Pediatr Blood Cancer 2015;62:1481–1484
BRIEF REPORT A Child With Li–Fraumeni Syndrome: Modes to Inactivate the Second Allele of TP53 in Three Different Malignancies Brigitte Schlegelberger, MD,1* Hans Kreipe, MD,2 Ulrich Lehmann, Dr. rer. nat.,2 Doris Steinemann, Dr. rer. nat.,1 Tim Ripperger, MD, PhD,1 Gudrun Go¨hring, MD,1 Kathrin Thomay, Dr. rer. nat.,1 Andreas Rump, Dr. rer. nat.,3 Nataliya Di Donato, MD,3 and Meinolf Suttorp, MD4 Here we report on a child with Li–Fraumeni syndrome with a de novo TP53 mutation c.818G>A, who developed three malignancies at the age of 4 months, 4 and 5 years, respectively. We show that (i) in the choroid plexus carcinoma, the germline mutation was detected in a homozygous state due to copy-neutral LOH/uniparental disomy, (ii) in the secondary AML, a complex karyotype led to loss of the wild-
type TP53 allele, (iii) in the Wilms tumor, the somatic mutation c.814G>A led to compound heterozygosity. The findings show that the complete inactivation of TP53 by different mechanisms is an important step towards tumorigenesis. Pediatr Blood Cancer 2015; 62:1481–1484. # 2015 Wiley Periodicals, Inc.
Key words: choroid plexus carcinoma; leukemia; Li–Fraumeni syndrome; sarcoma; TP53; uniparental disomy (UPD)
INTRODUCTION Li–Fraumeni syndrome (LFS), a syndrome caused by germline mutations in TP53 [1,2], is associated with the development of soft tissue sarcomas, osteosarcoma, pre-menopausal breast cancer, brain tumors, adrenocortical cancer, and leukemia [3–5]. The prevalence of deleterious TP53 germline mutations is estimated to be 1 in 5,000 [6]. TP53 mutations may arise de novo or be inherited from parents, and can be passed on to offspring in an autosomaldominant fashion. Up to 30% of children carrying a TP53 germline mutation will suffer from cancer. Besides early cancer onset, there is an increased risk to develop multiple cancers, in particular for those patients with LFS, who developed a primary cancer at a young age [7,8]. Moreover, in line with the role of TP53 as a key regulator in the response to DNA damage, an increased risk for radiotherapyassociated secondary cancers was reported [9]. Although LFS was already described in 1969 [3], and it is well accepted that a heterozygous TP53 germline mutation is a first and important step towards tumorigenesis, molecular mechanisms triggering the development of overt malignant tumors are complex.
RESULTS
CD117, CD15, CD13, and CD45) in the bone marrow, acute myeloid leukemia (AML) subtype FAB M4/M5 without central nervous system (CNS) involvement was diagnosed. Cytogenetic analyses showed a complex aberrant karyotype involving chromosomes 5, 7, 11, 13, 16, 17, and 22 (Fig. 1C,D) including a TP53 deletion consistent with secondary AML. Given the poor prognosis of secondary AML, unrelated single HLA C-locus mismatched peripheral blood stem cell transplantation (SCT) from a female donor was performed with a rather uneventful clinical course. At 5.6 years, she presented with severe transfusion-dependent hematuria due to a tumor in the left kidney without metastases. Total left nephrectomy was performed. Histologically, the tumor was classified as high-grade malignant nephroblastoma (SIOP stage I) with diffuse anaplasia (Wilms tumor, WT) (Fig. 1E). No neoadjuvant or adjuvant chemotherapy was administered after balancing the risk of reoccurrence of a biologically aggressive tumor versus the context of a germline TP53 mutation. At the age of 6.9 years, the child is in ongoing complete remission. Thus, our patient suffering from three aggressive malignancies so far has had a surprisingly good outcome. Additional Supporting Information may be found in the online version of this article at the publisher’s web-site. 1
Here we report on a female child with LFS, who developed three different malignancies: a choroid plexus carcinoma (CPC) at the age of 4 months, a secondary acute myeloid leukemia (AML) by 4 years, and a Wilms tumor at 5 years. The occurrence of three different tumors arising in a single child within an interval of 6 years is extremely rare and has been reported so far only once [10]. The 4-month-old female, the first child of healthy unrelated parents, presented with a tumor originating from the right lateral ventricle of the brain. Histological examination revealed a papillary and partially solid proliferation of pleomorphic epithelial cells and necrosis (Fig. 1A,B). The cells were positive for cytokeratin and vimentin consistent with a diagnosis of choroid plexus carcinoma (CPC) WHO grade 3. Due to suspected LFS, the tumor was treated without irradiation by chemotherapy. At the age of 4.2 years, the child developed progressive pancytopenia. Due to 62% myeloid blasts (positive for CD34 and CD33, partially positive for CD4,
C
2015 Wiley Periodicals, Inc. DOI 10.1002/pbc.25486 Published online 18 March 2015 in Wiley Online Library (wileyonlinelibrary.com).
