Li–Fraumeni syndrome in Tunisian carriers with different and rare tumor phenotype: genotype–phenotype correlation

Li–Fraumeni syndrome (LFS) is a rare autosomal hereditary predisposition to multiples cancers, mainly affecting young individuals. It is characterized by a broad tumor spectrum. To our best knowledge, only one Tunisian study with a confirmed LFS was published. Our study focused on the clinical, histopathological and genetic results of two patients with rare tumor phenotype and tried to establish genotype–phenotype correlation. The clinical diagnosis was based on Chompret-Bonaiti criteria relative to LFS. Molecular study was assessed using Sanger sequencing of the hotspot germline variants of TP53 gene. We report 2 Tunisian families fulfilling the clinical criteria of Chompret-Bonaiti. The tumor phenotype was bilateral breast cancer (BC) in 27-year-old woman and multiple tumors for the second proband, with an onset age of 14, 35 and 36 yo for osteosarcoma, BC and esophageal cancer respectively. Each of them had a rare histological type of breast cancer associated with LFS, phyllode tumor and intralobular carcinoma. Both patients had cancer family history. The molecular study showed deleterious heterozygous germline TP53 variants in each index case: The first had a well-known hotspot missense variation c.742C>T p.(R248W) with a rare histological association, explaining genotype phenotype correlation. The second case had a nonsense variation c.159G>A p.(W53*), rare worldwide, extending the phenotype spectrum in LFS. Immunohistochemistry study in tumor samples confirmed the lack of p53 protein expression. Conclusively, germline TP53 testing is primordial in patients with a family history suggestive of LFS for clinical practice avoiding genotoxic treatments and adapting the surveillance. National database in LFS listing clinical and mutational data is important to set, particularly for variants rarely reported worldwide. Experience from different countries must be integrated to harmonize global protocols for cancer surveillance in LFS.

Sassi et al. BMC Medical Genomics (2022) 15:44 screening guidelines are continuously updated. It's characterized by high phenotypic heterogeneity in onset age of cancers and a genetic predisposition to broad tumor spectrum comprising bone and soft-tissue sarcoma, brain tumors, premenopausal breast cancers (BC), adrenocortical tumors, plexus choroid tumors, leukemias and lung cancers [1][2][3][4]. LFS results mainly from heterozygous germline variant in TP53 tumor suppressor gene (OMIM# 191170), with a detection rate varying from 18 to 35% of families suggestive of LFS [5][6][7][8]. TP53 encodes for the transcription factor p53, known as the "guardian of the genome" because of its role in the preservation of DNA integrity. It is implicated in the cell cycle arrest, DNA repair, genomic stability, senescence, cell differentiation, autophagy, angiogenesis and apoptosis [9,10]. To our best knowledge, one Tunisian family has been published with a confirmed LFS [11]. The aim of our study is to present the clinical and genetic particularities of LFS through two confirmed Tunisian families and set a genotype-phenotype correlation.

Clinical data
The radiation oncology department referred two proband patients from two different families to the oncogenetic department for clinical suspicion of LFS. For each family, we applied the Chompret-Bonaiti revised criteria elaborated by the French LFS working group and the 2015 updated version [5,12] and congregated all cancers with its histopathological diagnosis reported in an expanded pedigree.

Clinical data Family 1 (F1)
The index case was diagnosed with a bilateral BC at the age of 27 years old (yo). The Patey's radical left mastectomy showed a grade I phyllode tumor associated with an invasive ductal carcinoma of 0.3 cm long axis, SBR grade II with negative axillary lymph nodes. Ductal in situ carcinoma was found in the conservative right breast surgery. Immunohistochemistry studies on FFPE (formalin-fixed paraffin-embedded) tissue sections showed HER2 molecular subtype and proliferation index Ki67 at 30% bilaterally. She had radiation therapy combined with chemotherapy and trastuzumab as adjuvant treatment.
The genetic survey, one year later, revealed that the proband (III.13) had a familial history of tumors belonging to LFS tumor spectrum (Fig. 1A).

