Rapid Research Autopsy is a Stealthy but Growing Contributor to Cancer Research

Precis

Rapid autopsy has been shown to be a powerful tool for advancing research at essentially no risk and with no distress to the contributing patient. Academic and health centers with a substantial investment in cancer research should consider using this emerging research support modality.

Introduction

Although novel personalized therapeutic approaches, such as targeted therapies and immunotherapies, are revolutionizing treatment of patients with advanced malignancies^1,2,3, most patients are still not cured by these treatments. It remains critical to study and understand resistance mechanisms to existing standard and targeted therapies, modes of immune evasion to immunotherapies, and fundamental alterations related to the molecular pathogenesis of cancer, including local invasion and metastatic spread^4,5. Biopsy tissue of metastatic lesions in pre- and post-treatment settings is fundamental to tailoring the best therapy for patients with advanced malignant tumors. However, in the clear majority of patients, it is simply not practical to obtain tissues from multiple metastatic sites and ore limited sampling may bias future treatments. Although cancer cells in multiple metastatic sites often share clonal origins from the primary tumor, these sites can be quite heterogeneous in terms of subclonal somatic alterations and phenotypic features^6,7,8. In addition, some sites, such as bone lesions in prostate and breast cancer patients, are nearly impossible to sample adequately, whether or not patients are critically ill.

Rapid autopsy (RA) is a unique methodology that can overcome the necessary limitations of tissue sampling in living patients and has contributed substantially to the understanding of metastatic spread of cancer. Rapid autopsies are post-mortem examinations performed on an urgent basis (measured in hours) after the death of the patient. Sampling tissues during a rapid autopsy from patients with advanced malignancies provides unique research opportunities. First, tumor tissues can be procured in large quantities from many separate body sites. Secondly, neoplastic tissues can be sampled after disease resistance has occurred, allowing for studies to be performed on tissues after the time point of aggressive local spread or metastasis. In these instances, biopsy/surgical tissue is often not available or accessible in sufficient quantity. Finally, different and often unique tissue samples for multiple researchers/research projects may be collected during a single case^9,10. Parallel specimens may be collected for comparison of gross and microscopic pathology and molecular analysis of tumor clones^{11, 12}.

Demonstrated Value of Rapid Autopsy (RA)

Rapid autopsy studies have demonstrated mutational heterogeneity from different regions of a primary tumor and across metastatic sites in single patients, including different inactivating mutations of tumor suppressor genes in renal cell and prostate cancer^{13,14,15,16,17}. Collection as soon as possible after the death of the patient means that rapid autopsy tissue quality can, in many instances, be considered comparable to fresh surgical biopsy tissue¹⁸.

Tissues harvested during rapid autopsies have successfully been utilized for DNA sequencing, RNA expression analysis (including in situ hybridization), proteomic approaches and immunohistochemical studies in the field of prostate^{19, 20, 21, 22}, pancreas²³, breast cancer^10,24 and brain tumors²⁵. In addition, these tissues have provided the opportunity to develop patient derived animal models recapitulating the end stage of metastatic disease and to utilize molecular studies that have become milestones in understanding intratumor heterogeneity^12,26. Studies have traced the origins of particular metastases to lower grade regions of cancer primaries¹⁷ and through qualitative and quantitative analysis of subclones, have delineated the time course from an initial founder cell to development of metastases²⁷.

Several important contributions that have depended upon the ability to sample multiple metastatic sites include: 1) a study of diffuse intrinsic pontine glioma showed 100% DNA and 63% RNA suitable for genome-wide analysis in 38 cases with average postmortem intervals of 7.7 hours²⁸, 2) RNA from autopsy heart tissue can be extracted and reproducibly distinguish between failing and nonfailing hearts, even at 24 hours of postmortem interval²⁹, 3) a New England Journal of Medicine study utilized postmortem tissue to demonstrate mutational heterogeneity from different regions of a primary tumor and across metastatic sites in single patients, including different inactivating mutations of tumor suppressor genes¹³, and 4) A 2014 follow-up study to 3) which demonstrated 73% to 75% subclonal driver mutations, most in spatially separated metastasis. Interestingly, in the renal cancer study it was shown that the more biopsies were taken, the more subclones were identified, calling into question often far reaching conclusions about tumor biology based on single needle core biopsies alone¹⁴.

