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

Lack of EGFR-activating mutations in European patients with triple-negative breast cancer could emphasise geographic and ethnic variations in breast cancer mutation profiles

INTRODUCTION: Triple-negative breast cancers (TNBCs) are characterised by lack of expression of hormone receptors and epidermal growth factor receptor 2 (HER-2). As they frequently express epidermal growth factor receptors (EGFRs), anti-EGFR therapies are currently assessed for this breast cancer subtype as an alternative to treatments that target HER-2 or hormone receptors. Recently, EGFR-activating mutations have been reported in TNBC specimens in an East Asian population. Because variations in the frequency of EGFR-activating mutations in East Asians and other patients with lung cancer have been described, we evaluated the EGFR mutational profile in tumour samples from European patients with TNBC. METHODS: We selected from a DNA tumour bank 229 DNA samples isolated from frozen, histologically proven and macrodissected invasive TNBC specimens from European patients. PCR and high-resolution melting (HRM) analyses were used to detect mutations in exons 19 and 21 of EGFR. The results were then confirmed by bidirectional sequencing of all samples. RESULTS: HRM analysis allowed the detection of three EGFR exon 21 mutations, but no exon 19 mutations. There was 100% concordance between the HRM and sequencing results. The three patients with EGFR exon 21 abnormal HRM profiles harboured the rare R836R SNP, but no EGFR-activating mutation was identified. CONCLUSIONS: This study highlights variations in the prevalence of EGFR mutations in TNBC. These variations have crucial implications for the design of clinical trials involving anti-EGFR treatments in TNBC and for identifying the potential target population

[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

In situ hybridization for the assessment of urokinase plasminogen activator and plasminogen activator inhibitor type‑1 in formalin‑fixed paraffin‑embedded breast cancer specimens

International audienceUrokinase plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor type 1 (PAI‑1), have been reported as prognostic and predictive biomarkers in breast cancer, particularly in patients with node‑negative tumors. uPA and PAI‑1 expression levels classify patients into a poor‑prognosis subgroup, requiring adjuvant chemotherapy and a favorable‑prognosis subgroup, which can be considered for de‑escalation. However, the clinical use of these two biomarkers remains limited, since fresh‑frozen/fresh tumor samples are currently required for their quantification. The aim of the present study was to compare PLAU and SERPINE1 mRNA expression levels (corresponding to uPA and PAI‑1 proteins, respectively), assessed using in situ hybridization in 83 formalin‑fixed paraffin‑embedded (FFPE) breast tumor samples, with uPA and PAI‑1 protein expression assessed using immunometric assay with paired fresh‑frozen breast cancer samples. The results from the two methods significantly correlated as regards uPA quantification; however, >30% of the samples were discordant, according to the clinically validated threshold. Concordance between the two analytical methods was less prominent for PAI‑1 protein and SERPINE1 mRNA. Taken together, the results of the present study indicate that although PLAU and SERPINE1 mRNA may be reliably detected in FFPE samples using in situ hybridization, this technology cannot be used as a substitute for the replacement of the immunometric assay‑derived quantification on fresh‑frozen samples

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

Serum NSE, MMP-9 and HER2 extracellular domain are associated with brain metastases in metastatic breast cancer patients: predictive biomarkers for brain metastases?

International audienceBreast cancer (BC) is the second most common cause of brain metastases (BM). Predictive factors for BM have been widely studied in metastatic BC; however, there is no known serum tumor marker to accurately predict BM. Elevated serum S100ß protein and neuron-specific enolase (NSE) could reflect the brain damages induced by BM. Matrix metalloproteinase 9 (MMP-9) is involved in tumor invasion and metastatic dissemination, including BM. Also, HER2-amplified BC were shown to have a particular tropism for central nervous system (CNS). This study evaluated the correlation of these biomarkers with the presence of BM in metastatic BC patients. In this case-control study, 88 consecutive metastatic BC patients with BM (BM group) treated in our institution (2008-2015) were retrospectively selected, based on the availability of frozen serum samples for tumor marker determination. Patients were matched by age, tumor biology and number of previous metastatic chemotherapy lines to 162 metastatic BC patients without CNS involvement (control group). Serum NSE, MMP-9 and HER2 extracellular domain (ECD) levels were significantly higher in the BM group (p = 0.0051, p = 0.0062 and p = 0.0057, respectively). In multivariate analysis, serum HER2 and MMP-9 levels accurately discriminated patients with BM: odds ratios 4.4 (p < 0.001; 95%CI: 1.9-9.6) for HER2 ECD and 3.5 (p = 0.005; 95%CI: 1.5-8.4) for MMP-9. In multivariate analysis, HER2 ECD and NSE serum levels were independent prognostic factors in patients with BM. Serum HER2 ECD and MMP-9 appear to be associated with BM in metastatic BC patients. Their predictive value for BM still needs to be evaluated in further prospective studies

A homogeneous resonance energy transfer-based assay to monitor MutS/DNA interactions.

International audienceProbing the interactions of the DNA mismatch repair protein MutS with altered and damaged DNA is of great interest both for the understanding of the mismatch repair system function and for the development of tools to detect mutations. Here we describe a homogeneous time-resolved fluorescence (HTRF) assay to study the interactions of Escherichia coli MutS protein with various DNA substrates. First, we designed an indirect HTRF assay on a microtiter plate format and demonstrated its general applicability through the analysis of the interactions between MutS and mismatched DNA or DNA containing the most common lesion of the anticancer drug cisplatin. Then we directly labeled MutS with the long-lived fluorescent donor molecule europium tris-bipyridine cryptate ([TBP(Eu(3+))]) and demonstrated by electrophoretic mobility shift assay that this chemically labeled protein retained DNA mismatch binding property. Consequently, we used [TBP(Eu(3+))]-MutS to develop a faster and simpler semidirect HTRF assay

Clinicopathological Correlates of γδ T Cell Infiltration in Triple-Negative Breast Cancer

International audienceThe prognostic impact of the different tumor-infiltrating lymphocyte (TIL) subpopulations in solid cancers is still debated. Here, we investigated the clinicopathological correlates and prognostic impact of TILs, particularly of γδ T cells, in 162 patients with triple-negative breast cancer (TNBC). A high γδ T cell density (>6.625 γδ T cells/mm2) was associated with younger age (p = 0.008), higher tumor histological grade (p = 0.002), adjuvant chemotherapy (p = 0.010), BRCA1 promoter methylation (p = 0.010), TIL density (p < 0.001), and PD-L1 (p < 0.001) and PD-1 expression (p = 0.040). In multivariate analyses, γδ T cell infiltration (cutoff = 6.625 γδ T cells/mm2) was an independent prognostic factor (5-year relapse-free survival: 63.3% vs. 89.8%, p = 0.027; 5-year overall survival: 73.8% vs. 89.9%, p = 0.031, for low vs. high infiltration). This prognostic impact varied according to the tumor PIK3CA mutational status. High γδ T cell infiltration was associated with better survival in patients with PIK3CA wild-type tumors, but the difference was not significant in the subgroup with PIK3CA-mutated tumors. Altogether, these data suggest that high γδ T cell infiltrate is correlated with immune infiltration and might represent a candidate prognostic tool in patients with TNBC