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Tumours and their genomes are neither homogenous nor static; it is appreciated that they may change and evolve with time.1 This process might contribute to the failure of clinical systemic treatments, and therefore remains an important clinical challenge to overcome, and biological phenomenon to understand.2 During tumour evolution, changes to the genome (mutations) and its gene expression lead to metastatic spread and promotes the survival of the ‘fittest’ of a genomically heterogenous mix of cancer cell ‘clones’ that can be disseminated across the body. Research into tumour evolution has been enabled by the advent of next-generation sequencing providing a better appreciation of the genomic complexity of the tumour cells that are present within an individual patient. This builds on an already appreciated histological understanding of morphological heterogeneity between a primary tumour and its seeded metastases.

Tumour heterogeneity can occur between different lesions from a single patient, but also within each lesion with different cells within the tumour evolving different genotypes and having varying potential to metastasise.3 Important advancements in understanding the genomic landscapes of many tumour types have been made through global sequencing programmes such as the International Cancer Genome Consortium.4 However, these programmes have been less valuable for understanding the genomic heterogeneity of tumours within individual patients. Nevertheless, in the clinic, understanding tumour heterogeneity within each patient can sometimes contribute to appropriate treatment decisions. For example, the presence of multiple distinct tumour cell clones within the tumour(s) of an individual could lead to treatment resistance where the ‘fittest’ cells survive and evade therapy or require a different treatment approach. Similarly, specific clones may selectively metastasise to areas of the body that are less amenable to systemic therapy, such as beyond the blood brain barrier. However, these processes remain incompletely understood.

Researchers appreciating the clinical importance of tumour heterogeneity within individual patients have made significant gains in the laboratory. Approaches often involve the analysis of multiple solid tissue samples from an individual, for example for pancreatic or renal carcinomas, taken at different times during their care and/or from different sites in their body.5,6 This temporal and spatial information has been invaluable in better understanding the molecular changes that occur as a tumour evolves to evade therapy.7,8 However, solid tumours, particularly within internal organs, are a challenge to sample unless surgically resected. Invasive tissue biopsies are seldom taken for purely research purposes due to the risks to the patients, making the study of progressive disease difficult in patients where surgical intervention is no longer clinically helpful. Circulating free DNA (cfDNA) has been proposed as a surrogate for multi-organ sampling, but it is unknown whether cfDNA can represent all tumours in the body, or if there is a selective elucidation of individual clones or favouring of particular anatomical locations. To facilitate study of metastastatic cancer in single patients, research hospitals and biobanks around the globe have established routine ‘Rapid Autopsy’ programmes, allowing patients to donate tissues through autopsy at their point of natural death for use by research groups to better understand tumour heterogeneity and evolution. Such established programmes at over a dozen institutes worldwide have collected invaluable samples, leading to improved understanding across the spectrum of cancers.9,10 Several well-documented programmes are located in the US (eg, Johns Hopkins Legacy Gift Rapid Autopsy Programme,11,12 Fred Hutchinson Institute at University of Washington13 and University of Michigan14) and Australia (CASCADE programme, Peter MacCallum Cancer Centre15). No established programme of this kind for cancer research exists in New Zealand.

Our New Zealand-based research group, NETwork!, brings together clinical, epidemiological and genomic information to build improved biological understanding of neuroendocrine tumours (NETs). The NETwork! programme was established in 2012 and works closely with patients, collecting tissues for use in research with the ultimate aim of improving care for people diagnosed with NETs in New Zealand. The team is truly multidisciplinary with expertise ranging from clinical oncology through to computational biology. In early 2016, a patient with a metastastic broncho-pulmonary NET made a direct approach to her oncologist, a principal investigator of the NETwork! team. The patient had multiple metastases from her original tumour, and wished to donate her tumour tissues through rapid autopsy on her natural death. Her hope was to contribute to biological understanding which might help those who followed her. Initially, the research team were reluctant to pursue this request and declined, not having established the ethical or logistical framework to effectively conduct such a procedure. However, the patient was persistent and returned on her next appointment accompanied by two of her children, who were both highly supportive. Given this level of determination on the part of the patient, and the strong support from her immediate family, the team felt that they had a duty to at least ascertain whether or not such a procedure would be possible.

In this viewpoint article we summarise the considerable efforts taken to develop and coordinate a process that could meet the challenge initiated by this selfless act. This undertaking has allowed us to assemble and analyse a unique tissue resource, and has given us a valuable understanding of the legal, ethical and logistical considerations needed to carry out rapid autopsy research in New Zealand. We also highlight the value of this partnership between patient, family and researchers to better understanding the biology of neuroendocrine tumour evolution.

Feasibility assessment

After the initial approach from the patient to her treating oncologist, the first step was to assess the feasibility of sample collection through a rapid autopsy procedure. Considerations were made on scientific, logistical, ethical and financial bases. This was performed in parallel through multiple discussions with the lead of the Forensic Pathology Department at Auckland City Hospital, with the Health and Disability Ethics Committee (HDEC), DHB clinicians responsible for her care, and with the laboratory researchers within the NETwork! group. These discussions took a number of months, with ongoing consultation needed throughout the pre-autopsy period in order to plan and later refine the processes. A further consideration for the research team was to balance the scientific value of the samples with the financial cost of collection and subsequent analysis. The study, the autopsy, sample collections and processing were funded as part of the existing NETwork! programme. A research project was clearly designed around the potential use of the samples before committing to the autopsy to ensure a scientifically beneficial study was possible with the types of tissues that could be collected. These consultations concluded with the decision to proceed with organising the rapid autopsy process, promising best efforts to the family to collect the samples on the day of her natural death. Ongoing communication with the family was important to clarify expectations and balance the likelihood of the autopsy going ahead.

Ethical and legal consent

Upon confirmation from all parties that the process was feasible, ethical approval was sought as an amendment on an existing ethical approval for prospective collection of NET tissue from clinically indicated medical procedures (eg, surgery and biopsy). The patient and their family described their wish to participate with no issues raised during this conversation regarding religious or cultural beliefs. Informed signed donor consent was obtained for the tissues to be collected, stored and used for genomics research after death via an autopsy process and was co-signed by family members. Legal consent was also received to perform an autopsy. The HDEC amendment was submitted alongside letters of support from the donor and her family. This step in the approval followed extensive previous community consultation regarding tissue collection for other parts of the project, and took just three weeks to obtain. When obtaining this approval, we undertook to ensure that the patient’s participation in this study would have no impact on her clinical care.

Sampling documentation plan

Spatial information is vital to meaningfully model and interpret tumour metastasis and clonal evolution. In order to accurately map every lesion and site sampled, a documentation process was developed in conjunction with a radiologist and an anatomical pathologist. Cross-sectional images from computed tomography (CT) scans conducted as part of clinical care or follow-up were carefully interrogated prior to the autopsy, and every visible individual lesion localised and anatomic location stated and mapped. This information would then be adjusted according to findings made by the forensic pathologist and technician during the autopsy. The plan would require the manual completion of paper-based forms to record anatomical specimen location, assign naming codes and to sketch diagrams of where each sample was taken from within a specimen. A coding system was developed whereby each excised specimen was assigned a number, and the individual samples derived from this specimen were assigned either a letter or numerical code if to be stored as formalin fixed paraffin embedded (FFPE) or fresh frozen tissues respectively; to be recorded using the paper forms. Further, a photography plan was developed to incorporate in-situ and resected large-scale photographs of each specimen and the samples derived from it. As we wanted to use samples for genomic analysis in combination with morphological analysis it was imperative that the quality of the nucleic acids was maximised in the stored tissues. The decision was made to store both formalin fixed and snap frozen tissues in order to ‘future-proof’ the collection for use in multiple downstream molecular analyses.

Preparing the team

In order to ensure that enough people were available on the day of autopsy, remembering that it could happen at any time of any day, a roster was established to ensure that all areas of expertise would be available. A hierarchical phone-call system was also established to be used to contact people when needed. The autopsy required people with clinical training; oncologists, surgeons and pathologists (forensic and anatomical); as well as tissue banking scientists from the Auckland Regional Tissue Bank and the NETwork! laboratory team experienced in preparation of samples for genomic analyses. All members of the team generously volunteered their time to assist with the project.

Assembling the kit

The collections required extensive surgical and laboratory consumables and ready access to liquid nitrogen and dry ice stores. Documentation forms and sampling tubes (fresh frozen samples) and formalin-filled pots (FFPE samples) were pre-labelled with alpha-numeric codes. All items were assembled on a large trolley ready to be transferred from the research laboratory at the University of Auckland Grafton Campus across the road to the Auckland City Hospital Mortuary when required.

The autopsy

On an early morning in mid-2017, now 14 months following her initial request, our donor passed away from a natural death. She had since moved from her own home to care in a private nursing hospital, and further discussions and collaboration had been fostered with her carers, GP and institution administrators. The family had chosen a funeral director with whom they had an existing relationship, and discussion and planning for the process required had been made in advance. The treating oncologist received a phone call from the private hospital nurse at 4am and the roster phone calls were initiated and cascaded to pathologists and the research team. A member of the patient’s family met with the oncologist at the bedside, acknowledged her life and her gift, farewelled their mother, and the process began. End-of-life paperwork was completed.

