Search

Recent studies have suggested that the presence of a c-Kit/c-Kit ligand autocrine loop may be an important regulator of proliferation and progression of human colorectal cancer, capable of affecting the prognosis of these patients. If present, the c-Kit alteration may provide a suitable target for therapy, similar to what has been observed in gastrointestinal stromal tumors. To determine the incidence of c-Kit expression in human colorectal carcinomas, we studied the immunohistochemical c-Kit expression in a selection of 126 colorectal carcinomas of different stage, using stage-oriented human cancer tissue microarrays. Formalin-fixed, paraffin-embedded tissues of each case were immunostained using the avidin-biotin-peroxidase method and the antihuman c-Kit (CD117) rabbit polyclonal antibody. High cytoplasmic c-Kit staining (Allred score of 7-8) was observed in 1.6% of the colon carcinoma patients evaluated. The c-Kit-positive tumors were poorly differentiated carcinomas arising at the anorectal junction. The remaining tumors revealed no detectable expression of c-Kit. Twenty-seven non-neoplastic tissues, normal colonic mucosa, and adenomas were also CD117 negative. We show the rare expression of c-Kit in normal and neoplastic colorectal tissues, suggesting that routine screening for c-Kit by immunohistochemistry to identify c-Kit-positive carcinomas may be cost ineffective. However, further study of c-Kit expression in poorly differentiated colon cancers may be useful since these generally chemoresistant tumors may respond to therapy with inhibitor compounds directed against c-Kit.

Src has been implicated in the development and progression of human colon cancer. Because the capacity for tumor cells to dissociate from the primary tumor is a critical step in the development of metastases, the effect of a naturally occurring, activated Src-531 on intercellular adhesion was examined. Homotypic adhesion was assessed using dissociation assays on Src-transformed rat fibroblasts and human colon cancer cell lines. The data indicate that both rodent and human cells expressing the mutant Src protein display up to 7-fold less homotypic adhesion than do wild-type cells (P0.01). Experiments demonstrated that cadherin was phosphorylated in cells transfected with activated Src and that cadherin/catenin complexes were disrupted as a result. Experiments using dominant negative (DN) Src or an Src-specific inhibitor (PD 180970), demonstrated that adhesion was restored when Src activity was inhibited in Src-531 transfectants, confirming that Src is a causal factor in the decreased homotypic adhesion observed. In addition, DN Ras, DN focal adhesion kinase (FAK), but not Stat3beta, restored intercellular adhesion, which suggested that Ras and FAK may be downstream effectors of Src-mediated homotypic adhesion. Collectively, these data support a role for Src, Ras, and FAK in the regulation of intercellular adhesion, which may in turn regulate metastatic potential of human colon cancer cells.

We used a classical rodent model of transformation to understand the transcriptional processes, and hence the molecular and cellular events a given cell undergoes when progressing from a normal to a transformed phenotype. Src activation is evident in 80% of human colon cancer, yet the myriad of cellular processes effected at the level of gene expression has yet to be fully documented. We identified a Src 'transformation fingerprint' within the gene expression profiles of Src-transformed rat 3Y1 fibroblasts demonstrating a progression in transformation characteristics. To evaluate the role of this gene set in human cancer development and progression, we extracted the orthologous genes present on the Affymetrix Hu95A GeneChip (12k named genes) and compared expression profiles between the Src-induced rodent cell line model of transformation and staged colon tumors where Src is known to be activated. A similar gene expression pattern between the cell line model and staged colon tumors for components of the cell cycle, cytoskeletal associated proteins, transcription factors and lysosomal proteins suggests the need for co-regulation of several cellular processes in the progression of cancer. Genes not previously implicated in tumorigenesis were detected, as well as a set of 14 novel, highly conserved genes with here-to-fore unknown function. These studies define a set of transformation associated genes whose up-regulation has implications for understanding Src mediated transformation and strengthens the role of Src in the development and progression of human colon cancer. Supportive Supplemental Data can be viewed at http://pga.tigr.org/PGApubs.shtml.

Background: Genetic aberration is responsible for the development of neoplastic potential in a number of malignancies. These DNA alterations result in significant changes in gene expression that may now be measured and catalogued. The microarray technique screens and identifies expressed genes that may be responsible for tumorigenesis. Methods: The authors review the application of the microarray technique in malignant melanoma. Results: Candidate melanoma suppressor genes have been identified in melanoma cell lines using this technique. Furthermore, molecular classification using gene expression profiling may improve the accuracy of the staging system for determining prognosis. Conclusions: The microarray technique is in its initial development for clinical application in a variety of tumor models. Melanoma is an ideal system to study the genetic changes associated with the stepwise progression of malignancy. It may be possible to efficiently screen the entire human genome to identify the particular aberrations in gene expression responsible for tumorigenesis in melanoma.

Introduction: Metastasis is thought to be a clonal event whereby a single cell initiates the development of a new tumor that reproduces the primary tumor at a distant site. We hypothesized that primary tumors and their derivative metastases might be genetically identical. To test this hypothesis, we used deep nextgen sequencing to assess the molecular composition of primary:metastatic tumor pairs. Methods: Nearly 20,000 tumors were molecularly profiled as part of a large personalized medicine initiative. Of these, 468 colorectal tumor samples were further assessed by targeted exome sequencing of 1321 individual genes. 16 patients produced genomic profiles paired primary:metastatic specimens; 2 patients produced paired metastases. Non-synonymous, deleterious, single nucleotide variants were identified as potential driver or passenger mutations using the BWA/GATK pipeline. Results: An average of 33.3 mutations/tumor was concordant (shared), with common drivers identified (APC, KRAS, TP53). An average of 2.3 mutations/tumor was discordant (unshared) in the paired primary and metastatic sites. Thus, the overall concordance rate for mutations was 93.5%; however, nearly all (17/18 (94.4%)) paired tumors showed at least one mutational discordance: TTN, the largest gene (5 discordant pairs); ADAMTS20, APC, MACF1, RASA1, TP53, and WNT2 (2 discordant pairs); SMAD2, SMAD3, SMAD4, FBXW7, and ∼66 others (1 discordant pair). Conclusions: Primary and metastatic tumors displayed little variance overall supporting clonality; however, in ∼50% of cases, co-evolution produced incremental, potential driver mutations in both. These results suggest analysis of the primary lesion alone may be insufficient to precisely tailor therapy. Citation Format: Timothy J. Yeatman, Jamie Teer, Danielle Grenawalt, Richard Kim, Michael J. Schell. Co-evolution of primary and metastatic colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-188. doi:10.1158/1538-7445.AM2014-LB-188

386 Background: Recent data has demonstrated that regular ASA use improves overall and cancer-specific survival in the subset of CRC patients harboring PIK3CA mutations. However, as this series only analyzed 15 PIK3CA mutant CRC patients with metastatic disease at diagnosis, it remains uncertain whether the survival benefit associated with regular ASA use extends to patients with metastatic disease. We combined data from two large academic institutions to explore the association between regular ASA use and survival in metastatic CRC. Methods: Patients with PIK3CA mutated CRC were identified at Moffitt Cancer Center (MCC) in Tampa, FL and Royal Melbourne Hospital (RMH) in Australia. Prospective clinicopathological data (including age, sex, site of disease) and survival data were available. At MCC, PIK3CA mutations were identified by exome sequencing using an Illumina Next Generation Sequencing platform with 50-100X coverage. At RMH, Sanger sequencing was used to identify PIK3CA mutations. Survival analyses were conducted using Cox regression. Results: We identified 187 CRC patients who harbored a PIK3CA mutation. Median age was 72 years and median follow up was 48 months. 49 (26%) patients used ASA regularly. 47 (25%) patients had metastatic disease at diagnosis. In univariate analyses, regular ASA use was not associated with improved overall survival (HR 0.87, p = 0.60), although there was a trend towards improved cancer-specific survival (HR 0.48, p = 0.06). In patients with stage-II or stage-III disease, regular ASA use did not improve overall, cancer-specific or recurrence-free survival. However, in stage-IV patients, regular ASA use was significantly associated with improved overall (HR 0.35, p = 0.04) and cancer-specific (HR 0.28, p = 0.02) survival in a univariate analysis. Conclusions: Our study demonstrates that in patients with metastatic CRC harboring a PIK3CA mutation, regular ASA use is associated with a significant overall and cancer-specific survival advantage. However, we were not able to confirm the survival advantage across all stages. To our knowledge, our study is the largest to examine ASA use in PIK3CA mutated CRC.

