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Gemcitabina; Cáncer avanzado Gemcitabina; Càncer avançat Gemcitabine; Advanced cancer Purpose: This was a Phase I/II trial of the novel checkpoint kinase 1 (Chk1) inhibitor SRA737 given in combination with gemcitabine. Its objectives were to establish the safety profile, recommended Phase 2 dose (RP2D), pharmacokinetics profile, and clinical activity of SRA737. Patients and Methods: Patients with advanced solid tumors were enrolled into dose-escalation cohorts and treated in 28-day cycles with oral SRA737 on days 2, 3, 9, 10, 16, and 17, and intravenous gemcitabine on days 1, 8, and 15. Treatment was continued until progression. Each expansion cohort included up to 20 patients with specific genetically defined tumors. Results: The RP2D was determined to be 500 mg SRA737 combined with low-dose (250 mg/m2) gemcitabine. Of 143 enrolled patients, 77 were treated at doses of at least 500 mg SRA737 combined with 250 mg/m2 gemcitabine. Common toxicities of nausea, vomiting, fatigue, and diarrhea were primarily mild to moderate, and rarely led to treatment discontinuation. Anemia, neutropenia, and thrombocytopenia were grade ≥3 in 11.7%, 16.7%, and 10% of patients treated at the RP2D, respectively. The objective response rate (ORR) was 10.8% overall and notably the ORR in anogenital cancer was 25%. Partial tumor responses were observed in anogenital cancer, cervical cancer, high-grade serous ovarian cancer, rectal cancer, and small cell lung cancer. Conclusions: SRA737 in combination with low-dose gemcitabine was well tolerated with lower myelotoxicity than has been seen at standard doses of gemcitabine or with other combinations of Chk1 inhibitors with gemcitabine. Tumor responses were observed in anogenital and other solid tumors. The trial was sponsored by Sierra Oncology, Inc. Medical writing support was provided by Tina Ippolito, an independent consultant funded by Sierra Oncology. Andrew Dye, an employee of Sierra Oncology, also provided medical writing support and data curation. Bryan Strouse, an employee of Sierra Oncology, also contributed to data curation for this report. UK clinical trial sites acknowledge infrastructural funding from the Experimental Cancer Medical Center and National Institute of Health and Care Research Biomedical Research Centers. The ICR/RMH in addition acknowledges Cancer Research UK funding to Cancer Centre Funding and funding to the Cancer Therapeutics Unit. U. Banerji is a recipient of the NIHR RP-2016–07–028. The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes. Significance: Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

Supplementary Figure from Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma

Supplementary Table from Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma

Supplementary Data from Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma

Ovarian high-grade serous carcinoma (HGSC) prognosis correlates directly with presence of intratumoral lymphocytes. However, cancer immunotherapy has yet to achieve meaningful survival benefit in patients with HGSC. Epigenetic silencing of immunostimulatory genes is implicated in immune evasion in HGSC and re-expression of these genes could promote tumor immune clearance. We discovered that simultaneous inhibition of the histone methyltransferases G9A and EZH2 activates the CXCL10–CXCR3 axis and increases homing of intratumoral effector lymphocytes and natural killer cells while suppressing tumor-promoting FoxP3+ CD4 T cells. The dual G9A/EZH2 inhibitor HKMTI-1–005 induced chromatin changes that resulted in the transcriptional activation of immunostimulatory gene networks, including the re-expression of elements of the ERV-K endogenous retroviral family. Importantly, treatment with HKMTI-1–005 improved the survival of mice bearing Trp53−/− null ID8 ovarian tumors and resulted in tumor burden reduction. These results indicate that inhibiting G9A and EZH2 in ovarian cancer alters the immune microenvironment and reduces tumor growth and therefore positions dual inhibition of G9A/EZH2 as a strategy for clinical development.

SV loads in non-BRCA1/2 HR genes per sample by SV type

Supplementary Data from Multiomic Characterization of High-Grade Serous Ovarian Carcinoma Enables High-Resolution Patient Stratification

SNVs in non-BRCA1/2 HR genes

SVs at BRCA1/2

Genes in KEGG HR pathway

Supplementary methods and figures

Supplementary tables 3a and 3b. Germline and somatic SNVs/indels at BRCA1/2.

