Evaluation of Cytologic Sample Preparations for Compatibility With Nucleic Acid Analysis.

OBJECTIVES: In this study, the influence of several key elements of the cytologic sample workflow on DNA and RNA content was evaluated. METHODS: The A549 cell line, patient-derived organoids, and pleural effusions were used to investigate the effect of (1) several collection media and delayed time to processing; (2) cytology specimens; (3) cytologic staining; and (4) formalin-fixed, paraffin-embedded (FFPE) cell block processing on nucleic acid quality and quantity as determined by fragment analyzer, Qubit analysis (Thermo Fisher Scientific), and quantitative polymerase chain reaction-based analysis on the Idylla platform (Biocartis). RESULTS: Alcohol-based collection media (CytoRich Red [Thermo Fisher Scientific] and EtOH95%) displayed high DNA and RNA preservation capacity, while phosphate-buffered saline and, to a lesser extent, formalin were associated with high RNA quality. Cytospin and smear cytology specimens were subject to DNA and RNA loss. Cytologic staining had no further impact on sample quality, hence destaining is not necessary. Both H&E-stained and unstained FFPE sections are compatible with nucleic acid analysis, despite a strong decrease in DNA and RNA quality. CONCLUSIONS: Although several key elements of the cytologic sample workflow have an influence on DNA and RNA quality and quantity, the selection of these elements is also dependent on the downstream (ancillary) testing methods.

Clinical Impact of Circulating Tumor RAS and BRAF Mutation Dynamics in Patients With Metastatic Colorectal Cancer Treated With First-Line Chemotherapy Plus Anti-Epidermal Growth Factor Receptor Therapy.

PURPOSE: RAS and BRAF mutations can be detected as a mechanism of acquired resistance in circulating tumor (ct) DNA in patients with metastatic colorectal cancer treated with anti-epidermal growth factor receptor therapy. METHODS: RAS and BRAF mutational status was assessed in ctDNA in a baseline plasma sample and a serum sample collected at the time of the last available determination (named secondary extraction) from patients with KRAS exon 2 wild-type metastatic colorectal cancer treated in two first-line prospective biomarker-designed clinical trials (PULSE, ClinicalTrials.gov identifier: NCT01288339; and POSIBA, ClincialTrials.gov identifier: NCT01276379). RESULTS: Analysis of extended RAS and BRAF in tissue and plasma from 178 patients with KRAS exon 2 wild-type metastatic colorectal cancer showed a sensitivity of 64.1% and a specificity of 90%. The median overall survival (OS) of baseline patients with RAS and BRAF mutations in ctDNA was 22.3 months (95% CI, 15.6 to 29 months) and 8.9 months (95% CI, 6.3 to 11.4 months), respectively, which was significantly inferior to the median OS of 40.4 months (95% CI, 35.9 to 44.9 months) in two patients with wild-type disease (P < .001). Acquisition of RAS/BRAF mutations occurred in nine of 63 patients (14%) with progressive disease (PD; ie, blood draw within 1 month before PD or after PD) compared with six of 73 patients (8%) with no PD or blood extraction for ctDNA analysis before 1 month of PD (P = .47). Median OS in patients with RAS/BRAF acquisition was 23.9 months (95% CI, 19.7 to 27.9 months) compared with 40.6 months (95% CI, not reached to not reached) in patients who remained free of mutations (P = .016). CONCLUSION: Our results confirm that baseline RAS and BRAF testing in ctDNA discriminates survival. The emergence of RAS/BRAF mutations has limited relevance for the time to progression to anti-epidermal growth factor receptor therapy.

Key differences between 13 KRAS mutation detection technologies and their relevance for clinical practice.

