Banked Specimens Research Articles

Abstract PD5-5: Phase 2 study of single agent MLN8237 (alisertib), an investigational aurora A kinase (AAK) inhibitor, in patients (pts) with relapsed/refractory breast cancer (BrC)

Abstract Background: AAK, a key mitotic regulator, is frequently amplified/overexpressed across a spectrum of tumors, including BrC. AAK overexpression is associated with poor prognosis. MLN8237 is an oral selective AAK inhibitor under evaluation in pts with advanced cancer as a single agent and in combination therapy. A phase 1/2 study (NCT01045421) evaluated MLN8237 in pts with different solid tumors; phase 2 data for the Brc cohort are presented. Methods: Females aged ≥18 y with relapsed/refractory BrC including HR+, HER2+ and triple-negative histological subtype, ECOG PS 0–1, measurable disease by RECIST v1.1 and ≤4 prior cytotoxic chemotherapy regimens (not including adjuvant, neo-adjuvant; no limitation on prior hormonal or HER2 targeted, immunological or biological agents) were enrolled. Symptomatic brain metastases were excluded (treated stable metastasis allowed). A Simon's optimal 2-stage design was used; 20 pts were initially enrolled, expansion proceeded if ≥2 objective responses were observed in these response-evaluable pts. Pts received MLN8237 50 mg BID for 7 days in 21-day cycles. Primary objective: overall response rate (ORR). Secondary objectives: safety, duration of response (DOR) and progression-free survival (PFS). An exploratory study was performed to assess clinical responses in relation to candidate biomarker dysregulation (mutation, amplification, and deletion) in banked tumor specimens. Results: As of April, 2013, 53 pts were enrolled: median age was 60 y (range 33–81), median of 4 cycles (range 1–21). 49 pts (92%) were response-evaluable (HR+, n = 26; HER2+, n = 9; triple negative, n = 14). 33% of pts received treatment for ≥6 months (HR+, n = 11 [69%]; HER2+, n = 3 [19%]; triple negative, n = 2 [12%]). ORR (all pts) was 18% and median PFS was 5.42 months. Efficacy data per subgroup are shown in the table. All 53 pts were included in the safety population; drug-related adverse events (AEs) were reported in 51 pts (96%), most frequent were neutropenia (55%), alopecia (49%) and diarrhea (45%). 38 pts (72%) had grade ≥3 drug-related AEs, including neutropenia (49%), leukopenia (21%) and febrile neutropenia (4%). G-CSF use was 32%. 2 pts (4%) discontinued due to AEs (sepsis [grade 4], n = 1; neutropenia [grade 3], n = 1); no on-study deaths were reported. Treatment is ongoing in 3 HR+ pts. Whole-exome sequencing of selected tumor samples was completed. Correlative analysis is ongoing to identify potential genetic markers/mutated pathways associated with clinical response. Preliminary results will be presented. Conclusions: MLN8237 appears to have a generally manageable toxicity profile and shows signs of single agent antitumor activity in pts with heavily pretreated (different molecular subgroups) of relapsed/refractory BrC supporting further evaluation of MLN8237 in this tumor type in different combination strategies. Efficacy HR+ (n = 26)HER2+ (n = 9)Triple-negative (n = 14)Best response,%ORR (PR)23227Stable disease653336Progressive disease124457Median DOR, months4.2-*-**Median PFS, months7.94.11.5*2 events, **1 event Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD5-5.

Vitamin D status of human immunodeficiency virus-positive patients with advanced liver disease enrolled in the solid organ transplantation in HIV: Multi-site study

An optimal vitamin D status may benefit liver transplantation (LT) patients. Higher levels of 25-hydroxyvitamin D [25(OH)D] mitigate steroid-induced bone loss after LT, correlate with better hepatitis C virus treatment responses, and increase graft survival. This study investigated 25(OH)D levels and assessed strategies for vitamin D deficiency prevention in human immunodeficiency virus (HIV)–positive patients with advanced liver disease who were enrolled in the Solid Organ Transplantation in HIV: Multi-Site Study. 25(OH)D was measured in banked specimens from 154 LT candidates/recipients with the DiaSorin assay; deficiency was defined as a 25(OH)D level < 20 ng/mL. Information about vitamin D supplement use after LT was obtained from medication logs and via surveys. Logistic regression, Cox regression, and linear repeated measures analyses were performed with SAS software. We found that none of the 17 academic medical centers in the United States routinely recommended vitamin D supplements before LT, and only a minority (4/17) recommended vitamin D supplements to all patients after LT. Seventy-one percent of the 139 patients with pre-LT values had vitamin D deficiency, which was significantly associated with cirrhosis (P = 0.01) but no other variable. The vitamin D status improved modestly after LT; however, the status was deficient for 40% of the patients 1 year after LT. In a multivariate linear repeated measures model, a higher pre-LT 25(OH)D level (P < 0.001), specimen collection in the summer (P < 0.001), a routine vitamin D supplementation strategy after LT (P < 0.001), and the time elapsing since LT (P = 0.01) were significantly associated with increases in the post-LT 25(OH)D level; black race was associated with a decreased level (P = 0.02). In conclusion, the majority of patients awaiting LT were vitamin D deficient, and approximately half were vitamin D deficient after LT. More extensive use of vitamin D supplements, more sun exposure, or both are needed to prevent this deficiency in HIV-positive LT candidates and recipients. Liver Transpl 20:156-164, 2014. © 2013 AASLD.

