Intra-tumour diversification in colorectal cancer at the single-cell level.

Every cancer originates from a single cell. During expansion of the neoplastic cell population, individual cells acquire genetic and phenotypic differences from each other. Here, to investigate the nature and extent of intra-tumour diversification, we characterized organoids derived from multiple single cells from three colorectal cancers as well as from adjacent normal intestinal crypts. Colorectal cancer cells showed extensive mutational diversification and carried several times more somatic mutations than normal colorectal cells. Most mutations were acquired during the final dominant clonal expansion of the cancer and resulted from mutational processes that are absent from normal colorectal cells. Intra-tumour diversification of DNA methylation and transcriptome states also occurred; these alterations were cell-autonomous, stable, and followed the phylogenetic tree of each cancer. There were marked differences in responses to anticancer drugs between even closely related cells of the same tumour. The results indicate that colorectal cancer cells experience substantial increases in somatic mutation rate compared to normal colorectal cells, and that genetic diversification of each cancer is accompanied by pervasive, stable and inherited differences in the biological states of individual cancer cells.

Limited wedge resection for T1 colon cancer (LIMERIC-II trial) - rationale and study protocol of a prospective multicenter clinical trial.

BACKGROUND: The sole presence of deep submucosal invasion is shown to be associated with a limited risk of lymph node metastasis. This justifies a local excision of suspected deep submucosal invasive colon carcinomas (T1 CCs) as a first step treatment strategy. Recently Colonoscopy-Assisted Laparoscopic Wedge Resection (CAL-WR) has been shown to be able to resect pT1 CRCs with a high R0 resection rate, but the long term outcomes are lacking. The aim of this study is to evaluate the safety, effectiveness and long-term oncological outcomes of CAL-WR as primary treatment for patients with suspected superficial and also deeply-invasive T1 CCs. METHODS: In this prospective multicenter clinical trial, patients with a macroscopic and/or histologically suspected T1 CCs will receive CAL-WR as primary treatment in order to prevent unnecessary major surgery for low-risk T1 CCs. To make a CAL-WR technically feasible, the tumor may not include > 50% of the circumference and has to be localized at least 25 cm proximal from the anus. Also, there should be sufficient distance to the ileocecal valve to place a linear stapler. Before inclusion, all eligible patients will be assessed by an expert panel to confirm suspicion of T1 CC, estimate invasion depth and subsequent advise which local resection techniques are possible for removal of the lesion. The primary outcome of this study is the proportion of patients with pT1 CC that is curatively treated with CAL-WR only and in whom thus organ-preservation could be achieved. Secondary outcomes are 1) CAL-WR's technical success and R0 resection rate for T1 CC, 2) procedure-related morbidity and mortality, 3) 5-year overall and disease free survival, 4) 3-year metastasis free survival, 5) procedure-related costs and 6) impact on quality of life. A sample size of 143 patients was calculated. DISCUSSION: CAL-WR is a full-thickness local resection technique that could also be effective in removing pT1 colon cancer. With the lack of current endoscopic local resection techniques for > 15 mm pT1 CCs with deep submucosal invasion, CAL-WR could fill the gap between endoscopy and major oncologic surgery. The present study is the first to provide insight in the long-term oncological outcomes of CAL-WR. TRIAL REGISTRATION: CCMO register (ToetsingOnline), NL81497.075.22, protocol version 2.3 (October 2022).

Colonoscopic-Assisted Laparoscopic Wedge Resection for Colonic Lesions: A Prospective Multicenter Cohort Study (LIMERIC-Study).