Institute of Human Genetics, Hannover Medical School, Hannover, Germany; 2Institute of Pathology, Hannover Medical School, Hannover, Germany; 3Institute for Clinical Genetics, Medical Faculty Carl Gustav Carus, TU Dresden, Germany; 4Department of Pediatric Hematology and Oncology, University Hospital Dresden, Germany Grant sponsor: Cluster of Excellence Rebirth funded by the DFG (Rebirth 2, Units 9.3a and b); Grant sponsor: German Cancer Aid (German Consortium for Hereditary Breast and Ovarian Cancer) Conflict of interest: Nothing to declare. Brigitte Schlegelberger and Hans Kreipe contributed equally to this work. Correspondence to: Prof. Dr. med. Brigitte Schlegelberger, Institute of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail: [email protected]
Received 25 July 2014; Accepted 2 February 2015
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Fig. 1. Histopathological and cytogenetic findings. A: Plexus carcinoma with partially blurred papillary pattern and focal necrosis. B: Plexus carcinoma with area of solid proliferation, high nuclear pleomorphism, and more than 10 mitoses/10 high power fields. C: Karyogram of bone marrow AML cells, fluorescence R-banding. D: Karyogram of bone marrow AML cells, multicolor fluorescence in situ hybridization (mFISH) analysis to further identify the origin of the complex chromosomal aberrations. The unknown material in the long arm of chromosome 11 could be identified as derived from chromosome 22, in the long arm of chromosome 13 from chromosomes 11 and 22 and in the long arm of chromosome 22 from chromosome 11. There is an unbalanced translocation between chromosomes 5 and 17. In addition, we detected a low-level amplification of the MLL locus. The karyotype was finally described as: 4044,XX,der(5)t(5;17)(q11;q11),der(11)qdp(11)(q14q24)t(11;22)(q24;q12), ins(13;11) (q31;q12q24),-16,-17,der(22)t(11;22)(?;p13)[21]. nuc ish 11q23(MLLx4-8)[66/100,]17p13.1(P53 1)[72/100]. E: Nephroblastoma with marked nuclear enlargement and hyperchromasia, proliferation of pleomorphic spindle cells with a more sarcomatous rather than blastemal pattern and diffuse anaplasia. Foci of epithelial cells were observed, forming solid nests or tubular structures. Epithelial cells and spindle cells were immunohistochemically positive for vimentin and intensely positive for TP53; spindle cells revealed focal desmin expression and were negative for WT1.
