De novo germline pathogenic variant in Lynch Syndrome: A rare event or the tip of the iceberg?

Lynch Syndrome is an autosomal dominant cancer predisposition syndrome caused by germline pathogenic variants or epimutation in one of the DNA mismatch repair genes. De novo pathogenic variants in mismatch repair genes have been described as a rare event in Lynch Syndrome (1-5%), although the prevalence of de novo pathogenic variants in Lynch Syndrome is probably underestimated. The de novo pathogenic variant was identified in a 26-year-old woman diagnosed with an adenocarcinoma of the caecum with mismatch repair protein deficiency at immunohistochemistry and a synchronous neuroendocrine tumor of the appendix with normal expression of mismatch repair proteins. DNA testing revealed deletion of exon 6 of the MLH1 gene. It appeared to be a de novo event, as the deletion was not detected in the patient's parents. The presence of a mosaicism in the patient was excluded and haplotype analysis demonstrated the paternal origin of the chromosome harboring the deletion. The de novo deletion probably originated either from a very early postzygotic or a single prezygotic mutational event, or from a gonadal mosaicism. In conclusion, the identification of de novo pathogenic variants is crucial to allow proper genetic counseling and appropriate management of the patient's family.

Exploring the association with disease recurrence of miRNAs predictive of colorectal cancer.

INTRODUCTION: Disease recurrence after surgery is a crucial predictor of poor prognosis in colorectal cancer, where disseminated disease at the time of intervention can also be observed in localized early-stage cases. We evaluated the ability to predict disease recurrence of miRNAs from two signatures that we have found linked to the presence of colorectal cancer (CL signature) or adenoma (HgA signature) in higher-risk subjects. METHODS: miRNAs from the signatures were studied longitudinally by quantitative real-time polymerase chain reaction in plasma from 24 patients with resectable colorectal cancer collected at the time of surgery and during scheduled follow-up across 36 months. Patients either showed relapse within 36 months (alive with disease (AWD)), or remained disease-free (no evidence of disease (NED)) for the same period. RESULTS: Although the signatures did not predict recurrence, expression of the miRNAs from the CL signature decreased 1 year after surgery, and one miRNA of the signature, miR-378a-3p, almost reached significance in the NED subgroup (Wilcoxon signed-rank test: p-value = 0.078). Also, miR-335-5p from the HgA signature was higher in AWD patients before surgery (Kruskal-Wallis test: p-value = 0.019). CONCLUSIONS: These data, although from a small cohort of patients, support the possible use of miRNAs as non-invasive biomarkers in liquid biopsy-based tests to identify patients at risk of relapse and to monitor them during follow-up.

Plasma miRNA-based signatures in CRC screening programs.

Colorectal cancer (CRC) screening programs help diagnose cancer precursors and early cancers and help reduce CRC mortality. However, currently recommended tests, the fecal immunochemical test (FIT) and colonoscopy, have low uptake. There is therefore a pressing need for screening strategies that are minimally invasive and consequently more acceptable to patients, most likely blood based, to increase early CRC identification. MicroRNAs (miRNAs) released from cancer cells are detectable in plasma in a remarkably stable form, making them ideal cancer biomarkers. Using plasma samples from FIT-positive (FIT+) subjects in an Italian CRC screening program, we aimed to identify plasma circulating miRNAs that detect early CRC. miRNAs were initially investigated by quantitative real-time PCR in plasma from 60 FIT+ subjects undergoing colonoscopy at Fondazione IRCCS Istituto Nazionale dei Tumori, then tested on an internal validation cohort (IVC, 201 cases) and finally in a large multicenter prospective series (external validation cohort [EVC], 1121 cases). For each endoscopic lesion (low-grade adenoma [LgA], high-grade adenoma [HgA], cancer lesion [CL]), specific signatures were identified in the IVC and confirmed on the EVC. A two-miRNA-based signature for CL and six-miRNA signatures for LgA and HgA were selected. In a multivariate analysis including sex and age at blood collection, the areas under the receiver operating characteristic curve (95% confidence interval) of the signatures were 0.644 (0.607-0.682), 0.670 (0.626-0.714) and 0.682 (0.580-0.785) for LgA, HgA and CL, respectively. A miRNA-based test could be introduced into the FIT+ workflow of CRC screening programs so as to schedule colonoscopies only for subjects likely to benefit most.

Bone Metastasis Phenotype and Growth Undergo Regulation by Micro-Environment Stimuli: Efficacy of Early Therapy with HGF or TGFß1-Type I Receptor Blockade.

