Rikke Fredslund Andersen

The Aarhus statement: improving design and reporting of studies on early cancer diagnosis

Early diagnosis is a key factor in improving the outcomes of cancer patients. A greater understanding of the pre-diagnostic patient pathways is vital yet, at present, research in this field lacks consistent definitions and methods. As a consequence much early diagnosis research is difficult to interpret. A consensus group was formed with the aim of producing guidance and a checklist for early cancer-diagnosis researchers. A consensus conference approach combined with nominal group techniques was used. The work was supported by a systematic review of early diagnosis literature, focussing on existing instruments used to measure time points and intervals in early cancer-diagnosis research. A series of recommendations for definitions and methodological approaches is presented. This is complemented by a checklist that early diagnosis researchers can use when designing and conducting studies in this field. The Aarhus checklist is a resource for early cancer-diagnosis research that should promote greater precision and transparency in both definitions and methods. Further work will examine whether the checklist can be readily adopted by researchers, and feedback on the guidance will be used in future updates.

The importance of KRAS mutations and EGF61A>G polymorphism to the effect of cetuximab and irinotecan in metastatic colorectal cancer

BACKGROUND The effect of anti-epidermal growth factor receptor (EGFR) antibodies (mAb) in metastatic colorectal cancer seems limited to KRAS wild-type (wt) tumours, but still a major fraction of KRASwt patients are nonresponders and supplementary selection criteria are needed. We investigated methodological aspects of KRAS testing and the predictive and prognostic value of KRAS status combined with three EGFR-related gene polymorphisms [single-nucleotide polymorphisms (SNPs)] in patients treated with cetuximab and irinotecan. PATIENTS AND METHODS The study included 71 patients referred to third-line cetuximab-irinotecan. Blood samples were analysed for SNPs. KRAS analysis was carried out by sequencing analysis and quantitative PCR (DxS kit) in primary tumour and distant metastases. RESULTS There was a clear correlation between KRAS status in primary tumours and metastasis. The DxS kit presented the highest sensitivity. Response was confined to KRASwt patients (40% response rate versus 0%, P < 0.1(-3)), which translated into a significant difference in PFS. The EGF61A>G polymorphism showed relation to clinical outcome. A combined biomarker analysis showed a 19% progression rate in KRASwt-EGF61 homozygote patients and 60% in the EGF61A/G patients (P = 0.006) and a significant increase in overall survival (17.1 versus 5.9 months, log-rank, P = 0.002). CONCLUSION The combined biomarker analysis maybe an attractive approach to selection of patients for third-line treatment including anti-EGFR mAbs.

The prognostic value of KRAS mutated plasma DNA in advanced non-small cell lung cancer.

BACKGROUND Lung cancer is one of the most common malignant diseases worldwide and associated with considerable morbidity and mortality. New agents targeting the epidermal growth factor system are emerging, but only a subgroup of the patients will benefit from the therapy. Cell free DNA (cfDNA) in the blood allows for tumour specific analyses, including KRAS-mutations, and the aim of the study was to investigate the possible prognostic value of plasma mutated KRAS (pmKRAS) in patients with non-small cell lung cancer (NSCLC). MATERIAL AND METHODS Patients with newly diagnosed, advanced NSCLC eligible for chemotherapy were enrolled in a prospective biomarker trial. A pre-treatment blood sample was drawn and subsequently DNA was extracted and pmKRAS analysed. The patients received carboplatin (AUC5) i.v. day 1 and vinorelbine (30mg/m(2) i.v. day 1 and 60mg/m(2) p.o. day 8) for a maximum of six cycles. Response to chemotherapy was evaluated according to RECIST v.1.0 by CT scans of the chest and upper abdomen. The presence of pmKRAS at baseline was assessed by an in-house qPCR method. The primary endpoint was overall survival (OS). Secondary end-points were progression free survival (PFS) and overall response rate. RESULTS The study included 246 patients receiving a minimum of 1 treatment cycle, and all but four were evaluable for response according to RECIST. Forty-three patients (17.5%) presented with a KRAS mutation. OS was 8.9 months and PFS by intention to treat 5.4 months. Patients with a detectable plasma-KRAS mutation had a significantly shorter OS and PFS compared to the wild type (WT) patients (median OS 4.8 months versus 9.5 months, HR 1.87, 95% CI 1.23-2.84, p=0.0002 and median PFS 3.0 months versus 5.6 months, HR 1.60, 95% CI 1.09-2.37, p=0.0043). A multivariate Cox regression analysis confirmed the independent prognostic value of pmKRAS in OS but not in PFS. The response rate to chemotherapy was significantly lower in the group of patients with a mutation compared to WT (p<0.0001). CONCLUSION The presence of KRAS mutations in plasma may be a marker of poor prognosis and may also hold predictive value. Further validation in an independent cohort is highly needed.

