Turkish Neurosursery 12: 17 - 21, 2002 Uqnr; Trnltsferril1 Rt'ceptor~ ill ll/lL'lIil/siml1{/~

ExpressionMeningiomas

Transferrin Receptorin Intracranial

intrakranial Menenjiomalarda TransferrinReseptor Diizeyleri ve Prognastik Onemi

TANJU Ue;AR, iNANe; GURER, MAHMUT AKYUZ, ELip PESTERELi

Akdeniz University Medical School, Neurosurgery Department (TU, MA), Antalya, TurkeyAkdeniz University Medical School, Pathology Department (iG, EP), Antalya, Turkey

Received: 24.1.2002 <:::> Accepted: 5.3.2002

Abstract: Objective: Recent studies of iron mechanism inthe brain have indicated that there may be a relationshipbetween transferrin receptor (TfR) expression andhistological malignancy._ TfR expression may also reflectcell proliferation. We investigated TfR staining intensityin intracranial meningiomas, and assessed whether thiswas related to recurrence and/ or malignant proliferation.Methods: Immunohistochemical techniques were used toassess TfR expression in different types of intracranialmeningiomas (11=15) that had been surgically removed.All were primary tumors. The specimens weresemiquantitatively scored. for staining intensity andproportions of cells stained, and the relationship betweenTfR immunoscore and recurrence/proliferation wassubjectively evaluated.Results: High levels of TfR expression were observed inall the meningioma subtypes. Three of the 15 casesrecurred, and two of these patients died due to intracranialpathology.Couclusioll: Patients 'with meningiomas that have high TfRimmunoscores must be carefully followed for recurrenceand/or malignant transformation.

Ozet: A11la(;: Beyinde demir metabolizmasl vefonksiyonlan uzerinde son y!llarda yapJian <;ah~mal<1rdaTransferrin reseptorleri (TfR) ile histolojik malignitearasmda bir ili~ki oldugu vurgulanm!~t!r. Bu <;ah~madapostoperatif olarak tams! konmu~ ve yi.iksek TfRboyanmas! gosteren meningiom olgulannda boyanmayogunlugu dikkate almarak, bununla rekkurens ve maligntransformasyon arasll1da bir ili~ki olup olmadlglara~tmlm!~hr.YOlltelll ve Bulgular: C;:ah~mada 15 primer intrakranialmeningiom olgusunda TfR boyanmaslm gostermekamaClyla immunohistokimya teknikleri kullal1llml~tlr.SOtIll(;: Him meningiom tiplerinde TfR belirgin olarakyuksek duzeyde boyanma gostermi;;tir.TfR yogunboyanma gosteren olgulardan 3 tanesi nuks nedeniylereopere edilmi~, rekkurens gosteren olgulann 2 si ti.imorebagh nedenlerden dolay! kaybedilmi~tir. TfR seviyeleriyuksek olan intrakranial meningiom olgulanmnrekkurens ve malign dejenerasyon a<;lsll1dan takipedilmeleri gereklidir.

Key words: Meningioma, recurrence, transferrin receptor(TfR)

Anahtar kelimeler: Meningioma, rekkurens, transferrinreseptorleri

INTRODUCTION

Iron is essential for oxygen transfer in the body.It is a cofactor for many enzyme systems that are

specific to the brain, and is vitally important for braincell function and viability 0,5,15,19,24,27). In recentyears, more researchers have focused on exploringthe mechanisms of iron function in the nervous

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TlIrkish NeuroslIrgery 12: 17 - 21, 2002

system. Transferrin (Tf) is an 80 KDa glycoprotein inplasma that is capable of binding iron, and functions as acarrier for iron to the cells via the bloodstream. Tf is

recognized as an essential growth factor in cellproliferation, and both iron and Tf are required for DNAsynthesis and cell division. A number of studies havedemonstrated a close link between the quantity oftransferrin receptors (TfRs) on the cell surface and the rateof cell proliferation (30). TfRs are not found in all brainregions, but large numbers are expressed inoligodendrocytes, cerebrospinal fluid, and capillaryendothelial cells of the brain (4,13,14,15,23).

Various types of cell surface receptors, includingepideTI11algrowth factor receptors, interleukin receptors,and TfRs, have been identified on the surface of malignantbrain tumor cells (10).The level ofTfR expression in normalhealthy brain tissue is insignificant compared to that inneoplastic tissue (3). Recent experimental findings havesuggested that TfR expression is closely associated withdegree of histological malignancy; thus, it may be anindicator of cell proliferation (8). In 'liw studies havedemonstrated the presence ofTfRin the human melanomacell line, and in a cell line derived from glioblastomamulti forme (GBM) (26).

