Predictors of brain metastases in patients with oligometastatic solid tumours treated with stereotactic body radiation therapy.

PURPOSE: In patients with oligometastatic disease (OMD) treated with stereotactic body radiation therapy (SBRT), those who develop brain metastases (BrM) may have poor outcomes. We aimed to investigate variables associated with BrM development in this population. METHODS: Patients with ≤ 5 extracranial metastases from solid tumors treated with SBRT from 2008 to 2016 at Sunnybrook Odette Cancer Centre were included. We investigated the association between covariates and CIBrM (cumulative incidence of BrM) using Fine-Gray analysis, and progression-free survival (PFS) and overall survival (OS) using Cox regression. We investigated the association between extracranial progression and CIBrM using time-based conditional analysis. RESULTS: Among 404 patients, the most common primary sites were lung, colorectal, prostate, breast and kidney. Median follow-up was 49 months. Median PFS was 25 months. Median OS was 70 months. 58 patients developed BrM, and 5-year CIBrM was 16%. On multivariable analysis, number of extracranial metastases, location of metastases, total planning target volume (PTV), and time from primary diagnosis to OMD were not associated with CIBrM, although several of these variables were associated with extracranial PFS and OS. Primary site was associated with CIBrM, with colorectal and prostate cancer associated with lower CIBrM compared to lung cancer. Widespread extracranial progression (≥ 5 sites) within 24, 36, 48 and 60 months of OMD diagnosis was independently associated with higher CIBrM. CONCLUSION: In patients with OMD treated with SBRT, baseline variables related to extracranial disease burden and distribution were not associated with BrM development, while primary site and widespread extracranial progression were associated with BrM development.

Patterns of treatment and outcomes of patients with brain-only metastatic breast cancer.

BACKGROUND: We characterized the risk factors and survival of metastatic breast cancer (MBC) patients with brain metastases (BrM) as the first and only site of disease in a large, retrospective cohort. METHODS: MBC patients treated for BrM with radiation at a quaternary institution between 2005 and 2019 were identified. MBC patients with BrM but without concurrent extracranial metastases (ECM) or leptomeningeal disease (LMD) were classified as brain-only. Factors associated with brain-only MBC, brain-specific progression free survival (bsPFS) and overall survival (OS) were investigated. RESULTS: A total of 691 patients with MBC and BrM were analyzed. Among them, 67 patients (9.7%, n = 67/691) presented with brain-only MBC without concurrent ECM/LMD. Within this subgroup, 40 patients (5.8%, n = 40/691) remained free of any ECM or LMD, while 17 patients (2.5%) developed LMD, and 10 patients (1.4%%) developed ECM with a median follow-up of 8 months (IQR 2-35). Patients with brain-only MBC were more likely to have a single BrM [OR 3.41 (1.62-7.19), p = 0.001] and either HER2+ [OR 3.3 (1.13-9.65), p = 0.03] or TNBC [OR 4.09 (1.42-11.74), p = 0.009] subtypes. Patients who presented with brain-only MBC also had significantly longer OS [HR 0.45, (0.22-0.86), p = 0.008] and a trend toward longer bsPFS [HR 0.67 (0.44-1.03), p = 0.05] compared to those with concurrent ECM/LMD. CONCLUSION: Patients with brain-only MBC had a longer bsPFS and OS than those with ECM. Patients with HER2+ and TNBC were more likely to have brain-only disease compared to those with HR+/HER2- MBC.

Demographics, pattern of practice and clinical outcomes in rectal squamous cell carcinoma.

