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Purpose: Patients who inherit a pathogenic loss-of-function genetic variant involving one of the four succinate dehydrogenase (SDH) subunit genes have up to an 86% chance of developing one or more cancers by the age of 50. If tumors are identified and removed early in these high-risk patients, they have a higher potential for cure. Unfortunately, many alterations identified in these genes are variants of unknown significance (VUS), confounding the identification of high-risk patients. If we could identify misclassified SDH VUS as benign or pathogenic SDH mutations, we could better select patients for cancer screening procedures and remove tumors at earlier stages.Experimental Design: In this study, we combine data from clinical observations, a functional yeast model, and a computational model to determine the pathogenicity of 22 SDHA VUS. We gathered SDHA VUS from two primary sources: The OHSU Knight Diagnostics Laboratory and the literature. We used a yeast model to identify the functional effect of a VUS on mitochondrial function with a variety of biochemical assays. The computational model was used to visualize variants' effect on protein structure.Results: We were able to draw conclusions on functional effects of variants using our three-prong approach to understanding VUS. We determined that 16 (73%) of the alterations are actually pathogenic, causing loss of SDH function, and six (27%) have no effect upon SDH function.Conclusions: We thus report the reclassification of the majority of the VUS tested as pathogenic, and highlight the need for more thorough functional assessment of inherited SDH variants. Clin Cancer Res; 23(21); 6733–43. ©2017 AACR.

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TIFF file for supplemental figure 1

Video for Biochemical, molecular, and clinical characterization of SDHA VUS from Biochemical, Molecular, and Clinical Characterization of Succinate Dehydrogenase Subunit A Variants of Unknown Significance

Supplemental tables 1 and 2, supplemental figure legends, supplemental video legend

The goal of this study was to develop a high-plex assay to simultaneously quantitate 27 established and novel breast cancer (BC)-related, immune protein and phosphoprotein biomarkers using the GeoMx® Digital Spatial Profiler (DSP). The custom assay performance was compared to standard clinical BC biomarker assays (e.g.ER, PR, HER2, Ki67) across the spectrum of BC subtypes and in multiple laboratories. Commercially available antibodies to 27 BC-related protein biomarkers, including ER, PR, HER2, Ki-67, AR, immune-related (e.g. PD-L1) and several cell cycle/proliferation markers were oligonucleotide-tagged, and verified by immunohistochemistry for performance against untagged antibodies. The tagged antibodies were combined with 3 isotype controls and 2 housekeeping proteins into a custom BC high-plex assay for DSP. Confirmation of target specificity was done on a custom tissue microarray (TMA) (Run control) composed of (un)treated cancer cell lines and normal tissues. For clinical BC samples, each biopsy had 4x600 µm regions of interest segmented into pan-Cytokeratin+ tumoral epithelium and pan-Cytokeratin- adjacent stroma segments. With targeted UV light, oligonucleotides were collected from each segment sequentially and quantitated with nCounter. Raw counts were geomean normalized for analysis. Intra-site and inter-site assay reproducibility was assessed with the TMA and serial FFPE sections from BC biopsies. Quantitation of proteins showed high reproducibility within sites (e.g. Run 1 v. Run 2, p>0.05 for all protein targets) and the high-plex custom assay showed good concordance between 3 participating laboratories. GeoMx DSP-derived protein data correlated well with orthogonal methodologies performed on a sample subset including immunohistochemistry, reverse phase protein array, DNA and RNA-sequencing. For example, DSP protein counts for p53 protein were higher in tumors with TP53 point mutation than those with truncating TP53 alterations. Expanded sample testing is underway with 120 well-characterized breast cancer biopsies of various histologies, immune subtypes (e.g. TIL scores) and clinical biomarker phenotypes (e.g. ER+/HER2-/Ki-67 high BC; ER+/HER2-/Ki-67 low BC; HER2+ BC; ER-/PR-/HER2- BC).Our preliminary data demonstrate that this custom high-plex BC assay can quantitate protein biomarkers across a wide dynamic range with high intra-lab and inter-lab reproducibility. The assay requirement of a single 5-µm tissue section facilitates complex biomarker profiling in biopsies with limited material. The custom assay alone or in combination with other targeted DSP protein modules can simultaneously interrogate the biomarker profile, immune-based and other drug target profile, and immune microenvironment of BC specimens, providing a novel approach for actionable tumor subtyping. For research use only. Not for use in diagnostic procedures. Citation Format: Christopher Corless, Amber Bridgeman, Jinho Lee, Gary Geiss, Sarah Warren, Sarah Church, Joseph M. Beechem, E Aubrey Thompson, Jodi Carter. Development of a custom high-plex GeoMx digital spatial profiler breast cancer protein biomarker assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3484.

Mechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities.

Comprehensive characterization of an individual’s cancer using multi-omic analyses and an expanding list of targeted therapies is providing an opportunity to uncover therapeutic vulnerabilities and rationally target multiple driving alterations in the tumor. To leverage this opportunity, a multi-disciplinary team is required to unravel the complexity of tumor behavior and genomic variant information, integrate data from multi-omic assays, and exploit potential synergies from combination therapy while avoiding toxicity, all of which occurs within the context of the patient’s clinical history and comorbidities. We report the preliminary experience of OHSU Knight Cancer Institute’s SMMART (Serial Measurements of Molecular and Architectural Responses to Therapy) Treatments Program Multi-omics Tumor Board (MTB). The role of the SMMART MTB, to date, has been to develop and optimize procedures in a feasibility protocol for future application in the clinical setting where we will provide personalized combinatorial treatment suggestions. Here we report our preliminary experience over the last 15 months with multi-disciplinary tumor boards for metastatic breast cancer. A deep analysis of data, in many cases from serial biopsies, enabled identification of personalized, multi-targeted therapy. Clinical and laboratory data are explored individually and collectively by oncologists, oncology pharmacists, molecular pathologists, cancer biologists, and computational biologists. Data from CLIA-certified analytics include, but are not limited to, a 124 gene targeted panel, whole exome sequencing, MSI, gene-fusion, RNAseq, and clinical IHC assays that provide status information about receptors (ER/PR/AR/HER2), proliferation rate, and tumor immunogenicity along with reflexive assays for expression of relevant proteins such as p16, RB, and PTEN. A customized LabKey® system allows for visual display of a patient’s clinical timeline with information about diagnoses, treatments, biopsies events, radiographic changes, and blood biomarkers; thereby providing MTB participants with a holistic view of the patient’s case summary. Our experience with several example breast cancer case scenarios identified therapeutic vulnerabilities that would not have been considered by a standard clinical tumor board with genomic data alone. Recent cases have included observations such as HER2 status switching, a rare pathogenic germline mutation, high PD-L1 expression, and gained expression of the androgen receptor. These cases further emphasize the value and importance of MTB discussions to oncologists for interpreting and analyzing complex multi-omic data and uncovering therapeutic options for patients. This experience will be utilized in the future for employing SMMART MTBs in a clinical setting as a platform to triage patients into different multi-targeted combinatorial treatment options. Citation Format: Jamie M. Keck, Swapnil Parmar, Ben Kong, Zahi Mitri, Christopher Corless, Annette Kolodzie, Allison Creason, Jeremy Goecks, Patrick Leyshock, Kiara Siex, Brett E. Johnson, Janice Patterson, Laura Heiser, Anastasiya Olson, Matt Viehdorfer, Georgia Mayfield, Jennifer Laverdure, Joe W. Gray, Gordon B. Mills, Raymond C. Bergan. Leveraging multi-omics tumor boards for precision medicine in the OHSU SMMART Treatments Program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4929.

