Marlene B. Zuraek

Fractional Genomic Alteration Detected by Array-Based Comparative Genomic Hybridization Independently Predicts Survival after Hepatic Resection for Metastatic Colorectal Cancer

Purpose: Although liver resection is the primary curative therapy for patients with colorectal hepatic metastases, most patients have a recurrence. Identification of molecular markers that predict patients at highest risk for recurrence may help to target further therapy. Experimental Design: Array-based comparative genomic hybridization was used to investigate the association of DNA copy number alterations with outcome in patients with colorectal liver metastasis resected with curative intent. DNA from 50 liver metastases was labeled and hybridized onto an array consisting of 2,463 bacterial artificial chromosome clones covering the entire genome. The total fraction of genome altered (FGA) in the metastases and the patients clinical risk score (CRS) were calculated to identify independent prognostic factors for survival. Results: An average of 30 ± 14% of the genome was altered in the liver metastases (14% gained and 16% lost). As expected, a lower CRS was an independent predictor of overall survival (P = 0.03). In addition, a high FGA also was an independent predictor of survival (P = 0.01). The median survival time in patients with a low CRS (score 0-2) and a high (≥20%) FGA was 38 months compared with 18 months in patients with a low CRS and a low FGA. Supervised analyses, using Prediction Analysis of Microarrays and Significance Analysis of Microarrays, identified a set of clones, predominantly located on chromosomes 7 and 20, which best predicted survival. Conclusions: Both FGA and CRS are independent predictors of survival in patients with resected hepatic colorectal cancer metastases. The greater the FGA, the more likely the patient is to survive.

Homeodomain transcription factor NKX2.2 functions in immature cells to control enteroendocrine differentiation and is expressed in gastrointestinal neuroendocrine tumors

The homeodomain transcription factor NKX2.2 is necessary for neuroendocrine (NE) differentiation in the central nervous system and pancreas. NE tumors derived from the gut are defined by their NE phenotype, which is used for diagnosis and contributes to tumorigenicity. We hypothesized that NKX2.2 is important for NE differentiation in normal and neoplastic gut. NKX2.2 and NE marker expression was investigated in the small intestine of embryonic and adult mice using immunofluorescence (IF). To determine the role of NKX2.2 in NE differentiation of the intestine, the phenotype of Nkx2.2 (-/-) mice was examined by IF and real-time (RT)-PCR. NKX2.2 and NE marker expression in human NE tumors of the gut and normal tissues were evaluated by immunohistochemistry and qRT-PCR. NKX2.2 expression was detected in the intervillus/crypt regions of embryonic and adult mouse intestine. Co-expression of Nkx2.2 with neurogenin3 (NEUROG3) and hormones was observed in the adult intestinal crypt compartment, suggesting NKX2.2 functions in NEUROG3-positive endocrine progenitors and newly differentiated endocrine cells. In the intestine of Nkx2.2 (-/-) mice, we found a dramatic reduction in the number of cells producing numerous hormones, such as serotonin, gastrin, cholecystokinin, somatostatin, glucagon-like peptide 1 (GLP-1), and secretin, but an increase in cells producing ghrelin. NKX2.2 was expressed in most (24 of 29) human NE tumors derived from diverse primary sites. We conclude NKX2.2 functions in immature endocrine cells to control NE differentiation in normal intestine and is expressed in most NE tumors of the gut, and is therefore a novel target of diagnosis for patients with gastrointestinal NE tumors.

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. Clin Cancer Res; 19(20); 5777–87. ©2013 AACR.

The ETS oncogene family transcription factor FEV identifies serotonin-producing cells in normal and neoplastic small intestine

Neuroendocrine (NE) or carcinoid tumors of the small intestine (SI) frequently metastasize and produce the hormone serotonin, causing significant morbidity and mortality. A member of the ETS oncogene family of transcription factors, Fev, acts with the homeodomain transcription factor Nkx2.2 in the development of serotonin neurons in mice. In this study, we investigated the role of Fev in normal and neoplastic SI. In NE tumors (NETs) of the SI, serotonin stimulates tumor growth and causes debilitating symptoms, such as diarrhea, flushing, wheezing, and right-sided valvular heart disease (i.e. carcinoid syndrome). Compared with those in the matched normal human SI, FEV expression levels were significantly elevated in primary NETs (20-fold, P<0.0001), lymph node metastases (35-fold, P=0.004), and NET liver metastases (22-fold, P<0.0001) resected from patients with serotonin excess. Fev is expressed in the wild type but not in Nkx2.2 (-/-) mouse SI, in which cells producing serotonin are absent. Using recombination-based cell lineage tracing, we found that FEV-positive cells give rise to serotonin-producing cells in the SI. In Fev (-/-) mouse SI, we observed no difference in the number of cells producing serotonin or other hormones. We conclude that FEV expression identifies serotonin-producing cells in normal and neoplastic SI and is a novel target for diagnosis of patients with NETs of the SI.

