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AKT S473 distribution based on ER expression measured by IHC for 31 lesions included of the discovery set. ER was measured in a binary scale (Panel A) and in continuous scale using the H-Score (Panel B).

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K–AKT–mTOR pathway is associated with specific sites of breast cancer metastasis.Experimental Design: Next-generation sequencing–based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K–AKT–mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K–AKT–mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions.Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined.Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K–AKT–mTOR signaling network. Clin Cancer Res; 23(16); 4919–28. ©2017 AACR.

Supplementary material legend

Concordance between PIK3CA, AKT, and PTEN mutation status and AKT (S473) and p70S6 (T389) activation.

Purpose:PNOC003 is a multicenter precision medicine trial for children and young adults with newly diagnosed diffuse intrinsic pontine glioma (DIPG).Patients and Methods:Patients (3–25 years) were enrolled on the basis of imaging consistent with DIPG. Biopsy tissue was collected for whole-exome and mRNA sequencing. After radiotherapy (RT), patients were assigned up to four FDA-approved drugs based on molecular tumor board recommendations. H3K27M-mutant circulating tumor DNA (ctDNA) was longitudinally measured. Tumor tissue and matched primary cell lines were characterized using whole-genome sequencing and DNA methylation profiling. When applicable, results were verified in an independent cohort from the Children's Brain Tumor Network (CBTN).Results:Of 38 patients enrolled, 28 patients (median 6 years, 10 females) were reviewed by the molecular tumor board. Of those, 19 followed treatment recommendations. Median overall survival (OS) was 13.1 months [95% confidence interval (CI), 11.2–18.4] with no difference between patients who followed recommendations and those who did not. H3K27M-mutant ctDNA was detected at baseline in 60% of cases tested and associated with response to RT and survival. Eleven cell lines were established, showing 100% fidelity of key somatic driver gene alterations in the primary tumor. In H3K27-altered DIPGs, TP53 mutations were associated with worse OS (TP53mut 11.1 mo; 95% CI, 8.7–14; TP53wt 13.3 mo; 95% CI, 11.8–NA; P = 3.4e−2), genome instability (P = 3.1e−3), and RT resistance (P = 6.4e−4). The CBTN cohort confirmed an association between TP53 mutation status, genome instability, and clinical outcome.Conclusions:Upfront treatment-naïve biopsy provides insight into clinically relevant molecular alterations and prognostic biomarkers for H3K27-altered DIPGs.

Supplementary Figure from Upfront Biology-Guided Therapy in Diffuse Intrinsic Pontine Glioma: Therapeutic, Molecular, and Biomarker Outcomes from PNOC003

Stereotactic body radiation therapy (SBRT) has been shown to be safe and effective for delaying systemic treatment change among patients with metastatic renal cell carcinoma (mRCC). In this study, we sought to assess the genomic signatures of patients with mRCC who underwent SBRT for oligoprogression. A total of 30 patients with oligoprogressive disease were identified, the majority of whom had clear cell renal cell carcinoma (83.3%) and were receiving first-line treatment (53.3%). Genomic and transcriptomic sequencing were available in 20 and 16 patients, respectively. Duration of systemic treatment (DOT) was categorized as that prior (DOT[P]) and subsequent (DOT[S]) to radiation treatment. The median DOT(P) and DOT(S) were 15.1 and 18.3 mo, respectively, with a median DOT(S)/DOT(P) ratio of 1.4. Patients who had a DOT(S)/DOT(P) ratio of ≥1 had increased expression in pathways related to cell proliferation and development. In contrast, among patients with a ratio of ≤1, the reactive oxygen species pathway was enriched. This study highlights the potential role of genomics and transcriptomics to refine radiation treatment selection in patients with mRCC. PATIENT SUMMARY: In this study, we looked at mutations and genomic expressions among kidney cancer patients who responded better to stereotactic body radiotherapy. We found that enriched expression of certain pathways might play a role in response to radiotherapy.

