Postmortem Tissue Donation Research Articles

Socioeconomic Factors Associated With Participation in Postmortem Tissue Donation in Pediatric Central Nervous System Tumors.

The gift of postmortem tissue donation is critical to pediatric central nervous system (CNS) research. As a "Center of Excellence" for the Gift from a Child Program, our institution has a well-established postmortem tissue donation program. Our objective was to determine if differences exist between patients who participated in and those that declined research-based autopsy. We performed a single-institution retrospective chart review of pediatric patients with CNS malignancies who died from their disease between January 1, 2021 and December 31, 2022. Individual clinical, demographic, and socioeconomic data were assessed. Population-level data were estimated using Zip Code Tabulation Areas. Descriptive statistics were used to compare categorical data. Among the 23 patient families approached during the study time frame, 8/23 (35%) consented to participation. In the consented (C) versus declined (D) group, there was a higher percentage of White, non-Hispanic/Latino patients by self-reported race (C: 88% vs. D: 55%), and no patients who identified as Asian or Hispanic/Latino (C: 0% and 0% vs. D: 13% and 27%). Of all patients approached, two required interpreters (9%), and both families declined participation. The rate of private insurance was higher in the consented group (C: 75% vs. D: 47%) compared to Medicaid as primary insurance in the declined group (C: 13% vs. D: 53%). CONCLUSIONS: Future research should aim to understand and improve identified disparities to ensure research advancements benefit all children with CNS malignancies. One area we plan to address is improved communication with non-English-speaking families by partnering with our interpreter services.

Stromal tumor infiltrating lymphocytes in hormone receptor positive/HER2 negative metastatic breast cancer

The immune landscape of hormone receptor positive, HER2-negative metastatic breast cancer (HR+/HER2- mBC), the most common subtype of BC, remains understudied. This is mainly due to reduced sample acquisition opportunities from metastases as compared to primary tumors. In this study, we explored stromal tumor-infiltrating lymphocytes (sTIL) in metastatic samples collected through our post-mortem tissue donation program UPTIDER (NCT04531696). sTIL were scored as a continuous parameter according to international guidelines on 427 metastases and 38 primary untreated tumors acquired from 20 patients with HR+/HER2- mBC. ER-status was evaluated on 362 metastases with a cut-off for positivity set at 1% according to ASCO/CAP guidelines. Our analyses show that 54% and 15% of metastases had sTIL levels >1% and >5% respectively. sTIL levels tended to be lower in metastases as compared to their respective primary tumor (Estimate: -2.83, 95%CI: -5.77-0.11, p:0.07). sTIL levels were lower in metastases from invasive lobular carcinoma than in metastases from invasive breast carcinoma of no special type (Estimate: -1.67, 95%CI: -2.35--0.98, p:<0.001). A loss of ER expression was observed in 14% of all metastases, yet a negative ER-status was not significantly associated with increased sTIL levels. Finally, sTIL levels were significantly higher in lung and axillary lymph node metastases compared to all metastases. While these analyses were conducted on multiple metastases obtained at the end of life after several lines of treatment, the data provides novel and valuable insights into the state of immune infiltration in patients with metastatic HR+/HER2- BC.

OTHR-17. FACILITATING THE POSTMORTEM DONATION OF WHOLE BRAIN AND SPINAL CORD IN PEDIATRIC PATIENTS WITH CENTRAL NERVOUS SYSTEM CANCERS

Abstract BACKGROUND Postmortem tissue donation is often presented to families during end-of-life discussions. The sensitive nature requires every step to be handled with care, leading with empathy and delicately balancing scientific research and humanity. The Children’s National Hospital Postmortem Tissue Donation Workflow (The Workflow) provides a step-by-step guide for coordinating a successful donation. It outlines the order in which clinical research teams communicate with patient care teams, families and collaborators to make the donation possible. This program, established in 2010, aims to acquire more pediatric brain and spinal cord tumor specimens to further understand the disease biology. OBJECTIVE This paper aims to provide a template for coordinating a successful postmortem tissue donation. Ultimately, to broaden the network of tissue donation across all hospitals and institutions, thus furthering scientific research. METHOD In order to create The Workflow, Clinical Research Coordinators from Children’s National Hospital conducted numerous postmortem donations and noted the failures and successes of each case. This information was compiled into a stepwise workflow including email templates and checklists. It begins with contacting the care team, and the following hours to days are spent obtaining a pathologist for tissue procurement, and facilitating the shipment of samples. While these steps must be completed in a timely manner for sample integrity, we also prioritize the wishes of the family and act with compassion at the forefront. RESULTS/DISCUSSION Children’s National Hospital was designated as a Center of Excellence for postmortem coordination by the Swifty Foundation in 2018. Our team has since established and standardized a workflow for the donation process. The Children’s National Hospital Postmortem Workflow allows our research team to study brain tumors including DMGs, High and Low Grade Gliomas, and Medulloblastomas. The generosity of the families and hard work of each collaborator allows us to expand our knowledge of tumor biology.