Lack of NKX2.2 Expression in Bronchopulmonary Typical Carcinoid Tumors: Implications for Patients with Neuroendocrine Tumor Metastases and Unknown Primary Site

BACKGROUND Patients with neuroendocrine tumors (NETs) may have metastatic disease and unknown primary site. NETs commonly arise from the bronchopulmonary (BP) and gastrointestinal (GI) tract. The largest subgroups of well-differentiated BP-NETs are typical carcinoids (TCs). The homeodomain transcription factor NKX2.2 regulates development of gut serotonin cells and is a marker of GI-NETs. Previous work on a limited number of samples suggested that BP-TCs do not express NKX2.2. We hypothesized that lack of NKX2.2 expression in BP-TCs might be useful to distinguish BP- from GI-NETs, and evaluated NKX2.2 expression in a larger number of BP-TCs. METHODS Archived formalin-fixed, paraffin-embedded tissues were obtained from 13 previously undescribed patients with BP-TCs. Expression of NKX2.2, serotonin, and the NE marker chromogranin A (CgA) were assessed by immunohistochemistry. RESULTS CgA expression was robust in all 13 BP-TCs, confirming the NE phenotype. Serotonin expression was less frequent (9/13; 69%). Two patients with BP-TCs in which serotonin expression was absent exhibited Cushings syndrome due to ectopic ACTH production. NKX2.2 expression was not observed in any of the 13 tumors. CONCLUSIONS Bronchopulmonary TCs uniformly express CgA but not NKX2.2. Because most of these tumors express serotonin, our findings suggest that NKX2.2 may not be required for serotonin production by BP-TCs. We conclude that the presence or absence of NKX2.2 expression may assist in the determination of the primary tumor site in patients with NET metastases of unknown origin. NET metastases that are CgA-positive/NKX2.2-negative would suggest a BP primary, whereas those that are CgA-positive/NKX2.2-positive would suggest a GI primary.

Prognostic importance of superior diaphragmatic adenopathy at computed tomography in patients with resectable hepatic metastases from colorectal carcinoma.

Purpose: To determine the prognostic importance of superior diaphragmatic adenopathy at CT in patients with resectable hepatic metastases from colorectal carcinoma. Materials and Methods: We retrospectively identified 85 patients who underwent contrast-enhanced abdominal computed tomography (CT) at our institution before surgical resection of hepatic metastases from colorectal carcinoma. The study group consisted of 45 men and 40 women with a mean age of 60 years (range, 27-89 years). The presence, size, and number of superior diaphragmatic nodes were recorded on preoperative CT images. Kaplan-Meier analysis was used to investigate the association between the presence, number, and size of superior diaphragmatic nodes and postoperative outcome. Results: One or more superior diaphragmatic nodes were seen on preoperative CT in 43 (51%) of 85 patients, and 29 (34%) patients had nodes of 5 mm or more in short-axis diameter. After a median follow-up of 599 days (range, 3-1960 days), 49 patients were alive, and 36 were dead. Kaplan-Meier analysis showed no association between the presence (P = 0.67), size (P = 0.74), or number (P = 0.95) of superior diaphragmatic nodes and patient outcome. Conclusions: The presence, size, or number of superior diaphragmatic nodes at preoperative CT are unrelated to postoperative outcome in patients with resectable hepatic metastases from colorectal carcinoma, suggesting that superior diaphragmatic adenopathy in this setting may be reactive rather than metastatic.

A novel interaction of genotype, gender, and adjuvant treatment in survival after resection of stage III colon cancer: Results of CALGB 89803.

452 Background: The p53 tumor suppressor gene is frequently mutated in colorectal cancer, but reports on the effect of p53 mutations on response to adjuvant chemotherapy and survival are inconclusive. This study investigates whether p53 mutational status (wild-type, zinc or non-zinc binding mutations) impacts survival following adjuvant therapy containing fluorouracil/leucovorin with or without irinotecan (5FU/LV or IFL) in women and men with stage III colon cancer. METHODS As part of a retrospective analysis of prospectively accrued data, p53 mutational status was determined for 609 patients with stage III colon cancer who were randomized on CALGB 89803, a phase III adjuvant chemotherapy trial. p53 exons 5-8 were analyzed by direct sequencing or sequencing by hybridization. p53 mutations were identified in 276 tumors (45%), of which 134 were in the zinc binding and 142 were in the non-zinc binding regions of the core domain. Cox regression was used to study the impact of p53 mutational status, sex, and adjuvant chemotherapy on disease-free (DFS) and overall survival (OS). RESULTS p53 mutational status did not predict differential survival or response to adjuvant therapy among the 609 patients assessed. However, a significant sex by treatment interaction was observed for both DFS (Pinteraction=0.008) and OS (Pinteraction=0.002). Significant differences in DFS by p53 mutational status were observed among women (logrank P = 0.009). No such differences were observed among men (logrank P = 0.33). Similar results were observed for OS. There was marginal evidence of a treatment-related impact on the interaction between sex and p53 mutational status for both DFS and OS (DFS Pinteraction = 0.07; OS Pinteraction = 0.11). There was a trend toward improved OS when women with zinc binding mutations received IFL versus 5FU/LV (P = 0.08) and toward worse DFS when women with non-zinc binding mutations were treated with IFL versus 5FU/LV (P =0.08). CONCLUSIONS This exploratory subset analysis suggests that p53 mutational status may be used to predict prognosis in a sex- and potentially chemotherapeutic regimen-specific manner.

Distinct mTOR Pathway Activity in Neuroendocrine Tumors of the Pancreas and Small Intestine Revealed by Immunohistochemical Analysis

1 Department of Surgery, Division of Surgical Oncology, University of California, San Francisco, CA 94143 2 Department of Pathology, University of California, San Francisco, CA 94143 3 Diabetes Center, University of California, San Francisco, CA 94143 4 Department of Medicine, Division of Hematology/Oncology, University of California, San Francisco, CA 94143 5 UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA 94143