The timing was fortuitous; a quiet early weekday morning allowed easy access to the mortuary and a complete, experienced group of people were available to assist. The donor arrived at the mortuary two hours after her passing. Eleven people worked for six hours to collate over 300 carefully annotated tissue samples. The team was divided into two key groups: the post-mortem team and the sampling team. The post-mortem team included a forensic pathologist, an oncologist, a surgeon and a forensic technician. Another research team member was the link between the two teams, moving between the autopsy room to courier samples and information to the sampling team. The sampling team was led by an anatomical pathologist, alongside two sampling excision staff, two annotation/recording staff and one sample preservation staff member. The sampling team were located in a separate laboratory, away from the autopsy. This enabled separation for non-clinical staff and allowed for the donor’s privacy and dignity to be preserved.

At the donor’s last CT imaging scan, over 90 distinct lesions had been identified, ranging from small 5mm subcutaneous nodules to replacement of complete organs with tumour tissue. All lesions but one were successfully sampled. Prior to removal, each lesion was photographed by the team member acting as courier, labelled with a unique number, excised and placed in a pre-labelled dish with orientation noted, before being transferred to the sampling team in an adjacent laboratory. Here, the excised lesions were again photographed and sampled into small cubes for snap freezing or placed into formalin for fixation. Where tissues were large and potentially heterogeneous, multiple samples from different regions were taken; see Figure 1 for an example of the complex sampling in the thyroid gland, compared to sampling of a smaller subcutaneous lesion. Every sample code was noted onto the specimen documentation forms (in written and diagramatic form) in order to record the location of each specimen relative to the others. The stored samples averaged 5mm3 for the frozen tissues and 20mm3 for the formalin fixed tissues. No tissues were stored that would be unlikely to be used in later research and all surplus tissues were ‘returned to the body’ for cremation, which had been the donor’s wish. Participation in the autopsy did not change the timing or nature of her funeral arrangements.

Figure 1: Comparison between sampling methods for large and small lesions.

c

For large lesions, such as the thyroid tumour shown, the lesion was first sliced into 10mm thick sections, sections were carefully laid flat in order, photographs were taken of all sections lying flat and individual sections were sampled, taking some samples for fresh frozen and some for FFPE fixation. All sites were carefully annotated on paper drawings, which were later transferred onto the photographs of sections, and unused tissue was returned to the body. For small lesions, such as the 8mm subcutaneous lesion shown here, the lesion was bisected, photographed, and each side received either fresh frozen or FFPE fixation.

Sample processing

After the day of autopsy, 141 small specimen containers of tissues fixed in formalin were transferred into ethanol after adequate fixation. Where too large to be blocked individually, tissues were divided, totalling 187 samples, and blocked in paraffin with further photographs taken to record specimen orientation and relative location. This process took a number of weeks, and was carefully documented. A small number of vertebral specimens required decalcification and were placed into a gentle EDTA buffer for up to 12 weeks in order to try and retain the nucleic acid quality and tissue morphology, following an appropriate decalcification protocol to optimise preservation of nucleic acids.16 Finally, all data and photographs were transferred to digital storage on a password-secured server and stored in a non-identifiable manner.

Speed was essential to maximise the quality of nucleic acids for later genomics. The samples were collected over the course of six hours and timing of freezing or fixation carefully documented as samples were sequentially collected. The complexity of the case, ie, the extensive number of sampled lesions throughout the body defined the timeframe. This extended timeframe to fixation could affect sample quality, causing hypoxia and necrosis driven changes within the RNA and methylation profiles in the tissues.17 Careful documentation of sampling has been important to monitor for these effects in resultant genomic data.

Invaluable tissue samples

Tissue samples are only as valuable as their annotation. This includes the histopathology, the spatial and organ location as well as a detailed summary of clinical features. The clinical history of the donor is summarised in Figure 2, alongside a summary of the breadth of information collected during this project in Figure 3. The donor’s tumour progression and clinical care is shown overlaid with the samples collected both for diagnostics and for research, imaging dates, and the research project consultations and collaborations. Since its inception, this project has required input from close to 100 people, each contributing their expertise in areas including pathology, radiology, surgery, oncology, tissue banking, genomics and evolutionary mathematics. It has provided a valuable training opportunity for a PhD student who is working to coordinate new collaborations with evolutionary biologists to better model the progression of the lesions. Indeed the experience was a profoundly moving and unique experience for all members of the laboratory research team. A year and a half after her passing, the NETwork! group presented the first preliminary results of the evolutionary genomics model to the audiences at the New Zealand Society of Oncology and Queenstown Research Week annual meetings. The overwhelming feedback from these presentations has highlighted the generosity, value and incredible opportunity provided from the donation. The patient’s family continue to provide advice, undertake lab visits and receive project updates. It is interesting to consider processes we may alter, should we complete another similar study. Our collection resulted in a large number of tissue samples from a range of complex tissue types, all found to be suitable for the prespecified genomic analyses. We believe the strengths of the project are the relationship with the patient and family, the focus on carefully designing the types of analyses to be undertaken prior to tissue collection, and completing necessary process personalisation with respect to the clinical case, geographical location and timing. Above all, research value, impetus and research time to analyse the samples must be considered in order to accept invaluable patient donations.

Figure 2: Overall clinical and research history of the donor.

c

The top two rows indicate the patient’s clinical history, including chemotherapy regimes (CAP-TEM refers to combination Capecitabine and Temozolomide chemotherapy), and computed tomography (CT) scan timings, overlaid on the timeline in row three. Row four indicates the tissue samples collected for clinical care and research. Row five and six show when the rapid autopsy consultation occurred in relation to clinical events, and the initiation of consultations and collaborations currently underway.

Figure 3: Summary of all data types collected and available for integration in evolutionary analyses.

c

The collection of tissues at autopsy enabled generation of a wealth of genomic and histopathological data, which will be interpreted alongside clinical imaging, photographs and sample annotation to form spatial models and augmented reality representations to better understand tumour evolution in this donor.  

The New Zealand context

While this work may be the first tumour collection tissue banking rapid autopsy of its kind in New Zealand, or at least the most extensive effort documented thus far, it is certainly not the first research tissue rapid autopsy collection in New Zealand. Among other initiatives, the now-named Neurological Foundation Douglas Human Brain Bank has been collecting donated human tissue at post-mortem for over 20 years, and underpins many of the research successes of the Centre for Brain Research at University of Auckland, like providing new evidence that adult human brain cells were capable of regeneration.18 The success of the Brain Bank, and indeed our one-off rapid autopsy collection, indicate that it is possible to undertake rapid autopsy tumour collections for use in research, but extending this out to a routine tumour collection rapid autopsy programme for our small country is still a large leap. Limitations in New Zealand include the facilities and resources available for completing a research autopsy as well as the funding required to staff, process and suitably conduct research around the collected specimens.

Our programme was tailor-made in response to this individual donor and her family, with the research project designed around the tumour tissues to be collected—an unorthodox way to design a research programme in the era of competitive funding driving research. While we envy permanent rapid autopsy programmes in operation in the northern hemisphere and Australia, we believe that there are key advantages to the personalised approach we have employed here. The number of samples collected, the extent of sampling of each lesion, the degree of annotation of these samples, the bespoke preservation strategies for unusual sample types, the close relationship with the donor and her family, and the commitment from the entire team to honouring the wishes of our donor, we believe are all important aspects to what makes this tumour collection unique and valuable; and indeed more difficult to achieve as part of a large formal programme. We argue that there is great value in the tailoring that is possible with one-off programmes, even when accounting for the increased time investment. In small countries like New Zealand, hoping to contribute on the same scale as large laboratories and institutions across the world will always be challenging, but instead of focusing on throughput, perhaps an emphasis on quality, completeness and annotation proves the real value that our approach can provide.

Digital developments

This bespoke project has provided our research group with experiences beyond the value of the tissue itself. Aside from providing the opportunity for student training within the genomics field, the study has also initiated a unique collaboration between the NETwork! team, the School of Architecture and the Centre for eResearch at the University of Auckland to build an augmented reality model of the donor’s tumours and how they changed over time, to enable interpretation of the genomic and evolutionary patterns in 3D and improve our spatial appreciation of complex genomic data. Further, it has provided impetus for members of the research team to develop a digital application for enhanced recording of sampling for use during tissue banking; one of the changes that we might implement if the process was repeated.

Reflection from patient’s family (anonymous)

When our mother first suggested donating her tumours to medical research on her death, we were all very supportive of her decision to hopefully benefit others in the future who contract the type of cancer that had slowly ravaged her body—and we still are. The team that undertook this task have been fantastic right from Mum’s initial donation suggestion and we can’t thank them enough for not only the work they are currently doing, but also ensuring we have been kept in the loop as Mum’s tumours have been analysed. Late last year we saw the first virtual replication of Mum’s body and her tumours and it was mind-blowing—she would have been (and may still be) thrilled to see what her donation has led to and there is still a long way to go. We are all very proud of Mum’s decision and hope the ongoing study of her tumours by a great team of specialists will help to increase the understanding of her cancer, making a difference in the future.

