Ovarian cancer is the fifth leading cause of cancer death among women and the most lethal gynecologic malignancy. One of the leading causes of death in high-grade serous ovarian cancer (HGSOC) is chemoresistant disease, which may present as intrinsic or acquired resistance to therapies. Here we discuss some of the known molecular mechanisms of chemoresistance that have been exhaustively investigated in chemoresistant ovarian cancer, including drug efflux pump multidrug resistance protein 1 (MDR1), the epithelial–mesenchymal transition, DNA damage and repair capacity. We also discuss novel therapeutics that may address some of the challenges in bringing approaches that target chemoresistant processes from bench to bedside. Some of these new therapies include novel drug delivery systems, targets that may halt adaptive changes in the tumor, exploitation of tumor mutations that leave cancer cells vulnerable to irreversible damage, and novel drugs that target ribosomal biogenesis, a process that may be uniquely different in cancer versus non-cancerous cells. Each of these approaches, or a combination of them, may provide a greater number of positive outcomes for a broader population of HGSOC patients. Ovarian cancer remains a devastating diagnosis with an overall survival rate of ~40%, making it the fifth leading cause of cancer death in women and the most lethal gynecologic malignancy ,2,3. OveFirst-line therapeutic interventions in ovarian cancer have evolved over the last few decades from a single nitrogen mustard alkylating agent to the current standard of care: cytoreductive surgery followed by combination taxane-platinum treatment ,4,5. AboHistorically, the first major studies that encountered acquired resistance to therapy were the clinical trials in 1965 in pediatric hematopoietic malignancies by Frie et al. . In usinBroadly speaking, resistance to therapies is categorized into intrinsic or acquired resistance, although distinguishing between these two mechanisms can be difficult. Intrinsic resistance is the innate ability of the cancer cells to maintain and persist through their first exposure to treatment. Acquired resistance is the evolution of cancer cells, following treatment exposure, to an unaffected and persistent state whereby cells maintain and expand in the presence of subsequent therapies ,20,21. AThe inherent ability of cells to survive chemotherapy can be mediated through several distinct mechanisms. Drug efflux pumps (adenosine triphosphate (ATP) binding cassette (ABC) transporters) lower drug concentrations within cells  transporters have been extensively studied in cancer chemoresistance. They are a diverse group of ATP-dependent efflux pumps containing 48 different family members with a common structural theme: a variable transmembrane domain and a conserved nucleotide binding domain ,41. In aMDR1 is responsible for the efflux of a truly staggering number of toxic compounds with diverse properties . SeveralFirst generation small molecule inhibitors of MDR pumps have had limited success due to these unforeseen consequences and overall toxicity problems, with clinical trials being marred by poor tolerability ,54,55. STargeting the MDR1 mRNA for degradation with small interfering RNA/antisense oligonucleotide (siRNA/ASO) is also being investigated as a method to ensure target specificity, and combining this with nanoliposomes that target cancer cells has been attempted in an effort to limit toxicity ,54. Yang50s) an order of magnitude lower than typical concentrations seen with paclitaxel, and also has less neuropathies and other side effects in patients [The development of novel tubule stabilizers that are not substrates for MDR1 efflux, such as the epothilone family, has shown efficacy in cells and PDX models previously resistant to paclitaxel ,63,64,65patients . Positivpatients . These rpatients .While some success has been observed in targeting MDR1 to overcome resistance, any real clinical gain has remained elusive. A recent whole genome sequencing study of a cohort of chemoresistant ovarian cancer has shown MDR1 to be rearranged in some chemoresistant populations, with promoter fusion driving upregulation. The promiscuous nature of the ABC transporters combined with the diverse side effects and toxicity problems has prompted some investigators to call for a change in tactics. Moving upstream of MDR1 to the epithelial–mesenchymal transition (EMT) pathway ties together multiple mechanisms associated with the acquisition of resistance, and EMT has a special position in ovarian cancer progression.EMT is the process of epithelial cells losing their apical-basal polarization, detaching through loss of adhesion, and becoming mesenchymal-like in both appearance and invasiveness ,68. TherChemoresistant phenotypic changes in ovarian cancer appear strongly correlated with EMT and the presence of subpopulations of cancer stem cells ,71,74,78DNA damage response pathways are very well characterized and generally signal through checkpoint kinase 1 and checkpoint kinase 2 (CHK1/CHK2) to determine the amount of damage present, amplify the repair signal to enhance repair efficiency, and delay the cell cycle to allow time for repair . Damage Platinums are the drug of choice for treating many aggressive cancers, and most HGSOC are quite sensitive to its toxicity . The mec50), showing promising results [TP53 mutant cells are thought to have a deficient G1 arrest ability with an enhanced G2 delay, allowing them to cope with genetic damage ,98. Canc results ,102. PD0 results ,104. Wit results . Other mAngiogenesis has long been a hallmark of most malignancies and it is well characterized in HGSOC. Anti-angiogenic therapies for HGSOC have been FDA approved for use in the recurrent setting, and while showing an increase in progression free survival (PFS) there has been no change to overall survival (OS) ,107. TarRecent phase III clinical trials in HGSOC have shown a significant increase in PFS with the same lack of OS increase in platinum resistant HGSOC with both bevacizumab and trebananib ,113. WhiUsing poly(ADP-ribose) polymerase (PARP) inhibitors for treating breast cancer 1 (BRCA) or HR deficient HGSOC is a highly active area of research with several FDA-approved therapies in the recurrent and maintenance setting . PARP isResistance to PARP inhibitors develops rapidly in some patients and occurs in surprising ways (reviewed in ). ReversExosomes are extracellular vesicles <100 nm in diameter that were initially thought of as cellular waste disposal units . ResearcExosomes have been found to play a role in resistance to chemotherapy , and in The idea that exosomes are also capable of sending out tumor specific signals begs the question: can we use exosomes as a cancer specific targeting system for drug delivery? Evidence is building for this being a promising way to deliver toxic therapies directly to the tumor  with HadThe most commonly-used therapeutic strategy in HGSOC today is combined platinum/taxane treatment after surgery. Several other options are becoming more clinically viable. The discovery of immune system checkpoint inhibition, like programmed cell death-1 (PD-1), has revolutionized the treatment of several types of cancers and is being actively explored in HGSOC ,145.The immune system has a dedicated surveillance network for identifying and destroying cells displaying neoplastic changes, neoantigens or other signals of dysfunction. Tumors, as they form, generally set off various checkpoint alarms and inflammatory processes that signal to the immune effector cells. For any growing lesion to form a viable tumor it has to evade immune system detection. PD-1 is one of a host of cell surface receptors used to identify cells to the immune system as “self” to stop normal tissues from being attacked. Many types of tumors have used this as a mechanism to hide from immune system detection, and blocking this escape tactic has shown efficacy in a diverse set of cancers. In HGSOC, trials have shown positive results using both anti-PD-1 antibody therapies as well as antibodies directed at programmed death-ligand 1 (PD-L1), its normal ligand ,147. HGSResistance to checkpoint inhibitors is both intrinsic and acquired, with intense research underway determining sensitivities in many tumor types. The recognition system appears to have multiple compensatory factors interacting, and blockade of a single receptor has been shown to compensate with upregulation of parallel pathways . SeveralOne of the first identified hallmarks of cancer, dating back to the late 1800’s, was enlarged and/or pronounced nucleoli . The sizRecently the idea of targeting ribosomal biogenesis has come forward as a viable strategy for treating various forms of cancer ,157 and RNA polymerase I is only responsible for the transcription of the pre-ribosomal RNA, so targeting it with small molecule inhibitors appears to be the most appealing strategy for specifically shutting down ribosomal synthesis ,161. TwoIn summary, while the understanding of mechanisms underlying intrinsic and acquired chemoresistance in ovarian cancer is expanding rapidly, overall therapeutic options remain limited. Promising results generated in vitro showing the therapeutic potential of targeting MDR1 have fallen short in practical application in the clinic due to toxicity. Alterations in EMT targets have had minimal success given there are several mechanisms remaining active that allow the tumor to adapt. As well, agents that try to exploit high levels of DNA damage may only be clinically applicable in contexts where tumors are also HR deficient.The pitfalls and shortcomings of translating these novel targets and associated drugs to the clinic further highlight the essential need for approaches to treatment that go beyond targeting single mechanisms. Mechanisms targeted previously only represent a handful of players in a complex and heterogeneous system that comprises chemoresistance in ovarian cancer. With all the new information on mechanisms of resistance to cytotoxic agents, specific resistance mechanisms to emerging targeted therapies and whole genome characterization of HGSOC personalized medicine may be on the horizon, and required for durable cures. Molecular characterization of the tumor followed by prediction of optimal therapeutic combinations with inhibitors blocking the expected paths of resistance may be the only way to achieve an increase in overall cure rates. The novel approach of targeting ribosomal biogenesis may prove more useful than previous approaches in improving outcomes for a larger proportion of patients with chemoresistant ovarian cancer in the future; however, previous research provides hope that it is feasible to identify the most important mediators of chemoresistance, while exploring the therapeutic potentials of current novel targets, so a broader population of patients with chemoresistant ovarian cancer will experience positive outcomes. As the understanding of resistance with tumor heterogeneity is enhanced, it is hopeful that targeting the specific chemoresistant population with upfront or consolidation therapy will lead to absolute cures, rather than just modest responses to therapy.


