Bone metastases often appear in advanced stages of lung cancer. They are the result of modulation of bone metabolism
by tumor cells that migrated into bone microenvironment and degraded bone organic matrix. Measurement of C-terminal
telopeptide of type I collagen (ICTP) in the serum of subjects with lung cancer with and without bone metastases and
healthy population is the way to explore bone resorption. In 343 subjects included in this research ICTP level was significantly
higher in the bone metastasis than other two groups. The existence of pathologic fracture significantly increased
ICTP level. ICTP showed sensitivity of 66.0% in bone metastases at 95.0% specificity in lung cancer stages IA and IB.
ICTP is a good diagnostic marker in detection of bone metastasis of lung cancer. Its level can distinguish lung cancer
with and without bone involvement and can be used as an addition to standard techniques used in diagnostics of bone
metastasis

Ana Franjević

Gordana Bubanović

Radomir Pavićević

English

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Coll. Antropol. 35 (2011) 1: 43–47Original scientific paperICTP in Bone Metastases of Lung CancerAna Franjevi}, Radomir Pavi}evi} and Gordana Bubanovi}University of Zagreb, Zagreb University Hospital Center, »Jordanovac« Clinic for Pulmonary Diseases, Department for ClinicalApplication of Lung Tumor Markers, Laboratory of Cancer Genetics, Zagreb, CroatiaA B S T R A C TBone metastases often appear in advanced stages of lung cancer. They are the result of modulation of bone metabolismby tumor cells that migrated into bone microenvironment and degraded bone organic matrix. Measurement of C-termi-nal telopeptide of type I collagen (ICTP) in the serum of subjects with lung cancer with and without bone metastases andhealthy population is the way to explore bone resorption. In 343 subjects included in this research ICTP level was signifi-cantly higher in the bone metastasis than other two groups. The existence of pathologic fracture significantly increasedICTP level. ICTP showed sensitivity of 66.0% in bone metastases at 95.0% specificity in lung cancer stages IA and IB.ICTP is a good diagnostic marker in detection of bone metastasis of lung cancer. Its level can distinguish lung cancerwith and without bone involvement and can be used as an addition to standard techniques used in diagnostics of bonemetastasis.Key words: bone matrix, lung neoplasms, osteoclasts, osteolysisIntroductionLung cancer frequently develops bone metastases inadvanced stages of disease. At initial diagnosis 16% ofnon-small cell lung cancer (NSCLC) and 38% of small celllung cancer (SCLC) patients present with bone meta-stasis1. At the time of death, the majority of patientshave bone metastases2.Lung cancer metastases to bone are predominantlyosteolytic. Osteolysis results from the increased numberand activity of osteoclasts in bone remodeling process.Once caught in the bone marrow vasculature, tumorcells adhere to endothelial cells, and extravasate to bonemicroenvironment3. The attachment to bone matrix isfacilitated by bone sialoprotein (BSP), often overexpres-sed in lung cancer cells4–7. It is assumed that tumor cellsbind to BSP through the integrin avb3 expressed on theirsurface whilst glutamate-rich regions of BSP attach tocrystals of hydroxyapatite8.In the bone microenvironment tumor cells secreteparathyroid hormone-related peptide (PTHrP) which sha-res 70% homology with parathyroid hormone (PTH) andhas similar biologic function binding to PTH/PTHrPreceptors9–12. PTHrP stimulates the production of recep-tor activator of nuclear factor-kB ligand (RANKL) on thesurface of stromal cells and osteoblasts. RANKL thenbinds to receptor activator of nuclear factor-kB (RANK)on osteoclast progenitors. Activation of transcription fac-tors AP1 and NF-kB in osteoclast progenitors leads totheir differentiation13.Simultaneously, PTHrP binds to PTH/PTHrP recep-tors in kidneys and stimulates the tubular resorption ofcalcium what causes hypercalcaemia which is one of thetypical