The study population consisted of long-acting bronchodilator naive COPD patients, initiating inhalation therapy with mono-LABAs . Analyses were performed using pharmacy dispensing data from 1994 to 2012, obtained from the IADB.nl database. Study outcomes were 1-year persistence and switching patterns. Results were adjusted for initial prescriber, initial medication, dosing regimen and relevant comorbidities.P=0.99). Over 80% re-started or switched medication.In all, 575 patients initiating LABAs were included in the final study cohort. Among them, 475 (83%) initiated a multiple-dose inhaler and 100 (17%) a single-dose inhaler. Further, 269 (47%) initiated formoterol, 9 (2%) indacaterol and 297 (52%) salmeterol. There was no significant difference in persistence between users of multiple-dose or single-dose inhalers  on COPD patients’ persistence with LABAs. Over 80% of patients who initially seemed to discontinue LABAs, re-started their initial medication or switched inhalers or medication within 1 year. Furthermore, these trial populations do not fully represent the characteristics of the average COPD population.5 Both factors contribute to a real-life adherence to COPD medication that is far beyond adherence reported in clinical trials.7 Recently, non-adherent patients were highlighted as a target for interventions because of the significant clinical and economic impact of non-adherence on patients with COPD.8 Factors associated with non-adherence with COPD medication include age, comorbidity, disease severity and class of medication.10 However, only a few factors may be modified by the prescribing clinician. Compared to the abundant number of adherence studies in asthma patients, studies on patients with COPD are limited.11–13 In particular, studies assessing the impact of the type of inhaler device on medication adherence in patients with COPD are lacking.14 With a growing availability of different types of medication and inhaler devices, additional evidence is required to assist clinicians in prescribing the optimal medication in relation to their patients’ adherence. Adherence does encompass compliance (the extent of acting according to the prescribers’ advice) and persistence (duration of time from initiation till discontinuation of therapy).15 The objective of this study is to assess the impact of the type of inhalation device on COPD patients’ persistence with, and switching between, long-acting respiratory medication. The focus on persistence was chosen as this could be objectively measured using a dispensing database, whereas compliance would require face-to-face assessment of patients. In particular, we focused on persistence with long-acting β2-agonists (LABAs), where we compared single-dose versus multiple-dose inhalers.Chronic obstructive pulmonary disease (COPD) places a high burden on both public health and health-care budgets of patients and the government.http://www.iadb.nl). The IADB.nl database covers pharmacy-dispensing data from over 500,000 patients from 55 community pharmacies located in the Northern Netherlands between 1994 and 2012. Dispensing data contain information on drug name, product specifications, anatomical therapeutic chemical code, prescriber, amount of delivered drug units, daily dose and patient characteristics such as date of birth, gender and socioeconomic status, regardless of health-care insurance status of the patients. Thus, the database is representative for the real-world situation in the Netherlands. Medication dispensed in hospitals is not included. The IADB.nl database has been validated for drug utilisation studies16 and has been used in previous persistence studies.18 In the Netherlands, patients usually redeem their prescriptions at the same pharmacy allowing a complete longitudinal follow-up.Data were obtained from the InterActionDatabase , formoterol (R03AC13), or indacaterol (R03AC18)). The group of LABAs was chosen as for this group both dry powder inhalers (DPIs) and pressurised metered dose inhalers are available. Moreover, within the DPI group, both single-dose as well as multiple-dose inhalers are available, making this the group of long-acting bronchodilators where the influence of inhaler device could be most extensively tested. Starters were defined as not having used any long-acting respiratory medications  or long-acting muscarinic antagonists) for at least 2 years before treatment initiation. To make sure that a complete overview of history and follow-up was available, patients needed to be registered in the database at least 2 years before and 1 year after initiation of therapy. We only included patients who started one type of inhaled medication at treatment initiation . To minimise the possibility to include (late onset) asthma patients, we excluded patients using typical asthma medication (antihistamines (R06), anti-allergics (R03BC), leukotriene receptor antagonists (R03DC), and omaluzimab (R03DX05)) within 1 year after long-acting beta agonist initiation.As the database lacked data on clinical diagnosis, COPD patients were selected based on initiation of inhaled respiratory medication above the age of 55 years, in line with previous studies.7Continuous drug use in the first year was calculated by the total number of doses dispensed from therapy initiation, divided by the prescribed daily dose. A gap of maximal 60 days without medication coverage within the period of continuous use was allowed to consider a patient being still persistent to medication, that is, patients who did not have any gaps exceeding 60 days in the 1-year period after therapy initiation was considered persistent. This 60-day gap is a common cutoff and was used in various previous studies assessing persistence with COPD medication.vice versa), a switch to a LABA-pressurised metered dose inhaler, a switch to a different bronchodilator class (LABA/ICS or long-acting muscarinic antagonist), a restart with initial type of medication/inhaler or permanent discontinuation of any long-acting bronchodilator.Part of the nonpersistent patients were those who did discontinue their initial inhaler or medication but did not completely discontinue the use of any long-acting medication at all . Therefore, follow-up of non-persistent  patients was monitored by assessing patients’ medication profiles after the date of discontinuation of their initial therapy. For all patients, follow-up was at least 1 year from the date of treatment initiation; however, to be able to assess switching patterns in the year after the date of treatment discontinuation, follow-up needed to be longer. Therefore, we defined a subcohort where patients needed to have a follow-up of at least 2 years. After discontinuation of initial treatment, we assessed the following possibilities: a switch to a different inhaler within the DPI class  that can be taken successively until the device runs out of doses. Examples include the Diskus, the Turbuhaler and the Novolizer. We hypothesised that the use of single-dose inhalers required more complex and time-consuming handlings in order to perform proper inhalation and would therefore decrease treatment persistence.Two cohorts were defined based on the type of LABA-DPI initiated by the patients. We made a distinction between single-dose and multiple-dose DPI inhalers. Single-dose inhalers require loading of separate capsules before using the device.P<0.05) between the two cohorts.Although this study focused on the medication and inhaler devices, persistence may be altered by several other parameters. The following parameters were compared between the two cohorts: age, gender, socioeconomic status, initial prescriber, comorbidity (described in more detail below), number of short courses of oral corticosteroids, use of short-acting bronchodilators, initial medication and dosing regimen. We performed a multivariate analysis to correct for parameters that significantly differed . Heart failure and ischaemic heart disease were identified by prescription of digoxin or loop diuretics (C01AA05 or C03C) and C01DA, respectively. Patients not using previously mentioned anatomical therapeutic chemical codes but using other cardiovascular drugs  were classified as patients suffering from other cardiovascular disorders. Other diseases included osteoporosis (M05B), anxiety (N05B and N05C), dementia (N06B), depression (N06A), rheumatic arthritis (M01 and M02) and hypothyroid disease (H03). Patients needed to have at least two of these prescriptions within 1 year after COPD treatment initiation to be considered a chronic user of these comedications.Included comorbidities were selected based on frequently reported comorbidities from previous studies.U-tests, where appropriate.Parameters of the two cohorts were compared using Fisher’s exact tests or Mann–Whitney P<0.05). Statistics were performed with IBM SPSS Statistics 22 .Kaplan–Meier survival analyses were used to visualise patients’ persistence over a 1-year-time period, comparing patients initiating multiple-dose inhalers and single-dose inhalers. In multivariate Cox proportional hazard analyses, results were adjusted for parameters that differed between the two cohorts with a 95% confidence level  in the year after treatment initiation, that is, one-time users  were excluded.In all, 1,838 potential COPD patients initiated a LABA of interest  between 1996 and 2011. After exclusion of likely asthma patients (223), 1,615 patients remained. Of these, 595 patients had no complete information records on essential parameters to calculate persistence  and were excluded, leaving 1,020 patients. Among them, 207 started two long-acting inhalers on the same date and were excluded, leaving 813 patients. Of those, 238 (29%) started with a pressurised metered dose inhaler and 575 (71%) started with a DPI. Of all the DPI users, 475 (83%) initiated a multiple-dose inhaler and 100 (17%) a single-dose inhaler.P<0.05) were the initial prescriber, comorbidities , the initial medication and the dosing regimen.A flow diagram is provided in P=0.74). In the multivariate analysis, results did not change .Overall, 1-year persistence in the basecase scenario  was 14.4%. Persistence between users of multiple-dose and single-dose inhalers did not significantly differ . Persistence with initial medication over time is graphically shown in initial inhaler within 60 days after the theoretical end date of their previous prescription (when used as prescribed). Take into account that the dosing regimen varied between one and four times per day. The time point of non-persistence is the theoretical end date of their last prescription before their first gap.Including only patients with two prescriptions or more resulted in a cohort of 375 patients initiating LABAs. Sixty-six (18%) patients initiated a single-dose inhaler and three hundred nine (82%) a multiple-dose inhaler. Overall persistence was 21.9%. There was no significant difference between persistence of users of single-dose or multiple-dose inhalers  with at least two dispensings for long-acting bronchodilators  and a follow-up of at least 2 years from treatment initiation. In this cohort, 38 out of 53 single-dose inhaler users were non-persistent and 203 out of 254 multiple-dose inhaler users. From This study showed no significant impact of inhaler device on COPD patients’ persistence with LABAs: persistence of patients initiating multiple-dose inhalers was comparable to patients initiating single-dose inhalers. Whereas overall persistence with LABAs seemed relatively low at first sight, this study showed that ~40% of all patients  re-started their initial therapy within 1 year. Only 15% of the patients who seemed to discontinue based on a 60-day treatment gap did indeed permanently discontinue any type of long-acting respiratory therapy. A considerable percentage of patients (about one-third) switched to fixed dose inhalers of LABA and ICS. Of the single-dose inhaler users, 11% switched to multiple-dose inhalers of the same medication, whereas none of the multiple-dose inhaler users switched to single-dose inhalers with