Constitutive activation of the PI3K-AKT-mTOR pathway (mTOR pathway) underlies megalencephaly in many patients. Yet, prevalence of the involvement of the PI3K-AKT-mTOR pathway in patients with megalencephaly remains to be elucidated, and molecular diagnosis is challenging. Here, we have successfully established a combination of genetic and biochemical methods for diagnosis of mTOR pathway-associated megalencephaly, and have attempted to delineate the clinical characteristics of the disorder.Thirteen patients with an increased head circumference and neurological symptoms participated in the study. To evaluate the activation of the mTOR pathway, we performed western blot analysis to determine the expression levels of phosphorylated S6 ribosomal protein (phospho-S6 protein) in lymphoblastoid cell lines from 12 patients. Multiplex targeted sequencing analysis for 15 genes involved in the mTOR pathway was performed on 12 patients, and whole-exome sequencing was performed on one additional patient. Clinical features and MRI findings were also investigated.AKT3, 1 patient; PIK3R2, 2 patients; PTEN, 3 patients) of the 13 patients. Increased expression of phospho-S6 protein was demonstrated in all five mutation-positive patients in whom western blotting was performed, as well as in three mutation-negative patients. Developmental delay, dysmorphic facial features were observed in almost all patients. Syndactyly/polydactyly and capillary malformations were not observed, even in patients with AKT3 or PIK3R2 mutations. There were no common phenotypes or MRI findings among these patients.We identified pathogenic mutations in six  patients with megalencephaly, indicating a major contribution of the pathway to the pathogenesis of megalencephaly. Our combined approach could be useful to identify patients who are suitable for future clinical trials using an mTOR inhibitor. Megalencephaly is accompanied by hyperplasia of the brain parenchyma, and is defined by a head circumference greater than +2 SD from the mean of the general population. Various diseases involve development of the condition, including metabolic diseases, such as Alexander disease and Canavan disease, and syndromes, such as Sotos syndrome and Noonan syndrome .AKT3 and PIK3R2 and a postzygotic mutation in PIK3CA in patients with MCAP, MPPH, and overlapping phenotypes of MCAP and MPPH  ] was identified in two patients, and PTEN heterozygous mutations  were identified in three patients ], and was found to be negative ] was not reported previously. This de novo mutation was located in the C2 domain that is involved in binding to phospholipids in biological membranes  always presented clinical symptoms of MPPH. PIK3CA mutations were identified at multiple sites, and all but one case showed MCAP. AKT3 mutations were found in patients with overlapping phenotypes or MPPH  detected in our study has also been previously reported in Bannayan syndrome [PTEN mutation also had no major anomaly other than megalencephaly and dysmorphic facial features. Neither patient had complications with tumors. While all patients showed a +2 SD or larger head circumference, the head circumference at birth was not necessarily large, indicating that megalencephaly in our patients was progressive. While developmental delays were observed in all patients, patients with AKT3 or PIK3R2 mutations had no meaningful words and had a more severe disease phenotype than those with PTEN mutations. Overall, no apparent differences in phenotype could be identified between patients with or without the involvement of the mTOR pathway and between patients with or without identified pathogenic mutations, suggesting genetic heterogeneity of the disorder.In a report from Riviere et al., a syndrome . Howeversyndrome , but hamAKT3 or PIK3R2 mutations, and seemed indicative of mTOR pathway involvement. One mutation-negative patient, however, did exhibit these features, and thus these findings are not sufficiently specific to indicate all underlying pathogenesis. No abnormal MRI findings were observed in patients with PTEN mutations. The lack of a cortical anomaly has been reported for PTEN macrocephaly [PTEN involvement.Regarding head MRI findings, enlargement of the ventricle, polymicrogyria, and white matter abnormalities were observed in all patients with ocephaly , and it PIK3R2 in particular is low at 79.2%), and the Ion PGM method offers only limited indel detection. Nevertheless, our target sequencing is useful in early diagnosis in clinical settings due to the lower costs and shorter turn-around time than WES. Secondly, our strategy is limited when analyzing cases of mosaicism. Indeed, Riviere et al. performed targeted ultra-deep sequencing  of the PIK3CA mutation sites in mutation-negative affected individuals, as well as in known mutation carriers and control individuals [In our study, target sequencing was performed primarily on 15 genes involved in the mTOR pathway. Therefore, the first conceivable limitation is the possibility of mutations in other genes that were not covered by our gene panel. In addition, our gene panel do not cover the entire exon region (the coverage ratio of mTOR inhibitors are effective treatment for not only cancer, but for the brain and renal tumors of tuberous sclerosis . They arA combination of genetic and biochemical methods was able to identify the involvement of the mTOR pathway in approximately 70% of patients with megalencephaly of an unknown etiology. Our combined approach could be useful to identify patients who are suitable for future clinical trials using an mTOR inhibitor.


The rising interest in human induced pluripotent stem cell (hiPSC)-derived organoid culture has stemmed from the manipulation of various combinations of directed multi-lineage differentiation and morphogenetic processes that mimic organogenesis. Organoids are three-dimensional (3D) structures that are comprised of multiple cell types, self-organized to recapitulate embryonic and tissue development in vitro. This model has been shown to be superior to conventional two-dimensional (2D) cell culture methods in mirroring functionality, architecture, and geometric features of tissues seen in vivo. This review serves to highlight recent advances in the 3D organoid technology for use in modeling complex hereditary diseases, cancer, host–microbe interactions, and possible use in translational and personalized medicine where organoid cultures were used to uncover diagnostic biomarkers for early disease detection via high throughput pharmaceutical screening. In addition, this review also aims to discuss the advantages and shortcomings of utilizing organoids in disease modeling. In summary, studying human diseases using hiPSC-derived organoids may better illustrate the processes involved due to similarities in the architecture and microenvironment present in an organoid, which also allows drug responses to be properly recapitulated in vitro. The global rise in the number of patients suffering from chronic and/or degenerative diseases has not only led to a tremendous increase in the demand for an unlimited source of transplantable tissues, but also sustainable human disease models that will allow for in-depth investigations into disease pathogenesis. Harvesting cadaveric donor tissues, which in itself is a limited source, for both transplantation and disease modeling is highly inefficient and logistically challenging, often resulting in low yield of functional cells/tissues. This then has fueled the advancement of cellular regenerative therapy of which the primary focus lies in tapping into the pluripotency of human embryonic stem cells (hESCs) (and even extending towards the use of organ-restricted adult stem cells) to generate large quantities of transplantable somatic cell types in vitro in the presence of defined culture conditions; a process also known as directed differentiation. Since hESCs are proliferative and capable of self-renewal, they can be used to generate transplantable cells, as well as different cell types that can be manipulated to model human diseases.Ethical and immuno-compatibility issues faced due to the use of hESCs and its derivatives for clinical transplantation have been overcome by the advent of the human induced pluripotent stem cell (hiPSC) technology where patient-specific somatic cells  can be directly reprogrammed by defined factors to induce pluripotency . These hUsing hiPSCs to model diseases has provided an opportunity to bridge the gap between fundamental genetic studies and disease pathogenesis. Researchers can now readily generate hiPSCs from patients to launch in-depth investigations into the underlying disease mechanisms of complex diseases and even study developmental defects by recreating embryonic development through directed differentiation of those hiPSCs; such investigations were virtually impossible prior to the availability of the hiPSC technology.Recent advancements in genome editing, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (CRISPR-Cas9) genome engineering tool, has enabled researchers to efficiently manipulate genomic sequences in hESCs and hiPSCs . SeveralRobust directed differentiation of hESCs and hiPSCs into somatic cell types in 2D monolayer cell culture systems have advanced disease-modeling efforts over the past decade. Many researchers have been successful in gathering novel and crucial insights into complex diseases. However, the field has grown to realize that these conventional 2D models, such as cells obtained from monolayer differentiation methods and homogenous primary/patient cell line cultures, are still relatively limited in providing a comprehensive understanding of complicated processes such as embryonic development, cellular differentiation, tissue regeneration, and disease development . CurrentWhilst the 3D organoid technology is still in its infancy, it has prevailed to be superior as compared to its traditional 2D counterparts. The organoids that can be obtained from well-established differentiation protocols have the capacity to self-organize and generate sophisticated 3D structures that accurately mimic the morphology of organs ,15,16. IUnsurprisingly, this has spurred recent reports of various differentiation protocols to obtain a myriad of organoid types (modeling different tissues) in vitro, such as intestinal, kidney, brain, retinal, pancreatic, and liver organoids. While organoids have been derived from several tissues or organs, cardiac organoids have yet to be reported, although several groups have reported generation of 3D cardiac tissues in vitro . By direIn this review, we will discuss how hiPSCs can differentiate into different types of organoids and how genetic manipulation of genes using advanced genome editing tools can be used in understanding normal embryonic and disease development and, eventually, provide avenues for disease modeling in vitro. The knowledge gathered from such disease models can subsequently be translated into therapeutic options. Herein, we have also highlighted some strengths of modeling human diseases in 3D and address the current limitations that are plaguing this culture system. Perhaps while 3D organoid technologies are still developing and improving, utilizing this modern methodology to model diseases in tandem with the use of more traditional approaches such as animal models will be a powerful and holistic approach in modeling numerous types of complex human diseases.The 2D cell culture models still predominantly used place cells in a non-natural environment with cells growing in a homogeneous monolayer and adhering themselves on a bed of artificial matrix that attempts to mimic the extracellular matrix (ECM). Though such an experimental setup proves to