Colorectal cancer is the third most common site for cancer in men and women in the United States. It is estimated that there will be 36,400 new diagnoses of rectal cancer and 8,600 deaths from rectal cancer in the year 2000. 1 The current standard treatment for distal rectal cancer is abdominoperineal resection (APR), low anterior resection, or resection with coloanal anastomosis. These operations are associated with significant rates of death and complications, and local or distant recurrences occur in 10% to 65% of patients. 2 The complications associated with radical rectal surgical procedures include urinary dysfunction in 10% to 70%, sexual dysfunction in 13% to 70%, and anastomotic leaks in 5% to 17%, with death rates of 2% to 6%. 3–10 Compared with a radical resection for distal rectal cancer, local excision avoids a laparotomy, permanent colostomy, and the complications associated with pelvic dissection. The incidence of local recurrence even after radical surgery ranges from 10% to 29%. 11–14 A recent review of published series reported an 18.5% overall local recurrence rate after APR. The incidence of local recurrence increased with advancing stage: 8.5%, 16.3%, and 28.6% for Dukes A, B, and C, respectively. 11 Extrapelvic and distant recurrences occur in approximately 30% of patients. These patients likely present with occult metastatic disease and would not be expected to benefit from the more radical operations. The 5-year disease-free survival rate of patients with node-positive rectal cancers is 30% to 40%. 2 Therefore, most patients with advanced rectal cancers are not cured by radical resection of the tumor. For these reasons, treatment alternatives for distal rectal cancers are of interest. Historically, local excision for distal rectal cancers has been approached with caution because of the high rates of local recurrence. Local therapy alone for rectal cancer has been used for patients with significant comorbid conditions that make a more radical surgery prohibitive. With newer techniques in adjuvant radiation therapy and advancements in chemotherapy, it has been possible to explore the option of multimodality treatment schemas to improve local control rates and allow better functional outcomes in a select group of patients with distal rectal cancer. In the United States, initial studies of preoperative radiation treatment for rectal cancer were influenced by the lack of efficacy of low-dose (2,000–3,000 cGy) radiation. However, data from Europe suggest that preoperative radiation alone reduces local recurrence rates and improves overall survival compared with surgery alone 15 and was more effective than postoperative radiotherapy. 16 Combination therapy using moderate-dose (4,000–4,500 cGy) and high-dose (>5,000 cGy) chemoradiation has allowed downstaging of tumors in 59% to 76% of patients, with complete pathologic response rates of 20% to 44%. 17–20 These studies have encouraged the use of preoperative chemoradiation and expanded the realm of surgical options to include sphincter preservation. We report on a highly select group of patients with advanced distal rectal cancers who had good responses to preoperative chemoradiation therapy and were treated with transanal local excisions.

Microarray gene expression technology has recently made it feasible to characterize the RNA expression of thousands of genes across numerous tissue samples. We hypothesized that the warm ischemia commonly associated with the surgical extirpation of human tissue would have significant effects on gene expression profiles. To quantitate the effects of warm ischemia on human tissue, we rapidly dissected normal mucosa from a human colon cancer specimen. The specimen was divided and maintained at room temperature until snap-frozen in liquid nitrogen. Aliquots of tissue were frozen at times 5, 10, 15, 20, 40, and 60 min after extirpation. Spotted microarrays composed of 2400 distinct elements were used to assay mRNA derived from each time point in triplicate. Eisen's hierarchical clustering methodology and Bayesean statistical methods were then used to assay the effects of warm ischemia on gene expression. Application of time-course statistical models suggest that three patterns were induced by ischemia, accounting for 68.2, 17.8, and 13.4% of the evaluable genes, respectively. Pattern I corresponds to an average change of 27% over 60 min from 5 min baseline level of expression and 63.8% of the genes with at least 80% probability of membership in this pattern show average increases in expression over 60 min. The remainder decrease on average. Pattern II genes show the least ischemia-related effects, demonstrating an average change of only 12% over 60 min. In contrast to pattern I, we find that 67.5% of the genes with at least 80% probability of membership in this pattern are decreasing in expression on average over time. The remaining 32.5% in this pattern increase an average of 12% over 60 min. Finally, pattern III genes (13.4% of the sample) show the greatest sensitivity to ischemia, changing an average of 50% over 60 min, with about the same number increasing as are decreasing. Fold changes in RNA over- or under-expression were observed up to greater than 20-fold. Warm ischemia associated with the surgical extirpation of human tissues has significant effects on gene expression. These data support the careful monitoring of ischemic time for tissues harvested for the purposed of gene profiling.

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3β protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c -myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3β protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF → Src → Stat3 → Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.

The NCCN Colorectal Cancer Guidelines panel believes that a multidisciplinary approach is necessary for the management of the patient with colorectal cancer. The panel endorses the concept that treatment of patients in a clinical trial has priority over standard or accepted therapy. The recommended surgical procedure for resectable colon cancer is an en bloc resection; laparoscopic surgery should be done only in the context of a clinical trial. For patients with stage III disease, 5-FU-based adjuvant therapy is recommended. A patient who has metastatic disease in the liver or lung should be considered for surgical resection if he or she is a candidate for surgery and if surgery can extend survival. Surgery should be followed by adjuvant chemotherapy. The panel advocates a conservative post-treatment surveillance program for colon and rectal carcinoma patients. Serial CEA determinations are appropriate if the patient is a candidate for aggressive surgical resection, should recurrence be detected. Abdominal and pelvic CT scans should be utilized only when there are clinical indications of possible recurrence. Patients whose disease progresses during 5-FU-based therapy should be treated with irinotecan or encouraged to participate in a phase I or phase II clinical trial.

Since the original identification of a transmissible agent responsible for the development of tumors in chickens, now known to be a retrovirus encoding the v-src gene, significant progress has been made in defining the potential functions of its human homolog, SRC. The product of the human SRC gene, c-Src, is found to be over-expressed and highly activated in a wide variety of human cancers. The relationship between Src activation and cancer progression appears to be significant. Moreover, Src may have an influence on the development of the metastatic phenotype. This review discusses the data supporting a role for c-Src as a critical component of the signal transduction pathways that control cancer cell development and growth, and provides the rationale for targeting Src in drug discovery efforts.