traits of bone metastasis14.Degradation of bone matrix which is rich with growthfactors causes their release into the bone microenviron-ment. The release of TGF-b subsequently induces the sig-nal transduction of Smad proteins15–17. This process sti-mulates the proliferation of tumor cells and their produc-tion of PTHrP which closes the »vicious circle« of theosteolytic bone metastasis.In optimal pH from 4,7 to 6,8 cathepsin K from osteo-clasts can cleave collagen fibers on several sites on triplehelix, but primarily on telopeptide region generatingfragments from 70 to 80 kDa18–20. Its concentration in os-teoclasts is the highest relative to cathepsins S, L andB21.Osteoclasts strongly express matrix-metalloproteina-se 9 (MMP-9) which is beside MMP-2 an enzyme that has43Received for publication October 11, 2010an ability to cleave denaturated collagen type I betweenresidues 775 and 776 thus generating N and C-terminalfragments in optimally neutral pH19,22–24. The positivecorrelation between serum levels of MMP-9 and ICTPhas been proven by Ylisirniö et al.25.Normally, the pH value in subosteoclastic resorptionzone ranges from 3.5 and 4.5, but it is hypothesized thatthe buffer activity of dissolved salts from the bone (Ca2+and PO43–) shifts the pH value towards the neutral18,20.The C-terminal telopeptide of type I collagen (ICTP)is found only in C-terminal part of the mature collagentype I in which the crosslinks between fibrils have beenformed. Since the 90% of bone organic matrix is com-posed of collagen type I that makes ICTP good potentialmarker of bone resorption process.Increased ICTP levels can be found in bone metasta-ses of breast, prostate, lung cancer and other malig-nancies26,27. ICTP levels can discriminate lung cancerwith and without bone metastasis26. Also, ICTP level re-flects the extent of skeletal involvement and the associ-ated bone pain26,27. It has been proven to be a good prog-nostic marker in lung cancer elevated level implyinghigher risk of death than those below cut-off value25.The aim of this study is to investigate the level ofICTP in lung cancer with proven bone metastases, lungcancer in stage IA or IB prior to surgery and phenotypichealthy individuals, and to test the differences in ICTPlevel between studied groups.The clinical application of ICTP depends on popula-tion characteristics, patient medical history, sample sizeA. Franjevi} et al.: ICTP in Bone Metastases of Lung Cancer, Coll. Antropol. 35 (2011) 1: 43–4744TABLE 1CHARACTERISTICS OF THE STUDY POPULATIONPatients with bonemetastasisPatients in stageIA/IBPhenotypic healthyindividualsNumber 144 99 100GenderMale 101 74 31Female 43 25 69Age 64.0±10.2 62.8±9.9 46.6±12.4Histological type oflung cancerAdenocarcinoma 60 27 –Squamous cell carcinoma 38 46 –Small cell lung cancer 12 2 –Adenosquamous or other mixed types ofnon-small cell lung cancer10 3Large cell carcinoma 3 7 –Carcinoids 1 11Other (other mixed types, non-differentiated) 20 3 –StageIA – 16 –IB 83Pathologic fracturePresent 19 – –Not present 125 – –Months after initial diagnosis of lung cancer 0–84.0±13.0 – –Number of affectedmetastatic sites1 104 – –2 17 – –3 12 – –4 7 – –5 and more 4 – –Location of bonemetastasisRib 69 – –Vertebra 64 – –Pelvis 20 – –Femur 18 – –Scapula 14 – –Humerus 10 – –Calvarial bones 8 – –Sternum 5 – –Clavicle 4 – –Other 15included in research, sampling time in relation to otherdiagnostic invasive procedures, and clinically definedcontrol groups that are expected to have a different dy-namics of ICTP level than the healthy population.Subject selectionSamples from 144 lung cancer patients with bone me-tastasis were obtained at the time of the first evidence ofosteolytic lesions proven by scinthigraphy and x-ray scans,before treatment of bone metastases by irradiation andbiphosphonates.Second group is consisted of 99 lung cancer patientswho underwent surgical treatment (lobectomy or pneu-monectomy). Samples were collected at the time of diag-nosis prior to surgery, and patient selection has beenmade according to the pathologic