Human DNA sequencing protocols have revolutionized human biology, biomedical science, and clinical practice, but still have very important limitations. One limitation is that most protocols do not separate or assemble (i.e., "phase") the nucleotide content of each of the maternally and paternally derived chromosomal homologs making up the 22 autosomal pairs and the chromosomal pair making up the pseudo-autosomal region of the sex chromosomes. This has led to a dearth of studies and a consequent underappreciation of many phenomena of fundamental importance to basic and clinical genomic science. We discuss a few protocols for obtaining phase information as well as their limitations, including those that could be used in tumor phasing settings. We then describe a number of biological and clinical phenomena that require phase information. These include phenomena that require precise knowledge of the nucleotide sequence in a chromosomal segment from germline or somatic cells, such as DNA binding events, and insight into unique cis vs. trans-acting functionally impactful variant combinations-for example, variants implicated in a phenotype governed by compound heterozygosity. In addition, we also comment on the need for reliable and consensus-based diploid-context computational workflows for variant identification as well as the need for laboratory-based functional verification strategies for validating cis vs. trans effects of variant combinations. We also briefly describe available resources, example studies, as well as areas of further research, and ultimately argue that the science behind the study of human diploidy, referred to as "diplomics," which will be enabled by nucleotide-level resolution of phased genomes, is a logical next step in the analysis of human genome biology.

Background: Children with relapsed CNS tumors, neuroblastoma, sarcomas, and other rare solid tumors face poor outcomes. Here we describe a study to determine the feasibility of leveraging genomic profiling results within a molecular tumor board (MTB) to make real-time treatment decisions for children with relapsed/refractory solid tumors. Methods: Subjects were divided to 3 strata: Strata 1: Relapsed/refractory neuroblastoma, Strata 2: Relapsed/refractory CNS tumors, and Strata 3: Relapsed/refractory rare solid tumors. Samples were sent for tumor/normal whole exome (WES) and tumor whole transcriptome sequencing, and the genomic data were used in a MTB to make real-time treatment decisions. The MTB recommended plan allowed for a combination of up to 4 agents. Feasibility was measured by time to completion of genomic sequencing, MTB review and initiation of treatment. Response was assessed after every 2 cycles using Response Evaluation Criteria in Solid Tumors (RECIST). Patient benefit was calculated by the sum of the CR, PR, SD, and NED subjects divided by the sum of CR, PR, SD, NED, and PD subjects. Grade 3 and higher related and unexpected adverse events (AEs) were tabulated for safety evaluation. Results: 144 eligible subjects were enrolled with 144 evaluable for safety and 124 evaluable for response. Tumor types included neuroblastoma (n=31), CNS tumors (n=41), and rare tumors (n=72). Sarcomas (n=40) were the most common tumor type in the rare tumor stratum. The average time from biopsy to completion of DNA/RNA sequencing was 10 days (range 2-31 days); to completed analysis and drug prediction report, 17 days (8-41); 23 days (10-66) to MTB decisions; and 38 days (18-146) to initiation of the 4-drug combination agreed upon by the MTB. Treatments were selected on DNA and RNA findings in 19.5% of cases and in 80.5% on RNA alone. Patient benefit was exhibited in 70% of all subjects, 85% of neuroblastoma subjects, 73% of CNS tumor subjects, and 62% of rare tumor subjects. AEs occurred in Conclusions: It has been well-established that comprehensive genomic sequencing is the future of precision medicine. Here, we have demonstrated the feasibility, efficacy, and safety of a comprehensive sequencing model to guide targeted therapy for patients with any relapsed/refractory solid malignancies. Targeted therapy was well tolerated, and the response benefit rate of 70% in these heavily pretreated populations suggests that this treatment could be an effective option for relapsed and refractory pediatric cancers. Citation Format: Giselle L. Saulner Sholler, Jacqueline Kraveka, Genevieve Bergendahl, Elizabeth Lewis, William Ferguson, Abhinav Nagulapally, Karl Dykema, Valerie Brown, Michael Isakoff, Joseph Junewick, Deanna Mitchell, Don Eslin, Virginia Harrod, William Roberts, Javier Oesterheld, Randy Wada, Jawhar Rawwas, Devang Pastakia, Kevin Ginn, Raya Saab, Kevin Bielamowicz, Jason Glover, Eugenia Chang, Gina Hanna, Daniel Enriquez, Tyler Izatt, Rebecca Halperin, Sara Byron, William Hendricks, Jeffrey Trent. Molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancers: a Beat Childhood Cancer Research Consortium trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT089.

