5FU Chemotherapy Research Articles

Abstract B083: The splicing factor SMNDC1 facilitates alternative RNA splicing, contributing to therapy resistance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive and lethal cancers, mainly due to its high resistance to existing treatments. The standard treatment for PDAC involves chemotherapy, either with Gemcitabine (GEM) combined with paclitaxel or the FOLFIRINOX regimen (5-fluorouracil (5FU), leucovorin, irinotecan, oxaliplatin). However, PDACs rapidly develop resistance to these therapies. Similar resistance is observed with new treatments targeting the KRAS-MAPK pathway, which is crucial in driving PDAC. Our preliminary studies have shown that Survival Motor Neuron Domain Containing 1 (SMNDC1) promotes PDAC tumor growth by facilitating exon retention, specifically cassette exon 4 (E4) of MAPK3, linking RNA splicing to KRAS-MAPK signaling. To uncover the mechanisms underlying PDAC therapeutic resistance, we have developed novel in vitro models of resistance to chemotherapy and MAPK inhibitors (MAPKi). Using these models, we found that SMNDC1 and MAPK3 E4 retention is significantly increased in GEM, 5FU (chemotherapy agents), Adagrasib, Sotorasib (KRAS inhibitors; KRASi G12C), and Selumetinib (MEK inhibitor; MEKi) resistant PDAC cells. Additionally, PDAC lines acutely treated (24h) with trametinib or selumetinib (MEKi) showed a doubling in the expression of SMNDC1 and MAPK3 E4 retention. Protein expression analysis via western blotting revealed that GEM, 5FU, Adagrasib, MRTX1133 (KRASi G12D), and Selumetinib resistant PDAC cells upregulated SMNDC1 expression compared to their sensitive counterparts. To determine the role of SMNDC1 in MAPK resistance, we tested SMNDC1 proficient and deficient PDAC cells with KRASi or MEKi. SMNDC1-deficient PDAC cells were more sensitive to these inhibitors than isogenic cells expressing SMNDC1. Furthermore, to show that increased E4 retention of MAPK3 drives resistance to KRASi or MEKi, we treated SMNDC1 proficient PDAC cells with these inhibitors and MAPK3 E4 specific antisense morpholino oligonucleotide (ASO), which increased sensitivity compared to cells treated with the inhibitor and control ASO. To determine if increased E4 retention is a resistance mechanism specific to therapies targeting upstream of ERK1, we treated PDAC cells, proficient and deficient in SMNDC1, with ERK inhibitors (MK8353, Temuterkib). No changes in sensitivity were observed, as these inhibitors target the ERK1 protein downstream of MAPK3 splicing. Additionally, in an orthogonal model, we found that mutant EGFR lung adenocarcinoma cells resistant to the third-generation tyrosine kinase EGFR inhibitor (Osimertinib) also had higher incorporation of E4 of MAPK3. This suggests that upstream inhibition of ERK in the MAPK pathway leads to increased MAPK3 E4 retention. These findings indicate that PDACs use a "splicing switch" to enhance ERK kinase activity, thereby resisting targeted therapies and chemotherapy in cancers driven by mutant KRAS-MAPK signaling pathways. Citation Format: Md Afjalus Siraj, Yushan Zhang, Deanne Yugawa, Gilbert Giri, Prabir Chakraborty, Grant Goda, Geeta Rao, Daniel Dominguez, Stefan Kubicek, Resham Bhattacharya, Luisa Escobar-Hoyos, Priyabrata Mukherjee. The splicing factor SMNDC1 facilitates alternative RNA splicing, contributing to therapy resistance in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B083.

Feasibility of Hepatic Artery Infusion Chemotherapy for Colorectal Liver Metastasis in an Indian Setting.

Hepatic artery infusion chemotherapy (HAIC) is a popular treatment modality for the treatment of colorectal liver metastasis (CRLM). The aim of this study was to determine the feasibility of HAIC for high-risk resected CRLM delivered using repeated femoral puncture and delivering 5-fluorouracil infusional chemotherapy along with systemic adjuvant chemotherapy. The present study is a retrospective review of a prospectively maintained database. All patients who underwent HAIC for colorectal liver metastases between July 2022 and July 2023 were included. A total of 12 patients were included in the study of which 11 completed four sessions as planned. The median age was 47 (29-73) years with nine male (81%) and two female (18%) patients. Rectum (n = 7, 63%) was the most common primary location. All patients received systemic chemotherapy with 5-fluorouracil-based regimens prior to HAIC (median 12 cycles). The median number of metastasis was 2 (1-8). Eight patients had metastasis in unilobar distribution (73%). On completion of HAIC treatment, nine patients (64%) were completely disease free with a median follow-up of 8 months. None of the patients experienced any immediate adverse events during or after completion of the procedure. Conventional HAIC comes with various challenges such as unavailability of the agent floxuridine and the specialized HAIC pump. Percutaneous HAIC has a lower chance of infection. The delivery of HAIC using repeated femoral punctures and 5FU chemotherapy was successful in over 90% of the patients making it a feasible option in the treatment of CRLM.

Association Between Microsatellite Instability (MSI) based on MLH1 and PMS2 Marker with Clinicopathological Features of Colorectal Cancer Patients

Background: Microsatellite instability (MSI) is one of the important pathways involved in development of colorectal cancer (CRC). MSI occurs due to mutations or hypermethylation of negative MMR proteins MLH1 and PMS2. The accumulation of mutations at microsatellite locus accelerated the development of CRC. Characteristics of CRC patients with MSI are not sensitive to 5FU chemotherapy and have a good clinical outcomes that can be used as a prognostic factor and a predictor of therapy. Therefore MSI detection is needed. Method: A retrospective cross-sectional study of 80 CRC slides obtained from DR. Sardjito Hospital Yogyakarta and clinical laboratory in 2010-2016. Immunohistochemical staining with anti-MLH1 and anti PMS2 antibodies to see MSI status. Negative MLH1 expression is called MSI MLH1 positive and PMS2 negative expression is called positive MSI PMS2. The association between MSI MLH1 and PMS2 with clinicopathology parameters was analyzed using Chi square. Results: Colorectal cancer patients MSI MLH1 as much as 44 (61.1%), MSI PMS2 as much as 21 (29.2%) and, MSI MLH1 and PMS2 simultaneously as much as 18 (25.0%). MSI MLH1 and PMS2 positive are found in ≥50 years old. The number of male patients is more than women. Most of the patients had a T3-T4 tumor size with advanced stage and well differentiated. MSI MLH1 associated with tumor differentiation (p = 0.011). Conclusion: MSI MLH1 is associated with tumor differentiation (p = 0.011), but, no association with the other clinicopathology parameters. MSI PMS2 is not associated to the overall clinicopathology parameters. MSI MLH1 and PMS2 are not simultaneously associated with all clinicopathology parameters (p> 0.05)

Immunotherapy in colorectal cancer: an unmet need deserving of change.

