A Phase I trial to evaluate cisplatin, gemcitabine and the mTOR inhibitor temsirolimus for first-line treatment of participants with transitional cell urothelial cancer, or advanced solid cancer

A Phase I trial to evaluate cisplatin, gemcitabine and the mTOR inhibitor temsirolimus for first-line treatment of participants with transitional cell urothelial cancer, or advanced solid cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Phase I trial to evaluate cisplatin, gemcitabine and the mTOR inhibitor temsirolimus for first-line treatment of participants with transitional cell urothelial cancer, or advanced solid cancer Simon Pacey, Javier Garcia-Corbacho, Richard Baird, Tracie Madden, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5333803/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Gemcitabine plus cisplatin (GC) is effective in urothelial and other cancers. We investigated GC combined with escalating doses of temsirolimus (T), intravenous mTOR inhibitor. Adults with histologically confirmed solid tumours were enrolled. The primary endpoint was safety of GC with T, including dose limiting toxicity. Exploratory endpoints included pharmacokinetics and circulating tumour DNA (ctDNA) analyses. Fourteen participants (6 bladder cancer) were enrolled. Four dose levels were evaluated, adverse events were common and led to dose reductions, delays and omissions. Plasma concentrations of gemcitabine metabolites and cisplatin were unaffected by temsirolimus. ctDNA was detected in all samples taken from a sub set of patients. Levels of ctDNA changed earlier and correlated with imaging, while biologically relevant mutations e.g. PIK3CA were detected. Our results confirm the poor tolerability of three-drug combinations including GC and T but highlight the potential for ctDNA monitoring in Phase 1 cancer trials. Health sciences/Oncology/Cancer/Cancer therapy/Drug development Biological sciences/Cancer/Cancer therapy/Drug development mTOR temsirolimus cisplatin gemcitabine bladder ctDNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The five-year survival rates for patients with metastatic urothelial cancer (UC) remain low compared to those with other cancers ( 1 ). Despite this UC is regarded as a chemo-sensitive disease since response rates of 50% are reported with methotrexate / vinblastine / doxorubicin & cisplatin (MVAC). Gemcitabine and cisplatin (GC) become a standard of care given similar survival outcomes with an improved tolerability profile compared to MVAC ( 2 , 3 ). Further improvements in survival rates exploring cytotoxic combinations have been limited however, and novel therapies including immune checkpoint inhibitors, antibody-drug conjugates targeting e.g. nectin 4 as well as small molecule inhibitors against targets such as FGFR have entered clinical use ( 4 , 5 ). TCGA data reveals the PI3/AKT/mTOR pathway is commonly perturbed in urothelial cancers ( 6 ), including PIK3CA mutations, PTEN loss, and alterations in TSC1 ( 7 ) with implications for urothelial cancer growth and progression ( 8 ). Inhibitors of mTOR e.g. temsirolimus or everolimus ( 9 ) have shown preclinical and clinical efficacy in several cancers ( 10 – 13 ). These data include anti-proliferative effects in in-vitro urothelial cell line models. These anti-proliferative effects appear to be enhanced by gemcitabine or cisplatin combination therapy ( 14 – 16 ) Clinical data in, biomarker unselected, platinum-resistant UC patients report anti-tumour control as a single agent. In a phase II study of temsirolimus four out of fourteen patients experienced stable disease (SD) ( 17 ). Insimilar patient populations, treated with everolimus one study reported two partial responses (PR) and 8 SD out of 37 ( 18 ). A second trial noted 23 patients were progression free at 2 months with two PR out of 45 enrolled ( 19 ). To improve on these response rates, combination approaches have been tested. Both sirolimus, rapamycin( 20 ) and temsirolimus( 21 ) have been combined with gemcitabine and are reported to be tolerable. Three-drug combinations are reported using alternate (lower) dose schedules for gemcitabine and cisplatin (GC) e.g. with everolimus ( 22 ) or poorly tolerated eg GC-sunitinib ( 23 ). To our knowledge, no trials have combined cisplatin, gemcitabine and temsirolimus Patients and Methods Study design This was an investigator-initiated, multi-centre, Phase I/II single-arm trial in which participants were enrolled from two UK centres. Written informed consent was obtained from all participants prior to the commencement of trial-specific activities. The primary objective was to determine the safety profile of temsirolimus in combination with cisplatin and gemcitabine (GC-T) and establish the recommended phase 2 dose (RP2D). A single-arm, 3 + 3 dose-escalation, design was used (Table 2 ). Planned dose escalation involved addition of temsirolimus IV 10 mg on day 15 (cohort 1), day 8 and 15 (cohort 2) and day 1, 8 and 15 (cohort 3) to standard 21-day cycles of GC - cisplatin (70mg/m 2 IV day 1 of each cycle), gemcitabine (1000mg/m 2 IV day 1 and 8) – up to a maximum of 6 cycles. However, emerging toxicity data reviewed by Safety Review Committee (SRC) led to a protocol amendment and an alternative dose escalation schedule (cohort 3b: temsirolimus days 2, 9 and 15) to assess whether dose limiting toxicity (DLT) seen in cohort 3 might be due to interaction between temsirolimus and gemcitabine on day 8. Further increments of 5mg/week of temsirolimus were planned for subsequent cohorts. Detailed investigations of the pharmacokinetic (PK) profile of GC-T and pharmacodynamic (including circulating tumour DNA monitoring in some patients) were incorporated. The study was registered on clinicaltrials.gov (NCT01090466) and performed in accordance with the protocol, principles of Good Clinical Practice, the Declaration of Helsinki, and UK Clinical trial regulations as well as being approved by an independent ethics committee (Wales 09/MRE09/30). An independent steering committee supervised the conduct of the study and an independent SRC reviewed accumulating safety data and approved all dose escalation decisions. Participants Eligible patients were ≥ 16 years of age, with histologically confirmed locally-advanced and/or metastatic solid cancer not amenable to curative treatment with surgery or radiotherapy and for which GC-T was a suitable treatment option. Any number of previous lines of therapy were allowed except for advanced UC (no prior systemic therapy for locally advanced or metastatic disease, neoadjuvant or adjuvant chemotherapy allowed). Additional requirements were: included WHO performance status 0–2; minimum life expectancy of 3 months; not suitable for radical radiotherapy or curative surgery. For further detail see Supplementary Table 1. Study assessments Baseline and weekly blood draws included full blood count, biochemistry and coagulation. Serum creatinine was measured within 24 hours prior to dosing of cisplatin and used to calculate GFR according to the Cockcroft and Gault formula ( 27 ). Radiological evaluation of tumour response to therapy was carried out by CT scan at baseline and after cycles 3 (week 9), and 6 (week 18) then at 6 and 12 months after commencement of treatment, assessed according to RECIST version 1.1. Adverse Events (AEs) were monitored throughout the study and for 28 days after the last treatment with laboratory monitoring for toxicity recorded at baseline and every 4 weeks. AEs were graded in accordance with National Cancer Institute Common Terminology Criteria for AEs version 4.03. DLT were assessed during the dose escalation and defined as any of the following treatment- emergent AEs, occurring during the first cycle: complicated grade 4 neutropenia (absolute neutrophil count (ANC) 38°C and/or haemodynamic compromise; grade 4 thrombocytopenia (platelets < 25 x 10 9 /L) lasting at least 7 days; grade 3 or 4 thrombocytopenia (platelets < 50 x 10 9 /L) with active bleeding; any grade 3 or 4 non-haematological toxicity other than incompletely treated nausea and vomiting; and delay of commencement of cycle 2 by 15 days due to drug related toxicity. Serial blood samples were collected for PK analysis (at time-points detailed in supplementary table 2). Plasma concentrations of temsirolimus and its active metabolite (rapamycin/sirolimus) in the absence and presence of GC therapy to establish the effects on area under curve (AUC), C max , and half life. We also measured plasma concentrations of the active metabolite gemcitabine (dFdU) and cisplatin (total platinum). Results were determined by a combination of liquid chromatography tandem mass spectrometry (LC/MS/MS) (temsirolimus/gemcitabine) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (cisplatin) using validated methods. PK parameters were determined by non-compartmental analysis using validated WinNonlin software (Version 4.0.1) (Certara, CA.). Samples for ctDNA analysis were collected from patients who consented under an additional observational trial protocol at one participating centre (CALIBRATE, NCT02994511). Shallow WGS: ctDNA was isolated from plasma samples using the QIAsymphony DSP Circulating DNA Kit_V2.0(Qiagen,UK) following the manufacturer’s protocol. 