Faculty Publications

Specimen collection is an integral component of clinical research. Specimens from subjects with various stages of cancers or other conditions, as well as those without disease, are critical tools in the hunt for biomarkers, predictors, or tests that will detect serious diseases earlier or more readily than currently possible. Analytic methodologies evolve quickly. Access to high-quality specimens, collected and handled in standardized ways that minimize potential bias or confounding factors, is key to the "bench to bedside" aim of translational research. It is essential that standard operating procedures, "the how" of creating the repositories, be defined prospectively when designing clinical trials. Small differences in the processing or handling of a specimen can have dramatic effects in analytical reliability and reproducibility, especially when multiplex methods are used. A representative working group, Standard Operating Procedures Internal Working Group (SOPIWG), comprised of members from across Early Detection Research Network (EDRN) was formed to develop standard operating procedures (SOPs) for various types of specimens collected and managed for our biomarker discovery and validation work. This report presents our consensus on SOPs for the collection, processing, handling, and storage of serum and plasma for biomarker discovery and validation.

OBJECTIVE: Tp53 mutation is frequent and associates with malignant, high-grade ovarian cancer. However, the details about the progression of Tp53 mutation from heterozygous to homozygous, and association between genotypes and morphological transformation are not clear. We further investigated the expression and mutation of Tp53 and associated markers such as p21 and HDM2 in ovarian cancer. METHOD: Areas of contiguous ovarian surface epithelia linking morphological normal monolayer to multilayer neoplastic cells were analyzed for the correlation of Tp53 pathway alteration in relation to morphological transformation, by immunostaining and sequencing of Tp53 gene in cells from laser captured microdissection. RESULTS: Consistent with previous reports, Tp53 staining is positive in 78% of the tumors. The staining of p21 is positive in about 12%, and HMD2 is positive in only 1% of the tumors. In 9 out of 10 cases of p21-positive tumors, p53 is also positive. In the majority of cases of epithelial histological transitions, overexpression of Tp53 correlates with morphological transformation: Tp53 is negative in monolayered cells and positive in neoplastic lesions. Morphological transformation also closely correlates with cell proliferation as indicated by Ki-67 staining and loss of p21 expression. We detected heterozygous mutation of Tp53 in the monolayers adjacent to neoplastic cells. CONCLUSIONS: p21 expression is an indicator of a wild type Tp53 and lack of p21 in the presence of Tp53 expression predicts an inactivated Tp53. Tp53 inactivation immediately precedes morphological transformation of the ovarian surface epithelium in most cases, and the histological transitional epithelia containing a heterozygous Tp53 mutation are thus pre-neoplastic lesions. We propose that the loss of a second allele of Tp53 leading to the loss of p21 expression, and subsequent cell proliferation, compose a sequence of events that lead to morphological transformation and instigation of ovarian epithelial tumor development.

Context The established relationship between lymph node metastasis and prognosis in colorectal cancer suggests that recurrence in 25% of patients with lymph nodes free of tumor cells by histopathology ( pN0) reflects the presence of occult metastases. Guanylyl cyclase 2C ( GUCY2C) is a marker expressed by colorectal tumors that could reveal occult metastases in lymph nodes and better estimate recurrence risk. Objective To examine the association of occult lymph node metastases detected by quantifying GUCY2C messenger RNA, using the reverse transcriptase - polymerase chain reaction, with recurrence and survival in patients with colorectal cancer. Design, Setting, and Participants Prospective study of 257 patients with pN0 colorectal cancer enrolled between March 2002 and June 2007 at 9 US and Canadian centers ( 7 academic medical centers and 2 community hospitals) provided 2570 fresh lymph nodes measuring 5 mm or larger for histopathology and GUCY2C messenger RNA analysis. Patients were followed up for a median of 24 months ( range, 2- 63 months) for disease recurrence or death. Main Outcome Measures Time to recurrence ( primary outcome) and diseasefree survival ( secondary outcome) relative to expression of GUCY2C in lymph nodes. Results Thirty- two patients ( 12.5%) had lymph nodes negative for GUCY2C ( pN0 [ mol-]), and all but 2 remained free of disease during follow- up ( recurrence rate, 6.3%; 95% confidence interval [ CI], 0.8%- 20.8%). Conversely, 225 patients ( 87.5%) had lymph nodes positive for GUCY2C ( pN0 [ mol +]), and 47 developed recurrent disease ( 20.9%; 95% CI, 15.8%- 26.8%) ( P=. 006). Multivariate analyses revealed that GUCY2C in lymph nodes was an independent marker of prognosis. Patients who were pN0 ( mol +) exhibited earlier time to recurrence ( adjusted hazard ratio, 4.66; 95% CI, 1.11- 19.57; P=. 04) and reduced disease- free survival ( adjusted hazard ratio, 3.27; 95% CI, 1.15-9.29; P=. 03). Conclusion Expression of GUCY2C in histologically negative lymph nodes appears to be independently associated with time to recurrence and disease- free survival in patients with pN0 colorectal cancer.

