Total circulating cell-free DNA as a prognostic biomarker in metastatic colorectal cancer before first-line oxaliplatin-based chemotherapy.

BACKGROUND: Metastatic colorectal cancer (mCRC) is a heterogeneous disease where prognosis is dependent both on tumor biology and host factors. Total circulating cell-free DNA (cfDNA) has shown to harbor prognostic information in mCRC, although less is known about the biological correlates of cfDNA levels in this patient group. The primary objective was to evaluate the prognostic value of pretreatment cfDNA in patients receiving the first-line oxaliplatin-based chemotherapy for mCRC, by using a predefined upper limit of normal (ULN) from a cohort of presumed healthy individuals. The secondary objective was to model cfDNA levels as a function of predefined tumor and host factors. PATIENTS AND METHODS: This was a retrospective post hoc study based on a prospective multicenter phase III trial, the NORDIC-VII study. DNA was purified from 547 plasma samples and cfDNA quantified by a droplet digital PCR assay (B2M, PPIA) with controls for lymphocyte contamination. Main clinical end point was overall survival (OS). RESULTS: cfDNA was quantified in 493 patients, 54 were excluded mainly due to lymphocyte contamination. Median cfDNA level was 7673 alleles/ml (1050-1 645 000) for B2M and 5959 alleles/ml (555-854 167) for PPIA. High cfDNA levels were associated with impaired outcome; median OS of 16.6 months for levels above ULN and 25.9 months for levels below ULN (hazard ratio = 1.83, 95% confidence interval 1.51-2.21, P < 0.001). The result was confirmed in multivariate OS analysis adjusting for established clinicopathological characteristics. A linear regression model predicted cfDNA levels from sum of longest tumor diameters by RECIST, the presence of liver metastases and systemic inflammatory response as measured by interleukin 6 (F(6, 357) = 62.7, P < 0.001). CONCLUSION: cfDNA holds promise as a minimally invasive and clinically relevant prognostic biomarker in mCRC before initiating first-line oxaliplatin-based chemotherapy and may be a complex entity associated with tumor burden, liver metastases and systemic inflammatory response. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00145314.

Differential expression of miRNAs in pancreatobiliary type of periampullary adenocarcinoma and its associated stroma.

Periampullary adenocarcinomas can be of two histological subtypes, intestinal or pancreatobiliary. The latter is more frequent and aggressive, and characterized by a prominent desmoplastic stroma, which is tightly related to the biology of the cancer, including its poor response to chemotherapy. Whereas miRNAs are known to regulate various cellular processes and interactions between cells, their exact role in periampullary carcinoma remains to be characterized, especially with respect to the prominent stromal component of pancreatobiliary type cancers. The present study aimed at elucidating this role by miRNA expression profiling of the carcinomatous and stromal component in twenty periampullary adenocarcinomas of pancreatobiliary type. miRNA expression profiles were compared between carcinoma cells, stromal cells and normal tissue samples. A total of 43 miRNAs were found to be differentially expressed between carcinoma and stroma of which 11 belong to three miRNA families (miR-17, miR-15 and miR-515). The levels of expression of miRNAs miR-17, miR-20a, miR-20b, miR-223, miR-10b, miR-2964a and miR-342 were observed to be higher and miR-519e to be lower in the stromal component compared to the carcinomatous and normal components. They follow a trend where expression in stroma is highest followed by carcinoma and then normal tissue. Pathway analysis revealed that pathways regulating tumor-stroma interactions such as ECM interaction remodeling, epithelial-mesenchymal transition, focal adhesion pathway, TGF-beta, MAPK signaling, axon guidance and endocytosis were differently regulated. The miRNA-mRNA mediated interactions between carcinoma and stromal cells add new knowledge regarding tumor-stroma interactions.

AGXT and ERCC2 polymorphisms are associated with clinical outcome in metastatic colorectal cancer patients treated with 5-FU/oxaliplatin.

The objective of the study was to investigate whether specific single nucleotide polymorphisms (SNPs) with influence on drug transport, biotransformation and repair mechanisms are associated with treatment outcome and toxicity in metastatic colorectal cancer (mCRC). We genotyped blood samples from 519 mCRC patients treated with first-line 5-fluorouracil and oxaliplatin +/- cetuximab for 17 SNPs in 10 genes involved in membrane transport (ABCC1 and ABCC2), drug biotransformation (GSTP1 and AGXT) and DNA repair (ERCC1, ERCC2, XRCC1, XRCC3, XPG and MSH6). The AGXT-rs34116584 and the ERCC2-rs238406 polymorphisms were significantly associated with progression-free survival (P=0.002 and P=0.001, respectively). Associations between 18 toxicity variables and SNPs were identified, although none were significant after Bonferroni correction for multiple comparisons. The study identified SNPs of potential use as markers of clinical outcome in oxaliplatin-treated mCRC patients. If validated in other studies, they could improve the selection of therapy in mCRC.

