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Frequency of the TP53 p.R337H mutation in a Brazilian cohort of pediatric patients with solid tumors
José Antonio da Silva Feitosa1 . Pablo Ferreira das Chagas2 . Graziella Ribeiro de Sousa2 . Rosane Gomes de Paula Queiroz1 . Gustavo Alencastro Veiga Cruzeiro1,3 . Luiz Gonzaga Tone1,2 . Kleiton Silva Borges1,4,5,6 (D . Elvis Terci Valera1
Received: 13 April 2020 / Accepted: 8 July 2020 Springer Nature B.V. 2020
Abstract
TP53 p.R337H germline mutation is highly prevalent in the Southern region of Brazil. We sought to investigate TP53 p.R337H mutation in pediatric tumor samples from a population settled in a geographic area of high prevalence for this variant. Mutation assessment and genetic counseling for carriers/relatives were provided. 6/57 tumor samples were het- erozygous for TP53 p.R337H. As expected, a high frequency was observed within adrenocortical tumors (3/3) and choroid plexus carcinomas (2/2). Interestingly, the TP53 R337H mutation was found in one case of pediatric rhabdomyosarcoma with Li-Fraumeni pedigree. Our finding expands the spectrum of childhood cancer associated with this germline mutation.
Keywords Pediatric cancer · Li-Fraumeni syndrome · Tumor genetics · Rhabdomyosarcoma
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11033-020-05655-5) contains supplementary material, which is available to authorized users.
José Antonio da Silva Feitosa and Pablo Ferreira das Chagas are sharing co-first authorship.
Kleiton Silva Borges and Elvis Terci Valera have contributed equally to this work.
☒ Kleiton Silva Borges kleiton.silvaborges@childrens.harvard.edu
1 Department of Pediatrics, University of São Paulo, Ribeirão Preto, Brasil
2 Department of Genetics Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brasil
3 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, USA
4 Division of Endocrinology, Boston Children’s Hospital, Boston, USA
5 Department of Pediatrics, Harvard Medical School, Boston, USA
6 Center for Life Sciences, 16030.17, 3 Blackfan Circle, Boston, MA 02115, USA
Introduction
Childhood cancer is the second most common cause of death in children aged 1-14 years in the United States of Amer- ica [1]. Germline pathogenic variants are strongly associ- ated with a greater susceptibility to develop tumors in the pediatric age group; among these disorders the Li-Fraumeni syndrome (LFS) stands out [2, 3]. The LFS is characterized as a rare hereditary disease of an autosomal dominant trait. Diagnosis is set through the confirmation of germline muta- tions of the tumor suppressor gene TP53 located on chromo- some 17 [3-6]. Tumors associated with LFS include breast cancer [7], brain tumors [8], leukemia [9] and adrenocortical carcinoma [10] among others.
Pathogenic variations of the TP53 gene are often mis- sense, with nonsense mutations or those that originate a truncated protein more rarely observed [3, 11]. The TP53 p.R337H is a pathogenic variant with incomplete clinical penetrance that alters the tetramer of the p53 protein and is highly prevalent in the Southeast and South of Brazil [12-14]. Several pediatric tumors are associated with this mutation, particularly Adrenocortical Tumors (ACT) and choroid plexus carcinomas (CPC) [15, 16]. However, the extent to which the p.R337H variant correlates with other pediatric tumors remains to be determined. This report aimed to evaluate a cohort of several different pediatric
tumors for the presence of TP53 p.R337H, in a population settled at a high prevalence geographic area for this germline mutation in Brazil.
Materials and methods
Genomic DNA was extracted from frozen tumor specimens using a Dneasy Blood and Tissue kit (Qiagen, Valencia, CA, USA) of all 57 patients. Genomic DNA was also extracted from peripheral blood from four patients, according to stand- ard protocols. The entire exon 10 was amplified by PCR using the following intronic primers: 5’-GCTGTATAG GTACT-TGAAGTGCAG-3’ and 5’-GATGAGAATGGA ATCCTATG-3’ [17]. Analyzes of whole TP53 gene in sam- ples with p.R337H mutation also were done as described previously [18] (Primers sequence in the Supplementary Table 2). The PCR products were directly sequenced using the BigDye terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA) in an ABI PRISM 310 automatic sequencer (Perkin-Elmer Corp.). The sequence analyzes were performed using the Codon Code Aligner software and paired with a reference human genome.
Results
Case series
This was a retrospective, cross-sectional, descriptive and experimental study. Fifty-seven non-consecutive samples of pediatric patients (0-21 years) treated at the Ribeirão Preto Medical School, University of São Paulo (Pediatric Oncology Unit) between 2015 and 2019 were evaluated. Exclusion criteria included lack of cryopreserved tumor sample, poor DNA quality derived from tumor sample and absence of informed consent. Patient’s samples were col- lected, identified, cryopreserved and microdissected. Tumor
histologies were classified and assigned as brain (33.3%), bone and soft tissue tumors (21%), abdominal (22.8%) and mesenchymal tumors (5.2%), pediatric lymphomas (5.2%) as well as germ-cell lesions (5.2%) and other rare pediatric tumors (7.3%). Supplemental Table 1 describes all cases in clinical details. Patients/legal guardians granted written informed consent to use samples kept at tumor bioreposi- tory. This study was approved by Institutional Review Board (IRB) (CAAE:09821219.0.0000.5440).