the same medication.25 Specific studies in COPD patients assessing the impact of inhaler device on adherence or persistence are lacking.14 In general, medication persistence in our study was far short of optimal, for both multiple-dose as well as single-dose inhalers. This relatively low persistence is in line with findings from previous studies;7 however, this study showed that over 80% of patients who initially seemed to discontinue LABAs, based on a 60-day treatment gap, re-started their initial medication or switched to different types of inhaler or medication within 1 year after discontinuation. The high percentage of switching may suggest that for some prescribers it takes some time to find the optimal inhaler and medication to suit their patients’ preferences regarding persistent use. However, whereas an optimal inhaler may favour patients’ persistence, improved disease control and better clinical outcomes are not guaranteed. Even though in controlled clinical trial settings comparing inhaler devices no differences in outcomes were observed,26 in daily practice, many problems with inhalers have been described.27 Main problems are related to poor inhalation technique,28 resulting in suboptimal deposition in the lungs, oropharyngeal side effects29 and ultimately poor disease control.30 Instruction on inhalation techniques has been shown to not only improve adherence but was also associated with better health-related quality of life.31 Apart from inhalation technique and lung deposition issues, one can argue whether improved health outcomes are solely achieved by adherence to respiratory medication or that COPD patients’ (self-management) skills and behaviour determine an important part of improved outcomes. Notably, good adherers with COPD placebo inhalers showed comparable outcomes on mortality and hospitalisation rates as good adherers to real medications.32 Vestbo et al.32 describe this phenomenon as the ‘healthy adherer effect’. It is suggested that COPD patients who are good adherers to their medication have a healthier lifestyle in general . In other words, medication non-adherence may be an indicator of patients’ shortage of disease- and self-management skills. This reveals an important opportunity for the health-care provider: actively search for non-adherent COPD patients  and target your interventions to these patients. Using this strategy, not only health-care providers’ time is more efficiently spent but also the cost effectiveness of disease management interventions is expected to increase as non-adherence has a marked effect on worsened economic outcomes.8This study is the first describing the relationship between inhaler devices and persistence with COPD maintenance medication. In asthma, DPI devices have been linked to improved adherence compared with pressurised metered dose inhalers.By using real-world and up-to-date data, the results of this study are expected to be representative for the current situation in the community. However, whereas relative results are expected to be generalisable between countries, absolute results may be less generalisable because of intensive monitoring and lung revalidation programmes currently running in the Netherlands, which may encourage more adherent behaviour. Some late onset asthma patients may be incorrectly classified as COPD patients as the database does not provide a diagnosis. In addition, some severe exacerbations  may have been missed as the database only collects data from community pharmacies. Although we tried to use the dispensing of short courses of oral corticosteroids as a proxy for disease severity, findings could still be confounded by COPD severity, as neither lung function data nor a full set of proxies for COPD severity were available. The last two limitations are related to the size of the sample. It might be possible that the observed non-difference in persistence between the two inhaler types resulted from an insufficient power to detect the persistence difference. Another possibility is that the persistence difference might only exist, for example, in those COPD patients using their inhaler four times per day, and only a small proportion of the studied subjects used the inhaler at this high frequency.33 Besides, factors relating to optimal prescription, taking into account patients’ preferences34 and interventions on behaviour modification , are equally important and may add to patients’ capacity of acting in a more adherent behaviour.35 In addition, enhancing health-care providers’ education and communication skills may be beneficial.36 Targeting these interventions on non-adherent patients is expected to improve cost effectiveness.37In order to optimise patients’ persistence with COPD medication, we recommend a choice of inhaler depending on patients’ individual abilities to handle the inhaler.The choice of an appropriate inhaler device is an important issue regarding COPD patients’ persistence; however, regarding the inhaler being a multi-dose or a single-dose device does not seem to affect patients’ persistence with LABAs. Over 80% of patients, who initially seemed to discontinue LABAs, re-started their initial medication or switched to a different type of inhaler or medication within 1 year.


Bats have been recognized as the natural reservoirs of a large variety of viruses. Special attention has been paid to bat coronaviruses as the two emerging coronaviruses which have caused unexpected human disease outbreaks in the 21st century, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), are suggested to be originated from bats. Various species of horseshoe bats in China have been found to harbor genetically diverse SARS-like coronaviruses. Some strains are highly similar to SARS-CoV even in the spike protein and are able to use the same receptor as SARS-CoV for cell entry. On the other hand, diverse coronaviruses phylogenetically related to MERS-CoV have been discovered worldwide in a wide range of bat species, some of which can be classified to the same coronavirus species as MERS-CoV. Coronaviruses genetically related to human coronavirus 229E and NL63 have been detected in bats as well. Moreover, intermediate hosts are believed to play an important role in the transmission and emergence of these coronaviruses from bats to humans. Understanding the bat origin of human coronaviruses is helpful for the prediction and prevention of another pandemic emergence in the future. Alphacoronavirus and Betacoronavirus, which mainly infect mammals, 7 out of the 15 currently assigned viral species have only been found in bats [Bats, with extensive geographical distribution and capability of flight, constitute the second largest group of mammalian species and have been documented as natural hosts of a large number of diverse viruses such as lyssaviruses, paramyxoviruses and filoviruses , 2. In t in bats . It is pIn this review, we focus on the emerging coronaviruses putatively linked to a zoonotic origin from bats, represented by SARS coronavirus (SARS-CoV) and MERS coronavirus (MERS-CoV). We present an overview of current evidence for bat origin of these two viruses and also discuss how the spillover events of coronavirus from animals to humans may have happened. Considering that bats have been known to harbor more coronaviruses than any other species, it is likely that SARS-CoV and MERS-CoV won’t be the only bat coronaviruses to jump among species and cause human infections. Bat coronaviruses should be seriously regarded in light of their potential risks to public health.http://www.who.int/csr/sars/country/table2004_04_21/en/). The second outbreak in 2004 only caused 4 infections with no mortality nor further transmission [SARS first emerged in late 2002 in Guangdong Province, southern China, as a novel clinical severe disease  marked by fever, headache and subsequent onset of respiratory symptoms including cough, dyspnea and pneumonia. Being highly transmissible among humans, SARS rapidly spread to Hong Kong and other provinces across China and then to other 28 countries , 7. By JThe MERS epidemic emerged in the Kingdom of Saudi Arabia (KSA) since June 2012, with a similar clinical syndrome to SARS but seemingly less transmissible. In addition to respiratory illness, renal failure was identified in some severe cases –11. UnliBetacoronavirus  in the characteristic gene order in the 5’ to 3’ direction: ORF1a and 1b which comprise two-thirds of the genome and encode the nonstructural polyproteins, and four ORFs downstream that encode structural proteins: spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N). Some coronaviruses have a hemagglutinin-esterase (HE) gene between ORF1b and S. Besides the coronavirus-conserved genes, the SARS-CoV genome contains a number of specific accessory genes including ORF3a, 3b, ORF6, ORF7a, 7b, ORF8a, 8b and 9b –15. CompThe S protein of coronaviruses is a surface-located trimeric glycoprotein consisting of two subunits: the N-terminal S1 subunit and the C-terminal S2 subunit. The S1 subunit specializes in recognizing and binding to the host cell receptor while the S2 region is responsible for membrane fusion. Compared with the S2, the S1 subunit shows much higher variability . Owing tAngiotensin-converting enzyme 2 (ACE2) was identified to be the functional receptor of SARS-CoV –24. A 19Dipeptidyl peptidase 4  was identified as a functional receptor for MERS-CoV  and it iEpidemiological survey showed that early cases of SARS in 2002–2003 and all 4 cases in 2003–2004 had a history of animal contact through animal trade in wet markets or in restaurants where live animals were kept in Guangdong Province. Molecular detection and virus isolation studies suggested that the pandemic-causing SARS-CoV originated from traded civets in wet markets. This was indirectly confirmed by the massive culling of market civets, which was believed to play a major role in efficiently containing the SARS pandemics and no further SARS case was reported after 2004 –42.However, subsequent extensive epidemiology studies did not find SARS-CoV in farmed or wild-caught civets, indicating that other animal(s) was involved in SARS-CoV transmission in the animal market or other trading activities and civets are unlikely the natural reservoir of SARS-CoV –45.Rhinolophus) in China, which were termed SARS-like coronavirus (SL-CoV) [Rhinolophus species of a wider geographic range in China. The provinces or regions where SL-CoV-positive bats were captured included Hong Kong, Guangxi, Hubei, Shandong, Guizhou, Shaanxi and Yunnan [Several years before the outbreak of SARS, two other zoonotic viruses, Nipah virus and Hendra virus, emerged in Asia and Australia and they were both known to be originated from bats , 47. The(SL-CoV) , 49. Thed Yunnan –53. 