This paper assembles current thoughts on the concepts and mechanisms behind the DOHaD as they relate to maternal nutrition, and highlights specific contributions made by macro- and micronutrients.Chronic diseases such as type 2 diabetes and cardiovascular disease are the leading cause of death and disability worldwide. Although the metabolic syndrome has been defined in various ways, the ultimate importance of recognizing this combination of disorders is that it helps identify individuals at high risk for both type 2 diabetes and cardiovascular disease. Evidence from observational and experimental studies links adverse exposures in early life, particularly relating to nutrition, to chronic disease susceptibility in adulthood. Such studies provide the foundation and framework for the relatively new field of developmental origins of health and disease (DOHaD). Although great strides have been made in identifying the putative concepts and mechanisms relating specific exposures in early life to the risk of developing chronic diseases in adulthood, a complete picture remains obscure. To date, the main focus of the field has been on perinatal  The metabolic syndrome has become a major public health challenge with an estimated 22% of US adults having this condition . A conseIn the 1970s Forsdahl, using official statistical data on Norwegian counties, reported that poverty during adolescence, followed by prosperity, was positively correlated with risk of death from coronary heart disease . AlthougHistorically, investigations on the fetal origins hypothesis have focused on maternal undernutrition and specific nutrient deficiencies. Today, the world faces the dual burden of malnutrition that encompasses both under- and overnutrition . It is eMicrotus pennsylvanicus) to illustrate programming [in utero [For most organs and systems the critical period of plasticity is during intrauterine development . Developgramming . Meadow gramming . This ocgramming . A thickin utero . Barker in utero . Humans in utero . Programin utero . Those tin utero , 23. Thein utero . In most cases programming is beneficial for the health and survival of the organism. However, the problem of “mismatch” occurs when individuals developmentally adapted to one environment are exposed to another . Revisitβ cells, nephrons, and cardiomyocytes [β cell hypoplasia). Such adaptations may increase the chance of fetal survival by means of “brain sparing” but result in difficulty in coping with nutritional abundance as an adult. The “thrifty phenotype” hypothesis, put forth by Hales and Barker, proposes that poor fetal and early postnatal nutrition imposes mechanisms of nutritional thrift upon the growing individual. In conditions of severe intrauterine deprivation the developing fetus may lose functional and structural units such as pancreatic myocytes . Such chmyocytes . The thrmyocytes . From thβ cells that leads to impaired glucose tolerance, but rather it is that the fetus develops peripheral insulin resistance. It is this peripheral insulin resistance that ensures that adequate amounts of glucose are delivered to essential organs such as the brain with subsequent reduced delivery to nonessential tissues such as skeletal muscle. Support for the fetal salvage hypothesis comes from intrauterine growth restriction studies in rats. In these rodent models, glucose transport (glucose uptake and glucose transporter mRNA and protein levels) is reduced in lung and skeletal muscles of growth-restricted fetuses, whereas glucose transport is unaffected in brain [Interestingly, the thrifty phenotype hypothesis has been challenged by the “fetal salvage” hypothesis which offers a different explanation for the insulin resistance seen in those affected by intrauterine growth restriction . In thisin brain , 33. in utero, and no new nephrons are formed after birth; therefore a nephron deficit present at birth persists throughout life [The kidney is another organ thought to be affected by nutritional thrift. In both human and animal studies small offspring, due to maternal undernutrition and other causes, have reduced numbers of nephrons which has been strongly linked to hypertension , 34, 35.out life , 36. In out life . This reout life , 34. Catch-up growth, also known as compensatory growth, is where children return to their genetic trajectory for size after a period of growth delay or arrest. It may occur at any stage of growth but is most commonly observed in the first 2 years of life . Studies In the Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC) birth cohort, children who showed catch-up growth, as calculated by specified gains in standard deviation scores, between 0 and 2 years of age, were heavier, taller, and fatter  at 5 years of age than other children . A studyCianfarani and colleagues speculate that the tremendous effort to recover lost growth shortly after birth involves insulin and insulin-like growth factors (IGF). Infants affected by intrauterine growth restriction have low concentrations of insulin and IGF-1 at birth, and normalization of these parameters occurs in the postnatal period , 43. It The implication of catch-up growth is that rapidly enhancing early childhood growth by a nutrient enriched diet may cause harm overtime and that encouraging slower growth rates may actually be beneficial. In a study that provides evidence of the benefits of steady  growth, investigators measured fasting concentrations of 32-33 split proinsulin, a marker of insulin resistance, in adolescents born preterm who had participated in randomized intervention trials of neonatal nutrition . Fastingβ cells are particularly sensitive to reactive oxygen species because they are low in enzymatic antioxidant defense equipment [β cells to oxidative stress, it is believed that early and ongoing exposures to oxidative insults could result in the eventual manifestations of the metabolic syndrome and related disorders [Excessive reactive oxygen species can cause modulation of gene expression and/or direct damage to cell membranes and other molecules at critical developmental windows. Many believe that oxidative stress is the primary link between adverse fetal growth and later elevated risks of the metabolic syndrome, type 2 diabetes, and other disorders . Smokingquipment . It has quipment . With thisorders . Elevated oxidative stress has been observed among human infants born small for gestational age (SGA) as compared to those appropriate for gestational age (AGA) and among preterm as compared to term infants. It is important to note that preterm (birth at <37 weeks of pregnancy) infants are generally of low birth weight but are not necessarily growth restricted or small for gestational age. A study using cord blood to compare the status of oxidative stress between SGA infants born to undernourished mothers and AGA infants born to healthy mothers found elevated oxidative stress, as determined by increased quantities of malondialdehyde (one of the major products of lipid peroxidation), reduced quantities of the antioxidant glutathione, and decreased activity of the antioxidants superoxide dismutase and catalase in the SGA infants as compared to the AGA controls . A human study determined vitamin A, C, and E levels in the umbilical cord blood of term and preterm infants and the blood of their mothers taken at the time of delivery . MaternaGestational diabetes, a common cause of macrosomia, is associated with oxidative stress which could have effects on the developing infant. A study evaluating oxidative and antioxidative status in pregnant diabetic  women between 26 and 32 weeks gestation demonstrated increased oxidative stress in those who were diabetic . Levels ad libitum or offspring from dams undernourished for 20 days [An increase in circulating glucocorticoids may play a role in early programming of adult disease. Experimental studies in animals and epidemiological studies in humans have suggested an altered set-point of the hypothalamic-pituitary-adrenal (HPA) axis as an important long-term change that occurs in association with reduced fetal growth. Pregnant animal models have shown that exposure to a variety of stressors, including nutrient restriction, results in the birth of offspring with elevated basal or stress-induced glucocorticoid secretion , 55. It  20 days . An epidemiological study in humans involving three populations found that adults who had lower birth weights had elevated fasting plasma concentrations of cortisol and that cortisol concentrations positively correlated with current blood pressure . The autThe hypothalamus plays a critical role in the regulation of appetite and body composition by way of responding to cues from neuropeptides. A series of studies, primarily in rodents, have explored the possibility that maternal nutrition during pregnancy may alter the level of energy intake in the offspring through inducing changes in the hypothalamic circuitry and in the expression, localization, and action of specific neuropeptides. Central and peripheral neuropeptides function in the regulation of appetite. Appetite stimulating neuropeptides include neuropeptide Y (NPY), agouti-related peptide (AgRP), and ghrelin. Conversely, appetite suppressing neuropeptides include cocaine and amphetamine-related transcript (CART), melanocyte-stimulating hormone (MSH), serotonin, insulin, and leptin , 62. Of Studies in rats have found that exposure to overnutrition in the fetal or neonatal period can result in permanent changes in body fat mass and in the hypothalamic neuronal circuitry regulating appetite in the adult brain. Dörner, Plagemann, and colleagues found immunocytochemical and morphometric aberrations in the hypothalamus of offspring of diabetic dams or in those exposed to milk from diabetic dams. In one of their early studies, offspring of diabetic dams had permanent hypoplasia of the VMN, decreased insulin responsiveness to glucose, impaired glucose tolerance, and increased susceptibility to diabetes . A laterob/Lepob) mice have demonstrated permanently disrupted neural projection pathways from the ARC [adult Lepob/Lepob mice, leptin treatment did not reverse these neuroanatomical defects; however treatment of neonatal Lepob/Lepob mice with exogenous leptin rescued the development of ARC projections [Leptin has received a considerable amount of attention in regard to early programming of appetite and body composition. In a rodent study to establish whether neonatal  leptin treatment can alleviate postnatal obesity and the associated metabolic sequelae that occur in the offspring of undernourished dams, leptin treatment reversed the programmed phenotype . The ben the ARC . In the jections . Epigenetics refers to all modifications to genes other than changes in the DNA sequence itself and includes DNA methylation and histone modifications , 72. DNAAy or Avy develop the complex set of traits collectively referred to as the yellow obese syndrome. The yellow obese syndrome encompasses the pleiotropic effects of yellow fur, obesity, hyperinsulinemia, hyperglycemia, and increased susceptibility to neoplasia [a/a mice fed a methyl-supplemented diet with extra folate, vitamin B-12, choline, and betaine 2 weeks prior to mating with male Avy/a agouti mice and throughout pregnancy and lactation passed along the agouti gene to their offspring intact, yet demonstrated a shift in distribution towards having more offspring with the pseudoagouti phenotype [Avy locus [β cell. Rats faced with intrauterine growth restriction had permanently reduced expression of Pdx1 in β cells and developed type 2 diabetes in adulthood [Every cell in the body has the same genetic information; what makes cells, tissues, and organs different is that different sets of genes are turned on or expressed. An increasing body of evidence supports the role of environmentally-induced epigenetic changes in disease susceptibility. Experimental studies in agouti mice suggest a role for maternal diet in inducing epigenetic changes in the offspring , 76. Agoeoplasia . Female henotype . This shdulthood , 79. Reddulthood . in utero exposure to famine on birth weight in the subsequent generation persisted after control for potential confounding and intervening variables [The Dutch famine of 1944 has been used by various investigators as an equivalent to an experimental study to investigate the effects of perinatal undernutrition in humans . The ongariables . α (RXRA) chr9: 136355885+ (in cohort 1 and 2) and endothelial nitric oxide synthase (eNOS) chr7: 150315553+ (in cohort 1 only) at birth was correlated with greater adiposity in later childhood. Although the data is correlative and does not prove causality between DNA methylation at birth and adiposity in childhood, the observation suggests that epigenetics is involved in fetal programming of later obesity [In a study of 2 prospective cohorts, Godfrey and colleagues used DNA extracted from umbilical cord tissue obtained at birth in children who were later assessed for adiposity at 9 years of age to measure methylation status of CpGs in the promoters of candidate genes . Five ca obesity .