The holy grail of cancer therapy in the near future could relate to the identification of biomarkers that might enable personalized therapy for cancer—finding the right drug for the right patient. There is a growing list of examples supporting the feasibility of biomarker selection to predict therapeutic response. Biomarkers may fall into the category of response or nonresponse signatures; they may be single gene or multigene predictors. Specific, fairly straightforward examples include the estrogen receptor predicting response to tamoxifen, c-Kit activation predicting response to imatinib, or HER2 amplification predicting response to trastuzumab. More recently, RAS mutation has been found to be a nonresponse predictor for cetuximab. To clarify, the presence of a wild-type RAS does not predict response, but a mutant RAS is associated with the high likelihood of nonresponse. To date, it has been difficult to reliably identify biomarkers that enable drug therapy. Although HER2 and RAS may have been predicted to select or de-select patients for specific therapy, the discoveries were made after clinical trial observations. The more common use of gene profiling technology, however, has made it feasible to begin genome-wide searches for biomarkers that might predict response or nonresponse to therapy. This approach attempts to correlate the expression of upwards of 30,000 expressed genes (and many more proteins) to clinical outcomes after therapy. Thousands of microarray studies have been performed validating the potential of the technology to create molecular signatures linked to phenotypic end points. There are numerous examples of microarray-based molecular signatures falling into the prognostic or predictive category. Prognostic signatures have been identified for many common diseases such as breast cancer, colon cancer, lung cancer, and even hepatocellular cancer. Similarly, a number of predictive signatures have been identified. Only a handful of signatures, however, have been successfully validated, commercialized, and brought forward to the clinic. Many of the signatures still reside in the developmental phase, requiring substantially more validation before clinical application. To be clinically useful, a molecular signature should have independent predictive value superior to clinicopathologic staging or other molecular means for predicting prognosis or response to therapy. To be effective, signatures must be developed in independent training and test sets, with assurances that the two data sets have not been shared in the analysis. The inherent problem associated with microarray data sets is termed “overfitting” the data. This occurs when many elements (genes) are correlated with a few clinical end points (survival, recurrence, and metastasis). Thus, it is easy to fathom how a small number of genes from a list of 30,000 might be found to correlate in expression, by random chance, with a single clinical end point such as response to therapy or survival. The article by Debucquoy et al begins to address a significant problem in rectal cancer therapy—can patient selection for therapy be improved? Forty-one patients with rectal cancer received preoperative radiotherapy in combination with capecitabine and cetuximab in association with a clinical trial designed to examine molecular profiles of patients before and during therapy. Tumor biopsies and blood samples were obtained before initiation of cetuximab therapy and 1 week after induction before initiation of radiotherapy. These biopsies were applied to Affymetrix GeneChips (Santa Clara, CA) and differential gene expression associated with therapy was identified. The study identified a putative set of dysregulated genes linked to a single dose of cetuximab therapy. Genes involved in proliferation and invasion were downregulated, and genes linked to inflammation were upregulated. Endothelial growth factor receptor (EGFR) upregulation was linked to better disease-free survival. Interestingly, plasma transforming growth factor–alpha, but not EGF or EFGR, was upregulated in patients after cetuximab therapy. Previously reported epiregulin and amphiregulin overexpression were not linked to response in this study. Similarly, RAS mutations did not seem to confer resistance to therapy in the current study. The current study, although enticing, falls short of confirming or validating the identified gene signature as being predictive or clinically useful. The difficulty in obtaining carefully controlled clinical trial data with molecular end points, however, should not be underestimated. This study, although too small to validate any derivative gene signatures, was able to create testable hypotheses. These hypotheses will need to be validated with additional data sets derived on a single, standardized platform to ensure that the identified genes were not the simple product of overfitting the data. This sort of problem will likely not be reduced by the introduction of nextgen gene sequencing technology or proteomics technology, each producing hundreds of thousands of new data points for correlative studies. It is clear that large genome-wide assessments linked to carefully curated longitudinal clinical data and performed on standardized analysis platforms are sorely needed. Even the largest microarray studies often involve only several hundred patients, when close to a thousand might be preferred, to account for the inherent heterogeneity of cancer. Only this sort of longitudinal effort may produce the sample sizes required to properly validate tangible gene signature hypotheses such as that proposed by Debucquoy et al and ultimately find the right patient for the right drug. It is now just a matter of time.

The significance of breast cancer lymph node micrometastasis has been addressed by numerous studies. Although the results of these studies are mixed, most of the larger trials strongly suggest that the presence of a lymph node micrometastasis, in the absence of other nodal disease, will have a negative impact on disease free survival and on overall survival. The focus of this article is to analyze the biologic and therapeutic significance of micrometastatic disease in the axillary lymph nodes of patients with breast cancer.

Lymphatic mapping techniques have the potential of changing the standard of surgical care of breast cancer patients. This paper reports a prospective study documenting the safety and efficacy of sentinel lymph node biopsy in 167 breast cancer patients and reviews the world literature on the procedure.One hundred sixty-seven patients with newly diagnosed breast cancers underwent a prospective trial of intra-operative lymphatic mapping using a combination of vital blue dye and filtered technetium-labeled sulfur colloid. A sentinel lymph node (SLN) was defined as a blue node and/or "hot" node with a 10/1 ex-vivo gamma-probe ratio of SLN to non-SLN. All SLN were bi-valved, step-sectioned, and examined with routine HE stains and immunohistochemical stains for cytokeratin. Cytokeratin-positive SLN were defined as any SLN with a defined cluster of positive staining cells which could be confirmed histologically on HE sections. Finally, a review of the worldwide data was undertaken using a uniform analytical method to compare the rates of sensitivity, diagnostic accuracy, and false negatives of SLN mapping.In 167 patients, 337 SLN were harvested, for an average of 2.01 SLN/patient. Fifty-two (31.1%) of the patients had metastasis in the SLN. In the 115 patients with negative SLN, 1 was found to have tumor in higher axillary nodes, for a false negative rate of 0.88%. Fifty-nine (37.8%) of the patients were diagnosed by fine-needle aspiration, 89 (53.3%) by excisional biopsy, and 19 (11.4%) by core biopsy. Positive SLN were identified in 1/17 (5.9%) patients with DCIS. Metastasis was found in 33/115 (28.7%) of the patients with infiltrating ductal tumors and in 11/19 (57.9%) of the patients with infiltrating lobular tumors. Positive SLN were identified in 7/16 (43.7%) of the patients with mixed cellularity tumors. Metastasis in the SLN was detected in 7/55 (12.7%) of the 59 patients with T1a-T1b tumors and in 21/58 (36.2%) of the patients with T1c tumors. Positive SLN were found in 17/30 (56.7%) of the patients with T2 tumors and in 6/7 (85.7%) of the patients with T3 tumors. A literature review of 731 patients (including this study) demonstrates a sensitivity rate of 95% and a diagnostic accuracy rate of 98%. The overall false negative rate is 3.1%.This study demonstrates that SLN biopsy is a highly sensitive and accurate method of predicting axillary nodal status. It is a reproducible technique that is easily learned. The future addition of more sensitive methods such as PCR evaluation of nodal involvement may reduce the need for widespread use of adjuvant chemotherapy with its high cost and attendant morbidity and mortality. We believe that this technique will eventually become the standard of care in the treatment of breast cancer, particularly for T1 and T2 lesions and perhaps also for high-grade DCIS tumors.

