Martin R. Johnson

Rapid Identification of Dihydropyrimidine Dehydrogenase Deficiency by Using a Novel 2-13C-Uracil Breath Test

Purpose: Dihydropyrimidine dehydrogenase (DPD)-deficient cancer patients have been shown to develop severe toxicity after administration of 5-fluorouracil. Routine determination of DPD activity is limited by time-consuming and labor-intensive methods. The purpose of this study was to develop a simple and rapid 2-13C-uracil breath test, which could be applied in most clinical settings to detect DPD-deficient cancer patients. Experimental Design: Fifty-eight individuals (50 “normal,” 7 partially, and 1 profoundly DPD-deficient) ingested an aqueous solution of 2-13C-uracil (6 mg/kg). 13CO2 levels were determined in exhaled breath at various time intervals up to 180 min using IR spectroscopy (UBiT-IR300). DPD enzyme activity and DPYD genotype were determined by radioassay and denaturing high-performance liquid chromatography, respectively. Results: The mean (±SE) Cmax, Tmax, δ over baseline values at 50 min (DOB50) and cumulative percentage of 13C dose recovered (PDR) for normal, partially, and profoundly DPD-deficient individuals were 186.4 ± 3.9, 117.1 ± 9.8, and 3.6 DOB; 52 ± 2, 100 ± 18.4, and 120 min; 174.1 ± 4.6, 89.6 ± 11.6, and 0.9 DOB50; and 53.8 ± 1.0, 36.9 ± 2.4, and <1 PDR, respectively. The differences between the normal and DPD-deficient individuals were highly significant (all Ps <0.001). Conclusions: We demonstrated statistically significant differences in the 2-13C-uracil breath test indices (Cmax, Tmax, DOB50, and PDR) among healthy and DPD-deficient individuals. These data suggest that a single time-point determination (50 min) could rapidly identify DPD-deficient individuals with a less costly and time-consuming method that is applicable for most hospitals or physicians’ offices.

The Role of Pharmacogenetics and Pharmacogenomics in Cancer Chemotherapy with 5-Fluorouracil

There is increasing evidence supporting the important role of genetics in determining the effect (response and toxicity) to cancer chemotherapy. This has included both pharmacogenetics, where the alteration of a gene coding for an important drug metabolizing enzyme results in increased toxicity (and occasionally altered efficacy), and pharmacogenomics, where knowledge of the expression of genes critical to the action of the cancer chemotherapy drug can be used to individualize therapy. This manuscript focuses on the widely used cancer chemotherapy drug 5-fluorouracil (5-FU) to illustrate the following concepts: (1) The effect of the pharmacogenetic syndrome known as dihydropyrimidine dehydrogenase (DPD) deficiency on 5-FU pharmacology; (2) the role of pharmacogenomics in individualizing 5-FU therapy, and (3) the potential value of pharmacogenomics in designing new drugs.

Neuronal Repellent Slit2 Inhibits Dendritic Cell Migration and the Development of Immune Responses

One of the essential functions of dendritic cells is to take up Ags in peripheral tissues and migrate into secondary lymphoid organs to present Ags to lymphocytes for the induction of immune responses. Although many studies have demonstrated that the migration of dendritic cells is closely associated with the development of immune responses, little is known about factors that inhibit dendritic cell migration and control the extent of immune responses to Ag stimulation. We show that Slit2, a neuronal repellent factor, is up-regulated in the skin by allergen sensitization and down-regulates the migration of Langerhans cells. The effect is mediated by direct interaction of Slit2 with cells that express a Slit-specific receptor, Robo1. Slit2-mediated inhibition of Langerhans cell migration results in suppression of contact hypersensitivity responses. These findings provide insights into a novel mechanism by which Slit2 functions as an anti-inflammatory factor for the initiation of immune responses.

Phase II study of capecitabine with concomitant radiotherapy for patients with locally advanced pancreatic cancer: up-regulation of thymidine phosphorylase.

Purpose:The objectives of this phase II study were to evaluate the effect of radiation (XRT) on thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and tumor necrosis factor-&agr; (TNF-&agr;) and the efficacy of capecitabine-XRT in patients with locally advanced pancreatic cancer. Patients and Methods:Twenty patients received 50.4 Gy XRT with capecitabine 1,600 mg/m2 on Monday through Friday for 6 weeks determined from our phase I study (Saif MW, Eloubeidi MA, Russo S, et al. J Clin Oncol. 2005;23:8679–8687). After capecitabine-XRT, stable and responding patients received capecitabine 2,000 mg/m2 for 14 days every 3 weeks till progression. Restaging was performed every 9 weeks. Tumor specimens were procured with endoscopic ultrasound-fine needle aspiration before and at week 2 after capecitabine-XRT was started to evaluate the effect of XRT on TP, DPD, and TNF-&agr; mRNA levels, determined by reverse transcriptase-polymerase chain reaction. Results:Among 20 patients, 4 (20%) had a partial response and 13 (65%) had stable disease. Two patients underwent surgical resection (10%). The 6-month survival rate was 84%, and the 1-year survival was 58%. Grade ≥3 toxicities included nausea/vomiting (5%), thrombosis (5%), hyperbilirubinemia (5%), and grade 3 gastrointestinal bleeding (5%). TP was elevated during week 2 when compared with the pre-XRT TP (P = 0.01). However, no such effect of XRT was found either on DPD (P = 0.22) or on TNF-&agr; (P = 0.6). No correlation between TP and TNF-&agr; was noticed. Also, no association between TP/DPD ratio and efficacy of capecitabine was identified. Conclusions:This phase II study further confirms our phase I results and suggests that capecitabine-XRT is an effective, tolerable, and convenient alternative to an infusional 5-fluorouracil regimen for patients with pancreatic cancer. Although results support the use of capecitabine-XRT and TP was up-regulated, there appears to be additional genes associated with the response to capecitabine.

