Critical appraisal of quantitative PCR results in colorectal cancer research: can we rely on published qPCR results?

The use of real-time quantitative polymerase chain reaction (qPCR) in cancer research has become ubiquitous. The relative simplicity of qPCR experiments, which deliver fast and cost-effective results, means that each year an increasing number of papers utilizing this technique are being published. But how reliable are the published results? Since the validity of gene expression data is greatly dependent on appropriate normalisation to compensate for sample-to-sample and run-to-run variation, we have evaluated the adequacy of normalisation procedures in qPCR-based experiments. Consequently, we assessed all colorectal cancer publications that made use of qPCR from 2006 until August 2013 for the number of reference genes used and whether they had been validated. Using even these minimal evaluation criteria, the validity of only three percent (6/179) of the publications can be adequately assessed. We describe common errors, and conclude that the current state of reporting on qPCR in colorectal cancer research is disquieting. Extrapolated to the study of cancer in general, it is clear that the majority of studies using qPCR cannot be reliably assessed and that at best, the results of these studies may or may not be valid and at worst, pervasive incorrect normalisation is resulting in the wholesale publication of incorrect conclusions. This survey demonstrates that the existence of guidelines, such as MIQE, is necessary but not sufficient to address this problem and suggests that the scientific community should examine its responsibility and be aware of the implications of these findings for current and future research.

Microsatellite and chromosomal stable colorectal cancers demonstrate poor immunogenicity and early disease recurrence.

OBJECTIVE: Colorectal cancers may demonstrate chromosomal instability (CSI) or microsatellite instability (MSI-H). A third group of microsatellite and chromosome stable (MACS) colorectal cancer has been described more recently. Patients with MSI-H colorectal cancers demonstrate improved outcome and a pronounced inflammatory infiltrate. Enhanced host immune response and increased immunogenicity might explain these observations. This study aims to further characterize colorectal cancer immunogenicity. METHOD: Microsatellite stability status was determined in resected tumour samples. Microsatellite stable (MSS) tumour samples were stratified by DNA ploidy status, as determined by flow cytometry into aneuploid MSS (CSI) and diploid MSS (MACS) cancers. Lymphocyte proliferation, quantified by bromodeoxyuridine incorporation assays assessed tumour protein immunogenicity and ELISA assays quantified inflammatory cytokine release. Kaplan-Meier survival curves and multivariate analyses were used to determine prognostic value. RESULTS: Patients with MSI-H colorectal cancer had improved outcome but those with MACS cancers undergoing curative surgery had significantly poorer disease-free survival (P = 0.002). The MACS phenotype was an independent predictor of poor outcome (HR = 2.44, 1.33-4.47, P = 0.004). Lymphocyte proliferation assays confirmed enhanced immunogenicity of MSI-H proteins and reduced immunogenicity of MACS proteins (P < 0.0001). In vitro levels of IFN-gamma (P = 0.004) and IL-18 (P < 0.0001) mirrored these differences in lymphocyte activity. CONCLUSIONS: Stratification of colorectal cancer by MSI and ploidy status may have prognostic value in patients undergoing curative surgery. MSI-H cancers display enhanced immunogenic properties but the immune response to MACS cancers appears to be absent and this may contribute to their poor prognosis.

Altered monocyte cyclo-oxygenase response in non-obese diabetic mice.

Monocytes infiltrate islets in non-obese diabetic (NOD) mice. Activated monocyte/macrophages express cyclo-oxygenase-2 (COX-2) promoting prostaglandin-E(2) (PGE(2)) secretion, while COX-1 expression is constitutive. We investigated in female NOD mice: (i) natural history of monocyte COX expression basally and following lipopolysaccharide (LPS) stimulation; (ii) impact of COX-2 specific inhibitor (Vioxx) on PGE(2), insulitis and diabetes. CD11b(+) monocytes were analysed for COX mRNA expression from NOD (n = 48) and C57BL/6 control (n = 18) mice. NOD mice were treated with either Vioxx (total dose 80 mg/kg) (n = 29) or methylcellulose as control (n = 29) administered by gavage at 4 weeks until diabetes developed or age 30 weeks. In all groups, basal monocyte COX mRNA and PGE(2) secretion were normal, while following LPS, after 5 weeks of age monocyte/macrophage COX-1 mRNA decreased (P < 0.01) and COX-2 mRNA increased (P < 0.01). However, diabetic NOD mice had reduced COX mRNA response (P = 0.03). Vioxx administration influenced neither PGE(2), insulitis nor diabetes. We demonstrate an isoform switch in monocyte/macrophage COX mRNA expression following LPS, which is altered in diabetic NOD mice as in human diabetes. However, Vioxx failed to affect insulitis or diabetes. We conclude that monocyte responses are altered in diabetic NOD mice but COX-2 expression is unlikely to be critical to disease risk.