Pediatr Blood Cancer DOI 10.1002/pbc
Inactivation of TP53 in Li–Fraumeni Syndrome
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Fig. 2. Inactivation of TP53 in the germline and in the three different tumors. A: Sequence analysis of TP53, exon 8. Electropherograms showing nucleotides c.811 to c.828 of TP53, exon 8 (ENST00000269305). Sequences obtained are displayed in accordance with the IUPAC code. The nucleotides c.814 and c.818 are highlighted by a circle and square respectively (black, wild-type; red, mutant). Sequencing results obtained from the analyses of DNA from a peripheral blood sample (i) at the time of secondary AML and a fibroblast culture (ii). In both samples, the heterozygous mutation c.818G>A; p.(Arg273His) was detected. Besides the known germline mutation analysis of Wilms tumor, DNA additionally displayed the heterozygous missense mutation (c.814G>A; p.(Val272Met). Subsequent investigations following TOPO TA cloning of the PCR product of the Wilms tumor DNA proved that the acquired mutation affects the second TP53 allele. Representative sequencing results of individual bacterial clones display the germline mutation c.818G>A (iii) and the somatic mutation c.814 (iv) in trans. B: Schematic presentation of biallelic inactivation of TP53 in the three different tumors. The heterozygous germline mutation c.818G>A (red circle) was detected in our patient. As a result of loss of the wild-type allele (green circle) and a duplication of the mutant allele due to copy-neutral LOH/uniparental disomy, the known germline mutation was detected in a homozygous state in the choroid plexus carcinoma. In the secondary AML showing a complex karyotype, FISH displayed a loss of TP53 in 72% of the analyzed nuclei, which most probably led to a loss of the wild-type allele. Identification of somatically acquired mutation c.814G>A (violet circle) leading to compound heterozygosity in the Wilms tumor.
Due to the presence of multiple tumors belonging to the LFS tumor spectrum, diagnosed in early childhood, the patient reported here fulfils the Chompret criteria II and III [8]. Indeed, in germline DNA isolated from fibroblasts, sequence analysis of exons 1–11 of TP53 (ENST00000269305) showed the missense mutation c.818G>A; p.(Arg273His) (rs28934576) as described according Pediatr Blood Cancer DOI 10.1002/pbc
to the Human Genome Variation Society (www.hgvs.org) (for details see Supplemental Table SI). According to the IARC (International Agency for Research on Cancer) database (http:// p53.iarc.fr/TP53), this missense mutation is located within the structural motif L1/S/H2 affecting DNA binding and is classified as a deleterious mutation. This mutation has already been described as
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a germline mutation in patients with Li–Fraumeni syndrome [11]. Sequencing of TP53 showed normal results in both parents as well as in the younger sister of the index patient. Therefore, we concluded that the germline mutation occurred de novo. The heterozygous mutation was present in leukemic blasts and in peripheral blood at the timepoint of hematological remission as well as in fibroblasts (Fig. 2Ai and ii). The CPC tumor cells are homozygous for TP53 c.818G>A. One allele of TP53 is lost in leukemic blasts. The malignant nephroblastoma is compound heterozygous for TP53 c.818G>A and c.814G>A (ts-sensitive, loss-of-function) (Fig. 2Aiii and iv). Morphologically inconspicuous non-neoplastic kidney tissue isolated by manual microdissection is heterozygous for TP53 c.818G>A, while c.814G>A was not detected. FISH analysis on both solid tumor specimens revealed no abnormalities of chromosome 17p.
DISCUSSION TP53 is one of the key factors responsible for the maintenance of genetic integrity. After cellular stress or DNA damage, TP53 can trigger G1 arrest, senescence or apoptosis. Concerning its role as “guardian of the genome” [12], it is intriguing that monoallelic inactivation of TP53 by germline mutations leads to an increased cancer risk in LFS. The missense mutation c.818G>A detected in our patient is located within the DNA binding domain of TP53, that is, the L1/S/H2 motif. It is known as a contact mutation, preventing wild-type transcriptional activity, itself leading to an inactivation of the second allele, and thereby confers gain-of-function properties with a more aggressive phenotype than mutations resulting in loss of TP53 expression [13–15]. It shows a dominant-negative effect due to the formation of heterotetramers consisting of mutant and wild-type TP53. Furthermore, it leads to aggregation of wild-type TP53 into cellular inclusions and also to co-aggregation with the TP53 family members p63 and p73. These findings are in line with the early onset of cancer in our patient and a previous report showing that the mean age of onset of primary cancer is 9 years earlier in LFS due to missense mutations in TP53 compared to LFS due to TP53 deletions [16]. Recent whole exome sequencing in tumors with somatic TP53 mutations showed a surprising paucity of mutations in other genes [17]. However, genome sequencing of pediatric medulloblastoma linked catastrophic DNA rearrangements to TP53 mutations [18]. We did not have the chance to perform whole exome sequencing in the three tumors of our patient. However, the three different modes of inactivation in each of the three malignancies (Fig. 2B) indicate an increased genomic instability. In the choroid plexus carcinoma, the homozygous mutation without a deletion can be explained by uniparental disomy or copy-neutral LOH due to loss of the normal and duplication of the mutant allele. In the AML, the complex karyotype clearly demonstrates the
Pediatr Blood Cancer DOI 10.1002/pbc
increased genomic instability resulting in, amongst other changes, the loss of TP53 due to a deletion of 17p. In the Wilms tumor, the somatic TP53 mutation Val272Met was detected in trans to the germline mutation Arg273His, thus leading to compound heterozygosity. These findings underline that a complete inactivation of TP53 by uniparental disomy, compound heterozygosity or deletion may be the decisive step towards tumorigenesis.
