Consumers receive over 27 million antibiotic prescriptions annually in Australian primary healthcare. Hence, consumers are a key group to engage in the fight against antibiotic resistance. There is a paucity of research pertaining to consumers in the Australian healthcare environment. This study aimed to investigate the perspectives, attitudes and behaviours of Australian consumers on antibiotic use and antibiotic resistance, to inform national programs for reducing inappropriate antibiotic consumption.Semi-structured interviews with 32 consumers recruited via convenience and snowball sampling from a university population in South East Queensland. Interview transcripts were deductively and inductively coded. Main themes were identified using iterative thematic analysis.Three themes emerged from the analysis, to elucidate factors affecting antibiotic use: (a) prescription type; (b) consumer attitudes, behaviours, skills and knowledge; and (c) consumer engagement with antibiotic resistance. Consumers held mixed views regarding the use of delayed antibiotic prescriptions, and were often not made aware of the use of repeat antibiotic prescriptions. Consumers with regular general practitioners were more likely to have shared expectations regarding minimising the use of antibiotics. Even so, advice or information mediated by general practitioners was influential with all consumers; and helped to prevent inappropriate antibiotic use behaviours. Consumers were not aware of the free Return of Unwanted Medicines service offered by pharmacies and disposed of leftover antibiotics through household waste. To engage with mitigating antibiotic resistance, consumers required specific information. Previous public health campaigns raising awareness of antibiotics were largely not seen by this sample of consumers.Australian consumers have specific information needs regarding prescribed antibiotics to enable appropriate antibiotic use behaviours. Consumers also have expectations for high quality general practice consults conducted in a manner that increases consumer confidence in the treatment decision, regardless of whether an antibiotic is prescribed. To reduce inappropriate consumption of antibiotics and to more fully engage Australian consumers in mitigating antibiotic resistance, changes in health policy and practice are required. Every dose of antibiotic used increases the likelihood of antibiotic resistance . AntibioIn Australian primary healthcare over 27 million antibiotic prescriptions are issued annually . Hence, Consumers are aware of the link between antibiotic use and antibiotic resistance . HoweverTo date, qualitative studies have predominantly been conducted with consumers in Europe and the USA , 21–24, This study aimed to investigate the perspectives, attitudes and behaviours of Australian consumers recruited from a university campus in South East Queensland in May and June (early winter) 2015 about antibiotic use and antibiotic resistance, to inform national programs for reducing inappropriate antibiotic consumption. The findings of this study are pertinent to Objective 1 of Australia’s national antimicrobial resistance strategy — to “increase awareness and understanding of antimicrobial resistance, its implications, and actions to combat it through effective communication, education and training” .Pragmatism, understood as a problem-driven approach, was the underpinning philosophy for this study . The oneEligible participants were consumers between 18 and 54 years of age, residing or working within a 1 h drive from the Brisbane Central Business District. This age range was specified as it represents the peak years of the Australian workforce ; and 65%Individual interviews were conducted face-to-face and audio-recorded in May and June 2015, either at participants’ workplace or in a university meeting room. The length of interviews was between 30 and 59 min . Ethics approval was granted by the Queensland University of Technology. Informed written consent from participants was obtained prior to the interview. Participants were not paid for their time, but were offered appropriate incentives in line with university guidelines. Participants were also offered a summary report of research interviews.Interviews were conversational following Rubin and Rubin’s responsive interviewing technique . ParaphrThe quality of the co-created knowledge in an interview is contingent on the skill of the researcher (EL); where co-creation invokes the deliberate practice of the researcher in leading and being led by the participant as a conversation partner. Previous experience as a clinical pharmacist, skills in educational visiting, and active listening were used in the preparation and conduct of these interviews. The co-creation of knowledge via interviews minimises researcher bias as the backgrounds and worldviews of both researcher and participants, shape the research.Interviews were transcribed verbatim from the audio recording, using an adaptation of the Jeffersonian Transcription Notation . De-idena priori for deductive coding based on the main interview questions. An eclectic combination of coding methods was used for inductive coding: descriptive, initial, In Vivo, and theming the data  just like, ‘Oh just keep it in case you need it [delayed prescription].’ I’m like, what- ((makes an incredulous face)) … Well, how will I know if I need it?” Some consumers interpret the provision of a delayed prescription to mean that the GP was leaning towards the judgment that antibiotics were warranted. In such cases, consumers reported that they filled the prescription immediately (thus negating the GP’s intent for the delayed prescription) and commenced treatment without delay; unless the GP had given explicit instructions not to do so.Consumers not in favour of a delayed antibiotic prescription were overwhelmingly uncomfortable that the final decision whether to use antibiotics fell on them, rather than the GP. Although highly educated, these consumers did not want such responsibility. They were concerned that GPs did not, or were not able to, provide precise and definitive instructions on when to use the delayed antibiotic prescription:A repeat antibiotic prescription enables a patient to obtain another course of antibiotics without having to consult a GP. Repeat antibiotic prescriptions are authorised by the GP when issuing the original antibiotic prescription. These prescriptions are usually only issued if: (a) the GP intentionally prescribes a duration of treatment which requires more than one course of antibiotics, (b) when the dosage required means a repeat prescription is needed to obtain the correct quantity of tablets/capsules for a course of treatment, or (c) when a standby antibiotic prescription is needed in the event of an acute relapse/exacerbation e.g. patients suffering from chronic obstructive pulmonary disease.“I finish[ed] one [prescription] and I thought that’s all over you know, but I see that I’ve got a repeat. And so, you know, ‘Oh wait, do I need to do this?’ [fill the repeat prescription and take the antibiotics]. And then you call up [the clinic] and then by the time you get to the pharmacy again, sometimes you’ve lost time. To me, that was kind of annoying, when that happened. … I ended up getting the repeat. But it wasn’t until about three days [later], so there was a 3-day gap.” .Unused repeat prescriptions create a problematic “reservoir” of antibiotics accessible to consumers, much like unused delayed antibiotic prescriptions. Many consumers retained these prescriptions for future use, discarding them only when the prescription can no longer be dispensed. Antibiotic prescriptions are currently valid for dispensing within 12 months from the date of prescribing.When GPs do not explicitly discuss what to do with repeat antibiotic prescriptions — assuming one was given intentionally, let alone those issued unintentionally due to default settings in computer-generated prescriptions — consumers are left to decide for themselves how to act. Undesirable consequences may ensue, such as truncated treatment or an unintended gap in treatment.This theme groups together key sub-themes related to consumer attitudes, behaviours, skills and knowledge which influence antibiotic consumption: expectations for antibiotics, how consumers use antibiotics, self-management strategies for respiratory tract infections, and consumer perception of risks associated with antibiotic use.“But if you’re going to miss more than 1 day [of work] then I’ll go and see a doctor to get a medical certificate. … I’ll say to them to make clear that I don’t want antibiotics or anything. I don’t need to say that if I see my GP. Because she knows that.” Overwhelmingly, when describing what they expected from a GP consultation, consumers said they wanted to be listened to. Consumers also expected the GP to conduct a thorough clinical examination, explain their findings, treatment options, and decisions, and to answer any questions. These elements of the consultation taken together, increased consumer confidence in the professionalism, competence and trustworthiness of the GP. Most consumers would accept the GP’s decision not to prescribe an antibiotic if it was clearly explained:“I’d be disappointed that it couldn’t be treated [with antibiotics]. But I also understand that there’s no point in treating some things with antibiotics. … So if that was clearly explained, I think I’d be less disappointed in the care that I receive from the doctor. But of course, I would always be disappointed in the fact that you just have to like, tough it out through a sickness, because there’s no easy fix.” Consumers who have regular GPs tend to have shared expectations regarding overall management of their health and health outcomes. Informed consumers with established doctor-patient relationships reported having had preliminary conversations regarding their preference to avoid antibiotic use where possible. Although it was not apparent whether these conversations were GP or consumer initiated, it provided a basis for future GP consultations whenever the need for antibiotic use came into question:“… [the] doctor doesn’t comment much about when you stop it ). So, the assumption is, you stop when you feel good.” Middle-of-the-day doses were especially challenging to remember for antibiotics requiring a dosing frequency of three or more times a day. Some handled missed doses by working it in within the same 24-h period, doubling the antibiotic dose for their next dose, or skipping the missed dose resulting in taking longer to complete the course:“… I have no idea what effect that has, but I figured- I guess it’s better to finish it.” Others were proactive in anticipating the likelihood of forgetting doses and implemented strategies, such as phone apps as reminder systems.Consumers struggled to adhere to prescribed antibiotics. The most common behaviour was not completing the course of antibiotics, either due to a conscious decision to cease taking, or forgetting to take when they felt better as the main driver of adherence (feeling ill) is lost. Given that most consumers rely on GPs’ instructions to guide them on appropriate use of antibiotics, GPs who omit to provide clear advice may inadvertently contribute to misuse. For example, omission to provide advice on the duration of treatment led to non-completion of the prescribed course of antibiotics for this consumer:"Just keep [the leftovers]. Like next time somebody gets sick you start with that, you don’t even go to the doctor ((chuckles))." " … like when I was