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Results of College Exam 2018 (Council Meeting 27 September 2018)

Fellowship Assessment:

Membership Examination:

Results of College Written Exam in Chemical Pathology, Clinical Microbiology & Infection, and Haematology 2018
 

Candidates who failed in written exam cannot proceed further in Fellowship Assessment in 2018.
Passed candidates can proceed further in Membership or Fellowship Assessment in 2018.

Antimicrobial resistance – a global health crisis

Volume 13, Issue 2 July 2018  (download full article in pdf)

Editorial note

With increasing prominence of the threat of antimicrobial resistance both internationally and locally, awareness and knowledge on the problem and prospects are essential, in order for rational application of control measures and monitoring of their effectiveness. In this issue of the Topical Update, Dr. Dominic Tsang and Dr. Christopher Lai present an updated overview of this important subject. We welcome any feedback or suggestion. Please direct them to Dr. Janice Lo (e-mail: janicelo@dh.gov.hk), Education Committee, The Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists. 

Dr. Dominic TSANG and Dr. Christopher LAI
Consultant Microbiologist and Associate Consultant, Department of Pathology, Queen Elizabeth Hospital, Hong Kong 

End of modern medicine as we know it

The World Health Organization (WHO) described in 2001 antimicrobial resistance (AMR) as a global problem and an impending crisis. The apocalyptic term “Post-antibiotic era” was mentioned.¹ The situation did not improve since. In fact, it deteriorated. In the United Kingdom (UK) government-commissioned “Review of Antimicrobial Resistance” by Lord Jim O’Neill, it was estimated that 700,000 people died from AMR infections in 2016 in the UK alone, and that drug-resistant infections could cause 10 million human deaths annually by 2050, costing the world up to $100 trillion.² The Chief Medical Officer for England, Dame Professor Sally Davies, has predicted that unless tackled now, AMR could lead to the end of modern medicine as we know it. It could lead to routine operations and even childbirth becoming increasingly dangerous without the required antibiotics. In the UK, over 25,000 deaths a year are attributed to drug resistant infections.³ European Commission estimated the costs associated with AMR infection at €1.5 billion annually.⁴

The driving force behind emergence and dissemination of AMR is directly related to the use of antibiotics, i.e. the antibiotic selection pressure.⁵ It is recognized that non-human indiscriminate use of antibiotics in agriculture and animal husbandry to promote growth in animals and the consequential persistence of antibiotics in soil and aquatic environment select for AMR that could be disseminated widely.⁶ Human overuse nonetheless needs to be controlled by dedicated efforts on strengthening regulations on over-the- counter purchase of prescription only antibiotics, enhancing training in antibiotic prescriptions, monitoring compliance with antibiotic guideline and antibiotic stewardship programme (ASP).⁷

Concerted effort against AMR

Antibiotics on one hand is an agent required for life saving. But on the other, its usage and the resultant selective pressure have been recognized as the main drivers for AMR. Therefore, its use in human and non-human settings should be balanced against the risk of driving AMR. As such, antibiotic usage data has been linked to AMR surveillance data in the AMR containment strategy.^8,9

There are already in place a few surveillance systems on AMR, covering healthy animals, diseased animals, food and humans in countries such as Canada, Denmark, Finland, Germany, Italy, Japan, USA, the Netherlands, Norway, France, and Sweden.¹⁰

The UK and China will establish the Global AMR Research Innovation Fund and encourage further investment from other governments and the private sector, helping to address AMR. The new fund will invite bids from industry, academia and other bodies. It aims to create international partnerships to build a global response and support new research to reduce the spread of antibiotic resistance.

The World Health Assembly in May 2015 endorsed a WHO global action plan to tackle antimicrobial resistance to ensure continuity of successful treatment and prevention of infectious diseases with effective and safe medicines.¹¹ The plan sets out five strategic objectives including: to improve awareness and understanding of antimicrobial resistance; to strengthen knowledge through surveillance and research; to reduce the incidence of infection; to optimize the use of antimicrobial agents; and to develop the economic case for sustainable investment that takes account of the needs of all countries, and increase investment in new medicines, diagnostic tools, vaccines and other interventions. Under each of the objectives, specific actions were listed out for member states as well as international partners to implement.

Hong Kong Strategy against Multidrug resistant organisms (MDROs)

The Hong Kong Strategy and Action Plan on Antimicrobial Resistance 2017-2022¹² was released in July 2017 with the goals to develop a territory-wide network on surveillance on AMR and antimicrobial use, promote appropriate therapeutic use of antimicrobials in human and animals and to promote research on diagnostic and related interventions. Six key areas were targeted in the plan, including strengthening knowledge, optimizing antimicrobial use, reducing infections, improving awareness, promoting research and fostering partnerships among stakeholders.

In terms of AMR surveillance, the overall resistance profiles of MDROs have all along been closely monitored in public hospitals under the Hospital Authority in Hong Kong¹³ Among the concerned MDROs, Gram positive organisms include methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE). Gram negative organisms include extended spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) and the WHO top priority organisms of carbapenem-resistant Enterobacteriaceae (CRE), carbapenem- resistant Acinetobacter baumannii (CRAB), and carbapenem-resistant Pseudomonas aeruginosa (CRPA).¹⁴

Trends in antibiotic resistant organisms

MRSA: In Hong Kong, MRSA bloodstream infection was made a key performance indicator (KPI) to gauge the performance of infection control practices in all public hospitals.¹⁵ The corresponding MRSA bacteraemia rate was 0.18- 0.19 per 1,000 acute bed days when the monitoring began in 2008 and declined gradually to 0.144 per 1,000 acute bed days in 2017. At the same time, MRSA constituted 43.1% among S. aureus isolated from local HA hospitals.

VRE: The total number of VRE cases detected remains below 50 a month since 2015, after an upsurge and successful control in the Queen Elizabeth Hospital in 2013 when the peak number of cases detected in a month reached 300.¹⁶

CRAB/multi-drug resistant Acinetobacter baumannii (MDRA) is commonly associated with patients with prolonged hospital stay and who required ventilator-assisted ventilation. These patients are also usually put on multiple antibiotics for the treatment of underlying infections.¹⁷ Substantial environmental contamination with the resistant bacteria is frequent as a result of nursing care procedures, leading to explosive outbreaks which are difficult to abort unless attention is given to patient segregation and effective disinfection of instruments and environmental surfaces.^18,19

ESBL-E began to emerge in late 1980s and peaked in UK in 2006 with 12% E. coli isolated from bacteraemic cases being ESBL positive. ESBL-E has been common locally, at 22% in 2017 compared to 24.3% since 2012. They are especially important as they commonly caused community-acquired as well as hospital-acquired infections. In fact, a local report showed 62% of imported chicken were found to be contaminated with ESBL-E.²⁰ Community carriage rates for ESBL-E are high in East Mediterranean areas and South East Asia, from 30-60%.^21,22 For treatment of ESBL-E infections, imipenem has been universally effective since its launch in 1987, and the same applies to meropenem, another carbapenem antibiotic which was launched in 1996.

