Macaca fascicularis), in a series of studies primarily designed to characterize the influence of the dopaminergic ventral midbrain on auditory processing. Compared to its role in motor behavior, little is known about the sensory response properties of neurons in the red nucleus (RN); particularly those concerning the auditory modality. Sites in the RN were recognized by observing electrically evoked body movements characteristic for this deep brain structure. In this study we applied brief monopolar electrical stimulation to 118 deep brain sites at a maximum intensity of 200 μA, thus evoking minimal body movements. Auditory sensitivity of RN neurons was analyzed more thoroughly at 15 sites, with the majority exhibiting broad tuning curves and phase locking up to 1.03 kHz. Since the RN appears to receive inputs from a very early stage of the ascending auditory system, our results suggest that sounds can modify the motor control exerted by this brain nucleus. At selected locations, we also tested for the presence of functional connections between the RN and the auditory cortex by inserting additional microelectrodes into the auditory cortex and investigating how action potentials and local field potentials (LFPs) were affected by electrical stimulation of the RN.The response of neurons in the Red Nucleus pars magnocellularis (RNm) to both tone bursts and electrical stimulation were observed in three cynomolgus monkeys ( In chloralose-anesthetized cats, neurons in the rostral RN have been found to exhibit short-latency responses to clicks and tones  from the RN, and to characterize neuronal responses to clicks and pure tones. At selected locations, we also tested for the presence of functional connections between the RN and the auditory cortex by inserting additional microelectrodes into the auditory cortex and analysing how action potentials and LFPs were affected by electrical stimulation of the RN.Macaca fascicularis); monkey E  monkey W , and monkey M . Each monkey was fitted with a head holder and a recording chamber (18 mm diameter), which was positioned in the right (monkeys E and M) and the left (monkey W) temporal regions of the skull. Details of the surgery are given elsewhere .Through these electrodes, we simultaneously recorded multiunit activity using a band-pass filter set between 1 to 7 kHz, and LFPs with a band-pass of 0.1 to 250 Hz, using the systems SUA-02 and LFP-03 (Thomas Recording). All signals were fed into an AD data-acquisition system . The LFP sampling rate was 658 Hz, and for multiunit activity it was 50 kHz. We only stored time stamps of multiunit activity when spike amplitudes exceeded noise thresholds.Initially, MRI images were used to plan electrode trajectories toward the RN. As can be seen in Figure Multiunit activity and LFPs were recorded from the auditory cortex using a 16-channel microdrive . The microdrive was oriented slightly off the dorsoventral plane (10°). Electrodes entered the cortex between stereotactic coordinates A5 to A9 and D7.5 to D12.5 and subsequently were moved through the parietal cortex into the core fields of the auditory cortex.Monopolar electrical stimulation was delivered using a Multichannel Systems STG 400-4 stimulator, programmed to generate 50 ms trains of square edged biphasic pulses. These were presented at a frequency of 100 Hz, and a pulse width of 700 μs and at a maximum intensity of 200 μA, to evoke minimal body movements only. We presented acoustic stimulation in the form of clicks and tone bursts. Clicks were produced by a waveform generator  and presented every 2 s during each of the electrode tracks. Tone bursts were generated using a computer coupled to an array processor , which converted the signal from digital to analog at a sample rate of 100 kHz. Each frequency  was repeated 10 times, in pseudorandom order, from 0.11 to 27.2 kHz, covering a range of 8 octaves spaced by 0.2 of an octave. The tone burst envelope was 100 ms in duration, ramped by 5 ms with an inter tone interval of 900 ms. The auditory stimulus gain was increased using an amplifier , then fed to two bilaterally placed free-field loudspeakers (Canton Karat 720.2), positioned 1.1 m from the subject, at intensities not exceeding 65 dB (re. 20 μPa). The sound pressure level was measured with a free-field 1/2′ microphone , located near to the monkey's head.Custom written MATLAB  programs were used for the off-line analyses of multiunit and LFP recordings. In the RN, tuning curves were generated from each multiunit recording of responses to the tone bursts which were presented at 40 different frequencies, as described elsewhere . Figure When we recorded action potentials from these 118 stimulation sites, we found that 18 sites (14 in monkey E and 4 in monkey W) responded to the click stimulation, which was presented every 2 s to the monkeys. With one exception, the auditory responses were found exclusively at the 86 sites where electrical stimulation evoked upper body movements. Thus, auditory responses were found at 19.7% of the sites in the RN. We also tested the click stimulation at another 335 sites (312 in monkey E and 23 in monkey W) at which electrical stimulation was not applied directly to reduce the number of electrical stimulations delivered to the monkeys, but which fell within 200 μm from a site from which upper body and face movements were elicited. Of these sites, most are presumed to be located in the RN; 67 sites  did respond to the click stimulation. Figure Indication for auditory responses in the RN was also obtained at 23 of 51 sites in a third monkey (monkey M). From this monkey, neuronal recordings were obtained from a brain region that corresponded to that tested in monkeys E and W, and following the same stereotactic coordinates used on the two monkeys. Electrical stimulation was omitted so long as the electrode was estimated to be within the RN, in order to reserve electrical stimulation exclusively for locations within the ventral tegmental area where the monkey would receive brain stimulation reward for bar pressing. In this experiment, the presence of auditory responses around the estimated depth of the RN was actually used as a landmark to determine the position of the proximal ventral tegmental area.Auditory sensitivity of RN neurons was analyzed more thoroughly at 15 sites (12 in monkey E and 3 in monkey W), where strong auditory responses to clicks were observed and from where upper body movements could be electrically evoked, or which fell within 200 μm from such a site. To achieve this, we presented 400 tone bursts over a range of frequencies from 0.11 to 27.2 kHz. Figures The auditory sensitivity of the RN was also detected in the LFPs that were recorded in parallel with the spikes. Figure In our sample, RN neurons had best frequencies that were distributed between 0.16 and 17.75 kHz  in the auditory cortex, electrical stimulation in the RN even resulted in an elevated firing of the neurons at a latency of around 50 ms and a duration of 90 ms  revealed that such stimulation always resulted in an electrically evoked potential in the auditory cortex, which is similar to the representative evoked potentials shown in Figure s Figure .This is the first time that auditory responses have been recorded from the RN of primates, and reveals that groups of neurons that control upper body movement also respond to acoustic signals. The auditory sensitivity observed here is generally in good agreement with that previously observed in chloralose-anesthetized cats (Massion and Albe-Fessard, The existence of responses to auditory and electrical stimulation in these brainstem nuclei is of considerable use, especially as a landmark when targeting proximal “harder to detect” structures, such as the ventral tegmental area. Our study also suggests a functional connection from the RN to the auditory cortex. Electrical stimulation of the RN-evoked post-synaptic potentials in the auditory cortex, which were suprathreshold in some neurons, thereby causing them to fire action potentials. The latter finding together with the short response latency of ~30 ms, suggests the presence of a projection from the RN to the auditory cortex that has a slow conduction velocity in the order of 1 mm/ms although the existence of this pathway still needs to be anatomically demonstrated.Our findings corroborate previous accounts that the RN provides a place for audiomotor interactions (Courville, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycobacterium tuberculosis (Mtb) proteins or immune epitopes has the potential to become the new frontier in the TB vaccine development field. Researchers have addressed this approach by fusing the immune epitopes of their vaccines with molecules such as interleukins, lipids, lipoproteins, and immune stimulatory peptides, which have the potential to enhance the immune response. The fused molecules are being tested as subunit vaccines alone or within live attenuated vector contexts. Therefore, the objectives of this review are to discuss the association of Mtb fusion proteins with adjuvants; Mtb immunogens fused with adjuvants; and cytokine fusion with Mtb proteins and live recombinant vectors expressing cytokines. The incorporation of adjuvant molecules in a vaccine can be complex, and developing a stable fusion with proteins is a challenging task. Overall, the fusion of adjuvants with Mtb epitopes, despite the limited number of studies, is a promising field in vaccine development.Several approaches have been developed to improve or replace the only available vaccine for tuberculosis (TB), BCG . The development of subunit protein vaccines is a promising strategy because it combines specificity and safety. In addition, subunit protein vaccines can be designed to have selected immune epitopes associated with immunomodulating components to drive the appropriate immune response. However, the limited antigens present in subunit vaccines reduce their capacity