taking them [antibiotics] all the time, I would keep them [leftovers] because I would expect to take them very soon. … and then they run out of date … and I just had a big clean out last year ((chuckles)) and I just chucked them all out. And I probably should have taken them to the pharmacy but I didn’t, I just chucked it." Antibiotic mixtures/syrups were disposed of in household sinks. Most consumers were not aware of the Return of Unwanted Medicines (RUM) program for safe destruction of medicines offered at no charge by community pharmacies.Consumers reported the following undesirable behaviours: using leftover antibiotics from the last unfinished course the next time they were unwell with similar symptoms, sharing unused medications with other people, and disposing of expired unused/leftover antibiotics as part of household waste:"He [husband] would just say- ‘coz I guess he’s very against all the antibiotics and everything, and I think that’s probably another influence on me that I have shifted from that, because … I would come back from  with a big sack of medication ((smiles)). And he said, 'Oh you shouldn’t be doing this you know, you have to go and see the doctor' … and he said, 'you know the more you take [antibiotics], it’s not going to work anymore…' " The GP was another important influence, particularly for consumers who had established good doctor-patient relationship with a regular GP. For example, consumers reported that they were more likely to complete the course of antibiotics if the GP had explicitly instructed them to do so. Another consumer recounted that patient education from her GP, enabled her to recognise and resist inappropriate behaviours i.e. not accepting shared antibiotics from family and not using her own leftover antibiotics.Social influences were important in shaping consumer approach and behaviours in antibiotic use. Influences from family — parents (while growing up), friends and partner/spouse, were cited.“Because often I guess, those types of remedies like nasal sprays and … you know all those other types, they can … be a lot of them and they can be expensive [to purchase]. So whereas, antibiotics can just be quite affordable, in terms of treatment options … Like even just like cold and flu tablets are more expensive than antibiotics themselves.” The relatively higher cost of products for symptomatic management of coughs and the common cold may motivate some consumers to seek antibiotic treatment, which costs less. This was true of consumers for whom costs of healthcare was a key concern. For example, these consumers would prefer to use bulk-billing clinics as far as possible to reduce out-of-pocket costs.Consumers used the following strategies for symptomatic management of respiratory tract infections: home remedies, commercially available natural remedies, immune boosters, over-the-counter cough and cold products, increased rest and increased fluids. Consumers with underlying respiratory conditions such as asthma were especially mindful of following their asthma management plan during periods of intercurrent illness.Workplace culture can shape self-care behaviours. Some workplaces expect staff to “power through” minor illnesses such as coughs and the common cold, whereas other organisations deem it acceptable for staff to take a few days off to recuperate at home, and to minimise the spread of infections to colleagues.Consumers were realistic about feeling miserable and unwell during a common cold or cough, and would tolerate their symptoms for up to 3 weeks before seeking a GP consult. Self-care and self-management strategies were used in the meantime to alleviate their symptoms.“… so it would depend on my symptoms, but also what I have to do [life context]. So if I’m going through a period where I don’t have a lot on, then I wouldn’t mind so much suffering through symptoms. But if I have something that I need to do like a run [athletic event] or something important job-wise or something [like that], then I might seek out healthcare earlier. … I’ll just say persistence of the symptoms.” Consumers were more likely to seek a GP consult if: they were unable to take time off work/study to recover, they had to maintain a high level of functionality (both work and family commitments), they have an important upcoming event, they need a medical certificate to take time off work to recover, and persistence or worsening of symptoms.:“I’m pretty hesitant to take them [antibiotics], only because of the lack of information that I have received about what they actually do. And I prefer to find an alternative method to fixing what ails me ((chuckles)).” Cultivating a healthy immune system was another reason cited by consumers who were mindful of using antibiotics only when required:“… from a while ago I got the idea in my head that in order to build up your immune system well, you need to give it a chance to fight things on its own. It’s only if it’s clearly going to lose the battle that you should really help it with medication.” Consumers who had negative experiences with antibiotics were more reluctant to use them unless necessary. They reported experiencing side effects such as: adverse impact to digestive system for a prolonged period, feeling physically tired, and vaginal thrush. Consumers who had neutral experiences with taking antibiotics, for example those with no troublesome side effects and recovered with the treatment, were open to using antibiotics.Many consumers perceived antibiotics to be safe medicines; and hence, would lean toward taking the antibiotic if a delayed prescription was given. Others expressed their preference to avoid antibiotics where possible due to being unsure how the antibiotics worked, unwanted effects it could have on the body, and being concerned about becoming “resistant”. Many of these consumers considered themselves low users of medicines overall.This theme comprises three sub-themes which has an impact on consumer engagement with the fight against antibiotic resistance.Quote 1: “But it seems to be the same one that’s prescribed. So it would just be good if they [the GP] could provide the information of how that actually helps an ear infection and chest infection. You know … because they’re completely different. To me they’re completely different areas [the infections].” Consumers interviewed recognised that individuals can contribute to the fight against antibiotic resistance through responsible use of antibiotics. To help them do this, consumers wanted the following types of information: how the antibiotic worked; the rationale for selection of an antibiotic (quote 1); the duration of treatment, including when or whether the repeat antibiotic prescriptions (if issued) should be used; whether they can have alcohol while on antibiotics (quote 2); and the rationale for finishing the course of antibiotics or the consequences of not doing so, to encourage adherence to the treatment. Consumers noted that their information needs were often not voluntarily met by GPs or community pharmacists.Quote 2: “And so I looked it up because I have heard competing information. … And all of the information that I could find said, most antibiotics, doesn’t really do anything when you drink alcohol [not necessary to avoid alcohol], but these particular like, maybe two strains or something … stop you from processing alcohol properly … so … you only need to have one drink or less, and you can start vomiting and fainting and be really sick ((chuckles)). So the doctor didn’t tell me that much detail. He just said, don’t drink alcohol.” Consumers interviewed were largely not aware of the public campaigns, whether current or past, run by Government or Commonwealth funded agencies. Some were generally sceptical about news reported in mainstream media, and assumed that the issue of antibiotic resistance had been sensationalised. However, consumers were consistent in acknowledging that antibiotic resistance is a difficult topic with which to engage the public, as it is “invisible” and inconsequential for most people currently:“It’s tough to convince people about that [the importance of addressing antibiotic resistance]. It’s invisible … And it is real, but it’s invisible. So it’s very difficult to push the message across, people don’t see it you know. They think like, it doesn’t matter, I’m better today, that’s all that matters to me.” Consumer understanding of antibiotic resistance was conceptualised in four ways: (a) as a property of the body — body becomes resistant to antibiotics; (b) as a property of the medication — antibiotic is no longer effective; (c) as a property of the bacteria — bacteria is resistant to the antibiotic; and (d) as a property of a collective — society is immune to antibiotics. The following quotes illustrate the different ways consumers described antibiotic resistance.“… it’s [the antibiotic] just not effective for the body anymore. That’s obviously, you know, it’s like the body’s built up this immunity to it [the antibiotic] actually working.” Expressing antibiotic resistance as a property of the bacteria:“… bacteria … is getting stronger. … People don’t finish the doses … so … the first part of the treatment there are more weak bacteria [that] died, and then the stronger ones live and as you don’t finish [the course of antibiotics], only the stronger ones [survive] and genetically would be the best bacteria, and that’s what’s happened.” Expressing antibiotic resistance as a property of a collective:“… if it’s [antibiotics] prescribed for reasons which aren’t very serious, then as a society we get immune to the effects of those drugs [antibiotics].” Other misconceptions about antibiotics and antibiotic resistance gleaned from this consumer sample were: (a) many were not aware that antibiotic resistance could occur at both an individual and societal level, having assumed it was one or the other, (b) a few were unaware that using antibiotics inappropriately for the common cold and/or coughs can contribute to antibiotic resistance, and in time cause these antibiotics to be ineffective for the treatment of other more serious infections; and (c) most were unaware that resistant bacteria could be transferred from person to person or from food-producing animals to people.Expressing antibiotic resistance as both a property of the body and of the antibiotic:“I’m actually quite worried. ‘Coz even healthy people, when you travel around, one of the main reasons for spread of all these drug resistant strains has been human movement. … people can carry them [resistant strains of bacteria] with them, and not- [be sick], just a carrier. And then, you know, you go to a region that’s endemic for … these drug resistant bacteria, pick them up and you come back, and you disseminate it. And that’s how it spread[s]. So, yeah, I’m, I’m quite worried. And I hope if I do get a bacterial infection, it’s not drug resistant.” .On the other hand, some consumers were optimistic and confident that medical technologies to address or overcome resistant bacteria would soon be found:“I guess medicine and technology is advancing so quickly that they may be able to stamp them [resistant bacteria] all out …” ; and“Scientists out there will come up with something and they’re really clever, so I don’t worry too much because I think somebody’s solving the problem.” Consumers who had lived and/or worked overseas felt that Australia had been managing the issue of antibiotic resistance rather well. They note that antibiotics are regulated prescription medicines in Australia; they surmised and were optimistic that GPs acted as effective gatekeepers for antibiotic use in the community. There was also a sense of complacency, of being safe in a “first-world” country which conferred a false sense of impermeability to issues such as antibiotic resistance.Consumers were concerned as to whether antibiotic resistance could be adequately addressed in a timely manner; pointing out that preventing/reducing antibiotic resistance is preferable to having to find ways to resolve it. A well-informed minority were very concerned about the issue:Many consumers felt that GPs should play a more proactive role e.g. prescribe less antibiotics, educate patients when antibiotics are not required, and not succumb to patient demands for antibiotics. However, they acknowledged that consumers needed to be part of the solution. At the personal/individual level, consumers felt that by avoiding unnecessary antibiotics they were not worsening the problem of antibiotic resistance. When antibiotics were prescribed, consumers recognised that socially responsible behaviour on their part would constitute completing the course of antibiotics, so as not to encourage the growth of resistant bacteria. At the societal level, consumers reported attempting to influence their social and familial circle, albeit with mixed results.“… So we need to look after what we have, and not just hope that the next breakthrough is just around the corner. I certainly hope that it is. But … where does it stop? You might find a new antibiotic, well that one becomes resistant … so I think it’s a continual thing.” Consumers maintained a realistic view of having to tackle antibiotic resistance from multiple angles, highlighting the need for conservation of antibiotics now through individual efforts, while at the same time pursuing innovative approaches:This study adds to current literature in the field the perspectives, attitudes and behaviours of Australian consumers toward antibiotic use and antibiotic resistance. In particular, consumer information needs regarding prescribed antibiotics, consumer expectations and perceived trustworthiness of a GP consult, the importance of GP-mediated advice on antibiotic use behaviours, and consumer views and behaviours in relation to delayed antibiotic prescriptions.Australian consumers expect to be given information regarding prescribed antibiotics which would enable appropriate use and motivate adherence. Consumers want information on how the antibiotic worked; the GP’s rationale for antibiotic selection; treatment duration; the rationale for completing the course of treatment or the consequences of not doing so; and advice regarding alcohol consumption while on antibiotics. Consumers sought other avenues of information when not enough detail was provided as evidenced by a retrospective analysis of an Australian medicines helpline where over 40% of antibiotic-related calls were due to inadequate information provision . CommuniThe expectations of Australian consumers of a GP consult are made explicit in this study. Consumers want to be listened to; to be given a thorough clinical examination; to have the GP explain their findings, treatment options, and decisions including the decision not to treat with antibiotics when it is not warranted; and to have their questions answered. These elements of the consultation, when present, increased consumer confidence in the professionalism, competence and trustworthiness of the GP. Most consumers would accept the GP’s decision not to prescribe an antibiotic if it was clearly explained, which complements findings from other studies where patient satisfaction with clinic visits were not necessarily related to getting antibiotics , 37. ManGP-mediated advice exerted an important influence in encouraging desirable antibiotic use behaviours in consumers and preventing inappropriate behaviours, even as consumer behaviours in self-care and antibiotic use were shaped through the social influences of significant others (partner and family), friends, and workplace culture. Hence, GPs need to be empowered and skilled to communicate confidently and unambiguously to patients, especially when conveying the decision that an antibiotic is not warranted and discussing other management options. Public health campaigns can be used to support GPs in their antibiotic stewardship role, by reframing public perception as to what constitutes a “good” GP, contextualised for conservation of antibiotics. In this study, consumer expectations of a GP consult outlined earlier, mirrored the qualities of a “good” GP reported by Australian GPs — someone who: has the skills to deal with uncertainty; practices evidence-based medicine where evidence is available; has good communication skills; has the ability to establish rapport to build robust doctor-patient relationships; and makes treatment decisions in the best interest of the patient . Hence, Regarding delayed antibiotic prescriptions, consumers had mixed views on the use of this strategy when there was uncertainty in diagnosis. While some welcomed the convenience of this avenue of accessing antibiotics, others did not want the responsibility of being the final decision maker. Overall, if such prescriptions were issued consumers wanted specific instructions from GPs on when/whether to use the prescription; a finding which corroborates that of a recent Australian study focussed on delayed antibiotic prescribing .Consumer interpretation of the GP’s intention through the provision of delayed antibiotic prescriptions led some to assume that the GP was leaning towards the judgment that antibiotics were warranted. Such interpretation prompted consumers to immediately fill the prescription. This behaviour highlights the differences between consumer and GP interpretation and tolerance of clinical uncertainty; and negates the GP’s intent to take a “watch and wait” approach. Hence, while there is evidence that delayed antibiotic prescriptions could reduce antibiotic consumption , policy Unused antibiotic prescriptions create a problematic “reservoir” of antibiotics in the community, which could potentially be misused. Consumers reported retaining unused antibiotic prescriptions for future use, discarding them only when the prescription can no longer be dispensed. Hence, regulatory changes to the national medicines subsidy scheme to remove oral antibiotic repeats and to reduce the period of prescription validity should be enacted ; while eDespite the consumer sample being highly educated, there was variation in the level of knowledge, awareness and concerns regarding antibiotic use and antibiotic resistance; some of which were erroneous indicating that consistently clear messages from health professionals, public health campaigns, and media are needed. Australian consumers conceptualised antibiotic resistance in four ways. Three conceptualisations are similar to consumers in Europe , 15 and Given the findings of this study, questions remain on the efficacy and cost-effectiveness of public health campaigns in influencing consumer behaviour; and the extent of behaviour change possible through the re-engineering of social and cultural memes. Research in these areas should be supported.Semi-structured interviews captured participants’ knowledge, lived experience and views, in their own words; which helped overcome potential researcher bias and resulted in the co-creation of knowledge , 42. IntThe use of convenience and snowball sampling meant that only respondents with interest in the topic volunteered to participate. Other consumers may hold different views. Recruitment was limited to the university population which is not likely to be representative of the general population in terms of educational levels achieved. However, the university is a microcosm of diversity as demonstrated by the consumer sample i.e. multi-ethnic, multi-cultural, and includes people from rural/regional areas, ensuring that a variety of views were captured. This study is relevant to the Australian context; and as is the case with other qualitative studies, generalisability of the findings was not intended.Australian consumers expect information on prescribed antibiotics which enable appropriate use, and a GP consult conducted in a manner that increases consumer confidence in the treatment decision. To more fully engage consumers as partners in mitigating antibiotic resistance, consumer information needs regarding prescribed antibiotics must be addressed; shared expectations between consumers and GPs in avoiding the use of antibiotics should be encouraged; resources such as the Return of Unwanted Medicines service should be widely promoted; and the use of clearer terminology and the development of new emphases suggested by this study for public health campaigns should be supported. Regulatory changes to the national medicine subsidy scheme to remove oral antibiotic repeats and to reduce the period of validity for oral antibiotic prescriptions should be enacted. This study provided useful insight into the perspectives, attitudes and behaviours of Australian consumers towards antibiotic use and antibiotic resistance, and presented pertinent suggestions for Australian public health policy and practice.


The etiology of Parkinson’s disease (PD) may involve endogenous and exogenous factors. 1-Benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ), which was shown to be neurotoxic for dopaminergic neurons, is one of such factors, thus it can be used to construct an animal model of PD. In contrast, 1,2,3,4-tetrahydroisoquinoline (TIQ) and 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) produce neuroprotective effects acting as monoamino oxidase (MAO) inhibitors and free radical scavengers that reduce oxidative stress in the mammalian brain. In this study, we aimed to investigate the effects of neuroprotective compounds, TIQ and 1MeTIQ, on the dopamine release in vivo in an animal model of PD induced by chronic administration of 1BnTIQ (25 mg/kg i.p.). Using an in vivo microdialysis methodology, we measured the impact of both acute and chronic treatment with TIQ and 1MeTIQ (50 mg/kg i.p.) on 1BnTIQ-induced changes in dopamine release in the rat striatum. Additionally, the behavioral test was carried out to check the influence of repeated administrations of the investigated compounds on the locomotor activity of rats. The behavioral studies showed that the chronic administration of 1BnTIQ produced a significant elevation of exploratory locomotor activity, and both the investigated amines, TIQ and 1MeTIQ, administered together with 1BnTIQ completely prevented 1BnTIQ-produced hyperactivity. The in vivo microdialysis studies demonstrated that the chronic treatment with 1BnTIQ caused a significant and long-lasting increase in the dopamine release (approximately 300 %) to the extracellular space in the rat striatum, which was demonstrated in the basal samples 24 h after 1BnTIQ injection. The combined chronic administration of 1BnTIQ and the investigated compounds, TIQ or 1MeTIQ, completely antagonized the 1BnTIQ-induced essential disturbances of the dopamine releasing to the extracellular space in the striatum. In conclusion, we suggest that higher concentrations of 1BnTIQ in the brain produced distinct impairment in the dopamine release, whereas TIQ and 1MeTIQ  completely prevented 1BnTIQ-induced abnormalities in the function of dopamine neurons and restored the dopamine release to the control values. The rats used for the microdialysis study after surgery were housed for several days individually. The experiments were carried out between 09:00 and 16:00 h. Control rats were treated with