Purpose To determine the impact of adjuvant androgen deprivation therapy (ADT) for patients who have node-positive prostate cancer in the prostate-specific antigen (PSA) era. Patients and Methods We used linked Surveillance, Epidemiology and End Results-Medicare data to construct a cohort of men who underwent radical prostatectomy ( RP) between 1991 and 1999 and who had positive regional lymph nodes. We classified men as receiving adjuvant ADT if they received ADT within 120 days of RP, and we compared them to the men who had not received adjuvant ADT. We used propensity scores to balance potential confounders of receiving adjuvant ADT (ie, tumor characteristics, extent of nodal disease, demographics, receipt of radiation therapy) and Cox proportional hazard methods to measure the impact of adjuvant ADT on overall survival ( OS), stratified by propensity score quintile. We conducted a sensitivity analysis that used 90, 150, 180, and 365 days as the definition for adjuvant ADT. Results A total of 731 men were identified, 209 of whom received ADT within 120 days of RP. There was no statistically significant difference in OS between the adjuvant ADT and non-ADT group (HR, 0.97; 95% CI, 0.71 to 1.27). There was no statistically significant survival difference with 90, 150, 180, and 365 days as the adjuvant ADT definition. Conclusion Deferring immediate ADT in men with positive lymph nodes after RP may not significantly compromise survival. Because observational studies should be considered hypothesis-generating studies, these results should be validated in a prospective fashion in a similar patient population.

Purpose MDM2 regulates p53, which controls cell cycle arrest and apoptosis. Both proteins, along with Ki-67, which is an established strong determinant of metastasis, have shown promise in predicting the outcome of men treated with radiation therapy (RT) with or without short-term androgen deprivation (STAD). This report compares the utility of abnormal expression of these biomarkers in estimating progression in a cohort of men treated on RTOG 92-02. Patients and Methods Adequate tissue for immunohistochemistry was available for p53, Ki-67, and MDM2 analyses in 478 patient cases. The percentage of tumor nuclei staining positive (PSP) was quantified manually or by image analysis, and the per-sample mean intensity score (MIS) was quantified by image analysis. Cox regression models were used to estimate overall mortality (OM), and Fine and Gray's regressions were applied to the end points of distant metastasis (DM) and cause-specific mortality (CSM). Results In multivariate analyses that adjusted for all markers and treatment covariates, MDM2 overexpression was significantly related to DM (P = .02) and OM (P = .003), and Ki-67 overexpression was significantly related to DM (P < .0001), CSM (P = .0007), and OM (P = .01). P53 overexpression was significantly related to OM (P = .02). When considered in combination, the overexpression of both Ki-67 and MDM2 at high levels was associated with significantly increased failure rates for all end points (P < .001 for DM, CSM, and OM). Conclusion Combined MDM2 and Ki-67 expression levels were independently related to distant metastasis and mortality and, if validated, could be considered for risk stratification of patients with prostate cancer in clinical trials.