OTHR-15. POSTMORTEM TISSUE BIOBANKING AND COLLABORATIVE EFFORTS: A SINGLE INSTITUTION REPORT

Abstract BACKGROUND Post-mortem tissue donation holds significant value to researchers, clinicians and bereaved families however it is an underutilized tool. Initiatives in pediatric neuro-oncology allow new insights into tumors such as DIPG and is becoming more widespread. Barriers include treating physicians lack of knowledge, lack of resources, religious beliefs and more. Postmortem tissue gives insight into the evolving biology of cancer and failed treatments. Postmortem tissue donation offers some understanding to families and empower them to help others. Surgical tissue collection used for diagnosis, leaves limited amounts for research. METHODS Since 2012, we have contributed to pediatric cancer research by offering postmortem donation to 48 patients. We have consented and facilitated 35 autopsies. In 2020, Arnold Palmer Hospital for Children became a lead institution for the COG’s Project:EveryChild. In 2021, we became a Center of Excellence for Gift From A Child. Our local Translational Protocol, IRB-approved, allowing us to collect and store our surgical and postmortem samples. To date, we have collected over 839 postmortem samples from 35 patients. In our Translational Laboratory, we have access to 207 postmortem samples from 13 patients. Through collaborative research initiatives, we have successfully sent over 368 samples from 11 patients and 14 parents to GFAC. Through Project:EveryChild we have shared 218 samples from 7 patients. RESULTS To explore the quality of postmortem tissues, we tested the integrity of our samples through DNA and RNA sequencing. The DNA quality is excellent across specimens and equivalent to surgical specimens however the RNA quality from autopsy specimens has degraded 7.5% RINe factor &amp;gt; 8 (excellent) and 38% RINe factor &amp;gt; 6 (mildly degraded) compared to surgical specimens, 85% with RINe factor &amp;gt; 8, 14.3% RINe factor &amp;gt; 6. CONCLUSION The platform of postmortem tissue donation can lead to further advancements in understanding the evolution and resistance of cancer.

DEI-01. CLINICAL AND SOCIOECONOMIC FACTORS ASSOCIATED WITH PARTICIPATION IN POST-MORTEM TISSUE DONATION IN PEDIATRIC BRAIN TUMORS: A RETROSPECTIVE CHART REVIEW

Abstract BACKGROUND The gift of post-mortem tissue donation is a critical resource in the discovery and interrogation of pathobiological mechanisms of pediatric brain tumors. Broad generalizability of research findings requires a representative population to participate in pre-clinical studies. As a “Center of Excellence” within the Gift from a Child initiative, our institution has a well-established post-mortem tissue donation program funded by the Swifty Foundation. Our primary objective was to determine if clinical and socioeconomic differences exist between patients that participated in and those that declined research-based autopsy. METHODS We performed a single-institution retrospective chart review of pediatric patients with central nervous system (CNS) malignancies who died from their disease between 1/1/2021 and 12/31/2022. Individual clinical, demographic, and socioeconomic data were assessed. Population-level data were estimated using Zip Code Tabulation Areas. Descriptive statistics were used to compare categorical data. RESULTS Among 26 patients who died secondary to disease in the study timeframe, 23 (88%) were approached and 8/23 (35%) consented to participation. In the consented group (C) vs. declined group (D), there was a higher percentage of White, Non-Hispanic/Latino patients by self-reported race (C: 88% vs. D: 55%) and no patients who identified as Asian or Hispanic/Latino (C: 0% &amp; 0% vs. D: 13% &amp; 27%). Of all patients approached, 2 required interpreters (9%) and both families declined participation. The rate of private insurance was higher in the consented group (C: 75% vs D: 47%) compared to Medicaid as primary insurance in the declined group (C: 13% vs D: 53%). CONCLUSIONS Patients from racial and ethnic minorities are underrepresented in tissue donation programs. Future research should aim to understand and improve identified disparities to ensure advancements benefit all children with CNS malignancies. One area we plan to address is improved communication with non-English speaking families by educating interpreter services prior to discussion.

METB-12. GIFT FROM A CHILD POSTMORTEM SPECIMEN DATASET EXTENSION OF THE PEDIATRIC BRAIN TUMOR ATLAS EMPOWERS PEDIATRIC BRAIN TUMOR RESEARCH

Abstract BACKGROUND Pediatric brain and spinal cord tumors are the leading cause of cancer-related mortality in children. An incomplete understanding of brain tumor biology and associated limited access to high-quality biological samples for research are the main factors driving the lack of clinical therapeutic development for pediatric brain tumors that recur or progress. Post-mortem tissue donation provides an unprecedented resource for addressing some of these limitations. METHODS The Gift from a Child (GFAC) program by the Swifty Foundation has a unique mission to increase post-mortem pediatric brain tissue donations through advocacy as well as the education of clinicians and families. Through GFAC’s strategic collaboration with the Children’s Brain Tumor Network (CBTN), CBTN has leveraged postmortem tissue to expand the Pediatric Brain Tumor Atlas (PBTA), a cross-histology multi-omics atlas resource. As part of the effort CBTN has sequenced and released data for over 350 post-mortem pediatric brain tumor specimens including multiple brain region sampling cases with specimen and sequencing quality metrics. RESULTS Here we present an assessment of postmortem samples and available multi-omic data on postmortem samples within the PBTA dataset. Data have been harmonized and released with no publication embargo. To access data, researchers can utilize existing open source data resources and platforms including PedCbioPortal and OpenPedCan to: (1) Identify tumor spatial and temporal specific alterations (2) Establish tumor evolution trajectory leading to therapeutic resistance and tumor progression; (3) Understand tumor heterogeneity longitudinally across multiple ‘omics layers; and (4) Identify and request specimens and derived tumor models. CONCLUSIONS Together, we present the largest deeply characterized cohort of postmortem pediatric brain tumor samples as powerful expansion of the PBTA cohort of &amp;gt;3,000 pediatric brain tumors. CBTN’s open-science model supported by the GFAC mission highlights the value and utility of autopsy-based specimen collection on behalf of improving outcomes for children with brain tumors.

