Li-Fraumeni Syndrome Patients Research Articles

Pediatric Head and Neck Tumors Associated with Li-Fraumeni Syndrome.

Cancer predisposition syndromes are germline pathogenic variants in genes that greatly raise the risk of developing neoplastic diseases. One of the most well-known is Li-Fraumeni syndrome (LFS), which is due to pathogenic variants in the TP53 gene. Children with LFS have higher risks for multiple malignancies before adulthood, often with rare and aggressive subtypes. To examine head and neck manifestations of LFS in children treated at a tertiary children's hospital over a 20-year period. A retrospective review of LFS children with neoplastic disease presenting in traditional Otolaryngologic head and neck subsites from 2000 to 2019, with patient charts reviewed for relevant clinical, imaging, and operative data. Of the 40 LFS patients initially identified, 27 neoplastic tumors were identified in 20 children within this cohort (20 primary, 7 second primary). Head and neck subsites aside from the brain or orbit were involved in 22% (6/27) of these tumors, representing 20% (4/20) of primary tumors and 29% (2/7) of second primary tumors. Both second primaries within the head and neck were within the radiation fields of the first primary tumor. The mean ages at primary and second primary diagnosis were 4.6 years (SD 3.5) and 12 years (SD 1.4), respectively. The male/female ratio was 1:6 among all patients with head and neck tumors. All 6 head and neck tumors were sarcomas. Rhabdomyosarcoma (N = 3, 50%) was the most common pathology, and the other 3 demonstrated rare tumor pathological subtypes (synovial cell sarcoma, pleomorphic myxoid liposarcoma, mandibular osteosarcoma). The neck was the most common subsite (75%) within this group for primary tumor presentation. This study identifies a high potential for head and neck involvement in children with LFS, which has not been previously described in the literature. Otolaryngological care should be included in a multidisciplinary care team surveilling these patients.

22. Masked hypodiploid B-Cell Lymphoblastic Leukemia (B-ALL) characterized by SNP microarray in a Li Fraumeni syndrome patient with post therapy karyotype changes

Cases of B-ALL with chromosome counts of >60 and tetrasomic for chromosomes 6, 18, 21 and X are suspicious for “masked hypodiploidy” in that the theoretic hypodiploid stemline is present as a doubled clone only. To characterize and define these cases when FISH and chromosomes show no evidence of the hypodiploid clone, our laboratory utilizes the IlluminaⓇ Cyto-SNP 850K microarray to recognize the loss of heterozygosity (LOH) of the disomic chromosomes. We report an 18yo female with a known diagnosis of Li Fraumeni syndrome, referred to Cytogenetics with pancytopenia and circulating blasts. Cytogenetic results at diagnosis revealed three hyperdiploid clones with counts of 62-64, tetrasomy for X, 1, 6, 18, and 21, with additional trisomies and structural rearrangements including a derivative 17p with 11q. No evidence for monosomy was observed by FISH or chromosomes. Microarray results confirmed loss of heterozygosity (LOH) for those chromosomes present in 2 copies. Genes associated with low hypodiploid B-ALL include TP53, IKZF1 and RB1 which were present in 2 copies on the isodisomic chromosomes in this specimen. This patient was in remission for several months, but with reemergence of B-ALL, underwent CD19 directed CAR-T therapy which resulted in deep remission (by NGS-MRD). Recent surveillance studies showed a karyotype change to loss of 5q and TP53 by FISH and a dic(5;17)(q11.2;p11.2) consistent with MDS. Microarray studies confirm the MDS genetic findings and were negative for the masked hypodiploid findings present at diagnosis. This strategy has been useful to report chromosome results with confidence for cases of masked hypodiploidy that may have been otherwise misinterpreted.

Refractory alveolar rhabdomyosarcoma in an 11-year-old male.

Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li–Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.

Genetic and stochastic influences upon tumor formation and tumor types in Li-Fraumeni mouse models.

p53 is the most frequently mutated gene in human cancers. Li-Fraumeni syndrome patients inheriting heterozygous p53 mutations often have a much-increased risk to develop cancer(s) at early ages. Recent studies suggest that some individuals inherited p53 mutations do not have the early onset or high frequency of cancers. These observations suggest that other genetic, environmental, immunological, epigenetic, or stochastic factors modify the penetrance of the cancerous mutant Tp53 phenotype. To test this possibility, this study explored dominant genetic modifiers of Tp53 mutations in heterozygous mice with different genetic backgrounds. Both genetic and stochastic effects upon tumor formation were observed in these mice. The genetic background of mice carrying Tp53 mutations has a strong influence upon the tissue type of the tumor produced and the number of tumors formed in a single mouse. The onset age of a tumor is correlated with the tissue type of that tumor, although identical tumor tissue types can occur at very different ages. These observations help to explain the great diversity of cancers in different Li-Fraumeni patients over lifetimes.