Rapid advances in molecular diagnostics have led to a growing need for sophisticated and adaptive biospecimen collection methodologies that employ careful acquisition, preservation, and allocation of limited biopsy tissue and blood specimens. To facilitate the comprehensive characterization of serial biospecimens from patients participating in real-time adaptive treatment precision medicine clinical trials, we designed a multi-omic and multi-imaging analytical platform and sought to maximize the quality and suitability of tissue for each downstream analytic. We optimized this biospecimen management system over 38 research-protocol driven biopsies on 33 patients with late stage metastatic breast, prostate, and pancreatic cancer. Importantly, no biopsy-related adverse events occurred. All tissue samples and derivatives therefrom were barcoded and tracked using a custom database built on the LabVantage platform. Here, we report successful strategies for coordination between medical and surgical oncology, interventional radiology, pathology, and clinical and research laboratories. We developed a biopsy prioritization schema that considers patient safety, site selection, procedure type, and amount of tissue required. We evaluated specimens from a variety of biopsy procedures, including radiologically-guided needle cores, as well as laparoscopic, video-assisted thoracoscopic, and excisional biopsies. Radiologically-guided biopsies using 18 gauge needles yielded a median of 5 (1 cm) cores with 70% tumor cellularity, and was the best substrate to rapidly allocate known quantities of tissue into a variety of preservation formats. We also examined enhanced biospecimen preservation methods that improved performance of downstream analytics. For example, optimization of the formalin fixation process included transferring tissue from bedside to formalin within 2 minutes, maintaining low temperature throughout fixation, and standardizing the time spent in formalin. These efforts resulted in recovery of higher quality RNA from FFPE blocks, less tissue required for RNA sequencing, and opened the door to immunofluorescent staining of phosphoproteomic markers. Our protocol uses several preservation methods, including: FFPE for immunohistochemical and cyclic immunofluorescent staining as well as targeted panel, whole exome, and transcriptomic sequencing; OCT freezing for multiple single cell sequencing methods; flash freezing for proteomic profiling; disaggregation of live cells for 2D and 3D culturing; and a specialized fixative for electron microscopy. In conclusion, we demonstrate the feasibility of collecting high quality biospecimens for a variety of downstream analytical platforms with diverse sample requirements. These methods can be used to comprehensively characterize limited amounts of valuable tissue in molecularly driven clinical trials. Citation Format: Brett E. Johnson, Swapnil Parmar, Kiara Siex, Anastasiya Olson, Jennifer Laverdure, Jamie Keck, Annette Kolodzie, Alexander R. Guimaraes, Christopher Corless, Joe Gray, Gordon Mills, Raymond Bergan. Biospecimen management for the serial measurement of molecular and architectural responses to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 480.

Gastric metastasis from breast cancer is uncommon and typically occurs in patients with disseminated disease. The vast majority of patients with gastric lesions have a known preexisting diagnosis of breast cancer. In contrast, we describe a case in which a minimal breast cancer was found to be the primary tumor during the workup of a patient first diagnosed with carcinoma of unknown primary and subsequently presumed to have metastatic gastric cancer. Our case illustrates that a diagnosis of breast cancer metastatic to the stomach may require a high index of suspicion, as well as a meticulous breast workup. It also emphasizes that even tiny breast cancers have a small but real risk of metastatic spread. Determination of the correct primary source in these cases may not be only an academic exercise, since the treatment and prognosis of metastatic breast cancer (especially receptor positive) and metastatic gastric cancer are different.

The expression of ASPP2 (53BP2L), a proapoptotic member of a family of p53-binding proteins, is frequently suppressed in many human cancers. Accumulating evidence suggests that ASPP2 inhibits tumor growth; however, the mechanisms by which ASPP2 suppresses tumor formation remain to be clarified. To study this, we targeted the ASPP2 allele in a mouse by replacing exons 10–17 with a neoR gene. ASPP2 −/− mice were not viable because of an early embryonic lethal event. Although ASPP2 +/− mice appeared developmentally normal, they displayed an increased incidence of a variety of spontaneous tumors as they aged. Moreover, γ-irradiated 6-week-old ASPP2 +/− mice developed an increased incidence of high-grade T cell lymphomas of thymic origin compared with ASPP2 +/+ mice. Primary thymocytes derived from ASPP2 +/− mice exhibited an attenuated apoptotic response to γ-irradiation compared with ASPP2 +/+ thymocytes. Additionally, ASPP2 +/− primary mouse embryonic fibroblasts demonstrated a defective G 0 /G 1 cell cycle checkpoint after γ-irradiation. Our results demonstrate that ASPP2 is a haploinsufficient tumor suppressor and, importantly, open new avenues for investigation into the mechanisms by which disruption of ASPP2 pathways could play a role in tumorigenesis and response to therapy.