Differentially expressed genes in HR deficiency

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes.Significance:Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

Number of samples with BRCA1/2 SVs

Purpose:The abundance and effects of structural variation at BRCA1/2 in tumors are not well understood. In particular, the impact of these events on homologous recombination repair deficiency (HRD) has yet to be demonstrated.Experimental Design:Exploiting a large collection of whole-genome sequencing data from high-grade serous ovarian carcinoma (N = 205) together with matched RNA sequencing for the majority of tumors (N = 150), we have comprehensively characterized mutation and expression at BRCA1/2.Results:In addition to the known spectrum of short somatic mutations (SSM), we discovered that multi-megabase structural variants (SV) were a frequent, unappreciated source of BRCA1/2 disruption in these tumors, and we found a genome-wide enrichment for large deletions at the BRCA1/2 loci across the cohort. These SVs independently affected a substantial proportion of patients (16%) in addition to those affected by SSMs (24%), conferring HRD and impacting patient survival. We also detail compound deficiencies involving SSMs and SVs at both loci, demonstrating that the strongest risk of HRD emerges from combined SVs at both BRCA1 and BRCA2 in the absence of SSMs. Furthermore, these SVs are abundant and disruptive in other cancer types.Conclusions:These results extend our understanding of the mutational landscape underlying HRD, increase the number of patients predicted to benefit from therapies exploiting HRD, and suggest there is currently untapped potential in SV detection for patient stratification.

Supplementary methods and supplementary figures

Purpose: Eftilagimod alpha (efti) is a soluble LAG-3 protein that binds to a subset of MHC class II molecules to mediate antigen presenting cell (APC) activation and then CD8 T-cell activation. The stimulation of the dendritic cell network and subsequent T cell recruitment with efti may lead to stronger anti-tumor responses than observed with pembrolizumab alone. Combining an APC activator with an immune checkpoint inhibitor aims to increase efficacy without additional toxicity. We hereby report initial results of a phase II trial (NCT03625323). Methods: A predefined number of patients (pts) are recruited into this umbrella trial irrespective of PD-L1 expression. In part A: 1st line, PD-X naïve NSCLC; in part B: 2nd line, PD-X refractory NSCLC and in part C: 2nd line PD-X naive HNSCC. The study has a Simon's 2-stage design, with objective response rate (ORR) as primary endpoint. Secondary endpoints include progression free and overall survival, PK, PD and immunogenicity. Additional pts (N2) will be recruited for each part if the pre-specified threshold for ORR is met. Up to 109 pts will be enrolled. Efti is administered as 30 mg subcutaneous injection every 2 weeks for 8 cycles and then every 3 weeks for 9 cycles. Pembrolizumab is administered at a standard dose (200 mg intravenous infusion every 3 weeks for up to 2 years). The study was approved by ethic committees and institutional review boards. Results: Between 04 Mar 19 and 17 Jan 2020, 46 pts were enrolled and evaluated for safety. The median age was 66 years (range 48-84) and 74 % were male. The ECOG was 0 in 52 % and 1 in 48 % of the pts, respectively. Pts received a median of 5 (7) pembrolizumab (efti) administrations until data cut-off in Jan 2020. The most common (≥ 10%) adverse events (AEs) being cough (28 %), asthenia (24 %), dyspnea (17 %), decreased appetite (17 %), fatigue (15 %), diarrhea (15 %), non-cardiac chest pain (11 %), neck pain (11 %) and hypothyroidism (11 %). Two pts discontinued any study treatment due to AEs. All pts part A (1st line NSCLC) stage 1 (n=17) are evaluable for efficacy. Eight pts (47 %) had a partial response (iPR) and six (35 %) had stable disease according to iRECIST leading to an ORR of 47 %. Ten (10; 59 %) pts are still under therapy (7+ months). In part C (2nd line HNSCC) stage 1 13/18 pts are evaluable and five (39 %) had an iPR so far. Conclusions: Efti s.c. administered every 2 weeks in combination with pembrolizumab is safe and shows encouraging antitumor activity in all comer PD-L1 1st line NSCLC and 2nd line HNSCC. Stage 2 for both parts has been opened and stage 1 for part B is still recruiting. Citation Format: Martin Forster, Enriqueta Felip, Bernhard Doger, Margarita Majem, Enric Carcereny, Julio Peguero, Pawan Bajaj, Tim Clay, Matthew Krebs, Patricia Roxburgh, Leora Horn, Frederic Triebel. Initial results from a phase II study (TACTI-002) in metastatic non-small cell lung or head and neck carcinoma patients receiving eftilagimod alpha (soluble lag-3 protein) and pembrolizumab [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT202.