Abstract B15: Investigating the contribution of mTORC1-dependent stromal signaling to cancer onset in Li-Fraumeni syndrome

Abstract In this study, we are investigating the role of stromal mTORC1 signaling in the development of malignancy in individuals with Li-Fraumeni syndrome (LFS). LFS is a cancer predisposition syndrome that is associated with germline mutations in TP53. Individuals with LFS are faced with a significant lifetime risk of developing a wide spectrum of early-onset malignancies, thus necessitating the identification of pharmacologic agents to prevent tumor onset. Inhibitors of the mTORC1 pathway, namely rapamycin and metformin, have been shown to promote lifespan extension and delay or even prevent tumor onset in LFS mouse models. These findings are unsurprising—since wild-type p53 inhibits mTORC1—yet noteworthy, since they suggest that mTORC1 signaling plays a central role in favoring tumorigenesis in LFS. mTORC1 signaling has been implicated in promoting the protumorigenic senescence-associated secretory phenotype (SASP). The SASP constitutes the release of soluble factors, such as cytokines, growth factors, and proteases from senescent cells into the microenvironment, thus promoting oncogenesis in a paracrine manner. Interestingly, an increased incidence of senescence has been reported in LFS patients, and mutant p53 has been shown to promote the secretion of a quantitatively and qualitatively more protumorigenic SASP. As such, we believe that mTORC1 signaling in LFS stromal cells favors the production and secretion of protumorigenic factors, which may induce changes in the surrounding tissue to promote development of a precancerous niche, thus favoring tumorigenesis. Our objectives are 1) to investigate the contribution of stromal cells and stromal secreted factors in favoring tumorigenesis in LFS, and 2) to investigate the role of mTORC1 signaling in favoring these altered stromal phenotypes. To address these objectives, we co-injected patient-derived LFS fibroblasts with tumor cells of a common LFS subtype (rhabdomyosarcoma [RMS]) into immunocompromised mice and observed that tumors formed from the co-injection of RMS cells with LFS fibroblasts grew to be larger than tumors formed from co-injection with non-LFS fibroblasts. To determine whether LFS fibroblasts secrete more protumorigenic cytokines than non-LFS fibroblasts, we performed protein arrays with conditioned media (CM) from fibroblast cultures and saw that LFS fibroblasts secrete markedly higher levels of protumorigenic, SASP-related cytokines. To investigate the protumorigenic effects of fibroblast secreted factors, we treated spheroids formed from tumor cells of a common LFS subtype (osteosarcoma) with CM from LFS and non-LFS fibroblast cultures. LFS fibroblast CM had a robust proproliferative effect on spheroids, while non-LFS fibroblast CM inhibited spheroid growth. Thus far, we have observed that LFS stromal constituents favor tumor cell proliferation in vitro and in vivo, and based on the current literature, we believe that mTORC1 signaling plays a role in favoring these processes. Citation Format: Camilla M. Giovino, Nish Patel, Sangeetha Paramathas, David Malkin, Ran Kafri. Investigating the contribution of mTORC1-dependent stromal signaling to cancer onset in Li-Fraumeni syndrome [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B15.

Radiation therapy and secondary malignancy in Li-Fraumeni syndrome: A hereditary cancer registry study.

BackgroundLi‐Fraumeni Syndrome (LFS) is a rare cancer‐predisposing condition caused by germline mutations in TP53. Conventional wisdom and prior work has implied an increased risk of secondary malignancy in LFS patients treated with radiation therapy (RT); however, this risk is not well‐characterized. Here we describe the risk of subsequent malignancy and cancer‐related death in LFS patients after undergoing RT for a first or second primary cancer.MethodsWe reviewed a multi‐institutional hereditary cancer registry of patients with germline TP53 mutations who were treated from 2004 to 2017. We assessed the rate of subsequent malignancy and death in the patients who received RT (RT group) as part of their cancer treatment compared to those who did not (non‐RT group).ResultsForty patients with LFS were identified and 14 received RT with curative intent as part of their cancer treatment. The median time to follow‐up after RT was 4.5 years. Fifty percent (7/14) of patients in the curative‐intent group developed a subsequent malignancy (median time 3.5 years) compared to 46% of patients in the non‐RT group (median time 5.0 years). Four of seven subsequent malignancies occurred within a prior radiation field and all shared histology with the primary cancer suggesting recurrence rather than new malignancy.ConclusionWe found that four of14 patients treated with RT developed in‐field malignancies. All had the same histology as the primary suggesting local recurrences rather than RT‐induced malignancies. We recommend that RT should be considered as part of the treatment algorithm when clinically indicated and after multidisciplinary discussion.

