text:
Investigating Court Accommodations for Persons with Severe Communication Disabilities: Perspectives of International Legal Experts
Authors: {'first_name': 'Robyn', 'last_name': 'White'},{'first_name': 'Ensa', 'last_name': 'Johnson'},{'first_name': 'Juan', 'last_name': 'Bornman'}
Globally, persons with disabilities, specifically individuals with severe communication disabilities, require a range of court accommodations to enable them access to the court system, thereby realizing their right to access to justice. This study aimed to investigate the perspectives of nine international experts on possible universal court accommodations for these individuals. An asynchronous, online focus group discussion with four questions was conducted over five days. Through deductive thematic analysis, four themes related to Article 13 of the CRPD were identified: Accommodations related to procedural fairness (e.g., testifying via CCTV camera); accommodations related to ensuring equality (e.g., specific international and local legislation); accommodations related to non-discrimination (e.g., developing appropriate questioning techniques); and accommodations related to legal practitioners (e.g., disability sensitivity training). The range of identified court accommodations could act as the impetus needed to ensure access to justice, a basic human right, for persons with disabilities internationally.
Keywords: Convention on the Rights of Persons with Disabilities (CRPD), court accommodations, disability, equality, human rights, severe communication disabilities, procedural justice
DOI: http://doi.org/10.16993/sjdr.779
Accepted on 13 Aug 2021 Submitted on 18 Nov 2020
For centuries, minority groups, such as persons with disabilities, have faced discrimination, inequality, and countless barriers when attempting to claim their basic human rights, such as access to justice, healthcare, and education (Beqiraj, Mcnamara & Wicks 2017; Bossuyt 2015; White et al. 2020b). Within the already marginalised sphere of disability, the most vulnerable group are those individuals with severe communication disabilities as they are unable to rely on spoken language not only to make their needs and wants known, but also to protect themselves and to be safe (Bryen 2014). The term severe communication disabilities (also known as complex communication needs or being nonverbal) describes persons from different socio-economic, ethnic, and racial backgrounds, across the age range, whose disabilities stem from a range of diagnoses, such as autism spectrum disorder, cerebral palsy, traumatic brain injury, sensory disability, and intellectual disability (Beukelman & Light 2020; Camilleri & Pedersen 2019; Doak & Doak 2017). These individuals typically require accommodations and adaptive supports to communicate effectively across a wide range of different contexts, including specific communication contexts, such as in court.
For persons with severe communication disabilities, access to justice is an essential tool to counter the discrimination and violence they commonly face (Beqiraj et al. 2017; Bornman et al. 2016). Regrettably, these individuals are often denied access to justice—especially participation in the court system—due to factors such as their own limited communication skills, a lack of information (on the side of both persons with disabilities and legal practitioners), legal practitioners' insufficient training and inadequate knowledge, as well as limited resources in the court system. Collectively these factors contribute to a lack of awareness of court accommodations and inevitably result in such accommodations not being available (Cremin 2016; Dagut & Morgan 2003; Flynn 2016; Gooding et al. 2017).
In cases where accommodations are available, a one-size-fits-all approach seems to prevail, despite evidence supporting the opposite (Holness 2014). For example, providing a sign language interpreter to all individuals who are deaf may not necessarily be an appropriate accommodation to provide the best outcome (Kermit, Mjøen & Olsen 2011; Olsen & Kermit 2015). In a recent South African case, the accused did not use standard South African Sign Language (SASL) and could therefore not benefit from the use of an interpreter who used SASL (Kruse v S (A100/2018) [2018] ZAWCHC 105; [2019] 4 All SA 287 (WCC) (27 August 2018). Moreover, persons with severe communication disabilities may have multiple disabilities and may therefore need more than one accommodation to achieve equal participation in court (White et al. 2020a). Although the literature discusses some accommodations (e.g., intermediaries or augmentative and alternative communication (AAC) strategies) these accommodations barely scrape the surface.
The nexus of procedural justice and Article 13 of the CRPD
The United Nation's Convention on the Rights of Persons with Disabilities (CRPD) (United Nations 2006)—an international treaty—has highlighted the importance of equality and non-discrimination for persons with (severe communication) disabilities. Article 13 of the CRPD specifically describes the roles and responsibilities of State institutions, such as the courts, to ensure effective access to justice for all persons with disabilities on an equal basis with others through the provision of procedural and age-appropriate accommodations to facilitate their participation in all legal proceedings (Celik 2017; United Nations 2006). As such, the CRPD states substantive equality for persons with disabilities as its purpose through exemplifying the interrelationship of all human rights (Degener 2016; Lord & Brown 2011), while also analysing the role of State institutions in terms of the legal obligations embedded within a substantive equality framework (Lord & Brown 2011). Ergo the CRPD can be applied as a useful framework to deliver positive outcomes for persons with disabilities (Chan et al. 2012).
When focusing on the type of court accommodations that could assist persons with severe communication disabilities to access the court, it is important to consider procedural justice as a central driving force (Dorfman 2017). This thoroughly researched construct, also known as procedural fairness, is rooted in the notion that the manner in which disputes are handled by the courts has an important influence upon how individuals evaluate their experiences in the court system (Bowen & LaGratta 2014; Ellem & Richards 2018; Pennington 2015; Tyler, Goff & MacCoun 2015; Wood, Tyler, & Papachristos 2020). The basis of procedural justice is that in people's contact with the justice system, not only do they care about the outcome of their case, but also they value the way in which it was handled (Brems & Lavrysen 2013). This highlights the subjective experience of the process through which human rights decision making is achieved, rather than only its outcomes. Therefore, procedural justice is valued not because of the way it facilitates a desired outcome, but rather because it portrays a sense of procedural fairness and respect for the individuals involved (Brems & Lavrysen 2013).
In a recommendation for court accommodations, the legal scholar Tyler (2008) distinguishes four procedural justice principles that courts should take into account when assisting persons with severe communication disabilities (Bowen & LaGratta 2014; Brems & Lavrysen 2013): i) the 'having a voice' principle, which requires that legal practitioners support persons with severe communication disabilities to actively participate in court by allowing their 'voice' to be heard, irrespective of the means or modes of communication that are used (e.g., gestures, sign language, speech-generating devices, writing, symbol-based communication boards); ii) the 'treated with respect' principle, which requires that legal practitioners engage with persons with severe communication disabilities in a respectful manner, thereby implying courtesy and dignity towards them and recognising the individual and their disability; iii) the 'using objective criteria for decision-making' principle, which requires that legal practitioners use objective, legitimate criteria to make decisions and apply fairness in all decisions; and iv) the 'understanding the court language' principle, which requires that legal practitioners focus on the individuals with severe communication disabilities' ability to understand the language used in court in order to build trust (Bowen & LaGratta 2014; Brems & Lavrysen 2013; Tyler 2008).
From the above discussion it is thus clear that the court system is found to be lacking in providing court accommodations that focus on procedural justice and fairness to enable persons with severe communication disabilities to participate equally in the court system. Perspectives of international experts who work in the court system may be able to suggest valuable and relevant recommendations of court accommodations for persons with severe communication disabilities.
The aim of the current study was to investigate the perspectives of international experts on possible universal court accommodations that could enable persons with severe communication disabilities from across the globe to participate equally in the court system so as to ensure access to justice for them. This was done by applying an international treaty, namely the CRPD, as the bedrock for this research with a further focus on procedural justice principles. The research question was formulated to read as follows: What court accommodations should be provided for persons with severe communication disabilities to participate equally in the court system?
The first author moderated an asynchronous online focus group with a panel of nine international experts while the second and third authors acted as observers. An asynchronous online focus group is a selected group of individuals (or experts) who volunteer to participate in a moderated, structured, online discussion to explore a particular topic for the purpose of research (Jensen et al. 2017; Williams et al. 2012). Furthermore, this type of online discussion allows participants to read and reply to each other's postings at a time that suits them (Williams et al. 2012).
The choice of platform for hosting an online focus group is a crucial consideration for this type of methodology to ensure that the participants feel comfortable and safe to share information (Cortini, Galanti & Fantinelli 2019; Johansson 2019). Three criteria were set for the platform: it had to ensure the participants' safety and confidentiality; it had to be easy to understand and use; and it had to allow for asynchronous discussion. The learning platform, Blackboard Learn, was selected as it allowed a degree of customisation and was relatively easy to use by the moderator, observers, and participants (Stewart & Shamdasani 2017). It could also ensure confidentiality and between-participant anonymity, while making it possible to capture the content of the discussion in an easy-to-follow manner (Stewart & Shamdasani 2017). Adhering to the principles established in a face-to-face focus group, a facilitation script was developed (Tates et al. 2009) that specifically examined participants' perspectives about court accommodations for persons with communication disabilities.
