The Microsatellite instability phenotype in human colorectal carcinoma

Il carcinoma del colon-retto e' una delle neoplasie con maggior incidenza nella popolazione generale ed una delle maggiori cause di morte per neoplasia, in entrambi i sessi. L’acquisizione di una diffusa instabilità genetica e' essenziale per il suo sviluppo. Instabilità a livello dei microsatelliti o MSI, e' comunemente causata da un’alterazione a livello dei geni del MisMatch Repair (MMR) system, meccanismo multienzimatico deputato al mantenimento della stabilità dell'informazione genetica, che corregge i disappaiamenti di base e le anse di inserzione/delezione che si originano durante la replicazione del DNA. Instabilità microsatellitare si osserva nella Sindrome di Lynch, la forma più comune di carcinoma del colon-retto su base ereditaria, causata da difetti nella linea germinale di uno dei geni del MMR, e nel 10-15% dei carcinomi sporadici del colon-retto per silencing epigenetico del gene hMLH1. Nella Syndrome di Lynch la carcinogenesi procede attraverso la sequenza adenoma-carcinoma. Tuttavia, sebbene il numero di adenomi in pazienti con Lynch sia simile a quello della popolazione generale, questi si osservano in età più precoce, hanno una predilezione per il colon prossimale, sono più voluminosi, classificati istologicamente come villosi, e crescono rapidamente e progrediscono in una forma invasive in meno di tre anni. Uno dei maggiori problemi in individui con sospetta Sindrome di Lynch e' l’assenza di tessuto tumorale su cui effettuare tests di instabilità microsatellitare. Nella presente tesi, utilizzando una popolazione altamente selezionata di individui affetti da Sindrome di Lynch, dimostriamo come l’analisi molecolare di adenomi colo-rettali possa avere un ruolo nella diagnostica. Utilizzando un’analisi di instabilità dei microsatelliti e la valutazione immunoistochimica dell’espressione delle proteine codificate dai geni del MMR su tessuto tumorale incluso in paraffina, abbiamo infatti identificato un difetto nel MMR system nel 73% degli adenomi asportati. Le singole metodiche hanno identificato alterazioni nel 58% e 70% dei casi, rispettivamente. Così nel work-up di individui con sospetta Lynch, e’ ragionevole iniziare con studi di MSI ed IHC su adenomi, ed i nostri dati suggeriscono che l’IHC da sola e’ sensibile quanto un approccio combinato. Sebbene un risultato positivo può dirigere la successiva analisi genetica, un risultato negativo su un adenoma deve essere interpretato con cautela e non dovrebbe essere utilizzato per escludere la diagnosi se altre caratteristiche cliniche suggeriscono la presenza di Lynch. Tumori del colon-retto con instabilità microsatellitare sono caratterizzati da una prognosi particolarmente favorevole ma, ad oggi, le basi molecolari di tale fenomeno non sono note. Il gene più frequentemente mutato in tumori con MSI e’ il recettore di tipo II per il transforming growth factor-β (TGFBR2), il cui signaling media effetti soppressori nelle fasi precoci di sviluppo tumorale, mentre nelle fasi più tardive incrementa la progressione tumorale anche attraverso l'induzione di una transizione epitelio-mesenchima (EMT). Nella presente tesi, utilizzando una serie di linee cellulari a differente stato microsatellitare e con un recettore wild-type o mutato dimostriamo come linee con instabilità microsatellitare ed un recettore mutato, al seguito del trattamento con TGF-β, non vengono indotte in EMT, al contrario di linee con MSI ma senza mutazioni in TGFBR2. Inoltre, nella nostra analisi di 129 casi di tumori del colon-retto con o senza instabilità microsatellitare, la perdita di marcatori epiteliali e l’acquisizione di marcatori mesenchimali sono associati non solo con variabili clinicopatologiche di cattiva prognosi ma anche con l'assenza di instabilita’ microsatellitare. Collettivamente, i nostri dati in vitro ed in campioni umani suggeriscono che mutazioni nel TGFBR2 interferiscono con l’induzione di EMT da parte del TGF-β e pertanto riducono le capacita’ migratorie ed invasive delle cellule tumorali. Oltre a fornire una spiegazione a base molecolare per la prognosi favorevole costantemente osservata in tumori del colon-retto con instabilità microsatellitare, questi risultati suggeriscono un razionale per l’inibizione terapeutica del signaling del TGF-β in tumori del colon-retto senza MSI.Colorectal cancer is the second to the fourth most common cancer in