CLINICAL STUDY -PATIENT STUDY
Allelic loss on chromosomes 1p32,9p21,13q14,16q22,17p,
and 22q12in meningiomas associated with meningioangiomatosis and pure meningioangiomatosis
Na Rae Kim ÆSeong Jin Cho ÆYeon-Lim Suh
Received:1December 2008/Accepted:19March 2009/Published online:5April 2009ÓSpringer Science+Business Media,LLC.2009
Abstract Meningioangiomatosis (MA)is a rare lesion appearing sporadically or as a part of neurofibromatosis 2.The occurrences of meningiomas arising from MA (MA-M)have raised doubts about the traditional concept of a ham-artomatous origin for MA.Cytogenetic or molecular studies on MA,with or without meningiomas,are limited because of the rarity of MA.The current study was to evaluate the loss of heterozygosity (LOH)in seven cases of MA-M and two cases of pure MA.LOH on six chromosomes (1p32,9p21,13q14,16q22,17p,and 22q12)were investigated using 13sets of microsatellite markers,including D1S193,D1S463,D22S193,D22S929,D22S282,TP53,D17S796,D16S421,D16S512,D13S118,D13S153,D9S162,and
D9S104.PCR was performed using each marker and polymorphic analysis was accomplished by silver staining.Immunohistochemical stain for Ki-67was carried out and labeling index was measured by using a semiquantitative manual counting method.The meningioma portions of MA-Ms showed LOH for loci on chromosomes 22q12,9p21,and 1p32in 57.1%(4/7),28.6%(2/7),and 28.6%(2/7)of cases,respectively.The MA portions of MA-M had a LOH for loci on 22q12in 28.6%(2/7)of cases,whereas each pure MA harbored one LOH on either chromosome 22q12or
9p21.The proliferation indices of MA-Ms were signifi-cantly higher in the meningioma than in the MA compo-nents.Our data suggest that both the meningioma and the MA undergo the same overlapping clonal process,with the MA-M while undergoing additional genetic alterations that confer a greater proliferative potential.
Keywords Chromosome 1p32ÁChromosome 9p21ÁLoss of heterozygosity ÁMeningioangiomatosis ÁMeningioma ÁNF2gene
Introduction
Meningioangiomatosis (MA)is accompanied by neurofi-bromatosis 2or occurs sporadically,and its biological nature has long been debated whether it is a hamartoma or a neoplasm [1,2].Meningioma
s derived from MA (MA-M)in individuals without neurofibromatosis 2have rarely been reported [3].The most common association of meningiomas with MA suggested the possibility that MA may be neoplastic in nature.This suggestion was supported by demonstration of loss of 22q12in an each case of MA and MA-M [4,5].However,recent largest genetic series demonstrated no NF2gene mutation in 24sporadic MAs and two neurofibromatosis-associated MAs.These obser-vations supported the hypothesis of the hamartomatous nature of MA [6,7].Perry et al.[6]demonstrated identical NF2gene deletions and protein loss in both components of MA-M patients.Their data suggest that the MA component of MA-M cases may represent cortical perivascular spread of an overlying meningioma,rather than an underlying hamartoma.However,their data on proliferation index on MA-Ms were against this hypothesis because Ki-67labeling index was consistently higher in the meningioma
N.R.Kim
Department of Pathology,Gil Medical Center,Gachon University of Medicine and Science,Incheon,Korea S.J.Cho
Department of Pathology,Kangdong Sacred Heart Hospital,Hallym University College of Medicine,Seoul,Korea Y.-L.Suh (&)
Department of Pathology,Samsung Medical Center,
Sungkyunkwan University School of Medicine,50Irwon-Dong,Gangnam-Gu,Seoul 135-710,Korea e-mail:ylsuh76@skku.edu
J Neurooncol (2009)94:425–430DOI 10.1007/s11060-009-9879-3
rather than MA components of MA-M cases.Also,it cannot be explained by the lack of EMA-positive men-ingothelial cells in perivascular areas in some MA cases [8].These different observations among the previous studies may be partly caused by the limited numbers of samples as well as genetic loci such as NF2,4.7B gene (chromosome18p),and chromosome1p.We,therefore, investigated genetic alterations of MA-M and pure MA, through loss of heterozygosity(LOH)studies with expanding markers to gain insight into the histogenesis of MA-M.
In general,early genetic alterations occur in polymor-phic markers on22q in30–70%of the meningiomas[9, 10],and chromosome1p has been reported as the second most commonly deleted region;it is the critical step for meningioma progression,and is highly correlated with recurrence[11].Cytogenetic alterations of chromosomes 6q,10q,14q,and17q are also known to be related to progression-associated genes[12].Markers used in this study included chromosomes9p21,
13q14,16q22,17p, 22q12and1p32.
