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Background: Previous studies have shown that rapid autopsies (RA) provide a unique opportunity for tissue collection from patients who succumb to the disease. Because cancer patients are unable to donate their organs to other people, this program provides the patient an opportunity to leave a legacy by donating their body to research. These donations are vital for advancing breast cancer research. The UPMC/Pitt RA group revamped an existing program in 2018 through the formation of a larger multidisciplinary team that includes breast cancer laboratory and clinical researchers, pathologists, nurses, bioinformaticians, and tissue bankers. Because recruitment to the RA program was a challenge, we recently added patient advocates to the team to provide their essential perspective, and a dedicated research coordinator who serves as an ambassador for the program. Methods: Autopsy is performed by the Autopsy and Forensic Pathology Center of Excellence/Decedent Affairs Service of UPMC. Samples are banked in the Pitt Biospecimen Core (PBC), in addition to immediate processing including preparing of samples for sequencing and growing of organoids in the laboratory. Immunohistochemical (IHC) analysis is performed by UPMC/Magee Pathology. Results: The research coordinator quickly became an integral part of the program and closely interacts with care providers, patients and their families, pathologists on call, and manages interactions with transport services. Five breast cancer advocates have been instrumental in advising on additional changes to the program. The advocates attend regular team meetings and have formulated patient considerations for the the RA program, including appropriate and sensitive recruitment of patients, the role of physicians in decision making by the patient, registration for more than one RA program, potential issues with transporting a body across state lines and more. The advocates also developed the name for the program - “Hope for OTHERS” with Others standing for “Our Tissue Helping Enhance Research & Science”. As of June 2022, the team has completed 26 autopsies, and an additional 20 patients have consented to the program. The completed autopsies include patients with breast tumors representing different molecular and histological classes, ethnicities, and genders. The average disease-free survival and overall survival of patients that underwent autopsy was 81.6 and 127.8 months, respectively. Most patients passed outside the hospital (86%), with 62% in home hospice and 24% in inpatient hospice. Average time between death and start and end of autopsy was 4.56 hrs and 7.09 hours, respectively. The most common metastatic sites from which specimens are collected are liver, lung and lymph nodes. Per patient we collect on average specimens from 4 different organs. In addition to the metastatic lesions, we have access to primary tumor tissue and liquid biopsies obtained during the breast cancer disease progression for 44% and 73% of the patients, respectively. For a subset of the patients, tissue has been grown as patient-derived organoids or xenograft models. Preliminary IHC and sequencing analysis has provided insight into inter- and intra-patient and intra-tumor heterogeneity. Further molecular studies are ongoing. Conclusion In summary, over the last 5+ years, we have established a successful post-mortem tissue collection program, by addressing a series of barriers through the formation and work of a multi-disciplinary well-coordinated team. We are currently expanding our omics studies using state-of-the-art technologies to improve understanding how intra- and inter-tumor heterogeneity play a role in the clinical course of advanced breast cancer, to increase diversity of the patients enrolled in the RA program, and to support the successful implementation of other RA programs nationwide and worldwide. Citation Format: Steffi Oesterreich, Lori Miller, Margaret Q. Rosenzweig, Tanner L. Bartholow, Megan Yates, Ashuvinee Elangovan, Laura Savariau, Allison N. Casey, Nolan Priedigkeit, Kai Ding, Abdalla Wedn, Jie Bin Liu, Daniel D. Brown, Tara Hyder, Geoffrey Pecar, Neil Carleton, Humberto Trejo Bittar, Daniel Geisler, Oscar Lopez-Nunez, Amanda M. Clark, Alan Wells, Partha Roy, Shannon Puhalla, Naomi Howard, Christine Needles, Susan Trent, Stephanie Walker, Christine Hodgdon, Rohit Bhargava, Jennifer M. Atkinson, Adrian V. Lee. Hope for OTHERS – An organ donation program for metastatic breast cancer research [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 P6-14-02.

