Radio-chemotherapy feasibility for biopsy-only unresectable IDH wild-type glioblastomas (BO-GBM).

Background: "Biopsy-only" glioblastoma (BO-GBM) is a heterogeneous, understudied group of patients associated with a poor outcome. Our objective was to explore the pattern of care and prognosis associated with BO-GBM in our center. Methods: Patients with IDH wild-type BO-GBM included in a prospective regional cohort initiated in 2014 and closed in 2017 were retrospectively reviewed for patient characteristics, MRI findings, treatment allocation, and delivery. Results: Of 535 patients included in the cohort, 137 patients were included in the present analysis. The median age was 66 years old and the median KPS was 70. Forty-six patients (33.6%) were referred to radiotherapy and chemotherapy (RT-TMZ) regimen, 75 (54.7%), considered unfitted for RT, received chemotherapy upfront (CT) and 16 (11.7%) were referred to palliative care (PC). Regarding the first group, 91% of patients completed the RT-TMZ. In the CT group, 11 of 75 patients (14.7%) underwent radiotherapy after chemotherapy upfront. Median overall survival was 12.3 months (95% CI, 15.30-24.16), 5.7 months (95% CI, 6.22-9.20), and 1.9 months (95% CI, 1.43-5.08) in RT-TMZ, CT, and PC groups, respectively. In multivariate analyses, progression-free survival was impacted by baseline KPS (P < .001) and MGMT status (P = .004). Overall survival was impacted by baseline KPS (P < .001) and age (P = .030). Conclusion: BO-GBM constitute a large and heterogeneous population in which one-third of patients is amenable to the standard of care, with survival outcome close to one of the patients who underwent surgery. Reliable criteria are needed to help select patients for adequate treatment while new strategies are warranted for BO-GBM unfit for RT.

Cytological Samples: An Asset for the Diagnosis and Therapeutic Management of Patients with Lung Cancer.

BACKGROUND: Lung cancer has become the leading cause of cancer death for men and women. Most patients are diagnosed at an advanced stage when surgery is no longer a therapeutic option. At this stage, cytological samples are often the less invasive source for diagnosis and the determination of predictive markers. We assessed the ability of cytological samples to perform diagnosis, and to establish molecular profile and PD-L1 expression, which are essential for the therapeutic management of patients. METHODS: We included 259 cytological samples with suspected tumor cells and assessed the ability to confirm the type of malignancy by immunocytochemistry. We summarized results of molecular testing by next generation sequencing (NGS) and PD-L1 expression from these samples. Finally, we analyzed the impact of these results in the patient management. RESULTS: Among the 259 cytological samples, 189 concerned lung cancers. Of these, immunocytochemistry confirmed the diagnosis in 95%. Molecular testing by NGS was obtained in 93% of lung adenocarcinomas and non-small cell lung cancer. PD-L1 results were obtained in 75% of patients tested. The results obtained with cytological samples led to a therapeutic decision in 87% of patients. CONCLUSION: Cytological samples are obtained by minimally invasive procedures and can provide enough material for the diagnosis and therapeutic management in lung cancer patients.

Adrenomedullin Secreted by Melanoma Cells Promotes Melanoma Tumor Growth through Angiogenesis and Lymphangiogenesis.

INTRODUCTION: Metastatic melanoma is an aggressive tumor and can constitute a real therapeutic challenge despite the significant progress achieved with targeted therapies and immunotherapies, thus highlighting the need for the identification of new therapeutic targets. Adrenomedullin (AM) is a peptide with significant expression in multiple types of tumors and is multifunctional. AM impacts angiogenesis and tumor growth and binds to calcitonin receptor-like receptor/receptor activity-modifying protein 2 or 3 (CLR/RAMP2; CLR/RAMP3). METHODS: In vitro and in vivo studies were performed to determine the functional role of AM in melanoma growth and tumor-associated angiogenesis and lymphangiogenesis. RESULTS: In this study, AM and AM receptors were immunohistochemically localized in the tumoral compartment of melanoma tissue, suggesting that the AM system plays a role in melanoma growth. We used A375, SK-MEL-28, and MeWo cells, for which we demonstrate an expression of AM and its receptors; hypoxia induces the expression of AM in melanoma cells. The proliferation of A375 and SK-MEL-28 cells is decreased by anti-AM antibody (αAM) and anti-AMR antibodies (αAMR), supporting the fact that AM may function as a potent autocrine/paracrine growth factor for melanoma cells. Furthermore, migration and invasion of melanoma cells increased after treatment with AM and decreased after treatment with αAMR, thus indicating that melanoma cells are regulated by AM. Systemic administration of αAMR reduced neovascularization of in vivo Matrigel plugs containing melanoma cells, as demonstrated by reduced numbers of vessel structures, which suggests that AM is one of the melanoma cells-derived factors responsible for endothelial cell-like and pericyte recruitment in the construction of neovascularization. In vivo, αAMR therapy blocked angiogenesis and lymphangiogenesis and decreased proliferation in MeWo xenografts, thereby resulting in tumor regression. Histological examination of αAMR-treated tumors showed evidence of the disruption of tumor vascularity, with depletion of vascular endothelial cells and a significant decrease in lymphatic endothelial cells. CONCLUSIONS: The expression of AM by melanoma cells promotes tumor growth and neovascularization by supplying/amplifying signals for neoangiogenesis and lymphangiogenesis.