INTRODUCTION: This study assessed KRAS mutation detection and functional characteristics across 13 distinct technologies and assays available in clinical practice, in a blinded manner. METHODS: Five distinct KRAS-mutant cell lines were used to study five clinically relevant KRAS mutations: p.G12C, p.G12D, p.G12V, p.G13D and p.Q61H. 50 cell line admixtures with low (50 and 100) mutant KRAS allele copies at 20%, 10%, 5%, 1% and 0.5% frequency were processed using quantitative PCR (qPCR) (n=3), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) (n=2), next-generation sequencing (NGS) (n=6), digital PCR (n=1) and Sanger capillary sequencing (n=1) assays. Important performance differences were revealed, particularly assay sensitivity and turnaround time. RESULTS: Overall 406/728 data points across all 13 technologies were identified correctly. Successful genotyping of admixtures ranged from 0% (Sanger sequencing) to 100% (NGS). 5/6 NGS platforms reported similar allelic frequency for each sample. One NGS assay detected mutations down to a frequency of 0.5% and correctly identified all 56 samples (Oncomine Focus Assay, Thermo Fisher Scientific). One qPCR (Idylla, Biocartis) and MALDI-TOF (UltraSEEK, Agena Bioscience) assay identified 96% (all 100 copies and 23/25 at 50 copies input) and 92% (23/25 at 100 copies and 23/25 at 50 copies input) of samples, respectively. The digital PCR assay (KRAS PrimePCR ddPCR, Bio-Rad Laboratories) identified 60% (100 copies) and 52% (50 copies) of samples correctly. Turnaround time from sample to results ranged from ~2 hours (Idylla CE-IVD) to 2 days (TruSight Tumor 15 and Sentosa CE-IVD), to 2 weeks for certain NGS assays; the level of required expertise ranged from minimal (Idylla CE-IVD) to high for some technologies. DISCUSSION: This comprehensive parallel assessment used high molecular weight cell line DNA as a model system to address key questions for a laboratory when implementing routine KRAS testing. As most of the technologies are available for additional molecular biomarkers, this study may be informative for other applications.

BRAF Mutation Testing in Cell-Free DNA from the Plasma of Patients with Advanced Cancers Using a Rapid, Automated Molecular Diagnostics System.

Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis for diagnostics and response monitoring. In this study, plasma-derived cfDNA samples from patients with progressing advanced cancers or malignant histiocytosis with known BRAF(V600) status from formalin-fixed paraffin-embedded (FFPE) tumors were tested using a prototype version of the Idylla BRAF Mutation Test, a fully integrated real-time PCR-based test with turnaround time about 90 minutes. Of 160 patients, BRAF(V600) mutations were detected in 62 (39%) archival FFPE tumor samples and 47 (29%) plasma cfDNA samples. The two methods had overall agreement in 141 patients [88%; κ, 0.74; SE, 0.06; 95% confidence interval (CI), 0.63-0.85]. Idylla had a sensitivity of 73% (95% CI, 0.60-0.83) and specificity of 98% (95% CI, 0.93-1.00). A higher percentage, but not concentration, of BRAF(V600) cfDNA in the wild-type background (>2% vs. ≤ 2%) was associated with shorter overall survival (OS; P = 0.005) and in patients with BRAF mutations in the tissue, who were receiving BRAF/MEK inhibitors, shorter time to treatment failure (TTF; P = 0.001). Longitudinal monitoring demonstrated that decreasing levels of BRAF(V600) cfDNA were associated with longer TTF (P = 0.045). In conclusion, testing for BRAF(V600) mutations in plasma cfDNA using the Idylla BRAF Mutation Test has acceptable concordance with standard testing of tumor tissue. A higher percentage of mutant BRAF(V600) in cfDNA corresponded with shorter OS and in patients receiving BRAF/MEK inhibitors also with shorter TTF. Mol Cancer Ther; 15(6); 1397-404. ©2016 AACR.

BRAF mutation testing with a rapid, fully integrated molecular diagnostics system.

Fast and accurate diagnostic systems are needed for further implementation of precision therapy of BRAF-mutant and other cancers. The novel IdyllaTMBRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those obtained by a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory employing polymerase chain reaction-based sequencing, mass spectrometric detection, or next-generation sequencing. In one set of 60 FFPE tumor samples (15 with BRAF mutations per Idylla), the Idylla and cobas results had an agreement of 97%. Idylla detected BRAF V600 mutations in two additional samples. The Idylla and MiSeq results had 100% concordance. In a separate set of 100 FFPE tumor samples (64 with BRAF mutation per Idylla), the Idylla and CLIA-certified laboratory results demonstrated an agreement of 96% even though the tests were not performed simultaneously and different FFPE blocks had to be used for 9 cases. The IdyllaTMBRAF Mutation Test produced results quickly (sample to results time was about 90 minutes with about 2 minutes of hands on time) and the closed nature of the cartridge eliminates the risk of PCR contamination. In conclusion, our observations demonstrate that the Idylla test is rapid and has high concordance with other routinely used but more complex BRAF mutation-detecting tests.

