Plasma Of Colorectal Cancer Patients Research Articles

Mass Spectrometry Profiling of the Protein Composition of Blood Plasma of Colorectal Cancer Patients

The objective of the study is themass spectrometry analysis of the protein composition in blood plasma samples of large intestine cancer patients. Protein profiles of 58 blood plasma samples of large intestine cancer patients and healthy volunteers are analyzed using ultrahigh-resolution panoramic mass spectrometry. It is shown that the coincidence of identifications in the protein composition samples of intestine cancer patients and healthy men is 60–80%. Samples of intestines cancer patients contain proteins involved in the interaction with ribonucleic acid, proteins, and lowmolecular substrata.

Elevated circulating miR-182 acts as a diagnostic biomarker for early colorectal cancer.

PurposeGlobally, colorectal cancer (CRC) is one of the most common cancers with high mortality. Although CRC patients in stages I–II are curable after surgical resection, due to the lack of sensitive and specific biomarkers, many patients are in the advanced stages when diagnosed. This study aimed to investigate whether circulating miRNAs in plasma could act as biomarkers for early CRC diagnosis.Patients and methodsAll healthy subjects and patients were from Nanjing First Hospital. We first selected 2 differential miRNAs by integrated analysis of 4 Gene Expression Omnibus (GEO) data sets and The Cancer Genome Atlas (TCGA) database. Next, the expression of these 2 miRNAs in tissue and plasma samples were examined through quantitative real-time polymerase chain reaction. Training phase and validation phase were designed to investigate the diagnostic utility of these differential miRNAs using receiver operating characteristic (ROC) curve analysis.ResultsAfter integrated analysis of 4 GEO and TCGA databases, upregulated miR-182 and miR-20a were selected to further investigate their diagnostic potential for CRC. We discovered that miR-182 and miR-20a were upregulated in CRC tissue and plasma and that circulating miR-182 and miR-20a in the plasma of CRC patients were tumor derived. The area under the ROC curve (AUC) of circulating miR-182 was 0.929 (95% CI 0.875–0.983) in the training phase and 0.891 (95% CI 0.821–0.961) in the validation phase. The AUC of circulating miR-20a expression was 0.801 (95% CI 0.695–0.906) in the training phase and 0.736 (95% CI 0.631–0.842) in the validation phase.ConclusionCirculating miR-182 is a novel potential biomarker for early CRC diagnosis.

Clinical value of quantification for cell-free DNA in plasma of colorectal cancer patients

Objective To determine the diagnostic and prognostic value of quantitative estimates of cell-free DNA (cfDNA) in coloretal cancer (CRC). Methods The study included 178 patients at different stages of CRC and 56 healthy donors. The plasma cfDNA was determined using real-time quantitative polymerase chain reaction(Real-time PCR) amplification of the β-actin gene (99 bp). Receiver operating characteristic (ROC) curve was applied to analyze the diagnostic value of cfDNA level. The patients of stage Ⅳ were followed up to determine the prognostic value of cfDNA. Results The cfDNA levels were higher in CRC patients than those in health controls [(63.07±65.38) vs. (22.85±8.88) ng/ml, P=0.000]. The cfDNA levels were also higher in CRC patients of Ⅲ-Ⅳ stage than those in health controls [(39.59±31.56) and (107.32±102.85) ng/ml vs. (22.85±8.88) ng/ml, P=0.000 for all]. Association was observed among plasma cfDNA level and histologic grading [(29.42±18.29), (54.69±75.05), (9.67±103.18) ng/ml, P=0.000] as well as TNM stage (P=0.000). Survival analysis confirmed that high cfDNA levels were clear indicators of a poor over survival. Conclusion This study shows that the baseline cfDNA levels in plasma of CRC patients has no value in the early diagnosis of CRC, but is an effective prognosis biomarker. Key words: Colorectal cancer; Cell-free DNA; Real-time quantitative polymerase chain reaction

Plasma Exosomal Long Non-Coding RNAs Serve as Biomarkers for Early Detection of Colorectal Cancer