AIM: The aim of this study was to describe the baseline clinical features, treatment patterns and outcomes in rectal squamous cell carcinoma (SCC). METHOD: This is a retrospective study of patients with rectal SCC treated at the Princess Margaret Cancer Centre (Toronto, Canada) between 1 January 1995 and 31 December 2020. Clinical factors associated with locoregional failure (LRF), distant metastases (DM), disease-free survival (DFS) and overall survival (OS), such as age, sex, HIV status, T-category, nodal status, grade and primary treatment, were investigated with univariate analysis (UVA). RESULTS: Twenty nine patients with rectal SCC were analysed with a median follow-up of 7.4 years (range 0.3-20.4 years). The median age at diagnosis was 52 years, with the majority presenting with clinical T3 disease or higher (n = 21, 72%) and positive regional lymph nodes (n = 16, 55%), while more than quarter of patients (28%) had metastatic disease. Definitive chemoradiation was the treatment modality of choice in more than half of all cases (n = 17, 59%) with a response rate of 100%. The 10-year cumulative incidence of LRF and DM was, respectively, 12% (95% CI 1.8%-32.9%) and 31% (95% CI: 12.0%-52.6%). The 5- and 10-year OS was 82% (95% CI 66.1%-100%). UVA revealed a trend towards an association of male gender (hazard ratio = 4.65, 95% CI 0.9%-24.1; p = 0.067) and primary surgical treatment (hazard ratio = 0.76, 95% CI 0.09-6.34; p = 0.061) with DFS. CONCLUSION: Definitive chemoradiation is an effective and preferred treatment for rectal SCC allowing for sphincter preservation with complete clinical response observed in all patients.

Trastuzumab emtansine increases the risk of stereotactic radiosurgery-induced radionecrosis in HER2 + breast cancer.

INTRODUCTION: In this study, we investigate factors associated with radionecrosis (RN) in HER2 + (human epidermal growth factor receptor 2) patients with brain metastases (BrM) treated with stereotactic radiosurgery (SRS). METHODS: Patients with HER2 + breast cancer BrM treated with SRS (2010-2020) were identified from an institutional database. The incidence of RN was determined per treated BrM according to serial imaging and/or histology. Factors associated with RN such as age, RT dose, BrM volume, and initiation of Trastuzumab Emtansine (T-DM1) were investigated with univariate and multivariable analyses (MVA). RESULTS: 67 HER2 + patients with 223 BrM were identified. 21 patients (31.3%) were treated with T-DM1 post-SRS, including 14 patients (20.9%) who received T-DM1 within 12 months of SRS. The median follow-up was 15.6 (interquartile range (IQR) 5.4-35.3) months. The overall probability of RN post-SRS was 21.6% (95% confidence interval (CI) 2.7-10.7), and the 1 and 2 year risk was 6.7% (95% CI 2.7-10.7) and 15.2% (95% CI 9.2-21.3). MVA identified T-DM1 treatment post-SRS (hazard ratio (HR) 2.5, 95% CI 1.2-5.3, p = 0.02) and equivalent dose in 2 Gy fractions (EQD2) > 90 Gy2 (HR 2.4, 95% CI 1.1-5.1, p = 0.02) as predictors of RN. Patients treated with T-DM1 and SRS had a 29.9% (95% CI 15.3-44.6%) probability of RN, with a 25.2% (95% CI 12.8-37.6%) risk at 1- and 2 years post-T-DM1. The majority of RN were symptomatic (71%), with a median time to RN of 4.8 months. CONCLUSION: T-DM1 exposure post-SRS was associated with a higher risk of RN among patients with HER2 + BrM.

Treatment Patterns and Outcomes of Women with Symptomatic and Asymptomatic Breast Cancer Brain Metastases: A Single-Center Retrospective Study.

