A R T I C L E
Therapy-induced malignant neoplasms in Nf1 mutant mice
Richard C. Chao,1,8,11Urszula Pyzel,1Jane Fridlyand,2Yien-Ming Kuo,1,3Lewis Teel,1Jennifer Haaga,1
Alexander Borowsky,9Andrew Horvai,4Scott C. Kogan,5,6Jeannette Bonifas,1Bing Huey,6Tyler E. Jacks,10
Donna G. Albertson,5,6,7and Kevin M. Shannon1,6,*
1Department of Pediatrics, University of California, San Francisco, San Francisco, California 94143
2Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, California 94143
3Department of Medicine, University of California, San Francisco, San Francisco, California 94143
4Department of Pathology, University of California, San Francisco, San Francisco, California 94143
5Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California 94143
6Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California 94143
7Cancer Research Institute, University of California, San Francisco, San Francisco, California 94143
8Department of Medicine, Division of Hematology/Oncology, Department of Veterans’ Affairs Medical Center, San Francisco,
9Medical Pathology Center for Comparative Medicine, University of California, Davis, Davis, California 95616
10Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
11Present address: Pfizer Global Research and Development, 10578 Science Center Drive (B95), San Diego, California 92121
Therapy-induced cancers are a severe complication of genotoxic therapies. We used heterozygous Nf1 mutant mice as a
sensitized genetic background to investigate tumor induction by radiation (RAD) and cyclophosphamide (CY). Mutagen-
exposed Nf1+/−mice developed secondary cancers that are common in humans, including myeloid malignancies, sarco-
mas, and breast cancers. RAD cooperated strongly with heterozygous Nf1 inactivation in tumorigenesis. Most of the
solid tumors showed loss of the wild-type Nf1 allele but retained two Trp53 alleles. Comparative genomic hybridization
demonstrated distinct patterns of copy number aberrations in sarcomas and breast cancers from Nf1 mutant mice, and
tumor cell lines showed deregulated Ras signaling. Nf1+/−mice provide a tractable model for investigating the pathogene-
sis of common mutagen-induced cancers and for testing preventive strategies.
Therapy-induced malignant neoplasms, also known as second
malignant neoplasms (SMNs), are a severe complication of
genotoxic cancer treatments including radiation (RAD) and
chemotherapeutic agents (Bhatia and Sklar, 2002; Matesich
and Shapiro, 2003; Smith et al., 2003). SMNs have a substan-
tial public health impact, as they account for most of the
w90,000 new cancers that are diagnosed in the United States
each year in persons who had a previous histologically distinct
malignancy (Bhatia and Sklar, 2002). Whereas early reports
emphasized the risk of SMNs in patients with Hodgkin’s dis-
ease and other hematopoietic malignancies (Le Beau et al.,
1986; Rowley et al., 1977; Tucker et al., 1988), these cancers
are increasingly recognized after intensive treatment for breast
cancer and other solid tumors (Matesich and Shapiro, 2003;
S I G N I F I C A N C E
Cancers induced by genotoxic treatments are a major clinical problem; however, the long-term mutagenic potential of specific
therapeutic regimens may not be known until many years from now. We used Nf1 mutant mice to recapitulate the dynamic interac-
tion between mutagen exposure and tumorigenesis that underlies the development of human therapy-induced malignancies. These
animals develop a similar spectrum of malignancies as human patients who are treated with radiation and alkylating agents, and
provide a tractable system for performing mechanistic studies, for comparing the mutagenic potential of different regimens, and
for testing preventive strategies. Our data also have translational implications for assessing the potential risks of genotoxic modalities,
particularly in persons with neurofibromatosis type 1.
