MEM: An Algorithm for the Reliable Detection of Microsatellite Instability (MSI) on a Small NGS Panel in Colorectal Cancer.

PURPOSE: MEM is an NGS algorithm that uses Expectation-Maximisation to detect the presence of unstable alleles from the NGS sequences of five microsatellites (BAT-25, BAT-26, NR-21, NR-24 and NR-27). The purpose of this study was to compare the MEM algorithm with a reference PCR method (MSI-PCR) and MisMatch Repair protein immunohistochemistry (MMR-IHC). METHODS: FFPE colorectal cancer samples from 146 patients were analysed in parallel by MSI-PCR and NGS using the MEM algorithm. MMR-IHC results were available for 133 samples. Serial dilutions of an MSI positive control were performed to estimate the limit of detection. RESULTS: the MEM algorithm was able to detect unstable alleles of each microsatellite with up to a 5% allelic fraction. Of the 146 samples, 28 (19.2%) were MSI in MSI-PCR. MEM algorithm results were in perfect agreement with those of MSI-PCR, at both MSI status and individual microsatellite level (Cohen's kappa = 1). A high level of agreement was noted between MSI-PCR/MEM algorithm results and MMR-IHC results (Cohen's kappa = 0.931). CONCLUSION: the MEM algorithm can determine the MSI status of colorectal cancer samples on a small NGS panel, using only five microsatellites approved by international guidelines, and can be combined with screening for targetable mutations.

Circulating Tumor DNA Early Kinetics Predict Response of Metastatic Melanoma to Anti-PD1 Immunotherapy: Validation Study.

The ability of early (first weeks of treatment) ctDNA kinetics to identify primary resistance to anti-PD1 immunotherapies was evaluated with a validation cohort of 49 patients treated with anti-PD1 for metastatic BRAF or NRAS-mutated melanoma, alone and pooled with the 53 patients from a previously described derivation cohort. BRAF or NRAS mutations were quantified on plasma DNA by digital PCR at baseline and after two or four weeks of treatment. ctDNA kinetics were interpreted according to pre-established biological response criteria. A biological progression (bP, i.e., a significant increase in ctDNA levels) at week two or week four was associated with a lack of benefit from anti-PD1 (4-month PFS = 0%; 1-year OS = 13%; n = 12/102). Patients without initial bP had significantly better PFS and OS (4-month PFS = 78%; 1-year OS = 73%; n = 26/102), as did patients whose ctDNA kinetics were not evaluable, due to low/undetectable baseline ctDNA (4-month PFS = 80%; 1-year OS = 81%; n = 64/102). ctDNA detection at first-line anti-PD1 initiation was an independent prognostic factor for OS and PFS in multivariate analysis. Overall, early ctDNA quantitative monitoring may allow the detection of primary resistances of metastatic melanoma to anti-PD1 immunotherapies.

Circulating Tumour DNA Is an Independent Prognostic Biomarker for Survival in Metastatic BRAF or NRAS-Mutated Melanoma Patients.

Circulating tumour DNA (ctDNA) can be used to identify gene alterations. The purpose of this study was to determine whether the detection of ctDNA, based on the identification of BRAF and NRAS mutations before systemic treatment initiation, was associated with the prognosis of metastatic melanoma. In total, 68 BRAF or NRAS-mutated stage IV or unresectable stage III metastatic cutaneous melanoma patients were included and tested for the presence of BRAF and NRAS mutations in circulating DNA before treatment initiation, using the Cobas BRAF/NRAS Mutation Test (Roche). The expected mutation was detected in the plasma of 34/68 patients (50% sensitivity). ctDNA detection was associated with AJCC stage, along with the number and nature of metastases. ctDNA was less frequently detected in NRAS-mutated than in BRAF-mutated melanoma (36% and 66%, respectively). At initiation of first-line treatment, ctDNA detection was associated with poor prognosis in Progression Free Survival (PFS) and Overall Survival (OS) in univariate analysis (log-rank: p = 0.002 and p < 0.0001, respectively). In multivariate analysis, ctDNA detection was an independent factor of poor prognosis in OS, after adjustment for AJCC stage, number and nature of metastases and gender (HR = 4.384; 95% CI: (1.308; 14.699); p = 0.017).