Hepatocyte growth factor (HGF) and transforming growth factor ß1 (TGFß1) are biological stimuli of the micro-environment which affect bone metastasis phenotype through transcription factors, but their influence on the growth is scarcely known. In a xenograft model prepared with 1833 bone metastatic cells, derived from breast carcinoma cells, we evaluated mice survival and Twist and Snail expression and localization after competitive inhibition of HGF with NK4, or after blockade of TGFß1-type I receptor (RI) with SB431542: in the latter condition HGF was also measured. To explain the in vivo data, in 1833 cells treated with SB431542 plus TGFß1 we measured HGF formation and the transduction pathway involved. Altogether, HGF seemed relevant for bone-metastatic growth, being hampered by NK4 treatment, which decreased Twist more than Snail in the metastasis bulk. TGFß1-RI blockade enhanced HGF in metastasis and adjacent bone marrow, while reducing prevalently Snail expression at the front and bulk of bone metastasis. The HGF accumulation in 1833 cells depended on an auxiliary signaling pathway, triggered by TGFß1 under SB431542, which interfered in the transcription of HGF activator inhibitor type 1 (HAI-1) downstream of TGFß-activated kinase 1 (TAK1): HGF stimulated Twist transactivation. In conclusion, the impairment of initial outgrowth with NK4 seemed therapeutically promising more than SB431542 chemotherapy; a functional correlation between Twist and Snail in bone metastasis seemed to be influenced by the biological stimuli of the micro-environment, and the targeting of these phenotype biomarkers might inhibit metastasis plasticity and colonization, even if it would be necessary to consider the changes of HGF levels in bone metastases undergoing TGFß1-RI blockade.

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor.

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Oxidative stress activates a specific p53 transcriptional response that regulates cellular senescence and aging.

Oxidative stress is a determining factor of cellular senescence and aging and a potent inducer of the tumour-suppressor p53. Resistance to oxidative stress correlates with delayed aging in mammals, in the absence of accelerated tumorigenesis, suggesting inactivation of selected p53-downstream pathways. We investigated p53 regulation in mice carrying deletion of p66, a mutation that retards aging and confers cellular resistance and systemic resistance to oxidative stress. We identified a transcriptional network of ~200 genes that are repressed by p53 and encode for determinants of progression through mitosis or suppression of senescence. They are selectively down-regulated in cultured fibroblasts after oxidative stress, and, in vivo, in proliferating tissues and during physiological aging. Selectivity is imposed by p66 expression and activation of p44/p53 (also named Delta40p53), a p53 isoform that accelerates aging and prevents mitosis after protein damage. p66 deletion retards aging and increases longevity of p44/p53 transgenic mice. Thus, oxidative stress activates a specific p53 transcriptional response, mediated by p44/p53 and p66, which regulates cellular senescence and aging.

An autoinflammatory neurological disease due to interleukin 6 hypersecretion.

Autoinflammatory diseases are rare illnesses characterized by apparently unprovoked inflammation without high-titer auto-antibodies or antigen-specific T cells. They may cause neurological manifestations, such as meningitis and hearing loss, but they are also characterized by non-neurological manifestations. In this work we studied a 30-year-old man who had a chronic disease characterized by meningitis, progressive hearing loss, persistently raised inflammatory markers and diffuse leukoencephalopathy on brain MRI. He also suffered from chronic recurrent osteomyelitis of the mandible. The hypothesis of an autoinflammatory disease prompted us to test for the presence of mutations in interleukin-1-pathway genes and to investigate the function of this pathway in the mononuclear cells obtained from the patient. Search for mutations in genes associated with interleukin-1-pathway demonstrated a novel NLRP3 (CIAS1) mutation (p.I288M) and a previously described MEFV mutation (p.R761H), but their combination was found to be non-pathogenic. On the other hand, we uncovered a selective interleukin-6 hypersecretion within the central nervous system as the likely pathogenic mechanism. This is also supported by the response to the anti-interleukin-6-receptor monoclonal antibody tocilizumab, but not to the recombinant interleukin-1-receptor antagonist anakinra. Exome sequencing failed to identify mutations in other genes known to be involved in autoinflammatory diseases. We propose that the disease described in this patient might be a prototype of a novel category of autoinflammatory diseases characterized by prominent neurological involvement.

Differentiation-associated microRNAs antagonize the Rb-E2F pathway to restrict proliferation.