An advantage of asynchronous online focus groups is that it enables access to hard-to-reach populations (e.g., experts) and to a more diverse participant group from a larger geographical area, which is challenging when using traditional research techniques (Reisner et al. 2018; Skelton et al. 2018). The use of asynchronous online focus groups is particularly advantageous when investigating sensitive topics, such as court accommodations for persons with severe communication disabilities, as it allows the participants to choose which aspects of their experience they feel comfortable disclosing. Online focus groups are also more cost effective than traditional face-to-face ones because there are no costs related to facility rental, equipment, and transportation (Lijadi & Schalkwyk 2015; Stewart & Shamdasani 2017). Research has also shown that the content was virtually the same between synchronous and asynchronous focus groups despite obvious differences in the data collection format (Biedermann 2018; Reisner et al. 2018).
However, a disadvantage of online focus groups is that comments are not elaborated on in detail, as participants might say less when they need to type their responses. It may also take longer to respond; therefore, they might only give a shallow response (Biedermann 2018). Careful attention was thus given to each participant's response, and the moderator asked additional questions if clarity was needed.
Expert perspectives may provide an accessible source of information that can be harnessed relatively quickly to provide opinions and knowledge when there is a paucity of research evidence regarding a specific topic (Baker, Lovell & Harris 2006). Consequently, when investigating relatively new areas of research, experts can make a significant contribution based on their extensive experience in this specific focus area (Bornman & Naude 2019; Etikan, Musa & Alkassim 2016).
A purposive sampling technique was used to identify participants to ensure that they could be considered experts on the research topic and that they would be able to provide thick and rich data regarding possible court accommodations (Creswell & Plano Clark 2018). These experts also had to be practising in a country that is a signatory of the CRPD. When defining an expert, different criteria can be used. For the purpose of the current study, three criteria (as based on Baker et al. 2006) were employed: knowledge (articulated by qualifications and publications in the field of the current study), experience, and influence (articulated by whether they had informed policy or were involved in policy revision) (see Table 1). Each of these criteria were then scored using specific parameters. For example, experience was used, with number of years used as the proxy. Experience of between 1 and 5 years yielded a score of 1; 6 to 10 years yielded a score of 2; 11 to 15 years a score of 3, and 16 years or more a score of 4. (See Table 1 for the scoring that was used for the knowledge and influence criteria). A minimum score of 7 was required to ensure that potential participants met the minimum criteria for consideration as experts related to the specific topic at hand. A total of 16 potential participants were identified, of whom 9 consented to participate in the expert online focus group. The seven non-consenting potential participants cited prior commitments, high workload, maternity leave, and health challenges.
Participant Description (N = 9).
Has a disability
Knowledge (qualifications) weighting: Bachelors = 1 Honours = 2 Master's = 3 PhD = 4
Knowledge (publications) weighting: 1 to 5 = 1 6 to 10 = 2 11 to 15 = 3 16+ = 4
Experience (in years) weighting: 1 to 5 = 1 6 to 10 = 2 11 to 15= 3 16+ = 4
Influence (informed policy or policy revision) weighting Yes = 1 No = 0
Total weighting score
1 Female 37 Shona No Attorney 3 2 2 1 8
2 Female 74 English No Professor Emerita (disability) 4 4 4 1 13
3 Female 65 Germany No Professor (Special needs education and rehabilitation) 4 4 4 1 13
4 Female 59 Hebrew No Speech Therapist 1 4 4 1 10
5 Female 42 English Yes, Cerebral Palsy Tribunal Member 2 1 3 1 7
6 Female 50 English No Associate Professor (Criminology) 4 4 4 1 13
7 Female 44 English Yes, Speech disability Compliance Officer 3 3 4 1 11
8 Female 61 English No Associate 3 3 3 1 10
9 Male 54 English No Forensic Psychologist 4 3 3 1 11
The nine participants were well-known, influential, published scholars in their respective fields. The majority were female, and their ages ranged from 37 to 74 years (average age of 54 years). The participants practised in Australia, Canada, England, Germany, Israel, the USA, and Zimbabwe. All participants met the 3 criteria, with weighted scores for inclusion ranging from 7 to 13 points, with an average weighting of 11 points.
Before recruitment commenced, ethics approval was obtained from the Research Ethics Committee at the tertiary institution concerned (Ethics approval number: GW20180718HS). The potential participants were emailed letters of informed consent with details about the research topic, what was expected of them, as well as potential risks and benefits. To ensure confidentiality, each participant was given a unique login name and password with which they could anonymously (between participants) access their discussion group forum for 5 days, 24 hours a day. The only persons who could be identified by name were the moderator (first author) and observers (second and third authors) who played a similar role as in face-to-face focus groups, for example by asking clarifying questions and encouraging group discussions (Williams et al. 2012).
The discussions took place from Monday to Friday with no holidays or public holidays that could influence the frequency of participants' postings (Skelton et al. 2018). Instead of introducing all questions at the start of the online focus group discussion, the facilitation script enabled the moderator to post a question daily, aiming to achieve optimal group discussion as recommended in previous research (Tates et al. 2009). The following four questions were posted:
Please introduce yourself to the group by referring to your experience with people with communication disabilities in the criminal justice system.
Please describe the specific court accommodations that you have asked for/recommended in court.
Please describe any specific legislation/laws that you are aware of in your country that can assist a person with a communication disability to equally access the court.
In your opinion, what may facilitate the process for a person with a communication disability to be able to access and equally participate in the court system and process?
Although no question was posted on the last day, participants had the opportunity to view all the responses in the discussion thread and to review their own responses. Furthermore, all the questions remained open for responses during the whole week. To ensure all participants participated in the online discussion, a reminder was sent to those participants who had not yet joined the discussion on the second day. Thereafter, apart from one participant (who only responded to two questions), all the other participants participated daily. On conclusion of the online focus group, the discussions (questions and responses) were exported to Microsoft Word and the original formatting was removed and replaced with standard document formatting. The Word document was emailed to all the participants for verification as part of member checking and to enhance the trustworthiness and credibility of the data (Nowell et al. 2017).
ATLAS.ti 8, a computer-assisted qualitative data analysis software (CAQDAS), was employed to conduct a thematic analysis using a deductive coding approach (Braun & Clarke 2020; Nowell et al. 2017). This deductive thematic analysis approach was selected as it allows for a recursive process, with movement back and forth between different phases involving distinct steps (Braun & Clarke 2019). First, the authors familiarized themselves with the data by exploring the text of each posting. Next, the text was divided into preliminary codes, based on an existing structured codebook developed from Article 13 of the CRPD (Access to Justice) (White et al. 2020b). Following reflective and critical analysis, the researchers adapted the existing codebook and combined Article 13 with procedural justice principles, which then became auditable evidence to support the trustworthiness of the study (Braun & Clarke 2020; Tyler 2008). This deductive type of coding allowed for the text to reflect codes based on the theoretical interests guiding the research. Codes were then grouped within main themes that reflected the most prominent ideas represented in each code category (e.g., accommodations related to procedural fairness). The data was coded and analysed by the first author, after which the second and third authors independently checked the codes to increase inter-coder reliability and agreement of the data (Campbell et al. 2013). Thereafter, codes and themes were reviewed, defined, and named (Braun & Clarke 2020).
This research focusses on specific legislations/laws related to court accommodations that the experts were aware of and that they considered should be made available to persons with severe communication disabilities to facilitate equal participation in the court. As the research questions thus focused on possible facilitators, it might create the incorrect impression that these accommodations are implemented in court. Theory does not necessarily equate to practice. The findings should therefore be read keeping the research aim in mind, without interpreting the findings to mean that these accommodations are in fact provided and/or implemented.
Four main themes were extrapolated from the data: accommodations related to procedural fairness; accommodations related to ensuring equality; accommodations related to non-discrimination; and accommodations related to legal practitioners. Within the theme, 'accommodations related to ensuring equality', the following codes were generated: treated with respect, understanding the court language, having a voice, and using objective criteria for decision-making. Within the theme 'accommodations related to ensuring equality', the following codes were generated: follow legal process, international laws that apply, national laws that apply, case law, and specific narrative examples that apply, barriers related to equality, and facilitators related to equality. Within the theme 'accommodations related to non-discrimination', the following codes were generated: discrimination based on disability definition, discrimination based on level/type of disability, discrimination based on fitness to stand trial, discrimination based on identification/screening, role of the defendant, roles of the family and legal guardians. Within the theme 'accommodations related to legal practitioners', the following codes were generated: importance of legal practitioners' training, responsibilities of legal practitioners, and no cross-referencing and collaboration between disciplines.
Accommodations related to procedural fairness
Accommodations related to procedural fairness were mentioned the most frequently by the participants. The accommodations were categorised into four specific codes that resonate with the procedural justice constructs: treated with respect, understanding court language, having a voice, and using objective criteria for decision-making.
Under the 'treated with respect' code, Expert 6 reiterated specific court accommodations that were currently available for persons with severe communication disabilities:
There are typical accommodations within the criminal law such as testifying behind a screen [and]having a support person….
Other 'treated with respect' accommodations related to procedural fairness that were identified by the experts included providing extra time for clients who appear literate but still cannot understand letters sent from the court (Expert 7), using a stress ball (Expert 9), and giving testimony via CCTV camera so that the complainant does not have to testify in the same room as the accused person (Expert 1).
