Rapid autopsy holds ‘great power’ for deciphering tumor heterogeneity in various cancers

Rapid autopsy programs have the potential to greatly inform cancer research by unearthing how a tumor unfolds over time at different disease sites.

A rapid autopsy — typically performed anytime up to 18 hours postmortem — allows for samples to be extracted and preserved to undergo genetic sequencing, measurement of RNA expression and other molecular analyses to determine tumor origin, characteristics and other potential resistance factors. Programs arrange and initiate autopsies on a prompt basis to collect bodily tissues and biofluids before they degrade and are no longer viable for research purposes.

In the era of precision medicine, in-depth inspection of individual patient tumors at cellular and molecular levels is fundamental to create and apply treatment approaches for patients with cancer.

“Cancer science has really shown tumor heterogeneity; tumor characteristics vary widely from primary to metastasis and between metastatic sites in the body,” Jody E. Hooper, MD, director of the Legacy Gift Rapid Autopsy program at Johns Hopkins University and deputy director of the autopsy service, told HemOnc Today.

Source: Johns Hopkins Pathology Photography.

Currently, tumor analysis is mainly limited to the initial biopsy or surgery. As a result, obtaining specific tumor tissue during treatment to understand potential resistance is harder to accomplish on a living patient.

Therefore, it remains a challenge to understand why some cancers do or do not respond to treatment.

“We can’t do all the science that we need to on a tiny biopsy,” Hooper said. “We have to be able to compare different sites and different timepoints of a tumor, which is a great power that rapid autopsy can give.”

Despite these clinical benefits, rapid autopsy programs are only available in select institutions and medical centers due to logistical challenges, lack of funding or staff, or a perceived lack of benefit.

HemOnc Today spoke with pathologists, oncologists and researchers about existing rapid autopsy programs and logistical challenges in developing them, the substantial knowledge that can be gained from such programs, and how expanding efforts to increase the number of programs and patients who participate is crucial for the future of cancer treatment.

Program basics

Rapid autopsy has been in practice at various institutions and medical centers since the early 1990s.

A rapid autopsy program can cater to one specific specialty or to all patients with cancer enrolled in clinical trials. For instance, University of Washington initiated its autopsy program in 1991 for prostate cancer and, in 2015, it initiated a bladder cancer rapid autopsy program.

Michael A. Hollingsworth, PhD, led a team of oncologists, surgical oncologists, pathologists, a research nurse and volunteers to expand efforts to better obtain high-quality primary, metastatic and unaffected human tissues by developing The University of Nebraska Medical Center (UNMC) Rapid Autopsy Program for Pancreas in 2002.

“This collaborative effort has been developed into a comprehensive torso-based autopsy program that includes volunteers from six labs at UNMC, with collection of samples within 2 to 3 hours after death from 10 to 14 donors per year,” Paul M. Grandgenett, PhD, director of the rapid autopsy program at UNMC, said in an interview. “The program currently stores approximately 140,000 grams of tissue and more than 43 liters of biofluids from 121 pancreas cancer donors and five normal donors.”

When the rapid autopsy program at Johns Hopkins began in the early 2000s, it focused on prostate, pancreas and breast cancers.

“Now, it is a little bit different,” Hooper said. “Our Legacy Gift Rapid Autopsy program began when I arrived at Johns Hopkins in 2014, and now we are centralized and work with researchers in all the different organ systems.”

Programs differ based on interest and funding.

“As you look at programs around the country, you will see that some of them do all different kinds of tissues and some specialize in certain areas,” Hooper said. “Also, many rapid autopsy programs are run by oncology researchers rather than by pathologists, so the types of samples taken may depend on what the institution or oncologist/researcher is interested in.”

The rapid autopsy program at The Ohio State University began nearly 2 years ago in conjunction with the divisions of autopsy services, led by Patricia Allenby, MD, within the department of pathology.

“We wanted a more systematic way to study tumor heterogeneity,” Hui-Zi Chen, MD, PhD, medical oncology fellow in the divisions of hematology and oncology at The Ohio State University Wexner Medical Center, said in an interview. “In our precision medicine cancer clinic, we see any patient who is involved in any clinical trial for any cancer type. We have narrowed focus on three cohorts of patients — small cell lung cancer, bile duct cancer and cancer of unknown primary — but we offer rapid autopsy to any patient in a clinical trial.”

