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Genetic variation in the activation of the prodrug cyclophosphamide (CP) by cytochrome P450 (CYP) enzymes has been shown to influence outcomes. However, CYP are also subject to phenoconversion due to either the effects of comedications or cancer associated down-regulation of expression. The aim of this study was to assess the relationship between CP bioactivation with CYP2B6 and CYP2C19 genotype, as well as CYP2C19 phenotype, in breast cancer patients. CP and the active metabolite levels were assessed in breast cancer patients (n = 34) at cycle 1 and cycle 3 of treatment. Patients were genotyped for a series of SNP known to affect CYP2B6 and CYP2C19 function. The activity of CYP2C19 was also assessed using a probe drug. We found a significant linear gene-dose relationship with CYP2B6 coding SNP and formation of 4-hydroxycyclophosphamide. A possible association with CYP2C19 null genotype at cycle 1 was obscured at cycle 3 due to the substantial intra-individual change in CP bioactivation on subsequent dosing. Comedications may be the cause for this inter-occasion variation in bioactivation of cyclophosphamide and the ensuing phenoconversion may account for the conflicting reports in the literature about the relationship between CYP2C19 genotype and CP bioactivation pharmacokinetics. Trial registration ANZCTR363222 (6/11/2012, retrospectively registered).

Standard dosages of fluoropyrimidine chemotherapy result in severe toxicity in a substantial proportion of patients, however, routine pre-therapeutic toxicity prediction remains uncommon. A thymine (THY) challenge test can discriminate risk of severe gastrointestinal toxicity in patients receiving fluoropyrimidine monotherapy. We aimed to measure endogenous plasma uracil (U) and its ratio to dihydrouracil (DHU), and assess the performance of these parameters compared with the THY challenge test to evaluate risk of severe toxicity. Plasma samples, previously collected from 37 patients receiving 5-fluorouracil (5-FU) or capecitabine monotherapy for a THY challenge test (ACTRN12615000586516; retrospectively registered), were assessed for endogenous plasma concentrations of U and DHU using a validated LC–MS/MS method. Renal function was estimated from blood creatinine, and patients with ≥ grade 3 toxicity (CTCAE v4.0) were classified as cases. There were no differences in median endogenous U plasma concentrations or U/DHU ratios between severe toxicity cases and non-cases. Significant differences between cases and non-cases were noted when these measures were normalised to the estimated renal function (CrCL), Unorm p = 0.0004; U/DHUnorm p = 0.0083. These two parameters had a sensitivity of 29%, compared with 57% for the THY challenge test in the same patients. Genotyping for clinically relevant DPYD variants was inferior to either of these pyrimidine phenotyping tests (sensitivity of 14%). The endogenous uracil-based parameters, adjusted to CrCL, were more predictive of increased risk of severe fluoropyrimidine toxicity than DPYD genotyping. However, endogenous U measurement detected fewer cases of severe toxicity than the THY challenge test.

Fluorouracil (5-FU), a chemotherapeutic agent widely used in the treatment of numerous common malignancies, causes oral mucositis in a proportion of patients. The contribution of drug transport processes to the development of this toxicity is currently unknown. This work aimed to establish and optimise a simple phenotyping assay for 5-FU uptake into primary buccal mucosal cells (BMC). The uptake kinetics of radiolabelled 5-FU were determined in pooled BMC freshly collected from healthy volunteers. The inter- and intra-individual variability in 5-FU uptake was then assessed across a cohort that included both healthy volunteers and cancer patients. 5-FU uptake into pooled primary BMC was both time and concentration dependent. An Eadie–Hofstee analysis suggested two components; a high-affinity (KM = 3.3 µM) low-capacity ( $$V_{\text{MAX}}$$ = 57.8 pmol min−1 105 viable cells−1) transporter, and a high-capacity ( $$V_{\text{MAX}}$$ = 1230 pmol min−1 105 viable cells−1) low-affinity (KM = 3932 µM) transporter. There was 180-fold variation in the rate of 5-FU uptake into BMC (0.10–17.86 pmol min−1 105 viable cells−1) across the 34 subjects (healthy participants N = 24, cancer patients N = 10). Notably, retesting of a subset of these participants (N = 16) multiple times over a period of up to 140 days demonstrated poor stability of the uptake phenotype within individuals. The uptake of 5-FU into healthy oral mucosal cells is a highly variable process facilitated by membrane transporters at pharmacologically relevant concentrations. This bioassay is simple, minimally invasive, and suitable for phenotypic analysis of drug transport in healthy primary cells.

