Search

Immunotherapy (IO) agents have led to significant improvements in patient outcomes across many tumor types. There have been great efforts to introduce immune checkpoint inhibitors into the treatment paradigm of esophagogastric cancers as well. A number of randomized phase III trials, which will be reviewed here, established the role of these agents in both early-stage and advanced-stage disease. Adjuvant nivolumab is US Food and Drug Administration–approved after neoadjuvant chemoradiation and resection of esophageal and gastroesophageal junction cancers on the basis of the phase III CheckMate 577 trial. In the advanced setting, patients with programmed death receptor ligand-1–positive tumors should be recommended IO in combination with chemotherapy in the first-line setting on the basis of the results from KEYNOTE 590, CheckMate 649, and CheckMate 648. Across trials, chemotherapy continues to play a critical role in the first-line setting and should be offered to all patients who are eligible for systemic therapy, including those with biomarker select tumors. In the later lines of treatment, IO has modest activity, and prior studies have grown largely irrelevant because of the enrollment of IO-naive patients. Similar to other disease types, patients with microsatellite unstable (microsatellite instability high) tumors represent a unique cohort that is more sensitive to IO. However, there are no randomized studies evaluating how best to apply IO in early or advanced stages specifically for the treatment of patients with microsatellite instability high upper GI tumors. Questions remain how to best select patients who benefit from IO treatments, how to augment IO activity in programmed death receptor ligand-1–negative tumors, and how to incorporate IO in late-line settings or for recurrent disease that has been treated with IO-containing regimens during early stages.

SummaryFibrolamellar carcinoma (FLC) is a rare and lethal cancer that afflicts young individuals. The tumor arises in the background of a healthy liver, and patients typically present with advanced cancer at the time of diagnosis. Unfortunately, for these patients with advanced or recurrent cancer, no proven systemic therapies exist resulting in only 30-45% of patients surviving to 5 years. Investigations into the molecular underpinning of FLC have revealed a unique gene fusion between heat shock protein 40 (DNAJB1) and the catalytic subunit alpha of protein kinase A (PRKACA), leading to the formation of an oncoprotein (DNAJ-PKAc) that retains kinase activity and is a proven tumor-causing event in FLC. To uncover potential therapeutic targets, we engineered an FLC cell line by introducing theDNAJB1-PRKACAoncogene rearrangement into human hepatocellular cells using CRISPR/Cas9. We identified aberrant cell cycle progression, and follow-up molecular analysis revealed evidence of enhanced cyclin dependent kinase 7 (CDK7) activation in theDNAJB1-PRKACAexpressing FLC cells. These findings were confirmed in human samples of FLC. In turn, targeting CDK7 with selective inhibitors demonstrated efficacy in several patient-derived models of FLC, with minimal toxicity to normal liver. Collectively, this work uncovers a novel candidate therapeutic vulnerability in FLC.

Supplementary Figure S1 presents the viability of SW48 and SW48PK cells after treatment with BEZ235 and TAK-228 at the 100-400nM concentrations.

PIK3CA mutations are common in clinical molecular profiling, yet an effective means to target these cancers has yet to be developed. MTORC1 inhibitors are often used off-label for patients with PIK3CA mutant cancers with only limited data to support this approach. Here we describe a cohort of patients treated with cancers possessing mutations activating the PI3K signaling cascade with minimal benefit to treatment with the MTORC1 inhibitor everolimus. Previously, we demonstrated that dual PI3K/mTOR inhibition could decrease proliferation, induce differentiation, and result in a treatment response in APC and PIK3CA mutant colorectal cancer. However, reactivation of AKT was identified, indicating that the majority of the benefit may be secondary to MTORC1/2 inhibition. TAK-228, an MTORC1/2 inhibitor, was compared with dual PI3K/mTOR inhibition using BEZ235 in murine colorectal cancer spheroids. A reduction in spheroid size was observed with TAK-228 and BEZ235 (−13% and −14%, respectively) compared with an increase of >200% in control (P < 0.001). These spheroids were resistant to MTORC1 inhibition. In transgenic mice possessing Pik3ca and Apc mutations, BEZ235 and TAK-228 resulted in a median reduction in colon tumor size of 19% and 20%, respectively, with control tumors having a median increase of 18% (P = 0.02 and 0.004, respectively). This response correlated with a decrease in the phosphorylation of 4EBP1 and RPS6. MTORC1/2 inhibition is sufficient to overcome resistance to everolimus and induce a treatment response in PIK3CA mutant colorectal cancers and deserves investigation in clinical trials and in future combination regimens.

Background: Therapeutic screening Pancreatic Ductal Adenocarcinoma (PDAC) relies on well-level assessment for high throughput response evaluation. Patient-derived cancer organoids (PCOs) model subclonal populations, however the significance of resistant populations is uncertain when characterized with well-level response. Using a high-throughput screening assay, we present an automated alignment algorithm to characterize populations of organoid growth as compared to validated well-level therapeutic response assays. Methods: High content imaging was performed in low volume (10uL), 96-well angiogenesis plating format (Ibidi, Inc) at 4x objective with 5-frame Z-stack (600um) with brightfield imaging captured at 0h and 72h. Images underwent processing using Gen5 suite (Biotek, Inc) including Z-projection to render organoids into single two-dimensional planes. Baseline objects were defined between 50µm and 750µm, and filtration based on circularity defined as >0.4. Object alignment was performed based on root-mean-square-deviation (RMSD) between all combinations of objects to optimize match determination. This analysis was performed in drug screen of 80 independent agents in early clinical trials in combination with CDK7 inhibitor, SY-5609. Well level viability was performed using standardized 3D CellTiterGlo (CTG, Promega Inc.) (33% v/v). Response was assessed using descriptive statistics, effect size (Glass’s Δ), and Therapeutic Sensitivity Index (TSI) defined as the weighted average between elements with growth from media control versus treated population. Results: Z projection of 600um in Low-volume plating (10µL) of matrix suspension yielded 1.68 organoids per µL relative to the traditional hanging drop design (50µL) 0.75 organoids per µL (p5% reduction in of matched objects, while a decrease in circularity by 0.1 yielded a Conclusion: We provide a method for high fidelity alignment of PCOs in low-volume format for matrix-based screening applications. These techniques can be adapted to existing staining protocols to characterize subclonal response in the context of both molecular heterogeneity and clinical outcomes. Citation Format: Ethan Samuel Lin, Md Shahadat Hossan, Austin Stram, Eleanor E. Riedl, Luke J. Koeppel, Jaimie M. Warner, Jeremy D. Kratz. Automated organoid alignment for clonal response characterization in pancreatic ductal adenocarcinoma. [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 5327.