Family 2 (F2)
The proband had multiple primary tumors, two of which belong to LFS tumor spectrum and first of which occurred before age 46 yo (Fig. 1B). She was followed since the age of 14 yo for an osteosarcoma of the right lower limb for which she underwent surgery followed by adjuvant radiochemotherapy. Lobular carcinoma in the right breast was diagnosed at the age of 35. Immunohistochemical study showed loss of E-cadherin expression in neoplastic cells and positive expression of hormonal receptors ( Fig. 2A). She was treated by neoadjuvant chemotherapy followed by radical mastectomy and adjuvant radiochemotherapy. Postoperative follow-up revealed 9 months later an esophageal differentiated epidermoid carcinoma with a circumferential margin less than 1 mm, for which she underwent subtotal esophagectomy.
Family history found besides multiple tumors in proband, two relatives with malignant BC (Fig. 1B).

Genetic analysis and confrontation to histological data
DNA sequencing in F1 index case revealed heterozygous missense germline variant NM_000546.5: c.742C>T replacing an arginine for a tryptophan at codon 248 p.(R248W) (Fig. 2B). Her relatives (III.33 and IV.6) carried the same variant.

Discussion
In F2, the deleterious germline variant p.(W53*) affects the N-terminal transactivation domain. This nonsense variation was not referenced in online IARC TP53 database as germline variant. It was only reported in one child with adrenocortical carcinoma [5] while our patient F2 had multiple tumors, osteosarcoma, lobular breast carcinoma and esophageal epidermoid carcinoma expanding the clinical phenotype. The p.(W53*) has been reported in somatic variations (N = 19) in less than 1% in each site, cited in decreasing order of frequency: vulva, head and neck, oropharynx, ovary, brain, colon and lung cancers (IARC database).
Multiple primary tumors (n = 2 to 6) were reported in 43% of cases (139/322) and were metachronous (83%, 116/139) with a mean gap between the first and the second tumor of 10,7 years (2-26) after radiotherapy [5]. The role of p53 in response to DNA damage could explain the high risk of radio and chemo-induced tumor [9,10]. The LFS was confirmed late after the third cancer in F2 index case. The esophageal epidermoid carcinoma is probably radio induced. We underline the interest of addressing patients to oncogenetic consultation as soon as possible, to confirm the LFS early. An appropriate genetic counselling to these families could be given with regular monitoring according to recent recommendations among genetic carriers and personalized care in case of cancers by avoiding genotoxic radiotherapy and chemotherapy [25].
It is insufficient to set a genotype phenotype correlation in the germline variant p.(W53*), since it was only reported in two cases. So the functional impact of these variants would be helpful. The p.(R248W) has a strong dominant negative (DNE) property and a drastically altered transcriptional activity of 0,09% showing a nonfunctional impact (PHANTM classifier) which could explain the phenotype severity [26,27]. Furthermore, this variant is one of the major gain-of-function (GOF) variant suggested by mouse models of LFS with interaction of GOF mutants p53 with multiple proteins, such as the nuclease Mre1 leading to inhibit the interaction of MRN complex to DNA double-stranded breaks and induce genetic instability by inactivating ATM protein, contributing to drive and promote tumorigenesis [28,29]. The p.(W53*) leads to a loss of function effect. Initially, DNE property was not related to the early onset of tumors but correlated the occurrence in type of cancer [26]. In fact, DNE-variants carriers in TP53 show twice more bone sarcomas and BC [26], which is consistent with F1. Carriers of DNE variant are reported to have severe clinical phenotype with an early onset tumor than those carrying loss of function variant (mean age: 21.3 vs. 27 years) with a statistically significant difference (p = 0.0042) [5]. This was not consistent with age of tumor onset in our patients, F1 at 27 yo with a DNE variant and F2 at 14 yo with loss of function variation. DNE variants are observed in 40% of children with osteosarcoma [5], while F2 proband had loss of function variation. Loss of function are known to have less severe phenotype with later tumor onset [26,27]. These previous arguments could not conclude about the clinical severity of p.(W53*) in F2 proband, but we underline and should be aware of the possibility of childhood cancer in this case, ACC already reported and osteosarcoma in F2 [5].
