Background

Common mutations arise from MAPK and PI3K pathways are RAS, BRAF and PIK3CA. These mutations are focus for targeted inhibitors but also serve as integrators to drug resistance occurrence. Activated MAPK signaling will co-activate PI3K pathway which serves as by-pass mechanisms for drug resistance development. Many studies demonstrated that dual-inhibition of both pathways is a better strategy to overcome drug resistance. Our research investigates this rationale and tests if palbociclib (P; CDK4/6 inhibitor) can act synergistically with either gedatolisib (G; P13K/mTOR dual inhibitors) or PD0325901 (PD; MEK 1/2 selective inhibitor) to offer a highly potent antitumour efficacy in mutant CRC cell lines.

Methods

Five human CRC cells lines were profiled for mutations using Agena Massarray. The antiproliferative response to P, G and PD alone and in combination was assessed in each cell line. Finally, IC₅₀ and combination indexes at effective dose 50 (CI) were calculated using CalcuSyn.

Results

Table 19P:

Common mutations identified in each cell line and corresponding CI values for both drug combinations. The CI values <0.9 are synergistic, whereas CI values >1.0 are antagonistic. P+PD=palbociclib/PD0325901; P+G=palbociclib/gedatolisib; NR=not reached

Conclusions

Drug combination P+G has clear synergistic antiproliferative effect in CRC cell lines with common mutations arising from MAPK & P13K pathways except for BRAF_V600E. Combination P+G is also a potential option in CRC cells harbouring KRAS with(out) activating PIK3CA mutation, which associated with MEK inhibitor resistance. Our invitro data provide good rationale for further invivo evaluation and potential clinical drug development of P+G combination as future beneficial therapy in patients with treatment-resistant colorectal cancers.

Legal entity responsible for the study

Oncology Molecular Medicine, Royal College of Surgeons in Ireland (RCSI), Dublin.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Colorectal cancer is the fourth leading cause of cancer-related deaths worldwide. Current adjuvant chemotherapy regimens often lead to significant toxicities, so novel targets and targeted therapies are of great interest. Nischarin (NISCH, IR1, IRAS) is a non-adrenergic imidazoline-1 receptor protein that has been described as a tumor suppressor in breast and ovarian cancer. NISCH expression and its biological role in other types of cancer have not been investigated to date. Importantly, NISCH is a druggable target for which chemical agonists and antagonists have already been developed. The aim of this study was to examine the expression of nischarin in colon cancer and determine its potential role in disease progression and patient outcome.

Methods

The web resource for analyzing cancer transcriptome data from The Cancer Genome Atlas (TCGA) was used to analyze the NISCH mRNA expression levels in normal and colon cancer samples. Patients from the TCGA colon cancer database (n = 440) were divided into "high" and "low" groups based on mean NISCH expression and Kaplan-Meier survival analysis was performed. Gene Set Enrichment Analysis (GSEA) was performed on several publicly available datasets using the Hallmarks and KEGG gene sets to find gene expression signatures that correlate with NISCH expression.

Results

In the TCGA database there was no significant difference in expression of NISCH between normal and tumor tissue (p > 0.05). Survival analysis showed that patients with higher NISCH tumor levels had significantly shorter overall survival (p = 0.0141, Log-rank test). In the GSE41258 dataset, levels of NISCH were higher in primary tumors of patients with M1 status compared to tumors of patients without metastasis (p = 0.027, unpaired t test). GSEA revealed that gene expression sets of epithelial-mesenchymal transition, angiogenesis, focal adhesion assembly and extracellular matrix-receptor interaction, all of which are associated with metastasis, were significantly correlated with NISCH expression in primary tumors.

Conclusions

In colon cancer, unlike in breast and ovary, higher NISCH expression might be associated with poor prognosis. Given that nischarin is a druggable target, it is potentially a new therapeutic target for selective treatment of patients with advanced colon cancer.