Prospective evaluation of two screening methods for molecular testing of metastatic melanoma: Diagnostic performance of BRAF V600E immunohistochemistry and of a NRAS-BRAF fully automated real-time PCR-based assay.

Screening for theranostic biomarkers is mandatory for the therapeutic management of cutaneous melanoma. BRAF and NRAS genes must be tested in routine clinical practice. The methods used to identify these alterations must be sensitive to detect mutant alleles in a background of wild type alleles, and specific to identify the correct mutation. They should not require too much material, since in some cases the available samples are small biopsies. Finally, they should also be quick enough to allow a rapid therapeutic management of patients. Sixty five consecutive formalin-fixed paraffin-embedded (FFPE) melanoma samples were prospectively tested for BRAF mutations with the VE1 (anti-BRAF V600E) antibody and for both BRAF and NRAS mutations with the Idylla NRAS-BRAF-EGFR S492R Mutation Assay cartridges. Results were compared to our routine laboratory practice, allele specific amplification and/or Sanger sequencing and discordant cases confirmed by digital PCR. Excluding discordant by-design-mutations, system failures and DNA quantity or quality failures, BRAF IHC demonstrated an overall concordance of 89% for BRAF V600E mutation detection, the Idylla system gave a concordance of 100% for BRAF mutation detection and of 92.1% for NRAS mutation detection when compared to our reference. When discrepancies were observed, all routine results were confirmed by digital PCR. Finally, BRAF IHC positive predictive value (PPV) was of 82% and negative predictive value (NPV) of 92%. The Idylla cartridges showed a PPV and NPV of both 100% for BRAF mutation detection and a PPV and NPV of 100% and 87% respectively, for NRAS mutation detection. In conclusion, BRAF V600E immunohistochemistry is efficient for detecting the V600E mutation, but negative cases should be further evaluated by molecular approaches for other BRAF mutations. Since 3 NRAS mutations have not been detected by the Idylla NRAS-BRAF-EGFR S492R Mutation Assay, these cartridges should not be used as a substitute for traditional molecular methods in the conventional patient therapeutic care process without the expertise needed to have a critical view of the produced results.

Quantitative monitoring of circulating tumor DNA predicts response of cutaneous metastatic melanoma to anti-PD1 immunotherapy.

Immunotherapies have changed the medical management of metastatic melanoma. However, the early detection of patients who do not respond to these treatments is a key issue. We evaluated the quantitative monitoring of circulating tumor DNA (ctDNA) as an early predictor of response to anti-PD1. Patients treated with anti-PD1 for metastatic mutated melanoma were selected. The somatic alteration detected on the tumor tissue was quantified on plasma DNA by digital PCR (dPCR) at treatment initiation, after 2 and 4 weeks of treatment, and then every 4 weeks until progression. The absence of biological response (defined as a significant decrease in the amount of ctDNA relative to the baseline level) after 2 weeks of treatment was associated with a lack of clinical benefit under anti-PD1. In the presence of a biological response at week 2, detection of subsequent biological progression (significant increase in the amount of ctDNA relative to its nadir) was 100% predictive of progressive disease, on average 75 days prior to radiological detection. Patients with a persistent biological response beyond week 16 did not experience any progressive disease and exhibited sustained responses. In conclusion, we show that quantitative monitoring of ctDNA, using criteria accounting for dPCR measurement imprecision, allows the early and specific detection of patients who do not respond to anti-PD1 therapy.

BRAF mutations might be more common than supposed in vulvar melanomas.

