine was measured by a modifiedJaffe method on Dimension system

23 Statistical Analysis For statistical analysis of the resultsSPSS software release 170 (SPSS Inc Chicago IL USA) wasused Mann-Whitney U test was used to compare differencesin cystatin C levels between independent groups and theWilcoxon matched pair test was used to compare cystatinC level measured at the time of diagnosis and before fourthcycle of chemotherapy Correlations of cystatinCwith clinicalstage of disease IL-6 CRP 120573

2M and IPI were defined by

Spearman rank correlation analysis In all tests two-sided Psbelow 005 were considered significant

3 Results

In patients who achieved partial or complete remissionafter three cycles of chemotherapy the level of cystatin C

ISRN Oncology 3

Table 1 Comparison of cystatin C levels measured at the time of diagnosis and before IV cycle of chemotherapy in patients with partial orcomplete remission

Group No Median and range (ngmL) Wilcoxon Z PTime of diagnosis Before IV

chemotherapy

All patientsa 40 94463(56247ndash159430)

89007(6180ndash141520) minus2 245lowast 0025

Aggressive 34 94463(56247ndash159430)


Extranodal 19 93130(56247ndash159430)

99576(69838ndash141520) minus1087 0277

Nodal 15 95796(59945ndash145160)

82336(6180ndash118290) minus1874 0061

lowastStatistically significant differenceaTotal group of patients

Table 2 Correlation of cystatin C with IL-6 1205732M and CRP

Parameter Group Time at diagnosis Before IV chemotherapyNumber of patients Correlation coefficient P value Number of patients Correlation coefficient P value

IL-6

All patientsa 31 0056 0766 26 minus0268 0186Aggressive 26 0079 0701 20 minus0138 0561

Extranodal 13 0082 0789 11 0345 0298Nodal 13 0110 0721 9 minus0500 0170

1205732M

All patientsa 50 0 332lowast 0019 36 0 460lowastlowast 0005Aggressive 39 0286 0077 31 0 488lowastlowast 0005

Extranodal 22 0201 0370 18 0418 0084Nodal 17 0 522lowast 0032 13 0 626lowastlowast 0022

CRP

All patientsa 52 0107 0451Aggressive 41 0181 0258

Extranodal 23 0381 0073Nodal 18 minus0028 0913

lowastStatistically significant correlationaTotal group of patientslowastlowastStatistically highly significant correlation

significantly decreased in total patientrsquos group and in groupwith aggressive lymphoma (Table 1)

Out of 40 patients with disease remission twenty-four(60) patients had lower cystatin C level before fourth cycleof chemotherapy compared to the level measured at the timeof diagnosis In the group of aggressive lymphomas (119873 = 34)twenty-one (618) patients had the lower cystatin C levelbefore fourth cycle of therapy

Out of total patientrsquos number (119873 = 52) IL-6 wasdetected in 31 patients at the time of diagnosis (median529 pgmL range 175ndash9269 pgmL) In the group of aggres-sive lymphomas it was detected in 26 patients (median525 pgmL range 175ndash9269 pgmL) in 13 patients withextranodal (median 486 pgmL range 332ndash2574 pgmL)and in 13 patients with nodal disease presentation (median533 pgmL range 175ndash9269 pgmL) The results of correla-tion between cystatin C and IL-6 are presented in Table 2In all groups of patients these two parameters showed nocorrelation

Before fourth cycle of chemotherapy IL-6 was detected in26 patients (median 595 pgmL range 088ndash2573 pgmL)In the group of aggressive lymphomas it was detected in20 patients (median 539 pgmL range 188ndash2573 pgmL)in 11 patients with extranodal (median 501 pgmL range325ndash2573 pgmL) and in 9 patients with nodal diseasepresentation (median 649 pgmL range 188ndash2317 pgmL)The results of correlation between cystatin C and IL-6 beforefourth cycle of chemotherapy are presented in Table 2 Inall groups of patients these two parameters showed nocorrelation