β cell mass at birth and reduced insulin secretion in later life presumably due to dietary-induced reduction in proliferation rate and increased apoptosis of β cells [A sizable number of experimental studies, predominantly in rats, have explored the effects of protein restriction on fetal growth and later health. As discussed in the section on the thrifty phenotype hypothesis, protein restriction during pregnancy has been found to produce small offspring that have reduced numbers of nephrons which potentially contributes to the development of hypertension in adulthood , 34, 35. β cells . Weanlin β cells . In addi β cells . Protein β cells . When gi β cells . Although the literature on protein restriction and disease risk is quite extensive, it is largely based on rodent models with few having assessed the specific role of protein restriction in human risk of disease. One study of men and women in Scotland sought to determine how diet of the mother in pregnancy influences blood pressure in the 40-year-old adult offspring . The autAll dietary carbohydrates can be converted into glucose . In bothData derived from the Camden Study of adolescent pregnancy implicated high sugar consumption with a twofold increased risk for delivering small for gestational age infants . AdditioOur laboratory has performed multiple experimental studies aimed at determining the role of high fat diet in maternal-fetal health. In one such study, mice exposed to a high saturated fat diet during gestation and lactation developed adult obesity, hyperglycemia, insulin resistance, and hypertension, despite being fed a standard rodent diet post-weaning . Given tHuman observational studies have also found high fat intakes to be related to pregnancy and birth outcome as well as long-term health of the offspring. In a case-control study of 912 women admitted to obstetric hospitals for spontaneous abortion, the risk of miscarriage was directly associated with consumption of the main types of fats, butter and oil, while a significant inverse relationship was established between the risk of spontaneous abortion and the consumption of green vegetables, fruits, milk, cheese, eggs, and fish . Resultsβ-hydroxysteroid dehydrogenase-2, an enzyme that protects the fetus from overexposure to maternal glucocorticoids [ Although micronutrient deficiencies are known causes of several well-characterized diseases, such as scurvy and rickets , the rolrticoids . As mentrticoids , 115. Corticoids , 115. Few human studies have explored the role of early micronutrient deficiencies in the development of adult metabolic disorders. Of the existing reports, results are often conflicting and the significance is ambiguous. In two randomized controlled trials, both in Nepal, one found that daily multiple micronutrient supplementation in pregnant women resulted in slightly lower blood pressure in the offspring at 2.5 years of age , while t It is now widely accepted that certain chronic diseases of adulthood may have their origins in the womb. Studies discussed in this paper provide evidence that a mother's diet during pregnancy can exert major effects on the short- and long-term health of her children including programming of the metabolic syndrome. The challenges at present are to identify common mechanisms and pathways involved in disparate perinatal malnutrition paradigms, deciphering physiological and/or pathological roles of specific nutrients, and to determine which components of the maternal diet may be best modified to optimize maternal health, placental integrity, birth outcome, and lifelong health of the offspring. The implications of this avenue of research, particularly to obstetric and preventative medicine, dictate that effective interdisciplinary communication and knowledge transfer occur and that the information generated is disseminated to the general public. A recent review provides encouraging evidence that prenatal nutritional advice appears effective in reducing the risk of preterm birth and that balanced energy and protein supplementation seem to improve fetal growth and may reduce the risk of stillbirth and infants born small for gestational age . β cells produce fetal insulin prior to birth, thus increasing glucose transport and growth prenatally, which does not occur in the rodent, whose endocrine pancreas becomes functional postnatally [Whilst the evidence in support of the DOHaD is compelling, there are many caveats and controversies in the field including species differences, body weight verses body composition, and twin studies. First, species differences  should be considered when results derived from animal studies are extrapolated to humans. Rodents are particularly important to reproductive and obstetric studies because of the strong similarities between human and murine placentas –123; howtnatally , 125. Setnatally . The inctnatally , 29. Twitnatally , 29, 126


AbstractAlthough multimodal treatment has brought important benefit, there is still great heterogeneity regarding the indication and response to chemotherapy in Stage II and III, and individual variations related to both overall survival and toxicity of new therapies in metastatic disease or tumor relapse. Recent research in molecular biology led to the development of a large scale of genetic biomarkers, but their clinical use is not concordant with the high expectations. The Aim of this review is to identify and discuss the molecular markers with proven clinical applicability as prognostic and/or predictive factors in CRC and also to establish a feasible algorithm of molecular testing, as routine practice, in the personalized, multidisciplinary approach of colorectal cancer patients in our country. Despite the revolu¬tion that occurred in the field of molecular marker research, only Serum CEA, Immunohistochemical analysis of mismatch repair proteins and PCR testing for KRAS and BRAF mutations have confirmed their clinical utility in the management of colorectal cancer. Their implementation in the current practice should partially resolve some of the controversies related to this heterogenic pathology, in matters of prognosis in different TNM stages, stage II patient risk stratification, diagnosis of hereditary CRC and likelihood of benefit from anti EGFR therapy in metastatic disease. The proposed algorithms of molecular testing are very useful but still imperfect and require further validation and constant optimization. In the absence of a national screening program in CRC, most cases are diagnosed in advanced stages – 25% are presented as emergencies  and only a small percent are diagnosed in Stage I.Colorectal cancer (CRC) is one of the most frequent malignancies in Romania, with an incidence of 23,57/100000, more than 4000 new cases being diagnosed annually, representing 8% of all cancers. CRC is the third most frequent malignancy in our country, both in men and women after bronchopulmonary and gastric cancer and breast and uterine cancer, respectively. 90% of CRC are diagnosed after 50 years of age. The risk of developing CRC after 50 years is 5%, death related to CRC occurring in approximately 2.5% . The clinical management of CRC is currently based on clinicopathological factors such as TNM staging introduced by the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer (UICC), histology, tumor margin status and performance status. Additional histopathological features such as lymphovascular inva-sion, peritoneal involvement and degree of differentiation along with clinical features such as obstruction and perfora¬tion at the time of diagnosis are currently used to better define the poor prognostic subsets of patients [3]. Although it has brought great benefit, the multimodal treatment based on TNM staging remains imperfect. Individual patients with same stage tumors may have different long-term prognosis and response to therapy. Stages IIB and IIC (AJCC) have lower survival rates than the more advanced Stage IIIA [4]. TNM staging alone cannot identify the high-risk group of 20–30% of patients with locally restricted stage II (UICC) colon cancer who will suffer from disease relapse. This group of patients may actually benefit from adjuvant therapy in addition to surgical treatment, even though chemotherapy is not usually recommended, according to current guidelines [5].  Most patients presenting with stage I, II, or III disease (75%) can be treated with surgery alone, or in combination with chemotherapy  and radiotherapy , and have a 5-year survival rate of 93.2%, 82.5% and 59.5% respectively, compared with only 8.1% survival rate in stage IV disease [6].  Despite the extensive use of chemotherapy, mechanisms involved in clinical response remain elusive. A significant proportion of patients receiving various protocols of chemotherapy in metastatic disease do not derive any advantage. It becomes essential to identify subgroups of patients who may benefit from specific, targeted agents used in combinations such as FOLFOX , XELOX , FOLFIRI (Irinotecan + 5 F U + Leucovorin) or biological agents such as Epidermal Growth Factor Receptor Inhibitors  and Vascular Endothelial Growth Factor (Bevacizumab), in order to avoid a potentially toxic over treatment and an unprofitable financial burden for the health care system [7]. Clinical research of molecular biology in the last two decades confirmed CRC as a heterogeneous complex of diseases through the accumulation of distinctive genetic and/or epigenetic alterations. It is believed that CRC may arise from at least three interlinked mechanisms: Chromosomal Instability (CIN), CpG island methylation phenotype (CIMP) and Microsatellite Instability (MSI) . The current thinking behind CRC tumor genesis led to the development of numerous potential prognostic and predictive biomarkers. Unlike other types of cancer, with the exception of KRAS mutation, few of the studied markers have entered the clinical management of colorectal cancer so far, due to inconclusive studies or lack of current clinical applicability although they are widespread available in molecular biology laboratories [10]. The Aim of this review is to identify and discuss the molecular markers with proven clinical applicability such as prognostic and/or predictive factors in CRC and also to establish a feasible algorithm of molecular testing, as routine practice, in the personalized, multidisciplinary approach of colorectal cancer patients in our country.  Thus, research and validation of new prognostic factors, defined as any parameter evaluated at diagnosis (or surgery), which is associated with treatment outcome  and predictive factors, defined as any parameter which evaluates the response or lack of response to specific treatment is the key towards an optimized and personalized treatment of CRC [ CEA 11]. An ideal way to assess tumor status would be a serum marker. Serum markers allow a minimally invasive method of CRC screening that could easily be integrated into regular health checks. Currently, CEA is the only serum biomarker in a widespread clinical use for CRC that can be quantitatively measured. CEA is a normal cell product that is overexpressed by adenocarcinomas, primarily of the colon, rectum, breast and lung [12]. Smokers have a higher circulating CEA concentration (13.6%) than non-smokers (1.8%), but there are no significant effects of age, sex, or ethnic group on the normal range. In metastatic disease, 30% of patients may have normal CEA. Moderate to significant elevations of serum CEA can be observed in a variety of chronic and acute inflammatory diseases, including alcoholic cirrhosis, cholelithiasis, obstructive jaundice, cholangitis, liver abscess, emphysema, bronchitis, gastric ulcer, gastritis, diverticulitis, diabetes and collagen vascular diseases [13]. Serum Carcinoembryonic Antigen (CEA) is a complex serum glycoprotein produced by 90% of CRCs that functions as a homotypic intercellular adhesion molecule that promotes the aggregation of human CRC cells. CEA may facilitate metastasis of CRC cells to the liver and lung [14]. However, because of its lack of sensitivity in the early stages of CRC, CEA testing alone is an unsuitable modality for population screening. Wild et al found that the combination of six biomarkers had a sensitivity equal to fecal immunohistochemical testing for the early detection of colorectal cancer, CEA showing the best sensitivity at 95%, with a specificity of 43.9% followed by seprase (42.4% sensitivity), CYFRA 21-1 (35.5%), osteopontin (30.2%), ferritin (23.9%) and anti-p53 (20.0%) [15]. In some studies, the presence of elevated preoperative CEA (>5 ng/ml) correlates with poor prognosis and reduced overall survival in Stage II colon cancer . CEA could therefore be used in identifying high-risk Stage II patients who might benefit from