Data on BRAF, NRAS and KIT mutations are scarce in patients with vulvo-vaginal melanomas and are associated with important therapeutic issues. We investigated their prevalence in a cohort of patients with female lower genital tract melanomas between 2003 and 2017. Of the 22 patients, 5 (22.7%) harboured a BRAF mutation, which was much higher than the rate of 5% reported in the literature. One patient, who was tested negative on the primary melanoma, had a NRAS mutation in a cutaneous metastasis. Our data provide a rationale for prospective and repeated mutations testing in female lower genital tract melanomas.

Circulating tumour DNA: analytical aspects and clinical applications for metastatic melanoma patients.

The management of metastatic melanoma has evolved since the onset of treatments with BRAF inhibitors. In order to predict which patients are likely to respond to these treatments, the therapeutic strategy is now conditioned by the search for the activating mutations of the BRAF gene. Tumor genotyping is routinely performed from DNA extracted from tissue or cellular specimens from the primary tumor, metastases, or neoplastic effusions. Due to their invasiveness, these specimens are rarely repeated during the management. In addition, the analysis of the tumor material requires a pretreatment of the sample (formalin fixation, paraffin inclusion, preparation of tissue sections) and may take up to several weeks, making emergency treatment with BRAF inhibitors impossible. Circulating tumor DNA (ctDNA), released by cancer cells in the blood stream, appears as an alternative to tissue sampling. The pre-analytical conditions are now well defined, and several technological approaches can be used to demonstrate the desired molecular alterations. ctDNA is less affected by tumor heterogeneity, can be collected in a minimally invasive manner and analyzed rapidly. Furthermore, ctDNA can be repeatedly analyzed during follow-up, which makes it possible to envisage its use as a specific tumor marker, in order to monitor the response to the treatment and to detect treatment failure.

Efficient treatment of a metastatic melanoma patient with a combination of BRAF and MEK inhibitors based on circulating tumor DNA analysis: a case report.

BACKGROUND: Fixed tissues are the standard samples used in routine practice for molecular testing. But sometimes tissues are lacking or difficult to obtain. In these cases, circulating tumor DNA released from tumor cells can be used as an alternative source of tumor DNA. CASE PRESENTATION: We present the case of a 63-year-old Caucasian woman with a metastatic melanoma and a very poor performance status. A plasma sample was tested and the BRAF p.V600E mutation was detected. Based on this result, a treatment combining a BRAF inhibitor and a MEK inhibitor was immediately started. This patient achieved a complete response. In addition, by repeating the plasma test, we could obtain a precise kinetic of release of mutated BRAF DNA in plasma. CONCLUSIONS: We report here for the first time the efficient treatment of a metastatic melanoma patient on the basis of circulating tumor DNA analysis. This urgent treatment provided a dramatic response in a patient with a very poor initial condition. The kinetic data most likely reflect treatment efficacy.

Clinical significance of BRAF mutation status in circulating tumor DNA of metastatic melanoma patients at baseline.

Circulating tumor DNA is a promising non-invasive tool for cancer monitoring. The main objective of our work was to investigate the relationship between mutant BRAF DNA in plasma and clinical response. Thirty-eight stage IV patients with a V600 mutated BRAF melanoma were included prior to any treatment. DNA was extracted from plasma and mutant DNA was detected using the amplification-refractory mutation system method. Before the beginning of any treatment, the corresponding BRAF mutation was detected in 29 of the 38 tested plasma samples (76.3% positive per cent agreement). We observed a strong correlation between the presence of circulating mutated DNA and overall survival (OS; P=.02), and with the number of metastatic sites (P=.01). The presence of circulating mutated DNA was also strongly correlated with serum LDH activity (P<.01) and S100 protein concentration (P<.01). Finally, seven patients presented discordant BRAF status in different tumor sites. In all these patients, the test performed on ctDNA was positive, suggesting that ctDNA analysis might be less sensitive to tumor heterogeneity. Altogether, these results suggest that plasmatic mutant BRAF DNA is a prognostic factor of OS, correlated with tumor burden. In addition, it represents an interesting alternative source of DNA to detect BRAF mutations before treatment.

