Plasma ctDNA RAS status selects patients for anti-EGFR treatment rechallenge in metastatic colorectal cancer: a meta-analysis.

BACKGROUND: Recently one randomized trial and several phase II studies underscored that patients with metastatic colorectal cancer who progressed after an initial clinical benefit from anti-epidermal growth factor receptor (EGFR) treatment may further benefit from rechallenge with anti-EGFR therapy. Testing circulating tumor DNA (ctDNA) RAS status prior to anti-EGFR rechallenge seems a promising non-invasive method to predict and monitor response to anti-EGFR readministration. AIM: To assess the capability of liquid biopsy ctDNA in exploring RAS status and in predicting outcome of metastatic colorectal cancer patients treated with anti-EGFR monoclonal antibody rechallenge. MATERIALS AND METHODS: Systematic review of literature and meta-analysis of the available evidence. RESULTS: Data from four studies involving 117 patients were available. All patients harbored RAS wild type tumors and derived benefit from first line anti-EGFR therapy. Of these, 65 underwent plasma ctDNA before anti-EGFR treatment rechallenge and were eligible for analyses: 35 patients had RAS wild type ctDNA, and 30 RAS mutated, indicating that 46% of patients underwent RAS status conversion after primary anti-EGFR therapy. Anti-EGFR rechallenge among patients with plasma ctDNA RAS wild type status was associated with a consistent benefit in progression free survival (hazard ratio (HR) 0.40, 95% confidence interval (CI) 0.22-0.70; p = 0.001; I2 = 0) and overall survival (HR 0.37, 95% CI 0.16-0.85; p = 0.02; I2 = 74%) when compared to its use among patients with plasma ctDNA RAS mutation. Patients with plasma ctDNA RAS wild type profile also performed statistically better in term of disease control rate, risk for disease progression at 3 and 6 months, and risk for death at 6 and 12 months. CONCLUSION: RAS status assessment continues to be useful in predicting benefit for anti-EGFR treatment.

REMARRY and PURSUIT trials: liquid biopsy-guided rechallenge with anti-epidermal growth factor receptor (EGFR) therapy with panitumumab plus irinotecan for patients with plasma RAS wild-type metastatic colorectal cancer.

BACKGROUND: Previous clinical trials have demonstrated the potential efficacy of rechallenge with anti- epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) for patients with RAS/BRAF V600E wild-type metastatic colorectal cancer (mCRC). Moreover, post hoc biomarker analyses of clinical trials has suggested that RAS status in circulating tumor DNA (ctDNA) has a high probability to select patients who could benefit from anti-EGFR mAb rechallenge. METHODS: This trial is composed of 2 phases: a monitoring phase (REMARRY) and a trial phase (PURSUIT). A monitoring phase, the REMARRY study, aims to evaluate the dynamics of plasma RAS status during the subsequent treatments after refractory to anti-EGFR therapy in patients with mCRC with RAS/BRAF V600E wild-type tumors who have progressed after a response to previous anti-EGFR therapy, using a highly sensitive digital polymerase chain reaction OncoBEAM RAS CRC kit in a central laboratory (Sysmex, Japan). A trial phase, the PURSUIT trial, is a multicenter, single-arm phase II trial to assess the efficacy and safety of rechallenge therapy with panitumumab plus irinotecan in patients without RAS mutations in ctDNA (plasma RAS negative) in the REMARRY study. Key eligibility criteria of the PURSUIT trial include RAS/BRAF V600E wild-type mCRC in tumor tissue refractory or intolerant to fluoropyrimidine, oxaliplatin, and irinotecan; progression after complete or partial response to previous anti-EGFR therapy; plasma RAS negative (defined as plasma mutant allele frequencies [MAF] of all RAS ≤ 0.1%) within 28 days prior to enrollment; 4 months or more between the last administration of previous anti-EGFR mAb and the start of protocol treatment; and Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) ≤ 1. The primary endpoint is the confirmed objective response rate (ORR). The target sample size of the PURSUIT trial is 50 patients. Biomarker analyses will be performed in parallel using the OncoBEAM RAS CRC kit and a next-generation sequencing-based ctDNA analysis (Guardant360). DISCUSSION: Our trial aims to confirm the clinical benefit of anti-EGFR mAb rechallenge therapy in patients with plasma RAS negative. Moreover, through biomarker analyses, our trial will shed light on which patients would benefit from rechallenge in addition to being plasma RAS negative. TRIAL REGISTRATION: The REMARRY study: UMIN, UMIN000036424 . Registered date: April 5, 2019. The PURSUIT trial: jRCT, jRCTs031190096 . Registered date: October 1, 2019.