There is increasing evidence that prolactin (PRL), a hormone/cytokine, plays a role in breast, prostate, and colorectal cancers via local production or accumulation. Elevated levels of serum PRL in ovarian and endometrial cancers have been reported, indicating a potential role for PRL in endometrial and ovarian carcinogenesis. In this study, we show that serum PRL levels are significantly elevated in women with a strong family history of ovarian cancer. We show dramatically increased expression of PRL receptor in ovarian and endometrial tumors as well as in endometrial hyperplasia, signifying the importance of PRL signaling in malignant and premalignant conditions. PRL mRNA was expressed in ovarian and endometrial tumors, indicating the presence of an autocrine loop. PRL potently induced proliferation in several ovarian and endometrial cancer cell lines. Binding of PRL to its receptor was followed by rapid phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, mitogen-activated protein kinase/ERK kinase 1, signal transducer and activator of transcription 3, CREB, ATF-2, and p53 and activation of 37 transcription factors in ovarian and endometrial carcinoma cells. PRL also activated Ras oncogene in these cells. When human immortalized normal ovarian epithelial cells were chronically exposed to PRL, a malignant transformation occurred manifested by the acquired ability of transformed cells to form clones, grow in soft agar, and form tumors in severe combined immunodeficient-beige mice. Transformation efficiency was diminished by a Ras inhibitor, providing proof that PRL-induced transformation uses the Ras pathway. In summary, we present findings that indicate an important role for PRL in ovarian and endometrial tumorigenesis. PRL may represent a risk factor for ovarian and endometrial cancers. [Cancer Res 2009;69 (12):5226-33]

Purpose: Data from two randomized phase III trials were analyzed to evaluate prognostic factors and treatment selection in the first-line management of advanced non-small cell lung cancer patients with performance status (PS) 2. Patients and Methods: Patients randomized to combination chemotherapy (carboplatin and paclitaxel) in one trial and single-agent therapy (gemcitabine or vinorelbine) in the second were included in these analyses. Both studies had identical eligibility criteria and were conducted simultaneously. Comparison of efficacy and safety was performed between the two cohorts. A regression analysis identified prognostic factors and subgroups of patients that may benefit from combination or single-agent therapy. Results: Two hundred one patients were treated with combination and 190 with single-agent therapy. Objective responses were 37 and 15%. respectively. Median time to progression was 4.6 months in the combination arm and 3.5 months in the single-agent arm (p < 0.001). Median survival times were 8.0 and 6.6 months, and 1-year survival rates were 31 and 26%, respectively. Albumin <3.5 g, extrathoracic metastases, lactate dehydrogenase >= 200 IU, and 2 comorbid conditions predicted outcome. Patients with 0-2 risk factors had similar outcomes independent of treatment, whereas patients with 3-4 factors had a nonsignificant improvement in median survival with combination chemotherapy. Conclusion: Our results show that PS2 non-small cell lung cancer patients are a heterogeneous group who have significantly different outcomes. Patients treated with first-line combination chemotherapy had a higher response and longer time to progression, whereas overall survival did not appear significantly different. A prognostic model may be helpful in selecting PS 2 patients for either treatment strategy.

OBJECTIVES: To determine the proportion of patients with tumor response, the proportion who survived progression-free for at least 6 months (progression-free survival &gt;or= 6 months), and the frequency and severity of toxicities of patients with recurrent squamous cell carcinoma of the uterine cervix treated with erlotinib. METHODS: This was a multicenter, open-label, single-arm trial evaluating the toxicity and efficacy of oral erlotinib at an initial dosage of 150 mg daily until progressive disease or adverse effects prohibited further therapy. RESULTS: Twenty-eight patients with squamous cell carcinoma were enrolled onto this trial. Twenty-five patients were evaluable. There were no objective responses, with 4 (16%) patients achieving stable disease; only 1 patient had a progression-free survival of 6 months (4%) or more. The 1-sided 90% confidence interval for response was 0.0% to 8.8%. The 2-sided 90% confidence interval for the proportion of patients surviving progression-free for at least 6 months is 0.2% to 17.6%. Erlotinib was well tolerated, with the most common drug-related adverse events being gastrointestinal toxicities, fatigue, and rash. CONCLUSIONS: Erlotinib is inactive as monotherapy in patients with recurrent squamous cell carcinoma of the uterine cervix.