Comparison between Immunocytochemistry, FISH and NGS for ALK and ROS1 Rearrangement Detection in Cytological Samples.

The detection of ROS1 and ALK rearrangements is performed for advanced-stage non-small cell lung cancer. Several techniques can be used on cytological samples, such as immunocytochemistry (ICC), fluorescence in situ hybridization (FISH) and, more recently, next-generation sequencing (NGS), which is gradually becoming the gold standard. We performed a retrospective study to compare ALK and ROS1 rearrangement results from immunocytochemistry, FISH and NGS methods from 131 cytological samples. Compared to NGS, the sensitivity and specificity of ICC were 0.79 and 0.91, respectively, for ALK, and 1 and 0.87 for ROS1. Regarding FISH, the sensitivity and specificity were both at 1 for ALK and ROS1 probes. False-positive cases obtained by ICC were systematically corrected by FISH. When using ICC and FISH techniques, results are very close to NGS. The false-positive cases obtained by ICC are corrected by FISH, and the true-positive cases are confirmed. NGS has the potential to improve the detection of ALK and ROS1 rearrangements in cytological samples; however, the cost of this technique is still much higher than the sequential use of ICC and FISH.

Adult H3K27M mutated thalamic glioma patients display a better prognosis than unmutated patients.

BACKGROUND: Adult thalamic gliomas are a rare entity whose management is challenging for physicians. The aim of this study is to describe the characteristics and prognostic factors of thalamic gliomas in adult patients. METHODS: We retrospectively analyzed the clinical, neuro-radiological, histological, and molecular characteristics of all cases of adult thalamic glioma in our regional center. RESULTS: We included 38 adult patients. Median age at diagnosis was 56.5 years old (range, 24-80). Median KPS at diagnosis was 70%. Two-thirds of patients presented with tumor necrosis on MRI. Bithalamic lesions were present in four patients. The median volume of enhancement associated with lesions was relatively small (14 mm3). Two patients had undergone partial surgical resection. All other patients underwent biopsy. Median PFS was 7.1 months (95% CI [3.7-10.5]) and median OS was 15.6 months (95% CI [11.7-19.6]). Among 20 patients with available tumor samples for molecular analyses, only 4 (20%) presented with H3K27M mutation. Patients with H3K27M mutation had longer survival compared to those without. Finally, we identified a long-term survivor population characterized by a younger age, no cognitive impairment, low steroid dose treatment and the presence of H3K27M mutation. CONCLUSION: Thalamic adult glioma differs from bithalamic glioma in children with regards to its clinical, radiological and molecular profiles. Long-term survival is observed in young patients with limited symptoms and H3K27M mutation. A larger prospective cohort is needed to validate these findings.

Machine learning random forest for predicting oncosomatic variant NGS analysis.

