Mutation status and prognostic values of KRAS, NRAS, BRAF and PIK3CA in 353 Chinese colorectal cancer patients.

Mutations in KRAS exon 2, BRAF and PIK3CA are commonly present in colorectal cancer (CRC) worldwide, but few data about RAS mutations outside KRAS exon 2 are available for Chinese CRCs. We, therefore, determined the mutation frequencies and prognostic values of KRAS exon 2, 3 and 4, NRAS exon 2 and 3, PIK3CA exon 9 and 20, and BRAF exon 15 by PCR and direct sequencing in 353 CRC patients from two Chinese clinical centers. KRAS exon 2, BRAF, PIK3CA mutations were identified in 42.2%, 4.5%, 12.3% of the cases, respectively. We found "rare mutations" in RAS genes in nearly 14% of CRCs-i.e., in almost a quarter (24.0%) of KRAS exon 2 wild type CRCs, including 2.3% in KRAS exon 3, 8.2% in KRAS exon 4 and 3.4% in NRAS. Stage I-III patients with PIK3CA or NRAS mutations developed more distant metastases (3-year risk in PIK3CA mutated and wild type patients: 23.3% vs 11.5%, P = 0.03; multivariate Hazard ratio (HR) = 3.129, P = 0.003; 3-year risk in NRAS mutated and wild type patients: 40.0% vs 12.2%, P = 0.012; multivariate HR = 5.152, P = 0.003). Our data emphasizes the importance of these novel molecular features in CRCs.

Pivotal role of glutamine synthetase in ammonia detoxification.

Glutamine synthetase (GS) catalyzes condensation of ammonia with glutamate to glutamine. Glutamine serves, with alanine, as a major nontoxic interorgan ammonia carrier. Elimination of hepatic GS expression in mice causes only mild hyperammonemia and hypoglutaminemia but a pronounced decrease in the whole-body muscle-to-fat ratio with increased myostatin expression in muscle. Using GS-knockout/liver and control mice and stepwise increments of enterally infused ammonia, we show that ∼35% of this ammonia is detoxified by hepatic GS and ∼35% by urea-cycle enzymes, while ∼30% is not cleared by the liver, independent of portal ammonia concentrations ≤2 mmol/L. Using both genetic (GS-knockout/liver and GS-knockout/muscle) and pharmacological (methionine sulfoximine and dexamethasone) approaches to modulate GS activity, we further show that detoxification of stepwise increments of intravenously (jugular vein) infused ammonia is almost totally dependent on GS activity. Maximal ammonia-detoxifying capacity through either the enteral or the intravenous route is ∼160 µmol/hour in control mice. Using stable isotopes, we show that disposal of glutamine-bound ammonia to urea (through mitochondrial glutaminase and carbamoylphosphate synthetase) depends on the rate of glutamine synthesis and increases from ∼7% in methionine sulfoximine-treated mice to ∼500% in dexamethasone-treated mice (control mice, 100%), without difference in total urea synthesis. CONCLUSIONS: Hepatic GS contributes to both enteral and systemic ammonia detoxification. Glutamine synthesis in the periphery (including that in pericentral hepatocytes) and glutamine catabolism in (periportal) hepatocytes represents the high-affinity ammonia-detoxifying system of the body. The dependence of glutamine-bound ammonia disposal to urea on the rate of glutamine synthesis suggests that enhancing peripheral glutamine synthesis is a promising strategy to treat hyperammonemia. Because total urea synthesis does not depend on glutamine synthesis, we hypothesize that glutamate dehydrogenase complements mitochondrial ammonia production. (Hepatology 2017;65:281-293).

Quantitative assessment of TIM-3 polymorphisms and cancer risk in Chinese Han population.