Molecular signatures of mRNAs and miRNAs as prognostic biomarkers in pancreatobiliary and intestinal types of periampullary adenocarcinomas.

Periampullary adenocarcinomas include four anatomical sites of origin (the pancreatic duct, bile duct, ampulla and duodenum) and most of them fall into two histological subgroups (pancreatobiliary and intestinal). Determining the exact origin of the tumor is sometimes difficult, due to overlapping histopathological characteristics. The prognosis depends on the histological subtype, as well as on the anatomical site of origin, the former being the more important. The molecular basis for these differences in prognosis is poorly understood. Whole-genome analyses were used to investigate the association between molecular tumor profiles, pathogenesis and prognosis. A total of 85 periampullary adenocarcinomas were characterized by mRNA and miRNA expressions profiling. Molecular profiles of the tumors from the different anatomical sites of origin as well as of the different histological subtypes were compared. Differentially expressed mRNAs and miRNAs between the two histopathological subtypes were linked to specific molecular pathways. Six miRNA families were downregulated and four were upregulated in the pancreatobiliary type as compared to the intestinal type (P < 0.05). miRNAs and mRNAs associated with improved overall and recurrence free survival for the two histopathological subtypes were identified. For the pancreatobiliary type the genes ATM, PTEN, RB1 and the miRNAs miR-592 and miR-497, and for the intestinal type the genes PDPK1, PIK3R2, G6PC and the miRNAs miR-127-3p, miR-377* were linked to enriched pathways and identified as prognostic markers. The molecular signatures identified may in the future guide the clinicians in the therapeutic decision making to an individualized treatment, if confirmed in other larger datasets.

Plasma microRNAs predicting clinical outcome in metastatic colorectal cancer patients receiving first-line oxaliplatin-based treatment.

The conventional first-line chemotherapy for metastatic colorectal cancer (mCRC) consists of fluorouracil (5-FU) in combination with either oxaliplatin or irinotecan. We have explored microRNAs (miRNAs) in plasma as potential predictive markers to oxaliplatin-based chemotherapy. The expression of 742 miRNAs was examined in plasma samples from 24 mCRC patients (12 responders and 12 non-responders) before onset and after four cycles of 5-FU/oxaliplatin. The top differentially expressed miRNAs between responders and non-responders were selected for further analysis in a validation cohort of 150 patients. In the validation cohort, there was a significant overrepresentation of miRNAs with higher mean expression in the non-responder group than in the responder group before treatment (p < 0.002). Moreover, we found three miRNAs (miR-106a, miR-484, and miR-130b) to be significantly differentially expressed before treatment (p = 0.008, 0.008, and 0.008, respectively). All three miRNAs were upregulated in non-responders. High expression of miR-27b, miR-148a, and miR-326 were associated with decreased progression-free survival (Hazard ratios (HR) of 1.4 (95% CI 1.1-1.8, p = 0.004), 1.3 (95% CI 1.1-1.6, p = 0.007), and 1.4 (95% CI 1.1-1.8, p = 0.008), respectively). miR-326 was also associated with decreased overall survival (HR 1.5 (95% CI 1.1-2.0, p = 0.003)). There were no significantly differentially expressed miRNAs in association with clinical outcome after four cycles of chemotherapy. The present study demonstrates that plasma miRNAs analyzed before treatment may serve as non-invasive markers predicting outcome in mCRC patients treated with 5-FU and oxaliplatin-based chemotherapy.

Genetic susceptibility and environmental estrogen-like compounds.

Environmental chemicals with estrogenic activities have been suggested to be able to interact with the endocrine system. Endogenous estrogen is synthesized in the ovarian theca cells of premenopausal women or in the stromal adipose cells of the breast of postmenopausal women and minor quantities in peripheral tissue. These cells, as well as breast tissue, express all the necessary enzymes for this synthesis, CYP17, CYP11a, CYP19, 17-beta-hydroxysteroid hydrogenase, steroid sulfatase as well as enzymes further hydroxylating estradiol, such as CYP1A1, CYP3A4, CYP1B1, catechol-o-methyltransferase (COMT). Polymorphisms in these enzymes may have a possible role in the link between environmental estrogens and hormone-like substances and the interindividual risk of breast cancer.

p53 mutations in defined structural and functional domains are related to poor clinical outcome in non-small cell lung cancer patients.