A comparative analysis of translated dihydropyrimidine dehydrogenase cDNA; conservation of functional domains and relevance to genetic polymorphisms.

A pharmacogenetic syndrome caused by molecular defects in the dihydropyrimidine dehydrogenase gene (DPYD ) results in partial to complete loss of dihydropyrimidine dehydrogenase (DPD) enzyme activity with patients exhibiting life-threatening toxicity following administration of routine doses of 5-fluorouracil. To date, more than 19 reported mutations have been putatively associated with DPD deficiency with 16 occurring within the open reading frame of the cDNA. The purpose of this study was to examine the conservation of functional domains (including the uracil, flavine adenine dinucleotide and NADPH binding sites) across three phyla (Chordata, Arthropoda and Nematoda) and the conservation of regions corresponding to the previously reported mutations. Comparative analysis of the uracil and NADPH binding sites in mammals and invertebrates demonstrated 100% amino acid identity between mammals and Drosophila melanogaster. Caenorhabditis elegans demonstrated 89% and 88% identity in these domains, respectively. The mammalian sequences demonstrated 100% identity in two iron sulphur motifs (amino acids 953-964 and 986-997) with significant conservation in D. melanogaster (92% and 92% identity, respectively) and C. elegans (100% and 92% identity, respectively). Comparative amino acid analysis revealed non-conservation in the loci of four DPYD mutations [DPYD*12 (R21Q), DPYD*5 (I543V), DPYD*6 (V732I), DPYD*9A (C29R)]. Seven mutations occurred in highly conserved regions [M166V, DPYD*8 (R235W), DPYD*11 (V335l), DPYD*4 (S534N), DPYD*9B (R886H), D949V, DPYD*10 (V995F)]. In summary, this comparative analysis identified conserved regions which may be critical to enzyme structure and/or function. The conservation of loci where DPYD mutations occur was also examined to evaluate their functional significance on DPD enzyme activity. These data should prove useful in the evaluation of newly discovered mutations in the DPYD gene.

Abstract 563: Novel role of VEGF and neuropilin-1 in breast cancer tumor-stromal interactions

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Human breast cancers contain high levels of fibroblastic stroma as a critical feature of desmoplasia. The complex interactions between tumor and stromal cells play important roles in tumor growth, metastasis and resistance to anticancer treatments. Our previous in vivo studies showed that increased levels of tumor-released VEGF were associated with increased desmoplastic responses and resulted in a hormonal resistance phenotype of ER+ breast tumor. Based on our findings, a clinical trial conducted at UAB with a new combinational treatment targeting both ER and VEGF signalings has achieved significant objective response in ER+ breast cancer patients. In our current studies, we further studied the role of VEGF in mediating tumor-stromal interactions and initiation of desmoplasia. Our results have shown that tumor released VEGF interacts with stromal fibroblasts which express VEGF receptors R1, R2 and Neuropilin-1 (NRP1), thereby stimulating their infiltration to promote desmoplastic response. Down regulation of NRP1 expression by siRNA significantly abolished the fibroblast chemo-attractive motility towards VEGF, while the VEGFR1 and VEGFR2 blocking antibodies had no effects on their migration mobility, suggesting that NRP1 expressed in fibroblast is involved in tumor-stroma cell interactions in breast cancer. Our studies using breast cancer xenograft mouse model with regulated tumor VEGF expression revealed that tumor VEGF expression was positively correlated with expression of NRP1 and alpha smooth muscle actin (α-SMA, a transformed/myo fibroblast marker) in the tumor stroma. In addition, levels of tumor VEGF, stromal NRP1 and α-SMA were all positively correlated with level of tumor stromal content and tumor growth. Blocking tumor VEGF with a neutralizing antibody (Avastin) as a treatment modality in xenograft mice reversed the VEGF increased desmoplasia. Our study results reveal a novel role of VEGF and NRP1 in tumor-stromal interactions and desmoplasia, indicating NRP1 can be a therapeutic target for breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 563.