adjuvant therapy [3]. Current ASCO guidelines recommend preoperative determination of CEA and also regular (every 2–3 months) CEA monitoring for 2–3 years follow-up in patients with stage II and stage III CRC who might be amenable for metastatic surgery upon recurrence and also in monitoring patients with advanced disease, receiving palliative chemotherapy [18]. CEA is also associated with Microsatellite Instability of CRC, as shown in another study, in which Stage III cancers with MSI had an elevated preCEA more often than those without MSI  [19]. A failure of the CEA to return to normal levels after surgical resection is indicative of inadequate resection , or of occult systemic disease, as reported for elevated postoperative CEA levels [20]. Other studies found that CEA levels above zero were indeed more predictive than any level within the normal range or above. Thus, absent CEA after surgery appears to confer a favorable prognosis. In 2006, Park et al. [19] investigated the role of serum CEA level in predicting the response to preoperative radiotherapy in rectal cancer. CEA level (>5 ng/mL) was significantly associated with both node infiltration and poor response to radiotherapy. In 562 patients with non-metastatic rectal cancer, who received pRCT and underwent total mesorectal excision, Das et al. [21] confirmed that CEA level (>0.25 ng/mL) predicted tumor downstaging along with circumferential extent and distance of tumor from the anal verge. Moreover, Perez et al. [22] found that a post-radiotherapy CEA level <5 ng/mL was a favorable prognostic factor for rectal cancer and was associated with increased rates of earlier disease staging and complete tumor regression. In a previous study, we found that a positive rate of serum CEA was significantly higher in advanced stages of disease, also in poorly differentiated colorectal cancer than in well and moderately-differentiated types [23]. However, the test often adds little useful information in the follow-up of CRC. Most evaluations of treatment success or failure will be made on the basis of imaging studies, such as CT or MRI scans. In general, CT or MRI should trump CEA changes, and a patient should not have treatment changed on the basis of a CEA alone, unless the patient has a relatively uncommon circumstance, such as predominantly peritoneal metastases, in which CT or MRI evaluation is extremely limited. If the CT shows clear improvement or worsening and the CEA result disagrees, the CEA result is going to be  ignored.  Serial CEA measurements can detect recurrent CRC with a sensitivity of roughly 80% and speciﬁcity of roughly 70%, and can provide a lead time of approximately 5 months . Microsatellites (MS) are stretches of DNA in which short sequences  are repeated. Given their repetitive nature they are liable for errors that can occur during DNA replication. Mismatch repair genes (MMR) play a critical role in the identification and correction of these errors. Failure of the mismatch repair apparatus leads to persistence of errors and an alteration in the length of a microsatellite sequence, a process described as microsatellite instability. Persistence of such errors in the critical areas of genes responsible for cell growth regulation leads to frameshift mutations with loss of the normal function of these genes and the promotion of tumorgenesis. MMR deficiency may result from the inheritance of a MMR gene mutation, somatic (noninherited) MMR gene alterations, epigenetic suppression of MMR gene expression or a combination of these factors [24]. The DNA mismatch repair system requires the cooperation of many genes including MLH1, MSH2, MSH3, MSH6 and PMS2. Germline mutations in one of the MMR genes are responsible for a genetic predisposition to CRC, known as Lynch syndrome, previously referred to as hereditary nonpolyposis colorectal cancer (HNPCC). In sporadic CRCs, MSI appears to arise owing to an epigenetic phenomenon, namely silencing of the MLH1 gene by hypermethylation of CpG islands in its promoter region . Samples with instability in two or more of these markers were defined as MSI-H (for high-frequency MSI), whereas those with one unstable marker were designated MSI-L (for low-frequency MSI). Samples with no instability in any of the markers were considered to be microsatellite stable (MSS). In 2004, the revised Bethesda guidelines included another panel of five quasimonomorphic mononucleotide repeats  [25]. There are no conclusive molecular or clinicopathological differences between MSL-L and MSS tumors, as studies reported [26], thus from the clinician point of view, only positive testing for MSI-H tumors should be considered.  (1)The PCR products can be separated electrophoretically and differences in fragment sizes from tumor-derived DNA versus DNA from normal tissue are scored, leading to an assessment of instability. At first, there was a great variability of the microsatellite loci selected. A National Cancer Institute (NCI)-sponsored workshop in 1998 recommended a panel of five microsatellite markers, known as the Bethesda markers, which consisted of two mononucleotide (BAT-25 and BAT-26) and three dinucleotide  repeats [27] .  (2)IHC analysis of MMR proteins, widely available as part of the routine services in general pathology, has become a popular alternative to detect MSI in the clinical setting. DNA mismatch repair proteins are normally expressed in normal human tissues, however in MSI-CRC a complete loss of protein expression of at least 1 of the MMR genes appears. This loss of protein expression is pinpointed by the absence of immunohistostaining using a panel of 4 antibodies to the protein product of the 4 genes most frequently involved in MSI-CRC: MLH1, MSH2, MSH6, PMS2 [28].If all 4 proteins are expressed, then we have a MSS phenotype. The interpretation of the results should follow a good understanding of the interaction of MMR proteins as MLH1 binds to PMS2 to the site of DNA repair and the same happens with MSH2 and MSH6 [29]. If the MLH1 gene is affected, either by germline mutation or CpG hypermethylation, then normal expression of MLH1, and also PMS2 will be lost, with preserved MSH2 and MSH6. If MSH2 gene is affected by germline mutation, then MSH2 and MSH6 expression will be lost. In contrast, in the rare cases of germline mutations of either PMS2 or MSH6, only the protein affected is lost [30], similar with MSI PCR testing. As studies confirmed, MSI testing and IHC appear to be almost equivalent strategies for identifying subjects with affected MMR genes – For the clinician, MSI-H phenotype at MSI testing and dMMR at immuhistostaining should be equivalent and considered as MSI-H [31]. However, identifying a MSI-H phenotype in CRC by MSI-PCR or by IHC is not enough to differentiate between an inherited and a sporadic lesion (See the diagnosis of Lynch Syndrome below). Both inherited and sporadic MSI-H tumors have very distinct clinical characteristics: proximal tumor location, female gender dominance, mucinous histology, lymphatic infiltration, high number of peritumoral lymph nodes, undifferentiated histology [32]. In a comparative study, the predictive value of normal IHC for an MSS/MSI-L phenotype was 96.7%, and the predictive value of abnormal IHC was 100% for an MSI-H phenotype , resulting in a sensibility of 94% and specificity of 100% in identifying MSI-H tumors . The 2-hit hypothesis of tumorigenesis applies to Lynch syndrome, where the first hit is represented by the germline mutation in 1 copy of 1 MMR and the “second hit is a somatic inactivation of the wild type allele”. The inherited defect results in a lifetime risk of 80% of developing early-onset and even multiple CRC in the setting of relatively few polyps . The syndrome has an incidence of approximately 1:1000 in the general population and accounts for 3–5% of CRCs. These patients also have an increased risk of extracolonic malignancies; endometrial cancer is the most common extracolonic cancer (40-60%) followed by gastric, small intestinal, hepatic, pancreaticobiliary, ovarian, ureteral, and brain tumors . The terms Lynch syndrome and hereditary nonpolyposis colorectal cancer have largely been used interchangeably over the years. The definition of HNPCC is primarily based on family history. Without knowledge of the family history, a diagnosis of HNPCC cannot be made. Lynch syndrome, on the other hand, is defined by the documentation of inherited inactivating mutations in the DNA MMR system and thus, without a molecular genetic testing of DNA from peripheral blood or normal tissue of the MMR genes to identify a germline mutation, a diagnosis of Lynch syndrome cannot be made . However, full gene sequencing in all CRC patients is highly inefficient. Various versions of sequential testing algorithms have been proposed for the diagnosis of Lynch Syndrome in which family history, clinical and pathological criteria correlated with IHC or PCR MSI testing, narrow considerably the number of patients that should undergo genetic testing, and in many cases, can even eliminate the need of further testing [40-42] .  Lynch Syndrome is a familial autosomal dominant condition characterized by a germline mutation in one of the MMR genes  [43]. These criteria (now known as Amsterdam criteria I) which addressed only personal and family histories of CRC were set to be the basis of the diagnosis of HNPCC. At the 1998 group meeting, these criteria were updated to include also the known extracolonic tumors associated with Lynch Syndrome (now known as Amsterdam criteria II – Table 1) [44].The first major clinical criteria were set by the International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer meeting held in Amsterdam in 1990 . Possible explanations for this lack of reliability include small family size, the physicians’ unfamiliarity with the syndrome, lack of documentation or reduced penetrance of the MMR gene mutation in the family. Also, the specificity of the Amsterdam II criteria is low. 50% of patients whose families fulfill the Amsterdam criteria fail to demonstrate MSI on tumor testing or germline mutations in any of the MMR genes. Such families are unlikely to have Lynch syndrome and may represent an unknown hereditary colon cancer syndrome. These families have an increased risk of developing CRC compared with the general population, but compared with Lynch syndrome patients, the risks are lower, with an older age of onset and no increased risk of extracolonic cancers [47].Since then, the growing understanding of the molecular basis of Lynch Syndrome permitted numerous studies to demonstrate that the sensibility of the Amsterdam II criteria is quite low (40%)  incorporate broader age and family history criteria, but also include the unique histopathological features associated with Lynch tumors  as a fifth criteria (Table 2 ). The novelty of the Bethesda Guidelines was the introducing of MSI testing in colorectal tumors that fulfill the criteria as a mandatory step in the diagnosis of Lynch Syndrome.  The Bethesda Guidelines formulated in 1996 and revised in 2004 .In 2007, Jenkins et al. published the MsPath model that used easily assessable clinicopathological characteristics to identify colorectal cancers with microsatellite instability (MSI-H), presenting in patients younger than 60 years, the age group most likely to be associated with LS, while ruling out colorectal cancers that are highly unlikely to be MSI-H. This model considers six clinicopathological features (with the corresponding coefficient): age at diagnosis (<50-year -0.7), anatomical site , histologic type , grade (poorly differentiated -0.6), Crohn-like reaction (present -0.5), and tumor-infiltrating lymphocytes (TILs) (present -2.1). Authors have recommended a cutoff MsPath score of 1.0 to maximize the specificity while maintaining a high sensitivity, because it is important not to miss MSI-H cases [50]. Studies tried to evaluate the practical validity of revised Bethesda criteria, MSI histology, and MsPath model [9] for detection of MSI-H phenotype in CRC and found a sensibility of 79% , and a specificity of 77%  for the Bethesda guidelines. MSPATH score achieved 93% sensitivity and 55% specificity . Deng et al. found that this specific BRAF mutation occurred in 87% of sporadic tumors with hypermethylated MLH1, whereas it was not present in any MSI tumor with a germline MLH1 mutation [55], hence, a ‘‘positive’’ BRAF mutation virtually excludes the possibility of Lynch syndrome. If the BRAF mutation is negative, then germline mutation analysis should be performed .Our approach is to apply the Amsterdam II and the revised Bethesda criteria and also the MsPath model (cutoff >1) to all CRC patients. The fulfillment of either one is an indication for IHC-MMR analysis . Patients with MSH2, MSH6 or PMS2 protein loss at imunohistostaining are considered highly suspects for Lynch Syndrome and referred for specific germline mutation testing and genetic