Materials and methods
Selected cases were collected from four tertiary centers: four cases were from Seoul National University Hospital (cases1,2,3,and4),three cases from Samsung Medical Center(cases5,8,and9),one case from Daejeon St.Mary’s Hospital of the Catholic University of Korea(case6),and one case from Gil Medical Center of the Gachon University of Medicine and Science(case7)in Korea.Cases1,2,3,4, and9had been previously reported[13,14].All the tumors werefixed in10%buffered formalin and stained with hematoxylin–eosin.The immunohistochemical stainability of Ki-67(prediluted;DAKO,Glostrup,Denmark)was investigated by using a semiquantitative manual counting method i-Solution7.5(version7.5,IMT i-Solution,Co-quitlam,Canada).The proliferation indices were deter-mined as follows;at least1,000nuclei were counted and the Ki-67proliferation indices were defined as the percentage of positively stained nuclei.Differences of proliferation indices between MA and meningioma were estimated using the paired samples t test.
The formalin-fixed,paraffin-embedded tissue was used for molecular analysis.All the tumor specimen
s were mi-crodissected with the aid of a light microscope.Genomic DNA from the dissected tissue was extracted using a standard method[15].Briefly,DNA was extracted from the meningioma and from peripheral blood lymphocytes of the patient by standard methods as described.The allelic alterations were assessed by13microsatellite markers of chromosomes22q12,1p32,13q14,9p21,16q22,and 17p13(D1S193,D1S463,D22S193,D22S929,D22S282, TP53,D17S796,D16S421,D16S512,D13S118,D13S153, D9S162,and D9S104).The primers for these markers are commercially available(GIBCO,Carlsbad,CA,USA). PCR was carried out in thermal cycler(Perkin Elmer Cetus 9700,USA).PCR amplifications were performed in10l l containing2l l DNA template,0.25l l each primer, 1.25mM NTP with1/2dCTP,1.5mM MgCl2,0.6U Taq polymerase0.07l l,and109PCR buffer.Every cycle consist of denaturation at94°C for30s,annealing at55–60°C for30s and extension at72°C for40s,followed by the last extension at72°C for10min.Amplified PCR products3l l were analyzed by12%polyacrylamide gel electrophoresis.By using silver staining,the gain or loss was assessed as described previously[16].The band intensity of two alleles of each case was determined by scanning densitometric analysis(Pharmacia,San Fran-cisco,CA,USA).LOH in the tumor samples were deter-mined by loss of one allele compared with those from adjacent non-neoplastic cerebral tissue.Scanning densi-tometry was performed to determ
ine the allelic status of the markers analyzed,and LOH in an informative case was defined as a greater than50%reduction in band intensity relative to the non-tumorous tissue control.PCR was per-formed using each marker and polymorphic analysis was accomplished by silver staining.
Results
Clinical results
Seven cases of MA-M includedfive males and two females.Pure MAs involved one male and one female. Gross total resection was done in seven cases[7/9(77.8%)] and subtotal resection was achieved in two cases(22.2%). The ages of the patients were ranged from3to23years (mean,10.3years).All except one patient,who presented with acute intracranial hemorrhage(case4),were accom-panied by long-standing intractable seizures and well-defined calcified masses demonstrated on brain computed tomography.During the follow-up period,a recurrence occurred in one case which had been subtotally resected. One patient died due to an unrelated cause.The clinical data are summarized in Table1.
Pathological and immunohistochemical results
MA-M had the following histologic subtypes:cases2and5 were transitional types;cases3,6and7were meningo-thelial types;and cases1and4werefibrous types. Meningioma portion of case2showed focal increased cellularity with mitotic activity(up to3per20high power
fields).All the meningiomas belonged to WHO grade I tumors.The tumors had surrounding MAs that were com-posed of elongated spindle cells around haphazardly arranged vessels (Fig.1).Calcification was present in all cases except cases 8and 9.Case 9was accompanied by co-existing Taylor-type focal cortical dysplasia type IIA,as well as MA.
Ki-67labeling indices of MA ranged from 0.1to 0.91%(mean 0.35%),whereas those of meningiomas ranged from 1.1to 10.1%(mean 4.7%).In MA-M,proliferation indices of meningioma portions were significantly higher than those of MA portions (P =0.01).The results are summa-rized in Table 1and shown in Fig.2.