The function of a heterozygous p53 mutation in a Li-Fraumeni syndrome patient.

p53 is one of the most extensively studied proteins in cancer research. Mutations in p53 generally abolish normal p53 function, and some mutants can gain new oncogenic functions. However, the mechanisms underlying p53 mutation-driven cancer remains to be elucidated. Our study investigated the function of a heterozygous p53 mutation (p.Asn268Glufs*4) in a Li-Fraumeni syndrome (LFS) patient. We used episomal technology to perform somatic reprogramming, and used molecular and cell biology methods to determine the p53 mutation levels in patient-originated induced pluripotent stem (iPS) cells at the RNA and protein levels. We found that p53 protein expression was not increased in this patient's somatic cells compared with those of a healthy control. p53 mutation facilitates the proliferation of tumor cells by inhibiting apoptosis and promoting cell division. It can inhibit the efficiency of somatic reprogramming by inhibiting OCT4 expression during reprogramming stage. Moreover, not all p53 mutant iPS cell lines have mutant p53 RNA sequences. A small percentage of mutant p53 mRNA is present in the somatic cells from the patient and his mother. In summary, this p53 mutation can promote tumor cell proliferation, inhibit somatic reprogramming, and exhibit random p53 allelic expression of heterozygous mutations in the patient and iPS cells which may be one of the reasons why the people with p53 mutations develop cancer at random. This finding suggested that mutant p53 allelic expression should be added to the risk forecasting of cancer.

Frequency of radiation-induced malignancies post-adjuvant radiotherapy for breast cancer in patients with Li-Fraumeni syndrome.

Women with Li-Fraumeni syndrome (LFS), a cancer predisposition syndrome caused by germline mutations in TP53, have an over 50% risk of developing breast cancer by age 70. Patients with LFS are at risk for radiation-induced malignancies; however, only small case series have prior investigated radiation risks in the treatment of breast cancer. We therefore aimed to investigate the risk of malignancy in breast cancer patients with LFS following adjuvant radiotherapy. A single-institution retrospective chart review was conducted for female breast cancer patients with confirmed germline TP53 mutation. The frequency of radiation-induced malignancies in LFS patients was compared to non-LFS breast cancer cases reported in the Penn Medicine Cancer Registry via statistical analyses. We identified 51 female LFS breast cancer patients with 74 primary diagnoses. Fifty-seven% had a history of breast cancer only, and 25% had breast cancer as their presenting diagnosis of LFS. LFS-associated breast cancers were predominantly invasive ductal carcinoma (48%) and HER2+ (58%). Twenty patients underwent adjuvant radiotherapy with a median follow-up of 12.5 (2-20) years. Of 18 patients who received radiation in a curative setting, one (6%) patient developed thyroid cancer, and one (6%) patient developed sarcoma in the radiation field. This risk for radiation-induced malignancy associated with LFS was higher for both sarcoma and thyroid cancer in comparison with thecontrol cohort. We found a lower risk of radiation-induced secondary malignancies in LFS breast cancer patients than previously reported in the literature (33% risk of radiation-induced sarcoma). These findings suggest that LFS may not be an absolute contraindication for radiotherapy in breast cancer. The potential risk for locoregional recurrence without radiotherapy must be weighed against the long-term risk for radiation-induced malignancies in consideration of adjuvant radiotherapy for LFS breast cancer patients.

Abstract P4-12-33: Frequency of radiation-induced malignancies post-adjuvant radiotherapy for breast cancer in patients with Li-Fraumeni syndrome

