Immunoscore Is Prognostic in Low-Risk and High-Risk Stage III Colon Carcinomas Treated With Adjuvant Infusional Fluorouracil, Leucovorin, and Oxaliplatin in a Phase III Trial.

PURPOSE: The recommended duration of adjuvant fluoropyrimidine and oxaliplatin chemotherapy for patients with stage III colon cancer is based on tumor classification into clinically low-risk (T1-3 N1) and high-risk (T4 or N2) groups. We determined whether Immunoscore can enhance prognostication within these risk groups. MATERIALS AND METHODS: Patients with stage III colon carcinomas (N = 600) were randomly selected from the infusional fluorouracil, leucovorin, and oxaliplatin arm of adjuvant trial NCCTG N0147 (Alliance for Clinical Trials in Oncology). Tumors were evaluated for Immunoscore that quantifies CD3+ and CD8+ T-cell densities in the tumor center and invasive margin by digital image analysis. Disease-free survival (DFS) by Immunoscore was analyzed using a multivariable Cox regression model in each risk group with adjustment for covariates including KRAS, BRAFV600E, and mismatch repair status. RESULTS: Of 559 cancers with Immunoscore data, 299 (53.5%) were classified as clinically low-risk (T1-3 N1) and 260 (46.5%) as clinically high-risk (T4 and/or N2). Among patients with low-risk tumors, those with Immunoscore-Low versus Immunoscore-High tumors had significantly worse 5-year DFS rates (77.5% v 91.8%; hazard ratio, 1.70; 95% CI, 1.03 to 2.79; P = .037). Among patients with high-risk tumors, those with Immunoscore-Low versus Immunoscore-High tumors also had significantly worse DFS (55.3% v 70.3%; hazard ratio, 1.65; 95% CI, 1.11 to 2.47; P = .013). Tumors that were low-risk/Immunoscore-Low had similar outcomes as did tumors that were high-risk/Immunoscore-High (P = .174). Prognostication was significantly improved in multivariable models where Immunoscore was added to clinical risk parameters and limited biomarkers (likelihood ratio test P = .0003). CONCLUSION: Immunoscore can refine patient prognosis beyond clinical risk group classification, suggesting its potential utility for adjuvant decision making.

Association of Adiponectin and Vitamin D With Tumor Infiltrating Lymphocytes and Survival in Stage III Colon Cancer.

Background: Adipocyte-derived adiponectin may play a role in the host inflammatory response to cancer. We examined the association of plasma adiponectin with the density of tumor-infiltrating lymphocytes (TILs) in colon cancers and with vitamin D, clinicopathological features, and patient survival. Methods: Plasma adiponectin and 25-hydroxyvitamin D [25(OH)D] were analyzed by radioimmunoassay in 600 patients with stage III colon cancer who received FOLFOX-based adjuvant chemotherapy (NCCTG N0147 [Alliance]). TIL densities were determined in histopathological sections. Associations with disease-free survival (DFS), time to recurrence, and overall survival were evaluated by multivariable Cox regression adjusting for potential confounders (ie, body mass index, race, TILs, and N stage). All statistical tests were 2-sided. Results: We found a statistically significant reduction in adiponectin, but not 25(OH)D, levels in tumors with high vs low TIL densities (median = 6845 vs 8984 ng/mL; P = .04). A statistically significant reduction in adiponectin was also observed in obese (body mass index >30 kg/m2) vs nonobese patients (median = 6608 vs 12 351 ng/mL; P < .001), in men vs women (median = 8185 vs 11 567 ng/mL; P < .001), in Blacks vs Whites or Asians (median = 6412 vs 8847 vs 7858 ng/mL; P < .03), and in those with fewer lymph node metastases (N1 vs N2: median = 7768 vs 9253 ng/mL; P = .01). Insufficiency of 25(OH)D (<30 ng/mL) was detected in 291 (48.5%) patients. In multivariable analyses, neither adiponectin nor 25(OH)D were associated with a statistically significant difference in DFS, overall survival , or time to recurrence in models adjusted for potential confounders. We found a statistically significant association of TILs with prognosis, yet no such interaction was observed for the association of adiponectin with TILs for DFS. Conclusions: Lower circulating adiponectin levels were associated with a statistically significant increase in TIL densities in colon cancers, indicating an enhanced antitumor immune response. In contrast to TILs, neither adiponectin nor 25(OH)D was independently prognostic.