to stimulate a complete immune response compared with vaccines composed of live attenuated or killed microorganisms. This deficiency can be compensated by the incorporation of adjuvants in the vaccine formulation. The fusion of adjuvants with  Mycobacterium bovis BCG) did not have the capacity to eradicate the illness, and it persists as the second leading cause of deaths by infectious diseases, behind only AIDS , the development of a vaccine  and its host, the understanding of the bacteria’s immunogenic repertoire is of utmost importance for the development of an efficient vaccine. Antigens that are recognized by the host cells during active TB, when the bacilli are replicating, or during latent infection as well as those involved in the immunologic evasion mechanisms or the elicitation of CD4+ and CD8+ specific T cells are potential targets for immunologically controlling infections , several types of receptors have been described . The TLR family is the most abundant and diversified and present on antigen-presenting cells (APCs) and many other cell types not related to the immune system. Signaling through TLRs can result in two possible cascades: the first is dependent on the molecule MYD88 (for myeloid differentiation factor 88) and related to TLR1, TLR2, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9; the second is dependent on TIF (TIR-domain-containing adaptor-inducing interferon-β) and is associated with TLR3 and TLR4 . The recMtb antigens to PAMPs  is a substance purified and fractionated from the bark of the South American tree Quillaja saponaria. The known action of MPL is through TLR4, whereas QS-21 has no related TLR agonistic action  in combination with AS01B or AS02A, both vaccine formulations were shown to be safe and immunogenic, with memory cell generation (persistency was followed for 3 years) and the production of cytokines protective against Mtb by several cell populations, generating similar immune responses  and Mtb39 (Rv0125)] uses the adjuvants AS01B™ and AS02A™(an oil-in-water emulsion) was developed by GlaxoSmithKline (GSK). The abovementioned adjuvants are composed of MPL (3-deacylated monophosphoryl lipid A) and the detergent QS-21. MPL is a detoxified derivative of lipid A from the Gram-negative bacteria c action . In animesponses –30.Two other fusion proteins (Hybrid 4 and Hybrid 56), which are currently in clinical trials, are combined with the adjuvant IC31™. This adjuvant is made of two components, a TLR9 agonist (the oligodeoxynucleotide ODN1a) and an artificial antimicrobial cationic peptide (KLKL5KLK), which serves as a vehicle. Its mechanism of action is related to TLR activation within endosomes, and as such, IC31 is a good adjuvant for use in vaccines against intracellular microorganisms. This adjuvant was shown in several animal models to aid the skewing of the immune response toward Th1 and Th17, which is most likely associated with its adjuvant effect on dendritic cells, enhancing the expression of co-stimulatory molecules  and the expression of IL-12p40 .Mtb infection in mice, guinea pigs, and cynomolgus monkeys  in a stable emulsion] as an adjuvant. Baldwin et al.  reported monkeys . A pepti monkeys . HoweverMtb. TDB arose from changes in TDM, also known as cord factor, which is a potent inducer of Th1 type responses with restricted use in humans because of its toxicity. The effect of TDB was evaluated with the H1 fusion protein (Ag85B-ESAT-6) and shown to be a powerful aid in stimulating the cellular response of Th1 and Th17 populations as well as humoral immune responses  has drawn attention because it is a less toxic analog of TDM , a critical component of the cell wall of esponses , 37. Theesponses  and TDM esponses . It has esponses , 41.Mtb is a great source of PAMPs, as TLR2 is responsible for recognizing most of the mycobacterial lipid antigens such as lipoproteins, lipoarabinomannan (LAM), and other glycolipids. In addition to its pro-inflammatory action, TLR2 activation can result in the production of cathelicidin, a peptide with microbicidal function that also acts against intracellular bacteria  whose recognition is related to increased production of IL-12, which in turn stimulates T lymphocytes and NK cells to produce and secrete IFN-γ, the most important inducer of reactive oxygen and nitrogen species, which are key effector molecules to kill the bacilli   and CD44hi CD62Llo (effector memory) CD4+ T cells. Last, L91 was more capable than BCG of protecting vaccinated guinea pigs from Mtb challenge by reducing the bacterial load and the pathology -cysteine (Pam2Cys), has also presented favorable characteristics for a fusion vaccine against TB. Pam2Cys is a potent dendritic cell stimulator and has the ability to induce antigen cross-presentation. This synthetic molecule was derived from mycoplasma MALP-2 (macrophage-activating lipopeptide) . When a Pseudomonas aeruginosa can bind to TLR3/TLR4, and it has been demonstrated to be capable of supporting a strong Th1 response with the production of IFN-γ and TNF-α as well as the induction of IgG2a in a Leishmania major model of infection (P. aeruginosa OprI was fused to antigen 85A (Ag85A) from Mtb and introduced in Escherichia coli, which has the machinery necessary to N-acylate OprI. This fusion was used in a prime-boost strategy following vaccination with Ag85A DNA or BCG. The intranasal administration of the fusion with the DNA vaccine enhanced the immune response by potentiating the production of antibodies against Ag85A and enhancing IL-2- and IFN-γ-producing cells, but it was not able to improve its protective capacity. Similarly, the OprI-Ag85A fusion improved the Th1 response induced by BCG, but it was unable to increase its protective efficacy .Mtb antigens with cytokines have been shown to be immunogenic and to improve the response to BCG, only one of the studies we reviewed, using IL-15/Ag85B, induced levels of protection against bacteria that were better than those induced by the current vaccine. Additionally, two studies did not evaluate the protection against Mtb, which prevents a more thorough assessment of the real potential of the examined fusion vaccines because there is no consensus in the scientific community of a biomarker for protection.When using cytokines as immune stimulation components in vaccines, one expects that they will enhance the immune response due to their participation in the regulation of both innate and adaptive immune responses. In the published literature, IL-2, IL-12, IL-15, and GM-CSF have been reported as potential immune response stimulators. However, only the Hsp65 antigen fusion with IL-2 was used without being in the context of the BCG vector, and in that case, it was necessary to use a dimethyl dioctadecyl ammonium bromide (DDA) and MPL emulsion as an adjuvant to generate an efficient immune response, demonstrating the inability of a single cytokine to support an antigen-elicited response. Although fusions of Among the major obstacles faced in the search for adjuvants capable of stimulating Th1 type responses is the immunotoxicity generated by some molecules. Therefore, an adjuvant should be effective in assisting the generation of protective immune responses while inducing few side effects . HoweverMtb proteins or immunodominant epitopes have been evaluated as subunit vaccines for TB. Some adjuvants have been incorporated in the vaccine formulation without a physical association with the recombinant fusion proteins, while others have been incorporated in the backbone of the subunit vaccines. Other studies have evaluated recombinant BCG vaccines expressing both Mtb proteins and adjuvant molecules. The majority of the vaccine formulations were able to induce higher levels of Th1 and IgG2a responses, although not all of the vaccines discussed here presented better protection against Mtb than BCG.Several fusions of Mtb antigens with adjuvants can interfere with the induction of specific immune responses, but in most of the reviewed articles, the vaccine formulations did not offer improvements over the protection conferred by BCG. Thus, further studies are needed to develop an effective adjuvant with low toxicity to be used in vaccine formulations to control TB.The fusion of Ana Paula Junqueira-Kipnis designed the review and critically wrote and edited the manuscript. Lázaro Moreira Marques Neto critically wrote the draft. André Kipnis critically wrote the manuscript and edited the manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This phase 2 trial was designed to evaluate ixazomib, an orally bioavailable proteasome inhibitor, in patients with myeloma who have limited prior exposure to bortezomib. Thirty-three patients with relapsed multiple myeloma were enrolled. Ixazomib was given at 5.5 mg weekly for 3 of 4 weeks. Dexamethasone was added for lack of a minor response (MR) by end of cycle 2 or lack of a partial response (PR) by end of cycle 4 or for disease progression at any time. Median age was 69 years; patients had a median of two prior therapies (range 1–7). A grade 3 or 4 adverse event considered at least possibly related to drug was seen in 19 (59%) and 6 (19%) patients, respectively. The most common adverse events were thrombocytopenia, fatigue, nausea and diarrhea. Dexamethasone was initiated in 22 (67%) patients, 17 for not reaching the desired response and 5 for progression. Response (⩾PR) to single agent was seen in five patients within four cycles of therapy including three patients with PR, one patient with complete response (CR) and one patient with stringent CR. Six additional patients with either an MR (2) or SD (4) achieved a PR after addition of dexamethasone, translating to an overall response rate of 34%. However, the need for a clinic visit for subcutaneous bortezomib or intravenous carfilzomib adds to the disease-related burden for patients, especially those on long-term therapy.Proteasome inhibition has become an important therapeutic