CANCER CELL : OCTOBER 2005 · VOL. 8 · COPYRIGHT © 2005 ELSEVIER INC.DOI 10.1016/j.ccr.2005.08.011337
Smith et al., 2003). Myeloid leukemia, lymphoma, and sarcoma
are the most common SMNs found in survivors of hematologic
and nonhematologic cancers (Bhatia and Sklar, 2002; Matesich
and Shapiro, 2003; Smith et al., 2003). With prolonged follow-
up, cancer survivors are also at elevated risk of developing epi-
thelial tumors of the breast, uterus, and gastrointestinal tract
(de Vathaire et al., 1989a, 1989b; Hawkins et al., 1987; Tucker
et al., 1988). Importantly, many SMNs are resistant to treat-
ment, and previous exposure to cytotoxic agents may limit the
use of intensive salvage regimens. The lack of relevant animal
models of SMNs has impeded efforts to understand how muta-
genic cancer therapeutics induce tumors in vivo, and to test
Studies of familial cancer syndromes have provided funda-
mental insights into mechanisms that underlie tumorigenesis.
In addition to markedly increasing the incidence of primary ma-
A R T I C L E
lignancies, some inherited cancer predispositions confer a high
risk of SMNs. For example, persons with the Li-Fraumeni syn-
drome carry germline TP53 mutations, which leads to an ele-
vated risk of both primary malignancies and SMNs (Li and
Fraumeni, 1982; Malkin et al., 1992; Nichols et al., 2001). Simi-
larly, most children with a germline RB1 mutation develop one
or more retinoblastoma tumors and are predisposed to osteo-
sarcoma and other cancers later in life (Wong et al., 1997). The
incidence of osteosarcoma is dramatically increased in areas
exposed to RAD in the course of treating the initial retinoblas-
toma (Wong et al., 1997). As in de novo cancers, investigating
how heritable mutations cooperate with genotoxic cancer ther-
apies will likely provide mechanistic insights that are relevant
to tumorigenesis in individuals who develop SMNs without a
known genetic predisposition.
Mutations in the NF1 tumor suppressor gene cause neurofi-
bromatosis type 1 (NF1), an inherited cancer syndrome that
affects 1 in 3500 persons (Cichowski and Jacks, 2001; Das-
gupta and Gutmann, 2003). NF1 encodes neurofibromin, a
GTPase-activating protein that negatively regulates Ras signal-
ing (Boguski and McCormick, 1993; Donovan et al., 2002b).
Affected individuals are predisposed to specific benign and
malignant tumors, particularly in tissues derived from the em-
bryonic neural crest (Side and Shannon, 1998). In addition, the
incidence of juvenile myelomonocytic leukemia (JMML) and
other myeloid malignancies is increased 200- to 500-fold in
children with NF1 (Stiller et al., 1994). Clinical data suggest that
persons with NF1 are also predisposed to SMNs. Maris and
coworkers (Maris et al., 1997) reported five children with NF1
who developed myeloid malignancies and performed a sys-
tematic review of 64 children with NF1 who received chemo-
therapy and/or RAD to treat a primary cancer. This study re-
vealed an 11% incidence of SMNs, with an especially high risk
in children who had a primary embryonal cancer. Two adults
with NF1 also developed therapy-related myelodysplastic syn-
drome (MDS) after treatment for de novo acute myeloid leuke-
mia (AML) (Papageorgio et al., 1999). These reports suggested
that therapeutic exposure to genotoxic agents might cooperate
with germline NF1 mutations in the genesis of common SMNs
found in the general population, namely myeloid leukemia
Based on these clinical observations, we reasoned that het-