Contribution of Immunoscore and Molecular Features to Survival Prediction in Stage III Colon Cancer.

BACKGROUND: The American Joint Committee on Cancer staging and other prognostic tools fail to account for stage-independent variability in outcome. We developed a prognostic classifier adding Immunoscore to clinicopathological and molecular features in patients with stage III colon cancer. METHODS: Patient (n = 559) data from the FOLFOX arm of adjuvant trial NCCTG N0147 were used to construct Cox models for predicting disease-free survival (DFS). Variables included age, sex, T stage, positive lymph nodes (+LNs), N stage, performance status, histologic grade, sidedness, KRAS/BRAF, mismatch repair, and Immunoscore (CD3+, CD8+ T-cell densities). After determining optimal functional form (continuous or categorical) and within Cox models, backward selection was performed to analyze all variables as candidate predictors. All statistical tests were two-sided. RESULTS: Poorer DFS was found for tumors that were T4 vs T3 (hazard ratio [HR] = 1.76, 95% confidence interval [CI] = 1.19 to 2.60; P = .004), right- vs left-sided (HR = 1.52, 95% CI = 1.14 to 2.04; P = .005), BRAF V600E (HR = 1.74, 95% CI = 1.26 to 2.40; P < .001), mutant KRAS (HR = 1.66, 95% CI = 1.08 to 2.55; P = .02), and low vs high Immunoscore (HR = 1.69, 95% CI = 1.22 to 2.33; P = .001) (all P < .02). Increasing numbers of +LNs and lower continuous Immunoscore were associated with poorer DFS that achieved significance (both Ps< .0001). After number of +LNs, T stage, and BRAF/KRAS, Immunoscore was the most informative predictor of DFS shown multivariately. Among T1-3 N1 tumors, Immunoscore was the only variable associated with DFS that achieved statistical significance. A nomogram was generated to determine the likelihood of being recurrence-free at 3 years. CONCLUSIONS: The Immunoscore can enhance the accuracy of survival prediction among patients with stage III colon cancer.

Relationship Between Metformin Use and Recurrence and Survival in Patients With Resected Stage III Colon Cancer Receiving Adjuvant Chemotherapy: Results From North Central Cancer Treatment Group N0147 (Alliance).

BACKGROUND: Preclinical and epidemiological data suggest that metformin might have antineoplastic properties against colon cancer (CC). However, the effect of metformin use on patient survival in stage III CC after curative resection is unknown. The survival outcomes were comparable regardless of the duration of metformin use. PATIENTS AND METHODS: Before randomization to FOLFOX (folinic acid, 5-fluorouracil, oxaliplatin) with or without cetuximab, 1,958 patients with stage III CC enrolled in the N0147 study completed a questionnaire with information on diabetes mellitus (DM) and metformin use. Cox models were used to assess the association between metformin use and disease-free survival (DFS), overall survival (OS), and the time to recurrence (TTR), adjusting for clinical and/or pathological factors. RESULTS: Of the 1,958 patients, 1,691 (86%) reported no history of DM, 115 reported DM with metformin use (6%), and 152 reported DM without metformin use (8%). The adjuvant treatment arms were pooled, because metformin use showed homogeneous effects on outcomes across the two arms. Among the patients with DM (n = 267), DFS (adjusted hazard ratio [aHR], 0.90; 95% confidence interval [CI], 0.59-1.35; p = .60), OS (aHR, 0.99; 95% CI, 0.65-1.49; p = .95), and TTR (aHR, 0.87; 95% CI, 0.56-1.35; p = .53) were not different for the metformin users compared with the nonusers after adjusting for tumor and patient factors. The survival outcomes were comparable regardless of the duration of metformin use (<1, 1-5, 6-10, ≥11 years) before randomization (ptrend = .64 for DFS, ptrend = .84 for OS, and ptrend = .87 for TTR). No interaction effects were observed between metformin use and KRAS, BRAF mutation status, tumor site, T/N stage, gender, or age. CONCLUSIONS: Patients with stage III CC undergoing adjuvant chemotherapy who used metformin before the diagnosis of CC experienced DFS, OS, and TTR similar to those for non-DM patients and DM patients without metformin use. IMPLICATIONS FOR PRACTICE: The present study did not find any relationship between metformin use or its duration and disease-free survival, time to recurrence, and overall survival in a large cohort of patients with resected stage III colon cancer receiving adjuvant FOLFOX (folinic acid, fluorouracil, oxaliplatin)-based chemotherapy. This relationship was not modified by KRAS or BRAF mutation or DNA mismatch repair status. Metformin use did not increase or decrease the likelihood of chemotherapy-related grade 3 or higher adverse events.