Under the 'understanding court language' code, Expert 4 reflected on her professional experience:
For the people that did have a communication board we usually added vocabulary to their boards, vocabulary that will help them answer questions in the investigation.
The 'having a voice' code highlighted the use of intermediaries as one of the most frequently used accommodations. Expert 8 specifically highlighted the benefit of this accommodation:
That said, global interest in the role of [the] intermediary and the many examples of where individuals, children and adults [with disabilities], have been enabled to access justice is greatly encouraging.
Other accommodations mentioned included asking whether the witness may be provided with access to a pen and paper to write their answers down if they do not wish to speak them aloud in court, as well as the use of AAC. The latter included unaided forms of communication (e.g., gestures, fingerspelling, and sign language) as well as aided forms (e.g., pictures and written words displayed on communication boards or on speech-generating devices). Expert 4 shared her experience relating to her specific country's law regarding the use of AAC:
After application of the Investigation and Testimony Procedural Act (Accommodations for Persons with Mental or Intellectual Disabilities) of 2005, children's investigators were trained and given new authorisations for special investigations according to the law. The authorisation was developed in such a way so that the investigators themselves are considered a 'tool of the investigation' [and] made accessible, which is to say, the investigators learned how to conduct an interview (linguistic simplification, relating to times and quantities, using open and closed questions, etc.). Alongside training, the need arose for additional tools and media that will be at the disposal of the investigator and used at his or her discretion. As such, a special AAC kit was developed, aiding investigations with the assistance of a speech language pathologist funded by the Ministry of Welfare and Social Services, just as translators for sign language have aided in investigations and testimonies for several years.
Several accommodations were identified by the experts under the 'using objective criteria for decision-making' code. For instance, experts mentioned that every investigation should be recorded by two video cameras, one focusing on the person investigated and one on the communication board; judges and attorneys should not be in formal attire; and the court should have the discretion to forbid a criminal defendant from single-handedly cross-examining a witness with intellectual disabilities.
Accommodations related to ensuring equality
Accommodations related to equality that were identified by the experts were important international (the CRPD) and regional (country-specific) laws that could assist persons with severe communication disabilities in their pursuit of justice. Specific regional laws that were mentioned by the participants were from Canada (Criminal Code of Canada); Germany (Guidelines for Criminal and Administrative Summary Fine Proceedings (Richtlinien für das Straf- und Bußgeldverfahren, RiSTBV) referring to the German Criminal Code (Strafgesetzbuch, StgB); South Africa (Children's Act 38 of 2005, Criminal Procedure Act 51 of 1977); Israel (Israel's Investigation and Testimony Procedural Act, Accommodations for Persons with Mental or Intellectual Disabilities, 2005); United Kingdom (Youth Justice and Criminal Evidence Act 1999, The Police and Criminal Evidence Act 1984 (PACE) Code of Practice); and the USA (Americans with Disabilities Act of 1990).
Expert 4 spoke in detail about Israel's Investigation and Testimony Procedural Act (Accommodations for Persons with Mental or Intellectual Disabilities, 2005) that promotes equality for persons with severe communication disabilities:
In Israel, there is a very good law for access to justice. People with disabilities have the possibility to be in court with all the accessibility they need, stated by law. The main advantage of this law is that special investigators conduct investigations of people with disabilities in the criminal cases. The investigators are social workers, specialised in investigating people with disabilities. They belong to the ministry of welfare with authority like the police.
Expert 2 underscored the importance of including a variety of strategies that could be used in terms of ensuring equality for persons with severe communication disabilities:
In my opinion, there needs to be a variety of strategies, with each strategy focused on different stakeholders. National or international legislation that mandates equal opportunity/non-discrimination for people with disabilities, including those who have complex communication needs, in accessing the justice system is a good starting point.
Accommodations related to non-discrimination
Accommodations related to non-discrimination that were highlighted by the experts were procedures that should be put in place that could identify at the earliest point when a person with a communication disability enters the system (Expert 8), developing appropriate questioning techniques for children with intellectual or psychiatric difficulties (Expert 1), and people with severe communication disabilities and their families benefiting from being educated about their legal rights within the criminal justice system (Expert 2).
Expert 8 mentioned that
England's Police and Criminal Evidence Act of 1984 makes provision for the support role titled 'Appropriate Adult' (Home Office: National Appropriate Adult Network 2011) which directly benefits defendants with severe communication disabilities who come into contact with the court system. This appropriate adult should be called if the suspect is either younger than 17 years of age or an adult whom the custody sergeant considers to be 'mentally disordered or otherwise mentally vulnerable'. Also, an appropriate adult is called to the police station as an important safeguard, providing independent support to defendants to ensure that they understand what is happening at the police station during the police interview (Home Office and National Appropriate Adult Network 2011).
This support role of the Appropriate Adult as a possible accommodation is a positive example of a non-discriminatory practice for defendants and could be of great benefit to vulnerable populations, such as defendants with severe communication disabilities, not only prior to court, for example at the police station, but also in court.
Expert 1 also described in detail certain accommodations related to non-discrimination that could assist persons with severe communication disabilities in accessing the court system:
For instance, the person who conducts the assessment to determine the individual's communication needs has to understand that it is more than just a matter of a medical diagnosis. What matters most is the individual's needs relating to communication. Secondly, the needs assessment has to be carried out at the appropriate time, that is at the investigative stage to determine the person's needs as early as possible.
Accommodations related to legal practitioners
Almost all of the experts highlighted the importance of training legal practitioners regarding disability awareness and knowledge. Expert 2 made a comment on which specific legal practitioners should be considered for training:
… this includes training of judges, attorneys, police and victim's assistance professionals.
Court accommodations that highlight procedural justice principles can contribute to feelings of self-worth and satisfaction for persons with severe communication disabilities (Brems & Lavrysen 2013; White et al. 2020a). There is existing case law where accommodations related to procedural fairness have successfully been implemented. For example, giving testimony via CCTV camera so that the complainant does not have to testify in the same room as the accused, as was used in Donnelly v Ireland [1998] 1 IR 321 and in White v Ireland [1995] 1 IR 268); employing intermediaries was used in People v Miller, 530 N.Y.S.2d 490 (City Ct. Rochester City. 1988, R v Watts, [2010] EWCA Crim 1824, [2011] 1 Crim LR 58 at 61 and R (on the application of C) v Sevenoaks Youth Court [2010] 1 All ER 735; and utilizing AAC as was done in R v Watts, [2010] EWCA Crim 1824, [2011] 1 Crim LR 58 at 61, Com v. Tavares, 555 A.2d 199 (Pa. Super. Ct. 1989) and People v Webb 157 Misc.2d 474 (1993) 597 N.Y.S.2d 565).
Within the broader disability spectrum, individuals with severe communication disabilities have heightened vulnerabilities associated with the range and severity of their impairments. They may well be disadvantaged by discrimination on the basis of these vulnerabilities when attempting to access the court (Satz 2008). However, procedural justice principles can guide legal scholars and practitioners on how to combat these discriminatory practices and how to identify accommodations that can be used in court to allow for these individuals' voices to be heard (using a variety of communication modes and means) and for them to be treated with dignity and respect (Bowen & LaGratta 2014).
A strong connection between the principles of procedural justice and the perceived legitimacy given to legal institutions, such as the court system, exist (Dorfman 2017). When individuals believe that they have been treated in a procedurally fair and neutral manner, they are more likely to think highly of the institutions they have dealt with in terms of respect, loyalty, and compliance (Dorfman 2017). The accommodations related to procedural fairness identified in this research (e.g., the use of intermediaries and AAC) could assist persons with severe communication disabilities in perceiving that they are being treated in a fair and neutral manner by legal practitioners. This could, in turn lead to them showing reciprocal respect and compliance towards the court system.
Another theme extrapolated from the data dealt with accommodations related to ensuring equality. As such, the CRPD provides for transformative equality (Degener 2016; Goldschmidt 2017) that requires not only the removal of barriers to inclusion, but also the implementation of positive measures to initiate real change that addresses institutional and State power relations (Degener 2016). For transformative equality to be achieved, certain rules, laws, and procedures need to be revised and changed to include specific accommodations for persons with communication disabilities in order to enable them to participate effectively in the court system (Flynn 2016; Minkowitz 2017). The courts, regardless of country or jurisdiction, need to be transformed so they no longer remain grounded in historically determined patterns of power that used to exclude people with severe communication disabilities (Minkowitz 2017). When procedural justice principles, such as having a voice, being treated with respect, using objective criteria for decision-making, and understanding the court language, are used to identify potential court accommodations, research shows that these principles can improve transformative equality among persons with severe communication disabilities (who may have low perceptions of the court system) (Bowen & LaGratta 2014; Tyler 2008).