The efficacy of immunotherapy in colorectal cancer is currently restricted to patients with metastatic colorectal cancer presenting deficient mismatch repair (dMMR) or microsatellite instability (MSI). So far, neither immunotherapy agents as monotherapy nor immunotherapy-based combinations have shown benefit for metastatic colorectal cancer with proficient mismatch repair (pMMR) or microsatellite stability (MSS), which are mostly immunologically cold. 1 Ganesh K Stadler ZK Cercek A et al. Immunotherapy in colorectal cancer: rationale, challenges and potential. Nat Rev Gastroenterol Hepatol. 2019; 16: 361-375 Crossref PubMed Scopus (480) Google Scholar However, the immune landscape of colorectal cancer is complex and heterogenous, and the development of these treatment strategies must consider, not only the histological context, but also its potential distinctive features. 2 Guinney J Dienstmann R Wang X et al. The consensus molecular subtypes of colorectal cancer. Nat Med. 2015; 21: 1350-1356 Crossref PubMed Scopus (2403) Google Scholar In The Lancet Oncology, Carlotta Antoniotti and colleagues 3 Antoniotti C Rossini D Pietrantonio F et al. Upfront FOLFOXIRI plus bevacizumab with or without atezolizumab in the treatment of patients with metastatic colorectal cancer (AtezoTRIBE): a multicentre, open-label, randomised, controlled, phase 2 trial. Lancet Oncol. 2022; (published online May 27.)https://doi.org/10.1016/S1470-2045(22)00274-1 Summary Full Text Full Text PDF PubMed Google Scholar report the results of the addition of atezolizumab to front-line FOLFOXIRI (fluorouracil, leucovorin, oxaliplatin, and irinotecan) plus bevacizumab (the atezolizumab group) compared with FOLFOXIRI plus bevacizumab (the control group) in patients with metastatic colorectal cancer from the phase 2 AtezoTRIBE trial, irrespective of tumour microsatellite status. The superiority of FOLFOXIRI plus bevacizumab over FOLFIRI (leucovorin, fluorouracil, and irinotecan) plus bevacizumab as front-line treatment of metastatic colorectal cancer was previously demonstrated in the phase 3 TRIBE trial. Drawing from this rationale, in this multicentre, academic study, the investigators raised the question of whether the restoration of the anticancer immunity through inhibition of the PD-1–PD-L1 axis enhances efficacy of an upfront intensified chemotherapy regimen. The underlying hypothesis was that chemotherapy can induce tumour cells to release more neoantigens, leading to immunogenic cell death and activation of CD8 T lymphocytes, while bevacizumab-mediated vasculature normalisation upon VEGF or VEGFR blockade promotes increased tumour-infiltrating lymphocytes and activation of effector immune cells. 4 Kanterman J Sade-Feldman M Biton M et al. Adverse immunoregulatory effects of 5FU and CPT11 chemotherapy on myeloid-derived suppressor cells and colorectal cancer outcomes. Cancer Res. 2014; 74: 6022-6035 Crossref PubMed Scopus (113) Google Scholar , 5 Terme M Pernot S Marcheteau E et al. VEGFA–VEGFR pathway blockade inhibits tumor-induced regulatory T-cell proliferation in colorectal cancer. Cancer Res. 2013; 73: 539-549 Crossref PubMed Scopus (413) Google Scholar Given that no significant benefit has been derived from the combination of immunotherapy with non-intensified regimens, 6 Grothey A Tabernero J Arnold D et al. Fluoropyrimidine (FP) + bevacizumab (BEV) + atezolizumab vs FP/BEV in BRAFwt metastatic colorectal cancer (mCRC): findings from Cohort 2 of MODUL—a multicentre, randomized trial of biomarker-driven maintenance treatment following first-line induction therapy. Ann Oncol. 2018; 29: viii714-viii715 Google Scholar , 7 Lenz HJ Parikh AR Spigel DR et al. Nivolumab (NIVO) + 5-fluorouracil/leucovorin/oxaliplatin (mFOLFOX6)/bevacizumab (BEV) versus mFOLFOX6/BEV for first-line (1L) treatment of metastatic colorectal cancer (mCRC): phase 2 results from CheckMate 9X8. Proc Am Soc Clin Oncol. 2022; 40 (abstr).: 8 Crossref PubMed Google Scholar the use of FOLFOXIRI is a rational partner in an untreated RAS-mutated enriched metastatic colorectal cancer population. Upfront FOLFOXIRI plus bevacizumab with or without atezolizumab in the treatment of patients with metastatic colorectal cancer (AtezoTRIBE): a multicentre, open-label, randomised, controlled, phase 2 trialThe addition of atezolizumab to first-line FOLFOXIRI plus bevacizumab is safe and improved progression-free survival in patients with previously untreated metastatic colorectal cancer. Full-Text PDF

Microbial metabolite restricts 5-fluorouracil-resistant colonic tumor progression by sensitizing drug transporters via regulation of FOXO3-FOXM1 axis.