Vaccines that elicit targeted tumor antigen-specific T-cell responses have the potential to be used as adjuvant therapy in patients with high risk of relapse. However, the responses induced by vaccines in cancer patients have generally been disappointing. To improve vaccine function, we investigated the possibility of exploiting the immunostimulatory capacity of type 1 Natural killer T (NKT) cells, a cell type enriched in lymphoid tissues that can trigger improved antigen-presenting function in dendritic cells (DCs). In this phase I dose escalation study, we treated eight patients with high-risk surgically resected stage II-IV melanoma with intravenous autologous monocyte-derived DCs loaded with the NKT cell agonist α-GalCer and peptides derived from the cancer testis antigen NY-ESO-1. Two synthetic long peptides spanning defined immunogenic regions of the NY-ESO-1 sequence were used. This therapy proved to be safe and immunologically effective, inducing increases in circulating NY-ESO-1-specific T cells that could be detected directly ex vivo in seven out of eight patients. These responses were achieved using as few as 5 × 105 peptide-loaded cells per dose. Analysis after in vitro restimulation showed increases in polyfunctional CD4+ and CD8+ T cells that were capable of manufacturing two or more cytokines simultaneously. Evidence of NKT cell proliferation and/or NKT cell-associated cytokine secretion was seen in most patients. In light of these strong responses, the concept of including NKT cell agonists in vaccine design requires further investigation.

CYP2C19 contributes to the metabolism of several chemotherapeutic agents. The CYP2C19 homozygous null function genotype strongly predicts activity phenotype in healthy populations. An additional acquired loss of function has been reported in up to one-third of cancer patients. It is not known whether this phenomenon also occurs in patients with earlier stage or in resected disease. This study investigated whether acquired loss of CYP2C19 function was detectable in patients with stage III–IV or resected gastrointestinal cancer. CYP2C19 genotype was determined in 49 patients, and subjects were probed for CYP2C19 activity on three test occasions. An acquired loss of CYP2C19 activity was observed in 20 % of stage III–IV and 17 % of resected patients at the first test. Significant (p

There is no standard treatment for recurrent glioblastoma multiforme (GBM). Retreatment with temozolomide (TMZ) is one treatment option. We reasoned this could be more effective if combined with a vaccine that preferentially targeted TMZ-resistant cells. To test the feasibility and safety of such an approach, a phase 1 trial was conducted in which patients with GBM tumors relapsing after standard chemoradiotherapy were retreated with TMZ in combination with a vaccine consisting of monocyte-derived dendritic cells (DC) pulsed with autologous tumor cells that had previously been exposed to TMZ in vivo in the course of primary treatment. Of 14 participants, nine patients completed the initial phase of priming vaccinations and two cycles of TMZ, one proved to have radionecrosis, one rapidly progressed, and in three the yield of DC vaccine was insufficient to proceed with treatment. Other than expected toxicities related to TMZ, there were no adverse events attributable to the combined treatment. Two patients had objective radiological responses. Six month progression-free survival was 22 %, similar to retreatment with TMZ alone. Anti-tumor immune responses were assessed in peripheral blood mononuclear cells using interferon-γ ELISpot, with two patients meeting criteria for a vaccine-induced immune response, one of whom remained disease-free for nearly three years. Another patient with an anti-tumor immune response at baseline that was sustained post-vaccination experienced a 12-month period of progression-free survival. In summary, the combined treatment was safe and well-tolerated but feasibility in the recurrent setting was marginal. Evidence of immune responses in a few patients broadly correlated with better clinical outcome.

Breast reduction is a common procedure used to improve physical and aesthetic factors associated with breast hypertrophy. This study investigated how surgical technique alone affects the risk factors for complications and profiled differences between techniques. Complications were assessed by the use of time-to-event methods.Patient information was extracted from a cohort of 283 patients. Demographic, surgical, and follow-up information was analyzed for patients undergoing surgical procedures using the inferior pedicle Wise pattern (IPWP) and modified Hall-Findlay (MHF) techniques. The patients managed with the IPWP technique were considered control subjects. The failure rates were described using the Kaplan-Meier failure estimator to provide a true estimate of the experienced complication rates.Overall, few differences were noted between the groups except for total tissue removed. The overall failure (complication) rate at 6 months was 18.8%, with 9% of all the patients experiencing a major complication that required operative intervention/revision. As expected, the period with the greatest risk of complication was the first month after surgery. Surgical technique, total tissue removed, and age were nonpredictive of complications. Overall, the IPWP group had significantly more total tissue removed than the MHF group (median difference, 227 g; P=0.002). There was no evidence of a learning curve when an experienced surgeon moved from the one technique to the other.At 6 months after surgery, 19% of patients are expected to have experienced a complication. There appears to be few differences in outcomes between the techniques of breast reductions used, and the success or otherwise almost certainly relates to factors independent of surgical technique and includes patient selection, operative skill, and experience. Time-to-event analysis provides a precise assessment and description of the complication profile.This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors at www.springer.com/00266.