be both simple and efficient, it does not provide cells with the natural biophysical and biochemical environmental cues that have a significant impact in cellular activities and functionalities . The monMany recent studies have shown that the attributions of the surfaces that cells grow on in 2D models influence the cellular and molecular characteristics of the cells, such as cell development, proliferation, differentiation, apoptosis, and cell migratory behaviors . The micCurrent organoid systems have reproducibility concerns as there is little to no control over how cells self-organize into the organoids. Therefore, these protocols are unable to guarantee exact replications of organoid spheres of the same dimensions , cellular composition, phenotypic and molecular characteristics . The autResearchers have begun to recognize the importance of generating vascularized organoids. Vascularization will allow for oxygen and nutrients to be supplied throughout the mass, encouraging better development of cells into tissue-like structures and for the cells that are within the mass to be able to survive and function as well as cells on the periphery . From a However, while promoting neo-vascularization of organoids is no simple task, great strides have been made in devising methods to vascularize 3D cellular models. Vascularized 3D cancer models have been generated to study tumorigenesis and angiogenesis . VasculaWhile existing 3D organoid protocols have revolutionized our approach to disease modeling and brought much success in recreating the intricate interactions with various cell types in a spherical micro-environment, these models are still unable to recapture the complex and dynamic inflammation niche that occurs in vivo and that involves a multitude of cell types  and cellular processes  published within the last decade have attempted to model inflammatory skin diseases, which are the most common dermatological pathologies. These models have attempted to address the perplexing task of modeling inflammation through the utilization of various techniques that are technically demanding. One of the earlier models made use of a layering technique to create a multi-layered 3D skin structure supported by collagen matrices and exposed to an air–liquid culturing interface to mimic actual skin  . The autOne of the earlier works on engineering blood perfusion in organoids was published by Sefton in 2006 where they allowed for modular assembly of endothelial cells with “solidified cells (in collagen gel rods)” that were assembled into a larger tube where fluid or whole blood could pass through . HoweverMurine models have been extremely helpful in disease modeling prior to the advent of the organoid technology. Immunodeficient mice such as the nude mouse and severe combined immunodeficiency (SCID) mouse have been commonly used as recipients of human cells or tissues as they accept foreign cells relatively easily due to a lack of host immunity. In contrast, humanized mice are rodents that have been removed from an innate immune system and completely re-populated with human immune cells by human hematopoietic stem cell transplantation . Such huConsidering the limitations together, the 2D culture systems may no longer be useful in human disease modeling. In addition, the use of animals for disease modeling, drug testing, and therapeutic development are not only costly and time consuming but may not faithfully represent biological responses in humans due to species differences. Hence, whenever possible, it is a strategic choice to use organoid-based methods to investigate regulatory and pathological mechanisms at a molecular level. The wide-array of biomedical applications of which 3D organoids can be utilized is illustrated in Organoid culture is a powerful tool for modeling neurodevelopmental disorders, such as primary microencephaly, and can be employed as a drug-screening platform. Due to an increasing incidence of infants born with severe microencephaly, many studies have presented evidence of prior/premature exposure to Zika virus ,14,74. ITLR3) gene upon ZIKV infection, which was associated with perturbed cell fate differentiation and reduced organoid volume, both of which physiologically mimic clinical manifestations of microencephaly [3D models of neural stem cells (NSCs) in the form of neurospheres and brain organoids were used to explore implications of Zika virus (ZIKV) infection during neurogenesis and growth . Interesncephaly .SOX1 and PAX6 were observed [BF1 and SIX3) remained highly expressed whereas hindbrain markers (KROX20 and ISL1) decreased [2+ oscillations in single cells. Treatment of these cells obtained from the organoid showed that tetrodotoxin (TTX)-induced action potential blockade dampened calcium surges, thus indicating calcium spikes were dependent on neuronal activity. Finally, the authors validated the functionality of glutamatergic receptor activity of the developing neural tissue [Cerebral organoids have been able to express heterogeneity and regionalization of several regions of the brain, including the cerebral cortex, ventral forebrain, midbrain–hindbrain boundary, and hippocampus . Upon suobserved . Cerebraecreased . Lancastecreased . More rekers KROX and ISL1ecreased . Neuronsl tissue . CerebraCollectively, brain region-specific organoids can model neurodevelopmental disorders and possible compound testing for ZIKV antiviral drugs as demonstrated by Zhou et al. Their group identified two potential drug compounds, hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ), which can inhibit ZIKV infection in human pluripotent stem cell-derived cortical neural progenitors cells (hNPCs), as well as rescue the effects of ZIKV-induced growth and differentiation defects in hNPCs and human fetal-like forebrain organoids .Organoids derived from adult liver and pancreas also provide a platform for regenerative medicine and disease modeling . Takebe Induced human intestinal organoids (iHIOs) can be generated to mimic the human intestine through exposure to a series of growth factors to mimic embryonic intestinal development ; ActivinThe ability to generate iHIOs greatly opens the field of translational cell-based therapy for diseases such as necrotizing enterocolitis, inflammatory bowel disease, and short gut syndromes . EarlierHelicobacter pylori and Salmonella enterica, where anti-viral/bacterial therapy testing can be done on such models [Helicobacter pylori in the human stomach and its progression to stomach cancer [The induced human intestinal organoids can also model other host–microbe interactions with pathogens such as h models . Recentlh cancer .New frontiers of modeling cancer in vitro have included the use of patient cell-derived tumor organoids. Tumor organoids can be efficiently generated from either circulating tumorigenic cells, cancer cell lines, or cells extracted from tumors, and are highly expandable ,84, thusPTEN loss, TMPRSS2-ERG interstitial deletion, SPOP mutation, SPINK1 overexpression, FOXA1 mutation, and CHD1 loss, were also observed in the organoid models [Earlier prostate cancer organoids (PCOs) were generated from patient biopsy samples and circulating metastatic cancer cells . These od models . This imd models .LGR5 recapitulated several properties of the original tumor architecture, cell composition, and self-renewing capabilities. Tumor organoids generated from healthy epithelium and tumor-derived organoid cultures provide a comparative model for investigating the causal role of genetic mutations leading to colorectal cancer pathogenesis. While wild-type intestinal organoids expressed goblet cell genes such as MUC1, MUC4, and CA2 (colonocyte marker), tumorigenic organoids were enriched with cancer-associated genes such as PROX1, BAMBI, PTCH1 and APCDD1 [Patient-derived intestinal stem cell-derived tumor organoids marked by d APCDD1 . Additiod APCDD1 .APC, P53, KRAS, SMAD4) [In addition, Drost et al. recently demonstrated that CRISPR/Cas9-mediated genome editing was capable of generating human intestinal cancer stem cells by inducing four most commonly mutated colorectal cancer genes  . Mutant , SMAD4) .Fallopian tube epithelium (FTE) has been described as one of the sites of origin of high-grade serous ovarian cancer (HGSOC). Traditional 2D FTE culture systems have failed to recapitulate the architecture and geometrical features of tissues in vitro, as well as gradients of nutrients, oxygen, and carbon dioxide that are the fundamental tissue environments in vivo. On the other hand, 3D organoid-derived FTE reported by Lawrenson et al. demonstrated polarized columnar epithelium containing the desired cell types and convoluted luminal architecture, which closely represents the structure and organization of the fallopian tube in vitro ,86. The The derivation of fallopian tube organoids (FTOs) from adult stem cells came only recently when Kessler et al. described the formation of FTOs using stem cell-like cells obtained from gynecological FTE tissue samples . The FTOFOXG1 could potentially be used as a biomarker of severe ASD [FOXG1 gene predominant in these cortical organoids provides an understanding of the alterations in the dynamics of brain growth and differentiated neurons. A separate study by Mariani et al. reported that organoids displayed organized layers of radial glia, intermediate progenitors, and neurons, and that several pathological features in these organoids were observed, which correlates to earlier studies suggesting an increased number of neurons [Autism spectrum disorder (ASD) is a neurodevelopmental disorder that can be modeled using neural cortical organoids generated from iPSCs obtained from individuals with severe idiopathic ASD. These cortical organoids exhibited a decreased cell cycle duration, indicative of perturbed cell cycle potential, and showed an overproduction of GABAergic inhibitory neurons, providing critical insight into the pathogenesis of ASD that was previously unclear. The success of cortical organoids in modeling ASD is largely due to the ability to model embryonic telecephalic development seen in the third trimester of human development, as well as recapturing the regulatory networks of GABAergic neuron production. The authors identified gene expression of vere ASD . Dysregu neurons  and an i neurons  and syna neurons  in ASD i neurons .CFTR) gene mutation. The CFTR gene encodes for the ATP-binding cassette transporter and CFTR mutations affect the liver, lung, pancreas, and intestine [CFTR mutation using CRISPR/Cas9-mediated gene editing in primary adult intestinal stem cells fully restored the functional phenotype in intestinal organoids in culture [Intestinal organoids are not only capable of modeling host–microbe interactions  [Whilst hepatic organoids (HOs) may be useful in drug screening and toxicology studies (as mentioned in and JAG1 . Clinicaand JAG1 . Nicholaand JAG1 . More reand JAG1 . Patientand JAG1 . ALGS ornd CFTR) .rev) and introduced two ALGS mutations (C1mu and C2mu) in iPSCs generated from two different control individuals [rev rescued the disease phenotype and recovered their capacity to form well-organized structures with bile ducts [mu and C2mu formed less compact and well-organized bile duct structures compared to wild-type HOs [As a proof of concept, the authors generated an isogenic control through the correction of ALGS1 mutation  to form organoids in vitro. As such, while researchers are still juggling between the strengths and weaknesses of 2D and 3D cellular models in disease modeling (as summarized in