A rapid autopsy program requires a highly collaborative, interdisciplinary effort involving pathologists, medical oncologists, technicians and volunteers. A pathologist is a critical piece of the team and should be heavily involved in the sampling process, according to Hooper.

“There is a lot of judgment involved in picking out which part of a tumor to take,” Hooper said. “It is an advantage when a pathologist is present to assist in identifying what tissue should and shouldn’t be taken.”

Timing also is important.

“We want to perform these within 18 hours after death. The rapid autopsy allows us to go in and procure samples of the cancer from all different sites or organs involved with the disease,” Chen said, adding that the tissue is then used to perform a variety of studies. “We can study the tissue in a way that otherwise would not be possible from a single specimen tumor biopsy from a living patient.”

All rapid autopsy programs follow a specific protocol.

“At Hopkins, a majority of patients who undergo rapid autopsy die in hospice or at home, so we have mechanisms in place ahead of time to have them transported here after they die,” Hooper said. “We also obtain a study consent, which gives permission to perform procedures on the sampled tissue such as genetic testing and growth of cell lines.”

“The study consent is signed while patients are alive, but in Maryland we also must have an autopsy consent signed by the legal next of kin after the patient dies” Hooper says. “In some states the autopsy consent can also be signed before death and/or by the patients themselves.”

Still, rapid autopsy programs can differ between institutions.

“All [rapid autopsy programs] provide invaluable resources to the institutions they are associated with and investigators around the world,” Grandgenett said. “These programs differ in the amount and types of tissues taken, the approaches they use to target specific tissues, and the time allowed after death to sample collection. Thus, they all provide a unique collection of samples that differ from institution to institution.”

Although similar, standard and rapid autopsies do differ.

“The difference to emphasize is that rapid autopsy is really an autopsy for the purpose of procuring tumor tissues,” Chen said. “When this is being performed, their goal isn’t like a traditional autopsy to determine cause of death. We know why they died. It’s a very focused procedure during which we look for cancer, and usually it’s very easy to distinguish which tissue is cancer.”

Rapid autopsy holds great benefit for other diseases, as well.

“We at UW Medicine, and several other centers, have had rapid autopsy programs for neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease for many years,” Lawrence D. True, MD, leader of GU Pathology at University of Washington School of Medicine said. “A rapid autopsy program could accelerate our learning mechanistic molecular pathways of other noncancer diseases for which little is known, such as idiopathic interstitial fibrosis of the lung and multiorgan diseases such as systemic lupus erythematosus.”

A rapid autopsy not only helps the science community, but families and patients who participate.

“It’s a way for their spirit to keep going and contributing to cancer research when they are no longer here,” Chen said.

Logistical challenges

Despite the benefits, a number of logistical challenges must be considered when developing a rapid autopsy program.

“Specifically, disease interests, sample quantity collected, salaried and volunteer personnel, clinical integration, availability of patients, administrative support and budgets are all critical elements that determine the processes and practices used,” Grandgenett said. “Although the answers to these questions vary among medical centers, the UNMC program has developed a comprehensive program and has established standard operating procedures for all aspects of site preparation, sample collection, archiving, quality control and distribution.”

The definition of “rapid” can vary among centers and the types of specimens that each center acquires are not necessarily identical.

“We perform tissue acquisition and blood collection typically within 4 hours after the patient is deceased,” Ming Lam, MD, assistant professor and researcher in the department of urology at University of Washington School of Medicine, said in an interview. “We have two physicians, including a pathologist, onboard to ensure we identify and acquire these precious specimens in a timely fashion. This is instrumental for maintaining RNA integrity and the development of patient-derived xenografts, which are invaluable tools for patient-relevant, novel precision targeting.”

Lam added their program also acquires systematic bone samples, because most prostate cancers metastasize to bone.

Not all rapid autopsy programs are able to have on-call staff available 24 hours a day, 7 days a week, but The Ohio State University and University of Washington both have staff within their programs available at any time, day or night.

“Teamwork and coordination are hard to pull off, which is why rapid autopsy programs aren’t available everywhere,” Sameek Roychowdhury, MD, PhD, medical oncologist at The Ohio State University Comprehensive Cancer Center, said in an interview.

“The key for existing programs is that they’ve had champions for them,” Roychowdhury added. “A champion is the one person to spearhead the initiative and to be the glue to keep a team together. Without one, it is just not possible.”

