specific chromosomal abnormalities in malignant human...

(CANCER RESEARCH 88, 405-411. January 15. 1988]

Specific Chromosomal Abnormalities in Malignant Human Gliomas1

Sandra H. Bigner,2 Joachim Mark, Peter C. Burger, M. Stephen Mahaley, Jr., Dennis E. Bullan!,

Lawrence H. Muhlbaier, and Dareil D. BignerPreuss Laboratory for Brain Tumor Research [D. D. B.J, Departments of Pathology fS. H. B., P. C. B., D. D. BJ. Surgery [D. E. B.J and Community and FamilyMedicine [L. H. M.], Duke University Medical Center, Durham, North Carolina 27710; Central Hospital, SkÃ³vde, Sweden fj. M.]; and Department of Neurosurgery,University of Alabama Medical Center, Birmingham, Alabama 35294 {M. S. M.J

ABSTRACT

Karyotypic analysis of 54 malignant human gliomas (5 anaplasticasina-)tomas, 43 glioblastoma multiformes, 3 gliosarcomas, 2 giant cell

glioblastomas, 1 anaplastic mixed glioma) has demonstrated that 12tumors contained normal stemlines or only lacked one sex chromosome.The 42 tumors with abnormal karyotypes included 38 tumors which couldbe completely analyzed. Six of these 38 cases had near-triploid or near-tetraploid stemlines and 32 had near-diploid stemlines. Statisticallysignificant numerical deviations in the near-diploid group were gains ofchromosome 7 (26 of 32; P < 0.001), and losses of chromosome 10 (19of 32; P < 0.001). Double minutes occurred in 18 of 32 near diploidrumors. The distribution of structural abnormalities was analyzed statistically by comparing the incidence of breakpoints in each chromosomalarm to the expected value based on chromosomal arm length. Thisanalysis demonstrated that structural abnormalities of 9p and 19q weresignificant statistically (P < 0.005 and P = 0.02, respectively). Althoughchromosome 1, 6p, the centromeric region of chromosome 11, 13q, and15q were also frequently involved in structural abnormalities, the incidence of these breaks did not reach statistical significance. This demonstration of specific chromosomal abnormalities in near-diploid gliomasprovides the basis for the investigation of genes which may be quantitatively or qualitatively altered in these neoplasms.

INTRODUCTION

Glioblastoma multiforme is the most common malignantprimary brain tumor of adults. It is also one of the most lethalwith a median posttreatment survival of less than 1 year (1).This poor prognosis reflects in large part the paucity of information about the neoplasm's basic biology, particularly the

mechanisms which control cellular growth and the factors thatmake it resistant to current therapies.

In recent years, cytogenetic analysis of certain types of leukemia, lymphoma, and solid tumors have revealed specificchromosomal abnormalities. Some aberrations such as the 9;22translocation of chronic granulocytic leukemia, the 8; 14, 2;8,and 8;22 translocations of Burkitt's lymphomas, and the 15;17

translocation of acute promyelocytic leukemia are sufficientlyspecific to be useful in clinical diagnosis (2). Furthermore,subgrouping individual diseases according to chromosomal pattern as has been done with childhood acute lymphocytic leukemia has been useful in separating patients with different meansurvivals (3). More recently, the chromosomal locations ofstructural abnormalities have predicted rearrangements or differences in expression of genes that regulate cellular proliferation or differentiation. For example, breakpoints at 14ql3 inlymphoid disorders are often associated with rearrangementsof the T-cell receptor gene in T-cell disorders (4), the c-abloncogene is displaced from its normal location on 9q in thecharacteristic translocation of chronic granulocytic leukemia

Received 6/26/87; revised 10/1/87; accepted 10/26/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This investigation was supported in part by CA-11898 and CA-43722 fromthe National Cancer Institute, P01-NS-20023 from NINCDS, and the SwedishCancer Society.

2To whom requests for reprints should be addressed.

(5), and the specific translocations of Burkitt's lymphoma bringc-myc into the vicinity of the immunoglobulin chain loci (6, 7).

Progress in elucidating genetic mechanisms has generallybeen slower in solid tumors than in leukemia and lymphomas.Among solid tumors, however, those of nervous system originhave been best studied. For example, meningiomas characteristically have losses or deletions of chromosome 22 (8). Neu-roblastomas often have deletions of l p and frequently containamplified N-myc and the accompanying cytogenetic manifestations (9, 10). In addition, loss of heterozygosity for the region13ql4 through either deletion or loss of the entire chromosomewith duplication of the other alÃelehas lead to isolation of theputative retinoblastoma gene (11).