Loss of p53 and p21WAF1 expression have previously been reported in pancreatic adenocarcinoma. Despite these findings in several reports of oncogene and tumor suppressor gene alterations in pancreatic cancer, the clinical significance of these changes is still poorly understood. In an attempt to detect molecular prognostic markers for pancreatic carcinoma, we studied the immunohistochemical expression of p53, p21WAF1, and TGF-beta1 proteins in 42 pancreatic adenocarcinomas of the ductal type. The results were correlated with clinicopathologic findings to identify the markers with prognostic significance. p53 nuclear immunoreactivity was seen in 20 (48%) of the cases, and it was strong to moderate in 14 (33%) of them. p21WAF1 cytoplasmic positivity was found in 16 (38%) of the tumors, with 72% staining strong to moderate. TGF-beta1 stained the cytoplasm of the tumor cells in 13 (31%). Of the p53-negative cases, 12 (54%) exhibited p21WAF1 expression. In 3 (30%) of cases, TGF-beta1 reactivity was seen in the absence of p53 and p21WAF1 p53 positivity identified tumors of higher grade, but did not correlate with stage or survival. TGF-beta1 expression, however, identified low-grade tumors and patients with longer survival. No correlation was found between the expression of any of these molecular markers and smoking history. We report a significant correlation between TGF-beta1 reactivity and low-grade tumors and between TGF-beta1 and better survival. This is a novel finding pointing to TGF-beta1 as a possible new stage-independent predictor of tumor survival in pancreatic ductal adenocarcinoma. In agreement with others, we also found p53 mutation in 20 (48%) of the tumors.

OBJECTIVE: To define preliminary guidelines for the use of lymphatic mapping techniques in patients with breast cancer. SUMMARY BACKGROUND DATA: Lymphatic mapping techniques have the potential of changing the standard of surgical care of patients with breast cancer. METHODS: Four hundred sixty-six consecutive patients with newly diagnosed breast cancer underwent a prospective trial of intraoperative lymphatic mapping using a combination of vital blue dye and filtered technetium-labeled sulfur colloid. A sentinel lymph node (SLN) was defined as a blue node and/or a hot node with a 10:1 ex vivo gamma probe ratio of SLN to non-SLN. All SLNs were bivalved, step-sectioned, and examined with routine hematoxylin and eosin (H&E) stains and immunohistochemical stains for cytokeratin. A cytokeratin-positive SLN was defined as any SLN with a defined cluster of positive-staining cells that could be confirmed histologically on H&E sections. RESULTS: Fine-needle aspiration (FNA) or stereotactic core biopsy was used to diagnose 195 of the 422 patients (46.2%) with breast cancer; 227 of 422 patients (53.8%) were diagnosed by excisional biopsy. The SLN was successfully identified in 440 of 466 patients (94.4%). Failure to identify an SLN to the axilla intraoperatively occurred in 26 of 466 patients (5.6%). In all patients who failed lymphatic mappings, a complete axillary dissection was performed, and metastatic disease was documented in 4 of 26 (15.4%) of these patients. Of the 26 patients who failed lymphatic mapping, 11 of 227 (4.8%) were diagnosed by excisional biopsy and 15 of 195 (7.7%) were diagnosed by FNA or stereotactic core biopsy. Of interest, there was only one skip metastasis (defined as a negative SLN with higher nodes in the chain being positive) in a patient with prior excisional biopsy. A mean of 1.92 SLNs were harvested per patient. Twenty percent of the SLNs removed were positive for metastatic disease in 105 of 440 (23.8%) of the patients. Descriptive information on 844 SLNs was evaluated: 339 of 844 (40.2%) were hot, 272 of 844 (32.2%) were blue, and 233 of 844 (27.6%) were both hot and blue. At least one positive SLN was found in 4 of 87 patients (4.6%) with noninvasive (ductal carcinoma in situ) tumors. A greater incidence of positive SLNs was found in patients who had invasive tumors of increasing size: 18 of 112 patients (16%) with tumor size between 0.1 mm and 1 cm had positive SLNs. However, a significantly greater percentage of patients (43 of 131 [32.8%] with tumor size between 1 and 2 cm and 31 of 76 [40.8%] with tumor size between 2 and 5 cm) had positive SLNs. The highest incidence of positive SLNs was seen with patients of tumor size greater than 5 cm; in this group, 9 of 12 (75%) had a positive SLN (p < 0.001). CONCLUSIONS: This study demonstrates that accurate SLN identification was obtained when all blue and hot lymph nodes were harvested as SLNs. Therefore, lymphatic mapping and SLN biopsy is most effective when a combination of vital blue dye and radiolabeled sulfur colloid is used. Furthermore, these data demonstrate that patients with ductal carcinoma in situ or small tumors exhibit a low but significant incidence of metastatic disease to the axillary lymph nodes and may benefit most from selective lymphadenectomy, avoiding the unnecessary complications of a complete axillary lymph node dissection.

Current models suggest that colon cancer initiation and progression are secondary to both the activation of oncogenes and the deletion of tumor suppressor genes. The role of each, however, is still poorly understood, particularly with regard to the induction of metastasis. We hypothesized that genetic differences exist between tumors that metastasize distantly and those that do not, and that oncogenes and tumor suppressor genes participate equally in this process. To address this hypothesis, human tumor specimens from localized [tumor-node-metastasis (TNM) stage I-III] and primary colon cancers (n = 10) were directly compared with metastatic (TNM stage IV) lesions (n = 10) using comparative genomic hybridization analysis. Although several alterations were shared equally between primary tumors and metastases (+7q, +19q, and +20q), two patterns of distinguishing alterations were observed: (a) alterations that were more extensive in liver metastases than in primary tumors (+8q, +13q, -4p, -8p, -15q, -17p, -18q, -21q, and -22q); and (b) alterations that were unique to metastatic lesions (-9q, -11q, and -17q). Overall, genetic losses were more common than gains, and, more importantly, the number of losses/tumor was significantly higher for metastases than for primary tumors (9.3 + 1.3 versus 4.1 + 0.7; P = 0.00062, Wilcoxon's rank-sum test). The distinct predominance of genetic losses in the metastatic lesions when compared with the primary localized tumors provides evidence that the metastatic phenotype is induced by the deletion of tumor suppressor genes and permits the construction of physical maps targeting regions where novel tumor suppressor genes are likely to exist.

Recent data suggest that signal transduction may have a critical role in the development and regulation of the metastatic phenotype. Here, we investigated the role of c-Src activation in the process of human colon cancer metastasis to the liver. Our data, derived from two different sets of human colon cancer cell line metastatic variants, suggest that not only do highly-metastatic cells display constitutively elevated c-Src protein kinase activity when compared to poorly metastatic cells, but also that receptor tyrosine kinases participate in the ligand-activation of c-Src above basal levels. Specifically, the epidermal growth factor receptor (EGFR), p185HER2/Neu and the hepatocyte growth factor receptor (c-Met) appear to be linked to the process because they preferentially activate c-Src in highly-metastatic cells. EGFR was found to associate with c-Src in colon cancer cells and specific inhibitors of the EGFR resulted in a reduction of c-Src activity to basal levels. In addition, c-Src transfectants displayed partially-activated EGFRs, suggesting a feedback role for c-Src in the regulation of the EGFR. p185HER2/Neu was also identified in immunocomplexes of c-Src following ligand activation of the EGFR, but only in highly-metastatic cells. Collectively, these observations suggest a paradigm whereby c-Src interacts with multiple cell-surface growth factors in a catalytic fashion for the development of tumor cells with metastatic potential.

Whereas genetic paradigms are now defined for the development of human colon cancer, little is known regarding the mechanisms that regulate development of the metastatic phenotype. Recent reports have indirectly linked the expression and activation of c-Src to the process of human colon cancer metastasis. Whereas v-Src, a highly activated mutational derivative of c-Src, has been shown to induce metastasis, normal c-Src has not been tested for this property. We hypothesized that c-Src overexpression in the milieu of a poorly metastatic cancer cell might permit the development of a highly metastatic cell. Two poorly metastatic human colon cancer cell lines were stably transfected with expression vectors encoding normal human c-Src. Clones producing 4-10-fold more c-Src than controls were injected s.c. and intrasplenically into the nude mouse to assess primary tumor growth and liver metastatic potential. Whereas metastatic potential was unaffected, primary tumor growth in vivo was significantly enhanced by c-Src overexpression. No effects on rates of tumor cell proliferation were seen in vitro. Our findings suggest that normal c-Src may be necessary but is insufficient for the induction of the metastatic phenotype.