7 cod Yunnan .Rhinolophus blasii in Bulgaria was highly divergent from Chinese isolates, displaying major sequence differences in several genes including ORF3b and ORF6 and lacking the coding region of ORF8 in its genome [Hipposideros and Chaerophon species from Ghana, Kenya and Nigeria. However, compared with Asian and European SL-CoVs, these viruses of non-rhinolophid origin were phylogenetically distant to SARS-CoV. The Western African isolates even formed a potential new lineage of Betacoronavirus in the phylogenetic tree  amino acid identity to civet SARS-CoV SZ3 [R. ferrumequinum in Hubei Province, of which the ORF8 shared 80.4 % amino acid identity to SZ3 [R.sinicus and R.ferrumequinum and it has been suggested that the ancestor of civet SARS-CoV probably acquired ORF8 from R.ferrumequinum SL-CoVs by recombination [Despite the fact that Rs3367 or WIV1 is unprecedently close to SARS-CoV in terms of RBD region and genome identity, still there are gaps between them and the immediate ancestor of SARS-CoV. ORF8 is a highly variable gene and remarkable differences can be observed among SARS-CoVs and SL-CoVs of different host origins. Isolates from civets and from early phase of the 2002/2003 pandemic contained a single long ORF8, while in the human SARS-CoV isolates from the middle and late phase of the pandemic the ORF8 was disrupted into two ORFs, ORF8a and ORF8b, as a result of the acquisition of a 29-nt deletion after interspecies transmission to humans , 62. The-CoV SZ3 . This isy to SZ3 . Potentibination .As with SARS-CoV, most early MERS cases had contact history with animals, e.g., dromedary camels , 65. MERTylonycteris bat coronavirus HKU4 (BtCoV-HKU4) in Tylonycteris bats and Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5) in Pipistrellus bats in China [Eptesicus isabellinus bats in Spain [Pipistrellus pipistrellus bats in the Netherlands [Betacoronavirus. After the outbreak of MERS, MERS-CoV related coronaviruses were found in more bat species and countries [Prior to the emergence of MERS-CoV, a group of bat coronaviruses had been reported including in China , 84, 85,in Spain  and N.noherlands . Based oountries –96. Amonountries . Other bountries , while HAlthough NeoCoV is closer to MERS-CoV than other bat coronaviruses at genomic level, the phylogenetic analysis of the spike protein showed that HKU4 is the most closely related to MERS-CoV among all currently known bat coronaviruses, sharing 67 % sequence identity Fig. . This isBoth SARS-CoV and MERS-CoV are emerging zoonotic pathogens that crossed the species barriers to infect humans , 53, 99.Hipposideros caffer ruber in Ghana termed Hipposideros/GhanaKwam/19/2008 was genetically related to HCoV-229E. Its RdRp fragment shared 92 % nucleotide sequence identity with HCoV-229E and they were predicted to share a most recent common ancestor (MRCA) only 200 years ago [HCoV-229E was found in the 1960s and causes comparatively mild common colds worldwide . A bat cears ago . A recenears ago .Perimyotis subflavus) in the US showed close relationship with HCoV-NL63. The MRCA for HCoV-NL63 and ARCoV.2 was predicted to have existed 563 to 822 years ago [HCoV-NL63 was first isolated from babies suffering of pneumonia and bronchiolitis in 2004 . To dateears ago , 107. FuAlthough the study of bat-borne coronaviruses has only started just about 10 years ago, the scientific community has already learnt a great deal of useful lessons which will be instrumental in mitigating, predicting, and preventing future zoonotic coronavirus outbreaks. Some of these lessons are summarized below.Bats harbor coronaviruses with great genetic diversity. It is believed that most, if not all, currently circulating alphacoronaviruses and betacoronaviruses in different mammals are evolutionally linked to ancestral coronaviruses originated from bats. Different species of rhinolophid bats in China carry genetically diverse SARS-like coronaviruses, some of which are direct ancestors of SARS-CoV and hence have the potential to cause direct interspecies transmission to humans. Meanwhile, different coronavirus species closely related to MERS-CoV are circulating in bats. Bats are likely natural reservoirs of MERS-CoV or an ancestral MERS-like CoV. It is hypothesized that bat MERS-like CoV jumped to camels or some other as yet unidentified animal several decades ago. The virus evolved and adapted with accumulating mutations in camels and then was transmitted to humans very recently. It took almost a decade from the first discovery of SL-CoV in bats to the final isolation of the SARS-CoV ancestral virus from bats, so continuing surveillance is vital to uncover the origin of MERS-CoV and bats should certainly be a priority of research. Besides, as the spike protein and host receptor are key factors of cross-species transmission of coronaviruses, characterization of the receptor and key binding sites of the spike protein will be important in estimating host tropism of bat coronaviruses and predicting spillover risk.With human activity increasingly overlapping the habitats of bats, diseases outbreaks resulted from spillover of bat coronaviruses will continue to occur in the future despite the fact that direct transmission of bat coronaviruses to humans appears to be rare. To better prepare ourselves in predicting and preventing the next emergence of a coronavirus disease, it is necessary to maintain our vigilance in long-term coronavirus surveillance studies in bats as well as in other wildlife and livestock. Combined with other laboratory-based studies such as receptor specificity, pathogenesis and animal infection, a focus on continued surveillance will help us to improve risk assessment as well as to reveal the potential intermediate hosts that may play an important role in the interspecies transmission of various known and as yet unknown bat coronaviruses.


The worldwide incidence of kidney cancer is estimated at 337,860 new cases per year in the International Agency for Research on Cancer's GLOBOCAN 2012 update, with an estimated 143,369 deaths annually. Over the past 10 years, there have been significant advances in the treatment of advanced/metastatic renal cell carcinoma, including the development of targeted therapies. Currently recommended first-line treatments include sunitinib, temsirolimus, bevacizumab plus interferon, and pazopanib, or high-dose interleukin-2 or sorafenib for selected patients. Recommended second-line treatments include all of the above agents, as well as everolimus and axitinib. Unfortunately, combination therapies have generally resulted in increased toxicity and little improvement in efficacy. Recent studies focused on identification of predictive biomarkers for responses to specific targeted therapies and have not been successful to date. Despite recent advances in targeted treatment for metastatic renal cell carcinoma, important questions regarding biomarkers of efficacy, and optimal combination and sequencing of agents remain to be answered. This paper reviews literature concerned with first-and second-line treatment of metastatic renal cell carcinoma and will discuss key issues in Latin America. Renal cell carcinoma (RCC) arises primarily from the proximal tubular epithelium and accounts for ~85% of all kidney cancers, with the remainder consisting of renal pelvis cancer and other rare malignancies . Many RCThe age-standardized rates (ASRs) for kidney cancer incidence are similar in Latin America and the Caribbean  to those in North America and Europe , while the ASR for mortality is slightly lower in Latin America and the Caribbean  compared with that in North America and Europe   developed a model for dividing patients with advanced disease in low-, intermediate-, and high-risk categories . PatientDespite considerable research, there are currently no validated biomarkers for use in the clinical management of mRCC, and only histology, staging, and clinical/laboratory characteristics can guide physicians in defining therapy and predicting patients’ outcomes. Nevertheless, biomarkers related to the VHL tumor suppressor gene, hypoxia-inducible factor (HIF), tumor-promoting genes responsive to HIF , the mammalian target of rapamycin kinase (mTOR) pathway, the tumor suppressor gene phosphatase and tensin homolog, Akt, and phosphorylated S6K are all being explored . Single This review is focused on patients with advanced or mRCC. Stages I–III kidney cancers are managed with partial or radical nephrectomy, active surveillance or ablative techniques for non-surgical candidates . At thisCurrently, the National Comprehensive Cancer Network (NCCN) guidelines’ recommended first-line treatments for metastatic and surgically unresectable RCC, supported by category 1 evidence, including sunitinib, temsirolimus (for poor-prognosis patients), bevacizumab plus IFN-α, and pazopanib ; 5. SuniDespite differences in mechanism of action, the safety profiles of all four treatments share some similarities, with asthenia/fatigue (20-63%), nausea (26-52%), diarrhea (20-63%), and anorexia (22-37%) among the most commonly reported adverse events (AEs) , 17, 18.An expanded-access program was established to provide sunitinib to patients with mRCC who were ineligible for ongoing sunitinib clinical trials and/or before regulatory approval in their countries , 21. TheTo date, there are few direct comparisons of the safety and efficacy of currently recommended first-line treatments for advanced RCC. The COMPARZ trial recently showed that pazopanib was non-inferior to sunitinib with respect to PFS, with an HR of 1.05 (95% CI: 0.90-1.22), and OS was similar   17). Th. Th17). There are still unanswered questions related to treatment selection for patients with advanced RCC (see below).Current recommendations for second-line treatment of advanced RCC following a prior tyrosine kinase inhibitor include everolimus and axitinib, and following prior cytokine therapy include axitinib, sorafenib, sunitinib, and pazopanib ; 5. The In a phase III study comparing axitinib with sorafenib as second-line treatment in 723 patients with clear-cell mRCC, median PFS was 6.7 months for axitinib and 4.7 months for sorafenib (p<0.0001)  24) and and24) aTo explore the efficacy of temsirolimus after VEGF inhibitor therapy, the INTORSECT trial compared temsirolimus with sorafenib as second-line treatment for patients with disease progression after sunitinib . PFS didMany of the established and emerging treatments for mRCC have similar or overlapping biologic actions and we need more information about how they might influence each other's efficacy and the mechanisms underlying resistance to each . At presIdentifying the optimum treatment for advanced RCC requires increased understanding of the tumor biology and patient characteristics predictive of response to specific treatments. A retrospective analysis based on pooled efficacy (n=544) and safety (n=4917) data from four clinical trials showed that sunitinib-induced hypertension was associated with significantly improved clinical outcomes . For patA gene expression profiling study identified a 20-gene signature predicting response to sunitinib with 68.5% accuracy , and micOne consideration when using targeted agents to treat Latin American patients with mRCC is their different racial/ethnic mix compared with patients from North America. Results from a survey of 508 patients with RCC in Brazil indicated that 78.9% of patients were white, 6.5% were black, and 14.0% were mixed race . In contDrug availability is one of the key points to improved survival in patients with mRCC, as has been shown by the impact of post-progression therapy on OS in the clinical trial setting. In the AVOREN phase III trial of bevacizumab plus IFN-α, 63% of patients in the control arm (i.e. those randomized to IFN-α only) received at least one post-protocol therapy, comprising either sunitinib or sorafenib in 37% of cases . In thisThese data show the positive impact of targeted therapy on survival in patients with advanced RCC. Nonetheless, with the exception of temsirolimus in poor risk patients, median OS, unlike PFS, was not significantly increased with targeted therapies compared with standard therapies or placebo in the phase III trials of first-line therapy ; however