Since 2017, we have used IonTorrent NGS platform in our hospital to diagnose and treat cancer. Analyzing variants at each run requires considerable time, and we are still struggling with some variants that appear correct on the metrics at first, but are found to be negative upon further investigation. Can any machine learning algorithm (ML) help us classify NGS variants? This has led us to investigate which ML can fit our NGS data and to develop a tool that can be routinely implemented to help biologists. Currently, one of the greatest challenges in medicine is processing a significant quantity of data. This is particularly true in molecular biology with the advantage of next-generation sequencing (NGS) for profiling and identifying molecular tumors and their treatment. In addition to bioinformatics pipelines, artificial intelligence (AI) can be valuable in helping to analyze mutation variants. Generating sequencing data from patient DNA samples has become easy to perform in clinical trials. However, analyzing the massive quantities of genomic or transcriptomic data and extracting the key biomarkers associated with a clinical response to a specific therapy requires a formidable combination of scientific expertise, biomolecular skills and a panel of bioinformatic and biostatistic tools, in which artificial intelligence is now successful in developing future routine diagnostics. However, cancer genome complexity and technical artifacts make identifying real variants challenging. We present a machine learning method for classifying pathogenic single nucleotide variants (SNVs), single nucleotide polymorphisms (SNPs), multiple nucleotide variants (MNVs), insertions, and deletions detected by NGS from different types of tumor specimens, such as: colorectal, melanoma, lung and glioma cancer. We compared our NGS data to different machine learning algorithms using the k-fold cross-validation method and to neural networks (deep learning) to measure the performance of the different ML algorithms and determine which one is a valid model for confirming NGS variant calls in cancer diagnosis. We trained our machine learning with 70% of our data samples, extracted from our local database (our data structure had 7 parameters: chromosome, position, exon, variant allele frequency, minor allele frequency, coverage and protein description) and validated it with the 30% remaining data. The model offering the best accuracy was chosen and implemented in the NGS analysis routine. Artificial intelligence was developed with the R script language version 3.6.0. We trained our model on 70% of 102,011 variants. Our best error rate (0.22%) was found with random forest machine learning (ntree = 500 and mtry = 4), with an AUC of 0.99. Neural networks achieved some good scores. The final trained model with the neural network achieved an accuracy of 98% and an ROC-AUC of 0.99 with validation data. We tested our RF model to interpret more than 2000 variants from our NGS database: 20 variants were misclassified (error rate < 1%). The errors were nomenclature problems and false positives. After adding false positives to our training database and implementing our RF model routinely, our error rate was always < 0.5%. The RF model shows excellent results for oncosomatic NGS interpretation and can easily be implemented in other molecular biology laboratories. AI is becoming increasingly important in molecular biomedical analysis and can be very helpful in processing medical data. Neural networks show a good capacity in variant classification, and in the future, they may be useful in predicting more complex variants.

Critical Assessment in Routine Clinical Practice of Liquid Biopsy for EGFR Status Testing in Non-Small-Cell Lung Cancer: A Single-Laboratory Experience (LPCE, Nice, France).

BACKGROUND: The introduction of liquid biopsy using PCR-based assays into routine practice has had a strong impact on the treatment of EGFR-mutated lung adenocarcinoma and is now commonly used for routine testing of EGFR mutations in certain clinical settings. To assess whether the claimed benefits of PCR-based assays hold true in daily practice at a multicenter clinical institution, we assessed how treatment decisions are affected by PCR-based assays for the analysis of EGFR mutations from plasma samples in a centralized laboratory (LPCE, Nice, France). PATIENTS AND METHODS: A total of 345 samples were analyzed using the US Food and Drug Administration-approved Cobas EGFR Mutation Test v2 and 103 using the Therascreen EGFR Plasma RGQ PCR Kit over 3 years (395 samples from 324 patients). Eleven plasma samples were validated independently using Cobas at 3 institutions, and 130 samples were analyzed using Stilla digital PCR. Clinical data were collected for 175 (54%) of 324 patients. RESULTS: Cobas was superior to the Therascreen assay and demonstrated 100% reproducibility. Digital PCR showed only 48%, 83%, and 58% concordance with Cobas for exon 19 deletions, L858R mutations, and T790M mutations, respectively. Liquid biopsies helped inform and change treatment when resistance occurred and enabled the detection of EGFR mutations in patients when biopsy tissue results were unavailable. CONCLUSION: PCR-based assays are a fast and convenient test, allowing the detection of primary and secondary EGFR mutations from plasma. Cobas proved to be a reliable test, whereas digital PCR produced too many inconclusive results to be currently recommended as a principal testing device.

High MET Overexpression Does Not Predict the presence of MET exon 14 Splice Mutations in NSCLC: Results From the IFCT PREDICT.amm study.