Alcohol consumption and colon cancer prognosis among participants in north central cancer treatment group phase III trial N0147.

Alcohol consumption is associated with a modest increased risk of colon cancer, but its relationship with colon cancer survival has not been elucidated. Using data from a phase III randomized adjuvant trial, we assessed the association of alcohol consumption with colon cancer outcomes. Patients completed a risk factor questionnaire before randomization to FOLFOX or FOLFOX + cetuximab (N = 1984). Information was collected on lifestyle factors, including smoking, physical activity and consumption of different types of alcohol. Cox models assessed the association between alcohol consumption and outcomes of disease-free survival (DFS), time-to-recurrence (TTR) and overall survival (OS), adjusting for age, sex, study arm, body mass, smoking, physical activity and performance status. No statistically significant difference in outcomes between ever and never drinkers were noted [hazard ratio (HR)DFS = 0.86, HRTTR = 0.87, HROS = 0.86, p-values = 0.11-0.17]. However, when considering alcohol type, ever consumers of red wine (n = 628) had significantly better outcomes than never consumers (HRDFS = 0.80, HRTTR = 0.81, HROS = 0.78, p-values = 0.01-0.02). Favorable outcomes were confirmed in patients who consumed 1-30 glasses/month of red wine (n = 601, HR = 0.80-0.83, p-values = 0.03-0.049); there was a suggestion of more favorable outcomes in patients who consumed >30 glasses/month of red wine (n = 27, HR = 0.33-0.38, p-values = 0.05-0.06). Beer and liquor consumption were not associated with outcomes. Although alcohol consumption was not associated with colon cancer outcomes overall, mild to moderate red wine consumption was suggestively associated with longer OS, DFS and TTR in stage III colon cancer patients.

Association between DPYD c.1129-5923 C>G/hapB3 and severe toxicity to 5-fluorouracil-based chemotherapy in stage III colon cancer patients: NCCTG N0147 (Alliance).

Severe (grade≥3) adverse events (AEs) to 5-fluorouracil (5-FU)-based chemotherapy regimens can result in treatment delays or cessation, and, in extreme cases, life-threatening complications. Current genetic biomarkers for 5-FU toxicity prediction, however, account for only a small proportion of toxic cases. In the current study, we assessed DPYD variants suggested to correlate with 5-FU toxicity, a deep intronic variant (c.1129-5923 C>G), and four variants within a haplotype (hapB3) in 1953 stage III colon cancer patients who received adjuvant FOLFOX±cetuximab. Logistic regression was used to assess multivariable associations between DPYD variant status and AEs common to 5-FU (5FU-AEs). In our study cohort, 1228 patients (62.9%) reported any grade≥3 AE (overall AE), with 638 patients (32.7%) reporting any grade≥3 5FU-AE. Only 32 of 78 (41.0%) patients carrying DPYD c.1129-5923 C>G and the completely linked hapB3 variants c.1236 C>G and c.959-51 T>C showed at least one grade≥3 5FU-AE, resulting in no statistically significant association (adjusted odds ratio=1.47, 95% confidence interval=0.90-2.43, P=0.1267). No significant associations were identified between c.1129-5923 C>G/hapB3 and overall grade≥3 AE rate. Our results suggest that c.1129-5923 C>G/hapB3 have limited predictive value for severe toxicity to 5-FU-based combination chemotherapy.