The penetrance in germline TP53 variants was variable regarding the variation type and the sex. The highest Table 1 Germline TP53 variants associated with phyllode tumors or lobular carcinoma reported in literature   [30]. Penetrance is higher in men who tend to develop more tumors especially brain neoplasia and sarcomas before age 30 [30]. In F1, six members were diagnosed with brain tumors including three men, the youngest of which was a three-year-old boy. Contrasting with p.(W53*) which seems to have lower penetrance. The penetrance may be explained by the loss of transcriptional activity [26]. Thus, in routine practice, gradient of severity could be established depending on the type of variant, its functional classification, its penetrance and the early onset of tumors. These data are reliable to set the surveillance protocols. In fact, due to the severity of DNE variants, presymptomatic testing and annual total body magnetic resonance imaging are mandatory and recommended since childhood (National Comprehensive Cancer Network). Contrasting with lower severity variants, frequent in adulthood, where presymptomatic testing are reserved to adults and justify annual breast MRI from the age of 20 years (National Comprehensive Cancer Network). Our data is an example of the exception to the rule, to be taken into account with families harboring p.(W53*), predisposing to early bone cancer and ACC, for whom we propose presymptomatic testing to children and whole body MRI. The first limitation of this study was that we have tested only the proband in family F2 and should investigate the affected members. The second limitation is that we only did a germline sequencing of probands. Reviewing the literature about tumor sequencing in LFS found only some case reports. Sugawara et al. reported a LFS case with p.(R273H) variant associated with an aggressive phenotype, two rhabdomyosarcomas at the ages of 18 months and 21 years [31]. An arraybased analysis revealed in the second tumor an amplicon at 5q11.2 including MAP3K1 gene and the second one at 11q22.2 containing genes: YAP1 and BIRC2/3, known for its anti-apoptosis function, allowing a comprehensive view of cancer progression [31]. An Italian study tried to find genetic modifiers that accelerate tumor development in LFS families other than MDM2 promoter polymorphisms SNP309T>G (rs2279744) or shortness of telomere length by performing whole-exome analysis (WES) of the trio [32,33]. The Italian proband developed at 4 year-old, an adrenocortical carcinoma. His mother had an in-situ carcinoma of the right breast at the age of 37 and his father was healthy. He inherited from his mother TP53: c.266_269del, p.(S90fs) related to LFS and nonsense variation c.1720C>T, p.(R574*) in ERCC3 from his father, a potential candidate modifier gene that encodes an ATP-dependent DNA helicase and could explain the earlier onset age [32]. Zureick et al. reported a 14-year-old boy with a rare entity of glioblastoma, a gigantocellular-type glioblastoma multiforme, who carried a germline deletion in exons 1 and 2 of TP53 gene [34]. The family history was made of leiomyosarcoma among his father and grandfather. The proband's tumoral DNA WES and RNA sequencing showed a homozygous deletion of PAK1, a variant p.(R611W) in TSC2 gene, copy gain of chromosomes 1, 7, 9, 18 and X and copy loss of chromosomes 19 and 22. They highlighted the clinical benefit of precision medicine with a maintain of complete remission after treatment with everolimus during 25 months [34]. These cases provide an insight into targetable pathways that may be involved in LFS cases that we need to study with multicentric projects.

Conclusions
Tunisian patients adhere to the dichotomous presentation of LFS tumor spectrum with mainly female BC in adults and osteosarcoma in childhood and early adulthood. Our patients showed degrees of phenotypic severity with distinctive types of variants, confirming the heterogeneity of LFS in Tunisian patients. The Tunisian experience allowed us to extend the phenotype spectrum of LFS. After fifty years of the identification of LFS, we are continuously changing and adapting the personalized care of germline TP53 variant's carriers regarding the surveillance protocols of cancers, stratified according to the type of variants.