Many countries, other than the ones represented by the experts, also have regional laws that, when enforced, can provide the necessary court accommodations for persons with severe communication disabilities. For example, Sweden's Social Services Act (SFS 2001:453) provides persons with disabilities with the support of requesting a special contact person or support person to assist them with their personal and/or legal matters (Kuosmanen & Starke 2015). The local Swedish social services can appoint such a contact person, for example, to support persons with severe communication disabilities in their dealings with different authorities, such as the court system, based on their individual needs (Kuosmanen & Starke 2015). Accommodations related to non-discrimination was another theme that emerged from the data. Non-discrimination, together with equality, are considered fundamental principles of the CRPD and, given their interconnectedness with human dignity, form the cornerstones of all human rights (United Nations 2006). Although not the focus of the current study, the findings showed that discriminatory practices still exist in the court system. Examples that were mentioned include the lack of early identification of disability, the combining of certain disorders and disabilities under a single umbrella term, and unequal access to support in court.
'Discrimination on the basis of disability' is defined in the CRPD as
…exclusion or restriction on the basis of disability which has the purpose or effect of impairing or nullifying the recognition, enjoyment or exercise, on an equal basis with others, of all human rights and fundamental freedoms in the political, economic, social, cultural, civil or any other field. It includes all forms of discrimination, including denial of reasonable accommodation (United Nations 2006: 4).
The CRPD further highlights that it is the State's duty to take specific concrete measures to achieve de facto equality and non-discrimination for persons with disabilities (both witnesses and defendants). This is to ensure that they can in fact enjoy all human rights and fundamental freedoms (Committee on the Rights of Persons with Disabilities 2018).
One such concrete measure that could be taken is to ensure that the assessments of persons with severe communication disabilities are conducted as early as possible in the court process (investigative stage) and that assessments of the individual's ability should only be made for the purpose of determining what accommodations they need in order to participate effectively in court (Msipa 2015).
The final theme extrapolated from the data looked at accommodations related to legal practitioners. Article 13 of the CRPD stresses the importance of training legal practitioners by clearly stating the following: 'In order to help to ensure effective access to justice for persons with disabilities, State Parties shall promote appropriate training for those working in the field of administration of justice, including police and prison staff' (United Nations 2006: 11).
Celik (2017: 950), a jurist, explains how the CRPD places persons with disabilities in an empowered position, as their 'autonomy needs to be respected and supported through institutions; institutions which owe their very existence for the protection of vulnerability'. This statement also underscores the importance of training legal practitioners across the entire judiciary system, including the courts. This could potentially enable them to support and accommodate individuals with severe communication disabilities as soon as they enter court (White, Bornman & Johnson 2018).
A recent study that encouraged police officers to adopt procedural justice policing strategies emphasised respect, neutrality, and transparency in the exercise of authority as an example of also reflecting on what happens prior to court. It also stressed the importance of providing opportunities for citizens to explain their side of events, which proved to be effective as complaints against police were reduced (Wood et al. 2020). Using procedural justice guidelines to train legal practitioners in court accommodations for persons with severe communication disabilities could be just as effective. It will allow these individuals effective access to the court system and will ensure that they receive the same treatment as their able-bodied equals.
Critical reflection and limitations
The current study focused on identifying a universal range of specific court accommodations for individuals (children and adults) with severe communication disabilities. Our findings could well serve as a blueprint for other vulnerable populations, such as children (Murphy 2014). Although our focus was on international experts, only seven countries were represented. It must be noted that each country's laws and jurisdiction differ, and therefore court accommodations mentioned in this study would have to be considered in line with the specific law of each country. The findings should be considered as illustrating certain principles, rather than attempting to suggest an exhaustive list of court accommodations.
Furthermore, only the perspectives of legal practitioners were captured, and therefore obtaining multiple stakeholder perspectives is suggested for future research. For example, including the voices of persons with severe communication disabilities who have been in contact with the court system themselves (either as witnesses or as defendants) or those of legal practitioners with disabilities (e.g., jurors, attorneys, judges, or judicial officers) would produce rich data and might offer a different perspective. Furthermore, this study focused only on one stage of the justice process, namely attending and participating in court. Future research could focus on accommodations needed for other stages, such as after the trial (i.e., participating in counselling or in prison reform programmes). As this research study's focus was one of a universal outlook and a starting point for court accommodations for persons with severe communication disabilities, future research could also focus on the distinct roles in the justice system, for example the witness with a severe communication disability or the defendant with a severe communication disability, as each role is different and requires different processes and court accommodations that would need to be individualised per role and per court (criminal, civil, mental health).
The court accommodations identified in this research could assist persons with severe communication disabilities, their families, and legal practitioners when accessing the court system. Traditionally, the human right of access to justice has notoriously been violated for persons with severe communication disabilities, as accommodations to participate in court have typically been unknown to legal practitioners and hence been unavailable to persons in need. If, or when, these individuals and their families arrive at court, they often encounter little or no support in the form of accommodations or resources that can assist them in their pursuit of justice. The identified court accommodations could act as the impetus needed to assist the court system and relevant legal practitioners to ensure that persons with severe communication disabilities are treated in a fair and equal manner and, ultimately, that these individuals can have the realisation of their human rights fulfilled.
Data Accessibility Statement
The data that supports the findings of this study is available on request from the corresponding author. The data is not publicly available due to confidentiality restrictions.
The authors would like to thank all the experts for participating in the research, despite extremely busy work schedules. The authors would also like to thank the journal referees for their valuable feedback.
The first author would like to thank her doctoral scholarship funders, the National Institute for the Humanities and Social Sciences (NIHSS), in collaboration with the South African Humanities Deans Association (SAHUDA) for the financial assistance. Opinions expressed and conclusions arrived at are those of the author and are not necessarily to be attributed to the NIHSS and SAHUDA.
The authors have no competing interests to declare.
All authors contributed to the conceptualisation of the study, development of methodology, data collection, analysis of data, summary of findings, and writing of the original draft and the subsequent versions.
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text:
Back to Journals » OncoTargets and Therapy » Volume 11
Measurement of serum PODXL concentration for detection of pancreatic cancer
Authors Taniuchi K, Tsuboi M, Sakaguchi M, Saibara T
Received 27 October 2017
Published 15 March 2018 Volume 2018:11 Pages 1433—1445
DOI https://doi.org/10.2147/OTT.S155367
Editor who approved publication: Professor Jianmin Xu
Keisuke Taniuchi,1,2 Makiko Tsuboi,2 Masahiko Sakaguchi,3,4 Toshiji Saibara1,2
1Department of Endoscopic Diagnostics and Therapeutics, Kochi Medical School, Kochi University, Nankoku, Kochi, Japan; 2Department of Gastroenterology and Hepatology, Kochi Medical School, Kochi University, Nankoku, Kochi, Japan; 3Integrated Center for Advanced Medical Technologies, Kochi Medical School, Kochi University, Nankoku, Kochi, Japan; 4Cancer Prevention and Control Division, Kanagawa Cancer Center Research Institute, Yokohama, Kanagawa, Japan
Background: The aim of this study was to investigate the use of podocalyxin (PODXL) and secretoglobin family 1D, member 2 (SCGB1D2) expressions in whole blood as diagnostic biomarkers to distinguish between patients with pancreatic cancer and control participants, in comparison with serum cancer antigen 19-9 (CA19-9), which is the current clinical standard.
Patients and methods: Flow cytometric analysis was performed to determine the expressions of PODXL and SCGB1D2 on the surface of cultured pancreatic cancer cells. Immunoblotting was performed to determine whether PODXL and SCGB1D2 were detectable in the media of cultured pancreatic cancer cells. A discovery-stage clinical study was performed in a cohort of 23 patients with pancreatic cancer and 51 control individuals without pancreatic disease who had been treated in the Department of Gastroenterology and Hepatology at Kochi Medical School Hospital from April 2014 to January 2016. Serum PODXL and SCGB1D2 levels were measured by enzyme-linked immunosorbent assay (ELISA).
Results: PODXL and SCGB1D2 accumulated in the protrusions of cultured pancreatic cancer cells, and they were detectable both on the cell surface and in the cultured media from these cells. The discovery-stage clinical study showed that the area under the receiver-operating characteristic curve (AUC) was 0.96 (95% confidence interval [CI] 0.91–1.000) for PODXL, 0.80 (95% CI 0.67–0.94) for SCGB1D2, and 0.78 (95% CI 0.66–0.90) for CA19-9. The AUC for PODXL was thus significantly higher than that for CA19-9 (P = 0.006). The combination of SCGB1D2 with CA19-9 did not significantly increase the AUC (0.83; 95% CI 0.70–0.96) compared with the AUC for either SCGB1D2 or CA19-9 alone (P = 0.563).
Conclusion: PODXL may be a novel, non-invasive diagnostic biomarker for the detection of pancreatic cancer.
Keywords: podocalyxin-like protein, secretoglobin, pancreatic cancer, diagnostic marker
Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in the Western world.1 The prognosis is poor, with 1- and 5-year survival rates of only 20% and 6%, respectively.2 Early diagnosis of PDAC is difficult, and there are no blood biomarkers that can assist in the identification of such patients at an early stage.3 Therefore, the discovery of blood biomarkers that can improve the diagnosis of PDAC and provide prognostic information for these individuals would greatly assist patient management.