Lam agreed logistical challenges have limited the number of available programs.

“Apart from the funding pressure to support the infrastructure, a dedicated team of scientists, coordination of different parties involved in the program, being geographically located in a reasonably large catchment area to ensure patient number, and maintaining and characterizing specimens present in the biorepository are a few of the challenges that we face,” Lam said.

It is also crucial to maintain enough staff and volunteers on hand to make a successful rapid autopsy program possible. Strong, collaborative relationships among the researchers, medical and surgical oncologists, pathologists and volunteers are essential so all envision the same goal, Grandgenett said.

Additionally, funding for supplies, facilities and effort also are potential mitigating factors that can limit program development.

“It is essential to build a large collaborative group that can see the importance of the program to not only their research, but also the broader picture of overall disease study,” Grandgenett said. “Long-term sustainability requires strong institutional support and funding mechanisms that allow for the archiving and distribution of samples.”

Lack of physician knowledge regarding such programs also may present a challenge.

Rajagopal and colleagues from UPMC presented a poster at San Antonio Breast Cancer Symposium in 2016 based on a survey of 98 clinical oncology faculty at their cancer institute regarding familiarity with autopsies, awareness of the existing program at UPMC, whether they personally asked patients their thoughts on rapid autopsy, whether they think rapid autopsy can identify new sites of metastatic disease and how they would recommend raising awareness for the rapid autopsy program, among others.

Survey results showed 45% of faculty were not aware of rapid autopsy at UPMC prior to the survey, although a majority agreed or strongly agreed rapid autopsy held benefits for patients.

Further, 85% of respondents said they would be willing to ask patients about rapid autopsy in the future.

“Advancing rapid autopsy will require independent education of patients, as well as physicians, to advance buy-in from the oncology community,” the researchers wrote.

Understanding treatment resistance

Understanding how tumors respond, fail to respond and evolve during treatment is critical to the advancement of cancer care.

Findings from rapid autopsies can determine why a patient’s treatment may not have worked.

A study by Pisapia and colleagues showed a next-generation sequencing-based autopsy program in conjunction with a premortem precision medicine pipeline created the opportunity to discover more about clonal evolution metastasis and alterations to underlying treatment.

Researchers procured 346 frozen samples from 15 rapid autopsies of patients with metastatic carcinoma (n = 10), melanoma (n = 1) and glioma (n = 4). The study involved whole-exome sequencing on 113 frozen tumor samples from multiple anatomic sites, as well as nonneoplastic tissue. Researchers also performed RNA sequencing on a subset of 72 samples.

The overall postmortem time interval ranged from 0.5 to 18 hours. Transportation, administrative wait times or accommodation requests by family members attributed to any delays.

Investigators achieved successful cell strain, tumor organoid and/or patient-derived xenograft development from four samples, including an inoperable pediatric glioma. Mutational profiles of primary tumors and metastases provided candidate mediators of metastatic spread and organotropism, including CUL9 and PIGM in metastatic ependymoma and ANKRD52 in metastatic melanoma to the lung. RNA sequencing data identified novel gene fusion candidates.

“We have demonstrated that analysis of whole-exome sequencing data of rapid autopsy material in the context of precision medicine clinical trial permits the identification of genes that become altered at biologically significant branch points in tumor evolution, including those that may contribute to metastatic potential or treatment resistance,” Pisapia and colleagues wrote.

Ultimately, identifying such mutations is the goal of rapid autopsy programs.

“It’s often very hard to obtain tissue to research at the timepoint of aggressive local spread or metastasis,” Hooper said. “With rapid autopsy, we can sample anywhere the consent allows us to. We can compare the tumor that is in the liver with the one in the lung and leg, or any other site, all at the same time.”

An important advantage is the likelihood of reducing cell destruction and, in turn, deriving more tumor cultures for analysis.

A study by Walmsley and colleagues concluded rapid autopsy allowed for gathering “invaluable samples” used to assemble information on tumor heterogeneity, evolutionary dynamics and possible resistance to targeted therapies.

Researchers reviewed samples taken during rapid autopsies on 51 patients with genomically defined advanced solid tumors. All samples were recorded in a centralized database linked to patient’s treatment history and any tissue and liquid biopsies previously collected. Researchers collected a median 12 (range, 5-24) tissue samples per autopsy after a median 2 hours and 50 minutes (range, 32 minutes-9 hours) postmortem.