Cyclo-oxygenase-2 (COX-2) mRNA expression and hormone receptor status in breast cancer.

AIMS: The purpose of this study was to evaluate COX-2 mRNA expression with known clinical prognostic features of breast cancer, oestrogen/progesterone receptor status, tumour size and grade. METHODS: Total RNA was extracted from 45 frozen breast tumour (invasive) and 22 normal breast tissue samples. COX-2 mRNA transcription was quantified using a real time RT-PCR assay and expressed as copy number/microg total RNA. All specimens were assessed for tumour grade, size, nodal status and presence of vascular invasion and oestrogen and progesterone receptor status. RESULTS: COX-2 mRNA was detected in all samples with a median copy number of 1.15 x 10(7) for tumours and 6.5 x 10(6) for normal samples. Expression was significantly higher in oestrogen receptor negative tumours compared to the receptor positive group. There was no correlation between COX-2 mRNA levels and tumour size, grade, nodal status and presence of vascular invasion. CONCLUSIONS: COX-2 mRNA expression is increased in oestrogen and progesterone receptor negative breast cancers.

Differential expression of IGF-binding protein-3 in normal and malignant colon and its influence on apoptosis.

IGF-binding protein-3 (IGFBP-3) has been reported to exert a protective influence on the pathogenesis of colorectal cancer. This may reflect its modulation of IGF-I bioactivity as well as IGF-I-independent effects on cell proliferation and apoptosis. Although local expression of IGF-I in the colon is increasingly recognised as having important regulatory consequences, the role of locally expressed IGFBP-3 remains unknown. The aims of the present study were: (i) to quantify and localise the expression of IGFBP-3 in human normal and malignant colon; (ii) to relate this expression to that of other components of the IGF-I axis; and (iii) to investigate the effects of IGFBP-3 on colonic epithelial cell proliferation and apoptosis. RNA was extracted from 46 paired samples of normal and malignant colonic tissue. IGFBP-3, IGF-I, IGF-I receptor and GH receptor mRNA levels were quantified using real-time RT-PCR. Laser-capture microdissection of the same samples was used to isolate mRNA from epithelium and stromal components and localise mRNA expression. Expression was confirmed at a protein level by immunohistochemistry. Human colorectal cancer HT-29 and CaCo-2 cells were cultured with IGFBP-3 (200 ng/ml), +/- IGF-I (20 ng/ml), +/- sodium butyrate (5 mM). Cell number was assessed by an MTS assay (a modification of the MTT assay), and apoptosis assessed by cell morphology and FACS analysis using both annexin and propidium iodide staining. UO146, a MAP kinase inhibitor, and wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI-3K) pathway, were used to determine the contribution of these signalling pathways on the effects of IGFBP-3. IGFBP-3 mRNA was detected in all samples (mean copy number/mug total RNA in normal colon, 2.6 x 10(6) compared with 1.3 x 10(7) in the cancers; P < 0.0001). Immunohistochemistry confirmed the expression and showed it to be equally distributed between epithelial and stromal components in normal tissue, but to be mainly restricted to the stromal component of malignant tissue. This differential expression was confirmed by RT-PCR of RNA from laser-capture microdissected samples. IGF-I mRNA was detected in 31 samples of normal colon; mean IGFBP-3 copy number was higher in the IGF-I-positive samples compared with IGF-I-negative samples. IGFBP-3 on its own induced apoptosis in HT-29 cells (P < 0.001). Co-incubation of 200 ng/ml IGFBP-3 with butyrate (5 mM) resulted in the potentiation of its apoptosis (P < 0.0001), which was not rescued by co-incubation with IGF-I (P < 0.0001). The addition of UO126 caused a decrease in cell number and increased the effects of IGFBP-3. IGFBP-3 is differentially expressed between stromal and epithelial components of normal and malignant colon, which may reflect its pro-apoptotic, IGF-I-independent effect on colonic epithelial cells. These effects are mediated in part by the PI-3K pathway in contrast to the MAP kinase pathway used by IGF-I.

Quantitative real-time RT-PCR--a perspective.

