Differential neuroglycan C expression during retinal degeneration in Rpe65-/- mice.

PURPOSE: An increased mRNA expression of the genes coding for the extracellular matrix proteins neuroglycan C (NGC), interphotoreceptor matrix proteoglycan 2 (IMPG2), and CD44 antigen (CD44) has been observed during retinal degeneration in mice with a targeted disruption of the Rpe65 gene (Rpe65-/- mouse). To validate these data, we analyzed this differential expression in more detail by characterizing retinal NGC mRNA isoform and protein expression during disease progression. METHODS: Retinas from C57/Bl6 wild-type and Rpe65-/- mice, ranging 2 to 18 months of age, were used. NGC, IMPG2, and CD44 mRNA expression was assessed by oligonucleotide microarray, quantitative PCR, and in situ hybridization. Retinal NGC protein expression was analyzed by western blot and immunohistochemistry. RESULTS: As measured by quantitative PCR, mRNA expression of NGC and CD44 was induced by about 2 fold to 3 fold at all time points in Rpe65-/- retinas, whereas initially 4 fold elevated IMPG2 mRNA levels progressively declined. NGC and IMPG2 mRNAs were expressed in the ganglion cell layer, the inner nuclear layer, and at the outer limiting membrane. NGC mRNA was also detected in retinal pigment epithelium cells (RPE), where its mRNA expression was not induced during retinal degeneration. NGC-I was the major isoform detected in the retina and the RPE, whereas NGC-III was barely detected and NGC-II could not be assessed. NGC protein expression was at its highest levels on the apical membrane of the RPE. NGC protein levels were induced in retinas from 2- and 4-month-old Rpe65-/- mice, and an increased amount of the activity-cleaved NGC ectodomain containing an epidermal growth factor (EGF)-like domain was detected. CONCLUSIONS: During retinal degeneration in Rpe65-/- mice, NGC expression is induced in the neural retina, but not in the RPE, where NGC is expressed at highest levels.

Signal peptide and helical bundle domains of virulent canine distemper virus fusion protein restrict fusogenicity.

Persistence in canine distemper virus (CDV) infection is correlated with very limited cell-cell fusion and lack of cytolysis induced by the neurovirulent A75/17-CDV compared to that of the cytolytic Onderstepoort vaccine strain. We have previously shown that this difference was at least in part due to the amino acid sequence of the fusion (F) protein (P. Plattet, J. P. Rivals, B. Zuber, J. M. Brunner, A. Zurbriggen, and R. Wittek, Virology 337:312-326, 2005). Here, we investigated the molecular mechanisms of the neurovirulent CDV F protein underlying limited membrane fusion activity. By exchanging the signal peptide between both F CDV strains or replacing it with an exogenous signal peptide, we demonstrated that this domain controlled intracellular and consequently cell surface protein expression, thus indirectly modulating fusogenicity. In addition, by serially passaging a poorly fusogenic virus and selecting a syncytium-forming variant, we identified the mutation L372W as being responsible for this change of phenotype. Intriguingly, residue L372 potentially is located in the helical bundle domain of the F(1) subunit. We showed that this mutation drastically increased fusion activity of F proteins of both CDV strains in a signal peptide-independent manner. Due to its unique structure even among morbilliviruses, our findings with respect to the signal peptide are likely to be specifically relevant to CDV, whereas the results related to the helical bundle add new insights to our growing understanding of this class of F proteins. We conclude that different mechanisms involving multiple domains of the neurovirulent A75/17-CDV F protein act in concert to limit fusion activity, preventing lysis of infected cells, which ultimately may favor viral persistence.

Immunohistochemistry as a valuable tool to assess CD30 expression in peripheral T-cell lymphomas: high correlation with mRNA levels.

The extended use of brentuximab-vedotin was reported for CD30(+) nonanaplastic peripheral T-cell lymphomas (PTCLs) with promising efficacy. CD30 status assessment is thus a critical factor for therapeutic decision, but the reliability of immunohistochemistry (IHC) in evaluating its expression remains to be defined. This prompted us to investigate the correlation between semiquantitative CD30 protein assessment by IHC and messenger RNA (mRNA) assessment by microarrays in a cohort of 376 noncutaneous PTCLs representative of the main entities. By IHC, CD30 expression was heterogeneous across and within entities and significantly associated with large tumor cell size. In addition to 100% anaplastic large-cell lymphomas, 57% of other PTCL entities were CD30-positive at a 5% threshold. CD30 protein expression was highly correlated to mRNA levels. mRNA levels were bimodal, separating high from low CD30-expressing PTCL cases. We conclude that IHC is a valuable tool in clinical practice to assess CD30 expression in PTCLs.