INTRODUCTION: MET proto-oncogene (MET) exon 14 splice site (METex14) mutations were recently described in NSCLC and has been reported to correlate with efficacy of MET tyrosine kinase inhibitors. High diversity of these alterations makes them hard to detect by DNA sequencing in clinical practice. Because METex14 mutations induce increased stabilization of the MET receptor, it is anticipated that these mutations are associated with MET overexpression. We aim to determine whether NSCLC with high MET overexpression could define a subset of patients with a high rate of METex14 mutations. METHODS: From The French Cooperative Thoracic Intergroup PREDICT.amm cohort of 843 consecutive patients with a treatment-naive advanced NSCLC who were eligible for a first-line therapy, 108 NSCLC samples with high MET overexpression defined by an immunochemistry score 3+ were tested for METex14 mutations using fragment length analysis combined with optimized targeted next-generation sequencing. MET copy number analysis was also derived from the sequencing data. RESULTS: METex14 mutations were detected in two patients (2.2%) who also displayed a TP53 mutation and a PIK3CA mutation, respectively. An MET gene copy number increase was observed in seven additional patients (7.7%). Next-generation sequencing analysis revealed inactivating mutations in TP53 (52.7%) and PTEN (1.1%), and oncogenic mutations in KRAS (28.6%), EGFR (7.7%), PIK3CA (4.4%), BRAF (4.4%), NRAS (2.2%), GNAS (1.1%), and IDH1 (1.1%). CONCLUSIONS: The rate of METex14 mutations in NSCLC with high MET overexpression was similar to that found in unselected NSCLC. Moreover, we observed a high frequency of driver alterations in other oncogenes. Consequently these findings do not support the use of MET immunohistochemistry as a surrogate marker for METex14 mutations.

Efficacy of Immune Checkpoint Inhibitors in KRAS-Mutant Non-Small Cell Lung Cancer (NSCLC).

INTRODUCTION: KRAS mutation is the most frequent molecular alteration found in advanced NSCLC; it is associated with a poor prognosis without available targeted therapy. Treatment options for NSCLC have been recently enriched by the development of immune checkpoint inhibitors (ICIs), and data about its efficacy in patients with KRAS-mutant NSCLC are discordant. This study assessed the routine efficacy of ICIs in advanced KRAS-mutant NSCLC. METHODS: In this retrospective study, clinical data were extracted from the medical records of patients with advanced NSCLC treated with ICIs and with available molecular analysis between April 2013 and June 2017. Analysis of programmed death ligand 1 (PD-L1) expression was performed if exploitable tumor material was available. RESULTS: A total of 282 patients with ICI-treated (in the first line or more) advanced NSCLC (all histological subgroups) who were treated with ICIs (anti-programmed death 1, anti-PD-L1, or anti-cytotoxic T-lymphocyte associated protein 4 antibodies), including 162 (57.4%) with KRAS mutation, 27 (9.6%) with other mutations, and 93 (33%) with a wild-type phenotype, were identified. PD-L1 analysis was available for 128 patients (45.4%), of whom 45.3% and 19.5% had PD-L1 expression of 1% or more and 50%, respectively (49.5% and 21.2%, respectively, in the case of the 85 patients with KRAS-mutant NSCLC). No significant difference was seen in terms of objective response rates, progression-free survival, or overall survival between KRAS-mutant NSCLC and other NSCLC. No significant differences in overall survival or progression-free survival were observed between the major KRAS mutation subtypes (G12A, G12C, G12D, G12V, and G13C). In KRAS-mutant NSCLC, unlike in non-KRAS-mutant NSCLC, the efficacy of ICIs is consistently higher, even though not statistically significant, for patients with PD-L1 expression in 1% or more of tumor cells than for those with PD-L1 expression in less than 1% of tumor cells, and this finding is especially true when PD-L1 expression is high (PD-L1 expression ≥50%). CONCLUSION: For patients with KRAS-mutant NSCLC (all mutational subtypes), the efficacy of ICI is similar to that for patients with other types of NSCLC. PD-L1 expression seems to be more relevant for predicting the efficacy of ICIs in KRAS-mutant NSCLC than it is in other types of NSCLC.

Proof of concept: prognostic value of the plasmatic concentration of circulating cell free DNA in desmoid tumors using ddPCR.

Since desmoid tumors (DT) exhibit an unpredictable clinical course, with stabilization and/or spontaneous regression, an initial "wait-and-see" policy is the new standard of care-thus, the actual challenge is to identify early factors of progression. We present a method of detection of CTNNB1 mutations using a targeted digital droplet PCR (ddPCR) on cell-free DNA (cfDNA) extracted from blood samples of 31 DT patients. Furthermore, we analyzed the correlation between DT evolution and plasmatic concentration of total and mutated cfDNA at the time of diagnosis. Circulating copies of CTNNB1 mutants (ctDNA) were detected in the plasma of 6 patients (33%) but their concentration was not correlated with evolution of the tumor. Concentration of total cfDNA was higher in the plasma of patients with progressive desmoids (p = 0,0009). Using a threshold <900 copies/mL of plasma to detect indolent desmoid and a threshold >1375, it was possible to predict desmoid evolution for 65% of patients by measuring the quantity of circulating DNA in their plasma as early as the time of diagnosis. Albeit showing that the detection of CTNNB1 mutants is possible in the plasma of patients harboring a desmoid tumor, the results of this preliminary study raise the hypothesis that most of the circulating DNA detected in their plasma is derived from non-neoplastic cells, most likely normal neighboring tissues being actively invaded. Our results open the perspective of using cfDNA as a biomarker to predict prognosis at the time of diagnosis and assess tumor dynamics to optimize the treatment strategy.

