Improving the Quality of Molecular Genetics Testing and Reporting

ESTABLISHMENT OF QUALITY ASSURANCE ACTIVITIES

FINAL REPORT MAY 2016

A project funded under the Australian Government’s Quality Use of Pathology Program

Table of ContentsBackground 2

Need 2

Benefits 3

Overview of The Project 4

Aims 4

Key Aspects of the Project 4

Somatic Cancer Module Development 4Acquisition of samples 5

Laboratory pre-testing (Method development, optimisation) 5

Nanodrop vs Fluorometer (Quantus, Promega) 7

Multiplex PCR optimisation 8

Establishment of an advisory committee for somatic cancers 10

Comparison between patient-derived and cell-line derived tissue samples 10

Completion of a homogeneity and stability study 10

Information Technology Development 11

Sample Exchange 15Establishment and development of relationships with tumour banks 15

Training Sessions 16Current situation 18

Problems encountered 18

Lessons learned 20

Future work 20

Funding / Sustainability plans 20

Appendices 22

A - Reference Testing – Somatic Cancer Module 22

B - Preliminary Report – Somatic Cancer Module 23

C - Stability pre-testing – Somatic Cancer Module 24

D - Homogeneity pre-testing – Somatic Cancer Module 25

E - Pathologist slide review – Somatic Cancer Module 26

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BackgroundGenetic testing by its very nature is highly complex, constantly changing, and therefore still under development for some new and novel techniques. Conventional models of quality assurance used in other pathology disciplines are not always appropriate for genetics. The results of genetic testing is however often critical and highly sensitive, and it is important that this testing be quality assured to the best extent possible.

The use of genetic testing as an aid in diagnosing disease, in predicting future disease risk, and by assessment in targeted therapy (including cancer) has grown at a rapid rate. Some genetic tests are still performed in research laboratories and a very large (and increasing) number of genetic markers have been identified, most of them performed in one or only a few specialist laboratories. Quality assurance of these tests are very difficult, albeit essential, in ensuring that the results are accurate, which is directly associated with patient safety and positive health outcomes.

Genetic testing often requires specialised equipment which can further add to the cost in both time and performance to produce a single result. In addition, there can be considerable time and effort involved in reporting that result to the referring clinician. The high costs and time required to produce these results therefore make it important that each genetic test be performed to the highest possible standard.

NeedQuality assurance programs for genetics are either rare or non-existent locally. Many laboratories are having to utilise international programs, but samples are difficult to bring into the country owing to customs and quarantine restrictions. Such feedback has been received from numerous laboratories that contacted the Molecular Genetics Quality Assurance Programs enquiring if it would be possible to set up quality assurance exchanges or modules to address the Australasian needs. In addition, it is a National Association of Testing Authorities (NATA) requirement for laboratories to participate in a quality assurance module if it is available locally. It is not mandatory for laboratories to enrol in quality assurance modules from international vendors. There therefore existed an important unmet need in the area of quality assurance for genetics.

Leading on from this, somatic cancers are becoming progressively more prevalent and owing to this we need to ensure that all molecular diagnostic laboratories are well equipped to deal with the increasing demand of testing. Many of these somatic cancer mutations have associated companion diagnostics. The need for detecting the

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correct mutation therefore becomes even more crucial in this context. This has just recently become even more important with resistance mutations being found in genes such as the epidermal growth factor receptor (EGFR).

In terms of somatic cancer testing, several aspects of the process needed to be quality assured to ensure laboratories were skilled in all areas from pre-analytical assessment of the tumour sections to post-analytical reporting of the mutation and the significance thereof to the referring clinician (Appendix A [Figure A]).

In addition there are numerous rare and infrequently tested genes for which no form of quality assurance exists. Establishing quality assurance sample exchanges for such markers were imperative to ensure some degree of quality metrics could be captured.