Abstract Background. Women with Li-Fraumeni syndrome (LFS), a cancer predisposition syndrome caused by germline mutations in TP53, have an over 50% risk of developing breast cancer (BC) by age 70. Providers often avoid adjuvant radiotherapy to treat BC in LFS patients due to a reported high risk of radiation-induced malignancies of over 30%. We aimed to investigate the characteristics of LFS-associated BC and the risk of subsequent malignancy in BC patients with LFS following adjuvant radiotherapy. Methods. A single institution retrospective chart review was conducted for female BC patients with a confirmed germline TP53 mutation. Statistical analyses were performed to compare the frequency of radiation-induced malignancies in LFS patients to non-LFS BC cases reported in the Penn Medicine Cancer Registry (PMCR) (n=6607 patients total, 3863 who received radiation). Results. Among 95 patients with LFS, we identified 51 female BC patients with 74 primary BC diagnoses. Of 51 patients, 57% had a history of BC only, and 25% had BC as their presenting diagnosis of LFS. LFS-associated BCs were predominantly invasive ductal carcinoma (48%) and HER2+ (58%). We analyzed 20 LFS BC patients who underwent adjuvant radiotherapy with an average follow up of 11.1 (2-20) years. Of 18 patients who received radiation in a curative setting, one (6%) patient developed thyroid cancer and one (6%) patient developed sarcoma in the radiation field. The incidence of thyroid cancer did not significantly differ between LFS-associated and non-LFS BCs. The incidence of radiation-induced sarcoma in patients with LFS is significantly higher than in non-LFS BC patients (0.03%, p=0.01). Conclusion. We found a 6% (one in 18 patients) risk of radiation-induced sarcoma in LFS BC patients, lower than the previously reported rate of 33%. Adjuvant radiotherapy should be considered in LFS BC patients when the potential risk for locoregional recurrence (LRR) or the mortality benefit to radiation is greater than 6%. Citation Format: Anh N Le, Jacquelyn Powers, Kristin Zelley, Angela Bradbury, Payal Shah, Gary Freedman, Katherine Nathanson, Susan M Domchek, Suzanne P MacFarland, Kara N Maxwell. Frequency of radiation-induced malignancies post-adjuvant radiotherapy for breast cancer in patients with Li-Fraumeni syndrome [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-12-33.

Challenges of screening germline predispositions in children

Genetic predisposition is a major cause of childhood cancer. Multiple cancer-predisposing syndromes have been identified, including Li-Fraumeni syndrome (LFS), neurofibromatosis type 1, APC-related adenomatous polyposis, Beckwith-Wiedemann syndrome, multiple endocrine neoplasia 1, ataxia telangiectasia, RUNX1 deficiency, Fanconi anemia, Bloom syndrome, and PTEN hamartoma tumor syndrome. LFS is a prototypical genetically predisposing condition. Accordingly, individualized therapy, surveillance, risk reduction, and family counseling are needed when a patient is diagnosed with LFS. More ethically important problems are encountered in a pediatric LFS patient, including the identification of patients requiring screening, the age at screening, the process of obtaining informed consent from children, and the responsibility of following a pediatric patient with a genetic predisposition. Therefore, it is crucial to determine whether planned genetic testing has direct benefits for pediatric patients. In this context, TP53 testing may be justified in a pediatric cancer patient with suspected LFS, given the importance of decisions such as the use of radiotherapy and the screening of family members as hematopoietic stem cell transplantation donors, the surveillance of subsequent cancers, and counseling for family members. In this review article, I have discussed these issues and indicated some consensus among various clinicians, including adult hematologists.

Radiotherapy-induced malignancies in breast cancer patients with TP53 pathogenic germline variants (Li-Fraumeni syndrome).

The risk of radiotherapy-induced malignancies (RIMs) is a concern when treating Li-Fraumeni syndrome (LFS) or Li-Fraumeni Like (LFL) patients. However, the type of TP53 pathogenic germline variant may possibly influence this risk. TP53 p.R337H mutation is particularly prevalent in Brazil. We aimed to evaluate the outcomes of patients with pathogenic TP53 variants treated for localized breast cancer in a Brazilian cohort. We evaluated retrospectively a cohort of patients with germline TP53 pathogenic variants treated for localized breast cancer between December 1999 and October 2017. All patients were followed by the Hereditary Cancer Group of an academic cancer center. Our primary objective was to evaluate the occurrence of RIMs after adjuvant radiotherapy. Sixteen patients were evaluated; 10 (62.5%) had a germline TP53 p.R337H pathogenic variant. Median age was 39.8years. Thirteen patients had invasive ductal carcinoma: 8 (61.5%) were hormone receptor-positive; 6 (46.1%), human epithelial growth factor receptor 2 (HER2)-amplified. Three patients had ductal carcinoma in situ. Most patients (N = 12/16, 75%) received adjuvant radiotherapy. After a median follow-up of 52.5months, 2 patients (2/12, 16.6%) had RIMs. One had a fibrosarcoma and the other, a low-grade leiomyosarcoma. In the group treated with radiotherapy, one distant recurrence was diagnosed (1/12), and no loco-regional recurrence occurred. Among 4 patients who did not receive radiotherapy, 2 presented with loco-regional recurrence. In this cohort of patients with LFS enriched in TP53 p.R337H pathogenic variant, the incidence of RIMs after treatment of localized breast cancer was lower than previous literature. Nevertheless, rates of RIMs were still alarming. Early molecular diagnosis and careful evaluation of treatment risks and benefits are essential for these patients.