Background: Cancer therapy has witnessed great progress over the last 50 years with advancements in diagnostic tools to enable more precise treatment. With the development of next-generation sequencing, the field of precision oncology has been expanded to tailor treatments to a patient's mutational profile, however, there is a need for additional information such as RNA Differential Expression (DE) to match more patients to potentially beneficial drugs. We have previously described our PATRIOT platform, which integrates RNA DE analysis, NGS, and high throughput patient-derived organoid models to identify new therapeutic targets. In our previous study, we tested the PATRIOT platform on a patient with predicted KRAS and IDHI inactivation resulting in inhibition of DNA damage repair pathway. Patient-derived tumor organoids showed a significant response to the suggested treatment, Olaparib, supporting the reliability of PATRIOT prediction. To further validate this platform, we interrogated the predictive efficacy of PATRIOT in cohorts of patients characterized by CDKN2A downregulation or MET overexpression. Methods: Following PATRIOT analysis, patients with predicted CDKN2A downregulation or MET overexpression consisting primarily of colon and pancreatic cancer were grouped into two cohorts. Patient-derived tumor organoids were tested for sensitivity to drugs matched to the predicted targets as single agent or in combination, using a 4-fold dilution followed by 72h viability assay. Drug cytotoxicity and Bliss Synergy were analyzed using GraphPad Prism and Combenefit. All screenings were subjected to stringent quality control measures and normalized against untreated organoids. Results: Our analysis identified patients characterized by MET overexpression suggesting crizotinib, a small-molecule tyrosine kinase inhibitor of the c-MET receptor, as sequencing-matched therapy. Tumor organoids showed sensitivity to crizotinib (IC50 5.74 M+/- 3.2), validating our initial predictions. In a separate cohort, tumor organoids generated from patients characterized by downregulation of CDKN2A were treated with Palbociclib, a CDK4/6 inhibitor. Tumor organoids showed limited response to treatment (IC50 57.75 M+/- 25.1), which has been reported in an earlier clinical trial (NCT01740427). However, recent studies have shown that Pablociclib treatment enhances cisplatin cytotoxic effect in several solid tumor models through the CDK4/6-cyclin D1-RB-E2F pathway (NCT02897375). When tumor organoids were co-treated with Palbociclib and Cisplatin, significant synergy was observed (moderate-strong). In summary, PATRIOT analysis provided bioinformatic predictions of pathway based therapeutic options that were validated using tumor organoid platforms. Citation Format: Anna Larson, Taylor Bargenquast, Sara Byron, Anish Raju, Avijit Podder, Shelby Rheinschmidt, Tithi Ghosh Halder, Samuel Sampson, Sydney Adamson, Serina Ng, Kate Gutowsky, Alexis Weston, Trason Thode, Justin Moser, Erkut Borazanci, Mohan Kaadige, Nicholas Schork, Raffaella Soldi, Sunil Sharma. A novel PAThway based RNA and DNA Integration with Tumor Organoid Testing (PATRIOTTM) platform predicts therapeutic options in cohorts with MET overexpression and CDKN2A downregulation in patients with colon and pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1069.