We have previously described two glycoproteins, podocalyxin (PODXL) and secretoglobin family 1D, member 2 (SCGB1D2), which promote the motility and invasiveness of PDAC cells.4,5 PODXL is a highly glycosylated and sialylated transmembrane protein.6 High PODXL expression is associated with a significantly higher risk of death from PDAC,4,7 as well as poor differentiation, perineural invasion, and perivascular invasion.7 PODXL contributes to the formation of additional membrane protrusions in PDAC cells through the recruitment of gelsolin to filamentous actin, resulting in increased motility and invasiveness.4 PODXL is thus an important marker of poor prognosis in PDAC.4
SCGB1D2, also termed lipophilin B, is a member of the secretoglobin (SCGB) superfamily, a group of small, secretory, rarely glycosylated, dimeric proteins with unclear physiological functions, mainly expressed in mucosal tissues.8 Mature SCGBs are secreted proteins composed of homo- and heterodimers of SCGB polypeptides.9 SCGB1D2 is expressed in a high proportion of breast cancers,10 and it is more frequently expressed in estrogen receptor-positive tumors.11 We recently reported that, like PODXL, SCGB1D2 is also a biomarker of poor prognosis in PDAC and plays a role in the formation of cell protrusions important for cancer cell migration.5
Although it was discovered >30 years ago, serum cancer antigen 19-9 (CA19-9) remains the gold standard serum marker for patients with PDAC. In a cohort where approximately half of the patients had Stage IV disease, CA19-9 was found to have a median sensitivity of 79% (70%–90%) and a median specificity of 82% (68%–91%) for PDAC diagnosis; however, the marker is not useful in the mass screening of asymptomatic subjects.12 Moreover, inadequate sensitivity and specificity limit the use of CA19-9 in the early diagnosis of PDAC.13 Several other potential serum or tissue markers for PDAC, such as TIMP-1 and ICAM-1, are currently being evaluated.14 Overall, however, in current clinical practice, biomarkers have a limited role to play in diagnosing PDAC.
The aim of the present study was to assess the utility of serum PODXL and SCGB1D2 as diagnostic markers for differentiating PDAC from control participants without pancreatic disease. The differential expressions of PODXL and SCGB1D2 in serologic samples of patients with early-stage (0, I, and II) and late-stage (III and IV) PDAC are also reported.
The human PDAC cell line S2-013, a subline of SUIT-2, was obtained from Dr T Iwamura (Miyazaki Medical College, Miyazaki, Japan).15 The human PDAC cell line, PANC-1, was purchased from the American Type Culture Collection (Manassas, VA, USA). HPNE immortalized normal pancreatic epithelial cells were a kind gift from Dr Ouellette.16 The ethics review board of Kochi Medical School approved the use of the gifted cell lines. All cells were grown in Dulbecco's Modified Eagle's Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal calf serum at 37°C in a humidified atmosphere saturated with 5% CO2.
Confocal immunofluorescence microscopy
Immunocytochemistry was carried out, as previously described.17 Briefly, coverslips were coated with 10 μg/mL of fibronectin (Sigma-Aldrich Co., St Louis, MO, USA) for 1 h at room temperature. Cells were seeded on fibronectin-coated glass coverslips, incubated for 5 h, and then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Blocking solution (3% bovine serum albumin/PBS) was added, followed by incubation with anti-PODXL antibody (PAB20111; Abnova, Taipei, Taiwan) or anti-SCGB1D2 antibody (sc-48327; Santa Cruz Biotechnology Inc., Dallas, TX, USA) for 1 h. After washing, Alexa488- and Alexa594-conjugated secondary antibody (Molecular Probes, Carlsbad, CA, USA) was applied. Each specimen was visualized using a Zeiss LSM 510 META microscope (Carl Zeiss Meditec AG, Jena, Germany).
S2-013 and HPNE cells (1 × 106 cells) were incubated with anti-PODXL antibody (NBP2-27219; Novus Biologicals, Littleton, CO, USA) or anti-SCGB1D2 antibody (sc-48327) at 4°C for 1 h (0.34 mg/mL). Mouse IgM for PODXL and goat IgG for SCGB1D2 were used as isotype controls (0.34 mg/mL, Santa Cruz Biotechnology Inc.). The cells were washed in PBS and then incubated with Alexa488-conjugated secondary antibodies (Molecular Probes) at 4°C for 30 min. The cells were washed in PBS and analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) and ModFit (Verity Software House Inc., Topsham, ME, USA). Mean fluorescence intensity was calculated as relative signal intensity, ie, cells treated with anti-PODXL/cells treated with mouse IgM isotype control or cells treated with anti-SCGB1D2/cells treated with goat IgG isotype control.
Immunoblot analysis of cell lysates
Cell pellets were resuspended in 20 mM HEPES (pH 7.4), 100 mM KCl, 2 mM MgCl2, 0.5% Triton X-100, protease inhibitor cocktail tablets (Hoffman-La Roche Ltd., Basel, Switzerland), and a phosphatase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). The bicinchoninic acid (BCA) assay was used to determine the protein concentration of each lysate; an aliquot of each lysate was then diluted with sample buffer (50 mM Tris, 2% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, and 10% glycerol) to a final concentration of 1–2 μg/μL, and it was then analyzed by SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting using anti-PODXL (PAB20111), anti-SCGB1D2 (sc-48327), and anti-GAPDH (014-25524; Wako Pure Chemical Industries, Ltd., Osaka, Japan) antibodies.
Immunoblot analysis of culture conditioned media
S2-013 and HPNE cells were grown to 70% confluency, washed three times with PBS, and incubated for 48 h in the presence of conditioned media supplemented with 1% heat-inactivated fetal calf serum. Culture-conditioned media were collected, and cell cultures were trypsinized and analyzed for the presence of necrotic cells using trypan blue staining. Culture-conditioned media harvested from S2-013 and HPNE cells were centrifuged using a Beckman Coulter Allegra X-15R centrifuge at 300× g at 4°C for 10 min to remove detached cells. Supernatant was collected and filtered through 0.22 μm filters (Merck Millipore, Billerica, MA, USA) to remove contaminating apoptotic bodies, microvesicles, and cell debris. Next, the samples were concentrated using an Amicon Ultra 10K Centrifugal Filter (Merck Millipore), according to the manufacturer's instructions. The concentrations of PODXL and SCGB1D2 in these concentrated conditioned media were determined by immunoblotting. An aliquot of each conditioned medium was diluted with sample buffer (50 mM Tris, 2% SDS, 0.1% bromophenol blue, and 10% glycerol) and analyzed by SDS-PAGE and Western blotting using anti-PODXL (PAB20111), anti-SCGB1D2 (sc-48327), anti-GAPDH (014-25524), and anti-α-tubulin (017-25031; Wako Pure Chemical Industries, Ltd.) antibodies.
Human serum PDAC samples for enzyme-linked immunosorbent assay (ELISA)
The serum PODXL and SCGB1D2 levels and the clinicopathological data were analyzed retrospectively. All serum samples analyzed by ELISA were collected in the Kochi Medical School Hospital from April 2014 to January 2016. Serum samples from PDAC patients were selected for the study on the basis of the following criteria: 1) patients who were newly diagnosed and previously untreated, and 2) those with tumors pathologically diagnosed as PDAC. Tumors were classified (Stages I–IV) according to the classification of the International Union against Cancer (Table 1).18 Clinicopathological parameters were classified according to the pancreatic carcinoma criteria of the Japan Pancreas Society.19 All individuals classified as controls were diagnosed with benign gastrointestinal diseases such as gastric and colonic polyps while being evaluated for non-pancreatic diseases. The study was approved by the ethics review board of Kochi Medical School (approval number: ERB-101891), and the methods were carried out in accordance with the approved guidelines. Written informed consent was obtained from each patient. All serum samples were obtained at the time of diagnosis and stored at −80°C until use.
Table 1 Summary of characteristics
Note: aClassified according to the classification of the International Union against Cancer.
Abbreviations: CA19-9, cancer antigen 19-9; IQR, interquartile range; PDAC, pancreatic ductal adenocarcinoma.
Serum levels of PODXL and SCGB1D2 were measured by ELISA using commercially available enzyme test kits (SEA768Hu [CLOUD-CLONE, Katy, TX, USA] for PODXL and CSB-EL020814HU [CUSABIO, Hubei Province, China] for SCGB1D2). All assays used a quantitative sandwich enzyme immunoassay technique. Serum samples were diluted 1 in 10 in assay buffer, and then 100 μL of the diluted sample was incubated in pre-coated ELISA 96-well plates, along with standards, for 2 h at 37°C. After washing the wells, 100 μL of biotin-labeled polyclonal secondary antibody (detection reagent A) was added and incubated for 1 h at 37°C. After washing, 100 μL of avidin-conjugated horseradish peroxidase (detection reagent B) was added and incubated for 30 min at 37°C. After a final washing step, 90 μL of tetramethylbenzidine (TMB) substrate was added to each well and incubated for ~15 min in the dark at 37°C, until the second lowest standard could be distinguished from the blank by a change in color. Stopping solution (50 μL of sulfuric acid) was then added, and the absorbance at 490 nm was analyzed using the SpectraMax 190 Microplate Reader (Molecular Devices LLC, Sunnyvale, CA, USA). Levels of CA19-9 in serum were measured by ELISA using a commercially available enzyme test kit (TM-CA-19-9 ELISA KIT; DRG International Inc., Springfield, NJ, USA), following the same protocol as mentioned earlier.
