Role of contrast-enhanced ultrasound (CEUS) in paediatric practice: an EFSUMB position statement

The use of contrast-enhanced ultrasound (CEUS) in adults is well established in many different areas, with a number of current applications deemed off-label, but the use supported by clinical experience and evidence. Paediatric CEUS is also an off-label application until recently with approval specifically for assessment of focal liver lesions. Nevertheless there is mounting evidence of the usefulness of CEUS in children in many areas, primarily as an imaging technique that reduces exposure to radiation, iodinated contrast medium and the patient-friendly circumstances of ultrasonography. This position statement of the European Federation of Societies in Ultrasound and Medicine (EFSUMB) assesses the current status of CEUS applications in children and makes suggestions for further development of this technique

Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR

<p>Abstract</p> <p>Background</p> <p>Circulating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited.</p> <p>Methods</p> <p>We quantified by RT-qPCR <it>CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+</it>, and <it>mammaglobin </it>gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer.</p> <p>Results</p> <p>RT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, <it>CK-19 </it>was detected in 42.4%, <it>HER-2 </it>in 13.6%, <it>MAGE-A3 </it>in 21.2%, <it>hMAM </it>in 13.6%, <it>TWIST-1 </it>in 42.4%, and <it>hTERT α+β+ </it>in 10.2%. In 26 patients with verified metastasis, <it>CK-19 </it>was detected in 53.8%, <it>HER-2 </it>in 19.2%, <it>MAGE-A3 </it>in 15.4%, <it>hMAM </it>in 30.8%, <it>TWIST-1 </it>in 38.5% and <it>hTERT </it>α⁺β⁺in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch.</p> <p>Conclusions</p> <p>Molecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.</p

Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

<p>Abstract</p> <p>Background</p> <p><it>HER-2 </it>gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC) and fluorescence <it>in situ </it>hybridization (FISH). These procedures permit correlation between <it>HER-2 </it>expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic <it>in situ </it>hybridization) is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas.</p> <p>Methods</p> <p>To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH.</p> <p>Results</p> <p>The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of <it>HER-2 </it>status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4%) and <it>HER-2 </it>transcript overexpression (20%). Moreover, 2+ immunostaining cases presented nonamplified status (50%) by CISH and <it>HER-2 </it>downexpression (38.5%) by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between <it>HER-2 </it>downexpression and the involvement of less than four lymph nodes (<it>P </it>= 0.0350).</p> <p>Conclusion</p> <p>Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the <it>HER-2 </it>gene.</p

Detection of Mammaglobin A-mRNA-positive circulating tumor cells in peripheral blood of patients with operable breast cancer with nested RT-PCR

Objectives: The development and validation of a nested RT-PCR methodology for the detection of Mammaglobin A-mRNA-positive circulating tumor cells in peripheral blood of patients with operable breast cancer and evaluation of its prognostic significance. Design and methods: Different combinations of specific primers were in silico designed and selected, so that false positive results due to genomic DNA contamination were avoided. The specificity of the primers used was evaluated in 30 healthy individuals, 20 patients with colorectal cancer and 20 patients with non-small cell lung cancer. The method was applied in 101 patients with operable breast cancer before the administration of adjuvant chemotherapy and 39 patients with metastatic breast cancer. Results: Mammaglobin A-mRNA-positive cells were detected in 14/101 (13.9%) of early breast cancer patients but not in the control population studied (0%); 9 of them (64.3%) relapsed during the follow-up period. Mammaglobin A was detected in 7/39 (17.9%) of patients with verified metastasis. Multivariate analysis revealed the detection of Mammaglobin A-mRNA-positive cells, as an independent risk factor for reduced DFI. Conclusions: Mammaglobin A is a highly specific molecular marker for the detection of circulating tumor cells in operable breast cancer, with important prognostic applications. © 2006 The Canadian Society of Clinical Chemists

A highly specific real-time RT-PCR method for the quantitative determination of CK-19 mRNA positive cells in peripheral blood of patients with operable breast cancer