Comparison of FOLFIRI with or without cetuximab in patients with resected stage III colon cancer; NCCTG (Alliance) intergroup trial N0147.

BACKGROUND: Two arms with FOLFIRI, with or without cetuximab, were initially included in the randomized phase III intergroup clinical trial NCCTG (North Central Cancer Treatment Group) N0147. When other contemporary trials demonstrated no benefit to using irinotecan as adjuvant therapy, the FOLFIRI-containing arms were discontinued. We report the clinical outcomes for patients randomized to FOLFIRI with or without cetuximab. PATIENTS AND METHODS: After resection, patients were randomized to 12 biweekly cycles of FOLFIRI, with or without cetuximab. KRAS (Kirsten rat sarcoma viral oncogene homolog) mutation status was retrospectively determined in a central lab. The primary end point was disease-free survival (DFS). Secondary end points included overall survival (OS) and toxicity. RESULTS: One hundred and six patients received FOLFIRI and 40 received FOLFIRI plus cetuximab. Median follow-up was 5.95 years (range, 0.1-7.0 years). The addition of cetuximab showed a trend toward improved DFS (hazard ratio [HR], 0.53; 95% CI, 0.26-1.1; P = .09) and OS (HR, 0.45; 95% CI, 0.17-1.16; P = .10) in the overall group, regardless of KRAS status, and in patients with wild type KRAS. Grade ≥ 3 nonhematologic adverse effects were significantly increased in the cetuximab versus FOLFIRI-alone arm (68% vs. 46%; P = .02). Adjuvant FOLFIRI resulted in a 3-year DFS less than that expected for FOLFOX. CONCLUSION: In this small randomized subset of patients with resected stage III colon cancer, the addition of cetuximab to FOLFIRI was associated with a nonsignificant trend toward improved DFS and OS. Nevertheless, considering the limitations of this analysis, FOLFOX without the addition of a biologic agent remains the standard of care for adjuvant therapy in resected stage III colon cancer.

Effect of oxaliplatin, fluorouracil, and leucovorin with or without cetuximab on survival among patients with resected stage III colon cancer: a randomized trial.

CONTEXT: Leucovorin, fluorouracil, and oxaliplatin (FOLFOX) is the standard adjuvant therapy for resected stage III colon cancer. Adding cetuximab to FOLFOX benefits patients with metastatic wild-type KRAS but not mutated KRAS colon cancer. OBJECTIVE: To assess the potential benefit of cetuximab added to the modified sixth version of the FOLFOX regimen (mFOLFOX6) in patients with resected stage III wild-type KRAS colon cancer. DESIGN, SETTING, AND PARTICIPANTS: A randomized trial of 2686 patients aged 18 years or older at multiple institutions across North America enrolled following resection and informed consent between February 10, 2004, and November 25, 2009. The primary randomized comparison was 12 biweekly cycles of mFOLFOX6 with and without cetuximab. KRAS mutation status was centrally determined. The trial was halted after a planned interim analysis of 48% of predicted events (246/515) occurring in 1863 (of 2070 planned) patients with tumors having wild-type KRAS. A total of 717 patients with mutated KRAS and 106 with indeterminate KRAS were accrued. The 2070 patients with wild-type KRAS provided 90% power to detect a hazard ratio (HR) of 1.33 (2-sided α = .05), with planned interim efficacy analyses after 25%, 50%, and 75% of expected relapses. MAIN OUTCOME MEASURES: Disease-free survival in patients with wild-type KRAS mutations. Secondary end points included overall survival and toxicity. RESULTS: Median (range) follow-up was 28 (0-68) months. The trial demonstrated no benefit when adding cetuximab. Three-year disease-free survival for mFOLFOX6 alone was 74.6% vs 71.5% with the addition of cetuximab (HR, 1.21; 95% CI, 0.98-1.49; P = .08) in patients with wild-type KRAS, and 67.1% vs 65.0% (HR, 1.12; 95% CI, 0.86-1.46; P = .38) in patients with mutated KRAS, with no significant benefit in any subgroups assessed. Among all patients, grade 3 or higher adverse events (72.5% vs 52.3%; odds ratio [OR], 2.4; 95% CI, 2.1-2.8; P < .001) and failure to complete 12 cycles (33% vs 23%; OR, 1.6; 95% CI, 1.4-1.9; P < .001) were significantly higher with cetuximab. Increased toxicity and greater detrimental differences in all outcomes were observed in patients aged 70 years or older. CONCLUSION: Among patients with stage III resected colon cancer, the use of cetuximab with adjuvant mFOLFOX6 compared with mFOLFOX6 alone did not result in improved disease-free survival. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00079274.

