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.
In this study, we evaluated the effect of RNA degradation on the quantification of the relative expression of nine genes (18S, ACTB, ATUB, B2M, GAPDH, HPRT, POLR2L, PSMB6 and RPLP0) 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 EGFR, HER2 and HER3 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.
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.
Abstract New therapeutic strategies for ovarian cancer include the identification of involved signaling pathways that could potentially serve as a source of biomarkers for early stages of the ...disease. In this study, we show that the embryonic male prostaglandin D synthase (Pgds)/SOX9 pathway is expressed at both the RNA and protein levels in different types of human ovarian tumors, pointing to Pgds and SOX9 as possible diagnostic markers for ovarian carcinomas. Using ovarian cancer cell lines, we found, first, that components of the Pgds/SOX9 pathway are expressed in these cells, and second, that treatment of these cells with prostaglandin D2 (PGD2) can inhibit their growth via its DP1 receptor and induce apoptosis. Finally, using siRNA and overexpression strategies, we demonstrate that SOX9 expression is induced by PDG2 and is responsible for PDG2-mediated growth inhibition. Accordingly, as stimulating the PGD2/DP1 signal transduction pathway upregulates SOX9 expression, either activators of this pathway or DP1 agonists may be useful as new therapeutic agents.
Aberrant activation of the HER signaling pathways plays a critical role in the invasive and metastatic potential of tumors. The aim of this study was to address whether, in rectal cancer, alterations ...of these pathways could have a value as prognostic factors to be used to identify patients who are at risk of distant metastases. Therefore, the mRNA expression of the four members of the HER family as well as the frequency of PTEN allelic loss and KRAS/BRAF mutations were determined in pretreatment biopsies from a series of 100 locally advanced rectal cancers and then their ability to predict distant metastases was evaluated. Over‐expression of EGFR (p = 0.021), HER2 (p = 0.011) and HER3 (p = 0.020) was significantly associated with worse metastasis‐free survival in univariate analysis. In multivariate analysis, both over‐expression of EGFR (p = 0.028) and HER3 (p = 0.011) remained independent prognostic factors for distant metastasis. In conclusion, quantification of EGFR and HER3 mRNA expression in pretreatment biopsies may be useful to identify patients who are at risk of developing metastases.
Reproductive techniques such as prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD), although debated, are legally forbidden in France in case of Lynch syndrome. The preference of ...mutation carriers about their reproductive options is not systematically considered in France. We aimed to prospectively assess the reproductive preferences of mismatch repair mutation carriers consulting in our institution (2003–2010,
n =
100). We also considered the short- and long-term post-disclosure psychological impact using the Impact of Events Scale-Revised questionnaire to measure the prevalence of posttraumatic stress disorder (PTSD) in those patients. Complete data were obtained for 34 respondents (17 males, 17 females, median age of 33.5 years 22–59). Seventeen respondents (57 %) preferred spontaneous natural conception versus 28 % and 35 % choosing PND and PGD, respectively. At results disclosure, respondents mainly explained their distress by fear of premature death (43 %) and transmitting mutated genes (42 %). One year later, this last fear remained predominant in 55 % of subjects. None of the main socio-demographical, psychological or medical variables (including fear of transmitting mutations) was significantly associated with the reproductive preferences. Results disclosure had a real and time-decreasing psychological impact on mutation carriers. Reproductive techniques, expected to decrease the hereditary risk, were not significantly preferred to natural conception.
An emerging hypothesis suggests that cytokines could play an important role in cancer as potential modulators of angiogenesis and leucocyte infiltration.
A novel multiplexed flow cytometry technology ...was used to measure the expression of 17 cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p70, IL-13, IL-17, granulocyte colony-stimulating factor CSF, granulocyte-macrophage CSF, IFN-gamma, monocyte chemoattractant protein MCP-1, macrophage inflammatory protein MIP-1beta, tumour necrosis factor TNF-alpha) at the protein level in 105 breast carcinoma. B lymphocyte, T lymphocyte and macrophage levels were determined by immunohistochemistry.
Fourteen of the 17 cytokines were expressed in breast carcinoma, whereas only nine cytokines could be detected in normal breast. Most cytokines were more abundant in breast carcinoma than in normal breast, with IL-6, IL-8, granulocyte CSF, IFN-gamma, MCP-1 and MIP-1beta being very abundant. IL-2, IL-6, IL-8, IL-10, IFN-gamma, MCP-1, MIP-1beta and TNF-alpha, and to a lesser extent IL-1beta and IL-13 exhibited levels of expression that were inversely correlated to oestrogen receptor and progesterone receptor status. Most cytokines were not correlated with age at cancer diagnosis, tumour size, histological type, or lymph node status. However, IL-1beta, IL-6, IL-8, IL-10, IL-12, MCP-1 and MIP-1beta were more abundant in high-grade tumours than in low-grade tumours. In addition, IL-8 and MIP-1beta were expressed to a greater degree in HER2-positive than in HER2-negative patients. The expression of most of the studied cytokines was correlated to levels of activator protein-1, which is known to regulate numerous cytokines. Overexpression of MCP-1 and MIP-1beta were linked to B lymphocyte, T lymphocyte and macrophage infiltration, whereas high levels of IL-8 were correlated with high macrophage content in tumour. Moreover, IL-8 positive tumours exhibited increased vascularization.