ACKNOWLEDGMENTS The authors wish to express their gratitude to Norbert Graf, MD (Homburg, Germany) for helpful discussions and advice concerning the clinical management of the Wilms tumor.
REFERENCES 1. Malkin D, Li FP, Strong LC, Fraumeni JF Jr., Nelson CE, Kim DH, Kassel J, Gryka MA, Bischoff FZ, Tainsky MA, Friend SH. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 1990;250:1233–1238. 2. Srivastava S, Zou ZQ, Pirollo K, Blattner W, Chang EH. Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome. Nature 1990;348:747–749. 3. Li FP, Fraumeni JF Jr. Soft-tissue sarcomas, breast cancer, and other neoplasms. A familial syndrome? Ann Intern Med 1969;71:747–752. 4. Li FP, Fraumeni JF Jr., Mulvihill JJ, Blattner WA, Dreyfus MG, Tucker MA, Miller RW. A cancer family syndrome in twenty-four kindreds. Cancer Res 1988;48:5358–5362. 5. Ruijs MW, Verhoef S, Rookus MA, Pruntel R, van der Hout AH, Hogervorst FB, Kluijt I, Sijmons RH, Aalfs CM, Wagner A, Ausems MG, Hoogerbrugge N, van Asperen CJ, Gomez Garcia EB, MeijersHeijboer H, Ten Kate LP, Menko FH, van ’t Veer LJ. TP53 germline mutation testing in 180 families suspected of Li-Fraumeni syndrome: Mutation detection rate and relative frequency of cancers in different familial phenotypes. J Med Genet 2010;47:421–428. 6. Lalloo F, Varley J, Ellis D, Moran A, O’Dair L, Pharoah P, Evans DG, Group EOBCS. Prediction of pathogenic mutations in patients with early-onset breast cancer by family history. Lancet 2003;361: 1101–1102. 7. Nichols KE, Malkin D, Garber JE, Fraumeni JF Jr., Li FP. Germ-line p53 mutations predispose to a wide spectrum of early-onset cancers. Cancer Epidemiol Biomarkers Prev 2001;10:83–87. 8. Tinat J, Bougeard G, Baert-Desurmont S, Vasseur S, Martin C, Bouvignies E, Caron O, Bressac-de Paillerets B, Berthet P, Dugast C, Bonaiti-Pellie C, Stoppa-Lyonnet D, Frebourg T. 2009 version of the Chompret criteria for Li Fraumeni syndrome. J Clin Oncol 2009;27:e108–e109; author reply e110. 9. Henry E, Villalobos V, Million L, Jensen KC, West R, Ganjoo K, Lebensohn A, Ford JM, Telli ML. Chest wall leiomyosarcoma after breast-conservative therapy for early-stage breast cancer in a young woman with Li-Fraumeni syndrome. J Natl Compr Canc Netw 2012;10:939–942. 10. Allen AM, Kelly DR, Watts RG. Three sequential malignancies in a child. Clin Pediatr (Phila) 2011;50:879–881. 11. Malkin D, Jolly KW, Barbier N, Look AT, Friend SH, Gebhardt MC, Andersen TI, Borresen AL, Li FP, Garber J, Strong LC. Germline mutations of the p53 tumor-suppressor gene in children and young adults with second malignant neoplasms. New Engl J Med 1992;326:1309–1315. 12. Lane DP. p53, guardian of the genome. Nature 1992;358:15–16. 13. Muller PA, Vousden KH. Mutant p53 in cancer: New functions and therapeutic opportunities. Cancer Cell 2014;25:304–317. 