CPE are members of the Enterobacteriaceae including E. coli and Klebsiella species which possess the gene on plasmids coding for the enzymes such as KPC, NDM and OXA-48 under the Ambler Classification on molecular class A, B or D respectively which enable them to inactivate carbapenem antibiotics and also other beta- lactams. CPE are usually multiply antibiotic resistant, therefore rendering limited options in treatment of their infections.²³ The emergence and subsequent spread of CPE followed human movement closely as exemplified by the clustering of KPC-CPE in New York, North-West England, Israel and the spreading of NDM-CPE to Sweden by a returning patient from New Delhi, India.^24-26 In Hong Kong, CPE cases were being increasingly detected, from 19 cases in 2011 to 473 cases in 2017. Active bacterial screening (ABS) of patients based on a set of consensus criteria in public hospitals including admission screening of all patients who have stayed in overseas hospitals in the past 12 months, suffering from antibiotic associated diarrhoea, staying in the same cubicle of a known CPE case, etc., helped to pick up an increasing number of CPE. Fortunately, most of the cases (90%) were asymptomatic carriers and did not require specific treatment. Of note, the number of confirmed CPE isolates in England in 2016 was more than 2,500 with KPC, NDM and OXA-48 predominant. Effective treatment options for CPE are few. The activities of ceftazidime and aztreonam with and without avibactam were tested against a large, contemporary, international collection of carbapenemase-producing Gram negative bacilli (CP-GNB) with diverse resistance mechanisms. Aztreonam-avibactam was active against all isolates except two NDM producers with elevated MICs of 8/4 and 16/4 mg/litre; ceftazidime- avibactam was active against all KPC-, IMI-, SME-, and most OXA-48 group-producing isolates (93%) but not metallo-β-lactamase producers. Among the older and contemporary antimicrobials, the most active were colistin, tigecycline, and fosfomycin, with overall susceptibilities of 88%, 79%, and 78%, respectively.²⁷ Among local CPE isolates, the resistance rate of colistin is around 5.8%.

The “Find and Confine” Control strategy of AMR in HA hospitals

“Find”: This is the single most important control measures aiming to uncover the carriage state of any asymptomatic carrier, especially of VRE and CPE, who could shed the MDRO in the excreta. This is commonly done by ABS in the form of admission screening based on high risk factors such as hospitalization in recent months, a history of exposure to a known case, prolonged hospital stay (e.g. for 14 days or more), development of antibiotic associated diarrhoea etc. The yield of ABS at present is not high, ranging from 0.6% to 1.4% (average 1.0%) based on 2017 CPE ABS data in one of the hospitals, but ABS has undoubtedly provided a substantial impact in mitigating otherwise uninterrupted dissemination of VRE and CPE in the hospital setting. The availability of commercial agar media with the function to differentiate CPEs after overnight incubation has greatly facilitated ABS. Coupled with other rapid enzyme detection and PCR confirmation of CPE, which are also available commercially, the time to detection of CPE carriage in patients has been shortened to one to two days.

“Confine”: It is an important part of standard precautions (SP) plus contact precautions (CP) in the care of a known MDRO case. A room with en- suite is the preferred placement especially for those who suffer from diarrhoea and therefore at a higher risk of transmission of MDROs. This is widely practiced in developed countries. When en-suite rooms are not available, segregation of MDRO carriers by “cohorting” patients carrying the same MDRO is a pragmatic alternative.

Hand hygiene (HH): Healthcare workers (HCWs) strictly observing proper HH, e.g. WHO 5 moments, is undoubtedly the most important and effective measure in aborting the dissemination of MDROs in healthcare setting. The use of alcohol handrub in place of hand washing in situations where soiling is minimal has greatly improved HH compliance. However, HH is an action governed by human behavior which unfortunately also suffers from all factors that affect our behavior, such as physical fatigue, forgetfulness, motivation, persistence, peer influence, etc. For the same reason, the reliability of HH compliance monitoring by direct observation is also limited by the well-known “Hawthorn effect” in that, as humans, we tend to perform when we are aware of being observed.²⁸ Further, in periods of high bed occupancy like influenza seasons, it is very hard to expect HCWs to fully comply with HH and as a consequence, these periods are more prone to cross transmission and outbreaks of MDROs. Apart from HCWs, patients and visitors are equally important in observing HH in order to avoid cross transmission. Similar moments of HH are being promoted for patients in local hospitals. In the long run, the development of a positive and motivated infection control culture and HH habit would not only maintain the cross transmission of MDROs at a low level but also mitigate the risk of other cross infections.

Environmental hygiene: The inanimate environment plays an important role inperpetuating the dissemination of MDROs in the hospital settings, especially VRE and MDRA.^29,30 High-touch (frequently touched) surfaces such as patients’ privacy curtain, bed rails, door knobs, nursing trolleys, drip stands are often contaminated with the MDROs during outbreaks.³¹ The conventional cleansing and disinfection by using diluted sodium hypochlorite solution, although effective, is labour intensive. Also, to ensure the cleaning procedure is meticulously performed, it is commonly monitored by the use of a surrogate marker (e.g. UV-fluorescent marker) to ensure satisfactory performance.³² To circumvent such drawback, there are now plenty of effective new products on environmental disinfection, ranging from self- disinfecting surface coating sprays, hydrogen peroxide vapor, UV-C device, 2-in-1 disinfectant wipes to antimicrobial privacy curtains that could achieve effective decontamination with less labour. In a multi-centre study, 42.7% of standard hospital curtains were contaminated with MRSA and 42.3% with CRAB.³³ The use of disposable antibacterial privacy curtains, e.g. nanoparticle silver or quaternary ammonium impregnated curtains, have been shown to prolong the time to contamination and reduce the bio-burden even after extended usage in acute care setting.³⁴

Reducing overuse of antibiotics: It is imperative to maintain the use of antibiotic at the minimum essential level in order to prevent the emergence of resistance.34 Education and training at an early stage of the medical curriculum is critical in establishing the concept and skills in prudent antibiotic use. Guideline such as the local IMPACT guideline on antibiotic use is indispensable in providing the guidance on the right indications, choice, dose, route, and duration of antibiotic use. In addition, antibiotic stewardship program (ASP) has been widely practiced and proven to be effective in ensuring appropriate use of antibiotics.³⁵ Locally, a multi- disciplinary team of clinical microbiologists, physicians, pharmacists and infection control nurses has been put in place in all HA hospitals to provide concurrent feedback on the use of targeted “big-gun” antibiotics. The percentage of appropriate use stays at above 80% in general and feedbacks on antibiotic use are welcomed in most of the cases.

Another very important aspect in the control of AMR is diagnostic support in infection.³⁶ The rapid isolation and identification of an aetiological agent lends strong support in the continuation of antibiotic treatment, or its discontinuation in the absence of any evidence of infection. This is made possible with the introduction of molecular platforms such as 16S ribosomal RNA PCR and metagenomic studies.³⁷

Surrogate biomarker of infection, in particular procalcitonin (PCT) which exhibits greater specificity than other proinflammatory markers such as C reactive protein (CRP) helps in identifying patients with sepsis and can be used for diagnosing infections, especially ventilator- associated pneumonia (VAP).^38,39 The short half- life (25-30 hours in plasma) of PCT and its absence in healthy state make it the preferred biomarker for bacterial infections. PCT levels

The outlook of the challenges from AMR

The outcome in our battle against MDROs depends on how successful we are in preserving the efficacy of our existing antibiotics for treatment of infections and the result of our search for new antibiotics. Both require resources, efforts and dedication. While preserving existing effective antibiotics demands the aforementioned One Health approach, research breakthroughs arguably provide us the only hope in winning the battle against MDROs and to ensure effective treatment of infections for the continual practice of modern medicine.