strategy in multiple myeloma (MM), for newly diagnosed as well as relapsed disease, and particularly in patients with certain cytogenetic abnormalities associated with aggressive disease behavior.15 Ixazomib citrate is a modified peptide boronic acid and is the citrate ester of ixazomib (MLN2238), the biologically active moiety. Ixazomib citrate rapidly hydrolyzes to ixazomib upon contact with aqueous solution or plasma. Ixazomib preferentially binds the β5 site of the 20 S proteasome at lower doses, with inhibition of the β1 and β2 sites at higher concentrations. Compared with bortezomib, nonclinical studies have shown that ixazomib has a faster dissociation rate from the proteasome. Ixazomib has demonstrated antitumor activity in a range of tumor xenograft models, including MM models.17 Preclinical studies have shown activity in myeloma cells resistant to bortezomib as well as synergistic anti-myeloma activity when combined with dexamethasone and lenalidomide. In clinical trials, ixazomib has shown promising activity as a single agent in patients with relapsed and refractory MM, with very low rates of peripheral neuropathy observed in the single-agent trials.20 Given that the majority of patients in the early trials had been exposed previously to bortezomib, we designed this trial to better understand the efficacy of single agent ixazomib in patients with relapsed MM with limited exposure to bortezomib and also to examine the utility of adding dexamethasone to ixazomib.Ixazomib citrate (MLN9708) is an investigational inhibitor of the 20S proteasome that represents the first orally bioavailable proteasome inhibitor to be evaluated for the treatment of MM.www.clinicaltrials.gov as NCT01415882.This open-label phase 2 study evaluated the safety, tolerability and efficacy of weekly oral ixazomib citrate in patients with relapsed MM who either had no exposure to proteasome inhibitors or had limited (no more than six cycles) exposure to a bortezomib-containing regimen. It also explored the utility of adding weekly dexamethasone to ixazomib in patients with suboptimal response to single agent ixazomib. The study enrolled patients from January 2012 to October 2012. The study was performed in accordance with the provisions of the Declaration of Helsinki, the International Conference on Harmonization, and the Guidelines for Good Clinical Practice, and with approval of the Mayo Clinic Institutional Review Board. The study was registered at The primary objective of the study was to determine the confirmed overall response rate (ORR) ) of ixazomib, used as a single agent in patients with relapsed MM, who were proteasome inhibitor naïve or had received less than six cycles of therapy with bortezomib, and were not refractory to bortezomib. The secondary objectives included assessment of ORR of ixazomib in combination with dexamethasone, when dexamethasone was added for lack of response or for disease progression, and measurement of event-free survival and overall survival following treatment with ixazomib with dexamethasone added for lack of response or progression.3, platelets ⩾75 000/mm3), hepatic  and renal (creatinine clearance ⩾30 ml/min) function. Patients with grade ⩾3 peripheral neuropathy or grade 2 with pain, grade >1 diarrhea, or who had major surgery or serious infection within 14 days prior to start of therapy were excluded. Patients receiving systemic treatment with strong CYP1A2 inhibitors or strong inhibitors/inducers of CYP3A within 14 days were excluded. Other factors that precluded participation in the trial included uncontrolled cardiovascular conditions , known human immunodeficiency virus positivity, known hepatitis B surface antigen-positive status, or known or suspected active hepatitis C infection, and known allergy to any of the study medications, their analogues, or excipients in the various formulations. Other comorbidities or severe pre-existing illness that in the treating physician's opinion could interfere with oral absorption and/or tolerance of ixazomib citrate excluded patients from participation.The study enrolled patients, 18 years of age or older, with MM that had relapsed after at least one previous therapy. Patients were required to have measurable disease , Eastern Cooperative Oncology Group performance status of 0–2, adequate hematologic  by end of cycle 2, lack of a PR by end of cycle 4 or if there was disease progression at any time. Dose modifications were made for ixazomib-related toxicities with successive reductions in its dose to 4, 3, 2 mg followed by discontinuation if the 2 mg dose was not tolerated. Patients who had confirmed progression despite addition of dexamethasone were taken off study.Prophylactic anti-emetics were not initially planned, but given the incidence of nausea among the initial 12 patients, the study was amended to allow prophylactic 5 HT3 antagonists prior to each dose of ixazomib. Prophylactic antidiarrheals were not used; however, the administration of antidiarrheals was allowed after infectious causes were excluded. Topical steroids and other symptomatic measures were permitted for management of any skin rash.Adverse events (AEs) were graded using the National Cancer Institute's Common Terminology Criteria for AEs, version 4.0. Myeloma disease response was done in accordance with the International Myeloma Working Group uniform criteria, incorporating the additional category of MR. All response categories required confirmation of the required tests with the exception of the bone marrow used for complete response (CR) determination. At any point in treatment, patients suspected of progressive disease had response assessments repeated to confirm disease progression, done at least 1 week apart.The primary endpoint of this study was the ORR with single agent ixazomib, where a success was defined as sCR, CR, VGPR or PR noted as the objective status on two consecutive evaluations while receiving single agent ixazomib. All patients meeting the eligibility criteria, who signed a consent form and received at least one dose of the drug were evaluable for response, with the exception of patients who are determined to be a major treatment violation. The sample size for the study was calculated using one-stage binomial design. With 29 evaluable patients, the study provided 91% power to test the null hypothesis that the ORR is at most 10% versus the alternative hypothesis that the ORR is at least 30%, with a one-sided significance level of α=0.06. An additional four patients were enrolled to account for ineligible patients and protocol violations, for a total of 33 patients. For toxicity assessment, all patients who received at least one dose of study drug were included in the analysis. Overall survival was defined as the time from study entry to death due to any cause. Event-free survival was defined as the time from study entry to disease progression while receiving ixazomib and dexamethasone, death due to any cause or subsequent treatment for myeloma. Patients who went off study but never received dexamethasone were censored at the off-study date.Thirty-three patients were enrolled, and one patient was considered ineligible and excluded from all analysis. The median age was 69 years and 53% were male. The median duration from diagnosis was 57 months (range 14 months to 12.3 years) and patients had a median of two prior therapies (range 1–7). The baseline characteristics at study entry are described in Overall, 11 (34%) patients achieved a PR or better across the entire trial, with or without the addition of dexamethasone, including one patient each with CR and sCR. An additional two patients achieved a MR. Dexamethasone was initiated in 22 (67%) patients, 17 for not reaching the desired response (MR by end of two cycles and PR by end of four cycles) and in five patients dexamethasone was added for progression. The timing of dexamethasone and the responses before and after addition of dexamethasone is detailed in The median event-free survival was 11.5 months  and 6 month overall survival was 93% . The evePatients received a median of eight cycles of therapy  across the trial; 19 and 16 patients received at least four and eight cycles, respectively and 12 patients stayed on trial for more than 12 cycles. Overall, 336 cycles of treatment were delivered, with a dose reduction required for 21 (6%) cycles. A median of 100%  of the intended dose of ixazomib was delivered, whereas a median of 100%  of intended dose of dexamethasone was delivered among the 22 patients started on dexamethasone. The median dose (per cycle) of ixazomib at the time of discontinuation among the 27 patients who have gone off therapy was 12 mg .An AE of any grade, that was considered at least possibly related, was reported in 100% of the patients. A grade 3 or 4 AE considered at least possibly related to drug was seen in 19 (59%) and 6 (19%) patients, respectively; there were no deaths on study. The most common AEs observed included thrombocytopenia, fatigue, nausea and diarrhea. Peripheral neuropathy possibly related to the drug was seen in eight patients (grade 1) and five patients (grade 2), respectively. Overall, 22 patients had dexamethasone added per protocol and 173 of the 336 cycles administered contained dexamethasone. We compared the toxicity profile between the cycles containing dexamethasone and those with ixazomib as a single agent. Less hematologic and gastrointestinal (GI) toxicity was seen in patients receiving the combination than in those receiving ixazomib alone .20 Subsequent studies have explored the combination of ixazomib with lenalidomide in newly diagnosed myeloma, where the combination had high efficacy as well as adequate tolerability to allow for long-term therapy as a maintenance agent.19 The current study was designed to ask two important questions: what is the single-agent activity of ixazomib among patients with limited prior exposure to proteasome inhibitors and the value