industrialized countries. Baseline mutation rates are insufficient to account for the multiple mutations that are required for cancer to develop. Genomic instability is now recognized as an essential cellular feature that accompanies the acquisition of these mutations. In colon cancer, at least 3 distinct pathways of genomic instability have been described, the chromosomal instability, microsatellite instability (MSI), and the so-called CpG island methylator phenotype pathways, each with distinctive tumor genotypes and phenotypes. MSI is commonly caused by loss of the DNA mismatch repair (MMR) system, which normally recognizes and repairs mismatched nucleotides and insertion/deletion loops caused by slippage of DNA polymerase. MSI occurs in hereditary as well as sporadic CRC. In Lynch syndrome, responsible for 2-5% of all CRC cases, a germline mutation in a MMR gene accounts for more than 90% of cases, whereas in 10-15% of sporadic CRCs MSI is due to loss of expression of a MMR gene (most commonly hMLH1) caused by epigenetic silencing. In Lynch syndrome, carcinogenesis proceeds through the adenoma-carcinoma sequence. Although the number of polyps in Lynch patients appears to be similar to the general population, the polyps are more likely to occur at a younger age, have a predilection for the proximal colon, be larger, display villous features or high-grade dysplasia, and most importantly, grow rapidly and progress to invasive cancer in less than 3 years. As the recognition of Lynch is increasing in the population, many individuals with suspected Lynch now undergo routine colonoscopic screening with polypectomy. In such a scenario, there is no colon cancer tissue available for MSI and IHC testing. In this thesis, we present the results of a study that was undertaken to test the hypothesis that MSI testing and IHC analysis in pre-cancerous colorectal adenomas instead of colorectal cancers may be an alternative approach to screen for Lynch syndrome. MSI analysis, IHC staining for MMR proteins or both detected DNA repair deficiency in 58%, 70%, and 73% of the Lynch-associated adenomas, respectively, and this included adenomas smaller than 5 mm. Thus, in the workup of patients suspected to have a germline MMR gene mutation, it is reasonable to begin with MSI and IHC analyses of adenomas, and our data suggest that IHC testing alone is nearly as sensitive as a combined approach. Positive results can be utilized to direct germline genetic testing. However, a negative MSI or IHC test result in an adenoma should be interpreted cautiously and cannot be used to formally exclude the diagnosis of Lynch syndrome if other clinical features suggest the diagnosis. Nevertheless, this approach would expand the diagnostic testing options in cases with suspected Lynch and increase the opportunities to recognize the syndrome before the development of invasive cancer. The clinical behavior of MSI tumors is distinctive, and the most intriguing and consistently described feature is the enhanced survival benefit that does not appear to be attributable to differences in therapeutic response. The molecular basis for the prognostic advantage due to MSI is not clearly established. The most commonly mutated gene in tumors with MSI is the transforming growth factor-β receptor II (TGFBR2) gene, which harbors an (A)10 microsatellite that undergoes a frame shift. This mutation leads to a disruption in the TGF-β signaling, which plays a dual role in tumorigenesis: in early stages it mediates tumor-suppressive effects whereas, paradoxically, at later may enhance tumor progression due to its ability to inhibit cell death from growth factor deprivation, suppress immune function, and induce an epithelial to mesenchymal transition (EMT). In this thesis we provide a potential molecular explanation for the favorable outcome observed in MSI tumors. We show that TGFBR2 mutations, observed in up to 90% of CRCs with MSI, interfere with TGF-β-induced EMT, an important component of cancer progression, and therefore reduce the migratory and invasive capabilities of cancer cells. Tumors with MSI but without TGFBR2 mutations undergo EMT in response to TGF-β1, suggesting that TGFBR2 genotype and not MSI status per se may be the key determinant of the EMT response and ultimately, prognosis. In addition, these findings suggest a rationale for the therapeutic inhibition of TGF-β signaling in MSS colorectal tumors