Interaction network of proteins associated with unfavorable prognosis in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant disorder of hematopoietic stem and progenitor cells, characterized by accumulation of immature blasts in the bone marrow and peripheral blood of affected patients. Standard induction therapy leads to complete remission in approximately 50% to 75% of patients. In spite of favorable primary response rates, only 20% to 30% of patients enjoy long-term disease free survival. Identifying proteins involved in prognosis is important for proposing biomarkers that can aid in the clinical management of the disease. The aim of this study was to construct a protein-protein interaction (PPI) network based on serum proteins associated with unfavorable prognosis of AML, and analyze the biological pathways underlying molecular complexes in the network. We identified 16 candidate serum proteins associated with unfavorable prognosis (in terms of poor response to treatment, poor overall survival, short complete remission, and relapse) in AML via a search in the literature: IL2RA, FTL, HSP90AA1, D2HGDH, PLAU, COL18A1, FGF19, SPP1, FGA, PF4, NME1, TNF, ANGPT2, B2M, CD274, LGALS3. The PPI network was constructed with Cytoscape using association networks from String and BioGRID, and Gene Ontology enrichment analysis using the ClueGo pluggin was performed. The central protein in the network was found to be PTPN11 which is involved in modulating the RAS-ERK, PI3K-AKT and JAK-STAT pathways, as well as in hematopoiesis, and in the regulation of apoptotic genes. Therefore, a dysregulation of this protein and/or of the proteins connected to it in the network leads to the defective activation of these signaling pathways and to a reduction in apoptosis. Together, this could cause an increase in the frequency of leukemic cells and a resistance to apoptosis in response to treatment.

Emergence of RAS mutations in patients with metastatic colorectal cancer receiving cetuximab-based treatment: a study protocol.

BACKGROUND: In the management of patients with RAS wild-type metastatic colorectal cancer (mCRC), anti-epidermal growth factor receptor (EGFR) therapies have demonstrated a clinical benefit, with longer survival. However, the correlation between the emergence of circulating RAS mutations and secondary resistance to anti-EGFR therapies requires further elucidation. In this study, we aim to examine evolutionary changes in RAS mutations through liquid biopsy in patients with mCRC during and after anti-EGFR therapy. METHODS: A total of 120 patients diagnosed with RAS wild-type mCRC will be enrolled in this study. Patients will receive a cetuximab-based infusional 5-fluorouracil regimen as first-line treatment. Cetuximab-based treatment is expected to continue until disease progression, intolerable toxic effects, or withdrawal of consent. Blood samples from enrolled patients will be collected before and then every 3 months during cetuximab-based treatment and also at disease progression. These blood samples will be evaluated for RAS resistance mutations by using the MassARRAY platform. The primary endpoint is the percentage of RAS mutations detected in circulating DNA from patients during cetuximab treatment. The correlation between the tumor response and survival outcomes of these patients and the emergence of circulating RAS mutations will be further analyzed. DISCUSSION: Liquid biopsy is a powerful technology that can represent tumor heterogeneity in a relatively noninvasive manner. Because RAS mutations play a major role in resistance to anti-EGFR therapy for mCRC, examining evolutionary changes in these mutations during such treatment through liquid biopsy would be useful. After comprehensively analyzing the emergence of circulating RAS mutations and its clinical relevance in this study, our results should provide practical guidance on anti-EGFR therapy for mCRC. TRIAL REGISTRATION: The date of trial registration ( NCT03401957 ) in this study was January 17, 2018.

Plasma Dynamics of RAS/RAF Mutations in Patients With Metastatic Colorectal Cancer Receiving Chemotherapy and Anti-EGFR Treatment.

