Future perspectives of circulating tumor DNA in colorectal cancer.

Tumor biopsy is currently the gold standard for diagnosis and in determining cell signaling pathways involved in the development of treatment resistance. However, there are major challenges with this technique, including the need for serial sampling to monitor treatment resistance, which is invasive and also has the potential for selection bias due to intra-tumoral and inter-tumoral heterogeneity. These challenges highlight the need for more effective methods for obtaining Tumor samples. Liquid biopsy analyzes genetic material or tumor cells shed into the blood from the primary tumor and metastatic sites and consequently provides a comprehensive, real-time picture of the tumor burden in an individual patient. Indeed, liquid biopsy has the potential to revolutionize cancer management. Here, we review recent studies on the potential clinical applications of liquid biopsy using circulating tumor DNA in colorectal cancer, including screening, diagnosis, detection of minimal residual disease after surgery, detection of recurrence, prognosis, predicting treatment response, monitoring tumor burden or response during treatment, and tracking resistance. We also discuss recent data demonstrating the utility of detecting KRAS-mutated circulating tumor DNA, both at diagnosis to determine an appropriate treatment strategy and during anti-epidermal growth factor receptor therapy to predict treatment resistance. The future integration of liquid biopsy into clinical practice is discussed, together with alternative approaches and key questions that need to be answered in future clinical studies before this technology can be implemented and used routinely.

Insulin-like growth factor receptor polymorphism defines clinical outcome in estrogen receptor-positive breast cancer patients treated with tamoxifen.

Compelling evidence points to a key role for insulin-like growth factor 1 (IGF1) signaling in breast cancer development and progression. In addition, IGF1 receptor (IGF1R) expression has been correlated and functionally linked with estrogen receptor (ER) signaling. Recent translational studies support a cross talk between IGF1R and ERα at different levels and data suggest enhanced IGF1R signaling as a causative mechanism of tamoxifen (TAM) resistance. We tested whether functional germline variations in the IGF pathway are associated with clinical outcome in ER-positive primary invasive breast cancer patients, who were treated with surgery and adjuvant TAM. Tissue samples of 222 patients with ER+ primary invasive breast cancer, who had undergone surgery at Charing Cross Hospital, London, UK between 1981 and 2003, were analyzed. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue samples and six functional IGF1 pathway polymorphisms were analyzed using direct DNA sequencing and PCR-restriction fragment length polymorphism. In multivariable analysis, patients with primary invasive breast cancer carrying IGF1R_rs2016347 G allele had a significantly increased risk of early tumor progression (hazard ratio (HR) 2.01; adjusted P=0.004) and death (HR 1.84; adjusted P=0.023) compared with patients carrying G/T or T/T, independent of established clinicopathological determinants. This association remained significant after adjusting for multiple testing. In addition, we were able to demonstrate that IRS1_rs1801123 and IGFBP3_rs2854744 were significantly associated with lymph node involvement and tumor size, respectively. We provide the first evidence for IGF1R_rs2016347 as an independent prognostic marker for ER+ breast cancer patients treated with TAM and support a rational for combined treatment strategies.

Pharmacogenetic profiling of CD133 is associated with response rate (RR) and progression-free survival (PFS) in patients with metastatic colorectal cancer (mCRC), treated with bevacizumab-based chemotherapy.

Recent studies suggest CD133, a surface protein widely used for isolation of colon cancer stem cells, to be associated with tumor angiogenesis and recurrence. We hypothesized that gene expression levels and germline variations in CD133 will predict clinical outcome in patients with metastatic colorectal cancer (mCRC), treated in first-line setting with 5-fluorouracil, oxaliplatin and bevacizumab (BV), and we investigated whether there is a correlation with gene expression levels of CD133, vascular endothelial growth factor (VEGF) and its receptors. We evaluated intra-tumoral gene expression levels by quantitative real-time (RT) PCR from 54 patients and three germline variants of the CD133 gene by PCR-restriction-fragment length polymorphism from 91 patients with genomic DNA. High gene expression levels of CD133 (>7.76) conferred a significantly greater tumor response (RR=86%) than patients with low expression levels (7.76, RR=38%, adjusted P=0.003), independent of VEGF or its receptor gene expression levels. Gene expression levels of CD133 were significantly associated with VEGF and its receptors messenger RNA levels (VEGFR-1 (P<0.01), -2 and -3, P<0.05). Combined analyses of two polymorphisms showed a significant association with progression-free survival (PFS) (18.5 months vs 9.8 months, P=0.004) in a multivariate analysis as an independent prognostic factor for PFS (adjusted P=0.002). These results suggest that CD133 is a predictive marker for standard first-line BV-based treatment in mCRC.