DEI-06. IMPROVING ACCESS TO POST-MORTEM BRAIN TUMOR TISSUE DONATION BEYOND THE INPATIENT HOSPITAL SETTING

Abstract Between May 2021 and December 2023, approximately 33% of patients were being treated inpatient at the time of consent for post-mortem tissue donation through Gift From A Child. In the past, a major obstacle preventing families from donating post-mortem tissue was the inability to leave the hospital. Care teams and patient families are often concerned that in order to donate, the patient would have to pass away at the hospital. This creates a situation where the desire to donate comes at the cost of patient care and comfort. Through GFAC, children can pass at home, hospital, or hospice and still donate tissue successfully. The ability to donate post-mortem tissue is independent of patient location, ensuring that the focus of the family and care team remains on the child’s care rather than donation logistics. Additionally, patients can successfully donate tissue independent of status at the time of inquiry (declining, stable, urgent). The ability to quickly access a resource at a low burden to the family has been crucial in allowing tissue donation to happen. This also improves patient equity by eliminating access to hospital resources as a reason for a patient family to be required to live near a hospital. This is oftentimes a tremendous financial difficulty for patient families. Through establishing standard operating procedures and appointing tissue navigators, GFAC has streamlined what was previously an extremely complicated process mired in logistical obstacles. Patient families now have access to an efficient process for tissue donation. Previously, families would need to navigate complicated logistics unsupported, now they (or someone on their behalf) only need to contact GFAC or a Research Center of Excellence via phone or email and consent for donation. This ensures that post-mortem tissue can be donated easily, efficiently, and from wherever the patient family chooses.

Abstract PO1-06-06: Underestimation of metastatic spread in patients with lobular breast cancer: results from the post-mortem tissue donation program UPTIDER

Abstract Background: Invasive lobular carcinoma (ILC) accounts for 15% of all invasive breast cancers (BC) and has a peculiar metastatic spread in comparison to BC of no-special type. Since ILC is less likely to disrupt normal tissue architecture and is usually non-mass forming, imaging of ILC lesions entails many challenges. Insights into pathologic versus radiological metastatic invasion can lead to better diagnostic and monitoring tools for patients with ILC. Here, we compare microscopical findings during autopsy with clinical findings prior to death in patients with metastatic ILC included in our post-mortem tissue donation program UPTIDER (NCT04531696). Methods: UPTIDER was started in November 2020 in University Hospitals Leuven/KU Leuven with inclusion of patients with stage IV BC who were willing to participate. One of the predefined subgroups consisted of patients with primary pure (i.e. not mixed) ILC. Samples were taken from different macroscopically invaded and non-invaded sites. Clinical data on disease progression, imaging, biochemistry and treatment regimens were extracted from patient files. The number of samples that were preregistered as pathological served as a surrogate for lesions that were seen on imaging performed during the treatment of the patient. These samples are compared to microscopical findings of the autopsy. Results: Since the start of UPTIDER, an autopsy has been performed on 6 patients with pure ILC (26.1% of all autopsies). Median age at initial diagnosis was 52 years (range: 37 – 80 years). Three patients (50.0%) had stage IV disease at diagnosis, the others relapsed on average 162.7 months (range: 55 – 358 months) after initial diagnosis. The average time between clinical occurrence of metastases and death was 44.8 months (range: 15 – 83 months). Median number of treatment lines for stage IV disease was 5 (median endocrine lines 2; median chemotherapy regimens 3.5). To follow disease evolution, computed tomography of thorax and abdomen was used for 4 (66.7%) patients and whole-body diffusion-weighted magnetic resonance imaging (WB-DWI/MRI) for the remaining 2 (33.3%). Median time between last premortem imaging and death was 5.9 weeks (range: 1.6 – 16.6 weeks). At autopsy, a median of 26 unique metastases (range: 12-36) were sampled per patient. Table 1 gives an overview on the unique microscopically invaded metastases that were sampled per patient. Only 47.3% of the sampled unique metastases was preregistered. Of all unique metastases, 26.7% appeared macroscopically normal during autopsy. Tissues that appeared normal but turned out to be microscopically infiltrated included liver, stomach, adrenal glands, heart, pericardium, visceral and subcutaneous fat tissue. There were 2 patients with normal appearing, microscopically infiltrated livers. In these patients, elevation of γGT and transanimases was seen in the months prior to death. Conclusions: The disease burden of metastatic ILC reported on premortem imaging does not reflect the microscopical findings at autopsy. One of the priorities of metastatic ILC research should be the development of diagnostic tools to better estimate the extent of the disease. Future analyses of the performed postmortem MRIs in our study can aid in improving the interpretation of WB-DWI/MRI for patients with ILC. For now, clinicians should consider that unexplained clinical and/or biochemical findings might indicate progression of ILC. Table 1: patient overview on performed imaging and unique metastases sampled during autopsy CT = computed tomography; ER = estrogen receptor; HER2 = human epidermal growth factor receptor 2; pt = patient; WB-DWI MRI = whole-body diffusion-weighted magnetic resonance imaging *exclusion of samples of patient 2012 since no preregistration was performed for this patient due to unexpected death Citation Format: Karen Van Baelen, Gitte Zels, Maxim De Schepper, Marion Maetens, Josephine Van Cauwenberge, Tatjana Geukens, Kristien Borremans, François Richard, Amena Mahdami, Ha-Linh Nguyen, Sophia Leduc, Anirudh Pabba, Ann Smeets, Ines Nevelsteen, Patrick Neven, Hans Wildiers, Vincent Vandecaveye, Wouter Van Den Bogaert, Giuseppe Floris, Christine Desmedt. Underestimation of metastatic spread in patients with lobular breast cancer: results from the post-mortem tissue donation program UPTIDER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-06.