counseling. IHC showing loss of MLH1 expression could be seen in either sporadic MSI-CRC or Lynch syndrome. In these cases, an additional layer of testing may be performed on the tumor before proceeding to germline analysis. These tests include MLH1 promoter methylation and/or BRAF mutation analysis. Given the technical difficulty related to methylation analysis, testing for BRAF mutations is more commonly performed. The V600E hotspot mutation in exon 15 of the BRAF gene, a member of the RAF family of kinases, has demonstrated utility in distinguishing whether the loss of MLH1 staining is due to somatic hypermethylation of MLH1 or a germline mutation [55]. However, the selection of which gene to test is dependent on the abnormality identified on the initial screening test. Once the diagnosis of Lynch syndrome has been established, patient genetic counseling and discussion of cancer risks and of the pros and cons of surgical options is crucial. An intensive surveillance program should be initiated and close family members can proceed with testing for the identified specific germline mutation. Current guidelines, both in Europe and in the USA, recommend colonoscopy starting by the age of 20–25 years every 2 years until the age of 40 years, at which point examinations should be performed annually. When a diagnosis of CRC is made in a MMR gene mutation carrier, subtotal colectomy with ileorectal anastomosis is recommended, owing to the 16% risk for developing a second primary CRC within 10 years [56]. Endometrial cancer in Lynch syndrome is usually recognized early (stage I), and the overall 5‑year survival rate is 88%. Annual transvaginal ultrasound and endometrial aspiration biopsies beginning between age of 25 and 35 years can be considered. The demonstration of a germline mutation is the gold standard for the diagnosis of Lynch syndrome. Germline testing is performed on DNA isolated from peripheral blood mononuclear cells. More than 90% of Lynch syndrome cases that have been genetically characterized show germline mutations in MSH2 or MLH1 [57]. The lifetime risks of developing stomach, ureter, renal pelvis, small bowel, bile duct and brain cancers are lower. Surveillance with upper endoscopy is offered to all Lynch syndrome patients every 2 years, starting from the age of 30–35 years. Annual renal ultrasound, urinalysis and urine cytology can be considered in families with a history of urinary tract tumors [58].Increased risks of ovarian cancer have also led some to propose annual transvaginal ultrasound with CA-125 testing beginning at the age of 30 years. Risk-reducing hysterectomy and bilateral salpingo-oophorectomy should also be presented as an option when childbearing is completed [Microsatellite instability as a Prognostic Marker 26]. In addition, MSI-H cancers are preferably right-sided with a decreasing prevalence of the MMR-D phenotype from proximal to distal locations, so that only around 4% of rectal cancers are MSI-H. One of the key clinical features of MSI-CRC is their good prognosis and non-aggressive biology in spite of a commonly found undifferentiated histology. One of the consequences of this behavior is that the prevalence of MSI-H colorectal cancers is higher in earlier compared with later tumor stages. Specifically, MSI-H tumors account for up to 22% of all stage II colon cancers, but only for 12% and about 5% of colorectal cancers diagnosed as stage III and IV, respectively [59]. These results have been confirmed by many subsequent studies, including a meta-analysis by Popat et al. of 32 studies with more than 7000 CRC patients, approximately 1300 of whom demonstrated MSI. The HR estimate for overall survival associated with MSI was 0.65 [60] More recent results from large individual randomized adjuvant trials (PETACC-3 [61] and QUASAR [62]) clearly validated the prognostic implication of microsatellite instability with hazard ratios for relapse-free survival (RFS), disease-free survival (DFS), and overall survival between 0.16 and 0.70. More interesting is the fact that both trials trial demonstrated a very strong prognostic effect of MSI-H compared with non–MSI-H for stage II colon cancers but only an attenuated prognostic effect in stage III tumors. In 2000, Gryfe et al. reported a population based study in which MSI was associated with a significant survival advantage, independent of all standard prognostic factors, including tumor stage . Furthermore, regardless of the depth of tumor invasion, MSI CRC had a decreased likelihood of metastasizing to regional lymph nodes  or to distant organs  [62]. In conclusion, the MSI phenotype has unanimously been recognized as a marker of good prognosis. The risk of recurrence of a MSI-H stage II colon cancer is in the range of 3%–6% within the first 3 years, even without any adjuvant therapy [Microsatellite instability as a Predictive Marker62].  The relationship between MSI status and response to chemotherapy has been more controversial. Although early nonrandomized studies indicated a potentially higher activity of 5-FU–based chemotherapy in MSI-H than in MSS CRC, data from randomized clinical trials consistently suggested quite the contrary: patients with tumors exhibiting the MSI-H phenotype did not derive any benefit from 5-FU–based adjuvant chemotherapy, and might even have a detrimental effect. [63] who pooled the individual patient data from five randomized adjuvant trials which compared a 5-FU–based adjuvant chemotherapy against surgery alone in stage II and stage III colon cancer. There was a statistically significant detriment in overall survival in stage II MSI-H tumors treated with 5-FU–based adjuvant chemotherapy compared to the untreated cohort . This negative effect of adjuvant therapy was not found in stage III colon cancers. Therefore, the overall consensus has been that MSI is a predictor of nonresponse to 5-FU in stage II colon cancers. The prognostic and predictive value of MSI has notable implications for clinical practice. The mainstay of treatment for stages II and III CRC is surgical resection, with preoperative radiotherapy in rectal cancers. In stage III colon cancer, postsurgical adjuvant fluoropyrimidine-based chemotherapy has been the standard of care since the 1980s when 2 landmark studies demonstrated that fluorouracil plus levamisole reduced mortality by approximately 30% in patients with lymph node-positive (stage III) cancer . Special attention in this context received Sargent et al. [65]. Based upon the MOSAIC trial, 6 months of combination chemotherapy with the FOLFOX regimen is now the recommended adjuvant treatment for patients with stage III colon cancer, with no concern of the MSI status [66] . In 2004, the MOSAIC trial showed that the addition of oxaliplatin to 5-fluorouracil and leucovorin (FOLFOX) reduced relapse by comparison with fluorouracil and leucovorin alone with hazard ratio (HR) 0.76  [67]. However, not all of them were confirmed by subsequent studies. In fact, in the QUASAR study, only T4 and inadequate lymph node sampling (<12) were confirmed as independent poor prognostic factors, while high grade of tumor, paradoxically, was a positive prognostic factor (perhaps due to its strong association with mismatch repair protein deficiency) [62]. The use of adjuvant chemotherapy in patients with curatively resected stage II remains controversial, since not all patients are likely to receive any benefit. Current consensus guidelines require a risk stratification of patients based upon the presence of clinical, pathological and molecular risk factors. The high-risk features included in the current guidelines of the National Comprehensive Cancer Network (NCCN) are: bowel obstruction, grade 3-4 histology, T3 tumors with localized perforation, lymphatic or vascular Invasion, T4 Tumors, close, indeterminate or positive margins, less than 12 lymph nodes examined [6]. In Fig. 3, we present our decision algorithm in the approach toward adjuvant therapy in all CRC stages. We consider high risk Stage II, patients presenting one of the following: T4, less than 12 lymph nodes examined and CEA>5ng/ml. These patients should follow the same postoperative protocols as in stage III CRC, as highlighted in the MOSAIC trial . The same trial found that Stage II with none of the high risk features received no recurrence or survival benefit with the use of FOLFOX by comparison with fluorouracil plus leucovorin alone so these patients should be spared from the toxicity of oxaliplatin treatment [65]. These patients are further stratified into low risk (those who exhibit MSI-H/MMR-D phenotype) with good prognosis and no need for postoperative chemotherapy and intermediate risk (MSS phenotype), that should be considered for 5FU based treatment. In addition, as seen above, preoperative CEA>5ng/dl can be regarded as a high risk feature in stage II patients . Ras proteins occupy a key position in the Epidermal Growth Factor Receptor (EGFR) signaling pathway, which is involved in cell differentiation, proliferation, and angiogenesis. KRAS is one of the most studied proto-oncogenes. A review of large trials reveals that approximately 30%-50% of metastatic CRC tumors harbor KRAS mutations. The mutations predominantly involve codons 12 and 13 of the KRAS gene and appear in an equal distribution at all sites of the colorectum [ The introduction of EGFR-targeted monoclonal antibodies (cetuximab and panitumumab) in the treatment of metastatic colorectal cancer transformed KRAS mutation testing from a fundamental research to an important predictive marker with proven clinical applicability.69], CRYSTAL [70], in which tumors were also evaluated for KRAS status. Studies found significant improvement in response rate (RR) and progression-free survival (PFS) in both associations , but only with patients with wild type KRAS. Patients with a KRAS mutation had a significant worsening in outcome with the addition of Cetuximab to both FOLFOX and FOLFIRI, demonstrating that mutated KRAS is a negative predictive factor for anti EGFR therapy. The same negative predictive value of KRAS mutation status was found on Cetuximab or Panitumumab monotherapy as second-line treatment of refractory metastatic CRC [72]. In February 2009, ASCO published a provisional clinical opinion unequivocally recommending KRAS gene testing in patients with metastatic CRC to select patients appropriate for therapy with Cetuximab or Panitumumab [71]. The National Comprehensive Cancer Network (NCCN) has also added a recommendation for KRAS testing to their revised 2009 Clinical Practice Guidelines for Colon Cancer, stating that patients with known KRAS mutations should not be treated with Cetuximab or Panitumumab, either alone or in combination with other agents [67]. Knowing the mutational status of a patient’s tumor before prescribing an EGFR-targeting MoAbs must now be considered the standard of care in metastatic CRC. At present, there are no clinical decisions that are impacted by KRAS mutation status in patients with stage I, II, or III colorectal cancer .  Combination cytotoxic regimens such as FOLFOX and FOLFIRI have typically been the chemotherapy backbones for first line treatment for metastatic colorectal cancer. The adding of Cetuximab to FOLFOX or FOLFIRI was studied into two large trials – OPUS [74]. Cappuzzo et al. [73] showed that patients whose CRC harbored BRAF mutation showed no response to anti- EGFR therapy, and they also showed a negative trend for both time to progression and survival. Currently, KRAS mutations are identified by widely available PCR kits applied on formalin-fixed, paraffin-embedded tissue specimens from resected primary tumors or needle or endoscopic biopsies in unresectable metastatic CRC. In addition to the role in differentiating between sporadic and hereditary MSI-CRC (discussed above), BRAF mutation was found to have poor prognostic and predictive value in metastatic CRC. BRAF mutations are found in 10% of CRC; recent studies found that KRAS and BRAF mutation occur in a mutually exclusive manner [ In conclusion, BRAF V600E mutation PCR testing is indicated in CRCs that are negative for KRAS mutation, when the patient is being considered for anti-EGFR therapy .Although it remains a controversial issue in oncology practice and should be interpreted with caution, on a case by case basis, in correlation with stage, degree of differentiation and MSI status, CEA is a useful prognostic biomarker in CRC, due to its validated clinical applicability, wide availability and cost effectiveness. Despite the revolu¬tion that occurred in the field of molecular marker research, only Serum CEA, Immunohistochemical analysis of mismatch repair proteins and PCR testing for KRAS and BRAF mutations have confirmed their clinical utility in the management of colorectal cancer. Their implementation into current practice should partially resolve some of the controversies related to this heterogenic pathology, in matters of prognosis in different TNM stages, stage II patient risk stratification, diagnosis of hereditary CRC and likelihood of benefit from 5-FU chemotherapy or anti EGFR therapy in metastatic disease. The proposed algorithms of molecular testing are very useful but still imperfect and require further validation and constant optimization; clinical judgement must be exercised in all cases. In order to fully exploit the potential of molecular markers towards patients’ benefit, CRC should be treated in a multidisciplinary team of surgeons, pathologists and oncologists, in which knowledge transfer and feedback between team members is essential. In the current environment in which cost-effectiveness in medicine is an appropriate concern, the promising new markers being researched should be incorporated into clinical practice only after their prognostic and/or predictive value was demonstrated by randomized clinical trials and providing they will impact decision-making in a patient’s care. ACKNOWLEDGEMENT:  This paper is supported by the Sectorial Operational Programme Human Resources Development (SOP HRD) 2007-2013, financed from the European Social Fund and by the Romanian Government under the contract number POSDRU/107/1.5/S/82839