At the time of diagnosis the median level of CRP in totalpatientrsquos group was 760mgL (range 030ndash25450mgL)in the group of aggressive lymphomas CRP was 880mgL(range 080ndash25450mgL) in the group with extranodaldisease presentation CRP was 770mgL (range 080ndash7880mgL) and in the groupwith nodal disease presentationCRP was 1365mgL (range 190ndash25450mgL) The cystatinC sera levels were compared with the CRP levels at the time

4 ISRN Oncology

of diagnosis (Table 2) In all groups of patients these twoparameters showed no correlation

At the time of diagnosis the median level of 1205732M in

total patientrsquos group was 186mgL (range 051ndash425mgL)in the group of aggressive lymphomas 120573

2M was 182mgL

(range 051ndash425mgL) in the group with extranodal diseasepresentation 120573

2M was 167mgL (range 051ndash385mgL)

and in the group with nodal disease presentation 1205732M was

189mgL (range 113ndash425mgL)In patients with partial or complete remission after three

cycles of chemotherapy the median 1205732M was in the total

patientrsquos group 18mgL (range 10ndash429) in the group ofaggressive lymphomas 183mgL (range 10ndash429mgL) inthe group with extranodal disease presentation 185mgL(range 10ndash429mgL) and in the group with nodal diseasepresentation 172mgL (range 110ndash398mgL)

At the time of diagnosis cystatin C showed significantcorrelation with 120573

2M in total group of patients and in the

group of nodal aggressive lymphomas (Table 2) Consideringthe obtained correlations and the fact that the increased levelsof 1205732M indicate the patients with the aggressive lymphomas

who will experience relapse [13] we analyzed correlationbetween cystatin C and 120573

2M in patients with partial or com-

plete remission after three cycles of chemotherapy (Table 2)Significant correlation was obtained in total group of patientswith disease remission and in group with aggressive nodallymphoma In patients who did not experience diseaseremission after three cycles of chemotherapy correlationbetween cystatin C and 120573

2M was not calculated because of

small number of patients per groupFor the measurement of LDH two methods were used

and it was not possible to correlate LDH and cystatin C levelsInstead patients were divided in two groups a group withnormal and a group with elevated LDH level (Table 3) Thenthe cystatin C values of these two groups were comparedfor each type of lymphoma In patients with aggressive lym-phoma the groupwith elevated LDHhad significantly higherlevel of cystatin C than the group with normal LDH activity

Based on the presence or absence of B symptoms (weightloss over 10 for a six month night sweats fever andunexplained itching) patients were divided in two groupswith and without symptoms for each type of lymphomaThen the values of cystatin C of these two groups werecompared (Table 3) In all types of lymphoma no statisticalsignificant difference was obtained

Based on the age patients were divided in two groupsge60 years and lt60 years old (Table 3) Then the values ofcystatin C of these two groups were compared for each typeof lymphoma In total patients group those ge60 years old hadsignificantly higher inhibitor level than the patients lt60 yearsold

The correlation between cystatin C and clinical stageof disease at the time of diagnosis was significant in thegroup of aggressive lymphomas and in subgroup with nodalpresentation (Table 4)

The correlation between cystatin C and IPI at the time ofdiagnosis was significant in total group of patients and in thegroup with nodal aggressive lymphomas (Table 4)

Table 3 Comparisons of cystatin C levels measured at the time ofdiagnosis between groups of patients with normal and elevated LDHlevel groups of absent and present B symptoms and groups underand over 60 years of age

Parameter Number of patientsAll patientsa Aggressive Extranodal Nodal

LDHNormal 33 23 13 10Elevated 19 18 10 8U 225 120lowast 38 21P value 0093 0022 0101 0101

B symptomsNo 12 9 6 3Yes 40 32 17 15U 150 94 29 16P value 0051 0120 0135 0498

Agele60 years 29 23 11 12gt60 years 22 17 11 6U 191lowast 124 42 21P value 0015 0051 0243 0180


