CD44v6 Defines a New Population of Circulating Tumor Cells Not Expressing EpCAM.

Circulating tumor cells (CTCs) are promising diagnostic and prognostic tools for clinical use. In several cancers, including colorectal and breast, the CTC load has been associated with a therapeutic response as well as progression-free and overall survival. However, counting and isolating CTCs remains sub-optimal because they are currently largely identified by epithelial markers such as EpCAM. New, complementary CTC surface markers are therefore urgently needed. We previously demonstrated that a splice variant of CD44, CD44 variable alternative exon 6 (CD44v6), is highly and specifically expressed by CTC cell lines derived from blood samples in colorectal cancer (CRC) patients. Two different approaches-immune detection coupled with magnetic beads and fluorescence-activated cell sorting-were optimized to purify CTCs from patient blood samples based on high expressions of CD44v6. We revealed the potential of the CD44v6 as a complementary marker to EpCAM to detect and purify CTCs in colorectal cancer blood samples. Furthermore, this marker is not restricted to colorectal cancer since CD44v6 is also expressed on CTCs from breast cancer patients. Overall, these results strongly suggest that CD44v6 could be useful to enumerate and purify CTCs from cancers of different origins, paving the way to more efficacious combined markers that encompass CTC heterogeneity.

Limitation of Screening of Different Variants of SARS-CoV-2 by RT-PCR.

Since January 2021, the diffusion of the most propagated SARS-CoV-2 variants in France (UK variant 20I/501Y.V1 (lineage B.1.1.7), 20H/H501Y.V2 (lineage B.1.351) and 20J/H501Y.V3 (lineage P.1)) were urgently screened, needing a surveillance with an RT-PCR screening assay. In this study, we evaluated one RT-PCR kit for this screening (ID SARS-CoV-2/UK/SA Variant Triplex®, ID Solutions, Grabels, France) on 2207 nasopharyngeal samples that were positive for SARS-CoV-2. Using ID Solutions kit, 4.1% (92/2207) of samples were suspected to belonged to B.1.351 or P.1 variants. Next-generation sequencing that was performed on 67.4% (62/92) of these samples confirmed the presence of a B.1.351 variant in only 75.8% of the samples (47/62). Thirteen samples belonged to the UK variant (B.1.1.7), and two to A.27 with N501Y mutation. The thirteen with the UK variant presented one mutation in the S-gene, near the ΔH69/ΔV70 deletion (S71F or A67S), which impacted the detection of ΔH69/ΔV70 deletion. Using another screening kit (PKampVariantDetect SARS-CoV-2 RT-PCR combination 1 and 3® PerkinElmer, Waltham, MA, USA) on the misidentified samples, we observed that the two mutations, S71F or A67S, did not impact the detection of the UK variant. In conclusion, this study highlights the limitations of the screening strategy based on the detection of few mutations/deletions as well as it not being able to follow the virus evolution.

Circulating tumour cells from patients with colorectal cancer have cancer stem cell hallmarks in ex vivo culture.

OBJECTIVE: Although counting of circulating tumour cells (CTC) has attracted a broad interest as potential markers of tumour progression and treatment response, the lack of functional characterisation of these cells had become a bottleneck in taking these observations to the clinic. Our objective was to culture these cells in order to understand them and exploit their therapeutic potential to the full. DESIGN: Here, hypothesising that some CTC potentially have cancer stem cell (CSC) phenotype, we generated several CTC lines from the blood of patients with advanced metastatic colorectal cancer (CRC) based on their self-renewal abilities. Multiple standard tests were then employed to characterise these cells. RESULTS: Our CTC lines self-renew, express CSC markers and have multilineage differentiation ability, both in vitro and in vivo. Patient-derived CTC lines are tumorigenic in subcutaneous xenografts and are also able to colonise the liver after intrasplenic injection. RNA sequencing analyses strikingly demonstrate that drug metabolising pathways represent the most upregulated feature among CTC lines in comparison with primary CRC cells grown under similar conditions. This result is corroborated by the high resistance of the CTC lines to conventional cytotoxic compounds. CONCLUSIONS: Taken together, our results directly demonstrate the existence of patient-derived colorectal CTCs that bear all the functional attributes of CSCs. The CTC culture model described here is simple and takes <1 month from blood collection to drug testing, therefore, routine clinical application could facilitate access to personalised medicine. CLINICAL TRIAL REGISTRATION: ClinicalTrial.gov NCT01577511.

