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Skeletal remodelling is a continuous process during life and is still active also in extreme senescence. In the elderly, bone resorption often prevails over bone formation, causing bone loss and fragility. Elderly subjects are exposed to the risk of fractures, and loss of self-sufficiency, if considering that the proximal femur is the most frequently involved site. Bone remodelling can maintain circulating calcium within physiological ranges, at the expense of a substantial loss of this ion from the skeleton, particularly during senescence. Calcium metabolism is regulated at cellular/molecular level by a network of cytokines, growth factors, systemic hormones that act on bone in paracrine/autocrine/systemic fashion. Among the molecules involved in bone metabolism, parathyroid hormone (PTH) and vitamin D present some peculiar aspects during senescence. The osteometabolic features in a consistent group of centenarians have been evaluated. It results that a severe hypovitaminosis D was present in 99 out of 104 centenarians (25-OH vitamin D below 5 nmol/L), and that it plays an important role as a factor inducing a vicious circle involving hypocalcemia, secondary hyperparathyroidism, together with biochemical features indicating a consistent bone loss. Serum C-terminal cross-linking telopeptide, a specific marker of bone resorption was elevated in 92% of these subjects. Moreover, it has been found that several femoral fractures had occurred after 90 years of age. These data offer a rational for the possible prevention of elevated bone turnover, bone loss and consequently the reduction of osteoporotic fractures and fractures-induced disability, in the oldest olds, through the simple supplementation with calcium and vitamin D.

The Italian multicentric study on centenarians (IMSC) was aimed at assessing the level of preservation of the sense of taste, and at estimating to what extent the recognition of various gustative stimuli can give satisfaction and information regarding the surrounding environment for the centenarians. Taste sensitivity has been qualitatively established in a group of 126 Italian centenarians (mean age 101.9+/-1.4 years) and compared to that of a group of 100 elderly subjects (mean age 70.5+/-5.0 years). All the individuals included in the study groups had a mini mental state examination (MMSE) score larger than 19. The results revealed that taste sensitivity is significantly reduced in the centenarians; as a matter of fact, the elderly control subjects are able to perceive taste stimuli in 84.25+/-3.40% of the testing, while the centenarians were successful only in 74.23+/-6.19% of the experiments (P

Life expectancy has dramatically expanded, the global population has aged unprecedentedly and the number of centenarians has significantly increased. The analysis of autopsy series, cancer registries data, vital statistics and surveys specific to this age group allows unique observations with respect to incidence and prevalence, cause of death by cancer, frequency of primary tumours, metastatic patterns, occurrence of incidental cancers and of multiple primary tumours.Data analysis demonstrates how cancer incidence and cause of death present a threefold decrease after age 90 and reach 0-4% above age 100. In addition, the number of metastatic sites are remarkably less and incidental malignant tumours or multiple primary cancers are more frequent, indicating that cancer in centenarians carries a more indolent behaviour.The unique features of malignant tumours in this population is hereby presented and discussed following a systematic review of the available literature.Cancer in the very elderly is relatively uncommon as a disease and as a cause of death. It is characterized by a slow growth and a modest life-threatening potential. © 2011 Elsevier Ireland Ltd. 83 1 145 152

The increasing use of genomics to define the pattern of actionable mutations and to test and validate new therapies for individual cancer patients, and the growing application of liquid biopsy to dynamically track tumor evolution and to adapt molecularly targeted therapy according to the emergence of tumor clonal variants is shaping modern medical oncology., In order to better describe this new therapeutic paradigm we propose the term "Liquid dynamic medicine" in the place of "Personalized or Precision medicine". Clinical validation of the "Liquid dynamic medicine" approach is best captured by N-of-1 trials where each patient acts as tester and control of truly personalized therapies. © 2017 The Author(s).