4 Discussion

Our previous study showed that the level of cystatin Cwas significantly higher in patients who experienced relapsecompared to patients newly diagnosed with non-Hodgkinlymphomas Thus we assumed that the level of this inhibitorcan serve as a marker of relapse In this studyrsquos framework itwas not possible to reach the minimal number of patients forstatistical analysis with sample taken at diagnosis of diseasein clinical remission and at diagnosis of relapse becausesuch approach would require the inclusion of several clinicalcenters numerous researchers and years of research For thisreason we evaluated the predictive value of cystatin C indisease monitoring in two ways

The first method involved the separation of group ofpatients who were in partial or complete remission beforeadmission of fourth cycle of chemotherapy In this groupthen we compared the levels of the inhibitor measuredat the time of diagnosis with the levels measured beforefourth cycle of chemotherapy It was found that the level ofinhibitor significantly decreases with the disease remissionAggressive lymphomas almost completely contributed tostatistical significance of complete sample (Table 1) Doesthis mean that there is a specific mechanism in aggressivelymphomas not present in patients with indolent lymphomawhich contributes to decreased levels of cystatin C in diseaseremission It could not be precisely determined due to thesmall sample of patients with indolent lymphomas (119873 = 6)who get into remission after the third cycle of chemotherapy

Another way to test the predictive value of cystatin C indisease monitoring was to compare its level with establishedbiochemical (CRP 120573

2M and LDH levels) clinical parameters

ISRN Oncology 5

Table 4 Correlation of cystatin C with clinical stage of the disease and IPI

Group No Clinical stage Correlation coefficient (r) P valueI II III IV

All patientsa 52 3 24 21 4 0250 0074Aggressive 41 2 19 17 3 0 313lowast 0046

Extranodal 23 2 12 7 2 0242 0265Nodal 18 mdash 7 10 1 0 476lowast 0046

IPI stage0 1 2 3 4

Aggressive 41 3 13 13 7 5 0 488lowastlowast 0001Extranodal 23 mdash 8 8 3 4 0398 0060Nodal 18 4 4 5 4 1 0 575lowast 0013


of disease progression (the presence of B symptoms clinicalstage age and IPI) and with the levels of IL-6 as prognosticfactor in patients with lymphoma [9]

Since the 1205732M is a light chain of antigens A B and C of

the main histocompatibility complex the rate at which it isreleased into the circulation is determined by the activationand proliferation of lymphocytes [14] Previous studies haveshown that 120573

2M correlated with the clinical stage of the

disease in non-Hodgkin lymphomas [15 16] Johnson andcolleagues [17] found that the outcome of chemotherapy inpatients with aggressive non-Hodgkin lymphomas correlateswith initial values of 120573

2M in the group with normal values

70 of patients reached a clinical remission and in the groupof patients with elevated 120573

2M only 37 of patients achieved

a clinical remission (119875 lt 0001) In addition patients withnormal initial 120573

2M values had significantly longer period of

clinical remission compared to patients with elevated 1205732M

(followup six years expressed as a median) About 70 ofpatients with normal serum 120573

2M did not develop the disease

the next 6 years in contrast to patients with elevated 1205732M

where only 35 of patients did not develop the disease forthe same period Our results show that cystatin C correlateswith 120573

2M in the total group of patients and in the group of

aggressive nodal lymphoma (Table 2) When we correlate thelevels of cystatin C and 120573

2M in patients who after the third

cycle of therapy experienced partial or complete remissionresult is more pronounced correlation in the total sampleof patients in the group of aggressive lymphomas and inthe subgroup of aggressive nodal lymphoma (Table 2) Basedon the results of correlation of cystatin C and 120573

2M and on

the basis of previous studies indicating the importance of1205732M as a prognostic factor in aggressive lymphomas it may

be concluded that cystatin C could serve as an indicator ofcourse and prognosis of the disease