Patient-derived cancer organoids (PDCOs) are organotypic 3D cultures grown from patient tumor samples. PDCOs provide an exciting opportunity to study drug response and heterogeneity within and between patients. This research can guide new drug development and inform clinical treatment planning. We review technologies to assess PDCO drug response and heterogeneity, discuss best practices for clinically relevant drug screens, and assert the importance of quantifying single-cell and organoid heterogeneity to characterize response. Autofluorescence imaging of PDCO growth and metabolic activity is highlighted as a compelling method to monitor single-cell and single-organoid response robustly and reproducibly. We also speculate on the future of PDCOs in clinical practice and drug discovery.Future development will require standardization of assessment methods for both morphology and function in PDCOs, increased throughput for new drug development, prospective validation with patient outcomes, and robust classification algorithms.

Representative models are needed to screen new therapies for patients with cancer. Cancer organoids are a leap forward as a culture model that faithfully represents the disease. Mouse-derived cancer organoids (MDCOs) are becoming increasingly popular, however there has yet to be a standardized method to assess therapeutic response and identify subpopulation heterogeneity. There are multiple factors unique to organoid culture that could affect how therapeutic response and MDCO heterogeneity are assessed. Here we describe an analysis of nearly 3500 individual MDCOs where individual organoid morphologic tracking was performed. Change in MDCO diameter was assessed in the presence of control media or targeted therapies. Individual organoid tracking was identified to be more sensitive to treatment response than well-level assessment. The impact of different generations of mice of the same genotype, different regions of the colon, and organoid specific characteristics including baseline size, passage number, plating density, and location within the matrix were examined. Only the starting size of the MDCO altered the subsequent growth. These results were corroborated using ~ 1700 patient-derived cancer organoids (PDCOs) isolated from 19 patients. Here we establish organoid culture parameters for individual organoid morphologic tracking to determine therapeutic response and growth/response heterogeneity for translational studies.

Tumor heterogeneity is predicted to confer inferior clinical outcomes, however modeling heterogeneity in a manner that still represents the tumor of origin remains a formidable challenge. Sequencing technologies are limited in their ability to identify rare subclonal populations and predict response to the multitude of available treatments for patients. Patient-derived organotypic cultures have significantly improved the modeling of cancer biology by faithfully representing the molecular features of primary malignant tissues. Patient-derived cancer organoid (PCO) cultures contain numerous individual organoids with the potential to recapitulate heterogeneity, though PCOs are most commonly studied in bulk ignoring any diversity in the molecular profile or treatment response. Here we demonstrate the advantage of evaluating individual PCOs in conjunction with cellular level optical metabolic imaging to characterize the largely ignored heterogeneity within these cultures to predict clinical therapeutic response, identify subclonal populations, and determine patient specific mechanisms of resistance.

Representative models are needed to screen new therapies for patients with cancer. Cancer organoids are a leap forward as a culture model that faithfully represents the disease. Mouse-derived cancer organoids (MDCOs) are becoming increasingly popular, however there has yet to be a standardized method to assess therapeutic response and identify subpopulation heterogeneity. There are multiple factors unique to organoid culture that could affect how therapeutic response and MDCO heterogeneity are assessed. Here we describe an analysis of nearly 3,500 individual MDCOs where individual organoid morphologic tracking was performed. Change in MDCO diameter was assessed in the presence of control media or targeted therapies. Individual organoid tracking was identified to be more sensitive to treatment response than well-level assessment. The impact of different generations of mice of the same genotype, different regions of the colon, and organoid specific characteristics including baseline size, passage number, plating density, and location within the matrix were examined. Only the starting size of the MDCO altered the subsequent growth. Here we establish organoid culture parameters for individual organoid morphologic tracking to determine therapeutic response and growth/response heterogeneity for translational studies using murine colorectal cancer organoids.

Background: Therapeutic development in pancreatic ductal adenocarcinoma (PDAC) requires accurate models for assessing response. Patient-derived cancer organoids (PCOs) have been shown to faithfully recapitulate genomic features of advanced PDAC. Mechanisms of subclonal resistance or intratumoral heterogeneity cannot be characterized using well-level viability assays. Methods: A high-content imaging platform was adapted in low volume, 96-well format for using Cytation 5 (Biotek). PDAC PCOs were plated from organoid suspension in 1:1 reduced growth factor Cultrex plating 10uL per well. After 24h, physiologic chemotherapy was applied to match pharmacologic exposure of FOLFIRINOX (FFX) and gemcitabine/nab-paclitaxel (g/n-pac). Initial response was assessed by normalized change in PCO area at 72h. Subsequent viability staining was performed for single organoid level response for apoptosis (caspase-3/7-FITC) and necrosis (TO-PRO-3). Analyses were automated in Gen5.12 (Biotek) with selection for elements using circularity (>0.4) and alignment by location within 50% which represented 17.8% of g/npac and 18.5% of FFX treated PCOs. Individual organoids with mean area staining positive for apoptosis was increased in g/npac +67.3% (GD=2.96) and FFX +65.1 (GD=2.84) versus background control +12.8%. Individual organoids with mean with area staining positive for necrosis was increased in g/npac +47.3% (GD=1.88) as compared to both FFX +34.9 (GD=1.20, p Citation Format: Md Shahadat Hossan, Ethan S. Lin, Ellie E. Riedl, Austin Stram, Jamie M. Warner, Jeremy D. Kratz. Multiplexed therapeutic sensitivity of patient derived pancreatic cancer organoids after neoadjuvant chemotherapy by high content imaging [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A073.

4034 Background: Neoadjuvant CRT followed by surgery is the standard of care for patients (pts) with stage II/III E/GEJ cancer, yet recurrence rates remain high. Immunotherapy has demonstrated activity in advanced E/GEJ cancer and was recently approved for adjuvant treatment of early stage disease. This trial evaluated the safety and efficacy of avelumab with perioperative CRT in resectable E/GEJ cancer. Methods: This is a two part phase I/II trial. Phase I was a safety run-in of 6 pts. Phase II planned to enroll an additional 18 pts in an expansion cohort. Pts with E/GEJ adenocarcinoma or squamous cell cancer received CRT (41.4 Gy in 23 fractions) with weekly carboplatin and paclitaxel. Three doses of avelumab (10 mg/kg IV, q14 days) were administered starting on day 29 of treatment, to coincide with the last chemotherapy dose. Surgery was performed 8-10 weeks after CRT completion. Pts received 6 doses of avelumab after resection (10 mg/kg IV, q14 days). The primary endpoint of the Phase 1 was safety and tolerability. The primary endpoint of the Phase II was pathologic complete response (pathCR) rate, assessing patients from the safety run in and expansion cohorts. Results: Between 6/2018 and 10/2021, 22 pts (20 males, median age 64) enrolled in the study. Enrollment was stopped after 16 patients in the expansion cohort due to accrual delays and changes in standard treatment. 19/22 patients (86%) had adenocarcinoma; 15/22 (68%) had lymph node positive disease at diagnosis. 19 pts underwent successful resection while on study. 3 pts went off study before resection due to grade 3 avelumab-related infusion reaction (1), patient preference (1), and non-adherence (1). There were no unexpected surgical complications. 4 pts (21%) had R1 resection with 3/4 having positive radial margin and 1/4 positive proximal margin. At resection, 5 pts (26%) had pathCR (3/16 adenocarcinomas, 2/3 squamous cell), 4 ypT1N0 disease, and 14/19 were ypN0. 42% had tumor regression score of 0 or 1. The combination of CRT and avelumab had an acceptable toxicity profile. No grade ≥3 immune-related AEs were observed. Immune-related hypothyroiditis was seen in 2 patients (grade 2). Three patients had grade 2 infusion-related reaction, but were able to continue with treatment. 21/22 pts had reversible grade ≥3 lymphopenia; 13/22 grade ≥ 3 wbc decrease; 6/22 grade 3 neutropenia. As of data cutoff on 2/1/2022, 1 patient remains on study treatment, 15 in follow up, 5 expired, 1 off study. Additional efficacy data is being collected. Correlative studies are ongoing. Conclusions: Perioperative CRT with avelumab is well tolerated with no unexpected toxicities. Neoadjuvant chemoradiation with immunotherapy is a promising approach for patients with E/GEJ tumors. Additional safety, efficacy and correlative analysis from this study will be presented at the meeting. Clinical trial information: NCT03490292.

