Cognitive outcomes in patients treated with neuromuscular electrical stimulation after coronary artery bypass grafting.

Objective: Mechanisms of neurocognitive injury as post-operative sequelae of coronary artery bypass grafting (CABG) are not understood. The systemic inflammatory response to surgical stress causes skeletal muscle impairment, and this is also worsened by immobility. Since evidence supports a link between muscle vitality and neuroprotection, there is a need to understand the mechanisms by which promotion of muscle activity counteracts the deleterious effects of surgery on long-term cognition. Methods: We performed a clinical trial to test the hypothesis that adding neuromuscular electrical stimulation (NMES) to standard rehabilitation care in post-CABG patients promotes the maintenance of skeletal muscle strength and the expression of circulating neuroprotective myokines. Results: We did not find higher serum levels of neuroprotective myokines, except for interleukin-6, nor better long-term cognitive performance in our intervention group. However, a greater increase in functional connectivity at brain magnetic resonance was seen between seed regions within the default mode, frontoparietal, salience, and sensorimotor networks in the NMES group. Regardless of the treatment protocol, patients with a Klotho increase 3 months after hospital discharge compared to baseline Klotho values showed better scores in delayed memory tests. Significance: We confirm the potential neuroprotective effect of Klotho in a clinical setting and for the first time post-CABG.

Rationale and design of the CV-PREVITAL study: an Italian multiple cohort randomised controlled trial investigating innovative digital strategies in primary cardiovascular prevention.

INTRODUCTION: Prevention of cardiovascular disease (CVD) is of key importance in reducing morbidity, disability and mortality worldwide. Observational studies suggest that digital health interventions can be an effective strategy to reduce cardiovascular (CV) risk. However, evidence from large randomised clinical trials is lacking. METHODS AND ANALYSIS: The CV-PREVITAL study is a multicentre, prospective, randomised, controlled, open-label interventional trial designed to compare the effectiveness of an educational and motivational mobile health (mHealth) intervention versus usual care in reducing CV risk. The intervention aims at improving diet, physical activity, sleep quality, psycho-behavioural aspects, as well as promoting smoking cessation and adherence to pharmacological treatment for CV risk factors. The trial aims to enrol approximately 80 000 subjects without overt CVDs referring to general practitioners' offices, community pharmacies or clinics of Scientific Institute for Research, Hospitalization and Health Care (Italian acronym IRCCS) affiliated with the Italian Cardiology Network. All participants are evaluated at baseline and after 12 months to assess the effectiveness of the intervention on short-term endpoints, namely improvement in CV risk score and reduction of major CV risk factors. Beyond the funded life of the study, a long-term (7 years) follow-up is also planned to assess the effectiveness of the intervention on the incidence of major adverse CV events. A series of ancillary studies designed to evaluate the effect of the mHealth intervention on additional risk biomarkers are also performed. ETHICS AND DISSEMINATION: This study received ethics approval from the ethics committee of the coordinating centre (Monzino Cardiology Center; R1256/20-CCM 1319) and from all other relevant IRBs and ethics committees. Findings are disseminated through scientific meetings and peer-reviewed journals and via social media. Partners are informed about the study's course and findings through regular meetings. TRIAL REGISTRATION NUMBER: NCT05339841.

Patient-Specific Analysis of Ascending Thoracic Aortic Aneurysm with the Living Heart Human Model.

In ascending thoracic aortic aneurysms (ATAAs), aneurysm kinematics are driven by ventricular traction occurring every heartbeat, increasing the stress level of dilated aortic wall. Aortic elongation due to heart motion and aortic length are emerging as potential indicators of adverse events in ATAAs; however, simulation of ATAA that takes into account the cardiac mechanics is technically challenging. The objective of this study was to adapt the realistic Living Heart Human Model (LHHM) to the anatomy and physiology of a patient with ATAA to assess the role of cardiac motion on aortic wall stress distribution. Patient-specific segmentation and material parameter estimation were done using preoperative computed tomography angiography (CTA) and ex vivo biaxial testing of the harvested tissue collected during surgery. The lumped-parameter model of systemic circulation implemented in the LHHM was refined using clinical and echocardiographic data. The results showed that the longitudinal stress was highest in the major curvature of the aneurysm, with specific aortic quadrants having stress levels change from tensile to compressive in a transmural direction. This study revealed the key role of heart motion that stretches the aortic root and increases ATAA wall tension. The ATAA LHHM is a realistic cardiovascular platform where patient-specific information can be easily integrated to assess the aneurysm biomechanics and potentially support the clinical management of patients with ATAAs.