For the in vitro experiments, StarFlex software (version 6; YUMIT, Osaka, Japan) and SAS software (version 9.1.3; SAS Institute Inc., Cary, NC, USA) were used for statistical analysis. Student's t-test was used for the comparison of continuous variables. P-values < 0.05 were considered significant, and all tests were two-tailed.
In the discovery stage clinical study, statistical analysis was performed using R (version 3.3.3; The R Foundation, Vienna, Austria). The pROC package (The R Foundation) was used for receiver operating characteristic (ROC) curve analysis. Patient sample characteristics, including PODXL, SCGB1D2, and CA19-9 expressions, were compared using the Wilcoxon rank sum test. Pearson's correlation coefficients were calculated for PODXL, SCGB1D2, and CA19-9. The predictive performances of PODXL and SCGB1D2 were evaluated using ROC analysis, the area under the receiver-operating characteristic curve (AUC), and the corresponding 95% confidence intervals (CIs). DeLong's test was used for differences in AUC values.20 Multivariate logistic regression was used to establish the diagnostic mathematical model. The variance inflation factor (VIF) was used to check for multicollinearity using the DAAG package (The R Foundation). Diagnostic values of PODXL and SCGB1D2 were evaluated based on AUCs, with sensitivity and specificity evaluated at various cutoff values. The three cutoff values were calculated as: 1) the highest Youden index,21 2) the highest sensitivity with specificity not <80%, and 3) the highest specificity with sensitivity not <80%. The logit model was used to determine cutoff values for combinations of multiple biomarkers.22 In all analyses, P-values < 0.05 were considered significant.
Extracellular localization of PODXL and SCGB1D2 from cultured PDAC cells
Immunocytochemistry was used to determine the subcellular localization of PODXL and SCGB1D2 in the moderately differentiated PDAC cell line (S2-013), the poorly differentiated PDAC cell line (PANC-1), and the immortalized normal pancreatic epithelial cell line (HPNE). Western blotting showed that S2-013 and PANC-1 cells expressed higher levels of PODXL and SCGB1D2 than HPNE cells (Figure 1A). Confocal microscopy showed that PODXL and SCGB1D2 were present in the cytoplasm of the cell bodies and in membrane protrusions in S2-013 and PANC-1 cells, which had many peripheral actin structures (Figure 1B). In contrast, there was no obvious staining of PODXL and SCGB1D2 in HPNE cells (Figure 1B).
Figure 1 Subcellular localization of PODXL and SCGB1D2 in PDAC and immortalized normal pancreatic epithelial cells.
Notes: (A) Western blotting of total cell lysates of S2-013, PANC-1, and HPNE cells was performed using anti-PODXL and anti-SCGB1D2 antibodies. Loading control: GAPDH. (B) Confocal immunofluorescence microscopic images of S2-013, PANC-1, and HPNE cells labeled with anti-PODXL antibody (green) or anti-SCGB1D2 (green), and phalloidin (red; actin filaments); arrows, PODXL and SCGB1D2 localized to cell protrusions; blue, DAPI (nuclear) staining; bars, 10 μm. (C) Expressions of PODXL and SCGB1D2 on the surface of S2-013 and HPNE cells, as evaluated by flow cytometry. Cells treated with anti-PODXL or anti-SCGB1D2 antibody (red) vs cells treated with the corresponding isotype control (blue). (D) Western blotting of total cell lysates and culture media of S2-013 and HPNE cells was performed using anti-PODXL and anti-SCGB1D2 antibodies. Loading control: α-tubulin (an intracellular protein marker) and GAPDH.
Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDAC, pancreatic ductal adenocarcinoma; PODXL, podocalyxin; SCGB1D2, secretoglobin family 1D, member 2.
The cell surface expressions of PODXL and SCGB1D2 were assessed in S2-013 and HPNE cells by flow cytometry. This demonstrated that PODXL and SCGB1D2 antibodies bound to the surface of S2-013 cells and that PODXL and SCGB1D2 were expressed on the surface of S2-013 cells (Figure 1C). PODXL and SCGB1D2 were not detectable in HPNE cells. Because PODXL is a transmembrane glycoprotein whose extracellular domain is modified heavily by O-linked glycosylation and the addition of highly charged sialic acid residues,23 and the extracellular domain of SCGB1D2 is thought to be secreted,8 immunoblotting was next used to examine the presence of these proteins in the culture media of S2-013 cells. The anti-PODXL antibody (PAB20111) recognized the extracellular domain of PODXL. Western blotting showed that PODXL and SCGB1D2 were both detected in media from S2-013 cultures (Figure 1D). In contrast, PODXL and SCGB1D2 were not detectable in media from HPNE cells (Figure 1D). We used two loading control markers, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and α-tubulin. GAPDH with the same molecular mass as the intracellular GAPDH is detected in the conditioned medium of mammalian cultured cell lines,24 and α-tubulin is an intracellular protein marker. GAPDH was abundantly present in media from S2-013 cultures, and α-tubulin was not detectable in media from S2-013 and HPNE cultures (Figure 1D).
Serum levels of PODXL
Because the in vitro findings suggested the potential use of PODXL as a novel tumor maker for PDAC, whether PODXL is present in the serum of patients with PDAC was investigated. The discovery-stage clinical study included a total of 74 serologic samples from patients with PDAC and control individuals without pancreatic disease. For individuals with no prior therapy for PDAC, disease staging (0–IV) for PDAC was performed at the time of specimen procurement. Clinical characteristics at the time of specimen procurement are summarized in Table 1. Of the individuals with PDAC, seven (30.4%) had early-stage disease (0/I/II) at the time of specimen procurement.
The PODXL levels in serum samples from both PDAC patients and control individuals were detected by ELISA experiments; the median serum level of PODXL in PDAC patients was 8.74 ng/mL (interquartile range [IQR] 7.1–12.4), while in control individuals it was significantly lower (0.15 ng/mL; IQR −1.01 to 0.86) (Figure 2A, P < 0.001). The detection sensitivity of the PODXL ELISA kit was 0.156 ng/mL. The relationships between serum PODXL concentration and clinicopathological features were analyzed by the Wilcoxon rank sum test (Table 2). However, there were no significant associations between serum PODXL concentration and the clinical characteristics such as age, gender, tumor size, clinical stage, and serum CA19-9 levels in PDAC patients.
Figure 2 The performance of PODXL, SCGB1D2, CA19-9, and the combination of SCGB1D2 with CA19-9 determined by ELISA for distinguishing PDAC patients from control individuals.
Notes: (A) Distribution of PODXL and SCGB1D2 in sera from PDAC patients and control individuals. The horizontal line in the middle of each box indicates the median, whereas the top and bottom borders of the box mark the 75th and 25th percentiles, respectively. The upper whisker is the 75th percentile + (1.5 × IQR). The lower whisker is the 25th percentile − (1.5 × IQR). Differences are significant between PDAC patients and control individuals (*P = 0.001, Mann–Whitney U test). (B) ROC curves of the levels of PODXL, SCGB1D2, and CA19-9 in sera from PDAC patients and control individuals; X-axis, 1 – specificity; Y-axis, sensitivity. (*P = 0.001, Mann–Whitney U test) (C) Relationships between serum levels of PODXL, SCGB1D2, and CA19-9 (X-axis, CA19-9 concentration; Y-axis, PODXL or SCGB1D2 concentration). (D) ROC curves for the performance of SCGB1D2 and CA19-9 alone, the combination of SCGB1D2 with CA19-9, the combination of PODXL with CA19-9, and the combination of PODXL and SCGB1D2 with CA19-9.
Abbreviations: CA19-9, cancer antigen 19-9; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IQR, interquartile range; PDAC, pancreatic ductal adenocarcinoma; PODXL, podocalyxin; ROC, receiver operating characteristic; SCGB1D2, secretoglobin family 1D, member 2.
Table 2 Correlation between serum PODXL level and clinicopathological parameters in PDAC
Abbreviations: IQR, interquartile range; PDAC, pancreatic ductal adenocarcinoma; PODXL, podocalyxin.
Serum levels of SCGB1D2
The SCGB1D2 levels in serum samples of both PDAC patients and control individuals were detected by ELISA experiments; the median serum level of SCGB1D2 in PDAC patients was 238.87 ng/mL (IQR 115.11–683.74), while in control individuals it was 71.09 ng/mL (IQR 13.08–104.51) (Figure 2A, P < 0.001). The relationship between SCGB1D2 levels and clinicopathological features is shown in Table 3. Serum SCGB1D2 level was significantly correlated with gender and serum triglyceride (P < 0.05). However, there were no significant associations between serum SCGB1D2 levels and other clinicopathological features such as tumor size, clinical stage, and serum CA19-9 levels in PDAC patients.