Though many advancements in personalized medicine have been made, better methods are still needed to predict treatment benefit for patients with colorectal cancer. Patient-derived cancer organoids (PDCOs) are a major advance towards true personalization of treatment strategies. A growing body of literature is demonstrating the feasibility of PDCOs as an accurate and high-throughput preclinical tool for patient treatment selection. Many studies demonstrate that these cultures are readily generated and represent the tumors they were derived from phenotypically and based on their mutation profile. This includes maintenance of the driver muatations giving the cancer cells a selective growth advantage, and also heterogeneity, including molecular and metabolic heterogeneity. Additionally, PDCOs are now being utilized to develop patient biospecimen repositories, perform high to moderate-throughput drug screening, and to potentially predict treatment response for individual patients that are undergoing anti-cancer treatments. In order to develop PDCOs as a true clinical tool, further studies are required to determine the reproducibility and accuracy of these models to predict patient response.

186 Background: Distinct molecular subgroups of colorectal cancer (CRC) have been afforded with use of next-generation sequencing (NGS) as standard in clinical practice for advanced disease. We have previously demonstrated that disease bulk predicts clinical resistance to EGFR inhibition in RAS/RAF-wildtype (WT) CRC. We hypothesized bulky disease would predict advanced subclonal heterogeneity by circulating tumor DNA (ctDNA) in RAS/RAFWT CRC. Methods: Following IRB-approval, a retrospective review of molecular profiles in advanced CRC (n = 965) were compiled from the Veteran Administration’s (VA) National Precision Oncology Program (NPOP) and University of Wisconsin Precision Medicine Molecular Tumor Board (MTB). Disease bulk was defined as the longest diameter of metastatic disease or short axis for advanced lymphadenopathy. Molecular profiling was performed using commercially available platforms including Strata Oncology (MTB) and FoundationOne (NPOP). Bulky was compared as categorical (> 35 cm) and continuous variable against the count of pathologic variants. Results: The population was largely representative of advanced CRC with alterations in TP53 (80.5%), KRAS (44.8%), PIK3CA (22.0%) and BRAF (12.8%). Veterans had increased frequency of alterations in PIK3CA (22.7% v. 13.0%, p < 0.02) and BRAF (13.3% v. 6.9%, p < 0.05). There was no difference in metastatic bulk at the time of NGS for tissue biopsy between MTB and NPOP populations (t = 0.80). Disease bulk did not predict the number of pathologic variants from tissue sampling in RAS/RAFWT CRC (n = 96, t = 0.24). RAS/RAFMT cancers had increased frequency of subclonal alterations by ctDNA (9.1±4.0) v. RAS/RAFWT (4.5±3.4, p < 0.0001). Using ctDNA, bulky disease in RAS/RAFMT CRC was not predictive of increased pathologic variants (8.8±3.5 v. 9.5±4.8, t = 0.62). Bulky disease (> 35mm) in RAS/RAFWT CRC predicted increased subclonal variants (6.2±3.6 v. 3.5±2.9, p < 0.02). As a continuous variable, disease bulk predicted the number of pathologic variants in RAS/RAFWT CRC (R = 0.51). Conclusions: These data indicate that metastatic bulk is a predictor of subclonal heterogeneity by ctDNA in RAS/RAFWT CRC. Molecular profiling of tissue alone did not predict differences in subclonal heterogeneity when stratified by disease bulk in RAS/RAFWT CRC. Limited subclonal heterogeneity in non-bulky cancers support ongoing prospective investigations to select non-bulky cancers for early incorporation of anti-EGFR inhibition (NCT04587128).

603 Background: The Veteran Health Administration’s (VHA) National Precision Oncology Program was established to provide comprehensive molecular profiling for US military veterans with advanced cancers. There is an urgent need for precision strategies in pancreatic ductal adenocarcinoma (PDAC), as it is a leading cause of cancer-related mortality. We hypothesized that contributions of molecular alterations in PDAC would fail to stratify overall survival (OS), as current strategies are largely dependent on the activity of cytotoxic chemotherapy. Methods: A retrospective, multicenter cohort of 342 veterans with PDAC were identified from January 2016 to March 2021 who underwent comprehensive next-generation sequencing of tumor using FoundationOne CDx (UW IRB#2020-0696). Subjects were stratified by localized (L) or metastatic (M) disease at the time of diagnosis. Molecular alterations were compared by disease presentation using chi-squared analysis, and the clinical outcomes of overall survival (OS) were evaluated using Student’s t-test. Results: Baseline characteristics were representative of the VA population across 80 independent sites. The cohort was male-dominant (97%) with a median age of 69 years at diagnosis. Of this sample, 55% had M disease (n=189) compared to 45% with L disease (n=153). Median OS for M PDAC was 8.9±10.2 months (mo) v. L PDAC with median OS 22.5±18.0 mo (p