Conclusion

Donated human tissue is a valuable resource in medical research, and post-mortem tissue is particularly valuable when it allows collection of tissues not usually available through standard clinical procedures. This generous donation will enable the study of fundamental questions plaguing cancer biology, tumour evolution and heterogeneity, and leave a legacy in the form of a unique tissue resource. Here we have outlined the process by which a post-mortem collection procedure can be built from the ground up. While considerable effort is required to coordinate the legal, ethical, scientific and logistical considerations needed to carry out a bespoke rapid autopsy in New Zealand, the value of tissue collected and its scientific utility well outweigh these considerations when collected as part of a carefully designed research study. The latter point is the key to balancing whether to undertake a bespoke autopsy process; to ensure that the gift is indeed used for the purpose that it was given. The donor handed responsibility to the research team to use this gift for the good of others, and this ethos forms the basis for our ongoing work. We honour the incredible foresight and generosity shown by the donor and her family in championing this research.

Summary

Abstract

Genomic analysis of tissues from rapid autopsy programmes has transformed our understanding of cancer. However, these programmes are not yet established in New Zealand. Our neuroendocrine tumour research group, NETwork!, received a request from a patient wishing to donate tumour tissues post-mortem. This viewpoint article summarises the ethical, logistical and social process undertaken to accept this patient s generous donation, and highlights the scientific and educational value of such a gift.

Aim

Method

Results

Conclusion

Author Information

- Cherie Blenkiron, Senior Research Fellow, Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland; The Maurice Wilkins Centre for Biodiscovery, The University of Auckland, Auckland; Tamsin Robb, P

Acknowledgements

Correspondence

Dr Cherie Blenkiron, Senior Research Fellow, Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland.

Correspondence Email

c.blenkiron@auckland.ac.nz

Competing Interests

All authors report grants from Translational Medicine Trust during the conduct of the study.

  1. McGranahan N, Swanton C. Clonal Heterogeneity and Tumor Evolution: Past, Present, and the Future. Cell 2017; 168(4):613–28. doi: 10.1016/j.cell.2017.01.018
  2. Burrell RA, Swanton C. Tumour heterogeneity and the evolution of polyclonal drug resistance. Mol Oncol 2014; 8(6):1095–111. doi: 10.1016/j.molonc.2014.06.005
  3. Burrell RA, McGranahan N, Bartek J, et al. The causes and consequences of genetic heterogeneity in cancer evolution. Nature 2013; 501(7467):338–45. doi: 10.1038/nature12625
  4. Gundem G, Van Loo P, Kremeyer B, et al. The evolutionary history of lethal metastatic prostate cancer. Nature 2015; 520(7547):353–57. doi: 10.1038/nature14347
  5. Saygin C, Matei D, Majeti R, et al. Targeting Cancer Stemness in the Clinic: From Hype to Hope. Cell Stem Cell 2019; 24(1):25–40. doi: 10.1016/j.stem.2018.11.017
  6. Campbell PJ, Yachida S, Mudie LJ, et al. The patterns and dynamics of genomic instability in metastatic pancreatic cancer. Nature 2010; 467(7319):1109-13. doi: 10.1038/nature09460
  7. Ascierto ML, Makohon-Moore A, Lipson EJ, et al. Transcriptional Mechanisms of Resistance to Anti-PD-1 Therapy. Clin Cancer Res 2017; 23(12):3168–80. doi: 10.1158/1078-0432.CCR-17-0270
  8. Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing. New England Journal of Medicine 2012; 366(10):883–92. doi: 10.1056/NEJMoa1113205
  9. Fan J, Iacobuzio-Donahue CA. The Science of Rapid Research Autopsy. In: Hooper JE, Williamson AK, eds. Autopsy in the 21st Century: Best Practices and Future Directions. Cham: Springer International Publishing 2019:151–66.
  10. Hooper JE, Duregon E. Performance of Rapid Research Autopsy. In: Hooper JE, Williamson AK, eds. Autopsy in the 21st Century: Best Practices and Future Directions. Cham: Springer International Publishing 2019:167–85.
  11. Yachida S, Jones S, Bozic I, et al. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature 2010;467(7319):1114–7. doi: 10.1038/nature09515
  12. Liu W, Laitinen S, Khan S, et al. Copy number analysis indicates monoclonal origin of lethal metastatic prostate cancer. Nat Med 2009;15(5):559–65. doi: 10.1038/nm.1944
  13. Kumar A, Coleman I, Morrissey C, et al. Substantial interindividual and limited intraindividual genomic diversity among tumors from men with metastatic prostate cancer. Nat Med 2016; 22(4):369–78. doi: 10.1038/nm.4053
  14. Grasso CS, Wu YM, Robinson DR, et al. The mutational landscape of lethal castration-resistant prostate cancer. Nature 2012; 487(7406):239–43. doi: 10.1038/nature11125
  15. Patch AM, Christie EL, Etemadmoghadam D, et al. Whole-genome characterization of chemoresistant ovarian cancer. Nature 2015; 521(7553):489–94. doi: 10.1038/nature14410
  16. Choi SE, Hong SW, Yoon SO. Proposal of an appropriate decalcification method of bone marrow biopsy specimens in the era of expanding genetic molecular study. J Pathol Transl Med 2015; 49(3):236–42. doi: 10.4132/jptm.2015.03.16
  17. Zhou JH, Sahin AA, Myers JN. Biobanking in genomic medicine. Arch Pathol Lab Med 2015; 139(6):812–8. doi: 10.5858/arpa.2014-0261-RA
  18. Curtis MA, Penney EB, Pearson AG, et al. Increased cell proliferation and neurogenesis in the adult human Huntington’s disease brain. Proc Natl Acad Sci U S A 2003; 100(15):9023–7. doi: 10.1073/pnas.1532244100

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Tumours and their genomes are neither homogenous nor static; it is appreciated that they may change and evolve with time.1 This process might contribute to the failure of clinical systemic treatments, and therefore remains an important clinical challenge to overcome, and biological phenomenon to understand.2 During tumour evolution, changes to the genome (mutations) and its gene expression lead to metastatic spread and promotes the survival of the ‘fittest’ of a genomically heterogenous mix of cancer cell ‘clones’ that can be disseminated across the body. Research into tumour evolution has been enabled by the advent of next-generation sequencing providing a better appreciation of the genomic complexity of the tumour cells that are present within an individual patient. This builds on an already appreciated histological understanding of morphological heterogeneity between a primary tumour and its seeded metastases.

Tumour heterogeneity can occur between different lesions from a single patient, but also within each lesion with different cells within the tumour evolving different genotypes and having varying potential to metastasise.3 Important advancements in understanding the genomic landscapes of many tumour types have been made through global sequencing programmes such as the International Cancer Genome Consortium.4 However, these programmes have been less valuable for understanding the genomic heterogeneity of tumours within individual patients. Nevertheless, in the clinic, understanding tumour heterogeneity within each patient can sometimes contribute to appropriate treatment decisions. For example, the presence of multiple distinct tumour cell clones within the tumour(s) of an individual could lead to treatment resistance where the ‘fittest’ cells survive and evade therapy or require a different treatment approach. Similarly, specific clones may selectively metastasise to areas of the body that are less amenable to systemic therapy, such as beyond the blood brain barrier. However, these processes remain incompletely understood.

Researchers appreciating the clinical importance of tumour heterogeneity within individual patients have made significant gains in the laboratory. Approaches often involve the analysis of multiple solid tissue samples from an individual, for example for pancreatic or renal carcinomas, taken at different times during their care and/or from different sites in their body.5,6 This temporal and spatial information has been invaluable in better understanding the molecular changes that occur as a tumour evolves to evade therapy.7,8 However, solid tumours, particularly within internal organs, are a challenge to sample unless surgically resected. Invasive tissue biopsies are seldom taken for purely research purposes due to the risks to the patients, making the study of progressive disease difficult in patients where surgical intervention is no longer clinically helpful. Circulating free DNA (cfDNA) has been proposed as a surrogate for multi-organ sampling, but it is unknown whether cfDNA can represent all tumours in the body, or if there is a selective elucidation of individual clones or favouring of particular anatomical locations. To facilitate study of metastastatic cancer in single patients, research hospitals and biobanks around the globe have established routine ‘Rapid Autopsy’ programmes, allowing patients to donate tissues through autopsy at their point of natural death for use by research groups to better understand tumour heterogeneity and evolution. Such established programmes at over a dozen institutes worldwide have collected invaluable samples, leading to improved understanding across the spectrum of cancers.9,10 Several well-documented programmes are located in the US (eg, Johns Hopkins Legacy Gift Rapid Autopsy Programme,11,12 Fred Hutchinson Institute at University of Washington13 and University of Michigan14) and Australia (CASCADE programme, Peter MacCallum Cancer Centre15). No established programme of this kind for cancer research exists in New Zealand.