Prostaglandin E2 drives cyclooxygenase-2 expression via cyclic AMP response element activation in human pancreatic cancer cells.

Cyclooxygenase-2 (COX-2) is constitutively expressed in most human primary carcinomas and with its synthesized product, prostaglandin E2 (PGE2), appears to play important roles in tumor invasion, angiogenesis, resistance to apoptosis and suppression of host immunity. However, the molecular mechanisms that control COX-2 expression are unclear. The purpose of this study was to clarify the mechanism of basal and PGE2-mediated COX-2 expression in the highly metastatic L3.6pl human pancreatic cancer cell line. Using RNA interference to disrupt the expression of CREB and the NF-kappaB p65 subunit, we found that both are involved in maintaining basal COX-2 expression in L3.6pl cells. We also demonstrated that PGE2 increased the cyclic AMP concentration, thereby activating protein kinase A (PKA), which in turn phosphorylated the cyclic AMP response element binding protein (CREB), leading to interaction with the cyclic AMP response element in the promoter region of the COX-2 gene. Immunocytochemical analysis confirmed that PGE2 stimulated the translocation of PKA to the nucleus and increased the immuno-reactivity of phosphorylated CREB. Pretreatment with the PKA selective inhibitor H 89 and the E-prostanoid receptor 2 inhibitor AH 6809 reduced COX-2 upregulation by PGE2. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay results further suggested a role for CREB in COX-2 transcriptional control. Understanding the pathways that control COX-2 expression may lead to a better understanding of its dysregulation in pancreatic carcinomas and facilitate the development of novel therapeutic approaches

High glomerular filtration rate is associated with impaired arterial stiffness and subendocardial viability ratio in prediabetic subjects.

BACKGROUND AND AIMS High glomerular filtration rate (HGFR) is associated with cardiovascular damage in the setting of various conditions such as obesity and diabetes. Prediabetes was also associated with increased GFR, however, the association between prediabetes, HGFR and cardiovascular damage has not been investigated. In this study, we investigated the association between HGFR and early markers of cardiovascular disease in subjects with prediabetes. METHODS AND RESULTS Augmentation pressure (Aug), augmentation index (AIx), subendocardial viability ratio (SEVR), pulse wave velocity (PWV), intima-media thickness (IMT) and estimated GFR (eGFR) were evaluated in 230 subjects with prediabetes. The eGFR was assessed using the Chronic Kidney Disease Epidemiology Collaboration formula. HGFR was defined as an eGFR above the 75th percentile. Prediabetic subjects were divided into two groups according to presence/absence of HGFR: 61 subjects with HGFR and 169 subjects without HGFR. Subjects with HGFR showed higher Aug, AIx and lower SEVR compared with prediabetic subjects with lower eGFR (14.1 ± 7.2 vs 10.8 ± 6.2, 32.9 ± 12.7 vs 27.6 ± 11.7, 153.5 ± 27.8 vs 162 ± 30.2, p < 0.05). No differences were found in PWV and IMT values between the two groups. Then, we performed multiple regression analysis to test the relationship between Aug, SEVR and several cardiovascular risk factors. In multiple regression analysis Aug was associated with age, systolic blood pressure (BP), HOMA-IR and eGFR; the major determinants of SEVR were systolic BP, HOMA-IR and eGFR. CONCLUSION Subjects with prediabetes and HGFR exhibited an increased Aug, AIx and a reduced SEVR. These alterations are associated with eGFR, insulin resistance and systolic BP

Systematic versus on-demand early palliative care: results from a multicentre, randomised clinical trial

Background Early palliative care (EPC) in oncology has been shown to have a positive impact on clinical outcome, quality-of-care outcomes, and costs. However, the optimal way for activating EPC has yet to be defined. Methods This prospective, multicentre, randomised study was conducted on 207 outpatients with metastatic or locally advanced inoperable pancreatic cancer. Patients were randomised to receive ‘standard cancer care plus on-demand EPC’ (n&nbsp;=&nbsp;100) or ‘standard cancer care plus systematic EPC’ (n&nbsp;=&nbsp;107). Primary outcome was change in quality of life (QoL) evaluated through the Functional Assessment of Cancer Therapy – Hepatobiliary questionnaire between baseline (T0) and after 12 weeks (T1), in particular the integration of physical, functional, and Hepatic Cancer Subscale (HCS) combined in the Trial Outcome Index (TOI). Patient mood, survival, relatives' satisfaction with care, and indicators of aggressiveness of care were also evaluated. Findings The mean changes in TOI score and HCS score between T0 and T1 were −4.47 and −0.63, with a difference between groups of 3.83 (95% confidence interval [CI] 0.10–7.57) (p&nbsp;=&nbsp;0.041), and −2.23 and 0.28 (difference between groups of 2.51, 95% CI 0.40–4.61, p&nbsp;=&nbsp;0.013), in favour of interventional group. QoL scores at T1 of TOI scale and HCS were 84.4 versus 78.1 (p&nbsp;=&nbsp;0.022) and 52.0 versus 48.2 (p&nbsp;=&nbsp;0.008), respectively, for interventional and standard arm. Until February 2016, 143 (76.9%) of the 186 evaluable patients had died. There was no difference in overall survival between treatment arms. Interpretations Systematic EPC in advanced pancreatic cancer patients significantly improved QoL with respect to on-demand EPC