Family Health Leaders: Lessons on Living with Li-Fraumeni Syndrome across Generations.

Li-Fraumeni Syndrome (LFS) is a hereditary disorder that confers an approximately 90% lifetime risk of cancer and requires comprehensive lifetime cancer screening. We explored healthcare roles for managing LFS-related cancer risks and treatments that were assumed by parents, adolescents, and adult children. Semi-structured interviews were conducted with 23 families. Family groupings were comprised of 2-5 members, with the younger generation in each family ranging in age from 7 to 40years. Using grounded theory methods, we conducted open and focused coding of interview transcript content. Family members described how the role of health leader was implemented in their family, as well as factors such as maturation of a child or death of a member that determined who assumed particular roles and how these roles shifted over time. They often expressed collective responsibility for helping relatives understand LFS and implement appropriate cancer risk management. Members demonstrated their health role by attending others' medical appointments for support or information gathering. The health leader role was intergenerational and provided the family necessary support in navigating complicated healthcare decisions. Our findings provide insight into healthcare providers regarding how LFS patients and their relatives develop unique medical decision-making and caring roles influenced by the hereditary nature of LFS, and how these roles change over time. Providers who are attuned to family role dynamics may be better able to meet relatives' psychosocial and medical needs by understanding how living with LFS influences the family system's functioning and facilitating members' support for each other.

No Significant Association between Radiation Therapy and Subsequent Malignancies in Patients with Li-Fraumeni Syndrome: A Multi-Institutional Hereditary Cancer Registry Study

Li-Fraumeni Syndrome (LFS) is a rare cancer-predisposing disease caused by germline mutations in a potent tumor suppressor gene calledTP53. Accordingly, LFS patients carry a high lifetime risk of developing multiple primary cancers. Conventional wisdom and prior work have suggested that the risk of developing a subsequent malignancy is increased by the use of radiation therapy (RT) during primary or secondary cancer treatment. This risk, however, is not well characterized. Here we describe the risk of subsequent malignancy and cancer-related death in LFS patients after undergoing RT for a first or second primary cancer. We reviewed a cohort of patients in a multi-institution hereditary cancer registry with germline TP53 mutations. We assessed the rate of subsequent malignancy and death in patients who received RT (RT group) as part of their cancer treatment against those who did not (non-RT group). We further evaluated outcomes after RT with respect to p53 mutation type. Among 41 LFS patients (14 males and 28 females from 28 different families), 22 TP53mutations were present. 61% (25) and 7% (3) of patients had missense mutations in the DNA binding domain and tetramerization domain of p53, respectively, and 27% (11) had truncating mutations caused by nonsense (3), frameshift (1), splice site (2), or deletion (5) mutations. The median age of first cancer diagnosis was 22 years. 13 patients received RT with curative intent as part of their cancer treatment which included 3 for locally recurrent disease. The median time to follow up after RT was 5.0 years. 61% of patients in the RT group compared to 40% of patients in the non-RT group developed a subsequent malignancy (P=0.31). The median time to subsequent malignancy in the RT group and non-RT group was 3.2 years and 4.3 years respectively. Although 4 patients developed a subsequent malignancy within the radiation field, all were of the same initial histology and none of the cancers had features of an RT-induced secondary malignancy. To date, 8 deaths have occurred in the RT group (median age 36.5 years) and 2 deaths in the non-RT group (median age 30.5 years). The 5-year overall survival (OS) for the RT group was 54% compared to 95% in the non-RT group (P=0.033). Notably, there was no association between the type of p53 mutation and the rate of subsequent malignancy after RT. LFS patients who received RT for a primary cancer suffered from subsequent malignancies and death at a higher rate than those who did not receive RT. However, these results must be interpreted with caution as the majority of patients within the RT-treated group had more aggressive cancers with a higher risk of recurrence. We found that all 4 in-field malignancies in the RT-group likely represent locally recurrent disease and not RT-induced malignancies. Accordingly, the higher rate of mortality in the RT-group is likely a reflection of more aggressive cancer diagnoses and not their treatment.