VEGFR1 single nucleotide polymorphisms associated with outcome in patients with metastatic renal cell carcinoma treated with sunitinib - a multicentric retrospective analysis.

BACKGROUND: There are no validated markers that predict outcome in metastatic renal cell cancer (mRCC) patients treated with sunitinib. Recently, single nucleotide polymorphism (SNP) rs9582036 in VEGFR1 has been proposed as a predictor of progression-free survival (PFS) and overall survival (OS) to bevacizumab in patients with pancreatic cancer and rs7993418 in VEGFR1 as predictor for PFS in mRCC-patients treated with bevacizumab. Here, we aim to study the impact of these SNPs in mRCC patients treated with sunitinib. METHODS: We included patients with mRCC treated in 15 institutions in France and Belgium. Patients received sunitinib as first-line targeted therapy. We assessed response, time-to-tumor progression (TTP), OS, and clinical and biochemical parameters associated with outcome. We genotyped rs9582036 and rs7993418 as well as three other surrounding SNPs in VEGFR1: rs9554320, rs9554316 and rs9513070. Association between SNPs and treatment outcome were studied by univariate analysis and by multivariate Cox regression using relevant clinical factors associated with TTP and OS as covariates. FINDINGS: Ninety-one patients were included. We found that mRCC patients with the CC-variant in rs9582036 in VEGFR1 have a poorer response rate (RR) (0% vs. 46%, p = 0.028), a poorer PFS (10 vs. 18 months, p = 0.033 on univariate and 0.06 on multivariate analysis) and a poorer OS (14 vs. 31 months, p = 0.019 on univariate and 0.008 on multivariate analysis) compared to patients with the AC- and AA-genotypes. mRCC patients with the AA-variant in rs9554320 in VEGFR1 have a poorer PFS (12 vs. 21 months, p = 0.0066 on univariate and 0.005 on multivariate analysis) and a poorer OS (22 vs. 34 months, p = 0.019 on univariate and 0.067 on multivariate analysis) compared to patients with the AC- and CC-genotypes. Interpretation. mRCC patients with the CC-genotype in VEGFR1 SNP rs9582036 have a poorer response rate, PFS and OS when treated with sunitinib. These findings are in agreement with the association of rs9582036 and outcome observed in bevacizumab treated pancreatic cancer patients. Prospective validation of this SNP is warranted.

Somatic mutation profiling and associations with prognosis and trastuzumab benefit in early breast cancer.

BACKGROUND: Certain somatic alterations in breast cancer can define prognosis and response to therapy. This study investigated the frequencies, prognostic effects, and predictive effects of known cancer somatic mutations using a randomized, adjuvant, phase III clinical trial dataset. METHODS: The FinHER trial was a phase III, randomized adjuvant breast cancer trial involving 1010 women. Patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer were further randomized to 9 weeks of trastuzumab or no trastuzumab. Seven hundred five of 1010 tumors had sufficient DNA for genotyping of 70 somatic hotspot mutations in 20 genes using mass spectrometry. Distant disease-free survival (DDFS), overall survival (OS), and interactions with trastuzumab were explored with Kaplan-Meier and Cox regression analyses. All statistical tests were two-sided. RESULTS: Median follow-up was 62 months. Of 705 tumors, 687 were successfully genotyped. PIK3CA mutations (exons 1, 2, 4, 9, 13, 18, and 20) were present in 25.3% (174 of 687) and TP53 mutations in 10.2% (70 of 687). Few other mutations were found: three ERBB2 and single cases of KRAS, ALK, STK11/LKB1, and AKT2. PIK3CA mutations were associated with estrogen receptor positivity (P < .001) and the luminal-A phenotype (P = .04) but were not statistically significantly associated with prognosis (DDFS: hazard ratio [HR] = 0.88, 95% confidence [CI] = 0.58 to 1.34, P = .56; OS: HR = 0.603, 95% CI = .32 to 1.13, P = .11), although a statistically significant nonproportional prognostic effect was observed for DDFS (P = .002). PIK3CA mutations were not statistically significantly associated with trastuzumab benefit (P(interaction): DDFS P = .14; OS P = .24). CONCLUSIONS: In this dataset, targeted genotyping revealed only two alterations at a frequency greater than 10%, with other mutations observed infrequently. PIK3CA mutations were associated with a better outcome, however this effect disappeared after 3 years. There were no statistically significant associations with trastuzumab benefit.