Lymph node metastatic involvement persists to be among the most important predictors of recurrence and survival in breast carcinoma (BC). This study is aimed at investigating possible gene expression differences in primary BC between patients with or without lymph node involvement at the time of diagnosis. In a retrospective study, we investigated the potential prognostic role of 9 candidate biomarkers at the mRNA level in a cohort of 305 breast cancer patients, 151 lymph node-negative (LN-) and 154 lymph node-positive (LN+) individuals. The analyzed genes belonged to the RAS pathway (RAF1, ERBB2, PIK3CB, AKT1, AKT2, and AKT3), RB pathway (RB1 and CDK2), and cellular differentiation (KRT8). Their expression profiles were investigated by RT-qPCR and were correlated to immunohistochemically based molecular subtypes and BC clinical and pathological features. The differential expression of several genes in the primary tumor tissue was related to the LN involvement. Some of those genes, including PIK3CB, RB1, and AKT3, were more expressed in LN- BC patients, while some others, notably ERBB2 and AKT1, in LN+ ones. Among the candidate biomarkers, the expression levels of AKT isoforms influenced also patients’ survival rates. In detail, higher expression levels of AKT1 and AKT2 negatively influenced overall patients’ survival, and in particular, AKT2 expression levels defined a group of luminal B BC patients with shorter cancer-specific survival. On the contrary, longer cancer-specific survival was recorded in luminal A BC patients with higher expression levels of AKT3. That finding was also confirmed by Cox multivariate analysis. The same AKT3 resulted to be a possible candidate predictive biomarker for Tamoxifen response. In conclusion, our study highlighted the complex regulation of the PI3K/AKT pathway in BC and its differences in BC patients with and without lymph node involvement.

Summary Next Generation Sequencing (NGS) is increasingly used in diagnostic centers for the assessment of genomic alterations to select patients for precision oncology. The Italian Society of Anatomic Pathology and Diagnostic Cytopathology (SIAPEC) through the Molecular Pathology and Predictive Medicine Study Group (PMMP) has been following the progressive development of centers that have adopted NGS technology in diagnostics over time. In July 2017, a study network on massive parallel sequencing was activated in Italy and recognized as the NGS SIAPeC National Network by the SIAPeC Scientific Society Board. Since then, activities have been implemented within the network that provide for alignment of laboratories through diagnostic concordance analysis and monitoring of centers adhering to the Network. Recently, considering the growing need for extended genomic analyses, the PMMP distributed a national survey to assess activities related to the use of genomic diagnostics in oncology within the NGS SIAPEC National Network. Thirty centers participated in the survey. Eighty percent of the centers are laboratories within Pathology Departments. The distribution of laboratories in the country, the diagnostic laboratory/population ratio, the staff dedicated, the type and number of sequencing and mechatronics platforms available, the genomic panels utilized, and the type and number of diagnostic tests carried out in the last year in each center, are reported. The centers were also asked whether they participated in a multidisciplinary Molecular Tumor Board (MTB) for management of patients. Thirty percent of the centers had a MTB that was ratified by regional decree. The professionals most frequently involved in the core team of the MTB are the pathologist, oncologist, molecular biologist, geneticist, pharmacologist, and bioinformatician. The data from this survey indicate that NGS diagnostics in Italy is still heterogeneous in terms of geographical distribution and the characteristics of laboratories and diagnostic test performed. The implementation of activities that favors harmonization, the logistics and the convergence of biological material in reference centers for molecular analyses is a priority for the development of a functional laboratory network.

