Cadaveric Study of Colon-Lengthening Maneuvers After Sigmoidectomy.

BACKGROUND: Creation of a tension-free colorectal anastomosis after left colon resection or low anterior resection is a key requirement for technical success. The relative contribution of each of a series of known lengthening maneuvers remains incompletely characterized. OBJECTIVE: The aim of this study was to compare technical procedures for lengthening of the left colon before rectal anastomosis. DESIGN: A series of lengthening maneuvers was performed on 15 fresh cadavers. Mean distance gained was measured for each successive maneuver, including 1) high inferior mesenteric artery ligation, 2) splenic flexure takedown, and 3) high inferior mesenteric vein ligation by the ligament of Treitz. SETTING: Cadaveric study. MAIN OUTCOME MEASURES: The premobilization and postmobilization position of the proximal colonic end was measured relative to the inferior edge of the sacral promontory. Measurements of the colonic length relative to the sacral promontory were taken after each mobilization maneuver. The inferior mesenteric artery, sigmoid colon, and rectum specimen lengths were measured. The distance from the inferior border of the sacral promontory to the pelvic floor was measured along the sacral curvature. RESULTS: Mean sigmoid colon resection length was 34.7 ± 11.1 cm. Before any lengthening, baseline reach was -1.3 ± 4.2 cm from the sacral promontory. Inferior mesenteric artery ligation yielded an additional 11.5 ± 4.7 cm. Subsequent splenic flexure takedown added an additional 12.8 ± 9.6 cm. Finally, inferior mesenteric vein ligation added an additional 11.33 ± 6.9 cm, bringing the total colonic length to 35.7 ± 14.7 cm. BMI and weight negatively correlated with length gained. LIMITATIONS: The study was limited by nature of being a cadaver study. CONCLUSIONS: Stepwise lengthening maneuvers allow significant additional reach to allow a tension-free left colon to rectal anastomosis. See Video Abstract . ESTUDIO CADAVRICO DE MANIOBRAS DE ALARGAMIENTO COLNICO TRAS UNA SIGMOIDECTOMA: ANTECEDENTES:La creación de una anastomosis colorrectal libre de tensión tras una resección de colon izquierdo o tras una resección anterior baja es un requisito clave para el éxito relacionado con la técnica quirúrgica. La relativa contribución de las diversas maniobras de alargamiento permanece caracterizada de manera incompleta.OBJETIVO:El propósito de este estudio fue la de comparar procedimientos técnicos de alargamiento del colon izquierdo previo a la anastomosis rectal.DISEÑO:Una serie de maniobras de alargamiento fueron realizados en 15 cadáveres frescos. La distancia promedio ganada fue medida para cada maniobra sucesiva, incluyendo (1) ligadura alta de la arteria mesentérica inferior, (2) descenso del ángulo esplénico, (3) ligadura alta de la vena mesentérica interior mediante el ligamento de Treitz.AJUSTES:Estudio cadavérico.PRINCIPALES MEDIDAS DE RESULTADO:La posición premobilizacion y postmobilizacion del extremo proximal del colon fue medido tomando en cuenta el borde inferior del promontorio sacro. Las mediciones de la longitud colónica en relación al sacro fueron tomadas luego de cada maniobra de movilización. Fueron tomadas así mismo las longitudes de la arteria mesentérica inferior, el colon sigmoides y recto. Las distancias desde el borde inferior del promontorio sacro al suelo pelvico fueron medidas a lo largo de la curvatura sacra.RESULTADOS:Average sigmoid colon resection length was 34.7 ± 11.1 cm. Prior to any lengthening, baseline reach was -1.3 ± 4.2 cm from the sacral promontory. Inferior mesenteric artery ligation yielded an additional 11.5 ± 4.7 cm. Subsequent splenic flexure takedown added an additional 12.8 ± 9.6 cm. Finally, inferior mesenteric vein ligation added an additional 11.33 ± 6.9 cm, bringing the total colonic length to 35.7 ± 14.7 cm. BMI and weight negatively correlated with length gained.LIMITACIONES:Este estudio tuvo como limitación la naturaleza de haber sido un estudio cadavérico.CONCLUSIONES:Maniobras de alargamiento permiten un alcance adicional significativo permitiendo de esta manera una anastomosis de colon izquierdo a recto libre de anastomosis. (Traducción-Dr Osvaldo Gauto ).

Pathologic Outcomes of Short-Course and Long-Course Radiotherapy for Locally Advanced Rectal Cancers Treated With Total Neoadjuvant Therapy.