Impact of baseline culture condition on the growth and treatment response of mouse derived cancer organoidsAuthors: Autumn Olson, Rebecca A. DeStefanis, Alyssa DeZeeuw, Samantha Anderson, Gioia Sha, Jeremy Kratz, Cheri Pasch, Linda Clipson, Dustin A. DemingIntroduction: Colorectal cancer (CRC) is a leading cause of cancer related deaths and there has been an increased focus on developing therapies targeted to the mutational profile of individual patients. Mouse derived cancer organoids (MDCOs) are used as a model for therapeutic drug testing. How the culture conditions can impact the determination of treatment response from MDCOs has been understudied and is the focus of these investigations. Here, we examine how baseline size, passage number, plating density, location within the Matrigel droplet, and lineage impact MDCO growth and treatment response.Methods: CRC MDCOs were derived from multiple tumors across several Apc and Pik3ca mutant mice (Fc1 Apcfl/+ Pik3caH1047R/+) totaling 3,152 individual MDCOs. Untreated MDCO growth was analyzed across 902 MDCOs. Additionally, the PI3K pathway inhibitors, AZD2014, Everolimus, BEZ235, MLN0128, Palbociclib, and Copanlisib were analyzed across 2,250 MDCOs. In each study, baseline brightfield images of the MDCOs were taken and following 48 hours. Individual MDCOs were evaluated for the impact of baseline size, passage number (passage 1-15), plating density, location within the Matrigel droplet, and the generation of MDCOs across different mice on MDCO growth and treatment response. A change point analysis was used to assess the impact of baseline size. The percent relative change in diameter over 48 hours was compared to each other culture condition by calculating an R2 value.Results: A change in the organoid baseline growth rate was observed if the MDCOs were greater than 0.373 mm in baseline was identified. There were no significant differences in growth due to changes in the other culture conditions for the untreated MDCOs. The R2 values comparing the percent relative change in diameter over 48 hours to the culture conditions of passage number, plating density, location within the Matrigel droplet, and from which mouse the MDCOs were derived from were 0.0103, 0.0243, 0.0001, and 0.0295, respectively. There were no significant differences in the response to PI3K pathway inhibition of the MDCOs related to passage number, plating density, location within the Matrigel droplet, and from which mouse the MDCOs were derived from (R2 values: 0.1174, 0.0979, 0.0221, and 0.1267, respectively).Conclusions: Mouse derived cancer organoids are a useful tool in modeling drug response of cancers with specific mutational profiles. While the baseline size of MDCOs can change the growth rate, the growth rate and treatment response of MDCOs is not impacted by the passage number, plating density, location within the Matrigel, and from which mouse the MDCOs were derived. Citation Format: Autumn Olson, Rebecca A. DeStefanis, Alyssa DeZeeuw, Samantha Anderson, Gioia Sha, Jeremy Kratz, Cheri Pasch, Linda Clipson, Dustin A. Deming. Impact of baseline culture condition on the growth and treatment response of mouse derived cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2969.

PURPOSE OF REVIEW: Treatment options for patients with metastatic colorectal cancer continue to advance as the therapeutic implications of the molecular subtypes of this disease are becoming better understood. DNA sequencing and mismatch repair assessment are now standard of care analyses for patients with metastatic colorectal cancer Thi review describes important aspects of the biology of the clinically relevant molecular subtypes of colorectal cancer based on the current standard of care testing. In addition, the clinical treatment strategies available now and potentially in the future for these colorectal cancer subtypes are discussed. RECENT FINDINGS: Currently for metastatic colorectal cancer, standard of care molecular testing is done for mutations in exons 2, 3, and 4 of KRAS and NRAS, and BRAF V600E. Testing for mismatch repair (MMR) deficiency/microsatellite instability (MSI) status is also done. These aberrations are well known to change the clinical prognosis and guide patients’ treatment strategies. Additionally, three new subtypes have emerged: PIK3CAmut, HER2 amplified, and NTRK fusions. With the addition of these emerging subtypes, tumor heterogeneity further validates the need to examine mCRC as a heterogeneous disease. Here we present recent exciting data from translational research and clinical trials exhibiting possible distinct treatment strategies for these different subtypes. SUMMARY: Altogether these data show promising treatment strategies for many of these well-known and emerging subtypes of mCRC. In addition, these also give better clinical prognostic and predictive information. We believe that as molecular testing expands PIK3CA mutation, HER2 amplification, and NTRK fusion molecular testing will be included in standard of care analyses. This incorporation of testing in clinical practice will generate further information regarding prognostic and therapeutic options for these and other CRC subtypes in the future.

68 Background: HER2 amplification is an emerging biomarker in colorectal cancer (CRC) with increased copy number associated with improved clinical outcomes to HER2 targeting. RAS/RAF wildtype CRC also benefit from use of epidermal growth factor receptor inhibition (EGFRi). The sequencing of EGFRi versus HER2 inhibition in low copy number HER2 amplified CRC remains uncertain. Patient-derived cancer organoids (PDCOs) allow an ex vivo method to assess treatment sensitivity. We examined treatment sensitivity of a HER2 amplified PDCO at baseline and following resistance to panitumumab and trastuzumab. Methods: Following IRB-approval, fresh CRC tissue was cultured to maturation. After expansion, subcultures were treated with stepwise (20%) increase to physiologic Cmax of panitumumab (230ug/mL) and trastuzumab (180ug/mL). Threshold for escalation was median relative growth of +20% at 96h. Sensitivity was assessed on primary culture (RC1), panitumumab resistance (RC1-P) and trastuzumab resistance (RC1-T) using 96h of physiologic Cmax panitumumab, trastuzumab, and combination trastuzumab/pertuzumab. Individual sphere response was assessed for change in mean NADH autofluorescence intensity and ratio of NADH/FAD signal. Response was assessed at 96h in comparison to control using effect size of Glass’s Delta (GΔ). Results: Molecular profiling revealed HER2 copy number of 14 with no concurrent alterations in RAS, RAF, or PIK3CA. Time to resistance was similar between panitumumab (55 days) and trastuzumab (51 days). RC1 had baseline growth (+116%) which was reduced with single agent panitumumab (+17%, GΔ=1.40) with intermediate sensitivity to trastuzumab (+48%, GΔ=0.95) and trastuzumab/pertuzumab (46%, GΔ=0.99). Normalized NADH/FAD ratio revealed significant metabolic response to panitumumab (-20%, GΔ=0.66) and trastuzumab/pertuzumab (-35%, GΔ=1.16) with insignificant effect of single agent trastuzumab (-14%, GΔ=0.46). Following resistance to panitumumab, RC1-P had persistent growth with trastuzumab (+68%) which improved in combination trastuzumab/pertuzumab (+34%, GΔ=1.16). Following resistance to trastuzumab, RC1-T was insensitive to EGFRi with panitumumab including persistent growth (+58%, GΔ=0.70) and unchanged metabolism (+2%, GΔ=-0.10). Conclusions: Therapeutic dose escalation in a single PDCO of HER2 amplified CRC suggests improved sensitivity to EGFRi and dual HER2 targeting with trastuzumab/pertuzumab. Resistance to EGFRi resulted in persistent sensitivity to dual HER2 inhibition using trastuzumab/pertuzumab, however resistance to single agent trastuzumab. Resistance to trastuzumab resulted in future insensitivity to EGFRi. Molecular profiling at resistance revealed no pathologic alterations in EGFR or ERBB2 signaling, with ongoing analysis of transcriptional changes by RNAseq.

Over the past several decades, tremendous advances have been made in the understanding, diagnosis, and treatment of coronary artery disease (CAD). However, with shifting demographics and evolving risk factors we now face new challenges that must be met in order to further advance are management of patients with CAD. In parallel with advances in our mechanistic appreciation of CAD and atherosclerosis, nanotechnology approaches have greatly expanded, offering the potential for significant improvements in our diagnostic and therapeutic management of CAD. To realize this potential we must go beyond to recognize new frontiers including knowledge gaps between understanding atherosclerosis to the translation of targeted molecular tools. This review highlights nanotechnology applications for imaging and therapeutic advancements in CAD.