Not long ago, researchers have identified from a soil sample a new cell wall inhibitor, teixobactin, from a previously unknown Gram-negative bacterium that lives in soil but which cannot be cultured in the laboratory using standard technique.⁴⁰ The researchers used the “Ichip”, an isolation chamber, in which a soil sample is diluted with agar and a single bacterial cell in a chamber is then placed in soil where the bacteria could access to nutrients and growth factors. The teixobactin identified has excellent activity against Gram-positive pathogens including MRSA, Clostridium difficile, Bacillus anthracis and Mycobacterium tuberculosis. Another recent breakthrough was reported⁴¹ on accessing hidden natural products (NP) made by bacteria not by culturing but by sequencing, bioinformatics analysis and heterologous expression of biosynthetic gene clusters captured on DNA extracted from environmental samples. Such technique has led to the discovery of malacidins, a distinctive class of antibiotics which are active against MRSA infections without selection for resistance under the laboratory conditions. These discoveries are definitely good news given the great potential for more to be discovered by using these innovative technologies. Of course, there are still tests to be done before they could become clinically useful, but at least these discoveries through our human creative and innovative minds are holding promise in the battle against AMR. Meanwhile, we must not be distracted away from the momentum in tackling the rapidly deteriorating AMR situation.

Reference

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24. Woodford N, Tierno PM Jr, Young K, Tysall L, Palepou MF, Ward E, Painter RE, Suber DF, Shungu D, Silver LL, Inglima K, Kornblum J, Livermore DM. Outbreak of Klebsiella pneumoniae producing a new carbapenem- hydrolyzing class A beta-lactamase, KPC-3, in a New York Medical Center. Antimicrob Agents Chemother [Internet]. 2004 [cited 2004 Dec];48(12):4793-9. In: Ovid MEDLINE(R) [Internet].

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26. Khong WX, Xia E, Marimuthu K, Xu W, Teo YY, Tan EL, Neo S, Krishnan PU, Ang BS, Lye DC, Chow AL, Ong RT, Ng OT. Local transmission and global dissemination of New Delhi Metallo-Beta-Lactamase (NDM): a whole genome analysis. BMC Genomics [Internet]. 2016 [cited 2016 06 13];17452. In: Ovid MEDLINE(R) [Internet].

27. Vasoo S, Cunningham SA, Cole NC, Kohner PC, Menon SR, Krause KM, Harris KA, De PP, Koh TH, Patel R. In Vitro Activities of Ceftazidime-Avibactam, Aztreonam-Avibactam, and a Panel of Older and Contemporary Antimicrobial Agents against Carbapenemase-Producing Gram-Negative Bacilli. Antimicrob Agents Chemother [Internet]. 2015 [cited 2015 Dec];59(12):7842-6. In: Ovid MEDLINE(R) [Internet].

28. Srigley JA, Furness CD, Baker GR, Gardam M. Quantification of the Hawthorne effect in hand hygiene compliance monitoring using an electronic monitoring system: a retrospective cohort study. BMJ Qual Saf [Internet]. 2014 [cited 2014 Dec];23(12):974-80. In: Ovid MEDLINE(R) [Internet].

29. Chemaly RF, Simmons S, Dale C Jr, Ghantoji SS, Rodriguez M, Gubb J, Stachowiak J, Stibich M. The role of the healthcare environment in the spread of multidrug-resistant organisms: update on current best practices for containment. Ther. adv. infect. dis. [Internet]. 2014 [cited 2014 Jun];2(3-4):79-90. In: Ovid MEDLINE(R) In- Process & Other Non-Indexed Citations [Internet].

30. Tan TY, Tan JS, Tay H, Chua GH, Ng LS, Syahidah N. Multidrug-resistant organisms in a routine ward environment: differential propensity for environmental dissemination and implications for infection control. J Med Microbiol [Internet]. 2013 [cited 2013 May];62(Pt 5):766-72. In: Ovid MEDLINE(R) [Internet].

31. Dancer SJ. Controlling hospital-acquired infection: focus on the role of the environment and new technologies for decontamination. Clin Microbiol Rev [Internet]. 2014 [cited 2014 Oct];27(4):665-90. In: Ovid MEDLINE(R) [Internet].

32. Ling ML, Apisarnthanarak A, Thu le TA, Villanueva V, Pandjaitan C, Yusof MY. APSIC Guidelines for environmental cleaning and decontamination. Antimicrob. resist. infect. control [Internet]. 2015 [cited 2015];458. In: Ovid MEDLINE(R) In-Process & Other Non-Indexed Citations [Internet].

33. Shek K, Patidar R, Kohja Z, Liu S, Gawaziuk JP, Gawthrop M, Kumar A, Logsetty S. Rate of contamination of hospital privacy curtains on a burns and plastic surgery ward: a cross-sectional study. J Hosp Infect [Internet]. 2017 [cited 2017 May];96(1):54-58. In: Ovid MEDLINE(R) [Internet].

34. Canton R, Morosini MI. Emergence and spread of antibiotic resistance following exposure to antibiotics. FEMS Microbiol Rev [Internet]. 2011 [cited 2011 Sep];35(5):977-91. In: Ovid MEDLINE(R) [Internet].

35. Plachouras D, Hopkins S. Antimicrobial stewardship: we know it works; time to make sure it is in place everywhere. Cochrane Database Syst Rev [Internet]. 2017 [cited 2017 02 09];2ED000119. In: Ovid MEDLINE(R) Revisions [Internet].

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37. Bogaert D, van Belkum A. Antibiotic treatment and stewardship in the era of microbiota-oriented diagnostics. Eur J Clin Microbiol Infect Dis [Internet]. 2018 [cited 2018 05];37(5):795-798. In: Ovid MEDLINE(R) In- Process & Other Non-Indexed Citations [Internet].

38. Stolz D, Smyrnios N, Eggimann P, Pargger H, Thakkar N, Siegemund M, Marsch S, Azzola A, Rakic J, Mueller B, Tamm M. Procalcitonin for reduced antibiotic exposure in ventilator- associated pneumonia: a randomised study. Eur Respir J [Internet]. 2009 [cited 2009 Dec];34(6):1364-75. In: Ovid MEDLINE(R) [Internet].

39. Schuetz P, Wirz Y, Sager R, Christ-Crain M, Stolz D, Tamm M, Bouadma L, Luyt CE, Wolff M, Chastre J, Tubach F, Kristoffersen KB, Burkhardt O, Welte T, Schroeder S, Nobre V, Wei L, Bucher HC, Bhatnagar N, Annane D, Reinhart K, Branche A, Damas P, Nijsten M, de Lange DW, Deliberato RO, Lima SS, Maravic-Stojkovic V, Verduri A, Cao B, Shehabi Y, Beishuizen A, Jensen JS, Corti C, Van Oers JA, Falsey AR, de Jong E, Oliveira CF, Beghe B, Briel M, Mueller B. Procalcitonin to initiate or discontinue antibiotics in acute respiratory tract infections. Cochrane Database Syst Rev [Internet]. 2017 [cited 2017 10 12];10CD007498. In: Ovid MEDLINE(R) In- Process & Other Non-Indexed Citations [Internet].

40. Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A, Schaberle TF, Hughes DE, Epstein S, Jones M, Lazarides L, Steadman VA, Cohen DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K. A new antibiotic kills pathogens without detectable resistance. Nature [Internet]. 2015 [cited 2015 Jan 22];517(7535):455-9. In: Ovid MEDLINE(R) [Internet].

41. Hover BM, Kim SH, Katz M, Charlop-Powers Z, Owen JG, Ternei MA, Maniko J, Estrela AB, Molina H, Park S, Perlin DS, Brady SF. Culture- independent discovery of the malacidins as calcium-dependent antibiotics with activity against multidrug-resistant Gram-positive pathogens. Nat. microbiol. [Internet]. 2018 [cited 2018 Apr];3(4):415-422. In: Ovid MEDLINE(R) In-Process & Other Non-Indexed Citations [Internet].