Background: Most GISTs express mutant KIT or PDGFRA oncoproteins, which are targets of tyrosine kinase inhibitors (TKIs), such as front-line imatinib (IM) or second-line sunitinib (SU). GIST clinical resistance to IM or SU is commonly associated with the acquisition of heterogeneous secondary mutations in the KIT/PDGFRA ATP-binding pocket (ABP) or activation loop (AL), which maintain the constitutively activated state of these kinases. We therefore asked whether the heterogeneous IM-resistant KIT oncoproteins in GIST are uniformly HSP90 clients, and whether they can be inhibited by STA-9090, a synthetic small molecule HSP90 inhibitor that is structurally unrelated to the first-generation natural product-derived ansamycin HSP90 inhibitor 17-AAG. Methods: KIT and PDGFRA were genotyped in up to 15 metastases from each of 10 patients whose metastatic GIST had progressed after IM therapy. Kinase mutants were biochemically profiled for IM and STA-9090 sensitivity in: 1. Ba/F3 cells transformed by mutant KIT constructs; 2. GIST cell lines (IM-sensitive and IM-resistant); and 3. a novel assay measuring inhibition of kinase phosphorylation after drug treatment in GIST48B (KIT-negative) GIST cells transfected with mutant KIT constructs. Drug effects on proliferation, apoptosis and cell cycle were evaluated in five GIST cell lines, including a KIT-dependent GIST subline (GIST882B) that is resistant to 17-AAG. Results: As many as 8 different secondary KIT IM-resistance mutations (both ABP and AL) were detected in individual patients whose GISTs progressed after IM therapy. All mutations were sensitive to STA-9090. STA-9090 was 5-15 fold more potent than 17-AAG against these IM-resistant KIT secondary mutations, and was at least as effective against the primary + secondary (IM-resistant) mutations in combination, as compared to the primary IM-sensitive mutation alone (Table). STA-9090 also potently inhibited the 17-AAG resistant GIST882B cell line. Conclusions: STA-9090 was more potent than 17-AAG against a panel of KIT mutations found in TKI-resistant GISTs. STA-9090 potency was undiminished against combination activating KIT mutations (including secondary kinase-domain resistance mutations), as typically occur in IM-resistant GIST clones. Based on these results, we hypothesize that STA-9090 might have broad clinical activity against IM-resistant GIST. Table: IC50 (nM) for GIST viability: HSP90-inhibition vs. imatinib GIST Line STA-9090 17-AAG Imatinib GIST882: KIT Ex 13 40 200 300 GIST882B 35 >1000 300 GIST430: KIT Ex 11 + V654A (ABP) 20 300 >1000 Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B184.