an appropriate solvent (0.9 % NaCl).1BnTIQ was administered at a dose of 25 mg/kg intraperitoneally (i.p.) and chronically for 14 consecutive days. In the mixed group, TIQ or 1MeTIQ (50 mg/kg i.p.) was administered once 20 min before the last 1BnTIQ administration or was administered chronically 20 min before each 1BnTIQ injection.All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Bioethics Commission as compliant with the Polish Law. All experimental procedures were approved by the Local Bioethics Commission of the Institute of Pharmacology, Polish Academy of Sciences in Kraków.1BnTIQ and 1MeTIQ were synthesized (according to Cannon and Webster 1958) at the Department of Drug Chemistry of the Institute of Pharmacology, the Polish Academy of Sciences in Krakow. The purity of the compounds was verified by measuring the melting point, and the homogeneity was assessed on a chromatographic column. TIQ  was obtained commercially. The compounds were dissolved in a 0.9 % NaCl solution.Locomotor activity was assessed in actometers  linked on-line to a compatible IBM PC. Each cage (43 × 44 × 25 cm) perimeter was lined with an array of 15 × 15 photocell beams located 3 cm from the floor. Interruptions of the photocell beams were counted as a measure of horizontal locomotor activity which was defined as the distance traveled (in cm). Rats were given 25 mg/kg 1BnTIQ i.p. as their last dose (14-day chronic treatment group). In the combined groups, TIQ or 1MeTIQ (50 mg/kg i.p.) was given chronically 20 min before each 1BnTIQ administration. Subsequently, the animals were transferred to experimental cages, and locomotor activity  was recorded for 90 min and analyzed using the Auto-Track Software Program . Each group comprised seven animals.2, 1 mM MgCl2, 0.3 mM NaH2PO4, and 1.7 mM Na2HPO4 (pH 7.4), at a flow rate of 1.5 μl/min with a microinfusion pump . Samples were collected from freely moving rats in 20-min intervals after a 3-h wash-out period. 1BnTIQ was injected chronically (for 14 consecutive days) in a 25 mg/kg i.p., and dialysis samples were collected for 180 min. In the mixed groups, 1MeTIQ or TIQ (50 mg/kg i.p.) was administered 20 min before the last dose of 1BnTIQ (acute) or 20 min before each 1BnTIQ injection (chronic). All dialysates were immediately frozen on the dry ice (−70 οC), until they were used in a biochemical assay.Rats were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) and secured in a stereotaxic frame . Vertical microdialysis guide cannulas  were implanted in the striatum (STR) according to the following stereotaxic coordinates: A/P +1.0, L/M +2.5, and V/D −3.5 mm from bregma and the dura (G. Paxinos and C.H. Watson). Fourteen days after surgery, microdialysis probes were inserted into the cannulas, and the striatum was perfused with an artificial cerebrospinal fluid (aCSF), which consisted of 140 mM NaCl, 2.7 mM KCl, 1.2 mM CaClLevels of dopamine (DA) and its extraneuronal metabolite, 3-methoxytyramine (3-MT), were assayed in dialysates (20 μl) using HPLC with electrochemical detection as described below. The chromatograph Dionex Ultimate 3000  was equipped with C18 columns . The mobile phase comprised 0.05 M citrate–phosphate buffer (pH 3.5), 0.1 mM EDTA, 1 mM sodium octyl sulfonate, and 3.5 % methanol. The flow rate was maintained at 0.75 ml/min. Chromatographic data were processed using the Chromeleon Dionex computer programme (Germany). Dopamine and its metabolites were quantified by chromatograph peak height and compared with standards run on the day of the analysis. At the end of the experiment, frozen brains were examined histologically for correct probe placement. Each group consisted of six animals.Data obtained from the locomotor activity and microdialysis studies were analyzed by a two-way analysis of variance (ANOVA) for repeated measures, followed by Duncan’s post hoc test, if significant differences were detected.p < 0.01), while chronic administration of TIQ (50 mg/kg i.p.) induced a significant reduction in the locomotor activity of rats . In the combined group, TIQ completely antagonized 1BnTIQ-induced hyperactivity  administrations produced a significant increase in the horizontal locomotor activity of rats  the locomotor activity, while the chronic administration of 1MeTIQ (50 mg/kg i.p.) over 14 days induced a significant  decrease in this parameter. In the joint 1BnTIQ + 1MeTIQ treatment group, rats showed the response similar to that for TIQ, i.e., 1MeTIQ completely antagonized the 1BnTIQ-induced hyperactivity  in dopamine concentration in the synaptic cleft in the basal samples (from −60 to 0 min.) 24 h after chronic 1BnTIQ (25 mg/kg i.p.) administration. The last dose of 1BnTIQ fortified this effect approximately 200 %, and after 60 min, the concentration of dopamine returned to the value which was observed in the basal samples but it still was elevated about 300 % in comparison with the control group  . A statistical analysis demonstrated a significant increase  of the basal samples  Fig. a. A postles Fig. a. Howeveles Fig. a.p < 0.01) , but it increased the dopamine release after the last dose of TIQ (up to 500 %)  increase in the concentration of 3-MT in the rat striatum (up to 450 %). This effect was strongly potentiated in the combined treatment group (chronic 1BnTIQ + acute TIQ),   increase in the concentration of 3-MT in the rat striatum (up to 3500 %)   administration produced a huge and significant  of the basal samples  was observed Fig. b. IntereDuncan’s test indicated that chronic 1BnTIQ administration did not change the level of 3-MT Figs. a, b.Fig.p < 0.05) increase in the concentration of 3-MT in the rat striatum (up to 500 %). This effect was strongly potentiated in the joint treatment group when 1MeTIQ was given with the last dose of 1BnTIQ   increase in the concentration of 3-MT in the rat striatum (up to 2000 %)   administrations produced a huge and significant  damaged the dopamine storage mechanisms causing its non-physiological rise in the extracellular space in the rat striatum  inhibitor) significantly depleted striatal dopamine (Wąsik et al. In the combined treatment group, an acute dose of TIQ given before the last dose of 1BnTIQ only slightly reduced the effect of 1BnTIQ, whereas acute administration of 1MeTIQ produced a clear decrease in dopamine release Figs. a, 4a. InInterestingly, in both combined treatment groups, chronic treatment with TIQ or 1MeTIQ completely antagonized the 1BnTIQ-induced pathological increase in dopamine release in the basal samples Figs. b, 4b. SiSumming up the results, based on the in vivo microdialysis study presented in this paper, we suggest that both endogenous amines TIQ and 1MeTIQ can antagonize 1BnTIQ-induced clear disorders of dopamine release in vivo in rat striatum. Furthermore, these amines were able to restore the physiological functions of striatal dopamine neurons disturbed by chronic application of 1BnTIQ.


Mesenchymal–epithelial transition factor (MET) is expressed in gastric cancer and associated with poor clinical outcomes. We assessed activity, safety, and pharmacokinetics of emibetuzumab, a bivalent monoclonal anti-MET antibody that blocks ligand-dependent and ligand-independent MET signaling.This non-randomized, single-arm, Phase 2 study enrolled Asian patients with MET diagnostic positive advanced gastric adenocarcinoma. Emibetuzumab  was given on days 1 and 15 (28-day cycle). The primary endpoint was 8-week progression-free survival rate. Secondary objectives included safety, pharmacokinetics, overall survival, and change in tumor size.Tumors from 65 patients were immunohistochemically screened to enroll 15 MET diagnostic positive patients . Eight-week progression-free survival rate was 0.47 . Disease control rate was 40% . Median overall survival was 17.1 weeks . No serious emibetuzumab-related adverse events or new safety signals emerged. Grade ≥ 3 possibly drug-related adverse events were hyperkalemia, hyponatremia, and hyperuricemia (one each). Emibetuzumab’s pharmacokinetics profile was similar to that observed previously. MET expression and clinical outcomes were not obviously associated.Emibetuzumab was well tolerated with limited single-agent activity in advanced gastric adenocarcinoma. To determine serum concentrations of emibetuzumab, blood samples were collected before drug infusion and at the end of drug infusion on day 1 and day 15 during each of the first four cycles.The primary efficacy measure of the study was the PFS rate at 8 weeks + 3 days. The timepoint for PFS rate was set at 8 weeks + 3 days, because tumor measurement at 8 weeks was done 3 days before or after 8 weeks, but it is referred to hereafter as 8 weeks. The secondary measures were: disease control rate (DCR), overall response rate, duration of response, overall survival (OS), PFS, best percentage change in tumor size, drug safety [assessed by AEs], and pharmacokinetic properties of the drug (serum concentrations at each sampling point).H-score = 1 × (% of cells staining at 1 +) + 2 × (% of cells staining at 2 +) + 3 × (% of cells staining at 3 +) [H-score > 210 in the membrane; > 30% of cells staining at 3 + intensity in the membrane; H-score > 210 in the membrane; ≥ 80% of cells staining at ≥ 2 + intensity in the membrane; and 100% of cells staining at ≥ 2 + intensity in the membrane.For the exploratory biomarker analysis, MET protein expression in MET diagnostic positive patients was further categorized as high MET or low MET by different cut points for high expression. Levels of MET protein expression were assessed using a semi-quantitative scoring scheme that incorporated both the staining intensity and the percentage of cells displaying each level of staining intensity. These assessments were used to calculate the H-score for the cell membrane, as follows:  at 3 +) . The cutc-met and epidermal growth factor receptor gene (EGFR) amplification of tumor samples using two different definitions of each. First, the number of cells with amplification was analyzed and tumor samples were considered amplified if ≥ 40% of the cells counted had ≥ 4 signals. Second, the ratio of cells with amplification was measured and tumor samples were considered amplified if the average number of signals divided by the average number of signals from the chromosome enumeration probe (CEP7) was ≥ 2.Fluorescence in situ hybridization (FISH) was used to assess A sample size of 15 was determined, based on feasibility and the design of a previous Phase 1 study of emibetuzumab conducted in mostly Caucasian patients with advanced solid tumors (Study JTBA) . This saClinical activity and safety analyses were conducted for all patients who received at least one dose of emibetuzumab. For clinical activity parameters, point estimates and confidence intervals (CIs) were obtained. PFS rate was calculated using the Kaplan–Meier method. To address the primary objective, the lower limit of the two-sided 70% CI of the PFS rate at 8 weeks was compared with the threshold value of 0.40 to determine whether this limit was greater than the threshold value. For safety, AEs were summarized as the number and percentage of patients for whom each event was reported. SAS 9.2  was used for all statistical analyses.In total, 65 patients from 12 study sites  were prescreened for diagnosis of MET protein expression status. Of these patients, 22 were identified as being MET diagnostic positive (≥ 60% of tumor cells staining at 2 + or 3 + intensity for MET)—a MET diagnostic positive rate of 34% (22 of 65 patients). Seven of these 22 patients were not enrolled owing to patient decision (two