Abstract 1639: Predictive modeling of cancer-type in Li-Fraumeni syndrome

Abstract Objective: Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition syndrome associated with a germline TP53 mutation. Individuals with LFS are prone to developing a wide spectrum of tumors. This heterogeneity in tumor type makes it difficult to provide patient specific surveillance protocols. As a result, there is an immediate need to develop robust, evidence-based stratification strategies to tailor surveillance protocols to an individual patient’s risk of cancer. Germline TP53 mutations themselves do not explain specific cancer phenotypes in LFS, nor the collective clinical heterogeneity. Hence, it is important to consider genomic level data in the development of predictive algorithms in these patients. The proposed study is two-fold—to identify germline modifiers predictive of tumor type in LFS, and to use these modifiers to develop a predictive model for the early detection of cancer type. Methods: This study consists of blood-derived DNA methylation and whole genome sequencing (WGS) from a cohort of LFS patients (n=134); a subset of this cohort was held out as a test set. The first objective was to identify germline modifiers: single nucleotide variants (SNVs), insertions and deletions (indels), structural variants (SVs), copy number variation (CNV) and differentially methylated regions (DMRs) predictive of cancer type. Blood DNA methylation was generated using Illumina HumanMethylation450 BeadChip array. The top statistically significant DMRs were identified between cancer types by performing pairwise comparisons using a linear model. CNV, SNVs, indels, and SVs were detected from WGS using benchmarked tools: CNVnator, ERDS, GATK, and Delly. Custom filtering pipelines, and a curated list of cancer genes were established to determine high quality, biologically relevant modifiers. The second objective was to develop a model using the identified modifiers to predict cancer type in LFS patients. A generalized linear model was implemented using elastic net regularization to estimate the probability of getting a particular cancer type. Results: The classifier can accurately identify the cancer type of all the individuals in the test set. The model can determine with > 90% accuracy whether an individual has cancer and can differentiate between cancer types among affected individuals with 42%-92% probability. Pathway analysis of the predictors highlight the TGF-β signaling pathway as a possible therapeutic target for personalized treatment in LFS. Conclusion: This project is the first comprehensive molecular analysis of LFS and uses the largest LFS cohort to-date. It illustrates the contribution of genetic changes in LFS beyond TP53 and highlights the existence of molecular differences within LFS patients that contribute to phenotypic differences. Ultimately, this study will allow for the early detection of tumor-onset in LFS patients to assist clinicians in developing personalized surveillance protocols. Citation Format: Vallijah Subasri, Nicholas Light, Benjamin Brew, Nathaniel Anderson, Adam Shlien, Anna Goldenberg, David Malkin. Predictive modeling of cancer-type in Li-Fraumeni syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1639.

Abstract 979: Programmed death-ligand 1 expression and tumor-infiltrating immune cells in cancers associated with Li-Fraumeni syndrome

Abstract Purpose: Li-Fraumeni syndrome (LFS), due to TP53 germline mutations, is characterized by high incidence of multiple cancers. Dysregulation of the TP53 pathway is associated with changes in chemokine production and reduced tumor immune infiltration in some cancer types. Recent works also suggested that TP53 negatively regulates PDL1 expression through the non-coding RNA miR-34. To better understand immune evasion of LFS spectrum cancers and to explore the feasibility of PD1/PDL1 targeted therapies as an alternative to genotoxic treatments in LFS patients, we investigated a range of LFS-related tumor types for PDL1 expression and tumor-infiltrating immune cells. Methods: We retrospectively collected a total of 72 tumor samples (osteosarcoma=32, rhabdomyosarcoma=11, adrenocortical carcinoma=11 and breast carcinoma=18) from 60 pediatric and adult patients identified through the LFS French database and direct contact with oncologic centers. Immunochemistry analyses were performed on whole slide sections. PDL1 staining was performed using the clone E1L3N (Cell Signaling Technology) according to the standard automated protocols. Immune infiltrate was characterized by the following antibody panel: CD8, FOXP3 and CD68. For CD8+ and FoxP3+ T cells, cell density per surface unit was determined by cell counting; for CD68+ macrophages, a semi-quantitative scoring (from 0 to 4) was performed. Results: Median age of patients was 13 years (range: 6-23) for osteosarcoma, 1.7 years (range: 0.8-5) for rhabdomyosarcoma, 2.3 years (range: 0.4-6) for adrenocortical carcinoma and 27.5 years (range: 22-54) for breast carcinoma. Seven patients presented ≥2 metachronous tumors. Biopsy was performed at diagnosis for 68 patients, at relapse for 3 patients and at early progression for one patient. None of the samples presented any detectable PDL1 expression neither on tumor cells nor on tumor microenvironment. Low/very low amount of CD8+ cytotoxic T-cells was displayed, regardless of the histology. FoxP3+ regulatory T-cell infiltration varied among tumor types. Thirty-five/72 tumors (49%) did not contain regulatory T cells. Breast carcinomas presented significant amounts of regulatory T cells (16/18, 89%), but at low density for 10 out of 16 samples (62%). Sixty-two/72 tumors (86%) presented CD68+ macrophage infiltrates, with high density in 37/62 (60%) of them (score ≥ 2+). Conclusion: The absence of PDL1 expression on tumor cells and microenvironment as well as the low amount of regulatory T cells do not support the hypothesis of PDL1 induction through p53 inactivation in LFS. The role of innate immunity needs to be explored to identify alternative immunological targets. Citation Format: Emilie Saucier, Claudia Pasqualini, Louise Galmiche, Frédérique Larousserie, Marie Karanian, Véronique Minard-Colin, Birgit Geoerger, Dominique Valteau-Couanet, Laurence Brugières, Jean-Yves Scoazec. Programmed death-ligand 1 expression and tumor-infiltrating immune cells in cancers associated with Li-Fraumeni syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 979.