of combining ixazomib with dexamethasone. The latter is particularly relevant, as most of the currently used combinations include corticosteroids, and such a two-drug regimen can have value in situations that calls for less intense and more convenient therapies as in older or frailer patient.Proteasome inhibitors have become an integral part of myeloma therapies in the upfront setting as well as in the relapsed setting. Increasingly, this class of drugs is being utilized in combination with other myeloma drugs, both new and old. Introduction of an oral proteome inhibitor can have a significant impact on the management of myeloma, as this would allow for all-oral combination regimens incorporating proteasome inhibitors and immunomodulatory drugs (IMiDs). Ixazomib has been studied as a single agent, given once or twice weekly, in the initial phase 1 studies.18 It is further highlighted by the deep responses of sCR and CR seen in one patient each. It is difficult to directly compare the proportion of responses between the two studies, given that patients in the current study had dexamethasone added for lack of adequate responses by two or four cycles of therapy or progression at any time. The response rates also need interpretation in the context of initial trials of bortezomib, where it was used as a single agent in patients with relapsed MM. In the phase 3 APEX trial, patients receiving bortezomib on days 1, 4, 8 and 11 of a 3-week cycle had overall best response rate of 38%, representing the best response across the trial in a group of patients with median of two prior therapies, which primarily included alkylators, anthracyclines, high dose therapy or thalidomide.6 Majority of the patients in the current study in contrast had been refractory to lenalidomide and had prior high dose therapy. The approach of adding dexamethasone for suboptimal response was also evaluated in the SUMMIT trial, where patients received bortezomib on days 1, 4, 8 and 11 of a 3-week cycle with dexamethasone for progressive disease after two cycles or stable disease after four cycles.21 The ORR was 27% with monotherapy and 18% of the 78 patients who had dexamethasone added had a MR or better. In the current study, we had more stringent criteria for addition of dexamethasone leading to earlier addition, and among the 22 patients, who received dexamethasone eventually, an additional four patients had a PR and another four had an MR. The ORR among the entire cohort with this on-demand approach of adding dexamethasone was 34% with a clinical benefit rate of 48% including the four patients with an MR. Responses did not differ significantly depending on prior exposure to bortezomib, but the overall numbers are low and it is important to note that both patients achieving a CR had no prior bortezomib exposure.The current study confirms the single-agent activity of ixazomib that has been observed in the initial phase 1 trial, where 24% of the patients treated at the 5.5 mg weekly dose of ixazomib had a PR or better.20 We noticed more nausea in the beginning of the study and instituted prophylactic anti-emetic prior to each dose, which abrogated this problem in the majority of the patients. Interestingly, the overall GI toxicity was less in the cycles that also included dexamethasone, which may reflect the anti-emetic effect of the drug or the fact that dexamethasone was added in later by which time the GI toxicity may have been managed more efficiently. The thrombocytopenia was typically noted mid-cycle and recovered prior to initiation of a new cycle in the vast majority of patients, and no cumulative effect was seen across the study. Overall, the frequency of dose reduction was low at 6% across the 336 cycles over the study. Rate of peripheral neuropathy was low and predominantly grade 1 or 2; eight patients with grade 1 and 5 patients with grade 2.The main toxicities that we observed here are in line with the previous experience with this drug, and included nausea, thrombocytopenia and fatigue.In conclusion, ixazomib has promising single-agent activity in relapsed myeloma along with a favorable toxicity profile. Addition of dexamethasone significantly enhances the response rates demonstrating an important role for this two-drug combination. Given the convenience of oral route and once weekly dosing, this regimen can have a role in the management of the older patients and the more frail patient as well as in patients with more indolent relapses. Given the results with delayed addition of dexamethasone, it is likely that initiation of therapy along with dexamethasone can lead to higher response rates as well as deeper responses. Another cohort of patients is being enrolled currently, to examine the utility of adding dexamethasone from the beginning.


Falls while walking are frequent in patients with muscular dysfunction resulting from neurological disorders. Falls induce injuries that may lead to deconditioning and disabilities, which further increase the risk of falling. Therefore, an early gait stability index would be useful to evaluate patients in order to prevent the occurrence of future falls. Derived from chaos theory, local dynamic stability (LDS), defined by the maximal Lyapunov exponent, assesses the sensitivity of a dynamic system to small perturbations. LDS has already been used for fall risk prediction in elderly people. The aim of the present study was to provide information to facilitate future researches regarding gait stability in patients with neurological gait disorders. The main objectives were 1) to evaluate the intra-session repeatability of LDS in patients and 2) to assess the discriminative power of LDS to differentiate between healthy individuals and neurological patients. Eighty-three patients with mild to moderate neurological disorders associated with paresis of the lower extremities and 40 healthy controls participated in the study. The participants performed 2×30 s walking wearing a 3D accelerometer attached to the lower back, from which 2×35 steps were extracted. LDS was defined as the average exponential rate of divergence among trajectories in a reconstructed state-space that reflected the gait dynamics. LDS assessed along the medio-lateral axis offered the highest repeatability and discriminative power. Intra-session repeatability (intraclass correlation coefficient between the two repetitions) in the patients was 0.89 and the smallest detectable difference was 16%. LDS was substantially lower in the patients than in the controls . LDS measured in short over-ground walking tests seems sufficiently reliable. LDS exhibits good discriminative power to differentiate fall-prone individuals and opens up the possibility of future clinical applications for better prediction of fall risk in neurological patients. In patients with muscular dysfunction associated with a central nervous system pathology, the maintenance of optimal mobility in daily living activities is of utmost importance The occurrence of a single fall can trap the patient in a vicious circle: the injuries due to the fall can lead to transient disabilities, deconditioning, and fear of falling, which decrease walking capabilities and hence further increase the risk of falling. Consequently, it is of paramount importance to identify patients at high risk of falling even before falls are reported. Furthermore, practical and efficient early detection must rely on rapid and simple tests that can be administered by medical staff in clinical settings. An interesting candidate to fulfill those requirements is local dynamic stability (LDS). This index, derived from chaos theory, assesses the sensitivity of gait to small perturbations that occur naturally during walking, due to either internal causes (neuromuscular noise), or small external disturbances (such as floor irregularities). LDS can be estimated from a simple measure of gait dynamics, such as trunk acceleration The objective of the present study was to provide preliminary information to facilitate future research on gait stability –assessed with LDS– in patients with neurological gait disorders. We studied 40 healthy individuals and 83 patients with mild to moderate neurological gait disorders associated with paresis of the lower extremities. Tridimensional trunk acceleration was measured during short over-ground walking tests. First, in order to complement the results obtained in treadmill walking Patients who underwent a gait analysis between July 2007 and November 2013 were retrospectively selected from the database of the clinical gait analysis laboratory at the Clinique romande de réadaptation . The patients consulted because of gait disorders related mainly to paresis of various etiologies. In addition to a short walking test, the patients underwent several analyses , carried out by the same team of physiotherapist and supervised by two skilled neurologists. Inclusion criteria were (1) less than 65 years of age; (2) diagnosed with a central nervous system disorder; (3) reduced walking capabilities; and (4) able to walk independently and continuously for one minute at a minimal cadence of 84 steps/minute, which corresponds to 35 steps in 25 seconds. Among 260 eligible patients, 83 met the inclusion criteria. The reasons for exclusion were (in order of importance): (1) incomplete data ; (2) insufficient walking capability ; (3) age; (4) inappropriate pathology . The included patients, whose characteristics are summarized in In order to compare the patients with healthy controls, we analyzed 40 healthy individuals; their characteristics are also reported in During a short walking test, trunk accelerations were recorded with a tri-axial accelerometer that was attached with an elastic belt at the level of the L3–L4 spinous process and connected to a lightweight data logger . The accelerometer measured the body accelerations along three axes: medio-lateral (ML), vertical (V) and antero-posterior (AP). The