Introduction: p53 is an important tumor suppressor, and loss of p53 pathway function is a common event in human cancer. ASPP2 is a damage-inducible p53 binding protein that enhances apoptosis, at least in part, by selective stimulation of p53 pro-apoptotic target genes. Low ASPP2 expression occurs in many human tumors and as we have previously demonstrated, correlates with poor clinical outcome in patients with B-cell lymphomas. However, the mechanisms by which ASPP2 suppresses tumor formation remain unknown. Methods: To rigorously study ASPP2 in vivo function, we targeted the ASPP2 allele in a mouse model by homologous recombination by replacing exons 10–17 with a neoR gene. Aging mice were followed for spontaneous tumor formation. Additionally, six week old mice were irradiated at different doses and followed for tumor development. To explore the functional consequences of low ASPP2 expression, ASPP2+/− and ASPP2+/+ thymocytes were subjected ex vivo to 5Gy ionizing irradiation and apoptosis was assessed by Annexin-V/PI staining and flow cytometry. We also irradiated ASPP2+/+ and ASPP2+/−MEFs (mouse embryonic fibroblasts) with 5Gy and performed cell cycle analysis. Furthermore, we conducted global gene expression profiling between the unirradiated and irradiated ASPP2+/+ and ASPP2+/−MEFs using an Affymetrix GeneChip® Mouse Gene 1.0 ST Array. Moreover, phosphoproteomic analysis was performed on unirradiated and irradiated ASPP2+/+ and ASPP2+/−MEFs using 2-dimensional gel electrophoresis, fluorescent phosphoprotein dye Pro-Q Diamond staining, and liquid chromatography tandem mass spectroscopy. Results: We were unable to generate ASPP2−/− mice due to an early embryonic lethal defect. However, ASPP2+/− mice appear developmentally normal and reproduce. We observed an increased formation of spontaneous tumors in aging ASPP2+/− mice compared to ASPP2 +/+ mice (43% vs. 22%, at 115 weeks, p=0.011). Additionally we were able to show that after ionizing radiation, ASPP2+/− mice develop high-grade lymphomas in a dose-dependent manner at a significantly higher incidence at 50 weeks compared to ASPP2+/+ mice (p = 0.024 and p = 0.045, 6 Gy and 10.5 Gy respectively). Immunophenotyping demonstrated that these were high-grade T-cell lymphomas of thymic origin. Since ASPP2 is damage-inducible and promotes apoptosis, we wished to determine the extent to which reduction in ASPP2 expression attenuated the cellular damage response. We therefore gamma-irradiated ex vivo ASPP2+/+ and ASPP2+/−thymocytes in short-term culture and found a two-fold reduction in apoptosis in ASPP2+/− thymocytes compared to ASPP2+/+ thymocytes. Additionally, after 5 Gy gamma-irradiation, ASPP2+/− MEFs exhibited an attenuated G0/G1 checkpoint compared to ASPP2+/+ MEFs. Preliminary analysis of global gene expression in ASPP2+/+ and ASPP2+/− MEFs shows specific differences in gene expression patterns after damage. Likewise, preliminary analysis of phosphoproteomics between ASPP2+/+ and ASPP2+/− MEFs after damage, demonstrate differences in the phosphophorylation status of 170 proteins. Conclusions: ASPP2 is a haploinsufficient tumor suppressor, and reduction in ASPP2 expression attenuates both cell cycle checkpoints and apoptosis-induction after damage. These results suggest that reduction of ASPP2 levels modulate the cellular damage response, possibly at transcriptional as well as post-transcriptional levels, and provide a functional explanation for the increased tumor incidence in ASPP2+/− mice---since attenuated damage-response thresholds would lead to an impaired ability to eliminate cells that have accumulated tumorigenic mutations. Our study provides important insights into the p53-ASPP2 axis, and opens new avenues for investigation into its role in tumorigenesis and response to therapy.