Fast accumulation of sequencing data on plant virus genomes and plant transcriptomes demands periodic re-evaluation of current views on the genome evolution of viruses. Here, we substantiate and further detail our previously mostly speculative model on the origin and evolution of triple gene block (TGB) encoding plant virus movement proteins TGB1, TGB2, and TGB3. Recent experimental data on functional competence of transport gene modules consisting of two proteins related to TGB1 and TGB2, as well as sequence analysis data on similarity of TGB2 and TGB3 encoded by a viral genome and virus-like RNAs identified in a plant transcriptomes, suggest that TGB evolution involved events of gene duplication and gene transfer between viruses. In addition, our analysis identified that plant RNA-seq data assembled into RNA virus-like contigs encode a significant variety of hydrophobic proteins. Functions of these orphan proteins are still obscure; however, some of them are obviously related to hydrophobic virion proteins of recently sequenced invertebrate (mostly insect) viruses, therefore supporting the current view on a common origin for many groups of plant and insect RNA-containing viruses. Moreover, these findings may suggest that the function of at least some orphan hydrophobic proteins is to provide plant viruses with the ability to infect insect hosts. In general, our observations emphasize that comparison of RNA virus sequences in a large variety of land plants and algae isolated geographically and ecologically may lead to experimental confirmation of previously purely speculative schemes of evolution of single genes, gene modules, and whole genomes. Plant viruses often induce significant rearrangements of the endomembrane system in infected cells to facilitate viral multiplication and cell-to-cell movement . The plaMembrane MPs have been first identified in two viral transport gene modules, triple gene block (TGB) coding for an RNA-binding helicase TGB1 and two small hydrophobic proteins TGB2 and TGB3  and doubThe potyvirus-encoded 6K2 protein, not considered as a true MP, is a membrane protein required for the formation of replicative vesicles, which contain viral RNA polymerase and are capable of trafficking toward the plasma membrane, association with plasmodesmata, and even translocation to neighboring cells . In a siSince viral multi-gene transport modules typically encode membrane proteins, the evolutionary origin and phylogenetic links of such proteins are of great importance for understanding the genesis of currently existing plant virus transport systems, as well as general mechanisms of new gene acquisition by plant virus genomes.Recently, we have proposed a two-step helicase gene-centered scenario of TGB evolutionary origin. According to this model, a gene of progenitor accessory helicase (future TGB1 protein), which could have functions of silencing suppression and viral cell-to-cell movement, first evolved to acquire an overlapping gene for a membrane protein TGB2 facilitating the TGB1 movement function, and then the TGB3 gene emerged in the genomic block consisting of the TGB1 and TGB2 genes . Our newHibiscus green spot virus , we have recently identified a novel transport gene module termed ‘binary movement block’ (BMB) encoding two proteins, a TGB1-like helicase BMB1 and a membrane protein BMB2 exhibiting a distant relation to TGB2 proteins  with a gene array coding for polypeptides most closely related to proteins encoded by viruses of the genus Carlavirus (family Betaflexiviridae) . In the Ap-VLRA, a gene block consisting of TGB1 and TGB2 is immediately followed by a downstream gene of viral capsid protein, and no TGB3-like gene (or any other ORF) could be identified downstream of the TGB2 gene (data not shown). Moreover, recent sequence analyses of cassava-infecting viruses of the genus Potexvirus (family Alphaflexiviridae) revealed that Cassava virus X (CsVX) and Cassava new alphaflexivirus (CsNAV), both having a gene arrangement typical for potexviruses, lack the TGB3 gene  can occur in the absence of TGB3, although the efficiency of transport is greatly decreased (Alternanthera mosaic virus (AltMV) containing a premature stop codon in TGB3 gene exhibits a limited transport between adjacent epidermal cells  resembling a plant virus genome fragment encoding TGB proteins related to the respective proteins of the genus Benyvirus . Deschampsia antarctica (Poaceae) and Colobanthus quitensis (Caryophyllaceae) represent the only two vascular plants species in the Antarctica coast flora isolated from the rest of the world for approximately 20 million years . In a similar manner, the TGB3 protein of Beet soil-borne mosaic virus  shows a detectable similarity of its central region to TGB2 proteins . These observations indicate that at least in benyviruses the TGB3 gene could arise by a duplication of TGB2 gene.We previously suggested that the TGB3 gene could emerge in the transport module consisting of TGB1 and TGB2 as a result of overprinting, when a coding region became translatable in a different reading frame, horizontal gene transfer (HGT), or duplication of the TGB2 gene and subsequent divergence of the two copies . Our addon years . Thus itBeet necrotic yellow vein virus (BSBMV)] is lower that 28%, whereas sequence identity between the genera Hordeivirus, Pomovirus, and Pecluvirus is higher than 33% (data not shown). Thus, benyvirus TGB3 proteins show a high degree of sequence diversity, and some of these proteins have sequence similarity to TGB2 proteins (see above). These observations could indicate that the benyvirus TGB3-encoding genes underwent a considerable divergence after evolutionarily recent duplication of the TGB2 gene.It should be noted that previously we have distinguished two types of TGB, namely potex-like and hordei-like TGB, which differ in both domain organization of TGB1 and the structure of TGB3 proteins, but have similar TGB2 proteins with two transmembrane domains and a highly conserved central region between them . Among hAsplenium nidus VLRA exhibits a sequence similarity to TGB2 proteins of potex-like TGB, whereas its TGB2 belongs to the hordei-like type . This observation might imply that TGB3 gene origin could be a result of HGT from a different virus genome. Thus, both gene duplication and HGT could be considered as possible mechanisms of TGB3 gene origin in the TGB evolution. On the other hand, the absence of any sequence similarity between potex-like and hordei-like TGB3 proteins could indicate that potex-like TGB3 either evolved independently, or emerged as a result of an extreme divergence with hordei-like TGB3   .Chronic bee paralysis virus (CBPV) virions (1) and 1KP . Interestingly, a dozen of plant virus-like RNA assemblies (VLRAs) encoding SP24-like proteins and having diverse ORF organizations was found. All new plant VLRA-encoded SP24-like proteins are only moderately similar to the previously described plant virus SP24 hydrophobic proteins  in animal hosts . We propFigure 2B) and possessing the features typical for a virion glycoprotein . plants . Together with the occurrence of similarly positioned transmembrane segments (data not shown), this suggests a common origin of these proteins in different plants and arthropods.Another hydrophobic protein (ORF2 protein) is encoded by CBPV RNA2. This protein showed an obvious similarity to the polypeptides encoded by insect negeviruses; however no similarity to plant virus polypeptides, even those of viruses coding for SP24 homologs, was detected . The regoprotein . Search Membrane proteins encoded by genes acquired by viral genomes in the course of co-evolution with their hosts can be involved in essential processes such as replication, intercellular movement in plants, and spread in insect vectors. Currently available data suggest that the evolution of such genes could involve events of gene duplication and HGT between genomes of plant viruses, as well as between genomes of plant and insect viruses. These evolutionary events apparently account, together with gene shuffling and divergence, for the current diversity of plant virus genomes. Future studies involving newly sequenced plant virus genomes and plant transcriptomes will undoubtedly unravel a complex picture of a non-collinear evolution of viral genome elements and reveal functions of yet uncharacterized viral genes, including those of integral membrane proteins. Ñonsidering TGB evolution, one suggestion for future experimental directions might be to estimate the impact of VRC compartmentalization and functioning in virus intercellular movement. It is quite important to examine this aspect of TGB-mediated transport in comparative studies of related viruses and even virus isolates diverged because of the geographical separation and host range expansion.AS and SM designed and planned the research, SM performed database searches, AS performed sequence comparisons and prepared figures, AS and SM wrote the manuscript and approved the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vulvodynia refers to pain in the vulva of at least 3 months’ duration in the absence of a recognized underlying cause. Provoked, localized vestibulodynia is the term used to describe superficial pain confined to the vulvar vestibule, provoked by touch. This review will focus on provoked vestibulodynia with regard to its suggested causative factors and will discuss the role of inflammation, vulvovaginal infections, mucosal nerve fiber proliferation, hormonal associations, central pain mechanisms, pelvic floor muscle dysfunction, and genetic factors. Clinical observations, epidemiological studies, and data from basic research emphasize the heterogeneity of vulvar pain syndromes. There is a critical need to perform prospective, longitudinal studies that will allow better diagnostic criteria and subgrouping of patients that would lead to improvements in our understanding of provoked vestibulodynia and its treatment. It refers to ‘pain in the vulva’ of at least 3 months’ duration, in the absence of a recognized underlying cause, and can consist of various clinical features. Some women with this diagnosis have continuous, diffuse vulvar pain , while others experience localized pain, usually provoked by touch. The current nomenclature2. However, owing to its separate clinical presentation, this communication will focus only on PVD. In some women, their vestibular pain can be caused by minimal touch (sitting or tight-fitting clothing), whereas in others it is provoked only by vaginal penetration during sexual intercourse, tampon insertion, or gynecological examination, resulting in dyspareunia or a complete inability to have intercourse.The vulvar pain syndrome currently known as provoked, localized vestibulodynia (PVD) is the term used to describe superficial pain confined to the vulvar vestibule, provoked by touch. Some researchers suspect that PVD and generalized, unprovoked vulvodynia may represent a continuum of the same conditionSome women have a primary form of PVD (PVD1), experiencing pain at first introital touch, while others describe a period of pain-free vaginal penetration before the onset of symptoms and are defined as having secondary PVD (PVD2). The pain is often described as a burning or cutting sensation and may be located throughout the vestibule or confined to the lower vestibule. Erythema may or may not be found and is no longer considered a defining criterion.4 rather than by an evidence-based approach from randomized clinical trials5.No single causative factor of PVD has yet been identified, and its etiology is considered multifactorial. The accepted theory is that PVD represents a diverse group of disorders, causing similar symptoms. The clinical diagnosis of PVD is defined by subjective signs and symptoms of entry dyspareunia and vestibular tenderness to gentle touch after the exclusion of defined disorders . Current