Yvonne Wettergren

A Study of the MTHFR Gene Polymorphism C677T in Colorectal Cancer

PURPOSEnThe aim of this study was to examine the clinical significance of the methylenetetrahydrofolate reductase (MTHFR) gene polymorphism C677T in colorectal cancer (CRC). The hypothesis was that the genotype could affect the risk of cancer development and the results of cancer treatment.nnnPATIENTS AND METHODSnGenotyping was made for a random 30% (n = 544) of all patients treated for CRC at our unit from 1999 to 2006 (n = 1812). Basic clinical and pathologic factors were analyzed by genotype group and also compared with those of the entire cohort. Tolerability of chemotherapy and possible side effects were analyzed by genotype. Survival was analyzed by genotype for all stages for patients treated between 1999 and 2003. The genotype prevalence was also compared with a control material of healthy blood donors.nnnRESULTSnNo genotype was associated with an increased risk of CRC or higher cancer stage. The patients with CT/TT genotype had significantly greater risk of suffering side effects from fluoropyrimidine (5-fluorouracil) treatment (P < .05). In stage III colon cancer, the patients with CT/TT genotype had a poorer prognosis than those with the CC genotype. The difference was significant in univariate (P < .003) and multivariate (P < .040) analysis. Though the genotype-associated side effect risks remained in stage IV, the effect on survival was not significant (P < .1).nnnCONCLUSIONnThe MTHFR polymorphism C677T does, in our material, not affect the risk of CRC; however, it can affect the sensitivity to chemotherapy and the risk of side-effects and therefore survival in stage III and possibly stage IV colon cancer. It could be a future predictive factor in the choice of a treatment regimen.

Molecular determinants of efficacy for 5‐FU‐based treatments in advanced colorectal cancer: mRNA expression for 18 chemotherapy‐related genes

5‐Fluorouracil (5‐FU)‐based regimens remain a cornerstone in the treatment of colorectal cancer (CRC). However, the attendant toxicity prevents these regimens from reaching maximum therapeutic potential. In this retrospective analysis, we examined the pretreatment expression of 18 genes in archival tumor bank samples from patients with advanced CRC to determine if one or more of the selected genes showed promise as either a prognostic or predictive marker of 5‐FU‐based treatment outcomes. One hundred and forty‐four CRC patient samples (collected from 1983 to 2004) were analyzed via real‐time PCR for gene expression. Univariate analyses were used to correlate gene expression with efficacy and time‐to‐event variables. Low thymidine phosphorylase (TP), dihydrofolate reductase, dihydropyrimidine dehydrogenase (DPD), excision repair cross‐complementing 1 (ERCC1) and thymidylate synthase gene expression were associated with better time‐to‐progression in the entire population. Low TP, DPD and ERCC1 expression were independently associated with improved overall survival. Low TP gene expression was also predictive of response. This study suggests that TP gene expression in particular is a predictive as well as a prognostic biomarker for advanced CRC patients. Gene panels assessing pretreatment TP, DPD, ERCC1, dihydrofolate reductase and thymidylate synthase gene expression may help improve the therapeutic potential of 5‐FU‐ or other novel antifolate‐based regimens. Further analysis of the prognostic or predictive value of these genes in prospective trials in CRC patients seems warranted.

Pretherapeutic uracil and dihydrouracil levels of colorectal cancer patients are associated with sex and toxic side effects during adjuvant 5-fluorouracil–based chemotherapy†

In Nordic countries, the standard treatment of colorectal cancer (CRC) in the adjuvant setting is bolus 5‐fluorouracil (5‐FU) plus leucovorin alone or in combination with oxaliplatin. 5‐FU competes with the natural occurring pyrimidine uracil (Ura) as a substrate for dihydropyrimidine dehydrogenase (DPD; enzyme commission number 1.3.1.2). Low DPD activity is associated with toxicity during treatment. Pretherapeutic detection of DPD deficiency could prevent severe toxicity otherwise limiting drug administration. Assays showing that DPD deficiency impairs breakdown of Ura to dihydrouracil (UH2) seem promising for clinical use.