Statistical Shape Analysis of Ascending Thoracic Aortic Aneurysm: Correlation between Shape and Biomechanical Descriptors.

An ascending thoracic aortic aneurysm (ATAA) is a heterogeneous disease showing different patterns of aortic dilatation and valve morphologies, each with distinct clinical course. This study aimed to explore the aortic morphology and the associations between shape and function in a population of ATAA, while further assessing novel risk models of aortic surgery not based on aortic size. Shape variability of n = 106 patients with ATAA and different valve morphologies (i.e., bicuspid versus tricuspid aortic valve) was estimated by statistical shape analysis (SSA) to compute a mean aortic shape and its deformation. Once the computational atlas was built, principal component analysis (PCA) allowed to reduce the complex ATAA anatomy to a few shape modes, which were correlated to shear stress and aortic strain, as determined by computational analysis. Findings demonstrated that shape modes are associated to specific morphological features of aneurysmal aorta as the vessel tortuosity and local bulging of the ATAA. A predictive model, built with principal shape modes of the ATAA wall, achieved better performance in stratifying surgically operated ATAAs versus monitored ATAAs, with respect to a baseline model using the maximum aortic diameter. Using current imaging resources, this study demonstrated the potential of SSA to investigate the association between shape and function in ATAAs, with the goal of developing a personalized approach for the treatment of the severity of aneurysmal aorta.

Shear Stress and Aortic Strain Associations With Biomarkers of Ascending Thoracic Aortic Aneurysm.

BACKGROUND: This study aims to investigate the association of wall shear stress (WSS) and aortic strain with circulating biomarkers including matrix metalloproteinases (MMP), tissue inhibitors of metalloproteinase (TIMP), and exosomal level of microRNA (miRNA) in ascending aortic aneurysms of patients with bicuspid or tricuspid aortic valve. METHODS: A total of 76 variables from 125 patients with ascending aortic aneurysms were collected from (1) blood plasma to measure plasma levels of miRNAs and protein activity; (2) computational flow analysis to estimate peak systolic WSS and time-average WSS (TAWSS); and (3) imaging analysis of computed tomography angiography to determine aortic wall strain. Principal component analysis followed by logistic regression allowed the development of a predictive model of aortic surgery by combining biomechanical descriptors and biomarkers. RESULTS: The protein activity of MMP-1, TIMP-1, and MMP-2 was positively correlated to the systolic WSS and TAWSS observed in the proximal ascending aorta (eg, R = 0.52, P < .001, for MMP-1 with TAWSS) where local maxima of WSS were found. For bicuspid patients, aortic wall strain was associated with miR-26a (R = 0.55, P = .041) and miR-320a (R = 0.69, P < .001), which shows a significant difference between bicuspid and tricuspid patients. Receiver-operating characteristics curves revealed that the combination of WSS, MMP-1, TIMP-1, and MMP-12 is predictive of aortic surgery (area under the curve 0.898). CONCLUSIONS: Increased flow-based and structural descriptors of ascending aortic aneurysms are associated with high levels of circulating biomarkers, implicating adverse vascular remodeling in the dilated aorta by mechanotransduction. A combination of shear stress and circulating biomarkers has the potential to improve the decision-making process for ascending aortic aneurysms to a highly individualized level.

Simulation study of transcatheter heart valve implantation in patients with stenotic bicuspid aortic valve.

