There is a species difference in the function of the preautonomic LC. In diurnal animals, the α2-adrenoceptor agonist clonidine stimulates mainly autoreceptors on SymPN, causing miosis, whereas in nocturnal animals it stimulates postsynaptic α2-arenoceptors in the EWN, causing mydriasis. Noxious stimulation activates SympPN in diurnal animals and ParaPN in nocturnal animals, leading to pupil dilation via sympathoexcitation and parasympathetic inhibition, respectively. These differences may be attributed to increased activity of excitatory LC neurones due to stimulation by light in diurnal animals. This may also underlie the wake-promoting effect of light in diurnal animals, in contrast to its sleep-promoting effect in nocturnal species. The hub of the serotonergic pathway is the dorsal raphe nucleus that is light-sensitive, both directly and indirectly (via an orexinergic input). The light-stimulated pathways mediate a latent mydriatic effect of light on the pupil that can be unmasked by drugs that either inhibit or stimulate SympPN in these pathways. The noradrenergic pathway has widespread connections to neural networks controlling a variety of functions, such as sleep/arousal, pain, and fear/anxiety. Many physiological and psychological variables modulate pupil function via this pathway.Pupil dilation is mediated by a sympathetic output acting in opposition to parasympathetically mediated pupil constriction. While light stimulates the parasympathetic output, giving rise to the light reflex, it can both inhibit and stimulate the sympathetic output. Light-inhibited sympathetic pathways originate in retina-receptive neurones of the pretectum and the suprachiasmatic nucleus (SCN): by attenuating sympathetic activity, they allow unimpeded operation of the light reflex. Light stimulates the noradrenergic and serotonergic pathways. The hub of the noradrenergic pathway is the locus coeruleus (LC) containing both excitatory sympathetic premotor neurones (SympPN) projecting to preganglionic neurones in the spinal cord, and inhibitory parasympathetic premotor neurones (ParaPN) projecting to preganglionic neurones in the Edinger-Westphal nucleus (EWN). SympPN receive inputs from the SCN via the dorsomedial hypothalamus, orexinergic neurones of the latero-posterior hypothalamus, wake- and sleep-promoting neurones of the hypothalamus and brain stem, nociceptive collaterals of the spinothalamic tract, whereas ParaPN receive inputs from the amygdala, sleep/arousal network, nociceptive spinothalamic collaterals. The activity of LC neurones is regulated by inhibitory α The basic autonomic mechanism controlling the pupil is straightforward: pupil constriction is mediated via parasympathetic activation of the circular sphincter pupillae muscle, and dilation via sympathetic activation of the radial dilator pupillae muscle . The autIn order to unravel the complexity of central autonomic regulation, it has been suggested to consider central autonomic control in terms of the functional organization of autonomic pathways –5, 7. OrAs the fundamental function of the autonomic innervation of the pupil is to transmit the effect of light, it is proposed that the effect of light be used as the organizational principle in the case of pupil-controlling autonomic pathways. While light has a robust stimulatory effect on parasympathetic outflow, it has a dual (inhibitory/stimulatory) effect on sympathetic outflow. Thus the parasympathetic output is controlled by a light-stimulated pathway, whereas the sympathetic outflow is controlled by separate light-inhibited and light-stimulated pathways. The light-inhibited sympathetic pathway is yoked to the light-stimulated parasympathetic pathway mediating the pupillary light reflex: as the pupil is constricted by stimulation of the parasympathetic pathway, sympathetically mediated pupil dilation is withdrawn . The actEarly work has shown that light inhibits neuronal activity in efferent peripheral sympathetic fibers, recorded from both preganglionic (“sympathetic nerves”) –11 and pEarly work by Okada et al.  providedTwo groups of light-sensitive neurones have been identified in the pretectum: one group in the olivary pretectal nucleus (“luminance detectors”) that is stimulated by light, and another one (“darkness detectors”) in the posterior pretectal nucleus that is inhibited by light . An attrMore recent experimental evidence supports the existence of a neural link between the pretectal area and sympathetic premotor neurones. A direct link has been identified between the anterior pretectal nucleus and the rostral ventrolateral medulla , a majorThe PAG functions as an integrative relay nucleus . SympathTherefore, it is likely that, in the case of this light-inhibited sympathetic pathway, an excitatory output from the light-sensitive cells of the pretectum is converted into an inhibitory signal by the PAG Figure .The PVN has been identified as a major sympathetic premotor nucleus , and itsThere is an overlap between the sympathetic controls of melatonin synthesis by the pineal gland and that of pupil dilation by the dilator muscle of the iris. In the case of both functions, the preganglionic neurones are located in the C8-T2 segments of the IML, and project to the SCG. This overlap is highlighted by a clinical observation: bilateral oculo-sympathetic paresis (Horner's syndrome) resulting from injury to the lower cervical/upper thoracic spinal cord leads to the cessation of nocturnal melatonin secretion .The neuronal pathway controlling melatonin synthesis is well established: it runs from the SCN to the PVN, that projects to the preganglionic neurones in the IML  Figure . Light ePremotor neurones in the PVN involved in pupillary control are likely to be separate from those controlling melatonin synthesis since there is no evidence that pupil control is subject to the same circadian regulation as melatonin synthesis. However, these neurones, like those controlling melatonin synthesis, may also be susceptible to the direct inhibitory effect of light relayed via the SCN. Thus the SCN may give rise to a light-inhibited sympathetic pathway controlling pupillary function Figure . On the It is well established that light constricts the pupil by stimulating the parasympathetic output to the constrictor pupillae muscle via the light reflex pathway, and that pupil constriction is facilitated by the concurrent inhibition of the sympathetic output to the dilator pupillae muscle . Indeed,1-adrenoceptors : α2-adrenoceptor agonists  “switch off” the activity of these neurones, whereas α2-adrenoceptor antagonists  enhance it eptors”) , whereaseptors”) . Stimula2-adrenoceptor in/or associated with the preautonomic LC: autoreceptors on , the αn the LC , 43, 63 eceptors , 85, andeceptors .2-adrenoceptors in the EWN  and crepuscular (cat) animals , 43, 63  the EWN , innervaThere is a robust projection from the LC to the spinal cord , demonstrated in the rat . This paExcitatory LC neurones also operate in the sleep/arousal network: they project to other wake-promoting neurones and the cerebral cortex .Latero-posterior hypothalamus. The orexinergic neurones in the lateral hypothalamic area/perifornical area (LHA/PFA) play an important role in the control of both arousal , and darkness is wake-promoting in nocturnal animals, including rodents used in laboratory research , 100–102In diurnal animals, luminance information is channeled via the SCN → DMH route to the LC leading to activation of not only wake-promoting but also sympathetic premotor neurones. The latter activation leads to an increase in sympathetic outflow in general, including cardiovascular activity, and increased sympathetic stimulation of the iris manifesting as pupil dilation [see Figure 9 in ]. ActivaCollaterals from spinothalamic tract. Pain signals are carried, via the somatosensory nucleus of the thalamus, to the somatosensory area of the cerebral cortex, by the spinothalamic  applied topically to the cornea  applied systemically  , 119, ore cornea , or α2-aemically .1-adrenoceptor antagonists , and potentiated by the α2-adrenoceptor antagonist RS79948, administered systemically (1-adrenoceptor antagonists may have blocked α1-adrenoceptors at two sites (IML and iris) in the noradrenergic sympathetic pathway originating from the LC, whereas the α2-adrenoceptor antagonist may have antagonized inhibitory autoreceptors in the LC. The α1- and α2-adrenoceptor antagonists failed to affect reflex dilation in the eye whose sympathetic input had been sectioned , and potentiated by α2-adrencoptor agonists . They concluded that noxious stimulation in cats and rats activated a noradrenergic pathway inhibiting the EWN, leading to a reduction in parasympathetic outflow, appearing as pupil dilation  .Latero-posterior hypothalamus. Orexinergic neurones in the LHA/PFA project to the LC where they innervate both sympathetic and parasympathetic premotor neurones (see Connections of Excitatory Sympathetic Premotor Neurones). Electrical stimulation of the lateral hypothalamus in experimental animals leads to pupil dilation due to inhibition of the EWN via an inhibitory noradrenergic input  (or (CRF) . Both thor (CRF)  and the or (CRF)  have beeor (CRF) , 136.Anxious states amenable for experimental study can be generated by the paradigm of fear conditioning: pairing of a noxious stimulus  with a neutral stimulus  leads to the development of the ability of the neutral stimulus to evoke the response to the noxious stimulus. Using this paradigm, it was possible to modulate two physiological reflexes, the acoustic startle reflex and the pupillary light reflex, by the anticipatory anxiety associated with the procedure . Interes1-adrenoceptor antagonist dapiprazole, while a small associated increase in pupil diameter can (In the case of the modulation of both reflexes by fear (“anticipatory anxiety”), the amygdala and the LC play a joint role. The activation of the amygdala by stressful stimulation is transmitted to the LC, leading to potentiation of the noradrenergic facilitation of striated muscle contraction, in the startle reflex paradigm, and enhancement of the noradrenergic inhibition of the EWN, leading to inhibition of the light reflex . As the eter can , the feaCollaterals from spinothalamic tract. Collaterals from the spinothalamic tract project to parasympathetic premotor neurones in the LC in nocturnal animals, such as rats ..1-adreno2-adrenoceptor agonists  and the μ opiod receptor agonists . The α2-adrenoceptor agonists “switch off” LC activity by stimulating inhibitory autoreceptors on LC neurones (see α2-Adrenoceptors Associated With Premotor Autonomic Neurones). The μ opiod receptor agonists also suppress LC activity since μ opiod receptors are co-localized with α2-adrenoceptors in the membrane of LC neurones and operate via a shared signaling mechanism   may influence LC activity either directly or indirectly via inputs from the sleep/arousal network. Directly acting sedative drugs are the αhannels) . Exampleeceptors , whereasneurones .As the LC is not only a wake-promoting, but also a pre-autonomic