Targeted Methylation Sequencing of Plasma Cell-free DNA for Cancer Detection and Classification

Abstract

Background

Targeted methylation sequencing of plasma cell-free DNA (cfDNA) has a potential to expand liquid biopsies to patients with tumors without detectable oncogenic alterations, which can be potentially useful in early diagnosis.

Patients and methods

We developed a comprehensive methylation sequencing targeting 9,322 CpG sites consistently hypermethylated according to The Cancer Genome Atlas. Next, we performed a clinical validation of our method using plasma cfDNA samples from 78 patients with advanced colorectal cancer, non-small cell lung cancer (NSCLC), breast cancer or melanoma and compared results to patients' outcomes.

Results

Median methylation scores in plasma cfDNA samples from patients on therapy were lower than from patients off therapy (4.74 vs. 85.29; p=0.001). Of 68 plasma samples from patients off therapy, methylation scores detected the presence of cancer in 59 (86.8%), and methylation-based signatures accurately classified the underlying cancer type in 45 (76.3%) of these. Methylation scores were most accurate in detecting colorectal cancer (96.3%), followed by breast cancer (91.7%), melanoma (81.8%) and NSCLC (61.1%), and most accurate in classifying the underlying cancer type in colorectal cancer (88.5%), followed by NSCLC (81.8%), breast cancer (72.7%) and melanoma (55.6%). Low methylation scores versus high were associated with longer survival (10.4 vs. 4.4 months, p<0.001) and longer time to treatment failure (2.8 vs. 1.6 months, p=0.016).

Conclusions

Comprehensive targeted methylation sequencing of 9,322 CpG sites in plasma cfDNA from patients with common advanced cancers detects the presence of cancer and underlying cancer type with high accuracy. Methylation scores in plasma cfDNA correspond with treatment outcomes.

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Total iron-binding capacity is a novel prognostic marker after curative gastrectomy for gastric cancer

Abstract

Background

Patients with gastric cancer (GC) are affected by changes in iron status. Before surgery, GC patients are likely to have iron-deficiency anemia; and after gastrectomy, patients suffer from low nutritional status and low iron. This study investigated preoperative iron status associated with prognosis after curative gastrectomy for gastric cancer.

Methods

We evaluated preoperative serum hemoglobin (Hgb), Fe and total iron-binding capacity (TIBC) in 298 patients who underwent curative gastrectomy for GC without preoperative chemotherapy, and analyzed these factors' associations with prognosis after surgery.

Results

Of the 298 patients, 129 (43.2%) had low Hgb levels, and 33 (11.1%) had low TIBC (< 260 µg/dl) that was not associated with Hgb or Fe level. Patients with low TIBC were significantly associated with older age (≥ 65 years old; P = 0.0085), low albumin (< 3.9 g/dl; P = 0.0388) and high CRP (≥ 0.15 mg/dl; P = 0.0018) in multivariate analysis. Low Fe (< 60 µg/dl) was not associated with disease-free survival (DFS) or overall survival (OS); however, low Fe was associated with longer cancer-specific survival in Stage III GC patients (P = 0.0333). Both low Hgb and low TIBC were significantly associated with shorter DFS (Hgb: P = 0.0433; TIBC: P < 0.0001) and shorter OS (Hgb: P = 0.0352; TIBC: P < 0.0001). Low TIBC were significantly associated with shorter DFS (HR 2.167, 95% CI 1.231–3.639, P = 0.0086) and shorter OS (HR 2.065, 95% CI 1.144–3.570, P = 0.0173) in multivariate Cox hazard regression analysis.

Conclusions

Preoperative serum TIBC level of GC patients who undergo curative gastrectomy is a novel prognostic marker in univariate and multivariate analyses.

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Chemosensitizing Activity of Histone Deacetylases Inhibitory Cyclic Hydroxamic Acids for Combination Chemotherapy of Lymphatic Leukemia

Background: Anti-tumor effect of hydroxamic acid derivatives is largely connected with its properties as efficient inhibitors of histone deacetylases, and other metalloenzymes involved in carcinogenesis.

Objective: The work was aimed to (i) determine the anti-tumor and chemosensitizing activity of the novel racemic spirocyclic hydroxamic acids using experimental drug sensitive leukemia P388 of mice, and (ii) determine the structure-activity relationships as metal chelating and HDAC inhibitory agents.

Method: Outbreed male rat of 200-220 g weights were used in biochemical experiments. In vivo experiments were performed using the BDF1 hybrid male mice of 22-24 g weight. Lipid peroxidation, Fe (II) -chelating activity, HDAC fluorescent activity, anti-tumor and anti-metastatic activity, acute toxicity techniques were used in this study.

Results: Chemosensitizing properties of water soluble cyclic hydroxamic acids (CHA) are evaluated using in vitro activities and in vivo methods and found significant results. These compounds possess iron (II) chelating properties, and slightly inhibit lipid peroxidation. CHA prepared from triacetonamine (1a-e) are more effective Fe (II) ions cheaters, as compared to CHA prepared from 1- methylpiperidone (2a-e). The histone deacetylase (HDAC) inhibitory activity, lipophilicity and acute toxicity were influenced by the length amino acids (size) (Glycine

Conclusion: It was shown that the use of combination of non-toxic doses of cisplatin (cPt) or cyclophosphamide with CHA in most cases result in the appearance of a considerable anti-tumor effect of cytostatics. The highest chemosensitizing activity with respect to leukemia Р388 is demonstrated by the CHA derivatives of Valine 1c or 2c.

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FOXP2 Promotes Tumor Proliferation and Metastasis by Targeting GRP78 in Triple-negative Breast Cancer

Background: FOXP2, a member of the forkhead box P (FOXP) family, has been reported to be important in breast cancer. However, its exact mechanisms and pathways remain unclear.