INTRODUCTION: Total neoadjuvant therapy (TNT) for patients with locally advanced rectal cancer (LARC) is now the standard of care. Randomized trials suggest the use of short-course radiotherapy (SCRT) and long-course radiotherapy (LCRT) are oncologically equivalent. OBJECTIVE: To describe pathologic outcomes after surgical resections of patients receiving SCRT versus LCRT as part of TNT for LARC. PARTICIPANTS: All patients with LARC treated at a single tertiary hospital who underwent proctectomy after completing TNT were included. Patients were excluded if adequate details of TNT were not available in the electronic medical record. RESULTS: A total of 53 patients with LARC were included. Thirty-nine patients (73.5%) received LCRT and 14 (26.4%) received SCRT. Forty-nine patients (92.5%) were clinical stage III (cN1-2) prior to treatment. The average lymph node yield after proctectomy was 20.9 for SCRT and 17.0 for LCRT (P = .075). Of the 49 patients with clinically positive nodes before treatment, 76.9% of those who received SCRT and 72.2% of those who received LCRT achieved pN0 disease after TNT. Additionally, there were no significant differences in rates of pathologic complete response between patients who received SCRT and LCRT, 7.1% and 12.8%, respectively (P = .565). CONCLUSION: Pathologic outcomes of patients with LARC treated with SCRT or LCRT, as part of TNT, may be similar. Further prospective trials are needed to assess long-term clinical outcomes and to determine best treatment protocols.

Assessing the Association Between Endoscopic Tattooing and Lymph Node Yield in Rectal Cancer.

INTRODUCTION: Preoperative endoscopic tattooing is an effective tool for intraoperative tumor localization in colon cancer. Endoscopic tattooing in rectal cancer may have unidentified benefits on lymph node yield, making it easier for pathologists to identify nodes during histopathologic assessment. There remains concern that tattoo ink may alter anatomical planes, increasing surgical difficulty. METHODS: Retrospective chart reviews from 2016 to 2021 of n = 170 patients presenting with rectal cancer were divided into two groups: with (n = 79) and without (n = 91) endoscopic tattoos. Demographics, operative details, tumor characteristics, prior chemoradiation, and pathologic details were collected. Primary outcome was total lymph node yield. Secondary outcomes were rates of adequate (> 12) nodes, margin status, and operative variables including operative time. RESULTS: No differences between pathologic stage, tumor height, high inferior mesenteric artery ligation, operative times, conversion rate, or surgical approach (open versus minimally invasive) were noted between groups. Receipt of neoadjuvant chemoradiation was less frequent in the endoscopic tattooing group (53.2% versus 76.9%, P ≤ 0.001). Total node number and rate of adequate lymph node yield were higher with endoscopic tattooing (20.5 ± 7.6 versus 16.8 ± 6.6 lymph nodes and 100.0% versus 83.5% adequate lymph node harvest, both P ≤ 0.001). Rates of positive circumferential and distal margins and complete total mesorectal excision were also similar. Regression analysis identified endoscopic tattooing (Incidence Risk Ratio 1.17, 95% confidence interval 1.04-1.31) and operative time more than 300 min (Incidence Risk Ratio 0.88, 95% confidence interval 0.77-0.99) had significant effects on lymph node harvest. Removal of patients with inadequate lymph node yield resulted in similar rates of total and positive lymph nodes. CONCLUSIONS: Endoscopic rectal tattooing is associated with increased lymph node yield (including after neoadjuvant chemoradiotherapy) without sacrificing oncologic or perioperative outcomes, although this effect is inconsistent when only considering patients with an adequate lymph node yield.

"Unresectable" polyp management utilizing advanced endoscopic techniques results in high rate of colon preservation.