Rapid method for relative gene expression determination in human tissues using automated capillary gel electrophoresis and multicolor detection.

The aim of this study was to evaluate a direct and automated post-polymerase chain reaction (PCR) detection system to simultaneously determine the relative gene expression levels of nine cancer-related human genes. Total RNA was prepared from flash-frozen biopsies derived from human colorectal tumors or normal mucosa and reverse-transcribed to cDNA which was PCR-amplified using primer pairs corresponding to the studied genes. In each reaction, the forward primer was labeled with a fluorescent dye. The PCR products were pooled and an internal size standard with a uniquely colored fluorescent dye was added. The samples were then subjected to automated capillary gel electrophoresis. Fragment analysis software was used to calculate the relative gene expression using beta-actin as the reference gene. We found that automated capillary gel electrophoresis with multicolor detection is a rapid, accurate and highly reproducible method for separation and quantification of PCR-amplified cDNA.

p37δ is a new isoform of PI3K p110δ that increases cell proliferation and is overexpressed in tumors

The phosphatidylinositol 3-kinases (PI3Ks) regulate cell growth, proliferation and survival, and are frequently affected in human cancer. PI3K is composed of a catalytic subunit, p110, and a regulatory subunit, p85. The PI3K catalytic subunit p110δ is encoded by PIK3CD and contains p85- and RAS-binding domains, and a kinase domain. Here we present an alternatively spliced PIK3CD transcript encoding a previously unknown protein, p37δ, and demonstrate that this protein is expressed in human ovarian and colorectal tumors. p37δ retains the p85-binding domain and a fraction of the RAS-binding domain, lacks the catalytic domain, and has a unique carboxyl-terminal region. In contrast to p110δ, which stabilizes p85, p37δ promoted p85 sequestering. Despite the truncated RAS-binding domain, p37δ bound to RAS and we found a strong positive correlation between the protein levels of p37δ and RAS. Overexpressing p37δ, but not p110δ, increased the proliferation and invasive properties of HEK-293 cells and mouse embryonic fibroblasts. Cells overexpressing p37δ showed a quicker phosphorylation response of AKT and ERK1/2 following serum stimulation. Ubiquitous expression of human p37δ in the fruit fly increased body size, DNA content and phosphorylated ERK1/2 levels. Thus, p37δ appears to be a new tumor-specific isoform of p110δ with growth-promoting properties.

Folate levels measured by LC–MS/MS in patients with colorectal cancer treated with different leucovorin dosages

PurposeCalcium folinate (leucovorin), which is converted in vivo into biologically active folate, enhances the potency of 5-fluorouracil (5-FU)-based chemotherapy in colorectal cancer. A common dosage of leucovorin in adjuvant and palliative settings is 60xa0mg/m2. The aim was to determine the levels of tetrahydrofolate (THF), 5,10-methylenetetrahydrofolate (methyleneTHF), and 5-methyltetrahydrofolate (methylTHF) in tumour and mucosa of colorectal cancer patients who received different dosages of leucovorin intravenously at time of surgery.MethodsEighty patients scheduled for colorectal resection with indication of colorectal cancer were randomised into four groups to receive leucovorin at 0, 60, 200, or 500xa0mg/m2, respectively. Blood samples were taken 10 and 30xa0min after leucovorin administration. Biopsy samples from tumour and mucosa were collected and snap-frozen at surgery. The levels of THF, methyleneTHF, and methylTHF in tumour and mucosa were assessed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC–MS/MS) and the results were related to clinical diagnosis and therapeutic regimens.ResultsThe folate levels in tissue revealed extensive inter-individual variability. The mean methyleneTHF value for the four treatment groups were 880, 1,769, 3,024 and 3,723xa0pmol/gww. Only half of the patients who received 60xa0mg/m2 leucovorin had higher levels of methyleneTHF in tumour than patients who received 0xa0mg/m2 leucovorin. Rectal cancer patients had significantly lower levels of methyleneTHF compared with colon cancer patients.ConclusionsThere was a large inter-patient variability of tissue folate levels in colorectal cancer patients after supplementation with leucovorin at standardised dosage. High leucovorin doses were needed to exceed baseline methyleneTHF values, especially in rectal cancer patients. The results indicate that the standardised leucovorin dose may be insufficient to attain the full antitumour effect of 5-FU. Further studies are needed to establish whether higher dosage yields a better treatment response.