The unique avifauna of West Polynesia  has been subjected to prehistoric human-caused extinctions but little was previously known about this topic in the Lau Group. We expected that the degree of human disturbance would be a strong determinant of tree species composition and habitat quality for surviving landbirds, while island area would be unrelated to bird diversity.We examined species composition of forest and bird communities in relation to environmental and human disturbance gradients on Lakeba  than to environmental gradients . Our point counts (73 locations in 1 or 2 seasons) recorded 18 of the 24 species of landbirds that exist on the three islands. The relative abundance and species richness of birds were greatest in the forested habitats least disturbed by people. These differences were due mostly to increased numbers of columbid frugivores and passerine insectivores in forests on Lakeba and Aiwa Levu. Considering only forested habitats, the relative abundance and species richness of birds were greater on the small but completely forested (and uninhabited) island of Aiwa Levu than on the much larger island of Lakeba.All trees >5 cm diameter were measured and identified in 23 forest plots of 500 mForest disturbance history is more important than island area in structuring both tree and landbird communities on remote Pacific islands. Even very small islands may be suitable for conservation reserves if they are protected from human disturbance. The 25,000+ islands in the tropical Pacific Ocean represent discrete terrestrial habitats that vary greatly in size, isolation, origin, and age 2) totaling 376 km2 of land. The plant communities of the Lau Group have been described qualitatively This paper concerns the Lau Group in Eastern Fiji , which lQuantitative descriptions of WP forest communities exist for Tonga Because prehistoric and historic deforestation has been a major driver of vertebrate extirpations and extinctions in WP We also expected, based on our previous findings in Tonga 2; talasiga  or, in recent decades, by pine plantations 2) of Lakeba in patches on coastal limestone outcrops and in small ravines on the volcanic hillsides Lakeba  is 28 km NW of Lakeba  and Aiwa Lailai . They are composed entirely of limestone, surrounded by extensive reefs, and separated from each other by a narrow (∼100 m), shallow passage Lying at 18°19′ S, 178°42′ W (12 km SSE of Lakeba) are the twin islets of Aiwa Levu  of the 21 species that occur on Lakeba, the largest island in Lau and the tenth largest in Fiji, are endemic to Fiji. Of the 24 species of landbirds that inhabit the entire Lau Group today, the only one endemic to Lau is Mayrornis versicolor, a monarch confined to Ogea Levu [1: The modern avifauna of the Lau Group is dominated by species that are widespread in WP. The Lau Group lacks most of the species of landbirds found on the six largest, western Fijian islands. Only two  is adequate to capture patterns of tree species composition in small forest patches, and is similar to the plot size used in forest vegetation sampling elsewhere in Polynesia Plot locations reflected gradients of human disturbance (different distances from habitation and cultivation) and environment . The plot size of 500 mTree species accumulation with total surveyed area was examined to estimate the extent to which the sample (23 plots) captured the forest community species pool, in order to confirm that the small sample is representative of compositional variation. An asymptotic species accumulation curve would suggest that the sample adequately described the community. Clustering Species and environmental data were then subjected to constrained multivariate ordination using partial Canonical Correspondence Analysis Collocalia spodiopygia vocalizes irregularly and is much easier to detect  in open habitats than in forests, we do not include its data in the total relative abundance values.Our point-count methods were identical or very similar to those used elsewhere in the region Each point-count station was at least 150 m apart from any other. Each point count was done within ca. 3 hr of sunrise . While we report all of these data, we provide interisland comparisons only for the February-March 2000 (wet season) data of Lakeba vs. Aiwa Levu, thereby eliminating the potential effects of seasonality and interannual variability. Avian taxonomy and nomenclature follow that of Steadman (2006).rarefy in the package vegan of the R software We examined the variation in species richness and abundance of landbirds by season (fixed effect) and habitat type (nested within seasons) for Lakeba using generalized linear mixed effects Poisson models (for count data). Because these estimates of species richness per habitat are biased by unequal sample sizes (point counts per habitat), we also carried out rarefaction analysis to interpolate estimated species richness to equal sample sizes. We used the function We also compared abundances of functional (feeding) guilds of landbirds, including columbid frugivores/granivores, passerine insectivores, and nectarivores, between seasons (fixed effect) and among habitats (nested within season) on Lakeba using Poisson generalized linear mixed effects models for count data. Then we compared the species richness and abundance of forest-obligate guilds (passerine insectivores and columbids) on Lakeba vs. Aiwa Levu based only on the 2000 wet season data (collected during the same months on both islands) and only for point counts in native forest habitat , using non-parametric Kruskal-Wallis tests.Collocalia spodiopygia which, as already mentioned, was excluded from the comparisons.These were the only statistical comparisons of bird data we made owing to the unbalanced sampling ; other patterns are described qualitatively. Although the comparisons of abundances of species or guilds among habitats and islands may be affected by species-specific detectability that can vary by habitat We recorded 85 species of trees >5 cm DBH in the 23 Lau forest plots, including eight of indeterminate identity  of large size  and low (∼50 m elevation), a strong littoral  component to this forest is seen in the occurrence of species such as Guettarda speciosa, Neisosperma oppositifolium, Pandanus tectorius, and Barringtonia asiatica.Aiwa Levu supports a unique forest type, distinctive from those on the other two islands. Group 1 corresponds to Coastal Forest and includes four plots  with shorter canopies, and occurring at lower elevations and on gentler slopes than in the other plots . Group 1Alphitonia zizyphoides, Dysoxylum richii, and Pleiogynium timoriense, with Melicope cucullata abundant only in one plot  were marginally significant among seasons (P = 0.083) and highly significantly among habitat types on Lakeba (P≪0.001), with more individuals recorded in the dry season, and more found in Mid-successional Forest than in any of the more disturbed habitats including Secondary Forest  was significantly greater in the dry season (P = 0.020), with more found in Mid-successional Forest than in either Pure Pine Woodland or Coastal Coconut Plantation  was not significantly different between seasons (P = 0.53) but significant differences among habitats (P≪0.001) were the result of much higher abundance in Coastal Coconut Plantation than in any other habitat on Lakeba , average richness per point differed among habitats (P≪0.001) with a pattern that mirrored overall abundance , i.e., rPtilinopus porphyraceus, swiftlet Collocalia spodiopygia, and honeyeater Foulehaio carunculata). The latter was abundant in Coastal Coconut Plantation  because the coconut trees were flowering heavily in October. The overall scarcity of birds in Mid-successional Forest on Nayau stands in stark contrast to the situations in similar habitats on Lakeba and Aiwa Levu , Gyrocarpus americanus and Dysoxylum spp. , or Ficus prolixa . Alphitonia zizyphoides is an important early-successional tree in the region Cryptocarya hornei, in contrast with the low frequency of Macaranga spp., suggests that Group 2 Secondary Forest sites have been regenerating for at least several decades. Species of Cryptocarya are typically shade tolerant Macaranga are short lived, fast growing pioneers, and dominate only in the first decade or two after disturbance Alphitonia, Cryptocarya, and Macaranga, the two Group 4 plots represent the most mature forest sampled on Lakeba. Group 3 Mid-successional Forest shares its strong dominance by deciduous Gyrocarpus americanus with dry forest described for western Fiji All of the 19 plots surveyed on Lakeba and Nayau fall within the Limestone Forest community . We diffSyzygium (Myrtaceae) [Intsia bijuga, Burckella richii, and Callophyllum vitiense are selectively cut on Nayau for house and boat building, so the forest there continues to lose its old growth characteristics.While it is commonly recognized that forests on small islands can sustain endemic and endangered species of vertebrates The relative abundance and species richness of birds on Lakeba are greatest in Mid-successional Forest, the habitat least affected by people. This is mostly due to increased numbers of columbids and passerine insectivores. By contrast, the relative abundance of passerine nectarivores is greatest in disturbed habitats with many coconut trees cf. . In the Gallus gallus), Pacific rats (Rattus exulans), black rats (R. rattus), cats (Felis catus), dogs (Canis familiaris), horses , pigs (Sus scrofa), and cows (Bos taurus). Aiwa Levu is uninhabited, 100% forested, and lacks non-native mammals except Pacific rats and goats (Capra hircus). To what extent the differences in non-native vertebrates affects the abundance and diversity of landbirds is not known. Columbids and passerine insectivores are more abundant per unit area on Aiwa Levu than on Lakeba  seem to be extirpated on Lakeba; Myzomela jugularis is the only species found in native forest on Lakeba that we did not detect on Aiwa Levu.Habitat quality influenced the landbird communities more than island area. Both richness and abundance of birds in forest were greater on very small Aiwa Levu than on Lakeba. This is in spite of the fact that Aiwa Levu's forest is more coastal in composition than Lakeba's , and typn Lakeba . Two speDucula latrans (absent on Lakeba and Aiwa Levu) and an apparent island-wide absence or rarity of the passerines Lalage maculosa, Mayrornis lessoni, Clytorhynchus vitiensis, and Myiagra vanikorensis, which inhabit the other islands (except that we did not detect C. vitiensis on Lakeba).Nayau is mostly deforested and has 430 people as well as chickens, rats (species uncertain), cats, dogs, horses, pigs, and cows. From our brief visit in October 2001, the most distinctive aspects of Nayau's landbird fauna are the presence of the pigeon 2, where the prehistoric number of bird species varied little with island area before human impact [shown by rarefaction–based species–area calculations; Many bird populations on Pacific islands have been extirpated as a result of prehistoric human impacts We identified variation in the composition of forest in Lau that corresponds to a gradient of habitat quality and degree of human disturbance. We discovered that the level of forest maturity is more important than island area in shaping the forest landbird communities of Lau. This effect is especially strong for pigeons and doves . The estimated number of species in the sampled forests is 112-127 using first and second-order jackknife estimates (see text).(DOC)Click here for additional data file.Table S12 in area.Basal area by species (square m/ha) and environmental and stand variables for 23 forest plots in Lau, each 500 m(DOC)Click here for additional data file.