Our New Zealand-based research group, NETwork!, brings together clinical, epidemiological and genomic information to build improved biological understanding of neuroendocrine tumours (NETs). The NETwork! programme was established in 2012 and works closely with patients, collecting tissues for use in research with the ultimate aim of improving care for people diagnosed with NETs in New Zealand. The team is truly multidisciplinary with expertise ranging from clinical oncology through to computational biology. In early 2016, a patient with a metastastic broncho-pulmonary NET made a direct approach to her oncologist, a principal investigator of the NETwork! team. The patient had multiple metastases from her original tumour, and wished to donate her tumour tissues through rapid autopsy on her natural death. Her hope was to contribute to biological understanding which might help those who followed her. Initially, the research team were reluctant to pursue this request and declined, not having established the ethical or logistical framework to effectively conduct such a procedure. However, the patient was persistent and returned on her next appointment accompanied by two of her children, who were both highly supportive. Given this level of determination on the part of the patient, and the strong support from her immediate family, the team felt that they had a duty to at least ascertain whether or not such a procedure would be possible.

In this viewpoint article we summarise the considerable efforts taken to develop and coordinate a process that could meet the challenge initiated by this selfless act. This undertaking has allowed us to assemble and analyse a unique tissue resource, and has given us a valuable understanding of the legal, ethical and logistical considerations needed to carry out rapid autopsy research in New Zealand. We also highlight the value of this partnership between patient, family and researchers to better understanding the biology of neuroendocrine tumour evolution.

Feasibility assessment

After the initial approach from the patient to her treating oncologist, the first step was to assess the feasibility of sample collection through a rapid autopsy procedure. Considerations were made on scientific, logistical, ethical and financial bases. This was performed in parallel through multiple discussions with the lead of the Forensic Pathology Department at Auckland City Hospital, with the Health and Disability Ethics Committee (HDEC), DHB clinicians responsible for her care, and with the laboratory researchers within the NETwork! group. These discussions took a number of months, with ongoing consultation needed throughout the pre-autopsy period in order to plan and later refine the processes. A further consideration for the research team was to balance the scientific value of the samples with the financial cost of collection and subsequent analysis. The study, the autopsy, sample collections and processing were funded as part of the existing NETwork! programme. A research project was clearly designed around the potential use of the samples before committing to the autopsy to ensure a scientifically beneficial study was possible with the types of tissues that could be collected. These consultations concluded with the decision to proceed with organising the rapid autopsy process, promising best efforts to the family to collect the samples on the day of her natural death. Ongoing communication with the family was important to clarify expectations and balance the likelihood of the autopsy going ahead.

Ethical and legal consent

Upon confirmation from all parties that the process was feasible, ethical approval was sought as an amendment on an existing ethical approval for prospective collection of NET tissue from clinically indicated medical procedures (eg, surgery and biopsy). The patient and their family described their wish to participate with no issues raised during this conversation regarding religious or cultural beliefs. Informed signed donor consent was obtained for the tissues to be collected, stored and used for genomics research after death via an autopsy process and was co-signed by family members. Legal consent was also received to perform an autopsy. The HDEC amendment was submitted alongside letters of support from the donor and her family. This step in the approval followed extensive previous community consultation regarding tissue collection for other parts of the project, and took just three weeks to obtain. When obtaining this approval, we undertook to ensure that the patient’s participation in this study would have no impact on her clinical care.

Sampling documentation plan

Spatial information is vital to meaningfully model and interpret tumour metastasis and clonal evolution. In order to accurately map every lesion and site sampled, a documentation process was developed in conjunction with a radiologist and an anatomical pathologist. Cross-sectional images from computed tomography (CT) scans conducted as part of clinical care or follow-up were carefully interrogated prior to the autopsy, and every visible individual lesion localised and anatomic location stated and mapped. This information would then be adjusted according to findings made by the forensic pathologist and technician during the autopsy. The plan would require the manual completion of paper-based forms to record anatomical specimen location, assign naming codes and to sketch diagrams of where each sample was taken from within a specimen. A coding system was developed whereby each excised specimen was assigned a number, and the individual samples derived from this specimen were assigned either a letter or numerical code if to be stored as formalin fixed paraffin embedded (FFPE) or fresh frozen tissues respectively; to be recorded using the paper forms. Further, a photography plan was developed to incorporate in-situ and resected large-scale photographs of each specimen and the samples derived from it. As we wanted to use samples for genomic analysis in combination with morphological analysis it was imperative that the quality of the nucleic acids was maximised in the stored tissues. The decision was made to store both formalin fixed and snap frozen tissues in order to ‘future-proof’ the collection for use in multiple downstream molecular analyses.

Preparing the team

In order to ensure that enough people were available on the day of autopsy, remembering that it could happen at any time of any day, a roster was established to ensure that all areas of expertise would be available. A hierarchical phone-call system was also established to be used to contact people when needed. The autopsy required people with clinical training; oncologists, surgeons and pathologists (forensic and anatomical); as well as tissue banking scientists from the Auckland Regional Tissue Bank and the NETwork! laboratory team experienced in preparation of samples for genomic analyses. All members of the team generously volunteered their time to assist with the project.

Assembling the kit

The collections required extensive surgical and laboratory consumables and ready access to liquid nitrogen and dry ice stores. Documentation forms and sampling tubes (fresh frozen samples) and formalin-filled pots (FFPE samples) were pre-labelled with alpha-numeric codes. All items were assembled on a large trolley ready to be transferred from the research laboratory at the University of Auckland Grafton Campus across the road to the Auckland City Hospital Mortuary when required.

The autopsy

On an early morning in mid-2017, now 14 months following her initial request, our donor passed away from a natural death. She had since moved from her own home to care in a private nursing hospital, and further discussions and collaboration had been fostered with her carers, GP and institution administrators. The family had chosen a funeral director with whom they had an existing relationship, and discussion and planning for the process required had been made in advance. The treating oncologist received a phone call from the private hospital nurse at 4am and the roster phone calls were initiated and cascaded to pathologists and the research team. A member of the patient’s family met with the oncologist at the bedside, acknowledged her life and her gift, farewelled their mother, and the process began. End-of-life paperwork was completed.

The timing was fortuitous; a quiet early weekday morning allowed easy access to the mortuary and a complete, experienced group of people were available to assist. The donor arrived at the mortuary two hours after her passing. Eleven people worked for six hours to collate over 300 carefully annotated tissue samples. The team was divided into two key groups: the post-mortem team and the sampling team. The post-mortem team included a forensic pathologist, an oncologist, a surgeon and a forensic technician. Another research team member was the link between the two teams, moving between the autopsy room to courier samples and information to the sampling team. The sampling team was led by an anatomical pathologist, alongside two sampling excision staff, two annotation/recording staff and one sample preservation staff member. The sampling team were located in a separate laboratory, away from the autopsy. This enabled separation for non-clinical staff and allowed for the donor’s privacy and dignity to be preserved.

At the donor’s last CT imaging scan, over 90 distinct lesions had been identified, ranging from small 5mm subcutaneous nodules to replacement of complete organs with tumour tissue. All lesions but one were successfully sampled. Prior to removal, each lesion was photographed by the team member acting as courier, labelled with a unique number, excised and placed in a pre-labelled dish with orientation noted, before being transferred to the sampling team in an adjacent laboratory. Here, the excised lesions were again photographed and sampled into small cubes for snap freezing or placed into formalin for fixation. Where tissues were large and potentially heterogeneous, multiple samples from different regions were taken; see Figure 1 for an example of the complex sampling in the thyroid gland, compared to sampling of a smaller subcutaneous lesion. Every sample code was noted onto the specimen documentation forms (in written and diagramatic form) in order to record the location of each specimen relative to the others. The stored samples averaged 5mm3 for the frozen tissues and 20mm3 for the formalin fixed tissues. No tissues were stored that would be unlikely to be used in later research and all surplus tissues were ‘returned to the body’ for cremation, which had been the donor’s wish. Participation in the autopsy did not change the timing or nature of her funeral arrangements.

Figure 1: Comparison between sampling methods for large and small lesions.

c

For large lesions, such as the thyroid tumour shown, the lesion was first sliced into 10mm thick sections, sections were carefully laid flat in order, photographs were taken of all sections lying flat and individual sections were sampled, taking some samples for fresh frozen and some for FFPE fixation. All sites were carefully annotated on paper drawings, which were later transferred onto the photographs of sections, and unused tissue was returned to the body. For small lesions, such as the 8mm subcutaneous lesion shown here, the lesion was bisected, photographed, and each side received either fresh frozen or FFPE fixation.

Sample processing

After the day of autopsy, 141 small specimen containers of tissues fixed in formalin were transferred into ethanol after adequate fixation. Where too large to be blocked individually, tissues were divided, totalling 187 samples, and blocked in paraffin with further photographs taken to record specimen orientation and relative location. This process took a number of weeks, and was carefully documented. A small number of vertebral specimens required decalcification and were placed into a gentle EDTA buffer for up to 12 weeks in order to try and retain the nucleic acid quality and tissue morphology, following an appropriate decalcification protocol to optimise preservation of nucleic acids.16 Finally, all data and photographs were transferred to digital storage on a password-secured server and stored in a non-identifiable manner.

