Lynch syndrome is an autosomal-dominant hereditary condition predisposing to the development of specific cancers, because of germline mutations in the DNA-mismatch repair (MMR) genes. Large genomic ...deletions represent a significant fraction of germline mutations, particularly among the MSH2 gene, in which they account for 20% of the mutational spectrum. In this study we analyzed 13 Italian families carrying MSH2 exon 8 deletions, 10 of which of ascertained Sardinian origin. The overrepresentation of Sardinians was unexpected, as families from Sardinia account for a small quota of MMR genes mutation tests performed in our laboratory. The hypothesis that such a result is owing to founder effects in Sardinia was tested by breakpoint junctions sequencing and haplotype analyses. Overall, five different exon eight deletions were identified, two of which recurrent in families, all apparently unrelated, of Sardinian origin (one in eight families, one in two families). The c.1277-1180_1386+2226del3516insCATTCTCTTTGAAAA deletion shares the same haplotype between all families and appears so far restricted to the population of South-West Sardinia, showing the typical features of a founder effect. The three non-Sardinian families showed three different breakpoint junctions and haplotypes, suggesting independent mutational events. This work has useful implications in genetic testing for Lynch syndrome. We developed a quick test for each of the identified deletions: this can be particularly useful in families of Sardinian origin, in which MSH2 exon 8 deletions may represent 50% of the overall mutational spectrum of the four MMR genes causing Lynch syndrome.
The
MLH1
c.2252_2253delAA mutation was found in 11 unrelated families from a restricted area south-west of Turin among 140 families with mutations in the mismatch repair genes. The mutation is ...located in the highly conserved C-terminal region, responsible for dimerization with the PMS2 protein. Twenty-five tumour tissues from 61 individuals with the c.2252_2253delAA mutation were tested for microsatellite instability (MSI) and protein expression. We compared the clinical features of these families versus the rest of our cohort and screened for a founder effect. All but one tumours showed the MSI-high mutator phenotype. Normal, focal and lack of
MLH1
staining were observed in 16, 36 and 48 % of tumours, respectively. PMS2 expression was always lost. The mutation co-segregated with Lynch syndrome-related cancers in all informative families. All families but one fulfilled Amsterdam criteria, a frequency higher than in other
MLH1
mutants. This was even more evident for AC II (72.7 vs. 57.5 %). Moreover, all families had at least one colon cancer diagnosed before 50 years and one case with multiple Lynch syndrome-related tumours. Interestingly, a statistically significant (
p
= 0.0057) higher frequency of pancreatic tumours was observed compared to families with other
MLH1
mutations: 8.2 % of affected individuals versus 1.6 %. Haplotype analysis demonstrated a common ancestral origin of the mutation, which originated about 1,550 years ago. The mutation is currently classified as having an uncertain clinical significance. Clinical features, tissue analysis and co-segregation with disease strongly support the hypothesis that the
MLH1
c.2252_2253delAA mutation has a pathogenic effect.
Large TSC gene rearrangements are not rare findings in tuberous sclerosis. Interestingly, all deletions, duplications and inversions so far described involve TSC2, none being associated with TSC1. In ...order to shed light on the structural basis of the preferential DNA rearrangements in TSC2 over TSC1 and to assess, in an unselected patient population, the prevalence of large re-arrangements in both TSC loci, we screened 202 tuberous sclerosis patients consecutively referred at our center. Southern blot analysis on EcoRI+HindIII double-digested DNA identified 19 partial or full-length gene deletions: three involved TSC1 and sixteen TSC2. The breakpoint sequence of seven internal deletions, three in TSC1 and four in TSC2, allowed us to speculate on the mechanism favoring TSC2 unequal recombinations and to identify a deletion hot spot that lies in TSC1 and that may be relevant in the routine genetic testing of tuberous sclerosis. Briefly, three major features appear to distinguish TSC1 from TSC2 deletions: (1) deletion size: all TSC1 deletions are within the transcriptional unit, whereas 12 of the 16 TSC2 deletions have at least one external breakpoint; (2) location within the gene: all TSC1 deletions are confined to the 3'end of the gene (all three 5' breakpoints being located in intron 20) thus resulting in the same frameshift mutation following amino acid K875, whereas the TSC2 internal breakpoints appear to be scattered along the gene; (3) preference for recombinatorial sequences: six out of eight internal TSC2 breakpoints map within Alu repeats, whereas none of the three TSC1 deletions appear to be Alu-mediated. Indeed, in the latter gene, unique structural features (a purine-rich tract flanked by pyrimidine-rich segments) surrounding one of the two identified breakpoint cluster regions might play a role in promoting inappropriate recombinations.