Animal studies suggest that renal tissue hypoxia plays an important role in the development of renal damage in hypertension and renal diseases, yet human data were scarce due to the lack of noninvasive methods. Over the last decade, blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI), detecting deoxyhemoglobin in hypoxic renal tissue, has become a powerful tool to assess kidney oxygenation noninvasively in humans. This paper provides an overview of BOLD-MRI studies performed in patients suffering from essential hypertension or chronic kidney disease (CKD). In line with animal studies, acute changes in cortical and medullary oxygenation have been observed after the administration of medication  or alterations in sodium intake in these patient groups, underlining the important role of renal sodium handling in kidney oxygenation. In contrast, no BOLD-MRI studies have convincingly demonstrated that renal oxygenation is chronically reduced in essential hypertension or in CKD or chronically altered after long-term medication intake. More studies are required to clarify this discrepancy and to further unravel the role of renal oxygenation in the development and progression of essential hypertension and CKD in humans. Hypertension is a major public health burden and affects about 1 billion people worldwide. Hypertension increases the risk of coronary heart disease and cerebrovascular accidents . HyperteAnimal studies have suggested that renal tissue hypoxia might be another common mechanism of renal damage in hypertension and renal diseases . Indeed,2) as low as 10–20 mmHg in the medulla and 50 mmHg in the renal cortex [2 is determined by the oxygen delivery  and by the O2 consumption, which is driven essentially by the glomerular filtration rate and the active tubular transport. Renal kidney perfusion is characterized by preglomerular oxygen diffusion shunting, partly explaining the low tissue pO2 levels [+, K+-ATPase and partly on passive transport via paracellular pathways [2, resulting in a cortical pO2 around 50 mmHg [2, and local pO2 is as low as 10–15 mmHg under normal circumstances, which renders them highly susceptible to ischemic injury [Fifty years ago, Aukland and Krog were the first to report the unique oxygenation pattern of kidneys using microelectrodes sensitive to oxygen in dogs and rats. They measured levels of tissue partial pressure of oxygen  and HIF-1 regulated genes. The activation of these genes will not only lead to an increased erythropoiesis and to neoangiogenesis through activation of vascular endothelial growth factor and inducible nitric oxide synthase, but also to the stimulation of growth factors that cause endothelial dysfunction, activation of inflammatory cells, and oxidative stress, which will lead to tissue injury and interstitial fibrosis , 23. WheT2*-weighted MR images. Deoxygenated hemoglobin can therefore be used as an endogenous contrast agent. The ratio of deoxyhemoglobin to oxyhemoglobin concentrations is proportional to the pO2 of blood, and blood pO2 is supposed to be in equilibrium with the surrounding tissue. Hence, BOLD signal estimated by transverse relaxation rate R2* (=1/T2*) can be considered as a sensitive indicator of tissue pO2 [R2* as measured by BOLD-MRI has been shown to correlate well with tissue pO2 [A relatively new technology called ‘Blood Oxygenation Level-Dependent Magnetic Resonance Imaging (BOLD-MRI), offers now possibilities to assess renal tissue oxygenation non-invasively in humans , 26. Thessue pO2  and in hssue pO2 . R2* as ssue pO2 , 29. An Since 1996, BOLD-MRI has been used in humans to evaluate, amongst others, the effect of water diuresis on renal medulla and cortex in healthy volunteers , 35 and R2* values is low when performed by an experienced investigator and when using a standardized breath-hold technique [R2* signal (see below). The BOLD-MRI technique itself has been criticized by some , 41, argechnique . An idenEssential hypertension is often characterized by an increased sodium retention, either in the proximal or distal nephron segments, a process requiring active sodium transport which might lead to increased tubular oxygen consumption , 44. BesR2* levels in 20 African-American hypertensive subjects, as compared to 20 Caucasians suggesting a lower medullary oxygenation in hypertensives [Indeed, studies in hypertensive rats using oxygen microelectrodes found pronounced medullary and cortical hypoxia in spontaneously hypertensive animals as compared to normotensive controls . Most sttensives . This obtensives . HoweverT2*, suggesting a decrease in cortical oxygenation. Unfortunately, medullary oxygenation was not assessed in this study, and since hypertension was artificially induced, the results cannot be translated to the hypertensive population [Schachinger et al. showed that intravenous infusion of angiotensin II to six healthy volunteers leads to an acute rise in blood pressure (BP) and a simultaneous decrease of cortical pulation .Finally, in our research unit, we have investigated ten young normotensive and eight untreated hypertensive men with BOLD-MRI after one week of a high sodium (>200 mmol/day) and again after one week of a low sodium diet (<100 mmol/day). Simultaneously, renal hemodynamics and renal sodium handling were assessed using, respectively, inulin-, PAH-, and endogenous lithium-clearances . The maiR2* values than normotensive subjects, suggesting an increased rather than a decreased medullary oxygenation in young hypertensive men, again possibly due to reduced medullary oxygen consumption as a result of increased proximal sodium reabsorption. Interestingly, at any sodium intake, hypertensive patients had slightly lower medullary It is important to note that this study was performed in young hypertensive men, under extremely low or high sodium-intake conditions, and the oxygenation pattern might be different in older patients with hypertension-induced organ damage. Hence, acquired data in humans, so far, are not in line with animal studies and show no clear difference between normotensive and hypertensive subjects or even higher medullary oxygenation in hypertension as compared with normotensive controls. Whether this is due to less active medullar sodium transport, adaptations in medullar microcirculation or altered arterial venous shunting as compared to normal subjects merits further investigations. All presented data should be interpreted with caution, due to the small sample size, and confirmation in larger prospective studies is needed.β which on his turn stimulates the formation of interstitial fibrosis, the development of proteinuria, and worsening of tissue hypoxia [In clinical practice, renal ischemia has become a well-accepted cause of acute renal failure in situations of circulatory shock, accounting for almost 50% of cases of acute renal insufficiency . However hypoxia , 53.Accumulating data from animal studies supports indeed, a pathogenic role of tissue hypoxia in the chronic deterioration of kidney function. The most convincing evidence probably comes from Manotham et al., who have shown in a remnant kidney model (which is a representative model of CKD) that the number of hypoxic tubules was markedly increased 4 and 7 days after subtotal (5/6) nephrectomy, as compared to a sham-operated control group . HypoxiaR2* levels) in diabetics as compared with controls [BOLD-MRI can also be used to estimate renal tissue oxygenation in CKD patients , and sevcontrols ; the othR2* values and eGFR , it seems that renal oxygenation is kept constant over a broad range of physiological and pathological conditions. As such, the findings of Michaely et al. put into question whether “chronic renal hypoxia” truly exists in humans or whether acute episodes of hypoxia are “corrected” by, for example, alterations in renal microcirculation and HIF-induced interstitial fibrosis. In line with this hypothesis, Juillard et al. have described acute increases in cortical and medullary R2* levels the first days after subtotal clipping of the renal artery [The largest study so far assessing renal oxygenation at different degrees of kidney dysfunction was recently reported by Michaely et al. . This st formula ), and R2l artery . Yet, fol artery . The use of blockers of the renin-angiotensin system (RAS blockers) as antihypertensive and antiproteinuric medication has been particularly effective in slowing the progression of renal disease in type 2 diabetics , and allR2* levels, suggesting increased oxygenation, in nine healthy volunteers two hours after the intake of 50 mg of losartan [Animal studies have suggested that administration of RAS blockers leads to an acute increase in renal tissue oxygenation , 53. In losartan . R2* levels were not altered after one month of enalapril (20 mg qd) nor after one month of candesartan (16 mg qd) as compared with baseline . Acute changes in R2* levels were not assessed in this study. However, in combination with the study of Djamali et al., one might conclude that RAS blockers induce acute yet not chronic increases in renal oxygenation.We recently performed a cross-over pilot study in twelve patients with type 2 diabetes and CKD . Patients were either already on treatment with an ACEI or ARB or had a formal indication to start one albuminuria, or both). In this study, cortical and medullary R2* levels in healthy volunteers, in diabetics, and in patients with renal artery stenosis suggesting a diuretic-induced increase in renal oxygenation [The administration of furosemide has been shown to induce in an acute drop of medullary and to a lesser degree cortical genation , 46. TheTaken together, the BOLD-MRI technique has opened an exciting new field of research that allows for the first time the assessment of renal oxygenation non-invasively in humans. The changes in renal oxygenation observed in response to furosemide or in association with dietary sodium intake suggest that renal sodium handling is one of the main determinants of renal tissue oxygenation.  So far, no studies have convincingly demonstrated that renal oxygenation is reduced in essential hypertension, diabetes, or CKD. These findings are in contrast with most animal studies. First of all, it might be that BOLD-MRI is not sensitive enough or simply not as good a tool to assess renal oxygenation. Indeed, the assumption that tissue oxygenation varies with blood oxygenation might not always hold in kidneys that are characterized by profound arteriovenous oxygen shunting. Nonetheless, several animal studies have validated this technique. Another possibility is that animal studies provide excellent models to study short-term changes in oxygenation but that they are suboptimal in simulating the long-term changes and adaptations in the kidney that might have occurred after several decades of exposure to chronic disease states such as diabetes or hypertension. Whatever the truth may be, BOLD-MRI and other new radiological techniques that study renal functioning non-invasively merit the full attention of clinicians, and their development will certainly help to further unravel the role of renal oxygenation in the development and progression of essential hypertension and CKD in humans.