High grade serous ovarian carcinomas (HGSOCs) are molecularly and clinically heterogeneous malignancies. Currently, molecular stratification of patient care is limited to tumors rendered homologous recombination deficient (HRD) by BRCA1/2 mutation (BRCAm). Here we explore the overlap, interplay and clinical impact of molecular subgrouping layers in a cohort of 362 FFPE HGSOCs. We overlay genomic subgrouping and tumor-infiltrating lymphocyte (TIL) burden with the three transcriptionally-defined subtypes (Immune, Angio and AngioImmune) we have previously reported as associated with survival and bevacizumab sensitivity. The BRCAm group, the Immune subtype and those with high CD8+ TIL burden demonstrated prolonged overall survival (OS) as previously reported. The OS benefit of CD8+ TILs was abrogated in the context of gross residual disease following debulking [multivariable hazard ratio (mHR)=0.51 (0.34-0.78) vs 0.93 (0.76-1.52)]. BRCAwild-type patients demonstrating high expression of EMSY, encoding the BRCA2-binding protein EMSY, demonstrated prolonged OS [mHR=0.48 (0.29-0.79)]. BRCA2m and high-EMSY patients displayed greater chemosensitivity (first-line CA125-CR 94.4% and 81.3% vs 48.2%, P There was significant enrichment and depletion of BRCAm and CCNE1-gain (CCNE1g) in the Immune subtype (28.9% vs 6.3%, P=0.001 and 9.1% vs 17.4%, P=0.050). The Angio subtype harboured far fewer CD8+ TILs compared to the Immune and AngioImmune subtypes (P The clinical impact of CCNE1g was modulated by transcriptomic subtype: in the Immune group, CCNE1g was associated with poor OS [mHR=3.32 (1.49-7.41)], while in the Angio group CCNE1g cases had favourable outcome [PFS mHR=0.26 (0.10-0.68)]. CD8+ TIL burden was not associated with outcome in CCNE1g cases. Missense TP53 mutation was associated with better outcome versus TP53-null mutations in BRCAm patients [PFS mHR=0.43 (0.14-0.82)], but not in the context of HR proficiency [mHR=0.90 (0.67-1.22)]. Integrated classification using the consensus of favourable and unfavourable HR-centric (HRD favourable, HR-proficient unfavourable) and transcriptomic (Immune favourable, Angio/AngioImmune unfavourable) subgrouping yielded three groups with distinct OS [favourable vs unfavourable mHR=0.49 (0.34-0.74); favourable vs no-consensus mHR=0.64 (0.42-0.98); unfavourable vs no-consensus mHR=1.31 (1.00-1.70)]. Together, these data paint a more granular picture of the clinical impact of HGSOC subgroups and demonstrate novel candidate interactions between subgrouping layers. The poorest outcome groups represent those with most to gain from trials of novel treatment regimens. Citation Format: Robert L. Hollis, Alison M. Meynert, Michael Churchman, Tzyvia Rye, Patricia Roxburgh, Daniel Stetson, Athena Matakidou, Brian Dougherty, J. Carl Barrett, Ruth E. March, Colin A. Semple, C. Simon Herrington, Charlie Gourley. Multi-layer molecular characterization of high grade serous ovarian carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 749.