treatment strategies follow a “trial-and-error” approach, guided mainly by expert opinion and strategies used in other pain disordersMuch effort has been made to find pathophysiological changes characteristic of PVD. A range of abnormalities in different systems  as well as in different pathways  have been identified in groups of subjects. This review will focus on our current understanding of the suggested etiologies of PVD.6. One hypothesis suggests that the enhancement of peripheral pain perception may be caused by changes in the biochemical environment, modifying the conduction of nerve fibers, further lowering the threshold in nociceptors2. Another suggested mechanism is hyperinnervation of the vestibular mucosa. Increased vestibular nerve fiber density was first described by Westrom and Willen7 and was thereafter confirmed by others13. Immunohistochemical methods defined these nerve endings as nociceptors14. This hyperinnervation corresponds to heightened mechanical allodynia (pain from light touch) and hyperalgesia (enhanced pain perception) and is termed “neuroproliferation”. Neuroproliferation is hypothesized to be either congenital or acquired.Hypersensitivity of the vulvar vestibule is one of the defining characteristics of PVD. Quantitative sensory testing of vestibular mucosa indicates peripheral sensitization to both mechanical and thermal stimuliet al.13 showed that PVD1 patients have significantly greater neural hypertrophy as compared to those with PVD2, supporting the hypothesis that PVD1 and PVD2 may have distinct histopathologic pathways rather than representing different stages of the same disease.Leclair17 and was attributed to dysfunctional regulation of the hypothalamic–pituitary–adrenal axis18. Recent studies in animal models found evidence that early life stress in mice induced increased vaginal sensitivity18 and that chemical neonatal vaginal irritation led to permanent vaginal hypersensitivity19.It is possible that PVD1 may be congenital, or that mucosal hypersensitivity may be acquired in early life, initially being recognized in adolescence. A correlation between early life adverse events and PVD in adults has been found in case-control studies21. Embryologically, the vestibule is of endodermal origin, derived from the urogenital sinus, and neuroproliferation may represent a congenital anomaly. In such cases, the neural hypersensitivity is primary and may also be present in other tissues derived from the urogenital sinus. This may explain the coexistence of PVD and interstitial cystitis in some women22, as well as the significantly higher level of umbilical sensitivity in women with PVD123. Acquired PVD has also been attributed to neural proliferation in response to an inflammatory process (see below) or endocrine factors25.It was also suggested that PVD may be congenital in some women26 and attribute the enhanced pain perception to neurogenic inflammation26. In addition, and in contrast to what one might expect, it is well accepted that reduced, and not increased, intraepidermal nerve fiber density is associated with an elevated risk of developing neuropathic pain27. However, an additional observation supporting the contribution of peripheral pain mechanisms to PVD is that 80% of patients undergoing surgical excision (“vestibulectomy”), in which the hyperinnervated region is removed, experience symptomatic relief. However, it is important to recognize the lack of randomized trials and variation in the definition of successful outcome.The significance of nociceptor hypertrophy is controversial; some researchers consider neuroproliferation as a non-specific reaction to previous mucosal trauma or inflammation28. In vulvar biopsies obtained from PVD patients, the subepithelial part of the lamina propria is infiltrated by inflammatory cells, mainly T lymphocytes. This is often described as non-specific chronic inflammation30. However, in later studies, a similar inflammatory infiltrate was also found in the vestibule of healthy women31. Consequently, the initial inflammatory theory has been abandoned and the condition is now regarded as a pain syndrome32.PVD was initially regarded as a chronic local inflammatory condition and was therefore called “vulvar vestibulitis syndrome”33, and persistent elevation of pro-inflammatory substances35. These changes in the biochemical milieu could alter the ion channel activity of peripheral pre-terminal axons, which leads to a lowered mechanical, thermal, or chemical threshold in the primary nociceptors2. Consequently, even light touch can result in exaggerated release of pro-inflammatory mediators by sensitized nerve fibers36. This, in turn, activates neuroendocrine cells and mast cells to release additional pro-inflammatory compounds. This self-perpetuating process of neurogenic inflammation is thought to play a key role in the maintenance of local inflammation in PVD37.Nevertheless, multiple studies suggest that inflammation may play a role in the development of PVD. The suggested mechanism hypothesizes that persistent inflammation in the vestibular mucosa promotes hyperplasia of nociceptive c-fibers, secondary to the production of nerve growth factor, altered receptor expression38, while others reported inflammation without mast cell predominance30 or the absence of inflammatory cells31.Studies that have evaluated inflammatory characteristics in PVD have shown contradictory results. In some histological studies, mast-cell-predominant inflammation was demonstrated39 and increased levels of the neurokine CGRP14, while others found lower levels of TNF-α and similar levels of IL-1b among patients and controls40. Nitric oxide synthase and cyclooxygenase 2 were not upregulated in vestibular mucosa biopsies from PVD patients34, which is in opposition to ongoing cell-mediated inflammation26. Laser Doppler perfusion imaging showed increased superficial blood flow in the mucosa, further supporting the neurogenic inflammation theory35.Assays for pro-inflammatory molecules in vulvovaginal samples have also shown inconsistent results, with some studies reporting an elevation in the pro-inflammatory cytokines interleukin (IL)-1b and tumor necrosis factor (TNF)-α41. A systematic review by Chalmerset al. published recently42 highlights the lack of a consistent inflammatory profile in PVD patients.Levels of systemic interferon (IFN)-α and IFN-γ were similar in PVD patients and controls43, as the history of RVVC has often been based on self-report and the presence of the yeast was not confirmed by culture. Nevertheless, various studies described findings associated with a possible deficient immune response that resulted in RVVC and subsequent development of PVD. It has been postulated that an inability to clear vulvovaginal infections and the resulting chronic inflammation may lead to PVD development.Vulvovaginal infections are frequently cited as an inciting inflammatory event triggering the development of PVD. Often, PVD patients report a history of recurrent vulvovaginal candidiasis (RVVC), and they frequently relate the onset of their symptoms to a symptomatic vaginal candidiasis. Whether this represents an accurate association or is mainly a misinterpretation of patients’ symptoms is not clear44. Other observations imply a possible genetic variability causing a predisposition to RVVC47. Additionally, an increased cutaneous hypersensitivity toCandida albicans organisms was reported in women with PVD48. In an RVVC mouse model49, chronic vulvar pain and increased vulvar innervations were also observed.Circulating natural killer cells, a predominant factor in vaginal defense against Candida infections, are significantly lower in PVD patientset al. reported that vulvar fibroblasts produce high levels of IL-6, IL-8, and prostaglandin E2 (PGE2) following stimulation by irritants in both women with PVD and controls51. Vestibular fibroblasts released elevated levels of IL-6 and PGE2 compared to fibroblasts isolated from non-painful vulvar sites. Furthermore, pro-inflammatory mediator production was elevated in PVD fibroblasts compared with controls. Similar findings were reported when fibroblasts were challenged with live yeast species51, and the response was highly predictive of clinically measured pain thresholds. The authors concluded that vulvar tissue of women with and without PVD can be differentiated by the degree of naturally occurring inflammation52. They suggested that the vestibule of PVD patients is inherently more sensitive to yeast and that even a subclinical infection may trigger a maladaptive immune response in these fibroblasts.Foster53. They found that vestibular fibroblasts from PVD patients express elevated levels of Dectin-1, a surface receptor that bindsC. albicans. They also showed that blocking the function or expression of Dectin-1in vitro resulted in a significant decrease in IL-6 and PGE2 production.In recently published work, the same group evaluated the signaling pathways involved in the recognition of yeast components that are present during chronic infectionIn summary, our understanding of inflammation as a possible contributing factor to PVD development is still evolving. Recent studies have revisited this potential origin, suggesting that inflammation is likely to play a role in PVD.57. Results showed a 6.6 relative risk of PVD for ever-users of HCs compared to non-users, rising with increased duration of use (at least up to 2–4 years of use) and first use at a young age (<16 years)56. The relative risk was higher when the product used was progestogenic and androgenic but low in estrogenic potency56. The use of low-estrogen HCs (≤20 mcg ethinylestradiol) was significantly more common in women with PVD than in the general population of HC users57. Burrows and Goldstein58 described a case series of 50 women who developed PVD while on HCs and who were successfully treated with topical estradiol and testosterone. However, these findings were not confirmed in two epidemiologic studies60.A possible correlation between hormonal contraception preparations (HCs) and PVD has been investigated primarily in epidemiological studies. Several reports have demonstrated that HCs increase the risk of developing PVD261. This effect may contribute to the decreased mechanical pain thresholds reported in healthy women using HCs62. These morphological alterations may influence mechanical properties by thinning the epithelium and causing nerve endings to become more superficially located, thus altering the transduction of mechanical pressure to the receptors without affecting nerve fibers.The effect of HCs on vestibular mucosa is probably multifactorial. HCs modify the morphological pattern of the vestibular mucosa, with the appearance of shallow and sparse dermal papillae65. Results from studies investigating the expression of estrogen receptor α (ERα) in PVD patients are contradictory. Evaet al.65 reported a decrease in vestibular ERα in women with PVD, while Johannessonet al.63 reported an increased amount of vestibular ERα in patients who were past HC users compared to controls. Healthy HC users displayed a higher amount of ERβ in the vestibular stroma compared with healthy non-users64.HCs can also affect the vestibular epithelium through interaction with hormone receptors or alteration of receptor expression66, causing introital pain. It was also found that androgen receptors (ARs) are significantly lowered in vestibular tissue and cells of the minor vestibular glands in HC users67. Additionally, some HCs contain synthetic progestins that act as testosterone antagonists at the AR68. Goldsteinet al.69 identified a genetic polymorphism in the AR in PVD patients and concluded that an inefficient AR combined with lowered free