The epithelial to mesenchymal transition (EMT) is implicated in many processes, ranging from tissue and organogenesis to cancer and metastatic spread. Understanding the key regulatory mechanisms and mediators within this process offers the opportunity to develop novel therapeutics with broad clinical applicability. To date, several components of EMT already are targeted using pharmacologic agents in fibrosis and cancer. As our knowledge of EMT continues to grow, the potential for novel therapeutics will also increase. This review focuses on the role of EMT both as a necessary part of development and a key player in disease progression, specifically the similarity in pathways used during both processes as targets for drug development. Also, the key role of the tumor microenvironment with EMT is outlined, focusing on both co-factors and cell types with the ability to modulate the progression of EMT in cancer and metastatic disease. Lastly, we discuss the current status of clinical therapies both in development and those progressed to clinical trial specifically targeting pathologic EMTs including small molecule inhibitors, non-coding RNAs, exogenous co-factors, and adjunctive therapies to current chemotherapeutics. The epithelial to mesenchymal transition (EMT) was first described in the context of being a fundamental component in the differentiation of tissues and organs within the developing embryo . Beyond EMT is contemporarily classified into 3 classes: type 1 referring to embryogenesis; type 2 referring to wound healing; type 3 referring to its role in cancer and metastases. A hallmark of each is the process through which epithelial cells lose their cell-cell junctions, apical-basal polarity, and re-organize their cytoskeleton by e-configuring their gene expression. These processes are highly regulated in the setting of type 1 and 2 EMT. In type 3 EMT, dysregulation leads to diseased states .On a molecular level, EMT is defined by several intermediates responsible for influencing gene expression. These include transcription factors, multiple interacting signal pathways, promoter sequence modifications, and autocrine signaling. Of these, the principal regulators of EMT are transcription factors classified into 3 families: SNAIL, TWIST, and zinc-finger E-box-binding (ZEB). The fundamental event in EMT is the repression of epithelial cadherin (E-cadherin), which is responsible for maintaining adherens junctions and cell-cell adhesion. SNAIL, TWIST, and ZEB factors all can repress E-cadherin while also activating key mesenchymal genes including N-cadherin (cell-cell adhesion), vimentin (an intermediate filament protein that regulates cell motility), and fibronectin 1 (cell growth and migration). Together, these transcription factors orchestrate the activation of mesenchymal gene expression while repressing epithelial genes through diverse downstream targets.The same mediators used for modifying gene expression are used as biomarkers to identify a cell as epithelial or mesenchymal. For example, expression of E-cadherin is used to describe a cell as being in an epithelial state whereas expression of N-cadherin, vimentin, and/or fibronectin 1 are used to describe the mesenchymal phenotype . Finally, discussing the areas within the spectrum of EMT which allow for novel therapeutics and the current progress towards clinical application.EMTs are pathways followed during normal development; most organs and adult tissues are generated via these transitions embryologically. Each of these transitions rely on signal cascades with multiple regulatory and feedback loops allowing both plasticity and cooperation during development. EMT plays a prominent role during gastrulation and many of the key molecular players during these processes have been noted to be important in disease progression during adulthood. For example, members of the TGFβ superfamily are powerful inductors of gastrulation . SNAIL1 The importance of key EMT mediators in early gastrulation is further emphasized in SNAIL-mutant mice where cells are unable to migrate from the primitive streak, effectively halting development . TGFβ siEMT has a role in several disease states including pathologic organ degradation (ie fibrosis) and cancer. How EMT fits into these processes will be reviewed, including the initiating signal pathways, downstream targets, and mechanisms for regulating these pathologic EMTs.In all pathologic EMTs, a series of steps are conserved and are crucial for defining an epithelial cell’s conversion to a mesenchymal phenotype. Epithelial cells are broadly characterized as those positioned on the basement membrane (either as single layered or multilayered tissues), show polarity, adhere, and communicate using cell-cell junctions . Loss ofOne such example can be seen in the pathways regulating cell-cell junctions and their dissolution during EMT. Cell-cell junctions are critical for maintaining basement membranes and the integrity of epithelial layers. In vertebral cells, tight junctions, gap junctions, and desmosomes are the primary regulators of these connections ,12]. Se. Se12]. Fibrotic tissues are characterized by an up-regulation of extracellular matrix (ECM) protein deposition eventually leading to compromise of organ function, and in the most severe cases, organ failure . The celInflammatory cytokines, specifically TGFβ and IL-1, are critical in the differentiation of cells from any site into myofibroblasts . In wounAlveolar epithelial cells, hepatocytes, lens epithelium, endothelial cells, mesothelial cells, and cardiomyocytes all have the potential to undergo EMT and resemble myofibroblasts, subsequently contributing to organ fibrosis . In pulmIn renal fibrosis, mice that do not express Smad3 (a target of TGFβ) are protected from development of fibrosis. The crucial role of TGFβ in renal fibrosis was taken a step further by Zeisberg et al. who demonstrated that inhibiting TGFβ with an antagonist (BMP7) actually could revert renal fibrosis in mice .The pathologic potential of EMT is seen in organ fibrosis and similar mechanisms are seen in the development of cancer, evolution of disease, and metastatic progression. In cancer, EMTs rely on: 1) exogenous induction agents acting through transcriptional control 2) Non-coding RNAs and 3) creating and maintaining cancer stem cells .1. Transcriptional control mechanismsThe discovery of the transcription factor SNAIL1 interacting with the CDH1 promoter to repress the production of E-cadherin began our understanding of how EMT is regulated ,31]. Nu. Nu31]. The association between disease progression and solid tumor oxygenation is seen in various malignancies. It is apparent that tumor hypoxia contributes to several features of aggressive cancers by enhancing invasive growth, local spread, and distant deposits . The mecCholestatic disorders seem to promote hepatic fibrosis and to the progression of carcinogenesis in cholangiocytes and hepatocytes. Studies have shown that chendeocycholic acid (CDCA), a bile acid, can activate EGFR to mediate gene expression. In a cDNA microarray, CDCA increased the expression of SNAIL to repress E-cadherin both in vitro and in vivo in liver cancer models, initiating an EMT program . The abiAnother substrate which activates EMT through a similar mechanism is nicotine. Specifically, in breast and lung cancer cell lines , treatment with nicotine increased the proliferation, invasion, and migration of both lung and breast cancer cells in a dose-dependent fashion. Notably, expression of E-cadherin was repressed while the expression of fibronectin and vimentin, both mesenchymal markers, increased. The mechanism for these changes occurred through signaling by nicotinic acetylcholine receptors (nAChRs) .Recent data demonstrates a pathway for cigarette smoke to stimulate EMT in small bronchi of patients demonstrating a mechanism contributing to the progression of disease. In primary human bronchial epithelial cells (HBEC), exposure to cigarette smoke led to expression of EMT markers by increased production of intracellular ROS, release of TGFβ, and TGFβ activation of the transcription factors Smad3 and ERK1/2 . This ki2. Non-coding RNAsNon-coding RNAs and, in particular, microRNAs (miRNAs) have gained significant attention for their ability to modify gene expression allowing for quick cellular adaptation to new environments and conditions ,40]. Th. Th40]. Since that time, numerous miRNAs have been found to interact either directly or indirectly with EMT machinery in several cell models . InteresTwo major classes of miRNAs have been studied extensively in the setting of EMT: the miR-200 family and the miR-34 family. Other families include the miR-205, miR-9, and miR-10 families, respectively. The miR-200 family has five named members located on human chromosome 1  and 12 . Both the miR-200 and miR-34 families are typically considered to be “tumor suppressive” as their members all stabilize the epithelial phenotype .The interplay between miR-200 and the ZEB family of transcription factors to maintain E-cadherin expression has been validated in several systems. In a TGFβ-induced model of EMT in a murine mammary cell line, miR-200 family member expression was repressed. This same cell line was transfected with synthetic pre-miRNAs to stimulate overexpression of corresponding miR-200 members in vitro. The result was inhibiting EMT in the transfected cell lines, when compared to control, by increasing the expression of E-cadherin. Furthermore, using an elegantly designed luciferase assay, miR-200 family members were noted to interact directly with the transcription factors ZEB1 and ZEB2 . ZEB1 anThe clinical relevance of miR-200 family members can be seen in malignant breast, prostate, and colon cancer cells. Silencing of the miR-200 family induces early transformation of epithelial cells leading to EMT, reinforcing the role of these miRNAs in tumor suppression .An interesting link between the p53 tumor suppressor gene, SNAIL, and the miR-34 was described by Siemans et al. in 2011. In colon cancer cells, activation of p53 notably induced the expression of miR-34 family genes, resulting in the inhibition of SNAIL activation. As would be predicted, a correlate decrease in EMT markers was seen in the absence of SNAIL activation in this cell line. Treatment of cells with miR-34 halted TGFβ dependent EMT. It was noted, however, that the action of miR-34 family members was not unidirectional. The miR-34 family promoter sequences contain E-boxes which can be bound by SNAIL, resulting in decreased expression of miR-34. This double negative feedback loop between miR-34 and SNAIL offers an interesting regulatory mechanism for EMT creating unique challenges in drug development targeting either of these factors .In contrast to the tumor suppressive functions of miR-200 and miR-34, the miR-9 family appears to be tumor activating through a direct interaction with the CDH1 promoter which subsequently initiates EMT . miR-9 eIt must be emphasized that work with non-coding RNAs is far from complete and their relevance to cancer (and cancer therapy) remains imprecise. While the miR-200 family appears to have compelling data for a tumor suppressive function, as discussed above, there is are also data indicating a pro-metastatic niche for miR-200 family members.In samples from colon cancer patients, there is lowered expression of miR-200 within the primary tumor which fits the impression that miR-200 is tumor suppressive. However, in metastatic lesions, miR-200 expression is high ,50]. Th. Th50]. Another important miRNA family is the miR-10 group including miR10b. In the study of metastatic breast cancer cells, miR-10b was noted to be highly expressed and positively regulate cell invasion. It was also able to initiate tumor formation and played a role in distant metastases. Taken together, these properties are highly suggestive that miR-10b is implicated in EMT .3. Cancer stem cellsThe concept of the cancer stem cell is based on the hypothesis that a single cell type or subset of cells can act as tumor-initiators in various cancers ranging from hematopoietic to solid organ. By definition, these cancer stem cells (CSC) have features which give them “stem-like” properties allowing for self-renewal and differentiation leading to the histologic heterogeneity displayed by tumors .+,CD38-), were able to proliferate, disseminate, and maintain an AML phenotype similar to the patients they were derived from after transplantation into severe combined immune-deficient (SCID) mice . Ma. Ma91]. Another dietary agent EGCG, the major polyphenolic compound in green tea, demonstrates anti-proliferative effects in several cancers . EGCG inThe translation of bench findings to clinical trial for both resveratrol and EGCG has been challenging. At this stage, resveratrol has been studied in phase 1 clinical trial and EGCG remains in pre-clinical study. Other dietary and herbal chemopreventative agents also have been noted to inhibit EMT through non-TGFβ-dependent mechanisms. β-elemene, an active component of the herbal medicine Curcuma wenyujin, acts to stop EMT through Smad3 .Curcumin, the active component of the spice turmeric, is an inhibitor of EMT by altering the SNAIL1 transcription factor . 