patients), physician decision (two patients), sponsor decision , death due to study disease (one patient), or meeting the exclusion criterion of having an active fungal, bacterial, and/or known viral infection (one patient).All 15 enrolled patients had an initial diagnosis of gastric adenocarcinoma; the majority were male, and the median patient age was 63 years (Table The median number of cycles of emibetuzumab given (one or both of the day 1 and day 15 doses administered) was 2 (range 1–10), and the median number of cycles completed (day 1 and day 15 doses both administered) was 2. Six patients completed one cycle, three completed two cycles, two completed three cycles, two completed five cycles, and one completed nine cycles; only one patient did not complete any cycles. The study treatment was discontinued for all 15 patients, either because of PD (14 patients) or death due to study disease (one patient). Six patients had post-discontinuation therapy; for five patients, this was systemic therapy, and for one patient, it was radiotherapy.The PFS rate at 8 weeks—the primary endpoint of this study—was 0.47 . No complete responses (CRs) or partial responses (PRs) according to RECIST were observed. The best percentage change in tumor size ranged from − 22.4 to 32.1%, and shrinkage in target lesions was observed in three patients  experienced ≥ 1 AEs that were considered possibly study drug-related Table . The mosNo AEs led to treatment discontinuation or death. Serious AEs were observed in six patients (40%), of which none were considered related to emibetuzumab. For three patients (20%), there was a dose delay due to an AE, including one AE that the investigator considered at least possibly related to emibetuzumab . An abnormal value of QTcF (Fridericia’s QT correction) exceeding 500 ms and an increase of QTcF > 60 ms from baseline was observed in two Japanese patients at a single timepoint (one pre-dose and one post-dose). Both prolongations were observed at only one visit and no coinciding clinical symptoms related to QTc prolongation were reported .The serum concentration of emibetuzumab reached steady state in about 2–3 months  was categorized as high for four of these patients. Using FISH ratio and FISH count thresholds for c-met amplification , c-met was amplified for three of the four patients with high MET expression by IHC, and c-met was unamplified for all five of those with low MET expression by IHC. The association between high/low MET status and amplified/unamplified c-met status was significant, irrespective of whether the ratio or count definition was used for determining FISH amplification status .Tumor samples from nine patients were tested by both IHC and FISH. MET expression by IHC . This finding suggests that both FISH and IHC might be appropriate methods for identifying a similar patient population depending on the cut point used. However, exploratory biomarker analyses assessing different dichotomous cut points for MET expression did not reveal any clear statistically significant relationship between MET expression and clinical outcomes. In part, this may be because of the small sample size of 15 patients in this study, but could also reflect the need for additional or complementary biomarkers for patient selection.In our study, we observed a statistically significant association between c-met amplification and corresponding high MET protein expression (H-score ≥ 210), two did not show any reduction in target lesions following emibetuzumab treatment  has been reported in up to 6% of gastric cancer cases [c-met amplification, to overcome primary and acquired drug resistance, include combination therapies that target MET and EGFR, as well as therapies utilizing tetraspecific antibodies [c-met amplification, but no EGFR molecular alterations. In contrast, concomitant MET/EGFR inhibition resulted in complete tumor regression and prevented the onset of resistance in this cohort of patient-derived xenografts with c-met amplification, but no EGFR molecular alterations [Concomitant c cancer , 20. Amper cases , 22. Thetibodies –20. For erations .In addition, combination of standard of care chemotherapy regimens and antibodies targeting MET signaling have been studied. However, the results of late stage trials for the anti-HGF antibody rilotumumab and the monovalent anti-MET antibody onartuzumab in combination with chemotherapy did not show a survival benefit , 23, 24.Emibetuzumab had a favorable safety profile in our study as in the previous studies and there were no new unexpected safety findings. The reported emibetuzumab-related AEs were mostly mild or moderate in intensity and the most common were hypoalbuminemia (a known class effect for agents targeting MET or HGF) –27 and cThe pharmacokinetic profile of emibetuzumab in this Asian study population was similar to the profiles observed in the previous studies of emibetuzumab monotherapy, which were conducted in the United States with mostly Caucasian patients and supported a 2-week dosing schedule , 30. AnaOne limitation of this report is the small sample size, and the non-randomized, single-arm study design, which has known inferential deficiencies. However, the study design was appropriate for assessing the primary endpoint of PFS rate at 8 weeks in a limited number of patients to explore putative evidence of antitumor activity in the selected patient population.In conclusion, although the study did not meet its primary endpoint, emibetuzumab was well tolerated and showed limited single-agent activity in Asian patients with advanced gastric adenocarcinoma. Further investigation, including complementary biomarkers to identify patients with gastric cancer who are most likely to benefit from emibetuzumab single-agent therapy, is warranted. Given the favorable safety profile, combination strategies with other therapeutic agents in patients with MET-positive gastric cancer appear feasible and might provide strategies for improving the activity of emibetuzumab in this patient population in the future.


It has been suggested that ultra-rare diseases should be recognized as distinct from more prevalent rare diseases, but how drugs developed to treat ultra-rare diseases (DURDs) might be distinguished from drugs for ‘other’ rare diseases (DORDs) is not clear. We compared the characteristics of DURDs to DORDs from a health technology assessment (HTA) perspective in submissions made to the CADTH Common Drug Review. We defined a DURD as a drug used to treat a disease with a prevalence ≤ 1 patient per 100,000 people, a DORD as a drug used to treat a disease with a prevalence > 1 and ≤ 50 patients per 100,000 people. We assessed differences in the level and quantity of evidence supporting each HTA submission, the molecular basis of treatment agents, annual treatment cost per patient, type of reimbursement recommendation made by CADTH, and reasons for negative recommendations.We analyzed 14 DURD and 46 DORD submissions made between 2004 and 2016. Compared to DORDs, DURDs were more likely to be biologic drugs , to have been studied in uncontrolled clinical trials , and to have a higher annual treatment cost per patient . Also, submissions for DURDs were associated with a less robust evidence base versus DORDs, as DURD submissions were less likely to include data from at least one double-blinded randomized controlled trial  and have smaller patient cohorts in clinical trials . Furthermore, DURDs are less likely to receive a positive reimbursement recommendation , and low level of evidence was the major contributor for a negative recommendation.The results suggest that DURDs could be viewed as distinct category from an HTA perspective. Applying the same HTA decision-making framework to DURDs and DORDs might have contributed the higher rate of negative reimbursement recommendations made for DURDs. Recognition of DURDs as a distinct subgroup of DRDs by explicitly defining DURDs based on objective criteria may facilitate the implementation of HTA assessment process that accounts for the issues associated with DURD. Treatments for rare diseases (RDs) are increasingly the focus of drug developers, as reflected by the strong market growth of the ‘orphan drug’ sector . HoweverAn RD prevalence threshold of, for example, ≤ 50 per 100,000 people equally includes diseases that affect as few as 1 per 100,000 individuals as well as much rarer diseases that affect only a few individuals per million. Therefore, populations with a disease defined as ‘rare’ by the aforementioned prevalence threshold may differ in size by 500%. In a previous paper, we described the characteristics of all drugs for RDs that fit the definition of a prevalence of less than 50 per 100,000 people . SubsequIn this study, we sought to empirically compare the key characteristics related to clinical data, drug cost, and rates of negative reimbursement recommendations of submissions for drugs to treat RDs (DRD) made to the CDR in Canada from 2004 until 2016. With this study, we aim to enrich the discussion on the potential need to recognise DURD as a separate category from other types of RD for HTA purposes.www.cadth.ca) for all submissions made to the CDR in the study period. We excluded submissions for treatments of diseases with a prevalence of > 50 per 100,000 people. Where disease prevalence was not specified in a CADTH recommendation report, we sourced prevalence figures from Orphanet (www.orpha.net). Where a prevalence range was reported, we used the mean. Submissions that were withdrawn, under review at the time data collection, or for which a CDR recommendation was not available were also excluded.We conducted a search to update the dataset of DRD that we collected and reported on in a previous paper . BrieflyFor the purpose of this study, drugs for rare diseases (DRDs) were defined in line with the commonly used definition of DRDs, or ‘orphan drugs’, that treat diseases affecting ≤ 50 per 100,000 people , 9.TableWe extracted information relating to the following parameters from each submission that met the inclusion criteria:We determined whether the treatment was a small molecule or a biologic molecule, and what the mechanism of action is based on, e.g., whether the drug is an analog or an inhibitor of a particular molecule.orpha.net).Prevalence was determined from the reccomendation report or, if not available, using values for the disease type obtained from Orphanet , randomized vs. non-randomized, comparative vs. non-comparative, controlled vs. non-controlled), and whether an active comparator or placebo was included.The average cost of treating one patient for 1 year was captured if this information was available publicly. Where price or annual treatment costs per patient were not available, we calculated an average annual treatment cost based on the unit cost cited in the submissions and dosing schedules specified in the relevant product monograph. Where unit cost was not available, a web search of Canada’s public plans formularies or the manufacturer media releases using the generic or the trade name of the drug was conducted to determine if a treatment cost in Canada was publicly available. In addition, published incremental cost per quality adjusted life year figures were collected from the CDEC recommendations.Clinical only: in cases where high uncertainty and major limitations in the clinical evidence were the driving factors, or where there is methodologically sound evidence but the outcome may not show an incremental clinical benefit over available alternatives, or the lack of validation on surrogate outcomes prevents an assessment