testosterone predisposes to PVD. This has not been confirmed.Another possible mechanism involves alteration of serum hormone levels. In women on HCs, there is suppression of ovarian testosterone production, reduced ovarian estradiol, and increased sex hormone binding globulin (SHBG) synthesis. This combination leads to low calculated free testosterone and low estradiol. Decreased estradiol may further contribute to vestibular atrophy found in patients with PVD270. Endogenous pain modulation was found to be less effective in HC users71, and a persistent genital hyperinnervation via a direct effect of synthetic progesterone on unmyelinated sensory nociceptor neurons has been described in animal models24.Other potential mechanisms refer to estrogen and progesterone as endogenous pain modulatorsIn summary, the association between HC use and the development of PVD is possible. The actual prevalence and susceptibility factors remain incompletely elucidated and it is thus not possible to make clinical recommendations. It is also not clear whether termination of HC usage alone can reverse PVD and which additional treatments may also be necessary.72.The muscles of the pelvic floor (PFM) consist of three layers: superficial, intermediate, and the deeper muscles known collectively as the “levator ani”. The PFM participate in multiple activities, including mechanical support of the pelvic organs, trunk stability and mobility, defecation, urination, closure of the urinary and anal orifices, and enhancement of sexual pleasure72. Muscle contraction in response to pain has been referred to as guarding, while persistent states of muscle overactivity have been described as spasticity or hypertonus and are more frequently associated with amplification in neurological tone rather than as a response to pain72.PFM dysfunction (PFMD) generally refers to disorders of laxity (hypotonus) or to muscles’ overactivity (hypertonicity). PFMD can result from various causes, including musculoskeletal factors, inflammation, trauma, pregnancy, vaginal delivery, abdominal or pelvic surgery, neuropathic pain, past or current psychological, physical, and/or sexual abuse, and anxiety in addition to a variety of other factors. Pelvic pain disorders and dyspareunia are usually associated with pelvic floor overactivity75, increased contractile responses to painful stimuli75, decreased flexibility, lower relaxation capacity75, lower pain thresholds76, and lesser strength73 compared with controls. Reissinget al. reported that 90% of the women in their study diagnosed with PVD demonstrated PFMD73.It has long been recognized that PVD is often associated with some degree of PFMD, such as elevated resting tone72. Reissinget al.73 demonstrated that PVD patients displayed considerably higher PFM tone in the superficial layer, and these findings were less remarkable at the deeper PFM layers. They concluded that the absence of generalized hypertonicity may indicate that PFM hypertonus could result from, rather than cause, PVD. They hypothesized that muscle tension starts as a protective response to vestibular pain, and this response later results in increased resting muscle tone75. Increased PFM contraction causes an enhancement of pressure on the vestibule during penetration, further mounting both pain and the protective guarding reaction. Hypertonicity may, consequently, act to maintain as well as to exacerbate PVD. Recently, transperineal three-dimensional real-time ultrasound imaging has been used to investigate the morphology and function of the PFM in PVD patients78. Because this method does not involve vaginal penetration  and is therefore pain-free, it has the advantage of limiting bias caused by the participant’s pain and anxiety during the procedure79. In accordance with previous studies, ultrasound imaging showed higher PFM tone and lesser contractile capacities among PVD patients79. These findings suggest that PVD patients display PFM impairments which are not limited to a defense reaction but are rather chronic78.The mechanisms associating PFM hypertonus and PVD are not fully understood. Vaginal closure is assisted by the bulbospongiosus and puborectalis muscles, as well as activation of the levator ani80. This may contribute to sensitization of muscle pain receptors, which, in turn, through a development of a central sensitization process, diminish sensory pain thresholds. This hypothesis suggests a “vicious cycle” of inflammation and further muscle contraction82.Alternatively, it has been suggested that PFMD may result from a chronic inflammatory process in the vestibular mucosa. According to this theory, mucosal inflammation or trauma may induce hypersensitivity and contraction of the underlying PFM36. The mechanism suggested to be involved in such a pathway is an abnormal neuropathic output state due to changes in the peripheral and central nervous systems secondary to noxious stimuli over a prolonged period of time83. It was also hypothesized that trigger points within the PFM may refer pain to the region of the vestibule79 or that hypertonicity of the muscles that insert at the posterior vestibule  can lead to allodynia in the posterior vestibule due to altered neurodynamics and neural and tissue hypoxia20.Another hypothesis proposes the opposite pathway, i.e. an underlying PFMD may act as an initiator of sensory changes in susceptible mucosa84.In summary, the PFMD commonly documented in PVD patients, although clinically similar, could, in fact, be driven by distinctly different pathophysiological processes. These aforementioned theories have yet to be fully substantiated at the foundational level. Understanding the pathophysiological pathways will enable clinicians to target therapies to specific causes of vulvar pain, mucosal or muscular, as well as to recommend direct physical therapy regimens according to the type of pain and its contributors85. Patients often suffer from other bodily pain and have lower pain thresholds in regions remote from the vestibule6. In addition, an increased number of painful tender points were reported86 and enhancement of a post-capsaicin pain response was found87, suggesting central sensitization. Moreover, functional magnetic resonance imaging, performed during painful vestibular pressure, revealed similar activation in cerebral pain centers as in other chronic pain conditions88, and grey matter density was increased in pain modulatory and stress-related areas of the brain compared to controls89.Among etiological factors participating in PVD development, the alteration of central pain perception has also been suggested based on various observations. Comorbidity of PVD with other pain syndromes is often reported, most notably with irritable bowel syndrome, fibromyalgia, interstitial cystitis, and orofacial pain90 or alternatively can be viewed as a reflection of an intrinsic defect in mechanisms of pain regulation92. As PVD1 patients exhibit greater sensitivity to thermal pain in comparison to women with PVD2, it was suggested that subgroups of PVD patients are different with regard to extragenital pain thresholds, implying differences in underlying pathophysiological mechanisms93. Consideration of the contribution of central pain regulatory mechanisms may help to explain the variations in the clinical presentation and treatment outcomes in subgroups of PVD patients93.Taken together, these findings indicate that an abnormal feature of pain amplification may exist in PVD patients. This pain sensitivity may be attributed to either the chronicity of pain 94. These psychosocial and emotional factors influence the expression, clinical presentation, and response to therapy. It is unknown whether these disorders have a causal role in PVD, whether they represent a response to a chronic disabling medical disorder, or if they share a dysfunction in central-regulatory systems with chronic pain and, as such, coexist95.In addition to comorbidity with pain syndromes, the incidence of depression, anxiety, stress, somatization, phobic symptoms, and catastrophizing is higher in PVD patients than in control women- Polymorphisms in genes regulating the inflammatory response: variability in the DNA sequence of genes regulating immune recognition or magnitude and direction of the response to infections may explain an altered inflammatory response in patients with PVD (see above). Such genetic variations, termed “polymorphisms”, were described with regard to the IL-1 receptor antagonist97, IL-1b98, mannose-binding lectin (MBL)46, NALP-347, and the melanocortin-1 receptor genes97. These polymorphic genes were suggested to influence susceptibility to PVD, severity of symptoms, or both.- Polymorphisms in genes associated with an increased sensitivity to pain: the serotonin receptor gene5HT-2A99- Polymorphisms in genes involved in the effect of hormonal changes caused by HCs69Several studies point to a possible genetic involvement in PVD based on several mechanisms:et al.100 evaluated whether PVD is more common in female relatives of women diagnosed with PVD using population-based genealogy-coded data. They found that the relative risk of vestibulectomy was elevated in first-, second-, and third-degree relatives and concluded that this familial clustering supports a genetic predisposition for PVD and warrants further studies to identify the specific genes involved.In addition to these case-control studies, a recent study by Morgan5. Inconclusive data regarding etiology, epidemiology, mucosal characteristics, and treatment results can be explained by grouping patients who in reality have different underlying conditions under a single diagnosis and then evaluating the efficacy of a single intervention that might be relevant to only one subset of patients. This can lead to an apparent lack of significance or effect due to dilution caused by the heterogeneous patient population.Clinical observations and epidemiological studies emphasize the heterogeneity of vulvar pain syndromes. In addition, data from basic research suggest different mechanisms relevant to the division of women with PVD into subgroups. However, in clinical practice, the relative contributions of different triggering or persistent factors remain poorly understood, and current diagnostic criteria are based on highly subjective measures, while treatment proceeds on a trial-and-error approach. The result is that many forms of therapeutic interventions have been used, yet the evidence remains largely inconclusive, the response rate varies between 40 and 85%, and many women with PVD do not respond to any of the treatments101: hormonally mediated PVD, hypertonic pelvic floor dysfunction, congenital neuroproliferative PVD, and acquired neuroproliferative PVD (secondary to inflammation), based on a patient’s history and physical examination. According to our personal experience, this classification provides better outcomes compared to the trial-and-error approach we used before (higher response rates as well as shortening the period for significant improvement). However, objective data are missing to support this algorithm.Constructing an algorithm that may enable the allocation of patients into PVD subgroups will advance both basic science and clinical research and improve future medical management. Although a large research effort has been made in this area, only one such clinical algorithm has been suggested, distinguishing between four subtypes of PVDGiven the high incidence of PVD in the population and its huge effect on patients’ lives, there is a critical need to perform prospective, longitudinal studies that will allow better diagnostic criteria and subgrouping of patients that would lead to improvements in our understanding of PVD and its treatment.