The real-time reverse transcription polymerase chain reaction (RT-PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the nucleic acid amplification and detection steps into one homogeneous assay and obviates the need for gel electrophoresis to detect amplification products. Use of appropriate chemistries and data analysis eliminates the need for Southern blotting or DNA sequencing for amplicon identification. Its simplicity, specificity and sensitivity, together with its potential for high throughput and the ongoing introduction of new chemistries, more reliable instrumentation and improved protocols, has made real-time RT-PCR the benchmark technology for the detection and/or comparison of RNA levels.

Tumour-infiltrating lymphocytes in colorectal cancer with microsatellite instability are activated and cytotoxic.

BACKGROUND: Patients with colorectal cancer that display high-level microsatellite instability (MSI-H) appear to have a better prognosis. This may be explained by the pronounced T cell infiltrate seen in MSI-H tumours that is related to a specific antigen-driven immune response. The nature of tumour-infiltrating lymphocytes in colorectal cancers was investigated using quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. METHODS: Quantitative fluorescent hydrolysis probe-based reverse transcriptase-PCR assays were used to detect levels of mRNA specifying T cell markers in fresh frozen colorectal tissue from MSI-H tumours and those with little or no microsatellite instability (microsatellite stable (MSS) tumours). In addition, immunohistochemistry was performed on paraffin-embedded sections to compare expression of the same T cell markers and the activation markers granzyme B and interleukin 2 receptor alpha-subunit (IL-2Ralpha) in MSI-H and MSS tumours. RESULTS: MSI-H tumours contained higher ratios of CD8/CD3 mRNA copy numbers than MSS tumours (P = 0.016), confirming the cytotoxic nature of lymphocyte infiltrates in this subset of colorectal cancers. Furthermore, immunohistochemistry confirmed that MSI-H tumours contained more infiltrating lymphocytes than MSS tumours, as shown by increased expression of CD3 (P = 0.003) and CD8 (P = 0.008). Consistent with other studies, the lymphocytes in MSI-H tumours were activated as indicated by significantly higher granzyme B counts (P = 0.020) and a significantly higher level of expression of IL-2Ralpha (P = 0.017). CONCLUSION: The results support the hypothesis that MSI-H colorectal cancers may be more immunogenic than MSS tumours.

Identification of differentially expressed genes associated with colorectal cancer liver metastasis.

The molecular mechanisms underlying successful metastasis of primary colorectal tumour to the liver remain unknown. We have used suppression subtractive hybridisation (SSH) and reverse Northern dot blot analysis to profile the mRNA expression patterns of a primary colorectal cancer and its liver metastasis. After SSH and reverse Northern dot blot analysis, differential expression was confirmed in 17 clones from the forward, and 13 clones from the reverse subtracted cDNA library. Four clones showed no significant sequence identities with any known sequences in the GenBank data base and likely to represent novel genes whose up- or down-regulation is associated with colorectal liver metastasis. Interestingly, one of the 13 down-regulated clones displayed 99% sequence identity with the BRCA1 tumour suppressor gene. Since promoter methylation is a direct cause of transcription silencing of the BRCA1 gene in approximately 10-20% of human breast cancer we further investigated its promoter methylation status in ten primary colorectal tumour samples, but revealed no evidence of promoter methylation.

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.

The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

Expression of 25-hydroxyvitamin D-1-alpha-hydroxylase mRNA in individuals with colorectal cancer.

Vitamin D prevents proliferation, promotes differentiation, and induces apoptosis of colon cells, and reduced intake or insufficiency of the vitamin in the body are associated with increased risk of colorectal cancer. Results of previous studies have suggested that mRNA that codes for 25-hydroxyvitamin D-1-alpha-hydroxylase (1 alpha OHase), which converts 25-hydroxyvitamin D to its active metabolite, might be up regulated in human colon carcinomas. We used real-time reverse transcription PCR assays to measure absolute 1 alpha OHase mRNA concentrations in the colonic mucosa of 44 individuals without cancer, and in paired healthy colon and cancerous colon samples taken from 27 individuals with the disease, to ascertain whether or not such up regulation takes place. Our results suggest that concentrations of 1 alpha OHase mRNA in tumour samples and in healthy colon samples from individuals without cancer are similar, but that concentrations are significantly lower in the paired, phenotypically healthy mucosa of individuals with cancer.

The growth hormone-insulin-like growth factor-I axis and colorectal cancer.

The growth hormone (GH)-insulin-like growth factor (IGF)-I axis is an important modulator of growth and development, but in addition to their classical role as endocrine hormones, its components also regulate a wide range of biological functions through paracrine and autocrine mechanisms. Their potent mitogenic and anti-apoptotic effects play a critical role in the regulation of rapidly renewing epithelial cell populations such as those found in the colon. Recent evidence suggests an association between inappropriate regulation of the GH-IGF-I axis and the development of colorectal cancer. However, the molecular mechanisms and signalling pathways responsible are only beginning to be unravelled, as are the relative contributions of the endocrine and autocrine or paracrine effects.