Speed was essential to maximise the quality of nucleic acids for later genomics. The samples were collected over the course of six hours and timing of freezing or fixation carefully documented as samples were sequentially collected. The complexity of the case, ie, the extensive number of sampled lesions throughout the body defined the timeframe. This extended timeframe to fixation could affect sample quality, causing hypoxia and necrosis driven changes within the RNA and methylation profiles in the tissues.17 Careful documentation of sampling has been important to monitor for these effects in resultant genomic data.

Invaluable tissue samples

Tissue samples are only as valuable as their annotation. This includes the histopathology, the spatial and organ location as well as a detailed summary of clinical features. The clinical history of the donor is summarised in Figure 2, alongside a summary of the breadth of information collected during this project in Figure 3. The donor’s tumour progression and clinical care is shown overlaid with the samples collected both for diagnostics and for research, imaging dates, and the research project consultations and collaborations. Since its inception, this project has required input from close to 100 people, each contributing their expertise in areas including pathology, radiology, surgery, oncology, tissue banking, genomics and evolutionary mathematics. It has provided a valuable training opportunity for a PhD student who is working to coordinate new collaborations with evolutionary biologists to better model the progression of the lesions. Indeed the experience was a profoundly moving and unique experience for all members of the laboratory research team. A year and a half after her passing, the NETwork! group presented the first preliminary results of the evolutionary genomics model to the audiences at the New Zealand Society of Oncology and Queenstown Research Week annual meetings. The overwhelming feedback from these presentations has highlighted the generosity, value and incredible opportunity provided from the donation. The patient’s family continue to provide advice, undertake lab visits and receive project updates. It is interesting to consider processes we may alter, should we complete another similar study. Our collection resulted in a large number of tissue samples from a range of complex tissue types, all found to be suitable for the prespecified genomic analyses. We believe the strengths of the project are the relationship with the patient and family, the focus on carefully designing the types of analyses to be undertaken prior to tissue collection, and completing necessary process personalisation with respect to the clinical case, geographical location and timing. Above all, research value, impetus and research time to analyse the samples must be considered in order to accept invaluable patient donations.

Figure 2: Overall clinical and research history of the donor.

c

The top two rows indicate the patient’s clinical history, including chemotherapy regimes (CAP-TEM refers to combination Capecitabine and Temozolomide chemotherapy), and computed tomography (CT) scan timings, overlaid on the timeline in row three. Row four indicates the tissue samples collected for clinical care and research. Row five and six show when the rapid autopsy consultation occurred in relation to clinical events, and the initiation of consultations and collaborations currently underway.

Figure 3: Summary of all data types collected and available for integration in evolutionary analyses.

c

The collection of tissues at autopsy enabled generation of a wealth of genomic and histopathological data, which will be interpreted alongside clinical imaging, photographs and sample annotation to form spatial models and augmented reality representations to better understand tumour evolution in this donor.  

The New Zealand context

While this work may be the first tumour collection tissue banking rapid autopsy of its kind in New Zealand, or at least the most extensive effort documented thus far, it is certainly not the first research tissue rapid autopsy collection in New Zealand. Among other initiatives, the now-named Neurological Foundation Douglas Human Brain Bank has been collecting donated human tissue at post-mortem for over 20 years, and underpins many of the research successes of the Centre for Brain Research at University of Auckland, like providing new evidence that adult human brain cells were capable of regeneration.18 The success of the Brain Bank, and indeed our one-off rapid autopsy collection, indicate that it is possible to undertake rapid autopsy tumour collections for use in research, but extending this out to a routine tumour collection rapid autopsy programme for our small country is still a large leap. Limitations in New Zealand include the facilities and resources available for completing a research autopsy as well as the funding required to staff, process and suitably conduct research around the collected specimens.

Our programme was tailor-made in response to this individual donor and her family, with the research project designed around the tumour tissues to be collected—an unorthodox way to design a research programme in the era of competitive funding driving research. While we envy permanent rapid autopsy programmes in operation in the northern hemisphere and Australia, we believe that there are key advantages to the personalised approach we have employed here. The number of samples collected, the extent of sampling of each lesion, the degree of annotation of these samples, the bespoke preservation strategies for unusual sample types, the close relationship with the donor and her family, and the commitment from the entire team to honouring the wishes of our donor, we believe are all important aspects to what makes this tumour collection unique and valuable; and indeed more difficult to achieve as part of a large formal programme. We argue that there is great value in the tailoring that is possible with one-off programmes, even when accounting for the increased time investment. In small countries like New Zealand, hoping to contribute on the same scale as large laboratories and institutions across the world will always be challenging, but instead of focusing on throughput, perhaps an emphasis on quality, completeness and annotation proves the real value that our approach can provide.

Digital developments

This bespoke project has provided our research group with experiences beyond the value of the tissue itself. Aside from providing the opportunity for student training within the genomics field, the study has also initiated a unique collaboration between the NETwork! team, the School of Architecture and the Centre for eResearch at the University of Auckland to build an augmented reality model of the donor’s tumours and how they changed over time, to enable interpretation of the genomic and evolutionary patterns in 3D and improve our spatial appreciation of complex genomic data. Further, it has provided impetus for members of the research team to develop a digital application for enhanced recording of sampling for use during tissue banking; one of the changes that we might implement if the process was repeated.

Reflection from patient’s family (anonymous)

When our mother first suggested donating her tumours to medical research on her death, we were all very supportive of her decision to hopefully benefit others in the future who contract the type of cancer that had slowly ravaged her body—and we still are. The team that undertook this task have been fantastic right from Mum’s initial donation suggestion and we can’t thank them enough for not only the work they are currently doing, but also ensuring we have been kept in the loop as Mum’s tumours have been analysed. Late last year we saw the first virtual replication of Mum’s body and her tumours and it was mind-blowing—she would have been (and may still be) thrilled to see what her donation has led to and there is still a long way to go. We are all very proud of Mum’s decision and hope the ongoing study of her tumours by a great team of specialists will help to increase the understanding of her cancer, making a difference in the future.

Conclusion

Donated human tissue is a valuable resource in medical research, and post-mortem tissue is particularly valuable when it allows collection of tissues not usually available through standard clinical procedures. This generous donation will enable the study of fundamental questions plaguing cancer biology, tumour evolution and heterogeneity, and leave a legacy in the form of a unique tissue resource. Here we have outlined the process by which a post-mortem collection procedure can be built from the ground up. While considerable effort is required to coordinate the legal, ethical, scientific and logistical considerations needed to carry out a bespoke rapid autopsy in New Zealand, the value of tissue collected and its scientific utility well outweigh these considerations when collected as part of a carefully designed research study. The latter point is the key to balancing whether to undertake a bespoke autopsy process; to ensure that the gift is indeed used for the purpose that it was given. The donor handed responsibility to the research team to use this gift for the good of others, and this ethos forms the basis for our ongoing work. We honour the incredible foresight and generosity shown by the donor and her family in championing this research.

Summary

Abstract

Genomic analysis of tissues from rapid autopsy programmes has transformed our understanding of cancer. However, these programmes are not yet established in New Zealand. Our neuroendocrine tumour research group, NETwork!, received a request from a patient wishing to donate tumour tissues post-mortem. This viewpoint article summarises the ethical, logistical and social process undertaken to accept this patient s generous donation, and highlights the scientific and educational value of such a gift.

Aim

Method

Results

Conclusion

Author Information

- Cherie Blenkiron, Senior Research Fellow, Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland; The Maurice Wilkins Centre for Biodiscovery, The University of Auckland, Auckland; Tamsin Robb, P

Acknowledgements

Correspondence

Dr Cherie Blenkiron, Senior Research Fellow, Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland.

Correspondence Email

c.blenkiron@auckland.ac.nz

Competing Interests

All authors report grants from Translational Medicine Trust during the conduct of the study.