Volume 27, Issue 1 (click here to download the full pdf version)

Message from the President

The Year of 2018 would be a year of celebration as our College has had a very good start!

After months of deliberation and negotiation in the Genetics and Genomics Taskforce, the Training and Examinations Committee as well as the College Council, various pathology specialties including Anatomical Pathology, Chemical Pathology, Haematology and Immunology have finally come to a consensus on the draft training programme for Genetic and Genomic Pathology. On 17 January 2018, the College held an Open Forum to introduce the background and the draft training programme to all Fellows and to receive comments and suggestions from them. It turned out to be a success! With the blessings from our Fellows, the Genetic and Genomic Pathology training programme was approved at one of the Hong Kong Academy of Medicine’s Council Meetings on 12 April 2018. It would be a 2-year post- fellowship training programme with knowledge-based, cross-college and specialty-based components. This new Genetic and Genomic Pathology programme will be implemented within 24 months of the date of the Academy’s approval. To accommodate the changes, the College has also updated all the fellowship training programmes, which again were endorsed at the Academy’s Council Meeting on 15 March 2018.

The College has recently established a Young Fellows’ Chapter to encourage our younger generations to increase their participation in College activities. During their participation, various generations of Fellows can sit down together and exchange their views and opinions. In addition, one of the Young Fellows, Dr. Elaine Au, would like to share a Topical Update on immune-mediated demyelinating disease.

To celebrate the ILPP(International Liaison of Pathology Presidents) International Pathology Day 2017, and to promote the image of Pathologists to the public, the College organised a workshop at the Prince of Wales Hospital to receive over 100 senior secondary school students to enjoy some interactive laboratory experiments and demonstrations.

Lastly, Dr. NG Wai Fu would like to share his hobby that can make him young and fit!

Dr. CHAN Ho Ming
June 2018

Results of College Supplementary Exam 2017 (Council Meeting 25 January 2018)

An overview of NMO Spectrum Disorder and the diagnostic utility of anti-NMO antibodies

Volume 13, Issue 1 December 2017  (download full article in pdf)

Editorial note

NMOSD is an immune mediated demyelinating disease. Though its clinical presentation may overlap with multiple sclerosis, distinguishing these two entities is important in view of differences in treatment. Anti-NMO antibodies play a crucial role in the workup and diagnosis of NMOSD. In this review, Dr Elaine Au provided an overview of the NMOSD condition and the use of anti-NMO antibody assays. We welcome any feedback or suggestions. Please direct them to Dr Elaine Au (email: ayl436@ha.org.hk) of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists. 

Dr Au Yuen Ling Elaine
Associate Consultant, Division of Clinical Immunology, Department of Pathology, Queen Mary Hospital 

NMO is an idiopathic immune mediated demyelinating disease that predominantly affects optic nerves and spinal cord. The prevalence range from 0.3 to 4.4 per 100000 people, with Asian and African-American more affected than Caucasian, where multiple sclerosis is more common in the white population (1-5). The condition has been named as Devic’s disease in the past (6), which described a monophasic disorder presenting with simultaneous bilateral optic neuritis and transverse myelitis. With the availability of specific serological marker, antibodies that targeted the water channel aquaporin-4 (AQP4), the clinical and neuroimaging spectrum of NMO disease is broadened. Instead of being a monophasic disorder, NMO antibodies positive patients with recurrent attacks are not uncommonly found. Moreover, the clinical presentations are more variable than the traditional Devic disease. There is no pathognomonic clinical feature of spectrum disorder (NMOSD), though certain clinical presentations are particularly suggestive of the disorder. These include simultaneously bilateral optic neuritis, complete spinal cord syndrome and area postrema clinical syndrome.

Multiple sclerosis is an important differential diagnosis of this condition in view of the overlapping clinical features of these two conditions. In NMOSD, optic neuritis tends to be more severe, more often with simultaneous bilateral involvement or sequential in rapid succession, compare to multiple sclerosis. Complete spinal cord syndrome, with longitudinally extensive transverse myelitis in MRI, is more suggestive of NMOSD than multiple sclerosis. Differentiating NMOSD from other demyelinating disease, i.e. multiple sclerosis is important since the treatment is different. Indeed, some multiple sclerosis therapies may aggravate NMO disorders (7-10).

Diagnostic Criteria

In 2006, the serological marker was first incorporated into the revised NMO diagnostic criteria. In 2007, NMOSD was introduced to include seropositive patients who do not follow the classical monophasic bilateral optic neuritis and transverse myelitis. Lately, the International Panel for NMO Diagnosis (IPND) has further revised the diagnostic criteria (11). For anti-NMO antibodies positive cases, presenting at least one core clinical characteristic of the disease is required for diagnosis. Core clinical characteristics include optic neuritis, acute myelitis, area postrema syndrome (episode of otherwise unexplained hiccups or nausea and vomiting), acute brainstem syndrome, narcolepsy or acute diencephalic syndrome with typical Magnetic Resonance Imaging (MRI) lesions and symptomatic cerebral syndrome with typical MRI lesions. On the other hand, more stringent clinical criteria, with additional neuroimaging findings, are required in seronegative patients to fulfill the diagnostic criteria (See appendix). 

For the workup of the disease, MRI, cerebral spinal fluid (CSF) exam and serological test for anti-NMO antibodies are important. In some patients, CSF pleocytosis, usually in the form of monocytosis and lymphocytosis, are present. Increased CSF protein levels are noted in 46-75% of cases (12, 13). Nevertheless, presence of CSF oligoclonal bands is uncommon in NMOSD, and at most transient, in contrast to the presence of persistent CSF oligoclonal bands in the case of multiple sclerosis. Finally, visual evoked potentials, somatosensory evoked potentials and brainstem acoustic evoked potentials examination may also be helpful in the workup.

Clinical course and prognosis

In NMOSD syndromes affecting regions other than optic nerve and spinal cord, not uncommonly patients will relapse with more classical involvement in subsequent attacks. Majority of NMOSD patients suffer from recurrent attacks (80- 90%), less frequently monophasic attack (10-20%) (14), while gradual progressive course with neurological deterioration is very rare (15). Relapses usually occur in clusters, but unpredictable intervals. In the Mayo Clinic series, the second relapse occurred within 1 year in 60% of cases, and within 3 years in 90% of cases (16). Repeated NMO attacks not uncommonly lead to accumulation of neurological impairment. NMO is associated with more adverse outcome than MS in general(14).

NMOSD has been shown to be frequently associated with other autoimmune disorders, including lupus, Sjogren’s syndrome, etc (17-19). On contrary, NMOSD is not common in rheumatic disease patients.

Anti-NMO antibodies

Among the different investigations, NMO antibodies test is central to the workup. Anti-NMO antibodies are pathogenic. The third extracellular loop of AQP4 is the major epitope for the anti- NMO antibodies. Biopsy and autopsy tissue obtained from seropositive patients demonstrate loss of AQP4 immunoreactivity. Perivascular complement activation in actively demyelinating lesions is also happened. In the central nervous system (CNS), AQP4 is expressed at the foot processes of astrocytes, near the basement membranes, in the optic nerve, in a subpopulation of ependymal cells, in hypothalamic nuclei and in the subfornical organ (20, 21). Truncated astrocyte processes or cell loss were found in demyelinating lesions (11). In rat models, passive transfer of the antibodies leaded to the development of disease (22, 23). These pathological findings distinguish NMOSD from multiple sclerosis.