Abstract 4512: Identifying novel mechanisms of p53-mediated tumor suppression

Abstract TP53 is the most frequently mutated gene in human cancer. Classically, p53-mediated tumor suppression is thought to occur via transactivation of canonical targets that induce cell cycle arrest and apoptosis. However, this model has been challenged by studies using mouse p53 mutants. The mouse transactivation domain mutant, p5325,26, is unable to activate most canonical p53 targets and fails to induce cell cycle arrest or apoptosis in response to DNA damage. However, it retains the ability to induce cellular senescence and suppresses tumor development in mouse models. Indeed, some human p53 mutants found in Li-Fraumeni syndrome as well as cancer retain transactivation, while some p53 mutants are not found in cancer despite loss of transactivation. The goal of our study is to identify novel p53 functions that drive tumor suppression by evaluating p53 mutations where transactivation is uncoupled from tumor suppression activity. To identify human TP53 missense mutants with tumor suppressive function dissociated from the ability to activate canonical p53 pathways, we first utilized the results of a 2003 saturation mutagenesis screen performed in yeast which stratified human p53 mutants by their ability to activate 8 canonical p53 targets. We then cross-referenced these results with tumor sequencing data from the IARC, the TCGA PanCancer Atlas, and the MSK-IMPACT Sequencing Cohort. We identified panels of human p53 missense mutants which either 1) retained transactivation ability in yeast, yet had been identified in sequenced human tumors or 2) were unable to transactivate p53 targets in yeast, but had not been identified in sequenced human tumors. To study these p53 mutants compared to wt p53, we cloned their sequences into doxycycline-inducible pLVX-TetOne-Puro plasmids and puromycin-selected for cell lines stably expressing the construct in p53 null H1299 and HCT116 cells. Using qPCR and western blots we validated the expression of mutant or wt p53 and assessed for transactivation of canonical targets p21, mdm2, puma, gadd45α, and 14-3-3. The IncuCyte Live Cell Analysis system and XTT Cell Proliferation assays were used to evaluate p53-mediated growth arrest of human cancer cells following induction of each mutant. From these experiments, we selected three mutants for further study. Missense p53 mutants E224D and R290H are found in human cancers and in the germline of Li-Fraumeni syndrome patients yet retain the capacity to transactivate p53 targets and arrest tumor cell growth in vitro. In contrast, p53 mutant G262A which is not found in cancer lacks the ability to activate p53 targets and fails to arrest tumor cell growth in vitro. We are currently using soft agar assays to assess the transformation potential of the identified p53 mutants in FSF-KRAS ; p53fl/+ MEFs. To dissect tumor suppressive function we are developing transgenic mice expressing the p53 mutants. The results of this ongoing study may uncover novel p53 tumor suppressor mechanisms. Citation Format: Nathan H. Leisenring, Robert W. Floyd, David G. Kirsch. Identifying novel mechanisms of p53-mediated tumor suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4512.

Oncogenic role of SFRP2 in p53-mutant osteosarcoma development via autocrine and paracrine mechanism

Osteosarcoma (OS), the most common primary bone tumor, is highly metastatic with high chemotherapeutic resistance and poor survival rates. Using induced pluripotent stem cells (iPSCs) generated from Li-Fraumeni syndrome (LFS) patients, we investigate an oncogenic role of secreted frizzled-related protein 2 (SFRP2) in p53 mutation-associated OS development. Interestingly, we find that high SFRP2 expression in OS patient samples correlates with poor survival. Systems-level analyses identified that expression of SFRP2 increases during LFS OS development and can induce angiogenesis. Ectopic SFRP2 overexpression in normal osteoblast precursors is sufficient to suppress normal osteoblast differentiation and to promote OS phenotypes through induction of oncogenic molecules such as FOXM1 and CYR61 in a β-catenin-independent manner. Conversely, inhibition of SFRP2, FOXM1, or CYR61 represses the tumorigenic potential. In summary, these findings demonstrate the oncogenic role of SFRP2 in the development of p53 mutation-associated OS and that inhibition of SFRP2 is a potential therapeutic strategy.