EGFR and KRAS Mutations Predict the Incidence and Outcome of Brain Metastases in Non-Small Cell Lung Cancer.

BACKGROUND: Lung cancer is the leading cause of brain metastases (BM). The identification of driver oncogenes and matched targeted therapies has improved outcome in non-small cell lung cancer (NSCLC) patients; however, a better understanding of BM molecular biology is needed to further drive the process in this field. METHODS: In this observational study, stage IV NSCLC patients tested for EGFR and KRAS mutations were selected, and BM incidence, recurrence and patients' outcome were assessed. RESULTS: A total of 144 patients (142 Caucasian and two Asian) were selected, including 11.27% with EGFR-mutant and 33.10% with KRAS-mutant tumors, and 57.04% patients had developed BM. BM incidence was more frequent in patients with EGFR mutation according to multivariate analyses (MVA) (Odds ratio OR = 8.745 [1.743-43.881], p = 0.008). Among patients with treated BM, recurrence after local treatment was less frequent in patients with KRAS mutation (OR = 0.234 [0.078-0.699], p = 0.009). Among patients with untreated BM, overall survival (OS) was shorter for patients with KRAS mutation according to univariate analysis (OR = 7.130 [1.240-41.012], p = 0.028), but not MVA. CONCLUSIONS: EGFR and KRAS mutations have a predictive role on BM incidence, recurrence and outcome in Caucasian NSCLC patients. These results may impact the routine management of disease in these patients. Further studies are required to assess the influence of other biomarkers on NSCLC BM.

Changes in PlGF and MET-HGF expressions in paired initial and recurrent glioblastoma.

Angiogenesis is one of the key features of glioblastoma (GB). However, the use of anti-angiogenic therapies directed against vascular endothelial growth factor (VEGF) is limited by primary or acquired resistance. MET/HGF and PlGF signaling are involved in potential alternative escape mechanisms to VEGF pathway. Our objective was to explore the potential changes of MET/HGF and PlGF expression, comparing initial diagnosis and recurrence after radiotherapy-temozolomide (RT/TMZ). Paired frozen tumors from both initial and recurrent surgery after radio-chemotherapy were available for 28 patients. RNA expressions of PlGF, MET, and HGF genes were analyzed by RT-qPCR. PlGF expression significantly decreased at recurrence (p = 0.021), and expression of MET showed a significant increase (p = 0.011) at recurrence. RNA expressions of MET and HGF significantly correlated both at baseline and recurrence (baseline: p = 0.005; recurrence: p = 0.019). Evolutive profile (increasing versus decreasing expression at recurrence) of MET was associated with PFS (p = 0.002) and OS (p = 0.022) at recurrence, while the evolutive profile of HGF was associated with PFS at relapse (p = 0.049). Recurrence of GB after chemo-radiation could be associated with a variation in PlGF and MET expression. These results contribute to suggest a modification of the GB angiogenic process between initial diagnosis and recurrence.

Molecular heterogeneity of glioblastomas: does location matter?

Glioblastomas in adults are highly heterogeneous tumors that can develop throughout the brain. To date no predictive-location marker has been identified. We previously derived two glioblastoma cell lines from cortical and periventricular locations and demonstrated distinct transcriptomic profiles. Based on these preliminary results, the aim of this study was to correlate glioblastoma locations with the expression of ten selected genes (VEGFC, FLT4, MET, HGF, CHI3L1, PROM1, NOTCH1, DLL3, PDGFRA, BCAN). Fifty nine patients with newly diagnosed glioblastomas were retrospectively included. Tumors were classified into cortical and periventricular locations, which were subsequently segregated according to cerebral lobes involved: cortical fronto-parietal (C-FP), cortical temporal (C-T), periventricular fronto-parietal (PV-FP), periventricular temporal (PV-T), and periventricular occipital (PV-O). Gene expression levels were determined using RT-qPCR. Compared to cortical glioblastomas, periventricular glioblastomas were characterized by a higher expression of two mesenchymal genes, VEGFC (p = 0.001) and HGF (p = 0.001). Among cortical locations, gene expressions were homogeneous. In contrast, periventricular locations exhibited distinct expression profiles. PV-T tumors were associated with higher expression of two proneural and cancer stem cell genes, NOTCH1 (p = 0.028) and PROM1 (p = 0.033) while PV-FP tumors were characterized by high expression of a mesenchymal gene, CHI3L1 (p = 0.006). Protein expression of NOTCH1 was correlated with RNA expression levels. PV-O glioblastomas were associated with lower expression of VEGFC (p = 0.032) than other periventricular locations, whereas MET overexpression remained exceptional. These data suggest a differential gene expression profile according to initial glioblastoma location.