Legal entity responsible for the study

Institute for Oncology and Radiology of Serbia.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Cancer, a heterogeneous disease is caused by genetic and epigenetic mutation. Irinotecan was indicated for treatment of colorectal cancer either alone or in combination with other chemotherapeutic drug. Due to associated toxicity, irinotecan was less explored in cancer therapy research. In recent years, magnetic nanoparticles (MNPs) were well explored for different biomedical applications including targeted drug delivery.

Methods

We hypothesized that the anticancer activity of irinotecan would improve when attached to the surface of MNPs. Magnetic nanoparticles were developed using in house developed method. Fourier transform infrared (FT-IR) spectra and scanning electron microscopy (SEM) were recorded to characterize the irinotecan MNPs. In Vitro and in vivo evaluations were perfomed to assess the feasibility of this novel formulation using colon cancer cell lines.

Results

Irinotecan MNPs were successfully developed with an average diameter of the magnetic core of 22 ± 5 nm and the thickness of the shell is around 7 ± 4 nm. Using colon cancer culture (HT-29 cells), we evaluated the effects of MNPs on cancer cell viability and apoptosis. Treatment of cancer cells with the irinotecan MNPs caused a greater decrease in cell viability and a higher rate of apoptosis compared with treatment using free irinotecan.

Conclusions

Although further clinical study is needed, the targeted uptake of irinotecan MNPs by colon cancer cells associated with their ability to eliminate and prevent further cancer cell growth indicates its potential for targeted and personalized cancer therapy with lower toxicity in patients with colon cancer.

Editorial acknowledgement

Not Applicable

Legal entity responsible for the study

Akhilesh Vikram Singh.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.

Background

Matricellular proteins are a subset of extracellular matrix (ECM) proteins which are dynamically expressed and serve many regulatory roles. Among matricellular proteins, the CCN family, a group of secreted proteins are known to regulate cell adhesion, migration, proliferation, differentiation, apoptosis, survival, senescence, and gene expression. Connective tissue growth factor (CTGF), also known as CCN2, is a member of the CCN family. CTGF is known to have many roles in biological processes such as cell proliferation, migration, adhesion, and angiogenesis.

Methods

To investigate the function of CTGF in triple-negative breast cancer cells (TNBC), we silenced CTGF in TNBC cell lines using short hairpin RNA. Knockdown of CTGF was verified by western blot, qPCR, and immunostaining. The effect of CTGF knockdown on TNBCs was examined by cell proliferation assay, adhesion assay, migration assays, metabolism assays, and cell cycle analysis.

Results

Knockdown of CTGF decreased cell proliferation, adhesion, migration, glucose uptake, ATP production and lactate production. Since CTGF is a secreted protein, we gave recombinant human CTGF (rhCTGF) to the cells and found that rhCTGF induced activation of the Src/FAK/MAPK pathway and led to expression of proteins related to cell cycle progression. Also, when CTGF-specific antibodies were given to the cells, they expressed cytotoxicity by neutralizing the extracellular CTGF and decreasing CTGF-mediated signalling.

Conclusions

We suggest that the secreted CTGF mediates tumor cell progression via modulation of cell proliferation, adhesion, migration and metabolism and could possess a potential for being a therapeutic target.

Legal entity responsible for the study

Hanyang University.

Funding

National Research Foundation of Korea.

Disclosure

All authors have declared no conflicts of interest.

Background

CCN3(NOV) is a matricellular protein belonging to the CCN family and is known to be involved in various cellular signaling processes including tumorigenesis. Previous studies have shown that CCN3 can promote cell migration in sarcoma, glioblastoma, prostate and renal cancer cells. In-silico analysis using the TCGA database shows CCN3 was highly amplified in breast cancer patients especially in TNBC (triple-negative breast cancer) patients. Accordingly, we confirmed that the expression of CCN3 is up-regulated in the TNBC cell lines. Based on these findings, it was thought that CCN3 would participate in TNBC tumorigenesis.

Methods

We generated CCN3 knockdown TNBC cell lines by using shRNA. Proliferation assays and wound healing assay were performed to check the phenotype changes in the established cell line, and western blotting and RT-qPCR were used to monitor changes in intracellular protein expression. In addition, in-silico analysis using the TCGA database confirmed gene sets affected by CCN3 expression.