Preoperative multimodality therapy improves disease-free survival in patients with carcinoma of the rectum: NSABP R-03.

PURPOSE: Although chemoradiotherapy plus resection is considered standard treatment for operable rectal carcinoma, the optimal time to administer this therapy is not clear. The NSABP R-03 (National Surgical Adjuvant Breast and Bowel Project R-03) trial compared neoadjuvant versus adjuvant chemoradiotherapy in the treatment of locally advanced rectal carcinoma. PATIENTS AND METHODS: Patients with clinical T3 or T4 or node-positive rectal cancer were randomly assigned to preoperative or postoperative chemoradiotherapy. Chemotherapy consisted of fluorouracil and leucovorin with 45 Gy in 25 fractions with a 5.40-Gy boost within the original margins of treatment. In the preoperative group, surgery was performed within 8 weeks after completion of radiotherapy. In the postoperative group, chemotherapy began after recovery from surgery but no later than 4 weeks after surgery. The primary end points were disease-free survival (DFS) and overall survival (OS). RESULTS: From August 1993 to June 1999, 267 patients were randomly assigned to NSABP R-03. The intended sample size was 900 patients. Excluding 11 ineligible and two eligible patients without follow-up data, the analysis used data on 123 patients randomly assigned to preoperative and 131 to postoperative chemoradiotherapy. Surviving patients were observed for a median of 8.4 years. The 5-year DFS for preoperative patients was 64.7% v 53.4% for postoperative patients (P = .011). The 5-year OS for preoperative patients was 74.5% v 65.6% for postoperative patients (P = .065). A complete pathologic response was achieved in 15% of preoperative patients. No preoperative patient with a complete pathologic response has had a recurrence. CONCLUSION: Preoperative chemoradiotherapy, compared with postoperative chemoradiotherapy, significantly improved DFS and showed a trend toward improved OS.

Hereditary colorectal cancer syndromes and the role of the surgical oncologist.

The expanding understanding of the genetic basis to hereditary colon cancer syndromes is dismantling previously conceived categorizations and shedding light on why those schemes often failed in past. This review highlights evolving concepts regarding the genetic diagnosis and clinical management of the more commonly inherited colorectal cancer syndromes, including a discussion of recently described familial syndromes. This review also addresses clinician responsibilities in recognition of familial syndromes and provision of counseling.

Aesthetic outcomes in breast conservation therapy.