BACKGROUND: Breast cancer is the most common cancer among women worldwide and the second leading cause of brain metastases (BrM). We assessed the treatment patterns and outcomes of women treated for breast cancer BrM at our institution in the modern era of stereotactic radiosurgery (SRS). MATERIALS AND METHODS: We conducted a retrospective analysis of women (≥18 years of age) with metastatic breast cancer who were treated with surgery, whole brain radiotherapy (WBRT), or SRS to the brain at the Sunnybrook Odette Cancer Centre, Toronto, Canada, between 2008 and 2018. Patients with a history of other malignancies and those with an uncertain date of diagnosis of BrM were excluded. Descriptive statistics were generated and survival analyses were performed with subgroup analyses by breast cancer subtype. RESULTS: Among 683 eligible patients, 153 (22.4%) had triple-negative breast cancer, 188 (27.5%) had HER2+, 246 (36.0%) had hormone receptor (HR)+/HER2-, and 61 (13.3%) had breast cancer of an unknown subtype. The majority of patients received first-line WBRT (n = 459, 67.2%) or SRS (n = 126, 18.4%). The median brain-specific progression-free survival and median overall survival (OS) were 4.1 months (interquartile range [IQR] 1.0-9.6 months) and 5.1 months (IQR 2.0-11.7 months) in the overall patent population, respectively. Age >60 years, presence of neurological symptoms at BrM diagnosis, first-line WBRT, and HER2- subtype were independently prognostic for shorter OS. CONCLUSION: Despite the use of SRS, outcomes among patients with breast cancer BrM remain poor. Strategies for early detection of BrM and central nervous system-active systemic therapies warrant further investigation. IMPLICATIONS FOR PRACTICE: Although triple-negative breast cancer and HER2+ breast cancer have a predilection for metastasis to the central nervous system (CNS), patients with hormone receptor-positive/HER2- breast cancer represent a high proportion of patients with breast cancer brain metastases (BrM). Hence, clinical trials should include patients with BrM and evaluate CNS-specific activity of novel systemic therapies when feasible, irrespective of breast cancer subtype. In addition, given that symptomatic BrM are associated with shorter survival, this study suggests that screening programs for the early detection and treatment of breast cancer BrM warrant further investigation in an era of minimally toxic stereotactic radiosurgery.

Assessment of TP53 Polymorphisms and MDM2 SNP309 in Premenopausal Breast Cancer Risk.

Germline polymorphic variants in cancer predisposition genes such as TP53 have been shown to impact the risk of premenopausal cancer. Accordingly, the aim of this study was to assess the spectrum of polymorphisms in TP53 and its negative regulatory gene, MDM2 (SNP309:T>G) in patients with premenopausal breast cancer. Our findings in a cohort of 40 female patients demonstrate no significant correlation between the studied polymorphisms and risk of premenopausal breast cancer. Although one polymorphism is found in high frequency in this cohort (rs1800372:A>G, 9.0%), it was not associated with the risk of developing cancer before the age of 35 years in an extended cohort of 1,420 breast cancer cases. Functional studies of the rs1800372:A>G polymorphic allele reveal that it does not affect p53 transactivation function. Further study of variants or mutations in other cancer susceptibility genes is warranted to refine our understanding of the germline contribution to premenopausal breast cancer susceptibility.

Super-Transactivation TP53 Variant in the Germline of a Family with Li-Fraumeni Syndrome.

Li-Fraumeni Syndrome (LFS) is a rare autosomal dominant familial cancer syndrome, characterized by multiple malignancies and frequent germline alterations in TP53. In this study, we highlight four unclassified exonic TP53 variants detected in patients with a suspected diagnosis of LFS. Most intriguing was the discovery of a "super-transactivation" variant within Exon 10 of TP53 (c.1079G>T/p.G360V). Functional analysis of this novel variant revealed a paradoxical "super-transactivation" effect on tp53 response elements and a corresponding tumor suppressive effect on colony formation and apoptosis. While unlikely to be disease-causing, we propose that this variant may represent a novel tp53 polymorphism and potential phenotypic modifier in LFS. In the future, the enhanced transactivation effects of p.G360V-tp53 may also prove useful in designing more efficacious tp53-based gene therapies.

A narrative review of oncologic emergencies in patients with head and neck cancers: initial management and the role of radiation therapy.