of potential clinical benefits.Cost only: in cases where cost-effectiveness is not demonstrated or cost is too high, as determined by the committee.Clinical and cost: in cases where a combination of both factors lead to the negative recommendation.If the recommendation was to ‘list’, ‘list with criteria and/or conditions’, ‘reimburse’, or ‘reimburse with criteria and/ or conditions’, it was classified as positive. If the recommendations was to ‘do not list’, ‘do not list at the submitted price’, or ‘do not reimburse’ it was classified as negative.Each negative recommendation was classified according to the reason(s) stated in the recommendation document published by CADTH, referring to insufficient clinical data and/or unacceptable treatment costs.P values were two-sided and values < 0.05 were considered as statistically significant. If a DRD submission was labeled as a ‘resubmission’, we excluded the original submission from the statistical analysis, resulting in the analysis of only unique submissions, in order to preserve independence of statistical observation and to prevent skewing of the results by duplication of observations. Observations with missing data were excluded from the specific analysis. Correlation between prevalence and annual cost was tested through the use of Spearman Rank Correlation.Descriptive statistics were used to characterize continuous variables, using mean and standard deviation (SD) for normally distributed data and, for data that was not normally distributed, the median and range were calculated. Categorical variables are presented as percentages. We used the Mann-Whitney U test to compare medians for continues variables. Categorical variables were compared using the odds ratio (OR), conditional maximum likelihood estimates and Fisher’s Exact test. In the study period of 2004 to 2016, we identified 67 submissions made to the CADTH CDR for drugs to treat diseases with a prevalence of ≤ 50 per 100,000 people, i.e. for DRDs, for which recommendations were issued. Of these, 16 (23.2%) were submissions for DURDs; the remaining 50 (76.8%) submissions were for DORDs. The relative proportions of the number of submissions made for DORDs and DURDs are illustrated in Fig. The annual number of submissions for DRDs (including resubmissions) for the period 2004 through 2016 is presented in Fig. Comparison of the variables studied in DURD and DORD submissions is shown in Table The total number of clinical studies considered in the CDR review for each submission was similar for DORDs and DURDs, ranging from 1 and 3 studies for the majority of submissions in both categories (85%). Nearly all submissions for DORDs (91.3%) contained data from at least one double-blind RCT, compared to only 57.1% of DURDs submissions, a statistically significant difference. Almost half of all DURDs submissions (42.9%) included data from non-randomized uncontrolled trials as their best level evidence, whereas only one submission (2.2%) in the DORD category contained a similarly low level of evidence. In addition to relying largely on non-randomized uncontrolled trial data, more than a third (35.7%) of DURDs submissions contained only evidence from clinical trials without a comparator, and one submission (7.1%) contained data generated with a historical control group. In contrast, only one submission (2.2%) in the DORDs category contained data from clinical studies without a comparator. We also found that half of DURDs submissions included evidence generated from clinical trials with placebo control. The median of the largest study size reported in each submission was statistically significantly smaller in the DURDs group  compared to the DORDs group . The median difference in the size of the largest study in the DURD compared to DORDs was −108 (95%CI -234 to −50) and statistically significant. Cost information was available for 13 (92.9%) DURDs submissions and 29 (63.0%) DORDs submissions, with a median annual cost per patient of CAN$330,395  for DURDs and CAN$52,596  for DORDs respectively. The difference in annual cost per patient between DURDs and DORDs was statistically significant, with a median difference of CAN$243,787 . Incremental cost per quality adjusted life-year was available for 2 (14.3%) DURDs submissions and 22 (47.8%) DORDs submissions, with a median inctemental cost per quality adjusted life-year of CAN$2,680,000  for DURDs and CAN$165,923.5 . The median difference and 95% confidence interval for in the incremental cost per quality adjusted life-year between DURDs and DORDs was not calculated due to limited number of obsrvations in the DURDs. Overall, submissions for DURDs received more negative recommendations than DORD submissions , and differences in the recommendation types were statistically significant.P value of 0.0003. Figure N = 42, missing = 18). The trend line for the annual cost increases sharply at the threshold of the disease prevalence range that corresponds to the definition we used for DURDs in this study.The Spearman’ rank correlation coefficient (Rho) between disease prevalence and annual cost per patient was −0.51 (95%CI -0.71 to −0.25), with a statistically significant Reasons for negative reimbursement recommendations are shown in Table The current study, compared data from submissions for DRDs made to the CADTH CDR in Canada between 2004 through to 2016 to identify potential differentiating factors between DURDs and DORDs that could be utilized to optimize HTA of DRDs. A summary of the distinguishing characteristics between DORDs and DURDs are listed in Table Our study has shown that there are significant differences between submissions for DURDs and DORDs. In particular, efficacy evidence submitted for DURDs tended to derive from smaller clinical studies, often of an uncontrolled design. Notwithstanding that more than half of DURD submissions did include a double-blind, randomized placebo-controlled trial, the quality of supporting clinical data was often cited as the reason for a higher rate of negative recommendations than for DORD submissions. These observations are likely a reflection of the difficulty in recruiting patients to clinical studies of ultra-rare diseases due to very low disease prevalence and suggest that the level of clinical evidence required for DURDs should be less exacting than that for DORDs. Our findings suggest that it may be inappropriate to apply the same appraisal standards to DURDs and DORDs. The current practice of using the same HTA appraisal processes for DURDs and DORDs could account for the relatively higher rate of negative reimbursement recommendations for DURD submissions observed in this study.Thus, a distinct HTA process for DURDs that also considers benefits of a DURD which may not currently be captured in a cost-effectiveness analysis could be more appropriate to fully characterize the added value of a DURD. Several major international jurisdictions have recently introduced specific HTA frameworks for DRDs alongside established HTA processes for non-rare diseases –10. In CIf DURDs were to be classed as a category of drugs that is distinct from DORDs, the definition of what constitutes a DURD would be a crucial factor. In this study, we defined DURDs as diseases that affect ≤ 1 in 100,000 people, which differs from the prevalence threshold cited in other published definitions  (8-10). However, we found that the disease prevalence in the DRD submissions examined for this study were generally classifiable around the 1 in 100,000. We, therefore, considered a prevalence threshold of 1 in 100,000 individuals the most appropriate and practicable cut-off for the definition of a DURD. The consideration of prevalence alone to define DURDs would nevertheless be problematic for several reasons, all of which apply to current definitions of DRDs. First, prevalence is essentially an arbitrary metric. Second, prevalence for a disease may vary among different geographic locations both within and among countries . Third, The parameters included in the definition of a DRD are of wider relevance: to date, DRDs have been considered by some stakeholder groups as being largely exempt from cost-related restrictions commonly placed on drugs for non-RDs, including ‘traditional’ cost-effectiveness thresholds –17. InstRawson  reanalyzAlthough a distinct HTA review process for DURDs might lead to the expectation of improved access to DURDs, the same dilemma would face policy makers of not having access to high-level evidence to support the clinical effectiveness of a DURD. In their decision-making about the commissioning of a potentially high-cost life-long therapy, policy makers and public payers strive towards minimising uncertainty with respect to the clinical effectiveness and cost data. In addition, an ethical question might arise in that accepting low level evidence could expose patients to harm from potential adverse events whilst having little certainty of clinical benefits . Also, iOur study has several limitations. First, we limited our review to submissions made to the CADTH CDR in Canada. Therefore, our results might not be generalizable to other jurisdictions, although it should be noted that the clinical evidence used to support reimbursement submissions in Canada is largely identical to that used in other countries, and Canadian pricing rules usually ensure that drug prices are within the range of international prices. Therefore, the CADTH CDR is a reasonable proxy for other HTA agencies, although similar studies of other HTA bodies would be needed to confirm our findings.Second, the number of DRD submissions that we identified was relatively small and therefore decreases the robustness of the comparative statistics. However, this limitation reflects the nature of DRD reimbursement assessments: while the number of DRD reimbursement submissions has been continuously on the rise in recent years, there are still relatively few DRD submissions compared to submission for technologies that treat less rare diseases.Third, as noted above, prevalence for a disease may vary across different geographic locations both within and across countries; therefore, a disease classified as rare in our study might not be considered rare elsewhere. Similarly, there might be diseases that are considered rare in some regions that were not classified as rare in our study.Finally, as we used an explicit and arbitrary prevalence-based threshold to define DRDs in our study, application of a different threshold may have resulted in different findings. Therefore, our results might not be applicable to jurisdictions that have a substantively different definition of ‘rare disease’.DORDs and DURDs are similar in terms of the number of clinical studies used to support HTA submissions. In contrast, HTA submissions for DURDs differ from those for DORDs in some key areas: study sizes for DURDs are smaller, reliance on uncontrolled trials is more frequent, DURDs are more likely to be complex molecules (biologics), and the cost of DURDs is higher. These factors may all have contributed to a higher rate of negative reimbursement recommendations observed for DURDs compared to DORDs. Recognition of DURDs as a distinct subgroup of RDs may facilitate the development of HTA assessment processes that appropriately account for the inherent limitations that appear to be unique to DURDs. Based on the prevalence threshold applied in our study, we suggest that DURDs could be defined as diseases that affect ≤ 1 patients per 100,000 people, and that this prevalence threshold is combined with additional objective and descriptive criteria.