The median tumor cellularity of the samples was 60%, which indicated the samples could be used for subsequent genomic analyses. Full analysis of three samples showed molecular alterations that drove resistance to PI3K-alpha inhibitors, CDK4/6 inhibitors and FGFR inhibitors among patients with PIK3CA-altered metastatic breast cancer and FGFR2 fusion-positive cholangiocarcinoma.

Juric and colleagues used a rapid autopsy to determine why a patient with metastatic breast cancer with the PIK3CA mutation became resistant to a PI3K-alpha inhibitor.

Researchers collected tissue from 14 different metastatic sites within the patient. When compared with the pretreated tumor, sequencing showed all lesions had a copy loss of phosphatase and tensin homolog. As a result, the lesions became refractory to the treatment and developed different genetic alterations.

The PIK3CA mutations found in the primary tumor were no longer detected at time of disease progression.

“We conclude that parallel genetic evolution of separate metastatic sites with different phosphatase and tensin homolog genomic alterations leads to a convergent phosphatase and tensin homolog-null phenotype resistant to PI3K-alpha inhibition,” Juric and colleagues wrote.

Driving cancer advances

To improve patient outcomes for various cancers, new biomarkers and targets are urgently needed for drug and diagnostic development.

Tumor tissue extracted from living patients is often suboptimal for research purposes. Rapid collection of tissues and biofluids after death ultimately provides the resources necessary for various investigative approaches.

Rapid autopsy biorepositories are vital to cancer research as they provide investigators around the world access to previously unavailable sample sets for full review.

“Programs such as the UNMC rapid autopsy program captures a complete staging of the disease that includes the primary lesion, all metastatic sites and associated unaffected tissues, thus allowing for studies that directly compare all these tissues within one patient and between patients,” Grandgenett said. “These unique collections represent lesions that responded to treatment, as well as those that were nonresponsive, therefore providing a complete genetic and cellular history of disease evolution within the patient.”

Researchers and volunteers are able to learn and be a part of something extraordinary, Grandgenett said.

“Contextual training for cancer-focused doctoral and medical students and participating clinicians provides a unique opportunity for volunteers to experience firsthand the devastating effects of cancer on the body,” he said. “The differences in disease spread and presentation heterogeneity serve to instill a higher level of understanding for these students, post-docs and faculty, and reminds them of the human impact of these diseases.”

Lam and colleagues were able to develop the first patient-derived xenograft of metastatic bladder cancer from tissues derived during rapid autopsy.

Researchers performed 10 bladder cancer rapid autopsies — performed 2 to 6 hours after death — and acquired 105 metastatic and 45 normal specimens. They collected frozen and formalin-fixed paraffin-embedded (FFPE) specimens to perform pathological evaluation, and they also implanted tumors subcutaneously into mice to establish patient-derived xenografts.

Using this process, researchers were able to create xenografts of omentum and liver bladder metastases, the first of their kind.

“This program allows simultaneous collection of multiple metastases upon treatment resistance, providing unprecedented opportunity to study emerging disease phenotype and tumor heterogeneity within and across patients,” Lam said. “The program provides a sufficient number of specimens for sharing to allow multiple biological studies using different derivatives — DNA, RNA, FFPE, fresh tissue and frozen tissues. Patient-derived xenografts provide a valuable tool to test novel targets that identified in these aggressive, treatment-resistant patients.”

Patient participation

The success of a rapid autopsy program is primarily dependent upon the willingness of patients and their families. Without them, researchers would not have this opportunity to gain knowledge that can potentially benefit future patients.

“I’ve had the privilege of meeting many patients whose autopsies I eventually performed, and they truly see it as such a privilege and find it so rewarding to contribute to science in this way,” Hooper said. “It’s wonderful to see.”

As long as these programs are discussed with patients, most are willing to participate.

“It’s as simple as asking our patients, but also at the right place and time.” Roychowdhury said. “We offer this earlier on, and we’ve been pleasantly surprised by the amount of interest by patients. We offer it to as many people as we can if we think they would be a good fit for the study.”

Although a majority of patients are open to rapid autopsy, certain challenges may prevent them from getting involved.