BACKGROUND: Since the National Surgical Adjuvant Breast and Bowel Project B06 (NSABP-B06) trial demonstrated equivalent survival outcomes between patients with breast cancer undergoing modified radical mastectomy versus lumpectomy and radiation, an increasing number of patients are seeking breast conservation therapy. Traditionally, only patients who have undergone total mastectomy have been referred for reconstruction. OBJECTIVE: The purpose of the study was to determine the number of dissatisfied patients treated with breast conservation therapy who have suboptimal cosmesis and should be referred for reconstruction. METHODS: After obtaining approval from the Institutional Review Board and patient consent, patients identified as more than 1 year posttreatment from breast conservation therapy (1999-2004) were interviewed and photographed. Data were gathered by use of a questionnaire that included patient aesthetic score, patient satisfaction, and change in body image. Photographs were shown to a surgical oncologist, a general surgeon, and a plastic surgeon for a physician aesthetic score. RESULTS: Thirteen of 46 patients (28.3%) were dissatisfied with their cosmetic result. Women who were dissatisfied with their cosmetic result were more likely to have a negative change in their body image when compared with patients who were satisfied with their cosmetic result (46.2 % vs 6.1%, P = .02). Additionally, dissatisfied patients were more likely to rate their cosmetic result as poor (15.4 % vs 0%, P = .007) and were more likely to consider reconstruction (46.2% vs 9.1%, P = .01) when compared with satisfied patients. Risk factors to predict dissatisfaction in our patient population included age younger than 52 years and the resection of tumor from the upper inner quadrant. CONCLUSIONS: Twenty-eight percent of patients in this study were dissatisfied with their cosmetic result. Furthermore, a large portion of these patients would consider reconstruction if it were offered. Although this study only identified a few broad risk factors for suboptimal cosmetic outcome, it confirms our hypothesis that many patients who have undergone breast conservation therapy should be referred for plastic surgery consultation.

The implications of colorectal cancer molecular biology in clinical practice.

Colorectal cancer (CRC) is the third most common malignancy in the United States. Advances in molecular biology have enhanced the understanding of colorectal carcinogenesis. Approximately 75% of CRCs are sporadic; the rest are hereditary or belong to a familial syndrome. Identification of familial forms of CRC have enabled the development of several models of carcinogenesis and made CRC a well-studied malignancy in terms of molecular pathogenesis. Pathways containing multiple mutations and genetic alterations that play a role in hereditary CRC pathogenesis have been elucidated. Many of the molecular changes seen in these pathways also are involved in the development of sporadic cancers.

Actinomycotic infection of the abdominal wall mimicking a malignant neoplasm.

BACKGROUND: Actinomyces infections can create diagnostic dilemmas and be mistaken for malignant tumors. METHODS: Case report and review of the pertinent English-language literature. RESULTS: A 44-year-old man who had undergone an appendectomy for perforated appendicitis presented three years later with an abdominal wall mass. He had been treated postoperatively for an abdominal wall infection with Actinomyces. Given his poor response to antibiotics and a diagnostic work-up that could not definitively rule out a malignant tumor, a wide local excision was performed. Microscopic examination revealed sulfur granules consistent with Actinomyces. CONCLUSIONS: Although the current recommendation is to treat Actinomyces infections with antibiotics, there may be a role for surgical intervention if the response to antibiotics has been suboptimal or a malignant process cannot be ruled out.

Preoperative chemotherapy: updates of National Surgical Adjuvant Breast and Bowel Project Protocols B-18 and B-27.

PURPOSE: National Surgical Adjuvant Breast and Bowel Project (NSABP) Protocol B-18 was designed to determine whether four cycles of doxorubicin and cyclophosphamide (AC) administered preoperatively improved breast cancer disease-free survival (DFS) and overall survival (OS) compared with AC administered postoperatively. Protocol B-27 was designed to determine the effect of adding docetaxel (T) to preoperative AC on tumor response rates, DFS, and OS. PATIENTS AND METHODS: Analyses were limited to eligible patients. In B-18, 751 patients were assigned to receive preoperative AC, and 742 patients were assigned to receive postoperative AC. In B-27, 784 patients were assigned to receive preoperative AC followed by surgery, 783 patients were assigned to AC followed by T and surgery, and 777 patients were assigned to AC followed by surgery and then T. RESULTS: Results from B-18 show no statistically significant differences in DFS and OS between the two groups. However, there were trends in favor of preoperative chemotherapy for DFS and OS in women less than 50 years old (hazard ratio [HR] = 0.85, P = .09 for DFS; HR = 0.81, P = .06 for OS). DFS conditional on being event free for 5 years also demonstrated a strong trend in favor of the preoperative group (HR = 0.81, P = .053). Protocol B-27 results demonstrated that the addition of T to AC did not significantly impact DFS or OS. Preoperative T added to AC significantly increased the proportion of patients having pathologic complete responses (pCRs) compared with preoperative AC alone (26% v 13%, respectively; P < .0001). In both studies, patients who achieved a pCR continue to have significantly superior DFS and OS outcomes compared with patients who did not. CONCLUSION: B-18 and B-27 demonstrate that preoperative therapy is equivalent to adjuvant therapy. B-27 also showed that the addition of preoperative taxanes to AC improves response.