Bicuspid aortic valve (BAV) anatomy has routinely been considered an exclusion in the setting of transcatheter aortic valve implantation (TAVI) because of the large dimension of the aortic annulus having a more calcified, bulky, and irregular shape. The study aims to develop a patient-specific computational framework to virtually simulate TAVI in stenotic BAV patients using the Edwards SAPIEN 3 valve (S3) and its improved version SAPIEN 3 Ultra and quantify stent frame deformity as well as the severity of paravalvular leakage (PVL). Specifically, the aortic root anatomy of n.9 BAV patients who underwent TAVI was reconstructed from pre-operative CT imaging. Crimping and deployment of S3 frame were performed and then followed by fluid-solid interaction analysis to simulate valve leaflet dynamics throughout the entire cardiac cycle. Modeling revealed that the S3 stent frame expanded well on BAV anatomy with an elliptical shape at the aortic annulus. Comparison of predicted S3 deformity as assessed by eccentricity and expansion indices demonstrated a good agreement with the measurement obtained from CT imaging. Blood particle flow analysis demonstrated a backward blood jet during diastole, whereas the predicted PVL flows corresponded well with those determined by transesophageal echocardiography. This study represents a further step towards the use of personalized simulations to virtually plan TAVI, aiming at improving not only the efficacy of the implantation but also the exploration of "off-label" applications as the TAVI in the setting of BAV patients. Graphical abstract Computational frameworks of TAVI in patients with stenotic bicuspid aortic valve.

Usefulness of regional right ventricular and right atrial strain for prediction of early and late right ventricular failure following a left ventricular assist device implant: A machine learning approach.

BACKGROUND: Identifying candidates for left ventricular assist device surgery at risk of right ventricular failure remains difficult. The aim was to identify the most accurate predictors of right ventricular failure among clinical, biological, and imaging markers, assessed by agreement of different supervised machine learning algorithms. METHODS: Seventy-four patients, referred to HeartWare left ventricular assist device since 2010 in two Italian centers, were recruited. Biomarkers, right ventricular standard, and strain echocardiography, as well as cath-lab measures, were compared among patients who did not develop right ventricular failure (N = 56), those with acute-right ventricular failure (N = 8, 11%) or chronic-right ventricular failure (N = 10, 14%). Logistic regression, penalized logistic regression, linear support vector machines, and naïve Bayes algorithms with leave-one-out validation were used to evaluate the efficiency of any combination of three collected variables in an "all-subsets" approach. RESULTS: Michigan risk score combined with central venous pressure assessed invasively and apical longitudinal systolic strain of the right ventricular-free wall were the most significant predictors of acute-right ventricular failure (maximum receiver operating characteristic-area under the curve = 0.95, 95% confidence interval = 0.91-1.00, by the naïve Bayes), while the right ventricular-free wall systolic strain of the middle segment, right atrial strain (QRS-synced), and tricuspid annular plane systolic excursion were the most significant predictors of Chronic-RVF (receiver operating characteristic-area under the curve = 0.97, 95% confidence interval = 0.91-1.00, according to naïve Bayes). CONCLUSION: Apical right ventricular strain as well as right atrial strain provides complementary information, both critical to predict acute-right ventricular failure and chronic-right ventricular failure, respectively.

Patterns of ascending aortic dilatation and predictors of surgical replacement of the aorta: A comparison of bicuspid and tricuspid aortic valve patients over eight years of follow-up.

BACKGROUND: Predictors of thoracic aorta growth and early cardiac surgery in patients with bicuspid aortic valve are undefined. Our aim was to identify predictors of ascending aorta dilatation and cardiac surgery in patients with bicuspid aortic valve (BAV). METHODS: Forty-one patients with BAV were compared with 165 patients with tricuspid aortic valve (TAV). All patients had LV EF > 50%, normal LV dimensions, and similar degree of aortic root or ascending aorta dilatation at enrollment. Patients with more than mild aortic stenosis or regurgitation were excluded. A CT-scan was available on 76% of the population, and an echocardiogram was repeated every year for a median time of 4 years (range: 2 to 8 years). Patterns of aortic expansion in BAV and TAV groups were analyzed by a mixed-effects longitudinal linear model. In the time-to-event analysis, the primary end point was elective or emergent surgery for aorta replacement. RESULTS: BAV patients were younger, while the TAV group had greater LV wall thickness, arterial hypertension, and dyslipidemia than BAV patients. Growth rate was 0.46 ±â€¯0.04 mm/year, similar in BAV and TAV groups (p = 0.70). Predictors of cardiac surgery were aorta dimensions at baseline (HR 1.23, p = 0.01), severe aortic regurgitation developed during follow-up (HR 3.49, p 0.04), family history of aortic aneurysm (HR 4.16, p 1.73), and history of STEMI (HR 3.64, p < 0.001). CONCLUSIONS: Classic baseline risk factors were more commonly observed in TAV aortopathy compared with BAV aortopathy. However, it is reassuring that, though diagnosed with aneurysm on average 10 years earlier and in the absence of arterial hypertension, BAV patients had a relatively low growth rate, similar to patients with a tricuspid valve. Irrespective of aortic valve morphology, patients with a family history of aortic aneurysm, history of coronary artery disease, and those who developed severe aortic regurgitation at follow-up, had the highest chances of being referred for surgery.