PURPOSE: "Endoscopically unresectable" benign polyps identified during screening colonoscopy are often referred for segmental colectomy. Application of advanced endoscopic techniques can increase endoscopic polyp resection, sparing patients the morbidity of colectomy. This retrospective case-control study aimed to evaluate the success of colon preserving resection of "endoscopically unresectable" benign polyps using advanced endoscopic techniques including endoscopic mucosal resection, endoscopic submucosal dissection, endoluminal surgical intervention, full-thickness laparo-endoscopic excision, and combined endo-laparoscopic resection. METHODS: A prospectively maintained institutional database identified 95 patients referred for "endoscopically unresectable" benign polyps from 2015 to 2018. Cases were compared to 190 propensity score matched controls from the same database undergoing elective laparoscopic colectomy for other reasons. Primary outcome was rate of complete endoscopic polyp removal. Secondary outcomes included length of stay, unplanned 30-day readmission and reoperation, 30-day mortality, and post-procedural complications. RESULTS: Advanced endoscopic techniques achieved complete polyp removal without colectomy in 66 patients (70%). Failure was most commonly associated with previously attempted endoscopic resection and occult malignancy. Compared with matched colectomy controls, endoscopic polyp resection resulted in significantly shorter hospital length of stay (1.13 ± 2.41 vs 3.89 ± 4.57 days; p < 0.001), lower unplanned 30-day readmission (1.1% vs 7.7%; p < 0.05), and fewer postoperative complications (4.2% vs 33.9%; p < 0.001). Unplanned 30-day reoperation (2.1% vs 4.4%; p = 0.34) and 30-day mortality (0% vs 0.6%; p = 0.75) trended lower. CONCLUSIONS: Endoscopic resection of complex polyps can be highly successful, and it is associated with favorable outcomes and decreased morbidity when compared with segmental colon resection. Attempting colon preservation using these techniques is warranted.

Full-Thickness Laparoendoscopic Excision for Management of Complex Colon Polyps.

BACKGROUND: Benign colon polyps are increasingly being detected because of improved colonoscopic screening and early detection of masses on the adenoma-to-carcinoma pathway. Full-thickness laparoendoscopic excision is a colon-preserving technique for endoscopically unresectable polyps consisting of endoscopically guided nonanatomic wedge colectomy. OBJECTIVE: This study aimed to evaluate the safety and success of full-thickness laparoendoscopic excision compared to segmental colectomy for complex polyps not amenable to endoscopic resection. DESIGN: This is a retrospective case-control study. SETTINGS: This study was conducted at a tertiary academic center. PATIENTS: A prospectively maintained institutional database identified 22 patients with benign complex polyps managed with full-thickness laparoendoscopic excision from 2015 to 2020. These patients were compared with 22 propensity score-matched controls from the same database that underwent laparoscopic segmental colectomy. MAIN OUTCOME MEASURES: Primary outcome was inpatient length of stay. Secondary outcomes included operative details and postoperative morbidities. RESULTS: Full-thickness laparoendoscopic excision was successful in all patients. Patients had a median age of 64 years (41-85), and 82% were men. Final pathology revealed complete excision of benign lesions in 20 of 22 patients and adenocarcinoma in 2 of 22. For the adenocarcinomas, 1 patient underwent subsequent elective colectomy without complications, and 1 patient declined surgery. Propensity score matching was successful for age, sex, BMI, ASA score, colon location, and prior abdominal surgery. Compared with controls, cases had significantly shorter operative time (89.5 minutes (46-290) vs 122 minutes (85-200), p = 0.009), length of stay (1 day (0-17) vs 3 days (1-8), p < 0.001), and reduced blood loss (5 mL (2-15) vs 25 mL (10-150), p < 0.001). Thirty-day morbidity (9.1% vs 27.3%, p = 0.240) was not significantly different. An unplanned 30-day reoperation was performed in 1 patient for suspected small-bowel obstruction. There was 1 mortality due to decompensated cirrhosis in the treatment group. LIMITATIONS: This study was limited by its single-institution retrospective design. CONCLUSIONS: Full-thickness laparoendoscopic excision is safe and successful compared with corresponding segmental colectomy for complex polyps. Favorable postoperative outcomes, including decreased operative time, length of stay, and blood loss, make it a useful approach for managing complex polyps throughout the colon.

Prevalence of mesothelin expression in peritoneal disease from colorectal and appendiceal cancers.

BACKGROUND: Mesothelin is a cell surface glycoprotein overexpressed in 28%-58% of colorectal cancer (CRC). We hypothesized that CRC mesothelin expression contributes to peritoneal spread and that it is selectively overexpressed in those with peritoneal metastasis versus distant metastasis. METHODS: This case-controlled study involved mesothelin immunohistochemistry staining of tumor specimens from patients with metastatic CRC/appendiceal cancers between 2017 and 2019. Staining reactivity was graded from trace to 4+ (low ≤1+; high >1+). Staining patterns were characterized on global (focal/patchy/diffuse) and cellular (apical/cytoplasmic) levels. Immunostaining of normal mesothelial cells served as internal control. RESULTS: Thirty-one patients were identified: 11 peritoneal (study) and 20 distant metastasis (control). The control group did not include appendiceal cancers. The study group had greater proportion of high staining reactivity (55% vs. 5%; odds ratio [OR] = 20.4, 95% confidence interval [CI] 1.96-211.8). The study group had more diffuse (36% vs. 0%; OR = 22.2, 95% CI 1.1-465.3) and cytoplasmic staining patterns (73% vs. 28%; OR = 6.9, 95% CI 1.3-37.2). CONCLUSION: Mesothelin expression is higher in CRC/appendiceal cancers with peritoneal metastasis than those with distant metastasis. Immunohistochemistry staining patterns suggestive of propensity towards peritoneal metastasis include diffuse and cytoplasmic staining. Mesothelin may be a potential target for novel treatments of CRC/appendiceal carcinoma with peritoneal involvement.