Table 3 Correlation between serum SCGB1D2 level and clinicopathological parameters in PDAC
Abbreviations: IQR, interquartile range; PDAC, pancreatic ductal adenocarcinoma; SCGB1D2, secretoglobin family 1D, member 2.
ROC curve analyses
ROC curves were used to evaluate the performance of PODXL and SCGB1D2 in distinguishing patients with PDAC from control individuals (Figure 2B). For the detection of PODXL and SCGB1D2 in PDAC and control individuals without pancreatic disorders, the AUC was 0.96 (95% CI 0.91–0.99) and 0.80 (95% CI 0.67–0.94), respectively. The levels of CA19-9 were determined using a commercial ELISA kit, and the AUC of CA19-9 was 0.78 (95% CI 0.66–0.90). The performance of PODXL in distinguishing PDAC from control individuals was found to be superior to that of CA19-9 (P = 0.006). However, SCGB1D2 was not superior to CA19-9 (P = 0.802).
The sensitivity and specificity of PODXL for PDAC diagnosis were analyzed using different cutoff values (Table 4). Three cutoff values for each marker were obtained by minimizing the total prediction error, as described in the "Patients and methods" section. The sensitivity of PODXL ranged from 91.3% to 100%, and the specificity varied from 86.3% to 92.2%, for the different cutoff values. Specifically, when a threshold of 6.27 ng/mL was used, a sensitivity of 91.3%, a specificity of 92.2%, and an accuracy of 91.9% were achieved (Table 4). The sensitivity of SCGB1D2 ranged from 69.6% to 82.6%, and the specificity varied from 47.1% to 94.1% for the different cutoff values. Specifically, when a threshold of 185.58 ng/mL was used, a sensitivity of 69.6%, a specificity of 94.1%, and an accuracy of 86.5% were achieved (Table 4). Using the same method for CA19-9, instead of using the standard cutoff value (37.0 U/mL), when a threshold of 66.0 U/mL was used, a sensitivity of 56.5%, a specificity of 96.1%, and an accuracy of 83.8% were achieved (Table 4). Decreasing the cutoff value to 50.0 U/mL improved the sensitivity for the detection of PDAC to 60.9%, but lowered the specificity to 88.2% and the accuracy to 79.7%.
Table 4 Sensitivity and specificity for PODXL, SCGB1D2, and CA19-9 at specified cutoff values
Abbreviations: AUC, area under the curve; CA19-9, cancer antigen 19-9; CI, confidence interval; LR, likelihood ratio; PODXL, podocalyxin; SCGB1D2, secretoglobin family 1D, member 2.
Combination with SCGB1D2 and CA19-9
There were significant correlations between the serum levels of PODXL and CA19-9 (Pearson's correlation coefficient [R] = 0.38, 95% CI 0.17–0.56, P = 0.007), between SCGB1D2 and CA19-9 (R = 0.31, 95% CI 0.09–0.50, P = 0.007), and between PODXL and SCGB1D2 (R = 0.72, 95% CI 0.588–0.81, P < 0.001) (Figure 2C). To take account of multicollinearity, VIF was employed. None of the VIF values for the two models reached a value of 10, meaning that there was no collinearity in the model. VIF values for CA19-9, PODXL, gender, and age were 1.02, 1.08, 1.07, and 1.01, respectively. VIF values for CA19-9, SCGB1D2, gender, and age were 1.05, 1.25, 1.24, and 1.04, respectively.
SCGB1D2 and CA19-9 were combined in an attempt to improve the sensitivity and specificity for the detection of PDAC. ROC analysis showed that the AUC for the combination of SCGB1D2 and CA19-9 was 0.83 (95% CI 0.70–0.96), which was not significantly different from the AUC of SCGB1D2 alone (P = 0.213) or CA19-9 alone (P = 0.563) (Figure 2D). The sensitivity of the combination of SCGB1D2 and CA19-9 ranged from 73.9% to 82.6%, the specificity ranged from 80.4% to 90.2%, and the accuracy ranged from 81.1% to 85.1% for the different cutoff values (Table 5). When a threshold of 0.306 was used, a sensitivity of 73.9%, a specificity of 90.2%, and an accuracy of 85.1% were achieved (Table 5). With the combination of PODXL and CA19-9, the AUC of this combination was not significantly different from the AUC of PODXL alone (Figure 2D), indicating that PODXL was independently able to distinguish PDAC patients from control individuals.
Table 5 Sensitivity and specificity for combination with SCGB1D2 and CA19-9 at specified cutoff values
Association of serum tumor marker levels with the risk of PDAC
Next, the significance of serum tumor marker levels for the diagnosis of PDAC was evaluated using logistic regression to obtain crude odds ratios (ORs) (Table 6). To exclude the possible effects of age and gender, crude ORs were then adjusted for the effects of age and gender (adjusted OR [aOR] values). The results showed that among PODXL, SCGB1D2, and CA19-9, the serum level of PODXL was the most highly correlated with the risk of PDAC. The combination of PODXL with CA19-9 did not improve PDAC detection compared to the use of PODXL alone (aOR of CA19-9 = 1.00, 95% CI 0.99–1.01, P = 0.227, for the multivariate regression model of PODXL and CA19-9).
Table 6 Univariate and multivariate logistic regression analyses
Notes: Model 1: Odds ratio adjusted for CA19-9. Model 2: Odds ratio adjusted for CA19-9, age and gender.
Abbreviations: AUC, area under the curve; CA19-9, cancer antigen 19-9; CI, confidence interval; OR, odds ratio; PODXL, podocalyxin; SCGB1D2, secretoglobin family 1D, member 2.
Detection of early-stage PDACs using PODXL, SCGB1D2, or CA19-9
The level of PODXL in serum samples from patients in the early stages of PDAC (Stages 0, I, and II) was significantly higher than the level in controls, as well as the level of CA19-9 in these patients (Figure 3A). ROC curves for PODXL, SCGB1D2, and CA19-9 are shown in Figure 3B. The AUC values of PODXL used to distinguish patients with Stage 0/I/II and Stage III/IV PDAC from controls were 0.96 and 0.96, respectively (Table 7). For SCGB1D2, the AUC values used to distinguish patients with Stage 0/I/II and Stage III/IV PDAC from controls were 0.66 and 0.87, respectively (Table 7). For CA19-9, the AUC values to distinguish patients with Stage 0/I/II and Stage III/IV PDAC from controls were 0.79 and 0.77, respectively (Table 7). The performance of PODXL in distinguishing PDAC at Stage 0/I/II from control individuals was found to be superior to that of CA19-9 (P = 0.048).
Figure 3 Determination of PODXL, SCGB1D2, and CA19-9 by ELISA in Stage 0/I/II and Stage III/IV PDACs.
Notes: (A) PODXL, SCGB1D2, and CA19-9 levels in sera from Stage 0/I/II PDACs, Stage III/IV PDACs, and control individuals. The horizontal line in the middle of each box indicates the median, whereas the top and bottom borders of the box mark the 75th and 25th percentiles, respectively. The upper whisker is the 75th percentile + (1.5 × IQR). The lower whisker is the 25th percentile − (1.5 × IQR). (B) ROC curves for the performance of PODXL, SCGB1D2, and CA19-9 from Stage 0/I/II PDACs, Stage III/IV PDACs, and control individuals.
Abbreviations: CA19-9, cancer antigen 19-9; ELISA, enzyme-linked immunosorbent assay; IQR, interquartile range; PDAC, pancreatic ductal adenocarcinoma; PODXL, podocalyxin; ROC, receiver operating characteristic; SCGB1D2, secretoglobin family 1D, member 2.
Table 7 Concentration and AUC of PODXL, SCGB1D2, and CA19-9 at each clinical stage of PDAC
Abbreviations: AUC, area under the curve; CA19-9, cancer antigen 19-9; CI, confidence interval; IQR, interquartile range; PDAC, pancreatic ductal adenocarcinoma; PODXL, podocalyxin; SCGB1D2, secretoglobin family 1D, member 2.
Kaplan–Meier plots and univariate and multivariate analyses of pathological findings have previously shown that high expressions of PODXL and SCGB1D2 are significant prognostic factors associated with worse survival after PDAC resection.4,5 Expression levels of PODXL and SCGB1D2 were evaluable in all 102 PDAC cases, and these cases were classified into low-expressing (70.6% for PODXL and 63.7% for SCGB1D2) and high-expressing (29.4% for PODXL and 36.3% for SCGB1D2) groups.4,5 Therefore, an increase in PODXL and SCGB1D2 within tumors would be expected to affect signaling pathways relevant to cell proliferation, invasiveness, and metastasis in PDAC. Since sensitive and specific biomarkers to identify patients with PDAC at an early stage are needed, it is important to investigate whether serum PODXL and SCGB1D2 are useful in the diagnosis of PDAC, as was done in the present study.