Molecular results
Four cases of MA-M showed LOH for loci on chromosome 22q12[4/7(57.1%)]in meningioma components;allelic loss was observed in three cases with D22S193,one with D22S282,and one with D22S929.Two cases of MA-M had LOH on chromosome 9p21in meningioma compo-nents [2/7(28.6
%)]:one was LOH at locus D9S162and the other one LOH at locus D9S104.Two cases of MA-M had LOH for loci on chromosome 1p32in meningioma com-ponents [2/7(28.6%)]:one had LOH at locus D1S193and the other one had LOH at locus D1S463.The MA com-ponent of two MA-M cases had a LOH at loci D22S282
Table 1Clinical summary of nine cases of MA with or without meningiomas Case no.Age (years),gender Site
Procedure Histologic type
of meningioma Ki-67proliferation index (%)Association with NF2Outcome during follow-up MA Meningioma 13,M Fr-P GTR Fibrous 0.32 1.1–NR,alive,LFU (7year)26,M T STR Transitional 0.210.1–R,alive (4year)39,M Fr GTR Meningothelial 0.1 1.3–NR,alive (13month)44,M Fr GTR Fibrous 0.1 2.2–NR,alive (1year)56,F PO Fr GTR
赛马Transitional 0.368.53–NR,alive (1year)
小说月报2012
69,F T STR Meningothelial
0.38
7.62
–NR,dead due to unrelated cause 710,M T GTR Meningothelial 0.1 2.2
–NR,alive (14month)823,F P GTR No meningioma 0.66No meningioma –NR,alive (5month)9
23,M
T
GTR
No meningioma 0.91
No meningioma
宜人贷上市
–
NR,alive (5year)
MA Meningioangiomatosis,GTR gross total resection,STR subtotal resection,M male,F female,Fr-P fr
onto-parietal lobe,PO parieto-occipital lobe,T temporal lobe,Fr frontal lobe,NF2neurofibromatosis type 2,NR no recurrence,R recurrence,LFU lost to follow
up
Fig.1Cases 4(a )and 5(b )show transition from MA (left )to meningioma (right ).The majority of the cal
cific mass consists of the MA component with proliferation of small blood vessels and perivascular spindle cells (Hematoxylin–eosin stain)
and D22S193[2/7(28.6%)],and two cases of pure MA had a LOH at loci D22S929and D9S162[2/2(100%)].Two MA-M cases (cases 3and 7)had the same allelic loss at locus D22S193or D22S282in both meningioma and MA components [2/7(28.6%)],of which one (case 3)had additional allelic loss on locus D9S104other than locus at 22q12in meningioma component.None of the normal portions showed allelic losses.These results are summa-rized in Table 2and Fig.3.
Discussion
In the present study,allelic loss was observed in the meningioma component of six (85.7%)cases of MA-M,and in the MA component of two (28.6%)out of seven MA-Ms at least one of 13microsatellite markers was analyzed.Two pure MAs included in this study demon-strated the allelic loss at one microsatellite marker.The number of specific loci lost ranged from one to three.The microsatellite markers used in this study are specific loci on chromosomes 22q12(D22S193,D22S282,D22S929),1p32(D1S463,D1S193),13q14(D13S118,D13S153),9p21(D9S162,D9S104),16q22(D16S421,D16S512),and 17p13(TP53,D17S796).The current study demonstrated allelic loss at 22q12,1p32,and 9p21in the m
eningioma and/or MA components.The frequency of allelic loss at 22q12,9p21,and 1p32in meningioma components of MA-Ms was 57%,28.6%,and 28.6%,respectively.The
frequency of allelic loss at 22q12in this study is similar to that (56%;5/9MA-Ms)observed in the largest series of Perry et al.[6].This frequency also parallels that of molecular genetic findings in meningiomas unrelated to MA.Approximately half of meningiomas have allelic losses that involve chromosome 22q12[9,10].