Expression of HLA class II in colorectal cancer: evidence for enhanced immunogenicity of microsatellite-instability-positive tumours.

Microsatellite-instability-positive (MSI+) colorectal cancers are characterised by an accumulation of deletion and insertion mutations at simple repeated sequences caused by inactivation of mismatch repair proteins. They share several clinicopathological features, including a better prognosis and a pronounced stromal inflammatory reaction. We have used suppression subtraction hybridisation between normal colonic epithelium and paired carcinomas to generate differential gene expression profiles in MSI+ and MSI- tumours. Following reverse northern blotting analysis, 11 genes were found to be up-regulated in the MSI+ tumour, and one of these specified the HLA-DM gene. This was in contrast to the MSI(-) tumour screen, where none of the clones analysed expressed this class of gene. Our results confirm that MSI(+) tumours may have a greater potential for the efficient presentation of antigens to the helper arm of the immune system and lend further support to the theory that the better prognosis of patients with MSI+ tumours may be linked to an enhanced immunogenicity of these tumours.

Expression of the Ca2+-activated chloride channel genes CLCA1 and CLCA2 is downregulated in human colorectal cancer.

The role of ion channels in carcinogenesis and tumor progression remains unclear. We have used suppression subtractive hybridization of mRNA from paired normal colon epithelium and tumor, followed by quantitative kinetic RT-PCR, to demonstrate that the transcription of two members of a novel Ca(2+)-dependent chloride channel family, CLCA1 and CLCA2, was significantly downregulated in approximately 80% of colorectal carcinomas. This figure rose to >90% when expression was adjusted for tumor cell proliferation. In normal colon epithelium, CLCA1 mRNA levels were significantly associated with c-myc transcription but became decoupled in the tumor samples. There was no association between CLCA2 and either CLCA1 or c-myc mRNA levels. Transcription of both genes in three colorectal cancer cell lines, T84, HT29, and Caco2, was barely detectable. Illegitimate transcription of CLCA1 was detected in 12 of 15 blood samples taken from healthy volunteers, making its use as a marker for the detection of tumor spread unreliable. Our results suggest that CLCA1 could specify a new tumor suppressor and that, as in breast cancer, CLCA2 may function as a tumor suppressor in colorectal cancer.

Increased levels of insulin-like growth factor binding protein-2 in sera and tumours from patients with colonic neoplasia with and without acromegaly.

OBJECTIVE: Patients with acromegaly are at increased risk of developing colorectal carcinoma and premalignant tubulovillous adenoma. The pathogenesis of these neoplasms could involve a stimulatory effect of serum growth factors on colonic epithelial cell proliferation. The aim of this study was to evaluate changes in (1) serum IGF-I, IGF-II, IGFBP-3 and IGFBP-2 and (2) changes in local expression of IGFBPs and p53 in colonic epithelium in patients with colonic neoplasia with and without acromegaly. DESIGN: A cross-sectional retrospective study was performed. Fasting serum samples were obtained at the time of colonoscopy for patients with acromegaly and at the time of surgery for patients with colonic neoplasia without acromegaly. MEASUREMENTS: Serum IGF-I, IGF-II, IGFBP-2 and IGFBP-3 were measured using specific immunoassays. Tissue expression of IGFBP-2, IGFBP-3 and p53 status were determined by immunohistochemistry. PATIENTS: Group 1: 26 age- and sex-matched control subjects (range 40-69 years); group 2: 18 patients with acromegaly without colonic neoplasia (range 39-68 years); group 3: 18 patients with acromegaly and colonic neoplasia (range 41-74 years, 11 = adenoma, seven = carcinoma); group 4: 19 patients with colonic neoplasia without endocrine disease (range 43-91 years, four = adenoma, 15 = carcinoma). Immunohistochemical staining of colonic biopsies was performed for IGFBP-2, IGFBP-3 and p53 in groups 3 and 4. RESULTS: Mean serum IGF-I and IGFBP-3 levels were significantly elevated in group 2 (371 +/- 131 microg/l and 6.5 +/- 1.8 mg/l, respectively) and group 3 (379 +/- 174 microg/l and 5.8 +/- 1.6 mg/l, respectively), and significantly reduced in group 4 (103 +/- 36 microg/l and 2.4 +/- 1 mg/l) compared to controls (165 +/- 40 microg/l and 4.7 +/- 1 mg/l; P < 0.0001, P < 0.001, respectively). However, median serum IGFBP-2 levels were significantly elevated in group 3 (P < 0.01) and group 4 (P < 0.0001). Immunostaining for IGFBP-2 showed strong areas of immunoreactivity in the cytoplasm of malignant colonic epithelium compared to benign epithelium. IGFBP-3 immunostaining showed strong areas of immunoreactivity in the cytoplasm and in the nucleus of malignant and benign colonic epithelium compared to the normal epithelium. Nuclear staining for p53 was observed in three patients from group 3 (two carcinoma, one adenoma) and four patients from group 4 (all carcinoma). CONCLUSION: Our results describe changes in IGFBP-2 expression in colonic neoplasia in patients with and without acromegaly, which suggest that this binding protein may regulate local bioavailability of IGF, which in turn could modulate colonic cell proliferation and/or differentiation.