Growing tumor cells and proliferating cells bothexhibit higher levels ofTfR expression than normal restingcells (3,8,26). Clinical studies have detected TfRs in most

types of carcinomas, sarcomas, cerebral gliomas, non-glialtumors, meningiomas, and in Hodgkin's and non­Hodgkin's lymphoma (6,7,8). Meningioma is one of themost common brain tumors. Although most meningiomasare benign, recurrence after complete resection is relativelycommon. Various predictors of merungioma recurrenceor malignant proliferation have been identified, includingimmunohistochemical markers such as Ki-67,

neuroradiological findings, and degree of tumor removal.However, there are no detailed reports on how TfRexpression relates to tumor proliferation and prognosisin meningioma patients. In this study, weimmunohistochemically evaluated TfR expression invarious types of intracranial meningiomas, and thensubjectively assessed for links to outcome

MATERIALS AND MElHODS

Fifteen surgically removed intracranialmeningiomas were irnmunohistochemical1y studied. Thesubtypes varied, and all were primary tumors. In eachcase, the degree of tumor removal was scored accordingto the Simpson grading system (28). Histologicalclassification and malignancy grading were perfonnedaccording to the system recommended by the WorldHealth Organization in 1993. Patients were followed for

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LT(llr: TmllsJerrill Receptors ill Mellillgiollll1s

varying periods, and the case outcomes were categorizedas no recurrence, recurrence, or exitus.

HistoJXIthological and Immunohistochemical Metllorls

Tissue from each tumor was fixed in 10%

f0TI11aldehydesolution, and the histological diagnosis wasestablished from preparations stained with hematoxylinand eosin. For immunohistochemical determination ofTfR levels, tissues were incubated in a 1:50 dilution of

TfR mouse anti-cd73 antibody (Biogenex-mu-354-uc)in a DAKO Techmate SOD-plus automatedimmunohistochemistry device with a microwavestreptoimmunoperoxidase program. Staining intensitiesand proportions of positively stained cells were used assemiquantitative determinants of TfR immunoreactivity(18). Cases were graded "a" for no staining, "I" for mildstairung, "2" for moderate staining, and "3" for intensestaining. Regarding the proportions of peroxidase-stainedcells, the cases were graded "a" if no cells were stained;"I" if the stained cells covered less than one-third of the

processed area; "2" if the stained cells covered one- to two­thirds of the area; and "3" if the stained cells covered two­thirds or more of the area. For each case, these two scores

were added together to give an overall "immunostainingscore" between a and 6.

RESULTS

Table I lists the features and immunostaining scoresfor each of the 15 intracranial meningioma cases studied.The mean TfR irnmunoscore was 3.6±1.7 (mean±SD), andthe follow-up times ranged from 2 weeks to 44 months(mean, 24.3 months).

The neoplasm recurred in three cases. In tvvo of thesepatients (CasesNo.2and No. 9), the primary and reccurentexpressed very high levels of TfR. The times from initialsurgery to first recurrence in these cases were 27 and 38months, respectively.

As the table shows, both of these patients had veryhigh TfR immunoscores when they died. One individual(Case No.9) was a pediatric patient whose primarytumor was histopathologically diagnosed asmeningotheliomatous meningioma. The first operation(Simpson grade 1) was in 1996, and the TfR immunoscoreat that time was 3 (Figure 1). This patient required twoother surgeries (Simpson grade 2) in 1997 and 1999 due torecurrence, and he died 2 months after his last operation.The final histopathologic diagnosis was malignantmeningioma, and the TfR immunoscore for that masswas 5 (Figure 2).

The other patient (Case No.2) was in herniation

Turkish Neurosurgery 12: 17 - 21, 2002

Table I: Case features and transferrin receptor (TfR) immunoscores.