The international prognostic index is widely acceptedprognostic index for patients with aggressive lymphoma [18]Our results show a correlation of cystatin C with IPI inthe group of aggressive lymphomas and in the subgroup ofaggressive nodal lymphoma (Table 4) In addition the levelsof the inhibitor were compared with three individual factors

that are included in the IPI as age serum LDH activity andclinical stage of disease

In the total sample serum cystatin C was significantlyhigher in patients over 60 years of age compared with thoseunder 60 years (Table 3) Elevated levels of cystatin C inpatients over 60 years of age may be the result of theweakening of kidney function due to aging [19] However itshould be noted that all the patients involved in the study hadnormal serum creatinine

Cystatin C levels were significantly elevated in patientswith elevated LDH activity compared to those with normalLDH activity in a group of aggressive lymphomas (Table 3)

In addition our results show that cystatin C at the timeof diagnosis as well as 120573

2M correlates with clinical stage of

disease A statistically significant correlation was found inthe group of aggressive lymphomas and in the subgroup ofaggressive nodal lymphoma (Table 4) These results suggestthe possibility that there is a specificmechanism in aggressivenodal lymphomas that drives increased gene expression andsecretion of cystatin C in body fluids Moreover cystatin Cis produced by all nucleated body cells so the correlation ofthe inhibitor level and clinical stage of disease in aggressivenodal lymphomas can be the result of the general responseof organism to the increased proteolytic activity in theinfiltrated lymph nodes

Study of Preti et al [9] showed that IL-6 is an importantindependent prognostic factor in patients with diffuse largecell lymphoma and a high value of serum interleukin 6 priorto the initiation of therapy correlates with poor responseto therapy and shorter survival However the correlationbetween cystatin C and IL-6 was not determined neitherat the time of diagnosis or before the fourth cycle ofchemotherapy (Table 2)

Cystatin C shows no correlation with CRP (Table 2)which is to be expected because the biosynthesis of CRP as anacute phase reactant in the liver is under the control of IL-6[20] As the inhibitor level does not correlate with the levelof IL-6 there is also no correlation with CRP In additionthe presence of B symptoms is associated with elevatedinflammatory proteins including CRP [21] and cytokines

6 ISRN Oncology

such as IL-6 [22] Since cystatin C shows no correlation withthese parameters the result of comparison of the cystatin Clevels of the group with B symptoms and of the cystatin Clevels of the group without symptoms of disease is expected

Our results show that cystatin C should be taken intoconsideration in lymphoma monitoring However we expectthat the disease-free and overall survival analysis will givethe definitive answer about the reliability of cystatin C as anindicator of course of aggressive B non-Hodgkin lymphomasThis particularly refers to a group of patients with nodaldisease presentation which almost completely contributes tocorrelation of cystatin C level and IPI

Conflict of Interests

The author and coauthors did not have a direct financialrelation with the trademarks mentioned in the paper thatmight lead to a conflict of interests

Acknowledgments

This work was supported by Grants from Federal Ministryof Education and Science of Bosnia and Herzegovina (03-39-5980-55-108) and Research Agency of the Republic ofSlovenia (P4-0127 J4-0123)

References

[1] M Abrahamson A J Barrett G Salvesen and A GrubbldquoIsolation of six cysteine proteinase inhibitors from humanurine Their physicochemical and enzyme kinetic propertiesand concentrations in biological fluidsrdquo Journal of BiologicalChemistry vol 261 no 24 pp 11282ndash11289 1986

[2] Y M C Henskens E C L Veerman and A V N AmerongenldquoCystatins in health diseaserdquoBiological Chemistry Hoppe-Seylervol 377 no 2 pp 71ndash86 1996

[3] P Pierre and I Mellman ldquoDevelopmental regulation of invari-ant chain proteolysis controlsMHC class II trafficking inmousedendritic cellsrdquo Cell vol 93 no 7 pp 1135ndash1145 1998