Cutaneous melanoma (CM) incidence is rising worldwide and is the primary cause of death from skin disease in the Western world. Personal risk factors linked to environmental ultraviolet radiation (UVR) are well-known etiological factors contributing to its development. Nevertheless, UVR can contribute to the development of CM in different patterns and to varying degrees. The present study aimed at investigating whether altitude of residence can contribute to the development of specific types of CM and/or influence its progression. To this aim, 306 formalin-fixed and paraffin-embedded (FFPE) tissues from primary CM diagnosed in different geographical areas were submitted to B-RAF proto-oncogene serine/threonine kinase (BRAF) and N-RAS proto-oncogene GTPase (NRAS) mutational status detection and mRNA and miRNA profiling by qPCR. Genes were chosen for their functions in specific processes, such as immune response (CD2, PDL1, or CD274) and pigmentation (MITF, TYRP1, and TRPM1). Furthermore, four microRNAs, namely miR-150-5p, miR-155-5p, miR-204-5p, and miR-211-5p, were included in the profiling. Our results highlight differences in the gene expression profile of primary CM with respect to the geographical area and the altitude of residence. Melanoma-specific survival was influenced by the gene expression of mRNA and miRNAs and varied with the altitude of patients&rsquo, residence. In detail, TYRP1 and miR-204-5p were highly expressed in patients living at higher altitudes, unlike miR-150-5p, miR-155-5p, and miR-211-5p. Since miRNAs are highly regulated by reactive oxygen species, it is possible that different regulatory mechanisms characterize CMs at different altitudes due to the different environment and UVR intensity.

In clinical practice, patients&rsquo, tissues are fixed and paraffin-embedded in order to enable histological diagnosis. Nowadays, those tissues are also used for molecular characterization. Formalin is the most used fixative worldwide, and Bouin&rsquo, s solution in some worldwide institutions. Among molecular targets, micro RNAs (miRNAs), the single-stranded non-coding RNAs comprised of 18 to 24 nucleotides, have been demonstrated to be resistant to fixation and paraffin-embedding processes, with consequent possible application in clinical practice. In the present study, let-7e-5p, miR-423-3p, miR-92a-1-5p, miR-30d-5p, miR-155-5p, miR-200a-3p, and miR-429 were investigated in formalin and matched Bouin&rsquo, s solution-fixed tissues of high grade serous ovarian cancers by means of real-time and droplet digital PCR (ddPCR). Micro RNAs were detectable and analyzable in both formalin- and Bouin&rsquo, s-fixed specimens, but on average, higher Ct values and lower copies/&micro, L were found in Bouin&rsquo, s-fixed samples. Data from formalin-fixed samples correlated significantly for most targets with Bouin&rsquo, s ones, except for let-7e-5p and miR-155-5p. This study shows that miRNAs are analyzable in both formalin- and Bouin&rsquo, s-fixed specimens, with the possibility, after proper data normalization, to compare miRNA-based data from formalin-fixed samples to those of Bouin&rsquo, s-fixed ones.

Metformin, the drug of choice in the treatment of type 2 diabetes mellitus (DM2), in addition to aspirin (ASA), the drug prescribed for cardioprotection of diabetic and non-diabetic patients, have an inhibitory effect on cancer cell survival. The present population-based study conducted in the province of Trieste (Italy), aimed to investigate the prevalence of DM2 in patients with colorectal adenocarcinoma (CRC) and survival for CRC in diabetic and nondiabetic patients. All permanent residents diagnosed with a CRC between 2004 and 2007 were ascertained through the regional health information system. CRC-specific and relative survival probabilities were computed for each group of patients defined by CRC stage, presence or absence of DM2 treated with metformin, and presence or absence of daily ASA therapy. A total of 515 CRC patients without DM2 and 156 with DM2 treated with metformin were enrolled in the study. At the time of CRC diagnosis, 71 (14%) nondiabetic and 39 (25%) diabetic patients were taking ASA daily. The five-year relative survival for stage III CRC was 101% [95% confidence interval (CI)=76–126] in the 18 patients with DM2 treated with metformin and ASA, 55% (95% CI=31–78) in the 23 without DM2 treated with ASA, 55% (95% CI=45–65) in the 150 without DM2 not taking ASA, and 29% (95% CI=13–45) in the 43 with DM2 treated with metformin, however not with ASA. The findings support the hypothesis of a possible inhibitory effect of metformin and ASA on CRC cells. Randomized controlled trials are required to verify this hypothesis.