Overexpression of adenylate cyclase-associated protein 1 is associated with metastasis of lung cancer

Lung cancer ranks first in both prevalence and mortality rates among all types of cancer. Metastasis is the main cause of treatment failure. Biomarkers are critical to early diagnosis and prediction and monitoring of progressive lesions. Several biomarkers have been identified for lung cancer but none have been routinely used clinically. The present study assessed the diagnostic and prognostic value of cyclase-associated protein 1 (CAP1) for lung cancer. CAP1 mRNA abundance and protein content were determined by real-time PCR and western blot analysis and/or immunostaining in biopsy specimens (24 neoplastic and 6 non-neoplastic) freshly collected at surgical lung resection, in 82 pathologically banked lung cancer specimens and in cultured non-invasive (95-C) and invasive (95-D) lung cancer cells. Multivariate regression analysis was performed to correlate immunoreactive CAP1 signal with cancer type and stage. In vitro cell migration was performed to determine the effect of RNA interference-mediated CAP1 gene silencing on invasiveness of 95-D cells. These analyses collectively demonstrated that: i) both CAP1 mRNA abundance and protein content were significantly higher in neoplastic compared to non-neoplastic specimens and in metastatic compared to non-metastatic specimens but not different between adenocarcinoma and squamous cell carcinoma; ii) immunoreactive CAP1 signal was significantly stronger in metastatic specimens and 95-D cells compared to non-metastatic specimens and 95-C cells; and iii) RNA interference-mediated CAP1 gene silencing adequately attenuated the invasive capacity of 95-D cells in vitro. These findings suggest that overexpression of CAP1 in lung cancer cells, particularly at the metastatic stage, may have significant clinical implications as a diagnostic/prognostic factor for lung cancer.

MiRNA expression signature for potentially predicting the prognosis of ovarian serous carcinoma

One of the best prognostic predictors for patients with epithelial ovarian cancer is the Federation of Obstetrics and Gynecology (FIGO) stage at diagnosis. Advanced-stage ovarian serous carcinoma (OSC) generally have poor prognosis. The goal of this study is to develop and validate a miRNA expression profile that can differentiate the OSC at early and advanced stages and study its correlation with the prognosis of OSC. To identify a unique microRNA (miRNA) pattern associated with the progression of OSC at early and advanced stages, a miRNA microarray was performed using Chinese tumor bank specimens of patients with OSC stage I or III in a retrospective analysis. The expression of four dysregulated miRNAs was validated using quantitative real-time polymerase chain reaction (qRT-PCR) in an external cohort of 51 cases of OSC samples at stages I and III. Kaplan-Meier analysis was performed to analyze the correlation between the expression of some miRNAs and prognosis. Of the 768 miRNAs analyzed in the microarray, 26 miRNAs were significantly either up- or downregulated, with at least a 2-fold difference, in OSC stage I compared with stage III. The qRT-PCR results showed that miR-510, miR-509-5p, and miR-508-3p were significantly downregulated and that miR-483-5p was upregulated in stage III OSC compared with stage I, which was consistent with the microarray results. Kaplan-Meier analysis showed low miR-510 expression, low miR-509-5p expression, and advanced FIGO stage, and chemotherapy resistance were significantly associated with poorer overall survival (P < 0.05). Our results suggest that miRNAs may play a role in the progression of OSC, and miR-510 and miR-509-5p may be considered novel-candidate clinical biomarkers for predicting OSC outcome.

25-Hydroxyvitamin D in Obese Youth Across the Spectrum of Glucose Tolerance From Normal to Prediabetes to Type 2 Diabetes

OBJECTIVETo 1) determine if plasma 25-hydroxyvitamin D (25[OH]D) concentrations differ among obese youth with normal glucose tolerance (NGT) versus prediabetes versus type 2 diabetes and 2) assess the relationships between 25(OH)D and in vivo insulin sensitivity and β-cell function in this cohort.RESEARCH DESIGN AND METHODSPlasma 25(OH)D concentrations were examined in banked specimens in 9- to 20-year-old obese youth (n = 175; male 42.3%, black 46.3%) (NGT, n = 105; impaired glucose tolerance [IGT], n = 43; type 2 diabetes, n = 27) who had in vivo insulin sensitivity and secretion measured by hyperinsulinemic-euglycemic and hyperglycemic clamp techniques and had an assessment of total body composition and abdominal adiposity.RESULTSThe mean age and BMI of the subjects were 14.3 ± 2.1 years and 35.7 ± 5.6 kg/m2, respectively. BMI, plasma 25(OH)D, and the proportion of vitamin D–deficient and –insufficient children did not differ across the three groups. Furthermore, there was no association between 25(OH)D and in vivo insulin sensitivity or β-cell function relative to insulin sensitivity (disposition index) in all groups combined or in each group separately.CONCLUSIONSOur data in obese youth show 1) no differences in plasma 25(OH)D concentrations across the glucose tolerance groups and 2) no relationship between 25(OH)D and in vivo insulin sensitivity and β-cell function relative to insulin sensitivity in any of the groups. It remains uncertain if enhancement of the vitamin D status could improve pathophysiological mechanisms of prediabetes and type 2 diabetes in obese youth.