erozygous Nf1 mutant mice (Nf1+/−) might be harnessed to
investigate the pathogenesis of SMNs in vivo. Nf1+/−mice
spontaneously develop pheochromocytoma and a myeloprolif-
erative disorder (MPD) that resembles JMML with incomplete
penetrance (Jacks et al., 1994b). In a previous study, exposing
these mice to the alkylating agent cyclophosphamide (CY) in-
creased the incidence of MPD and reduced the latency (Mah-
goub et al., 1999). Here, we show that RAD alone or in combi-
nation with CY induces a spectrum of SMNs in Nf1+/−mice
that includes soft tissue sarcomas and breast carcinomas. The
normal Nf1 allele is inactivated in most of these solid tumors,
and some also demonstrate loss of heterozygosity (LOH) at the
Trp53 locus. Comparative genomic hybridization (CGH) uncov-
ered tumor-specific patterns of copy number aberrations,
which implies the existence of distinct pathways of cooperat-
ing genetic lesions in different cancers. Biochemical investiga-
tion of cell lines developed from a subset of these malignant
tumors revealed deregulated Ras signaling. Nf1+/−mice pro-
vide a tractable in vivo model for understanding how RAD and
CANCER CELL : OCTOBER 2005
alkylating agents induce cancer, and for testing preventive stra-
CY and RAD induce reversible myelosuppression
We selected CY for investigation because this alkylating agent
is a component of many front-line therapeutic regimens. To
model the myelosuppression that occurs in human patients,
we intercrossed wild-type C57Bl/6 and 129/Sv mice to gener-
ate cohorts of five to ten F1 animals that were exposed to dif-
ferent CY doses. Mice injected with a weekly intraperitoneal
CY dose of 200 mg/kg for 6 consecutive weeks reproducibly
developed anemia and leukopenia that resolved after the drug
was discontinued (data not shown). This regimen was not
otherwise associated with obvious morbidity. A single RAD
dose of 3 Gy, which was selected on the basis of previous data
showing that this dose was leukemogenic in CBA mice (Major,
1979; Major and Mole, 1978; Mole et al., 1983), was adminis-
tered 2 weeks after the last dose of CY. In a pilot experiment
that assessed the combination of CY at 200 mg/kg/week for 6
weeks followed by 3 Gy of RAD, we found that mice tolerated
sequential treatment without significant toxicity (data not
Based on these preliminary data, 192 wild-type and Nf1+/−
mice were assigned to one of four groups at 8–17 weeks of
age: no treatment, CY only, RAD only, or CY followed by RAD.
Treatment with CY, RAD, or the combination resulted in revers-
ible myelosuppression (Figure S1 in the Supplemental Data
available with this article online). Mice that received six weekly
injections of CY developed anemia with decreases in hemoglo-
bin concentration (from 16.2 ± 1.0 g/dl to 12.3 ± 1.3 g/dl; p <
0.00001) and white blood cell count (from 7.4 ± 3.0 × 103to
1.5 ± 0.7 × 103cells/?l; p < 0.00001). Animals assigned to
receive RAD alone entered the study cohort concurrently and
were irradiated at the same time as mice that had been treated
with CY. RAD induced a significant reduction in leukocyte
counts and a modest fall in the hemoglobin concentration that
was not statistically significant (Figure S1). Myelosuppression
was similar in wild-type and Nf1+/−mice, and peripheral blood
cell counts recovered quickly after cessation of CY and/or
RAD. The only early treatment-related deaths occurred 19 and
20 days after RAD in two wild-type mice that received both CY
and RAD (w1% of the cohort).