Focused Ultrasound Effects on Osteosarcoma Cell Lines.

MRI guided Focused Ultrasound (MRgFUS) has shown to be effective therapeutic modality for non-invasive clinical interventions in ablating of uterine fibroids, in bone metastasis palliative treatments, and in breast, liver, and prostate cancer ablation. MRgFUS combines high intensity focused ultrasound (HIFU) with MRI images for treatment planning and real time thermometry monitoring, thus enabling non-invasive ablation of tumor tissue. Although in the literature there are several studies on the Ultrasound (US) effects on cell in culture, there is no systematic evidence of the biological effect of Magnetic Resonance guided Focused Ultrasound Surgery (MRgFUS) treatment on osteosarcoma cells, especially in lower dose regions, where tissues receive sub-lethal acoustic power. The effect of MRgFUS treatment at different levels of acoustic intensity (15.5-49 W/cm2) was investigated on Mg-63 and Saos-2 cell lines to evaluate the impact of the dissipation of acoustic energy delivered outside the focal area, in terms of cell viability and osteogenic differentiation at 24 h, 7 days, and 14 days after treatment. Results suggested that the attenuation of FUS acoustic intensities from the focal area (higher intensities) to the "far field" (lower intensities) zones might determine different osteosarcoma cell responses, which range from decrease of cell proliferation rates (from 49 W/cm2 to 38.9 W/cm2) to the selection of a subpopulation of heterogeneous and immature living cells (from 31.1 W/cm2 to 15.5 W/cm2), which can clearly preserve bone tumor cells.

Assessment of "grading" with Ki-67 and c-kit immunohistochemical expressions may be a helpful tool in management of patients with flat epithelial atypia (FEA) and columnar cell lesions (CCLs) on core breast biopsy.

It is essential to reach a better understanding of "flat epithelial atypia/columnar cell lesions" (FEA/CCLs) in breast core biopsies. Our aim was to explore their biological nature, in order to predict the likelihood of an upgrade to carcinoma. "Cytological grading" has been specially focused, in view of its possible utility in the choice of management. One hundred thirty of a total of 900 cases core needle (CN)/vacuum-assisted biopsies (VABs), with diagnoses of "hyperplasia" and "atypia" were retrospectively re-evaluated. Pathological findings of further excision biopsies (FEBs) performed in 40/75 patients with follow-up were compared with the previous diagnoses. In all cases, both Ki-67 and c-kit immunoreactivities were explored and compared with both normal breast tissues and subsequently documented cancers, with special reference to the hyperplastic FEA/CCLs, with "mild" atypia (FEA/CCHAm). Sixteen cases were re-diagnosed as "usual ductal hyperplasia" (UDH), 60 as "columnar cell hyperplasia" (CCH), and 54 as FEA/CCHA, 30 of which FEA/CCHAm and 24 FEA/CCHAh (with high atypia). Significantly, the Ki-67 index proved to be on the increase and c-kit expression on the decrease in FEA/CCHA lesions, mainly in the FEA/CCHAh group and in the subsequently observed cancers, compared with either benign tissues or the FEA/CCH cases. It was also significant that most of the carcinomas were found in FEBs within the FEA/CCHAh group. In this study cytological grading, together with Ki-67 and c-kit indices, proved to be helpful in FEA/CCLs evaluation. With regard to FEA/CCHAm lesions, an adequate surveillance appears to be a more appropriate management tool than FEB, as a result of their biological nature and behavior.

Is BRCA1-5083del19, identified in breast cancer patients of Sicilian origin, a Calabrian founder mutation?