Cell-free DNA in healthy individuals, noncancerous disease and strong prognostic value in colorectal cancer

The purpose was to investigate total cell‐free DNA (cfDNA) in colorectal cancer (CRC) patients during treatment with second‐line chemotherapy and in healthy controls and patients with different comorbidities. Patient treated with second‐line irinotecan for metastatic CRC (n = 100), a cohort of healthy controls with and without comorbidity (n = 70 and 100, respectively) were included. cfDNA was quantified by an in‐house developed quantitative polymerase chain reaction from plasma samples drawn prior to the first cycle of chemotherapy and at time of progression. cfDNA levels were significantly higher in CRC compared to controls, with a clear capability for discriminating between the groups (receiver operation curve analysis; area under the curve 0.82, p < 0.0001). Patients with high levels had a shorter survival from irinotecan compared to those with lover levels. The cohort independent upper normal limit divided patients into high and low risk groups. The progression‐free survival (PFS) was 2.1 months [95% confidence interval (CI) 2.0–3.4] and 6.5 (95% CI 4.2–7.2) months [hazard ratio (HR) 2.53; 95% CI 1.57–4.06, p < 0.0001] and overall survival (OS) 7.4 months (95% CI 4.3–8.7) and 13.8 months (95% CI 11.9–18.9; HR 2.52; 95% CI 1.54–4.13, p < 0.0000), respectively. Cox regression multivariate analysis showed a PFS HR of 1.4 (95% CI 1.1–1.7) for each increase in cfDNA quartile, p = 0.03 and 1.6 (1.3–2.0) for OS, p < 0.0001, respectively. A combined marker analysis with plasma KRAS mutations added further prognostic impact, which was consistent when performed on the samples drawn at time of progression. In conclusion, cfDNA measurement holds important clinical information and could become a useful tool for prediction of outcome from chemotherapy in mCRC.

Microvessel density and the association with single nucleotide polymorphisms of the vascular endothelial growth factor receptor 2 in patients with colorectal cancer

The measurement of microvessel density (MVD) is a widely accepted method for assessing the neoangiogenetic activity in neoplasia. The aim of the present study was to compare MVD with single nucleotide polymorphisms (SNPs) in the vascular endothelial growth factor receptor (VEGFR)-1 and VEGFR-2 genes and, furthermore, with quantitative measurements of the receptors in colorectal cancer (CRC) tissue. Prognosis was also assessed. Blood and tissue were collected from 110 patients surgically resected for CRC. SNPs were analysed from genomic DNA by polymerase chain reaction. MVD was assessed by immunohistochemistry using CD34 and CD105 combined with caldesmon in order to identify also immature vessels. Microvessels were counted in three fields of vision, and the mean MVD was used for statistical analysis. The VEGFR-2 1192 C/T and −604 T/C SNPs were associated with the MVD assessed by CD105. The median MVD score for the 1192 CC genotype was significantly lower compared to the CT + TT genotypes (p = 0.002). The median MVD score for the −604 CC genotype was significantly higher compared to the TT + TC genotypes (p = 0.009). A possible association, although non-significant, was demonstrated for the CD34-positive microvessels. The 1192 CC genotype and the −604 TT + TC genotypes correlated with improved survival. This is the first report on correlations between SNPs in the VEGF receptor genes and MVD in patients with CRC. Associations were shown between two SNPs in the VEGFR-2 gene and the CD105-positive microvessels indicating an impact on neoangiogenesis. Moreover, an association between the SNPs and survival was demonstrated. The clinical implications of these findings need further investigations.