BACKGROUND AND OBJECTIVE: Head and neck cancers (HNCs) encompass a complex group of malignancies with high morbidity, often leading to critical emergencies such as pain crises, airway obstruction and hemorrhage. This review aims to outline an evidence-based approach to the multidisciplinary management of HNC oncologic emergencies with a focus on the role of emergent radiotherapy (RT). METHODS: A literature search was performed using Medline, Embase and the Cochrane Central Register of Controlled Trials databases with a focus on three common oncological emergencies using the following keywords: "head and neck cancer", "radiation OR radiotherapy", "pain", "bleeding OR haemorrhage", and "airway obstruction". All English language articles published up to April 2022 were screened to identify studies pertaining to the management of oncologic emergencies in HNC. KEY CONTENT AND FINDINGS: The management of oncologic emergencies in HNC present a unique set of challenges that require early recognition and aggressive treatment. In this narrative review, we summarize the evidence supporting the role of RT in the management of HNC patients presenting with pain crisis, malignant airway obstruction and acute haemorrhage. We demonstrate that while RT can be used as a primary or adjunct therapy, optimal management depends on the involvement of a multi-disciplinary team that includes head and neck surgeons, interventional radiology and palliative care. CONCLUSIONS: RT plays a critical role in the multidisciplinary management of HNC oncological emergencies. Further prospective and comparative studies are needed to assess optimal management strategies.

Stereotactic Body Radiotherapy for Extracranial Oligometastatic Disease from Head and Neck Primary Cancers: A Systematic Review and Meta-Analysis.

INTRODUCTION: Stereotactic body radiotherapy (SBRT) is increasingly used to treat disease in the oligometastatic (OM) setting due to mounting evidence demonstrating its efficacy and safety. Given the low population representation in prospective studies, we performed a systematic review and meta-analysis of outcomes of HNC patients with extracranial OM disease treated with SBRT. METHODS: A systematic review was conducted with Cochrane, Medline, and Embase databases queried from inception to August 2022 for studies with extracranial OM HNC treated with stereotactic radiotherapy. Polymetastatic patients (>five lesions), mixed-primary cohorts failing to report HNC separately, lack of treatment to all lesions, nonquantitative endpoints, and other definitive treatments (surgery, conventional radiotherapy, and radioablation) were excluded. The meta-analysis examined the pooled effects of 12- and 24-month local control (LC) per lesion, progression-free survival (PFS), and overall survival (OS). Weighted random-effects were assessed using the DerSimonian and Laird method, with heterogeneity evaluated using the I2 statistic and Cochran Qtest. Forest plots were generated for each endpoint. RESULTS: Fifteen studies met the inclusion criteria (639 patients, 831 lesions), with twelve eligible for quantitative synthesis with common endpoints and sufficient reporting. Fourteen studies were retrospective, with a single prospective trial. Studies were small, with a median of 32 patients (range: 6-81) and 63 lesions (range: 6-126). The OM definition varied, with a maximum of two to five metastases, mixed synchronous and metachronous lesions, and a few studies including oligoprogressive lesions. The most common site of metastasis was the lung. Radiation was delivered in 1-10 fractions (20-70 Gy). The one-year LC (LC1), reported in 12 studies, was 86.9% (95% confidence interval [CI]: 79.3-91.9%). LC2 was 77.9% (95% CI: 66.4-86.3%), with heterogeneity across studies. PFS was reported in five studies, with a PFS1 of 43.0% (95% CI: 35.0-51.4%) and PFS2 of 23.9% (95% CI: 17.8-31.2%), with homogeneity across studies. OS was analyzed in nine studies, demonstrating an OS1 of 80.1% (95% CI: 74.2-85.0%) and OS2 of 60.7% (95% CI: 51.3-69.4%). Treatment was well tolerated with no reported grade 4 or 5 toxicities. Grade 3 toxicity rates were uniformly below 5% when reported. CONCLUSIONS: SBRT offers excellent LC and promising OS, with acceptable toxicities in OM HNC. Durable PFS remains rare, highlighting the need for effective local or systemic therapies in this population. Further investigations on concurrent and adjuvant therapies are warranted.