BACKGROUND: RAS and RAF mutations in colorectal cancer (CRC) hold value in precision medicine. Liquid biopsy is an alternative to tumor tissue biopsy, and circulating tumor DNA (ctDNA) has been intensively investigated, but the clinical relevance of RAS and RAF mutations in plasma is yet to be determined. This study aimed to investigate the clinical aspects of RAS/RAF mutations during combination treatment. PATIENTS AND METHODS: Patients with RAS/RAF tumor wild-type metastatic CRC treated with combination chemotherapy and an EGFR inhibitor were included. Blood samples were collected at baseline and every treatment cycle and analyzed for 31 RAS, RAF, and EGFR mutations until progressive disease or censoring using droplet digital PCR. RESULTS: Forty-six patients were prospectively enrolled onto the study. At baseline, 7% had detectable RAS/RAF mutations in ctDNA. During the treatment course, the fraction of patients with mutated ctDNA increased to 22%. The emergence of mutations did not correlate with response or risk of progression while receiving treatment (P = 1.0). CONCLUSION: Emergence of plasma RAS/RAF mutations was not correlated with the effect of combination chemotherapy and EGFR inhibition in patients with RAS/RAF wild-type metastatic CRC.

GWAS implicated risk variants in different genes contribute additively to increase the risk of coronary artery disease (CAD) in the Pakistani subjects.

BACKGROUND: Coronary artery disease (CAD) remains the single most important cause of mortality worldwide. Many candidate and GWAS genetic variants have been identified in the recent years. In the current study, we selected six SNPs from various genes that have originally been identified in GWAS studies and examined the association of SNPs individually and as a genetic risk score (GRS) with CAD and blood lipid levels in the Pakistani subjects. METHODS: Six hundred twenty-four (404 cases and 219 controls) subjects were genotyped for variants rs10757274 in CDKN2A gene, rs17465637 in MIA3 gene, rs7025486 in DAB2IP gene, rs17228212 in SMAD3 gene, rs981887 in MRAS gene and rs1746048 in CXCL12 gene, by TaqMan and KASPar allele discrimination techniques. Serum lipid parameters were measured using commercially available kits. Statistical analyses were done using SPSS version 22. RESULTS: Individually, the single SNPs were not associated with CAD (p < 0.05). However, the combined GRS of 6 SNPs was significantly higher in cases than controls (4.89 ± 0.11 vs 4.58 ± 0.08, p = 0.024). Among blood lipids, GRS showed significant positive association with serum triglycerides levels (p = 0.022). CONCLUSION: The GRS was quantitatively associated with CAD risk and showed association with serum triglycerides levels, suggesting that the mechanism of these variants is likely to be in part at least through creating an atherogenic lipid profile in subjects carrying high numbers of risk alleles.

The prognostic value of simultaneous tumor and serum RAS/RAF mutations in localized colon cancer.

The impact of RAS/RAF mutations in localized colon cancer needs clarification. Based on analysis of tumor-specific DNA, this study aimed at elucidating the prognostic influence of mutational status in tumor and serum using an extended panel of mutations. The study retrospectively included 294 patients with curatively resected stage I-III adenocarcinoma of the colon. Mutations in tumor and serum were determined at time of surgery. Analyses were performed with droplet digital PCR technology. Hazard ratio (HR) for the association between mutational status and survival was estimated in multivariate analysis taking known prognostic factors into account. Mutational status in tumor did not on its own have significant prognostic impact (P = 0.22). Patients with a RAS mutation simultaneously in tumor and serum had a significantly worse prognosis, overall survival (OS) (HR = 2.30, 95% CI = 1.27-4.15, P = 0.0057), and disease-free survival (DFS) (HR = 2.18, 95%CI = 1.26-3.77, P = 0.0053). BRAF mutation in the serum and proficient mismatch repair (pMMR) protein in tumor also indicated significantly worse prognosis, OS (HR = 3.45, 95% CI = 1.52-7.85, P = 0.0032) and DFS (HR = 3.61, 95% CI = 1.70-7.67, P = 0.0008). In conclusion, RAS mutations in serum, and BRAF mutation in serum combined with pMMR in tumor were strong independent prognostic factors in patients with RAS/RAF mutated tumors.

Risk of statin therapy in elderly.

A meta-analysis of younger patients included in randomized trials found good evidence that statins reduce vascular events and mortality in people with high cholesterol and the risk of coronary heart disease. The use pf statin in the elderly prevents the disease but can exert considerable side effects.

Analysis of circulating DNA and protein biomarkers to predict the clinical activity of regorafenib and assess prognosis in patients with metastatic colorectal cancer: a retrospective, exploratory analysis of the CORRECT trial.