O-0012 - Updated Results of the Randomized Phase 2 Impact Trial: Maintenance with TLR-9 Agonist Mgn1703 Vs Placebo in Patients with Metastatic Colorectal Carcinoma (MCRC)

Background: The PRIME study reported significantly improved progression-free survival (PFS) and overall survival (OS) with panitumumab + FOLFOX vs FOLFOX in patients with WT RAS (exons 2, 3 and 4 of KRAS and NRAS) mCRC in a prospective-retrospective analysis (unpublished data). The PEAK study, presented here, was an estimation study of the treatment effect of FOLFOX6 + panitumumab or bevacizumab in 1st-line wild-type (WT) KRAS mCRC. Methods: This prospective-retrospective analysis was designed to assess the effect of FOLFOX6 + panitumumab or bevacizumab on PFS (primary endpoint) and OS in WT RAS (exons 2, 3 and 4 of KRAS and NRAS) mCRC in the PEAK study. WT KRAS exon 2 tumors were required for study entry. Transgenomic SURVEYOR/WAVE™ analysis and bidirectional Sanger sequencing were conducted independently in banked specimens to detect mutations in KRAS exon 3 (codons 59/61), exon 4 (codons 117/ 146); NRAS exon 2 (codons 12/13), exon 3 (codons 59/61), exon 4 (codons 117/146); BRAF exon 15 (codon 600). Results: Of the 285 WT KRAS (exon 2) mCRC patients randomized, 278 received treatment. The RAS ascertainment rate is currently 75%. For patients with WT RAS, treatment HRs (pmab:bev) were 0.63 (95% CI, 0.43-0.94; p = 0.02) for PFS and 0.55 (95% CI, 0.30-1.01; p = 0.06) for OS (Table). Worst grade 3-5 adverse event rates were consistent with the primary analysis. Conclusion: PFS and OS HRs favored panitumumab + FOLFOX6 relative to bevacizumab + FOLFOX6 in this 1st-line estimation study in WT RAS mCRC, suggesting that activating RAS mutations appear to be predictive for panitumumab treatment effect. Safety profiles for both arms were consistent with previously reported studies.

Analysis of KRAS/NRAS mutations in PEAK: A randomized phase II study of FOLFOX6 plus panitumumab (pmab) or bevacizumab (bev) as first-line treatment (tx) for wild-type (WT) KRAS (exon 2) metastatic colorectal cancer (mCRC).

3631 Background: PEAK estimated the tx effect of FOLFOX6 with pmab or bev in 1st-line WT KRAS mCRC. The PRIME study showed significantly improved progression free survival (PFS) and overall survival (OS) with pmab + FOLFOX vs FOLFOX in pts with WT RAS (KRAS/NRAS exons 2, 3, 4) mCRC in a prospective-retrospective analysis (unpublished data). Methods: This prospective-retrospective analysis of PEAK was designed to assess the effect of pmab + FOLFOX6 or bev + FOLFOX6 on PFS (primary endpoint) and OS in WT RAS (KRAS/NRAS exons 2, 3, 4) mCRC. Pts were required to have WT KRAS exon 2 tumors. Bidirectional Sanger sequencing and Transgenomic SURVEYOR/WAVE analysis were independently conducted to detect mutations in KRAS exon 3 (codons 59/61), exon 4 (codons 117/146); NRAS exon 2 (codons 12/13), exon 3 (codons 59/61), exon 4 (codons 117/146); BRAF exon 15 (codon 600) in banked specimens. Results: 285 WT KRAS (exon 2) mCRC patients (pts) were randomized, 278 received tx. The current RAS ascertainment rate is 75%. Tx HRs (pmab:bev) for pts with WT RAS were 0.63 (95% CI, 0.43-0.94; p = 0.02) for PFS and 0.55 (95% CI, 0.30-1.01; p = 0.06) for OS (Table). The incidence of worst grade 3-5 adverse events was consistent with the primary analysis. Updated OS and BRAF results will be presented. Conclusions: In this 1st-line estimation study in WT RAS mCRC, PFS and OS HR favored pmab + FOLFOX6 relative to bev + FOLFOX6, suggesting that activating RAS mutations appear to be predictive for pmab tx effect. The safety profile for both arms was consistent with previously reported studies. Clinical trial information: NCT00819780. [Table: see text]

High-dose interleukin-2 (HD IL-2) in the treatment of advanced melanoma: The University of Pittsburgh experience.