Somatic profiling of the epidermal growth factor receptor pathway in tumors from patients with advanced colorectal cancer treated with chemotherapy ± cetuximab.

PURPOSE: To study the somatic molecular profile of the EGF receptor (EGFR) pathway in advanced colorectal cancer, its relationship to prognosis, the site of the primary and metastases, and response to cetuximab. EXPERIMENTAL DESIGN: We used Sequenom and Pyrosequencing for high-throughput somatic profiling of the EGFR pathway in 1,976 tumors from patients with advanced colorectal cancer from the COIN trial (oxaliplatin and fluoropyrimidine chemotherapy ± cetuximab). Correlations between mutations, clinicopathologic, response, and survival data were carried out. RESULTS: Sequenom and Pyrosequencing had 99.0% (9,961/10,063) genotype concordance. We identified 13 different KRAS mutations in 42.3% of advanced colorectal cancers, 2 BRAF mutations in 9.0%, 4 NRAS mutations in 3.6%, and 5 PIK3CA mutations in 12.7%. 4.2% of advanced colorectal cancers had microsatellite instability (MSI). KRAS and PIK3CA exon 9, but not exon 20, mutations cooccurred (P = 8.9 × 10(-4)) as did MSI and BRAF mutations (P = 5.3 × 10(-10)). KRAS mutations were associated with right colon cancers (P = 5.2 × 10(-5)) and BRAF mutations with right (P = 7.2 × 10(-5)) and transverse colon (P = 9.8 × 10(-6)) cancers. KRAS mutations were associated with lung-only metastases (P = 2.3 × 10(-4)), BRAF mutations with peritoneal (P = 9.2 × 10(-4)) and nodal-only (P = 3.7 × 10(-5)) metastases, and MSI (BRAF(WT)) with nodal-only metastases (P = 2.9 × 10(-4)). MSI (BRAF(WT)) was associated with worse survival (HR = 1.89, 95% CI 1.30-2.76, P = 8.5 × 10(-4)). No mutations, subsets of mutations, or MSI status were associated with response to cetuximab. CONCLUSIONS: Our data support a functional cooperation between KRAS and PIK3CA in colorectal tumorigenesis and link somatic profiles to the sites of metastases. MSI was associated with poor prognosis in advanced disease, and no individual somatic profile was associated with response to cetuximab in COIN.

High-throughput interrogation of PIK3CA, PTEN, KRAS, FBXW7 and TP53 mutations in primary endometrial carcinoma.

OBJECTIVE: Endometrial cancer patients may benefit from systemic adjuvant chemotherapy, alone or in combination with targeted therapies. Prognostic and predictive markers are needed, however, to identify patients amenable for these therapies. METHODS: Primary endometrial tumors were genotyped for >100 hot spot mutations in genes potentially acting as prognostic or predictive markers. Mutations were correlated with tumor characteristics in a discovery cohort, replicated in independent cohorts and finally, confirmed in the overall population (n=1063). RESULTS: PIK3CA, PTEN and KRAS mutations were most frequently detected, respectively in 172 (16.2%), 164 (15.4%) and 161 (15.1%) tumors. Binary logistic regression revealed that PIK3CA mutations were more common in high-grade tumors (OR=2.03; P=0.001 for grade 2 and OR=1.89; P=0.012 for grade 3 compared to grade 1), whereas a positive TP53 status correlated with type II tumors (OR=11.92; P<0.001) and PTEN mutations with type I tumors (OR=19.58; P=0.003). Conversely, FBXW7 mutations correlated with positive lymph nodes (OR=3.38; P=0.045). When assessing the effects of individual hot spot mutations, the H1047R mutation in PIK3CA correlated with high tumor grade and reduced relapse-free survival (HR=2.18; P=0.028). CONCLUSIONS: Mutations in PIK3CA, TP53, PTEN and FBXW7 correlate with high tumor grade, endometrial cancer type and lymph node status, whereas PIK3CA H1047R mutations serve as prognostic markers for relapse-free survival in endometrial cancer patients.