OBJECTIVE: The aim of this study was to evaluate the safety and efficacy of a modified CAL-WR. SUMMARY BACKGROUND DATA: The use of segmental colectomy in patients with endoscopically unresectable colonic lesions results in significant morbidity and mortality. CAL-WR is an alternative procedure that may reduce morbidity. METHODS: This prospective multicenter study was performed in 13 Dutch hospitals between January 2017 and December 2019. Inclusion criteria were (1) colonic lesions inaccessible using current endoscopic resection techniques (judged by an expert panel), (2) non-lifting residual/recurrent adenomatous tissue after previous polypectomy or (3) an undetermined resection margin after endoscopic removal of a low-risk pathological T1 (pT1) colon carcinoma. Thirty-day morbidity, technical success rate and radicality were evaluated. RESULTS: Of the 118 patients included (56% male, mean age 66 years, standard deviation ± 8 years), 66 (56%) had complex lesions unsuitable for endoscopic removal, 34 (29%) had non-lifting residual/recurrent adenoma after previous polypectomy and 18 (15%) had uncertain resection margins after polypectomy of a pT1 colon carcinoma. CAL-WR was technically successful in 93% and R0 resection was achieved in 91% of patients. Minor complications (Clavien-Dindo i-ii) were noted in 7 patients (6%) and an additional oncologic segmental resection was performed in 12 cases (11%). Residual tissue at the scar was observed in 5% of patients during endoscopic follow-up. CONCLUSIONS: CAL-WR is an effective, organ-preserving approach that results in minor complications and circumvents the need for major surgery. CAL-WR, therefore, deserves consideration when endoscopic excision of circumscribed lesions is impossible or incomplete.

Long-term Outcome of Radical Excision Versus Phenolization of the Sinus Tract in Primary Sacrococcygeal Pilonidal Sinus Disease: A Randomized Controlled Trial.

BACKGROUND: Phenolization of pilonidal sinus disease has been shown to have advantages over radical excision with regard to short-term outcome; however, long-term outcomes are essentially lacking. OBJECTIVE: The aim of this randomized controlled trial was to compare the long-term outcome of pit excision and phenolization of the sinus tracts vs radical excision with primary wound closure in pilonidal sinus disease. DESIGN: Single-center, randomized controlled trial. SETTINGS: A primary teaching hospital in the Netherlands. PATIENTS: The study population included patients with primary pilonidal sinus disease presented between 2013 and 2017. INTERVENTIONS: Patients were randomly assigned to either pit excision with phenolization of the sinus tract(s) or excision with primary off-midline wound closure. MAIN OUTCOME MEASURES: The main outcomes included recurrence, quality of life (Short-Form 36), and patient's satisfaction. RESULTS: A total of 100 patients were randomized. Seventy-four patients (77.1%) were available for long-term follow-up. The mean (±SD) time to follow-up was 48.4 (±12.8) months for the phenolization group and 47.8 (±13.5) months for the excision group. No significant difference was found between both groups regarding quality of life. Two patients in the phenolization group (5.6%) and 1 in the excision group (2.6%) developed a recurrence ( p = 0.604). The impact of the whole treatment was significantly less after phenolization ( p = 0.010). LIMITATIONS: The response rate was almost 80% in this young patient population, patients and assessors were not blinded for the type of surgery, and the results are only applicable to primary pilonidal sinus disease. CONCLUSIONS: Because of the previously shown favorable short-term results and the currently reported comparable long-term recurrence rate and quality of life between phenolization and excision, phenolization should be considered the primary treatment option in patients with pilonidal sinus disease. See Video Abstract at http://links.lww.com/DCR/C27 . DUTCH TRIAL REGISTER ID: NTR4043. RESULTADO A LARGO PLAZO DE LA ESCISIN RADICAL FRENTE AL TRATAMIENTO CON FENOL DEL TRACTO SINUSAL EN LA ENFERMEDAD DEL SENO PILONIDAL SACRO COCCGEO PRIMARIO UN ENSAYO ALEATORIO CONTROLADO: ANTECEDENTES:El tratamiento con fenol de la enfermedad del seno pilonidal ha demostrado tener ventajas sobre la escisión radical con respecto al resultado a corto plazo; sin embargo, los resultados a largo plazo aún se encuentran escasos.OBJETIVO:El objetivo de este ensayo aleatorio controlado fue comparar el resultado a largo plazo de la escisión de la fosa del quiste y el tratamiento con fenol de los trayectos sinusales frente a la escisión radical con cierre primario de la herida en la enfermedad del seno pilonidal.DISEÑO:Ensayo aleatorio controlado de un solo centro.AJUSTES:Hospital de enseñanza primaria en los Países Bajos.PACIENTES:Pacientes con enfermedad primaria del seno pilonidal presentados entre 2013 y 2017.INTERVENCIONES:Los pacientes fueron asignados de manera aleatoria a la escisión de la fosa del quiste y posterior administración de fenol de los tractos sinusales o a la escisión con cierre primario de la herida fuera de la línea media.PRINCIPALES MEDIDAS DE RESULTADO:Recurrencia, calidad de vida (Short-Form 36) y satisfacción del paciente.RESULTADOS:Un total de 100 pacientes con enfermedad primaria del seno pilonidal fueron aleatorizados; 50 pacientes fueron sometidos al tratamiento con fenol y 50 a la escisión radical. Eventualmente, 74 pacientes (77,1%) estuvieron disponibles para seguimiento a largo plazo; 36 pacientes después del uso del fenol y 38 después de la escisión. El tiempo medio (± desviación estándar) de seguimiento fue de 48,4 (± 12,8) y 47,8 (± 13,5) meses, respectivamente. No hubo diferencia significativa entre ambos grupos con respecto a la calidad de vida. En el grupo tratado con fenal, dos pacientes (5,6%) desarrollaron recurrencia y un paciente (2,6%) en el grupo de escisión ( p = 0,604). El impacto de todo el tratamiento fue significativamente menor después del uso del fenol (p = 0,010).LIMITACIONES:La tasa de respuesta fue de casi el 80% en esta población de pacientes jóvenes, los pacientes y los evaluadores no estaban cegados por el tipo de cirugía, los resultados son solo aplicables a la enfermedad primaria del seno pilonidal.CONCLUSIONES:Debido a los resultados favorables a corto plazo descritos y a la tasa de recurrencia a largo plazo y la calidad de vida comparables actualmente informadas entre la administración de fenol y la escisión con cierre primario de la herida para la enfermedad primaria del seno pilonidal, la administración de fenol del tracto sinusal debe considerarse como opción de tratamiento primario en pacientes con enfermedad del seno pilonidal. Consulte Video Resumen en http://links.lww.com/DCR/C27 . (Traducción-Dr. Osvaldo Gauto )Registro de prueba holandés-ID:NTR4043.