2-HydroStudying miRNAs and their potential role in cancer is an extremely fast-growing area of research. In parallel, several research groups are working on ways to modulate the effect of miRNAs in tumor biology with the intent of developing new and novel therapeutic targets. Antagomirs and miRNA sponges each show promise for future clinical application and will be discussed.1. AntagomirsAs described earlier, miRNAs can play a role in cancer progression and metastases leading to them often being referred to as “onco-miRs” . AntagomThe same group responsible for determining the important role played by miR-10b in breast cancer invasion and metastatic progression also utilized an antagomir (antagomir-10b) to silence miR-10b in vitro and in vivo. Interestingly, antagomir-10b did not reduce the bulk or size of the primary mammary tumor. However, it significantly suppressed the formation of metastatic lesions in a mouse. Overall it was well tolerated by treated animals laying the groundwork for further study of antagomirs as anti-cancer drugs ,99]..99].2. miRNA spongesmiRNA sponges are another tool increasingly utilized to modify the activity of miRNAs . These sAs illustrated, this remains a relatively young area of work with few advancements to the clinical setting to date. However, strategies including the development of antagomirs and miRNA sponges have the potential to make an impact on a patient level.As discussed, EMT signaling pathways have important roles in the transformation of epithelial cells into CSCs. Several groups have started the process towards drug development to inhibit this process using large-scale screening techniques ,104]. T. T104]. Another strategy for eliminating CSCs is specifically targeting EMT pathways important for their generation and maintenance. Physiologic factors already exist to prevent mesenchymal transition, likely secreted by epithelial cells in order to remain in a fixed state. These autocrine factors include Dickkopf-1 (DKK1) and secreted frizzle-related protein 1 (SFRP1), both known inhibitors of Wnt pathways. These proteins could be pharmacologically mimicked to produce a similar effect in diseased states .1. Small molecule inhibitorsWnt pathway inhibitors currently in development for the purpose of inhibiting CSC formation include LGK974 and CWP232291  and the tumor – including the microenvironment as a stimulator of EMTs – is important in cancer progression . TargetiThe TMEN is a specialized extracellular matrix (ECM) wherein a cancer cell proliferates . The comThe cytokines IL-1, IL-8, IL-10, and TGFβ all demonstrate effects on either tumor growth or metastasis through their interactions with cancer cells. The major cell types responsible for these mediators in the TMEN include the primary tumor cell and stromal cells including macrophages, adipocytes, and cancer associated fibroblasts (CAFs) . A key cMesenchymal stem cells (MSCs) have the ability to induce EMTs in several models and play a role in both pre-metastatic and metastatic sites. Breast cancer cell lines  cultured with MSCs increase expression of EMT markers  with concomitant decrease in E-cadherin expression. This increase in EMT markers corresponded with an increase in protein expression as well, with increased Vimentin and SNAIL compared to untreated cells. Gene expression assays also revealed upregulation of TGFβ-receptor implicating TGFβ mechanistically as a regulator of this EMT. This occurred with only co-culturing these breast cancer cell lines with MSCs and no further co-stimulatory factors .A similar role for MSCs initiating EMT is seen in hepatocellular carcinoma (HCC). HCC cell lines (SMMC-7721 and Hep-3B) expressed EMT markers after exposure to IFNy and TNF-alpha stimulated MSCs. The mechanism of action appeared to be via TGFβ expressed by MSCs, similar to results seen in breast cancer cell lines. These findings were correlated to clinical samples revealing over-expression of SSEA-4  was found to confer a shorter cancer-free interval and worse overall survival .While MSCs have the ability to initiate EMTs, they also differentiate into other cell types within the TMEN to potentiate their action as tumor promoters. One particular cell type, the cancer-associated fibroblast (CAF) has garnered significant attention as a target for cancer therapy . CAFs arThe most significant challenge of designing therapies to eliminate CAFs are their similarities with myofibroblasts – which are essential for wound healing. Predictably, the pathways used in cancer progression are also essential for wound healing . OsteopoDespite these inherent challenges, strategies to inhibit CAFs have been tried at numerous potential points of intervention. Imatinib mesylate, a tyrosine kinase inhibitor currently used in the clinical setting for treatment of numerous malignancies, decreased the viability of CAFs in vitro by inhibiting expression of alpha-SMA . AnotherAnother potential site for intervention within the TMEN include blocking the interaction between MSCs and cancer cells by preventing MSC recruitment to tumor sites. Zoledronic acid (ZA), a bisphosphonate, appears to show promise in this role. Specifically, in a breast cancer model, treatment with ZA prevented MSC migration by decreasing the expression of RANTES and IL-6, both markers for MSC migration. These data suggest a role for ZA as an adjunctive therapy to conventional chemotherapeutics .EMT plays a considerable role both in normal development and diseased states. Understanding the interplay between EMT and development, both embryologically and in wound healing, has offered unique perspectives in determining the key regulatory steps for progression to disease. Shared molecular pathways amongst all EMTs allows for the development of novel therapies to potentially halt or reverse EMT prior to metastatic spread. Small molecule inhibitors and exogenous compounds appear to offer the most promise at this stage.Additionally, the interplay between the TMEN and induction of EMT creates yet another opportunity to design targeted therapies in cancer. Modulating the key effectors within the tumor stroma is an area ripe for drug development. Undoubtedly, EMTs and their regulators have significant clinical implication across the oncologic spectrum.EMT: Epithelial-mesenchymal transitionTGF: Transforming growth factorEGFR: Epidermal growth factor receptorECM: Extracellular matrixIL: InterleukinMAPK: Mitogen-activated protein kinasenAChRs: Nicotinic acetylcholine receptorROS: Reactive oxygen speciesmiR: micro-RNAALK5: TGFβ type 1 receptorCK2: Protein kinase 2INF: InterferonThe authors declare that they have no competing interests.AK and PK conceived the format for the review and participated in its design and drafting. Literature and data acquisition were conducted by AK. Final manuscript editing and review were conducting by PK. All authors read and approved the final manuscript.


Aberrant levels of circulating miRNAs are potential biomarkers for the early detection of colorectal cancer (CRC). However, no previous systematic study has examined miRNAs in various specimen types from the same patient to evaluate their clinical utility. In this study, we compiled information from ∼450 articles published before 2012, and selected the 46 most frequently reported CRC-related miRNAs as candidates. We then established a 46-miRNA multiplex RT-qPCR method, and efficiently examined two clinically accessible samples: stool from fecal occult blood test and EDTA plasma. A total of 62 tissue, 447 stool, and 398 plasma samples were collected from CRC patients and healthy controls. Good correlations of detectable miRNAs were noticed in paired tumor tissues, stool, and plasma samples of 62 CRC patients. Using these 62 CRC patients and 62 matched healthy controls as the training set, 5 and 11 differentially expressed miRNAs achieved the area under the ROC curve (AUC) greater than 0.7 in stool and plasma samples, respectively. The selected miRNAs was subsequently validated using the remaining enrolled samples as the test cohort; 4 miRNAs in stool and 6 miRNAs in plasma were maintained discriminating powers for CRC patients. After examining the complementary effect, combined analysis of miR-223 and miR-92a, which were commonly present in stool and plasma samples, yielded the highest sensitivity of 96.8% and the specificity of 75% for CRC (AUC = 0.907). These results allowed us to establish a two-miRNA biosignature in two types of CRC clinical specimens with a high sensitivity for CRC detection. Colorectal cancer (CRC) has a high worldwide prevalence rate and a 5-year survival rate around 60% . CRC ariMicroRNAs (miRNAs) are a group of short non-coding RNA molecules that are encoded in genomes. Each miRNA negatively regulates hundreds to thousands of protein-coding genes to govern various biological processes, including development, differentiation, and proliferation . AberranSince 2001, more than 500 studies have examined CRC-related miRNAs. Beginning in 2008, some studies began to explore the potential of using miRNAs in blood and stool for the early detection of CRC and the prediction of its prognosis and therapeutic responsiveness . Most suIn the present study, we comprehensively and critically surveyed the published literature related to CRC-associated miRNAs, selected 46 potential miRNA candidates, and assembled a panel for CRC detection. We established a multiplex RT-qPCR platform and used it to quantify the 46 miRNAs in paired CRC and adjacent normal tissues. We linked the expression level of each miRNA in plasma and stool samples with that in the primary tumor, and selected the most cancer-relevant miRNAs. We then evaluated the performances of the selected miRNAs in retrospective case-control cohort samples. To minimize cost and maximize the clinical applicability, we excluded miRNAs that failed to show any mutual complementary effect in enhancing the rate of cancer detection. Finally, we used a logistic regression algorithm to integrate the expression levels of the selected miRNAs in plasma and stool samples, to aid in sensitively identifying patients at high risk for having CRC.To select CRC-related miRNA candidates from the literature, we conducted an extensive review, selected the most frequently reported miRNAs, and verified them with clinical specimens from CRC and control groups, using training and test steps -PCR, with externally spiked-in cel-miR-238 applied as an internal control for between-sample normalization of expression levels. The linearity of the quantitative curves for the 46 miRNAs were evaluated from 5 to 5×10ue of 40 . No crosue of 40 . The effue of 40 . The dupue of 40 , demonstTo examine the expression signatures of the 46 selected CRC-relevant miRNAs and their correlations in different sample types, paired tumor/adjacent normal tissue, stool and plasma samples were collected from 62 CRC patients. As controls, 62 stool and plasma samples were collected from 84 age- and gender-matched healthy individuals with negative colonoscopy results. In the training set, the distributions of age and gender were similar between the cancer and control groups Table . AnotherThe levels of the 46 candidate miRNAs were measured in 62 paired tumor and adjacent normal tissues. Principle of component analysis (PCA) revealed that the expression patterns of the miRNAs in tumor tissues could be easily distinguished from those in their normal counterparts Figure . CompareR = 0.73), tissue and plasma (R = 0.72), and plasma and stool (R = 0.88). This rationalizes that such circulating miRNAs might be clinically applicable for CRC detection.We also compared the expression levels of the 46 miRNAs in stool and plasma samples obtained from the same patients. Interestingly, when we ranked the expressions of these miRNAs based on the average copy number across the 62 samples, the rankings were similar across the three specimen types Figure . MiRNA-9The mean level, fold-change and area under the curve (AUC) for the receiver-operating characteristic (ROC) curve of 28 detectable miRNAs in stool and 41 detectable miRNAs in plasma for discriminating CRC patients from healthy controls in training set were calculated. Table To further clarify if there could be a complementary effect of detecting miR-223 and miR-92a in the stool and plasma samples of a single patient, we investigated their expressional distribution in 62 CRC patients and 40 healthy controls for whom paired stool and plasma samples were available. Figure In the present study, we collected a myriad of CRC-relevant miRNA biomarkers published in the literature and explored their clinical applicability for CRC detection. Forty-six miRNAs were carefully selected, and a multiplex RT-qPCR test was successfully developed to quantify the expression levels of these miRNAs in non-invasively obtained specimens. Using paired-tissue, stool and plasma samples from 62 CRC patients, we show for the first time that there is a strong correlation of the relative concentrations of these 46 miRNAs in the three specimen types. Using case-control cohort samples, we found that two common miRNAs, miR-223 and miR-92a, performed best as biomarkers for detecting CRC. This suggests the novel concept that the detectable miRNAs in stool and plasma samples may be complementary in their relevance as clinical biomarkers.via an interaction with its target, PTEN [Numerous markers have been reported for CRC detection, but few have moved into clinical application. One of the major concerns is whether the biomarkers are derived from the CRC tumors. Our present study solves this by: (1) establishing the expression profiles of the 46 candidate miRNAs and showing that they can be used to discriminate CRC tumors versus adjacent normal tissues, which suggests that these miRNAs may be strongly associated with CRC carcinogenesis; (2) showing that the expression levels of these miRNAs are correlated in paired-tissue, stool, and plasma samples from the same patients, which supports the feasibility of detecting CRC by assessing such specimens for the selected miRNAs; and (3) validating our method in clinical samples that are convenient to access in the current clinical setting, which provides evidence that our technique could be moved into clinical application. MiR-223 and miR-92a were initially selected as candidates because they were previously demonstrated to participate in CRC carcinogenesis, and have been reported as diagnostic biomarkers in either stool or plasma. MiRNA-223 is an onco-miRNA; it has been shown to target survival- and death-related genes, and its knockdown was shown to decrease cell proliferation, migration and invasion in CRC cells , 12. MiRet, PTEN . Severalet, PTEN , 15 or set, PTEN -18 in CRvia exosomes or apoptotic processes [As miRNAs may be secreted from the tumor site into the bloodstream rocesses , high barocesses , 21 or frocesses , which crocesses . This coThe amount and stability of miRNAs are two major factors that contribute to the success of miRNA measurements, especially in stool samples. We found that the concentrations of detectable miRNAs were 10- to 100-fold lower in plasma and stool compared to the sampled tissues. Thus, it is important to select more abundant  miRNAs when seeking to identify biomarkers. Wu's group recently reported that miR-31 and miR-135b were the most highly upregulated miRNAs in CRC tissues, and that miR-135b in stool yielded a CRC detection rate of 76% . These tUnder most circumstances, age is a major determinant for cancer screening; the elderly usually have a higher positive rate, and should be given priority for screening . In our Compared to other available non-invasive CRC screening tests, the 96.8% sensitivity obtained by examining the combination of miR-223 and miR-92a in stool and plasma is superior to those obtained using the FOBT (around 70∼75% ) or the In summary, we herein propose and support the use of a two-miRNA panel consisting of miR-223 and miR-92a. Quantitation of these two miRNA levels in both stool and plasma can cost effectively yield a high detection rate for CRC. To further validate the effectiveness of this miRNA panel in the future, an independent prospective cohort of patients with advanced polyps should be recruited.This study included 291 patients with CRC diagnosed between February 2012 and October 2013 at Chang Gung Memorial Hospital in Taiwan. Patients with family history and concomitant tumors were excluded. Tumors were staged according to the 2009 American Joint Committee on Cancer (AJCC) staging criteria (7th edition) . PatholoFor the control group, we recruited 452 volunteers from the Health-Check Center at Chang Gung Memorial Hospital. All participants were self-reported as negative for any colorectal cancer history or inflammatory bowel disease, and reported having negative colonoscopy results . Due to the younger age of the control group, we tentatively selected 84 elder healthy individuals to match the age and gender distribution in the CRC training group. Among them, 40 participants provided both stool and plasma specimens; these paired normal samples were used to evaluate the specificity of the combined use of miRNAs in stool and plasma. The remaining participants were placed in the test group; 247 provided stool samples and 121 provided plasma samples, with no overlap. All patients and healthy individuals provided written informed consent. The study was approved by the institutional review board of Chang Gung Memorial Hospital .g for 10 min, and the obtained plasma was aliquoted into several tubes for further use. Stool samples were dipped with a designated FOBT swab and inserted into preservation buffer in FOBT tube . After being vortexed thoroughly, buffered stool samples were squeezed out of the device and collected for storage. Fresh tumor and adjacent normal (at least 5 cm away from the tumor) tissue samples were taken from CRC patients during surgery. In the control groups, EDTA-plasma samples left over from the routine complete blood count test and residual samples from the FOBT were collected within 4 h after routine laboratory examination. All samples were stored at −80°C until use.EDTA blood or fresh stool samples were collected from CRC patients prior to bowel preparation and surgery. Blood samples were centrifuged at 20003) were placed in tubes containing QIAzol Lysis Reagent and 7-mm stainless steel beads  and homogenized using a TissueLyser II (Qiagen) at 30 Hz until the tissue debris became invisible. Plasma samples with a hemolysis titer higher than “trace,” as inspected by the color chart, were discarded. Plasma and stool samples were subjected to high-speed centrifugation at 12000g for 5 min to remove debris. 300 μL supernatants of centrifuged stool and plasma samples were subsequently added with 107 copies of synthetic RNA corresponding to Caenorhabditis elegans miR-238 (5′-UUU GUA CUC CGA UGC CAU UCA GA-3′)  as the spike-in control and 700 μL QIAzol Lysis Reagent. Total RNA was extracted from above pretreated stool, plasma, or homogenized tissue samples by miRNeasy Mini Kit according to the manufacturer's protocol (Qiagen). The obtained total RNA was dissolved in 30°C μL RNase-free water and stored at −80°C.Tissue samples (2 mmhttp://www.mirbase.org). Tissue RNA (500 μg), stool RNA or plasma RNA (5.4 μL each) were subjected to RT using a TaqMan miRNA Reverse Transcription Kit and TaqMan® MicroRNA Assays  according to a modified version of the manufacturer's protocol. Briefly, RT primers corresponding to the 47 miRNAs from TaqMan® MicroRNA Assays  were mixed together to convert the miRNAs into their corresponding cDNAs in a single reaction, and individual PCR was performed using the following cycling conditions: 16°C for 30 min, followed by 50 cycles at 20°C for 30 s, 42°C for 30 s, and 50°C for 1 s, and a final step of 70°C for 10 min. The products were diluted 5-fold with 0.1X TE buffer prior to quantitative PCR (qPCR).To quantify miRNA, we used the stem-loop reverse transcription-polymerase chain reaction (RT-PCR) principle  and miRN™ 12K flex real-time PCR system (Applied Biosystems). After qPCR, the Ct value of each miRNA was normalized to that of the spike-in control, cel-miR-238. The copy numbers of individual miRNAs were calculated from a standard curve plotted using 5 to 5 × 106 copies of synthetic cDNA oligonucleotides. The analytical detection limit was defined as a Ct value of 40; miRNAs with Ct > 40 were considered undetectable.Quantification of individual miRNAs was performed with the TaqMan Human MiRNA Assay (Applied Biosystems) according to the manufacturer's instructions, using a QuantStudiot tests and Mann-Whitney tests, and correlations between two groups were assessed by Spearman correlation. All statistical analyses, including plotting of whisker boxes, calculation of the area under the curve (AUC) of the receiver operating characteristic (ROC) curve for specific miRNA, and logistic regression, were conducted using PASW Statistics 18.0 . A P-value less than 0.05 (two-tailed) was considered statistically significant. A heatmap of miRNA expression in CRC tumor and adjacent normal tissues was constructed using non-supervised hieratical clustering and principal component analysis (PCA), as applied with Partek Genomics Suite 6.6 .This was a cross-sectional case-control cohort study. Descriptive statistics are summarized with means, standard deviations and percentages. Intergroup comparisons were conducted by paired


There have been major recent advancements in the understanding and management of multiple myeloma. Diagnostic criteria have been revised and former ultra-high-risk smoldering multiple myeloma is now considered multiple myeloma in need of treatment. Understanding clonal progression, evolution, and tides not only has helped elucidate the disease behavior but might help expand therapeutic choices in order to select appropriate treatment for patients. Unprecedented response rates with modern triplet induction therapies containing proteasome inhibitor and immunomodulators have made this approach standard for initial treatment. The US Food and Drug Administration approved four new drugs  in 2015 in relapsed/refractory multiple myeloma and these drugs along with the other already-available drugs have now increased the choices of regimens. Even drugs without single-agent activity, such as panobinostat and elotuzumab, have an important role, especially in the proteasome inhibitor refractory setting. Recent studies done in the context of novel agent induction suggest that high-dose therapy followed by autologous transplant continues to improve response rates and progression-free survival, thus underscoring their role in transplant-eligible patients. Evolving paradigms in the treatment of multiple myeloma include newer promising immune approaches, such as adoptive cellular therapies, vaccines, or antibody-based immune manipulations. Though multiple myeloma is still considered incurable, it is clear that with the improved understanding of disease biology and clonal architecture of relapse combined with the availability of multi-targeted approaches, we are ever closer to a lasting cure or transformation into indolent and long-lasting disease courses or both. In 2015, four new drugs—the first two monoclonal antibodies and two oral agents—were approved by the US Food and Drug Administration (FDA). Importantly, these drugs have improved the outcomes in relapsed and heavily pre-treated patients.Multiple myeloma (MM) is a clonal expansion of malignant plasma cells resulting in end organ damage, including lytic bone lesions, anemia, renal failure, or hypercalcemia. Both the understanding of MM and its management have evolved rapidly over recent years. Concomitant with the increasing incidence of MM to almost 30,000 patients annually in the US, mortality has trended down in the last decade, resulting in an increased prevalence of MM2. Currently, SMM is defined by the presence of serum M protein of at least 3 g/dl or 10 to 60% of clonal bone marrow plasma cells (or both) with no evidence of target organ damage, myeloma-defining