Measles virus (MV) is one of the most transmissible microorganisms known, continuing to result in extensive morbidity and mortality worldwide. While rare, MV can infect the human central nervous system, triggering fatal CNS diseases weeks to years after exposure. The advent of crucial laboratory tools to dissect MV neuropathogenesis, including permissive transgenic mouse models, the capacity to manipulate the viral genome using reverse genetics, and cell biology advances in understanding the processes that govern intracellular trafficking of viral components, have substantially clarified how MV infects, spreads, and persists in this unique cell population. This review highlights some of these technical advances, followed by a discussion of our present understanding of MV neuronal infection and transport. Because some of these processes may be shared among diverse viruses, comparisons are made to parallel studies with other neurotropic viruses. While a crystallized view of how the unique environment of the neuron affects MV replication, spread, and, ultimately, neuropathogenesis is not fully realized, the tools and ideas are in place for exciting advances in the coming years.

Measles virus (MV) is one of the most transmissible microorganisms known, continuing to result in extensive morbidity and mortality worldwide. While rare, MV can infect the human central nervous system, triggering fatal CNS diseases weeks to years after exposure. The advent of crucial laboratory tools to dissect MV neuropathogenesis, including permissive transgenic mouse models, the capacity to manipulate the viral genome using reverse genetics, and cell biology advances in understanding the processes that govern intracellular trafficking of viral components, have substantially clarified how MV infects, spreads, and persists in this unique cell population. This review highlights some of these technical advances, followed by a discussion of our present understanding of MV neuronal infection and transport. Because some of these processes may be shared among diverse viruses, comparisons are made to parallel studies with other neurotropic viruses. While a crystallized view of how the unique environment of the neuron affects MV replication, spread, and, ultimately, neuropathogenesis is not fully realized, the tools and ideas are in place for exciting advances in the coming years.

BACKGROUND: The benefit of breast MRI for newly diagnosed breast cancer patients is uncertain. This study characterizes those receiving MRI versus those who did not, and reports on their short-term surgical outcomes, including time to operation, margin status, and mastectomy rate. STUDY DESIGN: All patients seen in a multidisciplinary breast cancer clinic from July 2004 to December 2006 were retrospectively reviewed. Patients were evaluated by a radiologist, a pathologist, and surgical, radiation, and medical oncologists. RESULTS: Among 577 patients, 130 had pretreatment MRIs. MRI use increased from 2004 (referent, 13%) versus 2005 (24%, p=0.014) and 2006 (27%, p=0.002). Patients having MRIs were younger (52.5 versus 59.0 years, p &lt; 0.001), but its use was not associated with preoperative chemotherapy, family history of breast or ovarian cancer, presentation, or tumor features. MRI was associated with a 22.4-day delay in pretreatment evaluation (p=0.011). Breast conserving therapy (BCT) was attempted in 320 of 419 patients with complete surgical data. The odds ratio for mastectomy, controlling for T size and stage, was 1.80 after MRI versus no MRI (p=0.024). Patients having MRIs did not have fewer positive margins at lumpectomy (21.6% MRI versus 13.8% no MRI, p=0.20), or conversions from BCT to mastectomy (9.8% MRI versus 5.9% no MRI, p=0.35). CONCLUSIONS: Breast MRI use was not confined to any particular patient group. MRI use was not associated with improved margin status or BCT attempts, but was associated with a treatment delay and increased mastectomy rate. Without evidence of improved oncologic outcomes as a result, our study does not support the routine use of MRI to select patients or facilitate the performance of BCT.

Materials and methods: Patients who had unresectable/metastatic squamous cell carcinoma or adenocarcinoma of the esophagus, measurable disease, Eastern Cooperative Oncology Group performance status of zero to two, and normal bilirubin were eligible. Tumor assessment was carried out every three cycles. Results: We enrolled 29 chemotherapy-naive (CN) and 15 chemotherapy-exposed (CE) eligible patients. Principal toxic effects were diarrhea, neutropenia, and hyperglycemia. There were no toxic deaths. There was one early death, from myocardial infarction. Among 26 CN and assessable patients, there were seven (26.9%) with a partial response (PR) and one (3.8%) with a complete response (CR). There were two PRs and one CR among the patients with CE disease. Median time to progression for CN patients was 4.0 months and for CE patients 3.5 months. Median survival for CN eligible patients was 9.0 months and for CE patients 11.4 months. Conclusions: Docetaxel-irinotecan combination given on a weekly x 2 of 3 schedule is promising in the treatment of advanced esophageal cancer.

