Background: Rapid Autopsy Programs offer an opportunity to collect tissue from patients immediately after death, providing critical biological material necessary to develop more effective therapies and improve patient outcomes. Here, we present a step-by-step guide to build a cancer-focused Rapid Autopsy Program, based on our own experiences building “The Legacy Project” at the City of Hope Comprehensive Cancer Center. Methods: The linear timeline of events is separated into four phases: 1) Building the Infrastructure, 2) Recruiting and Consenting, 3) Preparing for Death, and 4) Tissue Collection and Follow up. Important considerations and methods for adaptation are discussed throughout the protocol. Discussion: Using these methods, we successfully collected a total of 533 specimens from 9 subjects. The average time from death to last specimen acquisition was 6.1 hours (range: 4.03 – 7.66 hours; median: 5.71 hours). A diverse team with various areas of expertise is critical for successful program implementation. Our goal herein this protocol is to provide a comprehensive framework and foundation for other institutions to use as a model.
Metastatic disease is understudied largely because of inaccessibility to quality specimens for research use. The Legacy Project, a rapid or “warm”, autopsy program at City of Hope, seeks to overcome this challenge by collecting tissue from metastatic patients immediately (within 6 hours) after their death. This paradigm serves as a specimen resource to address many clinically relevant questions, such as disease heterogeneity and mechanisms driving disease progression. Using this model, we uncovered clinically relevant disease information that is normally unavailable while a subject is alive. In this study, 9 metastatic breast cancer patients and their families were approached and consented prior to death. The cohort includes a diversity of clinical presentations in terms of disease subtype, progression history, organ involvement, and final cause of death. A total of 533 specimens were collected across 9 subjects. The average time from death to specimen acquisition was 6.1 hours (range: 4.03 - 7.66 hours; median: 5.71 hours). Total number of specimens collected from each participant ranged from 38-75, with an average of 60 across all patients; the mean number of tumor-positive specimens collected was 29 (range 12-46); the mean number of non-cancer specimens collected was 31 (range 25-45). In patients with primary estrogen receptor (ER) positive disease, we observed variable heterogeneity in estrogen, progesterone, and ki67 status across metastatic lesions. Furthermore, we observed a profound shift in disease phenotype towards end of life, trending towards complete loss of hormone receptor expression and stark increase of Ki67 levels. At the time of procurement, one third of subjects exhibited clinically unidentified diseased sites in organs not commonly associated with breast cancer metastases a, including ovary, kidney, and pancreas. In two other instances, “resolved” bone specimens (as measured by absence of FTG uptake in PET/CT imaging) were later determined to be >30% tumor positive when assessed by H&E. While these preliminary findings generate more questions than answers regarding mechanisms of metastatic progression and resistance to therapy, they highlight the utility of rapid autopsy in a research setting. We suggest that many unanswered clinical questions can be addressed through interrogation of post-mortem tissues and we urge research institutions to thoughtfully consider adoption of the “rapid autopsy” model. Citation Format: Eliza R Bacon, Kena Ihle, Colt Egelston, Weihua Guo, Diana Simons, Peter P Lee, James Waisman. Utility of rapid autopsy in cancer research: Unexpected findings and lessons learned from warm autopsies of metastatic breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-16.
Immune composition in the tumor microenvironment (TME) of patient tumors has proven to play a central role in the development of metastases and response to therapy. Evidence has suggested that the metastatic TME is immune aberrant, however difficulty in obtaining biopsies of metastatic tumors has made assessment of the immune TME difficult. Here we utilize a rapid autopsy tissue collection protocol to assess the infiltration and composition of the immune TME in numerous metastatic tissue sites, paired disease-free tissue sites, and the associated tissue draining lymph nodes. Post-mortem tissues were collected from nine metastatic breast cancer patients shortly after death through City of Hope’s “Legacy Project for Rapid Tissue Donation” Program. The average post-mortem interval (PMI) for tissue collection was 6 hours. Collected specimens include metastatic lesions and paired non-cancer samples from every cancer-involved organ, disease-free specimens from non-involved major organs, distant and tumor-draining lymph nodes (both cancer-infiltrated and disease free), as well as blood and spleens. Immediately following collection, specimens were processed into single cell suspension for flow cytometry. Over 80 immune cell phenotypes were assessed, including CD8+ and CD4+ T cell subsets, B cell subsets, natural killer (NK) cells, tumor associated macrophages (TAMs), dendritic cell subsets, and other immune cells. Tumor infiltrated tissues were found to have comparable immune cell densities and composition compared to paired disease-free tissues of the same organ type. However, immune cell densities in metastatic tissues and disease-free tissues were significantly different between organ types, with lung immune infiltration consistently being greater than liver, brain, and skin tissues. Differences in immune composition between tissue sites were also observed. Notably, liver tissues favored the presence of IL-2 producing central memory CD8+ T cells, while lung tissues favored the presence of CD8+ tissue resident memory T cells and CD16+ NK cells. Relative to disease-free lung tissues, tumor infiltrated lungs contained diminished frequencies of CD8+ tissue resident memory T cells and altered B cell and monocyte phenotypes. Increased levels of targetable immune pathways were observed, including increased PD-L1 and CTLA-4 expressing cells in skin metastases, and increased GARP+ B cells in bone marrow metastases. These data suggest that immune monitoring and trafficking of metastatic tissues site is dictated by organ type, which can be altered in composition by tumor infiltration. Further studies such as these may reveal organ-specific mechanisms of response to therapeutic interventions. Identity of organ location for tumor metastases may guide choices for immunotherapeutic interventions. Citation Format: Colt Egelston, Weihua Guo, Eliza R. Bacon, Kena Ihle, Diana Simons, Christian Avalos, JIayi Tan, Jian Ye, Lei Wang, Minhui Lim, James R. Waisman, Peter P Lee. Metastatic tissues display organ specific immune infiltration archetypes; lessons from a rapid autopsy tissue collection study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-45.