Results

We identified that CCN3 knockdown cell lines exhibited decreased proliferation, migration ability and colony-forming ability compared with control cell lines. Then we performed immunoblotting to find altered signaling by CCN3 knockdown. Phosphorylation level of FAK and EGFR were decreased in CCN3 knockdown cell lines.

Conclusions

We have shown that CCN3 can be involved in proliferation and migration ability in TNBC cell lines. We thought that this regulation may involve EGFR, which is known to be overexpressed in TNBC, or phosphorylation of FAK through integrin signaling. This study shows that CCN3 expressed in TNBC affects tumorigenesis of TNBC and may be used as a possible molecular therapeutic candidate to target TNBC.

Legal entity responsible for the study

Hanyang University.

Funding

National Research Foundation Korea.

Disclosure

All authors have declared no conflicts of interest.

Background

Hypoxia plays an important role in ovarian cancer and understanding the role of reactive oxygen species and VEGF may identify patients who benefit the most from targeted therapies. The aims of the study were to determine the intensity of oxidative stress in ovarian cancer patients and to establish a connection between the presence of the reactive oxygen species (ROS) and VEGF.

Methods

Forty-one patients diagnosed with epithelial ovarian carcinoma stage II-IV between 2010 and 2017, who underwent multimodality treatment (surgery and chemotherapy) were included in the study. ROS measured in dynamic (four determinations between every cycle) were malondialdehyde to evaluate the lipid peroxidation, ceruloplasmin, SH- albumin thiols groups and total antioxidants.

Results

There was an increase in the value of ROS: malondialdehyde mean value was 8.5 μmol/100 ml (normal value 4 μmol/100 ml); ceruloplasmin mean value was 147.8U.I. (normal value 120U.I), both showing an active oxidative process in patients with ovarian cancer. A small decrease of the value of thiols (397 vs. 450 μmol/l) and a small increase of total antioxidants was noticed (1.45 vs. 1.4 μmol). All four compounds decreased between the first determination and the fourth one. There was a strong correlation between lipid peroxides levels and ceruloplasmin (Pearson correlation 0.315 p = 0.005) and between lipid peroxides and thiols groups (Pearson correlation 0.23 p = 0.039). There was a correlation between thiols and antioxidants (Pearson correlation 0.33 p = 0.003). Lipid peroxidation and ceruloplasmin were significantly higher in patients with residual disease (p = 0.039, p = 0.046) emphasizing that the tumor is a generator of oxidative stress. Mean VEGF levels were elevated 983.42 pg/ml, and VEGF levels statistically significant corelates with malondialdehyde (Pearson correlation 0.439, p = 0.036).

Conclusions

Tumours produce ROS in excess in patients with advanced ovarian adenocarcinoma. Those ROS are corelated between each other and with VEGF and act as signalling molecules.

Legal entity responsible for the study

National Institute of Oncology Al. Treistioreanu.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Recently the texture analysis has been applied to help predict the clinical outcome and treatment response of various tumor types. This study investigates the relationship between the contrast enhanced computed tomography (CECT) texture features of small cell lung cancer (SCLC) tumors tissue, and the survival of the patients.

Methods

Eighty-eight patients diagnosed with unresectable SCLC (Extended stage, 57; Limited stage, 31), and treated with platinum-based chemotherapy at West China hospital between January 2010 and 2015 were enrolled. All the patients are followed for at least 18 months or until death. The texture features of tumor tissues were extracted from CECT images taken prior to treatment. Based on the receiver operating characteristics curve analysis, the optimal cutoff value of each texture parameter was obtained for further calculation. The Kaplan-Meier analysis was performed to compare the eighteen-month overall survival (OS) rates and six-month event-free survival (EFS) rates of different patient subgroups. The multivariate Cox regression analysis was performed to determine the prognostic value of texture features on CECT images in patients with unresectable SCL C.