Supplementary Figure Legend

Esophageal adenocarcinoma is an increasingly common disease with a dismal 5-year survival rate of 10% to 15%. In the first systematic evaluation of the PD-1 pathway in esophageal adenocarcinoma, we identify expression of PD-L2 in cancer cells in 51.7% of esophageal adenocarcinomas. Epithelial PD-L1 was expressed on only 2% of cases, although PD-L1+ immune cells were observed in 18% of esophageal adenocarcinomas. We also evaluated expression in the precursor lesion of esophageal adenocarcinoma, Barrett's esophagus, which emerges following gastric reflux–induced esophageal inflammation, and found PD-L2 expression in Barrett's esophagus but not in non–Barrett's esophagus esophagitis. Because the progression from squamous esophagitis to Barrett's esophagus is accompanied by a transition from a TH1 to TH2 immune response, we hypothesized that the TH2 cytokines IL4/IL13 could contribute to PD-L2 induction. We confirmed that these cytokines can augment PD-L2 expression in esophageal adenocarcinoma cell lines. These results suggest that the inflammatory environment in Barrett's esophagus and esophageal adenocarcinoma may contribute to the expression of PD-L2. Furthermore, the potential for PD-1 receptor blockade to be effective in esophageal adenocarcinomas with epithelial PD-L2 or immune cell PD-L1 expression should be evaluated in clinical trials. Cancer Immunol Res; 3(10); 1123–9. ©2015 AACR.

PD-L2 expression in patient derived xenografts.

Supplementary Methods

qRT-PCR primer sequences

Purpose:Lung squamous cell carcinoma (LSCC) is a deadly disease for which only a subset of patients responds to immune checkpoint blockade (ICB) therapy. Therefore, preclinical mouse models that recapitulate the complex genetic profile found in patients are urgently needed.Experimental Design:We used CRISPR genome editing to delete multiple tumor suppressors in lung organoids derived from Cre-dependent SOX2 knock-in mice. We investigated both the therapeutic efficacy and immunologic effects accompanying combination PD-1 blockade and WEE1 inhibition in both mouse models and LSCC patient-derived cell lines.Results:We show that multiplex gene editing of mouse lung organoids using the CRISPR–Cas9 system allows for efficient and rapid means to generate LSCCs that closely mimic the human disease at the genomic and phenotypic level. Using this genetically defined mouse model and three-dimensional tumoroid culture system, we show that WEE1 inhibition induces DNA damage that primes the endogenous type I IFN and antigen presentation system in primary LSCC tumor cells. These events promote cytotoxic T-cell–mediated clearance of tumor cells and reduce the accumulation of tumor-infiltrating neutrophils. Beneficial immunologic features of WEE1 inhibition are further enhanced by the addition of anti–PD-1 therapy.Conclusions:We developed a mouse model system to investigate a novel combinatory approach that illuminates a clinical path hypothesis for combining ICB with DNA damage–inducing therapies in the treatment of LSCC.

Figures S1-9

Somatic alterations in cancer genes are being detected in normal and premalignant tissue, placing greater emphasis on gene-environment interactions that enable disease phenotypes. By combining early genetic alterations with disease-relevant exposures, we developed an integrative mouse model to study gastric premalignancy. Deletion of Trp53 in gastric cells confers a selective advantage and promotes the development of dysplasia in the setting of dietary carcinogens. Organoid derivation from dysplastic lesions facilitated genomic, transcriptional, and functional evaluation of gastric premalignancy. Cell cycle regulators, most notably Cdkn2a, were upregulated by p53 inactivation in gastric premalignancy, serving as a barrier to disease progression. Co-deletion of Cdkn2a and Trp53 in dysplastic gastric organoids promoted cancer phenotypes but also induced replication stress, exposing a susceptibility to DNA damage response pathway inhibitors. These findings demonstrate the utility of mouse models that integrate genomic alterations with relevant exposures and highlight the importance of gene-environment interactions in shaping the premalignant state.