Abstract ED02-01: Biological peculiarities of ILC

Abstract One in 8 women will be diagnosed with breast cancer in her lifetime and approximately 15% of them have invasive lobular breast cancer (ILC). ILC differs in many ways from the most common BC subtype, called non-special type (NST) (Van Baelen et al. Ann Oncol 2022). The microscopic hallmark of ILC is the presence of single files of relatively small discohesive tumor cells, which stems from a dysfunctional E-cadherin-catenins complex at the intercellular junctions, a feature that is uncommon in NST. More than 90% of ILC express the estrogen (ER) and/or progesterone receptor (PR) and lack HER2 amplification, while this is only the case in ~65% of NST. Patients with ILC also show a distinct clinical behavior as compared to NST: generally, a poor response to chemotherapy, more late recurrences, and a peculiar metastatic pattern. The first part of the presentation will focus on presenting the biological knowledge we have accumulated over the years about primary ILC. We will review the studies which compared ILC and NST at the genomic, transcriptomic and immune level. The second part of the presentation will be focused on metastatic breast cancer. Here we will review the published evidence regarding the metastatic dissemination of ILC, its genomic landscape and the contribution of the local tumor microenvironment. We will also dedicate attention to the important biological and clinical knowledge which can be gained by studying samples collected from patients with metastatic ILC who consented to post-mortem tissue donation. Finally, the last part of the presentation will be dedicated to mixed NST/ILC tumors. These tumors account for up to 18% of all invasive breast cancers and represent an even more under investigated area of research as pure ILC. Citation Format: C. Desmedt. Biological peculiarities of ILC [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr ED02-01.

Rapid autopsies to enhance metastatic research: the UPTIDER post-mortem tissue donation program

Research on metastatic cancer has been hampered by limited sample availability. Here we present the breast cancer post-mortem tissue donation program UPTIDER and show how it enabled sampling of a median of 31 (range: 5-90) metastases and 5-8 liquids per patient from its first 20 patients. In a dedicated experiment, we show the mild impact of increasing time after death on RNA quality, transcriptional profiles and immunohistochemical staining in tumor tissue samples. We show that this impact can be counteracted by organ cooling. We successfully generated ex vivo models from tissue and liquid biopsies from distinct histological subtypes of breast cancer. We anticipate these and future findings of UPTIDER to elucidate mechanisms of disease progression and treatment resistance and to provide tools for the exploration of precision medicine strategies in the metastatic setting.

Global post-mortem tissue donation programmes to accelerate cancer research.

Global post-mortem tissue donation programmes to accelerate cancer research.

Research autopsy programmes in oncology: shared experience from 14 centres across the world.

While there is a great clinical need to understand the biology of metastatic cancer in order to treat it more effectively, research is hampered by limited sample availability. Research autopsy programmes can crucially advance the field through synchronous, extensive, and high-volume sample collection. However, it remains an underused strategy in translational research. Via an extensive questionnaire, we collected information on the study design, enrolment strategy, study conduct, sample and data management, and challenges and opportunities of research autopsy programmes in oncology worldwide. Fourteen programmes participated in this study. Eight programmes operated 24 h/7 days, resulting in a lower median postmortem interval (time between death and start of the autopsy, 4 h) compared with those operating during working hours (9 h). Most programmes (n = 10) succeeded in collecting all samples within a median of 12 h after death. A large number of tumour sites were sampled during each autopsy (median 15.5 per patient). The median number of samples collected per patient was 58, including different processing methods for tumour samples but also non-tumour tissues and liquid biopsies. Unique biological insights derived from these samples included metastatic progression, treatment resistance, disease heterogeneity, tumour dormancy, interactions with the tumour micro-environment, and tumour representation in liquid biopsies. Tumour patient-derived xenograft (PDX) or organoid (PDO) models were additionally established, allowing for drug discovery and treatment sensitivity assays. Apart from the opportunities and achievements, we also present the challenges related with postmortem sample collections and strategies to overcome them, based on the shared experience of these 14 programmes. Through this work, we hope to increase the transparency of postmortem tissue donation, to encourage and aid the creation of new programmes, and to foster collaborations on these unique sample collections. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract A004: Comparison of ctDNA detection in seven different types of body liquids from patients with metastatic breast cancer