The surgical evolution of breast cancer treatment over the past century from Halsted through Haagensen and Urban has demonstrated the utility of radical and ultra-radical surgery. Patey, Meyer, and subsequently Fisher’s studies have initiated the movement toward surgical breast conservation. The continued progress toward lesser surgery has brought the value of axillary lymph node dissection (ALND) under close scrutiny (Frazier et al. 1977; Silverstein et al. 1987; Balch et al. 1993).

Carcinoembryonic antigen (CEA) has been recently implicated in the process of human colon cancer liver metastasis by means of an adhesion mechanism. Based on the strong sequence and structural homology of biliary glycoprotein (BGP) to CEA, we hypothesized that BGP might be overexpressed at the RNA and protein level in tumor cells with high metastatic potential. We have found the BGP messenger RNA derived from highly metastatic colon cancer cells is constitutively overexpressed—nearly fourfold greater than poorly metastatic cells—and that BGP expression is induced by interferon-gamma. Similarly, we have demonstrated that BGP protein levels were constitutively elevated in highly metastatic human colon cancer cells when compared to poorly metastatic cells. Collectively these results suggest that the basal and interferon-stimulated expression of BGP transcripts may be regulated in a manner similar to CEA and that a potential role in the process of metastasis may be inferred.

Computed tomographic arterial portography (CTAP) has been shown to be the most sensitive preoperative test for determining resectability of hepatic lesions but we have shown it to have low specificity. Intraoperative ultrasound (IOUS) evaluation of the liver has also been proposed as an accurate means of assessing resectability. We sought to compare the effectiveness of the two modalities. Fifty-six patients who had been deemed candidates for liver resection based on CTAP findings underwent systematic exploration, liver mobilization, and IOUS examination. Ultrasound findings were compared with results of CTAP. In 46 patients the IOUS findings were in complete agreement with those of CTAP. In 10 patients CTAP lesions could not be verified by IOUS and these patients did not undergo resection. Follow-up of these 10 patients revealed eight who did not have progression of malignancy at the CTAP-predicted site (CTAP false positive). Two patients did have progression at a CTAP-positive IOUS-negative site (IOUS false negative). Sensitivity for CTAP and IOUS was 100% and 96%, respectively. Specificity for IOUS was 100%. These findings demonstrate the high sensitivity of CTAP and the high sensitivity and specificity of IOUS. CTAP may "overcall" hepatic lesions but IOUS can correctly identify these false positives in most instances. Because CTAP is useful for determining which patients might benefit from surgical exploration, we conclude that the two modalities are complementary for the assessment of resectability of hepatic lesions. The false positive rate for CTAP implies that caution must be used when declining to operate on patients on the basis of this test.

3644 Background: Recent work has shown an association between longer survival and aspirin use in colorectal cancer (CRC) patients with mutated PIK3CA. It has been hypothesized that this survival advantage could occur via blocking the PI3K pathway and allowing apoptosis of mutated cancer cells. The goal of this work is examine the use of aspirin and outcome in CRC patients with PI3KCA mutation. Methods: PIK3CA mutation status was assessed in paraffin-embedded tumor samples from 471 CRC patients between 1998-2010. PIK3CA mutation was assessed by exome sequencing using an Illumina Next Generation (NGS) platform with 50-100X coverage on all patients. The BWA/GATK pipeline was used to identify variants and indels. Because matched normal samples were not available for comparison to identify somatic mutations, filtering of normal variants was performed using 1000 Genomes. The usage of aspirin was collected retrospectively with electronic chart review. Results: Out of 471 patients, 73 were found to have unique PIK3CA mutations by NGS (15.4%). The most common mutations were found at codon 9 (38%) and codon 20 (21%). Patients had a median follow up of 47 months. Initial stage at diagnosis for PIK3CA mutants were as follows: 11 pts were stage I, 31 pts were stage II, 24 pts were stage III and 16 patients were stage IV. Of patients who died, those taking ASA had a 5% cancer related mortality compared to 23% cancer related mortality in non-ASA users. In contrast, the non-cancer related mortality was 25% in ASA users and only 8% in non-ASA users. Cancer specific rates for five year survival were 90% in the ASA group and 57% in the non-ASA group for all stages. In stage IV patients, there was 80% five year survival in the ASA users and 32% in the non-ASA group. Conclusions: There was a trend toward improvement in five year survival for colorectal cancer patients with PIK3CA mutations who used ASA. This trend persisted even in stage IV patients. Notably, non-cancer related deaths were higher in the ASA users, most likely secondary to medical comorbidities that necessitated ASA use. As follow up continues and this data set matures, future work will focus on validating these preliminary results and relating specific PIK3CA mutations to ASA response.

G A A b st ra ct s CDC5L expression was found in the nuclei of normal murine intestinal epithelial cells. Areas of the colonic epithelium in AhCre+Apcfl/fl and ApcMin/+ mice which showed evidence of Wnt signalling by nuclear localisation of beta catenin showed increased nuclear expression of SFRS2 and cytoplasmic localisation of CDC5L. CDC5L knockdown in HCT116 and HT29 cells resulted in significant inhibition of cell proliferation by both SRB and clonogenic assays (p,0.05). SFRS2 knockdown also resulted in increased nuclear expression of CDC5L in these cell lines. The malignant colonic lesions that developed in ApcMin/+Pten-/mice showed loss of nuclear beta catenin expression and reversal of the phenotype observed in ApcMin/+ mice, with nuclear CDC5L localisation once again. Conclusion Apc inactivation results in increased nuclear expression of SFRS2 in colonic epithelial cells in vivo. This coincides with translocation of CDC5L from the nucleus into the cytoplasm, which limits its ability to regulate the cell cycle and splice pre-mRNA. Acquisition of an additional mutation in Pten alters Wnt signalling further, causing reduced nuclear accumulation of beta catenin and allowing CDC5L to translocate back into the nucleus, where it possibly stabilises the tumor phenotype.

Background: Intraductal papillary mucinous neoplasms (IPMNs) are cystic pancreatic cancer (PC) precursors that are increasingly being detected incidentally by cross-sectional imaging. IPMNs harbor potential for invasive malignancy depending on the degree of histologic dysplasia which ranges from low to high grade, yet the only way to determine disease severity is through surgical resection and histopathologic investigation. Mi(cro)RNAs, non-coding RNAs that regulate one-third of protein coding genes, are attractive candidate biomarkers of early pancreatic malignancy because they are detectable in tissue and blood in a stable form, making them amenable to reliable measurement, and they have been implicated in the development and progression of PC. The goal of our pilot project was to discover and characterize a miRNA signature that accurately differentiates between low-grade IPMNs that merit continued surveillance and high-grade IPMNs that warrant immediate surgical resection. Methods: In our discovery phase, we microdissected formalin-fixed paraffin-embedded tissue (FFPE) and isolated total RNA from 28 pathologically-confirmed IPMNs (9 low-grade and 19 high-grade) surgically resected at our institution between 1999 and 2011, and evaluated the expression of 378 mature miRNAs using high-throughput Taqman Low Density Arrays. After normalization using the endogenous control RNU44, a rank-sum test was performed to compare the expression between the two groups for each miRNA. Results: Using a false discovery rate of 10%, there were 13 differentially expressed miRNAs with statistical significance. The top candidates include miR-100 (P=1.6 x10−3), miR-99b and miR-99a (P=2.7 x10−3), miR-342-3p and miR-126 (P = 3.7 x10−3), and miR-130a (P = 3.7 x10−3). Several of these miRNAs were highlighted in a recent investigation of FFPE tissue and cyst fluid from patients with IPMNs and other pancreatic cysts, demonstrating consistency of findings. Furthermore, the expression level of the top candidate miRNAs was down-regulated in high-grade compared to low-grade IPMN tissue, in line with recent data supporting a role for these miRNAs as tumor suppressors and regulators of key genes that contribute to cell proliferation and invasion in pancreatic and other malignancies. Analysis is underway to evaluate the expression of the top candidate miRNAs in an independent set of high- and low-grade IPMN tissue specimens, and to correlate miRNA expression with other possible clinical predictors of malignant potential. Conclusions: Although preliminary, our findings suggest that miRNAs may serve as a diagnostic adjunct for stratifying patients with IPMNs for continued surveillance or surgical resection. Citation Format: Jennifer Permuth Wey, Susan McCarthy, Y. Ann Chen, Kate Fisher, Agnieszka Kasprzak, Mark Lloyd, Xiaotao Qu, Timothy Yeatman, Jason Klapman, Domenico Coppola, Mokenge Malafa. Tackling a clinical challenge: Using microRNAs to differentiate between low-and high-risk Pancreatic Cysts . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-70. doi:10.1158/1538-7445.AM2013-LB-70