Oxidative stress contributes to the pathophysiology of a variety of diseases, and circulating biomarkers of its severity remains a topic of great interest for researchers. Our peptidomic strategy enables accurate and reproducible analysis of circulating proteins/peptides with or without post-translational modifications. Conventional wisdom holds that hydrophobic methionines exposed to an aqueous environment or experimental handling procedures are vulnerable to oxidation. However, we show that the mass spectra intensity ratio of oxidized to non-oxidized methionine residues in serum tryptic proteins can be accurately quantified using a single drop of human serum and give stable and reproducible results. Our data demonstrate that two methionine residues in serum albumin (Met-111 and Met-147) are highly oxidized to methionine sulfoxide in patients with diabetes and renal failure and in healthy smokers versus non-smoker controls. This label-free mass spectrometry approach to quantify redox changes in methionine residues should facilitate the identification of additional circulating biomarkers suitable for predicting the development or progression of human diseases. Because the former have very short half-lives and show high chemical reactivity, free radical processes have been monitored by detecting the latter. The identification of clinical biomarkers that accurately evaluate the severity of oxidative stress remains an important unmet challengeOxidative stress is a common component of the pathophysiology of a variety of human conditions and diseases, including aging1In vitro Met oxidation is a reversible process1012141410Methionine (Met) oxidation is a mechanism by which proteins perceive oxidative stress and function in redox signalingWe have established a series of new technologies to enrich and analyze circulating low-molecular-weight peptides suitable for mass spectrometry analysis and have comprehensively identified native and tryptic peptides in the human peripheral circulation202122E. coli β-galactosidase peptides ) during the processes of reductive alkylation, trypsin digestion and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS). We quantified signal intensities of peptides with Met(O) ([Met(O)]) and those with corresponding unoxidized Met ([Met]) using extracted ion chromatograms of LC-MS analysis and found that there were no significant differences in [Met(O)]/[Met] ratios when quantified before and after processing of human serum samples containing tryptic peptides .We next determined whether [Met(O)]/[Met] for serum tryptic peptides is stable and can be quantified reproducibly using the study workflow summarized in To accurately quantify the frequency of Met(O) residues relative to their corresponding unoxidized residues, complete trypsin digestion is mandatory prior to LC-MS analysis. Trypsin treatment reduced the number of undigested products as a function of time, at least during the initial period (data not shown), but from 18 hours onwards, mis-cleavage products were undetectable and did not significantly affect [Met(O)]/[Met] in any of the four candidate serum tryptic peptides . TherefoWe next tested whether the rate of spontaneous oxidation of Met residues in serum proteins would be affected by the length of clotting time necessary to obtain serum, given that withdrawn blood samples are left for at least 30–60 min at room temperature for a clot to form and residual cellular elements and other contaminants to be removed. To investigate this, blood samples were either immediately centrifuged after blood withdrawal, or allowed to clot at room temperature for 30 min to 6 hours before centrifugation, and the sera obtained were processed for reductive alkylation, trypsin digestion and subsequent LC-MS analysis. We found that [Met(O)]/[Met] of the four tryptic peptides were very similar, irrespective of the time the blood samples were left at room temperature .We also compared [Met(O)]/[Met] of the four peptides before and after freeze/thaw procedures. Repetition of freeze-thaw up to four times did not affect [Met(O)]/[Met] levels in any of the peptides . Thus, sBaseline [Met(O)]/[Met] of the four candidate serum tryptic peptides in healthy non-smokers ranged from 0.252 ± 0.057% ) to 2.511 ± 0.814% (Igγ1 chain C region (Met-135)), while that of complement C3 (Met-1118) was always higher than 99.9% in all subjects. Thus, distinct baseline oxidative statuses are apparent for each methionine residue. [Met(O)]/[Met] levels of the other four peptides determined nine times a day  in four healthy volunteers showed minimal diurnal fluctuations .We determined [Met(O)]/[Met] of these four peptides in six healthy smokers before and after cigarette smoking. Acute smoking did not affect [Met(O)]/[Met] of any of the four peptides . In addiWe next measured [Met(O)]/[Met] levels in 23 patients with type 2 diabetes, 12 patients with diabetes-related chronic renal failure and nine healthy smokers, and compared these with levels in 18 healthy non-smokers . InteresBecause high glycemic variability induces more oxidative stress than continuous high glucoseMet is readily oxidized to Met(O), and this reaction is believed to be reversibly catalyzed by the methionine sulfoxide reductases272930Three lines of evidence support this proposal. First, [Met(O)]/[Met] in some serum tryptic proteins were accurately determined using mass spectrometry in a highly reproducible manner. Although baseline steady-state [Met(O)]/[Met] levels detected in the current study widely varied among particular methionine residues, the values were neither affected by clotting time at room temperature for serum separation, nor by serum handling procedures, such as reductive alkylation, trypsin digestion, repeated freeze-thaw, and subsequent mass spectrometry analysis. Second, [Met(O)]/[Met] in all four candidate serum tryptic proteins showed limited diurnal fluctuation and negligible acute changes after smoking or ingestion of vitamin C, a well-known physiological antioxidant. Third, two Met residues in serum albumin showed elevated [Met(O)]/[Met] in patients with type 2 diabetes, diabetes-associated chronic renal failure and smokers, reflecting the altered oxidative stress status present in these conditions. Thus, some Met residues in serum proteins may undergo oxidation far more easily than other residues in response to underlying diseases or conditions.Experience in many areas of redox research highlights how challenging it can be to identify clinical biomarkers reflecting endogenous oxidative stress status. The experimental and analytical performance of [Met(O)]/[Met] measurement was reproducible and robust, irrespective of the time of blood withdrawal, the time the sample was left at room temperature before centrifugation or repeated freeze/thaw cycles. Such results may be attributable to a previously reported feature of the ubiquitously expressed enzymes regulating Met(O) levels. Met(O) reductase, for example, may not function in the presence of circulating proteins19Our LC-MS approach has overcome well-recognized technical difficulties associated with quantifying redox changes in circulating proteins. Incomplete digestion of serum proteins affected [Met(O)]/[Met] due to differential digestion efficiency between unoxidized Met and Met(O), but we have achieved complete trypsin digest by using phase-transfer surfactant plus Lys-COur data showed distinct variation in the baseline steady-state oxidation status between candidate Met residues. For example, Met-111 and Met-147 of serum albumin showed far lower baseline [Met(O)]/[Met] than Met-1181 of complement C3, but these residues were clearly more oxidized in patients with type 2 diabetes and renal failure. Met-111 of serum albumin showed distinctly higher [Met(O)]/[Met] in diabetic patients with greater glycemic excursion than those with minimal variability. These results indicate that these Met residues are susceptible to oxidation by factors that are associated with the pathophysiology of such diseases. Accumulating evidence indicates elevated oxidative stress in diabetes3435373840In conclusion, we have developed a novel label-free mass spectrometry approach to quantify Met oxidation in proteins present in the human peripheral circulation. Oxidized Met levels were accurately and reproducibly determined in a single drop of human serum. [Met(O)]/[Met] levels of two residues in serum albumin were elevated in type 2 diabetes, diabetes-associated chronic renal insufficiency and chronic smoking. This strategy could also be applied to identify other Met-containing serum proteins whose status reflects the severity of pathophysiological conditions or the likelihood of a patient developing a major disease in the future.The study population consisted of 18 healthy non-smokers (9 men and 9 women), 23 patients with type 2 diabetes (14 men and 9 women), 12 patients with non-dialyzed chronic renal failure associated with type 2 diabetes (9 men and 3 women), and 9 healthy smokers (9 men) . Type 2 44g for 15 min at room temperature. Aliquots were stored at −30 °C until use.Venous blood samples were collected from healthy volunteers and patients with type 2 diabetes with and without impaired renal function. Type 2 diabetic patients provided an additional serum sample at the time of scheduled blood sampling on the last morning of 72-h CGM recording using the CGMS system GOLD 4643Trypsin digestion of serum proteins was performed essentially as described18 0.075 × 20 mm trap column  and then eluted onto a C18 0.075 × 120 mm analytical column , configured to an EASY-nLC 1000 HPLC system (Thermo Fisher Scientific). The flow rate of the mobile phase was 300 nL/min; mobile phase A consisted of 0.1% formic acid (FA) and mobile phase B consisted of 0.1% FA/90% ACN. The mobile phase gradient was programmed as follows: 5–25% B (0–48 min), 25–50% B (48–58 min), 50–95% B (58–60 min), and 95% B (60–70 min). Separated peptides were introduced from the HPLC to a Q-Exactive (Thermo Fisher Scientific) operating in data-dependent mode, to automatically switch between full-scan MS and MS/MS acquisition. Full-scan MS spectra  were acquired in an Orbitrap instrument with a mass resolution of 70,000 at m/z 200, after accumulation of ions to a 1 × 105 target value. Fractions representing the 12 most intense full-scan peaks with charge state 2–4 were selected using an isolation window of 2.4 Da and fragmented in the high-energy collisional dissociation cell at a normalized collision energy of 27%. MS/MS spectra were acquired by the Orbitrap mass analyzer with a mass resolution of 17,500 at m/z 200, after accumulation of ions to a 1 × 105 target value. The ion selection threshold was 2 × 104 counts, and the maximum allowable ion accumulation times were 120 ms for full MS scans and 200 ms for MS/MS spectra. Typical mass spectrometric conditions were as follows: spray voltage 2 kV, no sheath or auxiliary gas flow, capillary temperature heated to 250 °C and dynamic exclusion time 15 s.Tryptic digests of serum samples were injected onto a CDatabase searches were performed using the SEQUEST algorithm incorporated into Proteome Discoverer 1.4.0.288 software (Thermo Fisher Scientific). The