Colorectal cancer (CRC) is a leading cause of cancer-related death. PIK3CA mutations are common, leading to a constitutively active phosphoinositide-3 kinase (PI3K). An effective means to target this pathway has yet to be identified. We investigated the use of a panel of inhibitors targeting the PI3K pathway including copanlisib (dual PI3K/mTOR), BYL-719 (alpha isomer specific PI3K), GDC-0941 (pan PI3K), and TAK-228 (MTORC1/2). To test the efficacy of these inhibitors in CRC, murine organotypic cancer spheroids (MDOCS) were generated from the invasive adenocarcinomas of Apc and Pik3ca transgenic mice. These inhibitors were investigated at clinically relevant doses (100-400nM). Copanlisib and TAK-228 were the only inhibitors to result in a significant reduction in the size of the MDOCS (200nM; 27% p-value Citation Format: Rebecca A. DeStefanis, Susan N. Payne, Devon Miller, Cheri A. Pasch, Christopher Babiarz, Alyssa DeZeeuw, Stephanie L. Fricke, Carley Sprackling, Alexander E. Yueh, Demetra P. Korkos, Dana R. Van De Hey, Gioia Sha, Aurora Greane, Jeremy D. Kratz, Linda Clipson, Kristina A. Matkowskyj, Michael A. Newton, Dustin A. Deming. MTORC1/2 inhibition as a treatment strategy for PIK3CA mutant colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B27.

Background: Patient-derived cancer organoids (PDCOs) are emerging as an in vitro model to recapitulate the molecular and phenotypic features of colorectal cancer (CRC). Heterogeneity in established CRC PDCOs has been observed in the underlying molecular profiles and growth characteristics at the individual organoid level. Here, we present a dedicated assessment of individual organoid growth as a function of experimental culture parameters. Methods: CRC PDCO cultures were established from patient biopsy/resection specimens following patient consent to an IRB-approved protocol. Growth was assessed by relative change in diameter at 48h. Baseline size was compared to relative growth at 48h using coefficient of determination (R). Interquartile sets were compared by effect size of Glass's Delta (GΔ) to compare change difference in average growth normalized to standard deviation and defined with significance 1.0. Impact of density was assessed by manual count of spheroids and lines plated across varied densities and compared to 48h relative growth. Growth rates were compared as both absolute and relative passage number. Results: 22 unique cultures were established from fresh tissue and representative of CRC including pathologic alterations in APC, KRAS, NRAS, BRAF, PIK3CA TP53, MTOR and PTEN. Pairwise spheres were assessed at baseline and 48h for analysis (n=1714) with mean relative growth rate of 27.1% (range -40.0, 156.2%). Baseline size did not predict relative growth at 48h (R=0.023) with insignificant interquartile effect size [0.10, 0.07, 0.32]. Replicates (n=63) across a range of passages [1, 36] including line-specific relative passage number [0-14] did not predict change in relative growth (R Conclusions: Following culture maturation, CRC PDCOs have heterogeneous growth rates at the organoid level. These studies demonstrate that the growth rate is independent of baseline organoid size, passage number, or culture plating density. Understanding the effect of culture variation helps to define meaningful population effects in response and resistance to therapy and supports the translation of this technology as a future predictive biomarker. Citation Format: Aishwarya Sunil, Jeremy D. Kratz, Sarbjeet K. Makkar, Suhjah Rehman, Amani A. Gillette, Katherine A. Johnson, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Melissa C. Skala, Dustin A. Deming. Etiologies of patient-derived colorectal cancer organoid growth heterogeneity across multiple patient samples and culture conditions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1494.

e16035 Background: Advances in the understanding of colorectal cancer (CRC) biology, including clonal evolution are leading to novel treatment strategies. Epidermal growth factor receptor inhibition (EGFRi) with cetuximab or panitumumab has shown significant clinical utility in KRAS/NRAS/BRAF wildtype (wt) CRC. These inhibitors are most commonly used in the third-line treatment setting in the United States. Upon the development of resistance to EGFRi, withdrawal of the EGFR inhibitor can lead to decay of resistant clones allowing for EGFR inhibitor retreatment in additional lines of treatment. Earlier-line use of single agent EGFRi could allow more patients to utilize a retreatment strategy, however no large prospective clinical trials have assessed the clinical activity of second line EGFRi in the setting of molecular selection. Here, we assess the clinical outcomes of EGFRi monotherapy for advanced KRAS wt CRC after initial progression on a single line 5FU based chemotherapy. Methods: A retrospective chart review examined patients who received single agent EGFRi in KRAS wt CRC in the second line setting. Line of therapy was defined at time of metastatic disease or recurrence within 6 months of adjuvant therapy. Clinical outcomes of overall response rate (ORR) and progression free survival (PFS) were assessed per RECIST v1.1. Results: 20 cases were identified who received single agent EGFRi (panitumumab or cetuximab) as monotherapy in the second line setting. All pts received prior 5FU based chemotherapy. Reasons for using EGFR inhibitor monotherapy in this setting included to avoid toxicities from prior chemotherapy (largely immunosuppression and GI toxicities), bridge from recent operative management, or maintain functional performance status. Patients had median age of 62 [45, 89] and majority with ECOG PS 0-1 (78.5% of those reported). Best responses included complete response 7%, partial response 21%, stable disease 64% and progressive disease 7%, and a disease control rate of 93%. The 6 and 8 month PFS were 70% and 50%, respectively. PFS ranged from 1.5 to 17.3 months with median PFS 8.2 ± 3.6 months. Conclusions: This work demonstrates preliminary efficacy data to use single agent EGFRi in the second-line setting for KRASwt patients with CRC. Future investigations will aim to prospectively validate the benefit of this treatment strategy to facilitate EGFRi retreatment and improve patient survival.

177 Background: No current clinical tool can predict the efficacy of cancer therapeutics for patients with colorectal cancer (CRC). We recently demonstrated the feasibility of using patient-derived cancer organoids (PDCOs) to examine therapeutic response and tumor heterogeneity for individual patients with CRC via optical metabolic imaging (OMI). Here we expand these analyses in a cohort of patients with clinical outcomes. Methods: CRC tissue was collected from patients on IRB-approved protocols. PDCOs were matured and treated with chemotherapy regimens concurrent with patient treatment. Previously established effect size response thresholds were used for diameter ( > 1.5) and OMI ( > 0.5) following 48 hours of treatment. OMI measures the intrinsic autofluorescence of NAD(P)H and FAD using 2-photon microscopy without specific reagents or dyes. Clinical outcomes were prospectively collected by manual chart review. Results: 12 CRC PDCOs were established from patients with CRC. PDCOs were collected from initial diagnosis and advanced setting of both primary and metastatic sites by core needle biopsy and surgical resection. Differential growth rates were observed across lines. PDCOs with RAS/RAF alterations had more uniform growth, while PDCOs without these alterations demonstrated more heterogeneous growth and metabolism. Clinical correlation of PDCOs response with recurrence of disease in the adjuvant setting will be presented. Cases with prior 5-FU-based chemotherapy at the time of PDCO collection had intermediate sensitivity. For PDCOs collected pre-treatment, PDCO response predicted clinical response for 5 of 6 cases using predefined sensitivity thresholds. In the case that overall PDCO response did not predict clinical response, a heterogenous response was observed with distinct sensitive and resistant populations. Across PDCOs, greater post-treatment heterogeneity was observed in resistant lines compared to those with treatment sensitivity. Conclusions: Tumor heterogeneity in treatment response can be assessed using PDCOs growth and metabolism. The utility of PDCOs to predict clinical outcomes for patients with CRC deserves further prospective validation.