Abstract Background. Liquid biopsies represent a less invasive alternative to tissue biopsy to characterize the disease in patients with metastatic cancer. Blood remains the most frequently investigated body liquid with regards to detection, quantification and characterization of the circulating tumor DNA (ctDNA). However, it might not capture the full disease profile, and other sources of body liquids may be complementary. The aim of the present study is to compare ctDNA detection in different types of body liquids. Patients and methods. Sixteen patients from the post-mortem tissue donation program UPTIDER (NCT04531696) were included in this study. The receptor status of their primary tumor was: estrogen receptor positive, Human Epidermal Growth Factor Receptor 2 non-amplified (ER+/HER2-) (n=12), ER-/HER2- (n=3) and ER+/HER2+ (n=1). Seven types of liquids were collected: blood, saliva, ascites, pleural fluid (PFL), cerebrospinal fluid (CSF), pericardial fluid and urine. Fluids were collected at study inclusion (blood, saliva, urine, and ascites whenever possible) and at autopsy (except for saliva). In total, 281 liquid samples were collected and processed according to standard protocols. All extracted cfDNA (supernatant), and the 16 matched germline DNA (buffy coat) samples underwent shallow whole genome sequencing. Log2 coverage ratios were computed with CNVkit, and good quality profiles were co-segmented per patient using the copynumber R package. Purity and ploidy were assessed by ABSOLUTE and manually reviewed. Associations between organ involvement and ctDNA yield were assessed by Wilcoxon rank-sum tests. Results. At the patient level, the proportion of liquid types in which ctDNA was detected was highly variable (median: 62%, IQR: 25-89%). ctDNA was detected in ascites of all patients where investigation was possible, in 92% of PFL, 77% of CSF, 69% of blood, 30% of pericardial fluid and in 10% of urine samples. No ctDNA was detected in the saliva samples. At autopsy, ctDNA could not be identified in blood in 4 out of the 16 patients but was detected in at least one of the other fluids for 3 of these patients. ctDNA levels tended to be higher in PFL and CSF in case of pleural and central nervous system (CNS) metastases, respectively. 3 patients had CSF ctDNA detected with no documented involvement of the CNS. In ascites, ctDNA levels were independent of peritoneal invasion. Conclusion. ctDNA was detected in 6 out of the 7 investigated body liquids and its level was partially associated with metastases in surrounding organs, except for ascites. In 3 patients, blood was not contributive but other liquids were, encouraging the evaluation of additional body fluids, when possible, in patients with metastatic breast cancer. These results open new avenues for the clinical monitoring and characterization of the disease. Citation Format: François Richard, Tatjana Geukens, Maxim De Schepper, Amena Mahdami, Karen Van Baelen, Marion Maetens, Ha-Linh Nguyen, Anirudh Pabba, Sophia Leduc, Edoardo Isnaldi, Maysam Mohammadzadeh Hajipirloo, Emily Vanden Berghe, Imane Bachir, Sigrid Hatse, Peter Vermeulen, Evy Vanderheyden, Bram Boeckx, Diether Lambrechts, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Hans Wildiers, Wouter Van Den Bogaert, Jonas Demeulemeester, Elia Biganzoli, Giuseppe Floris, Christine Desmedt. Comparison of ctDNA detection in seven different types of body liquids from patients with metastatic breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A004.

Pediatric Postmortem Tissue Donation in the Confines of a Pandemic: A Model of Collaboration

BackgroundObtaining postmortem tissue from pediatric oncology patients is critical to research and may help grieving families heal. Since 2019, the national Gift from a Child program has made significant progress in collecting postmortem tissue from pediatric patients with central nervous system tumors to advance research. This progress was at risk during the onset of the severe acute respiratory syndrome coronavirus 2 pandemic, when some autopsy programs came to a halt. MethodsWe retrospectively reviewed autopsies of four patients treated at Memorial Sloan Kettering Cancer Center who underwent postmortem examination at Weill Cornell Medicine from June 2020 to March 2021. We collected patient demographics, Do not resuscitate status, time of death and procedure, restrictions due to the coronavirus disease 2019 (COVID-19) pandemic, and results of the tissue analysis. ResultsThree of four specimens were processed within 12 hours of the time of death. Two families required interpreter services to obtain consent. In all cases, tumor aliquots were flash frozen for further study. Cell line generation was successful in one case. All families expressed gratitude both for the opportunity to participate and for the handling of the procedures. ConclusionsDespite the sensitive nature of these cases and the challenges presented by COVID-19 restrictions, clinicians should offer the option of a rapid autopsy to caregivers of pediatric patients based on the scientific need and the positive effect it has on grieving families. This article outlines the logistic efforts required for these donations to take place and provides a framework for providers to offer rapid autopsy as an option for families through this program.

OTHR-11. THE IMPORTANCE AND INFLUENCE OF PATIENT FAMILY INVOLVEMENT IN PEDIATRIC NEURO-ONCOLOGY RESEARCH

Abstract The involvement of patient families in clinical research in pediatric brain cancer is an underutilized tool that benefits all those involved. These families are ultimately responsible for making decisions on treatment, as well as end-of-life decisions such as post-mortem tissue donation. As demonstrated by organizations such as Gift from a Child, listening to family input on how to best have the conversation surrounding post-mortem tissue donation has led to an increase in total donations, as well as parents stating they are overwhelmingly happy with their decision to donate. Tissue donation is just one example of a large, complex hindrance in the field that had not previously been addressed successfully. Due to the rarity and devastating impact of pediatric brain tumors, the family community is tight-knit and passionate. There is incredibly impactful communication that happens between families influencing decision-making for treatment, funding, and advocacy. Partnering with families allows care teams and researchers both the ability to understand the decision-making process as well as educate and discuss with the families. Surveys of families overwhelmingly show satisfaction with the decision to donate post-mortem and dissatisfaction when they are deprived of the option. Frustration with the lack of treatment has led families to become more proactive in both their child’s treatment and future research. Increasingly, family advocates are becoming more sophisticated in their support of research opportunities. An example of this spearheading the creation of an open access, safe, anonymized data bank with clinical data paired to the patient’s tumor tissue. Family foundations are actively partnering with researchers and industry to make it happen. It is clear that this type of partnership is a strong force for change that benefits researchers, clinicians, and most importantly, patients and their families.