Previous studies have investigated the associations of TIM-3 polymorphisms (-1516G/T, -574G/T, and +4259T/G) with cancer risk in Chinese Han population, but the results remain conflicting. Therefore, we conducted a meta-analysis to derive a more precise estimation of the associations. The pooled data showed that TIM-3 polymorphisms (-1516G/T, -574G/T, and +4259T/G) were significantly associated with an increased risk of overall cancer in Chinese Han population. Subgroup analyses based on cancer system showed that TIM-3 -1516G/T polymorphism was only associated with an increased risk of digestive system cancer in Chinese Han population. TIM-3 -574G/T polymorphism was associated with an increased risk of digestive system cancer and other cancer in Chinese Han population. TIM-3 +4259T/G polymorphism was only associated with an increased risk of other cancer in Chinese Han population. In summary, our results indicated that TIM-3 polymorphisms (-1516G/T, -574G/T, and +4259T/G) were associated with the increased risk of cancer in Chinese Han population.

Developmental expression and localization of MHC class I molecules in the human central nervous system.

Recent animal studies have found neuronal expression of major histocompatibility complex (MHC) class I in the central nervous system (CNS). However, the developmental expression profiles of MHC class I in human CNS remain unclear. Here, we systemically evaluate the expression and subcellular localization of MHC class I molecules during human CNS development using immunohistochemistry and immunofluorescence. Between the age of 20-33 gestational weeks (GW), MHC class I expression was relatively absent in the cerebral cortex with the exception of a few neurons; however, expression increased rapidly in the cochlear nuclei and in the cerebellar cortical Purkinje cells while increasing slowly in the substantia nigra. Expression was also detected in some nuclei and nerve fibers of the brain stem including the ambiguus nucleus, the locus coeruleus and the solitary tract as early as 20 GW and persisted through 33 GW. These early-stage neural cells with MHC class I protein expression later developed neuronal morphology. 30-33 GW is an important period of MHC class I expression in neurons, and during this period, MHC class I molecules were found to be enriched not only in neuronal cell bodies and neurites but also in nerve fibers and in the surrounding stroma. No expression was detected in the adult brain with exception of the cerebrovascular endothelium. MHC class I molecules displayed greater postsynaptic colocalization in cerebellar Purkinje cells, in the lateral geniculate nucleus and in the cochlear nuclei. These results demonstrate diverse spatiotemporal expression patterns for MHC class I molecules in the prenatal human CNS and strongly support the notion that MHC class I molecules play important roles in both CNS development and plasticity.

Enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA on human brain metastatic breast cancer.

Novel molecularly targeted agents that block the development and metastasis of human brain metastatic breast cancer hold great promise for their translational value. In this study, we constructed a novel targeting composite peptide BRBP1-TAT-KLA comprising of three elements: a brain metastatic breast carcinoma cell (231-BR)-binding peptide BRBP1, a cell penetrating peptide TAT, and a proapoptotic peptide KLA. This composite peptide efficiently internalized in 231-BR cells and consequently induced mitochondrial damage and cellular apoptosis. Exposure of 231-BR cells to BRBP1-TAT-KLA significantly decreased cell viability and increased apoptosis compared with the cells treated with the control peptides. In vivo relevance of these findings was further corroborated in the 231-BR tumor-bearing mice that demonstrated significantly delayed tumor development and metastasis following administration of BRBP1-TAT-KLA compared with those treated with TAT-KLA alone. Interestingly, BRBP1-TAT-KLA inhibited the formation of both large and micro-metastases, while TAT-KLA alone failed to significantly reduce micro-metastases in the breast cancer brain metastasis mice. BRBP1-TAT-KLA selectively homed to the tumors in vivo where it induced cellular apoptosis without significant toxicity on non-tumor tissues. Our findings therefore demonstrated the enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA, providing insights toward development of a potential therapeutic strategy for brain metastatic breast cancer.

BRAF V600E mutation and KRAS codon 13 mutations predict poor survival in Chinese colorectal cancer patients.