Previous karyotypic studies of MHG3 have demonstrated that

the most prevalent changes are numerical deviations of wholechromosomes, particularly gains of No. 7 and losses of No. 10(12). The most frequent structural abnormalities are deletionsand translocations of 9p and the presence of DMs (13). Herewe report the karyotypic findings in 26 new cases of MHG. Wehave analyzed 54 tumors, these 26 together with 28 previouslyreported tumors, to demonstrate statistically significant chromosomal abnormalities in MHG and to determine if thesechromosomal changes are indicative of differences in histology,survival, and clinical characteristics among patients with MHG.

MATERIALS AND METHODS

Selection of Cases. All primary untreated MHG operated on fromFebruary 1981 through March 1986 at Duke University Medical Center, Durham Veterans Administration Medical Center, or North Carolina Memorial Hospital for which sufficient fresh tumor was availablewere subjected to chromosomal analysis. Karyotypic analyses of thefirst 28 cases (D-212MG through D-280MD) have been reportedpreviously (12-14). Karyotypes of the remaining 26 cases (D-282MGand higher) are described here. All histolÃ³gica! sections from theresected tumors were reviewed by S. H. B. Tumors were classifiedaccording to the WHO brain tumor scheme (15) with the modificationthat the presence of necrosis was a major distinguishing factor betweenAA and GBM (16). Only malignant astrocytic tumors including AA,AMG, GBM, GCGBM, and GS were included.

Chromosomal Analysis. Sterile tumor tissue was obtained from theoperating room and immediately taken to the laboratory where it wasdissected free of necrosis, blood, and grossly normal tissue, using aseptictechniques. Tumor tissue was finely minced and the first 51 tumorswere enzymatically dissociated using Hanks' balanced salts solution

(pH 7.0) containing 0.02% collagenase (125 units/mg), 0.05% Pronase(45 PKU/mg B grade), and 0.02% DNase (7 x IO4 domase units/mgof DNase I, B grade) at 37'C for approximately 45 min. These cellsuspensions were centrifuged and the pellets resuspended in Richter's

(17) improved minimal essential zinc option medium (Grand IslandBiological Co., Grand Island, NY) supplemented by 20% fetal calfserum. Cell and trypan blue viability counts were done and cells wereplated in 2 or 4 one hundred mm dishes at 5 x Id" viable cells/dish.

Two dishes were used for direct chromosomal preparations as described

3The abbreviations used are: MHG, malignant human glioma; DMs, double

minutes; AA, anaplastic astrocytoma; GBM, glioblastoma multiforme; AMG,anaplastic mixed glioma; GCGBM, giant cell glioblastoma; GS, gliosarcoma; G-banded, giemsa-trypsin banded; BANF, bilateral acoustic neurofibromatosis.

405

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CHROMOSOMES IN CHOMAS

previously (12). Two dishes were incubated at 37Â°Cin a 5% CO2

atmosphere. Cultures were observed daily and chromosomal analysiswas performed as described previously when cultures entered exponential growth (12). For the last 3 tumors the dissociation method wasaltered to a 4- to 16-h incubation in serum free zinc option mediumcontaining 8 mg/ml of collagenase. G-banded preparations were utilized routinely, but C banding was supplemented as necessary to demonstrate the Y chromosome and in identification of markers.

G-banded slides were examined and adequately spread metaphasesphotographed using xlOOO oil immersion with a Zeiss photo microscope and enlarged. Modal numbers were determined by counting alladequately spread metaphases in each preparation. Each metaphasewas examined microscopically for the presence and number of DMs.Karyotypes were determined by arranging all photographed metaphaseswhich were technically satisfactory according to the International System for Human Cytogenetic Nomenclature (1985) scheme (18). Normaland abnormal chromosomes have been designated according to thisclassification and karyotypes are expressed as recommended under thissystem with the exception that numerical deviations which are notconsistently present are underlined. During the time period that these54 cases were accumulated, 7 additional tumors (3 AA, 4 GUM), wereprocessed but contained no metaphases.

Controls. Heparinized peripheral venous blood was obtained from33 of 54 glioma patients prior to radiation and chemotherapy. G-banded slides were prepared from phytohemagglutinin-stimulated lymphocytes using standard procedures. The constitutional karyotype ofthe individuals whose mitogenic response was sufficient for analysiswas determined by counting the chromosome number for at least 20metaphases/patient and arranging at least 4 G-banded spreads according to the International System for Human Cytogenetic Nomenclature(1985). All cases revealed normal 46,XX or 46,XY cells.