EGFR T790M resistance mutation in non small-cell lung carcinoma.

Lung cancer patients carrying sensitive epidermal growth factor receptor (EGFR) mutations show dramatic responses to tyrosine kinase inhibitors (TKIs). However, the majority of patients whose disease responds to drugs eventually develop resistance to these EGFR-TKIs. The T790M gatekeeper mutation in the EGFR tyrosine kinase domain accounts for half of resistance to these drugs. In some patients, this mutation is also detected as a primary event before drug exposure, at a frequency that is highly dependent on the technique used. This review will focus on the methods that have been used to detect the T790M mutation, and its potential clinical applications both in TKI naïve patients and in patients with an acquired resistance.

PPARγ is functionally expressed in clear cell renal cell carcinoma.

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been demonstrated to exert an inhibitory effect on cell growth in several tumor models, including clear cell renal cell carcinoma (CCRCC). PPARγ has therefore been proposed to be a potential therapeutic target. Thus, the PPARγ gene must be expressed and not altered in cancer cells. We have therefore analyzed tumor specimens collected from 63 patients with CCRCC who underwent partial or total nephrectomy. The multiplex ligation-dependent probe amplification (MLPA) assay was used to detect deletions in the PPARγ gene. The majority of the tumors (48/63; 76.2%) did not present alterations. Two samples (3.2%) presented a deletion of the non-coding exon A1. Nine samples (14.3%) showed large heterozygous deletions in chromosome 3p including PPARγ. Potential mutations were analyzed by DNA sequencing of the 6 coding exons of the PPARγ gene. No mutation was found in exons 1-5. In exon 6, a silent polymorphism was detected in 14 samples (22.2%). CCRCC were found to express the PPARγ1 isoform. The expression level of PPARγ was measured by real-time quantitative PCR. A significantly reduced transcript level was associated with an elevated Fuhrman grade. Finally, we analyzed the expression of angiopoietin-like 4, a known PPARγ target gene, in CCRCC cell lines cultured in the presence of rosiglitazone, a PPARγ agonist. A strong induction was found in the 3 cell lines tested, indicating that PPARγ is functional in all these cell lines. In conclusion, we show here that PPARγ is expressed and functional in CCRCC, prerequisites for being a potential target for CCRCC treatment.

Loss of expression of TIMP3 in clear cell renal cell carcinoma.

AIMS: In clear cell renal cell carcinoma (CCRCC), vascular endothelial growth factor (VEGF) represents the central positive mediator of tumour angiogenesis while VEGF receptor (VEGFR) is the primary target of anti-angiogenic therapies. TIMP3 is a physiological VEGFR-2 antagonist and thus could be considered as an anti-angiogenic factor. We therefore determined the status of this physiological inhibitor in CCRCC. PATIENTS AND METHODS: Archival tumour from 105 patients was studied. TIMP3 expression was analysed using immuno-histochemistry and real-time RT-PCR. Results were correlated with clinicopathological variables. To analyse the mechanisms of gene silencing involved, we performed Multiplex Ligation-dependent Probe Amplification (MLPA) and methylation-specific MLPA (MS-MLPA). At last, we evaluated the main upstream pathway described implicating TGFbetaRII, which induces TIMP3 expression. RESULTS: A down-expression of TIMP3, determined by immunohistochemistry, affected 100/105 renal cancers (95.2%). TIMP3 mRNA levels were significantly lower in high-grade tumours. Loss of heterozygosity of the TIMP3 gene was observed in 8 tumours (7.6%) and the 5'CpG island of the TIMP3 promoter was found to be methylated in 25 tumours (23.8%). A down-expression of TGFbetaRII was found in 85/105 CCRCCs (80.9%). A significant correlation was found between TIMP3 expression and TGFbetaRII expression. CONCLUSIONS: This is the first demonstration that the loss of TIMP3 expression is observed in almost all CCRCCs. This loss of expression is a common molecular event in CCRCC. It may be an important initiation step for tumour development in a complex process implicating loss of heterozygosity on chromosome 22q, promoter hyper-methylation and inactivation of the TGFbetaRII pathway.