5ng of isolated ctDNA was used to prepare libraries for shallow whole genome sequencing (sWGS) using the Thruplex DNA Seq HV kit (Takara Bio). Prepared libraries were sequenced on Illumina NextSeq instrument. Tumour fraction estimates were obtained using the Broad Institute’s ichorCNA tool, which employs a probabilistic model to derive tumour fraction, sample ploidy, and large-scale copy number aberrations from ultra-low coverage sWGS (0.1X) data. For SNV calling on ctDNA samples: Typically 30 ng of isolated ctDNA was used to prepare libraries using a custom 350 gene panel (Total Size: 1.46 Mb) from TWIST Biosciences using the manufacturer’s protocol and were sequenced on an Illumina NextSeq 2000. All exons of 350 genes and flanking sequences (+/- 20bp) are targeted with this panel. (See Supplementary table 1 for list of genes). Sequencing data was analysed using an in-house bioinformatics pipeline, aligning to hg38, which utilizes the following main algorithms: SNVs/INDELS identified following GATK4/MuTect2 best practices from Broad Institute. The minimum coverage to call variants is 100x, and the targeted coverage is 500x. The limit of detection of the assay is 3% variant allele frequency. The algorithm may not detect complex deletions, duplications, and large genomic rearrangements. All known benign variants and SNPs present at > 1% frequency in population databases (e.g. GnomAD, ExAC) were filtered out. All called variants were visually inspected in Integrative Genomics Viewer (IGV) and false positive calls were removed. Statistical Aspects A maximum of 42 participants evaluable for toxicity were required to complete all planned dose levels for Phase I. All participants were analysed on an intention to treat basis. All participants starting cycle 1 treatment were evaluable for toxicity. Participants who did not complete the first cycle of GTC, for reasons other than toxicity, were replaced at the current dose level for DLT evaluation. Categorical data were summarised by numbers and percentages. Continuous data were summarised by median and range if data were normally distributed or by median, IQR and range if data were skewed. Compliance was assessed for each participant. Results Participants and treatment Seventeen participants were screened between December 2012 and August 2015. Two progressed before completing screening and one was ineligible. Fourteen received at least 1 dose of temsirolimus, across four temsirolimus dosing schedules and all 14 were evaluable for safety. Baseline participant characteristics are summarised in Table 1. Six of the 14 participants (42.9%) had urothelial cancer (3 in cohort 1 and 1 in each of cohorts 2, 3 and 3b). Only 4 (28.4%) of the participants had received prior cisplatin, in the adjuvant or neoadjuvant setting. Three participants were treated in cohort 1 (temsirolimus 10mg, d15 only) and further 3 in cohort 2 (temsirolimus 10mg, days 8 and 15). A fourth patient was recruited in cohort 2 but was ineligible due to inadequate renal function and was withdrawn before starting treatment. Four participants were treated in each of cohorts 3 (temsirolimus 10mg, days 1, 8 and 15) and 3b (temsirolimus 10mg, days 2, 9 and 15), see Figure 1 Table 1: baseline participant characteristics n=14 Median age (range) 63.5 (40 - 80) Sex Male Female 5 (35.7%) 9 (64.3 %) WHO Performance status 0 1 9 (64.3 %) 5 (35.7 %) Histology Urothelial Cancer Non-Urothelial Cancer - Lung - Gallbladder - Uterus - Thyroid - Biliary Tract - Ovarian 6 (42.9 %) 8 (57.1 %) 3 (21.4 %) 1 (7.1 %) 1 (7.1 %) 1 (7.1 %) 1 (7.1 %) 1 (7.1 %) Metastatic disease (TNM staging) M0 M1 1 (7.1 %) 13 (92.9 %) Prior chemotherapy Yes No 9 (64.3 %) 5 (35.7 %) Prior cisplatin Yes No 4 (28.6 %) 10 (71.4 %) Prior radiotherapy Yes No 2 (14.3 %) 12 (85.7 %) Safety No DLTs were observed per protocol in the first 2 cohorts (temsirolimus on day 15 and on days 8,15 respecitvely). However, only 1/3 patients in cohort 2 received all three drugs as planned. In cohort 3, the addition of a third dose of temsirolimus (day 1,8,15) led to DLT in 2 participants (both complicated Grade 4 neutropenia). To mitigate potential PK or pharmacodynamic interaction between gemcitabine and temsirolimus, the trial protocol was amended to explore an alternative temsirolimus dosing schedule (cohort 3b), with temsirolimus dosing separated from gemcitabine and cisplatin (moved to D2 and 9, rather than D1 and 8), see Table 2. In cohort 3b two participants experienced DLT (Grade 3 hypokalaemia and Grade 3 macular rash). Twelve serious adverse events (SAE)/ reactions (SAR) were reported, occurring in eight patients. These were vomiting, infections, neutropenia, hypertension, vasovagal reaction, hypomagnesaemia, thrombocytopaenia and low potassium (Table 2). Detailed adverse event listings are provided in Supplementary Table 3. Table 2: Summary of dose limiting toxicity, serious adverse events (AE), serious adverse reactions and AE leading to temsiroimus discontinuation all listed by cohort: gemcitabine (G): 1000 mg/m 2 IV, cisplatin (C): 70 mg/m2 IV and temsirolimus (T): 10 mg IV Day Cohort 1 (n=3) Cohort 2 (n=3) Cohort 3 (n=4) Cohort 3b (n=4) Total (n=14) 1 GC GC GCT GC 2 T 8 G GT GT G 9 T 15 T T T T Any AE 207 140 169 213 729 AE ≥Grade 3 12 14 18 14 58 Dose Limiting Toxicities 0 0 2 2 4 Complicated Gr 4 neutropenia 0 0 2 0 2 Any Gr 3 or 4 toxicity 0 0 0 2 Hypokalaemia Gr3 Macular Rash Gr 3 2 Serious Adverse Events / Reactions (1 patient per box) 4 2 5 1 12 Vomiting Gr 3 (1) Lung infection Gr 3 (1) Hypertension Gr 3 (1) Low Potassium Gr 3 (1) Ano-rectal infection Gr 2 (1) Neutropenia fever Gr 3 (1) Neutropenic Sepsis Gr 3 (1) Thrombocytopenia Gr 4 (1) Vasovagal reaction Gr 3 (1) Hypomagnesaemia Gr 2 (1) Sepsis Gr 5 (1) Hypomagnesaemia Gr 2 (1) While no DLT were reported from cohort 1 or 2, dose reductions and delays were commonly required beyond the DLT period from cohort 2 (summarised in Figure 2), as is commonly seen with GC. Within the first 3 cycles: there were no missed doses in level 1 (one participant required delay of C2D1 only); in cohort 2, each of the three participants missed at least 1 dose of temsirolimus and two of them required a gemcitabine dose reduction; all cohort 3 and 3b participants missed at least one dose of temsirolimus, 7 out of 8 required gemcitabine dose reduction or missed doses, and start of cycle 2 was delayed in. Overall, 66.7%, 0%, 0% and 25% of patients received at least 3 cycles of treatment with no miseed visits and treatment per protocol forcohort 1, 2, 3 or 3b respectively. Cycles 4-6 were poorly tolerated with participants from all dose levels missing doses. Six of the 14 participants completed 6 cycles of platinum treatment. Data are summarised for each patient in Figure 2. Following review, by the TSC and sponsor, it was proposed that the study did not enter the pre-planned Phase II given that it was not feasible to deliver temsiroluimus doses with GC consistent with likely improved clinical outcomes. Therefore, as an investigator initiated study, with limited funding, the TMG ratified a decision to halt further patient recruitment. Efficacy Of the fourteen participants included in the safety analysis, eight underwent repeat imaging after cycle 6 to assess response to treatment: five with PR, three SD and two confirmed PD. Three patients had withdrawn/ missed the follow up visit, while one patient died from sepsis. Overall, the best response observed after 6 cycles were five PR (6/14, ORR = 42.8 %). Most of these were in patients with UC (4 PR, 4/6, ORR = 66.6 %, Supplementary Table 4) consistent with historical response rates in this group. At six-month follow-up scanning, one patient achieved CR, one additional patient had SD. All others either had PD (seven) or did not undergo the follow up visit (inlcuding the patient who died from sepsis and four that missed visit or withdrew). The single complete response (having received GCT and radiotherapy), was maintained at the time of last reported CT scan (57 months). This patient was included in an exploratory genetic mutation analysis (labelled 11 within their mansuscript), reported by Pritchard and colleagues (24). Pharmacokinetics The plasma concentrations of temsirolimus and its major metabolite (rapamycin) were similar whether administered alone (days 2, 9 or 15) or in combination with gemcitabine (d8) or gemcitabine and cisplatin (d1), as shown in Fig 3A,B and Supplementary Table 5. In addition extrapolated plasma concentrations are similar to historical data in a population predicted model for temsirolimus and its metabolite following IV doses of temsirolimus at 25mg (25, 26) and 10mg (27). The plasma concentrations of the major metabolite of gemcitabine (dFdU) appear unaffected by the administration of temsirolimus and are comparable to those seen in the FIESTA trial (28) as shown in Fig 3D and Supplementary Table 6. Co-administration of the three drugs did not have any measureable impact on steady state plasma concentrations of total platinum on day 1 and on day 8 were similar in the presence of gemcitabine with or without temsirolimus (Fig 3C and Supplementary Figure 1). Circulating tumour DNA (ctDNA) ctDNA was detected in all four plasma samples available from a subset of patients (Figure 4). A fall in total ctDNA (by IchorTF) correlated with PR on CT scan in one patient (Fig 4A) with urothelial cancer. However, ctDNA levels subsequently increased, prior to progression being detected on CT one month after completing study. Subtle increases in ctDNA levels in another patient with cholangiocarcinoma (Fig 4B) preceded PD on a scxan at week 18, following SD at 9 weeks of treatment. While a reduction in ctDNA levels were seen in a patient with non-small cell lung cancer, prior to imaging showing SD after 6 cycles. A fourth patient, with clear cell ovarian cancer (Fig 4D-F), had radiological SD (and stable CA125 tumour marker levels) after 3 cycles (Fig 4D, F). Corresponding stable levels of overall ctDNA were seen, as assessed by IchorTF. Interestingly, however, quantitation of levels of individual mutated PIK3CA and TP53 genes showed decreased levels at week 4, followed by increases to slightly above base-line at the end of study treatment (week 25). Conclusions The ToTEM study was conceived and designed before recent advances in therapy for metastatic UC including immune checkpoint inhibitors, ADCs and molecularly targeted therapy ie FGFR inhibitors. However, the data remain relevant given the continued importance of cisplatin and gemcitabine chemotherapy treating both patients with urothelial and other cancer types. In addition, we present analysis of circulating tumour DNA (ctDNA) that add to our interpretation of toxicity and pharmacokinetic data in this phase 1 study. To the best of our knowledge, the study presented here is the first report of a clinical trial which combines gemcitabine, cisplatin and the mTOR inhibitor, temsirolimus. In this Phase 1 study the majority of AE reported were haematological (neutropenia and thrombocytopenia). Non haematological toxicities were also reported (most commonly, nausea, vomiting, hypokalaemia or thromboembolic events). No DLTs were observed in the first two cohorts. However, the addition of a third temsirolimus dose (days 1, 8 and 15) in cohort 3 resulted in DLTs for 2/4 participants (complicated grade 4 neutropenia and thrombocytopenia). Despite adjusting the schedule in cohort 3b (temsirolimus day 2, 9 and 15) 2/4 patients experienced DLT (Grade 3 hypokalaemia and skin rash). In light of the poor tolerability we attempted to define a Recommended Dose for Sustained Tolerability (RDST) – a dose level at which at least 3 of 6 evaluable patients were treated for at least 3 cycles without unacceptable toxicity - since Grade 3 toxicities were seen in all dose levels from cycle 1. Although most AEs were Grade 1 or 2, toxicities were common during all six cycles and frequently led to dose delays