Deciphering Brain Metastasis Stem Cell Properties From Colorectal Cancer Highlights Specific Stemness Signature and Shared Molecular Features.

BACKGROUND & AIMS: Brain metastases (BMs) from colorectal cancer (CRC) are associated with significant morbidity and mortality, with chemoresistance and short overall survival. Migrating cancer stem cells with the ability to initiate BM have been described in breast and lung cancers. In this study, we describe the identification and characterization of cancer stem cells in BM from CRC. METHODS: Four brain metastasis stem cell lines from patients with colorectal cancer (BM-SC-CRC1 to BM-SC-CRC4) were obtained by mechanical dissociation of patient's tumors and selection of cancer stem cells by appropriate culture conditions. BM-SC-CRCs were characterized in vitro by clonogenic and limiting-dilution assays, as well as immunofluorescence and Western blot analyses. In ovo, a chicken chorioallantoic membrane (CAM) model and in vivo, xenograft experiments using BALB/c-nude mice were realized. Finally, a whole exome and RNA sequencing analyses were performed. RESULTS: BM-SC-CRC formed metaspheres and contained tumor-initiating cells with self-renewal properties. They expressed stem cell surface markers (CD44v6, CD44, and EpCAM) in serum-free medium and CRC markers (CK19, CK20 and CDX-2) in fetal bovine serum-enriched medium. The CAM model demonstrated their invasive and migratory capabilities. Moreover, mice intracranial xenotransplantation of BM-SC-CRCs adequately recapitulated the original patient BM phenotype. Finally, transcriptomic and genomic approaches showed a significant enrichment of invasiveness and specific stemness signatures and highlighted KMT2C as a potential candidate gene to potentially identify high-risk CRC patients. CONCLUSIONS: This original study represents the first step in CRC BM initiation and progression comprehension, and further investigation could open the way to new therapeutics avenues to improve patient prognosis.

Association between Iron Deficiency and Survival in Older Patients with Cancer.

BACKGROUND: iron deficiency (ID) is frequent in older patients. PURPOSE: to evaluate the association between ID and survival in patients ≥ 75 years old with confirmed solid tumors. METHODS: a retrospective monocentric study including patients between 2009 and 2018. ID, absolute ID (AID) and functional ID (FID) were defined according to the European Society for Medical Oncology (ESMO) criteria. Severe ID was defined by a ferritin level < 30 µg/L. RESULTS: in total, 556 patients were included, the mean age was 82 (±4.6) years, 56% were male, the most frequent cancer was colon cancer (19%, n = 104), and metastatic cancers were found in 38% (n = 211). Median follow-up time: 484 [190-1377] days. In anemic patients, ID and FID were independently associated with an increased risk of mortality (respectively, HR 1.51; p = 0.0065 and HR 1.73; p = 0.0007). In non-anemic patients, FID was independently associated with better survival (HR 0.65; p = 0.0495). CONCLUSION: in our study, ID was significantly associated with survival, and with better survival for patients without anemia. These results suggest that attention should be paid to the iron status in older patients with tumors and raise questions about the prognostic value of iron supplementation for iron-deficient patients without anemia.

[Intention of geriatric interns to train and go into private practice]. / Intention des internes de gériatrie à se former et à s'installer en libéral.

Geriatrics is a recent medical specialty in France, and private practice, possible, is still a minority. The specialized studies diploma, created in 2017, provides for the possibility of completing a semester of internship in private practice. According to our study, 62% of geriatric interns in training would consider doing a semester outside the hospital and 42.7% would consider setting up in private practice. Proportions which vary according to the year in which the national qualifying tests were taken and the internship subdivision.

Role of antibiotic use, plasma citrulline and blood microbiome in advanced non-small cell lung cancer patients treated with nivolumab.