Intra-patient and inter-metastasis heterogeneity of HER2-low status in metastatic breast cancer

IntroductionAnti-HER2 antibody-drug conjugates (ADCs) have shown important efficacy in HER2-low metastatic breast cancer (mBC). Criteria for receiving ADCs are based on a single assay on the primary tumour or a small metastatic biopsy. We assessed the intra-patient inter-metastasis heterogeneity of HER2-low status in HER2-negative mBC. Patients and MethodsWe included samples of 10 patients (7 ER-positive and 3 ER-negative) donated in the context of our post-mortem tissue donation program UPTIDER. Excisional post-mortem biopsies of 257 metastases and 8 breast tumours underwent central HER2 immunohistochemistry (IHC), alongside 41 pre-mortem primary or metastatic samples. They were classified as HER2-zero, HER2-low (HER2-1+ or HER2-2+, in situ hybridisation [ISH] negative) or HER2-positive (HER2-3+ or HER2-2+, ISH-positive) following ASCO/CAP guidelines 2018. HER2-zero was further subdivided into HER2-undetected (no staining) and HER2-ultralow (faint staining in ≤10% of tumour cells). ResultsMedian post-mortem interval was 2.5 h. In 8/10 patients, HER2-low and HER2-zero metastases co-existed, with the proportion of HER2-low lesions ranging from 5% to 89%. A total of 32% of metastases currently classified as HER2-zero were HER2-ultralow. Intra-organ inter-metastasis heterogeneity of HER2-scores was observed in the liver in 3/6 patients. Patients with primary ER-positive disease had a higher proportion of HER2-low metastases as compared to ER-negative disease (46% versus 8%, respectively). At the metastasis level, higher percentages of ER-expressing cells were observed in HER2-low or -ultralow as compared to HER2-undetected metastases. ConclusionsImportant intra-patient inter-metastasis heterogeneity of HER2-low status exists. This questions the validity of HER2-low in its current form as a theranostic marker.

Abstract P5-05-06: ctDNA detection in seven different types of body liquids in patients with metastatic breast cancer

Abstract Background. Liquid biopsies represent a less invasive alternative to tissue biopsy to characterize and possibly monitor the disease in patients with metastatic breast cancer. So far, blood remains the most frequently investigated body liquid in this context and the investigations mainly focus on the detection, quantification and characterization of the circulating tumor DNA (ctDNA). However, since blood might not capture the full disease profile, other sources of body liquids may have the potential to complement the information obtained from blood. The aims of the present study are therefore to assess whether: (i) ctDNA can be detected in different types of body liquids, and, (ii) the levels of ctDNA in a given liquid are associated with metastases in specific organs. Patients and methods. Twelve patients from the post-mortem tissue donation program UPTIDER (NCT04531696) were included in this study. The receptor status of their primary tumor was: estrogen receptor negative, HER2 non-amplified (ER+/HER2-) (n=9), ER-/HER2- (n=2) and ER+/HER2+ (n=1). Median time between inclusion and death of the patient was 1.6 months (Interquartile range: [0.4-3.4]). Seven types of liquids were collected: blood, saliva, ascites, pleural fluid (PFL), cerebrospinal fluid (CSF), pericardial fluid and urine. Fluids were collected at study inclusion (blood, as well as saliva, urine, and ascites whenever possible) and at autopsy (except for saliva). In total, 108 liquid samples were collected and immediately centrifuged according to standard protocols. Cell free DNA (cfDNA) was extracted from the supernatant. All extracted cfDNA as well as germline DNA extracted from the 12 matched buffy coat samples underwent shallow whole genome sequencing. Log2 ratios were computed with CNVkit, and co-segmented per patient using the copynumber R package. Purity and ploidy were assessed by ABSOLUTE. Associations between organ involvement and ctDNA yield were assessed by Wilcoxon rank-sum tests. Samples at study inclusion and at autopsy were considered together unless otherwise specified. Results. At the sample level, ctDNA could be identified in 54% of the samples. At the patient level, the proportion of liquid types in which ctDNA was detected was highly variable (median: 58%, IQR: 34-77%, Table 1). CtDNA was detected in ascites of all patients when investigated, in 78% of PFL, 73% of CSF, 67% of blood and 37% of pericardial fluid. Only for one patient with invasive lobular carcinoma, ctDNA was detected in saliva and urine, the latter most likely explained by invasion of the bladder. Of note, in 4/12 patients ctDNA could not be identified in blood but was detected in at least one of the other fluids for 3 of these patients. At autopsy, ctDNA levels tended to be higher in PFL, ascites, and CSF in case of pleural, peritoneal, and central nervous system (CNS) metastases respectively, reaching statistical significance only for PFL. In CSF, two patients have CSF ctDNA detected with no documented involvement of the CNS. No brain autopsy was however performed for these patients. Conclusion. We have shown that ctDNA can be detected in all 7 different body liquids that were investigated in this study. The ctDNA levels in a given liquid can be associated with the presence of metastases in specific organs. Since ctDNA was not detected in 4 of our patients in blood but detectable for 3 of them in other liquids, the evaluation of additional sources of body fluids should be further investigated in patients with metastatic breast cancer. These results therefore open new avenues for the clinical monitoring and characterization of the disease. Table 1. Summary of ctDNA detection per liquid type at the patient level based on the 108 evaluated samples. Histo.= Histological, ILC= Invasive lobular carcinoma, NA= not available, nr= number, NST= non-special type Citation Format: François Richard, Tatjana Geukens, Maxim De Schepper, Amena Mahdami, Karen Van Baelen, Marion Maetens, Ha-Linh Nguyen, Anirudh Pabba, Sophia Leduc, Edoardo Isnaldi, Maysam Hajipirloo, Emily Vanden Berghe, Imane Bachir, Sigrid Hatse, Peter Vermeulen, Evy Vanderheyden, Bram Boeckx, Diether Lambrechts, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Hans Wildiers, Wouter Van Den Bogaert, Elia Biganzoli, Giuseppe Floris, Christine Desmedt. ctDNA detection in seven different types of body liquids in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-06.