14. Xu J, Reumers J, Couceiro JR, De Smet F, Gallardo R, Rudyak S, Cornelis A, Rozenski J, Zwolinska A, Marine JC, Lambrechts D, Suh YA, Rousseau F, Schymkowitz J. Gain of function of mutant p53 by coaggregation with multiple tumor suppressors. Nat Chem Biol 2011;7:285–295. 15. Zerdoumi Y, Aury-Landas J, Bonaiti-Pellie C, Derambure C, Sesboue R, Renaux-Petel M, Frebourg T, Bougeard G, Flaman JM. Drastic effect of germline TP53 missense mutations in Li-Fraumeni patients. Hum Mutat 2013;34:453–461. 16. Bougeard G, Sesboue R, Baert-Desurmont S, Vasseur S, Martin C, Tinat J, Brugieres L, Chompret A, de Paillerets BB, Stoppa-Lyonnet D, Bonaiti-Pellie C, Frebourg T. French LFS working group. Molecular basis of the Li-Fraumeni syndrome: An update from the French LFS families. J Med Genet 2008;45: 535–538. 17. Kulasekararaj AG, Smith AE, Mian SA, Mohamedali AM, Krishnamurthy P, Lea NC, Gaken J, Pennaneach C, Ireland R, Czepulkowski B, Pomplun S, Marsh JC, Mufti GJ. TP53 mutations in myelodysplastic syndrome are strongly correlated with aberrations of chromosome 5, and correlate with adverse prognosis. Br J Haematol 2013;160:660–672. 18. Rausch T, Jones DT, Zapatka M, Stutz AM, Zichner T, Weischenfeldt J, Jager N, Remke M, Shih D, Northcott PA, Pfaff E, Tica J, Wang Q, Massimi L, Witt H, Bender S, Pleier S, Cin H, Hawkins C, Beck C, von Deimling A, Hans V, Brors B, Eils R, Scheurlen W, Blake J, Benes V, Kulozik AE, Witt O, Martin D, Zhang C, Porat R, Merino DM, Wasserman J, Jabado N, Fontebasso A, Bullinger L, Rucker FG, Dohner K, Dohner H, Koster J, Molenaar JJ, Versteeg R, Kool M, Tabori U, Malkin D, Korshunov A, Taylor MD, Lichter P, Pfister SM, Korbel JO. Genome sequencing of pediatric medulloblastoma links catastrophic DNA rearrangements with TP53 mutations. Cell 2012;148:59–71.
BRIEF REPORT A Child With Li–Fraumeni Syndrome: Modes to Inactivate the Second Allele of TP53 in Three Different Malignancies Brigitte Schlegelberger, MD,1* Hans Kreipe, MD,2 Ulrich Lehmann, Dr. rer. nat.,2 Doris Steinemann, Dr. rer. nat.,1 Tim Ripperger, MD, PhD,1 Gudrun Go¨hring, MD,1 Kathrin Thomay, Dr. rer. nat.,1 Andreas Rump, Dr. rer. nat.,3 Nataliya Di Donato, MD,3 and Meinolf Suttorp, MD4 Here we report on a child with Li–Fraumeni syndrome with a de novo TP53 mutation c.818G>A, who developed three malignancies at the age of 4 months, 4 and 5 years, respectively. We show that (i) in the choroid plexus carcinoma, the germline mutation was detected in a homozygous state due to copy-neutral LOH/uniparental disomy, (ii) in the secondary AML, a complex karyotype led to loss of the wild-
type TP53 allele, (iii) in the Wilms tumor, the somatic mutation c.814G>A led to compound heterozygosity. The findings show that the complete inactivation of TP53 by different mechanisms is an important step towards tumorigenesis. Pediatr Blood Cancer 2015; 62:1481–1484. # 2015 Wiley Periodicals, Inc.
