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Purpose: Reovirus is a wild-type oncolytic virus that is ubiquitous in the environment; most patients are therefore preimmune. Therapeutic administration leads to an increase in neutralizing antireovirus antibody (NARA) titer. We hypothesized that if NARA limited reovirus antitumor activity, the effect might be attenuated by coadministration of cyclophosphamide.Experimental design: In a phase I study, patients with advanced cancer received cyclophosphamide 3 days before intravenous reovirus serotype 3 Dearing (RT3D). The primary objective was to reduce the resulting rise in NARA titer. Cyclophosphamide dose was escalated from 25–1,000 mg/m2 through nine cohorts; we aimed to define a well-tolerated immunomodulatory dose.Results: The combination was well tolerated in 36 patients, with grade 3/4 toxicities only seen at or above the maximum tolerated dose of cyclophosphamide, which was 800 mg/m2 combined with reovirus. Immunosuppressive effect, defined as maintaining NARA titer rise below a predefined threshold, was observed in only one patient. Furthermore, despite expected myelosuppression seen at higher cyclophosphamide doses, no changes in T-cell subsets, including Tregs, occurred with dose escalation. Viable virus was detected in association with peripheral blood mononuclear cells (PBMC) from 14% of patients 10 days after the last RT3D injection, despite high plasma NARA titer, demonstrating a potential mechanism for prolonged evasion of neutralization by reovirus.Conclusions: Coadministration of cyclophosphamide with reovirus is safe, but does not attenuate host antiviral responses. Alternative immunomodulation approaches should be explored, but association with PBMCs may allow reovirus to persist and evade even high levels of neutralizing antibodies. Clin Cancer Res; 21(6); 1305–12. ©2014 AACR.

Supplementary Tables S1-S4 and Figures S1-S3. Tables S1/S2: % tumor response in mice with HCT116 and A2058 tumor xenografts following treatment; Table S3: Diagram showing dose-decision guidelines utilized in the MK-2206 clinical study based on observed toxicities; Table S4: number of patients with drug-related AEs in all courses of treatment; Figure S1/S2: Line graph showing the relative tumor volume and body weight observed in patients over time expressed in terms of days following treatment; Figure S3: Patient presenting with Grade III rash

Supplementary Figure 1. Total white blood cell numbers were reduced by higher doses of the cytotoxic CP in later cohorts

Purpose: KRAS is the most commonly mutated oncogene in human tumors. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies.Experimental Design: We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumors. Recommended dosing schedules were defined as MK-2206 at 135 mg weekly and selumetinib at 100 mg once daily.Results: Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug–drug interactions. Clinical antitumor activity included RECIST 1.0–confirmed partial responses in non–small cell lung cancer and low-grade ovarian carcinoma.Conclusion: Responses in KRAS-mutant cancers were generally durable. Clinical cotargeting of MEK and AKT signaling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). Clin Cancer Res; 21(4); 739–48. ©2014 AACR.