diagnosis made aftersurgical treatment, so only the patients in stage IA or IBwere selected in order to be positive there was no diseasespreading outside the thoracic wall.Third group is composed of 100 healthy individualswho had no visible symptoms of any disease nor hadknowledge about any disease or disorder.Marker assayBlood samples were collected by standard venipun-cture into sterile tubes containing clot activator. Aftercentrifugation sera were stored at –20°C until analysis.ICTP measurement was done using commerciallyavailable immunoassay Uniq ICTP EIA (Orion Diag-nostica, Finland), according to the manufacturer’s in-structions. ICTP concentration is given in mg/L.Statistical analysisDifferences between groups were tested by Mann--Whitney U-test or Kruskal-Wallis ANOVA on Statistica7.1 software package (Statsoft, USA). ROC curves werecalculated using Analyse-it 1.73 (Analyse-it Software,UK).P-value below 0.05 was considered significant.ResultsThe characteristics of 343 subjects enrolled in thestudy are presented in Table 1.Highest median ICTP levels are seen in lung cancerpatients with bone metastasis (Table 2).Comparison between groups revealed significant dif-ferences between the bone metastasis population andother two groups (Figure 1). Also, significant differenceshave been found between the population of patients instage IA and IB and phenotypic healthy individuals, butnot to the extent as in comparison with bone metastasispatients.A. Franjevi} et al.: ICTP in Bone Metastases of Lung Cancer, Coll. Antropol. 35 (2011) 1: 43–4745TABLE 2SERUM LEVEL OF ICTP IN THREE GROUPS OF STUDIED POPULATIONPatients with bone metastasis Patients in stage IA/IB Healthy individualsMedian ICTP (mg/L) 12.0 5.8 4.3Inter-quartile range 9.1–20.5 4.7–7.2 3.2–5.5BM IA/IB PHP020406080100120p=0.0001p=0.0001p=0.0466Fig. 1. Comparison of ICTP levels between groups. BM=patientswith bone metastasis, IA/IB=patients in stage IA or IB, PHP=phenotypic healthy population.b)Not present Present020406080100120Pathologic fracturep=0.0305a)AD SQC LCC OtherNSCLCC SCLC Other020406080100120NSFig. 2. a) Comparison in ICTP level among various histologicaltypes of lung cancer with bone metastasis; b) Differences in ICTPlevel between patients without and with pathologic fracture.NS=non-significant, AD=adenocarcinoma, SQC=squamous cellcarcinoma, LCC=large cell carcinoma, Other NSCLC= adeno-squamous and other mixed types of non-small cell lung cancer,C=carcinoid, SCLC=small cell lung cancer.The comparison of ICTP levels according to the his-tological type of lung cancer in bone metastasis group didnot reveal any significant correlation (Figure 2a). On thecontrary, Mann-Whitney U-test showed significant dif-ferences in ICTP level between subjects with and with-out pathologic fracture as a result of bone metastasis(Figure 2b). Obtained median ICTP value in patientswithout the fracture is 11.7 mg/L and with pathologicfracture 20.1 mg/L.Furthermore, ROC curve is constructed using twodatasets; truly positive=patients with proven bone me-tastasis, and truly negative=lung cancer stage IA or IB(Figure 3).The cut-off value of 9.9 mg/L is selected from the ROCcurve which corresponds to 66.0% of sensitivity and95.0% of specificity. That gives positive predictive valueof 95.0% and negative predictive value of 65.7%.DiscussionThe results obtained in this research show signifi-cantly higher ICTP values in lung cancer with bone me-tastasis than in lung cancer in stages IA and IB andphenotypic healthy population. This confirms the mech-anism of osteolyis in lung cancer bone metastasis result-ing predominantly from the increased osteoclast matura-tion and activation and subsequent bone organic matrixdegradation13,25. The median ICTP value in patients instage IA and IB is closer to the one measured in healthypopulation suggesting the absence of increased bone re-sorption in the early stages of lung cancer.All three groups compared together showed signifi-cant differences between lung cancer with bone meta-stases and other two groups. Such