Beta-catenin Status in P?aediatric Medulloblastomas: correlation of immunohistochemical expression with mutational status, genetic profiles, and clinical characteristics.

Medulloblastoma is the most frequent malignant paediatric brain tumour. The activation of the Wnt/beta-catenin pathway occurs in 10-15% of medulloblastomas and has been recently described as a marker for favourable patient outcome. We report a series of 72 paediatric medulloblastomas evaluated for beta-catenin protein expression, CTNNB1 mutations, and comparative genomic hybridization. Gene expression profiles were also available in a subset of 40 cases. Immunostaining of beta-catenin showed extensive nuclear staining (>50% of the tumour cells) in six cases and focal nuclear staining (<10% of cells) in three cases. The other cases either exhibited a signal strictly limited to the cytoplasm (58 cases) or were negative (five cases). CTNNB1 mutations were detected in all beta-catenin extensively nucleopositive cases. The expression profiles of these cases documented strong activation of the Wnt/beta-catenin pathway. Remarkably, five out of these six tumours showed a complete loss of chromosome 6. In contrast, cases with focal nuclear beta-catenin staining, as well as tumours with negative or cytoplasmic staining, never demonstrated CTNNB1 mutation, Wnt/beta-catenin pathway activation or chromosome 6 loss. Patients with extensive nuclear staining were significantly older at diagnosis and were in continuous complete remission after a mean follow-up of 75.7 months (range 27.5-121.2 months) from diagnosis. All three patients with focal nuclear staining of beta-catenin died within 36 months from diagnosis. Altogether, these data confirm and extend previous observations that CTNNB1-mutated tumours represent a distinct molecular subgroup of medulloblastomas with favourable outcome, indicating that therapy de-escalation should be considered. International consensus on the definition criteria of this distinct medulloblastoma subgroup should be achieved.

JNK3 maintains expression of the insulin receptor substrate 2 (IRS2) in insulin-secreting cells: functional consequences for insulin signaling.

We have recently shown that silencing of the brain/islet specific c-Jun N-terminal Kinase3 (JNK3) isoform enhances both basal and cytokine-induced beta-cell apoptosis, whereas silencing of JNK1 or JNK2 has opposite effects. While it is known that JNK1 or JNK2 may promote apoptosis by inhibiting the activity of the pro-survival Akt pathway, the effect of JNK3 on Akt has not been documented. This study aims to determine the involvement of individual JNKs and specifically JNK3 in the regulation of the Akt signaling pathway in insulin-secreting cells. JNK3 silencing strongly decreases Insulin Receptor Substrate 2 (IRS2) protein expression, and blocks Akt2 but not Akt1 activation by insulin, while the silencing of JNK1 or JNK2 activates both Akt1 and Akt2. Concomitantly, the silencing of JNK1 or JNK2, but not of JNK3, potently phosphorylates the glycogen synthase kinase3 (GSK3β). JNK3 silencing also decreases the activity of the transcription factor Forkhead BoxO3A (FoxO3A) that is known to control IRS2 expression, in addition to increasing c-Jun levels that are known to inhibit insulin gene expression. In conclusion, we propose that JNK1/2 on one hand and JNK3 on the other hand, have opposite effects on insulin-signaling in insulin-secreting cells; JNK3 protects beta-cells from apoptosis and dysfunction mainly through maintenance of a normal IRS2 to Akt2 signaling pathway. It seems that JNK3 mediates its effects mainly at the transcriptional level, while JNK1 or JNK2 appear to mediate their pro-apoptotic effect in the cytoplasm.

Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons.

