ONCOGRAM: study protocol for the evaluation of therapeutic response and survival of metastatic colorectal cancer patients treated according to the guidelines of a chemosensitivity assay, the Oncogramme®

Background Colorectal cancer is a major public concern, being the second deadliest cancer in the world. Whereas survival is high for localized forms, metastatic colorectal cancer has showed poor prognosis, with a 5-year survival barely surpassing 11%. Conventional chemotherapies against this disease proved their efficiency and remain essential in first-line treatment. However, the large number of authorized protocols complexifies treatment decision. In common practice, such decision is made on an empirical basis, by assessing benefits and risks for the patient. In other words, there is currently no efficient means of predicting the efficacy of any chemotherapy protocol for metastatic colorectal cancer. Methods/design The use of a chemosensitivity assay, the Oncogramme®, should help clinicians administer the best chemotherapy regimen to their patients. We hypothesize it would ultimately improve their survival. In this multicentred, prospective trial (ONCOGRAM), eligible patients with metastatic colorectal cancer are randomized to determine whether they will receive an Oncogramme®. For clinicians whose patients benefited from the assay (arm A), results are used as a decision support tool. Patients not undergoing the Oncogramme® procedure are treated according to current practice, without the assistance of the assay (arm B). Primary outcome is 1-year progression-free survival. Secondary outcomes include response rates, as well as 6-month and 1-year survival rates. Discussion This study aims at investigating the clinical utility of the Oncogramme® as a decision support tool for the treatment of patients with metastatic colorectal cancer. If the Oncogramme® positively influenced patient overall survival and/or progression-free survival, it would be of great value for clinicians to implement this assay within the current landscape of personalized medicine tools, which include genomics and biomarker assays. Trial registration ClinicalTrials.gov identifier NCT03133273. Registered on April 28, 2017.

CAVIN2 is frequently silenced by CpG methylation and sensitizes lung cancer cells to paclitaxel and 5-FU

Aim: To explore the biological functions and clinical significance of CAVIN2 in lung cancer. Materials & methods: Methylation-specific PCR was used to measure promoter methylation of CAVIN2. The function of CAVIN2 was tested by Cell Counting Kit-8, colony formation, Transwell, flow cytometric analysis, acridine orange/ethidium bromide, chemosensitivity assay and xenograft assay. Results: CAVIN2 is significantly downregulated by promoter methylation in lung cancer. CAVIN2 overexpression inhibits lung cancer cell migration and invasion. Furthermore, ectopic expression of CAVIN2 inhibits cell proliferation in vivo and in vitro by inducing G2/M cell cycle arrest, which sensitizes the chemosensitivity of lung cancer cells to paclitaxel and 5-fluorouracil, but not cisplatin. Conclusion: CAVIN2 is a tumor suppressor in non-small-cell lung cancer and can sensitize lung cancer cells to paclitaxel and 5-fluorouracil.

Enolase 1 Regulates Stem Cell-Like Properties in Gastric Cancer Cells by Stimulating Glycolysis

Background: Recent studies have demonstrated that gastric cancer stem cells (CSCs) are a rare sub-group of gastric cancer (GC) cells and play an important role in promoting the tumor growth and progression of GC. In the present study, we demonstrated that the glycolytic enzyme Enolase 1 (ENO1) was involved in the regulation of the stem cell-like characteristics of GC cells.Methods: Self-renewal, Chemosensitivity and invasion of GC cells were evaluated by sphere formation, Chemosensitivity assay and invasion assay respectively. Glycolysis level was examined by the Seahorse XFe/XF Analyzer. ENO1 expression was determined in 83 GC specimens by immunohistochemistry. Results: The expression of ENO1 in sphere cells markedly increased as compared to the parental cell lines PAMC-82 and SNU16. We then observed that ENO1 could enhance stem cell-like characteristics, including self-renewal capacity, cell invasion and migration, chemoresistance, and even the tumorigenicity of GC cells. ENO1 is known as an enzyme that is involved in glycolysis, and our results showed that ENO1 could markedly promote the glycolytic activity of cells. Furthermore, inhibiting glycolysis activity using 2-Deoxy-D-glucose treatment significantly reduced the stemness of GC cells. Therefore, ENO1 could improve the stemness of CSCs by enhancing the cells’ glycolysis. Subsequently, to further confirm our results, we found that the inhibition of ENO1 using AP-III-a4 (ENOblock) could reduce the stemness of GC cells to a similar extent as the knockdown of ENO1 by shRNA. Finally, increased expression of ENO1 was related with poor prognosis in GC patients. Conclusion: Taken together, our results demonstrated that ENO1 is a significant biomarker associated with the stemness of GC cells.