The survival rate of colorectal cancer patients is adversely affected by the selection of tumors resistant to conventional anti-cancer drugs such as 5-fluorouracil (5FU). Although there is mounting evidence that commensal gut microbiota is essential for effective colon cancer treatment, the detailed molecular mechanisms and the role of gut microbial metabolites remain elusive. The goal of this study is to decipher the impact and mechanisms of gut microbial metabolite, urolithin A (UroA) and its structural analogue, UAS03 on reversal of 5FU-resistant (5FUR) colon cancers.Methods: We have utilized the SW480 and HCT-116 parental (5FU-sensitive) and 5FUR colon cancer cells to examine the chemosensitization effects of UroA or UAS03 by using both in vitro and in vivo models. The effects of mono (UroA/UAS03/5FU) and combinatorial therapy (UroA/UAS03 + 5FU) on cell proliferation, apoptosis, cell migration and invasion, regulation of epithelial mesenchymal transition (EMT) mediators, expression and activities of drug transporters, and their regulatory transcription factors were examined using molecular, cellular, immunological and flowcytometric methods. Further, the anti-tumor effects of mono/combination therapy (UroA or UAS03 or 5FU or UroA/UAS03 + 5FU) were examined using pre-clinical models of 5FUR-tumor xenografts in NRGS mice and azoxymethane (AOM)-dextran sodium sulfate (DSS)-induced colon tumors.Results: Our data showed that UroA or UAS03 in combination with 5FU significantly inhibited cell viability, proliferation, invasiveness as well as induced apoptosis of the 5FUR colon cancer cells compared to mono treatments. Mechanistically, UroA or UAS03 chemosensitized the 5FUR cancer cells by downregulating the expression and activities of drug transporters (MDR1, BCRP, MRP2 and MRP7) leading to a decrease in the efflux of 5FU. Further, our data suggested the UroA or UAS03 chemosensitized 5FUR cancer cells to 5FU treatment through regulating FOXO3-FOXM1 axis. Oral treatment with UroA or UAS03 in combination with low dose i.p. 5FU significantly reduced the growth of 5FUR-tumor xenografts in NRGS mice. Further, combination therapy significantly abrogated colonic tumors in AOM-DSS-induced colon tumors in mice.Conclusions: In summary, gut microbial metabolite UroA and its structural analogue UAS03 chemosensitized the 5FUR colon cancers for effective 5FU chemotherapy. This study provided the novel characteristics of gut microbial metabolites to have significant translational implications in drug-resistant cancer therapeutics.

Adipocytic Glutamine Synthetase Upregulation via Altered Histone Methylation Promotes 5FU Chemoresistance in Peritoneal Carcinomatosis of Colorectal Cancer.

The development of resistance to 5-fluorouracil (5FU) chemotherapy is a major handicap for sustained effective treatment in peritoneal carcinomatosis (PC) of colorectal cancer (CRC). Metabolic reprogramming of adipocytes, a component of the tumor microenvironment and the main composition of peritoneum, plays a significant role in drug resistance of PC, with the mechanisms being not fully understood. By performing metabolomics analysis, we identified glutamine (Gln), an important amino acid, inducing resistance to 5FU-triggered tumor suppression of CRC-PC through activating mTOR pathway. Noteworthily, genetic overexpression of glutamine synthetase (GS) in adipocytes increased chemoresistance to 5FU in vitro and in vivo while this effect was reversed by pharmacological blockage of GS. Next, we showed that methionine metabolism were enhanced in amino acid omitted from CRC-PC of GS transgenic (TgGS) mice, increasing intracellular levels of S-carboxymethy-L-cys. Moreover, loss of dimethylation at lysine 4 of histone H3 (H3k4me2) was found in adipocytes in vitro, which may lead to increased expression of GS. Furthermore, biochemical inhibition of lysine specific demethylase 1 (LSD1) restored H3k4me2, thereby reducing GS-induced chemoresistance to 5FU. Our findings indicate that GS upregulation-induced excessive of Gln in adipocytes via altered histone methylation is potential mediator of resistance to 5FU chemotherapy in patients with CRC-PC.

Hematologic Toxicity Patterns In Patients Treated With Chemoradiation For Anal Squamous Cell Carcinoma

The standard of care for patients with squamous cell carcinoma (SCC) of the anus is chemoradiation (CRT) with 5-fluorouracil (5FU) and mitomycin-c (MMC). Hematologic toxicity and blood count nadirs are a common toxicity of treatment. This analysis sought to correlate CRT dosage with hematologic toxicities as well as the trends and specific time points of toxicities, nadirs, and recovery from nadirs. Our cohort consisted of patients with anal SCC treated in the Department of Radiation Oncology at Stanford University with radiation, concurrent MMC, and either 5FU or CAPE. Patients were treated from October of 2009 to October of 2019. Data collection included radiation and chemotherapy treatment details, weekly labs during CRT, and follow up labs. Lymphocyte, absolute neutrophil (ANC), white blood cell (WBC), platelet, and hemoglobin (Hb) counts were analyzed during the initial 5-6 weeks of CRT and for patient’s 1, 3, 6, 9, and 12 month follow-up appointments. Blood count nadirs as well as trends to and recovery from nadirs were calculated. Time-to-events were calculated from the first week of CRT. Toxicities were graded using the Common Terminology Criteria for Adverse Events (CTCAE) v5.0. This analysis included 53 patients with anal SCC treated with radiation and concurrent MMC (n = 50), and either 5FU (n = 21) or CAPE (n = 29). Three patients were excluded from chemotherapy analysis due to an absence of chemotherapy, administration of only CAPE, or due to incomplete data. Median dose to primary tumor was 54Gy (39-66Gy) and the average length of treatment was 41 days. Hematologic toxicity was not significantly correlated with additional CAPE or 5FU chemotherapy agent used, or to total radiation does to primary tumor. Lymphocyte, ANC, platelet, and Hb levels changed significantly throughout the course of treatment (p<0.0001). Lymphocyte and platelet counts dropped sharply starting at a mean of 14 days after starting treatment, reaching a nadir at a mean of 28 days, and recovered after day 28. ANC dropped from initial maximum values at the beginning of treatment and reached a nadir at a mean of 10 days into treatment, recovered after day 10, dropped again at a mean of 30 days, reaching a second nadir at a mean of 38 days, and lastly recovered at a mean of 40 days into treatment, which coincided with the MMC infusions. WBC and Hb curves showed initial decreases in counts, but these trends were not significant. Lymphocyte, platelet, and ANC levels displayed distinct trends in the time-points to and recovery from nadirs. Quantification of these high-risk periods may allow clinicians to anticipate toxicities and prescribe effective supportive measures. This information may have further clinical significance for immunotherapy and in combating and preventing immunosuppressed states in patients receiving CRT.

A Novel Nomenclature for Repeat Motifs in the Thymidylate Synthase Enhancer Region and Its Relevance for Pharmacogenetic Studies.

Inhibition of thymidylate synthase (TS) is the primary mode of action for 5-fluorouracil (5FU) chemotherapy. TS expression is modulated by a variable number of tandem repeats in the TS enhancer region (TSER) located upstream of the TS gene (TYMS). Variability in the TSER has been suggested to contribute to 5FU-induced adverse events. However, the precise genetic associations remain largely undefined due to high polymorphism and ambiguity in defining genotypes. To assess toxicity associations, we sequenced the TSER in 629 cancer patients treated with 5FU. Of the 13 alleles identified, few could be unambiguously named using current TSER-nomenclature. We devised a concise and unambiguous systematic naming approach for TSER-alleles that encompasses all known variants. After applying this comprehensive naming system to our data, we demonstrated that the number of upstream stimulatory factor (USF1-)binding sites in the TSER was significantly associated with gastrointestinal toxicity in 5FU treatment.