Benefits (How this Activity will contribute to improving the overall quality and standardisation of genetic testing in Australian laboratories)

One of the benefits we have seen from developing the somatic cancer modules was more discussion between laboratories and at conferences on both genotyping accuracy and interpretation. This is a crucial part of the process and needed to be addressed as a matter of urgency. We have enlisted the help of key opinion leaders in pathology practice to assist with these modules and we have received positive feedback from laboratories on the content and educational components of the modules. The program has steadily progressed from incorporating only patient derived tissues samples to including cell-line derived synthetic samples. This is a significant development as laboratories now have the ability to determine their mutation load from a sample where the allelic frequency/mutation load was determined by digital PCR with high accuracy, which adds to the overall quality of testing.

These genetic tests utilise complex technology derived from research-based processes and developed in-house in research based environments. Hence they are more difficult to quality assure than many other more standardised tests within the laboratory. There was a strong need to introduce more rigorous quality assurance processes to ensure that this testing is done accurately and effectively as the results of the genetic test are now seen to have critical implications, i.e., identifying the impact of drugs associated with specific gene mutations. As an example, we have included a detailed methods section into our worksheet to gather important information on the techniques and instruments being used to detect somatic gene mutations as there is an awareness of the sensitivities of methods used for a specific laboratory and this is collated in a generic report, which is sent out after all surveys

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have been assessed and completed. Where there have been anomalies in the results (i.e., a wildtype/normal sample comes back with a mutation from some laboratories) from laboratories using a specific instrument, it has been noted in the assessment reports, thereby allowing laboratories the opportunity to address their specific methodology and to discuss concerns with the equipment manufacturer. This leads to increased exposure of those laboratories and manufacturers that produce results that are incorrect.

In addition, several laboratories that develop assays for rare and infrequently tested genes have provided positive feedback on the benefit they have derived from us providing the sample exchange service through the Royal College of Pathologists Australasia Quality Assurance programs (RCPAQAP). This ensured the laboratories are independently assessed. Previously, where sample exchanges were set up by laboratories, the scientists that established the exchange was privy to all the laboratories results, as samples were not blinded or masked, which is not ideal for various reasons including confidentiality.

Overview of the ProjectAimsThe project aimed to establish an expanded range of external quality assessment modules with a focus on methodological exercises designed to assess proficiency in processes that are common to many assays.

Key Aspects of the ProjectThere were four aspects to the project.

i) Development of modules with specific reference to somatic cancers.

ii) Establishment of samples exchanges for rare diseases and those on the Medicare Benefit Schedule.

iii) Generation of software for data analysis (this tied in to some extent with module development)

iv) Design and implementation of training programs. The details of each aspect will be presented below.

With respect to development of external quality assessment modules for somatic cancers, the key objectives have been met, namely, i) external quality assessment programs to evaluate the diagnostic performance for new genetic tests and other external quality assessment exercises developed by this activity, and ii) quality

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assurance processes that have been developed by this activity. Details of each are provided below:

Somatic Cancer Module Development (External quality assessment programs to evaluate the diagnostic performance for new genetic tests and other external quality assessment exercises developed by this activity)

This served as the most significant, primary objective in the project. We set out to further develop three gene specific modules, namely EGFR (epidermal growth factor receptor), KRAS (V-ki-ras2 kirsten rat sarcoma viral oncogene homologue) and BRAF (V-raf murine sarcoma viral oncogene homologue b1) (Appendix B [Figure B]). These genes were of particular significance owing to mutation-specific companion drugs available. Further to this we set out to establish quality assurance capability for new markers such as NRAS (neuroblastoma RAS viral (v-ras) oncogene homologue) and either ROS1 (V-ros UR2 sarcoma virus oncogene homologue 1 (avian)) (if there is sufficient interest) or testing of KIT (Hardy-Zuckerman 4 feline sarcoma viral oncogene homologue) in gastrointestinal stromal tumours, for which there is currently no quality assurance program. A survey has been sent out to prospective participating laboratories to gauge interest in ROS1 and another survey is being prepared for gastrointestinal stromal tumours.

A comprehensive planning document was constructed to outline all stages of the module production process. This included: acquisition of samples, microtomy, a laboratory phase (including homogeneity and stability testing), advisory committee involvement, clinical scenario work-up, data analysis following return of results and report generation.