We previously reported that intracellular PODXL promotes PDAC cell motility and invasion by physically binding to the cytoskeletal protein gelsolin, and PODXL is a significant prognostic factor that predicts the overall survival of patients with PDAC.4 Since membranous PODXL is rarely expressed in migrating PDAC cells, intracellular PODXL bound to gelsolin may be more important for the promotion of cell motility and invasion than membranous PODXL.4 The main function of PODXL is to regulate cell morphology and adhesion by mediating the connection between intracellular proteins and extracellular ligands.25 The present study demonstrated that PODXL accumulates in the protrusions of PDAC cells and localizes to the cell surface. In addition, it was shown that PODXL is present in the media of PDAC cell cultures; however, the molecular differences (both in terms of amino acid sequence and post-translational modifications) between membrane-bound PODXL and extracellular PODXL, as well as the functions of extracellular PODXL, are currently unknown. Future studies should thus evaluate the structure of the soluble form of PODXL that can be detected in culture-conditioned media from PDAC cells.
Tumor-specific transmembrane or secretory proteins have significant advantages as biomarkers because they are present on the cell surface, within the extracellular space, or in the blood and are thus easily accessible.26 Like PODXL, SCGB1D2 was also found to accumulate in the protrusions of migrating PDAC cells, and it was detected both on the cell surface and in the culture media of PDAC cells. Western blotting using anti-PODXL antibody that recognized the extracellular domain of PODXL showed two bands of PODXL (30 kDa and 45 kDa) in the cell lysate of PDAC cells and a single band of PODXL (35 kDa) in the culture media of PDAC cells (Figure 1D). PODXL contains a potential NH2-terminal signal peptide, a single COOH-terminal membrane-spanning region, and the extracellular domain containing multiple potential sites for N- and O-linked glycosylation.23 It is possible that 30 kDa and 40 kDa PODXLs were present due to post-translational modifications, most likely glycosylation, in PDAC cells. The molecular weight of a portion of the intracellular domain of PODXL is ~10 kDa,23 and, thus, a portion of the extracellular domain of PODXL could be detected in the culture media of PDAC cells. There were also high levels of PODXL and SCGB1D2 in serum samples from PDAC patients. It is possible that the extracellular domain of PODXL was present in the serum obtained from PDAC patients. Serum SCGB1D2 was correlated with gender and serum triglyceride levels, but other clinicopathological factors were not significantly correlated with SCGB1D2 serum levels. The reasons for the observed associations between serum SCGB1D2, gender, and serum triglyceride levels are unknown; however, this should be further examined in future clinical studies.
To validate the feasibility of using serum levels of PODXL and SCGB1D2 as diagnostic tools, the sensitivity and specificity of these markers were compared with those of the current standard serum marker, CA19-9. PODXL performed better than serum CA19-9 when using AUC to discriminate between PDAC and control individuals; however, the increase in AUC was not significant for SCGB1D2. It was also found that the combination of SCGB1D2 and CA19-9 did not significantly increase the AUC (0.83; 95% CI 0.70–0.96) compared with either SCGB1D2 (0.80; 95% CI 0.67–0.94) or CA19-9 alone (0.78; 95% CI 0.66–0.90). Importantly, these results suggest that increased expression of serum PODXL is more accurate for the diagnosis of PDAC than serum CA19-9. However, the combination of SCGB1D2 and CA19-9 does not appear to be particularly useful as a serum biomarker.
A multicenter trial comparing resection and chemoradiotherapy for locally invasive resectable PDAC (Stages IIA and IIB) showed significantly better outcomes with surgery, despite the premature termination of the trial.27,28 The circumferential encasement of the celiac axis, the hepatic artery, or the superior mesenteric artery with suspected arterial tumor infiltration (T4 or Stage III) is categorized as non-resectable in most cases.28,29 Not only could better markers improve the early diagnosis of PDAC and allow more patients to undergo curative surgical resection but they could also potentially be used for patients at high risk of developing pancreatic cancer, to identify precancerous lesions while they are amenable to cure.30 The present study included seven cases of Stage 0/I/II PDAC, and the AUC value for PODXL in the detection of these tumors was significantly higher than that for CA19-9 (P = 0.048). This study included only one patient in Stage 0-IB; therefore, additional validation with a larger set of serum samples covering various clinical stages, especially Stage 0-IB, is necessary to validate that PODXL is a potential biomarker for the screening of early-stage PDAC.
The use of serum PODXL as a biomarker achieved a sensitivity of 94.6% and a specificity of 84.3% for the detection of PDAC, at a cutoff value of 2.68 U/mL. All 23 patients with PDAC (100%) and all seven patients with Stage 0/I/II PDAC (100%) were correctly diagnosed as positive, whereas 13.7% of the 51 control individuals were falsely diagnosed as positive. Although it was quantitatively demonstrated that the detection of PODXL in serum may provide a novel, non-invasive approach for distinguishing PDAC from control participants, the present findings are preliminary until more elaborate studies clarify that PODXL is superior to CA19-9. Further studies are required to elucidate whether PODXL is useful as a diagnostic marker for the detection of Stage 0/I/II PDACs, as well as how much additional benefit it may add in comparison to the use of serum CA19-9. To this end, we have started a prospective clinical validation study (UMIN000021938) in the Department of Gastroenterology and Hepatology at Kochi Medical School Hospital to assess the accuracy of increased expression of PODXL in the diagnosis of PDAC and the benefit of PODXL as a potential diagnostic marker for the early detection of PDACs in comparison to CA19-9. This prospective clinical study also includes patients with chronic pancreatitis and a non-cancerous pancreatic lesion, such as intraductal papillary mucinous neoplasm, to assess the utility of serum PODXL as a diagnostic marker for differentiating PDAC from other pancreatic disorders.
The authors would like to thank Miki Nishigawa and Rieko Takahashi for their excellent technical assistance. This study was supported by a Grant-in-Aid for Scientific Research (KAKENHI; 15K14396).
All authors contributed toward data analysis, drafting and critically revising the paper and agree to be accountable for all aspects of the work.
The authors report no conflicts of interest in this work.
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redpajama_set_name: RedPajamaCommonCrawl


Notes ex (114) 3710AO Moreland Group - Phillip Island Bus Lines, Cowes, Victoria; ex (10) 3710AO Phillip Island Bus Lines, Cowes, Victoria; ex 1335AC Linga Longa Tours, Yarrawonga, Victoria; ex (45) 572IKF (QLD) Surf City Coaches, Southport, QLD; ex WJL193 (NSW) BAE Systems Australia, Kapooka, NSW; ex Army, Australia.
13/07/2013 body Austral Denning "Majestic" Austral Denning "Majestic"
13/07/2013 notes ex XMT494 (SA) Hanley's Passenger Service, Gladstone, SA; ex TV3889 (NSW) Jones Bros, Lithgow, NSW; ex Army, Australia. ex (10) 3710AO Phillip Island Bus Lines, Cowes, Victoria; ex XMT494 (SA) Hanley's Passenger Service, Gladstone, SA; ex TV3889 (NSW) Jones Bros, Lithgow, NSW; ex Army, Australia.
31/07/2016 notes ex (10) 3710AO Phillip Island Bus Lines, Cowes, Victoria; ex XMT494 (SA) Hanley's Passenger Service, Gladstone, SA; ex TV3889 (NSW) Jones Bros, Lithgow, NSW; ex Army, Australia. ex (10) 3710AO Phillip Island Bus Lines, Cowes, Victoria; ex TV3889 (NSW) Jones Bros, Lithgow, NSW; ex Army, Australia.
31/07/2016 notes ex (10) 3710AO Phillip Island Bus Lines, Cowes, Victoria; ex TV3889 (NSW) Jones Bros, Lithgow, NSW; ex Army, Australia. ex (10) 3710AO Phillip Island Bus Lines, Cowes, Victoria; ex 1335AC Linga Longa Tours, Yarrawonga, Victoria; ex (45) 572IKF (QLD) Surf City Coaches, Southport, QLD; ex WJL193 (NSW) BAE Systems Australia, Kapooka, NSW; ex Army, Australia.
13/01/2019 body Austral Denning "Majestic" Austral Denning "Majestic"
13/01/2019 notes ex (10) 3710AO Phillip Island Bus Lines, Cowes, Victoria; ex 1335AC Linga Longa Tours, Yarrawonga, Victoria; ex (45) 572IKF (QLD) Surf City Coaches, Southport, QLD; ex WJL193 (NSW) BAE Systems Australia, Kapooka, NSW; ex Army, Australia. ex (114) 3710AO Moreland Group - Phillip Island Bus Lines, Cowes, Victoria; ex (10) 3710AO Phillip Island Bus Lines, Cowes, Victoria; ex 1335AC Linga Longa Tours, Yarrawonga, Victoria; ex (45) 572IKF (QLD) Surf City Coaches, Southport, QLD; ex WJL193 (NSW) BAE Systems Australia, Kapooka, NSW; ex Army, Australia.