Sorafenib inhibits proliferation and invasion in desmoid-derived cells by targeting Ras/MEK/ERK and PI3K/Akt/mTOR pathways.

Desmoid tumors (DTs) are unusual neoplasms of mesenchymal origin that exhibit locally invasive behavior. Surgical resection is the initial treatment of choice for DTs. For patients with recurrent or unresectable disease, however, medical options are limited. Sorafenib is a multikinase inhibitor with known antitumor activity in various cancers via suppression of the PI3K/Akt/mTOR pathway. Here, we examined the effects of sorafenib on patient-derived DT cell lines, with the aim of characterizing the efficacy and molecular mechanism of action. Early passage DT-derived cells were treated with increasing doses of sorafenib (0-10 µM) and demonstrated up to 90% decrease in proliferation and invasion relative to controls. Signaling arrays identified multiple potential targets of sorafenib in the Ras/MEK/ERK and PI3K/Akt/mTOR signaling cascades. Immunoblot analysis revealed that sorafenib inhibited Akt, MEK and ERK phosphorylation, and this effect correlated with inhibition of total Akt and total MEK, while total ERK levels remained unchanged. Sorafenib also inhibited 4E-BP1 phosphorylation, and this effect correlated with decrease of p-eIF4E and total eIF4E. Finally, in combination with the mammalian target of rapamycin (mTOR) inhibitor everolimus, sorafenib decreased phosphorylation of the ribosomal protein and mTOR effector S6K in an additive manner. Taken together, our results suggest that sorafenib suppresses DT proliferation and invasion via inhibition of Ras/MEK/ERK and PI3K/Akt/mTOR signaling pathways with additional effects on translation. Sorafenib may be a promising therapeutic option in the treatment of DTs. Additional studies in DT patients are warranted to examine the efficacy of combination therapy using sorafenib.

Cytoreductive Surgery for Metastatic Gastrointestinal Stromal Tumors Treated With Tyrosine Kinase Inhibitors: A 2-institutional Analysis.

OBJECTIVE: To refine treatment recommendations for patients with metastatic gastrointestinal stromal tumors (GISTs) treated with tyrosine kinase inhibitors (TKIs) and surgery. BACKGROUND: Early reports suggested that patients with metastatic GIST responding to TKIs treated with surgery may have favorable outcomes. However, identification of prognostic factors was limited by small cohorts. METHODS: Progression-free survival (PFS) and overall survival (OS) from time of surgery and from start of initial TKI was determined. Multivariate analysis was conducted on all patients undergoing GIST metastasectomy between 2001 and 2014 at 2 institutions. RESULTS: We performed 400 operations on 323 patients with metastatic GIST on TKIs. Radiographic response at time of surgery was classified as responsive disease (RD, n = 64, 16%), stable disease (SD, n = 100, 25%), unifocal progressive disease (UPD, n = 132, 33%), and multifocal progressive disease (MPD, n = 104, 26%). For patients on imatinib before surgery, radiographic response was predictive of PFS from time of surgery (RD 36 months, SD 30 months, UPD 11 months, MPD 6 months; P < 0.001) and from imatinib initiation (RD 71 months, SD 51 months, UPD 47 months, MPD 33 months; P < 0.001). Radiographic response was predictive of OS from time of surgery (RD not reached, SD 110 months, UPD 59 months, MPD 24 months; P < 0.001), and from imatinib initiation (RD not reached, SD 144 months, UPD 105 months, MPD 66 months; P = 0.005). Radiographic response was not predictive of PFS/OS for patients on sunitinib. Metastatic mitotic index ≥5/50 HPF, MPD, and R2 resection were prognostic of worse PFS/OS; primary mutation was not. CONCLUSIONS: Surgery in metastatic GIST patients in the absence of MPD on imatinib is associated with outcomes at least comparable with second-line sunitinib and may be considered in select patients.

A Study of Thymidylate Synthase Expression as a Biomarker for Resectable Colon Cancer: Alliance (Cancer and Leukemia Group B) 9581 and 89803.