Acquisition of samplesA procedure is in place to obtain tumour blocks from Liverpool hospital. Meetings were held with Associate Professor Shantay Ryan and Professor Jim Yong at the outset. Dr. Tao Yang from the hospital has also been assisting with the provision of samples. Initially there was a system in place to obtain a single block, however over the past two years the enrolments have increased substantially (from 18 to >30) thus making it essential for at least two or three blocks to be provided. This has been an onerous task, however, with assistance from other laboratories including Sullivan Nicolaides Pathology (Queensland), Royal Prince Alfred Hospital (New South Wales), Queensland Medical Laboratory Pathology (Queensland), Canberra Hospital (New South Wales) and Pathwest Laboratory Medicine (Western Australia), we have been successful in obtaining the necessary number of tumour blocks for the quality assurance modules. Further to this, Professor Sandra O’Toole from New South Wales

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Pathology has been instrumental in providing samples, particularly for the BRAF and NRAS modules.

Laboratory pre-testing (Method development, optimisation)A defined plan was put together which included the following method development:

DNA extraction methods including both in-house and commercially available kits

Enzyme testing including those of high fidelity/high processivity activity from different manufacturers

Optimisation of multiplex polymerase chain reactions (PCRs)

Four different DNA extraction kits from Promega, Machery-Nagel and Qiagen were trialled (Figure 1). A separate DNA extraction method also trialled was an in-house based crude preparation Chelex method. The kit based methods appear to have a higher purity (260/280 ratio) (Figure 2). The in-house Chelex method showed the highest DNA yield before purification, however, DNA yield was dramatically reduced after DNA purification (Figure 3). All methods were consistent with DNA yield ranging from 12.90 to 15.47 µg (Figure 4). In terms of the kit based methods, in 2014, a new Qiagen GeneRead kit was manufactured specifically for next generation sequencing. We trialled this specific kit (Figure 2) and have since used it in preference. All our future samples will therefore be extracted using the Qiagen GeneRead kit.

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Figure 1 Comparison between different commercial kits for DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue.

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Figure 2. Comparison between different commercial kits for DNA extraction from FFPE Tissue and the in-house Chelex method of extraction.

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Figure 4. Comparison between total DNA yields obtained from commercial kits and the Chelex method (after DNA purification).Nanodrop vs Fluorometer (Quantus, Promega)DNA from formalin-fixed paraffin-embedded (FFPE) tissue is known to be inherently degraded. On account of this, it is important to determine how much of intact DNA is present. A Nanodrop spectrophotometer usually provides an indication of not only double-stranded DNA but also single-stranded nucleic acids including RNA. After conducting specific literature reviews on this, a fluorometer instrument was purchased as it has been shown to more accurately reveal the amount of DNA present in a sample (Figure 5, Figure 6, Figure 7). This is important for FFPE tissue samples as one can then accurately determine how much of sample to include in a multiplex PCR.

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Figure 5. Nanodrop vs Quantus Fluorometer measurements of DNA concentration, where DNA was extracted from blood samples.

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Figure 6. Nanodrop vs Quantus Fluorometer measurements of DNA concentration, where DNA was extracted from FFPE tissue samples.

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Figure 7. Nanodrop vs Fluoromenter DNA measurements shown, when a comparison between QIAamp and GeneRead (Qiagen) kits was undertaken. Multiplex PCR optimisationWith respect to optimisation of multiplex PCRs, a collection of six high-fidelity, proof-reading and robust Taq DNA polymerases were tested. Four Taq DNA polymerases were trialled as good reviews were obtained and they were provided as samples by the companies in question. These included 2 X platinum Taq (Platinum, Pfx), 1 X Amplitaq Gold, 1 X Phusion Taq, 2 X Bioline (MyTaq HS, MyFi). Previously, Amplitaq Gold was successful in producing the 5 expected bands of the multiplex PCR using DNA from blood samples as the template and only 4 bands were obtained when DNA from FFPE tissue samples were used. When the largest 600 bp band was tested as a single-plex using DNA from FFPE tissue, the Amplitaq Gold was successful in amplifying the product therefore suggesting that the problem lay with the conditions. The other Taq capable of producing 5 bands was the Bioline Taq. The Bioline Taq and Amplitaq were therefore suitable to amplify DNA from all tissue samples and Bioline was used as the Taq of choice for all samples in the module.