HtrA3 is regulated by 15-deoxy-Delta12,14-prostaglandin J2 independently of PPARgamma in clear cell renal cell carcinomas.

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been shown to possess anti-proliferative effects in many types of cancer. In clear cell renal cell carcinoma (CCRCC), the targets involved in these effects are not known. In this study, we demonstrated that, in CCRCC cell lines, the endogenous PPARgamma ligand 15-deoxy-Delta12,14-prostaglandin J2 (15dPGJ2) induces the expression, both at the mRNA and the protein levels, of the HtrA3 gene. This gene belongs to the High-Temperature Requirement Factor A family of serine proteases that repress signaling by TGF-beta family members and inhibit cell migration. Rosiglitazone or ciglitazone, synthetic PPARgamma agonists, did not induce HtrA3 expression, and the PPARgamma antagonist GW9662 did not prevent 15dPGJ2 induction, suggesting that the up-regulation of HtrA3 by 15dPGJ2 is independent of PPARgamma. The MEK/ERK inhibitor PD98059 dramatically repressed HtrA3 induction. Altogether, these data indicate that 15dPGJ2 is able to stimulate the expression of HtrA3 through an indirect mechanism involving the MEK/ERK pathway but independent of PPARgamma. Our results provide a better understanding of the mechanisms involved in the regulation of HtrA3, a potential tumor suppressor gene.

KLF4-dependent, PPARgamma-induced expression of GPA33 in colon cancer cell lines.

The glycoprotein A33 (GPA33) is a colon cancer antigen. Phase I trials with 131I and 125I monoclonal antibody A33 in colon carcinoma patients showed excellent localization to colorectal cancer and some evidence of tumor response. Using DNA microarrays, we have identified the GPA33 gene as a target of PPARgamma in HT29-Cl.16E colon cancer cells. Treatment of HT29-Cl.16E, Caco2, SW1116 and LS174T colon cancer cells with the PPARgamma agonist GW7845 induced a 2- to 6-fold increase in GPA33 mRNA as determined by real-time PCR. This induction was also found in HT29-Cl.16E cells treated with rosiglitazone and ciglitazone and was prevented by cotreatment with the PPARgamma antagonist GW9662, indicating that this regulation was PPARgamma dependent. No canonical PPAR responsive element was found in the GPA33 promoter. We therefore analyzed the expression of transcription factors involved in GPA33 expression. CDXl, CDX2 and KLF5 expression was not modified by PPARgamma activation. By contrast, a significant increase in KLF4 was seen, both at mRNA and protein levels. Furthermore, chromatin immunoprecipitation studies demonstrated that an increased amount of KLF4 protein was bound to the GPA33 promoter in cells treated with rosiglitazone. Finally, downregulation of KLF4 expression by siRNA reduced rosiglitazone-induced GPA33 expression. This indicates that PPARgamma activation induces KLF4 expression, which in turn increases GPA33 expression. We also demonstrate that PPARgamma activation leads to increased (p21WAF1/Cip1 and keratin 19) or decreased (cyclin D1) expression of known KLF4 targets, suggesting that KLF4 is a nodal player in a network of PPARgamma-regulated genes.

Cooperative binding of TIA-1 and U1 snRNP in K-SAM exon splicing activation.