Comprehensive mutation analysis in colorectal flat adenomas.

BACKGROUND: Flat adenomas are a subgroup of colorectal adenomas that have been associated with a distinct biology and a more aggressive clinical behavior compared to their polypoid counterparts. In the present study, we aimed to compare the mutation spectrum of 14 cancer genes, between these two phenotypes. METHODS: A consecutive series of 106 flat and 93 polypoid adenomas was analyzed retrospectively for frequently occurring mutations in "hot spot" regions of KRAS, BRAF, PIK3CA and NRAS, as well as selected mutations in CTNNB1 (ß-catenin), EGFR, FBXW7 (CDC4), PTEN, STK11, MAP2K4, SMAD4, PIK3R1 and PDGFRA using a high-throughput genotyping technique. Additionally, APC was analyzed using direct sequencing. RESULTS: APC mutations were more frequent in polypoid adenomas compared to flat adenomas (48.5% versus 30.3%, respectively, p = 0.02). Mutations in KRAS, BRAF, NRAS, FBXW7 and CTNNB1 showed similar frequencies in both phenotypes. Between the different subtypes of flat adenomas (0-IIa, LST-F and LST-G) no differences were observed for any of the investigated genes. CONCLUSION: The lower APC mutation rate in flat adenomas compared to polypoid adenomas suggests that disruption of the Wnt-pathway may occur via different mechanisms in these two phenotypes. Furthermore, in contrast to previous observations our results in this large well-defined sample set indicate that there is no significant association between the different morphological phenotypes and mutations in key genes of the RAS-RAF-MAPK pathway.

VEGF pathway genetic variants as biomarkers of treatment outcome with bevacizumab: an analysis of data from the AViTA and AVOREN randomised trials.

BACKGROUND: No biomarkers that could guide patient selection for treatment with the anti-VEGF monoclonal antibody bevacizumab have been identified. We assessed whether genetic variants in the VEGF pathway could act as biomarkers for bevacizumab treatment outcome. METHODS: We investigated DNA from white patients from two phase 3 randomised studies. In AViTA, patients with metastatic pancreatic adenocarcinoma were randomly assigned to receive gemcitabine and erlotinib plus either bevacizumab or placebo. In AVOREN, patients with metastatic renal-cell carcinoma were randomly assigned to receive interferon alfa-2a plus either bevacizumab or placebo. We assessed the correlation of 138 SNPs in the VEGF pathway with progression-free survival and overall survival in a subpopulation of patients from AViTA. Significant findings were confirmed in a subpopulation of patients from AVOREN and functionally studied at the molecular level. FINDINGS: We investigated DNA of 154 patients from AViTA, of whom 77 received bevacizumab, and 110 patients from AVOREN, of whom 59 received bevacizumab. Only rs9582036, a SNP in VEGF receptor 1 (VEGFR1 or FLT1), was significantly associated with overall survival in the bevacizumab group of AViTA after correction for multiplicity (per-allele hazard ratio [HR] 2·1, 95% CI 1·45-3·06, p=0·00014). This SNP was also associated with progression-free survival (per-allele HR 1·89, 1·31-2·71, p=0·00081) in bevacizumab-treated patients from AViTA. AC and CC carriers of this SNP exhibited HRs for overall survival of 2·0 (1·19-3·36; p=0·0091) and 4·72 (2·08-10·68; p=0·0002) relative to AA carriers. No effects were seen in placebo-treated patients and a significant genotype by treatment interaction (p=0·041) was recorded, indicating that the VEGFR1 locus containing this SNP serves as a predictive marker for bevacizumab treatment outcome in AViTA. Fine-mapping experiments of this locus identified rs7993418, a synonymous SNP affecting tyrosine 1213 in the VEGFR1 tyrosine-kinase domain, as the functional variant underlying the association. This SNP causes a shift in codon usage, leading to increased VEGFR1 expression and downstream VEGFR1 signalling. This VEGFR1 locus correlated significantly with progression-free survival (HR 1·81, 1·08-3·05; p=0·033) but not overall survival (HR 0·91, 0·45-1·82, p=0·78) in the bevacizumab group in AVOREN. INTERPRETATION: A locus in VEGFR1 correlates with increased VEGFR1 expression and poor outcome of bevacizumab treatment. Prospective assessment is underway to validate the predictive value of this novel biomarker. FUNDING: F Hoffmann-La Roche.

Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial.

BACKGROUND: In the Medical Research Council (MRC) COIN trial, the epidermal growth factor receptor (EGFR)-targeted antibody cetuximab was added to standard chemotherapy in first-line treatment of advanced colorectal cancer with the aim of assessing effect on overall survival. METHODS: In this randomised controlled trial, patients who were fit for but had not received previous chemotherapy for advanced colorectal cancer were randomly assigned to oxaliplatin and fluoropyrimidine chemotherapy (arm A), the same combination plus cetuximab (arm B), or intermittent chemotherapy (arm C). The choice of fluoropyrimidine therapy (capecitabine or infused fluouroracil plus leucovorin) was decided before randomisation. Randomisation was done centrally (via telephone) by the MRC Clinical Trials Unit using minimisation. Treatment allocation was not masked. The comparison of arms A and C is described in a companion paper. Here, we present the comparison of arm A and B, for which the primary outcome was overall survival in patients with KRAS wild-type tumours. Analysis was by intention to treat. Further analyses with respect to NRAS, BRAF, and EGFR status were done. The trial is registered, ISRCTN27286448. FINDINGS: 1630 patients were randomly assigned to treatment groups (815 to standard therapy and 815 to addition of cetuximab). Tumour samples from 1316 (81%) patients were used for somatic molecular analyses; 565 (43%) had KRAS mutations. In patients with KRAS wild-type tumours (arm A, n=367; arm B, n=362), overall survival did not differ between treatment groups (median survival 17·9 months [IQR 10·3-29·2] in the control group vs 17·0 months [9·4-30·1] in the cetuximab group; HR 1·04, 95% CI 0·87-1·23, p=0·67). Similarly, there was no effect on progression-free survival (8·6 months [IQR 5·0-12·5] in the control group vs 8·6 months [5·1-13·8] in the cetuximab group; HR 0·96, 0·82-1·12, p=0·60). Overall response rate increased from 57% (n=209) with chemotherapy alone to 64% (n=232) with addition of cetuximab (p=0·049). Grade 3 and higher skin and gastrointestinal toxic effects were increased with cetuximab (14 vs 114 and 67 vs 97 patients in the control group vs the cetuximab group with KRAS wild-type tumours, respectively). Overall survival differs by somatic mutation status irrespective of treatment received: BRAF mutant, 8·8 months (IQR 4·5-27·4); KRAS mutant, 14·4 months (8·5-24·0); all wild-type, 20·1 months (11·5-31·7). INTERPRETATION: This trial has not confirmed a benefit of addition of cetuximab to oxaliplatin-based chemotherapy in first-line treatment of patients with advanced colorectal cancer. Cetuximab increases response rate, with no evidence of benefit in progression-free or overall survival in KRAS wild-type patients or even in patients selected by additional mutational analysis of their tumours. The use of cetuximab in combination with oxaliplatin and capecitabine in first-line chemotherapy in patients with widespread metastases cannot be recommended. FUNDING: Cancer Research UK, Cancer Research Wales, UK Medical Research Council, Merck KGgA.

Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis.