Background/Aims: Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the second leading cause of cancer-related deaths worldwide. Thus, methods for early diagnosis of CRC are urgently needed. We aimed to identify potential long non-coding RNAs (lncRNAs) in circulatory exosomes that may serve as biomarkers for the detection of early-stage CRC. Methods: Exosomes from the plasma of CRC patients (n = 50) and healthy individuals (n = 50) were isolated by ultracentrifugation, followed by extraction of total exosomal RNAs using TRIzol reagent. Microarray analysis was used for exosomal lncRNA profiling in the two groups, and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine the expression level of lncRNAs in all patients and healthy subjects. Results: The expression of six lncRNAs (LNCV6_116109, LNCV6_98390, LNCV6_38772, LNCV_108266, LNCV6_84003, and LNCV6_98602) was found to be significantly up-regulated in CRC patients compared with that in healthy individuals by qRT-PCR. The receiver operating characteristic curve was used to verify their diagnostic accuracy. The values of the area under the curve for these lncRNAs were 0.770 (LNCV6_116109), 0.7500 (LNCV6_98390), 0.6500 (LNCV6_38772), 0.6900 (LNCV_108266), 0.7500 (LNCV6_84003), and 0.7200 (LNCV6_98602). Conclusion: Our study suggested that the expression of these six exosomal lncRNAs (LNCV6_116109, LNCV6_98390, LNCV6_38772, LNCV_108266, LNCV6_84003, and LNCV6_98602) was significantly up-regulated in the plasma of CRC patients, and that they may serve as potential non-invasive biomarkers for early diagnosis of CRC.

Aberrant SEPT9 methylation in plasma cell-free DNA of CRC patients

Colorectal cancer (CRC) is one of the most common cancers worldwide. CRC develops from precancerous polyps in the colon or rectum and is preventable by an early diagnosis and with the removal of precursor lesions. Numerous genetics and epigenetic alterations transform benign polyps to malignant tumors by affecting different pathways. Over the past decade, increasing evidence represent the utility of cell-free DNA as a ‘liquid biopsy’ to supplement non-invasive biopsies for genetic and epigenetic characterization and monitoring of solid cancers. One of the epigenetic biomarkers that has gained more attention in CRC is aberrant DNA methylation of Septin 9 gene. In this study we try to evaluate the methylation of Septin 9 gene status in the cfDNA of the plasma in colorectal cancer patients. Plasma cell-free DNA samples were extracted from 30 patients with background of tumors or polyps and 30 samples from healthy individuals. Septin 9 methylation analysis was performed by using the bisulfite specific high resolution melting analysis. The result showed a sensitivity and specificity of 10% and 53.33%, respectively. In conclusion, our results demonstrated that Septin 9 DNA methylation in plasma determined by THP and BSP-HRM had not have sufficient accuracy.

Surface Enhanced Raman Spectroscopy (SERS) for the Multiplex Detection of Braf, Kras, and Pik3ca Mutations in Plasma of Colorectal Cancer Patients.

In this paper, we discuss the use of a procedure based on polymerase chain reaction (PCR) and surface enhanced Raman spectroscopy (SERS) (PCR-SERS) to detect DNA mutations.Methods: This method was implemented by first amplifying DNA-containing target mutations, then by annealing probes, and finally by applying SERS detection. The obtained SERS spectra were from a mixture of fluorescence tags labeled to complementary sequences on the mutant DNA. Then, the SERS spectra of multiple tags were decomposed to component tag spectra by multiple linear regression (MLR).Results: The detection limit was 10-11 M with a coefficient of determination (R2) of 0.88. To demonstrate the applicability of this process on real samples, the PCR-SERS method was applied on blood plasma taken from 49 colorectal cancer patients to detect six mutations located at the BRAF, KRAS, and PIK3CA genes. The mutation rates obtained by the PCR-SERS method were in concordance with previous research. Fisher's exact test showed that only two detected mutations at BRAF (V600E) and PIK3CA (E542K) were significantly positively correlated with right-sided colon cancer. No other clinical feature such as gender, age, cancer stage, or differentiation was correlated with mutation (V600E at BRAF, G12C, G12D, G12V, G13D at KRAS, and E542K at PIK3CA). Visually, a dendrogram drawn through hierarchical clustering analysis (HCA) supported the results of Fisher's exact test. The clusters drawn by all six mutations did not conform to the distributions of cancer stages, differentiation or cancer positions. However, the cluster drawn by the two mutations of V600E and E542K showed that all samples with those mutations belonged to the right-sided colon cancer group.Conclusion: The suggested PCR-SERS method is multiplexed, flexible in probe design, easy to incorporate into existing PCR conditions, and was sensitive enough to detect mutations in blood plasma.