Results

Of all CT texture features analyzed, six (GLCM-Contrast, GLCM-Dissimilarity, HISTO-Energy, HISTO-Entropy, HISTO-Skewness and Std-value) were shown to be significantly related to 18-month OS and could be considered as independent predictors of patients’ survival. Two features (GLCM-Energy and GLCM-Entropy) were shown to be significantly correlated with the 6-month EFS but could not be taken as independent predictors according to the multivariate Cox regression analysis.

Conclusions

Texture features of contrast-enhanced computed tomography could potentially serve as prognostic predictors for SCLC patients taken platinum-based chemotherapy. In view of the present study and previous reports, the CECT-based teture analysis atlas can be recommended in the clinical practice of SCLC.

Legal entity responsible for the study

West China Hospital, Sichuan University.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

There is increasing interest in the use of circulating tumour DNA (ctDNA) to identify targetable genomic alterations for therapy selection. However, the feasibility of next generation sequencing on ctDNA in BC patients with varying disease burden merits further investigation.

Methods

The cohort of 35 BC patients included 30 metastatic cases with paired primary and metastatic specimens in addition to plasma taken prior to commencement of a new line of palliative systemic therapy (all subtypes), + 5 patients (3 stage III, 2 stage II) about to commence neoadjuvant systemic therapy. DNA from tumour, buffy coat and plasma was sequenced on a 77-gene capture panel customised for BC. Matched tumour/normal samples were processed to discover somatic alterations using a standard GATK pipeline. Plasma samples were processed using unique molecular identifiers to identify potential alterations at low frequency.

Results

Among the entire cohort, 74% (26/35) of patients had mutation(s) in at least 1 gene detected from ctDNA: 60% (3/5) for the neoadjuvant cases, 77% (23/30) for the metastatic cases. The most frequently mutated genes from ctDNA analysis were TP53 (17/35; 49%) and PIK3CA (8/35; 23%), with HER2, HER3, JAK2, NF1, MAGI3 and TSC2 mutations observed in 2 patients each (6%). There was no obvious correlation between detection of ctDNA mutation and disease burden, serum CA-15.3 tumour marker or circulating tumour cell levels using a microfluidic platform. Out of 35 patients, 21(60.0%) had ≥1 concordant mutation via both ctDNA and tumour genotyping. Concordance between the plasma and primary and/or metastatic lesions was observed for 59% (39/66) of the mutations detected in ctDNA. Among the metastatic cases, 14/37(38%) of the concordant mutations were shared between the plasma, primary and metastatic specimens, while 5(14%) were shared between the plasma and the primary only, and 18/37(49%) shared between the plasma and metastatic lesions only.

Conclusions

Detection of genomic alterations from ctDNA is feasible in BC patients, but concordance of mutations in ctDNA is better with the metastatic than the primary lesion. This is likely due to suboptimal quality of DNA from archived tissue, and spatial, temporal heterogeneity.

Legal entity responsible for the study

National Cancer Centre Singapore and Genome Institute of Singapore.

Funding

SingHealth Foundation.

Disclosure

Y.S. Yap: Personal financial interests, honoraria for consultancy and talks, travel support: AstraZeneca, Eisai, Lilly, Novartis, Pfizer, Roche. All other authors have declared no conflicts of interest.

Background

Recent studies have demonstrated of utility of quantifying circulating proteins for early disease detection, prediction of therapeutic response, and treatment monitoring. Although the utility is clear, standardization of measurement methods for a large number of proteins from plasma remains challenging. Here, plasma proteins are detected using hyper-reaction monitoring (HRM) mass spectrometry in samples from normal and pancreatic cancer subjects. The data is acquired and analyzed simultaneously with unbiased label-free quantification of all proteins and absolute quantification of proteins from a > 500-protein panel constructed from stable-isotope standard (SIS) peptides.

Methods

Plasma samples from subjects with Stage IV pancreatic cancer (PC, n = 6) and age matched healthy donors (n = 15) were prepared for mass spectrometry. Prior to analysis, a panel of SIS peptides, covering 582 plasma proteins, was spiked into each sample. All samples were acquired using nano-flow liquid chromatography with separation over a one-hour gradient coupled online to a Thermo Scientific Q Exactive HF mass spectrometer. Protein data was extracted using Spectronaut (Biognosys) and statistical analysis was conducted to identify disease associated biomarker candidates.