Various studies have been published in Italy regarding the different BRCA1 mutations, but only the BRCA1-5083del19 mutation is recurrent and specific to individuals of Italian descent with a founder effect on the Calabrian population. In our previous study, BRCA1-5083del19 mutation carriers were found in four index cases of 106 Sicilian patients selected for familial and/or hereditary breast/ovarian cancers. The high frequency rate of this mutation identified in the Sicilian population led us to perform haplotype analysis in all family carriers. Five highly polymorphic microsatellite markers were used (D17S1320, D17S932, D17S1323, D17S1326, D17S1325) to establish whether or not all these families had a common ancestor. This analysis showed that all mutation carriers of these families had a common allele. None of the non-carriers of the mutation or of the 50 healthy Sicilian controls showed this haplotype. This allelotype analysis highlighted the presence of a common allele (ancestor), thus suggesting the presence of a founder effect in the Sicilian population. Our results are in contrast with other studies but only the allelotype analysis of all the BRCA1-5083del19 mutation carriers of two neighboring regions of the south of Italy (Calabria and Sicily) will make it possible to identify the real ancestor of this mutation.

Targeting apoptosis in solid tumors: the role of bortezomib from preclinical to clinical evidence.

The ubiquitin-proteasome pathway is the main proteolytic system present in the nucleus and cytoplasm of all eukaryotic cells. Apoptosis activation induced by ubiquitin-proteasome pathway inhibition makes the proteasome a new target of anticancer therapy. Bortezomib is the first proteasome inhibitor to be approved by the US FDA; in 2003 as a third line and in 2005 as a second line therapy for the treatment of multiple myeloma only. This review focuses on the use of bortezomib, not only in its therapeutic role but also, more specifically, in its biologic role and discusses the most recent applications of the drug in solid tumors, both at a preclinical and clinical level.

BRCA1 genetic testing in 106 breast and ovarian cancer families from Southern Italy (Sicily): a mutation analyses.

PURPOSE: To evaluate the contribution of germline BRCA1 mutations in the incidence of hereditary and familial Breast Cancer (BC) and/or Ovarian Cancer (OC) in patients from Southern Italy (in the region of Sicily) and to identify a possible association between the higher frequency of BRCA1 mutations and a specific familial profile. EXPERIMENTAL DESIGN: A consecutive series of 650 patients with BC and/or OC diagnosed between 1999 and 2005 were recruited from the Southern Italian region of Sicily, after interview at the "Regional Reference Centre for the Characterization and Genetic Screening of Hereditary Tumors" at the University of Palermo. Genetic counselling allowed us to recruit a total of 106 unrelated families affected with breast and/or ovarian cancer screened for mutations occurring in the whole BRCA1 gene by automatic direct sequencing. RESULTS: Germline BRCA1 mutations were found in 17 of 106 (16%) Sicilian families. The HBOC profile had a major frequency (66%) of mutations (P < 0.01). A total of 28 sequence variants was identified. Seven of these were pathogenic, 5 unknown biological variant (UV) and 16 polymorphisms. We also identified a pathological mutation (4843delC) as a possible Sicilian founder mutation. CONCLUSIONS: The present study is the first BRCA1 disease-associated mutations analysis in Southern Italian families. The early age of onset of such tumors and the association with the HBOC familial profile could be two valid screening factors for the identification of BRCA1 mutation carriers. Finally, we identified a BRCA1 mutation with a possible founder effect.

TP53 and p16INK4A, but not H-KI-Ras, are involved in tumorigenesis and progression of pleomorphic adenomas.

The putative role of TP53 and p16(INK4A) tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex-PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific-PCR (MS-PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H-Ras and K-Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K-Ras. p16(INK4A) promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16(INK4A) promoter methylation, but not alterations in the H-Ras and K-Ras genes, might be involved in the malignant progression of PA into carcinoma.

TP53 mutations and S-phase fraction but not DNA-ploidy are independent prognostic indicators in laryngeal squamous cell carcinoma.

To prospectively evaluate the prognostic significance of TP53, H-, K-, and N-Ras mutations, DNA-ploidy and S-phase fraction (SPF) in patients affected by locally advanced laryngeal squamous cell carcinoma (LSCC). Eight-one patients (median follow-up was 71 months) who underwent resective surgery for primary operable locally advanced LSCC were analyzed. Tumor DNA was screened for mutational analysis by PCR/SSCP and sequencing. DNA-ploidy and SPF were performed by flow cytometric analyses. Thirty-six patients (44%) had, at least, a mutation in the TP53 gene. Of them, 22% (8/36) had double mutations and 3% (1/36) had triple mutations. In total, 46 TP53 mutations were observed. The majority (41%) of these occur in exon 5 (19/46), while the mutations in exons 6, 7, and 8 were represented in 14, 7, and 6 patients, respectively (31%, 15%, and 16%). Five LSCC patients (6%) showed a mutation in H-Ras gene. Sixty-three percent of the cases (51/81) were DNA aneuploidy, 14% of these (7/51) were multiclonal. Thirty-nine patients (48%) had an high SPF value. At Univariate analysis, the DNA aneuploidy, high SPF (>15.1%), TP53 mutations and, in particular, the mutations that occur in exons 5 and 8 were significantly related to quicker disease relapse and short OS. At Multivariate analysis, the major significant predictors for both disease relapse and death were high SPF and any TP53 mutations. While histological grade G3 was an independent factor only for relapse. In conclusions, any TP53 mutations and high SPF are important biological indicators to predict the outcome of LSCC patients.