Statistical Analysis. Analysis of numerical chromosomal deviationswas done using a x: test '<"" goodness of fit, assuming that each

autosome had an equal probability of being gained or lost to obtain theexpected values for the test. A similar goodness of fit test was performedfor the chromosomal breakpoint analysis, except that the expectedvalues for the number of breakpoints were obtained from a publishedreport containing the percentage of the total human genome represented by each chromosome arm (19).

Differences in demographic characteristics and mortality distributions between patients in the 3 ploidy groups (45,XO or normal versusnear diploid stemline versus polyploid stemline) were limited to patientswith GBM and GS tumor types as cases of AA, GCGBM, and AMGwere too few for statistical evaluation. Patients with GBM and GS weregrouped together for these analyses (designated GBM/GS) because GSis considered a subtype of GBM according to the WHO classificationscheme (15) and in previously published survival analyses of gliomapatients (16, 20). Comparison of demographic characteristics amongthe 3 ploidy group was done using the Wilcoxon Rank Sum test (21)for age and the x2 or Fisher's Exact Test (21) for gender. Differences

in tumor location among patients with tumors in the 3 ploidy groupswere analyzed for right versus left using Fisher's Exact Test (21) andfor lobe of the brain by the x2 test (21). Mortality distributions wereplotted using the Kaplan-Meier method (22). The survival analyses todetermine the effect of ploidy, age, gains of chromosome 7, losses ofchromosome 10, breaks in 9p, and DMs were done using the Coxproportional hazards model (23).

RESULTS

Karyotypes were obtained on 54 untreated malignant humangliomas consisting of 5 AA, 43 GBM, 3 GS, 2 GCGBM, andone AMG (Table 1). Normal stem lines with variant cells or45,XO stemlines were identified in 12 tumors (4 AA, 8 GBM);abnormal stemlines characterized the remaining 42 tumors.

The 42 tumors with abnormal kayrotypes included 4 cases(D-262MG, D-238MG, D-301MG, and D-332MG) in whichstructurally abnormal chromosomes (sometimes DMs) wereseen but that the chromosomes were too short, dark, and poorly

banded to allow complete characterization. In the remaining 38tumors, the banding patterns and number of mitoses weresufficient to delineate numerical and structural deviations.Thirty-two of these 38 tumors had stemlines in the near-diploidregion. In 2 of these tumors, both of which were GCGBM,near-haploid sidelines were identified comprising exactly one-half of the stemline complement. Six tumors had stemlines inthe triploid-tetraploid region although frequently near-diploidsidelines were seen as well.

Tumors with Near-Diploid Stemlines. The most prevalentabnormalities among the 32 near-diploid tumors were gains ofwhole copies of chromosome 7 (26 cases) followed by losses ofchromosome 10 with 19 cases (Figs. 1 and 2). Statistical analysis assuming that each autosome has an equal likelihood ofbeing gained or lost showed these 2 deviations to be highlystatistically significant (P< 0.001). Seventeen tumors containedboth +7,-10, but the incidence of each change was so high that

a true association between these 2 features could not be demonstrated (P= 0.135). The next most frequent numerical deviations were gains of chromosomes 19 and 20 which were eachseen in 5 tumors. The incidences of these gains, however, werenot statistically significant (P = 0.08).

One of the most frequent structural abnormalities was thepresence of DMs (Fig. 2). These structures occurred in 18 of32 near-diploid tumors which were completely characterizedand in one of the 2 near-diploid tumors with incomplete characterization. There was a wide range of size and number ofDMs both with and among tumors ranging from only scatteredDMs in one cell (designated "rare" in Table 1) to more than 50

DMs in virtually every ell examined. In contrast to the highincidence of DMs, homogeneously staining regions were notseen in any tumor.

Statistical analysis of the breakpoint incidence which correctsfor the differences in chromosomal arm length demonstratedthat the breaks in 9p were significant statistically (P < 0.005)(Fig. 3). Although 15 breaks involved chromosome 1, they wererandomly distributed throughout the length of the chromosome(P > 0.20). There was clustering of breakpoints to 6p, thecentromeric region of chromosome 11, and distal 13q, 15q, and19q. Among these chromosomes only breaks in 19q (Figs. 2and 3) reached statistical significance (P = 0.02). This observation should be regarded as tentative, however, because it wasnot always possible to be absolutely certain that the break wasin 19ql3 rather than 19pl3.