Bleeding from Small Intestine: No Man's Land.

Bleedings from small intestine account for 5% of all gastrointestinal bleeding. With advanced endoscopic tools, such as video capsule endoscopy and deep enteroscopy, accurate diagnosis and treatment is possible in majority of cases with low mortality and morbidity. Nonoperative management includes endoscopic hemostasis and angiographic embolization. Recurrence after initial treatment is relatively common. Surgery is reserved for the cases that are refractory to endoscopic or angiographic treatment, bleeding from tumor or mass lesions, or hemodynamic instability. At the time of surgical exploration, unless the lesion has been marked by endoscopic tattoo or clip, intraoperative enteroscopy is often necessary to localize the lesion.

Engineering the shape and structure of materials by fractal cut.

In this paper we discuss the transformation of a sheet of material into a wide range of desired shapes and patterns by introducing a set of simple cuts in a multilevel hierarchy with different motifs. Each choice of hierarchical cut motif and cut level allows the material to expand into a unique structure with a unique set of properties. We can reverse-engineer the desired expanded geometries to find the requisite cut pattern to produce it without changing the physical properties of the initial material. The concept was experimentally realized and applied to create an electrode that expands to >800% the original area with only very minor stretching of the underlying material. The generality of our approach greatly expands the design space for materials so that they can be tuned for diverse applications.

The intestinal epithelial cell differentiation marker intestinal alkaline phosphatase (ALPi) is selectively induced by histone deacetylase inhibitors (HDACi) in colon cancer cells in a Kruppel-like factor 5 (KLF5)-dependent manner.

The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect.

Intestinal epithelial-specific PTEN inactivation results in tumor formation.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of phosphatidylinositol 3-kinase (PI3K) signaling that is frequently inactivated in colorectal cancer through mutation, loss of heterozygosity, or epigenetic mechanisms. The aim of this study was to determine the effect of intestinal-specific PTEN inactivation on intestinal epithelial homeostasis and tumorigenesis. PTEN was deleted specifically in the intestinal epithelium, by crossing PTEN(Lox/Lox) mice with villin(Cre) mice. PTEN was robustly expressed in the intestinal epithelium and maximally in the differentiated cell compartment. Targeted inactivation of PTEN in the intestinal epithelium of PTEN(Lox/Lox)/villin(Cre) mice was confirmed by genotyping, immunohistochemistry, and qPCR. While intestinal-specific PTEN deletion did not have a major effect on cell fate determination or proliferation in the small intestine, it did increase phosphorylated (p) protein kinase B (AKT) expression in the intestinal epithelium, and 19% of animals developed small intestinal adenomas and adenocarcinomas at 12 mo of age. These tumors demonstrated pAKT and nuclear ß-catenin staining, indicating simultaneous activation of the PI3K/AKT and Wnt signaling pathways. These findings demonstrate that, while PTEN inactivation alone has a minimal effect on intestinal homeostasis, it can facilitate tumor promotion upon deregulation of ß-catenin/TCF signaling, further establishing PTEN as a bona fide tumor suppressor gene in intestinal cancer.

Ex vivo bone morphogenetic protein-2 gene delivery using gingival fibroblasts promotes bone regeneration in rats.