A new germline mutation in BRCA1 gene in a sicilian family with ovarian cancer.

A group of 103 sicilian patients with hereditary and familiar breast and/or ovarian cancer were screened for Breast Cancer 1 gene (BRCA1) mutations by direct sequencing PCR products spanning the coding region and partial intronic regions of the BRCA1 gene. In this study, we report a new germline mutation in BRCA1 gene, not previously reported in the BIC database, in a woman with ovarian cancer at 46 years old. Mother's proband has been diagnosed the same histotype of ovarian cancer at 42 age. The mutational analyses that shown a 4843delC frameshift mutation in exon 16 of BRCA1 gene was extended to other family members including the proband's brother and her two sons. Direct automatic sequencing of DNA extracted from the lymphocytes showed exactly the same 4843delC frameshift mutation only in the brother. In conclusion, the characterization of this mutation could help in the identification of a founder mutation of sicilian area and this may provide significant advantages for genetic counselling.

Pharmacogenomics in colorectal carcinomas: future perspectives in personalized therapy.

The recent introduction of new drugs such as capecitabine, irinotecan, and oxaliplatinum has greatly improved the clinical outcome of patients with advanced/metastatic colorectal cancer. Nevertheless, some patients may suffer from the adverse drug reactions which will probably be the main cause of chemotherapy failure. The goal of pharmacogenomics is to find correlations between therapeutic responses to drugs and the genetic profiles of patients; the different responses to a particular drug are due, in fact, not only to the specific clinico-pathological features of the patient or to environmental factors, but also to the ethnic origins and the particular individual's genetic profile. Genes which codify for the metabolism enzymes, receptor proteins, or protein targets of chemotherapy agents often present various genetic polymorphisms. The main aim of this review is to provide an overview of the known polymorphisms present in the genes which codify for factors (thymidylate synthase dihydropyrimidine dehydrogenase, uridine diphosphate (UDP)-glucuronosyl-transferase 1A1, enzymes implicated in DNA repair) involved in the action mechanisms of the drugs now utilized in chemotherapeutic treatment of colorectal carcinoma, such as fluoropyrimidines, irinotecan, and platinum agents.

Laser pressure catapulting (LPC): optimization LPC-system and genotyping of colorectal carcinomas.

Genotype analysis is becoming more and more useful in clinical practice, since specific mutations in tumors often correlate with prognosis and/or therapeutic response. Unfortunately, current molecular analytical techniques often require time-consuming and costly steps of analysis, thus making their routine clinical use difficult. Moreover, one of the most difficult problems arising during tumor research is that of their cell heterogeneity, which depends on their clear molecular heterogeneity. SSCP analysis discriminates by means of aberrant electrophoresis migration bands, mutated alleles which may represent as little as 15-20% of their total number. Nevertheless, in order to identify by sequencing the type of alteration revealed by this technique, only the mutated allele must be isolated. The advent of laser microdissection is a procedure which easily solves these problems of accuracy, costs, and time. The aims of this study were to perfect the system of laser pressure catapulting (LPC) laser microdissection for the assessment of the mutational status of p53 and k-ras genes in a consecutive series of 67 patients with colorectal carcinomas (CRC), in order to compare this technique with that involving hand-dissection and to demonstrate that since the LPC system guarantees more accurate biomolecular analyses, it should become part of clinical routine in this field. The LPC-system was perfected with the use of mineral oil and the LPC-membrane. To compare the techniques of hand- and LPC-microdissection, alcohol-fixed, paraffin-embedded tissue from 67 cases of CRC were both hand- and laser-microdissected. In either case, dissected samples were analyzed by SSCP/sequencing and direct sequencing for k-ras and p53 gene mutations. LPC-microdissection made it possible to pick up mutations by direct sequencing or SSCP/sequencing, whereas hand-microdissection mutations were identified only by means of SSCP followed by sequencing; direct sequencing did not reveal any mutation. In the 67 patients examined by either method, 36% (24/67) showed p53 mutations, 32 of which identified. Seventy-eight percent (25/32) were found in the conserved areas of the gene, while 12% (4/32) were in the L2 loop, 50% (16/32) were in the L3 loop, and 12% (4/32) in the LSH motif of the protein. Moreover, of the 67 cases examined, 40% (27/67) showed mutations in k-ras, with a total of 29 mutations identified. Of these, 14 (48%) were found in codon 12 and 15 (52%) in codon 13. The modifications which we brought to the LPC system led to a vast improvement of the technique, making it an ideal substitution for hand-microdissection and guaranteeing a considerable number of advantages regarding facility, accuracy, time, and cost. Furthermore, the data obtained from the mutational analyses performed confirm that the LPC system is more efficient and rapid than hand-microdissection for acquiring useful information regarding molecular profile and can therefore be used with success in clinical routine.