BACKGROUND: Tumour mutational status is an important determinant of the response of metastatic colorectal cancer to targeted treatments. However, the genotype of the tissue obtained at the time of diagnosis might not accurately represent tumour genotype after multiple lines of treatment. This retrospective exploratory analysis investigated the clinical activity of regorafenib in biomarker subgroups of the CORRECT study population defined by tumour mutational status or plasma protein levels. METHODS: We used BEAMing technology to identify KRAS, PIK3CA, and BRAF mutations in DNA obtained from the plasma of 503 patients with metastatic colorectal cancer who enrolled in the CORRECT trial. We quantified total human genomic DNA isolated from plasma samples for 503 patients using a modified version of human long interspersed nuclear element-1 (LINE-1) quantitive real-time PCR. We also measured the concentration of 15 proteins of interest-angiopoietin 2, interleukin 6, interleukin 8, placental growth factor, soluble TIE-1, soluble VEGFR1, VEGF-A, VEGF-C, VEGF-D, VEGF-A isoform 121, bone morphogenetic protein 7, macrophage colony-stimulating factor, stromal cell-derived factor-1, tissue inhibitor of metalloproteinase 2, and von Willebrand factor-in plasma samples from 611 patients. We did correlative analyses of overall survival and progression-free survival in patient subgroups based on mutational status, circulating DNA concentration, and protein concentrations. The CORRECT trial was registered with ClinicalTrials.gov, number NCT01103323. FINDINGS: Tumour-associated mutations were readily detected with BEAMing of plasma DNA, with KRAS mutations identified in 349 (69%) of 503 patients, PIK3CA mutations in 84 (17%) of 503 patients, and BRAF mutations in 17 (3%) of 502 patients. We did not do correlative analysis based on BRAF genotype because of the low mutational frequency detected for this gene. Some of the most prevalent individual hot-spot mutations we identified included: KRAS (KRAS G12D, 116 [28%] of 413 mutations; G12V, 72 [17%]; and G13D, 67 [16%]) and PIK3CA (PIK3CA E542K, 27 [30%] of 89 mutations; E545K, 37 [42%]; and H1047R, 12 [14%]). 41 (48%) of 86 patients who had received anti-EGFR therapy and whose archival tumour tissue DNA was KRAS wild-type in BEAMing analysis were identified as having KRAS mutations in BEAMing analysis of fresh plasma DNA. Correlative analyses suggest a clinical benefit favouring regorafenib across patient subgroups defined by KRAS and PIK3CA mutational status (progression-free survival with regorafenib vs placebo: hazard ratio [HR] 0·52, 95% CI 0·35-0·76 for KRAS wild-type; HR 0·51, 95% CI 0·40-0·65 for KRAS mutant [KRAS wild type vs mutant, pinteraction=0·74]; HR 0·50, 95% CI 0·40-0·63 for PIK3CA wild-type; HR 0·54, 95% CI 0·32-0·89 for PIK3CA mutant [PIK3CA wild-type vs mutant, pinteraction=0·85]) or circulating DNA concentration (progression-free survival with regorafenib vs placebo: HR 0·53, 95% CI 0·40-0·71, for low circulating DNA concentrations; HR 0·52, 95% CI 0·40-0·70, for high circulating DNA concentrations; low vs high circulating DNA, pinteraction=0·601). With the exception of von Willebrand factor, assessed with the median cutoff method, plasma protein concentrations were also not associated with regorafenib activity in terms of progression-free survival. In univariable analyses, the only plasma protein that was associated with overall survival was TIE-1, high concentrations of which were associated with longer overall survival compared with low TIE-1 concentrations. This association was not significant in multivariable analyses. INTERPRETATION: BEAMing of circulating DNA could be a viable approach for non-invasive analysis of tumour genotype in real time and for the identification of potentially clinically relevant mutations that are not detected in archival tissue. Additionally, the results show that regorafenib seems to be consistently associated with a clinical benefit in a range of patient subgroups based on mutational status and protein biomarker concentrations. FUNDING: Bayer HealthCare Pharmaceuticals.

Characterizing the patterns of clonal selection in circulating tumor DNA from patients with colorectal cancer refractory to anti-EGFR treatment.