252 Background: ASCO defined meaningful trial endpoints in colorectal cancer (CRC) to include OS HR ≤0.67 (Ellis, JCO 2014). This measure is limited in identifying treatment benefit for subgroups from heterogeneous populations. Effect size (Glass’s Δ) calculates the absolute difference in median clinical outcomes normalized to the control group standard deviation. We hypothesized that durable effect sizes ≥2 would be useful in predicting which trials possess subgroup populations of clinical significance despite a HR > 0.67. Methods: Prospective phase II-III trials in metastatic CRC from the ASCO Meeting Library (2016-2019) were cataloged by clinical outcomes of PFS and OS. Effect size was calculated from trials reporting confidence intervals and compared with absolute difference in clinical outcome, hazard ratio and therapeutic intervention. Trials with an indeterminant HR, yet effect size > 2 were reviewed in subgroup analyses. Results: 385 abstracts were reviewed with 99 clinical analyses available for effect size calculation. Absolute difference in PFS correlated with effect size (R = 0.64) and was inversely proportional to HR (R = -0.63). The absolute difference in OS correlated with effect size (R = 0.69) and was inversely proportional to HR (R = -0.57). When stratified by clinically significant HR (defined ≤0.67), median effect size for PFS was 13.7±13.3 (SD) which was significantly different from HR > 0.67 with median effect size 1.0±3.8 (p < 0.001). Median effect size for OS when stratified by HR ≤0.67 was 3.7±2.5 which was significantly different when compared to endpoints with HR > 0.67 with median effect size 0.9±1.4 (p < 0.003). Subgroup populations with survival benefit included combination checkpoint blockade durvalumab/tremelimumab vs supportive care with effect size 3.1 (HR 0.72; NCT02870920). First-line PFS benefit was predicted in KRAS wildtype liver-limited CRC treated with FOLFOX+cetuximab vs FOLFOX+bevacizumab by effect size of 3.2 (HR 0.80; NCT01836653). Conclusions: Effect size holds potential as a measure to delineate improved clinical outcomes from heterogeneous populations and could identify those trials for which further subgroup analysis should be explored.

Metastatic breast cancer is a very heterogeneous disease. Recent advances in genomic sequencing have revealed genetic diversity between patients and across distinct subclonal cell populations within the same patient that may evolve across metastatic tumor sites and during treatment. With the increasing availability of commercial and laboratory-developed tests that can detect genomic alterations from patient tumor and blood samples, translating this knowledge into improved clinical care remains a challenge. The goals of this review are to outline the clinical relevance of tumor genomic heterogeneity and clonal evolution, to help clinicians understand how to interpret genomic testing reports, and to provide an overview of recurrent genomic alterations that may be relevant for clinical trials with investigational drug treatments.

Metastatic colorectal cancer (CRC) is now being better understood as a collection of diseases with each distinguished by distinct molecular profiles. BRAF mutant cancers are an important CRC subtype composing about 10% of metastatic CRCs. Of those with a mutation in the BRAF gene, 80% possess the V600E mutation (1).

Gynaecomastia is a common clinical presentation that varies from benign presentations in stages of human development to hormonal pathology, mainly due to hepatic dysfunction, malignancy, and adverse pharmacologic effects. We describe the development of significant bilateral gynaecomastia after starting treatment for pulmonary tuberculosis (TB) in two males with WHO stage III Human Immunodeficiency Virus (HIV) infection on stable antiretroviral regimens. Emerging reports suggest that distinct hepatic impairment in efavirenz metabolism modulates oestrogenic activity, which may be potentiated by antituberculosis therapy. Clinical application includes early recognition of efavirenz-induced gynaecomastia, especially after commencing tuberculosis treatment. To avoid decreased adherence resulting from the distressing side effect of gynecomastia, transition to an alternative ART regimen over the course of tuberculosis treatment should be considered.

Background: Colorectal cancer (CRC) remains the second leading cause of cancer-related mortality with limited precision strategies. The human epidermal growth factor receptor (HER2) is amplified in 5% of patients with metastatic CRC. Therapeutic studies have demonstrated activity of HER2 targeting regimens in the late-line treatment setting though little characterization has been done in the setting of not having prior anti-epidermal growth factor receptor antibody therapy or in the first-line treatment setting. Methods: Patient with metastatic colorectal cancer underwent next-generation sequencing (NGS) panel testing by Strata Oncology through the University of Wisconsin Precision Medicine Molecular Tumor Board. Patients were treated per multi-disciplinary recommendation across multiple lines of therapy and tracked by prospective registry (IRB#UW15068). HER2 copy numbers >/= 10 were used to determine amplification. A subset of patients with HER2-amplified CRC were treated with single agent trastuzumab. Results: HER2 amplifications were identified in 4 of 78 cases of metastatic CRC (5.1%). All biopsies were performed prior to exposure to anti-epidermal growth factor receptor inhibitors. Median estimated copy number (CN) was 55.5 [range 13-178]. There was no concurrent alterations in extended RAS, BRAF, PIK3CA, AKT or MTOR. All cases included concurrent mutations in TP53 at R175H (2), C135W, or C135F. Single agent trastuzumab was administered to two patients at standard dosing (trastuzumab 8mg/kg intravenous day 1 cycle 1 followed by 6 mg/kg intravenous day 1 cycle 2+ on a 21 day cycle). One patient was treated in the treatment refractory setting. They had resolution of bowel obstructive symptoms and a minor response on imaging. Additionally, a patient with a HER2 amplification (CN 178) was treated in the first-line setting with single agent trastuzumab. The patient had grade 1 fever with the first cycle and no other treatment related adverse events thereafter. The patient’s CEA decreased from 7,506 to 2,525 ng/mL with the first cycle of therapy and overall reduced to 6.5 ng/mL after 12 cycles of therapy. The patient has had a partial response per RECIST v1.1 response criteria with a reduction of cross-sectional diameter of their disease by 60%. The patient currently continues on this therapy without evidence of disease progression. Conclusions: HER2 amplified colorectal cancers are an important subtype of colorectal cancer. Here we describe the potential benefit of HER2 targeting with single agent trastuzumab and even demonstrate single agent activity in the first-line setting. Further studies should be done to further characterize the benefit of this low toxicity treatment option for patients with metastatic HER2 amplified colorectal cancer. Citation Format: Jeremy Kratz, Nataliya Uboha, Kayla Lemmon, Hannah Houtler, Mark Burkard, Dustin Deming. Durable response to first-line Trastuzumab in HER2 amplified colorectal cancer [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 1414.