There are many logistical issues that present challenges, such as patient enrollment or the feasibility of securing and transporting the body of a patient who has recently passed to an academic facility.

“It’s not usually a matter of patients not wanting to do it or even not wanting to discuss it, the most difficult part is just getting it done,” Hooper said.

Cultural and religious factors also may play a role in whether a patient consents; however, this is not often the case.

“They want to participate,” Hooper said. “Typically, it has been my experience that patients and families are eager to be involved.”

Still, effort to accommodate the wishes of the patient and their family with respect to postmortem care, timing of the procedure and which samples they can take, is an important piece of a successful program.

Rapid autopsy protocols require both practical and ethical considerations. Ethically, the rapid autopsy team needs to ensure that the rights of a deceased patient are respected.

“It is important to bear in mind that the patient’s rights are just as important when they are alive as when they have died,” Grandgenett said. “For this reason, it is critical that investigators be mindful of patients’ preferences and willingness to participate in autopsy programs.”

Researchers noted the importance of knowing a rapid autopsy does not interfere with the funeral or families wishes.

“We always treat our patients with respect,” Hooper said.

Practically, ensuring the deceased patient’s body is available and accessible may also interfere, and the process is time-consuming.

“We process numerous valuable tissue specimens that are acquired, annotate and archive the specimens, perform quality control of specimens, and conduct biological characterization of the deidentified specimens,” Lam said. “These processes require a robust infrastructure of human capital and funds.”

Due to all of these hurdles that must be overcome prior to biopsy, “sometimes a case feels like a battle because of all these things that have to happen correctly,” Hooper said. “But, when it does, we are representatives, in a sense, for the patients. We are carrying the torch for them and going forward with it and providing it, in the form of the tissue, to the scientists.”

Despite positive response from patients, misconceptions about the program have lingered.

“People often think it is easier than it looks,” Roychowdhury said. “The actual idea of doing it in principle seems simple and straightforward and people might not appreciate the amount of time and teamwork that is needed.”

A lack of knowledge about the program could be the catalyst.

“We shouldn’t assume patients don’t want to hear about it though,” Hooper said. “It’s an exciting line of work to be in and a great privilege to do and I’m so happy to see it expanding.”

Educating the public on the importance of rapid autopsies and tumor donation is crucial, Grandgenett said.

“While organ donation and medical school body donation are relatively well-known avenues for donating to science, tumor and biofluid donation is essential to the continued understanding and development of new detection and treatment strategies,” he said.

The fact that researchers can now study what happens to cancer after drug resistance develops and tumor heterogeneity is a huge step forward, according to Roychowdhury.

“We’ve asked things like how cancers form at different parts of the body, how do they relate to cancer cells circulating in the blood — because a liquid biopsy is a big deal now — and other important questions,” Roychowdhury said. “These are the amazing questions no one has been able to answer before and now we can.”

It is also important to step back and recognize the importance of these donations, Grandgenett said.

“This selfless act will contribute to a better understanding of the disease and may help develop earlier detection and more comprehensive treatments for future generations,” he said. – by Melinda Stevens

Click here to read the , “Should rapid autopsy be requested for every participant in a clinical trial?”

References:

Juric D, et al. Nature. 2015;doi:10.1038/nature13948.

Lam H-M, et al. J Clin Oncol. 2018;doi:10.1200/JCO.2018.36.6_suppl.478.

Pisapia DJ, et al. JCO Precis Oncol. 2017;doi:10.1200/PO.16.00038.

Rajagopal PS, et al. P1-14-02. Presented at: San Antonio Breast Cancer Symposium; Dec. 6-10, 2016; San Antonio.

Walmsley CS, et al. Ann Oncol. 2017;doi:10.1093/annonc/mdx390.001.

For more information:

Hui-Zi Chen, MD, PhD, can be reached at hui-zi.chen@osumc.edu.

Paul M. Grandgenett, PhD, can be reached at pgrandgenett@unmc.edu.

Jody E. Hooper, MD, can be reached at jhooper9@jhmi.edu.

Ming Lam, MD, can be reached at minglam@uw.edu.

Sameek Roychowdhury, MD, PhD, can be reached at sameek.roychowdhury@osumc.edu.

Lawrence D. True, MD, can be reached at ltrue@vw.edu.

Disclosures: Chen, Grandgenett, Hooper, Lam, Roychowdhury and True report no relevant financial disclosures.