Outcomes for oligometastatic head and neck cancer treated with stereotactic body radiotherapy: Results from an international multi-institutional consortium.

BACKGROUND: We report the results of an international multi-institutional cohort of oligometastatic (OMD) head and neck cancer (HNC) patients treated with SBRT. METHODS: Patients with OMD HNC (≤5 metastases) treated with SBRT between 2008 and 2016 at six institutions were included. Treated metastasis control (TMC), progression-free survival (PFS), and overall survival (OS) were analyzed by multivariable analysis (MVA). RESULTS: Forty-two patients with 84 HNC oligometastases were analyzed. The TMC rate at 1 and 2 years were 80% and 66%, with a median time to recurrence of 10.1 months. The median PFS and OS were 4.7 and 23.3 months. MVA identified a PTV point maximum (BED)10 > 100 Gy as a predictor of improved TMC (HR = 0.31, p = 0.034), and a cumulative PTV > 48 cc as having worse PFS (HR = 2.99, p < 0.001). CONCLUSION: Favorable TMC and OS was observed in OMD HNCs treated with SBRT.

Individualized risk assessment of distant metastases in oral cavity carcinoma: a validated predictive-score model.

BACKGROUND: We aimed to develop and validate a risk-scoring system for distant metastases (DMs) in oral cavity carcinoma (OCC). METHODS: Patients with OCC who were treated at 4 tertiary cancer institutions with curative surgery with or without postoperative radiation/chemoradiation therapy were randomly assigned to discovery or validation cohorts (3:2 ratio). Cases were staged on the basis of tumor, node, and metastasis staging according to the eighth edition of the American Joint Committee on Cancer/Union for International Cancer Control guidelines. Predictors of DMs on multivariable analysis in the discovery cohort were used to develop a risk-score model and classify patients into risk groups. The utility of the risk classification was evaluated in the validation cohort. RESULTS: Overall, 2749 patients were analyzed. Predictors (risk score coefficient) of DMs in the discovery cohort were the following: pathological stage (p)T3-4 (0.4), pN+ (N1: 0.8; N2: 1.0; N3: 1.5), histologic grade (G) 3 (G3, 0.7), and lymphovascular invasion (0.4). The DM risk groups were defined by the sum of the following risk score coefficients: high (>1.7), intermediate (0.7-1.7), and standard risk (<0.7). The 5-year DM rates (high/intermediate/standard risk groups) were 30%/15%/4% in the discovery cohort (C-index = 0.79) and 35%/16%/5% in the validation cohort, respectively (C-index = 0.77; both P < .001). In the whole cohort, this predictive model showed excellent discriminative ability in predicting DMs without locoregional failure (29%/11%/1%), later (>2 year) DMs (11%/4%/2%), and DMs in patients treated with surgery (20%/12%/5%), postoperative radiation therapy (34%/17%/4%), and postoperative chemoradiation therapy (39%/18%/7%) (all P < .001). The 5-year overall survival rates in the overall cohort were 25%/51%/67% (P < .001). CONCLUSIONS: Patients at higher risk for DMs were identified by use of a predictive-score model for DMs that included pT3-4, pN1/2/3, G3, and lymphovascular invasion. Identified patients may be evaluated for individualized risk-adaptive treatment escalation and/or surveillance strategies.

Development and Validation of an Oral Cavity Cancer Outcomes Prediction Score Incorporating Patient-Derived Xenograft Engraftment.