Survival and tumorigenesis in wild-type and Nf1+/−mice
Pathologic analysis was performed on 91% of the study co-
hort, including 97 of 104 wild-type mice and 77 of 86 Nf1+/−
mice. Three wild-type and four Nf1+/−mice were considered
evaluable without complete pathologic analysis, including two
mice with treatment-related mortality, two mice with massive
splenomegaly in which there was no histologic analysis of
other organs, and three mice with tumors of the Harderian
gland, which secretes lipid and porphyrins over the eye. Eleven
mice that died unexpectedly could not be analyzed for tumor
formation. Heterozygous inactivation of Nf1 was strongly asso-
ciated with an increased risk of premature death following
treatment with a survival rate of only 30% after 15 months in
Nf1+/−mice compared with 78% in wild-type littermates (Figure
1, left panel; p < 0.001). Death was due to cancer in 96% of
evaluable mice. We identified 51 malignancies in 81 Nf1+/−ani-
A R T I C L E
and malignant peripheral nerve sheath tumor. An immunohistochemical
study of sporadic and NF1-associated tumors. Am. J. Clin. Pathol. 106,
Halperin, E.C., Greenberg, M.S., and Suit, H.D. (1984). Sarcoma of bone
and soft tissue following treatment of Hodgkin’s disease. Cancer 53, 232–
Hawkins, M.M., Draper, G.J., and Kingston, J.E. (1987). Incidence of second
primary tumours among childhood cancer survivors. Br. J. Cancer 56,
Huvos, A.G., Woodard, H.Q., Cahan, W.G., Higinbotham, N.L., Stewart,
F.W., Butler, A., and Bretsky, S.S. (1985). Postradiation osteogenic sarcoma
of bone and soft tissues. A clinicopathologic study of 66 patients. Cancer
Imamura, N., Abe, K., and Oguma, N. (2002). High incidence of point muta-
tions of p53 suppressor oncogene in patients with myelodysplastic syn-
drome among atomic-bomb survivors: a 10-year follow-up. Leukemia 16,
Ingram, D.A., Yang, F.C., Travers, J.B., Wenning, M.J., Hiatt, K., New, S.,
Hood, A., Shannon, K., Williams, D.A., and Clapp, D.W. (2000). Genetic and
biochemical evidence that haploinsufficiency of the Nf1 tumor suppressor
gene modulates melanocyte and mast cell fates in vivo. J. Exp. Med. 191,
Inwards, C.Y., and Unni, K.K. (1995). Classification and grading of bone
sarcomas. Hematol. Oncol. Clin. North Am. 9, 545–569.
Jacks, T., Remington, L., Williams, B.O., Schmitt, E.M., Halachmi, S., Bron-
son, R.T., and Weinberg, R.A. (1994a). Tumor spectrum analysis in p53-
mutant mice. Curr. Biol. 4, 1–7.
Jacks, T., Shih, S., Schmitt, E.M., Bronson, R.T., Bernards, A., and Wein-
berg, R.A. (1994b). Tumorigenic and developmental consequences of a tar-
geted Nf1 mutation in the mouse. Nat. Genet. 7, 353–361.
Jain, A.N., Tokuyasu, T.A., Snijders, A.M., Segraves, R., Albertson, D.G.,
and Pinkel, D. (2002). Fully automatic quantification of microarray image
data. Genome Res. 12, 325–332.
Johannessen, C.M., Reczek, E.E., James, M.F., Brems, H., Legius, E., and
Cichowski, K. (2005). The NF1 tumor suppressor critically regulates TSC2
and mTOR. Proc. Natl. Acad. Sci. USA 102, 8573–8578.
Kogan, S.C., Ward, J.M., Anver, M.R., Berman, J.J., Brayton, C., Cardiff,
R.D., Carter, J.S., de Coronado, S., Downing, J.R., Fredrickson, T.N., et al.
(2002). Bethesda proposals for classification of nonlymphoid hematopoietic
neoplasms in mice. Blood 100, 238–245.
Kushner, B.H., Heller, G., Cheung, N.K., Wollner, N., Kramer, K., Bajorin, D.,
Polyak, T., and Meyers, P.A. (1998). High risk of leukemia after short-term
dose-intensive chemotherapy in young patients with solid tumors. J. Clin.
Oncol. 16, 3016–3020.
Laskin, W.B., Silverman, T.A., and Enzinger, F.M. (1988). Postradiation soft
tissue sarcomas. An analysis of 53 cases. Cancer 62, 2330–2340.
Le Beau, M.M., Albain, K.S., Larson, R.A., Vardiman, J.W., Davis, E.M.,
Blough, R.R., Golomb, H.M., and Rowley, J.D. (1986). Clinical and cytoge-
netic correlations in 63 patients with therapy-related myelodysplastic syn-
dromes and acute nonlymphocytic leukemia: further evidence for character-
istic abnormalities of chromosomes no. 5 and 7. J. Clin. Oncol. 4, 325–345.
Li, F.P., and Fraumeni, J.F., Jr. (1982). Prospective study of a family cancer
syndrome. JAMA 247, 2692–2694.