AIM: The aim of the present study was to investigate bone regeneration following ex vivo bone morphogenetic protein-2 (BMP-2) gene delivery using human gingival fibroblasts (HGFs) in rat calvarial defects. MATERIALS AND METHODS: An 8 mm craniotomy defect was created in Sprague-Dawley rats. The animals were divided into four groups: (1) non-grafted group, the defect was left empty; (2) collagen matrix group, the defect was filled with collagen matrix only; (3) HGF group, the defect was filled with non-transduced HGFs on collagen matrix; (4) BMP-2/HGF group, the defect was filled with BMP-2 gene-transduced HGFs on collagen matrix. Animals were sacrificed at 2 and 4 weeks after surgery, and micro-computed tomographic and histologic observations were performed. RESULTS: The BMP-2/HGF group showed promoted osseous healing of calvarial defects, as compared with the other groups. At both 2 and 4 weeks, regenerated bone area was significantly greater in the BMP-2/HGF group than the other three groups. Quite a few number of transplanted HGFs were observed within the regenerated bone tissues. CONCLUSIONS: The results of this study suggest that ex vivo BMP-2 gene delivery induces prominent bone regeneration in vivo and HGFs may be useful as target cells for ex vivo gene therapy.

An A13 repeat within the 3'-untranslated region of epidermal growth factor receptor (EGFR) is frequently mutated in microsatellite instability colon cancers and is associated with increased EGFR expression.

Colorectal cancers (CRC) with microsatellite instability (MSI) have clinical, pathologic, genetic, and epigenetic features distinct from microsatellite-stable CRC. Examination of epidermal growth factor receptor (EGFR) mRNA and protein expression levels in a panel of colon cancer cell lines identified strong expression of EGFR in multiple cell lines with MSI. Although no relationship between EGFR overexpression and the length of a CA dinucleotide repeat in intron 1 was observed, a variant A13/A14 repeat sequence within the 3'-untranslated region (3'-UTR) of the EGFR gene was identified, which was mutated by either mononucleotide or dinucleotide adenosine deletions in 64% of MSI cell lines and 69% of MSI colon tumors. Using a Tet-Off system, we show that this mutation increases EGFR mRNA stability in colon cancer cells, providing a mechanistic basis for EGFR overexpression in MSI colon cancer cell lines. To determine whether this mutation is a driver or a bystander event in MSI colon cancer, we examined the effect of pharmacologic and molecular inhibition of EGFR in EGFR 3'-UTR mutant MSI cell lines. Cell lines with an EGFR 3'-UTR mutation and that were wild-type (WT) for downstream signaling mediators in the Ras/BRAF and PIK3CA/PTEN pathways were sensitive to EGFR inhibition, whereas those harboring mutations in these signaling mediators were not. Furthermore, in cell lines WT for downstream signaling mediators, those with EGFR 3'-UTR mutations were more sensitive to EGFR inhibition than EGFR 3'-UTR WT cells, suggesting that this mutation provides a growth advantage to this subset of MSI colon tumors.

G3139, an anti-Bcl-2 antisense oligomer that binds heparin-binding growth factors and collagen I, alters in vitro endothelial cell growth and tubular morphogenesis.

PURPOSE: We examined the effects of G3139 on the interaction of heparin-binding proteins [e.g., fibroblast growth factor 2 (FGF2) and collagen I] with endothelial cells. G3139 is an 18-mer phosphorothioate oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA. A randomized, prospective global phase III trial in advanced melanoma (GM301) has evaluated G3139 in combination with dacarbazine. However, the mechanism of action of G3139 is incompletely understood because it is unlikely that Bcl-2 silencing is the sole mechanism for chemosensitization in melanoma cells. EXPERIMENTAL DESIGN: The ability of G3139 to interact with and protect heparin-binding proteins was quantitated. The effects of G3139 on the binding of FGF2 to high-affinity cell surface receptors and the induction of cellular mitogenesis and tubular morphogenesis in HMEC-1 and human umbilical vascular endothelial cells were determined. RESULTS: G3139 binds with picomolar affinity to collagen I. By replacing heparin, the drug can potentiate the binding of FGF2 to FGFR1 IIIc, and it protects FGF from oxidation and proteolysis. G3139 can increase endothelial cell mitogenesis and tubular morphogenesis of HMEC-1 cells in three-dimensional collagen gels, increases the mitogenesis of human umbilical vascular endothelial cells similarly, and induces vessel sprouts in the rat aortic ring model. CONCLUSIONS: G3139 dramatically affects the behavior of endothelial cells. There may be a correlation between this observation and the treatment interaction with lactate dehydrogenase observed clinically.

Angiogenesis alteration by defibrotide: implications for its mechanism of action in severe hepatic veno-occlusive disease.