Long-term Results of a Randomized Double-blinded Prospective Trial of a Lightweight (Ultrapro) Versus a Heavyweight Mesh (Prolene) in Laparoscopic Total Extraperitoneal Inguinal Hernia Repair (TULP-trial).

OBJECTIVE: The aim of the randomized clinical trial was to compare the 2 years of clinical outcomes of a lightweight (Ultrapro) vs a heavyweight (Prolene) mesh for laparoscopic total extraperitoneal (TEP) inguinal hernia repair. BACKGROUND: Lightweight meshes reduce postoperative pain and stiffness in open anterior inguinal hernia repair. The discussion about a similar benefit for laparoscopic repair is ongoing, but concerns exist about higher recurrence rates. METHODS: Between March 2010 and October 2012, male patients who presented with a primary, reducible unilateral inguinal hernia who underwent day-case TEP repair were eligible. Outcome parameters included chronic pain, recurrence, foreign body feeling, and quality of life scores. RESULTS: During the study period, 950 patients were included. One year postoperatively the presence of relevant pain (Numeric Rating Score 4-10) was significantly higher in the lightweight mesh group (2.9%) compared with the heavyweight mesh group (0.7%) (P = 0.01), and after 2 years this difference remained significant (P = 0.03). There were 4 (0.8%) recurrent hernias in the heavyweight mesh group and 13 (2.7%) in the lightweight group (P = 0.03). No differences in foreign body feeling or quality of life scores were detected. CONCLUSIONS: In TEP hernia surgery, there was no benefit of lightweight over heavyweight meshes observed 2 years postoperatively.

Male infertility after endoscopic Totally Extraperitoneal (Tep) hernia repair (Main): rationale and design of a prospective observational cohort study.