Circulating cell-free DNA in plasma of colorectal cancer patients - A potential biomarker for tumor burden

BackgroundMeasurement of cell-free DNA (cfDNA) in plasma – the so called liquid biopsy - is a novel method for early detection of cancer. Necrotic cancer cells release various DNA fragments that can be detected in plasma or serum. The aim of our study was to investigate the concentration of circulating ALU115, LINE79 and LINE297 fragments in plasma from venous and arterial blood of colorectal cancer (CRC) patients before, during and 5 days after surgical intervention. Patients and methodsThirty patients (16 female, 14 male, median age 56 years), undergoing surgery for colorectal and appendix cancer, and 17 healthy volunteers were included in this study. Plasma samples were collected from patients and healthy individuals. Qualitative polymerase chain reaction (PCR) and quantitative real-time PCR analyses were conducted using specific primers for ALU115, LINE79 and LINE297. ResultsThe concentration of ALU115 was significantly increased in plasma of CRC patients compared to the control group (p = 0.002). Interestingly, the concentration of LINE297 was significantly higher in healthy individuals than patients (p = 0.031). We did not find any difference regarding LINE79 between the two groups (p = 0.893). The total cfDNA concentration was slightly increased in plasma after the surgery (p < 0.056), however, the difference was not significant. Interestingly, no correlation was detected between the peritoneal carcinosis index (PCI) and conventional tumor markers. ConclusionAccording to our results, the concentration of ALU115 in cfDNA could be a potential biomarker for diagnosis of CRC. LINE79 or the conventional tumor markers CEA or CA19-9 do not seem useful for the detection of malignant tumors. Whether the amount of LINE297 in cfDNA represents a reliable biomarker for early diagnosis has yet to be confirmed.

GPC1 exosome and its regulatory miRNAs are specific markers for the detection and target therapy of colorectal cancer.

Colorectal cancer (CRC) is the second leading cause of cancer‐related deaths worldwide. However, a biomarker for a sensitive and simple diagnostic test and highly effective target therapy of CRC is still clinically unavailable. This study is to investigate the evidence and significance of plasma GPC1 positive exosomes as a biomarker of CRC. Results showed that GPC1+ exosomes were successfully isolated from tissues and plasma. The percentage of GPC1+ exosomes and the GPC1 protein expression in exosomes from tumour tissues and plasma of CRC patients before surgical treatment was significantly elevated compared to that in the peritumoural tissues and the plasma of healthy controls. miR‐96‐5p and miR‐149 expression in tumour tissues and plasma of CRC patients as well as in the GPC1+ exosomes from CRC patients were significantly decreased compared to that in the peritumoural tissues and the plasma of healthy controls. Two months after surgical treatment, levels of all tested markers significantly normalized. Overexpression of miR‐96‐5p and miR‐149 significantly decreased GPC1 expression in HT‐29 and HCT‐116 cells, xenograft tumours, plasma in mice bearing HT‐29 and HCT‐116 tumours, and the secretion of GPC1+ exosomes from the HT‐29 and HCT‐116 cells and xenograft tumours. Overexpression of miR‐96‐5p and miR‐149 significantly decreased cell viability and increased cell apoptosis in HT‐29 and HCT‐116 cells, and inhibited the growth of xenograft HT‐29 and HCT‐116 tumours. In conclusion, the increased plasma GPC1+ exosomes and reduced plasma miR‐96‐5p and miR‐149 expression are specific markers for the diagnosis of CRC and targets for the therapy of CRC.