Refinement of the diagnostic approach for the identification of children and adolescents affected by familial hypercholesterolemia: Evidence from the LIPIGEN study

Background and aims: We aimed to describe the limitations of familiar hypercholesterolemia (FH) diagnosis in childhood based on the presence of the typical features of FH, such as physical sings of cholesterol accumulation and personal or family history of premature cardiovascular disease or hypercholesterolemia, comparing their prevalence in the adult and paediatric FH population, and to illustrate how additional information can lead to a more effective diagnosis of FH at a younger age.Methods: From the Italian LIPIGEN cohort, we selected 1188 (&gt;= 18 years) and 708 (&lt;18 years) genetically-confirmed heterozygous FH, with no missing personal FH features. The prevalence of personal and familial FH features was compared between the two groups. For a sub-group of the paediatric cohort (N = 374), data about premature coronary heart disease (CHD) in second-degree family members were also included in the evaluation.Results: The lower prevalence of typical FH features in children/adolescents vs adults was confirmed: the prevalence of tendon xanthoma was 2.1% vs 13.1%, and arcus cornealis was present in 1.6% vs 11.2% of the cohorts, respectively. No children presented clinical history of premature CHD or cerebral/peripheral vascular disease compared to 8.8% and 5.6% of adults, respectively. The prevalence of premature CHD in first-degree relatives was significantly higher in adults compared to children/adolescents (38.9% vs 19.7%). In the sub-cohort analysis, a premature CHD event in parents was reported in 63 out of 374 subjects (16.8%), but the percentage increased to 54.0% extending the evaluation also to second-degree relatives.Conclusions: In children, the typical FH features are clearly less informative than in adults. A more thorough data collection, adding information about second-degree relatives, could improve the diagnosis of FH at younger age

Identification of a novel locus on chromosome 2q13, which predisposes to clinical vertebral fractures independently of bone density.

OBJECTIVES: To identify genetic determinants of susceptibility to clinical vertebral fractures, which is an important complication of osteoporosis. METHODS: Here we conduct a genome-wide association study in 1553 postmenopausal women with clinical vertebral fractures and 4340 controls, with a two-stage replication involving 1028 cases and 3762 controls. Potentially causal variants were identified using expression quantitative trait loci (eQTL) data from transiliac bone biopsies and bioinformatic studies. RESULTS: A locus tagged by rs10190845 was identified on chromosome 2q13, which was significantly associated with clinical vertebral fracture (P=1.04×10-9) with a large effect size (OR 1.74, 95% CI 1.06 to 2.6). Bioinformatic analysis of this locus identified several potentially functional SNPs that are associated with expression of the positional candidate genes TTL (tubulin tyrosine ligase) and SLC20A1 (solute carrier family 20 member 1). Three other suggestive loci were identified on chromosomes 1p31, 11q12 and 15q11. All these loci were novel and had not previously been associated with bone mineral density or clinical fractures. CONCLUSION: We have identified a novel genetic variant that is associated with clinical vertebral fractures by mechanisms that are independent of BMD. Further studies are now in progress to validate this association and evaluate the underlying mechanism

Lipoprotein(a) Genotype Influences the Clinical Diagnosis of Familial Hypercholesterolemia