Potential Contributions of Rapid Autopsy

An overview of research methodologies that have been associated with and supported by rapid autopsy tissue are given in Figure 1. Also summarized in Figure 1 are methodologies expected to succeed when using such tissue as well as those that have not yet been evaluated using rapid autopsy tissue. Proven methods have addressed such biological questions as time course and sequence of metastases as well as effectiveness of established and newly developed treatments using animal models. Single cell DNA and RNA sequencing to explore cell lineage relationships and epigenomics are expected to yield results using postmortem tissue. Areas of new exploration will include the effect of tumor environments including tissue, metabolic, and microbiologic milieu, facilitated by the ability to sample areas near to and distant from cancer sites as well as the metastases themselves.

Figure 1:

Research methodologies that can be supported by rapid research autopsy tissue resources

Organization of Rapid Autopsy Programs

There are currently a relatively small number of RA programs in the United States with an additional few existing in Canada, Australia, and very recently in Europe as well. Some of these programs are focused just on one tumor type (like prostatic cancer⁹), while others accept more wide varieties of patients. These programs may be directed by pathologists (usual), oncologists (frequent), or other researchers (occasional), though pathologist involvement is always important to a properly executed autopsy procedure as well as for selection of best quality tissue for sampling. Most programs are on-call 24/7, often with at least two rotating teams, that cover one or multiple week shifts. However, successful programs may also be run by one team only that is on-call for a restricted number of hours every day (e.g. 5AM-8PM).

Patients can be either be referred by clinicians or nurses/social workers, usually when the they are admitted in a hospice care or during the enrollment in a clinical trial, but sometimes they may even self-refer. The study consenting process in which the patient or his legal next of kin signs and gives his consent to collect and use his/her tissues for future research is very important. Autopsy consenting is usually separate from a study consent and, in some states, may only be completed by the legal next of kin after the patient’s death.

A minimum rapid autopsy case team should include a Study Coordinator who could assist with patient communication and consenting, a Pathologist, and an Autopsy Assistant (diener) to perform autopsy dissection. In addition, two specimen technicians, one to perform sterile dissection during a case and one to handle non-sterile specimen collection duties are also desirable to achieve the collection of the highest quality tissue. Participating researchers may also provide additional support personnel to assist during the rapid autopsy.

In the “post-case” setting, continuous exchange and feedback between the researchers who are using the specimens collected and the RAP team is absolutely necessary in order to critique successes and failures, identify evolving needs, and implement new strategies and approaches for future cases.

Funding of RAPs

Institutional rapid autopsy activities often begin with autopsies performed by individual pathologist researchers who are utilizing the tissues collected for their own cancer work and who may be supporting these activities through individualized grant funding. Then, when these initial activities expand and become centralized, initial start-up funding may be provided by Oncology and Pathology departments. At Johns Hopkins University, our RAP eventually became an NIH funded Cancer Clinical Core serving the Sidney Kimmel Comprehensive Cancer Care Center. RA programs can also participate in disease specialty grants such as supplements to SPORE grants (JHU has participated in supplements to Pancreas and Prostate SPOREs) or perhaps secure support from cancer and other private disease foundations. Successful programs may eventually be fortunate enough to receive generous donations from families and friends of patients as well.

The amount of centralized infrastructure necessary to establish and support a RAP can vary among programs, however, even in a full coverage program, initial and ongoing equipment and supply costs are relatively low (freezers, sterile supplies, containers and specimen media). Helpful support may already be available in hospitals with already active autopsy services. Of course, expertise, skilled labor, and transportation of patients comprise significantly more of the total cost of a RAP than any other items. Most RAPs do not charge families for participation and transportation of deceased patients.