Whole-body magnetic resonance imaging of Li-Fraumeni syndrome patients: observations from a two rounds screening of Brazilian patients

BackgroundLi-Fraumeni syndrome (LFS) is an autosomal dominant disease that is associated with germline TP53 mutations and it predisposes affected individuals to a high risk of developing multiple tumors. In Brazil, LFS is characterized by a different pattern of TP53 variants, with the founder TP53 p.R337H mutation being predominant. The adoption of screening strategies to diagnose LFS in its early stages is a major challenge due to the diverse spectrum of tumors that LFS patients can develop. The purpose of this study was to evaluate two rounds of whole-body magnetic resonance imaging (WB-MRI) which were conducted as a screening strategy for LFS patients.MethodsOver a 4-year period, 59 LFS patients underwent two rounds of WB-MRI. Each MRI was characterized as positive or negative, and positive cases were further investigated to establish a diagnosis. The parameters used to evaluate the WB-MRI results included: positive rate, number of invasive investigations of positive results, and cancer detection rate.ResultsA total of 118 WB-MRI scans were performed. Positive results were associated with 11 patients (9.3%). Seven of these patients (11.8%) were identified in the first round of screening and 4 patients (6.7%) were identified in the second round of screening. Biopsies were performed in three cases (2.5%), two (3.4%) after the first round of screening and one (1.7%) after the second round of screening. The histopathological results confirmed a diagnosis of cancer for all three cases. There was no indication of unnecessary invasive procedures.ConclusionsWB-MRI screening of LFS carriers diagnosed cancers in their early stages. When needed, positive results were further examined with non-invasive imaging techniques. False positive results were less frequent after the first round of WB-MRI screening.

Association Between the Oligomeric Status of p53 and Clinical Outcomes in Li-Fraumeni Syndrome.

Li-Fraumeni syndrome (LFS) is a rare hereditary cancer disorder with highly variable clinical outcomes that results from germline mutations in the TP53 gene. Here we report that the quaternary structure of p53 is an important factor affecting cellular functions and the clinical outcomes of LFS patients (n = 87). Specifically, carriers of monomeric p53 mutants (n = 56) exhibited complete penetrance, with a 2.11-fold greater risk of cancer-related death (95% confidence interval [CI] = 1.07 to 4.30) and a statistically significantly lower median survival age as compared with carriers of multimeric (dimeric or tetrameric, n = 31) p53 mutants (33 years, 95% CI = 30 to 50, vs 51 years, 95% CI = 40 to NA, respectively, two-sided P = .03), who presented incomplete penetrance. Cellular functional assays using p53-null H1299 cells expressing clinically relevant p53 mutants confirmed that the cellular effects observed upon loss of p53 oligomerization are associated with clinical outcomes of LFS patients. The association between p53 oligomeric state and clinical phenotype suggests that TP53 mutations are not all equivalent and supports the implementation of new genotype-adapted guidelines for the management of LFS patients with TP53 mutations in the oligomerization domain.

Li-Fraumeni Syndrome-related Malignancies Involving the Genitourinary Tract: Review of a Single-institution Experience

ObjectiveTo report a case of pelvic angiosarcoma in a 27-year-old man with Li-Fraumeni Syndrome (LFS) and evaluate the presentation and timeline of genitourinary (GU) tract involvement in LFS patients. MethodsWe retrospectively identified 39 LFS patients treated at our institution between 2000 and 2014; 7 (18%) had experienced a GU malignancy or an LFS-related malignancy involving the GU tract. Clinical characteristics, including dates of onset of first GU tract malignancies; pathologic findings; multimodal management; and familial history of LFS were reviewed. ResultsMedian age at first malignancy was 14.0 years (interquartile range [IQR] 5.5-24.0). There was a slight male predominance (4 of 7). Median time between first malignancy and the malignancy involving the GU tract was 10.1 years (IQR 8.0-19.5). Six of the 7 patients (86%) had a form of sarcoma involving the GU tract; 1 developed adrenocortical carcinoma. The cancer pedigree of all patients showed LFS-associated malignancies in family members. Multimodal management included surgical resection in 6 patients with adjuvant chemotherapy or radiotherapy in 1 patient each. One patient received chemotherapy only. Following diagnosis of malignancy involving the GU tract, 5 of the 7 patients developed additional primary malignancies. At a median follow-up of 4.7 years (IQR 3.0-12.1), 2 patients are alive, 3 died of disease, and 1 died of unknown cause. One patient was lost at follow-up. ConclusionContinued follow-up of LFS cancer patients aimed at the determination of optimal screening, management, and surveillance protocols is recommended and may result in longer survival expectations.