BACKGROUND: To describe the rationale and design of an observational cohort study analyzing the effects of endoscopic Totally Extraperitoneal (TEP) hernia repair on male fertility (MAIN study). METHODS AND DESIGN: The MAIN study is an observational cohort study designed to assess fertility after endoscopic TEP hernia repair. The setting is a high-volume single center hospital, specialized in TEP hernia repair. Male patients of 18-60 years of age, with primary, reducible, bilateral inguinal hernias and no contraindications for endoscopic TEP repair are eligible for inclusion in this study. Patients with an ASA-classification≥III and patients with recurrent and/or scrotal hernias and/or a medical history of pelvic surgery and/or radiotherapy, known fertility problems, diabetes and/or other diseases associated with a risk of fertility problems, will be excluded. The primary outcome is the testicular perfusion before and 6 months after TEP hernia repair (assessed by means of a scrotal ultrasonography). Secondary endpoints are the testicular volume (Ultrasound), semen quality and quantity and the endocrinological status, based on serum levels of the sexual hormones follicle-stimulating hormone (FSH), luteinizing hormone (LSH), testosterone and inhibin B before and 6 months after TEP hernia repair. DISCUSSION: The use of polypropylene mesh is associated with a strong foreign body reaction which could play a role in chronic groin pain development. Since the mesh in (endoscopic) inguinal hernia repair is placed in close contact to the vas deferens and spermatic vessels, the mesh-induced inflammatory reaction could lead to a dysfunction of these structures. Relevant large and prospective clinical studies on the problem are limited. This study will provide a complete assessment of fertility in male patients who undergo simultaneous bilateral endoscopic TEP hernia repair, by analyzing testicular perfusion and volume, semen quantity and quality and endocrinological status before and 6 months after TEP repair. TRIAL REGISTRATION: The MAIN study is registered in the Dutch Trial Register (NTR2208).

Early laparoscopic cholecystectomy improves outcomes after endoscopic sphincterotomy for choledochocystolithiasis.

BACKGROUND & AIMS: Patients with choledochocystolithiasis generally undergo endoscopic sphincterotomy (ES) followed by laparoscopic cholecystectomy (LC). However, many patients receive this surgery 6-8 weeks after ES. There is a high conversion rate of elective LC after ES, and patients can develop recurrent biliary events during the waiting period. We investigated whether the timing of surgery influences outcome. METHODS: We performed a randomized trial of patients with choledochocystolithiasis who underwent successful ES. Patients were randomly assigned to groups that received early LC (within 72 hours after ES, n = 49) or delayed LC (after 6-8 weeks, n = 47), based on an expected difference in conversion rate of 25% vs 5%, respectively. Conversion rate, biliary events during follow-up, duration and difficulty of surgeries, postoperative morbidity, and hospital stay were scored. Intention-to-treat analyses were performed. RESULTS: Groups were comparable in age, sex, and comorbidity. There was no difference between groups in conversion rate (4.3% in early vs 8.7% in delayed group) nor were there differences in operating times and/or difficulties or hospital stays. During the waiting period for LC, 17 patients in the delayed group (36.2%) developed recurrent biliary events compared with 1 patient in the early group (P < .001). CONCLUSIONS: In a randomized trial to evaluate timing of LC after ES, recurrent biliary events occurred in 36.2% of patients whose LC was delayed for 6-8 weeks. Early LC (within 72 hours) appears to be safe and might prevent the majority of biliary events in this period following sphincterotomy.

Oncogenic K-Ras turns death receptors into metastasis-promoting receptors in human and mouse colorectal cancer cells.