In 293 cells, splicing of the human fibroblast growth factor receptor-2 K-SAM alternative exon is inefficient, but can be made efficient by provoking TIA-1 binding to the U-rich IAS1 sequence downstream from the exon's 5' splice site. We show here that TIA-1 domains known to interact with U1 snRNP and to recruit it to 5' splice sites in vitro are required for TIA-1 activation of K-SAM exon splicing in vivo. We further show that tethering downstream from the K-SAM exon a fusion between the U1 snRNP component U1C and the bacteriophage MS2 coat protein provokes IAS1-dependent exon splicing, and present evidence that the fusion functions after its incorporation into U1 snRNP. Our in vivo data, taken together with previous in vitro results, show that K-SAM splicing activation involves cooperative binding of TIA-1 and U1 snRNP to the exon's 5' splice site region.

OPG/membranous--RANKL complex is internalized via the clathrin pathway before a lysosomal and a proteasomal degradation.

The members of the OPG/RANK/RANKL (osteoprotegerin/receptor activator of nuclear factor kappaB/RANK ligand) triad are involved in various osteolytic pathologies such as bone tumors. Although many studies described the use of OPG during the treatment of bone diseases, its bioavailability and the mechanism by which the cells control the extracellular OPG remains blurred. The present work uses a strongly RANKL expressing cellular model to assess the becoming and the bioavailability of exogenous OPG in the context of its interactions with RANKL. The human kidney cell line 293, which initially expresses neither OPG nor RANKL, was stably transfected by the full length of mouse transmembranous form of RANKL (293RL). When OPG is incubated with 293RL cells, the extracellular concentration of OPG was strongly decreased in a time-dependent manner. The OPG disappearance was not inhibited by the addition of several proteases inhibitors, thus excluding any extracellular protease degradation. Contrary to previous results obtained on myeloma cells, which strongly express syndecan-1, the OPG disappearance was unaffected by the use of an antibody against syndecan-1. However, this event was abolished by an antibody against RANKL. These results, not necessarily conflicting, could be in relation with the expression level of the receptors in the two cellular models. In this context, an internalization process was put forward. Confocal microscopy demonstrated via the clathrin pathway an internalization of OPG mediated by RANKL. After being internalized, OPG was then degraded by the proteasome and the lysosome. A similar internalization phenomenon was also observed in osteoblast cells expressing physiologically RANKL, thus validating our data observed on 293RL cells. Western blotting analysis revealed that the half-life of RANKL was greatly reduced in the presence of OPG, pointing out that OPG binding to RANKL induces an enhancement of the ligand internalization. By the light of these results, the inhibitory effect of OPG on bone resorption can be explained not only by a decoy receptor function, competitor inhibitor of the RANK/RANKL binding, but also by the modulation of the RANKL half-life induced by OPG. Reciprocally, this modulation contributes to reduce the bioavailability of OPG.

The molecular triad OPG/RANK/RANKL: involvement in the orchestration of pathophysiological bone remodeling.

The past decade has seen an explosion in the field of bone biology. The area of bone biology over this period of time has been marked by a number of key discoveries that have opened up entirely new areas for investigation. The recent identification of the receptor activator of nuclear factor kappaB ligand (RANKL), its cognate receptor RANK, and its decoy receptor osteoprotegerin (OPG) has led to a new molecular perspective on osteoclast biology and bone homeostasis. Specifically, the interaction between RANKL and RANK has been shown to be required for osteoclast differentiation. The third protagonist, OPG, acts as a soluble receptor antagonist for RANKL that prevents it from binding to and activating RANK. Any dysregulation of their respective expression leads to pathological conditions such as bone tumor-associated osteolysis, immune disease, or cardiovascular pathology. In this context, the OPG/RANK/RANKL triad opens novel therapeutic areas in diseases characterized by excessive bone resorption. The present article is an update and extension of an earlier review published by Kwan Tat et al. [Kwan Tat S, Padrines M, Theoleyre S, Heymann D, Fortun Y. IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology. Cytokine Growth Factor Rev 2004;15:49-60].