Results

Analysis of the PQ500 panel enabled absolute quantification of 282 proteins across all samples. Univariate statistical testing identified 29 candidate proteins (27 up- and 2 down-regulated; q-value > 0.05 and fold change > 1.5). Key dysregulated proteins include CRP, SAA1, and C9. With unbiased, label-free quantification, 414 proteins were quantified, with 87 significantly regulated. In addition to the acute phase protein candidates identified in the PQ500 panel, 12 adhesion related protein candidates were identified (e.g. TLN1, MYH9, TPM4), 9 of which were decreased in PC. Notably, membrane associated ICAM1 and VCAM1, both potential therapeutic targets, were increased in PC.

Conclusions

Combining PQ500 with unbiased label-free quantification enables comprehensive characterization of the plasma proteome, while at the same time providing absolute quantification for a large subset of well annotated, clinically relevant proteins.

Legal entity responsible for the study

Biognosys AG, Schlieren, Switzerland.

Funding

Biognosys AG, Schlieren, Switzerland.

Disclosure

H. Yu, J. Vowinckel, C. Escher, D. Heinzmann, N. Dupuis: Employee: Biognosys AG.

Background

Esophageal squamous cell carcinoma (ESCC) is the most important pathological type of esophageal cancer with high incidence and limited efficient therapies in China and worldwide. Understanding of ESCC genomic features is advantageous for exploring clinical therapeutic strategies.

Methods

Deep sequencing targeting 450 cancer genes was performed on FFPE and matched blood samples collected from 90 Chinese ESCC patients(pts) with a median age of 60 years old. Genomic alterations (GAs) including single nucleotide variations, short and long indel, gene rearrangements and copy number variations were analyzed. Tumor mutation burden (TMB) was assessed in all samples by standard NGS algorithms. The expression of PD-L1 in 44 samples was evaluated by IHC (28-8 Ab).

Results

The most commonly found GAs were mutations in TP53 (94.4%), FGF3 (43.3%), CCND1 (43.3%), FGF4 (42.2%), FGF19 (42.2 %), CDKN2A (36.7%), NOTCH1 (25.6%), PIK3CA (22.2%), and ERBB2 amp(2.2%).Compared with TCGA data, the frequency of TP53 and ERBB2 amp mutations was similar, while the frequency of NOTCH1 mutations was significantly higher (25.6% vs 11%) and the frequency of EGFR amp was lower (5.6% vs 19%) in Chinese cohort. In addition, 4 of 90 samples harbored germline mutations (SDHA, RAD51C, PALB2 and BCL2L11). Among these GAs, 73.3% mutations were in cell cycle (62.2%) and PI3K/AKT (42.2%) pathway. In Chinese ESCC pts, the median TMB was 7.0 muts/Mb, and some pts showed high TMB (33.3%pts≥10 muts/Mb,11.1%pts≥20 muts/Mb). 15.6% pts (TMB≥10 muts/Mb) harbored chromosome 11q13 (FGF3/4/19 and CCND1) amp, which may be related to immunotherapy hyper-progression. Based on the IHC analysis, 10 of 44 Chinese pts showed positive (TPS≥1%) PD-L1 expression, and 7 of them also showed high TMB (≥10 muts /Mb). However, no correlation was found between high TMB and PD-L1 expression.

Conclusions

In this study, we found that the frequencies of GAs, such as EGFR and NOTCH1 alterations, were different between Chinese and western ESCC pts. Compared to Western ESCC pts, the positivity of PD-L1 expression was similar, while TMB was higher in Chinese ESCC pts. These findings suggested potential therapeutic targets for targeted and immunotherapies of Chinese ESCC pts.

Legal entity responsible for the study

OrigiMed.

Funding

Has not received any funding.

Disclosure

M. Yao, A. Liu, Y. Zhou: Employee: OrigiMed. All other authors have declared no conflicts of interest.

Background

The clinical behavior of GISTs is highly variable. This study aims at detection of different histo-pathological tumor features and correlation with different clinical aspects after treatment with imatinib, based on C-KIT exon mutation status.