Neoplastic conversion of human colon smooth muscle cells: No requirement for telomerase.

The role of telomerase as an essential requirement for the neoplastic conversion of human cells has been controversial. In the model of conversion of normal human cells to cancer cells by the combination of simian virus 40 (SV40) early region genes and oncogenic Ras (H-Ras(G12V)), telomerase (hTERT) was originally described as essential in conjunction with these other genes. Here we used primary cultures of colon smooth muscle cells isolated from surgical specimens. SV40 large T antigen (TAg) and oncogenic Ras(G12V) were introduced into the cells by retroviral transduction and cells were rapidly transplanted into the subrenal capsule space in immunodeficient mice, without selection in culture. Malignant tumors were formed from transduced cells. Extensive invasion into the kidney occurred even when tumors were small; in contrast, at the same tumor size, oncogene-expressing fibroblasts did not show much invasion. Increased invasiveness was also observed in vitro. However, cells in these cancers showed morphological evidence of crisis, consistent with their lack of telomerase. These experiments on human colon smooth muscle cells support the concept that Ras(G12V) and SV40 TAg form a minimal set of genes that can convert normal human cells to cancer cells without a requirement for hTERT.

Flavonoid, silibinin, inhibits proliferation and promotes cell-cycle arrest of human colon cancer.

BACKGROUND: Anti-oxidative extracts from the milk thistle plant (Silybum marianum) have been shown to have antiproliferative effects in several tumor types. Silibinin is the primary active component isolated from the crude seed extract, silymarin. It has been used as a dietary supplement for hepatoprotection for over 2000 years. Silibinin has been shown to be safe in multiple animal models and has had no significant adverse events in human studies. We investigated the potential for this nontoxic flavolignan to inhibit proliferation of human colon cancer. MATERIALS AND METHODS: Three well-characterized cell lines, Fet, Geo, and HCT116, were studied. The MTT cell-viability assay was performed to study the effect of silibinin on proliferation. Fluorescence-activated cell sorter (FACS) analysis was used to determine the effects of silibinin on cell cycle and apoptosis. 4', 6'-diamidine-2'-phenylindole (DAPI) staining with confocal microscopy was used to morphologically confirm these results. Poly ADP-ribose polymerase (PARP) cleavage and expression levels of p21, p27, cyclins B1/D1, and CDK-2 were measured. Cyclooxygenase-2 (COX-2) levels were also measured. The experiments were performed in triplicate and reported as mean values with standard errors. Means were contrasted using analysis of variance with Dunnet's correction for multiple testing. All statistical testing was two-sided with a significance level of 5%. RESULTS: The MTT assay revealed a strong dose-dependent inhibitory effect. Treatment with 75 microg/mL resulted in 50% inhibition of cell-viability (IC-50) in Fet and Geo lines at 72 h. An IC50 dose of 40 ug/mL was obtained in HCT116, a poorly-differentiated cell line, at 72 h. FACS analysis demonstrated statistically significant cell-cycle arrest in all cell lines. G(2)-M phase arrests in Fet and Geo cell lines (P < 0.001) and a G1 arrest in HCT116 (P = 0.005) were noted. Trivial increases in early apoptotic rates (2% to 3%) for Geo and HCT116 were noted on FACS analysis via annexin V-propidium iodide technique (P < 0.05), but no evidence for apoptosis was seen on Western blot for PARP cleavage or DAPI. Cyclin B1/D1 and CDK-2 levels were inhibited. Increased expression of cell cycle inhibitors, p21 or p27, was noted, and there was no effect on COX-2 expression. CONCLUSIONS: Silibinin significantly inhibits proliferation through cell-cycle arrest via inhibition of cyclin-CDK promoter activity. Despite its antioxidant profile, there is no effect on COX-2 expression. Apoptosis does not appear to be greatly increased in human colon cancer cell lines Fet, Geo, and HCT116. Rather, inhibition of cell cycle regulatory proteins plays a fundamental role in silibinin's mechanism of action, and this may serve as a basis for combined use with conventional chemotherapeutics.