Background: There are limited clinical tools for predicting the effectiveness of cancer therapies. We aim to prospectively predict patient treatment response using patient-derived organotypic cancer spheroids (PDOCS) as an in vitro model which recapitulates the genetic characteristics and 3D organization of the patient’s tumor. Using optical metabolic imaging (OMI) to analyze single cells, we can determine heterogeneous subpopulations in response to drug treatment. Further clinical validation of these techniques and analysis methods are needed before clinical translation. Methods: Tissue biopsies and gross tissue resections were acquired through the University of Wisconsin Precision Medicine Molecular Tumor Board (IRB#UW15068) and UWCCC TSB Biobank. Next-generation sequencing (NGS) from the biopsies was performed to determine molecular profiling. In alignment with the patient’s treatment course, PDOCS were treated with physiologic doses of chemotherapy or targeted therapy. Treatment response was evaluated by measuring change in diameter in conjunction with optical metabolic imaging (OMI) using a multiphoton microscope to measure the fluorescence and redox ratio of NAD(P)H and FAD as an indication of cellular metabolism. Diameter changes between control and treatment groups were compared using Glass’s delta; resistance to therapy was indicated by a Glass’s delta score of below 1.5. The optical redox ratios determined by OMI were compared using Glass’s delta, and resistance was indicated below 0.5. Clinical response was measured using RECIST v1.1 standard response assessment criteria. Results: PDOCS were successfully isolated from colorectal (CRC), lung, gastrointestinal stromal tumor (GIST), ovarian, and breast cancers. These biopsies were all obtained in the treatment refractory setting. PDOCS were generated for seven patients and treated with the same pharmacologic treatment as the patient from which the PDOCS were generated. Multiple treatments were able to be tested both in vitro and clinically for a subset of patients. Treatments included: 5-fluouracil, oxaliplatin, gemcitabine, paclitaxel, olaparib, panitumumab, osimertinib, fulvestrant, and palbociclib. In this cohort, two treatments resulted in stable disease and seven treatments resulted in disease progression. Change in spheroid diameter correlated with clinical treatment outcomes with an effect size (Glass’s delta) threshold of 1.5. OMI predicted response for all patients imaged with an effect size threshold of 0.5 which correlated with the size change analyses. Treatment heterogeneity of OMI was observed in many of the samples. Conclusions: In this largely prospective cohort of patients across disease types, changes in PDOCS size and OMI indices predict treatment benefit for individual patients. Studies on a larger scale are needed to further validate these findings. Citation Format: Carley M. Sprackling, Jeremy D. Kratz, Peter F. Favreau, Mohammad R. Karim, Christopher P. Babiarz, Cheri A. Pasch, Amani A. Gillette, Linda Clipson, Kristina A. Matkowskyj, Jens C. Eichoff, Kayla K. Lemmon, Hannah K. Houtler, Mark E. Burkard, Devon Miller, Melissa C. Skala, Dustin A. Deming. Predicting treatment response using patient derived organotypic cancer spheroids [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 3143.

e15104 Background: ASCO defined clinically meaningful trial endpoints in colorectal cancer (CRC) as absolute overall survival (OS) of 3-5 months and OS hazard ratio (HR) ≤0.67. Few tools predict clinically distinct subgroups from heterogeneous populations. Effect size (Glass’s delta) calculates the absolute difference in clinical outcomes relative to the standard deviation of control group. We hypothesized that effect sizes ≥1 would be useful in predicting subgroup populations of clinical significance for trials with an indeterminate HR. Methods: Prospective phase II-III trials in metastatic CRC were queried from clinicaltrials.gov and cataloged by clinical outcomes of PFS and OS. Effect size was back-calculated from trials reporting 95% confidence intervals and compared with absolute difference in clinical outcome and hazard ratio. Results: 46 prospective trials were evaluable including 49% with biomarker selection, 57% in the first-line setting, and more commonly studied with targeted therapy over chemotherapy (70 v. 30%; p < 0.001). EGFR inhibitors were studied in 34% and VEGF inhibitors in 23%. Both effect size and HR correlated similarly with PFS (correlation coefficients, R = 0.65 vs. 0.76) and OS (R = 0.83 vs. 0.84) across these studies. Of studies with an indeterminate HR (0.69-0.86; n = 24), 10 studies had a significant effect size (Glass’s delta > 1), indicating the potential for sub-populations with improved clinical benefit within the experimental treatment group. These included 4 trials with planned biomarker analyses. Remaining trials were enriched for studies investigating anti-angiogenic therapies (83% vs. 23%, p = 0.001), including bevacizumab, aflibercept, regorafenib, and ramucirumab. Conclusions: Effect size holds potential as a measure to indicate the presence of subpopulations of patients benefiting in clinical trials. Those trials with a significant effect size despite an indeterminate HR should be examined closely for such populations. In CRC, effect size indicates the potential of a subgroup of patients who benefit significantly from anti-angiogenic agents. Further investigations are needed to validate effect size as a tool to delineate improved clinical outcomes from heterogeneous populations.

569 Background: Epidermal growth factor receptor inhibitors (EGFRi) have improved clinical outcomes in patients (pts) with metastatic colorectal cancer (mCRC). Molecular profiling and primary tumor sidedness are used to predict population benefit. Our group has recently demonstrated that patient-derived organotypic cancer spheroids (PDOCS) and optical metabolic imaging (OMI) can predict in vivo chemotherapy response. Translational tools are needed to characterize targeted therapeutic response to predict clinical outcomes. Methods: PDOCS were generated from patients with mCRC at time of molecular profiling. Following culture maturation, PDOCS were treated with physiologic doses of EGFRi panitumumab. Response was evaluated by change in sphere diameter and OMI to exploit intrinsic autofluorescence of NAD(P)H and FAD at spheroid and single-cell level. Effect size was calculated using Glass’s delta (GΔ) defined as differences in means between treatment groups normalized to control standard deviation with comparison to predetermined sensitivity thresholds. Results: PDOCS from pts with mCRC were generated from tissue biopsies, surgical specimens, and malignant effusions (n = 38). Mutational profiles were stratified by RAS status from next-generation sequencing. Six PDOCS were evaluable for experimental and clinical response. KRAS mutation predicted primary resistance to EGFRi with no difference in diameter (GΔ = -0.01) or single cell response by OMI (GΔ = 0.02). RAS wild-type PDOCS had significant response with decreased diameter with EGFRi (P < 0.001). Gaussian fit of single-cell analyses revealed heterogeneity in EGFRi sensitivity. Differential sensitivity to EGFRi in RAS wild-type population correlated with clinical response. Conclusions: PDOCS predict response to panitumumab in these preliminary investigations. Diameter and OMI analyses provide complementary information of line-specific sensitivity. Further studies are warranted to characterize the molecular profiles underlying intratumor heterogenity. Prospective investigations are needed to understand the predictive role of this technique in targeted therapeutic response.