MiR-542-3p suppresses colorectal cancer progression through targeting survivin

Background: miR-542-3p has been reported to be a tumor suppressor in several tumor types, while its role in colorectal cancer (CRC) has not been fully understood. This study aimed to investigate the expression of miR-542-3p and its potential role in human CRC. Methods: Real-time PCR was used to detect the expression of miR-542-3p in tissues and plasma of CRC patients. The impact of miR-542-3p on the aggressive phenotypes of CRC cells were evaluated by in vitro functional assays. Luciferase activity assay was conducted to confirm the direct binding of miR-542-3p to survivin. Results: miR-542-3p was decreased in CRC cell lines that derived from metastatic sites. Among the 65 CRC patients enrolled in this study, 63.08% (41/65) had a decreased miR-542-3p expression in cancerous tissues. miR-542-3p expression was associated with lymphovascular invasion (P=0.008), distant metastasis (P=0.006), tumor stage (P=0.034) and patients’ survival (P=0.027). A decreased expression of miR-542-3p in plasma was detected in stage IV patients. In vitro and in vivo experiments showed that miR-542-3p could inhibit the aggressive phenotypes of CRC cells. Finally, survivin was identified as a direct target of miR-542-3p in CRC. Conclusions: Decreased expression of miR-542-3p in CRC patients was associated with unfavourable clinicopathological features of the patients. miR-542-3p inhibits the aggressive phenotypes of CRC cell lines. Survivin is a direct target of miR-542-3p in CRC.

Poster

Poster

KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients.

KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy”) by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker.

Isolation and characterization of circulating micro(nano)vesicles in the plasma of colorectal cancer patients and their interactions with tumor cells.

Micro(nano)vesicles (MV) are regarded as important messengers in cell-to-cell communication. There is also evidence for their pivotal role in cancer progression. Circulating MV are of different body cells origin, including tumor cell‑derived MV (TMV) in cancer patients. Determination of circulating TMV is of importance because of their potential diagnostic and therapeutic applications. In the present study, an analysis of circulating MV in colorectal cancer (CRC) patients was undertaken. Plasma from healthy donors was used as the control. In order to define MV characteristics, two plasma fractions: obtained by sequential centrifugation at 15,000xg (MV15) and 50,000xg (MV50) were used for analysis. The two fractions possessed a large range of sizes: 70(80)-1,300(1,400)nm and the most common particles with sizes 70-90nm, both in patients and controls. Atomic force microscopy images of MV50 revealed a heterogeneous population of particles with different shapes and sizes. MV15 contained an increased level of CD41+ and CD61+ particles, suggesting their platelet origin. No difference between patients and controls was observed. A more precise analysis of MV50 showed the increased level of particles expressing EGFR (HER-1/ErbB1), HER-2/neu and Mucin1 (MUC1), suggesting their tumor origin. The total level of MV50‑expressing EGFR, HER-2/neu and MUC1 was enhanced in CRC patients. MV50 both of patients and controls attached to a colon cancer cell line (SW480) and to isolated blood monocytes at 2h and were engulfed at 24h. This uptake showed the lack of specificity. Thus, apart from the direct delivery of MV to the tumor site by plasma, monocytes carrying MV may also be involved in their transportation. Taken together, the presented data indicate that MV15 contain mainly platelet‑derived particles, while MV50 from CRC patients are enriched in TMV. Interaction of MV with cancer cells may pin-point their role in communication between tumor cells, resulting in molecular cargo exchange between them.

Circulating pEGFR is a candidate response biomarker of cetuximab therapy in colorectal cancer.

The lack of secreted biomarkers measurable by noninvasive tests hampers the development of effective targeted therapies against cancer. Our hypothesis is that cetuximab (an anti-EGFR mAb) induces a specific secretome in colorectal cancer cells that could be exploited for biomarker discovery. Considering the strong correlation between mutated KRAS and a lack of response to cetuximab therapy, we addressed whether performing secretome-based proteomics on isogenic colorectal cancer cells sharing the KRAS mutations found on patients would yield candidate-secreted biomarkers useful in the clinical setting. Because 2D culture did not optimally model the sensitivity/resistance to cetuximab observed in colorectal cancer patients, we moved to 3D spheroids, developing a methodology for both cell-based assays and quantitative proteomics. A large comparative quantitative proteomic analysis of the 3D secretomes of colorectal cancer isogenic cells treated with cetuximab uncovered an EGFR pathway-centric secretome found only when cells grow in 3D. The validation of the secretome findings in plasma of colorectal cancer patients, suggests that phosphorylated-EGFR (pEGFR) is a candidate-secreted biomarker of response to cetuximab. We have proved that 3D spheroids from colorectal cancer cells generate secretomes with a drug-sensitivity profile that correlates well with patients with colorectal cancer, illustrating molecular connections between intracellular and extracellular signaling. Furthermore, we show how the secretion of pEGFR is associated with the sensitivity of colorectal cancer cells to cetuximab and the response of patients with colorectal cancer to the drug. Our work could allow the noninvasive monitoring of anti-EGFR treatment in patients with colorectal cancer.