The clinical management of pancreatic ductal adenocarcinoma (PDAC) is hampered by the lack of reliable biomarkers. This study investigated the value of soluble stroma‐related molecules as PDAC ...biomarkers. In the first exploratory phase, 12 out of 38 molecules were associated with PDAC in a cohort of 25 PDAC patients and 16 healthy subjects. A second confirmatory phase on an independent cohort of 131 PDAC patients, 30 chronic pancreatitis patients, and 131 healthy subjects confirmed the PDAC association for MMP7, CCN2, IGFBP2, TSP2, sICAM1, TIMP1, and PLG. Multivariable logistic regression model identified biomarker panels discriminating respectively PDAC versus healthy subjects (MMP7 + CA19.9, AUC = 0.99, 99% CI = 0.98–1.00) (CCN2 + CA19.9, AUC = 0.96, 99% CI = 0.92–0.99) and PDAC versus chronic pancreatitis (CCN2 + PLG+FN+Col4 + CA19.9, AUC = 0.94, 99% CI = 0.88–0.99). Five molecules were associated with PanIN development in two GEM models of PDAC (PdxCre/LSL‐KrasG12D and PdxCre/LSL‐KrasG12D/+/LSL‐Trp53R172H/+), suggesting their potential for detecting early disease. These markers were also elevated in patient‐derived orthotopic PDAC xenografts and associated with response to chemotherapy. The identified stroma‐related soluble biomarkers represent potential tools for PDAC diagnosis and for monitoring treatment response of PDAC patients.
Synopsis
Seven stroma‐related circulating biomarkers that discriminate between healthy subjects and pancreatic ductal adenocarcinoma (PDAC) in two different cohorts of patients have been identified and suggested as a potential diagnosis tool to monitor treatment efficacy in patients.
Panels of biomarkers that improve the performance of CA19.9 in distinguishing i) PDAC from healthy subjects and ii) PDAC from chronic pancreatitis have been developed. Multivariable models confirmed their predictive accuracy at early stages of disease.
The association of these biomarkers with the development of pre‐invasive disease in GEM models confirmed their potential to detect early stage lesions.
The correlation of biomarker levels with tumor burden and drug response in patient‐derived PDAC xenograft models indicate their value to monitor treatment response and efficacy.
Seven stroma‐related circulating biomarkers that discriminate between healthy subjects and pancreatic ductal adenocarcinoma (PDAC) in two different cohorts of patients have been identified and suggested as a potential diagnosis tool to monitor treatment efficacy in patients.
Abstract
Inflammation is an important biological process involved in many target organ toxicities. However, there has been little consensus on how to represent inflammatory processes using the ...adverse outcome pathway (AOP) framework. In particular, there were concerns that inflammation was not being represented in a way that it would be recognized as a highly connected, central node within the global AOP network. The consideration of salient features common to the inflammatory process across tissues was used as a basis to propose 3 hub key events (KEs) for use in AOP network development. Each event, “tissue resident cell activation”, “increased pro-inflammatory mediators”, and “leukocyte recruitment/activation,” is viewed as a hallmark of inflammation, independent of tissue, and can be independently measured. Using these proposed hub KEs, it was possible to link together a series of AOPs that previously had no shared KEs. Significant challenges remain with regard to accurate prediction of inflammation-related toxicological outcomes even if a broader and more connected network of inflammation-centered AOPs is developed. Nonetheless, the current proposal addresses one of the major hurdles associated with representation of inflammation in AOPs and may aid fit-for-purpose evaluations of other AOPs operating in a network context.