Background: MicroRNAs (miRs) are small non-coding RNAs involved in cell homeostasis. miR dysregulation has been linked with activation of oncogenic pathways, cancer progression and clinical outcome in mCRC. Chemo-refractory mCRC patients are often treated with regorafenib, a multi-tyrosine kinase inhibitor with anti-angiogenic effect. Given the limited clinical benefits of regorafenib in unselected patient populations, there is an unmet need for better patient stratification and identification of mechanisms of resistance. Methods: Serial liquid biopsies were obtained at baseline (BL) and monthly until disease progression (PD) in 43 patients treated with regorafenib for chemo-refractory mCRC in the context of a phase II clinical trial (PROSPECT-R). Tissue biopsies were obtained at BL, after 2 months and at PD within the same trial and used to establish Patient-Derived Organoids (PDOs) and for molecular analyses. PDOs co-cultures and PDO-xenotransplants were generated to study primary and acquired resistance to regorafenib. Liquid biopsies were also obtained from an additional cohort (n=97) of mCRC patients treated with regorafenib. MiR profiling was performed on baseline seras using NanoString nCounter platform and significant miRs were validated with digital droplet (dd)PCR in serum, plasma, PDOs and by In Situ Hybridization (ISH) in matching tissue biopsies. Functional experiments were performed in PDOs, PDO co-cultures and PDO-xenotransplants. Results: MiR expression was tested in 43 BL in the PROSPECT-R trial. Up-regulation of miR-652-3p was associated with poor PFS and OS. These results were validated by ddPCR on the same serum samples, matching plasmas and organoids. ISH confirmed upregulation of this miR in sequential tissues biopsies, PDOs and PDO-xenotransplants of patients with primary and acquired resistance to regorafenib. The same findings were confirmed in the validation cohort. Functional experiments showed that miR-652-3p upregulation has significant effects on cancer cell migration. Up and down-regulation of miR-652-3p upon regorafenib treatment translated in a significant effect on cell viability in PDO co-cultures and liver PDO-xenotransplants. RNA-sequencing analysis of miR-652-3p over-expressing organoids showed downregulation of several components of the serine synthesis pathway. Among them, phosphoserine aminotransferase (PSAT1) was validated as a miR-652-3p direct target. Rescue experiments confirmed that PSAT1 over-expression and silencing lead to increase sensitivity and resistance to regorafenib respectively via cell and non-cell autonomous regulation of autophagy. Conclusions: Our data suggest that miR-652-3p may be uses as a prognostic/predictive biomarker for the selection of treatment and provide mechanics insight on regorafenib resistance. Citation Format: Somaieh Hedayat, Andrea Lampis, George Vlachogiannis, khurum Khan, Silvia Marchetti, Matteo Fassan, Michele Ghidini, Ruwaida Begum, Marta Schirripa, Rodolfo Passalacqua, David Cunningham, Fotios Loupakis, Nicola Valeri. MicroRNA deregulation of the serine synthesis pathway controls intrinsic and non-cell autonomous mechanism of resistance to Regorafenib in metastatic colorectal cancer (mCRC) [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 5720.

Purpose: KRAS is the most commonly mutated oncogene in human tumors. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies. Experimental Design: We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumors. Recommended dosing schedules were defined as MK-2206 at 135 mg weekly and selumetinib at 100 mg once daily. Results: Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug–drug interactions. Clinical antitumor activity included RECIST 1.0–confirmed partial responses in non–small cell lung cancer and low-grade ovarian carcinoma. Conclusion: Responses in KRAS-mutant cancers were generally durable. Clinical cotargeting of MEK and AKT signaling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). Clin Cancer Res; 21(4); 739–48. ©2014 AACR.

MicroRNAs (miRs) are small non-coding RNAs involved in cell homeostasis and carcinogenesis and control multiple oncogenic pathways. Numerous miRs are aberrantly expressed in colorectal cancer (CRC) and their deregulation is associated with clinical outcome and cancer progression. Chemo-refractory metastatic CRC (mCRC) patients are often treated with regorafenib, a multi-tyrosine kinase inhibitor with anti-angiogenic effect. Given the limited clinical benefits of regorafenib in unselected patient populations, there is an unmet need for better patient selection and identification of mechanisms of resistance. miRs are highly stable and have shown encouraging value as potential biomarkers for CRC detection and prognosis. Here we aimed to identify circulatory miRs that might be exploited for the upfront selection of patients’ candidate to regorafenib treatment. We ran a translational phase II trial of regorafenib in chemo-refractory mCRC patients. Serum, plasma and tissue biopsies were obtained at baseline (BL), every four weeks and at disease progression (PD). Patient Derived Organoids (PDOs) and PDO-xenotransplants were generated to study primary and acquired resistance to regorafenib. MiR profiling was performed in baseline serum of all patients by NanoString nCounter platform of 800 genes and validated with digital droplet (dd)PCR in serum, plasma, PDOs and by In Situ Hybridization (ISH) in matching tissue biopsies. Progression Free Survival (PFS) was measured from date of registration to date of first progression/relapse or death from cancer progression. Overall Survival (OS) was measured from date of randomisation to death from cancer. Further validation was performed by ddPCR in 97 patients from an independent patient’s cohort. MiR expression was tested in 43 BL sera and dysregulation in 28 miRs was associated with PFS and OS. Up-regulation of miR-652-3p was associated with worse PFS and OS. These results were validated by ddPCR on the same serum samples, and matching plasmas. ISH confirmed upregulation of this miR in sequential tissues biopsies, PDOs and PDO-xenotransplants of patients with primary and acquired resistance to regorafenib. Validation in an independent patient’s cohort confirmed direct association of miR-652-3p dysregulation with PFS and OS. Functional experiments to define miR652-3p mediated resistance have shown that there is a decrease of tumour growth and migration in miR-652-3p inhibition in PDOs. We provide initial evidence suggesting that circulating miR-652-3p might work as a negative predictive biomarker for the upfront selection of patients’ candidate to regorafenib treatment. Citation Format: Somaieh Hedayat, Khurum Khan, David Cunningham, George Vlachogiannis, Andrea Lampis, Silvia Marchetti, Matteo Fassan, Ruwaida Begum, Marta Schirripa, Fotios Fotios Loupakis, Nicola Valeri. Circulating miR-652-3p as a biomarker of resistance to regorafenib in metastatic colorectal cancer patients [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 LB-305.