Mutations in CHMP2B, an ESCRT‐III (endosomal sorting complexes required for transport) component, are associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Neurodegenerative disorders including FTD are also associated with a disruption in circadian rhythms, but the mechanism underlying this defect is not well understood. Here, we ectopically expressed the human CHMP2B variant associated with FTD (CHMP2BIntron5) in flies using the GMR‐GAL4 driver (GMR>CHMP2BIntron5) and analyzed their circadian rhythms at behavioral, cellular, and biochemical level. In GMR>CHMP2BIntron5 flies, we observed disrupted eclosion rhythms, shortened free‐running circadian locomotor period, and reduced levels of timeless (tim) mRNA—a circadian pacemaker gene. We also observed that the GMR‐GAL4 driver, primarily known for its expression in the retina, drives expression in a subset of tim expressing neurons in the optic lobe of the brain. The patterning of these GMR‐ and tim‐positive neurons in the optic lobe, which appears distinct from the putative clusters of circadian pacemaker neurons in the fly brain, was disrupted in GMR>CHMP2BIntron5 flies. These results demonstrate that CHMP2BIntron5 can disrupt the normal function of the circadian clock in Drosophila.

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.

Purpose: Lung squamous cell carcinoma (LSCC) is a deadly disease for which only a subset of patients responds to immune checkpoint blockade (ICB) therapy. Therefore, preclinical mouse models that recapitulate the complex genetic profile found in patients are urgently needed. Experimental Design: We used CRISPR genome editing to delete multiple tumor suppressors in lung organoids derived from Cre-dependent SOX2 knock-in mice. We investigated both the therapeutic efficacy and immunologic effects accompanying combination PD-1 blockade and WEE1 inhibition in both mouse models and LSCC patient-derived cell lines. Results: We show that multiplex gene editing of mouse lung organoids using the CRISPR–Cas9 system allows for efficient and rapid means to generate LSCCs that closely mimic the human disease at the genomic and phenotypic level. Using this genetically defined mouse model and three-dimensional tumoroid culture system, we show that WEE1 inhibition induces DNA damage that primes the endogenous type I IFN and antigen presentation system in primary LSCC tumor cells. These events promote cytotoxic T-cell–mediated clearance of tumor cells and reduce the accumulation of tumor-infiltrating neutrophils. Beneficial immunologic features of WEE1 inhibition are further enhanced by the addition of anti–PD-1 therapy. Conclusions: We developed a mouse model system to investigate a novel combinatory approach that illuminates a clinical path hypothesis for combining ICB with DNA damage–inducing therapies in the treatment of LSCC.

Summary paragraph Synthetic lethality, an interaction whereby the co-occurrence of two or more genetic events lead to cell death but one event alone does not, can be exploited to develop novel cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets since many cancers exhibit an impaired DNA repair pathway, which can lead these cells to become dependent on specific repair proteins2. The success of poly (ADP-ribose) polymerase 1 (PARP-1) inhibitors in homologous recombination-deficient cancers highlights the potential of this approach in clinical oncology3,4. Hypothesizing that other DNA repair defects would give rise to alternative synthetic lethal relationships, we asked if there are specific dependencies in cancers with microsatellite instability (MSI), which results from impaired DNA mismatch repair (MMR). Here we analyzed data from large-scale CRISPR/Cas9 knockout and RNA interference (RNAi) silencing screens and found that the RecQ DNA helicase WRN was selectively essential in MSI cell lines, yet dispensable in microsatellite stable (MSS) cell lines. WRN depletion induced double-strand DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models specifically required the helicase activity, but not the exonuclease activity of WRN. These findings expose WRN as a synthetic lethal vulnerability and promising drug target in MSI cancers.