events (MDEs), or amyloidosis2. Patients with high-risk SMM have a 25% risk of progression to MM in 2 years, whereas those with low risk have a 5% risk per year3. Multiple prognostic markers have been identified as surrogates for high-risk SMM9. Some of these include SMM with serum M protein of at least 3 g/dl, IgA isotype, involved/uninvolved free light chain ratio of at least 8 (but less than 100), bone marrow clonal plasma cells of 50 to 60%, t, del(17p), or 1q gain and Bence-Jones proteinuria. Management of high-risk SMM remains controversial after treatment with lenalidomide was shown to improve overall survival (OS) compared with a strategy of observation until target organ damage10. It is possible that SMM represents a curable stage of disease wherein early intervention approaches with limited toxicity may be curative and delay or avoid end organ damage provided that treatment does not lead to the outgrowth of more resistant clones. When compared with symptomatic MM, high-risk SMM can achieve a deeper response, as measured by minimal residual disease (MRD), with 11 out of 12 patients developing MRD negativity11. A longer follow-up is necessary to determine the duration of response, comparing it with the financial and physical burden of therapy. Thus, these treatments should be performed in clinical trials exploring the timing, the benefits, and the type of therapy in high-risk SMM.Smoldering MM (SMM) was traditionally defined on the basis of laboratory criteria—monoclonal (M) protein level of more than 3 g/dl or a clonal marrow plasmacytosis of more than 10%—and the absence of end organ damage. The ability to identify persons with SMM who are likely to progress rapidly to symptomatic MM has led to the revision of the criteria that define treatment initiation. Ultra-high-risk SMM patients with an 80% risk of progression to MM in 2 years are now considered in the updated criteria to have MM requiring treatment8.The revised International Myeloma Working Group criteria for the diagnosis of MM requires, in addition to the presence of either 10% or more clonal plasma cells or a biopsy-proven plasmacytoma, one or more MDEs. MDE includes CRAB features  and three specific biomarkers: (a) clonal plasma cells of at least 60%, (b) involved/uninvolved light chain ratio of at least 100, and (c) more than one focal lesion on magnetic resonance imaging. Each of these biomarkers was associated with an 80% risk of progression to symptomatic MM in 2 years in independent studies12.Staging of MM was recently updated to reflect the advances in treatment of MM as well as improved detection of high-risk disease. Revised-International Staging System (R-ISS) stage I disease includes ISS stage I with a normal lactate dehydrogenase (LDH) level and absence of high-risk cytogenetics (del(17p), t, or t). R-ISS III includes ISS stage III with either high-risk cytogenetics or elevated LDH. R-ISS II includes all others. The 5-year OS rates were 82% in R-ISS I, 62% in R-ISS II, and 40% in R-ISS III13. The progression to MM involves additional oncogenic events, including activation of oncogenesK-Ras,N-Ras,FAM46C,MYC, andBRAF and loss of function ofp53, epigenetic changes with histone methylation/deacetylation, and genomic instability15. No one common driver mutation has been identified in MM, although recurrent variants have been identified. Deregulation of the RAS/MAPK, NF-κB pathway, and apoptotic response are involved, but only the inability for effective apoptotic response to DNA damage has prognostic value15. A second study confirmed that the presence of the aforementioned oncogenes did not influence OS or progression-free survival (PFS)16. In MM, at least four distinct evolutionary patterns can be discerned between initial treatment phase and subsequent relapses. The simplest situation is no change in the clonal and sub-clonal composition between various time points in the natural course of disease, which represents re-emergence of the original clonal plasma cell population at relapses. About one-third of patients in this group have stable genomes and are typically associated with hyperploidy and favorable outcomes. The other three scenarios include “either differential clonal response or clonal tides” where in sub-clones are identified at different time points, but their relative proportions change over time or linear clonal evolution where a new (previously undetectable) sub-clone emerges later or a branching evolution pattern corresponding to the emergence of new clones with the disappearance of old ones17. In these three groups, selective therapeutic pressures can suppress responsive clones, allowing resistant clones to become dominant. Persons with high-risk disease are more likely to exhibit linear evolution or clonal tides. Clones with inherent genomic instability, detected at low levels at diagnosis, may undergo further genomic shifts when exposed to DNA-damaging agents, emerging after multiple relapses as the fatal clone. Understanding the clonal evolution pattern will become increasingly relevant as therapeutic choices expand in order to select appropriate treatment for patients. Re-emergence of a previously sensitive sub-clone would warrant the use of the same combination therapy at relapse. Alternatively, the emergence under treatment-induced selection pressure of a newly dominant linearly evolved or pre-existing resistant sub-clone should lead to alternate drugs.The initial event leading to malignant plasma cells occurs in the germinal center during isotype class switching and somatic hypermutation. These mutations are present in monoclonal gammopathies of unknown significance but are not sufficient for evolution to MMP = 0.0001) and OS 18. In newly diagnosed patients, the KRD triplet had a response rate of 98%, a complete response rate of 56%, and MRD negativity rate by next-generation sequencing of 58%, and no discontinuation due to adverse events11. Benefits of carfilzomib include rapid time to response, excellent tolerability (15% discontinued due to adverse events), efficacy in high-risk disease, and the low rate of carfilzomib-induced peripheral neuropathy. Challenges with carfilzomib include twice-weekly intravenous administration, the risk for tumor lysis syndrome, and the possibility for cardiac events and dyspnea. One retrospective study identified that more than 20% of patients developed systolic heart failure or arrhythmias. Previous irradiation to the thoracic spine and concomitant administration of doxorubicin increased the risk for cardiac dysfunction19. To overcome these challenges, recent trials have demonstrated tolerability and response with escalated doses20, whereas others are investigating efficacy of once-weekly dosing.Two new proteasome inhibitors (PIs) have been approved in recent years: carfilzomib (K) and ixazomib (I). Carfilzomib is an intravenous, irreversible PI with impressive outcomes in both newly diagnosed MM and relapsed MM. In patients with early relapse (one to three prior regimens), when compared with lenalidomide and dexamethasone (RD), carfilzomib in combination with lenalidomide and dexamethasone (KRD) improved PFS (hazard ratio (HR) 0.69,21. It has modest single-agent activity and response to bortezomib-exposed persons when combined with dexamethasone22. The combination of ixazomib with lenalidomide and dexamethasone (IRD) compared with RD shortened the time to response, improved the overall response rate, and increased median PFS 23. Early signal also suggests that it may overcome the negative impact of cytogenetic alterations. Major toxicities include gastrointestinal (GI) toxicity, peripheral neuropathy, and cyclical thrombocytopenia. The tissue distribution and dissociation half-life have a theoretical benefit with extramedullary disease, although this has not yet been demonstrated in a clinical trial. Ixazomib can be used as the proteasome backbone for combination oral therapies, reducing the need for frequent infusion visits. IRD is being investigated in newly diagnosed, transplant-ineligible persons with MM in a multi-center clinical trial, but results are not yet available. The availability of an oral PI also increases the feasibility of oral PI-based maintenance/consolidation regimens after transplant. Although it has been approved by the FDA, European regulators are waiting for additional positive data.Ixazomib is an orally bioavailable, reversible PI with higher tissue distribution and a shorter dissociation half-life than bortezomib25. Pomalidomide is an oral third-generation IMID approved for relapsed refractory MM after at least two prior regimens. In lenalidomide-refractory patients, including 75% who were dual refractory, the response rate for pomalidomide and weekly dexamethasone was 30% with a 4-month PFS26. In high-risk cytogenetics, patients with del(17p) had higher response rates, duration of response, and time to progression than those with t27. Pomalidomide is more effective earlier in the disease course28, although approximately 40% of bortezomib- and lenalidomide-refractory patients can have a prolonged PFS29. Toxicities, including cytopenias, rash, and peripheral neuropathy, are unpredictable30. Unlike prior IMIDs, pomalidomide causes less endothelial stress and may be associated with lower rates of venous thromboembolic events, although patients should be on prophylactic anti-platelet therapy or anticoagulation31. Its limited toxicity allows for combination with PIs, cyclophosphamide, and monoclonal antibodies, improving its efficacy.Immunomodulatory drug (IMID) can bind cereblon on the E3 ubiquitin ligase complex, enhancing the ubiquitination and degradation of two zinc finger transcription factors32. In bortezomib-refractory patients, the clinical benefit rate was better than 50% with a 4.2-month PFS. As high rates of GI distress and thrombocytopenia are seen with this agent, tolerability is a major impediment to its use and requires intense clinic/nursing support for continuous use. Dose interruptions occur in more than 50%, and relative dose intensity is only 73%. In a less heavily pre-treated population, response rates and PFS are improved, but the difficulty in maintaining dose intensity persists33. Nonetheless, panobinostat has a novel mechanism of action, is an oral agent, and continues to have an important role in the management of MM, especially in the PI-refractory setting.Panobinostat, a pan-histone deacetylase inhibitor (HDAC), targets HDAC enzymes involved in the aggresome protein degradation pathway, working synergistically with PIs. In a heavily pre-treated patient population, including 73% who were bortezomib-refractory, the response rate of panobinostat, bortezomib, and dexamethasone was 35% with a 5.4-month PFS35. In a phase III trial of patients with RRMM, the addition of elotuzumab to RD significantly improved PFS 35. In addition, it improves the PFS of the subsequent therapy, suggesting a lasting effect on natural killer (NK) cells. Single-agent daratumumab produced a response rate of approximately 30% in heavily pre-treated patients and little toxicity other than infusion reactions34. Another CD38 antibody, isatuximab (SAR650984), has shown similar single-agent activity in RRMM but is not commercially available36. Both elotuzumab and daratumumab are being investigated in the front-line setting, and given the unprecedented single-agent activity, it is likely that daratumumab might be the backbone of MM treatment, akin to rituximab in B-cell lymphomas. Emerging data point to impressive responses for anti-CD38 antibodies in combination with IMIDs and PIs.The FDA has recently approved two monoclonal antibodies in relapsed or refractory MM (RRMM) or both—elotuzumab and daratumumab—which target the signaling lymphocytic activation molecule F7 (SLAMF7) and CD38, respectively38. A recent study has shown that bortezomib-lenalidomide and dexamethasone (VRD) has superior OS compared with RD39. Bortezomib-thalidomide and dexamethasone (VTD) has shown superior response rates than thalidomide and dexamethasone (TD) and bortezomib-dexamethasone