Purpose: PNOC003 is a multicenter precision medicine trial for children and young adults with newly diagnosed diffuse intrinsic pontine glioma (DIPG). Patients and Methods: Patients (3–25 years) were enrolled on the basis of imaging consistent with DIPG. Biopsy tissue was collected for whole-exome and mRNA sequencing. After radiotherapy (RT), patients were assigned up to four FDA-approved drugs based on molecular tumor board recommendations. H3K27M-mutant circulating tumor DNA (ctDNA) was longitudinally measured. Tumor tissue and matched primary cell lines were characterized using whole-genome sequencing and DNA methylation profiling. When applicable, results were verified in an independent cohort from the Children's Brain Tumor Network (CBTN). Results: Of 38 patients enrolled, 28 patients (median 6 years, 10 females) were reviewed by the molecular tumor board. Of those, 19 followed treatment recommendations. Median overall survival (OS) was 13.1 months [95% confidence interval (CI), 11.2–18.4] with no difference between patients who followed recommendations and those who did not. H3K27M-mutant ctDNA was detected at baseline in 60% of cases tested and associated with response to RT and survival. Eleven cell lines were established, showing 100% fidelity of key somatic driver gene alterations in the primary tumor. In H3K27-altered DIPGs, TP53 mutations were associated with worse OS (TP53mut 11.1 mo; 95% CI, 8.7–14; TP53wt 13.3 mo; 95% CI, 11.8–NA; P = 3.4e−2), genome instability (P = 3.1e−3), and RT resistance (P = 6.4e−4). The CBTN cohort confirmed an association between TP53 mutation status, genome instability, and clinical outcome. Conclusions: Upfront treatment-naïve biopsy provides insight into clinically relevant molecular alterations and prognostic biomarkers for H3K27-altered DIPGs.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a fatal brain tumor. Herein, we report on novel prognostic and predictive genomic biomarkers identified in PNOC003, a multi-center precision medicine trial for children and young adults diagnosed with DIPG. METHODS: Patients aged 3-25 years were enrolled on PNOC003 based on radiographic diagnosis of DIPG. Pre-treatment tumor biopsies were analyzed using tumor-normal whole-exome sequencing and mRNA-tumor sequencing to determine biology-informed, multi-agent therapy following radiation therapy (RT). Whole-genome sequencing was performed as an exploratory study aim. Genomic biomarkers were investigated to identify predictors of RT response and overall survival (OS) in patients with confirmed H3K27M-altered DIPG. Prognostic biomarkers were verified in a retrospective, H3K27M-altered diffuse midline glioma cohort (n=22) from the Children’s Brain Tumor Network (CBTN). RESULTS: Thirty patients enrolled on PNOC003 met molecular criteria for H3K27M-altered DIPG. TP53 was the most frequently altered driver gene (73%). Somatic alterations in PTEN>TP53>PDGFRA were independently associated with OS (P

Glioblastoma (GB) is the most aggressive adult brain tumor with a devastating median survival time of about fourteen months post-surgery and standard of care therapy with radiation and temozolomide. The low incidence of GB is a dis-incentive to develop novel therapies. To overcome that obstacle, we investigated the efficacy of repurposing four FDA approved drugs known to cross the BBB, minocycline, propranolol, chlorpromazine, and metformin, to inhibit signaling and metabolism in GB cells. Minocycline is a tetracycline class broad spectrum antibiotic commonly used to treat severe acne and other skin infections. Propranolol is a beta blocker type heart medication primarily used to treat high blood pressure and irregular heartbeat. Chlorpromazine is a phenothiazine antipsychotic usually used for schizophrenia. Metformin is the most widely used first-line oral treatment for type-2 diabetes. Based on a literature survey, minocycline is expected to prevent the phosphorylation of STAT3, a transcription factor downstream of EGFR; propranolol is expected to disrupt EGFR trafficking; chlorpromazine is expected to target the mTOR/AKT pathway; metformin is believed to exploit vulnerabilities in cancer cell metabolism. Efficacy of minocycline in inhibiting EGFR-driven STAT3 activation was investigated using western blot analysis. Our results demonstrate that Minocycline effectively inhibits activation of EGFR-driven STAT3 in U373 glioma cells at 100μM. The ability of chlorpromazine to inhibit the mTOR/AKT pathway was similarly tested via western blot, which showed inhibition of phosphorylated Akt and S6 at 10μM. Efficacy of propranolol in perturbing EGFR trafficking was evaluated using flow cytometry and immunofluorescence, which depicted altered membrane-associated EGFR abundance. Finally, concentration-dependent inhibition of colony formation was tested for all four drugs. Propranolol and minocycline showed potential stimulatory effects at 10μM, but all drugs inhibited cell growth at 50μM and higher. Efficacy of these drugs in treatment of GB is being further evaluated using in vitro neurosphere cultures from patients identified as having the cellular vulnerabilities potentially targeted by these drugs. Successful completion of this project will lead to in vivo efficacy testing of these four drugs in orthotopic GB PDX models. Citation Format: Tristan Neal, Nanyun Tang, George Reid, Sara Byron, Harshil Dhruv, Michael Berens. Evaluating efficacy of repurposed drugs in treatment of glioblastoma [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 2189.