Dear Sir,Hydatid disease is a common occurrence in the Indian subcontinent, however involvement of kidneys constitutes only about 2-4% of cases.[1] Isolated renal involvement without involvement of liver or lung is even more rare. Associated hydatiduria is seen in only 10-20% of all cases of renal hydatidosis and involvement of ureter is rarely reported.[2] Though, due to slow evolvement and asymptomatic nature, hydatid disease is rarely seen in children, one such isolated hydatid disease of right kidney and ipsilateral ureter is reported here in an adolescent girl who presented with recurrent renal colic and gross hydatiduria.A fifteen year old girl referred to our institute for complaints of colicky pain in right lumbar region and dysuria with passage of small pale ruptured-grape like structures in urine for the last 6 months. Her general physical examinations and routine investigations were in normal limits. Ultrasonography of abdomen was suggestive of multiloculated cystic lesion, with typical spoke-wheel appearance, in the mid pole of right kidney and ELISA test was positive for echinococcus antibodies. CT scan obtained which revealed 11.5 cm X 11.0 cm multiseptated cystic lesion in mid and lower pole of right kidney and upper ureter with typical “cyst within the cyst” appearance , without any contrast excretion from right kidney. With four weeks of albendazole therapy, surgical exploration done by flank extraperitoneal approach and right nephroureterectomy done as almost entire kidney was converted into a bag of cysts with multiple cysts filled up in the upper ureter as well . Postoperatively, patient was put on albendazole (10 mg/kg daily) for eight weeks. Histopathological examination was consistent with hydatid disease. Follow-up ultrasound abdomen at 6 months showed no recurrence.Renal hydatid cysts primarily develop when the larvae, which are not filtered in the liver, pass through the pulmonary circulation and travel to the kidney.[1] Presenting symptoms depend on the kind of cyst. Closed cyst that contains all 3 layers, pericyst, ectocyst, and endocyst, usually remain asymptomatic unless large enough to stretch renal capsule. Once the pericyst is lost, exposed cyst expands rapidly and presents as chronic dull flank pain. In communicating cysts, all the three layers of the cyst rupture with direct communication into the collecting system, causing hydatiduria and acute colic or obstruction but it is seen in only 10-20% of renal hydatidosis and is usually microscopic in nature.There is no pathognomonic serological test for hydatid disease. Sonography is a sensitive method for the detection of membranes, septa, and hydatid sand within the cyst. Gharbi et al classified them into five types.[3] Present case, as having multiseptated daughter cysts, is a Type III cyst. Contrary to this, the most common variety reported in different paediatric series are Type I cysts, that have appearance of simple renal cysts but with double-contoured thick wall and hydatid sand as “falling snowflakes” . The CT scan has a higher accuracy and sensitivity to demonstrate the cyst wall and the daughter cysts.Medical management with oral albendazole alone, is mostly unreliable and radiological intervention in the form of Percutaneous Aspiration Injection Re-aspiration followed by Percutaneous drainage (PAIR-PD) has limited success in only 70% of cases of unilocular cysts with vested fear of spillage and anaphylactic shock. Surgery remains the mainstay of treatment and a variety of surgical procedures are tailored to suit individual cases. Kidney sparing surgery i.e. marsupialization, closed total cystectomy, partial pericystectomy, or capitonnage with or without omentoplasty are possible in most (75%) cases. In other 25% cases, where the kidney is almost destroyed, nephrectomy is the only option. In recent years, laparoscopic approach has also been used, but, the major problem with large renal hydatid cysts is the spillage of contents during dissection and the extent of the incision for the delivery of specimen.[1] Pre and postoperative one-month courses of albendazole should be considered in order to sterilize the cyst and to decrease the tension in the cyst wall that reduces the risk of spillage during surgery.[2] Though treatment is mostly surgical, it must be individualized.Source of Support: NilConflict of Interest: None declared


Cancer is nowadays considered to be both a genetic and an epigenetic disease. The most well studied epigenetic modification in humans is DNA methylation; however it becomes increasingly acknowledged that DNA methylation does not work alone, but rather is linked to other modifications, such as histone modifications. Epigenetic abnormalities are reversible and as a result novel therapies that work by reversing epigenetic effects are being increasingly explored. The biggest clinical impact of epigenetic modifying agents in neoplastic disorders thus far has been in haematological malignancies, and the efficacy of DNMT inhibitors and HDAC inhibitors in blood cancers clearly attests to the principle that therapeutic modification of the cancer cell epigenome can produce clinical benefit. This paper will discuss the most well studied epigenetic modifications and how these are linked to cancer, will give a brief overview of the clinical use of epigenetics as biomarkers, and will focus in more detail on epigenetic drugs and their use in solid and blood cancers. It has been thirty years since the “war on cancer” was declared, yet in 2008, the most recent year for which incidence and mortality rates are available, almost 12.7 million people were diagnosed with cancer and more than 7.5 million died of the disease [genetic and an epigenetic disease [Traditionally, the development of cancer is thought to be largely due to the accumulation of genetic defects such as mutations, amplifications, deletions, and translocations affecting the cancer cell machinery and providing the cancer cell with the advantage to survive and metastasize. In addition, interactions between cancer cells and their microenvironment further support these processes . Of equa disease , 6. EpigThere are many chemical modifications that affect not only DNA, but also RNA and proteins, and create different epigenetic layers. The most well studied epigenetic modification in humans is DNA methylation; however, it becomes increasingly acknowledged that DNA methylation does not work alone, but rather is linked to other modifications, such as histone modifications. This paper will discuss the most well studied epigenetic modifications and how these are linked to cancer, give a brief overview of the clinical use of epigenetics as biomarkers, and focus in more detail on epigenetic drugs and their use in solid and blood cancers.οDe nov DNA methylation is brought about by DNA methyltransferases (DNMT) 3A and 3B that convert cytosine residues of CpG dinucleotides into 5-methylcytosine, whereas DNA methylation is maintained by DNMT1. 5-methylcytosine can be further converted into 5-hydroxymethyl-2′-deoxycytidine by the Ten-Eleven-Translocation (TET) family enzymes [DNA methylation consists of the addition of a methyl group to carbon 5 of the cytosine within the dinucleotide CpG. Regions of DNA in the human genome, ranging from 0.5 to 5 kb, that are CG rich are called CpG islands and are usually found in the promoters of genes. Approximately half of all gene promoters have CpG islands that when methylated lead to transcriptional silencing.  enzymes . The fun enzymes  as well  enzymes . writers that place histone modifications, the erasers that can remove these modifications, and finally the readers that recognize the histone modifications and can deliver nucleosome, histone, or DNA-modifying enzymes.Histones are proteins around which DNA winds to form nucleosomes. A nucleosome is the basic unit of DNA packaging within the nucleus and consists of 147 base pairs of genomic DNA wrapped twice around a highly conserved histone octamer, consisting of 2 copies each of the core histones H2A, H2B, H3, and H4. Histones, however, are not only packaging elements, but also critical regulators of gene expression. Histone tails may undergo many posttranslational chemical modifications, such as acetylation, methylation, phosphorylation, ubiquitylation, and sumoylation that constitute a code, named the “histone code.” These modifications can alter the chromatin structure, from an open to a closed, condensed form and vice versa. Histone modifications act, except for chromatin packaging, on various other biological processes including transcriptional repression, gene activation, and DNA repair . Three cε-amino group of the lysine residue on the histone protein and use acetyl-CoA as a cofactor. As a result chromatin adopts a more relaxed form (euchromatin) allowing the recruitment of transcription factors. HDACs reverse the acetylation of lysine residues and the local chromatin architecture becomes condensed (heterochromatin). Acetylation of lysine 16 of histone 4 (H4K16) appears to be crucial in chromatin folding and in the switch from the euchromatin state to heterochromatin [Histone acetylation occurs at either arginine-(R) or lysine-(K) residues and is a dynamic and reversible process that is regulated by two enzyme families, histone acetyltransferases (HAT) and histone deacetylases (HDAC). HATs catalyse the transfer of an acetyl group to the hromatin . Histonehromatin . Histones can also be methylated at their lysine-(K) and arginine-(R) residues. Lysine residues can be monomethylated, dimethylated, or trimethylated whereas arginine residues can be mono- or dimethylated. Methyl marks are written by S-adenosylmethionine (SAM)- dependent methyltransferases and erased by either the Jumonji family of demethylases  or the lBRCA1 [hMLH1 [VHL [BIK [MGMT [In cancer, a global process of genomic hypomethylation occurs mostly at DNA-repetitive regions which results in activation of genes with growth and tumour promoting functions and loss of genome stability and imprinting . In contBRCA1 , hMLH1 [1 [hMLH1 , VHL [20VHL [BIK , and MGMIK [MGMT , 23. Cancer contains not only DNA methylation aberrations, but also major disruption of the histone modification landscape . HistoneDNMT3A [DNMT3B in pancreatic and breast cancer cells [DNMT3A mutations have been described in approximately 20% of acute myeloid leukemia (AML) patients, especially in those with an intermediate risk cytogenetic profile and although they did not affect the 5-methylcytosine content [DNMT3A mutations on 5-methylcytosine content is linked to an otherwise poor clinical outcome is not yet fully understood. It has been suggested that the R882 DNMT3A mutations alter functions of DNMT3A such as its ability to bind other proteins involved in transcriptional regulation and localization to chromatin regions containing methylated DNA [TET2 mutations were also identified in myeloid neoplasms in 20–30% [Deregulation of the epigenetic machinery can also occur due to activation or inactivation of the epigenetic regulatory proteins. In other words, the enzymes that maintain and modify the epigenome are themselves frequent targets for mutation and/or epimutation in neoplasia ; for exaDNMT3A  