search parameters were as follows: enzyme, trypsin; variable modification, oxidation of Met residue; variable modification, carbamidomethylation of Cys residue; peptide ion mass tolerance, 6 ppm; fragment ion mass tolerance, 0.02 Da; peptide charges, +2 to +8. The identified peptides were searched for in the decoy database and the false discovery rate was set as 0.01 using Percolator scoring with posterior error probability validation. Peptide quantitation was also performed using Proteome Discoverer 1.4.0.288.m/z 300–2,000) were acquired using an Orbitrap instrument at a mass resolution of 30,000 at m/z 400.To quantify [Met(O)]/[Met], digested peptides were injected onto a 2.0 mm (inner diameter) × 50 mm CAPCELL PACK MGIII-H S3 column attached to a Nanospace SI-2 HPLC system . The column temperature was maintained at 45 °C. The flow rate of the mobile phase was 200 μL/min; mobile phase A consisted of 0.05% FA and mobile phase B consisted of 0.05% FA/90% ACN. The mobile phase gradient was programmed as follows: 0% B (0–3 min), 0–55.5% B (3–40 min), 55.5–80% B (40–40.1 min), and 80% B (40.1–45 min). Peptides were introduced from the HPLC to an LTQ-Orbitrap Discoverer. Full-scan MS spectra ]/[Met] value as an objective variable. All analyses were performed with JMP ver. 5.0.1a . P < 0.05 was considered statistically significant.Data are expressed as mean ± SD unless stated otherwise. Differences among groups or time-course changes in [Met(O)]/[Met] levels were analyzed using ANOVA. How to cite this article: Suzuki, S. et al. Methionine sulfoxides in serum proteins as potential clinical biomarkers of oxidative stress. Sci. Rep.6, 38299; doi: 10.1038/srep38299 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


ARQ 197-215 was a randomized placebo-controlled phase II study testing the MET inhibitor tivantinib in second-line hepatocellular carcinoma (HCC) patients. It identified tumor MET as a key biomarker in HCC.MNNG-HOS transforming gene) and circulating  biomarkers in second-line HCC. Tumor MET-High status was centrally assessed by immunohistochemistry. Circulating biomarkers were centrally analyzed on serum samples collected at baseline and every 4-8 weeks, using medians as cut-off to determine High/Low status. Tumor MET, tested in 77 patients, was more frequently High after (82%) versus before (40%) sorafenib. A significant interaction (p = 0.04) between tivantinib and baseline tumor MET in terms of survival was observed. Baseline circulating MET and HGF (102 patients) High status correlated with shorter survival , while the association between AFP (104 patients) or VEGF (103 patients) status and survival was non-significant.Aim of this research was to study the prognostic and predictive value of tumor (MET, the receptor tyrosine kinase encoded by the homonymous Conclusions: Tumor MET levels were higher in patients treated with sorafenib. Circulating biomarkers such as MET and HGF may be prognostic in second-line HCC. These results need to be confirmed in larger randomized clinical trials. Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related death , and sorMNNG-HOS transforming gene (MET) encoding the receptor tyrosine kinase for hepatocyte growth factor (HGF) is involved in cancer progression and metastasis formation, and suggests poor prognosis in early stage and second-line HCC patients [The patients -8. Hypoxpatients , 9, 10, patients , 12. METpatients . MET is patients , 15.Tivantinib is an oral, ATP-independent inhibitor of MET, binding to its inactive form , 17; itsFollowing promising phase I study results with tivantinib as monotherapy and in combination with sorafenib in HCC , 33, theConsidering the lack of accepted biomarkers in HCC and realizing the necessity of placebo-controlled studies, we analyzed the prognostic and predictive values of tumor (MET) and circulating , vascular endothelial growth factor (VEGF)) biomarkers in the ARQ 197-215 study.Tumor samples of sufficient quality for tumor MET analysis at immunohistochemistry (IHC) were submitted for a total of 77 patients, 49 who were randomized to tivantinib and 28 to placebo [N = 55), and 82% if obtained after sorafenib  were found to be MET-High. Biopsy date was known for 72 samples. When samples were analyzed with respect to prior therapy, the chance of being MET-High was 40% if the biopsy was obtained before sorafenib . Tivantinib was ineffective in patients with MET-Low tumors , suggesting that tivantinib may offset the negative prognostic impact of high MET expression . Correlation between MET and other baseline characteristics was not detected except for a slight overlap between MET-High patients and patients with a baseline AFP higher than the median.The test for interaction between treatment and MET status showed a statistical significance at an alpha level of 0.05 for OS . The median MET concentration at baseline was 13.26 ng/mL, with a range of 1.29-49.8 ng/mL. Eighty-six patients  who were evaluable for survival were also evaluable for changes in circulating MET over the course of the study. Baseline characteristics were generally balanced between groups, except for higher prevalence of hepatitis C virus (HCV) infection  in the circulating MET-High compared to the MET-Low group  and 3.8 months on placebo (N = 15), . The OS in circulating MET-Low patients was 7.5 months on tivantinib (N = 32) and 9.4 months on placebo (N = 19), . Such an advantage was evident by week 8 of therapy . No such trend was observed in patients receiving placebo . The baseline median HGF concentration was 2307 pg/mL, with range of 421-58080 pg/mL. Eighty-six patients  who were evaluable for survival were also evaluable for changes in circulating HGF over the course of the study. Baseline characteristics were generally balanced between groups  but not for patients on placebo . Furthermore, the test for interaction shows no correlation between HGF and OS benefit with tivantinib.Patients with a baseline HGF lower than the median survived longer than patients with a higher baseline HGF regardless of the therapy . No difference in OS was observed when comparing patients with and without HGF reduction on tivantinib  or placebo . In patients with stable disease past the first tumor assessment, median change from baseline HGF was +28.2%  regardless of treatment.Patients with a reduction over time by at least 10% in circulating HGF survived longer than patients with no or minimal reduction . The baseline median AFP concentration was 186 IU/mL, with range of 1.5-440008 IU/mL. Baseline characteristics were generally balanced between groups . To understand if higher AFP values have a greater prognostic impact, an additional analysis was performed using an arbitrary cut-off value equal to the AFP 75th percentile (third quartile (Q3); 3507.50 IU/mL) resulting in longer OS for patients with baseline values lower than Q3 .A trend towards better outcome was seen in patients with a baseline AFP lower than the median, regardless of treatment allocation  had baseline values ≥20 IU/mL, had samples at multiple time points, and were evaluable for OS; therefore, no analysis was performed on AFP changes over time.versus placebo by any AFP status was comparable, with the test for interaction resulting non-significant.Survival of patients on tivantinib N = 51), 5.0 months in patients with VEGF-High (N = 52) ; no trend was observed by treatment arm. Survival of patients with a VEGF reduction over time by at least 10% tended to be longer than survival of patients with no or minimal reduction , regardless of treatment.Circulating VEGF was measured in 103 patients also evaluable for OS . Median baseline circulating VEGF was 160 pg/mL. Baseline characteristics were generally balanced between groups  enrolled only patients with tumor MET-High HCC. Such studies may clarify the role of MET, HGF, and AFP in this patient population.MET expression was evaluated by IHC assay, on the most recent sample available, using the CONFIRM™ anti-total MET(SP44) antibody , analyzed at an independent Clinical Laboratory Improvement Amendments (CLIA) certified laboratory by a board certified pathologist after randomization and prior to study un-blinding. MET-High was defined as a majority (≥50%) of tumor cells with moderate or strong (2+ or 3+) staining intensity per ArQule's protocol. A negative control slide from each patient sample was run as well as positive control from colon adenocarcinoma ensuring that proper antibody and reagent dispensing took place. Additionally, results of MET testing were reported as an H-score, calculated by multiplying the percentage of cells staining by the intensity of the stain .Serum biomarker concentrations were measured by ICON Central Labs, Farmingdale, NY, using commercial materials.MET, HGF and VEGF serum samples were collected before the first dose on cycle 1 day 1, and post dose every 4 weeks thereafter, and at treatment end. One blood collection tube was used for MET/HGF testing and another for VEGF testing.For circulating MET measurement, Kamiya Biomedical K-ASSAY Human MET ELISA kit , Automated ELISA System (Dynex Technologies), and the Spectramax reader (Molecular Devices) were used. The Quantikine Human HGF Immunoassay  was used to measure HGF levels in serum using the Spectramax reader (Molecular Devices). For VEGF, QuantiGlo Human VEGF immunoassay kit from R&D  using the Spectramax Plus 384 reader was used.AFP serum samples were collected at screening/pre-study, and every 8 weeks after randomization, as well as at the end of treatment. AFP levels were tested using automated analyzer Immulite 2000 .th percentile. As for the changes in biomarker concentrations, the best change (highest reduction or the lowest increase) during the treatment period for each patient was calculated as the percent change from baseline, and 10% was arbitrarily chosen as cut-off value. Survival analysis by biomarker reduction was also performed restricting the sample to patients who survived to a pre-specified arbitrary landmark time of 8 weeks after the date treatment was started. This latter analysis was specifically undertaken to eliminate guarantee-time bias and was proposed by others in similar settings [All statistical analyses were performed using SAS statistical software. Biomarker baseline values and their changes over the course of treatment were dichotomized and analyzed with respect to OS probability. Median OS and the 95% CI were estimated using the Kaplan-Meier method and the logrank test was used to compare the survival distributions. The Cox regression model was used to obtain the HR. The interaction between biomarkers and the treatment was evaluated using the Cox proportional hazards model with an interaction term. Multivariate analysis was not attempted due to the small sample size of available biopsies for tumor MET biomarker assessment. The correlation between the biomarkers was assessed using Fisher's exact test. The median concentrations of the serum baseline biomarkers were used as the cut-off values to differentiate the respective Low and High groups for the study of prognostic and predictive effects. However, in the case of AFP, an additional arbitrary cut-off was set at the 75settings , 48.