The role of KRAS, when activated through canonical mutations, has been well established in cancer1. Here we explore a secondary means of KRAS activation in cancer: focal high-level amplification of the KRAS gene in the absence of coding mutations. These amplifications occur most commonly in esophageal, gastric and ovarian adenocarcinomas2-4. KRAS-amplified gastric cancer models show marked overexpression of the KRAS protein and are insensitive to MAPK blockade owing to their capacity to adaptively respond by rapidly increasing KRAS-GTP levels. Here we demonstrate that inhibition of the guanine-exchange factors SOS1 and SOS2 or the protein tyrosine phosphatase SHP2 can attenuate this adaptive process and that targeting these factors, both genetically and pharmacologically, can enhance the sensitivity of KRAS-amplified models to MEK inhibition in both in vitro and in vivo settings. These data demonstrate the relevance of copy-number amplification as a mechanism of KRAS activation, and uncover the therapeutic potential for targeting of these tumors through combined SHP2 and MEK inhibition.

Esophageal adenocarcinoma is an increasingly common disease with a dismal 5-year survival rate of 10% to 15%. In the first systematic evaluation of the PD-1 pathway in esophageal adenocarcinoma, we identify expression of PD-L2 in cancer cells in 51.7% of esophageal adenocarcinomas. Epithelial PD-L1 was expressed on only 2% of cases, although PD-L1+ immune cells were observed in 18% of esophageal adenocarcinomas. We also evaluated expression in the precursor lesion of esophageal adenocarcinoma, Barrett's esophagus, which emerges following gastric reflux–induced esophageal inflammation, and found PD-L2 expression in Barrett's esophagus but not in non–Barrett's esophagus esophagitis. Because the progression from squamous esophagitis to Barrett's esophagus is accompanied by a transition from a TH1 to TH2 immune response, we hypothesized that the TH2 cytokines IL4/IL13 could contribute to PD-L2 induction. We confirmed that these cytokines can augment PD-L2 expression in esophageal adenocarcinoma cell lines. These results suggest that the inflammatory environment in Barrett's esophagus and esophageal adenocarcinoma may contribute to the expression of PD-L2. Furthermore, the potential for PD-1 receptor blockade to be effective in esophageal adenocarcinomas with epithelial PD-L2 or immune cell PD-L1 expression should be evaluated in clinical trials. Cancer Immunol Res; 3(10); 1123–9. ©2015 AACR.

Oesophageal squamous cell carcinoma is a deadly disease where systemic therapy has relied upon empiric chemotherapy despite the presence of genomic alterations pointing to candidate therapeutic targets, including recurrent amplification of the gene encoding receptor tyrosine kinase epidermal growth factor receptor (EGFR). Here, we demonstrate that EGFR-targeting small-molecule inhibitors have efficacy in EGFR-amplified oesophageal squamous cell carcinoma (ESCC), but may become quickly ineffective. Resistance can occur following the emergence of epithelial–mesenchymal transition and by reactivation of the mitogen-activated protein kinase (MAPK) pathway following EGFR blockade. We demonstrate that blockade of this rebound activation with MEK (mitogen-activated protein kinase kinase) inhibition enhances EGFR inhibitor-induced apoptosis and cell cycle arrest, and delays resistance to EGFR monotherapy. Furthermore, genomic profiling shows that cell cycle regulators are altered in the majority of EGFR-amplified tumours and a combination of cyclin-dependent kinase 4/6 (CDK4/6) and EGFR inhibitors prevents the emergence of resistance in vitro and in vivo. These data suggest that upfront combination strategies targeting EGFR amplification, guided by adaptive pathway reactivation or by co-occurring genomic alterations, should be tested clinically., Oesophageal squamous cell carcinoma often develop resistance to EGFR tyrosine kinase inhibitors. Here, the authors demonstrate that inhibition of cell cycle regulators CDK4/6 or MAPK blockade enhances the efficacy of EGFR inhibitors for these tumours in mice.

In the Supplementary Information originally published with this article, a lane was missing in the β-actin blot in Supplementary Fig. 2. The lane has been added. The error has been corrected in the Supplementary Information associated with this article.