patients were instructed to walk straight ahead, while barefoot, at a self-selected comfortable walking speed, along a 70-meter hallway for a period of at least 30 seconds. The measurement was performed twice, wearing the accelerometer continuously in order to ensure a consistent placement of the sensor. The same procedure was carried out with the healthy controls. The subsequent data analysis was performed with Matlab R2013a .The first 5 s of the raw acceleration data were discarded to rule out starting effects. In order to mitigate sensor placement variability among participants, the 3D-acceleration signals were reoriented according to a standard procedure 0.5) or over the duration of one stride (λ1). Because of the resampling to a uniform sample length, a constant number of samples corresponding to one step (114) or one stride (228) was used for the linear fitting.We thoroughly described the theoretical background behind LDS methodology in a recent study 0.5) in ML, V and AP directions; and LDS computed over one stride (divergence exponent λ1) in ML, V and AP directions.Seven dependent variables were analyzed in the patients (N = 83) and the healthy controls (N = 40): cadence (or step frequency); LDS computed over one step  and absolute  intra-session repeatability were assessed for each dependent variable, separately for healthy controls and patients . The ICCThe two within-subject repetitions were then averaged for the other analyses. Boxplots (median and quartiles), means and standard deviations (SD) were used to describe the distribution of the dependent variables among participants  and 3. T0.5) exhibits a higher repeatability than LDS measured over one stride (λ1), both in terms of relative reliability  and in terms of absolute reliability . The neurological patients presented higher ICCs than the healthy controls, mainly due to the larger inter-individual variability among them , and particularly low SDD in the controls and patients .The reliability results  show thathem see  and 3. TAs showed in 0.5, which indicates that the dynamic stability was reduced by 33% in the neurological patients. On the other hand, λ1 exhibited a smaller difference, with, for example, only 10% of relative difference in the vertical direction.2 is 6%. Furthermore, over 12 correlations only five were significant. Nonetheless, a trend to a positive association between cadence and LDS is apparent in healthy controls. It is also possible that in the patients, there was a negative association between the LDS measured along the vertical axis and the cadence.The correlation analysis  highligh0.5-ML) is likely the most suitable stability index, given the reliability  wearing a 3D accelerometer that measured trunk acceleration. Gait stability was assessed using a methodology derived from chaos theory . In summary, the results indicate a good repeatability of the LDS  tract. This clinical picture is found in a substantial part of the neurologically impaired population with gait disorders; however, other pathologies  should be investigated in future studies.Several studies have highlighted decreased gait stability in older age 0.510.5 exhibited better precision. The use of λ0.5 is further supported by the evidence presented here: it is confirmed , there is no correlation between cadence and LDS in patients (r = −0.08). In other words, if a patient exhibits a high cadence , nothing can be predicted about his/her dynamic stability. Therefore, LDS is effective for measurement and cadence does not constitute a confounding factor. This finding should be put into perspective with the results of our recent study, which showed that LDS was more responsive (larger effect size) than cadence or speed in discriminating therapeutic effects in patients with multiple sclerosis more stable than individuals, who walk with a faster cadence. Similar speed dependence has been described in treadmill experiments less stable  the measure should be simple to perform and the computing should be straightforward, and (2) the results should be reliable , (3) valid (representative of the investigated issue) and (4) easy to interpret . Given those requirements, the present study provides the following information.Short walking tests are easy to perform, and are short enough (about one minute) not to exacerbate pain and to keep a low level of fatigue; furthermore, the accelerometer is easy to attach (simple belt) and to use (start/stop button). However, the computing of LDS still requires custom software and programing skills.The reliability of LDS, estimated in healthy controls as well as in neurological patients, seems sufficient to assess small differences between groups. However, longer walking tests (more repetitions) are needed to detect small changes at the individual level. Furthermore, given the substantial day-to-day variability of LDS Although no true validation analysis was performed , the results show that LDS can differentiate between healthy individuals and neurological patients with a high confidence (effect size 2.3). Furthermore, as highlighted by the correlation results (cadence vs. LDS), kinematic variability among individuals does not seem to constitute a relevant confounding factor.0.5) in the medio-lateral direction is the best candidate to provide a single value to evaluate gait stability. Provided that the same methodology is used , the results (It is likely that short-term LDS measured over one step (λ results  could beIt has been shown that specific management programs and exercise interventions can reduce fall risk in older individuals Due to ethical policy, the access to raw individual data has been restricted. However, the data can be made available upon request through academic cooperation.


Mapping of the great saphenous vein is very important for planning of peripheral and coronary bypass surgery. This study investigated mapping of the great saphenous vein as an adjunct to peripheral MR angiography using a blood pool contrast agent in patients who were referred for evaluation of peripheral arterial occlusive disease and bypass surgery.38 patients with peripheral arterial occlusive disease  underwent peripheral MR angiography using the blood pool contrast agent Gadofosveset trisodium. Apart from primary arterial assessment images were evaluated in order to determine great saphenous vein diameters at three levels: below the saphenofemoral junction, mid thigh and 10 cm above the knee joint (usability: diameter range: >3 and <10 mm at one level and >3.5 and <10 mm at a neighboring level). Duplex ultrasound was performed by an independent examiner providing diameter measurements at the same levels. Additionally, vessel usability was determined intraoperatively by the vascular surgeon during subsequent bypass surgery.P = 0.207/0.806/0.518). Subsequent surgery was performed in 27/38 patients. A suitable saphenous vein was diagnosed in 25 and non-usability was diagnosed in 2 of the 27 patients based on MR angiography/duplex ultrasound, respectively. Usability was confirmed by intraoperative assessment in all of the 24 patients that received a venous bypass graft in subsequent bypass surgery. In 1 case, in which the great saphenous vein was assessed as useable by both MR angiography and duplex ultrasound, it was not used during subsequent bypass surgery due to the patients clinical condition and comorbidities.Mean venous diameters for MR angiography/duplex ultrasound were 5.4±2.6/5.5±2.8 mm (level 1), 4.7±2.7/4.6±2.9 mm (level 2) and 4.4±2.2/4.5±2.3 mm (level 3), respectively, without significant differences between the modalities (Simultaneous mapping of the great saphenous vein as an imaging adjunct to peripheral MR angiography with a blood pool contrast agent is an alternative to additive duplex ultrasound in patients undergoing subsequent peripheral bypass grafting. The first bypass surgery with an autologous saphenous vein was carried out by Jean Kunlin in 1948 Since the 1970 s, the preoperative assessment of the saphenous vein regarding its anatomic configuration, venous abnormalities and pathologic changes has been proven successful in evaluating its suitability as a bypass graft in arterial reconstructions The advantages of the preoperative saphenous vein mapping, i.e. reduction in morbidity, operation time and hospital stay The major quantitative parameter which has to be determined during venous mapping and has been identified as the predominant technical factor in predicting primary patency, primary assisted patency and secondary patency is the internal venous diameter B-mode ultrasound has been established as the gold standard in the preoperative mapping of the great saphenous vein (GSV) Contrast-enhanced magnetic resonance angiography (CE-MRA) is considered an alternative to digital subtraction angiography (DSA) in the diagnosis and assessment of patients with peripheral arterial occlusive disease The basis of this work was the hypothesis that peripheral MRA in the steady state using a blood pool contrast agent to image the size and quality of great saphenous veins would be a useful adjunct to arterial phase MRA to improve the performance of peripheral bypass surgery in patients with PAOD. Thus, the purpose of this study was to prospectively determine the accuracy of high-spatial-resolution steady-state MR angiography using the blood pool contrast agent Gadofosveset trisodium in comparison to color-coded duplex ultrasound (DUS) as the standard of reference in order to assess the usability of autologous saphenous veins for subsequent peripheral bypass surgery. Results were correlated with intraoperative findings.The study protocol was approved by the institutional ethic committee of the University Hospital Bonn, Germany . All patients provided written informed consent.This prospective, intraindividual comparative study included 38 consecutive patients    with perClinical inclusion criteria were PAOD stage IIb-IV (Fontaine classification) and