CORONARY VASOSPASM ASSOCIATED WITH 5-FLUOROURACIL CHEMOTHERAPY

SESSION TITLE: Medical Student/Resident Cardiovascular Disease Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: 5-Fluorouracil (5FU) is the third most commonly used chemotherapeutic agent in the treatment of solid malignancies, particularly colorectal and pancreatic neoplasms. (1,2) Coronary vasospasm associated with 5FU is a known complication. (3) Cardiotoxicity occurs in 1% to 18% of patients receiving 5FU, and the incidence of this complication is on the rise.(4) The objective of this report is to highlight the characteristics of this uncommon complication. CASE PRESENTATION: A 66-year-old male with a past medical history of coronary artery disease status-post 4-vessel coronary artery bypass graft surgery and stage 4 colon adenocarcinoma with metastasis to the liver, presented to the emergency department (ED) with complaints of sudden onset of burning deep sub-sternal chest pain with no radiation. The chest pain began approximately eight hours after his first round of 5FU, leuocorin and eloxatin (FOLFOX) chemotherapy. In the ED he was given aspirin and heparin. EKG was concerning for ST-segment elevation myocardial infarction in the inferior leads. Troponins were negative (<0.01). A subsequent left heart catheterization revealed patent grafts and normal coronary arteries. An echocardiogram showed no acute wall motion abnormalities and an ejection fraction of 65%. The 5FU was discontinued and the patient’s chest pain improved. He was discharged with sublingual nitroglycerin and diltiazem. Two weeks later, the patient returned with similar chest pain after his second cycle with 5FU, leuocorin and eloxatin (FOLFOX). The symptoms of his coronary artery vasospasm were believed to be due to 5FU chemotherapy. He was further instructed to go to the cardiovascular ICU to receive nitroglycerine infusions before each cycle of FOLFOX. Unfortunately, the patient expired two months after starting chemotherapy; from cardiopulmonary arrest secondary to sepsis. DISCUSSION: This is a case of a 66-year-old male with a past medical history of coronary artery disease and stage 4 colon adenocarcinoma on 5-Fluorouracil, leuocorin and eloxatin (FOLFOX) chemotherapy presenting for coronary vasospasm likely from 5-Fluorouracil. Cardiotoxicity related to 5FU is poorly understood. Leading theories of the mechanism of 5FU-induced coronary vasospasm seem to involve 5FU's modification of smooth muscle tone. (5,6) Patients on 5FU with coronary vasospasm may have ECG findings suggestive of occlusion, (3) similar to our patient. Treatment of affected patients is determined by confirmation of 5FU as the definitive cause of the coronary vasospasm, as well as the requirement of 5FU as the only suitable chemotherapeutic agent. If possible, care providers should consider switching to an acceptable chemotherapeutic substitute. CONCLUSIONS: In conclusion, consideration should be given for patients with coronary artery disease starting 5-Fluorouracil chemotherapy. Reference #1: 1. Grem JL. 5-Fluorouracil: forty-plus and still ticking. A review of its preclinical and clinical development. Invest New Drugs 2000; 18: 299-313. 2. Myers CE. The pharmacology of the fluoropyrimidines. Pharmacol Rev 1981; 33: 1-15. 3. de Forni M, Malet-Martino MC, Jailais P, et al. Cardiotoxicity of high-dose continuous infusion fluorouracil: a prospective clinical study. Clin Oncol 1992; 10: 1795-1801. 4. Lestuzzi C, Vaccher E, Talamini R, et al. Effort myocardial ischemia during chemotherapy with 5-fluorouracil: an underestimated risk. Ann Oncol 2014; 25: 1059-1064. 5. Bonetti PO, Lerman LO and Lerman A. Endothelial dysfunction: a marker of atherosclerotic risk. Arterioscler Throb Vasc Biol 2003, 23: 168-175. 6. Reddy KG, Nair RN, Sheehan HM, et al. Evidence that selective endothelial dysfunction may occur in the absence of angiographic or ultrasound atherosclerosis in patients with risk factors for artherosclerosis. I Am Coll Cardio! 1994; 23: 833-843. DISCLOSURES: No relevant relationships by Ahmad ababneh, source=Web Response No relevant relationships by HANAD BASHIR, source=Web Response No relevant relationships by Ahmad JABRI, source=Web Response No relevant relationships by Gauranga Mahalwar, source=Web Response

Observational study of ocular surface squamous neoplasia: Risk factors, diagnosis, management and outcomes at a tertiary eye hospital in South Africa

BackgroundOcular surface squamous neoplasia (OSSN) is the most common ocular surface tumour. Diagnosis and management have traditionally been by excision biopsy. Recently there has been success with the use of topical chemo or immunotherapy, which has resulted in a move from invasive diagnosis by histology to an array of non-invasive diagnostic tests.MethodsThis observational study aims to describe the characteristics of patients with OSSN at St John Eye Hospital in Johannesburg, South Africa. Non-invasive diagnostic tests (impression cytology, anterior segment-OCT, methylene blue staining) will be compared to the gold standard, histology. Treatment success, recurrence and adverse events will be documented between three treatment options that include: surgical excision, topical 5-Fluorouracil (5FU) chemotherapy, and topical 5FU with retinoic acid therapy.DiscussionThere is a trend to the use of less invasive diagnosis and management for OSSN. Minimally invasive diagnostic tests include cytology, anterior-segment OCT and methylene blue staining. The study will compare these to the gold standard histology, thereby providing evidence for their use in clinical practice. Interferon alpha 2b is commonly used as immunotherapy for OSSN. The cost of this medication is prohibitive to its adoption in a developing country. We therefore decided to use 5FU as the chemotherapeutic agent of choice in this study. The success, adverse events and recurrence rates with this agent may provide additional evidence for its use in the management of OSSN. Overall, if diagnosis and management can be implemented with good success in the outpatient environment, care can be improved for this condition in a developing country.

New CXCR4 Antagonist Peptide R (Pep R) Improves Standard Therapy in Colorectal Cancer.

