Complete Or Partial Homozygosity of Chromosome 13 in Primary Retinobiastoma'

Home , Chromosome 13

[CANCER RESEARCH 47, 4189-4191. August 1, 1987] Complete or Partial Homozygosity of Chromosome 13 in Primary Retinobiastoma'

William F. Benedict,2 Eri S. Srivatsan, Corey Mark, Ashutosh Banerjee, Robert S. Sparkes, and A. Linn Murphree3 Clayton Ocular Oncology Center, Departments of Ophthalmology and Pediatrics [W. F. B., E. S. S., C. M., A. B., A. L. M.], Children's Hospital of Los Angeles, Los Angeles, California 90027, and Departments of Medicine, Pediatrics, and Psychiatry [R. S.], University of California, Los Angeles, Los Angeles, California 90024

ABSTRACT retinoblastoma to document that two apparently normal No. 13 chromosomes were present. Sixteen such tumors were ran Sixteen ret ino Mast ornas were examined with chromosome 13 poly domly selected from patients with both unilateral and bilateral morphic probes to determine the frequency of homozygosity for the disease. Our results using various polymorphisms on chromo chromosome in the tumors. Each of the tumors had two cytogenetically normal appearing No. 13 chromosomes. Nontumorous cells from the some 13 indicate that 12 of the 16 tumors became homozygous same patients were heterozygous for the various polymorphic chromo for at least a portion of one chromosome 13. some 13 probes used. At least partial homozygosity for a single chro Additional information on another tumor from a patient mosome 13 was observed in 75% of the tumors. These studies confirm previously described (13) is also presented. Rapid clonal evo and extend previous studies which suggest that homozygosity or hemi- lution occurred in this tumor resulting in a change from hemi zygosity at RBI occurs in the majority of retinoblastomas. We also zygosity to homozygosity for one chromosome 13. demonstrate in an additional tumor that rapid clonal evolution from hemizygosity to homozygosity can occur in the tumor. MATERIALS AND METHODS