Figure 8 shows the multiplex PCR results using the Bioline MyTaq HS and DNA templates obtained using different DNA extraction kits.

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A. Qiagen QIAamp FFPE tissue kit. B. Qiagen GeneRead FFPE tissue kit.

C. Chelex (in-house), Reliaprep (Promega) & QIAamp kits. D. Nucleospin (MN) kits.

Figure 8 (A-D). Multiplex PCR results obtained using DNA templates obtained by extracting FFPE tissue samples from different commercial kits and the in-house Chelex method. In all instances the optimised MyTaq DNA polymerase (Bioline) was utilised (see also Appendix C [Figure C]).

Establishment of an advisory committee for somatic cancers An advisory committee was created for the somatic cancer modules with the inaugural meeting held in September 2013. Teleconference meetings have been held

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at least once a year since the establishment of the committee with a distinctive education component forming part of every meeting. The committee comprises of Professor John Christodoulou (Chair of the Molecular Genetics Advisory Committee), Dr. Beena Kumar (Anatomical Pathologist), Dr. Muhammad Alamgeer (Clinical Oncologist), Professor Sandra O’ Toole (Anatomical Pathologist), Dr. Bing Yu (Head of Laboratory and Senior Scientist), Dr. Kerryn Garrett (Senior Scientist in Charge, St John of God Pathology, Western Australia), Dr. Cleo Robinson (Medical Scientist in Charge – Pathwest, Western Australia), Dr. Karen Ambler (Senior Scientist, South Australia Pathology, South Australia). Dr. Glenice Cheetham also served as a member in 2014 and unfortunately resigned on account of her retirement. Oncologists Dr. Weng Ng and Dr. Bavanthi Balakrishnar have also played active roles in the committee as they assisted significantly with the generation of clinical scenarios and report review. Overall, the committee serves as a working party that reviews, assesses and comments on participants’ results. Further to this, meetings have been held with individual committee members such as Professor Sandra O’Toole, Dr. Beena Kumar, Dr. Weng Ng and Dr. Bavanthi Balakrishnar.

Comparison between patient-derived and cell-line derived tissue samplesThe National Measurement Institute (NMI) provided a synthetically produced BRAF DNA mutant (c.1799T>A) sample for testing purposes. The aim was to use this sample in the in BRAF module to assist laboratories with mutation load. A specific amount of mutant was provided in a background on wildtype (normal) genomic DNA. The samples were sent to specific laboratories for reference testing purposes and all laboratories were successful in detecting the c.1799T>A mutation. Unfortunately the workup required to produce this reference material was very high and the samples were very expensive and we elected to not use the sample in a module. At that point, another commercial avenue was pursued. Dr. Hallwirth Pillay contacted Horizon Diagnostics and they provided samples for testing purposes. At the outset, DNA samples were tested, however, it was decided that the FFPE tissue samples were more suitable as they closely mimicked the patient derived tissue samples included in the somatic cancer samples. This stage of the project is currently underway.

Completion of a homogeneity and stability study

This aspect of the project has been completed. Anatomical pathologists were enlisted to assist with determination of homogeneity prior to module send out. The pathologists reviewed representative slides and provided an indication of the amount of tumour was present. This was cross referenced with tumour content data provided by laboratories (Appendix D [Figure D] and Appendix E [Figure E]).

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Information Technology Development (Quality assurance processes that have been developed by this activity)

A review of the existing data handling software was undertaken by Dr. Hallwirth-Pillay and the information technology (IT) manager in early 2014. A new online data entry and reporting structure was discussed at length. The online data entry was based on already established company quality assurance program formats.

