Unraveling the potential clinical utility of circulating tumor DNA detection in colorectal cancer-evaluation in a nationwide Danish cohort.

BACKGROUND: Increasingly, circulating tumor DNA (ctDNA) is proposed as a tool for minimal residual disease (MRD) assessment. Digital PCR (dPCR) offers low analysis costs and turnaround times of less than a day, making it ripe for clinical implementation. Here, we used tumor-informed dPCR for ctDNA detection in a large colorectal cancer (CRC) cohort to evaluate the potential for post-operative risk assessment and serial monitoring, and how the metastatic site may impact ctDNA detection. Additionally, we assessed how altering the ctDNA-calling algorithm could customize performance for different clinical settings. PATIENTS AND METHODS: Stage II-III CRC patients (N = 851) treated with a curative intent were recruited. Based on whole-exome sequencing on matched tumor and germline DNA, a mutational target was selected for dPCR analysis. Plasma samples (8 ml) were collected within 60 days after operation and-for a patient subset (n = 246)-every 3-4 months for up to 36 months. Single-target dPCR was used for ctDNA detection. RESULTS: Both post-operative and serial ctDNA detection were prognostic of recurrence [hazard ratio (HR) = 11.3, 95% confidence interval (CI) 7.8-16.4, P < 0.001; HR = 30.7, 95% CI 20.2-46.7, P < 0.001], with a cumulative ctDNA detection rate of 87% at the end of sample collection in recurrence patients. The ctDNA growth rate was prognostic of survival (HR = 2.6, 95% CI 1.5-4.4, P = 0.001). In recurrence patients, post-operative ctDNA detection was challenging for lung metastases (4/21 detected) and peritoneal metastases (2/10 detected). By modifying the cut-off for calling a sample ctDNA positive, we were able to adjust the sensitivity and specificity of our test for different clinical contexts. CONCLUSIONS: The presented results from 851 stage II-III CRC patients demonstrate that our personalized dPCR approach effectively detects MRD after operation and shows promise for serial ctDNA detection for recurrence surveillance. The ability to adjust sensitivity and specificity shows exciting potential to customize the ctDNA caller for specific clinical settings.

MET amplification and epithelial-to-mesenchymal transition exist as parallel resistance mechanisms in erlotinib-resistant, EGFR-mutated, NSCLC HCC827 cells.

Although many epidermal growth factor receptor (EGFR)-mutated lung cancer patients initially benefit from the EGFR-inhibitor erlotinib, all acquire resistance. So far, several mechanisms implicated in resistance have been identified, but the existence of multiple resistance mechanisms in parallel have only been sparsely investigated. In this study, we investigated parallel resistance mechanisms acquired by HCC827, an EGFR-mutated adenocarcinoma cell line dependent on EGFR activity and sensitive to erlotinib. The cell line was treated with erlotinib by stepwise escalation of the drug-concentration and erlotinib-resistant (HCC827ER) cells created. HCC827ER cells depicted a mixed epithelial and mesenchymal phenotype. To clarify potential parallel resistance mechanisms, 14 resistant subclones were established by limited dilution. Interestingly, all HCC827ER subclones harbored either a MET-amplification (6/14) or underwent EMT (8/14), mechanisms both found in previous studies, but not in co-occurrence. Both subclone-types were resistant to erlotinib, but only MET-subclones responded to the MET-inhibitors crizotinib and capmatinib. EMT-subclones on the other hand had markedly increased FGFR1 expression and responded to the FGFR-inhibitor AZD4547, whereas MET-subclones did not. Monitoring gene expression through the development of HCC827ER revealed upregulation of FGFR1 expression as an early response to erlotinib. In addition, FGFR1 expression increased upon short-term erlotinib treatment (48 h) identifying a physiological role immediately after erlotinib exposure. The high FGFR1 expression seen in EMT-subclones was stable even after five passages without erlotinib. Here we show, that parallel resistance mechanisms appear during erlotinib-resistance development in EGFR-mutated NSCLC cells and highlight a role for FGFR1 expression changes as an early response to erlotinib as well as a bypass-signaling mechanism.

Evaluation of VEGF A expression and microvascular density as prognostic factors in extrahepatic cholangiocarcinoma.

OBJECTIVES: Angiogenesis is essential for tumor growth and metastasis. An association between microvessel density, a measure of tumor angiogenesis, and conventional prognostic variables has been shown for many different tumor entities. In extrahepatic cholangiocarcinoma, the VEGF expression and microvessel density have rarely been investigated. METHODS: Paraffin-embedded specimens from 51 resected adenocarcinomas of the extrahepatic bile duct were immunostained for vascular endothelial growth factor A (VEGF A) and CD 34 to evaluate the microvessel density (MVD). VEGF A staining was evaluated by combining intensity and percentage of positive tumor cells, as low (expression equal or below the median), or high (above the median). Microvessel density was assessed using a method published by Weidner et al. RESULTS: Median disease free survival (DFS) of the study group was 12.5 months (range, 1-66.3 months). DFS was calculated in the 39 patients with complete resection. It was significantly better in patients with low microvessel density than DFS in patients with high microvessel density (33 months (range, 3-66.3 months) vs. 21.8 months (range, 1.6-31.6 months); p=0.022). In contrast, VEGF A expression did not correlate with survival. There was a trend toward a higher VEGF A expression in highly vascularized tumors (p=0.08), but failed to reach statistic significance. CONCLUSIONS: The present study indicates, that vascularisation has an important impact on survival of extrahepatic cholangiocarcinoma patients. Other molecules than VEGF A are probably involved in neovascularization in extrahepatic cholangiocarcinoma.