9075 Background: IL-2 is a T-cell growth factor tested in a variety of regimens for advanced melanoma (MEL) and renal cell carcinoma (RCC). High-dose IL-2 (600,000-720,000 IU/kg administered intravenously every 8 hours for up to 14 consecutive doses) was approved by FDA for advanced MEL and RCC in 1998 based upon the durability of responses observed. Early studies of HD IL-2 reported overall (OR) and complete response (CR) rates of 16% and 8% respectively. Severe toxicity limited use to specialized centers with standardized protocols, either intensive care (ICU) or oncology specialty settings. The U Pittsburgh has treated 1022 patients with IL-2 at any dosage and we here present outcomes of 550 MEL pts treated with HD IL-2 in an oncology specialty non-ICU setting. Methods: Clinical and radiological data were collected on all pts treated with IL-2 using the UPCI Cancer Registry and Medical Archival System (MARS). Pharmacy records were reviewed for dosing details. The influence of baseline characteristics on treatment outcomes was assessed using Cox proportional hazards analysis. Results: A total of 848 pts received HD IL-2, of which 298 pts had RCC while 550 had MEL. Detailed pharmacy dosing records were reviewed from 176 pts treated over the past 12 years (2000-2012) who received a total of 3738 cycles. Of 165 pts evaluable for response, OR was documented in 24 pts (14.8%) and CR in 5 pts (3.0%). Median overall survival (OS) was 10.0 mos for all patients and 21.5 mos for responders (CR+PR). Median number of doses per cycle was 7. Toxicity was consistent with prior reports. HD IL-2 required ICU transfers in 5% and 1 death was attributed to HD IL-2. Pts with higher baseline lactate dehydrogenase (LDH) had poorer OS (p &lt; 0.05). Conclusions: In this large and uniformly treated series of recent patients treated with IL-2 OR/CR rates with HD IL-2 are 14.8% and 3.0% respectively. Higher LDH is associated with poorer outcome. Biomarkers of response are currently being evaluated in banked clinical specimens collected from patients under the SPORE in Skin Cancer (P50 CA121973).

Abstract 5530: Rejuvenation of dissociated cryopreserved human ovarian papillary serous tumor cells in an in vivo model.

Abstract Banked tumor tissues are a vital source of research material; however, current preservation techniques rely on flash-freezing. While this storage technique maintains the integrity of the nucleic acids and stable proteins, it does not allow for the recovery of live cells from the banked specimen. Serous carcinomas are the most common and deadliest form of gynecologic cancers. These tumors are heterogeneous with a wide array of genetic alterations. Our lab has developed a method which allows banking of live cells derived from primary ovarian serous papillary carcinoma specimens. Here we show that viable cell populations of interest can be isolated from banked cryopreserved tumor samples and grown in vivo as a potential source for analysis of individual tumors. Primary serous tumor samples were subjected to mechanical disruption and enzymatic digestion to produce dissociated cell suspensions. Samples were cryopreserved using controlled temperature reduction and stored in liquid nitrogen vapor. Viability of banked human tumor specimens was evaluated as the ability to establish a xenograft that histologically resembled the primary tumor. Serous tumor epithelia were isolated by flow cytometry using a cell surface marker, epithelial cell adhesion molecule (EpCAM), from this banked cryopreserved cellular population. Xenografts were generated by subcutaneous injection into NOD-scid IL2Rgammanull mice. Growth was monitored by palpation and regenerated tumors were analyzed for histology and marker expression profile. Nine serous tumor samples were banked using this method. To prove the viability of this cryopreservation process, 3 dissociated tumor cell preparations were thawed and injected into NOD-scid mice. Within 6 months, all 3 samples grew as xenografts that closely resembled the primary serous tumor. We then tested if viable cellular fractions of interest could be isolated from these banked serous tumors cells using fluorescent activated cell sorting. A subpopulation of tumors cells expressing EpCAM was collected from the cryopreserved tumor cell population and injected to establish xenografts. In this in vivo assay, xenografts grew as epithelial tumors in 7 of 9 cases. Our findings demonstrate that cryopreservation of dissociated primary tumor cells is a viable option for the generation of live cell tumor banks for papillary serous carcinoma. The advantages of a live cell tumor bank are numerous: 1) characterization of subpopulations of tumor cells in each patient's tumor sample at the RNA, DNA, and protein levels and 2) defining heterogeneity of a serous tumor with a special focus on identifying cancer initiating populations in each patient. We foresee that characterization of each patient's tumor sample using this live tissue bank may help individualize therapeutic strategies in patients with serous cancers. Citation Format: Daniel Y. Paik, Deanna Janzen, Sanaz Memarzadeh. Rejuvenation of dissociated cryopreserved human ovarian papillary serous tumor cells in an in vivo model. [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 5530. doi:10.1158/1538-7445.AM2013-5530

Abstract 21: Survival prognostic significance of TNFRII, TGFa, TIMP1 &amp; CRP in high-risk melanoma patients.