During the course of 2014, an external software development company was enlisted to produce online data entry and reporting capability. For Molecular Genetics, much work was put into developing the model and content for online data entry. This was a successful endeavour, however, the amount of resources it would have taken to establish reporting capability was too high if this were to be rolled out to the entire company. The company therefore decided to utilise internal resources to undertake this task as a company-wide project. One of the software developers was able to quickly mimic the online data model produced in collaboration with the manager of the genetics department and the external company, Proquest. This was implemented in 2015 for two Molecular genetics modules, mitochondrial myopathy (Figure 9) and Fragile X syndrome (Figure 10A and Figure 10B ) using the current quality assurance program desktop software, which is used by all other departments in the company.

Following the implementation of online data entry, feedback was received that the character limit set for data entry was insufficient for participants to enter the full results. This was quickly resolved by the IT department.

Figure 9A

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Figure 9B

Figure 9A and Figure 9B. Preliminary work undertaken by the external Proquest software development team for establishment of quality assurance online data entry capability for Molecular Genetics modules. A data input section (A) and a data reference section (B) are illustrated. Figure 10A

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Figure 10B

Figure 10A and Figure 10B Snapshot of the data entry screen for Fragile X syndrome. Final outcome of the IT project for developing Molecular Genetics quality assurance online data entry capability by the internal RCPAQAP Software development manager. This went live in 2015.

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Sample Exchange (External quality assessment programs to evaluate the diagnostic performance for new genetic tests and other external quality assessment exercises developed by this activity)

Establishment and development of relationships with tumour banks (the establishment of a sample exchange bank of test materials for rare genetic conditions)

Some difficulty was experienced with successfully implementing this aspect of the project. Meetings were held with the Manager of the tumour bank at the Children’s Hospital at Westmead (Professor Dan Catchpoole) and Monash Health Biobank (Zdenka Prodanovic and Dr. Beena Kumar). Monash Health Biobank provided a small number of samples on slides for testing purposes early in the project. Unfortunately, owing to them adopting a cost recovery model for services rendered, this source could not be used long term. A proposal, followed by an agreement document was put together for the Children’s Hospital at Westmead tumour bank. Following meetings with the head of the tumour bank, a decision was taken by the tumour bank governance department where it was revealed that the Sydney Children’s Hospital Network and RCPAQAP would need to enter into a full contract and sign a Material Transfer Agreement as quality assurance fell outside of the standard avenue of sample provision for research purposes. This is bound to become highly complex and it was recommended that the Molecular Genetics department approach individual pathology laboratories for donation of samples. This is already in place. Therefore, despite the effort that was put into this aspect of the project we were not successful in establishing a sample exchange bank of test materials from the tumour bank.

Table I shows the samples exchanges that were produced in 2014 and 2015. This was a successful undertaking and laboratory feedback indicated that benefit was gained from establishing this program.

Table I. Sample exchanges that were instituted in 2014 and 2015 at the Molecular Genetics Discipline of the RCPAQAP.

# Sample Exchange Sample Type

1 ALK Translocation in NSCLC FFPE slide

2 IL28B Genotyping DNA

3 Kennedy’s Disease DNA

4 TPMT Genotyping DNA

5 IDH1/ IDH2 Sequencing FFPE slide

6 CYP2C19 Genotyping DNA

7 CYP2C9 Genotyping DNA

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# Sample Exchange Sample Type