Compensatory increases in Her-2/neu activation in response to EGFR tyrosine kinase inhibition in colon cancer cell lines.

BACKGROUND: Tyrosine kinase receptors of the ErbB family have become promising targets for anti-neoplastic drugs, but mechanisms of resistance are incompletely understood. To investigate such pathways, we applied a small-molecule, selective EGFR inhibitor, OSI-774, to three well-characterized colon cancer cell lines and studied the alterations of expression and activation of receptors in the erbB family. METHODS: MTT assays were performed to determine the IC(50)s of GEO, FET, and HCT 116 human colorectal cancer cell lines treated with OSI-774. Plated cells were then exposed to either DMSO control or 7 microm of OSI-774 for treatment durations of 1, 3, 5, 7, 10, 14, 28, and 56 days. Cell lysates were evaluated by Western blotting, evaluating both total and phosphorylated levels of EGFR, Her-2/neu, and erbB-3. RESULTS: IC(50) values for GEO, FET, and HCT 116 cell lines exposed to OSI-774 were 12.0, 16.0, and greater than 100 microm, respectively. In all treated cell lines, OSI-774 diminished EGFR activation but did not affect total expression compared with controls. In contrast, Her-2/neu activation was increased in all cell lines. These changes in EGFR and Her-2/neu were identified within 24 h but peaked later in the treatment cycle. ErbB-3 expression and activation did not follow a consistent pattern between cell lines. CONCLUSIONS: Inhibition of EGFR led to increased activation of Her-2/neu. This result suggests a possible mechanism by which cells might escape the proapoptotic signals resulting from EGFR blockade. Our findings suggest concurrent inhibition of multiple members of the erbB family may yield stronger apoptotic responses than single receptor blockade alone.

Colorectal cancer in Hispanics: a population at risk for earlier onset, advanced disease, and decreased survival.

BACKGROUND: While colorectal cancer (CRC) incidence and mortality rates have declined slightly over the past decade, there remain marked differences by ethnicity. Our aim was to investigate ethnic differences in occurrence, clinical presentation and outcome of CRC at a tertiary university center that serves a predominantly Hispanic population. METHODS: Prospectively collected data from the tumor registry on patients diagnosed with colorectal cancer from 1985 through 2001 was examined. Age at diagnosis, mode of presentation, sex, tumor location, ethnicity, TNM stage, and survivals were assessed and ethnic differences were sought. RESULTS: Records from 453 patients with CRC were reviewed. There were 296 (65%) patients that were Hispanics, 112 (25%) non-Hispanic Whites, 37 (8%) African Americans, and 8 (2%) of other or unknown ethnicity. Compared with non-Hispanic Whites, Hispanics presented at a younger age (58.5 +/- 14 versus 53.6 +/- 12.73, respectively; P < 0.01), with a significantly greater incidence of stage IV disease (19% versus 32%, respectively; P = 0.02). They had significantly poorer age-adjusted survival (median survival of 92 months for <55 years and 77 months for >55 years versus 48 months for <55 years and 48 months for >55 years, respectively; adjusted log rank P = 0.045). There were no differences in tumor location, mode of presentation or adjuvant treatment received. CONCLUSIONS: Hispanic patients with CRC in our catchment area present at a younger age with more metastatic disease and have a poorer survival than non-Hispanic Whites. Modification of screening criteria and treatment paradigms may be required for Hispanics.