Apoptosis Stimulating Protein of p53 (ASPP2) binds the tumor suppressor protein p53 and promotes damage-induced apoptosis in part through stimulation of p53 apoptotic function. We have previously demonstrated that low ASPP2 levels correlate with poor clinical outcome in patients with diffuse large B-cell lymphoma treated with anthracycline-based chemotherapy, which is consistent with its role as a tumor suppressor. We targeted the ASPP2 allele in a mouse model by homologous recombination using a knockout vector that replaced exons 10–17 with a neoR gene. Although ASPP2−/− is an early embryonic lethal event, ASPP2+/– mice appeared developmentally normal and reproduce. Importantly, ASPP2+/– mice developed tumors spontaneously at a higher frequency than sibling ASPP2+/+ controls. We now show that after ionizing irradiation, ASPP2+/– mice develop high grade lymphatic malignancies compared to ASPP2+/+ mice. ASPP2+/+ and +/– mice were irradiated, with either 6 Gy or 10,5 Gy, 8 weeks after birth. Following irradiation, mice were monitored weekly by blood smears and flow cytometry if suspicious cells were observed. Tumor formation was detected as early as 17 weeks post-irradiation. In the 10,5 Gy group, ASPP2+/– mice developed thymic lymphomas (7/31), while no tumors were detected in ASPP2+/+ mice (0/14). Tumor-free survival was statistically significant (p= 0,045, log-rank test). In the 6 Gy dose group 9 out of 28 of the ASPP2+/– mice developed high-grade thymic lymphomas compared to 0/20 ASPP2+/+ mice. Tumor-free survival was statistically significant (p=0,024, log-rank test). Immunophenotyping and flow cytometry on mice with circulating tumor cells from whole unfractionated gated viable bone marrow (Bm) cells revealed that the Bm was typically almost completely replaced by CD4neg, CD5+, CD8+ leukemic/lymphoma cells that are negative for B-cell (B220) and myeloid markers (Mac-1/Gr-1). Preliminary analysis demonstrated that MEFs (mouse embryonic fibroblasts) from ASPP2+/– mice treated with doxorubicin, serum starvation or irradiation had an attenuated G1/G0 checkpoint arrest. Ongoing experiments are being conducted in thymocytes prepared from mice of different ASPP2 and p53 genotypes. Our data demonstrates that ASPP2 plays an important role in suppressing the development of lymphomas after irradiation in a mouse model. Consequently, disruption of the p53-ASPP2 axis may be important in lymphomagenesis and response to therapy. Furthermore, our data provides a rationale for the future study of ASPP2 expression in patients during the formation of secondary hematologic malignancies after therapies with DNA damaging agents.

ASPP2 interacts with the tumor suppressor protein p53 and promotes damage-induced apoptosis in part through stimulation of p53-mediated apoptosis. We have previously demonstrated that low ASPP2 levels correlate with poor clinical outcome in patients with diffuse large B-cell lymphoma treated with anthracycline-based chemotherapy. Moreover, reduced ASPP2 expression has been demonstrated in other tumor types. These findings led us to hypothesize that ASPP2 may function as a tumor suppressor. To further explore this, we targeted the ASPP2 allele in a mouse by homologous recombination using a knockout vector that replaced exons 10–17 with a neoR gene. Two separate ES clones were used for blastocyst injections to generate several chimeras that were used to generate ASPP2 heterozygous mice. ASPP2+/− mice appear developmentally normal and reproduce. However ASPP2−/− mice could not be generated. Genotype analysis as early as Ed 6.5 did not detect ASPP2−/− embryos---which implies an early embryonic lethal defect in the homozygote. ASPP2+/− (n=135) and ASPP2+/+ (n=63) sibling mice were observed for spontaneous tumor formation. Overall median tumor-free survival was 117 weeks in the ASPP2+/− mice verses 125 weeks in the ASPP2+/+mice (p = 0.035 log-rank test). Overall tumor incidence (at 115 weeks) for ASPP2+/− and ASPP2+/+ mice was 43% and 22%, respectively. The incidence of tumor types, from all tumors detected, was similar between ASPP2+/− and ASPP2+/+ mice: 34% versus 33% (lymphoma), 18% versus 14% (sarcoma), and 47% versus 52% (carcinoma), respectively. Compound p53+/−;ASPP2+/− mice did not exhibit accelerated tumor formation relative to p53+/−;ASPP2+/+ mice. Additionally, a tet-Myc:ASPP2+/− lymphoma mouse model did not exhibit accelerated lymphomagenesis. However, preliminary data suggests that ASPP2+/− mice may have an increased incidence of irradiation-induced leukemia/lymphoma when compared to ASPP2+/+ mice, and confirmatory studies are ongoing. In response to ionizing radiation, doxorubicin, or serum-starvation, preliminary analysis reveals a G0/G1 checkpoint defect in ASPP2+/− MEFs compared to ASPP2+/+ MEFs. Our results provide in vivo evidence that ASPP2 can function as a tumor suppressor. Further studies are underway to determine the mechanism of this observation.