The excitatory neurotransmitter glutamate has been reported to have a major impact on brain energy metabolism. Using primary cultures of rat hippocampal neurons, we observed that glutamate reduces glucose utilization in this cell type, suggesting alteration in mitochondrial oxidative metabolism. The aquaglyceroporin AQP9 and the monocarboxylate transporter MCT2, two transporters for oxidative energy substrates, appear to be present in mitochondria of these neurons. Moreover, they not only co-localize but they interact with each other as they were found to co-immunoprecipitate from hippocampal neuron homogenates. Exposure of cultured hippocampal neurons to glutamate 100 μM for 1 h led to enhanced expression of both AQP9 and MCT2 at the protein level without any significant change at the mRNA level. In parallel, a similar increase in the protein expression of LDHA was evidenced without an effect on the mRNA level. These data suggest that glutamate exerts an influence on neuronal energy metabolism likely through a regulation of the expression of some key mitochondrial proteins.

Enhanced cerebral expression of MCT1 and MCT2 in a rat ischemia model occurs in activated microglial cells.

Monocarboxylate transporters (MCTs) are essential for the use of lactate, an energy substrate known to be overproduced in brain during an ischemic episode. The expression of MCT1 and MCT2 was investigated at 48 h of reperfusion from focal ischemia induced by unilateral extradural compression in Wistar rats. Increased MCT1 mRNA expression was detected in the injured cortex and hippocampus of compressed animals compared to sham controls. In the contralateral, uncompressed hemisphere, increases in MCT1 mRNA level in the cortex and MCT2 mRNA level in the hippocampus were noted. Interestingly, strong MCT1 and MCT2 protein expression was found in peri-lesional macrophages/microglia and in an isolectin B4+/S100beta+ cell population in the corpus callosum. In vitro, MCT1 and MCT2 protein expression was observed in the N11 microglial cell line, whereas an enhancement of MCT1 expression by tumor necrosis factor-alpha (TNF-alpha) was shown in these cells. Modulation of MCT expression in microglia suggests that these transporters may help sustain microglial functions during recovery from focal brain ischemia. Overall, our study indicates that changes in MCT expression around and also away from the ischemic area, both at the mRNA and protein levels, are a part of the metabolic adaptations taking place in the brain after ischemia.

Connexin 43 expression in human hypertrophied heart due to pressure and volume overload.

Left ventricular hypertrophy (LVH) is due to pressure overload or mechanical stretch and is thought to be associated with remodeling of gap-junctions. We investigated whether the expression of connexin 43 (Cx43) is altered in humans in response to different degrees of LVH. The expression of Cx43 was analyzed by quantitative polymerase chain reaction, Western blot analysis and immunohistochemistry on left ventricular biopsies from patients undergoing aortic or mitral valve replacement. Three groups were analyzed: patients with aortic stenosis with severe LVH (n=9) versus only mild LVH (n=7), and patients with LVH caused by mitral regurgitation (n=5). Cx43 mRNA expression and protein expression were similar in the three groups studied. Furthermore, immunohistochemistry revealed no change in Cx43 distribution. We can conclude that when compared with mild LVH or with LVH due to volume overload, severe LVH due to chronic pressure overload is not accompanied by detectable changes of Cx43 expression or spatial distribution.

Clic4, a novel protein that sensitizes β-cells to apoptosis.

OBJECTIVES: Chloride intracellular channel protein 4 (Clic4) is a ubiquitously expressed protein involved in multiple cellular processes including cell-cycle control, cell differentiation, and apoptosis. Here, we investigated the role of Clic4 in pancreatic β-cell apoptosis. METHODS: We used βTC-tet cells and islets from β-cell specific Clic4 knockout mice (βClic4KO) and assessed cytokine-induced apoptosis, Bcl2 family protein expression and stability, and identified Clic4-interacting proteins by co-immunoprecipitation and mass spectrometry analysis. RESULTS: We show that cytokines increased Clic4 expression in βTC-tet cells and in mouse islets and siRNA-mediated silencing of Clic4 expression in βTC-tet cells or its genetic inactivation in islets β-cells, reduced cytokine-induced apoptosis. This was associated with increased expression of Bcl-2 and increased expression and phosphorylation of Bad. Measurement of Bcl-2 and Bad half-lives in βTC-tet cells showed that Clic4 silencing increased the stability of these proteins. In primary islets β-cells, absence of Clic4 expression increased Bcl-2 and Bcl-xL expression as well as expression and phosphorylation of Bad. Mass-spectrometry analysis of proteins co-immunoprecipitated with Clic4 from βTC-tet cells showed no association of Clic4 with Bcl-2 family proteins. However, Clic4 co-purified with proteins from the proteasome suggesting a possible role for Clic4 in regulating protein degradation. CONCLUSIONS: Collectively, our data show that Clic4 is a cytokine-induced gene that sensitizes β-cells to apoptosis by reducing the steady state levels of Bcl-2, Bad and phosphorylated Bad.

Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes.

Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses.

Continuous quantification of HER2 expression by microfluidic precision immunofluorescence estimates HER2 gene amplification in breast cancer.

Chromogenic immunohistochemistry (IHC) is omnipresent in cancer diagnosis, but has also been criticized for its technical limit in quantifying the level of protein expression on tissue sections, thus potentially masking clinically relevant data. Shifting from qualitative to quantitative, immunofluorescence (IF) has recently gained attention, yet the question of how precisely IF can quantify antigen expression remains unanswered, regarding in particular its technical limitations and applicability to multiple markers. Here we introduce microfluidic precision IF, which accurately quantifies the target expression level in a continuous scale based on microfluidic IF staining of standard tissue sections and low-complexity automated image analysis. We show that the level of HER2 protein expression, as continuously quantified using microfluidic precision IF in 25 breast cancer cases, including several cases with equivocal IHC result, can predict the number of HER2 gene copies as assessed by fluorescence in situ hybridization (FISH). Finally, we demonstrate that the working principle of this technology is not restricted to HER2 but can be extended to other biomarkers. We anticipate that our method has the potential of providing automated, fast and high-quality quantitative in situ biomarker data using low-cost immunofluorescence assays, as increasingly required in the era of individually tailored cancer therapy.

Angiotensin II favors progression to heart failure in diabetic hearts: role of myocardial fatty acid oxidation downregulation

Purpose: Diabetic myocardium is particularly vulnerable to develop heart failure in response to chronic stress conditions including hypertension or myocardial infarction. We have recently observed that angiotensin II (Ang II)-mediated downregulation of the fatty acid oxidation pathway favors occurrence of heart failure by myocardial accumulation of lipids (lipotoxicity). Because diabetic heart is exposed to high levels of circulating fatty acid, we determined whether insulin resistance favors development of heart failure in mice with Ang II-mediated myocardial remodeling.Methods: To study the combined effect of diabetes and Ang II-induced heart remodeling, we generated leptin-deficient/insulin resistant (Lepob/ob) mice with cardiac targeted overexpression of angiotensinogen (TGAOGN). Left ventricular (LV) failure was indicated by pulmonary congestion (lung weight/tibial length>+2SD of wild-type mice). Myocardial metabolism and function were assessed during in vitro isolated working heart perfusion.Results: Forty-eight percent of TGAOGN mice without insulin resistance exhibited pulmonary congestion at the age of 6 months associated with increased myocardial BNP expression (+375% compared with WT) and reduced LV power (developed pressure x cardiac output; -15%). The proportion of mice presenting heart failure was markedly increased to 71% in TGAOGN mice with insulin resistance (TGAOGN/Lepob/ob). TGAOGN/Lepob/ob mice with heart failure exhibited further increase of BNP compared with failing non-diabetic TGAOGN mice (+146%) and further reduction of cardiac power (-59%). Mice with insulin resistance alone (Lepob/ob) did not exhibit signs of heart failure or LV dysfunction. Myocardial fatty acid oxidation measured during in vitro perfusion was markedly increased in non-failing hearts from Lepob/ob mice (+380% compared with WT) and glucose oxidation decreased (-72%). In contrast, fatty acid and glucose oxidation did not differ from Lepob/ob mice in hearts from TGAOGN/Lepob/ob mice without heart failure. However, both fatty acid and glucose oxidation were markedly decreased (-47% and -48%, respectively, compared with WT/Lepob/+) in failing hearts from TGAOGN/Lepob/ob mice. Reduction of fatty acid oxidation was associated with marked reduction of protein expression of a number of regulatory enzymes implied in fatty acid oxidation.Conclusions: Insulin resistance favors the progression to heart failure during chronic exposure of the myocardium to Ang II. Our results are compatible with a role of Ang II-mediated downregulation of fatty acid oxidation, potentially promoting lipotoxicity.