nucleus, it couples arousal and autonomic activity  , 68. ConDuring transition from wakefulness to sleep (drowsiness or sleepiness), there is likely to be instability between opposing excitatory wake-promoting and inhibitory sleep-promoting inputs impinging on the LC, leading to fluctuations in LC activity. Due to the close association between LC activity and pupillary diameter (see Noradrenergic Pathway), the drowsiness-related fluctuations of LC activity are transmitted to the pupil, leading to fluctuations in pupil diameter. Thus instability between opposing excitatory and inhibitory inputs may be the basis for the appearance of pupillary oscillations in darkness, termed “fatigue waves” or “sleepiness waves” , 141. ThThe PST has also been used to assess the sedative and alerting properties of centrally acting drugs , 146–148A receptors , may withdraw the dopaminergic activation of EWN neurones. Consistent with this model, it has been shown that amisulpiride, a D2 dopamine autoreceptor antagonist, evokes an effect opposite to that of pramipexole: it potentiates the light reflex    However,As discussed above , there is evidence supporting the view that sympathetic and parasympathetic premotor neurones in the LC form separate populations, and, in many situations, operate independently. However, the sympathetic and parasympathetic divisions of the autonomic nervous system do not function in isolation. Examples of cross-talk between the two divisions have been described in the medulla oblongata and the PVN in the hypothalamus .There is also evidence of cross-talk between the two populations of premotor autonomic neurones in the LC. The anatomical basis of such cross-talk may be the gap junctions between LC neurones, through which cells can communicate with each other via eletrotonic transmission . Electro2-adrenoceptor agonists) (Pupillary Effects of Noradrenergic Drugs), may influence autonomic function by interacting with both contingents of noradrenergic premotor neurones in the LC.The following two subsections discuss how two variables, light  and noradrenergic drugs . However, in addition to its sympatho-excitatory effect, light has also been reported to evoke a parasympatholytic effect , 159. Th2-adrenoceptor agonists.Drugs acting at noradrenergic neurones may modify the activity and/or the transmitted effects of both sympathetic and parasympathetic premotor neurones, leading to alterations in both sympathetic and parasympathetic pupil control. Three classes of such drugs will be considered: vesicular monoamine transporter (VMAT) inhibitors (“monoamine depletors”), noradrenaline reuptake inhibitors, and αMonoamines are accumulated in synaptic vesicles of the nerve terminals by an active membrane pump, the vesicular monoamine transporter (VMAT). The form of VMAT accumulating noradrenaline is termed VMAT2. Drugs that inhibit VMAT2 in noradrenergic nerve terminals, such as reserpine and tetrabenazine, lead to depletion of noradrenergic neurones of noradrenaline . These dNoradrenaline reuptake inhibitors, including a number of antidepressants, enhance the effect of released noradrenaline, by blocking its reuptake into noradrenergic nerve terminals . They, l2-adrenoceptor agonists, like clonidine and dexmedetomidine, are selective for sympathetic premotor neurones in man and other diurnal species (see α2-Adrenoceptors Associated With Premotor Autonomic Neurones). The basis for this selectivity is likely to be a difference in the baseline activities of sympathetic and parasympathetic premotor neurones. Sympathetic premotor neurones are likely to have high baseline activity due to their stimulation by light via an input from the retina , whereas the baseline activity of parasympathetic premotor neurones is likely to be low. Therefore, while inhibitory α2 adrenergic autoreceptors are likely to occur on both populations of premotor autonomic neurones, the low baseline of parasympathetic premotor neurones does not allow the conversion of their stimulation into an inhibitory response .In contrast to the VMAT inhibitors and noradrenaline reuptake inhibitors, αresponse . Indeed,response , 166, co2A receptors. The premotor autonomic neurones in the DRN contain inhibitory 5HT1A receptors: the stimulation of these receptors by serotonin, released from recurrent serotonergic axon terminals, inhibits the activity of the serotonergic neurones. The existence of parasympathetic premotor neurones has been postulated: these neurones, via an inhibitory output to the EWN, would inhibit the light reflex. However, although the DRN inhibits the parasympathetic output to the pupil, this is likely to be via an indirect route . The DRN also sends an excitatory output to the cerebral cortex where it stimulates 5HT2A receptors. The DRN receives afferents from the retina, both directly and indirectly, via the LHA/PFA.This pathway is displayed in Figure 2 receptors on sympathetic preganglionic neurones  is one of the major monamine neurotransmitters that, like noradrenaline, is involved in the regulation of both arousal , 167 andneurones . There ineurones .1 receptors are inhibitory, and occur both in presynaptic and postsynaptic locations, whereas 5HT2 and 5-HT3 receptors are excitatory, and occur postsynaptically (1A and the 5-HT2A receptor (1A autoreceptors on serotonergic neurones play an important role in the regulation of serotonergic neurotransmission (Serotonin interacts with a large array of presynaptic (auto) and postsynaptic receptors that can mediate both excitatory and inhibitory effects. 5-HTptically . The mosreceptor . Inhibitsmission .During the past decade or so, an anatomical link has been identified between the retina and the DRN (“retino-raphe projection”) in a number of rodent species. These species include the rat –179, theApart from the direct link described above, an indirect link via the orexinergic neurones of the LHA/PFA has also been reported. Orexinergic neurones may be directly light-sensitive via an input from the retina , or may 1A and 5-HT2 receptor. The role of these receptors in controlling pupil function has been explored using selective 5-HT1A receptor agonists and 5-HT2 receptor antagonists.Serotonergic neurones in the DRN operate via stimulating serotonin receptors both in the DRN and the targets innervated by it. The two most important receptor types are the 5-HT1A receptors occur both presynaptically (autoreceptors) and postsynatically where they mediate an inhibitory action. The autoreceptors are usually more sensitive than the postsynaptic receptors, and their role in controlling serotonergic neuronal function is analogous to that of the α2-adrenoeptors in controlling noradrenergic neurone function (see α2-Adrenoceptors Associated With Premotor Autonomic Neurones). 5-HT1A autoreceptors are abundant on serotonergic neurones in the DRN . In diurnal species, these drugs evoke miosis, whereas in nocturnal animals they cause mydriasis.5-HT1A receptor agonists buspirone, lesopitron, and 8-OH-DPAT evoked dose-dependent miotic responses in rabbit   . The lig1A receptor agonist 8-OH-DPAT evoked consistent dose-dependent mydriatic responses in mice , but also by α2-adrenoceptor antagonists . These observations argue against the existence of a direct serotonergic inhibitory input to the EWN operating via 5-HT1A receptors  and two pathways mediating a paradoxical stimulatory effect (“light-stimulated sympathetic pathways”) are described.While the inhibitory effect of light on the sympathetic output to the pupil was demonstrated in the 1960s and 1970s, little experimental work has been done since then. Okada et al.  demonstrThe two light-stimulaed sympathetic pathways are based on well-established connections of their “hub” nuclei, the LC and the DRN. Both these nuclei are light sensitive, either directly (DRN) and/or indirectly (DRN and LC), and there is evidence of their roles in both the sympathetic and parasympathetic controls of the pupil. Light has a manifest sympatho-excitaroy effect on functions  controlled by the thoraco-lumbar sympathetic outflow. However, at the levels of C8-T2, the sympatho-excitatory effect of light may be superseded by its powerful inhibitory effect required for the operation of the light reflex and control of melatonin synthesis. Therefore, pupil dilation resulting from the stimulation of the sympathetic output to the iris would be masked by the pupil-constricting effect of light. The latent mydriasis can be unmasked by drugs that modulate the activity of the hub nuclei. Drugs that inhibit LC activity  or DRN activity  potentiate light-evoked pupil constriction, while drugs that enhance LC activity  antagonize light-evoked pupil constriction. The light-stimulated sympathetic pathways, by attenuating light-evoked pupil constriction, may enable diurnal animals to function in daylight, when light may cause pinpoint pupils in nocturnal animals .The activity of the light-stimulated pathways appears to be related to age. The monotonic decline in pupil diameter with increasing age in humans –205 may The noradrenergic light-stimulated sympathetic pathway has widespread connections via sympathetic and parasympathetic premotor neurones in the LC, and via these connections it is integrated into the wider central autonomic network , 44. It There is a remarkable species difference in the operation of light-stimulated sympathetic pathways: diurnal animals respond differently from nocturnal animals to light, noxious stimulation, and autoreceptor agonist drugs . A tentative explanation for the species difference may be that it is related to regular exposure to light in diurnal animals that may lead to proliferation and/or a raised baseline activity of sympathetic premotor neurones in the LC and DRN. Therefore autoreceptor agonists and pain signals may affect sympathetic premotor neurones preferentially, as compared to parasympathetic premotor neurones, in diurnal animals. These observations suggest that the effects of some non-luminance-related variables  may be influenced by the luminance-exposure history of the species, determined by the “temporal niche.”Apart from transmitting slow time-course (“tonic”) changes in pupil diameter in response to light, the LC is also involved in mediating non-luminance-related fast time-course (“phasic”) pupil dilations in response to cognitive load . It has Unraveling the multiple sympathetic pathways controlling the pupil suggests that the sympathetic has roles beyond fading away in the background when the light reflex operates. While the parasympathetic pathway mediating the light reflex has one robust dedicated function, the sympathetic pathways, through their connections, are multifunctional, integrating pupil function with a wide range of autonomic, neuroendocrine, physiological, and psychological functions.