BACKGROUND: Following the discovery that mutant KRAS is associated with resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the tumours of patients with metastatic colorectal cancer are now profiled for seven KRAS mutations before receiving cetuximab or panitumumab. However, most patients with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS selection era. METHODS: 1022 tumour DNA samples (73 from fresh-frozen and 949 from formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab between 2001 and 2008 were gathered from 11 centres in seven European countries. 773 primary tumour samples had sufficient quality DNA and were included in mutation frequency analyses; mass spectrometry genotyping of tumour samples for KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, progression-free survival (PFS), and overall survival in molecularly defined subgroups of the 649 chemotherapy-refractory patients treated with cetuximab plus chemotherapy. FINDINGS: 40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% (108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive benefit compared with wild types, with a response rate of 6.7% (17/253) versus 35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS of 12 weeks versus 24 weeks (hazard ratio [HR] 1.98, 1.66-2.36; p<0.0001), and a median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly lower response rate than did BRAF and NRAS wild types, with a response rate of 8.3% (2/24) in carriers of BRAF mutations versus 38.0% in BRAF wild types (124/326; OR 0.15, 95% CI 0.02-0.51; p=0.0012); and 7.7% (1/13) in carriers of NRAS mutations versus 38.1% in NRAS wild types (110/289; OR 0.14, 0.007-0.70; p=0.013). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were associated with a worse outcome compared with wild types, with a response rate of 0.0% (0/9) versus 36.8% (121/329; OR 0.00, 0.00-0.89; p=0.029), a median PFS of 11.5 weeks versus 24 weeks (HR 2.52, 1.33-4.78; p=0.013), and a median overall survival of 34 weeks versus 51 weeks (3.29, 1.60-6.74; p=0.0057). Multivariate analysis and conditional inference trees confirmed that, if KRAS is not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) gives additional information about outcome. Objective response rates in our series were 24.4% in the unselected population, 36.3% in the KRAS wild-type selected population, and 41.2% in the KRAS, BRAF, NRAS, and PIK3CA exon 20 wild-type population. INTERPRETATION: While confirming the negative effect of KRAS mutations on outcome after cetuximab, we show that BRAF, NRAS, and PIK3CA exon 20 mutations are significantly associated with a low response rate. Objective response rates could be improved by additional genotyping of BRAF, NRAS, and PIK3CA exon 20 mutations in a KRAS wild-type population. FUNDING: Belgian Federation against Cancer (Stichting tegen Kanker).

Vitamin D deficiency is highly prevalent in COPD and correlates with variants in the vitamin D-binding gene.

INTRODUCTION: Vitamin D deficiency has been associated with many chronic illnesses, but little is known about its relationship with chronic obstructive pulmonary disease (COPD). OBJECTIVES: Serum 25-hydroxyvitamin D (25-OHD) levels were measured in 414 (ex)-smokers older than 50 years and the link between vitamin D status and presence of COPD was assessed. The rs7041 and rs4588 variants in the vitamin D-binding gene (GC) were genotyped and their effects on 25-OHD levels were tested. RESULTS: In patients with COPD, 25-OHD levels correlated significantly with forced expiratory volume in 1 s (FEV(1)) (r=0.28, p<0.0001). Compared with 31% of the smokers with normal lung function, as many as 60% and 77% of patients with GOLD (Global Initiative for Obstructive Lung Disease) stage 3 and 4 exhibited deficient 25-OHD levels <20 ng/ml (p<0.0001). Additionally, 25-OHD levels were reduced by 25% in homozygous carriers of the rs7041 at-risk T allele (p<0.0001). This correlation was found to be independent of COPD severity, smoking history, age, gender, body mass index, corticosteroid intake, seasonal variation and rs4588 (p<0.0001). Notably, 76% and 100% of patients with GOLD stage 3 and 4 homozygous for the rs7041 T allele exhibited 25-OHD levels <20 ng/ml. Logistic regression corrected for age, gender and smoking history further revealed that homozygous carriers of the rs7041 T allele exhibited an increased risk for COPD (OR 2.11; 95% CI 1.20 to 3.71; p=0.009). CONCLUSION: Vitamin D deficiency occurs frequently in COPD and correlates with severity of COPD. The data warrant vitamin D supplementation in patients with severe COPD, especially in those carrying at-risk rs7041 variants.

Thrombomodulin mutations in atypical hemolytic-uremic syndrome.