Role of microRNAs in the pathogenesis of Ewing's sarcoma

SummaryEwing's sarcoma family tumors (ESFT) are the second most frequent cancer of bone in adolescents and young adults. ESFT are characterized by a chromosomal translocation that involves the 5' segment of the EWSR1 gene and the 3' segment of an ets transcription factor family member gene. In 85% of cases the chromosomal translocation generates the fusion protein EWSR1-FLI-1. Recent work from our laboratory identified mesenchymal stem cells (MSC) as the putative cell of origin of ESFT and characterized a CD133+ subpopulation of ESFT cells with tumor initating and self-renewal capacity, known as cancer stem cells (CSC). MicroRNAs (miRNAs) are small non-coding RNA that regulate protein expression at the post-transcriptional level by either repressing translation or destabilizing mRNA. MiRNAs participate in several biological processes including cell proliferation and differentiation. We used miRNA expression profile comparison between MSC and ESFT cell lines and CD133+ ESFT cells and CD133" ESFT cells to investigate the role of miRNAs in ESFT pathogenesis. MiRNA expression profile comparison of MSC and ESFT cell lines identified 35 differentially expressed miRNAs. Among these was down-regulation of let-7a which results, in part, by the direct repression of let-7a-l promoter by EWSR1-FLI-1. Overexpression of let-7a in ESFT cells blocked ESFT tumorigenesis through an High-motility group AT-hook2 (HMGA2)-mediated mechanism.MiRNA profiling of CD133+ ESFT and CD 133" ESFT cells revealed a broad repression of miRNAs in CD133+ ESFT mediated by down-regulation of TARBP2, a central regulator of the miRNA maturation pathway. Down-regulation of TARBP2 in ESFT cell lines results in a miRNA expression profile reminescent of that observed in CD133+ ESFT and associated with increased tumorigenicity. Enhancement of TARBP2 activity using the antibiotic enoxacin or overexpression of miRNA-143 or miRNA-145, two targets of TARBP2, impaired ESFT CSC self-renewal and block ESFT tumorigenicity. Moreover in vivo administration of synthetic let- 7a, miRNA-143 or miRNA-145 blocks ESFT tumor growth.Thus, dysregulation of miRNA expression is a key feature in ESFT pathogenesis and restoration of their expressions might be used as a new therapeutic tool.RésuméLe sarcome d'Ewing est la deuxième tumeur osseuse la plus fréquente chez l'enfant et le jeune adolescent. Le sarcome d'Ewing est caractérisé par une translocation chromosomique qui produit une protéine de fusion EWSR1-FLI-1. Des récents travaux ont identifié les cellules mésenchymateuses souches (MSC) comme étant les cellules à l'origine du sarcome d'Ewing ainsi qu'une sous-population de cellules exprimant le marqueur CD 133, dans le sarcome d'Ewing connu comme les cellules cancéreuses souches (CSC). Ces cellules ont la capacité d'initier la croissance tumorale et possèdent des propriétés d'auto-renouvellement. Les microRNAs (miRNAs) sont de petits ARN qui ne codent pas pour des protéines et qui contrôlent l'expression des protéines en bloquant la traduction ou en dégradant l'ARNm. Les miRNAs participent à différents processus biologiques comme la prolifération et la différenciation cellulaires.Le but de ce travail est d'étudier le rôle des miRNAs dans le sarcome d'Ewing. Un profil d'expression de miRNAs entre les MSC et des lignées cellulaires de sarcome d'Ewing a mis en évidence 35 miRNAs différemment exprimés. Parmi ceux-ci, la répression de let-7a est liée à la répression directe du promoteur de let-7a-l par EWSR-FLI-1. La sur-expression de let-7a dans des lignées cellulaires de sarcome d'Ewing inhibe leur croissance tumorale. Cette inhibition de croissance tumorale est régulée par la protéine high-motility group AT-hook2 (HMGA2).Un profil d'expression de miRNAs entre les cellules du sarcome d'Ewing CD133+ et CD133" montre une sous-expression d'un grand nombre de miRNAs dans les cellules CD133+ par rapport aux cellules CD133". Cette différence d'expression de miRNAs est due à la répression du gène TARBP2 qui participe à la maturation des miRNAs. La suppression de TARBP2 dans des cellules d'Ewing induit un profil d'expression de miRNAs similaire aux cellules CD133+ du sarcome d'Ewing et augmente la tumorigenèse des lignées cellulaires. De plus l'utilisation d'enoxacin, une molécule qui augmente l'activité de TARBP2 ou la sur- expression des miRNA143 ou miRNA-145 dans les CSC du sarcome d'Ewing bloque l'auto- renouvellement des cellules et la croissance tumorale. Finalement, l'administration de let-7a, miRNA-143 ou miRNA-145, dans des souris bloque la croissance du sarcome d'Ewing. Ces résultats indiquent que la dysrégulation des miRNAs participe à la pathogenèse du sarcome d'Ewing et que les miRNAs peuvent être utilisés comme des agents thérapeutiques.