An individual coding polymorphism and the haplotype of the SPARC gene predict gastric cancer recurrence.

The 5-year survival rate for gastric adenocarcinoma (GA) remains only 40% and biomarkers to identify patients at high risk of tumor recurrence are urgently needed. Secreted protein acidic and rich in cysteine (SPARC) is an extracellular matrix glycoprotein that mediates cell matrix interactions, and upregulation of SPARC can promote tumor progression and metastasis. This study investigated whether single-nucleotide polymorphisms (SNPs) in SPARC impact the prognosis of GA. Blood or formalin-fixed, paraffin-embedded tissues were obtained from 137 GA patients at the University of Southern California and Memorial Sloan-Kettering Cancer Center medical facilities. DNA was isolated and five SNPs in the SPARC 3'-untranslated region (UTR) were evaluated by DNA sequencing or PCR-restriction fragment length polymorphism. Associations between SNPs and time to tumor recurrence (TTR) were analyzed using Kaplan-Meier curves, log-rank tests, and likelihood-ratio test within logistic or Cox regression model as appropriate. Patients carrying at least one G allele of the SPARC rs1059829 polymorphism (GG, AG) showed a median TTR of 3.7 years compared with 2.1 years TTR for patients with AA (hazard ratio (HR) 0.57; P=0.033). In a multivariate analysis adjusted for T and N category as covariates and stratified by race, hospital and chemotherapy, patients with at least one SPARC rs1059829 G allele (GG, AG) remained significantly associated with superior TTR than patients with AA genotype (adjusted P=0.026). In addition, patients harboring the G-A-A haplotype had the highest risk of tumor recurrence (HR 1.892; adjusted P=0.016). Our findings suggest that SPARC 3'-UTR SNPs may be useful in predicting GA patients at increased risk of recurrence.

ANO5-muscular dystrophy: clinical, pathological and molecular findings.

BACKGROUND AND PURPOSE: Anoctamin 5 (ANO5) is a putative intracellular calcium-activated chloride channel. Recessive mutations in ANO5 cause primary skeletal muscle disorders (limb-girdle muscular dystrophy 2L and distal muscular dystrophy), which are phenotypically similar to dysferlinopathy, a muscular dystrophy due to dysferlin-encoding gene (DYSF) mutations. METHODS: This study reports the phenotype and genotype of seven unrelated patients with ANO5-muscular dystrophy. RESULTS: Three patients had amyloid deposition in muscle and two had cardiac involvement. An additional patient without skeletal muscle amyloidosis had cardiac involvement with septal hypokinesis and supraventricular tachycardia requiring ablation. Amyloid subtyping using laser capture microdissection and mass spectrometry-based proteomic analysis did not identify ANO5 or any fragment of ANO5 in the amyloid deposits, but detected other known amyloidogenic proteins. Three patients had myotonic discharges without clinical myotonia. Four ANO5 mutations are novel, including a heterozygous 0.4 Mb deletion involving the entire ANO5 gene. CONCLUSIONS: The results of the present study suggest that ANO5 mutations can be associated with amyloid deposition in muscle, but the nature of the amyloid deposits remains indeterminate, as does their relationship with cardiac involvement. ANO5 analysis should be considered in cases of muscle amyloid deposition of indeterminate etiology. Electrical myotonia can accompany ANO5-muscular dystrophy.

TS and ERCC-1 mRNA expressions and clinical outcome in patients with metastatic colon cancer in CONFIRM-1 and -2 clinical trials.

