Reverse transcription-quantitative polymerase chain reaction: description of a RIN-based algorithm for accurate data normalization

<p>Abstract</p> <p>Background</p> <p>Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard technique for mRNA quantification, but appropriate normalization is required to obtain reliable data. Normalization to accurately quantitated RNA has been proposed as the most reliable method for in vivo biopsies. However, this approach does not correct differences in RNA integrity.</p> <p>Results</p> <p>In this study, we evaluated the effect of RNA degradation on the quantification of the relative expression of nine genes (<it>18S</it>, <it>ACTB</it>, <it>ATUB</it>, <it>B2M</it>, <it>GAPDH</it>, <it>HPRT</it>, <it>POLR2L</it>, <it>PSMB6</it> and <it>RPLP0</it>) that cover a wide expression spectrum. Our results show that RNA degradation could introduce up to 100% error in gene expression measurements when RT-qPCR data were normalized to total RNA. To achieve greater resolution of small differences in transcript levels in degraded samples, we improved this normalization method by developing a corrective algorithm that compensates for the loss of RNA integrity. This approach allowed us to achieve higher accuracy, since the average error for quantitative measurements was reduced to 8%. Finally, we applied our normalization strategy to the quantification of <it>EGFR</it>, <it>HER2 </it>and <it>HER3 </it>in 104 rectal cancer biopsies. Taken together, our data show that normalization of gene expression measurements by taking into account also RNA degradation allows much more reliable sample comparison.</p> <p>Conclusion</p> <p>We developed a new normalization method of RT-qPCR data that compensates for loss of RNA integrity and therefore allows accurate gene expression quantification in human biopsies.</p

Biomarqueurs prédictifs de la réponse à la radio ( ou radiochimiothérapie néo-adjuvante dans les cancers du rectum)

Le cancer du rectum, de par l'importance du nombre de personnes qu'il atteint, constitue un problème majeur de santé publique. Pour les stades avancés de la maladie, le traitement repose généralement sur la résection chirurgicale, précédée par une radiochimiothérapie. Ce traitement néo-adjuvant permet de diminuer le taux de récidives locales, d augmenter les chances d effectuer une chirurgie avec conservation du sphincter, et, dans certain cas, d obtenir une réponse pathologique complète. Cependant, pour les tumeurs résistantes à la radiochimiothérapie, des traitements alternatifs ou plus intensifs seraient requis. L objectif de ce projet était de mettre en évidence des facteurs prédictifs de la réponse à la radio- ou radiochimiothérapie néo-adjuvant dans les cancers du rectum. Pour cela, nous avons procédé à l analyse de marqueurs génétiques sur deux populations de patients, l une traitée en néo-adjuvant par radiothérapie exclusive, et l autre par radiothérapie et chimiothérapie. Les marqueurs étudiés correspondent à des variations génétiques germinales ou somatiques (polymorphismes, mutations, pertes d allèles, différences d expression transcriptionnelle) au niveau de plus de 80 gènes candidats. La valeur prédictive de ces marqueurs, a été déterminée en réalisant des analyses d association avec la réponse pathologique au traitement néo-adjuvant, évaluée selon le grade de régression tumorale de Dworak. Les résultats obtenus ont permis de montrer que des polymorphismes de base unique localisés au niveau des gènes CCND1, ERCC4, SOD2, IL13 et MLH1 sont des marqueurs potentiels qui pourraient permettre d adapter la stratégie thérapeutique à chaque patient.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

[Considerations for normalisation of RT-qPCR in oncology]

International audienceGene expression analysis has many applications in the management of cancer, including diagnosis, prognosis, and therapeutic care. In this context, the reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become the "gold standard" for mRNA quantification. However, this technique involves several critical steps such as RNA extraction, cDNA synthesis, quantitative PCR, and analysis, which all can be source of variation. To obtain biologically meaningful results, data normalisation is required to correct sample-to-sample variations that may be introduced during this multistage process. Normalisation can be carried out against a housekeeping gene, total RNA mass, or cell number. Careful choice of the normalization method is crucial, as any variation in the reference will introduce errors in the quantification of mRNA transcripts. By reviewing the different methods available and their related problems, the aim of this article is to provide recommendations for the set up of an appropriate normalisation strategy for RT-qPCR data in oncology

Immunohistochemical staining for p16 and BRAFV600E is useful to distinguish between sporadic and hereditary (Lynch syndrome-related) microsatellite instable colorectal carcinomas

International audienceDNA mismatch repair (MMR) protein analysis by immunohistochemistry (IHC) can identify colorectal cancer (CRC) with microsatellite instability (MSI). As MLH1-deficient CRC can be hereditary or sporadic, markers to distinguish between them are needed. MLH1 promoter methylation assay is the reference method; however, sometimes, it is challenging on formalin-fixed paraffin-embedded tissue samples. We assessed by IHC the expression of BRAFV600E, p16, MGMT, and CDX2 in 55 MLH1-deficient MSI CRC samples (of which 8 had a germline MLH1 mutation) to determine whether this panel differentiates between sporadic and hereditary CRCs. We also analyzed MLH1 promoter methylation by methylation-specific PCR and pyrosequencing and BRAF status by genotyping. None of the hereditary CRCs showed MLH1 methylation, BRAF mutation, BRAFV600E-positive immunostaining, or loss of p16 expression. We detected MLH1 promoter methylation in 67 % and a BRAF mutation in 42 % of CRC, all showing MLH1 promoter methylation. BRAFV600E IHC and BRAF genotyping gave concordant results in all but two samples. Loss of expression of p16 was found in 30 % of CRC with methylation of the MLH1 promoter, but its expression was retained in all non-methylated and part of MLH1-methylated tumors (100 % specificity, 30 % sensitivity). CDX2 and MGMT expression was not associated with MLH1 status. Thus, BRAFV600E and p16 IHC may help in differentiating sporadic from hereditary MLH1-deficient CRC with MSI. Specifically, p16 IHC might be used as a surrogate marker for MLH1 promoter methylation, because all p16-negative CRCs displayed MLH1 methylation, whereas hereditary CRCs were all p16-positive