Tumors with Near-Triploid, Near-Tetraploid Stemlines. EightMHG had stemlines in the triploid-tetraploid region. DMswere seen in 6 of these tumors including 2 tumors which couldnot be completely characterized. The number of cases in thisgroup was too small for statistical analysis of specific numericaland structural abnormalities. It is interesting, however, thatgains of chromosome 7 were seen in only one of 6 tumors andloss of 2 copies of chromosome 10 was seen twice, although 2additional tumors contained deletions of chromosome 10. Incontrast, loss of 2 copies of chromosome 22 characterized 4 of6 tumors in this group.

HistolÃ³gica! and Clinical Parameters (Table 1). Patients withGBM/GS in the different ploidy groups (45,XO or normalstemline versus near-diploid stemline versus polyploid stemline)had similar mean ages (59.0, 60.6, and 60.1 years, respectively;P = 0.95). There was a male preponderance in the overall seriesdue in part to the contribution of cases from a Veterans Administration Hospital which predominantly serves men. The45,XO normal group contained 75% males and the near-diploidgroup contained 63% males, while the patients with polyploid

406



CHROMOSOMES IN GLIOMAS

Table 1 Clinical characteristics, histopathologic diagnosis, and tumor karyotypefor 54 MHG patients

Tumor no.Age (yr),and sex Location

Survival(mo) Diagnosis Karyotype

D-223 MG"D-244MGD-248MGD-251 MGD-261 MGD-264 MG*D-285MG*D-302 MGD-315MGD-326MGD-342 MG7D-343MG

D-225MGD-245MGD-247 MGD-249MG

D-250MGD-256MGD-259MGD-262 MGD-263MG

D-266MGD-267MGD-268MG

D-270MGD-271 MGD-272MGD-273MG

D-274MGD-280MGD-290MG

D-298 MG

D-299MGD-301 MGD-304MG

D-308MGD-311MGD-316MGD-317MGD-320MG

D-336MG

D-338MG

D-344MGD-348MG

D-238 MGD-279 MG

55* F

33 M26 M56 M81 F32 M45 M81 M67 M43 M61 M30 F

58 F70 M80 F75 F

64 M60 M46 M63 M71 F

63 M60 F76 M

42 M54 M43 F43 F

49 F59 M62 F

46 M

54 M72 M75 M

67 M54 F55 F64 M55 F

68 M

66 M

55 M67 M

D-212MG 11 F

D-282 MG 60 F

D-222 MG 48 F

44 M66 F

RT*

RTLPOLFRPTLFRGBLTPRPLTLFRF

RORFTPLPLF

RPLFRTLFLT

LTLTRTP

RPLFRTRF

RTFPLTLP

RTP

RFRTRT

RTLTRPLFRF

LP

LP

LFRT

RP

RF

RF

LTRT

\4J

411657

33677

Alive (15)2

Alive

49 days8

13

4131037

157

12 days

156

1313

624

16

1032

13 daysAlive (20)Alive (21)

8Alive (18)

6

5

Alive (7)Alive (4)

20

22

10

183 days

Normal stemlines or 45,XOGBM' 45.XO with variant cell/

AA 46,XY/45,XOGBM 46.XY with variant cellsGBM 45,XO/46,XYGBM 46,XX with variant cellsAA 46,XY with variant cellsGBM 46XY/45,XOGBM 45.XOGBM Random loss from 46,X YAA 46,XYGBM 46,XYAA 46.XX with variant cells

Near-diploid stemlines

GBM 47,XX,+7,-10,+20,+DMsGBM 49,XY,+l,+7,+19,t(15;17)(q22;q25),+DMsGS 46.XX,+7,-10GBM 47,XX,-10,del(4)(pl5),del(5)(ql4),+del(7)(q33),t(l;16)(q21;pl3),t(5;9)(qll;ql1),

der(6)t(4;6)(pl5;q23),+der(6)t(4;6)(pl5;q23),der(9)t(6;9)(q23;p24)GBM 47,XY,+7,-10,+19,-21,del(l)(pl3p35)+derl7,t(9;17)(pl3;q22),+DMsGBM 44,XY,-6,+7,-10,-22,t(9;19)(pl3;ql3),der(9)t(6;9)(pll;pIl),+DMsGBM 46,XY,+7,-10,+DMsGBM 46,XY,(with markers),+DMs (cannot be further characterized)GBM 43-44,XO,-X,-4,-17,del(22)(ql3),t(9;9)(pl3;p24),der(l I)del(ll)(pl2)t(l l;?Kq22;?),