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Evidence from clinical trials suggests that the addition of bevacizumab to chemotherapy in the first-line treatment of patients with HER2-negative metastatic breast cancer improves progression-free survival (PFS) but not overall survival (OS). However, a retrospective analysis of real-world data from the French Comprehensive Cancer Centers (FCCC) through the Epidemiological Strategy and Medical Economics (ESME) Research Program, suggested that in this setting, the addition of bevacizumab may confer a significant benefit in terms of both PFS and OS. A cost-effectiveness analysis was performed to determine the cost-effectiveness of bevacizumab plus paclitaxel versus paclitaxel alone in the first-line treatment of HER2-negative metastatic breast cancer at specialist oncology centers in France.N = 3426 HER2-negative metastatic breast cancer patients treated with bevacizumab plus paclitaxel or paclitaxel alone. The analysis was performed from a third party payer perspective over a 10-year time horizon; future costs and clinical outcomes were discounted at 4% per annum.The analysis was performed using a three-state Markov model and clinical input data from In the overall population, the addition of bevacizumab to paclitaxel led to incremental gain of 0.72 life years and 0.48 quality-adjusted life years (QALYs) relative to paclitaxel alone. The incremental lifetime cost of the addition of bevacizumab was EUR 27,390, resulting in an incremental cost-effectiveness ratio (ICER) of EUR 56,721 per QALY gained for bevacizumab plus paclitaxel versus paclitaxel alone. In a subgroup of triple negative patients the ICER was EUR 66,874 per QALY gained.The analysis indicated that the combination of bevacizumab plus paclitaxel is likely to be cost-effective compared with paclitaxel alone for the first-line treatment of HER2-negative metastatic breast cancer in specialized oncology centers in France. In Europe, breast cancer is the most commonly occurring type of cancer in women , and accremains a valuable treatment option for patients suffering from metastatic breast cancer” . Th. Th19]. The proportion of patients in each state was estimated at each model cycle based on survival curves for PFS and OS, and extrapolated beyond the follow-up time of the ESME MBC retrospective analysis on bevacizumab. Survival data for parametric extrapolation, using the least squares method, were adjusted for duration of pre-chemotherapy period, treatment period, grade, age at diagnosis of metastatic disease, type of metastases, number of metastatic sites, prior treatment history and contribution of site to the ESME MBC database Fig. .Fig. 2MaThe adjustment of survival data based on prognostic factors prevented the calculation of survival based on the maximum likelihood method. The chosen option consisted of linearizing the survival function and then fitting using the least squares method. Not all survival distributions could be linearized, as such, Weibull and log logistic distributions were investigated and the OS, PFS and duration of treatment were fitted using this method. The distribution with the highest determination coefficient was selected for implementation in the model.Data relating to the proportion of patients in each arm experiencing adverse events were sourced from the E2100 randomized controlled trial , and datThe methodology used by the ESME Research Program has been acknowledged as relevant and robust by the French Health Authorities .Costs accounted in the analysis included direct medical and non-medical costs including drug acquisition and administration costs for first-line treatment (based on treatment dosages sourced from the summary of product characteristics as these data were not available in the ESME study), adverse event costs, medical transport costs, follow-up and monitoring costs and costs associated with progression and treatment. Pharmacy costs were sourced from the BdM_IT CNAMTS database , costs fHealth state utilities were a function of age, response to treatment, progression and chemotherapy-based adverse events and were sourced from a UK-based study . The utiA series of one-way sensitivity analyses were performed to determine key drivers of outcomes. Sensitivity analyses were performed around discount rates (1.5 and 6% per annum for both future costs and outcomes compared with 4% per annum in the base case), time horizon (5 years and 15 years compared with 10 years in the base case), as well as treatment and management costs (+/− 20%). Sensitivity analysis was also performed around the survival estimates using the upper and lower bounds of the 95% confidence intervals (CIs) around the point estimates for survival. Additional scenarios investigated included a scenario in which 100% patients in both treatment arms remained progression-free  and a scenario that permitted the sharing of bevacizumab vials, rather than single use, in the bevacizumab-treated arm. Probabilistic sensitivity analysis (PSA) was also performed (1000 iterations were run). In terms of distributions used for the PSA, for utilities/disutilities gamma distributions were used and for the progression-free state, progression state costs, administration costs and number of adverse events log normal distributions were used. It was not possible to include survival data in the PSA as the use of the least square method for extrapolation prevents the calculation of a covariance matrix for the coefficient of the survival distributions, thereby meaning that the PSA did not evaluate uncertainty around survival. Consequently, scenario analyses based on the fit of the 95% confidence intervals for survival were performed instead.In the base case analysis for the overall population of HER2 negative patients with metastatic breast cancer, treatment with bevacizumab plus paclitaxel was associated with an incremental gain of 0.48 quality-adjusted life years (QALYs) relative to the use of paclitaxel alone . However, total lifetime costs were also higher for the bevacizumab arm  resulting in an incremental cost-effectiveness ratio (ICER) of EUR 56,721 per QALY gained for bevacizumab plus paclitaxel versus paclitaxel alone . Similarly, for TNBC patients treated with paclitaxel alone quality-adjusted life expectancy was 1.02 QALYs . The lower quality-adjusted life expectancy in both arms resulted in a smaller incremental benefit with bevacizumab, resulting in an ICER of EUR 66,874 per QALY gained for bevacizumab plus paclitaxel versus paclitaxel alone.A cost-effectiveness acceptability curve was plotted based on the PSA, which showed that the likelihood of bevacizumab plus paclitaxel being considered cost-effective in the overall population or patients with HR+ disease was between 40 and 50% at a willingness-to-pay threshold of EUR 50,000 per QALY gained Fig. . HoweverSensitivity analyses showed that the findings of the base case analysis were relatively robust Table . ChangesIn the current analysis, based on real world data, the combination of bevacizumab plus paclitaxel was associated with ICERs of EUR 38,003 per life year gained and EUR 56,721 per QALY gained compared with paclitaxel alone for the first-line treatment of patients with HER2-negative metastatic breast cancer in France. Subgroup analyses indicated that first-line treatment with bevacizumab plus paclitaxel was most cost-effective in patients with HER2-negative HR-positive metastatic breast cancer where the ICER was below EUR 50,000 per QALY gained and EUR 35,000 per life year gained for bevacizumab plus paclitaxel versus paclitaxel alone. Cost-effectiveness was also assessed in a subgroup of patients with TNBC, which represents approximately 15–20% patients with breast cancer . CurrentFor any drug used in oncology, the ability to identify patients more likely to respond well to treatment, and therefore take a more individualized approach to treatment may increase the cost-effectiveness of an intervention, although, for bevacizumab such data are currently lacking. In the AVADO trial, VEGF-A and VEGFR-2 were identified as potential biomarkers predictive of response to bevacizumab efficacy . HoweverThe current analysis is one of the first cost-effectiveness analyses of bevacizumab in HER2-negative metastatic breast cancer, and the first one in the French setting, to utilize clinical input data from a real-world environment. Important differences exist between the environments in clinical practice and clinical trials that can influence the mean effectiveness and in turn cost-effectiveness of interventions. In particular in clinical trials exclusion criteria frequently preclude the enrolment of patients with severe co-morbid conditions or poor performance status and patients aged > 65 are typically under-represented in many trials. Real world studies may subject to selection bias, but they do also provide an accurate representation of the patient population encountered in routine clinical practice. As a result, there is now an interest from payers for data relating to real-world effectiveness and cost-effectiveness as parameters such as effectiveness of treatment, adverse event rates, quality of life, adherence and medical resource utilization may differ between clinical practice and the clinical trial environment , 38.n = 33 treated with bevacizumab plus a taxane and n = 29 treated with a taxane alone) treated in the Netherlands, with the relative effectiveness estimates for PFS and OS sourced from the E2100 trial [n = 2127 treated with bevacizumab plus paclitaxel and n = 1299 treated with paclitaxel alone) and utilized real-world OS and PFS data [Recently, van Kampen et al. used real-world data to examine the cost-effectiveness of bevacizumab in HER2-negative metastatic breast cancer in the Netherlands . In theiPFS data . The autPFS data .The analysis of Delagoge et al. is among the first to report a significant improvement in OS for patients who initiated first-line treatment with bevacizumab . In clinWhilst there is no formal willingness-to-pay threshold in France, in oncology a willingness-to-pay threshold of USD 100,000 per QALY gained is a commonly cited benchmark in US-based analyses , 43. SimThe current analysis is associated with a number of limitations. In particular, clinical input data for OS relied on the extrapolation of data from adjusted Kaplan-Meier curves: however, in the absence of available long-term data the approach used represents one of the best available proxies. Additionally, clinical input data were sourced from a retrospective observational study and there are limitations that are inherent in this type of study design. These include the potential for selection bias as in a real-world setting baseline patient and disease characteristics such as performance status may be important drivers of treatment decisions . In addiRecent real-world data from specialist oncology centers in France suggest that in this setting, the combination of bevacizumab plus paclitaxel confers a significant advantage in terms of both PFS and OS relative to paclitaxel alone. The findings of the cost-effectiveness analysis performed using this real-world data suggests that in France, the addition of bevacizumab to paclitaxel relative to paclitaxel alone is likely to represent a cost-effective treatment for patients with HER2-negative metastatic breast cancer, particularly in patients with TNBC.