Circulating free DNA as biomarker and source for mutation detection in metastatic colorectal cancer.

Background Circulating cell-free DNA (cfDNA) in plasma has shown potential as biomarker in various cancers and could become an importance source for tumour mutation detection. The objectives of our study were to establish a normal range of cfDNA in a cohort of healthy individuals and to compare this with four cohorts of metastatic colorectal cancer (mCRC) patients. We also investigated the prognostic value of cfDNA and analysed the tumour-specific KRAS mutations in the plasma. Methods The study was a prospective biomarker evaluation in four consecutive Phase II trials, including 229 patients with chemotherapy refractory mCRC and 100 healthy individuals. Plasma was obtained from an EDTA blood-sample, and the total number of DNA alleles and KRAS mutated alleles were assessed using an in-house ARMS-qPCR as previously described. Results Median cfDNA levels were higher in mCRC compared to controls (p <0.0001). ROC analysis revealed an AUC of 0.9486 (p<0.00001). Data showed impaired OS with increasing levels of baseline cfDNA both when categorising patients by quartiles of cfDNA and into low or high cfDNA groups based on the upper normal range of the control group (Median OS 10.2 (8.3–11.7) and 5.2 (4.6–5.9) months, respectively, HR 1.78, p = 0.0006). Multivariate analysis confirmed an independent prognostic value of cfDNA (HR 1.5 (95% CI 1.3–1.7) for each increase in the cfDNA quartile). The overall concordance of KRAS mutations in plasma and tissue was high (85%). Conclusions These data confirm the prognostic value of cfDNA measurement in plasma and utility for mutation detection with the method presented.

Changes in mutational status during third‐line treatment for metastatic colorectal cancer—Results of consecutive measurement of cell free DNA, KRAS and BRAF in the plasma

KRAS and BRAF mutations are responsible for primary resistance to epidermal growth factor receptor (EGFR) MoAbs in metastatic colorectal cancer (mCRC), but it is unknown what causes wildtype (wt) patients to develop resistance during treatment. We measured circulating free DNA (cfDNA), KRAS and BRAF in plasma and report the changes during third line treatment with cetuximab and irinotecan. One‐hundred‐and‐eight patients received irinotecan 350 mg/m2 q3w and weekly cetuximab (250 mg/m2) until progression (RECIST) or unacceptable toxicity. cfDNA and number of mutated KRAS/BRAF alleles in plasma at baseline and before each cycle was analyzed by an in‐house qPCR. cfDNA and pKRAS levels decreased from baseline to cycle three and increased at time of progression (p = 0.008). The decrease was larger in responding patients than in non‐responding (p < 0.05). Two patients with primary mutant disease had different types of mutations detected in the plasma, including synchronous KRAS and BRAF. Twelve patients had a primary KRAS mutant tumor, but wild‐type disease according to baseline plasma analysis, eight of these obtained stabilization of disease. In five patients with primary wt disease a mutation appeared in plasma before radiological evidence of progression. Loss of mutations may explain observed benefit of treatment in primary mutant disease, whereas appearance of mutations during therapy may be responsible for acquired resistance in primary wt disease. Benefit from EGFR MoAbs may be influenced by the quantitative level of mutational alleles rather than by mutational status alone, and plasma levels of cfDNA, KRAS and BRAF could be used to monitor patients during treatment.

Prognostic importance of vascular endothelial growth factor-A expression and vascular endothelial growth factor polymorphisms in epithelial ovarian cancer.