der(13)t(13;?)(q22;?),+DMsGBM 44,XY,-10,-15,+DMsGBM 48,XX,+7,-10,+19,+22,de!(9)(p24),?der(15)t(15;?)(q24;?)GBM 46-48,XY,+3,-9,-10,-H2,-21,del(6)(ql5),der(7)t(7;?)(p22;?),+der(7?)t(7?;?)(p?;?),

der(13)t(l3;?)(q22;?),+mar,+marGBM 45,XY,-9,+DMs/44,XY,-6,-9,+DMsGBM 44,XY,+7,-9,-10,-18,del(l)(q32q43),t(6;16)(pll;q23-4)AA 43,XX,-6,+7,-10,-13,-16,del(18)(q21),der(9)t(9;16)(qll;qll),+DMs(rare)GBM 44,der(X)t(X;?)(q26-28;?)-X,-5,-14,-15,-17,-22,del(ll)(q22),del(12)(pll),der(3)t(3;?)

(q13;?),der(11)del(11)(p14)t(11;?)(q23;?)+mar,+mar,+mar,+frag,+DMsGBM 41-42,XX,-10,-ll,-12,-14,-16,-17,del(9)(pl3),t(l;l)(p36;q21),der(13)t(13;14)(pll;q32)GBM 46,t(X;9)(q26-28;pl2)Y,t(7;10)(q31;q22)GBM 45,XO,-X,+7,-8,-16,-21,del(6)(pl2)(ql5);del(13)(q21),t(l;19)(q21;ql3),der(12)

t(12;?)(q22;?)+ring,+frag,+DMsGS 45,XY,-10,-17,-18,+20,del(1)(p22),del(15)(q22-3),t(5;11)(q34-5;q13),der(2)t(2;7)(p16-

21;p12-13),+der(7)t(7;14)(p12-13;q32),der(14)t(2;14)(p16-21;q32),der(14)t(2;14)(p16-21;q32),+DMs (with random loss)

GBM 46,XY,+7,-10,-17,i(6p),+del(ll)(qllql2),der(19)t(17;19)(qll;ql3),+DMs (rare)GBM 48,XY,del(l)(q?) (cannot be further characterized)AMG 43,XY,+7,-10,-13,-19,-21,del(l)(p21-2),del(6)(ql3-14),del(9)(pl3),+DMs(rare)/44,XY,+7,

- 10,- 13,-2 1,del(6)(q 13-14),del(9)(p 13)/45,XO,-YGBM 44,XY,+7,+7,-8,-10,-13,-15,-22(?),+del(ll?)(pll),t(2;4)(pl6;pl6),+DMs(rare)GBM 46,XX,+7,-10,-ll,-16,+19,+20,del(18)(q21),(i(3p)?),der(5)t(3?;5)(q21-23;q33-35),+DMsGBM 46,XX,-6,+7,+7,-10,der(4)t(4;6)(pl4;qll-12),der(19)t(10;19)(qll;qll),+DMsGBM 45,XY,-5,+7,+7,-8,-10,+ll,-15,-22,del(9)(p21),t(l;l)(p36;p32),dup(6)(pll;pl2),+DMsGBM 47,XX,-9,-10,-f4T+20,del(5)(qT5-21),+del(7)(pl2),del(22)(ql2),der(2)t(2;14)(p25Ã¯il3),

+der( 19)t(5; 10; 19)(q 15-2 1;q11-q26;q 13),+r(9; 10)(9p24-Â»9q34-.l Op14-Â»lOq11)GS 50,XO,-Y,+6,+7,+del(3)(q23),+del(3)(q23),del(13)(q21),+der(9)t(9;12)(qll-12;pll-12),

der(12)t(9;12)(qll-12;pll-12)GBM 50,XY,+3,+7,-18,-H9,+del(l)(p31)(q31),del(8)(p21),del(9)(pllpl3),der(l)t(l;13)(p36)ql4),

der(2)t( 1;2; 13)(2pterâ€”2q34-37:: 1p3 1â€”1p36:: 13q 14-.1 3qter),+der( 13)t( 1;13)(13pter-Â»13ql4::lqter-Â»lq31)

GBM 48,XY,+Y,+7,+ 12,-14,del(15)(ql2q21)/45,XO,-YGBM 46,XY,+7,-10

Near-diploid stemlines with near-haploid sidelines

GCGBM 50,XX,+l,+ l,+ 18,+18,-20,der(7)t(7;?)(p22;?)+der(7)t(7;?)(p22;?)/26X,+ l,+7,-15,+ 18,-t-der(9)t(9; 15)(q 11,q 11),der( 11)t( 11;?)(q24;?)