The cancer-associated loss of microRNA (miRNA) expression leads to a proliferative advantage and aggressive behavior through largely unknown mechanisms. Here, we exploit a model system that recapitulates physiological terminal differentiation and its reversal upon oncogene expression to analyze coordinated mRNA/miRNA responses. The cell cycle reentry of myotubes, forced by the E1A oncogene, was associated with a pattern of mRNA/miRNA modulation that was largely reciprocal to that induced during the differentiation of myoblasts into myotubes. The E1A-induced mRNA response was preponderantly Retinoblastoma protein (Rb)-dependent. Conversely, the miRNA response was mostly Rb-independent and exerted through tissue-specific factors and Myc. A subset of these miRNAs (miR-1, miR-34, miR-22, miR-365, miR-29, miR-145, and Let-7) was shown to coordinately target Rb-dependent cell cycle and DNA replication mRNAs. Thus, a dual level of regulation-transcriptional regulation via Rb-E2F and posttranscriptional regulation via miRNAs-confers robustness to cell cycle control and provides a molecular basis to understand the role of miRNA subversion in cancer.

A serum circulating miRNA diagnostic test to identify asymptomatic high-risk individuals with early stage lung cancer.

Lung cancer is the first cause of cancer mortality worldwide, and its early detection is currently the main available strategy to improve disease prognosis. While early diagnosis can be successfully achieved through tomography-based population screenings in high-risk individuals, simple methodologies are needed for effective cancer prevention programs. We developed a test, based on the detection of 34 microRNAs (miRNAs) from serum, that could identify patients with early stage non-small cell lung carcinomas (NSCLCs) in a population of asymptomatic high-risk individuals with 80% accuracy. The signature could assign disease probability accurately either in asymptomatic or symptomatic patients, is able to distinguish between benign and malignant lesions, and to capture the onset of the malignant disease in individual patients over time. Thus, our test displays a number of features of clinical relevance that project its utility in programs for the early detection of NSCLC.

Common breast cancer susceptibility alleles and the risk of breast cancer for BRCA1 and BRCA2 mutation carriers: implications for risk prediction.

The known breast cancer susceptibility polymorphisms in FGFR2, TNRC9/TOX3, MAP3K1, LSP1, and 2q35 confer increased risks of breast cancer for BRCA1 or BRCA2 mutation carriers. We evaluated the associations of 3 additional single nucleotide polymorphisms (SNPs), rs4973768 in SLC4A7/NEK10, rs6504950 in STXBP4/COX11, and rs10941679 at 5p12, and reanalyzed the previous associations using additional carriers in a sample of 12,525 BRCA1 and 7,409 BRCA2 carriers. Additionally, we investigated potential interactions between SNPs and assessed the implications for risk prediction. The minor alleles of rs4973768 and rs10941679 were associated with increased breast cancer risk for BRCA2 carriers (per-allele HR = 1.10, 95% CI: 1.03-1.18, P = 0.006 and HR = 1.09, 95% CI: 1.01-1.19, P = 0.03, respectively). Neither SNP was associated with breast cancer risk for BRCA1 carriers, and rs6504950 was not associated with breast cancer for either BRCA1 or BRCA2 carriers. Of the 9 polymorphisms investigated, 7 were associated with breast cancer for BRCA2 carriers (FGFR2, TOX3, MAP3K1, LSP1, 2q35, SLC4A7, 5p12, P = 7 × 10(-11) - 0.03), but only TOX3 and 2q35 were associated with the risk for BRCA1 carriers (P = 0.0049, 0.03, respectively). All risk-associated polymorphisms appear to interact multiplicatively on breast cancer risk for mutation carriers. Based on the joint genotype distribution of the 7 risk-associated SNPs in BRCA2 mutation carriers, the 5% of BRCA2 carriers at highest risk (i.e., between 95th and 100th percentiles) were predicted to have a probability between 80% and 96% of developing breast cancer by age 80, compared with 42% to 50% for the 5% of carriers at lowest risk. Our findings indicated that these risk differences might be sufficient to influence the clinical management of mutation carriers.

Myc-binding-site recognition in the human genome is determined by chromatin context.

Large-scale chromatin immunoprecipitation (ChIP) studies have been effective in unravelling the distribution of DNA-binding transcription factors along eukaryotic genomes, but specificity determinants remain elusive. Gene-regulatory regions display distinct histone variants and modifications (or marks). An attractive hypothesis is that these marks modulate protein recognition, but whether or not this applies to transcription factors remains unknown. Based on large-scale datasets and quantitative ChIP, we dissect the correlations between 35 histone marks and genomic binding by the transcription factor Myc. Our data reveal a relatively simple combinatorial organization of histone marks in human cells, with a few main groups of marks clustering on distinct promoter populations. A stretch of chromatin bearing high H3 K4/K79 methylation and H3 acetylation (or 'euchromatic island'), which is generally associated with a pre-engaged basal transcription machinery, is a strict pre-requisite for recognition of any target site by Myc (whether the consensus CACGTG or an alternative sequence). These data imply that tethering of a transcription factor to restricted chromatin domains is rate-limiting for sequence-specific DNA binding in vivo.