Li, H., Velasco-Miguel, S., Vass, W.C., Parada, L.F., and DeClue, J.E. (2002).
Epidermal growth factor receptor signaling pathways are associated with
tumorigenesis in the Nf1:p53 mouse tumor model. Cancer Res. 62, 4507–
Liapis, H., Marley, E.F., Lin, Y., and Dehner, L.P. (1999). p53 and Ki-67 prolif-
erating cell nuclear antigen in benign and malignant peripheral nerve sheath
tumors in children. Pediatr. Dev. Pathol. 2, 377–384.
Ling, B.C., Wu, J., Miller, S.J., Monk, K.R., Shamekh, R., Rizvi, T.A., Decour-
ten-Myers, G., Vogel, K.S., DeClue, J.E., and Ratner, N. (2005). Role for the
CANCER CELL : OCTOBER 2005 347
epidermal growth factor receptor in neurofibromatosis-related peripheral
nerve tumorigenesis. Cancer Cell 7, 65–75.
Lothe, R.A., Smith-Sorensen, B., Hektoen, M., Stenwig, A.E., Mandahl, N.,
Saeter, G., and Mertens, F. (2001). Biallelic inactivation of TP53 rarely con-
tributes to the development of malignant peripheral nerve sheath tumors.
Genes Chromosomes Cancer 30, 202–206.
Mahgoub, N., Taylor, B., Le Beau, M., Gratiot, M., Carlson, K., Jacks, T., and
Shannon, K.M. (1999). Myeloid malignancies induced by alkylating agents in
Nf1 mice. Blood 93, 3617–3623.
Major, I.R. (1979). Induction of myeloid leukaemia by whole-body single
exposure of CBA male mice to x-rays. Br. J. Cancer 40, 903–913.
Major, I.R., and Mole, R.H. (1978). Myeloid leukaemia in x-ray irradiated
CBA mice. Nature 272, 455–456.
Malkin, D., Jolly, K.W., Barbier, N., Look, A.T., Friend, S.H., Gebhardt, M.C.,
Andersen, T.I., Borresen, A.L., Li, F.P., Garber, J., et al. (1992). Germline
mutations of the p53 tumor-suppressor gene in children and young adults
with second malignant neoplasms. N. Engl. J. Med. 326, 1309–1315.
Maris, J.M., Wiersma, S.R., Mahgoub, N., Thompson, P., Geyer, R.J., Hur-
witz, C.G., Lange, B.J., and Shannon, K.M. (1997). Monosomy 7 myelodys-
plastic syndrome and other second malignant neoplasms in children with
neurofibromatosis type 1. Cancer 79, 1438–1446.
Marsche, E. (1922). Tuberkulose und Sarkom. Zentralbl. Chir. 49, 1057–
Matesich, S.M., and Shapiro, C.L. (2003). Second cancers after breast can-
cer treatment. Semin. Oncol. 30, 740–748.
Menon, A.G., Anderson, K.M., Riccardi, V.M., Chung, R.Y., Whaley, J.M.,
Yandell, D.W., Farmer, G.E., Freiman, R.N., Lee, J.K., Li, F.P., et al. (1990).
Chromosome 17p deletions and p53 gene mutations associated with the
formation of malignant neurofibrosarcomas in von Recklinghausen neurofi-
bromatosis. Proc. Natl. Acad. Sci. USA 87, 5435–5439.
Mertens, F., Rydholm, A., Bauer, H.F., Limon, J., Nedoszytko, B., Szadow-
ska, A., Willen, H., Heim, S., Mitelman, F., and Mandahl, N. (1995). Cytoge-
netic findings in malignant peripheral nerve sheath tumors. Int. J. Cancer
Mole, R.H., Papworth, D.G., and Corp, M.J. (1983). The dose-response for
x-ray induction of myeloid leukaemia in male CBA/H mice. Br. J. Cancer
Murray, E.M., Werner, D., Greeff, E.A., and Taylor, D.A. (1999). Postradiation
sarcomas: 20 cases and a literature review. Int. J. Radiat. Oncol. Biol. Phys.