GCGBM 50,XX,+7,+7,+ 20,+20,+frag (with random loss)/25,X,+7,+20

Near triploid-near-tetraploid stemlines

GBM 89-9 1,XXXX,- 14+20,+20,-2 1,-22,-22,del( 10)(q24),del( 10)(q24),del( 14)(q23),del( 14)(q23),+DMs

GBM 92-95.XXYY, +DMs (cannot be further characterized)GBM 79,XXXX,-4,-4,-9,-9,- 10,- 10,- 11,- 13,- 13,- 13,- 14,- 16,-22,-22,del(6Xq 15),

del( 12)(p 11),del( 12)(p 11),del( 14)(q27),der(5)t(5; 13)(q22;q2 1-2),der(5)t(5; 13)(q22;q2 1-2),der(l l)t(7?;9?,l l)(ql l-q32;ql3;q21)+ring,+DMs

" Laboratory number in which D indicates that the preparation was initiated at Duke and MG that it was a malignant human glioma.* Age at time of diagnosis.* R, right; L, left; F, frontal lobe; P, parietal lobe; T, temporal lobe; O, occipital lobe; CB, cerebellum. All tumors are supratentorial unless otherwise indicated.d Survival from diagnosis (initial resection of tumor). Patients still living at the time of publication are so indicated along with the length of follow-up.'' Histopathological diagnosis according to the WHO classification (15).â€¢^Stemlineor modal tumor karyotype expressed as recommended by the International System for Human Cytogenetic Nomenclature (17). Numerical deviations

which are not consistently present are underlined.* Only a preparation cultured for more than 96 h was available for evaluation. In all other cases a direct preparation and/or a preparation cultured for less than or

equal to 96 h was available.

407




Table 1â€”Continued

Tumorno.D-292MG

D-295 MGAge

(yr),andsex72

M

71 MSurvival

Location (mo) DiagnosisKaryotypeLT

LO10 2GBM GBM96,XXYY,+20,+20,+20,+20,der(ll)t(ll;?)(pl5;?),der(ll)t(ll;?)(pl5;?),+DMs

(withloss)/45,XOrandom

D-303 MG

D-332 MGD-340 MG

63F

67 F56 M

LF

Kl-RT

16 GBM

5 GBMAlive (10) GBM

-18, -18, -19, -"50, -22, -22, del(l)(p21-22), +(?)del(3)(qll), +(?)dÃ¨TÃŽÃ•OKq2"2-23),

+(?)del(10)(p22-3)86-88,XXXX, +7, +7, -10, -10, -13, -13, -15, -15, -19, -19, -21, -21, -21, del(3)(p 11), del(3)

79-83,+DMs (with markers) (cannot be further characterized)

-19, -19,'"^

der(ll)t(ll;19)(cen;ql3)

GAIN

LVVKV4^^^XX3

I 2 3 4 5 6 7 8 9 10 II 12 13 14 19 16 17 18 19 20 21 22 X Y

LOSSFig. 1. Gains or losses of whole chromosomes in 32 MHGs with abnormal

stemline karyotypes in the near-diploid range. Gains of chromosome 7 occurredin 26 tumors (/' < 0.001) and losses of chromosome 10 occurred in 19 tumors (/'

< 0.001). Gains and losses of the other chromosomes were randomly distributed.

tumors were equally divided between men and women. Thesedifferences between gender distributions in the 3 groups werenot statistically significant (P = 0.058). Tumor locations wereequally divided between the right and left cerebral hemispheresin patients in all 3 karyotypic groups. There were no statisticallysignificant differences in tumor locations by lobe of the brainamong the 3 karyotypic groups (P = 0.11).

An analysis of histolÃ³gica! tumor type by karyotypic patternrevealed that AAs were more likely than GBMs to contain45,XO or normal stemlines (4 of 5 AAs versus 8 of 43 GBMs),but there were no obvious karyotypic differences between GBMsand GSs. In addition, the 2 GCGBMs were the only 2 tumorsin the entire series which contained near-haploid sidelines. Dueto the small numbers of AA, GS, AMG, and GCGBM thesepossible relationships between histolÃ³gica! tumor type and kar-yotype could not be evaluated statistically.

Survival (Table 1). Age at diagnosis was a statistically significant predictor of survival among patients with GBM/GS (P =0.009). Patients with AA, GCGBM, and AMG were too fewfor analysis. Age-adjusted mortality distributions for patientswith GBM/GS revealed no differences in survival among the 3ploidy groups (P = 0.50). The presence of +7,-10 and 9p

abnormalities was each evaluated individually for correlationswith survival using the Cox proportional hazards survival model

and none was demonstrated (P > 0.20 for all). Patients whosetumors contained DMs survived longer (median survival =10months) than those without these structures (median survival= 6 months) but these differences were not statistically significant (P = 0.095).