Thread as a Low-Cost Material for Microfluidic Assays on Intact Tumor Slices

In this paper we describe the use of thread as a low-cost material for a microfluidic chemosensitivity assay that uses intact tumor tissue ex vivo. Today, the need for new and effective cancer treatments is greater than ever, but unfortunately, the cost of developing new chemotherapy drugs has never been higher. Implementation of low-cost microfluidic techniques into drug screening devices could potentially mitigate some of the immense cost of drug development. Thread is an ideal material for use in drug screening as it is inexpensive, widely available, and can transport liquid without external pumping hardware, i.e., via capillary action. We have developed an inexpensive microfluidic delivery prototype that uses silk threads to selectively deliver fluids onto subregions of living xenograft tumor slices. Our device can be fabricated completely for less than $0.25 in materials and requires no external equipment to operate. We found that by varying thread materials, we could optimize device characteristics, such as flow rate; we specifically explored the behavior of silk, nylon, cotton, and polyester. The incremental cost of our device is insignificant compared to the tissue culture supplies. The use of thread as a microfluidic material has the potential to produce inexpensive, accessible, and user-friendly devices for drug testing that are especially suited for low-resource settings.

Predicting Response to BET Inhibitor in Combination with Palbociclib / Sorafenib Using a Computational Model and Its Validation: A Beat AML Project Study

Background: In AML, leukemic transformation causes clonal expansion of immature cells through de-regulated cell division cycles. CDK4/CDK6 regulates neoplastic progression, which might represent an effective strategy for treating AML. But current clinical data shows either limited efficacy or elusive results. Bromodomain and extra-terminal (BET) inhibitors interferes with transcriptional complexes and disrupting gene transcription of key oncogenes such as MYC. Also, there is need to explore usage of other receptor tyrosine kinase inhibitors. Therefore, a drug combination strategy should be explored to overcome limited clinical efficacy. Aims: To create digital drug models for BET inhibition (BETi), CDK4/6 inhibition (CDKi), FLT3 inhibition (FLT3i) and validate the predicted responses in AML patient samples with ex vivo chemosensitivity testing. Furthermore, to validate the identified combination of BET inhibitor with CDK4/6 inhibitor or FLT3 inhibitor. Methods: The Beat AML project (supported by the Leukemia & Lymphoma Society) collects clinical data and bone marrow specimens from AML patients. Bone marrow samples are analyzed by conventional cytogenetics, whole-exome sequencing, RNAseq, and an ex vivo chemosensitivity assay. 33 patients were randomly chosen. Every available genomic abnormality was inputted into a computational biology model (CBM, Cell Works Group Inc.) that uses PubMed and other online resources to generate patient-specific protein network maps of activated and inactivated pathways. Digital drug simulations with BETi (JQ1), CDKi (palbociclib), FLT3i (sorafenib) were conducted by quantitatively measuring drug effect on a composite AML disease inhibition score (i.e., cell proliferation, viability, and apoptosis). Paired comparisons between the computational predictions and the sample's ex vivo chemosensitivity IC50 values were conducted. Results: None of the 33 AML patients showed ex vivo chemosensitivity to palbociclib alone, and the CBM method was highly accurate (94%) in predicting this lack of response (Table 1). Through CBM mapping the following genetic mutations were identified as potentially contributing to palbociclib resistance: loss of function (LOF) of RB1 (Retinoblastoma 1), LOF of PTCH1 (patched 1), LOF of FBXW7 (F-box and WD repeat domain containing 7), gain of function (GOF) of CCNE1/2 (Cyclin E1/2) or LOF in NPM1 (nucleophosmin 1). Additionally, the CBM method showed that NPM1 mutated AML cases that were resistant to palbociclib also showed a better response to the combination of palbociclib and JQ1. In 28/33 (85%) patients, this combination of palbociclib and BETi (JQ1) was toxic to AML cells in ex vivo chemosensitivity assay (Table 2). The CBM method predicted that 31/33 (94%) of AML patients would respond to palbociclib and JQ1 (Table 2), and the accuracy of matching between CBM and ex vivo chemosensitivity assay was 91% (Table 2). Another combination with high proportion of responding patients was FLT3i and BETi (Table 3), with accuracy of matching between CBM and ex vivo assay of 91% (Table 3). Furthermore, computational analysis of the combination of BETi and FLT3i revealed that patients with mutation of NPM1 along with FLT3 TKD/ITD mutation showed high degree of synergy at lower drug concentrations. Conclusion: Digital drug simulations of inhibitions of CDK4/6, BET, and FLT3 using AML patient genomic data accurately matched ex vivo chemosensitivity results. The integration of computational biology modeling and Beat AML data led to the identification of potential palbociclib resistance pathways in AML, which led to the rational design of new multi-drug regimens. This integrated system enabled novel inferences that informs future clinical trials for patients with AML. Disclosures Tyner: Janssen: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Leap Oncology: Equity Ownership; Gilead: Research Funding; Syros: Research Funding; Seattle Genetics: Research Funding; Agios: Research Funding; Aptose: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding. Druker:Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Patient True Talk: Consultancy; Celgene: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Bristol-Meyers Squibb: Research Funding; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Oregon Health & Science University: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Research Funding; Monojul: Consultancy; ARIAD: Research Funding; Fred Hutchinson Cancer Research Center: Research Funding. Vidva:Cellworks Research India Private Limited: Employment. Narvekar:Cellworks Research India Private Limited: Employment. Agrawal:Cellworks Research India Private Limited: Employment. Gera:Cellworks Research India Private Limited: Employment. Prasad:Cellworks Research India Private Limited: Employment. Shyamasundar:Cellworks Research India Private Limited: Employment. Tunwer:Cellworks Research India Private Limited: Employment. Abbasi:Cell Works Group Inc.: Employment. Vali:Cell Works Group Inc.: Employment. Cogle:Celgene: Other: Steering Committee Member of Connect MDS/AML Registry.

