Fossum, Croix C.

Abstract Background: Treatment-related AML (t-AML) accounts for 10 to 20% of all AML cases and carries an especially poor prognosis (Kayser et al. 2011, Godley et al. 2008). Patients diagnosed with t-AML are likely to have abnormal cytogenetic profiles with chromosome changes that are predictive of an aggressive malignancy, poor response to therapy, and decreased overall survival(Smith et al. 2003). The simultaneous use of multiple chemotherapeutic agents of different classes makes it increasingly difficult to predict risk for developing t-AML and determining disease course. An updated analysis of predictive factors for t-AML is needed so clinicians can more accurately inform patients of their prognosis. The aim of this study was to classify t-AML according to primary malignancy, previous chemotherapy exposure, and cytogenetic profile. Methods: A retrospective chart review of patients that were diagnosed with AML at Mayo Clinic from 7/1/1990 to 5/13/2015 was performed following IRB approval. AML patients found to have a previous malignancy treated with chemotherapy were classified as t-AML. Chemotherapeutics were classified as alkylating agents, antimetabolites, anti-tubulin agents, and topoisomerase II inhibitors. Patients diagnosed with a myelodysplastic or myeloproliferative disorder prior to development of AML were excluded from this study. Previous chemotherapy exposures, duration of chemotherapy exposure, complete blood count, chromosome abnormalities, and survival data were collected for t-AML cases. Cytogenetic changes were classified as favorable, intermediate, and adverse according to the system used by Kayser et al. 2011. JMP 10.0 was used for statistical analysis. Results: Out of 584 patients, 64 patients (11%) had a primary malignancy that was treated with chemotherapy prior to being diagnosed with AML. The most common primary malignancies were breast cancer (31%), non-Hodgkin lymphoma (27%), colorectal cancer (8%), and Hodgkin lymphoma (8%). Laboratory findings showed median hemoglobin 9.6 g/dL (4.7-13.8), median white blood cells 3.2 x109 (0.6-126), median platelets 50x109 (3-320), median peripheral blood blasts of 8% (0-91), and median bone marrow blasts 38% (1-94). 95% of patients diagnosed with t-AML had been previously treated with an alkylating agent. Additional exposure to an anti-metabolite trended towards a more adverse cytogenetic profile (χ2=5.0, p=0.08) but there was not a statistically significant decrease in overall survival (KM analysis, p=0.31). The median overall survival for patients diagnosed with t-AML was 10.2 months compared to 19.2 months for patients with de-novo AML (KM analysis, p=0.04). Adverse cytogenetic profiles were associated with decreased survival (KM analysis, p <0.0001). However, there was no difference in overall survival between patients with t-AML that had intermediate cytogenetics and those with de-novo AML (KM analysis, p=0.36). None of the chemotherapy classes other than antimetabolites were associated with poor cytogenetics or survival when combined with an alkylating agent. Conclusion: Over half of all patients classified as having t-AML in this study received prior chemotherapy for breast cancer or non-Hodgkin lymphoma. Cytogenetic classification of t-AML into favorable, intermediate and adverse groups is useful in predicting disease course. Interestingly, t-AML patients with intermediate risk cytogenetics had similar overall survival to patients with de-novo AML. This suggests that the poor outcomes observed in patients with t-AML is predominantly due to the subset with adverse cytogenetics. Thus, cytogenetic analysis remains the best indicator of overall survival regardless of chemotherapy exposure. Additional work is needed to delineate the risk associated with the aforementioned chemotherapy classes. Disclosures Al-Kali: Celgene: Research Funding.