Barriers to Entry for New Programs

Table 1 gives existing rapid autopsy programs within the U.S., identified through internet and publication searches as well as telephone and other interactions with colleagues. As demonstrated in Table 1, hospitals/health systems currently supporting RA programs have several demographic characteristics in common: 1) a location in major metropolitan area with a large proximate population, 2) recognition as a distinguished cancer center (recognized by U.S. News and World Report) with many oncologists and oncology researchers, and 3) existence of a resource rich environment with infrastructure for cancer research activity and experience with obtaining grant funding. These very characteristics imply potentially significant barriers of entry for possible new programs. A large nearby population is necessary for there to be a sufficient source of patients close enough to the center to facilitate transportation within a reasonable number of hours. Recognition as a distinguished cancer center seems necessary to have a sufficient audience of researchers willing and able to put the rapid autopsy tissue collected to immediate productive use. Experience and success in obtaining grant funding is important because most successful RAPs are or are becoming “self-sustaining” through grant funding. In addition to all of these characteristics, a RAP is a logistically complex and labor-intensive structure in which a dedicated and knowledgeable “champion” or founder is a key to success.

Table 1.

Demographic Characteristics of Current U.S. Rapid Autopsy Programs

Associated City	Metropolitan Area Population	Type/Size of Hospital (beds)	Program Administered By	Recognition (see legend below)
Tucson AZ	4,700,000	Private health system	Organizational Administrator	3
Baltimore MD	2,800,000	University/1,177	Pathologist	1/2
Boston MA	4,700,000	University/777	Pathologist	1/2
New York NY	20,300,000	University/473	Pathologist	2
Ann Arbor MI	5,300,000	University/998	Internal Medicine/ Pathologist	1/2
Omaha NE	975,000	University/809	Geneticist	3
Bethesda MD/ Washington DC	6,200,000	National Clinical Research Center/200	Researcher	N/A
Chapel Hill/Durham NC	2,000,000	University/803	Pathologist	2
Columbus OH	2,100,000	University/1,506	Oncologist	2
Philadelphia PA	7,100,000	University/695	Ob/Gyn	1/2
Pittsburgh PA	2,400,000	University/770	Oncologist	1/2
Seattle WA	3,500,000	University/442	Pathologist	2
New York NY	20,300,000	University/2410	Pathologist	2

¹= U.S. News 2018 – 2019 Best Hospitals Honor Roll (Top 20)

²= U.S. News 2018 Hospital Rankings by Specialty – Cancer (Top 50)

³= U.S. News 2018 Hospital Rankings by Specialty – Cancer (Higher Performance)

RAP Opportunities

The demand from oncology researchers, family members and patients themselves for programs to utilize postmortem tissue in research continues to grow creating the need for new program development at universities across the country. Many patients who want to participate in this type of program are not within reach of an operating program. In order to help facilitate the expanding use of this invaluable postmortem tissue resource, alternative strategies may need to be employed. Hospitals unable to mount or support a full program might still participate in rapid specimen collection during daytime hours and then ship specimens to researchers at different centers (our program has shipped specimens for cell lines and xenografts to other institutions with good success). Centers can liaison with oncologists and pathologists in nearby communities for referral of patients and successful performance of autopsies using available specialized specimen collection and shipment kits.

Conclusion

Without being able to obtain high quality tissue samples by rapid autopsy, some studies of clinical and biological importance would be virtually impossible. For example, the extent of molecular heterogeneity of different metastases within a single patient is based on the ability to acquire high quality tissue samples of the type that rapid autopsy can provide. In the era of personalized medicine and as understanding of inter-cancer and intra-tumoral heterogeneity grows, rapid autopsy has been shown to be a powerful tool for advancing research at essentially no risk and with no distress to the contributing patient Indeed, families of donors have very often testified that their experience in postmortem donation is a rewarding way for them to feel a contribution to science³⁰. Academic and health centers with a substantial investment in cancer research should consider using this emerging research support tool and assess to what extent a RAP might contribute to their cancer research mission. As rapid autopsy becomes more widely accepted and utilized, it will be essential for those involved in these programs to share the results of their investment in this powerful tool with institutions interested in participating. Over time this new research-oriented 21^st century purpose for autopsy seems positioned to reinvigorate this historically significant area of medicine and, as a result, programs organized to collect these unique specimens are poised to proliferate and support research throughout the United States and internationally.