indication for MRA. Exclusion criteria were contraindications for 3D contrast-enhanced MR angiography . The indication for MRA was defined clinically by the vascular surgeons. Every patient who was enrolled into the study also underwent DUS. 3D contrast-enhanced MR angiographic and ultrasound examinations were carried out in random order. MR-angiographic venous mapping was performed as an adjunct to the BPCA-MRA which was done in order to assess arterial occlusive disease. A possible surgical intervention was not delayed for any patient because of his or her participation in this study.MR imaging was performed as previously described −1 sec−1 at 1.5 T and 37°C in plasma) and an extended plasma half-life as compared to standard Gadolinium-containing contrast agents. A dose of 0.03 mmol/kg has been proven safe and effective for imaging of peripheral vascular disease Gadofosveset trisodium  was the first intravascular contrast agent approved for use with MRA in the European Union, Switzerland, Turkey, Canada and Australia The maximum venous vessel diameter of the GSV (area of enhancing vessel lumen) was measured on cross-sectional images perpendicular to the flow axis by one radiologist (10 years of experience in vascular radiology) on multiplanar reformats of steady-state MR angiograms on a post-processing workstation  –5. Each Examinations were performed with the patient in supine position. A HD 11 ultrasound system  with a 5–10 MHz ultrasonographic transducer was used to visualize the saphenous vein in transverse scans. Starting from the groin, the main trunk was tracked distally to the knee joint. Analogously to the MR angiographic image analysis, three independent measurements were taken from the cross-sectional scan at each level –5 and thThe preparation of the GSV was carried out through an inguinal incision and a few auxiliary incisions along the saphenous vein. The vein was dissected and measured with a ruler as seen in P value of less than 0.050 was considered to indicate statistical significance. Correlation between the MR-tomographic and sonographic data was analyzed using Pearsońs correlation coefficient. All statistical analyses were performed with the Statistical package for Social Sciences (SPSS) version 17.0 .Students t-test for paired samples was used to test the differences between the diameters measured by DUS and MR angiography based on a venous segment level. A 3D steady-state MR angiography was successfully completed in 38 patients (76 assessable legs). Legs, which offered no measureable saphenous vein due to stripping of the saphenous vein (14/76), amputation at the level of mid-thigh (1/76) and absent definability in MRA as well as in sonography (no available level for comparison) (3/76) were excluded. Finally a total of 58 veins were available for intraindividual comparison at level 1, 54 veins for comparison at level 2, and 49 veins for comparison at level 3. The difference between the number of measurements available at the different levels is due to the fact that in some cases a segment (i.a. a level) of the vein was not included in the scanned volume (5/13) or certain levels were not clearly definable in MRA and/or DUS (8/13). All segments containing the measuring points were satisfactorily displayed on steady-state MR angiography as well as with DUS. Altogether, 161 comparative measurements were performed .P = 0.207/0.806/0.518, df = 57/53/48)  . The ven27 of the 38 patients underwent subsequent bypass surgery. In all of these 27 patients, the MR-angiographic assessment and the independent sonographic evaluation showed the same results regarding the suitability of the vein which was considered as bypass conduit. A suitable saphenous vein was diagnosed in all of the 24/27 patients that received a venous bypass conduit. Intraoperative findings confirmed usability in these 24 patients . Non-usability was found in 2/27 patients based on MRA/DUS: In one of these patients the diameter of the respective saphenous vein was too small, the second patient had nodular varicosities. A prosthetic graft was used for reconstruction in both patients. In 1/27 patients a prosthetic graft was utilized despite the usability of the great sapehnous vein based on MRA and DUS findings. This decision was taken by the surgeons due to the patients clinical condition and comorbidities (including coronary heart disease) in order to minimize the duration of the operation as well as to save the saphenous vein for a potential coronary bypass surgery. In 11 patients no bypass surgery was performed.Patients suffering from PAOD often present with concomitant venous disease In some patients that are evaluated for bypass surgery, veins or parts of them have already been removed due to varicosities or previous arterial surgery. Furthermore, the correct identification of a suitable vein helps to prevent unnecessary large dissections in already ischemic limbs Our study demonstrates the diagnostic accuracy of MRA with a BPCA in comparison to DUS in the assessment of the luminal diameter of the GSV. Venous mapping was included in the MR-angiographic examination for arterial stenosis grading without the need of a change of examination parameters or any additional diagnostic step in the patient management. We compared sonographic and MR-angiographic measurements and tested the hypothesis that both modalities provide the same results concerning the usability of the GSV as a bypass conduit.The visualization of the veins in the equilibrium phase of the MRA with Gadofosveset trisodium offered reliable results with regard to the luminal diameter of the GSV in our study population. Statistical analysis showed no significant differences between sonographic and MR-angiographic measurements. Usability (25/27) and non-usability (2/27) were diagnosed consistently by DUS as the standard of reference and MRA. Additionally, usability was verified by the intraoperative assessment of the vein in all patients that received a venous bypass conduit in subsequent bypass surgery.MRA with the BPCA Gadofosveset trisodium has been proven to be safe, well-tolerated and highly efficient in patients with vascular disease The sequences needed for first pass and steady state MRA with a BPCA can be easily applied at any MR imaging system and allow for both arterial stenosis grading and the evaluation of venous diameters within the same scan with just a single-dose of contrast agent The accuracy of CE-MRA with a BPCA in arterial stenosis grading as compared with digital subtraction angiography (DSA) has already been demonstrated Our study has several limitations. In the first place, the comparison of the mean venous diameters for the three levels as well as the comparison of the single measurements point out, that MRA in comparison to DUS tends to somewhat over- or underestimate the vessel diameter. However, the fact that the diagnosis of usability versus non-usability by MRA and DUS was consistent in all cases indicates that these slight differences of BPCA-MRA and DUS can practically be neglected. Still, it has to be taken into consideration that a larger study population will increase statistical power. Secondly, the fact that the MRA was carried out as a routine examination for the evaluation of the arterial system, in some cases resulted in the MR technologist accidently excluding the saphenous vein from the scan volume, leading to a reduced number of available segments for comparisons of level 2 (54/76) and 3 (49/76) diameters in comparison to level 1 (58/76). In relation to DUS In this context, the benefits of DUS are well known: it is a noninvasive, relatively cheap, mobile and adjustable procedure free of ionizing radiation and contraindications and risks related to strong magnetic fields Another limitation of this study was due to the surgical practice that a vein identified as unusable by DUS did not undergo an additional intraoperative evaluation. An intraoperative evaluation of such veins would generally not yield an assessment differing from the ultrasonographic evaluation, which renders them unsuitable for bypass surgery. Its intraoperative exploration would therefore cause unnecessary trauma to the patient.A final limitation of our study is the fact that in the bypass surgeries performed, the use of the GSV was often not limited to its supragenual segment. In several cases the length of the bypass required resulted in the additional intraoperative dissection and use of the infragenual segment of the GSV. Intraoperatively, in three patients the diameter of the infragenual segment of the vein turned out to be too small to be used as a bypass conduit. In these cases the GSV of the contralateral lower leg, the small saphenous vein of the contralateral lower leg and a prosthetic graft were used respectively to complement the supragenual GSV in order to acquire a bypass conduit which is long enough. This observation does not hamper the validity or significance of our results but retrospectively highlights that an extension of our preoperative mapping to the infragenual GSV could have presented useful additional information for the surgeons.In conclusion, the MR-angiographic assessment of the suitability of the saphenous vein as a bypass conduit corresponded with the sonographic evaluation in all of the patients who underwent peripheral MRA and subsequent bypass surgery. With BPCA-MRA, a reliable assessment of the venous diameter of the GSV is possible and shows no significant differences in comparison to DUS as the standard of reference. Consequently, in patients with PAOD who in the preoperative workup undergo peripheral MRA to evaluate the arterial status, the inherent MR-angiographic imaging of autogenous saphenous veins can offer additional information regarding the suitability of the veins as a bypass conduit without the need of an additive ultrasound examination.Table S1Diameters of the great saphenous vein.(DOC)Click here for additional data file.