[4] O Simonsen A Grubb and H Thysell ldquoThe blood serumconcentration of cystatin C (120574-trace) as a measure of theglomerular filtration raterdquo Scandinavian Journal of Clinical andLaboratory Investigation vol 45 no 2 pp 97ndash101 1985

[5] J Kos M Krasovec N Cimerman H J Nielsen I J Chris-tensen and N Brunner ldquoCysteine proteinase inhibitors stefinA stefin B and cystatin C in sera from patients with colorectalcancer relation to prognosisrdquo Clinical Cancer Research vol 6no 2 pp 505ndash511 2000

[6] I Zore M Krasovec N Cimerman et al ldquoCathepsin BcystatinC complex levels in sera from patients with lung and colorectalcancerrdquo Biological Chemistry vol 382 no 5 pp 805ndash810 2001

[7] J Kos B Stabuc A Schweiger et al ldquoCathepsins B H and Land their inhibitors stefin A and cystatin C in sera of melanomapatientsrdquo Clinical Cancer Research vol 3 no 10 pp 1815ndash18221997

[8] A Mulaomerovic A Halilbasic E Cickusic T Zavasnik-Bergant L Begic and J Kos ldquoCystatin C as a potential markerfor relapse in patients with non-Hodgkin B-cell lymphomardquoCancer Lett vol 248 pp 192ndash197 2007

[9] H A Preti F Cabanillas M Talpaz S L Tucker J F Seymourand R Kurzrock ldquoPrognostic value of serum interleukin-6 indiffuse large-cell lymphonardquo Annals of Internal Medicine vol127 no 3 pp 186ndash194 1997

[10] N Cimerman P M Brguljan M Krasovec S Suskovic andJ Kos ldquoSerum cystatin C a potent inhibitor of cysteineproteinases is elevated in asthmatic patientsrdquo Clinica ChimicaActa vol 300 no 1-2 pp 83ndash95 2000

[11] L Risch R Herklotz A Blumberg and A R Huber ldquoEffectsof glucocorticoid immunosuppression on serum cystatin Cconcentrations in renal transplant patientsrdquo Clinical Chemistryvol 47 no 11 pp 2055ndash2059 2001

[12] T Gruev K Chakalarovski O Stojceva-Taneva A Grueva andK Trenceva ldquoEffects of glucocorticoid immunosuppression onserum cystatin C levelsrdquo Journal of Medical Biochemistry vol28 no 3 pp 191ndash196 2009

[13] A Aviles B R Narvaez J C Dıaz-Maqueo R Guzman ATalavera and E L Garcıa ldquoValue of serumbeta 2microglobulinas an indicator of early relapse in diffuse large cell lymphomardquoLeukemia amp Lymphoma vol 9 no 4-5 pp 377ndash380 1993

[14] J Azocar M Essex and A Watson ldquoChanges in the expressionof HLA and 1205732-microglobulin by cultured lymphoid cellsrdquoHuman Immunology vol 5 no 4 pp 283ndash293 1982

[15] H Hagberg A Killander and B Simonsson ldquoSerum 1205732-microglobulin in malignant lymphomardquo Cancer vol 51 no 12pp 2220ndash2225 1983

[16] M Legros J Ferriere Y Bignon P Chollet G Gaillard and RPlagne ldquoSerum120573

2microglobulin a good prognosis indicator in

non-Hodgkinrsquos lymphomardquo Proceedings of the American Societyof Clinical Oncology (ASCO) vol 6 article 748 1987

[17] PWM Johnson JWhelan S Longhurst et al ldquo120573-2microglob-ulin a prognostic factor in diffuse aggressive non-Hodgkinrsquoslymphomasrdquo British Journal of Cancer vol 67 no 4 pp 792ndash797 1993

[18] M A Shipp ldquoPrognostic factors in aggressive non-Hodgkinrsquoslymphoma who has ldquohigh- riskrdquo diseaserdquo Blood vol 83 no 5pp 1165ndash1173 1994