Background: The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis, and resistance to chemotherapy. The EGFRi class of reagents are thought to target tumors with the epithelial phenotype. Hypothesis: We hypothesized that reversing this biological process through a mesenchymal to epithelial transition (MET), using a recently identified miR-147, might also lead to a reversal of resistance to EGFR inhibitors (EGFRi). Methods/Results: Following a screen of > 400 miRs, we recently identified miR-147 as highly correlated with an “intrinsic” and prognostic EMT gene expression signature derived from an unsupervised analysis of >2000 colorectal cancers. This suggested miR-147 might regulate the EMT program. We have shown that miR-147 is capable of reversing the EMT phenotype in vitro and converting EGFRi-resistant cells to sensitive cells. To further understand the mechanism underpinning this observation, we analyzed the genes affected by miR-147 over-expression in colon cancer cells using a global gene expression survey (Affymetrix). Among significantly altered genes, Akt expression was down-regulated (>2 fold) and Stat3 expression was up-regulated (>2 fold) by miR-147. Subsequent knock down of Akt with siRNA did induce MET and significantly increased EGFRi sensitivity. Western analysis showed increased Stat3 protein levels and total Stat3 phosphorylation with miR-147 transfection. Interestingly, knockdown of Stat3 inhibited the capacity of miR147 to reverse native EGFRi resistance. Conclusion: miR-147 induced MET and reversed resistance to EGFRi. This observation appears to be related to both the inhibition of Akt and to the induction of Stat3 activity. Understanding the precise mechanisms by which miRs regulate chemosensitivity and resistance will be important in designing more effective EGFRi therapies. Citation Format: Chang Gong Lee, Michael Gruidl, Cindy R. Timmee, Susan McCarthy, Timothy J. Yeatman. MiR-147 is a novel tumor suppressor that reverses EMT and native resistance to EGFR inhibitors by inactivating Akt and activating Stat3. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-43. doi:10.1158/1538-7445.AM2013-LB-43

345 Background: It is well known that KRAS mutations limit the efficacy of anti-EGFR therapy in patients with mCRC. Therefore, accurate testing of KRAS is needed to ensure that appropriate patients receive anti-EFGR therapy. Most clinical institutions conduct KRAS testing in CLIA approved labs using standard DNA sequencing methods. The purpose of this study is to correlate KRAS mutation detection by Illumina KRAS gene sequencing to standard KRAS testing performed with CLIA. Methods: We analyzed tumor samples collected from 471 patients between 1998-2010. Patients were chosen randomly based on availability of sufficient tissue for DNA extraction. We performed targeted exome sequencing using an Illumina NGS platform with 50-100X coverage of KRAS. The BWA/GATK pipeline was used to identify variants and indels. Because matched normal samples were not available for comparison to identify somatic mutations, filtering of normal variants was performed using 1000 Genomes. Variants identified in 1000 Genomes with an MAF < 0.01 were filtered. Results: Out of 471 patients, 83 pts had KRAS testing done both by exome sequencing & CLIA approved labs. The concordance rate between the two testing methods was 89%. 39 pts (47%) were KRAS mutation negative (wild type) and 35 pts (46%) harbored KRAS mutation as determined by both methods. Of these, 31 pts had codon 12 mutations and 4 pts had codon 13 mutations. However, 6 pts (7%) were found to have no KRAS mutation using CLIA but were found to have KRAS mutations by NGS (two had A146V mutations, the others had Q61 mutations). In addition, 3 pts (4%) were KRAS mutants using CLIA but wild type by NGS. Thus, 10% of samples tested showed a discrepancy in outcome between the NGS & CLIA testing. Conclusions: Standard DNA sequencing methods (CLIA) was able to identify a majority of the KRAS mutants detected by NGS. However 10% of patients had either false positive or false negative results. The main explanation for this discrepancy is that CLIA approved labs generally only test for codons 12 & 13. The clinical role for NGS in tumor KRAS mutation detection should be further investigated to ensure that patients with mCRC receive appropriate therapy.

Understanding the genetic elements controlling the process of tumor metastasis to distant organ sites such as the liver may be the key to improving survivorship from colon cancer. By using standard cytogenetic techniques in combination with comparative genomic hybridization, multiple genetic imbalances within three human colon cancer cell lines previously selected for differences in liver-metastatic behavior were identified. The entire genome of one poorly metastatic cell line (KM12C) was compared directly with that of two highly metastatic cell lines (KM12SM, KM12L4A) derived from it. A number of chromosomal gains (8q, 12q15, 20q11.2) and losses (5p13, 6p21.3, 18) were common to all three cell lines and are likely related to early tumor development rather than to the selection process used to generate cell lines of increased metastatic potential. Chromosomal imbalances detected only in the highly metastatic cell lines were also observed. KM12SM showed losses of portions of 2p22, 2q24.3--2q32.2, 4p15.3--cen, 4q24 without the 13q and 15q22.3 gains noted for KM12C. Both gains (1p31.3--1p21, 2q22--2q33, 3cen--3q26.2, 5q14--5q23, 6cen--6q23) and losses (16p, 17p, 17q 19p, 19q 22q) were observed for KM12L4A but not for the other two cell lines. Identification of these alterations provides valuable insight into the process of experimental liver metastasis and is a first step towards mapping genes linked to the terminal phases of human colon cancer progression.

Breast cancer is considered a chronic disease in most women. This belief is based on natural history data that suggest that some patients with untreated, advanced disease may survive for up to and beyond 20 years after diagnosis. These data are corroborated by studies of treated patients in whom breast cancer recurred up to 15 to 20 years postmastectomy. Conversely, there are also patients who die rapidly after presentation. These divergent observations suggest that there are at least two subpopulations of patients with breast cancer-one that co-exists with the disease and one that rapidly succumbs to it. This heterogeneous biologic behavior is likely related to divergent tumor cell growth rates that have been documented as well as to other unidentified factors. These two subsets of patients, unfortunately, are quite difficult to distinguish. With the promise of the ongoing genetic revolution, the hope is that genes associated with rapidly progressing disease states can be identified. It is important to be cognizant of the prolonged natural history of this disease whenever we attempt to draw conclusions regarding a promising new treatment and we must make every attempt to ensure that patients are improving because of, rather than despite, a therapeutic intervention. Patients entered into trials examining survival as an endpoint should make every attempt to follow patients for 20 to 30 years after treatment. These considerations will ensure that patients requiring therapy are treated to derive benefit, whereas those who would normally not benefit or fare just as well without treatment are not exposed to unnecessary morbidity and mortality. Finally, it must be concluded as Bloom et al have stated that "the value of treatment of primary breast cancer cannot be measured entirely by survival statistics." The quality of a patient's remaining life once the diagnosis of breast cancer has been made should be considered. From untreated natural history data, we know that patients may suffer a painful death without intervention, and we are aware as well that overtreatment may impart untoward symptomatic consequences in the final stages of life when quality is of paramount importance to both the patient and the family.