Introduction: Vascular endothelial growth factors (VEGFs) play a central role in angiogenesis and consequently, in various steps of ovarian carcinogenesis. Gene polymorphisms within the VEGF system have revealed a correlation with prognosis in some malignancies. The aim of the present study was to examine the possible importance of 2 VEGF polymorphisms and VEGF-A expression in ovarian cancer. Methods: We investigated 2 single nucleotide polymorphisms VEGF +405G/C and VEGF −460C/T by polymerase chain reaction and also analyzed VEGF-A expression by immunohistochemistry in 159 women with ovarian cancer. Results: Vascular endothelial growth factor-A expression revealed a significant correlation with survival in a Cox proportional hazards regression model (P = 0.012). Germline polymorphisms were not correlated with clinicopathological parameters such as stage, type, and histology. Heterozygous genotype in VEGF +405G/C predicted a better survival compared with homozygous genotypes (P = 0.034), and the heterozygous genotype in VEGF −460C/T pointed to the same direction. A multivariate analysis also indicated that heterozygosity of either of the 2 polymorphisms held independent prognostic significance (P = 0.001). Conclusions: Vascular endothelial growth factor polymorphisms +405G/C and VEGF expression seem to have independent prognostic importance.

The predictive value of genetic variations in the vascular endothelial growth factor A gene in metastatic colorectal cancer.

Single-nucleotide polymorphisms (SNPs) in the vascular endothelial growth factor A (VEGF-A) gene may have clinical implications. The aim of this study was to investigate the possible predictive value of the VEGF-A SNPs, in patients with metastatic colorectal cancer (mCRC) treated with first-line capecitabine and oxaliplatin (XELOX). The study included 72 patients with mCRC. Genomic DNA was isolated from whole blood, and SNPs were analyzed by PCR. SNPs were correlated with response and progression-free survival (PFS). Haplotypes were estimated using the PHASE program. Response was observed in 21% of the patients with the −2578 CA genotype compared with 59% of the patients with CC+AA, P=0.002, in 26% of the patients with the −460 CT genotype compared with 57% with CC+TT, P=0.01, and in 27% of the patients with the +405 GC genotype compared with 54% with GG+CC, P=0.02. Two SNPs were significantly related to PFS. A haplotype with a significant relationship to response was identified. The results demonstrated obvious relationships between genetic variations in the VEGF-A gene and response to first-line XELOX in patients with mCRC, which translated to a significant difference in PFS. The results call for validation in a larger cohort of patients.

EGF61A>G polymorphism as predictive marker of clinical outcome to first-line capecitabine and oxaliplatin in metastatic colorectal cancer.

BACKGROUND The purpose of the present study was to investigate polymorphisms related to the metabolism of fluoropyrimidine and oxaliplatin, thymidylate synthase (TS) and excision repair cross-complementing gene 1 (ERCC1) 118, in metastatic colorectal cancer patients treated with capecitabine and oxaliplatin (XELOX). We also investigated the importance of the EGF61A>G polymorphism, which holds a functional influence on the tyrosine kinase receptor regulation. MATERIALS AND METHODS We included 68 patients treated with first-line XELOX. Polymorphism analyses were carried out on pretreatment blood samples. Response was evaluated according to the RECIST. Survival analysis was described by the Kaplan-Meier method and log-rank testing. RESULTS The overall response rate was 38% and the median overall survival 19.4 months. A favorable outcome was seen in patients with the EGF61A/G genotype compared with the combined group of A/A and G/G, with response rates of 57% and 18%, respectively (P = 0.001). There was a significantly different progression-free survival (P = 0.018) in favor of the A/G group. The TS and ERCC1 genotypes failed to provide any significant impact on the outcome. CONCLUSION Polymorphism analysis of a simple blood sample is a feasible approach to biomarker analysis and the EGF61A>G polymorphism may influence the effect of first-line XELOX. Consequently, this marker deserves further investigation.