BACKGROUND: Recent data suggested a role of gut microbiota and antibiotic use on immune checkpoint inhibitors efficacy. We aimed to evaluate the impact of early use of antibiotic (EUA), blood microbiome and plasmatic citrulline (marker of the intestinal barrier) on nivolumab efficacy in non-small cell lung cancer (NSCLC). METHODS: We included all consecutive patients with advanced NSCLC treated with nivolumab in our Department between 2014 and 2017. Blood microbiome was analyzed at month (M) M0 and M2. Citrulline rates were evaluated at M0, M2, M4 and M6. RESULTS: Seventy-two patients were included (EUA in 42%). Overall survival (OS) was longer without EUA (median 13.4 months) than with EUA (5.1 months, p = 0.03). Thirty-five patients (49%) had plasma samples available. High citrulline rate (≥20 µM) at M0 was associated with tumor response (p = 0.084) and clinical benefit (nivolumab > 6 months) (p = 0.002). Median progression-free survival (PFS) was 7.9 months (high citrulline) vs 1.6 months (low citrulline) (p < 0.0001), and median OS were respectively non reached vs 2.2 months (p < 0.0001). Patients with EUA had lower median citrulline rates at M0: 21 µM (IQR 15.0-30.8) vs 32 µM (IQR 24.0-42.0) without EUA (p = 0.044). The presence of specific bacterial DNA in blood at M0 was associated with response and clinical benefit (Peptostreptococcae, Paludibaculum, Lewinella) or with tumor progression (Gemmatimonadaceae). Multivariate analyses on PFS and OS confirmed the prognostic role of citrulline and blood microbiome. CONCLUSIONS: EUA is associated with shorter OS with nivolumab and lower citrulline rates. Plasma citrulline and blood microbiome appear to be promising predictive factors of nivolumab efficacy.

Predictive role of plasmatic biomarkers in advanced non-small cell lung cancer treated by nivolumab.

Immune checkpoint inhibitors, as nivolumab, are used in advanced non-small cell lung cancer (NSCLC). However, no associated biomarker is validated in clinical practice with this drug. We investigated herein immune-related blood markers in patients with advanced NSCLC treated with nivolumab. Plasma of 43 consecutive patients were prospectively collected at time of the diagnosis of cancer, at the initiation of nivolumab and at the first tumour evaluation (2 months). Concentrations of PD-L1 (sPD-L1), soluble PD-L2 (sPD-L2), Interleukine-2 (sIl-2), Interferon-gamma (sIFN-γ), and Granzyme B (sGranB) were quantified by ELISA. Cell free RNA was quantified by Reverse Transcriptase -PCR), and plasmatic microRNAs (miRNAs) were evaluated by targeted sequencing. Expression of PD-L1 on tumour biopsies was performed by immunohistochemistry using E13LN. High sPD-L1 at 2 months and increase of sPD-L1 concentrations were associated with poor response and absence of clinical benefit (nivolumab treatment less than 6 months). The variation of sPD-L1 concentrations were confirmed by RNA quantification. sPD-L1 concentrations were not correlated with PD-L1 expression on corresponding tumour samples. Low sGranB at nivolumab initiation was also associated with poor response. High sPD-L1 and low sGranB were associated with poor progression-free survival (PFS) and overall survival (OS). Low sPD-L2, low sIl-2 and high sIFN-γ were associated with grade 3-4 toxicities. Finally, miRNA screening showed that patients with clinical benefit (n = 9) had down-expression of miRNA-320b and -375 compared to patients with early progression at 2 months (n = 9). In conclusion, our results highlight the interest of circulating biomarkers in patients treated with nivolumab.

Circulating tumor DNA evaluated by Next-Generation Sequencing is predictive of tumor response and prolonged clinical benefit with nivolumab in advanced non-small cell lung cancer.

Nivolumab is an anti-PD1 antibody, given in second-line or later treatment in advanced non-small cell lung cancer (NSCLC). The objective of this study was to describe the predictive value of circulating tumor DNA (ctDNA) on the efficacy of nivolumab in advanced NSCLC. We prospectively included all consecutive patients with advanced NSCLC treated with nivolumab in our Department between June 2015 and October 2016. Plasma samples were obtained before the first injection of nivolumab and at the first tumor evaluation with nivolumab. ctDNA was analyzed by Next-Generation Sequencing (NGS), and the predominant somatic mutation was followed for each patient and correlated with tumor response, clinical benefit (administration of nivolumab for more than 6 months), and progression-free survival (PFS). Of 23 patients, 15 had evaluable NGS results at both times of analysis. ctDNA concentration at the first tumor evaluation and ctDNA change correlated with tumor response, clinical benefit and PFS. ROC curve analyses showed good diagnostic performances for tumor response and clinical benefit, both for ctDNA concentration at the first tumor evaluation (tumor response: positive predictive value (PPV) at 100.0% and negative predictive value (NPV) at 71.0%; clinical benefit: PPV at 83.3% and NPV 77.8%) and the ctDNA change (tumor response: PPV 100.0% and NPV 62.5%; clinical benefit: PPV 100.0% and NPV 80.0%). Patients without ctDNA concentration increase >9% at 2 months had a long-term benefit of nivolumab. In conclusion, NGS analysis of ctDNA allows the early detection of tumor response and long-term clinical benefit with nivolumab in NSCLC.

Comparative oncogenomics identifies tyrosine kinase FES as a tumor suppressor in melanoma.