Objective: To investigate the effect of FOXP2 on tumor proliferation and metastasis in triplenegative breast cancer (TNBC) and study its underlying molecular mechanism.

Methods: We first used qRT-PCR to detect FOXP2 expression in TNBC cell lines and tissues. Then we conducted cell proliferation assays, colony formation assays, and transwell assays to analyze the effects of FOXP2 expression in TNBC cells. Mouse xenograft model was performed to further confirm the role of FOXP2 in TNBC. Moreover, we used qRT-PCR and Western blot to access the effect of FOXP2 on GRP78 expression and qRT-PCR to analyze GRP78 expression in TNBC tissues. We conducted IHC analysis to detect both FOXP2 and GRP78 expressions in transplanted tumors and used the correlation analysis to further analyze the link between them.

Results: FOXP2 was found to be highly expressed in TNBC cell lines and tissues. FOXP2 knockdown attenuated the growth and invasiveness of TNBC in vitro as well as tumor progression and metastasis in vivo. Moreover, FOXP2 knockdown downregulated glucose-regulated protein of molecular mass 78 (GRP78) expression in TNBC cells and transplanted tumors. Correlation analysis showed that GRP78 expression was positively associated with FOXP2 expression in TNBC cells.

Conclusion: FOXP2 plays a crucial role in TNBC, partly through modulating GRP78, and could act as a potential target for TNBC treatment.

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DNA Fragmentation, Cell Cycle Arrest, and Docking Study of Novel Bis Spiro-cyclic 2-oxindole of Pyrimido[4,5-b]quinoline-4,6-dione Derivatives Against Breast Carcinoma

Background: Recently, it is reported that heterocycles containing pyrimidoquinoline moiety show a broad spectrum of medicinal and pharmacological properties including anticancer, anti-microbial, anti-inflammatory activities, analgesic and antiviral. In additions, spirocyclicoxindole containing compounds represent an important class of compounds that exhibit wide range of biological properties. The asymmetric chiral spiro carbon is considered to be the main criteria of the bioactivities. Spirooxindole structures represent the main skeleton for various alkaloids and pharmaceutically important compounds. Among them, the naturally occurring pyrrolidinylespirooxindole alkaloid, horsifiline that exhibits anticancer activity against human brain cancer cell lines.

Objective: The objective of this study is the synthesis of novel bis spiro-cyclic 2-oxindole of pyrimido[4,5-b]quinoline derivatives and evaluate the anticancer activity of new compounds for synergistic purpose. Different genetic tools were used in an attempt to know the mechanism of action of this compound against breast cancer.

Method: An efficient one pot synthesis of bis spiro-cyclic 2-oxindole derivatives of pyrimido[4,5- b]quinoline-4,6-dione using 6-aminouracil, bis-isatin and dimedone has been developed. The cytotoxic effect against different human cell lines MCF7, HCT116 and A549 cell lines was evaluated. The derivative 6a, was found the most encouraging compound in this series and it was selected for molecular studies against MCF7.

Results: Our data indicated that compound 6a is an attractive target for breast cancer, as it inhibits proliferation, cell cycle progression and induces apoptosis of tumor cells. This inhibition is mediated by fragmentation of genomic DNA, up-regulation of [caspase-3, tumor suppressor gene p53, and pro-apoptotic gene BAX], and down-regulation of anti-apoptotic BCL2 gene. In additions it caused cell cycle arrest in S phase. This work provides an evidence of the potent effect of the new compound 6a and assists in the progress of new healing agents for cancer.

Conclusion: We have developed an efficient method for the synthesis of novel bioactive bis spirocyclic 2-oxindole derivatives incorporating pyrimido[4,5-b]quinoline derivatives. Most of our new derivatives give potent cytotoxic effect more than the standard drug Fluorouracil (5-FU) especially, compound 6a which was the most active and promising one in this series against MCF7, HCT116, and A549 cell lines.

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Neoisoliquiritigenin Inhibits Tumor Progression by Targeting GRP78-β- catenin Signaling in Breast Cancer

Background: Breast cancer mortality has been stable or decreasing in the world, its incidence and recurrence rates have sharply risen worldwide in the recent years.

Objective: To investigate the clinicopathological significance and potential function of GRP78 in the development and progression of breast cancer. To explore the effects of neoisoliquiritigenin (NISL) in breast cancer and the underlying mechanism.

Method: GRP78 was detected by immunohistochemistry (IHC) using breast cancer tissue microarrays (TMAs), and the association between GRP78 levels and clinicopathological factors and prognosis was analyzed. The functional effects of GRP78 on breast cancer were validated by an MTT assay, foci formation assay, Matrigel invasion assay and mouse xenograft assay. The effects of NISL were tested by an MTT assay, apoptosis assay and mouse xenograft assay. A LigandFit algorithm, ATPase activity assay, western blot and IHC assay were used to discover the underlying mechanism of the effects of NSIL.

Results: GRP78 was highly expressed in breast cancer cell lines and tissues. In addition, high expression of GRP78 was correlated to poor outcomes and distant metastasis. Functional experiments showed that GRP78 promoted breast cancer proliferation and invasion in vitro and in vivo. NISL inhibited cell proliferation and induced cell apoptosis in breast cancer by directly binding to GRP78 to regulate the β-catenin pathway.

Conclusion: Taken together, these results highlighted the significance of GRP78 in breast cancer development and suggested NISL as a natural candidate to inhibit breast cancer by targeting GRP78 and β-catenin signaling.

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Vaccine options for influenza: thinking small

Bert Schepens | Dorien De Vlieger | Xavier Saelens

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