The chemokine receptor CXCR4 is overexpressed and functional in colorectal cancer. To investigate the role of CXCR4 antagonism in potentiating colon cancer standard therapy, the new peptide CXCR4 antagonist Peptide R (Pep R) was employed. Human colon cancer HCT116 xenograft-bearing mice were treated with chemotherapeutic agents (CT) 5-Fluorouracil (5FU) and oxaliplatin (OX) or 5FU and radio chemotherapy (RT-CT) in the presence of Pep R. After two weeks, CT plus Pep R reduced by 4-fold the relative tumor volume (RTV) as compared to 2- and 1.6-fold reductions induced, respectively, by CT and Pep R. In vitro Pep R addition to CT/RT-CT impaired HCT116 cell growth and further reduced HCT116 and HT29 clonal capability. Thus, the hypothesis that Pep R could target the epithelial mesenchyme transition (EMT) process was evaluated. While CT decreased ECAD and increased ZEB-1 and CD90 expression, the addition of Pep R restored the pretreatment expression. In HCT116 and HT29 cells, CT/RT-CT induced a population of CD133+CXCR4+ cells, supposedly a stem-resistant cancer cell population, while Pep R reduced it. Taken together, the results showed that targeting CXCR4 ameliorates the effect of treatment in colon cancer through inhibition of cell growth and reversal of EMT treatment-induced markers, supporting further clinical studies.

Natural Anthraquinone Emodin May Protect Immune and Gastrointestinal Health During Chemotherapy Treatment

The clinical utility of 5‐fluorouracil (5FU) chemotherapy remains hampered by hematopoietic and gastrointestinal toxicities. Emodin, a natural anthraquinone, has been shown to have anti‐inflammatory potential and may be an effective complementary therapeutic. The purpose of this study was to investigate the ability of emodin to reduce non‐specific toxicities, especially inflammation, associated with 5FU treatment. We evaluated acute and chronic exposure to 5FU utilizing tumor‐free mice. In the chronic phase, we randomized male C57BL/6 mice into four groups; Control + Vehicle (n=10), Control + Emodin (n=10), 5FU + Vehicle (n=15), 5FU + Emodin (n=15). 5FU was administered via I.P. for 3 cycles (5 consecutive days of 5FU at 35mg/kg followed by 9 days of recovery). Emodin was administered via oral gavage 3x/week at 40mg/kg throughout the entire 3 cycles of 5FU treatment. 5FU + Emodin treated mice showed improved grip strength (p&lt;0.05), run‐to‐fatigue time (p&lt;0.05), and reduced sensitivity to peripheral nociception (p&lt;0.05) compared to 5FU + Vehicle mice. Gene expression of the small intestine showed increased TNFα expression in 5FU + Vehicle treated mice which was decreased (p&lt;0.05) with emodin treatment. 5FU + Emodin treated mice show decreased colonic NOS2 expression (p&lt;0.05) but did not decrease MCP‐1, IL‐6, and TNFα expression compared to 5FU + Vehicle treated mice. To investigate the acute benefits of Emodin, male and female mice (n=125/sex) were randomized into four groups; 5FU + Vehicle, 5FU + 20mg/kg, 5FU + 40mg/kg, 5FU + 80mg/kg. Emodin treatment prevented body weight loss (p&lt;0.05) during one cycle of 5FU and was able to prevent performance loss during run‐to‐fatigue (p&lt;0.05). In male mice only, we observed a maintenance of the hematopoietic system, specifically in circulating lymphocytes, with increased emodin dosage (p&lt;0.05). Complementary to this trend, we found that bone marrow cells of 5FU + Emodin treated mice had greater percentage of cells in S (p&lt;0.05) and G2 (p&lt;0.05) phases, compared to 5FU + Vehicle mice. Flow cytometric analysis of spleen, mesenteric lymph node, and colon lamina propria did not show differences in total macrophages or neutrophils. Gene expression analysis of the colon did not show significant differences between all groups. Chronic exposure to 5FU causes increased production of Muc5ac, an indication of dysplasia in the colon, which is ameliorated with complementary emodin. Despite this, we did not see decreased Muc5ac from emodin treatment acutely. However, utilizing 16S rRNA FISH, we can see that 5FU can cause pockets of mucus disruption, gut barrier damage, and alterations in bacterial spatial arrangement, all of which was mitigated with increased dosage of emodin treatment. Taken together, emodin treatment may contribute to improved GI health by reducing damaging inflammation which may improve overall quality of life. Future analysis will expand on the inflammatory signaling pathways involved in emodin signaling and will investigate the potential role of the gut microbiome in reducing GI inflammation in this model of 5FU treatment.Support or Funding Information(AT009964) (F31AT009820)

Abstract P1-10-17: cGAS-STING driven immune activation predicts response to neoadjuvant chemotherapy and suggests rational IO combination therapies: Results of the Neo-DDIR study

Abstract Introduction: We previously identified an immune-driven gene expression signature recognizing loss of the Fanconi Anemia/BRCA DNA repair pathway - the DNA damage immune response (DDIR) assay. This 44-gene expression-based assay predicted response to DNA damaging chemotherapy in breast cancer, with a 5-year relapse-free survival hazard ratio of 0.37 post adjuvant treatment in assay positive patients. We demonstrated constitutive activation of the cGAS-STING pathway as the underlying mechanism driving immune gene expression in this subgroup, as a result of aberrant cytoplasmic DNA due to deficient DNA repair. We sought to evaluate efficacy of this immune-based signature in predicting response to neoadjuvant DNA-damaging chemotherapy (NAC) in breast cancer. Methods: Patients with early breast cancer were recruited from 2 Northern Ireland centres. All received 3 cycles of 5FU, epirubicin and cyclophosphamide (FEC) chemotherapy initially, with subsequent taxane/HER2 targeted therapies as indicated. Formalin fixed paraffin embedded tumor samples were obtained at baseline, post 3 cycles of FEC and at resection. Chemotherapy response was assessed by residual cancer burden (RCB) scoring. Gene expression data was obtained from all baseline samples and 18 midpoint samples containing sufficient residual tumor. DDIR assay call was determined using a predetermined cut-off. Tumor infiltrating lymphocytes (TILs) were assessed at each time point on H&amp;E stained sections. In addition, gene expression signatures (n=62) encompassing 6 hallmarks of cancer (Immune response, Genome instability, Angiogenesis, EMT, Cell death and Proliferation) were reported using the Almac ClaraT platform. Results: 46 patients were recruited - 26 were assay positive, with high immune gene expression, and 20 assay negative. The DDIR assay predicted NAC response with an odds ratio for RCB 0-1 of 4.67 (95% CI 1.13 - 15.09; p = 0.03). In patients with DDIR positive tumors, there was a strong association with immune response and immune checkpoint response signatures (p &amp;lt; 0.0001), as well as TILs in baseline biopsies (p =0.0262). Matched baseline and mid-point (after 3 cycles of FEC chemotherapy) gene expression analysis was available for 17 non-responders and 4 responders. In responding tumors a trend to reduced immune gene expression was seen, with an associated reduction in TILs (p = 0.0573), probably reflecting eradication of DDIR positive tumor cells (decreased tumor content on midpoint specimens in responders was noted compared to non-responders (p = 0.0257)). Conversely, increased DDIR signature score was observed in non-responders post FEC chemotherapy (p = 0.0332), with a trend to increased TIL infiltration (p = 0.0792). Immune checkpoint gene expression also increased following NAC in these patients. DDIR negative non-responding tumors demonstrated upregulation of angiogenesis signatures at baseline (p &amp;lt; 0.01), along with higher scores for TGFβ driven immune resistance signatures (p &amp;lt; 0.05), suggesting other potential therapeutic approaches for these patients. Conclusions: The cGAS-STING driven DDIR gene expression assay is predictive of response to neoadjuvant DNA damaging chemotherapy and is associated with high baseline immune infiltration. In keeping with the recent TONIC trial, we note anthracycline-based treatment as a potent activator of innate immunity. In non-responders, although TIL infiltration and immune gene expression is induced by DNA damaging chemotherapy, tumors do not respond, presumably due to intact DNA repair pathways and immune checkpoint gene induction. Therefore, there is a rationale to offer this population induction anthracycline-based chemotherapy followed by immune checkpoint targeting treatment to improve outcome. Citation Format: Stuart A McIntosh, Kienan I Savage, Colin James, Tong Lioe, Steven Walker, Keith Lowry, Laura Knight, Andrena McGavigan, Gemma Logan, Jane Hurwitz, Stephen J Kirk, Paul Harkin, Richard D Kennedy, Eileen E Parkes. cGAS-STING driven immune activation predicts response to neoadjuvant chemotherapy and suggests rational IO combination therapies: Results of the Neo-DDIR study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-17.