in randomized trials37. Bortezomib-cyclophosphamide and dexamethasone (VCD) was recently shown to have lower response rates than VTD40  has also shown excellent response rates in phase II trials42, but additional data and regulatory approval are necessary prior to broader use. Conversely, lenalidomide-based triplets failed to show a PFS or OS benefit over lenalidomide-based doublets in an elderly population43. When compared with doublets, triplet therapies are associated with increased toxicity and usually longer PFS but not necessarily OS. The upfront use of the most active agents may limit treatment choices in future lines of therapy, possibly shortening the second and subsequent PFS periods. Given the prolonged survival in patients with myeloma as well as rapidly changing treatment modalities, this debate may be difficult to resolve.When compared with doublets, triplet induction therapy containing a PI and an IMID results in a higher response rate and improved PFS and is preferred whenever possible in fit patients, especially given the synergism between these two drug classes VTD40 . Althoug44. Frail patients have shorter OS irrespectively of staging, treatment types, or physician-assessed performance status. Even when adjusted for International Staging System and chromosomal abnormalities, frail patients have shorter OS and PFS and higher rates of non-hematologic adverse events and treatment discontinuation and are likely to benefit from dose-adjusted therapy. Other validated frailty indexes evaluate similar factors as well as cytogenetics, renal function, and Karnofsky Performance Status45.Management of RRMM remains controversial, although the field is moving toward combination triplet therapy at first relapse in fit patients. In the US, an abundance of second-line therapies are available, allowing individualization of therapy to both the patient and the disease. In older persons with RRMM, a geriatric assessment should be performed to determine a frailty score, evaluating the Katz Activities of Daily Living, the Lawton Instrumental Activity of Daily Living, and the Charlson Comorbidity Index46. The addition of bortezomib (intravenous) to TD prolongs time to progression  in previously transplanted patients and results in a trend toward improved 24-month survival 46. However, this population is not typical for the US, since only 20% were previously treated with bortezomib and 8% with thalidomide. Although VTD may have a limited role in the US, this study does illustrate the benefit of triplet therapy. The addition of carfilzomib to lenalidomide and dexamethasone improves not only response rates and PFS  but also 24-month survival 11. The data from other recent trials of triplets in early relapsed disease, though clearly demonstrating PFS benefit, did not show an OS advantage.The addition of a third drug to an IMID steroid combination improves response rates and PFS, although an OS benefit is not demonstrated consistentlyThe choice of treatment at relapse is determined by the type, response, and duration of initial therapy as well as disease factors, patient factors, and patient preference. The ideal sequence of therapies is not known, but switching drug classes is likely beneficial, especially in higher-risk disease, given the evidence for clonal tides. For instance, if there is disease progression while on lenalidomide maintenance, a triplet containing lenalidomide would likely provide a suboptimal response. If available, carfilzomib/IMID triplet should be considered in aggressive relapses, given the excellent data with rapid induction of response and high rates of MRD negativity. Those who relapse following a short duration of remission after upfront therapy, including autologous stem cell transplant, or symptomatic relapses with significant clinical symptoms or disease relapse in high-risk cytogenetics are generally considered to be aggressive relapses. Elotuzumab-based treatment could be considered in individuals who are frail or have underlying cytopenias. An ixazomib-based regimen is an option if an oral regimen is desired and, based on early trends, may be considered, especially in del(17p) or extramedullary disease. A pomalidomide-based regimen could also be considered in del(17p). Panobinostat has a limited role in first relapse because of the toxicity profile unless used to restore PI sensitivity in those relapsing after prior PI regimens.48. In one of the studies designed to address the question of early versus late ASCT49, patients undergoing ASCT were found to have longer time without symptoms and treatment, and thus the patients with ASCT were found to have improved quality of life.High-dose therapy followed by autologous stem cell transplantation (ASCT) was the standard of care for transplant-eligible patients before the era of novel agents. In pre-novel agent randomized trials, ASCT was uniformly associated with improved response rates and PFS, and about half of the studies showed an OS benefit and superior quality of life52. Novel agents have significantly improved the rate of complete response and MRD, both surrogates for improved survival54. The early versus late ASCT was investigated in a number of randomized trials56, most recently in an international study conducted in the context of modern therapy with VRD induction, ASCT, and lenalidomide maintenance57. Superior OS benefit was seen in one study56 whereas in the other two studies, early ASCT was found to have better PFS with no transplant-related deaths57. Maintenance therapy after ASCT is gaining increased acceptance. Two randomized trials demonstrated a PFS benefit to lenalidomide maintenance, but only the Cancer and Leukemia Group B (CALGB) trial demonstrated an OS benefit59. The difference in results could be explained by the use of two cycles of consolidation chemotherapy after ASCT and only one year of maintenance lenalidomide in the IFM (Intergroupe Francophone du Myelome) trial. In addition, the majority of subjects in the CALGB study, unlike the IFM trial, received a lenalidomide-based induction regimen. In addition, an unplanned subset analysis in the CALGB trial failed to show an OS benefit in those who did not receive a lenalidomide-based induction regimen. Likewise, bortezomib maintenance can improve PFS and possibly OS60. Further support for maintenance comes from the observation that maintenance lenalidomide, despite ASCT and consolidation therapy, can further deepen response, when measuring for MRD by flow61. Last, newer data demonstrate the benefit to continuous therapy. A meta-analysis of three trials, one of which included ASCT as part of consolidation, demonstrated improved PFS1, PFS2 (time to second progression of death), and OS with continuous therapy. In fact, a prolonged PFS1 did not reduce PFS262. In conclusion, novel agent-based triplet induction, front-line early ASCT, and maintenance therapy remain the preferred strategy for transplant-eligible patients.Given the unprecedented response rates with novel agents, the role of ASCT as front-line therapy has been questioned63. A series of prospective randomized studies, both in the US and Europe, explored the role of a tandem ASCT and reduced-intensity conditioning allogeneic stem cell transplants from a matched sibling or unrelated donor65. The results of these studies have been discordant, especially in the front-line treatment of MM68. Nonetheless, younger eligible patients with well-defined high-risk features should be considered for allogeneic transplant, especially within a clinical trial and early in the clinical course (front-line or first relapse). The risks of chronic graft-versus-host disease (cGVHD) and the need for long-term immunosuppression remain major challenges. A number of studies suggest that immune therapy works in MM and these include demonstration of clinically meaningful GVM effect by donor lymphocyte infusions in patients relapsing after allogeneic transplant69, complete remission after immunosuppression withdrawal62, progression after allogeneic transplant, and the presence of cGVHD correlating with freedom from progression after allogeneic transplant64.The best-established immunotherapy in MM is allogeneic transplantation with a potent and often sustained graft-versus-myeloma (GVM) effect70, redirected T cells with chimeric antigen receptors72, and tumor-based vaccines to induce myeloma-specific immunity in the context of enhanced antigen presentation73. A phase I/II study showed that adoptive transfer of tumor antigen vaccine-primed and co-stimulated T cells leads to augmented and accelerated cellular and humoral immune reconstitution, including antitumor immunity, after ASCT74. In another phase II trial, idiotype-pulsed antigen-presenting cells immunotherapeutic called APC8020 (Mylovenge), which was given after ASCT for MM, appeared to be associated with improved OS75. In a recent report, deep sustained response to anti-CD19 chimeric antigen receptor T cells was reported in a patient with relapsed/refractory disease76. Several promising antigenic targets have been identified for the development of anti-MM chimeric antigen receptors, such as B-cell maturation antigen, CD138, kappa light chains, and CS-1. Inhibitors of PD-1 and PDL-1 are being studied as a means of breaking down MM immune tolerance77. Though the anti-PD1 agent nivolumab was unimpressive78, promising response rates were observed in combination with lenalidomide79. NK cell therapy has generated interest in the treatment of MM and a number of studies are exploring this option. These include modulation of NK activity using anti-KIR Ab IPH2101  to establish MM-specific immunity80 safety and efficacy study of autologous-, allogeneic-, and cord-derived NK cells82. A clinical trial involving the haploidentical allo-stem cell transplant followed by planned NK cell infusion attempts to use donor-recipient KIR ligand mismatch and NK cell reactivity to facilitate long-term remission (NCT02100891). Another promising area is the use of tumor vaccine based on the study in which patients receiving a patient-specific dendritic cell/myeloma fusion vaccine demonstrated the expansion of MM-specific T cells as well as upgrading of response in a subgroup of patients73. A randomized phase II trial (BMT CTN 1401) with ASCT followed by lenalidomide maintenance with or without vaccination using the dendritic cell/myeloma fusion vaccine is currently accruing patients.A variety of novel post-ASCT or allogeneic transplant immunotherapeutic strategies are now being explored in MM. These include cellular approaches such as myeloma-specific T cells (via T-cell expansion), marrow-infiltrating lymphocytes79. Additionally, lymphopenia resulting from ASCT facilitates elimination of tolerogenic antigen-presenting cells and induces cytokine release that generates a more favorable environment for adoptive T-cell therapy. Indirect evidence suggests that the immune system can contribute to the clinical benefits of ASCT 83.Hematopoietic cell transplantation provides an ideal platform for additional immune-based therapies. The recovery phase from ASCT (or other lymphodepleting therapy) represents a favorable platform for adoptive cellular therapy. The homeostatic lymphocyte proliferation following lymphopenia is a context in which immune checkpoint blockers may also be able to reverse MM-associated T-cell exhaustion84. It is clear that with the improved understanding of disease biology and clonal architecture of relapse, combined with the availability of multi-targeted approaches, we are closer to a lasting cure than ever before. Nonetheless, many questions remain unanswered. Will the better understanding of myeloma biology guide the intensity of treatment, with high-risk disease necessitating aggressive therapy? With an increasing number of drug classes as well as new drugs in each class, the combination and sequencing of drugs are largely unresolved.Although MM has always been considered incurable, many investigators now believe that a significant fraction of patients currently undergoing therapy may be cured