INTRODUCTION: KRAS and EGFR ectodomain-acquired mutations in patients with metastatic colorectal cancer (mCRC) have been correlated with acquired resistance to anti-EGFR monoclonal antibodies (mAbs). We investigated the frequency, co-occurrence, and distribution of acquired KRAS and EGFR mutations in patients with mCRC refractory to anti-EGFR mAbs using circulating tumor DNA (ctDNA). PATIENTS AND METHODS: Sixty-two post-treatment plasma and 20 matching pretreatment archival tissue samples from KRAS (wt) mCRC patients refractory to anti-EGFR mAbs were evaluated by high-sensitivity emulsion polymerase chain reaction for KRAS codon 12, 13, 61, and 146 and EGFR 492 mutations. RESULTS: Plasma analyses showed newly detectable EGFR and KRAS mutations in 5/62 [8%; 95% confidence interval (CI) 0.02-0.18] and 27/62 (44%; 95% CI 0.3-0.56) samples, respectively. KRAS codon 61 and 146 mutations were predominant (33% and 11%, respectively), and multiple EGFR and/or KRAS mutations were detected in 11/27 (41%) cases. The percentage of mutant allele reads was inversely correlated with time since last treatment with EGFR mAbs (P = 0.038). In the matching archival tissue, these mutations were detectable as low-allele-frequency clones in 35% of patients with plasma mutations after treatment with anti-EGFR mAbs and correlated with shorter progression-free survival (PFS) compared with the cases with no new mutations (3.0 versus 8.0 months, P = 0.0004). CONCLUSION: Newly detected KRAS and/or EGFR mutations in plasma ctDNA from patients refractory to anti-EGFR treatment appear to derive from rare, pre-existing clones in the primary tumors. These rare clones were associated with shorter PFS in patients receiving anti-EGFR treatment. Multiple simultaneous mutations in KRAS and EGFR in the ctDNA and the decline in allele frequency after discontinuation of anti-EGFR therapy in a subset of patients suggest that several resistance mechanisms can co-exist and that relative clonal burdens may change over time. Monitoring treatment-induced genetic alterations by sequencing ctDNA could identify biomarkers for treatment screening in anti-EGFR-refractory patients.

KRAS mutations in tumor tissue and plasma by different assays predict survival of patients with metastatic colorectal cancer.

BACKGROUND: The optimal laboratory assay for detecting KRAS mutations in different biospecimens from patients with metastatic colorectal cancer (mCRC), and the clinical relevance of these gene alterations is still in question. We analyzed the prognostic-predictive relevance of KRAS status, determined in tumor and plasma DNA by two different assays, in a large mono-institutional series of mCRC patients. METHODS: DNA sequencing and peptide-nucleic-acid-mediated-polymerase chain reaction clamping (PNA-PCR) were used to determine KRAS status in 416 tumor and 242 matched plasma DNA samples from mCRC patients who received chemotherapy only. Relationships with outcomes were analyzed with respect to the different assays and tissue types. RESULTS: PNA-PCR was significantly more sensitive in detecting KRAS mutations than sequencing (41% vs. 30%, p < 0.001). KRAS mutations were more frequent in tumor tissue than in plasma (sequencing, 38% vs. 17%, p < 0.001; PNA-PCR, 47% vs. 31%, p < 0.001). Median OS was consistently shorter in KRAS-mutated patients than KRAS wild-type patients, independent from the assay and tissue tested; the largest difference was in plasma samples analyzed by PNA-PCR (KRAS mutated vs. wild-type: 15.7 vs. 19.1 months, p = 0.009). No association was observed between KRAS status and other outcomes. When tumor and plasma results were considered together, median OS in patients categorized as tissue/plasma KRAS negative/negative, tissue/plasma KRAS discordant, and tissue/plasma KRAS positive/positive were 21.0, 16.9 and 15.4 months, respectively (p = 0.008). CONCLUSIONS: KRAS mutation status is of prognostic relevance in patients with mCRC. KRAS mutations in both tumor tissue and plasma are a strong prognostic marker for poor outcomes.

COPD-related adenocarcinoma presents low aggressiveness morphological and molecular features compared to smoker tumours.