Mutation finding on specimens
TP53 p.R337H mutation was found in six samples, namely: three cases of ACT (out of 3 adrenal tumors examined), two cases of CPC (in a total of 2 cases) and one case of naso- pharyngeal rhabdomyosarcoma (RMS) (Table 1). The three patients with ACT are alive with no evidence of disease (NED), one patient with CDC died and other is alive with disease (AWD) and the RMS patient is alive with NED. Four positive tumor samples for TP53 p.R337H mutation were confirmed germline, all with concordant results. All TP53 p.R337H mutations were heterozygous. Mutation evaluation and genetic counseling for carriers/relatives were provided following the modified version of the “Toronto Protocol” for LFS [3].
A novel LFS pedigree with TP53 p.R337H and pediatric RMS: index case and family cluster description
A 5-year-old female was referred due to a short-term com- plaint of throat pain and stridor, which rapidly progressed to an obstructive respiratory failure. At oroscopy, it was observed left hypoglossal nerve palsy, with tongue con- traction at the right side (Fig. 1a). Nasofibroscopic exam revealed a large obstructive nasopharyngeal mass extend- ing to the hypopharynx. She was submitted to an emer- gency tracheostomy procedure, followed by a biopsy of the
| Cancer type | Anatomical location | Gender | Age at diagnosis, years | Outcome | Treatment | TP53 mutation |
|---|---|---|---|---|---|---|
| Adrenocortical carcinoma | Adrenal | F | 2 | NED | Adrenalectomy | p.R337H |
| Adrenocortical carcinoma | Adrenal | F | 2 | NED | Adrenalectomy | p.R337H |
| Adrenocortical carcinoma | Adrenal | F | 3 | NED | Adrenalectomy | p.R337H |
| Choroid plexus carcinoma | Cerebral ventricle | M | 3 | DOD | Resection tumor followed by RT+CT | p.R337H |
| Choroid plexus carcinoma | Cerebral ventricle | F | 3 | AWD | Resection tumor followed by RT+CT | p.R337H |
| Embryonal rhabdomyosarcoma | Nasopharynx | F | 5 | NED | Partial tumor resection followed by RT+CT | p.R337H |
F female, M male, y years, RT radiotherapy, CT chemotherapy, DOD died of disease, AWD alive with disease, NED no evidence of disease, p.R337H arginine-to-histidine mutation at codon 337
A
B
C
D
TP53 R337H/WT
A
G
A
1 CC G
T G
G
G
C
G
1
G AG CAGC
C GAG
AT TGT TCC G
MERCEDESa
E
Unknown cancer 90y
Intestine cancer 60y
Pancreatic cancer 60y
Gastric cancer 50y
Sarcoma 58y
Prostate cancer 64y
Intestine cancer 60y
Breast cancer 48y
Legend
Liver cancer 56y
Breast cancer 37y
Breast cancer 31y
Affected by cancer
R337H
Death
? Unknown cancer
Neonatal cancer Newborn
Neonatal cancer Newborn
Embryonal rhabdomyosarcoma 5y
lesion. Magnetic resonance image (MRI) depicted a heter- ogeneous expansive formation centered at the nasopharynx and extending to the left parapharyngeal space; the lesion was hyperintense on T2- and hypointense on T1-weighted image acquisition and showed strong heterogeneous con- trast enhancement and marked restriction to diffusion (Fig. 1b, c). The tumor measured 7.0×4.6×3.1 cm and obliterated the air column of the rhino and nasopharynx. Pathological exam revealed an undifferentiated tumor composed of small-round cells. Immunohistochemistry study demonstrated positivity for smooth muscle actin, myosin and MyoD1; staining was negative for AE1AE3, leukocyte common antigen (LCA), protein S100, PAX5, myogenin and synaptophysin. The final diagnosis was of an embryonal rhabdomyosarcoma (ERMS) of nasopharynx with parameningeal extension. Image workups, as well as bone marrow aspirates were negative for metastatic dis- ease. The child was treated following the Brazilian Proto- col for Soft Tissue Sarcomas, which included upfront local radiotherapy, vincristine, dactinomycin, cyclophospha- mide, doxorubicin, ifosfamide and etoposide. The patient has recently ended treatment and remains in complete clinical remission for 18 months from diagnosis. TP53
heterozygous G to A transition at codon 337, corresponding to the p.R337H mutation. e Family pedigree of the index case: each affected family member is labeled by cancer diagnosis, age of diagnosis and clinical status (alive or dead). The arrow indicates the EMRS index case. Square =male; circle =female. The numbers following cancer sites indicate age at diagnosis
p.R337H mutation was detected both in blood and tumor samples (Fig. 1d).