The prognostic value of p53 status in non-small cell lung cancer has been investigated in 148 patients with clinical stage I-IIIB disease. Tumor tissues were examined for mutations in exons 4-9, with emphasis on defined structural and functional domains. Eighty-four mutations were detected in 83 (54%) of the patients. Eighty-eight percent of the mutations were within exons 5-8, and 12% of the mutations were within exons 4 and 9. Missense mutations occurred in 67% of the tumors, and 30% were null mutations (10% stop mutations, 15% frameshift mutations, and 5% splice site mutations). Patients with mutations in p53 had a significantly higher risk for lung cancer-related death and for death from all causes than those with wild-type p53 [hazard ratio (HR) = 2.09 and 95% confidence interval (CI) = 1.20-3.64 and HR = 1.69 and 95% CI = 1.06-2.70, respectively]. Mutations in p53 related to even still poorer lung cancer-related prognosis were found at the following locations: (a) exon 8 (HR = 3.5; 95% CI, 1.59-7.71)]; (b) the structural domains L2 + L3 (HR = 2.36; 95% CI, 1.18-4.74), and (c) codons involved in zinc binding (HR = 11.7; 95% CI, 3.56-38.69). Together, the biologically functional group of severe flexible mutants (codons 172, 173, 175, 176, 179, 181, 238, 245, and 267) and severe contact mutants (248, 282) were significantly related to shorter lung cancer-related survival (HR = 4.16; 95% CI, 1.93-8.97). Squamous cell carcinoma was the dominant histological type in tumors involved in poor prognosis in exon 8 (HR = 3.19; 95% CI, 1.07-9.45). These results indicate that mutations in defined structural and functional domains of p53 may be useful molecular biological markers for prognosis and treatment strategy in non-small cell lung cancer patients.

p53 gene mutation analysis in tumors of patients exposed to alpha-particles.

The p53 gene was examined for point mutations in archived, alpha-radiation-associated lung and liver cancers. Lung tumors of 50 uranium miners in Germany were screened by restriction fragment length analysis for the putative hotspot mutation at codon 249 (Arg-->Met) previously detected in a significant fraction of miners from the Colorado Plateau, USA. This mutation has been proposed as a marker of radon exposure. None of the tumors we examined harbored the hotspot mutation. Five of the 50 tumors, however, did indeed harbor exon 7 mutations, as determined by subsequent mutation analysis of exon 7. These mutations were dispersed among various codons and may be attributable to heavy tobacco smoking in this cohort. In support of this interpretation, we found no mutations in exons 5-8 of the p53 gene in 13 iatrogenic liver cancers induced by injection of Thorotrast, an alpha-emitting radiocontrast agent. We propose that if the p53 tumor suppressor gene is a target for the carcinogenic action of alpha-particle radiation, loss of suppressor function may occur preferentially by mechanisms such as intrachromosomal deletions, rather than by base substitution mutations.

Benzo(a)pyrene-albumin adducts in humans exposed to polycyclic aromatic hydrocarbons in an industrial area of Poland.

OBJECTIVES: The interaction of benzo(a)pyrene with serum albumin was measured in an attempt to identify the actual exposure and to evaluate albumin adduct measurements as biomarkers for exposure monitoring. METHODS: Benzo(a)pyrene-diol-epoxide (BPDE)-albumin adducts were measured by competitive enzyme linked immunosorbent assay (ELISA) in plasma of coke oven plant workers from three plants and from people living in a highly industrialised area of Silesia in Poland. Due to the high air concentrations of polycyclic aromatic hydrocarbons (PAHs) in this area, a control group was selected from a rural non-industrialised area in Poland. Breathing zone air measurements of PAHs were collected from some of the participants. RESULTS: Coke oven plant workers and non-occupationally exposed people had similar concentrations of albumin adducts whereas the rural controls were significantly lower (2.74 fmol adducts/microgram albumin (SEM 0.124)). The mean concentration of BPDE-albumin adduct in plasma of both the occupational and the environmental groups were significantly higher in the summer samples (4.34 fmol adducts/microgram albumin (SEM 0.335) and 4.55 fmol adducts/microgram albumin (SEM 0.296), respectively) than in the winter samples (3.06 fmol adducts/microgram albumin (SEM 0.187) and 3.04 fmol adducts/microgram albumin (SEM 0.184), respectively) even though the air measurements showed higher concentrations of PAHs in the winter. The statistical analysis did not show any effects of air exposures on concentrations of BPDE-albumin adduct. CONCLUSIONS: A multiple regression analysis of the measured concentrations of BPDE-albumin adducts for all the groups, during both seasons, indicates that occupational exposures do not contribute significantly to the formation of adducts. In general, the concentrations of albumin adducts found vary within relatively small limits for the two seasons and between the various groups of participants. No extreme differences were found.

p53 mutations in lung tumours: relationship to gender and lung DNA adduct levels.