PURPOSE: Tumor levels of thymidylate synthase (TS), a target of 5-fluorouracil (5-FU)-based chemotherapy for colorectal cancer, have been studied as a predictive or prognostic biomarker with mixed results. PATIENTS AND METHODS: Tumor TS levels were prospectively evaluated in two adjuvant therapy trials for patients with resected stage II or III colon cancer. TS expression was determined by standard immunohistochemistry and by automated quantitative analysis. Tumor mismatch repair deficiency (MMR-D) and BRAF c.1799T > A (p.V600E) mutation status were also examined. Relationships between tumor TS, MMR-D, and BRAF mutation status, overall survival (OS), and disease-free survival (DFS) were investigated in the subset of stage III patients. RESULTS: Patients whose tumors demonstrated high TS expression experienced better treatment outcomes, with DFS hazard ratio (HR) = 0.67, 95% confidence interval (CI) = 0.53, 0.84; and OS HR = 0.68, 95% CI = 0.53, 0.88, for high versus low TS expression, respectively. No significant interaction between TS expression and stage was observed (DFS: interaction HR = 0.94; OS: interaction HR = 0.94). Tumors with high TS expression were more likely to demonstrate MMR-D (22.2% vs. 12.8%; p = .0003). Patients whose tumors demonstrated both high TS and MMR-D had a 7-year DFS of 77%, compared with 58% for those whose tumors had low TS and were non-MMR-D (log-rank p = .0006). Tumor TS expression did not predict benefit of a particular therapeutic regimen. CONCLUSION: This large prospective analysis showed that high tumor TS levels were associated with improved DFS and OS following adjuvant therapy for colon cancer, although tumor TS expression did not predict benefit of 5-FU-based chemotherapy. The Oncologist 2017;22:107-114Implications for Practice: This study finds that measurement of tumor levels of thymidylate synthase is not helpful in assigning specific adjuvant treatment for colorectal cancer. It also highlights the importance of using prospective analyses within treatment clinical trials as the optimal method of determining biomarker utility.

MRE11-deficiency associated with improved long-term disease free survival and overall survival in a subset of stage III colon cancer patients in randomized CALGB 89803 trial.

PURPOSE: Colon cancers deficient in mismatch repair (MMR) may exhibit diminished expression of the DNA repair gene, MRE11, as a consequence of contraction of a T11 mononucleotide tract. This study investigated MRE11 status and its association with prognosis, survival and drug response in patients with stage III colon cancer. PATIENTS AND METHODS: Cancer and Leukemia Group B 89803 (Alliance) randomly assigned 1,264 patients with stage III colon cancer to postoperative weekly adjuvant bolus 5-fluorouracil/leucovorin (FU/LV) or irinotecan+FU/LV (IFL), with 8 year follow-up. Tumors from these patients were analyzed to determine stability of a T11 tract in the MRE11 gene. The primary endpoint was overall survival (OS), and a secondary endpoint was disease-free survival (DFS). Non-proportional hazards were addressed using time-dependent covariates in Cox analyses. RESULTS: Of 625 tumor cases examined, 70 (11.2%) exhibited contraction at the T11 tract in one or both MRE11 alleles and were thus predicted to be deficient in MRE11 (dMRE11). In pooled treatment analyses, dMRE11 patients showed initially reduced DFS and OS but improved long-term DFS and OS compared with patients with an intact MRE11 T11 tract. In the subgroup of dMRE11 patients treated with IFL, an unexplained early increase in mortality but better long-term DFS than IFL-treated pMRE11 patients was observed. CONCLUSIONS: Analysis of this relatively small number of patients and events showed that the dMRE11 marker predicts better prognosis independent of treatment in the long-term. In subgroup analyses, dMRE11 patients treated with irinotecan exhibited unexplained short-term mortality. MRE11 status is readily assayed and may therefore prove to be a useful prognostic marker, provided that the results reported here for a relatively small number of patients can be generalized in independent analyses of larger numbers of samples. TRIAL REGISTRATION: ClinicalTrials.gov NCT00003835.