Recurrence of glioblastoma after radio-chemotherapy is associated with an angiogenic switch to the CXCL12-CXCR4 pathway.

Angiogenesis is one of the key features of glioblastoma (GBM). Our objective was to explore the potential changes of angiogenic factors in GBM between initial diagnosis and recurrence after radiotherapy-temozolomide (RT/TMZ). Paired frozen tumors from both initial and recurrent surgery were available for 29 patients. Screening of genes expressions related to angiogenesis was performed using RT- PCR arrays on 10 first patients. Next, RNA expressions of the selected genes were analyzed on all samples. Protein expression was examined by immunohistochemistry. The anti-tumor effect of AMD3100 (anti-CXCR4) was tested in GBM explants. In the screening step, the initial-recurrence expression changes contributed to a selection of seven genes (VEGFA, VEGFR2, VEGFR1, CXCL12, CXCR4, uPA HIF1α). By quantitative RT-PCR, RNA expressions of CXCR4 (p = 0.029) and CXCL12 (p = 0.107) were increased while expressions of HIF1α (p = 0.009) and VEGFR2 (p = 0.081) were decreased at recurrence. Similarly, CXCL12 protein expression tended to increase (p = 0.096) while VEGFR2 staining was decreased (p = 0.004) at recurrence. An increase of anti-tumoral effect was observed with the combination of AMD3100 and RT/TMZ versus RT/TMZ alone in GB explants. Recurrence of GB after chemo-radiation could be associated with a switch of angiogenic pattern from VEGFR2-HIF1α to CXCL12-CXCR4 pathway, leading to new perspectives in angiogenic treatment.

Expression of adrenomedullin in human colorectal tumors and its role in cell growth and invasion in vitro and in xenograft growth in vivo.

Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through calcitonin receptor-like receptor/receptor activity-modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). In this study, real-time quantitative reverse transcription demonstrated a significant expression of AM mRNA in tumor samples from colorectal cancer (CRC) patients in clinical stage II, III, and IV when compared with normal colorectal tissue. AM, CLR, RAMP2, and RAMP3 proteins were immunohistochemically localized in the carcinomatous epithelial compartment of CRC tissue. Tissue microarray analysis revealed a clear increase of AM, CLR, RAMP2, and RAMP3 staining in lymph node and distant metastasis when compared with primary tumors. The human colon carcinoma cells HT-29 expressed and secreted AM into the culture medium with a significant increase under hypoxia. Treatment of HT-29 cells with synthetic AM stimulated cell proliferation and invasion in vitro. Incubation with anti-AM antibody (αAM), anti-AM receptors antibodies (αAMR), or AM antagonist AM22-52 inhibited significantly basal levels of proliferation of HT-29 cells, suggesting that AM may function as an autocrine growth factor for CRC cells. Treatment with αAM significantly suppressed the growth of HT-29 tumor xenografts in vivo. Histological examination of αAM-treated tumors showed evidence of disruption of tumor vascularity with decreased microvessel density, depletion of endothelial cells and pericytes, and increased tumor cell apoptosis. These findings highlight the potential importance of AM and its receptors in the progression of CRC and support the conclusion that αAM treatment inhibits tumor growth by suppression of angiogenesis and tumor growth, suggesting that AM may be a useful therapeutic target.

Pharmacogenetic profiling and cetuximab outcome in patients with advanced colorectal cancer.