Aedes aegypti GnRH-related neuropeptide receptors have been characterized and functionally deorphanized. Individually they exhibit high specificity for their native ligands, prompting us to investigate the contribution of ligand structures in conferring receptor specificity for two of these receptors. Here, we designed a series of analogs based on the native ACP sequence and screened them using a heterologous system to identify critical residues required for ACP receptor (ACPR) activation. Analogs lacking the carboxy-terminal amidation, replacing aromatics, as well as truncated analogs were either completely inactive or had very low activities on ACPR. The polar threonine (position 3) and the blocked amino-terminal pyroglutamate are also critical, whereas ACP analogs with alanine substitutions at position 2 , 5 (serine), 6 (arginine), and 7 (aspartate) were less detrimental including the substitution of charged residues. Replacing asparagine (position 9) with an alanine resulted in a 5-fold more active analog. A naturally-occurring ACP analog, with a conserved substitution in position two, was well tolerated yet displayed significantly reduced activity compared to the native mosquito ACP peptide. Chain length contributes to ligand selectivity in this system, since the endogenous octapeptide Aedae-AKH does not activate the ACPR whereas AKH decapeptides show low albeit significant activity. Similarly, we utilized this in vitro heterologous assay approach against an A. aegypti AKH receptor (AKHR-IA) testing carefully selected naturally-occurring AKH analogs from other insects to determine how substitutions of specific residues in the AKH ligand influence AKHR-IA activation. AKH analogs having single substitutions compared to Aedae-AKH revealed position 7 (either serine or asparagine) was well tolerated or had slightly improved activation whereas changes to position 6 (proline) compromised receptor activation by nearly 10-fold. Substitution of position 3 (threonine) or analogs with combinations of substitutions were quite detrimental with a significant decrease in AKHR-IA activation. Collectively, these results advance our understanding of how two GnRH-related systems in A. aegypti sharing the most recent evolutionary origin sustain independence of function and signaling despite their relatively high degree of ligand and receptor homology.Adipokinetic hormone (AKH), corazonin (CRZ), and the AKH/CRZ-related peptide (ACP) are neuropeptides considered homologous to the vertebrate gonadotropin-releasing hormone (GnRH). All three  Gryllus bimaculatus regulates the concentration of carbohydrates and lipids in the haemolymph (In invertebrates there exist three neuropeptide systems of which the mature peptides as well as their cognate G protein-coupled receptors (GPCRs) are structurally similar, and collectively, they are related to the vertebrate gonadotropin releasing hormone (GnRH) system and suggested to form a large peptide superfamily –4. Theseemolymph .Aedes aegypti for a number of reasons. Firstly, A. aegypti is an infamous disease vector for pathogens such as Yellow and Dengue fever, Chikungunya and, as latest addition, Zika arboviruses; summarily these are responsible for affecting approximately 50–100 million people per year  of the human promiscuous G protein, Gα16, which indiscriminately couples a wide variety of GPCRs to calcium signaling  with Kozak translation initiation sequence had been previously prepared in pcDNA3.1+ ; however+  whereby ignaling  was inse+  from dose-response curves from 3 to 4 independent biological replicates.Functional activation of the AedaeACPR and AedaeAKHR-IA receptors was carried out following a previously described mammalian cell culture system involving a Chinese hamster ovary (CHO)-K1 cell line stably expressing aequorin . Cells wPeptide analogs based on the native ACP sequence were designed and synthesized by Pepmic Co., Ltd.  at a purity of over 90%. All synthetic peptides were initially prepared as stock solutions at a concentration of 1 mM in dimethyl sulfoxide. All stocks of arthropod AKH analogs were prepared similarly to the ACP synthetic peptide analogs and were commercially synthesized by either Pepmic Co., Ltd.  or by Synpeptide Co.  at a purity of over 90% verified by HPLC and mass spectrometry. Specific sequence information for each of the peptides  used on the ACP and AKH receptors examined in this study are provided in A. aegypti ACP receptor (ACPR) expressed using a heterologous system. Carboxy-terminal amidation of ACP was found to be critical for bioactivity as were the two aromatic residues of ACP, including the phenylalanine in the fourth position or tryptophan in the eighth position, since analogs modifying these key features demonstrated no activation of ACPR up to the highest tested concentration of 10 μM (Gryllus bimaculatus (Grybi-ACP) was used to further examine the importance of the second position valine in Aedae-ACP, which is replaced by an isoleucine in G. bimaculatus. Grybi-ACP was quite active compared to Aedae-ACP but nonetheless demonstrated nearly ~14-fold reduced activity and failed to reach full ACPR activation even when the highest concentration was tested , which represents a reduced efficacy of at least five orders of magnitude (Libellula auripennis (Libau-AKH), with substitutions at positions two and three only, had a nearly 2-fold improved activation over that achieved by Grybi-AKH; however, the activity of this peptide on the A. aegypti AKHR-IA was still reduced by 548-fold compared to Aedae-AKH (Pyrrhocoris apterus (Pyrap-AKH) that has two substitutions in positions three and seven, both asparagine, is better tolerated than Libau-AKH and Grybi-AKH but is still less active on the A. aegypti AKHR-IA being compromised by 337-fold relative to Aedae-AKH (Erythemis simplicicollis (Erysi-AKH), which is only different to Aedae-AKH at position three , having glycine at position nine, glutamine at position 10 and a glycine at position seven (instead of the serine in Aedae-AKH), revealed activation of AKHR-IA was reduced by only 11-fold , had only an 8-fold reduced activity , had an activation that partially exceeded (~30% improved efficacy) the activity of the native mosquito AKH . By examining the activity of a variety of ACP analogs on the activation of the A. aegytpi ACP receptor (ACPR), including single alanine substitutions along with truncated or extended analogs, a few observations can be highlighted. Firstly, the overall charge of the peptide does not appear to play a role with regards to its influence on receptor activation since substitution of the basic (arginine) or acidic (aspartic acid) residues did not lead to a highly detrimental effect. Specifically, one would assume that the ACP receptor may prefer a neutral ligand given that the native ACP has no net charge  since it has both a single basic and acidic residue. However, the modified analogs which replace the basic (position 6) or acidic (position 7) residues with alanine, create negatively or positively charged molecules, respectively, with both being quite active and so neither proved to be detrimental substitutions, suggesting that the extra carboxy- or amino group is not involved in forming a salt bridge with the receptor. Secondly, with the exception of the aromatic tryptophan residue in the eighth position, the C-terminal residues of ACP do not appear to be highly important since alanine substitutions in positions five through seven only marginally impacted activity between ~16 to 45-fold whereas alanine substitution in position nine resulted in this analog having 5-fold increased activity. Such a compound may be a good lead substance for the development of a superagonist in future studies, with the eventual goal to design an active non-peptidergic mimetic. Amidation of the C-terminal residue was critical since the free acid analog exhibited no activity; however, it is noteworthy that the C-terminally extended peptide having an amidated glycine was also quite effective with ACPR activation compromised by only 6-fold. Previous studies examining free acid analogs of AKH (including both in vitro and in vivo assays) have demonstrated that in some cases the absence of a blocked C-terminus is less critical , or even simply glutamate or glutamine that can spontaneously undergo cyclization , whose sequence matches that of native Aedae-AKH except for positions two to three  that are instead shared with Grybi-AKH. Indeed, our results confirmed that the substitutions in this N-terminal region are much more critical and are not well tolerated by AKHR-IA in A. aegypti since Libau-AKH displayed only a marginal improvement  compared to Grybi-AKH. Supporting the notion that N-terminal residues are most important for AKHR-IA activation, two additional natural AKH analogs were examined having substitutions in either the third position alone (Erysi-AKH) or both the third and seventh position residue (Pyrap-AKH). In comparison to Aedae-AKH, Erysi-AKH had a similar reduction in activity as Pyrap-AKH indicating that the residue in the third position is essential within the natural AKH ligand since it is established that a seventh position substitution has a negligible effect. In light of this evidence indicating substitutions in positions two and three are not well tolerated by the A. aegypti AKHR-IA, we aimed to discern which of these two sites were the absolute most critical. Comparing the activity V2-PeramCAH-II with that of Pyrap-AKH demonstrates that the valine substitution for leucine in the second position is far better tolerated compared to the asparagine substitution for threonine in the third position, indicating this latter position is indeed the most critical.To corroborate that the substitutions in positions two to three were of greatest importance, we examined the activity of a natural AKH analog from D. melanogaster and A. gambiae AKH receptors, which the authors described was due to this core region of the peptide forming a predicted β -strand necessary for receptor interaction , nonetheless exhibit a β-turn structure and interact with the same residues within the single locust AKH receptor  was quite active on the A. aegypti AKHR-IA, the longer length of ACP is unlikely to be the cause of its inactivity on the AKHR-IA.Integrating the novel data from the present study analyzing structural characteristics of the two most recently diverged GnRH-related family members in arthropods, we can infer with some degree of confidence how these ligands act only upon their respective receptors. The ACP sequence in many insect species often contain charged residues in positions six, seven or nine, which in instances where only a single basic or acidic residue occurs, provide an overall charge to the peptide , 61. Howmericana , 64, 65. members , whereasA. aegypti AKH has no activity on the ACP receptor. As we saw with the AKHR-IA, substitution of valine for leucine in position two led to nearly a 70-fold reduction in receptor activity. On the other hand, the removal of valine and its replacement with alanine in the ACP sequence led to a similar 70-fold reduction in activity on the ACPR. In contrast, substitution of valine in position two with another branched-chain amino acid  found in the Grybi-ACP analog, only marginally reduced activity. Thus, the presence of valine in position two is needed for optimal activity for ACPR whereas its absence in the same position (but on the AKH peptide) is necessary for optimal activation of AKHR-IA. Finally, one clear finding from these studies is that ACP analogs that are too short (nonapeptide or octapeptide), even if they contain all the hallmark features, as demonstrated by the C-terminally truncated analogs for example, fail to activate ACPR. Thus, the length of the ligand is most relevant and the binding pocket of ACPR requires at least a decapeptide leaving the A. aegypti AKH, an octapeptide, too short for ACPR binding and activation, while comparatively some decapeptide AKH analogs demonstrated low  levels of activity on ACPR.Our findings also set the framework toward understanding why the paluzzi@yorku.ca).The datasets generated for this study are available on request to the corresponding author (J-PP: GG and J-PP designed the synthetic analogs and wrote the manuscript. AW performed all the experiments and AW, J-PP, and GG analyzed the data. All authors have contributed toward the revisions and have granted approval of the final manuscript submitted for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