BACKGROUND: Mutations in KRAS, BRAF and PIK3CA are the most common somatic alterations found in the colorectal cancer (CRC) patients from Western countries; but their prevalence and prognostic value have not been adequately assessed in Asian patients. The aim of this study was to determine the mutation frequencies of these genes in Chinese CRC patients and to investigate their impact on prognosis. METHODS: The sequences of exon 2 of KRAS, exon 15 of BRAF and exons 9 and 20 of PIK3CA were evaluated by PCR and direct sequencing using DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues from primary CRC tumors of 214 patients (colon/rectum: 126/88). RESULTS: KRAS, BRAF and PIK3CA mutations were identified in 44.9% (96/214), 4.2% (9/214) and 12.3% (26/212) CRCs, respectively. The most frequent mutations in KRAS, BRAF and PIK3CA were G12D, V600E and H1047R, respectively. All BRAF and 80.8% PIK3CA mutations were from colon cancer patients. BRAF V600E was associated with advanced TNM (P < 0.001), more distant metastases (P = 0.025), and worse overall survival (OS, P < 0.001; multivariate HR = 4.2, P = 0.004) in colon cancer patients. Compared with KRAS wt/BRAF wt CRC patients (N = 109), those with KRAS codon 13 mutations (N = 25) had significantly worse OS (P = 0.016; multivariate HR = 2.7, P = 0.011), whereas KRAS codon 12-mutated cases were not significantly associated with survival. Among the three most common KRAS mutations, G13D (N = 23) showed significant association with poor OS (P = 0.024; multivariate HR = 2.6, P = 0.016) compared with KRAS wt/BRAF wt patients. CONCLUSION: Our findings indicate that PI3K/RAS-RAF signaling pathway genes are frequently mutated in Chinese CRC patients, but have different characteristics than found in Western patients. BRAF V600E is an independent prognostic factor for Chinese patients. Our finding that KRAS codon 13 mutations (in particular G13D) are associated with inferior survival in BRAF wild-type CRCs in Chinese patients was not reported thus far. Our data emphasizes the importance of prospective evaluation of molecular features in CRC patients, because a single mutation type may represent a distinct biologic effect and clinical implication.

Identification and characterization of a novel phage display-derived peptide with affinity for human brain metastatic breast cancer.

A novel peptide, BRBP1 (MYPWTEPSYLSN), was identified using an in vitro phage biopanning strategy against human brain-seeking breast carcinoma cells (231-BR cells).The peptide-phage clone, BRBP1-M13 displaying BRBP1 sequence, specifically bound to 231-BR cells and the binding could be competitively abolished by BRBP1. In vivo distribution studies showed that BRBP1-M13 preferentially homed to the 231-BR tumors. Fluorescently-labeled BRBP1, BRBP1-K(5-TAMRA), preferentially bound to 231-BR cells in a dose-dependent and energy-dependent manner and it was efficiently internalized into the cells after 2 h incubation. Near-infrared fluorophores imaging demonstrated the accumulation of Cy5.5-conjugated BRBP1 peptide in the tumors in vivo. Thus, BRBP1 is a promising peptide binding to human brain metastatic breast cancer and it may be applied to targeted delivery of cytotoxic agents to the intended tumor.

Interaction of parental KIR and fetal HLA-C genotypes with the risk of preeclampsia.

OBJECTIVE: The aim of this study is to investigate whether certain combination of maternal killer cell immunoglobulin-like receptors (KIR) and fetal human leukocyte antigen-C (HLA-C) is risk for preeclampsia in the Chinese Han population. METHODS: A case-control study was conducted in 47 pregnant women with preeclampsia and 54 normal pregnant women. Twelve KIR genes were genotyped by PCR-sequence-specific primer in mothers. High-resolution HLA-C genotyping was performed in couples and fetuses by a routine sequencing-based typing method. RESULTS: The frequency of KIR2DS1 was decreased (p = 0.028) and AA genotype was increased (p = 0.017) in preeclampsia compared with controls. More women with KIR AA genotype have fewer C2 genes than their fetuses in preeclampsia than controls. CONCLUSION: Women with KIR AA genotype and fewer C2 genes than their fetuses were at risk for preeclampsia in the Chinese Han population, supporting that maternal-fetal KIR-HLA-C interaction plays an important role in preeclampsia development.