Uqnr: Trallsferrm Receptors ill M~lIillsiollln"

No AgeSexTfRLocationHistopathologyResection GradeFollow-upOutcome

(year)

score (Simpson)(month)(*)

1

85d" 3Convexity Transitional 129a

2

64d"3Ant. fossa Meningotheliomatous2271

2r

66d"5Ant. fossa Meningothelioma tous30.52

3

56~6Post. fossaTransitional 1270

4

62~3ParasagittalAngioblastic 133a

5

48d'4Middle fossaFibrous 131a

6

69~6Convexity Meningothelioma tous225a

7

55d"5Ant. fossa Angioblastic 1250

8

62~3Falx Transitional 217a

9

7d'3Convexity Meningotheliomatous1381

9r

10d'5Convexity Malignant 222

10

43d'4Tentorial Transitional 110a11

38~2Convexity Angioblastic 1260

12

57~4Olfactory Fibrous 1231

groove 12r59~2Olfactory Fibrous 126a

groove 1346~aPara sagittalTransitional 131a

14

51~2Convexity Transitional 129a16

48d'2Ant. fossa Fibrous 1440

(0) 0: no recurrence, 1: recurrence, 2: death) ("r" indicates recurrence)

status on admission to hospital. His first operation(Simpson grade 2) took place in 1998, and the TfRimmunoscore at that time was 3. Simpson grade 3removal of a recurrent mass was performed 27 monthslater, but the patient died 2 weeks after this operation.The final histopathological diagnosis wasmeningotheliomatous meningioma and, as in Case No.9, the final TfR immunoscore was 5.

Figure 1: TfR expression in meningioma: This specimenfrom Case NO.9 shows mild TfR staining. (X40)

DISCUSSION

TfR is a specific cell surface antigen that is mainlyexpressed in tumoral tissue (11,25,26,31). As mentionedabove, experimental studies have documented high levelsofTfR expression on proliferating cells and cells that haveundergone malignant transformation. Clinical shldies

Figure 2: A specimen from the same patient obtained afterrecurrence 3 years later shows malignanttransformation and intense staining. (X40)

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TlIrkish Nell ros II rger!/ 12: 17 - 21, 2002

have also yielded evidence that supports these links.Immw10histochemical investigations have detected TfRson human surgical tissue from breast carcinomas,sarcomas, pituitary adenomas, GBM, and certain othertumor types (6,10,12,30). Recht and co-workers assessed27 brain tumor biopsy specimens, and compared thenumbers ofTfRs in nom1al and neoplastic tissue (26). Theyfound that GBM exhibited the most intense TfR staining,and reported that approximately 60% of the meningiomasshowed less than 25% staining for TfR. A similar studyrevealed no detectable TfR expression in slow-growingbrain tumors such as cerebellar astrocytomas and acousticneuromas (10). Some investigators have found that theintensity of TfR staining correlates with tumorhistopathology (12,25).In accord with this, several in vitroand in vivo studies have shown that GBM exhibits

more intense TfR staining than other brain neoplasms (11).

Our immunohistochemical analysisdemonstrated significant TfR expression in all typesof meningiomas. Although we anticipated that wemight see high levels ofTfR expression in histologicallyatypical and anaplastic tissues, the intense staining inother meningioma subtypes was a surprise. Asmentioned previously, most meningiomas are benign,but the rate of recurrence even after complete excisionis considerable. The highest rates of meningiomarecurrence are associated with incomplete tumorremoval or higher degrees of histologicalaggressiveness (2,21,22). However, in all three of ourcases that recurred, the patients had undergoneSimpson grades 1 and 2 removal. Similar to what hasbeen observed in many malignant neoplasms, ourstudy results suggest thatJhe levels of TfR expressionin meningioma are directly related to recurrence anddegree of malignancy.

Four of our patients had initial TfR immunoscoresof 2 or less, and none of these individuals developedserious problems or tumor recurrence/malignanttransformation. Three of the 15 cases recurred, and oneof them showed malignant proliferation. Two of the15 patients died at 2 weeks and 2 months, respectively,after removal of tumor material that was

immunoscored as 5. Although positive TfR stainingappears to reflect prognosis in meningioma cases, it isclearly not the only valuable indicator of recurrenceand malignant proliferation. Simpson resection grade,peri tumoral edema, tumor location, and tumor shapeare some other important prognostic indicators.Nakasu et al. stressed the importance of radiologicalfeatures for predicting malignancy in meningioma(21) The same study revealed a strong correlationbetween peri tumoral edema and higher rates of

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ll~nr: TmllsJarill neaplor" ill Mellillgiollll'"

proliferation and recurrence in meningioma cases. Werecognize the relevance and significance of theseconventional indicators, but believe thatimmunohistochemical markers should also be

considered critical for assessing meningiomarecurrence. Further, upregulation of TfR in braintumors provides an alternative route for deliveringanti-cancer drugs and/or toxins to the proliferatingcells. Tumor-toxin targeted therapy in brain tumors,especially GBM and medulloblastoma, has been welldocumented (9,12,16,17,20,26,29,32). In our opinion,TfR upregulation is one of the best candidates for futuremeningioma immunotherapy.