CpG island methylator phenotype is associated with response to adjuvant irinotecan-based therapy for stage III colon cancer.

BACKGROUND & AIMS: The CpG island methylator phenotype (CIMP), defined by a high frequency of aberrantly methylated genes, is a characteristic of a subclass of colon tumors with distinct clinical and molecular features. Cohort studies have produced conflicting results on responses of CIMP-positive tumors to chemotherapy. We assessed the association between tumor CIMP status and survival of patients receiving adjuvant fluorouracil and leucovorin alone or with irinotecan (IFL). METHODS: We analyzed data from patients with stage III colon adenocarcinoma randomly assigned to groups given fluorouracil and leucovorin or IFL after surgery, from April 1999 through April 2001. The primary end point of the trial was overall survival and the secondary end point was disease-free survival. DNA isolated from available tumor samples (n = 615) was used to determine CIMP status based on methylation patterns at the CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1 loci. The effects of CIMP on survival were modeled using Kaplan-Meier and Cox proportional hazards; interactions with treatment and BRAF, KRAS, and mismatch repair (MMR) status were also investigated. RESULTS: Of the tumor samples characterized for CIMP status, 145 were CIMP positive (23%). Patients with CIMP-positive tumors had shorter overall survival times than patients with CIMP-negative tumors (hazard ratio = 1.36; 95% confidence interval: 1.01-1.84). Treatment with IFL showed a trend toward increased overall survival for patients with CIMP-positive tumors, compared with treatment with fluorouracil and leucovorin (hazard ratio = 0.62; 95% CI: 0.37-1.05; P = .07), but not for patients with CIMP-negative tumors (hazard ratio = 1.38; 95% CI: 1.00-1.89; P = .049). In a 3-way interaction analysis, patients with CIMP-positive, MMR-intact tumors benefited most from the addition of irinotecan to fluorouracil and leucovorin therapy (for the interaction, P = .01). CIMP was more strongly associated with response to IFL than MMR status. Results for disease-free survival times were comparable among all analyses. CONCLUSIONS: Patients with stage III, CIMP-positive, MMR-intact colon tumors have longer survival times when irinotecan is added to combination therapy with fluorouracil and leucovorin.

Predictive and prognostic analysis of PIK3CA mutation in stage III colon cancer intergroup trial.

BACKGROUND: Somatic mutations in PIK3CA (phosphatidylinositol-4,5-bisphosphonate 3-kinase [PI3K], catalytic subunit alpha gene) activate the PI3K-AKT signaling pathway and contribute to pathogenesis of various malignancies, including colorectal cancer. METHODS: We examined associations of PIK3CA oncogene mutation with relapse, survival, and treatment efficacy in 627 stage III colon carcinoma case subjects within a randomized adjuvant chemotherapy trial (5-fluorouracil and leucovorin [FU/LV] vs irinotecan [CPT11], fluorouracil and leucovorin [IFL]; Cancer and Leukemia Group B 89803 [Alliance]). We detected PIK3CA mutation in exons 9 and 20 by polymerase chain reaction and pyrosequencing. Cox proportional hazards model was used to assess prognostic and predictive role of PIK3CA mutation, adjusting for clinical features and status of routine standard molecular pathology features, including KRAS and BRAF mutations and microsatellite instability (mismatch repair deficiency). All statistical tests were two-sided. RESULTS: Compared with PIK3CA wild-type cases, overall status of PIK3CA mutation positivity or the presence of PIK3CA mutation in either exon 9 or 20 alone was not statistically significantly associated with recurrence-free, disease-free, or overall survival (log-rank P > .70; P > .40 in multivariable regression models). There was no statistically significant interaction between PIK3CA and KRAS (or BRAF) mutation status in survival analysis (P(interaction) > .18). PIK3CA mutation status did not appear to predict better or worse response to IFL therapy compared with FU/LV therapy (P(interaction) > .16). CONCLUSIONS: Overall tumor PIK3CA mutation status is not associated with stage III colon cancer prognosis. PIK3CA mutation does not appear to serve as a predictive tumor molecular biomarker for response to irinotecan-based adjuvant chemotherapy.

