Eva Gross

Analysis of the DPYD Gene Implicated in 5-Fluorouracil Catabolism in a Cohort of Caucasian Individuals

Purpose: Complete or partial loss of dihydropyrimidine dehydrogenase (DPD) function has been described in cancer patients with intolerance to fluoropyrimidine drugs like 5-fluorouracil (5-FU) or Xeloda. The intention of this population study is to assess and to evaluate gene variations in the entire coding region of the dihydropyrimidine dehydrogenase gene (DPYD), which could be implicated in DPD malfunction. Experimental Design: A cohort of 157 individuals was genotyped by denaturing high-performance liquid chromatography; 100 of these genotypes were compared with functional studies on DPD activity and mRNA expression. Results: Twenty-three variants in coding and noncoding regions of the DPYD gene were detected, giving rise to 15 common haplotypes with a frequency of >1%. Rare sequence alterations included a frameshift mutation (295-298delTCAT) and three novel point mutations, 1218G>A (Met406Ile), 1236G>A (Glu412Glu), and 3067C>T (Pro1023Ser). DPD enzyme activity showed high variation in the analyzed population and correlated with DPD mRNA expression. In particular, the novel variants were not accompanied with decreased enzyme activity. However, a statistically significant deviation from the median DPD activity of the population was associated with the mutations 1601G>A (Ser534Asn) and 2846A>T (Asp949Val). Conclusion: This work presents an analysis of DPYD gene variations in a large cohort of Caucasians. The results reflect the genetic and enzymatic variability of DPD in the population and may contribute to further insight into the pharmacogenetic disorder of DPD deficiency.

Clinical relevance of DPYD variants c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity: a systematic review and meta-analysis of individual patient data

BACKGROUND The best-known cause of intolerance to fluoropyrimidines is dihydropyrimidine dehydrogenase (DPD) deficiency, which can result from deleterious polymorphisms in the gene encoding DPD (DPYD), including DPYD*2A and c.2846A>T. Three other variants-DPYD c.1679T>G, c.1236G>A/HapB3, and c.1601G>A-have been associated with DPD deficiency, but no definitive evidence for the clinical validity of these variants is available. The primary objective of this systematic review and meta-analysis was to assess the clinical validity of c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity. METHODS We did a systematic review of the literature published before Dec 17, 2014, to identify cohort studies investigating associations between DPYD c.1679T>G, c.1236G>A/HapB3, and c.1601G>A and severe (grade ≥3) fluoropyrimidine-associated toxicity in patients treated with fluoropyrimidines (fluorouracil, capecitabine, or tegafur-uracil as single agents, in combination with other anticancer drugs, or with radiotherapy). Individual patient data were retrieved and analysed in a multivariable analysis to obtain an adjusted relative risk (RR). Effect estimates were pooled by use of a random-effects meta-analysis. The threshold for significance was set at a p value of less than 0·0167 (Bonferroni correction). FINDINGS 7365 patients from eight studies were included in the meta-analysis. DPYD c.1679T>G was significantly associated with fluoropyrimidine-associated toxicity (adjusted RR 4·40, 95% CI 2·08-9·30, p<0·0001), as was c.1236G>A/HapB3 (1·59, 1·29-1·97, p<0·0001). The association between c.1601G>A and fluoropyrimidine-associated toxicity was not significant (adjusted RR 1·52, 95% CI 0·86-2·70, p=0·15). Analysis of individual types of toxicity showed consistent associations of c.1679T>G and c.1236G>A/HapB3 with gastrointestinal toxicity (adjusted RR 5·72, 95% CI 1·40-23·33, p=0·015; and 2·04, 1·49-2·78, p<0·0001, respectively) and haematological toxicity (adjusted RR 9·76, 95% CI 3·03-31·48, p=0·00014; and 2·07, 1·17-3·68, p=0·013, respectively), but not with hand-foot syndrome. DPYD*2A and c.2846A>T were also significantly associated with severe fluoropyrimidine-associated toxicity (adjusted RR 2·85, 95% CI 1·75-4·62, p<0·0001; and 3·02, 2·22-4·10, p<0·0001, respectively). INTERPRETATION DPYD variants c.1679T>G and c.1236G>A/HapB3 are clinically relevant predictors of fluoropyrimidine-associated toxicity. Upfront screening for these variants, in addition to the established variants DPYD*2A and c.2846A>T, is recommended to improve the safety of patients with cancer treated with fluoropyrimidines. FUNDING None.

