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Purpose Although the link between mutant TP53 and human cancer is unequivocal, a significant knowledge gap exists in clinically actionable molecular targets in Li-Fraumeni syndrome (LFS), a highly penetrant cancer predisposition syndrome associated with germline mutations in TP53. This study surveyed the epigenome to identify functionally and clinically relevant novel genes implicated in LFS. Patients and Methods We performed genome-wide methylation analyses of peripheral blood leukocyte DNA in germline TP53 mutation carriers (n = 72) and individuals with TP53 wild type in whom histologically comparable malignancies developed (n = 111). Targeted bisulfite pyrosequencing was performed on a validation cohort of 30 TP53 mutation carriers and 46 patients with TP53 wild type, and candidate sites were evaluated in primary tumors from patients with LFS across multiple histologic tumor types. Results In 183 patients, distinct DNA methylation signatures were associated with deleterious TP53 mutations in peripheral blood leukocytes. TP53-associated DNA methylation marks occurred in genomic regions that harbored p53 binding sites and in genes encoding p53 pathway proteins. Moreover, loss-of-function TP53 mutations were significantly associated with differential methylation at the locus encoding microRNA miR-34A, a key component of the p53 regulatory network (adjusted P < .001), and validated in an independent patient cohort (n = 76, P < .001). Targeted bisulfite pyrosequencing demonstrated that miR-34A was inactivated by hypermethylation across many histologic types of primary tumors from patients with LFS. Moreover, miR-34A tumor hypermethylation was associated with decreased overall survival in a cohort of 29 patients with choroid plexus carcinomas, a characteristic LFS tumor (P < .05). Conclusion Epigenetic dysregulation of miR-34A may comprise an important path in TP53-associated cancer predisposition and represents a therapeutically actionable target with potential clinical relevance.