or omissions. For example, only 27% of participants received at least three cycles of planned treatment (two patients in cohort 1 and one in cohort 3b). The study was closed and no further patients recruite, in part, because it was unlikely that a tolerable GC-T dose with adequate exposure to temosirolimus would be identified. Clinical activity was observed in our study, the overall best response (CR/PR/SD) rate was 42.8% for all patients and 66.7% for patients with urothelial cancer. However, our small sample size and termination of the study prior to the planned Phase II meant that it is not possible to draw conclusions or comment on whether temsirolimus increased the likelihood of responses compared with GC treatment. However, given that the recommended single-agent dose for temsirolimus in advanced renal cell carcinoma is 25mg, it is debatable whether the doses explored here were likely to add substantially to the efficacy of the GC combination. Triple-drug combinations are often limited by toxicities or require adjustment to cisplatin and/or gemcitabine dose and schedule. For example, Abida and colleagues recruited patients with treatment naive UC, utilising a lower dose of gemcitabine (800mg/m 2 ) and cisplatin (35mg/m 2 ) on D1 and 8, combined with everolimus using a 21 day schedule. Haematological DLT limited escalation, with dose level one (everolimus 5mg daily) declared the RP2D (29). Overall, our experience suggests that this triple-drug combination is not likely to be routinely able to deliver at temsirolimus doses sufficient to achieve meaningful additional efficacy without substantial toxicity. To investigate whether toxicities seen might be due to a pharmacokinetic interaction, we undertook an extensive sampling strategy to determine the plasma concentrations of all three drugs. Temsirolimus and rapamycin concentrations were similar (when extrapolated) in a population predicted model to those seen in other PK studies of intravenous temsirolimus monotherapy. Plasma concentrations of dFdU, the major metabolite of gemcitabine, and cisplatin were not substantially affected by the administration of temsirolimus and vice versa. We have not explored the alternative possibility that toxicity might result from an increased pharmacodynamic interaction on sensitive non-tumour tissues. However, we have attempted to explore pharmacodynamics of this novel combination on tumour cells via ctDNA analyses. Some interesting observations have emerged on how ctDNA analysis may impact the conduct of early phase clinical trials (30). Patient selection in early phase trials of molecularly targeted therapy may be aided by ctDNA detecting and tracking biological relevant mutations for the drug(s) being tested (31). Furthermore, ctDNA measurements may support conversations with patients around the risk-benefit of clinical trial participation, especially in phase 1 studies where response rates are uncertain. We were able to measure ctDNA levels in all plasma samples taken from a sub-set of patients (4/14) who were also enrolled into a parallel translational sampling study. Data presented here suggest potential value for UC patients in tracking PIK3CA mutation levels in ctDNA (patient 189-962) and for assessment of total levels of ctDNA as an adjunct to radiological response assessment. Data from two of our patients suggest additional benefit for ctDNA analysis in refining the benefit:cost assessment of treatment in patients with advanced disease. In one patient who experienced significant side effects (Patient 189-964) total ctDNA levels fell and stable disease persisted to 6 months. Conversely in another patient who had significant toxicity and required dose reductions (Patient 189-963) there was no fall in ctDNA levels before progression was detected on CT scan. Detailed translational (PK and PD) studies have added value to the interpretation of our results. Specifically, our data suggest that the combination of ctDNA analyses with traditional toxicity and efficacy assessments may enable a more complete assessment in successfully targeting the PI3K/AKT/mTOR pathway, aiding patient selection and improving results of future clinical trials (32). We report examples of the utility of plasma ctDNA measurements for patient management in early clinical trials to aid patient selection and inform prompt conversations with patients around the risk-benefit (33) in their decision making on continuing trial participation with confidence that benefits may outweigh risks of toxicity or pursuing alternative therapeutic approaches which minimise avoidable exposure to the risk of further toxicity. References Siegel RL, Miller KD, Wagle NS, Jemal A (2023) Cancer statistics, 2023. CA Cancer J Clin 73(1):17–48 Sternberg CN, Yagoda A, Scher HI, Watson RC, Herr HW, Morse MJ et al (1988) M-VAC (methotrexate, vinblastine, doxorubicin and cisplatin) for advanced transitional cell carcinoma of the urothelium. J Urol 139(3):461–469 von der Maase H, Hansen SW, Roberts JT, Dogliotti L, Oliver T, Moore MJ et al (2023) Gemcitabine and Cisplatin Versus Methotrexate, Vinblastine, Doxorubicin, and Cisplatin in Advanced or Metastatic Bladder Cancer: Results of a Large, Randomized, Multinational, Multicenter, Phase III Study. J Clin Oncol 41(23):3881–3890 Jones RJ, Crabb SJ, Linch M, Birtle AJ, McGrane J, Enting D et al (2024) Systemic anticancer therapy for urothelial carcinoma: UK oncologists' perspective. Br J Cancer 130(6):897–907 Lopez-Beltran A, Cookson MS, Guercio BJ, Cheng L (2024) Advances in diagnosis and treatment of bladder cancer. BMJ 384:e076743 Robertson AG, Kim J, Al-Ahmadie H, Bellmunt J, Guo G, Cherniack AD et al (2018) Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer. Cell 174(4):1033 Platt FM, Hurst CD, Taylor CF, Gregory WM, Harnden P, Knowles MA (2009) Spectrum of phosphatidylinositol 3-kinase pathway gene alterations in bladder cancer. Clin Cancer Res 15(19):6008–6017 Huan J, Grivas P, Birch J, Hansel DE (2022) Emerging Roles for Mammalian Target of Rapamycin (mTOR) Complexes in Bladder Cancer Progression and Therapy. Cancers (Basel). ;14(6) Dancey J (2010) mTOR signaling and drug development in cancer. Nat Rev Clin Oncol 7(4):209–219 Chan S, Scheulen ME, Johnston S, Mross K, Cardoso F, Dittrich C et al (2005) Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer. J Clin Oncol 23(23):5314–5322 Atkins MB, Hidalgo M, Stadler WM, Logan TF, Dutcher JP, Hudes GR et al (2004) Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma. J Clin Oncol 22(5):909–918 Hudes G, Carducci M, Tomczak P, Dutcher J, Figlin R, Kapoor A et al (2007) Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med 356(22):2271–2281 Galanis E, Buckner JC, Maurer MJ, Kreisberg JI, Ballman K, Boni J et al (2005) Phase II trial of temsirolimus (CCI-779) in recurrent glioblastoma multiforme: a North Central Cancer Treatment Group Study. J Clin Oncol 23(23):5294–5304 Schedel F, Pries R, Thode B, Wollmann B, Wulff S, Jocham D et al (2011) mTOR inhibitors show promising in vitro activity in bladder cancer and head and neck squamous cell carcinoma. Oncol Rep 25(3):763–768 Pinto-Leite R, Arantes-Rodrigues R, Ferreira R, Palmeira C, Colaço A, Moreira da Silva V et al (2014) Temsirolimus improves cytotoxic efficacy of cisplatin and gemcitabine against urinary bladder cancer cell lines. Urol Oncol 32(1):41e11–41e22 Garcia JA, Danielpour D (2008) Mammalian target of rapamycin inhibition as a therapeutic strategy in the management of urologic malignancies. Mol Cancer Ther 7(6):1347–1354 Gerullis H, Eimer C, Ecke TH, Georgas E, Freitas C, Kastenholz S et al (2012) A phase II trial of temsirolimus in second-line metastatic urothelial cancer. Med Oncol 29(4):2870–2876 Seront E, Rottey S, Sautois B, Kerger J, D'Hondt LA, Verschaeve V et al (2012) Phase II study of everolimus in patients with locally advanced or metastatic transitional cell carcinoma of the urothelial tract: clinical activity, molecular response, and biomarkers. Ann Oncol 23(10):2663–2670 Milowsky MI, Iyer G, Regazzi AM, Al-Ahmadie H, Gerst SR, Ostrovnaya I et al (2013) Phase II study of everolimus in metastatic urothelial cancer. BJU Int 112(4):462–470 Martin-Liberal J, Gil-Martín M, Sáinz-Jaspeado M, Gonzalo N, Rigo R, Colom H et al (2014) Phase I study and preclinical efficacy evaluation of the mTOR inhibitor sirolimus plus gemcitabine in patients with advanced solid tumours. Br J Cancer 111(5):858–865 Karavasilis V, Samantas E, Koliou GA, Kalogera-Fountzila A, Pentheroudakis G, Varthalitis I et al (2018) Gemcitabine Combined with the mTOR Inhibitor Temsirolimus in Patients with Locally Advanced or Metastatic Pancreatic Cancer. A Hellenic Cooperative Oncology Group Phase I/II Study. Target Oncol 13(6):715–724 Costello BA, Borad MJ, Qi Y, Kim GP, Northfelt DW, Erlichman C et al (2014) Phase I trial of everolimus, gemcitabine and cisplatin in patients with solid tumors. Invest New Drugs 32(4):710–716 Geldart T, Chester J, Casbard A, Crabb S, Elliott T, Protheroe A et al (2015) SUCCINCT: an open-label, single-arm, non-randomised, phase 2 trial of gemcitabine and cisplatin chemotherapy in combination with sunitinib as first-line treatment for patients with advanced urothelial carcinoma. Eur Urol 67(4):599–602 Pritchard JJG, Hamilton G, Hurst CD, Fraser S, Orange C, Knowles MA et al (2020) Monitoring of urothelial cancer disease status after treatment by digital droplet PCR liquid biopsy assays. Urol Oncol 38(9):737. .e1-.e10 Danesi R, Boni JP, Ravaud A (2013) Oral and intravenously administered mTOR inhibitors for metastatic renal cell carcinoma: pharmacokinetic considerations and clinical implications. Cancer Treat Rev 39(7):784–792 Boni JP, Hug B, Leister C, Sonnichsen D (2009) Intravenous temsirolimus in cancer patients: clinical pharmacology and dosing considerations. Semin Oncol 36(Suppl 3):S18–25 Zhang XL, Li A, Shi X, Kelley R, Huang RK, Simple YA, Sensitive (2012) LC–MS/MS Method for Simultaneous Determination of Temsirolimus and Its Major Metabolite in Human Whole Blood. Chromatographia 75:1405–1413 Jones RADJ, Crabb R, Hussain S, Birtle S, Spiliopoulou A, Robinson P, Knowles T, McLellan M, Jones L, Boylan H, Landers Z, Atuah D, Sherratt K, Brown D, Flanagan S, Loadman L, Twelves P, Chester C (2016) A phase Ib and pharmacokinetic trial of AZD4547 in combination with gemcitabine and cisplatin. J Clin Oncol 34:4521 Abida W, Milowsky MI, Ostrovnaya I, Gerst SR, Rosenberg JE, Voss MH et al (2016) Phase I Study of Everolimus in Combination with Gemcitabine and Split-Dose Cisplatin in Advanced Urothelial Carcinoma. Bladder Cancer 2(1):111–117 Wan JCM, Massie C, Garcia-Corbacho J, Mouliere F, Brenton JD, Caldas C et al (2017) Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer 17(4):223–238 Rothwell DG, Ayub M, Cook N, Thistlethwaite F, Carter L, Dean E et al (2019) Utility of ctDNA to support patient selection for early phase clinical trials: the TARGET study. Nat Med 25(5):738–743 Sun SY (2021) mTOR-targeted cancer therapy: great target but disappointing clinical outcomes. why? Front Med 15(2):221–231 Mackley MP, Fernandez NR, Fletcher B, Woolcott CG, Fernandez CV (2021) Revisiting Risk and Benefit in Early Oncology Trials in the Era of Precision Medicine: A Systematic Review and Meta-Analysis of Phase I Trials of Targeted Single-Agent Anticancer Therapies. JCO Precis Oncol 5:17–26 Additional Declarations There is NO Competing Interest. Supplementary Files Supp.