Editorial on the Research TopicExploring Cancer Metabolic Reprogramming through Molecular ImagingOncogene-driven reprogramming of energy metabolism has been added in 2011 to the list of general cancer hallmarks originally introduced by Hanahan and Weinberg to rationalize the complexities of neoplastic diseases  and MRI, forming the mainstay of patient assessment, from diagnosis to treatment monitoring. However, the need for further improvements in specificity and sensitivity, coupled with imaging techniques that are reaching their limit of clinically attainable spatial resolution, has resulted in the emergence and growing use of imaging techniques with additional functional read-outs, such as 2-deoxy-2[18F]fluoro-d-glucose (18FDG)-PET and multiparametric MRI. These techniques add a new dimension to our understanding of the biological behavior of tumors, allowing a more personalized approach to patient management.As noted in the opinion article by Challapalli and Aboagye. In addition to providing us with opportunities for examining the complex regulation of reprogrammed energy metabolism in living subjects, the PET methods open up opportunities for monitoring pharmacological activity of new therapies that directly or indirectly inhibit tumor cell metabolism.Compared to normal differentiated cells, cancer cells require a metabolic reprogramming to support their high proliferation rates and survival. A rewiring of energy metabolism through the Warburg effect is essential to generate the required biomass, including membrane biosynthesis, and to overcome bioenergetic and redox stress. Both established and evolving radioprobes developed in association with PET to detect tumor cell metabolism, and effects of treatment have been reviewed by 1H-MRS measurements have also been used to investigate tumor metabolism for diagnostic purposes. However, clinical applications of MRS have been hampered by low sensitivity and consequently low spatial and temporal resolution. Nuclear spin hyperpolarization of 13C-labeled substrates by dynamic nuclear polarization (DNP) radically increases the sensitivity of these substrates to detection by 13C MRS  on 13C MRS and LC-MS flux analysis of tumor intermediary metabolism presented the first validated metabolic network model for analysis of flux through key pathways of tumor intermediary metabolism including glycolysis, the oxidative and non-oxidative arms of the pentose pyrophosphate shunt, and the TCA cycle, as well as its anaplerotic pathways, pyruvate malate shuttling, glutaminolysis, and fatty acid biosynthesis and oxidation. Two models, respectively called bonded cumomer analysis for application to 13C MRS data and fragmented cumomer analysis for mass spectrometric data, are refined and efficient forms of isotopomer analysis that can be readily expanded to incorporate glycogen, phospholipid and other pathways, thereby encompassing all the key pathways of tumor intermediary metabolism. Results validated with melanoma and lymphoma cell models suggest the potential translation of these methods to in situ investigations on human cancer reprogramming using MRS with stable 13C isotopically labeled substrates on instruments operating at high magnetic fields (≥7 T), possibly in combination with FDG-PET and hyperpolarized 13C MRS methods.An original research article by Glickson and colleagues  provides a timely overview of the metabolic changes observed in mutant IDH cells and the various molecular imaging methods that have been used to characterize these changes. The review describes how metabolic imaging has helped shed light on the basic biology of mutant IDH cells and how this information can be leveraged to identify new therapeutic targets and develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.Mutations in metabolic enzymes involved in cell bioenergetics but not directly responsible for aerobic glycolysis may also play an important role in cancer metabolic reprogramming. Notably, mutations in the metabolic enzyme isocitrate dehydrogenase (IDH), whose wild-type form catalyzes the interconversion of isocitrate to α-ketoglutarate, have recently been identified as drivers in the development of several tumor types. In particular, cytosolic IDH1 is mutated in 70–90% of low-grade gliomas and secondary glioblastomas, and mitochondrial IDH2 is mutated in about 20% of acute myeloid leukemia cases. An article by Ronen and colleagues , recent evidence suggests that cancer cells undergo metabolic reprogramming beyond aerobic glycolysis and bioenergetic rewiring, in the course of tumor development and progression. Starting from pioneering studies at the end of the last century  as well as tumor growth and vascular features. The review summarizes the current efforts to clarify how non-invasive multimodal imaging can help to characterize tumor–stroma interaction and understand its role in the development, progression, and treatment response of tumors.Besides aerobic glycolysis and altered choline metabolism, tumors are often characterized by peculiar microenvironment features such as hypoxia, vascular abnormalities, and low extracellular pH (pHe). The impact of these tumor characteristics has been investigated extensively in the context of tumor development, progression, and treatment response, resulting in a number of non-invasive imaging biomarkers. As highlighted by Indraccolo and Mueller-Klieser. Tumor heterogeneity at the genetic level has been illustrated by a multitude of studies on the genomics of cancer, but whether tumors can be heterogeneous at the metabolic level is an issue that has been less systematically investigated so far. A burning related question is whether the metabolic features of tumors can change following either natural tumor progression  or therapeutic interventions. In this regard, recent findings by independent teams indicate that antiangiogenic drugs cause metabolic perturbations in tumors, as well as metabolic adaptations associated with increased malignancy. ImBI is an imaging technique that enables detection of key metabolites associated with glycolysis, including lactate, glucose, pyruvate, and ATP in tumor sections. Signals captured by imBI can be used to visualize the topographic distribution of these metabolites and quantify their absolute amount. ImBI can be very useful for metabolic classification of tumors and to track metabolic changes in the glycolytic pathway associated with certain therapies. Imaging of the metabolic changes induced by antiangiogenic drugs in tumors by imBI or other emerging technologies is a valuable tool to uncover molecular sensors engaged by metabolic stress and offers an opportunity to understand how metabolism-based approaches could improve cancer therapy.The potential of induced metabolic bioluminescence imaging (imBI) to uncover metabolic effects of antiangiogenic therapy in tumors has been overviewed by Podo et al.) provides an overview of recent findings on functional and metabolic features of PC-PLC in breast and ovarian cancer cells in terms of (a) activation, protein overexpression, and subcellular redistribution of this enzyme in cancer cells compared with non-tumoral counterparts; (b) relative contributions of ChKα and PC-PLC to the intracellular MRS-detected phosphocholine pool; (c) interaction of PC-PLC with ErbB receptors’ family members such as human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 1 ; and (d) effects of PC-PLC inhibition on HER2 overexpression, cell proliferation, and cell differentiation , was considered as a radiological response to cytotoxic chemotherapies. In addition to RECIST-based dimensional measurements, a metabolic response to cytotoxic drugs can be assessed by PET using 18F-fluoro-thymidine (18FLT) as a radioactive tracer for drug-disrupted DNA synthesis. The decreased 18FLT-PET uptake is often seen concurrently with increased apparent diffusion coefficients by diffusion-weighted MRI (DWI) due to chemotherapy-induced changes in tumor cellularity. Recently, the discovery of molecular origins of tumorigenesis led to the introduction of novel signal transduction inhibitors (STIs). STIs are targeted cytostatic agents; their effect is based on a specific biological inhibition with no immediate cell death. As such, tumor size is no longer a sensitive end point for a treatment response to STIs; novel physiological imaging end points are needed. For receptor tyrosine kinase inhibitors, as well as modulators of the downstream signaling pathways, an almost immediate inhibition in glycolytic activity (the Warburg effect) and phospholipid turnover (the Kennedy pathway) has been seen by metabolic imaging in the first 24 h of treatment. The quantitative imaging end points by MRS and metabolic PET  provide an early treatment response to targeted STIs, before a reduction in tumor burden can be seen.The unique capabilities of metabolic imaging to assess treatment response to cytotoxic and cytostatic agents were reviewed by in vivo tissues especially in breast , ovarian , and prostate cancers .A series of 13 original articles or minireviews focused on MRI and metabolic imaging studies on cell-based models, dissected tissue specimens, and in vivo using novel ER-targeted contrast agents (Pais and Degani); identification of key players in choline metabolic reprogramming in triple negative breast cancer ; influence of the tumor microenvironment on choline and lipid metabolism ; impact of freezing delay time on tissue samples for metabolomic studies ; monitoring of metabolic changes induced by gamma irradiation on breast tumor spheroids using 1H NMR spectroscopy and microimaging ; identification of 31P MRS phosphodiester signals of human fibroglandular breast tissue at ultrahigh field ; and evaluation of the potential of DWI in the characterization of malignant, benign, and healthy breast tissues and molecular subtypes of breast cancer . An article entitled “Clinical breast MR using MRS or DWI: Who is the winner?” by Sardanelli et al. provides a critical summary of secondary evidence on two different in vivo non-contrast molecular imaging approaches, 1H MRS and DWI, with special focus on the translational perspective toward clinical feasibility and applicability.The reported studies on breast cancer highlight relevant issues concerning development of quantitative molecular imaging methods that specifically detect estrogen receptor (ER) Bagnoli et al.). The article shows that ChKα inhibition, besides reducing ovarian cancer aggressiveness, increases disease sensitivity to drug treatment sparing normal cells and therefore opening a new therapeutic window. An abnormal tCho profile along with altered levels of other metabolites has also been detected in human EOC xenografts, as reviewed by Canese and colleagues . This molecular imaging study provides a more extensive picture of tumor metabolism in EOC models in vivo, potentially opening the way to a multiple metabolic targeting. Furthermore, DWI is suggested as a potential tool for better differentiating malignant from benign tissues and possibly distinguishing cytotoxic from cytostatic treatment effects. New therapeutic strategies are urgently needed to improve survival of ovarian cancer patients. The effect of pantethine (precursor of vitamin B5 and active moiety of coenzyme A) on ovarian tumor progression and choline metabolism has been investigated by  using MRI and high-resolution 1H MRS in a orthotopic ovarian cancer model. Pantethine treatment resulted in slower tumor progression, decreased levels of phosphocholine and phosphatidylcholine, and reduced metastases and ascites occurrence.Regarding epithelial ovarian carcinoma (EOC), a highly heterogeneous and lethal malignancy characterized by late diagnosis, frequent relapse, and development of chemoresistance, Bagnoli and colleagues reviewed the role of ChKα in sustaining the cancer “cholinic phenotype” . Last, but not least, Testa and colleagues reviewed the recent literature regarding molecular imaging methods developed and used to improve diagnosis and staging of prostate cancer . The encouraging progress of in vivo metabolic imaging approaches nowadays points to the need of harmonized and shared protocols to increase the applicability of these technologies to a clinical setting. Furthermore, the use of well-characterized preclinical models that closely mirror the pathogenesis of human prostate cancer  allows for further progress in the comparative evaluation of DWI versus other molecular imaging approaches in assessing different stages of this disease (MRI can portray spatial variations in tumor heterogeneity, architecture, and tumor microenvironment, key biological features of prostate cancer. An original research article by Selnaes et al. focused on the relationships between MRI parameters measured on prostate cancer patients  disease .All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, SB, declared a shared affiliation and a past coauthorship with one of the authors, ZB, to the handling editor, who ensured that the process nevertheless met the standards of a fair and objective review.