Strong Association of a Common Dihydropyrimidine Dehydrogenase Gene Polymorphism with Fluoropyrimidine-Related Toxicity in Cancer Patients

Background Cancer patients carrying mutations in the dihydropyrimidine dehydrogenase gene (DPYD) have a high risk to experience severe drug-adverse effects following chemotherapy with fluoropyrimidine drugs such as 5-fluorouracil (5-FU) or capecitabine. The pretreatment detection of this impairment of pyrimidine catabolism could prevent serious, potentially lethal side effects. As known deleterious mutations explain only a limited proportion of the drug-adverse events, we systematically searched for additional DPYD variations associated with enhanced drug toxicity. Methodology/Principal Findings We performed a whole gene approach covering the entire coding region and compared DPYD genotype frequencies between cancer patients with good (n = 89) and with poor (n = 39) tolerance of a fluoropyrimidine-based chemotherapy regimen. Applying logistic regression analysis and sliding window approaches we identified the strongest association with fluoropyrimidine-related grade III and IV toxicity for the non-synonymous polymorphism c.496A>G (p.Met166Val). We then confirmed our initial results using an independent sample of 53 individuals suffering from drug-adverse-effects. The combined odds ratio calculated for 92 toxicity cases was 4.42 [95% CI 2.12–9.23]; p (trend)<0.001; p (corrected) = 0.001; the attributable risk was 56.9%. Comparing tumor-type matched sets of samples, correlation of c.496A>G with toxicity was particularly present in patients with gastroesophageal and breast cancer, but did not reach significance in patients with colorectal malignancies. Conclusion Our results show compelling evidence that, at least in distinct tumor types, a common DPYD polymorphism strongly contributes to the occurrence of fluoropyrimidine-related drug adverse effects. Carriers of this variant could benefit from individual dose adjustment of the fluoropyrimidine drug or alternate therapies.

Routine dihydropyrimidine dehydrogenase testing for anticipating 5-fluorouracil-related severe toxicities: hype or hope?

5-Fluorouracil (5-FU) is a mainstay for treating colorectal cancer, alone or more frequently as part of combination therapies. However, its efficacy/toxicity balance is often limited by the occurrence of severe toxicities, showing in about 15%-20% of patients. Several clinical reports have shown the deleterious effect of dihydropyrimidine dehydrogenase (DPD) genetic polymorphism, a condition that reduces the liver detoxification step of standard dosages of 5-FU, in patients undergoing fluoropyrimidine-based therapy. Admittedly, DPD deficiency accounts for 50%-75% of the severe and sometimes life-threatening toxicities associated with 5-FU (or oral 5-FU). However, technical consensus on the best way to identify patients with DPD deficiency before administrating 5-FU is far from being achieved. Consequently, no regulatory step has been undertaken yet to recommend DPD testing as part of routine clinical practice for securing the administration of 5-FU. This review covers the limits and achievements of the various strategies proposed so far for determining DPD status in patients scheduled for 5-FU therapy.

Germline mutations in the PALB2 gene are population specific and occur with low frequencies in familial breast cancer

The Partner and Localizer of BRCA2 (PALB2) protein has been linked to Fanconi anemia and breast cancer predisposition. Here we present data of a comprehensive mutation screening of the PALB2 gene in 818 familial cases of breast cancer from Germany. By analysing the entire coding region of PALB2, we found seven truncating mutations (six of them novel) in families tested negative for BRCA1/2‐mutations. In addition, two novel potentially disease causing missense mutations were found. Remarkably, only one mutation reported previously in other populations, was also identified in the German population. No PALB2 mutation carriers were identidfied in 450 unaffected controls. Thus, our observations indicate a low prevalence of deleterious PALB2 mutations and a specific mutation profile within the German population. As PALB2‐deficient tumors were shown to be sensitive to Poly(ADP‐ribose) Polymerase (PARP) inhibitors, our study has implications for newly developed, favourable treatment options in familial breast cancer.

Tumor suppressor KAI1 affects integrin αvβ3-mediated ovarian cancer cell adhesion, motility, and proliferation

The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin alphavbeta3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin alphavbeta3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with beta1-integrins, also colocalizes with integrin alphavbeta3. Functionally, elevated KAI1 levels drastically increased integrin alphavbeta3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin alphavbeta3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin alphavbeta3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.

Downregulation of Serine Protease HTRA1 Is Associated with Poor Survival in Breast Cancer

HTRA1 is a highly conserved serine protease which has been implicated in suppression of epithelial-to-mesenchymal-transition (EMT) and cell motility in breast cancer. Its prognostic relevance for breast cancer is unclear so far. Therefore, we evaluated the impact of HTRA1 mRNA expression on patient outcome using a cohort of 131 breast cancer patients as well as a validation cohort including 2809 publically available data sets. Additionally, we aimed at investigating for the presence of promoter hypermethylation as a mechanism for silencing the HTRA1 gene in breast tumors. HTRA1 downregulation was detected in more than 50% of the breast cancer specimens and was associated with higher tumor stage (p = 0.025). By applying Cox proportional hazard models, we observed favorable overall (OS) and disease-free survival (DFS) related to high HTRA1 expression (HR = 0.45 [CI 0.23–0.90], p = 0.023; HR = 0.55 [CI 0.32–0.94], p = 0.028, respectively), with even more pronounced impact in node-positive patients (HR = 0.21 [CI 0.07–0.63], p = 0.006; HR = 0.29 [CI 0.13–0.65], p = 0.002, respectively). Moreover, HTRA1 remained a statistically significant factor predicting DFS among established clinical parameters in the multivariable analysis. Its impact on patient outcome was independently confirmed in the validation set (for relapse-free survival (n = 2809): HR = 0.79 [CI 0.7–0.9], log-rank p = 0.0003; for OS (n = 971): HR = 0.63 [CI 0.48–0.83], log-rank p = 0.0009). In promoter analyses, we in fact detected methylation of HTRA1 in a small subset of breast cancer specimens (two out of a series of 12), and in MCF-7 breast cancer cells which exhibited 22-fold lower HTRA1 mRNA expression levels compared to unmethylated MDA-MB-231 cells. In conclusion, we show that downregulation of HTRA1 is associated with shorter patient survival, particularly in node-positive breast cancer. Since HTRA1 loss was demonstrated to induce EMT and cancer cell invasion, these patients might benefit from demethylating agents or histone deacetylase inhibitors previously reported to lead to HTRA1 upregulation, or from novel small-molecule inhibitors targeting EMT-related processes.