BACKGROUND & AIMS: Death receptors expressed on tumor cells can prevent metastasis formation by inducing apoptosis, but they also can promote migration and invasion. The determinants of death receptor signaling output are poorly defined. Here we investigated the role of oncogenic K-Ras in determining death receptor function and metastatic potential. METHODS: Isogenic human and mouse colorectal cancer cell lines differing only in the presence or absence of the K-Ras oncogene were tested in apoptosis and invasion assays using CD95 ligand and tumor necrois factor-related apoptosis-inducing ligand (TRAIL) as stimuli. Metastatic potential was assessed by intrasplenic injections of green fluorescent protein- or luciferase-expressing tumor cells, followed by intravital fluorescence microscopy or bioluminescence imaging, and confocal microscopy and immunohistochemistry. Ras-effector pathway control of CD95 output was assessed by an RNA-interference and inhibitor-based approach. RESULTS: CD95 ligand and TRAIL stimulated invasion of colorectal tumor cells and liver metastases in a K-Ras-dependent fashion. Loss of mutant K-Ras switched CD95 and TRAIL receptors back into apoptosis mode and abrogated metastatic potential. Raf1 was essential for the switch in CD95 function, for tumor cell survival in the liver, and for K-Ras-driven formation of liver metastases. K-Ras and Raf1 suppressed Rho kinase (ROCK)/LIM kinase-mediated phosphorylation of the actin-severing protein cofilin. Overexpression of ROCK or LIM kinase allowed CD95L to induce apoptosis in K-Ras-proficient cells and prevented metastasis formation, whereas their suppression protected K-Ras-deficient cells against apoptosis. CONCLUSIONS: Oncogenic K-Ras and its effector Raf1 convert death receptors into invasion-inducing receptors by suppressing the ROCK/LIM kinase pathway, and this is essential for K-Ras/Raf1-driven metastasis formation.

Phenolisation of the Sinus Tract in Recurrent Sacrococcygeal Pilonidal Sinus Disease: A Prospective Cohort Study.

Purpose Phenolisation is a minimally invasive treatment option in patients with primary pilonidal disease. However, most studies focus on patients with primary pilonidal sinus disease, while data of patients with recurrent pilonidal disease are very scarce. The purpose of this study was to evaluate phenolisation of the sinus tract in patients with recurrent pilonidal sinus disease after previous surgery for SPSD. Methods This single-center prospective cohort study included 60 patients with recurrent pilonidal disease. Loss of days of normal daily activities, surgical site infection, wound epithelization, quality of life, and complaints related to pilonidal disease were postoperatively assessed. Results A total of 57 patients (95%) were treated with phenolisation and the median loss of days of normal daily activities was 5.0 (1.0 - 12.0) days. Fifty-one patients (89.5%) resumed normal daily activities after two weeks. Surgical site infection occurred in five patients (8.8%). Compared to preoperative scores, quality of life was significantly higher 12 weeks postoperatively (p=0.014) and pain and itch scores were lower after six and 12 weeks (p ≤ 0.005). Wounds were completely healed in 45 of 51 patients (89.8%) who were available after 12 weeks of follow-up. Conclusion Phenolisation for recurrent pilonidal disease is safe with a median complete return to daily activities within five days and complete wound healing after three months in 90%. Therefore, phenolisation should be considered as a treatment option in patients with recurrent pilonidal sinus disease.

Sexual Function in Patients Suffering from Sacrococcygeal Pilonidal Sinus Disease.

Introduction Sexual function is one of the aspects upon which quality of life (QoL) is based. Although previous studies have evaluated the influence of sacrococcygeal pilonidal sinus disease (SPSD) on QoL, no data are available on the influence of SPSD on sexual function in a highly active sexual population based on the age range. The aim of this prospective study was to evaluate whether SPSD has a negative impact on sexual function and whether this is influenced by the surgical treatment of SPSD. Methods  Sexual function was pre- and postoperatively assessed by the Sexual Self-Consciousness Scale (SSCS; score range 0-48), subdivided into the sexual embarrassment (SE; score range 0-24) and sexual self-focus subscale (SFF; score range 0-24). The higher the score, the higher is the sexual dysfunction. Patients were also asked whether SPSD influenced their sexual functioning. Results  A total of 88 male patients who underwent surgical treatment for SPSD were included in the study. The mean (±SD) preoperative SSCS score was 14.5±9.1 and 13.9±8.4 two weeks postoperatively (p=0.394). Six and twelve weeks after surgery, there was a significant reduction to 12.2±9.0 (p=0.002) and 12.3±8.8 (p=0.013), respectively. SE decreased from 5.5±5.1 preoperatively to 5.1±4.6 (p=0.258), 4.2±4.7 (p=0.004) and 4.0±4.6 (p=0.013) two, six, and twelve weeks after surgery. For SFF, there was a decrease from 9.0±5.0 to 8.9±4.9 (p=0.717), 7.8±5.2 (p=0.004) and 8.2±5.3 (p=0.168), respectively. Preoperatively, 70% of the patients totally or partially disagreed that SPSD influenced their sexual functioning, and this increased to 80% of the patients 12 weeks after surgery. Conclusion  This prospective study showed a significant decrease in sexual dysfunction, both six and twelve weeks after surgery, compared to preoperatively in patients suffering from SPSD.

Single-cell derived tumor organoids display diversity in HLA class I peptide presentation.

Tumor heterogeneity is a major cause of therapeutic resistance. Immunotherapy may exploit alternative vulnerabilities of drug-resistant cells, where tumor-specific human leukocyte antigen (HLA) peptide ligands are promising leads to invoke targeted anti-tumor responses. Here, we investigate the variability in HLA class I peptide presentation between different clonal cells of the same colorectal cancer patient, using an organoid system. While clone-specific differences in HLA peptide presentation were observed, broad inter-clone variability was even more prevalent (15-25%). By coupling organoid proteomics and HLA peptide ligandomics, we also found that tumor-specific ligands from DNA damage control and tumor suppressor source proteins were prominently presented by tumor cells, coinciding likely with the silencing of such cytoprotective functions. Collectively, these data illustrate the heterogeneous HLA peptide presentation landscape even within one individual, and hint that a multi-peptide vaccination approach against highly conserved tumor suppressors may be a viable option in patients with low tumor-mutational burden.

[Chronic pilonidal sinus disease: overview of treatment options]. / Chronische sinus pilonidalis.

Chronic pilonidal sinus disease: overview of treatment options Pilonidal sinus disease is a common disease in predominantly young men; this condition can have a significant socio-economic impact. Although excision with primary or secondary wound healing is often applied, there is no consensus on the treatment of first choice for pilonidal sinus disease. Minimally invasive techniques, such as phenolisation, laser, and endoscopic treatments are increasingly applied, since these are associated with less pain, faster wound healing and facilitating a more rapid return to work for patients. Skin advancement flap techniques, considering they cause morbidity and large wounds, should only be considered in case of recurrent pilonidal sinus disease. Laser hair removal at the natal cleft after surgical treatment appears to reduce the risk of recurrence.

Differential Notch and TGFbeta signaling in primary colorectal tumors and their corresponding metastases.

BACKGROUND: Loss of epithelial morphology and the acquisition of mesenchymal characteristics may contribute to metastasis formation during colorectal tumorigenesis. The Wnt, Notch and TGFbeta signaling pathways control tissue homeostasis and tumor development in the gut. The relationship between the activity of these pathways and the expression of epithelial and mesenchymal markers was investigated in a series of primary colorectal tumors and their corresponding metastases. METHODS: Tissue samples of primary colorectal tumors, normal colonic mucosa, and regional and systemic metastases were processed for immunohistochemistry in a tissue microarray format. The expression of mesenchymal (vimentin, fibronectin) and epithelial (E-cadherin) markers was related to markers of Wnt (beta-catenin), Notch (HES1) and TGFbeta (phospho-SMAD2) signalling. In addition, the KRAS mutation status was assessed. RESULTS: When compared to normal mucosa, primary colorectal tumors showed a marked increase in the levels of cytoplasmic vimentin and nuclear beta-catenin, phospho-SMAD2 and HES1. Increased vimentin expression correlated with the presence of oncogenic KRAS and with nuclear beta-catenin. The corresponding liver, lymph node, brain and lung metastases did not express vimentin and displayed significantly lower levels of nuclear phospho-SMAD2 and HES1, while retaining nuclear beta-catenin. CONCLUSIONS: Primary colorectal carcinomas display aberrant expression of vimentin, and have activated Notch and TGFbeta signaling pathways. Surprisingly, many regional and distant metastases have lost nuclear HES1 and pSMAD2, suggesting that the activity of the Notch and TGFbeta pathways is reduced in secondary colorectal tumors.

KRAS(D13) Promotes apoptosis of human colorectal tumor cells by ReovirusT3D and oxaliplatin but not by tumor necrosis factor-related apoptosis-inducing ligand.

Colorectal tumors frequently contain activating mutations in KRAS. ReovirusT3D is an oncolytic virus that preferentially kills tumor cells with an activated Ras pathway. Here we have assessed the contribution of endogenous mutant KRAS in human colorectal cancer cell lines to ReovirusT3D replication and to tumor cell oncolysis. In addition, treatment combinations involving ReovirusT3D, oxaliplatin, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were tested for their efficacy in tumor cell killing. The mutation status of KRAS did not predict the sensitivity of a panel of human colorectal cancer cell lines to ReovirusT3D. Virus replication was observed in all cell lines tested regardless of KRAS status and was not affected by deletion of endogenous mutant KRAS(D13). However, deletion of KRAS(D13) or p53 did reduce apoptosis induction by ReovirusT3D whereas deletion of beta-catenin(DeltaS45) had no effect. Likewise, KRAS(D13)- or p53-deficient cells display reduced sensitivity to oxaliplatin but not to death receptor activation by TRAIL. Finally, the treatment of colorectal cancer cells with ReovirusT3D combined with either oxaliplatin or TRAIL resulted in a nonsynergistic increase in tumor cell killing. We conclude that oncolysis of human tumor cells by ReovirusT3D is not determined by the extent of virus replication but by their sensitivity to apoptosis induction. Oncogenic KRAS(D13) increases tumor cell sensitivity to activation of the cell-intrinsic apoptosis pathway without affecting ReovirusT3D replication.

Dual effect of Kras(D12) knockdown on tumorigenesis: increased immune-mediated tumor clearance and abrogation of tumor malignancy.

Activating mutations in the human KRAS proto-oncogene are acquired during the earliest stages of colorectal cancer development. If mutant KRAS is to be used as a target for therapy in colorectal cancer, tumor growth should depend on its continued presence. Here, we report that stable knockdown of Kras(D12) in murine C26 colorectal cancer cells by RNA interference resulted in loss of transformed properties in vitro. The incidence of subcutaneous tumor formation was reduced by 60% and the lag time was increased sevenfold. Kras(D12)-knockdown tumors grew noninvasively and did not cause morbidity. Remarkably, some of the Kras(D12)-knockdown tumors regressed spontaneously, which rendered these mice resistant to parental C26 tumor growth. In immune-deficient hosts, the incidence of tumor formation by Kras(D12)-knockdown cells was 100%. None of these tumors regressed spontaneously. We conclude that the reduced incidence of tumor formation by Kras(D12)-knockdown cells is due to tumor cell clearance by the host immune system, but not to an intrinsic inability of these cells to grow out as tumors. Interestingly, Kras(D12) knockdown resulted in increased production of interleukin 18 (Il-18), an immune-stimulatory cytokine that has been implicated in limiting colorectal tumor formation. Thus, mutant Kras(D12) suppresses Il-18 production in colorectal tumor cells, which may contribute to evasion of the local immune system during tumor development.

Control of colorectal metastasis formation by K-Ras.

Mutational activation of the K-Ras proto-oncogene is frequently observed during the very early stages of colorectal cancer (CRC) development. The mutant alleles are preserved during the progression from pre-malignant lesions to invasive carcinomas and distant metastases. Activated K-Ras may therefore not only promote tumor initiation, but also tumor progression and metastasis formation. Metastasis formation is a very complex and inefficient process: Tumor cells have to disseminate from the primary tumor, invade the local stroma to gain access to the vasculature (intravasation), survive in the hostile environment of the circulation and the distant microvascular beds, gain access to the distant parenchyma (extravasation) and survive and grow out in this new environment. In this review, we discuss the potential influence of mutant K-Ras on each of these phases. Furthermore, we have evaluated the clinical evidence that suggests a role for K-Ras in the formation of colorectal metastases.

Cyclooxygenase-2 is a target of KRASD12, which facilitates the outgrowth of murine C26 colorectal liver metastases.

PURPOSE: Mutational activation of the KRAS oncogene and overexpression of cyclooxygenase-2 (COX-2) contribute to colorectal carcinoma (CRC) development, but the relationship between these two events is unclear. This study was designed to clarify that relationship and to assess the contribution of KRAS-dependent COX-2 to the seeding of CRC cells in the liver and to their outgrowth as liver metastases in an experimental mouse model. EXPERIMENTAL DESIGN: The effect of RNA interference-mediated KRAS knockdown on COX-2 expression and activity was tested in murine C26 CRC cells. The contribution of KRAS-dependent COX-2 to early metastatic tumor cell seeding (by intravital microscopy) and outgrowth of metastases in the liver (by bioluminescence imaging) was studied by using parecoxib, a novel and highly selective liver-activated COX-2 inhibitor. Intratumoral cell proliferation, apoptosis, and tumor-associated angiogenesis were assessed by immunohistochemistry on liver tissue sections. RESULTS: Stable knockdown of mutant KRAS(D12) in murine C26 CRC cells by RNA interference lead to a dramatic reduction of COX-2 synthesis and prostaglandin E2 production. Inhibition of host or tumor cell COX-2 activity had no effect on early metastatic cell seeding in the liver but greatly reduced intrahepatic tumor cell proliferation and the rate of liver metastasis outgrowth. COX-2 inhibition had no effect on early tumor vascularization or on tumor cell apoptosis. CONCLUSIONS: The high levels of COX-2 enzyme and prostaglandin production in C26 CRC cells are primarily caused by the presence of endogenous mutant KRAS(D12). Furthermore, COX-2 inhibition affects the tumoral rather than the vascular compartment during the early stages of C26 liver metastasis outgrowth.

Combined surgery for primary colorectal cancer and synchronous pulmonary metastasis: a pilot experience in two patients.

BACKGROUND: Pulmonary metastasectomy in patients with pulmonary metastases from primary colorectal cancer seems to improve survival in properly selected patients. Therefore, pulmonary metastasectomy has been incorporated widely into the management of colorectal pulmonary metastases. Generally, in patients presenting with primary colorectal cancer and synchronous pulmonary metastases, the primary colorectal cancer is resected first, followed by pulmonary metastasectomy during a second-stage procedure. In the current paper we describe our pilot experience with laparoscopic resection of primary colorectal cancer and thoracoscopic pulmonary metastasectomy during the same operative session. PATIENTS AND METHODS: The results of two patients who underwent laparoscopic resection of primary colorectal cancer and thoracoscopic pulmonary metastasectomy during the same operative session are described. RESULTS: Both patients were healthy women, 60 and 81 years old, respectively, and without severe comorbidities. In both patients, the colorectal resection was performed first by a laparoscopic approach. Subsequently, thoracoscopic resection of a single pulmonary metastasis followed in both patients. The operative procedure and postoperative course were uneventful and the patients could be discharged within 1 week after surgery. Both the primary colorectal cancer and the pulmonary metastasis were radically removed in both patients. Current follow-up, 14 and 8 months after surgery, respectively, showed no signs of disease recurrence on computed tomographic scan of the abdomen and chest in both patients. CONCLUSION: The outcome in these two patients suggests that simultaneous resection of primary colorectal cancer and pulmonary metastasectomy using minimal invasive surgery is safe and might lead to both a decrease in costs and benefit to patients. This simultaneous approach could therefore be considered as an alternative for a two-stage approach in properly selected patients. However, these results should be validated in a larger series.

Enterobius vermicularis infection of the liver in a patient with colorectal carcinoma with suspected liver metastasis.

A 68-year-old man diagnosed with cT3N2 adenocarcinoma of the rectum presented with a synchronous solitary liver metastasis on CT scan. Neoadjuvant chemoradiotherapy was started to downstage the primary tumour. Resection of the rectal tumour followed 3 months after the last radiotherapy session and primary resection of the isolated liver lesion was performed in the intervening period. Histopathological assessment of the liver lesion, however, showed no malignancy, but did reveal a necrotic infection due to Enterobius vermicularis. This parasite is frequently found in the intestines, but only rarely infects the liver. The patient was subsequently treated with the anthelmintic drug mebendazole 100 mg once a week for 2 weeks. Histopathological assessment of the rectal specimen showed complete regression after neoadjuvant chemoradiotherapy without evidence of remaining E. vermicularis, suggesting pinworm eradication. The patient recovered promptly after both surgical procedures.