TP53 in gastric cancer: mutations in the l3 loop and LSH motif DNA-binding domains of TP53 predict poor outcome.

The aim of this study was to clarify whether specific p53 mutations may have biological relevance in terms of disease relapse or death in gastric carcinomas (GC). Resected specimens from a consecutive series of 62 patients with GC undergoing potentially curative surgery were prospectively studied. The mutational status of exons 5-8 of the p53 gene was investigated in 62 cases using the PCR-SSCP and sequencing. Presence of microsatellite instability (MSI) was evaluated in 56 cases by analyzing loci highly sensitive of MSI. Twenty mutations of p53 were detected in 17 of the 62 cases analyzed (27%). Ten mutations (50%) occurred in highly conserved domains. According to the p53 specific functional domains: 4/20 mutations (20%) were in the L3 loop and 3/20 (15%) in LSH motif. Eight of the 56 GC resulted MSI-H, 5 (9%) MSI-L, and 43 (77%) MSI stable (MSS). None of the 8 (14%) MSI-H GC showed p53 mutations. p53 mutations were associated with intestinal histotype. Moreover, specific mutations in functional domain (L3 and LSH), together with advanced TNM stage, node involvement, depth of invasion, diffuse histotype, proved to be significantly related to quicker relapse and to shorter overall survival. Specific mutations in p53 functional domains, rather than any mutations in this gene, may be biologically more significant in terms of patients outcome, indicating that these mutations might have biological relevance to identify subgroups of patients at higher risk of relapse or death who might benefit from a more aggressive therapeutic approach.

Nm23-H1 expression does not predict clinical survival in colorectal cancer patients.

The gene Nm23, which encodes for a nucleoside diphosphate kinase, has been defined as a metastasis-suppressor gene because of the inverse correlation between its expression and the metastatic capacity of the tumor cells. For colorectal cancer, however, the findings are equivocal. The aim of our study was to assess, in 160 patients undergoing surgery for colorectal cancer (CRC), the expression of the Nm23-H1 protein and to evaluate its possible associations with traditional clinicopathologic variables, with DNA-ploidy and proliferative activity (S-phase fraction, SPF), and with disease-free and overall survival of patients. Nm23-H1 expressions were evaluated on paraffin-embedded tissue by immunohistochemistry; DNA-ploidy and SPF on frozen tissue by flow-cytometric analysis. The median follow-up time in our study group was 71 months (range 34-115 months). No association was observed between Nm23-H1 protein expression and clinicopathological variables, S-phase fraction and DNA-ploidy. Furthermore, no significant differences were observed in the survival of patients with either moderate or strong Nm23-H1 expression. The major significant predictors for both disease relapse and death were advanced Dukes' stage, DNA aneuploid tumors and high SPF, while lymphohematic invasion was the only independent factor for relapse and non-curative resection for death. Our results indicate that Nm23-H1 activity is tissue-specific and that in CRCs the expression of the protein is not associated with tumor progression and patient prognosis, although further studies are required in order to throw more light on the possible clinical significance of the overexpression of the protein Nm23-H1 in such tumors.