With the intention to gain support for the hypothesis that incident ischemic complications of atherosclerotic disease involve a stochastic aspect, we performed a histological, qualitative evaluation of the epidemiology of coronary atherosclerotic disease in a cohort of aortic valve donors.Donors  were dichotomized into a non-cardiovascular (non-CVD) and a cardiovascular disease death (CVD) group. Consecutive 5 mm proximal left coronary artery segments were Movat stained, and the atherosclerotic burden for each segment was graded (revised AHA-classification).p<1.10−16, p<0.0001, and p<0.05, respectively).Non-CVD and CVD groups showed steep increase of atherosclerosis severity beyond the age of 40, resulting in an endemic presence of advanced atherosclerosis in men over 40 and women over 50 years. In fact, only 19% of the non-CVD and 6% of the CVD donors over 40 years were classified with a normal LCA or a so called non-progressive lesion type. Fibrous calcified plaques (FCP), the consolidated remnants of earlier ruptured lesions, dominated in both non-CVD and CVD donors. Estimates of the atherosclerosis burden  were all higher in the CVD group (Dominance of consolidated FCP lesions in males over 40 and females over 50 years, show that plaque ruptures in the left coronary artery are common. However, the majority of these ruptures remain asymptomatic. This implies that the atherosclerotic process is repetitive. A relative difference in disease burden between CVD and non-CVD donors supports the concept that complications of atherosclerotic disease involve a stochastic element. Ischemic heart disease remains the leading cause of death worldwide. While thIncident ischemic coronary events result from a complex sequence of events that involves atherosclerotic lesion formation and destabilization as the initial provocative factor. Actual development (or absence) of clinical manifestations relates to the occurrence or absence of thrombo-embolic complications. Development of thrombo-embolic complications and its clinical consequences reflect a complex interplay of pro- and anti-thrombotic factors, the fibrinolytic system, ischemia times,  coronary lumen size, tissue oxygen demand, pre-existing collateral vascular networks etc.–12 In thThe various aspects of the atherosclerotic process  are best appreciated by histology as only this technique allows for assessment of the different stages of the disease. Discriminative plaque characteristics are captured by the AHA consensus classification scheme.,14 This We reasoned that if the assumption ‘that atherosclerosis reflects an asynchronous, repetitive process with thrombo-embolic complications as a stochastic  element’ is correct, this would translate in relative rather than in absolute differences in atherosclerotic lesion load in patients dying from cardiovascular causes and those from non-cardiovascular causes, with accrual of consolidated lesions  and a hiTo test this hypothesis, we performed a systematic evaluation of the atherosclerotic load of the left main coronary artery, a primary predilection place for atherosclerotic disease, of young and middle-aged deceased Dutch tissue donors who donated their heart for aortic valve procurement. Two separate groups were created: one group dying from atherosclerosis-related (cardiovascular) causes, and a second group of donors who died from non-atherosclerotic related causes. The atherosclerosis load is evaluated and reported separately for each group.Use of the donor material for scientific purposes is approved by the Medical and Ethical Committee of the Leiden University Medical Center, The Netherlands. Sample collection and handling was performed in accordance with the guidelines of the Medical and Ethical Committee of the Leiden University Medical Center, and with the code of conduct of the Dutch Federation of Biomedical Scientific Societies. In all cases permission for transplantation orientated research was given or permission was inherent to donation.http://www.federa.org/codes-conduct). More specific information such as cholesterol or CRP levels were not available for the donors. Information on smoking (history) relied on hetero-anamnesis.Due to national regulations, only data relevant for transplantation was available for research  segments from as many Dutch donors who donated their heart for aortic valve procurement in the period 2011–2017. The age limit for aortic valve donation is 65 years, as such the maximum age of individuals in the study is 65 years. Further main exclusion criteria for donation include (a history of) malignancy, sepsis and/or risk of transferable disease , (suspected) connective tissue disorders or vasculitis/myocarditis. Moreover, donation criteria exclude men over 50 years with a history of diabetes, COPD or abdominal aortic aneurysm.The LCA segments were collected during the aortic valve dissection. Valve procurement was performed within 40hrs after death at the Euro Heart Valve Bank . In short: the aortic valves were carefully removed from the intact heart, freed from the surrounding pericardial fat, and banked for transplantation. The surrounding pericardial fat tissue containing a segment of the proximal LCA (approximately 2-3cm) was formaldehyde fixed and used in this study. This collection procedure did not interfere with the actual valve dissection or the pathological analysis of the heart necessary for the final release of the banked valves.Two study groups were created: a non-cardiovascular death (non-CVD) group and a cardiovascular death (CVD) group. The non-CVD group included all donors whose cause of death was not atherosclerosis-related . The CVD group included all individuals who died from atherosclerosis-related causes .Histological lesion classification.The formaldehyde-fixed tissue was decalcified (Kristensen's solution) in order to facilitate proper sectioning. Decalcification does not interfere with  scoring, as histological footprints of earlier calcium deposits remain present after the process of decalcification  as proposed by Virmani et al. , Table 1For each donor, the section of the tissue segment showing the most advanced lesion type was used in this study (reference-section). Many donors presented with multiple consolidated end-stage lesions ) in the reference-section. These sections were further sub-classified on basis of the sum of separate FCP lesions present in the reference-section . The Virmani atherosclerosis grading  used is In order to avoid interpretation problems due to over-plotting, data points in the graphs have been jittered.  IndividuThis study includes data for the proximal LCA of 698 aortic valve donors (376 men and 322 women). The 65-year upper age limit was dictated by the eligibility criteria for valve donation. Donors were sub-classified on basis of their cause of death in a non-CVD group (n = 335) and a group that died from atherosclerosis-related causes . Reportedly, approximate 24% of all deaths (46.7% in the CVD group) were related to an acute myocardial infarction. Three patients in the CVD-death group had an intracoronary stent in their LCA, and could therefore not be graded. These individuals were excluded from the analysis.Baseline characteristics for the two groups are provided in p<1.10−7) and a slightly higher BMI (p< 0.02). Proportions of individuals treated for hypercholesterolemia and/or hypertension were similar in the CVD and non-CVD group.In those over 40, the data for the CVD group showed a clear dominance of male sex (The age distribution of atherosclerosis (defined by most advanced lesion type present (grading summarized in p<0.0001) of the non-CVD donors was classified with a FCP in the reference cross-section. Many donors presented with multiple FCP lesions in the reference cross-section  in two donors. The prevalence of so-called vulnerable (culprit) lesions (i.e. thin cap fibro-atheroma (TCFA), ruptured plaque (RP) or healed rupture (HR)) was 3.5% (95% CI: 1.8–6.3%) in men and 1.6% (95% CI: 0.6–3.9%) in women over 40.For both groups, the age of 40 marks a transition after which the more advanced lesion types become prominent . This re-section . The numThis systematic, qualitative evaluation of coronary atherosclerosis burden of the proximal left coronary artery of young and middle-aged tissue donors shows a relative rather than an absolute difference in disease burden between individuals dying from cardiovascular- and those dying from non-cardiovascular causes. Accumulation of consolidated FCP lesions in men over 40 and women over 45 implies that incident plaque rupture, and subsequent healing and scarring is a common phenomenon.This comparative qualitative study is performed on material from a unique and large biobank of proximal LCAs from (deceased) Dutch donors who donated their heart for aortic valve replacement procedures. The LCA is an established predilection place for (coronary) atherosclerosis. In fact, intravascular ultrasound studies show a mild gradient with the higher ‘plaque burden’ for the more proximal aspects of the coronary arterial tree. , 19qualitative aspects of the atherosclerotic process. [14 better incorporates the natural history of the atherosclerotic process; viz. plaque initiation, lesion (lipid core) formation and -progression, plaque destabilization, and ultimate rupture. In the absence of fatal complications, rupture is followed by plaque healing and lesion consolidation. [Multiple studies have shown that the manifestations and complications of atherosclerotic disease essentially relate to the qualitative rather than quantitative (i.e. degree of occlusion) aspects of the process. , 21 Accoprocess.  More speprocess.  This modidation. , 16 Considation. Although the data from this evaluation confirms observations from earlier quantitative reports showing that atherosclerosis manifests early in life, –28 this The age of 40 years marks a critical tipping point beyond which advanced atherosclerotic lesion types become endemic. In fact, this study indicates dominance of the FCP lesion types , 16 in tThe higher FCP load in CVD-death group supports the concept that thrombo-embolic complications of coronary atherosclerosis involve a stochastic element. That is to say, each bout of plaque rupture comes with a risk for a threatening thrombo-embolic complication, and consequently the more plaque ruptures, the higher the chance of a fatal life-threatening event. A further noteworthy observation was the common presence of multiple adjacent, or even stacked FCP lesions in a single LCA cross-section. This observation implies that atherosclerosis is a repetitive process.The data further indicates a low prevalence of classic, advanced atherosclerotic lesion types (EFA and LFA) and vulnerable lesions. , 16 ThisIt came to our attention that the apparent repetitive nature of the atherosclerotic process with asynchronous cycles of atherosclerotic lesion initiation and evolution provides a rationale for the observed delays in benefit reported for clinical cardiovascular risk management. , 32 In fLimitations: this study is based on the proximal LCA segment collected during aortic valve procurement for tissue donation. Consequently, information on other segments of the coronary arterial tree is missing. The proportion of patients dying from stroke is higher than expected,  an obserFor the younger individuals, a further bias may result from clinical competition between tissue , and organ (heart transplant) donation for the ‘healthier’ donors, and thus that LCAs from healthier young donors are underrepresented. This will obviously not impact the core conclusions of this study as these are essentially based on information from the donors over the age of 40 years. Moreover, we consider the potential impact minimal as we observed remarkable parallels between the atherosclerosis distribution in this LCA cohort, and our earlier published data for the peri-renal artery aorta (a predilection location for human atherosclerosis).  MaterialIn conclusion, this cohort of Dutch tissue donors shows that advanced atherosclerotic disease is endemic over the age of 40 (men) and 45 (women). Asymptomatic plaque ruptures appear common, supporting the concept that thrombotic-embolic complications after plaque rupture reflect a stochastic event.S1 TableLindeman et al Qualitative evaluation of coronary dataset.(XLS)Click here for additional data file.