: Background Evidence suggests that LPA risk genotypes are a possible contributor to the clinical diagnosis of familial hypercholesterolemia (FH). This study aimed at determining the prevalence of LPA risk variants in adult individuals with FH enrolled in the Italian LIPIGEN (Lipid Transport Disorders Italian Genetic Network) study, with (FH/M+) or without (FH/M-) a causative genetic variant. Methods and Results An lp(a) [lipoprotein(a)] genetic score was calculated by summing the number risk-increasing alleles inherited at rs3798220 and rs10455872 variants. Overall, in the 4.6% of 1695 patients with clinically diagnosed FH, the phenotype was not explained by a monogenic or polygenic cause but by genotype associated with high lp(a) levels. Among 765 subjects with FH/M- and 930 subjects with FH/M+, 133 (17.4%) and 95 (10.2%) were characterized by 1 copy of either rs10455872 or rs3798220 or 2 copies of either rs10455872 or rs3798220 (lp(a) score ≥1). Subjects with FH/M- also had lower mean levels of pretreatment low-density lipoprotein cholesterol than individuals with FH/M+ (t test for difference in means between FH/M- and FH/M+ groups &lt;0.0001); however, subjects with FH/M- and lp(a) score ≥1 had higher mean (SD) pretreatment low-density lipoprotein cholesterol levels (223.47 [50.40] mg/dL) compared with subjects with FH/M- and lp(a) score=0 (219.38 [54.54] mg/dL for), although not statistically significant. The adjustment of low-density lipoprotein cholesterol levels based on lp(a) concentration reduced from 68% to 42% the proportion of subjects with low-density lipoprotein cholesterol level ≥190 mg/dL (or from 68% to 50%, considering a more conservative formula). Conclusions Our study supports the importance of measuring lp(a) to perform the diagnosis of FH appropriately and to exclude that the observed phenotype is driven by elevated levels of lp(a) before performing the genetic test for FH

Beta-Blocker Use in Older Hospitalized Patients Affected by Heart Failure and Chronic Obstructive Pulmonary Disease: An Italian Survey From the REPOSI Register

Beta (β)-blockers (BB) are useful in reducing morbidity and mortality in patients with heart failure (HF) and concomitant chronic obstructive pulmonary disease (COPD). Nevertheless, the use of BBs could induce bronchoconstriction due to β2-blockade. For this reason, both the ESC and GOLD guidelines strongly suggest the use of selective β1-BB in patients with HF and COPD. However, low adherence to guidelines was observed in multiple clinical settings. The aim of the study was to investigate the BBs use in older patients affected by HF and COPD, recorded in the REPOSI register. Of 942 patients affected by HF, 47.1% were treated with BBs. The use of BBs was significantly lower in patients with HF and COPD than in patients affected by HF alone, both at admission and at discharge (admission, 36.9% vs. 51.3%; discharge, 38.0% vs. 51.7%). In addition, no further BB users were found at discharge. The probability to being treated with a BB was significantly lower in patients with HF also affected by COPD (adj. OR, 95% CI: 0.50, 0.37-0.67), while the diagnosis of COPD was not associated with the choice of selective β1-BB (adj. OR, 95% CI: 1.33, 0.76-2.34). Despite clear recommendations by clinical guidelines, a significant underuse of BBs was also observed after hospital discharge. In COPD affected patients, physicians unreasonably reject BBs use, rather than choosing a β1-BB. The expected improvement of the BB prescriptions after hospitalization was not observed. A multidisciplinary approach among hospital physicians, general practitioners, and pharmacologists should be carried out for better drug management and adherence to guideline recommendations

Prescription appropriateness of anti-diabetes drugs in elderly patients hospitalized in a clinical setting: evidence from the REPOSI Register

Diabetes is an increasing global health burden with the highest prevalence (24.0%) observed in elderly people. Older diabetic adults have a greater risk of hospitalization and several geriatric syndromes than older nondiabetic adults. For these conditions, special care is required in prescribing therapies including anti- diabetes drugs. Aim of this study was to evaluate the appropriateness and the adherence to safety recommendations in the prescriptions of glucose-lowering drugs in hospitalized elderly patients with diabetes. Data for this cross-sectional study were obtained from the REgistro POliterapie-Società Italiana Medicina Interna (REPOSI) that collected clinical information on patients aged ≥ 65 years acutely admitted to Italian internal medicine and geriatric non-intensive care units (ICU) from 2010 up to 2019. Prescription appropriateness was assessed according to the 2019 AGS Beers Criteria and anti-diabetes drug data sheets.Among 5349 patients, 1624 (30.3%) had diagnosis of type 2 diabetes. At admission, 37.7% of diabetic patients received treatment with metformin, 37.3% insulin therapy, 16.4% sulfonylureas, and 11.4% glinides. Surprisingly, only 3.1% of diabetic patients were treated with new classes of anti- diabetes drugs. According to prescription criteria, at admission 15.4% of patients treated with metformin and 2.6% with sulfonylureas received inappropriately these treatments. At discharge, the inappropriateness of metformin therapy decreased (10.2%, P &lt; 0.0001). According to Beers criteria, the inappropriate prescriptions of sulfonylureas raised to 29% both at admission and at discharge. This study shows a poor adherence to current guidelines on diabetes management in hospitalized elderly people with a high prevalence of inappropriate use of sulfonylureas according to the Beers criteria