Thioguanine Combined with Decitabine Can Overcome Resistance to Hypomethylating Agents: Final Results of a Phase I Trial of a Pharmacodynamically-Conceived Thioguanine/Decitabine Combination in Patients with Advanced Myeloid Malignancies

Background: Outcomes are poor for older patients with acute myeloid leukemia (AML), high-risk myelodysplastic syndromes (MDS), or relapsed/refractory disease, and new therapies are needed. Using a chemosensitivity screening assay, we previously demonstrated that combination treatment with thioguanine and decitabine can restore therapeutic efficacy in primary leukemia cells isolated from patients with relapsed/refractory AML. To test the safety and synergistic efficacy of this combination in patients with advanced myeloid malignancies, we performed a Phase I dose-escalation trial of thioguanine given with decitabine. Patients and Methods: Patients with untreated AML ≥60 years of age and ineligible for standard induction, relapsed/refractory AML, and high-risk or relapsed MDS were eligible. Two thioguanine dose levels were evaluated: 80 and 120 mg/m2/day, given on Days 1-12 of induction and Days 1-7 of maintenance. Decitabine at 20mg/m2 was administered on Days 3-12 during induction and on Days 3-7 during maintenance. The primary objective was to determine the maximum tolerated dose (MTD) of thioguanine when given with decitabine. Key secondary objectives were to evaluate the overall response rate (ORR) and progression-free survival (PFS). Patient-specific pharmacodynamic measures to assess the biologic activity of thioguanine-decitabine were also performed. These included an in vitro chemosensitivity assay, BH3 profiling to measure the degree to which the leukemic blasts were primed for apoptosis, and genome-wide analysis of DNA methylation changes. Results: Twelve patients (median age 67; range 56-83) with de novo AML (n=1), secondary AML (n=6), relapsed/refractory AML (n=4), and chronic myelomonocytic leukemia (CMML) (n=1) were treated. Three patients experienced dose-limiting toxicity (DLT), which were acute renal failure requiring hemodialysis (80 mg/m2), persistent grade 4 leukopenia and thrombocytopenia (120 mg/m2), and grade 4 sepsis preventing continued treatment (120 mg/m2). Thioguanine at 80 mg/m2 was determined to be the MTD. Eleven of the 12 patients completed the first induction cycle, and 6 patients completed a second, identical induction cycle. The median number of cycles administered was 3 (range 1-8). One patient experienced a DLT prior to the first response assessment and was removed from study. The ORR in this intent-to-treat study was 67% (8/12). Six patients achieved a CR or CRi, one obtained a morphologic leukemia-free state, and one patient had a PR. Responses were observed in all disease types. Five of the 8 responses, including 4 CR/CRi, were achieved with thioguanine at 80 mg/m2, suggesting no loss of efficacy at the MTD compared with the higher dose level. All 11 evaluable patients had ≥50% reduction in bone marrow blast percentages after induction therapy. Six patients had previously received single-agent hypomethylating therapy, and 5 (83%) of these patients responded, demonstrating that thioguanine-decitabine can rescue prior hypomethylating agent failure. Out of the 8 responders, four (50%) proceeded to allogeneic stem cell transplantation (SCT), two relapsed after CR or CRi, one had a CNS-only relapse after achieving a CR, and one patient experienced DLT and was removed from the study. Of the four patients who proceeded to allogeneic SCT, two patients died in CR from transplant-related toxicity, one relapsed, and one patient remains alive and in remission greater than 2 years. Median PFS in responding patients was 42 weeks (range, 10-not reached, weeks). In vitro pharmacodynamic studies currently have been completed on samples from the first 6 patients treated on this trial. The chemosensitivity assay results on pre-treatment mononuclear cells directly correlated with initial response. In addition, significant apoptotic priming of the blasts, as suggested from BH3 profiling, also corresponded to initial clinical response. Conclusions: Thioguanine-decitabine can be administered safely and induce remission, even among patients who had previously been treated with hypomethylating agents. Intriguingly, preliminary results from the chemosensitivity screening assay and BH3 profiling correlated well with clinical responses. Additional correlative studies including DNA methylation analysis are ongoing to better understand the mechanism of synergy between thioguanine and decitabine. A multi-center Phase II trial is planned. Disclosures Jurcic: Astellas: Research Funding. Letai:Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding.