Significance of Radial Margin in Patients Undergoing Complete Mesocolic Excision for Colon Cancer.

BACKGROUND: Comparable to circumferential resection margin in rectal cancer, radial margin is a potential prognostic factor in colon cancer that has just begun to be studied. No previous studies have investigated the influence of radial margin in the context of complete mesocolic excision. OBJECTIVE: This study aimed to examine the impact of radial margin on oncologic outcomes after complete mesocolic excision for colon cancer. DESIGN: We retrospectively reviewed patients with stage I to III colon cancer who underwent curative resection from October 2010 to March 2013. SETTINGS: This study was conducted using the prospective colorectal cancer registry of Severance hospital. PATIENTS: A total of 834 consecutive patients who underwent complete mesocolic excision for colon adenocarcinoma were included. INTERVENTIONS: We assigned patients into 3 groups according to radial margin distance: group A, radial margin ≥2.0 mm; group B, 1.0 ≤ radial margin < 2.0 mm; group C, radial margin <1 mm. MAIN OUTCOMES AND MEASURES: Overall survival and disease-free survival were estimated. RESULTS: On adjusted Cox regression analysis, only group C was predictive of reduced overall survival (HR, 1.90; 95% CI, 1.11-3.25; p = 0.018) and disease-free survival (HR, 1.93; 95% CI, 1.28-2.89; p = 0.001). We thereby defined radial margin threatening as radial margin <1 mm. Postoperative 5-fluorouracil (HR, 0.86; 95% CI, 0.35-2.10; p = 0.743) and FOLFOX (HR, 1.23; 95% CI, 0.57-2.64; p = 0.581) chemotherapy did not affect disease-free survival in patients with radial margin threatening. LIMITATIONS: This study has the limitations inherent in all retrospective, single-institution studies. CONCLUSIONS: Even with complete mesocolic excision, radial margin <1 mm was an independent predictor of survival and recurrence. This finding suggests that special efforts for obtaining a clear radial margin may be necessary in locally advanced colon cancer. See Video Abstract at http://links.lww.com/DCR/B125. IMPORTANCIA DEL MARGEN RADIAL EN PACIENTES SOMETIDOS A ESCISIÓN MESOCÓLICA COMPLETA PARA CÁNCER DEL COLON: Comparable al margen de resección circunferencial en cáncer rectal, el margen radial en cáncer de colon, es un factor pronóstico potencial, que recientemente comienza a estudiarse. Ningún estudio previo ha investigado la influencia del margen radial, en el contexto de la escisión mesocólica completa.Examinar en cáncer de colon, el impacto del margen radial en los resultados oncológicos, después de la escisión mesocólica completa.Revisión retrospectiva de pacientes con cáncer de colon en estadio I-III, sometidos a resección curativa de octubre 2010 a marzo 2013.Este estudio se realizó utilizando un registro prospectivo de cáncer colorrectal del hospital Severance.Se incluyeron un total de 834 pacientes consecutivos con adenocarcinoma de colon, sometidos a escisión mesocólica completa. Dividimos a los pacientes en 3 grupos según la distancia del margen radial: grupo A, margen radial ≥ 2.0 mm; grupo B, 1.0 ≤ margen radial <2.0 mm; grupo C, margen radial <1 mm.Se estimó la supervivencia general y la supervivencia libre de enfermedad.En el análisis de regresión de Cox ajustado, solo el grupo C fue predictivo de supervivencia global reducida (HR, 1.90; IC 95%, 1.11-3.25; p = 0.018) y supervivencia libre de enfermedad (HR, 1.93; IC 95%, 1.28-2.89; p = 0.001). Definimos como margen radial amenazante, un margen radial <1 mm. La quimioterapia posoperatoria con 5-FU (HR, 0,86; IC 95%, 0,35-2,10; p = 0.743) y FOLFOX (HR, 1,23; IC 95%, 0,57-2,64; p = 0,581), no afectó la supervivencia libre de enfermedad en pacientes con riesgo de margen radial.Este estudio tiene limitaciones inherentes a todos los estudios retrospectivos de una sola institución.Aun con la escisión mesocólica completa, el margen radial <1 mm fue un predictor independiente de supervivencia y recurrencia. Este hallazgo sugiere que pueden ser necesarios esfuerzos especiales para obtener un claro margen radial, en cáncer de colon localmente avanzado. Consulte Video Resumen en http://links.lww.com/DCR/B125.

Clinicopathological and biomolecular characteristics of stage IIB/IIC and stage IIIA colon cancer: Insight into the survival paradox.

BACKGROUND: A survival paradox of stage IIB/IIC and IIIA colon cancer has been consistently observed throughout revisions of the TNM system. This study aimed to understand this paradox with clinicopathological and molecular differences. METHODS: Clinicopathological characteristics of patients with pathologically confirmed stage IIB/IIC or IIIA colon cancer were retrospectively reviewed from a database. Publicly available molecular data were retrieved, and intrinsic subtypes were identified and subjected to gene sets enrichment analysis (GSEA). RESULTS: Among the 159 patients included in the clinicopathological analysis, those at stage IIB/IIC had worse 3-year disease-free and overall survival than those at stage IIIA (59.3% vs 91.7%, P < 0.001 and 82.7% vs 98.5%, P < 0.001, respectively), even after adjusting for confounding factors. Data of 95 patients were retrieved from public databases, demonstrating a higher frequency of the microsatellite instable subtype in stage IIB/IIC. The consensus molecular subtype distribution pattern differed between the groups. The GSEA further suggested the protumor inflammatory reaction might be more prominent in stage IIB/IIC. CONCLUSIONS: The survival paradox in colon cancer was confirmed and appears to be a multifactorial phenomenon not attributed to a single clinicopathologic factor. However, the greater molecular heterogeneity in stage IIB/IIC could contribute to the poor prognosis.

Brief Isolation Changes Nociceptive Behaviors and Compromises Drug Tests in Mice.

Herding with a litter is known to comfort rodents, whereas isolation and grouping with noncagemates provoke stress. The effects of stress induced by isolation and grouping with noncagemates on pain responses, and their underlying mechanisms remain elusive. We assessed the effect of isolation, a common condition during behavioral tests, and of grouping on defecation and pain behaviors of mice. Fecal pellets were counted 2 hours after exposure to the test chamber. It is significantly more in the isolated mice than in the grouped mice. Hindpaw withdrawal threshold and withdrawal latency were adopted as the indicatives of mechanical and thermal pain sensitivities, respectively. Interestingly, isolated mice showed higher pain thresholds than mice grouping with cagemates, and even those with noncagemates, indicating analgesic effects. Such effects were reduced by intrathecal injection of 0.01 mg/kg of naloxone (opioid receptor antagonist), atosiban (oxytocin and vasopressin receptor antagonist), and ketanserin (5-HT receptor antagonist). Intraperitoneal delivery of 1 mg/kg of naloxone and atosiban, but not ketanserin, also alleviated the isolation-induced analgesic effects. In contrast, these drugs at the same dose had no significant effect on the mice grouping with cagemates. In addition, the effect of morphine on thermal pain was more robust in the mice grouping with cagemates than in the isolated mice. These data demonstrated that brief isolation caused analgesia, mediated by endogenous opioidergic, oxytocinergic, and serotonergic pathways. These results indicate that isolation during pain behavioral tests can affect pain responses and the efficacy of drugs; thus, nociception tests should be conducted in grouping.

Drug screening by uniform patient derived colorectal cancer hydro-organoids.

As the importance of organoids increases, the need to develop organoid culture systems suitable for basic biological and clinical applications is being emphasized. However, there is still an unmet need to produce functionally complex and scalable uniform organoids. Here, we demonstrate a scalable organoid production platform with 8 well strips and a total of 8 × 9 microwells per strip using organoids derived from colorectal cancer tissue. The new culture platform is a format in which single cells are self-organized into organoids in culture medium supplemented with 2% Matrigel. It is functionally compatible with existing 96 well plates and Matrigel based conventional organoid culture methods. The consistency, uniformity and reproducibility of organoid produced on the new platform have been significantly improved compared to those of conventional plates. Importantly, Hydro-organoids are functionally identical to conventional Matrigel organoids, but show better consistency in drug screening. Our results show the possibility that Hydro-organoids can be used in high-throughput assays and incorporated into drug screening models to predict clinical outcomes.

A novel long noncoding RNA Linc-ASEN represses cellular senescence through multileveled reduction of p21 expression.

Long noncoding RNAs (lncRNAs) regulating diverse cellular processes implicate in many diseases. However, the function of lncRNAs in cellular senescence remains largely unknown. Here we identify a novel long intergenic noncoding RNA Linc-ASEN expresses in prematurely senescent cells. We find that Linc-ASEN associates with UPF1 by RNA pulldown mass spectrometry analysis, and represses cellular senescence by reducing p21 production transcriptionally and posttranscriptionally. Mechanistically, the Linc-ASEN-UPF1 complex suppressed p21 transcription by recruiting Polycomb Repressive Complex 1 (PRC1) and PRC2 to the p21 locus, and thereby preventing binding of the transcriptional activator p53 on the p21 promoter through histone modification. In addition, the Linc-ASEN-UPF1 complex repressed p21 expression posttranscriptionally by enhancing p21 mRNA decay in association with DCP1A. Accordingly, Linc-ASEN levels were found to correlate inversely with p21 mRNA levels in tumors from patient-derived mouse xenograft, in various human cancer tissues, and in aged mice tissues. Our results reveal that Linc-ASEN prevents cellular senescence by reducing the transcription and stability of p21 mRNA in concert with UPF1, and suggest that Linc-ASEN might be a potential therapeutic target in processes influenced by senescence, including cancer.

Impact of Adjuvant Chemotherapy Completion on Oncologic Outcomes in ypTNMstage 2 Rectal Cancer Patients.

PURPOSE: Adjuvant chemotherapy (aCT) in rectal cancer patients who have undergone curative resection after neoadjuvant chemoradiation (nCRT) is controversial. We aimed to investigate the benefits of using aCT and the clinical impact of completing aCT in ypstage 2 rectal cancer patients. METHODS: We retrospectively reviewed clinicopathological data from patients who had undergone radical resection after nCRT between January 2006 and December 2012. In total, 152 patients with ypT3/4N0M0 rectal cancer were included. Of these patients, 139 initiated aCT, while 13 did not receive aCT (no-aCT). Among those who received aCT, 132 patients completed their planned cycles (aCT-completion) whereas 7 did not (aCT-incompletion). All patients received longcourse chemoradiation; a 5-fluorouracil-based regimen was used for nCRT in most patients. The prognostic factors affecting disease-free survival (DFS) and overall survival (OS) were analyzed. RESULTS: The median follow-up duration was 41 months. Demographic data did not differ significantly among the 3 groups. In multivariate analysis, open surgery, a tumor size >2 cm, retrieval of <12 lymph nodes, circumferential resection margin (CRM) positivity and aCT incompletion were independent prognostic factors for poor DFS. Old age (≥60 years), open surgery, CRM positivity, aCT incompletion, and lack of aCT initiation compared to aCT completion were independent prognostic factors for poor OS. CONCLUSION: In ypstage 2 rectal cancer patients, aCT after nCRT and total mesorectal excision affected both DFS and OS; however, only patients who completed planned aCT exhibited survival benefits. Therefore, improving patients' compliance with the completion of aCT is desirable.

VEGF-A drives TOX-dependent T cell exhaustion in anti-PD-1-resistant microsatellite stable colorectal cancers.

Although immune checkpoint blockade therapies have demonstrated clinical efficacy in cancer treatment, harnessing this strategy is largely encumbered by resistance in multiple cancer settings. Here, we show that tumor-infiltrating T cells are severely exhausted in the microsatellite stable (MSS) colorectal cancer (CRC), a representative example of PD-1 blockade-resistant tumors. In MSS CRC, we found wound healing signature to be up-regulated and that T cell exhaustion is driven by vascular endothelial growth factor-A (VEGF-A). We report that VEGF-A induces the expression of transcription factor TOX in T cells to drive exhaustion-specific transcription program in T cells. Using a combination of in vitro, ex vivo, and in vivo mouse studies, we demonstrate that combined blockade of PD-1 and VEGF-A restores the antitumor functions of T cells, resulting in better control of MSS CRC tumors.

Organization of histamine-immunoreactive, tuberomammillary neurons projecting to the dorsal tier of the substantia nigra compacta in the rat.

Following the injection of a retrograde tracer, gold-conjugated and inactivated wheatgerm agglutinin-horseradish peroxidase (WGA-apo-HRP-gold), into the dorsal tier of substantia nigra compacta (SNCD), histamine immunostaining was performed for the tuberomammillary nucleus (TMN) in order to investigate the projection from the TMN to the SNCD. At the rostral pole of the TMN, the retrograde labeling in the dorsomedial subdivision following medial SNCD injections was predominantly ipsilateral (78%), whereas only a few cells were located bilaterally following lateral SNCD injections. Following tracer injections along the medio-lateral location along the SNCD, the labeling at the ventrolateral TMN was bilateral with slightly ipsilateral (58-61%) dominance. At rostral and caudal TMN levels, clusters of labeled neurons were localized within two discrete columns of the ventrolateral TMN. At rostral TMN level, a lateral column of cells was located at the lateral tip of the ventrolateral TMN just medial to the internal capsule, while the medial column was close to the protruded region along the ventral, pial border. At the caudal TMN level, two columns were located on either side of the lateral mammillary nucleus. Taken together, the present study suggests that ventrolateral as well as dorsomedial TMN might provide arousal-related information to medial, intermediate, and lateral regions of the SNCD, which in turn influence extrapyramidal, behavioral functions performed by the substantia nigra compacta.

The collateral projection from the dorsal raphe nucleus to whisker-related, trigeminal sensory and facial motor systems in the rat.

The primary goal of this study was to identify the collateral projection from the dorsal raphe (DR) nucleus to whisker-related, trigeminal sensory and facial motor systems in the rat. Following the injections of two retrograde tracers, gold-conjugated and inactivated wheatgerm agglutinin-horseradish peroxidase (WGA-apo-HRP-gold) and Fluorogold (FG) within vibrissae-related, sensory and motor areas at the cerebral cortical, thalamic, and medullary levels, the distribution of double-labeled neurons was examined within each subdivision of the DR. The major findings were: 1) the 5-HT-immunoreactive, DR neurons projecting to vibrissae-related, primary sensory and motor cortices were mainly observed in the ventromedial subdivision, with a few cells in the dorsomedial subdivision; 2) the DR neurons projecting to ventroposteromedial and ventrolateral thalamic nuclei were observed in the lateral wing subdivision ipsilateral to the injection sites; and 3) the DR neurons projecting to vibrissae-related, principal trigeminal and facial motor nuclei were also located mainly in the lateral wing subdivision ipsilateral to the injection sites. Taken together, these observations provide evidence that midline vs. lateral wing DR subdivisions have a differential functional organization with respect to their efferent projection systems and that individual DR neurons in each subdivision might preferentially send axon collaterals to sensory and motor whisker system targets, thus providing an anatomical substrate for coordination of whisker movement and tactile sensory coding.

Temporal changes in immune cell composition and cytokines in response to chemoradiation in rectal cancer.

We measured systemic changes in the immune response in 92 patients receiving preoperative chemoradiation therapy (CRT) and subsequent surgery for rectal cancer. The peripheral blood was sampled five times from the onset of CRT until surgery. Lymphocytes decreased continuously during CRT but increased after CRT. The increased lymphocyte population was predominantly CD8+ T lymphocytes, which accounted for a significantly larger proportion in patients without residual lymph node metastasis than in those with residual lymph node metastasis. Neutrophils and monocytes decreased during the initial two weeks of CRT but were maintained or increased afterwards. Neutrophil and monocyte counts were significantly lower in patients with a pCR (pathologic complete response) than in those without a pCR two weeks after CRT began but not at the initiation of CRT. All cytokines showed dramatic changes one month after the termination of CRT. Cytokines related to the antitumour immune response increased, and those related to tumour progression decreased. The predictive value of cytokines was not clear. In short, we observed that immune components in peripheral blood are affected by CRT and show dynamic changes over time. We identified biomarker candidates to predict the pathologic response in the future.

Juvenile obesity aggravates disease severity in a rat model of atopic dermatitis.

PURPOSE: There is increasing epidemiological evidence of an association between childhood obesity and atopic dermatitis, but little is known about the underlying mechanism(s). In the present study, we used a rat model of atopic dermatitis to assess whether juvenile obesity, induced by reduction of litter size, aggravated the signs of atopic dermatitis and, if so, whether this aggravation was associated with changes in plasma concentration of adipokines, such as leptin and adiponectin. METHODS: Dermatitis was induced by neonatal capsaicin treatment. Body weight, dermatitis score, serum IgE, skin nerve growth factor (NGF), serum leptin and adiponectin, and cytokine mRNA expression in the skin lesion were compared between small (SL, 5 pups) and large litters (LL, 15 pups). RESULTS: The body weight of juvenile rats up to 6 weeks of age was significantly heavier in the SL group, compared with those in the LL group. The SL group showed more robust development of dermatitis, and higher levels of serum IgE and skin NGF than the LL group. Additionally, the SL group demonstrated higher levels of leptin and pro-inflammatory cytokine mRNA but lower levels of adiponectin than the LL group. CONCLUSIONS: These results suggest a causal link between a decrease in immunological tolerance, induced by juvenile obesity, and aggravation of atopic dermatitis.

Chronically relapsing pruritic dermatitis in the rats treated as neonate with capsaicin; a potential rat model of human atopic dermatitis.

BACKGROUND: The pathophysiological mechanisms underlying chronic pruritic skin diseases, e.g. atopic dermatitis (AD), and effective therapies remain elusive due to the paucity of animal models. Recently, we rediscovered that injection of capsaicin into rat pups resulted in vigorous scratching behavior and chronically relapsing AD-like cutaneous lesions well into adulthood. OBJECTIVES: To characterize the chronic pruritic dermatitis induced by neonatal capsaicin treatment. METHODS: Capsaicin (50mg/kg) was given to rat pups subcutaneously within 48 h after birth, and then scratching behavior, dermatitis and pathophysiological changes of rat skin were investigated chronologically. RESULTS: Neonatal capsaicin treatment led to not only severe scratching and cutaneous lesions but also a large number of pathophysiological changes in the skin, such as histopathological changes including the deficiency of epidermal filaggrin expression, increases in the number of mast cells, levels of tissue NGF and Th2 cytokine mRNA, impaired skin barrier function and colonization with S. aureus. In addition, we observed the hyperproduction of serum IgE, which is clinically similar to the pathophysiology seen in the patients with atopic dermatitis. During the follow-up observation, the rats showed the alternative periods of relapsing and remitting skin lesions. CONCLUSION: Injection of capsaicin into rat pups results in chronically relapsing pruritic dermatitis, similar to human AD. Therefore, we think neonatal capsaicin treatment could be a useful model for studying human AD and for the development of novel therapeutic drugs.

Evaluation of recombinant adenovirus-mediated gene delivery for expression of tracer genes in catecholaminergic neurons.

Selective labeling of small populations of neurons of a given phenotype for conventional neuronal tracing is difficult because tracers can be taken up by all neurons at the injection site, resulting in nonspecific labeling of unrelated pathways. To overcome these problems, genetic approaches have been developed that introduce tracer proteins as transgenes under the control of cell-type-specific promoter elements for visualization of specific neuronal pathways. The aim of this study was to explore the use of tracer gene expression for neuroanatomical tracing to chart the complex interconnections of the central nervous system. Genetic tracing methods allow for expression of tracer molecules using cell-type-specific promoters to facilitate neuronal tracing. In this study, the rat tyrosine hydroxylase (TH) promoter and an adenoviral delivery system were used to express tracers specifically in dopaminergic and noradrenergic neurons. Region-specific expression of the transgenes was then analyzed. Initially, we characterized cell-type-specific expression of GFP or RFP in cultured cell lines. We then injected an adenovirus carrying the tracer transgene into several brain regions using a stereotaxic apparatus. Three days after injection, strong GFP expression was observed in the injected site of the brain. RFP and WGA were expressed in a cell-type-specific manner in the cerebellum, locus coeruleus, and ventral tegmental regions. Our results demonstrate that selective tracing of catecholaminergic neuronal circuits is possible in the rat brain using the TH promoter and adenoviral expression.

Collateral projection from the locus coeruleus to whisker-related sensory and motor brain regions of the rat.

The primary goal of this study was to examine whether the locus coeruleus (LC) provides collateral projections to whisker-related, sensorimotor brain regions. After injections of retrograde tracers into the primary sensory (S1) barrel field/primary whisker motor (M1) cortices, ventroposteromedial (VPM)/ventrolateral (VL) thalamic nuclei, or principal sensory trigeminal (Pr5)/facial motor (Mo7) nuclei, the distribution of double-labeled neurons within the LC was examined. Our observations indicated that a large number of individual LC cells provided axon collaterals to S1-M1 or VPM-VL regions, whereas only a few projected to Pr5-Mo7 nuclei. The laterality and the distribution of dual-projecting LC neurons were as follows. 1) The neurons projecting to the S1-M1 cortices were predominantly ipsilateral (96% +/- 0.7%). Labeled neurons were located ventrally at the rostral pole but were evenly distributed along the dorsoventral aspect of the principal LC. 2) The cells projecting to the VPM-VL nuclei were bilateral, with ipsilateral (68% +/- 2.3%) dominance. Neurons were observed at the rostrocaudal extent of the LC, where the labeling was most pronounced at the ventral, principal LC. 3) The neurons projecting to the Pr5-Mo7 regions exhibited slightly contralateral (56% +/- 2.9%) dominance, where labeled cells were confined within the ventral margin of the principal subdivision. Taken together, the present observations demonstrate that each subdivision of the LC possesses a differential functional organization with respect to its collateral projection to whisker-related sensorimotor targets, suggesting that the nucleus might play a modulatory role in vibrissal sensorimotor integration that allows the guidance of behavioral action essential for the survival of the animal.