The similar expression pattern of MHC class I molecules in human and mouse cerebellar cortex.

The major histocompatibility complex (MHC) class I molecules are considered to be important in the immune system. However, the results reported in the past decade indicate that they also play important roles in the central nervous system. Here we examined the expression of MHC I and ß2-microglobulin (ß2m) in human and mouse cerebellar cortex. The results show that MHC I molecules are expressed both in human and mouse cerebellar cortex during brain development. The expression of H-2K(b)/D(b) is gradually increased with the development of mouse cerebellar cortex, but finally decreased to a very low level. Similarly, the expression of HLA-B/C genes is increased in developing human cerebellar cortex, but decreased after birth. The spatial and temporal expression of ß2m overlaps mostly with that of HLA-B/C molecules, and they are co-expressed in Purkinje cells. Our findings provide a fundamental basis to reveal the functions of neuronal MHC class I molecules in the development of human cerebellum.

Combination of human leukocyte antigen and killer cell immunoglobulin-like receptor genetic background influences the onset age of hepatocellular carcinoma in male patients with hepatitis B virus infection.

To investigate whether killer cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) genetic background could influence the onset age of hepatocellular carcinoma (HCC) in patients with hepatitis B virus (HBV) infection, one hundred and seventy-one males with HBV-related HCC were enrolled. The presence of 12 loci of KIR was detected individually. HLA-A, -B, and -C loci were genotyped with high resolution by a routine sequence-based typing method. The effect of each KIR locus, HLA ligand, and HLA-KIR combination was examined individually by Kaplan-Meier (KM) analysis. Multivariate Cox hazard regression model was also applied. We identified C1C1-KIR2DS2/2DL2 as an independent risk factor for earlier onset age of HCC (median onset age was 44 for C1C1-KIR2DS2/2DL2 positive patients compared to 50 for negative patients, P = 0.04 for KM analysis; HR = 1.70, P = 0.004 for multivariate Cox model). We conclude that KIR and HLA genetic background can influence the onset age of HCC in male patients with HBV infection. This study may be useful to improve the current HCC surveillance program in HBV-infected patients. Our findings also suggest an important role of natural killer cells (or other KIR-expressing cells) in the progress of HBV-related HCC development.

The spatio-temporal expression of MHC class I molecules during human hippocampal formation development.

In the immune system, the major histocompatibility complex (MHC) class I molecules mediate both the innate and adaptive immune responses in vertebrates. There has been a dogma that the central nervous system (CNS) is immune privileged and healthy neurons do not express MHC class I molecules. However, recent studies have indicated that the expression and non-immunobiologic roles of MHC class I in mammalian CNS. But data referring to humans are scarce. In this study we report the expression and cellular localization of MHC class I in the human fetal, early postnatal and adult hippocampal formation. The expression of MHC class I was very low in the hippocampus at 20 (gestational weeks) GW and slowly increased at 27-33 GW. The gradually increased expression in the somata of some granular cells in dentate gyrus (DG) was observed at 30-33 GW. Whereas, a rapid increase in MHC class I molecules expression was found in the subiculum and it reached high levels at 31-33 GW and maintained at postnatal 55 days. No expression of MHC class I was found in hippocampal formation in adult. MHC class I heavy chain and ß2 microglobulin (ß2M) showed similar expression in some cells of the hippocampal formation at 30-33 GW. Moreover, MHC class I molecules were mainly expressed in neurons and most MHC class I-expressing neurons were glutamatergic. The temporal and spatial patterns of MHC class I expression appeared to follow gradients of pyramidal neurons maturation in the subiculum at prenatal stages and suggested that MHC class I molecules are likely to regulate neuron maturation. This article is part of a Special Issue entitled Priority to Publish.