Validation of an appropriate reference gene for normalization of reverse transcription-quantitative polymerase chain reaction data from rectal cancer biopsies.: Normalization of RT-qPCR data in rectal cancer

International audienceGene expression quantification using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) requires data normalization using an invariable reference gene. Here we assessed the stability of 15 housekeeping genes in 31 tumor and normal rectal samples to validate a reliable reference gene for rectal cancer studies. Our data show that 18S and 28S RNA are stably expressed in all samples. Moreover, when used for normalization, 18S, but not 28S, greatly reduced unspecific variations of gene expression due to RNA degradation. These results demonstrate that 18S is an appropriate reference gene for normalization of RT-qPCR data from rectal cancer samples

Correction: The p53 isoform delta133p53ß regulates cancer cell apoptosis in a RhoB-dependent manner.

[This corrects the article DOI: 10.1371/journal.pone.0172125.]

The p53 isoform delta133p53ß regulates cancer cell apoptosis in a RhoB-dependent manner.

The TP53 gene plays essential roles in cancer. Conventionally, wild type (WT) p53 is thought to prevent cancer development and metastasis formation, while mutant p53 has transforming abilities. However, clinical studies failed to establish p53 mutation status as an unequivocal predictive or prognostic factor of cancer progression. The recent discovery of p53 isoforms that can differentially regulate cell cycle arrest and apoptosis suggests that their expression, rather than p53 mutations, could be a more clinically relevant biomarker in patients with cancer. In this study, we show that the p53 isoform delta133p53ß is involved in regulating the apoptotic response in colorectal cancer cell lines. We first demonstrate delta133p53ß association with the small GTPase RhoB, a well-described anti-apoptotic protein. We then show that, by inhibiting RhoB activity, delta133p53ß protects cells from camptothecin-induced apoptosis. Moreover, we found that high delta133p53 mRNA expression levels are correlated with higher risk of recurrence in a series of patients with locally advanced rectal cancer (n = 36). Our findings describe how a WT TP53 isoform can act as an oncogene and add a new layer to the already complex p53 signaling network

Cyclin D1 gene G870A polymorphism predicts response to neoadjuvant radiotherapy and prognosis in rectal cancer.

PURPOSE: To investigate whether CCND1 genetic variations associated with a constitutive nuclear protein may influence either the pathologic response to preoperative RT or the prognosis in a series of rectal cancer patients. METHODS AND MATERIALS: Seventy rectal cancer patients treated by neoadjuvant radiotherapy were included in the study. CCND1 exon 5 mutations were screened, and the G870A polymorphism was assessed for correlation with clinical variables, tumor response, and patient outcome. RESULTS: No exon 5 mutation was found. Concerning the G870A polymorphism, the A/A variant was significantly associated with radiosensitivity (p = 0.022). Moreover, patients harboring the A allele were correlated with a lower risk of local failure (p = 0.017). Also, combination of the G870A polymorphism with the post-therapeutic lymph node status allowed the elaboration of a prognostic index, which accurately distinguished subgroups of patients with predictable recurrence-free (p = 0.003) and overall (p = 0.044) survival. CONCLUSIONS: Although CCND1 exon 5 mutations are rare in rectal cancer, G870A polymorphism is a frequent variation that may predict radiosensitivity and prognosis

Combined cetuximab and trastuzumab are superior to gemcitabine in the treatment of human pancreatic carcinoma xenografts.

International audienceBACKGROUND: Pancreatic carcinoma remains a treatment-refractory cancer with a poor prognosis. Here, we compared anti-epidermal growth factor receptor (EGFR) and anti-HER2 monoclonal antibodies (2mAbs) injections with standard gemcitabine treatment on human pancreatic carcinoma xenografts. MATERIALS AND METHODS: Nude mice, bearing human pancreatic carcinoma xenografts, were treated with either combined anti-EGFR (cetuximab) and anti-HER2 (trastuzumab) or gemcitabine, and tumor growth was observed. RESULTS AND CONCLUSION: In first-line therapy, mice survival was significantly longer in the 2mAbs group compared with gemcitabine (P < 0.0001 for BxPC-3, P = 0.0679 for MiaPaCa-2 and P = 0.0019 for Capan-1) and with controls (P < 0.0001). In second-line therapy, tumor regressions were observed after replacing gemcitabine by 2mAbs treatment, resulting in significantly longer animal survival compared with mice receiving continuous gemcitabine injections (P = 0.008 for BxPC-3, P = 0.05 for MiaPaCa-2 and P < 0.001 for Capan-1). Therapeutic benefit of 2mAbs was observed despite K-Ras mutation. Interestingly, concerning the mechanism of action, coinjection of F(ab')(2) fragments from 2mAbs induced significant tumor growth inhibition, compared with controls (P = 0.001), indicating that the 2mAbs had an Fc fragment-independent direct action on tumor cells. This preclinical study demonstrated a significant improvement of survival and tumor regression in mice treated with anti-EGFR/anti-HER2 2mAbs in first- and second-line treatments, compared with gemcitabine, independently of the K-Ras status