BACKGROUND: We analyzed the influence of 8 germinal polymorphisms of candidate genes potentially related to EGFR signalling (EGFR, EGF, CCND1) or antibody-directed cell cytotoxicity (FCGR2A and FCGR3A) on outcome of colorectal cancer (CRC) patients receiving cetuximab-based therapy. METHODS: Fifty-eight advanced CRC patients treated with cetuximab-irinotecan salvage therapy between 2001 and 2007 were analyzed (mean age 60; 50 PS 0-1). The following polymorphisms were analyzed on blood DNA: EGFR (CA repeats in intron 1, -216 G > T, -191C > A, R497K), EGF (A61G), CCND1 (A870G), FCGR2A (R131H), FCGR3A (F158V). Statistical analyses were conducted on the total population and on patients with wt KRas tumors. All SNPs were considered as ternary variables (wt/wt vs wt/mut vs mut/mut), with the exception of -191C > A EGFR polymorphism (AA patient merged with CA patients). RESULTS: Analysis of skin toxicity as a function of EGFR intron 1 polymorphism showed a tendency for higher toxicity in patients with a low number of CA-repeats (p = 0.058). CCND1 A870G polymorphism was significantly related to clinical response, both in the entire population and in KRas wt patients, with the G allele being associated with a lack of response. In wt KRas patients, time to progression (TTP) was significantly related to EGFR -191C > A polymorphism with a longer TTP in CC patients as compared to others, and to CCND1 A870G polymorphism with the G allele being associated with a shorter TTP; a multivariate analysis including these two polymorphisms only retained CCND1 polymorphism. Overall survival was significantly related to CCND1 polymorphism with a shorter survival in patients bearing the G allele, and to FCGR3A F158V polymorphism with a shorter survival in VV patients (in the entire population and in KRas wt patients). FCGR3A F158V and CCND1 A870G polymorphisms were significant independent predictors of overall survival. CONCLUSIONS: Present original data obtained in wt KRas patients corresponding to the current cetuximab-treated population clearly suggest that CCND1 A870G polymorphism may be used as an additional marker for predicting cetuximab efficacy, TTP and overall survival. In addition, FCGR3A F158V polymorphism was a significant independent predictor of overall survival.

Mutation analysis of BRAF exon 15 and KRAS codons 12 and 13 in Moroccan patients with colorectal cancer.

BACKGROUND: The RAS/RAF/MEK/MAP kinase cascade transduces signals from the cell surface to the nucleus in order to control cellular responses including proliferation, differentiation and survival. We investigated the occurrence of BRAF exon 15 and KRAS codon 12 and 13 mutations in Moroccan patients with colorectal cancer. METHODS: Sixty-two samples from patients with sporadic colorectal adenocarcinomas were studied for BRAF exon 15 and KRAS codon 12 and 13 mutations. DNA from paraffin-embedded tissue specimens was analyzed by a combination of polymerase chain reaction-high resolution melting and direct sequencing. RESULTS: Of the analyzed specimens, 29% exhibited KRAS codon 12 or 13 mutations and only 1.6% carried a BRAF codon 600 mutation. KRAS mutations were more often observed in women (35.5%) than in men (22.6%). Patients in the age range between 41 and 60 years were more likely to be carriers of this mutation. No KRAS mutations were detected in patients aged >60 years. CONCLUSION: Despite the limited study sample, our data suggest that KRAS mutations arise more frequently than BRAF mutations in Moroccan patients with colorectal carcinomas. The KRAS mutation status must be assessed in a large cohort of Moroccan patients to confirm these findings and to determine whether this mutation in combination with extrinsic, environmental or microenvironmental factors might be involved in the high frequency of colorectal cancer in middle-aged Moroccans.

Epidermal growth factor receptor in glioblastomas: correlation between gene copy number and protein expression.

Epidermal growth factor receptor is a transmembrane receptor involved in oncogenesis, including the development of glioblastoma. We studied the prognostic significance of epidermal growth factor receptor amplification as determined by fluorescence in situ hybridization, quantitative polymerase chain reaction, and protein expression by immunohistochemistry. Ninety-nine patients exhibiting glioblastoma were included. Immunohistochemistry was performed on microarray blocks with clone 25, which recognizes both epidermal growth factor receptor wild type and vIII, and scored using a semiquantitative approach. Quantitative polymerase chain reaction and fluorescence in situ hybridization techniques were performed on frozen section: 29.3% of cases had a high epidermal growth factor receptor immunohistochemistry score (score >/=200); and of these cases, 96.5% had gene amplification by fluorescence in situ hybridization and quantitative polymerase chain reaction. Conversely, of cases with a low immunohistochemistry score, 72.9% had normal karyotype or polysomy 7 by fluorescence in situ hybridization technique; but around 25% had gene amplification by fluorescence in situ hybridization and quantitative polymerase chain reaction. In the case of protein overexpression, quantitative polymerase chain reaction and fluorescence in situ hybridization could be avoided in first intention because their positive predictive value for amplification is 97%. In multivariate analysis, there was a trend toward an association between shorter overall survival time and epidermal growth factor receptor amplification as determined by fluorescence in situ hybridization analysis. However, cases with an immunohistochemistry score less 200 require further testing by fluorescence in situ hybridization or quantitative polymerase chain reaction.