INTRODUCTION Tumor Samples and Cytogenetic Analysis. We used only primary tumors or first to fifth passage cell cultures for our studies with the Retinoblastoma, the most common intraocular tumor of exception of LA-RB69 which was studied at various passages. When childhood, can be divided into distinct categories. Approxi ever possible, if early passage cells were utilized initially, the results mately 10% of patients with retinoblastoma have a family were confirmed using primary tumor. Primary tumor was prepared and history of the disease (familial) with an additional 30% having banded for chromosomal analysis as previously described (3). A mini bilateral tumors and no previous history of retinoblastoma mum of ten metaphases for each tumor was examined for the presence (sporadic hereditary). The remaining cases are unilateral, of of two normal appearing No. 13 chromosomes. Only these tumors were which it is estimated that nearly 85% are nonhereditary. Finally, studied further. at least 5% of both unilateral and bilateral cases have consti Recombinant DNA Probes and Esterase D Studies. Fibroblasts from tutional microscopic deletions in chromosome 13 which in each patient were examined initially with each DNA polymorphic probe available. The recombinant DNA probes used are all homologous to cludes chromosomal region 13ql4 (the location of the retino loci on chromosome 13 (9, 10, 14, 15). The restriction-fragment-length blastoma susceptibility locus, RBI). alÃelesreported are the same as previously published. The fibroblasts Although clinically the susceptibility to develop retinoblas were also screened to determine if they were polymorphic for the toma is inherited as an autosomal dominant trait, cytogenetic common Es D protein alÃeles,type 1 and type 2 (16). In individuals (1-3) and molecular biological (4-7) data strongly imply that found to be heterozygous for the Es D alÃeles,their primary tumor was the events occurring at RBI resulting in tumor formation are examined to determine whether the Es D polymorphism was main recessive, which is consistent with the initial hypothesis of tained within the tumor. Knudson (8) on the development of retinoblastoma. We have previously reported that 6 of 20 primary retinoblastomas ex RESULTS amined cytogenetically were missing one chromosome 13 or had a deletion in one chromosome 13 including 13ql4 (3). Cytogenetic Analysis. Direct metaphase preparations of all Thus, when primary tumors are examined carefully, a signifi primary tumors listed in Table 1 were analyzed for the presence cant number are hemizygous for all or part of one chromosome of two normal appearing No. 13 chromosomes. The karyotypic 13. The majority of retinoblastomas, however, contain cells patterns of LA-RB59, LA-RB62, LA-RB63, and LA-RB73 with two cytogenetically normal appearing No. 13 chromo have been previously reported (3) and were shown to contain somes. two cytogenetically normal No. 13 chromosomes. The remain The availability of several polymorphic DNA probes which ing tumors listed also had two normal No. 13 chromosomes in have been mapped to chromosome 13 (9, 10) as well as the Es the metaphases examined. Particular attention was given to D4 polymorphism which also has been mapped to 13ql4 (11) ensure that the No. 13 chromosome had a visible deletion of provide a means to examine the chromosomal status in these 13ql4. tumors. Initial results from studies using such probes indicate Loss of Heterozygosity at Chromosome 13 Loci. DNA samples that at least 50% of tumors become homozygous for all or a from the tumors listed in Table 1 were examined. These tumors portion of a chromosome 13 (4, 5, 12). and parallel fibroblast cultures from the same patient were We decided it was important to expand these observations hybridized with various recombinant DNA probes that were and to characterize only those tumors on which we had suffi homologous to unique loci on chromosome 13, namely 13ql2- cient chromosomal analysis on direct preparations of primary 13 (7F12 and HulO), 13ql4 (7D2), 13q21-22 (Hu26), 13q22 (9D11, IE8), 13q22-32 (HUBS), and 13q33-34 (9A7). We found Received 2/19/87; revised 5/4/87; accepted 5/6/87. that Tumors LA-RB62, LA-RB88, LA-RB94, LA-RBI09, LA- 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 in RBI 51, LA-RBI54, and LA-RBI57 became homozygous for accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by Grants EY 06195 and EY02715 from the NIH and by probes both above and below RBI (Fig. 1). These tumors either the Margaret Scott Bundy Trust. This work was done in conjunction with the represent cases in which there has been a loss of one chromo Clayton Foundation for Research. 2To whom requests for reprints should be addressed. some 13 and a duplication of the other chromosome or a mitotic 3 Research to Prevent Blindness Dolly Green Scholar. recombination proximal to any of the polymorphic probes ' The abbreviation used is: Es D, esterase D. presently available. Tumor LA-RBI33 became homozygous 4189 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1987 American Association for Cancer Research. HOMOZYGOSITY OF CHROMOSOME 13

Table 1 Chromosome 13 patterns in retinoblastomas studied LA - RB62 LA â€¢RB73 LA - RB88 LA - RB94

TumorLA-RB59LA-RB62LA-RB63LA-RB73LA-RB74LA-RB87LA-RB88LA-RB89LA-RB94LA-RBItypeUnilateralUnilateralBilateralBilateralBilateralUnilateralBilateralBilateralUnilateralBilateralBilateralBilateralUnilateralUnilateralBilateralUnilateralTumorpatternHeterozygousHomozygousHeterozygousHomozygousforconclusionEs NT NT NT NT DRFLPs'RFLPs 7F12 1.2 2,2 HU10 1.2 1,2 7F12 1,2 2.2 7F12 1.2 2.2 HU10 1.2 2.2 DRFLPsEsand Es HUBS 1.2 1,1 HUBS 1,2 2.2 9011 1,2 2,2 ESD 1.2 2.2 (MR)HomozygousHeterozygousHomozygousHeterozygousHomozygousHomozygousHomozygous Ã•ES 1.2 1,1 DEs HUB8 1,2 2.2 DRFLPsRFLPsRFLPs