1. TIF1-γ associated dermatomyositis

IntroductionThis is a case of a patient presenting at the age of 39 with dermatomyositis. Subsequent investigations revealed her to have a TIFI-γ antibody and a metastatic colon adenocarcinoma. She was initially steroid responsive and her malignancy simultaneously responded to chemotherapy. A relapse of her malignancy was preceded by a flare in her dermatomyositis which proved to be non-responsive to steroids and further chemotherapy.Case descriptionThe patient presented to rheumatology at the age of 39 with a few-week history of a photosensitive rash on her face, upper chest and hands. This was followed by rapidly progressive symmetrical proximal and truncal muscle weakness and pain. She described dysphagia to solids and shortness of breath on exertion. She had a very short history of weight loss, attributed to reduced oral intake. She had no relevant past medical history and was taking no medications. Examination revealed power of 3/5 in both upper and lower limbs, a heliotrope rash with Gottrons papules with normal cardiovascular and respiratory examinations.CK at presentation was 3709. Haematology and biochemistry was otherwise normal. MRI thighs showed extensive myositis. ANA was positive with a positive TIFI-γ antibody. CT scan showed a proximal sigmoid mass with local and mediastinal lymphadenopathy and a 6mm lung nodule. Histology from a mediastinal node confirmed a metastatic adenocarcinoma.Initially, the patient was treated as an autoimmue dermatomyositis with pulsed IV methylprednisolone followed by high dose prednisolone. She responded rapidly both clinically and biochemically to steroids.Following the diagnosis of malignancy, she underwent a hemicolectomy from which she made an uneventful recovery. She then completed 12 cycles of oxaliplatin and 5FU chemotherapy, with interval CT scanning showing good partial response to treatment and she returned to work.One month after completing chemotherapy, whilst still taking prednisolone her rash reoccurred. This rapidly progressed despite an increase in her steroids and quickly became associated with weakness and further shortness of breath. CT showed a progression of her malignancy with carcinomatosis lymphangiitis. She received one cycle of Irinotecan before being admitted with neutropenic sepsis and progression of her cancer. At this time she decided to withdraw all treatment and died shortly afterwards at the age of 40, 18 months after her initial presentation.DiscussionTIF1-γ antibodies were first identified in 2006 and are involved in cell regeneration, apoptosis and innate immunity. High levels of TIFI-Y are found in the nuclei of regenerating myofibres. They are associated with dermatomyositis and are found in between 13 – 31% of adults and 22 – 29% of children.There is a strong association with malignancy in those aged over 39 (positive predictive value of 58%, sensitivity 78%, specificity 89%). There are no case reports of malignancy associated with TIF1- Y antibodies in patients under the age of 39. Malignancy typically presents early in the course of dermatomyositis, being diagnosed at presentation or within 8 months. There are case reports of TIF1- Y antibodies co-existing with Mi2 antibodies, increasing malignancy risk.In younger patients this association with cancer is not seen but the antibody is associated with skin ulceration and chronic disease. It is hypothesised that differences in HLA regions and protein conformation may account for these different phenotypes. Patients typically have a lower CK and there is a higher incidence of amyopathic dermatomyositis compared with other myositis specific antibodies.Our patient was at the lower end of the risk spectrum for malignancy and had no localising symptoms for this. She was initially very steroid responsive, which again lowered our threshold for suspicion of malignancy. Indeed, her CT was requested on the basis of shortness of breath looking for interstitial lung disease rather than anything more sinister.Key learning pointsTIF1-γ antibodies have a strong association with malignancy in patients over the age of 39. Clinicians should have a high index of suspicion even in the absence of symptoms of malignancy. Malignancy associated dermatomyositis can be steroid responsive. A relapse of dermatomyositis should raise suspicion for a relapse of malignancy.Conflicts of interestThe authors have declared no conflicts of interest.