Human lung cancer exhibits a high frequency of transversion mutations at G:C base pairs of the p53 gene, possibly the result of DNA damage by cigarette smoke constituents, most notably benzo[a]pyrene. We have investigated gender differences in the p53 mutational spectrum and levels of hydrophobic DNA adducts. Tumour tissue was obtained from 115 non-small cell lung cancer tumours and examined for mutational alterations in the p53 gene (exons 4-9) using PCR and single-strand conformational polymorphism analysis. We have previously examined exons 5-8 in lung cancer. Sequence analysis of exons 4 and 9 revealed that almost 20% of the mutations were located in exons 4 and 9. The levels of hydrophobic DNA adducts in non-tumorous lung tissue of 55 of the patients were analyzed by the 32P-postlabelling assay. There were both a higher frequency of G:C-->T:A mutations and a higher average hydrophobic DNA adduct level in females than in male patients, even though the level of exposure to carcinogens from cigarette smoking was lower among the females than among the males. Frameshift mutations were more common in women than in men (30 versus 15%). These preliminary findings lend support to epidemiological evidence that women may be at greater risk than men of contracting tobacco-induced lung cancer.

Comparative synchronous fluorescence spectrophotometry and 32P-postlabeling analysis of PAH-DNA adducts in human lung and the relationship to TP53 mutations.

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were studied in human lung from 39 lung cancer patients by synchronous fluorescence spectrophotometric (SFS) and 32P-postlabeling assays. Regression analysis of the samples failed to detect any correlation between benzo[a]pyrene-diolepoxide (BPDE)-DNA adducts detected by SFS and the BPDE co-migrating spot detected by 32P-postlabeling. We have also analyzed the relationship between adduct levels and TP53 mutations. By postlabeling diagonal radioactive zone (DRZ) adducts were detected in 37 of 39 (95%) lung tissues from lung cancer patients and the adduct level ranged from 6.81 to 108.50 adducts/10(8) nucleotide. Thirty-three of 39 (85%) had detectable levels of BPDE-DNA adducts (> 1 adduct/10(9) nucleotide). Current heavy smokers (> 20 cigarettes/day) have significantly higher DRZ adduct levels compared to individuals smoking less than 20 cigarettes/day. By SFS combined with immunoaffinity column (IAC), 11 of 39 (28%) samples had detectable adduct levels, and 6 of 11 (55%) were detectable by SFS following purification of benzo[a]pyrene (BP)-tetrols by high pressure liquid chromatography (HPLC). Six of 33 (18%) samples were positive for BPDE-DNA adducts by both postlabeling and HPLC/SFS. No correlation was observed between the SFS and 32P-postlabeling assays for the detection of BPDE-DNA adducts. However, there was a good correlation between adduct levels detected by IAC/SFS and HPLC/SFS. We found a weak association between total PAH-DNA adduct levels in lung tissue and TP53 mutations.

Biological monitoring of polycyclic aromatic hydrocarbon exposure in a highly polluted area of Poland.

Air pollution in Poland and particularly in Silesia is among the worst in Europe. Many coal mines and coke oven plants are located in this area, representing a major source of carcinogenic polycyclic aromatic hydrocarbons (PAHs). We quantitated the PAH exposure level in air samples using personal sampling devices, collected urine samples from the same individuals, and measured 1-hydroxypyrene with high performance liquid chromatography. Samples were collected twice, once in February and once in September. Mean PAH level of samples collected at three different coke oven plants varied from 2.3 micrograms/m3 to 12.3 micrograms/m3; the lowest mean was in September. Mean levels of 0.15 micrograms/m3 (September) and 0.44 micrograms/m3 (February) were noted for the environmentally exposed group. Mean urinary 1-hydroxypyrene varied from 2.45 to 13.48 mumol/mol creatinine at the three coke oven plants. The corresponding variation between the three different environmentally exposed groups in Silesia was 0.41-1.54 mumol/mol creatinine. In the nonindustrialized area, the mean varied from 0.20 to 0.14 mumol/mol creatinine. Seasonal variation was found both at the coke oven plants and in the environmental exposed groups in Silesia. Both PAH levels and 1-hydroxypyrene varied seasonally among coke oven workers and the environmentally exposed group. Our study shows that PAH exposure in the industrialized area of Silesia is high compared to levels in Western Europe. 1-Hydroxypyrene excretion in environmentally exposed individuals in Poland is among the highest in Europe.