Allelic loss at 1p32that was observed in the meningioma component of MA-M cases in the present study has not been demonstrated in previous genetic or molecular studies.Our study showed no loss of D16S512,which is true of the previous studies [17].Losses of 16q22have not yet been detected in the meningiomas and are predominantly repor-ted in breast carcinomas or prostatic adenocarcinoma.In the present study,we have identified losses of D9S162in the meningioma component of a MA-M case and in a case of pure MA.Losses on 9p are often linked to alterations of CDKN2A that in turn occur predominately in grade II or III of sporadic meningiomas [18].However,there was no clinical and pathological difference from others without losses of D9S162in the present study.The present study revealed the same allelic loss at loci D22S193and D22S282in both MA and meningioma components of two MA-M cases,which is similar to those of Pe
rry et al.’s study [6].Perry et al.demonstrated dele-tion of NF2gene and protein losses between meningioma and MA components in 56%of nine MA-M cases,but no deletion in pure MAs by FISH and immunohistochemical study.They suggested that MA pattern may be perivascular spread of underlying meningioma rather than neoplastic transformation of MA in MA-M cases.However,this suggestion is not supported by our data on LOH at loci D22S929and D9S162in two pure MA cases or by a pre-vious report of MA with LOH on 22q12[4].The genetic alterations in pure MAs support the possibility that MA originates from the monoclonal meningothelial cells of neoplastic nature,although limited number of samples have been studied.Our two cases of MA-M had identical allelic loss in both components,of which one MA-M
had
Fig.2Case 5shows higher proliferation index in the meningioma component (right )than in MA portion (left )of MA-M (Ki-67immunostain)
Table 2Molecular results of LOH in nine cases Case no.MA portion Meningioma portion 1No LOH D1S193,D22S1932No LOH No LOH
3D22S282D22S282,D22S929,D9S1044No LOH D1S4635No LOH D22S1936No LOH D9S1627D22S193D22S1938D22S929–9
D9S162
–
–No tissue obtained,MA meningioangiomatosis
Fig.3a Microsatellite analysis
of MA-M(case3).The
meningioma portion(M)shows
LOH on D9S104(arrow).
b Microsatellite analysis of
MA-M(case7).Both the
meningioma(M)and MA show
LOH on D22S193.
c Microsatellite analysis of pure
MA(case8)shows allelic loss
on D22S929(arrow).N is the
PCR product from the non-
neoplastic cerebral tissue or
peripheral blood
Table3Summaries of the previous studies about meningiomas with or without MA
Authors Materials Association
with NF2Method of study Studied
markers
Summary of results
Takeshima et al.[4]MA(1case)–LOH(2
microsatellite
markers)
22q11-12LOH on chromosome22q in pure MA,
supporting neoplastic theory
Sinkre et al.[5]MA-M(1case)–FISH22q12region,
北京中国城
夜总会1p32region Loss of chromosome22q12in both components, supporting neoplastic theory
Perry et al.[6]MA-M
(10cases)
MA(14cases)?(2cases out
of14MAs)
FISH and ICH22q12.2
18p11.3
1.Gene deletions(NF2/4.7B)and protein losses
(merlin/protein4.1B)in both components,
supporting neoplastic theory
2.No gene deletions in pure MAs,supporting
hamartomatous nature of pure MA
Stemmer-Rachamimov et al.[7]MA(12cases)–SSCP NF2gene Absence of somatic mutation of the NF2gene,
supporting hamartomatous nature
Kim et al. (this study)MA-M(7cases)
MA(2cases)
–LOH(13
microsatellite
markers)
22q11-12
1p32
13q14
9p21
16q22
17p13
1.LOH on1p32,9p21,22q11-12in meningioma
component of MA-M,supporting neoplastic
porandtheory
2.LOH for loci on D22S929and D9S162
in pure MA,supporting neoplastic theory
NF2Neurofibromatosis type2,LOH loss of heterozygosity,FISHfluorescence in situ hybridization,ICH immunohistochemistry,SSCP single strand conformation polymorphism
additional allelic loss on chromosome9p21rather than chromosome22q12.These results are distinctly different from those of Perry et al.’s study.In our opinion,these findings support the evidence that meningioma develops from additional genetic alterations on the preexisting MAs having genetic alterations in itself.The previous studies are summarized in Table3.
In the literature pertaining to Ki-67proliferation indices of MAs,high indices were detected in the meningioma portions of MA-M,while low or absent indices were detected in the MA portions of MA-M or pure MA[5–7, 19].Absent Ki-67immunoreactivity or relatively low proliferation indices in pure MAs or MA-M support the clinical impression of a slow-growing lesion.In the present study,as measured by morphometric analysis,a similar pattern of Ki-67results was obtained in MA-M cases,and the proliferation indices of MA-M cases were significantly higher in the meningioma portion than in the MA com-ponents.The Ki-67labeling index of our cases are ranged in low grade meningiomas[20]except a case(case2)that showed higher labeling index(10.1%).That patient had a recurrent tumor but both original and recurrent tumors were histologically benign.
英国毒贩阿克毛
Microsatellite alterations on NF2,1p32and9p21might contribute to the progression from MA to meningiomas, similar to conventional de novo meningiomas.Our obser-vation on allelic losses on chromosome22q12or chro-mosome9p21in pure MAs,albeit a small series,suggests that the MA and MA-M may in part share the same genetic event and undergo the same overlapping clonal process, with the meningioma arising from MA probably undergo-ing additional and genetic alterations.These genetic alter-ations of MA may confer a more proliferative potential.It was also supported by the higher Ki-67-indices in the meningioma portions of MA-M.
Acknowledgment This article was partly supported by Gachon Biomedical Research Institutional Grant.
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