To validate established cutoff levels of thymidylate synthase (TS) and excision repair cross-complementing (ERCC-1) intratumoral mRNA expressions in tumor samples from metastatic colorectal cancer (mCRC) patients treated with PTK787/ZK222584 (PTK/ZK). From 122 samples of patients with mCRC enrolled in CONFIRM-1 (Colorectal Oral Novel Therapy for the Inhibition of Angiogenesis and Retarding of Metastases) or CONFIRM-2, mRNA was isolated of microdissected formalin-fixed paraffin-embedded samples and quantitated using TaqMan-based technology. Existing TS and ERCC-1 cutoff levels were tested for their prognostic value in first-line and second-line therapy. TS expression was associated with overall survival (OS) in first-line, but not second-line therapy. ERCC-1 was associated with OS in patients treated with first-line and second-line FOLFOX4. In first-line FOLFOX4, combination of high TS and/or high ERCC-1 was associated with shorter OS. A correlation was observed between ERCC-1 expression and benefit from PTK/ZK+FOLFOX4 treatment. TS and ERCC-1 expression is associated with clinical outcome in mCRC. Baseline TS and ERCC-1 levels may allow the selection of patients who benefit from FOLFOX4 chemotherapy.

GRP78 promoter polymorphism rs391957 as potential predictor for clinical outcome in gastric and colorectal cancer patients.

BACKGROUND: Recently, the analysis of gastric and colorectal tumor specimens determined that 78-kiloDalton glucose-regulated protein (GRP78), an endoplasmic reticulum chaperone, up-regulation serves as an efficient mechanism protecting cells against apoptosis and can confer drug resistance. We tested whether functional polymorphisms within the GRP78 gene are related to clinical outcome in gastric and colorectal cancer (CRC) patients. PATIENTS AND METHODS: Blood samples of 234 stage II/III CRC patients at the University of Southern California (USC) and formalin-fixed paraffin-embedded tissues of 137 patients with localized gastric adenocarcinoma (GA) at USC and Memorial Sloan-Kettering Cancer Centers were obtained. GRP78 polymorphisms analyzed on germline DNA were correlated with clinical outcome using univariate and multivariate analyses. RESULTS: GA patients with the combined GRP78 rs391957 C/T and T/T genotype were at higher risk for tumor recurrence and death [hazard ratio (HR) 2.61; P < 0.001 and HR 3.17; P < 0.001, respectively], than those with C/C. These findings were subsequently tested in a CRC cohort where patients with the homozygous T/T genotype were at highest risk for tumor recurrence (HR 2.61; P = 0.015). The results remained significant after adjusting for clinicopathologic determinants. CONCLUSION: These data provide the first evidence that the GRP78 rs391957 polymorphism can predict clinical outcome in localized GA and locally advanced CRC patients.

A let-7 microRNA-binding site polymorphism in 3'-untranslated region of KRAS gene predicts response in wild-type KRAS patients with metastatic colorectal cancer treated with cetuximab monotherapy.

PURPOSE: recent studies have found that KRAS mutations predict resistance to monoclonal antibodies targeting the epidermal growth factor receptor in metastatic colorectal cancer (mCRC). A polymorphism in a let-7 microRNA complementary site (lcs6) in the KRAS 3' untranslated region (UTR) is associated with an increased cancer risk in non-small-cell lung cancer and reduced overall survival (OS) in oral cancers. We tested the hypothesis whether this polymorphism may be associated with clinical outcome in KRAS wild-type (KRASwt) mCRC patients treated with cetuximab monotherapy. PATIENTS AND METHODS: the presence of KRAS let-7 lcs6 polymorphism was evaluated in 130 mCRC patients who were enrolled in a phase II study of cetuximab monotherapy (IMCL-0144). Genomic DNA was extracted from dissected formalin-fixed paraffin-embedded tumor tissue, KRAS mutation status and polymorphism were assessed using direct sequencing and PCR restriction fragment length polymorphism technique. RESULTS: KRAS let-7 lcs6 polymorphism was found to be related to object response rate (ORR) in mCRC patients whose tumors had KRASwt. The 12 KRASwt patients harboring at least a variant G allele (TG or GG) had a 42% ORR compared with a 9% ORR in 55 KRASwt patients with let-7 lcs6 TT genotype (P = 0.02, Fisher's exact test). KRASwt patients with TG/GG genotypes had trend of longer median progression-free survival (3.9 versus 1.3 months) and OS (10.7 versus 6.4 months) compared to those with TT genotypes. CONCLUSIONS: these results are the first to indicate that the KRAS 3'UTR polymorphism may predict for cetuximab responsiveness in KRASwt mCRC patients, which warrants validation in other clinical trials.