A rationally designed oligopeptide shows significant conformational changes upon binding to sulphate ions.

Oligopeptides that interact with oxoanions were developed by rational design methods. The substrate-binding site of the enzyme purine nucleoside phosphorylase served as a model for the design of the ionophores. The amino acids involved in the complexation of oxoanions were linked through flexible spacer residues. These spacers were chosen such that the relative orientation of the interacting amino acids was conserved. Several peptide sequences were preselected based on intermolecular H-bond frequencies. These frequencies were calculated from molecular dynamics trajectories of the corresponding peptide-anion complexes and used to score the binding properties of the peptides. The most promising peptides were prepared using solid phase peptide synthesis. Anion binding of the peptide ionophores was screened using circular dichroism (CD) and confirmed by NMR spectroscopy. CD measurements performed in methanol revealed a significant conformational change of a linear undecapeptide upon binding to sulphate ions. Two-dimensional-NMR experiments confirmed that a conformation with high helical content is formed in the presence of sulphate ions. These conformational changes induced by the anion stimulate the development of new transduction mechanisms in chemical sensors.

Mortality in the elderly and ambient ozone concentration during the hot summer, 1994, in Belgium.

Extensive investigations were carried out to study the relationship between daily mortality in the elderly, outdoor air temperature, and ozone concentration observed in Belgium during the hot summer, 1994. The two environmental variables were assessed through mean daily temperature and 24-hr ozone concentration, both measured the day before and averaged over the country. Data were stratified by terciles of mean daily temperature in order to reduce the degree of collinearity between the investigated environmental variables. In the first stratum, which ranged from 9.9 to 15.4 degrees C (41 days), mean daily temperature and 24-hr ozone concentration were not correlated while the mean number of daily deaths was higher when 24-hr ozone concentration increased from 45 to 55 micrograms/m3 (P < 0.05). In the second stratum, which ranged from 15.6 to 20.3 degrees C (42 days), mean daily temperature and 24-hr ozone concentration were strongly correlated (r = 0.54, P < 0.0001). In this stratum, the number of daily deaths did not depend on the mean daily temperature but increased linearly with 24-hr ozone concentration within the range 25 to 85.5 micrograms/m3 (P < 0.001). After having examined the possible confounding effect of sulfur dioxide, nitrogen dioxide, fine particulates, and humidity, ozone was found to be the only investigated variable contributing to the increased daily mortality. In the third stratum, which ranged from 20.4 to 27.6 degrees C (40 days), mean daily temperature and 24-hr ozone concentration were also strongly correlated (r = 0.71, P < 0.0001). Daily mortality, in this stratum, was correlated more with mean daily temperature (r = 0.68, P < 0.001) than with 24-hr ozone concentration (r = 0.55, P < 0.001). Nonparametric regression analyses were performed to model the number of daily deaths in the whole range of temperatures. These analyses confirmed the effect of 24-hr ozone concentration on daily mortality already uncovered by the least-squares regression analysis in the second stratum of mean daily temperature. In addition, at levels exceeding 20 degrees C, the effect of ozone concentration on daily mortality was enhanced by temperature owing to a positive interaction between these two variables. The present study thus demonstrated a statistical association between daily mortality, observed in the elderly during the hot summer, 1994, in Belgium, and ambient ozone concentration. This relationship was dependent on the range of temperatures.

Temperature, ambient ozone levels, and mortality during summer 1994, in Belgium.

The number of daily deaths, temperature, relative humidity, and 24-hr concentrations of main air pollutants observed during a heat wave (June 27-August 7, 1994) in Belgium were compared with those recorded before and after this heat wave. All these variables were averaged over the country. Expected mortality was calculated from daily deaths observed during the summers of 1985-1993. The influence of meteorological and air pollution variables on daily mortality was analyzed using generalized least-squares method. Mortality recorded during the heat wave was higher than expected: it increased by 9.4% in the age group 0-64 years (236 excess deaths; P < 0.001) and by 13.2% in the elderly (1168 excess deaths; P < 0.001). After the heat wave, mortality in the elderly was lower than expected (178 deficit deaths; P< 0.05); the net excess of mortality in the whole population amounted to 1226 deaths when accounting for this deficit. This increased mortality was associated with unusually high outdoor temperatures (range of daily mean: 15.3-27.5 degrees C) and elevated ozone levels (range of 24-hr concentration: 34.5-111.5 microg/m3). The duration of the ozone overexposure during the heat wave was also uncommon: half-hour concentrations of ozone exceeded, on an average, 100 microg/m3 for 8 consecutive hr. The number of daily deaths was mostly correlated with the mean daily temperature and 24-hr ozone concentration, both measured the day before. A synergy between temperature and ozone in their effects on mortality was also highlighted in both age groups. The product of the logarithm of temperature by the logarithm of ozone concentration, both measured the day before, contributed to 39.5% of the variance of the logarithm of daily deaths in elderly and to 4.5% in the age group 0-64 years. In conclusion, elevated outdoor temperatures combined with high ozone concentrations were assumed to be the likely cause of the important excess mortality observed in Belgium during the summer, 1994.