Abstract Background: The E1694 trial tested the GM2-KLH-QS21 (GMK) vaccine versus high dose interferon-alpha2b as adjuvant therapy for patients with operable high-risk stage IIB/III melanoma in relation to relapse-free survival (RFS) and overall survival (OS). Multiplex analysis of candidate serum analytes utilizing banked specimens from patients participating in this trial was evaluated to develop biomarkers of prognostic or therapeutic predictive value. Methods: The Aushon Multiplex Platform was used to simultaneously quantitate the serum levels of 115 candidate analytes selected based on previous data. Univariate proportional hazard (PH) model was used to assess the association between each marker and OS or RFS. The Benjamini and Hochberg's method was used to adjust for multiple testing. Baseline serum samples from 40 patients treated with the GMK vaccine were analyzed. The least absolute shrinkage and selection operator proportional hazard regression (LASSOPH) was used to select markers that are most informative for RFS and OS. We then fit the regular PH models using the markers selected by the LASSOPH. The survival ROC analysis was used to evaluate the ability of the models to predict 1 year RFS and 5 year OS. Leave-one-out cross validations (LOOCVs) were used to avoid over fitting. Results: A panel of four markers, Tumor Necrosis Factor alpha Receptor II (TNFRII), Transforming Growth Factor alpha (TGFa), Tissue Inhibitor of Metalloproteinases 1 (TIMP1), and C-reactive protein (CRP), at baseline was found to be most informative to OS after GMK vaccine treatment (high serum levels correlate with worse prognosis). The dichotomized risk score based on the four markers can separate the OS curves well (log rank test p-value=0.0005). When using the 4 marker PH model to predict the 5 year OS, we achieved an AUC of 89% (cross validated AUC=72%). High baseline TNFRII was also identified as the most informative marker of worse RFS after GMK vaccine. The RFS of patients with high (above median) baseline TNFRII was significantly lower than that of patients with low (below median) baseline TNFRII (log rank test p-value=0.01). Conclusions: Our modeling analysis yielded a panel of four serum biomarkers that is significantly prognostic of survival in patients with high-risk melanoma and warrants further investigation. Citation Format: Ahmad A. Tarhini, Yan Lin, William A. LaFramboise, Sandra Lee, John Kirkwood. Survival prognostic significance of TNFRII, TGFa, TIMP1 &amp; CRP in high-risk melanoma patients. [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 21. doi:10.1158/1538-7445.AM2013-21

The Sharing Partnership for Innovative Research in Translation (SPIRiT) Consortium: A Model for Collaboration across CTSA Sites

In 2006, the National Institutes of Health (NIH) created the Clinical and Translational Science Award (CTSA) program to “transform the local, regional and national environment for clinical and translational science, thereby increasing the efficiency and speed of clinical and translational research.”1 Since then, 61 CTSA-funded sites have reengineered their local research enterprises to develop cadres of highly trained translational scientists and provide them with a robust array of research resources. Now under the direction of the National Center for Advancing Translational Science (NCATS), the maturing CTSA program is poised to enter its second stage of development to provide “institutions with greater flexibility and more opportunities to build on institutional strengths.”2 This approach is supported by a new CTSA Funding Opportunity Announcement (FOA) that will enable CTSA sites to develop programs to address the specific needs of their investigators and local communities. As a result, institutional CTSA programs will likely evolve from a model that provides a set of common research resources locally to unique programs with a mix of core infrastructure (e.g., training, informatics) that is supplemented by institution-specific resources (e.g., drug discovery, comparative effectiveness research, and research implementation and dissemination cores). Leveraging complementary institutional capabilities through closer collaborations between CTSA sites is the logical next step for the National CTSA Consortium. During the formative years of the CTSA program, several sites forged collaborations by creating regional consortia based on geographic proximity to enable sharing of local resources and meetings of trainees.3 For example, the Chicago Consortium for Community Engagement represents a collaboration among the three Chicago CTSAs to advance coordinated and synergistic approaches to developing community-partnered research, disseminating research results, and providing training in community engagement methods to collaborators in academic and community-based organizations. The Ohio Consortium of three CTSA sites implemented a statewide central institutional review board model. In Texas, several institutions meet to exchange best practices. However, collaborative efforts that are constrained by geographic location may limit the benefits that can be derived from linking scientists, institutions, resources, and innovative tools across an array of institutions with complementary capabilities. Accordingly, six geographically dispersed CTSA sites (Johns Hopkins University, University of Chicago, University of Pennsylvania, University of Pittsburgh, Washington University at St. Louis, Yale University) formed a “virtual CTSA consortium” based on a shared vision for data sharing and prior collaborations focused on informatics initiatives. The initial goals of the six-site Sharing Partnership for Innovative Research in Translation (SPIRiT) Consortium were 1) to develop a data sharing infrastructure for biospecimen-based research and de-identified clinical research data, and 2) to establish inter-institutional collaborations related to pilot studies, regulatory support, core laboratory facilities, and education and career development. Over the past two years, the SPIRiT Consortium embarked on several initiatives to achieve these goals, including the deployment of an informatics software and regulatory infrastructure to enable discovery and sharing of banked pathology specimens, development of an inter-institutional pilot study program to link investigators, and validation of a novel technology to improve the diagnosis of malignant melanoma. These programs are described below.