Today, clinical evaluation of tumor heterogeneity is an emergent issue to improve clinical oncology. In particular, intra-tumor heterogeneity (ITH) is closely related to cancer progression, resistance to therapy, and recurrences. It is interconnected with complex molecular mechanisms including spatial and temporal phenomena, which are often peculiar for every single patient. This review tries to describe all the types of ITH including morphohistological ITH, and at the molecular level clonal ITH derived from genomic instability and nonclonal ITH derived from microenvironment interaction. It is important to consider the different types of ITH as a whole for any patient to investigate on cancer progression, prognosis, and treatment opportunities. From a practical point of view, analytical methods that are widely accessible today, or will be in the near future, are evaluated to investigate the complex pattern of ITH in a reproducible way for a clinical application.

// Serena Bonin 1, * , Alessia Parascandolo 2, * , Cinzia Aversa 1 , Renzo Barbazza 1 , Nobuo Tsuchida 3 , Maria Domenica Castellone 4 , Giorgio Stanta 1 and Giancarlo Vecchio 5, 6, 7 1 Dipartimento di Scienze Mediche, Universita di Trieste-Cattinara, Trieste, Italy 2 IRCSS SDN, Naples, Italy 3 Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan 4 Istituto di Endocrinologia e Oncologia Sperimentale G.Salvatore, CNR, Naples, Italy 5 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita di Napoli Federico II, Naples, Italy 6 Istituto Superiore di Oncologia, Naples, Italy 7 Istituto Superiore di Oncologia, Genoa, Italy * Co-first authors Correspondence to: Giancarlo Vecchio, email: vecchio@unina.it Keywords: breast cancer; luminal B; lymph node metastasis; α-L-Fucosidase-1; immunohistochemistry Received: October 06, 2017 Accepted: February 01, 2018 Epub: February 07, 2018 Published: March 16, 2018 ABSTRACT Background: The lysosomal enzyme α-L-Fucosidase-1 (FUCA-1) catalyzes the hydrolytic cleavage of terminal fucose residues. FUCA-1 gene is down-regulated in highly aggressive and metastatic human tumors as its inactivation perturbs the fucosylation of proteins involved in cell adhesion, migration and metastases. Results: Negativity to FUCA-1 was significantly related to the development of later recurrences in breast cancer patients with lymph node involvement at diagnosis. Cancer specific survival of luminal B LN+ patients was influenced by FUCA-1 expression as luminal B LN+ patients with positive expression had a longer cancer specific survival. FUCA-1 mRNA expression was inversely related to cancer stage and lymph node involvement. WB and qPCR analysis of FUCA-1 expression in breast cancer-derived cell lines confirmed an inverse relationship with tumor aggressiveness. Conclusions: This study shows that, within LN+ breast cancer patients, FUCA-1 is able to identify a sub-set of non recurrent patients characterized by the positive expression of FUCA-1 and that, within luminal B LN+ patients, the expression of FUCA-1 predicts longer cancer specific survival. Methods: We have analyzed FUCA-1 in 305 breast cancer patients by Immunohistochemistry (IHC), and by qPCR in breast cancer patients and in breast cancer cell lines.

We carried out a cohort study in the Italian province of Trieste (2001 population, 242,000) to ascertain whether the risk of a subsequent primary cancer among 265 individuals diagnosed with a first malignancy at ages up to 25 years between 1971 and 1993 differed from that in the general population and to evaluate the effect of cancer family history, quantified by the family risk index (FRI), on the occurrence of second primaries. During the follow-up (median duration = 10 years; 25th–75th percentile = 2–16), 15 cohort members developed a second cancer vs. 1.60 expected for a standardized incidence ratio (SIR) of 9.4 (p < 0.0001). The overall SIR fell to 4.7 (p = 0.004) after excluding the 8 patients with well-known cancer-predisposing conditions (SIR = 300.0; p < 0.0001) and the 50 with a positive family history (FRI ≥ 1.0) of malignant tumors (SIR = 20.0; p < 0.0001). Among 114 patients treated with radiotherapy and chemotherapy for their first neoplasms and not affected by predisposing disorders, 23 with a positive family history of cancer showed a 6.4-fold excess risk of second primaries (p = 0.008) compared with 91 with a negative history (FRI < 1.0). It is imperative that clinicians carefully and regularly evaluate cancer family history of young patients with malignancies. This would enable them to identify possible individual and familial features in patients at higher risk of multiple primaries and to adopt more suitable preventive and therapeutic measures. © 2005 Wiley-Liss, Inc.