Nakanishi, H., Tomita, Y., Myoui, A., Yoshikawa, H., Sakai, K., Kato, Y., Ochi,
T., and Aozasa, K. (1998). Mutation of the p53 gene in postradiation sar-
coma. Lab. Invest. 78, 727–733.
Neglia, J.P., Meadows, A.T., Robison, L.L., Kim, T.H., Newton, W.A., Ruy-
mann, F.B., Sather, H.N., and Hammond, G.D. (1991). Second neoplasms
after acute lymphoblastic leukemia in childhood. N. Engl. J. Med. 325,
Nichols, K.E., Malkin, D., Garber, J.E., Fraumeni, J.F., Jr., and Li, F.P. (2001).
Germ-line p53 mutations predispose to a wide spectrum of early-onset can-
cers. Cancer Epidemiol. Biomarkers Prev. 10, 83–87.
Olshen, A.B., Venkatraman, E.S., Lucito, R., and Wigler, M. (2004). Circular
binary segmentation for the analysis of array-based DNA copy number
data. Biostatistics 5, 557–572.
Papageorgio, C., Seiter, K., and Feldman, E.J. (1999). Therapy-related mye-
lodysplastic syndrome in adults with neurofibromatosis. Leuk. Lymphoma
Pendlebury, S.C., Bilous, M., and Langlands, A.O. (1995). Sarcomas follow-
ing radiation therapy for breast cancer: a report of three cases and a review
of the literature. Int. J. Radiat. Oncol. Biol. Phys. 31, 405–410.
Pinkel, D., Segraves, R., Sudar, D., Clark, S., Poole, I., Kowbel, D., Collins,
C., Kuo, W.L., Chen, C., Zhai, Y., et al. (1998). High resolution analysis of
DNA copy number variation using comparative genomic hybridization to
microarrays. Nat. Genet. 20, 207–211.
A R T I C L E
Plaat, B.E., Molenaar, W.M., Mastik, M.F., Hoekstra, H.J., te Meerman, G.J.,
and van den Berg, E. (1999). Computer-assisted cytogenetic analysis of 51
malignant peripheral-nerve-sheath tumors: sporadic vs. neurofibromatosis-
type-1-associated malignant schwannomas. Int. J. Cancer 83, 171–178.
Preston, D.L., Kusumi, S., Tomonaga, M., Izumi, S., Ron, E., Kuramoto, A.,
Kamada, N., Dohy, H., Matsuo, T., Matsui, T., et al. (1994). Cancer incidence
in atomic bomb survivors. Part III. Leukemia, lymphoma and multiple my-
eloma, 1950–1987. Radiat. Res. 137, S68–S97.
Remmelink, M., Decaestecker, C., Darro, F., Goldschmidt, D., Gebhart, M.,
Pasteels, J.L., Kiss, R., and Salmon, I. (1998). The in vitro influence of eight
hormones and growth factors on the proliferation of eight sarcoma cell lines.
J. Cancer Res. Clin. Oncol. 124, 155–164.
Rowley, J.D., Golomb, H.M., and Vardiman, J. (1977). Nonrandom chromo-
somal abnormalities in acute nonlymphocytic leukemia in patients treated
for Hodgkin disease and non-Hodgkin lymphoma. Blood 50, 759.
Sekyi-Otu, A., Bell, R.S., Ohashi, C., Pollak, M., and Andrulis, I.L. (1995).
Insulin-like growth factor 1 (IGF-1) receptors, IGF-1, and IGF-2 are ex-
pressed in primary human sarcomas. Cancer Res. 55, 129–134.
Side, L.E., and Shannon, K.M. (1998). The NF1 gene as a tumor suppressor.
In Neurofibromatosis Type 1, M. Upashyaya and D.N. Cooper, eds. (Oxford:
Bios Scientific Publishers), pp. 133–152.