Transoral robotic surgery and adjuvant therapy for oropharyngeal carcinomas and the influence of p16INK4aon treatment outcomes

To determine the prognostic influence of p16(INK4a) immunohistochemistry on the survival of resectable oropharyngeal carcinomas (OPSCC). Retrospective pathologic evaluation of a prospective single-arm cohort study at a tertiary referral center. There were 48 patients with resectable OPSCC who consented for transoral robotic surgery (TORS) and banked tissue specimen for assessment. TORS was with or without adjuvant radiation or chemoradiation. Main outcome measures were p16(INK4a) status, human papillomavirus status, local-regional disease control, and overall, disease-specific, and disease-free survival. p16(INK4a) and HPV positivity were identified in 73% and 74% of patients respectively. With a median follow-up of 38.8 months (2.5-63.3 months), only one local-regional relapse has occurred in both the p16(INK4a)-positive and p16(INK4a) -negative cohorts. No disease-specific, disease-free, and overall survival differences were observed between p16(INK4a) -positive and p16(INK4a)-negative patients (P = .446, P = .277, P = .643, respectively). p16(INK4a) was not prognostic in resectable OPSCC when treated with an initial TORS approach.

Differential HER2 expression in resected gastric cancer: Is there prognostic value?

54 Background: For advanced gastric cancer, the ToGA trial established HER2 as an important therapeutic target in the 20% of patients whose tumors exhibited HER2 overexpression or gene amplification. Others have reported that HER2-positive tumors are associated with poor survival in advanced disease. The expression profile and prognostic value of HER2 in resectable gastric cancer are unknown. Methods: 111 pts underwent curative intent resection of gastric adenocarcinoma between 1/00-6/11 and had tissue available for analysis. Immunohistochemistry (IHC) for HER2 was performed on banked tumor specimens and graded by two pathologists blinded to outcomes utilizing ToGA trial criteria. An IHC score of 0+ or 1+ was regarded as negative, 3+ as positive. Fluorescence in-situ hybridization (FISH) for HER2 was performed on equivocal (2+) IHC samples. Primary outcome was differential expression, secondary outcome was overall survival (OS). Results: Median age was 64 years, 54% were male. Median tumor size was 4 cm, 7.2% had a positive margin, 67.6% were poorly differentiated, 23.4% had perineural invasion, 35.1% had lymphovascular invasion, and 61.3% had nodal metastases. 24 patients had stage I disease (21.6%), 32 stage II (28.8%), and 55 stage III (49.6%). Mean follow-up was 28.9 months, median OS was 27.2 months. HER2 expression by IHC was negative in 61 (55%), equivocal in 37 (33.3%), and positive in 13 (11.7%). Of the 37 equivocal cases, FISH was positive in 8, for a total of 21 HER2-positive cases (18.9%) and 90 HER2-negative cases (81.1%). HER2 status did not correlate with T or N stage, tumor size or location, tumor grade, or perineural or lymphovascular invasion. HER2 status was not associated with OS (p=0.36). Conclusions: Resectable gastric cancer exhibits differential expression of HER2, similar to that of advanced disease. Despite reports suggesting HER2 positive status is associated with aggressive disease and worse outcomes in the advanced setting, HER2 status is not associated with adverse pathologic factors or survival in resectable disease. Although not prognostic, the predictive value of HER2 status for response to trastuzumab in the adjuvant setting requires further investigation.

Hepatitis E Virus Seroprevalence among Men Who Have Sex with Men, United Kingdom

Hepatitis E Virus Seroprevalence among Men Who Have Sex with Men, United Kingdom

Serum biomarkers of papillary thyroid cancer

ObjectiveTo identify serum biomarkers of papillary thyroid cancer.MethodsProspective analysis was performed of banked tumor and serum specimens from 99 patients with thyroid masses. Enzyme-linked immunosorbent assay (ELISA) was employed to measure levels of five serum proteins previously demonstrated to be up-regulated in papillary thyroid cancer (PTC): angiopoietin-1 (Ang-1), cytokeratin 19 (CK-19), tissue inhibitor of metalloproteinase-1 (TIMP-1), chitinase 3 like-1 (YKL-40), and galectin-3 (GAL-3). Serum levels were compared between patients with PTC and those with benign tumors.ResultsA total of 99 patients were enrolled in the study (27 men, 72 women), with a median age of 54 years. Forty-three patients had PTC and 58 cases were benign tumors. There were no statistically significant differences when comparing all five different biomarkers between PTC and other benign thyroid tumors. The p-values were 0.94, 0.48, 0.72, 0.48, and 0.90 for YKL-40, Gal-3, CK19, TIMP-1, and Ang-1, respectively.ConclusionSerum levels of four of the five proteins were elevated in patients with thyroid masses relative to normal values. However, the difference between benign and PTC was not significant. Two of the markers (Gal-3 & TIMP-1) displayed a greater potential difference, which may warrant further investigation. This study suggests that other serum markers should be sought. This is the first study to investigate potential serum biomarkers based on over-expressed proteins in thyroid cancer versus benign pathology.