Detection of methylated CDO1 in plasma of colorectal cancer; a PCR study.

BackgroundCysteine biology is important for the chemosensitivity of cancer cells. Our research has focused on the epigenetic silencing of cysteine dioxygenase type 1 (CDO1) in colorectal cancer (CRC). In this study, we describe detection of CDO1 methylation in the plasma of CRC patients using methylation specific PCR (Q-MSP) and extensive analysis of the PCR reaction.MethodsDNA was extracted from plasma, and analysed for methylation of the CDO1 gene using Q-MSP. The detection rate of CDO1 gene methylation was calculated and compared with that of diluted DNA extracted from “positive control” DLD1 cells. CDO1 gene methylation in the plasma of 40 CRC patients that were clinicopathologically analysed was then determined.Results(1) The cloned sequence analysis detected 93.3% methylation of the promoter CpG islands of the CDO1 gene of positive control DLD1 cells and 4.7% methylation of the negative control HepG2 CDO1 gene. (2) DLD1 CDO1 DNA could not be detected in this assay if the extracted DNA was diluted ∼1000 fold. The more DNA that was used for the PCR reaction, the more effectively it was amplified in Q-MSP. (3) By increasing the amount of DNA used, methylated CDO1 could be clearly detected in the plasma of 8 (20%) of the CRC patients. However, the percentage of CRC patients detected by methylated CDO1 in plasma was lower than that detected by CEA (35.9%) or CA19-9 (23.1%) in preoperative serum. Combination of CEA/CA19-9 plus plasma methylated CDO1 could increase the rate of detection of curable CRC patients (39.3%) as compared to CEA/CA19-9 (25%).ConclusionWe have described detection of CDO1 methylation in the plasma of CRC patients. Although CDO1 methylation was not detected as frequently as conventional tumor markers, analysis of plasma CDO1 methylation in combination with CEA/CA19-9 levels increases the detection rate of curable CRC patients.

Aberrant promoter methylation of PPP1R3C and EFHD1 in plasma of colorectal cancer patients.

Aberrant DNA methylation is a common epigenetic alteration involved in colorectal cancer (CRC). In our previous study, we performed methylated DNA immunoprecipitation-on-chip analysis combined with gene re-expression analysis by 5-aza-2′-deoxycytidine treatment, to identify methylation genes in CRC genome widely. Among these genes, 12 genes showed aberrant hypermethylation frequently in >75% of 149 CRC samples but did not in normal samples. In this study, we aim to find out any of these methylation genes to be utilized for CRC detection using plasma DNA samples. Primers for methylation-specific PCR and pyrosequencing were designed for seven of the 12 genes. Among them, PPP1R3C and EFHD1 were rarely hypermethylated in peripheral blood cells, but frequently hypermethylated in 24 CRC tissue samples and their corresponding plasma samples. In plasma samples, PPP1R3C was methylated in 81% (97/120) of CRC patients, but only in 19% (18/96) of noncancer patients (P = 6 × 10−20, Fisher's exact test). In combined analysis with EFHD1, both genes were methylated in 53% (64/120) of CRC patients, but only in 4% (4/96) of noncancer patients (P = 2 × 10−16), giving high specificity of 96%. At least one of the two genes was methylated in 90% (108/120) of CRC patients, and 36% (35/96) of control patients, giving high sensitivity of 90%. Compared with low sensitivity of carcinoembryonic antigen (17% at stage I, 40% at stage II) and CA19-9 (0% at stage I, 13% at stage II) for early-stage CRCs, sensitivity of aberrant methylation was significantly higher: PPP1R3C methylation at 92% (11/12) for stage I and 77% (23/30) for stage II, and methylation of at least one gene at 100% (12/12) for stage I and 87% (26/30) for stage II. PPP1R3C methylation or its combined use of EFHD1 methylation was highly positive in CRC plasma samples, and they might be useful in detection of CRC, especially for early-stage CRCs.