DRFLPsRFLPsRFLPsand Es 09LA-RBI LA-RB109 LA-RB133 LA-RB151 LA-RB154 33LA-RBI (MR)HomozygousHomozygousHomozygous 51LA-RBI DRFLPsRFLPsand Es 54LA-RB156LA-RBI 7F12HU10ESDÂ»9DI1IE89A7N.2.22.222T2.21.12.21.12.22.27F12iHUBSN1.21.2T2.21.27F12ESDi9A7NU1.21.2T2.21.12.27F127D2tHU269A7N'.21.21,21.2T2,21.1221.1 (MR)HomozygousHomozygousBasisDRFLPs7D2and Es 57LA-RBI 60Patient probe " RFLPs. DNA reslriction-fragment-length polymorphisms; MR, apparent mitolic recombinations. Other tumors which became homozygous for probes both above and below RUI may also represent cases of mitotic recombination which have occurred proximal to any probes we examined. only for a probe above RBI, whereas Tumors LA-RB73 and LA-RB156 LA-RB157 LA-RB160 LA-RBI56 were found to be homozygous only for a probe N T NT N T below RBI (Fig. 1). These latter three tumors apparently had 7F12 1.2 1.2 7F12 1,2 1,1 702 1.2 1.1 undergone a mitotic recombination, and in LA-RBI 33, a double HUIO 1.2 1.2 ESD 1.2 1.2 HU26 1.2 1.1 recombination likely occurred. Tumor LA-RB63 remained het 9A7 1.2 1.1 erozygous for both the 7F12 and 9D11 probes, and Tumor LA- 9A7 1.2 2.2 RB89 remained heterozygous for Probes HulO, 7D2, and IE8 Fig. l. Summary of status in various retinoblastomas for DNA and esterase D polymorphisms. All probes shown were polymorphic in the patient's fibroblasts (data not shown). (iV).The alÃelestatus for the same probes in the tumor (T) is depicted to the right Since Es D has been closely linked to RBI (11) eight tumors for each case. The esterase D and 7D2 polymorphisms are shown in bold type which were constitutionally heterozygous for the common Es since they are tightly linked to RBI (arrow). In general the probes are accurately D alÃeleswere studied to determine if reduction to homozygos- shown in relationship to their location either above or below RBI, although Es D has not been definitively mapped proximal to KKI ity had occurred. Four of the eight tumors became homozygous for a given Es D alÃele. Rapid Chromosomal Evolution from Hemizygosity to Hom- , ozygosity of Tumor LA-RB69. The tumor, LA-RB69, was ob tained from one eye of an individual with bilateral disease. Using cytogenetic criteria, the primary tumor was shown to I( have two distinct and independently derived cell clones each of which had lost one chromosome 13 (3, 13). Only one of the 13 1/13 1 two tumor clones was retained when passaged twice intraocularly in nude mice, which we designated as EP2 (13). This clone Fig. 2. Partial metaphase from a direct preparation of tumor cells passaged intraocularly three times in nude mice showing a normal No. 13 chromosome, a contained cells with an identical karyotype as found in the 1/13 translocated chromosome, and a normal No. 1 chromosome. The arrow to primary tumor including a missing No. 13 chromosome (13). the right of the translocated chromosome indicates the breakpoint. Almost an entire No. 13 chromosome has been translocated (region above arrow) to the long It also was shown that the tumor lost heterozygosity for recom arm of chromosome 1 (region below arrow). The location of the same locus where binant probes homologous to regions on chromosome 13 both the breakpoint occurred is also shown by an arrow in a normal No. 1 chromosome. above and below RBI, confirming that one of the 13 chromo Cells obtained from a cell line established from third-passage tumor cells also contained a normal and translocated chromosome 13 at all passages studied somes present in the constitutional cells was lost in the tumor (passages 2. 9. and 32). (4). Following an additional passage intraoculari) (EP3), a cul gosity to homozygosity already had begun in the primary tumor. ture of LA-RB69 cells was established. All metaphases exam The remaining nine metaphases examined had only one chro ined from this cell line (EP3TC) at various subsequent passages mosome 13 as previously reported for EP2 (13). However, all studied (passages 2, 9, and 32) contained one normal appearing ten metaphases contained similar other marker chromosomes, No. 13 chromosome as well as the marker chromosomes found proving that they had been derived from the same parent tumor in the previous tumor passage, EP2 (13). However, in addition cell. a chromosome 13 was found translocated to the long arm of chromosome 1 in every metaphase examined (Fig. 2). When direct chromosomal preparations of the EP3 tumor were stud DISCUSSION ied, the same chromosome 1/13 plus a normal appearing chro mosome 13 was found in all metaphases. The observation We have found that 9 of 16 retinoblastomas which contain 2 indicates that a marked selection occurred in vivo between eye normal appearing No. 13 chromosomes in their primary tumor passage 2 (EP2) and eye passage 3 (EP3) in which only a single cells became homozygous for various chromosome 13 poly cell or cells containing the chromosome 1/13 translocation morphic alÃelesboth above and below RBI (Fig. 1; Table 1). In grew. We also were able to document in one of ten additional these tumors there had either been a loss of one chromosome metaphases examined from first passage cells of primary tumor 13 and a duplication of the remaining chromosome 13 or a that both the normal chromosome 13 and the marker chromo mitotic recombination involving a region proximal to any poly some 1/13 were present. Thus clonal evolution from hemizy- morphic chromosome 13 probe now available. In addition, 3 4190