Toward understanding recurrent meningioma: the potential role of lysosomal cysteine proteases and their inhibitors.

OBJECT: The first aim of this study was to diagnose more aggressive and potentially recurrent meningiomas using an in vitro embryonic chick heart invasiveness assay in which lysosomal enzyme cathepsin B was used as the invasiveness marker. The second aim was to confirm if cathepsin B and/or cathepsin L and their endogenous inhibitors were also prognostic parameters in the clinical study of 119 patients with meningioma. METHODS: Primary meningioma cultured spheroids were "confronted" with embryonic chick heart spheroids in vitro, and cathepsin B was used as molecular marker to immunolabel the invasive tumor cells. In vitro invasion assays of the malignant meningioma cells were used to assess the invasive potential related to the cysteine cathepsins. As to the second aim, the possible association of cathepsin B along with selected molecular markers, cathepsin L, and endogenous cysteine protease inhibitors (stefins A and B and cystatin C) with meningioma malignancy was determined using enzyme-linked immunosorbent assays in tumor homogenates. Univariate and multivariate analyses were used to compare these parameters with established biological markers of meningioma recurrence in 119 patients with meningiomas. RESULTS: The more invasive tumors, which characteristically overgrew the normal tissue, were identified even within a group of histologically benign meningiomas. More intensive staining of cathepsin B in these tumors was not only found at the tumor front, but also in the invading pseudopodia of a single migrating tumor cells. Matrigel invasion of malignant meningioma cells was significantly altered by modulating cathepsin B activity and by stefin B silencing. In the clinical samples of meningioma, the levels of cathepsins B and L, stefin B, and cystatin C were highest in the tumors of higher histological grades, whereas stefin A and progesterone receptor were the only markers that were significantly increased and decreased, respectively, in WHO Grade III lesions. With respect to the prognosis of relapse, cathepsin L (p = 0.035), stefin B (p = 0.007), cystatin C (p = 0.008), and progesterone receptor (p = 0.049) levels were significant, whereas cathepsin B was not a prognosticator. As expected, WHO grade, age, and Simpson grade (complete tumor resection) were prognostic, with Simpson grade only relevant in the short term (up to 90 months) but not in longer-term follow-up. Of note, the impact of all these parameters was lost in multivariate analysis, due to overwhelming prognostic impact of stefin B (p = 0.039). CONCLUSIONS: The data indicate that the cysteine cathepsins and their inhibitors are involved in a process related to early meningioma recurrence, regardless of their histological classification. Of note, the known tumor invasiveness marker cathepsin B, measured in whole-tumor homogenates, was not prognostic, in contrast to its endogenous inhibitor stefin B, which was highly significant and the only independent prognostic factor to predict meningioma relapse in multivariate analysis and reported herein for the first time. Stefin B inhibition of local invasion was confirmed by in vitro invasion assay, although its other functions cannot be excluded.

High expression of cathepsin B and plasminogen activator inhibitor type-1 are strong predictors of survival in glioblastomas.

In contrast to pilocytic astrocytomas (WHO grade I gliomas) that are circumscribed and cured by surgical resection, invasion is a hallmark of grades II-IV gliomas. Proteases play a major role in the invasion process and correlations between glioma grading, survival and protease expression have been demonstrated. In this study, we have chosen to study using different technical approaches (Q-RT-PCR, in situ hybridization and immunohistochemistry) the expression of five molecules involved in extracellular matrix degradation (cathepsin B, MMP2, MMP9, uPA and PAI-1) in glioblastomas in order to determine their prognostic impact among grade IV gliomas. Pilocytic astrocytomas were used as controls. Q-RT-PCR showed that transcripts of uPA, PAI-1, cathepsin B and MMP9 were significantly more expressed in glioblastomas (n = 52), in comparison to pilocytic astrocytomas (n = 17) (P = 0.049, P < 0.0001, P = 0.03 and P < 0.0001, respectively). On both univariate and multivariate analyses, cathepsin B and PAI-1 were strong predictors of overall survival among the group of glioblastomas (P < 0.0001 and P = 0.01, respectively). Immunohistochemical expression of cathepsin B further confirmed its prognostic value in an independent cohort of patients with glioblastoma. In situ hybridization showed that uPA is detected at the invasive edge of glioblastomas, whereas PAI-1 is more abundant in microvascular proliferation and pseudo-palisading cells than at the infiltrative edges. These results suggest that cathepsin B and PAI-1 are important biomarkers for the stratification of glioblastoma patients with respect to survival.