8 PTEN Genotyping DNA

9 CDH1 Genotyping DNA

10 Coeliac Disease DNA

11 MLH1 Promoter Methylation DNA

12 MGMT Hypermethylation FFPE slide

13 Oligodendroglioma LOH 1p19q FFPE slide

14 TP53 Genotyping DNA

15 Von Hippel Lindau Syndrome DNA

16 SDHB Genotyping DNA

We indicated that an evaluation of the sample exchanges will be undertaken at the end of 2014 and one of the modules will be converted into a pilot quality assurance module. This activity was performed and owing to increased demand and positive feedback received, we elected to convert the anaplastic lymphoma kinase (ALK) fusion oncogene positive non-small cell lung cancer sample exchange into a pilot quality assurance module in 2015. This is a joint immunohistochemistry and fluorescent in-situ hybridisation module. This has been a difficult module to implement owing to the labile nature of the FFPE tissue samples. It was brought to our attention that these samples degrade in approximately 6-8 weeks post microtomy sectioning. We therefore ensured that the microtomist cut the FFPE samples within a week of sample dispatch and laboratories were given 4 weeks for testing in order to keep to the time frame of 6-8 weeks. The plan for 2016/2017 is to further develop this module by utilising expertise from our Anatomical Pathology Department and it is hoped that this will become a cross discipline module. It already has the support of the Chair of the Anatomical Pathology Department, Dr. David Moffat. A meeting has been held between Dr. Moffat, Mr. Martyn Peck and Dr. Hallwirth-Pillay to discuss potential new ways of producing the module such as the inclusion of tissue microarrays/composite blocks.

Training SessionsThere were a number of training sessions undertaken by the project officer who has been overseeing aspects of the project. Unfortunately, the envisaged European Molecular Genetics Quality Network (EMQN) training sessions were not possible as they do not provide such training. We indicated in the proposal that we will endeavour to improve on our modules and reporting and aspects will be based on the EMQN design. This has been implemented where possible (Table II).

Table II. Overview of the training undertaken by the project officer.

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Event Title Completed Date

Trainer

Molecular Genetics - Induction and Training

9/01/2014 Hallwirth-Pillay, Kumari

Meet the experts - What's new in 2014

24/02/2014 Cepheid

Centre of excellence seminar 27/03/2014 Theis, Torsten

Q-Pulse Training - Document Module

7/04/2014 Bateman, Alan

Genetic Solutions World Tour - Life Technologies

21/05/2014 Thermo Fisher Scientific

SmartDraw Software Webinar 6/06/2014 SmartDraw Software, LLC

Centre of excellence seminar 11/06/2014 Cohen, Paul

Medical Genetics Symposium 27/08/2014 NGS, Cancer Genetics

2014 AIMS National Scientific Meeting

6/09/2014 Australian Institute of Medical Scientists

Mutation Surveyor 30/09/2014 Softgenetics

Salesforce training - Requests and Q-Pulse training

2/10/2014 Eris, Christine

Coverslipper training 10/11/2014 Lal, Neeta

SOP-AP-11 Routine sample Staining - 5

25/11/2014 Lal, Neeta

QF-AP-31 Embedder Maintenance and Cleaning Checklist - 3

25/11/2014 Lal, Neeta

Pathology Update 2015 1/03/2015 The Royal College of Pathologists of Australasia

Exiqon 17/03/2015 miRNA - biofluid samples as biomarkers

Qiagen: Challenges of FFPE Sample Materials

9/04/2015 QIAGEN

Molecular quality control: using reference standards in next generation sequencing (NGS) pipelines

27/05/2015 Horizon Diagnostics

Special interest groups - genetics and molecular pathology meeting

26/06/2015 Hallwirth-Pillay, Kumari, Thermo Fisher Scientific

Special interest groups - genetics and molecular pathology meeting

30/07/2015 Hallwirth-Pillay, Kumari, Torsten Theis

Special interest groups - genetics and molecular pathology meeting

9/10/2015 Hallwirth-Pillay, Kumari, Torsten Theis, Kwang Tay

Qiagen NGS Webinar 23/10/2015 QIAGEN

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Current situation The single gene somatic cancer modules were a highly successful endeavour. We

plan to continue providing these modules in a more expanded format (i.e., multi-gene modules). However, funding for resourcing is being sought as the modules are not self-sustainable. This is due to the significant amount of work required to prepare the modules. Furthermore, the small participant number makes it difficult to be revenue generating. All the assessments are undertaken without IT assistance as Adobe forms are used to capture the data and not the internal IT system. Owing to the amount of data being captured it is not possible to establish IT capability for this module in the short term. It is envisaged (with additional funding) that this will be possible to develop IT online data entry capability in the next 12-18 months as a software developer is required.