[19] M-M Galteau M Guyon R Gueguen and G Siest ldquoDetermi-nation of serum cystatin C biological variation and referencevaluesrdquo Clinical Chemistry and LaboratoryMedicine vol 39 no9 pp 850ndash857 2001

[20] B Majello R Arcone C Toniatti and G Ciliberto ldquoConsti-tutive and IL-6-induced nuclear factors that interact with thehuman C-reactive protein promoterrdquo EMBO Journal vol 9 no2 pp 457ndash465 1990

[21] E Legouffe C Rodriguez M C Picot et al ldquoC-reactive proteinserum level is a valuable and simple prognostic marker in nonHodgkinrsquos lymphomardquo Leukemia and Lymphoma vol 31 no 3-4 pp 351ndash357 1998

[22] J F Seymour M Talpaz F Cabanillas M Wetzler and RKurzrock ldquoSerum interleukin-6 levels correlate with prognosisin diffuse large-cell lymphomardquo Journal of Clinical Oncologyvol 13 no 3 pp 575ndash582 1995

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 ISRN Oncology

levels compared to healthy controls In addition patients withrelapse of the disease had significantly higher cystatin C levelcompared to patients without relapse [8]

For sake of evaluation of the predictive value of cystatinC as a marker of course of the disease the cystatin C levelswere compared with established biochemical parameters (Creactive protein (CRP) beta-2 microglobulin (120573

2M) lactate

dehydrogenase (LDH) levelsactivities) with clinical parame-ters of disease progression (the presence of B symptoms clin-ical stage age and international prognostic index (IPI)) andwith suggested prognostic factor in patients with lymphoma[9] (the levels of cytokine interleukin 6 (IL-6))

2 Materials and Methods

21 Subjects Serum samples of patients with diagnosis of Bnon-Hodgkin lymphoma have been collected from February2008 until May 2011 at the Clinic for Oncology Hematologyand Radiotherapy of the University Clinical Centre of Tuzlawith the approval of the local ethical committee and thewritten consent of the participants Two samples of bloodfrom each patient were taken first at the time of diagnosisbefore therapy and the second immediately before fourthcycle of chemotherapy Thus it was possible to exclude thepossibility of increasing the cystatin C sera level due toinfluence of corticosteroids which are the common part ofchemotherapeutic protocols [10ndash12]

Fromfifty-two pairs of collected samples three pairs werefrom the patients with the relapse of the disease Elevenpatients had the indolent type of lymphoma and 41 hadthe aggressive form of those 18 with nodal and 23 withextranodal disease presentation

Thirty-six patients have been receiving CHOP regimen ofchemotherapy (cyclophosphamide doxorubicin hydrochlo-ride oncovin and prednisone) Among them thirty patientshave been receiving Rituximab and one patient bleomycinEight patients have been receiving CHOEP protocol (CHOP+ etoposide) and 6 of them with addition of immunotherapyThree patients have been receiving COP protocol (cyclophos-phamide oncovin and prednisone) 2 patients FCDprotocol(fludarabine cyclophosphamide and dexamethasone) onepatient high doses of methotrexate while 2 patients withhighly aggressive lymphomas have been treated by modifiedYugoslav protocol for acute lymphoblastic leukemia Theselast two patients have been included in group of aggressivelymphomas

For each patient his age type of lymphoma and IPI havebeen noted and his biochemical parameters like CRP 120573

2M

LDH and creatinine in serum have been measuredThe serum creatinine level has been used as a criterion for

selection of patients those with the creatinine level above theupper physiologic limit have not been included in the studybecause of possible renal dysfunction Also patients withautoimmune diseases asthma and other malignant diseaseshave been excluded from the study

Seven milliliters of blood per patient has been collectedThe blood was clotted at room temperature and subsequentlycentrifuged at 3000 rpm Serumwas separated aliquoted and

stored at minus20∘C For analysis delayed for a longer period oftime serum was stored at ndash80∘C

