Lee, Adam M

e22515 Background: Osteosarcoma is a skeletal malignancy affecting children, adolescents, and young adults. Surgical resection and chemotherapy are the mainstay of therapy with a three-drug regimen - cisplatin, doxorubicin, and methotrexate. However, prognosis remains poor mainly due to chemo-resistance and/or metastases. Emerging evidence has shown that long non-coding RNAs (lncRNAs) are implicated in drug sensitivity and cancer metastasis. Our lab has recently identified strong significant association between cellular sensitivity to cisplatin and doxorubicin and expression of a lncRNA ANRIL in a collection of osteosarcoma cell lines. Methods: SiRNA knockdown experiments were carried out in an osteosarcoma cell line, SAOS2. Cells were exposed to multiple concentrations of cisplatin or doxorubicin. Cellular sensitivity to these two drugs was quantified after siRNA experiment with siRNA targeting ANRIL and scramble control at 24, 48 and 72h. Clinical outcome analysis was performed using data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Results: We successfully knocked down ANRIL expression in SAOS2 cells with 88.5% efficiency. A significant decreased rate of proliferation was detected for cisplatin (7 uM, 72h) in knockdown cells (16.5% +/- 4.1) compared to scramble control (26.4% +/- 0.8). The cisplatin IC50 was decreased in the siRNA knockdown condition as compared to scramble control at 72h (p = 0.02). For doxorubicin, the IC50 was also significantly decreased in the siRNA knockdown condition as compared to scramble control (p = 0.002 and 0.007 at 24h and 72h, respectively). Survival outcomes, metastases at diagnosis, and percent necrosis (an indicator of treatment response) were evaluated in TARGET osteosarcoma patients (n = 121 and n = 43 for OS, percent necrosis, respectively) with varying degrees of ANRIL expression. Above the median ANRIL expression was associated with a greater number of death (p = 0.004) and higher chances of metastases at diagnosis (p = 0.013). Conclusions: Reducing ANRIL expression led to increased cellular sensitivity to cisplatin and doxorubicin, two key treatment agents for osteosarcoma. Higher ANRIL expression was significantly associated with higher rates of osteosarcoma death and metastases at diagnosis in a clinical cohort of patients. These findings may suggest ANRIL serves as a novel biomarker of cisplatin and doxorubicin sensitivity in osteosarcoma.

508 Background: Severe adverse events (AEs) to 5-fluorouracil (5-FU) constitute a major dilemma in CC treatment. We recently demonstrated that specific deleterious DPYD variants in the coding region were highly predictive of grade >3 (G3+) AEs in stage III CC pts receiving adjuvant 5-FU based combination chemotherapy [Lee et al. JNCI. In press]. Other studies have suggested potential associations between AEs and non-coding DPYD variants: a novel haplotype (HapB3) containing three intronic variants (c.483+18 G>A, c.680+139 G>A, and c.959-51 T>C), one synonymous variant (c.1236 G>A, E412E), and a deep intronic variant (c.1129-5923 C>G) that affects pre-mRNA splicing. However, these studies included a diversity of cancer types, stages and treatments. The findings require further evaluation in a refined population. Methods: A total of 2,134 Caucasian stage III CC pts without DPYD*2A, D949V, and I560S variants from NCCTG N0147 trial who received adjuvant FOLFOX or FOLFIRI +/- cetuximab were genotyped by multiplexed single-base extension assays on the Sequenom MassARRAY. G3+ AEs were recorded (CTCAE v3) and classified as common to 5-FU treatment (5FU-AEs). Logistic regressions were used to assess the univariate and multivariate associations. Results: Analyzed pts displayed the following characteristics: 54.3% male, median age 59 [19-85], proficient MMR 87.5%, PS-0 76.5%, + irinotecan 8.5%, and + cetuximab 46.5%. Pts carrying c.1129-5923 C>G were significantly associated with higher risk of G3+ 5FU-AEs (Odds Ratio [OR]=1.539, 95% confidence interval [CI]=1.001 – 2.367, p=0.049). Only marginal association was observed for one of the HapB3 variants, c.1236 G>A (p=0.058). A linearly increasing risk of G3+ 5FU-AEs was identified for HapB3, comparing pts with no, one or >1 variant present (unadjusted OR=1.539, 95% CI=1.001 – 2.367, p=0.041; adjusted OR=1.198, 95% CI=0.998-1.437, p=0.052). Conclusions: In the largest study to date, c.1129-5923 C>G and HapB3 were predictive of 5FU-AE risk in stage III CC patients without DPYD*2A, D949V, and I560S variants receiving 5-FU-based combination chemotherapy.

209 Background: Predictive markers of response to AA are not known. In a prior study, we found that 8 single nucleotide polymorphisms (SNPs) from 4 candidate hormone-related genes (JAK2, TRMT11, NKX3-1 and HSD17B12) were associated with response to AA in HSPC in a North American cohort of 519 subjects. We attempt to replicate these findings in a cohort of Portuguese patients. Methods: The Portuguese cohort consisted of 308 HSPC patients, and the primary endpoint was time to AA failure, defined as time from initiating continuous AA to two serial PSA increases over the nadir, initiation of chemotherapy for progressive disease, or clinical or imaging based progression, whichever came first. For each SNP, we calculated hazard ratios (HRs) and 95% confidence intervals (CI) for time to AA failure using a recessive model with adjustment for Gleason score (GS). Results: The median age of the Portuguese cohort was 69 years (range 43, 85). The GS distribution was 35% subjects with GS≥8; 35% with GS=7 and 30% with GS<7. Median time to AA failure for the cohort was 1.8 years (IQ range: 0.3, 3.6). Association of each SNP with time to AA failure is shown in the Table. None of the SNPs replicated. However, there was a suggestive inverse association for rs10503733 (NKX3.1) with time to AA failure (P=0.07). Conclusions: While we were not able to replicate our initial findings, there was an unexpected association with rs10503733 (NKX3.1 gene) that may warrant additional clinical validation. [Table: see text]