Table 2 Previous studies on chromosome 13 patterns in retinoblastoma they will be particularly valuable in understanding the mecha TumorRB-409RB-412RB-405RB-355RB-430HL-163RB-69ARB-247RB-267RB-KS2HRB-462E82-1751P83-1P83-6P82-15E82-2118E83-285E83-1218P83-10PatienttypeBilateralUnilateralUnilateralUnilateralUnilateralBilateralBilateralNotpatternHomozygousHomozygousforconclusionRFLPs" nism of tumorigenesis in retinoblastoma development as well andRFLPs DEs 114, as defining the Rb gene (18). (MR)HeterozygousHeterozygousHomozygousHomozygousHemizygousHeterozygousHomozygousandRFLPs DEs 114, andRFLPs DEs 114, andRFLPs DEs U4, ACKNOWLEDGMENTS andEsDCytogenetics 11113,4445566666666 DEs We wish to thank Dr. Webster Cavenee for his interest in and RFLPsRFLPsRFLP*RFLP*RFLPsRFLPsand contribution to this study. We are particularly indebted to the efforts givenNot of Dr. Marc Hansen who collaborated with us initially on many of the givenFamilialFamilialBilateralUnilateralBilateralUnilateralBilateralBilateralBilateralUnilateralTumor(?MR)Homozygous analyses on restriction fragment length polymorphisms. We also wish (?MR)Homozygous (MR)HomozygousHeterozygousHeterozygousHeterozygousHeterozygousHomozygousto thank C. Cook and M. Criss for their excellent technical assistance. DRFLPsRFLPsRFLPsRFLPsRFLPs'RFLPsand Es REFERENCES

1. Strong, L. C., Riccardi, V. M., Ferrell, R. E., and Sparkes, R. S. Familial (?MR)HomozygousHomozygousBasis retinoblastoma and chromosome 13 deletion transmitted via an insertional andRFLPs DEs translocation. Science (Wash. DC), 213: 1501-1503, 1981. andEs DRefs.4, 2. Balaban, G., Gilbert, F., Nichols, W., Meadows, A. T., and Shields, J. Abnormalities of chromosome 13 in retinoblastomas from individuals with * RFLPs, DNA restriction-fragment-length polymorphisms; MR, apparent mi- normal constitutional karyotypes. Cancer Genet. Cytogenet., 6: 213-221, totic recombinations. 1982. * Only one probe informative. 3. Benedict, W. F., Banerjee, A., Mark, C., and Murphree, A. L. Non-random c Only probes distal to RBI informative. chromosomal changes in direct preparations of primary retinoblastoma. Cancer Genet. Cytogenet., 10: 311-333, 1983. 4. Cavenee, W. K., Dryja, T. P., Phillips, R. A., Benedict, W. 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William F. Benedict, Eri S. Srivatsan, Corey Mark, et al.

Cancer Res 1987;47:4189-4191.

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