Pharmacogenetic profiling of Aurora kinase B is associated with overall survival in metastatic colorectal cancer.

Aurora kinases are conserved eukaryotic serine-threonine kinases, which serve as key regulators of mammalian mitosis. Several studies revealed a distinct correlation between inaccurate chromosome segregation, leading to chromosomal number instability, cancer progression and poor outcome. The aim of this study was to investigate the correlation of Aurora kinases A (AURKA) and B (AURKB) with overall survival (OS) by quantifying gene expression analysis and evaluation of single-nucleotide polymorphisms (SNPs) in human colorectal cancer samples and assessing the associations with clinicopathological features. We evaluated intratumoral gene expression levels and SNPs of AURKA and -B from 41 patients with metastatic colorectal cancer (mCRC). Patients with a high expression level of AURKB (>1.28) lived significantly shorter (n=11, median OS=6.4 months, 95% confidence interval (CI): 3.0-14.5 months) compared with patients with a low expression level (≤ 1.28) (n=30, median OS=18.4 months, 95% CI: 14.7-27.8 months, P=0.026, Wald's test). Patients harboring any G-allele in AURKB 885A>G showed a significantly decreased OS (P=0.05, log-rank test). We did not find any associations with clinicopathological variables and AURKA gene expression levels. Our results suggest a potential role for AURKB inhibition in patients with mCRC; thereby supporting its potential role as a target in mCRC.

Crustal seismic velocity responds to a magmatic intrusion and seasonal loading in Iceland's Northern Volcanic Zone.

Seismic noise interferometry is an exciting technique for studying volcanoes, providing a continuous measurement of seismic velocity changes (dv/v), which are sensitive to magmatic processes that affect the surrounding crust. However, understanding the exact mechanisms causing changes in dv/v is often difficult. We present dv/v measurements over 10 years in central Iceland, measured using single-station cross-component correlation functions from 51 instruments across a range of frequency bands. We observe a linear correlation between changes in dv/v and volumetric strain at stations in regions of both compression and dilatation associated with the 2014 Bárðarbunga-Holuhraun dike intrusion. Furthermore, a clear seasonal cycle in dv/v is modeled as resulting from elastic and poroelastic responses to changing snow thickness, atmospheric pressure, and groundwater level. This study comprehensively explains variations in dv/v arising from diverse crustal stresses and highlights the importance of deformation modeling when interpreting dv/v, with implications for volcano and environmental monitoring worldwide.

Sensory neuron degeneration in familial Kugelberg-Welander disease.

A 53 year old man developed symptoms of motor neuron disease in childhood. There was a family history of a similar disorder and it was felt to represent a form of Kugelberg-Welander disease. In addition to the motor deficits, sensory abnormalities in his legs were documented during life. Autopsy revealed anterior horn cell loss throughout the length of the spinal cord, with preservation of the phrenic nucleus. The lumbar dorsal root ganglia showed active degeneration of sensory neurons, with nuclear changes exceeding cytoplasmic ones. The fasciculus gracilis showed Wallerian degeneration. The findings provide direct evidence that sensory neurons can degenerate in some forms of motor neuron disease, and that the "demyelination" or "degeneration" of posterior columns sometimes seen in the various forms of motor neuron disease may actually be secondary to cell body disease in the dorsal root ganglia.

gamma-Aminobutyric acid (GABA) and sepsis-related encephalopathy.