Background. There are little data on causes of death in extreme aged. We compared, using autopsy findings, main cause of death, overall disease status, and accuracy rate of clinical diagnoses in extreme aged and persons dying at younger ages. Methods. We reviewed the complete clinical and autopsy records of 114 consecutive inpatients (97 women, 17 men, age range 97-106, mean 99, median 98) who died in Trieste, Italy, and represented 99% of all extreme-aged person deaths in the hospital and 70% in the area. The control group included 151 patients (66 women, 85 men, age range 65-74, mean 70, median 70) who died during the same period in that hospital. Results. Vascular and respiratory diseases together caused 84% of deaths in extreme aged. The main causes of death were pneumonia (n =40,35%), pulmonary embolism (n =16, 14%), stroke (n = 12, I 1%), and myocardial infarction (n =8, 7%). Cancer was responsible for 6% (7/1 14) of deaths in extreme aged and 42% (64/15 I) in the control group. In 5% of extreme aged, autopsy findings did not explain death. The premortem diagnostic accuracy rate for clinical diagnoses was good in 44% of extreme aged, sufficient in 18%, poor in 28%, and not evaluable in 10%, and was significantly different from controls. Pneumonia, pulmonary embolism, and myocardial infarction were markedly underestimated by clinicians in both groups. Conclusions. Extreme aged die mainly of cardiovascular and respiratory diseases and, in most cases, of acute events. Senescence is a rare cause of death. Death from cancer is substantially lower than in persons dying at younger ages. In contrast to no autopsy studies, most extreme aged in our study were found to have specific diseases that explained their deaths.

Purpose: Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy. Experimental Design: By using a kinase-directed short hairpin RNA library and HCT116p53KO drug-resistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression. Results: Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)–treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients. Conclusions: Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors. Clin Cancer Res; 19(14); 3820–31. ©2013 AACR.

The aim of this chapter is to provide a reliable method of obtaining DNA from formalin-fixed and paraffin-embedded (FFPE) tissue specimens. The application of this method allows the extraction of DNA from specimens of both biopsy and autopsy origin. The method described here is based on proteolytic digestion, phenol purification, and alcohol precipitation, using a point-to-point protocol with notes and references to guide the researcher into the laboratory practice. The DNA extracts obtained by the application of this method are suitable for most of the PCR analyses used in molecular pathology laboratories, ranging from single to complex multiplexed PCRs or B-/T-cell clonality PCR tests, as well as for PCR sequencing.

A fast and cheap strategy for an efficient extraction of proteins from formalin-fixed and paraffin-embedded (FFPE) tissues is illustrated in this chapter. The described method is based on the exposure of the tissue samples to high temperature in a SDS-containing-Laemmli buffer that allows a good formaldehyde cross-link reversal. The protocol herein enables efficient extraction of non-degraded proteins that are suitable for western blot and other proteomic analyses.

To determine whether the occurrence of first and second primary malignancies in first-degree relatives of cancer patients aged 0-25 years (probands) differed from that in the general population, a cohort study was carried out on 860 relatives of 265 probands living in the province of Trieste, Italy. During the follow-up period (median duration = 28 years, 25th-75th percentile = 20-34), the relatives developed 103 first primary cancers vs. 88.9 expected for a standardized incidence ratio (SIR) of 1.2 (p = 0.2). Significantly elevated risks were found for melanoma in the parents of probands aged 15-25 years with melanoma (SIR = 15.0, p = 0.002), for hemolymphatic malignancies in the fathers of probands aged 0-14 years with brain tumors (SIR = 13.3, p = 0.0005) and for hemolymphatic cancers in relatives as a whole of probands aged 15-25 years with lymphomas (SIR = 4.5, p = 0.01). During the follow-up period, 7 relatives with a first primary cancer had a subsequent malignancy vs. 4.2 expected for an SIR of 1.7 (p = 0.3). Our results indicate that young cancer patients per se should not to be considered as a factor that usually increases the risk of developing malignant tumors among their first-degree relatives, except when a known cancer family syndrome or predisposition is recognized.