Abstract P6-14-14: Advancing research on metastatic breast cancer: the UPTIDER post-mortem tissue donation program

Abstract Background. Research in metastatic breast cancer is hampered by limited sample availability. Post-mortem tissue donation programs can help to overcome this problem but are logistically challenging and have thus far mainly focused on histopathological and genomic research. We here present the UPTIDER program (NCT04531696), aimed at the multilevel characterization of advanced breast cancer and generation of tumour models. Patients and Methods. Patients with stage IV breast cancer receiving their last line(s) of treatment are eligible for participation. Blood, urine and saliva samples are collected upon inclusion. Upon death, a post-mortem MRI (when possible) followed by a rapid autopsy is performed. Liquid biopsies from all body fluids and tissue samples from all macroscopically identified metastatic sites are collected. Samples are processed as mirrored biopsies in different conditions, such as fresh frozen for omics analyses, formalin fixed paraffin-embedded for histopathology, and slowly frozen in freezing medium or fresh for generation of xenograft and organoid models. Results. Since approval by the local Ethical Committee in November 2020, 22 patients have been enrolled and 15 autopsies have been performed. Mean interval between death and start of autopsy was 3h (range 2-6h), mean duration of the autopsies was 6h (4-9h). A post-mortem MRI was performed in 6 patients. Peripheral blood, central blood and bone marrow were collected from all patients; urine, ascites, cerebrospinal, pericardial and pleural fluid all in more than 2/3 of patients. On average, 232 (range 90-406) tissue samples of which 164 (45-303) pathological from 42 (15 – 79) metastases were collected for each patient. Most often sampled metastatic sites were lymph nodes, liver, bones, pleura and peritoneum. Samples from the primary tumour could be retrieved from all patients, either during the autopsy (n=6) or from historical archives. In total, 133 tumour samples were sent to collaborating partners for patient-derived xenograft creation. Already some have been successfully established and stored, including models derived from a patient with invasive lobular carcinoma (ILC) and one with metaplastic squamous cell carcinoma. When correlating microscopic and macroscopic findings, patients could largely be divided into three main categories. Eleven patients presented with overt and extensive disease burden, often characterized by diffuse visceral, pleural, peritoneal, bone and lymph node involvement. Two patients, both with ILC, presented with underestimated yet extensive disease burden. While gross examination and cross sectioning of organs did not reveal clear involvement, microscopical invasion of stomach and liver, amongst others, was found. Lastly, limited disease burden was seen in two patients, both with leptomeningeal involvement. In those patients, massive tumoral infiltration in the subarachnoid space and along the blood-brain barrier was seen microscopically, with no grey matter invasion. Conclusion. We successfully launched a new and comprehensive post-mortem tissue donation program for patients with metastatic breast cancer, enrolling ~ 1 patient per month. Post-mortem tumour samples already resulted in successful establishment of some patient-derived xenografts. From a clinical point of view, vast underestimation of the disease extent on imaging during life as well as macroscopically during the autopsy was observed in some patients with metastatic ILC. For patients with leptomeningeal metastasis, we showed that the highly aggressive nature of their disease might be explained by extensive meningeal infiltration disrupting the blood-brain barrier. Further insights into disease progression and heterogeneity will be generated by the ongoing multi-omics analyses. Citation Format: Tatjana Geukens, Maxim De Schepper, Karen Van Baelen, François Richard, Marion Maetens, Amena Mahdami, Ha-Linh Nguyen, Edoardo Isnaldi, Anirudh Pabba, Sophia Leduc, Imane Bachir, Maysam Hajipirloo, Emily Vanden Berghe, Sigrid Hatse, Eleonora Leucci, Maria Francesca Baietti, Georgios Sflomos, Cathrin Brisken, Patrick Derksen, Colinda Scheele, Vincent Vandecaveye, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Elia Biganzoli, Hans Wildiers, Wouter Van Den Bogaert, Giuseppe Floris, Christine Desmedt. Advancing research on metastatic breast cancer: the UPTIDER post-mortem tissue donation program [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-14.

Abstract HER2-16: HER2-16 Inter-lesion heterogeneity of HER2-status in metastatic breast cancer: possible implications for treatment with anti-HER2 antibody-drug conjugates