Transrectal ultrasound-guided prostate biopsies are prone to detection errors. Multi-parametric MRI (MP-MRI) may improve the diagnostic pathway.PROMIS is a prospective validating paired-cohort study that meets criteria for level 1 evidence in diagnostic test evaluation. PROMIS will investigate whether multi-parametric (MP)-MRI can discriminate between men with and without clinically-significant prostate cancer who are at risk prior to first biopsy. Up to 714 men will have MP-MRI (index), 10–12 core TRUS-biopsy (standard) and 5 mm transperineal template mapping (TPM) biopsies (reference). The conduct and reporting of each test will be blinded to the others.PROMIS will measure and compare sensitivity, specificity, and positive and negative predictive values of both MP-MRI and TRUS-biopsy against TPM biopsies. The MP-MRI results will be used to determine the proportion of men who could safely avoid biopsy without compromising detection of clinically-significant cancers. For the primary outcome, significant cancer on TPM is defined as Gleason grade >/= 4 + 3 and/or maximum cancer core length of ≥ 6 mm. PROMIS will also assess inter-observer variability among radiologists among other secondary outcomes. Cost-effectiveness of MP-MRI prior to biopsy will also be evaluated.PROMIS will determine whether MP-MRI of the prostate prior to first biopsy improves the detection accuracy of clinically-significant cancer. Currently over 40,000 new cases are diagnosed every year in the UK The role of treatment, once diagnosed, has also been questioned. The Scandinavian Prostate Cancer Group-4 (SPCG-4) and the Prostate Cancer Intervention Versus Observation Trial (PIVOT) randomised controlled trials of radical prostatectomy compared to watchful waiting have shown that the benefit of treatment in terms of improved overall survival was restricted to those men with higher risk – clinically significant – prostate cancer In order to minimise over-diagnosis and over-treatment, the optimal diagnostic pathway should be able to discriminate reliably between men with and without clinically significant cancer.2Men deemed at risk of prostate cancer are those with an elevated PSA level, an abnormal digital rectal examination, a positive family history of prostate cancer or a specific ethnic profile 2.1A man who undergoes TRUS-biopsy has a 1 in 4 chance of being diagnosed with prostate cancer 2.2They have an estimated false negative rate of 30%–45% 2.3The random sampling error c can mea2.4It is associated with a number of complications, the most important being urinary tract infection (1–8%) that can result in life-threatening sepsis (1–4%). Haematuria (50%), haematospermia (30%), pain/discomfort (most), dysuria (most) and urinary retention (1%) can also be expected 3A novel pathway  in whichThis proposed new pathway might offer several advantages. Should the results of the trial be in favour of MP-MRI, we hypothesise three corrections in the current pathway that might occur. First, over-diagnosis might be reduced because fewer clinically insignificant prostate cancers will be detected by chance as only patients with positive MRIs will undergo a biopsy. Second, as a result there might be less over-treatment. Third, the detection of clinically significant prostate cancers might be improved. Fourth, there might be better treatment allocation to active surveillance or radical therapies due to more representative biopsy sampling and better risk stratification. Fifth, complications might be reduced if fewer men are biopsied At present multi-parametric MRI (MP-MRI) appears to have the desired attributes of a test that could be used in the prostate cancer diagnostic pathway Multi-parametric approaches in which sequences are combined together have found an advantage for using two or three MRI sequences rather than just one. When PROMIS started, none had evaluated the clinical validity of MP-MRI in the population of interest – men at risk – against an accurate and appropriate reference standard within a prospective multi-centre study  [46–56].a)biopsy artefact: studies mostly evaluate MRI after biopsy. This results in biopsy-related haemorrhage and inflammation, which can lead to false positives or negative results or scans which are not interpretable.b)Limited application: many studies evaluated only the peripheral zone of the prostate, ignoring up to one-third of prostate cancers that lie anteriorly.c)Segmentation: most studies segmented the prostate into a number of sectors for analysis in order to both achieve sufficient matched datasets. These studies classify each sector of analysis as ‘independent’. Segmentation is usually employed because the reference used is radical whole-mount prostatectomy.d)Inaccurate or inappropriate reference standard: most studies used radical prostatectomy (RP), leading to selection bias as men have to test positive on a biopsy first and then choose to have surgery. Those undergoing surgery tend to have burdens of cancer that are distinct from patients choosing other treatments There were a number of limitations to the early studies investigating the diagnostic accuracy of MRI for prostate cancer that probably account for the limited acceptance of MRI into the prostate cancer diagnostic pathway 44.1PROMIS is a validating paired cohort study representing level 1 evidence for diagnostic studies PROMIS is designed to overcome shortcomings highlighted in the current literature. First, since all patients will be biopsy-naïve and undergo MP-MRI prior to any biopsy, there will be no biopsy artefact. Second, MP-MRI will be evaluated for all anatomical zones of the prostate including peripheral and transition zones. Third, the study is powered so that the primary outcome will be derived using the whole prostate as the sector of analysis rather than segmented sectors of the prostate. Fourth, PROMIS employs an accurate reference test that can be applied to all men at risk.Furthermore, the study has been designed to ensure avoidance or minimisation of a number of biases that are inherent in the current literature. First, spectrum and selection biases will be avoided by recruiting men at risk of prostate cancer and applying all tests to all men. Second, work-up bias will be eliminated by ensuring that patients and clinicians remain blinded to all imaging test results until the biopsies have been carried out and reported. Third, reviewer/reporter bias will be avoided by ensuring that the radiologist is blinded to the reference test and the pathologist is blinded to the imaging. The radiology report in particular will be submitted prior to the biopsies. Last, incorporation bias is minimised by ensuring that TPM biopsies and TRUS-guided biopsies follow a standard accepted protocol.4.2Men who have never had a prostate biopsy before are eligible for the study if there is a clinical suspicion that they may be harbouring prostate cancer. This essentially includes men with an elevated PSA and/or a suspicious digital rectal examination, family history of prostate cancer or an ethnic risk group. Men with a PSA above 15 ng/ml are excluded from PROMIS as physicians are unlikely to use MP-MRI as a triage test to avoid a biopsy due to the higher incidence of prostate cancer in this sub-group. The inclusion and exclusion criteria are described in 4.34.3.1Although experts in the field generally regard the performance characteristics of MP-MRI of the prostate as promising As a result, MP-MRI in PROMIS will be standardised to the minimal requirements advised by a European consensus meeting Use of endorectal coils will be avoided, as there is no consensus on its role in minimal scanning requirements In order to maintain the quality of scans and ensure uniformity across all centres, optimization of the conduct of scans will be applied to all centres through a robust quality control process. This will be undertaken by a separate independent commercial sub-contractor  selected through an open competition compliant with the European Union guidelines on the tender process. Scans deemed of insufficient quality by the commercial partner or the reporting radiologist will be repeated.4.3.1.1In order to avoid variation in method of interpretation, a standardised operating procedure for MP-MRI reporting has been adopted in line with the recommendations of the European consensus meeting and the European Society of Uro-Radiology prostate MRI guidelines A 1 to 5 Likert scoring system With respect to the primary outcome, an overall score of 3 or more will be used to indicate the possible presence of clinically significant cancer . This reflects the level at which further tests  would be considered if MP-MRI were to be introduced into the diagnostic pathway in the future.4.3.1.2In order to establish if a diagnostic test can improve or change the diagnostic pathway in prostate cancer, it must be assessed for intra- and inter-observer variability. Thus, a subset of scans will be reported by another experienced central radiologist. A subset of scans will also be reported by the same reporter again at a different time-point to assess intra-observer variability.In order to make sure that the result of the MP-MRI does not influence the conduct of the biopsy, the results of the MP-MRI will not be revealed to either the men having the biopsies or to the clinicians undertaking the biopsies until after the results of the TRUS-biopsy and TPM biopsies are available (with the exceptions for un-blinding given below). This blinding is necessary to prevent the results of the MP-MRI influencing whether men are biopsied and if they are, how the biopsies are conducted.For safety purposes, the results of the MP-MRI can be un-blinded by the radiologist if the MP-MRI reveals an enlarged prostate > 100 ml in volume or there is evidence of T4 prostate cancer or involved lymph nodes or colorectal/bladder invasion. The presence of other cancers such as bladder or colorectal cancers will also be a criterion for withdrawal. This information will be provided to the treating clinician for appropriate clinical decision making.4.3.2TRUS-biopsy of the prostate is to be