Molecular cytogenetics as a clinical test for prognostic and predictive biomarkers in newly diagnosed ovarian cancer

BackgroundThere is a clinical need for routinely available genomic biomarker testing in newly diagnosed ovarian cancer. In the current study we performed molecular cytogenetics using a validated array based comparative genomic hybridization (array CGH) assay to screen for the presence of predictive and prognostic biomarkers in archival diagnostic tissue from ovarian cancer patients. We hypothesized that biomarkers of high-risk disease would be detectable in tumor samples from patients with treatment refractory, advanced disease, and would be detected less frequently in tumor samples from patients with more favorable outcomes. In addition, we predicted that the use of a genome-wide copy number analysis (CNA) testing platform would enable us to identify novel potentially targetable chromosomal alterations of therapeutic significance in a percentage of cases.MethodsFormalin-fixed paraffin-embedded tissue (FFPE) tumor bank specimens were retrieved from the initial surgical resection for 18 ovarian cancer patients. Molecular cytogenetics was performed by array CGH for the detection of somatic chromosomal alterations associated with high-risk disease including amplifications of the CCNE1 and HER2 genes. Genomic risk stratification results were correlated with available clinical data. CGH data from each patient’s tumor genome was also surveyed for the presence of potentially targetable aberrations. Relevant therapeutic agents and open studies for investigational drugs were reported for each patient.ResultsHigh-risk genomic alterations were identified in 12/18 (67%) of cases and all patients with high-risk markers had advanced, treatment refractory disease. Three tumors with minimal genomic changes had no high-risk markers and were from patients with Stage I/II disease that had been completely resected and under surveillance for recurrence. Eleven patients (61%) had at least one potentially targetable genomic alteration including CCNE1, HER2, KRAS gene amplifications, and somatic BRCA1 and/or BRCA2 gene deletions. Bi-allelic PTEN gene deletion was detected in one patient’s tumor.ConclusionsClinical genomic profiling of ovarian tumors by array CGH augments pathologic grade and stage to help stratify newly diagnosed ovarian cancer into high and low-risk disease. This personalized genomic information can also help guide treatment planning and disease monitoring by identifying novel potentially targetable genomic alterations that can be used by clinicians to choose rational directed therapies for patients with chemo-resistant disease.

Prediction of Venous Thromboembolism (VTE) in Patients with Pancreatic Cancer Using Clinical Data, Biomarkers, and VTE Risk Models