and DNMTer cells . Somatic content  they wer content , 31. Howated DNA . Loss-ofn 20–30% , 33 and n 20–30%  and bad n 20–30% . IDH) 1 and IDH2 genes [IDH1 and IDH2 mutations were mutually exclusive with TET2 mutations while they shared the similar epigenetic defects with the TET2 mutants. Epigenetic profiling revealed that AML patients with IDH1/2 mutations displayed global hypermethylation and a specific hypermethylation signature [EZH2 have been reported in B-cell lymphomas [EZH2 mutations [Genome sequencing has also revealed the presence of metabolic mutations in patients with myelodysplastic syndromes (MDS) and AML related to the isocitrate dehydrogenase  promoter has been shown to predict relapsed/refractory disease [FHIT has been shown to be an independent adverse prognostic factor [Several studies have explored the methylation status of gene promoters and its association with clinical parameters in primary patient samples from patients with haematological malignancies and solid tumours. Various methodologies have been used such as methylation-specific PCR (MSP), methylation-specific restriction enzyme digestion, HpaII tiny fragment enrichment by ligation-mediated PCR (HELP), bisulphite sequencing, and pyrosequencing. Either single genes or panels of genes in microarrays were studied. In MDS and AML methylation of several genes has been reported such as 3, SNRPN , Plk2 [5PN [Plk2 , cyclin-PN [Plk2 , and varPN [Plk2 . In multPN [Plk2  and meth disease , while mc factor .hMLH1 [Plk2 [MGMT in glioblastoma multiforme confers sensitivity to the alkylating agent temozolomide [In a study by Shen et al.  a panel hMLH1  and Plk2H1 [Plk2  in ovarizolomide  but as wNPM1 and FLT3 that are now widely used to risk classify AML patients. The list of genes reported to be methylated in haematological neoplasms is extensive, and although several have been linked to clinical parameters and have been associated with survival or response to treatment, none of these markers has been used so far in the clinic to guide diagnosis or treatment, as opposed to gene mutations such as One of the major goals of investigators in oncology is that of individualized cancer therapy. Investigators continue to identify genes whose transcriptional silencing affects sensitivity to chemotherapeutic agents. The challenge now is to translate these findings into clinically usable tests to inform optimal deployment of anticancer drugs. It remains unlikely that a single gene methylation test will be sufficiently informative to guide individual patient management and it is more likely that panels of genes will be required. Both epigenetic proteins and protein markers are good targets for the development of new anticancer treatments. The proof-of-concept for epigenetic therapies is the FDA and EMEA approval of demethylating agents and histone acetylase (HDAC) inhibitors for the treatment of MDS, AML and certain types of lymphomas, respectively. However, we should not forget that these agents are nonselective and their side effects are not clearly known. The two most well studied and in clinical use DNA methyltransferase inhibitors (DNMTi) are the azanucleosides azacytidine (5-azacytidine) and decitabine (5-aza′-2-deoxycytidine). Both are approved for use in the myelodysplastic syndromes and low-blast count AML and have improved the survival of patients with these diseases .MLH1, RASSF1A, HOXA10, and HOXA11 [Unfortunately, clinical trials with DNMTi in solid tumours did not have the same results. A phase 1 study of decitabine with interleukin-2 in melanoma and renal cell carcinoma showed that decitabine caused grade 4 neutropenia in most patients . Myelosud HOXA11 . It is pd HOXA11 . NT5E is overexpressed in aggressive metastatic melanomas, yet transcriptionally silenced by methylation in breast cancer with more favorable prognosis [A major likely reason for the disappointing activity of demethylating agents in solid tumours is limited incorporation into cells, which are proliferating relatively slowly. These limitations may be less relevant for newer DNMTis which are independent of replication for incorporation into DNA. A second explanation for these results is that agents such as azacytidine, which cause global hypomethylation, likely reactivate expression of multiple silenced genes including oncogenes and tumour suppressors in different cell types and in different cancers. Demethylation could therefore cause both therapeutic and deleterious effects. For example, the oncogene rognosis . A third and key possible explanation why DNMTi have advanced less rapidly in the clinic in solid tumours than in haematological malignancies is that of toxicity. Both decitabine and azacytidine are active in haematological malignancy at lower (less toxic) doses than are required for demethylation in epithelial malignancies. It is clearly of interest, therefore, that transient exposure of cells to low (relatively non-toxic) doses of these agents could induce a “memory” response with sustained reduction in CpG island methylation and reactivation of expression of previously silenced genes . These oNewer azanucleosides are zebularine, S-110, and SGI-1027 that have shown antiproliferative activity in cell lines , 64, butin vitro experiments, and valproic acid, a widely used antiepileptic, were identified. Valproic acid, in particular, has been used in combination with DNMTi and/or chemotherapy in patients with haematological malignancies [The HDACs catalyse removal of acetyl groups from lysine residues in the histones and functionally are transcriptional repressors. HDACs are divided into five classes: class I comprises HDAC1, HDAC2, HDAC3, and HDAC8; class IIa comprises HDAC4, HDAC5, HDAC7, and HDAC9; class IIb contains HDAC6 and HDAC10; class III comprises the sirtuins SIRT1-SIRT7 while class IV contains only HDAC11 . The disgnancies , 68.Currently HDACi that have been developed focus on class I and class II HDACs and can be further distinguished into chemically distinct subgroups based on their structure: aliphatic acids , benzamides (entinostat), cyclic peptides (romidepsin), and hydroxamates . Several HDACi are currently being tested in phase II-III trials, while two of them, vorinostat and romidepsin are the first FDA and EMEA approved agents for the treatment of progressive or recurrent cutaneous T cell lymphoma (CTCL) as second lines of treatment in 2006 and 2009, respectively , but conHDACi, however, do not deacetylate histones only. It becomes increasingly recognized that HDACi deacetylase other nonhistone proteins that are transcription factors, signal transducers, or even the products of oncogenes or TSG that are involved in oncogenesis . This coThe recognition that a subset of TSGs are silenced by a combination of CpG hypermethylation and histone hypoacetylation has prompted testing of combinations of the two classes of agents and trials of these are in progress. There is initial evidence to suggest that such combinations may greatly increase clinical efficacy without unacceptable toxicity. For example, in multiply pretreated metastatic non-small-cell lung cancer patients, the combination of azacytidine and the histone deacetylase inhibitor entinostat produced objective clinical responses and, importantly, four of 19 treated patients had therapeutic responses to further agents given immediately after epigenetic therapy . EvidencIn elderly previously untreated AML patients and high-risk MDS patients the combination of azacytidine and lenalidomide, an immunomodulator drug, is currently under investigation (NCT01442714). Both drugs as monotherapies have already shown efficacy in this group of patients so their combination seems very promising. Sequential treatment of azacytidine and lenalidomide in elderly patients with AML also showed encouraging clinical and biologic activity .In a recent Phase I study decitabine was combined with bortezomib for the treatment of elderly poor risk AML patients and the combination showed good preliminary activity since response rates were very encouraging .The use of epidrugs on the intent to restore sensitivity to cytotoxic or hormonal drugs is a major goal in the setting of solid tumors –87. RestGiven the complexity and heterogeneity of the cancer cell epigenome, it is highly likely that some form of epigenomic profiling of individual cancers will be required to inform optimal use of the available agents, which induce modification of the cancer cell epigenome. For example, it would clearly be important to determine the epigenome of chemotherapy resistant cancer cells, to identify potentially deleterious silenced genes, before deploying epigenetic therapeutic strategies in an attempt to pharmacologically reverse resistance. Malignant melanoma is an interesting example of such an approach. In this tumor type, loss of 5-hydroxymethylcytosine (5-hmC) has diagnostic and prognostic implications, which relates to downregulation of IDH2 and TET family enzymes. Reintroducing active TET2 or IDH2 was found to suppress melanoma growth and increase tumor-free survival in animal models .Identifying the epigenetically modified genes, which are principally involved in tumor resistance, can be achieved by comparative analysis of diagnostic (pretreatment) biopsy with a second biopsy at disease relapse. Such rebiopsying is rapidly becoming the standard of care in oncology, for example, in breast cancer . ASS1, which encodes arginine succinate synthetase, the rate-limiting enzyme in arginine biosynthesis, is silenced by methylation in some cancer types including renal cell carcinoma, hepatocellular carcinoma, malignant melanoma, glioblastoma multiforme (GBM), and platinum-resistant epithelial ovarian cancer. ASL encoding arginine succinate lyase (a second key enzyme in arginine biosynthesis) is also silenced by CpG island methylation in GBM [The ability of the physician to exploit therapeutic opportunities created by epigenetic changes in the cancer cell epigenome may also offer new approaches to cancer management. For example, n in GBM . Loss ofAs we previously discussed, several epigenetic modifiers such as EZH2, IDH1/2, and DNMT3A are genetically altered in cancer. These epigenetic modifiers provide now new therapeutic targets for clinical development. What seems to be needed though is a better selection of patients who will benefit from such treatments as well as identification of new druggable targets and compounds such as histone kinases  or inhibThe biggest clinical impact of epigenetic modifying agents in neoplastic disorders thus far has been in haematological malignancies and the efficacy of DNMTis and HDACi in blood cancers clearly attests to the principle that therapeutic modification of the cancer cell epigenome can produce clinical benefit. Although the efficacy of epigenetic therapy in solid tumours remains as yet unproven, there is every reason to believe that more rational use of existing agents, perhaps informed by individual patient epigenetic profiling, will improve the therapeutic index of this approach. Furthermore, an increasing number of viable new therapeutic targets are emerging from increased understanding of the epigenetic regulatory circuitry and its derangement in neoplasia.