  1. McGranahan N, Swanton C. Clonal Heterogeneity and Tumor Evolution: Past, Present, and the Future. Cell 2017; 168(4):613–28. doi: 10.1016/j.cell.2017.01.018
  2. Burrell RA, Swanton C. Tumour heterogeneity and the evolution of polyclonal drug resistance. Mol Oncol 2014; 8(6):1095–111. doi: 10.1016/j.molonc.2014.06.005
  3. Burrell RA, McGranahan N, Bartek J, et al. The causes and consequences of genetic heterogeneity in cancer evolution. Nature 2013; 501(7467):338–45. doi: 10.1038/nature12625
  4. Gundem G, Van Loo P, Kremeyer B, et al. The evolutionary history of lethal metastatic prostate cancer. Nature 2015; 520(7547):353–57. doi: 10.1038/nature14347
  5. Saygin C, Matei D, Majeti R, et al. Targeting Cancer Stemness in the Clinic: From Hype to Hope. Cell Stem Cell 2019; 24(1):25–40. doi: 10.1016/j.stem.2018.11.017
  6. Campbell PJ, Yachida S, Mudie LJ, et al. The patterns and dynamics of genomic instability in metastatic pancreatic cancer. Nature 2010; 467(7319):1109-13. doi: 10.1038/nature09460
  7. Ascierto ML, Makohon-Moore A, Lipson EJ, et al. Transcriptional Mechanisms of Resistance to Anti-PD-1 Therapy. Clin Cancer Res 2017; 23(12):3168–80. doi: 10.1158/1078-0432.CCR-17-0270
  8. Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing. New England Journal of Medicine 2012; 366(10):883–92. doi: 10.1056/NEJMoa1113205
  9. Fan J, Iacobuzio-Donahue CA. The Science of Rapid Research Autopsy. In: Hooper JE, Williamson AK, eds. Autopsy in the 21st Century: Best Practices and Future Directions. Cham: Springer International Publishing 2019:151–66.
  10. Hooper JE, Duregon E. Performance of Rapid Research Autopsy. In: Hooper JE, Williamson AK, eds. Autopsy in the 21st Century: Best Practices and Future Directions. Cham: Springer International Publishing 2019:167–85.
  11. Yachida S, Jones S, Bozic I, et al. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature 2010;467(7319):1114–7. doi: 10.1038/nature09515
  12. Liu W, Laitinen S, Khan S, et al. Copy number analysis indicates monoclonal origin of lethal metastatic prostate cancer. Nat Med 2009;15(5):559–65. doi: 10.1038/nm.1944
  13. Kumar A, Coleman I, Morrissey C, et al. Substantial interindividual and limited intraindividual genomic diversity among tumors from men with metastatic prostate cancer. Nat Med 2016; 22(4):369–78. doi: 10.1038/nm.4053
  14. Grasso CS, Wu YM, Robinson DR, et al. The mutational landscape of lethal castration-resistant prostate cancer. Nature 2012; 487(7406):239–43. doi: 10.1038/nature11125
  15. Patch AM, Christie EL, Etemadmoghadam D, et al. Whole-genome characterization of chemoresistant ovarian cancer. Nature 2015; 521(7553):489–94. doi: 10.1038/nature14410
  16. Choi SE, Hong SW, Yoon SO. Proposal of an appropriate decalcification method of bone marrow biopsy specimens in the era of expanding genetic molecular study. J Pathol Transl Med 2015; 49(3):236–42. doi: 10.4132/jptm.2015.03.16
  17. Zhou JH, Sahin AA, Myers JN. Biobanking in genomic medicine. Arch Pathol Lab Med 2015; 139(6):812–8. doi: 10.5858/arpa.2014-0261-RA
  18. Curtis MA, Penney EB, Pearson AG, et al. Increased cell proliferation and neurogenesis in the adult human Huntington’s disease brain. Proc Natl Acad Sci U S A 2003; 100(15):9023–7. doi: 10.1073/pnas.1532244100

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Tumours and their genomes are neither homogenous nor static; it is appreciated that they may change and evolve with time.1 This process might contribute to the failure of clinical systemic treatments, and therefore remains an important clinical challenge to overcome, and biological phenomenon to understand.2 During tumour evolution, changes to the genome (mutations) and its gene expression lead to metastatic spread and promotes the survival of the ‘fittest’ of a genomically heterogenous mix of cancer cell ‘clones’ that can be disseminated across the body. Research into tumour evolution has been enabled by the advent of next-generation sequencing providing a better appreciation of the genomic complexity of the tumour cells that are present within an individual patient. This builds on an already appreciated histological understanding of morphological heterogeneity between a primary tumour and its seeded metastases.

Tumour heterogeneity can occur between different lesions from a single patient, but also within each lesion with different cells within the tumour evolving different genotypes and having varying potential to metastasise.3 Important advancements in understanding the genomic landscapes of many tumour types have been made through global sequencing programmes such as the International Cancer Genome Consortium.4 However, these programmes have been less valuable for understanding the genomic heterogeneity of tumours within individual patients. Nevertheless, in the clinic, understanding tumour heterogeneity within each patient can sometimes contribute to appropriate treatment decisions. For example, the presence of multiple distinct tumour cell clones within the tumour(s) of an individual could lead to treatment resistance where the ‘fittest’ cells survive and evade therapy or require a different treatment approach. Similarly, specific clones may selectively metastasise to areas of the body that are less amenable to systemic therapy, such as beyond the blood brain barrier. However, these processes remain incompletely understood.

Researchers appreciating the clinical importance of tumour heterogeneity within individual patients have made significant gains in the laboratory. Approaches often involve the analysis of multiple solid tissue samples from an individual, for example for pancreatic or renal carcinomas, taken at different times during their care and/or from different sites in their body.5,6 This temporal and spatial information has been invaluable in better understanding the molecular changes that occur as a tumour evolves to evade therapy.7,8 However, solid tumours, particularly within internal organs, are a challenge to sample unless surgically resected. Invasive tissue biopsies are seldom taken for purely research purposes due to the risks to the patients, making the study of progressive disease difficult in patients where surgical intervention is no longer clinically helpful. Circulating free DNA (cfDNA) has been proposed as a surrogate for multi-organ sampling, but it is unknown whether cfDNA can represent all tumours in the body, or if there is a selective elucidation of individual clones or favouring of particular anatomical locations. To facilitate study of metastastatic cancer in single patients, research hospitals and biobanks around the globe have established routine ‘Rapid Autopsy’ programmes, allowing patients to donate tissues through autopsy at their point of natural death for use by research groups to better understand tumour heterogeneity and evolution. Such established programmes at over a dozen institutes worldwide have collected invaluable samples, leading to improved understanding across the spectrum of cancers.9,10 Several well-documented programmes are located in the US (eg, Johns Hopkins Legacy Gift Rapid Autopsy Programme,11,12 Fred Hutchinson Institute at University of Washington13 and University of Michigan14) and Australia (CASCADE programme, Peter MacCallum Cancer Centre15). No established programme of this kind for cancer research exists in New Zealand.

Our New Zealand-based research group, NETwork!, brings together clinical, epidemiological and genomic information to build improved biological understanding of neuroendocrine tumours (NETs). The NETwork! programme was established in 2012 and works closely with patients, collecting tissues for use in research with the ultimate aim of improving care for people diagnosed with NETs in New Zealand. The team is truly multidisciplinary with expertise ranging from clinical oncology through to computational biology. In early 2016, a patient with a metastastic broncho-pulmonary NET made a direct approach to her oncologist, a principal investigator of the NETwork! team. The patient had multiple metastases from her original tumour, and wished to donate her tumour tissues through rapid autopsy on her natural death. Her hope was to contribute to biological understanding which might help those who followed her. Initially, the research team were reluctant to pursue this request and declined, not having established the ethical or logistical framework to effectively conduct such a procedure. However, the patient was persistent and returned on her next appointment accompanied by two of her children, who were both highly supportive. Given this level of determination on the part of the patient, and the strong support from her immediate family, the team felt that they had a duty to at least ascertain whether or not such a procedure would be possible.

In this viewpoint article we summarise the considerable efforts taken to develop and coordinate a process that could meet the challenge initiated by this selfless act. This undertaking has allowed us to assemble and analyse a unique tissue resource, and has given us a valuable understanding of the legal, ethical and logistical considerations needed to carry out rapid autopsy research in New Zealand. We also highlight the value of this partnership between patient, family and researchers to better understanding the biology of neuroendocrine tumour evolution.

Feasibility assessment

After the initial approach from the patient to her treating oncologist, the first step was to assess the feasibility of sample collection through a rapid autopsy procedure. Considerations were made on scientific, logistical, ethical and financial bases. This was performed in parallel through multiple discussions with the lead of the Forensic Pathology Department at Auckland City Hospital, with the Health and Disability Ethics Committee (HDEC), DHB clinicians responsible for her care, and with the laboratory researchers within the NETwork! group. These discussions took a number of months, with ongoing consultation needed throughout the pre-autopsy period in order to plan and later refine the processes. A further consideration for the research team was to balance the scientific value of the samples with the financial cost of collection and subsequent analysis. The study, the autopsy, sample collections and processing were funded as part of the existing NETwork! programme. A research project was clearly designed around the potential use of the samples before committing to the autopsy to ensure a scientifically beneficial study was possible with the types of tissues that could be collected. These consultations concluded with the decision to proceed with organising the rapid autopsy process, promising best efforts to the family to collect the samples on the day of her natural death. Ongoing communication with the family was important to clarify expectations and balance the likelihood of the autopsy going ahead.