The Anti-NMO antibodies can be detected by indirect immunofluorescence staining on tissue slide using mouse cerebellum tissue section, cell- based assays, radioimmunoprecipitation assays and enzyme-linked immunosorbent assays (ELISA). Overall, cell-based assay is preferred in view of better assay sensitivity and specificity compared to other methods. Ideally, confirmatory testing with one or more techniques is suggested (11), especially in cases with atypical presentation or borderline results are obtained.

In tissue based indirect immunofluorescence test, NMO antibodies positive case is characterized by the binding to structures adjacent to microvasculature, the Virchow-Robin spaces (VRS) and pia mater (Fig.1).

Fig. 1. Mouse cerebellum tissue section stained with anti-NMO antibodies.

This assay allows the detection of any coexisting anti-neuronal antibodies, which may be important as differential diagnosis and workup. However, the method is observer dependent and subjective. The interpretation of antibody staining may easily be affected by non-specific background staining on the tissue. In some rare occasions, antibodies other than anti-NMO may mimic the staining pattern and lead to false positive results. Moreover, indirect tissue based immunofluorescence test has relative low sensitivity (63-64%) (24-27).

Cell-based assay utilize cell lines such as human embryonic kidney 293 (HEK293) cells or Chinese hamster ovary (CHO) cells that have been transfected with AQP4 gene expression vector, so that expressing much higher level of antigen comparing to normal tissues. Cell lines from different units may use different ratio of the two isoform of AQP4: M1 and M23 in order to obtain optimal antigen presentation. Cell-based assay can be assessed by indirect immunofluorescence staining or flow cytometry. For indirect immunofluorescence cell- based assay, slide with fixed AQP-4 gene transfected cells and non- transfected cells growing on different biochips are placed side by side for comparison. Therefore, false positivity is minimized with the inclusion of control non-transfected cells. A higher expression of antigen in the transfected cells also enhance the assay sensitivity compared to tissue-based indirect immunofluorescence testing. (Fig.2)

Fig.2. HEK 293 cells transfected with AQP-4 gene expression vector and stained positive with anti- NMO antibodies.

Overall, cell-based assay is the recommended assay in view of good sensitivity and specificity (mean sensitivity 76.7% in a pooled analysis; 0.1% false positivity in a multiple sclerosis cohort) (24- 27). Commercial kits for indirect immunofluorescence cell-based assay are available, which facilitate the assay setup in service laboratories. Nevertheless, indirect immunofluorescence method is semi-quantitative and observer dependent.

Protein-based assays, like ELISA and radioimmunoprecipitation assays, in general, have lower sensitivity compared to cell based assays. In addition, ELISA, in particular at low titer, may yield nonspecific results. However, these assays provide quantitative results which may potentially be used for serial monitoring (24).

Though NMO antibody testing in serum is well- established, the diagnostic role of testing the antibody in CSF is controversial. Most of the cases reported in literature are diagnosed by serum test, though there have been rare cases reported that were CSF positive but serum test negative (28, 29). When studying paired CSF and serum samples with antibody indices calculated, intrathecal production of the NMO antibody is rare (24). NMO antibodies can be present in patients few years before and after the disease presentations. Lately, there is increasing evidence that the antibody titre may reflect disease activity. Elevated antibody levels at relapse and decrease in titre after immunosuppressant treatment has been reported in literature (30-34). Therefore, serial monitoring may possibly facilitate management and medication adjustment. However, there is no general threshold value for clinical relapse and the absolute level varies with individual patients. Rising level may not predict relapse in all cases. In addition, some methodologies, like indirect immunofluorescence test, only provide semi-quantitative results, and inter-run reproducibility is another issue. Other factors including the frequency of test necessary to achieve meaningful disease status monitoring and the cost involved are also important consideration. Therefore, it remains to be determined whether the marker should be serially monitored for treatment response and disease activity monitoring.

Treatment

The treatment for classical Devic’s disease presentation and relapsing NMOSD presentation is no different. High dose steroid is commonly employed as first- line of treatment in acute presentation. Plasma exchange may be considered in treatment refractory cases. Immunomodulatory treatment with interferon β, which is a treatment option in multiple sclerosis, may exacerbate NMOSD disorders. Therefore, differentiating between these two conditions is important. Options of steroid sparing immunosuppressants to consider in NMOSD include azathioprine, methotrexate, mycophenolate mofetil, rituximab, etc (14).

Conclusion

NMOSD is a rare but increasingly recognized condition, which present as an inflammatory and demyelinating autoimmune disorder affecting the central nervous system. With the availability of a serological marker, anti-NMO antibody, the diagnosis and differentiating from related conditions is facilitated. Timely diagnosis and treatment is important for the management of these patients.

Reference

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3. Asgari N, Lillevang ST, Skejoe HP, Falah M, Stenager E, Kyvik KO. A population-based study of neuromyelitis optica in Caucasians. Neurology. 2011;76(18):1589-95.

4. Cossburn M, Tackley G, Baker K, Ingram G, Burtonwood M, Malik G, et al. The prevalence of neuromyelitis optica in South East Wales. European journal of neurology. 2012;19(4):655-9.

5. Mealy MA, Wingerchuk DM, Greenberg BM, Levy M. Epidemiology of neuromyelitis optica in the United States: a multicenter analysis. Archives of neurology. 2012;69(9):1176-80.

6. Jarius S, Wildemann B. The history of neuromyelitis optica. Journal of neuroinflammation. 2013;10:8.

7. Kimbrough DJ, Fujihara K, Jacob A, Lana- Peixoto MA, Leite MI, Levy M, et al. Treatment of Neuromyelitis Optica: Review and Recommendations. Multiple sclerosis and related disorders. 2012;1(4):180-7.

8. Kleiter I, Hellwig K, Berthele A, Kumpfel T, Linker RA, Hartung HP, et al. Failure of natalizumab to prevent relapses in neuromyelitis optica. Archives of neurology. 2012;69(2):239-45.

9. Min JH, Kim BJ, Lee KH. Development of extensive brain lesions following fingolimod (FTY720) treatment in a patient with neuromyelitis optica spectrum disorder. Multiple sclerosis. 2012;18(1):113-5.

10. Papeix C, Vidal JS, de Seze J,Pierrot- Deseilligny C, Tourbah A, Stankoff B, et al. Immunosuppressive therapy is more effective than interferon in neuromyelitis optica. Multiple sclerosis. 2007;13(2):256-9.

11. Wingerchuk DM, Banwell B, Bennett JL, Cabre P, Carroll W, Chitnis T, et al. International consensus diagnostic criteria for neuromyelitis optica spectrum disorders. Neurology. 2015;85(2):177-89.

12. O'Riordan JI, Gallagher HL, Thompson AJ, Howard RS, Kingsley DP, Thompson EJ, et al. Clinical, CSF, and MRI findings in Devic's neuromyelitis optica. Journal of neurology, neurosurgery, and psychiatry. 1996;60(4):382-7.

13. de Seze J, Stojkovic T, Ferriby D, Gauvrit JY, Montagne C, Mounier-Vehier F, et al. Devic's neuromyelitis optica: clinical, laboratory, MRI and outcome profile. Journal of the neurological sciences. 2002;197(1-2):57-61.

14. Sellner J, Boggild M, Clanet M, Hintzen RQ, Illes Z, Montalban X, et al. EFNS guidelines on diagnosis and management of neuromyelitis optica. European journal of neurology. 2010;17(8):1019-32.

15. Wingerchuk DM, Pittock SJ, Lucchinetti CF, Lennon VA, Weinshenker BG. A secondary progressive clinical course is uncommon in neuromyelitis optica. Neurology. 2007;68(8):603-5.