DISCUSSION

The present series of 54 MHG yielded 42 tumors withabnormal stemlines; 80% of these tumors were near-diploidand the remainder were near-triploid â€”Â»near-tetraploid. Statistically significant abnormalities of the near-diploid tumors weregains of whole copies of chromosome 7, losses of chromosome10, structural abnormalities of 9p and 19q, and the presence ofDMs. The high incidence of these numerical deviations andDMs and the demonstration of tumors containing only the+7,-10, DM abnormalities suggest that these changes are theearliest gross chromosomal deviations in near-diploid MHG.Structural abnormalities, consisting mainly of deletions andunbalanced translocations, seem to be superimposed on theseprimary changes. Although only 9p and 19q are involved at anincidence which reaches statistical significance in the presentseries, as larger numbers of glioma karyotypes become available,small subgroups of tumors representing separate evolutionarypathways may emerge. The polyploid tumors were too few forstatistical evaluation, but DMs were frequent and there was atrend toward loss of 2 copies of chromosome 22. The lowincidence of gains of chromosome 7 and the high incidence ofloss of chromosome 22 contrast sharply with tumors with near-diploid stemlines, suggesting that these polyploid tumors mayhave followed a separate evolutionary route from the near-diploid tumors.

The largest previously reported series of MHG karyotypesconsisted of 50 tumors studied without banding by Mark (24).This series established that approximately 75% of these tumorshad diploid or near-diploid stemlines and that near-tetraploidtumors were more than twice as common as near-triploidtumors. DMs were seen in 26% of cases and other structuralabnormalities were seen in approximately 75% of them. Thus,we have confirmed the general karyotypic profile demonstratedin Mark's original series and have extended these observations

to include nonrandom aberrations of specific chromosomes.The chromosomal deviations described here have been con

firmed in a number of other laboratories although some of thesereports are presented only as abstracts. Al Saadi and Latimer(25,26) observed gains of chromosome 7, losses of chromosome22, and losses of one sex chromosome in a large series ofastrocytomas and glioblastomas. Rey et al. (27) and Yamada etal. (28) have each described 2 gliomas with losses of chromosome 22. Shapiro et al. (29) and Shapiro and Shapiro (30) havedescribed losses of the sex chromosomes and chromosome 22

408




K M lÃ¬I u II i U 88

Fig. 2. Stemline karyotype of GBM, D-316MG is 46,XX,-6,+7,+7-10, der(4)-t(4;6)(p 14;q11-12), der( 19)t( 10;19)(q 11;q 11),+DMs; arrows, structural abnormalities.Giemsa-trypsin banding, x 1200.

il13 14

10 11 12

Il fil II II iÂ»15 16 17 18

Ãˆ19 1

Â»ft20

*ft

21 22 DMs

in suspension cultures from a small number of gliomas andillustrate one case which contains gains of chromosome 7, at(9;10)(pllqll), and DMs (29, 30).

The specific chromosomal abnormalities of MHG are potentially important clues for elucidating mechanisms which influence the growth of these tumors. For example, most gliomaswith DMs have been shown to contain amplification of the c-erbB gene which codes for the epidermal growth factor receptor;a few cases have revealed amplification of c-myc, N-wiyc, or gli(31-34). Although monosomy 22 characterizes only a smallproportion of MHG, partial or complete deletion of this chromosome is important in a number of other neurogenic tumors.Monosomy or deletion of chromosome 22 is a well-knownfinding in meningiomas (8, 35). In addition, Seizinger et al.(36-38) used restriction fragment length polymorphisms todemonstrate partial deletions of 22q in neurofibromas, meningiomas, and acoustic neuromas in patients with BANF and insporadic meningiomas and unilateral acoustic neuromas. Recently, Rouleau et al. (39) have used a genetic linkage analysisto demonstrate that the defective gene responsible for BANF islocated on chromosome 22. These findings suggest that sporadic neurogenic tumors with losses or deletions of chromosome 22 including unilateral acoustic neuromas, meningiomas,and gliomas have lost through somatic mutation the gene whichis defective in BANF patients (35). Furthermore, this geneprobably codes for a "tumor suppressor."