Key words: choroid plexus carcinoma; leukemia; Li–Fraumeni syndrome; sarcoma; TP53; uniparental disomy (UPD)
INTRODUCTION Li–Fraumeni syndrome (LFS), a syndrome caused by germline mutations in TP53 [1,2], is associated with the development of soft tissue sarcomas, osteosarcoma, pre-menopausal breast cancer, brain tumors, adrenocortical cancer, and leukemia [3–5]. The prevalence of deleterious TP53 germline mutations is estimated to be 1 in 5,000 [6]. TP53 mutations may arise de novo or be inherited from parents, and can be passed on to offspring in an autosomaldominant fashion. Up to 30% of children carrying a TP53 germline mutation will suffer from cancer. Besides early cancer onset, there is an increased risk to develop multiple cancers, in particular for those patients with LFS, who developed a primary cancer at a young age [7,8]. Moreover, in line with the role of TP53 as a key regulator in the response to DNA damage, an increased risk for radiotherapyassociated secondary cancers was reported [9]. Although LFS was already described in 1969 [3], and it is well accepted that a heterozygous TP53 germline mutation is a first and important step towards tumorigenesis, molecular mechanisms triggering the development of overt malignant tumors are complex.
RESULTS
CD117, CD15, CD13, and CD45) in the bone marrow, acute myeloid leukemia (AML) subtype FAB M4/M5 without central nervous system (CNS) involvement was diagnosed. Cytogenetic analyses showed a complex aberrant karyotype involving chromosomes 5, 7, 11, 13, 16, 17, and 22 (Fig. 1C,D) including a TP53 deletion consistent with secondary AML. Given the poor prognosis of secondary AML, unrelated single HLA C-locus mismatched peripheral blood stem cell transplantation (SCT) from a female donor was performed with a rather uneventful clinical course. At 5.6 years, she presented with severe transfusion-dependent hematuria due to a tumor in the left kidney without metastases. Total left nephrectomy was performed. Histologically, the tumor was classified as high-grade malignant nephroblastoma (SIOP stage I) with diffuse anaplasia (Wilms tumor, WT) (Fig. 1E). No neoadjuvant or adjuvant chemotherapy was administered after balancing the risk of reoccurrence of a biologically aggressive tumor versus the context of a germline TP53 mutation. At the age of 6.9 years, the child is in ongoing complete remission. Thus, our patient suffering from three aggressive malignancies so far has had a surprisingly good outcome. Additional Supporting Information may be found in the online version of this article at the publisher’s web-site. 1
Here we report on a female child with LFS, who developed three different malignancies: a choroid plexus carcinoma (CPC) at the age of 4 months, a secondary acute myeloid leukemia (AML) by 4 years, and a Wilms tumor at 5 years. The occurrence of three different tumors arising in a single child within an interval of 6 years is extremely rare and has been reported so far only once [10]. The 4-month-old female, the first child of healthy unrelated parents, presented with a tumor originating from the right lateral ventricle of the brain. Histological examination revealed a papillary and partially solid proliferation of pleomorphic epithelial cells and necrosis (Fig. 1A,B). The cells were positive for cytokeratin and vimentin consistent with a diagnosis of choroid plexus carcinoma (CPC) WHO grade 3. Due to suspected LFS, the tumor was treated without irradiation by chemotherapy. At the age of 4.2 years, the child developed progressive pancytopenia. Due to 62% myeloid blasts (positive for CD34 and CD33, partially positive for CD4,
C
2015 Wiley Periodicals, Inc. DOI 10.1002/pbc.25486 Published online 18 March 2015 in Wiley Online Library (wileyonlinelibrary.com).