Mutations in the TP53 tumor suppressor gene are the most common genetic aberrations across all human cancers. Germline TP53 mutations are also the hallmark genetic event in Li-Fraumeni syndrome (LFS), a highly penetrant human cancer susceptibility syndrome, conferring a predisposition to developing early-onset breast cancer, leukemias, bone and soft tissue sarcomas, brain tumors of various histologies, adrenocortical carcinomas, and a wide range of other malignancies. Although the link between mutant TP53 and human cancer is unequivocal, the mechanism by which this genetic aberration predisposes an individual to cancer remains to be elucidated. To address this gap, we surveyed the epigenome and describe herein the largest systematic analysis of DNA methylation in patients harboring germline TP53 mutations and TP53 wild-type individuals. Specifically, we performed genome-wide methylation analyses of peripheral blood leukocyte DNA in germline TP53 mutation carriers (n=72) and TP53 wild-type individuals who developed histologically comparable cancers (n=111). Targeted bisulfite pyrosequencing was performed on peripheral blood DNA in a validation cohort (n=76), and candidate sites were further evaluated in primary tumors from LFS patients. The differential methylation analysis demonstrates that in 183 patients, distinct DNA methylation signatures are associated with deleterious TP53 mutations. TP53 mutation-associated DNA methylation marks occur in genomic regions harboring known p53 binding sites and within genes encoding p53 pathway proteins. Moreover, loss-of-function TP53 mutations are significantly associated with differential methylation at the locus encoding miR-34A-a key component of the p53 regulatory network (adjusted p-value=3.1X10-15)-and validated in an independent patient cohort (n=76, 1.9X10-8). Targeted sequencing demonstrates that miR-34A is inactivated by hypermethylation across many different histologic types of primary tumors from LFS patients, such as brain tumors, osteosarcomas, rhabdomyosarcomas, and adrenocortical carcinomas. miR-34A promoter hypermethylation in tumors is also associated with decreased overall survival in a cohort of 29 patients with choroid plexus carcinomas, a characteristic LFS tumor (p This study refines the role of epigenetics in a cancer predisposition syndrome and is the first to implicate a microRNA, miR-34A, in human cancer susceptibility and provides a repository of genomic regions of deregulated methylation in the context of dysfunctional TP53. These findings suggest that deregulated DNA methylation at defined genomic loci may be an important hallmark of TP53-mediated cancer susceptibility. The most striking finding from this study is the relative miR-34A promoter hypomethylation at two adjacent CpG sites in peripheral blood from TP53 mutation carriers, confirmed in two independent cohorts and shown to cosegregate with TP53 mutations in LFS families. This result is remarkable since miR-34A is a central microRNA in the p53 network and the first microRNA identified as a direct proapoptotic target of the p53 pathway. The detection of miR-34A promoter hypomethylation in TP53 mutant cells that have not undergone malignant transformation supports a putative model whereby wild-type p53 may influence methylation patterns at this locus. In particular, in nontransformed cells that do not harbor mutations in TP53, wild-type p53 may be recruited to the miR-34A locus and sustain hypermethylation. We have performed a series of in vitro studies on primary patient-derived lymphoblastoid cell lines to corroborate this model. Conversely, in the setting of loss-of-function or deleterious mutations in TP53, mutant p53 may not able to maintain hypermethylation of the miR-34A promoter, leading to upregulation of miR-34A. Owing to the known redundant cellular roles of p53 and miR-34A, upregulation of miR-34A may be beneficial to cells harboring mutant p53 by supplementing the necessary basal tumor suppressive function that is lost when p53 is mutated. This mechanism may serve to guard against mutant p53, even when the wild-type allele remains. Accordingly, this may explain why miR-34A promoter hypermethylation is characteristic of TP53-mutant tumors that lack wild-type p53 because this microRNA serves a critical role in cell maintenance, and its loss may cooperate with other genetic and/or epigenetic events to drive malignancy. It is therefore not surprising that, akin to p53, somatic miR-34A deregulation is pervasive in human cancer and miR-34A inactivation by focal deletion or promoter hypermethylation has been reported in the literature to occur in a multitude of human malignancies. The precise mechanisms of how the miR-34A promoter undergoes somatic epimutation in tissues remains to be elucidated, and likely various pathways may converge to yield this outcome in different tissues. Given the high frequency of TP53 mutations in human malignancies, the relationship between mutant p53 and miR-34A has strong implications for the targeting of miR-34A in cancer. Encouragingly, studies have demonstrated in vivo the utility of miR-34A-based therapies in cancer, including intratumor or systemic delivery of lipid-formulated synthetic miR-34A. To further probe these intriguing findings, we conducted mechanistic studies aimed at functionally interrogating the the miR-34A-p53 axis. We utilized in vitro-based assays to modulate miR-34A levels in primary patient-derived fibroblast cell lines, and subsequently performed by RNA-sequencing of the transcriptional responses. Our results uncover a number of novel cellular roles for miR-34A in cell maintenance. Significantly, the transcriptional response to miR-34A inhibition revealed that this microRNA may be a crucial switch that can lead to numerous changes to noncoding RNA networks as well as known p53 pathways. Markedly increased expression of key components of the U12 (minor) spliceosome occurs when miR-34A expression is diminished, thereby identifying a novel putative role of miR-34A in modulating transcription of the U12 spliceosomal machinery. The majority of TP53 mutation-associated transcripts are involved in chromatin remodeling and nucleosome assembly, and are enriched for histone cluster 1 genes. These linker histones are crucial for maintaining higher-order chromatin structure and for regulating gene expression, demonstrating the interplay between genetic and epigenetic states. Lastly, miR-34A is associated with transcriptional regulation of a host of lincRNAs, including LINC-PINT, a p53-induced lincRNA. These results are the first to identify miR-34A as an important node in the transcriptional regulation of numerous noncoding RNAs and point to further study of these pathways. Taken together, these findings provide strong support for the impact of TP53 mutations on epigenetic dysregulation in human cancer susceptibility and demonstrate that miR-34A may be important in the pathogenesis of TP53-mediated cancer susceptibility. Moreover, miR-34A may be a putative novel therapeutic target and a marker for clinical prognostication. These studies also demonstrate that miR-34A is a central node in numerous p53-dependent and independent networks and provide further insight into the role of this critical tumor-suppressive microRNA. Further work aimed at refining our understanding of miR-34A-mediated pathways may yield additional molecular insight into the role of this microRNA in malignant transformation. Citation Format: Nardin Samuel, Gavin Wilson, Genevieve Deblois, Badr Id Said, Nicholas W. Fischer, Mathieu Lemire, Youliang Lou, Weili Li, Roumiana Alexandrova, Ana Novokmet, James Tran, Kim E. Nichols, Jonathan L. Finlay, Sanaa Choufani, Marc Remke, Vijay Ramaswamy, Florence M.G. Cavalli, Christine Elser, Lynn Meister, Michael D. Taylor, Uri Tabori, Meredith Irwin, Rosanna Weksberg, Jonathan D. Wasserman, Jean Gariepy, Mathieu Lupien, Daniele Merico, Andrew Paterson, Jordan R. Hansford, Maria Isabel W. Achatz, Thomas J. Hudson, David Malkin. TP53-mediated human cancer susceptibility is defined by epigenetic dysregulation of microRNA-34A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr NG05. doi:10.1158/1538-7445.AM2017-NG05