A collection of 16 sample exchange modules were produced in the Molecular Genetics department in 2014 and 2015. The modules were sent out twice in 2014 and once in 2015 owing to customer feedback and resourcing. Laboratories found that they needed a much longer lead time for these exchanges as they were rare disorders and the assays were batched in many instances. Unfortunately, the samples exchanges cannot be offered in 2016 as the grant funding ceases and this has left a void in the area of rare and infrequently tested genes. We hope that more funding is made available for us to continue to offer to crucial service in the latter part of 2016/2017. Feedback has already been received from some laboratories as they are unsure of how to adhere to the requirements of enrolling in a quality assurance sample exchange module without the RCPAQAP managing the service. One participant indicated that he has been able to optimise his assay owing to the information provided in the sample exchange report, which was highly beneficial to his laboratory.

Owing to customer demand the Molecular Genetics Department produced a pilot massively parallel sequencing (next generation sequencing, NGS) module even though our grant objective for this deliverable indicated that we would assess the need for such a module by familiarising ourselves with the platforms that are available and the type of NGS being performed (i.e., inherited diseases, somatic cancers, targeted approach and exome sequencing). The unmet need was clearly demonstrated by the increased customer input on the development of this module. The company utilised internal funds to initiate the pilot module. Discussions were held at advisory committee meetings, conferences and internal meetings.

Problems encountered (Difficulties encountered in performing the activity and the action that was taken to overcome those difficulties)

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Anaplastic lymphoma kinase non-small cell lung cancer/immunohistochemistry and fluorescent in-situ hybridisation module. The module preparation was undertaken well in advance to plan for homogeneity and stability testing. In addition, we utilised a casual staff member for microtomy as we had to work around his schedule. Unfortunately after the samples were prepared, it was brought to our attention that particular epitopes have just recently been shown in a publication to be highly labile therefore there is a short window in which all preparation as well as testing needs to be performed. This meant that the samples that were prepared earlier had to be discarded as the window for testing was too short for laboratories to meet the deadline. At that point, we acquired other samples and adhered to a very strict schedule for preparation, dispatch and testing.

Training sessions via the EMQN was not possible as they do not have a defined training plan. Other options were therefore explored and the staff were sent to symposia, conferences and meetings, as required. In addition, staff were sent to training sessions at Advisory Committee members’ laboratories to expand on their technical knowledge in a hand-on manner.

Sample exchanges: Some laboratories were not willing to provide samples to the exchanges even though they were aware that this was the process. This issue did not occur often, however it did hinder progress. In those instances, we either made the decision to include the laboratory that did not provide samples, if additional samples were available. If no additional samples were available, the laboratory was contacted and de-registered from the module.

In 2014, we elected to send out the Sample exchange modules twice a year, however this was extremely difficult to co-ordinate as laboratories were often late in submitting the results and this meant increased customer service and follow-up from RCPAQAP staff who were not associated with the grant deliverables. Feedback from some laboratories also confirmed the difficulty encountered in meeting the deadlines owing to the number of modules we produced (30 modules annually). These were therefore sent out once in 2015 and this alleviated some of pressure felt by laboratories and internally at the Molecular Genetics Department.

One of the genes, TP53 (Tumour protein p53), which we planned to establish as a pilot module did not draw enough enrolments as there were only three laboratories participating. This number was not high enough to warrant the time it would take to develop a quality assurance module. Dr. Hallwirth-Pillay therefore consulted with key opinion leaders on possible contenders for a pilot

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module, based on unmet needs and clinical utility and significance. Two genes were suggested, ROS1, where a fluorescent in-situ hybridisation (FISH) pilot module would need to be developed.

Lessons learned The time in which it takes to design, develop and implement the somatic

cancer modules was under-estimated. It took a significant amount of time to network with the appropriate laboratories and hospitals in order to acquire samples. This became more difficult as the enrolment numbers increased from 21 in 2013 to >30 in 2015 and two or three blocks were required to ensure adequate samples were provided. Further to this, reviewing the FFPE tissue blocks prior to microtomy was important in order to prevent a poor sample from being used. Also, hematoxylin and eosin staining should be implemented on the first few slides and cut to determine whether the samples are of good quality.