IMPORTANCE: Patient-derived xenografts (PDXs) offer the opportunity to identify patients with oral cavity squamous cell carcinoma (OSCC) who are at risk for recurrence and optimize clinical decision-making. OBJECTIVE: To develop and validate a prediction score for locoregional failure (LRF) and distant metastases (DM) in OSCC that incorporates PDX engraftment in addition to known clinicopathological risk factors. DESIGN, SETTING, AND PARTICIPANTS: In this retrospective cohort study, PDX models were generated from patients with OSCC treated with curative intent at Princess Margaret Cancer Centre (Toronto, Canada) between 2006 and 2018. The cohort included 288 patients (aged ≥18 years) with a new diagnosis of nonmetastatic (M0) OSCC whose tumor samples were available for engraftment under the skin of xenograft mice. Patients were scored as a nonengrafter if PDX formation did not occur within 6 months. Data analysis was performed between August 2006 and May 2018. INTERVENTIONS: All patients received up-front curative-intent surgery followed by either observation or postoperative radiation with or without concurrent chemotherapy based on institutional guidelines. MAIN OUTCOMES AND MEASURES: Main outcomes were LRF, DM, and overall survival (OS). Multivariable analysis (MVA) was used to identify predictors of LRF and DM. Factors retained in the final MVA were used to construct a prediction score and classify patients into risk groups. RESULTS: Overall, 288 patients (mean [SD] age at diagnosis, 63.3 [12.3] years; 112 [39%] women and 176 [61%] men) with OSCC were analyzed. The MVA identified pT3-4, pathologic extranodal extension, and engraftment as predictors of LRF and DM. Patients whose tumors engrafted (n = 198) were more likely to develop LRF (hazard ratio [HR], 1.98; 95% CI, 1.24-3.18) and DM (HR, 2.64; 95% CI, 1.21-5.75) compared with nonengrafters. A prediction score based on the aforementioned variables identified patients at high risk and low risk for LRF (43.5% vs 26.5%), DM (38.2% vs 8.4%), and inferior OS (34% vs 66%) at 5 years. Additionally, rapid engraftment was shown to be similarly prognostic, with rapid engrafters demonstrating higher rates of relapse and poor OS. CONCLUSIONS: In this cohort study, a prediction score using OSCC PDX engraftment, in conjunction with pT3-4 and pathologic extranodal extension, was associated with improved prognostic utility of existing clinical models and predicted patients at risk for LRF, DM, and poor survival.

Cancer Patient-Reported Preferences and Knowledge for Liquid Biopsies and Blood Biomarkers at a Comprehensive Cancer Center.

BACKGROUND: Blood-based biomarkers (liquid biopsy) are increasingly used in precision oncology. Yet, little is known about cancer patients' perspectives in clinical practice. We explored patients' depth of preferences for liquid vs tissue biopsies and knowledge regarding the role of blood biomarkers on their cancer. METHODS: Three interviewer-administered trade-off scenarios and a 54-item self-administered questionnaire were completed by cancer outpatients across all disease sites at the Princess Margaret Cancer Centre. RESULTS: Of 413 patients, 54% were female; median age was 61 (range 18-101) years. In trade-off scenario preference testing, 90% (n=372) preferred liquid over tissue biopsy at baseline; when wait times for their preferred test were increased from 2 weeks, patients tolerated an additional mean of 1.8 weeks (SD 2.1) for liquid biopsy before switching to tissue biopsy (with wait time 2 weeks). Patients also tolerated a 6.2% decrease (SD 8.8) in the chance that their preferred test would conclusively determine optimal treatment before switching from the baseline of 80%. 216 patients (58%) preferred liquid biopsy even with no chance of adverse events from tissue biopsy. Patients' knowledge of blood-based biomarkers related to their cancer was low (mean 23%); however, the majority viewed development of blood biomarkers as important. CONCLUSION: Patients had limited understanding of cancer-specific blood-based biomarkers, but 90% preferred liquid over tissue biopsies to assess biomarkers. There was little tolerance to wait longer for results, or for decreased test-conclusiveness. Developing accurate, low-risk tests for cancer diagnosis and management for blood biomarkers is therefore desirable to patients.