Ethical and legal consent

Upon confirmation from all parties that the process was feasible, ethical approval was sought as an amendment on an existing ethical approval for prospective collection of NET tissue from clinically indicated medical procedures (eg, surgery and biopsy). The patient and their family described their wish to participate with no issues raised during this conversation regarding religious or cultural beliefs. Informed signed donor consent was obtained for the tissues to be collected, stored and used for genomics research after death via an autopsy process and was co-signed by family members. Legal consent was also received to perform an autopsy. The HDEC amendment was submitted alongside letters of support from the donor and her family. This step in the approval followed extensive previous community consultation regarding tissue collection for other parts of the project, and took just three weeks to obtain. When obtaining this approval, we undertook to ensure that the patient’s participation in this study would have no impact on her clinical care.

Sampling documentation plan

Spatial information is vital to meaningfully model and interpret tumour metastasis and clonal evolution. In order to accurately map every lesion and site sampled, a documentation process was developed in conjunction with a radiologist and an anatomical pathologist. Cross-sectional images from computed tomography (CT) scans conducted as part of clinical care or follow-up were carefully interrogated prior to the autopsy, and every visible individual lesion localised and anatomic location stated and mapped. This information would then be adjusted according to findings made by the forensic pathologist and technician during the autopsy. The plan would require the manual completion of paper-based forms to record anatomical specimen location, assign naming codes and to sketch diagrams of where each sample was taken from within a specimen. A coding system was developed whereby each excised specimen was assigned a number, and the individual samples derived from this specimen were assigned either a letter or numerical code if to be stored as formalin fixed paraffin embedded (FFPE) or fresh frozen tissues respectively; to be recorded using the paper forms. Further, a photography plan was developed to incorporate in-situ and resected large-scale photographs of each specimen and the samples derived from it. As we wanted to use samples for genomic analysis in combination with morphological analysis it was imperative that the quality of the nucleic acids was maximised in the stored tissues. The decision was made to store both formalin fixed and snap frozen tissues in order to ‘future-proof’ the collection for use in multiple downstream molecular analyses.

Preparing the team

In order to ensure that enough people were available on the day of autopsy, remembering that it could happen at any time of any day, a roster was established to ensure that all areas of expertise would be available. A hierarchical phone-call system was also established to be used to contact people when needed. The autopsy required people with clinical training; oncologists, surgeons and pathologists (forensic and anatomical); as well as tissue banking scientists from the Auckland Regional Tissue Bank and the NETwork! laboratory team experienced in preparation of samples for genomic analyses. All members of the team generously volunteered their time to assist with the project.

Assembling the kit

The collections required extensive surgical and laboratory consumables and ready access to liquid nitrogen and dry ice stores. Documentation forms and sampling tubes (fresh frozen samples) and formalin-filled pots (FFPE samples) were pre-labelled with alpha-numeric codes. All items were assembled on a large trolley ready to be transferred from the research laboratory at the University of Auckland Grafton Campus across the road to the Auckland City Hospital Mortuary when required.

The autopsy

On an early morning in mid-2017, now 14 months following her initial request, our donor passed away from a natural death. She had since moved from her own home to care in a private nursing hospital, and further discussions and collaboration had been fostered with her carers, GP and institution administrators. The family had chosen a funeral director with whom they had an existing relationship, and discussion and planning for the process required had been made in advance. The treating oncologist received a phone call from the private hospital nurse at 4am and the roster phone calls were initiated and cascaded to pathologists and the research team. A member of the patient’s family met with the oncologist at the bedside, acknowledged her life and her gift, farewelled their mother, and the process began. End-of-life paperwork was completed.

The timing was fortuitous; a quiet early weekday morning allowed easy access to the mortuary and a complete, experienced group of people were available to assist. The donor arrived at the mortuary two hours after her passing. Eleven people worked for six hours to collate over 300 carefully annotated tissue samples. The team was divided into two key groups: the post-mortem team and the sampling team. The post-mortem team included a forensic pathologist, an oncologist, a surgeon and a forensic technician. Another research team member was the link between the two teams, moving between the autopsy room to courier samples and information to the sampling team. The sampling team was led by an anatomical pathologist, alongside two sampling excision staff, two annotation/recording staff and one sample preservation staff member. The sampling team were located in a separate laboratory, away from the autopsy. This enabled separation for non-clinical staff and allowed for the donor’s privacy and dignity to be preserved.

At the donor’s last CT imaging scan, over 90 distinct lesions had been identified, ranging from small 5mm subcutaneous nodules to replacement of complete organs with tumour tissue. All lesions but one were successfully sampled. Prior to removal, each lesion was photographed by the team member acting as courier, labelled with a unique number, excised and placed in a pre-labelled dish with orientation noted, before being transferred to the sampling team in an adjacent laboratory. Here, the excised lesions were again photographed and sampled into small cubes for snap freezing or placed into formalin for fixation. Where tissues were large and potentially heterogeneous, multiple samples from different regions were taken; see Figure 1 for an example of the complex sampling in the thyroid gland, compared to sampling of a smaller subcutaneous lesion. Every sample code was noted onto the specimen documentation forms (in written and diagramatic form) in order to record the location of each specimen relative to the others. The stored samples averaged 5mm3 for the frozen tissues and 20mm3 for the formalin fixed tissues. No tissues were stored that would be unlikely to be used in later research and all surplus tissues were ‘returned to the body’ for cremation, which had been the donor’s wish. Participation in the autopsy did not change the timing or nature of her funeral arrangements.

Figure 1: Comparison between sampling methods for large and small lesions.

c

For large lesions, such as the thyroid tumour shown, the lesion was first sliced into 10mm thick sections, sections were carefully laid flat in order, photographs were taken of all sections lying flat and individual sections were sampled, taking some samples for fresh frozen and some for FFPE fixation. All sites were carefully annotated on paper drawings, which were later transferred onto the photographs of sections, and unused tissue was returned to the body. For small lesions, such as the 8mm subcutaneous lesion shown here, the lesion was bisected, photographed, and each side received either fresh frozen or FFPE fixation.

Sample processing

After the day of autopsy, 141 small specimen containers of tissues fixed in formalin were transferred into ethanol after adequate fixation. Where too large to be blocked individually, tissues were divided, totalling 187 samples, and blocked in paraffin with further photographs taken to record specimen orientation and relative location. This process took a number of weeks, and was carefully documented. A small number of vertebral specimens required decalcification and were placed into a gentle EDTA buffer for up to 12 weeks in order to try and retain the nucleic acid quality and tissue morphology, following an appropriate decalcification protocol to optimise preservation of nucleic acids.16 Finally, all data and photographs were transferred to digital storage on a password-secured server and stored in a non-identifiable manner.

Speed was essential to maximise the quality of nucleic acids for later genomics. The samples were collected over the course of six hours and timing of freezing or fixation carefully documented as samples were sequentially collected. The complexity of the case, ie, the extensive number of sampled lesions throughout the body defined the timeframe. This extended timeframe to fixation could affect sample quality, causing hypoxia and necrosis driven changes within the RNA and methylation profiles in the tissues.17 Careful documentation of sampling has been important to monitor for these effects in resultant genomic data.

Invaluable tissue samples

Tissue samples are only as valuable as their annotation. This includes the histopathology, the spatial and organ location as well as a detailed summary of clinical features. The clinical history of the donor is summarised in Figure 2, alongside a summary of the breadth of information collected during this project in Figure 3. The donor’s tumour progression and clinical care is shown overlaid with the samples collected both for diagnostics and for research, imaging dates, and the research project consultations and collaborations. Since its inception, this project has required input from close to 100 people, each contributing their expertise in areas including pathology, radiology, surgery, oncology, tissue banking, genomics and evolutionary mathematics. It has provided a valuable training opportunity for a PhD student who is working to coordinate new collaborations with evolutionary biologists to better model the progression of the lesions. Indeed the experience was a profoundly moving and unique experience for all members of the laboratory research team. A year and a half after her passing, the NETwork! group presented the first preliminary results of the evolutionary genomics model to the audiences at the New Zealand Society of Oncology and Queenstown Research Week annual meetings. The overwhelming feedback from these presentations has highlighted the generosity, value and incredible opportunity provided from the donation. The patient’s family continue to provide advice, undertake lab visits and receive project updates. It is interesting to consider processes we may alter, should we complete another similar study. Our collection resulted in a large number of tissue samples from a range of complex tissue types, all found to be suitable for the prespecified genomic analyses. We believe the strengths of the project are the relationship with the patient and family, the focus on carefully designing the types of analyses to be undertaken prior to tissue collection, and completing necessary process personalisation with respect to the clinical case, geographical location and timing. Above all, research value, impetus and research time to analyse the samples must be considered in order to accept invaluable patient donations.

Figure 2: Overall clinical and research history of the donor.

c

The top two rows indicate the patient’s clinical history, including chemotherapy regimes (CAP-TEM refers to combination Capecitabine and Temozolomide chemotherapy), and computed tomography (CT) scan timings, overlaid on the timeline in row three. Row four indicates the tissue samples collected for clinical care and research. Row five and six show when the rapid autopsy consultation occurred in relation to clinical events, and the initiation of consultations and collaborations currently underway.

Figure 3: Summary of all data types collected and available for integration in evolutionary analyses.

c

The collection of tissues at autopsy enabled generation of a wealth of genomic and histopathological data, which will be interpreted alongside clinical imaging, photographs and sample annotation to form spatial models and augmented reality representations to better understand tumour evolution in this donor.  