Identification and functional validation of oncogenic drivers are essential steps toward advancing cancer precision medicine. Here, we have presented a comprehensive analysis of the somatic genomic landscape of the widely used BRAFV600E- and NRASQ61K-driven mouse models of melanoma. By integrating the data with publically available genomic, epigenomic, and transcriptomic information from human clinical samples, we confirmed the importance of several genes and pathways previously implicated in human melanoma, including the tumor-suppressor genes phosphatase and tensin homolog (PTEN), cyclin dependent kinase inhibitor 2A (CDKN2A), LKB1, and others. Importantly, this approach also identified additional putative melanoma drivers with prognostic and therapeutic relevance. Surprisingly, one of these genes encodes the tyrosine kinase FES. Whereas FES is highly expressed in normal human melanocytes, FES expression is strongly decreased in over 30% of human melanomas. This downregulation correlates with poor overall survival. Correspondingly, engineered deletion of Fes accelerated tumor progression in a BRAFV600E-driven mouse model of melanoma. Together, these data implicate FES as a driver of melanoma progression and demonstrate the potential of cross-species oncogenomic approaches combined with mouse modeling to uncover impactful mutations and oncogenic driver alleles with clinical importance in the treatment of human cancer.

An essential pathway links FLT3-ITD, HCK and CDK6 in acute myeloid leukemia.

CDK4/CDK6 and RB proteins drive the progression through the G1 phase of the cell cycle. In acute myeloid leukemia (AML), the activity of the CDK/Cyclin D complex is increased. The mechanism involved is unknown, as are the respective roles played by CDK4 or CDK6 in this process. Here, we report that AML cells carrying FLT3-ITD mutations are dependent on CDK6 for cell proliferation while CDK4 is not essential. We showed that FLT3-ITD signaling is responsible for CDK6 overexpression, through a pathway involving the SRC-family kinase HCK. Accordingly, FLT3-ITD failed to transform primary hematopoietic progenitor cells from Cdk6-/- mice. Our results demonstrate that CDK6 is the primary target of CDK4/CDK6 inhibitors in FLT3-ITD positive AML. Furthermore, we delineate an essential protein kinase pathway -FLT3/HCK/CDK6- in the context of AML with FLT3-ITD mutations.

Characterization of S628N: a novel KIT mutation found in a metastatic melanoma.

IMPORTANCE The KIT receptor is mutated in approximately 15%of acral, mucosal, and chronic, sun-damaged melanomas. The status of KIT mutations is of interest because they usually are mutually exclusive with N-RAS and B-RAF mutations and because of the availability of KIT kinase inhibitors in the clinic. Some recurrent KIT mutations are well characterized; others are poorly described.OBSERVATIONS We describe a novel KIT mutation in a patient with metastatic melanoma. The mutation, located in exon 13, resulted in S628N substitution in the KIT receptor. Using all-atom molecular dynamics simulations, biochemical assays, and cell-based assays, we showed that the mutation is a bona fide gain-of-function oncogenic mutation. Furthermore,we evaluated the sensitivity of the mutant to imatinib and dasatinib.CONCLUSIONS AND RELEVANCE We report a novel KIT gain-of-function mutation with S628N substitution (exon 13) and show that it is sensitive to imatinib in vitro. Therefore, patients with this mutation may be eligible for KIT kinase inhibitor­based therapy. Further studies are needed to evaluate the clinical benefit of such therapy.

Improvement of the quality of BRAF testing in melanomas with nationwide external quality assessment, for the BRAF EQA group.

BACKGROUND: Knowledge about tumour gene mutation status is essential for the treatment of increasing numbers of cancer patients, and testing quality has a major impact on treatment response and cost. In 2012, 4,629 tests for BRAF p.V600 were performed in France, in patients with melanomas. METHODS: Two batches of unstained melanoma sections were sent, in May and November 2012, to the 46 laboratories supported by the French National Institute of Cancer (INCa). An external quality assessment (EQA) evaluated mutation status, response times and compliance with INCa recommendations. RESULTS: All the French laboratories involved in testing participated in the EQA. Fourteen different methods were used to detect BRAF mutations, most consisting of combinations of in-house techniques. False responses were noted in 25/520 cases (4.8%), 11 of which concerned confusion between p.V600E and p.V600K. Thus, 2.7% of responses would have led to inappropriate treatment. Within six months, mean response times decreased from 22 to 12 days (P<0.001), and the percentage of samples evaluated by a pathologist for tumour cell content increased, from 75.2% to 96.9% (P<0.001). CONCLUSION: Despite the use of non-certified methods, the false response rate was low. Nationwide EQA can improve the quality of molecular pathology tests on tumours.