Considering our patients' TfR findings in relationto follow-up findings, we observed that a considerablenumber individuals with higher TfR immunoscoreshad poor outcomes and required re-opera tion.However, due to the limited number of cases we

studied and the relatively short duration of follow-up,we were unable to establish a statistical correlation

between score and outcome. Nevertheless, based on

these observations and results, we strongly recommendthat meningioma patients with TfR expression scoresof 3 or higher be carefully followed up for recurrenceand/ or malignant transformation. To further supportand clarify our conclusion, future studies shouldinclude larger numbers of patients.

Correspondence: Tanju U~arAkdeniz University MedicCiISchoolNeurosurgery Dept.AntCilyCi/ TurkeyFClx:0902422274490e-mClil:tucar@superonline.com

REFERENCES

1. Aisen P: Entry of iron into cells: A new role for thetrClnsferrin receptor in modulClting iron releCise fromtrClnsferrin.Ann Neurol 32: 62-68, 1992

2. Bello L,JiCinping2, Demetrios CN, Jon FS, Roberto MV,DClvidAC, Rona SC, Peter MB: C1va3C1ndC1va5integrinexpression in meningiomas. Neurosurgery 47(5): 1185­1195,2000

3. Colombatti M, Massimo B, Lorena DA, Massimo AG,Guiseppe T: Sensitivity of human glioma cells tocytotoxic heteroconjugates. Int J Cancer 42:441-448,1988

4. Connor JR,Fine RE:Development of transferrin positiveoligodendrocytes in the rat central nervous system.Journal of Neuroscience Research 17:51-59,1987

5. Connor JR, Brian SS, PCloioA, David AL, Peter LW: Aquantitative analysis of isoferritins in selected regionsof aged Parkinsonian and Alzheimer's diseased brClins.Journal of Neurochemistry 65(2): 717-724, 1995

6. FCiulkPW, BaiLH,Stevens PJ:TrClnsferrinand trClnsferrinreceptors in carcinoma of the breast. The LCincet23:390­392,1980

7. Feelders RA, Vreugdenhil G, Eggermont AM, Kuiper-

TlIrkislr NellroslIrgery 12: 17 - 21, 2002

Kramer PA, Van Eijk HG, Swaak AJ: Regulation of ironmetabolism in the acute phase response: interferongamma and tumour necrosis factor alpha inducehypoferraemia, ferritin production and decrease incirculating transferrin receptors in cancer patients. Eur JClin Invest 28(7):520-527, 1998

8. Gatter KC, Brown G, Strowbridge I, Woolston RE, MasonDY: Transferrin receptors in human tissues; theirdistribution and possible clinical relevance. J Clin Pathol36: 539-545, 1983

9. Hall WA: Targeted toxin therapy for malignantastrocytoma. Neurosurgery 46(3): 544-552, 2000

10. Hall WA, Merrill MJ, Walbridge S, Youle RJ: Epidermalgrowth factor receptors on ependymomas and otherbrain tumors. J Neurosurg 72: 641-646, 1990

11. Hall WA,Godal A, Juell S, Fodstad 0: In vitro efficacy oftransferrin-toxin conjuga tes against glioblastomamulti forme. J Neurosurg 76: 838-844, 1992

12. Hall WA: Transferrin receptor on glioblastomamultiforme. (letter) J Neurosurg 74: 313-314, 1991

13. Hill JM, Ruff MR, Weber RJ, Pert CB: Transferrinreceptors in rat brain: neuropeptide-like pattern andrelationship to iron distribution. Neurobiology 82: 4553­4557, 1985

14. Hulet SW, Hess EJ, Debinski W, Arosio P, Bruce K,Powers S, Connor JR: Characterization and distributionof ferritin binding sites in the adult mouse brain. JNeurochem 72(2): 868-874, 1999

15. Huwvler J, Froidevaux S, Roux F, Eberle A :Char~cterization of transferrin receptor in animmortalized cell line of ra t brain endothelial cells, RBE4.