Family had a significant history of tumors, many of them occurring in individuals before the age of 46. That included 2 pre-menopausal breast cancer cases and the index cases of ERMS. Also, second-degree family members aged from 47 to 60 were diagnosed with different malignant tumors from the liver, gastric, colon and pancreas. Figure 1 depicts the family pedigree in detail. The mother of the index case also tested positive for the mutation and she was referred to cancer prevention surveillance.
Discussion
The frequency of patients harboring TP53 p.R337H muta- tion in the general population of the South and Southeast of Brazil is estimated at 0.3% [19]. Although this specific TP53 variant is commonly referred to the Brazilian founder muta- tion, studies [5, 20] have occasionally reported individuals in other Countries harboring this mutation.
This investigation confirms the association of the TP53 p.R337H mutation with ACT in the pediatric
group [16] but also brings new insights into its occur- rence in other tumors. In children under 15 years of age, CPC is also strongly associated with germline mutations of TP53 p.R337H [20] in up to 63% of cases. In adults, TP53 p.R337H germline mutation is also associated with an increased risk for breast, prostate and gastric tumors [21]. Clinical penetrance for developing cancer for TP53 p.R337H carries varies and may be explained by second- ary genetic changes [22].
The most remarkable finding derived from this study was the identification of a 5-year-old female case diagnosed with parameningeal EMRS that harbors the TP53 p.R337H mutation. This family portrays a typical history of LFS. To reassure that this was the sole pathogenic TP53 variant pre- sent in this case, the complete sequencing of all 11 exons of the TP53 gene was carried out in constitutional DNA (total leukocytes) of the patient. We did not find any other patho- genic variants of the TP53 gene than the p.R337H mutation, reinforcing the involvement of this genetic variant in the LFS family phenotype. It is important to point out that somatic genomic testing is an integral part of care for patients with cancer since it may have an impact on managing and treating these patients. Moreover, germline testing is essential not only for optimal care of the patient with cancer but also to identify related at-risk family members who also may carry the familial mutation [23].
There are several clinical criteria utilized to guide the TP53 mutation detection during the screening of individuals and families prone to LFS. If only the classic LFS criteria had been applied to this case [24], it, as well as the other positive cases, would have been missed for TP53 mutation. Yet, by using Chompret’s criteria, a broad screening criteria [25], this family cluster would be eligible for TP53 mutation screening: index case with a tumor of LFS spectrum (sar- coma) before the age of 46, associated to a second-degree family member (aunt) with an LFS-related tumor (breast cancer at the age of 31). The cases of ACT and CPC, accord- ing to Chompret’s criteria, are isolated criteria for TP53 test- ing, regardless of family history of tumors. This is an impor- tant take-home message when evaluating families affected by multiple tumors through many generations.
The TP53 germline pathogenic variants outside R337 locus are frequently observed in RMS (particularly anaplas- tic subtype) [26] and may be directly related to the occur- rence of soft-tissue sarcomas in children with LFS. Yet, no reports on TP53 p.R337H germline mutations were found in pediatric RMS [27]. Although it is difficult to clearly infer the biological and clinical impact of the p.R337H germline mutation in this type of neoplasia, our finding strength- ens the possibility of this association for pediatric RMS, particularly when a family history for LFS or LFS-like is present, particularly for populations with Brazilian genetic background.
In summary, our study reaffirms the high frequency of the TP53 p.R337H mutation in pediatric ACT and CPC. Addi- tionally, a case of pediatric ERMS related to TP53 R337H was described for the first time in children. Larger cohorts and epidemiologic studies may provide a better understand- ing about the biological and clinical impact of the TP53 p.R337H mutation in pediatric sarcomas.
Author contribution E.T.V, J.A.S.F. and K.B.S. planned and conducted data analysis and drafted the manuscript. G.R.S. and P.F.C. wrote and organized the data, created the figures/tables, and edited and final- ized the manuscript. J.A.S.F, R.G.P.Q, G.A. V.C and L.G.T revised the text for important intellectual content. All authors critically read and approved the final manuscript.
Funding This collaborative study was supported by the Brazilian Pub- lic Research Agency Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP)-Grant 2014/20341-0, Conselho Nacional de Desen- volvimento Científico e Tecnológico (CNPq), Grant 457884/2014-2 and Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universi- dade de São Paulo (FAEPA).
Data availability All data generated from this case description are pre- sented in this article and supporting information material.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing interests.
Informed consent Parents gave written informed consent for genetic studies and for the publication of data (available upon request). This case report was also IRB approved by The Ethics Committee in Human Research at Ribeirão Preto General Hospital and Ribeirão Preto Medi- cal School (CAAE:09821219.0.0000.5440).
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