Quantification of maternal serum cell-free fetal DNA in early-onset preeclampsia.

The aim of this study was to determine whether the increased serum cell-free fetal DNA (cffDNA) level of gravidas developed into early-onset preeclampsia (EOPE) subsequently in the early second trimesters is related to prenatal screening markers. Serum was collected from 1011 gravidas. The level of cffDNA and prenatal screening markers were analyzed in 20 cases with EOPE and 20 controls. All fetuses were male. The maternal serum cffDNA level was assessed by amplification of the Y chromosome specific gene. Correlations between the variables were examined. (Logged) cffDNA in EOPE (median, 3.08; interquartile range, 2.93-3.68) was higher than controls (median, 1.79; interquartile range, 1.46-2.53). The increased level of (logged) cffDNA was correlated significantly with the increased human chorionic gonadotropin (HCG) level (r = 0.628, p < 0.001). Significant reciprocal correlations between cffDNA and babies' birth weight as well as gestation weeks at delivery were noted (r = -0.516, p = 0.001; r = -0.623, p < 0.001, respectively). The sensitivity and specificity of cffDNA to discriminate between the EOPE cases and the controls were 90% and 85%, respectively. CffDNA is a potential marker for EOPE, which had a significant reciprocal correlation with babies' birth weight and gestation weeks at delivery. Moreover, it may help in indicating the underlying hypoxic condition in the placenta.

Human leukocyte antigen class I alleles and haplotypes associated with primary hepatocellular carcinoma in persistent HBV-infected patients.

Many studies have shown that Human leukocyte antigen (HLA) class I alleles are associated with the development of various cancers. However, its role in hepatocellular carcinoma (HCC) is still unknown. To investigate whether HLA class I allelic polymorphism is related to the development of hepatitis B virus(HBV)-associated HCC, a total of 326 HBV-infected patients (138 individuals with HCC and 188 well-matched controls without HCC) were enrolled in this study. HLA-A, -B, and -C were genotyped by polymerase chain reaction-sequencing based typing (PCR-SBT) method. We identified HLA-B(∗)35:01:01G as a risk factor for HBV-related HCC development independent of our previous findings in HLA region (OR, 12.04; p, 0.0028; pc, 0.04). HLA-A(∗)11:01:01G, B(∗)58:01:01G, C(∗)03:02:01G and some of their extended haplotypes were found as potential susceptible factors for HCC development. HLA-B(∗)46:01:01G and some of its extended haplotypes were found as potential protective factors for HCC development. Our results support that specific HLA class I alleles and haplotypes may affect the risk of HBV-related HCC development. The findings may help to determine better approaches for prevention and treatment of HCC in these patients.

Involvement of c-Jun N-terminal kinase in reversal of multidrug resistance of human leukemia cells in hypoxia by 5-bromotetrandrine.

5-Bromotetrandrine (BrTet), a candidate multidrug resistance (MDR) modulator, is a potential compound for use in cancer therapy when combined with anticancer agents such as daunorubicin (DNR) and paclitaxel. The purposeof this study was to investigate the mechanism of reversal of P-glycoprotein (P-gp)-mediated MDR by BrTet and the involvement of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway in both adriamycin-sensitive K562 and adriamycin-resistant K562 (KA) leukemia cells in hypoxia. The combination of BrTet and DNR decreased both phosphorylated JNK1/2 and MDR1/P-gp levels under hypoxic conditions. Furthermore, a pharmacological inhibitor of JNK, SP600125, or small interfering RNA (siRNA) oligonucleotides to both JNK1 and JNK2 reversed BrTet- or DNR-induced JNK phosphorylation and MDR1/P-gp levels. We further demonstrated that the decreased JNK phosphorylation and MDR1/P-gp levels were associated with a significant increase in intracellular accumulation of DNR, which dramatically enhanced the sensitivity of drug-resistant KA cells to DNR, and led to cellular apoptosis through activation of the caspase-3 pathway. It is concluded that using BrTet in combination with other chemotherapeutic agents and pharmacological inhibitors of JNK can abrogate the P-gp-induced MDR in adriamycin-resistant K562 cells, which has potential clinical relevance in cancer therapy for chemotherapeutic-resistant human leukemia.