Introduction MDM2 inhibitor idasa aims to treat cancers retaining functional TP53, inactivated by binding of negative regulator, MDM2. Many CRPCs retain functional TP53. Pre-clinical data suggest addition of MDM2 inhibition to androgen deprivation enhances tumour regression & survival in xenografts. This phase 1b study explored safety, PK & PD of idasa with abi or enza to define optimum biological dose & combination. Methods Patients (pts) received oral (PO) idasa in a 3+3 dose escalation design with PO abi 1000mg once daily (OD) & PO pred 5mg twice daily (BD) (Part A) or PO enza 160mg OD (Part B) in 28 day cycles (C). In the first two cohorts (Part A), pts received a single dose of idasa on day (D) -7. Two different formulations for idasa were explored (microprecipitated bulk powder (MBP) and spray-dried powder (SDP)). Toxicity was assessed by CTCAE v4.03. Plasma idasa/M4 metabolite & plasma abi was measured (Part A). In Part B idasa/M4 metabolite & enza PK were examined. Macrophage inhibitory cytokine 1 (MIC-1) was measured to assess target modulation. Biochemical response was assessed monthly & radiological response 8 weekly. Results 24 pts were enrolled. In part A, 3 pts were treated in cohort 1 with idasa MBP 200mg, D -7 and D1-5, OD, q28; 2 pts experienced a suspected unexpected serious adverse reaction (SUSAR) (pulmonary embolism and myocardial infarction). The dose of idasa was de-escalated for cohort-1 (idasa MBP 200mg, D-7, D1-3, OD); no DLTs were reported (n=3). 3 pts treated in cohort 1* (idasa MBP 200mg, D1-5, OD) & 3 pts in cohort 2 (idasa MBP 400mg, D1-5, OD) had no DLTs. The formulation of idasa was switched to SDP (cohort 3) & a further 3 pts were treated at a dose equivalent to mono-agent MTD (MBP 500mg = SDP 250mg, D1-5, OD) & 1 DLT (grade 4 thrombocytopenia) was reported. A further 3 pts treated at this dose reported no DLTs. 4 additional SUSARs in 3 pts have been reported to date (grade 3 (G3) depression, G3 thromboembolic events, G3 vomiting and prolonged G4 thrombocytopenia/neutropenia). 4 serious adverse reactions (SAR)s have also been reported (G2 decreased platelet count, G3 atrial fibrillation, G3 lung infection & G4 decreased platelet count). In Part B pts received idasa SDP 250mg, D1-5, OD and no DLTs were reported. Dose escalation beyond this mono-agent MTD of idasa was not permitted. Idasa had a dose-proportional PK profile. There was no clinically significant PK interaction with abi, although idasa exposure was reduced when combined with enza (known strong CYP3A inducer). MIC-1 was induced by idasa & abi, but less so by idasa & enza. 7 pts remain on combination & 4 remain on hormonal monotherapy. Efficacy data are immature. Conclusions Idasa can be safely given with abi to achieve exposure comparable with previous data & sufficient to activate p53. Idasa can also be given with enza albeit with reduction in exposure to idasa and less robust p53 activation. Citation Format: Patricia Roxburgh, Dawn Currie, Paula Morrison, Caroline Kelly, Fiona Thomson, Carol McCormick, Alan Bilsland, Robert H. Jones, Amy Quinton, Elaine McCartney, Lorraine Barwell, Rob J. Jones. Safety, pharmacokinetics (PK) & pharmacodynamics (PD) of idasanutlin (idasa), combined with abiraterone (abi)/prednisolone (pred) or enzalutamide (enza) in castrate resistant prostate cancer (CRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT027.

The p53 protein is an important tumor suppressor that can function to inhibit cell growth through the activation of a number of different responses, including cell cycle arrest, senescence and apoptosis. Recent evidence has suggested that under conditions of low or transient stress, p53 can also contribute to the survival of stressed cells - allowing for the prevention or repair of damage. Each of these activities can restrain malignant progression, and most cancers show loss of the normal functions of p53. In many cancers this is due to a mutation in the p53 gene that leads to the expression of a mutant p53 protein. Interestingly, these tumor associated mutant p53s not only lose wild type p53 activity but can also acquire the ability to promote cell motility and migration, and so contribute to the development of metastases. We have found that tumor associated mutant p53s can promote invasion and loss of directionality when cells migrate in vitro. These activities are independent of the loss of wild type p53 function, and reflect activation of integrin and EGFR trafficking that depends on Rab-coupling protein and which results in constitutive activation of EGFR/integrin signalling. These findings open the possibility that blocking alpha5/beta1 integrin and/or the EGF receptor will have therapeutic benefit in mutant p53 expressing cancers. We are now proposing to extend these observations by testing whether this activity of mutant p53 is restricted to the EGFR, or may also promote the activity of other cell surface receptors too. Simultaneous loss of p53 and p63 recapitulates the phenotype of mutant p53, suggesting that this function of mutant p53 reflects, at least in part, the inhibition of p63. However, mutant p53 is likely to have additional functions that contribute to the ability to induce an invasive phenotype, and we are presently investigating the activity of other mutant p53 binding proteins. While mutant p53s are often expressed at very high levels in tumors, normal cells express mutant p53s at low level, like the wild type p53 protein. The turnover of mutant p53 in these cells is dependent on the function of the MDM2 ubiquitin ligase, which also regulates the stability of wild type p53, and deletion or inhibition of MDM2 leads to the stabilization of both mutant and wild type p53 proteins. A number of small molecule drugs that can inhibit MDM2's ability to target p53 for degradation have been described, with the hope that these will be useful in treating those tumors that retain wild type p53. However, studies have suggested that many apparently normal tissues harbour p53 mutations, leading to the possibility that systemic treatment with MDM2 inhibitors would result in the stabilization of these mutant p53s, with unanticipated, deleterious consequences. We are therefore investigating how mutant p53 turnover is regulated, with the view to testing whether the stabilization of mutant p53 would affect the behaviour of otherwise normal cells. Citation Format: Patricia Muller, Patricia Roxburgh, Patrick Caswell, Jim Norman, Karen H. Vousden. Functions of wild type and mutant p53 [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY29-01