We found that multiple cytokines were overexpressed in oestrogen receptor negative breast carcinoma, and that the three major cytokines--MCP-1, MIP-1beta and IL-8--were correlated with inflammatory cell component, which could account for the aggressiveness of these tumours.
Current histo-pathological prognostic factors are not very helpful in predicting the clinical outcome of breast cancer due to the disease's heterogeneity. Molecular profiling using a large panel of ...genes could help to classify breast tumours and to define signatures which are predictive of their clinical behaviour.
To this aim, quantitative RT-PCR amplification was used to study the RNA expression levels of 47 genes in 199 primary breast tumours and 6 normal breast tissues. Genes were selected on the basis of their potential implication in hormonal sensitivity of breast tumours. Normalized RT-PCR data were analysed in an unsupervised manner by pairwise hierarchical clustering, and the statistical relevance of the defined subclasses was assessed by Chi2 analysis. The robustness of the selected subgroups was evaluated by classifying an external and independent set of tumours using these Chi2-defined molecular signatures.
Hierarchical clustering of gene expression data allowed us to define a series of tumour subgroups that were either reminiscent of previously reported classifications, or represented putative new subtypes. The Chi2 analysis of these subgroups allowed us to define specific molecular signatures for some of them whose reliability was further demonstrated by using the validation data set. A new breast cancer subclass, called subgroup 7, that we defined in that way, was particularly interesting as it gathered tumours with specific bioclinical features including a low rate of recurrence during a 5 year follow-up.
The analysis of the expression of 47 genes in 199 primary breast tumours allowed classifying them into a series of molecular subgroups. The subgroup 7, which has been highlighted by our study, was remarkable as it gathered tumours with specific bioclinical features including a low rate of recurrence. Although this finding should be confirmed by using a larger tumour cohort, it suggests that gene expression profiling using a minimal set of genes may allow the discovery of new subclasses of breast cancer that are characterized by specific molecular signatures and exhibit specific bioclinical features.
Following the introduction of new adjuvant therapies we wanted to reappraise the prognostic and predictive value of uPA/PAI-1 in early breast cancer.
This monocentric retrospective study included 652 ...patients who had curative surgery between 2006 and 2011 and adjuvant treatment decision-making, taking into account uPA/PAI-1 tumor levels.
uPA and PAI-1 levels were associated with classical clinicopathological parameters and adjuvant chemotherapy decision, but not with peritumoral vascular invasion (PVI; also known as peritumoral vascular emboli). HER2 overexpression, PVI and uPA/PAI-1 levels were not significantly associated with relapse-free survival in univariate analysis. In multivariate analysis, T stage, N stage and progesterone receptors were the only independent relapse-free survival predictive factors.
The absence of an association between uPA/PAI-1 and PVI allows their concomitant consideration in adjuvant treatment discussion. The overall good prognosis of patients with high uPA/PAI-1 levels might be linked to the uPA/PAI-1 predictive value and the inclusion of these parameters in adjuvant guidelines.
Abstract Background HER2-targeted therapies have recently emerged as an option in the management of metastatic colorectal cancer (mCRC) overexpressing HER2. However, data regarding HER2 status in ...primary CRC and its corresponding liver metastases are limited, potentially influencing clinical decisions. Therefore, the aim of this study was to compare the HER2 status in primary CRC and paired liver metastases. Methods Patients with mCRC who were operated from their primary colorectal cancer and their corresponding synchronous or metachronous liver metastases, in the digestive surgery department of Besançon University Hospital, between April 1999 and October 2021, were included. Tissue microarrays were constructed from matched primary CRC and liver metastastic tissue samples. HER2 status was assessed by immunohistochemistry and in situ hybridization according to Valtorta’s criteria. Results A series of 108 paired primary CRC and liver metastases, including a series of multiple liver metastases originating from the same patients ( n = 24), were assessed. Among the primary CRC, 89 (82.4%), 17 (15.8%) and 2 (1.8%) cases were scored 0, 1 + and 2 + respectively. In liver metastases, 99 (91.7%), 7 (6.5%) and 2 (1.8%) were scored 0, 1 + and 2, respectively. Overall, there was a 19% discrepancy rate in HER2 status between primary CRC and metastases, which increased to 21% in cases with multiple synchronous or metachronous liver metastases in a given patient. No significant difference was found between metachronous and synchronous metastases regarding the HER2 status ( p = 0.237). Conclusions Our study highlights the temporal and spatial heterogeneity of HER2 status between primary CRC and corresponding liver metastases. These findings raise the question of a sequential evaluation of the HER2 status during disease progression, to provide the most suitable treatment strategy.
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