The prevalence of obesity among children, adolescents and adults has been dramatically increasing worldwide during the last several decades. The obesity epidemic has been recognized as one of the major global health problems, because its health hazard is linked to a number of common diseases including breast and prostate cancers. Obesity is caused by combination of genetic and environmental factors. While genetic contribution to obesity has been known to be significant, the genetic factors remain relatively unchanged. Recent studies have highlighted the involvement of environmental "obesogens", i.e. the xenobiotic chemicals that can disrupt the normal development and homeostatic control over adipogenesis and energy balance. Several lines of evidence suggest that increasing exposure to chemicals with endocrine-disrupting activities (endocrine-disrupting chemicals, EDCs) contributes to the increased obesity. The cellular and molecular mechanisms underlying obesogen-associated obesity are just now being appreciated. In this paper, we comprehensively reviewed current knowledge about the role of estrogen receptors alpha and beta (ER alpha and ER beta) in regulation of energy metabolism pathways, including glucose transport, glycolysis, tricarboxylic acid (TCA) cycle, mitochondrial respiratory chain (MRC), adenosine nucleotide translocator (ANT) and fatty acid beta-oxidation and synthesis, by estrogens; and then examined the disturbance of E-2/ER-mediated energy metabolism pathways by environmental obesogens; and finally, we discussed the potential implications of disturbance of energy metabolism pathways by obesogens in obesity and pointed out several key aspects of this area that need to be further explored. A better understanding of the cellular and molecular mechanisms underlying obesogen-associated obesity will lead to new approaches for slow down and/or prevention of the increased trend of obesity associated with exposure to obesogens. (C) 2009 Elsevier B.V. All rights reserved.

Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (Fc epsilon RI) resulting in release of proinflammatory mediators. The biochemical pathways linking IgE activation to calcium influx and cytoskeletal changes required for intracellular granule release are incompletely understood. We demonstrate, genetically, that Pak1 is required for this process. In a passive cutaneous anaphylaxis experiment, W-sh/W-sh mast cell-deficient mice locally reconstituted with Pak1(-/-) bone marrow-derived mast cells (BMMCs) experienced strikingly decreased allergen-induced vascular permeability compared with controls. Consistent with the in vivo phenotype, Pak1(-/-) BMMCs exhibited a reduction in Fc epsilon RI-induced degranulation. Further, Pak1(-/-) BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to Fc epsilon RI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases. (Blood. 2009;113:2695-2705)

The human CDKN2A locus encodes 2 distinct proteins, p16(INK4A) and p14(ARF) [mouse p19(Arf)], designated INK4A (inhibitor of cyclin dependent kinase 4) and ARF (alternative reading frame) here, that are translated from alternatively spliced mRNAs. Human ARF is implicated as a tumor suppressor gene, mainly in association with the simultaneous deletion of INK4A. However, questions remain as to whether loss of ARF alone is sufficient to drive tumorigenesis. Here, we report that mice deficient for Arf are susceptible to accelerated asbestos-induced malignant mesothelioma (MM). MMs arising in Arf (+/-) mice consistently exhibit biallelic inactivation of Arf, but, unexpectedly, do not acquire additional recurrent genetic alterations that we previously identified in asbestos-induced MMs arising in Nf2 (+/-) mice. Array CGH analysis was used to detect a recurrent deletion at chromosome 4C6 in MMs from Arf (+/-) mice. A candidate gene in this region, Faf1 (FAS-associated factor 1), was further explored, because it encodes a protein implicated in tumor cell survival and in the pathogenesis of some human tumor types. We confirmed hemizygous loss of Faf1 and down-regulation of Faf1 protein in a series of MMs from Arf (+/-) mice, and we then showed that Faf1 regulates TNF-alpha-mediated NF-kappaB signaling, a pathway previously implicated in asbestos-induced oncogenesis of human mesothelial cells. Collectively, these data indicate that Arf inactivation has a significant role in driving MM pathogenesis, and implicate Faf1 as a key component in the TNF-alpha/NF-kappaB signaling node that has now been independently implicated in asbestos-induced oncogenesis.