We have previously shown that methotrexate (MTX) conjugated to a cancer-specific poly amido amine (PAMAM) dendrimer has a higher therapeutic index than MTX alone. Unfortunately, these therapeutics have been difficult to advance because of the complicated syntheses and an incomplete understanding of the dendrimer properties. We wished to address these obstacles by using copper-free click chemistry to functionalize the dendrimer scaffolds and to exploring the effects of two dendrimer properties (the targeting ligand and drug linkage) on cytotoxicity. We conjugated either ester or amide-linker modified MTX to dendrimer scaffolds with or without folic acid (FA). Because of multivalency, the FA and MTX functionalized dendrimers had similar capacities to target the folate receptor on cancer cells. Additionally, we found that the ester- and amide-linker modified MTX compounds had similar cytotoxicity but the dendrimer-ester MTX conjugates were much more cytotoxic than the dendrimer-amide MTX conjugates. These results clarify the impact of these properties on therapeutic efficacy and will allow us to design more effective polymer therapeutics.

As treatment strategies for patients with colorectal cancer advance, there has now become an ever-increasing need for multidisciplinary teams to care for these patients. Recent investigations into the timing and duration of perioperative therapy, as well as, the rise of molecular profiling have led to more systemic chemotherapeutic options. The most efficacious use, in terms of timing and patient selection, of these therapies in the setting of modern operative and radiotherapy techniques requires the generation of care teams discussing cases at multidisciplinary conferences. This review highlights the role of multidisciplinary team conferences, advances in perioperative chemotherapy, current clinical biomarkers, and emerging therapeutic agents for molecular subtypes of metastatic colon cancer. As our understanding of relevant molecular subtypes increases and as data becomes available on treatment response, the treatment of colorectal cancer will become more precise and effective.

795 Background: Epidermal growth factor receptor (EGFR) antibodies, cetuximab (C) and panitumumab (P) have shown improvement in clinical outcomes for distinct molecular profiles in metastatic colorectal cancer (mCRC). In the first line setting, EGFR targeting of left-sided tumors has shown favorable clinical outcomes when compared to right-sided tumors. Here we examined the efficacy of EGFR therapy in the treatment refractory setting as well as the impact of tumor bulk on clinical outcomes. Methods: A retrospective cohort of 72 patients (pts) with KRAS wild-type mCRC were identified who received either C or P in the late-line setting. Tumor measurements were performed per RECIST v1.1. Disease bulk was defined as single lesion with longest diameter or lymph node with short axis > 3.5 cm. Right colon primary was defined proximal to splenic flexure. Results: In pts with treatment refractory right-sided disease the response rate (RR) was 16.7%, progression free survival (PFS) was 3.7 months (mo), and overall survival (OS) was 14.0 mo. This was compared to left sided disease with RR of 26.0%, PFS 6.2 mo (p < 0.05), and OS 15.0 mo. In the non-bulky cohort the RR was 32.4%, PFS 7.9 mo, and OS 18.4 mo. In the bulky cohort the RR was 5.3% (p < 0.01), PFS 4.0 mo (p < 0.02), and OS 6.6 mo. In the right-sided non-bulky cohort there was a RR of 33% v. 0% in the right-sided bulky cohort. In the left-sided non-bulky cohort there was a RR of 32.3% v. 7.7% in the bulky cohort (p < 0.05). Conclusions: These data indicate that tumor bulk can predict clinical outcomes for anti-EGFR targeting. In the late line setting, pts with left-sided cancers overall trended towards improvements in clinical outcomes, consistent with prior understanding in first line setting. Despite a limited cohort size, response was observed in pts with non-bulky right-sided disease. There was limited-to-no benefit of anti-EGFR targeting with right-sided bulky disease. Left-sided non-bulky patient received the greatest benefit from anti-EGFR targeting. Future prospective studies of targeted therapeutics should incorporate tumor bulk and sidedness when assessing clinical outcomes and tumor biology.

e15054 Background: Molecular testing and location of the primary tumor (right-sided (R) versus (vs) left-sided (L)) are useful in predicting the clinical benefit of the anti-epidermal growth factor receptor (EGFR) antibodies, cetuximab (cet) and panitumumab (pan), in metastatic colorectal cancer (mCRC). We hypothesized that tumor bulk might also be predictive of treatment benefit given the potential for increased intra-tumor heterogeneity and reduced penetration of antibodies into bulkier lesions. Methods: A single institution retrospective cohort of 69 patients (pts) with KRAS wild-type mCRC were identified who received either cet or pan in the late-line setting +/- chemotherapy. Metastatic sites were cataloged including independent review of CT imaging prior to initiation of anti-EGFR therapy. Disease bulk was defined categorically as single metastatic lesion with diameter measuring > 3.0 cm. Results: This cohort represents a diverse group having received varying prior lines of therapy and having assorted disease sites including mediastinal, pulmonary, hepatic, omental, and osseous lesions. When treated with anti-EGFR therapies, pts with pre-treatment metastases ≤3.0 cm in diameter had significant improvement in median progression free survival (mPFS) (6.2 months (mos)) vs pts with metastases > 3.0 cm (3.9 mos, p < 0.01). A trend towards improvement in overall survival was observed for pts with non-bulky (15.8 mos) vs bulky disease (8.6 mos, p = 0.08). A trend towards increased mPFS existed in L (5.1 mos) vs R cancers (2.5 mos, p = 0.11). In R cancers, no significant difference in mPFS was noted between bulky (2.5 mos) and non-bulky disease (2.9 mos, p = 0.53). Non-bulky L cancers had a significantly improved mPFS of 7.5 mos compared to 3.9 mos with L bulky disease (p < 0.01). Non-bulky L cancers trended towards improved overall survival of 17.0 mos compared to 9.6 mos with L bulky disease (p = 0.08). Conclusions: Despite limited small sample size, these data indicate that tumor bulk is a potential predictor of the therapeutic benefit of anti-EGFR agents in left-sided mCRCs across a heterogeneous pt population. Disease bulk deserves further evaluation in larger datasets of mCRC across targeted therapeutics.

RNA is growing in its importance as a drug target but current approaches used to identify protein-targeting small molecules are ill-suited for RNA. By docking small molecules onto an RNA dynamic ensemble constructed by combining Nuclear Magnetic Resonance (NMR) spectroscopy and computational molecular dynamics, we virtually screened small molecules that target the entire structure landscape of the transactivation response element (TAR) from the human immunodeficiency type 1 virus (HIV-1). We quantitatively predict binding energies for small molecules that bind different RNA conformations and report the de novo discovery of six compounds that bind TAR with near record affinity and inhibit its interaction with a Tat peptide in vitro (Kis = 710 nM–169 μM). One compound binds HIV-1 TAR with exceptional selectivity and inhibits Tat-mediated activation of the HIV-1 long terminal repeat by 81% in T cell lines and HIV replication in an HIV-1 indicator cell line (IC50 ~23.1 μM).