Abstract 3398 INTRODUCTION:VTE is common in patients with cancer and causes significant morbidity and mortality. Clinical risk models and biomarkers including C-reactive protein (CRP), soluble P-selectin (sPsel), and D-dimer have been used to predict VTE in diverse groups of cancer patients at varying risk for VTE. The applicability of these findings to specific high risk subtypes of cancer has not been established. Therefore, we sought to identify the value of clinical factors, plasma biomarkers, and risk models in predicting VTE in patients with pancreatic cancer, a malignancy with a high predilection for VTE. METHODS:Patients seen at the University of Michigan Comprehensive Cancer Center (UMCCC) and previously consented and enrolled in a prospective cohort study were eligible. Inclusion criteria are diagnosis of pancreatic adenocarcinoma, evaluation at UMCCC, no VTE within a month prior to cancer diagnosis, and documentation in the Electronic Medical Record (EMR) at least every 6 months until death. Primary objective was to identify factors predictive of VTE. Secondary objectives were to develop a VTE predictive model, assess the utility of published VTE risk models, and evaluate factors associated with overall survival (OS). Demographics, clinical data, and VTE (deep vein thrombosis [DVT], portal vein thrombosis [PVT], or pulmonary embolism [PE]) rate were obtained from the EMR. ELISAs were performed for CRP, D-dimer, Mac-2 binding protein, soluble E-selectin (sEsel), and sPsel using banked plasma specimens drawn at diagnosis. A retrospective cohort study was performed including univariate and multivariate regression analysis. The utility of predictive models by Khorana, et al (Blood, 2008. 111:4902–4907), which includes cancer site, body mass index (BMI), hemoglobin (Hb), platelet (plt) count, and white blood cell count, and the expanded model by the Vienna Cancer and Thrombosis Study (CATS) (Blood, 2010. 116:5377–5382), which additionally includes sPsel and D-dimer, were assessed. RESULTS:Between 2005 and 2011, 89 patients were eligible for analysis. Median follow-up was 268 (18–2433) days. Twenty (22%) cases had a VTE; 10 (50%) DVT, 2 (10%) PE, 4 (20%) PVT, and 4 (20%) multiple VTEs. Mean (SD) age was 63.4 (8.9) in cases and 65.3 (11.2) in controls. Women accounted for 55% of cases and 48% of controls.Higher BMI (median 28.8 [21.2–44.7] in cases vs. 25.4 [16.4–43.3] in controls, p=0.03) and lower plt count (median 241 [145–323] in cases vs. 289 [97–648] in controls, p=0.001) were associated with VTE on univariate analysis. On multivariate regression analysis, lower plt count (β −0.01, SE 0.004) and lower Hb (β −0.43, SE 0.20) were predictive of VTE after adjusting for BMI, tumor location, and treatment with surgery, chemotherapy or radiation (AUC 0.78). None of the biomarkers were significantly associated with VTE on univariate analysis, although there was a trend with D-dimer (p=0.09).The Khorana score was determined in 85 patients; 48 were intermediate (2 points) and 37 high risk (≥3 points) with VTE rates of 20.8% and 24.3%, respectively (p=0.70). The AUC of this model was 0.63. The risk score from CATS was calculated for 84 patients; 54 were intermediate (2 or 3 points), 17 high (4 points), and 13 highest risk (≥5 points). VTE incidence was not different among these groups and the AUC was 0.65.Factors associated with poor OS on univariate analysis were: age (per 10-year increment) (HR [95% confidence interval], p-value) (1.35 [1.07–1.71], 0.013), chronic kidney disease (5.67 [2.62–12.25], <0.0001), use of anticoagulation (3.14 [1.33–7.41], 0.009), stage III/IV vs. I/II pancreas cancer (2.05 [1.27–3.32], 0.003), and INR (1.65 [1.04–2.63], 0.035); elevated Hb (0.87 [0.76–0.99], 0.041) and sEsel (0.46 [0.29–0.72], 0.0007) were protective. CONCLUSIONS:Pancreatic cancer patients with higher BMI, lower plt count, and lower Hb were more likely to develop VTE. Other clinical variables and biomarkers did not add additional predictive information. Elevated sEsel, important for neutrophil trafficking to sites of inflammation, was found to be protective on survival analysis. The risk models developed by Khorana, et al and CATS in a diverse group of patients with cancer were not able to further differentiate VTE risk among this already high risk group. Additional work is needed to determine which patients with pancreatic cancer are at highest risk for VTE and who may benefit most from thromboprophylaxis. Disclosures:No relevant conflicts of interest to declare.

82nd Annual Meeting of the American Thyroid AssociationMEETING ABSTRACTS &amp; PROGRAM

82nd Annual Meeting of the American Thyroid AssociationMEETING ABSTRACTS &amp; PROGRAM

Comprehensive Genomic Studies

As part of the molecular revolution sweeping medicine, comprehensive genomic studies are adding powerful dimensions to medical research. However, their power exposes new regulatory, strategic, and quality assurance challenges for biorepositories. A key issue is that unlike other research techniques commonly applied to banked specimens, nucleic acid sequencing, if sufficiently extensive, yields data that could identify a patient. This evolving paradigm renders the concepts of anonymized and anonymous specimens increasingly outdated. The challenges for biorepositories in this new era include refined consent processes and wording, selection and use of legacy specimens, quality assurance procedures, institutional documentation, data sharing, and interaction with institutional review boards. Given current trends, biorepositories should consider these issues now, even if they are not currently experiencing sample requests for genomic analysis. We summarize our current experiences and best practices at Washington University Medical School, St Louis, MO, our perceptions of emerging trends, and recommendations.

Biobanking of Fresh-frozen Human Colon Tissues: Impact of Tissue Ex-vivo Ischemia Times and Storage Periods on RNA Quality

Biobanking plays an important role in translational cancer research. The impact of tissue ex-vivo ischemia time and storage period on RNA integrity is not well documented. Fresh-frozen colon tissues were collected in Taizhou Hospital of Zhejiang Province in China since 2004. Fifty-one colon cancer tissues with tumor cell content higher than 70 % and matched normal tissues during four storage periods (less than 15 months, 16-20 months, 21-25 months, and 26-40 months) were chosen to detect RNA quality. Fresh colon cancer tissues from 5 patients were cut into pieces and kept at room temperature or on ice for 0.5, 1, 2, and 4 h before snap freezing. RNA integrity was determined by microcapillary electrophoresis by the RNA integrity number (RIN) algorithm. Sixty-seven percent of normal colon tissues and 94 % of colon cancer specimens yielded RNA with a RIN of ≥7. Matched colon cancer and normal tissues showed significant difference in RNA quality. RNA remained stable in colon cancer tissues kept at room temperature and on ice for up to 4 h, and long-term storage of banked colon specimens did not negatively influence RNA quality (RNA with RIN of ≥7 banked less than 15 months, 83 %; 16-20 months, 78 %; 21-25 months, 77 %; 26-40 months, 90 %). Frozen colon tissues yield high-quality RNA in approximately 80 % of specimens. Ex-vivo ischemia times and storage periods did not adversely affect RNA quality. This study showed that standard operation protocols and the maintenance of high-quality tissue repositories were the keys to translational medicine research.