BACKGROUND: The hemolytic-uremic syndrome consists of the triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. The common form of the syndrome is triggered by infection with Shiga toxin-producing bacteria and has a favorable outcome. The less common form of the syndrome, called atypical hemolytic-uremic syndrome, accounts for about 10% of cases, and patients with this form of the syndrome have a poor prognosis. Approximately half of the patients with atypical hemolytic-uremic syndrome have mutations in genes that regulate the complement system. Genetic factors in the remaining cases are unknown. We studied the role of thrombomodulin, an endothelial glycoprotein with anticoagulant, antiinflammatory, and cytoprotective properties, in atypical hemolytic-uremic syndrome. METHODS: We sequenced the entire thrombomodulin gene (THBD) in 152 patients with atypical hemolytic-uremic syndrome and in 380 controls. Using purified proteins and cell-expression systems, we investigated whether thrombomodulin regulates the complement system, and we characterized the mechanisms. We evaluated the effects of thrombomodulin missense mutations associated with atypical hemolytic-uremic syndrome on complement activation by expressing thrombomodulin variants in cultured cells. RESULTS: Of 152 patients with atypical hemolytic-uremic syndrome, 7 unrelated patients had six different heterozygous missense THBD mutations. In vitro, thrombomodulin binds to C3b and factor H (CFH) and negatively regulates complement by accelerating factor I-mediated inactivation of C3b in the presence of cofactors, CFH or C4b binding protein. By promoting activation of the plasma procarboxypeptidase B, thrombomodulin also accelerates the inactivation of anaphylatoxins C3a and C5a. Cultured cells expressing thrombomodulin variants associated with atypical hemolytic-uremic syndrome had diminished capacity to inactivate C3b and to activate procarboxypeptidase B and were thus less protected from activated complement. CONCLUSIONS: Mutations that impair the function of thrombomodulin occur in about 5% of patients with atypical hemolytic-uremic syndrome.

PIK3CA mutations are not a major determinant of resistance to the epidermal growth factor receptor inhibitor cetuximab in metastatic colorectal cancer.

PURPOSE: It has been reported that activating KRAS mutations negatively affect response to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies in metastatic colorectal cancer. The mutation status of signaling molecules downstream of the EGFR target is thus crucial to predict clinical benefit to EGFR-targeted therapies. Other mechanisms of resistance to EGFR inhibitors could involve activating mutations of the other main EGFR effector pathway, i.e., the PI3K/PTEN/AKT pathway. EXPERIMENTAL DESIGN: We analyzed the PIK3CA and KRAS mutation status in a large group (n = 200) of chemorefractory metastatic colorectal cancers treated with cetuximab (Erbitux) in monotherapy or in combination with irinotecan, and correlated the mutation status with outcome. RESULTS: Twenty-three (12%) of the 200 samples carried 1 of the PIK3CA mutations included in our assay. We found no correlation between the presence of a PIK3CA mutation and impaired response to cetuximab. CONCLUSIONS: Our findings do not provide any evidence for a strong role of PIK3CA mutations as a single marker in determining response to cetuximab in chemorefractory metastatic colorectal cancer.

Novel role for vascular endothelial growth factor (VEGF) receptor-1 and its ligand VEGF-B in motor neuron degeneration.

Although vascular endothelial growth factor-B (VEGF-B) is a homolog of the angiogenic factor VEGF, it has only minimal angiogenic activity, raising the question of whether this factor has other (more relevant) biological properties. Intrigued by the possibility that VEGF family members affect neuronal cells, we explored whether VEGF-B might have a role in the nervous system. Here, we document that the 60 kDa VEGF-B isoform, VEGF-B(186), is a neuroprotective factor. VEGF-B(186) protected cultured primary motor neurons against degeneration. Mice lacking VEGF-B also developed a more severe form of motor neuron degeneration when intercrossed with mutant SOD1 mice. The in vitro and in vivo effects of VEGF-B(186) were dependent on the tyrosine kinase activities of its receptor, Flt1, in motor neurons. When delivered intracerebroventricularly, VEGF-B(186) prolonged the survival of mutant SOD1 rats. Compared with a similar dose of VEGF, VEGF-B(186) was safer and did not cause vessel growth or blood-brain barrier leakiness. The neuroprotective activity of VEGF-B, in combination with its negligible angiogenic/permeability activity, offers attractive opportunities for the treatment of neurodegenerative diseases.