Acute aortic syndromes (AAS) encompass a spectrum of life-threatening conditions characterized by acute aortic pain. AAS include acute aortic dissection, intramural hematoma, penetrating atherosclerotic ulcer, and aneurysm rupture. The prognosis of AAS is clearly related to prompt diagnosis and appropriate management. The different types of AAS cannot be reliably differentiated solely based on clinical presentation since the clinical features are indistinguishable. Multidetector-row computed tomography (MDCT) with electrocardiographic gating (ECG-gated MDCT) has been used in the acute emergency setting as a powerful clinical tool, which enables rapid and specific diagnosis of aortic pathologies. ECG-gated MDCT significantly reduces motion artifact and avoids potential pitfalls in the diagnosis of AAS. The aim of this review is to evaluate the role of MDCT imaging in the assessment of AAS and to discuss the differentiation of this spectrum of aortic diseases with reference to the key imaging findings. Acute aortic syndromes describe the acute presentation of patients with characteristic aortic pain caused by one of several life-threatening conditions of aortic diseases. AAS is an umbrella term that traditionally includes four aortic diseases: aortic dissection (AD), intramural hematoma (IMH), penetrating atherosclerotic ulcer (PAU), and rupture of an aortic aneurysm, often due to trauma -3. The iMultidetector-row computed tomography (MDCT) with electrocardiographic gating (ECG-gated MDCT), trans-esophageal echocardiography (TEE), and magnetic resonance imaging (MRI) are valuable tests in the diagnosis of AAS. TEE and MRI could be considered complementary modalities in defining particular diagnostic aspects, such as the functional evaluation of an aortic valve, heart function, floating thrombus, or in the case of a contraindication of iodinated contrast agent. The major advantage of echocardiography is the assessment of valve lesions, specifically aortic regurgitation in AAS . The rolIn this review, our aim is to illustrate the MDCT findings of the most frequently encountered non-traumatic aortic emergencies. The imaging technique and typical imaging findings of the AAS will be discussed in this review.MDCT angiography imaging technique in acute aortic syndromeThe MDCT imaging protocol depends on the technical characteristics of tomography available such as the number of detectors , rotation tube speed, and table feeding. Typically, axial reconstruction thickness should be between 1 mm and 3 mm, using 16 x 1.25 mm on 16-row scanners, 64 x 0.5 mm on 64-row scanners and 128 x 0.6 mm on the new 128-row scanners. Sagittal, coronal, and multiplanar reconstructions (MPRs) should be generated on three-dimensional (3D) workstations -13. The Non-contrast images provide important information concerning the presence of calcification and intramural hematoma, size of the aorta, general status of the lung parenchyma, mediastinum, and heart size, presence of pleural effusion, abdominal organs, bowel, intra, and retroperitoneal space, and fasciae fluid collections -15.The contrast agent administration protocol is based on the patient’s weight and potential abnormalities in kidney function. After a variable time of 10 to 30 seconds after contrast media injection, intravascular contrast enhancement increases and lumen opacification appears linearly correlated with dilution effect; this is influenced by several parameters, such as contrast media concentration, flow rate and pressure of infusion, cardiac output, scan parameters, and the presence of saline flush following the injection of contrast media when double injectors are used. Bolus timing is crucial in MDCT angiography; therefore, especially in critical patients, the automatic detection of bolus such as bolus tracking may be utilized. The protocol of contrast agent delivery depends on the characteristics of computed tomography available. Generally, non-ionic iodinated contrast agent at a high concentration (≥350 mg/ml) at a dosage max of 0.1- 0.2 ml/kg body weight with moderate-high flow rate (4-4.5 ml/s) with a bolus (30-50 ml) saline solution at the same flow rate following contrast injection using a double pump injector, could be considered the simplest and most efficient protocol, especially in emergency cases. The contrast agent dosage should be calculated in relation to the scan duration in order to avoid the scan time to exceed the infusion time delivery -18.ECG-gated MDCT of the thoracic aorta significantly reduces the motion artifacts when compared with non-ECG-gated studies -21. ECG Aortic dissectionAortic dissection is characterized by the separation of the aortic intima from the media, caused by the shearing forces of blood under high pressure, with variable longitudinal and circumferential extension, resulting in the formation of a double-channel aorta . High blThe clinical presentation of aortic dissection can be very misleading and findings on physical examination may be non-specific. Patients may present with a classic history of acute-onset central chest pain that radiates to the back. Syncope can result from acute dissections and occurs in 9% of cases; syncope may be caused by hypotension secondary to cardiac tamponade, aortic rupture, cerebral vessel obstruction, or the activation of cerebral baroreceptors -27.The intimal tear causes blood to enter the media from the vessel lumen. The blood-filled space within the medial layer creates a false lumen. This results in two lumina: a true and a false lumen with the false lumen having pressures greater than or equal to those in the true lumen . BecauseThe classification of aortic dissection is based on the location and extension of the dissection, and the DeBakey and Stanford classification systems are most commonly utilized -31. In gA widened mediastinum is the most common imaging finding on the radiography. In a study published in JAMA, a widened mediastinum was noted in 61.1% of aortic dissection cases, displacement of calcification of the aorta was reported in 14.1% of cases, with an abnormal cardiac contour being noted in 25.8% . TEE hasIn practice, ECG-gated MDCT should be preferred since it allows more precise delineation of the proximal extent of the intimal flap in relation to the aortic valve and coronary arteries and, more importantly, helps avoid the overdiagnosis of aortic dissections caused by the misinterpretation of a motion artifact as an intimal flap. Non-contrast MDCT images help obtain the extent of inward displacement of intimal calcification. The two most useful indicators of the false lumen are the beak sign and the cobweb sign ,5,27. ThContrast-enhanced magnetic resonance angiography is more available for the investigation of aortic dissection in medically stable patients or those with chronic dissections. It has several advantages over MDCT angiography, including a lack of non-ionizing radiation, multiplanar evaluation, and greater vessel coverage at high resolution. Three-dimensional magnetic resonance angiography can reveal a complete and dynamic display of aortic dissection and display the true and false lumina -35.Recently, triple rule-out MDCT protocol is used to assess the aorta, coronary arteries, and pulmonary arteries during a single scan with the use of several optimally timed boluses of contrast material and ECG gating in patients who are at low risk for an acute coronary syndrome. The main goal is to minimize the contrast material dose and radiation exposure while achieving optimal image quality, providing coronary artery image quality equivalent to that of dedicated coronary MDCT angiography, pulmonary artery image quality equivalent to that of dedicated pulmonary MDCT arteriography, and high-quality images of the thoracic aorta without pulsation artifact. In addition, the presence of acute coronary syndrome and aortic dissection can be evaluated via a triple rule-out technique . This teIntramural hematomaIntramural hematoma (IMH) is a variant of dissection and is characterized by the presence of hemorrhage into the aortic media from the vasa vasorum. Tears seen in classic aortic dissection are absent. IMHs are thought to comprise 10%-30% of all AAS -37. IMH The hyperdense crescent-shaped or ring-like thickening of the aortic wall is often detected on the non-contrast MDCT images, and precontrast imaging is essential in this protocol Figure . The absProgression to aortic dissection occurs in 28%-47% of patients with IMH . SimilarPenetrating atherosclerotic ulcerAn atherosclerotic plaque erodes the internal elastic lamina into the media of the aortic wall in patients with a penetrating atherosclerotic ulcer (PAU). These ulcers may be complicated by true aneurysm formation, erosion through the media to form a pseudoaneurysm or dissection ,42. PAU In extensive atherosclerosis, high-density hematoma surrounding the ulceration and variable size IMHs can be detected in non-contrast enhanced images. In contrast-enhanced images, ulceration of the atherosclerotic plaque can be seen and would show protrusion beyond the intimal level into the medial layer of the aortic wall, together with a focal outpouching of the outer aortic contour. The protrusion and focal contour change may differentiate the PAU from the common atheromatous ulcer Figure  36,44].,44.36,44Invasive treatments such as surgery and stent-grafting are required in acute and symptomatic cases, but course observation, including periodical evaluation using imaging techniques is recommended in asymptomatic or chronic cases ,45.Aortic aneurysm and ruptureAortic aneurysmal enlargement is defined as a permanent dilatation to at least 150% of the normal size. According to their contents, aneurysms are divided into two: true and false. “True” aneurysms include all the layers of the aortic wall whereas “false” aneurysms are contained ruptures and usually comprise just the adventitia, surrounded by fibrosis and hematoma. Localized aneurysms are usually divided into two: “saccular” and “fusiform,” with fusiform defined by more diffuse dilatation ,5,46. MoHyperdense thickening of the aortic wall that represents blood collection between partially disrupted wall layers and mediastinal hematoma is usually detected on MDCT angiography images. This mediastinal hematoma may extend from the site of the aortic lesion into the periaortic mediastinal fat. Pleural and rarely pericardial effusion can also be detected on MDCT images. MDCT angiography also may be helpful to depict signs of impending hypovolemic shock via presenting reduction in the caliber of central vessels and excessive contrast enhancement of the aorta relative to the injection parameters ,11,47-48Acute aortic syndromes are important emergency conditions of the aorta and can often lead to the patient’s death. Early diagnosis and treatment are essential for improving prognosis. MDCT allows the imaging of the entire aorta with rapid acquisition and data reconstruction to provide a prompt and accurate diagnosis, including all of the AAS types and helps identify relevant complications that may have an impact on surgical planning or management.