differences have beenpreviously found in breast, lung and prostate cancer withand without bone metastasis26,28–30. The ability of ICTPto discriminate between lung cancer with and withoutbone metastasis suggests its use as a potential marker ofbone collagen degradation in the advanced stages of lungcancer where there is suspicion about bone involvementbased on symptoms and available imaging techniques.The difference in ICTP levels between lung cancer ofstage IA and IB and phenotypic healthy population canbe explained by the destruction of extracellular matrixsurrounding primary lung tumor. Although not as highas the ones obtained by the comparison with the bonemetastasis group these differences suggest a certain levelof degradation of collagen fibers in the epithelial tissueand stroma built from the helical units of collagens I andIII31. These results have been previously described inlung cancer25.The histological type of lung cancer with bone metas-tasis did not significantly influence ICTP levels as themechanism of osteoclast activation being common to allpredominantly osteolytic bone metastasis.The existence of pathologic fracture influenced signif-icantly the ICTP level being higher than in bone metas-tasis without fracture. These results confirm that the in-crease in serum level of ICTP is mainly a consequence oftumor involvement of the bone organic matrix that ismore advanced in patients who have pathologic frac-tures.ROC curve revealed very good AUC and ratio of sensi-tivity and specificity. The high sensitivity in bone metas-tasis makes ICTP good diagnostic marker as an addi-tional tool to standard diagnostic techniques. ICTP canbe primarily used in monitoring of lung cancer patientswith the suspicion, increased risk or already presentbone metastasis because its level reflects bone resorptionresulting from modulation of bone metabolism by tumorcells.ConclusionThis research showed that ICTP is good diagnostictool that can detect bone metastasis of lung cancer withhigh sensitivity. Also, it can be used to distinguish lungcancer with and without bone metastasis. These resultssuggest its application as a complement to standard diag-nostic techniques in diagnosis and monitoring of the ad-vanced lung cancer with high risk of bone involvement oralready present bone metastasis.R E F E R E N C E S1. SCARANTINO CW, ORNITZ RD, WOODARD SA, Metastases anddisseminated disease. In: RUBIN, P (Ed) Clinical oncology: a multidis-ciplinary approach for physicians and students (W.B. 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Franjevi}University of Zagreb, Zagreb University Hospital Center, Department for Clinical Application of Lung Tumor Markers,»Jordanovac« Clinic for Pulmonary Diseases, Jordanovac 104, 10000 Zagreb, Croatiae-mail: ana.franjevic@zg.t-com.hrICTP KOD KO[TANIH METASTAZA RAKA PLU]AS A @ E T A KKo{tane metastaze ~esto su prisutne u uznapredovanim stadijima raka plu}a. One su rezultat modulacije ko{tanogmetabolizma od strane tumorskih stanica koje su migrirale u ko{tanu mikrookolinu i degradirale ko{tani organskimatriks. Mjerenje C-terminalnog telopeptida kolagena tipa I (ICTP) u serumu ispitanika sa i bez ko{tanih metastaza,te zdravoj populaciji, na~in je na koji je mogu}e ispitati ko{tanu resorpciju. Kod 343 ispitanika obuhva}enih istra-`ivanjem razina ICTP-a bila je zna~ajno vi{a kod ko{tanih metastaza, nego kod preostale dvije grupe. Postojanje pato-lo{ke frakture zna~ajno je utjecalo na pove}anje razine ICTP-a. ICTP je pokazao senzitivnost od 66,0% kod ko{tanihmetastaza koja odgovara 95,0%-tnoj specifi~nosti kod raka plu}a stadija IA i IB. ICTP je dobar dijagnosti~ki biljeg zadetekciju ko{tanih metastaza raka plu}a. Njegovom razinom mogu}e je razlikovati rak plu}a sa i bez zahva}enostikostiju, te se mo`e koristiti kao dodatak standardnim tehnikama dijagnostike ko{tanih metastaza.A. Franjevi} et al.: ICTP in Bone Metastases of Lung Cancer, Coll. Antropol. 35 (2011) 1: 43–4747

ICTP in Bone Metastases of Lung Cancer

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