Abstract Background. Trastuzumab deruxtecan (T-DXd) has shown promising activity in patients with HER2-low metastatic breast cancer. As the HER2-status can vary between the primary and its corresponding metastases, treatment decisions should ideally be based on HER2 assessment of a recent biopsy. However, limited data is available on intra-patient inter-metastatic heterogeneity in HER2-status, affecting representability of a single biopsy and potential therapeutic options and outcome. We therefore assessed HER2 status on multiple metastases from patients with primary ER-positive/HER2-non-amplified breast cancer in our prospective post-mortem tissue donation program UPTIDER (NCT04531696). Methods. Ninety-one metastatic samples retrieved during the autopsies of 6 patients (range: 13–16/patient) and their respective primary tumours were immunohistochemically (IHC) stained for HER2 (HercepTestTM, RTU, ISO-15189 accredited) in our institution. Consensus scoring was performed between two pathologists according to ASCO/CAP 2018 guidelines. The observers were blinded for patient ID. Reflex fluorescence in situ hybridization (FISH) testing was performed for samples with IHC score of 2+. HER2 status was categorized as HER2-zero (IHC 0), HER2-low (IHC 1+ or IHC 2+ with negative FISH), or HER2-positive (IHC 3+ or IHC 2+ with positive FISH). To assess stability of the performance of IHC scoring in the post-mortem setting, an additional 13 samples taken from 3 metastases at regular (every 1.5h) time intervals during the autopsy underwent HER2 IHC scoring. Results. Evaluation of HER2-status in the primary tumour showed 2 patients with HER2-zero disease and 4 with HER2-low disease. A discordance between HER2 status of the metastases and their respective primary was seen in all patients. Not a single lesion was found to be HER2-positive. For every patient, at least one HER2-low metastasis was observed, with the percentage being highly variable between patients and ranging between 7 and 100%. No association was observed between HER2 status and organ site: HER2-low as well as HER2-zero lesions were found in all organs evaluated in at least 4 patients (liver, bone, pleura, lymph nodes). For 5 patients, multiple lesions within the liver were evaluated: while HER2-zero versus HER2-low status was concordant in those lesions in 4 patients, a mix of HER2 IHC scores was seen in 3 of them. IHC scores were stable over time for tumour lesions assessed repeatedly. Discussion. Important inter-lesion heterogeneity in terms of HER2-low status was observed in patients with primary ER-positive/HER2-non-amplified breast cancer participating to our post-mortem tissue donation program. This observed heterogeneity is unlikely to be due to post-mortem changes in HER2 expression. HER2-low status was found in at least one distant lesion in all patients, complicating therapeutic decision-making based on a single biopsy. Of note, IHC 1+ and 2+ scores varied between metastases of each patient too, making assessment on a single biopsy less reliable for stratification in clinical trials. Further assessment on samples from UPTIDER-patients with ER-negative disease is currently ongoing and results will be available to be presented. Citation Format: Tatjana Geukens, Maxim De Schepper, François Richard, Marion Maetens, Karen Van Baelen, Amena Mahdami, Ha-Linh Nguyen, Edoardo Isnaldi, Sophia Leduc, Anirudh Pabba, Imane Bachir, Freya Mertens, Sara Vander Borght, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Hans Wildiers, Wouter Van Den Bogaert, Giuseppe Floris, Christine Desmedt. HER2-16 Inter-lesion heterogeneity of HER2-status in metastatic breast cancer: possible implications for treatment with anti-HER2 antibody-drug conjugates. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-16.

Abstract P6-14-12: Evaluation of changes in sequencing quality and transcriptomic profiles with increasing post-mortem interval: results from an optimization experiment within the UPTIDER tissue donation program

Abstract Background. Postmortem tissue donation programs can importantly enhance sample access for translational research on metastatic disease. However, this post-mortem setting poses logistical and technical challenges in terms of preserving nucleic acid quality and in particular RNA. Here we present the results of an experiment within our breast cancer tissue donation program UPTIDER (NCT04531696), aiming at assessing RNA degradation rates and expression profile changes in function of tissue type and sample-specific postmortem interval (ssPMI). Patients &amp; Methods. For 7 patients, bulk RNA sequencing was performed using the Lexogen protocol on fresh frozen samples from healthy or tumour tissues taken repeatedly (at 1.5h time intervals) during the autopsy. ssPMI was defined as the time between the death of the patient and the freezing of the sample. Quality threshold was set at 0.5 million (M) of assigned reads (AR). Other quality metrics included number of expressed genes, evolution of proliferation-, hypoxia-, stromal- and immune-related transcriptional signatures (PMID:18698033, 20087356) with increasing ssPMI. Associations between quality metrics and ssPMI were assessed by linear regressions for longitudinal data, with quality metrics as dependent variable, time as independent variable and accounting for the clustering of the data by patient and organ using the generalized estimating equation method. Three nested models - with constant, linear and non-linear relationship - were compared using ANOVA testing strategy. Non-linearity was rendered by a restrict cubic spline with three knots. All tests were performed by the Wald test on regression coefficients. Results. Ninety samples (67 healthy, 23 tumour) were analyzed. Median ssPMI was 7.50 hours (range: 3.07-11.12). Most (87%) samples passed quality thresholds, with median AR being 1.70M (interquartile range: [0.70M-3.57M]). No association was found between quality metrics and time in healthy samples. In tumor samples, regarding sequencing quality, negative associations with increasing time were found for AR and for number of expressed genes with an average decay of 242308 reads per hour (95 confidence interval (95CI): [94415.62-390200.50], p-value=.001) and 251 genes per hour (95CI: [17.30-485], p-value=.035), respectively. At the transcriptomic level, potential subtle changes were observed regarding immune (e.g. STAT1 signature: -0.02, 95CI: [-0.05;0.00]) and hypoxia-related signatures (e.g. PGAM1 signature: -0.03, 95CI [-0.05;-0.01]), while no effect of time was seen for the proliferation and stromal-related signatures. Sample size precluded organ specific analyses. Conclusion. A decrease in the number of AR and number of expressed genes with increasing ssPMI was found in tumor samples leading to subtle changes in few transcriptional programs. Healthy samples showed stable quality metrics over time possibly explained by a lower cell activity in healthy as compared to tumour cells. Knowledge derived from this study will be integrated in the upcoming transcriptomic analyses of the samples collected within UPTIDER. Citation Format: François Richard, Tatjana Geukens, Giuseppe Marano, Wouter Van Den Bogaert, Maxim De Schepper, Marion Maetens, Amena Mahdami, Karen Van Baelen, Ha-Linh Nguyen, Anirudh Pabba, Sophia Leduc, Edoardo Isnaldi, Maysam Hajipirloo, Imane Bachir, Evy Vanderheyden, Bram Boeckx, Diether Lambrechts, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Hans Wildiers, Elia Biganzoli, Giuseppe Floris, Christine Desmedt. Evaluation of changes in sequencing quality and transcriptomic profiles with increasing post-mortem interval: results from an optimization experiment within the UPTIDER tissue donation program [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-12.