J Recept Signal Transduct Res 19(1-4): 729-739, 199916. Johnson VG, Wrobel C, Wilson 0, Zovickian J,

Greenfield L, Oldfield EH, Youle R: Improved tumor­specific immunotoxins in the treatment of CNS andleptomeningeal neoplasia. J Neurosurg 70: 240-248, 1989

17. Laske OW, You Ie RJ, Oldfield EH: Tumor regressionwith regional distribution of the targeted toxin TF-CRM107 in patients with malignant brain tumors. Nat Med3(12): 1362-1368,1997

18. Lee CS, Redshaw A, Boag G: Nm 23-Hl proteinimmunoreactivity in laryngeal carcinoma. Cancer 77:2246-2250, 1996

19. Loeftler DA, Connor JR, Juneau PL, Snyder S, KanaleyL, DeMaggio AJ, Nguyen H, Brickman CM, LeWitt PA:Transferrin and iron in normal, Alzheimer's disease and

Parkinson's disease brain regions. Journal ofNeurochemistry 65(2): 710-716, 1995

U(ar: Trallsferrill ncceptors ill MellillS;Ol/lflS

20. Martell LA, Agrawal A, Ross DA, Muraszko KM:Efficacy of transferrin receptor-targeted immunotoxinsin brain tumor cell lines and pediatric brain tumorsCancer Res 15;53(6): 1348-1353, 1993

21. Nakasu S, Nakajima M, Matsumura K, Nakasu Y, HandaJ: Meningioma :prolifera ting poten tia I andc1inicoradiological features. Neurosurgery 37(6): 1049­1055, 1995

22. Nakasu S, Nakasu Y, Nakajima M, Matsuda M, HandaJ: Preoperative identification of meningiomas that arehighly likely to recur. J Neurosurg 90: 455-462, 1999.

23. Pardridge WM, EisenbergJ, YangJ: Human blood-brainbarrier transferrin receptor. Metabolism 36(9): 892-895,1987

24. Perl DP, Good PF: Comparative techniques fordetermining cellular iron distribution in brain tissues.Ann Neuro132: 76-81, 1992

25. Prior R, Reifenberger G, Wechsler W: Transferrinreceptor expression in tumours of the human nervoussystem: relation to tumour type grading and tumourgrowth fraction. Virchows Arch A Pathol AnatHistopathol 416(6): 491-496, 1990

26. Recht L, Torres CO, Smith TW, Raso V, Griffin TW:Transferrin receptor in normal and neoplastic braintissue; implications for brain tumor immunotherapy. JNeurosurg 72: 941-945, 1990

27. Roelcke U, Leenders KL, von Ammon K, Radu EW,Vontobel P, Gunther I, Psylla M: Brain tumor iron uptakemeasured with positron emission tomography and 52Fe­citrate. J NeurooncoI29(2): 157-165, 1996

28. Simpson D. The recurrence of intracranjalmeningiomasafter surgical treatment. J Neurol Neurosurg Psychiatry20:22-39,1957

29. Singh M: Transferrin as a targeting ligand for lipo50mesand anticancer drugs. Curr Pharm Des 1;5(6): 443-451,1999

30. Tampanaru SA, Stefaneanu L, Thapar K, KontogeorgosG, Sumi T, Kovacs K: Transferrin and transferrin receptorin human hypophysis and pituitary adenomas. Am JPa thol 152(2): 413-422, 1998

31. Wen DY, Hall WA, Conrad J, Godal A, Florene~ VA,Fodstad 0: In vitro and in vivo variation in transferrin

receptor expression on human medullablastoma cell line.Neurosurgery 36(6): 1158-1163, 1995

32. Zovickian J, Johnson VG, Youle RJ: Potent and specifickilling of human malignant brain tumor cells by an iJnti­transferrin receptor antibody ricin immlllllltoxin. JNeurosurg 66: 850-861, 1987

Mod PatllOl 2001 Mar; 14(3):197-201

Prostagla1ldi1l D synthase (beta-trace) 111 meningeal hemangiopel'icytoma.

Kawashima M, Suzuki SO, Yamashima T, Fukui M, Iwaki T.

The level of prostaglandin D synthase (PGDS), a major protein constituent of cerebrospinalfluid (CSF), is altered in various brain diseases, including meningitis. However, its role inthe brain remains unclear. PGDS is mainly synthesized in the arachnoid cells, thechoroid plexus and oligodendrocytes in the central nervous system. Among braintumors, meningiomas showed intense immunoreactivity to PGDS in the perinuclearregion. Thus, PGDS has been considered a specific cell marker of meningioma.

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