16. Wingerchuk DM, Hogancamp WF, O'Brien PC, Weinshenker BG. The clinical course of neuromyelitis optica (Devic's syndrome). Neurology. 1999;53(5):1107-14.

17. Jarius S, Jacobi C, de Seze J, Zephir H, Paul F, Franciotta D, et al. Frequency and syndrome specificity of antibodies to aquaporin-4 in neurological patients with rheumatic disorders. Multiple sclerosis. 2011;17(9):1067-73.

18. Pittock SJ, Lennon VA, de Seze J, Vermersch P, Homburger HA, Wingerchuk DM, et al. Neuromyelitis optica and non organ-specific autoimmunity. Archives of neurology. 2008;65(1):78-83.

19. Wandinger KP, Stangel M, Witte T, Venables P, Charles P, Jarius S, et al. Autoantibodies against aquaporin-4 in patients with neuropsychiatric systemic lupus erythematosus and primary Sjogren's syndrome. Arthritis and rheumatism. 2010;62(4):1198-200.

20. Graber DJ, Levy M, Kerr D, Wade WF. Neuromyelitis optica pathogenesis and aquaporin 4. Journal of neuroinflammation. 2008;5:22.

21. Tait MJ, Saadoun S, Bell BA, Papadopoulos MC. Water movements in the brain: role of aquaporins. Trends in neurosciences. 2008;31(1):37-43.

22. Kinoshita M, Nakatsuji Y, Moriya M, Okuno T, Kumanogoh A, Nakano M, et al. Astrocytic necrosis is induced by anti-aquaporin-4 antibody- positive serum. Neuroreport. 2009;20(5):508-12.

23. Kinoshita M, Nakatsuji Y, Kimura T, Moriya M, Takata K, Okuno T, et al. Neuromyelitis optica: Passive transfer to rats by human immunoglobulin. Biochemical and biophysical research communications. 2009;386(4):623-7.

24. Jarius S, Wildemann B. Aquaporin-4 antibodies (NMO-IgG) as a serological marker of neuromyelitis optica: a critical review of the literature. Brain pathology. 2013;23(6):661-83.

25. Waters PJ, McKeon A, Leite MI, Rajasekharan S, Lennon VA, Villalobos A, et al. Serologic diagnosis of NMO: a multicenter comparison of aquaporin-4-IgG assays. Neurology. 2012;78(9):665-71; discussion 9.

26. Sato DK, Nakashima I, Takahashi T, Misu T, Waters P, Kuroda H, et al. Aquaporin-4 antibody- positive cases beyond current diagnostic criteria for NMO spectrum disorders. Neurology. 2013;80(24):2210-6.

27. Pittock SJ, Lennon VA, Bakshi N, Shen L, McKeon A, Quach H, et al. Seroprevalence of aquaporin-4-IgG in a northern California population representative cohort of multiple sclerosis. JAMA neurology. 2014;71(11):1433-6.

28. Klawiter EC, Alvarez E, 3rd, Xu J, Paciorkowski AR, Zhu L, Parks BJ, et al. NMO-IgG detected in CSF in seronegative neuromyelitis optica. Neurology. 2009;72(12):1101-3.

29. Long Y, Qiu W, Lu Z, Peng F, Hu X. Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum. Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia. 2013;20(2):233-7.

30. Jarius S, Aboul-Enein F, Waters P, Kuenz B, Hauser A, Berger T, et al. Antibody to aquaporin-4 in the long-term course of neuromyelitis optica. Brain : a journal of neurology. 2008;131(Pt 11):3072-80.

31. Jarius S, Franciotta D, Paul F, Ruprecht K, Bergamaschi R, Rommer PS, et al. Cerebrospinal fluid antibodies to aquaporin-4 in neuromyelitis optica and related disorders: frequency, origin, and diagnostic relevance. Journal of neuroinflammation. 2010;7:52.

32. Jarius S, Franciotta D, Paul F, Bergamaschi R, Rommer PS, Ruprecht K, et al. Testing for antibodies to human aquaporin-4 by ELISA: sensitivity, specificity, and direct comparison with immunohistochemistry. Journal of the neurological sciences. 2012;320(1-2):32-7.

33. Kim W, Lee JE, Li XF, Kim SH, Han BG, Lee BI, et al. Quantitative measurement of anti- aquaporin-4 antibodies by enzyme-linked immunosorbent assay using purified recombinant human aquaporin-4. Multiple sclerosis. 2012;18(5):578-86.

34. Takahashi T, Fujihara K, Nakashima I, Misu T, Miyazawa I, Nakamura M, et al. Anti-aquaporin- 4 antibody is involved in the pathogenesis of NMO: a study on antibody titre. Brain : a journal of neurology. 2007;130(Pt 5):1235-43.

Appendix

NMOSD diagnostic criteria for adult patients

Diagnostic criteria for NMOSD with NMO-IgG

1. At least 1 core clinical characteristic

2. Positive test for NMO-IgG using best available detection method (cell-based assay strongly recommended)

3. Exclusion of alternative diagnoses

Diagnostic criteria for NMOSD without NMO-IgG or NMOSD with unknown NMO-IgG status

1. At least 2 core clinical characteristics occurring as a result of one or more clinical attacks and meeting all of the following requirements:

   - At least 1 core clinical characteristic must be optic neuritis , acute myelitis with LETM, or area postrema syndrome

   - Dissemination in space (2 or more different core clinical characteristics)

   - Fulfillment of additional MRI requirements, as applicable

2. Negative tests for NMO-IgG using best available detection method, or testing unavailable

3. Exclusion of alternative diagnoses

Core clinical characteristics

1. Optic neuritis

2. Acute myelitis

3. Area postrema syndrome: episode of otherwise unexplained hiccups or nausea and vomiting

4. Acute brainstem syndrome

5. Symptomatic narcolepsy or acute diencephalic clinical syndrome with NMOSD-typical diencephalic MRI lesions

6. Symptomatic cerebral syndrome with NMOSD-typical brain lesions

Additional MRI requirements for NMOSD without NMO-IgG and NMOSD with unknown NMO-Ig status

1. Acute optic neuritis: require brain MRI showing (a) normal findings or only nonspecific white matter lesions, OR (b) optic nerve MRI with T2-hyperintense lesion or T1-weighted gadolinium-enhancing lesion extending over >1/2 optic nerve length or involving optic chiasm

2. Acute myelitis: requires associated intramedullary MRI lesion extending over >=3 contiguous segments (LETM) OR >=3 contiguous segments of focal spinal cord atrophy in patients with history compatible with acute myelitis

3. Area postrema syndrome: requires associated dorsal medulla/ area postrema lesions 4. Acute brainstem syndrome: requires associated periependymal brainstem lesions

Neurology 2015;85:177-189

Clinical features and laboratory findings atypical for NMOSD, that need to consider alternative diagnoses

1. Progressive overall clinical course (neurologic deterioration unrelated to attacks: Consider MS)

2. Atypical time to attack nadir: less than 4 hours (consider cord ischemia/ infarction); continual worsening for more than 4 weeks from attack onset (consider sarcoidosis or neoplasm)

3. Partial transverse myelitis, especially when not associated with LETM MRI lesion (consider MS)

4. Presence of CSF oligoclonal bands (oligoclonal bands occur in < 20% of cases of NMO vs > 80% of MS

Comorbidities associated with neurologic syndromes that mimic NMOSD

1. Sarcoidosis, established or suggestive clinical, radiologic or laboratory findings thereof (e.g. mediastinal adenopathy, fever and night sweats, elevated serum angiotensin converting enzyme or interleukin-2 receptor level)