Despite stratification of only RAS wt CRCs for anti-EGFR antibody (aEa) therapy, many patients (pts) do not benefit and the molecular resistance (res) landscape remains incompletely understood. In order to decipher novel res mechanisms, we treated 40 RAS wt CRCs with single-agent aEa in a prospective trial and applied exome- and RNA-Seq to biopsies (bx) taken at baseline (BL) and at progression (PD). Among 20 BL bx from tumors with primary progression, 7 showed BRAF V600E mutations (mut), 3 harbored noncanonical BRAF D594K and/or KRAS L19F/A18D mut or KRAS amplifications (amp), 1 had a MEK1 mut and 1 was ERBB2 amp. Inactivating mut of the NF1 gene, a negative regulator of RAS, were found as novel mechanisms of primary res in 2 pts. No genetic drivers of primary res could be identified in 6/20 bx. Using transcriptomic analysis, 78% of pts with prolonged clinical benefit (≥16wks) displayed the Transit-Amplifying (TA) molecular subtype (Sadanandam et al., 2013). Stem-like (SL), goblet and inflammatory subtypes predominated (75%) among primary progressors. This significant enrichment in aEa sensitive CRCs (p=0.017) validates the TA subtype as a predictive biomarker. Exome- and deep Seq of PD bx from 13 pts who acquired res after prolonged clinical benefit detected KRAS mut or amp in only 2 cases. The FGFR2 ligand FGF10 was amplified in 1 PD bx and was validated in vitro as a novel mechanism of acquired res. No mut/amp of genes to which aEa res is usually attributed (RAS/RAF, EGFR, MET, ERBB2, MEK1) were found in the remaining 10 PD bx. Ultra-sensitive circulating tumor DNA-Seq at PD in 7 of these pts detected EGFR exodomain and RAS mut, including parallel evolution of multiple mut, in 3 cases. However, comparison to truncal TP53/APC mut showed that these res drivers were confined to small subclones and could therefore not explain the bulk of res. Overall, aberrations of RAS/RAF-pathway members and regulators were less abundant as drivers of acquired aEa res in this prospective trial than in reported retrospective series. Hence, we suspected additional novel mechanisms of acquired res. Molecular subtype switching from the aEa sensitive TA subtype to the comparatively insensitive SL subtype occurred in paired BL/PD bx from 3/7 pts who progressed after prolonged aEa benefit without detectable genetic res drivers in PD bx. Subtype switching was not observed in any of 6 analyzed BL/PD pairs from primary progressors. Considering the strong association of aEa sensitivity with TA subtype at BL, these data suggest subtype switching from TA to SL as a novel mechanism of acquired aEa res. This prospective trial revealed novel genetic (NF1 mut, FGF10 amp) and likely transcriptomic (TA to SL subtype switching) mechanisms of aEa resistance in CRC. These results should enable more precise aEa therapy stratification and may open opportunities to prevent res through SL subtype targeting strategies. Citation Format: Khurum Hayat Khan, Andrew Woolston, Georgia Spain, Louise Barber, Yatish Patil, Beatrice Griffiths, Reyes GonzalezExposito, Sonia Mansukhani, Matthew Davies, Sheela Rao, David Watkins, Francesco Sclafani, Jana Suntharanathan, Clare Peckitt, Ruwaida Begum, Isma Rana, Janet Thomas, Jacqui Oates, Annette Bryant, Andrew Wotherspoon, Nicos Fotiadis, Nasir Khan, Sebastian Guettler, Katharina von Loga, Naureen Starling, Ian Chau, Anguraj Sadanandam, David Cunningham, Marco Gerlinger. Molecular subtypes and novel genetic mechanisms of primary and acquired anti-EGFR resistance in colorectal cancer in the Prospect C biomarker trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4339.