Smith, P.G., and Doll, R. (1982). Mortality among patients with ankylosing
spondylitis after a single treatment course with x rays. Br. Med. J. (Clin.
Res. Ed.) 284, 449–460.
Smith, S.M., Le Beau, M.M., Huo, D., Karrison, T., Sobecks, R.M., Anastasi,
J., Vardiman, J.W., Rowley, J.D., and Larson, R.A. (2003). Clinical-cytoge-
netic associations in 306 patients with therapy-related myelodysplasia and
myeloid leukemia: the University of Chicago series. Blood 102, 43–52.
Snijders, A.M., Nowak, N.J., Huey, B., Fridlyand, J., Law, S., Conroy, J.,
Tokuyasu, T., Demir, K., Chiu, R., Mao, J.H., et al. (2005). Mapping segmen-
tal and sequence variations among laboratory mice using BAC array CGH.
Genome Res. 15, 302–311.
Stiller, C.A., Chessells, J.M., and Fitchett, M. (1994). Neurofibromatosis and
childhood leukemia/lymphoma: A population-based UKCCSG study. Br. J.
Cancer 70, 969–972.
CANCER CELL : OCTOBER 2005
Thirman, M.J., and Larson, R.A. (1996). Therapy-related myeloid leukemia.
Hematol. Oncol. Clin. North Am. 10, 293–320.
Tucker, M.A., Coleman, C.N., Cox, R.S., Varghese, A., and Rosenberg, S.A.
(1988). Risk of second cancers after treatment for Hodgkin’s disease. N.
Engl. J. Med. 318, 76–81.
van Leeuwen, F.E., Klokman, W.J., Hagenbeek, A., Noyon, R., van den Belt-
Dusebout, A.W., van Kerkhoff, E.H., van Heerde, P., and Somers, R. (1994).
Second cancer risk following Hodgkin’s disease: a 20-year follow-up study.
J. Clin. Oncol. 12, 312–325.
Vogel, K.S., Klesse, L.J., Velasco-Miguel, S., Meyers, K., Rushing, E.J., and
Parada, L.F. (1999). Mouse tumor model for neurofibromatosis type 1. Sci-
ence 286, 2176–2179.
Wang, J., Coltrera, M.D., and Gown, A.M. (1994). Cell proliferation in human
soft tissue tumors correlates with platelet-derived growth factor B chain
expression: an immunohistochemical and in situ hybridization study. Cancer
Res. 54, 560–564.
Watson, M.A., Perry, A., Tihan, T., Prayson, R.A., Guha, A., Bridge, J.,
Ferner, R., and Gutmann, D.H. (2004). Gene expression profiling reveals
unique molecular subtypes of Neurofibromatosis Type I-associated and
sporadic malignant peripheral nerve sheath tumors. Brain Pathol. 14, 297–
Weiss, S.W., Langloss, J.M., and Enzinger, F.M. (1983). Value of S-100 pro-
tein in the diagnosis of soft tissue tumors with particular reference to benign
and malignant Schwann cell tumors. Lab. Invest. 49, 299–308.
Wick, M.R., Swanson, P.E., Scheithauer, B.W., and Manivel, J.C. (1987).
Malignant peripheral nerve sheath tumor. An immunohistochemical study of
62 cases. Am. J. Clin. Pathol. 87, 425–433.
Wong, F.L., Boice, J.D., Jr., Abramson, D.H., Tarone, R.E., Kleinerman, R.A.,
Stovall, M., Goldman, M.B., Seddon, J.M., Tarbell, N., Fraumeni, J.F., Jr.,
and Li, F.P. (1997). Cancer incidence after retinoblastoma. Radiation dose
and sarcoma risk. JAMA 278, 1262–1267.
Yasui, K., Arii, S., Zhao, C., Imoto, I., Ueda, M., Nagai, H., Emi, M., and
Inazawa, J. (2002). TFDP1, CUL4A, and CDC16 identified as targets for
amplification at 13q34 in hepatocellular carcinomas. Hepatology 35,