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5333803","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":376809833,"identity":"afd2f2c1-6643-4dca-942f-da01a807ac97","order_by":0,"name":"Simon Pacey","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYJACgw8VcHYCcVoKZ5whVctnzjZStMi3nz24mXGenbxuA/PDD4xtaYS1GJzJSzYu3JZsuO0Am7EEY1sOEVokeMyMZ247wLjtAIMZA2NbBWEt8jN4zH/zzjlgv+0A+zfitDDc4DEw5m04kLjtAA/IFmIcdiYvwXDGseTkbYd5iiUSzhHhfWCIHTD4UGNnu+14+8YPH8qSiXAYAw+UZmYgOiJ5CCsZBaNgFIyCEQ4AHgg381zQdB8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3303-7577","institution":"University of Cambridge","correspondingAuthor":true,"prefix":"","firstName":"Simon","middleName":"","lastName":"Pacey","suffix":""},{"id":376809834,"identity":"823a032c-f773-4af2-8e3f-c8db21d8511f","order_by":1,"name":"Javier Garcia-Corbacho","email":"","orcid":"https://orcid.org/0000-0002-6109-8449","institution":"Hospital Clínic Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Javier","middleName":"","lastName":"Garcia-Corbacho","suffix":""},{"id":376809835,"identity":"b2f709de-2eca-48fe-b37e-6cacc2094587","order_by":2,"name":"Richard Baird","email":"","orcid":"https://orcid.org/0000-0001-7071-6483","institution":"Cancer Research UK Cambridge Centre","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Baird","suffix":""},{"id":376809836,"identity":"91c0207c-486d-4fd1-944c-4c9aaf05483b","order_by":3,"name":"Tracie Madden","email":"","orcid":"","institution":"Cardiff University","correspondingAuthor":false,"prefix":"","firstName":"Tracie","middleName":"","lastName":"Madden","suffix":""},{"id":376809837,"identity":"31051205-21da-46cf-bb65-40c202127f38","order_by":4,"name":"Angela Casbard","email":"","orcid":"","institution":"Cardiff University","correspondingAuthor":false,"prefix":"","firstName":"Angela","middleName":"","lastName":"Casbard","suffix":""},{"id":376809838,"identity":"60b7b1f9-58ba-4bc0-bef3-5660f83bffd7","order_by":5,"name":"Shubha Anand","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Shubha","middleName":"","lastName":"Anand","suffix":""},{"id":376809839,"identity":"09d129f2-155a-4793-b605-c52e99eb29a6","order_by":6,"name":"Katherine Honan","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Katherine","middleName":"","lastName":"Honan","suffix":""},{"id":376809840,"identity":"9bd81fa0-a6eb-4c43-8119-efd5f2788f41","order_by":7,"name":"Gareth Griffiths","email":"","orcid":"","institution":"University of Southampton","correspondingAuthor":false,"prefix":"","firstName":"Gareth","middleName":"","lastName":"Griffiths","suffix":""},{"id":376809841,"identity":"6e0a6bb6-ef85-49bb-85aa-d9e48262a019","order_by":8,"name":"Joanna Smith","email":"","orcid":"","institution":"Cardiff University","correspondingAuthor":false,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Smith","suffix":""},{"id":376809842,"identity":"f2a6cfd2-1034-4b53-b34b-47a75987abac","order_by":9,"name":"Robert Jones","email":"","orcid":"https://orcid.org/0000-0002-2904-6980","institution":"University of Glasgow","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Jones","suffix":""},{"id":376809843,"identity":"91cf0177-cf42-4aba-b3e2-00e16e45e5ec","order_by":10,"name":"Pavlina Spiliopoulou","email":"","orcid":"","institution":"Beatson West of Scotland Cancer Centre","correspondingAuthor":false,"prefix":"","firstName":"Pavlina","middleName":"","lastName":"Spiliopoulou","suffix":""},{"id":376809844,"identity":"22ddeacf-cf2d-4f16-b994-0cf6f2f8fb83","order_by":11,"name":"Paul Loadman","email":"","orcid":"https://orcid.org/0000-0002-4259-8616","institution":"University of Bradford","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Loadman","suffix":""},{"id":376809845,"identity":"4723c90e-c22f-4dea-8b9a-9dd8402d673f","order_by":12,"name":"John Chester","email":"","orcid":"","institution":"Alder Hey Childrens Hospital","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Chester","suffix":""}],"badges":[],"createdAt":"2024-10-25 16:45:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5333803/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5333803/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79318870,"identity":"3a4f5851-699f-4e9b-b3fa-adacf7760fb7","added_by":"auto","created_at":"2025-03-27 04:12:55","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummary diagram of participant recruitment\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5333803/v1/a9a17ea6fa722e1d868ccc10.jpeg"},{"id":79318869,"identity":"93687ac8-82a0-4a30-a455-e21f7495709e","added_by":"auto","created_at":"2025-03-27 04:12:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":123233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDose reductions and delays.\u003c/strong\u003e Delays in the commencement of cycles are shown by gaps in bars.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5333803/v1/e03c1397b74fe9841d9810ca.png"},{"id":79318867,"identity":"dd9a1cdc-93ce-4e15-bed8-3a79b90bf80a","added_by":"auto","created_at":"2025-03-27 04:12:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":52884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Temsirolimus (T) concentrations following administration on Day 1 gemcitabine (G), temsirolimus (T) and cisplatin (C) , Day 2 (T alone), Day 8 (GT), Day 9 (T alone) and Day 15 (T alone). \u003cstrong\u003eB\u003c/strong\u003e. Rapamycin concentrations on days 1,2,8,9 and 15, corresponding to temsirolimus levels in A. All timings in A and B are from start of the infusion (SOI) of temsirolimus. \u003cstrong\u003eC\u003c/strong\u003e. Concentrations of total plasma platinum on day 1 (GC) and Day 8 (GTC) following administration of gemcitabine (G), cisplatin (C) and temsirolimus (T). Comparison is made to platinum concentrations achieved in the FIESTA trial (28) with patients receiving the same platinum dose without temsirolimus. \u003cstrong\u003eD\u003c/strong\u003e. Concentrations of dFdU on day 1 (GC , n=10 and GTC , n=4) or day 8 (G, n=7 and GT , n=5) following administration of gemcitabine (G), cisplatin (C) and temsirolimus (T).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5333803/v1/99b79c5205123adaa45ffe21.png"},{"id":79318872,"identity":"1d3ba497-e16d-4cf3-bb4e-64cc16133c89","added_by":"auto","created_at":"2025-03-27 04:12:55","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":651269,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of change in ctDNA with time on treatment (weeks) in four selected patients labelled aligned with CT scan RECIST results (PR, SD, PD). A-D graphs show change in total ctDNA measured by IchorTF for individual patients. E. quantitative changes in individual genes (\u003cem\u003ePIK3CA, TP53 \u003c/em\u003emutations) relative to CT Scan (CT) findings at week 4 (W4) after start of treatment and End of Study (EoS) F. Representative abdominal CT scans at baseline and after Cycle 3 for same patient as in D and E, demonstrating stable liver lesion.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5333803/v1/ba60ccfb3357e8d7b4d2681a.jpeg"},{"id":79319908,"identity":"77c81f3d-c16a-47d1-ab5a-800ea58503bb","added_by":"auto","created_at":"2025-03-27 04:20:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1743294,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5333803/v1/9406a850-72b3-4cfe-810c-f8939774ec9e.pdf"},{"id":79318868,"identity":"05ef3d4e-32f0-44bd-a7f7-6f4f5b012b3d","added_by":"auto","created_at":"2025-03-27 04:12:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":101111,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.docx","url":"https://assets-eu.researchsquare.com/files/rs-5333803/v1/32650687f83b7c2c8f537953.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A Phase I trial to evaluate cisplatin, gemcitabine and the mTOR inhibitor temsirolimus for first-line treatment of participants with transitional cell urothelial cancer, or advanced solid cancer","fulltext":[{"header":"Background","content":"\u003cp\u003eThe five-year survival rates for patients with metastatic urothelial cancer (UC) remain low compared to those with other cancers (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Despite this UC is regarded as a chemo-sensitive disease since response rates of 50% are reported with methotrexate / vinblastine / doxorubicin \u0026amp; cisplatin (MVAC). Gemcitabine and cisplatin (GC) become a standard of care given similar survival outcomes with an improved tolerability profile compared to MVAC (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Further improvements in survival rates exploring cytotoxic combinations have been limited however, and novel therapies including immune checkpoint inhibitors, antibody-drug conjugates targeting e.g. nectin 4 as well as small molecule inhibitors against targets such as FGFR have entered clinical use (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTCGA data reveals the PI3/AKT/mTOR pathway is commonly perturbed in urothelial cancers (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), including \u003cem\u003ePIK3CA\u003c/em\u003e mutations, \u003cem\u003ePTEN\u003c/em\u003e loss, and alterations in \u003cem\u003eTSC1\u003c/em\u003e(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) with implications for urothelial cancer growth and progression (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Inhibitors of mTOR e.g. temsirolimus or everolimus (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) have shown preclinical and clinical efficacy in several cancers (\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). These data include anti-proliferative effects in \u003cem\u003ein-vitro\u003c/em\u003e urothelial cell line models. These anti-proliferative effects appear to be enhanced by gemcitabine or cisplatin combination therapy (\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) Clinical