Although the biological significance of the other numericaland structural deviations in MHG remains speculative, someof the changes are also typical of other types of human neoplasms. For example, trisomy 7 has been described in severalother types of solid tumors including carcinoma of the colon,bladder, and malignant melanoma (40-43). Although numericaldeviations of chromosome 10 are uncommon in human tumor

types other than gliomas, loss of one sex chromosome has beendescribed in a variety of benign and malignant conditions inaddition to its well-known occurrence in the bone marrow ofelderly men.

Likewise, many of the structural aberrations of MHG involvechromosomes which are abnormal in other tumor types and insome instances the breakpoints coincide with known fragilesites (44). For example, a (9;ll)(p21;q23) translocation characterizes a subgroup of patients with acute monocytic or mye-locytic leukemia, and deletions of all or part of 9p are seen insome patients with acute lymphocytic leukemia (45-47). In thislatter group the breakpoints are variable within the p arm as isthe case in the gliomas described here. Although a fragile sitehas been identified in 9p it occurs at 9p21 while the majorityof breaks in 9p in gliomas are in other bands (44). The clusteringof breakpoints at 19ql3 is of interest because this is the locationof a fragile site (48). Although only structural rearrangementsinvolving 9p and 19ql3 were seen in a high enough frequencyto be statistically significant, other breakpoint clusters such asthe centromeric region of chromosome 6 and 17q may prove tobe biologically significant in small subgroups of cases. Furthermore, our analyses have included only those abnormalities seenin the stemline karyotypes of these tumors. Analysis of minorsubpopulations and variant cells in gliomas may reveal a different set of chromosomal abnormalities as is suggested by Shapiroand Shapiro's (30) observations of deleted chromosomes 3 and

6 in rare glioma cells.It is well known that subclassification of MHG into "grades"

such as AA and GBM defines groups of patients that aresignificantly different into age, duration of symptoms, andlength of survival ( 16). It is also apparent that within specificgrades, such as the glioblastoma, patient age is a strong prognostic factor (20). Thus, in the present study MHGs were

409




13 14 15 16 17 18

*** -'s"8 1:

19 20 21 22Fig. 3. Distribution of breakpoints in 32 MHGs with abnormal stemline

karyotypes in the near-diploid range. Only abnormalities of chromosome 9 withbreakpoints in the p arm or centromeric region and chromosome 19 with breaksin the q arm were statistically significant (P < 0.005, P = 0.02, respectively).

subclassified histologically and individual cytogenetic parameters were examined for their ability to predict survival amongpatients with the same histolÃ³gica! grade both with and withoutcorrection for age. Using this method we were unable to demonstrate differences in survival between patients with GBM/GS in the three ploidy groups (normal or 45,XO stemlinesversus near-diploid versus polyploid). Furthermore, analysis ofindividual chromosomal abnormalities including +7,â€”10,

DMs, and abnormalities of 9p for their ability to predict survivalfailed to demonstrate a statistically significant relationship.Although there were too few patients with the other histologicalsubtypes for statistical analysis, it is interesting that mostpatients with AA have normal karyotypes or lack only one sexchromosome. This observation along with the well-describedlonger survivals of patients with AA as compared to GBM (16,20) may explain the association between normal karyotype orsex chromosome loss and longer survival reported by AI Saadiand Latimer (26).

Although no clear-cut relationship between karyotypic abnormalities and survival of patients with MHG emerged inthese studies, the retrospective construction prevented our controlling for additional factors which may influence prognosissuch as performance status, duration of symptoms, radio-

graphic appearance, tumor location, extent of resection, andprogram of radiation and/or chemotherapy. Thus, it is possiblethat karyotypic parameters may have more impact on survivalas a component of a multivariate analysis in a prospectivecooperative study setting than was demonstrated here. Furthermore, as the molecular events associated with these chromosomal abnormalities are dissected, subgroups of patients maybe identified for comparative analysis. For example, MHGcontaining DMs may have amplification of N-myc, gli, or theepidermal growth factor receptor gene (31). Amplifications ofthese various genes may have different effects on survival ormay influence response to various therapeutic regimens. Thesepossibilities emphasize the need for incorporation of cytogenetic and molecular analyses into cooperative studies evaluatingtherapies for patients with MHG.

ACKNOWLEDGMENTS

The authors wish to thank Janye Blivin, Patsy Elmore, and PamWatkins for their help in clinical correlations and Linda Cleveland forsuperb technical assistance. The authors also appreciate the secretarialassistance provided by Diane Evans.

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1988;48:405-411. Cancer Res Sandra H. Bigner, Joachim Mark, Peter C. Burger, et al. GliomasSpecific Chromosomal Abnormalities in Malignant Human

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