2. Cancer, established or with suggestive clinical, radiologic or laboratory findings thereof; consider lymphoma or paraneoplastic disease ( e.g. collapsing response mediator protein-5 associated optic neuropathy and myelopathy or anti-Ma- associated diencephalic syndrome)

3. Chronic infection, established or with suggestive clinical radiologic, or laboratory findings thereof ( e.g. HIV, syphilis)

Neurology 2015;85:177-189

Results of College Exam 2017 (Council Meeting 26 September 2017)

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Molecular alterations of gastrointestinal stromal tumor - Prognostic and therapeutic implications 

Volume 12, Issue 2 August 2017  (download full article in pdf)

Editorial note:

Gastrointestinal stromal tumor is the commonest mesenchymal tumor in the digestive system. It is a genetically heterogeneous disease with various mutations apart from classical activation mutations in KIT  and PDGFRA genes. In the topical update, Dr. Anthony Chan provided an overview of molecular alterations of gastrointestinal stromal tumor with emphasis on their prognostic and therapeutic significance. We welcome any feedback or suggestions. Please direct them to Dr. Anthony Chan (e-mail: awh_chan@cuhk.edu.hk) of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists. 

Dr. Anthony W.H. Chan
Clinical Associate Professor
Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong 

The gist of GIST

Gastrointestinal stromal tumor (GIST) is a rare tumor with the annual incidence rate of 10- 15/1,000,000, but it is the commonest mesenchymal tumor in the digestive system. It affects both sexes equally and presents at any age from children to elderly with the median age of mid 60s. Stomach (55.6%) is the most frequent primary tumor site followed by small intestine (31.8%), large intestine (6.0%) and esophagus (0.7%). Other uncommon primary sites, such as omentum, mesentery and liver, accounts for 5.5% of all GISTs.(1) Important milestones of GIST in diagnostic, prognostic and therapeutic aspects are briefly summarized in this section. 

In the past, GIST was regarded as leiomyoma, leiomyoblastoma or leiomyosarcoma before the era of wide availability of immunohistochemistry. In 1983, Mazur and Clark first applied the term "stromal tumor" to describe a group of gastric mesenchymal tumor lacking ultrastructural features of smooth muscle or schwann cells.(2) In 1989, a short-lived term, gastrointestinal autonomic nerve tumor (GANT), was used to describe a small subset of GIST featured by small intestinal location, epithelioid appearance and focal immunoreactivity towards neural markers (S100, neurofilament and synaptophysin).(3) In 1995, CD34 was found to be the first useful diagnostic immunohistochemical marker to differentiate GIST from leiomyoma and schwannoma although only 60-70% of all GISTs are immunoreactive to CD34.(4) In 1998, the hallmark constitutive activation mutation of KIT gene and overexpression of KIT/CD117 protein in GIST were discovered by Hirota et al.(5) This finding also suggested that GIST may be originated from interstitial cells of Cajal, pacemaker cells of intestine, which express KIT and CD34. However, activation mutation of KIT gene and overexpression of KIT are not consistently correlated. A subset of KIT positive GISTs was found to lack KIT mutation and this observation led to the subsequent discovery of gain-of-function mutation of platelet-derived growth factor receptor alpha (PDGFRA) gene in 2003.(6, 7) KIT and PDGFRA mutations are mutually exclusive. About 5-10% of GISTs, particularly those with PDGFRA mutation do not express KIT. In 2004, West et al. identified a novel gene, DOG1 (discovered on GIST-1), through cDNA microarray, and showed DOG1 protein was highly expressed in GISTs (97.8%), including those KIT negative GISTs.(8) KIT and/or DOG1 become crucial diagnostic immunohistochemical markers for GIST. A small subgroup of GISTs with immunoreactivity of KIT/DOG1 lack neither KIT or PDGFRA mutation was first designated as wild-type GISTs in the same year.(9) Wild-type GISTs are later shown to be a heterogeneous group with various mutations.(10- 13)

Prognosis of patients with GIST is shown to be correlated with tumor size and mitosis. The first consensus risk stratification was proposed by investigators in National Institutes of Health (NIH) in 2002 (Table 1).(14) Anatomical location of GIST is also an important prognostic factor and firstly integrated to the Armed Forces Institute of Pathology system in 2006 (Table 2)(15). Gastric GIST behaves more indolent than small and large bowel GIST with similar size and mitosis. Tumor rupture is an additional prognosticator for GIST patients and incorporated into the modified NIH system in 2008 (Table 3).(16) Finally, the most widely adopted tumor staging system, American Joint Committee on Cancer (AJCC), include GIST risk stratification composed of tumor size, mitosis, anatomical location, nodal and distant metastases in the 7th edition in 2010, which remains unchanged in the recently released 8th edition (Table 4 and 5).

Surgical resection remains the mainstay of curative therapy for GIST but a substantial portion of GIST patients present in advanced stage beyond surgical intervention. Imatinib, a multi-targeted tyrosine kinase inhibitor specific for c-abl, c-kit and PDGFR, was first used in a patient with metastatic GIST in 2001.(17) The dramatic clinical response from this patient and the subsequent successful phase II clinical trial in 2002 secured the first-line role of imatinib for patients with inoperable GIST and pioneered molecular targeted therapy for sarcoma.(18) Primary and acquired resistance to imatinib among GIST patients led to development of newer targeted agents. Two hallmark phase III randomized controlled trials on sunitinib (NCT00075218) and regorafenib (NCT01271712) for GIST were completed in 2006 and 2013, respectively.(19, 20) Sunitinib and regorafenib are indicated for patients with advanced GIST resistant or intolerant to imatinib. 

Table 1: NIH risk stratification for GIST (14) 

Table 2: AFIP risk stratification for GIST (15) 

Table 3: Modified NIH risk stratification for GIST (16) 

Table 4: AJCC staging system for gastric and omental GIST 

Table 5: AJCC staging system for small/large bowel, esophageal, mesenteric and peritoneal GIST 

Mutational landscape of GIST

KIT and PDGFRA mutations are major driver mutations in GIST tumorigenesis. Both genes encode type III receptor tyrosine kinases with similar structures: extracellular ligand binding domain and dimerization domain, a transmembrane sequence, a juxtamembrane domain and intracellular kinase domain (Figure 1). Binding of corresponding ligands, stem cell factor and PDGFA, to c-kit and PDGFRA receptor, respectively, dimerizes and activates receptor tyrosine kinases. In GIST, activation mutations in KIT and PDGFRA lead to uncontrolled ligand- independent receptor activation. Mutation hotspots of KIT gene are located at exons 9, 11, 13 and 17, whereas those of PDGFRA gene are situated at exons 12, 14 and 18. Mutation of extracellular domain of KIT encoded by exon 9 facilitate receptor dimerization. Mutations in the juxtamembrane domain, which is encoded by exon 11 of KIT and exon 12 of PDGFRA, allow dimerization of receptor without binding of ligands. Mutations of ATP binding region of kinase domain (encoded by exon 13 of KIT and exon 14 of PDGFRA) enhance kinase activity, while mutations of activation loop (encoded by exon 17 of KIT and exon 18 of PDGFRA) promote active conformation of kinase.(21) Table 6 and Figure 2 summarize the mutational landscape of GIST based on the data from population-based studies and clinical trials.(22-29) Frequencies of PDGFRA mutations are significantly lower among patients in clinical trials (mean 1.7%) than those in population-based studies (mean 14.9%) because GIST patients with PDGFRA mutations are associated with better prognosis and earlier stage and hence do not require systemic therapy.(9, 22, 23, 29)

KIT mutation accounts for 71.5% (64.8-89.1%) of mutations in GISTs.(24, 25, 27-29) Exon 11 mutation is the commonest mutation (61.1%, range: 56.1-77.1%). Deletion, substitution and duplication contribute to 23-28%, 2-20% and 2-7%, respectively. Deletion in exon 11 is associated with younger age, larger tumor size, higher mitotic count and poor prognosis, whereas duplication is associated with female and stomach predilection and better prognosis. Exon 9 mutation is found in 7.1-10.9% of GISTs, particularly in those arising from small and large intestine, and associated with poor prognosis. Exon 13 and exon 17 are rare mutation hotspots (<1-2%) in GISTs, which are almost exclusively spindle in morphology and more frequently developed in small intestine. GISTs with exon 13 and 17 mutants are associated good and intermediate prognosis, respectively.