data in, biomarker unselected, platinum-resistant UC patients report anti-tumour control as a single agent. In a phase II study of temsirolimus four out of fourteen patients experienced stable disease (SD) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Insimilar patient populations, treated with everolimus one study reported two partial responses (PR) and 8 SD out of 37 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). A second trial noted 23 patients were progression free at 2 months with two PR out of 45 enrolled (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). To improve on these response rates, combination approaches have been tested. Both sirolimus, rapamycin(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) and temsirolimus(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) have been combined with gemcitabine and are reported to be tolerable. Three-drug combinations are reported using alternate (lower) dose schedules for gemcitabine and cisplatin (GC) e.g. with everolimus (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) or poorly tolerated eg GC-sunitinib (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). To our knowledge, no trials have combined cisplatin, gemcitabine and temsirolimus\u003c/p\u003e"},{"header":"Patients and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThis was an investigator-initiated, multi-centre, Phase I/II single-arm trial in which participants were enrolled from two UK centres. Written informed consent was obtained from all participants prior to the commencement of trial-specific activities.\u003c/p\u003e \u003cp\u003eThe primary objective was to determine the safety profile of temsirolimus in combination with cisplatin and gemcitabine (GC-T) and establish the recommended phase 2 dose (RP2D). A single-arm, 3\u0026thinsp;+\u0026thinsp;3 dose-escalation, design was used (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Planned dose escalation involved addition of temsirolimus IV 10 mg on day 15 (cohort 1), day 8 and 15 (cohort 2) and day 1, 8 and 15 (cohort 3) to standard 21-day cycles of GC - cisplatin (70mg/m\u003csup\u003e2\u003c/sup\u003e IV day 1 of each cycle), gemcitabine (1000mg/m\u003csup\u003e2\u003c/sup\u003e IV day 1 and 8) \u0026ndash; up to a maximum of 6 cycles. However, emerging toxicity data reviewed by Safety Review Committee (SRC) led to a protocol amendment and an alternative dose escalation schedule (cohort 3b: temsirolimus days 2, 9 and 15) to assess whether dose limiting toxicity (DLT) seen in cohort 3 might be due to interaction between temsirolimus and gemcitabine on day 8. Further increments of 5mg/week of temsirolimus were planned for subsequent cohorts.\u003c/p\u003e \u003cp\u003eDetailed investigations of the pharmacokinetic (PK) profile of GC-T and pharmacodynamic (including circulating tumour DNA monitoring in some patients) were incorporated.\u003c/p\u003e \u003cp\u003e The study was registered on clinicaltrials.gov (NCT01090466) and performed in accordance with the protocol, principles of Good Clinical Practice, the Declaration of Helsinki, and UK Clinical trial regulations as well as being approved by an independent ethics committee (Wales 09/MRE09/30). An independent steering committee supervised the conduct of the study and an independent SRC reviewed accumulating safety data and approved all dose escalation decisions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eParticipants\u003c/h3\u003e\n\u003cp\u003eEligible patients were \u0026ge; 16 years of age, with histologically confirmed locally-advanced and/or metastatic solid cancer not amenable to curative treatment with surgery or radiotherapy and for which GC-T was a suitable treatment option. Any number of previous lines of therapy were allowed except for advanced UC (no prior systemic therapy for locally advanced or metastatic disease, neoadjuvant or adjuvant chemotherapy allowed). Additional requirements were: included WHO performance status 0\u0026ndash;2; minimum life expectancy of 3 months; not suitable for radical radiotherapy or curative surgery. For further detail see Supplementary Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eStudy assessments\u003c/h3\u003e\n\u003cp\u003eBaseline and weekly blood draws included full blood count, biochemistry and coagulation. Serum creatinine was measured within 24 hours prior to dosing of cisplatin and used to calculate GFR according to the Cockcroft and Gault formula (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRadiological evaluation of tumour response to therapy was carried out by CT scan at baseline and after cycles 3 (week 9), and 6 (week 18) then at 6 and 12 months after commencement of treatment, assessed according to RECIST version 1.1.\u003c/p\u003e \u003cp\u003eAdverse Events (AEs) were monitored throughout the study and for 28 days after the last treatment with laboratory monitoring for toxicity recorded at baseline and every 4 weeks. AEs were graded in accordance with National Cancer Institute Common Terminology Criteria for AEs version 4.03.\u003c/p\u003e \u003cp\u003eDLT were assessed during the dose escalation and defined as any of the following treatment- emergent AEs, occurring during the first cycle: complicated grade 4 neutropenia (absolute neutrophil count (ANC)\u0026thinsp;\u0026lt;\u0026thinsp;0.5 x 109/L) lasting for \u0026ge;\u0026thinsp;7 days and/or with fever\u0026thinsp;\u0026gt;\u0026thinsp;38\u0026deg;C and/or haemodynamic compromise; grade 4 thrombocytopenia (platelets\u0026thinsp;\u0026lt;\u0026thinsp;25 x 10\u003csup\u003e9\u003c/sup\u003e/L) lasting at least 7 days; grade 3 or 4 thrombocytopenia (platelets\u0026thinsp;\u0026lt;\u0026thinsp;50 x 10\u003csup\u003e9\u003c/sup\u003e/L) with active bleeding; any grade 3 or 4 non-haematological toxicity other than incompletely treated nausea and vomiting; and delay of commencement of cycle 2 by 15 days due to drug related toxicity.\u003c/p\u003e \u003cp\u003eSerial blood samples were collected for PK analysis (at time-points detailed in supplementary table 2). Plasma concentrations of temsirolimus and its active metabolite (rapamycin/sirolimus) in the absence and presence of GC therapy to establish the effects on area under curve (AUC), C\u003csub\u003emax\u003c/sub\u003e, and half life. We also measured plasma concentrations of the active metabolite gemcitabine (dFdU) and cisplatin (total platinum). Results were determined by a combination of liquid chromatography tandem mass spectrometry (LC/MS/MS) (temsirolimus/gemcitabine) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (cisplatin) using validated methods. PK parameters were determined by non-compartmental analysis using validated WinNonlin software (Version 4.0.1) (Certara, CA.).\u003c/p\u003e \u003cp\u003eSamples for ctDNA analysis were collected from patients who consented under an additional observational trial protocol at one participating centre (CALIBRATE, NCT02994511).\u003c/p\u003e \u003cp\u003eShallow WGS: ctDNA was isolated from plasma samples using the QIAsymphony DSP Circulating DNA Kit_V2.0(Qiagen,UK) following the manufacturer\u0026rsquo;s protocol. 5ng of isolated ctDNA was used to prepare libraries for shallow whole genome sequencing (sWGS) using the Thruplex DNA Seq HV kit (Takara Bio). Prepared libraries were sequenced on Illumina NextSeq instrument. Tumour fraction estimates were obtained using the Broad Institute\u0026rsquo;s ichorCNA tool, which employs a probabilistic model to derive tumour fraction, sample ploidy, and large-scale copy number aberrations from ultra-low coverage sWGS (0.1X) data.\u003c/p\u003e \u003cp\u003eFor SNV calling on ctDNA samples: Typically 30 ng of isolated ctDNA was used to prepare libraries using a custom 350 gene panel (Total Size: 1.46 Mb) from TWIST Biosciences using the manufacturer\u0026rsquo;s protocol and were sequenced on an Illumina NextSeq 2000. All exons of 350 genes and flanking sequences (+/- 20bp) are targeted with this panel. (See Supplementary table 1 for list of genes). Sequencing data was analysed using an in-house bioinformatics pipeline, aligning to hg38, which utilizes the following main algorithms: SNVs/INDELS identified following GATK4/MuTect2 best practices from Broad Institute. The minimum coverage to call variants is 100x, and the targeted coverage is 500x. The limit of detection of the assay is 3% variant allele frequency. The algorithm may not detect complex deletions, duplications, and large genomic rearrangements. All known benign variants and SNPs present at \u0026gt;\u0026thinsp;1% frequency in population databases (e.g. GnomAD, ExAC) were filtered out. All called variants were visually inspected in Integrative Genomics Viewer (IGV) and false positive calls were removed.\u003c/p\u003e\n\u003ch3\u003eStatistical Aspects\u003c/h3\u003e\n\u003cp\u003eA maximum of 42 participants evaluable for toxicity were required to complete all planned dose levels for Phase I. All participants were analysed on an intention to treat basis. All participants starting cycle 1 treatment were evaluable for toxicity. Participants who did not complete the first cycle of GTC, for reasons other than toxicity, were replaced at the current dose level for DLT evaluation. Categorical data were summarised by numbers and percentages. Continuous data were summarised by median and range if data were normally distributed or by median, IQR and range if data were skewed. Compliance was assessed for each participant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eParticipants and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeventeen participants were screened between December 2012 and August 2015. \u0026nbsp;Two progressed before completing screening and one was ineligible. \u0026nbsp; Fourteen received at least 1 dose of temsirolimus, across four temsirolimus dosing schedules and all 14 were evaluable for safety.