The New Zealand context

While this work may be the first tumour collection tissue banking rapid autopsy of its kind in New Zealand, or at least the most extensive effort documented thus far, it is certainly not the first research tissue rapid autopsy collection in New Zealand. Among other initiatives, the now-named Neurological Foundation Douglas Human Brain Bank has been collecting donated human tissue at post-mortem for over 20 years, and underpins many of the research successes of the Centre for Brain Research at University of Auckland, like providing new evidence that adult human brain cells were capable of regeneration.18 The success of the Brain Bank, and indeed our one-off rapid autopsy collection, indicate that it is possible to undertake rapid autopsy tumour collections for use in research, but extending this out to a routine tumour collection rapid autopsy programme for our small country is still a large leap. Limitations in New Zealand include the facilities and resources available for completing a research autopsy as well as the funding required to staff, process and suitably conduct research around the collected specimens.

Our programme was tailor-made in response to this individual donor and her family, with the research project designed around the tumour tissues to be collected—an unorthodox way to design a research programme in the era of competitive funding driving research. While we envy permanent rapid autopsy programmes in operation in the northern hemisphere and Australia, we believe that there are key advantages to the personalised approach we have employed here. The number of samples collected, the extent of sampling of each lesion, the degree of annotation of these samples, the bespoke preservation strategies for unusual sample types, the close relationship with the donor and her family, and the commitment from the entire team to honouring the wishes of our donor, we believe are all important aspects to what makes this tumour collection unique and valuable; and indeed more difficult to achieve as part of a large formal programme. We argue that there is great value in the tailoring that is possible with one-off programmes, even when accounting for the increased time investment. In small countries like New Zealand, hoping to contribute on the same scale as large laboratories and institutions across the world will always be challenging, but instead of focusing on throughput, perhaps an emphasis on quality, completeness and annotation proves the real value that our approach can provide.

Digital developments

This bespoke project has provided our research group with experiences beyond the value of the tissue itself. Aside from providing the opportunity for student training within the genomics field, the study has also initiated a unique collaboration between the NETwork! team, the School of Architecture and the Centre for eResearch at the University of Auckland to build an augmented reality model of the donor’s tumours and how they changed over time, to enable interpretation of the genomic and evolutionary patterns in 3D and improve our spatial appreciation of complex genomic data. Further, it has provided impetus for members of the research team to develop a digital application for enhanced recording of sampling for use during tissue banking; one of the changes that we might implement if the process was repeated.

Reflection from patient’s family (anonymous)

When our mother first suggested donating her tumours to medical research on her death, we were all very supportive of her decision to hopefully benefit others in the future who contract the type of cancer that had slowly ravaged her body—and we still are. The team that undertook this task have been fantastic right from Mum’s initial donation suggestion and we can’t thank them enough for not only the work they are currently doing, but also ensuring we have been kept in the loop as Mum’s tumours have been analysed. Late last year we saw the first virtual replication of Mum’s body and her tumours and it was mind-blowing—she would have been (and may still be) thrilled to see what her donation has led to and there is still a long way to go. We are all very proud of Mum’s decision and hope the ongoing study of her tumours by a great team of specialists will help to increase the understanding of her cancer, making a difference in the future.

Conclusion

Donated human tissue is a valuable resource in medical research, and post-mortem tissue is particularly valuable when it allows collection of tissues not usually available through standard clinical procedures. This generous donation will enable the study of fundamental questions plaguing cancer biology, tumour evolution and heterogeneity, and leave a legacy in the form of a unique tissue resource. Here we have outlined the process by which a post-mortem collection procedure can be built from the ground up. While considerable effort is required to coordinate the legal, ethical, scientific and logistical considerations needed to carry out a bespoke rapid autopsy in New Zealand, the value of tissue collected and its scientific utility well outweigh these considerations when collected as part of a carefully designed research study. The latter point is the key to balancing whether to undertake a bespoke autopsy process; to ensure that the gift is indeed used for the purpose that it was given. The donor handed responsibility to the research team to use this gift for the good of others, and this ethos forms the basis for our ongoing work. We honour the incredible foresight and generosity shown by the donor and her family in championing this research.

Summary

Abstract

Genomic analysis of tissues from rapid autopsy programmes has transformed our understanding of cancer. However, these programmes are not yet established in New Zealand. Our neuroendocrine tumour research group, NETwork!, received a request from a patient wishing to donate tumour tissues post-mortem. This viewpoint article summarises the ethical, logistical and social process undertaken to accept this patient s generous donation, and highlights the scientific and educational value of such a gift.

Aim

Method

Results

Conclusion

Author Information

- Cherie Blenkiron, Senior Research Fellow, Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland; The Maurice Wilkins Centre for Biodiscovery, The University of Auckland, Auckland; Tamsin Robb, P

Acknowledgements

Correspondence

Dr Cherie Blenkiron, Senior Research Fellow, Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland.

Correspondence Email

c.blenkiron@auckland.ac.nz

Competing Interests

All authors report grants from Translational Medicine Trust during the conduct of the study.

  1. McGranahan N, Swanton C. Clonal Heterogeneity and Tumor Evolution: Past, Present, and the Future. Cell 2017; 168(4):613–28. doi: 10.1016/j.cell.2017.01.018
  2. Burrell RA, Swanton C. Tumour heterogeneity and the evolution of polyclonal drug resistance. Mol Oncol 2014; 8(6):1095–111. doi: 10.1016/j.molonc.2014.06.005
  3. Burrell RA, McGranahan N, Bartek J, et al. The causes and consequences of genetic heterogeneity in cancer evolution. Nature 2013; 501(7467):338–45. doi: 10.1038/nature12625
  4. Gundem G, Van Loo P, Kremeyer B, et al. The evolutionary history of lethal metastatic prostate cancer. Nature 2015; 520(7547):353–57. doi: 10.1038/nature14347
  5. Saygin C, Matei D, Majeti R, et al. Targeting Cancer Stemness in the Clinic: From Hype to Hope. Cell Stem Cell 2019; 24(1):25–40. doi: 10.1016/j.stem.2018.11.017
  6. Campbell PJ, Yachida S, Mudie LJ, et al. The patterns and dynamics of genomic instability in metastatic pancreatic cancer. Nature 2010; 467(7319):1109-13. doi: 10.1038/nature09460
  7. Ascierto ML, Makohon-Moore A, Lipson EJ, et al. Transcriptional Mechanisms of Resistance to Anti-PD-1 Therapy. Clin Cancer Res 2017; 23(12):3168–80. doi: 10.1158/1078-0432.CCR-17-0270
  8. Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing. New England Journal of Medicine 2012; 366(10):883–92. doi: 10.1056/NEJMoa1113205
  9. Fan J, Iacobuzio-Donahue CA. The Science of Rapid Research Autopsy. In: Hooper JE, Williamson AK, eds. Autopsy in the 21st Century: Best Practices and Future Directions. Cham: Springer International Publishing 2019:151–66.
  10. Hooper JE, Duregon E. Performance of Rapid Research Autopsy. In: Hooper JE, Williamson AK, eds. Autopsy in the 21st Century: Best Practices and Future Directions. Cham: Springer International Publishing 2019:167–85.
  11. Yachida S, Jones S, Bozic I, et al. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature 2010;467(7319):1114–7. doi: 10.1038/nature09515
  12. Liu W, Laitinen S, Khan S, et al. Copy number analysis indicates monoclonal origin of lethal metastatic prostate cancer. Nat Med 2009;15(5):559–65. doi: 10.1038/nm.1944
  13. Kumar A, Coleman I, Morrissey C, et al. Substantial interindividual and limited intraindividual genomic diversity among tumors from men with metastatic prostate cancer. Nat Med 2016; 22(4):369–78. doi: 10.1038/nm.4053
  14. Grasso CS, Wu YM, Robinson DR, et al. The mutational landscape of lethal castration-resistant prostate cancer. Nature 2012; 487(7406):239–43. doi: 10.1038/nature11125
  15. Patch AM, Christie EL, Etemadmoghadam D, et al. Whole-genome characterization of chemoresistant ovarian cancer. Nature 2015; 521(7553):489–94. doi: 10.1038/nature14410
  16. Choi SE, Hong SW, Yoon SO. Proposal of an appropriate decalcification method of bone marrow biopsy specimens in the era of expanding genetic molecular study. J Pathol Transl Med 2015; 49(3):236–42. doi: 10.4132/jptm.2015.03.16
  17. Zhou JH, Sahin AA, Myers JN. Biobanking in genomic medicine. Arch Pathol Lab Med 2015; 139(6):812–8. doi: 10.5858/arpa.2014-0261-RA
  18. Curtis MA, Penney EB, Pearson AG, et al. Increased cell proliferation and neurogenesis in the adult human Huntington’s disease brain. Proc Natl Acad Sci U S A 2003; 100(15):9023–7. doi: 10.1073/pnas.1532244100

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