Reevaluation was carried out after three cycles ofchemotherapy by repeating the tests which had results out ofnormal range A complete response was defined as the disap-pearance of all clinical laboratory and radiographic evidenceof lymphoma Responding patients with minimal residualradiographic abnormalities were classified as showing partialresponse Lesser objective responses were classified as poorpartial response

22 Methods The levels of cystatin C in sera of lymphomapatients were measured by specific sandwich-type ELISAdeveloped at the Department of Biochemistry andMolecularBiology Jozef Stefan Institute and Krka dd as described[7] A microtiter plate was coated with rabbit affinitypurified anti-cystatin C polyclonal antibody Recombinanthuman cystatin C was used for preparation of calibrationcurves Sera from patients were diluted 1 100 prior tobeing applied to wells of microtiter plate As a detectionantibody 1A2 monoclonal antibody was used conjugatedto horseradish peroxidase A substrate for peroxidase was331015840551015840-tetramethylbenzidine liquid substrate system (TMBSigma) The reaction was stopped by adding of 2M H

2SO4

and the absorbance was measured at 450 nm on minireader(TECAN Sunrise) Measured values of diluted samples andcontrols were compared with the calibration curve andexpressed in ngmL of serum

The levels of IL-6 in sera of lymphoma patients weremeasured by commercial sandwich-type ELISA (Quan-tikine RampD Systems Inc Minneapolis USA) on minireader(TECAN Sunrise) ELISA assays were performed in dupli-cate and the mean value determined

Biochemical parameters were measured at the Polyclinicfor Laboratory Diagnostics University Clinical Centre TuzlaCRP was measured by immunoturbidimetric method onArchitect c8000 analyzer 120573

2M was measured by Micropar-

ticle Enzyme Immunoassay (MEIA) (Abbott) on AxSYMsystem LDH was measured by enzyme method (Abbott) onArchitect c8000 analyzer and enzyme method (Siemens) onDimension system Creatinine was measured by a modifiedJaffe method on Dimension system

23 Statistical Analysis For statistical analysis of the resultsSPSS software release 170 (SPSS Inc Chicago IL USA) wasused Mann-Whitney U test was used to compare differencesin cystatin C levels between independent groups and theWilcoxon matched pair test was used to compare cystatinC level measured at the time of diagnosis and before fourthcycle of chemotherapy Correlations of cystatinCwith clinicalstage of disease IL-6 CRP 120573

2M and IPI were defined by

Spearman rank correlation analysis In all tests two-sided Psbelow 005 were considered significant

3 Results

In patients who achieved partial or complete remissionafter three cycles of chemotherapy the level of cystatin C

ISRN Oncology 3



chemotherapy






99576(69838ndash141520) minus1087 0277

Nodal 15 95796(59945ndash145160)

82336(6180ndash118290) minus1874 0061




IL-6



1205732M



CRP









4 ISRN Oncology




2M was 182mgL


























4 Discussion





ISRN Oncology 5





IPI stage0 1 2 3 4






















2M and on












6 ISRN Oncology





Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















ISRN Oncology 3



chemotherapy






99576(69838ndash141520) minus1087 0277

Nodal 15 95796(59945ndash145160)

82336(6180ndash118290) minus1874 0061




IL-6



1205732M



CRP









4 ISRN Oncology




2M was 182mgL


























4 Discussion





ISRN Oncology 5





IPI stage0 1 2 3 4






















2M and on












6 ISRN Oncology





Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 ISRN Oncology




2M was 182mgL


























4 Discussion





ISRN Oncology 5





IPI stage0 1 2 3 4






















2M and on












6 ISRN Oncology





Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















ISRN Oncology 5





IPI stage0 1 2 3 4






















2M and on












6 ISRN Oncology





Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 ISRN Oncology





Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

The Predictive Value of Cystatin C in Monitoring of B Non ...downloads.hindawi.com/journals/isrn.oncology/2013/752792.pdf · ISRNOncology 5 Table4:CorrelationofcystatinCwithclinicalstageofthediseaseandIPI