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBaseline participant characteristics are summarised in Table 1. \u0026nbsp;Six of the 14 participants (42.9%) had urothelial cancer (3 in cohort 1 and 1 in each of cohorts 2, 3 and 3b). \u0026nbsp;Only 4 (28.4%) of the participants had received prior cisplatin, in the adjuvant or neoadjuvant setting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThree participants were treated in cohort 1 (temsirolimus 10mg, d15 only) and further 3 in cohort 2 (temsirolimus 10mg, days 8 and 15). A fourth patient was recruited in cohort 2 but was ineligible due to inadequate renal function and was withdrawn before starting treatment. \u0026nbsp; Four participants were treated in each of cohorts 3 (temsirolimus 10mg, days 1, 8 and 15) and 3b (temsirolimus 10mg, days 2, 9 and 15), see Figure 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: baseline participant characteristics\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"584\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 296px;\"\u003e\n \u003cp\u003e\u003cstrong\u003en=14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian age (range)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 296px;\"\u003e\n \u003cp\u003e63.5 (40 - 80)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 296px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5 (35.7%)\u003c/p\u003e\n \u003cp\u003e9 (64.3 %)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWHO Performance status\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 296px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9 (64.3 %)\u003c/p\u003e\n \u003cp\u003e5 (35.7 %)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHistology\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eUrothelial Cancer\u003c/p\u003e\n \u003cp\u003eNon-Urothelial Cancer\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e- Lung\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e- Gallbladder\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e- Uterus\u003c/p\u003e\n \u003cp\u003e- Thyroid\u003c/p\u003e\n \u003cp\u003e- Biliary Tract\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e- Ovarian\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 296px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6 (42.9 %)\u003c/p\u003e\n \u003cp\u003e8 (57.1 %)\u003c/p\u003e\n \u003cp\u003e3 (21.4 %)\u003c/p\u003e\n \u003cp\u003e1 (7.1 %)\u003c/p\u003e\n \u003cp\u003e1 (7.1 %)\u003c/p\u003e\n \u003cp\u003e1 (7.1 %)\u003c/p\u003e\n \u003cp\u003e1 (7.1 %)\u003c/p\u003e\n \u003cp\u003e1 (7.1 %)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetastatic disease (TNM staging)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eM0\u003c/p\u003e\n \u003cp\u003eM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 296px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1 (7.1 %)\u003c/p\u003e\n \u003cp\u003e13 (92.9 %)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrior chemotherapy\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 296px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9 (64.3 %)\u003c/p\u003e\n \u003cp\u003e5 (35.7 %)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrior cisplatin\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 296px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4 (28.6 %)\u003c/p\u003e\n \u003cp\u003e10 (71.4 %)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrior radiotherapy\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 296px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2 (14.3 %)\u003c/p\u003e\n \u003cp\u003e12 (85.7 %)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eSafety\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo DLTs were observed per protocol in the first 2 cohorts (temsirolimus on day 15 and on days 8,15 respecitvely). However, only 1/3 patients in cohort 2 received all three drugs as planned. \u0026nbsp;In cohort 3, the addition of a third dose of temsirolimus (day 1,8,15) led to DLT in 2 participants (both complicated Grade 4 neutropenia). \u0026nbsp;To mitigate potential PK or pharmacodynamic interaction between gemcitabine and temsirolimus, the trial protocol was amended to explore an alternative temsirolimus dosing schedule (cohort 3b), with temsirolimus dosing separated from gemcitabine and cisplatin (moved to D2 and 9, rather than D1 and 8), see Table 2. \u0026nbsp; In cohort 3b two participants experienced DLT (Grade 3 hypokalaemia and Grade 3 macular rash).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTwelve serious adverse events (SAE)/ reactions (SAR) were reported, occurring in eight patients. \u0026nbsp; These were vomiting, infections, neutropenia, hypertension, vasovagal reaction, hypomagnesaemia, \u0026nbsp; thrombocytopaenia and low potassium (Table 2). \u0026nbsp;Detailed adverse event listings are provided in Supplementary Table 3. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Summary of dose limiting toxicity, serious adverse events (AE), serious adverse reactions and AE leading to temsiroimus discontinuation all listed by cohort: gemcitabine (G): 1000 mg/m\u003csup\u003e2\u003c/sup\u003e IV, cisplatin (C): 70 mg/m2 IV and temsirolimus (T): 10 mg IV\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"6\" valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 3px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCohort 1\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n=3)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCohort 2 (n=3)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCohort 3\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n=4)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCohort 3b (n=4)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"6\" valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal (n=14)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 3px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003eGC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003eGC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eGCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eGC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 3px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 3px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003eG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003eGT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eGT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 3px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 3px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAny AE\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e207\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e169\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e729\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003eAE \u0026ge;Grade 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e58\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDose Limiting Toxicities\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003eComplicated Gr 4 neutropenia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003eAny Gr 3 or 4 toxicity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003cp\u003eHypokalaemia Gr3\u003c/p\u003e\n \u003cp\u003eMacular Rash Gr 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"4\" valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSerious Adverse Events / Reactions\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(1 patient per box)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003eVomiting Gr 3\u0026nbsp;(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003eLung infection Gr 3 (1)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eHypertension Gr 3 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eLow Potassium Gr 3 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003eAno-rectal infection Gr 2\u0026nbsp;(1)\u003c/p\u003e\n \u003cp\u003eNeutropenia fever Gr 3\u0026nbsp;(1)\u003c/p\u003e\n \u003cp\u003eNeutropenic Sepsis Gr 3\u0026nbsp;(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003eThrombocytopenia Gr 4 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eVasovagal reaction Gr 3 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eHypomagnesaemia Gr 2 (1)\u003c/p\u003e\n \u003cp\u003eSepsis Gr 5 (1)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eHypomagnesaemia Gr 2 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eWhile no DLT were reported from cohort 1 or 2, dose reductions and delays were commonly required beyond the DLT period from cohort 2 (summarised in Figure 2), as is commonly seen with GC. \u0026nbsp;Within the first 3 cycles: there were no missed doses in level 1 (one participant required delay of C2D1 only); \u0026nbsp;in cohort 2, each of the three participants missed at least 1 dose of temsirolimus and two of them required a gemcitabine dose reduction; \u0026nbsp; all cohort 3 and 3b participants missed at least one dose of temsirolimus, 7 out of 8 required gemcitabine dose reduction or missed doses, and start of cycle 2 was delayed in. \u0026nbsp;Overall, 66.7%, 0%, 0% and 25% of patients received at least 3 cycles of treatment with no miseed visits and treatment per protocol forcohort 1, 2, 3 or 3b respectively. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCycles 4-6 were poorly tolerated with participants from all dose levels missing doses. \u0026nbsp;Six of the 14 participants completed 6 cycles of platinum treatment. \u0026nbsp; Data are summarised for each patient in Figure 2. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing review, by the TSC and sponsor, it was proposed that the study did not enter the pre-planned Phase II given that it was not feasible to deliver temsiroluimus doses with GC consistent with likely improved clinical outcomes. \u0026nbsp;Therefore, as an investigator initiated study, with limited funding, the TMG ratified a decision to halt further patient recruitment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEfficacy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOf the fourteen participants included in the safety analysis, eight underwent repeat imaging after cycle 6 to assess response to treatment: five with PR, three SD and two confirmed PD. \u0026nbsp;Three patients had withdrawn/ missed the follow up visit, while one patient died from sepsis. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, the best response observed after 6 cycles were five PR (6/14, ORR = 42.8 %). \u0026nbsp;Most of these were in patients with UC (4 PR, 4/6, ORR = 66.6 %, Supplementary Table 4) consistent with historical response rates in this group. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt six-month follow-up scanning, one patient achieved CR, one additional patient had SD. \u0026nbsp;All others either had PD (seven) or did not undergo the follow up visit (inlcuding the patient who died from sepsis and four that missed visit or \u0026nbsp;withdrew).\u003c/p\u003e\n\u003cp\u003eThe single complete response (having received GCT and radiotherapy), was maintained at the time of last reported CT scan (57 months). \u0026nbsp; This patient was included in an exploratory genetic mutation analysis (labelled 11 within their mansuscript), reported by Pritchard and colleagues (24). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacokinetics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plasma concentrations of temsirolimus and its major metabolite (rapamycin) were similar whether administered alone (days 2, 9 or 15) or in combination with gemcitabine (d8) or gemcitabine and cisplatin (d1), as shown in Fig 3A,B and Supplementary Table 5. \u0026nbsp;In addition extrapolated plasma concentrations are similar to historical data in a population predicted model for temsirolimus and its metabolite following IV doses of temsirolimus at 25mg (25, 26) and 10mg (27). \u0026nbsp; The plasma concentrations of the major metabolite of gemcitabine (dFdU) appear unaffected by the administration of temsirolimus and are comparable to those seen in the FIESTA trial (28) as shown in Fig 3D and Supplementary Table 6. \u0026nbsp;Co-administration of the three drugs did not have any measureable impact on steady state plasma concentrations of total platinum on day 1 and on day 8 were similar in the presence of gemcitabine with or without temsirolimus (Fig 3C and Supplementary Figure 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCirculating tumour DNA (ctDNA)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ectDNA was detected in all four plasma samples available from a subset of patients (Figure 4). \u0026nbsp; A fall in total ctDNA (by IchorTF) \u0026nbsp;correlated with PR on CT scan in one patient (Fig 4A) with urothelial cancer. However, ctDNA levels subsequently increased, prior to progression being detected on CT one month after completing study. \u0026nbsp;Subtle increases in ctDNA levels in another patient with cholangiocarcinoma (Fig 4B) preceded PD on a scxan at week 18, following SD at 9 weeks of treatment. \u0026nbsp;While a reduction in ctDNA levels were seen in a patient with non-small cell lung cancer, prior to imaging showing SD after 6 cycles. \u0026nbsp;A fourth patient, with clear cell ovarian cancer (Fig 4D-F), had radiological SD (and stable CA125 tumour marker levels) after 3 cycles (Fig 4D, F). \u0026nbsp; Corresponding stable levels of overall ctDNA were seen, as assessed by IchorTF. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, however, quantitation of levels of individual mutated \u003cem\u003ePIK3CA\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e genes showed decreased levels at week 4, followed by increases to slightly above base-line at the end of study treatment (week 25). \u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe ToTEM study was conceived and designed before recent advances in therapy for metastatic UC including immune checkpoint inhibitors, ADCs and molecularly targeted therapy ie FGFR inhibitors. \u0026nbsp;However, the data remain relevant given the continued importance of cisplatin and gemcitabine chemotherapy treating both patients with urothelial and other cancer types. \u0026nbsp;In addition, we present analysis of circulating tumour DNA (ctDNA) that add to our interpretation of toxicity and pharmacokinetic data in this phase 1 study. \u0026nbsp;\u0026nbsp;To the best of our knowledge, the study presented here is the first report of a clinical trial which combines gemcitabine, cisplatin and the mTOR inhibitor, temsirolimus. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this Phase 1 study the majority of AE reported were haematological (neutropenia and thrombocytopenia). \u0026nbsp;Non haematological toxicities were also reported (most commonly, nausea, vomiting, hypokalaemia or thromboembolic events). \u0026nbsp;No DLTs were observed in the first two cohorts. \u0026nbsp; \u0026nbsp;However, the addition of a third temsirolimus dose (days 1, 8 and 15) in cohort 3 resulted in DLTs for 2/4 participants (complicated grade 4 neutropenia and thrombocytopenia). \u0026nbsp;Despite adjusting the schedule in cohort 3b (temsirolimus day 2, 9 and 15) 2/4 patients experienced DLT (Grade 3 hypokalaemia and skin rash). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn light of the poor tolerability we attempted to define a Recommended Dose for Sustained Tolerability (RDST) – a dose level at which at least 3 of 6 evaluable patients were treated for at least 3 cycles without unacceptable toxicity - since Grade 3 toxicities were seen in all dose levels from cycle 1. \u0026nbsp;Although most AEs were Grade 1 or 2, toxicities were common during all six cycles and frequently led to dose delays or omissions. \u0026nbsp;For example, only 27% of participants received at least three cycles of planned treatment (two patients in cohort 1 and one in cohort 3b). \u0026nbsp;The study was closed and no further patients recruite, in part, because it was unlikely that a tolerable GC-T dose with adequate exposure to temosirolimus would be identified.\u003c/p\u003e\n\u003cp\u003eClinical activity was observed in our study, the overall best response (CR/PR/SD) rate was 42.8% for all patients and 66.7% for patients with urothelial cancer. \u0026nbsp; However, our small sample size and termination of the study prior to the planned Phase II meant that it is not possible to draw conclusions or \u0026nbsp;comment on whether temsirolimus increased the likelihood of responses compared with GC treatment. \u0026nbsp;However, given that the recommended single-agent dose for temsirolimus in advanced renal cell carcinoma is 25mg, it is debatable whether the doses explored here were likely to add substantially to the efficacy of the GC combination.\u003c/p\u003e\n\u003cp\u003eTriple-drug combinations are often limited by toxicities or require adjustment to cisplatin and/or gemcitabine dose and schedule. For example, Abida and colleagues recruited patients with treatment naive UC, utilising a lower \u0026nbsp;dose of gemcitabine (800mg/m\u003csup\u003e2\u003c/sup\u003e) and cisplatin (35mg/m\u003csup\u003e2\u003c/sup\u003e) on D1 and 8, combined with everolimus using a 21 day schedule. \u0026nbsp; Haematological DLT limited escalation, with dose level one (everolimus 5mg daily) declared the RP2D (29). \u0026nbsp;Overall, our experience suggests that this triple-drug combination is not likely to be routinely able to deliver at temsirolimus doses sufficient to achieve meaningful additional efficacy without substantial toxicity.\u003c/p\u003e\n\u003cp\u003eTo investigate whether toxicities seen might be due to a pharmacokinetic interaction, we undertook an extensive sampling strategy to determine the plasma concentrations of all three drugs. \u0026nbsp; Temsirolimus and rapamycin concentrations were similar (when extrapolated) in a population predicted model to those seen in other PK studies of intravenous temsirolimus monotherapy. \u0026nbsp; Plasma concentrations of dFdU, the major metabolite of gemcitabine, and cisplatin were not substantially affected by the administration of temsirolimus and vice versa. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe have not explored the alternative possibility that toxicity might result from an increased pharmacodynamic interaction on sensitive non-tumour tissues. \u0026nbsp; However, we have attempted to explore pharmacodynamics of this novel combination on tumour cells via ctDNA analyses.\u003c/p\u003e\n\u003cp\u003eSome interesting observations have emerged on how ctDNA analysis may impact the conduct of early phase clinical trials (30). \u0026nbsp;Patient selection in early phase trials of molecularly targeted therapy may be aided by\u0026nbsp;ctDNA detecting and tracking biological relevant mutations for the drug(s) being tested\u0026nbsp;(31). \u0026nbsp;Furthermore, ctDNA measurements may support conversations with patients around the risk-benefit of clinical trial participation, especially in phase 1 studies where response rates are uncertain. \u0026nbsp;We were able to measure ctDNA levels in all plasma samples taken from a sub-set of patients (4/14) who were also enrolled into a parallel translational sampling study. \u0026nbsp;Data presented here suggest\u0026nbsp;potential value for UC patients in tracking \u003cem\u003ePIK3CA\u003c/em\u003e mutation levels in ctDNA (patient 189-962) and for\u0026nbsp;assessment of total levels of ctDNA as an adjunct to radiological response assessment. \u0026nbsp;Data from two of our patients suggest additional benefit for ctDNA analysis in refining the benefit:cost assessment of treatment in patients with advanced disease. \u0026nbsp; In one patient who experienced significant side effects (Patient 189-964) \u0026nbsp;total ctDNA levels fell and stable disease\u0026nbsp;persisted to 6 months. \u0026nbsp; Conversely in another patient who had significant toxicity and required dose reductions\u0026nbsp;(Patient 189-963)\u0026nbsp;there was no fall in ctDNA levels before progression was detected on CT scan. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDetailed translational (PK and PD) studies have added value to the interpretation of our results. \u0026nbsp; Specifically, our data \u0026nbsp;suggest that\u0026nbsp;the combination of ctDNA analyses with traditional toxicity and efficacy assessments may enable a more complete assessment\u0026nbsp;in successfully targeting the PI3K/AKT/mTOR pathway, aiding patient selection and improving results of future clinical trials (32). \u0026nbsp;We report examples of the utility of plasma ctDNA measurements for patient management in early clinical trials to aid patient selection and inform prompt conversations with patients around the risk-benefit (33) in their decision making on continuing trial participation with confidence that benefits may outweigh risks of toxicity or pursuing alternative therapeutic approaches which minimise\u0026nbsp;avoidable exposure to the risk of further toxicity. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Wagle NS, Jemal A (2023) Cancer statistics, 2023. CA Cancer J Clin 73(1):17\u0026ndash;48\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSternberg CN, Yagoda A, Scher HI, Watson RC, Herr HW, Morse MJ et al (1988) M-VAC (methotrexate, vinblastine, doxorubicin and cisplatin) for advanced transitional cell carcinoma of the urothelium. J Urol 139(3):461\u0026ndash;469\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evon der Maase H, Hansen SW, Roberts JT, Dogliotti L, Oliver T, Moore MJ et al (2023) Gemcitabine and Cisplatin Versus Methotrexate, Vinblastine, Doxorubicin, and Cisplatin in Advanced or Metastatic Bladder Cancer: Results of a Large, Randomized, Multinational, Multicenter, Phase III Study. J Clin Oncol 41(23):3881\u0026ndash;3890\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones RJ, Crabb SJ, Linch M, Birtle AJ, McGrane J, Enting D et al (2024) Systemic anticancer therapy for urothelial carcinoma: UK oncologists' perspective. Br J Cancer 130(6):897\u0026ndash;907\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLopez-Beltran A, Cookson MS, Guercio BJ, Cheng L (2024) Advances in diagnosis and treatment of bladder cancer. BMJ 384:e076743\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobertson AG, Kim J, Al-Ahmadie H, Bellmunt J, Guo G, Cherniack AD et al (2018) Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer. Cell 174(4):1033\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlatt FM, Hurst CD, Taylor CF, Gregory WM, Harnden P, Knowles MA (2009) Spectrum of phosphatidylinositol 3-kinase pathway gene alterations in bladder cancer. Clin Cancer Res 15(19):6008\u0026ndash;6017\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuan J, Grivas P, Birch J, Hansel DE (2022) Emerging Roles for Mammalian Target of Rapamycin (mTOR) Complexes in Bladder Cancer Progression and Therapy. Cancers (Basel). ;14(6)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDancey J (2010) mTOR signaling and drug development in cancer. Nat Rev Clin Oncol 7(4):209\u0026ndash;219\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChan S, Scheulen ME, Johnston S, Mross K, Cardoso F, Dittrich C et al (2005) Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer. J Clin Oncol 23(23):5314\u0026ndash;5322\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtkins MB, Hidalgo M, Stadler WM, Logan TF, Dutcher JP, Hudes GR et al (2004) Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma. J Clin Oncol 22(5):909\u0026ndash;918\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHudes G, Carducci M, Tomczak P, Dutcher J, Figlin R, Kapoor A et al (2007) Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med 356(22):2271\u0026ndash;2281\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalanis E, Buckner JC, Maurer MJ, Kreisberg JI, Ballman K, Boni J et al (2005) Phase II trial of temsirolimus (CCI-779) in recurrent glioblastoma multiforme: a North Central Cancer Treatment Group Study. J Clin Oncol 23(23):5294\u0026ndash;5304\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchedel F, Pries R, Thode B, Wollmann B, Wulff S, Jocham D et al (2011) mTOR inhibitors show promising in vitro activity in bladder cancer and head and neck squamous cell carcinoma. Oncol Rep 25(3):763\u0026ndash;768\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinto-Leite R, Arantes-Rodrigues R, Ferreira R, Palmeira C, Cola\u0026ccedil;o A, Moreira da Silva V et al (2014) Temsirolimus improves cytotoxic efficacy of cisplatin and gemcitabine against urinary bladder cancer cell lines. Urol Oncol 32(1):41e11\u0026ndash;41e22\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarcia JA, Danielpour D (2008) Mammalian target of rapamycin inhibition as a therapeutic strategy in the management of urologic malignancies. Mol Cancer Ther 7(6):1347\u0026ndash;1354\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerullis H, Eimer C, Ecke TH, Georgas E, Freitas C, Kastenholz S et al (2012) A phase II trial of temsirolimus in second-line metastatic urothelial cancer. Med Oncol 29(4):2870\u0026ndash;2876\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeront E, Rottey S, Sautois B, Kerger J, D'Hondt LA, Verschaeve V et al (2012) Phase II study of everolimus in patients with locally advanced or metastatic transitional cell carcinoma of the urothelial tract: clinical activity, molecular response, and biomarkers. Ann Oncol 23(10):2663\u0026ndash;2670\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMilowsky MI, Iyer G, Regazzi AM, Al-Ahmadie H, Gerst SR, Ostrovnaya I et al (2013) Phase II study of everolimus in metastatic urothelial cancer. BJU Int 112(4):462\u0026ndash;470\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin-Liberal J, Gil-Mart\u0026iacute;n M, S\u0026aacute;inz-Jaspeado M, Gonzalo N, Rigo R, Colom H et al (2014) Phase I study and preclinical efficacy evaluation of the mTOR inhibitor sirolimus plus gemcitabine in patients with advanced solid tumours. Br J Cancer 111(5):858\u0026ndash;865\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaravasilis V, Samantas E, Koliou GA, Kalogera-Fountzila A, Pentheroudakis G, Varthalitis I et al (2018) Gemcitabine Combined with the mTOR Inhibitor Temsirolimus in Patients with Locally Advanced or Metastatic Pancreatic Cancer. A Hellenic Cooperative Oncology Group Phase I/II Study. Target Oncol 13(6):715\u0026ndash;724\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCostello BA, Borad MJ, Qi Y, Kim GP, Northfelt DW, Erlichman C et al (2014) Phase I trial of everolimus, gemcitabine and cisplatin in patients with solid tumors. Invest New Drugs 32(4):710\u0026ndash;716\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeldart T, Chester J, Casbard A, Crabb S, Elliott T, Protheroe A et al (2015) SUCCINCT: an open-label, single-arm, non-randomised, phase 2 trial of gemcitabine and cisplatin chemotherapy in combination with sunitinib as first-line treatment for patients with advanced urothelial carcinoma. Eur Urol 67(4):599\u0026ndash;602\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePritchard JJG, Hamilton G, Hurst CD, Fraser S, Orange C, Knowles MA et al (2020) Monitoring of urothelial cancer disease status after treatment by digital droplet PCR liquid biopsy assays. Urol Oncol 38(9):737. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e.e1-.e10\u003c/span\u003e\u003cspan address=\"http://.e1-.e10\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDanesi R, Boni JP, Ravaud A (2013) Oral and intravenously administered mTOR inhibitors for metastatic renal cell carcinoma: pharmacokinetic considerations and clinical implications. Cancer Treat Rev 39(7):784\u0026ndash;792\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoni JP, Hug B, Leister C, Sonnichsen D (2009) Intravenous temsirolimus in cancer patients: clinical pharmacology and dosing considerations. Semin Oncol 36(Suppl 3):S18\u0026ndash;25\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang XL, Li A, Shi X, Kelley R, Huang RK, Simple YA, Sensitive (2012) LC\u0026ndash;MS/MS Method for Simultaneous Determination of Temsirolimus and Its Major Metabolite in Human Whole Blood. Chromatographia 75:1405\u0026ndash;1413\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones RADJ, Crabb R, Hussain S, Birtle S, Spiliopoulou A, Robinson P, Knowles T, McLellan M, Jones L, Boylan H, Landers Z, Atuah D, Sherratt K, Brown D, Flanagan S, Loadman L, Twelves P, Chester C (2016) A phase Ib and pharmacokinetic trial of AZD4547 in combination with gemcitabine and cisplatin. J Clin Oncol 34:4521\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbida W, Milowsky MI, Ostrovnaya I, Gerst SR, Rosenberg JE, Voss MH et al (2016) Phase I Study of Everolimus in Combination with Gemcitabine and Split-Dose Cisplatin in Advanced Urothelial Carcinoma. Bladder Cancer 2(1):111\u0026ndash;117\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan JCM, Massie C, Garcia-Corbacho J, Mouliere F, Brenton JD, Caldas C et al (2017) Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer 17(4):223\u0026ndash;238\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRothwell DG, Ayub M, Cook N, Thistlethwaite F, Carter L, Dean E et al (2019) Utility of ctDNA to support patient selection for early phase clinical trials: the TARGET study. Nat Med 25(5):738\u0026ndash;743\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun SY (2021) mTOR-targeted cancer therapy: great target but disappointing clinical outcomes. why? Front Med 15(2):221\u0026ndash;231\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMackley MP, Fernandez NR, Fletcher B, Woolcott CG, Fernandez CV (2021) Revisiting Risk and Benefit in Early Oncology Trials in the Era of Precision Medicine: A Systematic Review and Meta-Analysis of Phase I Trials of Targeted Single-Agent Anticancer Therapies. JCO Precis Oncol 5:17\u0026ndash;26\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"mTOR, temsirolimus, cisplatin, gemcitabine, bladder, ctDNA","lastPublishedDoi":"10.21203/rs.3.rs-5333803/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5333803/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGemcitabine plus cisplatin (GC) is effective in urothelial and other cancers. We investigated GC combined with escalating doses of temsirolimus (T), intravenous mTOR inhibitor.\u003c/p\u003e\n\u003cp\u003eAdults with histologically confirmed solid tumours were enrolled. The primary endpoint was safety of GC with T, including dose limiting toxicity. Exploratory endpoints included pharmacokinetics and circulating tumour DNA (ctDNA) analyses.\u003c/p\u003e\n\u003cp\u003eFourteen participants (6 bladder cancer) were enrolled. Four dose levels were evaluated, adverse events were common and led to dose reductions, delays and omissions. Plasma concentrations of gemcitabine metabolites and cisplatin were unaffected by temsirolimus. ctDNA was detected in all samples taken from a sub set of patients. Levels of ctDNA changed earlier and correlated with imaging, while biologically relevant mutations e.g. \u003cem\u003ePIK3CA\u003c/em\u003ewere detected.\u003c/p\u003e\n\u003cp\u003eOur results confirm the poor tolerability of three-drug combinations including GC and T but highlight the potential for ctDNA monitoring in Phase 1 cancer trials.\u003c/p\u003e","manuscriptTitle":"A Phase I trial to evaluate cisplatin, gemcitabine and the mTOR inhibitor temsirolimus for first-line treatment of participants with transitional cell urothelial cancer, or advanced solid cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 04:12:50","doi":"10.21203/rs.3.rs-5333803/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-medicine","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsmed","sideBox":"Learn more about [Communications Medicine](http://www.nature.com/commsmed)","snPcode":"43856","submissionUrl":"https://mts-commsmed.nature.com/cgi-bin/main.plex","title":"Communications Medicine","twitterHandle":"@commsmedicine","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"95aafafb-034e-4e2d-ad96-576b06ae4cd8","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":40112477,"name":"Health sciences/Oncology/Cancer/Cancer therapy/Drug development"},{"id":40112478,"name":"Biological sciences/Cancer/Cancer therapy/Drug development"}],"tags":[],"updatedAt":"2025-03-27T04:12:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-27 04:12:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5333803","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5333803","identity":"rs-5333803","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}