711 Background: For patients with stage IVA rectal cancer with liver metastases treated with curative-intent chemotherapy and surgery, the role of adjuvant pelvic radiotherapy (RT) is unclear. The purpose of this study was to evaluate the impact of pelvic RT on oncologic outcomes in this patient population. Methods: We retrospectively reviewed medical records of all patients with stage IVA rectal adenocarcinoma with liver metastases treated with curative-intent resection of the primary tumor and all liver metastases at our institution between 1991 and 2010. Patient and treatment characteristics were compared between patients who did or did not receive pelvic RT using chi-square and unpaired ttests. Survival and recurrence estimates were calculated from date of initial diagnosis using the Kaplan-Meier method. Local recurrence (LR) was defined as recurrence in the pelvis occurring at or before distant recurrence (DR). Cox regression was used to compare rates of freedom from LR (FFLR), freedom from DR (FFDR), and overall survival (OS) between patients who did or did not receive pelvic RT. Results: The analysis included 65 patients. Median patient age at diagnosis was 59 years (range, 27-87). Tumor stage was T2 (n = 4), T3 (n = 53), or T4 (n = 8). The median number of liver metastases was 2 (range, 1-14). Surgery was low anterior resection (n = 54) or abdominoperineal resection (n = 11). All patients received perioperative fluoropyrimidine based chemotherapy. Pelvic RT was administered to 35 patients (54%), either preoperatively (n = 22) or postoperatively (n = 13). Median RT dose was 50.4 Gy (range 25-58). Patient characteristics were similar for those who did or did not receive pelvic RT. Median follow-up was 3.7 years. The 3-year estimates of FFLR, FFDR and OS were 81% vs 58% (p =.056), 35% vs 29% (p =.75), and 66% vs 71% (p =.81) for RT vs no RT, respectively. Conclusions: In this analysis of patients with stage IVA rectal cancer with liver metastases undergoing curative intent therapy, pelvic RT (vs. no pelvic RT) was associated with a trend to lower rates of LR and similar rates of DR and OS.

Understanding the structure and function of the oropharynx is paramount for providing excellent patient care. In clinical oncology, the oropharynx is generally divided into four distinct components: (i) the base of the tongue; (ii) the soft palate; (iii) the palatine tonsillar fossa; and (iv) the pharyngeal wall. The oropharyngeal mucosa is distinct from other mucosal surfaces in the body, as it is composed of a reticulated epithelium with a discontinuous basement membrane, also known as lymphoepithelium. This review describes the anatomy, histology, immunology and surgical resection of the oropharynx as they relate to oncological care.

Abstract The pyrimidine analog 5-fluorouracil (5-FU) was developed in the 1950s and remains one of the most widely used chemotherapy drugs in use today. A significant portion of patients who receive 5-FU suffer severe adverse toxicities, and, in extreme cases, death. Dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene) rapidly degrades 85% of administered 5-FU, and as such, limits the amount of drug available for conversion into active metabolites. Genetic variations in DPYD have been suggested to increase the risk of 5-FU toxicity. Large-scale sequencing efforts have identified nearly 100 missense and nonsense DPYD variations, however only a handful of alleles have been studied for their association with 5-FU toxicity. Most studies have focused on a single allele (DPYD*2A), which only accounts for a small fraction of DPD deficiencies. Further complicating the interpretation of DPYD alleles, many individuals carry multiple variations within the DPYD gene. The purpose of this study was to identify missense variants that affect the enzyme function of DPD using a mammalian isogenic system of expression. Expression vectors encoding missense DPYD variations were generated by site directed mutagenesis and the expressed protein assayed for enzyme activity. Cells expressing selected variants were also teasted for sensitivity to 5-FU using a novel implementation of electrical impedance based realtime cellular analysis. The results of functional studies are compared to in silico predictions used to classify variants as deleterious or benign. An appreciable fraction of DPD variants showed enzyme activity that was significantly lower than that of wildtype protein. Within the deficient group of variants, a gradient of activities was noted, prompting the grouping of variants as mildly, moderately, and severely impaired. In addition to loss-of-function variations, a few hyperactive DPYD alleles were identified. This novel class of DPYD variants may be protective against 5-FU toxicity at the cost of treatment efficacy. The further evaluation of hyperactive alleles in clinical studies will be necessary to determine their impact on patient outcomes. Overall these data will facilitate the development of predictive tests for 5-FU toxicity by allowing researchers to prioritize variations for association studies. Citation Format: Steven M. Offer, Natalie J. Wegner, Croix Fossum, Robert D. Diasio. Classification of DPYD variations using an isogenic system of expression. [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 2202. doi:10.1158/1538-7445.AM2013-2202