Variation in drug disposition and response is associated with many anticancer agents, in part due to large inter-patient differences in pharmacokinetics (Petros and Evans, 2004). This may be the result of variable expression of hepatic cytochrome P450 (CYP), enzymes which play an important role in the metabolism and elimination of many chemotherapeutic drugs. CYP2C19 is an important member of the CYP family of enzymes and is involved in the metabolism of a diverse range of drug substrates such as the antiulcer agent omeprazole (OMP) (Desta et al, 2002) and the antimalarial drug proguanil (Ward et al, 1991). Recently, it has become apparent that CYP2C19 is also important in the disposition of a number of chemotherapeutic agents, including cyclophosphamide (Takada et al, 2004; Timm et al, 2005), thalidomide (Ando et al, 2002; Li et al, 2007) and bortezomib (Uttamsingh et al, 2005). There is a loss of function genetic polymorphism associated with the CYP2C19 enzyme, with the result that individuals who are homozygous variant (var/var) are ‘poor metabolisers' (PM) of drugs that are substrates for CYP2C19 (Desta et al, 2002). Approximately 3% of Caucasians and up to 30% of Asians are CYP2C19 PM (Xie et al, 1999). Numerous studies have shown a correlation between variant genotype and PM phenotype in healthy populations (Xie et al, 1999; Desta et al, 2002). Hence, the use of a genotyping test for CYP2C19 metaboliser status is often advocated as a substitute for direct phenotyping with validated probe drugs such as OMP. However, genetic variability is not the only factor that can affect functional activity of drug-metabolising enzymes; gender, age and co-medications can all play a role in variable CYP expression. In particular, co-medications can mimic the genetic variability observed for CYP2C19 with the result that a patient can become a PM ‘phenocopy' due to inhibition (competition) of drug metabolism. However, the importance of chronic disease as a contributor to variable drug disposition is often overlooked. There is increasing evidence that the activity of CYP is decreased in cancer. In a small study of patients (n=16) with advanced cancer (Williams et al, 2000), all individuals had normal CYP2C19 genotype but 25% of these patients were CYP2C19 PM phenocopies. However, the mechanism behind this genotype–phenotype discordance was not investigated. Previous studies have demonstrated that the activity of a different enzyme, CYP3A4, is also decreased in advanced cancer patients and that the decreased activity correlates with inflammatory response (Rivory et al, 2002). An inverse relationship between plasma cytokine (TNF-α and IL-6) concentrations and CYP2C19 activity has been demonstrated in patients with congestive heart failure (Frye et al, 2002). Moreover, CYP2C19 mRNA expression is reported to be downregulated by cytokines in human hepatocyte in vitro cultures (Aitken and Morgan, 2007). Hence, limited evidence indicates that advanced cancer patients may have decreased capacity to metabolise certain chemotherapeutic agents due to decreased CYP activity. The aim of this study was to confirm whether or not CYP2C19 activity is decreased in patients with advanced cancer and to determine whether a relationship exists between CYP2C19 metaboliser status and inflammatory markers such as cytokines and acute phase response proteins.

Despite its declining incidence, gastric cancer remains one of the leading causes of cancer-related death worldwide. The definitive management of localized gastric cancer has been the center of much international controversy over the years; surgery remains the mainstay, with debate centering on the required extent of lymph node resection. The role of adjuvant chemotherapy has been studied for decades, but because trials have been underpowered, it has been difficult to demonstrate a statistically significant benefit. Recently, four large meta-analyses have been published on adjuvant chemotherapy in gastric cancer. The first three have been criticized for their methodology, but the most recent meta-analysis was well conducted and showed a statistically significant benefit in favor of adjuvant chemotherapy. The heterogeneity of chemotherapy schedules included in this meta-analysis, however, makes it difficult to define the current standard. Randomized trials in the metastatic setting have shown that the combinations of epirubicin, cisplatin, and infusional fluorouracil (ECF) and docetaxel, cisplatin, and fluorouracil (TCF) have demonstrated superiority over other regimens. The role of preoperative chemotherapy is currently under investigation, with results from the MAGIC (Medical Research Council Adjuvant Gastric Infusional Chemotherapy) trial demonstrating a significant improvement in resectability, progression-free survival, and overall survival with perioperative therapy in patients with operable gastric cancer. Postoperative chemoradiotherapy has also been adopted as a standard of care in the US following the publication of the results of the Intergroup Study, INT-0116, although there remains debate over the relative benefits of more radical surgery versus the use of radiation with adjuvant treatment. It is clear that adjuvant therapy is now an appropriate addition to surgery, although there are still further questions regarding the optimal protocols.

Pancreatic adenocarcinoma (PAC) remains one of the most lethal cancers. The overall 5-year survival rate (all stages) remains