Chronic inflammatory diseases are increasing in developed societies, thus new anti-inflammatory approaches are needed in the clinic. Synthetic peptides complexes can be designed to mimic the activity ...of anti-inflammatory mediators, in order to alleviate inflammation. Here, we evaluated the anti-inflammatory efficacy of tethered peptides mimicking the interleukin-1 receptor antagonist (IL-1Ra) and the heat-shock protein 70 (HSP70). We tested their biocompatibility and anti-inflammatory activity in vitro in primary human monocytes and differentiated macrophages activated with two different stimuli: the TLR agonists (LPS + IFN-γ) or Pam3CSK4. Our results demonstrate that IL-1Ra and HSP70 synthetic peptides present a satisfactory biocompatible profile and significantly inhibit the secretion of several pro-inflammatory cytokines (IL-6, IL-8, IL-1β and TNFα). We further confirmed their anti-inflammatory activity when peptides were coated on a biocompatible material commonly employed in surgical implants. Overall, our findings support the potential use of IL-1Ra and HSP70 synthetic peptides for the treatment of inflammatory conditions.
Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC), the major lung cancer subtype, is characterized by high resistance to chemotherapy. Here we ...demonstrate that Tudor staphylococcal nuclease (SND1 or TSN) is overexpressed in NSCLC cell lines and tissues, and is important for maintaining NSCLC chemoresistance. Downregulation of TSN by RNAi in NSCLC cells led to strong potentiation of cell death in response to cisplatin. Silencing of TSN was accompanied by a significant decrease in S100A11 expression at both mRNA and protein level. Downregulation of S100A11 by RNAi resulted in enhanced sensitivity of NSCLC cells to cisplatin, oxaliplatin and 5-fluouracil. AACOCF(3), a phospholipase A(2) (PLA(2)) inhibitor, strongly abrogated chemosensitization upon silencing of S100A11 suggesting that PLA(2) inhibition by S100A11 governs the chemoresistance of NSCLC. Moreover, silencing of S100A11 stimulated mitochondrial superoxide production, which was decreased by AACOCF(3), as well as N-acetyl-L-cysteine, which also mimicked the effect of PLA(2) inhibitor on NSCLC chemosensitization upon S100A11 silencing. Thus, we present the novel TSN-S100A11-PLA(2) axis regulating superoxide-dependent apoptosis, triggered by platinum-based chemotherapeutic agents in NSCLC that may be targeted by innovative cancer therapies.
Juvenile myelomonocytic leukaemia (JMML) and chronic myelomonocytic leukaemia (CMML) are myelodysplastic myeloproliferative (MDS/MPN) neoplasms with unfavourable prognosis and without effective ...chemotherapy treatment. Trabectedin is a DNA minor groove binder acting as a modulator of transcription and interfering with DNA repair mechanisms; it causes selective depletion of cells of the myelomonocytic lineage. We hypothesised that trabectedin might have an antitumour effect on MDS/MPN.
Malignant CD14+ monocytes and CD34+ haematopoietic progenitor cells were isolated from peripheral blood/bone marrow mononuclear cells. The inhibition of CFU-GM colonies and the apoptotic effect on CD14+ and CD34+ induced by trabectedin were evaluated. Trabectedin's effects were also investigated in vitro on THP-1, and in vitro and in vivo on MV-4-11 cell lines.
On CMML/JMML cells, obtained from 20 patients with CMML and 13 patients with JMML, trabectedin - at concentration pharmacologically reasonable, 1-5 nM - strongly induced apoptosis and inhibition of growth of haematopoietic progenitors (CFU-GM). In these leukaemic cells, trabectedin downregulated the expression of genes belonging to the Rho GTPases pathway (RAS superfamily) having a critical role in cell growth and cytoskeletal dynamics. Its selective activity on myelomonocytic malignant cells was confirmed also on in vitro THP-1 cell line and on in vitro and in vivo MV-4-11 cell line models.
Trabectedin could be good candidate for clinical studies in JMML/CMML patients.
Thioredoxin, despite its function as an intracellular disulfide reducing enzyme and its lack of a signal sequence, has been found to play some roles extracellularly. Here we show that thioredoxin is ...actively secreted by a variety of normal and transformed cells, including fibroblasts, airway epithelial cells, and activated B and T lymphocytes. Neither brefeldin A nor dinitrophenol, two drugs that block transport through the exocytic pathway, inhibit secretion of thioredoxin, indicating that the latter does not follow the classical ER-Golgi route. The secretory mechanism for thioredoxin shares several features with the alternative pathway described for interleukin-1 beta, such as the potentiating effect on secretion of several unrelated drugs and the sensitivity to methylamine. However, unlike interleukin-1 beta, thioredoxin is not detected in membrane-bound compartments of secreting cells. In addition, when COS7 are transfected with plasmids encoding pro-interleukin-1 beta or thioredoxin, only the latter is detectable extracellularly.
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