The dihydrouracil/uracil ratio in plasma, clinical and genetic analysis for screening of dihydropyrimidine dehydrogenase deficiency in colorectal cancer patients treated with 5-fluorouracil.

A rapid and cost-effective reversed phase high performance liquid chromatography (HPLC) method for quantification of dihydrouracil to uracil ratio (UH2/U) in plasma has been developed and used to screen for dihydropyrimidine dehydrogenase (DPD) deficiency in nine patients treated with 5-fluorouracil (5-FU). This HPLC method is based on the use of a simultaneous UV detection at 205 and 268nm during the analysis run of the plasma extract and taking into account the particularity that UH2 shows no absorbance response at 268nm. The plasma UH2/U ratio values evaluated by the use of our HPLC assay were found to be highly correlated with the plasma 5-FU-half-life values and were significantly associated with the toxic side effects, whereas, data set provided from genetic analysis of the coding sequences of the DPD gene (DPYD) were found to be insufficient to explain all the cases of the 5-FU-related toxicity pattern. The proposed HPLC assay could be available for routine clinical use for DPD deficiency assessment in patients prior to 5-FU administration.

Alternative splicing of KAI1 abrogates its tumor-suppressive effects on integrin αvβ3-mediated ovarian cancer biology

Loss or downregulation of the tumor-suppressor KAI1 correlates with poor cancer patient prognosis. KAI1 functions by interacting with other proteins, including integrin cell adhesion and signaling receptors. We previously showed that KAI1 physically and functionally crosstalks with the tumor-biologically relevant integrin αvβ3, thereby suppressing ovarian cancer cell migration and proliferation. Interestingly, in metastases, a KAI1 splice variant had been identified, indicating poor patient prognosis. Thus, we here characterized differential effects of the two KAI1 proteins upon their cellular restoration. Opposite to KAI1, KAI1-splice reduced αvβ3-mediated cell adhesion, thereby inducing cell migration. This was accompanied by elevated αvβ3 levels and drastically elevated focal adhesion kinase activation, however, without any obvious colocalization with αvβ3, as observed for KAI1. Moreover, codistribution of KAI1 with the cell/cell-adhesion molecule E-cadherin was abrogated in KAI1-splice. Whereas KAI1 diminished cell proliferative activity, KAI1-splice prominently enhanced cell proliferation concomitant with elevated transcription and cell-surface expression of the epidermal growth factor receptor. Thus KAI1-splice does not only counteract the tumor-suppressive actions of KAI1, but - beyond that - promotes αvβ3-mediated biological functions in favor of tumor progression and metastasis.

Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers

Background:Genomic rearrangements at the fragile site FRA1E may disrupt the dihydropyrimidine dehydrogenase gene (DPYD) which is involved in 5-fluorouracil (5-FU) catabolism. In triple-negative breast cancer (TNBC), a subtype of breast cancer frequently deficient in DNA repair, we have investigated the susceptibility to acquire copy number variations (CNVs) in DPYD and evaluated their impact on standard adjuvant treatment.Methods:DPYD CNVs were analysed in 106 TNBC tumour specimens using multiplex ligation-dependent probe amplification (MLPA) analysis. Dihydropyrimidine dehydrogenase (DPD) expression was determined by immunohistochemistry in 146 tumour tissues.Results:In TNBC, we detected 43 (41%) tumour specimens with genomic deletions and/or duplications within DPYD which were associated with higher histological grade (P=0.006) and with rearrangements in the DNA repair gene BRCA1 (P=0.007). Immunohistochemical analysis revealed low, moderate and high DPD expression in 64%, 29% and 7% of all TNBCs, and in 40%, 53% and 7% of TNBCs with DPYD CNVs, respectively. Irrespective of DPD protein levels, the presence of CNVs was significantly related to longer time to progression in patients who had received 5-FU- and/or anthracycline-based polychemotherapy (hazard ratio=0.26 (95% CI: 0.07–0.91), log-rank P=0.023; adjusted for tumour stage: P=0.037).Conclusion:Genomic rearrangements in DPYD, rather than aberrant DPD protein levels, reflect a distinct tumour profile associated with prolonged time to progression upon first-line chemotherapy in TNBC.