PDGFRA mutation accounts for 14.9% (4.7-21.1%) of mutations in GISTs.(24, 25, 27-29) About 30- 40% of GISTs without immunoreactivity of KIT/CD117 harbour PDGFRA mutation. GISTs with PDGFRA mutation generally show predilection to gastric location (>90%) and epithelioid/mixed morphology, and favourable prognosis (except non-D842V exon 18 mutation).

Wild-type GIST, which express immunoreactivity of KIT/DOG1 but lack neither KIT or PDGFRA mutation, contributes to 13-18% of adult GISTs and 85% of pediatric GIST.(10-12) As previously mentioned, it is a genetically heterogeneous group (Figure 3). Wild-type GIST can be further stratified by using succinate dehydrogenase B (SDHB) immunohistochemistry and familial syndromes. On one hand, SDHB deficient wild-type GISTs accounts for about 5% of all GISTs, and can be sporadic or related to Carney triad and Carney- Stratakis syndrome. Carney triad is a constellation of GIST, paraganglioma and pulmonary chondroma with undetermined germline mutation, whereas Carney-Stratakis syndrome is an autosomal dominant disease with dyad of GIST and paraganglioma, and germline mutations in SDHB, SDHC or SDHD genes.(30) SDHB deficient wild-type GISTs are featured by female predominance (except for Carney-Stratakis syndrome), exclusive location in stomach, multifocality, epithelioid/mixed morphology, unpredictable clinical outcome by histology, indolent clinical course despite frequent nodal metastasis, and mutation id SDH subunits (except for Carney triad). On the other hand, SDHB proficient wild-type GISTs make up 10.5% of all GISTs, and are either sporadic (9%) or syndromic (1.5%). Syndromic SDHB proficient wild-type GISTs are associated with neurofibromatosis type 1, absence of sex/age predilection, small intestine in location, multifocality, spindle morphology, and favorable prognosis. Sporadic SDHB proficient wild-type GISTs can be further classified according to BRAF mutation. Sporadic SDHB proficient wild-type GISTs with BRAF mutation usually occur in 6th decade of age and small intestine with spindle morphology. Prognosis of this subgroup is inconclusive.(10, 29, 31, 32) Sporadic SDHB proficient wild-type GISTs without BRAF mutation are also known as quadruple wild-type GISTs without any mutation in KIT, PDGFRA, SDH and genes in RAS pathway (BRAF/NF1).(12, 13) They represent the commonest subgroup (7%) of wild-type GISTs and a genetically heterogeneous subgroup harboring ETV6-NTRK3 translocation, FGFR1-TACC1 translocation, mutation of MEN1 and MAX, and overexpression of COL22A1 and CALCRL.(12, 29, 33, 34) Due to complex genetic heterogeneity, clinicopathological features of this subgroup have not been well characterized.

Figure 1: Schematic diagram of the structures of KIT and PDGFRA receptor tyrosine kinases 

Figure 2: Mutational landscape of GIST 

Figure 3: Classification of wild-type GIST 

Table 6: Mutational landscape of GIST

Clinical implications of mutations in GIST

Different mutations in GIST have their own characteristic prognostic and therapeutic implications. Prognostic significance of individual mutations have been described by various investigators and briefly mentioned in the previous section. Rossi et al. recently systemically analyzed the prognostic impact of mutations among 451 patients with primary localized treatment-naive GISTs.(29) By multivariable Cox regression, mutational status was an independent prognosticator in addition to patient's age, tumor location, tumor size and mitotic count. Three molecular risk groups with prognostic significance were identified: Group 1 with the most favorable outcome is composed of mutations in KIT exon 13, PDGFRA exon 12 and BRAF; Group 2 with the intermediate outcome (hazard ratio 3.06) consists of KIT/PDGFRA/BRAF triple negative, and mutations in KIT exon 17, PDGFA exon 14 and 18 (D842V); and Group 3 with the most unfavorable outcome comprises mutations in KIT exon 9 and 11, and PDGFRA exon 18 (non-D842V).

Clinical response toward imatinib among GIST patients is closely related to tumor genotype. In a phase III clinical trial (SWOG S0033/CALGB 150105), the investigators demonstrated that patients with KIT exon 11 mutation (complete response [CR]/partial response [PR] 71.7%) had better response to imatinib than those with KIT exon 9 mutation (CR/PR 44.4%) and wild-type KIT (CR/PR 44.6%).(23) They also showed that doubling the dose of imatinib (from 400 mg to 800 mg) improved response rates for patients with exon 9-mutant tumors (CR/PR 17% vs. 67%). Double dose of imatinib did not offer any better response rate among patients with exon 11 mutant or wild- type KIT. A subsequent meta-analysis of 1,640 patients with advanced GIST receiving imatinib confirmed that double dose of imatinib improved progression-free survival and objective response rate, but not overall survival, among patients with KIT exon 9-mutant GIST.(35) PDGFA exon 18 (D842V) mutation and KIT/PDGFRA wild-type are responsible for primary resistance to imatinib.(36) Among patients with advanced GIST receiving imatinib, a substantial proportion of initial responders will develop acquired resistance. Secondary mutations in exon 11 (L576P and V559A), exon 13 (V654A), exon 14 (T670I), exon 17 and exon 18 (A829P) of KIT, and exon 18 of PDGFRA are related to acquired resistance to imatinib.(36)

Clinical response to sunitinib, the second line targeted therapy after imatinib failure, is also considerably affected by primary and acquired mutations of KIT. Patients with primary KIT exon 9 mutation or wild-type KIT had better overall and progression-free survival than those with KIT exon 11 mutation, whereas patients with acquired KIT exons 13 or 14 mutations had better outcome than those with KIT exon 17 or 18 mutations.(37) Similarly, clinical response to regorafenib, the third line therapy after imatinib and sunitinib failure, is significantly influenced by tumor genotype. Regorafenib provided better clinical outcome among patients with primary KIT exon 11 mutation and SDHB deficient GIST, (38) as well as those with secondary mutation of KIT exon 17, which are resistant to both imatinib and sunitinib.(39)

Summary

GIST is a genetically heterogeneous tumor. Genotypes and phenotypes are closely interrelated. Specific mutations have their characteristic clinicopathological features, prognostication and therapeutic implications. Genetic analyses KIT and PDGFRA are highly recommended especially among patients with advanced diseases undergoing targeted therapy. Wild-type GISTs are recommended to be further analysed by SDHB immunohistochemistry and BRAF mutation test. 

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Results of College Written Exam in Clinical Microbiology & Infection 2017
 

Candidates who failed in written exam cannot proceed further in Fellowship Assessment in 2017.
Passed candidates can proceed further in Fellowship Assessment in 2017.