The samples exchange modules should have only been dispatched once a year as the amount of time it took to prepare the samples and dispatch them was significant, which meant additional staffing resources were required. The RCPAQAP utilised internal resources to meet this objective.

In terms of the anaplastic lymphoma kinase non-small cell lung cancer/immunohistochemistry and fluorescent in-situ hybridisation module, microtomy services need to be on-hand to prepare all samples in a timely manner to ensure the samples are dispatched to laboratories within a strict timeframe to ensure the testing does not fail on account of sample lability. We do not have internal microtomy resources in our department. This has been brought to the attention of our Chief Executive and there is a plan to create a laboratory team over the next 12 months to help prevent such issues in future.

Future work This was seen as the start of a much larger project in the rapidly expanding

area of somatic cancers. The oncology program is our most successful program and we plan to expand on the service and educational components of these modules.

The conversion of the anaplastic lymphoma kinase sample exchange module into a pilot quality assurance module in 2015 was a good choice and fortuitous decision as this has recently been given an item number on the Medicare Benefits Schedule. This is an example of how essential the development of quality assurance services can be to the broader scientific/diagnostic

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community. In addition, owing to this being a pilot quality assurance module, there is now an immediate need to establish a more elaborate module given that the government requires a significant amount of “post implementation monitoring data” from quality assurance programs. The collection of such data requires additional resources for which we are planning to seek funding.

Funding / Sustainability plans Dr. Hallwirth-Pillay has invested a significant amount of time training staff and

we therefore do not want to lose the staff that have contributed to these project deliverables.

The company has supported the department in these endeavours and owing to its success we do not want to lose any momentum, which has been gained from offering these modules. In addition to quality assurance, the modules have succeeded in educational components to all participants.

A company-wide innovation oversight team has been established and Dr. Hallwirth-Pillay is a member. One of the team’s focus is funding. She is also a member of the sub-committee that is working on a project to find appropriate funding sources for niche areas in the company, including Molecular Genetics. This is currently underway.

Dr.Hallwirth-Pillay has been in contact with Labceutics (labceutics webpage ) . They have been instrumental in assisting diagnostic laboratories with establishing guidelines and other training programs internationally. They are keen to work with Dr. Hallwirth-Pillay in establishing some capability in the area of cancer testing. Their funding contribution is expected to be fairly minor as they have offered to provide funding for cell-line derived samples only.

Dr. Hallwirth-Pillay has held discussions with some companies that have manufactured drugs that are associated with specific gene mutations as quality in pathology practice is of interest and importance to them as well. There may be an opportunity for small amounts of funding for very specific areas such as developing quality assurance capability for specific gene (i.e., EGFR) associated mutations.

Funding will be sought for the design and development of a cross discipline Anatomical Pathology/Molecular Genetics, anaplastic lymphoma kinase non-small cell lung cancer/immunohistochemistry and fluorescent in-situ hybridisation quality assurance module in order to provide the government with post implementation monitoring data. The plan for this module has been initiated by Dr. Hallwirth-Pillay as she is overseeing a company-wide project to

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develop cross functional modules between different departments at the RCPAQAP.

AppendicesA - Reference Testing – Somatic Cancer Module

Figure A. Reference testing document provided to laboratories performing pre-testing of samples prior to module preparation.

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B - Preliminary Report – Somatic Cancer Module

Figure B. Preliminary report outlining the genotyping results of samples that were included the survey.

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C - Stability pre-testing – Somatic Cancer Module

Figure C. Stability testing documents providing a summary of the multiplex PCR results obtained from potential survey samples.

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D - Homogeneity pre-testing – Somatic Cancer Module

Figure D. Image showing the sample sets (kits) that are sent out to laboratories. This provides a good indication of the sample homogeneity however the actual tumour content is not obvious by such a macroscopic view. Virtual Microscopy is therefore also undertaken.

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E - Pathologist slide review – Somatic Cancer Module

Figure E. Homogeneity pre-resting document outlining the slide assessment undertaken by an anatomical pathologist prior to module dispatch, which aims to provide an assessment of the tumour content.

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