Exon 7 deletion in the bcr-abl gene is frequent in chronic myeloid leukemia patients and is not correlated with resistance against imatinib.

Chronic myeloid leukemia (CML) patients treated with imatinib develop frequent resistance generally due to a point mutation. Recently, large rearrangements of abl sequence have also been described. In this study, we focused on the complete deletion of exon 7. We screened for bcr-abl(delexon7) in 63 resistant patients by high-resolution melting (HRM) analysis and direct sequencing. Moreover, we analyzed expression of abl(delexon7) and bcr-abl(delexon7) in 17 CML patients at diagnosis, 32 patients at resistance, and 20 negative controls by quantitative PCR or fragment length analysis. bcr-abl(delexon7) was detected on 34 (54%) among 63 resistant patients by HRM, showing an increase in the sensitivity of screening, because only 3.2% could be detected by direct sequencing. This deletion was not associated with a point mutation (P = 0.3362). In addition, abl(delexon7) was found in all tested samples with the same pattern of expression, suggesting an alternative splicing mechanism. In the bcr-abl component, there was no statistical difference between CML patients at diagnosis and resistant patients (P = 0.2815) as regarding bcr-abl(delexon7) proportion, thus arguing against involvement of deletion in resistance. Moreover, among two patients harboring bcr-abl(delexon7) at diagnosis, one experienced a complete disappearance of this transcript, and the other decreased >75% at resistance. In conclusion, bcr-abl(delexon7) is frequently observed in CML patients when using sensitive techniques. It seems to be the result of an alternative splicing mechanism and to be independent from the occurrence of resistance.

Predicting outcomes for children with neuroblastoma using a multigene-expression signature: a retrospective SIOPEN/COG/GPOH study.

BACKGROUND: More accurate prognostic assessment of patients with neuroblastoma is required to better inform the choice of risk-related therapy. The aim of this study is to develop and validate a gene-expression signature to improve outcome prediction. METHODS: 59 genes were selected using an innovative data-mining strategy, and were profiled in the largest neuroblastoma patient series (n=579) to date using real-time quantitative PCR starting from only 20 ng of RNA. A multigene-expression signature was built using 30 training samples, tested on 313 test samples, and subsequently validated in a blind study on an independent set of 236 tumours. FINDINGS: The signature has a performance, sensitivity, and specificity of 85.4% (95% CI 77.7-93.2), 84.4% (66.5-94.1), and 86.5% (81.1-90.6), respectively, to predict patient outcome. Multivariate analysis indicates that the signature is a significant independent predictor of overall survival and progression-free survival after controlling for currently used risk factors: patients with high molecular risk have a higher risk of death from disease and higher risk of relapse or progression than patients with low molecular risk (odds ratio 19.32 [95% CI 6.50-57.43] and 3.96 [1.97-7.97] for overall survival and progression-free survival, respectively, both p<0.0001). Patients at an increased risk of an adverse outcome can also be identified in the current treatment groups, showing the potential of this signature for improved clinical management. These results were confirmed in the validation study, in which the signature was also independently statistically significant in a model adjusted for MYCN status, age, International Neuroblastoma Staging System stage, ploidy, International Neuroblastoma Pathology Classification grade of differentiation, and mitosis karyorrhexis index (odds ratios between 4.81 and 10.53 depending on the model for overall survival and 3.68 [95% CI 2.01-6.71] for progression-free survival). INTERPRETATION: The 59-gene expression signature is an accurate predictor of outcome in patients with neuroblastoma. The signature is an independent risk predictor, identifying patients with an increased risk of poor outcome in the current clinical-risk groups. The method and signature is suitable for routine laboratory testing, and should be evaluated in prospective studies. FUNDING: The Belgian Foundation Against Cancer, the Children Cancer Fund Ghent, the Belgian Society of Paediatric Haematology and Oncology, the Belgian Kid's Fund and the Fondation Nuovo-Soldati (JV), the Fund for Scientific Research Flanders (KDP, JH), the Fund for Scientific Research Flanders, the Institute for the Promotion of Innovation by Science and Technology in Flanders, Strategisch basisonderzoek, the Fondation Fournier Majoie pour l'Innovation, the Instituto Carlos III, the Italian Neuroblastoma Foundation, the European Community under the FP6, and the Belgian programme of Interuniversity Poles of Attraction.