Colorectal cancer (CRC) is a heterogeneous disease that is caused by the interaction of genetic and environmental factors. Although it is one of the most common cancers worldwide, CRC would be one of the most curable cancers if it is detected in the early stages. Molecular changes that occur in colorectal cancer may be categorized into three main groups: 1) Chromosomal Instability (CIN), 2) Microsatellite Instability (MSI), and 3) CpG Island Methylator phenotype (CIMP). Microsatellites, also known as Short Tandem Repeats (STRs) are small (1-6 base pairs) repeating stretches of DNA scattered throughout the entire genome and account for approximately 3 % of the human genome. Due to their repeated structure, microsatellites are prone to high mutation rate. Microsatellite instability (MSI) is a unique molecular alteration and hyper-mutable phenotype, which is the result of a defective DNA mismatch repair (MMR) system, and can be defined as the presence of alternate sized repetitive DNA sequences which are not present in the corresponding germ line DNA. The presence of MSI is found in sporadic colon, gastric, sporadic endometrial and the majority of other cancers. Approximately, 15-20 % of colorectal cancers display MSI. Determination of MSI status in CRC has prognostic and therapeutic implications. As well, detecting MSI is used diagnostically for tumor detection and classification. For these reasons, microsatellite instability analysis is becoming more and more important in colorectal cancer patients. The objective of this review is to provide the comprehensive summary of the update knowledge of colorectal cancer classification and diagnostic features of microsatellite instability. Colorectal cancer (CRC) is the third most prevalent cancer in humans and the third most common cause of cancer related deaths in both males and females that contributes to a significant public health problem worldwide . MSI-H This type of CRC is often considered sporadic and not any associated gene has been identified yet. People with a history of colorectal cancer in a first-degree relative are at increased risk of two to three times higher than the normal population .The hereditary type of CRC is divided into five subtypes:Familial adenomatous polyposis is the most common hereditary polyposis syndrome, which is an autosomal dominant disease caused by a mutation in the APC gene on chromosome 5q21. The APC gene is a tumor suppressor gene that produces APC protein, a multifunction protein which controls how quickly cells grow and prevent the development of tumors. The normal function of APC protein is the regulation of β-catenin by its degradation. β-catenin plays an important role in cell communication, Wnt signalling pathway, and growth by acting as a transcription factor for proliferation genes. Mutations in the APC gene lead to loss of APC function and result in an accumulation of β-catenin. Many different mutations  of the APC gene are described as a cause of FAP . In FAPMUTYH-associated polyposis is one of the hereditary polyposis syndromes which is an autosomal recessive disease and is caused by a biallelic germline mutation in MUTYH gene on chromosome 1p34.1. MUTYH gene encodes an enzyme called MYH glycosylase that is involved in a DNA repair system called Base Excision Repair (BER). The number of polyps in MAP disease is less than FAP and it is phenotypically similar to attenuated FAP . The avPeutz-Jeghers syndrome is a rare autosomal dominant disorder, which is one of the hereditary polyposis syndromes and is characterized by multiple benign hamartomatous polyps in the gastrointestinal tract, most often found in the small intestine. The number of polyps in PJS is less than MAP syndrome and those polyps are present from childhood . The maSerrated polyposis syndrome is a relatively rare syndrome characterized by multiple serrated polyps of the colon, which was previously known as the hyperplastic polyposis syndrome . This sLynch syndrome (LS), also was known as Hereditary non-polyposis colorectal cancer, is the most common hereditary colon cancer syndrome which is an autosomal dominant disease and is caused by germline mutations in one of several DNA mismatch repair (MMR) genes, including MSH2 on chromosome 2p16, MLH1 on chromosome 3p21, MSH6 on chromosome 2p16, and PSM2 on chromosome 7p22 . MSH2 aTo resolve inconsistencies between CRC classifications based on gene expressions reported and simplify clinical translation four Consensus Molecular Subtypes (CMSs) with distinct features have been reported. The CMS1 are , hypermutated, microsatellite unstable and are immunogenic. The CMS2 are , epithelial, marked WNT and MYC signalling activation and have the highest overall survival. The CMS3 are , epithelial and evident metabolic cancer phenotype; and the CMS4 , prominent transforming growth factor-β activation, stromal invasion and angiogenesis that have a worst survival. The Consensus Molecular Subtypes of CRC with clear biological interpretability may have a better prognosis, therapeutic response, and potential new treatment strategies . DNA mismatch repair system (MMR) corrects erroneous insertion, deletion, and base-base mismatches generated during DNA replication and recombination that have escaped the proofreading process . This rMicrosatellites, also known as Short Tandem Repeats (STRs) are small (1-6 base pairs) repeating stretches of DNA scattered throughout the entire genome (both in coding and non-coding regions) and account for approximately 3 % of the human genome. Due to their repeated structure, microsatellites are prone to high mutation rate . MicrosThe presence of MSI is found in the sporadic colon, gastric, sporadic endometrial and the majority of other cancers . DetermMSI is detected indirectly by analysis of MMR protein expression by Immunohistochemical (IHC) staining, or directly by PCR-based amplification of specific microsatellite repeats, which is the most common method to detect MSI . Immunohistochemical analysis can determine loss of expression of one or more of MMR proteins. Actually, IHC is correlates with MSI but it is not a perfect test for MSI determination. It is a test of expression of mismatch repair proteins in cells. In this method, antibodies against MMR proteins such as MLH1, MSH2, PMS2 and MSH6 provide information of the MMR system functionality. IHC analysis with PMS2 and MSH6 antibodies is able to detect most abnormalities in the corresponding encoding genes as well as mutations in MLH1 and MSH2; however, IHC assay with MLH1/MSH2 antibodies can detect a fraction of MLH1 or MSH2 abnormalities but not all of them. Therefore, IHC analysis with MSH6 and PMS2 antibodies has more diagnostic potential than analysis with MLH1 and MSH2 antibodies . The maFor MSI analysis by the fluorescent multiplex PCR-based method, we need DNA from tumor tissues and normal tissues, a series of primers one of which is fluorescently end labeled (the sense strand or antisense strand of each primer), a sequencer, and appropriate software. The principle of this method is to measure the presence of different lengths of specific microsatellite markers in tumor cells comparing to normal cells . In the first attempt to the diagnosis of MSI in CRC, a consensus conference recommended a panel of microsatellite markers included three dinucleotide repeats  and 2 mononucleotide repeats (BAT25 and BAT26). Three distinct MSI phenotypes have been described. If two or more microsatellite markers are mutated, the tumor is considered MSI-high (MSI-H); if only one is mutated, the tumor is defined as MSI-low (MSI-L); and if none of the examined loci demonstrate instability, the tumor will be considered Microsatellite Stable (MSS). This panel was known as the Bethesda panel .A few years later, it was found that mononucleotide markers have a better specificity and sensitivity than dinucleotide repeats (dinucleotide markers have a polymorphic nature)  and henThe uses of MSI status in the prediction of response to adjuvant chemotherapy is controversial although it has been confirmed that colorectal tumors displaying MSI have a better prognosis compared with MSS tumors. Antimetabolites (5-flourouracil), Alkylating agents and Topoisomerase Inhibitors are the three categories of chemotherapeutic agents that are used in CRC treatment . The chSince CRC is one of the most prevalent cancers in humans and causes a remarkable public health problem worldwide, identifying the ways of diagnosis and treatment of CRC is of most importance. MSI is a significant genetic marker in CRC that can be useful in diagnosis, prognosis, and prediction of chemotherapeutic treatment efficacy. Nowadays, molecular techniques have been developed for detection of MSI and drug development strategies are focused on specific tumor molecular characteristics.