Defibrotide (DF) is a mixture of porcine-derived single-stranded phosphodiester oligonucleotides (9-80-mer; average, 50-mer) that has been successfully used to treat severe hepatic veno-occlusive disease (sVOD) with multiorgan failure (MOF) in patients who have received cytotoxic chemotherapy in preparation for bone marrow transplantation. However, its mechanism of action is unknown. Herein, we show that DF and phosphodiester oligonucleotides can bind to heparin-binding proteins (eg, basic fibroblast growth factor [bFGF] but not vascular endothelial growth factor [VEGF] 165) with low nanomolar affinity. This binding occurred in a length- and concentration-dependent manner. DF can mobilize proangiogenic factors such as bFGF from their depot or storage sites on bovine corneal endothelial matrix. However, these molecules do not interfere with high-affinity binding of bFGF to FGFR1 IIIc but can replace heparin as a required cofactor for binding and hence cellular mitogenesis. DF also protects bFGF against digestion by trypsin and chymotrypsin and from air oxidation. In addition, DF binds to collagen I with low nanomolar affinity and can promote human microvascular endothelial cell-1 (HMEC-1) cell mitogenesis and tubular morphogenesis in three-dimensional collagen I gels. Thus, our data suggest that DF may provide a stimulus to the sinusoidal endothelium of a liver that has suffered a severe angiotoxic event, thus helping to ameliorate the clinical sVOD/MOF syndrome.

PIK3CA mutation/PTEN expression status predicts response of colon cancer cells to the epidermal growth factor receptor inhibitor cetuximab.

Cetuximab is a monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). Although approved for use in EGFR-overexpressing advanced colorectal cancer, recent studies have shown a lack of association between EGFR overexpression and cetuximab response, requiring the identification of novel biomarkers predictive of response to this agent. To do so, 22 colon cancer cell lines were screened for cetuximab response in vitro and sensitive and resistant lines were identified. In sensitive cell lines, cetuximab induced a G(0)-G(1) arrest without inducing apoptosis. Notably, cetuximab-sensitive but not cetuximab-resistant cell lines were preferentially responsive to EGF-stimulated growth. Whereas neither EGFR protein/mRNA expression nor gene copy number correlated with cetuximab response, examination of the mutation status of signaling components downstream of EGFR showed that cell lines with activating PIK3CA mutations or loss of PTEN expression (PTEN null) were more resistant to cetuximab than PIK3CA wild type (WT)/PTEN-expressing cell lines (14 +/- 5.0% versus 38.5 +/- 6.4% growth inhibition, mean +/- SE; P = 0.008). Consistently, PIK3CA mutant isogenic HCT116 cells showed increased resistance to cetuximab compared with PIK3CA WT controls. Furthermore, cell lines that were PIK3CA mutant/PTEN null and Ras/BRAF mutant were highly resistant to cetuximab compared with those without dual mutations/PTEN loss (10.8 +/- 4.3% versus 38.8 +/- 5.9% growth inhibition, respectively; P = 0.002), indicating that constitutive and simultaneous activation of the Ras and PIK3CA pathways confers maximal resistance to this agent. A priori screening of colon tumors for PTEN expression status and PIK3CA and Ras/BRAF mutation status could help stratify patients likely to benefit from this therapy.

A del T poly T (8) mutation in the 3' untranslated region (UTR) of the CDK2-AP1 gene is functionally significant causing decreased mRNA stability resulting in decreased CDK2-AP1 expression in human microsatellite unstable (MSI) colorectal cancer (CRC).

BACKGROUND: We have previously published results indicating that decreased expression of CDK2-AP1 in MSI human colorectal cancer is associated with deletion mutations in the poly (T) 8 repeat sequence within the 3'-UTR of the CDK2-AP1 gene. In this study, we test the hypothesis that the del T mutation results in decreased CDK2-AP1 expression by causing reduced mRNA stability. METHODS: We introduced wild-type and mutant 3'-UTR sequences fused to a green fluorescent protein (GFP) gene separately into human CRC cell lines and quantified the expression of the GFP gene. Native CDK2-AP1 mRNA stability was measured in human CRC cell lines, using an actinomycin D assay and the mRNA structure folding software mfold 3.2. RESULTS: Mutant GFP-3'-UTR samples demonstrated significantly reduced GFP expression compared with wild-type GFP-3'-UTR as measured by both FACS and real-time PCR. Both the actinomycin D assay and mfold software demonstrated significantly reduced mRNA stability for the del T poly (T) 8 transcript compared with the wild type. CONCLUSIONS: In summary, these novel results support our hypotheses that the del T poly (T) 8 observed in the 3'-UTR of the CDK2-AP1 gene in human MSI CRC is functionally significant and results in decreased CDK2-AP1 expression. The results also indicate the mechanism of this decreased expression is caused at least in part by decreased mRNA stability.