Prognostic impact of deficient DNA mismatch repair in patients with stage III colon cancer from a randomized trial of FOLFOX-based adjuvant chemotherapy.

PURPOSE: The association of deficient DNA mismatch repair (dMMR) with prognosis in patients with colon cancer treated with adjuvant fluorouracil, leucovorin, and oxaliplatin (FOLFOX) chemotherapy remains unknown. PATIENTS AND METHODS: Resected, stage III colon carcinomas from patients (N = 2,686) randomly assigned to FOLFOX ± cetuximab (North Central Cancer Treatment Group N0147 trial) were analyzed for mismatch repair (MMR) protein expression and mutations in BRAF(V600E) (exon 15) and KRAS (codons 12 and 13). Association of biomarkers with disease-free survival (DFS) was determined using Cox models. A validation cohort (Cancer and Leukemia Group B 88903 trial) was used. RESULTS: dMMR was detected in 314 (12%) of 2,580 tumors, of which 49.3% and 10.6% had BRAF(V600E) or KRAS mutations, respectively. MMR status was not prognostic overall (adjusted hazard ratio [HR], 0.82; 95% CI, 0.64 to 1.07; P = .14), yet significant interactions were found between MMR and primary tumor site (P(interaction) = .009) and lymph node category (N1 v N2; P(interaction) = .014). Favorable DFS was observed for dMMR versus proficient MMR proximal tumors (HR, 0.71; 95% CI, 0.53 to 0.94; P = .018) but not dMMR distal tumors (HR, 1.71; 95% CI, 0.99 to 2.95; P = .056), adjusting for mutations and covariates. Any survival benefit of dMMR was lost in N2 tumors. Mutations in BRAF(V600E) (HR, 1.37; 95% CI, 1.08 to 1.70; P = .009) or KRAS (HR, 1.44; 95% CI, 1.21 to 1.70; P < .001) were independently associated with worse DFS. The observed MMR by tumor site interaction was validated in an independent cohort of stage III colon cancers (P(interaction) = .037). CONCLUSION: The prognostic impact of MMR depended on tumor site, and this interaction was validated in an independent cohort. Among dMMR cancers, proximal tumors had favorable outcome, whereas distal or N2 tumors had poor outcome. BRAF or KRAS mutations were independently associated with adverse outcome.

Association of TP53 mutational status and gender with survival after adjuvant treatment for stage III colon cancer: results of CALGB 89803.

PURPOSE: The TP53 tumor suppressor is frequently mutated in colon cancer, but the influence of such mutations on survival remains controversial. We investigated whether mutations in the DNA-binding domain of TP53 are associated with survival in stage III colon cancer. EXPERIMENTAL DESIGN: The impact of TP53 genotype was prospectively evaluated in Cancer and Leukemia Group B 89803, a trial that randomized stage III colon cancer patients to receive adjuvant 5-fluorouracil/leucovorin (5FU/LV) or 5FU/LV with irinotecan (IFL). RESULTS: TP53 mutations were identified in 274 of 607 cases. The presence of any TP53 mutation did not predict disease-free survival (DFS) or overall survival with either adjuvant regimen when men and women were considered together or as separate groups. However, outcome differences among women became apparent when tumor TP53 genotype was stratified as wild-type versus zinc- or non-zinc-binding mutations in the TP53 DNA-binding domain. DFS at 5 years was 0.59, 0.52, and 0.78 for women with TP53 wild-type tumors, and tumors with zinc- or non-zinc-binding mutations, respectively. Survival at 5 years for these same women was 0.72, 0.59, and 0.90, respectively. No differences in survival by TP53 genotype were observed in men. CONCLUSIONS: The presence of any TP53 mutation within the DNA-binding domain did not predict survival in stage III colon cancer. However, TP53 genotype was predictive of survival in women following adjuvant therapy. Future colon cancer therapeutic trials, with inclusion of correlative molecular markers, should be designed to permit evaluation of survival and/or response to treatment in women separately from men.