Molecular quantification and mapping of lymph-node micrometastases in cervical cancer.

BACKGROUND: A proportion of patients with cancer and lymph nodes negative on histology will develop recurrence. Reverse-transcriptase PCR (RT-PCR) is a highly sensitive method for detection of lymph-node micrometastases, but accurate quantitative assessment has been difficult. METHODS: We studied primary tumours and 156 lymph nodes from 32 patients with cervical cancer (stage IA2, IB1, and IB2) and 32 lymph nodes from nine patients with benign disease. A fully quantitative, real-time RT-PCR assay was used to document absolute copy numbers of the epithelial marker cytokeratin 19. Primers and probe were designed not to amplify either of the two cytokeratin 19 pseudogenes. FINDINGS: All primary tumours and histologically involved lymph nodes (six) had more than 106 copies of cytokeratin 19 mRNA per microg total RNA. Expression of cytokeratin 19 (up to 1.1 x 10(5) copies per microg RNA) was detected in 66 (44%) of 150 histologically uninvolved lymph nodes, and in nodes from 16 of 32 patients with cervical cancer. 15 of these 16 patients with evidence of micrometastases had the highest cytokeratin 19 transcription level in a first lymph-node drainage station (three obturator, six internal, and six external iliac node). Transcription of cytokeratin 19 was found at a low level in just one of 32 lymph nodes obtained from nine patients with benign disease. Median copy number of cytokeratin 19 transcription was significantly higher (>10(3) copies) in association with adverse prognostic features. INTERPRETATION: The results suggest that about 50% of early-stage cervical cancers shed tumour cells to the pelvic lymph nodes. The amount of cytokeratin 19 expression was related to clinicopathological features. Further studies are required to document the clinical implications of molecular micrometastases.

The expression of the growth hormone secretagogue receptor ligand ghrelin in normal and abnormal human pituitary and other neuroendocrine tumors.

Ghrelin is a recently identified endogenous ligand of the GH secretagogue (GHS) receptor. It was originally isolated from the stomach, but has also been shown to be present in the rat hypothalamus. It is a 28-amino acid peptide with an unusual octanoylated serine 3 at the N-terminal end of the molecule, which is crucial for its biological activity. Synthetic GHSs stimulate GH release via both the hypothalamus and the pituitary, and the GHS receptor (GHS-R) has been shown by us and others to be present in the pituitary. We investigated whether ghrelin messenger ribonucleic acid (mRNA) and peptide are present in the normal human hypothalamus and in normal and adenomatous human pituitary. RNA was extracted from pituitary tissue removed at autopsy and transsphenoidal surgery (n = 62), and ghrelin and GHS-R type 1a and 1b mRNA levels were investigated using real-time RT-PCR. Both ghrelin and GHS-R mRNA were detected in all samples. Corticotroph tumors showed significantly less expression of ghrelin mRNA, whereas GHS-R mRNA levels were similar to those in normal pituitary tissue. Gonadotroph tumors showed a particularly low level of expression of GHS-R mRNA. Immunohistochemistry, using a polyclonal antibody against the C-terminal end of the ghrelin molecule, revealed positive staining in the homolog of the arcuate nucleus in the human hypothalamus and in both normal and abnormal human pituitary. Pituitary tumor ghrelin peptide content was demonstrated using two separate RIA reactions for the N-terminal and C-terminal ends of the molecule. Both forms were present in normal and abnormal pituitaries, with 5 +/- 2.5% octanoylated (active) ghrelin (mean +/- SD) present as a percentage of the total. We suggest that the presence of ghrelin mRNA and peptide in the pituitary implies that the locally synthesized hormone may have an autocrine/paracrine modulatory effect on pituitary hormone release.

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.

The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).