Store-operated Ca(2+) entry is the major route of replenishment of intracellular Ca(2+) in animal cells in response to the depletion of Ca(2+) stores in the endoplasmic reticulum. It is primarily mediated by the Ca(2+)-selective release-activated Ca(2+) (CRAC) channel, which consists of the pore-forming subunits ORAI1-3 and the Ca(2+) sensors, STIM1 and STIM2. Recessive loss-of-function mutations in STIM1 or ORAI1 result in immune deficiency and nonprogressive myopathy. Heterozygous gain-of-function mutations in STIM1 cause non-syndromic myopathies as well as syndromic forms of miosis and myopathy with tubular aggregates and Stormorken syndrome; some of these syndromic forms are associated with thrombocytopenia. Increased concentration of Ca(2+) as a result of store-operated Ca(2+) entry is essential for platelet activation. The York Platelet syndrome (YPS) is characterized by thrombocytopenia, striking ultrastructural platelet abnormalities including giant electron-opaque organelles and massive, multilayered target bodies and deficiency of platelet Ca(2+) storage in delta granules. We present clinical and molecular findings in 7 YPS patients from 4 families, demonstrating that YPS patients have a chronic myopathy associated with rimmed vacuoles and heterozygous gain-of-function STIM1 mutations. These findings expand the phenotypic spectrum of STIM1-related human disorders and define the molecular basis of YPS.

All forms of cutaneous T-cell lymphoma are rare in children. We describe an 11-year-old girl who had generalized exfoliative erythroderma, intense pruritus, peripheral lymphadenopathy, mycosis cells in the skin and lymph nodes, and Sézary cells in the peripheral blood. Results of a biopsy specimen of involved skin showed changes consistent with mycosis fungoides. A classic case of Sézary syndrome has not previously been reported in childhood or preadolescence.

Inactivation of wild-type p53 tumor suppressor function is the primary mechanism of tumor initiation in Li-Fraumeni syndrome (LFS) individuals with germline p53 mutations. Tumors derived from LFS patients frequently retain the normal p53 allele, suggesting that alternative mechanisms in addition to gene deletion must be involved in inactivating wild-type p53 protein. DNA tumor viruses, such as SV40, target p53 for inactivation through the action of viral oncoproteins. We studied the probands from two unrelated LFS families, each of whom presented with multiple malignant neoplasms. Patient 1 developed an embryonal rhabdomyosarcoma (RMS) and a choroid plexus carcinoma (CPC), while patient 2 developed a CPC and subsequently presented with both an osteosarcoma (OS) and renal cell carcinoma (RCC). We utilized DNA sequence analysis and immunohistochemistry to determine p53 gene status in the germline and tumors, as well as evidence for SV40 T-antigen oncoprotein expression. Each patient harbored a heterozygous germline p53 mutation at codons 175 and 273, respectively. In patient 1, the normal p53 gene was lost while the mutant p53 allele was reduced to homozygosity in the RMS. Both normal and mutant genes were maintained in the CPC. In patient 2, normal and mutant p53 alleles were retained in both the CPC and RCC. Both specific PCR and immunostaining detected SV40 T-antigen in both CPCs and the RCC. In addition to chromosomal alterations, epigenetic mechanisms may disrupt p53 function during tumorigenesis. In two LFS patients, we found SV40 DNA sequences and viral T-antigen expression that could account for inactivation of the normal p53 protein. Inactivation of p53 or other tumor suppressors by viral proteins may contribute to tumor formation in specific tissues of genetically susceptible individuals.

• A rare case of homozygous protein C deficiency occurred in a newborn. The patient presented with purpura fulminans in the first few hours after birth and showed multiple hemorrhagic lesions on computed tomography of the brain at 5 days of age. Neurologic symptoms developed at two weeks and the patient died at 37 weeks. His protein C level was less than 5%. Autopsy revealed thrombosis of the dural sinuses, multiple cortical infarcts, intraparenchymal hemorrhages, and hydrocephalus. The pathologic findings are correlated with the neurologic deficits and previously documented cases are reviewed.