Stratified mucin-producing intraepithelial lesion (SMILE) is a rare precursor lesion in the uterine cervix that is considered a variant of adenocarcinoma in situ (AIS). Although human papillomavirus (HPV) is thought to be related to the development of SMILE, there is little information available on the detection of HPV integrated into the lesion.A 30-year-old female underwent a routine uterine cervical cancer screening, and her Pap smear indicated the possible existence of atypical glandular cells. A cervical biopsy with endocervical curettage was performed. The histopathological analysis showed that she had SMILE and high-grade squamous intraepithelial lesion (HSIL) on her cervix. The lesion was found to be positive for HPV genotypes 52 and 68 by multiplex PCR. In situ hybridization with HPV RNA probes revealed that these HPV types were involved in the onset of HSIL and SMILE, respectively.Rare, high-risk HPV genotypes may contribute to the development of SMILE, and their detection can be useful for preventing the progression to carcinoma and ensuring adequate patient management. Uterine cervical cancer is the second most commonly diagnosed cancer and the third leading cause of cancer death among women in developed countries . AlthougStratified mucin-producing intraepithelial lesion (SMILE) is an uncommon premalignant lesion of the uterine cervix . It is tAlphapapillomavirus 9  or Alphapapillomavirus 7  [Studies over the last two decades have shown that persistent HPV infection is the main cause of cervical cancer development. Clinically validated HPV tests are recommended by the U.S. Preventive Services Task Force (USPSTF) and the Japan Society of Obstetrics and Gynecology for cervical pre-cancer screening, triage, and treatment follow-up in clinical practice , 14. Abo and 68) . However and 68)  with the and 68) . HPV RNA and 68) , 23. In A 30-year-old female  was referred to our hospital for routine uterine cervical cancer screening, and her Pap smear indicated the possible existence of atypical glandular cells. A colposcopic examination revealed dense white lesions in the 1 and 11 o’clock directions Fig. a as wellWe next examined lesions by single-molecule RNA fluorescent ISH using the RNAscope system   and specHistologically, SMILE is characterized by a multilayered atypical epithelium composed of cells with intracytoplasmic mucin in all cell layers. SMILEs are p16 positive and have a high MIB-1 proliferation index. However, SIL and AIS may coexist with SMILE, which is not surprising given their association with HPV infection. Moreover, a minority of invasive cervical carcinomas also exhibit both squamous and glandular features (referred to as adenosquamous carcinoma). Apart from the original description, there is little information on SMILEs, especially in relation to high-risk HPV infection . Most SMPrevious reports have suggested the preferential physical interaction of the virus with squamocolumnar junctions in the transformation zone of the basal epithelium that forms reserve cells with stem cell (SC) properties , 25. TheExamination of additional cases would be helpful in confirming the existence of CSCs in SMILEs. Therefore, we consider that most high-risk HPV types in cervical cancer are easily detectable, given their diffuse presence in the epithelium compared to that of high-risk HPV types, such as Alpha-7, that are predominant in the basal layer. Furthermore, the case implies that different HPV-infected cells individually define their disease phenotype as HSIL or SMILE. We speculate that SIL, AIS, and SMILE differ in terms of cellular origin with different HPV life cycles. Alpha-7 HPV types such as HPV18 and HPV68 may preferentially remain in the basal epithelium, unlike HPV16-related Alpha-9 HPV types, such as HPV52. In addition, complex cases of multiple HPV infection may exhibit distinct histopathology. Notably, our observations had certain limitations because of the study being a case report. Hence, further studies are needed to explore these possibilities. Nonetheless, our findings provide a basis for investigating multiple infection by Alpha-7 and -9 HPV types, the carcinogenicity of the rare HPV genotypes, and the outcome of SMILE in the cervix.


We aimed to synthesise the available data for the effect of stopping alpha-blocker therapy among men with lower urinary tract symptoms. The focus was on symptom, uroflowmetry and quality of life outcomes, but we also reviewed the adverse events (AEs) and the number of patients who restarted therapy.We searched MEDLINE/PubMed, EMBASE/Ovid and The Cochrane Central Register of Controlled Trials from inception to May 2018.We selected studies regardless of study design in which men were treated with an alpha-blocker for at least 3 months and in which the effects of alpha-blocker discontinuation were subsequently studied. Only controlled trials were used for the primary objective.Two reviewers independently extracted data and assessed the risk of bias for the controlled studies only using the Cochrane Collaboration’s tool for assessing risk of bias. Data were pooled using random-effects meta-analyses.We identified 10 studies (1081 participants) assessing the primary objective. Six studies (733 participants) assessed differences in AEs between continuation and discontinuation, and six studies (501 participants) reported the numbers of subjects that restarted treatment after discontinuation. No studies in primary care were identified. After discontinuing monotherapy, symptom scores increased and peak flow rates decreased at 3 and 6 months, but not at 12 months; however, neither parameter changed when alpha-blockers were stopped during combination therapy. Small differences in post-void residual volumes and quality of life scores were considered clinically irrelevant. We also found that 0%–49% of patients restarted after stopping alpha-blocker therapy and that AEs did not increase with discontinuation.Discontinuing alpha-blocker monotherapy leads to a worsening compared with continuing therapy. Discontinuing the alpha-blocker after combination therapy had no significant effects on outcomes in either the short or long term. Discontinuation may be appropriate for the frail, elderly or those with concomitant illness or polypharmacy. However, studies in primary care are lacking.CRD42016032648. This is the first systematic review that synthesises the literature concerning alpha-blocker discontinuation.The review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines.The number of studies that could be included was limited, and the risk of bias was high for most outcomes preventing the drawing of firm conclusions.Alpha-blockers are the first-choice treatment for men with moderate-to-severe lower urinary tract symptoms (LUTS) because of their proven, but small, superiority over placebo,A number of researchers have studied the effects of discontinuing alpha-blockers, but to date, there has been no synthesis of this literature.We performed a systematic review and meta-analysis to obtain data about the effect of discontinuing alpha-blockers on male LUTS. Our primary objective was to compare the effects of discontinuing therapy with those of continuing therapy. Secondary objectives were (1) to determine the proportion of men who restarted alpha-blocker therapy and (2) to determine the possible adverse effects (AEs) of both discontinuation and continuation.http://www.crd.york.ac.uk/PROSPERO/display_record.php?ID=CRD42016032648).We completed this review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and registered the protocol in the PROSPERO database (CRD42016032648)  and non-randomised trials (NRTs) that compared alpha-blocker discontinuation to continuation were selected. For the secondary objectives, we also included uncontrolled studies. At all stages, we excluded studies written in languages other than Dutch, English, French or German.The following outcomes were used for the primary objective: symptom scores, such as the International Prostate Symptom Score (IPSS); urinary flow rates; post-void residual urine volume (PVR); and quality of life (QoL). For the secondary objectives, we calculated the percentage of patients who restarted alpha-blocker therapy and the numbers of AEs in the continuation and discontinuation groups.We searched MEDLINE/PubMed, EMBASE/Ovid and The Cochrane Central Register of Controlled Trials using search terms covering LUTS, alpha-blockers and discontinuation  independently performed data extraction using standardised forms. We extracted the following data: (1) the participant characteristics; (2) the interventions used; (3) the primary and secondary outcomes, as well as the timing of the outcome assessment; and (4) the study design. If possible, we extracted data by allocated intervention to allow an intention-to-treat analysis. Discrepancies were resolved by re-examination and discussion of the full-text papers or by consultation with a third author (MHB).Two reviewers  independently assessed the risk of bias using the Cochrane Collaboration’s tool for assessing risk of bias.Data were analysed using Review Manager, V.5.3.2 statistic). We pooled data if we identified two or more studies with an I2 of <40%,Clinical heterogeneity was assessed by checking the characteristics of participants and interventions. Statistical heterogeneity was assessed by visual inspection of the forest plots and of the results of statistical testing for heterogeneity : 2.7 points for IPSS,This study was performed without patient involvement. The patients were not invited to comment on the study design and were not consulted to interpret the results. The patients were not invited to contribute to the writing or editing of this document for readability or accuracy.The searches yielded 1039 publications , of whicTwo of the included studies randomised patients into three groups: a discontinuation group, a continuation group and a third group that continued with alternate-day use of alpha-blockers.In another three studies, the data required for pooling were missing.Most of the included studies had risks of bias . The mosWe did not construct funnel plots to ascertain the existence of publication bias graphically, as the number of included studies in each meta-analysis was less than 10.Five studies of monotherapy (n=341)All but one studyBy 3 months after discontinuing alpha-blocker monotherapy, symptoms increased in the discontinuation group compared with the continuation group ,After 6 months, two RCTs and one NRT on monotherapy found a significant worsening of symptoms (differences varying from 2.0 to 5.8 points) in subjects that discontinued alpha-blockers.2=61%).After 12 months, the one study that looked at discontinuing monotherapy found a non-significant difference of 1.2 points between groups.Nine studies (804 patients) assessed peak urine flow rate (Q-max): five studies for monotherapy,After 3 months, Q-max was reduced by 2.59 mL/s ,2=68%).After 12 months, no differences were found in an NRT reporting on the effects of discontinuing monotherapy.2=70%).Data from two RCTs on monotherapy (84 patients) could not be pooled , with three measuring it after discontinuing monotherapyAfter 3 months, discontinuing monotherapy resulted in a PVR increase of 9.98 mL  also found a statistically significant difference 14.1 mL) in favour of continuing therapy..1 mL in At 12 months after discontinuing monotherapy, an NRT did not find a significant difference between groups.All five studies  that assessed