The aim of the present study was to decrease the incidence of false positives and to better characterize marginally cytokeratin-19 (CK-19) mRNA positive peripheral blood samples from patients with early stage breast cancer. A new set of highly specific primers for CK-19, which avoids amplification of contaminating genomic DNA, was designed and evaluated to improve the specificity and sensitivity of the previously described methodology. The primers were specifically designed to avoid amplification of contaminating genomic DNA and CK-19 pseudogenes. The breast cancer cell line MCF-7 was used as positive control for the development and analytical evaluation of the assay, while peripheral blood samples from 62 healthy female individuals and 160 patients with early breast cancer were used for the evaluation of the sensitivity and specificity of the new primer pair. The novel designed primer pair was highly sensitive, as it detects up to 1 MCF-7 cell, and specific as none of the healthy individuals had detectable CK-19 mRNA positive cells in their peripheral blood. CK-19 mRNA positive cells were detected in 33 out of 160 (20.6%) patients with early breast cancer. Results obtained by the proposed optimized real-time RT-PCR protocol correlated well with those obtained in the same samples by our previously reported quantitative real-time RT-PCR [concordance in 198/222 (89.2%), p = 0.0022, McNemar test]. The improved method eliminates the incidence of false positives and is highly sensitive and specific. The method could be used in a clinical setting in the near future for continuous monitoring and quantification of circulating epithelial cells in the peripheral blood of patients with operable breast cancer, provided that a quite larger number of clinical samples with a known follow-up will be analyzed. © 2006 Wiley-Liss, Inc

HER-2 DNA quantification of paraffin-embedded breast carcinomas with LightCycler real-time PCR in comparison to immunohistochemistry and chromogenic in situ hybridization

Objectives: To compare the detection of HER-2 status by real-time PCR, on paraffin-embedded breast carcinomas, in respect to immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). Design and methods: Paraffin-embedded breast carcinomas collected from 85 patients diagnosed with early stage breast cancer were analyzed for HER-2 gene amplification by real-time PCR and CISH, as well as for HER-2 protein expression by IHC. Results: HER-2 gene amplification was observed in 19 (22.4%) of 85 breast cancer patients by real-time PCR and in 19 (22.4%) of 85 patients by CISH. Strong (3+) HER-2 protein over-expression was observed in 13 (15.3%) out of 85 patients. Moreover, there were 4 out of 85 (4.7%) patients that had moderate (2+) HER-2 protein over-expression, while 68 out of 85 (80%) patients had no HER-2 protein over-expression by IHC. There were strong concordance rates between real-time PCR and IHC (79/85, 92.9%, p &lt; 0.0001) and real-time PCR and CISH (77/85, 90.6%, p &lt; 0.0001). The concordance rate between the three methods was 90.6% (p &lt; 0.0001). Conclusions: Our data show that the results obtained for amplification of HER-2 by real-time PCR on the LightCycler are comparable to those obtained by IHC and CISH. © 2006 The Canadian Society of Clinical Chemists

Prognostic value of the molecular detection of circulating tumor cells using a multimarker reverse transcription-PCR assay for cytokeratin 19, mammaglobin A, and HER2 in early breast cancer

Purpose: To investigate the prognostic value of the molecular detection of circulating tumor cells (CTCs) using three markers [cytokeratin 19 (CK19), mammaglobin A (MGB1), and HER2] in early breast cancer. Experimental Design; CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected using real-time (CK19) and nested (MGB1 and HER2) reverse transcription-PCR in the peripheral blood of 175 women with stage I to III breast cancer before the initiation of adjuvant chemotherapy. The detection of CTCs was correlated with clinical outcome. In 10 patients, immunofluorescence staining experiments were done to investigate the coexpression of cytokeratin, MGB1, and HER2 in CTCs. Results: CK19mRNA+, MGB1mRNA+, and HER2mNA+ cells were detected in 41.1%, 8%, and 28.6% of the 175 patients, respectively. Patients had one of the following molecular profiles: CK19mRNA+/MGB1mRNA+/HER2mRNA+ (n = 8), CK19mRNA+/MGB1mRNA+/ HER2mRNA- (n = 1), CK19mRNA+/MGB1mRNA-/HER2mRNA+ (n = 42), CK19mRNA+/MGS1mRNA-/HER2mRNA- (n = 21), CK19mRNA-/MGB1mRNA+/HER2mRNA- (n = 5), and CK19mRNA-/MGB1m RNA-IHER2mRNA- (n = 98). Double-immunofluorescence experiments confirmed the following CTC phenotypes: CK+/MGBJ+, CK+/MGB1-, CK-/MGB1+, CK+/ HER2+, CK+/HER2-, MGB1+/HER2-, and MGB1+/HER2+. In univariate analysis, the detection of CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells was associated with shorter disease- free survival (DFS; P « 0.001, P = 0.001, and P « 0.001, respectively), whereas the detection of CK19mRNA+ and MGB1mRNA+ cells was associated with worse overall survival (P = 0.044 and 0.034, respectively). In multivariate analysis, estrogen receptor-negative tumors and the detection of CK19mRNA+ and MGB1mRNA+ cells were independently associated with worse DFS. Conclusion: The detection of peripheral blood CK19mRNA+ and MGB1mRNA+ cells before adjuvant chemotherapy predicts poor DFS in women with early breast cancer. ©2008 American Association for Cancer Research