Emodin, a natural anthraquinone, may help protect gastrointestinal health during chemotherapy treatment by decreasing inflammation of the gastric mucosa and preserving gut morphology

The clinical utility of 5‐fluorouracil (5FU) chemotherapy remains hampered by hematopoietic and gastrointestinal toxicities. Emodin, a natural anthraquinone, has been shown to have anti‐inflammatory potential and may be effective as a complementary therapeutic used during chemotherapy treatments. The purpose of this study was to evaluate the efficacy of emodin on reducing the non‐specific toxicities associated with 5FU treatment. 50 C57BL/6 were randomized into four groups; Control + Vehicle (n=5), Control + Emodin (n=5), 5FU + Vehicle (n=20), 5FU + Emodin (n=20). 5FU was administered via I.P. over 3 cycles of the following regime: 5 consecutive days of 5FU at 35mg/kg followed by 9 days of recovery. Emodin was administered via oral gavage 3x/week at 40mg/kg through the entire 3 cycles of 5FU treatment. 5FU + Emodin treated mice showed improved muscular strength (p&lt;0.05), fun‐to‐fatigue time (P&lt;0.05), and peripheral nociception (p&lt;0.05) compared to 5FU + Vehicle treated mice. 5FU + Emodin treated mice did not show recovery of decreased circulating immune cells and red blood cells that is common with 5FU treatment. 5FU + Vehicle treated mice did show slightly increased splenic CD11b+ F4/80+ macrophages (6.8%) vs. control mice (4.3%), a phenotype that was not decreased in 5FU + Emodin treated mice (6.5%). 5FU treatment shows classic inflammatory damage to the gastric mucosa. 5FU + Vehicle treated mice show decreased villus length (p&lt;0.05) compared to control mice. 5FU + Emodin treated mice showed increased villus length (p&lt;0.05) compared to 5FU + Vehicle treated mice but remained shorter (p&lt;0.05) than control mice. Further, small intestine tissue shows increased TNFα expression in 5FU + Vehicle treated mice which was decreased (p&lt;0.05) in 5FU + Emodin treated mice. Similar increases in inflammatory gene expression are seen in the colon tissue of 5FU + Vehicle treated mice. 5FU + Emodin treated mice show decreased colonic TNFα expression (p&lt;0.05) and trending decrease in MCP‐1 (p=0.052) and NOS2 (p=0.065) expression compared to 5FU + Vehicle treated mice. Taken together, Emodin treatment may contribute to improved GI health by reducing damaging inflammation which may contribute to improved overall quality of life. Future analysis will expand on the inflammatory signaling pathways involved in emodin signaling and will investigate the potential role of the gut microbiome in reducing GI inflammation in this model of 5FU treatment.Support or Funding Information1F31AT009820 to ATS.1RO1CA218578 to EAM. 1R41AT009964 to EAM. 5R21CA191966 to EAM.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of 5 fluorouracil chemotherapy on gut inflammation, functional parameters, and gut microbiota

Emerging evidence suggests that gut microbiota may influence the response to chemotherapy. We sought to characterize the effects of 5 fluorouracil (5FU) chemotherapy on colon inflammation and functional measures in colorectal cancer (CRC) and to further determine whether gut microbiota can influence this response. 50 C57BL/6 were randomized into four groups; Control + Vehicle (n = 10), Control + 5FU (n = 10), AOM/DSS + Vehicle (n = 15), and AOM/DSS + 5FU (n = 15). CRC was induced chemically by a single 10 mg/kg injection of azoxymethane (AOM) followed by two cycles (2% and 1%) of dextran sodium sulfate (DSS). Mice were then treated with 3 cycles of vehicle or 5FU (cycle 1: 40 mg/kg, cycle 2 + 3: 20 mg/kg). Functional tests (grip strength and run-to-fatigue) were performed prior to 5FU treatment (baseline) and at the completion of the second cycle of 5FU. Following the third 5FU cycle, mice were euthanized and the colon was evaluated for expression of inflammatory genes using RT-qPCR and stool samples were profiled using 16S rRNA sequencing. A second experiment used fecal microbiota transplantation from 5FU treated mice to control mice (n = 10–15/group) to determine whether 5FU associated changes in the microbiota could influence functional measures and colon inflammation. 5FU reduced grip strength (p < 0.05) and caused a trending decrease in run-to-fatigue performance in cancer mice (p = 0.06). Select intestinal inflammatory genes were significantly elevated with 5FU treatment and this was further exacerbated with cancer (p < 0.05). Microbiota analysis revealed increased dissimilarity and alterations in bacterial taxonomy in 5FU and AOM/DSS-treated mice (p < 0.05). Fecal transplant from 5FU treated mice reduced functional performance (p < 0.05) and altered select colon inflammatory markers (p < 0.05). This study provides evidence of an effect of 5FU on inflammatory responses and functional measures in a mouse model of CRC and suggests that gut microbes may play a role in some, but not all, 5FU related perturbations.

DNA methylation signature predictive of benefit from neoadjuvant chemotherapy in esophageal adenocarcinoma: Results from the MRC OEO2 phase III trial.

43 Background: Platinum and 5-Fluorouracil (5FU) neoadjuvant chemotherapy followed by surgery is one of the standard approaches for patients with resectable EAC. To date, there are no predictive biomarkers of chemotherapy benefit. We hypothesize that DNA methylation of genes in key biologic and oncogenic pathways predict for chemotherapy benefit in EAC. Methods: In the OE02 trial, 802 patients with resectable esophageal carcinoma were randomised to surgery alone (S) versus two cycles of cisplatin and 5FU chemotherapy followed by surgery (CS). DNA was extracted from 213 EAC resection specimens (110 from the (CS) arm, 103 from the (S) arm). DNA methylation was analyzed at 1505 CpG sites within 807 genes using the Illumina GoldenGate platform. Cox proportional hazard analysis was performed to identify predictive markers of survival in (CS) arm; non-negative matrix factorization (NMF) was used to delineate methylation signatures. Results: Methylation status of 1505 CpG sites had no statistical difference between the (CS) and (S) arms. In the (CS) arm, 87 (5.7%) CpG sites were initially identified as promising candidates in univariate analysis (p &lt; 0.05 cutoff). NMF generated a 4 CpG site signature which divided patients into poor risk and good risk. Genes involved in the signature include RUNX1T1, CCND2, MST1R and MMP14. Survival was significantly different between poor risk and good risk in (CS) arm (HR 0.32, 95% CI: 0.21 to 0.52, p &lt; 0.0001). No difference in survival was detected in the surgery arm (HR 1.12, 95% CI: 0.76 to 1.80, p = 0.48), suggesting the signature served as a predictive and not prognostic biomarker. Methylation signature remained an independent predictor of survival in multivariate analysis with clinicopathologic factors (along with age and vascular invasion). Conclusions: Chemotherapy does not appear to change methylation status of EAC. Hypermethylation of RUNX1T1, CCND2 and hypomethylation of MST1R and MMP14 leads to significantly decreased benefit from chemotherapy in EA. We describe an epigenetic signature which may serve as a predictive biomarker for chemotherapy benefit using data form the largest bank of DNA methylation in EA reported to date.