Predictive and prognostic roles of BRAF mutation in stage III colon cancer: results from intergroup trial CALGB 89803.

PURPOSE: Alterations in the RAS-RAF-MAP2K (MEK)-MAPK signaling pathway are major drivers in colorectal carcinogenesis. In colorectal cancer, BRAF mutation is associated with microsatellite instability (MSI), and typically predicts inferior prognosis. We examined the effect of BRAF mutation on survival and treatment efficacy in patients with stage III colon cancer. METHODS: We assessed status of BRAF c.1799T>A (p.V600E) mutation and MSI in 506 stage III colon cancer patients enrolled in a randomized adjuvant chemotherapy trial [5-fluorouracil and leucovorin (FU/LV) vs. irinotecan (CPT11), FU and LV (IFL); CALGB 89803]. Cox proportional hazards model was used to assess the prognostic role of BRAF mutation, adjusting for clinical features, adjuvant chemotherapy arm, and MSI status. RESULTS: Compared with 431 BRAF wild-type patients, 75 BRAF-mutated patients experienced significantly worse overall survival [OS; log-rank P = 0.015; multivariate HR = 1.66; 95% CI: 1.05-2.63]. By assessing combined status of BRAF and MSI, it seemed that BRAF-mutated MSS (microsatellite stable) tumor was an unfavorable subtype, whereas BRAF wild-type MSI-high tumor was a favorable subtype, and BRAF-mutated MSI-high tumor and BRAF wild-type MSS tumor were intermediate subtypes. Among patients with BRAF-mutated tumors, a nonsignificant trend toward improved OS was observed for IFL versus FU/LV arm (multivariate HR = 0.52; 95% CI: 0.25-1.10). Among patients with BRAF wild-type cancer, IFL conferred no suggestion of benefit beyond FU/LV alone (multivariate HR = 1.02; 95% CI: 0.72-1.46). CONCLUSIONS: BRAF mutation is associated with inferior survival in stage III colon cancer. Additional studies are necessary to assess whether there is any predictive role of BRAF mutation for irinotecan-based therapy.

Microsatellite instability predicts improved response to adjuvant therapy with irinotecan, fluorouracil, and leucovorin in stage III colon cancer: Cancer and Leukemia Group B Protocol 89803.

PURPOSE: Colon cancers exhibiting DNA mismatch repair (MMR) defects demonstrate distinct clinical and pathologic features, including better prognosis and reduced response to fluorouracil (FU) -based chemotherapy. This prospective study investigated adjuvant chemotherapy containing FU and irinotecan in patients with MMR deficient (MMR-D) colon cancers. PATIENTS AND METHODS: Cancer and Leukemia Group B 89803 randomly assigned 1,264 patients with stage III colon cancer to postoperative weekly bolus FU/leucovorin (LV) or weekly bolus irinotecan, FU, and LV (IFL). The primary end point was overall survival; disease-free survival (DFS) was a secondary end point. Tumor expression of the MMR proteins, MLH1 and MSH2, was determined by immunohistochemistry (IHC). DNA microsatellite instability was also assessed using a panel of mono- and dinucleotide markers. Tumors with MMR defects were those demonstrating loss of MMR protein expression (MMR-D) and/or microsatellite instability high (MSI-H) genotype. RESULTS: Of 723 tumor cases examined by genotyping and IHC, 96 (13.3%) were MMR-D/MSI-H. Genotyping results were consistent with IHC in 702 cases (97.1%). IFL-treated patients with MMR-D/MSI-H tumors showed improved 5-year DFS as compared with those with mismatch repair intact tumors (0.76; 95% CI, 0.64 to 0.88 v 0.59; 95% CI, 0.53 to 0.64; P = .03). This relationship was not observed among patients treated with FU/LV. A trend toward longer DFS was observed in IFL-treated patients with MMR-D/MSI-H tumors as compared with those receiving FU/LV (0.57; 95% CI, 0.42 to 0.71 v 0.76; 95% CI, 0.64 to 0.88; P = .07; hazard ratio interaction between tumor status and treatment, 0.51; likelihood ratio P = .117). CONCLUSION: Loss of tumor MMR function may predict improved outcome in patients treated with the IFL regimen as compared with those receiving FU/LV.