Objective The purpose of this study was to characterize the biologic determinants that affect the behavior and management of infiltrating lobular cancer. Methods A prospectively accrued data base containing 1548 breast cancer cases was queried for specific pathologic and mammographic features. From this data base, 777 patients treated and followed-up at the H. Lee Moffitt Cancer Center were reviewed, and comparisons were made between the following three histologic subgroups : 661 infiltrating ductal (ID), 42 infiltrating ductal plus infiltrating lobular (ID + IL), and 74 infiltrating lobular (IL). Results Comparisons of the three histologic forms of breast cancer demonstrated the following : 1. At diagnosis, tumors with IL components were larger than those with ID components (p < 0.001) ; in addition, a greater percentage of IL cancers were T3 lesions (14.8%), compared with ID cancers (4.5%). 2. Sizes of IL tumors were underestimated frequently by mammographic examinations when compared with pathologic measurements (p < 0.001). 3. By comparison to ID tumors, increasing IL tumor size is less likely to be associated with an increased number of metastatic lymph nodes per patient (p = 0.09). 4. Infiltrating lobular cancers treated by lumpectomy with cytologic surgical margin analysis more often gave false-negative results than did ID cancers (p < 0.001). 5. Infiltrating lobular cancers treated by lumpectomy required conversion to mastectomy over 2 times more frequently than ID cancers treated by lumpectomy. 6. Mastectomy was performed more frequently than lumpectomy for the treatment of IL versus ID tumors (p = 0.039). Conclusions Infiltrating lobular cancers are biologically distinct from ID cancers. Although lumpectomy may be performed safely in selected patients, multiple difficulties exist in the management of IL cancer, particularly when breast conservation is chosen.

Colorectal cancer is a common disease that frequently produces limited survival in afflicted patients. Recently a genetic model for tumorigenesis has been defined and has led to a better understanding of the interrelationships between environmental mutagens and genes. Epidemiologic studies have identified multiple chemopreventive agents that appear to reduce the risk of developing colorectal cancer. Their molecular mechanisms of action, as related to the genetic model for colorectal cancer, are discussed.

In the United States and Europe, colorectal cancer (CRC) has the second highest mortality rate of all cancers. Early diagnosis allows for treatment and improved survival. Currently, colonoscopy is the standard of care for detection of early-stage CRC, which is an invasive procedure that requires a cathartic bowel preparation and general anesthesia. Consequently, less invasive screening methods are highly desirable. The objective of this study is to develop a targeted molecular imaging technique termed “molecular colonography,” that can non-invasively detect dysplastic and neoplastic colonic lesions with high sensitivity and conspicuity by oral administration of ligand-targeted probes containing image contrast agents followed by clearance and non-invasive colonography with MRI, CT or Fluorescence imaging. The first step in the development of molecular colonography is identification of cell-surface markers for CRC. To accomplish this goal, gene expression profiling of DNA microarray data from 432 adenocarcinomas, 39 adenomas and 16 inflammatory bowel disease; and unaffected patient tissue samples (178 colon, 6 small intestine, 4 stomach, 4 esophagus, 4 trachea, 3 oral mucosa and 3 tonsil) was performed. Data were filtered using a previously compiled list of 3,800 cell surface genes from the NCBI Gene Expression Omnibus database. Of these, 1085 genes were broadly expressed in CRC and were ranked by differential expression in adenomas and adenocarcinomas vs. normal tissues. Six cell-surface markers were identified that are highly and broadly expressed as mRNA in adenomas and adenocarcinomas relative to unaffected tissues: TLR4, GPR56, GRM8, LY6G6D, CLDN1 and SLC01B3. TLR4 and/or GRM8 were shown to be expressed in 100% of the colon adenomas and adenocarcinomas in this dataset. To confirm protein expression, immunohistochemistry of normal (n=15) and CRC (16 adenoma and 60 adenocarcinoma) patient tissue samples and 26 CRC cell lines was performed. All six markers were highly and broadly expressed in adenomas and adenocarcinomas compared to normal colon. Further validations by mRNA (qRT-PCR) and protein expression (Western blot and immunocytochemistry) of these markers were also performed in nine colon tumor cell lines (COLO-205, DIFI, HCT15, HCT116, HT-29, KM12C, Km12SM, SW480 and SW620). Development of specific binding ligands for selected targets is underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1156. doi:1538-7445.AM2012-1156

There is a large effort in the public domain to systematically perform DNA and RNA sequencing on large numbers of tumor samples. These efforts will bring us to a greater understanding of tumor biology and lead to identification of new tumor drivers. However, these efforts may fall short in allowing us to understand the progression of tumor resistance, relapse and metastasis, factors which make tumors difficult to treat and increase mortality rates. We are currently performing targeted DNA sequencing on 4000 tumor samples. We have selected 1321 genes, 5 GB of the genome to sequence. Genes were selected through a thorough review of the literature, mutation databases and key pathways in tumorigenesis such as growth factor signaling, DNA damage, p53 signaling, cell cycle and apoptosis. Our effort not only includes a diverse panel of breast, colon, ovarian, and kidney tumors but we have also sequenced pancreatic, liver, esophageal, cervix, endometrial, melanoma, CLL, DLBCL and carcinoid tumors. Our dataset also includes approximately 300 matched tumor-metastatic pairs and an additional 550 metastatic samples. Clinical phenotypes, treatment information, gene expression profiling and copy number variation data is available for all samples. To achieve the next level of care for cancer patients we must understand the biology of the tumors we are trying to treat, which are often metastatic and standard of care resistant. We believe that increasing our efforts in targeted DNA sequencing of key tumor and metastatic samples will allow us to achieve these goals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 435. doi:1538-7445.AM2012-435

Background & Aims Mutational inactivation of adenomatous polyposis coli (APC) is an early event in colorectal cancer (CRC) progression that affects the stability and increases the activity of β-catenin, a mediator of Wnt signaling. Progression of CRC also involves inactivation of signaling via transforming growth factor β and bone morphogenetic protein (BMP), which are tumor suppressors. However, the interactions between these pathways are not clear. We investigated the effects of loss of the transcription factor Smad4 on levels of β-catenin messenger RNA (mRNA) and Wnt signaling. Methods We used microarray analysis to associate levels of Smad4 and β-catenin mRNA in colorectal tumor samples from 250 patients. We performed oligonucleotide-mediated knockdown of Smad4 in human embryonic kidney (HEK293T) and in HCT116 colon cancer cells and transgenically expressed Smad4 in SW480 colon cancer cells. We analyzed adenomas from ( APC Δ1638/+ ) and ( APC Δ1638/+ ) × ( K19Cre ERT2 Smad4 lox/lox ) mice by using laser capture microdissection. Results In human CRC samples, reduced levels of Smad4 correlated with increased levels of β-catenin mRNA. In Smad4-depleted cell lines, levels of β-catenin mRNA and Wnt signaling increased. Inhibition of BMP or depletion of Smad4 in HEK293T cells increased binding of RNA polymerase II to the β-catenin gene. Expression of Smad4 in SW480 cells reduced Wnt signaling and levels of β-catenin mRNA. In mice with heterozygous disruption of Apc ( APC Δ1638/+ ), Smad4-deficient intestinal adenomas had increased levels of β-catenin mRNA and expression of Wnt target genes compared with adenomas from APC Δ1638/+ mice that expressed Smad4. Conclusions Transcription of β-catenin is inhibited by BMP signaling to Smad4. These findings provide important information about the interaction among transforming growth factor β, BMP, and Wnt signaling pathways in progression of CRC.