QoL used the IPSS QoL subscore.After 3 months, one study of combination therapy found no difference between groups (0 point).After 6 months, one study on monotherapy found a statistically significant difference of 0.2 point in favour of those who continued alpha-blocker therapy.2=60%).After 12 months, no differences in QoL scores (only 0.1 point in favour of discontinuation) were found in an RCT of 117 participants receiving combination therapy.Six studies (501 patients) reported data on restarting alpha-blockers after discontinuation.Three other studies provided indirect information about restarting alpha-blocker use.Nine studies provided no data on AEs during discontinuation, or if they did, provided data without a clear indication of the treatment group.Respiratory tract infection and urinary retention were the two most common AEs after discontinuing alpha-blockers , being reported in 1%–4% of patientsThe results of this systematic review indicate that discontinuing alpha-blocker monotherapy leads to a worsening of clinical symptoms and a decrease of urinary flow rates in the short term (3–6 months) compared with continuing therapy. However, after 1 year, no differences were found in these or other outcomes. Discontinuing the alpha-blocker after combination therapy had no significant effects on outcomes in either the short or the long term.The worsening of symptoms over the short term after stopping monotherapy was probably relevant to clinical practice. The reported differences in the IPSS between groups exceeded the MCID of 2.7 points.The worsening of symptoms noted by 3–6 months after discontinuing monotherapy was larger than the reported improvement of symptoms after initiating therapy, which was reported to be 2.55 points  based on 12 RCTs with a total of 9335 participants.Among patients receiving combination therapy, outcomes were not significantly different between those discontinuing and continuing alpha-blockers. Although 5-alpha-reductase inhibitors have no significant impact on LUTS severity after treatment initiation,The results for restarting a discontinued alpha-blocker were heterogeneous, ranging widely from 0% to 49%. These conflicting findings can be explained by the differences in instructions given to patients in these studies. Indeed, participants in some studies received explicit instructions about when to restart therapy, whereas in other studies, no instructions were given. Also, subjects in cohort studies who volunteered to discontinue therapy may have had greater freedom to restart therapy than those participating in an RCT.It was also shown that discontinuing alpha-blockers did not result in more AEs, including acute urinary retention.Interpretation of our findings is hampered by some limitations. For example, the limited numbers of studies and large amount of statistical heterogeneity limited data pooling. Heterogeneity, especially on IPSS outcomes after 6 months could be explained by differences in alpha-blockers studied and baseline symptom severity differences ranging from 12 to 19 in the included studies. The limited number of RCTs also precluded sensitivity analyses, and subgroup analyses, that were planned in the original review protocol. Another limitation related to the limited number of studies is the reduction in statistical power. It has been shown that at least five studies have to be pooled to achieve a greater power than the original studies independently.No firm conclusions can be drawn from this review because of the low quality of the available evidence. Overall, the data suggest that there is a short-term clinical worsening of LUTS after discontinuing alpha-blocker monotherapy, as assessed by symptom scores and urinary flow rates, but that this does not increase the risk of a complicated symptom course.Patients frequently discontinue alpha-blocker treatment in clinical practice. We have recently shown that men who continue to use alpha-blockers are typically unconcerned about stopping that therapy if advised to do so by a doctor.Our findings support the existing EAU guidance to consider discontinuing alpha-blockers in patients receiving combination therapy for 6 months.


Encouraging survival was observed in single arm and randomized phase 2 trials of patient-specific dendritic cell vaccines presenting autologous tumor antigens from autologous cancer cells that were derived from surgically resected metastases whose cells were self-renewing in vitro. Based on most advanced clinical stage and extent of tumor at the time of treatment, survival was best in patients classified as recurrent stage 3 without measurable disease. Next best was in stage 4 without measurable disease, and the worst survival was for measurable stage 4 disease. In this study, the survival of these patients was compared to the best contemporary controls that were gleaned from the clinical trial literature. The most comparable controls typically were from clinical trials testing other immunotherapy approaches. Even though contemporary controls typically had better prognostic features, median and/or long-term survival was consistently better in patients treated with this dendritic cell vaccine, except when compared to anti-programmed death molecule 1 (anti-PD-1). The clinical benefit of this patient-specific vaccine appears superior to a number of other immunotherapy approaches, but it is more complex to deliver than anti-PD-1 while equally effective. However, there is a strong rationale for combining such a product with anti-PD-1 in the treatment of patients with metastatic melanoma. The introduction of monoclonal antibody checkpoint inhibitors, especially the anti-programmed death molecule-1 (anti-PD-1) agents nivolumab and pembrolizumab, and anti-BRAF/MEK agents for patients with BRAF mutations, have revolutionized the treatment of metastatic melanoma. Anti-PD-1 agents have become the treatment of choice for the primary treatment of distant metastatic melanoma, and for the adjuvant treatment of high-risk surgically resected stage 3 and stage 4 melanoma, due to their curative potential ,3,4. In Some of the most encouraging clinical results have been reported for a DCV consisting of autologous dendritic cells (DC) that were loaded with autologous tumor antigens (ATA) from autologous tumor cells that were self-renewing in tissue culture, and administered in granulocyte-macrophage colony stimulating factor (GM-CSF) ,16,17,18Two of the major differences between these trials and most cancer vaccine trials is that the starting point for the preparation of the vaccine was surgical resection of tumor, and a short-term cell line had to be established as the source of ATA. Patients were typically referred for possible vaccine because they had surgically-resectable regionally recurrent stage 3 or distant stage 4 oligometastatic disease, or they were undergoing resection of a metastatic lesion for diagnostic or palliative reasons . It was ® Protocol #20090753). All patients provided written informed consent prior to treatment. Details regarding these trials and the 72-DCV-treated patients were previously published [The DCV-treated patients had metastatic melanoma and were enrolled in either a single-arm phase II trial , or a randomized phase II trial . Both clinical trials were conducted per the principles outlined in the Declaration of Helsinki and were approved by appropriate institutional review boards including the Hoag Hospital Institutional Review Committee for the Protection of Human Subjects (first approved 12 January 2000), the U.S. Food and Drug Administration , and reviewers for the National Cancer Institute’s Physician Data Query  and the Western Institutional Review Board  (first approved 01 February 2006, WIRBublished ,18,19.The manufacturing of the DCV-ATA product was previously described in detail ,18,19. BThe identification of comparable patient cohorts was achieved by a review of clinical trials in clinical trials.gov and articles that were identified in PubMed. An emphasis was placed on randomized trials that tested immunotherapy treatments. Preference was for studies conducted during 2000–2011, to coincide with the time when the patients of interest were treated with DCV.There were no comparative statistical tests performed on the available data, because there were no analyses that were appropriate to perform.All of the stage 3 patients that were treated with DC-ATA had recurrent stage 3 disease, and many had recurred despite prior therapy. A comparable control group could not be identified from other trials, but some trials were limited to patients with stage 3 disease that had recently been eliminated by surgery. The comparative results for patients whose most advanced stage was 3 and had no measurable disease at the time of immunotherapy treatment, is shown in There was one large placebo-controlled randomized trial that explored the use of HLA-2 restricted peptides as a vaccine, with or without GM-CSF in patients who had been rendered free of disease by surgery . One of There were no survival comparisons among patients with minimal or no prior treatment for metastatic melanoma because DC-ATA was only administered to patients who had failed previous systemic therapies. However, in the past few years, numerous trials were conducted that tested anti-PD-1 in previously untreated or minimally treated melanoma patients, rather than heavily treated patients, and they have become the treatment of choice for first-line treatment. The results from these trials, as summarized in The results for anti-PD-1 in patients with surgically resected stage 3 or stage 4 were not included in This study shows that the survival results for DC-ATA quite favorably compare with other immunotherapies that were available or being tested during 2000–2011. However, DC-ATA was clinically tested in melanoma patients in an era before the widespread availability of anti-PD-1 monoclonal antibodies or BRAF/MEK inhibitors. In fact, only one of the 72 DC-ATA-treated patients was ever treated with an anti-PD-1 during the five years of follow up. In more recent years, anti-PD-1 for metastatic melanoma patients ,34,35,36In addition to the checkpoint inhibitors, in recent years talimogene laherparepvec was approved for intralesional injections of metastatic melanoma . Most ofThe major strength of this analysis is the focus on patient cohorts defined by stage and disease measurability at the time of treatment with immunotherapies. The most obvious weakness of this analysis is that it relies on comparisons to historical controls that are not perfectly matched, rather than direct comparison in randomized trials. However, an effort was made to match patients as closely as possible by stage and whether the disease was measurable at the time of treatment. Another weakness is the reliance on specific points such as median survival and two-year or five-year survival rates rather than a direct comparison of survival curves. Finally, the numbers of DC-ATA-treated patients in each of the subsets is rather small.The survival outcomes for melanoma patients that were treated with DC-ATA vaccine compared favorably to other immunotherapies available or being tested at that time. In terms of convenience and rapidity of clinical benefit, this product does not appear to be as active as anti-PD-1 agents. There is a strong rational for combining DC-ATA vaccine with anti-PD-1 agents in concurrent or sequential treatment strategies due to the different mechanisms of action and toxicity.