OBJECTIVES: Adenocarcinoma comprises a group of diseases with heterogeneous clinical and molecular characteristics. COPD and lung cancer are strictly related; to date it is unknown if COPD-associated cancers have different features from tumours arising in non-COPD patients. Our aim was to study COPD-associated adenocarcinoma phenotypes mainly focusing on morphological and molecular aspects, in comparison to smoke-related cancer without COPD. MATERIALS AND METHODS: From 2010 to 2013, 54 patients with adenocarcinoma (20 COPD and 34 smokers) were prospectively studied. Each patient underwent a complete clinical and instrumental assessment. Morphological studies included analysis of growth pattern, cell proliferation (Ki-67/MIB1 expression) and parameters of intra- and peri-tumoral remodelling (inflammation, fibrosis and necrosis). Genetic analysis of EGFR and KRAS mutations was also performed. RESULTS: The two groups were comparable for the main demographic and biohumoral parameters except for increased blood basophil cell count in the COPD group. Compared to COPD, tumours of smokers presented an increased percentage of solid component (median: 20% vs 5%, p=0.02), a reduced percentage of lepidic pattern (median: 0% vs 10%, p=0.06) and higher Ki-67/MIB1 median value (55% vs 30%, p=0.02). In multivariate analysis lepidic and solid histological pattern were significantly influenced by clinical group (p=0.03 and 0.05, respectively). Concerning EGFR mutation, no differences were found between groups while KRAS mutation presented a trend of higher percentage in smokers compared to COPD (41% vs 20%, p=NS). Adenocarcinoma with KRAS mutation showed a higher value of Ki-67/MIB1 (65% vs 35%, p=0.048) and prevalent solid pattern (35% vs 10%, p=0.019) in comparison to wild-type form. CONCLUSIONS: COPD-related adenocarcinoma presents molecular and morphological features of lower aggressiveness (increased lepidic component, reduced solid pattern, lower cell proliferation and less frequent KRAS mutation) compared to smokers. Different molecular mechanisms could be associated with the development of COPD associated cancer.

Noninvasive diagnosis of actionable mutations by deep sequencing of circulating free DNA in lung cancer from never-smokers: a proof-of-concept study from BioCAST/IFCT-1002.

PURPOSE: Tumor somatic mutation analysis is part of the standard management of metastatic lung cancer. However, physicians often have to deal with small biopsies and consequently with challenging mutation testing. Circulating free DNA (cfDNA) is a promising tool for accessing the tumor genome as a liquid biopsy. Here, we evaluated next-generation sequencing (NGS) on cfDNA samples obtained from a consecutive series of patients for the screening of a range of clinically relevant mutations. EXPERIMENTAL DESIGN: A total of 107 plasma samples were collected from the BioCAST/IFCT-1002 lung cancer study (never-smokers cohort). Matched tumor DNA (tDNA) was obtained for 68 cases. Multiplex PCR-based assays were designed to target specific coding regions in EGFR, KRAS, BRAF, ERBB2, and PI3KCA genes, and amplicon sequencing was performed at deep coverage on the cfDNA/tDNA pairs using the NGS IonTorrent Personal Genome Machine Platform. RESULTS: CfDNA concentration in plasma was significantly associated with both stage and number of metastatic sites. In tDNA, 50 mutations (36 EGFR, 5 ERBB2, 4 KRAS, 3 BRAF, and 2 PIK3CA) were identified, of which 26 were detected in cfDNA. Sensitivity of the test was 58% (95% confidence interval, 43%-71%) and the estimated specificity was 87% (62%-96%). CONCLUSION: These data demonstrate the feasibility and potential utility of mutation screening in cfDNA using IonTorrent NGS for the detection of a range of tumor biomarkers in patients with metastatic lung cancer.

Microfluidic immunocapture of circulating pancreatic cells using parallel EpCAM and MUC1 capture: characterization, optimization and downstream analysis.