Vectors based on the adeno-associated virus (AAV) deliver therapeutic genes to muscle and heart at high efficiency and maintain transgene expression for long periods of time. Here we report about the synergistic effect on blood vessel formation of AAV vectors expressing the 165 aa isoform of vascular endothelial growth factor (VEGF165), a powerful activator of endothelial cells, and of angiopoietin-1 (Ang-1), which is required for vessel maturation. High titer AAV-VEGF165 and AAV-Ang-1 vector preparations were injected either alone or in combination in the normoperfused tibialis anterior muscle of rats. Long term expression of VEGF165 determined massive cellular infiltration of the muscle tissues over time, with the formation of a large set of new vessels. Strikingly, some of the cells infiltrating the treated muscles were found positive for markers of activated endothelial precursors (VEGFR-2/KDR and Tie-2) and for c-kit, an antigen expressed by pluripotent bone marrow stem cells. Expression of VEGF165 eventually resulted in the formation of structured vessels surrounded by a layer of smooth muscle cells. Presence of these arteriolae correlated with significantly increased blood perfusion in the injected areas. Co-expression of VEGF165 with angiopoietin-1-which did not display angiogenic effect per se-remarkably reduced leakage of vessels produced by VEGF165 alone.

Background/Aims: A twofold increased risk for breast cancer has been reported recently for women with late onset diabetes. Most studies showed that there were differences in serum concentrations of insulin-like growth factors and related proteins between women with and without diabetes who have breast cancer. This study investigated the expression of these markers at the cellular level in a cohort of women with and without type 2 diabetes who underwent biopsy because of a breast lump. Methods: Relative quantitative analysis of specific mRNA sequences was performed after extraction and reverse transcription polymerase chain reaction amplification from formalin fixed and paraffin wax embedded tissues. Sixty seven breast surgical specimens from women with and without diabetes who did not have cancer and from women with and without diabetes who did have cancer were studied for insulin-like growth factor I (IGF-I), the IGF-I receptor (IGF-IR), insulin-like growth factor binding protein 3 (IGFBP-3), and oestrogen receptor 1 gene expression. Results: The expression of IGF-I and IGF-IR was significantly lower in the cancer groups, whereas there was no significant difference for IGFBP-3 between women with and without cancer. Moreover, there was a good correlation between the expression of IGF-I and IGF-IR in women without cancer: this link was still present in breast tissue from patients with diabetes and cancer, whereas it was lost in patients without diabetes but with cancer. Conclusions: These differences in IGF-I/IGF-IR expression could contribute to the increased risk for breast cancer in women with type 2 diabetes.

We conducted a population-based cohort study in the province of Trieste, Italy, to assess whether the first-degree relatives of children with malignancies had an increased risk of cancer compared with the general population. We examined cancers occurring in all first-degree relatives of children who experienced malignancies under the age of 15 years between 1971 and 1993 (probands). A cohort of the 394 relatives of the 125 probands contributed 7,939 person-years of observation. Among the relatives as a whole, we found a statistically significant increased risk of developing all malignancies except non-melanoma skin carcinoma (21 observed relatives with cancer and 12.46 expected, for a standardized incidence ratio [SIR] of 1.69), of developing breast cancer (SIR = 3.09) and of developing haemolymphatic system neoplasms (SIR = 4.03). This was mainly due to the excess cancer risk in the relatives of probands with intracranial tumours, who showed a significant 3.1-fold risk for developing all cancers but non-melanoma skin tumours. Our findings and the previously reported steep rise in the incidence of childhood brain tumours in our area may imply that not only genetic factors but also shared environmental agents might be involved in the observed aggregation of cancer in the families of probands with intracranial tumours.