Cell-free DNA levels and correlation to stage and outcome following treatment of locally advanced rectal cancer

Accurate staging of rectal cancer remains essential for optimal patient selection for combined modality treatment, including radiotherapy, chemotherapy and surgery. We aimed at examining the correlation of cell free DNA with the pathologic stage and subsequent risk of recurrence for patients with locally advanced rectal cancer undergoing preoperative chemoradiation. We examined 75 patients with locally advanced rectal cancer receiving preoperative chemoradiation. Blood samples for translational use were drawn prior to rectal surgery. The level of cell free DNA was quantified by digital droplet PCR and expressed as copy number of beta 2 microglobulin. We found a median level of cell free DNA in the AJCC stages I-III of 3100, 8300, and 10,700 copies/mL respectively. For patients with 12 sampled lymph nodes or above, the median level of cell free DNA were 2400 copies/mL and 4400 copies/mL (p = 0.04) for node negative and node positive disease respectively. The median follow-up was 39 months and 11 recurrences were detected (15%). The median level for patients with recurrent disease was 13,000 copies/mL compared to 5200 copies/mL for non-recurrent patients (p = 0.08). We have demonstrated a correlation between the level of total cell free DNA and the pathologic stage and nodal involvement. Furthermore, we have found a trend towards a correlation with the risk of recurrence following resection of localized rectal cancer.

Hepatic deletion of p110α and p85α results in insulin resistance despite sustained IRS1-associated phosphatidylinositol kinase activity

Background: Class IA phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is an integral mediator of insulin signaling. The p110 catalytic and p85 regulatory subunits of PI3K are the products of separate genes, and while they come together to make the active heterodimer, they have opposing roles in insulin signaling and action. Deletion of hepatic p110α results in an impaired insulin signal and severe insulin resistance, whereas deletion of hepatic p85α results in improved insulin sensitivity due to sustained levels of phosphatidylinositol (3,4,5)-trisphosphate. Here, we created mice with combined hepatic deletion of p110α and p85α (L-DKO) to study the impact on insulin signaling and whole body glucose homeostasis. Methods: Six-week old male flox control and L-DKO mice were studied over a period of 18 weeks, during which weight and glucose levels were monitored, and glucose tolerance tests, insulin tolerance test and pyruvate tolerance test were performed. Fasting insulin, insulin signaling mediators, PI3K activity and insulin receptor substrate (IRS)1-associated phosphatidylinositol kinase activity were examined at 10 weeks. Liver, muscle and white adipose tissue weight was recorded at 10 weeks and 25 weeks. Results: The L-DKO mice showed a blunted insulin signal downstream of PI3K, developed markedly impaired glucose tolerance, hyperinsulinemia and had decreased liver and adipose tissue weights. Surprisingly, however, these mice displayed normal hepatic glucose production, normal insulin tolerance, and intact IRS1-associated phosphatidylinositol kinase activity without compensatory upregulated signaling of other classes of PI3K. Conclusions: The data demonstrate an unexpectedly overall mild metabolic phenotype of the L-DKO mice, suggesting that lipid kinases other than PI3Ks might partially compensate for the loss of p110α/p85α by signaling through other nodes than Akt/Protein Kinase B.