ANALYSIS OF NACL-MANNITOL HYDRATION ON RENAL FUNCTION OF HEAD AND NECK CANCER PATIENTS RECEIVING HIGH-DOSE CISPLATIN CHEMOTHERAPY COMBINATION

Cisplatin is one of platinum cytostatic drug for the medication of solid cancers, one of which is head and neck cancer. Adverse event that resulted during drug treatment was acute or chronic nephrotoxicity. Cisplatin concentration in proximal tubular epithelial cells is about 5 times the serum concentration. Platinum exposure on renal tubular cells bonding covalent complex which stimulate production of inflammatory factors that lead to apoptosis and necrosis cell. Cisplatin nephrotoxicity can be prevented by aggressive hydration or alternate method of administration. The aim of this study was to analyze the effectiveness of NaCl-Mannitol hydration on renal function of head and neck cancer patients receiving cisplatin 100 mg/m2 chemotherapy combination with 5FU or paclitaxel. This was a cohort, prospective, and observational study to analyze renal function of head and neck cancer patients receiving cisplatin 100 mg/m2 chemotherapy combination with 5FU or paclitaxel. Inclusion criteria were BUN 7-18 mg/dl and serum creatinine &lt; 2 mg/dl of any cycle. All patients received infuse NaCl-Mannitol hydration with term that provided in Surgeon Departement of Dr. Soetomo General Hospital. Data obtained were BUN, SCr, and eClCr Cockroft-Gault, each was measured pre- and post-hydration. In cisplatin and 5FU chemotherapy combination value BUN pre-hydration (11,99 + 4,62) mg/dl, value BUN post-hydration (12,14 + 4,74) mg/dl and value serum creatinine pre-hydration (0,97 + 0,34) mg/dl, value serum creatinine post-hydration (1,02 + 0,37) mg/dl. Meanwhile to the combination of cisplatin and paclitaxel chemotherapy, value BUN pre-hydration (10,19 + 2,58) mg/dl, value of BUN post-hydration (10,43 + 2,31) mg/dl and value of serum creatinine post- hydration (0,98 + 0,26) mg/dl. In conclusion, NaCl-Mannitol hydration administration is adequate which is shown by BUN and serum creatinine in pre- and post-hydration data within normal limits.

Comparison cisplatin with cisplatin plus 5FU in head and neck cancer patients received postoperative chemoradiotherapy

PurposeTo compare the treatment outcomes and toxicity of both cisplatin and cisplatin plus 5FU chemotherapy in head and neck cancer patients who have received surgery, in addition to postoperative chemoradiotherapy. Materials and methodsFrom May 1991 to December 2012, a total of 113 head and neck cancer patients who received surgery, along with postoperative chemoradiotherapy were analyzed. The primary sites were oral cavity (86), oropharynx (17), hypopharynx (4), and larynx (6). Thirty-nine patients received cisplatin (P), while 74 patients received cisplatin plus 5FU (PF). The endpoints were overall survival (OS), local failure-free survival (LFFS), and distant metastasis-free survival (DMFS). ResultsThe median follow up time was 43months, with a range of 4–222months. The 3-year rates of OS, LFFS, and DMFS were 62.1%, 71.3%, and 82.4%, respectively. The 3-year OS for P and PF were 71.3% and 57.5% (p=0.27).A multivariate analysis revealed that various chemotherapy regimens displayed no statistical difference for OS (Hazard Ratio [HR]=1.81; 95% Confidence Interval [CI]=0.963–3.408; p=0.065), LFFS (HR=0.98; 95% CI=0.458–2.127; p=0.973), and DMFS (HR=1.25; 95% CI=0.463–3.398; p=0.656).Grade 3 and 4 mucositis for P and PF group were 61.5% and 64.9%. A greater than grade 3 dermatitis for P and PF group were 7.7% and 14.9%. ConclusionPostoperative chemoradiotherapy with cisplatin alone appeared to have higher 3-year OS and lower severe mucositis and dermatitis than cisplatin plus 5FU.

The Development of an Umbrella Trial (PLATO) to Address Radiation Therapy Dose Questions in the Locoregional Management of Squamous Cell Carcinoma of the Anus

Purpose/Objective(s) Randomized controlled trials for patients with squamous cell carcinoma of the anus have been successfully performed, despite the rarity of the disease. The RTOG 9811 and ACT2 trials demonstrated no improvement in cancer outcomes when neoadjuvant or adjuvant cisplatin 5FU chemotherapy was used. To capture as many anal cancer patients as possible in future clinical trials, we developed an umbrella of trials testing efficacy and acceptable morbidity across different loco-regional risk strata. Materials/Methods We formed a network of UK and international multi-disciplinary trialists and identified the following priorities:- i] the need for national consensus guidance for the introduction of intensity modulated radiation therapy (IMRT) based on the successful principles and fractionation used in the ACT2 protocol prior to future trials; ii] to evaluate a strategy using selective chemoradiation therapy (CRT) after local excision of anal margin tumors; iii] to evaluate reduced dose CRT to the primary tumor in order to limit late effects whilst maintaining elective nodal irradiation in early stage disease; iv] to evaluate dose escalated CRT in locally advanced T3/4 and N+ stage disease. Results The PLATO (personalizing radiotherapy dose in anal cancer) umbrella trial comprising of the ACT3, 4 and 5 trials is funded by Cancer Research UK and is due to commence recruitment in Q3 2016.The ACT 3 trial (n = 90) is a non-randomized phase II study that will evaluate a strategy of local excision for T1N0 anal margin tumors with selective postoperative involved field CRT using 41.4 Gy in 23 fractions (F) and concurrent capecitabine reserved for patients with margins <=1mm. An exact single-stage A’Hern design is used. The ACT4 trial (n = 162) is a randomized phase II trial (2:1) comparing reduced dose CRT with 41.4 Gy in 23F to GTV with 50.4 Gy in 28F using concurrent capecitabine for T1-2(<4cm)N0 disease. An exact single-stage A’Hern design is used. The ACT5 trial (n = 677) is a seamless pilot (n = 60)/phase II (n = 140)/phase III trial (n = 672 total) that will compare 53.2 Gy with 58.8 Gy and 61.6 Gy using 28 fractions to GTV with either 5FU or capecitabine in T3/4 N1-3 disease. Only one of the dose escalated experimental arms will be evaluated for the phase III component. The primary end point for each trial is 3 year locoregional failure. Conclusion The PLATO trial concept allows different research questions across the locoregional disease spectrum to be addressed efficiently using a single protocol and clinical trial funding application. This type of trial design is increasingly important in the era of personalized medicine and the need for clinical studies to address different research questions within the same disease. Sharing the details of this concept should assist other investigators to develop similar future studies.