The expression patterns of MHC class I molecules in the developmental human visual system.

It has been considered that healthy neurons in central nervous system (CNS) do not express major histocompatibility complex (MHC) class I molecules. However, recent studies clearly demonstrated the expression of functional MHC class I in the mammalian embryonic, neonatal and adult brain. Until now, it is still unknown whether MHC I molecules are expressed in the development of human brain. We collected nine human brain tissues from fetuses aged from 21 to 31 gestational weeks (GW), one newborn of postnatal 55 days and one adult. The expression of MHC class I molecules was detected during the development of visual system in human brain by immunohistochemistry and immunofluorescence. MHC class I proteins were located at lateral geniculate nucleus (LGN) and the expression was gradually increased from 21 GW to 31 GW and reached high levels at 30-31 GW when fine-scale refinement phase was mediated by neural electric activity. However, there was no expression of MHC class I molecules in the visual cortical cortex during all the developmental stages examined. We also concluded that MHC class I molecules were mainly expressed in neurons but not in astrocytes at LGN. In the developing visual system, the expression of ß2M protein on neurons was not found in our study.

The expression pattern of classical MHC class I molecules in the development of mouse central nervous system.

Classical major histocompatibility complex (MHC) class I, first identified in the immune system, is also expressed in the developing and adult central nervous system (CNS). Although the MHC class I molecules have been found to be expressed in the CNS of different species, a necessary step to elucidate the temporal and spatial expression patterns of MHC class I molecules in the brain development has never been taken. Frozen sections were made from the brains of embryonic and postnatal C57BL/6 J mice, and the expression of H-2D(b) mRNA was examined by in situ hybridization. Immunofluorescence was also performed to define the cell types that express H2-D(b) in P15 mice. At E10.5, the earliest stage we examined, H2-D(b) was expressed in neuroepithelium of the brain vesicles. From E12.5 to P0, H2-D(b) expression was mainly located at cerebral cortex, neuroepithelium of the lateral ventricle, neuroepithelium of aquaeductus and developing cerebellum. From P4 to adult, H2-D(b) mRNA was detected at olfactory bulb, hippocampus, cerebellum and some nerve nuclei. The major cell types expressing H-2D(b) in P15 hippocampus, cerebral cortex and olfactory bulb were neuron. H2-K(b) signal paralleled that of H2-D(b) and the expression levels of the two molecules were comparable throughout the brain. The investigation of the expression pattern of H-2D(b) at both embryonic and postnatal stages is important for further understanding the physiological and pathological roles of H2-D(b) in the developing CNS.

Glutamine synthetase deficiency in murine astrocytes results in neonatal death.