Background: AE-mABs (cetuximab and/or panitumumab) have been approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC) patients (pts). Indeed, previous studies have identified mutations (MTs)/amplifications in RAS/RAF/MEK kinase pathway as the main genetic events promoting primary and acquired resistance to these mABs. RAS status is frequently established on archival material, as tumour re-biopsy may not be always feasible. PROSPECT-C is a prospective trial aiming to define novel and known mechanisms of resistance to AE-mABs by obtaining repeated biopsies and sequential bloods from pts receiving AE-mABs for chemo-refractory mCRC. Here we present the preliminary results of the PROSPECT-C liquid biopsy study where the goals were to: 1) confirm the ability of ctDNA to determine RAS status prior to treatment; 2) determine any discordance of RAS status between archival material and ctDNA; 3) confirm the ability of ctDNA to track emerging MTs in the RAS pathway. Materials and Methods: Plasma was collected at baseline (BL), every 4 weeks and at progression (PD). ctDNA was isolated using the QIAamp Circulating Nucleic Acid Kit (Qiagen) and analyzed by digital droplet PCR (QX200 Bio-Rad) for MTs in KRAS-G12D, KRAS-G13D, KRAS-G12V, KRAS-Q61HA>T, KRAS-Q61HA>C, and BRAF-V600E. In the next steps, plasma samples with no MTs in the first series of hotspots will be tested for the remaining KRAS hotspots, NRAS, EGFR, PIK3CA MTs and KRAS, c-MET and HER-2 amplifications (Data to be presented). Results: Twenty-four pts (all treated with AE-mAB monotherapy) with KRAS WT tumors (from archival tissue) have been treated on this ongoing study; 16/24 had ctDNA analysis (mean age 62 years, 62.5% males) so far. All pts were heavily pre-treated; 58.3%, 31.3% and 12.5% received 2, 3 and 4 lines of prior therapies respectively. Of 15 pts with BL samples, 5 had MTs at BL; 2 with KRAS-G12D, 1 with KRAS Q61HA>T and 2 pts with BRAF-V600E (in the latter case concordance was found with the archival material). Best response was found to be PD in 4/5 pts and stable disease in 1/5pt with BL MTs; 2 with BRAF MTs progressed in T (median PFS = 4.8 mo). 3/15 (20%) pts showed discordance in RAS MTs between archival material (WT) and baseline bloods (MT); all of them progressed within 3 mo. Conclusions: Liquid biopsies can be a useful tool in determining and tracking RAS MTs in pts undergoing anti-EGFR therapy for mCRC. ctDNA analysis to assess RAS mutational status prior to receiving AE-mABs in our series showed 20% discordance between the archival solid and BL liquid biopsies, which may account for resistance to these therapies. Citation Format: Khurum Khan, George Vlachogianis, David Cunningham, Jens Hahne, Mahnaz Darvish-Damavandi, Sarah Barton, Francesco Trevisani, Giulia Mentrast, Clare Peckitt, Andrea Lampis, Chiara Braconi, Nasir Khan, Ruwaida Begum, Naureen Starling, Sheela Rao, David Watkins, Annette Bryant, Ian Chau, Nicola Valeri. Validation of the role of circulating tumor DNA (ctDNA) in tracking mechanisms of resistance to anti-EGFR monoclonal antibodies (AE-mABs): preliminary results of the PROSPECT-C prospective trial. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3589. doi:10.1158/1538-7445.AM2015-3589