In order to determine whether disturbances in GABA homeostasis might play a role in the pathogenesis of sepsis-related encephalopathy, serum and brain tissue GABA concentrations from six areas of the brain (cortex, diencephalon, striatum, hippocampus, midbrain, and pons-medulla) were determined in a rat model of bacterial sepsis (cecal ligation and perforation). The results were compared to those obtained from sham operated control animals. All septic animals demonstrated clinical signs of encephalopathy and had elevated serum GABA levels (0.92 +/- 0.3 uM versus 0.48 +/- 0.15 in controls, p less than 0.01). GABA content in the specific subcompartments of the brain, however, were similar in the two groups. These results indicate that although serum GABA levels are elevated during sepsis, GABA is unlikely to play an important role in the pathogenesis of sepsis-related encephalopathy.

Adjuvant extension of chemotherapy after neoadjuvant therapy may not improve outcome in early-stage breast cancer.

INTRODUCTION: Neoadjuvant chemotherapy (NAC) is equivalent to adjuvant therapy (AdC) in terms of survival and disease-free interval. Many institutions add AdC after NAC and surgery. However, such extended chemotherapy (ExC) is not evidence based. Study aim was to investigate if ExC improved disease-free (DFS) and overall survival (OS). PATIENTS AND METHODS: From 1998 to 2006 356 consecutive patients received NAC (45 pts), AdC (221 pts) or ExC (90 pts). We analysed these 3 groups to determine effects of ExC and to identify patients who might benefit. NAC consisted in 93% of 3-6 cycles of epirubicin+docetaxel, AdC comprised EC+/-taxanes in 72%. Median age in the NAC, AdC, and ExC-groups was 54, 56 and 52 years with follow-up of 30, 57, and 55 months. RESULTS: After NAC, 35% achieved downstaging and 10% pathologic complete remission. Surprisingly ExC seemed to result in reduction of 5-year DFS: compared to 85% and 82% after NAC and AdC, DFS was 61% after ExC (p=0.001). OS was not significantly affected (79, 91, and 78% after NAC, AdC and ExC, p=0.13). In multivariate analysis after correction for age, menopausal status, stage, grading, hormone receptors, her2-status, radiotherapy and surgery, ExC seemed to adversely affect DFS (HR 2.15, p=0.008), loco-regional and distant recurrence-rates (HR 3.0, p=0.03 and HR 2.0, p=0.02). DISCUSSION: In this single-center analysis ExC could not show advantages in terms of DFS and OS. Because multivariate analyses of retrospective data cannot account for all potential biases, these data require confirmation in randomized clinical trials. Until then, extended chemotherapy should be considered carefully. As in previous studies, no differences were found between NAC and AdC groups.

Lipid profiles.

Lipid profiles are only part of the cardiovascular status of the patient. Treatment should be directed at the cause and not the lipid profile numbers. The minority of patients with serious problems should be defined and treated and the others should be left alone. There is no single magic drug and dietary advice can work well if given by experienced, personable professionals.

Imazaquin and chlorsulfuron resistance and cross resistance in mutants of Chlamydomonas reinhardtii.

Chlorsulfuron and/or imazaquin resistant mutants of Chlamydomonas reinhardtii strain CW15 have been obtained and shown to have actolactate synthase (ALS) with altered sensitivity to one or both of these herbicides. Herbicide resistance in the three mutants described is allelic, and resistance appears to result from a dominant or semidominant mutation in a single, nuclear gene. Imazaquin and chlorsulfuron resistant ALS from imazaquin and chlorsulfuron resistant mutants, together with single-gene Mendelian inheritance of these phenotypes, suggests that ALS is the sole site of action of the two herbicides in Chlamydomonas. A high degree of cross resistance between the two herbicides was found in only one mutant. This mutant (IMR-13) was selected for resistance to imazaquin and has a high level of in vitro resistance to both imazaquin (270-fold increased I50) and chlorsulfuron (900-fold increased I50). In another mutant selected for resistance to imazaquin (IMR-2), hyper-sensitivity to chlorsulfuron was found. A mutant selected for resistance to chlorsulfuron (CSR-5), had a substantial degree of resistance of chlorsulfuron (80-fold increased I50), but not to imazaquin (7-fold increased I50).