Institute of Human Genetics, Hannover Medical School, Hannover, Germany; 2Institute of Pathology, Hannover Medical School, Hannover, Germany; 3Institute for Clinical Genetics, Medical Faculty Carl Gustav Carus, TU Dresden, Germany; 4Department of Pediatric Hematology and Oncology, University Hospital Dresden, Germany Grant sponsor: Cluster of Excellence Rebirth funded by the DFG (Rebirth 2, Units 9.3a and b); Grant sponsor: German Cancer Aid (German Consortium for Hereditary Breast and Ovarian Cancer) Conflict of interest: Nothing to declare. Brigitte Schlegelberger and Hans Kreipe contributed equally to this work. Correspondence to: Prof. Dr. med. Brigitte Schlegelberger, Institute of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail: [email protected]
Received 25 July 2014; Accepted 2 February 2015
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Fig. 1. Histopathological and cytogenetic findings. A: Plexus carcinoma with partially blurred papillary pattern and focal necrosis. B: Plexus carcinoma with area of solid proliferation, high nuclear pleomorphism, and more than 10 mitoses/10 high power fields. C: Karyogram of bone marrow AML cells, fluorescence R-banding. D: Karyogram of bone marrow AML cells, multicolor fluorescence in situ hybridization (mFISH) analysis to further identify the origin of the complex chromosomal aberrations. The unknown material in the long arm of chromosome 11 could be identified as derived from chromosome 22, in the long arm of chromosome 13 from chromosomes 11 and 22 and in the long arm of chromosome 22 from chromosome 11. There is an unbalanced translocation between chromosomes 5 and 17. In addition, we detected a low-level amplification of the MLL locus. The karyotype was finally described as: 4044,XX,der(5)t(5;17)(q11;q11),der(11)qdp(11)(q14q24)t(11;22)(q24;q12), ins(13;11) (q31;q12q24),-16,-17,der(22)t(11;22)(?;p13)[21]. nuc ish 11q23(MLLx4-8)[66/100,]17p13.1(P53 1)[72/100]. E: Nephroblastoma with marked nuclear enlargement and hyperchromasia, proliferation of pleomorphic spindle cells with a more sarcomatous rather than blastemal pattern and diffuse anaplasia. Foci of epithelial cells were observed, forming solid nests or tubular structures. Epithelial cells and spindle cells were immunohistochemically positive for vimentin and intensely positive for TP53; spindle cells revealed focal desmin expression and were negative for WT1.
Pediatr Blood Cancer DOI 10.1002/pbc
Inactivation of TP53 in Li–Fraumeni Syndrome
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Fig. 2. Inactivation of TP53 in the germline and in the three different tumors. A: Sequence analysis of TP53, exon 8. Electropherograms showing nucleotides c.811 to c.828 of TP53, exon 8 (ENST00000269305). Sequences obtained are displayed in accordance with the IUPAC code. The nucleotides c.814 and c.818 are highlighted by a circle and square respectively (black, wild-type; red, mutant). Sequencing results obtained from the analyses of DNA from a peripheral blood sample (i) at the time of secondary AML and a fibroblast culture (ii). In both samples, the heterozygous mutation c.818G>A; p.(Arg273His) was detected. Besides the known germline mutation analysis of Wilms tumor, DNA additionally displayed the heterozygous missense mutation (c.814G>A; p.(Val272Met). Subsequent investigations following TOPO TA cloning of the PCR product of the Wilms tumor DNA proved that the acquired mutation affects the second TP53 allele. Representative sequencing results of individual bacterial clones display the germline mutation c.818G>A (iii) and the somatic mutation c.814 (iv) in trans. B: Schematic presentation of biallelic inactivation of TP53 in the three different tumors. The heterozygous germline mutation c.818G>A (red circle) was detected in our patient. As a result of loss of the wild-type allele (green circle) and a duplication of the mutant allele due to copy-neutral LOH/uniparental disomy, the known germline mutation was detected in a homozygous state in the choroid plexus carcinoma. In the secondary AML showing a complex karyotype, FISH displayed a loss of TP53 in 72% of the analyzed nuclei, which most probably led to a loss of the wild-type allele. Identification of somatically acquired mutation c.814G>A (violet circle) leading to compound heterozygosity in the Wilms tumor.