Previously we presented our initial findings from a 9-patient rapid autopsy pilot for metastatic breast cancer (MBC). At the time of procurement, one third of subjects exhibited clinically unidentified diseased sites in organs not commonly associated with breast cancer metastases, including ovary, kidney, and pancreas. In two other instances, “resolved” bone specimens (as measured by absence of FTG uptake in PET/CT imaging) were later determined to be >30% tumor positive when assessed by a pathologist. We now expand upon these findings in a more in-depth exploration of the presence of micro-metastases in presumed tumor-negative tissues. A subset of tumor-free tissues were selected from each patient (average of 10 specimens per patient). All selected specimens were negative by clinical imaging, appeared grossly normal at procurement, and were reported to be tumor negative by H&E assessment by a clinical pathologist. We included organs both commonly and uncommonly involved in MBC, including lung, bone, spleen, pancreas, kidney, and non-tumor draining lymph nodes. Tissues were stained for one or more of the markers, pan-cytokeratin, GATA-3, HMFG, MUC1, and ER (if patient was previously ER+), depending on tissue type. Of the 87 total specimens assessed, we identified micro-metastases in 13 specimens from 4 individual patients. Across these 4 patients, micro-metastases were found in lung, bone, pancreas, spleen, and several non-tumor draining lymph nodes. While lung and bone are commonly involved in MBC and these results are not entirely surprising, pancreas and spleen involvement is extraordinarily rare. Further surprising was the identification of micro-metastases in several lymph nodes that were not located anatomically downstream from a disease-involved organ. Image patterns demonstrate tumor cell infiltration into the lymph node within the subcapsular sinus. Presence of micro-metastases in tumor-negative tissue did not correlate with tumor hormone status or cancer type (e.g. lobular vs DCIS). Combined with our previous findings, we now report unexpected and clinically undiagnosed disease involvement in 6/9, or two-thirds, of our patients. Based on these findings, we hypothesize that cancer stem cells and/or micro-metastases are present throughout the body, in all tissue types, and that their ability to grow into tumors is regulated by the local immune microenvironment. Lastly, the differing roles and mechanics of lymphatic vs hematological spread in metastatic disease has long been discussed. Our findings provide strong evidence for cancer dissemination through the lymphatics system. Further study is necessary to better understand the timing of metastatic spread, whether systemic dissemination occurs early or later in disease, and if conducive metastatic or pre-metastatic niches are already present throughout the body at the time of primary diagnosis or if these permissive environments develop slowly overtime. Citation Format: Eliza R. Bacon, Kena Ihle, Colt Egelston, Weihua Guo, Diana Simons, Dan Schmolze, Christina Wei, Lusine Tumyan, Peter P Lee, James R. Waisman. Insights from rapid autopsy shed light on mechanisms of cancer dissemination in metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-06-01.
1032 Background: Immune composition in the tumor microenvironment (TME) of patient tumors has proven to play a central role in the propensity of tumors to metastasize and respond to therapy. Evidence has suggested that the metastatic TME is immune aberrant, however limited sample size and numbers has made assessment of the immune TME in the development of multi-organ metastases difficult. Here we utilize a rapid autopsy tissue collection protocol to assess the infiltration and composition of the immune TME in numerous metastatic tissue sites, paired disease-free tissue sites, and the associated tissue draining lymph nodes. Methods: Post-mortem tissues were collected from six metastatic breast cancer patients shortly after death through City of Hope’s “Legacy Project for Rapid Tissue Donation” Program. The average post mortem interval (PMI) for tissue collection was 6 hours. Collected specimens include metastatic lesions and paired non-cancer samples from every cancer-involved organ, disease-free specimens from non-involved major organs, distant and tumor-draining lymph nodes (both cancer-infiltrated and disease free), as well as blood. Immediately following collection, specimens were processed into single cell suspension for flow cytometry. Over 80 immune cell phenotypes were assessed, including CD8+ and CD4+ T cell subsets, B cell subsets, natural killer (NK) cells, tumor associated macrophages (TAMs), dendritic cell subsets, and other cells. Results: Tumor infiltrated tissues were found to have comparable immune cell densities and composition compared to paired disease-free tissues of the same organ type. However, immune cell densities in metastatic tissues and disease-free tissues were significantly different between organ types, with lung immune infiltration consistently being greater than liver tissues. Differences in immune composition between tissue sites were also observed. Notably, liver tissues favored the presence of central memory CD8+ T cells, while lung tissues favored the presence of CD8+ tissue resident memory T cells. Relative to disease-free lung tissues, tumor infiltrated lungs contained diminished frequencies of CD8+ tissue resident memory T cells and altered B cell phenotypes. Conclusions: These data suggest that immune monitoring and trafficking of metastatic tissues site is dictated by organ type, which can be altered in composition by tumor infiltration. Further studies such as these may reveal organ-specific mechanisms of response to therapeutic interventions.
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