Protein expression and gene polymorphism of CXCL10 in patients with colorectal cancer

Chemokines (chemotactic cytokines) promote leukocyte attraction to sites of inflammation and cancer. Certain chemokines promote and regulate neoplastic progression, including metastasis and angiogenesis. One such chemokine, CXCL10, was found to be expressed in colorectal cancer (CRC) tissue. To gain insight into the prognostic significance of CXCL10, we investigated whether the levels of this chemokine were altered in the colorectal tissue or plasma of CRC patients. Using Luminex technology for protein analyses, we observed a significantly higher CXCL10 protein level in cancer tissue compared to that in paired normal tissue. Moreover, significantly higher plasma levels of CXCL10 were detected in patients compared to those in control subjects and the plasma levels of CXCL10 in disseminated disease were found to be significantly higher compared to those in localized disease. The single-nucleotide polymorphism rs8878, which has been described in exon 4 in the 3′-untranslated region of the CXCL10 gene, was investigated using a TaqMan system. There were significant differences in genotype distribution and allelic frequencies between CRC patients and control subjects. In conclusion, altered CXCL10 protein concentrations in CRC tissues or plasma and the rs8878 genotype variant of CXCL10 may contribute to the prediction of clinical outcome.

Multiplex Picodroplet Digital PCR to Detect KRAS Mutations in Circulating DNA from the Plasma of Colorectal Cancer Patients

Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample. We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR. Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected. This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.

Pharmacokinetic/Pharmacodynamic Modeling of 5-Fluorouracil by Using a Biomarker to Predict Tumor Growth in a Rat Model of Colorectal Cancer

We developed a pharmacokinetic/pharmacodynamic (PK/PD) model with the value of the plasma ratio of dihydrouracil (UH2)/uracil (Ura), which is a possible surrogate biomarker of hepatic dihydropyrimidine dehydrogenase activity, determined before 5-fluorouracil (5-FU) treatment to simulate the growth of tumors after 5-FU treatment in rats with colorectal cancer (CRC). In the PK/PD model, the value of the elimination rate constant of 5-FU-ke -was estimated using the plasma UH2/Ura ratio observed before 5-FU treatment for simulating PKs of 5-FU and tumor growth. The PK/PD model with plasma UH2/Ura ratio effectively captured the features of tumor growth and the anticancer effect of 5-FU treatment, which provided reliable parameter estimates. In addition to an appropriate dosing regimen, pretherapeutic assessment of the UH2/Ura ratio in the plasma of CRC patients and PK/PD analysis with the plasma UH2/Ura ratio could enable the development of an optimal therapeutic scheme for each patient.

Abstract 163: Metabolomic profiling reveals a protective modulation on fatty acid metabolism in colorectal cancer patients following consumption of freeze-dried black raspberries.