Abstract Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme of the uracil catabolic pathway, being critically important for inactivation of the commonly prescribed anti-cancer drug 5-fluorouracil (5-FU). DPD impairment leads to increased exposure to 5-FU and, in turn, increased anabolism of 5-FU to cytotoxic nucleotides, resulting in more severe clinical adverse effects. Numerous variants within the gene coding for DPD, DPYD, have been described, although only a few have been demonstrated to reduce DPD enzyme activity. To identify DPYD variants that alter enzyme function, we expressed 80 protein-coding variants in an isogenic mammalian system and measured their capacities to convert 5-FU to dihydro-fluorouracil, the product of DPD catabolism. The M166V, E828K, K861R, and P1023T variants exhibited significantly higher enzyme activity than wild-type DPD (120%, P = 0.025; 116%, P = 0.049; 130%, P = 0.0077; 138%, P = 0.048, respectively). Consistent with clinical association studies of 5-FU toxicity, the D949V substitution reduced enzyme activity by 41% (P = 0.0031). Enzyme activity was also significantly reduced for 30 additional variants, 19 of which had &lt;25% activity. None of those 30 variants have been previously reported to associate with 5-FU toxicity in clinical association studies, which have been conducted primarily in populations of European ancestry. Using publicly available genotype databases, we confirmed the rarity of these variants in European populations but showed that they are detected at appreciable frequencies in other populations. These data strongly suggest that testing for the reported deficient DPYD variations could dramatically improve predictive genetic tests for 5-FU sensitivity, especially in individuals of non-European descent. Cancer Res; 74(9); 2545–54. ©2014 AACR.

Abstract Dihydropyrimidine dehydrogenase (DPD, encoded by DPYD) is the rate-limiting enzyme in the uracil catabolic pathway and has a pivotal role in the pharmacokinetics of the commonly prescribed anticancer drug 5-fluorouracil (5-FU). Deficiency of DPD, whether due to inadequate expression or deleterious variants in DPYD, has been linked to severe toxic responses to 5-FU. Little is known about the mechanisms governing DPD expression in the liver. In this report, we show increased accumulation of RNA-induced silencing complex (RISC) proteins on DPYD mRNA in cells overexpressing the highly homologous microRNAs (miRNA) miR-27a and miR-27b. These miRNAs were shown to repress DPD expression through two conserved recognition sites in DPYD. The IC50 of 5-FU for HCT116 cells overexpressing miR-27a or miR-27b was 4.4 μmol/L (both), significantly lower than that for cells expressing a nontargeting (scramble) control miRNA (14.3 μmol/L; P = 3.3 × 10−5 and P = 1.5 × 10−7, respectively). Mouse liver DPD enzyme activity was inversely correlated with expression levels of miR-27a (R2 = 0.49; P = 0.0012) and miR-27b (R2 = 0.29; P = 0.022). A common variant in the hairpin loop region of hsa-mir-27a (rs895819) was also shown to be associated with elevated expression of the miR-27a in a panel of cell lines (P = 0.029) and in a transgenic overexpression model (P = 0.0011). Furthermore, rs895819 was associated with reduced DPD enzyme activity (P = 0.028) in a cohort of 40 healthy volunteers. Taken together, these results suggest that miR-27a and miR-27b expression may be pharmacologically relevant modulators of DPD enzyme function in the liver. Furthermore, our data suggest that rs895819 may be a potential risk allele for 5-FU sensitivity. Mol Cancer Ther; 13(3); 742–51. ©2014 AACR.

Abstract In the 45 years since its development, the pyrimidine analog 5-fluorouracil (5-FU) has become an integral component of many cancer treatments, most notably for the management of colorectal cancer. An appreciable fraction of patients who receive 5-FU suffer severe adverse toxicities, which in extreme cases may result in death. Dihydropyrimidine dehydrogenase (DPD, encoded by DPYD) rapidly degrades 85% of administered 5-FU, and as such, limits the amount of drug available for conversion into active metabolites. Clinical studies have suggested that genetic variations in DPYD increase the risk for 5-FU toxicity, however, there is not a clear consensus about which variations are relevant predictors. In the present study, DPYD variants were expressed in mammalian cells, and the enzymatic activity of expressed protein was determined relative to wild-type (WT). Relative sensitivity to 5-FU for cells expressing DPYD variations was also measured. The DPYD*2A variant (exon 14 deletion caused by IVS14+1G&gt;A) was confirmed to be catalytically inactive. Compared with WT, two variants, S534N and C29R, showed significantly higher enzymatic activity. Cells expressing S534N were more resistant to 5-FU–mediated toxicity compared with cells expressing WT DPYD. These findings support the hypothesis that selected DPYD alleles are protective against severe 5-FU toxicity, and, as a consequence, may decrease the effectiveness of 5-FU an antitumor drug in carriers. In addition, this study shows a method that may be useful for phenotyping other genetic variations in pharmacologically relevant pathways. Cancer Res; 73(6); 1958–68. ©2013 AACR.