Efficient gene delivery and selective transduction of astrocytes in the mammalian brain using viral vectors.

Astrocytes are now considered as key players in brain information processing because of their newly discovered roles in synapse formation and plasticity, energy metabolism and blood flow regulation. However, our understanding of astrocyte function is still fragmented compared to other brain cell types. A better appreciation of the biology of astrocytes requires the development of tools to generate animal models in which astrocyte-specific proteins and pathways can be manipulated. In addition, it is becoming increasingly evident that astrocytes are also important players in many neurological disorders. Targeted modulation of protein expression in astrocytes would be critical for the development of new therapeutic strategies. Gene transfer is valuable to target a subpopulation of cells and explore their function in experimental models. In particular, viral-mediated gene transfer provides a rapid, highly flexible and cost-effective, in vivo paradigm to study the impact of genes of interest during central nervous system development or in adult animals. We will review the different strategies that led to the recent development of efficient viral vectors that can be successfully used to selectively transduce astrocytes in the mammalian brain.

Dukes B colorectal cancer: distinct genetic categories and clinical outcome based on proximal or distal tumor location.

PURPOSE: The aim of this study was to determine whether tumor location proximal or distal to the splenic flexure is associated with distinct molecular patterns and can predict clinical outcome in a homogeneous group of patients with Dukes B (T3-T4, N0, M0) colorectal cancer. It has been hypothesized that proximal and distal colorectal cancer may arise through different pathogenetic mechanisms. Although p53 and Ki-ras gene mutations occur frequently in distal tumors, another form of genomic instability associated with defective DNA mismatch repair has been predominantly identified in the proximal colon. To date, however, the clinical usefulness of these molecular characteristics remains unproven. METHODS: A total of 126 patients with a lymph node-negative sporadic colon or rectum adenocarcinoma were prospectively assessed with the endpoint of death by cancer. No patient received either radiotherapy or chemotherapy. p53 protein was studied by immunohistochemistry using DO-7 monoclonal antibody, and p53 and Ki-ras gene mutations were detected by single strand conformation polymorphism assay. RESULTS: During a mean follow-up of 67 months, the overall five-year survival was 70 percent. Nuclear p53 staining was found in 57 tumors (47 percent), and was more frequent in distal than in proximal tumors (55 vs. 21 percent; chi-squared test, P < 0.001). For the whole group, p53 protein expression correlated with poor survival in univariate and multivariate analysis (log-rank test, P = 0.01; hazard ratio = 2.16; 95 percent confidence interval = 1.12-4.11, P = 0.02). Distal colon tumors and rectal tumors exhibited similar molecular patterns and showed no difference in clinical outcome. In comparison with distal colorectal cancer, proximal tumors were found to be statistically significantly different on the following factors: mucinous content (P = 0.008), degree of histologic differentiation (P = 0.012), p53 protein expression, and gene mutation (P = 0.001 and 0.01 respectively). Finally, patients with proximal tumors had a marginally better survival than those with distal colon or rectal cancers (log-rank test, P = 0.045). CONCLUSION: In this series of Dukes B colorectal cancers, p53 protein expression was an independent factor for survival, which also correlated with tumor location. Eighty-six percent of p53-positive tumors were located in the distal colon and rectum. Distal colon and rectum tumors had similar molecular and clinical characteristics. In contrast, proximal neoplasms seem to represent a distinct entity, with specific histopathologic characteristics, molecular patterns, and clinical outcome. Location of the neoplasm in reference to the splenic flexure should be considered before group stratification in future trials of adjuvant chemotherapy in patients with Dukes B tumors.