Abstract Metabolic reprogramming which refers to altered nutrient uptake and use is thought to be essential for rapid cancer cell proliferation. Accelerated phospholipid biosynthesis is another metabolic signature of cancer because proliferating cells have a significant need for membrane production. Fatty acids that compose the hydrophobic tails of membrane phospholipids can regulate gene transcription through enzyme-mediated pathways, e.g., cyclooxygenase, lipoxygenase, and changing lipid raft composition that affect receptor-mediated signalings. We previously showed that dietary intervention with freeze-dried black raspberries (BRBs) decreased cell proliferation in colorectal tumors and IL-8, a pro-inflammatory cytokine, in plasma in colorectal cancer patients. The goal of the current study was to determine if BRBs affect fatty acid metabolism which may contribute to their anti-proliferative and anti-inflammatory activities. Plasma samples were collected from 28 colorectal cancer patients before and after oral consumption of BRB powder (60g/day) for 1-to-9 wks for metabolomic profiling analysis. 421 biochemicals were analyzed using UHPLC/Gas chromatography and mass spectrometry. When data from all 28 patients were combined, the top 30 ranking biochemicals suggest that berry intervention led to alterations mostly in lipids, following by carbohydrates, amino acids, and cofactors and vitamins. Both monounsaturated fatty acids (MUFAs), e.g., eicosenoate (20:1n9 or 11), and polyunsaturated fatty acids (PUFAs), e.g., linoleate (18:2n6), arachidonate (20:4n6), were lower in the post plasma samples. PUFAs can be synthesized from linoleate and they also can be released by phospholipase A from phospholipid membrane. Therefore, berry intervention might alter activities of phospholipase A, elongases, and desaturases which in turn reduce levels of PUFAs. Alternatively, berry intervention increased secondary bile acids, e.g., glycodeoxycholate, produced by the action of enzymes existing in the microbial flora of the colonic environment, suggesting dietary BRBs could alter colonic microflora. Berry intervention associated alternations in bile acid metabolism could affect fat absorption and subsequently impact fatty acid metabolism. In conclusion, our results suggest that dietary berry consumption protectively modulates enzymes associated with fatty acid metabolism in the host as well as in gut microflora leading to decreased proliferation and inflammation in colorectal cancer patients. Supported by R01 CA148818 to L-S Wang Citation Format: Li-Shu Wang, Matthew Young, Chieh-Ti Kuo, Mark Arnold, Edward Martin, Christine Sardo, Gary Stoner. Metabolomic profiling reveals a protective modulation on fatty acid metabolism in colorectal cancer patients following consumption of freeze-dried black raspberries. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 163. doi:10.1158/1538-7445.AM2013-163

Abstract LB-78: Detection of circulating cell-free DNA is enhanced by targeting short fragments: high concordance of the KRAS and BRAF mutation status from plasma analysis, with tumor-tissue analysis in colorectal cancer..

Abstract We investigated size distributions of circulating cell-free DNA (ccfDNA) to determine its value as a potential theranostic tool. CcfDNA quantification by Q-PCR was optimal when amplifying target lengths between 60 and 100 bp. CcfDNA sizes were previously believed to be greater than the length of the DNA per nucleosome (~180 bp). Q-PCR analyses of plasma samples from xenografted mice and from colorectal cancer (CRC) patients showed that tumor derived ccfDNA exhibited specific profiles of ccfDNA sizes with significantly higher ccfDNA fragmentation than was observed in samples from healthy individuals. Atomic Force Microscopy analysis confirmed these observations and, for the first time, enables visual representation of ccfDNA molecules. CcfDNA from plasma of CRC patients exhibited a median size of 135 bp and 88% of ccfDNAs are less than 180 bp in size. Based on these observations, we developed a refined single-step Allele Specific Q-PCR based method (Intplex) with improved specificity and sensitivity for enabling single-copy detection of variant alleles to a sensitivity of ≥ 0.005% mutant to WT ratio. We performed a blinded prospective study to compare KRAS and BRAF mutation status data by analysis of tumor tissue of CRC patients by standard methods and of plasma DNA (n=79). CcfDNA analysis showed 100% specificity and sensitivity for the BRAF V600E mutation. For the six tested KRAS point mutations, the method exhibited 100% specificity and 87% sensitivity with a concordance value of 96%. Mutation load expressed as the proportion of mutant alleles in ccfDNA, was highly variable (0.03% to 69%, median, 10.9%; n=128) and was determined in the plasma of every patient at stage I, II, III and IV (100% success rate). Tumor-derived ccfDNA did not correspond to "a tiny fraction" as it has been previously stated and it is likely not derived mainly from stroma cells. CcfDNA analysis could replace advantageously tumor-section analysis for determining KRAS and BRAF mutation status and expand the scope of personalized medicine for cancer patients. Citation Format: Florent Mouliere, Dalong Pang, Safia El Messaoudi, Denis Pezet, Muriel Mathonnet, Marc Ychou, Anatoly Dritschilo, Alain R. Thierry. Detection of circulating cell-free DNA is enhanced by targeting short fragments: high concordance of the KRAS and BRAF mutation status from plasma analysis, with tumor-tissue analysis in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-78. doi:10.1158/1538-7445.AM2013-LB-78