Methods

This is a retrospective study that included all cases diagnosed as GIST who presented to NCI from 2005 to 2017, The following data were collected from the patient’s files: age, gender, tumor site, size, mitotic rate, histological grade, capsular rupture, risk stratification by Joensuu criteria, treatment setting, date of start and end of treatment, dose and toxicity. KIT mutation was assessed on tumor tissues of all patients; clinical correlation between different clinical aspects after treatment with imatinib, based on C-KIT exon mutation status, was done.

Results

Eighty-nine cases of GIST presented to NCI in the period September 2005 to January 2017. Median age at diagnosis was 48 years old with a median follow up of 22 months. More than 75% of the patients had positive C-KIT mutation, while it was negative in 24.7 % of the patients. C-kit positive mutations were significantly associated with tumors more than 5 cm, high mitosis, and with high tumor risk stratification by Joensuu criteria in more than fifty percent of the patients. Exon 9 mutations had poor ORR (55.6 %) compared to those with exon 11(67.6%) (P = 0.33), with the latter having PFS of 55 months compared 120 months for exon 9 mutations, (P = 0.002). No statistically difference in OS was observed with exon 9 (70 months) compared to exon 11 mutations (77 months) (P = 0.55).

Conclusions

C-kIT-positive mutation per-se is an independent poor risk factor, associated with more aggressive tumor features whereas response to imatinib was affected by exon mutations with exon 11 having a tendency for better ORR, compared to exon 9 mutation, with the latter having longer PFS compared with exon 11, with no statistically difference in OS with exon 9 compared to exon 11 mutations.

Legal entity responsible for the study

NCI.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Metallothioneins (MTs) family is a intracellular and cysteine-rich proteins with a high affinity to metals. MT-3 could play important neuromodulatory and neuroprotective roles. MT-3 has been also found up-regulated in a number of cancers. Neuroblastoma (Nbl) is a cancer is the most common extra-cranial solid tumour of childhood. The main aim was to provide novel insights into the molecular mechanisms of hMT-3 up-regulation and to elucidate the effects beneath the MT-3 up-regulation in Nbl cells.

Methods

To increase the expression of MT-3 Nbl (UKF-NB-4) cells were transiently transfected with a plasmid containing MT-3 gene (pcDNA3.1-GFP-hMT-3-TOPO). Separations of tryptic digests were carried out on an Easy-nLC 1000 nano system. MS analysis was performed using a Q-Exactive MS. The mass spectrum *.raw file was searched against the human SwissProt 57.15 database using MASCOT search engine (version 2.3, Matrix Science).

Results

The efficiency of transfection analysed through a fluorescence of GFP tag expressed at the C-terminus of MT-3 showed more 70% transfection efficiency for both constructed plasmids (mock and MT-3). From the total of common proteins in dataset (hMT-3 vs. mock), 176, 20 and 1244 proteins were quantitatively identified with up-regulation, down-regulation, and no significant differences between hMT-3 and mock treatments. Noteworthy, the bioinformatical analyses revealed the exclusive expression (induced by MT-3) and up-regulation proteins of a number of proteins affecting biological pathways related to mitotic cell cycle, cellular responses to stress, positive regulation of proteolysis, negative regulation of cell cycle and programmed cell death.

Conclusions

Our proteomic data shed some light on the proteins involved in inducing senescence and apoptosis in tested Nbl cells with up-regulated MT-3. Organisms with renewable tissues had to evolve mechanisms to prevent the development of cancer. Cellular senescence and apoptosis are among those mechanisms. Further experiments will be performed to functionally verify these data to provide novel insights into the Nbl biology. These might be useful to develop novel therapeutic protocols utilizing MT-3 as prognostic biomarker or therapeutic target.

Legal entity responsible for the study

Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, CZ.

Funding

European Research Council (ERC) under the European Uniońs Horizon 2020 Research and Innovation Programme (grant agreement No. 759585).

Disclosure

All authors have declared no conflicts of interest.