performed after TPM biopsies, under the same general/spinal anaesthetic. This helps ensure that results are obtained for the reference test in an optimal fashion in a biopsy naïve gland that has not undergone swelling and distortion. It also theoretically minimises the risk of infection as the potential for faecal contamination is restricted to the end of the procedure. The surgeon performing the biopsy procedure will be blind to the MRI results so no targeting of suspicious areas will occur. TRUS-guided biopsies incorporate 10–12 core biopsies taken as per international guidelines 4.3.34.3.3.1Transperineal Template Prostate Mapping (TPM) biopsies  has beenTPM biopsies produce a histological map of the entire prostate in 3-dimensions with an estimated sensitivity and negative predictive value (NPV) in the order of 95% for clinically significant cancers when assessed against radical prostatectomy 4.3.3.2The expected side-effects of combining both procedures is detailed in the patient information sheet which is discussed with all patients prior to registration . Our rat4.4Despite the high accuracy of TPM biopsies in detecting clinically significant disease there is no widespread agreement on the criteria to define clinically significant prostate cancer on TRUS-biopsies or TPM biopsies. Whilst some definitions exist, such as the Epstein criteria 4.54.5.1The primary outcomes in this trial are of fundamental importance to decisions regarding the future use of MP-MRI in the diagnostic pathway for the prostate cancer. First, they include the proportion of men who could safely avoid biopsy as determined by specificity and negative predictive values (NPVs) for clinically significant cancer. Second, the proportion of men correctly identified by MP-MRI to have clinically significant prostate cancer as determined by sensitivity and positive predictive values (PPV). For the primary outcomes, UCL definition one criteria will be used to set the target definition of clinically significant prostate cancer on TPM biopsy and a score of 3 or more on MP-MRI will be used to define a positive index test. Further, the accuracy of TRUS-biopsy will also be reported in terms of sensitivity, specificity, NPV, and PPV as listed in the section below. In addition, a head-to-head comparison of the accuracy of MP-MRI versus TRUS-guided biopsy (current standard) will be performed. All primary and secondary outcomes are presented in 4.6PROMIS is ideal for assessing the utility of biomarkers (from urine and blood) to identify men with clinically significant prostate cancer. This is the first time that a broad spectrum of men at risk will be evaluated using an optimal biopsy technique that accurately characterises the presence, size and grade of prostate cancer. We will collect, process and store a comprehensive bank of tissue samples  from men prior to biopsy, to analyse urinary and serum biomarkers with respect to the detection of clinically significant prostate cancer on TPM biopsy.4.7The study has been set up to run in two stages: the pilot phase, followed by the main phase. The pilot study has already completed and recruited 50 patients over one year to allow testing of safety and recruitment. At the end of the pilot in May 2013, few safety concerns emerged and the Trial Steering Committee recommended continuation of the study into the main phase If the combination of TRUS-biopsy and TPM biopsies leads to more than a 4% risk of sepsis at any time, there could be cause for concern and a requirement for modification of the study design. If deemed appropriate, recruitment to the study will be suspended until any safety concerns have been resolved.4.8The long-term outcomes of the PROMIS cohort will be of interest and contribute to our understanding of the epidemiology of prostate cancer. Men who specifically consent to long-term data collection will be flagged and followed up using the Office for National Statistics and NHS databases. For example, linkage to Hospital Episode Statistics (HES) may give valuable information on further diagnoses, treatments and outcomes beyond the timeframe of the study for future analyses. Consenting men may additionally be contacted in the future to assess their willingness to respond to questionnaires. This allows the potential for research that would complement the planned long-term follow-up in terms of health status, for example picking up future biopsies not included in HES, and allows assessment of quality of life.4.94.9.1Power calculations were performed in relation to: (1) precision around the estimates for the accuracy of MP-MRI in terms of the joint primary outcomes of specificity and sensitivity, and (2) a head-to-head comparison of the MP-MRI versus TRUS. The largest sample size from (1) and (2) was 714 (as detailed below) and this was taken as the maximum number of men required to have all 3 tests .4.9.2For all calculations we have assumed 4.9.3Assuming a specificity of 77%, in order to demonstrate that the lower 95% confidence interval of specificity is at least 70% or greater, we would require 407 cases of negative or clinically insignificant prostate cancer. This is equivalent to a total of 479 men for UCL definition one and 543 men for UCL definition two.4.9.4647 men for UCL definition one and 388 men for UCL definition two. Since the number of men without clinically significant prostate cancer will be much higher than the number with, the precision for estimating specificity and NPV is much greater.Assuming a sensitivity of 75%, in order to demonstrate that the lower 95% confidence interval of sensitivity is at least 60% or greater, we would require 97 cases of clinically significant prostate cancer. This is equivalent to a total of 4.9.5We have assumed that TRUS-biopsy detects 48% of clinically significant prostate cancer 4.9.6A model will be populated from the study as well as a review of secondary sources of epidemiological, clinical and economic evidence together with appropriately elicited expert opinion We will also collect data on the costs of tests and the management of adverse events, and the health-related quality of life (HRQL) implications of any adverse events experienced with tests. The latter will be assessed using the EQ-5D instrument as part of the main clinical study. This is a widely used generic measure of HRQL which can be used to derive quality adjusted life years (QALYs) 4.10The study abides by the principles of the Declaration of Helsinki and the UK Research Governance Framework version 2 and received UK Research Ethics Committee approval on 16th March 2011 by the NRES Committee London—Hampstead. PROMIS is published on clinical trials.gov [96]5The PROMIS protocol has some potential limitations. First, the thresholds we have used for clinically significant disease are open to debate as no universally accepted definition exists. It is widely accepted that some prostate cancer lesions are clinically significant and others are not Second, TPM biopsies may not be as accurate as whole-mount prostatectomy, but a number of studies point to its accuracy being sufficiently high to use as a reference test for the specific population we will recruit. Indeed, for men with no cancer diagnosis or for those not choosing surgery, it is the best available reference standard.Third, the sequence of TPM biopsy first followed by TRUS-guided biopsy might compromise the standard test. However, the decision for this sequence was one primarily based around safety — inoculation of bacteria by TRUS-guided biopsy into the gland followed by numerous TPM biopsies may theoretically increase sepsis risk. We also wanted to ensure that the reference test was not compromised by swelling caused by the TRUS-guided biopsy if the latter was performed first. Further, because TRUS-guided biopsies were being performed under general/spinal anaesthetic with rectal cleansing performed using 2% chlorhexidine solution might actually be better than standard care in terms of sepsis and test accuracy.6PROMIS will determine whether introduction of MP-MRI prior to biopsy can safely allow men to avoid a biopsy and its associated harms. It will also determine whether MP-MRI can better identify men with clinically significant disease that requires a biopsy to confirm diagnosis using accurate targeting to the lesion. The evidence produced by PROMIS will aid current research interest investigating the possibility of directing biopsies only to the suspicious areas on MRI without deploying systemic biopsies.Much research has focused on developing and validating novel imaging and tissue biomarkers for early detection of clinically significant prostate cancer. These programmes of research have used TRUS-biopsy as the reference test with any volume, grade and risk of cancer taken as a ‘positive’. PROMIS aims to overcome the problems of TRUS-biopsy as a reference test by using TPM biopsies that have a very high degree of accuracy and can be applied to all eligible men. It therefore represents an opportunity to develop and validate numerous imaging and tissue biomarkers in their performance characteristics to discriminate between men at risk who have absence of clinically significant cancer and those men who have clinically significant cancer.Currently PROMIS is open for recruitment across 11 centres in the United Kingdom with 3 more undergoing site setup . We are National Institute of Health Research — Health Technology Assessment: Project Number 09/22/67.Department of Health Disclaimer:The views and opinions expressed therein are those of the authors and do not necessarily reflect those of the health technology assessment programme, NIHR, NHS or the Department of Health.Prostate Cancer UK: funding blood and urine collection and processing for the translational aspect of PROMIS (PROMIS-T).PROMIS has been fortunate to receive input and advice from a wide range of experts in their respective fields detailed in Study concept and initial design: Parker, Emberton, Ahmed.Study design and statistical analysis: Kaplan, Brown, Gaibe.Acquisition of data and Data analysis and interpretation: Hindley, El-Shater Bosaily.