Rearrangement bursts generate canonical gene fusions in bone and soft tissue tumors.

Sarcomas are cancers of the bone and soft tissue often defined by gene fusions. Ewing sarcoma involves fusions between EWSR1, a gene encoding an RNA binding protein, and E26 transformation-specific (ETS) transcription factors. We explored how and when EWSR1-ETS fusions arise by studying the whole genomes of Ewing sarcomas. In 52 of 124 (42%) of tumors, the fusion gene arises by a sudden burst of complex, loop-like rearrangements, a process called chromoplexy, rather than by simple reciprocal translocations. These loops always contained the disease-defining fusion at the center, but they disrupted multiple additional genes. The loops occurred preferentially in early replicating and transcriptionally active genomic regions. Similar loops forming canonical fusions were found in three other sarcoma types. Chromoplexy-generated fusions appear to be associated with an aggressive form of Ewing sarcoma. These loops arise early, giving rise to both primary and relapse Ewing sarcoma tumors, which can continue to evolve in parallel.

Transcriptome-wide characterization of the endogenous miR-34A-p53 tumor suppressor network.

microRNA-34A is a critical component of the p53 network and expression of miR- 34A is down-regulated by promoter hypermethylation or focal deletions in numerous human cancers. Although miR-34A deregulation may be an important driver in cancer, the endogenous role of this microRNA in cellular homeostasis is not well characterized. To address this knowledge gap, we aimed to determine the transcriptional landscape of the miR-34A-p53 axis in non-transformed cells. Using primary skin-derived fibroblast cell lines from patients who developed childhood cancers, and who harbor either germline TP53 mutations or are TP53 wild type, we sought to characterize the transcriptional response to miR-34A modulation. Through transcriptome-wide RNA-Sequencing, we show for the first time that in human non- transformed cells harboring TP53 mutations, miR-34A functions in a noncanonical manner to influence noncoding RNA networks, including RNA components of the minor (U12) spliceosome, as well as TP53-dependent and independent epigenetic pathways. miR- 34A-regulated transcripts include known cell cycle mediators and abrogation of miR-34A leads to a TP53-dependent increase in the fraction of cells in G2/M. Collectively, these results provide a framework for understanding the endogenous role of the miR-34A signaling axis and identify novel transcripts and pathways regulated by the essential miR-34A-p53 tumor suppressor network.

Genome-Wide DNA Methylation Analysis Reveals Epigenetic Dysregulation of MicroRNA-34A in TP53-Associated Cancer Susceptibility.

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.

A functional variant in miR-605 modifies the age of onset in Li-Fraumeni syndrome.

Li-Fraumeni syndrome (LFS) is a rare cancer predisposition syndrome, typically associated with germline mutations in the TP53 gene. Despite the high penetrance of TP53 mutations, LFS patients display striking phenotypic differences, suggesting the presence of secondary risk loci. To date, all genetic modifiers in LFS have been shown to map to either TP53 or its principal negative regulator, MDM2. Given this strong association, we set out to investigate the contribution of miR-605, a recently described microRNA (miRNA) regulator of the p53-MDM2 loop. We hypothesized that, if functional, the miR-605 gene and its variant (rs2043556) could impact the cancer risk profile of TP53 mutation carriers. Consistent with this proposition, the variant G-allele of miR-605 was associated with a 10-year acceleration in the mean age of LFS tumor onset (P = 0.04) and caused a 2.6-fold reduction in the processing levels of its host miRNA (P < 0.05). We also demonstrate that miR-605 overexpression leads to a decrease in cell proliferation, clonogenicity, and migration in two rhabdomyosarcoma cell lines carrying hotspot TP53 mutations. Together, our results implicate miR-605 as a novel modifier gene of the LFS phenotype and a promising therapeutic target in TP53 mutant cancers.