Glutamine synthetase (GS) is a key enzyme in the "glutamine-glutamate cycle" between astrocytes and neurons, but its function in vivo was thus far tested only pharmacologically. Crossing GS(fl/lacZ) or GS(fl/fl) mice with hGFAP-Cre mice resulted in prenatal excision of the GS(fl) allele in astrocytes. "GS-KO/A" mice were born without malformations, did not suffer from seizures, had a suckling reflex, and did drink immediately after birth, but then gradually failed to feed and died on postnatal day 3. Artificial feeding relieved hypoglycemia and prolonged life, identifying starvation as the immediate cause of death. Neuronal morphology and brain energy levels did not differ from controls. Within control brains, amino acid concentrations varied in a coordinate way by postnatal day 2, implying an integrated metabolic network had developed. GS deficiency caused a 14-fold decline in cortical glutamine and a sevenfold decline in cortical alanine concentration, but the rising glutamate levels were unaffected and glycine was twofold increased. Only these amino acids were uncoupled from the metabolic network. Cortical ammonia levels increased only 1.6-fold, probably reflecting reduced glutaminolysis in neurons and detoxification of ammonia to glycine. These findings identify the dramatic decrease in (cortical) glutamine concentration as the primary cause of brain dysfunction in GS-KO/A mice. The temporal dissociation between GS(fl) elimination and death, and the reciprocal changes in the cortical concentration of glutamine and alanine in GS-deficient and control neonates indicate that the phenotype of GS deficiency in the brain emerges coincidentally with the neonatal activation of the glutamine-glutamate and the associated alanine-lactate cycles.

Glutamine synthetase in muscle is required for glutamine production during fasting and extrahepatic ammonia detoxification.

The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a approximately 3-fold higher escape of ammonia revealed the absence of GS in muscle. However, after 20 h of fasting, GS-KO/M mice were not able to mount the approximately 4-fold increase in glutamine production across the hindquarter that was observed in control mice. Instead, muscle ammonia production was approximately 5-fold higher than in control mice. The fasting-induced metabolic changes were transient and had returned to fed levels at 36 h of fasting. Glucose consumption and lactate and ketone-body production were similar in GS-KO/M and control mice. Challenging GS-KO/M and control mice with intravenous ammonia in stepwise increments revealed that normal muscle can detoxify approximately 2.5 micromol ammonia/g muscle.h in a muscle GS-dependent manner, with simultaneous accumulation of urea, whereas GS-KO/M mice responded with accumulation of glutamine and other amino acids but not urea. These findings demonstrate that GS in muscle is dispensable in fed mice but plays a key role in mounting the adaptive response to fasting by transiently facilitating the production of glutamine. Furthermore, muscle GS contributes to ammonia detoxification and urea synthesis. These functions are apparently not vital as long as other organs function normally.

PIK3CA mutations predict local recurrences in rectal cancer patients.

PURPOSE: Identifying rectal cancer patients at risk for local recurrence would allow for refinement in the selection of patients who would benefit from preoperative radiotherapy. PIK3CA, KRAS, and BRAF mutations are commonly found in colon cancers, but their prevalence has not been clearly assessed in rectal cancer. In this study, we aim to determine the mutation frequencies of PIK3CA, KRAS, and BRAF and to investigate whether a mutation may be used as a prognostic parameter in rectal cancer patients. EXPERIMENTAL DESIGN: We evaluated DNA mutations in PIK3CA, KRAS, and BRAF in 240 stage I to III rectal tumors obtained from nonirradiated patients from the Dutch Total Mesorectal Excision trial. RESULTS: PIK3CA, KRAS, and BRAF mutations were identified in 19 (7.9%), 81 (33.9%), and 5 (2.1%) rectal cancers. Patients with PIK3CA mutations developed more local recurrences (5-year risks, 27.8% versus 9.4%; P = 0.006) and tended to develop these recurrences more rapidly after surgery (median local recurrence-free interval since surgery: 7.9 versus 19.6 months; P = 0.07) than patients without PIK3CA mutations. In multivariate analysis, PIK3CA mutations remained as an independent predictor for the development of local recurrences (hazard ratio, 3.4; 95% confidence interval, 1.2-9.2; P = 0.017), next to tumor-node-metastasis stage. CONCLUSION: PIK3CA mutations can be used as a biomarker in identifying rectal cancer patients with an increased risk for local recurrences. Currently, our findings suggest that prospective evaluation of PIK3CA mutation status could reduce overtreatment by preoperative radiotherapy for the low-risk patients who might otherwise only experience the side effects.