Modulation of tumor formation and intestinal cell migration by estrogens in the Apc(Min/+) mouse model of colorectal cancer.

Epidemiological studies suggest that post-menopausal hormone replacement therapy (HRT) reduces colorectal cancer (CRC) incidence. Phytoestrogens, including the soy isoflavone genistein and coumestrol, are used by many women as alternatives to HRT. Previous studies showed that ovariectomy induced a 77% increase in intestinal adenoma number in the C57BL/6J-Min/+ (Min/+) mouse, an animal model of adenomatous polyposis coli (APC)-associated CRC. Replacement of estradiol (E(2)) in ovariectomized Min/+ mice reduced tumor number to baseline and up-regulated the expression of estrogen receptor beta (ERbeta). We hypothesized that the phytoestrogens genistein and coumestrol would inhibit intestinal tumorigenesis in ovariectomized Min/+ mice. Min/+ and Apc(+/+) (WT) mice were ovariectomized and assigned to either a control diet or treatment with E(2), genistein or coumestrol. Treatment of ovariectomized Min/+ (Min/+ OX) mice with genistein resulted in a non-significant reduction in tumor number. Min/+ OX mice treated with coumestrol had significantly fewer tumors than untreated Min/+ OX controls and the same number of tumors as non-ovariectomized Min/+ mice. Bromodeoxyuridine migration assays also demonstrated that treatment with E(2) or coumestrol improved enterocyte migration rate. Immunoprecipitation and immunohistochemistry analyses showed that impaired association of the adherens junction proteins E-cadherin and beta-catenin in Min/+ mice was improved by treatment with either E(2) or coumestrol. Immunoblot analyses also showed that expression of ERbeta was elevated in enterocytes of Min/+ OX mice treated with E(2) or coumestrol as compared with those of untreated Min/+ OX mice. In conclusion, both coumestrol and E(2) prevent intestinal tumorigenesis and ameliorate enterocyte migration and intercellular adhesion in the Apc(Min/+) mouse model of CRC.

Prognostic significance of vascular endothelial growth factor and cyclooxygenase 2 expression in patients receiving preoperative chemoradiation for esophageal cancer.

OBJECTIVE: Both vascular endothelial growth factor and cyclooxygenase 2 overexpression have been associated with poor prognosis in a variety of human malignancies. In this study we assessed the effect of preoperative chemotherapy and radiation on expression levels of vascular endothelial growth factor and cyclooxygenase 2 in patients with esophageal cancer and determined whether these markers were associated with treatment response and overall survival. METHODS: Expression levels of vascular endothelial growth factor and cyclooxygenase 2 were measured in a cohort of 46 patients with esophageal cancer receiving preoperative chemoradiation followed by surgical resection. Immunohistochemical stains were performed on both pretreatment biopsy specimens and posttreatment resection specimens for each patient. Differences in vascular endothelial growth factor and cyclooxygenase 2 expression before and after treatment were measured, and pretreatment expression levels were correlated with treatment response and overall survival. RESULTS: We found that preoperative chemotherapy and radiation induced expression of cyclooxygenase 2 in stromal cells and induced vascular endothelial growth factor expression in both tumor and stromal cells. Pretreatment vascular endothelial growth factor expression did not correlate with treatment response, and cyclooxygenase 2 expression correlated with treatment response only in the subset of patients with squamous cell carcinoma. Although patients whose tumors expressed high levels of vascular endothelial growth factor and cyclooxygenase 2 tended to have shorter overall survival times, this trend did not reach statistical significance. CONCLUSIONS: Neither vascular endothelial growth factor nor cyclooxygenase 2 are strong predictors of treatment response and survival in patients undergoing preoperative chemoradiation for esophageal cancer. This lack of prognostic significance might be explained by changes in the expression levels of these markers during treatment.