We previously found that cell surface molecules of approximately 70, approximately 35, approximately 32, approximately 22, and approximately 14 kDa from liver-metastatic murine RAW117 large-cell lymphoma cells bound to target liver microvessel endothelial cells. Isolation and sequencing of the approximately 35-kDa component revealed it to be annexin II, a Ca(2+)-binding molecule involved in cytoskeletal and membrane interactions. Annexin II antibodies inhibited the adhesion of RAW117 tumor cells to live or fixed liver endothelial cells, and purified tumor cell surface fractions containing the approximately 35-kDa component inhibited partially RAW117 cell-endothelial cell adhesion, suggesting a role for annexins in tumor cell-endothelial cell adhesion. In the present study we identified the 70-kDa cell surface component that binds to hepatic sinusoidal endothelial cells in a Ca(2+)-dependent manner as annexin VI. Cytofluorographic analysis indicated that annexin VI was expressed on the cell surface in slightly higher amounts on highly metastatic RAW117 cells, and it was not removable by EDTA treatment. Anti-annexin VI antibodies inhibited the adhesion of RAW117 cells to fixed or unfixed murine hepatic sinusoidal endothelial cells by approximately 40%, indicating a role for annexin VI in mediating a portion of the Ca(2+)-dependent RAW117 cell adhesion to target liver microvessel endothelial cells.

Using murine monoclonal antibodies, we have detected the novel signal transducing enzyme, phospholipase C-gamma 1, on the surface of cultured human colorectal cancer cells. We have also demonstrated the presence of this enzyme on the surface of fresh human tumor cells derived from primary and metastatic colorectal tumors. This enzyme has previously been described to be associated only with the inner face of the plasma membrane and the cell cytosol. The finding of an enzyme critical to the signal transduction pathway may have important implications for additional functions of this protein.

e14058 Background: The Notch pathway is activated in colorectal cancer. Downstream signaling is mediated via cleavage of intracellular Notch (ICN) by a γ-secretase complex. Our gene-expression micr...

Introduction. The human cellular apoptosis susceptibility (hCAS) gene product has been shown to be involved in cellular proliferation, apoptosis, invasion and metastasis. CSE1 is a yeast gene with high homology to hCAS (CSE1-like or CSE1L) which has been shown to be involved in chromosome segregation during mitosis. hCAS has also been shown to be a key player in cytoplasm-to-nuclear transport by shuttling importin-alpha from the nucleus back out into the cytoplasm. We hypothesized that the expression of hCAS may be altered during the progression of human colorectal cancer (CRC). Methods. In order to examine hCAS at the gene expression level, we surveyed three different datasets from studies in which Affymetrix U133+gene chips had been performed on patients with CRC, as well as patients with adenomas and normal individuals. We examined relative gene expression levels for 3 probe sets on the U133+ gene chip that closely align to the refseq for the hCAS gene. hCAS protein expression level was semiquantitatively measured using immunohistochemistry (IHC) and the tissue microarray (TMA) technique. A CRC TMA had been previously created at the Moffitt Cancer Center using resection specimens of primary CRC of different stages, and inclusive of adenomas and normal tissues samples from the same patients. Results. hCAS gene expression was increased in all adenoma and CRC samples tested by microarray, compared to histologically normal colonic mucosa. However, the difference in hCAS mRNA levels between adenoma and CRC samples was not statistically different. No correlation was found between hCAS and cancer stage, grade, vascular invasion, sex and/or age. Interestingly, in the invasive CRCs, intense hCAS proteins expression was localized to both nucleus and cytoplasm. Conversely, early stage lesions showed predominantly cytoplasmic hCAS staining. Conclusions. Our results demonstrate that hCAS protein is expressed early and across all stages of CRC development. Because of its early expression hCAS may represent a marker for early detection of CRC. In addition, because of its involvement in a variety of important cellular processes, this gene product might represent a new potential target for prevention and treatment of CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1679. doi:10.1158/1538-7445.AM2011-1679

Background Colon cancer has been classically described by clinicopathologic features that permit the prediction of outcome only after surgical resection and staging. Methods We performed an unsupervised analysis of microarray data from 326 colon cancers to identify the first principal component (PC1) of the most variable set of genes. PC1 deciphered two primary, intrinsic molecular subtypes of colon cancer that predicted disease progression and recurrence. Results Here we report that the most dominant pattern of intrinsic gene expression in colon cancer (PC1) was tightly correlated (Pearson R = 0.92, P < 10-135) with the EMT signature-- both in gene identity and directionality. In a global micro-RNA screen, we further identified the most anti-correlated microRNA with PC1 as MiR200, known to regulate EMT. Conclusions These data demonstrate that the biology underpinning the native, molecular classification of human colon cancer--previously thought to be highly heterogeneous-- was clarified through the lens of comprehensive transcriptome analysis.

Using fixed microvessel endothelial cell monolayers the molecules involved in the adhesion of liver-preferring murine RAW117 large cell lymphoma cells to murine liver-derived microvessel endothelial cells were identified by affinity isolation. Detergent lysates obtained from poorly (P) or highly (H10) liver-metastatic cells inhibited RAW117-H10 cell adhesion to hepatic sinusoidal endothelial (HSE) cell monolayers. Allowing detergent lysates of cell surface-labeled RAW117 cells to bind to fixed HSE cell monolayers and eluting the bound components indicated that several tumor cell surface molecules (approximately 70, approximately 35, approximately 32, approximately 22, and approximately 14 kDa) might be involved in RAW117 cell-HSE cell adhesion. The approximately 35 kDa component was cation dependent in its binding to target HSE cells. Increasing detergent concentration had no effect on binding of the approximately 35 kDa component to HSE cell monolayers, whereas treatment with 0.5 M NaCl resulted in its selective elution from HSE cells. Incubation of the HSE cell monolayers with detergent lysates from cell surface-labeled RAW117-H10 cells resulted in selective depletion of the approximately 35 kDa component, suggesting that the binding is saturable. This divalent cation-dependent molecule is one of the major tumor cell surface components bound by several types of endothelial cells and murine hepatocytes, whereas there was poor binding of this component to unfixed or fixed human red blood cells. The purified, partially (approximately 40%) sequenced molecule had amino acid sequence identity with murine but not bovine annexin II, indicating that it was not bound from the bovine serum used to grow RAW117 cells. Using antibodies specific for annexin II flow cytometry indicated equivalent amounts of annexin II are expressed on RAW117 cell surfaces in the absence or presence of excess EDTA, whereas annexin I was only found in low amounts on the surfaces of RAW117 cells. Annexin II antibodies inhibited by approximately 40-50% the adhesion of RAW117 tumor cells to live or fixed endothelial cells, and purified tumor cell surface fractions containing the approximately 35 kDa component partially inhibited (approximately 35%) RAW117 cell-HSE cell adhesion. The data indicate that annexin II is expressed on the extracellular surface of RAW117 cells, and cell surface-annexin II mediates a portion of the Ca(2+)-dependent RAW117 cell adhesion to liver microvessel endothelial cells.