Due to the presence of multiple tumors belonging to the LFS tumor spectrum, diagnosed in early childhood, the patient reported here fulfils the Chompret criteria II and III [8]. Indeed, in germline DNA isolated from fibroblasts, sequence analysis of exons 1–11 of TP53 (ENST00000269305) showed the missense mutation c.818G>A; p.(Arg273His) (rs28934576) as described according Pediatr Blood Cancer DOI 10.1002/pbc
to the Human Genome Variation Society (www.hgvs.org) (for details see Supplemental Table SI). According to the IARC (International Agency for Research on Cancer) database (http:// p53.iarc.fr/TP53), this missense mutation is located within the structural motif L1/S/H2 affecting DNA binding and is classified as a deleterious mutation. This mutation has already been described as
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a germline mutation in patients with Li–Fraumeni syndrome [11]. Sequencing of TP53 showed normal results in both parents as well as in the younger sister of the index patient. Therefore, we concluded that the germline mutation occurred de novo. The heterozygous mutation was present in leukemic blasts and in peripheral blood at the timepoint of hematological remission as well as in fibroblasts (Fig. 2Ai and ii). The CPC tumor cells are homozygous for TP53 c.818G>A. One allele of TP53 is lost in leukemic blasts. The malignant nephroblastoma is compound heterozygous for TP53 c.818G>A and c.814G>A (ts-sensitive, loss-of-function) (Fig. 2Aiii and iv). Morphologically inconspicuous non-neoplastic kidney tissue isolated by manual microdissection is heterozygous for TP53 c.818G>A, while c.814G>A was not detected. FISH analysis on both solid tumor specimens revealed no abnormalities of chromosome 17p.
DISCUSSION TP53 is one of the key factors responsible for the maintenance of genetic integrity. After cellular stress or DNA damage, TP53 can trigger G1 arrest, senescence or apoptosis. Concerning its role as “guardian of the genome” [12], it is intriguing that monoallelic inactivation of TP53 by germline mutations leads to an increased cancer risk in LFS. The missense mutation c.818G>A detected in our patient is located within the DNA binding domain of TP53, that is, the L1/S/H2 motif. It is known as a contact mutation, preventing wild-type transcriptional activity, itself leading to an inactivation of the second allele, and thereby confers gain-of-function properties with a more aggressive phenotype than mutations resulting in loss of TP53 expression [13–15]. It shows a dominant-negative effect due to the formation of heterotetramers consisting of mutant and wild-type TP53. Furthermore, it leads to aggregation of wild-type TP53 into cellular inclusions and also to co-aggregation with the TP53 family members p63 and p73. These findings are in line with the early onset of cancer in our patient and a previous report showing that the mean age of onset of primary cancer is 9 years earlier in LFS due to missense mutations in TP53 compared to LFS due to TP53 deletions [16]. Recent whole exome sequencing in tumors with somatic TP53 mutations showed a surprising paucity of mutations in other genes [17]. However, genome sequencing of pediatric medulloblastoma linked catastrophic DNA rearrangements to TP53 mutations [18]. We did not have the chance to perform whole exome sequencing in the three tumors of our patient. However, the three different modes of inactivation in each of the three malignancies (Fig. 2B) indicate an increased genomic instability. In the choroid plexus carcinoma, the homozygous mutation without a deletion can be explained by uniparental disomy or copy-neutral LOH due to loss of the normal and duplication of the mutant allele. In the AML, the complex karyotype clearly demonstrates the
Pediatr Blood Cancer DOI 10.1002/pbc
increased genomic instability resulting in, amongst other changes, the loss of TP53 due to a deletion of 17p. In the Wilms tumor, the somatic TP53 mutation Val272Met was detected in trans to the germline mutation Arg273His, thus leading to compound heterozygosity. These findings underline that a complete inactivation of TP53 by uniparental disomy, compound heterozygosity or deletion may be the decisive step towards tumorigenesis.
ACKNOWLEDGMENTS The authors wish to express their gratitude to Norbert Graf, MD (Homburg, Germany) for helpful discussions and advice concerning the clinical management of the Wilms tumor.
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