We have developed and optimized a microfluidic device platform for the capture and analysis of circulating pancreatic cells (CPCs) and pancreatic circulating tumor cells (CTCs). Our platform uses parallel anti-EpCAM and cancer-specific mucin 1 (MUC1) immunocapture in a silicon microdevice. Using a combination of anti-EpCAM and anti-MUC1 capture in a single device, we are able to achieve efficient capture while extending immunocapture beyond single marker recognition. We also have detected a known oncogenic KRAS mutation in cells spiked in whole blood using immunocapture, RNA extraction, RT-PCR and Sanger sequencing. To allow for downstream single-cell genetic analysis, intact nuclei were released from captured cells by using targeted membrane lysis. We have developed a staining protocol for clinical samples, including standard CTC markers; DAPI, cytokeratin (CK) and CD45, and a novel marker of carcinogenesis in CPCs, mucin 4 (MUC4). We have also demonstrated a semi-automated approach to image analysis and CPC identification, suitable for clinical hypothesis generation. Initial results from immunocapture of a clinical pancreatic cancer patient sample show that parallel capture may capture more of the heterogeneity of the CPC population. With this platform, we aim to develop a diagnostic biomarker for early pancreatic carcinogenesis and patient risk stratification.

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.

Mutant ras elevates dependence on serum lipids and creates a synthetic lethality for rapamycin.

The conversion of normal cells to cancer cells involves a shift from catabolic to anabolic metabolism involving increased glucose uptake and the diversion of glycolytic intermediates into nucleotides, amino acids, and lipids needed for cell growth. An underappreciated aspect of nutrient uptake is the utilization of serum lipids. We investigated the dependence of human cancer cells on serum lipids and report here that Ras-driven human cancer cells are uniquely dependent on serum lipids for both proliferation and survival. Removal of serum lipids also sensitizes Ras-driven cancer cells to rapamycin-indicating that the enhanced need for serum lipids creates a synthetic lethal phenotype that could be exploited therapeutically. Although depriving humans of serum lipids is not practical, suppressing uptake of lipids is possible. Suppressing macropinocytosis in Ras-driven cancer cells also created sensitivity to suppression of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1). It is speculated that this property displayed by Ras-driven cancer cells represents an Achilles' heel for the large number of human cancers that are driven by activating Ras mutations.

Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer.

Exosomes are small vesicles (50-150 nm) of endocytic origin that are released by many different cell types. Exosomes in the tumor microenvironment may play a key role in facilitating cell-cell communication. Exosomes are reported to predominantly contain RNA and proteins. In this study, we investigated whether exosomes from pancreatic cancer cells and serum from patients with pancreatic ductal adenocarcinoma contain genomic DNA. Our results provide evidence that exosomes contain >10-kb fragments of double-stranded genomic DNA. Mutations in KRAS and p53 can be detected using genomic DNA from exosomes derived from pancreatic cancer cell lines and serum from patients with pancreatic cancer. In addition, using whole genome sequencing, we demonstrate that serum exosomes from patients with pancreatic cancer contain genomic DNA spanning all chromosomes. These results indicate that serum-derived exosomes can be used to determine genomic DNA mutations for cancer prediction, treatment, and therapy resistance.

Levels of cell-free DNA and plasma KRAS during treatment of advanced NSCLC.

Non-small cell lung cancer (NSCLC) is one of the most common malignant tumours in the western world and is associated with a poor prognosis. Biomarkers predicting prognosis and therapeutic effects are highly required, and cell-free DNA (cfDNA) may be a feasible option. Genetic mutations can be analysed in plasma and may increase the scientific use of such measurements. In the present study, we investigated: i) the dynamics of cfDNA and plasma mutated KRAS (pmKRAS) during the treatment of patients with advanced NSCLC; and ii) the prognostic value of baseline cfDNA and pmKRAS. Sixty­nine patients were included in a prospective biomarker trial. Inclusion criteria included advanced NSCLC, candidate for first-line treatment, no previous cancer within the five years prior to this study. Blood samples were drawn at baseline, day 8 and at progression. Analyses of cfDNA and KRAS mutations in plasma were performed using an in-house qPCR assay. Evaluation of the treatment effect and status at follow-up was performed according to RECIST 1.1. The median levels of cfDNA were significantly higher at progression (9,250 alleles/ml) than at baseline (5,450 alleles/ml). Overall survival and progression-free survival were both significantly shorter in patients with high levels of cfDNA (above the 75th percentile) compared to lower levels. Only few patients harboured KRAS mutations in plasma. Two patients had no KRAS mutations in plasma at baseline, but mutations appeared in the subsequent blood samples. High baseline levels of cfDNA indicate a poor prognosis. The level changes during the treatment course with a significant increase at progression, suggesting a possible predictive value of cfDNA. The plasma KRAS status may change during treatment with potential implications for treatment selection.